Gupta, Toxicology of Organophosphate and Carbamate Compounds This book brings together the expertise of leading scientists from around the world on the complex toxicology of anticholinesterase compounds (Organophosphates and Carbamates). It provides the most up-to-date and in-depth knowledge on various aspects of OP and CM compounds, including their use, classification, mechanism-based toxicity, and prophylactic and therapeutic measurements. Anticholinesterase compounds constitute the largest number of chemicals that are primarily used as insecticides in agriculture, industry, and around the home/garden. Some OPs (nerve agents) have been used both in chemical warfare and terrorist attacks; while other OPs and CMs have been recommended as therapeutic agents in human and veterinary medicine. Many chemicals of both classes are extremely toxic and lack selectivity, thus their inadvertent and accidental use continues to pose a threat to human and animal health, aquatic systems, and wildlife. These are some of the contributing factors that make this class of agents so very important and will make this book a crucial reference work for every researcher dealing with these agents. 9 Extensively covers pesticides, nerve agents, therapeutic drugs, and flame retardants 9 Describes epidemiology of the world's major disasters involving Organophosphates and Carbamates 9 Covers animal, human, aquatic, and wildlife toxicity of Anticholinesterases 9 Insights into in-depth cholinergic and noncholinergic mechanisms of toxicity 9 Describes recent advancements in cholinesterases, paraoxonases, carboxylesterases, oxidative stress, endocrine disruption, cardiac and pulmonary toxicity, and carcinogenesis 9 Provides in vitro and in vivo models for neurotoxicity testing 9 Integrates knowledge of studies in lab animals and humans 9 Offers risk/safety assessment and national/international guidelines for permissible levels of pesticide residues 9 Describes management of Anticholinesterase poisoning in humans
"SCIENCE IS THE GREAT ANTIDOTE TO THE POISON OF ENTHUSIASM AND SUPERSTITION" Adam Smith (1723-1790)
TOXICOLOGY OF ORGANOPHOS PHATE AND CARBAMATE C O M P O U N D S Edited by RAMESH C. GUPTA
AMSTERDAM PARIS
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Elsevier Academic Press 30 Corporate Drive, Suite 400; Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald's Road, London WC1X 8RR, UK This book is printed on acid-free paper. Copyright 9 2006, Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail:
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Working together to grow libraries in developing countries www.elsevier.com I www.bookaid.org I www.sabre.org
Dedicated to My Beloved parents, the late Chandra Gupta and Triveni Devi Gupta, My Beloved wife, Denise, and Dear daughter, Rekha
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Contents
Contributors Foreword Kai M. Savolainen
xv
Pharmacokinetics & Metabolism 9. Physiologically Based Pharmacokinetic Modeling of O r g a n o p h o s p h o r u s and Carbamate Pesticides Charles Timchalk
Uses, A b u s e s , & E p i d e m i o l o g y 1. Introduction Ramesh C. Gupta
10. Metabolism of O r g a n o p h o s p h o r u s and Carbamate Pesticides Jun Tang, Randy L. Rose, and Janice E. Chambers
2. Classification and Uses of O r g a n o p h o s p h a t e s and Carbamates Ramesh C. Gupta 3. Therapeutic Uses of Cholinesterase Inhibitors in Neurodegenerative Diseases Randall L. Woltjer and Dejan Milatovic 4. Coadministration of Mernantine with Acetylcholinesterase Inhibitors. Preclinical and Clinical Evidence Andrzej Dekundy
1 1. Interspecies Variation in Toxicity of Cholinesterase Inhibitors Stephanie J. Garcia, Michael Aschner, and Tore Syversen
127
145
25
Esterases, Receptors, M e c h a n i s m s , & Tolerance Development
35
12. Structure and Function of Cholinesterases Zoran Radic and Palmer Taylor
5. Cholinesterase Inhibitors as Chemical Warfare Agents: Community Preparedness Guidelines AnnettaWatson, KulbirBakshi, Dennis Opresko, RobertYoung,VeroniqueHauschild, and Joseph King
47
6. O r g a n o p h o s p h a t e s and the Gulf War Syndrome Linda A. McCauley
69
7. The Bhopal Accident and Methyl Isocyanate Toxicity Daya R. Varma and Shree Mulay
79
8. Global Epidemiology of Organophosphate and Carbamate Poisonings Tetsuo Satoh
103
13. Cholinesterase Pharmacogenetics Roberta Goodall 14. M e t h o d s for Measuring Cholinesterase Activities in Human Blood Elsa Reiner and Vera Simeon-Rudolf 15. Interactions of O r g a n o p h o s p h o r u s and Carbamate C o m p o u n d s with Cholinesterases Lester G. Sultatos 16. Structure, Function, and Regulation of Carboxylesterases Masakiyo Hosokawa and Tetsuo Satoh
89
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161
187
199
209
219
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Contents
17. Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets David A. Jett and Pamela J. Lein 18. Paraoxonase Polymorphisms and Toxicity of O r g a n o p h o s p h a t e s Lucio G. Costa, TobyB. Cole,Annabella Vitalone, and Clement E. Furlong 19. Tolerance Development to Toxicity of Cholinesterase Inhibitors Frode Fonnum and Sigrun Hanne Sterri
233
247
257
21. Developmental Neurotoxicity of Organophosphates: A Case Study of Chlorpyrifos Theodore A. Slotlcin
24. Behavioral Toxicity of Cholinesterase Inhibitors Philip J. Bushnell and Virginia C. Moser 25. Peripheral Nervous System Effects and Delayed Neuropathy Angelo Moretto and Marcello Lotti 26. Intermediate S y n d r o m e in O r g a n o p h o s p h a t e Poisoning Jan L. De Bleecker
30. Dermal Absorption/Toxicity of O r g a n o p h o s p h a t e s and Carbamates Jim E. Riviere
399
411
423
271 32. Reproductive Toxicity of O r g a n o p h o s p h a t e and Carbamate Pesticides Suresh C. Sikka and Nilgun Gurbuz
447
293
22. In Vitro Models for Testing Organophosphate-lnduced Neurotoxicity and Remediation 315 Evelyn Tiffany-Castiglioni,VijayanagaramVenkatraj, Yongchang Qian, and James R. Wild 23. Electrophysiological Mechanisms in Neurotoxicity of O r g a n o p h o s p h a t e s and Carbamates Toshio Narahashi
29. Approaches to Defining and Evaluating the Inhalation Pharmacology and Toxicology Hazards of Anticholinesterases Harry Salem and Bryan Ballantyne
31. Local and Systemic Ophthalmic Pharmacology and Toxicology of O r g a n o p h o s p h a t e and Carbamate Anticholinesterases Bryan Ballantyne
O r g a n Toxicity 20. Central Nervous System Effects and Neurotoxicity Carey N. Pope
28. Pulmonary Toxicity of Cholinesterase Inhibitors 389 Corey J. Hilmas, Michael Adler, and StevenI. Baskin
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33. Placental Toxicity of O r g a n o p h o s p h a t e and Carbamate Pesticides Olavi Pelkonen, Kirsi V~ihiikangas,and Ramesh C. Gupta
463
34. Endocrine Disruption by Organophosphate and Carbamate Pesticides 481 Shigeyuki Kitamura, Kazumi Sugihara, and Nariaki Fujimoto 35. Organophosphates, Carbamates, and the Immune System Raghubir P. Sharma
495
347
N o n s p e c i f i c Toxic Effects 361
371
27. Cardiovascular Toxicity of Cholinesterase lnhibitors 381 Csaba K. Zoltani, G. D. Thome, and Steven I. Baskin
36. Oxidative Stress in AnticholinesteraseInduced Excitotoxicity Wolf-D. Dettbarn, Dejan Milatovic, and Ramesh C. Gupta 37. DNA Damage, Gene Expression, and Carcinogenesis by O r g a n o p h o s p h a t e s and Carbamates Manashi Bagchi, ShirleyZafra, and Debasis Bagchi
511
533
Contents 38. Temperature Regulation in Experimental Mammals and Humans Exposed to Organophosphate and Carbamate Agents Christopher J. Gordon, Cina M. Mack, and Pamela J. Rowsey
44. WHO/FAO Guidelines for CholinesteraseInhibiting Pesticide Residues in Food P. K. Gupta
ix
643
549
Aquatic Life & Wildlife 39. Occupational Toxicology and Occupational Hygiene Aspects of Organophosphate and Carbamate Anticholinesterases with Particular Reference to Pesticides Bryan Ballantyne and Harry Salem
45. Aquatic Toxicity of Carbamates and Organophosphates Arun K. Ray and Manik C. Ghosh 567
46. Toxidty of Organophosphorus and Carbamate Insecticides Using Birds as Sentinels for Terrestrial Vertebrate Wildlife Spencer R. Mortensen
657
673
Risk A s s e s s m e n t & Regulations 40. Public Health Impacts of Organophosphates and Carbamates Daphne B. Moffett 41. Cumulative Effects of Organophosphorus or Carbamate Pesticides Stephanie Padilla 42. Federal Regulations and Risk Assessment of Organophosphate and Carbamate Pesticides Anna B. Lowit 43. Regulatory Considerations in Developmental Neurotoxicity of Organophosphorus and Carbamate Pesticides Susan L. Makris
Analytical & Biomarkers 599
607
47. Analysis of Organophosphate and Carbamate Pesticides and Anticholinesterase Therapeutic Agents Anant V. Jain 48. Biomarkers of Organophosphate Exposure Oksana Lockridge and LawrenceM. Schopfer
681
703
617 Therapeutic M e a s u r e s 49. Management of Organophosphorus Pesticide Poisoning Timothy C. Marrs and J. AllisterVale
715
633 Index
735
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Contributors
MICHAEL ADLER Neurotoxicology Branch, Pharmacology Division, USAMRICD (US Army Medical Research Institute of Chemical Defense), APG-AE, MD
STEPHANIE J. GARCIA Wake Forest University School of Medicine, Department of Pharamacology, Winston Salem, NC ROBERTA GOODALL Cholinesterase Investigation Unit, Department of Clinical Biochemistry, North Bristol Trust, The Lewis Laboratory, Southmead Hospital, Bristol, UK
MICHAEL ASCHNER Department of Pediatrics, Vanderbilt University, Nashville, TN DEBASIS BAGCHI Interhealth Research Center, Benicia, CA, and School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE
CHRISTOPHER I. GORDON US Environmental Protection Agency, Neurotoxicolgy Division, Research Triangle Park, NC MANIK C. GHOSH Department of Animal Physiology, Bose Institute, Calcutta, India
MANASHI BAGCHI Interhealth Research Center, Benicia, CA, and School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE
P. K. GUPTA Toxicology Consulting Services Inc., Bareilly, India
KULBIR S. BAKSHI National Academy of Sciences, Committee on Toxicology, Washington, DC
RAMESH C. GUPTA Toxicology Department, Breathitt Veterinary Center, Murray State University, Hopkinsville, KY
BRYAN BALLANTYNE Charleston, WV
NILGUN GURBUZ Department of Urology, Tulane University Health Sciences Center, New Orleans, LA
STEVEN I. BASKIN Biochemical Pharmacology Branch, Pharmacology Division, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD
VERONIQUE HAUSCHILD Office of Emergency Management, US Environmental Protection Agency, Washington, DC COREY I- HILMAS Neurotoxicology Branch, Pharmacology Division, USAMRICD (US Army Medical Research Institute of Chemical Defense), Aberdeen Proving Ground-AE, MD
PHILIP J. BUSHNELL US Environmental Protection Agency, Neurotoxicology Division B 105-04, Research Triangle Park, NC JANICE E. CHAMBERS Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS
MASAKIYO HOSOKAWA Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Chiba, Japan
TOBY B. COLE University of Washington, Seattle, WA LUCIO G. COSTA University of Washington, Department of Environmental and Occupational Health Sciences, Seattle, WA
ANANT V. JAIN Toxicology Section, The University of Georgia, College of Veterinary Medicine, Athens Diagnostic Laboratory, Athens, GA
]. L. DE BLEECKER Ghent University Hospital, Neurology Department, Ghent, Belgium
DAVID A. JETIr National Institutes of Health, NINDS, Bethesda, MD
ANDRZEJ DEKHNDY Merz Pharmaceuticals GmbH, Preclinical Research and Development, Frankfurt Am Main, Germany
IOSEPH KING US Army Environmental Center, Aberdeen Proving Ground, MD
WOLF-D. DETI'BARN Vanderbilt University School Medicine, Department of Pharmacology, Nashville, TN
of
SHIGEYUKI KITAMURA Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
FRODE FONNUM Group of Molecular Neurobiology, Department of Biochemistry, Institute of Basal Medicine, University of Oslo, Oslo, Norway
PAMELA J. LEIN Oregon Health & Science University, Portland, OR OKSANA LOCKRIDGE University of Nebraska Medical Center, Eppley Institute, Omaha, NE
NARIAKI FUJIMOTO Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
MARCELLO LO'I'i'I Universita di Padova, Departimento di Medicina Ambientale e Sanita Pubblica, Padova, Italy
CLEMENT E. FURLONG University of Washington, Seattle, WA xi
x ii
Contributors
ANNA B. LOWIT US Environmental Protection Agency, Office of Pesticide Programs, Washington, DC
PAMELA I. ROWSEY School of Nursing, University of North Carolina, Chapel Hill, NC
CINA M. MACK Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC
HARRY SALEM USA SBCCOM, Edgewood CB Center, Aberdeen Proving Ground, MD
SUSAN L. MAKRIS US Environmental Protection Agency, Office of Research and Development, National Center for Environmental Assessment, Washington, DC
TETSIlO SATOH Laboratory of Biochemical Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, and HAB Research Laboratories, Ichikawa, Chiba, Japan
TIMOTHY C. MARRS University of Central Lancashire, UK and National Poisons Information Service, (Birmingham Center, UK)
KAI SAVOLAINEN Finnish Institute of Occupational Health, Department of Industrial Hygeine & Toxicology, Helsinki, Finland
LINDA A. Mr School of Nursing, Office of Nursing Research, University of Pennsylvania, Philadelphia, PA
LAWRENCE M. SCHOPFER University of Nebraska Medical Center, Eppley Institute, Omaha, NE
DEIAN MILATOVIC University of Washington, Seattle, WA
RAGHIIBIR P. SHARMA The University of Georgia, Department of Physiology & Pharmacology, College of Veterinary Medicine, Athens, GA
DAPHNE B. MOFFETY CDR US Public Health Service, Agency for Toxic Substances and Disease Registry, US Department of Health and Human Services, Atlanta, GA ANGELO MORETrO Universita di Padova, Departimento di Medicina Ambientale e Sanita Pubblica, Padova, Italy SPENCER IL MORTENSEN Syngenta Crop Protection, Inc., Ecological Sciences, Greensboro, NC VIRGINIA C. MOSER US Environmental Protection Agency, Neurotoxicology Division, Research Triangle Park, NC SHREE MILLAY Department of Medicine, McGill University, Montreal, Canada TOSHIO NARAHASHI Northwestern University, The Feinberg School of Medicine, Department of Molecular Pharmacology and Biological Chemistry, Chicago, IL DENNIS OPRESKO Toxicology and Hazard Assessment Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN STEPHANIE PADILLA US Environmental Protection Agency, Neurotoxicolgy Division B 105-06, NHEERL, Office of Research and Development, Research Triangle Park, NC
SURESH C. SIKKA Tulane University Health Sciences Center, New Orleans, LA VERA SIMEON-RHDOLF Institute for Medical Research and Occupational Health, Ksaverska cesta 2, Zagreb, Croatia THEODORE A. SLOTKIN Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC SIGRIIN HANNE STERRI Division for Protection, Norwegian Defence Research Establishment, Institutt Veien, Kjeller, Norway KAZUM! SUGIHARA Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan LESTER GRANT SULTATOS UMDNJ, Pharmacology and Physiology, Newark, NJ
Department
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TORE SYVERSEN Norwegian University of Science & Technology, Department of Neuroscience, Trondheim, Norway ]I.IN TANG Cerep Inc., Redmond, WA
OLAVI PELKONEN University of Oulu, Department of Pharmacology and Toxicology, Oulu, Finland
PALMER TAYLOR University of California at San Diego, Department of Pharmacology, La Jolla, CA
CAREY N. POPE Oklahoma State University, Department of Physiological Sciences, College of Veterinary Medicine, Stillwater, OK
GEORGE THORNE US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD
YONGCHANG QIAN Department of Integrative Biosciences, Texas A&M University, College Station, TX
EVELYN C. TIFFANY-CASTIGLIONI Department of Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX
ZORAN RADIC University of California at San Diego, Department of Pharmacology, La Jolla, CA
CHARLES TIMCHALK Center for Biological Monitoring and Modeling, Pacific Northwest National Laboratory, Richland, WA
ARHN K. RAY Department of Animal Physiology, Bose Institute, Calcutta, India
KIRS! VAH,~,KANGAS University of Kuopio, Department of Pharmacology and Toxicology, Kuopio, Finland
ELSA REINER Institute for Medical Research and Occupational Health, Ksaverska cesta 2, Zagreb, Croatia
I. ALLISTER VALE National Poisons Information Service (Birmingham Centre) and West Midlands Poisons Unit, City Hospital, Birmingham, UK
IlM E. RIVIERE North Carolina State University, College of Veternary Medicine, Center for Chemical Toxicology Research & Phamacokinetics, Raleigh, NC RANDY L. ROSE Department of Molecular and Environmental Toxicology, North Carolina State University, Raleigh, NC
DAYA R. VARMA Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada VIJAYANAGARAM VENKATRAJ Department of Integrative Biosciences, Texas A&M University, College Station, TX
Contributors ANNABELLA VITALONE University of Bad, Bad, Italy ANNETrA WATSON Toxicology and Hazard Assessment Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN JAMES rc WILD Department of Biochemistry and Biophysics, Center for Environmental and Rural Health, Texas A&M University, College Station, TX
xiii
RANDALL L. WOLTIER Department of Pathology, Division of Neuropathology, University of Washington, Seattle, WA ROBERTYOUNG Toxicology and Hazard Assessment Group, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN SHIRLEY ZAFRA Interhealth Research Center, Benicia, CA CSABA K. ZOLTANI US Army Research Lab, MD
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Foreword KAI M. SAVOLAINEN Finnish Institute of Occupational Health, Helsinki, Finland
Consumers are protected by the setting of acceptable daily intake values. In the European Union, a separate communitylevel directive, 91/414/EC, is currently under revision for plant protection products according to which the safety of all pesticides is assessed. Similar pieces of legislation, rigorously enforced by health and other authorities, are in place in other industrialized countries. In the United States, the use of pesticides is regulated by the Federal Insecticide, Fungicide and Rodenticide Act, which originated in 1947, and the Food Quality Protection Act passed by the U.S. Congress in 1996 (Ecobichon, 2001). In developing countries, the situation is often much worse, and the information provided in this book is badly needed. There is a clear need to resort to OP and CM insecticides use in many developing countries since they are situated in climates that in addition to favoring the growth of crops also provide optimal breeding conditions for insects capable of destroying crops or causing communicable diseases such as malaria (Ecobichon, 2001). However, because there is such a lack of knowledge and no adequate legislation and regulations, OPs and CMs and other toxic pesticides are often misused, the protective measures are largely inadequate, and safe handling of crops is often inappropriate. In developing countries, there is also a lack of adequate infrastructure, including regulatory authorities, to enforce regulations and thus protect individuals who come into contact with pesticides, even when legislation and regulations are theoretically in place and the educational system does provide the necessary knowledge base to assess risks associated with the use of these Compounds (Rantanen et al., 2004). The literature on OPs and CMs is seemingly exhaustive (Ecobichon, 2001; Krieger, 2001). However, because ACHEinhibiting insecticides, as well as other pesticides, require marketing permission, most of the descriptive and much of mechanistic toxicology research has been carried out by the companies that manufacture these compounds. These data are often not publicly available, and much information
Highly toxic acetylcholinesterase (AChE)-inhibiting pesticides, organophosphates (OPs) and carbamates (CMs), are intensively used throughout the world and continue to be responsible for poisoning epidemics, especially in developing countries (e.g., Central American countries) (Wesseling et al., 2005). In industrialized countries, highly toxic OPs and CMs, and other toxic pesticides, are usually much better controlled, and the likelihood of occupational and other poisonings is relatively small. However, there is a continuous need to carefully assess the risks caused by the exposure and use in the occupational environment to workers as well as generally to consumers and other exposed groups. Although the literature on OP and CM insecticides is seemingly exhaustive and systematic, this is not the case. For example, there is really no truly comprehensive and in-depth analysis available on the toxicological data of the common AChE-inhibiting compounds, OPs and CMs. This book promises a welcome improvement by providing comprehensive coverage of the toxicology of these important group of pesticides. To my knowledge, this book is the first in-depth analysis of data on toxicology, risk assessment, and management, as well as the importance of OPs and CMs to society. I believe that this book will soon be on the bookshelves of researchers in academia and industry and risk assessors and managers within regulatory authorities. However, it would also be beneficial if we could convey the information contained in this book to the attention of policymakers and political and industrial decision makers and in this way multiply its impact in society and preserve human health. In this context, there are some issues that merit special consideration and that are relevant to this book in the field of toxicology and risk assessment of AChE inhibitors. In industrialized countries, highly toxic pesticides, including OPs and CMs, are regulated and controlled in the work environment via occupational exposure limits and by restrictions or bans on the use of the most toxic compounds. xv
xvi
Foreword
regarding toxicity of OPs and CMs is missing in the open literature. Therefore, there is a plethora of studies exploring mechanisms of acute and long-term toxicity of these compounds in experimental animals and man, but other areas are covered only vaguely, which means in effect that there is a very inconsistent database. In practice, this means that when an "old" AChE-inhibiting insecticide or another old pesticide is subjected to reevaluation for remarketing permission, the data available to the risk assessors are far from complete and additional studies are often required. Alternatively, the data may be old or scattered throughout the open literature and, hence, laborious to find. This book will improve the situation remarkably for evaluating the OP and CM pesticides. To carry out a comprehensive analysis of the often inconsistent and incomplete database on OPs and CMs is often a highly demanding task for risk assessors and decision makers. The situation is especially difficult if one wishes to combine analytical thinking with professional risk assessment and management that also takes into consideration societal impact, perception of risks, and the significance of these compounds to society. The editor, authors, and publisher of this book have decided to face this challenge by publishing this unique book dealing in an in-depth manner with various aspects of the toxicology of OPs and CMs. As indicated previously, there is a plethora of original papers and reviews on the toxicology of this group of compounds, especially on the mechanisms of immediate toxic actions of these compounds. Much of this information is based on studies dealing with AChE-inhibiting warfare agents, such as soman, tabun, and sarin (Savolainen, 2001). Although this information is important for protecting the general public from terrorist attacks, an even more important goal is to create a comprehensive systematic database on the toxicity of these compounds to allow a reliable and exhaustive assessment of their risks to workers and consumers and other exposed individuals. This book is unique in being able to provide a thorough assessment of all aspects of toxicology and risk assessment of OP and CM AChE-inhibiting compounds. The list of authors of the book is impressive - - the editor is to be congratulated for bringing together such a unique group of experts from various fields of OP and CM toxicology and risk assessment. The book is divided into nine sections that deal with different aspects of OP and CM toxicology and risk assessment. I am especially pleased by Section I, with its chapters on therapeutic uses, community preparedness, and epidemiology of OP and CM compounds. In Section III, noncholinesterase mechanisms of central and peripheral neurotoxicity, paraoxonase polymorphisms, and the development of tolerance are topics of special interest. The main body of the book (Section IV) discusses organ toxicity in especially interesting chapters dealing with in vitro testing, reproduction, placental toxicity, endocrinology, and effects on the immune system. In
Section V, special-interest areas are covered, and items of personal interest to me are those dealing with oxidative stress, DNA damage and gene expression, and occupational toxicology and hygiene. Special merits of the book, covered in Section VI, are the chapters that deal with in-depth risk assessment and risk management. These issues are crucial to ensure that the research conducted on the toxicology of OPs and CMs actually has an impact on human health and society at large. Another merit of the book is found in Section VII, in which issues dealing with ecotoxicology are reviewed in the context of human toxicology. Ultimately, human toxicology and ecotoxicology of OPs and CMs are interrelated and inseparable. A novel topic is introduced in Section VIII, a discussion on biomarkers of OP exposure. This topic has direct relevance both to human exposure and to effective assessment and, therefore, it holds major potential for prevention of risks induced by OPs. This book would not be a comprehensive presentation on issues relevant to OPs and CMs without a chapter on management of OP poisonings, and fortunately this issue is well covered in Section IX. The book is an extremely welcome addition to the literature on the toxicology and risk assessment of OPs and CMs. The chapters mentioned previously are personal choices and issues that I consider to be of special interest. As a whole, the book provides a thorough and analytical coverage of issues important in the field, without omitting anything of importance, and emphasizes new issues that have not been assessed in previous textbooks or reviews. Thus, it is a credit that this book has achieved its goal, to review the toxicology of OPs and CMs, in an indepth and comprehensive manner, including important and novel issues such as placental and reproductive toxicology and the effects of these compounds on the immune system. This book will be intensively used by not only scientists and teachers in academia, scientists in the industry, and regulators and decision makers but also students, who should be encouraged to study and learn from its wisdom.
References Ecobichon, D. J. (2001). Toxic effect of pesticides. In Casarett and Doull's Toxicology. The Basic Science of Poisons (C.D. Klaassen, Ed.), 6th ed., pp. 763-810. McGraw-Hill, New York. Krieger, R. (Ed.) (2001). Handbook of Pesticide Toxicology. Principles, 2nd ed. Academic Press, San Diego. Rantanen, J., Lehtinen, S., and Savolainen, K. (2004). The opportunities and obstacles to collaboration between the developing and developed countries in the field of occupational health. Toxicology 198, 63-74. Savolainen, K. (2001). Understanding the toxic actions of organophosphates. In Handbook of Pesticide Toxicology. Principles (R. Krieger, Ed.), 2nd ed., pp. 1013-1042. Academic Press, San Diego. Wesseling, C., Corriols, M., and Bravo, V. (2005). Acute pesticide poisoning and pesticide registration in Central America. Toxicol. AppL Pharmacol., 207, $697-$705.
Uses, Abuses, & Epidemiology
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CHAPTER
|
Introduction
RAMESH C. GUPTA Murray State University, Hopkinsville, Kentucky
By the turn of the 21st century, the development and use of organophosphate (OP) and carbamate (CM) compounds were greater than ever before. This trend will most likely continue because scientists are discovering new applications for these compounds. Both OPs and CMs are inhibitors of acetylcholinesterase (AChE) enzyme, which terminates the action of the neurotransmitter acetylcholine (ACh). Compounds with strong AChE-inhibiting potential are used as toxicants (e.g., pesticides and nerve agents), whereas those with weak ACHEinhibiting potential are used as prophylactic agents against nerve agent poisoning or as therapeutic agents in conditions such as glaucoma, myasthenia gravis, and Alzheimer's disease. In addition, some of these compounds are used as flame retardants, whereas others are misused in intentional and malicious poisonings. Currently, AChE-inhibiting compounds constitute the largest class of pesticides used in both industrialized and developing nations. The history and evolution of OPs and CMs is very interesting and intriguing. The earliest documentation of the synthesis of an OP compound, tetraethyl pyrophosphate, was in 1854 when Phillipe de Clermont presented a report to the French Academy of Sciences. Nearly 80 years later, in 1932, Lange and Kruger described the synthesis of dimethyl and diethyl phosphorofluoridate and noted that inhalation of their vapors produced dimness of vision and a choking sensation. Probably, these observations led Gerhard Schrader, a chemist, to the exploration of the OP class of compounds while he was engaged in the development of insecticides for I. G. Farbenindustrie. One of the earliest OP insecticides synthesized by Schrader was parathion, which is still commonly used worldwide. Prior to World War II (WWII), under the authority of the German Ministry of Defense, his priority shifted from insecticides to chemical warfare agents. The result was the development of diisopropyl phosphorofluoridate and then the development of considerably more toxic OP compounds of the G series (tabun, satin, and soman), which were intended to be used as nerve gases/agents. In the 1950s, agent VX was synthesized in the United Kingdom with a potency manyfold greater than nerve agents of the G series. At the same time, a compound of the V series (VR), with a supertoxicity, was synthesized by the Soviet military. In 1950, two separate accidental exposures occurred at Toxicology of Organophosphate and Carbamate Compounds
Dugway Proving Ground, wherein workers developed signs and symptoms, and laboratory evidence confirmed mild nerve agent exposure in a test area 3 days after a satin test. Since the 1980s, these agents have been used in wars and by dictators and terrorists. For example, sarin was used in Iraq against Kurdish villages in 1988 and in the Tokyo subway attacks in Japan in 1994 and 1995. Currently, many countries possess these deadly nerve agents, and in the current world situation the possibility exists that these compounds may be misused as chemical weapons of mass destruction (CWMD). In fact, OP nerve agents have received increasing attention as concerns about chemical warfare have intensified since the terrorist attack in the United States on September 11, 2001. After WWII, thousands of OP derivatives were synthesized worldwide in a search for compounds with species selectivity and less toxicity that could be used as insecticides more safely. In 1950, malathion was synthesized, and it has been the most popular insecticide for more than 50 years for use against certain insects, especially mosquitoes and medflies. Malathion has always been considered one of the safest OPs. However, in 1976 in Pakistan, out of 7500 spray men, 2800 became poisoned and 5 died from isomalathion that was produced during storage of formulated malathion. Many such poisoning incidences have occurred in the past with several other OPs due to their accidental or inadvertent use. Today, more than 100 OPs are in use for a variety of purposes, such as protection of crops, grains, gardens, and public health. Although OP insecticides are less toxic than the nerve agents, the illness they produce clinically resembles that produced by nerve agents. The knowledge of autonomic pharmacology, especially the cholinergic system, enabled us to understand the mechanism of toxicity of OPs and CMs. Subsequently, CMs such as aldicarb were synthesized based on the knowledge of ACh chemistry. These compounds were found to be the most toxic insecticides of the anti-AChE class. In the early 1950s, several nucleophilic agents (hydroxylamine, hydroxamic acid, and oximes) were developed as antidotes for reactivation of inhibited AChE against OPs. With a thorough understanding of the chemistry of ACh, AChE, and OPs, pralidoxime was synthesized, which was found to have 1 million times greater potency than hydroxylamine for reactivation of inhibited Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
4
S ECTI O N I 9Uses, Abuses, & Epidemiology
ACHE. Today, more than a dozen oximes are available, some of which are more effective against OP nerve agents and others are more effective against OP pesticides. The history of CMs is somewhat older than the history of OPs. In 1840, the Calabar bean (ordeal poison), from a perennial plant Physostigma venenosum, was brought to England from a tropical part of West Africa, where it was used for witchcraft. Approximately 25 years later, physostigmine (eserine alkaloid) was isolated by several investigators and used to treat glaucoma. Almost 50 years later, an aromatic ester of carbamic acid, neostigmine, was synthesized and used in the treatment of myasthenia gravis. It was not until the 1960s and 1970s that carbamates (esters of carbamic acid) were synthesized for pesticidal use. Today, CMs are preferred for pesticide use over OPs because some OPs have been found to be extremely toxic, whereas others induce delayed neuropathy in humans and animals. Carbaryl was the first CM compound used as an insecticide. Whereas OPs are irreversible ACHE inhibitors and extremely toxic, CMs are reversible ACHE inhibitors and therefore considered relatively less toxic. Although based on acute toxicity, some of the CMs, such as aldicarb, carbofuran, and many others, are extremely toxic. In 1984, an estimated 400,000 people were exposed to a toxic methyl isocyanate gas (used in the production of CM pesticides) that leaked from the Union Carbide plant in Bhopal, India. From this catastrophic incident, approximately 8000 humans and 4000 animals died. In 1985, an unprecedented outbreak of aldicarb poisoning occurred in which approximately 2000 California residents became sick due to consumption of contaminated melons. Since the early 1980s, both OPs and CMs have been used for multiple purposes, such as pesticides (crop and grain protection, indoors and around homes) and in veterinary (ectoparasiticides and endoparasiticides) and human medicine (in neurodegenerative diseases such as Alzheimer's disease). In human medicine, some of these compounds are also prescribed in myasthenia gravis and glaucoma and as prophylaxis to combat anticipated nerve agent poisoning. Like OPs, thousands of CMs have been synthesized, but not more than two dozen compounds have been used practically. In terms of volume, currently the use of CMs exceeds the use of OPs. OPs and CMs are the most commonly used pesticides throughout the world. This is partly due to their lack of residue persistence in the environment and in exposed individuals and also due to lesser resistance development in insects compared to the organochlorine pesticides. From the public health standpoint, in today's world the use of pesticides is a must rather than an option. For example, sporadic incidences of West Nile virus are reported in many countries, whereas malaria is still a major problem in developing countries. In both cases, the common vector is the mosquito. Without the use of pesticides against vectors of diseases, the impact on human and animal health would be devastating and the economic loss would be enormous. On the one hand, the world is greatly benefited from the use of
pesticides; on the other hand, pesticides are major contributors to environmental pollution. Many OPs and CMs are extremely toxic, and the majority of them lacks species selectivity and so, because of their global use, they constantly pose a threat to the environment, human and animal health, aquatic systems, and wildlife. It is important to note that OP and CM pesticides are encountered in intentional poisonings in humans and malicious poisonings in animals. Today, carbofuran is the pesticide most often associated with accidental and malicious poisoning in companion and domestic animals because of its widespread availability and extreme toxicity. Depending on the magnitude, frequency, and length of exposure, these compounds can produce minor health effects, such as mild discomfort or chest pain, or effects as severe as paralysis, coma, and death. The World Health Organization estimates that approximately 3 million people worldwide suffer from acute pesticide poisoning annually. By employing in vivo and in vitro models, these compounds are known to produce a variety of toxicological effects on the central nervous system, peripheral nervous system, cardiovascular, ocular, neurobehavioral, immunological, reproductive, placental, cutaneous, and other body systems, in addition to endocrine disruption, oxidative stress, and carcinogenesis. With the advent of sophisticated technologies, highly sensitive potentiometric and amperometric biosensors have been developed for qualitative and quantitative detection and monitoring of chemical warfare agents and OP and CM pesticides. Essentially, these biosensors aid in chemical and food safety, environmental monitoring, and agricultural production. During the past few years, investigators in the field of anticholinesterases have realized the need for a comprehensive compendium that can provide in-depth knowledge on various aspects of these compounds, including their use, toxicity, safety, regulations, and prophylactic and therapeutic measurements. This reference book, which is a collective work of approximately 100 subject experts from many countries, offers a plethora of cutting-edge knowledge on various aspects of OPs and CMs. The book is organized into nine sections with a total of 49 chapters. The editor and authors have made every effort to cite every important work in the field and avoid any duplication, but the possibility of some omissions and duplications certainly exists. Since OPs and CMs are used worldwide in agriculture, in gardens, in and around homes and offices, in therapeutic applications, in intentional and malicious poisonings, and possibly as CWMD, the book is intended for students and teachers; toxicologists; physicians; public health personnel and administrators; risk and safety assessors; local, state, federal, and international pesticide regulators and policy makers; industrial and agricultural watchdog groups; and medical, veterinary, and environmental advocacy groups. The editor truly appreciates the hard work and sincere efforts of each author, without which this book would not have been possible.
CHAPTER
Classification and Uses of O r g a n o p h o s p h a t e s and Carbamates RAMESH C. GUPTA Murray State University, Hopkinsville, Kentucky
I. INTRODUCTION Organophosphates (OPs) are a large class of chemicals. Since World War II, an estimated several thousand OPs have been synthesized for various purposes. The majority of these compounds are used as pesticides, whereas others are used as nerve agents, flame retardants, and parasiticides in veterinary medicine. Different OP compounds have structural similarities within classes. All OPs definitely share one thing in common: They all have a phosphorus atom and a characteristic phosphoryl bond ( ~ O ) or thiophosphoryl bond (P=S). Essentially, OPs are esters of phosphoric acid with varying combinations of oxygen, carbon, sulfur, or nitrogen attached. Of course, the chemistry of these compounds is much more complex and classification is somewhat confusing. In fact, complexity in classification of OPs arises due to different side chains attached to the phosphorus atom and the position at which the side chains are attached. More than 50 years ago, the Anglo-American system reached an agreement to adopt an "international nomenclature" instead of individual systems from four countries (British, Swedish, German, or American). However, none of the systems has ever been universally accepted. Compared to OPs, carbamate (CM) pesticides are of relatively recent origin and constitute another important group of pesticides. In addition to their use as pesticides, CMs are used as drugs of choice in human medicine against Alzheimer's disease, myasthenia gravis, and glaucoma and in veterinary medicine as parasiticides. Classification of CMs is simpler than classifying OPs. Some CMs have structural similarity with the neurotransmitter acetylcholine (ACh), and therefore they cause direct stimulation of ACh receptors, in addition to acetylcholinesterase (ACHE) inactivation. Although thousands of CMs have been synthesized, only a few dozen have practical utility. The classification of OPs and CMs presented in this chapter is based on their chemical structures and intended use or any syndrome they produce. Toxicology of Organophosphate and Carbamate Compounds
II. ORGANOPHOSPHATES Currently, there are hundreds of OP compounds in use, which are derivatives of phosphoric, phosphonic, or phosphinic acid. Throughout this chapter and the book, the term organophosphate is used as a generic term to include all the organic compounds containing phosphorus. These compounds are classified based on side chains and other elements attached to the phosphorus atom.
A. Types of Organophosphates There are at least 13 types of OPs, which are briefly presented in Table 1. The OPs that are derivatives of phosphoric or phosphonic acid possess anticholinesterase activity, unlike those that are derivatives of phosphinic acid. There are some OP compounds that do not conform to the structural requirement as shown in Table 1, but they possess anti-AChE activity. Usually, OP compounds have two alkyl substituents and an additional substituent group (leaving group), which is more labile to hydrolysis than the alkyl groups (Marrs, 1993). It is important to note that phosphorothioates (P---S) possess minimal or no anticholinesterase (anti-AChE) activity and require desulfuration to the analogous oxon before acquiring anti-AChE activity. Also, not all OPs exert anti-AChE activity, and therefore they are of low toxicity. For example, S,S,S-tributyl phosphorotrithioate and S,S,S-tributyl phosphorotrithioite (merphos), which are used as defoliants, and glyphosate and gluphosinate, which are used as herbicides, are of low mammalian toxicity.
B. OP Pesticides The majority of OP compounds are used as pesticides, and chemical descriptions for commonly used compounds are given in Table 2. Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
6
S ECTI O N I 9 Uses, Abuses, & Epidemiology
TABLE 1. Types of Organophosphates a Type
Chemical structure
Examples
O
Chlorfenvinphos Dichlorvos Monocrotophos Tri-o-cresyl phosphate Trichlorfon
Phosphates
II
RO~P~OR
I
OR O
Phosphonates
II
ROmPeR
I
OR O
Phosphinates
Glufosinate
II
R~P~R
I
OR
Phosphorothioates
(S--)
S
Bromophos Diazinon Fenthion Parathion Pirimiphos-methyl EPN Leptophos
II I
RO~P~OR OR
Phosphonothioates
(S--)
S
II I
ROmPeR OR
Phosphorothioates (S-substituted)
O
Demeton-S-methyl Echothiophate
II
RS~P~OR
I
OR
Phosphonothioates (S-substituted)
O
VX
II
RS~P~R
I
OR
Phosphorodithioates
O
II
RS~P~SR
or
I
OR S
II
RS~P~OR
I
OR O
Phosphorotrithioates
Azinphos-ethyl Azinphos-methyl Dimethoate Disulfoton Malathion Methidathion DEF (tribufos)
II
RS~P~SR
I
SR O
Phosphoramidates
II
/R
I OR
\R
RO ~ P ~ N
Phosphoramidothioates
S
II
/R
RO ~ P ~ N
I OR o
or
/a
I
\R
OR
Methamidophos Isofenphos
\R
II
RS ~ P ~ N
Fenamiphos
(continues)
CHAPTER 2 9Classification of OPs and CMs
7
TABLE 1. (continued) Type Phosphorofluoridates
Chemical structure O II RO~P~F
Examples Diisopropyl phosphorofluoridate (DFP)
I
OR
Phosphonofluoridates
O II
RO~P~F
I
R
Cyclosarin Sarin Soman
aAdapted fromMarrs (1993).
C. OP Nerve Agents/Gases Nerve agents of the OP group include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX. Soman, sarin, and cyclosarin are phosphonofluoridates, and VX is a phosphonothioate. Whereas soman has four isomers (C + P - , C - P - , C + P+, and C - P+), sarin and VX each have two isomers. V X is a mixture of two enantiomers resulting from the chiral center at the phosphorus atom, designated as P(+) and P ( - ) . There are significant differences in the reported toxicity and AChE inhibition rates of these isomers of nerve agents. Toxicological significance of stereoisomerism for other OPs, which have the potential of inhibiting ACHE, has been described (Battershill et al., 2004). This makes the ability to distinguish between them desirable for toxicological studies or the development of antidotal therapies (Benschop and De Jong, 1988; Smith, 2004; Ku~a and Kassa, 2004). Based on acute toxicity, VX is the most toxic compound among all the nerve agents. OP nerve agents are extremely toxic and have been used in wars and by terrorists on several occasions. They irreversibly inhibit the enzyme AChE at its active site. People who work at military sites where these nerve agents are stored may potentially be exposed. Soldiers and military personnel can be exposed to these compounds during war, and the general population can be exposed by accidental release from a military storage facility and during their transportation or destruction. For further details about OP nerve agents, see Chapter 5.
D. OPs Causing Delayed Neurotoxicity/ Neuropathy OP compounds that produce delayed neurotoxic effects are esters of phosphorus-containing acids. More than 35 years ago, tri-o-cresyl phosphate (TOCP) was known to produce delayed neurotoxic effects in humans and chickens characterized by ataxia and weakness of the limbs, developing 10-14 days after exposure (Johnson, 1969). This syndrome is called OP-induced delayed neuropathy (OPIDN). TOCP and certain other compounds have minimal or no anti-AChE
properties; however, they cause phosphorylation and aging (dealkylation) of a protein in neurons called neuropathy target esterase, and subsequently lead to OPIDN. Today, many compounds, such as diisopropyl phosphorofluoridate, N,N'diisopropylphosphorodiamidic fluoride (mipafox), tetraethyl pyrophosphate, paraoxon, parathion, o-cresyl saligenin phosphate, and haloxon, are known to produce this syndrome. For details of OPIDN syndrome, see Chapter 25.
E. OPs Causing Intermediate Syndrome OP insecticide-induced intermediate syndrome (IMS) was reported for the first time in human patients in Sri Lanka in 1987 (Senanayake and Karalliede, 1987), Since then, this syndrome has been diagnosed in OP-poisoned patients in South Africa (1989), Turkey (1990), Belgium (1992), the United States (1992), Venezuela (1998), France (2000), and elsewhere. IMS is usually observed in individuals who have ingested a massive dose of an OP insecticide either accidentally or in a suicide attempt. IMS is clearly a separate clinical entity from acute toxicity and delayed neuropathy. A similar syndrome has also been observed in dogs and cats poisoned maliciously or accidentally with massive doses of certain OPs. OPs that are known to cause IMS include bromophos, chlorpyrifos, diazinon, dicrotophos, dimethoate, fenthion, malathion, merphos, methamidophos, methyl parathion, monocrotophos, omethoate, parathion, phosmet, and trichlorfon. These compounds and IMS are discussed further in Chapter 26.
F. OPs Used as Flame Retardants Several OPs are used as fire retardants. Chemical structures of three commonly used compounds are as follows: 1. Tris (2-chloroethyl) phosphate (TCEP) O = P - (OCH2CH2C1)3 2. Tris (2-chloropropyl) phosphate (TCPP) O = P - (OCH2CHC1CH3)3 3. Tris (1,3-dichloroisopropyl) phosphate (TDCPP) O = P - (OCH(CH2C1)2)3
TABLE 2. A Brief Chemical Description of Commonly Used OP Pesticides ~
Chemical (CAS No.)
Chemical structure
Chemical name/ empirical formula
Molecular weight
Oral LD50 in rat (mglkg)
Dermal LD50 in rabbit (mglkg)
Acephate (30560- 19- 1)
0,S-dimethyl acetylamidothiophosphate C4Hl$rJ03PS
183.17
866
>2000
Azinphos-ethyl (2642-71-9)
0,O-diethyl S-[(4-0XO-1,2,3-benzotriazin3(4H)-yl) methyl] dithiophosphate
345.38
13
250
317.32
5
220
c12H16N303PS2 Azinphos-methyl (86-50-0)
0,O-dimethyl S-[(~-OXO1,2,3-benzotriazin3(4H)-yl) methyl] dithiophosphate ClOH12N303PS2
Bromophos (2104-96-3)
0-(4-bromo-2, 5dichlorophenyl) 0,O-dimethyl thiophosphate CgHgBrC1203PS
366.00
1600
2188
Cadusaphos (95465-99-9)
S,S-di-sec-butyl 0-ethyl dithiophosphate
270.40
391
143
342.87
6
22
c1OH2302PS2
Carbophenothion (78619-6)
S-{ [(4-chlorophenyl)thio]methyl} 0,O-diethyl dithiophosphate CllH16C102PS3 -i-
Chlorethoxyphos (54593-83-8)
Chlorfenvinphos (470-90-6)
0,O-diethyl 0-(1,2,2, 2-tetrachloroethyl) thiophosphate C6H11C1403PS
2-chloro-l-(2,4-dichloro-pheny1)
336.00
1.8
12.5
359.57
12
3200
350.59
135
2000
322.53
94 1
2000
362.77
13
314.27
125
vinyl diethyl phosphate
C12H14C1304P
Chlorpyrifos (2921-88-2)
0,O-diethyl
0-(3,5,6-trichloropyridin-2-y1)
thiophosphate CgHllC13N03PS
Q
Chlorpyriphos-methyl (5598-13-0)
0,O-dimethyl
0-(3,5,6-trichloropyridin-2-y1)
thiophosphate C7H7C13N03PS
Coumaphos (56-724)
Crotoxyphos (7700-17-6)
O-(3-chloro-4-methyl-2-oxo-
2H-chromen-7-yl) 0, 0-diethyl thiophosphate C14H16C105PS
1-phenylethyl (2E)-3-[(dimethoxyphosphory1)oxy]but-2-enoate
385
C14H1906P
(continues)
TABLE 2. (continued)
0
oI " \
0 -r
~ ~
r~
0-(4-~yanophenyl) 0,O-dimethyl thiophosphate CgHloN03PS
243.22
610
o
0,O-diethyl 0-[2-(ethy1thio)ethyll thiophosphate (C~H=JO)~PSOC~H~SC~H~
258.34
0,O-diethyl 0-(2-isopropyl6-methylpyrimidin-4-yl) thiophosphate
304.35
300 CD
379
220.98
25
~
59
237.19
22
~
223
229.26
250
400
r,.)
!
~ u
I 0
Z
~
c 12H21N203PS
0
co
"~.
d
2,2-dichlorovinyl dimethyl phosphate C4H7C1204P
m -r
o
ff \ I .0 O--n--O
0
o
0 y
II \
0
,.k, oi
Y-~
-1-
_~ o--z
oco
( 1 0 - 3-(dimethylamino)-
1-methyl-3-oxoprop-1-en-1-yl dimethyl phosphate o
I 0
z
0
~#o
C8H16N05P CD
0
0
C5H12N03PS2
.o
0,O-dimethyl S-[2-(methylamino)-2-oxoethyl] dithiophosphate ~ Eo~z
"I0 I "l-
00
m
I / O--n--O
z~(O
0/ \ "1I o-o o-I-
0 -I-
o
~9 o I
Dimethoate (60-51-5)
,-~
-r
\ /0
..%\ ~
o
o "1-
0
,.c:l~ 0
0
Dicrotophos (141-66-2)
9
o
-r"
0
0
#
0 I'T"
o ~ i"
"~
Dichlorvos (62-73-7)
8
o-J
/ \0
l..
"0 "T"
O" -r
I
e~
Diazinon (33341-5)
2.5
O0
co
=7~
5LJ
s 7
oV
N
i0 0
L
800
,Z
co
0 - - o ~:
o~
o
~o
e~
Demeton-0 (806548-3)
Dermal LD50 in rabbit (mgntg)
x:
Cyanophos (2636-26-2)
Chemical name/ empirical formula
=
Oral LD50 in rat (mg/kg)
0
"~ .=
Molecular weight
Chemical structure
@
Chemical (CAS No.)
"I0
274.40
2
o
~50-fold activity compared to BuChE. Phenserine has been tested in clinical trials for the treatment of AD, although no clinical data have been released. Ganstigmine (CHF 2819) is another novel orally active AChEI developed for the treatment of AD. It is a selective inhibitor of AChE (>115 times greater than against BuChE). Ganstigmine is also more selective for inhibition
TABLE 3. A Brief Chemical Description of Commonly Used CM Pesticides Chemical (CAS No.) Aldicarb ( 116-06-3)
Chemical structure/ empirical formula
Chemical name
(1E)-2-methyl-2-(methylthio)propanal 0-[(methyl-amino)carbonyl] oxime
Molecular weight 190.26
Oral LD50 in rat (mg/kg) 0.9
Dermal LD50 in rabbit (m€m)
5
C7H14N202S Aminocarb (2032-59-9)
4-(Dimethylamino)-3-methylphenyl methylcarbamate
208.26
30
275
223.23
34
566
410.53
138
>2000
422.87
340
4200
201.22
307
2000
CllH16N202 Bendiocarb (2278 1-23-3)
2,2-Dimethyl- 1,3-benzodioxo1-4-y1 methylcarbamate CllH13N04
L
OC,
Benfuracarb (82560-54-1)
2,3-Dihydro-2, 2-dimethyl-7-benzofuranyl N-[n[2-(ethylcarbonyl) ethyl]N-isopropyl sulfenamoyl]N-methylcarbamate C20H30N205S
BPMC (37 66-8 1-2)
2-sec-Butylphenyl N-methylcarbamate C12H17N02
Carbaryl (63-25-2)
1-Naphthyl methylcarbamate
C12H1lNO2
Carbofuran (1563-66-2)
2,2-Dimethyl-2, 3-dihydro-1-benzofuran-7-yl methylcarbamate
221.25
8
2550
380.55
209
>2000
225.31
200
1000
301.34
10,000
2000
193.24
450
225.31
15
C12H15N03
Carbosulfan (55285-14-8)
2,3-Dihydro-2, 2-dimethyl-7-benzofuranyl[(di-buty1amino)thioJmethyl carbamate C20H32N203S
Croneton (29973-1 3-5)
2-[(Ethylthio)methyl]phenyl methylcarbamate
CllH15N02S
Fenoxycarb (7 2490-0 1-8)
Ethyl [2-(4-phenoxyphenoxy)ethyl] carbamate C17H1gN04
Isoprocarb (2631-40-5)
Methiocarb (2032-65-7)
2-Isopropylphenylmethylcarbamate C11H15N03
3,5-Dimethyl-4-(rnethylthio)phenyl
2000
methylcarbarnate
CllH15N02S
(continues)
TABLE 3. (continued)
17
Dermal LD50 in rabbit (mg/kg) o o
162.21
"~..=
~
, _
o
"1-
co -r Ox- r Z
/
ko
O9
o--~
Methyl (lQ-N-{ [(methylamino) carbonylloxy ] ethanimidothioate
5000
C5H10N202S
268
i
165
0o c'q
~
-
3-Methylphenyl methylcarbamate o
Metolcarb (1129-4-5)
Oral LD50 in rat (mg/kg)
0
i.}
r,.)
Chemical name
=
~9-~
Methomyl (16752-77-5)
Molecular weight
=
e ~-=
Chemical structure1 empirical formula
Chemical (CAS No.)
.
~
C9H1I N 0 2
I ~
It~
0
219.26
~--
o o
5000
~
=~
I 0
C12H1SN202
r
Z --0
15
,--
/ Z -I-
0 09 -r
222.28
~
.
4-(Dimethylamino)-3, 5-dimethylphenyl methylcarbamate
~
0
o
Mexacarbate (315-184)
o
1-
Oco
20
'
"1~
710
=
o
~Z
5
o
-r 0
Methyl 2-(dimethyl-amino)N - { [(methyl-amino)carbonyl]oxy ) 2-oxoethan-imidothioate ~
Z
0
(-q
/
O9
1-
Oxamyl (23135-22-0)
z
CO
I
0
~0
"1-
C7H13N303S
"1-
>500
A
147
o o
238.29
,~
!
!
I
o z
/
"i-
o
o~
c1lH 1g N 4 0 2
4
r
O0
2-(Dimethylamino)-5, 6-dimethyl-pyrimidin-4-y l dimethy lcarbamate .. =
.0 0
Pirimicarb (23 103-98-2)
O9
-r 0
o
Z--O I
Promecarb (2631-37-0)
3-Isopropyl-5-methylphenyl methylcarbamate
207.27
61
>loo0
209.24
95
> 1000
193.24
125
>2000
179.22
542
179.22
384
C12H17N02
Propoxur (114-26-1)
2-Isopropoxyphenyl methylcarbamate CllH15N03
Trimethacarb (12407-86-2)
3,4,5-Trimethylphenyl methylcarbarnate CllH17N02
N XMC (2655-14-3)
3,s-Dimethylphenyl methylcarbamate CIOH13N02
Xylylcarb (2425-10-7)
3,4-Dimethylphenyl methylcarbarnate CgH10N402
22
S ECTI O N I 9Uses, Abuses, & Epidemioloooy O
H3~
O
~
CH3
H3C~O
OH3 DONEPEZIL
OH3
RIVASTIGMINE OH
O o"
H 3 c ~ O ~ "CH3 GALANTAMINE
H3C
/0
~CH3
NH2
OH
CI
CI
TRICHLORFON
TACRINE H3
O NH
~ O
~N
.H
.-
FIG. 1. Chemical structures of compounds indicated in Alzheimer's disease.
EPTASTIGMINE
of central (brain) AChE than peripheral (heart) AChE (Racchi et al., 2004). Chemical structures of some of the compounds that have been used in the past or are currently in use are shown in Fig. 1. These compounds are discussed in detail in Chapters 3 and 4.
B. Myasthenia Gravis From a pharmacological standpoint, postsynaptic disorders are treated with cholinesterase inhibitors (AChEIs), such as neostigmine, physostigmine, and pyridostigmine. However, AChEIs represent only symptomatic therapy, and they are of little aid in most cases of moderate to severe or progressive myasthenia gravis (MG), particularly if there is oropharyngeal or respiratory muscle involvement. Therefore, use of AChEIs as the mainstay of therapy for MG has been deemphasized. In the past, three commonly used AChEIs were physostigmine, pyridostigmine, and galantamine. Currently neostigmine, pyridostigmine, and ambenonium are the standard anti-AChE compounds used in the symptomatic treatment of MG for cholinergic crisis (Fig. 2). These compounds presumably counteract MG by compensating for lost ACh receptors through elevation of neurotransmitter
levels, resulting in increased neuromuscular transmission and improved muscular strength.
C. Glaucoma Physostigmine (eserine) and echothiophate (phospholine) are the two AChE-inhibiting compounds indicated for glaucoma. Both compounds are known to exert ocular side effects.
D. Urine Voiding Dysfunction Impairment of destrusor muscle contractibility appears to be one of the causes of voiding dysfunction in both men and women. The destrusor muscle becomes weak due to many factors, including aging, prostate hypertrophy, diabetes mellitus, and multiple sclerosis. AChE-inhibiting carbamates, such as physostigmine, distigmine bromide, and neostigmine bromide, seem to have the potential for correcting the problem. A study conducted on guinea pigs suggested that a novel anti-AChE compound, TAK-802, may be useful in the treatment of voiding dysfunction associated with impaired destrusor contractility (Nagabukuro et al., 2004).
CHAPTER 2 9Classification of OPs and CMs
H3C~ CH3
23
CH3 I H3C~~OyN~cH3 H3c/N~cH3
NEOSTIGMINE
H3C
__,,
+
c,
o
PYRIDOSTIGMINE
H3C~ 7
c,_ L...CH o/ /
N
CI.
I. CH
,30. c,
1
0
AMBENONIUM E. OP Nerve Agent Poisoning During the Persian Gulf War (Operation Desert Shield/Storm) in 1990, military personnel received a reversible AChE inhibitor, pyridostigmine bromide, as a prophylactic measure to combat anticipated deadly OP nerve agent exposure. Due to severe side effects, the drug has been discontinued for application in the setting of such military action (Keeler et al., 1991).
VI. ACHE INHIBITORS IN VETERINARY MEDICINE
A. Anthelmintics Six OP compounds have been used as anthelmintics in domestic animals: dichlorvos, trichlorfon, haloxon, coumaphos, naphthalophos, and crufomate. The first two were used in horses and the latter four in ruminants. These compounds affect parasites by ACh accumulation attributed to AChE inhibition, leading to interference with neuromuscular transmission and subsequently paralysis, followed by expulsion of parasites from the animal's body (Reinemeyer and Courtney, 2001).
H3C~N+---CH3
OH
EDROPHONIUM
FIG. 2. Chemical structures of anti-AChE compounds indicated in myasthenia gravis.
phosmet, pirimiphos-methyl, ronnel, tetrachlorvinphos, and trichlorfon. Unlike OPs, only two CMs (carbaryl and propoxur) are recommended for the control of ectoparasites. The mechanism of action of CMs is similar to that of OPs, except CMs reversibly inhibit ACHE.
VII. CONCLUSIONS Both OPs and CMs are synthesized compounds. Within each class, the chemicals have some similarities and some differences. Because of the differences, these compounds produce varying degrees of cholinergic and noncholinergic effects, and as a result, they have different applications. Although the majority of these chemicals are used as pesticides, some are used as chemical weapons of mass destruction. In addition, many of these compounds are used as therapeutic drugs in human and veterinary medicine. It is expected that in the future many more new OPs and CMs will be synthesized and novel applications will be discovered.
Acknowledgments I thank Mrs. Debra A. Britton and Mrs. Denise M. Gupta for their assistance and support in the preparation of this chapter.
B. Ectoparasiticides Currently, more than a dozen OP compounds are used as ectoparasiticides in veterinary medicine. These compounds are known to cause paralysis and death of insects by virtue of irreversible AChE inhibition and subsequent accumulation of ACh. These compounds include chlorfenvinphos, chlorpyrifos, coumaphos, cythioate, diazinon, dichlorvos, ethion, famphur, fenthion, malathion,
References Aston, L. S., Noda, J., Seiber, J. N., and Reece, C. A. (1996). Organophosphate flame retardants in needles of Pinus ponderosa in the Sierra Nevada Foothills. Bull. Environ. Contam. Toxicol. 57, 859-866. Battershill, J. M., Edwards, E M., and Johnson, M. K. (2004). Toxicological assessment of isomeric pesticides: A strategy for
24
S E CTI O N I 9 Uses, Abuses, & Epidemiology
testing of chiral organophosphorus (OP) compounds for delayed polyneuropathy in a regulatory setting. Food Chem. Toxicol. 42, 1279-1285. Benschop, H. P., and De Jong, L. P. A. (1988). Nerve agent stereoisomers: Analysis, isolation and toxicology. Acc. Chem. Res. 21, 366-374. Cuadra, G., Summers, K., and Giacobini, E. (1994). Cholinesterase inhibitor effects on neurotransmitters in rat cortex in vivo. J. Pharmacol. Exp. Ther. 270, 277-284. Darvesh, S., Walsh, R., Kumar, R., et al. (2003). Kinetic properties of human acetylcholinesterase and butyrylcholinesterase in the presence of drugs for Alzheimer's disease. Alzheimer Dis. Assoc. Disord. 17, 117-126. Green, J. (1993). A review of phosphorus-containing flame retardants. J. Fire Sci. 10, 470-487. Guillozet, A. L., Smiley, J. E, Mash, D. C., and Mesulam, M. M. (1997). Butyrylcholinesterase in the life cycle of amyloid plaques. Ann. Neurol. 42, 909-918. Johnson, M. K. (1969). Delayed neurotoxic action of some organophosphorus compounds. Br. Med. Bull. 25, 231-235. Keeler, J. R., Hurst, C. G., and Dunn, M. A. (1991). Pyridostigmine used as a nerve agent pretreatment under wartime conditions. J. Am. Med. Assoc. 266, 693-695. Kidd, H., and James, D. R. (1991). The Agrochemicals Handbook, 3rd ed. Royal Society of Chemistry Information Services, Cambridge, UK. Ku6a, K., and Kassa, J. (2004). Oximes-induced reactivation of rat brain acetylcholinesterase inhibition by VX agent. Hum. Exp. Toxicol. 23, 167-171. Marrs, T. C. (1993). Organophosphate poisoning. Pharmacol. Ther. 58, 51-66. Nagabukuro, H., Okanishi, S., and Doi, T. (2004). Effects of TAK-802, a novel acetylcholinesterase inhibitor, and various
cholinomimetics on the urodynamic characteristics in anesthetized guinea pigs. Eur. J. Pharmacol. 494, 225-232. Racchi, M., Mazzucchelli, M., Porrello E., Lanni, C., and Govoni, S. (2004). Acetylcholinesterase inhibitors: Novel activities of old molecules. Pharmacol. Res. 50, 441-451. Reinemeyer, C. R., and Courtney, C. H. (2001). Chemotherapy of parasitic diseases. In Veterinary Pharmacology and Therapeutics 8th ed., (H. R. Adams, Ed.), pp. 947-979. Blackwell, Ames, IA. Senanayke, N., and Karalliede, I. (1987). Neurotoxic effects of organophosphorus insecticides: An intermediate syndrome. N. Engl. J. Med. 316, 761-763. Smith, J. R. (2004). Analysis of the enantiomers of VX using normal-phase chiral liquid chromatography with atmospheric pressure chemical ionization-mass spectrometry. J. Anal. Toxicol. 28, 390-392. Sugimoto, H., Iimura, Y., and Yamanishi, Y. (2002). The new generation of acetylcholinesterase inhibitors: In Mapping the Progress of Alzheimer's and Parkinson's Disease (Y. Mizuno, A. Fisher, and I. Hanin, Eds.), pp. 193-198. Kluwer/Plenum, New York. Tomlin, C. D. S. (1997). A World Compendium. The Pesticide Manual, 1 l th ed. British Crop Protection Council, Farnham, Surrey, UK. U.S. Environmental Protection Agency (1988). Twenty-third report of the interagency testing committee to the administrator: Receipt of report and request for comments regarding priority list of chemicals. Fed. Reg. 53, 46244-46272. Weinstock, M., Poltyrer, T., Bejar, C., Sagi, Y., and Youdin, M. B. H. (2002). TV3326, a novel cholinesterase and MAO inhibitor and Parkinson's disease. In Mapping the Progress of Alzheimer's and Parkinson's Disease (Y. Mizuno, A. Fisher, and I. Hanin, Eds.), pp. 193-198. Kluwer/Plenum, New York.
CHAPTER
Therapeutic Uses of Cholinesterase Inhibitors in N e u r o d e g e n e r a t i v e Diseases RANDALL L. WOLTJERAND DEJAN MILATOVIC University of Washington, Seattle, Washington
for AD is the passage of time, which is currently inconveniently recapitulated in the laboratory setting, it is likely that we will continue to learn more about these diseases through the further characterization of clinical responses of AD and, increasingly, other age-related dementias to such therapies. This chapter describes the rationale for such therapies in AD, aspects of their application to clinical disease, and what we have learned and may continue to learn from the effects of these drugs in neurodegenerative disease.
I. I N T R O D U C T I O N The most common form of age-related neurodegenerative disease in the United States is Alzheimer's disease (AD), with approximately 4 million Americans afflicted; in the European Union, approximately 3 million people suffer from the disease. Furthermore, as the average life span continues to increase, it is anticipated that AD and other age-related neurodegenerative diseases will become increasingly major public health concerns. Already, the annual cost of disease (including paid and unpaid caregiver costs, as well as losses in productivity due to illness and premature mortality) of patients with AD in the United States alone has been estimated to be $100 billion (Leifer, 2003), and the prevalence of AD is expected to increase by approximately three-fold by the middle of this century. This represents a potentially staggering cost, both monetarily and in terms of lost human potential, to developed societies. A detailed understanding of the pathogenesis of AD, which might be useful in the rational development of strategies to prevent or treat disease, is not yet available, although substantial progress has been made in this area, especially in the past decade. Also, there is reason for considerable optimism, particularly regarding the possibility of treatments that may prevent or at least delay the onset of AD. However, AD is a complex disorder, with both genetic and environmental factors that affect the risk of disease, cognitive and neuropsychiatric manifestations that may vary between patients and over time, and, as is becoming increasingly apparent, only a part of a spectrum of age-related neurodegenerative disease that is still in the process of clinical, pathologic, and biochemical definition. The historical lack of an ideal animal model for AD has necessitated that at least some of this definition has derived from empirical observations of the effects of putative therapeutic agents, such as the acetycholinesterase inhibitors (AChEIs). Because the major nongenetic risk factor Toxicology of Organophosphate and Carbamate Compounds
II. THE CHOLINERGIC HYPOTHESIS Early investigations on postmortem tissue demonstrated a reduction of choline acetyltransferase (CHAT) activity and of cholinergic neurons in the basal forebrain of patients affected by AD (Davies and Maloney, 1976; Perry et al., 1977; Whitehouse et al., 1981). Decreases in presynaptic cholinergic neurons are also observed in the cerebral cortex and hippocampus as AD progresses (Bartus et al., 1982; Coyle et al., 1983). These observations led to the formulation, approximately 25 years, ago, of the "cholinergic hypothesis," which states that loss of cholinergic function in the cerebrum contributes significantly to cognitive dysfunction in AD (Barms, 2000). In addition to CHAT, cholinesterases, particularly butyrylcholinesterase (BuChE), have been associated with the pathogenesis and progression of AD (Guillozet et al., 1997; Darvesh et al., 2003). Based on these findings, it has been hypothesized that cholinesterase inhibitors that inhibit both AChE and BuChE stabilize disease progression better than those that inhibit only AChE in AD patients (Ballard, 2002; Giacobini, 2000). Importantly, the cholinergic hypothesis does not necessarily stipulate that cholinergic deficiency initiates or contributes to the progression of disease, although we deal with this possibility later. However, the goal of any medical hypothesis is to produce new, effective therapies for 25
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
26
S ECTI O N I 9Uses, A b u s e s , & E p i d e m i o l o g y
disease; in this light, the cholinergic hypothesis, which has brought several compounds with significant efficacy to the clinical treatment of AD, has been regarded as one of the successes of modern neuropharmacology (Bartus, 2000). We will next discuss the origins of the cholinergic hypothesis, the uses of AChEIs in the therapy of AD, determinations of their clinical efficacy, and what the use of these compounds has taught us about the disease.
III. C H O L I N E R G I C F A I L U R E , NEUROPATHOLOGICAL
CORRELATES,
AND THE SYMPTOMS OF AD Impairments in AD occur in both memory and other areas of cognition, such as language or visuospatial awareness. In addition, neuropsychiatric symptoms including psychosis and mood alterations such as depression, apathy, and agitation may present during the course of dementia, (Lyketsos et al., 2001). These perturbations reflect structural and neurochemical alterations in brain regions such as the hippocampus and cerebral cortex that house normal functions of memory and cognition. Hippocampal and cortical neurons are innervated by cholinergic afferents from the basal forebrain, the site of the nucleus basalis of Meynert, which contains approximately 80% of the cholinergic neurons of the central nervous system and is characterized by marked atrophy in advanced AD (Cummings and Back, 1998). In experimental rodent models, lesions of cholinergic pathways result in impairments in the performance of memory tasks (Dunnett et al., 1987). Taken together, these observations are consistent with the idea that cholinergic failure may contribute strongly to symptoms of AD. The cholinergic hypothesis has also been strengthened by observations of correlations between cholinergic failure and the degree of clinical dementia. Interestingly, however, cholinergic failure and dementia have also been found to correlate with the extent of extracellular senile plaques in brain tissue (Arendt et al., 1985; Etienne et al., 1986; Perry et al., 1978). Senile plaques are one of the pathologic hallmarks of AD; another characteristic lesion, the intracellular neurofibrillary tangle, is found in a topographically organized pattern in the gray matter of the limbic system and progressively in the neocortex in AD. Although this association has invited speculation that cholinergic failure may contribute substantially to the pathogenesis of AD, historically this has not been widely believed to be the case based on several observations. The first involves studies of patients with amnestic mild cognitive impairment (MCI), a condition of memory loss that is widely considered a paradigm of preclinical AD (Petersen, 2000; Petersen et al., 2001). In MCI, levels of ChAT have been reported to be increased, with a subsequent decrease to normal levels with the onset of clinical dementia (DeKosky et al., 2002; Fr61ich, 2002). In advanced AD,
the loss of cholinergic neurons results in up to a 90% reduction in the activity of CHAT, which is needed for the synthesis of the neurotransmitter ACh, and ACh levels decrease by 90%, especially in the cerebral cortex and hippocampus (Murphy et al., 1998). Furthermore, although cholinergic failure is most pronounced late in the course of AD, anticholinesterase therapies (as described later) have been best characterized and appear most efficacious in mild to moderate dementia, although studies on the use of AChEIs for longer term disease are ongoing. Finally, the pattern of memory loss in AD, in which more recent memories are lost first, with loss of older memories later, is not well recapitulated by models that invoke only neuronal loss, but it can be accounted for if changes in cholinergic activity that occur in MCI are viewed as responses to other instigators of brain dysfunction in AD (Small et al., 2001; Small, 2004).
IV. DETERMINATION OF T H E E F F E C T S O F A C h E I S O N S Y M P T O M S I N AD The effects of AChEI therapy on AD have proven to be modest, and the determination of the significance of effects of this magnitude required the development of standardized tools of measurement that encompass important aspects of AD symptomatology. Commonly used instruments include the Mini-Mental Status Examination (MMSE) (Folstein et al., 1975), which assays cognition with the use of 11 questions that produce a single score ranging from 0 (severe impairment) to 30 (no impairment). The second tool is the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog) (Rosen et al., 1984), designed to determine cognitive function in AD. This test also consists of 11 items that assess cognitive functions that are typically impaired specifically in AD, with an output score ranging from 0 (no impairment) to 70 (marked impairment); the average rate of score increase in patients with AD is 7-11 points per year (Kramer-Ginsberg et al., 1988; Stern et al., 1994). Neuropsychiatric symptoms described in Section III can likewise be quantified with the use of the Neuropsychiatric Inventory (Cummings et al., 1994) or a noncognitive subscale of the Alzheimer's Disease Assessment Scale (ADASnoncog). Recently, there has been an enlargement of the scope of assessment to include effects on so-called functional symptoms. Functional decline in AD involves the loss of ability to perform activities of daily living, such as the simple arithmetic of basic finances, driving, or using household tools such as the telephone, with progression of losses to the point that patients in a terminal state are no longer able to feed or bathe themselves. These losses increase the burden on patient caregivers, are highly correlated with the decision to place patients in institutional facilities, and contribute significantly to the financial burden of disease. Functional losses are also quantifiable through such tools as the
C H A P T E R 3 9Therapeutic Uses of AChE inhibitors
Alzheimer's Disease Functional Assessment and Change Scale and the Progressive Deterioration Scale (Reisberg et al., 1986).
V. A BRIEF HISTORY OF THE USE OF AChEIs IN AD AChEIs initially came to the attention of investigators concerned with dementias with the recognition of their capacity to enhance cognition in animals with scopolamineinduced amnesia (Bartus, 1978). With the discovery that ACh was depleted in the hippocampus (Smith and Swash, 1978) and initial reports of effects of physostigmine on cognition in both normal subjects and AD patients (Davis et al., 1978; Muramoto et al., 1979; Peters and Levin, 1979; Smith and Swash, 1979), the stage was set for trials of a variety of AChEIs as potential therapeutic agents in AD. Some drugs, such as galantamine, had long been used for other indications. Two compounds, velnacrine and eptastigmine, were halted in development due to the association of blood dyscrasias with their use. More typical limitations of a variety of agents center on drug tolerability, the response rate to a tolerated dose, and intersubject variability. Overall, the response of AD patients to AChEI therapy has been characterized as modest, with 3 or 4 point decreases in ADAS-cog scores compared with the yearly rate of cognitive decline in untreated controls. The U.S. Food and Drug Administration (FDA) uses a 4-point improvement in ADAS-cog scores as a criterion for a clinically significant response to t h e r a p y - an improvement also used by many investigators to define rate of response, which is typically 30-40% of AD patients undergoing treatment. The FDA has approved four AChEIs for the treatment of AD; in fact, these are currently the clinical mainstays of AD therapy.
A. Tacrine
NH2
Tacrine
In 1993, tacrine (aminoacridine, competitive unselective reversible inhibitor of cholinesterases) was the first anticholinesterase drug to receive FDA approval for the treatment of mild to moderate AD. The benefits of treatment were evident in end points that used ADAS as well as general clinical impressions, with 10-26% of recipients of high doses showing measurable improvement over the course of 30 weeks (Knapp et al., 1994). However, only a minority of patients were able to tolerate the maximally effective dose
27
(160 mg/day) due to hepatotoxicity manifested as asymptomatic transaminase elevations, as well as nausea and vomiting, diarrhea, and anorexia. Other limitations of tacrine, such as a relatively short half-life (2-3 hr) and significant interindividual variation in clearance rates that necessitated drug titration and plasma determinations, motivated the search for novel agents. It is available under the trade name Cognex, but is rarely prescribed.
B. Donepezil o
2
H3C // H3C~O Donepezil
Donepezil, a piperidine-based rapidly reversible noncompetitive AChEI, was approved by the FDA for treatment of cognitive dysfunction in AD in 1996. The drug was developed specifically for pharmacotherapy of AD (Bryson and Benfield, 1997; Barner and Gray, 1998) and has been found to have selectivity for brain AChE over peripheral forms of the enzyme (Kosasa et al., 2000). Donepezil is highly selective for ACHE, with significantly lower affinity for BuCHE. Major advantages over tacrine are its long half-life (70 hr) and uniform dosing in patients with renal or hepatic impairment. In the course of a study of the effects of 24 weeks of treatment with ADAS-cog, MMSE, and other end point determinations, cognitive function was found to be significantly improved by 12 weeks. The drug was generally well tolerated at low (5 mg/day) doses, with a slight but statistically significant increased incidence of diarrhea and vomiting at 10 mg/day that nevertheless resolved spontaneously without reduction in dosage (Rogers and Friedhoff, 1998; Rogers et al., 1998). The drug is marketed under the trade name Aricept.
C. Rivastigmine
0
~
H3C~N'~O I CH3
CH3 N~CH 3
CH3
Rivastigmine
Rivastigmine, approved by the FDA in 2000, is a noncompetitive pseudo-irreversible carbamate AChEI that was
28
SECTION I 9Uses, A b u s e s , & Epidemioloooy
selected for study based on its high affinity for brain AChE compared to peripheral forms of the enzyme (Weinstock et al., 1992, 2004; Enz et al., 1993). Rivastigmine selectively inhibits monomeric ACHE, especially in the cortex and hippocampus, and is thereby thought to facilitate cholinergic neurotransmission by slowing the degradation of ACh released by functionally intact cholinergic neurons (Polinsky, 1998; Ibach and Haen, 2004). The mechanism of action of rivastigmine differs from that of donepezil because donepezil is an AChE-selective inhibitor, whereas rivastigmine is a dual inhibitor of both AChE and BuChE. Furthermore, unlike donepezil, rivastigmine is bound more tightly to the active center of AChE than a naturally occurring choline ester. Rivastigmine was shown to improve cognition as determined by ADAS scores, participation in activities of daily living, and global evaluation scores in patients with mild to moderate AD in a multicenter trial of 28 weeks' duration (Rosler et al., 1999). As with donepezil, most side effects were found to be gastrointestinal and transient in nature. The drug, titrated up to 12 mg/day with two or three times daily dosing, yielded ADAS improvements in 24% of treated patients versus 16% for placebo controls. It is marketed under the trade name Exelon.
VI. T H E P E R S I S T E N C E O F A C h E I EFFICACIES The initial results for the previously discussed agents were generally based on trials of 6 months' duration or less. These and subsequent studies have indicated that treatment of AD with AChEIs tends to lead to an improvement in cognition that is maintained for up to approximately 1 year. This is typically followed by declines in cognition, but to levels that still improved relative to those of untreated controls. Several studies, typically continuations of trials under open-label conditions, demonstrate benefit of up to several years' duration with FDA-approved AChEIs (Knopman et al., 1996; Lilienfeld and Parys, 2000; Rogers et al., 2000; Doody et al., 2001; Rockwood et al., 2001; Tariot, 2001). The efficacy of a variety of agents with widely varying structures but that all share anticholinesterase properties, as well as the continuation of clinical efficacy of these drugs into the stage of AD that is characterized neurochemically by cholinergic failure, and in some instances initiated in more severely affected AD patients (Feldman et al., 2001), is widely seen to support the cholinergic hypothesis of AD.
VII. P U T A T I V E E F F E C T S O F A C h E I s O N AD P A T H O G E N E T I C M E C H A N I S M S
D. Galantamine OH O"--
H3c/O~ ~'~/~'~ N "CH3 Galantamine
Galantamine is a phenanthrene alkaloid that was initially isolated from the common snowdrop, Galanthus niralus. Galantamine acts as a rapidly reversible, competitive AChEI. The agent has been used for approximately 40 years in the treatment of myasthenia gravis and in reversal of pharmacologic neuromuscular blockade; hence, considerable familiarity with its pharmacokinetic and toxicologic properties existed prior to its approval by the FDA for treatment of symptoms of AD in 2001. Several trials have demonstrated efficacy in the treatment of cognitive symptoms in AD at a dose of up to 32 mg/day, as determined by ADAS scores and other end points (Rainer, 1997; Raskind et al., 2000; Tariot et al., 2000). Nausea appears t o b e the most common side effect. Stimulation of nicotinic receptors has been proposed to be an additional mechanism of action of galantamine, and there is evidence that such an effect may be relevant to AD (Dajas-Bailador et al., 2003). Galantamine is marketed under the trade name Reminyl.
Although the clinical indication of AChEIs is limited to symptomatic therapy for AD, increasing experience with their use, and the persistence of drug efficacy in particular, has led some to suggest that these agents may alter the natural history of disease (Farlow et al., 2000, 2003; Doraiswamy et al., 2002; Erkinjuntti et al., 2003; Stefanova et al., 2003). It has been hypothesized that normal activation of neurons in aging and during the course of AD may lead to preservation of neuronal function and/or promotion of survival of remaining neurons; this "cognitive reserve" or "use it or lose it" hypothesis has been proposed to account for the effects of education and other socioeconomic factors on the risk of development of cognitive impaLrment in general and AD in particular in a variety of populations (Yu et al., 1989; Brayne and Calloway, 1990; Moritz and Petitti, 1993; Stem et al., 1995; Evans et al., 1997; Hall et al., 2000; Qiu et al., 2001; Salemi et al., 2002; Karp et al., 2004). Additional proposed interactions of AChEIs with specific aspects of steps that are widely believed to be important in the pathogenesis of AD have been reviewed (Lane et al., 2004) and are discussed next.
A. Effects of AChEIs on Amyloid 13 Peptide Approximately 5% of cases of AD are attributable to mutations in known genes that act in an autosomal dominant fashion. Each of these is related to the metabolism of the amyloid precursor protein (APP) and its cleavage products, most prominently amyloid [3 (A[3). The remaining 95% of
CHAPTER 3 9Therapeutic Uses of AChE Inhibitors cases appear as sporadic disease with a relatively late onset and a complex etiology attributable to interactions between aging, environmental, and other genetic factors (Munoz and Feldman, 2000; Lahiri et al., 2004). The pathologic similarities between inherited and sporadic forms of AD, however, have led to the "amyloid hypothesis" that increased production or accumulation of A[3, with its subsequent aggregation and accumulation in cerebrum as senile plaques, provides the pathogenetic foundation for all forms of AD (Hardy and Selkoe, 2002). Efforts to understand the effects of A[3 have focused on its aggregation and reorganization to form progressively insoluble structures. This has been attributed to the presence in A[3 of an unstable domain that can readily adopt multiple conformations, some of which are prone to form aggregates that may propagate as filamentous structures with decreased solubility that are deposited in cerebrum as amyloid. This propensity appears to be key to the pathologic actions of A[3 because the uniform effect of genetic mutations in familial forms of AD is the promotion of A[3 aggregation and insolubility, either via alterations in the structure of A[3 (in the case of mutations in APP) or by increasing the relative amount of the more amyloidogenic 42-residue species relative to the 40-residue species (in the case of mutations in presenilin 1 or 2, which determine the carboxy-terminal cleavage of the A[3 peptide) (Marjaux et al., 2004). One effect of AChEIs may be mediated by ACh receptor-mediated activation of protein kinase C and mitogen-activated protein kinase pathways (Haring et al., 1998; Beach et al., 2001; Beach, 2002). Such activation promotes an alternate cleavage of APP that produces nonamyloidogenic forms of the peptide. Conceivably, the persistence of ACh in the context of AChEI treatment may promote this process. In addition to A[3, senile plaques contain a host of other proteins (Liao et al., 2004), including ACHE, which has been proposed to serve as a nucleating factor in the deposition of A[3 with reduced solubility, or otherwise promote the toxicity or aggregation state of A[3 in vivo (Inestrosa et al., 1996; Alvarez et al., 1997; De Ferrari et al., 2001; Rees et al., 2003). Other studies indicate that A[3 complexed with AChE is more toxic than A[3 species alone (Alvarez et al., 1998). A number of AChEIs have been reported to inhibit at least partially A[3 aggregation in the presence of AChE (Bartolini et al., 2003; Piazzi et al., 2003); the significance of this observation and its validity in the case of clinically widely used AChEIs remain unknown.
29
of kinases, especially glycogen synthase kinase-3 (GSK3). Kinase activities described previously that are promoted by ACh not only affect APP metabolism but also are associated with decreases in the activity of GSK3, leading to decreased tau phosphorylation and conceivably to a reduction in tangle generation (Forlenza et al., 2000). Consistent with this mechanism, a rivastigmine-treated AD group showed no change in cerebrospinal fluid (CSF) levels of tau after 1 year, whereas significant increases were observed in untreated patients (Stefanova et al., 2003). However, the CSF tau content of patients treated with tacrine resembled that of controls more closely, making interpretation of these results difficult.
C. Effects of AChEIs on Cerebrovascular Parameters Recently, the coexistence of vascular lesions and those of AD in cerebrum of patients with so-called mixed dementia have received increasing attention, along with the possibility of heretofore unanticipated interactions between pathophysiological factors (Langa et al., 2004). Some authors have suggested that factors that lead to the development of senile plaques and neurofibrillary tangles may be promoted by cerebrovascular disease (de la Torte, 2002; Honig et al., 2003; Casserly and Topol, 2004); conversely, deposition of amyloid in vessel walls compromises cerebrovascular function and, in the extreme, promotes the risk of hemorrhagic stroke. Interestingly, AChEI therapy for mixed dementia yields favorable, but modest, clinical results similar to those observed in the treatment of AD (Kumar et al., 2000; Erkinjuntti et al., 2002) and in some studies demonstrates efficacy in the treatment of dementia associated with cerebrovascular disease alone (Erkinjuntti et al., 2004). As in the case of AD, cholinergic deficiency has been demonstrated in vascular dementia and has been attributed to ischemic injury to cholinergic neurons, as supported by rodent models of ischemic injury (Togashi et al., 1994). However, effects of ACh on the vascular endothelium may account for a portion of both the interaction of AD and vascular dementia and the efficacy of AChEIs in both of these conditions. ACh at this site mediates the release of nitric oxide, a vasodilator that may account for increases in cerebral blood flow and glucose metabolism upon treatment with AChEIs (Minthon et al., 1993; Harkins et al., 1997; Lojkowska et al., 2003). Conceivably, such improvements could influence the natural history of vascular dementia and AD.
B. Effects of AChEIs on Tau According to the amyloid hypothesis, abnormalities involving A[3 peptides also lead to changes in the organization of tau to produce neurofibrillary tangles, the lesion in brain tissue that is more closely associated with the presence of clinical AD. Deposition of tau in tangles is enhanced by tau hyperphosphorylation, which can be accomplished by a variety
VIII. AChEIS IN T H E T R E A T M E N T OF O T H E R D E M E N T I A S Dementia associated with Parkinson's disease (PDD) and dementia with Lewy bodies (DLB) together comprise the second most common form of age-related dementia after
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SECTION 1 9 Uses, Abuses, & E p i d e m i o l o g y
AD. A recent body of literature suggests that AChEIs may be of utility in the treatment of these disorders, which have also been reported to be associated with cholinergic deficit (Aarsland et al., 2004). This deficit, as in the case of AD, has been attributed to basal forebrain degeneration, and this may be sufficient to account for any observed clinical efficacy of AChEIs. However, the interactions of AChE with A[3 in senile plaques described previously suggest other possibilities as well. Many neurodegenerative diseases are characterized by the presence of a variety of proteinaceous aggregates that are associated with neurotoxicity. As in the case of amyloid plaques, upon further examination these aggregates have been found characteristically to be constituted of numerous proteins that may or may not be predicted based on sequence structure analysis (Yoon and Welsh, 2004). Although the presence of AChE has not been reported in Lewy bodies of PDD or DLB, its association with these lesions would suggest the possibility of a more generalized role of AChE in the pathogenesis of age-related dementias. Whether treatments with AChEIs alter the course of these dementias, and, if so, by what mechanism, awaits further clinical studies and the further molecular characterization of these disorders.
IX. C O N C L U S I O N S Effective preventive and therapeutic strategies for AD and other age-related neurodegenerative diseases are the most pressing need in modem clinical neurological practice. Remarkable advances in our insight into the pathogenesis of AD in particular hold forth the prospect that manipulation of pathways of amyloid peptide synthesis, posttranslational processing, aggregation, degradation, interactions with other macromolecules, or the relation of A[3 to oxidative, inflammatory, or neuroexcitatory processes that may promote or be promoted by its presence in the cerebrum may profoundly alter the incidence or progression of AD in the future. The multifactorial nature of the etiology of sporadic AD seems to imply a likelihood that the most effective treatment strategies will target several or many of these processes. Indeed, many models of the progression of AD invoke self-reinforcing cycles of cerebral damage that may be checked in part at any one of a number of steps but that may be best approached by therapeutic strategies that target multiple aspects of disease pathogenesis. Definitive experimental evidence for a role of ACh in the pathogenesis of human neurodegenerative disease may prove elusive and perhaps become unambiguously manifest only in the context of cotreatments with AChEIs and agents that target other aspects of disease. In the meantime, the symptomatic effects alone of AChEIs dictate that they will remain important treatments for AD and, in all likelihood, for an expanding list of age-related dementing illnesses.
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CHAPTER ' 4
Coadministration of M e m a n t i n e with A c e t y l c h o l i n e s t e r a s e Inhibitors: Preclinical and Clinical Evidence
ANDRZEJ DEKUNDY Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany
in vivo showed that such a noncontingent activation of this receptor type also leads to impairment of neuronal plasticity (learning), which can be restored by therapeutic concentrations of memantine (Danysz and Parsons, 2003). These preclinical results were further confinned in multiple clinical trials. Memantine has been approved by the Food and Drug Administration as the first drug for moderate to severe AD and is also available in Europe. After the antidementia potential of memantine was widely acknowledged, it became obvious that possible effects of coadministration of this drug with the clinically available AChE inhibitors required investigation. This chapter presents the preclinical and clinical profile of memantine and discusses results of animal and human studies on its coadministration with various classes of AChE inhibitors.
I. INTRODUCTION The pathogenesis of Alzheimer's disease (AD) as a neurodegenerative disease involves more than one mechanism. Multiple studies in the past two decades revealed that degeneration of cholinergic nuclei localized in the basal forebrain occurs in the course of this disease. Impairment of the cholinergic system, which projects into large areas of the limbic system and the neocortex, is followed by disturbance of attentional processes and cognitive decline (Terry and Buccafusco, 2003). Consistent with these findings, some compounds that increase cholinergic neurotransmission were found to be effective in AD. Of the various cholinomimetic drug classes, only reversible acetylcholinesterase inhibitors (AChEIs) have been successfully used in AD patients (for review, see Ibach and Haen, 2004). The first compounds of this class tested in clinical trials were physostigmine (Stem et al., 1988; Thal et al., 1989) and tacrine (Summers et al., 1986). However, because of the short duration of action of the former and hepatotoxicity of the latter drug, their use in AD patients has been largely abandoned (Ibach and Haen, 2004). Tacrine was the first drug approved specifically for the treatment of AD (1993). This was followed by the development of compounds with improved specificity and tolerability: donepezil (1996), rivastigmine (2000), and galantamine (2001). Aside from the cholinergic hypothesis, it is generally agreed that compromised neuronal energy metabolism may occur in AD. Continuous mild activation of N-methylD-aspartate (NMDA) receptors under such conditions may render the cells susceptible to subsequent damage. Indeed, the NMDA antagonist memantine was shown to be efficient in animal models relevant to human neurodegenerative diseases and dementia. Preclinical studies in vitro and Toxicology of Organophosphate and Carbamate Compounds
II. PROPERTIES OF M E M A N T I N E Memantine (1-amino-3,5-dimethyl-adamantane hydrochlofide, ClzHz0N.HC1) was first synthesized in the early 1960s by Eli Lilly & Company as a derivative of amantadine, an anti-influenza agent. It was intended to become a hypoglycemic agent, but it was found to be void of such activity (Gerzon, 1963). Instead, the amino group in the three-ring (adamantane) structure (Fig. 1) has been shown to bind close to the magnesium site in the NMDA receptor channel (Blanpied et al., 1997; Sobolevsky et al., 1998; Kashiwagi et al., 2002). Memantine is a colorless to white crystalline substance readily soluble in water. Its oral bioavailability is nearly 100%. Memantine readily crosses the blood-brain barrier, and while in organism it shows linear, dose-proportional pharmacokinetics and weak protein-binding properties. 35
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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S ECTI O N I 9Uses, A b u s e s , & E p i d e m i o l o g y NH2
H3C
IV. C L I N I C A L U S E O F M E M A N T I N E
CH3
FIG. 1. The chemical structure of memantine.
Memantine's elimination half-life is approximately 60-80 hr; it is excreted mainly unchanged through the kidneys and undergoes limited metabolism in the liver. The concentrations of memantine in the cerebrospinal fluid (CSF) were shown to correlate well with its serum levels, with a CSF:serum ratio of 0.52 (Kornhuber and Quack, 1995). Memantine has also been shown to have minimal inhibitory effect on CYP450 enzymes (Periclou et al., 2004).
III. M E C H A N I S M OF MEMANTINE
OF ACTION
Serendipitous observation of beneficial effects of aminoadamantanes in Parkinson's disease patients undergoing antiviral therapy led to the conclusion that these compounds may possess dopaminomimetic or possibly cholinolytic properties. The fact that administration of clinically relevant doses of memantine results primarily in NMDA receptor antagonism was discovered only in the late 1980s (Bormann, 1989). Memantine is a specific, uncompetitive, voltagedependent NMDA receptor antagonist with moderate affinity, strong voltage dependency, and rapid blocking/ unblocking receptor kinetics (Fig. 2; Danysz et al., 2000). Under normal physiological resting conditions, magnesium ions block the NMDA receptor channels to inhibit calcium entry to the cell. Compromised neuronal energy metabolism is likely to occur in certain pathologic states, including AD. Continuous mild activation of NMDA receptors under such conditions may impair the magnesium block, rendering the cells more susceptible to subsequent damage (Greenamyre et al., 1988; Harkany et al., 2000; Winblad et al., 2002). Under such conditions, the moderate receptor affinity and fast blocking/unblocking kinetics give memantine a unique pharmacological profile. Memantine is able to prevent the pathogenic calcium influx caused by continuous mild activation by low-level glutamate. On the other hand, memantine allows the physiological activation of the NMDA channels by high concentrations of glutamate, a phenomenon necessary for synaptic plasticity underlying normal learning and memory (Parsons et al., 1999b). Thus, memantine can be compared to a "noise reduction system," filtering the necessary physiological "signal" from the unwanted "noise" disturbing the normal function (i.e., increasing the "signal-to-noise" ratio) (Danysz et al., 2000).
Subsequent to approval in Europe (2002), memantine was approved as the first drug for moderate to severe AD in the United States (2003). To date, seven double-blind, parallel group, placebo-controlled, randomized trials have been published in which memantine was administered as monotherapy to patients suffering from primary dementia (Ditzler, 1991; G6rtelmeyer and Erbler, 1992; Pantev et al., 1993; Winblad and Poritis, 1999; Wilcock et al., 2002; Orgogozo et al., 2002; Reisberg et al., 2003). The early, relatively small trials suggested memantine to be capable of exerting positive effects in unspecified dementia (Ditzler, 1991; G6rtelmeyer and Erbler, 1992). In a first trial applying recent methods, Winblad and Poritis (1999) assessed the clinical efficacy and safety of memantine in severe primary dementia in 166 patients, 49% of the Alzheimer type and 51% of the vascular type, after 12 weeks of treatment. In the prespecified AD subgroup, a total of 79 nursing home residents were randomized to receive either a conservative dose of 10 mg memantine (single daily dose) or placebo. Despite the small sample size, memantine had statistically significant favorable effects in all of the three main domains m that is, cognitive, functional, and globalmas measured by the cognitive subscore of the Rating Scale for Geriatric Patients [BGP (Beurteilungsskala ftir Geriatrische Patienten), based on the Stockton Geriatric Rating Scale], the care dependence subscore of the BGP, and the Clinical Global Impression of Change, respectively. Regarding the safety profile, no significant differences between treatment groups were observed (Graham et al., 2003). In the study of Reisberg et al. (2003), 252 outpatients with moderate to severe AD were randomly assigned to receive placebo or 20 mg of memantine daily for 28 weeks. Of these, 72% completed the study and were evaluated at week 28. Seventy-one patients discontinued treatment prematurely (42 taking placebo and 29 taking memantine). The primary efficacy variables were the Clinician's Interview-Based Impression of Change Plus Caregiver Input (CIBIC-Plus) and the Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory modified for severe dementia (ADCSADLl9 ). The secondary efficacy end points included the Severe Impairment Battery (SIB) and other measures of cognition, function, and behavior. Patients receiving memantine had statistically significantly better scores than those receiving placebo in cognitive functions and global outcomes as measured by the CIBIC-Plus, ADCS-ADL19, and SIB. Consistent with previous results, the frequency of adverse effects was similar to that for placebo (Reisberg et al., 2003). The results of these two trials provide further support to the hypothesis that memantine treatment leads to functional improvement and reduces care dependence in severely demented patients.
CHAPTER 4
9M e m a n t i n e and AChE Inhibitors
FIG. 2. (A) Schematic representation of glutamatergic synapse and the major ionotropic glutamate receptors, AMPA and NMDA. (B) The principle of synaptic plasticity in the central nervous system is detection of the relevant signal over the existing background noise. Such a signal, once detected, may lead to a long-lasting alteration in synaptic strength. NMDA receptors play a central role in such alterations and an endogenous "noise suppressant" is magnesium. Physiological mechanisms are fully capable of keeping very low glutamate levels in the synaptic cleft under normal conditions. (C) In neurodegenerative diseases such as Alzheimer's disease, contributing factors (e.g., malfunctioning uptake into the astroglia) lead to sustained enhanced concentrations of glutamate in the synaptic cleft and resulting partial depolarization. Under such conditions, magnesium is no longer capable of suppressing the "noise." This leads to malfunctioning of signal detection (cognitive functions) and, with time, to damage of vulnerable neurons. Reproduced from Danysz et al. (2000), copyright FP Graham Publishing Company.
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Two independent, placebo-controlled clinical trials conducted on approximately 900 patients with diagnosed mild to moderate vascular dementia (NINCDS-AIREN) showed that memantine (20 mg daily) significantly improved cognition relative to placebo as assessed by the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-cog) with no deterioration in global functioning and behavior. Memantine was considered well tolerated and safe (Wilcock et al., 2002; Orgogozo et al., 2002). Peskind et al. (2004) studied 403 patients older than 50 years of age with a diagnosis of mild to moderate AD. Patients were randomized to memantine 10 mg twice a day or to placebo and followed for 24 weeks. Over the course of 24 weeks, the patients taking memantine were shown to significantly improve in cognition (ADAS-cog) and remained improved compared to baseline at 24 weeks. In contrast, patients receiving placebo experienced small but significant steady declines in cognitive function over the 24-week study. Moreover, memantine was shown to significantly reduce societal as well as caregiver costs. Caregiver time for memantine-treated AD patients was assessed by means of the Resource Utilisation in Dementia Scale (Wimo et al., 1998). A significant reduction in mean monthly caregiver time by approximately 52 hr was demonstrated. In addition, a significant difference was found in the institutionalization status at week 28, favoring memantine treatment over placebo (Wimo et al., 2003). This finding may also indirectly point to the clinical efficacy of memantine. For more extensive reviews of clinical efficacy and safety trials, see Winblad and Jelic (2003), M6bius (2003), and Wilcock (2003).
discontinuations due to an adverse effect compared to placebo (7.4 vs 12.4%, respectively). Results of several postmarketing surveillance studies (PMS) of memantine in "dementia syndrome" are in accordance with the results of clinical trials. Rieke and Glaser (1996) followed 1420 patients treated with memantine (10-20 mg/day) for more than 1 year. At the end of the observation period, physicians appraised memantine as very well or well tolerated in 94% of cases. The most frequently reported adverse effects were restlessness (1.3%), nausea (0.9%), dizziness (0.8%), and fatigue/ tiredness or sleep disorders (0.4%). In another study of 531 care-dependent patients treated with memantine (up to 30 mg/day for a mean of 44 days), the drug was also well tolerated; 3% of patients reported adverse effects (Rt~ther et al., 2000). Noteworthy, a recent postmarketing surveillance study in 158 patients (median memantine dose, 20 mg/day), suffering mainly from Alzheimer's disease (77%) or vascular dementia (9%), and concurrently treated with memantine and AChE inhibitors revealed that the combination therapy was very well (56%) or well (42%) tolerated (Hartmann and M6bius, 2003). In both placebocontrolled and PMS trials, the nature of the memantine side effect profile appears to remain unaffected, irrespective of monotherapy or combination therapy with AChEIs. Based on the results of the previous studies, memantine may be considered a safe and well-tolerated drug. This is consistent with animal data showing that the therapeutic index for memantine is markedly higher than that for other NMDA antagonists (e.g., MK-801) (Parsons et al., 1999b).
VI. P R E C L I N I C A L P H A R M A C O L O G Y OF MEMANTINE V. S A F E T Y O F M E M A N T I N E The safety database of clinical trials with memantine comprises thousands of patients. The data derive from clinical trials of memantine carried out on patients suffering from primary dementia or other neurological disorders (e.g., peripheral neuropathy, Parkinson's disease, multiple sclerosis, and spasticity). A total of 2297 patients have been exposed to memantine, with 1244 patients receiving placebo, in 27 clinical trials. In double-blind, placebocontrolled dementia trials, comparable numbers of memantine patients and placebo patients experienced adverse effects, with dizziness, confusion, headache, and constipation among the most frequently reported. Most adverse effects were considered mild or moderate in severity and not related to the trial drug (M6bius et al., 2004). In a combination therapy trial, memantine was administered to patients receiving continuous donepezil therapy (Tariot et al., 2004). Daily doses of memantine (20 mg, added to donepezil) were very well tolerated, as indicated by fewer
A. In Vitro Profile of Memantine In in vitro studies, memantine displayed a Ki of approximately 1 IxM in NMDA competitive binding assays utilizing [3H]MK-801 in human and rat brain tissue (Komhuber et al., 1991; Bresink et al., 1995). Memantine is relatively selective for the [3H]MK-801 binding site. Memantine was shown to possess 5-HT3 receptor antagonistic properties in the low-micromolar range. Memantine's ability to block nicotinic receptors is approximately 6-10 times weaker than that to inhibit NMDA receptors (Danysz et al., 1997). Electrophysiological studies revealed an uncompetitive, voltage-dependent blockade of NMDA receptors with median inhibiting concentration (IC50) values of 1-3 txM (Parsons et al., 1993, 1999b). As mentioned previously, the hallmark of memantine that makes it unique among NMDA antagonists is its pronounced voltage dependence and rapid blocking/unblocking kinetics. It has been shown that memantine can block the toxicity of glutamate to cultured cortical neurons with an ICs0 of 1.4 txM (Parsons
CHAPTER 4 9Memantine and AChE Inhibitors et al., 1999a). On the other hand, hampering long-term potentiation requires much higher concentrations of memantine (ICs0 = 11.6 txM) (Frankiewicz et al., 1996; Frankiewicz and Parsons, 1999). This profile makes memantine capable of blocking pathological, but not physiological, activation of NMDA receptors. Unlike memantine, other NMDA receptor antagonists, such as MK-801 or ketamine, were shown to block physiological activation at their "therapeutic" doses (Parsons et al., 1999b). Neurofibrillary degeneration, considered to be a hallmark of AD and related tauopathies, is a consequence of abnormal hyperphosphorylation of tau protein. This in turn may be related to a decrease in protein phosphatase (PP-2A) activity found in the AD brain. Interestingly, memantine was shown to inhibit and reverse PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices (Li et al., 2004).
B. In Vivo Profile of Memantine Memantine has been demonstrated to exert protective effects in animal models relevant to human neurodegenerative diseases and dementia. Given as a food supplement for 10 days prior to acute intrahippocampal exposure to quinolinic acid, memantine prevented excitotoxic damage to hippocampal neurons (Keilhoff and Wolf, 1992). When delivered subcutaneously (sc) by osmotic minipump (20 mg/kg/day), it prevented the occurrence of short-term memory deficits induced by chronic intracerebroventricular (icv) administration of quinolinic acid, as measured in T-maze (Misztal et al., 1996), and it protected against the loss of cholinergic neurons of the nucleus basalis of Meynert resulting from inflammation caused by chronic icv infusion of lipopolysaccharides (Willard et al., 2000). Furthermore, continuous infusion of memantine (15 mg/kg/day, sc) protected against pathological changes and learning deterioration (measured by the T-maze test) induced by intrahippocampal injection of [3-amyloid (Miguel-Hidalgo et al., 2002). Memantine also exerted direct effects on learning, demonstrating the capability to restore the learning impairment in radial maze caused by lesioning the entorhinal cortex (Zajaczkowski et al., 1996) or direct activation of NMDA receptors (Zajaczkowski et al., 1997). Many patients with AIDS are likely to develop dementia. Macrophages infected with HIV are believed to release toxins producing neuronal damage, a phenomenon diminished by NMDA antagonists (Giulian et al., 1993). Indeed, memantine has been demonstrated to prevent neuronal degeneration caused by gp 120, the HIV envelope glycoprotein (Muller et al., 1992). Like many other NMDA antagonists, memantine has been shown to exert dose-dependent protective effects in the rat model of transient forebrain ischemia (Seif el Nasr et al., 1990). Memantine provided neuroprotection in the model of transient MCA occlusion after a single injection
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of 20mg/kg (Chen et al., 1998). At a daily dose of 20 mg/kg, memantine enhanced the ischemic tolerance induced by transient ischemia (Parsons et al., 2001). NMDA receptors have been implicated in sensitization of central pain pathways in neuropathic pain (Dubner and Ruda, 1992), and N M D A antagonists have been demonstrated to exhibit analgesic properties in a range of animal models of pain (Parsons et al., 1999b). Accordingly, memantine (10 mg/kg, ip) has been shown to block and reverse thermal analgesia in chronic constriction injury in the rat, a model of painful neuropathy (Eisenberg et al., 1995). Memantine (25-75 mg/kg, po or im) has been shown to be capable of reducing mechanical allodynia induced by a spinal nerve ligation in macaque monkeys (Carlton et al., 1994, 1998). Memantine was also shown to inhibit morphine selfadministration (Semenova et al., 1999) and to attenuate morphine place preference (Popik and Danysz, 1997; Popik et al., 2000), suggesting its potential effectiveness in treating morphine dependence. Moreover, memantine inhibited tolerance to the analgesic action of this opioid (Popik et al., 2000). Relapse drinking of ethanol in rats has been selectively inhibited by continuous sc infusion of memantine (Holter et al., 1996). Higher doses of memantine have been demonstrated to exert antiparkinsonian-like activity in animal models, such as haloperidol-induced catalepsy, reserpine-induced sedation, and rotation in rats with a unilateral lesion to the nigrostriatal system (Danysz et al., 1994; Karcz-Kubicha et al., 1999). Memantine also displayed a clear-cut, dose-dependent antidepressant-like activity in the Porsolt test (Moryl et al., 1993). For a detailed review of the preclinical profile of memantine, see Danysz et al. (1997, 2000).
VII. I N T E R A C T I O N O F M E M A N T I N E
WITH AChEIs A. Experimental Studies 1. IN VITRO STUDIES The first study to specifically address the issue of possible interference of memantine with AChE inhibition by drugs approved for the treatment of AD was an in vitro study performed by Wenk et al. (2000). The authors assessed the effects of preincubation of homogenates of the striata of untreated rats with memantine (1 or 5 IxM) on the inhibition of AChE activity produced by a clinically relevant concentration of galantamine, donepezil, and tacrine. Additionally, an irreversible organophosphate (OP) AChE inhibitor, diisopropyl fluorophosphate (DFP), was used as a positive control (McLean et al., 1992). Memantine alone, even at concentrations exceeding the therapeutically relevant ones, was not found to be capable of inhibiting AChE activity. The enzymatic inhibition by galantamine, tacrine, and
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donepezil was unaffected by memantine. However, memantine attenuated the inhibition produced by DFP, consistent with an earlier report investigating the nerve gas soman, an another irreversible OP inhibitor of AChE (McLean et al., 1992). It was suggested that the inhibitory effects of therapeutically relevant concentrations of memantine upon actions of AChEIs in vitro may be restricted to the irreversible ones (Wenk et al., 2000). McLean et al. (1992) demonstrated findings consistent with those of Wenk et al. (2000), demonstrating that memantine reduced AChE inhibition in crude brain homogenates by soman but not by an anionic site inhibitor (edrophonium) or a peripheral site inhibitor (decamethonium). The authors suggested that memantine may bind to some other, not yet characterized, modulatory site to protect ACHE. Enz and Gentsch (2004) attempted to determine in vitro whether memantine was able to prevent AChE inhibition by rivastigmine in a rat striatal preparation. After an initial preincubation period of 10 min, rivastigmine dose dependently inhibited AChE with an IC50 of 32 + 2 IxM. Memantine by itself did not inhibit the enzyme unless very high (millimolar) concentrations were used. After the 10-min preincubation, rivastigmine at a concentration of 30 txM caused approximately 45% inhibition of AChE in the rat striatal homogenate. Memantine (1, 5, 10, 50, or 100 IxM) did not influence the inhibition caused by rivastigmine, irrespective of whether it was applied before or after the AChE inhibitor (Enz and Gentsch, 2004). 2. IN VIVO STUDIES In vitro studies give important insight into the possible mechanisms of interaction of memantine. However, it is extremely important to prove whether and to what extent the results of in vitro experiments reflect the in vivo situation. Indeed, in a recent series of in vivo experiments, Gupta and colleagues investigated whether memantine may interact with inhibition of AChE activity by reversible (donepezil and rivastigmine) and irreversible (DFP and metrifonate) AChEIs in rat brain regions affected in AD (i.e., the cortex and the hippocampus). In preliminary experiments, a dose of each AChEI causing 40-50% inhibition of brain AChE was found. This level of AChE inhibition is well within the range previously shown to result from administration of therapeutic doses of donepezil and rivastigmine (Ibach and Haen, 2004). Then, in time course experiments, the time of maximal AChE inhibition was determined for each of the AChEIs studied. Further experiments per aimed to clarify whether memantine may interfere with AChE inhibition induced by donepezil, rivastigmine, metrifonate, or DFP (administered at the dose causing 40-50% AChE inhibition and twice that dose), measured at the time of maximum AChE inhibition. Memantine at a dose of 10mg/kg (i.e., two to four times greater than the therapeutically relevant dose) was
administered 15 min prior to donepezil (0.75 or 1.5 mg/kg), rivastigmine (0.35 or 0.7mg/kg), metrifonate (55 or 110 mg/kg), or DFP (1.5 or 3.0 mg/kg). All drugs were administered intraperitoneally (ip). Rats were sacrificed at the time of maximal AChE inhibition determined in the time course study (i.e., 15 min after donepezil, 30 min after rivastigmine or metrifonate, and 60 min after DFP) to determine AChE activity in the brain region homogenate. Neither memantine nor most of the AChEIs produced any toxic signs at any time during the study; however, metrifonate did produce muscle tremors at 110 mg/kg. Each AChEI studied produced a dose-dependent inhibition of ACHE. Memantine did not cause AChE inhibition in any brain area. The findings from those interaction studies revealed that memantine prevented AChE inhibition produced by DFP or metrifonate in both brain areas and at both dose levels. Memantine also prevented metrifonateinduced tremors. Finally, memantine did not interact with AChE inhibition by therapeutically used AChEIs, donepezil and rivastigmine, at either dose level. It was concluded that memantine can be applied concurrently with donepezil or rivastigmine (Gupta and Dekundy, 2005; Gupta et al., 2004; Dekundy et al., 2004). Enz and Gentsch (2004) compared AChE inhibition (measured ex vivo) following a 21-day chronic, oral administration of 6 Ixmol/kg rivastigmine or a combination of equimolar doses of rivastigmine and memantine (6 txmol&g perorally of either of the two compounds). Final administration of a drug or vehicle occurred 2 hr prior to decapitation of the rats. It was found that memantine, even at high brain levels of approximately 3500pmol/g, as achieved following the oral 6 txmol/kg dose, was unable to attenuate the inhibitory effect of rivastigmine on AChE activity in the rat brain. Following coadministration of memantine and rivastigmine for 21 days, AChE inhibition remained practically unaltered in comparison with that of the rivastigmineonly group. Whereas there are relatively few in vivo studies exploring a possible interaction of memantine with clinically available AChEIs, much more data have been published on the effects of memantine in animals treated with OP or carbamate (CM) insecticides used in agriculture or the household as well as in rodents experimentally intoxicated with the OP nerve gas soman. Pretreatment with a single dose of memantine (18 mg/kg, sc) alone or in combination with atropine (16 mg/kg, sc) prevented motor limbic seizures induced by a single sublethal dose of soman without causing any signs of sedation or ataxia. After seizure onset, memantine in combination with atropine, but not atropine alone, abolished the somaninduced seizures. AChE activities in the cortex, stem, striatum, and hippocampus were markedly reduced by soman but not by memantine, atropine, or a combination thereof. Preadministration of memantine and the combination of memantine and atropine in vivo significantly protected
CHAPTER 4 9Memantine and AChE lnhibitors AChE from inhibition by soman. However, when given after onset of soman-induced seizures, memantine and/or atropine did not reactivate ACHE, although seizures were controlled (McLean et al., 1992). Likewise, rats administered a single dose of an OP methyl parathion (5 mg/kg, ip) showed signs of hypercholinergic toxicity, including convulsions and depression of AChE activities in the cortex, stem, striatum, and hippocampus. Pretreatment with memantine (18 mg/kg, sc) 60 min and atropine (16 mg/kg, sc) 15 min before methyl parathion administration completely prevented the expected toxic signs and attenuated the induced inhibition of ACHE. Also, when given therapeutically, this combined treatment completely reversed the clinical evidence of methyl parathion toxicity and reduced AChE inactivation (Gupta and Kadel, 1990). Very similar results were shown in rats injected with a sublethal acute dose of the carbamate insecticide carbofuran (1.5 mg/kg, sc) (Gupta and Kadel, 1989). The results of these two studies suggest that memantine may counteract the acute toxicity of methyl parathion and carbofuran by protection of AChE from inhibition and rapid reactivation of inhibited ACHE. Additionally, memantine may increase rapid elimination of carbofuran and methyl parathion (Gupta and Kadel, 1989, 1990). A similar combination of memantine and atropine has been shown to reduce toxicity and AChE inhibition produced by other CM insecticides (i.e., aldicarb, methomyl, and oxamyl) (Gupta and Kadel, 1991; Gupta, 1994b). Interestingly, memantine was also demonstrated to exert beneficial effects in myotoxicity related to anticholinesterase intoxication. For instance, pretreatment with memantine (18 mg/kg, sc) together with atropine (16 mg/kg, sc) prevented necrotic lesions in skeletal muscles following administration of sublethal doses of different nerve agents (soman, satin, tabun, or VX). Attenuation of muscle AChE inhibition by anticholinesterases was implicated in the protective effects of memantine (Gupta and Dettbarn, 1992a). The beneficial effects of such antidotal treatment were shown in myotoxicity produced by administration of DFP (Gupta et al., 2002) and carbofuran (Gupta and Goad, 2000). Oxidative stress has been implicated in the pathology of myotoxicity, and pretreatment with memantine and atropine was demonstrated to prevent and/or reverse the increase in nitric oxide synthase and the decrease in high-energy phosphates induced by administration of DFP or carbofuran. These mechanisms may contribute to the well-documented favorable effects of memantine in AChEIinduced myopathology (Gupta and Goad, 2000; Gupta et al., 2002; Milatovic et al., 2005). On the other hand, Dai et al. (2004) studied the protective effect of memantine in dichlorvos-poisoned rats. Dichlorvos was applied at a dose of 25 mg/kg (ip) to three groups of rats, subsequently treated with memantine at doses of 5, 15, and 45 mg/kg. The activity of AChE and binding capacity of the NMDA receptor with [3H]MK-801 were determined 16 hr after dichlorvos injection. The low
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(5 mg/kg) and intermediate (15 mg/kg) doses of memantine were demonstrated to protect against the downregulation of NMDA receptor in rat brain. However, only the higher (45 mg/kg) dose of memantine slightly alleviated dichlorvos poisoning symptoms. The AChE activities in both blood and brain of memantine-treated groups were not significantly different from those in the dichlorvos-only group. The authors concluded that the observed protective effects of memantine in the dichlorvos-induced poisoning did not involve recovery of AChE activity (Dai et al., 2004).
B. Clinical Studies Schmitt et al. (2004) noted that the agents of at least six classes are approved for clinical use or are being tested or ready for phase III clinical trials for the treatment of AD. The authors listed AChEIs, NMDA receptor antagonists, antioxidants (including Gingko biloba preparations), antiinflammatory agents, neurotrophic factors, and anti-amyloid agents. Although all of these approaches may differentially modify the course of the disease and thus provide a rationale for the use of combination therapy, only nine clinical studies have investigated the effects of a combination regimen on cognitive performance or AD, and only five of these followed a randomized, controlled design. The authors emphasized the great need for further welldesigned studies on combination therapy in AD. The first publication to address the issue of combination treatment with memantine was a PMS study conducted among German physicians who, during routine clinical practice, treated demented patients with memantine in combination with an AChEI (Hartmann and M6bius, 2003). Most of the 158 surveyed patients (mean age, 74 years) were diagnosed with AD. However, other dementias were also included. Memantine was prescribed at a wide range of daily doses (5-60 mg) but the median dose was 20 mg/day, as recommended. Most patients received concomitant donepezil (84%), although 15% received rivastigmine and 1% received concomitant tacrine. In nearly all patients (98%), the combination therapy was well tolerated over the 4-month average observation period, during which the study participants were maintained at stable doses of both antidementia agents. The six reported adverse drug reactions resolved without sequelae and without drug discontinuation. Of these six events, two were considered unrelated to either drug, two mild events were probably related to memantine (10 mg/day), and one moderately severe event was probably related to donepezil (10 mg/day); drug relationship was not assessed by the physician in one case. No changes in blood chemistry were reported in 81% of patients (in 16%, no laboratory analyses were performed). Global clinical status was judged as improved or stable in 93% of patients (54% improved and 39% stable) over the observation period; worsening was observed in only 6%. In addition, improved communication abilities
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9Uses, Abuses,
& Epidemiology
and elevated mood were frequently reported by treating physicians. These findings suggested for the first time that memantine in combination with AChEIs is effective, safe, and well tolerated (Hartmann and M6bius, 2003). Results of the first randomized, double-blind, placebocontrolled, parallel group trial of memantine in patients receiving an AChEI have been published only recently (Tariot et al., 2004). In this study, the efficacy and safety of memantine were compared to placebo in 404 patients with moderate to severe AD already receiving stable treatment with donepezil. The study started with a 1- or 2-week single-blind placebo screening period followed by 24 weeks of double-blind treatment. A total of 322 patients (80%) completed the trial. Participants were randomized to receive memantine (starting dose 5 mg/day, increased to 20 mg/day; n = 203) or placebo (n = 201) for 24 weeks. In patients with moderate to severe AD already receiving donepezil (5 or 10 mg/day) for the immediate preceding 6 months and at a stable dose for at least 3 months prior to and during the study period, memantine resulted in significantly better outcomes than placebo on measures of cognition, activities of daily living, global outcome, and behavior. Patients treated with memantine/donepezil appeared to show improvement relative to baseline over the 24-week course of the study, whereas patients receiving placebo/ donepezil exhibited progressive cognitive decline over the same duration. The drug-placebo difference was statistically significant. In addition, memantine was found to be safe and well tolerated. An open-label, multiple-dose study in 24 healthy volunteers (aged 18-35 years) was aimed at determining whether an in vivo pharmacokinetic interaction exists between memantine and donepezil (Periclou et al., 2004). Furthermore, the effect of memantine coadministration on inhibition of red blood cell AChE activity by donepezil was examined. The subjects received memantine (10 mg, po) on day 1. Following a 14-day washout period, the subjects were administered donepezil at a single daily dose of 5 mg for 7 days, followed by 10 mg for 22 days. The last dose was administered concurrently with memantine 10mg on day 43. Pharmacokinetic and safety parameters were assessed. AChE inhibition was measured in red blood cells by radiolabeled enzyme assay following administration of donepezil alone or donepezil with a single memantine dose. Data from 19 subjects who completed the study indicated no significant pharmacokinetic interactions between a single dose of memantine and donepezil at steady state. Maximum inhibition of AChE activity from baseline by donepezil was not significantly altered by coadministration with a single dose of memantine. In addition, single memantine doses administered with donepezil at steady state were well tolerated (Periclou et al., 2004). The previously mentioned clinical studies indicate that memantine and donepezil can be safely coadministered, with no significant effects on the pharmacokinetic profile
of either drug and no significant alterations of donepezilinduced AChE inhibition. These findings support the potential for the combined use of memantine and AChEIs in patients with AD. Further controlled clinical studies are needed to confirm the available data and to extend them to other clinically available AChEIs.
VIII. M E C H A N I S T I C - B A S E D C L I N I C A L CONSIDERATIONS AD is a progressive neurodegenerative disease characterized primarily by memory loss, behavioral problems, and the inability to perform daily activities. AD is the most common cause of dementia, affecting 11% of the population between the ages of 80 and 85 years and 24% of the population over the age of 85 years (Grutzendler and Morris, 2001). In fact, in the next 50 years, the prevalence of AD is expected to quadruple, affecting an estimated 1 in 45 people. AD is characterized by neuronal loss, particularly affecting cholinergic neurons in the basal forebrain, which projects into the hippocampus and neocortex, the brain structures that play an important role in memory and cognitive function. The loss of cholinergic neurons results in a remarkable decrease in acetylcholine levels, especially in the cerebral cortex and hippocampus (Terry and Buccafusco, 2003). It is accepted that disturbances in the glutamatergic system also play an important role in dementia. Although glutamate mediates physiological neurotransmission, under certain conditions, prolonged and elevated glutamate levels lead to increased calcium influx into neurons and finally to neurodegeneration due to "excitotoxicity" (Rothman and Olney, 1986, 1995). This phenomenon may also take place in AD (Greenamyre et al., 1988; Harkany et al., 2000). Bearing in mind that disturbances of several neurotransmitter systems may underlie AD, future pharmacotherapy may combine drugs with different modes of action to optimize symptomatic treatment and to potentially slow disease progression. The first approved therapeutic principles comprise AChE inhibition and NMDA receptor antagonism. Clinically, AChE inhibitors may improve cognition by boosting brain acetylcholine level (Ibach and Haen, 2004). On the other hand, the first approved NMDA receptor antagonist, memantine, seems to exert its beneficial effects by improving glutamatergic neurotransmission and by protecting against glutamate-induced excitotoxicity (Jacobsson and Fowler, 1999; Parsons et al., 1999b; Danysz et al., 2000). It is conceivable that memantine combined with an AChEI may represent a fruitful path in the treatment of dementia by targeting both cholinergic and glutamatergic neurotransmission. However, many in vivo studies of animals have shown that memantine is able to attenuate AChE inhibition by several organophosphate or carbamate AChEIs, including
CHAPTER 4 9Memantine and AChE Inhibitors
reference substances, insecticides, and nerve agents (Gupta and Kadel, 1989, 1990; McLean et al., 1992; Gupta and Dettbarn, 1992; Gupta, 1994a,b; Gupta and Goad, 2000). The in vivo data are supported by in vitro studies (McLean et al., 1992; Antonijevic et al., 2002; Stojiljkovic et al., 2002). Although the doses of memantine used in these investigations exceeded the therapeutically relevant doses by several times, the results of the previous studies raised concerns about possible coadministration of memantine with AChEIs approved for the treatment of AD. This in turn triggered further research aimed at elucidating whether such interaction really does exist. In in vitro interaction studies, Wenk et al. (2000) showed that memantine does not influence AChE inhibition in rat brain induced by donepezil, tacrine, or galantamine. These findings have been strengthened by the observation that memantine is not able to prevent either rivastigmine- or donepezil-induced AChE inhibition both in vitro and in vivo (Gupta and Dekundy, 2005; Gupta et al., 2004; Dekundy et al., 2004; Enz and Gentsch, 2004). Nevertheless, studies have also confirmed the potential of the NMDA antagonist to prevent AChE inhibition by some irreversible inhibitors of this enzyme, namely metrifonate and DFP (Wenk et al., 2000; Gupta et al., 2004; Dekundy et al., 2004; Gupta and Dekundy, 2005). Numerous in vivo studies in rodents give clear evidence that memantine at doses up to 72 mg/kg does not cause AChE inhibition (Gupta and Kadel, 1990; Antonijevic et al., 2002; Stojiljkovic et al., 2002), a finding excluding the possibility of an interaction with memantine at the active center (anionic site) of ACHE. This would explain the lack of interaction of memantine with donepezil that binds to this site. On the other hand, rivastigmine is a pseudo-irreversible AChEI that binds to the esteratic binding site of the active center of the enzyme. Rivastigmine is bound firmly to the active center of ACHE, with a reported decarbamylation time for the rivastigmine-AChE complex of approximately 10 hr (Ibach and Haen, 2004). This finding implies that memantine does not interact with the esteratic binding site. It was suggested that the biochemical mechanism underlying the interaction of memantine with some AChEIs could be allosteric modulation of the active center of the enzyme, which in turn could lead to its hindrance and unavailability to AChEIs, such as DFP, methyl parathion, and nerve agents (Gupta and Kadel, 1990; McLean et al., 1992; Antonijevic et al., 2002; Stojiljkovic et al., 2002). However, this mechanism remains to be elucidated.
IX. C O N C L U S I O N S AND FUTURE DIRECTIONS Because memantine and AChEIs reduce dementia symptoms through distinct mechanisms of action (glutamate antagonism and cholinesterase inhibition, respectively),
43
there is growing interest in investigating whether this combination therapy may improve therapeutic benefit in demented patients. Results of animal studies and the first clinical trials suggest good efficacy and tolerability of the combination of the NMDA receptor antagonist and AChEIs. Future clinical research will optimize this approach.
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Gupta, R. C., and Dettbarn, W.-D. (1992). Potential of memantine, d-tubocurarine, and atropine in preventing acute toxic myopathy induced by organophosphate nerve a g e n t s - Soman, satin, tabun and VX. Neurotoxicology 13, 649-661. Gupta, R. C., and Goad, J. T. (2000). Role of high-energy phosphates and their metabolites in protection of carbofuraninduced biochemical changes in diaphragm muscle by memantine. Arch. Toxicol. 74, 13-20. Gupta, R. C., and Kadel, W. L. (1989). Prevention and antagonism of acute carbofuran intoxication by memantine and atropine. J. Toxicol. Environ. Health 28, 111-122. Gupta, R. C., and Kadel, W. L. (1991). Novel effects of memantine in antagonizing acute aldicarb toxicity: Mechanistic and applied considerations. Drug Dev. Res. 24, 329-341. Gupta, R. C., and Kadel, W. L. (1990). Methyl parathion acute toxicity: Prophylaxis and therapy with memantine and atropine. Arch. Int. Pharmacodyn. Ther. 305, 208-221. Gupta, R. C., Milatovic, D., and Dettbarn, W.-D. (2002). Involvement of nitric oxide in myotoxicity produced by diisopropylphosphorofluoridate (DFP)-induced muscle hyperactivity. Arch. Toxicol. 76, 715-726. Gupta, R. C., Dekundy, A., Danysz, W., and Quack, G. (2004). Memantine does not interfere with donepezil or rivastigmineinduced inhibition of AChE in rat cortex and hippocampus. J. Neurochem. 90(Suppl. 1), 43. Harkany, T., Abraham, I., Timmerman, W., Laskay, G., Toth, B., Sasvari, M., Konya, C., Sebens, J. B., Korf, J., Nyakas, C., Zarandi, M., Soos, K., Penke, B., and Luiten, E G. (2000). Beta-amyloid neurotoxicity is mediated by a glutamatetriggered excitotoxic cascade in rat nucleus basalis. Eur. J. Neurosci. 12, 2735-2745. Hartmann, S., and M6bius, H. J. (2003). Tolerability of memantine in combination with cholinesterase inhibitors in dementia therapy. Int. Clin. Psychopharmacol. 18, 81-85. Holter, S. M., Danysz, W., and Spanagel, R. (1996). Evidence for alcohol anti-craving properties of memantine. Eur. J. Pharmacol. 314, R1-R2. Ibach, B., and Haen, E. (2004). Acetylcholinesterase inhibition in Alzheimer's disease. Curr. Pharm. Design 10, 231-251. Jacobsson, S. O., and Fowler, C. J. (1999). Dopamine and glutamate neurotoxicity in cultured chick telencephali cells: Effects of NMDA antagonists, antioxidants and MAO inhibitors. Neurochem. Int. 34, 49-62. Karcz-Kubicha, M., Lorenz, B., and Danysz, W. (1999). GlycineB antagonists and partial agonists in rodent models of Parkinson's disease--Comparison with uncompetitive N-methyl-D-aspartate receptor antagonist. Neuropharmacology 38, 109-119. Kashiwagi, K., Masuko, T., Nguyen, C. D., Kuno, T., Tanaka, I., Igarashi, K., and Williams, K. (2002). Channel blockers acting at N-methyl-D-aspartate receptors: Differential effects of mutations in the vestibule and ion channel pore. Mol. Pharmacol. 61, 533-545. Keilhoff, G., and Wolf, G. (1992). Memantine prevents quinolinic acid-induced hippocampal damage. Eur. J. Pharmacol. 219, 451-454. Kornhuber, J., and Quack, G. (1995). Cerebrospinal fluid and serum concentrations of the N-methyl-D-aspartate (NMDA) receptor antagonist memantine in man. Neurosci. Lett. 195, 137-139.
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CHAPTER 5
Cholinesterase Inhibitors a s Chemical Warfare Agents: Community Preparedness Guidelines ANNE'I-FA WATSON, 1 KULBIR BAKSHI,2 DENNIS OPRESKO, 1 ROBERT YOUNG, 1 VERONIQUE HAUSCHILD, 3,* AND JOSEPH KING4 1Oak Ridge National Laboratory, Oak Ridge, Tennessee 2National Research Council, Washington, DC 3Aberdeen Proving Ground, Maryland 4U.S. Army Environmental Center, Aberdeen Proving Ground, Maryland
Destruction; Department of the Army (DA), 1988; Carnes, 1989; Carries and Watson, 1989; Munro et al., 1994]. Previous domestic (civilian) guidance regarding airborne agent vapor exposure was largely confined to "no adverse effect" limits established by the Centers for Disease Control and Prevention (CDC) for agent demilitarization workplaces and the general public [U.S. Department of Health and Human Services (DHHS) 1988, 2003]. Although the CDC guidelines provided necessary criteria for standing operating procedures at stockpile sites, the need to address potential civilian acute vapor exposure for a range of toxicological end points, as well as potential civilian incidental ingestion exposure, remained a concern. In furtherance of international treaty and congressional requirements to eliminate risks associated with continuous storage, the U.S. Federal Emergency Management Agency (FEMA) and the Chemical Materials Agency (CMA) of the U.S. Department of the Army have collaborated under the Chemical Stockpile Emergency Preparedness Program (CSEPP) to provide technical emergency planning and preparedness support as well as assistance to agencies and authorities representing host communities located near CW agent munition stockpile sites. Such support is being employed to develop and prioritize emergency response actions for use in the event of a release during CW agent destruction and munition disposal activities. Nerve agents comprise munition fill at six of the eight unitary stockpile sites in the domestic United States: Anniston Army Depot (Anniston, AL), Blue Grass Army Depot (Richmond, KY), Newport Chemical Depot (Newport, IN), Pine Bluff Arsenal (Pine Bluff, AR), Deseret Chemical Depot (Tooele, UT), and Umatilla Chemical Depot (Umatilla,
I. I N T R O D U C T I O N Recent global events have focused attention on the potential threat of international and domestic chemical terrorism as well as the possibility of chemical warfare (CW) proliferation. The need for preparedness is highlighted by the well-documented domestic terrorist use of an anticholinesterase compound during the Tokyo subway incident of March 1995, when commuters received toxic inhalation and dermal exposures to a nerve agent deliberately released within subway cars and stations (Lillibridge, 1995; Morita et al., 1995; Okumura et al., 1996; Sidell, 1996). Emergency preparedness plans and priorities for accidental or intentional release of CW agents were under development long before the Tokyo incident and are now in place for a number of U.S. and international communities as a consequence of (1) congressional mandate to destroy the existing U.S. unitary chemical munitions stockpile (PL 99-145, the Department of Defense Authorization Act of 1986 and subsequent acts); (2) congressional directives to examine and perform safe disposal of nonstockpile CW materiel (PL 102-484, the Defense Authorization Act of 1993), and (3) the April 1997 entry into force of the international chemical arms control treaty banning manufacture, use, stockpiling, and transfer of chemical weapons [Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on Their
*Present address: U.S. Environmental Protection Agency, Washington, DC.
Toxicology of Organophosphate and Carbamate Compounds
47
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
48
S E CTI 0 N I
9Uses, Abuses,
& Epidemiolooay
OR) (Carnes 1989; Army Chemical Materials Agency at www.cma.army.mil). "Unitary" munitions are loaded with undilute, finished CW agent, as opposed to "binary" munitions, in which agent precursors mix and react to form finished agent after the munition is fired. Non-stockpile chemical material (e.g., wastes from past CW agent disposal efforts, unserviceable munitions, contaminated containers, and "found rounds" that have been recently located after having been historically buried) are also undergoing disposal. Furthermore, any soil or groundwater that may be contaminated by potentially toxic agent residues at the sites where non-stockpile material has been found are remediated and subjected to the protocols of a formal cleanup program (Opresko et al., 1998, 2001; Bakshi et al., 2000). Responsible and efficient response during the crisis phase of a CW agent release as well as during the decontamination and restoration of potentially contaminated sites requires knowledge of key, agent-specific toxicological parameters to determine the most appropriate and protective responses. Such responses include, but are not limited to, determination of zones requiring shelter-in-place or evacuation, identifying geographic areas most likely to require immediate medical support, accurate diagnosis and treatment of agent intoxication, selection of protective clothing, and performance of site health risk assessments in support of installation restoration and site recovery. During release of a hazardous material, emergency phase decision making tends to focus on acute exposures to vapor plumes, and assessments generally do not center on "no effects criteria"; minimizing severe toxicological effects in populations most likely to be exposed is priority during this phase, and acute exposure guidelines are needed. In the case of site recovery and decontamination, exposure to potential residual CW agents in soil or water or on surfaces is a more critical concern (Opresko et al., 1998, 2001; Bakshi et al., 2000). Determining responsible cleanup standards in these efforts (as for other toxic substances) hinges on the existence of chronic reference doses so that public health may be properly safeguarded without defaulting to overly conservative actions (e.g., clean to "nondetect") that would divert limited resources without significant benefit. The necessary toxicological parameters have been examined and employed to develop guidelines for addressing both acute nerve agent vapor exposures [acute exposure guideline levels (AEGLs)] as well as incidental ingestion of residual nerve agents, such as on surfaces or in soils or water [reference doses (RfDs)]. These guidelines are in the process of being implemented by local, state, and federal agencies in preparation for possible accidental or intentional agent release. Fixed facilities and transportation carriers, as well as active or formerly used defense sites are being evaluated (Bakshi et al., 2000; Opresko et al., 1998, 2001, 2003; Krewski et al., 2004; Watson et al., 2006).
Development of these acute vapor and chronic ingestion CW agent exposure guidelines, initially performed to facilitate disposal of the U.S. stockpile of CW agent munitions as well as installation restoration or closure at sites where CW agents have historically been processed, has received new interest for homeland defense application since the events of September 2001. Due to their relatively recent finalization, there has been limited awareness among the scientific and emergency preparedness communities of the availability of these protective and technically sound guidelines derived under civilian protocols. As such, these guidelines allow agent-related public health issues to be considered within frameworks essentially equivalent to those involving more commonly encountered toxic industrial substances. This article represents the first time that the pertinent background, supporting logic, and guidelines for both acute vapor and chronic lifetime ingestion exposure to nerve agents have been published together in the same document. It is hoped that this wider distribution will encourage their greater use as the objective decision criteria for which they were intended.
II. HISTORY AND BACKGROUND A. Anticholinesterase Compounds as Threat Agents Sidell (1997) noted that a naturally occurring (botanical) anticholinesterase had long been known to west African tribal authorities, who employed the special properties of the Calabar bean ( P h y s o s t i g m a v e n e n o s u m ; Leguminosae) during local trials by ordeal during the 19th century (Koelle, 1975; Davis, 1985). At that time, the Calabar bean and its extracts were considered powerful "ordeal poisons." The active principle (physostigmine; C15H21N302) was isolated by various investigators in 1864 and 1865 (Koelle, 1975). Developments in synthetic chemistry begun during the mid-19th century resulted in the discovery of organophosphorous (e.g., tetraethyl pyrophosphate in 1854; Holmstedt, 1963) and carbamate (e.g., neostigmine in the 1930s) anticholinesterases and medicinal use of carbamates (e.g., for glaucoma; Koelle, 1975). By the mid-1930s, the expanding commercial chemical industry in Germany supported a significant research and development effort, which synthesized several organophosphate compounds with potent insecticidal properties. The first was tabun (in 1936; agent GA), the second was sarin (in 1937-1938; agent GB), followed by soman (in 1944; agent GD). Their toxicity attracted attention from the German Ministry of Defense for potential military application, and commercial production of tabun and sarin for weaponry use by the ministry was in progress at multiple German facilities by 1942 (Sidell, 1997; Harris and Paxman, 1982; Robinson, 1967). Robinson reported
CHAPTER 5 9Chemical Warfare Nerve Agent Guidelines that at least 12,000 tons of tabun, and smaller quantities of sarin, were produced by the German Ministry of Defense during World War II. Much smaller amounts of agent GD had been produced in German manufacturing facilities by the end of the war. There is no record of nerve agent weapon deployment by German forces during WWII (Sidell, 1997; Robinson, 1967). As a Cold War deterrent, nerve agents began to be manufactured and weaponized by the United States in the 1950s. By the time the U.S. CW agent production program was terminated by the Nixon presidential "Statement on Chemical and Biological Defense Policies" of November 1969 (National Security Decision Memorandum 35, see Stockholm International Peace Research Institute 1973), the U.S. stockpile of unitary munitions included bulk ("ton") containers, underwing spray tanks, projectiles, rockets, bombs, land mines, and rockets (Carnes, 1989; Sidell, 1997). All of the unitary munitions were filled with either one of three nerve agents (GA, GB, and VX) or vesicant (blister) agents (sulfur mustard agent or the organic arsenical, Lewisite). The U.S. CW stockpile was never employed, is obsolete, and is currently undergoing destruction and disposal by the CMA in compliance with the international treaty and to eliminate the risk of continued storage for these aging CW munitions. Of all the munitions stored in the U.S. unitary stockpile, it is generally recognized that the M-55 rockets (fully functional and each loaded with lethal concentrations of weapons-grade nerve agent GB or VX) pose the greatest risk of unintended release due to their potential for leakage (Carnes, 1989). A similar variety of nerve agent-containing unitary munitions is present in the CW agent stockpile of the former Soviet Union; this stockpile was also manufactured as a Cold War deterrent and is also scheduled for destruction. A disposal facility at Shchuch'ye, Russia, is under construction with U.S. assistance under the Cooperative Threat Reduction Program of the Army Chemical Materials Agency. Nevertheless, continued storage and safeguarding of the former Soviet CW munitions stockpile will be required until the facility becomes operational. Although there have been numerous instances of suspected nerve agent deployment during periods of civil unrest in various nations since WWII, there is little definite documentation of nerve agent deployment until the 1994-1995 incidents of chemical terrorism involving the nerve agent sarin in Japan. In both incidents, civilian populations were deliberately exposed to lethal satin concentrations by followers of a cult (Aum Shinrykyo) originally local to Japan but with members in other nations (Lillibridge, 1995; Morita et al., 1995; Okumura et al., 1996; Sidell, 1996). The first incident occurred in June 1994 in the central highland city of Matsumoto, where seven people died soon after exposure to a then unknown vapor later determined to be agent GB released into a residential area during the night
49
(Morita et al., 1995). The Matsumoto incident also resulted in 56 hospital admissions, as well as 253 cases in which the affected individuals sought medical consultation, plus reports of "mild symptoms" presented by 8 of 53 rescue personnel and 1 attending physician (Morita et al., 1995). Prompt deaths (n = 3) and those that occurred before arrival at the hospital (n = 4) appear to have been the result of respiratory insufficiency. At the time of the Morita et al. (1995) report, 1 patient remained "in a vegetative state because of anoxic encephalopathy." The second occurrence, widely known as "the Tokyo subway incident," took place on March 20, 1995. The same terrorist group responsible for the Matsumoto incident deployed individual sources of passive, evaporative release of nerve agent GB in each of five individual subway cars serving three separate subway lines during the morning commuter rush hour (Okumura et al., 1996; Sidell, 1996; Lillibridge, 1995). Of the 5510 people known to have been given medical attention, 8 died promptly; 4 more died later (hours to days). This "later" group included individuals who had initially presented with "critical" respiratory effects requiring mechanical ventilation and intensive care (Lillibridge, 1995). The total of 12 fatalities included commuters and subway transport employees receiving ocular, inhalation, or dermal exposures. Death appeared to be a result of respiratory insufficiency. On hospital day 28, a death occurred as a consequence of "severe hypoxic brain damage" sustained during the release incident (Okumura et al., 1996). This delayed fatality was a previously healthy woman 21 years of age who had presented without heartbeat or spontaneous respiration at the hospital but had been revived with CPR and treated with agent antidotes. Plasma and red blood cell cholinesterase had returned to normal within a period of days, but the patient eventually succumbed to hypoxic brain damage (Okumura et al., 1996).
B. Chemical and Physical Properties of Nerve Agents The standard threat nerve agents include the G series agents [GA (tabun), GB (sarin), GD (soman), GF (cyclosarin)] and nerve agent VX. These compounds are all toxic ester derivatives of phosphonic acid containing a cyanide (GA), fluoride (GD or GF), or sulfur (VX) substituent and are commonly termed "nerve" agents as a consequence of their anticholinesterase properties as well as their effects on both peripheral and central nervous systems (Table 1). The "G" series military nomenclature used by NATO member nations has historically been considered to be an abbreviation for "German," with the second letter of the code ("A," "B," etc.) identifying the order in which these compounds were found and analytically identified by Allied forces investigating materials found in captured German military facilities at the end of WWII (Sidell, 1997). Agent VX
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0 to 10 to be used when necessary to reflect a qualitative professional assessment of the critical study and the entire database. Default value for MF is 1.0. High confidence in an oral RfD can be achieved if the database is made up of at least one two-generation reproductive toxicity study, two chronic oral toxicity studies in two different species, and two developmental toxicity studies in different species. For compounds that pose only acute health hazards because low doses are degraded and/or excreted, chronic studies may not be as critical in deriving an RfD as special studies assessing specific end points, such as neurotoxicity (Cicmanec et al., 1996).
1. RfI) e DERIVATION FOR AGENT GB An example RfD e derivation for data-rich agent GB is provided here to illustrate the logic and protocol for deriving reference doses for nerve agents. Complete details of RfD e derivation for the other G agents and agent VX are documented in Bakshi et al. (2000) and Opresko et al. (1998,
2ool). a. Selection of Key Study The subchronic/chronic exposure evaluations conducted with GB consist of two reports: a 90-day study in which rats were given GB type I (Bucci et al., 1991) or GB type II (Bucci and Parker, 1992) by gavage and a 1-year study in which rats, mice, and dogs were exposed to GB by inhalation (Weimer et al., 1979). For the development of an oral RfD e, a study involving the same exposure pathway is preferred even though the exposure period may be less than chronic. Therefore, the subchronic rat gavage studies are considered to be more relevant than the inhalation studies for deriving this RfD e. Decreases in plasma and RBC AChE activity levels occurred in male and female CD rats dosed once per day, 5 days/week for 13 weeks with GB type II at 75, 150, or 300 Ixg GB/kg/day (Bucci and Parker, 1992). Inhibition of RBC ChE by GB type II was dose related for females in the two highest dose groups and for males in all dose groups. Maximum (48%) and significant (p 15 months), but that they were reversible and subclinical. Although not considered debilitating or permanent effects in themselves, SFEMG changes have been considered during the AEGL review as an early indicator of exposures that could potentially result in more significant effects. Selection of the SFEMG effect as a protective point of departure for determination of an AEGL-2 level has been considered appropriate given the steep dose-response toxicity curve of nerve agents (Aas et al., 1985, 1987; Mioduszewski et al., 2000, 2001, 2002b). The fact that AEGL-2 analyses for agent GB are based on the Baker and Sedgwick data from human volunteers precludes the use of an interspecies uncertainty factor. As in the case of the AEGL-1 estimations, a factor of 10 was applied for intraspecies variability (protection of susceptible populations exhibiting variable ChE and CarbE activity). A modifying factor is not
59
applicable. Thus, 10 is the total uncertainty factor for estimating AEGL-2 values for agent GB (Table 5). The temporal extrapolation used in the estimation of AEGL values for agent GB is based on a log-log linear regression of female SD rat miosis data following GB vapor exposures (Mioduszewski et al., 2002a) and a log-log linear regression of the LC01 lethality of GB to female SD rats (Mioduszewski et al., 2000, 2001, 2002b). Regression analysis of the LC01 values yields an n value of 1.93 with an r 2 of 0.9948, whereas regression analysis of the miosis data yields an n of 2.00 with an r 2 of 0.4335 (Opresko et al., 2003). Given that all mammalian toxicity end points observed in the data set for all nerve agents represent different points on the response continuum for anticholinesterase exposure, and that the mechanism of acute mammalian toxicity (ChE inhibition) is the same for all nerve agents, the experimentally derived n = 2 from the Mioduszewski et al. (2000, 2001, 2002a,b) rat lethality and miosis data sets has been used as the scaling function for all the nerve agent AEGL derivations. AEGL-1 and -2 values for other G agents and agent VX were derived from those of agent GB by a relative potency protocol (Opresko et al., 2003; see Table 3 for examples of end point-specific relative potency estimates for agent VX vs agent GB). This is considered a toxicologically acceptable approach given that all mammalian toxicity end points observed in the nerve agent data set represent different points on the response continuum for anticholinesterase effects, and that the principal mechanism of mammalian toxicity for the G agents and agent VX is ChE activity inhibition. As a consequence, target organ effects are expected to be identical but different in magnitude. Furthermore, there are no uncertainties regarding other toxic end points, such as reproductive or developmental effects or carcinogenicity. This concept has been previously applied in the estimation of G series nerve agent exposure limits by Mioduszewski et al. (1998). b. AEGL-3 Values AEGL-3 values for agent GB were derived from inhalation studies in which the lethality of GB vapor to female SD rats was evaluated for the time periods of 10, 30, 60, 90, 240, and 360 min (Mioduszewski et al., 2000, 2001, 2002b). Both experimental LC0m and LCs0 values were evaluated. The use of a rat data set resulted in selection of an inter' species uncertainty factor of 3; the full default value of 10 was not considered appropriate since the mechanism of toxicity in rats and humans is the same, and lethality is only one point on the response continuum for these anticholinesterase compounds. The full default value of 10 for intraspecies uncertainty was considered necessary to protect susceptible populations. Because a modifying factor is not applicable, the composite uncertainty factor for AEGL-3 determination for agent GB is equal to 30. For consistency with the AEGL-1 and -2 estimates described previously, AEGL-3 values for other G agents
60
SECTION
I 9 Uses, Abuses,
TABLE 5. Agent GA
GB
GD
GF
VXY
& Epidemiology
S u m m a r y of Final AEGL Values for N e r v e A g e n t s GA, GB, GD, GF, a n d VX (rag/m3) a
Classification
10 min
30 min
1 hr
4 hr
8 hr
AEGL- 1
0.0069
AEGL-2
0.087
AEGL-3
End point
0.0040
0.0028
0.0014
0.0010
Based on relative potency from GB. b ( E U F = 10)
0.050
0.035
0.017
0.013
Based on relative potency from GB. b ( E U F = 10)
0.76
0.38
0.26
0.14
0.10
Based on relative potency from GB. c ( E U F = 30)
AEGL- 1
0.0069
0.0040
0.0028
0.0014
0.0010
ECs0 for miosis observed in adult female SD rats receiving a range of GB vapor concentrations (0.01-0.48 mg G B / m 3) for 10-240 min (Mioduszewski et al., 2002a) and miosis data from supportive studies on marmosets (Van Helden et al., 2001, 2002) and humans (Harvey, 1952; Johns, 1952). ( E U F = 10)
AEGL-2
0.087
0.050
0.035
0.017
0.013
Miosis, dyspnea, R B C - C h E inhibition, single fiber electromyographic (SFEMG) changes in human volunteers receiving 0.5 mg G B / m 3 for 30 min (Baker and Sedgwick, 1996). ( E U F = 10)
AEGL-3
0.38
0.19
0.13
0.070
0.051
Experimental SD rat lethality data (LC01 and LCs0); whole body dynamic exposure to concentrations between 2 and 54 mg G B / m 3 for 3 - 3 6 0 min (Mioduszewski et al., 2000, 2001, 2002b). (EUF = 30)
AEGL-1
0.0035
0.0020
0.0014
0.00070
0.00050
Based on relative potency from GB. d ( E U F = 10)
AEGL-2
0.044
0.025
0.018
0.0085
0.0065
Based on relative potency from GB. d ( E U F = 10)
AEGL-3
0.38
0.19
0.13
0.070
0.051
Based on relative potency from GB. Supported by Wistar rat LCs0 from dynamic chamber exposures at 21 mg GD/m 3 for three time periods of -30 ppm) to kill so many people. Cyanide is not known to cause long-term health effects; it is a very fast killer, but if the dose is not high enough it spares the victim without causing long-term disability (Goldstein et al., 1968). On the other hand, some benefit from NTS injections days or months after the accident is very possible since NTS can improve tissue oxygenation by neutralizing cyanide normally present in the blood of smokers and in people living in polluted areas (Sriramachari, 2004; Varma, 1986, 1989; Varma and Varma, 2005). Moreover, MIC can cause cherry-red blood by interacting with hemoglobin (Sriramachari, 2004). Experimental studies noted the ineffectiveness of NTS against MIC (Alarie et al., 1987; Bucher et al., 1987; Nemery et al., 1985a; Varma et al., 1988); however, lack of antagonism between NTS and MIC does not rule out the presence of HCN in Bhopal.
III. T H E I S O C Y A N A T E F A M I L Y Isocyanates were first synthesized in 1849 and are primarily used for the manufacture of polyurethane resins; their production increased after World War II. Most of the commercially used isocyanates are mono- or diisocyanates. All isocyanates are toxic and their toxicity is greater following inhalation than following oral ingestion. MIC is the smallest member of the family and the most toxic (Varma, 1986). The general structure of isocyanates is R-N ~ - C - - O ; these are highly reactive members of the heterocumelene family and the presence of adjacent double bonds confers high reactivity to isocyanates by cumulative action. The molecular weight,
LC50 (mg/m3), and ceiling (mg/m 3) for MIC and commonly used diisocyanates are, respectively, as follows: MIC, 57, 12, and 0.05; hexamethylene diisocyanate, 168, 385, and 0.14; toluene diisocyanate (TDI), 174, 350, and 0.14; isophorone diisocyanate, 222, 260, and 0.18; and diphenylmethane diisocyanate, 250, 370, and 0.20. As early as 1956, more than 100 cases of illness and 4 cases of death due to TDI were recorded (Baader, 1956). If an isocyanate causes both sensory and pulmonary irritation, as observed for MIC (Alarie et al., 1987; Ferguson et al., 1986), its toxicity is greater than if it only causes sensory irritation (Weyel et al., 1982). Isocyanates can cause direct irritant effects on lungs, eyes, skin, and exposed mucosa as well as nonpulmonary effects; indirect toxicity of diisocyanates is secondary to lung damage and immunologic sensitization (Adams, 1970; Anonymous, 1966; Brugsch and Elkins, 1963; Hama, 1957; Karol et al., 1987; Munn, 1965). Diisocyanate toxicity may range from temporary reversible to long-term irreversible damage (Axford et al., 1976) as well as delayed appearance of complications (Le Quesne et al., 1976). With most isocyanates, with the exception of MIC, direct effects ensue a few hours after exposure. It should be noted that despite similarities, there are differences in the toxicity of different isocyanates (Anonymous, 1966; Munn, 1965).
IV. P H Y S I C O C H E M I C A L CHARACTERISTICS OF METHYL ISOCYANATE MIC (CH3-N = C = O) is the smallest member of the isocyanate family and the most toxic. MIC is a flammable colorless and odorless liquid; its properties and dispersion profile in Bhopal are presented in Table 1. The extremely
TABLE 1. Physicochemical Properties of Methyl Isocyanate (MIC) and Dispersal in Bhopal Molecular mass Specific gravity (water = 1) at 20 ~ Vapor density (air = 1) Boiling point (~ Vapor pressure (mbar) at 4 ~ Vapor pressure (mbar) at 20 ~ Vapor pressure (mbar) at 31 ~ Bhopal area exposed to MIC (km2) Estimated mean MIC concentration in Bhopal (ppm)a Estimated range of MIC concentration in Bhopal (ppm)a
57.05 0.96 1.97 39.1 267 464 800 40 27 0.12-86.5
aData on MIC concentration in Bhopal are from Dhara and Dhara (2002) and Varma(1986).
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity high vapor pressure of MIC can contribute to its high toxicity (Rye, 1973). MIC vapor settled on the ground level because it is heavier than air. The boiling point of MIC (39.1 ~ is lower than the daytime temperature in Bhopal on certain summer days. The concentration of MIC in Bhopal was well above its lethal concentration as determined in later studies (Bucher et al., 1987; Varma, 1987). The Occupational Safety and Health Administration of the United States has set an 8-hr exposure limit for MIC of 0.02 ppm (0.05 mg/m3), which is in conformity with other estimates (Alarie et al., 1987). A portion of MIC also degraded into nearly 20 products, including monomethylamine, dimethylamine, trrimethylamine, dimethylurea, trimethylurea, dimethylisocyanate, trimethyl isocyanate, trimethylbiuret, CO2, and cyanide; reaction between the products produced urethane, amides, and carbamates (Andersson et al., 1985; D'Silva et al., 1986; Vardarajan et al., 1985). It is very likely that some of these degradation products, which have a longer biological half-life than the parent compound, augmented the toxicity of MIC and may even account for some of the long-term effects. In any case, it is not possible to determine if the adverse effects of MIC observed in Bhopal were influenced by the degradation products. Also, the generation of other products in unspecified relative concentration in the MIC storage tank and possibly outside must have been influenced by several environmental factors. Factors such as temperature, humidity, and pollution in Bhopal, relatively long and unknown period of storage of MIC in tank No. 610 (MIC leaked from this tank), mixing of different batches of MIC in tank No. 610, reaction with an unknown quantity of water, and other specifics of the disaster could determine the generation of degradation product. Therefore, it is not possible to replicate the events of Bhopal at a test site as being planned (Crabb, 2004), and such an expensive operation, even if safe, will not yield any useful information.
V. GENERAL FEATURES OF MIC TOXICITY MIC can interact with a large number of inorganic and organic molecules and it is rapidly (minutes) degraded in aqueous media (Brown et al., 1987). The high chemical reactivity of MIC constituted the basis for the claim by Union Carbide physicians during a press conference in Bhopal on December 14, 1984, that MIC will be destroyed upon contact with body surface and will not produce any systemic effects (Varma, 1986); notwithstanding the high chemical reactivity of MIC and a short (2 min) biological half-life (Brown et al., 1987), subsequent studies (Bhattacharya et al., 1988; Ferguson et al., 1988; Karol and Kamat, 1988; Karol et al., 1987) disproved this claim. For example, administration of [14C]MIC by inhalation or injection into laboratory animals resulted in widespread
81
distribution of 14C, including to the brain and conceptus (Bhattacharya et al., 1988; Ferguson et al., 1988). Also, MIC-induced myelotoxicity (Hong et al., 1987), complement activation (Kolb et al., 1987), chromosomal aberration (Shelby et al., 1987), and anti-MIC antibody formation in animals and Bhopal residents (Karol and Kamat, 1988; Karol et al., 1987) imply transport of intact MIC molecule to internal organs. Note that the half-life of a number of clinically used peptides is in the range of 2-5 min. MIC can cause N-carbamoylation of several end-terminal amino acids of tissue proteins (Sriramachari, 2004). Isocyanates can react with hydroxyl, sulfhydryl, and imidazole groups under physiologic conditions (Brown et al., 1987), which would imply that a wide variety of physiological functions can be altered following exposure to MIC, for which now there is scientific evidence (Dhara and Dhara, 2002; Sriramachari, 2004; Varma and Guest, 1993; Varma and Varma, 2005). The ability of MIC to increase the affinity of hemoglobin for oxygen (Lee, 1976) and inhibit mitochondrial respiration (Jeevaratnam et al., 1992) can lead to tissue hypoxia. Unlike organophosphates, MIC is not a potent inhibitor of cholinesterases (Brown et al., 1987), and its main toxic effects cannot be attributed to be a result of an inhibition of cholinesterase. A glutathione conjugate of MIC, S-(N-methylcarbamoyl) glutathione, has been isolated from rat tissues injected with MIC (Pearson et al., 1990; Slatter et al., 1991). Cysteine and glutathione conjugates of MIC are cytotoxic and can carbamoylate DNA and proteins. Isocyanate-glutathione conjugates can release isocyanate at the cell membrane (Bruggeman et al., 1986; Guest and Varma, 1994; Guest et al., 1992; Pearson et al., 1991).
VI. TOXICITY OF MIC IN ANIMAL MODELS At the time of the Bhopal accident, there was a single report on the toxicity of MIC in rats, mice, guinea pigs, rabbits, and human volunteers (Kimmerle and Eben, 1964). Exposure of animals to 1-23 ppm MIC for 1-4 hr caused severe irritation and pulmonary edema; mortality was biphasic such that animals died within hours and several days after up to 18 days. Exposure of humans to 2-4 ppm MIC caused lacrimation and irritation of the nose and throat and higher concentrations forced volunteers to leave the area (Kimmerle and Eben, 1964). The paucity of data on MIC at the time of the Bhopal accident and extensive toxicity data on diisocyanates (Anonymous, 1966; Varma, 1986) prompted the prestigious journal L a n c e t (Anonymous, 1984a) to comment, "In a year's time we will have learned a lot more about methyl i s o c y a n a t e - at an appalling price." Indeed, we did learn a lot in a few years' time. The U.S. National Toxicology
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S ECTI O N I 9Uses, A b u s e s , & E,pidemiolooay
Program and the National Institute of Environmental Health Sciences, as well as the Indian Council of Medical Research, released funds for MIC toxicity studies. It should nonetheless be noted that most of the post-Bhopal animal studies (Alarie et al., 1987; Bucher et al., 1987; Dodd et al., 1987; Fowler et al., 1987; Nemery et al., 1985b), reviewed by others (Bucher, 1987; Nemery et al., 1987; Varma and Guest, 1993; Dhara and Dhara, 2002), merely confirm the results of the 1964 study by Kimmerle and Eben published in German, which was not taken note of by isocyanate toxicologists and even by the Union Carbide authorities. In some of the post-Bhopal studies, rats, mice, and guinea pigs were exposed to 1-30 ppm MIC for 1-3 hr to simulate the Bhopal situation (Alarie et al., 1987; Ferguson et al., 1986; Varma 1987; Varma et al., 1987, 1988, 1990); other studies used excessively high concentrations (25-3500 ppm) of MIC (Dodd et al., 1987; Fowler et al., 1987), which have little bearing on the Bhopal episode. Toxic effects of MIC degradation products have also been investigated (Guest and Varma, 1991, 1992, 1993; Guest et al., 1992, 1994). There is a significant correlation between the effects of MIC in animal models and those in the Bhopal population, especially with regard to the mortality pattern. For example, both in animal models and in Bhopal, there is a lag period between the exposure and onset of death. Also, effects of MIC on pulmonary function, eyes, reproductive function, and immune response in animals models broadly mimic clinical toxicity in Bhopal. Fetal malformations reported in Bhopal have not been observed in animal models. Unfortunately, the initial enthusiasm for laboratory investigation subsided rather quickly, and very few studies were done to determine the long-term toxicity of MIC in animal models.
A. General Effects of MIC and Mortality in Animal Models All experimental studies noted signs of restlessness, lacrimation, discharge from the nose and mouth, and respiratory distress during the exposure of animals to MIC (Alarie et al., 1987; Bucher et al., 1987; Dodd et al., 1987; Varma et al., 1988). Exposure to MIC produced a marked decrease in body weight and food intake; although animals started gaining weight after 2 or 3 days, body weights did not approach those of control animals for as long as 2 weeks (Varma et al., 1988). Mortality during and soon after exposure to MIC (minutes) is rare, even at concentrations as high as 3500 ppm (Dodd et al., 1987). At MIC concentrations of 10-30 ppm, animals die several hours and days after exposure; deaths occurred either within the first 2 days or after a lag period of 1 week, and by 20 days postexposure, approximately 80% of exposed animals had died (Bucher et al., 1987; Varma et al., 1988). The biphasic death pattern has also been reported in Bhopal, although it is difficult to translate 8-20 days in the life of a rat to that for humans. Mortality in mice exposed to 40 ppm MIC could be reduced by prior treatment with gluco-
corticoids but not by sodium thiosulfate and atropine (Varma et al., 1988); both these agents were used by Indian doctors in Bhopal. Subcutaneous injection of MIC into rabbits caused a decrease in arterial pressure and lactic acidosis (Jeevarathinam et al., 1988).
B. Pulmonary Toxicity of MIC in Animals MIC was found to be both a sensory and a pulmonary irritant; animals died of pulmonary damage and tissue hypoxia (Alarie et al., 1987; Dodd et al., 1987; Nemery et al., 1985b), and a decrease in lung function persisted in surviving animals (Alarie et al., 1987; Bucher et al., 1987; Boorman et al., 1987; Nemery et al., 1985b; Stevens et al., 1987; Tepper et al., 1987). Guinea pigs were more susceptible to the lethal effects of MIC than rats (Dodd et al., 1987), which points to the limitation of animal studies in terms of mortality in Bhopal. Exposure to MIC also produced degenerative damage to olfactory epithelium in rats and mice (Uraih et al., 1987) and an increase in lung weight suggestive of proliferative changes (Bucher et al., 1987; Varma et al., 1987).
C. Reproductive and Other Nonpulmonary Toxic Effects of MIC in Animals The exposure of mice to 9 and 15 ppm MIC on day 8 of gestation was found to cause resorption of more than 80% of implants (Varma, 1987; Varma et al., 1987, 1990), which was associated with a decrease in plasma progesterone to nonpregnant levels (Varma et al., 1990). Fetal toxicity seemed unrelated to maternal toxicity since suppression of pulmonary edema by dexamethasone or injections of progesterone did not increase fetal survival; also, exposure of embryos to MIC in culture caused a concentration-dependent decrease in growth (Varma et al., 1990). Repeated exposure of pregnant mice from day 14 to day 17 of gestation to 3 ppm MIC also caused significant fetal and neonatal deaths (Schwetz et al., 1987). Lens opacity (Harding and Rixon, 1985) and corneal damage (Salmon et al., 1985) in rats exposed to MIC have been reported, although negative data also exist (Gupta et al., 1987). Immunosuppression in rats exposed to MIC was found by Dwiwedi et al. (1988) but not by Tucker et al. (1987). In addition, myelotoxicity (Hong et al., 1987), inhibition of cell cycle in lymphocytes (Conner et al., 1987), and changes in chromosome structure (Shelby et al., 1987) have also been reported.
D. Toxicity of MIC Metabolites in Rats and Mice Injections of 5 mmol/kg body weight of trimethylamine into mice from day 1 to day 17 of gestation caused maternal mortality, decreased fetal body weight, and fetal deaths; no such effect was observed with monomethylamine and
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity dimethylamine, although all three methylamines were toxic to mouse embryos in culture (Guest and Varma, 1991). Also, trimethylamine inhibited macromolecular synthesis in embryos and caused neural tube defects (Guest et al., 1992, 1994). Interestingly, administration of trimethylamine into pregnant mice from day 6 to day 15 of gestation led to a selective decrease in the body weight of male fetuses and their growth for up to 8 weeks (Guest and Varma, 1993). S-(N-methylcarbamoyl) glutathione, a product generated by MIC (Pearson et al., 1990; Slatter et al., 1991), also caused a dose-dependent (0.1-2 mM) decrease of mouse embryo size in culture (Guest et al., 1992).
VII. C L I N I C A L T O X I C I T Y O F M E T H Y L
ISOCYANATE IN BHOPAL A. Mortality in Bhopal "The dead and dying arrived by the truckload, others came by rickshaw or were carried by relatives. For some the effort of the journey itself proved too much, and they died soon after arrival," wrote Sutcliffe (1985), a British medical student who arrived at Bhopal on the day before the accident to start her elective. By the early morning of December 3, nearly 50,000 patients had reached the three major hospitals. Soon, Bhopal became a city of death (Varma, 1986). The exact number of people who died within 48 hr of the accident is not known and perhaps will never be known. A conservative estimate puts the death toll at nearly 2500 people soon after the accident and more than 15,000 in the following days and months (Mehta et aL, 1990).
B. Pulmonary Toxicity Clinical evaluations by different investigators (Bhargava et al., 1987; Kamat et al., 1985, 1987, 1992; Misra and Nag, 1988; Misra et al., 1987; Patel et al., 1987; Sharma and Gaur, 1987) based on physical examination, review of patient charts, and radiological findings strongly suggest that the most common and severe problem in Bhopal was caused by damage to lungs. Predominant symptoms in the victims were cough, expectoration, chest pain, and breathlessness, and physical findings included necrotizing lesions in almost all parts of the respiratory tract, edematous lungs, consolidation, hemorrhage, bronchopneumonia, and bronchiolitis (Dhara and Dhara, 2002). Follow-up studies of 903 patients with radiological records found that approximately 25% had radiological and 39% had functional abnormalities 2-4 months after the disaster (Dhara and Dhara, 2002; Nemery, 1996). A survey conducted 10 years after the disaster also found respiratory symptoms in 81% of 474 exposed residents compared to 38% in a control cohort of 76 subjects (Cullinan et al., 1996). Spirometry of 74 subjects (Cullinan et al., 1997) revealed significant decreases
83
in 1-sec forced expiratory volume, forced vital capacity, and forced expiratory flow between 25 and 75%. These findings are in conformity with those reported by other investigations involving a sizable number of subjects (Kamat et al., 1992; Vijayan and Sankaran, 1996). Kamat et al. also noted a worsening of respiratory complications after a lag period of 1 year. Persistent airway hyperreactivity after a single exposure to a chemical irritant has been termed reactive airways dysfunction syndrome (RADS) (Brooks et al., 1985); whether or not Bhopal victims suffer from RADS needs to be established (Nemery, 1996). A study by Avashia et al. (1996) (Avashi was the medical director of the Union Carbide Institute at the West Virginia plant in 1984) concluded that prolonged low exposure of workers to MIC did not cause any pulmonary complications; the level of exposure was not quantified. However, this study has little bearing on the situation in Bhopal, in which the population was exposed to high concentrations of MIC. Indeed, there are reports that minor leaks of MIC were not uncommon in the Bhopal plant, which workers used to detect because of watering in their eyes (Varma, 1986). It would be of interest to locate workers who used to work in the Union Carbide plant and monitor their lung functions. The data of Avashia et al. (1996) reinforce a pharmacological principle that no chemical produces detectable toxicity at all doses.
C. Ocular Toxicity Irritation in the eyes and profuse lacrimation were the second most common symptoms in the Bhopal population; these were apparent even in areas farther from the plant, implying that irritation of the eyes can occur with much lower concentration than is needed for other complications. Major findings included ocular burning, watering, pain, and photophobia (Andersson et al., 1984, 1985, 1988; Dwivedi et al., 1985). Follow-up studies conducted 9 months to 2 years after the accident revealed persistent eye watering, itching, redness, photophobia, burning, Bitot spots, and even corneal opacity (Andersson et al., 1986, 1990; Khurrum and Ahmad, 1987; Raizada and Dwivedi, 1987). Given the poor living conditions of the majority of victims, the propensity for infection has been found to further complicate chronic ocular lesions (Crabb, 2004; Dhara and Dhara, 2002).
D. Reproductive Toxicity A survey involving 865 pregnant women from 3270 families within 1 km of the pesticide plant found that 43.8% did not deliver live babies; of the 486 live births, 14.2% died within 30 days, compared with an infant death rate of approximately 3% in the two preceding years (Varma, 1987, 1991). A study of 18,978 households also found that
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miscarriage rates were 23.6% in MIC-exposed area compared with 5.6% in an unaffected area of comparable sociological background (Bhandari et al., 1990). A decrease in fetal and placental weight following exposure to MIC in Bhopal has also been recorded (Kanhere et al., 1987).
E. Neurologic and Behavioral Toxicity Psychological problems resulting from the sheer horror of the accident could be anticipated and have been documented (Srinivasamurthy and Isaac, 1987). In addition, neuroses, anxiety state, and exacerbation of preexisting neurological problems have also been documented (Sethi et al., 1987). Kamat et al. (1992) did a follow-up of their original cohort of 113 subjects for up to 2 years and noted significant neurological problems, such as anxiety and depression, in a substantial number of subjects. Cognitive impairment in the MIC-exposed population 1 year after the accident has also been reported (Misra and Kalita, 1997).
F. Carcinogenic, Cytogenetic, and Immunotoxic Effects A significantly higher frequency of chromosomal aberrations (gaps, dicentrics, rings, and altered configurations) in MIC-exposed compared to a control population 3 years after the accident has been documented (Ghosh et al., 1990). Lymphocytes from gas-exposed victims revealed chromosomal aberrations (Goswami, 1986; Saxena et al., 1988). The carcinogenic potential of MIC is equivocal (Dikshit and Kanhere, 1999). Nevertheless, scientists in Bhopal anticipate a high rate of cancer in the coming years, as is the experience in smokers (Crabb, 2004). However, to relate an increase in cancer to MIC exposure would require follow-up of a large number of Bhopal residents and of people throughout India living under similar conditions. Anti-MIC antibodies belonging to IgG, IgM, and IgE classes have been detected in animals and Bhopal residents exposed to MIC (Karol and Kamat, 1988; Karol et al., 1987; Kamat et al., 1992). Although these data clearly indicate that a hapten as small as MIC interacted with some native protein to generate antibodies, the health implications of these findings need to be established; for example, could they cause RADS (Nemery, 1996)?
G. Effects on the Progeny of Victims Although long-term effects of the Bhopal disaster on different organ systems have received some attention as stated previously, there is much less information on the effect, if any, of exposure to MIC on the progeny. The long-term health monitoring program undertaken by the ICMR was terminated in 1994, and it is not known whether its mandate extends to the progeny of survivors. Anecdotal reports suggest a high rate of birth defects, such as syndactyly (fused
digits) and pigeon chest among the children of MICexposed parents (Crabb, 2004). Ranjan et al. (2003) conducted a study in the summer of 2001 on the growth patterns of boys and girls born before the disaster, soon after the disaster to exposed parents, or exposed to MIC in utero; the study included 104 families with 68 girls and 73 boys, with 71 of the adolescents exposed to the gases (mean age, 16.9 years) and 70 unexposed cohorts (mean age, 16.7 years). This study found that the height and mid-arm circumference of boys exposed to gases as infants or in utero or born to exposed parents were significantly less than those of comparable cohorts; in the case of boys exposed in utero or born to exposed parents, body weight, height, mid-arm circumference, as well as head circumference were less relative to control cohorts. The maximal decrease in height (13.5 cm) was for boys exposed in utero. No such effect on girls was noted in these studies. It is interesting that an earlier study in mice found that injections of MIC metabolite trimethylamine into pregnant mice caused selective retardation of male pups along with a decrease in serum testosterone (Guest and Varma, 1993). Preliminary unpublished data suggest that although the height of girls was not affected, they do have menstrual problems. The data of Ranjan et al. (2003) showing MIC exposure-linked growth retardation of boys need not be genetic. It would be of interest to follow the growth pattern of the next generation.
VIII. C O N C L U S I O N S The exposure of the Bhopal population to MIC caused thousands of deaths and long-term effects of varying severity in nearly 200,000 survivors. Although chemically induced acute respiratory distress syndrome was probably the cause of acute deaths, the magnitude and the underlying mechanisms of long-term effects with the exception of pulmonary complications have yet to be identified. Animal studies corroborate some of the clinical findings, but they neither offer an explanation nor disclose the underlying mechanism of long-term effects. Soon after the accident, the highly respected journal N a t u r e (Anonymous, 1984b) commented, "The anguish vividly carried round the world by the television cameras seems not to have matured into anger, even hysteria, there would have been had the accident occurred on the edge of a European city or in Connecticut [headquarters of Union Carbide]." Could this be the reason why the concern of the scientific community in the developed countries was short-lived? It is safe to say that the full dimension of the toxicity of a chemical (and also its therapeutic potential) cannot be predicted from its chemical structure but can be approximated by careful and painstaking studies. Such an enquiry into MIC would be well deserved not because it will disclose an astounding
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity
rational therapeutic approach (as often demanded by activists of nongovernmental organizations, who fail to recognize that the treatment, for example, of obstructive lung disease is the same regardless of what led to it), but because it will be a fitting conclusion to the speculative assertions one way or another. Ten years after the Bhopal disaster, Dhara and Kriebel (1993) felt a need for a "sound epidemiology"; the demand is still valid 20 years later. Also, there is a merit to the demand for the state control of hazardous corporate operations (Varma and Varma, 2005) and global monitoring of potentially toxic material (Sriramachari and Chandra, 1997).
Acknowledgments The studies of the authors, cited herein, were supported by the Canadian Institutes of Health Research a n d its predecessor, Medical Research Council of Canada, and by the Faculty of Graduate Studies, McGill University. The studies in Bhopal were facilitated by volunteers and the Sambhavana Clinic.
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Dhara, V. R., and Kriebel, D. (1993). The Bhopal gas disaster: It's not too late for sound epidemiology. Arch. Environ. Health 48, 436-437. Dikshit, R. P., and Kanhere, S. (1999). Cancer patterns of lung, oropharynx and oral cavity cancer in relation to gas exposure at Bhopal. Cancer Causes Control 10, 627-636. Dodd, D. E., Frank, E R., Fowler, E. H., Troup, C. M., and Milton, R. M. (1987). Biological effects of short-term, high concentration exposure to methyl isocyanate. I. Study objectives and inhalation exposure design.. Environ. Health Perspect. 72, 13-19. D'Silva, T. D. J., Lopes, A., Jones, R. L., Singhawangcha, S., and Chan, J. K. (1986). Studies of methyl isocyanate chemistry in the Bhopal incident. J. Org. Chem. 51, 3781-3788. Dwivedi, P. C., Raizada, J. K., Saini, V. K., and Mittal, P. C. (1985). Ocular lesions following methyl isocyanate contamination. Arch. Ophthalmol. 103, 1627. Dwiwedi, P. D., Mishra, A., Gupta, G. S. D., Dutta, K. K., Das, S. N., and Ray, P. K. (1988). Inhalation toxicity studies of methyl isocyanate (MIC) in rats. IV. Immunologic response of rats one week after exposure: Effect on body and organ weights, phagocytic and DTH response. Ind. J. Exp. Biol. 26, 191-194. Eckerman, I. (2005). The Bhopal Saga. Universities Press, Hyderabad, India. Everest, L. (1985). Behind the Poison Cloud. Banner Press, Chicago. Ferguson, J. S., Schaper, M., Stock, M. E, Weyel, D. A., and Alarie, Y. (1986). Sensory and pulmonary irritation with exposure to methyl isocyanate. Toxicol. Appl. Pharmacol. 82, 329-335. Ferguson, J. S., Kennedy, A. L., Stock, M. E, Brown, W. E., and Alarie, Y. (1988). Uptake and distribution of 14C during and following exposure to [14C]methyl isocyanate. Toxicol. Appl. Pharmacol. 94, 104-117. Fowler, E. H., Dodd, D. E., and Troup, C. M. (1987). Biological effects of short-term, high concentration exposure to methyl isocyanate. V. Morphologic evaluation of rat and guinea pig lungs. Environ. Health Perspecr 72, 39-44. Ghosh, B. B., Sengupta, S., Roy, A., Maity, S., Ghosh, S., Talukder, G., and Sharma, A. (1990). Cytogenetic studies in human population exposed to gas leak at Bhopal, India. Environ. Health Perspect. 86, 323-326. Goldstein, A., Aronow, L., and Kalman, S. M. (1968). Principles of Drug Action, pp. 399-402. Harper & Row, New York. Goswami, H. K. (1986). Cytogenetic effects of methyl isocyanate exposure in Bhopal. Hum. Genet. 74, 81-84. Guest, I., and Varma, D. R. (1991). Developmental toxicity of methylamines in mice. J. Toxicol. Environ. Health 32, 319-330. Guest, I., and Varma, D. R. (1992). Teratogenic and macromolecular synthesis inhibitory effects of trimethylamine on mouse embryos in culture. J. Toxicol. Environ. Health 36, 27-41. Guest, I., and Varma, D. R. (1993). Selective growth inhibition of the male progeny of mice treated with trimethylamine during pregnancy. Can. J. Physiol. Pharmacol. 71, 85-87. Guest, I., and Varma, D. R. (1994). Inhibition of mouse embryonic, yolk sac, and limb-bud functions by the methyl isocyanate metabolite S-(N-methylcarbamoyl)glutathione. Can. J. Physiol. Pharmacol. 72, 50-56. Guest, I., Baillie, T. A., and Varma, D. R. (1992). Toxicity of the methyl isocyanate metabolite S-(N-methylcarbamoyl)GSH on mouse embryos in culture. Teratology 46, 61-67.
' Guest, I., Cyr, D. G., and Varma, D. R. (1994). Mechanism of trimethylamine-induced inhibition of macromolecular synthesis by mouse embryos in culture. Food Chem. Toxicol. 32, 365-371. Gupta, B. N., Stefanski, S. A., Bucher, J. R., and Hall, L. B. (1987). Effect of methyl isocyanate (MIC) gas on the eyes of Fischer 344 rats. Environ. Health Perspecr 72, 105-108. Hama, G. M. (1957). Symptoms in workers exposed to isocyanates. Arch. Indust. Health 16, 232. Harding, J. J., and Rixon, K. C. (1985). Lens opacities induced in rat lenses by methyl isocyanate. Lancet 1, 762. Hong, H. L., Bucher, J. R., Canipe, J., and Boorman, G. A. (1987). Myelotoxicity induced in female B6C3F1 mice by inhalation of methyl isocyanate. Environ. Health Perspecr 72, 143-148. Jeevarathinam, K., Selvamurthy, W., Ray, U. S., Mukhopadhyay, S., and Thakur, L. (1988). Acute toxicity of methyl isocyanate, administered subcutaneously in rabbits: Changes in physiological, clinico-chemical and histological parameters. Toxicology 51, 223-240. Jeevaratnam, K., Vidya, S., and Vaidyanathan, C. S. (1992). In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration. Toxicol. Appl. Pharmacol. 117, 172-179. Kamat, S. R., Mahashur, A. A., Tiwari, A. K. B., Potdar, P. V., Gaur, M., Kolhatkar, V. P., Vaidya, P. R., Parmar, D., Rupwate, R., Chaterjee, T. S., Jain, K., Kelkar, M. D., and Kinare, S. G. (1985). Early observations on pulmonary changes and clinical morbidity due to the isocyanate gas leak at Bhopal. J. Postgrad. Med. (India) 31, 63-72. Kamat, S. R., Patel, M. H., Kolhatkar, V. P., Dave, A. A., and Mahashur, A. A. (1987). Sequential respiratory changes in those exposed to the gas leak in Bhopal. Indian J. Med. Res. 86(Suppl.), 20-38. Kamat, S. R., Patel, M. H., Pradhan, P. V., Taskar, S. P., Vaidya, P. R., Kolhatkar, V. P., Gopalani, J. P., Chaandarana, J. P., Dalai, N., and Naik, M. (1992). Sequential respiratory, psychologic, and immunologic studies in relation to methyl isocyanate exposure over two years with model development. Environ. Health Perspect. 97, 241-253. Kanhere, S., Darbari, B. S., and Shrivastava, A. K. (1987). Morphological study of expectant mothers exposed to gas leak at Bhopal. Indian J. Med. Res. 86(Suppl.), 77-82. Karol, M. H., and Kamat, S. R. (1988). The antibody response to methyl isocyanate: Experimental and clinical findings. Bull. Eur. Physiopathol. Respir. 23, 591-597. Karol, M. H., Taskar, S., Gangal, S., Rubanoff, B. E, and Kamat, S. R. (1987). The antibody response to methyl isocyanate: Experimental and clinical findings. Environ. Health Perspecr 72, 169-175. Khurrum, M. A., and Ahmad, H. S. (1987). Long-term follow up of ocular lesions of methyl isocyanate gas disaster in Bhopal. Indian J. Ophthalmol. 35, 136-137. Kimmerle, G., and Eben, A. (1964). Zur toxicitat von methylisocyanat und dessen quantitativer bestimung in der luft [Toxicity of methyl isocyanate and~ its quantitation in the air]. Arch. Toxikol. 20, 235-241. Kolb, W. P., Savary, J. R., Troup, C. M., Dodd, D. E., and Tamerius, J. D. (1987). Biological effects of short-term, high concentration exposure to methyl isocyanate. VI. In vitro and in vivo complement activation studies. Environ. Health Perspecr 72, 189-195. Lapierre, D., and Moro, J. (2001). It Was Five Past Midnight in Bhopal. Full Circle, Delhi.
CHAPTER 7 9The Bhopal Accident and Methyl Isocyanate Toxicity Lee, C. K. (1976). Methylisocyanate as an antisickling agent and its reaction with hemoglobin S. J. Biol. Chem. 251, 6226-6231. Lepkowski, W. (1985, December 2), Bhopal, Indian city begins to heal but conflicts remain. Chem. Eng. News, 18-32. Le Quesne, P. M., Axford, A. T., McKerrow, C. B., and Parry, J. A. (1976). Neurological complications after a single severe exposure to toluene diisocyanate. Br. J. Ind. Med. 33, 72-78. Marwick, C. (1985). Bhopal tragedy's repercussions may reach American physicians. J. Am. Med. Assoc. 253, 2001-2013. Mehta, P. S., Mehta, A. S., Mehta, S. J., and Makhijani, A. B. (1990). Bhopal tragedy's health effects: A review of methyl isocyanate toxicity. J. Am. Med. Assoc. 264, 2781-2787. Misra, U. K., and Kalita, J. (1997). A study of cognitive functions in methyl isocyanate victims one year after Bhopal accident. Neurotoxicology 18, 381-386. Misra, U. K., and Nag, D. (1988). A clinical study of toxic gas poisoning in Bhopal, India. Indian J. Exp. Biol. 26, 201-204. Misra, N. P., Pathak, R., Gaur, K. J. B. S., Jain, S. C., Yesikar, S. S. Manoria, P. C., Sharma, K. N., Tripathi, B. M., Asthana, B. S., Trivedi, H. H., Sharma, V. K., Malhotr, Y., Verma, A., Bhargava, D. K., and Batni, G. (1987). Clinical profile of gas leak victims in acute phase after Bhopal episode. Indian J. Med. Res. 86 (Suppl.), 11-19. Morehouse, W., and Subramaniam, M. A. (1986). The Bhopal Tragedy: What Really Happened and What It Means for American Workers and Communities at Risk. Council of International & Public Affairs, New York. Munn, A. (1965). Hazards of isocyanates. Ann. Occup. Hyg. 8, 163-169. Nemery, B. (1996). Late consequences of accidental exposure to inhaled irritants: RADS and the Bhopal disaster. Eur. Respir. J. 9, 1973-1976. Nemery, B., Dinsdale, D., and Sparrow, S. (1985a). Methyl isocyanate: Thiosulphate does not protect. Lancet 2, 1245-1246. Nemery, B., Dinsdale, D., Sparrow, S., and Ray, D. E. (1985b). Effects of methyl isocyanate on the respiratory tract of rats. Br. J. Ind. Med. 42, 799-805. Nemery, B., Dinsdale, D., and Sparrow, S. (1987). The toxicity of inhaled methyl isocyanate in experimental animals: A review of studies published less than two years after the Bhopal disaster. Bull. Eur. Physiopathol. Respir. 23, 315-322. Patel, M. H., Kolhatkar, V. P., Potdar, V. P., Shekhavat, K. L., Shah, H. N., and Kamat, S. R. (1987). Methyl isocyanate survivors of Bhopal: Sequential flow volume loop changes observed in eighteen months' follow-up. Lung (India) 2, 59-65. Pearson, P. G., Slatter, J. G., Rashed, M. S., Han, D. H., Grillo, M. P., and Baillie, T. A. (1990). S-(N-methylcarbamoyl)glutathione: A reactive S-linked metabolite of methyl isocyanate. Biochem. Biophys. Res. Commun. 166, 245-250. Pearson, E G., Slatter, J. G., Rashed, M. S., Han, D. H., and Baillie, T. A. (1991). Carbamoylation of peptides and proteins in vitro by S-(N-methylcarbamoyl)GSH and S-(N-methylcarbamoyl)cysteine, two electrophilic S-linked conjugates of methyl isocyanate. Chem. Res. Toxicol. 4, 436-444. Raizada, J. K., and Dwivedi, E C. (1987). Chronic ocular lesions in Bhopal gas tragedy. Indian J. Ophthalmol. 35, 453-455. Ranjan, N., Sarangi, S., Padmanabhan, V. T., Holleran, S., Ramakrishnan, R., and Varma, D. R. (2003). Methyl isocyanate exposure and growth patterns of adolescents in Bhopal. J. Am. Med. Assoc. 290, 1856-1857.
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Rye, W. A. (1973). Human responses to isocyanate exposure. J. Occup. Med. 15, 306-307. Salmon, A. G., Kerr Muir, M., and Andersson, N. (1985). Acute toxicity of methyl isocyanate: A preliminary study of the doseresponse for eye and other effects. Br. J. Ind. Med. 42, 795-798. Saxena, A. K., Singh, K. P., Nagle, S. L., Gupta, B. N., Ray, P. K., Srivastav, R. K., Tewari, S. P., and Singh, R. (1988). Effect of exposure to toxic gas on the population of Bhopal. IV. Immunological and chromosomal studies. Indian J. Exp. Biol. 26, 173-176. Schwetz, B. A., Adkins, B., Harris, M., Moorman, M., and Sloane, R. (1987). Methyl isocyanate: Reproductive and developmental toxicology studies in Swiss mice. Environ. Health Perspect. 72, 149-152. Sethi, B. B., Sharma, M., Trivedi, J. K., and Singh, H. (1987). Psychiatric morbidity in patients attending clinics in gas affected areas of Bhopal. Indian J. Med. Res. 86(Suppl.), 45-50. Sharma, D. C. (2005). Bhopal: 20 years on. Lancet 365, 111-112. Sharma, P. N., and Gaur, K. J. B. S. (1987). Radiological spectrum of lung changes in gas-exposed victims. Indian J. Med. Res. 86(Suppl.), 39--44. Shelby, M. D., Allen, J. W., Caspary, W. J., Haworth, S., Ivett, J., Kligerman, A., Luke, C. A., Mason, J. M., Myhr, B., Tice, R. T., Valencia, R., and Zeiger, E. (1987). Results of in vitro and in vivo genetic toxicity tests on methyl isocyanate. Environ. Health Perspect. 72, 183-187. Slatter, J. G., Rashed, M. S., Pearson, P. G., Han, D. H., and Baillie, T. A. (1991). Biotransformation of methyl isocyanate in the rat: Evidence of GSH conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities. Chem. Res. Toxicol. 4, 157-161. Srinivasamurthy, R., and Isaac, M. K. (1987). Mental health needs of Bhopal disaster victims and training of medical officers in mental health aspects. Indian J. Med. Res. 86(Suppl.), 51-58. Sriramachari, S. (2004). The Bhopal gas tragedy: An environmental disaster. Curr. Sci. 86, 905-920. Sriramachari, S., and Chandra, H. (1997). The lessons of Bhopal [toxic] MIC gas disaster scope for expanding global biomonitoring and environmental specimen banking. Chemosphere 34, 2237-2250. Stevens, M. A., Fitzgerald, S., M6nache, M. G., Costa, D. L., and Bucher, J. R. (1987). Functional evidence of persistent airway obstruction in rats following a two-hour inhalation exposure to methyl isocyanate. Environ. Health Perspect. 72, 89-94. Sufrin, S. C. (1985). Bhopal: Its Setting, Responsibility and Challenge. Ajanta, New Delhi. Sutcliffe, M. (1985). My student elective: An eyewitness in Bhopal. Br. Med. J. 290, 1883-1884. Tepper, J. S., Wiester, M. J., Costa, D. L., Watkinson, W. P., and Weber, M. E (1987). Cardiopulmonary effects in awake rats four and six months after exposure to methyl isocyanate. Environ. Health Perspect. 72, 95-103. Tucker, A. N., Bucher, J. R., Germolec, D. R., Silver, M. T., Vore, S. J., and Luster, M. I. (1987). Immunological studies on mice exposed subacutely to methyl isocyanate. Environ. Health Perspect. 72, 139-141. Uraih, L. C., Talley, E A., Mitsumori, K., Gupta, B. N., Bucher J. R., and Boorman, G. A. (1987). Ultrastructural changes in the nasal mucosa of F344/N rats and B6C3F1 mice following an acute exposure to methyl isocyanate. Environ. Health Perspect. 72, 77-88.
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SECTION I 9 Uses, Abuses, & E p i d e m i o l o g y
Vardarajan, S., Doraiswamy, L. K., Ayyangar, N. R., Iyer C. S. E, Khan, A. A., Lahiri, A. K., Muzumdar, K. V., Mashelkar, R. A., Mitra, R. B., Nambiar, O . G. B., Ramachandran, V., Sahasrabudhe, V. D., Sivaram, S., Sriram, M., Thyagarajan, G., and Venkataraman, R. S. (1985). A Scientific Enquiry into the Methyl Isocyanate Leak in Bhopal. Council of Scientific and Industrial Research, New Delhi. Varma, D. R. (1986). Anatomy of the methyl isocyanate leak in Bhopal. In Hazard Assessment of Chemicals (J. Saxena, Ed.), pp. 233-289. Hemisphere, Washington, DC. Varma, D. R. (1987). Epidemiological and experimental studies on the effects of methyl isocyanate on the course of pregnancy. Environ. Health Perspect. 72, 153-157. Varma, D. R. (1989). Hydrogen cyanide and Bhopal. Lancet 2, 557-558. Varma, D. R. (1991). Pregnancy complications in Bhopal women exposed to methyl isocyanate vapor. J. Environ. Sci. Health A26, 1437-1447. Varma, D. R., and Guest, I. (1993). The Bhopal accident and methyl isocyanate toxicity. J. Toxicol. Environ. Health 40, 513-529.
Varma, D. R., Ferguson, J. S., and Alarie, Y. (1987). Reproductive toxicity of methyl isocyanate in mice. J. Toxicol. Environ. Health 21, 265-275. Varma, D. R., Ferguson, J. S., and Alarie, Y. (1988). Inhibition of methyl isocyanate toxicity in mice by starvation and dexamethasone but not by sodium thiosulfate, atropine and ethanol. J. Toxicol. Environ. Health 24, 93-101. Varma, D. R., Guest, I., Smith, S., and Mulay, S. (1990). Dissociation between maternal and fetal toxicity of methyl isocyanate in mice and rats. J. Toxicol. Environ. Health 30, 1-14. Varma, R., and Varma, D. R. (2005). The Bhopal disaster of 1984. Bull. Sci. Technol. Soc. 25, 37--45. Vijayan, V. K., and Sankaran, K. (1996). Relationship between lung inflammation, changes in lung function and severity of methyl isocyanate exposure in victims of the Bhopal tragedy. Eur. Respir. J. 9, 1997-1982. Weyel, D. A., Rodney, B. S., and Alarie, Y. (1982). Sensory irritation, pulmonary irritation and acute lethality of polymeric isocyanate and sensory irritation of 2,6-toluene diisocyanate. Toxicol. Appl. Pharmacol. 64, 423-430.
CHAPTER
Global Epidemiology of Organophosphate and Carbamate Poisonings TETSUO SATOH Chiba University, Chiba, Japan
I. I N T R O D U C T I O N
chronic OP-induced neuropsychiatric disorder. The first evidence of this type of syndrome (delayed psychopathologic-neurologic lesions) was reported by Spiegelberg (1963), who worked on the production and handling of highly toxic nerve gases in Germany during World War II. Using the characteristic symptomatology, patients could be classified into two distinct groups. The first and largest group was characterized by persistently lowered vitality and ambition; defective autonomic regulation leading to cephalalgia and gastrointestinal and cardiovascular symptoms; premature decline in potency and libido; intolerance to alcohol, nicotine, and various medicines; and an impression of premature aging. The second group, in addition to the previously mentioned symptoms, showed one or more of the following: depressive or subdepressive disorders of vital function, cerebral vegetative (syncopal) attacks, slight or moderate amnestic or demential effects, and slight organoneurologic defects. These symptoms developed and persisted for 5-10 years following exposure to these most toxic OPs during the war years. The controversial paper of Gershon and Shaw (1961), who performed a study of 16 cases of pesticide applicators exposed primarily to OP insecticides for 10-15 years, reported a wide range of persistent signs of toxicity (Ecobichon, 2001). Although the results of other studies have been equivocal in their support of such an array of long-term signs and symptoms, there is a persistent recurrence of the symptomatology in a number of anecdotal and documented reports (Ecobichon, 1994; Marrs, 1993; Jamal, 1997). This chapter describes the mechanism of toxicity of OPs, intoxication and fatality rates associated with OPs, clinical aspects of OP intoxication, treatment of poisoning, and epidemiological studies of OP poisoning throughout the world.
Organophosphorus compounds (OPs) are a diverse group of chemicals, including insecticides (malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, and others) and nerve gases (soman, satin, tabun, and VX). OPs were first synthesized in the early 1800s, and in 1934, Lange in Berlin and Schrader (a chemist at Bayer AG) in Germany investigated the use of OPs as insecticides. However, the German military prevented the use of OPs as insecticides and instead developed an arsenal of chemical warfare agents (i.e., tabun, sarin, and soman). In 1941, during World War II, OPs were reintroduced worldwide for agricultural use, as originally intended. Serious poisonings due to misuse of OP insecticides have been reported for more than four decades. OPs are some of the most widely used insecticides in the world, and the agents comprising these insecticides have a common mechanism of action. Although the structures are diverse in nature, the mechanisms by which the OP insecticides elicit their toxicity are identical and are associated with the inhibition of the nervous tissue acetylcholinesterase (ACHE) (Chambers and Levi, 1992). The classical picture of anticholinesterase (anti-ChE) insecticide intoxication, first described by DuBois (DuBois, 1948; DuBois et al., 1949), has become more complicated in recent years due to the recognition of additional and persistent signs of neurotoxicity not previously associated with these chemicals. First and frequently associated with exposure to high concentrations of the insecticides are effects that may persist for several months following exposure involving neurobehavioral, cognitive, and neuromuscular functions (Marrs, 1993; Ecobichon, 1994, 1998; Jamal, 1997). Jamal described this phenomenon as a Toxicology of Organophosphate and Carbamate Compounds
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S ECTI O N I 9Uses, A b u s e s , & E p i d e m i o l o g y
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II. M E C H A N I S M O F T O X I C A C T I O N S OF OPs
A. AChE Inhibition The primary mechanism of action of OP pesticides is inhibition of ACHE. AChE is an enzyme found in the central nervous system (CNS) and the peripheral nervous system, and its normal physiologic action is to metabolize acetylcholine (ACh), a neurotransmitter. OPs inactivate AChE by phosphorylating the serine hydroxyl group located at the active site of ACHE. The phosphorylation occurs by loss of an OP leaving group and establishment of a covalent bond with ACHE. Although anti-ChE insecticides have a common mode of action, there are significant differences between OP and carbamate (CM) insecticides. The reaction between OP and serine residue at the active site in AChE results in the formation of a transient intermediate complex that partially hydrolyzes with the loss of the Z substituent group, leaving a stable, phosphorylated, and largely unreactive inhibited enzyme that can be reactivated only at a very slow rate (Fig. 1). With many OP insecticides, an irreversibly inhibited enzyme is formed, and the signs and symptoms of intoxication are prolonged and persistent. Without intervention, toxicity will persist until sufficient quantities of "new" AChE are synthesized 20-30 days later to destroy efficiently the excess ACh. The nature of the substituent groups at X, Y, and Z plays an important role in the specificity for the enzyme. Introduced OP insecticides (acephate, temephos, dichlorvos, etc.) are less tenacious inhibitors of nervous tissue ACHE, with the phosphorylated enzyme being more readily and spontaneously dissociated. In contrast, CM insecticides, which attach to the reactive site of ACHE, undergo hydrolysis in two stages. The first stage is the removal of the X substituent (an aryl or
Organophosphorus Ester XO E-OH
+
\
YO /
p
0 #. \Z
xo ~
+
O II X O C - NHCH 3
XO =
E-OH
YO ~ +
Carbamate Ester E-OH
\
E-O-P=O
\
YO /
p
0 9 \ OH
ZH
O II ~- E- O - C - NHCH 3 +
+
~ E-OH
O It + H O - C - NHCH 3
XOH
FIG. 1. Inhibition of AChE by organophosphorus and carbamate insecticides. The interaction between an organophosphorus or carbamate ester with the serine hydroxyl groups in the active site of acetylcholinesterase (E-OH). The intermediate, unstable complexes formed before the release of the "leaving" groups (ZH and XOH) are not shown. The dephosphorylation or decarbamylation of the inhibited enzyme is the rate-limiting step in forming free enzymes. Reproduced with permission from Ecobichon (2001).
alkyl group) with the formation of a carbamylated enzyme. The second stage is the decarbamylation of the inhibited enzyme with the generation of free, active enzyme (Fig. 1). CMs are poor substrate for serine-containing enzymes such as ACHE. A number of OP esters (phosphate, phosphonate, and phosphoramidate), chemical warfare agents (sarin, soman, and tabun), and a few other compounds, such as DFP, mipafox, and leptophos, have the ability to bind strongly to the active site of AChE and neuropathy target esterase to produce an irreversibly inhibited enzyme by a mechanism known as aging. The aging process is dependent on the size and configuration of the alkyl (R) substituent, with the potency of the ester increasing in the order of diethyl, diisopropyl, and dibutyl for such analogs as DFP and mipafox (Aldridge and Johnson, 1971).
B. Pesticide-Induced Oxidative Stress Abdollahi et al. (2004) reviewed the oxidative stress caused by pesticides. The widespread use of pesticides in public health and agricultural programs has caused severe environmental pollution and health hazards, including cases of severe acute and chronic human poisoning (Ellenhom et al., 1997; Abdollahi et al., 1997; Jalali et al., 2000; Pajoumand et al., 2002). The toxic pesticides have become an integral part of the ecosystem, although many of them are extremely toxic to mammals and other nontarget creatures. However, the implications of pesticide residues for human health have yet to be comprehensively documented. Free radicals play an important role in the toxicity of pesticides and environmental chemicals. Pesticides may induce oxidative stress by excess generation of free radicals, especially reactive oxygen species and reactive nitrogen species, and alteration in antioxidants and the scavenging system, causing lipid peroxidation (Banerjee et al., 1999; Dettbarn et al., 2001; Gupta et al., 2001a,b, 2002; Etemadi-Aleagha et al., 2002). The toxicology of various pesticides is noted in Table 1. Several studies have demonstrated oxidative stress induced by OPs in rats (Gultekin, 2000; Gupta et al., 2001a,b; Verma, 2001; Akhgari et al., 2003) and humans (Banerjee et al., 1999; Ranjbar et al., 2002; Dantoine et al., 2003). Lipid peroxidation is also evident in rat brains (Verma, 2001) and human erythrocytes (Gultekin, 2000; Dantoine et al., 2003). OP-induced seizures have been reported in association with oxidative stress (Gupta et al., 2001a,b). It has also been shown that the acute tubular necrosis that accompanies OP toxicity is related to reactive oxygen species and lipid peroxidation (Poovala et al., 1999). Gupta (2004) reviewed the importance of brain regional heterogeneity in relation to cholinergic and noncholinergic (oxidants/antioxidants) determinants, with particular reference to OP and CM pesticides and OP nerve agents.
CHAPTER 8 9Epidemiology of OP and CM Poisonings
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TABLE 1. Toxicological Characteristics of Organophosphate Poisoninga
Examples Diazinon, malathion, parathion, chlorpyrifos, dichlorvos
Site of toxicity
Major acute symptoms
Irreversible inhibition of red blood cell cholinesterase, acetylcholinesterase, plasma cholinesterase
Mild: fatigue, headache, blurred vision, dizziness, numbness of extremities, nausea, vomiting, excessive sweating and salivation, tightness in chest Moderate: weakness, difficulty talking, muscular fasciculations, miosis Severe: unconsciousness, flaccid paralysis, moist rales, respiratory difficulty, cardiac arrhythmias, cyanosis
aAdapted from Ellenhornet al. (1997). III. I N T O X I C A T I O N A N D F A T A L I T Y R A T E S
A. Mortality and Morbidity Worldwide mortality, studies report mortality rates of 3-25%. The compounds involved most frequently are malathion, dichlorvos, trichlorfon, and fenthion/malathion. Mortality rates depend on the type of compound used, amount ingested, general health of the patient, delay in discovery and transport, insufficient respiratory management, delay in intubation, and failure to wean off ventilatory support. Signs and symptoms of OP poisoning can be divided into three broad categories, including: muscarinic effects, nicotinic effects, and CNS effects. Complications include respiratory distress, seizures, and aspiration pneumonia. Respiratory failure is the most common cause of death.
B. Age Emerson e t al. (1999) reported that men aged 30-50 years were more likely to attempt suicide with OPs. In the study, 68 of 69 patients were men. Agarwal (1993) found that most OP poisonings occur in patients aged 21-30 years. The male-to-female ratio in the study was 2.1:1. Both Emerson et al. and Agarwal reported that accidental poisoning was more likely in children than in adults.
IV. C L I N I C A L A S P E C T S OF OP INTOXICATION
A. Physical Furtado and Chan (2004) reported that as the vital signs showing depressed respiratory rate, bradycardia, and hypotension are common. Hypothermia also can be observed. Paralysis due to OP intoxication is categorized into three types. Type I involves acute paralysis secondary to persistent depolarization at the neuromuscular junction. As the type II intermediate syndrome, was described in 1974, with an
incidence of 8-49%. It develops 24-96 hr after resolution of acute commonly cholinergic poisoning symptoms and manifests as paralysis and respiratory distress. This syndrome involves proximal muscle groups, with relative sparing of distal nuscle groups. Various degrees of cranial nerve palsies are also involved. Neuromuscular transmission defect and toxin-induced muscular instability play a role in intermediate syndrome. Intermediate syndrome persists for 4-18 days, can require intubation, and can be complicated by infections or cardiac arrhythmias. Type III involves OP-induced delayed polyneuropathy (OPIDN), occurs 2 or 3 weeks after exposure to large doses of certain OPs. Distal muscle weakness with relative sparing of the neck muscles, cranial nerves, and proximal muscle groups characterize OPIDN, and recovery can take up to 12 months. Neuropsychiatric effects associated with chronic OP intoxication include impaired memory, confusion, irritability, lethargy, and psychosis. Ophthalmic effects due to direct ocular exposure to OPs include optic neuropathy, retinal degeneration, defective vertical smooth pursuit, myopia, and miosis. Respiratory effects, including muscarinic, nicotinic, and central effects, contribute to respiratory distress in acute and delayed OP toxicity. Muscarinic effects, such as bronchospasm and laryngeal spasm, can lead to airway obstruction. Nicotinic effects can lead to weakness and paralysis of respiratory oropharyngeal muscles. Central effects can lead to cessation of respiration. Rhythm abnormalities include sinus tachycardia, sinus bradycardia, extrasystoles, atrial fibrillation, ventricular tachycardia, and fibrillation. Other cardiovascular effects include hypertension, hypotension, and noncardiogenic pulmonary edema. Gastrointestinal manifestations such as nausea, vomiting, diarrhea, and abdominal pain are the first to occur after OP exposure.
B. Cholinesterase Inhibition as an Extensively Used Biomarker Laboratory diagnosis of OP poisoning is based on the measurement of cholinesterase activity. Both erythrocyte and plasma cholinesterase levels can be used. Urinary
92
S E CTI O N I 9 Uses, Abuses, & Epidemiology
p-nitrophenol can be measured in parathion poisoning. Depressed cholinesterase levels only confirm the diagnosis of OP poisoning retrospectively. Measurement of red blood cell (RBC) and plasma ChE levels prior to treatment with 2PAM is of immense value. RBC AChE represents the AChE found in CNS gray matter, RBCs, and peripheral nerve, tissue, muscle, and brain. Plasma ChE is synthesized in the liver and eventually circulates in the blood plasma. It is found in the CNS white matter, pancreas, and heart. RBCChE is the more accurate of the two measurements, but plasma ChE is easier to assay and is more widely available. Mild poisoning is defined as depression of ChE activity to 20-50% of normal. Moderate poisoning occurs when activity is 10-20% of normal. Severe poisoning occurs at less than 10% of ChE activity. Small short-term exposures can depress ChE activity to very low levels with minimal symptoms. Levels do not always correlate with clinical illness. The level of ChE activity is relative and is based on population estimates. Neonates and infants are baseline level that are lower than those in adults. Because most patients do not know their baseline level, the diagnosis can be confirmed by observing a progressive increase in the ChE value until it plateaus over time. Falsely depressed levels of plasma ChE are observed in cases of liver dysfunction (e.g., cirrhosis), low protein conditions (e.g., malnutrition), neoplasia, and infectious hypersensitivity reactions. In addition, the use of drugs such as succinylcholine, codeine, and morphine renders falsely depressed plasma ChE levels. The first and second trimesters of pregnancy and genetic deficiency of plasma ChE are other causes. Other laboratory findings include leukocytosis with a normal differential consistent with a stress reaction, increased hematocrit from hemoconcentration due to fluid losses, anion gap acidosis due to poor tissue perfusion and hyperglycemia with hypokalemia, and hypomagnesemia due to catecholamine excess. Electrocardiogram (ECG) findings include prolonged QTc interval (most commonly up to 67%), elevated ST segments, inverted T waves, and prolonged PR interval (Chung et al., 1996).
TABLE 2.
C. Extremely Sensitive B i o m a r k e r of OP Exposure A novel biomarker of OP insecticide exposure, egasyn, is an accessory protein of [3-glucuronidase ([3G) in the liver microsomes (Swank and Paigen, 1973). Medda et al. (1987) reported that egasyn has esterase activity and it is one of the carboxylesterase (CarbE) isozymes. Later, Hosokawa and coworkers (1987, 1990) purified three CarbE isozymes (RL 1, RL2, and RH1) from rat liver microsomes, and egasyn was identified as RL2 isozyme. In 1998, Satoh and Hosokawa proposed a new classification of CarbE isozymes based on the molecular homology of the individual esterase isozymes. Figure 2 summarizes the localization of the egasyn-[3G complex and dissociation and release of [3G from liver microsomes into plasma. When animals as well as humans are intoxicated with OP, the OP is incorporated into the liver microsomes and metabolized to form the corresponding oxon. In order to study the release of [3G from the liver to plasma, rats and hamsters were treated with bis-p-nitrophenylphosphate, which is a specific CarbE inhibitor, and plasma [3G activities were determined. As shown in Table 2, plasma [3G activities
FIG. 2. Schematic representation of the release of [3-glucuronidase ([3G) from hepatocytes to blood by OP administration in animals. EG, egasyn; OP, organophosphate; ER, endoplasmic reticulum.
Changes in 13-Giucuronidase Activity after Administration of Insecticides in Ratsa
Time after administration (hr) Insecticide (mg/kg) EPN (30) Fenitrothion (100) Phenthoate (8.0) Carbaryl (200) Phenothrin (100) Allethrin (100)
3 3.42 4.22 0.15 4.56 0.042 0.050
__+0.43** (114.0) b _____0.51"* (114.0) _____0.07 (5.1) _____0.50** (152.0) __+0.005 (1.4) __+0.006 (1.6)
5 4.16 3.81 0.60 3.32 0.051 0.060
+ 0.51"* (138.6) • 0.42** (117.0) + 0.09* (20.1) __+0.53** (110.5) +__0.005 (1.7) __+0.006 (1.6)
24 0.05 0.04 0.04 0.03 0.03 0.040
_ 0.006 _ 0.007 _ 0.006 --- 0.007 +- 0.006 _ 0.006
(1.5) (1.3) (1.3) (1.0) (1.0) (1.3)
aNormal value of plasma [3-glucuronidase activity for control rats is 0.034 ___0.02 unit/ml. bNumbers in parentheses indicate the fold change from nontreated rats. Significantly different from nontreated rats: *p < 0.05, **p < 0.01.
CHAPTER 8 9Epidemiology of OP and CM Poisonings TABLE 3. Inhibition of Plasma Cholinesterase and Increase in Plasma 13-Glucuronidase after EPN Treatment in Ratsa
Dose (mg/kg) None 1.0 5.0 10.0 30.0
Plasma cholinesterase activity (unit/liter)
Plasma 13-glucuronidase activity (unit/ml)
29.5 + 3.66 (1.00)b 31.1 _+ 4.23 (1.05) 20.0 _+ 3.41 (0.65) 18.4 + 2.20* (0.62) 14.9 + 2.48* (0.50)
0.03 + 0.01 (1.00) 0.95 _+ 0.10" (31.7) 2.89 + 0.54** (96.3) 3.46 _+ 0.55** (115.3) 4.51 + 0.37** (150.3)
aAnimals were sacrificed 5 hr after EPN administration.Valuesrepresent mean __+SE from three to five rats. bNumbers in parentheses indicate the fold change from nontreated rats. Significantly different from nontreatedrats: *p < 0.05,**p< 0.01.
were increased after oral administration of EPN, fenitrothion, and carbaryl, but not pyrethroids, in a timedependent manner and reached maximum at 2.5 hr after administration. To compare the extent of plasma ChE inhibition, which is widely used as the marker of OP intoxication, and increase in plasma [3G activities, rats were treated with EPN and sacrificed 5 hr later. As shown in Table 3, plasma ChE activity was decreased to approximately 50% of the normal level after EPN administration, whereas plasma [3G activity was significantly increased approximately 150 times that of control. This indicates that the increase in plasma [3G activity is much more susceptible to exposure to OP insecticides than ChE inhibition. A similar increase in plasma [3G activities is observed in carbaryl intoxication but not in the case of pyrethroids. The reason for insensitivity of pyrethroids to OP seems to be due to the rapid metabolism by the enzyme to form the inactive metabolites. In summary, the increase in blood [3G activity after OP exposure is a much more sensitive and rapid biomarker than blood ChE inhibition.
V. T R E A T M E N T
OF POISONING
In order to secure the patient's airway, intubation is necessary in cases of respiratory distress from laryngospasm, bronchospasm, or severe bronchorrhea. Regular monitoring of neck muscle weakness, respiratory rate, arterial blood gas, and mental status is required to assess progression or decompensation. The tidal volume initiated by the patient can be used as a measure of disease severity in those who are intubated. Administration of atropine should be withheld until a cardiac monitor and a defibrillator are in place and the patient's airway is secured. Atropine can precipitate ventricular fibrillation in hypoxic patients. Continuous cardiac monitoring and an ECG are necessary. Electrical pacing is the treatment
93
of choice for ventricular tachycardia associated with a prolonged QTc. Atropine can reverse some cardiac manifestations. Electrolyte abnormalities may cause dysarrhythmias. Health care providers must avoid contaminating themselves while handling patients. Personal protective equipment, such as neoprene or nitrile gloves and gowns, should be used when decontaminating patients because hydrocarbons can penetrate nonpolar substances, such as latex and vinyl. Charcoal cartridge masks should be used for respiratory protection when decontaminating patients. The eyes of patients should be irrigated with ocular exposures using isotonic sodium chloride solution or lactated Ringer's solution. Morgan lenses can be used for eye irrigation. Activated charcoal (0.5-1gq4h) is used for gastric decontamination. Sorbitol can be used; however, many patients have increased gastrointestinal motility following OP poisoning. The mainstay of medical therapy in OP poisoning is atropine or glycopyrrolate 2-PAM, and diazepam which can be used for seizure control. In 1992, De Silva and Wijewickrema studied the treatment of OP poisoning with atropine and 2-PAM and, later in the same year, with atropine alone. They found that atropine seemed to be as effective as atropine plus 2-PAM in the treatment of acute OP poisoning. The controversy continued when other authors observed more respiratory complications and higher mortality rates with the use of high-dose 2-PAM. Low dose (1-2 g) 2-PAM is the current recommendation.
VI. E P I D E M I O L O G I C A L STUDIES OF OP-INDUCED POISONING More than 1.5 million tons of pesticides is manufactured every year. Most pesticides are hazardous. Extremely hazardous pesticides are easily available in many developing countries, and pesticide poisoning remains a serious problem worldwide. Internationally, pesticide poisonings are the most common mode of suicide in some developing countries (e.g., Sri Lanka). The World Health Organization (1999) estimated that more than 500,000 people died from self-harm in Southeast Asia and the west Pacific during 2000, and pesticides are among the most important methods of self-harm (Eddleston et al., 2002). In addition, 3 million pesticide poisoning cases occur worldwide every year, with 220,000 deaths, most of which are intentional.
A. India and Australia In a study of OP poisoning in India, Agarwal (1993) found that 67.4% of patients had suicidal intentions, 16.8% of the poisonings were caused by occupational exposures, and 15.8% of patients were poisoned accidentally. An
S E CTI O N I 9 Uses, Abuses, & Epidemiolooay
94
3.9% o f p o i s o n i n g s are due to insecticides. A c c o r d i n g to the E n v i r o n m e n t a l P r o t e c t i o n A g e n c y , cases r e p o r t e d in C a l i f o r n i a w i t h d o c u m e n t e d pesticide e x p o s u r e h a v e b e e n
A u s t r a l i a n study o f O P p o i s o n i n g p e r f o r m e d b y E m e r s o n et al. ( 1 9 9 9 ) f o u n d that o n l y 3 6 % o f patients h a d suicidal intentions c o m p a r e d to 6 5 - 7 5 % in d e v e l o p i n g countries.
p u b l i s h e d (Table 4).
B. U n i t e d S t a t e s C. J a p a n
The American Association of Poison Control Centers' " N a t i o n a l I n c i d e n c e R e p o r t " indicates that pesticide injuries r a n g e f r o m 7 0 , 0 0 0 to 80,000 annually. N a t i o n w i d e ,
T A B L E 4.
T h e National R e s e a r c h Institute o f Police Sciences ( N R I P S ) has published the annual report o f the epidemiological
Cases Reported in California with D o c u m e n t e d Pesticide Exposure S u m m a r i z e d by the Type of Illness and the Type of Pesticide, 2 0 0 2 a
Antimicrobials c
Type of illness b
Occupational d
Cholinesterase inhibitors c
Nonoccupational d
Occupational
Nonoccupational
Other pesticides c
Occupational
Nonoccupational
Total
Systemic Systemic with respiratory and topical effects
27
3
11
1
27
56
125
Systemic with respiratory effects
27
26
17
13
27
32
142
Systemic with topical effects
7
0
20
6
50
37
120
Systemic only
7
17
44
29
54
54
205
Respiratory with topical effects
18
9
4
1
18
52
102
Respiratory only
31
51
3
5
10
19
119
Eye only
94
14
10
3
198
85
404
Skin only
40
2
10
2
27
4
85
2
1
3
0
7
1
14
2
4
21
10
8
64
109
255
127
143
70
426
404
1425
Respiratory
Topical
Eye and skin
Asymptomatic Asymptomatic
Total
aData from the California Department of Pesticide Regulation, Pesticide Illness Surveillance Program (www.cdpr.ca.gov). Documented pesticide exposure includes cases classified as definitely, probably, or possibly related to pesticide exposure as well as cases of documented exposure that did not lead to symptomatology. Definite: High degree of correlation between the pattern of exposure and resulting symptomatology. Requires both medical evidence (such as measured cholinesterase inhibition, positive allergy tests, and characteristic signs observed by medical professional) and physical evidence. Probable: Relatively high degree of correlation exists between the pattern of exposure and the resulting symptomatology. Either medical or physical evidence is inconclusive or unavailable. Possible: Some degree of correlation evident. Medical and physical evidence is inconclusive or unavailable. bCategorization of the types of symptoms experienced. Systemic: Any health effects not limited to the respiratory, skin, and/or eye. Cases involving multiple illness symptom types including systemic symptoms are included in the systemic category. Respiratory: Health effects involving any part of the respiratory tree. Topical: Health effects involving only the eyes and/or skin. This excludes outward physical signs (miosis and lacrimation) related to effects on internal bodily systems. These signs are classified under Systemic. Asymptomatic: Exposure occurred but did not result in illness/injury. Cholinesterase depression without symptoms falls in this category. CType of pesticide is based on functional class. Antimicrobials: Pesticides used to kill or inactivate microbiological organisms (bacteria, viruses, etc.). Cholinesterase inhibitors: Pesticides known to inhibit the function of the cholinesterase enzyme. Other pesticides: Any pesticide that is not an antimicrobial or cholinesterase-inhibiting pesticide. dOccupational or nonoccupational: The relationship between the illness/injury and the individual's work. Occupational: Work related. The individual was on the job at the time of the incident. This includes both paid employees and volunteers working in a similar capacity as paid employees. Nonoccupational: Not work related. The individual was not on the job at the time of the incident. This category includes individuals on the way to or from work (before the start or after the end of their workday).
CHAPTER 8 9Epidemiology of OP and CM Poisonings
FIG. 3.
The incidence of various toxicants in Japan.
studies on criminal and suicidal events. The toxicants studied are carbon monoxide, cyanide and its derivatives, medicines, alkaloids, and pesticides, including paraquat and miscellaneous. As shown in Fig. 3, the numbers of the incidence of pesticide poisoning are approximately 22-30% of the total incidences between 1991 and 1996. The incidence of pesticide poisoning has been gradually declined during this time. This seems to be influenced by the decrease in the incidence of paraquat poisoning, which has extremely high toxicity. According to the NRIPS, there are many more cases of OP intoxication of OP than CM (Table 5). D. T a i w a n
Unlike the prevalence of drug poisoning exposures in most Western countries, pesticide poisoning exposures are numerous in Taiwan. Epidemiological studies of the
TABLE 5.
Mortality Rate of OP-Intoxicated Patients in Japan a Year
Insecticide
2000
2001
Organophosphate Malathion DDVP MEP Miscellaneous b Carbamates c Paraquat Miscellaneous d
31 39 40 127 129 293 74
37 27 42 123 105 216 55
aData from the National Research Institute of Police Science, Japan (2002) (www.nrips.go.jp). 9Fenitrothion, glyphosate,EPN, dipterex, diazinone, and acephate. CMethomyland others. dChlorpicrine and others.
95
National Poison Center in Taiwan indicate that the fatality rate is much higher in Taiwan than in most Western countries, with 5.65% of all poisoning exposures resulting in death. There were 6872 cases of pesticide intoxication among 223,436 total cases of various intoxications from 1985 to 1993 (Table 6), and these accounted for approximately 22-30% of the total incidences, from 1991 to 1996. The incidence of pesticide poisoning gradually declined during this time. Extremely toxic pesticides are readily available and pesticide poisoning is common. OP insecticides (n = 1854) were the most common offending agents, followed by the OP herbicide glyphosate (n = 983), pyrethrins and pyrethroids (n = 936), paraquat (n = 892), and CM insecticides (n -- 721). There were a total of 1325 fatalities (19.3%). Pesticide poisonings (n = 875) accounted for 60% of all fatalities during the same study period. Among them, paraquat was the leading cause of death (n = 485, 54.4%). As shown in Table 7, paraquat is the leading cause of death in Taiwan, followed by the OP herbicide glyphosate, amphetamine, and CMs, most of which are pesticides. Insecticides (5217 patients, 46.3%) accounted for nearly half of the poisoning exposures. In Taiwan, 11,269 patients were intoxicated with acute pesticide poisoning, and male patients (63.9%) outnumber female patients. After adjusting for measured baseline characteristics, gender is not associated with the risk of death among patients with acute pesticide poisoning' OPs were more toxic than CMs. Suicide (7511 patients, 66.7%) was the most common intent of exposure. The oral route (8930 patients, 79.2%) was the most common route of exposure. Table 8 shows increased age, suicide attempt, oral exposure, and concurrent exposure with a higher risk of fatality. In terms of age differences, people 19-39 years old comprised the largest number of injured patients. Acute pesticide poisoning mainly involved adults, and the mean age of patients was 43.3 _ 18.9 years. Acute pesticide poisoning was associated with a very high case fatality rate (14.5%), and there was a wide range of differences in clinical severity among various pesticides. E. T h a i l a n d
Poisoning is considered a serious problem in Thailand. According to the Division of Epidemiology of the Ministry of Public Health, overall morbidity due to poisoning from 1990 to 1995 was 30 per 100,000 population, with 300 deaths per year. The morbidity rate of reported pesticide poisoning in the country was 10 per 100,000 population. The National Environmental Board reported that there were 380 deaths of 5458 reported cases of pesticide poisoning in 1985. The incidence of poisoning at Ramathibodi Hospital, one of three medical schools in
SECTION I 9 Uses, Abuses, & Epidemioloooy
96
TABLE 6.
Distribution of Reported Human Poisoning Exposures to Poison Control Center in Taiwan, 1985-1993 Children
TABLE 7. Substance
Adults
Total
Category
No.
%
No.
%
No.
%
Pesticides Drugs Cleaning substances Solvents Animal bites and stings Rodenticides Cosmetics Insect repellents Chinese herds CO and toxic gases Food-borne toxins Hydrocarbon Plants Miscellaneous
633 1886 410 285 172 148 246 251 89 89 49 63 1376
10.9 32.4 7.1 4.9 3.0 2.5 4.2 4.3 1.9 1.5 1.5 0.8 1.1 23.7
6239 4874 1196 736 781 658 383 257 319 190 190 168 126 1366
35.4 27.7 6.7 4.2 4.4 3.7 2.2 1.5 1.8 1.1 1.1 1.0 0.7 7.8
6872 6760 1606 1021 953 806 629 508 430 279 279 217 189 2742
29.3 28.8 6.9 4.4 4.1 3.4 2.7 2.2 1.8 1.2 1.2 0.9 0.8 11.7
Total
5812
100.0
17624
100.0
223436
100.0
Clinical Severity by Pesticides and Fungicides among 11,269 Patients with Acute Poisoning in Taiwan Symptomless, No. (%)
Mild, No. (%)
Moderate, No. (%)
Severe, No. (%)
Death, No. (%)
Total, No. (%)
Paraquat Glyphosates Other herbicides Organophosphates Carbamates Pyrethrin and pyrethroids Mixed insecticides Other insecticides Fungicides Others
71 (5.3) 178 (10.3) 116 (15.3) 111 (4.2) 33 (4.3) 147 (11.3)
259 (19.3) 1168 (67.4) 508 (67.1) 1293 (49.2) 483 (63.6) 906 (69.4)
144 (10.7) 224 (12.9) 69 (9.1) 547 (20.8) 128 (16.8) 156 (12.0)
43 49 22 335 54 55
(3.2) (2.8) (2.9) (12.7) (7.1) (4.2)
825 (61.5) 115 (6.6) 42 (5.6) 344 (13.1) 62 (8.2) 41 (3.1)
1342 (11.9) 1734 (15.4) 757 (6.7) 2630 (23.3) 760 (6.7) 1305 (11.6)
8 (5.0) 52 (14.3) 106 (13.9) 161 (11.1)
69 (43.4) 236 (65.0) 526 (68.8) 907 (62.4)
23 (14.5) 45 (12.4) 82 (10.7) 157 (10.8)
13 (8.2) 16 (4.4) 15 (2.0) 116 (8.0)
46 (28.9) 14 (3.9) 36 (4.7) 113 (7.8)
159 (1.4) 363 (3.2) 765 (6.8) 1454 (12.7)
Total
983 (8.7)
6355 (56.4)
1575 (14.0)
718 (6.4)
1638 (14.5)
Bangkok, was 200 cases per year, which accounted for 4.1% of patients in the medical ward. At one provincial hospital at Nakornrachsima, the medical in-patient admission rate for OP poisoning alone was 2.8%, and OP poisoning was ranked the eighth most common illness requiring hospitalization. The Ramathibodi Poison Center (RPC) was established in 1996 under the auspices of the Faculty of Medicine, Ramathibodi Hospital. In the year of 2000, the RPC Toxic Exposure Surveillance System was supported by the Royal
11269
Thai Government W H O Collaboration Program. The system collects human exposures having consultation to RPC. The definition of terms in the system is adopted or modified from the IPCS INTOX data management system and the American Association of Poison Control Center data collection system. According to the RPC, in 2001, the total number of pesticide poisonings was 834. Insecticide exposure was 50.2% of all pesticide exposures, followed by herbicide (20.9%), rodenticide (15.5%), and miticide 6.7% (Table 9).
CHAPTER 8 9Epidemiology of OP and CM Poisonings TABLE 8.
Age Distribution of Fatal and Nonfatal Cases with Acute Pesticide Poisoning in Taiwan No. of fatal cases (n = 1638) (%)
No. of nonfatal cases (n = 9631) (%)
Age (years)
~18 19-29 30-39 40-49 50-59 60-69 -->70 Unknown
64 (8.2) 327 (15.9) 271 (12.3) 209 (12.4) 239 (13.8) 233 (15.4) 259 (23.3) 36 (19.2)
716 (91.8) 1725 (84.1) 1926 (87.7) 1481 (87.6) 1497 (86.2) 1283 (84.6) 851 (76.7) 152 (80.9)
Oral 1552 (17.4) Inhalation 22 (1.5) Skin/mucous membrane 18 (1.2) Other/unknown 63 (26.9)
7378 (82.6) 1473 (98.5) 1439 (98.8) 171 (73.1)
Route of exposure
Chemicals used as miticides were usually similar to insecticides. Among the 653 cases of insecticide exposure, the most common ones were exposure to CMs (25.7%), OPs (29.3%), and pyrethroids (19.1%). Products that were a combination of two or three-insecticides accounted for 9.5%. Organochlorine exposure was 6.6%. In herbicide exposure, paraquat, Chloroacetanilide, and chlorophenoxy compounds comprised 24.4, 8.4, and 9.3% respectively. In terms of final medical outcome after insecticide exposures, 714 of 821 patients (87%) were followed until completion; 92 of 821 patients (11.2%) died, 7.3% developed major effect, 4% developed moderate effect, 34.1% had only minimum effect, and 30.3% did not have any effect. Paraquat, methomyl, endosulfan, methyl parathion, and monocrotophos were the major substances responsible for fatality in this group.
VII. S A R I N V I C T I M S IN T O K Y O On March 20, 1995, a terrorist attack using sarin occurred on the Tokyo subway. The sarin was mixed in organic solvent and vaporized in the closed compartment of a train. Many people inhaled the sarin gas and collapsed. Eventually, 12 people died and more than 5000 were injured (Suzuki et al., 1995; Masuda et al., 1995; Nozaki et al., 1995). However, neither sarin nor its hydrolysis products were detected in the blood of almost all the sarin victims. It was concluded that satin passed through the blood-brain barrier and became distributed in brain tissue. These facts suggest that various brain
97
regions may show differing degrees of vulnerability to it. In 1998, Masuda and associates performed judicial autopsies on four sarin victims, and they detected sarin hydrolysis products bound to AChE on the erythrocyte membranes of all four victims. The plasma ChE activity of patients 2-4 was extremely low compared to the average activity of normal samples. The AChE activity in the brain cortices and blood of normal control subjects was 110.0 _ 8.1 U/g wet tissue and 5.00 ___ 1.20 U/ml, respectively. The brain AChE activity of all the victims was significantly lower than that of normal subjects. The AChE activity in the blood of patients 1 and 2 was low (89.8 and 90.0% decrease, respectively), whereas that of patients 3 and 4, who received PAM in the hospital, had partly recovered (32.8 and 24.4% decrease, respectively). In 1998, Masuda et al. attempted to detect the sarin hydrolysis products in the cerebellums of the victims, which had been stored in formalin fixative for 2 years. Sarin-bound AChE was solubilized~ purified by immunoaffinity chromatography, and digested with trypsin. The sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion and detected by gas chromatography mass spectrometry. Using these procedures, methylphosphonic acid was detected. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of the four sarin victims for approximately 2 years, probably because the isopropoxy group of sarin underwent chemical hydrolysis during storage. The procedureS used here are useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents such as sarin.
VIII. C O N C L U S I O N S OPs cause serious intoxication in both acute and chronic exposure. The epidemiological data in Taiwan and Thailand show that paraquat intoxication causes the most serious irreversible damage in patients. Although the mortality rate slightly decreased during the past decade, pesticide poisoning is still associated with a high mortality rate in Taiwan. Legislation to control the availability of extremely hazardous pesticides and further innovation in "effective" therapeutic measures are required to reduce the high mortality rate that accompanies acute pesticide poisoning. Increased use of a poison control center service may be helpful in reducing the risk of pesticide-related fatalities. In the 1995, 12 people died by terrorist attack using sarin, one of the most toxic OPs on the Tokyo subway, 12 people died. This is an example of acute intoxication of OP, in which some people are still hospitalized. Plasma 13G activity is a novel biomarker of OP exposure. Plasma 13G is rapidly and significantly increased after OP exposure, and this is much more sensitive to OP exposure than ChE inhibition.
98
SECTION I 9 Uses, Abuses, & E p i d e m i o l o g y TABLE.9
The most common of pesticides related in human poison exposure categorized by major group classification
Major/subgroup classification
1. Insecticide Carbamate Organophosphate Pyrethroid Combined Organochlorine Repellant Others Unknown 2. Herbicide Glycine Bipyridyl Others Chloroacetanilide Chlorophenoxy Unknown 3. Rodenticide Zinc phosphide Wafarin Long-acting anticoagulant Unknown 4. Miticide Combined Carbamate Pyrethroid Unknown Arsenic troxide Organochlorine Organophosphate 5. Plant hormone & growth regulator 6. Acaricide 7. Mollusicide 8. Poison dog 9. Fungicide 10. Synergist 11. Poison bird 12. Unknown pesticide Total
Number
419 102 86 92 57 30 11 17 24 174 74 41 22 18 16 3 129 51 66 5 7 56 23 12 10 6 2 2 1 19 12 8 5 5 2 2 3 834
%
50.2 24.3 20.5 22 13.6 7.2 2.6 4.1 5.7 20.9 42.5 23.6 12.6 10.3 9.2 1.7 15.5 39.5 51.2 3.9 5.4 6.7 41.1 21.4 17.9 10.7 3.6 3.6 1.8 2.3 1.4 1 0.6 0.6 0.2 0.2 0.4
Most common products or substances
Methomyl (63 events), Carbofuran (19 events) Parathion methyl (44 events), Methamidophos (10 events) Cypermethrin (36 events) Endosulfan (28 events)
Glyphosate (74 events) Paraquat (41 events) Quizalofop-p-tefuryl (5 events), Fenoxaprop-p-ethyl (4 events) Alachlor (11 events) 2,4-D (16 events)
100
Acknowledgments The author acknowledges the following coworkers for their significant contributions to this chapter: Dr. Anna Fan (Environmental Protection Agency, Sacramento, CA), Dr. Jou-Fang Deng (Veterans Administration Hospital, Taipei, Taiwan), Dr. ChenChang Yang (Veterans Administration Hospital, Taipei, Taiwan), Dr. Songsak Srianujata (Mahidol University, Bangkok, Thailand), and Dr. Takemi Yoshida (Showa University, Tokyo).
References Abdollahi, M., Jalali, N., Sabzevari, O., et al. (1997). A retrospective study of poisoning in Teheran. J. Toxicol. Clin. Toxicol. 35, 387-393. Abdollahi, M., Ranjbar, A., Shahin, S., et al. (2004). Pesticides and oxidative stress: A review. Med. Sci. Monit., RA141-147. Agarwal, S. B. (1993). A clinical, biochemical, neurobehavioral, and sociopsychological study of 190 patients admitted to
CHAPTER 8 9Epidemiology of OP and CM Poisonings hospital as a result of acute organophosphorus poisoning. Environ. Res. 62, 63-70.
Akhgari, M., Abdollahi, M., Kebryaeezadh, A., et al. (2003). Biochemical evidence for free radical-induced lipid peroxidation as a mechanism for subchronic toxicity of malathion in blood and liver of rats. Hum. Exp. Toxicol. 22, 205-211. Aldfidge, W. N., and Johnson, M. K. (1971). Side effects of organophosphorus compounds: Delayed neurotoxicity. Bull. WHO 44, 259-263. Banerjee, B. D., Seth, V., Bhattacharya, A., Pasha, S. T., and Chakraborty, A. K. (1999). Biochemical effects of some pesticides on lipid peroxidation and free radical scavengers. Toxicol. Lett. 107, 33-47. Chambers, J. E., and Levi, P. E. (1992). Organophosphates. Chemistry, Fate and Effects. Academic Press, New York. Chung, F. R., Jang, S. W., and Lin, J. L. (1996). QTc prolongation indicates a poor prognosis in patients with organophosphate poisoning. Am. J. Emerg. Med. 14, 451-453. Dantoine, T., Debord, J., Merle, I., and Charmes, J. P. (2003). Roles of paraoxonase 1 in organophosphate compounds toxicity and in atherosclerosis. Rev. Med. Interne 24, 436---442. Dart, R. C. (2000). Organophosphate insecticides. In The 5-Minute Toxicology Consult, pp. 554-555. Lippincott Williams & Wilkins, Philadelphia. Dettbarn, W.-D., Milatovic, D., Zivin, M., and Gupta, R. C. (2001). Oxidative stress, acetylcholine and excitotoxicity. In Antioxidants and Free Radicals in Health and Disease. (J. Marwah and A. Kanthasamy, Eds.), pp. 183-211. Prominent Press, Scottsdale, AZ. De Silva, H. J., and Wijewickrema, R. (1992). Does pralidoxime affect outcome of management in acute organophosphorus poisoning? Lancet 339, 1136. DuBois, K. P. (1948). New rodenticidal compounds. J. Am. Pharm. Assoc. 37, 307-310. DuBois, K. P., Doull, J., Salerno, P. R., and Coon, J. M. (1949). Studies on the toxicity and mechanisms of action of p-nitrophenyl-diethylthionophosphate (parathion). J. Pharmacol. Exp. Ther. 95, 75-91. Ecobichon, D. J. (1994). Organophosphorus ester insecticides. In Pesticides and Neurological Diseases (D. J. Ecobichon and R. M. Joy, Eds.), 2nd ed., pp. 171-249. CRC Press, Boca Raton, FL. Ecobichon, D. J. (1998). Biological monitoring: Neurophysiological and behavioral assessments. In Occupational Hazards of Pesticide Exposure, Sampling, Monitoring, Measuring
(D. J. Ecobichon, Ed.), pp. 209-230. Taylor & Francis, Philadelphia. Ecobichon, D. J. (2001). Toxic effects of pesticides. In Casarret & Doull's
Toxicology.
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(C. D. Klaassen, Ed.), 6th ed., pp. 763-810. McGraw-Hill, New York. Eddleston, M., et al. (2002). Pesticide poisoning in the developing world~A minimum pesticide fist. Lancet 360, 1163-1167. Ellenhorn, M. J., Schonwald, S., Ordog, G., and Wasserberger, J. (1997). Ellenhorn's Medical Toxicology: Diagnosis and Treatment of Human Poisoning, pp. 1614-1663. Williams & Wilkins, Baltimore. Emerson, G. M., Gray, N. M., and Jelinek, G. A. (1999). Organophosphate poisoning in Perth, Western Australia, 1987-1996. J. Emerg. Med. 17, 273-277.
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Etemadi-Aleagha, A., Akhgari, M., and Abdollahi, M. (2002). A brief review on oxidative stress and cardiac diseases. Middle East Pharmacol. 10, 8-9. Furtado, M., and Chan, L. (2004, August 7). Toxicity, organophosphate, eMedicine, 1-12. Gershon, S., and Shaw, E H. (1961). Psychiatric sequelae of chronic exposure to organophosphorus insecticides. Lancet 1, 1371-1374. Gultekin, E (2000). The effect of organophosphate insecticide chlorpyrifos-ethyl on lipid peroxidation and antioxidant enzymes (in vitro). Arch. Toxicol. 74, 533-538. Gupta, R. C. (2004). Brain regional heterogeneity and toxicological mechanisms of organophosphates and carbamates. Toxicol. Mechan. Methods 14, 103-143. Gupta, R. C., Milatovic, D., and Dettbarn, W.-D. (2001a). Depletion of energy metabolites following acetylcholinesterase inhibitor-induced epilepticus: Protection by antioxidants. Neurotoxicology 22, 271-282. Gupta, R. C., Milatovic, D., and Dettbarn, W.-D. (2001b). Nitric oxide modulates high-energy phosphates in brain regions of rats intoxicated with diisopropylphosphorofluoridate or carbofuran: Prevention by N-tert-oL-phenylnitrone or vitamin E. Arch. Toxicol. 75, 346-356. Hosokawa, M., Maki, T., and Satoh, T. (1987). Multiplicity and regulation of hepatic microsomal carboxylesterases in rats. Mol. Pharmacol. 31, 579-584. Hosokawa, M., Maki, T., and Satoh, T. (1990). Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277, 219-227. Jalali, N., Pajoumand, A., Abdollahi, M., and Shadnia, S. (2000). Epidemiological survey of poisoning mortality in Teheran during 1997-1998. Toxicol. Lett. Suppl. 116, 309. Jamal, G. A. (1997). Neurological syndromes of organophosphorus compounds. Adverse drug reaction. Pralidoxime (1 gm single bolus dose vs. 12 gm infusion) in the management of organophosphorus. Toxicol. Rev. 16, 133-170. Marrs, T. C. (1993). Organophosphate poisoning. Pharmacol. Ther. 58, 51-66. Masuda, N., Takatsu, M., Norinami, H., et al. (1995). Satin poisoning in Tokyo subway. Lancet 345, 1446-1447. Masuda, Y., Nagano, Y., Takatori, T., et al. (1998). Detection of the satin hydrolysis product in formalin-fixed brain tissues of victims of Tokyo subway terrorist attack. Toxicol. Appl. Pharmacol. 150, 310-320. Matkevich, V. A., Simonenkov, A. P., Ostapenko, L., Li, N., et al. (1995, May/June). Use of serotonin adipinate in acute oral poisoning. Anest. Reanimatol., 16-20. Medda, S., Takeuchi, K., Devore-Carter, D., et al. (1987). An accessory protein identical to mouse egasyn is complexed with rat [3-glucuronidase and is identical to rat esterase-3. J. Biol. Chem. 262, 7248-7253. Nozaki, H., Aikawa, N., Shinozawa, Y., et al. (1995). Satin poisoning in Tokyo subway. Lancet 345, 980-981. Pajoumand, A., Jalali, N., Abdollahi, M., and Shadnia, S. (2002). Survival following severe aluminum phosphide poisoning. J. Pharm. Pract. Res. 32, 297-299. Poovala, V. S., Huang, H., and Salahudeen, A. K. (1999). Role of reactive oxygen metabolites in organophosphateinduced renal tubular cytotoxicity. J. Am. Soc. Nephrol. 10, 1746-1752.
1 O0
S ECTIO N I 9 Uses, Abuses, & Epidemiology
Ranjbar, A., Pasalar, E, and Abdollahi, M. (2002). Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorus pesticide manufacturing workers. Hum. Exp. Toxicol. 21, 179-182. Satoh, T., and Hosokawa, M. (1998). The mammalian carboxylesterases: From molecules to functions. Annv. Rev. Pharmacol. Toxicol. 38, 257-288. Satoh, T., and Hosokawa, M. (2000). Organophosphates and their impact on the global environment. Neurotoxicology 21, 223-227. Spiegelberg, U. (1963). Psychopathologische-neurologische spat und dauerschaden nach geweblicher Intoxikation durch
Phosporsaeureester (alkylphosphate). Proc. 14th Int. Congr. Occup. Health Exerpta Med. Found. Int. Congr. Ser. 62, 1778-1780. Suzuki, T., Morita, H., Ono, K., et al. (1995). Sarin poisoning in Tokyo subway. Lancet 345, 980. Swank, R. T., and Paigen, K. (1973). Biochemical and genetic evidence for a macromolecular [3-glucuronidase complex in microsomal membranes. J. Mol. Biol. 77, 371-389. Verma, R. S. (2001). Chlorpyrifos-induced alterations in levels of thiobarbituric acid reactive substances and glutathione in rat brain. Indian J. Exp. Biol. 39, 174-177.
Pharmacokinetics & Metabolism
This Page Intentionally Left Blank
CHAPTER 9
Physiologically Based Pharmacokinetic Modeling of Organophosphorus and Carbamate Pesticides CHARLES TIMCHALK Pacific Northwest National Laboratory, Richland, Washington
insecticides in animals and humans and for the assessment of risk. The approach will not entail a comprehensive review of the extensive literature but, rather, a focused presentation highlighting important principles using specific examples for these classes of insecticides. OP and CM insecticides constitute two large families of pesticides that share a common mode of insecticidal and toxicological action associated with their ability to inhibit the enzyme AChE within nerve tissue (Ecobichon, 2001a,b; Sultatos, 1994). A major difference between these two insecticidal classes relates to the rate of AChE reactivation, which is measured in minutes for CMs versus days or even weeks for OP insecticides (Tobia et al., 2001; Ecobichon, 2001b). A comparison of the chemical structures for OP and CM insecticides is illustrated in Fig. 1. OP insecticides are structurally related, pentavalent phosphorus acids (Fig. 1, I). The "leaving group" (R1) is released upon phosphorylation of ACHE, R 2 and R3 are most commonly alkyloxy groups that are more stable constituents, whereas "X" represents either a sulfur or oxygen in association with a phosphate (H. W. Chambers et al., 2001; Chambers, 1992; Mileson et al., 1998). Carbamates (Fig. 1, II) are N-mono or-dimethylated carbamic acids, with R1 representing a range of alkyl or aryl substitutions that can result in substantial modification of the physiochemical properties of the CM (Ecobichon, 2001a,b).
I. I N T R O D U C T I O N Pharmacokinetics has and will continue to play an important role in assessing organophosphorus (OP) and carbamate (CM) insecticide dosimetry, biological response, and risk in humans exposed to these agents. These two major classes of pesticides share a common toxicological mode of action associated with their ability to target and inhibit acetylcholinesterase (ACHE). Pharmacokinetics is associated with the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. Pharmacokinetic studies provide important data on the amount of toxicant delivered to a target site as well as species-, age-, and gender-specific and dose-dependent differences in biological response. These studies have been conducted with OP and CM insecticides in multiple species, at various dose levels, and across different routes of exposure to understand how in vivo kinetics contributes to the observed toxicological response. Pharmacokinetic studies with these insecticides are also useful to facilitate extrapolation of dosimetry and biological response from animals to humans and for the assessment of human health risk. In this regard, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models are being utilized to assess risk and understand the toxicological implications of known or suspected exposures to various OP and CM insecticides. In this chapter, a number of examples are presented that illustrate the utility of pharmacokinetic studies to address human health concerns associated with these important insecticides.
A. Mode of Action OP and CM insecticides have a high affinity for binding to and inhibiting the enzyme ACHE, an enzyme specifically responsible for the destruction of the neurotransmitter acetylcholine (ACh) within nerve tissue (Wilson, 2001; Ecobichon, 2001b). Since the cholinergic system is widely distributed within both the central and peripheral nervous systems, chemicals that inhibit AChE are known to produce
II. B A C K G R O U N D This chapter focuses on the application of pharmacokinetic principles and in particular the development of PBPK/PD models to better understand the toxicology of OP and CM Toxicology of Organophosphate and Carbamate Compounds
103
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
104
SECTION
i
II
ii
/
0
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9Pharmacokinetics & Metabolism
RI.-.O--..C ..... R 1
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a broad range of well-characterized symptoms (for review, see Savolainen, 2001). A comparison of the AChE inhibition dynamics for the interaction of ACh, carbaryl (CM), or chlorpyrifos-oxon (OP) with AChE is illustrated in Fig. 2. All three substrates have relatively high affinities for AChE and will readily complex with the enzyme; however, the rates of hydrolysis and reactivation of AChE following carbamylation or phosphorylation of the active site will be drastically slower than for the hydrolysis of the acetylated enzyme (Ecobichon, 2001b). Specifically, the turnover time for the neurotransmitter ACh is on the order o f - 1 5 0 Ixsec, whereas the carbamylated enzyme tl/2 for hydrolysis is substantially slower (-15-30 min). The phosphorylated enzyme is highly stable (tl/2 -days), and further dealkylation of the phosphorylation group produces an "aged" AChE enzyme that is irreversibly inhibited (Taylor, 1980; Murphy, 1986; Ecobichon, 2001b; Sogorb and Vilanova, 2002).
B. Organophosphorus and Carbamate Pharmacokinetics Pharmacokinetics is the evaluation of those processes associated with the ADME of drugs and xenobiotics. The ADME of OP and CM insecticides in both animals and humans has been studied (Timchalk et al., 2002a, 2005; Poet et al., 2004; Tos-Luty et al., 2001; Wu et al., 1996; Moody and Franklin, 1987; Tomokuni et al., 1985; Nolan et al., 1984). Like all chemical contaminants, these insecticides can gain entry into the body, and based on the detection of low levels of metabolites in urine within human populations, there is good evidence for widespread although low-level exposures (Aprea et al., 1999; Shealy et al., 1997; Hill et al., 1995; Brouwer et al., 1993). These exposures can come from numerous sources, including ingestion of pesticide residues on foods or accidental as well as intentional ingestion of insecticides (Drevenkar et al., 1993). Dermal exposure represents a relevant route, particularly during the mixing, loading, and application of insecticides or from skin contact with contaminated
surfaces (Knaak et al., 1993). Likewise, inhalation of airborne insecticide is feasible either during the spraying of or from exposures associated with chemical drift (Vale and Scott, 1974). Once the pesticide arrives at a portal of entry, it is available for absorption, and based on the bioavailability for a given insecticide and exposure route, a systemic dose of the parent compound will enter the circulation. Although localized portal of entry metabolism (i.e., lung, intestines, and skin) is feasible (Poet et al., 2003), the bulk of the metabolic activation as well as detoxification reactions occur within the liver (Sultatos et al., 1984a,b; Sultatos, 1988). It is likewise clear from both tissue distribution and partitioning studies that OP and CM insecticides are generally well distributed in tissue throughout the body (Tos-Luty et al., 2001; Wu et al., 1996; Tomokuni et al., 1985). Finally, due to the extensive metabolism (see Section II, C) little if any parent insecticide is available for excretion; however, more stable degradation metabolites are readily excreted in the urine and are of potential utility as biomarkers of exposure (Colosio et al., 2002; Shealy et al., 1997; Iverson et al., 1975; Mticke et al., 1970).
C. Insecticide Biotransformation 1. ORGANOPHOSPHATES A more detailed overview of the metabolism of OP insecticides can be found in Calabrese (1991), Jakanovic (2001), Sogorb and Vilanova (2002), and Knaak et al. (2004). The three major classes of OP insecticides are the phosphorothionates, phosphorodithioates, and the phosphoroamidothiolates (Chambers, 1992; Mileson et al., 1998; J. E. Chambers et al., 2001). Phosphorothionate insecticides such as chlorpyrifos are weak inhibitors of ACHE, but once they undergo metabolic activation (desulfation) to their corresponding oxygen analogs (oxon), they become extremely potent inhibitors. This enhanced toxicity is due to the oxon having a higher affinity and potency for phosphorylating the serine hydroxyl group within the active site of AChE (Mileson et al., 1998; Sultatos, 1994). The toxic potency is dependent on the balance between a delivered dose to the target site and the rates of bioactivation and/or detoxification (Calabrese, 1991). In Fig. 3, the thionophosphate pesticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2pyridyl]-phosphorothioate) is utilized for illustration purposes, and based on a common mode of action, this scheme is readily extended to other structurally related organophosphorus insecticides. As previously mentioned, phosphorothionates do not directly inhibit AChE but must first be metabolized to the corresponding oxygen analog (chlorpyrifos-oxon) (Mticke et al., 1970; Iverson et al., 1975; Murphy, 1986; Sultatos, 1994). Activation to the oxon-metabolite is mediated by cytochrome P450 mixed function oxidases (CYP450)
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150 mg/kg). These PBPK/PD models provide a reasonably good prediction of dosimetry and biological response, but it is important to recognize that the predictive capabilities of these models are limited by the adequacy of the parameters and limitations of the experimental data (Timchalk et al., 2002b). However, it is anticipated that these basic structures can be used as a starting point for the development of other OP and CM insecticide models. Once validated, these models can then be used to understand complex mixture interactions, sensitive subpopulations, and the role of metabolic polymorphisms in altering dosimetry and biological response.
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Time (hrs) FIG. 17. Time course of chlorpyrifos (CPF) in the serum of a single poison victim who orally ingested a commercial insecticide product containing CPF (data from Drevenkar et al., 1993). The symbols represent observed data, whereas the line represents the model prediction. Adapted with permission from Timchalk et al. (2002).
V. SENSITIVE SUBPOPULATIONS: CHILDREN AND METABOLIC POLYMORPHISMS A. Children's Sensitivity There is currently a significant focus on and concern over the potential increased sensitivity of infants and children to the toxic effects of chemicals. The importance of this issue is highlighted by the National Research Council's report, "Pesticides in the Diets of Infants and Children," and the passage of the Food Quality Protection Act. It is recognized that children are not just "small adults" but, rather, a unique subpopulation that may be particularly vulnerable to chemical insult. Age-dependent changes in a child's physiology (i.e., body size, blood flow, and organ functions) and metabolic capacity (i.e., phase I and II metabolism) may significantly impact their response to a chemical insult, resulting in either beneficial or detrimental effects (Makri et al., 2004; Ginsberg et al., 2004; Johnson, 2003; Miller et al., 1997). Clear variability in the capacity to detoxify environmental chemicals has been established in both animals and humans. However, the current risk assessment paradigms may not adequately consider the implications of these differences on the risk to infants and children. Numerous studies have demonstrated that juvenile animals are more susceptible to the acute high-dose effects of OP insecticides than adults (Brodeur and DuBois, 1963; Benke and Murphy, 1975; Harbison, 1975; Gaines and Linder, 1986; Pope et al., 1991; Pope and Liu, 1997; Moser and Padilla, 1998). This greater sensitivity has primarily been attributed to the lack of complete metabolic competence during neonatal and postnatal development (Benke and Murphy, 1975). Several studies provide important perspective on this age- and dose-dependent sensitivity to OP insecticides. Collectively, they suggest that the age-dependent sensitivity in neonatal animals is associated with a lower CYP450 dearylation (detoxification) capacity and an age-dependent lower PON-1 and CarbE-mediated oxon hydrolysis capacity in neonates relative to adult animals (Atterberry et al., 1997; Li et al., 1997; Mortensen et al., 1996). These findings in animals are in agreement with observations in which newborn and young humans have lower metabolic capacity for CYP450 and PON-1 activity compared to adults (Johnson, 2003; Augustinsson and Barr, 1963; Mueller et al., 1983). The application of PBPK/PD modeling offers a unique opportunity to integrate age-dependent changes in metabolic activation and detoxification pathways into a comprehensive model that is capable of quantifying dosimetry and response across all ages (for review, see Corley et al., 2003). In this context, PBPK models are being extended to the modeling of chemical exposure in developing children (Price et al., 2003; Clewell et al., 2004) and in developing/neonatal animals (Fisher et al., 1990; Byczkowski et al., 1994; Sundberg et al., 1998).
CHAPTER 9 9PBPK/PD Organophosphates and Carbamates
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However, as illustrated in Fig. 19, the magnitude of brain AChE inhibition is clearly age- and dose-dependent, and although dramatically inhibited in neonatal rats (10 mg/kg response), the adults appear to be refractory to any AChE inhibition at these same dose levels. Although these simulations illustrate the ability to scale age-dependent changes within a species, the different time scales between development in neonatal animals and human infants create uncertainty in extrapolation across species (Ginsberg et al., 2004).
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B. Genetic Polymorphisms A number of human and experimental animal studies have demonstrated a wide range of variability in the metabolism of drugs and xenobiotics (Mackenzie et al., 2000; Tucker, 2000; Jones et al., 1995; Gonzalez and Gelboin, 1993). A human genetic polymorphism in the PON-1 detoxification of several OP insecticides, including the active metabolite of chlorpyrifos, chlorpyrifos-oxon, has been well established, resulting in the expression of a range of PON-1 enzyme activity within a segment of the population (Cowan et al., 2001; Furlong et al., 1998; Eckerson et al., 1983; Geldmacher-von Mallinckrodt et al., 1983). The PBPK/PD model for chlorpyrifos was used to assess the potential contribution of the human PON-1 (chlorpyrifos-oxonase) polymorphism on chlopyrifos dosimetry (Timchalk et al., 2002a), based on the distribution of chlorpyrifos-oxonase activities reported in human serum (Davies et al., 1996). A comparison of the dose-dependent
Based on the potential sensitivity of children to OP insecticides, there is a need to develop quantitative models that can be used to assess the risk associated with exposure in infants and children. The PBPK/PD model for chlorpyrifos has been modified to scale both the metabolism and the esterase levels based on the age of the animal. The time course for chlorpyrifos and the major metabolite trichloropyridinol in postnatal day 5 (PND 5) rats is illustrated in Fig. 18, and the time course of AChE inhibition as a function of dose and age is presented in Fig. 19. It is of interest that even in PND 5 animals, CYP450 activity is adequate to metabolize chlorpyrifos to trichloropyridinol and the PBPK/PD model provides a reasonable simulation.
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(continues)
CHAPTER 1 1 9Interspecies Variation in Anticholinesterases
TABLE 1. Compound Comparing compounds (Continued) Methyl parathion Dichlorvos Chlorpyrifos Carbaryl Diazinon Tri-o-cresyl phosphate Malathion Comparing route of exposure Aldicarb Aldicarb Aldicarb Aldicarb Aldicarb Carbaryl Carbaryl Carbaryl Carbaryl Carbaryl Parathion Parathion / Parathion Parathion Parathion Parathion Satin Sarin Sarin Sarin Sarin Sarin
147
(continued)
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al, CCOHS (2004); 2, Fahmy et al. (1970); 3, Ecobichon (2001b); 4, Hutson and Hathway (1967); 5, Sherman et al. (1967); 6, E1-Sebae et al. (1978); 7, Gaines (1969); 8, Storm et al. (2000); 9, Klimmer and Pfaff (1955); 10, Weiss and Orzel (1967); 11, Natoff and Reiff (1973); 12, Brodeur and Dubois, (1963); 13, Grob and Harvey (1958); 14, Gaines (1960); 15, Fleisher et al. (1963); 16, Brimblecombe et al. (1970); 17, Schoene et al. (1985).
Standardized laboratory guidelines are necessary for conducting various tests because even within the same species, different tests can lead to LOAELs that differ by up to 100-fold (Burbacher et al., 1990; Vidair, 2004). For an extensive review on tabulating NOAELs and LOAELs for a variety of end points in different species exposed to physostigmine, parathion, or diisopropyl fluorophosphate (DFP), see Raffaele and Rees (1990). NOAELs and LOAELs for neurotoxic endpoints generally are lower than LDsos for each of the compounds evaluated. However, the most sensitive endpoint differed among substances, reit-
erating compound-dependent effects when considering species variability (Raffaele and Rees, 1990). Table 1, compiled predominately from the R e g i s t r y o f Toxic Effects o f C h e m i c a l S u b s t a n c e s database [Canadian Centre for Occupational Health and Safety (CCOHS), 2004], summarizes some LDs0s for various compounds and species exposed via different routes, with the most sensitive (lowest LDs0 ) species and route listed accordingly. The LDs0, of course, depends on the compound in question, as is illustrated for rats exposed orally to 10 different compounds. It is generally accepted that birds are extremely
148
SECTION
II
9Pharmacokinetics
& Metabolism
susceptible to acute ChE inhibition by OPs, whereas fish and amphibians are relatively resistant and rodents are intermediate (Wallace, 1992). On the other hand, this is just a generalization and species sensitivity also depends on the compound exposure, as illustrated in Table 1 and by Johnson and Wallace (1987). Generally, birds are less sensitive than rodents to aldicarb and carbaryl but more sensitive to chlorpyrifos and diazinon, whereas mammals seem to be more resistant to chlorfenvinphos, carbaryl, and diazinon. For parathion, there is no obvious trend, with birds, rodents, and mammals (including human) intermixed (Table 1). Although rodents are more sensitive to parathion and paraoxon than sunfish and bullheads, fish in general are more sensitive to malathion and malaoxon compared to rodents (Johnson and Wallace, 1987). The greater sensitivity of rodent brain AChE to inhibition by paraoxon may contribute to the greater toxicity of parathion and paraoxon in rodents compared to fish, whereas the differences in malathion sensitivity may be due to greater carboxylesterase activity in rodents than in fish. Clearly, no hard and fast rules can explain interspecies variability to effects of antiChEs, but discussion of the pharmacokinetics and pharmacodynamics will shed some light on the contributing factors.
III. W H Y A R E S P E C I E S D I F F E R E N T I A L L Y AFFECTED? A. P h a r m a c o k i n e t i c s
A compound's action or effect within the body over a period of time is regulated by pharmacokinetics: absorption, distribution, metabolism, and elimination. These processes differ across species and provide some insight into species variability following exposure to anti-ChEs. In addition to species-dependent effects, signs and symptoms of ChE inhibition by OPs or CMs depend on the compound, dose, route, frequency and duration of exposure, as well as the time of observation relative to the time of peak effect (Table 1). Therefore, it is difficult to compare species across studies in which any one of these factors is not consistent, and the reader must bear this in mind when reviewing the literature for species-related differences. 1. ABSORPTION AND ELIMINATION Because the physiological response of an organism is dependent on the steady-state concentration of the compound at the site of action, differences in the relative rates of absorption and elimination of a compound in various species account, in part, for some of the interspecies variability of anti-ChEs. Oral exposures are subject to first-pass hepatic metabolism, which produces metabolites more or less toxic than the parent compound (Storm et al., 2000). Thus, to compare the effects of any compound across species, the same route of administration is required as well as the same endpoint. In the case of aquatic and
nonaquatic animals, presumably the lethal concentration that causes death in 50% of an aquatic population (LCs0) is sometimes correlated to the LDs0. Pharmacokinetic information provides insight into NOAEL and NOEL differences among exposure routes. For example, dichlorvos is rapidly absorbed, metabolized, and eliminated following both oral and inhalation exposures, possibly explaining its equivalent potency to inhibit red blood cell (RBC) AChE when exposure occurs via either route (Storm et al., 2000). On the other hand, malathion appears to be eliminated more rapidly following inhalation exposure compared to oral exposure, providing some context for the apparently higher inhalation NOEL, where RBC AChE is the endpoint (Storm et al., 2000). Inhibition of RBC AChE is not itself considered an adverse effect, although it is often measured as an indicator for the adverse inhibition of brain ACHE; thus, "NOEL" is used to describe this effect. In addition, clearance of the carbamate carbaryl is slower in rats than in hens, potentially contributing to the greater sensitivity of brain AChE inhibition in rats (Ehrich et al., 1992, 1995). Furthermore, dogs excreted this vinyl phosphate more rapidly than rats, reflective of the higher LDs0 in dogs versus rats (Table 1) (Hutson and Hathway, 1967; Natoff, 1971). Although differences in the relative rates of absorption and elimination explain some of the variability among species, they are insufficient to account for all interspecies variabilitymfor example, the insensitivity of fish compared to rodents to acute parathion toxicity (Hodson, 1985; Wallace, 1992). Nevertheless, in general, the LDs0 was lowest in rats following intravenous (iv) administration of an anti-ChE and highest following dermal exposure with intraperitoneal (ip) and oral LDs0s on the low end and subcutaneous (sc) LDs0s on the high end. The absorption rates depend on exposure route (iv > ip > sc > oral > dermal) and seem to correlate with acute toxicity (iv -> ip > oral > sc > dermal) (Table 1). 2. DISTRIBUTION Physiological response depends on bioavailability. The concentration of nonspecific binding sites affects the amount of compound that is free to bind target receptors and thus can serve as a reservoir to decrease the effective levels of anti-ChEs (Lauwerys and Murphy, 1969; Sultatos et al., 1984; Wallace, 1992). Plasma proteins such as albumin and soluble aliesterases bind OPs, decreasing toxicity. For instance, soluble aliesterases bind to the active metabolite of parathion, paraoxon, to limit toxicity (Chambers and Chambers, 1990; Lauwerys and Murphy, 1969; Wallace, 1992). As evidence, Lauwerys and Murphy demonstrated that tri-o-cresyl phosphate (TOCP), which inhibits nonspecific binding of OPs, potentiated paraoxon toxicity in rats. Additionally, albumin promotes nonenzymatic hydrolysis of OP esters, resulting in inactivation of the compound (Sultatos et al., 1984). Hutson and Hathway (1967) found that the blood concentration of chlorfenvinphos in dogs was one-fifth of that found in rats at similar
C H APT ER 1 1 oral dose levels and that it is taken up more readily by the brain of rats than dogs at similar blood levels, consistent with greater sensitivity of rats to acute toxicity (Table 1). A species comparison among rats, guinea pigs, and marmosets (primates) found that blood soman levels differed among these species following intravenous administration (Benschop and De Jong, 1991). This interspecies variability is probably due to decreasing amounts of binding sites (rats > guinea pigs > primates) that compete with ACHE, leading to increasing "toxicoavailability" (primates > guinea pigs > rats) (Benschop and De Jong, 1991). Contrary to these findings, guinea pigs were more sensitive to acute soman toxicity than rats following subcutaneous administration (Maxwell e t al., 1987). Since OPs are hydrophobic compounds, the lipid composition of an organism governs their hydrophobic partitioning (Wallace, 1992). The protective blood-brain barrier (BBB) is permeable to these pesticides. Likewise, the high lipid content of the brain results in a disproportionate amount of accumulation in the brain and partially explains TABLE 2.
Interspecies Variation in Anticholinesterases
149
the neurotoxicity of OPs. Mammalian P-glycoproteins are cell membrane ATP-dependent transporters expressed at blood-tissue barriers (e.g., BBB and blood-placenta barrier) that pump hydrophobic drugs out of the cell, protecting the brain and fetus (Fromm, 2004). I n v i v o studies suggest that chlorpyrifos oxon is a substrate for P-glycoprotein (Lanning e t al., 1996). Interspecies differences in P-glycoprotein expression have the potential to influence distribution of OP substrates; indeed, there are intraspecies differences within humans and rodents, as well as age-dependent and tissue-dependent differences in expression of this protein (Fromm, 2004; Rosati e t al., 2003; Warrington e t al., 2004). 3. METABOLISM Most studies comparing species variability focus on metabolism; activation and deactivation of the anti-ChEs within the body depend on several factors that vary among species. The amounts and activities of enzymes involved in metabolism must be considered (Table 2). For example, liver
Basal Enzyme Activity/Level in Various Species
Species
Site
Value
References a
Rabbit Guinea pig Mouse Human Rat Mouse Minnow Trout
Serum Serum Serum Serum Brain Brain Brain Brain
-300 nmol/ml x min -2900 nmol/ml x min -4800 nmol/ml x min --4800 nmol/ml X min 37.7 nmol/min/mg protein 49.9 nmol/min/mg protein 178.3 nmol/min/mg protein 88.8 nmol/min/mg protein
1 1 1 1 2 2 2 2
Carboxylesterase Carboxylesterase Carboxylesterase Carboxylesterase
Rabbit Human Guinea pig Mouse
Serum Serum Serum Serum
-300 nmol/ml x min -300 nmol/ml x min -2000 nmol/ml x min -3200 nmol/ml X min
1 1 1 1
Paraoxonase Paraoxonase Paraoxonase Paraoxonase Enzyme level Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450 Cytochrome P450
Guinea pig Mouse Human Rabbit
Serum Serum Serum Serum
- 100 nmol/ml x min -300 nmol/ml X min -300 nmol/ml X min -3400 nmol/ml x min
1 1 1 1
Chicken Cat Human Rat Mouse Rabbit Hamster Guinea pig
Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes Liver microsomes
0.13 nmol/mg protein 0.34 nmol/mg protein 0.28-0.53 nmol/mg protein 0.84 nmol/mg protein 1.1 nmol/mg protein 1.1 nmol/mg protein 1.26 nmol/mg protein 1.45 nmol/mg protein
3 4 5, 6 7 7 7 7 7
Enzyme activity Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase Cholinesterase
al, Kaliste-Korhonenet aL (1996); 2, Johnson and Wallace (1987); 3, Lasker et al. (1982);4, Kato (1966); 5, Nelson et al. (1971); 6, Pelkonen et al. (1973); 7, Chhabraet al. (1974).
150
SECTION II 9Pharmacokinetics & M e t a b o l i s m
microsomal cytochrome P450 levels and ChE, carboxylesterase, and paraoxonase (PON1) activities ,vary across species (Table 2) (Chhabra et al., 1974; Kato, 1966; Lasker et al., 1982; Nelson et al., 1971; Pelkonen et al., 1973). In a study to predict toxicity of OPs to four avian species, LDs0 correlated with rate of OP activation and brain AChE inhibition in the brain and serum carboxylesterase inhibition, supporting the contribution of OP metabolism to acute toxicity (Thompson et al., 1995). a. Organophosphate Activation CMs generally are not activated prior to AChE interaction. However, to inhibit ACHE, cytochrome P450 enzymes metabolize some OPs (e.g., chlorpyrifos, parathion, and malathion) to their more potent oxon via oxidative desulfuration (deBethizy and Hayes, 2001). Conversely, methyl parathion is highly toxic to rats, whereas its oxon is a relatively nonpotent inhibitor of brain AChE (Chambers, 1992). Parathion is metabolized to the more potent paraoxon, so a lower rate of activation--as in fish--is consistent with relative resistance to acute toxicity. On the other hand, a greater rate of activation in rodents compared to birds suggests that rodents are more sensitive, but this is not consistent with the general finding that birds are more sensitive to acute OP toxicity compared to rodents (Chambers, 1992; Hitchcock and Murphy, 1971; Thompson et al., 1995; Wallace, 1992; Wallace and Dargan, 1987). Thus, although differences in the rate of activation among species account for some species variability, this is not the definitive parameter in determining interspecies variability. b. Organophosphate Deactivation Although the rate of OP activation influences the toxic bioavailability of the oxon, the rate of deactivation is important in the protection of the organism. Both A- and B-esterases deactivate OPs. Not only do the amounts and activities of the esterases vary according to species and tissue but also their affinities for OPs are species, tissue, and compound specific (Kaliste-Korhonen et al., 1996). The A-esterases, including oxonases and arylesterases, hydrolyze the parent compound or oxon metabolite to produce inactive metabolites. These enzymes are present in plasma and tissue and are not inhibited by the oxon (Karanth and Pope, 2000; Moser et al., 1998; Padilla et al., 2000; Vidair, 2004). In contrast, the B-esterases, including carboxylesterases, aliesterase, ACHE, and butyrlcholinesterase (BuChE), bind OPs stoichiometrically, are irreversibly inhibited, and reactivate very slowly (Karanth and Pope, 2000; Moser et al., 1998; Padilla et al., 2000; Vidair, 2004). An important A-esterase involved in the detoxification of OPs is PON 1, which hydrolyzes the OP oxon metabolite. Accordingly, injected PON1 protects against OP poisoning in rodents (Cowan et al., 2001; Davies et al., 1996). PON1 is polymorphic in human populations, contributing to
intraspecies variability, whereas interspecies differences in PON1 activity are correlated with observed LDs0 values (Davies et al., 1996). In a very informative review, Wallace (1992) provides references to suggest that the sensitivity to acute cholinergic intoxication is inversely proportional to the serum arylesterase activity (Butler et al., 1985); likewise, the bimodal distribution of plasma A-esterases in humans may account for individual sensitivity to OP toxicity (Eckerson et al., 1983; La Du and Eckerson, 1984; Wallace, 1992). The plasma and tissue from fish, amphibians, birds, and mammals vary in A-esterase activity (Wallace, 1992). Fourteen species of birds tested have virtually undetectable plasma A-esterase activity, whereas rodents, sheep, and humans have abundant plasma arylesterase activity (Wallace, 1992). The low arylesterase activity in birds translates to a lower rate of paraoxon hydrolysis in their plasma and liver, correlating with the greater susceptibility of birds to OP intoxication (Walker and Mackness, 1987; Wallace, 1992). Interaction of OPs such as parathion and chlorpyrifos with carboxylesterase and aliesterase decreases the availability to interact with AChE to produce toxic effects, thus protecting against neurotoxicity (Storm et al., 2000). Conversely, inhibition of serum and lung carboxylesterases by cresylbenzodioxaphosphorin oxide potentiates the in vivo toxicity of some OPs (soman, satin, tabun, and paraoxon, but not dichlorvos or DFP) in rats and mice (Clement, 1984; Maxwell, 1992) and eliminates the soman LDs0 interspecies variability in rats, mice, guinea pigs, and rabbits (Maxwell et al., 1987). Moreover, Kaliste-Korhonen (1996) reported that human serum does not detoxify OPs as well as serum of rodents (mouse and guinea pig), making man potentially more sensitive to OPs than rodents. This is potentially related to the lower basal activity of carboxylesterase in man compared to mice and guinea pigs (Table 2) (KalisteKorhonen et al., 1996). Within animal classes, there are species and temporal variations in activity, level, and forms of carboxylesterases and ChEs, as noted in avian carnivore versus omni/herbivore serum B esterases (Thompson, 1993). Thus, it is challenging to generalize trends among classes such as birds, fish, and mammals for different compounds. Although it is tempting to offer a simplistic proposal regarding the role of OP deactivation and toxicity based on these findings, the relationship between hydrolyzing activity and in vivo sensitivity does not hold true for all OPs or for all species. Indeed, the lower arylesterase activity in fish compared to rodents is inconsistent with the relative resistance of fish to OP toxicity (Wallace, 1992; Wallace and Dargan, 1987). Although rodent susceptibility to OPs is generally intermediate between birds and fish, the activity of activating and deactivating enzymes is greater in rodents compared to these other species, making generalizing statements impractical. A number of other factors besides A- and B-esterase activities influence physiological responses to OPs
CHAPTER 1 1 among species; however the importance of the differences of these enzymes to determine interspecies variability remains controversial (Wallace, 1992).
B. Pharmacodynamics In addition to differences in pharmacokinetics, species also differ in their biochemical and physiological response to
lnterspecies Variation in Anticholinesterases
anti-ChEs; that is, the mechanism of action, in this case AChE inhibition, is subject to species-specific modification. 1. ACETYLCHOLINESTERASEINHIBITION Whereas CMs react with the serine group on ACHE, yielding carbamylation of the serine hydroxyl group, OPs phosphorylate the serine hydroxyl group (Fig. l a). Carbamylation of AChE is reversible and typically
A Organophosphorus
Ester
0 (or S) R10~
II
x
,
I
i
.....~ii;~'~i'~..... i~i~i:i~, OH .....~!ii~ii~~.ii~. i. . . . .:~,~i!i~.i~.i.i~..,~ ....~iii~ !!,,i~,~,~J~,~,/,i~ ~ i l i ~ ~,~,~,~,~,~ii~i ~,~;....... i~,i~,~ ...~,..~.',!i~ i~ i i~,ii........ i~
......
Acetylcholinesterase
Organophosphorus Ester + Free Acetyicholinesterase
Reversible Complex[ O
0 RIO~]I R20
~P~X
ka
,. R 1 0 ~
p[I_ _ x
R20
\
+ OH .......
.....
k. ~
0
'i~,~!,ill~
i~i~i~
I Free Enzyme I
+
O
R,O~ II p
\
(+ XH)
O ...~iiil ... iii~i~i~i~!~i~]i~!i~ii~i~.i.i.i2..ii.l
..%i' ..
i!
OH
I Phosphorylated Enzyme [
R20 /
....:'!~' i!~i
~i~~i~
R IO ~
151
0 II II
R~o -~P-~
0 R10 ~ . II O- /
P
\
(+ ROH)
O .....~ii,,',i~~ii~,~:~ilii,~iii!ii,~,,~~i,.~i.... ....~;i~ifi!ii~i,, ii{iii,.~!~iii!~,......... :;!
I Aged Enzyme (irreversible) I FIG. 1. An organophosphorus ester associates with the serine-hydroxyl moiety at the acetylcholinesterase active site (A) to form a reversible complex that is then inhibited via phosphorylation. The rates of association and phosphorylation are governed by ka and kp. The phosphorylated enzyme is then either reactivated via hydrolysis or becomes aged (irreversibly inhibited) via dealkylation (B).
15 2
SECTION II 9Pharmacokinetics & M e t a b o l i s m
hydrolyzed within minutes, whereas the phosphorylation of AChE is irreversible and reactivation can take several hours or days (Stevens and Breckenridge, 2001). During ChE inhibition, the inhibitor compound associates with AChE to form a reversible complex; following phosphorylation, the enzyme is either dephosphorylated, resulting in spontaneous reactivation of the enzyme, or dealkylated, resulting in the irreversible aging of the inhibited enzyme. The rate of association is governed by the association constant ka, whereas the rate of phosphorylation is mediated by the constant/c o (Fig. l b). There is some dispute as to whether species differences are due to differences in ka only (Andersen et al., 1977; Forsberg and Puu, 1984) or to differences in both ka and kp (Johnson and Wallace, 1987; Kemp and Wallace, 1990; Wallace, 1992; Wang and Murphy, 1982). Rates of spontaneous reactivation and aging of phosphorylated AChE differ among species and contribute, in part, to species variability. Whereas bird AChE reactivates rapidly, fish or insect AChE reactivates slowly, if at all (Wallace, 1992; Wallace and Herzberg, 1988), contradicting the general tenet that birds are more sensitive than fish to AChE inhibition. Furthermore, paraoxon- or DFP-inhibited AChE from frogs or fish ages slower compared to rat, mouse, or chicken AChE (Andersen et al., 1972; Wallace, 1992; Wallace and Herzberg, 1988). Finally, somaninhibited AChE in humans reactivates slowly but ages rapidly compared to bovine or rat AChE (de Jong and Wolfing, 1984, 1985; Wallace, 1992), suggesting that humans are relatively susceptible to irreversible AChE inhibition by soman. Species differ in their rate of recovery from AChE inhibition; indeed, oximes used as an antidote to reactivate AChE show differential species susceptibility (Eyer, 2003). There are species- and compound-dependent differences in the sensitivity of AChE to inhibition. Because brain AChE inhibition is the primary mechanism of acute toxicity, the sensitivity.of brain AChE to in vitro inhibition is an important determinant of acute toxicity. This is evident in correlations between the concentration required to inhibit 50% of AChE in vitro (IC50) and the dose that results in 50% lethality in vivo (LD50) (Wallace, 1992). Sensitivity of brain AChE to inhibition, and not the total AChE activity, potentially influences species differences (Johnson and Wallace, 1987; Wallace, 1992). The enzyme activity of 15 mammalian species surveyed ranged from approximately 2 to 10 ixmol/min/g wet tissue, whereas avian AChE activity from 44 species ranged from 10 to 30 ixmol/min/g (Blakley and Yole, 2002). The higher AChE activity in birds compared to mammalians is counterintuitive since birds are generally more susceptible to acute cholinergic toxicity than rodents. On the other hand, fish (minnow and trout) AChE activity is higher than that of rats (Table 2) (Johnson and Wallace, 1987), corresponding to relative resistance of fish to acute toxicity. Table 3 lists effective doses for ChE inhibition across species, including humans, for comparison among species
and compound. It is evident that plasma ChE is more sensitive to inhibition in humans than in dogs for physostigmine and DFP. For the same compounds, brain ChE is more sensitive to inhibition in rats compared to dogs. Chicken brain ChE is more sensitive than rat brain ChE for DFP and dichlorvos, but sensitivities are reversed for malathion and carbaryl (Table 3) (Raffaele and Rees, 1990). Whereas monkey brain AChE is more sensitive than chicken brain AChE to inhibition by DFP (Wallace, 1992; Wang and Murphy, 1982) human and chicken brain AChE sensitivities to inhibition by various OPs are comparable (within 30%) (Lotti and Johnson, 1978; Wallace, 1992). Conversely, BuChE (also known as pseudocholinesterase) inhibition does not provide a reliable indication of species differences in acute toxicity (Ecobichon, 2001a; Wallace, 1992). Although RBC AChE inhibition is often used as an indicator of OP exposure, it is unlikely that RBC AChE inhibition plays a major role in mammalian species differences among various OPs. Based on a literature review of 30 OP pesticides, Storm and colleagues (2000) provide an extensive table of adjusted 8-hr inhalation RBC AChE inhibition NOELs from humans, monkeys, dogs, and rats. This review focuses on occupational exposures, so exposure data from oral studies and inhalation studies were adjusted to provide an equivalent 8-hr inhalation exposure. The available data indicate that most OPs are equally potent RBC AChE inhibitors in different mammalian species (Storm et al., 2000). The adjusted 8-hr inhalation RBC AChE inhibition NOELs for the pesticides evaluated vary by a factor of two or three across species in most cases, when the exposure durations are approximately equivalent, although a few exceptions are noted. One exception, trichlorphon, appears to be 10-40 times more potent in primates than in dogs or rats (Storm et al., 2000). A contributing factor to this large variability may be relatively greater binding to nonspecific esterases in rats and dogs compared to primates (Anzueto et al., 1986; Storm et al., 2000), indicating that pharmacokinetic factors influence pharmacodynamics. 2. ALTERATIONSIN ACETYLCHOLINESTERASE STRUCTURE In addition to differences in enzyme kinetics contributing to species differences in sensitivity to AChE inhibition, alterations in the AChE enzyme impact its sensitivity to inhibition. Various interspecies, and possibly intraspecies, structural variants of mammalian AChE are synthesized by alternative splicing and posttranslational modification (Grisaru et al., 1999; Vidair, 2004). The distribution and molecular forms of AChE in the blood exhibit interspecies differences (Skau, 1985), although the significance to toxicity is not well appreciated. Differences in the physical or molecular properties of AChE influence enzyme and inhibitor interaction. Binding affinity of the parent compound and/or its metabolite for AChE is a major determinant of compound potency and is
CHAPTER 1 1 9lnterspecies Variation in Anticholinesterases TABLE 3.
153
Cholinesterase Inhibition in Various Species Exposed to Different Compounds
Compound
Species
Site
Value (mg/kg)
% ChE inhibition
Physostigmine Physostigmine Diisopropyl fluorophosphate Diisopropyl fluorophosphate Diisopropyl fluorophosphate Diisopropyl fluorophosphate Dichlorvos Dichlorvos Mipafox Mipafox Malathion Malathion Carbaryl Carbaryl
Rat Dog Chicken Rat Rat Dog Chicken Rat Rat Chicken Chicken Rat Rat Chicken
Physostigmine Physostigmine Diisopropyl fluorophosphate Diisopropyl fluorophosphate
Human Dog Human Dog
References a
Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain Brain
0.2 0.35 0.25 1.0 0.2 1.5 5 30 30 30 150 2000 160 300
40 40 63 66 17 12 26 21 81 76 53 56 47 60
1, 2 1, 3 4 4 1, 5 1, 3 4 4 4 4 4 4 4 4
Plasma Plasma Plasma Plasma
0.06 0.35 0.03 1.5
25 30 80 80
1, 2 1, 3 1, 6 1, 3
al, Raffaele and Rees (1990); 2, Becker and Giacobini (1988); 3, Paulet (1956); 4, Ehrich et aL (1995); 5, Chippendale et al. (1972); 6, Grob et aL (1947).
govemed by structural characteristics of AChE and the inhibitor (Storm et al., 2000). Wallace (1992) provides a detailed review of these properties. In brief, the steric properties and nucleophilic strength of the esteratic site, the presence and electronic strength of the anionic site, and the distance between the esteratic and anionic sites govern the interaction of the enzyme-inhibitor complex. The following properties confer relative resistance to AChE inhibition: (1) A smaller esteratic site, such as in fish, provides steric exclusion from the enzyme binding site; (2) weaker nucleophilic strength; (3) less effective allosteric regulation by the anionic site; and (4) a greater distance between the esteratic and anionic sites (Wallace, 1992). 3. NEUROTOXIC ESTERASE INHIBITION AND ORGANOPHOSPHATE-INDUCED DELAYED NEUROPATHY Exposure to some OPs produces a neuropathic response distinct from acute cholinergic toxicity, termed organophosphate-induced delayed neuropathy (OPIDN), and characterized by sensorimotor deficits affecting the distal extremities and reflecting selective degeneration of fibers in the spinal cord (CNS) and PNS (Cavanagh, 1963; Veronesi, 1992). Aside from species differences in acute cholinergic toxicity, there are also differences in OPIDN. Chickens are the sentinel species for studying OPIDN due to their sensitivity as a result of high accumulation rate and
low elimination rate relative to rodents (Abou-Donia, 1983). However, delayed neuropathy also is readily produced in cats and farm animals and is observed occasionally in humans as well (Veronesi, 1992). For purposes of human health risk, the cat may be a better model to extrapolate neurotoxicity results to humans because the exaggerated effects in chickens overestimate the risk to humans (Abou-Donia, 1983). Although rodents classically are considered insensitive to OPIDN due to their failure to develop hindlimb paralysis after exposure to neuropathic OPs, they do develop neuropathology and biochemical endpoints typical of OPIDN but without ataxia (Ehrich e t al., 1995; Veronesi, 1992). The species differences have been attributed to various notions, including differences in pharmacokinetics and qualitative differences in the target enzyme, generally accepted as neurotoxic esterase (NTE) (Abou-Donia, 1983; Hussain and Oloffs, 1979; Soliman e t al., 1982; Veronesi, 1992). NTE and AChE from both rat and chicken brain can be inhibited, albeit with varying sensitivities (Ehrich et al., 1995). Pretreatment with hepatic metabolic inhibitors increases rodent sensitivity to OPIDN, suggesting that interspecies differences in the metabolism of TOCP play a major role in neuropathy to this OP (Veronesi, 1992). Several species, including cats, chickens, humans, and rats, have been used to demonstrate that NTE inhibition can be used to predict neuropathic damage, distinct from
154
SECTION II 9P h a r m a c o k i n e t i c s & M e t a b o l i s m
acute cholinergic toxicity predicted from AChE inhibition (Veronesi, 1992). Mice also are sensitive to delayed neuropathy characterized by spinal cord pathology 2 or 3 weeks after TOCP exposure (Veronesi, 1992; Veronesi et al., 1991). Interspecies differences in the time course of NTE inhibition and recovery contribute to the species variability in sensitivity to OPIDN, with birds exhibiting a greater and longer duration NTE inhibition than rats despite an equipotent exposure to TOCP (Veronesi, 1992). The faster recovery of NTE inhibition in rats is due to reduced aging of the inhibited enzyme and/or more rapid synthesis of NTE (Clothier and Johnson, 1980; Soliman et al., 1982; Veronesi, 1992). A subsequent study found that rat and chicken brain NTE were similar with respect to inhibitor sensitivities, pH sensitivity, and molecular weight but differed in specific activity; hen brain NTE has approximately twice the specific activity as rat brain NTE (Novak and Padilla, 1986; Veronesi, 1992). Because the most striking difference in OPIDN between rats and chickens is the resistance of the rat to hindlimb ataxia compared to the chicken, interspecies neuroanatomical differences underlying modes of locomotion potentially account for species variability in sensitivity to OPIDN (Barnes and Denz, 1953; Cavanagh, 1954; Veronesi, 1992). Species such as chickens and humans become ataxic when fewer CNS neural tracts are damaged compared to rodents (Veronesi, 1992). Tracts housing the largest and longest nerve fibers are the most vulnerable to OPIDN, regardless of species, so that different tracts are damaged in various species. Spinal cord damage seen in rats differs from that in hens. For example, in humans and cats the most severely affected descending tracts are the pyramidal tracts, which are absent in the bird (Veronesi, 1992). On the other hand, the most vulnerable tracts in the hen are scattered throughout the lateral, ventral, and dorsal columns, whereas the large-diameter sensory fibers terminating in the dorsal columns of the upper cervical cord are most affected in the rat (Abou-Donia, 1981; Cavanagh, 1954; Veronesi, 1992). Thus, when considering species variability to nonacute toxicity, other target enzymes such as NTE are important as well as differences in neuroanatomy that could alter the outcome of OP exposure.
C. Age- and Gender-Dependent Effects Age is also an important factor to consider, and it is discussed elsewhere in this book. It is worth mentioning briefly here because some of the species-specific factors that contribute to interspecies variability also play a role in age-dependent effects of anti-ChEs. Relevant to this discussion is that young animals are more susceptible to OP toxicity than adults (Pope and Chakraborti, 1992; Pope et al., 1991; Vidair, 2004). Activities of brain AChE and liver aliesterase increase with age in rats; thus, both target (ACHE) and protective (aliesterase) esterases become more
abundant. However, sensitivity of brain AChE to paraoxon and chlorpyrifos-oxon-mediated inhibition does not change with age (Atterberry et al., 1997). Moreover, the similar maximal brain ChE inhibition in both neonatal and adult rats exposed to methyl parathion, parathion, or chlorpyrifos, coupled with the faster ChE synthesis and recovery in neonates (Pope and Chakraborti, 1992; Pope et al., 1991), suggests that target enzyme sensitivity is not key in age-related effects. In contrast, the greater sensitivity of neonates to acute OP toxicity correlates with lower levels of enzymes involved in OP deactivation compared to adults in both rats and humans (Vidair, 2004). For example, newborns have low levels of PON1, predicting greater sensitivity than adults to oxons hydrolyzed by PON1 (Davies et al., 1996). Similarly, gender differences in the overall rates of detoxification contribute to the greater sensitivity of female rats to some OPs (parathion, methyl parathion, and chlorpyrifos) compared to male rats (Chambers et al., 1994; Ma and Chambers, 1994; Neal and Dubois, 1965; Storm et al., 2000). Fetal enzyme isoforms differ from adult isoforms, and the ratio of phase I (e.g., cytochrome P450, glutathione peroxidase, and carboxylesterase) to phase II (e.g., sulfotransferase, glutathione S-transferase, and N-acetyltransferase) enzyme activities tends to increase with age (deBethizy and Hayes, 2001; Vidair, 2004). Primate fetuses contain a more developed set of toxicant-metabolizing enzymes compared to rodent fetuses (Dorman et al., 2001; Gow et al., 2001; Nau, 1986; Vidair, 2004), suggesting that primates are better equipped to protect against toxicity, although this is not always an accurate prediction. Furthermore, plasma binding proteins are lower in experimental animals compared to humans and are lower in human infants compared to adults (Nau, 1986; Vidair, 2004). Based solely on this, adult humans are expected to be protected from circulating OPs, whereas experimental animals and human infants are not expected to be protected. A host of noncholinergic developmental effects of OPs also contribute to age-related differences in toxicity (Slotkin, 1999, 2004; Pope, 1999). For instance, neurotransmitters, including serotonin and norepinephrine, in addition to ACh act as trophic regulators during development and are altered following developmental chlorpyrifos exposure (Aldridge et al., 2004; Dam et al., 1999a,b; Dreyfus, 1998; Lauder and Schambra, 1999; Raines et al., 2001; Weiss et al., 1998). Additionally, signaling cascades (e.g., adenylyl cyclase-cyclic AMP-protein kinase A cascade) involved in the proliferation and differentiation are disrupted following chlorpyrifos administration (Garcia et al., 2001; Huff et al., 1994; Olivier et al., 2001; Song et al., 1997; Ward and Mundy, 1996; Yanai et al., 2002; Zhang et al., 2002). Undeniably, developmental events render young animals, including humans, potentially more vulnerable to neurotoxicity from toxicants. In some cases,
CHAPTER 1 1 9Interspecies Variation in Anticholinesterases these age-dependent differences parallel species-dependent differences, as is the case for variable enzyme levels and activities as well as sensitivity to AChE inhibition.
IV. C O N C L U S I O N S A N D F U T U R E DIRECTIONS It is challenging to tease out a definitive source of interspecies variability; pharmacokinetic and pharmacodynamic parameters contribute dynamically to the sensitivity, response, and recovery of various species. Metabolism of anti-ChEs clearly plays a key role not only in species differences but also in age-dependent differences, whereas absorption, distribution, and elimination influence the bioavailability of the compound for metabolism. The route of exposure to an OP (or CM) significantly impacts the rate of absorption into the body and target organs and thus contributes to the toxic effects. Although anti-ChEs exert a common mechanism of action by inhibiting cholinesterases in blood and brain, the target enzyme is subject to species deviations. Whereas sensitivity to AChE inhibition contributes to the ultimate response, basal enzyme activity probably does not play a significant role in this sensitivity. Rather, the interaction of AChE and specific compounds is the predominant factor influencing sensitivity to inhibition. Structural AChE variants exist that can be more or less amenable to a "fit" from a specific compound. Finally, neuroanatomical differences among species may contribute to manifestations of toxicity, as proposed for the resistance of rodents to OPIDN compared to chickens. Interspecies comparisons of noncholinergic mechanisms are not well appreciated. Future studies should exploit both the similarities and the differences among species to delineate factors that are most influential on speciesspecific responses and to elucidate mechanisms of action beyond ChE inhibition. Although mammals are used for Environmental Protection Agency guideline toxicity tests (rats, mice, rabbits, dogs, and primates), a new corps of nonmammalian experimental animals, such as in vitro systems, have the potential to prove useful for addressing specific mechanistic questions. Nematodes, sea urchins, zebrafish, and avian embryos are being considered as models of mammalian neurotoxicity because they offer one or more of the following: more rapid screening, simplistic neural and neurotrophic systems, real'time in vivo observations during development, and elimination of confounding factors arising from maternal-fetal interactions (Buznikov et al., 2001; Cole et aL, 2004; Grunwald and Eisen, 2002; Slotkin, 2004; Yanai et al., 2004). Through pharmacokinetic and pharmacodynamic studies, we are closer to understanding the interspecies variability in response to anti-ChE intoxication.
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Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 114-125. Butterworth-Heinemann, Oxford. Veronesi, B., Padilla, S., Blackmon, K., and Pope, C. (1991). Murine susceptibility to organophosphorus-induced delayed neuropathy (OPIDN). Toxicol. AppL PharmacoL 107, 311-324. Vidair, C. (2004). Age dependence of organophosphate and carbamate neurotoxicity in the postnatal rat: Extrapolation to the human. Toxicol. Appl. Pharmacol. 196, 287-302. Walker, C., and Mackness, M. (1987). "A" esterases and their role in regulating the toxicity of organophosphates. Arch. Toxicol. 60, 30-33. Wallace, K. (1992). Species-selective toxicity of organophosphorus insecticides: A pharmacodynamic phenomenon. In Organophosphates: Chemistry, Fate, and Effects (J. Chambers and P. Levi, Eds.), pp. 79-105. Academic Press, New York. Wallace, K., and Dargan, J. (1987). Intrinsic metabolic clearance of parathion and paraoxon by livers from fish and rodents. Toxicol. Appl. Pharmacol. 90, 235-242. Wallace, K., and Herzberg, U. (1988). Reactivation and aging of phosphorylated brain acetylcholinesterase from fish and rodents. Toxicol. Appl. Pharmacol. 92, 307-314. Wang, C., and Murphy, S. (1982). Kinetic analysis of species difference in acetylcholinesterase sensitivity to organophosphate insecticides. Toxicol. Appl. Pharmacol. 66, 409-419. Ward, T. R., and Mundy, W. R. (1996). Organophosphorus compounds preferentially affect second messenger systems coupled to M2/M4 receptors in rat frontal cortex. Brain Res. Bull. 39, 49-55. Warfington, J., Greenblatt, D., and von Moltke, L. (2004). The effect of age on P-glycoprotein expression and function in the Fischer-344 rat. J. Pharmacol. Exp. Ther. 309, 730-736. Weiss, L., and Orzel, R. (1967). Some comparative toxicologic and pharmacologic effects of dimethyl sulfoxide as a pesticide solvent. Toxicol. Appl. Pharmacol. 11, 546-557. Weiss, E., Maness, P., and Lauder, J. (1998). Why do neurotransmitters act like growth factors? Perspect. Dev. Neurobiol. 5, 323-335. Yanai, J., Vatury, O., and Slotkin, T. A. (2002). Cell signaling as a target and underlying mechanism for neurobehavioral teratogenesis. Ann. NYAcad. Sci. 965, 473-478. Yanai, J., Beer, A., Huleihel, R., Izrael, M., Katz, S., Levi, Y., Rozenboim, I., Yaniv, S., and Slotkin, T. (2004). Convergent effects on cell signaling mechanisms mediate the actions of different neurobehavioral teratogens: Alterations in cholinergic regulation of protein kinase C in chick and avian models. Ann. N. Y. Acad. Sci. 1025, 595-601. Zhang, H. S., Liu, J., and Pope, C. N. (2002). Age-related effects of chlorpyrifos on muscarinic receptor-mediated signaling in rat cortex. Arch. Toxicol. 75, 676-684.
Esterases, Receptors, Mechanisms, & Tolerance Development
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CHAPTER
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2
Structure and Function of C h o l i n e s t e r a s e s
ZORAN RADI(~ AND PALMER TAYLOR University of California at San Diego, La Jolla, California
much better bind generally bulky ligands, including covalent inhibitors isoOMPA and bambuterol and reversible inhibitor ethopropazine, all well-known specific inhibitors of ChEs (Vellom et al., 1993; Radi6 et al., 1993). The site of catalysis in both enzymes is buried-~20/k deep in the center of the globular catalytic subunit. Tissue distributions and physiological functions of the two enzymes in higher organisms are different. AChE is mainly found in the central nervous systems, neuromuscular junctions, and the hematopoietic system of vertebrates, and it plays a key role in cholinergic neurotransmission. BuChE is found in liver, blood serum, and kidney. Its primary physiological role is not completely clear, but it may be involved in hydrolysis of dietary esters. Mice lacking the AChE gene and activity have normal BuChE activities and can survive more than 1 year but are more sensitive to organophosphate (OP) inhibitors, suggesting that BuChE activity may substitute at least in part for AChE activity (Adler et al., 2004). The mammalian cholinesterase profile contrasts with that of Drosophila, which lacks BuChE and in which AChE knockout results in embryonic lethality (Greenspan et al., 1980). In humans and most other vertebrate species, only one gene encodes ACHE. Insects and worms can have multiple genes encoding up to two (insects) or four (worms) AChEs.
I. I N T R O D U C T I O N From the beginning of the past century until the early 1970s, assay of the cholinesterases was based mainly on their capacity to catalyze the hydrolysis of acetylcholine and on the selectivity of compounds that interfere with the catalytic reaction (Dale, 1914). In the late 1960s and early 1970s, inhibitors and substrates were found to bind to a remote site(s), thereby allosterically interfering with the catalytic reaction (Changeux, 1966; Aldridge and Reiner, 1969; Taylor and Lappi, 1975). Improvements in protein purification techniques identified the active center and sufficient sequence to clone the gene encoding the enzyme (Schumacher et al., 1986). Five years later, the first threedimensional structure of a cholinesterase was solved (Sussman et al., 1991). Those events led to rapid accumulation of structural information on cholinesterases in the years to come, resulting in the determination of at least 125 primary structures and 61 three-dimensional structures of cholinesterases available today. In this chapter, we summarize the available structural information in the context of the evolutionary and functional relationships of the cholinesterases and related od[3 hydrolase fold proteins.
II. C H O L I N E S T E R A S E
FAMILY
A. Primary Structure
OF ENZYMES
Catalytic subunits of both AChE and BuChE consist of a single 500- to 600-amino acid-long peptide. The first amino acid sequence of an AChE was determined by Schumacher and colleagues in 1986 by cloning cDNA for the enzyme from fish Torpedo californica, whereas the first BuChE sequence was determined by sequencing purified enzyme protein isolated from human serum (Lockridge et al., 1987). A search of the SwissProt database revealed approximately 125 full-length or nearly full-length cholinesterase sequences, including approximately 108 AChE and 17 BuChE sequences. More than half of these refer to proteins
Two structurally and functionally very similar, yet distinct enzymes form the family of cholinesterases (ChEs). Acetylcholinesterase (ACHE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) both catalyze acetylcholine (ACh) hydrolysis with similarly high efficiency and only differ in efficiency to catalyze the hydrolysis of carboxylic acid esters of larger acyl group size, such as butyrylcholine or benzoylcholine. Larger substrates are hydrolyzed much better by BuChE due to small but significant differences in their structure that also allows BuChE to Toxicology of Organophosphate and Carbamate Compounds
161
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
162
SECTION III. Esterases, R e c e p t o r s , M e c h a n i s m s , & Tolerance D e v e l o p m e n t
whose function was not established directly but was inferred from sequence comparisons. The largest numbers of deposited sequences are of insect cholinesterases (51 sequences), including aphids (16 sequences), flies (15 sequences), and mosquitoes (10 sequences), followed by 19 entries of mammalian sequences. Also numerous are parasite (17 sequences) and worm (16 sequences) entries. The distribution of species with known cholinesterase sequences reflects an interest in studying enzymes targeted by OP and carbamate (CM) pesticides. Some deposited AChE sequences originate from species that developed resistance to pesticides used to eradicate them, most notably insects such as, mosquitoes, and flies. Cholinesterase activities have been documented in a number of plants and algae (Gupta and Gupta, 1997; Gupta et al., 1998; Fletcher et al., 2004), but their sequences are not known. Among the simpler life-forms, recently resolved genomic sequences of fungus Neurospora crassa (Galagan et al., 2003) and virus Mimivirus (Raoult et al., 2004) revealed cholinesterase-like sequences, suggesting utilization of cholinesterase activity in simple life cycles of primitive organisms. Table 1 summarizes full-length amino acid sequences of fish (ACES_TORCA; i.e., T. californica), insect (ACES_DROME; i.e., Drosophila melanogaster, 36% identity with Torpedo), human (ACES_HUMAN, 55% identity with Torpedo ACHE), and mouse (ACES_MOUSE, 56% identity with Torpedo ACHE) AChEs; human and mouse BuChEs; as well as four cholinesterases of very simple organisms m fungi (Q6MGI2 and Q872U5, 21 and 20% identity with Torpedo ACHE, respectively), tunicata Oikopleura dioica (Q675X9, 14% identity with Torpedo ACHE), and virus (Q5UR02, 21% identity with Torpedo ACHE). Out of approximately 50 serine residues found in cholinesterase sequences, only one is directly involved and essential in catalysis, Ser200, as confirmed by site-directed mutagenesis (Gibney et al., 1990). It is found in all sequences in Table 1, including viral and fungal proteins. A detailed comparison shown in Table 2 includes all 125 currently available cholinesterase sequences. The active Ser200 is conserved in 124 sequences and shifted by one residue C-terminal only in human hookworm Necator Americanus AChE (Q8IT86). It is not clear whether this protein is catalytically active, but similar one-position shifts in the active serine, although N-terminal, are found in sequences of structurally related, but catalytically inactive, neuroligins, a family of neuroadhesive proteins from human, mouse, and rat sharing approximately 30% amino acid identity with ACHE. Besides Ser200, five more serines are very well conserved throughout the family: Ser91, Ser205, Ser212, Ser226, and Ser428. The amino acid fragment (195-205) around the active Ser200 is well conserved in most cholinesterases. Of the 11 residues of the fragment, Gly202 appears to be strictly conserved in all 125 sequences, whereas the pattern Gly-X-Ser200-X-Gly-X-X-Ser
can be found in 123 sequences; exceptions are the simple marine organism Oikopleura dioica (Q675X9) and worm N. Americanus ACHE. Interestingly, 13 of 15 deposited fly AChE sequences have, starting with Ser200, the conserved pattern Ser-x-x-Ser-Ser-Ser, and one of the sequences (Q8MXC9) has a Ser-Ser'Gly-Ser-Ser-Ser pattern. Residues His440 and Glu327 were identified as the remaining two elements of the catalytic triad. Site-directed mutagenesis showed that substitution of His440Gln in T. californica AChE yielded inactive protein (Gibney et al., 1990), and Glu327 was identified as part of the catalytic triad only upon resolution of the first three-dimensional structure of an AChE (Sussman et al., 1991). The two residues are conserved in 123 (Glu327) and 124 (His440) sequences, suggesting that Oikopleura and one of two fungal proteins (Q6MGI2) in which residues other than Glu and His are found may not be catalytically active. Several other glutamates and histidines are well conserved in the cholinesterase family. Glu92 is conserved in all 125 sequences, and Glu199, Glu443, and His209 are conserved in almost all of them, whereas positions 172, 297, and 397 have either Asp or Glu always conserved. Besides the catalytic triad, additional residues are known to be critical for maintaining the catalytic activity of cholinesterases. The oxyanion hole stabilizes carbonyl oxygen of ACh during hydrolysis. It is formed by protein backbone amide nitrogen protons of residues Glyl 18, Gly119, and Ala201, as suggested by the positioning of trifluoroacetophenone, an ACh transition state analogue, in the threedimensional structure of Torpedo AChE (PDB code 1AMN; Harel et al., 1996). Two of three residues are likely sufficient to form a functional oxyanion hole entity, and all 125 sequences in Table 2 have at least two of three oxyanion hole residues conserved. The most conserved is Glyll8 (in 124 sequences), followed by Ala201 (in 120 sequences). The Ala201 is substituted with serine in 5 sequences. Gly 119, is conserved in 115 sequences with serine substitution in 6 of the remaining 10 sequences. Two additional sequence fragments exceptionally well conserved throughout the cholinesterase family are required for catalytic activity, although their involvement in the catalytic process is not fully understood. Residues 91-95 with sequence SEDCL (Ser-Glu-Asp-Cys-Leu) are conserved in 115 of 122 residues, motif xEDCL is conserved in 120 of 122 sequences, and motif xExxL is conserved in all 122 sequences. Two anionic residues in that fragment, Glu92 and Asp93, are involved in a salt bridge and a hydrogen bond formation with Arg44 and Tyr96, respectively. Both are conserved in number of cholinesterases, particularly Arg44, which is absent only in viral and O. dioica AChEs out of 125 sequences. Substitutions of Glu92Gln, Glu92Leu, Glu93Val, and Arg44Glu in single-site Torpedo AChE mutants resulted in the complete loss of catalytic activity, indicating their important role in folding and maintaining the three-dimensional structure of a disulfide loop.
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SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
FIG. 1. Three-dimensional structures of five ChEs. (A) Overlay of Ca backbone of all five structures. (B-F) Solvent-accessible surfaces of individual enzymes: (B) Torpedo ACHE, (C) mouse ACHE, (D) human ACHE, (E) Drosophila ACHE, and (F) human BuChE. Active center gorge is indentation in the center of each structure. Visualized by WebLabViewer software (Accelrys, San Diego). TABLE 5.
homogeneous for crystallization (Nachon et al., 2002; Nicolet et al., 2003). The protein backbones of the five mentioned ChE structures, four AChEs and one BuChE, overlay reasonably well, with only Drosophila AChE showing slightly larger deviations (Fig. 1A; Table 5). The common fold includes a twisted [3 pleated sheet (approximately 10 strands long) in the core of the enzyme molecule, surrounded by more than 12 longer and shorter oLhelices. Such unique distribution of elements of the secondary structure was recognized first in ChEs and lipases and then in a number of other proteins, and it was consequently termed the od[3 hydrolase fold (Cygler et al., 1993). Catalytically active residues in enzymes belonging to this fold are located almost in the center of a globular protein, at the bottom of a narrow, approximately 20/k deep active center gorge. The shape and size of the gorge are similar in Torpedo, mouse, and human AChEs, but despite similarity in the backbone fold, the respective volumes of gorges in human BuChE and Drosophila AChE are approximately 50-100% larger and 50% smaller than in mammalian and fish AChEs (Harel et al., 2000; Saxena et al., 1999). This significant structural difference~likely affects the ability of ChEs to effectively hydrolyze ACh (Fig. 2). Hydrolysis appears more efficient in the active center gorges of fish and mammalian AChEs than in either the smaller volume gorge of Drosophila .ACHE or the larger volume gorge of BuChE. To date, 61 cholinesterase structures have been deposited in the Protein Data Bank. Forty are of T. californica ACHE, nine of mouse ACHE, three of Drosophila ACHE, two of human ACHE, and seven of human BuCHE. Overlaying of all Torpedo ACHE structures reveals exceptional similarity in their protein backbone (Fig. 3A) and even in their side chain conformations (Fig. 3B). The mean value of the root mean square (RMS) deviation of the 40 Torpedo AChE structures from the alpha carbon trace of the highest resolution (1.8 ~), unliganded lea5 structure is only 0.26 _+ 0.10 ]k. Twelve
Structural Pairwise Overlays of Five Unliganded ChE Structures a RMS (Ca backbone) (~.)
ChE
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Drosophila melanogaster AChE Human BuChE
PDB ID No.
Resolution (A)
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Mouse AChE Chain A Chain B
Human AChE
Drosophila melanogaster AChE
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2.8 2.7
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CHAPTER 1 2 9Structure and Function of Cholinesterases
175
hAChE
3 kcat
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TAChE
2 mAChE hBuChE DAChE I
I
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0.1
1
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FIG. 2. Activity of ChEs (expressed as kcat) as a function of substrate acetylthiocholine (ATCh) concentration. Curves (except for DAChE) were calculated using Eq. (1) and literature values of catalytic constants: hAChE and hBuChE from Kaplan et al. (2001), TAChE from Radic et al. (1992), and mAChE from Radic et al. (1993). The DAChE curve was calculated using the equation and data from Stojan et al. (1998). All constants were determined at room temperature except for hAChE and hBuChE, which were determined at 27 ~ contributing to relatively higher kca t values compared to TAChE, mAChE, and DAChE. Unliganded AChEs are 0nly slightly more similar to the lea5 with a mean RMS value of 0.20 _+ 0.08 ,~ compared to an RMS of 0.28 _+ 0.10 A for the remaining 28 liganded structures. This is further emphasized in complexes of mouse ACHE, in which RMS variation between subunits of the same structure (mean RMS, 0.25/k) appeared even slightly larger than RMS deviation between the same protein chains of different structures (mean RMS, 0.21 ,~), indicating that the effect of packing identical subunits within the same structure perturbed the backbone conformation more than the presence of bound ligand. Even structures containing ligands forming a covalent bond with the AChE active serine did not differ in their RMS from noncovalent ones. The high degree of similarity and small RMS values found also for mouse and Drosophila AChEs and human BuChE suggest that binding of ligands to cholinesterases in general is not associated with large conformational changes of the protein backbone. However, dimensions of substrate molecules appear similar to or larger than the size of the AChE gorge that they need to traverse on the way to the active serine at its bottom. The fight entry of a substrate molecule into the narrow site of catalysis in the active center is thus possibly facilitated by a series of fast, small amplitude side chain motions, occurring repeatedly at time intervals far shorter than the catalytic cycle. The absence of structural evidence of large backbone movements in AChE structures may therefore result from evolutionary pressure to achieve and maintain their very fast catalytic tumover. Every AChE molecule (depending on the species) can tum over between 200 and 16,000 molecules of ACh every second or 1 molecule in as short as 63 Ixsec (Nolte
FIG. 3. Overlay of 40 Torpedo californica AChE structures using only CoL backbone atoms. (A) Trace of CoL backbone atoms with combined volume of all ligands found in structures represented as a solvent-accessible surface. (B) Representation of all enzyme atoms found in 40 overlaid structures. Structures were overlayed using SwissPDBViewer, software. Visualized by WebLabViewer software (Accelrys~ San Diego).
176
SECTION III. E s t e r a s e s ,
Receptors,
Mechanisms,
& Tolerance
Development
1. TOPOGRAPHY OF BINDING SITES IN C h E s
FIG. 4. Topography of binding sites in ChEs represented by selected residue side chains of Torpedo AChE structure. A molecule of ACh docked in the active center (taken from PDB entry 2ACE) and shown as solvent-accessible surface is given as a frame of reference. Binding sites are acyl pocket (residues 288 and 290), choline binding site (residues 84 and 330), and peripheral site (residues 70, 72, 121, and 279). Visualized by WebLabViewer software (Accelrys, San Diego).
et al., 1980). Continuous repetition of backbone movements would possibly require both more time and more energy invested per each hydrolytic cycle. These molecular motions have to be fast enough to allow diffusion-limited binding of relatively large ligands. Catalytic turnover of BuChEs is slower despite the much larger opening and larger volume of the active center gorge. Due to the absence of aromatic amino acid side chains lining the AChE gorge, approximately 20 water molecules found in either of the structures or substrate molecules may be retained along the gorge walls by hydrogen bonding and electrostatic interactions, This could inherently slow down catalysis, which may be even slower in the more water-accessible, and thus less hydrophobic, catalytic site of BuChE. Most of the volume of the AChE active center gorge seems to be well accessible to ligand binding judging from the combined volume of all ligands in active center gorges of 40 superimposed Torpedo AChE structures totaling approximately 1100/~3 (Fig. 3A) and filling practically 1000 ,&3 of the available gorge space (calculated by CASTp software; Liang et al., 1998).
In the immediate vicinity of catalytic triad residues at the base of the active center gorge, just above the active serine, is located the electrophilic oxyanion hole, which has the capacity to attract carbonyl oxygen of ACh and other substrates as well as phosphyl oxygen of covalent, OP inhibitors. Three backbone nitrogens well conserved throughout the cholinesterase family (Table 2) form the oxyanion hole, lending their amide protons for the interaction. In oxyanion holes of proteases, only two donor residues are involved. Site-directed mutagenesis of AChEs and use of substrates and inhibitors specific for interaction with either AChE or BuChE helped delineate specific locations of additional ligand binding sites within the enzyme gorge (Shafferman et al., 1992a; Vellom et al., 1993; Ordentlich et al., 1993; Radi6 et al., 1993). The acyl pocket and the choline binding site are located next to the active serine, at the base of the active center gorge (Fig. 4), controlling the size of ligands that can approach the site of catalysis. The space available for binding is generally smaller in AChEs in which several aromatic residues (14 in fish and mammalian AChEs; Sussman et al., 1991) line the walls of the gorge. In the acyl pocket, in-place of phenylalanines Phe288 and Phe290 found in AChEs smaller aliphatic residues are found in BuChEs, whereas in the choline binding site Phe330 (many AChEs have tyrosine at this position) in BuChEs is replaced by alanine (Table 6). The smaller residues and larger available space in BuChE enable preferential binding of the large substrates butyrylthiocholine and benzoylcholine in the acyl pocket and large inhibitors ethopropazine and isoOMPA in the choline binding site and acyl pocket, respectively. Some mutant insects (mosquitoes and flies) that developed resistance to pesticides have amino acid residues altered in this region in their AChEs, selectively preventing binding of pesticides to active serine while not compromising catalysis (Menozzi et al., 2004). In addition to the choline binding site and acyl pocket in the third binding domain of cholinesterases, the peripheral site is located at the rim of the gorge, approximately 14 /~ from the active serine. Formed by Tyr70, Tyrl21, and Trp 279, this aromatic cluster specifically binds cationic and aromatic inhibitors that are too large to enter the gorge, such as propidium, gallamine, or snake toxins fasciculins (Bourne et al., 1995, 2004), or long and slender bisquaternary ligands that extend from the bottom of the gorge, such as BW286c51, decamethonium, and a variety of bifunctional ligands including bis-tacrines, bishuperzines, and very high-affinity triazoles (Lewis et al., 2002; Bourne et al., 2004). In the vicinity of the aromatic cluster is Asp72, a specifically located anionic residue lending its stabilizing contribution to ligands binding to the -6 ~ proximal peripheral site and/or ,--8 A distal choline binding site. Devoid of an aromatic cluster in the peripheral site, BuChEs bind most bisquaternary and bifunctional inhibitors with three or four orders of magnitude lower
CHAPTER
T A B L E 6.
P e r i p h e r a l site I~
f~
f~
0~
~ oo
f~
~
~
00
00
BOVIN
Y
D
Y
W
V
Y
W
Y
F
W
F
R
HUMAN
Y
D
Y
W
V
Y
W
Y
F
W
F
R
ACES
AM
ACES
FELCA
ACES
MOUSE
AM
ACES
RAT
AMHUMANyt
Q86TM9
AMHUMAN2 AMONKEY AMONKEY ABCHICK
ACES
RABIT
Q86YX9
r-
Y Y Y Y Y Y
Q67BCI
Y
Q67BC2
ACES
CHICK
D D D D
T
D
G
D
D
061459
AF
ACES
TORCA
Y
D
AF
ACES
ELEEL
Y
D
AHAG F I S H
ACES
MYXGL
-
AF
061372
ACES
BRARE
AF
ACES
ASNAKE
ACES
ASQUID
097110
AIAPHID
Q6KAV3
TORMA BUNFA
ASPIDER
Q86CZ4
AVIRUS
QSUR02
AIAPHID
Q6KAV4
AIAPHID
Q65YU0
AIAPHID
Q65Z62
T F
Y
Y
W W
D
Y
D
Y
Y
V V
-
W
Y
F
V
Y
I
Y
W
W
I I Z
W
L
W
I
W
L
W
G
I
D
Y
W
F
Q65Z60
I
AIAPHID
Q65Z63
AIAPHID
Q66S79
AIAPHID
Q6BCH8
E
I
I
AIAPHID
Q66S75
E
AIAPHID
Q8MV35
I
AIAPHID
Q8MV36
E
AIAPHID
QgBMJI
I
AIAPHID
Q7YZP9
AIBEETLE
ACES
AIBOLLWORM
Q8MX85
AICULEX
Q75VX9
E
I
AIAPHID
Q6KAV5
I
AIAPHID
Q7YZQ0
E
AIBEE
Q9GQP7
Y
D
D Y
Y D
D Y
D
D
M
Y Y M M
Y Y M
Y Y
Y
M
E
Y
M
E
Y
E
Y
-
E E
-
Y Y
W
W
W
L
F
W
F
W
L
W
F
W
F
Y
W W
W W
W W
Y Y Y
Y
Y Y
Y
Y
M
W
L
W
M
W
M
W
M M
W W
I
L L
Y Y
Y Y
W
Y
L
Y
Y
W
Y
W
E
Y
Y
W
Y
Y
Q8MXC8
E
Y
W
Y
L
AIFLY
Q8MVZ4
Y
Y
W
W
L
AIFLY
Y
W
W
W
Y
Y
F
Y
Y
M
E
Y
Y
W
L
L
Y
Q7YZP7
Y
W
W
W
E
AIFLY
Y
V
M
M
M
Y
Y
-
W
W
W
Y
Y
Y
W
F
F
R R
R
F
R
S
Q
C
F
W
V
Y
G
W
E
E I
F
W
F
W
L
G
W
C
F
F
F S
W W W
C
L
C
F
W
C
F
W
L
F F S
W
W
W
F
C
F
C
L
S
W
C
F
F G
W
W
G
L
F S
F
C
G
F
G
F
F
W
L
G
F
W
L
G
F
W
F
W
F F
F
F
W W
W
W
L L L
L
L
C
G G G G
G
E
W
L
G
W
Y
F
W
L
S
W
Y
W
Y
W
Y
Y
W
F
E
F
Y
L
Y
W
L
E
Y
W
Y
Y
W
L
W
Y
L
F
F
Y
Y
Y
E
W
V
W
Q95P20
Q8MXC4
Y
Y
L
AIFLY
AIFLY
F
Y
L
M
Y
E
Y
W
W
Y
E
W
M
W
ACES
DROME
Y
Y
Y
AIFLY
P91954
F
W
M
R
Y
Y
L
F
W
F
R
R
R
Y
F
R
F
W
-
R
R
E
F
Y
F
R
L
F
F
F
R
W
W
Y
F
F
R
L
Y
W
L
W
Q75VY0
AIFLY
W
W
M
AICULEX
E
W
F
F
R
W
W
W
L
QSRLH9
D
W
W
AIBUDWORM
I
W W
F
F
F F
F
W
W W
W
F
F
F
Y
~
W
F
Y
F
W
Y
L
W
W
W
W
Y
Y
L
W
Y
F
W
W
Y
Y
L
W
W
Y
F
W
W
F
~
Y
N
M
W W
Y
Y
Y
Y
T
F
Y
W
W
V
T
W
Y
E
W
AIAPHID
Y
G
Y
Y
-
Y
V
Y
D
-
F
V
W
D
I
W
Y
F
W
I
I
-
W
Y
F
F
W
W
Y
W
Y
F
Y
Y
D
Y
W
Y
W
L
A
Y
W
Y
W
-
K
V
Y
~
V
W
Y
V
oq
W
Y
W
0~
I
E
Q86QW5
T
W
D
D
Q8T7U9
AIBOLLWORM
Y
W
W
G
AIAPHID
LEPDE
Y
W
W
D
E
Y
e~
D
M
V
Y
Y
D
G
Y
D
D
ACELEGANS
Q9NDG9
Y
Y
Y
M
~
D
ACE4
ACELEGANS
CAEBR
r-
ACBRIGSAE ACBRIGSAE
Acyl pocket
C~
AM
ACES
Choline b i n d i n g site
(~
SwissProt code
AM
177
O
ID
AM
9S t r u c t u r e a n d Function o f C h o l i n e s t e r a s e s
Elements of ChE Primary Structures Involved in Ligand Binding as Found in A l i g n m e n t of 125 ChE Structures a Sequence
AM
12
W W
Y Y
Y Y Y
Y
F F F
F F F
W W
W
W
W W
L
L
L
L
L L
S
S S
S
S
G
(continues)
178
SECTION
III. s
R e c e p t o r s , M e c h a n i s m s , & Tolerance D e v e l o p m e n t TABLE
Sequence
Peripherai site O
C~
Q8MXC9
E
Y
Q7YWJ9
E
Y
ID
SwissProt code
AIFLY AIFLY
AIFLY
AIFLY AIFLY AIFLY AIFLY
AIFLY ~
AIHOPPER
AIHOPPER
Q8MXC5 QSMXC6 QSMXC7 Q7YZP8
Q5WIL0 QgNJH6
ANOGA
AIMOSQTO
ACES
CULPI
AIMOSQTO AIMOSQTO
AIMOSQTO AIMOSQTO AIMOTH AIMOTH AIMOTH
APBOOPHILUS
E
QSMU94
ACE1
AIMOSQTO
e.
Q95WV7
AIMOSQTO
AIMOSQTO
ACES
ANOST
Q5XL61
Q6A2E2
E
'
E E
E
E
E
I
I
E
Q7RTM0
E
Q8MZL2
Q5S579 QSMZM0
E
E
I
E
V
Q7RTL7
E
APFLUKE
Q71SU7
P
APTICK
Q6XR73
APCIONA
APFLUKE
APFLUKE APTICK
APTICK
Q7RTL6 Q71SU5 Q86GL8 061864 Q9NFKI
I
~'-
f'1
~
~
C'~
~
r
M
W
L
Y
W
Y
F
M
W
L
Y
W
Y
F
M
Y Y
Y
Y
D
I
c~
Y Y
D D
M M M
M M M
Y Y Y
D
Y
Y
M
Y
M
D Y
Y D
Choline binding site
,~
M
I
I
(",I
Y
Y
Q7RTL9 QgTXll
e.
E
061987
APCIONA
(continued)
6.
Y
M
M
Y
W
W W W W
W W W W W W
~
L
L
L L L L L
r
Y
Y
Y Y Y
Y Y
L
Y
I
Y
I I
Y Y
W
I
Y
W
L
Y
W
L
W W
W W
I
L
Y Y
Y
L
Y
L
Y
Y
M
W
L
Y
D
Y
W
L
Y
D
T
Q
W
D
Y
D
L
Y
L
D
Y
P
Q
Y
V
D
Q
Y
P
v
D D
D
Y Y Y
Y
D D
I
Y
Y
P
D
Y
L
L
T
Q
T
Y
Y Y
Qo
W
W W W
W W W W
W W W W
Y
Y Y
Y, Y
Y Y Y Y Y
Y Y
S
Y
L
S
Y
L
S
Y
F
W W
L
S
Y
W
L
G
F
W
L
S
X
W
L
S
F
W
L
S
X
W
L
G
F
W
L
G
F
W
C
E
F
W
C
E
F
W
C
E
F
W
C
E
F
F F F
F F F F
F
F F
W
Y
F
W
W W
W W W W W
W W
Y Y Y Y W
Y Y Y Y
Y
W
Y
W
W
w
W
L
F,.
W
~ o (x) o~
W
Y Y
(D
F
W W
Acyl pocket
F
F F F F
F
F F F F
F
F
F
W
C
E
F
W
L
G
F
W
L
G
F
W
L
G
F
W
L
G
F
W
C
~E
F
W
L
G
F
V
D
F
W
F
D
F
F
D
F
W
F
s
v
F
S
V
W
F
S
V
v
D
F
W
V
D
F
V
D
F
W
V
D
F
I
D
F
I
H
A
L
W W W
W
W
F
W
W
F
W
APTICK
Q6WVH4
V
D
Y
T
Q
Y
W
W
APTICK
Q9NFK2
C
Q
S
N
F
Y
P
Y
APTICK
Q9NFK3
A
Y
T
W
N
I
APTICK
Q6XR75
Y
T
W
N
I
G
Y
F
W
Y
W
W
APTICK APTICK APTICK APTICK
AW
Q9NFK4
G
Q6XR74
G
062563
G
V
S
D
ACE1
CAEBR
ACE1
AW
061378
AW
CAEEL
AW AW
A
D
W
W
D
F
S
Q6XPY6
T
T
w
F
D
P
Y
W
Y
W
Y
F
D N
G
S
Q967G8
T
S
F
D D D D
D D
N
Y
F
F
,W ' Y
F
P
P
Y
Y Y
W
W
W W
V
Y
P
Y
W
V
Y
W
S
P
Y
W
P
Y
D D D
F
N
F
A
F
T
T
W
N
T
F
S
T
W
W
D D
Y
T
W
W
T.
D
Y
Y
Y
W
AW
QSIT86
D
Q
A
W
T
AW
D
Q
D
Q86GL7
Qgu640
V
S
AW AW
A
Q
T
Q71JB7
Q
V
Q6QDP4
QgXYA9
A
G
T
Q6QDP5
Q
Q
061587
AW
AW
T
V
T
096529
V
Y
G
061371
AW
AW
D
045210
AW
AW
V
I
P
P P
P
P
Y
w
Y
W
Y Y
Y
W
W W W
W
N
Y
F
F
I
I I
F
W
F
W
Y
W
Y
W
F
Y Y
F
W F
G
G
W W
G
D
F
G
D
F
M
T
F
M
T
F W
M
E
W
A
D
F
W
M
T
F
W
M
S
F
W
M
T
F
A
D
F
D
F
W
W'
,~
W
M
W
W W
W
F
W
F
G
W
F
F
G
F
W
M
S
F
M
E
W
W
M
E
W
L,
T
F
(continues)
CHAPTER
12
179
9 S t r u c t u r e a n d Function of C h o l i n e s t e r a s e s
TABLE 6. (continued) Sequence
P e r i p h e r a l site
ID
O
Ol
C4
O~ ~,-
T
D
F
Y
-
-
M
W
D
M
Q
A
N
FELCA
N
D
Q
HUM.AN
N
SwissProtcode
AW
QgGPL0
BAMPHIOXUS
CHLI CHL2
BAMPHIOXUS
BAMPHYOXUS
m w
076998
BAMPHYOXUS
076999
BFUNGUS
Q6MGI2
BBCHICK
Q872U5
BM BM
BM
BRALA
CHLE
CHLE
~
CHLE CHLE
BM
CHLE
BM
MOUSE
PANTT
C H L E ,,RABIT
QgNIN9
BMRAT
QgJKCI
QgGKJ6
BMPIG
BOIKOPLEURA
Q675X9
A
L
HORSE
BMHORSE
-
L
Q90ZK8
BFUNGUS
BM
BRALA
e-
D
e.
-
D D D
~
F
F
E
N
G
K
L
Q
D
Q
V
Q
V
N
D
-
N G
-
D T
~ f~
f~ f~
P
Y
W
F
W
W
L
Y
-
Y
F
W
W
Y
F
W
G
G
G
-
A
F
V
D~
Ni
O f~
V
N
-D
~
W
D
N
~ f~
m
V
W
V
m
Y
V
Y
W
P
R
G
V
V
F
Y
K
V
T
Y
Y
G
A
Q
F
I
Y
F
A
V
T
F
W
L
S
W
L
S
W
L
F
W W
F
W
F
W
A
F
Q
Y
F
A
F
H
-
W
Q
L
F
W
F
W
F
A
Y
S
D
W
V
A
O O~
A
Y
V
W
A
e~
M
O0
W
W
F
W
O~
eq
00
D
A
Y
Y
M
00
A
,,W
V
Q
S
m
W
Y
L
V
F
W
A
Q
V
F
m
W
,Y
V
C
W
m
Y
V
N
-
W
Y
A
Q
~
Y
L
Q
R
Acyl pocket
f~ f~
W
N
D
Q
e~
C h o l i n e b i n d i n g site
F
W
W
Y
C C P
L L
I
W
S
V
L
S
R
I
V
S
A
I
S
L
L
e~
S
V I
V V V
S
V
N
Y
S
I
aone hundred eighteen AChE sequences are listed first, followed by 17 BuCfiE sequences. Sequences areroughly clustered as mammals, fish, insects, pests, worms, fungi, and virus. Dashes indicate missing sequence fragments. Sequences were aligned using BioEdit software interface (Hall, 1999).
affinity than AChEs (Radid et al., 1993), whereas the a f f i n ity of fasciculins is up to eight orders of magnitude weaker than in corresponding AChEs (Radid et al., 1994). The absence of the aromatic cluster does not critically influence ACh hydrolysis in BuChEs, and its substitution w i t h . aliphatic residues in AChEs does not seem to affect catalytic parameters; however, substitution of Asp72 has a pronounced effect on substrate Km in both AChEs and BuChEs (Shafferman et al., 1992b; Radid et al., 1993; McGuire et al., 1989; Masson et al., 1997). The BuChE variant containing Asp70Gly substitution (corresponding to the Asp72 position in Torpedo ACHE) is naturally occurring in the human population. Individuals with Asp70Gly mutation appear unable to hydrolyze the muscle relaxant succinyldicholine efficiently and thus experience life-threatening apnea lasting from a few minutes to several hours. Binding of ligands to the various sites in Torpedo and mouse AChEs seems to have a comparatively small effect on the overall fold of the enzyme structure (Figs. 5A and 5B), the exception is the largest peripheral site ligand fasciculin that consistently caused the largest RMS deviations of the Ca fold (Figs. 6A and 6B) due to more than a 1100 ~2 contact area in the complex. The increased RMS was thus the cumulative result of a larger number of small, sub-,~ngstrom shifts associated with residues of the large f~ loop (Cys67-Cys94). The loop was slightly, but noticably (Figs. 5A and 5B), pressured by fasciculin into the space of the active center gorge. The Phe330 of the active center choline binding site, however, consistently and sig -~ 1/
nificantly changes its side chain conformation in all structures recorded to date, both in Torpedo and mouse AChEs (in which it is replaced by Tyr). Its unique position at the base of the gorge allows it to regulate available space for ligand binding by rotating around its Ce~-C[3 bond (Figs. 5C and 5D). Tacrine and its derivatives in Torpedo, mouse, and Drosophila structures are stabilized by an aromatic "sandwich" between Trp84, which is always fixed in the same conformation at the very bottom of the gorge, and Phe330 (or its Tyr equivalents in mouse and Drosophila). The latter residue assumes conformation roughly parallel to Trp84, thereby closing access to the lower part of the gorge. When bisquaternary ligands, decamethonium and BW286c51 are bound, the position of the Phe330 ring is nearly perpendicular to Trp84. thus opening the full length of the gorge. In the immediate vicinity of Phe330 lies catalytic triad His440. It has been implicated that this histidine 'might be "mobile" during catalytic reaction m that is, that it can repetitively revert between two or more conformational states during catalysis. The structural evidence of one of these hypothetical states is given in the threedimensional structure of the "aged" VX phosphonylated T. californica AChE with the nerve agent VX (Millard et al., 1999) (Fig. 7A). The analogous crystal structure solved with VX phosphonylated and aged human BuChE, however, did not reveal any movements in the His of the catalytic triad (Nachon et al., 2005). The largest conformational change in the peripheral 'site was observed in the complex of the tightest binding triazole, the femtomoiar
180
S E C T I O N I I I . Esterases, Receptors, M e c h a n i s m s ,
& Tolerance D e v e l o p m e n t
FIG. 5. Variations in conformations of "ligand-bound" and free AChEs found in 40 Torpedo californica structures and 18 mouse AChE molecules extracted from nine structures. Ligands found in overlaid structures are presented as dark sticks. Slight movement of the 1~ loop covering bound ligands was found in complexes of both AChEs with fasciculin 2. (A) Torpedo californica overlay. (B) Mouse AChE overlay. Side chain conformers of (C) Phe330 in TorpedoAChE and (D) Tyr337 in mouse AChE found in overlaid structures. Visualized by WebLabViewer software (Accelrys, San Diego).
FIG. 6. Quantitative representation of variations in Ca traces represented as RMS values of (A) 42 Torpedo AChEs using the unliganded lEA5 structure as a frame of reference and (B) 18 mouse AChEs using the 1J06 chain a as a frame of reference. Structures were overlaid and RMS deviations calculated using SwissPDBViewer software. Bar patterns represent the type of ligand found in each structure.
CHAPTER 1 2 9Structure and Function of Cholinesterases
FIG. 7. Conformational changes of AChEs found in (A) Torpedo AChE-DFP aged adduct. DFP is covalently attached to the active Ser200. The gray ribbon and selected side chain sticks represent unliganded Torpedo AChE structure. Aged DFP-AChE adduct is shown as black ribbon and sticks. (B) Mouse AChE conformations of Trp286 found in the peripheral sites of triazole syn-TZ2PA6 liganded (two conformations) and unliganded or liganded with other ligands (16 conformations). Visualized by WebLabViewer software (Accelrys, San Diego).
inhibitor syn-TZ2PA6 and mouse AChE (Bourne et al., 2004). Trp286 (Trp279 in Torpedo) flips in that structure by approximately 120 ~ (Fig. 7B) to enable a tight fit of the phenantridinium moiety of syn-TZ2PA6 in another "aromatic" sandwich between Trp286 and Tyr72 (Tyr70 in Torpedo). A substantial change in the backbone conformation has been reported in only two AChE structures, in both cases on the same acyl pocket loop (Trp279-Ser291). In the structure of Torpedo AChE phosphorylated by diisopropyl fluorophosphate (DFP) (Millard et al., 1999), and then aged, DFP is partially dealkylated, having only one of two isopropoxy groups bound to phosphorus. To fit between two phenylalanines of the AChE acyl pocket (Phe288 :and Phe290), the remaining isopropoxy group had to distort its backbone, causing the extended cationic side chain of Arg289 to flip by almost 90 ~ (Fig. 7A), facing the entrance of the gorge and incoming ligands. An even larger acyl pocket loop conformational change was reported for the complex between Torpedo AChE and a bifunctional galanthamine derivative (Greenblatt et al., 2004). Positioning of the ligand in this complex caused the acyl pocket loop (Trp279-Ser291) to assume a severely disordered and poorly defined conformation that was not included in the crystal structure coordinates.
C. Quaternary Structure Genetic information encoding AChE protein as documented for variety of species is contained in one gene, except for some insects and w o r m s , carying multiple genes for AChEs or BuChEs. The myriad of molecular forms includes covalent homodimers, either soluble or through a glycophospholipidinositol anchor attached covalently to mostly erythrocyte membranes; homotetramers formed as noncovalent dimers of covalent
181
dimers; and "readthrough" monomers expressed only during development and in mouse brain under stress as well as heteromeric associations of tetramers with structural peptides ColQ and PRIMA found in mammalian muscle and brain, respectively (Massoulie et al., 1993; Taylor and Radi6, 1994; Massoulie, 2002). All molecular forms contain identical catalytic subunits. Slightly different are C-terminal sequences that dictate oligomerization and cellular disposition of subunits. Sequence divergence of C-terminal end is achieved through 3' alternative splicing of mRNA (Gibney et al., 1988). Additional alternative splicing at the mRNAs 5' end was reported in tick AChEs (Baxter and Barker, 1998; Xu et al., 2003) but without affecting the sequence of the mature protein. In the vertebrate nervous system, AChE is found in tetrameric form associated through C-terminal peptides forming a four-helix amphipathic bundle around ColQ or PRIMA peptides. The regularly spaced Trp residues at the C-terminal (WAT domain) intercalate between Pro residues of PRAD (proline-rich amphipathic domain) and ColQ or PRIMA as described in a structural study on the WAT/PRAD tetramerization domain (Dvir et al., 2004) showing four parallel helices of WAT supercoiled around the antiparallel helix of PRAD. The naturally occurring monomeric form of fetal bovine serum ACHE, unlike the naturally occurring tetrameric form of fetal bovine serum, has a modified C-terminal end sequence, thus compromising its formation of tetramers (Saxena et al., 2003).
III. ChE VARIANTS IN THE H U M A N POPULATION Numerous structural variants of BuChE in humans consisting of one amino acid substitutions or frameshift mutations have been well documented in the literature (Kalow, 2004). Individuals carrying structural variants of very low or no BuChE activity in their tissues appear normal and are at risk only if given the muscle relaxant succinylcholine in preparation for surgery. On the other hand, variability of AChE structure in the human population is only starting to be discovered. To date, only approximately 13 single nucleotide polymorphisms (SNPs) have been identified in the AChE encoding DNA in the human population (Hasin et al., 2004). Eight are synonymous and they do not alter the protein sequence, and five cause amino acid substitutions in mature AChE protein. Substitutions are positioned on the AChE surface (Fig. 8) and are not likely to affect its catalytic function, but the ability of monomers to associate with other molecules may be influenced. In particular, SNP resulting in substitution located close to the C-terminal end (Pro561Arg in the mature human AChE sequence) could influence tetramerization of AChE and its anchoring in nervous tissue.
182
SECTION III- E s t e r a s e s , R e c e p t o r s , M e c h a n i s m s ,
& Tolerance Development
strate activation, respectively. A unique and simple reaction scheme can describe both kinds of interactions: kcat E +
+
S ~
S
FIG. 8. Variants of AChE structure, resulting in amino acid substitutions of the mature protein, found in the human population (data from Hasin et al., 2004). Sites and types of substitutions are labeled on the gray ribbon, with a molecule of ACh shown as a gray surface, docked in the active center. The fifth identified substitution, Pro561Arg located at the very C-terminal end, is not shown because the known three-dimensional structure extends only through residue 542. Visualized by WebLabViewer software (Accelrys, San Diego).
A. Reaction of Cholinesterases with Substrates The kinetics of substrate hydrolysis by both AChEs and BuChEs deviates from Michaelis-Menten kinetics. As implicated in Fig. 2 for acetylthiocholine, only at substrate concentrations lower than 1 mM does the rise in enzyme activity follow Michaelis-Menten kinetics. At higher concentrations, AChE activity decreases, inhibited by the excess substrate, whereas BuChE activity increases, activated by the excess substrate. Hence, the terms substrate inhibition and substrate activation are respective hallmarks of catalysis by AChE and BuChE. Both phenomena can be simply described as a consequence of the formation of a ternary complex between the enzyme and two substrate molecules and thus as an allosteric phenomenon. The ternary complex in AChE has reduced or no activity compared to the Michaelis-Menten complex, whereas it appears more active in BuChE hydrolysis. It is important to emphasize that this is a substrate-specific phenomenon. Not all AChE and BuChE substrates exhibit substrate inhibition and sub-
ES +
>E+P
S
ssl SE
~
ssll +
S ~
~ S E S ~ S E +
P
where E and S stand for the free enzyme and substrate molecules respectively; ES is the Michaelis-Menten complex; SE is substrate bound to an allosteric, peripheral site on the enzyme; and SES is the ternary complex having one substrate molecule bound to the active center and the other to the allosteric, peripheral site. Kss is the dissociation constant for substrate bound to an allosteric, peripheral site, and kca t and bkca t a r e turnover numbers for the Michaelis-Menten complex and the ternary complex, respectively. Kss constants for ChEs are always larger than Km. Thus, the previous reaction scheme differs from the simple Michaelis-Menten scheme in the assumption that S can bind to more than one site on the enzyme, influencing the enzyme turnover by the factor b. When b = 1, the reaction kinetics is indistinguishable from Michaelis-Menten kinetics and binding of S to the allosteric, peripheral site appears kinetically "silent." When b < 1, the enzyme is inhibited by excess substrate as most frequently found for hydrolysis of ACh or acetylthiocholine by AChEs. The relationship between enzyme activity and the log of the substrate concentration (Fig. 2) is bell-shaped, indicating substrate inhibition. When b > 1, the enzyme activity is increased by excess substrate, as found for hydrolysis of ACh or acetylthiocholine by BuChEs, and the relationship between enzyme activity and the log of the substrate concentration appears as a double sigmoidal substrate activation curve (Fig. 2). The kinetic equation derived from the previous scheme summarizes these relationships as follows (CE Radi6 et al., 1993): v = v ( 1 + b[S]/Ks~)/[(1 +
[S]/K,~)(1 + Km/[S])]
(1)
where v is the enzyme activity at concentration of substrate IS], V is maximal activity of the enzyme, Km is the Michaelis-Menten constant, and Kss is the dissociation constant for substrate bound to the allosteric, peripheral site of the enzyme. Although the previous relationships hold for most ChEs, there are exceptions. Drosophila ACHE, for example, exhibits slightly more complex catalytic behavior in which some substrate activation is observed at low substrate concentrations in addition to substrate inhibition observed at millimolar substrate concentrations (Fig. 2) in the same reaction profile. In order to describe the reaction mechanism more precisely in that case, a more complex reaction
CHAPTER 1 2 9Structure and Function of Cholinesterases mechanism has to be assumed (Stojan et al., 1998, 2004). The molecular basis for this complexity is likely related to the fact that the geometry of the active center gorge in Drosophila AChE differs significantly from the gorge geometries of other AChEs and BuChEs.
B. Reaction of Cholinesterases with Inhibitors The vast majority of molecules that inhibit ChE activity are either reversible inhibitors or "progressive," "irreversible" covalent inhibitors.
1. REVERSIBLE INHIBITORS Reversible inhibitors form noncovalent complexes with the enzyme at the bottom of the active center gorge, at the peripheral site at its rim, or they span between the two sites. Association rates of most reversible inhibitors with ChEs are diffusion limited, and traversing the path leading to the base of the enzyme active center gorge does not slow down their entry. The binding equilibrium for those complexes is established rapidly, and only the magnitude of their dissociation rates controls their binding affinity, with dissociation constants usually found in the micromolar to nanomolar range (Radi6 and Taylor, 2001). A separate group of reversible ChE inhibitors m fasciculins, triazoles, and trifluoroacetophenonesmdissociate from enzyme very slowly (requiting hours, days, or weeks for complete dissociation) while maintaining very fast, diffusion-limited association rates. Affinities of these fight binding inhibitors are exceptionally high, with KD constants in the low pM and fM range. Fasciculins are a family of peptidic toxins from snake venom (Fasciculinl, Fasciculin2, and Fasciculin3) (Marchot et al., 1993), 61 amino acids long with a characteristic three-finger shape. The central finger enters halfway into the AChE active center gorge, blocking access to the active serine, whereas the bulk of the toxin molecule resides at the enzyme peripheral site (Bourne et al., 1995; Harel et al., 1995). The mechanism of inhibition combines steric blockade with allosteric components (Rosenberry et al., 1999; Radic and Taylor, 2001). Triazoles are a family of small molecule inhibitors designed by the "in situ" cycloaddition reaction between azide and acetylene building blocks in the AChE active center gorge (Lewis et al., 2002; Krasinski et al., 2005; Manetsch et al., 2004). The enzyme is exposed to a library of building blocks, and the tightest binding products pedominate in the gorge. This exceptional strategy for the design of high-affinity ligands yielded multiple inhibitors with femtomolar KD in a single round of screening from a small library of building blocks having high nanomolar to micromolar KDS. The triazole inhibitors are bifunctional, spanning the peripheral site and the active center (Bourne et al., 2004), but the triazole ring, formed in the cycloaddition reaction, contributes to binding affinity as a separate pharmacophore as well (Manetsch et al., 2004).
183
In contrast to the previously discussed noncovalent but slowly dissociating inhibitors, trifluoroacetophenones are reversible ChE inhibitors that covalently bind to the active serine but maintain the same structure upon dissociation from the enzyme (Nair et al., 1994). Their structure resembles "extended" reactive conformation of ACh, mimicking the transition state in its reaction with ChEs. Trifluoroacetophenones share three points of stabilization with the enzyme. In addition to the formation of a covalent bond between the active serine and carbonyl carbon of a trifluoroacetophenone, its carbonyl oxygen is stabilized in the oxyanion hole and the positive charge of quaternary nitrogen is stabilized through cation-xr interactions by aromatic residues of the choline binding site (Harel et al., 1996).
2. PROGRESSIVE, IRREVERSIBLE ChE INHIBITORS These inhibitors are substrates of ChEs that deacylate extremely slowly from the active serine, thus causing prolonged occupation of the enzyme active center and its inability to hydrolyze acetylcholine (Aldridge and Reiner, 1972). OPs and CMs are common irreversible inhibitors of ChEs and are frequently used as pesticides (Taylor, 2005). In some pesticides, such as malathion and parathion, specificity of inhibition of insect AChE is achieved by in vivo oxidation of the pesticide by cytochrome P450 forming the active oxon (malaoxon and paraoxon). Some OPs are also used as nerve warfare agents. Similar to pesticides, nerve agents are usually small neutral esters of organophosphoric or organophosphonic acid that are prone to be volatile and efficiently penetrate tissues. Upon initial phosphorylation or phosphonylation of the active serine, many OPs undergo an additional reaction called aging (Barak et al., 2000). Aging is a loss of an additional alkyl substituent group of an OP-inhibited enzyme that renders inhibitory OP moiety negatively charged (Aldridge and Reiner, 1972): AChE
0II + RI'O'P-O'Rz R3
0
aging
II > AChE-O-P-O-R 2 I OR 1 + R3
>
0
II -AChE-O-P-O +R z I OR1
hydrolysis ~ H 2 0
AChE +
O II HO-P-O-R 2 OR 1
Although catalytic activity of phosphorylated or phosphonylated enzymes can be restored by spontaneous deacylation in water or by using nucleophiles stronger than water, such as oximes, means of reactivating aged enzyme have not been demonstrated. Aging of nerve agents such as soman is very fast and occurs in minutes, whereas aging of pesticides such as paraoxon or ethyldichlorvos can take days to complete (Nachon et al., 2005; Aldridge and Reiner, 1972). Intensive and repeated application of pesticides can lead to the development of pesticide resistance in some insects, as documented for mosquitoes and flies (Hemingway et al.,
184
SECTION I I I - Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
References
FIG. 9. Locations of residue substitutions found in insecticideresistant insect strains mapped onto Drosophila AChE structure. Eleven side chain positions (compiled by Menozzi et al., 2004) are given as black surface contours. A molecule of ACh docked in the active center is shown as a gray surface. Visualized by WebLabViewer software (Accelrys, San Diego).
2004; Menozzi et al., 2004). One of the mechanisms involved in this process is the alteration of the insect AChE structure in such a way so as to preserve its ability to hydrolyze ACh but minimize its ability to react with OPs (Fig. 9). This is possible since planar geometry around the carbonyl carbon of ACh and tetrahedral geometry around phosphorus are sufficiently different, as are the overall sizes of pesticide and ACh molecules.
IV. C O N C L U S I O N S This chapter summarizes the latest structural information on the cholinesterase family of enzymes and discusses their catalytic properties. Alignment of 125 full-length or nearly full-length ChE sequences demonstrates a high degree of similarity conserved in the family from fungal and viral enzymes to man. Sixty-one reported three-dimensional structures are analyzed herein, illustrating the high degree of similarity in protein fold conformation irrespective of the presence of bound ligands, and this argues for the absence of large conformational changes in the protein interaction with both inhibitors and substrates. At the same time, a plentitude of small-amplitude backbone and selected side chain movements illustrate that rapid fluctuations in conformational states of ChEs prevail in solution and ligands have the capacity to select those conferring higher affinity and lower energy of the respective complex.
Adler, M., Manley, H. A., Purcell, A. L., Deshpande, S. S., Hamilton, T. A., Kan, R. K., Oyler, G., Lockridge, O., Duysen, E. G., and Sheridan, R. E. (2004). Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice. Muscle Nerve 30, 317-327. Aldridge, W. N., and Reiner, E. (1969). Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: One the formation of phosphorylated enzyme and the other analogous to inhibition by substrate. Biochem. J. 115, 147-162. Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. North Holland, Amsterdam. Barak, D., Ordentlich, A., Kaplan, D., Barak, R., Mizrahi, D., Kronman, C., Segall, Y., Velan, B., and Shafferman, A. (2000). Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase. Biochemistry 39, 1156-1161. Baxter, G. D., and Barker, S. C. (1998). Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: Characterization and role in organophosphate resistance. Insect. Biochem. Mol. Biol. 28, 581-589. Behra, M., Cousin, X., Bertrand, C., Vonesch, J. L., B iellmann, D., Chatonnet, A., and Strahle, U. (2002). Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat. Neurosci. 5, 111-118. Bourne, Y., Taylor, E, and Marchot, E (1995). Acetylcholinesterase inhibition by fasciculin: Crystal structure of the complex. Cell 83, 503-512. Bourne, Y., Kolb, H. C., Radi6, Z., Sharpless, K. B., Taylor, E, and Marchot, E (2004). Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation. Proc. Natl. Acad. Sci. USA 101, 1449-1454. Bticht, G., Haggstrom, B., Radi6, Z., Osterman, A., and Hjalmarsson, K. (1994). Residues important for folding and dimerisation of recombinant Torpedo californica acetylcholinesterase. Biochim. Biophys. Acta 1209, 265-273. Changeux, J. E (1966). Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol. Pharmacol. 2, 369-392. Chothia, C., and Leuzinger, W. (1975). Acetylcholinesterase: The structure of crystals of a globular form from electric eel. J. Mol. Biol. 97, 55-60. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Doctor, B. E (1993). Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci. 2, 366-382. Dale, H. H. (1914). The action of certain esters of choline and their relation to muscarine. J. Pharmacol. Exp. Ther. 6, 147-190. Dvir, H., Harel, M., Bon, S., Liu, W. Q., Vidal, M., Garbay, C., Sussman, J. L., Massoulie, J., and Silman, I. (2004). The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix. EMBO J. 23, 4394-4405. Fletcher, S. E, Geyer, B. C., Smith, A., Evron, T., Joshi, L., Soreq, H., and Mor, T. S. (2004). Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants. Plant. Mol. Biol. 55, 33-43.
CHAPTER 1 2 9Structure and Function of Cholinesterases Galagan, J. E., Calvo, S. E., Borkovich, K. A., et al. (2003). The genome sequence of the filamentous fungus Neurospora crassa. Nature 422, 821-822. Gibney, G., MacPhee-Quigley, K., Thompson, B., Vedvick, T., Low, M. G., Taylor, S. S., and Taylor, E (1988). Divergence in primary structure between the molecular forms of acetylcholinesterase. J. Biol. Chem. 263, 1140-1145. Gibney, G., Camp, S., Dionne, M., MacPhee-Quigley, K., and Taylor, R (1990). Mutagenesis of essential functional residues in acetylcholinesterase. Proc. Natl. Acad. Sci. USA 87, 7546-7550. Greenblatt, H. M., Guillou, C., Guenard, D., Argaman, A., Botti, S., Badet, B., Thal, C., Silman, I., and Sussman, J. L. (2004). The complex of a bivalent derivative of galanthamine with torpedo acetylcholinesterase displays drastic deformation of the activesite gorge: Implications for structure-based drug design. J. Am. Chem. Soc. 126, 15405-15411. Greenspan, R. J., Finn, J. A., Jr., and Hall, J. C. (1980). Acetylcholinesterase mutants in Drosophila and their effects on the structure and function of the central nervous system. J. Comp. Neurol. 189, 741-774. Gupta, A., and Gupta, R. (1997). A survey of plants for presence of cholinesterase activity. Phytochemistry 46, 827-831. Gupta, A., Vijayaraghavan, M. R., and Gupta, R. (1998). The presence of cholinesterase in marine algae. Phytochemistry 49, 1875-1877. Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95-98. Harel, M., Kleywegt, G. J., Ravelli, R. B., Silman, I., and Sussman, J. L. (1995). Crystal structure of an acetylcholinesterase-fasciculin complex: Interaction of a three-fingered toxin from snake venom with its target. Structure 3, 1355-1366. Harel, M., Quinn, D. M., Nair, H. K., Silman, I., and Sussman, J. L. (1996). The X-ray structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase. J. Am. Chem. Soc. 118, 2340-2346. Harel, M., Kryger, G., Rosenberry, T. L., Mallender, W. D., Lewis, T., Fletcher, R. J., Guss, J. M., Silman, I., and Sussman, J. L. (2000). Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors. Protein Sci. 9, 1063-1072. Hasin, Y., Avidan, N., Bercovich, D., Korczyn, A., Silman, I., Beckmann, J. S., and Sussman, J. L. (2004). A paradigm for single nucleotide polymorphism analysis: The case of the acetylcholinesterase gene. Hum. Mutat. 24, 408-416. Hemingway, J., Hawkes, N. J., McCarroll, L., and Ranson, H. (2004). The molecular basis of insecticide resistance in mosquitoes. Insect. Biochem. Mol. Biol. 34, 653-665. Kalow, W. (2004). Human pharmacogenomics: The development of a science. Hum. Genom. 1, 375-380. Kaplan, D., Ordentlich, A., Barak, D., Ariel, N., Kronman, C., Velan, B., and Shafferman, A. (2001). Does "butyrylization" of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase? Biochemistry 40, 7433-7445. Krasinski, A., Radi6, Z., Manetsch, R., Raushel, J., Taylor, E, Sharpless, K. B., and Kolb, H. C. (2005). In situ selection of
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lead compounds by click chemistry: Target-guided optimization of acetylcholinesterase inhibitors. J. Am. Chem. Soc., 127, 6686-6692. Kryger, G., Harel, M., Giles, K., Toker, L., Velan, B., Lazar, A., Kronman, C., Barak, D., Ariel, N., Shafferman, A., Silman, I., and Sussman, J. L. (2000). Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II. Acta Crystallogr. D Biol. Crystallogr. 56, 1385-1394. Lewis, W. G., Green, L. G., Grynszpan, E, Radi6, Z., Carlier, E R., Taylor, E, Finn, M. G., and Sharpless, K. B. (2002). Click chemistry in situ: Acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem. Int. Ed. Engl. 41, 1053-1057. Liang, J., Edelsbrunner, H., and Woodward, C. (1998). Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design. Protein Sci. 7, 1884-1897. Lockridge, O., Bartels, C. E, Vaughan, T. A., Wong, C. K., Norton, S. E., and Johnson, L. L. (1987). Complete amino acid sequence of human serum cholinesterase. J. Biol. Chem. 262, 549-257. MacPhee-Quigley, K., Vedvick, T. S., Taylor, R, and Taylor, S. S. (1986). Profile of the disulfide bonds in acetylcholinesterase. J. Biol. Chem. 261, 13565-13570. Manetsch, R., Krasinski, A., Radi6, Z., Raushel, J., Taylor, E, Sharpless, K. B., and Kolb, H. C. (2004). In situ click chemistry: Enzyme inhibitors made to their own specifications. J. Am. Chem. Soc. 126, 12809-12818. Marchot, E, Khelif, A., Ji, Y. H., Mansuelle, R, and Bougis, R E. (1993). Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate. J. Biol. Chem. 268, 12458-12467. Masson, E, Legrand, E, Bartels, C. F., Froment, M. T., Schopfer, L. M., and Lockridge, O. (1997). Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase. Biochemistry 36, 2266-2277. Massoulie, J. (2002). The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11, 130-143. Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., and Vallette, E M. (1993). Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31-91. McGuire, M. C., Nogueira, C. E, Bagels, C. E, Lightstone, H., Hajra, A., Van der Spek, A. E, Lockridge, O., and La Du, B. N. (1989). Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase. Proc. Natl. Acad. Sci. USA 86, 953-957. Menozzi, R, Shi, M. A., Lougarre, A., Tang, Z. H., and Fournier, D. (2004). Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations. BMC Evol. Biol. 4, 4.
Millard, C. B., Kryger, G., Ordentlich, A., Greenblatt, H. M., Harel, M., Raves, M. L., Segall, Y., Barak, D., Shafferman, A., Silman, I., and Sussman, J. L. (1999). Crystal structures of aged phosphonylated acetylcholinesterase: Nerve agent reaction products at the atomic level. Biochemistry 38, 7032-7039. Nachon, E, Nicolet, Y., Viguie, N., Masson, R, Fontecilla-Camps, J. C., and Lockridge, O. (2002). Engineering of a monomeric
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SECTION I I I . Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
and low-glycosylated form of human butyrylcholinesterase: Expression, purification, characterization and crystallization. Eur. J. Biochem. 269, 630-637. Nachon, E, Asojo, O. A., Borgstahl, G. E., Masson, E, and Lockridge, O. (2005). Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: The crystal structure suggests two alternative mechanisms of aging. Biochemistry 44, 1154-1162. Nair, H. K., Seravalli, J., Arbuckle, T., and Quinn, D. M. (1994). Molecular recognition in acetylcholinesterase catalysis: Freeenergy correlations for substrate turnover and inhibition by trifluoro ketone transition-state analogs. Biochemistry 33, 8566-8576. Nicolet, Y., Lockridge, O., Masson, E, Fontecilla-Camps, J. C., and Nachon, E (2003). Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products. J. Biol. Chem. 278, 41141-41147. Nolte, H. J., Rosenberry, T. L., and Neumann, E. (1980). Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands. Biochemistry 19, 3705-3711. Ordentlich, A., Barak, D., Kronman, C., Flashner, Y., Leitner, M., Segall, Y., Ariel, N., Cohen, S., Velan, B., and Shafferman, A. (1993). Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket. J. Biol. Chem. 268, 17083-17095. Radi6, Z., and Taylor, E (2001). Interaction kinetics of reversible inhibitors and substrates with acetylcholinesterase and its fasciculin 2 complex. J. Biol. Chem. 276, 4622-4633. Radi6, Z., Gibney, G., Kawamoto, S., MacPhee-Quigley, K.,. Bongiorno, C., and Taylor, E (1992). Expression of recombinant acetylcholinesterase in a baculovirus system: Kinetic properties of glutamate 199 mutants. Biochemistry 31, 9760-9767. Radi6, Z., Picketing, N. A., Vellom, D. C., Camp, S., and Taylor, P. (1993). Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 12074-12084. Radi6, Z., Duran, R., Vellom, D. C., Li, Y., Cervenansky, C., and Taylor, E (1994). Site of fasciculin interaction with acetylcholinesterase. J. Biol. Chem. 269, 11233-11239. Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, E, Ogata, H., La Scola, B., Suzan, M., and Claverie, J. M. (2004). The 1.2-megabase genome sequence of Mimivirus. Science 306, 1344-1350. Raves, M. L., Harel, M., Pang, Y. E, Silman, I., Kozikowski, A. E, and Sussman, J. L. (1997). Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A. Nat. Struct. Biol. 4, 57-63. Rosenberry, T. L., Mallender, W. D., Thomas, R J., and Szegletes, T. (1999). A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate. Chem.-Biol. Interact. 119-120, 85-97. Saxena, A., Redman, A. M. G., Jiang, X., Lockridge, O., and Doctor, B. E (1999). Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase. Chem.-Biol. Interact. 119-120, 61-69. Saxena, A., Hur, R. S., Luo, C., and Doctor, B. E (2003). Natural monomeric form of fetal bovine serum acetylcholinesterase
lacks the C-terminal tetramerization domain. Biochemistry 42, 15292-15299. Schrag, J. D., Schmid, M. E, Morgan, D. G., Phillips, G. N., Jr., Chiu, W., and Tang, L. (1988). Crystallization and preliminary X-ray diffraction analysis of 11 S acetylcholinesterase. J. Biol. Chem. 263, 9795-9800. Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPheeQuigley, K., Taylor, S. S., Friedmann, T., and Taylor, E (1986). Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence. Nature 319, 407-409. Shafferman, A., Kronman, C., Flashner, Y., Leitner, M., Grosfeld, H., Ordentlich, A., Gozes, Y., Cohen, S., Ariel, N., Barak, D., Harel, M., Silman, I., Sussman, J., and Velan, B. (1992a). Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J. Biol. Chem. 267, 17640-17648. Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992b). Substrate inhibition of acetylcholinesterase: Residues affecting signal transduction from the surface to the catalytic center. EMBO J. 11, 3561-3568. Stojan, J., Marcel, V., Estrada-Mondaca, S., Klaebe, A., Masson, E, and Fournier, D. (1998). A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase. FEBS Lett. 440, 85-88. Stojan, J., Golicnik, M., and Fournier, D. (2004). Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism. Biochim. Biophys. Acta 1703, 53-61. Stok, J. E., Goloshchapov, A., Song, C., Wheelock, C. E., Derbel, M. B., Morisseau, C., and Hammock, B. D. (2004). Investigation of the role of a second conserved serine in carboxylesterases via site-directed mutagenesis. Arch. Biochem. Biophys. 430, 247-255. Sussman, J. L., Harel, M., Frolow, E, Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991). Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholine-binding protein. Science. 253, 872-879. Taylor, E (2005). Anticholinesterase agents. In The Pharmacological Basis of Therapeutics (J. G. Hardman and L. E. Limbird, Eds.), 10th ed., pp. 161-176. McGraw-Hill, New York. Taylor, E, and Lappi, S. (1975). Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. Biochemistry 14, 1989-1997. Taylor, E, and Radi6, Z. (1994). The cholinesterases: From genes to proteins. Annu. Rev. Pharmacol. Toxicol. 34, 281-320. Taylor, E, Jones, J. W., and Jacobs, N. M. (1974). Acetylcholinesterase from Torpedo: Characterization of an enzyme species isolated by lytic procedures. Mol. Pharmacol. 10, 78-92. Vellom, D. C., Radi6, Z., Li, Y., Picketing, N. A., Camp, S., and Taylor, E (1993). Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity. Biochemistry 32, 12-17. Xu, G., Fang, Q. Q., Keirans, J. E., and Durden, L. A. (2003). Cloning and sequencing of putative acetylcholinesterase cDNAs from the American dog tick, Dermacentor variabilis, and the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae). J. Med. Entomol. 40, 890-896.
CHAPTER |
Cholinesterase Pharmacogenetics ROBERTA GOODALL North Bristol NHS Trust, Bristol, United Kingdom
Until recently, pharmacogenetic studies focused on BuChE rather than AChE because the latter, although considered more important physiologically, has not been regarded as a DME. AChE has also been assumed to be a much less polymorphic enzyme than BuChE, with any variants likely to have severe clinical consequences. However, recent research has shown that AChE may be more polymorphic than previously thought (Hasin et al., 2004), and it may also have pharmacogenetic relevance, particularly with respect to the anticholinesterase drugs used to treat Alzheimer's disease and dementia. The genetic variation that causes the differing pharmacological effects of cholinesterase drug targets may also have resonance in causing differences in how individuals respond to exposure to anticholinesterases, including organophosphate (OP) and carbamate (CM) pesticides. This chapter focuses on the pharmacogenetics of BuChE and the clinical aspects thereof but also considers the possible pharmacogenetic implications of AChE variants.
I. I N T R O D U C T I O N Pharmacogenetics is a field of medicine concerned with inherited variations in drug metabolizing enzymes (DMEs) that result in an altered ability to metabolize their target drugs. In a clinical setting this translates to the identification of the causes and prevention of adverse drug reactions (ADRs) responsible for millions of dollars in potentially avoidable medical costs worldwide. The term, originally proposed by Vogel in 1959, was brought into common usage by Werner Kalow in the early 1960s (Kalow, 1962a) with respect to the plasma form of cholinesterase, butyrylcholinesterase (BuChE; EC 3.1.1.8). BuChE is a DME responsible for hydrolysis of the muscle relaxant drugs suxamethonium and mivacurium as well as certain drugs of abuse, such as cocaine and heroin. Its physiological function is unknown. Deficiencies in the enzyme arise from genetic polymorphism as well as from secondary and physiological causes. Deficiency from any cause can lead to prolonged apnea and paralysis following administration of suxamethonium and mivacurium. A highly polymorphic enzyme, it provided an early model for pharmacogenetic studies, with biochemical phenotyping being used to identify individuals and families at risk of prolonged paralysis. Other such inherited deficiencies in DMEs were observed at approximately the same time, for example, with respect to N-acetyltransferase ("acetylator") status and the antituberculosis drug isoniazid (Evans, 1989), but Kalow's was the first applied use of the term that is now in common use in the biomedical community. The two cholinesterase enzymes, acetyl (ACHE) and butyryl (BuChE), although closely related, show differences both in their occurrence in the body (leading to their older vernacular names of erythrocyte, red cell, or true cholinesterase in the case of AChE and plasma or pseudocholinesterase for BuChE) and in their substrate specificity. AChE is more correctly called acetylcholine acetylhydrolase (EC 3.1.1.7), and BuChE is more correctly called acylcholine acylhydrolase (EC 3.1.18). AChE is present in most vertebrates in several molecular forms, whereas BuChE is present in only c0ae, the tetrameric "T" form (Massouli6, 2002). Toxicology of Organophosphate and Carbamate Compounds
II. B U T Y R Y L C H O L I N E S T E R A S E A. Clinical Aspects Clinical and laboratory interest in BuChE has a different emphasis in different countries. Its considered value in the clinical arena varies from its use as a marker of exposure to OP pesticides to its utility as a marker of liver "function" (with decreased activity indicating impaired hepatic synthesis) and its pharmacological actions and role as a DME. Although both AChE and BuChE are inhibited by anticholinesterase agents and by OPs, it appears that BuChE activity falls more rapidly following exposure to pesticides than does ACHE, and for this reason BuChE measurements can be used as a marker of occupational exposure (Barnes and Davies, 1951). A DME, BuChE is involved in the metabolism of drugs such as cocaine and heroin (Lockridge et al., 1980), several local anesthetics such as procaine (Lockridge, 1990), and in hydrolyzing the short-acting muscle relaxants suxamethonium (Bourne et al., 1952) and mivacurium (Cook et al., 1989). 187
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Clinically, the main area of concern is with a decrease in enzyme levels, usually manifested as a decrease in the enzyme activity measured in vitro. Such decreases in enzyme activity can arise from a variety of factors, either by causing a reduction in concentration of the enzyme or by directly affecting its action (usually by inhibition). There are occasions when an increase in activity or concentration is observed that although uncommon, will have an effect on the drug metabolizing properties of the enzyme. The following are some of the factors leading to a decrease in activity: Disease 9 Liver disease (Orpollo, 1978; Schmidt and Schmidt, 1993) 9 Renal failure (Phillips and Hunter, 1992) 9 Malnutrition (Umeki, 1993) 9 Certain malignant diseases (Kaniaris et al., 1979) Physiological changes 9 Age; newborns have low levels, which gradually rise to a peak at approximately 6 years of age. Levels then fall, with adult levels reached by puberty (Hutchinson and Widdowson, 1952). 9 Pregnancy; levels fall during the first trimester and the lowest levels occur around, and just after, parturition (Robertson, 1966; Blitt et al., 1977). Iatrogenic 9 Anticholinesterase drugs (e.g., neostigmine) 9 Oral contraceptive pill (Robertson, 1967) Toxic inhibition 9 Pesticides, particularly OPs and CMs 9 Dietary inhibitors, particularly solanaceous glycoalkaloids, found in members of the plant family Solanaceae, such as potatoes, tomatoes, and aubergine (eggplant) (Krasowski et al., 1997; McGehee et al., 2000) Genetic variants of the enzyme, the focus of this chapter Many of the causes of decreased activity have been well reviewed by Whittaker (1989) and Davis et al. (1997). B. B i o c h e m i c a l Genetics Evidence for the genetic variability of BuChE originally came in the 1950s from individuals experiencing unexpected effects following the use of the newly introduced muscle relaxant drug suxamethonium (succinylcholine or "scoline"). Suxamethonium, a dicholine ester of succinic acid, is a depolarizing muscle relaxant with an action at the motor end-plate similar to that of acetylcholine (Ziamis and Head, 1976). Its effect is to cause a short-lived cascarinic or paralyzing effect that allows the performance of certain procedures, such as intubation, required during general anesthesia and surgery. In normal circumstances, the drug's effects last only minutes due to its rapid hydrolysis by the action of BuChE. However,
soon after the drug's introduction into clinical practice, it became clear that in some patients the paralyzing effect lasted much longer, in some cases many hours. This meant that for the duration of the drug's effect, affected individuals were apneic (i.e., they were unable to breathe for themselves) and required artificial ventilation. This prolonged apnea was referred to as "scoline apnea," a term still used today. The consequences of such a reaction were that for some individuals, a minor procedure became a significant event, whereby the sufferer required artificial ventilation in an intensive care unit, and may even have been life threatening if no such facility was available. This phenomenon was found to be frequently associated with reduced BuChE activity (Evans et al., 1952), and investigations began into the nature of this relationship. In vitro tests of the serum of patients demonstrating scoline apnea showed a greatly reduced affinity for succinylcholine in comparison to serum from individuals with a normal response. In fact, in some of the patients tested, the affinity was so low that it failed to hydrolyze any of the injected succinylcholine in vivo (Kalow, 1962b). However, it became clear that not all cases of prolonged apnea caused by low enzyme levels could be attributed to decreased enzyme synthesis arising from liver disease or malnutrition because it occurred in patients in whom neither of these situations applied. The first observations of certain cases of BuChE deficiency being a familial condition seemed to indicate that it was inherited in a recessive manner (Lehman, 1956). However, Kalow (1956) reported studies on the serum of deficient individuals and their families that pointed to a codominant mode of inheritance and he applied the terms "usual" (U) and "atypical" (A) to the two forms of BuChE. By inhibiting the enzyme, in vitro, with known cholinesterase inhibitors, notably the local anesthetic dibucaine, before determining the activity Kalow demonstrated a trimodal distribution of residual activity. That of the usual form of the enzyme was inhibited by 80%, but that from affected individuals was inhibited by only 20%; a third group of individuals showed an intermediate degree of inhibition of approximately 60%. This was evidence that homozygotes for the atypical form had enzyme that was dibucaine resistant, whereas that of usual homozygotes was not. The intermediate level of inhibition represented heterozygotes for the condition. 1. INHIBITION STUDIES AND PHENOTYPES Kalow and Staron (1957) developed a method that could be used for the routine determination of BuChE type with the use of inhibition studies employing precise concentrations of dibucaine. With this first analytical tool, the practice of monitoring patients for genetic variations in DME arrived and the concept of pharmacogenetics was born. Testing of the close relatives of individuals known to have a deficient enzyme is an important aspect of pharmacogenetic studies because it is this knowledge that can prevent future ADRs in those family members. In fact, Lehman suggested such
CHAPTER 1 3 9Cholinesterase Pharmacogenetics testing and the provision of "warning letters" to be shown to anesthetists, in 1956. He also suggested that affected individuals may be at risk should their employment bring them into contact with anticholinesterases. This early history of BuChE investigation is of interest not only because of its seminal role in establishing pharmacogenetic study but also because the methods of inhibition studies to derive a dibucaine number (DN) are still in use today in the routine biochemical characterization of BuChE phenotype. Work using other inhibitors of the enzyme has shown that the situation is rather more complex than was originally discovered and that there are more than two variants of BuChE. For example, fluoride ion at particular concentrations also inhibits the action of usual BuChE and analysis in its presence allows the calculation of the fluoride number (FN). Some patients who had suffered a moderate degree of scoline apnea were found to have an enzyme that resisted inhibition by fluoride, with a lower FN than the normal enzyme, and so the fluoride-resistant variant was discovered. Determination of both DN and FN allowed further classification of an individual's BuChE type (Harris and Whittaker, 1961). 2. GENETIC VARIANTS
a. Qualitative and Quantitative---Decreased Activity Variants Immunological examination of the atypical enzyme showed that it is produced in normal amounts but is functionally i m p a i r e d - it is a qualitative variant (Eckerson et al., 1983). It appeared that the fluoride-resistant enzyme is also a qualitative variant (see Section II.C.4). Using the two types of inhibition numbers as an analytical tool, it was possible to identify people at risk of scoline apnea and search for possible carriers in their families. Such work revealed that there were other variants. In some of the families investigated for BuChE deficiency, anomalies were discovered in the pedigrees in that some women, defined as being homozygote for the atypical allele on the basis of their DNs, had children with a usual, as opposed to a UA, phenotype (Kalow and Staron, 1957; Harris et al., 1960). Parentage was not in dispute in either report, and so it was proposed that another variant must exist that had no cholinesterase activity and so exerted no effect on the DN - - a "silent" (S) variant. Further evidence for the existence of such a variant came from reports of apparently healthy individuals who had no measurable BuChE activity, homozygotes for the S variant (Hart and Mitchell, 1962; Liddell et al., 1962) who demonstrated severe sensitivity to scoline. Because the first descriptions of the S variants were of individuals with apparently no enzyme activity, S variants were thought to be quantitative variants. However, since those early days of investigation into BuChE, it has been demonstrated that the S variant phenotype is a heterogeneous one, with some individuals lacking functional enzyme altogether, whereas that produced by other people
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with this phenotype lacks the appropriate structure to hydrolyze the choline ester bond. This has been shown to be due to the fact that the silent phenotype is produced by more than one polymorphism (see Section II.C.2). Thus, some silent phenotypes are now known to be due to qualitative variants of the enzyme (Primo-Parmo et al., 1996). The use of the term silent in this context leads to a rather unfortunate terminological problem. The early work on discriminating and characterizing BuChE variants was done based on clinical, biochemical, and immunological studies and before the molecular nature of the gene was determined and accepted terminology for such molecular variation developed. The expression "silent variant" was coined to describe those enzyme variants that showed little or no enzyme activity; however, in molecular biology the term is more commonly used for a mutation or polymorphism that has no effect on either the gene product's function or its amino acid sequence. In the case of BuChE, the word silent refers to a biochemical phenotype, and such a variant is certainly not associated with a silent mutation in the gene. For example, when Whittaker et al. (1990) published their immunological studies on the heterogeneity of the silent variant they referred to "the silent gene," whereas now it is more likely to be referred to as a silent "variant" or "allele." It is important that the terms are appropriately used, and the temptation to refer to a mutation or polymorphism leading to a silent variant/phenotype as a silent mutation is avoided, especially when reporting results. Three more quantitative variants that cause a decrease in activity (i.e., functionally normal enzyme that is produced in decreased amounts) have also been described that show varying degrees of reduced activity. The first was described by Garry et al. (1976), who named it the James or J variant, and it is associated with a 66% reduction in enzyme activity. The Kalow or K variant, named in honor of Werner Kalow, is associated with an approximately 30% reduction in activity (Rubenstein et al., 1978). Of the named quantitative variants determined initially by phenotypic studies, the most recent addition is that described by Whittaker and Britten (1987). This was named the Hammersmith, or H variant because it was first identified in two cases presenting at the Hammersmith Hospital in London. The reduced activity in these variants from reduced concentration of the enzyme was thought to be due to changes in the production, stability, or half-life of the enzyme. The quantitative variants demonstrate usual inhibition numbers in vitro and can only be identified using a third inhibitor, usually the carbamate Ro 02-0683 that gives the RO number (RN), and then only when they are present in families segregating for the atypical variant. In such circumstances, they demonstrate unusual inhibition numbers due to the combination of effects of reduced activity and decreased inhibition. Heterozygotes with the usual enzyme cannot be distinguished from usual homozygotes (or from each other) by inhibition studies (Table 1); the lower activity is the only indication.
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TABLE 1. Characteristic Inhibitor Number Patterns Demonstrated by the Different Biochemical Phenotypes (Based on the Reference Ranges in Use in the Author's Laboratory) and the Expected Degree of Suxamethonium Sensitivity Inhibition Nos. (mean values) Phenotype
DN
FN
RN
Usual UA
81 62
62 51
97 72
Atypical UF AF FS, FF AK
17 74 48 63 81 53
26 50 31 39 61 45
16 96 62 93 97 60
AJ
40
40
48
UK, UJ, JKa
Suxamethonium sensitivity Rare Rare (except pregnancy) Severe Rare Moderate Moderate Variable Moderate (25% of cases) Moderate
aThese types cannot be unequivocally identified biochemically in a single sample and should be regarded as "usual" phenotypes. They are included here as separate types because they are often reported on the basis of low activity and so may have a slightly higher risk of suxamethonium sensitivity.
The difficulty in identification can be further exacerbated by other effects on enzyme concentration and by the lack of a universally accepted or standardized analytical methodology. Examples of the former include intercurrent illness and intraindividual variations due to age or pregnancy, as described in Section II.A. The problems arising from the latter occur mainly in a clinical setting, as opposed to a research or academic one. A variety of different substrates are used to determine enzyme activity and inhibitor numbers along with inhibitors other than dibucaine, fluoride, and Ro 02-0683. The effect of such variation has been demonstrated in a National External Quality Assessment Scheme (Proficiency Testing Scheme) in which the identification of certain phenotypes proves difficult when certain combinations of substrate and inhibitors are used (Goodall, 2004).
b. Increased Activity Variants Individuals have been described with enzymes that showed increased activity in vitro, with the increase apparently inherited. One of these was discovered as an electrophoretic variant containing an additional fifth, slow-moving band, to the four bands seen in normal sera (Harris et al., 1962, 1963). This variant was called C5 and demonstrated increased enzyme activity. It was believed for some time that the C5 enzyme was encoded by a second genetic locus (see Section II.C. 1), but Masson and associates (1990) clearly demonstrated that its production is not caused by a second BuChE gene. It now seems clear that C5 is created by the association of normal, tetrameric BuChE with another protein. Unlike other macro enzymes, such as creatine kinase and amylase,
in C5 BuChE the complexing protein has been shown not to be immunoglobulin (Akizuki et al., 2004) but its exact nature remains unclear. Also unlike other macroenzymes, which often have normal physiological function, the C5 variant does have anesthetic consequences, with C 5 + individuals demonstrating a shortened neuromuscular blockade following suxamethonium administration (Sugimori, 1986), indicating that larger doses are required to achieve the desired clinical effect. However, although it had been clear for some time that the pharmacogenetics of BuChE was not completely straightforward, the true complexity began to emerge when investigation began at the molecular (DNA) level and the structural bases of the variants were determined.
C. M o l e c u l a r Genetics 1. THE BUCHE GENE, B C H E The observation that some individuals have higher than normal enzyme activity led to the idea that BuChE was encoded by two separate loci (called E1 and E2), with E2 being responsible for the higher levels. However, using linkage and in situ hybridization studies, Arpargus et al. (1990) demonstrated that BuChE is encoded by a single gene locus only (BCHE), which was then localized on the long arm of chromosome 3 at 3q26 (Allerdice et al., 1991). The gene is minimally 73 kilobases long with four exons separated by three introns. The gene product is the BuChE subunit, four of which are needed to make the active enzyme. Exon 2 is large and codes for 83% of the subunit,
CHAPTER 1 3 9Cholinesterase Pharmacogenetics including the active triad covering the esteratic serine site at amino acid 198 (Lockridge et al., 1987). Examination of the gene, for mutations/polymorphisms causing the observed biochemical phenotypes, resulted in the isolation of those responsible for all of the most widely known variants. 2. POLYMORPHISMSAND MUTATIONS The first variants to be identified were identified by screening libraries of genomic DNA j for comparison with known biochemical phenotypes; for example, in the case of the A variant, by looking at those known to be homozygous for the U and A traits and comparing the DNA sequences. Whereas only one mutation has been found to cause the A (McGuire et al., 1989) and K phenotypes (Bartels et al., 1992a), two have been assigned to the F type (Nogueira et al., 1990) and many (in excess of 30) cause the S variants (Yen et al., 2003). Most of the variant alleles described so far are the result of single nucleotide polymorphisms (SNPs). Using the A variant as an example, the mutation giving rise to it is a change/at nucleotide (nt) 209, where an adenine residue is substituted by a guanine and the codon thus changes from GAT to GGT. This changes the aspartate at position 70 to glycine and is designated D70G. In the S variant genotypes, however, there are several different types of mutation/polymorphism:
9 Single base deletions producing premature stop codons, leading to truncated proteins 9 Single base insertions leading to reading frameshifts that change the amino acid sequence of the remaining part of the protein and may also lead to a premature stop codon 9 Combinations of a point change and an insertion, leading to a frameshift 3. MUTATION FREQUENCY The issue of the frequency of the various mutations/polymorphism is still not completely resolved, but as more molecular genetic studies have been performed and more polymorphisms discovered, some issues have become clear. The K variant polymorphism appears to be the most common overall, especially in European and North American populations (La Du et al., 1990). The allele frequency is approximately 0.13, making 1 person in 65 a homozygote for this variant and therefore approximately 1 in 4 a heterozygote or carrier. The allele frequency for the A variant has been calculated at 0.017 (McGuire et al., 1989), although it appears that an isolated A mutation is rare in the North American population and that there is strong linkage disequilibrium with the K variant (Bartels et al., 1992b). However, work in my laboratory indicates it is slightly less uncommon in the United Kingdom, and in other areas of the world it is linkage with the K variant that is rare. Ehrlich et al. (1994) showed that in the Ashkenazi Jewish populations in Israel, although the A variant was
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relatively common (approximately 5.8%), there were no occurrences of the K allele. A study of 1000 Iranians found that as many as 70-80% of the people studied carried at least one atypical allele (Vahdati-Mashhadian et al., 2004). This study used DN rather than molecular analysis to identify the variant, but this is appropriate in the case of the atypical variant because it does demonstrate a unique DN range. In contrast, some populations demonstrate a complete absence of certain variants. Acuna et al. (2003) described work that demonstrated that certain tribes of native Amerindians show a complete absence of the atypical variant allele, whereas others show a frequency attributable to the degree of European admixture that indicated that this allele was absent before the arrival of Europeans. The overall frequency of silent variant polymorphisms was estimated in Caucasian populations by Hodgkin et al. in 1965 to be 0.003 using nonmolecular methods; however, as with the atypical allele, the silent phenotype has been shown to be more common in some populations and ethnic groups than in others. The phenotype appears in approximately 1% of the Inuit people of western Alaska (Gutsche et al., 1967) and ~in 2% of people in certain areas of India (Rao and Gopalam, 1979). The frequency of the phenotype is somewhat higher in groups of patients selected because they have experienced scoline apnea. Yen et al. (2003) deternfined that in a group of 65 patients referred for genotyping following scoline apnea, 52 had primary hypocholinesterasemia, of which 8% had previously described silent variants and 12% had new or rare mutations. This is indicative of the probability that although the combined frequency of all the mutations leading to a silent phenotype may be higher than originally thought, the frequency of individual polymorphisms may be too low to accurately calculate. A search of the literature for details of the numerous silent variant polymorphisms reveals that many of them may be "private I' to single families. This may also be true in the case of the J phenotype because it appears that the mutation causing it has only been described in the original, extended family in which it was first identified. There are relatively high occurrences of multiple polymorphisms, with the possession of the common K variant certainly not precluding an individual from also carrying one or more of the other variant polymorphisms. Genuine silent variant mutations (i.e., those that do not affect the enzyme's amino acid sequence or function) have also been reported (PrimoParmo et al., 1996), indicating that care has to be exercised when extrapolating from genotype to phenotype. The high degree of heterogeneity means that the possibility of further mutations (and variants) is high, as demonstrated in the work of Yen et al. (2003) described previously. In addition to differences in the frequency of the variants in different populations, there are several reports of variants that appear to be restricted to certain ethnic groups and races. This has been most frequently reported in Japanese populations, particularly with respect to silent variants
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S ECTI O N I I I 9Esterases, Receptors, M e c h a n i s m s , & Tolerance D e v e l o p m e n t
(Sudo et al., 1996; Maekawa et aL, 1997). Sudo et al. describe a fluoride-resistant variant that appears to be unique to the Japanese, whereas the polymorphisms producing this phenotype (F1 and F2) in North American and European populations are apparently absent. Therefore, there are definite ethnic and geographic differences in both gene frequency and allele linkage, a fact that has important consequences for those working in the clinical arena to identify the causes of scoline apnea in individual patients. 4. MOLECULAR BIOLOGY Not all DNA variations alter the function of a gene's protein product, and not all of those that do will exert the same effects. As with the types of polymorphisms found in BCHE, the effects of the variations are also somewhat heterogeneous. The likely effects of different polymorphisms can be extrapolated from knowledge of the structure of the gene product and from comparative studies with the corresponding genes of different species. The degree to which a particular codon or sequence is conserved across species and between similar proteins is a good indication of its importance and, therefore, of the likely severity of any variation. In the case of BuChE, the effects of the molecular changes on action can be explained for most of the variants, but for some they remain the subject of conjecture. a. Atypical (Dibucaine-Resistant) Variant The atypical variant shows reduced binding for the choline esters that bind to the anionic site of the enzyme. However, it has normal affinity for those neutral organic esters that interact primarily with the esteratic site. The aspartate at position 70, changed in the atypical variant, is located at the ligand binding site situated at the mouth of the active site gorge of the enzyme (Masson et al., 1997), indicating that the effect of the polymorphism is to change the binding, and therefore the access, of the substrate to the active site. This confirms the atypical variant as a qualitative, or low-affinity, variant. b. Fluoride-Resistant Variants That the fluoride-resistant variants exert an independent effect on the action of BuChE is demonstrated by the fact that individuals who are phenotypically heterozygous for the atypical and fluoride variants (AF) show a moderate degree of apnea, whereas UA heterozygotes do not (Viby-Mogensen and Hankel, 1981). By examining the pedigrees of known occurrences of the fluoride-resistant phenotype, Nogueira and associates (1990) identified two different SNPs (F1 and F2) leading to the variant in Caucasian populations but were unable to identify any linkage between them (i.e., they were mutually exclusive). The F1 SNP (T243M) affects the tripeptide recognition sequence for the glycosylation of the asparagine at position 241. Thus, the F1 polymorphism may lead to the loss of one of the nine carbohydrate chains present in the normal enzyme molecule, thereby reducing the molecular weight of the protein and altering its function.
The F2 SNP (G390V) leads to the loss of the glycine residue at position 390. Glycine 390 is important for substrate and ligand binding, and so the F2 variant is another lowaffinity (qualitative) variant, like the atypical but with less severe consequences. The Japanese fluoride-resistant variant, L330I, was demonstrated to have low activity when expressed in human fetal kidney cells and appears to be a low-affinity variant (Sudo et al., 1997). This is thought to be because leucine 330 is adjacent to the phenylalanine at position 329, close to the ligand binding site that is known to affect the enzyme's catalytic properties. c. Silent Variants As mentioned in Section II.C.2, there is a fair degree of heterogeneity in the polymorphisms producing the silent variant phenotype. The first to be described, and hence given the name S 1, arises from a frameshift at the glycine at position 117, where the codon GGT (gly) becomes GGAG (gly + 1) (Nogueira et al., 1990). This additional nucleotide shifts the reading frame in the gene, which leads to a premature stop codon 12 amino acids downstream. Consequently, only 22% of the normal mature protein is transcribed, and because the stop codon occurs upstream of the active site at serine 198, the gene product has no enzymatic activity. Other silent variants arise from straightforward single nucleotide polymorphisms; for example, a polymorphism that leads to no enzymatic activity is that for the $9 variant. An adenine-to-guanine change at nucleotide 198 (AGT ---> GGT) changes the active serine to a glycine (S198G) and therefore eliminates the enzyme's activity (Primo-Parmo et al., 1996). Unlike the S1 variant, however, $9 BuChE is produced in normal amounts, as judged by its immunoreactivity--it is simply inactive. d. Quantitative Variants Although some of the silent variants can be deemed quantitative, the term is usually reserved for the K, J, and H variants, the molecular bases of which were all published in the same year. As described in Section II.B.3.a, these variants all demonstrate reduced enzyme activity but cannot always be identified by normal biochemical phenotyping methods. Using pedigree analysis combined with DNA sequencing, Bartels et al. (1992b) determined that the K variant was caused by a SNP in exon 4 of the gene. A change at ntl615 (G--~ A) changes the alanine (GCA) at position 539 to a threonine (ACA) and the SNP is labeled A539T. The same group demonstrated that the enzyme produced had the same kinetic properties as the usual enzyme. It also exhibits the same heat stability as the usual enzyme, which argues against the theory that reduced molecular stability leads to the decrease in activity seen in this variant. Before the Bartels et al. study, that possession of this common polymorphism causes a reduction in activity (approximately 30%) had been well demonstrated (Whittaker and Brtitten, 1985) and shown to be due to the reduced number of circulating molecules (Rubenstein et al., 1978). How the A539T
CHAPTER 1 3 9Cholinesterase Pharmacogenetics change leads to this reduction remains unclear, although the alanine at 539 is well preserved across species. Given the high proportion of multiple polymorphisms in BCHE, and the degree of linkage, Bartels et al. proposed that the true cause of the decrease in activity may be another polymorphism/mutation elsewhere in the gene that is linked to A539T. However, such a mutation has not been reported. At the same time that they reported on the K variant, Bartels et al. (1992b) also described the SNP responsible for the J variant by investigating the pedigree of the extended family in whom Garry et al. (1976) had first reported the variant. They identified a SNP in exon 3, G497V, that segregated with the J phenotype, as determined by inhibition studies that included Ro 02-0683 inhibition, which is responsible for this variant. They also found that this SNP did not occur in isolation; it was always linked to the K variant in the same allele. Consequently, the J type is, in fact, JK. As with the K variant, Rubenstein et al. (1976) performed immunological studies on samples demonstrating the J variant phenotype and demonstrated a 66% reduction in circulating BuChE molecules (cf. the 33% reduction in K variant enzyme). It remains unknown whether the J variant produces the additional reduction, because it has never been found alone, or whether it acts in synergy with the K mutation. The third quantitative variant to be characterized was the H variant. This very rare variant is also only identifiable biochemically when present in a family segregating for the atypical variant. Jensen et al. (1992) demonstrated that the valine at position 142 is changed to a methionine (V142M). This amino acid is highly conserved in both cholinesterases in several species (Krejci et al., 1991), and its position in the folded protein indicates that it may be important in the folding and stability of the enzyme. There is overlap between some of the variants named silent and the H variant in that the 90% reduction in activity seen in the H variant is similar to the levels seen in some of the silent variants that do not result in the complete absence of function. This situation is indicative of the continuum of function and activity present in this enzyme due to the number and variety of genetic variants and the high number of multiple polymorphisms. When combined with the physiological changes that can occur, this can make interpretation of results in a clinical setting complex. 5. TERMINOLOGY Although DNA analysis has made it possible to identify specific BCHE polymorphisms, a routine clinical service for this is currently available in only a few centers, but it has identified difficulties in the previously accepted way of classifying patient samples by biochemical phenotypes. For example, it had been the practice to assign a phenotype on the basis of enzyme activity and inhibitor numbers with the assumption that a low activity and a usual pattern of inhibitor numbers indicated the presence of the silent variant and that the genotype was U/S (previously termed E lU E lS). The discovery of the K variant allele showed that not all lower activity samples would be due to the presence of
193
silent variants. It is only possible to biochemically identify the probable presence of this allele in a family or pedigree study in which the atypical allele is also present. However, such was the confidence in biochemical phenotyping that extrapolation from such pedigrees was made to provide reference ranges for enzyme activity and to subdivide samples with usual inhibitor numbers into specific phenotypes, such as UU, US, UK, or UJ. There is still some difference of opinion on this practice; however, unless all possible secondary causes of reduced activity can be excluded, it appears to be a potentially dangerous practice and such samples should be classified as having only a usual or U phenotype (La D u e t al., 1991) At this point, a description of the terminology involved may be useful. The use of the word "phenotype" refers, in this instance, to a biochemical classification rather than a true clinical phenotype and is that which is determined by the pattern of inhibitor numbers as listed in Table 1. Therefore, any sample with a pattern that fits the ranges for a particular type by definition has that phenotype. To standardize the terminology, La Du et al. (1991) proposed a system of nomenclature that is in almost universal usage today. Phenotypes are written, in upper case, as a pair of letters representing the expected variants: U for usual, A for atypical, F for the fluoride-resistant types, and so on (e.g., UA and AF). The exception is the usual and atypical phenotypes, where both alleles cannot be predicted. Genotypes are represented in two ways. The shorthand version uses the same letters as phenotypes but separates the letters by a forward stroke to indicate the alleles on the two chromosomes (e.g., U/A). The formal terminology for genotypes gives the gene name, B C H E , followed by the DNA change responsible for any polymorphism using standard genetic notation. In the example, the U/A genotype becomes B C H E / B C H E * 7 O G , with the forward stroke separating the alleles and the asterisk indicating a change at position 70 to the amino acid glycine. Genotyping of individuals who have suffered prolonged paralysis following suxamethonium or mivacurium administration has shown that any biochemical phenotype can arise from several possible genotypes, and that although the majority of samples fall into the defined range for a particular phenotype, there is not always the expected correlation when the genotype is determined. Table 2 shows some of the reported genotypes associated with currently defined phenotypes. In general, sensitivity to short-acting muscle relaxants is dependent on enzyme activity. Anyone with a very low activity, from whatever cause, is likely to suffer a severely prolonged paralysis. However, there are some individuals with a reasonably well-preserved activity who experience a more moderate reaction, and these individuals may have functionally impaired enzyme due to the presence of one of the qualitative variants. A further group has no determinable abnormality, in either activity or phenotype, but suffers prolonged paralysis that appears to be BuChE dependent. It is in these groups that genotyping can be
19 4
S ECTI O N I I I 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t TABLE 2.
Heterogeneity of Biochemical Phenotypes Determined by Molecular Genotyping a
r
Phenotype Usual Atypical UA UF AK AF AJ
Genotypes U/U, U/S, U/K, K/K, K/S, K/Sc, U/S, U/Sc, U/JK, K/JK A/AK, AK/AK, A/S, AK/KS U/A, U/AK, K/AK, A/K U/F2, K/F2 A/K, K/AK, U/A A/F1, A/F2, AK/F2 A/K, AK/F 1, AJJK
aData compiled mainlyfrom the author's own laboratorybut also from the literature. particularly useful. In some such individuals, it has been shown that there is at least one variant that is capable of hydrolyzing the substrates used in the standard methods for laboratory determination of activity but cannot hydrolyze suxamethonium in vivo. This variant, called the scoline or Sc variant, can be identified by DNA analysis and demonstrates reduced activity when succinylcholine is used as the substrate in a biochemical assay (Greenberg et al., 1995). As more genetic analysis is performed, it is likely that either phenotyping will be replaced by activity measurements plus genotyping or the acquired data on phenotype:genotype correlations will allow more accurate reference ranges for phenotyping.
III. PHARMACOGENETICS OF ACETYLCHOLINESTERASE Because of its physiological importance, it has long been assumed that effective mutations in the AChE gene would have severely deleterious consequences. The AChE gene (ACHE) has been mapped to the long arm of chromosome 7 (Zelinski et al., 1991) and localized to 7q22 (Getman et al., 1992), and the sequence has been determined. The gene is composed of six exons and the repertoire of isoforms is the result of alternate splicing producing three distinct polypeptides (Li et al., 1991). Since then, some polymorphisms have been found, but until recently, none appeared to affect the enzyme's normal action. For example, Bartels et al. (1993) reported that a mutation in the coding region of the gene was responsible for the human YT blood group. The YT blood group is defined, as are all blood groups, by antigens on red blood cells that are recognized by specific antibodies. Immunological evidence had implicated the erythrocyte AChE as the location for the two YT group antigens, YT1 and YT2 (Spring et al., 1992). By sequencing all the AChE exons and the intron/exon junctions in individuals showing all three YT genotypes (homozygous YT1 and YT2 plus heterozygotes), Bartels et al. identified three point
mutations. Two of these did not alter the amino acid sequence of the mature ACHE. One was a genuine silent mutation that did not lead to an amino acid change and the second changed the proline at position 561 to arginine, but this residue is not part of the mature, active protein in the AChE anchored to erythrocyte membranes. The third mutation resulted in a histidine (His)-to-asparagine (Asn) change at position 322, a residue located on the surface of the molecule and segregated with the different blood types, with YT1 having His322 and YT2 having Asn322. The presence of this polymorphism, although having possible consequences in blood transfusion, was shown to have no effect on the catalytic properties of the enzyme (Masson et al., 1994). Therefore, although it was apparent that the YT polymorphism conferred no genetic disadvantage or disease state, its discovery did raise the question that other genetic variants may exist that may be disease associated or, as in the case of BuChE, have pharmacogenetic consequences. The use of anticholinesterases as therapeutic agents in the treatment of myasthenia gravis and Alzheimer's disease has shown variation in individual responses to these agents, including some individuals who suffered severe hypersensitivity. Such observations have prompted a closer look at possible pharmacogenetic variability in ACHE. It was known that certain anticholinesterases promote overproduction of one of the splice variants, the readthrough AChE-R, with the resultant increase in enzyme activity, and thus scavenging ability, conferring a short-term protection to exposure to such chemicals (Kaufer et al., 1998, 1999). Conversely, it was possible that an impaired ability for such induction could result in a hypersensitivity to anticholinesterases. Shapira et al. (2000) were able to identify two polymorphisms in the distal promoter region of the gene. One of these, a 4-base pair deletion, was discovered in a woman who had displayed acute hypersensitivity to pyridostigmine. The deletion was shown to abolish a binding site for hepatocyte nuclear factor-3, which resulted in a constitutive increase in basal AChE expression, which in turn impaired the antiAChE-induced overexpression thus causing the sensitivity to the drug. This deletion was also found in
CHAPTER 1 3 9Cholinesterase Pharmacogenetics
other individuals who had also suffered intoxication following a subacute dose of pyridostigmine. In none of the cases studied was any abnormality of BuChE seen. The second mutation disrupts a probable glucocorticoid response element, but the consequences of this mutation are yet to be elucidated; however, there is surely some kind of effect. There had been no reports of any significant degree of polymorphism in the A C H E coding sequence until Hasin et al. (2004) reported on highly detailed studies in a cohort of 96 unrelated individuals. They examined four different ethnic populations and identified 13 different SNPs, of which 10 were new and 5 were likely to produce amino acid substitutions. Their contention was that the previously held belief that AChE was not a polymorphic enzyme was due to a combination of the technical difficulties encountered in amplifying the gene using the polymerase chain reaction and the limitation of previous studies to small numbers of single ethnic groups. The study identified 15 different haplotypes and 5 apparently ethnospecific polymorphisms. Although none of the SNPs identified appear likely to alter the catalytic action of ACHE, 3 of them are within the mature protein, mapping to the external surface, and so may have antigenic effects. This work seems to support the theory that polymorphisms affecting the enzyme's function may be too deleterious for survival. Conversely, it also demonstrates that AChE is polymorphic and that further work is needed to search for variations that may account for some of the adverse reactions seen in response to the therapeutic anticholinesterase drugs.
195
atypical BuChE or with the AChE promoter deletion, was a possible contributing factor or cause of Gulf War syndrome 9and indicates that individuals with variant cholinesterases would not benefit from pretreatment with anticholinesterases (Soreq and Seidman, 2001). Conversely, "artificial" mutations have also been shown in experimental animals to apparently confer resistance to the toxic effects of certain OPs (Wang et aL, 2004). It has been demonstrated that treatment with wild-type (usual) human BuChE with fetal bovine serum AChE can confer protection against OP agents such as soman in primates (Doctor et al., 1993). Because anticholinesterase prophylaxis appears to provide protection against certain agents (e.g., soman) but to increase toxicity to others such as satin (Abu-Qare and Abou-Donia, 2002), the use of purified enzyme in such instances appears to have far less risk, although the requirements are currently too large for practical application. There are no reports of how such a prophylaxis is likely to work in the presence of variant BuChE, and this would appear to be a useful area of study, given the fact that atypical and other variant enzymes occur in relatively high frequencies in certain populations. The cholinesterases have long been known to be important physiologically and pharmacologically. However, recent expansion in the use of anticholinesterases and cholinesterase inhibitors, both therapeutically and as weapons, means that the effect of genetic variation may be more relevant than previously thought.
References IV. C O N C L U S I O N S The genetic variability demonstrated by BuChE leads to a wide spectrum of in vivo activity with respect to its main pharmacological substrates, which can have severe clinical consequences. The same variability also produces differences in the degree of inhibition by a range of different substances, which allows biochemical characterization (phenotyping) to be performed. Recently, it has also been shown that genetic variation in AChE occurs, which may account for differences in responses to the therapeutic use of anticholinesterases. It is probable that this genetic variation will have an effect on individuals' responses to OP exposure. Case histories of severe consequences when individuals with variant BuChE have been exposed to OPs have been reported (LoewensteinLichtenstein et al., 1995). There is one case in the literature of a family of farmers exposed to OP pesticides who demonstrated a de novo amplification of B C H E (Prody et al., 1989), indicating that the consequences of OP poisoning may not be confined to exposed individuals but may have long-term genetic consequences. Because of the risk of chemical attack, anticholinesterases were used prophylactically during the Gulf War. It has been hypothesized that this, combined in some individuals with
Abu-Qare, A. W., and Abou-Donia, M. B. (2002). Sarin: Health effects, metabolism, and methods of analysis. Food Chem. Toxicol. 40, 1327-1333. Acuna, M., Eaton, L., Cifuentes, L., and Llop, E. (2003). Genetic variants of serum butyrylcholinesterase in Chilean Mapuche Indians. Am. J. Phys. Anthropol. 121(1), 81-85. Akizuki, S., Ohnisi, A., Kotani, K., and Sudo, K. (2004). Genetic and immunological analyses of patients with increased serum butyrylcholinesterase activity and its C5 variant form. Clin. Chem. Lab. Med. 42(9), 991-996. Allerdice, E W., Gardner, H. A. R., Galutira, D., Lockridge, O., La Du, B. N., and McAlpine, E J. (1991). The cloned butyrylcholinesterase gene (BCHE) maps to a single chromosome site, 3q26. Genomics 11, 452-454. Arpargus, M., Kott, M., Vatsis, K. E, Bartels, C. E, La Du, B. N., and Lockridge, O. (1990). Structure of the gene for human butyrylcholinesterase: Evidence for a single copy. Biochemistry 29, 124-131. Barnes, J. M., and Davies, D. R. (1951). Blood cholinesterase levels in workers exposed to organo-phosphorus insecticides. Br. Med. J. 2, 816-819. Bartels, C. E, James, K., and La Du, B. N. (1992a). DNA mutations associated with the human butyrylcholinesterase J-variant. Am. J. Hum. Genet. 50, 1104-1114. Bartels, C. E, Jensen, E S., Lockridge, O., van der Speck, A. E L., Rubenstein, H. M., and Lubrano, T. (1992b). DNA mutation
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Vol. 6, pp. 137-147. Macmillan, New York. Kalow, W., and Staron, N. (1957). On distribution and inheritance of atypical forms of human serum cholinesterase as indicated by dibucaine numbers. Can. J. Biochem. Physiol. 35, 1305-1317. Kaniaris, P., Fassoulaki, A., Liarmakopoulou, K., and Dermitzakis, E. (1979). Serum cholinesterase in patients with cancer. Anesth. Analg. 58, 82-84. Kaufer, D., Friedman, A., Seidman, S., and Soreq, H. (1998). Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393, 373-377. Kaufer, D., Friedman, A., and Soreq, H. (1999). The vicious circle: Long-lasting transcriptional modulation of cholinergic neurotransmission following stress and anti-cholinesterase exposure. Neuroscientist 5 173-183. Krasowski, M. D., McGehee, D. S., and Moss, J. (1997). Natural inhibitors of cholinesterases: Implications for adverse drug reactions. Can. J. Anaesth. 44, 525-534. Krejci, E., Duval, N., Chatonnet, A., Vincens, P., and Massoulie, J. (1991). Cholinesterase-like domains in enzymes and structural proteins: Functional and evolutionary relationships and identification of a catalytically essential aspartic acid. Proc. Natl. Acad. Sci. USA 88, 6647-6651. La Du, B. N., Bagels, C. E, Nogueira, C. P., Hajra, A., Lightstone, H., van der Speck, A., and Lockridge, O. (1990). Phenotypic and molecular biological analysis of human butyrylcholinesterase variants. Clin. Biochem. 23, 423-431. La Du, B. N., Bartels, C., Noqueira, C. P., Arpagaus, M., and Lockridge, O. (1991). Proposed nomenclature for human
CHAPTER 1 3 9Cholinesterase Pharmacogenetics
butyrylcholinesterase genetic variants identified by DNA sequencing. Cell. Mol. Neurol. 11, 79-89. Lehman, H. (1956). The familial incidence of low pseudocholinesterase level. Lancet 2, 124. Li, Y., Camp, S., Rachinsky, T. L., Getman, D., and Taylor, E (1991). Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue specific expression. J. Biol. Chem. 226, 23083-23090. Liddell, J., Lehman, H., and Silk, E. (1962). A silent pseudocholinesterase gene. Nature 193, 561-562. Lockridge, O. (1990). Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine. Pharmacol. Ther. 47, 35-60. Lockridge, O., Mottershaw-Jackson, N., Eckerson, H. W., and La Du, B. N. (1980). Hydrolysis of diacetylmorphine (heroin) by human serum cholinesterase. J. PharmacoL Exp. Ther. 215, 1-8. Lockridge, O., Bartels, C. E, Vaughan, T. A., Wong, C. K., Norton, S. E., and Johnson, L. L. (1987). Complete amino acid sequence of human serum cholinesterase. J. Biol. Chem. 262, 549-557. Loewenstein-Lichtenstein, Y., Scwartz, M., Glick, D., NCrgaardPedersen, B., Zakut, H., and Soreq, H. (1995). Genetic predisposition to adverse conditions of anti-cholinesterases in "atypical" BCHE. Nature Med. 1, 1082-1085 Maekawa, M., Sudo, K., Dey, D. C., Ishikawa, J., Izumi, M., Kotani, K., and Kanno, T. (1997). Genetic mutations of butyrylcholine esterase identified from phenotypic abnormalities in Japan. Clin. Chem. 43, 924-929. Masson, E, Chatonnet, A., and Lockridge, O. (1990). Evidence for a single butyrylcholinesterase gene in individuals carrying the C5 cholinesterase variant. FEBS Lett. 262(1), 115-118. Masson, E, Froment, M.-T., Sorenson, R. C., Bartels, C. E, and Lockridge, O. (1994). Mutation His322Asn in human acetylcholinesterase does not alter electrophoretic and catalytic properties of the erythrocyte enzyme. Blood 83, 3003-3005. Masson, P., Legrand, P., Bartels, C. E, Froment, M.-T., Schopfer, L. M., and Lockridge, O. (1997). Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase. Biochemistry 36, 2266-2277. Massouli6, J. (2002). The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11, 130-140. McGehee, D. S., Krasowski, M. D., Fung, D. L., Wilson, B., Gronert, G. A., and Moss, J. (2000). Cholinesterase inhibition by potato glycolalkaloids slows mivacurium metabolism. Anesthesiology 93, 510-519. McGuire, M. C., Nogueira, C. E, Bartels, C. E, Lightstone, H., Hajra. A., and Van der Spek, A. E L. (1989). Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase. Proc. Natl. Acad. Sci. USA 86, 953-957. Nogueira, C. E, McGuire, M. C., Graeser, C., Bartels, C. E, Arpargaus, M., and Van der Speck, A. E L. (1990). Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT----~GGAG). Am. J. Hum. Genet. 46, 934-942. Orpollo, A. T. (1978). Abnormal pseudocholinesterase levels in a surgical population. Anaesthesiology 48, 284-286. Phillips, B. J., and Hunter, J. M. (1992). Use of mivacurium chloride by constant infusion in the anephric patient. Br. J. Anaesth. 68, 492-498.
197
Primo-Parmo, S. L., Bartels, C. E, Wiersema, B., Van der Spek, A. E L., Innis, J. W., and La Du, B. N. (1996). Characterisation of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene. Am. J. Hum. Genet. 58, 52-64. Prody, C. A., Dreyfus, E, Zamir, R., Zakut, H., and Soreq, H. (1989). De novo amplification within a "silent" human cholinesterase gene in a family subjected to prolonged exposure to organophosphorous insecticides. Proc. Natl. Acad. Sci. USA 86, 690-694. Robertson, G. S. (1966). Serum cholinesterase deficiency, II: Pregnancy. Br. J. Anaesth. 38, 361-369. Robertson, G. S. (1967). Serum protein and cholinesterase changes in association with contraceptive pills. Lancet 1, 232-325. Rao, E R., and Gopalam, K. B. (1979). High incidence of the silent allele at cholinesterase locus 1 in Vysyas of Andhra Pradesh (S. India). Hum. Genet. 52, 139-141. Rubenstein, H. M., Dietz, A. A., Lubrano, T., and Garry, E J. (1976). ElJ, a quantitative variant at cholinesterase locus 1: Immunological evidence. J. Med. Genet. 13, 43-45. Rubenstein, H. M., Dietz, A. A., and Lubrano, T. (1978). E1k another quantitative variant at cholinesterase locus 1. J. Med. Genet. 15, 27-29. Schmidt, E., and Schmidt, E W. (1993). Enzyme diagnosis of liver diseases. Clin. Biochem. 26, 241-251. Shapira, M., Tur-Kaspa, I., Bosgraaf, L., Livni, N., Grant, A. D., Grisaru, D., Korner, M., Ebstein, R. E, and Soreq, H. (2000). A transcription-activating polymorphism in the ACHE promoter associated with acute sensitivity to anti-acetylcholinesterases. Hum. Mol. Genet. 9, 1273-1281. Soreq, H., and Seidman, S. (2001). Acetylcholinesterase - - New roles for an old actor. Nat. Rev. Neurosci. 2, 294-302. Spring, E A., Gardner, B., and Anstee, D. J. (1992). Evidence that the antigens of the Yt blood group system are located on human erythrocyte acetylcholinesterase. Blood 80, 2136-2141. Sudo, K., Maekawa, M., Kanno, T., Akizuki, S., and Magara, T. (1996). Three different point mutations in the butyrylcholinesterase gene of three Japanese subjects with a silent phenotype: Possible Japanese type alleles. Clin. Biochem. 29, 165-169. Sudo, K., Maekawa, M., Akizuki, S., Magara, T., Ogasawara, H., and Tanaka, T. (1997). Human butyrylcholinesterase L330I mutation belongs to a fluoride-resistant gene, by expression in human fetal kidney cells. Biochem. Biophys. Res. Commun. 240, 372-375. Sugimori, T. (1986). Shortened action of succinylcholine in individuals with cholinesterase C5 isozyme. Can. Anaesth. Soc. J. 33(3 Pt. 1), 321-327. Umeki, S. (1993). Biochemical abnormalities of the serum in anorexia nervosa. J. Nervous Mental Dis. 176, 503-506. Vahdati-Mashhadian, N., Hassenzadeh, M. K., Hosseini, J., and Saffareshargh, A. A. (2004). Ethnic differences in the frequency of distribution of serum cholinesterase activity in the Iranian population. Can. J. Physiol. Pharmacol. 82(5), 326-330. Viby-Mogensen, J., and Hankel, H. J. (1981). Succinylcholine neuromuscular blockage in subjects heterozygous for abnormal plasma cholinesterase. Anesthesiology 55, 231-235. Wang, Y., Boeck, A. T., Duysen, E. G., Van Keuren, M., Saunders, T. L., and Lockridge, O. (2004). Resistance to organophosporus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase. Toxicol. Appl. Pharmacol. 196(3), 356-366.
19 8
S E CTI O N I I I
9Esterases, Receptors,
Mechanisms, & Tolerance Development
Whittaker, M. (1989). Cholinesterase. Monographs in Human Genetics, Vol. 2. Karger, Basel. Whittaker, M., and Britten, J. J. (1985). Plasma cholinesterase variants: Family studies of the E1k gene. Hum. Hered. 35, 364-368. Whittaker, M., and Britten, J. J. (1987). E1k, a new allele at cholinesterase locus 1. Hum. Hered. 37, 54-58. Whittaker, M., Jones, J., and Brave, J. (1990). Heterogeneity of the silent gene for plasma cholinesterase. Immunological studies. Hum. Hered. 40, 153-158.
Yen, T., Nightingale, B. N., Burns, J. C., Sullivan, D. R., and Stewart, P. M. (2003). Butyrylcholinesterase (BChE) genotyping for post-succinylcholine apnea in an Australian population. Clin. Chem. 49, 1297-1308. Zelinski, T., Getman, D., Coghlan, G., and Phillips, S. (1991). Assignment of the YT blood group locus to chromosome 7q. Genomics 11, 165-167. Ziamis, E. J., and Head, S. (1976). Depolarizing neuromuscular blocking drugs. In Neuromuscular Junction (E. Ziamis, Ed.), pp. 1-18. Springer-Verlag, Berlin.
CHAPTER
/ 4
Methods for Measuring Cholinesterase Activities in H u m a n B l o o d ELSA REINER AND VERA SIMEON-RUDOLF Institute for Medical Research and Occupational Health, Zagreb, Croatia
I. I N T R O D U C T I O N
hydrolysis. When the substrate is acetylcholine or acetylthiocholine, EA is the acetylated enzyme E-O-C(O)CH3, P1 is choline or thiocholine, and P2 is the acetic acid. The constants kl, k-l, k2, and k3 are rate constants of the individual reaction steps. Acylation (Eq. 1) and deacylation (Eq. 2) take place on the hydroxyl group of serine, which is the key residue of the catalytic triad in the catalytic site of the enzyme. Cholinesterases also have a peripheral, allosteric site that can reversibly bind substrates and other ligands. This site has no catalytic activity. However, binding of a ligand to the site can cause either inhibition or activation of the enzyme. OPs and CMs are acylating inhibitors (ABs) of AChE and BuChE. Cholinesterases react with AB compounds in the same way as they react with substrates; that is, they acylate the hydroxyl group of serine in the catalytic site. However, there is a significant quantitative difference between substrates and AB compounds in the rates of the individual reaction steps. In the reaction with substrates, acylation and deacylation of the serine is very fast, whereas AB compounds quickly acylate the enzyme but very slowly deacylate from the enzyme, particularly when AB is an OP. The enzyme therefore stays acylated by AB compounds for a long time and cannot hydrolyze substrates during that time. Consequently, OP and CM compounds are inhibitors of cholinesterases. Acylation by AB compounds is termed progressive inhibition or irreversible inhibition (Eq. 3), and its time course follows (Eq. 4)"
Organophosphates (OPs) and carbamates (CMs) are toxic compounds primarily due to inhibition of the acetylcholinesterase (ACHE; EC 3.1.1.7) in the neural and neuromuscular synapses. Butyrylcholinesterase (BuChE; EC 3.1.1.8) and other serine esterases, such as carboxylesterase, trypsin, or chymotrypsin, are also inhibited by these compounds. Absorption of OPs and CMs can be detected by measuring the activity of cholinesterases in human blood, which contains both AChE and BuChE. Absorption of cholinesterase inhibitors can also be detected by determining the parent compound and/or its metabolite(s) in blood or urine. This approach is not generally applied, and no routine methods are available for these analyses. This chapter describes methods for determining AChE and BuChE activities and summarizes distribution profiles and interindividual variations of cholinesterase activities in human blood. The kinetics of interaction of cholinesterases with substrates, OPs, and CMs is briefly outlined.
H. I N T E R A C T I O N O F C H O L I N E S T E R A S E S
WITH SUBSTRATES, ORGANOPHOSPHATES, AND C A R B A M A T E S The hydrolysis of cholinesterase substrates proceeds in three steps: E+S
~
kl
k2
ES
~
EA+P1
E+AB
(1)
k3
EAB
~
In (eo / et) = ka" ab. t ,~ E + P2
(2)
(3)
(4)
where EAB is a Michaelis-type complex between AB and the enzyme; EA is the acylated enzyme - - that is, the phosphylated enzyme when AB is an OP compound and the carbamylated enzyme when AB is a carbamate; P1 is
where E and S are the enzyme and substrate, ES is the enzyme-substrate Michaelis complex, EA is the acylated enzyme, and P1 and P2 are the products of substrate Toxicology of Organophosphate and Carbamate Compounds
EA+P1
ka
k_l EA + H20
~
199
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
200
SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
the leaving group of AB; ka is the overall second-order rate constant of inhibition; eo and et are the enzyme concentrations at time zero and time t of inhibition, respectively; and ab is the concentration of AB. Deacylation of the inhibited enzyme (i.e., dephosphylation or decarbamylation) is termed spontaneous reactivation (Eq. 5), and its time course follows (Eq. 6):
EA+H20
~
E+P2
In (eao / eat) = kr" t
(5) (6)
where P2 is a phosphoric or carbamic acid, E is the reactivated enzyme, kr is the first-order rate constant of spontaneous reactivation, and eao and eat are the concentrations of the acylated enzyme at time zero and time t, respectively. Compounds with an oxime group in the molecule reactivate phosphylated cholinesterases much faster than water due to the powerful nucleophilicity of the oxime group. In the reactivation process, reactivated enzyme and phosphylated oximes are formed (Eq. 7), and the time course of reactivation follows (Eq. 8), where k' r is the second-order rate constant of oxime reactivation:
ktr EA + oxime
E + phosphylated oxime
In (eao / eat) = k'r" [oxime]- t
(7)
(8)
Due to their efficacy as reactivators, oximes are used as antidotes in the therapy of OP poisoning. Many publications extensively cover the structure and catalytic properties of cholinesterases (Giacobini, 2000; Inestrosa and Campos, 2004; Silman et al., 2004), the kinetics of interaction with ligands (Reiner and Radi6, 2000), and the role of oximes in OP poisoning (Eyer, 2003).
III. G E N E R A L O U T L I N E O F CHOLINESTERASE ACTIVITY
MEASUREMENTS IN HUMAN BLOOD AChE activity in human blood reflects the activity of synaptic ACHE, which is the target enzyme of anticholinesterases. However, many OPs or CMs inhibit plasma BuChE faster than erythrocyte ACHE, and the inhibition of plasma BuChE is for those compounds a more sensitive indicator of absorption. Consequently, more information about the exposure of an individual to OPs or CMs can be obtained if both AChE and BuChE are measured. Human blood contains BuChE in the plasma and AChE bound to the erythrocyte membrane. In order to determine the activity of each enzyme, erythrocytes and plasma either have to be separated or one has to apply selective substrates and/or inhibitors to assay only one enzyme in the presence of the other. There are no known substrates or inhibitors
that are fully selective for either AChE or BuChE. On the other hand, it is difficult to achieve full separation of erythrocytes from plasma without loss of some of the activity. Therefore, each approach to measuring AChE and BuChE activities in whole blood has its advantages and disadvantages, and any assay in whole blood is subject to some intrinsic error. BuChE activities are measured not only to evaluate inhibition by OPs or CMs but also to diagnose a wide range of physiological or pathological conditions reflected in BuChE activities. These measurements are done in serum or plasma and not in whole blood. Therefore, more data on BuChE than AChE activities are available, and more methods have been developed for the analysis in plasma or serum than in whole blood.
A. Collection and Storage of Blood Samples Blood samples are collected from finger pricks or earlobe pricks into capillaries and from venous blood into test tubes. Capillaries and test tubes should be heparinized (to prevent blood clotting) and dried (to prevent uncontrolled sample dilution). To prevent contamination of the samples by OPs and CMs during collection, the skin must be cleaned before sampling. Individuals exposed to anticholinesterases may have some unreacted OP or CM in the blood, and those treated with antidotes may also have some oximes in the blood. If cholinesterase activities cannot be measured immediately after sampling, some precautions should be undertaken to minimize changes in enzyme activities during storage of blood samples. Dilution of the sample will slow down further cholinesterase inhibition and further oxime reactivation because both reactions depend on the concentrations of the inhibitor and oxime (Eqs. 4 and 8). However, spontaneous reactivation will continue irrespective of dilution (Eq. 6). Because all reactions are temperature dependent, samples can be stored at a low temperature but not below zero if one wants to have nonhemolyzed erythrocytes. All three reactions are also pH dependent. Oximes reactivate when they are deprotonated, and lowering the pH therefore decreases rates of oxime reactivation. The pH profiles of inhibition and spontaneous reactivation depend on the structure of the inhibitor, but for the majority of compounds these rates decrease with decreasing pH. Blood samples can therefore be diluted with a buffer of pH between 5 and 6. If cholinesterase activities are measured in haemolysed blood, dilution with water is suggested, and these samples may be stored below zero.
B. Expression of Activities Activities of AChE and BuChE are expressed in micromoles hydrolyzed substrate per minute per volume of whole blood or plasma. AChE activities can also be expressed per
CHAPTER 1 4 9Methods for Measuring Cholinesterase Activities volume of packed erythrocytes, per hemoglobin content, or per iron content. Due to the wide interindividual variations of AChE and BuChE activities in human blood (see Section V), a single cholinesterase assay does not give information about the absorption of a cholinesterase inhibitor. To calculate the degree of cholinesterase inhibition due to absorption of an inhibitor, one has to know the AChE and BuChE activities before the exposure of an individual to OPs or CMs (preexposure activities).
IV. M E T H O D S
Enzyme activities are measured by determining either the decrease in substrate concentration or the increase in product concentration (i.e., the acid or the alcohol). These reactions are usually followed continuously over a time interval, which makes it ~possible to verify whether the increase or decrease in concentrations is linear over the time of assay. However, some methods are restricted to two measurements (end point methods) m one at the beginning and the other at the end of a specified time interval in which case the linearity of the reactions cannot be verified. To ensure a linear increase in product formation, the initial substrate concentrations should remain almost constant during the assay. On the other hand, this is not favorable if one measures the decrease of unhydrolysed substrate because the calculated activities are based on differences between two almost equal concentrations. Many methods have been developed for measuring cholinesterase activities, and readers are referred to several
TABLE 1.
comprehensive reviews that also cover some methods no longer in general use (Augustinsson, 1971; Evans, 1986; Hoffmann et al., 1999; Silver, 1974; Simeon, 1967; Whittaker, 1986; Wilson, 2001). Choline and thiocholine esters are good substrates of AChE and BuChE, and a selected list of compounds used for activity measurements is given in Table 1. Acetylcholine is the physiological substrate of ACHE, and BzCh is the suggested substrate for BuChE phenotyping. When activities are measured in whole blood that contains both AChE and BuChE, the following selective inhibitors are most often applied to differentiate between the two cholinesterases: ethopropazine, quinidine, and isoOMPA inhibit BuChE, and Huperzine A and BW2845c51 inhibit ACHE. iso-OMPA is an organophosphate, and the other compounds are reversible cholinesterase inhibitors. A. M e t h o d s B a s e d o n t h e D e c r e a s e in S u b s t r a t e Concentration
For the assay of ACh hydrolysis in whole blood, Winteringham and Disney (1964) introduced a radiometric method with [14C]ACh as substrate. This is an end point method intended to be used as a field method. Several modifications were later developed with [14C]ACh or [3H]ACh as substrates (Wilson, 2001), but no radiometric method was widely used. Disposal of radioactive waste material has been noted to be a problem. Another end point method for ACh concentrations is the spectrophotometric method of Hestrin (1949). ACh reacts with hydroxylamine to form acethydroxamic acid, which forms a colored complex with ferricions.
Structures, Names, and Abbreviations of Selected AChE and BuChE Substrates
Substrate
H3C-C(O)-OCH2CH2 N+ (CH3)3 X
Name and abbreviation -a
H3C-C(O)-SCH2CH2 N+ (CH3)3 XHsC2-C(O)-SCH2CH2N+ (CH3)3 XH7C3-C-C(O)'SCH2CH2N+ (CH3)3 XH5C6-C(O)-OCH2CH2 N+ (CH3)3 XCH2-C(O)-OCH 2 CH2N+ (CH3)3 I CH2-C(O)-OCH 2 CH2N+(CH3) 3 2XCH2-C(O)-SCH2 CH2N+ (CH3) 3 i CH2-C(O)-SCH2 CH2N+(CH3)3 2Xax- is an anion.
201
Acetylcholine ACh Acetylthiocholine ATCh Propionylthiocholine PTCh Butyrylthiocholine BTCh Benzoylcholine BzCh Succinyldicholine SuxCh Succinyldithiocholine SuxTCh
202
SECTION III. E s t e r a s e s ,
Receptors,
Kalow et al. (1956) introduced a spectrophotometric method for determining BuChE activities in plasma or serum by continuously measuring the decrease in BzCh concentration in the UV region. BzCh is a substrate of BuChE and is not hydrolyzed by ACHE. This substrate is used for phenotyping BuChE.
B. Methods Based on the Increase in Acid Concentration These methods are suitable for both AChE and BuChE and are applicable to any substrate because hydrolysis of an ester always results in acid formation. The increase in acid concentration can be measured potentiometrically by continuous titration with sodium or potassium hydroxide at a constant pH (pH-stat method) (Jensen-Holm et al., 1959), or one can measure the decrease in pH after a selected time of assay (ApH). The ApH method is an end point method introduced by Michel (1949), and it is used for routine or field measurements, pH can be measured colorimetrically with a pH indicator, which is a simple protocol.
C. Methods Based on the Increase in Choline Concentration Choline released during the hydrolysis of choline esters is oxidized with choline oxidase, and the formed hydrogen peroxide is reacted with peroxidase-phenol-4aminoantipyrine to give a chromofore measured at 500 nm (Abernethy et al., 1984, 1986, 1988; Faye and Evans, 1986). The method was developed for succinyldicholine and benzoylcholine as substrates and applied to identify patients sensitive to succinyldicholine, a neuromuscular relaxant used during anesthesia that is very slowly hydrolyzed by patients with the atypical BuChE variant. This method is not widely used, and sensitive patients are identified by phenotyping BuChE by other methods using BzCh (see Section IV.A) and, recently, succinyldithiocholine (a thio analogue of succinyldicholine) as substrates (Mosca et al., 2003).
D. Methods Based on the Increase in Thiocholine Concentration In 1961, a spectrophotometric method for the detection of low thiocholine concentrations was published by Ellman et al. The method is based on the reaction of thiocholine with the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). Due to its high sensitivity, reproducibility, and simplicity, this method became the most widely applied, and thiocholine esters the most widely used substrates, for measuring cholinesterase activities. Thiocholine released during hydrolysis reacts with the chromogenic DTNB to form the yellow dianion 5-thio-
Mechanisms,
& Tolerance
Development
2-nitrobenzoic acid (TNB). In the procedure of Ellman et al. (1961), the assay medium is 0.1 M phosphate buffer (pH 8.0) containing 0.33 mM DTNB. The enzyme activity (i.e., formation of TNB) is continuously measured at 412 nm for up to several minutes. The molar absorption coefficient (eM) of TNB reported by Ellman (1959) is 13,600M -1 cm -1 derived in 0.01 M phosphate buffer (pH 8.0) at unspecified temperature. This method has been applied to a variety of cholinesterase preparations, in different media at pH values other than 8, and over a wide temperature range. The activities were usually measured at 412 or 405 nm, and the eM stated previously was generally used for the calculation of cholinesterase activities. Since the method was published, slightly different eM values have been reported at 412 nm (Eyer et al., 2003). Furthermore, it has been suggested that in samples containing hemoglobin, activities should be measured at 436 nm (Worek et al., 1999). The absorbance of hemoglobin is high at 412 nm, which interferes with the assay, particularly when cholinesterase activities are low. Finally, it was shown that TNB reveals thermochromic properties" By increasing the temperature, the absorbance spectra of TNB are shifted toward longer wavelengths, whereas the absorbance maxima decrease (Worek et al., 1999). In reassessment studies, eM values were evaluated for temperatures between 10 and 50 ~ and wavelengths between 405 and 470 nm. Recommended eM values (M -1 cm -1) at 412 nm are 14,150 at 25 ~ and 13,800 at 37 ~ at 436 nm, the eM are 11,000 at 25 ~ and 11,280 at 37 ~ (0.1 M phosphate buffer, pH 7.4) (Eyer et al., 2003). Reassessment of eM values is required whenever experimental conditions are altered. DTNB reacts not only with thiocholine but also with other thiol compounds, thus producing TNB. When cholinesterase samples contain other thiol compounds, as in hemolyzed erythrocytes, DTNB should be added to the enzyme sample before the substrate. When the reaction of DTNB with the other thiols is completed, the substrate is added. Thiocholine esters react with oxime groups (oxymolysis), whereby thiocholine is produced (Primo~i~ et al., 2004; Skrinjarid-Spoljar et al., 1992). When samples contain oximes, the contribution of oximolysis should be measured in the absence of the enzyme and subtracted from the total thiocholine increase measured in the enzyme sample. However, when blood samples are taken from patients under oxime therapy, the samples may contain an unknown oxime concentration, and one has to be aware that due to oximolysis, the measured increase in thiocholine may lead to a false conclusion concerning enzyme activity.
E. Protocols Based on the Ellman Method Activities of BuChE are measured in plasma or serum at substrate concentrations in the millimolar range.
CHAPTER 14 9Methods for Measuring Cholinesterase Activities Thiocholine esters listed in Table 1 are good substrates of human BuChE, with activities decreasing in the order: PTCh > BTCh > ATCh. Activities in plasma (obtained from heparinized blood) are 4 or 5% lower than in serum (measured with ATCh and PTCh) (Reiner et al., 1996). ATCh is the preferred substrate of human ACHE. The hydrolysis of PTCh is slower, and that of BTCh is extremely slow. When AChE activities are measured in erythrocytes separated from plasma by centrifugation, they are measured either in unwashed erythrocytes or in erythrocytes washed with buffer or saline in order to remove residual plasma and residual inhibitors or oximes that may be present in the blood. Both separation from plasma and washing may cause some loss in activity. The pS curve for the hydrolysis of ATCh is bell-shaped with an optimum activity at approximately 1 mM ATCh, which is usually the suggested concentration for the assay. Several protocols have been published for measuring both AChE and BuChE in whole blood with no separation of the erythrocytes from plasma. These protocols require either more than one substrate or one substrate and a selective inhibitor (Reiner et al., 2000). Ethopropazine [(10-2-diethylaminopropyl) phenothiazine hydrochloride] is a reversible and almost selective inhibitor of BuChE (Meuling et al., 1992). The suggested concentration for the assay is 20 ~M ethopropazine, which inhibits 98% of the usual (UU) BuChE phenotype. However, the atypical (AA) phenotype and heterozygotes of the A variant are less inhibited (74-87%) (Simeon-Rudolf et al., 2001). Furthermore, at 20 txM ethopropazine, the AChE is also inhibited, although only 5-8% (Reiner et al., 2000; SimeonRudolf et al., 2001; Worek et al., 1999). Worek et al. (1999) published an improved method for AChE activities in whole blood with ATCh as substrate (0.45 mM) in the presence of 20 ~M ethopropazine, in 0.1 M phosphate buffer (pH 7.4). The activity is measured in hemolyzed whole blood at 436 nm and expressed per hemoglobin contents. The protocol proved very reliable even at very low AChE activities. For BuChE activities, the authors suggest a separate assay in plasma with BTCh as substrate (1.0 mM). Based on the previous protocol, Reiner et al. (2004) evaluated both AChE and BuChE activities in whole blood. The activities are measured in nonhemolyzed blood with ATCh as substrate (1.0 mM) in the absence (VT) and in the presence (VE) of 20 txM ethopropazine. The VE corresponds to the activity of ACHE, and the difference, VT-VE, corresponds to the activity of BuChE. The authors suggest that VE be corrected for the inhibition of AChE by ethopropazine. The authors consider this protocol suitable for screening both AChE and BuChE activities in whole blood (hemolyzed or nonhemolyzed) for routine and field tests. In the protocols of Wicki (1994) and Portmann and Hofmann (1998), activities are measured with two substrates
203
but no inhibitor. The activity of both enzymes in whole blood is first measured with ATCh (1.0 mM), and then BTCh (5.0 mM) is added to the same cuvette and the measurement continued. The calculation of AChE and BuChE activities is based on the assumption that BTCh is hydrolyzed only by BuChE, and that the addition of BTCh stops the hydrolysis of ATCh. In the protocol of Feaster et al. (2004), three substrates are used: ATCh, BTCh, and PTCh. The cholinesterase activities in whole blood are measured separately with each substrate, and the activities of AChE and BuChE are calculated by using the so-called sensitivity coefficients. These are determined prior to the assay by applying selective AChE and BuChE inhibitors to reference samples in order to evaluate the contribution of each enzyme to the hydrolysis of each of the three substrates.
F. Kits Based on the E l l m a n M e t h o d Several test kits have been produced for routine measurements of AChE and BuChE activities. Test kits comprise preweight reagents and instructions for dissolving the reagents before the assay. Test kits can be used either in automatic analyzers or in standard manual spectrophotometers or equipment modified with well microplate readers (Doctor et al., 1987). Several studies have reported on the validation of test kits (Wilson et al., 1995) using automatic and manual measurements. Lassiter et al. (2003) found good agreement between AChE activities measured with a Boehringer-Mannheim test kit on an automatic analyzer and a standard spectrophotometer with a 96-well microplate reader. Simeon (1989) studied the stability of dissolved reagents from two commercial test kits (Boehringer-Mannheim and Pliva) and reagents prepared in the laboratory. The three tested sets of reagents were equally stable at 4 ~ over several weeks. The demand for routine measurements of cholinesterase activities under field conditions resulted in the development of various field kits. The Test-mate field kit, which is currently widely used, is based on the Ellman method. It is a self-contained portable device comprising a set of reagents and instructions for use. The Test-mate kit measures AChE and BuChE activities and the hemoglobin content in a drop of blood; the activities are automatically normalized to 25 ~ (Wilson, 2001). Oliveira et al. (2002) tested three Test-mate kit models (on fetal bovine serum ACHE) and compared the results with those measured on a standard spectrophotometer with a 96-well microplate reader. The results from the three models showed a discrepancy, and there was also no good agreement between the results of the three models and those obtained from the standard photometric measurement. The authors strongly recommend the use of cholinesterase standards when activities are measured with field kits.
204
SECTION III. E s t e r a s e s ,
Receptors,
G. Cholinesterase Standards and Quality Control Measurements Several standards are available for intralaboratory and interlaboratory quality control studies, which also include day-to-day control of the precision and accuracy of withinrun and between-run imprecision of activity measurements. Commercially available standards also provide information on the activity of the standards for a given substrate at specified experimental conditions. Precinorm U and Precinorm S (Boehringer-Mannheim) are BuChE standards from lyophilized sera with activities within and below the normal range. Simeon (1989) compared the two standards with a pool of frozen native human sera and with a lyophilized US-NBS serum sample and concluded that a pool of native human sera, divided into aliquots and kept frozen, is a reliable and stable BuChE standard for quality control studies. Arrieta et al. (2003) produced a preparation from bovine red blood cell ghosts as a standard for AChE assays. The activity of this standard was stable at - 7 0 ~ for up to 3 years. Due to the widespread need for cholinesterase activity measurements, and for field kits, several attempts have been made to standardize and validate methods and protocols. Considerable effort toward this goal has been expended by the World Health Organization, the U.S. Environmental Protection Agency, and the Chemical and Biological Medical Treatment Symposia held regularly in Spiez, Switzerland, since 1994. So far, no general agreement has been obtained.
V. DISTRIBUTION PROFILES AND GENETIC VARIANTS OF HUMAN BLOOD CHOLINESTERASES Cholinesterase activities have a bell-shaped distribution profile, and the profile appears symmetrical for both AChE and BuChE. However, one statistical analysis of BuChE activities in plasma and serum showed that the distribution is slightly skewed toward high activities (Reiner et al., 1996). More data are available for BuChE activities in plasma or serum than for AChE activities in whole blood or packed erythrocytes. A selected list of data obtained with several commonly used substrates is given in Table 2. BuChE activities were usually measured in samples in which the BuChE was not phenotyped, but the activities presented in Table 2 most likely reflect the usual phenotype, which is most widely distributed. It follows from Table 2 that the range of activities is broad, and this indicates broad interindividual variations for both AChE and BuChE. The reported coefficients of variation (CV) are higher for BuChE than for AChE
Mechanisms,
& Tolerance
Development
activities, confirming larger interindividual variation for the BuChE. The intraindividual variations of AChE and BuChE activities (CV = 6-9%) are significantly smaller than the interindividual variations and are similar to variations reported for the day-to-day imprecision of measurements (Abernethy et al., 1988; Brock, 1990; Flegar-Me~tri6 et al., 1999). BuChE activities reflect, more than AChE activities, inherited genetic variants and some physiological conditions or some diseases. BuChE activities in serum are decreased in the last trimester of pregnancy. Impaired liver function and some other diseases decrease BuChE activities, whereas obesity and coronary arterial disease increase BuChE activities (Lockridge and Masson, 2000; Whittaker, 1986; Alc~ntara et al., 2002, 2003). BuChE activities also depend on age and sex. Children and adolescents (8-19 years) have significantly higher BuChE activities than adults. BuChE activities of women younger than 50 years are significantly lower than those of adult men; women older than 50 years have similar activity as men (Whittaker; 1986; Flegar-Me~tri6 et al., 1999). The BuChE is determined by a single B C H E gene that encodes the sequence of the enzyme. In addition to the wild-type allele producing the usual enzyme (UU), there are approximately 40 identified genetic variants resulting from mutations in coding regions of the B C H E gene. In populations screened throughout the world, atypical (A), fluoride-resistant (F), J, Kalow (K), H, and approximately 30 different silent variants were identified (G~itke et al., 2001; Liu et al., 2002; Lockridge and Masson, 2000; Primo-Parmo et al., 1996; Whittaker, 1986; Yen et al., 2003). The variants are characterized by different primary structures resulting either in different catalytic properties of the enzyme or in lower protein expression or partial or complete lack of catalytic capacity. All variants have lower activities than the usual enzyme. The BuChE variants give rise to 15 phenotypes that can be identified by standard biochemical methods coupled with family studies. The phenotypes can by identified by measuring activities with different substrates and by inhibition of the enzyme with dibucaine, sodium fluoride, dimethylcarbamate Ro 02-0683, and other inhibitors. The frequencies of the homozygous usual genotype (UU), atypical (AA), and silent (SS) are 1 person out of 1.3, 2500, and 110,000 individuals, respectively (Lockridge and Masson, 2000; Whittaker, 1986). Approximately 10% of Caucasians have the C5 + isozyme. This variant has an approximately 25% higher activity than the usual BuChE. It has been reported that farmers with the C5 + isozyme are more resistant to intoxication by anticholinesterase pesticides because BuChE is a scavenger of OPs and CMs (Akizuki et al., 2004; Fontoura-da-Silva and ChautardFreire-Maia, 1996; Lockridge and Masson, 2000). The biochemistry of BuChE variants and their clinical and toxicological significance are extensively described in
C H A P T E R 14
TABLE 2.
N 257 (M)
Substrate (mM) Temperature (~C) 1.0/ATCh (22-25)
151 (F) Eythrocytes d
205
AChE and B u C h E Activities in Male (M) and Female (F) Individuals Measured with Acetylthiocholine (ATCh), Butyrylthiocholine (BTCh), Propionylthiocholine (PTCh), and Benzoyicholine (BzCh) as Substrates a
Enzyme source Whole blood c
9M e t h o d s for M e a s u r i n g Cholinesterase Activities
30 (M)
0.50/ATCh
30 (F)
Activity range or mean 4_-_SD (Ixmol m i n - 1 m l - 1)
CV ( % )b
Reference
2.10-8.05
19
Wilhelm and Bradamante (1980)
2.45-6.65
19
8.71 + 1.39
16
8.54 + 1.24
15
Jones et al. (1991)
Whole blood e
894
5.4/ATCh
14.7 _+ 1.2
18
Wilson et al. (1998)
Serum BuChE-UU
295 (M + F)
5.0/ATCh (25)
0.82-4.93
23
Simeon-Rudolf et al. (1987)
0.050/BzCh (25)
0.33-1.54
23
Simeon-Rudolf et al. (1987)
Serum BuChE-UU
295 (M + F)
Simeon-Rudolf and Evans
4.0/PTCh (25)
Serum 506 (M + F)
3.89-8.13
18
BuChE-AA
83 (M + F)
0.52-2.50
33
BuChE-AMAS
341 (M + F)
0.30-2.10
38
BuChE-UU
Surina et al. (2004)
7.0/BTCh (30)
Serum
(2001)
BuChE-UU
359 (M + F)
3.99-16.9
21
BuChE-AS/AA
3 (M + F)
1.02-2.87
m
1.30-3.70
28
1.22-3.20
23
0.35-2.60
23
0.35-2.45
25
1.80-4.40
18
1.60-3.50
17
0.82-5.23
23
Simeon-Rudolf et al. (1987)
0.89-3.62
23
Simeon-Rudolf and Reiner (1991)
0.89-3.24
26
Serum
226 (M)
0.256/ATCh (25)
198 (F) Plasma
252 (M)
Serum
36 (M)
1.0/ATCh (22-25)
148 (F) 5.0/ATCh (25)
17 (F) Serum
180 (M)
5.0/ATCh (25)
154 (F) Plasma
134 (M)
von Prellwitz et al. (1976) Wilhelm and Bradamante (1980) Huizenga et al. (1985)
1.29-4.14 1.0/ATCh (25)
103 (F) Serum
89 (M + F)
1.0/ATCh (25)
0.92-3.24
23
Reiner et al. (1996)
Serum
226 (M)
6.0/BTCh (25)
2.27-7.40
28
von Prellwitz et al. (1976)
2.05-6.70
28
3.10-7.50
21
198 (F) Serum
221 (M)
20/PTCh (25)
173 (F)
von Prellwitz et al. (1976)
2.90-6.90
21
1.0/PTCh (25)
1.63-6.27
24
1.30-5.46
25
88 (M + F)
1.0/PTCh (25)
1.67-5.74
24
Reiner et al. (1996)
180 (M)
0.050/BzCh (25)
0.33-1.54
22
Simeon-Rudolf et al. 1987
0.35-1.1
23
Plasma
134 (M)
Serum
Serum
103 (F)
154 (F)
Simeon-Rudolf and Reiner (1991)
aData are from selected references. bCV is the coefficient of variation, i.e., the relative standard deviation of mean activities. CActivities measured in whole blood with 1.0 mM ATCh reflect primarily ACHE. CtMean + SD activity expressed as I~molmin-1 ml-1 packed erythrocytes. eMean _+ SD activity expressed as txmol min-1 mg-1 Hb. Activities measured in the presence of 0.2 mM quinidine, a selective BuChE inhibitor.
206
SECTION I I I . Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
several reviews (Evans, 1986; Lockridge, 1990; Lockridge and Masson, 2000; Pantuck, 1993; Whittaker, 1986). There are only a few reports on AChE polymorphism resulting from the mutations on the A C H E gene. It seems that these mutations have no impact on the catalytic properties of the enzyme but may affect protein stability and/or antigenicity (Bartels et al., 1993; Erlich et al., 1994; Hasin et al., 2004; Lockridge and Masson, 2000). However, their possibly different response to OPs and CMs has not been investigated.
VI. C O N C L U S I O N S AND F U T U R E DIRECTIONS OPs and CMs are used as pesticides, and some OPs are also potential warfare nerve agents. Pesticides are used worldwide, and warfare agents may also be used extensively. To detect absorption of anticholinesterases, a standardized protocol for measuring AChE and BuChE activities in human blood is needed that is simple in technical terms but reliable and well reproducible. This would enable a better comparison of data from different laboratories and an exchange of samples for analysis and confirmation of results. It seems that a protocol based on the Ellman method would meet the outlined requirements, but further studies including interlaboratory quality control are required, particularly when field methods are developed.
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CHAPTER 1 4 9Methods for Measuring Cholinesterase Activities
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207
cholinesterase activity. Internal document of NC Laboratory, Spiez, Switzerland. Primo-Parmo, S. L., Bartels, C. E, Wiersema, A. E L., Innis, J. W., and La Du, B. N. (1996). Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene. Am. J. Hum. Genet. 58, 52-64. Primo,S, I., Od~ak, R., Tomi6, S., Simeon-Rudolf, V., and Reiner, E. (2004). Pyridinium, imidazolium and quinuclidinium oximes: Synthesis, interaction with native and phosphylated cholinesterases, and antidotes against organophosphates. J. Med. Chem. Defense, online at jmedchemdef.org. Reiner, E., and Radi6, Z. (2000). Mechanism of action of cholinesterase inhibitors. In Cholinesterases and Cholinesterase Inhibitors (E. Giacobini, Ed.), pp. 103-119. Dunitz, London. Reiner, E., Simeon-Rudolf, V., and Buntic, A. (1996). Cholinesterase activities in human serum/plasma and their distribution profiles. Period. Biol.v98, 119-120. Reiner, E., Skrinjari6-Spoljar, M., Sinko, G., and Simeon-Rudolf, V. (2000). Comparison of protocols for measuring of human blood cholinesterases by the Ellman method. Arh. Hig. Rada. Toksikol. 51, 13-18. Reiner, E., Bosak, A., and Simeon-Rudolf, V. (2004). Activity of cholinesterases in whole blood measured with acetylthiocholine as substrate and ethopropazine as selective inhibitor of plasma buryrylcholinesterase. Arh. Hig. Rada. Toksikol. 55, 1-4. Silman, I., Soreq, H., Anglister, L., Michaelson, D., and Fisher, A. (Eds.) (2004). Cholinergic Mechanisms. Function and Dysfunction. Taylor & Francis, London. Silver, A. (1974). The Biology of Cholinesterases. Frontiers of Biology, Vol. 36. North-Holland, Amsterdam. Simeon, V. (1967). Methods for cholinesterase activity measurements [in Croatian]. Arh. Hig. Rada Toksikol. 18, 29-39. Simeon, V. (1989). Measurement of the serum cholinesterase activity: Comparison of commercial and laboratory test reagents, enzyme standards and statistical processing of the results [in Croatian]. Arh. Hig. Rada. Toksikol. 40, 183-189. Simeon-Rudolf, V., and Evans, R. T. (2001). Interlaboratory study into the proficiency of attribution of human serum butyrylcholinesterase phenotypes: Reference values of activities and inhibitor numbers. Acta Pharm. 51, 289-296. Simeon-Rudolf, V., and Reiner, E. (1991). Phenotypes of human serum esterases reacting with organophosphates, carbamates and other esters. In Ecogentics. Genetic Predisposition to the Toxic Effects of Chemicals (P. Grandjean, Ed,). Chapman & Hall, London. Simeon-Rudolf, V., Bunti6, A., Surina, B., and Flegar-Me~tri6, Z. (1987). Cholinesterase phenotyping and distribution of activity of sera of 346 individuals. Acta Pharm. Jugosl. 37, 107-114. Simeon-Rudolf, V., Sinko, G., Stuglin, A., and Reiner, E. (2001). Inhibition of human blood acetylcholinesterase and butyrylcholinesterase by ethopropazine. Croat. Chem. Acta 74, 173-182. Skrinjari6-Spoljar, M., Franciskovic, L., Radi6, Z., Simeon, V., and Reiner, E. (1992). Reaction of imidazolium and pyridinium oximes with the cholinesterase substrate acetylthiocholine. Acta Pharm. 42, 77-83. Surina, B., Nosso, D., Siftar, Z., Flegar-Me~tri6, Z., and SimeonRudolf, V. (2004). Cholinesterase unit establishment and issuing of warning cards for carriers of suxamethonium
208
SECTION I I I .
Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
sensitive serum butyrylcholinesterase variants. Coll. Antropol. 28(Suppl. 2), 297-304. von Prellwitz, W., Kapp, S., and Mtiller, D. (1976). Vergleich von Methoden zur Aktivit~it der Serumcholinesterasen (acylcholin-acethyhydrolase E.C.3.1.1.8) und deren diagnostische Wertigkeit. J. Clin. Chem. Clin. Biochem. 14, 93-97. Whittaker, M. (1986). Cholinesterase. In Monographs in Human Genetics (L. Beckman, Ed.), Vol. 11, Karger, Basel. Wicki, A. (1994). Monitoring of acetylcholinesterase and butyrylcholinesterase in human whole blood. In Proceedings of the Chemical and Biological Medical Treatment Symposium CBMTS, Spiez, Switzerland, pp. 1.18-1.24. Applied Science and Analysis, Portland (ME), USA. Wilhelm, K., and Bradamante, V. (1980). Blood cholinesterase activity in workers exposed to anticholinesterases, a ten year follow-up. Arh. Hig. Rada Toksikol. 31, 109-124. Wilson, B. W. (2001). Cholinesterases. In Handbook of Pesticide Toxicology (R. I. Krieger, Ed.), Vol. 2, pp. 967-985. Academic Press, San Diego. Wilson, B. W., Padilla, S., Sanborn, J. R., Henderson, J. D., and Billitti, J. E. (1995). Clinical blood cholinesterase
measurements for monitoring pesticide exposures. In Enzymes of the Cholinesterase Family (D. M. Quinn, A. B. Balasubramanian, B. E Doctor, and E Taylor, Eds.), pp. 329-386. Plenum, New York. Wilson, B. W., McCurdy, S. A., Henderson, J. D., McCarthy, S. A., and Billitti, J. E. (1998). Cholinesterase and agriculture. Humans, laboratory animals and wildlife. In Structure and Function of Cholinesterases and Related Proteins (B. E Doctor, E Taylor, D. M. Quinn, R. L. Rotundo, and M. K. Gentry, Eds.), pp. 539-546. Plenum, New York. Winteringham, E E W., and Disney, R. W. (1964). A radiometric method for estimating blood cholinesterase in the field. Bull. Worm Health Organization 30, 119-125. Worek, F., Mast, U., Kiderlen, D., Diepold, Ch., and Eyer, E (1999). Improved determination of acetylcholinesterase activity in human whole blood. Clin. Chim. Acta 288, 73-90. Yen, T., Nightingale, B. N., Bums, J. C., Sullivan, D. R., and Stewart, E M. (2003). Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population. Clin. Chem. 49, 1297-1308.
CHAPTER
15 Interactions of Organophosphorus and Carbamate Compounds with C h o l i n e s t e r a s e s
LESTERG. SULTATOS New Jersey Medical School, Newark, New Jersey
I. I N T R O D U C T I O N Cholinesterases are enzymes that are serine hydrolases that preferentially hydrolyze choline esters. Vertebrates are known to have two cholinesterases, referred to as acetylcholinesterase (ACHE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). These two cholinesterases have been differentiated historically on the basis of their substrate selectivity. AChE hydrolyzes acetylcholine (ACh) faster than butyrylcholine (BuCh) or propionylcholine, whereas BuChE metabolizes BuCh and propionylcholine faster than ACh. This difference in substrate selectivity is thought to arise from differences in acyl pocket structure, which in the case of BuChE allows access of larger substrates to the active site (Harel et al., 1992; Cygler et al., 1993; Kovarik et al., 2003). AChE is known to play a critical role in the termination of the action of ACh (Fig. 1) at synapses and neuromuscular junctions, whereas the exact function(s) of BuChE remains unclear. However, numerous potential roles for BuChE have been suggested, ranging from metabolism of lipoproteins (Kutty and Payne, 1994) to cell adhesion (Tsigelny et al., 2000), and the etiology of certain neurodegenerative diseases (Darvesh et al., 2003). Many organophosphorus (OP) and carbamate (CM) compounds exert their acute toxicity through inhibition of ACHE. These compounds include all of the OP and most of the CM pesticides, as well as certain chemical warfare agents. Additionally, a limited number of OP and CM drugs exert their therapeutic effects through inhibition of AChE (Taylor, 2001). Inhibition of this critical enzyme leads to an accumulation of ACh at synapses and neuroeffector junctions (Fig. 1), which in turn leads to a range of symptoms known as cholinergic crisis. Although inhibition of BuChE by these compounds has not been shown to result in a specific adverse event, BuChE is considered to represent an important nonenzymatic pathway for detoxification of
Toxicology of Organophosphate and Carbamate Compounds
@ ACh ACh
~
ACh 9 A + Ch ~ ~
ACh
ACh ACh
ACh
ACh
ACh -I~ A + Ch
,
FIG. 1. Role of AChE at cholinergic nerve endings. AChE catalytic subunits are represented by the shaded circles. Acetylcholine (ACh) stored in presynaptic terminals is released in response to depolarization of the nerve and diffuses across the synapse or junction to bind to and activate postjunctional cholinergic receptors. The postjunctional action of ACh is terminated primarily through its hydrolysis to acetate and choline by AChE. Inhibition of AChE by OP or CM compounds results in an accumulation of ACh within synapses and neuroeffectorjunctions, leading to cholinergic crisis. anticholinesterase compounds and is often used as a surrogate measure for inhibition of AChE elsewhere in the body.
II. I N H I B I T I O N OF C H O L I N E S T E R A S E S BY OPs A N D CMs The cholinesterases (ChEs) are serine hydrolases that catalyze the breakdown of ACh through an acyl-transfer, where water is the acceptor molecule to which the substrate acyl 209
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
2 10
SECTION III 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
moiety is transferred (Walsh, 1979). A serine oxygen of the active site gorge in ChEs carries out a nucleophilic attack on the electrophilic carbon of the carbonyl group of ACh, resulting in an acetylated enzyme intermediate and the release of choline (Fig. 2) (Wilson et aL, 1950; Froede and Wilson, 1984; Quinn, 1987). Deacylation occurs when an attacking water molecule (hydroxyl ion) acts as a more effective nucleophile, thereby releasing acetate (Fig. 2) (Walsh, 1979). The inhibition of AChE and BuChE by OPs and CMs can be viewed in many ways as a reaction analogous to that of the hydrolysis of acetylcholine by these same enzymes (Aldridge and Reiner, 1972) (Fig. 2). The molecular interactions between certain OPs and AChE have been investigated utilizing a variety of techniques, and much is known regarding how OPs bind to and phosphylate ACHE. Ordentlich et al. (1996) reported that the acyl pocket of the active gorge in AChE (Phe295 and Phe297) (all numbers in this chapter refer to amino acid positions in human AChE or BuChE) participates in the positioning of an OP molecule for the in-line attack by the catalytic
serine (Ser203), with Phe297 more important for branched alkoxy substituents, which are larger in volume (Fig. 3). This positioning can be interpreted as analogous to the formation of a Michaelis complex. Ordentlich et al. concluded that the ability to form these Michaelis complexes is extremely important in determining the reactivity of OPs toward AChE (and presumably BuChE as well). In addition to the acyl pocket, the peripheral anionic site as well as other subsites play important roles in the stereoselectivity toward enantiomers of the chemical warfare agent VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (Ordentlich et al., 2004) (Fig. 3). Replacement of Asp74 of the peripheral anionic site with asparagines (D74N) nearly eliminated the stereoselectivity of AChE toward the p s - v x enantiomer (Ordentlich et al., 2004). The charged leaving group of p s - v x interacts with the acidic Asp74 to promote formation of the Michaelis complex, thereby accounting for, at least in part, the greater reactivity of the p s - v x enantiomer compared to pR-vx (Ordentlich et al., 2004).
OH S~r I
lb
[la
,RI?
0II /CH3 CH3"- (~'-O--CH2--CH2- N - - C H 3 t ~'CH3
t I
O
,,CH 3
CH 3- (~ + NO - CH 2- CH 2- N ~, CH 3 I
O
CH3
sgr I
2c
,R,O CH 3- N - C + H O - R 2 I
O R2-O ,,~ R1-O " I + HO-R 3
?
Ser !
I
J
3c
3b
R,,O
oii
CH 3 - N - C~...OH OH-
Set
o s~r
-
I
OH
R 1- O
o
,R10 CH 3 - N - C I
OH
s~r I
OH-
4c
4b
o
ii CH 3 - C
;~o
R=-O
Sgr I
i
4a
I,
J
S~r
3a
OH
I
J,~
J
s~r
g;
OS~r
2a
I
oii 1R2-O-O~ - O -R3 R
CH 3- N - C - O - R 2
I
CH3- CI O
[,c
Organophosphate
Carbamate
Acetylcholine
OH
O R2-O ,,J' R 1-O "P
OH
OH
S~r I
FIG. 2. The interaction of ACh, CM, and OP compounds with the active site serine of AChE and BuChE. Reaction 1 represents the formation of a stable Michaelis complex and the beginning of the nucleophilic attack of the serine. Reaction 2 represents the acylation of the active site serine, coupled with the release of the first reaction product or leaving group. Reaction 3 begins the nucleophilic attack of a hydroxyl ion, which leads to the regeneration of active enzyme (reaction 4).
CHAPTER 1 5 9Interactions of OP and CM Compounds with Cholinesterases Peripheral Anionic I Binding Site
S CH 3-O ii
c. -o
~p37~_. 0 , Oxyanion Hole Acyl Binding Site
RiO
/
-0""
...
I
Ser203
O CH2CO2C2H5
Malathion
IC5o= 2.9 mM
OR 2
~
HN
/
0
N. . . . ~ " .o
His447 Catalytic Triad
Glu334
FIG. 3. Schematic drawing of the active site gorge of ACHE, with the entry of an OP molecule. R] and R2 on the OP are usually identical alkyl chains, whereas R 3 is the leaving group. The catalytic triad consists of Ser203, His447, and Glu334. The acyl binding site is likely important in positioning the inhibitor for the nucleophilic attack from Ser203 (Ordentlich et al., 1996), whereas the oxyanion hole may polarize the P=O bond, thereby facilitating the nucleophilic attack (Ordenflich et al., 1998). Binding of ligand to the peripheral anionic site can lead to inhibition or activation (Masson et al., 2004). Additionally, the peripheral anionic site plays an important role in the stereoselectivity of AChE toward methylphosphonates (Ordentlich et aL, 2004).
Ordentlich et al. (1998) suggested that the oxyanion hole subsite (Glyl21, Gly122, and Ala204) may polarize the P--O bond (or the C = O bond in the case of carbamates) during formation of the Michaelis complex and therefore activate the phosphorus, thereby promoting a nucleophilic attach by the active site serine oxygen. This nucleophilic attack is made possible through a proton transfer from Ser203 (ACHE) or Ser226 (BuChE) to a nitrogen in the imidazole ring of His447 (ACHE) or His466 (BuChE), which in turn transfers a proton from the second nitrogen of the imidazole ring to the carboxyl group of Glu334 (ACHE) or Glu353 (BuChE; Fig. 3) (Soreq and Seidman, 2001). The serine is phosphylated, and the remaining half of the molecule (the leaving group) disassociates from the enzyme. Although the acetylated enzyme intermediate in the case of AChE hydrolysis is rapidly broken down by a water molecule, the phosphylated intermediate is usually very stable and is only slowly destroyed by an attacking water molecule. The presence of the phosphyl moiety covalently bound to the active site serine prevents AChE and BuChE from hydrolyzing ACh, thereby leading to the accumulation of this transmitter and cholinergic crisis. It should be noted that most OP insecticides are generally poor inhibitors of AChE and BuChE since they contain a P = S moiety. However, they are metabolically activated by cytochromes P450 to produce the oxygen analogs, also known as oxons, that are very potent anticholinesterase compounds (Fig. 4). The greater capacity of the oxygen
211
LD5o = 2600 mg/kg
CH 3-O
CH2CO2C2Hs Malaoxon
IC5o= 700 nM LDso = 308 mg/kg
FIG. 4. Comparison of the anticholinesterase capacity and acute toxicity of the OP malathion (dicarbethoxyethyl-O,Odimethyldithiophosphate) with its oxon, malaoxon (O,O-dimethyl S-1,2-bis(ethoxycarbonyl)ethyl phosphorothioate). It should be noted that ICs0s were determined under identical conditions. These data were taken from Eto (1974).
analogs to inhibit AChE and BuChE results from the greater electronegativity of oxygen compared to sulfur, and therefore greater polarization of the P = O linkage compared to the P--S linkage. This in turn leads to a more electrophilic phosphorus in the oxons, which greatly facilitates the nucleophilic attack by the oxygen on the active site serine residue of the enzyme. Consequently, it is generally assumed that the inhibition of AChE and BuChE following exposure to an OP insecticide occurs as a result of the oxygen analog and not the parent compounds. The CM anticholinesterases are thought to inhibit AChE and BuChE in a manner similar to that of OPs. However, instead of an electrophilic phosphorus, as in the case of the OPs, CMs contain an electrophilic carbonyl carbon, which undergoes nucleophilic attack by the active site serine oxygen (Fig. 2). The resulting carbamylated enzyme intermediate inhibits enzyme activity until a water molecule attacks the carbonyl carbon to reactivate enzyme and produce a carbamic acid derivative (Fig. 2). This rate of reactivation is considerably faster than that of phospylated enzyme, although it is not as rapid as reactivation of the acetylated intermediate.
III. KINETIC SCHEMES FOR AChE METABOLISM AND INHIBITION BY OPs AND CMs For many decades, it has been thought that the interactions of the anticholinesterase OP and CM compounds with AChE and BuChE are kinetically analogous to those of ACh. In the absence of binding of ACh to the peripheral anionic site of AChE (the validity of this assumption is discussed later), hydrolysis of ACh by AChE or BuChE can be described by a Ping Pong Bi Bi kinetic scheme (Fig. 5). In this scheme, ACh represents the first substrate, whereas water represents the second. Additionally, choline is the first product formed, and it is released before the second
21 2
S E CTI O N I!I 9 Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
AB+E
k~
~
k_ 1
k~
E"AB
~
k_2
E-A
+
H20~
k3
k_3
+
B
AB+E
k~
~
k-1
E..AB
AB+E
E +
A
k~
-->E-A-->
k3
E
+
+
B
A
k,
"-> E - A m > E +
+
B
A
KD = K_I l K1 Ki= K2 / KD FIG. 5. Kinetic models descriptive of the interaction of CMs and OPs with AChE and BChE. The top panel represents an Ping Pong Bi Bi kinetic mechanism, with the following parameter definitions: AB is the CM or OP; E is free, active cholinesterase; E . . AB is the Michaelis complex between enzyme and CM or OP; E-A is the carbamylated or phosphylated intermediate; B is the first product released (leaving group); kl is the association rate constant for the formation of the Michaelis complex; k-1 is the dissociation rate constant for the Michaelis complex; k2 is the rate constant for acylation of the active site serine; k-2 is the rate constant for the reverse acylation step (negligible for CMs and OPs); k3 is the rate constant descriptive of the reactivation of enzyme by water; and k_ 3 is the rate constant for the reverse reactivation reaction (also negligible for CMs and OPs). As described by Segel (1975), since water is always present in excess, the Ping Pong Bi Bi reaction mechanism reduces to an Ordered Uni Bi reaction mechanism (middle). Note that k_ 2 and k_ 3 have been eliminated in the middle panel. The bottom panel represents the ki scheme, derived by Main (1964) from the Ordered Uni Bi reaction mechanism.
substrate binds. The second product is acetate. This reaction is extremely fast, with a turnover number greater than 104 sec -1 (Quinn, 1987). Because water is always present in excess, the Ping Pong Bi Bi reaction scheme can be simplified to an Ordered Uni Bi kinetic scheme (Fig. 5), in which there is one substrate with two products that are formed in a specific order (Segel, 1975). Note that the kinetic interactions of substrates with AChE and BuChE are in reality more complex than portrayed in Fig. 5. Both cholinesterases have been shown to display substrate inhibition and activation, depending on the incubation conditions (Masson et al., 2004), probably as a result of the presence of a binding site separate from the active site, termed the peripheral anionic site (Changeux, 1966; Taylor and Radi~, 1994; Barak et al., 1995; Soreq and Seidman,
2001; Bourne et al., 2003). Furthermore, occupation of the peripheral anionic site of AChE by ACh in vivo likely occurs since the evoked release of ACh at terminal endings can produce local concentrations in narrow synaptic clefts in the millimolar range (Van Der Kloot and Molg6, 1994). Moreover, BuChE has been shown to display an oscillatory hysteretic approach to steady state with some substrates under certain incubation conditions by an unknown molecular mechanism (Masson et al., 2004). However, because these kinetic complexities do not appear to affect the basic mechanism of the hydrolysis of ACh by the cholinesterases as outlined in Figs. 2 and 5 but instead alter the rates at which these events occur, the Ordered Uni Bi scheme can be said to represent the minimal catalytic mechanism for the hydrolysis of substrate by cholinesterases (Barnett and Rosenberry, 1977). Alternatively, more complex, comprehensive kinetic models of cholinesterases have been suggested in order to account for the documented substrate inhibition and activation (Barnett and Rosenberry, 1977; Radi~ et al., 1993; Stojan et al., 1998; Rosenberry et al., 1999; Goli~nik, 2001), as shown in Fig. 6. In these schemes, binding to a peripheral site may alter events at the active site gorge by a variety of putative mechanisms that may affect one or more steps outlined in the kinetic scheme shown in Fig. 5. Note that in most cases, the molecular mechanisms resulting in inhibition or activation of enzyme are not completely understood. The interactions of CM and OP anticholinesterase compounds with AChE and BuChE have also been viewed as a Ping Pong Bi Bi kinetic mechanism that simplifies to an Ordered Uni B i kinetic mechanism since water is always present in excess. The primary difference between hydrolysis of ACh and inhibition by OP and CM compounds is the rate at which the acylated intermediates are reactivated by water to form the second product and free, uninhibited enzyme (reaction 3 in Fig. 2 and k3 in Fig. 5). In the case of ACh, the acetylated enzyme exists for only
AB+E
qb AB + AB--E
"--~,---E-.AB /-E-A--->.E
+B
k~
+A
AB--E-'AB - - - ~ A B " E - A ~
+B
k:~
k4
AB-.E ~
+A
k-4
AB + E
FIG. 6. General kinetic scheme proposed for hydrolysis of ACh by AChE and BuChE. The minimal catalytic mechanism scheme (top line) is identical to that in Fig. 4. Binding to a modulatory site is represented in the bottom line by AB 9 9E. Those rate constants represented as k' designate steps that could be altered (either increased or decreased) as a result of ligand binding to the modulatory site. Adapted from Barnett and Rosenberry (1977) and Stojan et al. (1998).
CHAPTER 1 5 9Interactions of OP and CM Compounds with Cholinesterases a fraction of a second before a water molecule regenerates free enzyme and forms acetate. In contrast, spontaneous regeneration of a carbamylated enzyme intermediate usually requires several to many minutes depending on the structure of the carbamate moiety. For example, the halflife for the recovery of N-methylcarbamylated AChE is approximately 30 min (Fukuto, 1990). Still longer is the time required for the reactivation of a phosphylated enzyme intermediate formed by an OE The half-life for the recovery of a phosphylated intermediate can range from several hours to days, depending on the structure of the particular OE Moreover, certain phosphylated intermediates can undergo a process termed aging (see Section VI), after which regeneration cannot occur. Beginning with the Ordered Uni B i kinetic reaction scheme, Main (1964) derived a bimolecular inhibition rate constant, ki, which quantifies the inhibitory power of an OP toward AChE (the concept of k i can also be extended to BuChE). The k i is a function of both KD and k2 (Fig. 5) and has been considered the best estimate of inhibitory capacity of an OP (Fukuto, 1990). The larger the ki, the greater is the capacity of an individual molecule of OP to inhibit an individual molecule of enzyme. Although k i was derived based on OP interactions with ACHE, the term k i can be applied with the same meaning to the inhibition of enzyme by the CMs as well (O'Brien, 1968). Recent studies have raised questions about the accuracy of k i, and therefore the kinetic schemes in Figure 5, in describing the inhibitory capacity of AChE insecticides. Kardos and Sultatos (2000) reported that the k i for the OP paraoxon (and methyl paraoxon) changed over a wide range of inhibitor concentrations, suggesting that at low oxon concentrations an individual paraoxon molecule has a greater capacity to inhibit enzyme than an individual paraoxon molecule at high oxon concentrations. If viewed as an enzymatic reaction in which the oxon is hydrolyzed by ACHE, this phenomenon would be considered substrate inhibition. However, the stability of the phosphylated intermediate precludes the use of this terminology. The k i of an OP should be invariant, unless KD and/or k2 change as a function of oxon concentration (Fig. 5). Similar results were observed for chlorpyrifos oxon (Kousba et al., 2004). The authors suggested that these oxons may bind to a secondary site distinct from the active site and alter reactivity of the active site toward other oxon molecules through either steric hindrance or allosteric modification m a phenomenon that has been described in detail for occupation of the peripheral anionic site by certain ligands (Taylor and Radic, 1994). Consequently, just as the kinetic schemes in Fig. 5 represent the minimal catalytic mechanism for hydrolysis of substrate by ChEs, these same kinetic schemes probably represent the minimal inhibitory mechanism that describes, in part, the interactions of certain OPs with ACHE.
213
IV. S T R U C T U R E - A C T I V I T Y
RELATIONSHIPS The role of OP and CM structure in the inhibition of AChE has been studied intensively for decades. Structure-activity studies have often focused on the effects of chemical structure on reversible binding of inhibitor to enzyme, usually quantified by KD (Fig. 5) (formation of Michaelis complexes), and the phosphylation or carbamylation step, usually quantified by k2 (Fig. 5). Although investigators have sometimes debated the relative importance of reversible binding to the active site versus the phosphylation or carbamylation step in determining overall reactivity of inhibitor toward the enzyme, it is clear that both steps are critical in controlling inhibitor reactivity, and both steps are controlled by chemical structure and the structure of the active site gorge.
A. OPs For an OP to possess the capacity to phosphylate AChE or BuChE, the phosphorus must be electrophilic enough to accept a nucleophilic attack from the active site serine. As described by Fukuto (1990), structure-activity studies have found a direct relationship between the reactivity of the phosphorus atom and the capacity to phosphylate AChE and BuChE. As stated previously, the presence of a sulfur bonded to the phosphorus, such as can be found with most OP insecticides, renders the molecule a rather weak inhibitor, whereas replacement of the sulfur with an oxygen markedly increases the capacity to phosphylate AChE and BuChE due to the greater electron-withdrawing capacity of the oxygen (Fig. 4). The greater electronegativity of the oxygen therefore renders the phosphorus more electrophilic and more susceptible to a nucleophilic attack (Eto, 1974). Other substituents with varying electronwithdrawing capacity attached to the phosphorus will similarly affect anticholinesterase activity. The greater the electron-withdrawing capacity of the substituent, the greater the anticholinesterase activity of the OP (Eto, 1974). Systematic studies with paraoxon (diethyl p-nitrophenyl phosphate) and similar substituted phenyl phosphates have shown that their anticholinesterase activity is a direct function of their alkaline hydrolysis rates, which reflects phosphorus reactivity (Fukuto, 1990). The greater electron-withdrawing capacity of a particular substituent makes the P-O phenyl bond deficient in electron density and consequently makes the ester readily accept nucleophilic substitution on the phosphorus (Eto, 1974). Likewise, diethyl-substituted phenyl phosphates with little electron-withdrawing capacity has little or no anticholinesterase activity (Fukuto, 1990). Of equal importance to phosphorus reactivity are the steric properties of the OP with respect to the active site gorge of the enzyme. Unless an OP molecule can diffuse into the active site gorge and form a stable Michaelis complex,
2 14
S ECTI 0 N I II 9Esterases, Receptors, Mechanisms, & Tolerance Development
phosphylation will not have the opportunity to occur, regardless of phosphorus reactivity. Early studies demonstrated that the length of alkyl side chains was a critical determinant of anticholinesterase activity, with reduced inhibitory capacity associated with bulkier alkyl side chains (Fukuto and Metcalf, 1959; Hansch and Deutsch, 1966; Eto, 1974). These studies demonstrated that for anticholinesterase activity, the alkyl side chains should remain small (methyl or ethyl groups). In this regard, most OP insecticides possess either methyl or ethyl alkyl side chains, although some noninsecticidal OPs have isopropyl groups for side chains. Furthermore, the importance of steric properties was confirmed in recent studies in which specific AChE mutations that decreased the stability of Michaelis complexes of certain OPs decreased their anticholinesterase activity (Ordentlich et al., 1996). Additionally, the differences in the acyl pocket structure of AChE and BuChE allow the BuChE active site to better accommodate bulkier OPs such as iso-OMPA (N,N'diisopropyl phosphorodiamidic anhydride) and mipafox (N,N'-diisopropyl phosphorodiamidic fluoride). Iso-OOMPA has often been used as a selective inhibitor of BuChE experimentally (Austin and Berry, 1953; Harel et al., 1992). Although the acyl pocket is critical in determining the "fit" of OPs into the active site gorge, Ordenflich et al. (2004) documented the importance of the peripheral anionic site, as well as several additional sites, in determining the stereoselectivity of AChE toward enantiomers of methylphosphonates. B. C M s Extensive structure-activity relationships with CM insecticides have revealed general similarities with those of the OPs. Specifically, the anticholinesterase activity of a CM is primarily a function of the reactivity of the carbonyl carbon (rather than the phosphorus of OPs) and the ability of the CM to diffuse into the active site gorge and form Michaelis complexes (steric properties). Chemical modifications that reduce the electrophilic nature of the carbonyl carbon reduce the anticholinesterase activity, whereas the opposite is observed with substituents that increase the electrophilicity of this same carbon atom (Kuhr and Dorough, 1976; Fukuto, 1990). In contrast to the OPs, most (but not all) CMs can directly inhibit ChEs and do not require metabolic activation. In this regard, the reactivity of the carbonyl carbon for most methylcarbamate insecticides is so significant that the equilibrium constant for dissociation of the enzyme-inhibitor complex (Ko) correlated directly with the inhibitory capacity of the molecule (Fukuto, 1990). As is the case with the OPs, the steric properties of a CM modulate the access of the CM to the catalytic serine. Bulky substituents reduce access, whereas others increase access. For example, in phenyl N-methylcarbamates the placement of the substituent alkyl groups on the ring relative to the CM moiety influences anticholinesterase activity. Substitution at the 3-position produced the most active compounds,
probably because the distance between the CM group and the meta-substituent was most conducive to formation of a Michaelis complex (Khur and Dorough, 1976). Similary, additions of larger, bulkier substituents reduced anticholinesterase activity (Khur and Dorough, 1976). Only limited carbamate structure-activity relationship studies have been done with regard to comparisons of the inhibitory capacity toward AChE versus BuChE. However, based on structure-activity studies with five N-methyl carbamates, Loewenstein et al. (1993) concluded that the CM binding site on BuChE is less flexible than the same site on ACHE.
V. OXIME REACTIVATION OF I N H I B I T E D CHOLINESTERASES The classic studies of Wilson and Ginsburg (1955a,b) established that reactivation of phosphylated AChE could be accelerated by certain compounds more nucleophilic than water, such as N-methylhydroxylamine. Since that time, a variety of reactivators, termed oximes, have been synthesized for use in the treatment of subjects poisoned by OP anticholinesterases. In the United States, the oxime pralidoxime [N-methyl-(2-hydroxyaminoformylpyridinium)chloride] is the only reactivator approved for use (Fig. 7), whereas obidoxime (bis[4-hydroxyamino-
O
It R,-o.,p R1-O
O
3
O ~
S~r
11 I .,~--~ O-N=HCR2- O I,p R1-O O Sler
I
R1-O" I O
+
R2
Sir
Aged Form
t CH3
O
OH sler
I
R2- O " RI_O ~ P - O - N - H C -
I
CH3
FIG. 7. Reactivation and aging of phosphylated cholinesterase. Reaction 1 represents the nucleophilic attack of pralidoxime on the phosphate, followed by regeneration of free, active enzyme and the phosphylated oxime (reaction 2). Reaction 3 represents aging and therefore the formation of a monophosphylate ester that is resistant to reactivation by pralidoxime (and other oximes).
CHAPTER 1 5 9Interactions of OP and CM Compounds with Cholinesterases methyl-methylpyridinium] ether dichloride) is also available in other areas of the world (Kwong, 2002). Other experimental reactivators include HI-6 [1-(2-hydroxyiminomethyl1-pyridinium)-l-(4-carboxyaminopyridinium) dimethyl ether hydrochloride], TMB4 [1,1'-trimethylene bis(4-(hydroxyimino)-methyl) pyridinium dibromide], and LtiH6 [1,1'-(oxybismethylene) b i s ( 4 - ( h y d r o x y i m i n o ) m e t h y l ) p y r i dinium dichloride] (Rousseaux and Dua, 1989; Luo et al., 1999). Pralidoxime (and other reactivators) accelerates reactivation of phosphylated AChE as a result of a nucleophilic attack of the oxime on the phosphorus of the phosphylated enzyme to give phosphylated oxime and free, active enzyme (Fig. 7), provided that aging has not occurred (see Section VI). Aging greatly increases the resistance to reactivation, even by powerful reactivator oximes. The rate of reactivation of phosphylated enzyme by an oxime is dependent on the structure of the phosphyl moiety, the nature of the enzyme, and the structure of the oxime reactivator (Luo et al., 1998). Interestingly, certain phosphylated oximes have been shown to have the capacity to reinhibit reactivated ACHE, thereby reducing their potential as treatment of OP intoxication (Luo et al., 1998, 1999; Kideden et al., 2000). This inhibition can be prevented by certain drugs, such as edrophonium, that compete with the phosphylated oxime for the ACHE active site (Luo et al., 1998, 1999). Pralidoxime is not used for treatment of CM insecticide poisoning since it enhanced the toxicity of carbaryl (1-naphthyl N-methylcarbamate) (Harris et al., 1989). However, pralidoxime and the reactivator HI,6 reduced the lethality of the CM drug physostigmine in rats, providing a rational basis for the use of physostigmine or pyridostigmine pretreatment in conjunction with the standard therapy for exposure to the highly toxic nerve gases (Harris et al., 1989). Although the exact molecular interactions between phosphylated enzyme and oxime that lead to reactivation are not known, Ashon et al. (1995) and Wong et al. (2000), utilizing mutant forms of ACHE, identified a number of amino acid residues that are important in the reactivation of phosphylated ACHE. Wong et al. suggested that a major determinant of oxime-induced reactivation rates is simply the ability of the reactivator to "fit" into the active center gorge and gain access to the tetrahedral phosphorus. This fit is controlled by the structure of the oxime and the steric bulk of the intervening groups surrounding the tetrahedral phosphorus (Wong et al., 2000).
VI. AGING OF PHOSPHORYLATED CHOLINEsTERASE Following the phosphylation of serine at the active site of AChE or BuChE by certain OP compounds, a dealkylation of the phosphyl moiety can occur. This reaction, referred to
215
as "aging," results in the loss of an alkyl group from the phosphyl alkoxy substituent (Fig. 7), probably through the formation of a carbocationic transition state that forms a carbonium ion that rearranges to form alkene products (Smith and Usdin, 1966; Michael et al., 1967; Bencsura et al., 1995). This reaction is of great toxicological significance since the remaining negatively charged monophosphylate ester of serine is resistant to reactivation by nucleophilic oximes such as pralidoxime (Aldridge and Reiner, 1972; Worek et al., 1996). Consequently, the activity of aged cholinesterase in a patient poisoned with an anticholinesterase OP compound can only be regained through synthesis of new enzyme and not through treatment with the antidotal oximes. Thus, the recovery of aged red blood cell cholinesterase is longer than that of aged plasma cholinesterase since the turnover of red blood cells is much slower than the turnover of plasma cholinesterase (Mason, 2000; Mason et al., 2000). The rate of aging, and therefore the extent of aging, that occurs with phosphylated AChE or BuChE is a function of the chemical structure of the phosphyl moiety. Most OP insecticides have either two methoxy or two ethoxy side chains, and the methoxy compounds have been reported to age faster than the corresponding inhibitors with ethoxy side chains. For example, Worek et al. (1999) reported that the aging half-time of diethylphosphorylated AChE (31 hr) is approximately eight times longer than the aging halftime of dimethylphosphorylated ACHE. In contrast, aging of the nerve agents such as soman is markedly faster. Talbot et al. (1988) reported that the aging half-time for human red blood cell somanyl-AChE is approximately 1 min. Phosphylated BuChE has been reported to age at approximately the same rate or slightly faster than phosphylated AChE (Worek et al., 1999; Masson et al., 1997). Although the specific molecular events that lead to aging are not known with certainty, considerable progress has been made in understanding the details of aging of compounds such as soman and diisopropyl fluorophosphate through approaches combining site-directed mutagenesis, kinetic analyses, and molecular modeling studies. It is unlikely that aging of AChE or BuChE phosphorylated by OPs will be markedly different. In what has been referred to as a "push-pull" mechanism for aging, carbocation formation has been suggested to result from the strong negative electrostatic field of Glu202 (Kovach et al., 1997; Saxena et al., 1998). Shafferman et al. (1996) suggested that Glu202 and Phe338 contribute to aging by stabilizing the imidazolium of the catalytic triad His447, which has been proposed to act as an acid catalyst for the cleavage of the C-O bond (Ordentlich et al., 1993; Qian and Kovach, 1993). However, many other active site residues have also been implicated in the aging of somanyl-AChE, including Asp72, Trp86, Tyr133, Ser199, Glu202, and Glu450 (Shafferman et al., 1996; Masson et al., 1997).
21 6
S E CTI O N I I I
9Esterases, Receptors, Mechanisms,
In BuChE phosphylated by DFP, Trp82 seems to stabilize the carbonium ion released during aging, and Glu197 carboxylate appears to help stabilize the developing carbocation (Masson et al., 1997). Finally, Asp70 exerts allosteric control of dealkylation probably by altering the conformation state of Trp82 (Masson etal., 1997). As stated previously, the primary toxicological significance of aging is that aged phosphylated enzyme cannot be reactivated, even by the oximes. Resistance to reactivation has been attributed, at least in part, to two possible mechanisms. The first is an aging-induced conformation change of the inhibited enzyme, resulting in increased stability. Masson et al. (1997) suggested that such a change may result from the formation of a salt bridge between the catalytic protonated histidine and the negatively charged oxygen atom on the monophosphylate moiety. Second, aged ChEs may electrostatically repulse oximes as a result of the negatively charged oxygen of the monophosphylate moiety and the adjacent negatively charged Glul00 (Masson et al., 1997).
& Tolerance Development
inhibition profiles of wild-type and the atypical variant of BuChE toward five different N-methyl carbamates were not different. The atypical variant of BuChE they examined has approximately 30% lower activity toward succinylcholine than wild-type BuChE (Loewenstein et al., 1993; Neville et al., 1990). Finally, genetically engineered human BuChEs have been developed that are resistant to OP (and presumably CM) inhibition since the reactivation step by water (Fig. 2) is markedly accelerated in these mutants (Millard et al., 1995; Schopfer et al., 2004). Since human wild-type BuChE has been shown to act as a scavenger to protect against OP toxicity in animal models (Broomfield et al., 1991; Raveh et al., 1997), BuChE mutants with the capacity to hydrolyze OPs (and perhaps CMs) may eventually be utilized in the antidotal treatment for OP or CM intoxication.
References Aldridge, W. N., and Reiner, E. (1972). Enzyme Inhibitors as Substrates. Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland,
VII. GENETIC VARIANTS Genetic variants of AChE and BuChE have been identified. However, unlike AChE variants, BuChE variants display varied substrate activities. Since the classic work of Kalow and coworkers, who first investigated this phenomenon (Kalow and Staron, 1957; Kalow and Dunn, 1959), more than 40 variants of BuChE have been identified (Darvesh et al., 2003). The wild-type BuChE allelic frequency has been reported to be approximately 85%, whereas two variants, termed K and the atypical form, have reported allelic frequencies of 10 and 13%, respectively (Darvesh et al., 2003). The allelic frequencies of the remainder of the identified variants are extremely low. Although most of the BuChE variants have not been fully characterized, it is known that they generally have reduced enzymatic activity compared to the wild type (La Du et al., 1990; Darvesh et al., 2003), and at least 12 have no activity at all (Primo-Parmo et al., 1996). The functional significance of BuChE variants in the metabolism of certain drugs, such as succinylcholine, has been well characterized (Darvesh et al., 2003). However, their functional significance with respect to OPs and CMs is less clear. BuChE is thought to serve as a nonenyzmatic pathway for the detoxification of OPs and CMs. Given the lower activities of BuChE variants, it has been proposed that certain individuals with reduced BuChE activity will have a reduced capacity to "scavenge" anitcholinesterase OPs and CMs and therefore may have a genetic predisposition to the adverse effects of the anticholinesterase OPs and CMs (Loewenstein-Lichtenstein et al., 1995; Fontoura-da-Silva and Chautard-Freire-Maia, 1996; Lockridge and Masson, 2000). Although this is a reasonable proposition, it should be noted that Loewenstein et al. (1993) reported that the
New York. Ashon, Y., Radic, Z., Tsigelny, I., Vellom, D. C., Picketing, N. A., Quinn, D. M., Doctor, B. P., and Taylor, P. (1995). Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes. J. Biol. Chem. 270, 6370-6380. Austin, L., and Berry, W. K. (1953). Two selective inhibitors of cholinesterase. Biochem. J. 54, 695-700. Barak, D., Ordentlich, A., Bromberg, A., Kronman, C., Marcus, D., Lazar, D., Ariel, N., Velan, B., and Shafferman, A. (1995). Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite. Biochemistry 34, 15444-15452. Barnett, P., and Rosenberry, T. L. (1977). Cataysis by acetylcholinesterase. J. Biol. Chem. 252, 7200-7206. Bencsura, A., Enyedy, I., and Kovach, I. M. (1995). Origin and diversity of the aging reactions in phophonate adducts of serine hydrolase enzymes: What characteristics of the active site do they probe? Biochemistry 34, 8989-8999. Bourne, Y., Taylor, P., Radi~, Z., and Marchot, P. (2003). Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site. E M B O J. 22, 1-12. Broomfield, C. A., Maxwell, D. M., Solana, R. P., Castro, C. A., Finger, A. V., and Lenz, D. E. (1991). Protection by butyrylcholinesterase against organophosphorus poisoning in nonhuman primates. J. Pharmacol. Exp. Ther. 259, 633-638. Changeux, J. P. (1966). Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing agents. Mol. Pharmacol. 2, 369-392. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Doctor, B. P. (1993). Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related enzymes. Protein Sci. 2, 366-382. Darvesh, S., Hopkins, D. A., and Geula, C. (2003). Neurobiology of butyrylcholinesterase. Nat. Rev. Neurosci. 4, 131-138.
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La Du, B. N., Bartels, C. E, Nogueira, C. R, Hajara, A., Lightstone, H., van der Spek, A., and Lockridge, O. (1990). Phenotypic and molecular biological analysis of human butyrylcholinesterase variants. Clin. Biochem. 23, 423-431. Lockridge, O., and Masson, E (2000). Pesticides and susceptible populations: People with butyrylcholinesterase genetic variants may be at risk. Neurotoxicology 21, 113-126. Loewenstein, Y., Denarie, M., Zakut, H., and Soreq, H. (1993). Molecular dissection of cholinesterase domains responsible for carbamate toxicity. Chem.-Biol. Interact. 87, 209-216. Loewenstein-Lichtenstein, Y., Schwarz, M., Glick, D., NorgaardPedersen, B., Zakut, H., and Soreq, H. (1995). Genetic predisposition to adverse consequences of anti-cholinesterases in "atypical" BCHE carriers. Nat. Med. 1, 1082-1085. Luo, C., Ashani, Y., and Doctor, B. E (1998). Acceleration of oxime-induced reactivation of organophosphate-inhibited fetal bovine serum acetylcholinesterase by monoquaternary and bisquaternary ligands. Mol. Pharmacol. 53, 718-726. Luo, C., Saxena, A., Smith, M., Garcia, G., Radic, Z., Taylor, E, and Doctor, B. E (1999). Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium. Biochemistry 38, 9937-9947. Main, A. R. (1964). Affinity and phosphorylation constants for the inhibition of esterases by organophosphates. Science 144, 992-993. Mason, H. J. (2000). The recovery of plasma cholinesterase and erythrocyte acetylcholinesterase activity in workers after overexposure to dichlorvos. Occup. Med. 50, 343-347. Mason, H. J., Sains, C., Stevenson, A. J., and Rawbone, R. (2000). Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides. Hum. Exp. Toxicol. 19, 511-516. Masson, E, Fortier, E-L., Albaret, C., Froment, M.-T., Bartels, C. E, and Lockridge, O. (1997). Aging of di-isopropyl-phosphorylated human butyrylcholinesterase. Biochem. J. 327, 601-607. Masson, E, Goldstein, B. N., Debouzy, J.-C., Froment, M.-T., Lockridge, O., and Schopfer, L. M. (2004). Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate. Eur. J. Biochem. 271, 220-234. Michael, H. O., Hackley, B. E., Berkovitz, L., List, G., Hackley, E. B., Gillian, W., and Pankau, M. (1967). Aging and dealkylation of soman (pinacolylmethylphosphonofluoridate)-inactivated eel cholinesterase. Arch. Biochem. Biophys. 121, 29-34. Millard, C. B., Lockridge, O., and Broomfield, C. A. (1995). Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase. Biochemistry 34, 15925-15933. Neville, L. E, Gnatt, A., Padan, R., Seidman, S., and Soreq, H. (1990). Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations. J. Biol. Chem. 265, 20735-20738. O'Brien, R. D. (1968). Kinetics of the carbamylation of cholinesterase. Mol. Pharmacol. 4, 121-130. Ordentlich, A., Kronman, C., Barak, D., Stein, D., Ariel, N., Marcus, D., Velan, B., and Shafferman, A. (1993). Engineering resistance to "aging" of phosphylated human acetylcholinesterase: Role of hydrogen bond network in the active center. FEBS Lett. 334, 215• Ordentlich, A., Barak, D., Kronman, C., Ariel, N., Segall, Y., Velan, N., and Shafferman, A. (1996). The architecture of
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SECTION 1II 9 Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors. J. Biol. Chem. 271, 11953-11962. Ordentlich, A., Barak, D., Kronman, C., Ariel, N., Segall, Y., Velan, B., and Shafferman, A. (1998). Functional characteristics of the oxyanion hole in human acetylcholinesterase. J. Biol. Chem. 273, 19509-19517. Ordentlich, A., Barak, D., Sod-Moriah, G., Kaplan, D., Mizrahi, D., Segall, Y., Kronman, C., Karton, Y., Lazar, A., Marcus, D., Velan, B., and Shafferman, A. (2004). Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of ACHE. Biochemistry 43, 11255-11265. Primo-Parmo, S. L., Bartels, C. E, Wiersema, B., van der Spek, A. E, Innis, J. W., and La Du, B. N. (1996). Characterization of 12 silent alleles of the human butyrylcholinesterase (BCHE) gene. Am. J. Hum. Genet. 58, 52-64. Qian, N., and Kovach, I. M. (1993). Key active site residues in the inhibition of acetylcholinesterase by soman. FEBS Lett. 336, 263-266. Quinn, D. M. (1987). Acetylcholinesterase: Enzyme structure, reaction dynamics, and virtual transition states. Chem. Rev. 87, 955-979. Radi~, Z., Picketing, N. A., Vellom, D. C., Camp, S., and Taylor, P. (1993). Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32, 12074-12084. Raveh, L., Grauer, E., Grunwald, J., Cohen, E., and Ashani, Y. (1997). The stoichiometry of protection against soman and VX toxicity in monkeys pretreated with human butyrylcholinesterase. Toxicol. Appl. Pharmacol. 145, 43-53. Rosenberry, T. L., Mallender, W. D., Thomas, P. J., and Szegletes, T. (1999). A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate. Chem.-Biol. Interact. 119-120, 85-97. Rousseaux, C. G., and Dua, A. K. (1989). Pharmacology of HI-6 and H-series oxime. Can. J. Physiol. Pharmacol. 67, 1183-1189. Saxena, A., Viragh, C., Frazier, D. S., Kovach, I. M., Maxwell, D. M., Lockridge, O., and Doctor, B. P. (1998). The pH dependence of dealkylation in soman-inhibited cholinesterase and their mutants: Further evidence for a push-pull mechanism. Biochemistry 37, 15086-15096 Segel, I. H. (1975). Enzyme Kinetics. Wiley, New York. Schopfer, L. M., Boeck, A. T., Broomfield, C. A., and Lockridge, O. (2004). Mutants of human butyrylcholinesterase with organophosphate hydrolase activity: Evidence that HIS117 is a general acid base catalyst for hydrolysis of echothiophate. J. Med. Chem. Def. 2, 1-21. Shafferman, A., Ordentlich, A., Barak, A., Stein, D., Ariel, N., and Velan, B. (1996). Aging of phosphylated human acetylcholinesterase: Catalytic processes mediated by aromatic
and polar residues of the active centre. Biochem. J. 318, 833-840. Smith, T. E., and Usdin, E. (1966). Formation of nonreactivatible isopropylmethyl-phosphono-fluoridate-inhibited acetylcholinesterase. Biochemistry 5, 2914-2918. Soreq, H., and Seidman, S. (2001). Acetylcholinesterase - - New roles for an old actor. Nat. Rev. Neurosci. 2, 294-301. Stojan, J., Marcel, V., Estrada-Mondaca, S., Klaebe, A., Masson, P., and Fournier, D. (1998). A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase. FEBS Lett. 440, 85-88. Talbot, B. G., Anderson, D. R., Harris, L. W., Yarbrough, L. W., and Lennox, W. J. (1988). A comparison of in vivo and in vitro rates of aging of soman-inhibited erythrocyte acetylcholinesterase in different animal species. Drug Chem. Toxicol. 11, 289-305. Taylor, P. (2001). Anticholinesterase agents. In Goodman & Gilman's The Pharmacological Basis of Therapeutics (J. G. Hardman and L. E. Limbird, Eds.), 10th ed., pp. 175-191. McGraw-Hill, New York. Taylor, P., and Radi~, Z. (1994). The cholinesterases: From genes to proteins. Annu. Rev. Pharmacol. Toxicol. 34, 281-320. Tsigelny, I., Shindyalov, I. N., Bourne, P. E., Sudhof, T. C., and Taylor, P. (2000). Common EF-hand motifs in cholinesterases and neuroligins suggest a role for Ca 2+ binding in cell associations. Protein Sci. 9, 180-185. Van Der Kloot, W., and Molg6, J. (1994). Quantal acetylcholine release at the vertebrate neuromuscular junction. Physiol. Rev. 74, 899-991. Walsh, C. (1979). Enzymatic Reaction Mechanisms. Freeman, San Francisco. Wilson, I. B., and Ginsburg, S. (1955a). The reactivation of acetylcholinesterase inhibited by tetraethyl pyrophosphate and diisopropylfluorophosphate. J. Am. Chem. Soc. 77, 4286-4288. Wilson, I. B., and Ginsburg, S. (1955b). A powerful reactivator of alkyl phosphate-inhibited acetylcholinesterase. Biochim. Biophys. Acta 18, 168-170. Wilson, I. B., Bergmann, E, and Nachmansohn, D. (1950). Acetylcholinesterase X. Mechanism of the catalysis of acylation reactions. J. Biol. Chem. 186, 781-790. Wong, L., Radic, Z., Bruggermann, R. J. M., Hosea, N., Berman, H. A., and Taylor, P. (2000). Mechanism of oxime reactivation of acetylcholinesterase analyzed by chirality and mutagenesis. Biochemistry 39, 5750-5757. Worek, E, Kirchner, T., B~icker, M., and Szinicz, L. (1996). Reactivation by various oximes of human erythrocyte acetylcholinesterase inhibited by different organophosphorus compounds. Arch. Toxicol. 70, 497-503. Worek, E, Diepold, C., and Eyer, P. (1999). Dimethylphosphorylinhibited human cholinesterase: Inhibition, reactivation, and aging kinetics. Arch. Toxicol. 73, 7-14.
CHAPTER
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Structure, Function, and Regulation of Carboxylesterases MASAKIYO HOSOKAWA 1 AND TETSUO SATOH l,z 1Chiba, Institute of Science, Chiba, Japan eHAB Research Laboratories, Ichikawa, Chiba, Japan
dog intestine was very weak and produced no appreciable active band in a disk electrophoresis coupled with staining of esterase activity. On the other hand, esterase activities were observed in the intestines of other species (human, rat, mouse, guinea pig, and rabbit) and found to produce a few active bands in an electrophoretic assay. Since pharmacokinetic and pharmacological data of ester prodrugs obtained from preclinical experiments using various animals are generally used as references for human studies, it is important to clarify the biochemical properties of each CarbE isozyme, such as substrate specificity, tissue distribution, and transcriptional regulation. Recent developments have included more detailed biochemical characterization of mammalian CarbE enzymes and genes, leading to a better understanding of the biochemical significance and physiological role of CarbEs. This chapter deals primarily with the characteristics and the molecular cloning of the individual, recently identified CarbE isozymes.
I. I N T R O D U C T I O N The mammalian carboxylesterases CarbEs comprise a multigene family whose gene products are localized in the endoplasmic reticulum (ER) of many tissues. These enzymes efficiently catalyze the hydrolysis of a variety of ester- and amide-containing chemicals as well as drugs (including prodrugs) to the respective free acids. They are involved in detoxification or metabolic activation of various drugs, environmental toxicants, and carcinogens. Carboxylesterases also catalyze the hydrolysis of endogenous compounds, such as short- and long-chain acyl-glycerols, long-chain acyl-carnitine, and long-chain acyl-CoA esters. We have reviewed the characteristics of CarbEs in relation to the metabolism of xenobiotics (Satoh and Hosokawa, 1998). Multiple isozymes of hepatic microsomal CarbE exist in various animal species (Hosokawa et al., 1990; Satoh and Hosokawa, 1998), and some of these isozymes are involved in the metabolic activation of certain carcinogens, as well as being associated with hepatocarcinogenesis (Maki et al., 1991). Mammalian CarbEs are members of an oL,[3-hydrolasefold family and are found in various mammals (Hosokawa et al., 1990; Satoh and Hosokawa, 1998; Satoh et al., 2002). The expression of CarbEs is ubiquitous, with high levels in the liver, small intestine, kidney, and lung. CarbEs show such a broad range of substrate specificity that they can be involved in detoxification or biotransformation of many kinds of drugs as well as endogenous fatty acid esters. It has been suggested that CarbEs can be classified into four major groups according to the homology of the amino acid sequence (Hosokawa et al., 1990; Satoh and Hosokawa, 1998; Satoh et al., 2002), and the majority of CarbEs that have been identified belong to the CES1 or CES2 family. It has also been shown that striking species differences exist (Inoue et al., 1979; Hosokawa et al., 1990, 1994; Prueksaritanont et al., 1996; Zhu et al., 2000). For example, Inoue et al. showed that esterase activity in the Toxicology of Organophosphate and Carbamate Compounds
II. R O L E O F C a r b E I S O Z Y M E S IN D R U G M E T A B O L I S M
CarbEs are members of an oL,[3-hydrolase-fold family (Satoh and Hosokawa, 1998; Satoh et al., 2002) and they show ubiquitous tissue expression profiles with the highest levels of CarbE activity present in liver microsomes in many mammals (Satoh and Hosokawa, 1998; Satoh et al., 2002). Drug-metabolizing enzymes that are present predominantly in the liver are involved in biotransformation of both endogenous and exogenous compounds to polar products to facilitate their elimination. These reactions are categorized as phase I and phase II reactions. CarbEs are categorized as phase I drug-metabolizing enzymes that can hydrolyze a variety of ester-containing drugs and prodrugs, such as angiotensin-converting enzyme inhibitors (temocapril, cilazapril, quinapril, and imidapril) (Takai et al., 219
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220
SECTION 1II. Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
1997; Mori et al., 1999; Furihata et al., 2004a), antitumor drugs (CPT-11 and capecitabine) (Satoh et al., 1994; Tabata et al., 2004), and narcotics (cocaine, heroin, and meperidine) (Pindel et al., 1997; Zhang et al., 1999). In this regard, it is thought that CarbEs are one of the major determinants for pharmacokinetics and pharmacodynamics of ester drugs or ester prodrugs (Fig. 1). Actually, it has been shown that dog CES 1 isozyme was involved in a pulmonary first-pass effect in the disposition of a propranolol ester prodrug (Imai et al., 2003). It has also been shown that the expression level of human CarbE isozyme was correlated with the conversion ratio of CPT-11 to SN-38, the active metabolite, which is thought to be a key step for the chemotherapeutic action of this antitumor drug (Pindel et al., 1997; Zhang et al., 1999; Ohtsuka et al., 2003; Sanghani et al., 2003).
The CarbEs and the UDP-glucuronosyltransferase (UGT) families, the catalytic domains of which are localized in the luminal sides of the ER membrane, are two major enzyme groups responsible for phase I and phase II reactions (Fig. 2). The hydrolyzed products of CarbEs are also substrates for UGT, such as SN-38 from CPT-11. Thus, CarbE-UGT interaction in the luminal sides of the ER membrane is important for drug metabolism. Furthermore, hydrolyzed products of CarbEs have two kinds of chemical properties; one is the alcohol or phenol, which are substrates for UGT, and the other is organic anions, which are substrates for organic anion transporter such as multidrug resistance-associated protein 2 (MRP2) (Fig. 2). In this regard, CarBEs are one of the major drug-metabolizing enzymes for enzyme-enzyme interaction and enzyme- transporter interaction.
III. C L A S S I F I C A T I O N
AND NOMENCLATURE OF CarbEs
FIG. 1. Role of CarbE isozyme in drug metabolism. CES, carboxylesterase; CYP, cytochrome P450; UGT, UDP glucuronosyltransferase; MDR, multidrug resistance; MRP2, multidrug resistance-associated protein 2.
According to the classification of esterases by Aldridge (1993), the serine superfamily of esterases (i.e., acetylcholinesterase, butyrylcholinesterase, and CarbE) falls into the B-esterase group. CarbEs have very wide substrate specificity. It is becoming increasingly clear that esterases tend to have broad and overlapping substrate specificity toward amides and esters. A single esterolytic reaction is frequently mediated by several kinds of enzymes. Studies on esterases, as with other enzymes concerned with xenobiotic metabolism, have afforded evidence of multiple forms. It seems almost impossible to classify these CarbE isozymes
FIG. 2. CarbE-UGT interaction in the luminal sides of the ER membrane and CES-transporter interaction in the cell.
CHAPTER 1 6 9Structure and Function of CarbEs based on their substrate specificity using the IUB, International Union of Biochemistry classification because the individual hydrolases exhibit properties of CarbE, lipase, or both. Mentlein et al. (1984) proposed to classify these hydrolases as unidentified CarbE (EC 3.1.99.1 to 3.1.99.x). Table 1 summarizes the sequence identities of CES isozymes based on human liver CarbE (Satoh and Hosokawa, 1998). These esterases are markedly similar in terms of characteristics such as substrate specificity. The amino acid sequences of the isozymes of these esterases are highly homologous. Based on the high homology and similarity of the characteristics, we tried to classify CarbE isozymes into four families; CES1, CES2, CES3, and CES4 (Table 1) (Satoh and Hosokawa, 1998). The CES1 family includes the major forms of CES isozymes (more than 60% homology of human CES). Thus, they can be divided into three subfamilies: CES1A, CES1B, and CES1C. The CES 1A subfamily includes the major forms of human CarbEs and the major isoforms of rat, dog, rabbit, and mouse CarbE. The CES 1B subfamily includes RL1, ML1, and hydrolases B and C, which catalyze the long-chain acyl-CoA hydrolysis. Members of the CES 1C family, which share homology with
221
human CarbEs, are similar to the CES 1B family members. However, these families are all secretory type CarbEs. In contrast, the CES2 family includes human intestinal CarbE (hCE2, CES HU3), rat RL4 (rCES2), and rat intestinal CarbE. Mouse ML3(mCES2), rabbit form 2, and hamster AT51 are mainly expressed in the small intestines. CES3 includes ES-male, whose functions are not clear. The CES-4 family includes the novel 46.5-kDa CarbE isozymes, which have a different structure from other CarbE families. ES 46.5-kDa from mouse liver (Watanabe et al., 1993) and amido hydrolase of monkey liver (Kusano et al., 1996) probably belong to this family. These groupings are similar to those derived from phylogenetic analysis (Fig. 3).
IV. S T R U C T U R E - A C T I V I T Y RELATIONSHIPS OF SUBSTRATES W I T H CES1 A N D C E S 2 F A M I L I E S It has been suggested that CarbEs can be classified into four major groups according to the homology of the amino acid sequence as described previously (Satoh and
TABLE 1. Classification and Nomenclature of the Carboxylesterases Gene
Trivial name
Species
Homology (%)
Gene
Trivial name
Species
CES1A1
Macrophage HUla hCE-1 Hbrl
Human Human Human Human
100.0 99.9 99.5 99.6
CES1 C1
CESIA2
HUlb HUMLCEA HUMCES MoCE31 D1 hBr2
Human Human Human Monkey Dog Human
99.3 94.1 93.3 92.9 79.7 79.0
Serum Rat ES 1 Rat CE Hydrolase S Es-N MS1
Rat Rat Rat Rat Mouse Mouse
69.6 68.9 68.9 68.9 65.2 65.2
hBr3 MH1 pl 6.1 Rabbitl MouseCE Egasin ES3(Egasin)
Human Mouse Rat Rabbit Mouse Mouse Rat
76.8 77.9 77.4 77.8 73.2 75.5 74.6
hCE-2 HSiCE HU3 Rabbit 2 ML3 AT-51
Human Human Human Rabbit Mouse Hamster
46.8 46.8 46.8 46.9 43.5 45.7
CES3A1
Rat CE4.6 Rat ICES CES RL4 ES-male
Rat Rat Rat Mouse
42.7 44.1 44.0 41.1
CES4A1
46.5K
Human
31.3
ES4 Hydrolase B Hydrolase C RL1 ML1
Rat Rat Rat Rat Mouse
67.9 67.0 67.0 66.0 63.0
CESIC2 CESIA3
CESIA4
CES2A1
CES2A2 CES2A3
CESIA5 CESIA6
CESIA7
CES1B1
CES1B2
CES2A4 CES2A5
Homology (%)
222
SECTION III. E s t e r a s e s ,
Receptors, Mechanisms, & Tolerance Development D1 (dog liver) hBr2 (human brain)
( l~
C
hBrl (human brain) hCE/HU1 (human liver) hCE-1/Macrophage/ HUMCARA/ Rabbit1 (rabbit liver) hBr3 (human brain) MH1 (mouse liver)
CES1A
RH1 (rat liver) Egasyn (mouse liver) ES3 (Egasin) Hydrolase C (rat liver) RL1 (rat liver) ES4 (rat liver) Kidney (rat kidney) ML1 (mouse liver) Es-N (mouse liver) MS1 (mouse serum) RS1 (rat serum) RL4/rCES2 (rat liver) ratlCES (rat intestine) AT51 (hamster liver)
CES1B
CES1C
CES2 I HSiCE (human intestine) I hCE-2/HU3 (human liver/intestine) Rabbit2 (rabbit liver) ES-male (mouse liver) 46.5 kDa (human liver)
CES3 CES4
FIG. 3. Phylogenetic tree of the carboxylesterase superfamily using a simple unweighted pair-group method of analysis dendrogram. Hosokawa, 1998), and the majority of CarbEs that have been identified belong to the CES1 or CES2 families. Studies have shown that there are some differences between these families in terms of substrate specificity, tissue distribution, immunological properties, and gene regulation (Satoh and Hosokawa, 1998). For example, the preferential substrates for CES 1 (also called hCE1, hCE, or CES HU1) (Kroetz et al., 1993; Satoh and Hosokawa, 1998), a human CES1 family isozyme, are thought to be compounds esterified by small alcohol, whereas those for hCE-2, a human CES2 family isozyme, are thought to be compounds esterified by relatively large alcohol (Table 2). For drugs of abuse, heroin shows the highest rates of catalysis by both enzymes. CES 1, but not CES2, hydrolyzed the methyl ester of cocaine and the ethyl esters of meperidine and delapril (Kroetz et al., 1993; Pindel et al., 1997; Takai et al., 1997; Satoh and Hosokawa, 1998; Takayama et al., 1998; Zhang et al., 1999). In contrast to the specificity of CES 1 for the methyl ester of cocaine, only CES2 hydrolyzed the benzoyl ester of cocaine. For the remaining substrates that could be hydrolyzed by both enzymes, CES2 exhibited higher catalytic efficiency than CES1 for heroin; enzymatic conversion of 6-acetylmorphine to morphine was not known before the isolation and characterization of CES2 (Kamendulis et al., 1996). We reported that mouse MH1, a mouse CES 1 family isozyme, also hydrolyzed the temo-
capril, which esterified a small alcohol, similar to the human CES 1 isozyme (Moil et al., 1999, Fuilhata et al., 2004a). On the other hand, we also reported that rat rCES2 (also called CES RL4), a rat CES2 family isozyme, hydrolyzed methylprednisolone hemisuccinate, which esterified a large alcohol, similar to the human CES2 family (Furihata et al., 2005). It has been suggested that although these two CarbE families exhibit broad substrate specificity for ester, carbamate, or amide hydrolysis, these CarbE isozymes do exhibit distinct catalytic efficiencies that correlate with the relative size of the substrate substituents versus that of the enzyme active sites. Knowledge of these substrate structure-activity relationships and the tissue distribution of CarbE isoenzymes is critical for predicting the metabolism and pharmacokinetics and pharmacodynamics of ester drugs or prodrugs.
V. S T R U C T U R E A N D C A T A L Y T I C M E C H A N I S M OF CarbE I S O Z Y M E S It has been shown that several proteins of the ER lumen have the common carboxy-terminal sequence KDELCOOH, and the structural motif is essential for retention of the protein in the luminal site of the ER through KDEL receptor bound to the ER membrane (Pelham, 1990; Tang and Kalow, 1995).
CHAPTER 1 6 9Structure and Function of CarbEs TABLE 2. Substrate
223
Structure-Activity Relationship of Substrates with CESI and CES2 Families Alcohol substituent
Cocaine (methylester)
Acyl substituent
N~CH3 0
CH3OH
Substrate specificity
CES1
II
0
Meperidine
CH3CH2OH
CES1
N
[ ~
\c/'OII
0
Temocapril
C2HsOH
CES1
CPT- 11
CES2 > > > CES 1
HO
Heroin
HO
CH3COOH
CES2 > > > CES 1
HOOCmCH2mCH2mCOONa
CES2 > > > CES 1
O CHz
HoJ'--..~ Methylprednisolone 21-hemisuccinate
Korza and Ozols (1988) and Ozols (1989) established the primary structures of two microsomal esterases purified from rabbit liver and designated them as 60-kDa esterase form 1 and form 2. These two forms of CarbE have the consensus sequence of the ER retention tetra-peptide (HTEL or HIEL in the one-letter code) which recognized with the luminal side of the KDEL receptor. Robbi et al. (1990) reported cDNA cloning of rat liver pI 6.1 esterase (ES-10) and pI 5.5 esterase (ES-3, egasyn). This was the
first report to show that cDNA of liver CarbE has the consensus sequence of the ER retention tetrapeptide (HVELCOOH). Later, Robbi and Beaufay (1994) isolated a cDNA clone of another rat liver pI 5.5 esterase (ES-3, egasyn), which has the consensus sequence of the ER retention tetrapeptide (HTEL-COOH). In the case of mouse liver microsomal CarbE, the carboxyl-terminal amino acid sequence of clone Es-N is HTEHK-COOH, which differs from the consensus sequence of the ER retention signal.
224
SECTION III. E s t e r a s e s ,
Receptors,
Mechanisms,
The other clone encoded egasyn, an accessory protein of [3-glucuronidase in the liver microsomes (Medda et al., 1987). Egasyn is identical to CarbE, and it binds [3-glucuronidase via its CarbE active site. Thus, it contains the consensus sequence of the ER retention signal (HTELCOOH). Ovnic et al. (1991a) conducted genetic mapping and confirmed the location of an egasyn cDNA fragment in cluster 1 of the esterase region on chromosome 8. Shibata et al. (1993) found that the human CarbE gene spans approximately 30 kilobases (kb) and has 14 small exons. Many CarbEs have a signal peptide of 17-20 amino acid residues, including hydrophobic amino acid, for retention in the lumen of the ER (Fig. 4). In general, a bulky aromatic residue followed by a small neutral residue directly precede the cleavage site (von Heijne, 1983). Many CarbEs have four cysteines that may be involved in specific disulfide bonds. Among them, Cys98 is the most highly conserved residue in many CES isozymes (Fig. 4). Cygler et al. (1993) reported an important alignment of a collection of related amino acid sequences of esterase, lipase, and related proteins based on X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase. According to these authors, Ser203, Glu336, and His450 form a catalytic triad, and Gly124Gly125 may be part of an oxyanion hole. These residues are also highly conserved among CarbE isozymes. Thus, we performed mutation analysis (Satoh and Hosokawa, 1998). Site-specific mutation of Ser203 to The203, Glu336 to Ala336, or His450 to Ala450 greatly reduced the CES activity toward substrates. Therefore, the mutagenesis confirmed the role for Glu336 and His450 in forming a putative charge relay system with the active site Ser203 (Satoh and Hosokawa, 1998). Frey et al. (1994) found that the formation of low-barrier hydrogen bonds between His and Asp (Glu for CarbE) facilitates the action of nucleophilic attack by the [3-OH group of Ser on the acyl carbonyl group of peptide in chymotrypsin. The catalytic triad in the tetrahedral addition intermediate is stabilized by the low-barrier hydrogen bonds. According to their theory, we thought the low barrier hydrogen bond between Glu336 and His450 facilitates the action of nucleophilic attack by [3-OH group of Ser203 on the carbonyl group of substrate in CarbE (Figs. 5 and 6). In this mechanism, the His450 removes a proton from the Ser203 in the transition state for its addition to the acyl carbonyl group of substrate. In the tetrahedral intermediate, the formation of low-barrier hydrogen bonds between His450 and Glu336 and the transition state is stabilized by the low-barrier hydrogen bonds (Figs. 5 and 6). The low-barrier hydrogen bonds facilitated mechanism includes weak hydrogen bonds between the tetrahedral oxyanion and peptide N-H bonds contributed by Gly123 and Gly124, which stabilize the tetrahedral adduct on the substrate side of the transition state. Formation of the acyl-enzyme complex in the next step requires the removal of a proton from His450 so that the tetrahedral
& Tolerance
Development
intermediate is disrupted in the acyl-enzyme intermediate (Fig. 5). When the unbound portion of the alcohol group of the first product of substrate has diffused away, a second step occurs in which the deacylation step is essentially the reverse of the acylation step, with the water molecule substituting for the alcohol group of the original substrate. To clarify the catalytic mechanism of CarbE, mutation analysis of other structural domains, such as the sites of salt bridges, the substrate binding site, and glycosylation would be worthwhile. It is of interest that the sequences required for .the hydrolytic capability at the catalytic triad (Glu, His, and Ser) of CarbE, acetylcholinesterase, butyrylcholinesterase, and cholesterolesterase are highly conserved. This is a common structure of serine hydrolase superfamilies, which are responsible for the hydrolysis of endogenous and exogenous compounds. Furthermore, these elements are strongly conserved among orthologous CarbEs from mouse, rat, rabbit, monkey, and human. A three-dimensional model for human CarbE has been proposed based on the crystal structure coordinates of acetylcholine esterase and overlapping active sites with pancreatic lipase and CarbE (Alam et al., 2002). The modeled structure shares the overall folding and topology of the proteins identified in the published crystal structures of the rabbit (Bencharit et al., 2002) and human CarbE (Bencharit e t al., 2003a,b). CarbE has a threedimensional oL,[3-hydrolase fold that is a structural feature of all lipases (Wong and Schotz, 2002). In general, the structure of CarbE may be viewed as comprising a central catalytic domain surrounded by et,[3 and regulatory domains (Bencharit et al., 2002, 2003a,b). In essence, the oL,[3-hydrolase fold consists of a central [3 sheet surrounded by a variable number of oLhelices and accommodates a catalytic triad composed of Ser, His, and a carboxylic acid. The residues that comprise the catalytic domain of human CES 1 are very highly conserved among orthologous CES 1 proteins from different species (Fig. 4). This suggests that the catalytic function of these proteins is conserved across species. The catalytic triad is located at the bottom of a deep active site cleft in the molecule and comprises a large flexible pocket on one side of Ser203 and a small rigid pocket on the opposite side. The orientation and location of the active site provide an ideal hydrophobic environment for the hydrolysis of a wide variety of hydrophobic substrates. The small rigid active site pocket is adjacent to the oxyanion hole formed by Gly123-Gly124 and is lined by several hydrophobic residues (Bencharit et al., 2003a). Short acyl chains would be easily accommodated within the small rigid pocket. The larger flexible active site pocket is lined by several nonpolar residues and could accommodate larger or polycyclic molecules such as cholesterol. The large pocket is adjacent to a side door secondary pore that would permit small molecules (substrates and reaction products) to enter and exit the active site (Bencharit et al., 2003a). Longer acyl chains may be oriented for catalysis in
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CHAPTER 1 6 9Structure and Function of CarbEs
227
FIG. 6. A proposed mechanism of the formation of the tetrahedral intermediate. (A), Hydrolysis of substrate start with an attack by the oxygen atom of the hydroxy group of Ser203 on the carbonyl carbon atom of the substrate. (B), The carbon-oxygen bond of this carbonyl group becomes a single bond, and the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl carbon are arranged as in a tetrahedron. The formation of this transient tetrahedral intermediate from a substrate is made possibility by hydrogen bonds between the negative charged oxygen onion (called an oxyanion, and main-chain NH group (Fig. 5 and Fig. 6)). This site is called oxyanion hole.
such a way that they extend through the side door. Indeed, the presence of a hydrophobic residue at position 423 in mouse and 425 in human is necessary for efficient hydrolysis of hydrophobic substrates since mutation of Met present in position 423 of the related rat lung CarbE to lie increased the CarbE activity toward a more hydrophobic substrate without affecting activity toward short-chain esters (Wallace et al., 1999). According to the X-ray crystal structure of the human CES 1, this residue lines the flexible pocket adjacent to the side door (Bencharit et al., 2003a). Given the wide range of substrates that CarbEs are known to hydrolyze, the large flexible pocket confers the ability to hydrolyze many structurally distinct compounds, whereas the rigid pocket is much more selective with regard tO the substrates that may be accommodated.
VI. I N D U C T I O N O F CarbEs Much interest has been shown by both clinicians and researchers in the induction of expression of drugmetabolizing enzymes by chemicals, including medical agents, since this is one of the main reasons for drug-drug interaction that causes adverse effects and for the reduction in pharmacological potencies of drugs. Regarding CarbEs, it has been shown that rodent CarbE isozyme(s) is induced by phenobarbital (Hosokawa et al.,
1987), aminopyrine (Hosokawa et al., 1988), and peroxisome proliferators [clofibrate, di(2-ethylhexyl)phthalate, and perfluorinated fatty acids] (Hosokawa et al., 1994; Howarth et al., 2001; Furihata et al., 2003, 2004a). The mouse is one of the most widely used experimental animals in the process of drug development, and several mouse CarbE isozymes have been identified (Hosokawa et al., 1990; Ovnic et al., 1991a,b; Aida et al., 1993; Satoh and Hosokawa, 1998; Dolinsky et al., 2001; Xie et al., 2003; Furihata et al., 2003 2004a, 2005). However, information on the involvement of mouse CarbEs in drug metabolism is limited. We have reported that exposure of C57BL/6 mice to di(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator, through their diet resulted in a significant increase in the amount of CarbE protein concomitant with an increase in the level of hydrolytic activity toward xenobiotics in mouse liver microsomes (Hosokawa et al., 1994). We have also shown that one of the mouse CarbE isozymes induced by DEHP is mCES2/microsomal acyl carnitine hydrolase, a CES2 family isozyme (Furihata et aL, 2003). Our immunochemical study also suggested that mouse CES 1 isozymes were induced by DEHP treatment, but they remain to be identified. Recently, we identified a mouse CES 1 isozyme, mCES1, that was induced by DEHP. Purification, cDNA cloning, and baculovirus-mediated expression of mCES1 revealed that mCES 1 plays an important role in temocapril metabolism and that it belongs to the CES1A subfamily.
228
SECTION III. E s t e r a s e s ,
Receptors,
Collectively, our results show that mCES 1 is very similar to hCE-1. Therefore, mCES 1 is thought to be one of the critical determinants for pharmacokinetics and pharmacodynamic actions of ester prodrugs as well as ester drugs. This work provides Useful information for the study of metabolism and dispositions of ester prodrugs as well as ester drugs. Zhu et al. (2000) reported that dexamethasone caused a slight increase in human CES isozymes. Among the inducers, dexamethasone possesses a potent and interesting ability to affect CarbE expression in the rat liver. Hattori et al. (1992) reported that methylprednisolone hemisuccinate (MPHS) was hydrolyzed to methylprednisolone via CarbE in rat liver microsomes and that several clinically used glucocorticoids, including dexamethasone, caused a remarkable increase in the level of this hydrolytic activity. In contrast to the report of induction of CarbE activity, some researchers have shown that the level of microsomal p-nitrophenylacetate hydrolase activity was significantly decreased in rat liver microsomes. This apparent contradiction in the same animal is probably due to the different methods for determination of CarbE activity by different CarbE substrate. Therefore, it is hypothesized that the CarbE isozyme contributing to p-nitrophenylacetate hydrolysis in rat liver microsome is different from the one contributing to MPHS hydrolysis. It has been reported that dexamethasone decreased the levels of expression of rat CES1 isozymes (CES RH1, also known as ES-10 or hydrolase A, and CES RL1, also known as ES-4 or hydrolase B) in the rat liver and that the decrease in the expression levels of these enzymes was linked to the reduction in the level of p-nitrophenylacetate hydrolase activity (Furihata et al., 2005). On the other hand, rat CarbE isozymes responsible for MPHS hydrolysis in rat liver microsomes have not been identified. However, we identified a dexamethasone-induced CarbE isozyme that can hydrolyze MPHS in the rat liver and plasma as a member of the CES2 family, rCES2 (Furihata et al., 2005). The different biochemical properties of rCES2 from those of known rat CES1 isozymes, including its unique existence in plasma, will be useful for studies aimed at elucidating the functions of CarbEs in drug metabolism. In addition, we also identified the gene encoding rCES2 by cDNA cloning and functional expression in Sf9 cells. Since we demonstrated that the level of the corresponding mRNA expression was markedly increased, the identification of the coding gene is valuable for studies aimed at elucidating the molecular mechanisms by which dexamethasone induces rCES2 expression (Furihata et al., 2005).
VII. GENE STRUCTURE AND
REGULATION OF CarbE ISOZYMES Both the murine (Satoh and Hosokawa, 1998) and the human (Shibata et al., 1993; Langmann et al., 1997; Satoh et al., 2002) CES1 genes span approximately 30 kb and contain 14
Mechanisms,
& Tolerance Development
small exons. Recently, sequencing of the mouse and human genomes was completed, enabling detailed sequence comparisons. The previously published sequences of the individual exons, splice junctions, size of the introns, and restriction sites within the murine and human CarbE genes are consistent with theft respective genes sequenced by the mouse and human genome projects. Therefore, the organization of the CarbE gene is evolutionarily conserved in mice and humans. Previous studies have mapped the human carboxylesterase gene to chromosome 16 at 16q13-q22.1 (Zschunke et al., 1991; Kroetz et al., 1993). This region is syntenic to a region of mouse chromosome 8 at 8C5 (Zschunke et al., 1991). The murine CarbEs Es22 (Ovnic et al., 199 l a) and Es 1 (Ovnic et al., 1991b) have previously been mapped to chromosome 8. The completion of the mouse genome sequencing project unambiguously demonstrated that the murine CarbE gene was located on the minus strand of chromosome 8 at 8C5 in a cluster of six CarbE genes that spans 260.6 kb in total. These six CarbE genes are presumed to have originated from repeated gene duplications of a common ancestral gene that encoded a CarbE (Shibata et al., 1993) and subsequent evolutionary divergence occurred. Recent studies have shown that there are some differences between these families in terms of substrate specificity, tissue distribution, immunological properties, and gene regulation. Therefore, the 5'-flanking regions of the CES1 and CES2 genes were isolated from mouse, rat, and human genomic DNAs by polymerase chain reaction amplification. Two individual mouse CES genes (mCES MH1 and m C E S ML1) (Furihata et al., 2004a) and two individual human CarbE genes (CES H U l a and H U l b ) were found to belong to the CES1 family, and mouse m C E S 2 (Furihata et al., 2003), rat rCES2 (Furihata et al., 2004a), and human CES H U 3 genes were found to belong to the CES2 family. A TATA box does not precede the transcription start site of any of the CarbE promoters. CarbE promoters share several common binding sites for transcription factors among the same CarbE families, suggesting that orthologous CarbE genes have evolutionally conserved transcriptional regulatory patterns. Potential binding sites of CarbE promoters for transcriptional factors include Spl, Sp3, C/EBE USF1, NF-1, NF-KB, PPAR-~/, GR, SREBE HNF1, HNF3, and HNF4. In the case of human CES1 genes, we isolated two CarbE genes encoding human CES HU1, which were tentatively designated as CES H U l a and CES H U l b (Fig. 7). These genes are identical except for exon 1 and cis elements. Electrophoretic mobility shift assays and reporter gene assays demonstrated that SP1, C/EBE and NF-1 could bind to each responsive element of the CES H U l a promoter but that C/EBP could not bind to responsive elements of the CES H U l b promoter. On the other hand, the structure of the CES2 gene promoter was different from that of the CES1 gene promoter (Fig. 7). We have shown tissue expression profiles of mCES2 and parts of the mechanism by which transcription of the mCES2 gene is regulated (Furihata et al.,
CHAPTER 1 6 9Structure and Function of CarbEs
229
FIG. 7. Gene structure and 5' regulatory element of CES HUla (CESIA), CES HUlb (CESIA), and CES HU3 (CES2A) genes.
2004b). mCES2 is expressed in the liver, kidney, small intestine, brain, thymus, lung, adipose tissue, and testis. We have also shown that Spl, Sp3, and USF1 contribute to synergistic transactivation of the mCES2 promoter. Although the possibility of involvement of other transcription factors in the regulation of mCES2 gene expression cannot be ruled out and further studies are needed to elucidate the mechanism fully, our data indicate that Spl, Sp3, and USF1 are indispensable factors for transcription of the mCES2 gene. Our results have provided some clues for understanding the molecular mechanisms regulating mCES2 gene expression and represent an important step toward elucidation of physiological functions of mCES2 (Furihata et al., 2004b).
VIII. C O N C L U S I O N S AND F U T U R E DIRECTIONS Multiple CarbEs play an important role in the hydrolytic biotransformation of a vast number of structurally diverse drugs. These enzymes are a major determinant of the pharmacokinetic behavior of most therapeutic agents containing an ester or amide bond. There are several factors that influence CarbE activity, either directly or at the level of enzyme regulation. In the clinical field, drug elimination is decreased and the incidence of drug-drug interactions increases when two or more drugs compete for hydrolysis by the same CarbE isozyme. Exposure to environmental pollutants or to lipophilic drugs can result in induction of CarbE activity. Several drug-metabolizing enzymes, such as cytochrome P450, UGT and sulfotransferase have been extensively
studied to clarify the substrate specificity using molecular cloning and cell expersion system. Consequently, the novel findings obtained reveal that the substrate specificity of CarbE is, at least in part, explained by the differences in the nucleotide sequences of the individual CarbE isozymes. In addition, it is clear that membrane-bound type CarbE isozymes in microsomes are required to possess the KDEL tetrapeptide motif at the carboxy terminal of the molecule. Mammalian CarbE have been found to have acyl-glycerol, acyl-CoA, and acyl-carnitine hydrolyzing activities in vitro; however, physiological roles of CarbEs remain unclear. To clarify the substrate specificity of each CarbE isozymes, we have begun to study for search the substrate recognition site of each isozymes. In the future, we should be able to clarify the three-dimensional structure of CarbE by X-ray analysis and estimate the catalytic triad, oxyanion hole, salt bridge, substrate binding site, and so on. Also, we should be able to utilize in vivo experimental results to predict in vivo results. The substrate specificity of CarbE toward newly developed prodrugs under consideration may be examined using purified CarbE, mammalian cell expression systems and specific inhibitor. However, such in vitro experiments may not possible to predict in vivo results, except in particular cases. Therefore, we must obtain enough information for different pharmacokinetic parameters of prodrugs among mammalian species. We should clarify the inter-individual difference in human CarbE for the study of the prediction of pharmacodynamics. The expression levels of CarbE isozymes are extremely different in each liver. Thus, further investigations of the regulatory mechanism in CarbE may be able to clarify the cause of individual variation.
7_30
SECTION I I I . E s t e r a s e s , R e c e p t o r s , M e c h a n i s m s ,
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Hattori, K., Igarashi, M., Itoh, M., Tomisawa, H., Ozawa, N., and Tateishi, M. (1992). Purification and characterization of glucocorticoid-inducible steroid esterase in rat hepatic microsomes. Specific induction by glucocorticoids of steroid esterase in rat hepatic microsomes and its release into serum. Biochem. Pharmacol. 43, 1929-1937. Hosokawa, M., Maki, T., and Satoh, T. (1987). Multiplicity and regulation of hepatic microsomal carboxylesterases in rats. Mol. Pharmacol. 31, 579-584. Hosokawa, M., Maki, T., and Satoh, T. (1988). Differences in the induction of carboxylesterase isozymes in rat liver microsomes by xenobiotics. Biochem. Pharmacol. 37, 2708-2711. Hosokawa, M., Maki, T., and Satoh, T. (1990). Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277, 219-227. Hosokawa, M., Hirata, K., Nakata, E, Suga, T., and Satoh, T. (1994). Species differences in the induction of hepatic microsomal carboxylesterases caused by dietary exposure to di(2ethylhexyl)phthalate, a peroxisome proliferator. Drug Metab. Dispos. 22, 889-894. Howarth, J. A., Price, S. C., Dobrota, M., Kentish, E A., and Hinton, R. H. (2001). Effects on male rats of di-(2-ethylhexyl) phthalate and di-n-hexylphthalate administered alone or in combination. Toxicol. Lett. 121, 35-43. Imai, T., Yoshigae, Y., Hosokawa, M., Chiba, K., and Otagiri, M. (2003). Evidence for the involvement of a pulmonary first-pass effect via carboxylesterase in the disposition of a propranolol ester derivative after intravenous administration. J. Pharmacol. Exp. Ther. 307, 1234-1242. Inoue, M., Morikawa, M., Tsuboi, M., and Sugiura, M. (1979). Species difference and characterization of intestinal esterase on the hydrolizing activity of ester-type drugs. Jpn. J. Pharmacol. 29, 9-16. Kamendulis, L. M., Brzezinski, M. R., Pindel, E. V., Bosron, W. E, and Dean, R. A. (1996). Metabolism of cocaine and heroin is catalyzed by the same human liver carboxylesterases. J. Pharmacol. Exp. Ther. 279, 713-717. Korza, G., and Ozols, J. (1988). Complete covalent structure of 60-kDa esterase isolated from 2,3,7,8-tetrachlorodibenzo-pdioxin-induced rabbit liver. J. Biol. Chem. 263, 3486-3495. Kroetz, D. L., McBride, O. W., and Gonzalez, E J. (1993). Glycosylation-dependent activity of baculovirus-expressed human liver carboxylesterases: cDNA cloning and characterization of two highly similar enzyme forms. Biochemistry 32, 11606-11617. Kusano, K., Seko, T., Tanaka, S., Shikata, Y., Ando, T., Ida, S., Hosokawa, M., Satoh, T., Yuzuriha, T., and Horie, T. (1996). Purification and characterization of monkey liver amidohydrolases and its relationship to a metabolic polymorphism of E6123, a platelet activating factor receptor antagonist. Drug Metab. Dispos., in press. Langmann, T., Becker, A., Aslanidis, C., Notka, E, Ullrich, H., Schwer, H., and Schmitz, G. (1997). Structural organization and characterization of the promoter region of a human carboxylesterase gene. Biochim. Biophys. Acta 1350, 65-74. Maki, T., Hosokawa, M., Satoh, T., and Sato, K. (1991). Changes in carboxylesterase isoenzymes of rat liver microsomes during hepatocarcinogenesis. Jpn. J. Cancer Res. 82, 800-806.
CHAPTER 1 6 9Structure and Function of CarbEs Medda, S., Takeuchi, K., Devore-Carter, D., von Deimling, O., Heymann, E., and Swank, R. T. (1987). An accessory protein identical to mouse egasyn is complexed with rat microsomal beta-glucuronidase and is identical to rat esterase-3. J. Biol. Chem. 262, 7248-7253. Mentlein, R., Suttorp, M., and Heymann, E. (1984). Specificity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase, palmitoyl-carnitine hydrolase, and nonspecific carboxylesterase from rat liver microsomes. Arch. Biochem. Biophys. 228, 230-246. Moil, M., Hosokawa, M., Ogasawara, Y., Tsukada, E., and Chiba, K. (1999). cDNA cloning, characterization and stable expression of novel human brain carboxylesterase. FEBS Lett. 458, 17-22. Ohtsuka, K., Inoue, S., Kameyama, M., Kanetoshi, A., Fujimoto, T., Takaoka, K., Araya, Y., and Shida, A. (2003). Intracellular conversion of irinotecan to its active form, SN-38, by native carboxylesterase in human non-small cell lung cancer. Lung Cancer 41, 187-198. Ovnic, M., Swank, R. T., Fletcher, C., Zhen, L., Novak, E. K., Baumann, H., Heintz, N., and Ganschow, R. E. (1991a). Characterization and functional expression of a cDNA encoding egasyn (esterase-22): The endoplasmic reticulum-targeting protein of beta-glucuronidase. Genomics 11, 956-967. Ovnic, M., Tepperman, K., Medda, S., Elliott, R. W., Stephenson, D. A., Grant, S. G., and Ganschow, R. E. (199 l b). Characterization of a murine cDNA encoding a member of the carboxylesterase multigene family. Genomics 9, 344-354. Ozols, J. (1989). Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. J. Biol. Chem. 264, 12533-12545. Pelham, H. R. (1990). The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem. Sci. 15, 483-486. Pindel, E. V., Kedishvili, N. Y., Abraham, T. L., Brzezinski, M. R., Zhang, J., Dean, R. A., and Bosron, W. E (1997). Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin. J. Biol. Chem. 272, 14769-14775. Prueksaritanont, T., Gorham, L. M., Hochman, J. H., Tran, L. O., and Vyas, K. P. (1996). Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells. Drug Metab. Dispos. 24, 634-642. Robbi, M., and Beaufay, H. (1994). Cloning and sequencing of rat liver carboxylesterase ES-3 (egasyn). Biochem. Biophys. Res. Commun. 203, 1404-1411. Robbi, M., Beaufay, H., and Octave, J. N. (1990) Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum. Biochem. J. 269, 451-458. Sanghani, S. P., Quinney, S. K., Fredenburg, T. B., Sun, Z., Davis, W. I., Murry, D. J., Cummings, O. W., Seitz, D. E., and Bosron, W. E (2003). Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis. Clin. Cancer Res. 9, 4983-4991. Satoh, T., and Hosokawa, M. (1998). The mammalian carboxylesterases: From molecules to functions. Annu. Rev. Pharmacol. Toxicol. 38, 257-288.
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Satoh, T., Hosokawa, M., Atsumi, R., Suzuki, W., Hakusui, H., and Nagai, E. (1994). Metabolic activation of CPT-11, 7-ethyl-10-[4(1-piperidino)-l-piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxylesterase. Biol. Pharm. Bull. 17, 662-664. Satoh, T., Taylor, P., Bosron, W. E, Sanghani, S. P., Hosokawa, M., and La Du, B. N. (2002). Current progress on esterases: From molecular structure to function. Drug Metab. Dispos. 30, 488-493. Shibata, E, Takagi, Y., Kitajima, M., Kuroda, T., and Omura, T. (1993). Molecular cloning and characterization of a human carboxylesterase gene. Genomics 17, 76-82. Tabata, T., Katoh, M., Tokudome, S., Nakajima, M., and Yokoi, T. (2004) Identification of the cytosolic carboxylesterase catalyzing the 5'-deoxy-5-fluorocytidine formation from capecitabine in human liver. Drug Metab. Dispos. 32, 1103-1110. Takai, S., Matsuda, A., Usami, Y., Adachi, T., Sugiyama, T., Katagiri, Y., Tatematsu, M., and Hirano, K. (1997). Hydrolytic profile for ester- or amide-linkage by carboxylesterases pI 5.3 and 4.5 from human liver. Biol. Pharm. Bull. 20, 869-873. Takayama, H., Watanabe, A., Hosokawa, M., Chiba, K., Satoh, T., and Aimi, N. (1998). Synthesis of a new class of camptothecin derivatives, the long-chain fatty acid esters of 10-hydroxycamptothecin, as a potent prodrug candidate, and their in vitro metabolic conversion by carboxylesterases. Bioorg. Med. Chem. Lett. 8, 415-4 18. Tang, B. K., and Kalow, W. (1995). Variable activation of lovastatin by hydrolytic enzymes in human plasma and liver. 4. Eur. J. Clin. Pharmacol. 47, 449-451. von Heijne, G. (1983). Patterns of amino acids near signalsequence cleavage sites. Eur. J. Biochem. 133, 17-21. Wallace, T. J., Ghosh, S., and McLean Grogan, W. (1999). Molecular cloning and expression of rat lung carboxylesterase and its potential role in the detoxification of organophosphorus compounds. Am. J. Respir. Cell. Mol. Biol. 20, 1201-1208. Watanabe, K., Kayano, Y., Matsunaga, T., Yamamoto, I., and Yoshimura, H. (1993). Purification and characterization of a novel 46.5-kilodalton esterase from mouse hepatic microsomes. Biochem. Mol. Biol. Int. 31, 25-30. Wong, H., and Schotz, M. C. (2002). The lipase gene family. J. Lipid Res. 43, 993-999. Xie, M., Yang, D., Wu, M., Xue, B., and Yan, B. (2003). Mouse liver and kidney carboxylesterase (M-LK) rapidly hydrolyzes antitumor prodrug irinotecan and the N-terminal three quarter sequence determines substrate selectivity. Drug, Metab. Dispos. 31,21-27. Zhang, J., Burnell, J. C., Dumaual, N., and Bosron, W. F. (1999). Binding and hydrolysis of meperidine by human liver carboxylesterase laCE-1. J. Pharmacol. Exp. Ther. 290, 314-318. Zhu, W., Song, L., Zhang, H., Matoney, L., LeCluyse, E., and Yan, B. (2000). Dexamethasone differentially regulates expression of carboxylesterase genes in humans and rats. Drug Metab. Dispos. 28, 186-191. Zschunke, F., Salmassi, A., Kreipe, H., Buck, F., Parwaresch, M. R., and Radzun, H. J. (1991). cDNA cloning and characterization of human monocyte/macrophage sefine esterase-1. Blood 78, 506-512.
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17
Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets DAVID A. JETT1 AND PAMELA J. LEIN2 1National Institutes of Health, Bethesda, MD 2Oregon Health & Science University, Portland, OR
the brain (McDaniel and Moser, 2004). Perhaps some of the strongest evidence has been presented with AChE null mice in which cholinergic signs and lethality were comparable to those in wild-type mice (Duysen et al., 2001). Certain OP and CM pesticides interact directly with cholinergic receptors and alter their function. Importantly, these effects can occur at concentrations below those required to inhibit ACHE. Moreover, some compounds at extremely low doses may bypass the receptor altogether and target molecules that are involved in critical cellular functions. This chapter discusses the role of AChE inhibition in the toxicity of OP and CM pesticides, with particular emphasis on lower doses that are sublethal but induce changes in cholinergic receptors either by indirect modulation of ACh levels via AChE inhibition or by direct interaction with the receptor. Finally, case studies are presented that suggest that some of the toxicities associated with OP insecticides may not involve AChE or cholinergic receptors at all.
I. I N T R O D U C T I O N Organophosphorus (OP) and carbamate (CM) pesticides inhibit the catalytic function of acetylcholinesterase (ACHE; EC 3.1.1.7) by phosphorylating or carbamylating the esteratic site of the enzyme (Taylor, 1990). This effectively diminishes the capacity of the enzyme to catalyze its endogenous substrate acetylcholine (ACh). As a consequence, the hydrolysis of ACh is prevented, leading to accumulation of ACh in the synaptic cleft and overstimulation and subsequent desensitization of muscarinic and nicotinic ACh receptors. Acute poisonings are treated with atropine to inhibit central muscarinic effects, and an oxime, usually pralidoxime, is used to reactivate AChE. The consequences of the desensitization of cholinergic receptors by lethal exposures to OP and CM pesticides include respiratory failure and death; however, at sublethal doses that inhibit ACHE, exposure to these compounds may induce a compensatory downregulation of muscarinic receptors that allow for tolerance to some, but not all, of the toxic effects (Costa et al., 1982). In mammalian systems, it is believed that the acute toxicity of exposure to higher levels of OP and CM pesticides is derived from this anti-AChE activity and the subsequent parasympathomimetic effect. There is a general consensus that AChE inhibition represents a common mechanism of toxicity for OP pesticides (Mileson et al., 1998), and that unique affinities for other molecular targets in addition to AChE inhibition account for the range of toxicities among different OP compounds (Pope, 1999). However, it has become clear that the role of AChE inhibition in mediating toxicity following moderate and chronic low-level exposures to OP and CM pesticides is less certain, as suggested in subsequent sections of this chapter and by the lack of a good correlation between certain behavioral effects and the magnitude and regional selectivity of AChE inhibition in Toxicology of Organophosphate and Carbamate Compounds
II. C O R R E L A T I O N B E T W E E N CHOLINESTERASE INHIBITION AND NEUROTOXICITY The neurotoxicity of OPs has been extensively documented in accidental human poisonings, epidemiological studies, and animal models. From these studies, it is clear that OPs can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and host factors that influence susceptibility and sensitivity. These effects include acute cholinergic toxicity, a delayed ataxia known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), chronic neurotoxicity, and developmental neurotoxicity. As discussed in the following paragraphs, there is increasing evidence that OPs target molecules in 233
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S ECTI O N I II 9Esterases, Receptors, Mechanisms, & Tolerance D e v e l o p m e n t
addition to or other than AChE to give rise to this spectrum of neurotoxic effects. Most OP compounds cause acute toxicity following exposure to higher levels and this is believed to derive predominantly from AChE inhibition and the subsequent overstimulation of nicotinic and muscarinic receptors (Ecobichon and Joy, 1995). Thus, the symptoms of acute OP neurotoxicity, which include autonomic dysfunction (e.g., miosis and excessive secretions of the airways, excretory systems, salivary glands, and lacrimal glands), involuntary movements (tremors and convulsions), muscle fasciculations, and ultimately respiratory depression, are consistent with the initial stimulation and subsequent paralysis of cholinergic transmission that are observed following AChE inhibition, and they can be mostly reversed upon administration of agents that antagonize cholinergic receptors or reverse AChE inhibition (Ecobichon and Joy, 1995). However, several lines of evidence argue strongly for the involvement of molecular targets in addition to or other than AChE in acute OP neurotoxicity. This was first suggested by comparisons of the in vitro anticholinesterase potency of various OPs with their published LD50 values, which revealed a poor correlation between these parameters (Chambers, 1992). Thus, of six commonly used OP pesticides, methyl chlorpyrifos had the lowest acute toxicity as measured by the LD50 (-3 g/kg), yet its oxon was the most potent anticholinesterase in vitro. Although it is possible that the different ratios between in vitro and in vivo potencies of these OPs simply reflect differential rates of activation and deactivation in vivo, this seems unlikely since subsequent comparative in vivo neurotoxicity studies reported that different OPs gave rise to different toxicological profiles, even at doses that caused comparable AChE inhibition (Liu et al., 1999; McDaniel and Moser, 2004; Sheets et al., 1997). Perhaps the most convincing evidence for targets other than AChE in acute OP toxicity comes from studies of the AChE knockout mouse (Duysen et al., 2001; Li et al., 2000). Targeted deletion of four axons of the AChE gene reduces AChE expression by half in heterozygous (AChE +/-) mice and eliminates it entirely in nullizygous (AChE - / - ) mice. If AChE is the critical molecular target in OP acute neurotoxicity, then it might be predicted that neurotoxic effects following acute exposure to OPs would be attenuated or absent in mice nullizygous for the AChE gene relative to their wild-type littermates. However, experiments in which the OP nerve agent VX was administered to these mice subcutaneously revealed just the opposite: The LD50 value was 10-12 Ixg/kg in nullizygous, 17 Ixg&g in heterozygous, and 24 Ixg/kg in wild-type AChE mice (Duysen et al., 2001). Interestingly, the same cholinergic signs of toxicity were present in AChE - / - mice as in wild-type mice; however, wild-type, but not AChE - / - , mice were protected by pretreatment with atropine. This phenomenon is not unique to VX since similar observations were made following exposure of these mice to DFP (Li et al., 2000). These data, together with the comparative toxicity studies, strongly suggest that
mechanisms not mediated by AChE inhibition must be contributing to the acute neurotoxic effects of OPs. Another action of at least some OPs, which follows either single or repeated exposures, is a delayed effect known as OPIDN (Abou-Donia and Lapadula, 1990). OPIDN is characterized by a delayed onset of ataxia accompanied by a Wallerian-type degeneration of the axon and myelin in the most distal portion of the longest axon tracts in both the central nervous system (CNS) and the peripheral nervous system (PNS) (Cavanagh and Patangia, 1965). Early studies on the mechanisms of OPIDN focused on inhibition of AChE or butyrylcholinesterase by OPs; however, subsequent studies eliminated both esterases as targets for OPIDN (Aldridge and Barnes, 1966). Neurotoxic esterase (NTE) has since been proposed to be a critical molecular target in OPIDN because OP compounds that cause OPIDN preferentially inhibit its enzymatic activity (Johnson, 1969). However, inhibition of NTE is not sufficient for axonal degeneration (Abou-Donia, 2003; Abou-Donia and Lapadula, 1990), and reports that OPs exert neurotoxic effects in NTE knockout mice indicate that targets other than or in addition to NTE mediate OPIDN (Glynn, 2003). Potential alternate molecular targets include calcium/calmodulin-dependent protein kinase II (CaM kinase II). The evidence supporting this hypothesis, which has been reviewed by Abou-Donia (2003), includes observations that aberrant phosphorylation of cytoskeletal proteins is present in OPIDN and may be causally related to the OP-induced axonal degeneration and demyelination, and that CaM kinase II, which phosphorylates cytoskeletal proteins, is activated by OPs that cause OPIDN. There is increasing evidence that OPs may also cause a long-term, persistent chronic neurotoxicity following either a single acute high-dose exposure or repeated exposures to low-level, subclinical doses of OPs. The clinical and epidemiological data in support of OP-induced chronic neurotoxicity that is distinct from both cholinergic and OPIDN effects have been reviewed (Abou-Donia, 2003; Kamel and Hoppin, 2004; Ray and Richards, 2001) and are not discussed here. Chronic OP neurotoxicity presents with pathological lesions in both the PNS and CNS, but it is the latter that is primarily responsible for the presenting neurologic symptoms and changes in neurobehavioral performance, reflecting cognitive and psychomotor dysfunction. The most sensitive manifestation of chronic OP neurotoxicity is a general malaise lacking in specificity and related to mild cognitive dysfunction, similar to that described for Gulf War syndrome (Kamel and Hoppin, 2004). The mechanisms underlying these effects are not known, and the role of AChE inhibition is controversial (Abou-Donia, 2003; Kamel and Hoppin, 2004) and may vary depending on the exposure parameters. Chronic neurotoxicity subsequent to a single acute exposure to OPs may be triggered by AChE inhibition. It has been hypothesized that increased ACh levels following AChE inhibition activate glutamatergic neurons causing the release of glutamate, which ultimately results in excitotoxicity
CHAPTER 1 7 9Muscarinic Receptors and OP Neurotoxicity
via increased intracellular calcium and activation of nitric oxide synthase following NMDA receptor activation (de Groot et aL, 2001). The resultant necrosis in affected brain regions may thus contribute to the persistent neurologic and neurobehavioral deficits. There is support for this proposed mechanism of chronic OP neurotoxicity in that it has been demonstrated that nitric oxide synthesis inhibitors block OP-induced seizures (Aschner et al., 1999). However, the role of AChE inhibition in this sequence of events has yet to be established. In contrast, chronic OP neurotoxicity induced by repeated exposures to subclinical OP doses has been reported to occur in the absence of AChE inhibition (Abou-Donia, 2003; Kamel and Hoppin, 2004), suggesting that mechanisms other than anticholinesterase activity mediate the neurotoxic effects elicited by this exposure scenario. However, what these mechanisms are has yet to be established. A review of data relevant to this question suggests the intriguing possibility that repeated exposures to sublethal or subclinical doses of OPs increases apoptotic neuronal death via oxidative stress (Abou-Donia, 2003). Whether OP-induced apoptosis gives rise to long-term neurological and neurobehavioral deficits remains to be directly demonstrated. Another important neurotoxic sequel of OP exposure is developmental neurotoxicity. Since OP d e v e l o p m e n t a l neurotoxicity in both humans (Garry, 2004) and animal models (Izrael et aL, 2004) has been reviewed, we discuss only selected case studies to illustrate the conclusion that this neurotoxic effect of OPs can also occur independent of AChE inhibition. Initial studies indicated that the developing nervous system is far more sensitive to the acute cholinergic toxicity of OPs (Bushnell et al., 1991; Pope and Chakraborti, 1992), most likely due to age-related differences in the hepatic detoxification of the AChE-active metabolites of OPs (Atterberry et al., 1997; Benke and Murphy, 1975; Mortensen et al., 1996). However, subsequent studies
TABLE 1. Exposure period
235
suggested that OPs could also cause neurodevelopmental defects independent of AChE inhibition. Slotkin and colleagues reported decreased cell division and DNA and protein synthesis in the brains of animals treated with OPs and concluded that these effects are not mediated by AChE inhibition because they occur at doses that do not cause systemic cholinergic toxicity, and they are not reversed by cholinergic receptor antagonists (Campbell et al., 1997; Garcia et al., 2001; Johnson et al., 1998; Whitney et al., 1995). Jett et al. (2001) and Levin et al. (2002) demonstrated that exposure of developing rats to low levels of chlorpyrifos also impairs their cognitive function. We demonstrated that spatial learning in the Morris water maze was impaired in weanling rats exposed to chlorpyrifos during development (Table 1), and this cognitive deficit occurred in the absence of a significant effect on AChE or the downregulation of cholinergic receptors that typically accompanies AChE inhibition (Jett et al., 2001). Whether this OP interfered with neural development leading to cognitive impairment in the weanling rats is unknown. However, these in vivo studies are in agreement with in vitro studies indicating, for example, that chlorpyrifos inhibits axon outgrowth in primary neuronal cell cultures (Howard et al., 2005) or PC12 cells (Das and Barone, 1999) independent of AChE inhibition. The evidence indicates that although there is a correlation between AChE inhibition and the prevalence and/or severity of OP neurotoxicity, the spectrum of toxic effects elicited by any given OP cannot be attributed entirely to the inhibition of ACHE. If this is true, then OPs must react not only with AChE but also with other proteins, and it has been suggested that different OPs elicit different toxicological profiles because each interacts with a unique subset of molecular targets (Chiappa et al., 1995; Liu et al., 1999; Ray and Richards, 2001). In support of the hypothesis that OPs interact with proteins other than AChE, various OP
Performance in the Morris Water Maze in Chlorpyrifos-Exposed Weanling Ratsa Dosage (mg/kg)
N
Latency on last day of test (sec)
Time in training quadrant (sec) b
Swim speed (cm/sec) c
Preweaning
0 0.3 7.0
20 19 17
12.3 _+ 2.6 17.5 + 3.0 23.7 +_ 2.3*
13.8 _+ 1.2 12.2 +_ 1.0 7.7 _+ 1.0"
19.1 +_ 1.0 18.3 _+ 1.0 18.8 + 1.3
Postweaning
0 0.3 7.0
7 7 8
9.0 _+ 2.3 22.7 _+ 4.7* 22.3 _+ 4.4*
17.0 + 1.4 11.5 +_ 1.0" 12.2 _+ 1.4"
18.2 + 1.3 18.6 + 1.0 18.1 + 1.0
aRats in the preweaning group were injected before weaning on postnatal days 7, 11, and 16; rats in the postweaning group were injected after weaning on postnatal days 22 and 26. All rats were weaned on postnatal day 21 and tested from day 24 to day 28. Adapted from Jett et al. (2001). bin the probe test, rats were allowed to swim freely without the platform for 30 sec and the time spent in the quadrant of the pool that previously contained the platform during 5 days of prior training was recorded. CTotaldistance traveled during the probe test divided by 30 sec. *Value is different from control value at the 0.05 level of significance as determined by analysis of variance.
236
S ECTI O N III 9Esterases, Receptors, M e c h a n i s m s , & Tolerance D e v e l o p m e n t
compounds have been shown to bond covalently to a wide variety of proteins, including other cholinesterases (Casida and Quistad, 2004); serine hydrolases such as serum, liver, and brain carboxylesterases (Ray and Richards, 2001); muscarinic and nicotinic receptors; the cannabinoid CB 1 receptor (Bomser et al., 2002); and albumin (Peeples et al., 2005). The ongoing challenge is to determine which of these molecular interactions is toxicologically significant.
III, DIRECT AND INDIRECT E F F E C T S O N M U S C A R I N I C RECEPTORS
A. Tolerance Mediated by Muscarinic Receptor Downregulation Virtually all cholinergic synapses can be affected by exposure to anticholinesterase compounds such as OPs and CMs. These include autonomic postganglionic parasympathetic nerve endings, sympathetic and parasympathetic ganglia, motor end plates of skeletal muscle, and, of course, various regions of the CNS. Hyperactivity at these synapses due to accumulation of ACh causes a variety of symptoms mediated by overstimulation of muscarinic and nicotinic receptors. If laboratory animals are pretreated with sublethal doses of OPs and then treated with a higher, more toxic dose of the same compound, they are able to tolerate this dose to a much greater extent than naive rats given the same toxic dose. This adaptive response is a subsensitivity phenomenon and has been termed "tolerance." Tolerance to anti-AChE insecticides was first described by Barnes and Denz (1951) when they noticed that rats survived a long-term feeding trial with the highly toxic parathion. Although there are several factors that determine the sensitivity of an organism, or even a specific tissue, to AChE inhibitors, it is widely believed that the compensatory mechanism for the development of tolerance to prolonged inhibition of AChE is the downregulation of muscarinic cholinergic receptors in response to the accumulation of ACh at the synapse. Two important observations led to the discovery that cholinergic receptors mediated tolerance to anti-AChE compounds. First was the observation that cross-tolerance occurred between OPs, CMs (Costa and Murphy, 1983b), and nicotinic (Costa and Murphy, 1983a) and muscarinic (Brodeur and Dubois, 1964; Costa et al., 1981; Schwab and Murphy, 1981) cholinergic agonists. Second was that attenuation of AChE inhibition did not correlate well with tolerance. In fact, AChE activity can remain significantly depressed throughout the exposure period after the development of tolerance (Chippendale et al., 1972; Sivam et al., 1983). Importantly, these data showed that the metabolic disposition or differences in target tissue distribution of the AChE inhibitor may not play as prominent a role in the development of tolerance. Since these early studies, decreases in muscarinic receptors have become recognized universally as a hallmark of
exposure to OPs. The evidence for CM pesticides is not as strong, partially due to the rapidly reversible inactivation of AChE by these compounds compared to OPs; however, some studies have shown that muscarinic receptors are regulated by exposure to CMs (Jones et al., 1998). Downregulation of muscarinic receptors has been demonstrated repeatedly in a variety of experimental paradigms with several OPs (Abdallah et al., 1992; Costa et al., 1982; Russell and Overstreet, 1987). We have shown that the degree of AChE inhibition correlates well with decreases in muscarinic receptors (Jett et al., 1993). Stamper et al. (1988) found that rats exposed to parathion during development did not show any signs of overt toxicity, possibly due to tolerance as indicated by decreases in AChE and muscarinic receptors. However, these rats exhibited spatial memory deficits in the radial arm and T-mazes. This was one of the first studies to suggest that "compensatory" changes in receptors may result in tolerance to some effects (e.g., lethal) but may in fact be an underlying mechanism for other more subtle effects such as memory impairment. In light of the prominent role these receptors play in cognitive function, it is not surprising that changes in the availability of muscarinic binding sites due to OP-induced downregulation have been associated with altered behavior. Muscarinic receptors are a well-characterized group of five subtypes (M1-M5) belonging to the seven-transmembrane family of receptors coupled to signal transduction systems. Generally, the M1, M3, and M5 subtypes couple to Gq/11 protein, which activates phospholipase C, and M2 and M4 to G~0, which inhibits adenylyl cyclase. These receptors are distributed widely throughout the nervous system and are involved in many central and peripheral functions. They are the target for many therapeutics as well, such as Alzheimer's disease drugs. Changes in these receptors as a result of exposure to anti-AChEs may be restricted to one or two subtypes, which may not be detected by nonselective methods of identification. We have observed that protein and mRNA of certain subtypes may be more affected by OP exposure than others (Jett et al., 1993, 1994), and other laboratories have corroborated these findings (Yagle and Costa, 1996) and pointed to the M2 subtype as being especially vulnerable to OPs (Bakry et al., 1988; Katz and Marquis, 1989; Silveira et al., 1990).
B. Cholinesterase-Independent Effects on Muscarinic Receptors In addition to their indirect effects on muscarinic receptors through AChE-mediated changes in ACh levels, many OP and CM pesticides can affect their expression and function directly (Bakry et al., 1988; Eldefrawi et al., 1992). Paraoxon, dichlorvos, and tetraethyl pyrophosphate (TEPP) were found to be noncompetitive antagonists of muscarinic receptors in bovine caudate nuclei labeled with [3H]quinuclidinyl benzilate ([3H]-QNB) at concentrations that had no effect on AChE activity (Volpe et al., 1985). The
CHAPTER 1 7 9Muscarinic Receptors and OP Neurotoxicity CMs physostigmine and neostigmine were also tested in this study, but only physostigmine both decreased the number of receptors and enhanced their affinity for [3H]-QNB. The OP chemical weapon VX and the therapeutic agent echothiophate were found to inhibit high-affinity [3H]cis-methyldioxolane ([3H]-CD) binding at concentrations comparable to those that inhibit AChE in rat brain and N1E115 neuroblastoma cells (Bakry et al., 1988). It was found that the active metabolite of the pesticide chlorpyrifos, chlorpyrifos-oxon, bound to muscarinic receptors in rat striatum identified with [3H]-CD noncompetitively with an IC50 value of approximately 22 nM and resulted in a covalent modification of the receptor (Huff et al., 1994). [3H]-CD binds to M2 receptors (Huff and Abou-Donia, 1994) with high affinity, and thus it was suggested that direct actions on a subset of muscarinic receptors, in addition to their actions on ACHE, could account for some of the toxicity of OP compounds. Inhibition of [3H]-CD binding by OPs was observed at nanomolar to micromolar concentrations, with Ko.5 values of 3, 10, 40, 100, and 800 nM for VX, soman, satin, echothiophate, and tabun, respectively (Bakry et al., 1988). Other studies confirmed that binding of ligands to muscarinic receptors is inhibited by OPs at concentrations far below those that inhibit ACHE, as low as the picomolar range (Katz and Marquis, 1989; Silveira et al., 1990). Functional studies examining the effects of OPs on signaling events downstream of muscarinic receptor activation further support the hypothesis that OPs can interact directly with M2 receptors. Activation of M2 and M4 receptors generally reduces the activity of adenylyl cyclase, which decreases cAMP production, whereas activation of M1, M3, or M5 receptors increases phosphoinositide-specific phospholipase C activity, which increases release of inositol triphosphate. A comparative study of paraoxon, malaoxon, and chlorpyrifosoxon in slice cultures of rat frontal cortex indicated that all three OPs inhibited cAMP formation in a concentrationdependent manner (Ward and Mundy, 1996). In contrast, none of these OPs affected either basal or carbachol-stimulated phosphoinositide turnover. These data suggest that OPs activate M2/M4 receptors through direct interactions and not as the result of increased levels of endogenous ACh consequent to ACHE inhibition. Other in vitro studies of rat striatum predicted and confirmed that, generally, OPs act to stimulate M2/M4 receptor function (Axelrad et aL, 2002; Huff and Abou-Donia, 1994; Jett et al., 1991), with the exception of apparent inhibitory effects of chlorpyrifos-oxon on prejunctional M2/M4 receptors (Axelrad et al., 2002).
C. Direct Action on Muscarinic Receptors in the Lung In the lung, many of the normal physiological processes that govern airway function are mediated by muscarinic receptors. Cholinergic nerves in the vagi regulate airway tone and reactivity. These nerves release ACh onto M3 muscarinic
237
receptors causing contraction of airway smooth muscle, resulting in bronchoconstriction, and vagally induced bronchoconstriction is limited by autoinhibitory M2 muscarinic receptors on parasympathetic nerves (Fryer and Maclagan, 1984; Minette and Barnes, 1988). Previous studies have shown that neuronal M2 receptors are dysfunctional in animal models of asthma (Fryer and Wills-Karp, 1991; Gambone et aL, 1994; Jacoby and Fryer, 1991) and in patients with asthma (Minette et al., 1989). Loss of M2 receptor function leads to increased release of ACh from parasympathetic nerves resulting in potentiation of vagally mediated bronchoconstriction, which contributes to airway hyperreactivity. In part, these highly critical functions mediated by the cholinergic system within the lung suggest that pesticide exposure may be a contributing factor underlying the increased incidence of childhood asthma in the United States and other industrialized nations. A number of clinical and epidemiological studies have linked exposure to OPs to airway hyperreactivity and other symptoms of asthma (Deschamps et al., 1994; Hoppin et al., 2002; O'Malley, 1997). Many of the current pesticides, especially those that children may come in contact with, are anticholinesterases that may have adverse effects on the cholinergic control of vagally induced bronchoconstriction. To test the hypothesis that asthma may be exacerbated by low-level exposure to OPs, airway hyperreactivity was measured in guinea pigs exposed to chlorpyrifos, a widely used OP pesticide (Fryer et al., 2004). Electrical stimulation of the vagus nerves caused frequency-dependent bronchoconstriction that was significantly potentiated in animals 24 hr or 7 days after a single subcutaneous injection of either 390 or 70 mg/kg chlorpyrifos, respectively (Fig. 1). Mechanisms by which chlorpyrifos may have caused airway hyperreactivity include inhibition of ACHE, dysfunction of M3 muscarinic receptors on airway smooth muscle, or inhibition of autoinhibitory M2 muscarinic receptors on parasympathetic nerves in the lung. AChE activity in the lung was significantly inhibited 24 hr after treatment with 390 mg/kg chlorpyrifos but not 7 days after injection of 70 mg/kg chlorpyrifos (Fig. 2). The observations that subchronic exposure to 70 mg/kg potentiated vagally induced bronchoconstriction in the absence of significant AChE inhibition and that acute exposure to eserine (250 Ixg/kg) significantly inhibited lung AChE but did not potentiate bronchoconstriction (Figs. 1 and 2) suggest that this effect on respiratory function was not mediated by AChE inhibition. Neuronal M2 receptor function was tested using the M2 agonist pilocarpine, which inhibits vagally induced bronchoconstriction in control animals. In chlorpyrifostreated animals, pilocarpine dose-response curves were shifted significantly to the fight, demonstrating decreased responsiveness of neuronal M2 receptors (Fig. 3). In contrast, chlorpyrifos treatment did not alter methacholineinduced bronchoconstriction, suggesting that chlorpyrifos does not alter M3 muscarinic receptor function on airway
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SECTION IV- O r g a n Toxicity
neuronal migration (a secondary effect of damage to radial glia), and impaired synaptogenesis or altered synaptic function. Damage to neurons may produce developmental and/or late-onset cognitive dysfunction. An examination of Table 2, which describes most of the in vitro OP neurotoxicity studies presented in the literature, reveals that most studies have been carried out with clonal cell lines rather than primary cell cultures or tissue cultures. Four neuronal and one glial clonal cell line have been used for OP neurotoxicity testing in vitro. The characteristics of the neuronal cell lines that have made them useful for this purpose are briefly reviewed next, followed by selected comparisons with the results generated by studies in primary cultures.
A. Cell Lines and Primary Cultures Used in OP Neurotoxicity Testing Neuron-like cell lines are attractive models for exploring in vitro neurotoxicology because they proliferate rapidly in their undifferentiated state, and many of them can be induced to develop properties of more mature neurons (i.e., differentiate) with stimulants such as dibutyryl cyclic AMP (dBcAMP), retinoic acid, or NGE Several neuronal cell lines have been studied for more than 30 years and found to have neurobiological properties; these cells thus provide well-characterized models for specific toxicologic investigations. Mouse, rat, and human cell lines are commonly used for in vitro neurotoxicity testing because of their stability and hardiness. Three mouse neuroblastoma cell lines, cloned independently from the C1300 tumor, have been used in OP studies as neuronal cell models: NB2a (Klebe and Ruddle, 1969), NB41A3 (Augusti-Tocco and Sato, 1970), and N-18 (Amano et al., 1972). These cell lines proliferate rapidly, but are capable of developing properties of differentiated neurons. All three cell lines are characterized by a pseudotetraploid karyotype, a neuroblastoid morphology, a stable cadre of neurotransmitter enzymes and receptors, and the capacity to extend neurite-like processes. NB2A and NB41A3 clones display electrogenic membrane responses (Yogeeswaran et al., 1973). The NB2a (Harvey and Sharma, 1980; Flaskos et al., 1999; Fowler et al., 2001; Sachana et al., 2001; Axelrad et al., 2002, 2003) and N-18 cell lines (Henschler et al., 1992; Schmuck and Ahr, 1997) have been used in several OP toxicity studies because of their ability to extend neurite-like processes upon treatment with dBcAMP (Shea et al., 1991). Both cell lines express the 200-kDa neurofilament protein, which is the heavy subunit of neurofilaments localized to the cytoskeleton of neurons that provides a useful biochemical marker for neuronal differentiation. The NB41A3 cell line expresses AChE and NTE activities and can be induced to differentiate with retinoic acid. The AChE activities are considerably lower than the parallel AChE activities in mouse or hen brain. Specific activities of NTE correlate with those of mouse
brain (Ehrich and Veronesi, 1995), and this cell line has been used for comparisons of neurotoxic OP effects on murine vs human cells (Ehrich et al., 1995, 1997; Ehrich and Correll, 1998). PC12 cells, a neuron-like cell line established from a transplantable rat adrenal pheochromocytoma (Greene and Tischler, 1976), have been a neuron model in many neurobiological and neurochemical studies (Biocca et al., 1983; Levi et al., 1985). These cells have been used in several studies of OP neurotoxicity (Flaskos et al., 1994; Bagchi et al., 1995; Li and Casida, 1997, 1998; Song et al., 1998; Crumpton et al., 2000; Das and Barone, 1999; Qiao et al., 2001) because they express NGF receptors and become morphologically differentiated in response to NGE PC12 cells were originally characterized as resembling immature noradrenergic cells (Greene and Tischler, 1976). In addition, PC12 cells contain ACHE; synthesize, store, and release acetylcholine (ACh); and fail to synthesize epinephrine (Greene and Tischler, 1976; Green and Rein, 1977). The cell line also expresses monoamine oxidase type A (MAO-A) but not MAO-B. PC12 cells genetically expressing MAO-B display a dose-dependent increase in sensitivity to the toxicant MPTP (Wei et al., 1996). Ehrich and colleagues (Nostrandt and Ehrich, 1992, 1993; Veronesi and Ehrich, 1993) selected the SY5Y human neuroblastoma cell line as a model system for OP toxicity testing based on relatively high activities of NTE and AChE compared to other cell lines. After establishing that mipafox-induced NTE inhibition in SY5Y cells is similar to that of chicken brain, these investigators (Nostrandt and Ehrich, 1993) showed that mipafox-induced inhibition of NTE in SY5Y cells is decreased by aldicarb, a competitive inhibitor of NTE that protects hens from the development of OPIDN (Johnson, 1982; Lotti et al., 1993), and verapamil, a Ca 2+ channel blocker that reduces OPIDN effects in hens (E1-Fawal et al., 1990). Furthermore, NTE is inhibited in SY5Y cells by OP compounds that cause OPIDN in the hen test model, including DFP, phenyl saligenin cyclic phosphate, and tolyl saligenin phosphate. In contrast, several OP compounds that do not cause OPIDN in vivo, including paraoxon, parathion, malaoxon, malathion, dichlorvos, fenthion, and fenitrothion, do not significantly inhibit NTE in SY5Y cells (Ehrich et al., 1994). Thus, it is possible to use this cell line to examine the molecular mechanisms that distinguish acute and delayed neurotoxic effects of OP compounds. Ehrich et al. (1995) further determined NTE and AChE activities in SY5Y cells differentiated with retinoic acid or NGF after treatment with paraoxon, DFP, parathion, and mipafox. SY5Y cells differentiated with retinoic acid show similar AChE and NTE inhibition by DFP, paraoxon, and parathion compared to either undifferentiated or NGFdifferentiated cells. However, mipafox-induced inhibition is much lower in retinoic acid-differentiated cells. These findings suggest that SY5Y cells differentiated with NGF
CHAPTER 22 9In Vitro Neurotoxicity Testing would be a potentially valuable model for investigating OP-induced "neuropathy." In such a model system, the underlying mechanisms of OPIDN might also be examined, and these mechanisms might be distinguished from those associated solely with acute neurotoxicity. The cell line has been further studied as a model for OPIDN by our laboratory (Hong et al., 2003; Cho and Tiffany-Castiglioni, 2004). Several other types of culture systems have been used on a more limited basis to observe neuronal responses to OP compound exposure, including primary cultures and whole embryo cultures. These models exhibit more differentiated properties than those present in cell lines. Electrophysiological properties of synapses have been studied in primary cultures of neurons from fetal rat hippocampus, revealing several noncholinesterase-related effects of OP compounds. Acute exposure to low paraoxon levels increases the frequency of miniature postsynaptic currents mediated by GABA or glutamate in hippocampal neurons. These presynaptic effects are not mediated by actions on muscarinic or nicotinic receptors and are cholinesterase independent (Rocha et al., 1996). In the same culture system, the nerve agent VX directly interacts with presynaptic muscarinic receptors to block action potential-dependent release of GABA. VX also blocks the action potential-dependent release of glutamate, facilitating the action potential-independent release of both GABA and glutamate by cholinesterase-independent actions (Rocha et al., 1999). Long-term hippocampal slice cultures have not been examined for OP-induced neurotoxicity, although acutely isolated slices have been used to demonstrate that sarin inhibits the evoked release of GABA (Chebabo et al., 1999). In an interesting use of oocytes transfected to express the rat OL4[~2 nicotinic acetylcholine receptor, several OP compounds have been shown to interact directly with this receptor at concentrations several orders of magnitude lower than those that inhibit AChE (Smulders et al., 2004). Bovine adrenal chromaffm cells, which are of neuroepithelial origin, have been studied as an alternative model for neuronotypic cells because they have high carboxyl esterase activities, particularly NTE (Sogorb et al., 1996; Quesada et al., 2004). In these cells, acute treatment with a high concentration of triphenyl phosphite, but not paraoxon or diisopropyl phosphorofluoridate, reduces adenosine incorporation into ATP in mitochondria (Knoth-Anderson et al., 1992). Acute exposure to a high level of mipafox decreases depolarization-induced noradrenaline release and inhibits voltage-dependent calcium channel (VDCC) activity and nicotinic ionic currents. These results suggest that VDCC and nicotinic receptors are targets for mipafox leading to catecholamine release (Guti6rrez et al., 1996). These effects would be of interest to study at lower and repeated exposures to OP compounds. Whole rat embryos have been used in one study to morphologically examine the effects of OP compounds
329
on neuroepithelial development. At levels of chlorpyrifos (14 txM) that are below those causing dysmorphogenesis, the neuroepithelium of embryos shows increased apoptotic cell death, inhibition of mitosis, and displacement of mitotic cells. This model therefore appears to be sensitive to the detection of more subtle developmental abnormalities than those typically associated with teratogenesis (Roy et al., 1998).
B. Noncholinergic and Nonantiesterase End Points and Mechanisms As mentioned previously, OP compounds induce neurotoxicity by inhibition of AChE and NTE. Unlike whole organisms such as vertebrates, cells in culture do not require AChE activity for survival. This circumstance creates two operational conditions in vitro: (1) Cultured cells appear insensitive to cytotoxicity because molar concentrations in plasma that are lethal are not lethal in culture, and (2) noncholinergic mechanisms of toxicity (those that do not involve the inhibition of AChE and buildup of synaptic levels of ACh) become the focus of study in vitro. Increasing in vivo evidence on the importance of noncholinergic and nonantiesterase mechanisms, however, indicates that the latter is not a case of "looking under the lamppost" but a fortuitous circumstance in which cell culture models can be of particular value (Rocha et al., 1996, 1999; Schuh et al., 2002). Similarly, inhibition of esterases in general, including NTE, is not necessary for the induction of several biochemical alterations in cell and tissue cultures. It is of great interest that some studies listed in Table 2 show that protoxicant (nonesterase-inhibiting) forms of OP compounds, such as tri-o-tolyl phosphate (Carlson and Ehrich, 1999, 2001; Carlson et al., 2000), parathion (Carlson et al., 2000), and chlorpyrifos (Bagchi et al., 1995; Song et al., 1998; Crumpton et al., 2000; Das and Barone, 1999; Garcia et al., 2001; Schuh et al., 2002), exert biological effects on neural cells, albeit usually at high concentrations. Many in vitro studies suggest that the toxic effects of OP compounds on neural cells are unrelated to the inhibition of AChE because they occur at concentrations lower than those needed to inhibit ACHE. Some alterations are also uncorrelated with NTE inhibition. In vitro effects of OP compounds that appear to be affected via nonantiesterase and noncholinergic mechanisms include alterations in DNA synthesis (Song et al., 1998; Qiao et al., 2001), apoptosis (Carlson et al., 2000), transcription factor binding and expression (Crumpton et al., 2000; Garcia et al., 2001), signaling molecules and pathways (Garcia et al., 2001; Schuh et al., 2002; Hong et al., 2003), mitochondrial integrity (Knoth-Anderson et al., 1992; Carlson et al., 2000; Hong et al., 2003), stress responses (Garcia et al., 2001; Sachana et al., 2001), expression of cytoskeletal proteins (Schmuck and Ahr, 1997; Carlson and Ehrich, 2001; Sachana et al.,
330
SECTION IV. O r g a n T o x i c i t y
2001; Cho and Tiffany-Castiglioni, 2004), and major neurophysiological events such as synaptic or receptor function (Rocha et al., 1996, 1999; Smulders et al., 2004).
C. Neuronal Morphology and Differentiation It is interesting to note that all of the neuronal cell lines tested, including mouse, rat, and human clones of diverse origins, have been found to apparently distinguish OP compounds that cause OPIDN from those that do not, based on shortened neurites or alterations in the neuronal cytoskeleton (Henschler et al., 1992; Li and Casida, 1998; Fowler et al., 2001; Hong et al., 2003; Massicotte et al., 2003; Cho and Tiffany-Castiglioni, 2004). The observation that OPIDN-inducing compounds inhibit the initiation, outgrowth, or maintenance of neurites in cultured cells may have functional relevance to the phenomenon of dying-back neuropathy observed in vivo. Alternatively, or in addition, such changes in morphology may be relevant to changes in dendritic branching patterns observed in developing rat brains exposed to low or moderate levels of paraoxon (Santos et al., 2004). Three cell culture studies provide sufficient ranked numerical data for mipafox, chlorpyrifos or chlorpyrifosoxon, leptophos, and tri-o-cresyl phosphate to enable a comparison of relative potencies of the OP compounds on neurite extension and compare them with the in vivo hen model. The cell culture studies employed different treatment regimens, cell lines, and methods for assessing OP effects on neurite extension. In two studies, N-18 or NB2a mouse neuroblastoma cells were treated for 2 weeks with OP and then differentiated with dBcAMP for 6 days in the continued presence of OR In the case of N-18 cells, lengths of 50 processes per test group were measured microscopically and the ICs0 for inhibition of neurite extension was calculated (Henschler et al., 1992). In the case of NB2a cells, an enzyme-linked immunosorbent (ELISA) assay was used to measure reductions in levels of the neuronal cytoskeleton protein NF200 in lieu of a morphological assay, and the ECs0 was calculated (Schmuck and Ahr, 1997). In another study, PC 12 rat pheochromocytoma cells were differentiated with NGF in the absence of OP compounds for 5 days and then exposed to OP for 48 hr. Neurite lengths were measured microscopically (Li and Casida, 1998). Despite different treatment regimens and assays for neurite extension, the ranked order of potency for OP neurotoxicity is similar between the PC12 and NB2 cells in these studies, in which the order of potency is tri-o-cresyl phosphate > mipafox > chlorpyrifos or chlorpyrifos-oxon. Leptophos has a similar ECs0 as chlorpyrifos in NB2a; its effects were not measured in PC12 cells. In contrast, in N-18 cells, the order of potency is different: tri-o-cresyl phosphate, mipafox, and chlorpyrifos have similar ICs0 values and both are nearly three orders of magnitude lower than that of leptiphos. None of
these cell culture studies yielded the same rank order of potency for induction of OPIDN in hens in vivo, which is mipafox > leptophos > tri-o-cresyl phosphate > chlorpyrifos (Johnson, 1977). The lack of agreement between the hen animal model and rodent cell cultures suggests species or toxicokinetic differences because the mouse and rat are much more resistant to OPIDN than is the hen. The lack of agreement between two of the cell culture studies cannot be resolved without a standardized treatment regimen and method of assessment of neurite extension.
IV. E F F E C T S O F O P C O M P O U N D S ON GLIA Neuronal function and nervous tissue structure require the participation of neuroglia, or glial cells. The three main types of glia in the CNS are astroglia, oligodendroglia, and microglia. Astroglia possess active antioxidant systems that respond to stress and injury in the brain (Sagara et al., 1993; Bolanos et al., 1995; Tanaka et al., 1999; TiffanyCastiglioni and Qian, 2005). Astrocytes also integrate and modulate neuronal synaptic transmission through intrinsic signaling properties. These cells exhibit Ca 2+ excitability, possess functional neurotransmitter receptors that regulate intracellular Ca 2+ concentrations, demonstrate the ability to propagate Ca 2+ oscillations to neighboring cells through gap junctions, and facilitate the release of neuroactive transmitters to neurons (Araque et al., 2001; Carmignoto, 2000; Bezzi and Volterra, 2001). Radial glia and Bergmann glia, two specialized types of astroglia, provide scaffolding for neuronal migration during development (Rakic, 2003), Astroglia and radial glia may respond to toxicants by disruption of radial glial scaffolding in the developing nervous system, gliosis or glial activation, or altered metabolism. Oligodendroglia and Schwann cells myelinate axons in the CNS and PNS, respectively. Toxic effects on oligodendroglia may include demyelination, apoptosis succeeded by proliferation, and loss of oligodendroglial progenitor cells (Tiffany-Castiglioni et al., 2004). Microglias, which are of mesenchymal origin, mediate inflammatory responses in the CNS (Streit et al., 1988). Microglia have not been studied as primary targets for OP-induced neurotoxicity, and OP effects on other glia have received much less attention than neurons. The possibility that glia are targets for chlorpyrifos-induced neurotoxicity has been reviewed by Garcia et al. (2002).
A. C6 Rat Glioma Cell Line In vitro studies on OP-induced neurotoxicity effects on glia
have been conducted with the C6 rat glioma cell line as well as several primary culture types. C6 cells were derived from a rat brain tumor induced by N-nitrosomethylurea (Benda et al., 1968). These cells display properties of
CHAPTER 22 9In Vitro Neurotoxicity Testing oligodendrocytes, such as the expression of glucocorticoid receptors and the induction of glycerol phosphate dehydrogenase by glucocorticoids (de Vellis and Brooker, 1972; Beaumont, 1985), and astrocytes, such as the expression of glial fibrillary acidic protein (Bissell et al., 1975; Pishak and Phillips, 1980). Cells treated with dBcAMP extend thin cytoplasmic processes in a response believed to model either differentiation or glial reactivity, with the latter indicated by interleukin-6 production (Hamprecht et al., 1973; Slegers and Joniau, 1996). Cells induced to differentiate with norepinephrine or dBcAMP also express S-100 protein, a glial marker (Labourdette and Mandel, 1980; van Eldik and Zimmer, 1987). C6 cells are capable of producing and secreting NGF, which is stimulated by isoproterenol activation of [3-adrenergic receptors (Schwartz et al., 1977). In addition, C6 cells appear to express monoamine oxidase B (MAO-B) because (-)-deprenyl, a specific MAO-B inhibitor that has been used as an effective anti-Parkinsonian drug, can reduce GFAP expression. The results for the C6 cells are suggestive but not conclusive validation that glial cells are sensitive to OP neurotoxicants. Short-term exposure to chlorpyrifos at noncytotoxic levels arrests DNA synthesis in undifferentiated C6 cells and elicits reactive oxygen species formation in C6 cells differentiated with dexamethasone (Garcia et al., 2001). Furthermore, C6 cells are more sensitive than PC12 cells to inhibition of DNA synthesis by chlorpyrifos and other AChE inhibitors (Qiao et al., 2001). These results suggest that glia offer development-related targets of chlorpyrifos-induced toxicity. If so, the ramifications for chlorpyrifos exposure during brain development are significant because of the interdependence of neurons and glia during gliogenesis, neuronogenesis, neuronal migration, and synaptogenesis. However, other end points do not show greater sensitivity of C6 cells than neuronal cells to multiple exposures to OP compounds that do and do not induce OPIDN. For example, both dBcAMP differentiated N-18 mouse neuroblastoma cells and C6 rat glioma cells show a similar depression of "neurite-like" extension after 3-week exposures to chlorpyrifos or other OPIDN-inducing compounds. In general, strong inducers of OPIDN markedly depress cytoskeletal protein levels in both cell lines. In conjunction with NTE/AChE ratios, both cell lines appear to be able to detect the induction of OPIDN (Schmuck and Ahr, 1997).
B. Primary Culture Models Containing Glia In contrast to some of the previously mentioned results suggesting glial sensitivity to OP-induced neurotoxicity, studies with primary cultures have not detected either astroglial or oligodendroglial sensitivity to several OP compounds tested. The phosphorylation of the CaZ+/cAMP response element binding protein (pCREB) is a very sensitive response of primary embryonic rat cortical neurons
331
to chlorpyrifos and its metabolites chlorpyrifos-oxon or trichloropyridinol (Schuh et al., 2002). For example, pCREB levels are elevated at OP concentrations much lower than needed for AChE inhibition. However, chlorpyrifos exposure does not elevate pCREB in similarly exposed astrocyte cultures. The astrocyte cultures were treated in medium containing serum, whereas the neurons were in serum-free medium. One study showed that serum and albumin protect against chlorpyrifos toxicity in C6 and PC12 cultures (Qiao et al., 2001), which may therefore be a confounding factor in this study. Further study is needed on both genetic and differentiated end points in culture models in order to compare the relative sensitivities of glia and neurons to OP-induced neurotoxicity. Reaggregate cultures of dispersed cells from embryonic or fetal brain show developmental expression patterns that can be monitored for neuronal, astroglial, and oligodendroglial proteins in order to assess relative effects of toxicants on cell types (Zurich et al., 2004). Two such studies have been carried out with OP compounds. Funk et al. (1994) showed that brief exposure of chick brain reaggregate cultures to diisopropyl phosphorofluoridate (100 txM) or paraoxon (1 IxM) results in NTE and AChE inhibition patterns similar to those of the hen in vivo over 1 or 2 weeks. However, at the same OP compound concentrations, activity of the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) is not affected. This result has been confirmed in reaggregate cultures from fetal rat telencephalon and extended to include astrocytes (Monnet-Tschudi et al., 2000). In the rat culture study, activities of neuronal, oligodendroglial, and astroglial enzymes were monitored after a 10-day exposure to chlorpyrifos, parathion, or their oxon derivatives during immature vs differentiated developmental stages. Irrespective of culture age, AChE was inhibited at much lower concentrations than other neuronal enzymes (CHAT and glutamic acid dehydrogenase). Furthermore, activities of glutamine synthetase in astrocytes and CNP in oligodendrocytes were unchanged by treatment with OP compounds, except at concentrations of chlorpyrifos-oxon or paraoxon exceeding by 10- to 1000-fold those that reduce neuronal enzyme activities. These results indicate a much greater sensitivity of neuronal markers than glial markers to OP exposure. The latter result is in agreement with the finding that CNP activities are unchanged in the CNS of hens treated orally with an OPIDN-inducing dose of tri-o-tolyl phosphate (Luttrell et al., 1988).
C. OP Effects on Astrocyte-Endothelial Interactions Cells of the CNS directly interact with other cell types, notably the capillary endothelial cells that comprise the blood-brain barrier (BBB). Astrocytes are essential for the maintenance of B BB structural integrity and permeability
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characteristics (Janzer and Raft, 1987; Rubin et al., 1991). The BBB should be considered in two respects when discussing neurotoxicity: (1) the transport of toxicants across it to the brain parenchyma and (2) the direct effects of toxicants on the integrity of the barrier. Astroglial participation in OP-induced neurotoxicity has been explored in cell culture models of the BBB. Yang et al. (2001) measured the transendothelial permeability of chlorpyrifos across monolayers of the rat brain endothelium-4 (RBE4) cell line. When grown in astrocyte-conditioned medium, RBE4 cells showed a decreased permeability to [14C]-chlorpyrifos compared to those grown in control medium. This result was the first to demonstrate the positive effect of astroglial-derived soluble factors on reducing chlorpyrifos permeability across the BBB. In a second study, Parran et al. (2005) created an artificial BBB by culturing adult bovine microvascular endothelial cells (BMEC) and neonatal rat cortical astrocyte primaries on opposite surfaces of collagen-coated semipermeable membrane inserts. In this model, when chlorpyrifos was added to the BMEC side, it was metabolized by the endothelial cells and inhibited their carboxylesterase and cholinesterase activities. Furthermore, chlorpyrifos and its metabolites could cross the artificial B BB and reduced its electrical resistance. Astrocytes are a potential target for chlorpyrifos toxicity in this model. These two studies on astroglia-endothelial interactions in the BBB draw attention to the need to assess this important aspect of astrocyte physiology and OP-induced neurotoxicity.
V. FRONTIERS A. Prophylactic Detoxification of OP Compounds Assessed in Vitro OP neurotoxicants present major challenges for environmental remediation and for the protection of subjects who may be exposed to these compounds, such as farm workers and their children, pesticide applicators, and military or civilian personnel who may be exposed to chemical warfare agents. Although OP compounds are degraded environmentally, certain conditions, such as low pH, temperatures below 20 ~ and reduced natural light, allow them to persist in the environment. OP-based nerve agents have elicited worldwide attention because of the disarmament treaty of 132 countries in 1993 and the immediate need for a method to destroy these chemicals. Currently, the United States and Russia are seeking appropriate means to destroy large stockpiles of aging nerve agents. Incineration has not been practical because of operational safety and potential environmental impact concerns. Chemical neutralization of nerve agents, such as the application of sodium hydroxide, does not accomplish complete destruction and produces highly caustic and hazardous by-products. Therefore, biodegradation of OP compounds has been considered to be
a potentially reliable alternative method; however, exploration of alternatives has not been undertaken. The National Research Council (1993) recommended evaluation and potential development of this technology as a practical solution, but the current commitment of funds to the destruction of chemical warfare stockpiles by incineration appears to preclude altemative technology development that would address the primary destruction of OP neurotoxicants. A cell culture model possessing easily measured relevant toxicological end points would be a useful tool for evaluating enzymes and processes for the toxicological inactivation of OP compounds. Studies in our laboratory have begun to evaluate SY5Y cells for testing the potential of genetically engineered OPH to functionally biotransform OP neurotoxicants. In an initial study, interactive laser cytometry was used to compare the transient disruption of intracellular Ca 2+ (Ca2+i) homeostasis by paraoxon and its hydrolysis products (Hong et al., 2003). The highest paraoxon concentration used (1.8 mM) slightly exceeded the ICs0 (1.6 mM) for inhibition of AChE by paraoxon in SY5Y cells differentiated with retinoic acid but was below the ECs0 (2.6 mM) for cytotoxicity (Ehrich et al., 1997). Comparison of basal Ca2+i levels in cells treated acutely with paraoxon or OPH-hydrolyzed paraoxon revealed that OPH-hydrolyzed paraoxon produced qualitatively similar effects to those of paraoxon, indicating that the hydrolysis products, primarily p-nitrophenol, are no more toxic than paraoxon. Because OPH-hydrolyzed paraoxon has an effect like that of paraoxon, the effect on basal Ca2+i levels is likely not related to esterase inhibition. However, paraoxon invokes a sharp, transient increase in Ca 2+i levels, whereas hydrolyzed paraoxon invokes a slower, more sustained phase that persisted for 400 sec, suggesting some possible destabilization of the cell membrane by the hydrolysis products. Ca2+i homeostasis as an end point in the previously mentioned experiment was both cumbersome and insensitive. Therefore, we have examined other end points. The short-term effects of four OPH-treated OP compounds on AChE and NTE activities were measured in retinoic aciddifferentiated or undifferentiated SY5Y cells. This study revealed that there were delayed effects of OPH-treated paraoxon or mipafox on neuronal cytoskeletal proteins in NGF-differentiated cells. The anti-AChE activity of paraoxon (up to 3 ~M) and anti-NTE activity of mipafox (up to 250 ~zM) in SY5Y cells were prevented by biodegradation with OPH. Anti-AChE activities of mipafox, methyl parathion, and demeton-S were partially ameliorated, depending on OP concentration. Intracellular amounts of the 200-kDa neurofilament protein (NF200) were unchanged after treatment with OPH-treated or buffer-treated paraoxon, as expected, because this end point is insensitive to paraoxon. However, NF200 levels increased unexpectedly in cells treated during late differentiation with OPH-treated mipafox. This finding suggests the existence of a threshold concentration of mipafox below
CHAPTER 22 9In Vitro Neurotoxicity Testing
which SY5Y cells compensate for toxicity by cellular regeneration because biotransformation by OPH was incomplete and 4 IxM mipafox remained in the treatment medium. These results indicate that OPH can be used to enzymatically degrade OP compounds and remediate their neurotoxic effects in vitro. Furthermore, AChE and NTE were suitable detectors for OPH amelioration (Cho et al., 2005).
B. Extrapolation of in Vitro Results to Whole Animals and Humans Cell and tissue culture studies on OP-induced neurotoxicity are valuable if they provide valid insights into processes that occur in vivo. The extrapolation of in vitro results to animals and human populations requires careful attention to four elements of experimental design in vitro (Fig. 2). First, concentrations in vitro must be valid and comparable to toxicological doses. Unfortunately, little information is available on the brain concentrations of OP compounds that are
Operative dose to target cell:
9Bioavailability 9Metabolism in cell 9Cell defense
Functional endpoints:
9More sensitive than cytotoxicity 9Of potential value for screening and
I Phenotype of target cell: 9OP targets (enzymes, structural
~ J
~--I
proteins,
homeostatic mechanisms)
9Cell type 9Developmental stage
environment of target cell: I 9Secondary damage J to dependent cells [ 9Protection by other
remediation studies
FIG. 2. Critical interacting variables in the extrapolation of benchmark doses from in vitro to in vivo systems. In vitro models provide useful systems for dissecting the cellular and molecular mechanisms of adverse in vivo effects of OP compounds. They also help identify subtle biological effects of OP compounds that may be present in vivo but are masked by acute cholinergic effects or have gone unnoticed. Ideally, in vitro findings should be readily extrapolative to in vivo systems but, practically, this ideal has not been realized in neurotoxicology. The interacting variables that influence toxicity, as illustrated here, must first be better understood, and they present major challenges for cellular neuroscience and toxicology. Operative dose, for example, is influenced by the tissue context of a cell, such as the ability of other cells and tissues to metabolize or transport the OP compound. Toxicity is dependent on the cell having vulnerable OP targets, balanced against its ability to control the damage induced, which may be influenced by developmental maturity. The domino effects of secondary toxicity to cells that interact with the damaged cells must also be understood. Functional end points are key measurements for the extrapolation of in vitro findings to in vivo systems because they offer a rational possibility for the comparison of identical mechanistic events.
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associated with nonacute OP-induced neurotoxicity. For protoxicants, such as parathion and chlorpyrifos, the concentrations of biologically active toxicant should be integrated into the experimental design. Some studies have shown biological effects of protoxicants on neurons in culture. This information could reveal either desulfuration capacities in the cultures or protoxicant mechanisms of neurotoxicity that were previously unrecognized. Second, test targets should be selected for their toxicological value and plausibility. The most sensitive culture models should be selected in order to detect toxicant concentrations that have a biological effect. The sensitivities of target cells could be enhanced through the use of genetically engineered or transfected cells that express targets of interest and their polymorphisms. Third, OP compound exposure regimens must be valid. Exposures may be short term or long term, depending on the type of toxicity being modeled. However, although acute toxicity induced by inhibition of AChE is a sensitive marker of OP exposure in culture, more attention should be paid to longer-term and repeated, intermittent, noncytotoxic (nonlethal) exposures. Superimposed on long-term exposure regimens is the necessity for cultures to differentiate and age as they would in vivo. Fourth, the end points must be biologically plausible. As discussed, neurotoxicity of a compound is not always represented solely by its cytotoxicity, because functional alterations may be harmful, neutral, or beneficial. The following are suggested strategies for in vitro testing of OP compounds: 9 Evaluate the active metabolite(s) for secondary toxicity. 9 Determine and test at toxicologically relevant concentrations. 9 Use cell and tissue culture models that possess appropriate OP compound targets and relevant OP compound-metabolizing systems. 9 Include neurotransmitter receptors, signaling molecules, and metabolic enzymes. 9 Test functional biochemical end points in addition to cytotoxicity. 9 Test in multiple culture systems of different biological complexities.
VI. C O N C L U S I O N S
AND FUTURE
DIRECTIONS Information generated from in vitro studies is beginning to yield an improved understanding of the effects of OP compounds on neuronal and glial cells. Among the general conclusions from these studies and parallel in vivo studies are the following: 9 OP compounds affect neurons at toxicologically relevant concentrations in vitro. 9 Most OP compound effects in vitro are unrelated to the inhibition of AChE and many are unrelated to NTE
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activity, which suggests other nonesterase mechanisms of neurotoxicity. 9 Neurons are more sensitive to OP-induced biochemical damage or cytotoxicity than are astroglia and oligodendroglia, but developmental stages in the context of fetal bases for adult neurological disease need to be fully considered. 9 OP compounds should be tested at pharmacologic concentrations, although the repeated or sporadic nature of clinical levels is difficult to reproduce in culture. The relevance of high concentrations is not clear and should be carefully considered. 9 New assays based on the use of molecular markers have great potential to be more powerful and toxicologically relevant than conventional in vitro assays. Novel in vitro assays that would address the effects of OPIDN inducers on molecular markers of nervous tissue structure and function in differentiating neuronal and glial cells should continue to be explored. These culture systems are being developed with the concept that a battery of highly sensitive assays can be assembled that give a predictable set of responses (i.e., a signature that is unique for classes of OP compounds).
Acknowledgments The authors thank Dr. Marion Ehrich for her critical review of the manuscript and numerous helpful suggestions. The authors' work was supported by National Institutes of Health grants P42 ES04917, P30 ES09106, and T32 ES07273 and by ATSDR grant U61/ATU684505.
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Electrophysiological Mechanisms in Neurotoxicity of Organophosphates and Carbamates TOSHIO NARAHASHI Northwestern University Medical School, Chicago, Illinois
I. I N T R O D U C T I O N
of their medical use and OP nerve gases such as sarin, tabun, and VX because of the military concern. Therefore, although the primary purpose of this chapter is the electrophysiological mechanism of action of insecticidal OPs and CMs, noninsecticidal CMs and nerve gases are included to some extent because these data provide useful information for understanding the mechanism of action of insecticides. This chapter focuses on the mechanism of action of OP/CM insecticides as studied by electrophysiological techniques that are expected to provide the functional aspect of the toxic action. Survey of the literature has shown that data are quite sketchy and fragmental, and there are not any systematic long-term studies on this subject. The electrophysiological effects could be due to the direct action on the receptor or the indirect action via the sustained presence of ACh in the synaptic left as a result of ChE inhibition. The effects vary greatly depending on the kind of OPs/CMs, the dose, and the method of administration. There have been many descriptive or phenomenological studies of the effects of OPs/CMs on action potential conduction and synaptic transmission. Some studies that do not contribute to our knowledge of the mechanism of action are not included in this chapter. Many review articles have been published dealing with the mode of action of OPs and CMs. However, many of them do not address the mechanistic aspects or electrophysiological aspects. Readers who wish to obtain more comprehensive general aspects of OPs/CMs should consult the following articles: O'Brien (1967), Fukuto (1979), Jones et al. (1979), Woolley et al. (1979), Murphy et al. (1984), Chambers and Chambers (1989a,b), Bardin et al. (1994), and Koelle (1994).
There is general agreement that organophosphate (OP) and carbamate (CM) insecticides inhibit cholinesterase (ChE) and that this action is an important mechanism by which neurotoxicity is produced. The simplest notion of the ChE theory states that the accumulation of acetylcholine (ACh) at synaptic junctions as a result of ChE inhibition causes excess stimulation, thereby causing toxicity. However, additional effects not through ChE inhibition have been suspected since the introduction of parathion in 1944 (O'Brien, 1967). A variety of approaches have been used to elucidate the mechanism o f action of insecticides on insects and mammals. Electrophysiology is the most straightforward to characterize changes in the function of the nervous system. Ligand binding methods have been used to determine the site of binding of chemicals at the target receptor or channel but cannot follow the fast time course of changes. Behavioral techniques are sensitive to subtle changes in animal behavior caused by insecticide intoxication, but these do not elucidate the detailed mechanism of action. Histological approaches can find structural changes caused by insecticides, but these are usually questioned whether the observed changes are the cause of intoxication or the result of intoxication. Recently, molecular biological techniques such as point mutations of receptor amino acids have proven effective in locating the target site on the receptor/channel; however, these approaches remain descriptive at the molecular level and cannot elucidate the true molecular mechanism. Because OP/CM insecticides have been used extensively since parathion was developed, their mechanism of action has been the subject of intense investigations. Most of these mechanistic studies focused on ChE inhibition and metabolism in insects, mammals, and plants. Regarding the mechanism of functional changes, two groups of anti-ChEs other than insecticidal anti-ChEs were also studied extensively-that is, CMs such as physostigmine and neostigmine because
Toxicology of Organophosphate and Carbamate Compounds
II. E F F E C T S O N A C T I O N P O T E N T I A L CONDUCTION Parameters associated with the conduction of action potentials in the peripheral nerve can be measured relatively 339
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easily both in vivo and in vitro. Daily administration of trichlorfon to rats caused some changes in action potentials recorded from the sciatic nerve (Averbook and Anderson, 1983). Both the duration and the time to peak amplitude were shortened progressively over a 3-day period, and the rate of rise of the action potential was increased. The relative refractory period was shortened. In contrast, parathion had no effect on the action potential. However, acute exposure to trichlorfon did not cause any change in these action potential parameters. It was suggested that changes in nerve excitability may be a sensitive indicator of neurotoxicity. Nerve conduction in organophosphorus-induced delayed neuropathy (OPIDN) was studied using adult white leghorn hens treated with tri-2-o-cresyl phosphate or di-n-butyl2,2-dichlorovinyl phosphate (Robertson et al., 1987). Refractoriness was decreased in the tibial nerve but it was increased in the sciatic nerve, and the strength-duration threshold was elevated for both nerves. However, it is not known how these changes are related to the mechanism of OPIDN.
III. N E U R O M U S C U L A R
TRANSMISSION
In general, synaptic transmission is more vulnerable to drug action than action potential conduction. Thus, many electrophysiological studies of insecticide action were devoted to changes in synaptic transmission and various parameters associated with it. Soon after development of OP insecticides such as parathion, extensive electrophysiological studies were commenced for the purpose of elucidating the physiological mechanism of action of OPs/CMs on the insect nervous system. These studies were conducted almost at the same time as nonmedical CM anti-ChEs were examined. Many of these studies conducted in the late 1940s through the early 1960s involved vertebrate neuromuscular junctions and insect synaptic transmission. The effects of OPs/CMs on insect synapses are described later. Physostigmine, neostigmine (prostigmine), and diisopropyl fluorophosphate (DFP) were found to increase the endplate potential (EPP) duration and at high concentrations decreased the EPP amplitude of the frog (Eccles and McFarlane, 1949). Thus, this study clearly showed a postsynaptic block caused by the CMs. Presynaptic as well as postsynaptic actions were also demonstrated using rat diaphragm preparations in which paraoxon increased spontaneous miniature EPP (MEPP) frequency and half-decay time, but it caused neuromuscular block (Laskowski and Dettbarn, 1979). Rats injected with DFP showed neuromuscular weakness. In vitro experiments demonstrated that DFP prolonged the half-decay time of the MEPPs and nerve-evoked EPPs but had no effect on the amplitude of MEPPs and the quantal content of EPPs (Maselli and Soliven, 1991). oL-Tubocurarine had an antagonistic effect. It was concluded that the sustained endplate depolarization was
responsible for neuromuscular weakness of rats intoxicated with DFE However, it is not clear whether the direct action of DFP on the endplate is involved in addition to the effect caused by ChE inhibition. Involvement of the direct action on the endplate was indicated by the focal recording of miniature endplate currents (MEPCs) from frog sartorius muscle (Deana and Scuka, 1990). Neostigmine at 3 txM initially lengthened the decay phase of MEPCs and increased the amplitude of MEPCs probably due to ChE inhibition, but later it suppressed the amplitude and shortened the decay phase, suggesting a direct effect on the endplate. Tetanic contractions (Wedensky inhibition) induced by repetitive nerve stimulation in phrenic nerve-diaphragm preparations of mice was faded by 0.5-2 txM neostigmine (Chang et al., 1986). The fade was brought about by failure to elicit muscle action potentials, which was due to endplate depolarization and a decrease in transmitter release. Both effects were attributed to ACh accumulation as a result of ChE inhibition. Data that negate direct action on the muscle ACh receptor were also obtained using methamidophos, an OP insecticide (Camara et al., 1997). Despite its weak anti-ChE activity with an IC50 of --20 txM, ChE inhibition was long lasting, consistent with forming a covalent bond with the enzyme. Methamidophos increased the amplitude and prolonged the decay phase of nerve-evoked EPPs and MEPPs, but it did not affect transmitter release. There was no effect on whole cell currents induced by ACh, glutamate, or GABA in cultured hippocampal neurons. Thus, it was concluded that methamidophos acted as a selective anti-ChE. Albuquerque and associates conducted extensive studies of the mechanism of action of anti-ChEs at neuromuscular junctions and concluded that in addition to the effects via ChE inhibition, there were direct actions on the nAChRs. These studies are summarized in several papers (Albuquerque et al., 1984, 1985a,b, 1986, 1987, 1988, 1989). Furthermore, they showed that the antidotal effect of oximes is not simply due to carbamylation and reactivation of ChE but also due to the direct effect on the nACh receptors (Albuquerque et al., 1988b). Although their studies dealt with noninsecticidal OP compounds, such as pyridine-2-aldoxime (2-PAM), 1-(2-hydroxyiminomethyl1-pyridino)-3-(4-carbamoyl- 1-pyridino)-2-oxapropane (HI-6), and 1, l'-oxybis(methylene) bis-4-(1,1-dimethylethyl)-pyridinium (SAD-128), and CMs such as physostigmine, the data are worth summarizing because the studies are among the most extensive mechanistic studies. At the frog endplate, ( - ) physostigmine at low concentrations (0.2-2 txM) increased the peak amplitude of EPC and prolonged the decay phase of EPC. However, at high concentrations, the EPC peak amplitude was decreased, the EPC decay was accelerated, and single-channel lifetime was shortened (Shaw et al., 1985). DFP, dimethylphosphoramidocyanidic acid ethyl ester (tabun), O-ethyl S-2
CHAPTER 23 9Electrophysiological Mechanisms in Neurotoxicity diisopropylaminoethyl-methyl phosphonothiolate (VX), and ( - ) physostigmine interacted with pre- and postsynaptic regions of the glutamatergic neuromuscular synapse of locust muscle (Idriss et al., 1986). These agents initiated spontaneous EPSPs and muscle action potentials. Thus, this site is a new target of these anti-ChEs. In addition to ( - ) physostigmine, enantiomer (+) physostigmine also protected agonist lethality and myopathy caused by sarin, an effect most certainly not dependent on ChE inhibition but due to direct block of nACh receptors (Kawabuchi et al., 1988). The ChE reactivators 2-PAM and HI-6 increased the open probability of frog endplate channels that were activated by ACh. The oximes reduced mean channel open time and burst duration. EPC amplitude was increased by both of these oximes but depressed at their high concentrations. Thus, this study showed a direct interaction of 2-PAM and HI-6 with the nACh receptors (Alkondon et al., 1988). The nonoxime bispyridinium SAD-128 decreased the EPC peak amplitude and prolonged the EPC decay. At the single-channel level, SAD-128 reduced the mean open time and produced a blocked state evidenced as an additional phase in the closed time distribution. SAD-128 block of nACh receptors may underlie its efficacy in counteracting lethal effect of OP compounds (Alkondon and Albuquerque, 1989).
IV. S Y N A P T I C T R A N S M I S S I O N Studies on the effects of OPs/CMs have been performed with various preparations, including brain slices, cultured neurons, acutely isolated neurons, neuroreceptors expressed in a host cell, and neuronal cell lines. The electrophysiological techniques utilized are extracellular recording, intracellular recording, and patch clamp. Similar to the studies of neuromuscular functions, various compounds were tested. Thus, it is extremely difficult to obtain a unified picture regarding the mechanism of action of OPs/CMs on synaptic transmission. Many studies on the effects of OPs in brain slices have used slices containing only the hippocampus (Lebeda and Rutecki, 1985; Sarvey and Williamson, 1985; Williamson and Sarvey, 1985). In the evoked field potential, soman and other OPs induced a second population spike and spontaneous discharges. These effects are presumably caused by increases in ACh levels in the slice due to inhibition of ChE. However, there have been some suggestions of actions other than ChE inhibition (Lebeda and Rutecki, 1985; Williamson and Sarvey, 1985). A brain slice model has been developed that contains the entorhinal cortex as well as the hippocampus (Wood and Tattersall, 2001). The preparation allows the study of the spread of seizure discharges with the limbic system and the development of prolonged, sustained discharges that are rarely seen in the hippocampal slice preparation. In this preparation, 1 p.M soman induced a second
341
population spike in the evoked field potential in the CA1 or CA3 region, and a late repetitive discharge was also observed (Wood and Tattersall, 2001). Diazepam blocked these discharges. Although AP5 and MK-801, which are NMDA antagonists, had little or no effect on the discharges, CNQX and DNQX, which are AMPA/kainate antagonists, abolished the discharges. Effects of any chemicals on brain slice preparations could be caused by a direct or an indirect action. Therefore, the observed effects could be complicated. For example, 0.3-1 nM sarin decreased the amplitude of GABA-mediated postsynaptic currents in the CA1 pyramidal layer of the rat hippocampal slices. However, glutamatergic postsynaptic currents were not affected. It turned out that the observed effects were mediated by a direct interaction of the OP with muscarinic ACh receptors present on presynaptic GABAergic neurons and unrelated to ChE inhibition (Chebabo et al., 1999). Sarin had no effect on the amplitude or kinetics of GABA- or glutamate-mediated miniature postsynaptic currents, indicating that it does not interact with GABA or glutamate receptors. Another study using brain slices illustrates the difficulty in interpreting the data. Dichlorv0s exerted the opposite effect on somatosensory evoked potentials and hippocampal evoked population spikes (Papp et al., 1996). Only the effects on hippocampus could be explained in terms of an increased cholinergic activity. Friedman et al. (1998) studied the effects of several OPs and CMs on hippocampal slices using both electrophysiology and reverse-transcript polymerase chain reaction. DFP 1 txM or pyridostigmine 1 mM increased the mRNA levels of c-Fos and AChE and decreased the mRNA levels of choline acetyltransferase and vesicular ACh transporter. DFP, pyridostigmine, carbamylcholine, and physostigmine increased the amplitude of population spikes. When low stimulus intensities were used, pyridostigmine facilitated the second response but suppressed the response later. Both phases were prevented by atropine, indicating the dependence on muscarinic receptor activation. However, it remains to be determined how the changes in mRNA transcripts are related to electrophysiological changes. Chronic effects of sublethal injection of paraoxon on transmitter release were studied using intracellular recording of MEPPs and EPPs (Thomsen and Wilson, 1986). Tolerance was not due to a decrease in postsynaptic sensitivity but due to presynaptic changes. Transmitter release was suppressed due to a decrease in the transmitter store and mobilization ability. Thus, depression of quantal release of transmitter accounts for behavioral tolerance. Low-level, chronic dichlorvos treatment of rats caused alterations in all parameters measured, which included electrocorticogram, sensory cortical evoked potentials, conduction velocity, and refractory periods of peripheral nerve (D6si and Nagymajt6nyi, 1999). These parameters are sensitive biomarkers of the exposure at low-dose levels. Brain ChE was inhibited only at high doses.
342
SECTION IV. O r g a n
Toxicity
Coho salmon was highly sensitive to CMs and the effects of three CMs on the electroolfactogram were studied (Jarrard et al., 2004). The electroolfactogram was sensitive to brief exposure to two CMs: The effective nominal concentration for a 50% reduction (ECs0) in electroolfactogram amplitude was 10.4 i~g/liter for carbofuran and 1.28 #g/liter for the anti-sapstain IPBC. However, the fungicide mancozeb was less potent with an ECs0 of 2.05 mg/liter. The sensitivity of salmon olfactory neurophysiology to pesticides appears to be not only due to ChE inhibition but also due to other unknown mechanisms. Single neurons are a convenient material for detailed analysis of drug-receptor interactions. Sympathetic ganglion neurons of bullfrogs were used for the study of action of VX (Heppner and Fiekers, 1992). The amplitude of excitatory postsynaptic potentials (EPSPs) was increased, the membrane was depolarized, the input resistance was reduced, and the duration of the spike after hyperpolarization was shortened. The observed increase in neuronal excitability may be due to the decrease in after-hyperpolarization. The effects of
A Control
VX on cultured hippocampal neurons were also studied (Rocha et al., 1999). At a low concentration of 0.01 nM, VX decreased the amplitude of evoked GABAergic currents, and at higher concentrations (> 1 nM) it decreased the amplitude of glutamatergic currents as well. The VX effect on GABAergic currents was prevented by atropine, indicating an action via the cholinergic system. In the presence of tetrodotoxin, VX (->10 nM) increased the frequency of GABA- and glutamate-mediated miniature postsynaptic currents. This is unrelated to ChE inhibition and is due to alteration of transmitter release. Detailed patch clamp analyses were performed for nACh receptors of PC12 cells (Nagata et al., 1997). Carbaryl showed a biphasic effect: At 0.1 #M it greatly potentiated carbachol currents, and at 0.3-3 #M it suppressed the current. Single-channel experiments indicated that both carbaryl and neostigmine increased short closures or gaps during channel opening, decreased the mean open time and burst duration, but caused no change in single-channel conductance (Fig. 1). These effects appear to be exerted by direct
C 1 I~ carbaryl
close open -r. . . . . . . . . . .
~-':'~
....
~-'-~.~-'~-'=-~~ L v ~ ~
B 101~1 neostigmine
,,-"r-"'," r '"T-L~r,,,,-,.-,,-,
'' .... .---&-r~':;-:=-~--;'~
D 10 I~ carbaryl.
50 ms , , ,
FIG. 1. Single-channel currents induced by application of ACh and coapplications of ACh and neostigmine or carbaryl to cell-attached membrane patches of PC12 cells clamped at a membrane potential 40 mV more positive than the resting potential. (A) Currents induced by 30 ~M ACh occurred during brief isolated openings or during longer openings interrupted by a few short closures or gaps. (B) Coapplication of 30 ~M ACh and 10 I~M neostigmine. Channel openings occurred in bursts separated by brief closures or gaps. (C) Coapplication of 30 ~M ACh and 1 ~M carbaryl. Channel openings were separated by brief closures or gaps. (D) Coapplication of 30 ~M ACh and 10 ~M carbaryl. Channel openings were further separated by brief closures or gaps and occurred in bursts. A-D were obtained from different membrane patches. From Nagata et al. (1997).
I
5pA
CHAPTER 23 9Electrophysiological Mechanisms in Neurotoxicity
FIG. 2. Increase in synaptic after-discharge and eventual block during exposure to thiol-methyldemeton in cockroach nerve preparation. Postsynaptic action potentials were evoked by presynpatic nerve stimulation (A1 and A2). After-discharges were greatly intensified 8, 13, and 16 min after application of 500 txM thiol-methyldemeton and synaptic transmission was eventually blocked (B3, 21 min). Time marker of 20 msec applies to A2, A3, B 1, and B2, and 1-msec time marker applies to A1 and B3. From Narahashi and Yamasaki (1960).
343
block of nACh receptors. For the study of identified subunits of receptors, Xenopus oocyte expression is a powerful approach. The effects of OPs were studied on the OL4~2 nACh receptors expressed in oocytes (Smulders et al., 2004). Parathion-ethyl, chlorpyrifos, and disulfoton inhibited ACh currents in micromolar concentrations. Binding experiments showed that OPs noncompetitively interact with nACh receptors. Reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It appears that OPs interact directly with the OL4~2 nACh receptor, causing suppression. These two studies clearly indicate that OPs/CMs can directly block nACh receptors. Extensive studies were conducted on the effects of anti-ChE OPs and CMs on synaptic transmission of cockroaches. Stimulation of presynaptic cercal nerve evoked postsynaptic responses comprising the initial large action potentials followed by after-discharges with smaller amplitudes (Fig. 2, A1 and A2). After treatment with OPs or CMs, after-discharges were greatly increased in amplitude and prolonged in duration (Fig. 2, A3, B1, and B2), but synaptic transmission was eventually blocked (Fig. 2, B3). However, the effect reappeared soon, and this block-recovery cycle was repeated many times in the continuous presence of anti-ChEs (Roeder et al., 1947; Roeder, 1948; Roeder and Kennedy, 1955; Twarog and Roeder, 1957; Yamasaki and Narahashi, 1958, 1960; Narahashi and Yamasaki, 1960; Heppner et al., 1987). Obviously, such intense after-discharges are responsible for hyperactivity of insects intoxicated with anti-ChEs. The mechanism of hyperexcitatory action of anti-ChEs was further studied by recording the EPSPs from cockroach ganglia (Yamasaki and Narahashi, 1958, 1960). This was accomplished by placing an external electrode on the sixth abdominal ganglion while the other external electrode was in contact with the nerve cord between the second and third
FIG. 3. Augmentation and prolongation by physostigmine of the excitatory postsynaptic potential recorded from the sixth abdominal ganglion of the cockroach by external electrodes. Recording was made in urethane to block discharges of action potentials. (A) Control; (B) 8 min after application of 15 ~M physostigmine; (C) 50-Hz time marker for records A and B; (D) 12 min after application of physostigmine; 50-Hz time marker. Voltage calibration in C, 0.5 mV, applies to all records. From Narahashi (1965).
344
SECTION I V . O r g a n T o x i c i t y
abdominal ganglia. This arrangement allowed us to record changes in the membrane potential of the sixth abdominal ganglion as well as the action potentials. In the presence of urethane to step discharges, and EPSP could be elicited by presynaptic stimulation (Fig. 3A). The EPSP was augmented in amplitude and prolonged in duration after application of 15 IxM physostigmine (Fig. 3B). These effects were potentiated with time, and eventually a very large and greatly prolonged EPSP could be recorded (Fig. 3D). Since the synapses in the sixth abdominal ganglion were shown to be cholinergic, the effect of physostigmine was obviously due to accumulation of ACh as a result of ChE inhibition. Simultaneous measurements of postsynaptic responses and ChE activity were performed using the cockroach nerve cord, including the sixth abdominal ganglion intoxicated with parathion, thiol-demeton, thiol-methyldemeton, or thiono-methyldemeton (Yamasaki and Narahashi, 1960; Narahashi and Yamasaki, 1960). At the time when synaptic facilitation occurred, ChE was partially inhibited.
V. CONCLUSIONS OPs and CMs stimulate and then suppress synaptic and neuromuscular transmission. The effects are due partly to the inhibition of ChE and partly to the direct effect on the ACh receptors. The degree and time course of these effects varied considerably depending on the kind of OPs/CMs, dose, methods of administration, and the species of animals. However, the basic mechanism of action on mammals and insects appears to be the same.
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O'Brien, R. D. (1967). Insecticides: Action and Metabolism. Academic Press, New York. Papp, A., Gyorgyi, K., Nagymajtenyi, L., and Desi, I. (1996). Opposite short-term changes induced by an organophosphate in cortical and hippocampal evoked activity. Neurobiology 4, 431-440. Robertson, D. G., Schwab, B. W., Sills, R. D., Richardson, R. J., and Anderson, R. J. (1987). Electrophysiological changes following treatment with organophosphorus-induced delayed neuropathy-producing agents in the adult hen. Toxicol. Appl. Pharmacol. 87, 420429. Rocha, E. S., Santos, M. D., Chebabo, S. R., Aracava, Y., and Albuquerque, E. X. (1999). Low concentrations of the organophosphate VX affect spontaneous and evoked transmitter release from hippocampal neurons: Toxicological relevance of cholinesterase-independent actions. Toxicol. Appl. Pharmacol. 159, 3140. Roeder, K. D. (1948). Organization of the ascending giant fiber system in the cockroach (Periplaneta americana). J. Expl. Zool. 108, 243-261. Roeder, K. D., and Kennedy, N. K. (1955). The effect of certain tri-substituted phosphine oxides on synaptic conduction in the roach. J. Pharmacol. Exp. Ther. 114, 211-220. Roeder, K. D., Kennedy, N. K., and Samson, E. A. (1947). Synaptic conduction to giant fibers of the cockroach and the action of anticholinesterases. J. Neurophysiol. 10, 1-10. Sarvey, J. M., and Williamson, A. M. (1985). Diazepam and barbiturates antagonize the effect of an organophosphate anticholinesterase in the rat hippocampal slice. Proc. Med. Def. Biosci. Rev. 5, 421-423. Shaw, K.-E, Aracava, Y., Akaike, A., Daly, J. W., Rickett, D. L., and Albuquerque, E. X. (1985). The reversible cholinesterase inhibitor physostigmine has channel-blocking and agonist effects on the acetylcholine receptor-ion channel complex. Mol. Pharmacol. 28, 527-538. Smulders, C. J., Bueters, T. J., Vailati, S., van Kleef, R. G., and Vijverberg, H. P. (2004). Block of neuronal nicotinic acetylcholine receptors by organophosphate insecticides. Toxicol. Sci. 82, 545-554. Thomsen, R. H., and Wilson, D. E (1986). Chronic effects of paraoxon on transmitter release and the synaptic contribution to tolerance. J. Pharmacol. Exp. Ther. 237, 689-694. Twarog, B. M., and Roeder, K. D. (1957). Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. Ent. Soc. Am. 50, 231-237. Williamson, A. M., and Sarvey, J. M. (1985). Effects of cholinesterase inhibitors on evoked responses in field CAI of the rat hippocampus. J. Pharmacol. Exp. Ther. 235, 448-455. Wood, S. J., and Tattersall, J. E. (2001). An improved brain slice model of nerve agent-induced seizure activity. J. Appl. Toxicol. 21(Suppl. 1), $83-$86. Woolley, D. E., Chernobieff, J. R., and Reiter, L. W. (1979). Effects of parathion on the mammalian nervous system. In Neurotoxicology of Insecticides and Pheromones (T. Narahashi, Ed.), pp. 155-181. Plenum, New York. Yamasaki, T., and Narahashi, T. (1958). Synaptic transmission in the cockroach. Nature 182, 1805-1806. Yamasaki, T., and Narahashi, T. (1960). Synaptic transmission in the last abdominal ganglion of the cockroach. J. Insect Physiol. 4, 1-13.
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CHAPTER
24
Behavioral Toxicity of C h o l i n e s t e r a s e Inhibitors I
PHILIP J. BUSHNELL AND VIRGINIA C. MOSER U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
systemic toxicity caused by inhibition of cholinesterase activity and of treatment of acute poisoning from OPs followed subsequently (Minton and Murray, 1988; Marrs, 1993). Two reviews focusing on the behavioral effects of OPs were published in the early 1990s (Annau, 1992; D'Mello, 1993). It is interesting that these two reviews diverged greatly on the degree of concern warranted by the existing data. Thus, Annau concluded, "in all species examined and at all ages, exposure to these compounds can have deleterious and long-lasting, perhaps irreversible consequences." In contrast, D'Mello concluded that "very little confidence can be placed in existing descriptions of the behavioral effects of anticholinesterases (anti-ChEs) in humans," "data from animal experimental studies [yields] information [that] is relatively superficial," and "therefore, although much information is available, surprisingly little is known of the behavioural changes induced by anti-ChEs." Despite these differing conclusions, both authors called for improved and more systematic research into this contentious issue. The field has remained rife with energetic debates and contradictory conclusions, driven by the opposing concerns for agricultural benefit and public safety. The current debate centers around determining whether and under what conditions these compounds produce longlasting effects in humans. These questions have been examined descriptively in human clinical and epidemiological studies and experimentally in animal models. Reviews have flourished along with the debate; their authors have begun to organize the existing data in ways that bring the question in focus by classifying observed effects (acute vs long term) and attempting to categorize degrees of intoxication that may (or may not) be associated with each sort of effect. Section II reviews the acute behavioral effects of OP and carbamate (CM) pesticides in humans and animals. We describe the classic behavioral signs and symptoms experienced by humans poisoned with these compounds and illustrate ways in which animal models have been used
I. I N T R O D U C T I O N Because of the importance of acetylcholine (ACh) as a neurotransmitter in the mammalian nervous system, chemicals that inhibit acetylcholinesterase (ACHE) can exert profound effects on behaviors mediated by the cholinergic system. These effects may be either beneficial or detrimental, depending on the dose and degree of inhibition of the regulatory enzyme and the physiological condition of the treated individual. For example, the cholinesterase inhibitors tacrine and metrifonate have been used to treat the cognitive decline associated with aging under the assumption that insufficient cholinergic tone in the central nervous system (CNS) impairs cognitive function. Nonetheless, the widespread use of cholinesterase-inhibiting compounds as pesticides has generated concerns regarding their effects on public health. These concerns center around the acute adverse effects of high doses of these agents, how these effects may change with repeated exposure, and the possibility of long-term consequences of chronic, low-level exposure to them. This chapter examines these issues, focusing on the behavioral effects of cholinesteraseinhibiting pesticides as indicators of the impact of exposure to these beneficial products on public health. The effectsof these compounds have been the subject of many reviews since Gershon and Shaw (1961) described the anxiogenic effects of exposure to organophosphates (OPs) in humans. Early experimental work on these compounds in the 1970s focused on the mechanisms of action and tolerance to OPs (reviewed by Costa et al., 1982; Russell and Overstreet, 1987). More general reviews of the
1This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Toxicology of Organophosphate and Carbamate Compounds
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Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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Toxicity
to characterize them and to understand the mechanisms by which they are produced. In Section III, we review studies on persistent effects of exposure to cholinesterase inhibitors. The human epidemiological literature is examined for evidence that persistent behavioral effects of exposure can be measured in populations with histories of acute poisoning and/or chronic ongoing exposure. We then review behavioral data from animal models designed to characterize the behavioral changes caused by experimental treatments with cholinesteraseinhibiting pesticides and identify similarities with the literature on humans exposed occupationally to these compounds. Age-related differences in sensitivity to cholinesterase inhibitors are reviewed in Section IV. A considerable research effort has been expended to characterize the increased sensitivity of the young animal to these pesticides. We present data comparing the sensitivity of young and adult animals to the neurochemical and behavioral effects of these compounds and discuss the factors that appear to mediate differences in response during development.
II. A C U T E E F F E C T S
OF CHOLINESTERASE INHIBITORS The signs and symptoms of acute exposure to cholinesterase inhibitors are consistent with overstimulation of cholinergic receptors in the central and peripheral nervous systems due to the blockade of AChE and subsequent buildup of ACh. This includes stimulation of muscarinic receptors at effector organs and in the CNS, as well as stimulation and then desensitization of nicotinic receptors at autonomic ganglia and skeletal muscle (Taylor, 1985). The result is a dose-dependent mix of physiological, neuromuscular, and behavioral signs and symptoms. Most of these changes may be detected with measures of unconditioned behaviors (e.g., observational evaluations and tests of motor function); detection and characterization of more subtle changes require tests of conditioned behavior (e.g., tests of learning, memory, attention, and affect) in both human subjects and laboratory animals.
A. Changes in Unconditioned Behaviors Case reports of accidental and intentional poisoning with cholinesterase-inhibiting pesticides have identified common signs and symptoms of acute exposure in humans (Brown and Brix, 1998; Hayes, 1982; Morgan, 1989; O'Malley, 1997). Mild poisonings often present as flu-like symptoms headache, fever, sweating, respiratory congestion, nausea, vomiting, and abdominal cramping. Somewhat higher exposures produce more pronounced symptoms as well as dizziness, ataxia, gastrointestinal distress (including diarrhea and polyuria), muscle weakness, excessive salivation and lacrimation, constriction of the pupils (miosis), and
muscular twitching and fasciculations. Severe poisoning produces bronchospasm, irregular heartbeat, loss of reflexes, progressing to convulsions and coma in extremis. Table 1 lists the incidence of a range of symptoms reported in banana plantation workers (Wesseling et al., 2002), which clearly indicates that signs of mild toxicity are most often reported. Curiously, the frequency with which these signs are reported does not correlate well with the degree of cholinesterase inhibition, possibly due to differences in the sampled tissue (e.g., plasma, erythrocytes, whole blood, and serum), sampling time after exposure, assay method, specific pesticide involved, and the wide variation of cholinesterase activity in the human population (Bobba et al., 1996; Lessenger and Reese, 1999; Nouira et al., 1994). Given the nature of case reports, it is often difficult to identify which specific pesticide(s) may be involved, and it is even more difficult to specify the level of exposure. Such issues are not as pertinent to animal studies, for which the chemical, dose, and time course can be specified accurately. General descriptions of the effects of cholinesterase inhibitors in laboratory animals resemble the signs evident in humans, including ataxia, twitching and fasciculations, miosis, salivation, diarrhea, lacrimation, muscle weakness, depressed responses, and tremors (Ehrich et al., 1993; Mattsson et al., 1996; Moser, 1995; Moser et al., 1988, 1997; Nostrandt et al., 1997; Pope et al., 1991). Because all cholinergic receptors are involved, the autonomic signs of miosis, salivation, and hypothermia likely reflect muscarinic stimulation, whereas the tremors, gait changes, muscle weakness, and motor activity likely reflect suppressed nicotinic responsiveness from prolonged activation and desensitization of nicotinic receptors (Taylor, 1985). Other effects also appear to be cholinergically mediated, including decreased tail pinch response (indicating antinociception) (Iwamoto and Marion, 1993; Koehn and Karczmar, 1978; Pedigo et al., 1975), mouth smacking or chewing (Kelley et al., 1989; Rupniak et al., 1990; Salamone et al., 1986, 1990), and hypothermia (Gordon, 1994). To compare the acute effects of cholinesterase inhibitors in humans and animals more explicitly, we listed some of the measures used in animal studies and tallied the number of studies that showed effects on each measure. The data were taken from seven different chemicals, some tested more than once, to yield 19 dose-response curves (Mattsson et al., 1996; Moser, 1995, 1999; Moser et al., 1988, 1997; Nostrandt et al., 1997). The resulting incidence of signs is listed in Table 1, alongside the corresponding human sign. Some end points that are often reported in humans (e.g., lightheadedness, weakness, and salivation) correspond to signs that are often observed in rats as well. Some human signs cannot be measured in rats (e.g., nausea and sweating) and likewise there are some measures used in animals that either have no direct correlation to the human condition (e.g., antinociception) or were not reported in the
CHAPTER 24 9Behavioral Toxicity
TABLE 1.
349
Incidence of Signs and Symptoms of Acute Poisoning with ChE lnhibitors a
Human sign/symptom
Incidence (%)
Lightheadedness Nausea General weakness Abdominal pain Excessive sweating Salivation Headache Vomiting Blurred vision Muscle twitching Muscle cramps Difficulty breathing Involuntary loss of urine Seizures
94 91 91 89 83 79 78 75 75 67 56 51 6 3
Corresponding animal sign Ataxia, incoordination
Incidence (%) 100
b
Lowered activity, weakness
79
Salivation
84
Miosis, pupil response Twitching, fasciculations
74 68
Polyuria Convulsions, seizures Lacrimation Decreased tail pinch response Hypothermia
16 11 58 89 100
aHuman signs and symptoms are reported by 81 Costa Rican banana plantation workers (Wesseling et al., 2002). Corresponding animal signs are compilation of data from 19 studies with seven different ChE inhibitors. b indicateslack of corresponding end point.
specific paper cited (e.g., lacrimation). Although drawing conclusions with this comparison must be tempered with caution, it is clear that the animal model predicts the acute signs of toxicity of cholinesterase-inhibiting pesticides remarkably well. Rodents observed in test batteries reveal a wide range of signs in response to poisoning with pesticides and other chemicals. Domain analysis (Moser, 1991) shows that different cholinesterase-inhibiting pesticides tend to produce similar toxicities overall (Ehrich et al., 1993; Mattsson et al., 1996; Moser, 1995; Moser et al., 1988, 1997; Nostrandt et al., 1997). For example, all produce marked changes in the measures that evaluate autonomic function, activity levels, and neuromuscular ability. At high dose levels, tests of excitability and sensorimotor responsiveness are altered. A closer evaluation of the profile of behavioral effects, however, shows differences across pesticides in terms of the effects that appear at the lowest dose ("critical effects"). In one study, the critical effects of cholinesterase inhibitors varied by pesticide, and the slopes and maximum values of the dose-response curves varied for these effects (Moser, 1995, 1999). For example, comparing doses of aldicarb and methamidophos that produced similar levels of cholinesterase inhibition revealed that all
doses of aldicarb produced behavioral signs, but only the highest methamidophos dose was effective (Moser, 1999). This dissociation between behavioral effects and cholinesterase inhibition suggests the existence of other mechanisms of action, as proposed in the literature (e.g., direct actions on the receptor and altered reuptake of choline; McDaniel and Moser, 2004). The behavioral effects of cholinesterase-inhibiting pesticides may be at least partially mediated by other mechanisms (reviewed in Pope et al., 1995; Pope, 1999). These other actions may influence a pesticide's overall toxicity by modifying or enhancing the consequences of cholinergic overstimulation since some of these actions are observed at levels at or above those required to inhibit ACHE. For example, some OPs have been shown to act at the c i s - m e t h y l d i o x o l a n e sensitive population of muscarinic receptors and alter presynaptic choline uptake and ACh release (Bakry et al., 1988; Jett et al., 1991; Katz and Marquis, 1989; Liu and Pope, 1998; Silveira et al,, 1990; Van Den Beukel et al., 1997; Ward and Mundy, 1996; Ward et al., 1993). Furthermore, pesticides differ with respect to these noncholinesterase actions (Chaudhuri et al., 1993), which could provide an explanation for the different behavioral profiles observed. However, the precise role of these
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Organ Toxicity
differing influences has not been delineated. The implications of this dissociation include (1) that the degree of cholinesterase inhibition will not always predict the functional effect of a compound, and (2) that some of the low correspondence between cholinesterase activity and behavioral effect in human studies may be related to the specific inhibitors involved.
B. Changes in Conditioned Behaviors The clear and compelling importance of cholinergic neurotransmitter pathways in mediating sensory, motor, and cognitive functions in animals suggests that these functions should be sensitive to disruption by cholinesteraseinhibiting pesticides. Sensory and motor deficits, including general weakness, muscle twitching, blurred vision, tingling in the extremities, and difficulty breathing (Richter et al., 1992; Wessseling et al., 2002), are consistent with this supposition. On the other hand, complaints of cognitive impairment (e.g., confusion or forgetfulness)do not appear in the symptomatology of acute intoxication with these agents (Richter et al., 1992; Wessseling et al., 2002). In fact, assessment of choice behavior in animals after single doses of rapidly acting pesticides has not revealed evidence for cognitive impairment. Injection of rats with carbaryl or propoxur did not affect response accuracy in two tests of working memory - - delayed response (Heise and Hudson, 1985a) and continuous nonmatching (Heise and Hudson, 1985b). These negative data contrast with reductions in trial initiation caused by these compounds as well as with clear disruptions of working memory after injection of scopolamine in these tests. Similarly, a single injection of DFP in rats trained in a delayed matchingto-position/visual discrimination task showed no evidence of impaired working or reference memory (Bushnell et al., 1991). On the other hand, treatments that cause prolonged inhibition of cholinesterase activity have been shown to impair cognitive functions in rats. Prolonged inhibition of cholinesterase activity can also be achieved after a single subcutaneous injection of the OP chlorpyrifos (Pope et al., 1992). By this route, chlorpyrifos inhibits cholinesterase activity for 7 or 8 weeks and also causes significant downregulation of muscarinic receptors that recover in parallel with recovery of cholinesterase activity (Bushnell et al., 1994). In addition, this treatment impaired delayed matching accuracy (a measure of attention and working memory) and reduced response speed, but it spared discrimination accuracy (a measure of reference memory) (Bushnell et al., 1994). These effects suggest that prolonged inhibition of cholinesterase activity elicits compensatory changes in the CNS (e.g., downregulation of muscarinic receptors) that secondarily reduce the cognitive capacity of the animal. The behavioral effects of subcutaneous chlorpyrifos on delayed matching accuracy reported by Bushnell et al.
(1994) involved a parallel downward shift in the function relating matching accuracy to the delay between the sample presentation and the animals' choice. In other words, accuracy did not decrease faster as a function of delay in the treated rats: thus, these animals did not forget the sample information faster than controls, but they encoded it less accurately. This pattern suggests a deficit in attending to the sample information. To explore the potential effect of chlorpyrifos on attention, Bushnell et al. (2001) dosed rats with chlorpyrifos and quantified their behavior with a visual signal detection method that was designed to assess sustained attention (Bushnell, 1999). In addition, to evaluate the relative sensitivity of this operant test and a standard neurobehavioral screen, the animals were also tested with a functional observational battery plus motor activity. Acute treatment with chlorpyrifos transiently reduced accuracy and increased response time in the signal detection test, confirming that acute chlorpyrifos can impair attention. The signal detection task was not more sensitive to the effects of chlorpyrifos than was the neurobehavioral battery, which detected differences in the motor, reactivity, and sensorimotor domains at the same doses that affected signal detection. Thus, although the signal detection method did not provide an increase in sensitivity to chlorpyrifos exposure, it did specify the cognitive deficit resulting from the exposure. Two studies suggest that OPs may also be acutely anxiogenic in rats, a finding consistent with long-standing observations that have associated pesticide exposure with anxiety, depression, and suicide in humans, both after long-term exposure (Gershon and Shaw, 1961; Metcalf and Holmes, 1969; London et al., 2005) and associated with acute exposure (Richter et al., 1992; Stephens et al., 1995). First, S~inchez-Amate et al. (2001) reported that rats dosed with chlorpyrifos explored open arms in an elevated plusmaze less than did controls, in the absence of signs of acute toxicity. This pattern of behavior was also observed after injection with pentylenetetrazol, an anxiogenic drug, and the converse pattern of exploration was observed after injection with diazepam, an anxiolytic drug. Second, the same group reported that chlorpyrifos shares interoceptive stimulus properties with pentylenetetrazol in rats, as evaluated with a drug discrimination procedure, indicating that the internal cues generated by CPF resemble those of the anxiogenic drug (S~inchez-Amate et al., 2002). Thus, these animal models confirm reports of anxiety in acutely exposed humans. This evidence also suggests the importance of assessing affect in further work with OPs in animals and in humans. Whereas the time course of recovery from acute poisoning in humans depends on the chemical and the level of exposure, acute cholinergic signs of intoxication with cholinesterase inhibitors typically abate by 24 hr after treatment in rats. Reactivation of the AChE enzyme is much slower than this time course indicates, suggesting that
CHAPTER 24 9Behavioral Toxicity behavioral recovery cannot be due solely to resynthesis of the enzyme (Bushnell et al., 1994; Carr and Chambers, 1991; Moser and Padilla, 1998; Reiter et al., 1973). The mechanisms underlying this recovery have not been thoroughly explained, but they probably include changes in cholinergic receptor density as well as learned compensations associated with behavioral tolerance (Bushnell et al., 1994; Young and Goudie, 1995). In summary, these observations indicate that the acute signs of cholinesterase inhibition in humans are generally well understood and can be modeled usefully in rats. The functional domains affected by these compounds reflect the immediate effects of overstimulation of muscarinic and nicotinic cholinergic receptors in the central and peripheral nervous systems. These effects are well documented and serve as a basis for addressing the more vexing questions about the potential effects of repeated exposure to cholinesterase inhibitors and the potential for persistent effects after acute poisoning and after long-term, subclinical exposures. Future work would also profit by including assessments of affect induced by treatment with these compounds.
III. E F F E C T S O F R E P E A T E D E X P O S U R E TO C H O L I N E S T E R A S E I N H I B I T O R S
A. Clinical and Epidemiological Studies in H u m a n s Studies on the behavior of people occupationally exposed to cholinesterase inhibitors have yielded a range of findings that suggest that persistent behavioral deficits can follow from frank poisoning with these agents. Whether permanent deficits follow prolonged exposure to doses that do not induce signs or symptoms of poisoning ("subclinical" exposure) is far more controversial. This section considers epidemiological studies in this area and some animal studies that have addressed these issues experimentally. Kamel and Hoppin (2004) reviewed the epidemiological literature that explored potential neurotoxicity from chronic exposure to pesticides, including OPs, CMs, fungicides, and fumigants. Of 39 studies reported between 1974 and 2003, 27 focused on OP and CM insecticides; of these, 25 studies reported some association between exposure to the insecticide and an effect. The domains most consistently affected by OPs and/or CMs were psychomotor function, symptoms and affect, and cognitive function (Table 2). Vibration sensitivity, balance, tremor, and nerve function were less frequently measured and less frequently affected when assessed. Thus, there is compelling evidence that acute poisoning from pesticides is frequently, but not inevitably, associated with persistent behavioral changes of some kind, and that alterations in affective, cognitive, and motor functions are most commonly observed.
351
TABLE 2. Neurobehavioral Effects of Chronic Exposure to Cholinesterase-lnhibiting Pesticides (OPs and CMs) in Humans (Derived from Kamel and Hoppin, 2004) and in Animals (Compiled from 15 Papers on Repeated Exposures in Animals)
Domain Human studies Psychomotor Symptoms and affect Cognitive Vibration sensitivity Balance Tremor Animal studies Motor activity Neuromotor Cholinergic signs Vestibular function Cognitive
No. positive/No, studies
8/11 13/19 10/15 4/10 1/4 0/2 9/13 7/13 9/13 4/9 5/8 worsened 2/8 improved
% positive
73 68 67 40 25 0 69 54 69 44 63 25
However, these reports included studies of people overtly poisoned with pesticides as well as people exposed but not clinically affected. Studies of agricultural pesticide applicators from the early 1990s suggested that overt poisoning episodes were necessary for induction of persistent cognitive sequelae of exposure. For example, Nicaraguan pesticide applicators with a history of hospitalization for treatment of poisoning with OPs showed long-term deficits in cognitive and motor functions assessed approximately 2 years after the poisoning episode (Rosenstock et al., 1991). Similar results were obtained from poisoned workers in California (Steenland et al., 1994). In contrast, the neuropsychological performance of agricultural workers applying pesticides ~under well-controlled conditions, with less than 15% inhibition of cholinesterase activity (Karr et al., 1992), did not differ from that of controls when examined with a standardized neurobehavioral test battery (Daniell et al., 1992). Thus, the evidence for probable long,term effects of poisoning with cholinesterase inhibitors is quite convincing because it has also been documented after mild poisoning in workers from a number of occupations using a variety of neurobehavioral end points (Stallones and Beseler, 2002; see also reviews by Jamal et al., 2002a,b; Kamel and Hoppin, 2004). The more troublesome issue involves potential longterm effects from subsymptomatic exposures. A series of studies of farmers in the United Kingdom who regularly dip their sheep into solutions of OP pesticides for the control of parasites raised concern about persistent effects of subsymptomatic exposure. Stephens et al. (1995) showed that "sheep dippers" performed more poorly on
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SECTION IV. O r g a n
Toxicity
tests of sustained attention and information processing speed and were more vulnerable to psychiatric disorders than quarry workers who lacked exposure to pesticides. None of the farmers had experienced acute poisoning episodes, although they did experience symptoms of exposure during periods of dipping. Because the neurobehavioral tests were conducted 2 or more months after dipping, these effects were not related to recent pesticide exposure. In addition, a follow-up study showed that these effects of chronic exposure were not correlated with effects of acute exposure (Stephens et al., 1996). Subsequent studies of this population have focused on other effects, including sensory dysfunction (Beach et al., 1996; Jamal et al., 2002a) and peripheral neuropathy (Pilkington et al., 2001). As with the behavioral effects previously reported, a loss of somatotopic sensory acuity was related to the severity of symptoms during periods of acute exposure (Beach et al., 1996). Two studies of termiticide applicators in the United States support the conclusion that occupational exposure to OPs in the absence of frank poisoning episodes engenders risk of subclinical neurological dysfunction. Thus, commercial applicators of chlorpyrifos performed more poorly than unexposed controls on vestibulomotor tests and reported a higher frequency of symptoms of cognitive, emotional, and motor difficulties (Steenland et al., 2000). A small subgroup (eight men) who reported past poisoning episodes performed poorly on more tests than exposed men who did not report symptoms, confirming the additional risk of higher level exposure. In a separate follow-up analysis of subjects currently exposed by occupation, visual deficits were observed in addition to vestibular effects seen previously, suggesting a risk of other effects from acute exposure to the OP (Dick et al., 2001). These studies indicate that persistent neurological sequelae of exposure to OP pesticides can follow long-term exposure in the absence of acute poisoning episodes, although the severity of the sequelae appear to be related to the degree and currency of exposure, as assessed by symptom reporting (Jamal et al., 2002b) and careful analysis of subgroups stratified by recency of exposure and previous poisoning (Steenland et al., 2000; Dick et al., 2001). This conclusion is supported by reports of persistent deficits in other populations of exposed workers who did not require medical treatment for poisoning, including South African orchard workers (London et al., 1998) and Costa Rican banana workers (Wesseling et al., 2002). Recent evidence from large-scale studies of farm workers suggests that long-term exposure to pesticides may be associated with disorders of affect leading to suicide (London et al., 2005) rather than sensory, motor, or cognitive function per se. This finding is consistent with early studies of chronically exposed people (Gershon and Shaw, 1961; Metcalf and Holmes, 1969) and previously reported changes in the profile of effects after recovery from acute poisoning. That is, prospective studies of Nicaraguan
patients poisoned by OP pesticides showed that initial sensory, cognitive, and motor deficits tended to abate over a 2-year period and were replaced by an increase in neuropsychiatric symptoms even after mild poisoning episodes (Delgado et al., 2004; Miranda et al., 2002a,b). These studies suggest that further examination of affective disorders may be warranted for assessing the risk of exposure to OPs. A second study reported an increase in the prevalence of neurological symptoms in a large sample (-19,000) of U.S. pesticide applicators whose exposure was insufficient to cause acute symptoms (Kamel et al., 2005). In this study, the prevalence of nonspecific medical symptoms (i.e., symptoms not related to pesticide exposure) was associated with use of many pesticides, particularly OP insecticides, and was related to the degree of self-reported exposure. This study suggests that long-term, subsymptomatic exposure to OPs and other pesticides may exert important adverse effects on public health that escape detection in smaller studies. On the whole, however, epidemiological studies provide insufficient evidence to determine whether chronic, lowlevel exposure to cholinesterase-inhibiting insecticides, without acute poisoning episodes, results in persistent neurological or behavioral deficits in occupationally exposed humans (Kamel and Hoppin, 2004). Similarly, evidence for such effects in the general population is entirely lacking. Because of the difficulties of disentangling effects of chronic and acute exposures, as well as the impossibility of controlling all relevant variables in epidemiological studies, animal models have been developed to address the potential long-term effects of subsymptomatic exposure to these compounds.
B. Behavioral Effects of Repeated Exposure in Animals Perhaps because the question of the potential adversity of long-term, low-level exposure to cholinesterase-inhibiting pesticides in humans has only recently come into focus, few experimental animal studies of these compounds have addressed the issue. On the other hand, considerable work has been done to characterize the effects of repeated exposures, often with the goal of determining whether tolerance or sensitization occurs when exposure is prolonged or repeated. Some of this work bears on the question of the hazard of chronic exposure in humans, and a few studies have assessed the behavior of animals after the termination of dosing, when cholinesterase activity had returned to pretreatment levels. As noted previously, the neurobehavioral effects of chronic pesticide exposure in humans include psychomotor and cognitive functions, affect, vibration sensitivity, and balance. A survey of the animal literature revealed 15 papers on this topic in which exposure lasted at least 30 days;
CHAPTER 24 9Behavioral Toxicity these studies were classified according to outcome in each of several different domains (Table 2). Despite a lack of standardization in test measures and wide variation in route and duration of exposure, many of the effects can be combined into similar domains. For example, fighting reflex, ataxia, and balance beam behavior assess vestibular function, and grip strength, landing foot splay, and rotarod behavior assess neuromotor function (Moser, 1991). Motor activity, a commonly used and highly apical measure of nervous system function, was also analyzed. As Table 2 indicates, effects of cholinesterase inhibitors on activity and neuromotor changes were most commonly observed in those studies in which they were tested (Abdel-Rahman et al., 2004; Ivens et al., 1998; Llorens et al., 1993; Mattsson et al., 1996; Moser et al., 2005; Palumbo et al., 2001; Satin and Gill, 1998; Schulz et al., 1990; Sheets et al., 1997; Sobotka et al., 1986; Terry et al., 2003). These findings compare reasonably with the psychomotor alterations observed in humans and are also reported most frequently in human studies. Cholinergic signs in animals, and symptoms in humans, were also commonly observed (Bushnell et al., 1994; Drsi and Nagymajt6nyi, 1999; Ivens et al., 1998; Llorens et al., 1993; Mattsson et al., 1996; Maurissen et al., 2000; Moser et al., 2005; Sheets et al., 1997). Fewer studies evaluated vestibular function, but in both animals and humans, fewer than half of them showed adverse effects (Abdel-Rahman et al., 2004; Ivens et al., 1998; Moser et al., 2005; Sheets et al., 1997). Finally, cognitive function was worsened in a similar number of studies in humans and animals (Bushnell et al., 1994; Cohn and MacPhail, 1997; Llorens et al., 1993; Maurissen et al., 2000; Moser et al., 2005; Sarin and Gill, 1998). A few studies also reported improved cognitive function (Ivens et al., 1998; Palumbo et al., 2001), which likely reflects the memory-enhancing effects of low-level cholinesterase inhibition. This admittedly rough comparison suggests that animal models fairly represent the spectrum of effects reported in epidemiological studies of humans exposed to cholinesterase inhibitors and supports the contention that examination of the animal literature can provide insights into the hazard to be expected in human populations from long-term exposure to these compounds. As indicated, altered neuromotor function (decreased activity levels, impaired rotarod performance, slower responding, and decreased grip strength) corresponds to both the psychomotor and balance domains in Table 2. Effects of this nature have been observed duringtreatment with several pesticides administered in the diet, water, or parenterally, with durations ranging from 14 days to 4 months (Kobayashi et al., 1988; Llorens et al., 1993; Palumbo et al., 2001; Prendergast et al., 1997; Sheets et al., 1997; Schulz et al., 1990; Terry et al., 2003). In general, the largest and most prolonged motor effects are obtained during exposure to doses that markedly inhibit brain cholinesterase activity. On the other hand, when brain
353
cholinesterase inhibition is absent, is slight (e.g.,
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FIG. 4. Further modulation of the morphology of the AP occurs when cell swelling and ACh overflow are taken into account. Note the rise in the resting potential and the drastic shortening of the cycle length in comparison to the baseline AP.
I
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,
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,
[ms]
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FIG. 6. Pharmacological intervention, such as blocking of lCL,sw by an anionic blocker (dashed curve), can help to restore the tissue to homeostatic condition from that of the OP-affected condition (solid curve).
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FIG. 5. OP also causes deviation from the norm of potassium and calcium concentrations and its additional effect is shown in the ECG. Note the absence of the T wave, indicating modulation of the repolarization process.
therapeutic agent that acts on ionic concentrations, channels, and membrane currents. In addition, pretreatment with huperzine (Lallement et al., 2002) has been found to be effective. However, the widely used antidote, atropine, can also be toxic because it can cause VE Therefore, the strategy of choice is chemical blocking of OP-activated currents, such as Icl,sw or IKACh, and restoration of function of pumps and exchangers. Such an approach reconstitutes the morphology of the AP of cardiac myocytes and the ECG to baseline form.
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FIG. 7. Blocking the cell currents activated by cell swelling and ACh overload in OP-affected cardiac tissue (top dashed and solid curves) as contrasted with baseline conditions (bottom solid curve) in the simulated ECG.
IV. C O N C L U S I O N S DIRECTIONS
AND FUTURE
Computer simulations of OP-affected cellular processes can add considerable new insights into the mechanisms responsible for the modulation of cardiac electrophysiological processes. The identification of the importance of cell swelling-activated membrane current is a case in point. It was shown that inhibition of chloride transport can change overshoot of action potential and thus it can stop the AP
CHAPTER 27 firing. Process simulations can quantify the nature and magnitudes of the incurred changes and the deviation from the normal conditions. There is a need for additional cellular experimental data on membrane currents and ion concentrations in the various cellular compartments under pathological conditions. Such data will make the simulation of the cardiac cycle of the whole heart, from the start of systole to termination of diastole, possible and suggest pharmacological intervention strategies to overcome the effect of OP on cardiac tissue.
Acknowledgments We acknowledge the use of the computer assets of the Major Shared Resource Center of the U.S. Army Research Laboratory and the advice of Dr. John Pormann of Duke University on some software issues.
References Abraham, S., Oz, N., Sahar, R., and Kadar, T. (2001). QTc prolongation and cardiac lesions following acute organophosphate poisoning in rats. Proc. West Pharmacol. Soc. 44, 185-186. Alonso, M. J., Arribas, S., Marin, J., Balfagon, G., and Salaices, M. (1991). Presynaptic M2-muscarinic receptors on noradrenergic nerve endings and endothelium-derived M3 receptors in cat cerebral arteries. Brain Res. 567, 76-82. Antzelevitch, C., and Shimizu, W. (2002). Cellular mechanisms underlying the long QT syndrome. Curr. Opin. Cardiol. 17, 43-51. Balazs, T., and Ferrans, V. J. (1978). Cardiac lesions induced by chemicals. Environ. Health Perspect. 26, 181-191. Bar-Meir, A., Grubstein, A., Givoni, S., and Tadmor, B. (2001). Organophosphates "Continuous injury t o the heart" Harefuah 140, 764-769. Baskin, S. I., and Whitmer, M. P. (1991). The cardiac toxicolology of organophosphorous agents. In Principles of Cardiac Toxicology (S. I. Baskin, Ed.). CRC Press, Boca Raton, FL. Baumgarten, C. M., and Clemo, H. E (2003). Swelling-activated chloride channels in cardiac physiology and pathophysiology. Prog. Biophys. Mol. Biol. 82, 25-42. Britt, J. O., Martin, J. L., Okerberg, C. V., and Dick, E. J. (2000). Histopathologic changes in the brain, heart, and skeletal muscle of rhesus macaques, ten days after exposure to soman. Comp. Med. 50, 133-139. d'Anglemont de Tassigny, A., Souktani, R., Ghaleh, B., Henry, P., and Berdeaux, A. (2003). Structure and pharmacology of swelling-sensitive chloride channels, Icl,swell- Fundam. Clin. Pharmacol. 17, 539-553. Demir, S. S., Clark, J. W., and Giles, W. R. (1999). Parasympathetic modulation of sinoatrial node pacemaker activity in rabbit heart: A unifying model. Am. J. Physiol. 276, H2221-H2244. Dobrzynski, H., Marples, D. D. R., Musa, H., Yamanushi, T. T., Henderson, Z., Takagishi, Y., Honjo, H., Kodama, I., and Boyett, M. R. (2001). Distribution of the muscarinic K + channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart. J. Histochem. Cytochem. 49, 1221-1234.
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Dobrzynski, H., Janvier, N. C., Leach, R., Findlay, J. B., and Boyett, M. R. (2002). Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells. Am. J. Physiol. Heart Circ. Physiol. 283, H615-H630. Egan, T. M., and Noble, S. J. (1987). Acetylcholine and the mammalian "slow inward" current: A computer investigation. Proc. R. Soc. London B 230, 315-337. Filbert, M. G., Moore, D. H., and Adler, M. (1992). Relaxation of soman-induced contracture of airway smooth muscle in vitro. Drug Chem. Toxicol. 15, 203-215. Freeman, S. E., Dawson, R. M., and Keeghan, A. M. (1986). Modification of the effects of muscarinic agonists by reversible and irreversible anticholinesterase compounds in the guinea pig atrium. J. Mol. Cell Cardiol. 18, 231-239. Harvey, R. D., and Belevych, A. E. (2003). Muscarinic regulation of cardiac ion channels. Br. J. Pharmacol. 139, 1074-1084. Hu, C. Y., Hsu, C. H., and Robinson, C. P. (1991). Effects of soman on calcium uptake in microsomes and mitochondria from rabbit aorta. J. Appl. Toxicol. 11, 293-296. Karki, P., Ansari, J. A., Bhandary, S., and Koirala, S. (2004). Cardiac and electrocardiographical manifestations of acute organophosphate poisoning. Singapore Med. J. 45, 385-389. Kiss, Z., and Fazekas, T. (1982). Organophosphate poisoning and complete heart block. J. R. Soc. Med. 73, 138-139. Kiss, Z., and Fazekas, T. (1983). Organophosphate poisoning and complete heart block. J. R. Soc. Med. 76, 85. Kotev, G., and Paliev, B. (1968). Electrocardiographic changes in dogs exposed to soman and the effect of the antidote nemicol-3. Eksp. Med. Morfol. 7, 240-244. Lallement, G., Demoncheaux, J. P., Foquin, A., Baubichon, D., Galonnier, M., Clarencon, D., and Dorandeu, E (2002). Subchronic administration of pyridostigmine or huperzine to primates: Compared efficacy against soman toxicity. Drug Chem. Toxicol. 25, 309-320. Limaye, M. R. (1966). Acute organophosphorus compound poisoning. J. Indian M. A. 47, 492-498. Ludomirsky, A., Klein, H. O., Sarelli, P., Becker, B., Hoffman, S., Taitelman, U., Barzilai, J., Lang, R., David, D., DiSegni, E., and Kaplinsky, E. (1982). Q-T prolongation and polymorphous ("torsades de pointes") ventricular arrhythmias associated with organophosphorus insecticide poisoning. Am. J. Cardiol. 49, 1654-1658. Luo, C., and Rudy, Y. (1994). A dynamic model of the cardiac ventricular action potential: I. Simulations of ionic currents and concentration changes. Circ. Res. 74, 1071-1096. Nilius, B., and Droogmans, G. (2003). Amazing chloride channels: An overview. Acta Physiol. Scand. 177, 119-147. Opie, L. H. (1998). The Heart Physiology, from Cell to Circulation, 3rd ed. Lippincott Williams & Wilkins, Philadelphia. Osterrieder, W., Noma, A., and Trautwein, W. (1980). On the kinetics of the potassium current activated by acetylcholine in the SA node of the rabbit heart. Pfluegers Arch. 386, 101-109. Pimentel, J. M., and Carrington da Costa, R. B. (1992). Effects of organophosphates on the heart, In Clinical and Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.). Butterworth-Heinemann, Oxford. Povoa, R., Cardoso, S. H., Luna, F. B., Ferreira, E C., Ferreira, M., and Ferreira, C. (1997). Organophosphate poisoning and myocardial necrosis. Arq. Bras. Cardiol. 68, 377-380.
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Preston, E., and Heath, C. (1972). Depression of the vasomotor system in rabbits poisoned with an organophosphate anticholinesterase. Arch. Int. Pharmacodyn. Ther. 200, 245-254. Roth, A., Zellinger, I., Arad, M., and Atsmon, J. (1993). Organophosphates and the heart. Chest 103, 576-582. Saadeh, A. M., Farsakh, N. A., and A1-Ali, M. K. (1997). Cardiac manifestations of acute carbamate and organophosphate poisoning. Heart 77, 461-464. Sakmann, B., Noma, A., and Trautwein, W. (1983). Acetylcholine activation of single muscarinic K + channels in isolated pacemaker cells of the mammalian heart. Nature 303, 250-253. Shibata, O., Tsuda, A., Makita, T., Iwanaga, S., Hara, T., Shibata, S., and Sumikawa, K. (1998). Contractile and phosphatidylinositol responses of rat trachea to anticholinesterase drugs. Can. J. Anaesth. 45, 1190-1195. Shumaker, J. M., Clark, J. W., Giles, W. R., and Szabo, G. (1990). A model of the muscarinic receptor-induced changes in K +current and action potentials in the bullfrog atrial cell. Biophys. J. 57, 567-576. Stephenson, L. A., and Kolka, M. A. (1990). Acetylcholinesterase inhibitor, pyridostigmine bromide, reduces skin blood flow in humans. Am. J. Physiol. 258, R951-R957. Tan, H. L., Hou, C. J. Y., Lauer, M. R., and Sung, R. J. (1995). Electrophysiologic mechanisms of the long QT interval syndromes and torsades de pointes. Ann. Intern. Med. 122, 701-714. Thomsen, R. H., and Baskin, S. I. (1988). Effects of AF-DX 116, a cardioselective M-2 muscarinic antagonist on the negative inotropic action of acetylcholine. FASEB J. 2, A789. Tobin, T., Akera, T., Baskin, S. I., and Brody, T. M. (1973). Calcium ion and Na + + K+-ATPase: Its mechanism of inhibition and identification of the E1-P intermediate. Mol. Pharmacol. 9, 336-349. Tryphonas, L., Veinot, J. P., and Clement, J. G. (1996). Early histopathologic and ultrastructural changes in the heart of Sprague-Dawley rats following administration of soman. Toxicol. Pathol. 24, 190-198. Tseng, G. (1992). Cell swelling increases membrane conductance of canine cardiac cells: Evidence for a volume-sensitive C1 channel. Am. J. Physiol. 262, C 1056-C 1068. Tsuda, A., Shibata, O., Saito, M., Hashimoto, S., Iwanaga, S., Makita, T., and Sumikawa, K. (2001). A dose-response study
of anticholinesterase drugs on contractile and phosphatidylinositol responses of rat trachea. Anesth. Analg. 92, 100-105. Tsukahara, T., Hongo, K., Kassell, N. E, and Ogawa, H. (1989). Characterization of muscarinic cholinergic receptors on the endothelium and the smooth muscle of the rabbit thoracic aorta. J. Cardiovasc. Pharmacol. 13, 870-878. Vandenberg, J. I., Bett, G. C., and Powell, T. (1997). Contribution of a swelling-activated chloride current to changes in the cardiac action potential. Am. J. Physiol. Cell Physiol. 273, C541-C547. Viskin, S. (1999). Long QT syndromes and torsades de pointes. Lancet 354, 1625-1633. Viswanathan, P. C., and Rudy, Y. (2000). Cellular arrhythmogenic effects of congenital and acquired long-QT syndrome in the heterogeneous myocardium. Circ. Res. 101, 1192-1198. Wright, A. R., and Rees, S. A. (1998). Cardiac cell volume: Crystal clear or murky waters? A comparison with other cell types. Pharmacol. Ther. 80, 89-121. Yang, E, Zhang, B. H., and Hong, N. (1991). Anti-arrhythmia and vegetative nervous system effects of anisodamine. Acta Pharmacol. Sinica 12, 173-176. Yang, Z. K., Boyett, M. R., Janvier, N. C., McMorn, S. O., Shui, Z., and Karim, E (1996). Regional differences in the negative inotropic effect of acetylcholine within the canine ventricle. J. Physiol. (London) 492, 789-806. Ying, X. Y., Zhong, Y. X., and Ruan, J. X. (2003). Effect of anisodamine on the toxicokinetics of soman in rats. Toxicology 191, 28. Zoltani, C. K., and Baskin, S. I. (2000). Simulation of acetylcholine cardiac overload caused by soman, a cholinesterase inhibitor. In Proceedings of Comparative Cardiology 2000 (A. Murray, Ed.), Vol. 27, pp. 243-246. IEEE Press, Piscataway, NJ. Zoltani, C. K., and Baskin, S. I. (2002). Organophosphate-induced toxicity: Computer study of reentry in atrial tissue. In Proceedings of Comparative Cardiology 2002 (A., Murray, Ed.), Vol. 29, pp. 509-512. IEEE Press, Piscataway, NJ. Zoltani, C. K., Baskin, S. I., and Platoff, G. E. (2004). ECGs and metabolic networks: An in silico exploration of cyanide-caused cardiac toxicity. In Pharmacological Perspectives of Toxic Chemicals and Their Antidotes (S. J. S. Flora, J. A. Romano, S. I. Baskin, and K. Sekhar, Eds.). Narosa, New Delhi.
CHAPTER
~8
Pulmonary Toxicity of Cholinesterase Inhibitors COREY HILMAS, MICHAEL ADLER, AND STEVEN I. BASKIN U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland
that they originated in Germany. The A through F designation was based on their chronological order of synthesis. The first nerve agent to be synthesized was tabun (GA) in 1936 by Gerhard Schrader, a chemist at I. G. Farbenindustrie interested in developing OP compounds as insecticides (Harris and Paxman, 1982). This was followed by sarin (GB), named after the four scientists involved in its development (Schrader, Ambrose, Rudriger, and van der Linde) (Harris and Paxman, 1982; Sidell, 1997). The third nerve agent, soman, was synthesized by Richard Kuhn in Germany in 1944 and was termed GD rather than GC since the latter acronym had already been established in the medical literature. Cyclosarin (GF) was the fourth to be synthesized, but interest in this nerve agent declined in favor of the other OP compounds. The fifth agent (VX) was named for being venomous and was synthesized at Porton Down, England, in 1952. Due to their high toxicity in mammals and volatile nature, some of these fluoride (sarin and soman) and nitrile (tabun) containing OP compounds were further tested, manufactured, and stockpiled by the German military during World War II, but they were never deployed. Some experts believe that Hitler, a victim of a chlorine gas attack during World War I, disliked poison gas and would only use these agents as a last resort. Others speculate that the German High Command mistakenly believed the Allies had developed the nerve agents simultaneously and feared Allied retaliation as the Axis retreated. Nevertheless, tons of nerve agents in munitions were synthesized and stockpiled in Germany during World War II that the United States and Great Britain were not aware of at the time. German tabun production facilities, able to synthesize 100 tons a month, were in place near the end of the war (Saunders, 1957). The former Soviet Union captured an entire nerve agent production facility late in the war and moved it back to Russia, where it started to manufacture and stockpile these agents (Robinson, 1971). Allied forces found that the AChEI nerve agents were 15- to 100-fold more potent than the chemical agents used in World War I.
I. I N T R O D U C T I O N The lungs are a major organ system of entry into the body and a target for the toxic effects of organophosphorus (OP) compounds, potent inhibitors of the enzyme acetylcholinesterase (ACHE). In general, ACHE inhibitors (ACHEIs) were developed for a variety of indications, including military, medical, and insecticide applications. Nerve agents, OP chemicals with remarkable toxic activity, were first developed by Germany prior to World War II. Whereas nerve agents were produced primarily for military deployment, other cholinesterase inhibitors were used for treating conditions such as myasthenia gravis and as pretreatments for nerve agent exposure. As powerful inhibitors of ACHE, these compounds exhibit profound toxicity on multiple organ systems. This chapter discusses respiratory and pulmonary toxicity through direct inhalation of ACHEIs and indirect effects on all aspects of respiration through systemic toxicity. OP nerve agents can be disseminated as liquids or aerosols and are toxic by oral, dermal, or inhalational exposure. The lungs are one of the first organs affected following contact with aerosols and vapors. Lung toxicity by ACHEIs is due to the following: (1) parasympathetic muscarinic effects leading to increased glandular secretion throughout the respiratory tract and alveoli, (2) bronchoconstriction from contraction of airway smooth muscle, (3) nicotinic effects on respiratory muscles in the thorax and accessory muscles of the neck causing labored breathing and eventually flaccid paralysis, and (4) central effects resulting in a decrease in respiratory drive.
II. H I S T O R I C A L P E R S P E C T I V E The modem age of chemical warfare began during the past century with the development of the present-day vesicants and AChEIs (or OP class nerve agents). There are five OP compounds recognized as nerve agents, designated GA, GB, GD, GF, and VX by their North Atlantic Treaty Organization military abbreviation. The "G" series are named for the fact
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SECTION IV. Organ Toxicity
Since OPs were not used in World War II, the majority of cases of OP toxicity have come from accidental exposure in laboratories and agricultural exposure to OP insecticides. The earliest reported incident of OP toxicity from inhalation came from the laboratory of Willy Lange at the Friedrich-WilhelmsUniversity. In the early 1930s, Lange and his student, Gerde von Krueger, prepared dialkyl monofluorophosphates and noted their toxic fumes (Holmstedt, 1963; Sidell, 1997). They described the aromatic vapors as leading to dyspnea and laryngeal edema minutes later, followed by a lucid interval, diplopia, and photophobia. The symptoms of toxicity were noted to last several hours before subsiding. Similar clinical pictures have been reported for the OP compounds DFP (Grob et aL, 1947), satin and tabun (Grob and Harvey, 1953, 1958; Krop and Kunkel, 1954), as well as parathion (DuBois et al., 1949). Detailed clinical signs and symptoms have also been described in case reports of accidental exposures to G agents (tabun, sarin, and soman) (Craig and Comblath, 1953; Craig and Freeman, 1953; Sidell, 1974) and VX (Freeman et al., 1956; Lubash and Clark, 1960; Sidell, 1967).
toxicity to mammals at doses less than 1 mg/kg (Saunders, 1957; O'Brien, 1960). O-ethyl-S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX), an agent that is less volatile than the G agents, is the most potent AChEI. The first OP synthesized was B ladan, shown to be tetraethyl pyrophosphate (TEPP). The most studied of the OP compounds is diisopropyl fluorophosphate (DFP), originally synthesized by the British, who found it to be more potent than eserine (Adrian et al., 1947). The phosphorothioates and phosphorodithioates, shown in Table 1 of Chapter 2, were developed when the nerve agents were found to be too toxic and volatile for use in agriculture. These OP insecticides contain a P-S-alkyl and/ or a P - S group in their structure. The best known member of this class is parathion, the most widely used insecticide at one time and responsible for more cases of accidental poisoning and death than any other OP compound. The activation and conversion of this weak AChEI (parathion) to the more active and potent form (paraoxon) was demonstrated to take place in the liver (Diggle and Gage, 1951; Gage, 1953).
III. STRUCTURES OF OPs IV. RESPIRATORY PHYSIOLOGY The general formula and chemical structures of most OP compounds discussed in this chapter are shown in Table 1 of Chapter 2. The nerve agents tabun, sarin, and soman were the most potent compounds in the class, causing lethality to animals in the submilligram range. Their chemical structures are shown in Fig. 1. Originally developed for the agricultural industry, they contain either an F or CN substituent group and display
The respiratory system can be viewed as two components acting in tandem to facilitate gas exchange, namely the conducting and respiratory portions. The conducting portion supplies the lungs with warmed air on inhalation and allows gases to escape on exhalation, whereas the respiratory portion provides for actual gas exchange between the air and blood. The conducting portion consists of a
O 02H50
iiP ~ C N I N(CH3)2
Tabun (GA) Ethyl N-dimethylphosphoramido-cyanidate
O
il i CH3
(H3C)2HCO~ p~
Sarin (GB) Isopropyl methylphosphonofluoridate OH3
O
C2H50
ii I O02H5
..... P ~ O
O
IIP OC2H5 I OC2H5
Tetraethyl pyrophosphate (TEPP)
I
CH3
H
I
CH3
Soman (GD) Pinacolyl methylphosphonofluoridate H3C
O
I II HC~O~P~F I I CH 3 O H3C~C
HC~O-----P~F
O
ii I OCH2CH3
H3C~P~S
CH 3
Diisopropyl fluorophosphate (DFP)
\ CH~CH / CH2CH2--N \
CH~CH 3
H3C O-ethyl- S-(2-diisop ropylam inoethyl) methylphosphonothiolate (VX)
FIG. 1. Chemical structures of OP compounds and their common abbreviations.
3
CHAPTER 28 9Pulmonary Toxicity of AChEIs branching system of airways, including the nasal cavities, nasopharynx, larynx, trachea, mainstem bronchi, progressing to even smaller bronchi and eventually to bronchioles. This elaborate system of pipes conducts air into and out of the lungs as a result of respiratory movements of the thoracic intercostal musculature and diaphragm. Air reaches the alveolar ducts and finally the alveoli, the site of gas exchange. The most important factor of respiratory physiology to consider for pulmonary toxicity by AChEIs is the airway resistance through the conducting portion. Airflow through the conducting system from the trachea and mainstem bronchi to the small bronchioles can be characterized as airflow through a series of straight tubes or laminar flow. Jean Poiseuille, a French physician and physiologist, described the volume flow rate through straight circular tubes by the following equation, known as Poiseuille's law (West, 1995; Guyton and Hall, 2005): F = P'rrr4/8nl
where n is the coefficient of viscosity, P is the pressure difference across the length 1 of the tube, r is the radius of the tube, and F is the volume flow rate. Since the resistance to flow R is driving pressure P divided by flow F using the analogy of Ohm's law, we arrive at the following relationship for flow resistance R. R = 8nl/'rrr 4
When applied to airways, if the airway radius decreases by half its original diameter as a result of bronchoconstriction, the airway resistance increases 16-fold. In actuality, the airflow is a mixture of laminar and turbulent flow because the airways must branch to progressively smaller bronchi as they reach the lung periphery. The major site of airway resistance lies in the medium bronchi because the vast number of smaller airways negates any effect they might impose on airway resistance. Airway smooth muscle tone is under the control of the autonomic nervous system. Whereas sympathetic stimulation of adrenergic receptors causes bronchodilation, parasympathetic activity via acetylcholine (ACh) release causes bronchoconstriction. Excess ACh at smooth muscles surrounding airways due to ACHE inhibition by OPs produces significant increased airway resistance that is readily characterized on inspiratory and expiratory auscultation of the lungs (West, 1995).
V. C O N T R O L O F V E N T I L A T I O N Since the end result of OP-induced toxicity from lethal doses through inhalation or other routes is asphyxia secondary to respiratory failure, a brief summary of the mechanisms involved in control of ventilation is provided. Although ACHEIs affect several aspects of respiration, a detailed review of respiratory
391
physiology and ventilation can be found elsewhere (West, 1995; Guyton and Hall, 2005). The elements of the respiratory control system are the following: chemoreceptors, peripheral sensors, and central sensors, which monitor various measures of respiration to inform the brain; the effector muscles of respiration, which allow for ventilation; and the respiratory control centers in the brain, which integrate the information from the chemoreceptors and regulate the effector muscles. The lungs contain specialized receptor sensors: the pulmonary stretch receptors, the juxtacapillary or J receptors, and the irritant receptors. The J receptors, found in the alveolar walls close to the capillaries, are very sensitive to chemicals monitored in the pulmonary circulation. Receptor activation in this group leads to rapid, shallow breathing patterns and apneic episodes mediated through the vagus nerve. Furthermore, an increase in the interstitial fluid volume of the alveolar wall will activate these receptors, suggesting a possible role in late stages of OP toxicity after the onset of pulmonary edema. Clinical evidence of an abnormal pattern of breathing (Cheyne-Stokes respiration) has been noted to occur in patients exposed to nerve agents or pesticides. This is an unusual periodic breathing pattern characterized by long periods of apnea interspersed with episodes of hyperventilation (Taylor, 1996). This pattern is due to respiratory insufficiency, hypoxemia, and brain damage and is a grave clinical sign as the tidal volume gradually waxes and wanes prior to death. Direct brain damage from OPs and hypoxemia from respiratory insufficiency secondary to pulmonary congestion and flaccid paralysis of respiratory muscles probably contribute to this respiratory pattern. In addition, the lungs contain irritant receptors located between airway epithelial cells. These groups are activated by noxious fumes, gases, smoke, and dust, inducing a bronchoconstriction reflex that is thought to be responsible for the onset of asthma attacks. The effector muscles of respiration include diaphragmatic, intercostal, abdominal, and accessory muscles of respiration (e.g., sternocleidomastoid). They are very sensitive to the toxic effects of AChEIs. Accessory muscles of respiration are not a major contributor to the work of breathing under normal conditions, but they come into play during periods of labored breathing. OP compounds are toxic to these muscles of respiration through inhibition of ACHE, leading to an excess of ACh, excessive stimulation of nicotinic cholinergic synapses, and eventual flaccid paralysis. Subsequent expansion of the chest wall to inflate the lungs will not occur and respiration will cease. The respiratory centers are neuronal groups found primarily in the medulla and pons of the brain stem. The medullary respiratory center comprises a dorsal and ventral group, located in the reticular formation of the medulla below the fourth ventricle. They are believed to be central targets for OP toxicity through an unknown mechanism. Damage to these neuronal control centers will affect inspiration and expiration.
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S ECTI O N IV 9Organ Toxicity
VI. E V I D E N C E O F P U L M O N A R Y TOXICITY Nerve agents, extremely potent chemicals, are esters of phosphonic acid. A C t [the concentration C of agent vapor or aerosol in air (mg/m 3) multiplied by the time t of exposure (in minutes)] of 2 or 3 mg.min/m 3 of sarin is enough to produce symptoms in man (Johns, 1952). Derived from Haber's rule, the product of Ct is a constant such that this Ct for sarin can be attained with an exposure to a concentration of 2 or 3 mg/m 3 for 1 min or a concentration of 0.05-0.075 mg/m 3 for 40 min. Only a few milligrams of VX, absorbed through the skin, will cause clinical signs and symptoms of toxicity (Bowers et al., 1964; Craig et al., 1977). The initial signs and symptoms of exposure to small quantities of agent vapor are discussed later. Larger amounts will undoubtedly lead to loss of consciousness, seizure activity, respiratory and cardiac arrest, and death. Clinical effects are evident within minutes of exposure (Ward, 1962), and after a large exposure (Ct of 10-200 mg.min/m3), depending on the agent, death is inevitable in 10-15 min without medical intervention. After exposure to a sublethal amount on the skin (1-3 mg), the onset time for clinical effects is typically 1 or 2 hr (Bowers et al., 1964; Craig et al., 1977). The initial effect is usually vomiting, followed by muscular weakness. A lethal amount on the skin (10 mg) in the case of VX, the most toxic by percutaneous absorption, will cause clinical effects within several minutes and death soon thereafter.
A. Clinical Signs and Symptoms We have learned valuable information regarding the signs and symptoms observed after mild to moderate OP intoxication as it relates to the respiratory system. In most of these clinical cases, the exposure Ct is unknown. One study (Craig and Freeman, 1953) analyzed clinical cases of 53 individuals exposed accidentally to tabun (4 cases) or sarin (49 cases). Although miosis and rhinorrhea were the two most consistent signs of toxicity, occurring in 91 and 58% of reported cases, respectively, respiratory symptoms were recorded in 77% of cases of mild to moderate exposure. These cases were considered mild to moderate since symptoms were not severe enough to require atropine therapy, although atropine was administered in several cases. The types of respiratory symptoms described by patients were not consistent and included coughing, wheezing, increased exertional dyspnea, dyspnea at rest, inability to breathe deeply, and a sensation of pressure in the throat or chest. Rhinorrhea was shown to be accompanied by hyperemia of the nasal mucosa, persisting for the duration of clinical signs and reported symptoms. Since the nose is part of the upper airway, we consider rhinorrhea as part of pulmonary toxicity. Auscultation of the chest revealed prolongation of the expiratory phase and wheezy breath sounds (lung field
location unreported) for 5 days following exposure. In addition, the earliest symptom was recorded, and in the majority of cases, chest pressure and rhinorrhea were noted first, taking place 5 - 2 0 m in after exposure. Miosis and dim vision occurred next, with onset beginning 15-60 min after exposure. With regard to respiratory symptom severity, exertional dyspnea, wheezing, and cough became more severe over time, with no consistent pattern for regression of respiratory symptoms. Chest tightness, appearing early in the course of intoxication, was the first to disappear, and exertional dyspnea, reported later among cases, persisted the longest among all respiratory symptoms. Furthermore, an earlier onset for respiratory symptoms noted after exposure predicted a more uncomfortable and prolonged clinical course. This was associated with the suspected scenario in which the concentration was high and duration short. In contrast, individuals believed to have experienced more prolonged but lower concentrations of the agent developed symptoms more slowly. Among all cases, there was no correlation between cholinesterase activity and the clinical signs and symptoms, and no differences in symptoms were noted between those exposed to tabun or sarin. In contrast to GA and GB, VX contains a low vapor tension, resulting in absorption through the skin to produce systemic toxicity rather than inhalation via the respiratory tract. However, there is clinical evidence for pulmonary toxicity after dermal exposure to such agents. For example, case reports of accidental VX poisoning in humans have indicated evidence of respiratory toxicity from liquid agents without copious nasal secretions (Freeman et al., 1956). Since rhinorrhea is a hallmark of inhalational toxicity, this suggests an indirect toxicity of the respiratory system through a systemic route. In addition, respiratory symptoms of chest tightness were late appearing, beginning 24 hr after initial exposure and lasting 3 days, unlike the early respiratory symptoms experienced by patients exposed to the more volatile GA and GB agents (Craig and Cornblath, 1953; Craig and Freeman, 1953). At autopsy, evidence of pulmonary toxicity is quite pronounced, independent of the route of administration. Elevation of the diaphragm and collapse of the lungs are common findings. Further observations indicate pulmonary ischemia and congestion (O'Brien, 1960).
B. Nasal Airway Since the nasal passages constitute the initial conducting portion of the respiratory tract, we consider toxicity of the nasal mucosa here. The anterior aspect of the nose is the area most prone to toxic effects by any inhalants because it is the initial primary site of deposition of highly volatile vapors and particles (Stott and McKenna, 1984; Dahl and Bechtold, 1985). Rhinorrhea can be an early warning indicator of recent OP exposure because it is very common in case histories after inhalational exposure and is the earliest clinical sign, along with miosis, for the diagnosis of an acute exposure.
CHAPTER
Rhinorrhea or "runny nose" occurs because of muscarinic receptor activation from excess ACh secondary to inhibition of ACHE. Rhinorrhea usually occurs minutes after an exposure and secretions tend to be thin, clear, and serous in nature. Thick, rubbery secretions may be seen after atropine treatment. Even small amounts of OP vapor can set off a profound rhinorrhea, but the symptoms are typically dose related. One case report demonstrates the abundant rhinorrhea one might experience after exposure to inhaled vapors of sarin. The rhinorrhea was described by the patient to be like a "leaking faucett" (Sidell, 1997). A general increase in secretions from any glands, including nasal mucosa, intestinal, and salivary, can be triggered from dermal or inhalational exposure. From numerous clinical cases, it is known that low Cts will produce a triad of effects on the eyes, nose, and airways. Not only will bronchoconstriction in the airways contribute to dyspnea but also goblet cells and other secretory cells of the nasal mucosa and bronchi will contribute to the dyspnea experienced from OP exposure. Only during moderate exposure will one see deficits in ventilation, copious secretions, and severe dyspnea. Severe exposure will produce cyanosis, loss of consciousness, and convulsions.
C. Trachea and Bronchi Although the extent of signs and symptoms varies with the type of OP agent, concentration, time of exposure, and route of administration, the predominant signs of exposure include constriction of the airways and increased secretions, leading to various degrees of dyspnea (Taylor, 1996). Similar to rhinorrhea, low levels of exposure to nerve agents such as sarin at a Ct of 5-10 mg.min/m 3 will produce respiratory discomfort in the majority of patients, primarily due to bronchoconstriction (Sidell, 1997). The severity of pulmonary complaints will increase as the concentration of the agent or the time of exposure to the agent increases. Although pulmonary function studies have yielded mixed results on the importance of bronchoconstriction in subjects exposed to various low levels of satin (Cts up to 19.6 mg.min/m 3) (Clements et al., 1952), it is clear that pulmonary changes in airway resistance and tracheobronchial secretions are heard clinically upon auscultation of the lungs (Sidell, 1997). It is important to realize how sensitive lungs are to low levels of nerve agent vapors or aerosols because even a Ct exposure of 5 mg.min/m 3 of satin will produce signs and symptoms of toxicity (Marrs et al., 1996). Patients exposed to sufficient levels of OP compounds that are toxic to the respiratory system will indicate vague symptoms of chest tightness or pressure but show striking pulmonary signs on physical examination. Combinations of wheezing (expiratory, inspiratory, or both), rales, and rhonchi have all been reported in clinical cases. The pulmonary effects begin within seconds after inhalation. If the amount inhaled is large, the patient will exhibit signs of severe dyspnea, poor ventilation, cyanosis, and loss of consciousness.
28
9 Pulmonary Toxicity of AChEis
393
Although generalized systemic effects are sometimes present depending on the inhaled dose, local effects on the airways are always typically present and are the earliest symptoms recorded following inhalation of nerve agent vapors or aerosols (Craig and Freeman, 1953; Vojvodic, 1981). The airways are particularly vulnerable to the toxicity of AChEIs, considering that inhaled gases will be taken up at a rate of 8-14 breaths/min for an average adult human male. The involuntary smooth muscle that surrounds the airways of the bronchial tree is the target for AChEI activity, leading to bronchoconstriction with subsequent wheezing. Airway smooth muscles contain numerous excitatory cholinergic inputs (Suzuki et al., 1976) and a relative resistance to muscarinic receptor desensitization and muscle fatigue, making them highly vulnerable to AChE inhibition. One study investigating anti-ChE-induced constriction of isolated canine tracheal smooth muscle showed that a very low soman concentrations (10-9M) can increase the amplitude and prolong the half-relaxation time of contractions elicited by electric field stimulation (Adler et al., 1992). This was true for all AChEIs examined, including three OP (soman, satin, and paraoxon) and three carbamate (CM) ChE inhibitors (physostigmine, neostigmine, and pyridostigmine). Contractures of canine airway smooth muscle were detected when AChE activities were reduced by more than 52% (Fig. 2). When contractures are plotted as a function of AChE inhibition for the OPs and CMs, there is a linear rise in contracture amplitudes as AChE inhibition increases. This demonstrates that contracture depends on the degree of AChE inhibition and not on the nature of the inhibitor, suggesting that it is mediated by ACh accumulation. Another experiment compared the ability of two different oximes, pralidoxime (2-PAM) and HI-6, to relax soman- and sarin,induced contractures in canine tracheal smooth muscle (Fig. 3). For nerve agents that undergo rapid aging (soman), 2-PAM was unable to
100
o o o
,&
80 o ,,,_, tO
60
.E_ 40
0
~
~ ~ " O
X t~
20
Q Soman O Sarin A Paraoxon
0. .Physosti . . gmine ,8, Neostigmine Q Pyridostigmine
A 0
50
I
60
I
70
I
80
I
90
I
100
% AChE Inhibited
FIG. 2. Contractures plotted as a function of ChE inhibition for three OP and three carbamate AChEIs. Each symbol represents a single muscle strip on which both tension and AChE activity were determined.
394
SECTION IV- Organ Toxicity
E. Central Respiratory Center
FIG. 3. Relaxation of soman- or satin-induced contractures in isolated canine tracheal smooth muscle strips by the oximes HI-6 and 2-PAM. Incubation times for HI-6 (100 IxM) or 2-PAM (1 raM) were 30 min, and the oximes were added 15 min after soman (0.1 p.M) or satin (0.1 IxM) exposure. The symbols represent the mean +__SE of data from four to six muscle strips.
sufficiently reactivate ACHE. Surprisingly, the best known AChE reactivator, HI-6, was unable to reactivate AChE inhibited by soman in airway smooth muscle. Both oximes demonstrated an ability to reactivate AChE in the presence of satin, but HI-6 treatment led to more relaxation of satininduced contractures.
D. Bronchioles and Alveolar Cells One study of satin effects on rat lungs indicated increased cellular proliferation in the lungs with interstitial thickening 4 days after satin exposure (Pant et al., 1993). Signs of respiratory bronchiole damage, loss of alveolar spaces, and evidence of lung consolidation occurred 16 days after satin exposure. A typical combination therapy consisting of atropine, diazepam, and pralidoxime prevented these lung changes. There was significant interest in the toxic effects of trialkylphosphorothioates, contaminants formed during synthesis and storage of P=S phosphorothioate pesticides, because of their ability to produce pulmonary toxicity. Although these are classified as OPs (Clothier et al., 1981), these weak AChEIs do not produce cholinergic changes at doses causing visible lung pathology (Dinsdale, 1992). Trimethyl phosphorodithioate (OSSMeO), a representative of this class, was shown to produce selective type I alveolar pneumocyte damage in rats within 12 hr after oral administration of a lethal dose followed by consolidation of the lungs and alveolar edema (Dinsdale and Verschoyle, 1988). In addition, OSSMeO was shown to cause distortion of rat Clara cells of the bronchiolar epithelium. Similar changes in rat Clara cells, namely hypertrophy, distortion, and cell death, were demonstrated 24 hr after exposure to sublethal doses of OSSMeO (Imamura et al., 1983).
OP compounds affect important respiratory control centers in the brain stem. Although it was known early on that AChEIs cause death by respiratory failure (Modell et al., 1946; Freeman and Himwich, 1949), the majority of the actions were thought to be systemic-related effects on bronchi and respiratory muscles. Central nervous system effects of AChEIs on respiration were not considered a major mechanism until Douglas and DeCandole showed that anticholinesterases caused depression of the respiratory center (DeCandole and Douglas, 1949; Douglas, 1950). The first demonstration of decreased output from the respiratory center was shown by Krivoy and Marrazzi (Krivoy and Marrazzi, 1951; Krivoy et al., 1951). They showed that the output of the respiratory center, recorded as phrenic nerve potentials, was sensitive to the systemic effects of toxic DFP levels. It was also shown that recovery from this central respiratory depression could occur spontaneously or be induced with atropine. Further studies demonstrated similar findings in cats using TEPP (Douglas and Matthews, 1952) and in rabbits, cats, and monkeys using satin (Holmes, 1952, 1953).
VII. MECHANISM OF RESPIRATORY FAILURE FROM OP TOXICITY Although it was known that asphyxia from respiratory failure was the cause of death from OP intoxication, it was unclear which component (muscle paralysis, bronchoconstriction, or central respiratory drive) played a greater role. Respiratory failure was shown to be mostly due to failure of the central respiratory drive by Rickett and colleagues (Rickett, 1981; Ricket et al., 1986). They administered 1 LD50 of soman, satin, tabun, or VX every 15 min into a cat until the onset of respiratory arrest. Disruption of the normal firing pattern of the medullary respiratory-related neurons ensued first, followed by changes in phrenic nerve activity, diaphragmatic electromyogram, diaphragm contraction, and airflow. During respiratory arrest, the diaphragm muscle was still able to contract tetanically at 100 Hz for 500 msec, but the medullary respiratory-related units and the phrenic nerve stopped firing. An early technical report by DeCandole et al. (1953) showed similar findings of the importance of a central component in respiratory failure, but this depended on the species and the OP agent. DeCandole et al. investigated seven compounds against nine mammalian species and concluded that central respiratory failure predominated, but this depended on the species, the drug used, and the dosage administered. For example, central failure appeared to be the sole cause of respiratory arrest in monkeys. There were also differences between the two studies. Whereas the later study by Rickett et al. in cats showed the importance of central respiratory drive, the DeCandole report
CHAPTER 28 9Pulmonary Toxicity of AChEIs hinted at bronchoconstriction as the predominant feature occurring earliest in cats. Another study comparing the effects of bronchoconstriction in dogs and monkeys injected intravenously with satin (Johnson et al., 1958) supported the earlier findings (DeCandole et al., 1953) of weak bronchoconstriction in monkeys, but canine airway smooth muscle showed significant sensitivity to satin. In another study involving rabbits exposed to sarin, loss of central respiratory drive and neuromuscular block of diaphragmatic muscles were shown to be responsible for respiratory failure (Wright, 1954). From these studies, it can be concluded that central failure of respiration is most likely the predominant cause of death, which is aided by weakening of respiratory muscles and airway obstruction from increased secretions and bronchospasm. Furthermore, respiratory failure has been shown to precede significant cardiovascular depression (Wright, 1954; Rickett et al., 1986; Sidell, 1997), strengthening the importance of respiratory mechanisms as the primary cause of death.
VIII. T H E R A P E U T I C S T R A T E G I E S
FOR OP INTOXICATION OP nerve agent toxicity is due to their irreversible inhibition of the enzyme ACHE, present at all known cholinergic synapses (Taylor, 1996). AChE limits the duration of the activity of ACh and thus prevents its accumulation at synaptic junctions. Inhibition of AChE results in excessive stimulation of cholinergic synapses, which leads to bronchoconstriction, laryngospasm, muscle weakness, convulsion, and death (Ho and Hoskins, 1987). The standard U.S. military therapy for intoxication by OP compounds consists of administering atropine to antagonize excessive muscarinic stimulation and 2-PAM to reactivate the inhibited ACHE. For the nerve agents that undergo rapid aging, such as soman, 2-PAM is inadequate since AChE becomes resistant to reactivation within several minutes of exposure (Berman and Decker, 1986). In this case, the only practical strategy is to protect a critical pool of AChE from irreversible inhibition by pretreatment with the CM pyridostigmine bromide (PB) (Gordon et al., 1978; French et al., 1979). One study showed that when used as a pretreatment against the nerve agent soman, PB could rescue primates from respiratory failure and death (Kluwe et al., 1987). Low doses of PB have been shown to be effective in protecting against soman toxicity when combined with atropine and 2-PAM (Caldwell et al., 1989; Dawson, 1994; Marino et al., 1998). Since pyridostigmine does not enter the blood-brain barrier, peripheral protection of diaphragmatic muscle function and airway musculature is paramount to its mechanism. Although pretreatment with PB followed by treatment with atropine and an oxime represents a rational strategy for protection against soman exposure, newer pretreatment candidates for central
395
protection against OP toxicity would be useful therapeutic adjuncts.
IX. ORGANOPHOSPHATE USE AND ASTHMA Epidemiological studies have linked OP exposure to wheezing and symptoms related to a hyperactive airway (Deschamps et al., 1994; O'Malley, 1997; Salam, 2004). The establishment of this link is interesting because asthma prevalence has been increasing in the United States. OP insecticide use has increased in agrarian communities (Fenske et al., 2002; Koch et al., 2002) as well as urban populations (Lu et al., 2001; Berkowitz et al., 2003). Therefore, it is not surprising that the largest increase in asthma prevalence has occurred among youth in nonrural populations (Hartert and Peebles, 2000). The proposed mechanism of action to explain OP insecticide effects on asthma has been AChE inhibition, leading to excess ACh, resulting in activation of M3 muscarinic receptors on airway smooth muscle and subsequent bronchoconstriction (Roffel et al., 1990, 1994; Coulson and Fryer, 2003). Studies indicate that the OP insecticide chlorpyrifos induces airway hyperreactivity by a different mechanism in guinea pigs (Fryer et al., 2004; Lein and Fryer, 2005). It was shown that the OP insecticide chlorpyrifos could induce bronchoconstriction in guinea pig airways, and this was independent of AChE inhibition. Autoinhibitory M2 muscarinic receptors on parasympathetic nerves supplying airway smooth muscle prevent airway hyperreactivity (Minette and Barnes, 1988), but chlorpyrifos, parathion, and diazinon inhibit this M2 receptor population.
X. C O N C L U S I O N S AChEIs are toxic to multiple organ systems, but the main cause of death is through pulmonary toxicity. OP nerve agents cause toxicity to multiple aspects of breathing, including inhibition of central respiratory drive, constriction of airway smooth muscle leading to bronchospasm and bronchoconstriction, increased airway secretions, and neuromuscular block of diaphragmatic and intercostal muscles. Airway constriction, bronchospasm, and increased airway and nasal secretions compound the dyspnea experienced by the patient, but they can indicate early signs of mild OP exposure. Although death by asphyxiation is predominantly due to central respiratory failure, fatigue and flaccid paralysis of muscles responsible for expanding the chest wall in order to inflate the lungs are contributors to respiratory arrest, leading to hypoxemia, convulsions, brain damage, and death.
396
SECTION I V . Organ Toxicity
References Adler, M., Moore, D. H., and Filbert, M. G. (1992). Effects of anticholinesterases on airway smooth muscle. In Clinical & Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 149-155. ButterworthHeinemann, Oxford. Adrian, E. D., Feldberg, W., and Kilby, B. A. (1947). The cholinesterase inhibitory action of flurophosphonates. Br. J. Pharmacol. 2, 56-58. Berkowitz, G. S., Obel, J., Deych, E., Lapinski, R., Godbold, J., Liu, Z., Landrigan, P. J., and Wolff, M. S. (2003). Exposure to indoor pesticides during pregnancy in a multiethnic, urban cohort. Environ. Health Perspect. 111, 79-84. Berman, H. A., and Decker, M. M. (1986). Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography. J. Biol. Chem. 261, 10646-10652. Bowers, M. B., Goodman, E., and Sim, V. M. (1964). Some behavioral changes in man following anticholinesterase administration. J. Nervous Mental Dis. 138, 383-389. Caldwell, R. W., Lowensohn, H. S', Chryssanthis, M. A., and Nash, C. B. (1989). Interactions of pyridostigmine with cardiopulmonary systems and their relationships to plasma cholinesterase activity. Fundam. Appl. Toxicol. 12, 432-441. Clements, J. A., Moore, J. C., Johnson, R. P., and Lynott, J. (1952). Observations on airway resistance in men given low doses of GB by chamber exposure, MLR Report No. 122. Medical Laboratories Research, Edgewood Arsenal, MD. Clothier, B., Johnson, M. K., and Reiner, E. (1981). Interaction of some trialklphosphorothiolates with acetylcholinesterase. Characterisation of inhibition, aging and reactivation. Biochim. Biohys. Acta 660, 306-316. Coulson, E R., and Fryer, A. D. (2003). Muscarinic acetylcholine receptors and airway diseases. Pharmacol. Ther. 98, 59-69. Craig, A. B., Jr., and Comblath, M. (1953). Further clinical observations on workers accidentally exposed to "G" agents, MLR Report No. 234. Medical Laboratories Research, Edgewood Arsenal, MD. Craig, A. B., Jr., and Freeman, G. (1953). Clinical observations on workers accidentally exposed to "G" agents, MLR Report No. 154. Medical Laboratories Research, Edgewood Arsenal, MD. Craig, E N., Cummings, E. G., and Sim, V. M. (1977). Environmental temperature and the percutaneous absorption of a cholinesterase inhibitor, VX. J. Invest. Dermatol. 68, 357-361. Dahl, A. R., and Bechtold, W. E. (1985). Deposition and clearance of a water-reactive vapor, methylphosphonic difluoride (difluoro), inhaled by rats. Toxicol. Appl. Pharmacol. 81, 58-66. Dawson, R. M. (1994). Review of oximes available for treatment of nerve agent poisoning. J. Appl. Toxicol. 14, 317-331. DeCandole, C. A., and Douglas, W. W. (1949). The mechanism of respiratory arrest after GB poisoning. Porton Technical Paper No. 149. DeCandole, C. A., Douglas, W. W., and Evans, C. L. (1953). The failure of respiration in death by anticholinesterase poisoning. Br. J. Pharmacol. Chemother. 8, 466-475. Deschamps, D., Questel, E, Baud, E J., Gervais, P., and Dally, S. (1994). Persistent asthma after acute inhalation of organophosphate insecticide. Lancet 344, 1712.
Diggle, W. M., and Gage, J. C. (1951). Cholinesterase inhibition in vitro by O,O-diethyl O-p-nitrophenyl thiophosphate (parathion, E 605). Biochem. J. 49, 491-494. Dinsdale, D. (1992). Pulmonary toxicity of anticholinesterases. In Clinical & Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.), pp. 156-166. Butterworth-Heinemann, Oxford. Dinsdale, D., and Verschoyle, R. D. (1988). Comparative toxicity of two trialkylphosphorothioates to rat lung and the effects of atropine sulphate pretreatment. Arch. Toxicol. Suppl. 12, 432-434. Douglas, W. W. (1950). An effect of atropine in GB poisoning in the cat. Porton Technical Paper No. 195. Douglas, W. W., and Matthews, P. B. C. (1952). Acute TEPP poisoning in cats and its modifications by atropine and hyoscine. J. Physiol. 116, 202. DuBois, K. P., Doull, J., Salerno, P. R., and Coon, J. M. (1949). Studies on the toxicity and mechanisms of action of p-nitrophenyl diethyl thionosphosphate (parathion). J. Pharmacol. Exp. Ther. 95, 79-91. Fenske, R. A., Lu, C., Barr, D., and Needham, L. (2002). Children's exposure to chlorpyrifos and parathion in an agricultural community in central Washington State. Environ. Health Perspect. 110, 549-553. Freeman, A. M., and Himwich, H. E. (1949). DFP: Site of injection and variation in response. Am. J. Physiol. 156, 125. Freeman, G., Hilton, K. C., and Brown, E. S. (1956). V poisoning in man. CWL Report No. 2025. Chemical Warfare Laboratories, Edgewood Arsenal, MD. French, M. C., Wetherell, J. R., and White, P. D. (1979). The reversal by pyridostigmine of neuromuscular block produced by soman. J. Pharm. Pharmacol. 31,290-294. Fryer, A. D., Lein, P. J., Howard, A. S., Yost, B. L., Beckles, R. A1, and Jett, D. A. (2004). Mechanisms of organophosphate insecticide-induced airway hyperreactivity. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, L963-L969. Gage, J. C. (1953). A cholinesterase inhibition derived from O, O-diethyl O-p-nitrophenyl thiophosphate in vivo. Biochem. J. 54, 426-430. Gordon, J. J., Leadbeater, L., and Maidment, M. P. (1978). The protection of animals against organophosphate poisoning by pretreatment with a carbamate. Toxicol. Appl. PharmacoL 43, 207-216. Grob, D., and Harvey, A. M. (1953). The effects and treatment of nerve gas poisoning. Am. J. Med. 14, 52-63. Grob, D., and Harvey, J. C. (1958). Effects in man of the anticholinesterase compound satin (isopropyl methyl phosphonofluoridate). J. Clin. Invest. 37, 350-368. Grob, D., Lilienthal, J. L., Harvey, A. M., and Jones, B. E (1947). The administration of di-isopropyl fluorophosphates (DFP) to man. I. Effect on plasma and erythrocyte cholinesterase; General systemic effects; Use in study of hepatic function and erythropoiesis; And some properties of plasma cholinesterase. Bull. Johns Hopkins Hosp. 81, 217-244. Guyton, A. C., and Hall, J. E. (2005). Textbook of Medical Physiology, 1 lth ed. Saunders, Philadelphia. Harris, R., and Paxman, J. (1982). A Higher Form of Killing. Hill & Wang, New York. Hartert, T. V., and Peebles, R. S., Jr. (2000). Epidemiology of asthma: The year in review. Curr. Opin. Pulm. Med. 6, 4-9. Ho, I. K., and Hoskins, B. (1987). Biological and pharmacological aspects of neurotoxicity from and tolerance to organophosphorus cholinesterase inhibitors. In Handbook of
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Toxicology (T. J. Haley and W. O. Bemdt, Eds.), pp. 44-73. Hemisphere, Washington, DC. Holmes, R. (1952). The mechanism of respiratory failure in the rabbit poisoned with GB. Porton Technical Paper No. 275. Holmes, R. (1953). The cause of death from acute anticholinesterase poisoning in the rabbit, cat and monkey. Porton Technical Paper No. 356. Holmstedt, B. (1963). Structure-activity relationships of the organophosphorus anticholinesterase agents. In Cholinesterases and Anticholinesterase Agents (G. B. Koelle, Ed.). SpringerVerlag, Berlin. Imamura, T., Gandy, J., and Fukuto, T. R. (1983). An impurity of malathion alters the morphology of rat lung bronchiolar epithelium. Toxicology 26, 73-79. Johns, R. J. (1952). The effects of low concentrations of GB on the human eye, MRL Report No. 100. Medical Research Laboratory, Edgewood Arsenal, MD. Johnson, R. E, Gold, A. J., and Freeman, G. (1958). Comparative lung-airway resistance and cardiovascular effects in dogs and monkeys following parathion and sarin intoxication. Am. J. Physiol. 192, 581-584. Kluwe, W. M., Chinn, J. C., Feder, E, Olson, C., and Joiner, R. (1987). Efficacy of pyridostigmine pretreatment against acute soman intoxication in a primate model. In Proceedings of the Sixth Medical Chemical Defense Bioscience Review, Report No. AD B121516, pp. 226-234. U.S. Army Medical Research Institute for Chemical Defense, Aberdeen Proving Ground, MD. Koch, D., Lu, C., Fisker-Andersen, J., Jolley, L., and Fenske, R. A. (2002). Temporal association of children's pesticide exposure and agricultural spraying: Report of a longitudinal biological monitoring study. Environ. Health Perspect. 110, 829-833. Krivoy, W. A., and Marrazzi, A. S. (1951). Evaluation of the central action of anticholinesterases in producing respiratory paralysis. Fed. Proc. 10, 316. Krivoy, W. A., Hart, E. R., and Marrazzi, A. S. (1951). Further analysis of the actions of DFP and curare on the respiratory center. J. Pharmacol. Exp. Ther. 103, 351. Krop, S., and Kunkel, A. M. (1954). Observations on pharmacology of the anticholinesterases sarin and tabun. Proc. Soc. Exp. Biol. Med. 86, 530-533. Lein, E J., and Fryer, A. D. (2005). Organophosphorus insecticides induce airway hyperreactivity by decreasing neuronal M2 muscarinic receptor function independent of acetylcholinesterase inhibition. Toxicol. Sci. 83, 166-176. Lu, C., Knutson, D. E., Fisker-Andersen, J., and Fenske, R. A. (2001). Biological monitoring survey of organophosphorus pesticide exposure among preschool children in the Seattle metropolitan area. Environ. Health Perspect. 109, 299-303. Lubash, G. D., and Clark, B. J. (1960). Some metabolic studies in humans following percutaneous exposure to VX, CRDL Report No. 3003. Chemical Research and Development Laboratory, Edgewood Arsenal, MD. Marino, M. T., Schuster, B. G., Brueckner, R. E, Lin, E., Kaminskis, A., and Lasseter, K. C. (1998). Population pharmacokinetics and pharmacodynamics of pyridostigmine bromide for prophylaxis against nerve agents in humans. J. Clin. Pharmacol. 38, 227-235. Marts, T. C., Maynard, R. L., and Sidell, E R. (1996). Chemical Warfare Agents, Toxicology and Treatment. Wiley, New York. Minette, P. A., and Barnes, P. J. (1988). Prejunctional inhibitory muscarinic receptors on cholinergic nerves in
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CHAPTER ~ 9
Approaches to Defining and Evaluating the Inhalation Pharmacology and Toxicology Hazards of Anticholinesterases HARRY SALEM l A N D BRYAN BALLANTYNE z 1U.S. Army Chemical and Biological Center, Aberdeen Proving Ground, Maryland 2Charleston, West Virginia
I. I N T R O D U C T I O N
physically in a liquid state of variable vapor pressure and thus present differing vapor inhalation hazards (Table 1). When anti-ChEs are generated as dusts or liquid aerosols, the degree of respiratory tract exposure (total dose, depth of penetration, and distribution) will depend mainly on the particle size. The effects of inhaled anti-ChEs may, for descriptive purposes, be considered as local pharmacological effects on tissues within the respiratory tract, local toxicological effects on the respiratory tract, and systemic pharmacological and toxic effects that occur following absorption of anti-ChE into the systemic circulation. The nature of toxic effects also depends on whether exposure is by a single (acute) exposure or by repeated exposures. The acute lethal toxicity of anti-ChEs by inhalation exposure is usually the result of a combination of both local anti-ChE effects on the respiratory tract and systemic effects from absorbed anti-ChE. Acute lethal toxicity can be numerically expressed as either timed LCs0 (i.e., the concentration of material in the exposure atmosphere, calculated from the analytically measured exposure concentration-mortality data, that will be lethal to 50% of the species exposed for a set exposure time; e.g., mg m -3 for an exposure of x hours) or as the inhalation exposure dose (concentration • exposure time; CT), which is lethal to 50% of the exposed species, the L(CT)50. This is expressed as the product of exposure time and concentration (e.g., mg min m-3). The former method of citing lethality data is preferred, providing the exposure time is kept constant for the various exposure concentrations, since it gives a directly useable value for lethal hazard evaluation and permits a ready comparison between different materials. In the case of L(CT)50 values, however, this does not
The extensive use of anticholinesterases (anti-ChEs) as pesticides and their application in the form of dusts, land-based sprays, and by aerial spraying means that anti-ChE pesticide exposures by inhalation of the vapor and/or aerosols are frequent. Additionally, inhalation of vapor may be encountered in the intended lethal use of organophosphates (OPs) in chemical warfare operations and terrorist situations (Petrolanu et al., 2005). Inhalation studies are designed to determine (1) the nature, onset, and duration of adverse effects and hazards from acute, short-term repeated and long-term repeated respiratory exposure; (2) factors influencing the development of effects; and (3) the efficacy of antidotal treatment. They are also designed to aid in the development of protective and precautionary measures, including recommendations on appropriate airborne concentrations to ensure safe working conditions, such as Threshold Limit Values (American Conference of Governmental Industrial Hygiene, 2005) and Acute Emergency Guideline Levels (Hartmann, 2002). The development of pharmacological and potential short- and long-term toxicological effects from exposure to OP and carbamate (CM) anti-ChEs by the inhalation route of exposure depends on various factors, of which the most important a r e the physicochemical properties of the anti-ChE, whether exposure is to undiluted material or a formulation, and whether the material is physically and intentionally dispersed in the atmosphere for in-use application. Depending on the nature and required use of the material and its formulation, exposure by the inhalation route may be to vapor, liquid aerosol, or dust. Most neat CMs are solids of very low vapor pressure and inhalation hazards are generally low. The majority of OPs are Toxicology of Organophosphate and Carbamate Compounds
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SECTION IV. Organ Toxicity TABLE 1.
Representative Physical Properties of Anti-ChEs Relevant to Assessing Vapor Inhalation Hazards a
Anti-ChE Carbamate Aldicarb Carbaryl Carbofuran Methomyl Propoxur Organophosphate DFP Diazinon Dichlorvos Fenamiphos Malathion Parathion
Physical state
Vapor pressure (mmHg)
X X • x •
10 -5 10 -3 10 -5 10 -5 10 -6
(25 (26 (33 (25 (20
Water solubility (g dl-1)
Solid Solid Solid Solid Solid
9.8 5.0 2.0 5.0 6.5
~ ~ ~ ~ ~
0.6 (25 ~ 0.012 (30 ~ 0.07 (25 ~ 5.8 0.2 (25 ~
Liquid Liquid Liquid Solid Liquid Liquid
0.6 (20 ~ 1.4 • 10 -4 (20 ~ 1.2 x 10 -2 (20 ~ 7.5 X 10 -7 (30 ~ 4 x 10 -5 (30 ~ 3.8 X 10 -5 (20 ~
1.5 (25 ~ 0.004 (20 ~ 1.0 0.04 0.014 0.002
aData from Dodd (1992) and the World Health Organization (1986).
give a direct index of hazard for a given exposure time. The value cited could result from a variety of combinations of reciprocally related exposure times and concentrations (i.e., high concentration with short exposure time vs lower concentration with longer exposure period). Clearly, arithmetic manipulation of lethal inhalation exposure values could result in the calculation of a value for lethal concentration that in practice is too low to cause lethality at the corresponding exposure period to give the cited L(CT)50 value. Some have assumed that the inhalation exposure dosage (CT) for a given toxic end point is a constant that holds for a wide range of the reciprocal concentration values leading to this supposedly constant value. This is a gross misuse of the data resulting from a misunderstanding of factors governing toxic responses. For example, whereas in a limited time frame (usually a short time span) the constancy of CT may hold for local respiratory injury, outside this short time frame the relationship often does not hold for even direct injury to the respiratory tract. The inapplicability of CT as a constant is seen most obviously with those inhaled materials that become absorbed into the systemic circulation and then exert systemic toxicity. In such cases, toxicity will depend on the interaction of a variety of processes, which include the mechanism of uptake from the respiratory tract, tissue biodistribution, metabolism (metabolic activation/detoxification), the mechanism of toxicity in target organs, and elimination. Many of these processes are efficient at low concentrations but become saturated at higher concentrations, resulting in nonlinearity among concentration, exposure time, and target organ toxic effect (Hext, 1999). This is seen most obviously with the lethal toxicity of hydrogen cyanide vapor exerting its effects by absorption of inhaled material and subsequent systemic
toxicity (Ballantyne, 1994; Ballantyne and Salem, 2005). The variability in the inapplicability of L(CT)50 data across a wide range of conditions can be seen from some of the materials shown in Table 2. For example, with fensulfothion for both 1- and 4-hr exposures, the L(CT)50 values are within a close range of 113-118 m g h r m - 3 ; this accords
TABLE 2. Examples of Timed LC5o Values and Corresponding L(CI')5o Values for Rats Exposed to Several Organophosphates ~
Anti-ChE Carbamate Methomyl Propoxur Organophosphate DFP Diazinon Dichlorvos Fenamiphos Fensulfothion Mevinphos Parathion Satin Soman Tabun
LCs0 (mg -3)
Time
L(CT)50
(hr)
(mg hr m -3)
300 1440
4 1
1200 1440
360 3500 15 110-175 91-100 113 29.5 128 84 10.6 21 304
0.16 4 4 1 4 1 4 1 4 0.5 0.32 0.16
60 1400 600 110-175 364-400 113 118 128 336 5.3 6.7 51
aData from Dodd (1992) and the World Health Organization (1986).
CHAPTER 29 9Inhalation Toxicity of Anti-ChEs with the 1-hr LC50 being approximately four times greater than the 4-hr LCs0. However, with fenamiphos the 1-hr L(CT)50 values range from 110 to 175 mg hr m -3, whereas the 4-hr L(CT)50 values range from 364 to 400 mg hr m-3; thus, prediction of the 1-hr LC50 from the 4-hr data and vice versa would not be accurate and reliable.
II. F A C T O R S I N F L U E N C I N G T H E INHALATION AND RESPIRATORY TRACT DISTRIBUTION OF MATERIALS
A. Vapors The degree of respiratory tract exposure to vapors depends on a variety of factors, including vapor pressure of the material, vapor concentration in inspired air, the duration of exposure, breathing rate, and respiratory minute volume. Materials with peripheral sensory irritant properties will cause a decrease in breathing rate that will influence respiratory minute volume and hence inspired dose (Ballantyne, 1999, 2005). Differential distribution in the respiratory tract is largely dependent on the water solubility of the material. Substances of high water solubility may be absorbed in the upper respiratory tract and very little material may reach the deeper lung tissue. On the other hand, materials of low water solubility may reach the deep lung. Thus, the concentration of inspired material and its relative water solubility may affect the concentration gradient of material between the nasal cavity and alveolus and, therefore, the differential distribution and consequent local effects within the respiratory tract. For mixed inspired atmospheres (vapor and particulate), vapor may be adsorbed on the particles and the deposition pattern will then be determined by the influence of particle deposition. As discussed in detail later, the distribution of metabolizing capability within the respiratory tract may determine regional toxicity and uptake within the tract.
B. Aerosols An aerosol is a two-phase system consisting of finely divided particulate material suspended in a gaseous phase. The condensed particulate material may exist as a liquid, a solid, or a combination of the two. The most significant factor that determines the depth of penetration in the lung and the differential deposition of the material in the respiratory tract is particle size; this does not refer to physical size of the particles but, rather, to aerodynamic size. The aerodynamic diameter of a particle is defined as the diameter of a sphere of unit density having the same settling velocity as the particle being considered. The relationship between aerodynamic diameter and physical diameter is broadly covered by the following equation (Hext, 1999): Aerodynamic diameter = physical diameter • (density) ~
401
Thus, for example, a particle of 1 lxm physical diameter and density 4 g cm -3 will have an aerodynamic behavior in air similar to a unit density particle 2 txm in diameter. There may be species variations in the relation between the degree of penetration and differential deposition (Raabe et al., 1977; Schlesinger, 1985), which are related to factors such as differences in the regional anatomy and physiology (notably airflow pattern), of the respiratory tract. Thus, in the rat, an obligate nose breather with a complex turbinate system, there will be filtration of fine particles that would be expected to reach the alveolar region in man. In relation to airflow pattern, this is a function of volume and crosssectional diameter of the airway. As particles suspended in air pass through the dichotomous branching of the airway passages, the velocity of particles will decrease since the same volume of inspired air is passing through increasing surface area. This decrease in velocity continues as penetration becomes deeper in the respiratory system. Inhaled particles deposit in the respiratory tract by four processes: impaction, gravitational settlement, Brownian diffusion, and interception. Impaction occurs where there is a change in the direction of the airstream and is associated with both velocity and directional change. It thus occurs predominantly in the upper respiratory tract. Particles with aerodynamic size >0.5 Ixm may deposit by impaction. Gravitational settlement occurs principally in the lower respiratory tract, where velocities are low. Brownian diffusion occurs with particles 500.0
aData from Bellet and Casida (1973).
TABLE 6. Acute Lethal Inhalation Toxicity of Aerosols of Trimethyl Propane Phosphate and Trimethyl Propane Phosphite to Male and Female Rats
1-hr LCs0 (mg liter -1) with 95 % confidence limits Gender
Phosphate
Phosphite
Male Female
0.037 (0.033-0.040) 0.030 (0.027-0.034)
0.015 (0.013-0.017) 0.015 (0.013-0.017)
aData from Kimmerleet al. (1986).
polyurethane foams and some rigid polyurethane foams, but other rigid foams containing trimethylol propane polyols gave significant yields. The maximum yield was 0.2% by mass when the foam was decomposed at 500 ~ under nonflaming conditions in air. It was calculated that the equivalent concentration in air would be 0.12-0.06 mg liter-1. The 1-hr LCs0 for TMPP has been experimentally determined to be 0.03-0.07 mg liter -1 (Kimmerle et al., 1986). It was concluded that along with other toxic materials, such as CO and HCN, in the atmosphere, the TMPP yield could make major contributions to the combustion toxicology of polyurethane foams (Purser, 1992).
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Verschoyle, R. D., and Aldridge, W. N. (1987). The interaction between phosphorothionate insecticides, pneumotoxic trialkyl phosphorothiolates and effects on lung 7-ethoxycoumarin O-deethylase activity. Arch. Toxicol. 60, 311-318. Verschoyle, R. D., and Cabral, J. R. P. (1982). Investigation of the acute toxicity of some trimethyl and triethyl phosphorothioates with particular reference to those causing lung damage. Arch. Toxicol. 51, 221-231. Verschoyle, R. D., and Dinsdale, D. (1989). Protection against chemical-induced lung injury by inhibition of pulmonary cytochrome P-450. Environ. Health Perspect. 85, 95-100. Verschoyle, R. D., Aldridge, W. N., and Cabral, J. P. (1980). Toxicology of trimethyl and triethyl phosporothioates. In Mechanisms of Toxicity and Hazard Evaluation (B. Holmstedt, R. Lauwerys, and R. Mercier, Eds.), pp. 631-634. NorthHolland, Amsterdam. Vincent, J. H. (1995). Aerosol Science for Industrial Hygienists. Pergamon, Oxford. Warheit, D. B., Kelly, D. P., Carakostas, M. C., and Singer, A. W. (1989). A 90-day inhalation toxicity study with benomyl in rats. Fundam. Appl. Toxicol. 12, 333-345. Wedin, G. (1992). Anticholinesterase nephrotoxicity. In Clinical and Experimental Toxicology of Organophosphates and Carbamates (B. Ballantyne and T. C. Marrs, Eds.). Butterworth-Heineman, Oxford. Woolley, W. D., and Fardell, J. P. (1976). Formation of a highly toxic organophosphorus product (TMPP) during the decomposition of certain polyurethane foams under laboratory conditions, Borehamwood Fire Research Note No. 10060. U.K. Fire Research Station, Borehamwood, UK. World Health Organization (1986). Carbamate Pesticides. A General Introduction, Environmental Health Criteria No. 64. World Health Organization, Geneva. Wyman, J. E, Povaznik, M., and Serve, P. (1987). High temperature decomposition of military specification L-23699 synthetic aircraft lubricants. J. Fire Sci. 5, 162-177.
CHAPTER ~ 0
Dermal Absorption/Toxicity of Organophosphates and Carbamates JIM E. RIVIERE North Carolina State University, Raleigh, North Carolina
provide the only available human data with which to perform dermal absorption assessment. Dermal toxicity of pesticides is thus usually synonymous with dermal absorption since the critical adverse effect is classical OP systemic neurotoxicity (e.g., cholinesterase inhibition). The major mechanism of direct pesticide toxicity to skin is generally related to immunological mechanisms, either directly with skin immune components after topical absorption or indirectly after systemic administratio n .
I. INTRODUCTION Topical exposure to organophosphate (OP) and carbamate (CM) pesticides remains an important route for exposure to humans and animals. As in many subdisciplines in toxicology and other sciences, interpretation of pesticide dermal absorption data is highly dependent on the model systems used to generate the data. This chapter discusses studies of pesticide absorption in the context of the experimental and theoretical approaches used to assess absorption. This field was reviewed by Baynes and Riviere (2001), which should be consulted for further details. This review highlighted the overarching importance of experimental design and subsequent interpretation of pesticide data reported in the literature since these factors often overshadow differences in absorption and subsequent toxicity between individual pesticides. Topical pesticide exposure remains an important health issue. Epidemics of pesticide poisoning following cutaneous exposure have also been reported for nonoccupational uses (Ferrer and Cabral, 1993). These cases often involved accidental contamination of infant clothing or talcum powder with pesticides (Martin-Bouyer et al., 1983). Large-scale residential exposure to methyl parathion with resulting OP-induced toxicity occurred in 1994 and 1996 in Ohio, Illinois, and Mississippi (Rubin et al., 2002). Dermal exposure may occur secondary to processing of agricultural products such as tobacco (Lonsway et al., 1997). Fatal human pesticide exposures continue to occur (Rosenthal, 2003). Dermal absorption is the probable route of entry in 65-85% of all cases of occupational exposure to pesticides (Galli and Marinovich, 1987). Similarly, spray or dusting of pesticides can result in direct deposition of 20-1700 times the amount deposited in the respiratory tract after inhalational exposure, a loading that would potentiate absorption and direct irritation to the skin. Anecdotal case reports, coupled with dermal exposure estimated from various direct and indirect dosimetric experiments in outbreaks, often Toxicology of Organophosphate and Carbamate Compounds
II. M E C H A N I S M OF ABSORPTION The skin is composed of two primary layers--the epidermis, which includes the outermost stratum corneum barrier and underlying viable keratinocytes, and the dermis (MonteiroRiviere, 1991). Skin is relatively impermeable to most aqueous solutions and ions; however, it may be permeable in varying degrees to a large number of more lipophilic drugs or xenobiotics such as pesticides. The stratum corneum cell layer in humans (10-50 lxm) and pigs (15 ~zm) is nonviable and is considered the rate-limiting barrier in percutaneous absorption of many drugs and pesticides (Monteiro-Riviere, 1991). It is axiomatic that a topically applied chemical must first traverse the stratum corneum barrier before it is capable of eliciting any toxicological or immunological effect on subsequent cell layers, making absorption the primary factor in assessing the dermal effects of pesticides. Chemical absorption pathways can hypothetically involve both intercellular and intracellular passive diffusion across the epidermis and dermis and/or transappendageal routes via hair follicles and sweat pores. Transappendageal pathways are considered to contribute very little to the dermal transport of most drugs compared to transport across the epidermis (Barry, 1991). Most available research has concentrated on the stratum corneum as the primary barrier to absorption, although the viable epidermis (80 txm 411
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in humans and 60 txm in pigs) and dermis (3-5 mm in humans) may also contribute significantly to the percutaneous penetration of specific chemical classes, for example, when the true barrier to absorption is not diffusional but, rather, metabolic. The term barrier is thus often used to denote either a physical structure (e.g., stratum corneum) or a biological process (e.g., diffusional resistance, metabolism, and vascular uptake) that retards absorption of topically applied chemicals. The accepted hypothesis for dermal absorption is that the dominant pathway for chemicals, including pesticides, to traverse the stratum corneum is through the intercellular lipids. Lipophilic compounds diffuse through this lipid milieu while polar molecules traverse the aqueous region of the intercellular lipids. This intercellular region, described as the mortar in the "brick and mortar" model of the stratum corneum (Elias, 1981), is considered the most likely path for absorption of lipophilic drugs. Although this model is conceptually simple, the actual physical chemical environment of the intercellular lipids is complex. It is filled with neutral lipids (complex hydrocarbons, free sterols, sterol esters, free fatty acids, and triglycerides) that make up 75% of the total lipids, as well as other polar lipids (Magee, 1991; Monteiro-Riviere et al., 2001). These intercellular lipids are also inextricably linked to the outer cellular membranes of the corneocytes, making a relatively complex and fluid structure that is often modeled as a simple homogeneous lipid pathway. Successive tape stripping, delipidization techniques, and use of epidermis from heat or chemical separation techniques have been used by investigators to demonstrate the dominant influence that the stratum corneum and the lipid domain holds on penetration of hydrophilic and lipophilic chemicals. Percutaneous absorption through the intercellular pathway of the stratum corneum is driven by passive diffusion down a concentration gradient described at steady state by Fick's law of diffusion (Roberts et al., 1999; Wester and Maibach, 1983; Riviere, 1999); Flux = [(D. PC. SA)/H] ( ~ ) where D is the diffusion coefficient, PC is the partition coefficient, SA is the applied surface area, H is membrane thickness (or, more precisely, the intercellular path length), and zXx is the concentration gradient across the membrane. Since in vivo blood or in vitro perfusate concentrations after absorption are negligible compared to applied surface concentration, 2tx reduces to the concentration (C). It is this relationship that allows the prediction of compound flux across the skin to be correlated to factors predictive of D and PC (e.g., octanol/water partition coefficients). Flux is expressed in terms of applied surface area, often normalized to cm 2. The t e r m ( D . PC//-/) is compound dependent and is termed the permeability coefficient (Kp), reducing the
determination of flux to Kp 20( or Kp. C, a first-order pharmacokinetic equation (dx/dt = kX). Rearrangement of this equation yields the primary method used to experimentally determine Kp: Kp = steady state flux/concentration It must be stressed that both transdermal flux and Kp are not only chemical dependent but also tightly constrained by the membrane system studied as well as the method of topical application (neat compound, vehicle, length of experiment, etc.). The PC that is integral to Kp is the PC between the surface or applied vehicle and the stratum corneum. Different vehicles will thus result in different PCs. Similarly, skin from different species may result in different PCs due to differences in the stratum corneum lipids and intercellular path lengths. Since passive diffusion is the primary driving force behind dermal absorption, physicochemical factors such as molecular weight and structure, lipophilicity, pKa, ionization, solubility, partition coefficients, and diffusivity can influence the dermal absorption of various classes of pesticides. In addition, penetration of acidic and basic pesticides will be influenced by the skin surface, which is weakly acidic (pH ~ 5), since only the uncharged moiety of weak acids and bases is capable of diffusing through the lipid pathway. Several of these factors (e.g., molecular weight and PCs) have been used to predict absorption of various drug classes (Potts and Guy, 1992; Cleek and Bunge, 1993; Bunge and Cleek, 1995). This approach has not been widely applied to pesticides. The first such relationship widely used to assess chemical absorption is that of Potts and Guy (1992): log
kp =
0.71 log
PCoctanol/wate r -
0.0061 MW - 6.3
(R 2 = 0.67) where MW is the molecular weight. This equation was subsequently modified (Potts and Guy, 1995) to relate kp to molecular properties of the penetrants as log
kp -
0.0256 MV - 1.72 ZoLH2 -- 3.93 Z~H2 -- 4.85
(R 2 = 0.94) where MV is molecular volume, ~ OLH2 is the hydrogen bond donor acidity, and ~ H 2 is the hydrogen bond acceptor basicity. The most promising approach is to further extend this rationale using linear free energy relationships (LFERs) to relate permeability to the physical properties of the penetrant under defined experimental conditions (dose, membrane selection, and vehicle). Geinoz et al. (2004) critically reviewed most such quantitative structure permeability
CHAPTER 30 9Dermal Toxicity of OPs and CMs relationships (QSPeR) applied to dermal absorption. Abraham's LFER model is representative of the dermal QSPeR approaches currently available (Abraham and Martins, 2004). This model was selected since it is broadly accepted by the scientific community as being descriptive of the key molecular/physiochemical parameters relevant to solute absorption across skin. This basic model can be written as log kp = c + a~aH2 + b~13H2 + savH2 + rR2 + vV~ where 'rrH2 is the dipolarity/polarizability, R 2 represents the excess molar refractivity, Vx is the McGowan volume, and the other parameters are as described previously. The variables c, a, b, s, r, and v are strength coefficients coupling the molecular descriptors to skin permeability in the specific experimental system studied. In order to incorporate mixture effects, our laboratory has been exploring the use of an additional term operationally called the mixture factor (MF), yielding log kp : c + mMF + a~]aH2 + bE[3H2 + s~rH2+ rR2 + vV~ The nature of the MF is determined by examining the residual plot (actual - predicted log kp) generated from the base LFER equation based on molecular descriptors of the permeants against a function of the physical chemical properties of the mixture/solvents in which they were dosed (Riviere, 2006; Riviere and Brooks, 2005). The literature on QSPeR is exhaustive and rapidly growing. The limitation of applying these approaches to pesticide absorption is the lack of large and comparable databases of pesticide dermal absorption, as well as the lack of availability of molecular descriptors for many pesticides. As discussed later, data suitable for large-scale analyses must be rigorously controlled relative to the species studied, the nature of the experiments (in vitro vs in vivo), dose, surface area, vehicle, and method of sample collection and analyses. When dermal absorption data are not analyzed using the models described previously, data are often expressed as percentage dose absorbed. This is conceptually correct if one assumes that permeability is unchanged across dose because it represents a first-order pharmacokinetic process. It is also appropriate when comparing experimental treatments (e.g., temperature and vehicle) using the same applied dose. However, in many cases, topical dosing results in applying substantial amounts of chemical compared to what can be absorbed across the skin. In soils, thick layering where most soil is not in contact with skin, and thus not able to reach its surface to partition into, suggests that most of an applied dose is not actually available for absorption. In such studies, only a monolayer of soil is actually in contact with the skin. Unlike in fluid or gel matrices, compound generally does not diffuse from soil
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layers not in contact with the skin, unless water is added as another vehicle. In these cases, accounting for the applied dose as total dose in a multilayer system overestimates the available dose that artificially reduces the calculated percentage dose absorbed. Caking of heavy dermal formulations results in a similar layering phenomenon. Similarly, dose may bind to the application device and not be available. In these cases, a large fraction of dose may not be thermodynamically driving the diffusion process. Finally, when the dose is applied in solution, saturation may result in precipitation of chemical. Rapid evaporation of a volatile vehicle may also precipitate pesticide, decreasing its availability for absorption. All of these factors lead to a phenomenon often seen in dermal absorption studies in which percentage dose absorbed decreases with applied dose. Conducting a study at high applied doses may underestimate absorption of lower applied doses and vice versa. Thus, comparing percentage dose absorbed across pesticides exposed at different doses often provides more information on applied dose than the nature of the individual pesticide absorption. Unfortunately, this phenomenon is repeatedly encountered in the pesticide examples discussed later and limits the value of information gained on individual pesticide behavior.
III. EXPERIMENTAL D E R M A L ABSORPTION MODELS Assessment of dermal absorption for any topically applied chemical, including pesticides, can be classified based either on a model's level of biological complexity (in silico, in vitro, and in vivo) or on the specific species studied (human, laboratory rodent, monkey, and pig). The goal of the research must also be taken into consideration: Is the work being conducted to study the mechanism of absorption (e.g., identify a specific mathematical model or assess the effect of a vehicle) or to quantitatively predict absorption in humans? Another perspective is whether the study is designed to examine a local effect in skin versus a systemic effect after absorption; in the first case, skin concentrations are important, whereas in the later case flux of chemical across skin is needed. These end points determine both the level of model used and the appropriate species to employ. Model systems and approaches in use today to assess dermal absorption have been reviewed (Riviere, 2005). The primary approach to assess dermal absorption is the in vitro diffusion cell. In this model, skin sections (full thickness, dermatomed to a specific thickness) are placed in a two-chambered diffusion cell in which receptor fluid is placed in a reservoir (static cells) or perfused through a receiving chamber (flow-through cells) to simulate cutaneous blood flow. Chemical may either be dosed under ambient conditions neat or dissolved in a vehicle (Franz and Bronaugh cells) or in water (side-by-side diffusion
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cells), resulting in finite versus infinite dosing conditions, respectively. Selection of the receptor fluid (e.g., saline and albumin-based media) is also critical since absorption is only detected if the penetrating compound is soluble in the receptor fluid. This is particularly important for many lipophilic pesticides, making studies ignoring this factor difficult to interpret. Many studies of pharmaceutical compounds use saline as the receptor fluid due to the hydrophilic nature of many drugs, a choice that falsely suggests minimal absorption for lipophilic pesticides since they are not soluble in the receptor fluid and thus cannot be detected as absorbed. Steady-state flux is measured in these models and permeability calculated using the previously discussed relationship. In addition to perfusate composition, the temperature of perfusate is also controlled, with pharmaceutical investigators suggesting that studies be conducted at 35~ to mimic the surface temperature of skin. These techniques have been exhaustively reviewed (Riviere, 2005; Bronaugh and Stewart, 1984, 1985). The second major approach used to assess dermal absorption is in vivo. Many pesticide absorption studies referenced in the literature are full mass balance studies in which compound is dosed on the surface of an animal and total excreta (urine, feces, and expired air) are collected and analyzed for parent pesticide or metabolites. Radiolabeled compounds are often employed in these studies. These data are usually expressed as percentage dose absorbed per unit of surface area exposed. This method is best adapted to laboratory rodent models. Dose may be applied occluded (evaporation of dose prevented) or nonoccluded (dose site open to ambient environment). In calculating absorbed dose, all chemical at the dose site must be segregated from other tissues that would reflect absorbed chemical. This usually involves gently washing nonabsorbed chemical with a soapy solution. When larger animals (e.g., pigs and primates) or humans are studied and total mass balance is not possible (e.g., cannot collect feces and expired air), the fraction of a systemically absorbed compound excreted in the urine must first be determined using parenteral dosing. In some classic studies, this parenteral route correction factor was conducted in monkeys (Feldmann and Maibach, 1974) under the assumption that systemic distribution, metabolism, and elimination of these pesticides are similar in man and primate. In pigs, separate parenteral injections have been made to determine fractional excretion by other routes (Carver and Riviere, 1989). Finally, for many pharmaceutical compounds, absorption may be assessed by determining the area under the curve of the plasma concentration-time profile, much as it is for determining bioavailability from oral routes of administration. There are several perfused skin preparations with an intact functional microvasculature. The perfused rabbit ear model, perfused pig ear model, in situ sandwich skin flap in athymic rats, and the hybrid rat-human sandwich flap have been developed (Pershing and Krueger, 1987), but each
intuitively has severe limitations. The isolated perfused porcine skin flap (IPPSF) is a unique ex vivo skin preparation developed in our laboratory that has many advantages over other ex vivo models and most in vitro systems. The major advantage of such a perfused system is that subsequent systemic influences on absorbed chemical are not present, but the tissue is fully functional with an intact microcirculation, unlike simpler in vitro models. In addition to having an intact functional cutaneous microcirculation, predictions from IPPSF studies have correlated well with in vivo absorption data for several drugs and insecticides (Riviere et al., 1986, 1995; Wester et al., 1998). IPPSFs are physiologically and biochemically viable and therefore can be used to assess cutaneous toxicity of topically applied chemicals (Monteiro-Riviere, 1993). The latter is most important because cutaneous toxicity as well as dermal absorption of various pesticide formulations can be assessed simultaneously. For some pesticides, in vivo and in vitro data are comparable. An excellent example is the dermal absorption of malathion (6.8% dose at 24 hr and 8.2% dose at 5 days) in human volunteers (Feldmann and Maibach, 1974; Maibach and Feldmann, 1974), which is comparable to in vitro absorption (8.77% dose at 24 hr) in human skin (Wester et al., 1996), in vivo absorption of malathion in Yorkshire pigs (5.2% at 6 days) (Carver and Riviere, 1989), as well as predicted IPPSF absorption (5.9%) (Williams et al., 1990). Recent approaches have concentrated on developing noninvasive methods for assessing exposure. These include using postdosing stratum corneum tape stripping (Rougier et al., 1985), in which after calibration to the classical in vivo models just presented, estimates of absorption or exposure can be obtained by searching for the test compound in tape strips obtained after washing nonabsorbed chemical from skin. This is also used by some workers to document whether exposure has occurred in a field situation (Nylander-French, 2000). The mechanistic basis that allows for this to be used is that a chemical diffusing through the skin must first partition into the stratum corneum. At equilibrium (approximately 30 min), the concentration in the stratum corneum tape strips is the driving concentration that determines how much absorption will occur. The assumption is made that chemical absorbed into the stratum corneum may be absorbed into the systemic circulation. The alternate noninvasive approach is to assay collected urine for parent pesticide or metabolites (e.g., p-nitrophenol for parathion) that would be indicative of systemic absorption. If timed urine collections are not employed, the observed values are often corrected by assaying for creatinine to compensate for altered urine volumes. There is general consensus that for most risk assessment needs, in vivo models are preferred since they most closely match the exposure scenario to be modeled. However, in vivo models are also the most variable due to the myriad
CHAPTER 30 9Dermal Toxicity of OPs and CMs points of potential interactions in the absorption process. Thus, studies designed to examine the mechanism of pesticide absorption tend to use in vitro models to reduce the level of biological complexity so that specific mechanistic hypotheses can be evaluated. However, it is often not clear if these findings are extrapolatable to in vivo exposures.
IV. SPECIES DIFFERENCES There is an important phenomenological component to selecting animal models that is related to what the studies are trying to predict: toxicity or a pharmacological effect. Absorption studies designed to assess systemic toxicity after topical application generally attempt to overestimate absorption, whereas those trying to predict pharmacological effect in humans tend to try to use a model with a closer correlation to transdermal flux in humans. If the goal is to compare relative absorption by rank order of permeability, then the use of specific models may not be as crucial as long as absorption is representative across species. As long as the same model system is used, this generally also holds for comparing specific treatment effects (e.g., vehicle and occlusion) on dermal absorption. A good example related to pesticides is the comparative absorption study conducted on mice (Shah et al., 1981), in which absorption across a wide variety of compounds was greater than 90% of applied dose. In contrast, absorption of similar pesticides in humans or pigs was generally well below 25% (Riviere, 1996). Paraquat and diquat are two compounds that provide insight into interspecies comparisons. They are hydrophilic pesticides with fixed charged cations and remain dissociated at all pH values. Very little paraquat or diquat would therefore be expected to be absorbed across the lipoidal stratum corneum barrier. However, percutaneous absorption of paraquat has resulted in systemic effects and deaths in humans (Smith, 1988). Studies have determined that the in vitro permeability constants for paraquat in various animal species (rat, hairless rat, nude rat, mouse, hairless mouse, rabbit, and guinea pig) are 40-1600 times greater than those in humans (Walker etal., 1983). Dermal absorption studies in human volunteers demonstrated approximately 0.25% absorption from multiple sites (Wester et al., 1984). One in vivo study on rats suggested that greater paraquat absorption (3.5%) was attributable to an occlusive dressing or due to differences in skin thickness between species (Chui et al., 1988). Like paraquat, very little diquat is absorbed (0.3%) in the human forearm in vivo (Maibach and Feldmann, 1974). Diquat absorption increased to 1.4% with occlusion and to 3.8% with damaged skin. Topical application of 3-, 24-, and 200-mg doses of paraquat to IPPSFs for 8 hr resulted in total penetration (skin deposition as well as absorption into perfusate) of 0.91, 1.09, and 0.50%, respectively (Srikrishna et al., 1992). These absorption data are comparable to the human in vivo data.
415
Despite the limited amounts absorbed, they were sufficient to cause morphological and biochemical changes in the IPPSFs. This finding clearly indicates the importance of the phenomenological dilemma introduced earlier, in which although minimal systemic absorption and thus systemic toxicity would be expected to occur, sufficient paraquat was absorbed across the stratum corneum into the epidermis/ dermis to illicit a local dermatotoxic effect. The potential for local toxicological effects is present for topical dosing of these pesticides. The use of laboratory animal models may lead to overprediction of pesticide absorption in human skin, as shown for paraquat. Differences in permeability properties between human and laboratory animal skin can account for this overestimation. Inherent structural differences in skin biology, such as skin thickness and sebaceous secretions, make species-species extrapolation of dermal absorption data tenuous. Although the fundamental architecture of terrestrial mammalian skin is similar, well-documented differences in epidermal and dermal anatomy and physiology exist (Monteiro-Riviere, 1991; Monteiro-Riviere et al., 1990). It is plausible that a high density of hair follicles attenuates the thickness of the interfollicular epidermis, which may promote absorption. Alternatively, species differences in the size or width of epidermal cells would modulate path length across species, a critical parameter in the value of the permeability constant for a specific species. Species differences in stratum corneum lipid composition may be the overriding factor in determining the rate and extent of absorption. The skin of the domestic pig is functionally and structurally similar to that of humans (Monteiro-Riviere, 1991), supporting the observation that percutaneous absorption of toxicants through pig's skin mimics that through human skin. Studies have demonstrated that the range of percutaneous absorption of carbaryl, lindane, malathion, and parathion in pig skin in vivo (Carver and Riviere, 1989) or in vitro (Chang et al., 1994) is similar to that observed in humans (Feldmann and Maibach, 1974). This supports the close correlation of absorption across the IPPSF compared to in vivo humans previously discussed (Riviere et al., 1986, 1995; Wester et al., 1998). However, this was not the case when the permeability of hydrophilic chemicals (mannitol, water, and paraquat) and lipophilic chemicals (carbaryl, aldrin, and fluazifop-butyl) in pig ear skin was compared with human abdominal and rat dorsal skin (Dick and Scott, 1992). This study demonstrated that for hydrophilic chemicals, pig ear and rat skin overestimated permeability in human skin. It should be noted that pig ear skin is different from that of other body sites, being both thinner and closely associated with the underlying ear cartilage. Alternatively, confounding effects associated with using an in vivo model rather than the ex vivo IPPSF may weaken the inherent correlation between human and pig skin.
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Although permeability is generally higher in animal skin than in human skin for the lipophilic chemicals, permeability of carbaryl in human and pig skin was almost identical. Permeability of lipophilic chemicals in pig skin correlated better with data from human skin compared to permeability of hydrophilic chemicals. Bartek et al. (1972) demonstrated good agreement between human in vivo and pig in vivo dermal absorption data for lipophilic chemicals, such as butter yellow, DDT, haloprogin, lindane, and testosterone, and hydrophilic chemicals, such as caffeine, acetylcysteine, and cortisone. Further studies on nonhuman primates observed that lindane absorption from rhesus monkey forearm (18%) was approximately twice that for the ventral forearm in humans (9.3%) (Feldmann and Maibach, 1974) and for the ventral abdomen in pigs (7.7%) (Chang et al., 1994). Although all of these studies are taken into consideration for the majority of moderately lipophilic pesticides studied, the rank order of absorption is generally rabbits > rodents > weanling pig -- rhesus monkey -> human.
V. F A C T O R S A F F E C T I N G A B S O R P T I O N An additional variable that must be accounted for in dermal absorption studies that may overshadow the difference between chemicals is body site differences in absorption within a species. Regional variation in skin permeability at different body sites may be related to skin thickness, number of cell layers, cell size of the epidermis and stratum corneum, and distribution of hair follicles and sweat pores. Because of thick layers of stratum corneum, permeability in palmar and plantar skin is expected to be less than that in the scalp or forearm (Feldmann and Maibach, 1974). Data from several studies suggest that regional variation in vascular anatomy and blood flow should also be considered (Monteiro-Riviere et al., 1990; Qiao et al., 1993). Various studies have demonstrated regional variation in penetration of drugs and pesticides in pig skin (Qiao et al., 1993; Qiao and Riviere, 1995), rat skin (Bronaugh, 1985), rhesus monkey skin (Wester et al., 1980), and human skin (Wester et al., 1980, 1984), clearly demonstrating that regional variability is not limited to a single species. Qiao et al. (1993) demonstrated that parathion penetrated nonoccluded pig skin in the following order: back > shoulder > buttocks > abdomen; however, for occluded skin, the order was back > abdomen > buttocks > shoulder. This demonstrates the importance of experimental conditions for interpreting even carefully controlled comparative studies. Occlusion could mechanistically change the nature of both the partition coefficient and the diffusivity of a compound in skin, which could result in different mechanisms becoming rate limiting. Wester et al. (1994) demonstrated that pyrethrin absorption through human forearm was less than the predicted absorption in human scalp. This anatomical difference is somewhat consistent with lindane absorption
through the forearm (18%), forehead (34%), and palm (34%) of rhesus monkeys (Moody and Ritter, 1989). This anatomical range for lindane was also similar to that for dermal absorption of DEET (diethyl-m-toluamide) in rhesus monkeys (Moody et al., 1989). There is also significant data to suggest that dermal absorption of permethrin, aminocarb, DEET, and fenitrothion in monkey forehead is twice that in monkey forearm (Moody and Franklin, 1987; Moody et al., 1987; Sidon et al., 1988). In contrast, Moody et al. (1990, 1992) demonstrated no difference between the absorption of acid and amine forms of 2,4-D in rhesus monkey forearm and forehead or forearm and palm regions. The palmar absorption data are in conflict with the accepted dogma that absorption through palmar skin should theoretically be less than that in forearm skin because of the thickness of the stratum corneum in palmar skin (Maibach et al., 1971). It is proposed that because of the hydrophilic nature of 2,4-D-amine, absorption can occur through polar routes such as eccrine glands, which are more frequent in the palmar skin than in forearm skin. This anatomical difference does not explain the discrepancy with lindane, which is more lipophilic than 2,4-D and least likely to be absorbed via a polar route. Despite a 3-fold range in follicle area in the marmoset, no differences in absorption rates of paraquat, mannitol, water, and ethanol were observed between different body sites (Scott et al., 1991). However, among the different species examined in this study, there was an 80-fold range in follicle area, which correlated with observed differences in the rate of mannitol and paraquat absorption. The authors concluded that this correlation was only possible with relatively slowly absorbed test penetrants, such as paraquat and mannitol. Further work is needed to determine to what extent unique anatomical features at different body sites play a role in absorption and penetration of both lipophilic and hydrophilic pesticides. Another factor important in pesticide absorption is age. Topical application of a 2.5% chlorpyrifos spray to pigs resulted in almost uniform mortality when exposed at 3, 6, or 24 hr of age and no mortality at 36 hr (Scheidt et al., 1987). This study suggests that the barrier properties of newborn skin are not mature enough to prevent exposure immediately after birth. This could be secondary to the morphological (e.g., barrier integrity) or physiological (e.g., dermal blood flow) changes that occur as skin matures (Monteiro-Riviere and Stromberg, 1985; Monteiro-Riviere et al. 1991). Age is thus an important factor to assess when comparing absorption studies. Carbaryl is one of the most studied of all CM pesticides, and despite its low toxicity, it appears to penetrate human and animal skin more readily than most other pesticides. This increased permeability seen in most species, compared to most other pesticides, is most likely associated with its unique physicochemical characteristics. Almost complete penetration of carbaryl was observed when low-dose
CHAPTER 30 9Dermal Toxicity of OPs and CMs carbaryl (4 ixg/cm2) was dissolved in acetone and applied to the forearm and jaw angle of six human volunteers (Maibach et al., 1971; Feldmann and Maibach, 1974). The data from these studies demonstrated that 74% of the 24-hr applied dose was excreted in urine over 5 days. As discussed previously, percentage dose absorption for such a low applied concentration would be expected to be higher than for other doses. Utilizing deconvolution analysis of the same human data (skin-to-urine and blood-to-urine data), cumulative absorption over 5 days was estimated to be 63% of the applied dose, with 45% of this occurring 8 hr after onset of penetration (Fisher et al., 1985). When this analysis was performed with 11 other pesticides (e.g., parathion, aldrin, and diquat), more of the carbaryl dose was absorbed within 120 hr compared to other pesticides (0.3-20%). Only carbaryl had a lag time (3.5 hr) that was followed by rapid absorption. Approximately 50% of the 120-hr total absorption of parathion and dieldrin occurred in the first 4 hr. Therefore, although the absorption rate appeared to be less (due to the 3.5-hr lag time) with carbaryl compared to parathion in humans, the extent of absorption during 120 hr was greater for carbaryl (Shah et al., 1983; Hall et al., 1992). In a study comparing carbaryl absorption across formulations and vehicles, increased absorption was seen in some aqueous formulations compared to acetone (Baynes and Riviere, 1998), illustrating that formulation is an important variable that must be taken into consideration in dermal absorption studies. Knowledge of the vehicle or formulation in which the pesticide is dosed is crucial for interpreting dermal absorption studies. This is the primary confounding factor that makes comparisons across studies difficult. As discussed previously, the vehicle or complex formulation significantly affects the value of the partition coefficient, which in turn determines permeability. Vehicles are often not controlled in pesticide exposure studies, but they have repeatedly been shown to have a major effect on both the extent and the rate of pesticide absorption. This has been illustrated in our laboratory with carbaryl (Baynes and Riviere, 1998) as well as parathion (Qiao et al., 1996). In fact, co-administration of pesticides, in this case fenvalerate and parathion, modified absorption compared to single pesticide administration (Chang et al., 1995). Finally, first-pass biotransformation of topically applied pesticides may also occur, which complicates the comparison of the biological relevance of absorbed compound unless metabolism is specifically evaluated, a phenomenon clearly studied for parathion, in which both paraoxon and p-nitrophenol are produced by skin (Carver et al., 1990; Qiao et al., 1994). Insecticide efficacy, the stability of active ingredients, and programmed release of active ingredients from the vehicle/device are the most important characteristics controlled for when pesticides are formulated (Krenek and Rhode, 1988). Environmental Protection Agency (EPA) registration does not always require percutaneous absorp-
417
tion studies. For this reason, more efficacy data are available in the literature compared to dermal absorption or pharmacokinetic data. Furthermore, most of the available pesticide absorption data pertain to binary mixtures (pesticide plus vehicle), which, as described previously, may confound cross-pesticide comparisons. However, technicalgrade formulations are complex mixtures of formulation additives, making risk assessment based on data from exposure to binary mixtures inappropriate. Pesticides are usually formulated to contain active and inactive or inert ingredients. The latter component(s) can enhance the rate and extent of absorption or slow the release of the active ingredient and thus reduce the rate and extent of absorption (Waiters and Roberts, 1993). These "inert" ingredients can be classified as adjuvants, surfactants, preservatives, solvents, diluents, thickeners, and stabilizers. These pesticide additives were first controlled by the Food and Drug Administration and now by EPA regulation 40 CFR 180.1001 and also TSCA and FIFRA (Seaman, 1990). This increasing list of inert ingredients, as well as the prohibitive cost to obtain 40 CRF 180.1001 clearance of new ones, strongly supports the need to evaluate the influence of current and novel additives on the toxicology and dermal absorption of active ingredients in pesticide formulations. Early work by O'Brien and Dannelley (1965) showed that compared to benzene and corn oil, acetone was best at enhancing absorption of carbaryl. Recent studies have also demonstrated the differential effects of solvent systems on the absorption of carbaryl, p-nitrophenol, and 2,4-D (Moody et al., 1992; Brooks and Riviere, 1996; Baynes and Riviere, 1998). Many studies have demonstrated that commercial formulations are more effective than simple solvents at enhancing pesticide absorption. Methyl parathion absorption in vitro in human skin at 24 hr was 1.3% in acetone but was significantly increased to 5.2% in a commercial formulation (Sartorelli et al., 1997). Likewise, in vivo dermal exposure studies of lindane in humans found approximately 60% with white spirits formulation and 5% with acetone vehicle (Dick et al., 1997a,b). In these latter experiments, more of the lindane dose (79%) remained on the skin surface at 6 hr with acetone than with white spirits formulation (10.5%) and significant levels of lindane accumulated in the stratum corneum with white spirits (30%) and with acetone (14.3%) at 6 hr. These findings strongly suggest that the white spirits formulation enhanced lindane penetration with respect to acetone vehicle. The in vitro studies on human skin also demonstrated a similar pattern, although only 18 and 0.3% of the dose was absorbed into the perfusate at 6 hr for white spirits formulation and acetone vehicle, respectively. Topical application of 1% commercial lotion of lindane in vitro in human skin and guinea pig skin resulted in absorption levels as high as 71.72 and 35.31%, respectively, at 48-hr exposure (Franz et al., 1996). Based on the previous discussion of partition coefficients and permeability, vehicle effects are not surprising.
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SECTION IV 9O r g a n T o x i c i t y
Vehicles that are capable of solubilizing the pesticide, but that also favor pesticide partitioning into the stratum comeum, will promote absorption. The effect seen is thus dependent on both the inherent solubility properties of the pesticide relative to the vehicle and the relative solubility of the pesticide in the vehicle versus the stratum corneum. This makes easy generalizations very difficult. Skin hydration can be increased by occlusion with high relative humidity or immersion conditions (e.g., swimming/ bathing). Although it was once assumed that hydration only affects dermal absorption of polar compounds, there is significant data that suggest that at high relative humidity, this hydration effect becomes more important for nonpolar molecules such as pesticides and is most likely secondary to an increase in diffusivity of the penetrating molecule (Behl et al., 1980). Under relative humidity conditions greater than 80%, parathion absorption at three different doses (4, 40, and 400 lxg/cm2) was significantly increased in pig skin in vitro by as much as two or three times the value under standard conditions of 60% relative humidity (Chang and Riviere, 1991, 1993). Occlusion can change dermal absorption by various mechanisms, including reducing evaporative loss of volatile pesticides from the skin surface; enhanced skin hydration, which increases the pesticide's permeability; alteration of the water content of the dosing site, which would reflect dosing in an aqueous vehicle; changes in cutaneous metabolism; dermal irritation; and altered cutaneous blood circulation (e.g., vasodilation). Occlusion can increase hydration of the stratum comeum from as little as 5-15% to as much as 50% (Bucks et al., 1989), thereby modulating the absorption profile for the pesticide. The practical impact of occlusion is seen when exposure occurs through bathing or swimming or, more often, when pesticides get into and under the clothing of workers, creating the ideal conditions for penetration and absorption into the skin. Similarly, many in vitro studies prehydrate skin samples, a requirement in side-by-side and Franz-type diffusion cells, which results in all findings representing flux through fully hydrated skin, a condition not seen in many human exposure scenarios. Prolonged full hydration of skin (e.g., >24 hr) may lend to epidermal degradation (Monteiro-Riviere et al., 1987). One in vivo study on pigs (Qiao et al., 1997) demonstrated that occlusion significantly enhanced pentachlorophenol (PCP) absorption in a soil-based mixture from 29 to 85% of dose and changed the shape of the absorption profile in blood and plasma. The study also suggested that occlusion enhanced metabolism of PCP and resultantly the 14C partitioning between plasma and red blood cells. Occlusion was kinetically linked to modification of cutaneous biotransformation of topical parathion (Qiao and Riviere, 1995). Occlusion enhanced cutaneous metabolism of parathion to paraoxon and to p-nitrophenol as well as the percutaneous absorption and penetration of both parathion and p-nitrophenol. This probably resulted in the
enhanced absorption that was seen. Occlusion also reduced parathion and p-nitrophenol levels in skin, but it increased p-nitrophenol and p-nitrophenol glucuronide in blood. Other in vivo studies showed that dermal occlusion significantly enhanced the rate and extent of parathion absorption in pigs in the abdomen (44 vs 7.5%), buttocks (48 vs 16%), back (49 vs 25%), and shoulder (29 vs 17%) (Qiao et al., 1993). Although significant anatomical site differences were observed with nonoccluded skin, these site differences were concealed with occluded skin. Recall that in vitro studies with parathion also demonstrated that occlusion increased absorption from 0.5-7.7% to 1-17% at doses ranging from 4 to 400 txg/cm2 (Chang and Riviere, 1993). Pesticides can be transferred from cotton fabric onto and through human skin as demonstrated in several studies (Snodgrass, 1992; Wester et al., 1996), although it should be recognized that these studies were often conducted under wet and/or occlusive conditions. Dermal absorption of malathion was 4% with ethanol wet fabric and 0.6% with 2-day-treated cotton sheets (Wester et al., 1996). However, malathion absorption was increased to 7% when the 2-day treated/dried cotton fabric was wetted with aqueous ethanol. In the same study, absorption of glyphosphate was 1.4% in water solution, 0.7% when applied as wet cotton sheets, and 0.1% when applied as 2-day-treated/ dried cotton sheets. Absorption increased to 0.4% when the 2-day dried cotton sheets were wetted with water to simulate sweating and wet conditions. Military uniforms have been impregnated with permethrin as a defense against nuisance and disease-bearing insects. Application of fabric impregnated with permethrin to the backs of rabbits resulted in a 3% migration to the skin surface, with 2% of the impregnant being absorbed and 1% remaining on the skin surface after 7 days of continuous skin contact (Snodgrass, 1992). These interactions have implications for agricultural workers during pesticide application in humid climates and for military personnel under desert combat conditions. Occlusion is a primary experimental variable that may impact the magnitude of dermal absorption fluxes seen. Comparing fluxes for pesticides dosed under occluded versus nonoccluded conditions, or at different sites from the same species, does not necessarily provide data on the comparative absorption of different compounds but, rather, on the overarching effect of application methods.
VI. C O N C L U S I O N S As can be clearly appreciated from the previous presentation, there are numerous experimental variables that have a major impact on the assessment of dermal absorption of pesticides. This was clearly seen with studies involving different methods (in vitro vs in vivo), different species, different doses, or application conditions such as occlusion.
C H A PT E R 3 0 9Dermal Toxicity of OPs and CMs A similar conclusion was derived from analysis of how data are used in making pesticide risk assessments, where the authors concluded that the inability to match a specific exposure scenario to available data (e.g., species and duration of dosing) led to overestimation of absorption and thus risk (Ross et al., 2000). The "take home" lesson from this chapter is that the experimental conditions under which pesticide absorption studies are conducted often overshadow differences between individual compounds, as can easily be appreciated in the classic studies comparing absorption of pesticides in mice (Shah et al., 1981) versus humans (Feldmann and Maibach, 1974). Any comparison can only be conducted if all possible variables are controlled. These variables include, but are not necessarily limited to, 9 Dose 9 Area of skin dosed 9 Application site 9 Vehicle, impurities, and other formulation additives 9 Length of study 9 Occlusion 9 Species 9 Age of animal 9 In vitro vs ex vivo vs in vivo system 9 Assay method The major challenge facing a critical assessment of pesticide absorption is that very few data sets, either across pesticides or for dosing factors within a specific pesticide, are available for analysis. This is the major limitation to compiling large databases of comparative pesticide absorption because the previous factors confound any analysis. Building such a comprehensive dermal absorption database of pesticides would also facilitate development of more robust QSPeR models that ultimately would facilitate the risk assessment process.
Acknowledgments I thank Dr. Ronald Baynes for his invaluable input and guidance in tutoring me on the fine points of experimentally assessing dermal pesticide absorption.
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CHAPTER 30 Skin Pharmacokinetics (B. Shroot and H. Schaefer Eds.), pp. 57-69. Karger, Basel. Potts, R. O., and Guy, R. H. (1992). Predicting skin permeability. Pharm. Res. 9, 663-669. Potts, R. O., and Guy, R. H. (1995). A predictive algorithm for skin permeability: The effects of molecular size and hydrogen bond activity. Pharm. Res. 12, 1628-1633. Qiao, G. L., and Riviere, J. E. (1995). Significant effects of application site and occlusion on the pharmacokinetics of cutaneous penetration and biotransformation of parathion in vivo in swine. J. Pharm. Sci. 84, 425-432. Qiao, G. L., Chang, S. K., and Riviere, J. E. (1993). Effects of anatomical site and occlusion on the percutaneous absorption and residue pattern of 2,6-[ring-14C]parathion in vivo in pigs. Toxicol. Appl. Pharmacol. 122, 131-138. Qiao, G. L., Williams, P. L., and Riviere, J. E. (1994). Percutaneous absorption, biotransformation and systemic disposition of parathion in vivo in swine. I. Comprehensive pharmacokinetic model. Drug Metab. Disp. 22, 459-471. Qiao, G. L., Brooks, J. D., Baynes, R. E., Monteiro-Riviere, N. A., Williams, P. L., and Riviere, J. E. (1996). The use of mechanistically defined chemical mixtures (MDCM) to assess component effects on the percutaneous absorption and cutaneous disposition of topically-exposed chemicals. I. Studies with parathion mixtures in isolated perfused porcine skin. Toxicol. Applied. Pharmacol. 141, 473-486. Qiao, G. L., Brooks, J. D., and Riviere, J. E. (1997). Pentachlorophenol dermal absorption and disposition from soil in swine. Effects of occlusion and skin microorganism inhibition. Toxicol. Appl. Pharmacol. 147, 234-246. Riviere, J. E. (1996). In Models for Assessing Drug Absorption and Metabolism. Isolated purfused skin flap systems. (R. T. Borchardt, Ed.), pp. 387-407. Plenum, New York. Riviere, J. E. (1999). Comparative Pharmacokinetics: Principles, Techniques and Applications. Iowa State Univ. Press, Ames. Riviere, J. E. (2006). Dermal Absorption Models in Toxicology and Pharmacology. CRC Press, Boca Raton, FL. Riviere, J. E., and Brooks, J. D. (2005). Predicting skin permeability from complex chemical mixtures. Toxicol. Appl. Pharmacol., in press. Riviere, J. E., Bowman, K. F., Monteiro-Riviere, N. A., Carver, M. P., and Dix, L. P. (1986). The isolated perfused porcine skin flap (IPPSF). I. A novel in vitro model for percutaneous absorption and cutaneous toxicology studies. Fundam. Appl. Toxicol. 7, 444-453. Riviere, J. E., Monteiro-Riviere, N. A., and Williams, P. L. (1995). Isolated perfused porcine skin flap as an in vitro model for predicting transdermal pharmacokinetics. Eur. J. Pharm. Biopharm. 41, 152-162. Roberts, M. S., Anissimov, Y. G., and Gonsalvez, R. A. (1999). Mathematical models in percutaneous absorption. In Percutaneous Absorption (R. L. Bronaugh, and H. I. Maibach, Eds.), 3rd ed., pp. 3-55. Dekker, New York. Rosenthal, E. (2003). The tragedy of Tauccamarca: A human fights perspective on the pesticide poisoning deaths of 24 children in the Peruvian Andes. Int. J. Occup. Environ. Health 9, 53-58. Ross, J. H., Dong, M. H., and Krieger, R. I. (2000). Conservatism in pesticide exposure assessment. Regul. Toxicol. Pharmacol. 31, 53-58. Rougier, A., Dupuis, D., Lotte, C., and Roguet, R. (1985). The measurement of stratum corneum reservoir, a predictive method for
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in vivo percutaneous absorption studies: Influence of application time. J. Invest. Dermatol. 84, 66-68. Rubin, C., Esteban, E., Hill, R. H., and Pearce, K. (2002). I n t r o d u c t i o n - The methyl parathion story: A chronicle of misuse and preventable human exposure. Environ. Health Perspect. 110, 1037-1040. Sartorelli, P., Aprea, C., Bussani, R., Novelli, M. T., Orsi, D., and Sciarra, G. (1997). I n vitro percutaneous penetration of methyl-parathion from a commercial formulation through the human skin. Occup. Environ. Med. 54, 524-525. Scheidt, A. B., Long, G. G., Knox, K., and Hubbard, S. E. (1987). Toxicosis of newborn pigs associated with cutaneous application of an aerosol spray containing chlorpyfifos. J. Am. Vet. Med. Assoc. 191, 1410-1412. Scott, R. C., Corrigan, M. A., Smith, F., and Mason, H. (1991). The influence of skin structure on permeability: An intersite and interspecies comparison with hydrophilic penetrants. J. Invest. Dermatol. 96, 921-925. Seaman, D. (1990) Trends in the formulation of pesticides. Pestic. Sci. 29, 437-449. Shah, P. V., Monroe, R. J., and Guthrie, F. E. (1981). Comparative rates of dermal penetration of insecticides in mice. Toxicol. Appl. Pharmacol. 59, 414-423. Shah, P. V., Monroe, R. V., and Guthrie E E. (1983). Comparative penetration of insecticides in target and nontarget species. Drug Chem. Toxicol. 6, 155-179. Sidon, E. W., Moody, R. P., and Franklin, C. A. (1988). Percutaneous absorption of cis- and trans-permethrin in rhesus monkeys and rats: Anatomic site and interspecies variation. Toxicol. Environ. Health 23, 207-216. Smith, J. G. (1988). Paraquat poisoning by skin absorption: A review. Hum. Toxicol. 7, 15-19. Snodgrass, H. L. (1992). Permethrin transfer from treated cloth to the skin surface: Potential for exposure in humans. J. Toxicol. Environ. Health 35, 91-105. Srikrishna, V., Riviere, J. E., and Monteiro-Riviere, N. A. (1992). Cutaneous toxicity and absorption of paraquat in porcine skin. Toxicol. Appl. Pharmacol. 115, 89-97. Walker, M., Dugard, P. H., and Scott, R. C. (1983). In vitro percutaneous absorption studies: A comparison of human and laboratory species. Hum Toxir 2, 561-568. Waiters, K. A., and Roberts, M. S. (1993). Veterinary applications of skin penetration enhancers. In Pharmaceutical Skin Penetration Enhancement (K. A. Waiters and J. Hadgraft, Eds.) Dekker, New York. Wester, R. C., and Maibach, H. I. (1983). Cutaneous pharmacokinetics: 10 steps to percutaneous absorption. Drug Metab. Rev. 14, 169-205. Wester, R. C., Noonan, P. K., and Maibach, H. I. (1980). Variations in percutaneous absorption of testosterone in the rhesus monkey due to anatomic site application and frequency of application. Arch. Dermatol. Res. 267, 229-235. Wester, R. C., Maibach, H. I., Bucks, D. A. W., and Aufrere, M. B. (1984). In vivo percutaneous absorption of paraquat from hand, leg, and forearm of humans. J. Toxir Environ. Health 14, 759-762. Wester, R. C., Bucks, D. A., and Maibach, H. I. (1994). Human in vivo percutaneous absorption of pyrethrin and pipernyl butoxide. Food Chem Toxicol. 32, 51-53. Wester, R. C., Quan, D., and Maibach, H. I. (1996). In vitro percutaneous absorption of model compounds glyphosate and
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malathion from cotton fabric into and through human skin. Food Chem. Toxicol. 34, 731-735. Wester, R. C., Melendres, J., Sedik, L., and Maibach, H. I. (1998). Percutaneous absorption of salicylic acid, theophylline, 2,4-dimethylamine, diethyl hexylphthalic acid, and p-aminobenzoic acid in the isolated perfused procine skin
flap compared to man. Toxicol. AppL Pharmacol. 151, 159-165. Williams, P. L., Carver, M. P., and Riviere, J. E. (1990). A physiologically relevant pharmacokinetic model of xenobiotic percutaneous absorption utilizing the isolated perfused porcine skin flap (IPPSF). J. Pharm. Sci. 79, 305-311.
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Local and Systemic Ophthalmic Pharmacology and Toxicology of Organophosphate and Carbamate Anticholinesterases BRYAN BALLANTYNE Charleston, West Virginia
(extraocular) toxic or pharmacological effects. The various circumstances and conditions that determine the site and nature of effects are as follows:
I. I N T R O D U C T I O N Human subjects may be exposed to carbamate (CM) or organophosphate (OP) anticholinesterases (anti-ChEs) topically and/or systemically as a consequence of the wide range of their uses, which may result in exposures from deliberate, incidental, or accidental situations. These uses include the following:
1. Direct (local) contact of the substance with the external (visible) part of the eye (comea, conjunctivae, and palpebral surfaces); depending on the chemical and physical nature of the material, this may result in Injury and/or inflammation of the contaminated part of the eye and surrounding structures (local inflammation) A sensitization reaction (allergic conjunctivitis) A peripheral sensory irritant response Penetration of the substance through the surface structures, causing eye injury or pharmacological effect(s) deeper in the eye 2. Following local contact with the eye, there may be transport into the systemic circulation by absorption through pefiocular blood vessels, from the nasal mucosa after drainage into the nasal cavity through the nasolacrimal duct, or following swallowing into the alimentary tract. Materials with high toxic and/or pharmacological potency may exert systemic effects remote from the eye (extraocular) following such absorption (transocular systemic effects). 3. Materials (or their metabolites) absorbed through other routes of exposure may reach the eye after gaining access to the systemic circulation and exert toxic and/or pharmacological effects on the eye and its adnexa (systemic toxicity to the eye).
1. As pesticides in agriculture and horticulture, which may result in exposure through accidental contamination during their manufacture and in-use applications or incidental exposures through eating residues in treated crops (see Chapter 39). 2. Although the deliberate topical use of anti-ChE OP eye drops in the treatment of glaucoma has generally been replaced with other drug therapy having more effective and less toxic side effects, they are still used for producing short-term mydriasis in ophthalmic practice. Anfi-ChEs given by systemic dosing for conditions in which facilitation of cholinergic neuromuscular and central nervous system (CNS) cholinergic synaptic function is considered to be a therapeutic advantage, such as in Alzheimer's disease or parkinsonism. 3. Exposure may, and has, occurred in the context of the inhumane and vial use of potent OPs in chemical warfare, terrorism, and by some government "security" organizations for political assassinations. The eye may be a local target organ for toxic and/or pharmacological effects of anti-ChEs that are applied by direct (topical) contact to the eye or that reach the eye from the blood circulation. Additionally, the eye may be a route for absorption of anti-ChEs, which may result in distal Toxicology of Organophosphate and Carbamate Compounds
ChEs, both acetylcholinesterase (ACHE) and butyrylcholinesterase (BChE), are widely distributed in the eye. Activity is seen in the extraocular muscles, comeal 423
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epithelium, retina, choroid, and iris (Erickson-Lamy and Grant, 1992; Hikita et al., 1973). Many of the ocular pharmacological effects and some of the ocular toxic effects of anti-ChEs are attributable to their inhibitory effect on ChEs and the resultant cholinergic response, but some toxic effects may be related to other properties of the molecule that are not a function of its anti-ChE potency. Conjunctival hyperemia is characteristic of topical contamination of the eye, probably as a result of an anti-ChE activity (Chiou, 1992; Grant and Schuman, 1992). However, ChE inhibitors may produce varying types of corneal and ocular injury due to their chemical structure that is unrelated to anti-ChE activity, and for this reason it is necessary to undertake eye irritation studies on the OP or CM pesticides and medical products and their formulations. The nature of toxic and pharmacological effects produced by anti-ChEs as a result of their topical and/or systemic exposure of the eye, their investigation by laboratory in vitro and in vivo tests and by human clinical studies, and their practical and clinical implications are discussed in this chapter. For completeness, some reference is also given to cholinergic antagonists.
II. L O C A L O C U L A R I R R I T A N T E F F E C T S BY D I R E C T C O N T A C T
A. General Considerations Chemical injuries to the eye are common (Saunders et al., 1996). With anti-ChE pesticides the eye can become accidentally contaminated in the workplace (e.g., leakage in a manufacturing facility, and in the field/greenhouse there may be spray, droplet, or dust contamination), in the domestic environment (accidental spill, splash, or aerosol spray), or in transportation accidents. Many eye injuries result from misuse in the home, where protective and precautionary measures are often not strictly followed (e.g., eye protection). For example, in one study 84.4% of chemical eye injuries occurred in the home, and these were predominantly accidental exposures in children. In the same study, industrial eye accidents accounted for 14.2% of the eye accidents (Keres et al., 1987). With ophthalmic anti-ChE medication preparations, the eye is, by intended use, deliberately contaminated. The time to onset, severity, and duration of eye injury depend on a number of differing factors, all of which must be taken into account during the design, conduct, and interpretation of investigational laboratory studies and in considerations of in-use hazard and occupational hygiene. These include the chemistry and concentration of the active (anti-ChE) material, formulation (ingredients and concentrations), intended use pattern (e.g., liquid, aerosol, and dust), and likely mode and degree of exposure. In view of the likelihood for accidental and deliberate (medicinal) contamination of the eye, there is a need for
laboratory studies to be conducted in order to determine the eye injuring potential of active materials and formulations. The results of such studies should give practical information on the degree (severity) and nature of any induced eye injury; whether it is irreversible (permanent) or reversible (heals); if reversible, the duration for healing to occur; and whether first aid and medical treatment measures (e.g., eye irrigation) or the use of ocular anti-inflammatory agents are of therapeutic benefit. In many counties, there are mandated testing requirements for eye irritating potential; this covers domestic, agricultural, and industrial chemicals, including anti-ChE pesticides (Ballantyne and Marrs, 2004). There are several approaches to testing for eye irritation potential, which include the following: 1. In vivo direct eye irritation testing in which the material
to be tested is applied to the eye of an appropriate test species, and the eye is subsequently and periodically directly inspected for irritant (injurious) effects. Various ancillary techniques are available to allow a more precise evaluation of the nature and severity of the injury produced. 2. Because of the concern for possible severe discomfort to animals with conventional direct eye irritation testing, various in vivo alternatives have been developed to significantly reduce discomfort. These approaches in general employ lower concentrations (doses) and use noninvasive sensitive methods to detect (early) injury, often with provision for some degree of quantitation (in vivo irritant threshold approaches). 3. In vitro alternative methods, of differing degrees of specificity and reliability, have been developed in order to screen for eye irritating potential. These differing approaches use isolated ocular preparations and/or cytotoxicity assays. 4. Nonbiological models and computer-based analogy predictions have also been employed. Approaches to the various methods for assessing eye irritation potential are briefly summarized here. Details are discussed elsewhere (Ballantyne, 1999a; Ballantyne and Swanston, 1977; Green, 1999).
B. In Vivo Direct Eye Irritation Tests Topical contamination of the eye results in immediate contact with corneal epithelium. This tissue has not only high acetylcholinesterase (ACHE) activity but also choline acetyltransferase activity and the presence of the neurotransmitter acetylcholine (ACh) (Laties, 1969; Plestina and Piukovic-Plestina, 1978; van Alphen, 1957). These findings indicate the activity of a cholinergic physiology. Laties noted that OP anti-ChEs are bound in high concentration by the corneal epithelium.
CHAPTER 3 1 9Ocular Pharmacology and Toxicology Most studies are conducted as acute (single-exposure) investigations. For practical purposes, the formulation is normally tested, although for some registration and investigational needs the active ingredient alone is also tested. Ideally, the eye of an appropriate test species is exposed by the most likely in-use mode of contamination (e.g., droplet, aerosol spray, or vapor). However, many tests are routinely conducted by instilling a given volume (often 0.1 ml) into the inferior conjunctival sac of the test species (usually rabbit) and the eye is inspected sequentially and periodically to determine the nature, severity, time to onset, and duration of the ocular and periocular responses. The influence of varying dose on the effects produced can be studied by varying the concentration of material instilled (by serial dilution) and/or by reducing the volume of material instilled. The major ocular and periocular lesions that are searched for at the various inspection times are as follows: excess lacrimation, corneal injury (opacity/ulceration, vascularization, and area of involvement), blepharitis (hyperemia and thickness), inflammation of the conjunctivae and nictitating membrane (injection, congestion, chemosis, and sloughing), and iris (injection and congestion). For the purposes of convenient and standardized documentation of the effects, scoring systems for ocular lesions have been developed that give a numerical indication of the severity of lesions as a function of time; in this way, and in tabular or graphical form, the onset, duration, and resolution of individually noted lesions can be presented. This also permits a comparison of the eye irritating potential between different materials. Details of the conduct and scoring of local eye irritation tests have been discussed by Ballantyne (1999a) and Green (1999). In addition to macroscopic evidence for inflammation and injury, the eye is also examined for any pharmacological effects that may be produced (e.g., mydriasis and cycloplegia). With potentially potent biologically active materials, the test animals should be inspected for general (systemic) pharmacological and/or toxic effects following transocular absorption. Also, with some materials for which there may be occupational ocular exposure, or with medicinal eye drops, it may be required to perform studies on the peripheral sensory irritant potential of the material (Ballantyne, 1999b), In order to obtain a more precise evaluation of the presence and nature of eye injury in ocular irritation tests, the following ancillary procedures have been used. 1. FLUORESCEINSTAINING When the corneal epithelium is breached by injury, the barrier to fluorescein penetration is lost, and it can diffuse into the underlying corneal stroma. Thus, detection of the presence of fluorescein staining is valuable for the detection of early corneal injury. This is facilitated by examination under ultraviolet light or by using a blue filter in a slit-lamp biomicroscope.
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2. CORNEAL PERMEABILITY The detection of topically applied fluorescein in the aqueous humor can give an indication of whether corneal permeability has increased. This can be detected by the presence of a "fluorescein flare" in the aqueous humor when using a blue filter in the slit-beam path of a biomicroscope. Measurement of fluorescein has been used as the basis for the quantitative evaluation of corneal permeability (Maurice, 1967, 1968). Sulphorhodamine B is probably a better material for corneal permeability studies because of its lower lipid solubility at physiological pH, and hence decreased permeability with the undamaged cornea, and since the red wavelength emitted permits better quantifiable discrimination (Maurice and Singh, 1986). Using a fluorophotometer attached to a slit-lamp biomicroscope allows quantification of aqueous humor fluorescein and forms the basis for an objective test for eye injury, in which there is good agreement between fluorescein penetration and in vivo irritant potential (Easty and Mathalone, 1969; Etter and Wildhaber, 1985). 3. SLIT-LAMP BIOMICROSCOPY Slit-lamp examination of the eyes is valuable for detecting early and minimal changes in the cornea, and it permits a more precise evaluation of the structural integrity or otherwise of the cornea, iris, and lens and certain abnormalities in the aqueous humor, such as a flare due to the presence of excess protein (McDonald et al., 1972). A system for scoring was devised by Baldwin et al. (1973) for recording the effects and changes seen during biomicroscopy of the eye. 4. CORNEAL THICKNESS MEASUREMENT In vivo measurement of corneal thickness is an objective
approach for determining injury to the cornea, particularly during the early stages of the injury process. However, this approach may be more useful as an alternative method, rather than as an ancillary, for in vivo eye irritation studies. 5. CORNEAL EPITHELIUM HEALING RATE After injury of the corneal epithelium, reepithelialization occurs and the thickness of the cornea increases; however, with toxic substances there may be a retardation of the normal healing rate that forms the basis for a semiquantitive assessment of injury potential (Ubels et al., 1982). Combined, measurement of corneal thickness and healing rate can be used as an approach for the evaluation of eye irritants, notably with ophthalmic preparations that may be applied therapeutically to the injured eye. The method involves creating a standard epithelial wound using a trephine and then stripping the corneal epithelium. The material to be tested is then applied to the eye; the contralateral eye is also deepithelialized and used as a control. Corneal healing rate is assessed by fluorescein staining, photographing the injured area, and assessing its size
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Toxicity
by planimetry. Corneal thickness is measured before and sequentially following wounding of the epithelium. Several studies have demonstrated that chemicals of various categories delay the return to normal corneal thickness and retard epithelial healing rate (Fujihara et al., 1993; Green et al., 1989; Ubels et al., 1982). 6. HISTOLOGY At the end of the observation period for the progression and possible regression of eye Injury, it is valuable to remove some of the treated eyes for fixation and subsequent detailed histological observation in order to determine the completeness or otherwise of ocular healing. In some instances, adding additional animals allows for sequential sacrifice during the inspection period and thus permits a study of the pathogenesis of chemically induced ocular lesions. 9
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III. A L T E R N A T I V E S T O C O N V E N T I O N A L IN VIVO E Y E I R R I T A T I O N S T U D I E S In attempts to replace standard eye irritation tests with other approaches for assessing eye injuring potential that cause significantly less discomfort to test animals and that do not involve deliberately inducing eye injury, several methods have been developed, some of which have markedly reduced discomfort, and others have been proposed as alternatives or replacements to conventional in vivo eye irritation methodology. They are briefly considered here.
A. In Vivo Alternatives Although still employing whole animals, these methodologies significantly reduce discomfort by using lower concentrations or smaller volumes of test material, and they increase the sensitivity for detection of early injury by the use of noninvasive objective techniques. Most also have the advantage of allowing a quantitative approach. 1. MEASUREMENTOF CORNEAL THICKNESS (PACHYMETRY) This is a particularly sensitive approach for the early detection of corneal injury. Corneal thickness, hydration, and transparency are interrelated, with corneal thickness and hydration being linearly related (Medbys and Mishima, 1966). Following acute ocular chemical injury, there is increased corneal thickness, indicating the development of edema in the corneal stroma, which may reflect damage to the epithelium, endothelium, or limbic blood vessels (MacDonald et al., 1983). Such damage facilitates the passage of water into the stroma, resulting in an increase in corneal thickness. Thus, corneal edema can result from corneal epithelial or endothelial damage, an increase in intraocular pressure, and inhibition of Na+-K + ATPase
(Chan and Hayes, 1985). Measurement of corneal thickness has several advantages: (i) Quantitative data are obtained that can be statistically analyzed for the comparative evaluation of different substances, (ii) it is a noninvasive method and causes a minimum of discomfort, (iii) increases in corneal thickness usually occur at concentrations below those that cause macroscopic evidence of corneal injury, and (iv) examination of the concentration-response curve allows for the prediction of irritant potential (Ballantyne, 1999a; Ballantyne et al., 1975, 1976). Corneal thickness can be measured by a simple optical device attached to a slit-lamp biomicroscope or by means of ultrasonic probe devices (pachymeters), both of which show good reproducibility (Chan and Hayes, 1985; Martins et al., 1992; Myers et al., 1998). There is a predictive correlation between an increase in corneal thickness and the potential for eye irritancy (Ballantyne et al., 1975, 1976; Burton, 1972; Chun and Ballantyne, 1997; Conquet et al., 1977; Jacobs and Martens, 1989; Martins et al., 1992). The technique of confocal microscopy through focusing allows the quantitation of depth and thickness of different tissue layers and structures of the cornea, and it permits a threedimensional reconstruction of corneal thickness (Li et al., 1996; Maurer and Jester, 1999). Using this approach, Maurer et al. (1997) assessed epithelial cell size, epithelial and corneal thickness, and depth of keratinocyte necrosis following the application of various solvents and determined that it may be used to differentiate different degrees of irritation. 2. MEASUREMENTOF INTRAOCULARPRESSURE Increased intraocular pressure (IOP) can result from causes that include obstruction of the aqueous humor outflow tract, an increase in aqueous humor production, and increasing aqueous humor solute concentration (Chan and Hayes, 1985), and it is a frequent finding when irritant materials are applied to the eye (Ballantyne et al., 1977). The magnitude and duration of the increase in IOP depend on the severity of the ocular irritant response (Ballantyne et al., 1972, 1977; Walton and Heywood, 1978). Most appropriate for measurement of IOP changes are tonometric methods, which involve either measuring the amount of corneal deformation produced by a standard force applied to the corneal surface (indentation tonometry) or measuring the force required to produce a standard degree of corneal flattening (applanation tonometry); details of these techniques were discussed by Ballantyne et al. (1977). Due to the occurrence of circadian variations of IOP in experimental animals, it is necessary to undertake measurements at similar times in control and treated animals in comparative investigations (Ballantyne et al., 1977; Moore et al., 1995). For continuous and long-term recording of IOP changes a technique has been described that involves the implantation of a miniaturized pressure transducer radiotelemeter into the cervical subcutaneous tissues, from which a cannula
CHAPTER 3 1 9Ocular Pharmacology and Toxicology is tunneled to the superior conjunctival sac and inserted behind the corneoscleral junction into a midvitreous position (Percicot et al., 1996; Schnell et al., 1995, 1996). This approach has been used to investigate the effect of antiglaucoma eye drops on rabbits in which chronic ocular hypertension was induced by a posterior chamber injection of oL-chymotrypsin (Percicot et al., 1996). An increase in IOP is usually measurable within a few minutes of topical application of an irritant to the eye, and the time for pressure to return to control values varies from a few hours to several days. The magnitude of the increase in IOP and its duration depend on the concentration of substance applied, its irritant potential, and its ability to penetrate the cornea to produce deeper structural and functional injury (Ballantyne, 1999a). The magnitude and duration of the changes in IOP and the slope on the dose-response curve can be used to predict the likely irritant potential of a material (Ballantyne, 1999a; Ballantyne et al., 1977). 3. CORNEAL PERMEABILITY MEASUREMENTS The measurement of effects of substances on corneal permeability to fluorochromes can be used as a means for the early detection of corneal injury and is a predictor of eye irritating potential (Ballantyne, 1999a; Etter and Wildhaber, 1985; Maurice and Brooks, 1995).
B. In Vitro Alternatives 1. USE OF ENUCLEATED EYES A method was described by Burton et al. (1981) involving the direct application of irritant test substance to the cornea of eyes removed immediately after sacrifice of rabbits and subsequent evaluation of the cornea for swelling, opacity, and fluorescein staining. The eye is mounted on a clamp in a temperature-controlled superfusion chamber with isotonic saline dripped onto the surface of the cornea. After an equilibrium period of 30-45 min, the test material is applied to the surface of the cornea and then washed off. Eyes are inspected macroscopically. Then, with a slit-lamp biomicroscope, the corneal thickness is measured and any fluorescein staining noted. The predictive value of the method has been confirmed in several studies (Commission of the European Communities, 1991; Koeter and Prinsen, 1985; Price and Andrews, 1985; Whittle et al., 1992). By using corneal thickness measurement, fluorescein retention, and corneal opacity as criteria for ocular injury, the suitability of the chicken eye as an enucleated model was confirmed by Prinsen and Koeter (1993) and Prinsen (1996).
2. ISOLATED CORNEA AND CORNEAL CELL PREPARATIONS Isolated corneas from bovine eyes were used by Gautheron et al. (1992) to assess eye irritancy. The corneas were placed in holders with compartments in front of and behind the mounted corneas in a temperature- and humidity-
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controlled chamber. Corneal injury was subjectively assessed by corneal opacity and measured by light transmission and fluorescein permeability from the anterior to posterior chamber of the corneal mount holder. A good assessment of ocular irritancy was obtained. Using a bovine corneal opacity-fluorescein preparation, Vanparys et al. (1993) tested 50 materials and found a 77% concordance using a four-level irritation classification system (non, mild, moderate, and severe irritation) and a 95% correct classification using a two-level classification (nonirritant and irritant). Cassidy and Stanton (1997) also obtained a correct classification using the opacity/permeability approach, and they found these two monitors to accord with corneal histology. Kruszewski et al. (1995, 1997) used a cell line grown from human corneal epithelial cells, which were grown as a three-dimensional culture on a collagen membrane. Effects on the preparation were measured using fluorescein retention. A good correlation with Draize injury scores was obtained. Schneider et al. (1997) described a complex in vitro corneal preparation prepared by establishing primary and subcultures of endothelial, stromal, and epithelial cells from fetal pig eye. A three-dimensional in vitro corneal model was then established in culture plates by sequentially adding endothelial, stromal, and epithelial cells. The cytotoxic response of the preparation was assessed by measuring mitochondrial activity using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Based on calculation of 50% inhibition of mitochondrial capacity (Es0 values), the investigators obtained a good correlation between the E50 values and Draize classifications for various substances.
3. ISOLATED LENS Based on the fact that the lens is embryologically derived from the same ectodermal source as corneal and conjunctival epithelium, Sivak et al. (1992) used an automated scanning laser system to monitor spherical aberration and transmission of the lens in culture. They believed that this method of measuring lens damage compared favorably with standard Draize scores. 4. NONOCULAR TISSUES The chorioallantoic membrane (CAM) of the hen egg is highly vascular and has been used as a tissue to determine whether its irritant response can be employed as a predictor of eye irritant potential. It is a borderline in vivo/in vitro system and does not conflict with ethical and legal standards (Leupke, 1985). The method involves removing shell from around the air cell on day 12 of incubation. The vascular CAM is exposed by removing the inner egg membranes, and the test substance is dripped onto the membranes. Blood vessels and albumin are scored for hyperemia, hemorrhage, and coagulation at 0.5, 2, and 5 min posttreatment. A good correlation with Draize scoring was found by Leupke (1985), and Bagley et al. (1989) showed the
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method to have high sensitivity, specificity, and to be of predictive value. However, Blein et al. (1991) found the method to be too sensitive with undiluted materials, but at 10-fold dilutions there was a good correlation with in vitro eye tests and under these conditions it was possible to differentiate mild, moderate, and severe irritants. A combined CAM-excised bovine eye (opacity/fluorescein stain/ epithelial detachment) test was used by Martins et al. (1992) to simulate corneal injury (bovine eye) and mucosal response (CAM). They obtained limited correlation, with slightly less than 40% of the substances tested agreeing with in vivo findings. In contrast, Van Erp and Weterings (1990) obtained a good correlation between the combined assay and in vivo findings, and Gilleron et al. (1996) obtained a sensitivity of 80% and concordance with the Draize test of 80.4%. Using other nonocular tissues, Muir et al. (1983) obtained a good correlation between hemolysis of bovine erythrocytes and blocking of spontaneous contractions of ileum, an in vivo eye irritation. However, this model is biologically and mechanistically remote from the pathogenesis of ocular irritation. 5. CYTOTOXICITYASSAYS Cytotoxicity assays measure loss of some cellular or intercellular structure and/or functions, including lethal cytotoxicity. They thus give an indication of the potential to cause cell and tissue injury and as such have been used by some investigators to predict tissue injury, including eye injury. Predictability may be variable for a variety of reasons, including the fact that cytotoxicity assay systems are continuously exposed to test material and lack the biological protective mechanisms of mucosae. The choice of screening cytotoxicity assay(s) should include consideration of past experience, likely mechanism for the irritant response, and chemistry of the substance tested. Cell lines that have been used in cytotoxicity assays include corneal epithelial cells, lung fibroblasts, Chinese hamster ovary (CHO) cells, canine renal cells, HeLa cells, and microorganisms. When nonocular-derived cell lines are used, investigators have attempted to rationalize their choice. For example, canine renal cells were used by Shaw et al. (1991) on the basis that integrity of corneal epithelial cells depends on tight and desmosomal junctions, which are also observed in renal canine cells. Thus, they can be used to detect chemicals that may cause an increase in transepithelial permeability (e.g., by using fluorescein leakage to detect injury to tight junctions and neutral red to detect renal cell membrane injury). A large number of cytotoxicity assays have been developed, as listed in Ballantyne (1999a), and only a few illustrative examples are given here. Crystal violet staining uses lethal cytotoxicity as the end point (Itagaki et al., 1991). Cultured HeLa $3 cells or SIRC cells (an established line of rabbit corneal epithelial cells) are incubated with serial dilutions of the test substance, and then crystal violet is used to stain residual viable cells. The
concentration of test substance causing a 50% inhibition of growth (IC50) is calculated. A good correlation has been found between IC50 and maximum in vivo eye irritation for various surfactants (Itagaki et al., 1991). A silicon microphysiometer (a light-addressable potential sensor device) has been used to indirectly measure the rate of production of acidic metabolites from cells in a biosensor flow chamber. The end point calculated is the MRDs0, which is the concentration of test substance required to reduce the metabolic rate by 50% (Brunner et al., 1991a,b). Using mouse fibroblasts, a good correlation was found between the MRDs0 and in vivo eye irritation [Bagley et al., 1994; Rougier et al., 1992 (r 2 = 0.81-0.91); Catroux et al., 1993a,b (r = 0.89)]. Using human epidermal keratinocytes, Brunner et al. (1991a,b) obtained good agreement between MRDs0 values and in vivo eye irritation findings. Photobacterium phosphoreum luminescence has been used to assess eye irritation. Luminescence is generated through a process linked to respiration by NADH and ravin mononucleotide (Bulich, 1979), with light emission being measured photometrically before and after addition of the test substance and an ECs0 calculated (i.e., a value of 50% reduction in light emission). Bagley et al. (1992) found that test substances with the greatest in vivo eye irritation potential had the lowest ECs0 value. The plasminogen activation assay is based on the release of plasminogen activator from primary rabbit corneal epithelial cells as a quantitative index of toxicity. A high correlation between plasminogen activation and known in vivo eye irritating potential was demonstrated by Bagley et al. (1994). In the neutral red uptake assay, mouse fibroblasts or CHO cells are exposed to the test substance and then to neutral red, the retention of which indicates cell viability. Bagley et al. (1992) noted that, in general, the concentration of test substance required to reduce neutral red uptake decreased as the in vivo determined irritant potential of the test substance increased. However, B lein et al. (1991) found that correlation with pH extreme materials was underestimated due to buffering in the incubation medium, and volatile material irritant potential was also underestimated, probably as a consequence of volatilization of the test substance. Several intertest comparisons of cytotoxicity assays have been undertaken. Sina et al. (1992) compared the following assay procedures: leucine incorporation (a general cytotoxicity test), MTT dye reduction (an indicator of mitochondrial damage), and neutral red uptake. They found that none of the end points accurately predicted in vivo eye irritating potential, but the MTT dye reduction method gave the overall best correlation. Christian and Diener (1996) noted that the neutral red uptake assay had merit for ranking potential ocular irritation, particularly the weaker irritants.
CHAPTER 3 1 9Ocular Pharmacology and Toxicology
C. Nonbiological Model Predictors In general, these have not been widely applied. One example is synthetic protein membranes, the use of which is based on the fact that protein denaturation may be a mechanistic function in corneal eye injury with some materials. The reactive component is a synthetic protein-globulin matrix. In one series, the predictability of eye irritating potential was 89%, and in another it was 93% (Soto and Gordon, 1990). 1. PREDICTIVE APPROACHES BASED ON ANALOGY The most commonly used approaches that are based on analogy are structure-activity relationships (SARs) and prediction based on known irritant potential (by in vivo testing) in other mucosal surface or skin. Sugai et al. (1990) used a quantitative SAR (QSAR) to analyze the correlations between chemical structure and eye irritation in rabbits. They claimed 86.3% accuracy in classifying substances with respect to eye irritation. Barratt (1997) described an eye irritation QSAR model for neutral organic compounds. Based on the perturbation of ion transport across the cell membrane being related to dipole moments of the causative substance, the model parameters chosen were log Pow (octanol-water partition coefficient) and the inertial axes Ry and Rz (representing the crosssectional area of the molecule). The results were stated to provide support for the validity of the QSAR model. However, it is hoped that product safety evaluation will not be based solely on QSAR (Ballantyne, 1999a). Many authors who have recommended analogy with skin irritants as a predictive basis for classifying eye irritation potential note that materials that have been shown to be skin irritants in vivo will also be irritants to mucosal surfaces, including those of the eye. However, the converse situation is of much more concern: That is, substances shown not to produce skin irritation should not be assumed to be devoid of irritancy to the eye. Indeed, several studies have shown that there is not a good correlation between skin and eye irritating potential and that some eye irritants are not skin irritants (Dalbey et al., 1993; Kennedy and Banerjee, 1992; Rhodes, 1987; Williams, 1984). There is no simple relationship between skin and ocular irritancy, and in some cases it may be very misleading to attempt to predict one based on the other (Ballantyne, 1999a; Daston and Freeberg, 1991; Williams, 1984). 2. GENERAL COMMENTS ON ALTERNATIVESTO OCULAR IRRITANCY TESTS No single alternative test can completely predict the eye irritating potential of all materials, and expectations for a uniform test should be discouraged (Christian and Diener, 1996; Hutak and Jacarusco (1996) Reinhardt, 1990). However, an appropriately tiered scheme may be valuable in significantly reducing in vivo eye irritation testing (Jackson
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and Rutty, 1985; Reihardt, 1990). Schemes should include the following sequential approach: (i) consideration of the physicochemical characteristic of the substance to be tested, along with a comparison of other substances in the same chemical class whose eye irritating potential has been established; (ii) use of appropriate mechanistically based cytotoxicity studies, established on the prediction for specific chemical substances; (iii) confirmation of the potential for corneal injury by using in vitro enucleated and/or corneal preparation; and (iv) in situations in which there will be deliberate eye contamination (e.g., ophthalmic medications), it may be necessary to confirm the absence of ocular injuring potential by carefully controlled in vivo studies with objective measurements.
IV. S P E C T R U M O F O C U L A R PHARMACOLOGICAL EFFECTS P R O D U C E D BY A N T I C H O L I N E S T E R A S E S As discussed previously, toxic and pathological effects produced on the eye may cover a wide variety of end points and mechanisms of production depending on a number of factors, including the physical properties and chemical structure of the material, the duration and frequency of dosing, the magnitude of individual doses and total dose, and the route of exposure and whether this involves first-pass metabolism leading to enhancement of toxicity or detoxification. Pharmacological effects due to anti-ChEs are a consequence of cholinergic effects resulting from the inhibition of AChE at preganglionic nicotinic (N-) and muscarinic (M-) synapses, postganglionic M-receptors, somatic motor end-plates, and CNS N- and M-receptors (Koelle, 1994). In the eye and its adnexa, cholinergic activity due to anti-ChE activity is seen as the following: 1. Miosis due to contraction of the sphincter pupillae and that may be associated with reduced visual field (Rengstorff, 1985, 1994). 2. Ciliary muscle spasm due to contraction of the ciliary sphincter muscle, with accommodative spasm, myopia, and reduced visual acuity. 3. Reduction of IOP due to contraction of the sphincter pupillae and ciliary muscle. 4. Excess tear production due to cholinergic stimulation of the lacrimal gland. 5. Changes in visual sensitivity, dark adaptation, and visual fields. 6. Extraocular muscle weakness. These effects produced by anti-ChEs may be of clinical value (e.g., lowering of IOP in cases of glaucoma). In addition to their potentially lethal effects caused by paralysis of respiratory muscles, bronchoconstriction, increased tracheobronchial secretions, and depression of the CNS respiratory center, exposure to low concentrations can
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cause ocular effects that may be harassing. For example, in the context of potential harassment in the battlefield, it has been noted that a very small amount of nerve agent vapor can produce miosis, eye pain or headache, nausea, and a general sensation of being unwell, which may decrease the willingness and ability of a soldier to perform a task (Sidell, 1994).
V. I N T R A O C U L A R INJURY, T O X I C I T Y , AND P H A R M A C O L O G I C A L E F F E C T S BY P E N E T R A T I O N O F S U B S T A N C E S T O P I C A L L Y A P P L I E D TO T H E O C U L A R SURFACES Depending on their chemical and physical properties, after contact with the surface of the eye anti-ChEs may penetrate deeper into the eye by passing through the cornea and/or sclera. Penetration from the anterior ocular surface was studied in rabbits by examining the autoradiographic distribution of topically applied [3H]diisopropyl fluorophosphate (DFP) and by measuring inhibition of tissue ChEs following the administration of ecothiopate iodide eyedrops (S-2-dimethylaminoethyl diethyl phosphorothiolate methiodide) (Laties, 1969). From the cornea, anti-ChEs diffuse across the aqueous humor in the anterior chamber to the iris, the papillary surface of the lens, and the aqueous outflow system. After passing through the sclera, anti-ChEs enter the ciliary body. In this way, topically applied anti-ChEs can have pharmacological actions and possible toxicity in deeper eye structures. Thus, corneal and scleral penetration of eyedrop anti-ChEs into the anterior aqueous chamber is a major pathway for the development of lens opacities in patients with glaucoma.
VI. T H E E Y E AS A T A R G E T F O R SYSTEMICALLY MEDIATED TOXIC AND P H A R M A C O L O G I C A L E F F E C T S After absorption of anti-ChEs through routes of exposure remote from the eye, the parent substance or metabolite(s) may reach ocular and periocular tissues through the systemic circulation. Depending on the nature of the antiChE or metabolite(s), toxic and/or pharmacological effects may be exerted on the eye. The potentially hazardous effect on the human eye from uncontrolled exposure of humans to pesticides, including anti-ChE OPs, was emphasized by the identification in the 1950s and 1960s, notably in children, of a possible correlation between a broad spectrum of ocular complications and exposure to fields sprayed with OP pesticides in the Saku region of Japan. The range of adverse effects (Saku disease) included blurring of vision, progressive myopia, astigmatism, blood ChE inhibition, altered electroretinography
(ERG), optic nerve atrophy, optic disk edema, and retinal degenerative changes (Dementi, 1994; Ishikawa, 1972, 1978; Ishikawa and Ohto, 1972; Ohto, 1974). The OP insecticides used near where there were reported cases of Saku disease included dichlorvos (DDVP; 2,2dichlorovinyl dimethyl phosphate), diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl-phosphorothioate), O-ethyl-O-p-nitrophenyl-thiophosphonate (EPN), fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate), fenthion (O,O-dimethyl O-4-methylthio-m-tolyl phosphorothioate), malathion (diethyl [(dimethoxyphosphinothioyl)thio] butanedioate), parathion (O,O-diethyl O-(4-nitrophenyl) phosphorothionate), parathion-methyl (O,O-dimethyl O-4-nitrophenyl phosphorothioate), and trichlorofon (dimethyl 2,2,2-trichloro-l-hydroxyethylphosphonate) (Boyes et al., 1994). Although no other studies have been reported outside Japan that document the spectrum of ocular pathology seen in Saku disease, and some consider that the Japanese population studies are not conclusive, several experimental studies on OPs appear to have substantiated much of the ocular toxicity reported in Saku disease, and several epidemiological studies on the affected populations did establish a reasonable probability of the association between an increased incidence of adverse ocular effects and the greater use of OPs in Japan (Dementi, 1994; Ishikawa, 1973; Ishikawa and Miyata, 1980; Tamura and Mitsui, 1975). However, the possibility exists that there is an increased susceptibility of Oriental populations to anti-ChE-induced ocular toxicology and pathology. The potential for the eye to be a target for substances (and metabolites) distributed systemically following extraocular absorption stresses the necessity for inspection of the eye to detect toxic or pharmacological effects in general acute or repeated dosing studies by all routes of exposure, particularly for chemicals to be used medicinally, as food additives, and those with a potential for exposure in industrial or domestic environments (Ballantyne, 1999a; Barnett and Noel, 1969). Where screening studies suggest a potential for systemic effects on the eye, particularly if there is deliberate exposure of humans to the test substance (e.g., medicinal product or food additive) or a high probability of accidental exposure (e.g., chemicals employed domestically), in definitive general toxicology studies there should be consideration of inclusion of a "satellite" group of animals used specifically to determine ocular toxic and/or pharmacological effects. Ophthalmoscopy, slit-lamp biomicroscopy, tonometry, corneal pachometry, light microscopy, histocytochemistry, transmission electron microscopy, scanning electron microscopy, angiography, ERG, visual evoked potentials, and electrooculography may be required (Ballantyne, 1999a). The U.S. Environmental Protection Agency requires ocular toxicity testing in support of registration of OP pesticides (Hamernik, 1994). Monitors include plasma/erythrocyte/retinal (and possibly brain)
CHAPTER 3 1 9Ocular Pharmacology and Toxicology ChEs; routine gross eye examination and indirect ophthalmoscopy; slit-lamp biomicroscopy; fundus observations; tonometry; ERG; determination of objective refractivity, pupillary response, and tracking; and ocular histology (with possible electron microscopy). In some cases, experimental animal studies may be unable to detect certain forms of systemic ocular toxicity, and these may only be seen in carefully controlled human studies, during detailed clinical examination of exposed humans during in-use applications, or in epidemiological studies. Examples include dyschromatopsia from styrene overexposure (Gobba et al., 1991); impaired or defective color vision following overexposure to styrene (Fallas et al., 1992), n-hexane (Raitta et al., 1978), or carbon disulfide (Raitta et al., 198!), and marked overexposure to toluene (Sch~iper et al., 2004). The potential for substances to produce pharmacotoxic effects on the eye as a component of systemic distribution is related to a variety of factors, includeing total exposure dose absorbed and determinants, notably frequency, magnitude, and duration of exposure dose; route of exposure, which will determine the pharmacokinetics and metabolism of systemic distribution; and the biotransforming capacity of the eye (Watkins et al., 1991). Notable pharmacological effects produced by OPs reaching the eye by systemic distribution include miosis, enhancement of accommodation, and reduction of IOP.
VII. THE EYE AS A PORTAL FOR SYSTEMIC TOXICITY (TRANSOCULAR SYSTEMIC PHARMACOTOXICITY) In general, most substances that are deliberately applied to, or accidentally come into contact with, the eyes are unlikely to be absorbed in significant amounts and produce systemic toxicity because of the small volume of material that can be accommodated on the surface of the eye or in the conjunctival sac. Additionally, the protective reflexes of excess lacrimation and blepharospasm will reduce the degree of ocular contamination (Ballantyne, 1999b). However, substances of high toxicity or pharmacological potency, including certain anti-ChE CMs and OPs, may be absorbed in amounts sufficient to produce systemic toxicity or pharmacological effects. Indeed, this has led to the suggestion of the possibility of transocular systemic pharmacotherapy with certain biologically active materials. For example, facilitated by absorption enhancers, glucagon may be readily absorbed by ocular instillation (Chiou and Li, 1993; Chiou et al., 1990; Pillion et al., 1992), and with insulin the blood glucose was markedly reduced in both normal and hyperglycemic rabbits and rats (Chiou and Chuang, 1989; Chiou et al., 1990; Pillion et al., 1991; Yamamoto et al., 1989). The absence of significant local toxicity from these procedures was confirmed in human volunteer studies on normo-
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glycemic individuals having an ocular instillation of 50 Ixl of sterile saline solution containing various concentrations of porcine insulin, following which no anterior segment toxicity was seen by slit-lamp biomicroscopy (Bartlett et al., 1994).
A. Pathways of Absorption Substances coming into contact with the surface of the eye can be absorbed through conjunctival blood vessels, pass through the nasolacrimal drainage system into the nasal cavity and then through the naso- and oropharynx, and be swallowed. The relative contribution of ocular vascularity, nasal mucosa, and alimentary tract to the absorbed dose varies with the chemical species and its formulation. Absorption through the conjunctival blood vessels may be facilitated by materials also producing a conjunctival hyperemia. Absorption from the conjunctivae may also be modified by the rate of lacrimation, blepharospasm, and patency of the nasolacrimal duct. The large surface area and high vascularity of the nasal mucosa account for this site being a major component in the absorption of substances applied topically to the eye. It is well known that many pharmacologically active substances are absorbed following instillation into the nasal cavity as such (Chien, 1985), and for drugs with a significant first-pass metabolism by the peroral route, the nasal mucosa may offer a practical route for doing so. Hydrophobicity may be an important determinant for enhancing nasal mucosal absorption, whereas hydrophilicity may inhibit absorption (Duchateau et al., 1986; Hussain et al., 1980). The rate of drainage of substances from the conjunctival sac into the nasal cavity depends on a number of factors, including rates of lacrimation and blinking, both of which can be increased by irritant materials. Also, the volume of material is a determinant of the amount of materials transferred (Chrai et al., 1973). Since the drainage capacity of the eye exceeds the normal rate of lacrimation, drops instilled into the conjunctival sac may be rapidly transferred to the nasal cavity (Shell, 1992). Reducing the rate of lacrimal drainage may decrease systemic absorption. For example, Zimmerman et al. (1984) investigated the effects of eyelid closure and manual occlusion of the nasolacrimal duct on the absorption of the antiglaucoma drug timolol applied to the eye and on the permeability of the eye to topically applied fluorescein. It was found that with both eyelid and nasolacrimal duct occlusion, the plasma concentration of timolol was significantly decreased and anterior chamber fluorescein was increased. For timolol, Chang and Lee (1987) found that the nasal mucosa was approximately 2.5 times more effective than the conjunctival mucosa in contributing to total systemic absorption. That anti-ChEs applied topically to the eye may attain the alimentary tract and be absorbed there has been shown by several studies. For example, Wilensky et al. (1967) found that after ocular application of echothiophate or DFP, the greatest inhibition of ChE outside
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the eye was in the intestine. Also, anti-ChEs applied to the eye produced symptoms of intestinal pain and discomfort (Humphreys and Holmes, 1963).
B. Illustrative Examples Tropicamide [N-ethyl-N-(pyrid-4-ylmethyl)tropamide] is an M-receptor antagonist that produces transient mydriasis and cycloplegia. Because the paralysis of accommodation with 0.5-1.0% tropicamide is of short duration, its main use is in diagnostic examination or surgery of the eye requiring mydriasis of short duration. Although it has a low incidence of side effects, there have been reports of tropicamide causing a myasthenia gravis-like syndrome following topical application to the eye (Meyer et al., 1992). Plasma concentrations of tropicamide, and M-ACh receptor occupancy, were investigated following the conjunctival sac instillation of two 40 Ixl drops of 0.5% tropicamide in eight women (Vuori et al., 1994). Peak plasma concentrations of tropicamide were 2.8 + 1.7 ng m1-1 at 5 min postinstillation. There was rapid plasma clearance, with respective concentrations at 60 and 120 min of 0.46 + 0.51 ng m1-1 and 4 mg kg -~ increased the amplitude of the b-wave (Takeda et al., 1976). Von Bredow et al. (1971) reported that satin (10 mg in cat, intraperitoneally) caused an increase in the ERG b-wave, which was completely abolished by atropine. Imai (1974) showed that in the rat, four daily intramuscular injections (0.5 mg kg -1) of fenthion resulted in an increased amplitude and decreased latency and peak times of the ERG a- and b-waves. At higher doses (>25 mg kg -1) latency and peak times were increased and the amplitude of the a- and b-waves was decreased. Imai (1975a,b) demonstrated ERG changes in rats given an acute intramuscular injection of fenthion (5, 25, and 50mg kg-1). At the low dose, there was an increased amplitude of the a- and b-waves that persisted up to 10 days postinjection; thereafter, latency and peak times were lengthened and did not return to normal until 40 days postinjection. At the mid-dose, the a- and b-waves increased, and by postinjection day 4 the latency and peak times increased; the amplitudes recovered by 60 days, but the peak time remained increased. At the high dose, the a- and b-wave amplitudes were decreased and the latency and peak times were increased; no recovery of amplitudes was noted. Various studies, using both acute and repeated exposure dosing, have shown that OPs and, less frequently, CMs can be associated with effects on retinal ChE and the ERG and produce retinal histopathology and other ocular toxicity. Studies on several OPs in rats and dogs have suggested that they can produce abnormal ERGs and histopathological evidence of retinal degeneration (Boyes et al., 1994; Dementi, 1994). Studies on radiolabeled benomyl showed that it was concentrated in the mouse retina following systemic administration (Hellman and Latyea, 1990). Acute doses of parathion, malathion, and mevinphos (2-methoxycarbonyl-l-methylvinyl dimethyl phosphate) have produced ERG changes in mice (Carricabura and Lacroix, 1973; Carticabura et al., 1979, 1980, 1981). Fenthion at doses >50 mg kg-1 produced dose-dependent ERG changes, a decrease in retinal ChE activity, fundoscopic evidence of retinal degeneration, and histopathological changes in the pigmented epithelium and sensory retina (Imai, 1975a,b, 1977, 1978; Imai et al., 1973, 1983; Miyata et al., 1973). Details of some studies are presented here. The effects on the ERG and retinal AChE were studied in Wistar rats at acute subcutaneous dosages of
CHAPTER 3 1 9Ocular Pharmacology and Toxicology 0.005-500 mg kg -1. All rats dosed at 500 mg kg -1 died. At 50 and 100 mg kg -1, subnormal ERG activity was obtained; 25 mg kg -1 was a transitional dose (normal ERG), and at less than 25 mg kg -1 the ERG was supernormal. Minimal change was seen at the lowest dose of 0.005 mg kg -1, and thus a no observed effect level (NOEL) for the ERG was not obtained. Retinal and cerebellar AChE activity was inhibited in a dose-related manner in the 0.5-100 mg kg -1 range. Thus, ERG changes were detected at acute fenthion dosages below those causing inhibition of retinal AChE activity. Imai (1975a,b) also studied the effect of fenthion given acutely by subcutaneous injection at doses of 0, 5, 25, and 50 mg kg -1 to Wistar rats. At 5 mg kg -1, supernormal ERGs were recorded that increased until 10 days postdosing and decreased thereafter to normal by 2 months. A transitional ERG was conformed at 25 mg kg-1, and at 50 mg kg-1 a subnormal ERG was obtained that was still present at 66 days postdosing. Measured serum BChE activity decreased to approximately 15% 4 days postdosing. Retinal AChE activity was decreased for up to 49 days, and in the high-dose group the ERG changes corresponded with the retinal AChE recovery curve. Repeated exposure studies have been conducted with 50 mg kg -1 fenthion given subcutaneously every 4 days to Long-Evans black rats for 1 year (Imai, 1977; Miyata et al., 1979; Uga et al., 1979). Dosed animals showed signs of systemic toxicity (exophthalmos, shivering, and diarrhea). At 3 months, the ERG was found to be subnormal, and at 9 months the ERG a-waves disappeared. Fundoscopy revealed paleness of the papilla. Histology showed the disappearance of the retinal pigmented epithelial layer, outer and inner nodes, and outer granular layer. Ultrastructural examination showed complete disappearance of photoreceptor cells. Uga et al. (1976) studied the ultrastructure of the retina of dogs dosed with ethylthiometon at dosages of 0.5-1.5 mg kg -1 for 2 years. Degenerative changes were seen, principally in pigment epithelial cells in the area around the papilla, and myelin-like material was seen in retinal cells. Effects of fenthion on the retina of Long-Evans and Wistar rats were also studied by Imai et al. (1983) using a dosage of 50 mg kg -1 given subcutaneously twice a week for 1 year. ERGs were subnormal by 3 months and not recordable by 12 months. Retinal degeneration was seen in all Long-Evans (pigmented) rats and two-thirds of the Wistar rats at 1 year. Wistar rats dosed with chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) showed ERG changes of decreases in a- and b-wave amplitudes of more than 50% and increased latencies of 10-20%, which were associated with inhibition of plasma, erythrocyte, and brain ChE activities (Yoshikawa et al., 1990). Neuropathological lesions included optic nerve demyelination, partial necrosis of pigmented epithelial Cells, and cytoplasmic edema and degeneration of ciliary muscle myofilaments (Kono et al.,
437
1975; Mukuno and Imai, 1973; Mukuno et al., 1973). However, some repeated exposure studies have suggested that inhibition of retinal AChE may not be a major etiological factor in the production of retinal toxicity by anti-ChEs. For example, in a subchronic (6-month) study in which beagle dogs were given daily peroral doses of the OP ethyl parathion (2.4, 7.9, or 794 p~g kg -1 per day) by gelatin capsules, although plasma, erythrocyte, and retinal ChEs were inhibited at the high dose, there was no functional impairment of eye function during the 6-month period as assessed by ophthalmoscopy, slit-lamp biomicroscopy, refraction, tonometry, ERG, and ocular histology (Atkinson et al., 1996). In a chronic peroral study (1 year), beagle dogs were dosed with disufoton (O,O-diethyl S-2-ethyl thioethyl phosphorodithioate) in the diet with average daily consumptions of 0.0, 0.015, 0.121, and 0.321 mg kg -1 (Jones et al., 1999). ChE activity was measured in erythrocytes, plasma, brain, retina, cornea, ciliary body, and extraocular muscles. Neuroophthalmological monitors included task performance tests, gait and behavioral observations, ERG, refractivity, tonometry, and pachymetry. RBC, plasma, corneal, and brain ChEs were significantly decreased at 0.121 and 0.321 mg kg -1 per day, and ciliary body and retinal ChEs were significantly decreased at 0.321 mg kg -1 per day; the NOEL for ChE inhibition was 0.015 mg kg-1 per day. Despite the inhibition of ChE activity in various tissues, there were no adverse clinical neurological effects and no ophthalmologic findings by ERG, refractivity, tonometry, or pachymetry. It is of interest to note that a spontaneous retinal degeneration occurs in the rd mouse as a result of increased turnover of retinal cGMP phosphodiesterase (Farber et al., 1988). Perfused isolated cat eyes treated with inhibitors of phosphodiesterase activity cause ERG changes similar to those found in individuals with hereditary retinitis pigmentosa (Sandberg et al., 1987). These studies suggest the possibility, although theoretical, that OP-induced retinal degeneration may occur if a substantial inhibition of cGMP occurs in addition to inhibition of retinal ACHE. Tacrine (9-amino-l,2,3,4-tetrahydroacride hydrochloride hydrate) is a cholinesterase inhibitor of potential use in Alzheimer's disease, based on the ability to facilitate cholinergic function in the CNS via direct stimulation of M- and N-receptors through inhibition of AChE (Avery et al., 1997; Newhouse et al., 1997). Since ACh plays a fundamental role in visual function (Famiglietti, 1983; Hutchins, 1987; Ross et al., 1985), and because some visual symptoms and optic nerve degeneration occur in Alzheimer's disease patients (Hinton et al., 1986) andthe decrease in ACh found in the CNS may also occur in the retina (Strenn et al., 1991), it was considered appropriate by Alhomida et al. (2000) to investigate the effects of tacrine on human retinal AChE activity. In vitro studies on human retinal homogenates showed a concentrationdependent inhibition of AChE activity by tacrine, with an
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SECTION IV. O r g a n T o x i c i t y
IC50 of approximately 45 nM. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide by retinal AChE was 0.12 mM, which increased in the presence of tacrine. The Vmax was determined to be 2.23 ~mol hr-1 mg protein-1 and was decreased by tacrine treatment. Dixon and Lineweaver-Burk plots, and their secondary replots, indicated that the inhibitory process of tacrine on human retinal AChE was a combination of competitive and noncompetitive processes. Several human clinical and epidemiological studies have drawn attention to a potential for, or a suggestion for, retinal toxicity from exposure to anti-ChEs. A cross-sectional study of fenitrothion sprayers in India found macular degeneration i n 16% of workers compared to 3% of controls (Misra et al., 1985). Morse et al. (1979) described ocular toxicity, including a 22% incidence of macular degeneration, in workers exposed to methomyl [S-methylN-(methylcarbamoyloxy) thioacetimidate] in a pesticide manufacturing plant. Fluorescein angiography suggested that the macular lesion was a consequence of a lesion in the pigment epithelium. In a cohort study of licensed pesticide applicators from Iowa and North Carolina, there was evidence that retinal degeneration was associated with CM insecticides (Kamel et al., 2000). Schoolchildren from Saku, Japan, demonstrated a 65% incidence of optic neuritis and/or chorioretinal atrophy (Ishikawa et al., 1971). In Japanese farmers chronically exposed to pesticides including OPs, the incidence of optic neuritis was increased (Imaizumi et al., 1971).
F. Extraocular Muscles Extraocular muscles have the highest activities of AChE in the body (Hikita et al., 1973; Mukuno and Imai, 1973; Mukuno et al., 1973). Anti-ChE eyedrops in the treatment of glaucoma commonly cause brief (few minutes) eyelid twitching. Similar effects may be seen with systemic intoxication, which has been observed with tetraethyl pyrophosphate given intramuscularly in patients with myasthenia gravis (Grob and Harvey, 1949). Mukuno and Imai (1973) studied the extraocular muscles of ethylthiometon-dosed dogs. They found decreased ChE activity, and electron microscopically there was degeneration of nerve fiber bundles, neuromuscular junction, and muscle fibers, with mitochondrial degenerative changes. Hikita et al. (1973) studied ChE activity in the extraocular muscles of ethylthiometon chronically dosed dogs at 0 . 5 - 1 . 5 m g k g -1 per day. AChE and BChE were inhibited in the muscles in a dose-related manner. They also noted that retinal cholinesterase activity was decreased in a dose-dependent manner (3, 50, and 70% inhibition). Extraocular muscle effects have been reported as neuropathies from anti-ChE poisoning. Suicidal poisoning by the use of OPs has been reported to cause bilateral third or sixth paralyses in 8/200
patients and facial nerve paralysis in 12/200 patients (Wadia et al., 1974).
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SECTION I V .
Organ Toxicity
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electroretinogram in Alzheimer's disease. J. Neural Transmission Suppl. 33, 73-80. Sugai, S., Murata, K., Kitagaki, T., and Tomik, I. (1990). Studies on eye irritation caused by chemicals in rabbits: I. A quantitative structure-activity relationship approach to primary eye irritation of chemicals in rabbits. J. Toxicol. Sci. 15, 246-262. Suzuki, H., and Ishikawa, S. (1974). Ultrastructure of the ciliary muscle treated by organophosphate pesticide in beagle dogs. Br. J. Ophthalmol. 59, 931-940. Swan, K. C. (1964). Iris pigment nodules complicating miotic therapy. Am. J. Ophthalmol. 37, 886-889. Takeda, Y., Tsukahara, I., and Takori, S. (1976). Effects of chlorphenvinphos, an organophosphate pesticide, on afferent transmission in the central visual system. Jpn. J. OphthalmoL 20, 195-203. Tamura, O., and Mitsui, Y. (1975). Organophosphorus pesticides as a cause of myopia in schoolchildren: An epidemiological study. Jpn. J. Ophthalmol. 19, 250-253. Tarkkanen, A., and Karjalainen, K. (1966). Cataract formation during miotic treatment for chronic open-angle glaucoma. Acta Ophthalmol. 44, 932-939. Tokoro, T., Suzuki, K., Nakano, H., and Otsuka, J. (1973). Experimental studies of organic phosphors pesticide on beagle dogs: Long-term observations on the refraction and the intraocular pressure. Nippon Ganka Gakki Zasshi 77, 1237-1245. Ubels, J. L., Edelhauser, H. R., and Shaw, D. (1982). Measurement of corneal epithelial healing rates and corneal thickness for evaluation of ocular toxicity of chemical substances. J. Toxicol. Cutaneous Ocular Toxicol. 1, 133-146. Uga, S., Ishikawa, S., and Mukono, K. (1976). Microscopic changes in the retina of dogs administered organophosphorus for prolonged periods. Jpn. Rev. Clin. Ophthalmol. 70, 282-283. Uga, S., Imai, H., Miyata, M., and Ishikawa, S. (1979). Retinal degeneration in Long-Evans rats induced by fenthion intoxication: An electron microscopic study. Excerpta Medica Int. Congr. Ser. 450 1, 915-918. Upholt, W. M., Quinby, G. E., Batchelor, G. S., and Thompson, J. E (1956). Visual effects accompanying TEPP-induced miosis. Arch. Ophthalmol. 56, 128-135. Van Alphen, G. W. H. M. (1957). Acetylcholine synthesis in corneal epithelium. Arch. Ophthalmol. 58, 449-451. Van Erp, Y. H. M., and Weterings, E J. J. M. 1990). Eye irritation screening for classification of chemicals. Toxicol. in Vitro 4, 267-269. Vanparys, E L., Deknudt, G. H., Sysmans, M., Touns, G., Coussement, W., and Van Cauteren, M. H. (1993). Evaluation of the bovine corneal opacity-permeability assay as an in vitro alternative to the Draize eye irritation test. Toxicol. in Vitro 7, 471-476. Von Bredow, J., Bay, E., and Adams, N. (1971). Drug actions on the central nervous system as studied by the effects on the electroretinogram. Exp. Neurol. 33, 45-52. Von Sallam, L., and Dillon, B. (1947). The effects of di-isopropyl fluorophosphate on the capillaries of the anterior segment of the eye in rabbits. Am. J. Ophthalmol. 30, 1244-1262. Vuori, M.-L., Kaila, T., Iisalo, C., and Saari, K. M. (1994). Systemic absorption and anticholinergic activity of topically applied topicamide. J. Ocular Pharmacol. 10, 431-437. Wadia, R. S., Sadagopan, C., Amin, R. B., and Sradesai, H. V. (1974). Neurological manifestation of organophosphorus pesticide poisoning. J. Neurol. Neurosurg. Psychiatr. 37, 841-847.
CHAPTER 3 1 9Ocular Pharmacology and Toxicology
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CHAPTER
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Reproductive Toxicity of Organophosphate and Carbamate Pesticides SURESH C. SIKKA AND NILGUN GURBUZ Tulane University Health Sciences Center, New Orleans, Louisiana
expressed serious concerns about the estrogenic effects of environmental xenobiotic chemicals, such as polychlorinated biphenyls, dichlorodiphenyl trichloroethane (DDT), dioxin, and some pesticides, especially organophosphates (OPs) and carbamates (CMs) (Boris, 1965; Colborn et al., 1993; McLachlan and Arnold, 1996). The potential effects these chemicals may have on human health and ecological wellbeing include reproductive tract cancers, reduced fertility, and abnormal sexual development. Reproductive toxicity is expressed as alterations in sexual behavior and performance, infertility, and/or loss of the fetus during pregnancy. Exposure to chemical substances may include alterations to the female and male reproductive system; adverse effects on onset of puberty, gamete production and transport, reproductive cycle normality, sexual behavior, fertility, and parturition; and premature reproductive senescence or modifications in other functions that are dependent on the integrity of the reproductive systems of both female and male species (Colborn et al., 1993; Sharpe and Skakkebaek, 1993; Kelce et al., 1994; Kumar, 2004). Toxicants that target the male reproductive system can affect sperm production and their motion, morphology, and function, and can alter sexual behavior and performance. This can cause infertility, erectile dysfunction (ED), and reduced quality of life (Sikka, 1999a,b). Many of these chemicals mimic or otherwise disrupt the estrogens or the androgen balance in the body by binding to hormone receptors during fetal and neonatal development, and these are called "endocrine disruptors" or "gender benders." Newer tools for the detection of Y chromosome deletions have further strengthened the hypothesis that the decline in male reproductive health and fertility may be related to the presence of these toxic chemical compounds in the environment. The development of intracytoplasmic sperm injection (ICSI), a technique introduced in the early 1990s, is beyond doubt the most important recent breakthrough in the treatment of male infertility. This was made
I. I N T R O D U C T I O N Many environmentalists believe that the human species is approaching a fertility crisis based on evidence of the past 20-30 years that has shown disturbing trends in male reproductive health (Guillette et al., 1994). Many others, however, think that the available data are insufficient to deduce worldwide conclusions (Fisch et al., 1996). Although these assertions have been disputed, the fact remains that one in six couples have trouble conceiving, with males equally responsible for infertility. Reduced fertility in males is one of the major end points of reproductive toxicity and is the focus of this chapter. Interference with the action of androgen during development can cause male reproductive system abnormalities that include reduced sperm production and/or fertilizing capability. It is also possible that the genetic information of the sperm may potentially be altered prior to fertilization, which can result in birth defects in the offsprings (Parvinen et al., 1984). However, the evidence that such environmental chemicals cause infertility is still largely circumstantial. There are many missing links in the causal chain that would connect receptor binding to changes in reproductive health with decreased fertility. In the past decade, the occupational health and safety community has directed greater attention to pesticide exposures among workers and their families. Initiatives focused on minority workers, women, and the children of workers have also made pesticide exposure assessment a timely topic for scientific investigation and medical management. With discoveries of deformed frogs in Minnesota lakes and fertility problems in alligators found in Lake Apopka in Florida attributed to embryonic exposure to such pollutants (Guillette et aL, 1994), a myriad of environmental agents and pesticides have been classified as reproductive toxicants. This has been the subject of a number of reviews (Kavlock and Perreault, 1994; Sokol, 1994; Sikka, 1997; Lamb, 1997; Cheek and McLachlan, 1998). Several investigators have Toxicology of Organophosphate and Carbamate Compounds
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Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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possible by many well-controlled clinical studies and basic scientific discoveries in the physiology, biochemistry, and molecular and cellular biology of the male reproductive system. Despite these developments, the etiology, role of environmental toxicants, diagnosis, and treatment of male factor infertility remain a real challenge. Scientists have investigated the mouse embryonic stem cell approach using a precise combination of cellular growth factors to grow stem cells into mature sperm in the laboratory. This may lead to new approaches in understanding the pathophysiology and treatment for male infertility since the factors needed to grow mouse stem cells would probably also foster the growth of human sperm.
II. B A C K G R O U N D Pesticides are a heterogeneous group of chemicals developed to control a variety of pests and are generally categorized according to the type of pest for which they have been shown to be efficacious. The term "pesticides" is a general name that includes many chemicals, mostly in the classes of insecticides (OPs, organochlorines, CMs, and pyrethroids) and herbicides (bipyridyl compounds). Other categories include fungicides, termiticides, rodenticides, algaecides, repellents, miticides, and wood preservatives. OP and CM pesticides are an integral part of modem agriculture, whereas many organochlorine pesticides have been withdrawn from such usage due to their persistence in the environment. The use of OPs and CMs has soared due to their availability and quick degradation in the environment. Although OPs and CMs degrade quickly and are much less persistent in the environment, they are much more toxic to mammals, especially at high doses. Because of their adverse health effects, various governmental agencies set limits on allowable levels of pesticide residues in foods, animal feeds, and the environment. In order to enforce these allowable levels, pesticides are monitored in various types of samples. Most exposures are through respiration or dermal routes, although humans and animals may be poisoned accidentally or maliciously. The field of pesticide exposure assessment is complex and challenging. Exposures occur through multiple routes and are highly variable. Risks associated with pesticide handling differ substantially for different activities and from those experienced by agricultural reentry workers. Different assessment and control strategies are needed for each population. Families of pesticide handlers can be exposed to pesticides, and consideration of their children as a vulnerable subpopulation will likely lead to changes in the agricultural workplace that will reduce exposures for workers and families alike. Pesticides, which are designed to interfere with the function of the insect nervous system, interfere with the function of the mammalian nervous system at higher doses. In fact, the fetus and neonate are more vulnerable than the adult to insult by such pesticides because the drug-binding capacity of fetal
serum and tissue protein is significantly lower than that of the adult animal. Since the pituitary-gonadal axis differentiates during both prenatal and postnatal phases, it is vulnerable to toxic insult during a relatively long period of development. This may result in persistent endocrine dysfunction in the mature offspring leading to reproductive problems (Cranmer et al., 1978). OP and CM insecticides are widely used in both agricultural and landscape pest control. The potential for human exposure to this class of compounds is significant since OPs have the properties of low bioaccumulation and high rate of biodegradation (Sarkar et al., 2000; Tamura et al., 2001). In the mid-1970s, it was determined that dibromochloropropane (DBCP) exposure impaired fertility in the absence of any other clinical signs of toxicity, suggesting that the reproductive system was the most sensitive target organ. Reduced fertility, embryo/fetal loss, birth defects, childhood cancer, and other postnatal structural or functional problems were the most common outcomes from such exposures (Sokol, 1994). However, the database for establishing safe exposure levels or risk assessment for such outcomes remains very limited. Declining semen quality is not the only indicator that human reproduction is at risk. In fact, a marked increase in the incidence of testicular cancer in young men has been associated with other abnormalities (including undescended testis, Sertoli cell-only pattern, and hypospadias) that cause poor gonadal function and low fecundity rates. The human male produces relatively fewer sperm on a daily basis compared with many of the animal species used for toxicity testing. A less dramatic decrease in sperm numbers or semen quality in humans can have serious consequences for reproductive potential. In fact, in many men older than age 30, the lower daily sperm production rate already places them close to the subfertile or infertile range (Working, 1988). Decreased semen quality (low sperm number, motility, and structure) during the past 50 years has been attributed to environmental toxicants, many of which act as "estrogens" (Sharpe and Skakkebaek, 1993). This "estrogen hypothesis" has inspired a number of debates and serious investigations (Sharpe and Skakkebaek, 1993; Kelce et al., 1994). Does that make men less fertile? After all, it takes only one sperm to fertilize an egg. Problems in the production, maturation, and fertilizing ability of sperm are the single most common cause of male infertility. Even if produced in adequate numbers, sperm can have poor motility, viability, morphology, stay immature, and lack acrosome and other characteristics that will prevent them from fertilizing an oocyte. An environmental agent may disrupt reproductive function in the male at several potential target sites, most prominent being pituitary and testes that are under the control of the hypothalamus (Fig. 1). A dramatic increase in our knowledge of reproductive toxicity and infertility has resulted from advances in the understanding of gonadal function and dysfunction. Although any discussion of gonadal function and toxicity is of special relevance to man,
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases
449
FIG. 1. Scheme showing HPG axis in the male and potential target sites of action of organophosphate and carbamate pesticides.
much of the understanding of such target sites has been obtained from research using animal species and various experimental models (Maitra and Sarkar, 1996).
III. M A L E R E P R O D U C T I V E AS T A R G E T S I T E
1. SEMINIFEROUSTUBULES The proliferation of the mesenchyme separates the sex cords from the underlying coelomic epithelium by the seventh week of fetal development. These sex cords become the seminiferous tubules that develop a lumen after birth.
TRACT
A. Composition of Testis The male gonads in human usually exist in pairs and are the sites of spermatogenesis and androgen production. Spermatozoa are the haploid germ cells responsible for fertilization and species propagation. There are paracrine and autocrine regulations in various compartments of testis that are under the endocrine influences from the pituitary and hypothalamus. The testes develop abdominally at the renal level and descend into the scrotum. Embryonically, the testes develop from the testicular ridge located at the rear of the abdominal cavity. In the last months of fetal life, they begin a slow descent, passing out of the abdominal cavity through the inguinal canal and into the scrotum. The descent is usually complete by the seventh month. Primitive germ cells migrate to the medial surface from the yolk sac, cause the coelomic epithelial cells to proliferate, and form the sex cords that lead to the formation of major components of the testis. Approximately 80% of the testicular mass consists of highly coiled seminiferous tubules within which spermatogenesis takes place. The remaining 20% consists of Leydig cells and Sertoli cells, whose main job is to establish normal spermatogenesis.
2. RETE TESTIS During the fourth month, sex cords become U-shaped, and the ends anastomose to form the rete testis, which provides communication with the epididymis. The primordial sex cells are referred to as prespermatogonia and the epithelial cells of the sex cords as Sertoli cells. 3. LEYDIG CELLS These cells arise from interstitial mesenchymal tissue between the tubules during the eighth week of human embryonic development. They are located in the connective tissue between the seminiferous tubules. Leydig cells are the endocrine cells in testis that produce testosterone from cholesterol via a series of enzymatic pathways and steroidal intermediates under the control of luetinizing hormone (LH) from pituitary. The effects of testosterone can be grouped into the following categories: 1. Effects on the reproductive system before birth: Before birth, testosterone secretion by the fetal testes is responsible for masculinizing the reproductive tract and external genitalia and for promoting descent of the testes into the scrotum. After birth, testosterone secretion ceases, and the testes and remainder of the reproductive system remain small and nonfunctional until puberty.
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2. Effects on sex-specific tissues after birth: At puberty, the Leydig cells start secreting testosterone once again, and spermatogenesis is initiated in the seminiferous tubules for the first time. Testosterone is responsible for the growth and maturation of the entire male reproductive system as well as the libido. Ongoing testosterone secretion is essential for spermatogenesis, for maintaining a mature male reproductive tract throughout adulthood. 3. Other related effects: These include the development of libido at puberty; maintenance of the sex drive in the adult male; controlling the secretion of LH by the anterior pituitary via feedback mechanisms; the development and maintenance of male secondary sexual characteristics; and general protein anabolic effects, including bone growth and induction of aggressive behavior. Low levels of testosterone and decreased libido lead to ED. 4, SERTOLI CELLS Within the testicles are cells that envelop the developing sperm during spermatogenesis. These cells form a continuous and complete lining within the tubular wall and establish the blood-testis barrier by virtue of tight junctions. The luminal environment is both created and controlled by these Sertoli cells, also called "nurse cells." These Sertoli cells have several functions, including the following: 1. Provide nourishment for the developing sperm cells 2. Destruction of defective sperm cells 3. Secretion of fluid that helps in the transport of sperm into the epididymis 4. Release of the hormone inhibin, which helps regulate sperm production Thus, many irregularities of spermatogenesis due to gonadotoxicity may reflect changes in function of the Sertoli cell population, and not necessarily by pathology in the germ cells. The differentiation of Sertoli cells and the formation of a competent blood-testis barrier are essential to the establishment of spermatogenesis during puberty in all species.
B. Spermatogenesis and Spermiation Spermatogenesis is a chronological process spanning approximately 42 days in the rodent and 72 days in man (Sikka and Naz, 2002). Spermatogenesis can be divided into three distinct phases: mitosis, meiosis, and spermiogenesis. The first phase is referred to as spermatogonial proliferation and renewal. During this period, relatively undifferentiated diploid spermatogonia, the immature germ cells, undergo several mitotic divisions to generate a large population of cells called primary spermatocytes. In the second phase, the spermatocytes go through the process of two meiotic divisions leading to the formation of the haploid germ cells, spermatids. In the third phase, the spermatids go through a complex series of cytological transformations and
Spermatogonium i Mitosis I"
~MeiosisI " Prophase of
J
Primary Spermatoc~e
I Meiosiselted ~compl
: nesis Spermatoge(Meiosis) /
SecondarySpermatocytes I Meiosis tl
Spermiogenesis
L_
Spermatids
FIG. 2. Scheme of mammalian spermatogenesis showing the premeiotic and meiotic stages of spermatogenesis and postmeiotic spermiogenesis with the development and maturation of the spermatozoa.
dedifferentiate to form stem cells that cyclically develop into highly specialized spermatozoa (Fig. 2). Spermiogenesis is the transformation of spermatids into the elongated flagellar germ cells capable of motility. The release of mature germ cells is known as spermiation. The germ cells comprise the majority of testicular volume, which can be appreciated easily as a smaller size if testicular damage has occurred. A significant characteristic of mitotic arrest is that the gonocyte becomes acutely sensitive to toxic agents that may completely eradicate germ cells while causing little damage to developing Sertoli cells, thus creating a Sertoli cell-only testis.
IV. P E S T I C I D E S AND M A L E REPRODUCTION Many pesticides that are termed estrogenic pollutants (e.g., from agricultural products and industrial chemicals) have significant environmental consequences due to their multiple routes of exposure, their widespread presence in the environment, and their ability to bioaccumulate and resist biodegradation. Table 1 lists such possible agents and the reproductive adverse events caused by these agents.
CHAPTER 3 2 9Reproductive Toxicity of Anticholinesterases
TABLE 1. Effects of Hormonal Disruptors on Male Reproduction a Class Environmental Organochemicals and pesticides
Agent
Adverse events
DBCP DDT PCBs Dioxins Methyl chloride
~ fertility, ~ libido, embryo fetal loss, birth defects, cancer, estrogenic effects, poor semen quality HPG axis, $ spermatogenesis, CNS effects, testicular damage Germ cell and Leydig cell damage, steroidogenesis, 1' ROS, poor sperm morphology, ~, antioxidants, sperm function, LPO, 1' cytokines
aAbbreviafions used: DBCP, dibromochloropropane; DDT, dichlorodiphenyltrichloroethane; HPG,hypothalamic-pituitary-gonadalaxis; ROS, reactive oxygenspecies;LPO, lipid peroxidation.
A. Agricultural and Industrial Chemicals Agricultural chemicals implicated in male reproductive toxicity include DDT, epichlorhydrin, ethylene dibromide, kepone, and the dioxins (Worton et al., 1977). DBCP, a nematocide widely used in agriculture, is a testicular toxicant and induces hypergonadotropic hypogonadism (Mattison, 1983; Potashnik and Yanai-Inbar, 1987). DDT, a commonly used pesticide, and its metabolites (p,p'-DDT and p,p'-DDE) have estrogenic effects in males by blocking the androgen receptors (Kelce et al., 1994; McLachlan and Arnold, 1996). The levels of serum free/bound toxicant will influence the androgen-blocking capacity (Mattison, 1983). The plasma/tissue concentration of an estrogenic toxicant depends on the detoxification and elimination mechanisms in the organism. These agents can disrupt the hypothalamic-pituitary-gonadal axis shown in Fig. 1, thus affecting the endocrine and reproductive functions (Table 1).
hydrolyzes the neurotransmitter acetylcholine (ACh). The use of CM pesticides exceeds the use of OP and organochlofine pesticides. Some of the CMs, including carbofuran, are extremely toxic to the central nervous system (CNS) (Table 2). Overexposure of individuals involved in the production, transportation, and end use of these CMs can result in serious adverse health effects due to adverse events in the CNS and other vital organs. Similarly, carbofuran elicits the sign of acute intoxication by virtue of the reversible inhibition (carbamylation) of AChE at the synapses and neuromuscular junctions (Gupta, 2004; Goad et al., 2004). Cholinesterase has been detected in almost all major systems of the mammalian body, including the white matter of the brain, vascular system, respiratory system, digestive system, urogenital system, and certain endocrine systems. AChE is an enzyme located mainly in the nervous system and in the motor end plates of the skeletal muscle. Cholinesterase inhibitors were shown to modify the pituitary thyroid and pituitary-adrenal axes and to affect prolactin levels. When AChE is inhibited by cholinesterase inhibitors such as OP compounds, ACh accumulates at synapses, and parasympathetic overstimulation and adrenal stimulation may occur (Guven et al., 1999). In the presence of an inhibitor of AChE, synaptic acetylcholine may increase to abnormally high concentrations, which is postulated to precipitate a "cholinergic crisis" that can be debilitating and possibly fatal (Padilla et al., 1994; Fulton and Key, 2001). Sublethal doses of these pesticides lead to alterations in reproductive performance (Maitra and Sarkar, 1995, 1996; Sarkar et al., 2000). OP compounds have multiple effects on the animal endocrine system, but studies on the human endocrine system are inconclusive (Guven et al., 1999). All of these agents have numerous other chronic effects, including carcinogenesis, delayed polyneuropathy, immunotoxicity, and endocrine, developmental, and reproductive toxicity.
TABLE 2. Potential Sites of Toxic Effects of Pesticide Poisoning
Class Chlorinated hydrocarbons
Organophosphates
V. M E C H A N I S M ( S ) O F A C T I O N OF PESTICIDES Carbamates
A. Central Nervous System Effects In mammals, the primary sites of action of OP pesticides are the central and peripheral nervous systems because they inhibit acetylcholinesterase (AChE), the enzyme that
451
Agents Methoxychlor, iindane, toxaphene, chlordane Diazinon, malathion, parathion, chlorpyrifos dichlorvos Aldicarb, carbaryl, carbofuran
Site of action Neurotoxin,CNS, kidney, liver
Irreversible inhibition of red blood cell acetylcholinesterase and plasma cholinesterase Reversible inhibition of red blood cell acetylcholinesterase and plasma cholinesterase
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9Organ Toxicity
B. Effects on the H y p o t h a l a m i c - P i t u i t a r y - G o n a d a l Axis
Pesticides that are potential gonadotoxic agents can alter physiological control processes and affect the normal functioning of the reproductive system either by a direct chemical action of the agent on reproductive organs or indirectly via the metabolic products formed during the reaction process. These can interrupt the normal function of the male reproductive system at the level of the hypothalamic-pituitary axis; the testicular level; and/or by altering posttesticular events, such as sperm maturation, motility, and/or function. Any disruption of these events by toxicants may lead to hypogonadism, infertility, and/or decreased libido/sexual function (Wilson et al., 1990; Sikka, 1999a,b). The effect may be mild or severe, and the duration may vary from transient to severe dysfunction (Fig. 1). C. E n d o c r i n e - R e l a t e d Effects
Interactions involved in normal gonadal function and hormonal communication are very complex. Any of these loci may be involved mechanistically in a toxicant's endocrinerelated effect. Such impaired hormonal control may occur as a consequence of altered hormone synthesis, storage/release, transport/clearance, receptor recognition/binding, or postreceptor responses. 1. ALTERED HORMONE SYNTHESIS A number of pesticides possess the ability to inhibit the synthesis of various hormones by inhibiting specific enzymatic steps in the biosynthetic pathway. Some fungicides block estrogen biosynthesis by inhibiting aromatase activity. Some environmental estrogens and antiandrogens alter protein hormone synthesis induced by gonadal steroids. Both estrogen and testosterone have been shown to affect pituitary hormone synthesis directly or through changes in the glycosylation of LH and follicle-stimulating hormone (FSH) (Wilson et al., 1990). A decrease in glycosylation of these glycoproteins not only reduces the quantity but also reduces the biological activity of these hormones. Pesticides that mimic or antagonize the action of these steroid hormones could presumably alter glycosylation. 2. ALTERED HORMONE STORAGE AND/OR RELEASE Steroid hormones do not appear to be stored intracellularly within membranous secretory granules. For example, testosterone is synthesized by the Leydig cells of the testis and released on activation of the LH receptor. Thus, compounds that block the LH receptor or the activation of the 3',5'cyclic AMP-dependent cascade involved in testosterone biosynthesis can rapidly alter the secretion of this hormone. The release of many protein hormones is dependent on the activation of second messenger pathways, such as cAMP, phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5trisphosphate (IP3), tyrosine kinase, and intracellular calcium
[Ca2+]i . Interference with these processes will alter the serum levels and bioavailability of many hormones. 3. ALTERED HORMONE TRANSPORT AND CLEARANCE Hormones are transported from blood in the free or bound state. Steroid hormones are transported in the blood by specialized transport (carrier) proteins known as sex steroid hormone-binding globulin or testosterone-estrogen-binding globulin. Regulation of the concentration of these binding globulins in the blood is of practical significance because there may be either increases or decreases that could affect steroid hormone availability. For example, DDT analogs are potent inducers of hepatic microsomal monooxygenase activities in vivo (Sikka, 1999a). Induction of this monooxygenase activity by treatment with DDT analogs could possibly cause a decrease in testicular androgen as a result of enhanced degradation. Similarly, treatment with lindane (7-hexachlorocyclohexane) has been reported to increase the clearance of estrogen (Welch et al., 1971). 4. ALTERED HORMONE RECEPTOR RECOGNITION[BINDING Hormones elicit responses on their respective target tissues through direct interactions with either intracellular receptors or membrane-bound receptors. Specific binding of the natural ligand to its receptor is a critical step in hormone function. Intracellular (nuclear) receptors and response elements (such as those for sex steroids, adrenal steroids, thyroid hormones, vitamin D, and retinoic acid) regulate gene transcription in a ligand-dependent manner through their interaction with specific DNA sequences. A number of pesticides may alter this process by mimicking the natural ligand and acting as an agonist or by inhibiting binding and acting as an antagonist. The best known examples are DDT, some PCBs, alkylphenols (e.g., nonylphenols and octylphenols), methoxychlor, and chlordecone (Kepone), which can disrupt estrogen receptor function (Mueller and Kim, 1978; White et aL, 1994). The antiandrogenic action of the dicarboximide fungicide vinclozolin is the result of an affinity of this compound's metabolites for the androgen receptor (AR) (Sharpe and Skakkebaek, 1993). Interestingly, the DDT metabolite p , p ' - D D E has been found to bind also to the AR and block testosterone-induced cellular responses in vitro (Kelce et al., 1995). Some pesticides have the ability to interfere with steroid hormone receptors. These include chemicals such as the herbicide linuron (Gray et al., 1999c; McIntyre et al., 2000), metabolites of the fungicides vinclozolin (Gray et al., 1999b) and procymidone (Ostby et al., 1999), the insecticide methoxychlor (Gray et al., 1999a) and its metabolite HPTE (Maness et al., 1998), and the DDT metabolite p,p'DDE (Gray et al., 1999a). The structural diversity of these chemicals has heightened concern about the potential of other environmental chemicals to disrupt AR function and has led t o the development of models and strategies for predicting potential AR activity from chemical structure.
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases Tamura et al. (2001) demonstrated that fenitrothion is a competitive antagonist of the human AR and can inhibit androgen-dependent tissue growth in vivo. Inhibition of androgen-dependent tissue growth in vivo occurred with a dose of fenitrothion (15 mg/kg) that was not associated with a significant decrease in blood AChE activity, which is often used as a biomarker for human exposure to OP pesticides. Fenitrothrion represents one of the more potent environmental AR antagonists identified to date. Structural similarities between fenitrothrion and other OP compounds make it likely that additional OP insecticides will have antiandrogenic activity. Indeed, the OP pesticide parathion has been shown to inhibit DHT binding to the AR in the rat ventral prostate. Fenitrothrion inhibits brain AChE activity and induces signs of cholinergic stress (Tamura et al., 2001, 2003). In addition, Sarkar et al. (2000) reported that quinalphos, an OP insecticide, decreased the reproductive ability in males by affecting the hypothalamic-pituitary-gonadal axis. It was reported that fenitrothion bound to AR and acted as androgen antagonist. Many of the pesticides classified as environmental estrogens can actually inhibit binding to more than one type of intracellular receptor. For example, o,p-DDT and chlordecone can inhibit endogenous ligand binding to the estrogen and progesterone receptors, with each compound having ICs0s that are nearly identical for the two receptors. Receptors for protein hormones are located on and in the cell membrane. When these hormones bind to their receptors, transduction of a signal across the membrane is mediated by the activation of second messenger systems. These include (a) alterations in G protein/cAMP-dependent protein kinase A (e.g., after LH stimulation of the Leydig cell), (b) phosphatidylinositol regulation of protein kinase C and inositol triphosphate (e.g., after GnRH stimulation of gonadotrophs and thyrotropin-releasing hormone stimulation of thyrotrophs), (c) tyrosine kinase (e.g., after insulin binding to the membrane receptor), and (d) calcium ion flux through various calcium channels and their activation. Potent pesticides can disrupt such signal transduction mechanisms and interfere with one or more of these processes with toxic implications.
453
5. ALTERED HORMONE POSTRECEPTORACTIVATION Once the endogenous ligand or an agonist binds to its receptor, a cascade of events is initiated indicative of the appropriate cellular response. This includes the response necessary for signal transduction across the membrane or, in the case of nuclear receptors, the initiation of transcription and protein synthesis. A variety of environmental compounds can interfere with the membrane's second messenger systems. For example, cellular responses that are dependent on the calcium ion flux through the membrane (and the initiation of the calcium/calmodulin-dependent cellular response) are altered by a variety of environmental toxicants. Interestingly, the well-known antiestrogen tamoxifen also inhibits protein kinase C activity (O'Brian et al., 1985). Alternatively, the phorbol esters are known to mimic diacylglycerol and enhance protein kinase C activity. Steroid hormone receptor activation (including estrogen, progesterone, and glucocorticoid receptors) can be modified by indirect mechanisms, such as a downregulation of the receptor (temporary decreased sensitivity to ligand) as seen after TCDD exposure (Safe et al., 1991; Safe, 1995). Consequently, because of the diverse pathways of endocrine disruption, any assessment must consider the net result of all influences on hormone receptor function and feedback regulation. D. M e t a b o l i c Effects of Pesticides
Mahgoub and E1-Medany (2001) demonstrated that chronic administration of methomyl oxyethanimidothioate (lannate), a CM insecticide with anti-ChE activity, led to a significant reduction in the level of testosterone hormone and a significant increase in the level of FSH, LH, and prolactin. In addition, the decrease in succinic dehydrogenase, acid phosphatase, and esterase enzyme activities in the methomyl-treated male rats significantly affected mitochondrial metabolism and caused spermatogenic arrest (Fig. 3). The levels of acid phosphatase enzyme activity increased significantly in the Leydig and spermatogenic cells after such treatment (Mahgoub and E1Medany, 2001). Acid phosphatase enzyme plays an important role in cell metabolism, autolysis, differentiation, and many
T Membrane depolarization 11C 'oc rome c ox 0asel
,
I~ Creatinine kinase
TO em nma, rane a rxn0swen0 sru0, 0
~
~Organophosphoru~ Compounds
~ Bcl-2 levels
Alteration of mitochondrial oxygen uptake and respiration
I1 phosphorglation Oxidative I
FIG. 3. The metabolic effects of organophosphorus pesticides on mitochondrial metabolism.
454
SECTION IV. Organ Toxicity
related processes. The increase in acid phosphatase enzyme activity may be explained on the basis of enhancement of cell membrane permeability with disturbance in the transphosphorylation process as a result of cellular degeneration. In the same study, an increase in oL-esterase activity was observed in the interstitial Leydig cells that affected lipid metabolism induced by cellular membrane degeneration after insecticide intoxication (Afifi et al., 1991). Kackar et al. (1999) found that chronic exposure to mancozeb (a CM pesticide) in male rats produced a significant testicular dysfunction, as indicated by a marked reduction in serum testosterone level and sperm count. Goad and coworkers (2004) also demonstrated decreased serum testesterone levels in carbofuran-treated male rats due to reduced biosynthesis and release. Benomyl is a carbendazim-derivated insecticide that primarily affects the testis by sloughing effects of the chemical on microtubules and intermediate filaments of the Sertoli cells. These effects spread to dividing germ cells and also lead to abnormal development of the head of elongated spermatids. At higher dosages, it causes occlusion of the efferent ducts, blocking the passage of sperm from the rete testis to epididymis. The mechanism of occlusion appears to be related to fluid reabsorption, followed by leukocyte chemotaxis, sperm granulomas, fibrosis, and often the formation of abnormal microcanals. The occlusion results in a rapid swelling of the testis and ultimately seminiferous tubular atrophy and infertility. Benomyl may induce permanent testicular damage and a decrease in sperm production (Hess and Nakai, 2000). Afifi and coworkers (1991) reported that the administration of dimethoate (an OP compound) to male rats for 65 consecutive days caused suppression of testicular function with significant reduction in testosterone level and the number of motile sperm due to direct toxic effects of the insecticide on testicular tissue. OP insecticides, apart from inhibiting AChE activity, bind to the lipid component of mitochondrial membranes and alter mitochondrial function (Afifi et al., 1991). Such changes also include mitochondrial membrane depolarization, membrane disruption and matrix swelling, alterations in mitochondrial oxygen uptake and respiration, and inhibition of oxidative phosphorylation. In addition, inhibition of mitochondrial carboxylesterase, creatinine kinase and succinate dehydrogenase, cytochrome c oxidase, and NADH:cytochrome c reductase has been reported following exposure to OP compounds. These compounds increase mitochondrial transmembrane potantial and Bcl-2 levels (Fig. 3) (Carlson et al., 2000). Depending on the diminished level of ATP, intracellular sodium will accumulate with consequent gain of water associated with cell swelling and dilatation of the smooth endoplasmic reticulum in Sertoli, spermatogenic, and Leydig cells (Dunnick et al., 1984). Ezeasor (1990) correlated the dilatation of the endoplasmic reticulum in Leydig cells with the reduction in cellular activity and diminished androgen synthesis.
In addition, chlorpyrifos-methyl (CPM), another OP insecticide, is known to cause toxicity in adrenal gland, such as vacuolation of the zona fasicolata, which leads to low adrenal testosterone output (Breslin et al., 1996). Interestingly, Kang et al. (2004) observed that the relative weight of the adrenal gland was increased by the treatment of CPM in male castrated rats but not in immature female rats, suggesting compensatory activation of the androgen synthesis pathway in the adrenal gland.
E. Effects of OPs on Oxidative Stress and Apoptosis "Oxidative stress" is a condition associated with an increased rate of cellular damage induced by oxygen and oxygen-derived free radicals commonly known as reactive oxygen species (ROS). Exposure to many types of environmental contaminants can enhance this oxidative process both by increasing generation of free radicals and by decreasing antioxidant potential and thus causing gonadal damage (Sikka et al., 1995). Banks and Soliman (1997) showed that methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) induces lipid peroxidation and glutathione depletion in rats due to a significant increase in serum hydroperoxides and a significant decline in hepatic reduced glutathione (GSH) levels. In addition, N,Ndiphenyl-p-phenylenediamine (DPPD) and 21-aminosteroid (U74389G) blocked benomyl-induced lipid peroxidation and GSH depletion, thus confirming that in vivo toxicity of benomyl may be associated with increased oxidative stress to cellular membranes and that some degree of protection against this toxicity could be afforded by antioxidants. Such disturbance in the balance resulting in increased oxidative stress can influence male infertility (Fig. 4). The possible mechanism for loss of testicular and sperm function due to high oxidative stress has been shown to involve excessive generation of ROS (Aitken and Clarkson, 1987). Several studies have confirmed that oxidative stress is induced by OPs in rats (Gultekin et al., 2000; Gupta et al., 2001 a; Akhgari et al., 2003) and humans (Banerjee, 2001; Ranjbar et al., 2002). Small doses of OP compounds cause delayed neuronal cell death that involves free radical generation. OPs that cause mitochondrial damage and dysfunction due to depletion of ATP and increased generation of ROS can cause fetal depletion of mitochondrial energy (ATP), induction of proteolytic enzymes, and DNA fragmentation, leading to apoptotic death (Akhgari et al., 2003). Abdollahi et al. (2004a,b) demonstrated that inhibition of cholinesterase activity by malathion, a commonly used OP, is accompanied by induction of oxidative stress, suggesting use of saliva sampling as an alternative to plasma in OP toxicity monitoring. The phospholipid component of cell membranes is suggested as a site of toxic action of OP compounds. Similarly, the generation of nitric oxide (NO) and reactive nitrogen species has been found to have an astounding range
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases
Cellular Free Radical-Scavenging System Superoxide Dismutase Catalase Glutathione Peroxidase Vitamin E Carnitine Glutathione
Free Radical-Generating System Leukocytes and Inflammatory Components Toxicants Drug Radiation Immature Germ Cells
IT
It ~ T Protein Damage
Lipid Peroxidation
I
1
1
I
I Stress I T Biomembrane Damage
T Sperm Damage
l
455
INFERTILITY
of biological roles, including vascular tone, inflammation, and as a mediator of many cytotoxic and pathological effects. NO along with superoxide radicals induces endothelial cell injury, which may result in testicular dysfunction. This is primarily due to the vasoactive effects of NO- radical rather than the direct effects on testicular cells. Rao and coworkers (1999) investigated the effect of carbaryl, kepone, and malathion on NO synthase (NOS) activity in the rat brain. They showed that all three compounds inhibit NOS activity of rat brain in vitro in a concentration-dependent manner. In addition, they demonstrated that these insecticides inhibit calmodulin (CaM)-stimulated NOS activity without affecting the basal enzyme activity. According to their results, the inhibition of NOS activity by these insecticides may be due to their interaction with CaZ+/CaM (Rao et al., 1999). It is important to note that increased NOS activity, especially iNOS, due to OP exposure results in increased generation of NO free radical in inflalmmatory cells that usually combines with available superoxide ions to form highly toxic peroxynitrite radicals, resulting in increased oxidative insult and apoptotic damage (Fig. 5).
T
DNA Damage
FIG. 4. ROS generating and scavenging systems responsible for inducing oxidative stress to components of male reproductive axis leading to sperm damage and infertility.
Cytochrome c oxidase (COx), the terminal enzyme complex of the mitochondrial respiratory chain and the last site for ATP synthesis, has a critical function that if disturbed would seriously affect the energy production in the cell. Its dependency on the polyunsaturated phospholipid cardiolipin renders this enzyme especially vulnerable to peroxidative damage (Potashnik and Yanai-Inbar, 1987). Under conditions in which COx is well controlled, ROS formation is small; however, when the capacity of this enzyme is reduced, the risk for incomplete reduction of 02 and the formation of ROS increases (Staniek and Nohl, 2000). Lipid peroxidation, mitochondrial dyshomeostasis or dysfunction or damage, reduction of neuronal energy level, and reduced COx activity support the contention that AChEIs, such as diisopropyl phosphorofluoridate (an OP compound) and carbofuran (a CM compound) cause neuronal injury by excessive formation of ROS (Yang and Dettbarn, 1998; Gupta et al., 2001b). Carlson et al. (2000) observed significant OP compoundinduced increases in caspase-3 activation followed by increased nuclear fragmentation, characteristic of traditional apoptosis. High concentrations of PSP and TOTP (1 mM)
456
SECTION IV. Organ Toxicity
I Organophosphoruscompounds I
I
Mitochondrial damage/dysfunction I ATP depletion I
l Increasedgeneration of ROS
IReleaseof L-GlutamateI Stimulation of NMDA receptor sites I Activation of NOS I
1 I Increases of peroxynitrite I
I OXIDATIVESTRESS I I proteolytic Increases ~ enzymes
I DNA fragmentation I
Free radicals play an important role in toxicity of pesticides and environmental chemicals. Pesticide chemicals may induce oxidative stress, leading to generation of free radicals and alteration in antioxidants or the oxygen free radical scavenging system. This system includes SOD, CAT, gamma-glutamyl transpeptidase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, etc. (Datta et al., 1992; Banerjee et al., 1999; Sharkawy et al., 1994). Glucose-6-phosphate dehydrogenase catalyzes the initial step of the pentose phosphate pathway, whose most important function is the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH, which is used for the reduction of oxidized glutathione to the reduced state (GSH) and for the reduction of mixed disulfides of GSH and cellular proteins. This GSH is used for the detoxification of hydrogen peroxide (H202) and organic peroxides (Gurbuz et al., 2004). It has been indicated that the enzymes associated with such antioxidant defense mechanism are altered under the influence of pesticide and that lipid peroxidation is one of the molecular mechanisms involved in pesticide-induced cytotoxicity (Banerjee, 2001). It will be interesting to investigate the role of potential antioxidants in OP- and CM-induced toxicity.
FIG. 5. Mechanism of action of organophosphorus compounds on oxidative stress and apoptosis. VI. A S S E S S M E N T O F G O N A D O T O X I C I T Y induced caspase-3 activation and nuclear condensation. In addition, pretreatment with carbachol, cyclosporin A, protease inhibitors, and PMSF altered OP compound-induced caspase-3 activation and nuclear fragmentation. This suggested that multifunctional pathways such as muscarinic receptor activation or an increase in Bcl-2, mitochondrial permeability transition pore closure, receptor-mediated caspase activation, or serine protease activation were involved in OP-induced cytotoxicity (Carlson et al., 2000). The assumption that free radicals can influence male fertility has received substantial scientific support (Gagnon et al., 1991). The proposed mechanism for loss of testicular and sperm function due to oxidative stress has been shown to involve excessive generation of ROS (Aitken and Clarkson, 1987). Free radicals can damage DNA and proteins, either through oxidation of DNA bases (primarily guanine via lipid peroxyl or alkoxyl radicals) or through covalent binding to MDA resulting in strand breaks and cross-linking (Alvarez et al., 1987). ROS can also induce oxidation of critical SH groups in proteins and DNA, which will alter cellular integrity and function with an increased susceptibility to attack by toxicants. Oxidative stress is theoretically the result of an improper balance between ROS generation and intrinsic scavenging activities. Adequate levels of superoxide dismutase (SOD), catalase (CAT), and probably GSH peroxidase and reductase normally maintain the free radical scavenging potential in the testes. This balance can be referred to as oxidative stress status, and its assessment may play a critical role in monitoring testicular toxicity and infertility (Sikka, 1997).
Several methods are being evaluated for the assessment of the effects of toxicants on the male reproductive system. Essentially, any risk assessment usually has four components: hazard identification (Johnson, 1986), dose-response assessment (Mattison et al., 1990), human exposure assessment, and risk characterization (Cranmer et al., 1978). The hazard identification and dose-response data are developed from experimental animal studies that may be supplemented with data from in vitro studies. This information is then extrapolated and integrated to characterize and assess the risk to the human population. The most common approach to evaluate the effect of cytotoxic drugs on the testis has used testicular biopsy, semen analysis, and endocrine assessment of the hypothalamic-pituitary-testicular axis (Table 3). Research on testicular toxicology has been advanced significantly by the introduction of in vitro testing systems. In vivo systems, however, are still essential parts of the risk assessment process, and they are unlikely to be eliminated by in vitro models.
A. In Vitro Systems In vitro systems are uniquely suited to investigate specific
cellular and molecular mechanisms in the testis and thus improve risk assessment (Lamb and Chapin, 1993). These in vitro models can be used alone or in combination with each other to test hypotheses about testicular toxicity. A toxicant, its metabolites, the precursors, or selective inhibitors
CHAPTER 32 9Reproductive Toxicity of Anticholinesterases TABLE 3.
Potential site Testis Leydig cells
Sertoli cells
Seminiferous tubules
Evaluation of Effects of OP and CM in the Adult MaMa Effects
Evaluative tests
Necrosis LH/PRL, T biosynthesis/ secretion FSH/inhibin/ steroids, Sertoli/Leydig cell function
Weight, histopathology receptor analysis, RIA, in vitro production and hormone assay Receptor analysis, RIA, in vitro tests (coculture),bloodtestis barrier, morphology Germ cell count and % tubules without germ cells Spermatid counts and % tubules with luminal sperm Germ cell culture, morphology Histopathology, biochemical tests Pituitary cell culture, hypothalamus perfusion, histopathology, hormone challenge, accessory sex organ weights Spermatid counts, semen evaluation Hormones/ABP assays
Spermatogonial mitosis Spermatocyte meiosis
Epididymis
Spermatid differentiation Sperm maturation
Brain
Hypothalamicpituitary a x i s
Seminal fluid
Daily sperm production HPG a x i s
Blood
aAbbreviations used: LH, luteinizing hormone; PRL, prolactin; RIA, radioimmunoassay; FSH, follicle-stimulating hormone; ABP, androgen binding protein; HPG, hypothalamic-pituitary-gonadalaxis. can be individually administered to isolated cell types to evaluate specific toxicity mechanisms and to note the interaction of adjacent cell types. Numerous in vitro model systems are described in the literature, including Sertoli-germ cell cocultures (Gray, 1988), Sertoli cell-enriched cultures (Chapin et al., 1990; Steinberger and Clinton, 1993), germ cell-enriched cultures (Foster et al., 1987), Leydig cell cultures (Ewing et al., 1981), Leydig-Sertoli cell cocultures (Chapin et al., 1990), and peritubular and tubular cell cultures (Gray, 1988; Chapin et al., 1990). Use of these in vitro systems is the only way to directly compare human and animal responses and to screen a class of compounds for new product development. Although these in vitro systems are a valuable adjunct to the in vivo test system, they do not replace the in vivo data because they cannot provide all the facts essential for hazard assessment. Moreover, certain dynamic changes associated with spermatogenesis are diffi-
457
cult to model in vitro. For example, the release of elongated spermatids by the Sertoli cells (spermiation), which is commonly inhibited by boric acid and methyl chloride, can only be studied by specific in vivo systems.
B. In Vivo S y s t e m s In vivo methods are important tools to study the integrated male reproductive system. The complete in vivo assessment
of testicular toxicity involves multigenerational studies, now required by most regulatory agencies. These multigenerational studies have a complex design because testicular function and spermatogenesis are very complicated processes. The spermatogenic cycle is highly organized throughout the testis. In the rat, it requires 53 days. If a toxicant affects the immature spermatogonia, the effect may not be detectable as a change in mature sperm before 7 or 8 weeks. Effects on more mature germ cells would be detected sooner. To test the sensitivity of all stages of spermatogenesis, the exposure should last the full duration of the cycle. This cannot be achieved in vitro because germ cell differentiation and the physical relationship of stages within the tubules are lost in cell culture systems. The germ cells are entirely dependent on the Sertoli cells for physical and biochemical support. Complicated endocrine and paracrine systems control Sertoli cells, Leydig cells, and germ cells. Besides the loss of paracrine interactions, the altered metabolic activity of target or adjacent cells and difficulty in isolating and testing certain spermatogenic stages are other significant limitations of in vitro assessment of testicular toxicity (Soto et al., 1992). In addition, for accurate identification of stage-specific lesions of the seminiferous epithelium, critical evaluation of morphological structures is very important. Because germ cells are continuously dividing and differentiating, the staging of spermatogenesis has proven to be an extremely sensitive tool to identify and characterize even subtle toxicological changes. The effects of chronic sublethal doses ( 7 - 1 4 m g k g -1 daily for 15 days) of quinalphos were evaluated in adult male rats for changes in testicular morphology, circulatory concentrations of hormones (LH, FSH, prolactin, and testosterone), activities of AChE and angiotensin-converting enzyme, as well as metabolism of biogenic amines (dopamine, noradrenaline, and 5-hydroxytryptamine) in the hypothalamus and pituitary. It may be postulated that the initial effects of the pesticide are the result of increases in LH and testosterone concentrations, which affect spermatogenesis. Pesticideinduced inhibition of AChE in turn might increase concentrations of ACh in the pituitary and hypothalamus. This indicates that in pesticide toxicity, the hypothalamic-pituitary-gonadal axis is operational. Since many of the observed pesticide effects could be inhibited by estradiol, it is suggested that the pesticide acts directly on the gonadotrophins. In conclusion, quinalphos decreases fertility in adult male rats by affecting the pituitary gonadotrophins (Sarkar et al., 2000).
458
SECTION I V .
Organ Toxicity
C. Sperm Nuclear Integrity Assessment Chemicals may disrupt the structural stability of sperm nuclei, which depend on their unique packaging during either spermatogenesis or sperm maturation. Decondensation of an isolated sperm nucleus in vitro can be induced by exposure to disulfide reducing agents, and the time taken to induce extensive decondensation (assay end) is considered to be inversely proportional to the stability of the sperm nucleus. Attention has focused on assessments of sperm morphology and physiology as important end points in reproductive toxicology testing (Darney, 1991). Structural stability of sperm nuclei varies by species, appears to be enhanced by the oxidation of protamine sulfhydryl to inter- and intramolecular disulfide bonds, and is a function of the types of protamine present. This "sperm activation assay" is also useful in the evaluation of some cases of unexplained infertility (Brown et al., 1995). Human sperm decondenses most rapidly, followed by that of the mouse and hamster, whereas rat sperm nuclei show a slower decondensation (Perreault et al., 1988). Other tests, called DNA stability assay or sperm chromatin structure assay (SCSA), use direct evaluation of sperm chromatin integrity and may provide information about genetic damage to sperm. A shift in DNA pattern (from double-stranded intact DNA to denatured singlestranded) can be induced by a variety of mutagenic and chemical agents and evaluated either by DNA flow cytometric analysis or by SCSA (Evenson et al., 1986; Evenson, 1989). A single cell gel electrophoresis (Comet) assay, which uses fluorescence intensity measurements by microscopy and image analysis, has also been developed (Brown et al., 1995). A shift in the DNA pattern can also be evaluated by acridine orange staining, in which doublestranded DNA is stained green and single-stranded DNA is stained red. Animals exposed to known mutagens demonstrate increased amounts of single-stranded DNA, indicating an increase in genetic damage (Evenson et al., 1991; Ulbrich and Palmer, 1995). DNA flow cytometry is a very useful tool that permits rapid, objective assessment of a large number of cells but may not be readily available. Comet assay, when combined with centrifugal elutriation, can provide a useful in vitro model to study differences in metabolism and the susceptibility of different testicular cell types to DNA-damaging compounds. Thus, new findings using these systems should lead to greater knowledge about why a chemical or class of chemicals can cause testicular toxicity.
VII. S C I E N T I F I C D E B A T E In the wake of media coverage on possible reproductive health and cancer concerns (Raloff, 1994), a few toxicologists have questioned whether these adverse health effects can be attributed to environmental endocrine disruption
(Stone, 1994; Safe, 1995). Arguments for a demonstrable link between hormone-disruptive environmental agents and human reproductive health effects are supported by the fact that many pesticides and other agents with estrogenic or antiandrogenic activity operate via hormone receptor mechanisms. However, in the few studies on suspected weak estrogens, such as the alkylphenols, approximately 1000-10,000 times or up to 106 more agent is required to bind 50% of the estrogen receptor than estradiol (White et al., 1994). Of course, crucial to risk assessment is the need to know how many receptors must be occupied before activation of a response can ensue. For some hormones, such as human chorionic gonadotropin, as little as 0.5-5% receptor occupancy is required for full activation of response. For other hormones (those that require protein synthesis for expression of effect), higher levels of receptor occupancy are needed. Fluctuations of hormone concentration and receptor activities, by design, absorb some environmental and physiological challenges to maintain homeostasis in adults. Only when the equilibrium control mechanisms are overwhelmed do the deleterious effects occur. An important question is whether homeostatic mechanisms are operative in the embryo and fetus. Some investigators (Soto et al., 1992) have proposed the use of in vitro assays to screen for estrogenic or other hormonal activity. Although steroid receptors bound to their ligand act as transcription factors for gene expression in the target tissue, simple in vitro screening assays based on binding to a receptor are not sufficient for measuring hormone activity.
VIII. C O N C L U S I O N S Humans have experienced increased incidences of developmental, reproductive, and carcinogenic effects caused by pesticides acting to disrupt the endocrine system, which regulates these processes. In contrast, the hypothesis that the reported increased incidence of human cancers and reproductive abnormalities and infertility can be attributed to an endocrine disruption phenomenon is called into question for several reasons. First, secretion and elimination of hormones are highly regulated by the body, and mechanisms for controlling modest fluctuations of hormones are in place via negative feedback control of hormone concentrations. Therefore, minor increases following dietary absorption and liver detoxification of these xenobiotics may be inconsequential in disrupting endocrine homeostasis. Second, low ambient concentrations of chemicals along with low-affinity binding of purported xenobiotics to target receptors probably are insufficient to activate an adverse response in adults. Whether the fetus and the young are capable of regulating minor changes to the endocrine milieu is uncertain. Finally, the data are not available for mixtures such pesticides that may be able to affect endocrine function. At the same time, in the case of
CHAPTER 32
environmental estrogens as endocrine disrupters, it is known that competition for binding sites by antiestrogens in the environment may moderate estrogenic effects of some chemicals. Clearly, more research to fill data gaps and to remove the uncertainty in these unknowns is needed. With few exceptions (e.g., DES), a causal relationship between exposure to a specific environmental agent and an adverse effect on human health operating via an endocrine disruption mechanism has not been established. Short-term screening studies could be developed and validated in an effort to elucidate the mechanism. Through controlled dose-response studies, it appears that these compounds (e.g., alkyl phenol ethylates and their degradation products, chlorinated dibenzodioxins and difurans, and polychlorinated biphenyls) can induce irreversible induction of male sex characteristics on females (imposex), which can lead to sterility and reduced reproductive performance. There is an urgent need to characterize all the factors involved in such pesticide-induced reproductive toxicity and to develop reliable animal models of testicular disease. No major advances have been made regarding the medical management of poor sperm quality. The application of assisted reproductive techniques such as Y-chromosome deletion evaluation followed by ICSI regardless of cause does not necessarily treat the cause and may inadvertently pass on adverse genetic consequences.
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inhibitor-induced status epilepticus: Protection by antioxidants. NeuroToxicology 22, 271-282. Gupta, R. C., Milatovic, D., and Dettbarn, W. D. (2001b). Nitric oxide modulates high-energy phosphates in brain regions of rats intoxicated with diisopropylphosphorofluoridate or carbofuran: Prevention by N-tert-butyl-alpha-phenylnitrone or vitamin E. Arch. Toxicol. 75, 346-356. Gurbuz, N., Yalcin, O., Aksu, T. A., and Baskurt, O. K. (2004). The relationship between the enzyme activity, lipid perox. idation and red blood cells deformity in hemizygous and heterozygous glucose-6-phosphate dehydrogenase deficient individuals. Clin. Hemorheol. Microcirc. 31, 235-242. Guven, M., Bayram, F., Unluhizarci, K., and Kelestimur, F. (1999). Endocrine changes in patients with acute organophosphate poisoning. Hum. Exp. Toxicol. 18, 598-601. Hess, R. A., and Nakai, M. (2000). Histopathology of the male reproductive system induced by the fungicide benomyl. Histol. Histopathol. 15, 207-224. Johnson, E. M. (1986). Perspectives on reproductive and developmental toxicity. Toxicol. Ind. Health 2, 453-482. Kackar, R., Srivastava, M. K., and Raizada, R. B. (1999). Assessment of toxicological effects of mancozeb in male rats after chronic exposure. Indian J Exp. Biol. 37, 553-559. Kang, H. G., Jeong, S. H., Cho, J. H., Kim, D. G., Park, J. M., and Cho, M. H. (2004). Chlropyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats. Toxicology 1, 219-230. Kavlock, R. J., and Perreault, S. D. (1994) Multiple chemical exposure and risks of adverse reproduction function and outcome. In Toxicological of Chemical Mixtures: From Real Life Examples to Mechanisms of Toxicology Interactions (R. S. H. Yang, Ed.), pp. 245-297. Academic Press, Orlando, FL. Kelce, W. R., Monosson, E., Gamcsik, M. P., Laws, S. C., and Gray, L. E., Jr. (1994). Environmental hormone disruptors: Evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. Toxicol. Appl. Pharmacol. 126, 276-285. Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen, J. A., and Wilson, E. M. (1995). Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature 15, 581-585. Kumar, S. (2004). Occupational exposure associated with reproductive dysfunction. J. Occup. Health 46, 1-19. Lamb, D. J. (1997). Hormonal disruptors and male infertility: Are men at serious risk? Regul. Toxicol. Pharmacol. 26, 1-3. Lamb, J. C., and Chapin, R. E. (1993). Testicular and germ cell toxicity: In vitro approaches. Reprod. Toxicol. 1, 17-22. Mahgoub, A. A., and E1-Medany, A. H. (2001). Evaluation of chronic exposure of the male rat reproductive system to the insecticide methomyl. Pharmacol. Res. 44, 73-80. Maitra, S. K., and Sarkar, R. (1995). Morphological study of the testes in relation to the brain and testicular acetylcholinesterase activity in an organophosphate pesticide ingested wild passerine bird (Lonchura malabarica). Folia Biol. 43, 143-149. Maitra, S. K., and Sarkar, R. (1996). Influence of methyl parathion on gametogenic and acetylcholinesterase activity in the testis of whitethroated munia (Lonchura malabarica). Arch. Environ. Contamination Toxicol. 30, 384-389.
CHAPTER 3 2 9Reproductive Toxicity of Anticholinesterases Maness, S. C., McDonnell, D. R, and Gaido, K. W. (1998). Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells. Toxicol. Appl. Pharmacol. 151, 135-142. Mattison, D. R. (1983). The mechanisms of action of reproductive toxins. Am. J. Ind. Med. 4, 65-79. Mattison, D. R., Plowchalk, D. R., Meadows, M. J., al-Juburi, A. Z., Gandy, J., and Malek, A. (1990). Reproductive toxicity: Male and female reproductive systems as targets for chemical injury. Med. Clin. North Am. 74, 391-4 11. McIntyre, B. S., Barlow, N. J., Wallace, D. G., Maness, S. C., Gaido, K. W., and Foster, R M. D. (2000). Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat. Toxicol. Appl. Pharmacol. 167, 87-99. McLachlan, J. A., and Arnold, S. E (1996). Environmental estrogens. Am. Sci. 84, 452-461. Mueller, G. C., and Kim, U. H. (1978). Displacement of estradiol from estrogen receptors by simple alkyl phenols. Endocrinology 102, 1429-1435. O'Brian, C. A., Liskamp, R. M., Solomon, D. H., and Weinstein, I. B. (1985). Inhibition of protein kinase C by tamoxifen. Cancer Res. 45, 2462-2465. Ostby, J., Kelce, W. R., Lambright, C., Wolf, C. J., Mann, P., and Gray, L. E., Jr. (1999). The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro. Toxicol. Ind. Health 15, 80-93. Padilla, S., Wilson, V. Z., and Bushnell, P. J. (1994). Studies on the correlation between blood cholinesterase inhibition and "target tissue" inhibition in pesticide-treated rats. Toxicology 6,
11-25. Parvinen, M., Lahdetie, J., and Parvinen, L. M. (1984). Toxic and mutagenic influences on spermatogenesis. Arch. Toxicol. Suppl. 7, 128-139. Perreault, S. D., Barbee, R. R., Elstein, K. H., Zucker, R. M., and Keefer, C. L. (1988). Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry. Biol. Reprod. 39, 157-167. Potashnik, G., and Yanai-Inbar, I. (1987). Dibromochloropropane (DBCP): An 8-year reevaluation of testicular function and reproductive performance. Fertil. Steril. 47, 317-323. Raloff, J. (1994). The gender benders. Are environmental "hormones" emasculating wildlife? Sci. News 145, 24-27. Ranjbar, A., Pasalar, P., and Abdollahi, M. (2002). Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorous pesticide manufacturing workers. Hum. Exp. Toxicol. 21, 179-182. Rao, M. R., Kanji, V. K., and Sekhar, V. (1999). Pesticide induced changes of nitric oxide synthase in rat brain in vitro. Drug Chem. Toxicol. 22, 411-420. Safe, S., Astroff, B., Harris, M., Zacharewski, T., Dickerson, R., Romkes, M., and Biegel, L. (1991). 2,3,7,8 Tetrachlorodibenzop-dioxin (TCDD) and related compounds as antioestrogens: Characterization and mechanism of action. Pharmacol. Toxicol. 69, 400-409. Safe, S. H. (1995). Environmental and dietary estrogens and human health: Is there a problem? Environ. Health Perspect. 103, 346-351.
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CHAPTER
~
Placental Toxicity of Organophosphate and Carbamate Pesticides OLAVI PELKONEN, 1 KIRSI VAHAKANGAS, z AND RAMESH C. GUPTA3 1University of Oulu, Oulu, Finland 2University of Kuopio, Kuopio, Finland 3Murray State University, Hopkinsville, Kentucky
diseases, such as diabetes (Radaelli et al., 2003; Bjarneg~d et al., 2004) or hypertension (Sgambati et al., 2004; Ockleford et al., 2004), may have altered function, and these fetuses may be at an increased risk for toxic effects. This chapter provides some background information for the understanding and prediction of what pesticides can, in principle, do to the placenta and what the placenta does to pesticides. Also, a survey of methodologies employing human placenta as a tool to investigate and predict some aspects of developmental toxicity is presented. Finally, our current knowledge on the toxicokinetics and toxicodynamics of pesticides in the placenta and how these processes affect the fetus and pregnancy in general is reviewed.
I. I N T R O D U C T I O N During pregnancy, placenta serves many functions, including the production and release of hormones and enzymes; transport of nutrients and waste products; chemical information flow between mother and fetus; implantation; cellular growth and maturation; and, at the terminal phase of placental life, participation in delivery. The placental functions are carried out by two cell types, cytotrophoblasts and syncytiotrophoblasts, probably with overlapping tasks. These cells, together with vascular endothelial cells and connective tissue cells, form a layer of cells connecting and separating two genetically distinct individuals: the mother and the conceptus. In the assessment of placental toxicology of any foreign chemical substances, there are two major areas of concern: what the placenta does to xenobiotics and what xenobiotics do to the placema (Myllynen et al., 2005). In the former area; the major topics of concern are the entry and possible storage of substances in placental cells and through the placenta, aided perhaps by various transporters and efflux pumps; the distribution and binding of compounds in placental cells; and biotransformation of substances by intracellular enzymes. Metabolic activation and production of reactive intermediates by placental enzymes link these areas with toxicodynamics of placental toxicants. In the latter area, effects of compounds on placental blood flow and vasculature and the presence of membrane and intracellular receptors, enzymes, and other potential targets for foreign substances are important areas of inquiry for placental toxicity. The health of the placenta is a prerequisite for the health of the fetus. Consequently, any xenobiotic-induced damage to the placenta may cause damage to the fetus. Thus, it is very important to identify xenobiotics damaging the placenta and to elucidate their mechanisms of action associated with the toxicity. Furthermore, placentas of mothers with certain Toxicology of Organophosphate and Carbamate Compounds
II. S P E C I F I C A S P E C T S O F P L A C E N T A L TOXICITY STUDIES A. Species Differences in Placental Structure and Function Anatomically, the placental architecture is composed of cells derived from fetal membranes. Placentation differs among differentspecies and is therefore classified into four types: hemochorial (human, rat, and rabbit), endotheliochorial (cat and dog), syndesmochorial (sheep and ruminants), and epitheliochorial (pig and horse). The placenta has also been described as zonary in the dog, bidiscoid in the monkey, and multicotyledonary in the sheep. The placental thickness depends on the number of fetal and maternal cell layers. For example, the rat and rabbit have a single layer of cells, primates and humans have three layers, and pigs and horses have six layers. There are anatomical and qualitative physiological functional similarities and differences in placentas of different species that should be taken into consideration when studying placental toxicity. The human placenta is of 463
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SECTION IV. O r g a n T o x i c i t y
the hemochorial type, in which the fetal tissue is in direct contact with the maternal blood. The membrane separating the maternal and fetal compartments is thin and consists of the trophoblastic epithelium covering the villi, the chorionic connective tissue, and the fetal capillary endothelium. The average thickness of the barrier at term is 3.5 I~m, the average exchange area is approximately 11 m 2, and the placental blood flow rate is approximately 450 ml/min (Pacifici and Nottoli, 1995). It is well established that the intensity of passage of substances across the placenta is inversely proportional to the thickness of placental membranes. In hemochorial placenta, the intensity of exchange decreases at the end of gestation due to deposition of fibrinoid on the exchanging surface. Exchange involves not only physiological constituents but also substances or chemicals that represent a pathological risk for the placenta and/or fetus. Therefore, in assessing placental toxicology of chemicals, all physiological and functional variables should be taken into consideration. Note that the toxicological findings observed in the placenta of one species for a particular chemical should not be generalized to the placenta of another species. In other words, wide species variability exists in sensitivity to acetylcholinesterase (AChE)-inhibiting pesticide-induced placental toxicity. Furthermore, a wide interindividual variability for susceptibility exists during the gestation period.
TABLE 1.
Cell type
B. Life Span of the H u m a n Placenta The appreciation of the complexity of placenta as an organ has increased. During its lifetime of 9 months, it goes through a complicated development that requires highly proliferative and invasive capacities as well as an extremely tight control of the process (Chakraborty et aL, 2003). The expression of many factors is very different at different stages of pregnancy. For instance, the expression of Fas ligand, protecting placenta from cell-mediated immune response, decreases at term (Balkundi et al., 2000). Anatomical, physiological, and metabolic characteristics vary depending on the age of the placenta. Thus, it is important to study placentas from different stages of pregnancy. However, obtaining material for such studies is difficult for practical and ethical reasons. Since the placental thickness and the number of cell layers diminish toward the end of pregnancy, it is possible that the term placenta is the most sensitive to environmental agents. However, deposition of fibrinoid may be a counteracting factor. In term placenta, there are two cell layers (syncytium and endothelium) with basal laminae and connective tissue in between (Jinga et al., 2000). Information on the detailed cellular structure of the placenta is still emerging (Ockleford et al., 2004). Histologically, several cell types with their specialized functions can be recognized in the placental tissue (Table 1). Whereas some factors, such as endothelin,
Placental Cell Types and Their Functions a
Specific markers
Reported functions and characteristics
References
Anchorage and invasive functions, mononuclear, do not fuse Transplacental transfer, multinucleated endocrine functions
Ockleford et al. (2004), Old et al. (2004) Old et al. (2004), Malassine and Cronier (2002), Guyon et al. (2004)
Progenitor cells for syncytiotrophoblasts Hemostasis
Old et al. (2004), Guyon et al. (2004)
Extravillous trophoblast (EVT)
hPL, ErbB2, CAM
Syncytiotrophoblast (S cell)
hPL, ErbB 1, ErbB2, NPD kinase C
Villous cytotrophoblast (C cell)
ErbB 1, NDP kinase A and B Tissue factor Cytosolic PGES Cytosolic PGES vWf, ACE, BS-I lectin
Immunologic function Granular cytoplasm
Strong expression of vWf, ACE, UEA lectin, WGA lectin vWf, ACE
Long life span, higher proliferation than large placental vessels, rod-like cytoplasmic particles Granular cytoplasm
Jinga et al. (2000)
oL-Smooth muscle actin, [3-tropomyosin, h-caldesmon
Elongated cells in parallel rows
Leik et al. (2004)
Decidual cells Villous fibroblasts Villous macrophages Endothelial cells from large vessels Endothelial cells from villous microvessels Endothelial cells from umbilical cord Smooth muscle cells of arterial wall
aAbbreviations used: CAM, cell adhesion molecules; hPL, human placental lactogen; vWf, von Willebrand factor.
Lockwood et al. (2001) Meadows et al. (2004) Meadows et al. (2004) Jinga et al. (2000)
Jinga et al. (2000)
CHAPTER 33 9Placental Toxicity of OPs and CMs are produced by most of the cell types of the placenta (Chakraborty et al., 2003), others are strictly expressed by one cell type only, such as cytokeratin-7, which is specific for trophoblast (Oki et al., 2004). Nitric oxide, produced by endothelial cells, diffuses to vascular smooth muscle cells, where it regulates the vessel diameter by relaxing the smooth muscle cells (Bisseling et al., 2004). Such local mediators are important in the placenta because it lacks autonomic innervation.
III. G E N E R A L A S P E C T S O F P L A C E N T A L TOXICOKINETICS An attempt to encompass salient features of transplacental transfer and potential major factors governing various processes in the transfer is presented in Fig. 1. Our understanding of detailed placental toxicokinetic processes has advanced thus far because there is a need to understand the fate and effects of drugs in pregnant subjects (Ala-Kokko et al., 2000). Furthermore, studies on drugs use relatively high and fixed doses, obviating the analytical sensitivity problems. Studies on pesticides need to be performed at a somewhat more elementary level and only in experimental animals. Thus, extrapolation to the human in vivo situation, in which exposures are at a relatively low level, may pose considerable problems and uncertainties. Although some toxicokinetic processes (e.g., metabolism, plasma protein binding, and membrane permeation) can be studied in in vitro systems, these systems are often based on liver (e.g., metabolism), making extrapolation to the placental situation problematic.
465
A. Entry of Xenobiotics into Placental Cells A paradigm accepted practically without reservation until just a few years ago was that foreign chemicals are transferred through placental membranes by passive diffusion. The rate and extent of transfer were thought to be dependent on surface area, blood flow, concentration gradient across placental membranes, and physicochemical properties of a chemical, such as lipophilicity, molecular weight, and degree of ionization (Pacifici and Nottoli, 1995). Although this paradigm is still true to a certain extent, it has to be modified by the emerging appreciation of the role played by various transporters, which in some cases are able to also accept xenobiotic substances. Table 2 lists some general properties of transporters that affect the entry and transfer of at least a few xenobiotic chemicals. The demonstration of a chemical as a ligand for a particular transporter does not automatically guarantee that the particular transporter is important for the transfer of the compound in vivo. It is possible that passive transfer could still be far more important than active transport, depending on the relative importance of these two ways of transfer and on other factors such as concentration of the compound.
B. Distribution of Xenobiotics in the Placenta It has been demonstrated in a number of cases that some xenobiotic substances bind relatively tightly, although reversibly, to placental components. Good evidence on binding has been provided for several drugs (Ala-Kokko et al., 2000). In these cases, placenta serves as a kind of a sink, which has to be saturated before substances can be transferred along the concentration gradient to the opposite side of the placenta. In all of the examples mentioned by Ala-Kokko et al., it is not known which placental components are responsible for binding. However, it is a good guess that certain placental proteins and small molecular xenobiotics may have mutual affinity, which is then reflected as an attenuation of transplacental transfer.
C. Metabolism of Xenobiotics in the Placenta
FIG. 1. Scheme of potential factors in placenta that affect the transplacental transfer and ensuing effects on placenta and fetus. Although very little is known about the role of transporters, metabolizing enzymes, binding proteins, and cellular targets in pesticide kinetics and dynamics, there are a number of experimental tools available for the elucidation of these factors.
The presence of various classes of the xenobiotic-metabolizing enzymes in human placenta has been demonstrated convincingly. However, on closer inquiry one has to come to a conclusion that regarding the enzyme profile and metabolic capacity, placenta is far more restricted than the liver (Pasanen, 1999; Hakkola et al., 1998). First, the most important phase I enzymes, cytochrome P450 (CYP) enzymes, are relatively sparingly expressed in the placenta. It is clear that when the expression has been studied with sensitive reversetranscriptase polymerase chain reaction (RT-PCR) methods, some expression can be observed for many CYP enzymes belonging to families 1-3, which are mainly responsible for xenobiotic metabolism (Hakkola et al., 1996a,b). However, regarding the expression of proteins, only the expression of
466
SECTION I V .
TABLE 2.
Transporter
Organ Toxicity
Expression and Detection of MDR and MRP Proteins in Human Placenta during the First Trimester and at Term a
First trimester
Suggested transport function in human placenta
At term/location
MDR1
+
Brush-border membranes of absorptive cells
MRP1
+
Syncytiotrophoblast cells, trophoblasts, abluminal side of blood vessels in villi
MRP2
+
Apical syncytiotrophoblast membrane
MRP3
+
MRP5
+
Vascular endothelia with some evidence for expression in the apical syncytiotrophoblast mRNA+ (RT-PCR); Western blot +
MRP6-8
9
mRNA
Organic cation, steroid transporter; transfer of vinblastine, vincristine, and digoxin Organic anion, glutathione, and glucuronide conjugate transporter; transfer of unconjugated bilirubin to maternal circulation Promote the excretion of glutathione/ glucuronide metabolites in the maternal circulation? Promote the excretion of glutathione/ glucuronide metabolites in the maternal circulation? Nucleotide analog pump; transfer of unconjugated bilirubin to maternal circulation 9
aSee Pasanen (1999) and Myllynen et al. (2004). Semiquantitation: +, positive signal; - , negative results; ?, no data available.
CYP1A1 has been unequivocally demonstrated (Pasanen and Pelkonen, 1994). This CYP activity is also induced by maternal cigarette smoking and exposure to polychlorinated biphenyl and other Ah-receptor inducers. The presence of some major phase II enzymes at the activity level has been demonstrated. However, not much information is available on the expression of specific isoenzymes of, for example, UGT, GST, or SULT (see Table 3). What can be inferred from the results is that only a restricted set of isoenzymes is present in placenta, although individual activities can sometimes be relatively high.
IV. E X P E R I M E N T A L M E T H O D S U S E D TO STUDY THE PLACENTAL FATE OF XENOBIOTICS A. B a c k g r o u n d Human placenta differs more than any other organ between species (Faber, 1995; Leiser and Kaufman, 1994), which is a good reason to use human placental material to study placental functions. Also, since placenta is a large piece of tissue, usually thrown away after birth, it can be used for many purposesmfor example, as a model organ for human proteins and molecular pathways. Placenta and cord also contain stem cells for the fetus, which can be used for experimental as well as therapeutic purposes. Thus, the importance of human placenta in medicine and science has increased during the past 15 years, and it will continue to do so for the foreseeable future.
Individual variation of normal placenta occurs, and various conditions can cause placental dysfunction. For instance, processes such as intrauterine growth retardation (IUGR) cause increased apoptosis and possible changes in the structure of the placenta (Merchant et al., 2004). Much more research and development are needed before the placental functions can be controlled during such situations. Placenta, with its hormonal and xenobiotic metabolism, putative storage capacity of foreign compounds (Ala-Kokko et al., 2000), and rather poorly characterized extensive transport system, is an important target of studies. All different types of experimental systems, from isolated cells to whole tissue perfusion, have been used to study the placenta (Table 4). Since placenta is anatomically complex and highly polarized in its functions (Ganapathy et al., 2000; Cariappa et al., 2003), the method to be used has to be carefully considered based on the purpose of the studies. For a complete view of placental transporters (Ganapathy et al., 2000; Cariappa et al., 2003), it may be necessary to use more than one experimental system. Heikkila and coworkers (2002) demonstrated the integration of a gene construct in placental cells by combining placental perfusion and explant culture thereafter.
B. H u m a n Placental Perfusion M e t h o d For human placental perfusion, one cotyledon from term placenta after birth is usually used because the whole placenta is too large for practical equipment (Schneider and Proegler, 1988; Pienimaki et al., 1995; Ala-Kokko et al., 2000). Fetal
C H A PT E R 3 3 9Placental Toxicity of OPs and CMs
TABLE 3.
467
Xenobiotic-Metabolizing Enzymes Detected in Human Placenta According to Gestational Status a First trimester
At term
++
++
The only placental xenobiotic-metabolizing CYP form for which expression and inducibility have been demonstrated at all possible levels of detection
CYP1A2 CYP1B1
-
(+) (+)
Immunoreaction equivocal at term Marker activity negative No catalytic activity; immunodetection negative
CYP form
Cytochrome P450 (CYP) CYP1A1
CYP2B6
(+)
(+)
CYP2C
(+)
-
CYP2D6 CYP2E1
(+) (+)
(+)
CYP2F1 CYP3A4, 3A5, 3A7 CYP4B 1
Comments
No relevant enzymatic assays
No catalytic activity
+
+
Marker activity negative No function established
(+) +
(+) +
No function established
++ ?
(+) ++
Positive correlation with smoking status
++ ?
++ ?
Accounts for 85% of placental activity
++
++
No definite correlation with the chemical stress and EH expression at term
?
9
++
++
Identification of isoforms not certain; activities dependent on specific substrates
?
++
Obviously polymorphic
?
?
Several marker activities negative
UDP-glucuronosyl transferase (UGT) UGT1A UGT2B, 2B4, 2B7
Activity levels very variable
Glutathione transferase (GST) GSTPI-1 GSTA, M, T
Probably negligible contributions
Epoxide hydrolase (EH) EH mEH Sulfotransferase (ST, SULT) SULT N-acetyl transferase (NAT) NAT1 NAT2 Other drug-metabolizing enzymes NQO
?
++
QR NOS ADH ALDH
++ + ? ?
++ ++ ++ ++
No induction in vivo Effects of xenobiotics on placental NOS have not been studied
CYP19
++
++
Aromatase activity
aSee Pasanen (1999) and Myllynen et al. (2004). +, positive evidence at functional (activity) level; (+), positive evidence at mRNA level (RT-PCR) but no evidence of activity; - , negativeresults; ?, no data available.
artery and vein can be cannulated to simulate fetal circulation, whereas in the matemal side, circulation can be gained by placing cannulae through the decidual plate. This dual setting can be used for both once-through and recirculating experiments. The latter simulate normal physiological conditions and can be used to study xenobiotic metabolism in addition to transplacental transfer (Pienimaki et al., 1995). In dual perfusions, both transfer from the fetal compartment to the maternal or transfer from the maternal circulation to
the fetal can be studied. For short experiments, clear perfusion medium with plasma expander, glucose, and salts can be used. However, for longer experiments, cell culture medium is necessary (Heikkila et al., 2002). Perfusion up to 18 hr has been described in the literature (Miller et al., 1989; Boal et al., 1997; Heikkila et aL, 2002). Successful perfusion can be ensured by the production of placental hormones, use of oxygen and glucose, and transfer of antipyrine (Ala-Kokko et al., 2000). However,
468
S ECTI O N IV 9 O r g a n T o x i c i t y
TABLE 4.
Examples of Experimental Methods and Their Characteristics Used for Placental Studies
Method
Characteristics
Examples of studies used for
References
Pienimaki et al. (1995), Ala-Kokko et al. (2000), Myllynen et al. (2004) Heikkila et al. (2002), Polliotti et al. (1996) Heikkinen et al. (2000)
E x vivo perfusion of a placental cotyledon
Short-terma dual recirculating
Mimics physiological circulation
Transfer and metabolism of drugs
Long-term dual recirculating
Mimics physiological circulation Concentration of studied compound constant in perfusion medium
Integration of gene construct Transfer of infectious agents Transfer of physiological compounds and drugs
Include gap junctions
Microbial invasion
Short-term dual once-through
Explant cultures Villous explants
VEGF in preeclampsia Primary cell cultures Isolated extravillous trophoblasts (EVT) Cultured trophoblasts
Placental endothelial cells from microvessels Isolated membranes Microvillous membranes (MVM)
Basal membranes
Trophoblast cell lines BeWo
Jeg-3
Jar
Endothelial cell line HPEC-A1
Nishimura et al. (2004) Lecuit et al. (2004) Ahmad and Ahmed (2004)
Express markers specific for EVT Grow in monolayer, express corticotrophin-releasing factor Express markers specific for EC, Life span 45-50 pd
Hormonal effects on invasive potential Placental hormone secretion
Petraglia et al. (1989)
Isolation procedure
Jinga et al. (2000)
H/K ATPase, P-gp
Localization of proteins
MRP l, more susceptible to proteolytic cleavage than MVM
Localization of proteins
Nagashige et al. (2003), Jansson (2002), Johansson et al. (2004) Jansson (2002), Nagashige et al. (2003), Johansson et al. (2004)
Cytotrophoblasts, no differentiation to syncytium Derived from BeWo, form large multinucleated syncytia Resemble early placental trophoblasts, form syncytia
Ca uptake
Wadsack et al. (2003), Moreau et al. (2001)
Cholesteryl ester uptake
Wadsack et al. (2003)
Cholesteryl ester uptake
Wadsack et al. (2003)
Establishment and characterization
Schutz et al. (1997)
SV-40 transfected, express endothelial markers
Oki et al. (2004)
aUp to 6 hr.
comparison of various methods led Pienimaki and coworkers (1995) to conclude that the most useful method is to follow the leak from the maternal side to the fetal side. In the histology, typical change in the dual recirculating perfusion system after organ perfusion is tissue edema (V~ih~ikangas, 1981; Pienimaki et al., 1995). However,
compared to placental tissue explants, the viability of cells can be retained much better (Di Santo et al., 2003). Although human placental perfusion is the most tedious method to study placental transfer and metabolism, there are several good reasons to use it (Ala-Kokko et al., 2000). It is the only method that retains fully the structure of the
CHAPTER 33 9Placental Toxicity of OPs and CMs placenta. Interspecies differences in placental anatomy and physiology cause difficulties in species extrapolation. Ethical aspects are easier than in in vivo human studies. Also, it is self-evident that new drugs and toxic chemicals cannot be studied in vivo in humans. For instance, studies on the transplacental kinetics of drugs such as methadone are important but difficult, if not impossible, in vivo. Ex vivo placental perfusion has provided valuable information on the kinetics and putative mechanisms in the case of methadone (Nejhayeva et al., 2005).
469
cal for endothelial cells), and UEA lectin (but not BS-I lectin, typical of large placental vessels). The cultures growing in the monolayer represent two cell types with different cell organelle distribution (Jinga et al., 2000). The primary arterial cells isolated by Leik and coworkers (2004) were grown from explants of placental arteries by placing the vessel lumen down. Cells with uniform morphology appeared within 1 week and grew to confluency within 4 weeks. After gaining confluency, they expressed smooth muscle myofilament proteins and lacked expression for fibroblast-specific antigen.
C. Placental Explant Cultures Placental tissue can be retained as explant cultures (Siman et al., 2001; Merchant et al., 2004), although Di Santo and coworkers (2003) have shown that the trophoblast viability is very restricted despite culture conditions. Explants from normal and IUGR pregnancies were cultured by Merchant and coworkers to study matrix metalloproteinases. Placental explants can also be cultured after placental perfusion and have been used to show integration of a gene construct (Heikkila et al., 2002). Explant cultures have also been used to isolate specific cell types from the placenta. Leik et al. (2004) grew arterial smooth muscle cells from cultured small pieces of placental arteries from chorionic plate. Within 1 week, cells with uniform morphology expressing proteins similar to human aortic smooth muscle cells but clearly different from fibroblasts or endothelial cells grew out of the explants. These cells can be used as a general model for human arterial smooth muscle cells (Leik et al., 2004).
D. Primary Placental Cells Primary trophoblast cells (and also most cell lines of trophoblast origin) ~do not grow into confluent monolayers in culture (Cariappa et al., 2003). This inhibits studies on polarized transport of nutrients and other compounds. They are difficult to culture because of contamination by other cell types and poor viability (Choy et al., 2000). Villous and extravillous cytotrophoblasts differ in their function and characteristics. As studied by immunofluorescence, Ockleford and coworkers (2004) have shown a much higher cytokeratin expression in extravillous cytotrophoblasts compared to villous cytotrophoblasts. Placental venous endothelial cells have different characteristics depending on which part of the vasculature they have been isolated from. Isolation, purification, and culture of primary human placental endothelial cells (HPECs) from microvessels on the venous side were reported by Jinga and coworkers (2000). To gain pure cultures of HPECs, trypsin perfusion of the placental cotyledon was followed by Percoll gradient and sequential trypsinization of cultures. These cultures represent pure HPEC form microvessels by expressing typical patterns of markers: ACE, von Willebrand factor (typi-
E. Cell Lines of Placental Origin Immortalized trophoblast cell lines can be produced by transfection. Choy and coworkers (2000) were able to significantly increase the transfection efficiency by modifying a transfection protocol utilizing poly-L-ornithine. Choriocarcinoma cells resemble invasive trophoblasts morphologically and are used as a model to study trophoblast functions (Wadsack et al., 2003). Several choriocarcinoma cell lines exist (Table 2). Immortalized BeWo cells, which grow as a monolayer, are widely used because of their useful characteristics (van der Ende et al., 1990). Morphologically, they resemble normal trophoblasts and have similar biochemical marker enzymes. Interestingly, Cariappa and coworkers (2003) have shown that their characteristics are dependent on the material on which they are grown. Some of the growth factors typically expressed by these cells are only expressed when the cells are cultured on porous filters and not when cultured on plastic. The expression of these factors thus requires polarized membranes. BeWo cells appear to be an effective model for placental calcium transport (Moreau et al., 2001) but not as good as Jar or Jeg3 cells for selective cholesteryl ester supply to placental cells (Wadsack et al., 2003). This stresses the importance of comparing several models for any new studies to find the most suitable one.
V. P L A C E N T A L T O X I C O K I N E T I C S O F
ORGANOPHOSPHATES AND CARBAMATES From a search of the literature on pesticides and placental toxicokinetics, it is obvious that not much specific information is available. Most studies are on the effects of pesticide administration during pregnancy on experimental animals, mainly rabbits, rats, and mice, in which the fetal outcome and sometimes the presence of residues in the fetal tissues have been observed (Gupta, 1995). Naturally, if specific effects in the fetus, such as inhibition of cholinesterase or compound-related residues in the fetus and the placenta, have been observed, it is relatively safe to make the conclusion that the pesticide has been transferred from the mother to the fetus. Otherwise, very little direct information is available.
4 70
S ECTI O N IV 9Organ Toxicity
It is also important to stress that almost all studies have been performed in experimental animals and the extrapolation to human pregnancy should be made with caution.
A. Entry and Disposition of Organophosphates and Carbamates in the Placenta 1. THE PLACENTAAS A BARRIER FOR ANTICHOLINESTERASE PESTICIDES The placenta is a lipid membrane that permits bidirectional transfer of substances between maternal and fetal compartments. Essentially, the placenta is the entry (not a barrier) through which the fetus is exposed to xenobiotics, including organophosphate (OP) and carbamate (CM) pesticides. The two most common factors involved in transplacental transfer of common toxicants are physicochemical properties of the chemical and the type of placenta. In general, any chemical with a molecular weight < 1000 readily crosses the placenta, and most OPs and CMs have a molecular weight 90% of control after 96 hr of dosing. The authors found that diazinon, as a less toxic insecticide compared to methyl parathion (LDs0, 455 vs 67 mg/kg), caused less inhibition of AChE than that caused by methyl parathion alone or in combination with methyl parathion. Similar findings have been reported for CMs, including aldicarb, carbaryl, carbofuran, and pirimicarb (Cambon et al., 1980, 1979; Declume and Derache, 1977). These studies revealed AChE inhibition as the major biochemical mechanism of toxicity. Furthermore, subchronic prenatal exposure to methyl parathion in rats resulted in altered postnatal development of brain AChE and ChAT activities and selected subtle alterations in behavior (Gupta et al., 1985). To our knowledge, there are no systematic studies that have examined the effects of OPs and CMs on the human placental cholinergic system. However, since OPs and CMs are known to inhibit AChE activity, it is expected that exposure of a pregnant woman to these compounds can lead to enhanced levels of ACh, and thereby ACh can influence other components of the cholinergic system in the placenta. 2. PLACENTAL NEUROTOXIC ESTERASE
Neurotoxic esterase (NTE), which is a membrane protein, was discovered in chicken brain in relation to tri-ocresylphosphate-induced neuropathy (Johnson, 1974). In later studies, NTE, which is the putative target for OP-induced delayed neuropathy (OPIDN), was found in the neural tissue of a large number of different vertebrate species (Gurba et al., 1981). The same authors found NTE in preparations of human placenta similar to that found in the avian and human brain (Gurba and Richardson, 1983). The human placenta may be an ideal source of material for further purification and characterization of NTE, although in the context of OPIDN it appears to be insignificant in the placenta. 3. ACh-INDUCED RELEASE OF PLACENTAL HORMONES
Human placenta has been recognized as a source of chorionic gonadotropins and steroid hormones. Since the development of the placental cholinergic system follows
4 74
S ECTI O N IV 9Organ Toxicity
the development of the syncytiotrophoblast, it is interesting to determine the release of steroid hormones by ACh. Although the cytotrophoblasts, the source of gonadotropins in the placenta, are fully developed in the first 3 months of gestation, some cytotrophoblastic cells remain in full-term human placenta (Sastry, 1997). Harbison et al. (1976) reported that ACh stimulates placental release of chorionic somatomammotrophin. ACh increases the release of immunoreactive corticotrophin-releasing factor from human placental cell cultures in a dose-related manner, and its effect is reversed b y the cholinergic receptor antagonists atropine and hexamethonium.
C. Effects of Pesticides on Endogenous Steroid Synthesis Endocrine effects of pesticides have been known for some time. They can both interact with steroid hormone receptors and interfere with hormone synthesis. During pregnancy, the placenta is an important organ for the synthesis of steroid hormones, and it contains two important rate-limiting enzymes: CYPllA1, which catalyzes cholesterol side chain cleavage, and CYP19, an aromatase for the production of estrogens. CYP11A1 seems to be a rather stable and selective enzyme not affected to a great extent by environmental chemicals. On the other hand, CYP19 has a relatively nondiscriminatory binding site and a large number of inhibitors have been synthesized, some of which are in use in endocrine cancers. Regarding most OPs and CMs, it is not known whether they are inhibitors or activators of either one of these two important enzymes. Andersen and coworkers (2002) studied the endocrine effects of 24 pesticides in use in Danish greenhouses and showed that many had multiple effects. For instance, fenarimol acted as an estrogen agonist and androgen antagonist and, in addition, inhibited aromatase activity in human placental microsomes. Prochloraz inhibited aromatase even more drastically, and chlorothalonil more than 50% of the original activity. Others, such as methomyl and primicarb, weakly stimulated the placental aromatase. One molecular target of the endocrine effects in the placenta may be intracellular calcium homeostasis since estrogenic pesticides change calcium handling by trophoblasts and this effect may be endocrinally controlled (Derfoul et al., 2003).
D. General Effects on Protein and Nucleic Acid Synthesis by AChEs Placenta is a potentially important source of tissue for molecular studies in humans and animals because it is large, readily available, and responsive to environmental pollutants, including anti-AChE pesticides. Some of these pesticides are known to cause alterations in protein synthesis, DNA damage, mutagenesis, carcinogenesis, and teratogenesis. OPs have been shown to adversely influence protein
synthesis in both in vivo and in vitro studies (Marinovich et al., 1996; Koelle et al., 1977; Welsch and Dettbarn, 1971; Clouet and Waelsch, 1963). Gupta et al. (1984) examined the subchronic effects of methyl parathion administered throughout the period of organogenesis on in vivo protein synthesis in embryonic, fetal, and maternal tissues. The specific activity of [14C]valine in the free amino acid pool and protein-bound pool was significantly reduced in discrete regions of the maternal brain, maternal viscera (day 19), and placenta. The inhibitory effect of methyl parathion on net protein synthesis was dose dependent, greater on day 19 than on day 15 of gestation, and significantly greater in fetal than in maternal tissues. It is interesting to note that the inhibitory effect on protein synthesis was most pronounced at a dose causing overt maternal toxicity. Marinovich et al. also demonstrated that a mixture of OPs, including diazinon, dimethoate, and azinophos, was more toxic to protein synthesis than any of the single compounds alone. There are few studies in which OPs and CMs have been examined for their effects on embryonic development. It has been postulated that alkylation of nicotinamide adenine dinucleotide (NAD +) coenzymes by OPs may be a major factor in the induction of carcinogenesis (Schoental, 1977). In another study, malathion caused alterations in the levels of RNA, glycogen, sulfated mucopolysaccharides, and calcium in the developing tibiotarsus (Ho and Gibson, 1972).
VII. ABNORMAL PREGNANCY OUTCOMES DUE TO ANTICHOLINESTERASES Exposure to pesticides of the anticholinesterase class during pregnancy can cause several deleterious effects, such as stillbirths, fetal deaths, spontaneous abortions, low birth weights, and malformations at birth. Several OPs and CMs are known to exert direct embryocidal/fetocidal effects (Jayatunga et al., 1998a,b; Speilmann and Vogel, 1989; Deacon et al., 1980). Both OPs and CMs are known to readily cross the placenta and act on the cholinergic and noncholinergic components of the developing nervous system and other vital organs (Gupta and Sastry, 1999; Gupta et al., 1985; Gupta, 1995). The developing organism appears to be much more susceptible than the adult to the induction of functional neural deficits by OPs and CMs. The effects of maternal exposure on the fetus can vary from the production of severe anomalies to nondetectable differences from nontreated controls. Published studies have shown indications of elevated reproductive risk and exposure to pesticides, but the epidemiological evidence does not allow any clear inference to be drawn (Nurminen, 1995). Pesticides of both classes have been demonstrated to have potential for embryotoxicity, embryolethality, fetotoxicity, and teratogenesis. In general, these effects vary
CHAPTER 33 9Placental Toxicity of OPs and CMs depending on the particular OP or CM involved. Embryolethality is encountered so often that the expression of teratogenesis is rarely seen. In humans, infants born to mothers exposed to oxydemeton-methyl or mevinphos (both OPs) showed cardiac defects (ventricular and atrial septal defects), stenosis of the pulmonary artery and a patent ductus arteriosus, bilateral optic nerve colombo mass, microphthalmia of the eye, cerebral and cerebellar atrophy, and facial anomalies (Romero et al., 1989; Ogi and Hamada, 1965). In some studies, OPs have been shown to produce developmental alterations in rats, mice, hamsters, and rabbits. In a classical study, Khera (1979) discovered polydactyly in fetuses of cats treated with dimethoate (12 mg/kg/day) during days 14-22 of gestation. De Castro et al. (2000) reported that methamidophos, at the no-maternal toxicity dose (1 mg/kg, po), given to female rats during gestation (days 6-15) produced no lethal effect on embryos and did not cause congenital malformations at term; however, the embryo-fetal maturation process appeared to be affected. In a similar study conducted on rats with six OPs (tribufos, oxydemeton-methyl, azinophos-methyl, fenamiphos, isofenphos, and fenthion), Astroff and Young (1998) found no effects in fetuses at dose levels that elicited significant maternal effects. Among CM pesticides, carbaryl has been studied in detail for placental toxicity. Like OPs, CMs have a greater potential for embryolethality and fetotoxicity that precludes an expression of teratogenicity. A study conducted in beagle dogs showed dystocia due to atonic uterine musculature and evidence of terata in 21 of 181 pups. Fetal abnormalities included abdominal-thoracic fissures with varying degrees of intestinal agenesis and displacement, brachygnathia, ecaudate pups, failure of skeletal formation, and superfluous phalanges (Smalley et al., 1968). Carbaryl exposure during organogenesis produced terata in guinea pigs but not in hamsters and rabbits (Robens, 1969). Jayatunga et al. (1998a) demonstrated that exposure to carbofuran during early pregnancy in rats causes pregnancy loss and disruptions to neonatal development. In a detailed study, Jayatunga et al. (1998b) examined the antigestational effects of carbofuran (0.2, 0.4, and 0.8 mg/kg, po) on pregnancy outcome in rats following midterm exposure (days 7-14). Carbofuran was found to be injurious to the reproductive outcome because the findings revealed fetal deaths (in terms of postimplantation loss, viable uterine implants, litter index, and fetal survival ratio) and impaired prenatal development (size of uterine implants and interembryonic distance) and postnatal development (cranial length, craniosacral length, body weight, and time taken for the appearance of fur and opening of the eyes). In addition, there was a tendency for the pregnancy period to be prolonged and for female-biased litters to be produced. These effects of carbofuran appear to be mediated mostly via potentiation of postimplantational losses due to embryo/fetal toxicity. These authors observed a marked increase in the number of dead conceptuses at laparotomy
475
and by the pronounced reduction in the numbers of litters and of live pups at parturition. It is likely that the embryo/fetocidal action of carbofuran arose both directly and indirectly (via maternal toxicity). Carbofuran is estrogenic (Goad et al., 2004; Jayatunga et al., 1989a,b), and high levels of estrogens can induce fetal death by direct action (O'Neill et al., 1985). At birth, a predominance of female pups can possibly be attributed to the estrogenic activity of carbofuran and to its ability to inhibit food consumption. CMs such as propoxur and carbofuran have not been proved to be teratogenic. For further details on alterations in pregnancy outcomes following exposure to OPs and CMs, see Gupta and Sastry (1999) and Gupta (1995).
VIII. C O N C L U S I O N S AND F U T U R E
DIRECTIONS OP and CM pesticides are widely used and can expose pregnant mothers. The placenta is thus one of the targets for these pesticides, but both also convey deleterious effects to the developing fetus. Although pesticide-induced fetotoxicity has been studied in experimental animals and adverse effects have been observed, there is very little information concerning the toxicokinetic behavior of these pesticides (i.e., penetration, protein binding, distribution, and metabolism) in human placenta. Also, data on toxicodynamic mechanisms and consequences to the developing fetus mainly concern experimental animals, and human data are very scanty. In general, it is of considerable importance to elucidate the role of the placenta in contributing to developmental effects and fetotoxicity. Although there are large gaps in our knowledge of the placenta and pesticides, a variety of tools are available with which we may in principle obtain useful information for assessing the risks of pesticides to the placenta and developing fetus.
Acknowledgments Kirsi V~ih~ikangas is a member of the EU project CHILDRENGENONETWORK (QLRT-2001-02198). The research of Olavi Pelkonen has been supported by the DRUG2000 technology programme of the National Technology Agency of Finland (TEKES).
References Abu-Qare, A. W., and Abou-Donia, M. B. (2001). Inhibition and recovery of maternal and fetal cholinesterase enzyme activity following a single cutaneous dose of methyl parathion and diazinon, alone and in combination, in pregnant rats. J. Appl. Toxicol. 21, 307-316. Abu-Qare, A. W., Abdel-Rahman, A. A., Kishk, A. M., and AbouDonia, M. B. (2000). Placental transfer and pharmacokinetics of a single dermal dose of [14C]methyl parathion in rats. Toxicol. Sci. 53, 5-12.
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CHAPTER ~4
Endocrine Disruption by Organophosphate and Carbamate Pesticides SHIGEYUKI KITAMURA, KAZUMI SUGIHARA, AND NARIAKI FUJIMOTO Hiroshima University, Hiroshima, Japan
However, investigations of incidents involving misuse of OP and CM pesticides have revealed virtually no endocrine-disrupting activity of these chemicals (Flickinger et al., 1984). The putative endocrine-disrupting effect of OPs and CMs in wildlife is largely a prediction based on data obtained from laboratory animals. The influence of these pesticides on human health is also discussed here, based on observed endocrine-disrupting actions of OPs and CMs in vitro and in laboratory animals in vivo.
I. I N T R O D U C T I O N Many organophosphate (OP) and carbamate (CM) pesticides are used throughout the world to protect crops from insects and to protect farm and domestic animals from endo- and ectoparasites. They act as pesticides by inhibition of acetylcholinesterase (ACHE). OPs bind irreversibly with ACHE, whereas CMs bind reversibly. OPs and CMs are nonvolatile and chemically stable, but they are easily decomposed by carboxylesterase to nonactive metabolites. Because these pesticides, including their metabolites, are polar and water soluble, they are believed not to be bioconcentrated in aquatic species. Although certain OPs and CMs have been reported to accumulate to some extent in fish and invertebrates (Tsuda et al., 1990, 1996; Pereira and Hostettler, 1993; Deneer, 1994; Kitamura et al., 2000; Tilak et al., 2004), the risk of bioaccumulation is low due to rapid metabolism in the body and degradation in the environment. Indeed, fenthion sulfoxide, a metabolite of fenthion, was rapidly excreted but fenthion was moderately persistent in the body of fish, with a half-life of approximately 10 days after exposure to the pesticide (Kitamura et al., 2000). Estrogenic and/or antiandrogenic activities are exhibited by a number of pesticides, especially organochlorine pesticides. However, some OP and CM pesticides are also reported to be positive in in vitro assay systems. Antiandrogenic compounds so far reported include metabolites of vinclozolin, linuron, iprodione, chlozolinate, procymidone, flutamide, p,p'-DDE, a metabolite of p,p'-DDT, and ketoconazole (Kelce et al., 1995; Wong et al., 1995; Gray et al., 1999; Ostby et al., 1999; Lambright et al., 2000; McIntyre et al., 2000). Many pesticides, including OPs and CMs, have been found to bind to androgen receptor (AR) and to act as antagonists of AR. Pesticides such as DDT and methoxychlor are well known to act as endocrine disruptors (Guillette et al., 1994). Toxicology of Organophosphate and Carbamate Compounds
II. E N D O C R I N E D I S R U P T I O N BY OP AND CM I N S E C T I C I D E S IN VITRO
A. Endocrine-Disrupting Action of OPs I n V i t r o Many organochlorine insecticides can interact with estrogen, androgen, and progesterone receptors (Table 1) (Kelce et al., 1995; Vonier et al., 1996; Gray et al., 1999; Ostby et al., 1999; Lambright et al., 2000). Many results from in vitro screening tests indicate that tested OPs are not estrogenic. Sonnenschein and Soto (1998) showed that neither malathion nor parathion is estrogenic in the estrogen proliferative screening assay (E-screen assay) using MCF-7 human breast cancer cells. Chen et al. (2002) reported that some pyrethroid pesticides were positive in estrogen screening assay, E-screen assay, estrogen receptor (ER) competitive binding assay, or pS2 expression assay. However, all OP pesticides (phoxim, malathion, monocrotophos, dimethoate, and opunal) examined were negative in the concentration range of 1 X 10-11 to 1 X 10 - 6 M in these estrogen assays. Fenitrothion did not interact with ER in recombinant yeast expressing the human estrogen receptor (Sohoni et al., 2001). Fenthion and its oxidized products, fenthion sulfoxide and fenthion sulfone, did not 481
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
482
S E C T I O N I V . Organ T o x i c i t y
TABLE 1.
Endocrine-Disrupting Action of OPs In Vitro
Estrogenic and antiandrogenic OPs CI C2H501P- O
CI
c,~ ~_~ NO 2
Br
bromophos-ethyl
II H3CH2CCHNH- P - O
C2H50
'CH3
butamifos
CI C2H50\S_ O _ _ ( ~ C I C2H50IP dichlofenthion S
C2HsO/
-0"--0"--
No2
EPN
ethion
C2H50\S_o._(~ CI C3H7SIP Cl prothiofos S
CH30-P-O CH30
C2H50\S S II II ,,OC2H 5 P-SCH2S --p C2HsO i ,,OC2H5
c.,c.=~ OH3cH20/P
CH3 CHNH-~- O - ~ CH3-~ C2H50
--CH3
COOCH-.cH 3
isofenphos CI
~
~'N
quinalphos
leptophos
CI CH3 CI
tolclofos-methyl Estrogenic OPs
C2H50\S O - ~ C l 03H7SIPCI prothiofos CH3CH20\S/P-O .--~/ ~ CH3CH20 ~N quinalphos
S CI H3CO.IIH3CO,,P 0 ~--(~~Br Cl bromophos-methyl 1110ClcHs~ClBr S
Qo
P,,
leptophos
c.,o\~ c ~ CH30/P-O
CH3
Cl
tolclofos-methyl 1170 "OC2H5
CN
cyanofenphos (continues)
CHAPTER 34
(continued)
TA BLE 1.
CI C2H50\S_ O _ ~ C I C2H50IP
C2HsO/ - O ' - - ~
dichlofenthion
CH3CH20
9 Endocrine Disruption by OPs a n d CMs
NO2
C2H50\S SII ,,OC2H5 P-SCH2S--p C2HsOI "OC2Hs
EPN
P- O
NO2
s
Br CI
ethion
C2H50
bromophos-ethyl
s
H - P- O
C2H5~11 CH3
C2Hs/P-O~
butamifos
CH30\ S /p_ O._~T/CH3 CH30 N~,~ N~"C2H5 "C2H5
S
c.~.c..._~_o_0 CH3"" C2H50
"CH33 COOCH~CH
isofenphos
pirimiphos-methyl
isoxathion
CH30\ S
-S-CH CH30IP "COOC2H 5 phenthoate
N Cl C2H50\S_ O ~ C I
C2HsO~P Cl chlorpyrifos
Antiestrogenic OPs
C2H50\ S C2H501P- O ' - ~ -
NO2
parathion
Antiandrogenic OPs
c.~o..~, o-~c. CH30~P-
N CI
CI chlorpyrifos-methyl
S-c~-
CH30"II -O CH30..P
CH3 NO2
fenitrothion
~- -C~-
CH3
CH30" CH30,. O
SCH3
fenthion 9
(continues)
483
484
SECTION I V . Organ Toxicity
TABLE 1. (continued)
CI
H3CO\sll C~ P-S-CH3-CH3CO1 O
CI
C2H501 -O---~'-NO 2
anilofos
C2H50\SII C2H501P-O
EPN
NO2
parathion
H3co\S_0.__~__NO2 H3COIP
methyl parathion
o- c.
C3H5sIP-
prothiofos
CH30\S C ~ CH30/P-O CI
CH3
tolclofos-methyl NO 2
CH3CH2CH20\S iL~cH3 CH3CH2CH201P-S-CH2"CO
C2H50\S S / P-SCH2S--pIIsOC2H5 C2H50 "OC2H5
piperophos
ethion
C2HsO\s
O,~,O~cl
C2HsO ~ P - S - CH 2 - N - " ~
CI C2H50\~10--~CI C2H501P-
phosalone
I1,,O Br QP"o 3 CI leptophos
S CH3~CHNH-~-O---~ CH3"" C2H50 .'CH3 COOCH,,CH3
dichlofenthion
C2HsO\!_O~~~_Br C2HsOI Cl bromophos-ethyl
H3CH2CCHNH-P-O C2H50 II
CH3
butamifos
H3CO\S H3co/P- O ' - ~
CN
cyanophos
CH3CH20\S/p-o. _ ~ / N ~ CH3CH20 ~N quinalphos
CH30\IO I
CH30~P-O ' - - ~ C HNO2
isofenphos
show estrogenic or antiestrogenic activity in ERE-luciferase reporter-transfected MCF-7 cells (Kitamura et al., 2003b). Nishihara et al. (2000) also reported that no estrogenic activity of EPN, ethyl parathion, fenitrothion, fensulfothion, fenthion, malaoxon, malathion, or methidathion was observed at a higher concentration, 1 x 10-4M, even though 17-[3-estradiol (E2) was active at 3 x 10-1~ in a yeast two-hybrid assay. Vitellogenin (a biomarker of estrogens in fish) assay of OPs in fish also gave a negative result. Fenthion did not have estrogenic activity, based on
MEP oxon
the finding that levels of vitellogenin were not enhanced in the blood of male or female goldfish (Carassius auratus) kept in water containing fenthion or its oxidation products, fenthion sulfoxide and fenthion sulfone (3 mg/liter), for 5 days (Kitamura et al., 1999). Although parathion did not activate estrogen- or progesterone-responsive reporter genes in breast MCF-7 and endometrial (Ishikawa) cancer cells except for weak activation of a progesterone-responsive gene, the OP decreased Ee- or progesterone-induced activity (Klotz
CHAPTER 34 9Endocrine Disruption by OPs and CMs et al., 1997). Andersen et al. (2002) tested 22 pesticides, including 4 0 P s and 4 CMs, for effect on proliferation of MCF-7 cells and interference with the activation of ER and AR. They reported that chlorpyrifos and tolclofosmethyl exhibited estrogenic activity in cell proliferation assay and transactivation assay using MCF-7 cells in the concentration range of 1 x 10 -5 to 5 x 10 -5 M. At higher concentrations, there was a decreased response due to cytotoxicity. These investigators further examined the effect of 9 positive estrogenic pesticides (prochloraz, fenarimol, endosulfan, dieldrin, tolclofos-methyl, chlorpyrifos, pirimicarb, propamocarb, and methiocarb) on ER-oL and -[3 mRNA levels in the mammalian cancer fibroblast MCF7BUS cells using on-line reverse-transcriptase polymerase chain reaction (Grunfeld and Bonefeld-Jorgensen, 2004). The OPs listed previously interfered with the ER-oL and ER-[3 mRNA steady-state levels. In particular, coexposure to tolclofos-methyl and E 2 significantly increased ER-oL and -[3 mRNA levels. Kojima et al. (2004) examined the estrogenic activity of 56 OP pesticides in in vitro reporter gene assay using Chinese hamster ovary (CHO) cells. They reported that 16 OPs showed affinity for hER-oL. Prothiofos, bromophos-methyl, tolclofos-methyl, quinalphos, leptophos, cyanofenphos, dichlofenthion, EPN, ethion, and bromophos-ethyl showed affinity for ER-oL and ER-[3, but butamifos, isoxathion, pirimiphos-methyl, isofenphos, phenthoate, and chlorpyrifos lacked affinity for ER-[3. Butamifos showed the highest activity (20% of the activity of E2 at 6.7 X 10-7M) among positive compounds for ER-oL. A 10-fold higher concentration of o,p'-DDT was required for activity, but the concentration corresponded to that of methoxychlor. In this study, no antiestrogenic activity was observed with any of the pesticides tested. Chlorpyrifos-methyl has weak affinity for estrogen, progesterone, and androgen receptors in yeast transformants that express human steroid hormone receptors and steroid hormone response elements (Jeong et al., 2001). Androgenic activity has not been observed in any pesticide tested. However, antiandrogenic activity of OPs has been observed in vitro. Parathion inhibits dihydrotestosterone (DHT) binding to AR in rat ventral prostate (Shain et al., 1977). Kojima et al. (2004) reported that 19 OPs (fenitrothion, anilofos, EPN, prothiofos, parathion, methyl parathion, tolclofos-methyl, piperophos, ethion, butamifos, phosalone, dichlofenthion, fenthion, cyanophos, leptophos, bromopho-ethyl, quinalphos, isofenphos, and MEP oxon) showed inhibitory effects on the androgenic activity of DHT in a reporter gene assay using CHO cells transfected with hAR. Fenitrothion showed the highest activity, and the activity of DHT was inhibited by 20% at 1.8 X 10 -7 M and completely abrogated at 1 • 10 -5 M. The activity was higher than that of p,p'-DDE. Fenthion showed antiandrogenic activity against the androgenic activity of DHT in a reporter assay using NIH3T3 cells, but fenthion sulfoxide was negative (Kitamura et al., 2003b). In the literature,
485
fenitrothion and EPN, as well as fenthion, but not fensulfothion, trichlorfon, or malathion, are reported to be antiandrogenic. Fenitrothion was the most active. In our study, it was found that fenthion is markedly inactivated after oxidative metabolism by mixed function oxidase (MFO). In contrast, fenthion sulfoxide was activated by rat liver preparations. This is due to interconversion between fenthion and fenthion sulfoxide by rat liver preparations (Kitamura et al., 2000, 2003a). Tamura et al. (2001) reported that fenitrothion competitively inhibited DHT-dependent human androgen receptor activation in HepG2 cells. The potency of fenitrothion was approximately 8- to 35-fold higher than those of p,p'-DDE and linuron, well-known environmental antiandrogens, and approximately 50% of that of flutamide, which is a pharmaceutical antiandrogen (Maness et al., 1998; Mclntyre et al., 2000). Antiandrogenic activity of methyl parathion was also seen in a preliminary study. Sohoni et al. (2001) observed activity of fenitrothion in recombinant yeast expressing the human androgen receptor. The structure of fenitrothion closely resembles that of flutamide. Fang et al. (2003) reported that methyl parathion, parathion, and triphenyl phosphate were positive in a comparative binding assay to AR. A SAR study suggested that hydrophobic interactions of these pesticides are important for AR binding. Currently, the significant structural differences between antiandrogenic and non-antiandrogenic OP insecticides are difficult to understand. Further study of the structural requirements for estrogenic and antiandrogenic activities and of the mechanisms of action of OP insecticides is needed.
B. Endocrine-Disrupting Action of CMs In Vitro Some CM pesticides show endocrine-disrupting action, whereas others do not (Table 2). A CM pesticide, carbaryl, lacks binding affinity for estrogen receptor from rat uterus (Blair et al., 2000). Estrogenic activity of methomyl was not observed at 2 x 10 -4 M (Nishihara et al., 2000). Bendiocarb, carbofuran, and carbaryl are not estrogenic in E-screen~ assay (Sonnennschein and Soto, 1998). In contrast, Klotz et al. (1997) reported that aldicarb, baygon, bendiocarb, carbaryl, methomyl, and oxamyl weakly activated estrogen- or progesterone-responsive repo~er genes in breast MCF-7 and endometrial (Ishikawa) cancer cells at a concentration of 1 X 10 -7 M. However, these CMs (1 x 10 -7 M) decreased estrogen- or progesterone-induced reporter gene activity induced at the concentration of 1 x 10 -9 M to approximately one-third. In contrast, Kojima et al. (2004) examined the estrogenic activity of 22 CM pesticides and reported that methiocarb has an agonistic effect on hER-oL and ER-~. Furthermore, they found that the activity against ER-[3 was higher than that against ER-oL. In contrast, Andersen et al. (2002) reported that methiocarb exhibits estrogenic activity in cell proliferation assay and transactivation assay using MCF-7 human breast cancer cells. Methiocarb was estrogenic in the range of 1 X 10 -5
486
S E C T I O N IV 9 Organ Toxicity
T A B L E 2.
Endocrine-Disrupting Action of CMs In Vitro
Estrogenic and antiandrogenic CMs H3C
HaCS~}'-OCONHCH3 HaC methiocarb Estrogenic CMs
.30
O-CO_N'CH3 H3C,,,~ N "CH3 H 3 c / ~ N ~ " N--CH3 "CH 3
pirimicarb
H3C~ H3c~NCH2CH2CH2NHCOOCH2CH2CH
propamocarb OCONHCH3
CH3
I
H3CS~--(~/~'- OCONHCH3 H3C
methiocarb
OCONHCH3
CH3SNCCH = NOCONHCH3 I CH3 aldicarb
baygon
bendiocarb
C H 3 ( ~ NOCONHCH3 SCH3
H3C~ NCOC= NOCONHCH3 H3C ~ SCH3
methomyl
oxamyl
OCONHCH3
carbaryl Antiestrogenic CMs
OCONHCH3
CH3
I
CH3SNCCH-- NOCONHCH3 I CH3
aldicarb
OCONHCH3
O" CH3 baygon
bendiocarb
CH3C~NOCONHCH 3 SCH3
H3C~ NCOC=NOCONHCH3 H3C ~ SCH3
methomyl
oxamyl
OCONHCH3
carbaryl
(continues)
CHAPTER 34 9Endocrine Disruption by OPs and CMs
TABLE 2. Antiandrogenic CMs
.30
methiocarb
(continued)
~2SCON"C2H5 "C2Hs
H3CS~-'~/~-OCONHCH3 H3C
487
CI thiobencarb
Aromatase-stimulators
~2SCON "C2H5 *'C2H5 CI primicarb
H3C~, H3C" NCH2CH2CH2NHCOOCH2CH2CH 3 propamocarb
to 5 x 10 -5 M in MCF-7 cell proliferation and ER transactivation assays. Some chemicals are known to potentiate the estrogenic activity of E2. Primicarb and propamocarb exhibited no estrogenic response when tested alone but potentiated the activity of E2 (10 nM) in the range of 5 X 10 -7 to 5 X 10 -5 M in ER transcription assay. Grunfeld and Bonefeld-Jorgensen (2004) examined the effect of estrogenic CMs primicarb, propamocarb, and methiocarb on ER-oL and -[3 mRNA levels. They reported that pirimicarb weakly decreased the ER-oL mRNA level, whereas propamocarb and methiocarb had no effect. In contrast, chlorpyrifos weakly increased the ER-[3 mRNA level. Hofmeister and Bonefeld-Jorgensen (2004), using the same cell line, found that prochloraz had the potential to downregulate the expression of ER-oL and ER-[3 mRNAs as well as E 2, and the effect was abolished by cotreatment with the ER antagonists ICI 182 and 789. Methiocarb had no effect on the ER-oL mRNA level, whereas it increased the ER-[3 mRNA level. Andersen et al. (2002) reported that methiocrab acted as an AR antagonist as well as an ER agonist. Methiocarb inhibited the androgenic response to R1881 at the concentration of 20 IxM in transactivation assay using CHO cells. Birkhoj et al. (2004) examined the antiandrogenic activity of methiocarb alone or combined with other commonly used pesticides. This pesticide inhibited R1881-induced transcriptional activation (IC25, 5.8 ixM) in in vitro AR reporter assay using CHO cells, but it was negative in the in vivo Hershberger assay using castrated rats. Kojima et al. (2004) reported that methiocarb and thiobencarb inhibited the androgenic activity of DHT in CHO cells transfected with hAR at 2.8 X 10 -6 and 9.4 x 10 -6 M (IC20), respectively. When combined with four other pesticides (deltamethrine, prochloraz, simazine, and tribenuron-methyl), there was an additive effect in in vitro assay, but no significant effect was
seen in vivo except for a 25% reduction in the weight of the levator ani/bulbocavernosus muscle. Furthermore, urea-type pesticides propanil, linuron, and diuron, which are the main herbicides used throughout the world, have the ability to bind to AR (Cook et al., 1993; Bauer et al., 1998). Indeed, linuron is structurally related to the nonsteroidal antiandrogen flutamide, and its ICs0 for competition at the AR is approximately 3.5 times that of flutamide. The activity of its metabolite, 3,4-dichloroaniline, is higher than that of linuron.
C. Disruption of Steroidogenesis by OPs and CMs In Vitro Some pesticides may have an ability to modulate the effects of potent natural steroid hormones. Interaction with hormone receptors is one possible mechanism of hormonedisrupting action. Activation or inhibition of enzyme activities involved in steroid hormone synthesis may also alter endogenous hormone levels. Andersen et al. (2002) examined the effect of pesticides on CYP 19-aromatase, which catalyzes the conversion of C19 steroids to estrogens, and showed that the OPs tested had no effect on the activity but pifimicarb and propamocarb enhanced the activity at the concentration of 50 p~M. They also examined serum levels of testosterone, LH, T4, and prolactin after administration of pesticides and reported that methiocarb significantly reduced luteinizing hormone (LH) levels compared to those in testosterone-treated animals. Vinggaard et al. (2000) reported that chlorpyrifos, tetrachlorvinphos, and linuron did not affect aromatase activity. In contrast, a urea-type fungicide, prochloraz, inhibited the aromatase activity of human placental microsomes and human JEG-3 chofiocarcinoma cells (Mason et al., 1987; Vinggaard et al., 2000;
488
SECTION IV. O r g a n T o x i c i t y
Andersen et al., 2002). Linuron decreased accessory sex organ weights in sexually immature and mature rats treated with linuron. In linuron-treated mature rats, serum E 2 and LH levels were increased (Cook et al., 1993). Bisson and Hontela (2002) reported that diazinon (OP) and mancozeb (CM) inhibited ACTH- and dibutyryl-cAMP-stimulated cortisol secretion in adrenocortical cells of rainbow trout at concentrations of 50-500 IxM. The capacity of mancozeb to disrupt adrenal steroidogenesis was much greater than that of diazinon. They also suggested that the activity is due to ethylene thiourea, a degradation product in the medium.
III. E N D O C R I N E - D I S R U P T I N G A C T I V I T Y O F O P s A N D C M s IN A N I M A L S A. O P Pesticides 1. ESTROGEN- AND ANDROGEN-RELATED ENDOCRINE EFFECTS Quinalphos exhibited estrogen-like action in vaginal cornification and increased the uterine wet weight (uterotrophic assay; Clark et al., 1980) in immature and ovariectomized mature rats by the treatment of 1 mg/kg/day, sc, for 3 days, but the activity was weaker than that of aldrin (Chatterjee et al., 1992). However, Sohoni et al. (2001) reported that fenitrothion (15 mg/kg/day for 3 days) did not have estrogenic activity in the immature mouse uterotrophic assay. Chlorpyrifos-methyl, the most commonly used OP pesticide throughout the world, was also nonestrogenic in immature rat uterotrophic assay when given at 250 mg/kg/day for 20 days (Kang et al., 2004). Regarding antiandrogenic activity, fenthion was positive in vivo in the Hershberger assay (Hershberger et al., 1953) using castrated male rats (Kitamura et al., 2003a). When fenthion (25-50mg/kg/day) was subcutaneously dosed together with testosterone propionate (0.5 mg/kg/day) for 7 days, the effect of testosterone on the weights of the prostate and seminal vesicles was significantly suppressed. In contrast, both positive and negative results on the antiandrogenic activity of fenitrothion have been reported (Ashby and Lefevre, 2000; Sunami et al., 2000; Tamura et al., 2001; Sohoni et al., 2001). Antiandrogenic activity of fenitrothion has been reported in the Hershberger assay using castrated male rats (Tamura et aL, 2001). When fenitrothion was administered by gavage at 15-30 mg/kg/day for 7 days together with testosterone propionate (50 rag/day, sc), the tissue weights of the ventral prostate, seminal vesicle, and levator ani plus bulbocavernous muscles were significantly decreased compared to those of corn oil-dosed castrated male rats. The study concluded that the activity of fenitrothion is comparable in potency to that of the pharmaceutical antiandrogen flutamide. Curtis (2001) featured the previous result in parallel with multiple measurements of neurotoxicity in "Toxicological Highlights" of
Toxicological Sciences. However, Ashby and Lefevre (2000) reported a negative result in the peripubertal male rat assay, which is an altemative to the Hershberger assay. Fenitrothion (15 mg/kg/day) failed to cause a significant delay in preputial separation of peripubertal male rats. Furthermore, their group reported a negative result for fenitrothion in the Hershberger assay using castrated male rats (Sohoni et al., 2001). Sunami et al. (2000) also reported that it showed no antiandrogenic activity in the Hershberger assay in castrated male rats. They found that fenitrothion (0.075-3 mg/kg/day for 5 days) significantly suppressed cholinesterase activities in the brain and erythrocytes but did not decrease the androgenic activity of testosterone. Unfortunately, they did not conduct a higher dose experiment, despite using a 100 mg/kg/day dose of p,p'-DDE as a positive control. Later experiments in vitro showed that fenitrothion had the highest antiandrogenic activity among OPs tested (Kitamura et al., 2003b; Kojima et al., 2004). The significant antiandrogenic activity of fenitrothion in vitro supports the in vivo findings of antiandrogenic properties. Regarding anticholinesterase activity, OPs are metabolically activated, but OPs seem to exhibit endocrinedisrupting activity. In fact, fenthion lost its antiandrogenic activity after metabolic transformation to fenthion sulfoxide. On the contrary, some pesticides may be metabolically activated. Although chlorpyrifos-methyl has weak or no affinity for estrogen and androgen receptors in in vitro assay (Jeong et al., 2001), chlorpyrifos-methyl showed antiandrogenic activity when given by gavage at 50 mg/kg/day for 10 days in the Hershberger test using rats (Kang et al., 2004). This OP may be metabolically activated in the animal body. It may be necessary to conduct ER or AR affinity tests in the presence of the metabolic system of MFO. To investigate whether fenthion has an impact on androgen-dependent sexual differentiation, the effects of in utero exposure were further examined in rats (Turner et al., 2002). Pregnant Sprague-Dawley (SD) rats were administered fenitrothion by gavage from gestation day 12 to day 21. In male offspring maternally exposed to it at 25 mg/kg/day, a reduction in anogenital distance was evident on postnatal day (PND) l, although the effect was transient, and no abnormalities in male organ development were noted at PND 100. Administration of fenitrothion at high dosages (55 mg/kg for 3 days) has been reported to reduce testicular cytochrome P450 without affecting cytochrome b5 or NADPH-cytochrome c reductase (Closet al., 1994). A decrease in serum testosterone levels was also detected at this dosage. The ability of parathion to interfere with the metabolism of androgen, as well as the uptake, in male accessory organs in rats was described in the 1960s and 1970s (Kupfer, 1967; Schein and Thomas, 1976; Thomas and Schein, 1974). The oral administration of parathion (1.3-5.2 mg/kg/day) caused significant alterations in the metabolism of testosterone in mice. An early study suggested that this compound
CHAPTER 34 9Endocrine Disruption by OPs and CMs competitively inhibits androgen binding to its receptor (Schein et al., 1979). Parathion was reported to interfere with mouse spermatogenesis, which may be related to its prenatal toxicity in mammals. An organ culture study of mouse testis revealed that parathion directly suppresses normal testicular differentiation (Rojas et al., 1998). An increase in apoptosis in spermatogonia by this pesticide was also reported (Bustos-Obregon et al., 2001). Quinalphos is also known to have adverse effects on the testis and male accessory glands in rats (Ray et al., 1991, 1992). When Wistar rats were given this compound at 0.25 mg/kg/day (ip) for 26 days, massive degeneration of germ cells in the testis, a reduction in the sperm count, and a decrease in plasma follicle-stimulating hormone (FSH) level were evident (Ray et al., 1992). Since administration of human chorionic gonadotrophin partially prevented these toxicological effects, quinalphos probably acts on the hypothalamuspituitary axis to suppress gonadotrophin release. High-dose administration of quinalphos, however, may impact differently on the endocrine system in rats. Treatment of SD rats with sublethal doses at 7-14 mg/kg/day for 15 days resulted in increased serum LH/FSH and testosterone levels, along with a decrease in testicular weight and severe disruption of spermatogenesis. Malathion shows a relatively high toxicity to fish. Morphological abnormalities in African catfish larvae exposed to malathion were observed (Lien et al., 1997). Diazinon has no hormone-like activity but shows endocrinedisrupting potential in fish. In dispersed adrenocortical cells of rainbow trout, this compound suppressed cortisol secretion in response to ACTH in vitro (Bisson and Hontela, 2002). When bluegill fish were exposed to diazinon at 60 Ixg/liter in water, serum E 2 became undetectable within 24 hr and there was morphological disruption of ovarian follicles (Maxwell and Dutta, 2005). The antiandrogen mechanism of OPs is clearly based on their ability to bind to AR in competition with testosterone or to bind to ER as agonists. However, other mechanisms are also involved. Chlorpyrifos, for instance, induced gene expression of gonadotropin-releasing hormone in a hypothalamic cell line, GT1-7, suggesting that it may have an impact on the hypothalamic-pituitary-gonad axis (Gore, 2002; Kojima et al., 2004). It has also been reported that chlorpyrifos may interfere with testosterone metabolism, potentially leading to hormonal imbalance (Usmani et al., 2003). 2. THYROID HORMONE-RELATED ENDOCRINE EFFECTS Malathion can disrupt thyroidal activity. In young adult rats, administration of malathion at 60 ixg/rat/day for 21 days resulted in decreased serum T3 and T4 and increased thyroid-stimulating hormone levels (Akhtar et al., 1996). In tadpole, malathion affects development. A significant delay of growth was observed in tadpoles in water
489
containing 1 mg/liter malathion, suggesting a decrease in thyroid function (Fordham et al., 2001). In the freshwater catfish, Clarias batrachus, malathion decreased serum T3 level but accelerated T4 synthesis in the pharyngeal thyroid. Extrathyroidal conversion of T4 to T3 was also inhibited (Sinha et al., 1991b, 1992). Rawlings et al. (1998) reported that chlorpyrfos and dimethoate decreased serum T4 levels in ewes treated with these chemicals at 12.5 and 0.2 mg/kg, respectively, three times a week for 43 days. They also demonstrated that chlorpyrifos increased the serum cortisol concentration, and dimethoate decreased the basal LH concentration. The teleost fish, Channa punctatus (Bloch), exposed to 2-4 ppm cythion in water developed hypertrophy and hyperplasia in the follicular epithelium and a reduction of colloid content in the thyroid (Ram et al., 1989). A significant reduction of thyroidal iodine uptake was also observed. Treatment of bullfrog tadpoles (Rana catesbeiana) with malathion significantly delayed developmental progression (Fordham et al., 2001). Malathion inhibited T3 binding to transthyretin but did not bind to the ligand-binding domain of thyroid hormone receptor [3 in a study using recombinant of Japanese quail transthyretin and thyroid receptor (Ishihara et al., 2003). This affinity for transthyretin may account for the effect of malathion on serum thyroid hormone levels in vivo. For other OP pesticides, no data are available regarding interaction with thyroid hormone receptor, transthyretin, or other thyroid hormone binding proteins. B. C M Pesticides 1. ESTROGEN- AND ANDROGEN-RELATED ENDOCRINE EFFECTS The mechanisms of the toxicity of CMs to testis and ovaries are not clear. The toxicity may be due to a direct effect on the ovary or the hypothalamus-pituitary-ovarian axis, causing hormonal imbalance. However, carbaryl and carbofuran have been reported to affect the function and morphology of the reproductive organs as well as serum estrogen levels in rats and fishes. When a high dose of carbaryl was orally administered to male rats (100 mg/kg body weight, 5 days a week), marked histopathological changes in the testes were seen, with degeneration of spermatogenic cells (Pant et al., 1995). These effects were associated with declines in epididymal sperm count and percentage sperm motility and increased abnormal sperm morphology. Adult male Wistar rats fed laboratory chow containing 1.0, 6.3, or 203 ppm benomyl exhibited decreased ejaculate sperm counts, decreased testicular weight, and a lowered male fertility index (Barnes et al., 1983). In catfish, carbaryl exposure produced a reduction in the number of oocytes, deformity in oocytes at different stages, and a reduction in the gonadosomatic index (Kulshrestha and Arora, 1984). Carbofuran seems to be more toxic. When female Swiss mice were orally administered
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with this chemical at 1 mg/kg body weight/day, there was a significant decrease in the number of estrous cycles and in the duration of the phases of each cycle. There was a significant decrease in the number of healthy follicles (Baligar and Kaliwal, 2002). Rats dosed with carbofuran (1.5 mg/kg, sc) showed transient endocrine disruption; that is, the levels of progesterone, cortisol, and E2 were significantly increased, whereas the levels of testosterone were decreased. No significant change occurred in T3 or T4 (Goad et al., 2004). In fish, treatment with 0.5-1 mg/liter of carbofuran inhibited oocyte maturational processes in females (Chatterjee et al., 1997) and caused deleterious testicular changes, included necrosis of Leydig cells (Ram et al., 2001). In contrast, an impairment of the duration of estrous cycles in rats treated with mancozeb was observed (Mahadevaswami et al., 2000). Molinate, a thiocarbamate herbicide, showed testicular toxicity after metabolic activation because the sulfoxide, a major metabolite generated by testis microsomes, exhibited more potent toxicity (Jewell et al., 1998).
2. THYROID HORMONE-RELATED ENDOCRINE EFFECTS CMs disrupt serum thyroid hormone levels in rodents and fishes. In a study of SD rats fed aldicarb at 10ppb, metribzin at 10 ppm, or methomyl at 1 ppm in drinking water (6-16 weeks), serum thyroxin levels increased significantly (Porter et al., 1999). In catfish, carbaryl exposure (12 mg/liter) for 96 hr suppressed serum T4 but elevated serum T3 levels, whereas exposure (5 mg/liter) for 16 days decreased both T3 and T4 (Sinha et al., 1991a). Rawlings et al. (1998) reported that carbofuran caused a significant increase in serum thyroxin level, and triallate increased the basal LH level compared with that of control ewes. In the teleost fish, C. punctatus, treatment with carbaryl at 1.7 ppm for 30 days also caused a decline in T4 and an elevation in T3 (Ghosh et al., 1989). In the same species, long-term exposure (6 months) to carbofuran at 4.5 ppm led to histological abnormalities of the thyroid gland, including hypertrophy, hyperplasia, and degeneration of follicular epithelial cells, and a reduction in colloid content along with retardation of thyroid function. Another study also found a greater susceptibility of the younger group to chronic toxicity of carbofuran compared with adults in this species (Ram, 1988). Mancozeb can disrupt thyroid function, and its degradation product, ethylene thiourea, is causally related to thyroid cancer in animals (Steenland et al., 1997; Chhabra et al., 1992). The mechanisms of disruption of thyroid function have not been established. Direct interaction of CMs with thyroid hormone receptors or serum thyroid binding proteins seems unlikely but has not been investigated. The adverse histophysiological alterations in the thyroid and changes in serum thyroid hormone levels may be a result of CMs directly interfering with the iodide transporter or
the organic iodine formation process in the thyroid gland, or they may occur as a result of disruption at any point in the hypothalamus-pituitary-thyroid axis.
IV. I N F L U E N C E O F O P s A N D C M s ON HUMANS Human exposure to single endocrine-disrupting chemicals is generally considered to be low, and the compounds are much less potent than the natural hormones. However, humans are expected to be exposed to a mixture of potential endocrine-disrupting chemicals. Hence, potential additive or synergistic effects have to be considered to assess the human risk. Furthermore, in vivo potency is often different from in vitro potency since the mechanisms of hormonal action of chemicals may not be adequately reflected by the in vitro assays. OPs and CMs are widely used in agriculture and for pest control. OPs such as chlorpyrifos have been found in human blood in Spain (Pitarch et al., 2003), in human urine in the United States (Olsson et al., 2003), and in human breast milk in India (Sanghi et al., 2003), together with organochlorine pesticides. There are many opportunities for human exposure. Because the pesticides are believed to be readily absorbed through the skin, monitoring is an essential component of assessment of exposure. Cocker et al. (2002) reported that whereas in nonoccupationally exposed people the urinary alkyl phosphates do not exceed 72 lxmol/mol creatine, occupationally exposed people excrete alkyl phosphates in the urine at the average level of 122 ixmol/mol creatine. However, the authors indicated that the levels of alkyl phosphates in workers are unlikely to cause a significant reduction in blood cholinesterase activity. In a volunteer study of exposure to chlorpyrifos, diazinon, propetamphos, and diazinon, almost all of these pesticides were found to be excreted after an oral dose, but large amounts were recovered in the skin surface after a dermal dose (Cocker et al., 2002; Garfitt et al., 2002). Tuomainen et al. (2002) reported that after application of malathion in greenhouses, the level of the pesticide in urine of workers reached a maximum at 6 or 7 hr and then rapidly decreased. Colosio et al. (2002) measured the ethylenethiourea level in urine of workers in vineyards as an indicator of mancozeb, and they reported that the levels in the workers were much higher than in controls, being especially high in operators of open tractors. In contrast, infants and children may have other opportunities to be exposed to OPs and CMs. Gurunathan et al. (1998) reported that chlorpyrifos persistently accumulates on residential surfaces and toys accessible to children after household application. Exposure of children to OPs can be evaluated by measuring urinary biomarkers and their metabolites, and it may be related to their potential adverse health effects (Eskenazi et al., 1999).
CHAPTER 34
Environmental antiandrogens may affect the development of the male reproductive system (Kelce and Wilson, 1997). Because androgens initiate and maintain spermatogenesis, environmental antiandrogens may contribute to the decline in sperm content that is suspected to be occurring in humans and other animals. Furthermore, inhibition of androgen receptor-mediated actions during the embryonic stage may lead to developmental alteration of male external genitalia (Foster, 1997). In humans, although a causal relationship between exposure to endocrine-disrupting pesticides and reproductive disorders has not been established, some reports predict an effect on humans. Sherman (1996) reported birth defects of the central nervous system in children exposed in utero to chlorpyrifos. Weidner et al. (1998) noted an increased occurrence of cryptorchidism in sons of female gardeners. Abell et al. (2000) also reported reduced fecundity in female greenhouse workers. There are conflicting results concerning the association of exposure of pregnant women to pesticides with changes of fetal growth or length of gestation (Grether et al., 1987; Savitz et al., 1989; Fenster and Coye, 1990; Restrepo et al., 1990; Thomas et al., 1992; Willis et al., 1993; Kristensen et al., 1997; Xiang et al., 2000; Perera et al., 2003; Eskenazi et al., 2004). Further examination is needed to clarify the influence of OPs and CMs on humans and wildlife.
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Porter, W. R, Jaeger, J. W., and Carlson, I. H. (1999). Endocrine, immune, and behavioral effects of aldicarb (carbamate), atrazine (triazine) and nitrate (fertilizer) mixtures at groundwater concentrations. Toxicol. Ind. Health 15, 133-150. Ram, R. N. (1988). Carbofuran-induced histophysiological changes in thyroid of the teleost fish, Channa punctatus (Bloch). Ecotoxicol. Environ. Safety 16, 106-113. Ram, R. N., Joy, K. E, and Sathyanesan, A. G. (1989). Cythioninduced histophysiological changes in thyroid and thyrotrophs of the teleost fish, Channa punctatus (Bloch). Ecotoxicol. Environ. Safety 17, 272-278. Ram, R. N., Singh, I. J., and Singh, D. V. (2001). Carbofuran induced impairment in the hypothalamo-neurohypophysealgonadal complex in the teleost, Channa punctalus (Bloch). J. Environ. Biol. 22, 193-200. Rawlings, N. C., Cook, S. J., and Waldbillig, D. (1998). Effects of the pesticides carbofuran, chlorpyrifos, dimethoate, lindane, trillate, trifluran, 2,4-D and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes. J. Toxicol. Environ. Health A 54, 21-36. Ray, A., Chatterjee, S., Ghosh, S., Kabir, S. N., Pakrashi, A., and Deb, C. (1991). Suppressive effect of quinalphos on the activity of accessory sex glands and plasma concentrations of gonadotrophins and testosterone in rats. Arch. Environ. Contam. Toxicol. 21, 383-387. Ray, A., Chatterjee, S., Ghosh, S., Bhattacharya, K., Pakrashi, A., and Deb, C. (1992). Quinalphos-induced suppression of spermatogenesis, plasma gonadotrophins, testicular testosterone production, and secretion in adult rats. Environ. Res. 57, 181-189. Restrepo, M., Munoz, N., Day, N. E., Parra, J. E., de Romero, L., and Nguyen-Dinh, X. (1990). Prevalance of adverse reproductive outcomes in a population occupationally exposed to pesticides in Colombia. Scand. J. Work Environ. Health 16, 232-238. Rojas, M., Bustos-Obregon, E., Martinez-Garcia, E, Contreras, H., and Regadera, J. (1998). The effect of parathion on mouse testicular and epididymal development cultured in chicken allantochorion. Adv. Exp. Med. Biol. 444, 201-206. Sanghi, R., Pillai, M. K., Jayalekshmi, T. R., and Nair, A. (2003). Organochlorine and organophosphorus pesticide residues in breast milk from Bhopal, Madhya, Pradesh, India. Hum. Exp. Toxicol. 22, 73-76. Savitz, D. A., Whelan, E. A., and Kleckner, R. C. (1989). Self-reported exposure to pesticides and radiation related to pregnancy outcome m Results from National Natality and Fetal Mortality Surveys. Public Health Rep. 104, 473-477. Schein, L. G., and Thomas, J. A. (1976). Deldrin and parathion interaction in the prostate and liver of the mouse. J. Toxicol. Environ. Health 1, 829-838. Schein, L. G., Donovan, M. E, Thomas, J. A., and Felice, E R. (1979). Effects of pesticides on 3H-dihydrotestosterone binding to cytosol proteins from various tissues of the mouse. J. Environ. Pathol. Toxicol. 3, 461-470. Shain, S. A., Shaeffer, J. C., and Boesel, R. W. (1977). The effect of chronic ingestion of selected pesticides upon rat ventral prostate homeostasis. Toxicol. Appl. Pharmacol. 40, 115-130. Sherman, J. D. (1996). Chlorpyrifos (Dursban)-associated birth defects: Report of four cases. Arch. Environ. Health 51, 5-8.
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Sinha, N., Lal, B., and Singh, T. E (1991a). Carbaryl-induced thyroid dysfunction in the freshwater catfish Clarias batrachus. Ecotoxicol. Environ. Safety 21, 240-247. Sinha, N., Lal, B., and Singh, T. E (1991b). Pesticides induced changes in circulating thyroid hormones in the freshwater catfish Clarias batrachus. Comp. Biochem. Physiol. C 100, 107-110. Sinha, N., Lal, B., and Singh, T. E (1992). Thyroid physiology impairment by malathion in the freshwater catfish Clarias batrachus. Ecotoxicol. Environ. Safety 24, 17-25. Sohoni, E, Lefevre, E A., Ashby, J., and Sumpter, J. E (2001). Possible androgenic/anti-androgenic activity of the insecticide fenitrothion. J. Appl. Toxicol. 21, 173-178. Sonnenschein, C., and Soto, A. M. (1998). An update review of environmental estrogen and androgen mimics and antagonists. J. Steroid Biochem. Mol. Biol. 65, 143-150. Steenland, K., Cedillo, L., Tucker, J., Hines, C., Sorensen, K., Deddens, J., and Cruz, V. (1997). Thyroid hormones and cytogenetic outcomes in backpack sprayers using ethylenebis(dithiocarbamate) (EBDC) fungicides in Mexico. Environ. Health Perspect. 105, 1126-1130. Sunami, O., Kunimatsu, T., Yamada, T., Yabushita, S., Sukata, T., Miyata, K., Kamita, Y., Okuno, Y., Seki, T., Nakatsuka, I., and Matsuo, M. (2000). Evaluation of a 5-day Hershberger assay using young mature male rats: Methyltestosterone and p,p'-DDE, but not fenitrothion, exhibited androgenic or antiandrogenic activity in vivo. J. Toxicol. Sci. 25, 403-415. Tamura, H., Maness, S. C., Reischmann, K., Dorman, D. C., Gray, L. E., and Gaido, K. W. (2001). Androgen receptor antagonism by the organophosphate insecticide fenitrothion. Toxicol. Sci. 60, 56-62. Thomasl D. C., Petitti, D. B., Goldhaber, M., Swan, S. H., Rappaport, E. B., and Hertz-Picciotto, I. (1992). Reproductive outcomes in relation to malathion spraying in the San Francisco Bay Area, 1981-1982. Epidemiology 3, 32-39. Thomas, J. A., and Schein, L. G. (1974). Effect of parathion on the uptake and metabolism of androgens in rodent sex accessory organs. Toxicol. Appl. Pharmacol. 29, 53-58. Tilak, K. S., Veeraiah, K., and Rao, D. K. (2004). Toxicity and bioaccumulation of chlorpyrifos in Indian carp Catla catla (Hamilton), Labeo rohita (Hamilton), and Cirrhinus mrigala (Hamilton). Bull. Environ. Contam. Toxicol. 73, 933-941.
Tuomainen, A., Kangas, J. A., Meuling, W. J. A., and Glass, R. C. (2002). Monitoring of pesticide applicators for potential dermal exposure to malathion and biomarkers in urine. Toxicol. Lett. 134, 125-132. Turner, K. J., Barlow, N. J., Struve, M. E, Wallace, D. G., Gaido, K. W., Dorman, D. C., and Foster, E M. (2002). Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgen-dependent reproductive development in the Crl:CD(SD)BR rat. Toxicol. Sci. 68, 174-183. Tsuda, T., Aoki, S., Kojima, M., and Harada, H. (1990). Bioconcentration and excretion of diazinon, malathion and fenitrothion by carp. Comp. Biochem. Physiol. C 96, 23-26. Tsuda, T., Kojima, M., Harada, H., Nakajima, A., and Aoki, S. (1996). Accumulation and excretion of fenthion, fenthion sulfoxide and fenthion sulfone by killifish (Oryzias latipes). Comp. Biochem. Physiol. C 113, 45-49. Usmani, K. A., Rose, R. L., and Hodgson, E. (2003). Inhibition and activation of the human liver microsomal and human cytochrome P450 3A4 metabolism of testosterone by deployment-related chemicals. Drug Metab. Disp. 31, 384-391. Vinggaard, A. M., Hnida, C., Breinholt, V., and Larsen, J. C. (2000). Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. in Vitro 14, 227-234. Vonier, E M., Crain, D. A., McLachlan, J. A., Guillette, L. J., and Arnold, S. E (1996). Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator. Environ. Health Perspect. 104, 1318-1322. Weidner, I. S., Moller, H., Jensen, T. K., and Skakkebaek, N. E. (1998). Cryptorchidism and hypospadias in sons of gardeners and farmers. Environ. Health Perspect. 106, 793-796. Willis, W. O., de Peyster, A., Molgaard, C. A., Walker, C., and MacKendrick, T. (1993). Pregnancy outcome among women exposed to pesticides through work or residence in an agricultural area. J. Occup. Med. 35, 943-949. Wong, C., Kelce, W., Sar, M., and Wilson, E. M. (1995). Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270, 19998-20003. Xiang, H., Nuckolos, J. R., and Stallones, L. (2000). A geographic information assessment of birth weight and crop production patterns around mother's residence. Environ. Res. 82, 160-167.
CHAPTER ~ 5
Organophosphates, Carbamates, and the Immune System RAGHUBIR P. SHARMA The University of Georgia, Athens, Georgia
pesticides possess detrimental effects on various immune responses, information on the effects of these chemicals on the immune system of humans is very limited. It is controversial whether environmental or even occupational exposures to these pesticides are capable of affecting the immune system (Thomas, 1995). The level of exposure from the environment is negligible for anticholinesterase pesticides because of their short lives in the environment, including food. A greater concern of untoward effect with pesticide use is occupational exposures at the manufacturing, shipping and formulating, and application levels. In addition to pesticide exposure through environmental or occupational modes, people may also come in contact with cholinesterase inhibitors via therapeutic uses. Cholinesterase inhibitors have been employed in a variety of nervous system disorders, including Alzheimer's disease, Down syndrome, traumatic brain injury, and delirium (Giacobini, 2004). For the treatment of these disorders and cognitive enhancement, patients have to be treated for a long period (24 months or longer) with either specific acetycholinesterase inhibitors or butyrylcholinesterase inhibitors, sometime switching one with the other. A long-term treatment with such inhibitor is likely to cause various systemic effects, including those on the immune system. The possibility that OPs can be designed as selective immunosuppressors for therapeutic purposes has been considered (Becker, 1975). It was theorized that highly specific, highly active, relatively stable, and nontoxic chemicals can be prepared. Specificity against the first component of the complement system was selectively increased by adding a terminal amino group to the p-nitrophenyl ethyl pentyl phosphate. It is sometimes possible to separate the anticholinesterase activity from the protease inhibitor activity; however, this approach has had limited success. There is increasing concern regarding safety and risk assessment of chemicals on the developing immune system (Holsapple et al., 2004). The developing immune system is considered as susceptible to toxic effects; however, data supporting this assumption are limited. On the one hand,
I. I N T R O D U C T I O N Pesticides are toxic chemicals that are deliberately introduced in the environment and therefore exposure of people and animals to them is unavoidable. These chemicals are inherently toxic, especially to the organisms they are intended to eradicate. They also produce variable toxicity in all other organisms that come in contact with them. The use of pesticides is desirable because pests cause heavy losses to crops and other food commodities and they are indispensable for disease prevention in public health. Some of the original agents used as pesticides included metal compounds and chlorinated hydrocarbons. Due to the persistence and biomagnification of earlier pesticides in the food chain, their use is on the decline and they have largely been replaced by cholinesterase inhibitors and, recently, pyrethrins and analogs. Anticholinesterase agents are extensively used in agriculture because of both their effectiveness and their low cost; also, they are not persistent, so there is no concern of secondary exposures via food. Because exposure to prevalent cholinesterase-inhibiting insecticides is unavoidable, these compounds have been investigated in detail with regard to their toxic potential; however, their potential effects on the immune system are not so well characterized. Concern regarding the immunotoxic potential of chemicals in general is relatively new in the assessment of their safety. Only a few organophosphates (OPs) and carbamates (CMs) have been systematically investigated for their immunotoxic potential; studies have used different protocols and various parameters for measuring toxic outcomes. The immune system is important for defense against a variety of organic insults and is necessary for the well-being and even survival of host organisms. However, the immune system is highly complex, regulated by multistep control processes, and can be influenced by the status of other body systems and functions. There are a number of reviews on the immunotoxicity of OPs and CMs (Sharma, 1988; Sharma and Tomar, 1992; Galloway and Handy, 2003). Despite indications that these Toxicology of Organophosphate and Carbamate Compounds
495
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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SECTION IV. Organ Toxicity
the system undergoing development in neonates and children may be at greater risk due to differentiation and cellular proliferation occurring at this stage; on the other hand, adaptation and plasticity of developing systems suggest that the effects may not be long-lasting. However, various regulatory agencies are cautious and increasingly have been developing additional guidelines for risk assessment for developmental toxicology (Kimmel and Makris, 2001). New guidelines that involve additional information on effects in pregnant women and children are recommended in various areas of safety evaluation, including for immunotoxicity studies.
II. C O M P L E X I T I E S O F T H E I M M U N E SYSTEM AND LOCATIONS OF EFFECTS FOR TOXIC CHEMICALS The immune system is diffuse and distributed throughout the body. Various cells of this system, primarily macrophages and lymphocytes and their subpopulations, are found in the circulation and in most organs. The system possesses multistep control processes by which production and proliferation of immune-related cells are regulated depending on their need. Cells in the immune system require interaction and communication with each other or other systems to achieve their objectives. Immune functions include innate immunity, which is inherent in most tissues and is ready to neutralize invading organisms or antigens. This resident immune system in various organs employs macrophages, polymorphonuclear leukocytes, and natural killer (NK) lymphocytes. The other type is the adaptive immune system, which requires priming with specific antigens to mount an appropriate defense. The latter may be further divided into humoral immunitymthat is, production of specific antibodies [immunoglobulins (Igs)] by primed B cellsmand cell-mediated immunity carried out by T lymphocytes. Several subpopulations of T cells exist (e.g., cytotoxic T cells, helper T cells, and suppressor T cells). Some lymphocytes are NK cells, which generally require no priming or proliferation to exert their effects. The functions of immune competent cells are communicated either via cell-cell communications or through cytokines. Macrophages are derived from monocytes and are present in various body cavities, such as pulmonary alveoli and peritoneum; they are also found in the lymph nodes and liver as components of the innate immune system. Other peripheral leukocytes are often involved in various immunopathologic mechanisms. For acquired immunity, macrophages are phagocytes that concentrate antigens and confer specific immunological responses to various T or B cells and also remove cell debris. The immune system interacts with other systems and is profoundly influenced by the central nervous system, both directly via innervations of lymphatic organs and indirectly
via neuroendocrine mechanisms. The cells of the immune system produce factors that influence the nervous system. Hormones such as somatotrophin (growth hormone) and thymosin (thymic maturation factor) stimulate the immune responses, whereas steroids, including sex hormones, generally suppress the immune responses. Because the effective mounting of immune responses requires cell proliferation and production of signaling agents involving DNA and protein synthesis, chemical factors that interfere with cell cycling will modulate the r immune responses. This interference may occur at several steps in the immune functions. A schematic presentation of various locations where chemicals can interfere with the immune system is depicted in Fig. 1. This is a simplistic scheme and may not cover every aspect of either the immune system or other factors that regulate the system.
III. M E C H A N I S M S OF IMMUNOMODULATING ACTION OF ANTICHOLINESTERASES
A. Esterases and Immune Responses The immune system can be affected either by direct inhibition of esterases in effector cells or via alterations in neurotransmitters that regulate autonomic nervous system connections. Becker and Henson (1973) suggested that activated esterases may degranulate mast cells, releasing histamine, and may be involved in the process of chemotaxis by rabbit polymorphonuclear cells. Lymphocyte activation and subsequent division after antigen challenge are prerequisites for many immunological responses. It has been reported that serine esterases are activated after surface Ig cross-linking by anti-Ig. Diisopropylfluorophosphate (DFP; a potent anticholinesterase agent) inhibited the anti-Ig-induced activation of mouse B cells (Ashman, 1984). A direct role of esterases in immune responses was also suggested in the cytotoxic and NK cell-mediated cell lysis. The target cell lysis is associated with proteins stored in cytoplasmic granules of the effector cells, which are released on target cell recognition. Many of these proteins have been identified as serine esterases (Jenne and Tschopp, 1988). T cell killing was also inhibited by DFP and other serine esterase inhibitors (Ferluga et al., 1972). When mice were injected with the powerful cholinesterase inhibitor DFP, antigen processing and presentation by macrophages for immune processes requiting the cooperation of T cells were affected (Shek and Eastman, 1988). However, the response to macrophage-independent B cell antigens was not altered. DFP also interfered with the generation of memory cells that are responsible for clonal expansion when cells encounter a subsequent challenge to the antigen. Anticholinesterases may act directly on the cells possessing acetylcholine receptors located on lymphocytes.
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,-37.5 ~ Saadeh et al. (1996) described the clinical and sociodemographic data of 70 adults exposed acutely to OP or CM insecticides and found that 49% had a low-grade fever of 37.5-38.5 ~ with no evidence of infection. The onset and recovery from fever occurred between 1 and several days after exposure, with nearly half of the fevers persisting for 3 days. They noted that patients with fevers had received significantly more atropine than those without fevers. One might consider if the administration of atropine is in some way responsible for the fever since muscarinic antagonists have well-known hyperthermic effects in humans (Christoph, 1989). On the other hand, the authors noted that the fever persisted in 27% of the patients for 4 days despite discontinuing atropine therapy. A presumed terrorist attack with the nerve gas sarin in Matsumoto, Japan, led to the poisoning of approximately 600 residents (Morita et al., 1995). A mild, low-grade fever was reported in some subjects for up to 1 month after exposure, and one man had a low-grade fever 6 months after exposure. It is interesting to note that the aforementioned studies rarely mention whether standard antipyretics such as aspirin were prescribed for alleviating the fever. Overall, it appears that fever is the most frequent thermoregulatory response in humans acutely exposed to anti-ChEs. On the other hand, an acute hypothermic effect of OP and CM insecticides has occasionally been reported in humans poisoned with anti-ChEs. In one instance, a 16-year-old male suffering from OP poisoning
TABLE 2. Summary of Studies That Have Shown a Febrile or Hyperthermic Response in Humans Exposed to OP and/or CM Insecticides
Study Saadeh et al. (1996) Hirshberg and Lerman (1984)
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Morita et al. (1995)
561
Findings Fever was a symptom in 49% of 70 subjects exposed acutely to OP or CM insecticides Fever was a symptom in 25% of cases labeled as "complicated or delayed" in humans exposed to OP insecticides from 1958 to 1979 One of first studies to document delayed fevers in humans exposed to parathion or methylparthion Mild or low-grade fever reported in a number of subjects lasting up to 1 month following terrorist attack with satin nerve gas in Matsumoto, Japan
562
SECTION V .
N o n s p e c i f i c Toxic Effects
was admitted with a high blood pressure (152/102) but a rectal temperature of only 34.5 ~ (Cupp et al., 1975). One hour after a 17-year-old male was admitted to the hospital following ingestion of a large amount of malathion, his blood pressure was also high (170/80), and he was mildly hypothermic with a core temperature of 36.2 ~ (Hassan et al., 1981). A 58-year-old woman who ingested a large amount of diazinon had a low blood pressure and was markedly hypothermic with a core temperature of 34.4 ~ 1 hr after poisoning (Hassan et al., 1981). In another series of case reports on diazinon poisoning, there were incidences of transient hypothermia (34.4 ~ and delayed fever (38.9 ~ (Klemmer et al., 1978). A 39-year-old man was admitted following malathion poisoning with high blood pressure (140/90), profuse sweating, and a core temperature of 34 ~ (Meller et al., 1981). By 15 hr after admission, core temperature had recovered to normal. In the same study, an 81-year-old woman was also admitted with high blood pressure (180/100) and a core temperature of 35 ~ It is possible that the very young and old may be more susceptible to the hypothermic effects of these pesticides, but this has not been studied. The hypothermic effects of the anti-ChEs in humans are most likely mediated by the stimulation of muscarinic cholinergic pathways that lead to profuse sweating and peripheral vasodilation. However, it is difficult to determine whether the hypothermic response is regulated as it appears to be in rodents because whenever the hypothermia is reported during the course of treatment in the emergency room, measures are usually taken to raise the subject's core temperature to normal. Information on the subject's state of thermal comfort during the onset of poisoning would be useful to determine if the thermal set point is reduced in the same manner as in rodents subjected to acute antiChE exposure. That is, if the subjects felt warm during the period of hypothermia, then it would suggest, as is evident in the behavioral thermoregulatory response of rodents, that the anti-ChEs induce a regulated hypothermia. In addition, because of the large body mass and small surface area: body mass ratio of adult humans, the ability to lower core temperature in response to a toxicant is markedly attenuated with increasing body mass (Gordon, 2004). As the muscarinic effects of anti-ChE poisoning abate, either naturally or due to prophylactic intervention, it appears that thermoeffectors for increasing heat gain and/or reduction in heat loss take over, leading to a prolonged albeit low-grade fever.
XI. EXERCISE AND HEAT STRESS The factors that affect the biological dosage of an antiChE agent are also linked to thermal homeostatic mechanisms. Toxic agents can enter the body by three principal routes: respiratory surfaces, gastrointestinal tract, and
transcutaneously (Casarett and Doull, 1975). These routes of entry into the body are also intimately connected to ambient temperature and the organism's thermoregulatory state. That is, under ideal environmental conditions (e.g., thermoneutral environment), a stable core temperature is maintained with minimal strain on physiological systems. However, in the face of marked changes in ambient temperature, relative humidity, and/or workload, a constant core temperature is maintained but at the expense of activation of thermoeffectors and physiological stress. The surface of the respiratory tract and skin are integral for the operation of thermoeffectors for evaporative and dry heat loss. Hence, when a homeotherm is in an environment in which it must actively dissipate heat, it is likely to be more susceptible to lower doses and/or concentrations of certain types of toxicants. On the other hand, the increased demand for heat production in a cold environment results in an elevation in respiratory rate, thus increasing the intake of airborne toxicants and also raising susceptibility. The thermoregulatory system responds to heat stress and exercise by activating three key systems to dissipate excess heat: cardiovascular, respiratory, and sudomotor (sweating). The combination of peripheral vasodilation to increase skin blood flow and raise skin temperature along with sweating results in an effective mechanism to dissipate a heat load (Folk et al., 1998; Blatteis, 1998). True panting animals exhibit marked increases in breathing frequency during heat stress. Nonpanting homeotherms, including humans and rodents, also exhibit increases in breathing frequency and minute volume that contribute to a modest increase in evaporative water loss when heat stressed (Ingram and Mount, 1975). The added heat load of exercise will further increase ventilation and augment the total intake of airborne pollutants (Mautz, 2003). Sweating is the principal thermoeffector response in heat-stressed humans and some other mammals. Eccrine sweat glands in humans are activated by cholinergic pathways, and stimulation of these pathways generally occurs concurrently with an increase in skin blood flow. The flow of warm blood from the core to the surface combined with evaporative cooling from sweating is an effective mechanism to dissipate excess body heat. On the other hand, the combination of moisture, warm temperatures on bare skin, and increased skin blood flow also provides an ideal environment to accelerate the transcutaneous absorption of many types of pesticides (Chang et al., 1994; Wester et al., 1996). Both in vitro and in vivo studies suggest that activation of thermoeffectors during heat stress and/or exercise to dissipate heat will accelerate pesticide absorption in humans. An in vitro model of cutaneous absorption of parathion has been used to show how temperature, blood flow, and relative humidity affect the absorption of parathion. A small section of porcine skin positioned over a flow-through diffusion cell provides an ideal means to control air temperature, relative humidity, perfusate
CHAPTER 3 8 .
temperature (i.e., an indication of body temperature), and flow of the perfusate (i.e., an indication of the potential effects of blood flow) while studying the percutaneous absorption of a pesticide (Chang and Riviere, 1991). The absorption of radiolabeled parathion across porcine skin increases dramatically with an elevation in air and/or perfusate temperature. For example, a 5 ~ increase in air and perfusate temperature leads to a more than two-fold increase in parathion absorption. It is possible that skin warming can raise lipid fluidity and permeability of the dermal tissues, leading to increased penetration of the pesticide. The cutaneous absorption of parathion is directly affected by relative humidity and perfusate flow. The effects of humidity are profound, suggesting that increased moisture on the skin raises the permeability to parathion. Parathion is a lipophillic molecule, and it is thus not clear why percutaneous absorption would increase with additional moisture on the skin. Studies on humans performed decades ago showed how perspiration can accelerate the cutaneous absorption of OPs. Human volunteers were exposed to ambient temperatures of 14, 21, 28, and 40.5 ~ while their hand and arm were exposed for 2 hr to a 2% parathion dust (Funckes et al., 1963). The absorption of the insecticide was estimated by the quantity of paranitrophenol, a metabolite of parathion, that was excreted in the urine. The dermal absorption of parathion was mildly affected by skin warming at low temperatures and markedly affected at warm temperatures. Parathion absorption increased by 25% when the temperature of exposure was raised from 14 to 21 ~ however, from 21 to 28 ~ parathion absorption increased by only 17%. Raising ambient temperature from 28 to 40.5 ~ led to a 180% increase in absorption (Funckes et al., 1963). Although the rate of sweating was not measured, it was clear that subjects perspired profusely at the warmest ambient temperature. It is also interesting to note the increase in parathion absorption at the lower ambient temperatures despite a lack of sweating. The warmer skin temperature is likely to be a critical factor affecting parathion absorption even without sweating. The dose of parathion used in this study was relatively low because RBC and plasma cholinesterase activity was unaffected by the treatment. The dosage from exposure to anti-ChE pesticides should be exacerbated in a warm and humid environment because of cholil~ergic stimulation of sweating combined with greater transcutaneous absorption across moist skin. In another human study, volunteers had small amounts of the nerve gas VX [S-(2-diisopropylaminoethyl) o-ethyl methylphosphonothioate] applied topically to their cheek and forearm at ambient temperatures of - 1 8 , 2, 18, or 46 ~ (Fig. 9). The VX was left on the skin for 3 hr and its penetration into the body was estimated by measuring the inhibition of RBC ChE activity (Craig et al., 1977). The absorption of VX in humans was directly dependent on ambient temperature~ The decimal fraction of penetration
Anticholinesterase Agents and Thermoregulation
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of VX on the cheek was 0.04 at an extremely cold ambient temperature of - 1 8 ~ and 0.32 under conditions of extreme heat stress with an ambient temperature of 46 ~ For the skin on the forearm, the VX penetration was 0.004 at 18 ~ and increased to 0.029 at 46 ~ Overall, penetration across the cheek was much more effective than that on the forearm, with greater than 50% inhibition in ChE activity occurring at a dose of less than 10 Ixg/kg. It was postulated that after exposure to VX or a comparable agent, cooling the skin would delay absorption, thus allowing for safer decontamination of an exposed subject.
XII. S U M M A R Y The regulation of body temperature is an ideal benchmark to study the toxicity of the anti-ChE insecticides in experimental mammals and humans. The inhibition in AChE activity and subsequent stimulation of cholinergic pathways in the central and peripheral nervous systems is a key mechanism responsible for driving changes in body temperature and activation of thermoregulatory effectors. Body size and ambient temperature are perhaps the most critical factors governing the thermoregulatory effects of these toxicants. Small mammals with a relatively large surface area: body mass ratio undergo a marked increase in heat loss and become hypothermic when exposed to anti-ChEs. After recovery from the acute hypothermic effects, rats have been shown to exhibit a small but prolonged elevation in temperature manifested during the daytime that is similar in many ways to a fever. A fever predominates in large mammals such as humans following an acute exposure. The hypothermic response in large mammals is relatively small and transient compared to that seen in small rodents. Small mammals exposed to these agents lower their body temperature by selective activation of heat loss thermoeffectors. This hypothermic response is often protective
564
SECTION V . N o n s p e c i f i c Toxic Effects
and increases the likelihood of surviving the toxic insult. Large mammals such as humans are unable to take advantage of this hypothermic response, and it is not clear if a fever from these toxicants will affect the health effects of these agents. It does appear that exercise and/or heat stress will exacerbate the toxic effects of anti-ChE insecticides and nerve gas agents. The redistribution of blood to the skin combined with sweating will increase the cutaneous absorption of OP agents that are applied to the skin. The increase in pulmonary ventilation during exercise combined with higher tissue temperature is bound to increase the intake and toxicity of airborne anti-ChE agents.
XIII. FINAL PERSPECTIVE All life processes depend on chemical reactions that, in turn, are directly dependent on temperature, as based on the principles of the Arrhenius equation. The toxicology of the anti-ChE agents, like all other toxicants, is intimately related to temperature and the thermoregulatory system. We have shown that the intake through the lungs and skin, metabolic activation and deactivation, and physiological effects of anti-ChE agents can be affected by ambient, skin, and core temperature. The message of this chapter is that toxicologists working at the subcellular, cellular, in vitro, and/or in vivo level should always consider how temperature can influence the toxicological efficacy of anti-ChE agents.
Acknowledgments We thank Dr. Amir Rezvani and Andrew Geller for reviewing the manuscript.
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Bligh, J. (200]). Mammalian homeothermy: An integrative thesis. J. Thermal Biol. 23, 143-258. Casarett, L. J., and Doull, J. (1975). Factors influencing toxicology. In Toxicology. The Basic Sciences of Poisons, pp. 133-147. Macmillan, New York. Chang, S.-K., and Riviere, J. E. (1991). Percutaneous absorption of parathion in vitro in porcine skin: Effects of dose, temperature, humidity, and perfusate composition on absorptive flux. Fundam. Appl. Toxicol. 17, 494-504. Chang, S.-K., Brownie, C., and Riviere, J. E. (1994). Percutaneous absorption of tropical parathion through porcine skin: In vitro studies on the effect of environmental perturbations. J. Vet. Pharamcol. Ther. 17, 434-439. Christoph, R. A. (1989). Organophosphates and carbamates. In Manual of Toxicologic Emergencies. Year Book Chicago. Noji, E. K., Kelen, G. D., Goessel, T. K., eds. Clark, W. G., and Lipton, J. M. (! 985a). Changes in body temperature after administration of amino acids, peptides, dopamine, neuroleptics and related agents: II. Neurosci. Biobehav. Rev. 9, 299-371. Clark, W. G., and Lipton, J. M. (1985b). Changes in body temperature after administration of acetylcholine, histamine, morphine, prostaglandins and related agents: II. Neurosci. Biobehav. Rev. 9, 479-552. Clement, J. G. (1991). Variability of sarin-induced hypothermia in mice: Investigation into incidence and mechanism. Biochem. Pharmacol. 42, 1316-1318. Clement, J. G. (1993). Pharmacological nature of soman-induced hypothermia in mice. Pharmacol. Biochem. Behav. 44, 689-702. Costa, L. G., and Murphy, S. D. (1983). Unidirectional crosstolerance between the carbamate insecticide propoxur and the organophosphate disulfoton in mice. Fundam. Appl. Toxicol. 3, 483-488. Coudray-Lucas, C., Prioux-Guyonneau, M., Tassel, A., Coq, H. M., Cohen, Y., and Wepierre, J. (1981). Influence of intoxication by anticholinesterase agents on core temperature in rats: Relationship between hypothermia and acetylcholinesterase inhibition in different brain areas. Acta Pharmacol. Toxicol. 49, 215-222. Coudray-Lucas, C., Prioux-Guyonneau, M., Sentenac, H., Cohen, Y., and Wepierre, J. (1983). Brain catecholamine metabolism changes and hypothermia in intoxication by anticholinesterase agents. Acta Pharmacol. Toxicol. 52, 224-229. Craig, E N., Bales, E D., and Frankel, H. M. (1959). Lethality of sarin in a warm environment. J. Pharmacol. Exp. Ther. 127, 35-38. Craig, E N., Cummins, E. G., and Sum, V. M. (1977). Environmental temperature and the percutaneous absorption of a cholinesterse inhibitor, VX. J. Invest. Dermatol. 68, 357-361. Cupp, C. M., Kleiber, G., Reigart, R., and Sandfifer, S. H. (1975). Hypothermia in organophosphate poisoning and response to PAM. J. S. Carolina Med. Assoc. 71, 166-168. Doull, J. (1972). The effect of physical environmental factors on drug response. Essays Toxicol. 3, 37-63. Folk, G. E., Jr., Riedesel, M. L., and Thrift, D. L. (1998). Principles of Integrative Environmental Physiology. Austin & Winfield, Lanham, MD. Funckes, A. J., Hayes, G. R., Jr., and Hartwell, W. V. (1963). Urinary excretion of paranitrophenol by volunteers following dermal exposure to parathion at different ambient temperatures. J. Agr. Food Chem. 11, 455-457.
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9Anticholinesterase Agents and Thermoregulation
Gordon, C. J. (1993a). Temperature Regulation in Laboratory Rodents. Cambridge Univ. Press, New York. Gordon, C. J. (1993b). Acute and delayed effects of diisopropyl fluorophosphate on body temperature, heart rate, and motor activity in the awake, unrestrained rat. J. Toxicol. Environ. Health 39, 247-260. Gordon, C. J. (1994a). Thermoregulation in laboratory mammals and humans exposed to anticholinesterase agents. Neurotoxicol. Teratol. 16, 427-453. Gordon, C. J. (1994b). 24-Hour control of body temperature in the rat: II. Diisopropyl fluorophosphate-induced hypothermia and hyperthermia. Pharmacol. Toxicol. Behav. 49, 747-754. Gordon, C. J. (1996a). Homeothermy: Does it impede the response to cellular injury. J. Thermal Biol. 21, 29-36. Gordon, C. J. (1996b). Pharmacological analysis of diisopropyl fluorophosphate: Effects on core temperature, heart rate, and motor activity in the unrestrained rat. Pharmacol. Biochem. Behav. 55, 185-194. Gordon, C. J. (1997). Behavioral thermoregulatory response to chlorpyrifos in the rat. Toxicology 124, 165-171. Gordon, C. J. (2004). Temperature and Toxicology: An Integrative, Comparative, and Environmental Approach. CRC Press, Boca Raton, FL. Gordon, C. J., and Fogelson, L. (1993). Relationship between serum cholinesterase activity and the change in body temperature and motor activity in the rat: A dose-response study of the diisopropyl fluorophosphate. Neurotoxicol. Teratol. 15, 21-25. Gordon, C. J., and Grantham, T. A. (1999). Effect of central and peripheral cholinergic antagonists on chlorpyrifos-induced changes in body temperature in the rat. Toxicology 142, 15-28. Gordon, C. J., and Mack, C. M. (2001). Diurnal variation in thermoregulatory response to chlorpyrifos and carbaryl in the rat. Toxicology 169, 93-105. Gordon, C. J., and Mack, C. M. (2003). Influence of gender on thermoregulation and cholinesterase inhibition in the Long-Evans rat exposed to diazinon. J. Toxicol. Environ. Health A 66, 291-304. Gordon, C. J., and Padnos, B. (2002). Dietary exposure to chlorpyrifos alters core temperature in the rat. Toxicology 177, 215-226. Gordon, C. J., and Rowsey, P. J. (1999). Are circulating cytokines interleukin-6 and tumor necrosis factor e~ involved in chlorpyrifos-induced fever? Toxicology 134, 9-17. Gordon, C. J., Mohler, E S., Watkinson, W. E, and Rezvani, A. H. (1988). Temperature regulation in laboratory mammals following acute toxic insult. Toxicology 53, 161-178. Gordon, C. J., Fogelson, L., Lee, L., and Highfill, J. (1991). Acute effects of diisopropyl fluorophosphate (DFP) on autonomic and behavioral thermoregulatory responses in the Long-Evans rat. Toxicology 67, 1-14. Gordon, C. J., Grantham, T. A., and Yang, Y. (1997). Hypothermia and delayed fever in the male and female rat exposed to chlorpyrifos. Toxicology 118, 149-158. Gordon, C. J., Puckett, E., and Padnos, B. (2002). Rat tail skin temperature monitored noninvasively by radiotelemetry: Characterization by examination of vasomotor response with thermomodulatory agents. J. Pharmacol. Toxicol. Methods 47, 107-114.
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Gralewicz, S., and Socko, R. (1997). Persisting behavioural and electroencephalographic effects of exposure to chlorphenvinphos, an organophosphorous pesticide, in laboratory animals. Int. J. Occup. Med. Environ. Health 10, 375-394. Gupta, R. C., Goad, J. T., and Kadel, W. L. (1994). Energy related metabolic alterations in diaphragm muscle resulting from acute methomyl toxicity. Neurotoxicology 15, 321-330. Haque, N., Rizvi, S. J., and Khan, M. B. (1987). Malathion induced alterations in the lipid profile and the rate of lipid peroxidation in rat brain stem and spinal cord. Pharmacol. Toxicol. 61, 12-15. Hassan, R. M., Pesce, A. J., Sheng, P., and Hanenson, I. B. (1981). Correlation of serum pseudocholinesterase and clinical course in two patients poisoned with organophosphate insecticides. Clin. Toxicol. 18, 401-406. Hirshberg, A., and Lerman, Y. (1984). Clinical problems in organophosphate insecticide poisoning: The use of a computerized information system. Fundam. Appl. Toxicol. 4, $209-$214. Ingram, D. L., and Mount, L. E. (1975). Man and Animals in Hot Environments. Springer-Verlag, New York. Keplinger, M. L., Lanier, G. E., and Deichmann, W. B. (1959). Effects of environmental temperature on the acute toxicity of a number of compounds in rats. Toxicol. Appl. Pharmacol. 1, 156-161. Klemmer, H. W., Reichert, E. R., and Yauger, W. L., Jr. (1978). Five cases of intentional ingestion of 25 percent diazinon with treatment and recovery. Clin. Toxicol. 12, 435444. Kluger, M. J., Kozak, W., Leon, L. R., Soszynski, D., and Conn, C. A. (1995). Cytokines and fever, neuroimmunomodulation 2, 216-223. Kobayashi, H., Yuyama, A., Ohkawa, T., and Kajita, T. (1988). Effect of single or chronic injection with a carbamate, propoxure, on the brain cholinergic system and behavior in mice. Jpn. J. Pharmacol. 47, 21-27. Kozar, M. D., Overstreet, D. H., Chippendale, T. C., and Russell, R. W. (1976). Changes of acetylcholinesterase activity in three major brain areas and related changes in behaviour following acute treatment with diisopropyl fluorophosphates. Neuropharmacology 15, 291-298. Lim, D. K., Ito, Y., Stewart, T., Hoskins, B., and Ho, I. K. (1989). Toxicity study of continuous administration of physostigmine salicylate. Pharmacol. Biochem. Behav. 31, 627-631. Lipton, J. M., and Clark, W. G. (1986). Neurotransmitters in temperature control. Annu. Rev. Physiol. 48, 613-623. \ Maickel, R. P., Kinney, D. R., Ryker, D., and Nichols, M. B. (1988). Time course of physostigmine effects on neuroendocrine responding at varying environmental temperatures. Prog. Neuro-Psychopharmacol. Biol. Psychiatr. 12, 935-949. Maickel, R. P., Kinney, D. R., and Nichols, M. B. (1991). Antagonism of physostigmine induced hypothermia and neuroendocrine changes following exposure to different environmental temperatures. Prog. Neuro-Psychopharmacol. Biol. Psychiatr. 15, 873-884. Maurissen, J. P. J., Shankar, M. R., and Mattsson, J. L. (2000). Chlorpyrifos: Lack of cognitive effects in adult Long-Evans rats. Neurotoxicol. Teratol. 22, 237-246. Mautz, W. J. (2003). Exercising animal models in inhalation toxicology: Interaction with ozone and formaldehyde. Environ. Res. 92, 14-26.
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Meeter, E. (1969). The mode of action of cholinesterase inhibitors on the temperature regulation of the rat. Arch. Int. Pharmacodyn. 182, 416-419. Meeter, E., and Wolthuis, O. L. (1968). The effects of cholinesterase inhibitors on the body temperature of the rat. Eur. J. Pharmacol. 4, 18-24. Meeter, E., Woithuis, O. L., and Van Benthem, R. M. (1971). The anticholinesterase hypothermia in the rat: Its practical application in the study of the central effectiveness of oximes. Bull. World Health Organization 44, 251-257. Meller, D., Fraser, I., and Kryger, M. (1981). Hyperglycemia in anticholiesterase poisoning. Can. Med. Assoc. 124, 763-748. Morita, H., Yanagisawa, N., Nakajima, T., Shimizu, M., Hirabayshi, H., Okudera, H. O., Hohara, M., Midorikawa, Y., and Mimura, S. (1995). Sarin poisoning in Matsumoto, Japan. Lancet 346, 290-293. Moser, V. C. (1995). Comparisons of the acute effects of cholinesterase inhibitors using a neurobehavioral screening battery in rats. Neurotoxicol. Teratol. 17, 617-625. Myers, R. D. (1980). Hypothalamic control of thermoregulation, neurochemical mechanisms. In Handbook of the Hypothalamus (E J. Morgane and J. Panskeep, Eds.), Vol. 3, Part A, pp. 83-210. Dekker, New York. Namba, T., Nolte, C. T., Jackrel, J., and Grob, G. (1971). Poisoning due to organophosphate insecticides. Am. J. Med. 50, 475-492. Pope, C. N., and Chakraborti, T. K. (1992). Dose-related inhibition of brain and plasma cholinesterase in neonatal and adult rats following sublethal organophosphate exposures. Toxicology 73, 35-43. Ray, D. E. (1980). Selective inhibition of thermogenesis by tributyl S,S,S,-phosphorotrithioate (DEF). Br. J. Pharmacol. 69, 257-264.
Rowsey, R J., and Gordon, C. J. (1997). Tolerance to the hypothermic and hyperthermic effects of chlorpyrifos. Toxicology 121, 215-221. Rupniak, N. M., Tye, S. J., Brazell, C., Heald, A., Iversen, S. D., and Pagella, E G. (1992). Reversal of cognitive impairment by heptyl physostigmine, a long-lasting cholinesterase inhibitor, in primates. J. Neurol. Sci. 107, 246-249. Saadeh, A. M., A1-Ali, M. K., Farsakh, N. A., and Ghani, M. A. (1996). Clinical and sociodemographic features of acute carbamate and organophosphate poisoning: A study of 70 adult patients in North Jordan. Clin. Toxicol. 34, 45-51. Scimeca, J. A., Little, E J., and Martin, B. R. (1985). Relationship between the pharmacological effects and the biodisposition of [3H]diisopropylfluorophosphate in mice after inhalation. Toxicol. Appl. Pharmacol. 79, 502-510. Soininen, H., Unni, L., and Shillcutt, S. (1990). Effect of acute and chronic cholinesterase inhibition on biogenic amines in rat brain. Neurochem. Res. 15, 1185-1190. Stemler, E W., Corcoran, K. D., Parrish, J. H., Hurt, H. H., TezakReid, T., Kaminskis, A., and Jaeger, J. J. (1990). Effects of physostigmine on the cardiopulmonary system of conscious pigs. Fundam. Appl. Toxicol. 14, 96-103. Wang, L. C. H., and Lee, T. E (1989). Temperature regulation. In Psychoendocrinology pp. 437-539. Academic Press, New York. Brush, E R. and Levine, S. (eds.). Wester, R. C., Quan, D., and Maibach, H. I. (1996). In vitro percutaneous absorption of model compounds glyphosate and malathion from cotton fabric into and through human skin. Food Chem. Toxicol. 34, 731-735. Wheeler, T. G. (1989). Soman toxicity during and after exposure to different environmental temperatures. J. Toxicol. Environ. Health 26, 349-360.
CHAPTER ~ 9
Occupational Toxicology and Occupational Hygiene Aspects of Organophosphate and Carbamate Anticholinesterases with Particular Reference to Pesticides* BRYAN BALLANTYNE 1 AND HARRY SALEM z 1Charleston, West Virginia 2U.S. Army Chemical and Biological Center, Aberdeen Proving Ground, Maryland
hence the necessary protective and precautionary measures dictated by occupational hygiene considerations may be extensive and vary for differing chemical groups. The most geographically extensive and quantitatively greatest application of OPs and CMs is as pesticides used in agriculture, horticulture, public health protection, and domestically. Most of this chapter concerns these uses.
I. I N T R O D U C T I O N Organophosphate (OP) and carbamate (CM) anticholinesterases (anti-ChEs) have a wide spectrum of applications that includes pesticides in agriculture and horticulture, insecticides in domestic and public health applications, use in general commerce, use in therapeutic medicine, and as chemical warfare and terrorist agents. During normal civilian use, and when handled correctly with the appropriate recommended protective and precautionary measures, they can be used safely. However, because of their widespread and sometimes uncontrolled usage, there is a potential for misuse. Also, because of the high biological activity of many OPs and CMs, the likelihood for adverse effects occurring from accidental, and sometimes deliberate, exposure is high. Although the principal mechanism of action that underlies the practical use of OPs and CMs, namely inhibition of the cholinesterase group of enzymes, is also responsible for some of their known human toxicity, because of the wide range of chemical structures involved, the potential for numerous and differing toxic effects that are mediated by other mechanisms also exists (e.g., inflammation, immunotoxicity, myopathy, genetic toxicity, oncogenicity, and developmental and reproductive toxicity) (Ballantyne and Marrs, 1992). Thus, OPs and CMs may have a wide spectrum of potential occupational toxicity, and
II. S O U R C E S , P A T T E R N S , A N D R O U T E S OF E X P O S U R E Human adverse health effects from overexposure tO pesticides are, or should be, documented from carefully prepared clinical case notes of single and/or group poisonings, results from the findings of forensic toxicologists and pathologists in fatal cases of poisoning, the records and published works of poison control centers, and formal epidemiological studies. To the latter can be added the newer techniques of geographic processes for the capture, storage, retrieval, analysis, and display of spatial data (Clarke et al., 1996; Gunier et al., 2001; Ward et al., 2000). These information systems, which are automated, can be effectively utilized to study regional and temporal variations in the incidence of human symptomatic pesticide exposures (Sudakin et al., 2002). Occupational exposure to OP and CM pesticides occurs at different locations that include the workplace at production and formulation facilities, during transportation and distribution, during warehouse storage, and at in-use sites. The resultant potential health hazards and consequent published safety information
*The opinions and interpretations expressed in the chapter are those of the authors and do not necessarily represent the position of the U.S. Army and U.S. Department of Defense.
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sources, recommended safe handling practices, protective practices, and precautionary measures differ significantly for these different locations.
ing an accident, when there may be exposure of local inhabitants and others in the vicinity of the incident with a potential for adverse health effects and possible environmental effects on soil, rivers, and groundwater.
A. Production Facility Exposures can be to raw starting materials for the synthesis of pesticides, to intermediates in the manufacturing process, and to the OP or CM pesticides. Exposure to these various materials is generally limited to the workers at the facility ~ who produce and formulate the pesticide. However, the possibility for the escape of materials into the immediate area around a production plant needs to be considered in respect to possible local and general environmental and human adverse effects. This was most dramatically demonstrated in an incident involving the escape of the highly irritant material methyl isocyanate, used in the manufacture of the CM aldicarb, at a production facility in Bhopal, India. This resulted in the deaths of more than 2000 people (Lewinsohn, 1992) and more than 4000 animals (Gupta, 2004) in the surrounding area. Lethal adverse effects included pulmonary edema, bronchiolitis, and secondary pulmonary infections (Jain and Dave, 1986; Pandey, 1986). However, in general, losses of materials into the area around a production facility, where the general population could be exposed, are small in quantity and are controlled to "safe" levels by use of appropriate acute inhalation reference exposure levels and guidance levels (Collins et al., 2003). Formulators convert the pesticide into a desired physical state for use, such as a solution, emulsifiable concentrate, dust, powder, granule, and wettable powder. Thus, they may be exposed to various physical forms of the pesticide together with additional formulating materials of widely differing toxicity, which may significantly influence exposure considerations. Thus, these employees may represent a special health risk group requiring additional protective and precautionary measures. Exposure of workers in the production facility to starting materials, intermediates, and final product can be limited by good engineering controls and industrial hygiene practices, including the use of closed reactors, personal protective equipment (clothing and respirators), and precautionary measures that include medical surveillance, atmosphere and personnel monitoring, and ventilation for collective protection. Worker education, training sessions, the availability of appropriate first aid measures and emergency antidotes, and the provision for random audits of the health and safety program are all key elements for maintainence of a healthy working environment.
B. Transport Transportation does not usually result in direct exposure, although this may occur during loading and unloading of the road or rail tanker, for which monitoring techniques are available. Most problems with transportation occur follow-
C. In Use The in-use occupational applications of pesticides can be comparatively uncontrolled or difficult to control since they may occur in various and differing enclosed spaces (e.g., greenhouses) or open spaces (fields). Als0, the modes of applications may differ qualitatively and quantitatively, varying from hand application to land-based spray and powder applications and aerial applications. Since the atmospheric concentration of a pesticide cannot be controlled in these situations, particularly in open conditions, as it can by a plant workplace exposure guideline [e.g., Threshold Limit Value (TLV)], direct worker protection must be mainly by protective clothing and respirators. Also, there is a potential not only for those directly involved in applications to be contaminated but also for workers subsequently entering the treated areas before expiration of the reentry time to be contaminated. This may result in a significant degree of exposure. For example, the Centers for Disease Control (1999) reported an incident in California involving 34 farm workers who became ill after entry into a cotton field 2 hr after it had been aerially sprayed with a solution containing 0.26% carbofuran (N-methyl carbamate); the restricted entry interval for carbofuran is 48 hr. Approximately 4 or 5 hr after entering the sprayed field, they began to feel ill. The symptoms reported included nausea (7%), headache (94%), eye irritation (5%), muscle weakness (2%), tearing (68%), vomiting (79%), and salivation (56%); the most commonly observed signs were bradycardia (21%), diaphoresis (15%), and miosis (12%). Foliage samples showed carbofuran levels up to 0.77 ixgcm -2, and workers' clothing contained carbofuran residue up to 1 mg per item. Carbofuran metabolites were detected in urine samples obtained up to 11 days following exposure. Clearly, those who live in close proximity to the area being treated may also become contaminated from spray or dust drift ("farm proximity pathway"). For example, a study in Washington State (Fenske et al., 2002a) demonstrated that children of nonagricultural workers living close to farms (up to 200 ft), and who had been exposed to drift of azinphos-methyl, phosmet, chlorpyrifos, and ethyl parathion, had detectable amounts of urinary dimethyl OP metabolites similar to those in children of agricultural workers. Transient passers-by can also be subject to acute overexposure. Drift of pesticides after aerial application has been well documented (Ames and Stratton, 1991; Chester and Ward, 1984; Cone et al., 1994; Draper and Street, 1981; Draper et al., 1981). These considerations indicate the importance of various health and safety measures required with respect to the in-use application of pesticides; notably, monitoring of workers for exposure, medical surveillance,
C H APT ER 3 9 9Occupational Toxicology and Hygiene the use of physical protective measures, careful planning and timing of operations, and adherence to reentry times (Kahn, 1980; Lewinsohn, 1992; Spear, 1980). Drift also indicates the need to consider environmental protective measures, including adherence to derived exposure guidelines to protect the public (e.g., inhalation reference exposure levels, acute emergency guidance levels, short-term public emergency guidance levels, and emergency response planning guidelines) (American Industrial Hygiene Association, 1991; Collins et al., 2003; National Research Council, 2001). Considerable interest has been shown in the "take-home" (paraoccupational) exposure pattern, in which pesticide operatives contaminate members of their family with the material that is carried back to the home on worker clothing or skin (Baker et al., 2004). Thus, family members may face an increased risk of pesticide exposure (Fenske et al., 2002b; Quandt et al., 2004; Renner, 2002). A typical example is given by Fenske et al. (2002a), who studied children in the state of Washington and demonstrated that those from agricultural families compared to children from reference (control, nonexposed) families had a greater exposure to OPs as indicated by higher levels of urinary OP metabolites. In addition to the potential for occupational and paraoccupational exposure to pesticides, there may be significant and uncontrolled in-use exposures in nonoccupational settings, notably pesticide-treated general workplaces and residences. Also, OP and CM anti-ChEs have found a not insignificant use in deliberate (intentional) poisoning, both self-administered and for homicide (Ballantyne, 1992). Self-poisoning with anti-ChEs, notably OPs, is particularly prevalent in underdeveloped and developing countries. For example, in Turkey OPs are the most common agent employed for suicidal poisoning (Akgtir et al., 2003).
D. Routes of Exposure
The principal route(s) of potential exposure will depend on a variety of factors, including the chemistry and physical properties of the pesticide (e.g., solid, liquid, vapor pressure, and viscosity), its formulation and formulation constituents, and the mode of use and application (e.g., as liquid, emulsil-/ fiable concentrate, dust, aerosol, and spray). The route(s) of exposure together with the nature of the toxicity of the pesticide and its formulation constituents, the mode of exposure, and the degree of exposure will be major determinants of the potential hazards and the necessary protective and precautionary measures. The exposure routes discussed next may be encountered to varying degrees. 1. SKIN CONTACTAND PERCuTANEOUS ABSORPTION Clearly, unprotected skin may become contaminated during accidental spills and with the in-use application of pesticides during transfer of formulations, mixing and loading of applicators, and applications involving dusts and spray dispersions from land-based equipments or airplanes. This route
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also presents a problem for entry of unprotected agricultural workers into recently treated areas before the expiration of a recommended reentry interval. Contact with the skin may result in local irritancy and/or sensitization reactions; in California, 15-25% of adverse pesticide reports are due to skin reactions (O'Malley, 1997). More important with regard to OPs and CMs is the possibility for systemic toxicity following percutaneous absorption. The likelihood for absorption of pesticide across the skin, and its magnitude, will be determined by a variety of interacting factors, principal among which are the following: 1. Physicochemical properties, including molecular weight, charge, and hydrophilic/lipophilic characteristics. 2. The surface area of the skin contaminated (Maibach and Feldman, 1974); clearly, the larger the area contaminated, the greater the amount of material absorbed, i 3. The concentration of the material or amount of applied material; the higher the concentration, the greater the gradient to facilitate percutaneous absorption. 4. The time of contact will clearly influence the amount of pesticide absorbed, increasing with increasing duration of contact; hence the importance of early decontamination of skin. Related to this is the degree of occlusion i for example, material becoming trapped between skin and clothing (Semple, 2004). Clothing saturated with pesticide residue may promote continuing skin exposure and also percutaneous absorption (Freed et al., 1980; Webster and Maibach, 1985a,b). 5. The presence of formulation surfactants and solvents, which will facilitate skin penetration of the pesticide. Absorption rate is more effective for lipophilic materials. Some UV-absorbing chemicals can act as skin penetration enhancers, which may increase the percutaneous absorption of pesticides and other formulation chemicals (Morgan et al., 1998; Nakai and Chu, 1997). Thus, it has been shown that the application of sunscreens to the skin can increase the penetration of pesticides (Brand et al., 2002). 6. Anatomical site contamination. With many materials, including pesticides, the absorption rate through the skin of a specific material varies with the regional location of the skin area contaminated. For example, with parathion the absorption rate is faster through skin of the scrotum, axillae, and face than through skin of the hands and arms (Maibach et al., 1971). Also, Maibach and Feldman (1974) applied 4 txg cm -2 of parathion to the forearm, abdomen, and forehead of volunteers and found that the absorbed doses were 8.6, 18.5, and 36.3%, respectively. 7. The integrity of the absorption surface may be an important practical consideration. Thus, materials may be absorbed more effectively through diseased and abraded skin, particularly with more recently injured skin (Ballantyne, 1989; Ballantyne et al., 1999; Grissom and Shah, 1992). For example, parathion was absorbed 8.5 times more through damaged than through normal skin (Maibach and Feldman, 1975).
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Toxic Effects
8. Environmental factors, such as increased temperature and relative humidity, may facilitate skin penetration of a chemical (Ballantyne et al., 1999). All those factors emphasize the need, with the more toxic materials, for physical protection of skin (gloves, clothing, and face shields/respirators) and for decontamination of accidentally contaminated skin. Although cleansing of contaminated skin is advised to remove residual pesticide, a simple soap and water wash may not be sufficient since it has been shown that a considerable residuum may remain on or in the area of washed skin; most of the residual material remains in the stratum corneum (Fredriksson, 1961; Webster and Maibach, 1983; Zendzian, 1994, 2003). Although there is constant exfoliation of the stratum corneum, the turnover time is on the order of 14 days (Halpran, 1973). Thus, there is a potential for the residual pesticide in skin to contribute to potential local and/or systemic toxicity. This may be compounded by the fact that washing the contaminated site can lead to a transient increase in the absorption flux of the pesticide (Webster and Maibach, 1985a,b, 1999). The timing and the magnitude of any increase in absorption will vary with the specific pesticide and its physicochemical properties and the rate/magnitude of absorption in comparison with the rate/magnitude of excretion of the absorbed pesticide. Zendzian (2003) found that of 19 pesticides studied postwash in the rat, absorption continued for 15 at all dosages tested, in 2 it continued but not at all dosages, and in 2 absorption did not continue postwash. Of the 15 pesticides in which absorption continued postwash, only 9 had an increase in systemic concentration indicating a potential for increased toxicity.
2. RESPIRATORYTRACT EXPOSURES Inhalation of vapor is the more likely hazard with exposure to employees in plant production facilities, but with in-use applications there may inhalation exposure to vapor, dusts, mists, and aerosols. Vapor will be transported to the pulmonary alveoli, but the degree of penetration of dusts and aerosols into the respiratory tract will depend primarily on particle size (Dodd, 1992). Depending on the toxicity of the inhaled material, general effects that may be caused are irritation and inflammation within the respiratory tract, and possibly induction of an asthmatic response. OP and CM anti-ChEs will produce local respiratory tract toxicity particularly affecting the bronchiolar smooth muscle, resulting in bronchoconstriction and increased tracheobronchial secretions (Dodd, 1992). Systemic toxicity may result from absorption of OPs and CMs through the respiratory mucosa or, if the alveoli are reached, absorption into the pulmonary alveolar capillaries. The specific occupation of the worker may influence the degree of inhalation exposure. For example, Wolfe (1973) found that exposure of an operator of an air blast sprayer directing the spray upward into trees was 12 times higher than that of a boom operator directing
a comparable formulation down into rows of crops. Hayes (1975) reported that during an aerial application of tetraethyl pyrophosphate dust, the pesticide loader received approximately three times more exposure to the concentrate than did the airplane pilot and 4.5 times that received by the flagman. 3. GASTROINTESTINALTRACT Ingestion of OPs and CMs is not usually a major problem in the production facility, unless there is poor personal hygiene and unwrapped food or drink is kept in close proximity to anti-ChEs. Peroral exposure may also result from transfer of pesticide from contaminated hands (e.g., during eating or smoking). Worker ingestion of OPs and CMs is more likely to occur from swallowing saliva contaminated with pesticide inhaled during a dust or aerosol application session or from coughed mucus. Clearly, ingestion of foods from crops with pesticide residues is a general public health possibility and consideration. In some cases, this may present a significant acute hazard. Toxic illnesses caused by the CM aldicarb are typical; one case involved consumption of contaminated watermelons in Oregon (Hall and Rumack, 1992), and another was due to consumption of hydroponically grown cucumbers (Lewinsohn, !992). 4. EYE CONTACT Contamination of the eye with anti-ChE OPs and CMs may occur accidentally during pesticide manufacture and use and deliberately with therapeutic agents applied to the eye. This may result in both local and systemic toxicity.
III. G L O B A L A N D R E G I O N A L S C A L E S OF H U M A N H E A L T H P R O B L E M S D U E TO PESTICIDE POISONING Accidental and intentional poisoning of humans with CMs and particularly OPs is a worldwide occupational, public health, environmental, and forensic problem. Thus, acute pesticide poisoning represents a significant cause of morbidity and mortality in underdeveloped and developing counties. The World Health Organization (WHO) estimated that there are approximately 3 million cases of acute pesticide poisoning annually, with 220,000 deaths (Bryant et al., 2003; Jeyaratnam, 1990). The majority of these incidents occur in developing counties, notably Africa, Asia, and Central and South America (He, 2000; Van der Hook et al., 1998). A study in Zimbabwe from January 1998 to December 1999 showed that OPs and rodenticides were the leading cause of acute pesticide poisoning admissions to major hospitals; thus, of a total of 914 acute pesticide exposures, 42.2% were to anti-ChEs, mainly OPs (Tagwireyi et al., 2004). In Central America between 1992 and 2000, there was a significant increase in the importation of pesticides and this was associated with an increase in the incidence rate for acute
CHAPTER 39 9Occupational Toxicology and Hygiene poisoning from 6.3 per 100,000 to 19.5 per 100,000 population, and the mortality rate increased from 0.3 per 10,000 to 2.1 per 100,000 population (Hena and Arbelaez, 2000). A systematic randomized 10% retrospective screen was conducted of all hospital-referred poisoned patients from March 1993 to March 2000 in Imam Reza University Hospital of Mashhad, Iran; this country has no center for poison control and surveillance to gather information and analyze data (Afshari et al., 2004). There were 71,589 total cases, of which 7158 were selected for analysis. The annual crude rate of referral due to poisonings was 3.9 per 1000 population per year, with an annual crude rate of mortality of 2.3 per 100,000 population. Main poisoning groups in the analysis were pharmaceuticals (61.4%), chemicals (22.8%), and natural toxins (16.6%). For chemicals (industrial, domestic, and environmental), there were 1486 cases analyzed, of which OP and CM insecticides accounted for 250 (16.8% of group and 3.5% of the total poisonings analyzed). In Mexico, among 200 seasonal workers, 20% experienced acute pesticide poisoning (De Jesus et al., 1998), and in Costa Rica, an overall rate of pesticide poisoning of 5.3 per 100 workers per year was calculated based on data from the National Insurance Institute (Verga and Fuotes, 1998). Several major pesticide poisoning incidents have been reported from India. For example, more than 100 people died in Kerala in 1958 after consuming wheat flour contaminated with ethyl parathion (Karunakaran, 1958). In the same year in Kerala, 102 people died due to the careless handling and storage of wheat. Thirty-five cases of malathion poisoning were reported from Indore, of which there were 5 deaths (Sethuraman, 1977). In a report from Madhya Pradesh, 12 people who consumed wheat for 6 months that had been contaminated with aldrin and gammexane developed neurological symptoms that included myoclonic jerks, generalized clonic convulsions, and limb weakness (Gupta, 1975). In general terms, and with respect to the general population, in India there are currently approximately 145 pesticides registered for use, with an annual production of 85,000 metric tons (Gupta, 2004). The Poisons InformationCenter of the National Institute of Occupational Health in Ahmedabad has noted that OPs have been responsible for the largest number of poisonings (73%) among agricultural workers (Dewan and Sayed, 1998). Despite the fact that the consumption of pesticides is very low in India at 0.5 kg ha -1, there has been widespread contamination of foods with pesticide residues, basically due to the ill-informed use of pesticides. Thus, in India approximately 51% of food commodities are contaminated with pesticides, which the levels of 21% are higher than world accepted maximum residue levels (Gupta, 2004). Hence, in India there appear to be deficiencies in the understanding of pesticide health problems, safety in use, intelligent approaches to registration, and monitoring for overexposure. There is thus a need for local education at all strata in government, industry, and worker populations on the monitoring, correct handling, and protective measures neces-
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sary for the agricultural use of pesticides. Severe OP self-poisoning is a major clinical problem in the Asia Pacific region, where some hospitals treat 500-1000 patients annually, with a case fatality of more than 20% (Eddleston et al., 2004). Although the potential for adverse effects from working with agrochemicals in developed counties is generally well appreciated, there is still concern in such countries about the occurrence of pesticide-related illnesses from acute and/or repeated overexposure. It has been noted that working conditions in agriculture are among the most hazardous in the United States, and seasonal farm workers in particular are at a significant risk for occupational illness and injury (Arcury and Quandt, 1998; Bureau of Labor Statistics, 1999; Das et al., 2001; Griffith and Duncan, 1992; Mobed et al., 1992; National Institute for Occupational Safety and Health, 1996; Wilk, 1986). This applies notably for injury and illness resulting from pesticide exposure (Coye and Fenske, 1988; Moses, 1989). The greatest source of exposure is from dislodgeable residues (Fenske, 1997). Several authorities have noted that OPs continue to pose a risk to human health in the United States, and there is a clear need for continuous biological monitoring to regulate exposure (Jaga and Dharmani, 2003). An indication of the scale of the problem of exposure to pesticides in the United States is provide by the following examples. In 1999, more than 13,000 cases of OP poisoning were reported to poison control centers, there were more than 3000 cases seen in hospital emergency departments, and 83 fatalities occurred (Riegel, 2002). Data provided by the Toxic Exposure Surveillance System of the American Association of Poison Control Centers recorded there were 10,073 exposures to OP insecticides in adults in 2000, of which 9609 were unintentional and 276 were intentional (Litovitz et al., 2001). For 2003, the U.S. Bureau of Labor Statistics recorded 707 fatal work injuries under the heading of "agriculture, forestry, fishing, and hunting" out of a total of 5559 workplace fatalities for that year. In agriculture, the fatalities were crop production, 333 (of which 17 were classified as being due to exposure to harmful substances); vegetable and melon farming, 17; fruit and tree nut farming, 13; greenhouse, 18; and other crop farming (tobacco, cotton, sugarcane, hay, and miscellaneous), 77 (American College of Occupational and Environmental Medicine, 2004). Among a cohort of more than 50,000 pesticide applicators in the United States, 16% reported having a high pesticide exposure event (which did not always result in toxic effects) (Kiem and Alavanga, 2001); within the same cohort, and on the basis of nested case-control analysis, it was estimated that approximately 0.6% of the applicators had developed symptoms (Alavanja et al., 2001). In a study of acute occupational pesticide-related illnesses among adolescents in the United States, Calvert et al. (2003) concluded that there is still a need for greater effort to prevent such illnesses. They Suggested the need to improve surveillance in order to overcome the limitations of underreporting. Also, they advised that because of problems in
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relating signs and symptoms to acute pesticide overexposure, there is a need for vigilance on the part of health care professionals to consider the roles of environmental and occupational exposures. In an interview survey of Latino farm workers by Arcury et al. (2000), it was reported that farmers do not necessarily adhere to regulations mandating training and basic sanitation facilities. It was concluded that additional regulation by itself is not an advantageous starting point and the emphasis for intervention should include educating farmers as well as farm workers. A particular area of concern in the United States is morbidity from pesticide overexposure in hired migrant farm workers, many of whom are undocumented, have no legal authorization to work, are underpaid, have little or no formal education, may not be able to speak the language and thus have significant communication problems, and have barriers to health care access. It is therefore not surprising that they have a high incidence of systemic effects notably from OP and CM antiChEs, inorganic compounds, and pyrethroids (Das et al., 2001; Jaga and Dharmani, 2003).
TABLE 1. Clinical Signs and Symptoms of Anticholinesterase Intoxication a Muscarinic
Nicotinic
IV. M E D I C A L S U R V E I L L A N C E OF W O R K E R S P O T E N T I A L L Y EXPOSED TO OP AND CM ANTICHOLINESTERASES Details of the mechanism of action and clinical consequences of overexposure to OP and CM anti-ChEs are given in other chapters of this book. However, the possible adverse effects that may result from acute and repeated overexposure are briefly summarized here as a preliminary to the needs and requirements for surveillance of workers potentially exposed to OPs and CMs. Although the objectives of medical, and indeed any, surveillance programs are to detect the early onset of adverse effects and to confirm that health and safety measures are proceeding in a satisfactory, effective, objective, and planned manner, the ultimate goal is prevention of work-related morbidity and mortality (Schnitzer and Shannon, 1999).
A. Clinical Concerns Forming the Basis for Medical Surveillance 1. CHOLINERGIC EFFECTS The immediate adverse effects (type 1 syndrome) resulting from an acute overexposure to OP and CM anti-ChEs are summarized in Table 1. With OPs, these effects are the result of the OP binding with the catalytic center of ACHE, inhibiting its enzyme activity, and causing an accumulation of ACh at muscarinic receptors in skeletal neuromuscular sites, at nicotinic receptors in autonomic ganglia, and at central nervous system (CNS) cholinergic receptors (mainly muscarinic). The resultant increases in synaptic ACh at muscarinic, nicotinic, and CNS sites form the basis for the cholinergic signs and symptoms and the acute lethal
CNS
Excess lacrimation Rhinorrhea Hypersalivation Bronchorrhea Bronchoconstriction Hyperhidrosis Miosis Decreased visual acuity Urination Defecation Bradycardia Hypotension Mydriasis Tachycardia Hypertension Ataxia Weakness Skeletal muscle twitching Skeletal muscle fasciculations Skeletal muscle weakness Skeletal muscle paralysis Pallor Ataxia Lethargy Slurred speech Confusion Depression Tremors Convulsions Respiratory failure Hypothermia Coma
aAfter Ballantyneand Marrs (1992). toxicity of anti-ChEs. In general, because the carbamylation of AChE results in a more rapidly reversible inhibition complex with AChE than does phosphorylation, the clinical effects produced by OPs are generally more severe and more sustained than those caused by CMs. Also, since CMs penetrate the blood-brain barrier less effectively than do OPs, the CNS component is usually smaller with CMs (Ballantyne and Marrs, 1992).
2. ACUTE DELAYED-ONSET NEUROLOGICAL EFFECTS Following recovery from the cholinergic toxicity ("cholinergic crisis") of an acute overexposure to OPs, a proportion of patients may develop, after approximately 2 4 weeks, a delayed-onset peripheral neurotoxicity of the central peripheral-distal axonopathy type with secondary demyelination
CHAPTER 39 9Occupational Toxicology and Hygiene (Cavanagh, 1963; Davis et al., 1985; Genel et al., 2003). This is often referred to as organophosphorus-induced delayed-onset polyneuropathy (OPIDP). The onset of OPIDP is characterized by cramping muscle pains in the legs, rapidly followed by distal paresthesia and leg weakness with reduced deep tendon reflexes. In severe cases, the upper limbs can become affected and quadriplegia may develop; in these cases, pyramidal signs may occur because of spinal cord involvement (Lotti, 2003). Electrophysiological studies show reduced amplitude of compound muscle action potentials and delayed terminal latencies after supramaximal stimulation of motor nerves; maximal conduction velocity is generally normal or slightly reduced (Lotti et al., 1984). Electromyography reveals a denervation of affected muscles (fibrillation potentials and positive sharp waves) and reduced interference pattern (Lotti, 2001). Some functional recovery of peripheral nerves can occur. Mechanistically, OPIDP is a result of an inhibition of neurotoxic esterase (Johnson, 1992). Delayed-onset polyneuropathy in humans has been described as occurring with the following OP anti-ChEs: dichlorvos (Vasilescu and Florescu, 1980; Wadia et al., 1985), mipafox (Bidstrup et al., 1953), isofenphos (Moretto and Lotti, 2002), leptofos (Xintaras et al., 1978), methamidophos (Senanayake and Johnson, 1982), trichlorofon (Heirons and Johnson, 1978; Johnson, 1981), trichloronat (Jedrzejowska et al., 1980), Oethyl O-4-nitrophenyl phenylphosphonothioate (EPN; Xintaras and Burg, 1980), and chlorpyrifos (Lotti and Morreto, 1986; Lotti et al., 1986). Another delayed-onset neurological/myopathic condition is the "intermediate syndrome," identified by Senanayake and Karalliedde (1987). This appears after the cholinergic crisis but before the time anticipated for the development of delayed-onset peripheral neuropathy. It is characterized by weakness or paralysis of proximal limb muscles, neck flexors, respiratory muscles, and motor cranial nerves (Ballantyne and Marrs, 1992; Senanayake and Karalliedde, 1992). 3. NEUROPSYCHOBEHAVIORALEFFECTS
Following symptomatic acute overexposures to OP anti-ChEs particularly, there may be clinical evidence of neurobehavioral, psychological, and psychiatric effects, which may take months to regress. For example, Le~,in and Rodnitzky (1976) showed that when serum butyrylcholinesterase (BChE) or red blood cell (RBC) AChE activity is inhibited, some or all of the following may be impaired: (i) cognition m vigilance, information processing and psychomotor speed, and memory; (ii) speech, both performance and perception; (iii) psychic state m increased tendencies to depression, anxiety, and irritability; and (iv) electroencephalographic (EEG) records m tendency to faster frequencies and higher voltages. They concluded that EEG abnormalities were positively related to the degree of AChE activity inhibition in the early stages. Several authors have noted that neuropsychobehavioral effects may occur as a consequence of
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occupational overexposure to OPs. The effects that have been recorded include impaired cognition, memory deficits, impaired mental function, retrograde amnesia, increased vibrotactile threshold, depression, dizziness, irritability, anxiety, schizoid reactions, and exacerbation of preexisting psychiatric problems (Levin and Rodnitzky, 1976; Namba et al., 1971; Savage et al., 1988; Stephens et al., 1995). These abnormalities following acute intoxication were grouped together in a syndrome called COPIND 1 (chronic organophosphate-induced neuropsychiatric disorders) by Jamal (1997). However, Lotti (2001) and Ray (1998a) did not regard it as appropriate to include all these possible different effects together into a single syndrome such as COPIND 1. Also, they concluded that there is little evidence, in the absence of hypoxia and/or convulsions in the early cholinergic phase of OP intoxication, that acute OP poisoning results in late permanent neurological or psychiatric effects other than OPIDR Available evidence indicates that asymptomatic acute exposure to OPs is generally not a precursor to neuropsychiatric sequelae, but repetitive acute episodes of OP intoxication with clear signs of cholinergic symptoms are associated with long-term neuropsychobehavioral effects (Eyer, 1995). A long-term low-concentration/low-dosage exposure to OPs is associated with a number of psychiatric, neurobehavioral, and neurological effects, which may not be correlated with symptoms of acute exposure (Brown and Brix, 1998; European Center for Ecotoxicology and Toxicology, 1998; Lotti, 2001; Ray, 1998a,b; Salvi et al., 2003; Steenland, 1996). These were grouped under a syndrome called COPIDN phenomenon 2 by Jamal (1997), an approach that also has been subject to criticism by Lotti (2001). Most studies in which neurobehavioral tests were performed have given negative results, except when subjects were still exposed and had evidence of AChE inhibition (Gomes et al., 1998). Studies have been conducted to determine if there is an association between OP exposures and suicide rates or psychiatric disorders. The results were either negative (Pickett et al., 1998; Stoller et al., 1965) or inconclusive due to lack of information on either the actual exposure or confounding factors (Amr et al., 1997; Levin and Rodnitsky, 1976; Parr6n et al., 1996). However, there is a general consensus that exposure to low-level doses of OPs, as well as acute poisoning, can lead to persistent neurological and neurobehavioral effects, which cannot be explained by AChE inhibition alone (Smulders et al., 2004). It has been suggested that other, more sensitive brain proteins may be involved (Ray and Richards, 2001). Studies of the effects of several OPs (parathion-ethyl, chlorpyrifos, and disulfoton) on rat neuronal Ot.4[~2 nicotinic ACh receptors expressed in X e n o p u s laevis oocytes, using the two-electrode voltage clamp technique, showed that these OPs inhibited the ACh-induced ion current with potencies in the micromolar range (Smulders et al., 2004). The potency of inhibition increased with increasing concentrations of the agonist ACh. Comparison of the potency of inhibition of the nicotinic ACh
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Effects
receptor with that of AChE activity inhibition demonstrated that some OPs inhibit nicotinic ACh receptors more potently than AChE activity. The inhibitory effects of OPs on nicotinic ACh receptors can be described and explained by a sequential two-step mechanism, in which rapidly reversible OP binding to a separate binding site leads to inhibition followed by a stabilization of the blocked state or receptor desensitization. It was concluded that OPs interact directly with neuronal ot4~ 2 nicotinic ACh receptors to inhibit the agonist-induced response, and this implicates neuronal OL4~2 nicotinic ACh receptors as additional targets for some OPs (Smulders et al., 2004). These findings may have implications for explaining the mechanism of production of the neurobehavioral effects of OPs by repeated low-concentration exposure. Thus, mice lacking the oL4 nicotinic ACh receptor subunit exhibit a reduced antinociceptive effect of nicotine (Marubio et al., 1999) and elevated anxiety (Ross et al., 2000). Knockout of the ~2 nicotinic ACh receptor subunit showed that it is involved in the reinforcing properties of nicotine (Picciotto et al., 1998) and i n passive avoidance learning (CorderoErausquin et aL, 2000). It follows that repeated desensitization of Ot.4~2 nicotinic ACh receptors may result in some of the neurobehavioral deficits of OP poisoning. Related to this is the clinical observation that one of the effects resulting from long-term repeated exposure of workers to OPs is anxiety (Salvi et al., 2003). Kamel et al (2005) analyzed crosssectional data from licensed pesticide operators enrolled in an agricultural health study under the auspices of the National Institute of Health and Environmental Protection Agency. They found that prevelance of neurologic symptoms was associated with cumulative life time exposure to pesticides, particularly OPs and organochlorines. These associations were present in individuals with no history of pesticide poisoing or high exposure events, and were independent of recent exposures. The investigators consider them due to chronic moderate exposure, and that more attention should be paid to risks associated with moderate exposure. 4. ASTHMA There are differing reports on possible adverse respiratory effects of exposure to airborne pesticides. Some investigators have associated pesticide exposure with effects that include cough, bronchospasm, hypersensitivity pneumonitis, and pulmonary fibrosis (Do Pica, 1992; Schenker et al., 1991; Weisenburger, 1993). Others, however, have not found evidence for occupational asthma among workers with occupational exposure to pesticides, including serial pesticide applicators (Jones et al., 2003). However, since inhaled anti-ChEs have local pharmacological effects on the airways, including bronchospasm as discussed previously, there is reason to suspect that this group of pesticides could cause adverse airflow problems, including asthma, by aerial dispersion. In a cross-sectional study of Canadian farmers, Senthilselvan et al. (1992) found an association between self-reported asthma and CM insecticide use.
Several clinical and epidemiological studies have linked OP exposure to airways hyperreactivity and other symptoms of asthma (Bryant, 1985; Deschamps et al., 1994; Hoppin et al., 2002; O'Malley, 1997). In the respiratory tract, vagal cholinergic nerves mediate airway tone and reactivity by the release of ACh with stimulation of M3 muscarinic receptors resulting in bronchoconstriction. This vagally induced bronchoconstriction is limited by autoinhibitory M2 muscarinic receptors, and loss of M2 function leads to increased release of ACh from the parasympathetic nerves, resulting in potentiation of vagally induced bronchoconstriction. It is generally believed that inhibition of respiratory tract AChE is responsible for OP-induced bronchoconstriction by increasing ACh availability, and in this way contributing to the development of asthma (Ernst, 2002). However, experimental studies by Fryer et al. (2004) have shown that chlorpyrifos altered M2 receptor function in the lung at concentrations that did not inhibit ACHE. It was therefore postulated that OP-induced bronchoconstriction may be a consequence of blockade of M2 receptors resulting in increased ACh release from M3 parasympathetic fibers, and thus vagally induced bronchospasm is potentiated by disruption of the control of airway responsiveness. 5. MISCELLANEOUS Other less frequently described adverse effects that have been described as a consequence of overexposure to anti-ChEs include cardiomyopathy (Singer et al., 1981), rhabdomyolysis (Bright et al., 1991; Dettbarn, 1992; Futagami et al., 2001; Vanneste and Lison, 1993; Yeh et al., 1993; Young and Koplovitz, 1995), nephrotoxicity (Kaedtisuke et al., 1989; Wedin, 1992), and pancreatic injury (Hayes et al., 1978; 'Ma, 1983). 6. ANTI-ChE POTENTIATION Agricultural formulators and field workers may be exposed to more than one OP during a given time period. This is relevant because some OPs (e.g., carbophenothion, fenthion, and dioxathion) inhibit aliesterases, such as diethylsuccinase and tributyrinase, that detoxify other OPs and at doses lower than those inhibiting ChEs. Thus, such OPs are likely to enhance the toxicity of OPs that are normally detoxified by aliesterases and potentiate their activity. OPs that inhibit ChEs and aliesterases at the same rate are likely to cause additive rather than synergistic effects. A health and safety program designed to ensure safe working conditions for employees who may potentially be exposed to OPs and CMs should have a medically supervised surveillance system that involves an initial preemployment screen for suitability to work with the antiChEs, and for those who are actively employed, there should be provision for periodic medical examinations to exclude clinical evidence of short- or long-term adverse health effects from the OPs and CMs to which they may be potentially overexposed. Also, the medical surveillance program should
CHAPTER 39 9Occupational Toxicology and Hygiene be integrated into the general monitoring and industrial hygiene elements of the health and safety program.
B. Preemployment Medical Examination Before being employed in situations in which they may be exposed to OPs and CMs, and to ensure that they are medically, psychologically, and intellectually suitable for such employment, potential employees should have the following documented: 1. Detailed history and physical examination to exclude the presence of medical factors that may indicate a susceptibility of the individual to adverse effects, including precipitation of incipient disease(s), resulting from pesticide overexposure. This should pay particular attention to hepatorenal, respiratory, peripheral and central nervous, visual, cutaneous, and cardiovascular systems. This should include a determination of a history of allergies or asthma that could be aggravated by the anti-ChE or common formulation ingredients to which they may be exposed. Where there is reason to suspect a susceptibility, appropriate biochemical, physiological, and/or radiological investigations should be conducted. Potential employees with hepatorenal disease, glaucoma, cardiovascular disease, and CNS dysfunctions and those using anticholinergic drugs should be warned of the aggravation that could be caused by exposure to OPs and CMs. 2. Preemployment (preexposure) measurement of baseline (control) RBC AChE and serum BChE activities. 3. Exclusion of BChE (pseudo-ChE) deficiency or the presence of a genetically determined atypical pseudocholinesterase (BChE) variant (Bonderman and Bonderman, 1971; Ostergaard et al., 1992). 4. Confirm suitability to wear a respirator. Factors that may be considered as contraindicating respirator use include obstructive pulmonary disease, moderate cardiovascular disease, physical features that may result in poor fit (e.g., facial scars), and psychological problems such as claustrophobia and anxiety (Ballantyne, 1981).
C. Employee Periodic Medical Examination The periodic medical examination should ideally be conducted annually for full-time operatives (e.g., in pesticide manufacture) or at midseason and the end of the work season for part-time employees (e.g., field workers). However, in determining the periodicity of medical examinations and special studies for individual cases, the advising physician should take into account factors that include age, gender, frequency and duration of exposures, and potential exposure dosages. Additionally, employees should be advised, ideally at the preemployment medical examination, that if they believe that they are developing pesticide exposure-
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related symptoms and/or signs, they should immediately seek medical attention. Examinations should include the following, the results of which should be documented: 1. History to include questions concerning, but not limited to, headache, dizziness, chest tightness, muscle weakness, neurobehavioral effects, and disturbance of visual function, including difficulty with focusing. 2. Physical examination to exclude, particularly, hepatorenal, cardiorespiratory, neurological, and visual dysfunction. Where there is reason to suspect adverse effects, appropriate special investigations may need to be undertaken (biochemical, physiological, and/or radiological). Occasionally, there may be difficulty in clinically differentiating neurobehavioral effects from exposure to anti-ChE and those resulting as a consequence of chronic alcoholism. In such cases, the measurement of ~/-glutamyl transferase and erythrocyte mean corpuscular volume, with the use of the CAGE questionnaire, can assist in the differential diagnosis (Lewinsohn, 1992; WHO, 1987). 3. Measurement of RBC AChE and serum BChE to detect any overexposure to anti-ChE.
D. Monitoring for Exposure to OP and CM Anticholinesterases A number of methods are available to monitor workers for exposure to anti-ChE pesticides that differ in approach, sophistication, technical basis, sensitivity, specificity, and cost. Choice of the method(s) will depend on several factors, including the chemical(s) applied, size and nature of the operation, facilities available, reason for monitoring, and regulatory needs. Exposure of the skin to pesticides may estimated by the following methods: R e m o v a l methods: This involves measuring residues on skin and clothing that remain after a specified exposure time. It can be carried out by measuring residues on swabs taken by skin wipes, by hand rinses, by skin stripping techniques, and determining residues on clothing. For rinses, the hand is placed in a plastic container holding approximately 200 ml of 95% ethanol and the fingers are briskly rubbed against the thumb and palm to remove particles. Two hand rinses were found to remove 96% of parathion from one hand soon after exposure (Durham and Wolfe, 1962). Wipe sampling and wash techniques may show a high degree of variability in recovery and recovery efficiency. They may also be of limited use when the pesticide or formulation used is highly volatile or has a rapid percutaneous absorption (Brouwe et al., 2000). It has been stressed by Durham and Wolfe (1962) that the hands of the worker should be clean before entering the field or trial area. Tape stripping involves the
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physical removal of the outer layers of the epidermis and can be used to estimate the quantity of material that has been absorbed into the skin (Nylander-French, 2000). With removal methods, the skin wipe and hand washing approaches have been successfully employed in studies on premature reentry into sprayed lands, paraoccupational exposure, and contamination from the domestic use of pesticides (Ballantyne and Marrs, 2004; Quandt et al., 2004; Thompson et al., 2003). Surrogate skin techniques: These involve placing a collection medium against the skin or clothing and subsequently analyzing it for pesticide. Common approaches are the use of whole body suits or the "patch technique," which involves attaching sorbent pads to the skin or clothing and subsequently measuring the pesticide load of the patches. Although a simple technique with limitations, it does allow a semiqauntitative estimate of surface contamination. Bias may be introduced because of the fact that patches measure pesticide deposition over a selected area. Also, differences in the type of material used for patch sampling, the body location sampled, and patch substance interaction with pesticide will all increase the variability of measurements. Highly absorbent knit white cotton garments that cover the entire body area of interest may provide a more accurate estimate of exposure (e.g., gloves to estimate hand exposure and short-sleeved undershirts for upper torso). The patch technique may be employed in predictive studies for contamination during in-use applications of pesticide. Visualization methods: For practical qualitative assessment of skin exposure, fluorescent tracer methods can be used. Fluorescent tracer can be added to the material being handled or processed to assess the area of contamination (Cherrie et al., 2000). This approach can be combined with video imaging analysis to allow some degree of quantitation (Fenske, 1997). This is achieved by determining the amount of surface area exposed and correlated with image intensity (Semple, 2004). Biomonitoring: This can be employed as an indirect method to provide an estimate of actual exposure dose (or at least the cutaneously absorbed dose). It involves quantitative analysis of parent material and/or metabolites in expired air, blood, and urine; from these analyses it can be calculated how much material has been percutaneously absorbed (Honeycutt et al., 2001). Cutaneous exposure modeling" This approach utilizes statistical and deterministic methods to aid in estimating the amount of pesticide deposited on the skin. One conceptual model of cutaneous exposure (Schneider et al., 1999) divides the worker's environment into six compartments" the source, air, surface contaminant layer, outer clothing layer, inner clothing layer, and skin. The following transport processes then characterize movement of the chemical within the environment: emission, deposition, resuspension/evaporation, transfer,
removal, decontamination, penetration/permeation, and redistribution. Inhalation exposures to pesticides in the work area can be estimated from measurements of concentrations of vapor, aerosol, or dust in environmental air (Ballantyne and Marrs, 2004). This can be done using passive samplers in the general work area or personal samplers in the employees breathing zone (Griffith and Duncan, 1992). However, in field situations in which pesticide samples are not of uniform size, and there may be moving variable concentrations in the air, the estimates may be misleading.
V. BLOOD CHOLINESTERASE MEASUREMENTS Mechanistically, OPs act by covalently binding with ACHE, which involves a chemical reaction between the phosphoryl ( ~ O ) moiety of the OP and the active site serine hydroxyl group of the catalytic center of AChE to form a phosphorylated enzyme. The phosphorylation of the catalytic center occurs with varying affinity constants, depending on the OP involved. Phosphorothionates ( ~ S ) must first be converted to the more reactive oxon ( ~ O ) form via oxidative desulfuration or, less commonly, by isomerization (Gallo and Lawryk, 1991; Thompson, 1992). The phosphorylated enzyme is stable and the rate of spontaneous reactivation depends on the chemistry and chirality of the attached phosphoryl residue. In the process of aging, there is loss of an alkyl group leading to stabilization of phosphorylated ACHE. This inhibition of AChE underlies the toxicity of OPs, leading to an excess accumulation of ACh and producing a cholinergic crisis. It follows that the measurement of AChE is a mechanistic basis for monitoring exposure to OP anti-ChEs and is a biomarker of effect. A similar consideration applies to CMs, except that since carbamylated cholinesterase is spontaneously and rapidly reactivated, ChE measurements may be unreliable and misleading in diagnosing or confirming CM poisoning. The mechanism of inhibition of AChE by CMs is similar to the catalytic hydrolysis of ACh (Wilson et al., 1966). Like the reaction with substrate, the inhibitor first forms a Michaelis-type complex with the enzyme. The activated serine hydroxyl group in the catalytic triad of the active sites then reacts with the carbonyl group of the CM to form a carbamylated ACHE. The overall inactivation of the enzyme is characterized kinetically by the bimolecular rate constant of inhibition (ki), which provides a measure of the inhibitory potency of the compound. When first-order kinetics are observed, the 150 may be calculated from the ki by the relationship 150 = 0.693/(ki • t), where t is the time of preincubation of inhibitor and enzyme before the addition of substrate, and 150 is the concentration of inhibitor resulting in a 50% loss of the enzymatic activity after preincubating the enzyme for a fixed time (Thompson and
CHAPTER 39 9Occupational Toxicology and Hygiene Richardson, 2004). Kinetic determinations of inhibitory potency are preferable to fixed-time measurements because they yield a more complete understanding of the inhibitory process (Aldridge and Reiner, 1972; Richardson, 1992; Taylor and Radid, 1994). Anti-ChEs inhibit both erythrocyte (RBC) AChE [Enzyme Commission Number (E.C.) 3.1.1.7] and plasma/ serum BChE (pseudocholinesterase; E.C. 3.1.1.8). Serum BChE is inhibited more rapidly than RBC AChE and restored to normal (control) activity within 3 months. RBC AChE is inhibited less rapidly by OPs and takes several weeks to several months to retum to normal activity (Jaga and Dharmani, 2003). RBC AChE is the most frequently measured enzyme activity for assessing chronic OP exposure since activity may not be restored to control values for approximately 2 or 3 months, but in acute cases it is preferable to measure both RBC AChE and serum BChE since inhibition of RBC AChE activity may take a few hours to occur. Serum BChE is the more reliable enzyme determination for acute OP poisoning, but with decreased serum BChE activity there is a need to exclude acute/chronic inflammation, liver disease, malnutrition, and atypical pseudocholinesterase variant. An individual with symptoms of acute cholinergic poisoning may not necessarily have low RBC AChE activity during this phase, and the degree-of inhibition may not correlate with acute cholinergic symptoms. Several different analytical methodologies are available for measuring serum and RBC ChE, including visible spectrophotomettic, gasometric, fluorimetric, radiometric, electrometric (pH stat), and gas chromatography; they have been reviewed in detail by St. Omer and Rottinghaus (1992). A portable battery-operated test kit, based on a colorimetric approach, is available for field use (Higgins et al., 2001). A variety of factors may influence measured normal ChE activities and should be taken into consideration before the practical significance in relation to working conditions is interpreted. These include the methodology (technique) used for the measurement, intra- and interperson variation, age, race, gender, disease, and genetic factors: Methodology: Laboratory methods may account for 40% of the variability in RBC AChE activity and 24% in plasma BChE (Hartvig et al., 1980; Yager et al., 1976). Using the same methodology in the same laboratory can reduce this variability. Intraperson variation: Although there is less variability between samples taken serially from the same individual than when samples are compared between individuals (Sawitsky et al., 1948), a significant intraperson variation does exist. This variation is less for RBC AChE than for plasma BChE. For example, Callaway et al. (1951) found that the percentage standard deviation [coefficient of variation (C~V)] for an individual around his own RBC AChE was 10.7% and for plasma BChE was 22.8%. Similarly, Sawitsky et al. (1948) determined the CV for
577
RBC AChE to be 10.2%, and for plasma BChE it was 30.2%. Davies and Rutland (1950) determined the CV for RBC AChE to be 15.4% and for plasma BChE to be 25.8%. It was proposed by Hayes (1982) that in normal workers unexposed to OPs, the expected variations in RBC AChE would be in the range of 13-25% and in plasma BChE 20-23%. Interperson variation: A review of 10 studies comparing OP-exposed groups to nonexposed controls showed a CV of interperson plasma BChE ranging from 14.9 to 30.7% among unexposed controls (Duncan et al., 1986). For a group of unexposed individuals, Kilgore et al. (1977) suggested that the CV for plasma BChE values was 19-33% (mean, 25.9%), with a standard deviation of 5.9%; the RBC AChE CV for the group was 5.97-27.5% (mean, 16.2%), with a standard deviation of 1.06%. These data indicate that interperson variations in ChE activities are greater than intraperson variations. Genetic factors: The presence of an atypical ChE leading to a reduced plasma BChE is relatively rare but should be screened for at the preemployment medical examination of potential employees. Disease factors: Plasma BChE activity may be reduced in liver cirrhosis, hepatic parenchymal disease, protein malnutrition, low serum albumin, myocardial infarction, and dermatomyositis (Balistreri and Rej, 1994; Duncan and Griffith, 1992; Vorhaus and Kark, 1953). Age, sex, and race: RBC AChE decreases with increasing age in adults (Gage, 1967), but serum BChE is not so affected. In general, gender has little influence on ChE activity, although plasma BChE is somewhat lower in females and fluctuates during the menstrual cycle (Gage, 1967), and during the first trimester of pregnancy and days 2-7 postpartum plasma BChE is reduced (Evans and Wroe, 1980). Although race is not considered to be a factor in determining normal ChE activity, Reinhold et al. (1953) reported lower plasma BChE activities in black compared to white people of the same sex. The major objective of ChE screening programs is to detect potential overexposure to anti-ChEs before the onset of definitive poisoning. To this end, it is necessary for a "cutoff" point to be decided on that dictates the need for action to be taken to avoid further exposure of the affected individual(s) and to undertake reviews to determine the cause for the overexposure and institute corrective measures. Ideally, the most appropriate time for measurements to be made is as soon as possible after exposure; this is particularly important with CMs because of their relatively rapid reactivation, and thus prompt analysis is also needed. Clearly, there is a need to avoid contamination of blood with anti-ChEs from the environment or the skin during sampling. Interpretation of the significance of ChE measurement in relation to working conditions may differ somewhat between individual experts and authorities.
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However, a typical discussion would indicate the following for serum BChE: 1. When the BChE activity is 75-100% of baseline, the individual is asymptomatic and should not be restricted from working. 2. When activity is approximately 50% of normal, symptoms may be present. 3. Values in the range of 20-50% of baseline activity indicate there has only been mild overexposure, and minimal symptoms may be present. 4. In the range of 10-20%, there has been moderate exposure, and more marked effects may be present, including muscle fasciculation and miosis. 5. Less than 10% activity indicates there is severe poisoning, with life-threatening effects from an acute cholinergic crisis. A decrease in serum BChE activity may result from a variety of disease and other processes, which need to be considered in the differential diagnosis. These include hepatitis, hepatic cirrhosis, and various drugs. Similar considerations apply to inhibition of RBC AChE. Inhibition of RBC AChE to 20% on normal average values and of 10-20% on individual preexposure values can be considered significant (Lotti, 2001). The following surveillance protocol recommendations have been suggested: (i) If AChE activity is reduced by 30% or more of the preexposure level, repeat the test, (ii) decreases of 20-25% are diagnostic of exposure but not of hazard, and (iii) decreases of 30-50% are an indication for removal from further exposure with anfi-ChE until AChE returns to control levels (Lewinsohn, 1992). Inhibition of RBC AChE by dimethyl phosphates can be partially reversible; thus, this needs to be taken into account if a meas-
urement is not carried out immediately postexposure. Additionally, the rate of regain of AChE activity due to resynthesis of the enzyme occurs more rapidly in nervous tissue than in the RBC; animal data demonstrate a tl/2 in nervous issue of 5-7 days (Lotti, 1992) compared with reappearance of enzyme activity due to erythropoiesis at 1% per day (Lotti, 2001; Mason, 2000). Severe symptoms and mortality are generally believed to occur with RBC AChE inhibition of > 8 0 and >90%, respectively, in the absence of treatment (Moretto, 2004). The specific decreases in ChE activity that demand active intervention in employee work status vary among different authorities, but typical of the thresholds for removal of workers from exposure are those given by the California Health Services Department, which recommends removal of workers from further exposure when the RBC AChE activity reduces to 60% of baseline and serum BChE reduces to 50%; they are permitted to return to work when ChE activities increase to 75% of baseline (Ames et al., 1989; Jaga and Dharmani, 2003). For inhibition of ACHE, other values for intervention range from 30-60% against preexposure values to 50-70% against normal reference values (Jeyaratnam and Maroni, 1994). Ple~tina (1984) suggested waiting until RBC AChE returns to normal before allowing readmission to work. The World Health Organization (WHO, 1987) commented on the suggestion that a decrease of 30% in AChE activity from the baseline is a biological threshold for withdrawal of workers from anti-ChE exposure; they noted that this threshold has not been substantiated by existing medical and epidemiological information and may have been proposed based on the accuracy of the methods available at the time of its proposal. In the context of individuals with established OP poisoning who were admitted to the hospital within 6 hr, Aygun et al. (2002) found that low serum AChE levels (>50% of minimal normal value) supported a diagnosis of OP poisoning, but there was no significant association between serum ChE and the severity of poisoning on day 1, with the serum ChE activities of patients with severe poisoning not being statistically significantly different from those with mild poisoning. There was no significant difference in AChE levels between patients with delayed-onset neuropathy and those without, indicating that serum ChE is not useful as a predictor for delayed-onset neuropathy. However, patients who died did not show an increase in serum AChE between day 1 and time of death, in contrast with patients who survived, who showed a significant increase from day 1, indicating that serum AChE may be a useful predictor of prognosis of acute OP intoxication. Therefore, the investigators concluded that although a decrease in serum AChE activity during the cholinergic crisis of acute OP poisoning supports a diagnosis, it does not reflect the severity of poisoning, and although it is a useful indicator for following the acute prognosis, it is not a predictor for the development of
CHAPTER 39 9OccupationalToxicology and Hygiene delayed-onset polyneuropathy. CM anti-ChEs inhibit ChE activity by a reversible spontaneous hydrolysis and carbamylation of ACHE, with a rapid onset. ChE activity in workers exposed to CMs is as low as that with OPs, making it difficult to differentiate OP and CM poisoning on the basis of enzyme inhibition alone. Measurement of CM-inhibited AChE is limited by the rapid reversibility of the complex, and the time interval between sampling and analysis should be as short as possible. Blood samples should be kept cold and sample dilution during measurement should be kept to a minimum (Lotti, 1991).
VI. URINE/BLOOD METABOLITE MEASUREMENTS Monitoring anti-ChEs or their metabolites in urine and blood can be employed in two ways. The first is to give an accurate estimate of potential risk and requires a determination of the systemic dose received by the worker that can be equated to doses used in laboratory studies. This requires knowledge of the metabolism and toxicokinetics of the material and obtaining specific timed samples of blood and urine for analysis and accurate calculation of absorbed doses of the parent material. Second, for screening workers for exposure to an anti-ChE, there is a need to measure metabolite to qualitatively determine that there has or has not been exposure to the anti-ChE, or else to quantitatively measure the concentration of metabolite to ensure that it has not exceeded a specific level known to be associated with the development of pesticide-induced adverse health effects. Measurement of metabolites of anti-ChEs in urine or blood samples is an established method of monitoring for OP exposure and is a biomarker of exposure. For example, alkyl phosphates have been used to monitor exposure to OPs Carrier et al., (1999). However, the metabolism of OP compounds is variable, with some being extensively altered and others excreted mostly unchanged. The most common hydrolytic pathway is fission of the P-ester bond giving the alcoholic moiety ("leaving group") and the acidic moiety [alkyl(thio)phosphates]. Dimethylated OPs give dimethyl phosphates, dimethyl thiophosphates, and dimethyl phosphorothioates, whereas diethylated OPs give the corresponding diethylated metabolites (Coye et al., 1986; IPCS, 1986a). Since they can be derived from a large number of compounds, alkyl phosphates are nonspecific metabolites. Therefore, the compounds to which the employee is exposed must be known if a toxicological significance is to be attached to the analytical data. For example, dimethylated OPs have a wide range of acute toxicity and yet may give the same amount of dimethyl phosphates; the same consideration applies to diethylated OPs. Alkyl phosphate metabolites of OPs that have been extensively used as markers of occupational exposure to OPs include O,O-dimethyl phosphate (DMP), O,O-diethyl
579
phosphate (DEP), O,O-diethyl thiophosphate (DTP), O,O-dimethyl dithiophosphate (DMDTP), O,O-diethyl dithiophosphate (DEDTP), and O,O-dimethyl thiophosphate (Bravo et al., 2002; Hardt and Angerer, 2000; Linet al., 2002; Oglobline et al., 2001). Measurement of the urinary excretion of the alcoholic moiety may be more specific but is less frequently employed (Moretto, 2004). These include 3,5,6trichloro-2-pyridinol after exposure to chlorpyrifos-methyl and chlorpyrifos-ethyl (Nolan et al., 1984), malathion monoand dicarboxylic acids after exposure to malathion (Bradway and Shafik, 1977), and p-nitrophenol after exposure to parathion (Michalke and Daldrup, 1982; Morgan et al., 1977). Differences in the toxicokinetics of individual OPs and the route of exposure may further complicate the interpretation of data. Therefore, the timing of urine sampling is critical and has to be chosen according to the characteristics of the compound and the exposure route. In most field studies using urinary metabolites to assess OP exposure, there is insufficient data to allow a determination of any correlation between the amount of urinary metabolite and the degree of inhibition of RBC AChE and/or plasma BChE. In most published field studies, RBC AChE was found to be not or only minimally inhibited (Aprea et aL, 1997; Griffin et al., 1999; Jauhiainen et aL, 1991; Kraus et aL, 1977; Krieger and Thongsinthusak, 1993; Maroni et aL, 1990; Popendorf et aL, 1979; Spear et al., 1977). The only good correlation found has been between urinary p-nitrophenol and RBC AChE in workers exposed to parathion (Arterberry et al., 1961), which supports the biological exposure index (BEI) established by the American Conference of Governmental Hygienists (ACGIH, 2004) of 0.5 mg p-nitrophenol g-1 creatinine. Measurement of urinary p-nitrophenol is a relatively specific monitor for parathion exposure, and it is also a metabolite of parathion-ethyl and EPN. An approximate time interval of 8 or 9 hr is required from first exposure to parathion to peak p-nitrophenol excretion. Concentrations of urinary p-nitrophenol in the general population are 0.01-0.03 mg liter -1 (Baselt, 1980), and little or no decrease in ChE activity occurs when the urinary pnitrophenol concentration does not exceed 2.0 mg m1-1. For the rapid and sensitive quantitation of unchanged OP pesticide in urine and other biological samples, Tarbah et al. (2001) used a gas chromatograph with nitrogen/phosphorussensitive detection and electron impact gas chromatographymass spectrometry (GC-MS). A r a p i d and sensitive quantitative analysis for alkyl phosphate metabolites in urine, using GC-MS, has been developed by Kupfermann et al. (2004); they examined DMP, DEP, DMDTP, and DEDTP. A GC-MS method with deuterated DMP-d6 as an internal standard was developed and validated by Tarbah et al. (2004) for measurement of blood and urinary DMP. This is a metabolite of, for example, phosphamidon, mevinphos, monocrotophos, dicrotophos, dichlorvos, and trichlorofon. From the previous considerations, it follows that the measurement of plasma or urinary parent OP or its metabolite(s) can be used to monitor occupational exposure, providing the appropriate analytical
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Toxic Effects
techniques are available, but the resulting data can only be used to give a qualitative index of exposure rather than a toxicologically significant value (Moretto, 2004). However, this approach can be used to widely and noninvasively examine anti-ChE exposure in occupational situations, paraoccupational conditions, domestic applications, and exposure from food residues (Fenske, 1997; Hendorf et al., 2004). Many OPs are unstable in aqueous solution and, because of the presence of esterases, are not stable in blood. OPs are degraded more rapidly by esterases than by other biochemical mechanisms (Bouaid et al., 2001; Bowman and Sans, 1980; Drevenkar et al., 1983; O'Brien, 1960; Singh et al., 1985). Organophosphothioates, such as chlorpyrifos-methyl, are unstable in aqueous media but stable in blood for at least 2 hr, possibly due to stabilization of chlorpyrifos-methyl by proteins and lipids (Moriya et al., 1999). Stability of OPs in serum samples during storage was measured for 23 different OPs, which were mixed with serum containing 10 mg m1-1 EDTA and stored up to 10 days at 4 and - 2 0 ~ (Tarbah et al., 2001). Recovery rates of the OPs ranged from 50% (dimethoate) to 133% (dialifos). Stability studies on DMP have shown it to be stable over 2 weeks at 4 and - 2 0 ~ and additional studies showed no evidence of degradation when spiked in fresh blood and stored at 4 ~ for 1 week and stored in water for 10 months (Tarbah et al., 2004). In a case of selfpoisoning with phosphamidon by a 19-year-old female, the concentration of phosphamidon in serum decreased from 10 to 4.4 mg liter-1 after storage at - 2 0 ~ for 6 months; nearly complete degradation was found after 3 years (Tarbah et al., 2004). DMP was found in the body fluids stored at - 2 0 ~ for up to 3 years (respective values at 20 and 36 months of storage were blood, 3.9 and 4.9 mg liter-l; urine, 33.5 and 50.4mg liter-l; gastric fluid, 8.1 mg liter -1 and not detected). It is recommended that an examination for stable metabolites in suspected cases of OP poisoning should be performed. Unlike the situation with blood ChE inhibition measurements, the determination of CM metabolites may be useful to confirm and follow suspected CM-induced toxicity. CMs usually undergo extensive metabolism, particularly by carboxylesterases forming an aryl alcohol and a methylor dimethyl-amine. However, the rate of metabolism is dependent on the structure of the individual CM. Side chains may also undergo oxidation (e.g., hydroxymethylation), N-demethylation of secondary amines attached to the aryl moiety, or ring hydroxylation via the formation of an epoxide intermediate (Moretto, 2004). Thiocarbamates (e.g., aldicarb) may undergo S-oxidation forming the corresponding sulfane and sulfoxide. Urinary metabolites are mainly in the form of glucuronide or sulfate derivatives of the aryl groups. The parent compound may be found in small amounts in urine (IPCS, 1986b). Specific measurements for assessing worker exposure to CMs have included free and conjugated 1-naphthol from carbaryl (IPCS, 1986b, Lauwerys, 1982), 3-hydroxycarbofuran and
3-ketocarbofuran from carbofuran (Huang et al., 1989), 2- (di)methylamino-4-hydroxy-5,6-dimethylpyrimidime and other hydroxypyrimidimes from pirimicarb (Hardt et al., 1999; Verberk et al., 1990), and 2-isopropoxyphenol from propoxur (Brouwer et al., 1991, 1993). With carbaryl, in healthy nonexposed individuals the urinary 1-naphthol concentration is 4.0 mg liter -1 (Lauwerys, 1982). However, correlations of the chemical analyses of CMs or CM metabolites in urine have not been obtained with blood ChE measurements. Although sensitive GC and GC-MS methods are available for OP and CM measurements, they can be timeconsuming, require cleanup procedures, and involve the use of expensive equipment. Immunochemical assays may overcome some of these problems. Assays have been developed for parathion (Ercegovich et al., 1981) and paraoxon (Brimfield et al., 1985; Hunter and Lenz, 1982), as well as for certain CMs, including aldicarb (Mumma and Brady, 1987), benomyl, and carbendazim (Lukens et al., 1977; Newsome and Shields, 1981). These immunochemical assays have the advantages over traditional chemical analyses in that they require only minimal sample cleanup and permit high sample throughput, but they have the limitation that any one antibody allows only a very restricted set of structurally similar cross-reacting residues. Genetic engineering approaches for the development of monoclonal antibodies should enhance the potential for immunochemical techniques in qualitative and quantitative screening for anti-ChEs and metabolites.
VII. OTHER BIOCHEMICAL AND PHYSIOLOGICAL MONITORS OF OP/CM EXPOSURE Studies of greenhouse workers chronically exposed to antiChEs showed significant increases in acid phosphatase and [3-glucuronidase activities with decreased paraoxanase; the paraoxanase B allele was associated with a higher susceptibility to anti-ChE pesticides (Hermindez et al., 2004). Increases in creatine phosphokinase (CPK) may be an indication of myopathy resulting from exposures to anti-ChEs years previously. Friedman et al. (2003) described two patients who had elevations in CPK a decade after exposure to anti-ChEs. Both patients suffered from progressive generalized muscle weakness, chronic fatigue, myopathy, neuropathy, and marked neurobehavioral impairments. Measurement of lymphocytic neuropathy target esterase (Bertoncin et al., 1985) has been suggested as a monitor for exposure to neuropathic OPs, but its large interindividual variability, the different access of OPs to the nervous system, and the different rates of resynthesis between the nervous system and lymphocytes limit, its usefulness (Moretto, 2004).
C H APT ER 3 9 9Occupational Toxicology and Hygiene Neurophysiological monitors for peripheral effects include electromyography (EMG), nerve conduction studies, neuromuscular conduction techniques, and microelectrode studies. For central effects, techniques available are EEG and evoked potentials. Monitoring for delayed-onset neurotoxicity can be undertaken using EMG; with conventional EMG the motor units recorded are summated potentials of muscle fibers in a motor unit within a few millimeters of the recording electrode. EMG signals are analyzed for insertion activity, spontaneous activity, motor unit potential analysis, and recruitment pattern on voluntary muscle contraction. Some authorities regard EMG as a useful and sensitive technique (Jager et al., 1970; Roberts, 1976), but its sensitivity has been questioned by others (Le Quesne and Maxwell, 1981; WHO, 1986). EMG changes were found in 50% of workers exposed to dimethyl phosphate esters and consisted of low-voltage and repetitive activity (Jager et al., 1970). Motor nerve conductiqn involves stimulating the nerve, usually with a supramaximal square wave pulse, and recording the compound motor action potential using surface or needle electrodes. The evoked compound motor action potential is described by latency, amplitude, duration, and configuration. AChEs produce twitch potentiation, fasciculations, and tetanic fade. In twitch potentiation, there is potentiation of the tension developed by skeletal muscle in response to submaximal or maximal indirect stimulation at low frequency (Koelle and Gillman, 1949; Werner and Kuperman, 1963). Fasciculations are produced by OPs (Meer and Van der Meter, 1956) and CMs (Blaber and Goode, 1968) and result from intermittent synchronized contraction of muscle fibers of a motor unit, which is probably due to the initiation of an axon reflex allowing released ACh to have access to the first node of Ranvier and to depolarize it in the absence of nerve stimulation (Misra, 1992). Fasciculations occur within a limited range of AChE inhibition, disappearing when inhibition is >95% (Barnes and Duff, 1953). Indirect stimulation of skeletal muscle at >20 Hz for a brief period results in a sustained increase in tension, but following exposure to anti-ChEs the response to such stimulation consists of a rapid increase in tension followed by partial or complete relaxation while the stimulation is maintained; this is called tetanic fade (Blaber and Bowman, 1963). Tetanic fade is associated with inhibition of AChE (Misra, 1992) and caused mainly by postsynaptic block. Singlefiber EMG records from individual fibers within 300 lxm of the electrode, and the potential is biphasic, with a rise time of the negative phase of 200 lxsec, duration of 1 msec, and amplitude usually of 5-10 mV. Motor nerve stimulation propagates distally (orthodromically) and also proximally (antidromically) toward the anterior horn neuron cell bodies, which can be activated. The recurrent discharge produces small potentials after a delay of approximately 20-50 msec, which is called the F-response (Magladrey and McDougal, 1950). They provide information about
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conduction in the proximal segment of the motor fibers and may be a more sensitive index of peripheral nerve dysfunction than standard nerve conduction studies (Latchman et al., 1980). Sensory nerve conduction can be evaluated by stimulating and recording from a cutaneous nerve using surface or needle electrodes. Repetitive nerve stimulation techniques involve stimulating a muscle by repetitive supramaximal stimuli through the motor nerve. A decline in the amplitude of the evoked potential provides a measure of the degree of neuromuscular block. EEG changes in acute OP exposure have been reported to cause marked desynchronization and a triad of changes consisting of increased high-frequency activity, decreased low-frequency activity, and lowered background voltage. High-concentration exposures cause slowing of the EEG followed by the development of spike wave discharges that accompany convulsions (Burchfeil et al., 1976). The EEG is regarded by many as not being sufficiently sensitive or specific to discriminate between normal (control) subjects and neurobehaviorally affected OP-exposed individuals (Savage et al., 1988). Details on the use of neurophysiological monitors for exposure to anti-ChEs have been discussed in detail by Misra (1992) and WHO (1986).
VIII. WORKPLACE BIOMONITORING In addition to the control of workplace exposure to pesticides by the use of airborne exposure guidelines, BEIs may be recommended based on appropriate urine or blood analyses (Helath and Safety Execution, 1987). These BEIs can be applied not only to plant exposures but also to accident sites and where there are formulation and in-use applications. BEIs are guidance values for assessing biological monitoring results and represent the levels of determinants that are most likely to be observed in specimens collected from healthy workers who have been exposed to the chemical under examination to the same extent as workers with inhalation exposure at the TLV. Exceptions may be chemicals whose TLV is based on nonsystemic effects (e.g., irritation). The BEI in general indicates a concentration below which nearly all workers should not experience adverse health effects. They apply to 8-hr exposures for 5 days a week. Biological monitoring can assist the occupational health professional to determine absorbed dose of chemical and can assist in determining the efficacy of personal protective equipment, engineering controls, and general work practices. For example, the OP parathion has BEIs based on a urinary metabolite and on RBC ACHE. In urine, the determinant is total p-nitrophenol, with a BEI of 0.5 mg g-1 creatinine, and for RBC ACHE the BEI is 70% of the individual's baseline (ACGIH, 2004). Gosselin et al. (2005) developed a multicompartment model to describe the kinetics of parathion and its metabolites, p-nitrophenol and alkyl phosphates, in order to assess worker exposure and health risks. For percutaneous
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Toxic Effects
absorption, except for the cutaneous absorption fraction and \ absorption rate, which are known to show wide intraperson and interperson variability, a single set of parameter values for the internal body kinetics enabled the model to simulate accurately the available kinetic data. For dermal absorption to parathion, with a typical absorption rate of 0.085 hr-1, model simulations showed that it takes 20 hr to recover half of the total amounts of p-nitrophenol eventually excreted in urine and 30 hr for the alkyl phosphates. The model can be used to estimate the dose of parathion absorbed under different exposure routes and temporal scenarios, based on measurements of the amounts of metabolites accumulated in the urine over given time periods. Using the dose-excreta links and the human no observed effect level for parathion inhibition of cholinesterase activities, biological reference values can be established to prevent adverse health effects in exposed workers in the form of specific amounts of urinary metabolites excreted over selected periods of time.
IX. O C C U P A T I O N A L H Y G I E N E CONSIDERATIONS FOR RESTRICTION OF ANTICHOLINESTERASE EXPOSURES In view of the fact that exposure to pesticides occurs in a variety of circumstances and conditions, although there are factors common to all these, it is necessary to define occupational hygiene measures for each individual situation. For example, although workplace exposure guidelines such as TLVs are appropriate to use for worker protection in enclosed environments such as production plants, these cannot be applied to open-air situations. Occupational hygiene measures are developed and designed in order to give reliable protective and precautionary approaches against the development of morbidity and/or mortality in circumstances of potential overexposure.
X. G E N E R A L I Z A T I O N S A N D A C T I O N S COMMON TO ALL POTENTIAL OCCUPATIONAL OVEREXPOSURE CIRCUMSTANCES The following considerations apply to all situations in which OP and CM anti-ChEs may be manufactured, formulated, transported, and used in occupational environments.
A. Advance Planning This covers items such as ensuring that the least toxic material and formulation to achieve the desired pesticidal effect are used; ensuring that all necessary inspection and repairs have been carried out; and, where possible, determining the best time from a meterological perspective to undertake application (such as low wind speed to limit drift).
B. Education and Training It has been estimated that in the United States approximately two-thirds of farm workers have not received any information or training on pesticide safety (Arcury et al., 1999). Also, as noted previously, in a survey of Latino farm workers, Arcury et al. (2000) found that it was believed that not all farmers necessarily adhere to regulations mandating training and basic sanitation. They noted that regulation by itself is not an advantageous starting point, and intervention must include educating farmers as well as farm workers. For a developed country, it should be mandatory that everyone, at all levels of employment status, who may involved in work in which there is a potential for exposure to anti-ChEs should be adequately informed and trained about general aspects of anti-ChEs and specific aspects relevant to their occupation. Education and training sessions should at least cover the following: 1. The names, chemistry, and physicochemical properties of the anti-ChEs that may be encountered during work activities. 2. Routes of exposure and how overexposures to pesticides may occur. 3. The potential health hazards of pesticides and other chemicals that may be handled or encountered, and the signs and symptoms of intoxication. 4. Protective and precautionary measures needed to ensure safe working conditions, including protective clothing and equipment. There is a clear need for a respirator training program. 5. Emergency first aid measures (including controlled decontamination) and procedures to be adopted locally in the event of an accident leading to overexposure, including those who are to be immediately informed and how to obtain emergency medical information and care. The latter should include ensuring ready availability to telephone communications to a local hospital accident/ emergency center and the nearest poison control center. 6. Environmental and health hazards from contaminated clothing, spills, drift, and take-home path. 7. Warnings necessary, including oral and written reentry and warning notices, informing local inhabitants about applications. 8. Any relevant written information should be given to employees, who should be instructed to read manufacturers literature, material safety data sheets (MSDS), and container labels. Compound-specific MSDSs should be written by the pesticide manufacturer giving all necessary physicochemical, toxicology, medical, protective and precautionary, and regulatory information. These MSDSs should be made available for wide distribution to employees, users, poison control centers, and those who request them. Details on the intent, structure, and contents of the MSDS have been discussed elsewhere (Tyler and
CHAPTER 39 9Occupational Toxicology and Hygiene Ballantyne, 1988). Product labeling is a very important component in ensuring the safe use of an anti-ChE pesticide (Laughlin and Gold, 1988). Registration of a pesticide requires that the manufacturer provide an understandable and informative label for the user that must have the approval of the appropriate registration authority [e.g., the Environmental Protection Agency (EPA)]. The label should provide information on the following: active ingredient and concentration; approved uses; specific methods of handling the chemical; methods on preparation and application; guidance on storage and disposal of unused chemical; safe use, including protective and precautionary measures; statement on significant adverse health effects and of medical management and antidotes; first aid measures; and a manufacturer's emergency telephone number (ideally manned for 24 hr) from which expert advice on dealing with accidental situations or emergency health issues can be obtained (Ballantyne, 1975; Griffith and Duncan, 1992).
C. Audit of the Health and Safety Program There should be provision to undertake frequent, but random, audits of the following: 1. Educational and training sessions. 2. That collective and personal protective and precautionary measures are being strictly followed, including adherence to workplace exposure guidelines (e.g., TLVs). 3. Protective equipment and clothing are being kept clean and are readily available. 4. Respirators are being maintained, and respirator training and fitting sessions are adequate. 5. Engineering controls are being adhered to. This includes confirmation of the following: (i) Raw materials are delivered in bulk by railcar, pipeline, barge, or tanker truck and are mechanically unloaded, thus ensuring minimal handling by employees; (ii) raw materials are packaged in sealed containers; (iii) where possible, weighing and dispensing of raw materials are automated; (iv) mixing equipment, milling machines, and baggers should be enclosed, and emissions should be dealt with by other engineering controls; (v) a plant manufacturing potent anti-ChEs should not be sited ina heavily populated area; and (vi) there should be closed-transfer systems for use by employees mixing and loading liquid pesticide. 6. There is provision for atmosphere monitoring in situations in which a workplace exposure guideline is applicable (e.g., plant facility); the sampling times, sampling methods, and analysis should all be examined and judged to be adequate and appropriate. 7. Periodic medical surveillance is carried out, with all necessary health monitoring requirements for general health and to exclude anti-ChE overexposure.
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D. Supervision There should be one or more senior operatives who are knowledgeable and well trained in precautionary and protective measures and also compliance with pesticide regulations. They should be responsible for the day-to-day supervision of plant operations, applications in the field, etc. No person should work alone while handling or likely to be exposed to anti-ChEs. There should always be an additional person nearby, designated as safety officer, who can view the operation at a distance sufficient, if necessary, to raise an alarm, to allow protective clothing and equipment to be donned, and to undertake the initial rescue and possible first aid and resuscitation measures. Thus, ideally this person should be trained in the emergency measures for an anti-ChE leak, spill, and exposure, and it is desirable for him or her to be trained in first aid measures to treat OP and CM poisoning.
E. Protective Clothing Where there is likely to be direct skin contact with antiChEs, protective clothing should be available, and when the circumstances indicate the need, it should be donned. This includes loss of containment in a production facility; an accident during transportation; handling of the pesticide during formulation, mixing, or loading; and during in-use application. In such circumstances, there is a need for eye protection (goggles) and clothing that should cover the trunk, arms, hands, legs, feet, and face. Although face shields give protection of facial skin, they may provide only partial protection of the eyes. Thus, for full protection of the eyes, goggles are needed. Clothing should not have external cuffs or open pockets in which hazardous materials can accumulate, and sleeves should be of adequate length. Gloves should be chosen on the basis of impermeable characteristics and be carefully inspected for integrity before use. Boots or shoes should be mechanically sound and made of appropriate impermeable material. It is of absolute necessity to ensure that the recommended protective practices are followed; for example, agricultural workers frequently do not wear protective gloves even though they have been advised to do so (Webster and Maibach, 1985a,b).
F. Respirator Use Where there is likely to be inhalation overexposure to anti-ChE vapor, aerosols, or dusts, appropriate respiratory equipment should be available. There are, descriptively, two main types of respirators (Ballantyne and Schwabe, 1981). First are air-purifying respirators in which environmental air is drawn through a nonreturn valve and an appropriate absorbent filter material that removes atmospheric contaminants (particulates, vapor, or gas), and
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N o n s p e c i f i c Toxic Effects
expired air is eliminated through a nonreturn exhalation valve. This type of respirator is clearly of use for working in an atmosphere containing a toxic material for which an absorbent is available, but clearly not of use if there is also an oxygen-deficient atmosphere. Second are atmosphere-supplied respirators (breathing apparatus) in which an independent source of uncontaminated air or cylinder gas is introduced into the respiratory airspace. They can be used for prolonged periods and in many types of atmospheres. A decision regarding the type of respirator to be used needs to take into consideration various issues, including the type, toxicity, and physicochemical properties of the pesticide used; whether required for indoor or outdoor use; and whether they can be adequately stored and serviced at the user facility. A respirator-servicing program needs to be introduced to ensure that the respirators are periodically cleaned, inspected for damage, and checked for adequacy of function. A periodic check of individual respirator fit is needed to ensure continued adequacy of function with fit. As part of the preemployment medical, it should be determined if the potential employee can psychologically tolerate the wearing of a respirator and if there are any medical contraindications, such as respiratory disease. An employee training program should be introduced to ensure the continued appropriate maintenance and use of the equipment.
G. Good Personal Hygiene Good personal hygiene should be ensured to prevent contamination of the employee and avoid a take-home path. This includes frequent change of clothing, adequate washing facilities, and no eating, drinking, or smoking while there is a potential for exposure. In one survey, Griffith and Duncan (1983) found that 56% of field workers did not wash their hands within 15 min of leaving the work area. The provision of washing facilities, ideally with showers, is an important occupational hygiene necessity for agricultural workers.
H. Treatment of Established Poisoning If an on-site physician is not employed, there should be appropriately trained and qualified nursing or first aid staff who are promptly available and can undertake the initial elements in the management of acute anti-ChE poisoning. First responders should be familiar with the clinical presentation of acute anti-ChE poisoning, such that they are confident in the recognition of an individual with intoxication who requires treatment. The management of anti-ChE poisoning involves the following: 1. Decontamination. The person(s) undertaking decontamination should be suitably protected to avoid becoming contaminated from the victim (Abraham and Weinbrom,
2003). Generally, decontamination measures are removal of clothing and washing affected areas. 2. Maintenance of an adequate airway and breathing is necessary if there is obstruction resulting from bronchial secretions and bronchospasm. This may require the use of an oral airway (or insertion of an endotracheal tube if medical assistance is available) and assisted ventilation (e.g., mask with manual inflator). Oxygen is valuable because of the potential for hypoxia resulting from airway obstruction secondary to increased tracheobronchial secretions and bronchospasm (Munidasa et al., 2004). When hospitalized, endotracheal intubation and assisted ventilation may be required (Proudfoot and Vale, 1996). 3. Antidotes. Atropine, a muscarinic receptor antagonist, is particularly effective and important in the acute management of OP poisoning (Leenders et al., 2003). Full and early atropinization is essential to reverse cholinergic excess and improve respiratory function, heart rate, and blood pressure (Eddleston et al., 2004). Atropine should be given intramuscularly at the accident site and may be given by intravenous infusion in the hospital. Atropine is important in reducing tracheobronchial secretions, but it does not have a significant effect on nicotinic ACh receptors and therefore ventilatory muscle weakness persists. Early deaths result from respiratory failure and cardiovascular collapse (Eddleston et al., 2004; Munidasa et al., 2004). However, atropine does cross the blood-brain barrier and counteracts the convulsive effects of OPs. Because of the possibility for ventricular arrhythmias when atropine is used in the presence of cyanosis and/or an ischemic myocardium, oxygen should be administered in these situations. Dosage is best titrated on the basis of clinical signs, notably resolution/reduction of bronchospasm and bronchosecretion and a sinus tachycardia to approximately 80-100 beats/min (Leenders et al., 2003). Oximes are of importance for reactivation of phosphorylated AChE and used in combination with atropine (Eyer et al., 2003; Vucinic et al., 2003; Zilker, 2004; Zilker et al., 2004). Oximes are therapeutic by removing the phosphoryl group from AChE and thus restoring its catalytic sites (ChE reactivators). This antidotal action only occurs when the phosphorylated AChE has not undergone the intramolecular rearrangement of aging. Ideally, for the immediate treatment of OP poisoning, they would best be given intramuscularly by an autoinjector, but these are not readily available in a civilian first aid situation. In the context of occupational OP exposure, there are no clinically important differences between pralidoxime, obidoxime, and HI-6 (Marrs et al., 2003). Therapeutic benefit requires early and sustained treatment with high (effective) oxime doses (Lotti, 2003; Proudfoot and Vale, 1966; Thermann et al., 2003). Although the administration of oximes should occur at the earliest possible
CHAPTER 39 9Occupational Toxicology and Hygiene time after intoxication, their late administration can be beneficial in cases in which there is a possibility of prolonged absorption and/or high lipid solubility, such as may occur with fenitrothion, fenthion, and clofenthion (Burillo-Putze et al., 2004). Anti-ChE-induced centrally mediated seizures may develop into a clinical condition resembling status epilepticus. The administration of an anticonvulsant early in the management of intoxication is recommended (Ballantyne and Marrs, 1992). Diazepam and phenytoin have been recommended, although these may not provide optimal protection. Fosphenytoin, alone or in conjunction with diazepam, has little or no therapeutic anticonvulsant effectiveness for OP-induced convulsions (McDonough et al., 2004).
I. Environmental Issues In order to ensure that there is no harm to the environment from accidental discharges from plants or from drainage as a consequence of in-use applications, and for compliance with state and federal regulations, there should be frequent measurements of anti-ChEs in soil, water (rivers and groundwater), and surrounding air.
XI. O C C U P A T I O N A L HYGIENE CONSIDERATIONS IN CLOSED FACILITIES In the manufacture of pesticides, exposure of factory workers is probably negligible in well-organized plants with good industrial hygiene practices, but during formulation there may be additional factors to be considered, Overall, in closed facilities, the general considerations discussed previously need to be followed, and in production sites for anti-ChE pesticides, special attention is needed as follows.
A. Atmosphere Dust particles in the air of a closed environment can be inhaled or trapped in saliva or mucus and swallowed. Also, the volatility of liquids has to be taken into consideration during the design of engineering controls using local exhaust methods, which are the most widely used methods of atmosphere control in manufacturing facilities. They are designed to capture or contain emissions at their source before they can reach the workplace atmosphere. General ventilation systems provide clean air to, and remove air from, the workplace to provide frequent and adequate "tumover" of air and hence maintain contaminants at concentrations below those considered to represent a health hazard; however, this method is not generally considered appropriate to control exposures to antiChEs. Exhausted air may need to be treated to ensure that anti-ChE concentrations do not cause any local environmental or human population risks. In the atmosphere of a production
585
plant, there may be accumulation of anti-ChEs if there is a breakage of containment, if the engineering controls for ventilation fail, or if a spill occurs. Ideally, there should be an automatic warning system to indicate if this situation develops, and this should be maintained and tested periodically. A senior person at the site (ideally an industrial hygienist) should ensure that sampling is taken for routine measurements to confirm adherence to the recommended workplace exposure guidelines, such as the TLVs, with any qualifications proposed by ACGIH (2004); examples of these are given in Table 2. Govemment-mandated permitted exposure limits may apply in many countries. For example, in the United States, the Occupational Safety and Health Administration sets permissible exposure limits, and in the United Kingdom maximum exposure limits and occupational exposure standards are set under the Control of Substances Hazardous to Health regulations of the Advisory Committee on Toxic Substances and Working Group on the Assessment of Toxic Chemicals of the Health and Safety Executive. Clearly, these airbome workplace exposure guidelines are relevant to enclosed areas (factories, greenhouses, etc.) but cannot be applied to outdoor work situations (Ballantyne and Marrs, 2004). The acceptable occupational exposure level (AOEL) is defined by the Directive of the European Union 91/414 concerning the placing of plant protection products on the market as "the maximum amount of active substance to which the operator may be exposed without any adverse health effect" and it is expressed as mg kg -1 (body weight) day -1 of absorbed (by any route of exposure) dose available for systemic distribution (Council of Europe, 1991). AOELs for agricultural pesticides are derived from the toxicological database of the active substance involved. These levels are considered to be safe for workers exposed to the formulated product used as recommended. Therefore, worker exposure, either measured or estimated, must be compared to the established AOEL. In the European Union, an estimated exposure above the AOEL prevents the registration of the active ingredient or some of its formulations and/or uses. AOELs are intended for preregistration risk assessment purposes and not as a tool to control worker exposure. In this respect, AOELs differ from OELs used in the industrial setting, which are typically recommended for an 8-hr time-weighted average, based on a working life time of 40 hr per week, or 15-rain short-term exposure levels, which are used to qualify occupational exposure (Ballantyne and Marrs, 2004).
B. Respiratory Protection If a break in containment or a spill occurs in the plant manufacturing situation, then vapor may accumulate in the air at concentrations above the workplace exposure guideline and may reach toxic concentrations. Therefore, appropriate respiratory protective equipment should be available for emergency use. As a support for this, there should be a respirator training and fitting program.
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SECTION V . N o n s p e c i f i c Toxic Effects
TABLE 2. Examples of Workplace Exposure Guidelines (Threshold Limit Values) Assigned by ACGIH (2004) for OP and CM anti-ChEs a
Pesticide
Adopted value
Notation
Effects noted
Organophosphates Azinphos-methyl Chlorpyrifos
TWA8 0.2 mg m -3 (IV) TWA 8 0.1 mg m -3 (IV)
Skin: SENS: A4: BEI A Skin: BEIA: A4
Cholinergic Cholinergic
Demeton Demeton-S-methyl Diazinon Dichlorvos Dicrotophos Disulfoton EPN Ethion Malathion Methyl demeton
TWA8 0.05 g m -3 (IV) TWA80.05 mg m -3 (IV) TWA8 0.01 mg m -3 (IV) TWA8 0.1 g m -3 (IV) TWA8 0.05 mg m -3 (IV) TWA8 0.05 mg m -3 (IV) TWA8 0.1 mg m -3 (I) TWA8 0.05 mg m -3 (IV) TWA8 1 mg m -3 (IV) TWA8 0.5 mg m -3
Skin: BEIA Skin: SENS: A4: BEI A Skin: BEIA: A4 Skin: SENS: BEIA: A4 Skin: A4: BEIA Skin: A4: BEIA Skin: A4: BEIA Skin: A4; BEIA Skin: A4: BEIA Skin: BEIA
TWA8 0.2 mg m -3 TWA8 0.01 mg m -3 (IV) TWA8 0.0 mg m -3 TWA8 1 mg m -3 (I)
Skin: A4: BEIA Skin: A4: BEIA Skin: BEIA A4: BEIA
Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic Cholinergic, irritation Cholinergic Cholinergic Cholinergic Cholinergic
Carbaryl
TWA8 5 mg m -3
A4
Carbofuran Methomyl Propoxur
TWA8 0.1 mg m -3 (IV) TWA8 2.5 mg m -3 TWA 8 0.5 mg m -3
A4: BEIA A4: BEIA A3: BEIA
Methyl parathion Mevinphos TEPP Trichlorphon Carbamates
Cholinergic, reproductive Cholinergic Cholinergic Cholinergic
aAbbreviations used: EPN, O-ethyl-O-4-nitrophenylphenylphosphonothioate; TEPP, tetraethyl pyrophosphate; TWA8, time-weightedaverageconcentrationover 8 hr; I, inhalable fraction;V, vapor and aerosol; BEIA, Biological Exposure Index for cholinesterase-inhibitingpesticides (RBC AChE 70% of individual's baseline);A3, confirmed animal carcinogen with no known relevance to relevance to humans; A4, not classifiable as a human carcinogen; SENS, sensitizer(cutaneous or respiratory; animal and/or human data); skin, possibilityfor percutaneousabsorption and resultant systemictoxicity;TLV basis, critical effect(s).
C. Medical and Emergency Considerations The plant should have access to a full- or part-time occupational health physician supported by an occupational health nurse. They should be responsible for the conduct and recording of pre- and periodic medical surveillance and be available for any emergency overexposures and poisoning. Equipment and antidotes should be readily available in the event of an accidental poisoning (e.g., airways, oxygen, mask with manual inflator, and atropine and oxime).
XII. ADDITIONAL OCCUPATIONAL HYGIENE CONSIDERATIONS FOR IN-USE OPEN-AIR CONDITIONS The following discussion applies to the application of pesticides in open-air situations, such as fields and orchards.
A. Supervision During loading and application of pesticide, there should be supervision of employees by experienced people. The employees should be carefully instructed, before the working day starts, about the intended applications and where pesticide handling and exposure may occur. As a reminder, there should be posters and notices drawing attention to, and clearly defining the health hazards and risks from, overexposure to anti-ChEs. Provision should be made for preemployment medicals for new workers and for regular periodic medical examinations for established workers.
B. Protective Clothing Protective clothing, including gloves and a face shield, should be readily available where overexposure is likely. Although impermeable clothing provides the greatest
CHAPTER 39 9Occupational Toxicology and Hygiene protection against anti-ChEs (Griffith and Duncan, 1983), 100% cotton fabric reduces skin exposure more effectively than synthetic fibers (Griffith and Duncan, 1992). Clothing saturated with pesticide residue may promote continuing exposure of the skin (Freed et al., 1980; Maibach and Feldman, 1974; Wicker et al., 1979), which may enhance percutaneous absorption and the potential for systemic toxicity. Clearly, workers should wash and/or change clothing at least daily to prevent accumulation of potentially harmful residue (Finley et al., 1978). Additionally, washing facilities should be made available to employees.
C. Health Care Facilities When anti-ChEs are to be used, there should be provision for a trained medical or nursing professional to be on site during the application phase. They should have access to necessary emergency first aid and treatment needs (e.g., airways, oxygen, and antidotes). When there are no local health facilities nearby, there should be arrangements for transporting overexposed and poisoned workers to the nearest capable hospital.
D. Reentry Restrictions Entering a pesticide-treated zone before the material has decreased to nonhazardous levels can result in significant acute illness (Centers for Disease Control, 1999). After pesticide application, workers should be prevented from entering the treated zone until it is safe to do so; this is normally controlled by a restricted entry interval (REI). The duration of the REI depends on the specific pesticide used. However, a reentry interval established for intact residues of selected OPs may be of little value for those compounds that degrade to their more toxic analogues, such as parathion. The establishment of reentry intervals requires the following: (i) examination of dose-response data, (ii) an estimate of the relationship between surface residue and total body exposure, and (iii) examination of time versus residue data. These data should permit an estimate of the dose at which there is no effect or minimum risk (Zweig et al., 1984). California has set 48-hr worker safety reentry intervals and 96-hr harvest intervals based on, but not limited to, toxicity, rate of persistence and degradation curves, human exposure practices, usage patterns, frequency of documented poisoning cases, rate of pesticide application, formulation, concentrated or dilute application, and the possibility for potentiation (Knaak, 1980; Maddy, 1996). Reentry standards for field workers were first proposed in the United States by the EPA in 1974. The reentry interval or standard has been defined as the period of time, in hours or days, following pesticide application after which a worker may legally enter a treated field to engage in normal field activity resulting in prolonged contact with foliage (Kraus et al., 1981). Workers without appropriate personal protective
587
equipment and clothing should not enter the pesticidetreated area until expiration of the REI. Posted and oral warnings, based on the REI, should be made available immediately after pesticide application. Where reentry standards have been appropriately set and applied, there has been a reduction in systemic illness related to residues (Maddy, 1996).
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CHAPTER 40
Public Health Impacts of Organophosphates and Carbamates DAPHNE B. MOFFETT U.S. Department of Health and Human Services, Atlanta, Georgia
some OPs were developed as potential chemical warfare agents. The first OP to be used commercially was tetraethylpyrophosphate (TEPP); although effective, it proved chemically unstable and extremely toxic to all forms of life (Ecobichon, 2001). During the same time period, the first pesticidal carbamic acid esters were synthesized and marketed as fungicides. Because of TEPP's undesirable ubiquitous toxicity and due to poor insecticidal activity of the existing carbamic esters, insecticide development was directed toward the synthesis of more stable chemicals with moderate environmental persistence, which gave rise to parathion in 1944 and paraoxon at a later date (Ecobichon, 2001). In the mid-1950s, there was renewed interest in insecticides with anticholinesterase (anti-ChE) activity but reduced mammalian toxicity, thus leading to the synthesis of several potent aryl esters of methyl carbamic acid. The insecticidal CMs were synthesized as analogs of the drug physostigmine, a toxic anti-ChE alkaloid extracted from the seeds of Physostigma venenosum, the calabar bean (Ecobichon, 2001).
I. I N T R O D U C T I O N The public health impacts from the use of organophosphorus (OP) and carbamate (CM) pesticides are broad, ranging from benefits seen through control of vector-borne diseases (Le., diseases transmitted by insects or other arthropods) and increased survival rates, particularly in subtropical and tropical countries, to severe illness and death associated with acute OP poisoning. People of all ages are exposed to pesticides. Whether it is through diet (including breast milk and water), indoor spraying, aerial spraying, or outdoor activity, kids and adults are exposed. Occupational exposures are common, particularly among applicators and farm workers. The widespread use of pesticides feeds into the exposure cycle. In the United States, more than 18,000 products are licensed for use, and each year more than 2 billion pounds of pesticides are applied to crops, homes, schools, parks, and forests [U.S. Environmental Protection Agency (EPA), 2002]. Discussions of pesticide use and exposure, including the OP and CM classes, are often marked by ominous tones and monopolized emphasis on the negative contributions made to the environment and public health. The public health importance of the continued application of pesticides in integrated pest management and the control of outbreaks and vector-borne disease is overshadowed by the concern for human exposure and health effects, particularly in small children. In an effort to portray the positive and negative impacts of OPs and CMs in public health, two subtopics are reviewed in this chapter: uses of OPs and CMs in the control of vector-borne diseases and children's exposures to OPs and CMs.
A. Eradication of Vector-Borne Diseases The development of effective OPs and CMs historically played a critical role in the control of vector-borne diseases. Malaria, dengue, yellow fever, plague, filariasis, louse-borne typhus, trypanosomiasis, leishmaniasis, and other vector-borne diseases were responsible for more human disease and death in the 17th through the early 20th centuries than all other causes combined (Gubler, 1991). During the 19th and 20th centuries, vector-borne diseases prevented the development of large areas of the tropics, especially in Africa; it was not until these diseases were controlled that wider spread habitation and travel could occur. Once the disease transmission cycles were known, prevention and control programs became focused on vector control. These public health programs included the use of pesticides, initially organochlorine pesticides such as DDT and later OPs and CMs (Table 1).
II. C O N T R O L OF V E C T O R - B O R N E DISEASES OP insecticides were first synthesized in 1937 by a group of German chemists led by Gerhard Schrader (Ecobichon, 2001). Unfortunately, due to Nazi oversight in World War II, Toxicology of Organophosphate and Carbamate Compounds
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Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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SECTION VI 9Risk Assessment & Regulations TABLE 1. Selected Commonly Used OPs and CMs and Targeted Vector-Borne Diseases Chemical classification
Selected chemical
Organophosphates
Chlorpyrifos Fenitrothion Fenthion Malathion
Carbamates
Vector-borne disease
Naled Pirimiphos-methyl Temephos
Malaria, West Nile virus (WNV) Malaria, dengue, yellow fever, leishmaniasis WNV, dengue, yellow fever Malaria, WNV, dengue, yellow fever, leishmaniasis, head lice WNV, dengue, yellow fever Malaria, dengue, yellow fever Malaria, WNV, dengue, yellow fever, onchocerciasis
Bendiocarb Carbosulfan Propoxur
Malaria Malaria Malaria, leishmaniasis
In 1900, yellow fever in Cuba was the first vector-borne disease to be effectively controlled, followed quickly by yellow fever and malaria in Panama (Gubler, 1998). By the 1960s, vector-borne diseases were no longer considered major public health problems outside of Africa. Urban yellow fever and dengue, both transmitted by the mosquito Aedes aegypti, were effectively controlled in Central and South America and eliminated from North America; malaria was nearly eradicated in the Americas, the Pacific Islands, and Asia (Gubler, 1998). The effective use of residual insecticides from the 1940s through the 1960s contributed greatly to these successes. Major mosquito vectors responsible for some of the most widespread diseases include Culex genus, which is the vector for West Nile virus (WNV), filariasis, and Japanese encephalitis; Aedes of dengue, dengue hemorrhagic fever, and yellow fever; and Anopheles of malaria. DDT was first introduced for mosquito control in 1946. In 1955, the World Health Organization (WHO) assembly proposed the global eradication of malaria with DDT (Hemingway, 2003). DDT was the insecticide of choice globally for many years. It remains in use in developing nations because of its affordability and continued effectiveness in controlling diseases such as malaria. However, public concern over the environmental persistence of DDT and other organochlorine pesticides and their banning from manufacture and use in the United States in the 1970s led to the development and use of alternative insecticides, including OPs (malathion, pirimiphos-methyl, and fenitrothion) and CMs (bendiocarb, carbosulfan, and propoxur). The WHO Pesticide Evaluation Scheme identified and recommended these insecticides for indoor residual spraying (Chavasse and Vap, 1997). Since 1964, ultralow-volume (ULV) aerial applications of the OPs (malathion and naled) have been used many times in the United States and its territories for controlling mosquitoes in disaster areas and for controlling epidemics of mosquito-borne disease. The ULV method was used to
kill infected Culex pipiens quinquefaseiqtus during the Dallas, Texas, epidemic of St. Louis encephalitis; in 1967 to kill species of Aedes, Psorophora, Culex, and Anopheles in a 3-million acre flooded area in Texas; in 1969 in Ohio to kill Aedes vectors of LaCross encephalitis during an epidemic; in 1972 and 1974 in New England to kill species of Aedes, Coquillettidia, and Culiseta during an outbreak of eastern equine encephalitis; in 1975 in North Dakota and Minnesota to kill infected Culex tarsalis during an outbreak of western equine encephalitis; and in 1975 in Guam and Puerto Rico to control the vectors of dengue [U.S. Centers for Disease Control and Prevention (CDC), 2000]. With the dramatic appearance of epidemic WNV in the New York City area in 1999 and its subsequent spread to Canada and the eastern United States, ULV spraying of malathion was a frontline public health response to control the epidemic. B. R e e m e r g e n c e and R e s i s t a n c e of Vector-Borne Disease
The 1999 WNV emergence in the United States demonstrated that without sustained vector mosquito control in urban areas, even the world's most affluent cities are at risk for vectorborne disease. WNV, an Old World flavivirus related to St. Louis encephalitis virus and transmitted through mosquitoes, caused a serious outbreak (62 cases, with 7 deaths) and signaled the potential for similar outbreaks in the Western Hemisphere (Rose, 2001). This emergence or reemergence of vector-borne diseases thought to be under control was first observed in the 1970s in Asia and the Americas as rates of malaria and dengue began to climb (Gubler, 1998). The ease of travel and the increase in migrant laborers are two elements for the increased frequency in cases of dengue and malaria in the United States. Epidemic dengue was effectively controlled in the American region when the principal mosquito vector, Ae. aegypti, was controlled in the 1950s and 1960s. Unfortunately, the program was disbanded in the early 1970s
CHAPTER 40 9Public Health Impacts of OPs and CMs and the mosquito reinfested most countries of the region during the next 30 years (Gubler, 2001). According to Gubler (2001), global reports of dengue or dengue hemorrhagic fever were practically nonexistent in 1955. Data provided by decade show a dramatic increase during a 45-year span such that by 2000, more than 500,000 cases were reported. Each year, depending on the epidemic activity in the world, there are an estimated 50-100 million cases of dengue fever and several hundred thousand cases of the severe form of the disease, dengue hemorrhagic fever (Gubler, 2001). The resurgence of these diseases has been linked to demographic changes, societal changes, agricultural changes, and changes in public health practices. The use of insecticides has played a very important role in genetic changes in pathogens leading to increased epidemic potential. OP resistance has been recorded in all the major Culex vectors, which has ramifications for the control of WNV, Japanese encephalitis, and filariasis. OP and CM resistance occurs in the Aedes vector, which is the vector for dengue, dengue hemorrhagic fever, and yellow fever. The Simulium damnosum complex, vectors of onchocerciasis or "fiver blindness" have been under insecticide-based control in West Africa since 1974. Temephos (an OP insecticide) resistance prompted a switch to chlorphoxim, but resistance to this insecticide occurred within 1 year. Resistance in Simulium is currently being managed by a rotation of temephos, Bacillus thuringiensis, and permethrin (Hemingway, 2003). Investigations are under way in countries, provinces, and states to evaluate vector resistance to insecticides. A study in Brazil examined the resistance of the primary mosquito vector of dengue and yellow fever. Since 1967, public health programs have used mostly OPs in the control of Ae. aegypti. The mosquito vector was first eradicated from the country in 1955 following 8 years of a DDT application program. It was reintroduced into Brazil in 1967 as a DDT-resistant species, so temephos was used to eradicate it for a second time in 1973. The vector was reintroduced in 1976, and since then malathion and fenitrothion have been used for controls of adults concomitantly with the larvicide temephos (Lima et al., 2003). With the extensive use of temephos and malathion, dengue epidemics continue to emerge. The increasing insecticide resistance points to the need for new vector control strategies. Studies examining the resistance of Culex pipiens quinquefasciatus to propoxur, temephos, and chlorpyrifos in Martinique (Yebakima et al., 2004) and studies examining resistance of the same vector to malathion, chlorpyrifos, and other non-OP/CM insecticides in Alabama and Florida (Liu et al., 2004) have revealed important but not unanticipated results. In all cases, resistance and even cross-resistance to OP and CM insecticides and nonOP/CM insecticides were recorded in all species. Control of the reemergence of vector-borne disease and reversing the trend have become public health priorities. Controlling and preventing these diseases require strategies
601
including the application of larvicides and adult insecticides for mosquito control. For example, control of the main mosquito vector, Ae. aegypti, is currently the only option available for prevention and control of dengue, and it is carried out mainly by applying insecticides (generally OPs and CMs) to larval habitats, destroying unwanted containers, and educating the public. During epidemics, this is complemented by insecticide space spraying against adult mosquitoes (Corbel et al., 2004). Simultaneously, however, society is concerned with the effects of these chemicals on untargeted beneficial arthropods and vertebrates as well as their effects on mammals. Regulatory actions including the Food Quality and Protection Act of 1996 (FQPA) are intended to ensure that existing public health pesticide uses are not lost without economically effective alternatives. However, if FQPA results in cancellation of major agricultural uses of a pesticide that is also used in public health, it may become no longer profitable for the manufacturer to produce small quantifies for mosquito control, thus ending production of the pesticide (Rose, 2001). Reversing the trend of emergent/resurgent vector-borne disease is a major challenge. Vaccines are available for only a few vector-borne diseases (yellow fever, Japanese encephalitis, tick-borne encephalitis, tularemia, and plague) and are not widely used. Vaccine prospects for major vector-borne disease are not good. With the exception of malaria, few other vector-borne diseases have funding for vaccine research (Gubler, 1998). In the next decade, vector control will be required to interrupt transmission of most emergent/resurgent vector-borne diseases. Therefore, we must maintain the public health arsenal of effective, "safe" pesticides. This arsenal is likely to include a suite of traditional OP- and CM-based insecticides as well as newer pesticides and alternative approaches (e.g., biological control). Because adulticides used for mosquito control were registered decades ago, the data supporting their registrations may be insufficient to meet current requirements. None of the mosquito adulticides commonly used in the United States was developed recently; their registrations are up to 44 years old (Rose, 2001). Shrinking availability of insecticides as a result of resistance is exacerbated by removal from the market of insecticides no longer registered for public health use, especially in the past decade; the cost to keep certain compounds on the market is higher than can be recouped from such use. Local mosquito control programs would use an integrated program if resources were available, but often funding is limited to the extent that adulticiding trucks are the only means of mosquito intervention. Malathion, naled, and fenthion are examples of OP insecticides commonly used for adult mosquito control in the United States and for control of WNV (CDC, 2003a). Implementation of the public health pesticide provisions of FQPA must include comparative risk-benefit analyses of the sig~ficance of vector-borne disease impacts versus potential human and environmental toxic effects for pesticides used to control public health pests both
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in the United States and in other countries affected by EPA pesticide regulatory decisions (Rose, 2001).
III. CHILDREN'S EXPOSURES As discussed previously, pesticides are a part of the frontline public health defense :against vector-borne diseases such as malaria, dengue, WNV, and other major causes of infectious disease. OPs, CMs, and the broader group of pesticides assist in the control of food-borne and vector-borne diseases, which affect millions of children and adults and kill thousands annually in the United States (CDC, 2002; Gubler, 1998). Additionally, pesticide usage, including OPs and CMs, improves human nutrition through greater availability, greater crop diversity, and lower costs of food. The OP and CM insecticides are used to control insect pests that otherwise would lower crop yields significantly. In addition, OP/CM insecticides are inexpensive relative to many other pest control options. It has been argued that the benefits of OP/CM insecticide usage for crop pest control outweigh the countervailing risks to human health in large part due to the availability and affordability of nutritious fruits, vegetables, and grains that have been linked to chronic disease prevention (Taylor and Smith, 1999). Although there are benefits to the usage of OPs and CMs, there are also many public health dangers. Children reap the benefits from the use of OPs and CMs, but they also reap the negative consequences, often disproportionately compared to adults. Children's exposures to pesticides may be particularly increased because they have a tendency to explore their environments with their mouths and because their breathing zones are close to the ground where OP and CM residues accumulate, increasing inhalation to heavier-than-air toxicants and low-lying particulates. Children are susceptible populations in that they are still growing and developing (e.g., the brain grows rapidly during infancy and myelination is not completed until the second year of life). Children's vulnerabilities have fueled the public's escalating concerns about the large-scale use of pesticides (particularly AChE inhibitors, including OPs and CMs), resulting in the passage into law of FQPA, which requires the EPA to address risks to infants and children and requires pesticide tolerances to be safe, defined as "a reasonable certainty that no harm will result from aggregate exposure," including all exposure through the diet and other nonoccupational exposures, including drinking water, for which there is reliable information. As elements of their aggregate pesticide exposure, children's exposures will be explored by examining accidental poisonings, dietary exposures, indoor spraying, and agricultural exposures in the United States.
A. Accidental Poisonings In the United States, a total of 4.5 billion pounds of chemicals are applied annually as pesticides (Aspelin and Grube, 1999).
This total consists of 1.2 billion pounds of conventional pesticides (975 million pounds) and other pesticide chemicals, such as sulfur, wood preservatives, specialty biocides, and chlorine/hypochlorite compounds (Weiss et aL, 2004). OPs account for approximately half of the insecticides used in the United States, and CMs are widely used in homes and gardens (Weiss et al., 2004). The amount of OP insecticides used has declined nearly 45% since 1980, from an estimated 131 million pounds in 1980 to 73 million pounds in 2001. Since 1980, however, OP use as a percentage of total insecticide use has increased, from 58% in 1980 to 70% in 2001. The increase in use in 1999 was due mainly to the increased amount of malathion used as part of the U.S. Department of Agriculture-sponsored Boll Weevil Eradication Program. Malathion use in this program decreased during 2003 and 2004, resulting in a decline in total OP use. The estimates of OP insecticide use rely on the estimated amount used and changes in the amount of OPs used from public and proprietary EPA databases (Table 2). Since nearly 74 million households (or three-fourths of all U.S. households) use an estimated 76 million pounds of pesticides annually (Aspelin and Grube, 1999), the potential for children's exposure is high. Pesticides are often stored in relatively large quantities in garages, sheds, basements, barns, and other areas accessible to children. A study estimated that nearly half of all households with a child younger than 5 years old stored pesticide in an unlocked cabinet within reach of the child (Bass et al., 2001). Although these exposures are unintentional, they have significant immediate public health consequences. Accidental exposure to pesticides is a common cause of acute poisoning, particularly among young children. More than half (57%) of all reported pesticide poisonings in the United States occur in children younger than 6 years of age; this translates into approximately 50,000 children per year (Litovitz et al., 2002). Of these poisonings, at least 2000 are attributed to exposure to OPs and CMs (Watson et al., 2004). The number is potentially much greater due to cases reported in which the pesticide agent was not known. OPs are generally much more toxic than CMs. Unlike OPs, CMs do not irreversibly inhibit ACHE. Thus, their activity is quickly reversed after excretion of the pesticide. Acute, high-level exposure can result in immediate health effects potentially seen within minutes of exposure. The most frequent acute symptoms of OP poisoning in children include miosis, excessive salivation, nausea and vomiting, lethargy, muscle weakness, tachycardia, hyporeflexia and hypertonia, respiratory distress, and, in severe cases, death (Zwiener and Ginsburg, 1988). No studies have examined the long-term sequelae of acute pesticide poisoning in children.
B. Dietary Exposures Dietary exposures to OPs and CMs in children are a major pathway and main contributor to pesticide intake and body
CHAPTER 40 9Public Health Impacts of OPs and CMs TABLE 2.
603
Most Commonly Used OP Insecticide Active Ingredients, All Market Sectors J 2001 and 1999 Estimates a 2001
1999
Active ingredient
Rank
Range
Rank
Range
Malathion Chlorpyrifos Diazinon Terbufos Acephate Phorate Methyl parathion Phosmet Azinphos-methyl Dimethoate
1 2 3 4 5 6 7 8 9 10
23-32 11-16 4-7 3-5 2-3 2-3 1-3 1-2 1-2 1-2
1 2 4 3 7 6 5 9 8 10
30-38 13-19 4-7 5-7 2-3 2-3 2-4 1-2 1-2 1-2
Source: EPA estimates based on CroplifeAmerica annual surveys, USDA/NASS, and EPA proprietarydata aRanked by range in millions of pounds of active ingredient (www.epa.gov).
burden. In particular, dietary exposures to CMs are the primary route of exposure for children to this class of pesticide. Physiological differences between children and adults contribute to their increased risk of exposure to pesticides in food. As infants are weaned and progress to solid foods, they consume, per unit body weight, proportionally more fruit and more fruit juice than adults. Lu et al. (2005) determined the prevalence of dialkylphosphates (DAPs), which are metabolic products of most OP pesticides, in fruit juices. DAPs were found in both conventional and organic juices purchased from local grocery stores, and the original levels were higher, for both apple and orange juices, in conventional than in organic juices. The typical 1-year-old child drinks, per unit of body weight, 16 times as much apple juice as an adult (Wargo, 1998). The National Research Council (1993) reported that children's dietary exposures to pesticides differed from those of adults both quantitatively and qualitatively. The report noted that children experienced higher exposure to pesticides from agricultural crop residues because of their higher intake of fruits and vegetables, per body weight, relative to adults. The report estimated that 50% of lifetime pesticide exposure occurs during the first 5 years of fife. Fenske et al. (2002) conducted an assessment of OP exposures in the diets of children 2-5 years old in Washington State. Twenty-four-hour duplicate diet sampling was employed to examine the dietary OP exposures. Samples were collected from children living in the Seattle metropolitan area and from children living in counties in central Washington. A total of 88 food category samples were collected and analyzed for 15 OPs. Of the 15 targeted OPs, 6 were detected: azinphos-methyl, chlorpyfifos, malathion, methidathion, methyl parathion, and phosmet (Fenske et al., 2002). The fresh fruits and vegetables category had the most frequent pesticide determinations, followed by
beverages. No detections were above the legal tolerances for residues on produce; however, the acute population-adjusted reference dose for chlorpyrifos exposure of 1.7 ixg/kg/day was exceeded by one subject during one sampling event. This subject's cumulative daily dose of chlorpyrifos equivalents was estimated to be 2.5 lxg/kg/day. Since the family did not spray inside or outside the home, it was determined that his exposure was attributable to agricultural residues on food (Fenske et al., 2002). In 2003, the CDC published a national survey of human exposure to environmental chemicals based on laboratory analysis of blood and urine specimens obtained in 1999 and 2000 (CDC, 2003b). The report provides reference ranges for 116 chemicals, including selected pesticides, measured in a randomly selected subsample of participants in the National Health and Nutrition Examination Survey (NHANES), 6-59 years of age. Urine levels of dimethyl thiophosphate (a major metabolite of many OP pesticides) were approximately twice as high in children 6-11 years of age as in adults 20-59 years of age. Measurement of these metabolites in urine reflects exposure to OP pesticides that has occurred primarily in the past few days. The increased levels seen in children suggest that children in the United States have had higher levels of exposure to OPs than adults (Weiss et al., 2004). The long-term human health and children's health implications of this finding are not known, but further study is warranted. Exposure of the general population to CM pesticides occurs primarily from ingestion of food products or from residential use. In examining the CM metabolites also published in the CDC report, the levels of the carbaryl metabolite 1-naphthol seen in the 6- to 11-year age group were similar to those reported for Minnesota schoolchildren age 3-13 years (CDC, 2003b).
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FQPA explicitly requires the EPA to address risks to infants and children and to publish a specific safety finding before a pesticide tolerance can be established. It also provides for an additional safety factor (10-fold, unless reliable data show that a different factor will be safe) to ensure that tolerances are safe for infants and children and requires collection of better data on food consumption patterns, pesticide residue levels, and pesticide use. Diet is considered a significant source of aggregate pesticide exposure for children, and one aim of FQPA is to monitor pesticide exposure levels for children that are a result of agricultural residue from pesticide use and to adjust recommendations for pesticide use and/or registration in a manner that is protective of children's health. Although increased levels of OP metabolites were seen in children 6-11 years of age relative to levels in adults in the CDC cross-sectional study of the U.S. population, the meaning of this finding is not well understood, particularly as it relates to potential long-term health effects.
C. Indoor Spraying Exposures Recurrent broadcast spraying of pesticides in homes is another exposure pathway for children to OPs and CMs. Children's breathing zones are closer to the ground, where pesticide residues accumulate, increasing inhalation exposure to heavier-than-air toxicants and low-lying particulates. For example, higher chlorpyrifos concentrations have been demonstrated closer to the floor (25 vs 100 cm above the carpet) after the indoor broadcasting of Dursban (chlorpyrifos) (Fenske, 1997). Children are more likely to crawl around on the carpet and floors and to place their fingers in their mouths. Deposition of pesticides on toys and other objects has a greater impact on children's exposures because children frequently place objects in their mouths. Children are also less likely to wash their hands before eating, and they are less likely to use utensils when eating. Gurunathan et al. (1998) conducted a study examining residential treatment with broadcast spraying of chlorpyrifos. Peak levels on surfaces such as toys and furniture were measured 36 hr after the original application. The semivolatile pesticide accumulated on and in toys and other sorbant surfaces in a home via a two-phase physical process that continues for at least 2 weeks postapplication. The total dose of chlorpyrifos for a 3- to 6-year-old child whose home was treated depended on the frequency of that child's hand-to-mouth activity, but it was estimated to be 208 Ixg/ kg/day, which far exceeded the EPA's reference dose of 3 txg/ kg/day. The Gurunathan study examined the exposures from an indoor broadcast spray, but similar attention was given to a crack-and-crevice application of chlorpyrifos with very different results (Hore et al., 2005). The study was conducted to examine the distribution of chlorpyrifos within a home environment for 2 weeks after a routine professional crackand-crevice application and to determine the amount of the
chlorpyrifos that is absorbed by a 2- to 5-year-old child living within the home. The daily amount of chlorpyrifos estimated to be absorbed by the children postapplication ranged from 0.04 to 4.8 Ixg/kg/day. Comparison of results from the Hore et al. study and the Gurunathan et al. study suggests that selection of the application method will greatly influence the children's exposure and dose received from pesticides applied indoors. Another study assessed the exposure to OP pesticides by biomonitoring in epidemiologic studies of birth outcomes (Needham, 2005). The author evaluated three different exposure studies that had been conducted in California and New York. The New York studies (Berkowitz et al., 2004; Whyatt et al., 2004) involved recurrent indoor broadcast sprayings, primarily of chlorpyrifos. In the California study (Eskenazi et al., 2004), the primary exposure focus was agricultural applications of several pesticides, pesticide drift from the spraying, and the "bringing home" of the pesticides on workers' clothing; however, pesticides were also potentially used indoors, for landscape maintenance, for insect control, and for prevention of structural damage. The studies examined the relationship between pregnant women's exposures to OPs, particularly chlorpyrifos, and birth outcomes. Chlorpyrifos or its metabolite, 3,5,6-trichloro-2pyrinidol, were detected and measured in umbilical cord and maternal blood samples and in urine. Although the birth outcomes from these exposures were not identical across the studies, significant relations reported between their exposure assessments and birth outcomes included decreased birth size (Whyatt e t a l . , 2004), decreased head circumference with levels of paraoxonase-1 activity as a modifier (Berkowitz et al., 2004), and decreased gestational age at birth (Eskenazi et al., 2004). Although the birth outcomes may not be identical across the studies and may be difficult to interpret, clearly the application of indoor pesticides contributes to fetal and children's exposures to pesticides (including OPs), such that pregnant women should also be considered an at-risk population.
D. Exposures of Children of Agricultural Workers Children of agricultural families are very likely to be exposed to pesticides, even if they are not actively involved in farm activities. This same exposure risk applies to children of migrant farmworkers who are at increased risk of exposure to OPs because of "carry-home" transport processes and residential location (Lu et al., 2000). Simcox et al. (1995) studied 59 families in the Yakima Valley and compared levels of four OP pesticides in the homes of hired farmworkers, families residing on farms, and nonagricultural families. Chlorpyrifos was detected in 95% of the homes. House dust concentrations were consistently higher for agricultural families than for nonagricultural families, and pesticide applicators tended to have higher house dust concentrations compared to
CHAPTER 40
nonapplicators. There was a threefold difference in median chlorpyrifos house dust concentration between farmworkers who did not directly handle pesticides and reference families of non-farmworkers living in agricultural communities (median, 172 ng/g for farmworkers vs 53 ng/g for nonagricultural families). Bradman et al. (1997) conducted a pilot study of pesticide exposures to children of migrant farmworkers and nonfarmworkers living in California. Floor dust samples and child hand wipes were collected from the homes of 10 families, 5 of which had at least one resident farmworker. Higher levels of the OPs diazinon, chlorpyrifos, and malathion were found in house dust in farmworker homes. Residues of diazinon and chlorpyrifos were detected on the hands of two and three farmworker toddlers, respectively, who also lived in the homes with the highest dust concentrations. Overall findings from studies suggest that inadvertent carry-homes of occupational pesticides are occurring and that contamination in the homes of farm families is likely to be higher than in other homes. Furthermore, a significant source of exposure to farmworker families may derive from their residential proximity to fields (Eskenazi et al., 1999). As of 1999, studies of the effects of pesticide exposure on children's health were limited to those of birth defects, childhood cancer, and acute poisoning following~ingestion. Some case-control studies have associated parental exposure to pesticides or pesticide use in the home with childhood brain tumors, leukemia and lymphomas, testicular cancers, and other cancers. Other studies have reported that parental exposure to pesticides or application of pesticides in the home are associated with certain birth defects, including neural tube and other defects (Eskenazi et al., 1999). Evaluations of OP and CM exposures, levels of exposures, and health impacts to children are needed.
IV. C O N C L U S I O N S The public health impacts of OPs and CMs are historic and of current and future importance. They embody the frontline public health response for prevention and control of outbreaks of vector-borne diseases. Their applications have afforded society to enjoy a wide range of foods in large quantities at low costs. The nutritional benefits received from crop diversity are numerous. However, their use is somewhat of a "double-edged sword." At the same time the use of these chemicals is preventing disease, it is also promoting the mutation of vectors such that new diseases and reemergences of diseases once nearly eradicated are at epidemic levels in many tropical and subtropical regions of the world. At the same time they are used to protect public health from disease, they have been associated with undesirable chronic health effects in adult populations and developmental effects in children. At the same time their use provides diverse, low-cost food, it also taints breast milk and
9Public Health Impacts of OPs and CMs
605
foods commonly consumed by children. With the implementation of FQPA, regulatory and public health agencies will be charged with maintaining a delicate balance for use of OPs and CMs in the broader pesticide category. It will be necessary to address aggregate exposures and children's health and simultaneously maintain a cache of pesticides that are effective in the prevention and control of disease.
References Aspelin, A. L., and Grube, A. H. (1999). Pesticides Industry Sales and Usage: 1996 and 1997 Market Estimates. U.S. Environmental Protection Agency, Office of Pesticide Programs, Washington, DC. Bass, J. K., Ortega, L., Rosales, C., Petersen, N. J., and Philen, R. M. (2001). What's being used at home: A household pesticide survey. Pan Am. J. Public Health 9, 138-144. Bradman, M. A., Hamly, M. E., Draper, W., Seidel, S., Teran, S., Wakeham, D., and Neutra, R. (1997). Pesticide exposures to children from California's Central Valley: Results of pilot study. J. Exposure Anal. Environ. Epidemiol. 7, 217-234. Berkowitz, G. S., Wetmur, J. G., Birman-Deych, E., Obel, J., Lapinski, R. H., Godbold, J. H., Holzman, I. R., and Wolff, M. S. (2004). In utero pesticide exposure, maternal paraoxonase activity, and head circumference. Environ. Health Perspect. 112, 388-391. Chavasse, D. C., and Vap, H. H. (Eds.). (1997). Chemical Methods for the Control of Vectors and Pests of Public Health Importance, Document WHO/CTD/WHOPES/97.2. World
Health Organization, Geneva. Corbel, V., Duchon, S., Zaim, M., and Hougard, J.-M. (2004). Dinotefuran: A potential neonicotinoid insecticide against resistant mosquitoes. J. Med. Entomol. 41, 712-717. Ecobichon, D. J. (2001). Toxic effects of pesticides. In Casarett and Doull's Toxicology: The Basic Science of Poisons (C. D. Klassen, Ed.), 6th ed., pp. 763-810. McGraw-Hill, New York. Eskenazi, B., Bradman, A., and Castorina, R. (1999). Exposure of children to organophosphate pesticides and their potential adverse health effects. Environ. Health Perspect. 107, 409-419. Eskenazi, B., Harley, K., Bradman, A., Weltzien, E., Jewell, N. E, Barr, D. B., Furlong, C. E., and Holland, N. T. (2004). Association of in utero organophophate pesticide exposure and fetal growth and length of gestation in an agricultural population. Environ. Health Perspect. 112, 1116-1124. Fenske, R. (1997). Pesticide exposure assessment of workers and families. Occup. Med. 12, 221-237. Fenske, R. A., Kedan, G., Lu, C., Fisker-Andersen, J. A., and Curl, C. L. (2002). Asssessment of organophosphorous pesticide exposures in the diets of preschool children in Washington State. J. Exposure Anal. Environ. Epidemiol. 12, 21-28. Gubler, D. J. (1991). Insects in disease transmission. In Hunter Tropical Medicine (G. T. Strickland, Ed.), 7th ed., pp. 981-1000. Saunders, Philadelphia. Gubler, D. J. (1998). Resurgent vector-borne diseases as a global health problem. Emerging Infect. Dis. 4, 442-450. Gubler, D. J. (2001). Human arbovirus infections worldwide. Ann. N. Y. Acad. Sci. 951, 13-24. Gurunathan, S., Robson, M., Freeman, N., Buckley, B., Roy, A., Meyer, R., Bukowski, J., and Lioy, E J. (1998). Accumulation
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of chlorpyrifos on residential surfaces and toys accessible to children. Environ. Health Perspect. 106, 9-16. Hemingway, J. (2003). Insecticide resistance in insect vectors of human disease. In The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health Strategies for ContainmentmWorkshop Summary, pp. 79-106. National Academies Press, Washington, DC. Hore, P., Robson, M., Freeman, N., Zhang, J., Wartenberg, D., Ozkaynak, H., Tulve, N., Sheldon, L., Needham, L., Barr, D., and Lioy, P. J. (2005). Chlorpyrifos accumulation patterns for child-accessible surfaces and objects and urinary metabolite excretion by children for 2 weeks after crack-and-crevice application. Environ. Health Perspect. 113, 211-219. Lima, J. B. P., Da-Cunha, M. P., Da Silva, R. C., Jr., Galardo, A. K. R., Stares, S. D. S., Braga, I. A., Ramos, R. P., and Valle, D. (2003). Resistance of Aedes aegypti to organophosphates in several municipalities in the state of Rio de Janeiro and Espirito Santo, Brazil. Am. J. Trop. Med. Hyg. 68, 329-333. Litovitz, T. L., Klein-Schwartz, W., Rodgers, G. C., Jr., Cobaugh, D. J., Youniss, J., Omslaer, J. C., May, M. E., Woolf, A. D., and Benson, B. E. (2002). 2001 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am. J. Emerg. Med. 20, 391-401. Liu, H., Cupp, E. W., Micher, K. M., Gut, A., and Liu, N. (2004). Insecticide resistance and cross-resistance in Alabama and Florida strains of Culex quinquefaciatus. J. Med. Entol. 41, 408-4 13. Lu, C., Fenske, R. A., Simcox, N. J., and Kalman, D. (2000). Pesticide exposure of children in an agricultural community: Evidence of household proximity to farmland and take home exposure pathways. Environ. Res. A 84, 290-302. Lu, C., Bravo, R., Caltabiano, L. M., Irish, R. M., Weerasekera, G., and Barr, D. B. (2005). The presence of dialkylphosphates in fresh fruit juices: Implication for organophosphorus pesticide exposure and risk assessments. J. Toxicol. Environ. Health A 68, 209-227. National Research Council (1993). Pesticides in the Diets of Infants and Children. National Academy Press, Washington, DC. Needham, L. (2005). Assessing exposure to organophosphorous pesticides by biomonitoring in epidemiologic studies of birth outcomes. Environ. Health Perspecr 113, 494-498. Rose, R. I. (2001). Pesticides in public health: Integrated methods of mosquito management. Emerging Infect. Dis. 7, 17-23. Simcox, N. J., Fenske, R. A., Wolz, S. A., Lee, I. C., and Kalman, D. A. (1995). Pesticides in household dust and soil: Exposure pathways for children of agricultural families. Environ. Health Perspecr 103, 1126-1134.
Taylor, C. R., and Smith, H. A. (1999). Aggregate economic evaluation of banning organophosphate and carbamate pesticides, AFPC Policy Research Report 99-15. Agricultural and Food Policy Center, Texas A&M University, College Station, TX. U.S. Centers for Disease Control and Prevention (2000). Arboviral Encephalitis Cases Reported in Humans, by Type, United States, 1964-2000. U.S. Centers for Disease Control and Prevention, Atlanta. Available at www.cdc.gov/ncidod/dvbid/arbor/arbocase.htm. (accessed April 2, 2005). U.S. Centers for Disease Control and Prevention (2002). Disease Information: Food-Borne Illness Technical Information. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National center for Infectious Diseases, Division of Bacterial and Mycotic Diseases, Atlanta. Available at www.cdc.gov (accessed April 2, 2005). U.S. Centers for Disease Control and Prevention (2003a). Epidemic/Epizootic West Nile Virus in the United States: Revised Guidelines for Surveillance, Prevention, and Control U.S. Centers for Disease Control and Prevention, Atlanta. Available at www.cdc.gov (accessed April 2, 2005). U.S. Centers for Disease Control and Prevention (2003b). Second National Report on Human Exposure to Environmental Chemicals. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta. Available at www.cdc.gov (accessed April 2, 2005). U.S. Environmental Protection Agency, Office of Pesticide Programs (2002). FY 2002 Annual Report. U.S. Environmental Protection Agency, Washington, DC. Available at www.epa.gov (accessed April 2, 2005). Wargo, J. (1998). Our Children's Toxic Legacy: How Science and Law Fail to Protect Us from Pesticides, 2nd ed. Yale Univ. Press, New Haven, CT. Watson, W. A., Litovitz, T. L., Klein-Schwartz, W., Rodgers, G. C., Jr., Reid, N., Rouse, W. G., Rembert, R. S., and Borys, D. (2004). 2003 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am. J. Emerg. Med. 22, 335-404. Weiss, B., Amler, S., and Amler, R. W. (2004). Pesticides. Pediatrics 113, 1030-1036. Whyatt, R. M., Rauh, V., Barr, D. B., Camann, D. E., Andrews, H. E, Garfinkel, R., Hoepner, L. A., Diaz, D., Dietrich, J., Reyes, A., Tang, D., Kinney, P. L., and Perera, E P. (2004). Prenatal insecticide exposures, birth weight and length among an urban minority cohort. Environ. Health Perspecr 112, 1125-1132. Yebakima, A., Marquine, M., Rosine, J., Yp-Tcha, M. M., and Pasteur, N. (2004). Evolution of resistance under insecticide selection pressure in Culex pipiens quinquefasciatus (Diptera, Culcidae) from Martinique. J. Med. Entomol. 41, 718-725. Zwiener, R. J., and Ginsburg, C. M. (1988). Organophosphate and carbamate poisoning in infants and children. Pediatrics 81, 121-126.
CHAPTER 4
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Cumulative Effects of O r g a n o p h o s p h o r u s or Carbamate qr
Pesticides
STEPHANIE PADILLA U.S. Environmental Protection Agenc); Research Triangle Park, North Carolina
pesticide mixtures, and mixtures of OP and CM pesticides; and (iii)address future research priorities. Also, this chapter considers the N-methyl CMs or the cholinesteraseinhibiting CMs and not the thio- or dithiocarbamates, which have other mechanisms of toxicity. Therefore, only OP and CM pesticides that inhibit cholinesterase are considered. Very few investigators have ventured into the realm of mixture experiments during the past 40 years. These types of studies can be complicated and time-consuming. The overall design of these studies is usually one of two types: those that determine the toxicity of a given mixture or those that determine if the interaction of two or more OP or CM pesticides is synergistic (i.e., if the toxicity of a mixture is more toxic than the sum of the toxicity of the individual pesticides that make up the mixture).
I. I N T R O D U C T I O N Although there is no doubt that humans are exposed to low doses of mixtures of pesticides (Castorina et al., 2003; Duggan et al., 2003; Fenske et aL, 2002; S~inchez-Pefia et al., 2004), the question is whether mixtures are more toxic than the sum of the toxicity for each pesticide alone. In the literature describing the toxicity of mixtures of organophosphorus (OP) or carbamate (CM) pesticides, concern is expressed that exposure to mixtures of pesticides may be much more toxic than the toxicity of the individual pesticides. This concern about potentiated toxicity is all the more worrisome because data are lacking on the toxicity of pesticide mixtures, whether OP or CM, at less than lethal dosages. There is such scarcity of data, and the data are so disparate, that a science-based conclusion on the toxicity of OP or CM pesticide mixtures is not possible. There are some excellent review articles on various aspects of the toxicology of mixtures of chemicals (Calabrese, 1995; Krishnan and Brodeur, 1991), pesticides (Iyaniwura, 1990; Murphy, 1980), or OP compounds (Cohen, 1984; DuBois, 1961; Murphy, 1969, 1980). Many of these reviews, although not recent, provide a comprehensive overview of the possible mechanisms underlying interactions of chemicals in a mixture and, specifically, interactions of OP compounds in a mixture. This chapter does not provide an in-depth discussion of the mechanisms underlying interactions of OP or CM pesticides in a mixture. Rather, this chapter is designed to (i) address issues of experiment design of mixture studies; (ii) summarize the available literature on OP pesticide mixtures, CM
II. D E F I N E D M I X T U R E S Some investigators have chosen to study defined mixtures. The intention was not to determine the pattern of interaction of the OP or CM compounds in the mixture but to characterize the toxicity of a given mixture. For example, a basic adult rat neurotoxicity study was conducted using a 1:1 ratio of two cholinesterase-inhibiting metabolites of the N-methyl CM pesticide aldicarb: aldicarb sulfoxide and aldicarb sulfone (DePass et al., 1985). This study defined the dosage level that precipitated plasma, erythrocyte, or brain cholinesterase inhibition. The authors made no effort to assess the toxicity of either metabolite alone but were solely interested in the toxicity of the mixture. Another study used a mixture of the 15 most commonly used pesticides in Italy (not all were O P or CM compounds) to determine if that mixture was capable of precipitating adverse genetic effects in human lymphocytes (Dolara et al., 1994). It was found that the inclusion of benomyl in
*The views expressed are those of the author and do not necessarily reflect the views and policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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the mixture increased its toxicity markedly, leading the authors to suggest that removal of benomyl could reduce the potential for human toxicity due to pesticide contaminants in food. In related studies of mixtures based on probable human exposure (Ito et al., 1995a,b), rats were fed a mixture of either lx or 10x the Acceptable Daily Intake (ADI) of 10 OPs plus 1 organochlorine pesticide in an effort to determine if the mixture showed increased toxicity beyond the regulated levels. The authors were concerned with possible carcinogenic effects of a mixture of commonly used pesticides and therefore toxicity was defined as liver preneoplastic lesions. No lesions were found in the animals fed the pesticide mixture at lx the ADI, but the mixture at 100x the ADI did precipitate lesions. The authors concluded that humans were protected from possible carcinogenicity by current regulated pesticide exposure levels. In another study, the concentration of OP and CM pesticides in rainwater correlated well with the cholinesterase-inhibiting potency of the rainwater (Hamers et al., 2001). Because the in vitro anticholinesterase potency of the rainwater sample was directly related to its pesticide profile, it is very unlikely that the interaction of the pesticides in the mixture was more than additive. Although this is not a whole animal study, this study is especially relevant because the pesticide levels in the mixture were low and the end point, cholinesterase inhibition, was more sensitive than the commonly used lethal end points. Another important aspect of studies on defined mixtures involves interaction of the impurities with the pesticide in a formulation. One of the most well-known and wellstudied examples is the malathion epidemic poisoning of malaria pesticide appliers in Pakistan (Baker et al., 1978; Krishnan and Brodeur, 1994). Ordinarily, malathion has very low mammalian toxicity, but in this instance, many workers applying the pesticide became gravely ill and some died. The culprit was assumed to be higher than normal levels of a breakdown product of malathion, isomalathion, in the formulations used by the sprayers. In subsequent years, many investigators studied the toxicity of "contaminated" formulations (Aldridge et al., 1979; Toia et al., 1980; Umetsu et al., 1977, 1981). Two laboratories (Aldridge et al., 1979; Umetsu et al., 1977) confirmed potentiation of malathion toxicity by isomalathion and identified other contaminants that may also potentiate malathion toxicity. Although malathion tends to become more toxic if it is stored in a manner that promotes the production of isomalathion or trimethyl phosphorothioates, this is not the case for impurities found in other OP compounds, such as acephate (Umetsu et al., 1977) or fenthion (Toia et al., 1980). Some investigators have also studied the interaction of the pesticide with the other compounds found in pesticide formulations. For example, in a study comparing the
toxicity of a fenitrothion formulation (fenitrothion + emulsifier + cosolvent) with fenitrothion alone, Durham and coworkers (1982) found that the formulation did not appear to affect the toxicity of fenitrothion. In a more recent study using depressed neurite outgrowth in neuroblastoma cells as a measure of toxicity, some solvents found in commercial formulations appeared to potentiate the toxicity of chlorpyrifos (Axelrad et al., 2002). Obviously, the interaction potential for the formulation with a pesticide depends on the formulation and the pesticide because no consistent pattern can be ascertained from the scarce data.
lII. D E S I G N O F I N T E R A C T I O N S T U D I E S OP and N-methyl CM pesticides inhibit cholinesterase activity. It is assumed that the primary mechanism of toxicity is inhibition of cholinesterase activity, and this represents a common mechanism for grouping these pesticides for risk assessment (Mileson et al., 1998). There is no doubt, however, that these pesticides, especially the OP pesticides, have other mechanisms of toxicity (Reviewed in Pope, 1999). Nevertheless, for the purpose of assessing mixtures of these pesticides, the most reasonable, initial approach is to consider them as a "homergic" mixture (Wessinger, 1986; Woolverton, 1987) that is, having the same mechanism of toxicity. The appropriate manner to test for interactions of mixtures of homergic chemicals is to use a dose-additive (similar joint action or concentration addition) experimental design (Bliss, 1939; Plaa and V6zina, 1990" Wessinger, 1986; Wessinger and Evans, 1988; Woolverton, 1987). A dose-additive design assumes that the chemicals in the mixture have the same mode of toxicity and contribute to the same toxic response. An effect-additive model, on the other hand, assumes that the chemicals have different modes of toxicity and produce different physiological responses (Bliss, 1939; Plaa and V6zina, 1990; Wessinger, 1986; Wessinger and Evans, 1988; Woolverton, 1987). Unfortunately, only a small proportion of studies on mixtures of OP or CM pesticides appear to have used the appropriate, dose-additive experimental design. Most studies have instead used an effect-additive design (independent joint action and response-additive design). To illustrate the difference, consider the following example of a binary mixture of two chemicals, A and B" The LD25 for A is 25 mg/kg and that for B is 2.5 mg/kg. A doseadditive model would combine one-half of the LD25 dose of each chemical (i.e., 12.5 mg/kg of A + 1.25 mg/kg of B) and ask if the response of the animals to the mixture was an LDe5 (additive interaction), less than an LD25 (less-thanadditive interaction), or more than an LDe5 (more-thanadditive interaction). On the other hand, an effect-additive model would combine the LD25 dose of each (i.e.,
CHAPTER 41 25 mg/kg of A and 2.5 mg of B) and ask if the response of the mixture produced an LDs0. Of course, this is a simple example because compounds can be combined using either model in different ratios depending on their toxicity. An illustration of a dose-addition design for a binary mixture of OP compounds is shown in Fig. 1. Seume and O'Brien (1960b) characterized the interaction of O-ethyl O-p-nitrophenyl phenylphosphonothionate (EPN) and O,O-dimethyl S-(N-ethylcarbamoylmethyl) phosphorodithioate (CL 18706). After determining the LD10 in mice for the individual compounds, they created five mixtures of the two compounds using different proportions of the LD10 for each compound. If the interaction had been additive across all the mixtures, then each of the bars in Fig. 1 would have hovered around the 10% mark. Instead, most of the mixtures produced a marked morethan-additive interaction, with 80-100% of the animals dying. Only the 5% EPN + 95% CL 18706 showed the expected 10% lethality. These results illustrate two important points: There is a marked toxic interaction between these two OP compounds, and the toxicity profile is directly related to the proportion of each in the mixture. Other investigators working with either pesticides or nonpesticide chemicals have also noted that the ratio of the components in the mixture can influence the type of interaction (DuBois, 1961; Gessner, 1974; Gessner and Cabana, 1970; Wessinger, 1986). Unfortunately, remarkably few investigations have altered the pesticide proportions in mixtures. In summary, the study of the interaction of OP or CM pesticides should employ a dose-additiye experimental
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design varying ratios of the components to fully characterize the interaction profile.
IV. MIXTURES O F O R G A N O P H O S P H O R U S PESTICIDES One of the defining historical moments in pesticide mixture research occurred in 1957 when Frawley and coworkers showed the marked potentiation of malathion toxicity by EPN. When nonlethal dosages of both compounds were given to rats or dogs, 100% mortality was noted. These observations spurred a flurry of studies. The first group of studies identified one of the primary mechanisms underlying the potentiation of the malathion toxicity by EPN (Murphy and DuBois, 1957; Seume and O'Brien, 1960a): EPN inhibited the hydrolysis of malathion by carboxylesterases (CarbEs). Other investigators showed a potentiation between EPN and other OP compounds (Rosenberg and Coon, 1958; Seume and O'Brien, 1960b). Soon, these studies were followed by studies showing a more-than-additive interaction between other OP compounds that were potent inhibitors of CarbE activity, such as tri-o-cresylphosphate combined with malathion (Cohen and Murphy, 1971b; Lauwerys and Murphy, 1969; Murphy et aL, 1959; Polak and Cohen, 1969). In 1963, 112 OP compounds were assessed for their ability to lower the LDs0 of malathion in mice (Casida et al., 1963). Wide variation was obtained in the degree to which the compounds potentiated malathion toxicity; potentiation
EPN: % of Total Mixture CL18706: % of Total Mixture 100 90 80 -9 1= o
70 60 5O
o~ 4o 3o 20 10 0
Ep.oo.: 1 20.000 0o 1 23, i 33.000 000 1,2,01 0 00 1 156.750 0,2, t 165.000 0000 t 8.250 82.500 132.000
13L18706 Dose:
FIG. 1. Dose-additiveexperimental design exploring the interaction of two OP pesticides in female mice. Initially, an individual LD10 (ip) was determined for EPN and CL18706. The LD10 for EPN was 2.5 Ixg/g body weight and that for CL18706 was 165 ~g/g. The graph shows the results of the various mixtures of the two OP compounds, each as a percentage of its respective LD10. If the chemicals showed a purely additive interaction, then each of the mixtures should have shown lethality of 10%, no matter what the mixing ratio. The two compounds alone (solid black bar on left and solid gray bar on fight) produced only 10% lethality, but when the two were mixed, no matter what the ratio, 80-100% lethality was noted--a more-than-additive interaction. The only exception to this was the 5% EPN + 95% CL 18706, for which 10% lethality was noted. (Adapted from Table 4 of Seume and O'Brien, 1960b).
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varied considerably not only by class of compounds but also within a class of compounds. Further research has shown that CarbE inhibition does not totally explain the potentiation of OP toxicity by either EPN (Cohen and Murphy, 1971a) or tri-o-cresylphosphate (Lauwerys and Murphy, 1969; Polak and Cohen, 1969). It is thought that the potentiating chemical may interfere with the binding of the OP compound to nonspecific binding sites in nonvital tissues (Lauwerys and Murphy, 1969; Polak and Cohen, 1969) or that the potentiating compound may have other mechanisms for potentiation in addition to CarbE inhibition (Cohen and Murphy, 1971a). In addition, it has also been demonstrated that preexposure of rats to a CarbE inhibitor (iso-OMPA) also potentiated the toxicity of some CM pesticides (carbofuran, propoxur, or aldicarb) but not the toxicity of other CM compounds (physostigmine) (Gupta and Dettbam, 1993). Beyond the studies of CarbE inhibitors and potentiation of OP compounds, there are basic studies of the interaction of OP pesticides. Only a few of these studies used a dose-additive experimental design. One of the earliest and most extensive studies was described in DuBois's (1961) review that investigated 43 pairs of OP pesticides using a dose-additive design and lethality in female rats as an end point. Of the 43 pairs, 21 showed the expected additive interaction, 18 showed a less-than-additive interaction, and only 4 pairs (3 of the pairs contained malathion) showed a more-than-additive interaction [malathion + EPN, malathion + dipterex (Dylox; trichlorfon; O,O-dimethyl 1-hydroxyl-2,2,2-trichloroethylphosphonate), dipterex + Guthion (O, O-dimethyl S- [4-oxo- 1,2,3-benzotriazin3(4H)-ylmethyl] phosphorodithioate), and malathion + Co-Ral (O,O-dimethylO-[(1-methyl-2-methoxycarbonyl) vinyl] phosphate (oL-2-carbomethoxy-1-methylvinyl dimethyl phosphate)]. This study was followed by one of similar experimental design, except the end point was lethality in birds rather than rats (Kreitzer and Spann, 1973). Again, a more-than-additive interaction was found for the malathion + EPN and the malathion + trichlorfon mixtures; however, the malathion + Co-Ral and the trichlorfon + Guthion mixtures did not show a markedly more-thanadditive interaction as they had in rats. The phosphamidon + fenthion and malathion + parathion mixtures did not appear to show a substantial more-than-additive interaction. In another study using a dose-additive experimental design exploring the effects of pesticide mixtures on rainbowtrout lethality, the trichlorfon + Volaton [oL-([(diethyloxyphosphinothioyl)-ox]-imino) benzeneacetonitrile] produced only an additive interaction, whereas the trichlorfon + Guthion mixture may have produced a slightly more-than-additive interaction (Marking and Mauck, 1975). A dose-additive study of the mutagenic effects of mixtures of trichlorfon + methyl parathion or malathion or methyl azinphos in mice revealed that most end points were affected in a purely additive manner, with no potentiation noted (Degraeve et al., 1985).
In the studies that assessed the interaction of two OP compounds using a dose-additive design, the majority used lethality as an end point. Even when assessing that artificial, high-dose situation, many combinations did not produce a more-than-additive interaction. In fact, the majority of the binary mixtures of OP compounds produced a less-than-additive or additive interaction (DuBois, 1961). Only a few pairs of OP compounds definitively potentiated one another, such as malathion + EPN (Cohen and Murphy, 1971a; DuBois, 1961; Frawley et al., 1957; Kreitzer and Spann, 1973; Murphy and DuBois, 1957; Seume and O'Brien, 1960a)or malathion + trichlorfon (DuBois, 1961; Kreitzer and Spann, 1973), and the majority of other binary mixtures did not show definitive potentiation in these high-dose situations. A recent study of Moser & coworkers (2005) Moser et al. (2005), using a dose-additive design with mixtures of five commonly used OP pesticides (chlorpyrifos, diazinon, dimethoate, acephate, and malathion), showed a more-thanadditive interaction on multiple end points: blood and brain cholinesterase inhibition, motor activity, and gait score (tailpinch response did not show a more-than-additive interaction). This study is noteworthy because (i) relatively sensitive end points were used to test the toxic interaction of the OP pesticides, such as cholinesterase inhibition or depression of motor activity; (ii) more than two OP compounds were used in the mixture; and (iii) comprehensive statistical analyses of the data were performed. The pharmacokinetic interaction of two of the compounds in the mixture, chlorpyrifos and diazinon, has been studied in rats (Timchalk et al., 2004). The authors found that one compound did not affect the pharmacokinetics of the other unless high doses were given, concluding that a more-than-additive interaction is unlikely at environmentally relevant concentrations. In summary, there is some evidence that high doses of some but not all binary combinations of OP compounds show a more-than-additive interaction. In the one study using lower dosages of multiple OP compounds (Moser et al., 2005), a more-than-additive interaction was noted for multiple end points at low dosages.
V. M I X T U R E S O F C A R B A M A T E P E S T I C I D E S A perusal of the literature reveals virtually no studies of mixtures of CM pesticides in mammals. An in vitro study investigated the interaction of CM pesticides using an effectadditive model (Kok and Hasirci, 2004). In this investigation, an acetylcholinesterase biosensor was used to measure the cholinesterase-inhibiting potency of three single CM pesticides (aldicarb, carbaryl, and carbofuran) as well as two binary mixtures (aldicarb + carbofuran and aldicarb + carbaryl). The mixtures showed less cholinesterase inhibition than was predicted from the single compounds; it was unclear, however, what statistical comparison was used to reach this conclusion.
CHAPTER 41
VI. MIXTURES OF O R G A N O P H O S P H O R U S COMPOUNDS AND N-METHYL CARBAMATES Perhaps the most well-known CM/OP interaction studied in both laboratory animals and humans is CM protection against subsequent challenge with a nerve agent (Koster, 1946; reviewed in Bajar 2004; Gunderson et al., 1992; Lee, 1997). Normally, the prophylactic CM is the peripherally acting pyridostigmine; however, in an interesting study comparing the protective efficacy of pyridostigmine, mobam, physostigmine, and carbofuran, all four were able to protect guinea pigs against poisoning by sarin, tabun, or VX (Gordon et al., 1978). Pyridostigmine was however preferred because its protective effects were longer lasting. The generally accepted mechanism of this protection is based on the assumption that the CM reaches the cholinesterase molecule first and carbamylates the active site. This carbamylation does inhibit enzyme activity, but only for a matter of minutes to hours. Most important, this temporary occupation of the active site of the cholinesterase by the CM prevents the irreversible binding of the nerve agent to the active site. In time, the bond between the CM and the cholinesterase breaks and the cholinesterase activity is regenerated. If however, the CM had not occupied the active site of the cholinesterase, the nerve agent, which is much more reactive and potent than the CM, would have phosphorylated the active site of the cholinesterase, permanently inhibiting the enzyme activity for days. The temporary inhibition of cholinesterase by the CM is preferable to the permanent inhibition by the OP nerve agent. Beyond the nerve agent literature, there are few studies on the interaction of OP and CM compounds and these have produced mixed results. In one series of studies, pretreatment with fenthion (Miyaoka et al., 1984) or fenitrothion (Takahashi et al., 1984; Tsuda et al., 1984) markedly lowered the LD50 of a subsequent challenge with an N-methyl CM (BPMC; 2-sec-butylphenyl methylcarbamate) in mice. The increased lethality precipitated by pretreatment with the OP pesticides was correlated with an increase in the plasma concentration of BPMC (Miyaoka et al., 1984; Takahashi et al., 1984; Tsuda et al., 1984). A subsequent study (Takahashi et al., 1987), extending the work to various OP compounds (cyanophos, fenitrothion, malathion, and dichlorvos) and other structurally similar N-methyl CMs, found that cyanophos, fenitrothion, and malathion, but not dichlorvos, potentiated the toxicity of the N-methyl CMs and that the increase in plasma concentration of the N-methyl CM did not fully explain the potentiation by the OP compounds. An early investigation using a dose-additive design compared the expected to the actual LDs0 of a series of binary combinations of various pesticides in rats. (Keplinger and Deichmann, 1967). In the study, the expected:actual ratio for the carbaryl + malathion combination was 1.82, for the carbaryl + parathion combination it was 1.58, and for
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the carbaryl + diazinon combination it was 1.30. Because the actual LDs0 was less than expected, the results indicate a more-than-additive interaction between the N-methyl CM and OP pesticide. Two other studies investigated the carbaryl + malathion mixture with different results. Using lethality in female houseflies, the carbaryl + malathion mixture was reported to show a slight antagonism (less-than-additive interaction) (Kulkarni, 1976). A carbaryl + malathion mixture was also tested using teratological end points in rats (Lechner and Abdel-Rahman, 1984). The authors used an effectadditive design, so it is difficult to attribute interaction, but the effects of the mixture did not appear to differ markedly from the high dose of either compound alone. Thus, it appears that carbaryl + malathion mixtures produce various types of interactions, depending on the end point and the species. In an early in vitro investigation using human plasma or erythrocyte cholinesterase inhibition, the carbaryl + dichlorvos or physostigmine + dichlorvos combination produced slightly less cholinesterase inhibition than was expected from the individual IC50 curves (Carter and Maddux, 1974). Using fish mortality as an end point, carbaryl + methyl or ethyl parathion both exhibited an additive interaction pattern (Macek, 1975). Unfortunately, in both of the latter studies, it is difficult to determine whether the experimental design was dose- or effect-additive. In a study that did use a dose-additive design, two OP/CM mixtures exhibited lessthan-additive interaction when tested using a 96-hr LCs0 in rainbow trout (Marking and Mauck, 1975). In an interesting study that may shed some light on the disparate results described in this section, mixtures of carbaryl and an OP pesticide [phenthoate; ethyl-(dimethoxyphosphinothioyl)thio benzeneacetate] were tested using LC50 determinations for a fish (Channa punctatus) (Sambasiva Rao et al., 1985). The type of interaction varied according to the ratio of the chemicals in the mixture, with 3:1 (carbaryl:phenthoate) showing more-than-additive interaction and 1:3 showing moderate less-than-additive interaction. In summary, CM/OP interactions are not necessarily less than additive, as is the general expectation given the wellknown antagonism between CMs and nerve agents. In fact, there are quite a few indications that in some systems there is a more-than-additive interaction between the two. Moreover, the type of interaction likely depends on the proportion of each pesticide in the mixture.
VII. T I M I N G O F A D M I N I S T R A T I O N AND SEQUENCE OF ADMINISTRATION The timing of the administration of the pesticides also influences the nature of the interaction. For example, in a pioneering study in cats, Koster (1946) showed that physosfigmine protection against diisopropylfluorophosphate (DFP) toxicity was related to the time interval between the administration of physostigmine and DFP. Mortality increased as the time between the administration
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of physostigmine and DFP increased. Specifically, when physostigmine was administered minutes before DFP, mortality was only 12.5% (one of eight cats), but when physostigmine was given 5 or 6 hr before DFP, the mortality was 100%. Likewise, a detailed time course study of the interaction of malathion with tri-o-tolyl phosphate showed maximal potentiation at shorter intervals separating the two OP compounds, waning as the interval between administration of the two increased (Cohen and Murphy, 1971b). In this case, however, the time intervals were days rather than hours, as in the Koster study, and, of course, Koster was assessing protection, whereas Cohen and Murphy were assessing potentiation. Subsequently, other authors have delineated very detailed time courses for the interaction of two OP compounds, showing that the time course of the interaction depends on the compounds. In their investigation of the interaction of an N-methyl CM with various OP pesticides, Takahashi and coworkers (1987) mapped out the influence of the time between the two doses on the subsequent lethality (LDs0). In general, although the degree of potentiation of N-methyl CM lethality differed according to the OP compound, the shorter the time interval between the doses, the higher the lethality. There were substantial differences in how long the potentiation lasted, with cyanophos and fenitrothion showing potentiation for 1 or 2 days, whereas malathion showed potentiation for only 2 hr and dichlorvos did not show any potentiation. In all the previous examples, it appears that the shorter the time interval between the two anticholinesterases, the more potent the interaction; however, in an extensive study of the interaction of numerous OP compounds with malathion, Casida and coworkers (1963) demonstrated that this was not always the case. Specifically, in two of four compounds for which the interval between the two doses was varied between 0 and 120 hr, it was found that the maximal increase in malathion lethality was not achieved at the shorter interval but, rather, at an interval of 6-24 hr. In summary, one anticholinesterase pesticide can influence the toxicity of another without being administered to the animal at the same time. Therefore, they can be considered a mixture without necessarily physically occurring together. This can occur due to a pharmacodynamic interaction, as in the case of physostigmine and DFP (Koster, 1946), or a pharmacokinetic interaction, as with triorthotoyl phosphate and malathion (Cohen and Murphy, 1971a,b; Murphy et al., 1959). A caveat is that all of the previously mentioned time course studies used high doses and lethality as an end point, which makes one wonder if these same types of interactions would still be seen at lower, sublethal doses. In an interesting study relevant to the previous discussion, an interaction was found between first and second dosages of the same compound. When chlorfenvinphos was administered to rats in two separate dosages 24 hr apart, it was found that the toxicity of the second dosage was dampened by the previous administration of the same compound (Ikeda et al.,
1990). Both lethality and brain cholinesterase inhibition were lessened in the rats that had received the first dose of chlorfenvinphos 24 hr before the second dose. Exploring the mechanisms for this apparent protection, Ikeda and coworkers found that the first dose increased the hepatic clearance of the second dose of chlorfenvinphos (Ikeda et aL, 1992), possibly by the induction of the hepatic cytochrome P450s (Ikeda et al., 1991). This series of studies raises the interesting possibility that even the same compound may produce different toxic profiles depending on the time between successive exposures. Another consideration is the sequence of administration of two compounds. Some early studies explored how the order of administration of two different cholinesterase-inhibiting compounds affected their toxicity. Physostigmine given before DFP protected cats from lethality, but if DFP was given before physostigmine, there was lethality (Koster, 1946). In another study, tri-o-cresylphosphate given 24 hr before malathion killed all the dosed rats, but when the same dosages were given in reverse order (i.e., malathion given 24 hr before tri-o-cresylphosphate), none of the rats died or even showed toxic signs (Murphy et al., 1959). Karanth and coworkers explored the sequence of administration of chlorpyrifos and parathion (Karanth et al., 2001) or methyl parathion (Karanth et al., 2004). Chlorpyrifos given before either parathion or methyl parathion produced more lethality, toxic signs, and brain cholinesterase inhibition than when either parathion or methyl parathion was given before chlorpyrifos. As with the tri-o-cresylphosphate/malathion (Murphy et al., 1959), Karanth and coworkers interpreted these findings to be due to the first compound (either tri-o-cresylphosphate or chlorpyrifos) affecting the detoxification of the second compound (malathion, parathion, or methyl parathion), but not vice versa. In other words, tri-o-cresylphosphate was likely inhibiting the CarbEs that detoxify malathion (Murphy et al., 1959), and chlorpyrifos was likely inhibiting the carboxylesterases that detoxify parathion or methyl parathion (Karanth et al., 2001, 2004). Significant detoxification of chlorpyrifos is mediated via A-esterases (Li et al., 1995; Padilla et al., 2000, 2004), which cannot be inhibited by either parathion or methyl parathion. Obviously, at least at high doses, the order or sequence of administration of two OP compounds markedly affects toxicity.
VIII. FUTURE DIRECTIONS Many obvious research needs are apparent after a perusal of the anticholinesterase mixture literature. Most important is the need to conduct mixture testing in the low range of the dose-response relationship; researchers need to use sensitive end points such as cholinesterase inhibition :rather than lethality. Experimental design and statistical analyses are also very important to the interpretation of results. In their paper on the use of mixture data in risk assessment,
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Teuschler and Hertzberg (1995) suggest directions for future mixture research, emphasizing both experimental design and statistical analyses, with suggestions for each. Because the ratio of the OP or CM pesticides in the mixture, the sequence of administration, and the timing of the administration can all have a marked influence on the toxicity profile of a mixture, more research investigating these relationships is needed to define comprehensively the interaction profile of a group of pesticides. Only a very small percentage of the mixture studies in the literature used more than two anticholinesterase pesticides, whereas in the real world humans are routinely exposed to many more than two chemicals. More studies need to be conducted using multiple chemicals. There have been advances in experiment design and analysis so that this type of multiple chemical mixture study is much easier and more approachable (Gennings, 1995) than the traditional full factorial design. Another issue that has not been mentioned is the quality of the effects of a mixture. With anticholinesterase compounds, one always expects cholinesterase inhibition, but when investigating the effects of a mixture of anticholinesterase pesticides, one must always be watchful for novel effects that may not be manifested by the individual pesticides.
Acknowledgments The author thanks Michael Cummings of the U.S. EPA library for all his excellent and timely help finding copies of the cited articles and Dr. Robert MacPhail for his careful review of earlier versions of this chapter.
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Casida, J. E., Baron, R. L., Eto, M., and Engel, J. L. (1963). Potentiation and neurotoxicity induced by certain organophosphates. Biochem. Pharmacol. 12, 73-83. Castorina, R., Bradman, A., McKone, T. E., Barr, D. B., Harnly, M. E., and Eskenazi, B. (2003). Cumulative organophosphate pesticide exposure and risk assessment among pregnant woman living in an agricultural community: A case study from the CHAMACOS cohort. Environm. Health Perspect. 111, 1640-1648. Cohen, S. D. (1984). Mechanisms of toxicological interactions involving organophosphate insecticides. Fundam. Appl. Toxicol. 4, 315-324. Cohen, S. D., and Murphy, S. D. (1971a). Carboxylesterase inhibition as an indicator of malathion potentiation in mice. J. Pharmacol. Exp. Ther. 176, 733-742. Cohen, S. D., and Murphy, S. D. (1971b). Malathion potentiation and inhibition of hydrolysis of various carboxylic esters by triorthotolyl phosphate (TOTP) in mice. Biochem. PharmacoL 20, 575-587. Degraeve, N., Chollet, M.-C., and Moutschen, J. (1985). Mutagenic efficiency of organophosphorus insecticides used in combined treatments. Environ. Health Perspect. 60, 395-398. DePass, L. R., Weaver, E. V., and Mirro, E. J. (1985). Aldicarb sulfoxide/aldicarb sulfone mixture in drinking water of rats: Effects on growth and acetylcholinesterase activity. J. Toxicol. Environ. Health 16, 163-172. Dolara, P., Torricelli, E, and Antonelli, N. (1994). Cytogenetic effects on human lymphocytes of a mixture of fifteen pesticides commonly used in Italy. Murat. Res. 325, 47-51. DuBois, K. P. (1961). Potentiation of the toxicity of organophosphorus compounds. Adv. Pestic. Cont. Res. 4, 117-151. Duggan, A., Charnley, G., Chen, W., Chukwudebe, A., Hawk, R., Krieger, R. I., Ross, J., and Yarborough, C. (2003). Di-alkyl phosphate biomonitoring data: Assessing cumulative exposure to organophosphate pesticides. Regul. Toxicol. Pharmacol. 37, 382-395. Durham, H. D., Comeau, A. M., Cameron, P. H., and Ecohichon, D. J. (1982). Subacute toxicity in rats of orally administered fenitrothion alone and in a selected formulation. Toxicol. Appl. Pharmacol. 62, 455--464. Fenske, R. A., Kedan, G., Lu, C., Fisker-Andersen, J. A., and Cuff, C. L. (2002). Assessment of organophosphorous pesticide exposures in the diets of preschool children in Washington State. J. Exp. Anal. Environ. Epidemiol. 12, 21-28. Frawley, J. P., Fuyat, H. N., Hagan, E. C., Blake, J. R., and Fitzhugh, O. G. (1957). Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinesterase compounds. J. Pharmacol. Exp. Ther. 121, 96-106. Gennings, C. (1995). An efficient experimental design for detecting departure from additivity in mixtures of many chemicals. Toxicology 105, 189-197. Gessner, P. K. (1974). The isobolographic method applied to drug interactions. In Drug Interactions (P. L. Morselli, S. Garattini, and S. N. Cohen, Eds.), pp. 349-362~ Raven Press, New York. Gessner, P. K., and Cabana, B. E. (1970). A study of the interaction of the hypnotic effects and of the toxic effects of chloral hydrate and ethanol. J. Pharmacol. Exp. Ther. 174, 247-259. Gordon, J. J., Leadbeater, L., and Maidment, M. P. (1978). The protection of animals against organophosphate poisoning by
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pretreatment with a carbamate. Toxicol. Appl. Pharmacol. 43, 207-216. Gunderson, C. H., Lehmann, C. R., Sidell, E R., and Jabbari, B. (1992). Nerve agents: A review. Neurology 42, 946-950. Gupta, R. C., and Dettbarn, W.-D. (1993). Role of carboxylesterases in the prevention and potentiation of N-methylcarbamate toxicity. Chem.-Biol. Interact. 87, 295-303. Hamers, T., Smit, M. G. D., Murk, A. J., and Koeman, J. H. (2001). Biological and chemical analysis of the toxic potency of pesticides in rainwater. Chemosphere 45, 609-624. Ikeda, T., Kojima, T., Yoshida, M., Takahashi, H., Tsuda, S., and Shirasu, Y. (1990). Pretreatment of rats wtih an organophosphorus insecticide, chlorfenvinphos, protects against subsequent challenge with the same compound. Fundam. Appl. Toxicol. 14, 560-567. Ikeda, T., Tsuda, S., and Shirasu, Y. (1991). Metabolic induction of the hepatic cytochrome P-450 system by chlorfenvinphos in rats. Fundam. Appl. Toxicol. 17, 361-367. Ikeda, T., Tsuda, S., and Shirasu, Y. (1992). Pharmacokinetic analysis of protection by an organophosphorus insecticide, chlorfenvinphos, against the toxicity of its succeeding dosage in rats. Fundam. Appl. Toxicol. 18, 299-306. Ito, N., Hagiwara, A., Tamano, S., Hasegawa, R., Imaida, K., Hirose, M., and Shirai, T. (1995a). Lack of carcinogenicity of pesticide mixtures administered in the diet at acceptable daily intake (ADI) dose levels in rats. Toxicol. Lett. 82/83, 513-520. Ito, N., Hasegawa, R., Imaida, K., Kurata, Y., Hagiwara, A., and Shirai, T. (1995b). Effect of ingestion of 20 pesticides in combination at acceptable daily intake levels on rat liver carcinogenesis. Food Chem. Toxicol. 33, 159-163. Iyaniwura, T. T. (1990). Mammalian toxicity and combined exposure to pesticides. Vet. Hum. Toxicol. 32, 58-62. Karanth, S., Olivier, K., Jr., Liu, J., and Pope, C. (2001). In vivo interaction between chlorpyrifos and parathion in adult rats: Sequence of administration can markedly influence toxic outcome. Toxicol. Appl. Pharmacol. 177, 247-255. Karanth, S., Liu, J., Olivier, K., Jr., and Pope, C. (2004). Interactive toxicity of the organophosphorus insecticides chlorpyrifos and methyl parathion in adult rats. Toxicol. Appl. Pharmacol. 196, 183-190. Keplinger, M. L., and Deichmann, W. B. (1967). Acute toxicity of combinations of pesticides. Toxicol. Appl. Pharmacol. 10, 586-595. Kok, E N., and Hasirci, V. (2004). Determination of binary pesticide mixtures by an acetylcholinesterase-choline oxidase biosensor. Biosens. Bioelectron. 19, 661-665. Koster, R. (1946). Synergisms and antagonisms between physostigmine and di-isopropyl fluorophosphate in cats. J. Pharmacol. Exp. Ther. 88, 39-46. Kreitzer, J. E, and Spann, J. W. (1973). Tests of pesticidal synergism with young pheasants and Japanese quail. Bull. Environ. Contam. Toxicol. 9, 250-256. Krishnan, K., and Brodeur, J. (1991). Toxicological consequences of combined exposure to environmental pollutants. Arch. Complex Environ. Studies 3, 1-106. Krishnan, K., and Brodeur, J. (1994). Toxic interactions among environmental pollutants: Corroborating laboratory observations with human exposure. Environ. Health Perspecr 102, 11-17. Kulkarni, A. E (1976). Joint action of insecticides against houseflies. J. Toxicol. Environ. Health 1, 521-530.
Lauwerys, R. R., and Murphy, S. D. (1969). Interaction between paraoxon and tri-o-tolyl phosphate in rats. Toxicol. Appl. Pharmacol. 14, 348-357. Lechner, D. M. W., and Abdel-Rahman, M. S. (1984). A teratology study of carbaryl and malathion mixtures in rat. J. Toxicol. Environ. Health 14, 267-278. Lee, E. J. (1997). Pharmacology and toxicology of chemical warfare agents. Ann. Acad. Med. Singapore 26, 104-107. Li, W. E, Furlong, C. E., and Costa, L. G. (1995). Paraoxonase protects against chlorpyrifos toxicity in mice. Toxicol. Lett. 76, 219-226. Macek, K. J. (1975). Acute toxicity of pesticide mixtures to bluegills. Bull. Environ. Contam. Toxicol. 14, 648-652. Marking, L. L., and Mauck, W. L. (1975). Toxicity of paired mixtures of candidate forest insecticides to rainbow trout. Bull. Environ. Contam. Toxicol. 13, 518-522. Mileson, B. E., Chambers, J. E., Chen, W. L., Dettbarn, W., Ehrich, M,, Eldefrawi, A. T., Gaylor, D. W., Hamernik, K., Hodgson, E., Karczmar, A. G., Padilla, S., Pope, C. N., Richardson, R. J., Saunders, D. R., Sheets, L. E, Sultatos, L. G., and Wallace, K. B. (1998). Common mechanism of toxicity: A case study of organophosphorus pesticides. Toxicol. Sci. 41, 8-20. Miyaoka, T., Takahashi, H., Tsuda, S., and Shirasu, Y. (1984). Potentition of acute toxicity of 2-sec-butylphenyl N-methylcarbamate (BPMC) by fenthion in mice. Fundam. Appl. Toxicol. 4, 802-807. Moser, V. C., Casey, M., Hamm, A., Carter, J., W. H., Simmons, J. E., and Gennings, C. (2005). Neurotoxicological and statistical analyses of a mixture of five organophosphorus pesticides using a ray design. Toxicol. Sci., 86, 101-115. Murphy, S. D. (1969). Mechanisms of pesticide interaction in vertebrates. Residue Rev. 25, 201-222. Murphy, S. D. (1980). Assessment of the potential for toxic interactions among environmental pollutants. In The Principles and Methods in Modem Toxicology (C. L. Galli, S. D. Murphy, and R. Paoletti, Eds.), pp. 277-294. Elsevier/North-Holland, Amsterdam. Murphy, S. D., and DuBois, K. P. (1957). Quantitative measurement of inhibition of the enzymatic detoxification of malathion by EPN (ethyl p-nitrophenyl thionobenzenephosphonate). Proc. Soc. Exp. Biol. Med. 96, 813-818. Murphy, S. D., Anderson, R. L., and DuBois, K. E (1959). Potentiation of toxicity of malathion by triortotolyl phosphate. Proc. Soc. Exp. Biol. Med. 100, 483-487. Padilla, S., Buzzard, J., and Moser, V. C. (2000). Comparison of the role of esterases in the differential age-related sensitivity to chlorpyrifos and methamidophos. NeuroToxicology 21, 49-56. Padilla, S., Sung, H. J., and Moser, V. C. (2004). Further assessment of an in vitro screen that may help identify organophosphorus pesticides that are more acutely toxic to the young. J. Toxicol. Environ. Health A 67, 1477-1489. Plaa, G. L., and V6zina, M. (1990). Factors to consider in the design and evaluation of chemical interactions studies in laboratory animals. In Toxic interactions (R. S. Goldstein, W. R. Hewitt, and J. B. Hook, Eds.), pp. 3-30. Academic Press, New York. Polak, R. L., and Cohen, M. E. (1969). The influence of triorthocresylphosphate on the distribution of 32p in the body of the rat after the injection of 32p-sarin. Biochem. Pharmacol. 18, 813-820.
CHAPTER 41 Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health B 2, 161-181. Rosenberg, E, and Coon, J. M. (1958). Potentiation between cholinesterase inhibitors. Proc. Soc. Exp. Biol. Med 97, 836-839. Sambasiva Rao, K. R. S., Prasada Rao, K. S., Ahammad Sahib, I. K., and Ramana Rao, K. V. (1985). Combined action of carbaryl and phenthoate on a freshwater fish (Channa punctatus Bloch). Ecotoxicol. Environ. Safety 10, 209-217. Sfinchez-Pefia, L. C., Reyes, B. E., L6pez-Carrillo, L., Recio, R., Mor~in-Martinez, J., Cebrifin, M. E., and Quintanilla-Vega, B. (2004). Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers. Toxicol. Appl. Pharmacol. 196, 108-113. Seume, E W., and O'Brien, R. D. (1960a). Metabolism of malathion by rat tissue preparations and its modification by EPN. Agric. Food Chem. 8, 36-41. Seume, E W., and O'Brien, R. D. (1960b). Potentiation of the toxicity to insects and mice of phosphorothionates containing carboxylester and carboxyamide groups. Toxicol. Appl. Pharmacol. 2, 495-503. Takahashi, H., Miyaoka, T., Tsuda, S., and Shirasu, Y. (1984). Potentiated toxicity of 2-sec-butylphenyl methylcarbamate (BPMC) by O,O,-dimethyl O-(3-methyl-4-nitrophenyl)phosphorothioate (fenitrothion) in mice; Relationship between actue toxicity and metabolism of BPMC. Fundam. Appl. Toxicol. 4, 718-723. Takahashi, H., Kato, A., Yanashita, E., Naito, Y., Tsuda, S., and Shirasu, Y. (1987). Potentiations of N-methylcarbamate toxicities by organophosphorus insecticides in male mice. Fundam. Appl. Toxicol. 8, 139-146.
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Teuschler, L. K., and Hertzberg, R. C. (1995). Current and future risk assessment guidelines, policy, and methods development for chemical mixtures. Toxicology 105, 137-144. Timchalk, C., Poet, T. S., Hinman, M. N., Busby, A. L., and Kousba, A. A. (2004). Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat. Toxicol. Appl. Pharmacol. 205, 31-42. Toia, R. E, March, R. B., Umetsu, N., Mallipudi, N. M., Allahyari, R., and Fukuto, T. R. (1980). Identification and toxicological evaluation of impurities in technical malathion and fenthion. J. Agric. Food Chem. 28, 599-604. Tsuda, S., Miyaoka, T., Iwasaki, M., and Shirasu, Y. (1984). Pharmacokinetic analysis of increased toxicity of 2-secbutylphenyl methylcarbamate (BPMC) by fenitrothion pretreatment in mice. Fundam. Appl. Toxicol. 4, 724-730. Umetsu, N., Grose, E H., Allahyari, R., Abu-E1-Haj, S., and Fukuto, T. R. (1977). Effect of impurities on the mammalian toxicity of technical malathion and acephate. J. Agric. Food Chem. 25, 946-953. Umetsu, N., Mallipudi, N. M., Toia, R. E, March, R. B., and Fukuto, T. R. (1981). Toxicological properties of phosphorothioate and related esters present as impurities in technical organophosphorus insecticides. J. Toxicol. Environ. Health 7, 481-497. Wessinger, W. D. (1986). Approaches to the study of drug interactions in behavioral pharmacology. Neurosci. Biobehav. Rev. 10, 103-113. Wessinger, W. D., and Evans, E. B. (1988). Modeling multiple agent interactions in behavioral pharmacology. J. Am. Coll. Toxicol. 7, 953-962. Woolverton, W. L. (1987). Analysis of drug interactions in behavioral pharmacology. Adv. Behav. Pharmacol. 6, 275-302.
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CHAPTER 4
Federal Regulations and Risk A s s e s s m e n t of Organophosphate and Carbamate Pesticides A N N A B. LOWIT U.S. Environmental Protection Agency, Washington, DC
and technical analyses for specific risk assessments, can be found at www.epa.gov/pesticides/reregistration/status.htm and the EPA's pesticide docket and electronic EPA dockets Web site, www.epa.gov/edocket. Because this chapter focuses primarily on regulations, activities, and efforts in the United States, particularly those at the EPA, the reader may want to search other sources of information regarding risk assessment activities for the OPs and N-methyl CMs by other organizations, such as the World Health Organization's Joint FAO/WHO Meeting on Pesticide Residues, Canada's Pest Management Regulatory Agency, the Organization for Economic Co-operation and Development, and the California Department of Pesticide Regulation. People are exposed to OPs and N-methyl CMs through various pathways. OPs and N-methyl CMs are applied to various agricultural crops and therefore can be present in food. OPs and N-methyl CMs can be present in water. Some OPs and N-methyl CMs can be purchased by consumers for use in or around the home; thus, residential exposure to children and adults is possible. Agricultural workers and professional pesticide applicators are exposed to these pesticides through occupational activities. EPA-OPP develops risk assessments for many exposure pathways and scenarios, including exposures to food and water, residential use, and occupational exposure. Risk is composed of two major components: hazard assessment and exposure assessment. Aspects of chemical toxicity and actual environmental exposure impact potential risk to a particular agent(s). This chapter reviews the toxicology of OPs and N-methyl CMs; thus, hazard assessment of the OPs and N-methyl CMs is discussed in detail. There are many publicly available sources of information regarding exposure to OPs and N-methyl CMs, including the U.S. Department of Agriculture's pesticide data program, the Food and Drug Administration's food monitoring program, U.S. Geological Survey water monitoring programs, and the National Home and Garden Pesticide Use Survey. Specific
I. I N T R O D U C T I O N In the United States, pesticide chemicals are registered for use by the U.S. Environmental Protection Agency (EPA). Although some are no longer registered in the United States, there are more than 30 organophosphates (OPs) and more than 15 N-methyl carbamates (CMs). A complete, exhaustive review of the history and risk assessments for each of these chemicals is beyond the scope of this chapter. Instead, this chapter provides an overview of the federal regulations governing pesticide chemicals in the United States and the risk assessment practices and approaches used by the EPA's Office of Pesticide Programs (EPA-OPP) to evaluate the OP and N-methyl CM pesticides. The text focuses on aspects of the OP and N-methyl CM risk assessments that provide the most robust overview, particularly noncancer effects related to the inhibition of acetylcholinesterase (ACHE). This chapter highlights key milestones and accomplishments regarding risk assessments of the OP and N-methyl CM pesticides, particularly after the passage of the Food Quality Protection Act (FQPA) of 1996. It is important to note that while this chapter was being written the EPA was actively working on risk assessments for some OPs and N-methyl CMs and the cumulative risk assessments for both common mechanism groups. Reference doses and benchmark dose estimates provided in this chapter are subject to change as risk assessments develop or are refined and/or as new data and information become available for evaluation. The values listed reflect the best information as of January 1, 2005. For the most recent status of individual chemicals of interest and/or the cumulative risk assessments, the reader is referred to the EPA's Web site (www.epa.gov/ pesticides), which contains a wealth of information about many areas of pesticide regulation and risk assessment. Additional information, including supporting documentation
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exposure assessments for each pesticide can also be found at the EPA's Web site and/or at the EPA's e-docket.
II. F E D E R A L R E G U L A T I O N S GOVERNING P E S T I C I D E S IN T H E U N I T E D S T A T E S The first modern form of today's pesticide laws, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), was passed in 1947, and it has undergone significant revisions since that time. Under FIFRA, EPA registration is required prior to manufacture, transport, or sale in the United States. FIFRA also provides EPA with the authority to require data to be provided prior to registration. In 1984, extensive data requirements were published for toxicity testing in mammal and nonmammalian species, physical-chemical properties, environmental fate, and field trial studies (to evaluate potential food exposure). These requirements are listed in 40 CFR part 158 (www.access.gpo.gov/nara/cfr/index/html). Numerous toxicity studies in laboratory animals are required to evaluate metabolism and pharmacokinetics in addition to potential subchronic and chronic toxicity, neurotoxicity, developmental toxicity, carcinogenicity, and genotoxicity. Typically, toxicology studies are performed via oral administration; however, dermal and/or inhalation toxicology studies may be required in some cases, depending on the exposure pattern(s) of the particular pesticide, physical-chemical properties, and/or potential route-specific hazard concerns. EPA also regulates pesticides under the Federal, Food, Drug, and Cosmetic Act (FFDCA). The FFDCA provides EPA with the authority to set pesticide tolerances (maximum allowable pesticide residue levels) in food and feed. Food and feed are considered adulterated if they contain a pesticide residue greater than the tolerance or if they contain a pesticide not covered by an existing tolerance. The most recent revision to both FFDCA and FIFRA occurred in 1996 with the passage of the Food Quality Protection Act (FQPA). Among other things, FQPA required that EPA reevaluate all existing food tolerances by August 2006. This process is called "tolerance reassessment." By early 2005, EPA had reassessed more than two-thirds of all food tolerances. FQPA also imposed key requirements on EPA that substantially changed some approaches used by EPA-OPP in many of its pesticide risk assessments. Specifically, FQPA mandated that EPA (i) perform aggregate risk assessment in its single chemical risk assessment, (ii) perform cumulative risk assessment for groups of pesticides with a common mechanism of action, and (iii) apply an additional FQPA 10x safety factor specifically for infants and children. Aspects of each of these provisions are discussed in this chapter. As part of tolerance reassessment, Congress required EPA to prioritize tolerance reassessment by evaluating the "worst first." As such, many of the single chemical assessments for the OPs and N-methyl CMs were identified for development early in the tolerance reassessment process; many of the risk
assessments for the individual OPs and N-methyl CMs were complete or close to completion at the time of writing of this chapter. However, several chemicals, namely malathion, dimethoate, dichlorvos, formetanate HC1, aldicarb, and carbofuran, were not yet complete or finalized. Although available for OPs in a well-developed form, the cumulative risk assessments for neither common mechanism were finalized.
III. S I N G L E - C H E M I C A L , AGGREGATE
RISK ASSESSMENTS FOR OPs AND N-METHYL CMs Historically, EPA has focused on single pathways of exposure (e.g., pesticide residues in food, water, or residential/ nonoccupational uses) for individual chemicals and not on the potential for individuals to be exposed to multiple pathways or to multiple pesticides concurrently. The requirement by FQPA to perform aggregate and cumulative risk assessments substantially changed some approaches used by EPA-OPP. "Aggregate risk" is defined by EPA-OPP as the sum total of all exposure to pesticides through inhalation or dermal, oral, or optic contact (EPA, 2001a). Aggregate risk is the risk to a single pesticide chemical from multiple routes and pathways of exposure (e.g., food + water + residential).
A. Hazard Assessment and Hazard Identification Procedures When performing hazard assessment and hazard identification for aggregate risk assessment, dose-response aspects of all the potential toxicities for a single pesticide are considered. EPA's guidance documents for developing reference doses (RfDs) and reference concentrations (RfCs) (EPA, 1994, 2002a) provide a comprehensive discussion of performing dose-response analysis for risk assessment purposes. EPA-OPP uses two different risk metrics in its pesticide noncancer risk assessments: the RfD and the margin of exposure. In both methods, a point of departure (POD) is first established, followed by the identification of appropriate uncertainty and extrapolation factors. The information contained in EPA's RfD guidance document, the review of RfD procedures, and the benchmark dose (BMD) guidance (EPA, 1994, 2000, 2002a) together provide a thorough description of the history, purpose, and procedures for developing PoDs. The PoD is selected to be protective of the critical effect for a particular pesticide. This approach is based, in part, on the assumption that if the critical toxic effect is prevented, then all toxic effects are prevented. A PoD can be a BMD estimate (EPA, 2000), no-observed-adverse-effect-level (NOAEL), or a lowest-observed-adverse-effect-level (LOAEL). The types of effects that have been identified as critical effects for the single chemical assessments for OPs and N-methyl
CHAPTER 42 9Federal Regulations and Risk Assessment CMs include AChE inhibition, particularly in the blood and brain; behavioral and/or motor activity changes; and developmental toxicity or developmental neurotoxicity. When EPA evaluates AChE data for risk assessment purposes, measurements of AChE from the target tissue m the central or peripheral nervous system m are preferred. Toxicology studies submitted to EPA for pesticide registration typically measure plasma, red blood cell (RBC), and brain cholinesterase (ChE) inhibition and most often do not measure peripheral tissues. In the absence of such data, EPA considers blood measures as surrogates of peripheral tissues. The next step is to identify the appropriate uncertainty and extrapolation factors. Historically, EPA-OPP has used a 10X factor for interspecies extrapolation (e.g., extrapolation from animal to human) and a 10X factor for intraspecies extrapolation to account for human variability. In some cases, studies on human subjects are available and contain information regarding human pharmacokinetic (PK) or pharmacodynamic (PD) effects. In these cases, it may be appropriate to reduce or remove the interspecies extrapolation factor. The LOAEL to NOAEL uncertainty factor may be applied when a NOAEL is not identified in a toxicology study and the LOAEL must be used as the PoD. B MD modeling offers a preferred alternative to using NOAELs or LOAELs since the entire dose-response curve along with uncertainty are considered in the PoD development. When using B MD modeling, the LOAEL to NOAEL factor may not be necessary. A subchronic to chronic duration extrapolation factor may be applied when a chronic study is not available. In cases in which a key study (e.g., developmental toxicity) is not available in the database of toxicology studies, the database uncertainty factor may be used. When applying each of these factors, particular attention is paid to the mode or mechanism of action for a particular chemical. For example, as discussed later for the N-methyl CMs, the mechanism of action for the N-methyl CMs involves rapid recovery of AChE inhibition. For most N-methyl CMs, recovery occurs in minutes to hours. The toxic effects observed after exposure to N-methyl CMs generally do not increase with repeated days of exposure. Acute effects are the primary concern; the subchronic to chronic uncertainty factor may not be relevant for this class of pesticides. FQPA requires that EPA apply a 10X factor to account for added sensitivity of infants and children unless there are sufficient data to reduce this factor. This factor is called the FQPA 10X factor for the protection of infants and children. In single-chemical risk assessments, EPA-OPP evaluates the adequacy of the toxicology database for evaluating potential sensitivity in infants or children on a chemical-by-chemical basis. EPA published a data-call in notice (DCI) requiring developmental neurotoxicity (DNT) studies for each of the OPs with active pesticide registrations. In addition to the DNT study, a companion
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study to evaluate the comparative sensitivity for AChE inhibition in adult and juvenile animals was required. Chapter 42 describes aspects of the DNT and comparative AChE studies along with the DCI. Many of the DNT and comparative AChE studies for the OPs are under review by EPA. Several comparative AChE studies or DNT in several N-methyl CMs were planned or ongoing. As such, FQPA factors for some OPs and N-methyl CMs may be revised in the future; chemical-specific FQPA 10X factors are not provided in this chapter. Particularly through EPA's Integrated Risk Information System program, the agency typically develops RfDs and RfCs only for long-term or chronic exposures. However, because of the requirements of FQPA, EPA-OPP also develops risk assessments for exposure scenarios of varying duration and pathways of exposure. These include dietary exposure from food and water, residential exposure, and occupational activities. EPA-OPP also considers specific population groups (e.g., adults and children) in its risk assessments. In dietary risk assessment, EPA-OPP considers both acute (single exposures) and chronic exposures to food and drinking water. In residential and occupational risk assessments, EPA-OPP evaluates potential toxic effects that could result following dermal and/or inhalation exposure from applying pesticides in or around the home or from various activities such as playing on the lawn. Potential oral exposure to young children and toddlers from mouthing behaviors is also evaluated. When selecting PoDs, to the degree possible, EPA-OPP attempts to select end points from available toxicology studies that match the duration and route of exposure with specific exposure scenarios. For example, for acute dietary risk assessment, EPA prefers to use a single oral dosing study or a multiple dosing study in which clinical observations were made after an oral single exposure. Regarding the acute RfDs, AChE inhibition following single oral exposures is the critical effect for most OPs and N-methyl CMs. In cases in which developmental effects are observed in a developmental toxicity or developmental neurotoxicity study, EPA first considers the mechanistic information available that could characterize the progression of the noted teratogenic effect. In the absence of such mechanistic information, EPA may assume the observed developmental effect could potentially occur following a single exposure and may select the noted developmental effect for estimation of acute dietary risk. When estimating residential risk to a pesticide used to treat lawns, EPA evaluates potential dermal exposures of various durations. EPA prefers to use toxicity studies performed via the dermal route but again may consider developmental end points from oral studies in specific cases in which developmental toxicity provides the critical effect(s). When effects from an oral study are selected as the PoD, a dermal absorption factor is also identified to adjust for differential absorption between the oral and dermal absorption routes.
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B. Acute and Chronic Dietary Risk Assessment for OPs and N-Methyl CMs The oral route of administration provides the most robust database of toxicology studies and also provides the most extensive set of regulatory end points that can be used for comparison purposes. Regulatory end points used to develop dermal and inhalation risk assessments can be found for specific chemicals at EPA's Web site or e-docket. Tables 1 and 2 provide the PoDs identified for acute and chronic dietary exposure assessment, the selected uncertainty and/or extrapolation factors (FQPA 10• factors are not provided), and the derived acute and chronic RfDs for the OPs and N-methyl CMs. The mechanism of action for the N-methyl CMs involves rapid recovery of AChE inhibition, and the effects observed after exposure to N-methyl CMs generally do not increase with repeated exposure. Thus, for some N-methyl CMs, a chronic PoD has not been established. The acute PoD, along with uncertainty/extrapolation factors for these pesticides, is protective of effects observed following acute, subchronic, or chronic exposures. ChE inhibition provides the critical effect for acute and chronic RfDs for almost all the OPs and N-methyl CMs. Regarding acute exposures, the rat is most commonly used in acute neurotoxicity studies; thus, rat studies were selected most often for derivation of acute RfDs. Studies performed in rat and dog are commonly used for chronic RfD derivation.
IV. CUMULATIVE RISK ASSESSMENT FOR OPs AND N-METHYL CMs As mentioned previously, in addition to aggregate risk assessment, FQPA required EPA to consider "available evidence concerning the cumulative effects on infants and children of such residues and other substances that have a common mechanism of toxicity." Mechanism of toxicity is defined by EPA-OPP (EPA, 1999a) as the major steps leading to an adverse health effect following interaction of a pesticide with biological targets. All steps do not need to be specifically understood. Rather, it is the identification of the crucial events following chemical interaction that are required in being able to describe a mechanism of toxicity. Common mechanism of toxicity pertains to cases where two or more chemicals produce or may be expected to produce adverse effects by the same crucial steps(s). Thus, "cumulative risk" is defined as the risk that may result from dietary, residential, or other nonoccupational exposure to multiple chemicals that have a common mechanism of toxicity (i.e., cumulative risk). Cumulative risk assessments include multiple pathways and multiple chemicals. EPA-OPP has developed a guidance document for developing cumulative risks assessments under FQPA (EPA, 2002b). Single-chemical, aggregate assessments differ from cumulative risk assessments in focus and intent. Regarding hazard
assessment, single-chemical, aggregate assessments consider all potential toxicities; cumulative risk assessments focus on the common toxic effect for the common mechanism group. The first step in producing a cumulative risk assessment is to identify a group of chemicals that produce a common toxic effect(s) by a common mechanism of toxicity. EPA-OPP has developed a general framework for identifying the chemicals that belong to that group (EPA, 1999a). The cumulative guidance states that in determining this common mechanism group (CMG), careful attention should be given to a variety of factors, including the mechanism of toxicity, the time dimensions of the toxic effects and exposure, and the pesticide exposure patterns and treatment scenarios. Thus, assessing the potential for two or more pesticides to act by the same mechanism involves the consideration of the following principles: (i) They cause the same critical effect(s), (ii) they act on the same molecular target at the same target tissue, and (iii) they act by the same biochemical mechanism of action perhaps because they share a common toxic intermediate (Miteson et al., 1998). The OPs and N-methyl CMs have been identified as CMGs (EPA, 1999b, 2001b). The OP pesticides were grouped by a common mechanism based on their shared ability to inhibit AChE by phosphorylation of the active site of the enzyme. Regarding the CM pesticides, EPA distinguishes pesticides with the CM chemical structure that have the potential for inhibiting AChE and those CM pesticides that do not inhibit AChE (EPA, 1999b). Specifically, EPA separates CM pesticides into three distinct subgroups: N-methyl CMs, thiocarbamates, and dithiocarbamates. The following discussion focuses on the ChE-inhibiting N-methyl CMs; thiocarbamates and dithiocarbamates are not discussed further. The subgroup of N-methyl CMs share similar structural characteristics and share the ability to inhibit AChE by carbamylation of the serine hydroxyl group located in the active site of the enzyme (EPA, 2001c), leading to rapid recovery of ChE inhibition. Based on these similarities, the N-methyl CMs have been grouped into a CMG. Because the OPs and N-methyl CMs have been grouped into CMGs, these two classes of pesticides are subject to cumulative risk assessment.
A. OP Cumulative Risk Assessment Pesticides are determined to have a "common mechanism of toxicity" if they act the same way in the body; that is, if scientifically reliable data demonstrate that upon exposure to these chemicals, the same toxic effect occurs in or at the same organ or tissue by essentially the same sequence of major biochemical events. The OPs were the first common mechanism of toxicity group identified by EPA and are the first pesticides to undergo a full cumulative risk assessment. More than 30 OPs are included in the CMG. However, not all are included in the cumulative assessment group (CAG). After the CMG is established, the next step involves selecting a subset of these chemicals as the CAG. This subset
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24 hr up to 30 days >30 days up to approximately 10% of the life span in humans b Approximately 10% of the life span in humans
aFrom EPA (2002c). bMore than 30-90 days in typically used laboratory animal species.
approach was consistent with general OPP guidance on the application of the 10X FQPA factor (EPA, 2002a), as well as with the conclusions of a review of the reference dose (RfD) and reference concentration (RfC) setting process at EPA. This RfD/RfC report, conducted under the auspices of the Risk Assessment Forum, concluded that the absence of data or information that is reasonably expected to have the potential for lowering the reference value may indicate the need for the use of an additional uncertainty factor in the risk calculation (EPA, 2002c). For individual chemicals, such determinations are made following careful consideration of the entire toxicology database. EPA has committed resources to providing an updated peer-reviewed evaluation of the results of submitted DNT and comparative cholinesterase studies and their use and impact on individual chemical risk assessments. Although such reviews have been conducted in the past (Makris et al., 1998; Makris, 2004), the universe of submitted studies examined at the time of these earlier analyses did not include a broad sampling across chemical classes. In the 1998 analysis, DNT studies for only one OP (chlorpyrifos) and three CM (aldicarb, carbaryl, and carbofuran) pesticides were available. At the time of the 2004 review, only two additional OP DNT studies were available for inclusion (dimethoate and malathion). Although these analyses showed, in general, that the DNT study is a sensitive study compared to other studies in the toxicology database, and demonstrated the value of the DNT in risk assessment, the impact of the DNT study on any specific chemical class has not been determined. However, the database of DNT studies available for consideration and analysis increasingly includes a substantive number of OP and CM pesticides, in part as a result of the 1999 OP DNT DCI. Therefore, any future reviews should be able to expand on previous retrospective analyses of submitted DNT studies and examine the impact of the studies, including the comparative cholinesterase data, on risk assessment for the OP and CM pesticides. Additionally, these future reviews will be able to incorporate findings from cross-laboratory comparisons of DNT study methodologies and results previously conducted
by EPA scientists in an ongoing surveillance of agency study submissions. These cross-laboratory comparisons have focused on various neurobehavioral (Crofton et al., 2004; Raffaele et al., 2003, 2004; Sette et al., 2004) and neuropathological (Crofton et al., 2001; Raffaele et al., 2005) end points as well as general methodological considerations (Makris et al., 2005). Although they included information and data from all available OP and CM studies, they did not focus on specific chemical class issues. It is anticipated that information and conclusions derived from the evaluation and analysis of the OP and CM DNT and comparative cholinesterase studies will prove to be quite useful from a much broader chemical-class perspective.
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annual meeting of the Society of Toxicology, New Orleans, LA. Toxicologist 84(S- 1), 200. Reiter, L. W., Heavner, G. B., Dean, K. E, and Ruppert, P. H. (1981). Developmental and behavioral effects of early postnatal exposure to triethyltin in rats. Neurobehav. Toxicol. Teratol. 3(3), 285-293. Rodier, P. M., and Koeter, H. B. (1986). General activity from weaning to maturity in mice exposed to halothane or nitrous oxide. Neurobehav. Toxicol. Teratol. 8(2), 195-199. Rodier, P. M., Reynolds, S. S., and Roberts, W. N. (1979). Behavioral consequences of interference with CNS development in the early fetal period. Teratology 19(3), 327-336. Rodier, P. M., Kates, B., White, W. A., and White, A. L. (1991). The relationship of rat brain weight and pituitary weight to postnatal growth after prenatal exposure to methylazoxymethanol. Neurotoxicol. Teratol. 13(6), 583-590. Rodier, P. M., Ingram, J. L., Tinsdale, B., Nelson, S., and Romano, J. (1996). Embryological origin for autism: Developmental anomalies of the cranial nerve motor nuclei. J. Comp. Neurol. 370(2), 247-261. Sager, P. R., Aschner, M., and Rodier, P. M. (1984). Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure. Brain Res. 314(1), 1-11. Schull, W. L., Norton, S., and Jensh, R. P. (1990). Ionizing radiation and the developing brain. Neurotoxicol. Teratol. 12(3), 249-260. Sette, W., Crofton, K., Makris, S., Doherty, J., and Raffaele, K. (2004). Auditory startle reflex habituation in developmental neurotoxicity testing: A cross-laboratory comparison of control data, poster presentation No. 1341, 43rd annual meeting of the Society of Toxicology, Baltimore, MD. Toxicologist 78(S 1), 275. Stanton, M. E., and Spear, L. P. (1990). Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group I report: Comparability of measures of developmental neurotoxicity in humans and laboratory animals. Neurotoxicol. Teratol. 12(3), 261-268. Stine, K. E., Reiter, L. W., and Lemasters, J. J. (1988). Alkyltin inhibition of ATPase activites in tissue homogenates and subcellular fractions from adult and neonatal rats. Toxicol. Appl. Pharmacol. 94(3), 3394-406. Tilson, H. A., Jacobson, J. L., and Rogan, W. J. (1990). Polychlorinated biphenyls and the developing nervous system: Cross-species comparisons. Neurotoxicol. Teratol. 12(3), 239-248. Tilson, H. A., MacPhail, R. C., Moser, V. C., Becking, G. C., Cuomo, V., Frantik, E., Kulig, B. M., and Winneke, G. (1997). The IPCS collaborative study on neurobehavioral screening methods: VII. Summary and conclusions. NeuroToxicology 18(4), 1065-1070. Trasande, L., and Landrigan, P. J. (2004). Editorial. The National Children's Study: A critical national investment. Environ. Health Perspect. 112(14), A789-A790. Tyl, R. W., and Sette, W. (1990). Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, Work Group III report: Weight of evidence and quantitative evaluation of developmental neurotoxicity data. Neurotoxicol. Teratol. 12(3), 275-280. U.S. Environmental Protection Agency (1991a). Developmental neurotoxicity study, series 83-6, Addendum 10 (neurotoxicity), subdivision F: Hazard evaluation: Human and domestic animals, EPA Publication No. 540/09-91-123; NTIS Publication No. PB
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91-154617. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1991b). Guidelines for developmental toxicity risk assessment. Fed. Reg. 56, 63798-63826. U.S. Environmental Protection Agency (1992). Proceedings of the U.S. EPA Workshop on Cholinesterase Methodologies. March 1, 1992, Arlington, VA. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1996). Reproductive toxicity risk assessment guidelines. Fed. Reg. 61, 56274-56322. U.S. Environmental Protection Agency (1998a). Health effects test guidelines, OPPTS 870.6300, Developmental Neurotoxicity Study, EPA 712-C-98-239. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998b). Health effects test guidelines, OPPTS 870.3700, Prenatal Developmental Toxicity Study, EPA 712-C-98-207. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998c). Health effects test guidelines, OPPTS 870.6200, Neurotoxicity Screening Battery, EPA 712-C-98-238. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998d). Health effects test guidelines, OPPTS 870.3800, Reproduction and Fertility Effects, EPA 712-C-98-208. U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (1998e). Guidelines for neurotoxicity risk assessment. Fed. Reg. 63, 26926-26954. U.S. Environmental Protection Agency (2002a), (February 28). Determination of the appropriate FQPA safety factor(s) for use in the tolerance-setting process. Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2002b), June 10. Determination of the appropriate FQPA safety factor(s) in the organophosphorus pesticide cumulative risk assessment Evaluation of sensitivity and susceptibility to the common mechanism of toxicity, acetylcholinesterase inhibition. Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2002c). A review of the reference dose and reference concentration processes, risk assessment forum, EPA/630/P-02/002E U.S. Environmental Protection Agency, Washington, DC. U.S. Environmental Protection Agency (2005). The N-methyl carbamate cumulative risk assessment: Pilot analysis, Background document for FIFRA Scientific Advisory Panel, February 15-18. U.S. Environmental Protection Agency, Washington, DC. Voorhees, C. V., Klein, K. L., and Scott, W. J. (1982). Aspirinindflced psychoteratogenesis in rats as a function of embryonic age. Teratogen. Carcinogen. Mutagen. 2(1), 77-84. Weiss, B., Amler, S., and Amler, R. W. (2004). The vulnerability, sensitivity, and resiliency of the developing embryo, infant, child, and adolescent to the effects of environmental chemicals, drugs, and physical agents as compared to the adult pesticides. Pediatrics 113(4, Pt 2), 1030-1036. Wilson, B. W., Padilla, S., Henderson, J. D., Brimijoin, S., Dass, E D., Elliot, G., Jaeger, B., Lanz, D., Pearson, R., and Spies, R. (1996). Factors in standardizing automated cholinesterase assays. J. Toxicol. Environ. Health 48, 187-195.
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CHAPTER 44
WHO/FAO Guidelines for Cholinesterase-lnhibiting Pesticide Residues in Food P. K. GUPTA Toxicology Consulting Services, Bareilly, India
Agency (EPA) in 1970. The reorganization did not significantly alter the process of pesticide registration, but it did condense the functions of the previous two agencies (FDA and FDA) into a single governing body. Likewise, OECD guidelines for testing of chemicals and risks associated with pesticides have been issued (OECD, 1987). In general, when making recommendations international agencies consider the efforts and progress made in the area of international harmonization. For example, the conduct of toxicity studies [good laboratory practices (GLPs)] and agricultural practices [good agricultural practices (GAPs)] are governed by regulations and monitored by specific quality assurance functions. Such regulations specify the need for clear and consistent procedures (i.e., standard operating procedures) and documentation of studies such that all aspects can be re-created and verified. The precision and discipline are the characteristics frequently associated with safety assessment procedure; the interpretation and extrapolation process in toxicology is far from an exacting science. This chapter highlights guidelines and recommendations issued by various expert panels of the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) with particular reference to guidelines issued for OP and CM pesticide residues in food.
I. I N T R O D U C T I O N The banning of DDT and other commonly used organochlorine (OC) pesticides led to increased use of other, more acutely toxic synthetic compounds that were also effective in pest control but were much less persistent in the environment. Subsequently, several cases of pesticide poisoning, including the Bhopal disaster in India, have been reported (Gupta, 2004). Research efforts led to the development of different types of organic compounds, such as organophosphates (OPs) and carbamates (CMs). OP pesticides are typically much less persistent in the environment than the early OCs. In fact, broad-spectrum OP and CM pesticides were the foundation of insect control chemicals in the 1980s. OPs are toxic due to their inhibition of acetylcholinesterase (ACHE) activity of nervous tissue. Like the OPs, the mode of action of CMs is inhibition of ACHE. However, CMs are generally less toxic than OPs because inhibition is more rapidly reversed (Sandhu and Brar, 2000). For judicious use, the regulation of pesticides is covered by several legislative acts and enforced by several international, federal, and state agencies. The risks associated with the use of pesticides are evaluated by assessing the toxicity and by estimating the magnitude of exposure from sources in the workplace, the environment, food, and water. The need to balance the risk to man and the environment against benefits associated with using pesticides is an important issue. Various agencies, such as the U.S. Food and Drug Administration (FDA) and Organization for Economic Co-operation and Development (OECD), provide guidelines and maximum residue limits (MRLs) of pesticides. The FDA guidelines tend to coincide with those o f the OECD. Public concern of environmental and health issues as well as an interest in greater efficiency in regulation led to the formation of the U.S. Environmental Protection Toxicology of Organophosphate and Carbamate Compounds
II. W H O C L A S S I F I C A T I O N O F PESTICIDES BY HAZARDS In 1973, the WHO executive board took the initiative to develop a tentative classification of pesticides that would distinguish between the more and less hazardous forms of each pesticide. Taking into account the views of members of the WHO Expert Advisory Panel on Insecticides and other expert advisory panels with special competence and interest in pesticide technology, as well as the comments of WHO 643
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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Risk Assessment
&
Regulations
member states and international agencies, a proposal for a WHO-recommended classification of pesticides by hazard was prepared. The WHO Recommended Classification of Pesticides by Hazard was approved by the 28th World Health Assembly in 1975 and has since gained wide acceptance. Guidelines for this classification were first issued in 1978 and have since been revised and reissued at 2-year intervals [International Programme on Chemical Safety (IPCS), 2002]. In brief, part I of the document includes the classification as recommended by WHO. This part is not subject to periodic review, and the classification table and the text can only be changed by resolution of the WHO assembly. Part II includes guidelines to the classification of individual products in a series of tables, according to the oral or dermal toxicity of the technical product and its physical state. The tables are subject to review periodically. The hazard referred to in this recommendation is the acute risk to health (i.e., the risk of single or multiple exposures over a relatively short period of time) that may be encountered accidentally by any person handling the product in accordance with the directions for handling by manufacturers or in accordance with the rules for storage and transportation developed by competent international bodies. The classification is based primarily on the acute oral and dermal toxicity to the rat since these determinations are standard procedures in toxicology. The recommended classification is given in Table 1.
III. W H O / F A O ACTIVITIES
PESTICIDE
Additives (JECFA) and the Joint WHO/FAO Meeting on Pesticide Residues in Food (JMPR). The first meeting of JECFA was held in 1956. The WHO expert panel of JMPR provides information on toxicological evaluations. This has been accomplished through the establishment of acceptable daily intakes (ADIs). The FAO expert panel of JMPR is an authoritative voice on the levels of pesticides that can be ingested daily by humans without appreciable risk; this has been accomplished through the establishment of MRLs. The first meeting on pesticide residues was held in 1963. Since then, JMPR has been establishing ADIs and MRLs of pesticides in food commodities. In 1980, WHO established IPCS, which is a joint venture with the United Nations Environment Programme and the International Labor Organization. IPCS devotes significant resources to pesticides, primarily for their assessment but also for management support. The importance that IPCS places on these activities is based on the widespread use of pesticides, both in agriculture and in public health, and on a high level of public concern in both developed and developing countries because of the intrinsic toxicity of and potential exposure to the chemicals. The primary responsibilities of IPCS within WHO are risk assessment, provision of support for risk assessment, and holding the Joint Meeting on Pesticides. The detailed activities of WHO/IPCS are beyond the scope of this chapter. Since 1980, WHO activities concerned with the safety assessment of food chemicals have been incorporated into IPCS. Members invited by WHO review toxicological and related data and estimate, when possible, ADIs of the pesticides for humans. Members invited by FAO review pesticide use patterns (GAPs), data on chemistry composition of pesticides and methods of analysis for pesticide residues, and estimate MRLs that may occur as a result of the use of the pesticides according to GAPs. These MRLs serve as the basis for international standards established by the Codex Alimentarius Commission, which is administered
RESIDUE
In 1953, the World Health Assembly noted that the increasing use of various chemicals in the food industry had created a new public health problem. In response, WHO, in conjunction with FAO, initiated two series of annual meetings, the Joint FAO/WHO Expert Committee on Food
TABLE 1. WHO Classification of Pesticides by Hazard LD5o for the rat (mg/kg body weight) b
Class
Oral
Ia, extremely hazardous Ib, highly hazardous II, moderately hazardous III, slightly hazardous
Dermal
Solids a
Liquids a
Solids a
Liquids a
--500
-2000
- 1000 ~
-4000
aAdapted with permission from IPCS (2002). bThe terms solids and liquids refer to the physical state of the active ingredients being classified.
CHAPTER 44 9OP and CM Pesticide Residues in Food by the Joint FAOAVHO Food Standards Program. JMPR advises the Codex committee of the Codex that is responsible for recommending Codex MRLs, which is hosted by the government of The Netherlands. The objectives of this expert group also include the formulation of guiding principles for exposure limits, such as ADIs for food additives and pesticide residues, and tolerances for toxic substances in food, air, water, soil, and the working environment. Since 1963, JMPR has evaluated approximately 100 OP and CM pesticides.
IV. APPROACH FOR T O X I C O L O G I C A L EVALUATIONS This chapter reflects the broad guidelines of the panel of experts of JMPR that is involved with the toxicological assessment of pesticides. The WHO expert panel of J M P R is responsible for reviewing toxicological and related data and for estimating (where possible) an ADI for humans (WHO, 2000). Routine standard procedures are used for the assessment of the toxicity of pesticide residues and are briefly summarized here.
A. Biological Data The biological data are subgrouped under three headings: biochemical aspects, toxicological studies, and observations in humans. 1. BIOCHEMICALASPECTS These studies are required to measure the concentration/time profiles of the ingested substance and its metabolites in the various organs and tissues of the body. Human biochemical studies that fall into these categories are also included in this section, whereas other human studies are included under observations in humans. The evaluation of metabolism studies in plants is the responsibility of the FAO expert panel of JMPR:
Absorption, distribution, and excretion: These studies generally focus on hydrolysis/digestion of the parent compound and its products in the mammalian gastrointestinal tract. These studies also include bioavailability of the unchanged compound and hydrolysis or digestion products, the pattern and rate of distribution of absorbed substances, and the mode and excretion/elimination of parent as well as identified metabolites. Biotransformation: These studies include metabolism of the parent compound, jf absorbed as such, and of its products if they are not normal dietary or body constituents. Effects on enzymes and other biochemical parameters: The effects of absorbed substances and/or their metabolites on cellular and tissue enzyme production and morphology, chemical constitution, enzyme activity, and physiochemical
645
state are also evaluated while performing a toxicological evaluation of the compound. 2. TOXICOLOGICALSTUDIES These studies generally comprise routine acute studies, short-term feeding studies, long-term feeding studies, and studies on specific effects [e.g., carcinogenicity, genotoxicity, and reproduction toxicity (development toxicity and teratogenicity)]. Sometimes, these routine studies indicate the need to examine particular target organs or tissues; such studies are classified as special studies. Special studies are designed to test specific effects, such as neurotoxicity, immunotoxicity, allergenicity, cardiovascular effects, cataractogenicity, delayed neuropathy/neurotoxicity, acute neurotoxicity, development neurotoxicity, immune responses, macromolecular binding, photosensitization, and thyroid functions. Several parameters are of special consideration for OP and CM pesticides.
a. Delayed Neurotoxicity/Neuropathy Since 1974, delayed neurotoxicity has been discussed in a series of JMPR meetings. In 1987, JMPR recommended that in view of the presence of optical isomers of OP esters, two types of studies should be conducted on chemicals suspected of being neurotoxic (WHO, 1990). The first is the use of a suitable sensitive species (usually the adult hen), in which test substance is administered at two acute exposures (separated by 21 days) to atropineprotected animals at a level at or above the LDs0 of the compound. Observations on body weight, ataxia, and signs of delayed neurotoxicity are made while the animals are alive. At termination, usually 42 days after the first dose, histopathological examination of the brain, spinal cord, and proximal and distal sections of (usually) the sciatic nerve is performed. Data from this type of test suffer from two major drawbacks: The evaluation is often subjective, and a negative result cannot be graded. The second type of test is the determination of neurotoxic esterase activity (NTE). In its simplest form, this involves treatment of the adult hen with a single maximum tolerated dose of the test substance and subsequent assay of the brain enzyme after the time of peak inhibition but before substantial resynthesis of new enzyme has occurred (Johnson, 1982). The time of peak inhibition, which can be from 3 to 48 hr postdosing (and is determined by the pharmacokinetics of the compound), can often be assessed by observation of the time of onset of cholinergic signs. The threshold level of NTE inhibition at this early stage, which correlates with delayed neurotoxicity, is approximately 80%. No clinical signs are associated with an inhibition of 60% or less. When multiple determinations of NTE are made during chronic exposures, plateau levels are observed after 2 or 3 weeks. If inhibition of NTE in the brain and spinal cord is less than 50%, delayed neuropathy does not occur.
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b. Acute Neurotoxicity (ACHE Inhibition) It is established that the correlation between AChE inhibition (AChEI) in erythrocytes and that in the nervous system is usually unknown, and data on brain AChEI are considered to be of greater value than those on erythrocytes in assessing the cholinergic effects of cholinesterases (ChEs). However, in the absence of measurements of brain AChE, those of erythrocyte AChE serve as a better indicator of toxicity than those of plasma ChE activity. In vitro kinetic studies may be necessary for pesticides with antiesterase activity. Results of these studies in different species may be combined with in vivo study findings to establish ADIs for these compounds. Guidelines issued by JMPR have drawn attention to the currently used methods for the determination of ChE activity that may lead to erroneous conclusions when applied to rapidly reversible ChE inhibitions (e.g., N-methyl and N,Ndimethyl CMs). It has been suggested that in vitro kinetic studies should be made to elucidate the nature of reversible inhibition reactions and the results obtained from in vivo studies should be interpreted cautiously. The method for the determination of ChE inhibition of CMs is inadequate. Occasionally, data are inconsistent with respect to dose and the degree of ChE inhibition because CMs are reversible inhibitors of ChE with a short duration of action. Because of the reversible inhibition of the enzyme by dilution, as would occur during the preparation of the assay, inhibition cannot be accurately measured. JMPR has stressed that in order to permit evaluation of ChE inhibition by CMs in vivo, special care is required in reporting all details of such studies. CM-induced ChE inhibition studies should utilize minimal dilution during the preparation of the assay, minimal incubation times, and minimal times between blood sampling and assay (Ellman et al., 1967). It is recommended that 1. The information on delayed neurotoxicity appears to follow a dose-response relationship. Thus, with an adequate margin of safety, an ADI can be allocated. 2. Delayed neurotoxicity testing should be conducted routinely for OPs. However, it need not be done for monomethyl CMs, phosphinates, or sulfonates. 3. Tri-O-cresyl phosphate is recommended as a positive control substance only for OPs. 4. The NTE assay should be included in the database for OP evaluations. 5. While making assessment of effects on peripheral nervous tissues, comparison of the dose-response curves for erythrocyte AChE and brain AChEI and the occurrence of clinical signs may aid in the establishment of no-observable-adverse-effect levels (NOAELs). 6. For the purpose of establishing ADIs, the inhibition of plasma and brain butyrylcholinesterase (BuChE) is not a toxicologically significant effect because there is no evidence that BuChE inhibition has any adverse effect. It can be used as an indicator of absorption of the inhibitor
and, as such, is still a useful tool for monitoring occupational exposure. Data on statistically significant inhibition of BuChE activity should therefore always be induced. 7. For brain and erythrocyte ACHE, statistically significant inhibition by 20% or more represents a clear toxicological effect. Less than 20% or statistically insignificant inhibition indicates that a more detailed analysis of the data should be undertaken. The toxicological significance of these findings should be determined on a case-by-case basis. Considerations affecting such determinations include inter alia the shape of the dose-response curve, assay variability, and correlation with clinical signs. For neurotoxic compounds, an acute neurotoxicity study is required, unless short-term studies include a detailed neurotoxicological assessment.
c. Developmental Neurotoxicity In recent years, developmental neurotoxicity tests for assessing the safety of OP and CM pesticides to developing fetuses, infants, and children have been performed. The objective of these tests is to examine the use of such studies in the establishment of ADIs and acute reference dose (RfD). JMPR has recommended that if the toxicological profile of a chemical indicates developmental neurotoxicity end points, appropriate testing parameters should be incorporated into a multigeneration study of reproductive toxicity. While assessing such effects, the chances of introduction of artifacts due to stress resulting from directly dosing the pups and bolus (gavage) administration should be kept in mind (JMPR, 2002a).
3. OBSERVATIONSIN HUMANS Observations in humans are useful for assessing the relevance of the results of animal studies and for confirming ADIs. Studies dealing with epidemiological surveys, clinical experience, anecdotal observations, health effect studies relating to occupational exposure, reports of abuse, and volunteer studies measuring intolerance provide important observations.
V. A S S E S S M E N T OF E N D POINTS The overall objective of toxicological evaluation is to determine a NOAEL, based on consideration of the total toxicology database, that is utilized in conjunction with an appropriate safety factor to determine the ADI. The ADI has been defined as follows (JMPR, 1975): "ADI" of a chemical is the daily intake which, during an entire lifetime, appears to be without appreciable risk to the health of the consumer on the basis of all the known facts at the time of the evaluation of the chemical by the JMPR. It is expressed in milligrams of the chemical per kilogram of body weight. The initial stage of the evaluation is a critical examination of individual studies. In some cases, a study initially
CHAPTER 44 considered to be of marginal value in fact is useful when considered in the context of the entire database. Integration of the results from all studies permits an appraisal of the toxicity of the compound. Data from acute oral studies are rarely considered to be relevant to the establishment of the ADI. However, such data may provide information that permits a ranking of the sensitivity of different species and assist in the selection of dose levels in subsequent studies. Studies also indicate types of pharmacological activities, degree of absorption, or potential target organs. JMPR has occasionally required additional acute data to determine the relative toxicity of salts of a pesticide or required further metabolic studies to determine species differences in acute toxicity. Short-term feeding studies were considered to be the basis for the determination of ADIs for a number of compounds evaluated by JMPR before long-term toxicity studies were indicated to be an essential part of the toxicological database for evaluating the safety of pesticides. Currently long-term feeding studies have become an essential part of the toxicological database, and the major use of the data from short-term toxicity studies has been to determine suitable dose levels to be utilized in long-term and reproduction studies. However, multigeneration reproduction and teratogenicity studies have also been used for establishing ADIs for certain compounds (WHO, 1990). For carcinogenicity studies, a statistically significant difference between experimental and control groups is considered in light of its biological relevance. Thus, an increased incidence of a rare tumor type in treated animals may be of concern even if the incidence is not significantly different statistically from that in the concurrent control animals. Conversely, a statistically significant change in an isolated parameter (e.g., erythrocyte count) is usually not considered to be biologically relevant unless supported by changes in other parameters (e.g., bone marrow or spleen histopathology or methemoglobin formation). In interpreting carcinogenicity data, JMPR bases its evaluations on the threshold concept, which is the basis for evaluating most other toxicological effects (WHO, 1974). In assessing tumor incidence, benign and malignant tumors have been considered as separate entities in the majority of cases (WHO, 1987). Sometimes, the use of more than one species for the same type of toxicity study may complicate interpretation in those cases in which an effect occurs in one species but not in a second species or in which one species is much more sensitive to the agent than the other species. In such cases, it is often difficult to determine the most appropriate species for extrapolation to man. Generally, unless adequate data are available to indicate the most appropriate species (usually comparative pharmacokinetic or pharmacodynamic data), the most sensitive species (i.e., the species in which the adverse effect occurred at the lower dose) is used in determining the NOAEL and allocating the ADI.
9OP and CM Pesticide Residues in Food VI. E X T R A P O L A T I O N
647
O F DATA
AND DETERMINATION OF ADIs The objective of the safety evaluation of pesticide residues in food is to determine the ADIs of pesticides that will not result in adverse effects at any stage in the human life span. Since in the majority of cases data on humans are inadequate to permit such a determination, effects observed in other species must be extrapolated to man. This approach is used to determine the NOAEL from the experimental animal data or, preferably, from data in humans, if available. Generally, the 100-fold safety factor is used as the starting point for extrapolating animal data to humans and may be modified depending on the data that are available and the various concerns that arise when considering these data. The safety factors generally involved in establishing an ADI also serve to provide assurance that exposure exceeding the ADI for short time periods is unlikely to result in any deleterious effects on health. However, consideration should be given to the potentially acute toxic effects that are not normally considered in the assessment of an ADI. When relevant human data are available, the 10-fold factor for interspecies variability may not be necessary. However, relatively few parameters are studied in humans in the assessment of pesticide safety, and data on carcinogenicity, reproduction, and chronic effects are rarely available. Consequently, JMPR rarely utilizes safety factors as low as 10-fold. ADIs of selected OP and CM pesticides are summarized in Table 2. It is evident that certain factors may serve to increase and others to decrease the choice of the final safety factor. The total weight of evidence has to be considered when determining the appropriate safety factor to be used and the determination of safety factors must be considered on a case-by-case basis. If the metabolites in food commodities are qualitatively and quantitatively the same as those observed in laboratory test species, the ADI would apply to the parent compound as well as the metabolite(s). If the metabolite(s) is not identical or not present at the same order of magnitude, separate studies on the metabolite(s) may be necessary. When one or several pesticides are degradation products of another pesticide, a single ADI may be appropriate for the pesticide and its metabolites (e.g., oxydemeton-methyl, demeton-S-methyl sulfone, and demeton-S-methyl) (IPCS, 2003). In 1988, JMPR recommended that temporary acceptable daily intakes should not be allocated for new compounds and that an ADI should not be allocated in the absence of an adequate database (FAO/WHO, 1988). The monographs are published for all chemicals that are reviewed, regardless of whether an ADI is allocated. The data requirements are clearly specified for those chemicals with an inadequate database. The concept of the "conditional acceptable daily intake" adopted by the 1969 JMPR, was limited to those compounds for which the use was at that time considered essential but for which the toxicological database was
648
SECTION Vl
9 RiskAssessment &
TABLE 2.
Regulations
WHO/FAO Recommended Acceptable Daily Intakes (ADIs) of OP and CM Pesticides a
Compound
Year
ADI (mg/kg body weight)
Acephate
2002
0-0.01
N-acetyl glufosinate/NAGb
1999
0-0.02
Aldicarb
1995
0-0.003
Aminocarb
1979
No ADI
AMPA (metabolite of glyphosate)
1997
0-0.3
Azinphos-ethyl
1973
No ADI
Azinphos-methyl
1991
0-0.005
Bendiocarb
1984
0-0.004
Benomyl
1995
0-0.1
Bromophos
1977
0-0.04
Bromophos-ethyl
1975
0-0.003
Butacarboxim
1985
No ADI
Cadusaphos
1991
0-0.0003
Carbaryl
2001
0-0.008
Carbendazim
1995
0-0.03
Carbofuran
2002
0-0.002
Carbophenothion
1980
0-0.0005
Carbosulfan
1986
0-0.01
Chlorfenvinphos
1994
0-0.0005
Chlorpropham
2000
0-0.03
Chlorpyrifos
1999
0-0.01
Chlorpyrifos-methyl
2001
0-0.01
Chlorthion
1965
No ADI
Coumaphos
1990
No ADI
Crufomate
1968
0-0.1
Demeton
1984
No ADI
Demeton-S-methyl and related compounds c
1989
0-0.0003
Dialifos
1982
ADI withdrawn
Diazinon
2001
0-0.002
Dichlorvos
1993
0-0.004
Dimethoatea
1996
0-0.002
Dioxathion
1968
0-0.0015
Disulfoton
1996
0-0.0003
Edifenphos
1981
0-0.003
Ethephon
2002
0-0.05
Ethiofencarb
1982
0-0.1
Ethion
1990
0-0.002
Ethoprophos
1999
0-0.0004
Etrimfos
1986
0-0.003
Fenamiphos
2002
0-0.0008
Fenchlorphos
1968
0-0.01
Fenitrothion
2000
0-0.005
Fensulfothion
1982
0-0.0003
Fenthion
1997
0-0.007
Formothion
1996
ADI withdrawn
Glufosinate ammoniume
1999
0-0.02
(continues)
CHAPTER 44
TABLE 2.
9OP and CM Pesticide Residues in Food
(continued) ADI (mg/kg body weight)
Compound
Year
Glyphosate b
2004
0-1
Isofenphos
1986
0-0.001
Leptophos
1978
Temporary ADI withdrawn
Malathion
1997
0-0.3
Mecarbam
1986
0-0.002
Methacriphos
1990
0-0.006
Methamidophos
2002
0-0.004
Methidathion
1997
0-0.001
Methiocarb
1998
0-0.02
Methomyl
2001
0-0.02
Mevinphos
1997
0-0.0008
Monocrotophos
1995
0-0.0006
Omethoate
1996
ADI withdrawn
Oxamyl
2002
0-0.009
Oxydemeton-methyl/demeton-S-methyl
2002
0-0.0003
Parathion
1995
0-0.004
Parathion-methyl
1995
0-0.003
Phenthoate
1984
0-0.003
Phorate
2004
0-0.0007
Phosalone
2001
0-0.02
Phosmet
1998
0-0.01
Phosphamidon
1986
0-0.0005
Phoxim
1999
0-0.004
Primicarb
1983
0-0.02
Pirimiphos-methyl
1992
0-0.03
Profenofos
1990
0-0.01
Propamocarb
1986
0-0.1
Propham
1992
No ADI
Propoxur
1989
0-0.02
Pyrazophos
1992
0-0.004
Terbufos
1989
0-0.0002
Thiodicarb
2000
0-0.03
Thiometon
1979
0-0.003
Thiophanate-methyl
1998
0-0.08
Tolcfos-methyl
1994
O-0.07
Triazophos
2002
0-0.001
Trichlorfon (metrifonate)
2000
0-0.02
Trichlornate
1971
No ADI
Vamidothion
1988
0-0.008
sulfoxide c
aData from IPCS (2003) and JMPR (2004). bMetabolite of glufosinate ammonium. CGroup ADI, alone or in combination, evaluated under oxydemeton-methyl/demetonS-methyl sulfoxide/sulfone. dSum of dimethoate and omethoate as dimethoate. eADI applies to glufosinate, N-acetyl glufosinate, and 3-[hydroxy (methyl)phosphinoyl] propionic acid, alone or in combination.
649
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incomplete (FAO/WHO, 1970). This concept, which is unacceptable, has been abandoned. Long-term dietary intakes are expressed as percentage of the ADI for a 60-kg person, with the exception of the intake calculated for the Far East, for which a body weight of 55 kg is used (Codex, 1997b). The detailed procedure for calculations of long-term dietary intake can be found in Annexure 3 of JMPR (2002a).
VII. PREDICTING DIETARY INTAKE O F PESTICIDE RESIDUES The FAO expert panel of JMPR (WHO, 1997a) holds the responsibility for reviewing pesticide use pattems, data on the chemistry and composition of pesticides, and methods of analysis of pesticide residues and for recommending MRLs that may occur in food commodities following the use of pesticides according to GAPs. The MRL has been defined as follows: "MRL" is the maximum concentration of a pesticide residue (expressed as mg/kg), recommended by the Codex Alimentarius Commission (Codex) to be legally permitted in or on food commodities and animal feeds. MRLs are based on GAP data and foods derived from commodities that comply with the respective MRLs are intended to be toxicologically acceptable. Extraneous maximum residue limit (EMRL) refers to a pesticide residue or a contaminant arising from environmental sources (including former agricultural uses) other than the use of a pesticide or contaminant substance directly or indirectly on the commodity. It is the maximum concentration of a pesticide residue or contaminant that is recommended by the Codex (1997a) to be legally permitted or recognized as acceptable in or on a food, agricultural commodity, or animal feed. The concentration is expressed in milligrams of pesticide residue or contaminant per kilogram of the commodity. "GAP" includes the nationally authorized safe uses of pesticides under actual conditions necessary for effective and reliable pest control. It encompasses a range of levels of pesticide applications up to the highest authorized use, applied in a manner which leaves a residue, and is the smallest amount practicable. Authorized safe uses are determined at the national level and include nationally registered or recommended uses, which take into account public and occupational health and environmental safety considerations. Actual conditions include any stage in the production, storage, transport, distribution, and processing of food commodities and animal feed. MRLs which are primarily intended to apply in international trade are derived from estimations made by the JMPR following: (a) toxicological assessment of the pesticide and its residue; and (b) review of residue data from supervised trials and supervised uses including those reflecting national food agricultural practices. Data from supervised trials conducted at the highest nationally recommended, authorized, or registered uses are included. In order to
accommodate variations in national pest control requirements, Codex MRLs take into account the higher levels shown to arise in such supervised trials, which are considered to represent effective pest control practices. Estimates of dietary intake play an important role in ensuring safe food for consumers throughout the world. Consideration is given to the identity, purity, stability, quality of data, structure-activity relationship, and GAPs used during the application of pesticides. Generally, most treated crops contain residues well below the MRL at the harvest: Residues are usually reduced during storage, preparation, commercial processing, and cooking, and it is unlikely that every food for which MRL is proposed will have been treated with the pesticide over the lifetime of the consumer. Therefore, MRL represents the maximum residue level that is expected to occur in a commodity following the application of pesticide according to GAP. As per international standards, five regional diets (Middle Eastern, Far Eastern, African, Latin American, and European) are considered, and the average daily food consumption values are used in predicting pesticide residue intake for long-term hazard. Consideration of the various dietary residue estimates and determinations at both the national and international level in comparison with the ADI should indicate that foods complying with Codex MRLs are safe for human consumption.
A. MRLs for Milk and Milk Products For a "milk product" with a fat content less than 2%, the MRL applied should be half that specified for milk. The MRL for "milk products" with a fat content of 2% or more should be 25 times the MRL specified for milk, expressed on a fat basis. The residues in whole milk are usually expressed on a fat basis (4% fat).
B. MRLs for Meat and Meat Products Where adequate data are not available, MRLs are specified for the fat portion of animal products. Consumption data for mammalian and poultry meat are corrected on the assumption that mammalian meat contains 20% fat and poultry meat (with adhering skin) contains 10% fat. Dietary intake calculations are based on MRLs (fat) for fat-soluble pesticides and for non-fat-soluble pesticides. For mammalian animals, 20% of the meat consumption is used for fat-soluble pesticides and 80% of meat consumption is used for non-fat-soluble pesticides. For poultry, the corresponding values are 10 and 90% (JMPR, 2002a).
C. MRLs for Fruits and Vegetables For residues in whole commodities, including the edible portion, there is no universal distribution ratio because the amount of residue in the edible portion varies from pesticide
CHAPTER 44 to pesticide. To calculate the international estimated daily intake for assessing exposure to pesticide residues, the supervised trials median residue level (SMTR) is used. In practice, the STMR is estimated directly from residue data on the edible portion. Residue data on the edible portion are obtained to refine estimates of dietary intake for commodities such as citrus fruits, bananas, pineapples, kiwifruits, and other fruits with inedible peel; cereals and oilseeds; and cucurbits with inedible peel, such as melons. The consumption of the edible portion of food commodities should be used in estimating intakes rather than that of entire commodities. Correction factors currently used for the weight of the edible portion include 30% reduction for both citrus and bananas. In some countries, edible portions of some food commodities may vary, and their national governments may consider local dietary habits when using such data (WHO, 1997a). Risks associated with long-term dietary intake are calculated by multiplying the concentrations of residues (STMRs or STMR-P values or recommended MRLs) by the average daily per capita concentration estimated for each commodity on the basis of the GEMS/Food diet (WHO, 1997b, 1998). For risk assessment of long-term hazards, STMRs should be used together with mean reduction or mean concentration factors. However, data on the effects of processing on residues are needed.
D. MRLs and EMRLs in and on Spices There is a proposal to develop guidance for the submission of monitoring data for setting MRLs and EMRLs for spices. Since most of the spices moving in international trade are produced by millions of small farmers of less than 10 ha, and usually by intercropping, the presence of residues is frequently associated with products used for pest control on the main crop rather than on the spices. For estimation of MRLs in or on spices, it will be more meaningful if the commodities are subgrouped on the basis of parts of plants from which they are obtained (JMPR, 2004).
E. MRLs for Metabolite(s) of Parent Compounds Information is often available on the conversion of a parent compound to a metabolite or degradation product of toxicological concern that is produced or persists in processed food (e.g., oxygen analogs of phosphorothiolates). Estimates of dietary intake should be made on the basis of information on the consumption of processed commodities, provided an ADI or other assessment of toxicological properties of the metabolite products has been made.
9OP and CM Pesticide Residues in Food
651
VIII. EVALUATIONS O F M I X T U R E S The possibility of pesticide interaction was recognized as early as 1961 when the FAO/WHO Meeting on Principles Governing Consumer Safety in Relation to Pesticide Residues recognized that "different pesticides and other chemicals are often absorbed simultaneously during occupational use, or in food, by man or animals" (FAO/WHO, 1962). A survey of data indicates that residues of more than one pesticide may be detected in food. This gives rise to concern over the possibility of unanticipated interactions between such residues leading to adverse toxicological effects. There are, of course, a virtually unlimited number of combinations of pesticides on various crops. There are also a very large number of combinations of foods containing pesticide residues. Therefore, man could be exposed to unlimited chemicals leading to unlimited possibilities, and there is no special reason why the interactions of pesticide residues (which are at very low levels) should be highlighted as being of particular concern. Where isomeric mixtures exist, the ratio of isomers in the test material must be clearly specified since it has been amply documented that different isomers frequently have different toxicological activities. Second, very little data on these interactions are available, and the data obtained from acute potentiation studies are of little value in assessing ADIs for man. However, there is a need for further data on interactions of pesticides with each other and with other common contaminants (e.g., metals and mycotoxins) of food (FAO/WHO, 1988).
IX. ACUTE DIETARY RISK ASSESSMENT The establishment of an acute RfD forms the basis of shortterm intake and a short-term risk assessment. The acute RfD is defined as an estimate of the amount of a substance in food and/or drinking water, normally expressed on a body weight basis, that can be ingested in a period of 24 hr or less without appreciable health risk to the consumer on the basis of all known facts at the time of the evaluation. In other words, it is a short-term dietary intake assessment of pesticide residue intake over a single day (JMPR, 2002a). Since 1995, JMPR has established an acute RfD for a number of pesticides (Table 3). The meeting has followed the basic principle and an acute RfD has been considered on a case-by-case basis for all compounds that are evaluated. For this estimate, two population groups have been modeled: the general population and children (1-6 years of age). There may be certain use patterns that are unlikely to give rise to residues, and in such cases a full intake assessment may not be necessary. Most of the scientific concepts used for the establishment of ADIs apply equally to acute RfDs. The decision to establish an acute RfD is normally
652
S E CTI O N VI 9 Risk A s s e s s m e n t & Regulations
TABLE 3. WHO/FAO Recommended Acute Reference Dose (RE)) of OP and CM Pesticides a
Compound
Year
Acute RtI) (mg/kg body weight)
Acephate Aldicarb Carbaryl Carbofuran Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Demeton-S-methyl and related compounds Diazinon Disulfoton Ethephon Ethoprophos Fenitrothion Fenthion Glufosinate ammonium b Glyphosate Methamidophos Methidathion Methiocarb Methomyl Mevinphos Monocrotophos Oxamyl Oxydemeton-methyl/ demeton-S-methyl sulfoxide c Parathion Parathion-methyl Phorate Phosalone Phosmet Primicarb Thiodicarb Thiophanate-methyl Triazophos
2002 1995 2001 2002 2000 1999 2001
0.05 0.003 0.2 0.009 0.03 0.1 Unnecessary
2002 2001 1996 2002 1999 2000 1997 1999 2004 2002 1997 1998 2001 1997 1995 2002
0.002 0.03 0.003 0.05 0.05 0.04 0.01 Unnecessary Unnecessary 0.01 0.01 0.02 0.02 0.003 0.002 0.009
2002 1995 1995 2004 2001 1998 2004 2000 1998 2002
0.002 0.01 0.03 0.003 0.3 0.02 0.1 0.04 Unnecessary 0.001
aData from IPCS (2003) and JMPR (2004). bAcute RfD applies to glufosinate, N-acetyl glufosinate, and 3-[hydroxy (methyl)phosphinoyl]propionic acid, alone or in combination. CGroup acute RfD, alone or in combination, evaluated under demetonS-methyl and related compounds. based on toxicological grounds because an acute RfD is a toxicological reference value. Therefore, the establishment of an acute RfD is considered for all substances. In considering whether an acute RfD for a pesticide is necessary, it is not advisable to take into account current agricultural prac-
tice and related residues for existing crop use because with different application rates or new applications on other crops (or other crop groups), higher residue values and/or higher dietary intakes may occur.
A. Toxicological Effects Relevant for Derivation of the Acute RfD A number of effects could be due to a single exposure to a compound. The following list is not necessarily comprehensive and the omission of a toxicological effect does not mean that it should be discounted when considering the establishment of an acute RfD: The effects could be clinical signs, behavioral signs, pharmacological effects, or effects on target organs observed in single-dose studies (e.g., miosis) or early in studies with repeated doses such as acute neurotoxicity (e.g., acute delayed polyneuropathy or inhibition of AChE activity); clinical chemical or hematological effects (e.g., methemoglobin formation, hemolysis, or anemia); immunotoxicity, kidney toxicity, liver toxicity, and reproductive or development effects (e.g., teratogenicity or development neurotoxicity); or endocrine effects or other biochemical alterations observed in studies with repeated doses that may conceivably be elicited by a single exposure. Selection of the most relevant end point providing the lowest NOAEL is the most appropriate method (JMPR, 2004). Direct effects on the gastrointestinal tract should be assessed carefully to determine their relevance to human exposure. Are they due to irritation or a pharmacological action? Are they relating to the method of administration (present with bolus dosing but not by dietary admixture)? The relevance of these effects should be considered on a case-by-case basis. The route of administration should be considered carefully to minimize influences that are not relevant to the intake of residues (e.g., effects induced by gavage or the vehicle).
B. Determination of Acute RfD As discussed previously, toxicological effects will help to determine NOAELs. To determine acute RfD, safety factors higher or lower than the default values of 100 and 10 depending on animal or human data are used by JMPR (2000). It is considered inappropriate to have a higher ADI value than the acute RfD. If the previously derived ADI is higher than the acute RfD, then the complete database has to be reevaluated and the reference values need to be reconciled. Such a situation can occur for a number of reasons, such as the availability of additional studies or when compounds produce more severe effects when given by gavage than in the diet. Because short-term consumption data are for a 24-hr period, this is a precautionary approach for rapidly reversible effects (e.g., inhibitors of ChE activity by CM) for which the acute RfD is applicable to a shorter period.
CHAPTER 4 4
X. CONCLUSIONS The hazards associated with the use of OP and CM pesticides are evaluated by assessing the toxicity and by estimating the magnitude of exposure from sources in the workplace, the environment, food, and water. The need to balance the risk to man and the environment against benefits associated with pesticide use is an important issue. The conduct of toxicity studies (GLP) and agricultural practices (GAP) are important considerations for consistent procedures and documentation of studies so that all aspects can be recorded and verified. A joint panel of WHO/FAO (JMPR) is responsible for reviewing toxicological and residue data and estimating (where possible) an ADI and acute RfD for humans and MRLs for residues in foods. The overall objective of toxicological evaluation is to determine a NOAEL, based on consideration of the total toxicology database, that is utilized in conjunction with an appropriate safety factor to determine the ADI. The use of GAP helps to determine MRL values for pesticide residues in food. Integration of the results from all studies permits an appraisal of toxicity of the compound. The establishment of an acute RfD forms the basis of short-term intake and short-term risk assessment. Approximately 100 OP and CM pesticides have been evaluated to derive their MRL values in foods and ADIs for humans. Innumerable factors, such as crop pattern, food habits, processing conditions, species variations, and multiple uses of pesticides and toxic metabolites, complicate the determination; however, human data on a pesticide, whether from volunteer studies or from other investigations of human exposures in the workplace or environment, can be extremely valuable in placing the animal data in context and, when available, should always be evaluated even when they are not used to derive ADIs.
References Codex (1997a, June). Codex maximum residue limit for pesticides and extraneous residue limits. Twenty-second Session, Codex Alimentarius Commission report. World Health Organization, Geneva. Codex (1997b). Codex Alimentarius Commission report, CX/PR 98/5. World Health Organization, Geneva. Ellman, G. I., Courtney, K. D., Andres, V., Jr. and Featherstone, R. M. (1967). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Food and Agriculture Organization/World Health Organization (1962). Principles governing consumer safety in relation to pesticide residues, FAO Plant Production and Protection Division Report, No. PL/1961/ll; WHO Technical Report Series No. 240. World Health Organization, Geneva. Food and Agriculture Organization/World Health Organization (1970). Pesticide residues in food. Report of the 1969 joint meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Group on Pesticide Residues,
9OP and CM Pesticide Residues in Food
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FAO Agricultural Series No. 84; WHO Technical Report Series No. 458. World Health Organization, Geneva. Food and Agriculture Organization/World Health Organization (1988). Pesticide residues in f o o d - 1988. Report of the joint meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues, FAO Plant Production and Protection Paper 92. Food and Agriculture Organization, Rome. Gupta, E K. (2004). Pesticide exposure u Indian scene. Toxicology 198, 83-90. International Programme on Chemical Safety (2002). The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification. World Health Organization, Geneva. International Programme on Chemical Safety (2003). Inventory of IPCS and other WHO pesticide evaluations and summary of toxicological evaluations performed by the JMPR, WHO/PCS/02.3. World Health Organization, Geneva. Johnson, M. K. (1982). The target for initiation of delayed neurotoxicity by organophosphorus esters: Biochemical studies and toxicological applications. Rev. Biochem. Toxicol. 4, 141-212. Joint FAO/WHO Meeting on Pesticide Residues (1975). Report of the 1975 Joint FAO/WHO Meeting on Pesticide Residues, FAO Plant Production and Protection Series No. 1; WHO Technical Report Series No. 592. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2000). Proposed guidance for interpretation of data generated in studies with single oral doses (for use in establishing acute RfDs for chemical residues in food and drinking water), FAO/WHO Pesticide Residues in Food, Report 2000, Annex 5, pp. 197-198. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2002a). Pesticide residues in food. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticides Residues, Rome, September 16-25. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2002b). Pesticide residues in food. Evaluations (Part II-Toxicological) of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticides Residues, Rome, September 16-25. World Health Organization, Geneva. Joint FAO/WHO Meeting on Pesticide Residues (2004). Pesticide residues in food. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticides Residues, Rome, September 19-29. World Health Organization, Geneva. Organization for Economic Co-operation and Development (1987). Guidelines for Testing of Chemicals by Organization for Economic Co-operation and Development. Organization for Economic Co-operation and Development, Paris. Sandhu, H. S., and Brar, R. S. (2000). Textbook of Veterinary Toxicology. Kalyani, New Delhi. World Health Organization (1974). Assessment of the carcinogenicity and mutagenicity of chemicals, Report of a WHO Scientific Group, WHO Technical Report Series No. 546. World Health Organization, Geneva.
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World Health Organization (1987). IPCS Environmental Health Criteria 70: Principles for the safety assessment of food additives and contaminants in food. World Health Organization, Geneva. World Health Organization (1990). Principles for the toxicological assessment of pesticide residues in food, Environmental Health Criteria No. 104. World Health Organization, Geneva. World Health Organization (1997a). Guidelines for predicting dietary intake of pesticide residues, 2nd rev. edn., GEMS/Food document WHO/FSF/FOS/97.7. World Health Organization, Geneva.
World Health Organization (1997b). Food consumption and exposure assessment of chemicals, report of a FAO/WHO consultation, February 10-14. World Health Organization, Geneva. World Health Organization (1998). GEMS/Food regional diets. Food Safety Issues, WHO/FSF/98.3. World Health Organization, Geneva. World Health Organization (2000). Guidelines for the Preparation
of Toxicological Working Papers for the WHO Core Group of the JMPR. World Health Organization, Geneva.
Aquatic Life & Wildlife
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CHAPTER 4
5
Aquatic Toxicity of Carbamates and O rgano p hosp hates ARUN K. RAY AND MANIK C. GHOSH Bose Institute, Calcutta, India
environment cannot be ignored. The health hazards of pesticides are well recognized. It is a global problem for agricultural workers and rural communities. For the safeguard of human health, the potential adverse effects of different pesticides were studied in different mammalian models. There a~'e many routes by which pesticides can reach the aquatic environment Foy and Bingham, 1969; Nicholson, 1969; Edwards, 1973a,b):
I. I N T R O D U C T I O N Use of pesticides in the control of agricultural pests was a turning point in human civilization that ushered in the new horizon in production of food grain necessary for survival of humankind. Pest coevolution with the host plant sometimes seems to be more advanced and complex, interfering with and disrupting human civilization from time immemorial. The discovery of pesticides resulted in millions of tons of global food grains being saved every year. A wide spectrum of pesticides have contributed to the control of a variety of pests in the agricultural field. In modern times, toxicologists have been designing pesticides based on the physiological or anatomical characteristics of a particular pest so that use of a particular pesticide in the field kills a particular pest without harming the other fauna (O'Brein, 1967; Winteringham, 1969). The deciphering of the pesticidal properties of dichlorodiphenyltrichloroethane (DDT) by Paul Muller in 1939 marks the birth of modem insecticide chemistry. This served as the cornerstone for its subsequent development. Humans have faced difficulties in cohabitation with insects since the beginning of agriculture in 8000 BC. Sumerians first used insecticide as sulfur compounds in 2500 BC to combat insects and mites. In 1936, the German chemist Gerhard Schrader synthesized a variety of organophosphorus compounds with potential use as insecticides. After organochlorines were phased out, the organophosphate (OP) and carbamate (CM) pesticides became the preferred choice. This chapter focuses on the toxicity of OPs and CMs on aquatic system.
1. Surface runoff and sediment transported from treated soil 2. Industrial waste discharged from factory effluents 3. Direct application as aerial spray or granules to control pests inhabiting water 4. Spray drift from normal agricultural operation 5. Municipal waste discharge Runoff is generally considered to represent the major movement of pesticides in the aquatic environment. During runoff, pesticides remain suspended in the runoff water and are transported to the aquatic ecosystem (Malins and Ostrander 1991). Nicholson and coworkers conducted an extended study on the routes of pesticide movements into the water. During runoff, pesticides may be adsorbed on the eroding soil particles suspended in runoff water. Heavy rainfall immediately after application of pesticides has a higher potential for pesticide transport. The effects of pesticides on aquatic life may be acute, resulting in mass mortality of fishes, chronic changes in their behavior, or reductions in survival, growth, and reproduction (Malins and Ostrander, 1991). The problem of pesticide contamination is a unique environmental problem because pesticides are deliberately introduced into the environment for beneficial purposes. One of the most disastrous effects of runoff was reported in the Punarbhaba River in West Bengal, India, where several tons of commercially important fishes were killed. Pesticides in general are also toxic to many nontarget organisms and cause ecological imbalance by indiscriminate killing of
II. P E S T I C I D E S IN T H E A Q U A T I C ECOSYSTEM: ACCUMULATION AND BIOMAGNIFICATION Although the use of pesticides has become indispensable due to their beneficial effect on crop yield, forest protection, and domestic living, their potential threat to the Toxicology of Organophosphate and Carbamate Compounds
657
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
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SECTION Vll. Aquatic Life & Wildlife
aquatic insects, worms, and mollusks by contaminating the soil and water, thereby disturbing the general ecosystem. The distribution of pesticides in water influences the pathway of biological uptake. The quantity accumulated by each biological entity is dependent on the chemical properties of the pesticides and the physiology and behavior of the organisms. Laboratory studies have shown that 14C-labeled DDT can be absorbed from water quickly by Atlantic salmon and is distributed to the major organs in approximately 5 min (Premdas and Anderson, 1963). When pesticides are present for a short time in water, adsorption onto water and food organisms may play an important contributory role in determining the amount of residue found in fishes (Chadwick and Brocksen, 1969). Pesticides in the aquatic environment constitute both direct and indirect hazards to aquatic organisms and humans. The organism at the end of the food chain may be exposed to a higher concentration of the pesticide than originally found in the environment due to biomagnification. This sort of magnification mechanism is of greater importance in aquatic than in terrestrial organisms. All pesticides can be categorized as organochlorines, pyrethrins/pyrethroids, OPs, and CMs according to their chemical nature. Also, the mechanism of action varies depending on their chemical nature. Among them, CMs are the most widely used pesticides throughout the world since organochlorines have longlasting residue persistence (Parmar and Dureja, 1990; Hayes, 1982) and OPs are extremely toxic and pose a delayed neurotoxicity problem (Gupta, 1994). CM pesticides are preferred in agriculture more than OPs due to their rapid biodegradation, less bioaccumulation, and also due to their quick action compared to OPs and organochlorine (Corbett et al., 1984). Among the CMs, carbofuran is widely used in agriculture and forestry as a broad-spectrum systemic insecticide, nematicide, and acaricide that kills insects, nematodes, and mites on contact or after ingestion (Gupta, 1994). It is used for soil and foliar pests of field, fruits, vegetables, and forest crops. Carbofuran is available in liquid and granular form (Worthing, 1987; U.S. Environmental Protection Agency, 1991).
III. D E T E R M I N A T I O N O F P E S T I C I D E R E S I D U E IN W A T E R The determination of pesticide contents in water, organic substrates, sediments, and animal tissues depends on the chemical methods. The solid materials of plant and animal tissues are homogenized and extracted with acetone or hexane, evaporated to a small volume for microdetermination by various chromatographic methods. The concept of in situ bioassays is mainly based on exposure of test animals in the contaminated water and determination of
percentage survival. Most simple is the process of exposing fish to test the toxicity of water. Cages containing the experimental fish are hung in the contaminated water column or anchored at the bottom. Mortality is measured after exposure of 96 hr or longer.
IV. M E T H O D S T O D E T E C T A C U T E A N D DELAYED TOXICITY Several invertebrate organisms, among approximately 1 million species, have been selected to test the adverse effects of toxicants. These invertebrates include snail (Helisoma trivolvis), the clams (Spanerium, Corbicula, and Musculium), the amphipods (Hyalella azteca, Gammarus lacustris, and G. pulex fossarum), the burrowing mayfly larvae (Hexagenia rigida and H. bilineta), and larvae of the midge (Chironomus tentans). Rarely, marine amphipods or freshwater benthic oligochaetes are used (Swartz et al., 1982; Bailey and Liu, 1980). Approximately 25 fish species are also used to test waterborne pesticides. The most frequently used fishes are common carp (Cyprinus carpio), rainbow trout (Salmo gairdnari), brown trout (Salmo trutta), goldfish (Carassius auretus), zebra fish (Brachydanio rerio), and bluegill (Lepomis macrochirus). Nevertheless, any type of fish may be used for pesticide toxicity. Acute toxicity is generally expressed as LC50/48 hr or LC5/48 hr (Mayer and Ellersiek, 1986). In fish, most of the toxic effects are expressed through inhibition of some hydrolytic enzymes, mainly acetylcholinesterase. Acute or delayed toxic effects of pesticides are measured in bioassays in aquarium organisms as follows: 1. The hatch rate (cumulative percentage hatch per treatment) and initiation of hatching from eggs of live female mayfly, Hexagenia, at 20~ are tested with different doses of particular pesticide and LCs0s are reported. 2. In sediments, tests for pesticides are performed using the Chironomus adult emergence test. The test is performed with 10-day-old second-instar larva at 20~ and the end point is the number of emerged adults collected in an Erlenmayer flask trap (Nebecker et al., 1984). 3. In the Daphnia magma life cycle test, 5-day old Daphnia are exposed for 10 days through maturation and release of young (three broods). Experimental specimen is fed at the rate of 2 mg/liter of algae every other day. For water or sediment toxicity, the total number of survivors is reported. 4. Ramshorn snail (H. trivolvis) is used to measure mortality, growth, and fecundity in successive generations. The snails are cultured according to standard conditions and exposed to various concentrations of a required pesticide (Flannagan, 1974).
CHAPTER 45 9Aquatic Toxicity of CMs and OPs 5. Crayfish (Orconectes virilis) exposed to different concentrations of pesticides are used for measuring intermolt phase and hemolymph calcium. The absence of elevation of calcium concentration in specimens exposed to pesticides in laboratory defines failure to molt. Hemolymph samples are taken directly from the heart and analyzed calcium biochemically (Leonhard, 1974). 6. Most common effects of pesticides in fish are monitored through changes in respiratory rate, fin and body movements, heartbeat, and response to light and touch. Chronic or sublethal effects are monitored through long-term toxicity tests, such as growth and development, timing and duration of hatch, or even ovarian failure. Fish eggs are also used for toxicity tests (McKim, 1977; McDonald, 1979). 7. Fish serum and brain cholinesterase activity, serum glucose, serum protein, or total serum lipid contents are measured biochemically to monitor the strength of pesticide exposure in fish (Lockhart et al., 1951). Since most OP pesticides are known to inhibit cholinesterase, monitoring of this enzyme activity is of prime importance. 8. Zebra fish (B. rerio) can be used for its small size, egg-laying performance, and short life cycle of approximately 75 days to monitor chronic toxicity and bioaccummulation (Lillie et al., 1979).
V. A C U T E T O X I C I T Y ( M A M M A L S VS F I S H ) Carbofuran is highly toxic by inhalation and ingestion and moderately toxic by dermal absorption (Hayes, 1982) in mammalian species. The LDs0 in experimental rat by oral exposure was reported to be 5.0 mg/kg (Ben-Dyke et al., 1970) and by inhalation 85 mg/m 3 (Tobin, 1970), whereas intraperitoneal exposure of 2 mg/kg was found to be lethal (Gupta and Kadel, 1989a). Following oral and parenteral exposure to carbofuran, experimental laboratory animals exhibit the onset of toxic signs such as salivation, chewing, and fine tremors within 5-15 min with frequent propensity; signs of maximal severity, including muscle fasciculation and convulsion, are evident from 15 min to 1 hr and persist for approximatey 2 hr. In general, toxic manifestations are characteristics of acetylcholinesterase inhibition, with parasympathetic preponderance exhibiting profuse salivation, lacrimation, miosis, hypothermia, muscle twitch and fasciculations, body tremors, and convulsion. Overall, carbofuran elicits toxic manifestation of central and peripheral nervous systems origin by overstimulation of muscarinic and nicotinic acetylcholine (ACh) receptors (Cambon et al., 1979; Gupta and Kadel, 1989a,b). In humans, symptoms of carbofuran toxicosis include salivation, diaphoresis, abdominal pain, chest tightness, drowsiness, dizziness, vomiting, muscular twitching, convulsions, and coma
659
(Tobin et al., 1970). Similar clinical signs are observed in other species due to acute carbofuran poisoning. Although carbofuran is a rapidly degrading pesticide in soil and water, its residue may persist in soil and water for several weeks, months, or up to a 1 year, depending on the environmental conditions (Finlayson et al., 1991). Results from a study conducted by the Environmental Hazards Assessment Group of California, Department of Food and Agriculture, revealed the presence of carbofuran in the runoff water from rice and sugar beet fields in agricultural drain and in the Sacramento River (Nicosia et al., 1990). Thus, carbofuran reaches the aquatic ecosystem from the agricultural field and aquatic organisms face the toxicity when adsorbed. During the past 30 years, extensive studies have been conducted on various species, regarding carbofuran inhibition of AChE but most studies were targeted on mammalian species rather than fish or other invertebrates. This chapter focuses on the toxicity of carbofuran on aquatic organisms in retrospect to the effect in comparison to the mammalian system. Second, aquatic organisms encounter the toxic effect of this pesticide as the first consumers of the ecological food chain rather the second consumer such as mammals. Bhattacharyya (1985) reported carbofuran-induced acetylcholinesterase (ACHE) inhibition in the fish (Channa punctatus and Anabas testudineus). Brain ACHE activity decreased significantly when these fish were exposed to carbofuran in laboratory conditions. Carbofuran-induced inhibition of AChE in juvenile goldfish (Carassius auratus) was reported by Bretaud et al. (2000). Brain and muscle AChE were inhibited significantly by 50 txg/liter (19-28%) or 500 txg/liter (85-87%) of carbofuran. Carbofuran inhibits AChE significantly in C. carpio exposed to a sublethal dose (Dembele et al., 2000). This inhibited enzyme activity can be recovered in time, indicating the reversibility of binding of this pesticide with the enzyme (Dembele et al., 1999). Toxic and teratogenic effects of carbofuran to Cyprinus carpio communis were reported by Pawar (1994) when the pesticide was administered to the ambient water. The percentage of hatching and survival of hatching decreased with the increasing concentration of pesticide. Abnormalities observed included deformed body curvature, eye pigmentation, enlargement of pericardial sacs, circulatory failure, loss of balance, and abnormal behavior. Carbofuran causes a significant decrease in blood hemoglobin and remarkable changes in erythrocytes in fish (Bhakthavathasalam, 1991). Carbofuran-induced histopathological and biochemical changes in the liver of the teleost fish Channa punctatus (Bloch) were reported by Ram and Singh (1988). Channa punctatus exposed to a safe dose (4.5 ppm) of the commercial pesticide carbofuran for 6 months exhibited a varying degree of histopathological changes, including cytoplasmolysis, nuclear pyknosis, and necrosis in the liver. In some regions of the liver, extensive degeneration of proliferative hepatocytes and induction of the tumor were indicative
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of carcinogenic action of this pesticide (Gopalkrisna and Ram, 1994). Heath et al. (1992) simulated the environment of the Sacramento River, which is constantly exposed to rice pesticide originating as surface runoff from the agricultural field. They showed inhibition of AChE activity of striped bass (Morone saxatilis) larvae exposed to water mixed with parathion, carbofuran, and molinate. Carbofuran is well known for extensive field killing of fish, wildlife, and invertebrates, and therefore protection of such sensitive species is recommended. Anton et al. (1993) reported the ecotoxicity of carbofuran to freshwater fish and algae, which demonstrated heavy mortality of Carassius auratus L. (Cyprinidae) and Chlorella pyrenoidosa, the important species in the tropic chain of the Spanish freshwater ecosystem. Singh and Garg (1992) showed the localization of the carbofuran in the body of Clarius batracus after its exposure to the pesticide. They demonstrated that carbofuran accumulates in the gills, liver, and kidney of the fish, with a residue range of 0.01-0.095%. Heath et al. (1993) reported toxic effects of carbofuran on Japanese medaka (Oryzias latipes) larvae exposed for 4 days in the rice field. Pesticide concentration was half of the 96-hr LCs0 dose level simulating a condition of rice field water runoff. This sublethal dose resulted in impairment of swimming speed and inhibited AChE activity. Newly hatched fathead minnow (Pimephales promelas) larvae were exposed for 4 days to two pesticides in water in a simulated condition of the Sacramento River during the striped bass spawning season, which coincided with real pesticide use in adjacent rice culture. Carbofuran and molinate were tested at two concentrations a higher level approximating one-half of the LCs0 and a much lower level of LCs0 as found in the Colusa basin drain. The higher concentration of carbofuran and molinate caused a reduction in swimming capacity and inhibited AChE activity, whereas the lower concentration showed no significant changes. The study indicates that regulated use of pesticide in the agriculture field may restrict the deleterious effects on the fauna through surface runoff (Heath et al., 1997). Rice field surface runoff containing carbofuran caused a decline in the population of striped bass in the Sacramento River by either direct killing or reducing food items such as mycids (Finlayson etal., 1993). Since 1980, when molinate was demonstrated to kill carp in agricultural drain, an intensive research effort has been under way to assess the impact of rice pesticide on the aquatic system in the Sacramento River and its delta. Laboratory bioassay indicated the presence of carbofuran or methyl parathion and also bufencarb in the fiver water, which were responsible for the deaths of aquatic organisms and early life stages of striped bass. After 1981, management protocol for pesticide use was executed, which resulted in a dramatic decrease in rice pesticide levels in the fiver and delta water. The nontoxic level of pesticide in the fiver was commensurate with the recovery
of striped bass and Chinuk salmon (Byard, 1999). The effect of carbofuran on the fish reproductive system has been elaborately studied in our laboratory (Chatterjee et al., 1997). The study showed impairment of the hypothalamohypophysial-gonadal axis in Heteropneustes fossilis after treatment with carbofuran. The effects included retarded ovarian maturity, reduced gonadosomatic index, an increase in stage-1 oocyte and decrease in stage-3 oocytes numbers, atrophy of gonadotrophs in the pituitary glands, and increased total lipid in liver but its loss in ovary along with essential fatty acids. Carbofuran-induced atresia in freshwater fish Colisa lalia has been reported by Sukumar and Karpagaganapathy (1992). Saxena and Mani (1987) reported testicular recrudescence in freshwater murrel Channa punctatus by carbofuran treatment. Neuroendocrine dysfunction by carbofuran treatment in freshwater murrel Channa punctatus has been reported by Ram and Singh (1988). With prolong carbofuran exposure (4 months at 4.5 ppm), the thyroid gland showed abnormalities including hypertrophy, hyperplasia, degeneration of follicular cells, and reduction in colloidal material. Carbofuran, which is washed into the water system in small quantities, induces adverse histopathological alterations in thyroid, depending on the age and body weight of the animal, possibly by acting directly on thyroid or through the hypothalamuo-pituitary-thyroid axis. This pesticide may influence the growth and protein conversion efficiency in freshwater fish such as Oreochromis mossambicus, Mystus vittatus, and Channa striatus, as reported by Vasanthi et al., (1990). Carbofuran at sublethal concentration alters the adenylate parameters in the blue gill sunfish (Lepomis macrochirus) (Horneiter etal., 1991). Adenylate parameters (ATE total adenylate nucleotides, and adenylate energy charge) decreased in gill, liver, muscle, and stomach. The adenylate parameter in the stomach is very much susceptible to the concentration of the carbofuran (0.2-30 mg/liter). An important study was performed by Zayed et al. (1988) in which they showed the distribution of 14C-labeled carbofuran residue and the parent compound in fish exposed to paddy pisiculture for 90 days. Carbofuran residue was present in the rice plant after 30 days of application. Fish showed the presence of carbofuran in the body even after 30 days of exposure without any sign of accumulation of an appreciable amount. In water, the parent compound was decreased by 10% after 30 days. Analysis of residue in fish, plant, and soil showed the presence of several metabolites (60% of residue) apart from the parent compound. Among the degradation products, 3-hydroxycarbofuran and carbofuran phenol were identified as major metabolites. An excellent study was conducted on the transient carbofuran runoff from farmland and its effect on the bioassay organism, Gammarus pulex, by Matehiessen et al. (1995). One month after an application of carbofuran of 3 kg/ha as
CHAPTER 45 9Aquatic Toxicity of CMs and OPs broadcast granules or as broad granules to oilseed rape crop, a carbofuran concentration up to 26 mg/liter was measured in a nearby water reservoir after heavy rainfall. The majority of carbofuran translocates via field drain at a concentration up to 264 mg/liter. Maximum concentration persisted only for approximately 24 hr after the rainfall event, although measurable amounts could be detected for the next 4 days. An in situ bioassay of stream water that monitors the feeding rate of the bioassay species G. pulex showed that feeding stopped completely during the rainfall events, leading to the death of the organisms. Subsequently, laboratory studies demonstrated the LCs0 of carbofuran to be 21 mg/liter for G. pulex at 24 hr, which could reduce the feeding rate. Persistence of carbofuran in the fish of the Oconee River in central Georgia that receives surface runoff from adjacent agricultural orchard fields has been reported by Bush et al. (1986).
VI. M E C H A N I S M O F A C T I O N Carbofuran elicits acute intoxication by virtue of reversible inhibition (carbamylation) of AChE (Casida, 1963; O'Brien, 1967; Yu et al., 1972; Kuhr and Dorough, 1976; Gupta and Kadel, 1989a,b), which hydrolyzes ACh, a neurotransmitter. The inhibition of AChE consequently leads to excessive ACh accumulation at the synapses and neuromuscular junctions, resulting in overstimulation of ACh receptors (muscarinic and nicotinic), which could ultimately lead to death due to respiratory failure. When carbofuran mimics an alternate substrate, the alcohol moiety is cleaved, giving rise to the carbamylated ACHE. In contrast to the acetylated ACHE, carbamylated AChE is more stable (Wilson and Harrison, 1961). Sequestration of the AChE in carbamylated form thus precludes the hydrolysis of ACh, leading to ACh accumulation. Potent AChE inhibition results from rapid carbamylation (Yu et al., 1972; Kuhr and Dorough, 1976) because the carbamylation rate constant (kl) is directly correlated with toxicity (Fergusen et al., 1984). The IC50 value for AChE inhibition by carbofuran was reported to be 3.3 • 10 -8 (Dorough, 1968). In general, IC50 values for CMs decrease as the side chain becomes longer and bulkier (Soreq and Zakut, 1989). It should be noted that IC50 values vary in different tissues depending on experimental conditions, such as incubation time, temperature, and pH. Skeletal muscle, brain, heart, etc. are the main target organs for toxicity of carbofuran and other AChE-inhibiting pesticides (Petras, 1981). Recovery of AChE from carbofuran-induced inhibition (i.e., decarbamylation) is quite rapid since recovery simply requires dissociation of the methylcarbamyl moiety from the enzyme (O'Brien, 1967). The short enzyme recovery period is explained in part by kinetics of the reversible AChE-carbofuran complex. It has been reported that the
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in vivo carbofuran hydrolysis rate constant (krn 1 = 0.028 min -1, tl/2 = 25 min), representing all esterases active in carbofuran (Fergusen et al., 1984), is similar to that of the in vitro value (tl/2 = 20-63 min), suggesting rapid enzyme recovery (O'Brien et al., 1967; Aldridge, 1971). Enzyme recovery is faster in skeletal muscle than in the brain region (Gupta and Kadel, 1989a,b). Besides the inhibition of ACHE, carbofuran has a severe impact on other enzymes and compounds, such as butyrylcholinesterase (Gupta and Kadel, 1989a,b), lactate dehydrogenase (Gupta et al., 1991a,b; Gupta and Kadel, 1989b), neurotransmitter-like epinephrine, ~/-aminobutyric acid, protein, lipid and lipoprotein, and high-energy phosphates (Gupta et al., 1994) in rat or mice. It may also upset the immune system in laboratory rats (Burnett, 1980). The mechanism involved in toxicity of OPs is similar to that described for CMs (as described previously), except that OPs cause irreversible inhibition of AChE by phosphorylation, whereas CMs cause reversible AChE inhibition by carbamylation. Once OPs enter the body, they are mainly metabolized in the liver, gastrointestinal tract, malpighian tubules, and body fat by mixed function oxidases, which further increase their toxicity. Oxidative desulfuration of OPs leads to maximum anticholinesterase activity. To consider the possible toxic effect of OPs, it is necessary to take into account the type of chemical and the species in question because there are wide differences. For example, in vertebrates, after one single exposure to OP pesticides the recovery time from the anticholinesterase effects ranges from 1 to 3 weeks, whereas only approximately 1 day is necessary to recover from exposure to a CM pesticide. Birds and mammals have similar metabolic responses to anticholinesterase pesticides. However, birds tend to be more sensitive to their toxic action, probably due to lower levels of metabolizing enzymes. Based on their low LDs0s, most OPs and CMs are classified as "extremely toxic" to birds and mammals after a single dose. However, by virtue of their short half-life, they ~are quickly metabolized by the body and excreted, allowing for a fast recovery from the toxicity. A different outcome is observed when exposure occurs by means of continuous smaller doses. In this case, an accumulative anticholinesterase effect may be observed, even resulting in death after a few days. For aquatic animals, discrete studies have obtained no substantial information regarding the molecular mechanism of action of OPs.
VII. N E U R O T O X I C I T Y
OF ORGANOPHOSPHATES Several subacute and chronic syndromes can result from OP exposure, and these have been summarized by Jamal (1997) as the intermediate syndrome (IMS), the
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organophosphate-induced delayed neuropathy (OPIDN), and the chronic organophosphate-induced neuropsychiatric disorder (COPIND). Some OPs are known to produce IMS and it can occur within 3 or 4 days after poisoning. It is not responsive to atropine or oxime therapy. The patient usually recovers within 2 or 3 weeks, but this is usually an intermediary stage occurring prior to the delayed effects. OPIDN occurs usually 10 days to 3 weeks after exposure and is serious and irreversible. It consists of a distal symmetrical sensorimotor mixed peripheral neuropathy mainly affecting the lower limbs. It is unrelated to the effect of OPs on ACHE. It is thought to act by causing an aging (dealkylation) of an enzyme in nerve cells called neuropathy target esterase (NTE). This is only induced in certain species, including humans and chickens. Other induced abnormalities include axon transport and physicochemical changes of proteins and axon membrane integrity. The exact mechanism(s) is not fully understood. Evidence indicates that OPs can induce chronic effects on both the peripheral and central nervous systems following acute intoxication (Jamal, 1997). The mechanisms for this condition are not related to the inhibition of AChE or NTE. Two types of such disorders have been documented: type I, representing COPIND following an acute poisoning episode(s), and type II, representing COPIND following a chronic long-term exposure to subclinical doses. There is no distinction in the chemical nature of OPs for their apparent ability to cause COPIND. Several components of COPIND have been described, including neurobehavioral and cognitive changes, psychiatric and mental manifestations, chronic fatigue, peripheral neuropathy, neuromuscular junctional dysfunction, electroencephalographical changes, autonomic nervous system disturbances, frontal lobe syndrome, and abnormalities of cognitive evoked potentials (Jamal, 1997). Some patients have all markers, whereas others show a variety of combinations. However, no such studies at the neurophysiological level have been performed in aquatic animals. Toxicants, including OPs, often completely eliminate the performance behaviors that are essential to fitness and survival in the natural ecosystem (Scott and Sloman, 2004). The behavioral toxicity of many xenobiotics is unknown and warrants future study. Physiological effects of toxicants available in the literature have shed light on the disruption of sensory, hormonal, neurobiological, and metabolic systems that are likely to have profound effects on behavioral responses in fish. The environmental toxicants may possibly be linked with the disruption of behavioral responses in fish through cholinesterase inhibition, altered brain neurotransmitter levels, sensory deprivation, and impaired gonadal and thyroid hormone levels (Scott and Sloman, 2004).
VIII. M U T A G E N I C I T Y ,
CARCINOGENICITY, AND OXIDATIVE STRESS Nelson et al. (1981) reported the mutagenicity of N-nitroso derivatives of carbofuran and its toxic metabolites (3hydroxycarbofuran and 3-ketocarbofuran) by the Ames assay method with S. typhimurium strains TA98 and TA100. The nitroso derivatives of all three compounds were similar, giving a mutation ratio of 45 at 5 ixg/plate on TA100. In addition, all three compounds produced chromosomal aberration in Chinese hamster ovary cells. Nitrosocarbofuran and 3-hydroxynitrosocarbofuran were also found capable of inducing a large number of sister chromatid exchanges in the same cells. OPs are also known to induce tumorigenic risks (Brown etal., 1991; Soreq and Zakut, 1993). In a chronic study, isofenphos (OP) was shown to cause myeloid leukemia blast crisis and altered function of lymphocyte DNA. Several studies have demonstrated oxidative stress induced by OPs in rat brain and human erythrocytes (Abdollahi et al., 2004). OP-induced seizures have been reported to be associated with oxidative stress (Gupta et al., 2001).
IX. METABOLISM OF CARBOFURAN Metabolism of carbofuran has been studied in rats (Dorough, 1968; Lucier et al., 1972; Fergusen et al., 1984; Marshall and Dorough, 1977), mice (Metcalf et al., 1968), hens (Hicks et al., 1970), fish (Bruce, 1972; Chatterjee and Ghosh, 1995), and worms and houseflies (Metcalf et al., 1968; Dorough, 1968). Generally, in the body, carbofuran undergoes hydroxylation and oxidation reaction, producing 3-hydroxycarbofuran, 3-hydroxycarbofuran-7-phenol, 3-ketocarbofuran, and 3-ketofuran-7-phenol (Dorough, 1968; Metcalf et al., 1968). The key metabolites are produced by hydroxylation at the benzylic carbon to give 3-hydroxycarbofuran, which is oxidized to the 3-ketocarbofuran when not blocked by formation of conjugates. Dorough and Metcalf et al. first identified the metabolites with certainty: 3-hydroxycarbofuran, 3-ketocarbofuran, and their respective 7-hydroxy hydrolysis product. There is also evidence of the formation of N-CH2OH derivatives in mammals and insects produced from carbofuran or 3-keto derivatives. In an extensive investigation, Marshall and Dorough (1977) revealed that oral exposure of rats to 14C-labeled carbofuran resulted in enterohepatic circulation of some metabolites. Radiocarbon in the urine of carbofuran-treated rats consisted largely of sulfate and glucuronide conjugates of carbofuran hydrolytic derivatives. This enterohepatic cycling of glucuronides involves cleavage of conjugates in the gut and biliary
CHAPTER 45 9Aquatic Toxicity of CMs and OPs excretion, which may lead to increased systemic activity of toxic CM metabolites. 3-Hydroxycarbofuran and 3-ketocarbofuran as the main metabolites of carbofuran were identified in catfish (H. fossilis) liver by high-performance liquid chromatography (Chatterjee and Ghosh, 1995).
A. Cytochrome P450 and Pesticide Metabolism Among the CMs, carbofuran is one of the main pesticidal contaminants in the aquatic ecosystem and threatens the aquatic fauna and flora due to its detrimental effects associated with multipotent toxicity. The half-life of this pesticide is sufficient to exert its harmful effects on the living organism. The aquatic environment covers approximately 71% of the total earth surface (Goksoyr and Forlin, 1992), and 28,000 fish species inhabit this environment (Moyle and Cech, 1988), along with other innumerable living organisms. Deposition of large amounts of toxicants into the water occurs from a variety of anthropogenic sources, particularly in urban and industrial areas (Malins and Ostrander, 1991). Fish constitute the largest group of vertebrates in the aquatic ecosystem. They first come into contact with anthropogenic, industrial, or agricultural contaminants that enter the aquatic ecosystem through surface runoff. So fish face the challenge of pesticide-like carbofuran intoxication in water from agricultural fields. The integuments and the gills are the major sites of uptake of the contaminants into the body. These contaminants are rapidly distributed in the body through their efficient circulatory system. This poses a threat not only to the health of the fish but also to the consumers, including humans. Several attempts have been made to demonstrate the role of the cytochrome P450 monooxygenase system in metabolism of carbofuran in liver of catfish. Lee etal. (1998) reported the metabolism of ethyl carbamate by cytochrome P450 2El (CYP 2El) and carboxylesterase (hydrolase A) enzymes in murine liver microsomes. Binding of ethyl carbamate (ethyl 14C) to liver microsomes, followed by its metabolism in the microsomal system, has been found (Lee et al., 1998). Such involvement of cytochrome P450 2El in metabolism of ethyl carbamate was shown by Forkert and Lee (1997) in lung microsomes. Jeong et al. (1996) reported the role of P450 enzymes in the detoxification of ethyl carbamates in female BALB/c mice splenocytes in culture. Scientific interest in the cytochrome P450 system has increased dramatically since the unheralded discovery in 1958 of a hepatic microsomal carbon monoxide binding pigment with a maximum absorption at 450nm (Klingenberg, 1958; Garfinkel, 1957). Omura and Sato (1964) subsequently showed that the pigment had biochemical properties of a cytochrome and designated it P450 because of its absorption maxima in the CO-bound condi-
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tion at 450 nm (Omura and Sato, 1964). The cytochrome P450 superfamily is ancient and has expanded via divergent evolution. It is found from prokaryotes to human beings. The ancestral P450 gene probably appeared 1.5 billion years ago among the prokaryotes. The evolution of this gene among the eukaryotes is considered to be due to a defensive mechanism against the phytotoxin that accumulate during the ingestion of plants by herbivores. This gene later evolved in response to detoxification of drugs and lipophilic pollutants (Nebert and Gonzalez, 1987). Cytochrome P450 exists in bacteria, plants, and animals, and these hemoproteins catalyze the monooxygenation of a broad spectrum of lipophilic substrates (Guengerich and Shimada, 1991; Nelson etal., 1996; Mansuy, 1998). Biochemical analysis of cytochrome P450 has revealed the presence of multiple isozymes that often have broad and overlapping substrate specificity. Cytochrome P450 belongs to a superfamily of structurally and functionally related hemoproteins. The numerous CYP isozymes are classified on the basis of proposed evolutionary relationship as inferred from the degree of identity among amino acid sequences between the isozymes. Cytochrome P4501A has attracted attention because it is involved in the biotransformation of many foreign compounds, including polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons (PAHs) (Stegeman and Hahn, 1994; Parkinson, 1995). CYP1A converts the lipophilic xenobiotics by monooxygenation to more water-soluble metabolites, which is the first step toward excretion and detoxification. However, some intermediates are highly reactive and may interact with biological macromolecules, thereby appearing to be more toxic than the parent compounds (Parkinson, 1995). Purification and characterization of the P450 proteins in fish have progressed faster than the study of teleost CYP gene. Only a limited number of freshwater and marine fish have been studied. Multiple P450 forms have been purified from the freshwater fish rainbow trout (Oncorhynchus mykiss) and perch (Perca fluviatilis) and from the marine fish scup (Stenotomus chrysops and Gadus morhua). Many of these forms have been discussed in reviews (Buhler and Williams, 1988, 1989; Stegeman, 1989; Stegeman and Klopper-Sams, 1987; Stegeman et al., 1990). In some cases, the isoenzymes were purified from fish treated with a PAHtype inducer, such as [3-napthoflavone or 3-methylcholanthrene. The fish were taken directly from the environment. In mammals, the members of the CYP1A subfamily and their response to exogenous inducer, such as [3-napthoflavone, PAH, or 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), are among the best characterized and well studied of the P450 superfamily (Nebert and Gonzalez, 1987; Nebert et al., 1989) because of their role in metabolism and activation by aromatic hydrocarbons, carcinogens, and other toxic chemicals. Two genes, CYP1A1 and-1A2, being the two distinct protein products, are known to exist in all mammals studied to date.
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Observations reveal that typical CYP1A1 activities, such as aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin-odeethylase (EROD), and ethoxycoumarin-o-deethylase, may be differentially induced by different inducers, such as diesel oil and isosafarol (Leaver et al., 1993; Celander and Foflin, 1991). The characteristic feature of both mammalian and teleostean CYP1A forms is that they are inducible in nature (Nebert and Gonzalez, 1987; Nebert et al., 1989). The rate of gene transcription, affecting messenger RNA formation, new synthesis of cytochrome P450 protein, and subsequent processing, heme insertion, and folding lead to the availability of catalytically active enzyme. The previously mentioned process can be analyzed with suitable probes to detect their induction in fish (Goksoyr and Husoy, 1998; Stegeman and Hahn, 1994; Bucheli and Fent, 1995). The induction response is not fully characterized in all CYP families; the best studied family is the CYP1A subfamily. The induction response in this subfamily is known to occur via the high-affinity binding of aromatic hydrocarbons to an intercellular receptors complex (the Ah receptor), which remains associated with the 90-kDa heat shock protein (HSP90). Binding of ligand with the Ah receptor results in the dissociation of HSP90. Then, the ligandreceptor complex translocates to the nucleus with the help of aromatic hydrocarbon nuclear translocator protein (Arnt), where the heterodimer complex interacts with the xenobiotic responsive element of nucleus (XRE) to initiate the transcription of the CYP1A gene. Ligand for the Ah receptor includes the hydrophobic planar molecules that fit into the rectangular receptor binding site. The prototype ligand for the Ah receptor is TCDD, which is one of the potent CYP1A inducers (Safe, 1988; Hahn et al., 1994; Nebert et al., 1989; Stegeman and Hahn, 1994; Hankinson, 1995; Segner and Braunbeck, 1998). The inducibility of CYP1A by xenobiotic exposure led to its use as a biomarker in a pollution monitoring study of the aquatic environment (Whyte et al., 2000). EROD and AHH activities appear to be the most sensitive catalytic probes for determining response in fish. In most fish, they are highly inducible by a PAH-type inducer. Pollutants of major concern in the aquatic environment are PAH, polychlorinated dibenzo-p-dioxin and dibenzofuran, polyhalogenated biphenyls (PCB/PBB), and halogenated organic compounds and herbicides. The responsiveness of the CYP1A1 gene varies depending on the gradient of CYP1Ainducing chemicals, such as PAHs and PCBs. For instance, a complex mixture of inducing and noninducing xenobiotics as they are normally present in the environment can modify the CYP1A induction response (Croce et al., 1995; Beyer etal., 1997). Likewise, nonchemical exogenous parameters, such as temperature and season, and endogenous factors, such as hormone and physiological condition of the fish, can modulate the responsiveness of the CYP1A system (Achazi et al., 1994; Machala et al., 1997; Kopeonen et al., 1998; Lange et al., 1998.
X. A N A L Y T I C A L D E T E C T I O N O F C Y P EXPRESSION IN FISH For the assessment of CYP1A in fish, protein and catalytic activity can be used (Goksoyr and Husoy, 1998; Stegeman and Hahn, 1994; Brucheli and Fent, 1995). Mainly two enzymes translated from the CYP1A gene are used for assay: ethoxyresorufin-o-deethylase and AHH (Nebert and Gelboin, 1968; Burke and Mayer, 1974; Eggens and Galgani, 1992). CYP1A-associated catalytic activity in the fish tissue is conventionally analyzed on the microsomes prepared by ultracentrifugation from homogenized tissue. However, methods have been developed to study the activity directly from the cell without preparation of microsomes (Kennedy et al., 1995; Clemons et al., 1996; Hahn et al., 1996; Behrens et al., 1998). Assessment of CYP1A activity at the protein level was done as protein purification protocol for teleostean P450 protein was developed. CYP1A homologous protein has been purified from rainbow trout (Oncorhynchus mykiss; Williams and Buhler, 1984), and P450 LM4b/4a (Scholz et al., 1997) has been purified from scup (Stenotomus chrysops; Klotz etal., 1983), cod (Gadus morhua; Goksoyr, 1985), perch (Perca fluviatilis; Zhang etal., 1991), and tilapia (Oreochromis niloticus; Uang and Uang, 1995). For each of these proteins, polyclonal and monoclonal anti-CYP1A antibodies have been prepared (Williams and Buhler, 1984; Goksoyr, 1985; Park et al., 1986; Kloepper-Sams et al., 1987; Celander and Forlin, 1991; Husoy et al., 1996; Scholz et al., 1997). The available antibody toward fish CYP1A has generally shown broad interspecies cross-reactivity (Kloepper-Sams et al., 1987; Goksoyr, 1991; Goksoyr and Forlin, 1992). For immunochemical detection of teleostean CYP1A, the available antibody has been used in Western blotting, enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry. Immunochemical assay may be particularly advantageous in environmental monitoring programs because it does not rely on adequate conversion of the enzymatic activity of the samples (Goksoyr etal., 1991; Collier etal., 1995). Xenobiotics may inhibit CYP1A enzymatic activity in any assay in which such a compound may be present. Disadvantages of immunochemical CYP1A detection are that it is less sensitive compared to enzymatic assay (Scoltz et al., 1997) and problems in quantification (Goksoyr, 1991; Van Veld et al., 1997).
XI. C E L L AND T I S S U E D I S T R I B U T I O N O F C Y P 1 A IN T E L E O S T F I S H Intracellular immunochemical localization of CYP1A appears to be a potential target for the assessment of the degree of toxic action of xenobiotics and carcinogens. CYP1A is commonly expressed in many epithelial cells of many organs. The highest CYP1A activity is found in
CHAPTER 45 9Aquatic Toxicity of CMs and OPs liver, but it also occurs in extrahepatic organs, such as gallbladder, gonads, nervous tissue, and endocrine cells (Sarasquete and Segner, 2000). Although the biochemically detectable CYP1A level in extrahepatic organs is usually lower than that in liver, the prominent expression and induction of CYP1A in fish liver are consistent with the involvement of the organ in xenobiotic metabolism and excretion. Several studies have demonstrated the expression of CYP1A in hepatocytes of fish by means of immunohistochemistry (Reinecke and Segner, 1998; Sarasquete et al., 1999). In contrast to mammals, CYP1A shows heterogeneous distribution throughout the liver parenchyma and no zonation is observed in fish liver (Smolowitz et al., 1991). Subcellularly, in fish hepatocytes CYP1A is localized at the rough endoplasmic reticulum, the nuclear envelop, and in the plasma membrane of the microvillae of the bile canaliculi (Lester et al., 1993). Although the role of cytochrome P450 in the metabolism of carbofuran has not been explored in any system, the metabolism of ethyl carbamate by P450 2El has been reported by Lee et al. (1998). The study implicated cytochrome-P450 2El and carboxylesterase enzyme in the metabolism of carcinogens such as ethyl carbamate in the murine liver microsomal system. The fish taken from a lake in Newfoundland with a history of hydrocarbon pollution showed increased AHH activity (Payne and Penrose, 1975). Thus, many workers have studied the EROD activity of fish in laboratory conditions or naturally exposed samples as a molecular biomarker for aquatic pollution. These studies are reviewed by Whyte et al. (2000) and Goldfarb et al. (1998). In addition, EROD activity in carbofuran-treated fish has been measured in laboratory conditions. Carbofuran is a lipophilic PAH; therefore, it always accumulates in the lipid-lipid bilayer of the liver cell membrane (Gupta, 1994). Biophysical study of another pesticide, lindane, in relation to membrane partition has been performed by Madeira-Antues and Madeira (1985). This study suggests that lindane localizes in the hydrophobic part of the membrane phospholipid and decreases the midpoint temperature of the phase transition. It appeared that lindane tends to be excluded from the gel phase. The finding is reinforced by the fact that membrane cholesterol withdraws the lindane effectively from the membrane, and lindane thus takes a position in the region of the bilayer. The effects of the membrane fluidity and geometry have also drawn attention in the interpretation the cholesterol-related lindane withdrawal from the membrane. Such observations were made previously for parathion (Madeira-Antues and Madeira, 1984) and several other drugs. By virtue of its lipophilic nature, carbofuran accumulates in membrane lipid moieties en route to their target sites. Chatterjee and Ghosh (1995) showed that total cholesterol and phospholipid increased in the carbofuraninduced liver of fish compared to controls. In mice, treatment of Furadan (commercial name for carbofuran)
665
increased total lipid, cholesterol (free and esterified), triglyceride, and phospholipid and its fraction (lecithin, lysolecithin, phosphatidylethanolamine, and lysophosphatidylethanolamine) in liver, kidney, and serum. The elevation of cholesterol by carbofuran treatment can be justified by the inhibition of Ca2+-ATPase activity. Warren e t a l . (1975) stated that phospholipid annulus surrounding the Ca-2+ATPase enzyme is necessary for its catalytic activity. Many workers, such as Hunter et al. (1990, 1999), supported this finding. Ghiasuddin et al. (1982) reported that Ca2+-ATPase activity could be restored in the nerve by the addition of phosphatidylcholine. Ghiasuddin et al. (1982) showed inhibition of membrane Ca2+-ATPase activity by the pesticide treatment. Thus, changes in cholesterol and phospholipid spectrum due to the involvement of pesticide in the membrane induced an impetus for the measurement of cholesterol and phospholipid in the carbofuran-treated hepatocyte membrane that can be correlated with Ca2+-ATPase activity of the membrane. This study may provide information about the retention and persistence of the pesticide in the lipid bilayer. Currently, heat shock protein (HSP) is used as a biomarker in various ecotoxicological studies. HSP is the family of proteins with varying molecular weights, such as Hsp28, Hsp70, and Hsp90, which are induced during thermal stress. HSP functions as armor, which safeguards the native structure of another protein during temperature stress on the cell. However, it is established that HSP may be induced by any other cellular stress and act as a molecular chaperone, which plays a critical role during the stress, and the native structure of the cellular protein is threatened. Pyza et al. (1997) studied Hsp70 in centipede L i t h o b i u s m u t a b i l e s exposed to the insecticide dimethoate. Bagchi et al. (1996) demonstrated the induction of HSP in vivo and in vitro by selected pesticides. Regulation of P4501A1 (CYP1A1) through protein kinase C (PKC) has been reported by Stephen et al. (1997) in lymphoid tissue. Reiners (1993) studied the regulation of CYP1A1 in lclc7 murine hepatoma cells through PKC, in which treatment of cathepsin, staurosporine, or H7, the PKC blockers, suppressed the induction of EROD in that cell line. Lee and Dasmahapatra (1993) performed a similar type of study in the hepatocyte culture of rainbow trout (0. mykiss) in which they showed the regulation of EROD activity by PKC. This study suggests that pretreatment of 12-O-tetradecanoynl phorbol 13-acetate, a PKC activator, to the culture hepatocytes inhibits EROD activity with a decreased CYP1A1 protein level. Pretreatment of the cell with the PKC inhibitor staurosporine effectively blocked the EROD activity. The study indicated the role of PKC in the modulation of the CYP1A1 gene in fish liver by [3-napthoflavone. All these results indicate a change in the phosphorylation state of the cell during induction of the CYP1A1 gene. The role of PKC in regulating CYP1A1
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gene induction in the cultured human hepatoma cell 101L derived from the HEPG2 cell line has been studied. It was clearly shown that PKC was essential for the cellular and molecular events that controlled the induction of CYP1A1 gene transcription. Formation of PKC is an essential step in the signaling cascade initiated by the change of minor lipid spectrum, such as phosphatidyl-inositol diphosphate (PIP2), followed by the formation of diacylglycerol to activate the PKC (Hannun et al., 1985; Bonser et al., 1988). This study of PKC, which is a ubiquitous part of membrane signaling during stress, indicates the requirement to study prior cascade phenomenon, such as diacylglycerol or PIP2 and membrane phospholipid status. All these studies can examine signaling initiated in the membrane and culminating in the cytosol modulating the expression of HSP as well as aromatic hydrocarbon receptor protein. This may identify the details of the response of a cell under stress by the pesticide or related compound. Ding etal. (1998) studied the role of PKC in the expression of Hsp70 and showed that overexpression of Hsp70 inhibits the phosphorylation of heat shock protein-1 (HSF1) by activating protein phosphatase and inhibiting protein kinase activity. Sakai et al. (1997) and Holmberg et al. (1997) studied the effect of inhibitor and activator of the PKC in response to HSP in cultured mammalian cell. There are no reports in the fish system in this regard. HSP has been linked to CYP1A1 gene regulation. The involvement of PKC in regulation of the CYP 1A1 gene, which has been the subject of study by many workers in the past few years, has been analyzed by Ding et al. (1998). The study indicated the downregulation of PKC by HSF1, which is a transcription factor for production of HSP70. The transcription factor (HSF1) requires phosphorylation for initiation of transcription, in which PKC plays an indispensable role. However, overexpression of HSF1 downregulates PKC, which in turn can modulate CYP1A1 expression (Ding et al., 1998; Holmberg et al., 1997). It is well demonstrated that worldwide discharge of metal into the aquatic ecosystem has an impact on cytochrome P450, which must be considered in the role of regulation of this important protein. Considerable attention has been given to glutathione, which plays an important role in modulating the catalytic activity of P450 enzymes such as EROD (Otto et al., 1996). Copper is one of the main components among the discharges of metal into the aquatic ecosystem (Gumgum et al., 1994).
XII. BIOMARKERS AND TOXICITY ASSESSMENT The presence of carbofuran and its major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) in food, water, and air may be considered as the source of contamination. The presence of carbofuran and its metabolites in the urine,
feces, and bile or any other body tissues can be considered as the most specific biomarker of recent or ongoing exposure. The excretion of 3-hydroxycarbofuran has been found to be more associated with carbofuran exposure than 3-ketocarbofuran in urine. Huang et al. (1989) reported that the level of 3-hydroxycarbofuran in patients with acute carbofuran poisoning may increase to 80.64 ppb. Monitoring of AChE in erythrocytes gives a good measure of the effect of carbofuran exposure (He, 1993). Erythrocyte membrane AChE is more sensitive than plasma AChE (Vandekar etal., 1971; Wills, 1972). Hussain et al. (1990) demonstrated significant inhibition of red blood cell AChE in farmers exposed to carbofuran. Plasma AChE was not affected. The recovery of CM AChE inhibition is much faster than that of OP exposure because carbamylation of the enzyme is easily and rapidly reversed (Coye et al., 1986). AChE should be determined when assessing CM exposure. Blood samples in such cases should be drawn within 4 hr of exposure to CM (World Health Organization Task Group, 1986). This critical inhibition of AChE in blood, brain, and muscle in humans, animals, birds, or fish is indicative of poisoning due to either CM or OP. Cholinergic symptoms are usually associated in CM-exposed workers with a blood AChE lower than 70% of the individual's baseline level (Huang etal., 1989; He, 1993). Analysis of serum enzymes (creatine kinase, lactate dehydrogenase, and others) and their isozymes and subforms provides additional information on tissue-specific damage (Gupta etal., 1991 a,b, 1994), and these can be considered as markers of the effects.
XIII. EXPERIMENTS USING FISH HEPATOCYTES IN C U L T U R E Some interesting observations were noticed in the author's laboratory. Although carbofuran was present in all cellular compartments, it was highest in the membrane and lowest in the nucleus of the singi fish (Hetropneustes fossilis Bloch) hepatocytes in culture. Metabolites of carbofuran, such as 3-ketocarbofuran and 3-hydroxycarbofuran, also showed a qualitatively similar distribution as carbofuran. Hepatocytes incubated in the carbofuran-containing medium at different concentrations caused dose-dependent inhibition of Ca2+-ATPase activity in comparison to the untreated control. During carbofuran treatment, a higher amount of cholesterol was found in hepatocyte membranes. Indirectly, insertion of cholesterol to normal hepatocyte membranes also showed inhibition in Ca2+-ATPase activity depending on the amount of cholesterol inserted. Such effect has been studied by measuring EROD activity, a marker enzyme of CYP1A1, in different conditions of carbofuran treatment. Incubation of hepatocytes in different doses of carbofuran for 24 hr showed a dose-dependent increase in EROD
CHAPTER 45
activity, with the highest activity observed with 1 ~zM concentration of carbofuran in the medium. [3-Napthoflavone (BNF), a known inducer of CYP1A, also demonstrated a dose-dependent increase in EROD activity, ot-Napthoflavone (ANF), an effective blocker of the Ah receptor, demonstrated a dose-dependent inhibition of EROD activity in the hepatocytes maintained for 24 hr in ANF-containing medium. The carbofuran- and BNF-induced EROD activity in the cultured hepatocytes was counteracted by actinomycin-D and cycloheximide when either of the compounds was administered simultaneously with carbofuran or BNF in the medium. Interestingly, the same compounds showed a superinduction of EROD activity when added to hepatocytes pretreated with carbofuran or BNF for 12 hr. Phosphatidyl-inositol phosphate and PIP2 showed significant increases in carbofuran-treated hepatocytes in comparison to DMSO-treated control. Equimolar amounts of carbofuran or BNF used in the separate culture significantly increased the hepatocyte membrane PKC activity compared to the control. Use of staurosporine, a known inhibitor of PKC, counteracted the carbofuran- or BNF-induced increase in PKC activity. Interestingly, phorbol ester demonstrated an attenuation of the carbofuran- or BNF-induced increase in PKC activity, but it remained higher than the control value. Because PKC activity is related to the formation of HSP, this relationship has been examined in a hepatocyte culture system. Carbofuran or BNF increased the HSP70 content 8- to 11-fold higher than the control. Addition of Staurosporine (ST) or 12-Phorbol,13-myristate acetate (PE) to carbofuran or B NF attenuated the carbofuran- or B NFinduced increase in Hsp70 but maintained its level higher than that of the DMSO-treated control. Similar results were obtained for the EROD activity of the hepatocytes in the previously mentioned treatment conditions (Ghosh et al., 2000). Furthermore, it was demonstrated that carbofuran and BNF elevated the glutathione (GSH) level and EROD activity in hepatocytes in comparison to the control. Equimolar concentrations of carbofuran + BNF demonstrated a synergistic elevation of GSH level in the culture hepatocytes. Addition of Cu 2+ with carbofuran or BNF caused a decrease in GSH level and EROD activity (Ghosh et al., 2001). The catfish hepatic microsomal protein that contained EROD activity was purified up to approximately 82-fold and its authenticity was checked by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, Western blot, dot blot, capillary electrophoresis, and ELISA. Analysis of circular dichroism (CD) indicated functional aspects of oL and [3 helices in relation to the EROD activity of the purified protein (Ghosh et al., 2001). The study of the Indian catfish, Singhi, clearly indicated the impact of carbofuran in fish and evolutionary conservation of the detoxification system involving cytochrome P450. Further studies using OPs should be performed, which may show the major and minor differences in the
9Aquatic Toxicity of CMs and OPs
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molecular pathways of metabolism and detoxification in different organs in fish of the tropical and temperate zones.
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46
CHAPTER
Toxicity of O r g a n o p h o s p h o r u s and Carbamate Insecticides Using Birds as Sentinels for Terrestrial Vertebrate Wildlife SPENCER R. MORTENSEN Syngenta Crop Protection, Greensboro, North Carolina
I. I N T R O D U C T I O N an emphasis on those that have a U.S. registration. Two methods of estimating the relative toxicity of each compound to birds are also given: the HD5 method (Mineau et al., 2001), which is based on the avian acute oral toxicity test, and the U.S. Environmental Protection Agency's (EPA) category of toxicity to birds, which is also based on the avian acute oral toxicity test (EPA, 1994). Additionally, the number of avian incidents in the United States from 1968 to 2004 is provided where available, and a ranking of the top 10 OPs used in the United States in 2001 is given.
Although the use and number of registered organophosphorus (OP) and likely carbamate (CM) insecticides has steadily decreased during the past two decades (Fig. 1), the importance of these broad-spectrum classes of chemistry for pest control and integrated pest management programs remains unchanged. There are currently more than 40 OP and CM active ingredients and hundreds of formulations registered in the United States (Table 1). The purpose of this chapter is to summarize the OP and CM insecticides that are registered in the world, with
250
* u
A
y =-5.9138x + 11910 R2=0.8
OP Use (MM Ibs ai)
m Total Insecticide Use (MM Ibs ai)
200
. n
t~ o
~~ o
150
*-.....
~
--- 100
-
Z
y =-3.4248x + 6908.4 R2 = 0.9385 50-
0
1978
19'80
19'82
19'84
19'86
19'88
19'90
19'92
19'94
19'96
19'98
20'00
Year
FIG. 1. Decline in OP and total insecticide use in the United States from 1978 to 1998. Data from Kiely et al., (2004).
Toxicology of Organophosphate and Carbamate Compounds
673
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
674
SECTION V l l .
II. S U M M A R Y TABLE 1.
A q u a t i c Life & W i l d l i f e
DATA
Avian Toxicity and Incident Data for OP and CM Insecticides Registered in the United States and Other Areas of the World*
HD5 a
U.S. EPA category of toxicity b
Current U.S. registration? c
OP
18.52
3
Yes
OP
2.28
2
Yes
1
2921-88-2
OP
3.76
2
Yes
70
Chlorpyrifos-methyl
5598-13-0
OP
25.32
4
Yes
NRf
Coumaphos
56-72-4
OP
0.69
1
Yes
1
Diazinon
333-41-5
OP
0.59
1
Yes
390
Dichlorvos (DDVP)
62-73-7
OP
0.61
2
Yes
2
Dicrotophos
141-66-2
OP
0.42
1
Yes
1
Dimethoate
60-51-5
OP
5.78
2
Yes
8
Disulfoton
298-04-4
OP
0.81
2
Yes
NR
Ethion
563-12- 2
OP
1.06
3
Yes
1
Ethoprop
13194-48-4
OP
2.41
1
Yes
2
Fenamiphos
22224-92-6
OP
0.43
1
Yes
8
Fenitrothion
122-14-5
OP
3.37
3
Yes
NR
Fenthion
55-38-9
OP
0.87
1
Yes
54
Isofenphos
25311-71-1
OP
0.44
2
Yes
10
Malathion
121-75-5
OP
139.10
3
Yes
NR
Methamidophos
10265-92-6
OP
1.70
2
Yes
3
Methidathion
950-37-8
OP
3.53
2
Yes
NR
Methyl parathion
298-00-0
OP
2.13
2
Yes
2
Naled
300-76-5
OP
1.72
3
Yes
1
Oxydemeton-methyl
301 - 12-2
OP
13.96
3
Yes
1
Parathion
56-38-2
OP
0.40
1
Yes
46
Compound
CAS No.
OP or CM
Acephate
30560-19-1
Azinphos-methyl
86-50-0
Chlorpyrifos
No. of U.S. incidents d
Use rank, 2001 e
1
3
10
1
7
Phorate
298-02-2
OP
0.34
1
Yes
21
6
Phosmet
732-11-6
OP
1.24
3
Yes
NR
8
Pirimiphos-methyl
29232-93-7
OP
13.51
3
Yes
NR
Profenofos
41198-08-7
OP
NDg
3
Yes
NR
Propetamphos
31218-83-4
OP
7.09
3
Yes
NR
Tebupirimfos
96182-53-3
OP
2.36
2
Yes
NR
Temephos
3383-96-8
OP
8.68
3
Yes
NR
Terbufos
13071-79-9
OP
0.16
1
Yes
5
Tetrachlorvinphos
961-11-5
OP
25.32
5
Yes
1
Trichlorfon
52-68-6
OP
13.36
3
Yes
2
Aldicarb
116-06-3
CM
0.43
1
Yes
12
Aldoxycarb
1646-88-4
CM
ND
2
Yes
NR
Carbaryl
63-25-2
CM
30.05
4
Yes
6
Carbofuran
1563-66-2
CM
0.21
1
Yes
345
Methiocarb
2032-65- 7
CM
1.06
1
Yes
NR
Methomyl
16752-77-5
CM
8.46
2
Yes
2
Oxamyl
23135-22-0
CM
0.78
1
Yes
NR
Pirimicarb
23103-98-2
CM
6.78
2
Yes
NR
Propoxur
114-26-1
CM
1.31
2
Yes
3
Thiodicarb
59669-26-0
CM
234.96
5
Yes
NR
4
(continues)
CHAPTER 46
TABLE 1.
Compound
CAS No.
9Wildlife Toxicity of OPs and CMs
(continued)
OP or CM
HD5 a
U.S. EPA category of toxicity b
Current U.S. registration? c
No. of U.S. incidents d
Amidithion
919-76-6
OP
ND
ND
No
NR
Anilofos
64249-01-0
OP
ND
4
No
NR
Azamethiphos
35575-96-3
OP
3.98
2
No
NR
Azinphos-ethyl
2642-71-9
OP
1.53
1
No
NR
122-10-1
OP
0.25
1
No
NR
Bromophos
2104-96-3
OP
491.14
5
No
NR
Bromophos-ethyl
4824-78-6
OP
12.88
3
No
NR
Butonate
126-22-7
OP
40.00
3
No
NR
Bomyl
Cadusafos
95465-99-9
OP
6.33
3
No
NR
Carbophenothion
786-19-6
OP
2.00
3
No
NR
Chlorethoxyfos (Fortress)
54593-83-8
OP
3.25
2
No
NR
Chlorfenvinphos
470-9O-6
OP
2.73
2
No
NR
Chlormephos
24934-91-6
OP
25.10
3
No
NR
Chlorphoxim
14816-20-7
OP
11.61
3
No
NR
Chlorthiophos
60238-56-4
OP
ND
ND
No
NR
Crotoxyphos
7700-17-6
OP
14.23
3
No
NR
Cyanophos
2636-26-2
OP
0.83
ND
No
NR
Cyanthoate
3734-95-0
OP
ND
ND
No
NR
DAEP
13265-60-6
OP
ND
ND
No
NR
Demephion
8065-62-1
OP
ND
ND
No
NR
1
No
NR
Demeton
8065-48-3
OP
Demeton-methyl
867-27-6
OP
Dialifos
10311-84-9
OP
Diamidafos
1754-58-1
OP
1.04
7.24
2
No
NR
4
No
NR
3.37
2
No
NR
4.13
ND
No
NR
ND
Dicapthon
2463-84-5
OP
Dichlofenthion
97.17-6
OP
3
No
NR
Dioxabenzofos
3811-49-2
OP
ND
ND
No
NR
Dioxathion
78-34-2
OP
25.50
3
No
NR
ND
No
NR
7.54
DMCP
3309-87-3
OP
ND
Endothion
2778-04-3
OP
ND
ND
No
NR
EPBP
3792-59-4
OP
ND
ND
No
NR
EPN
2104-64-5
OP
1
No
NR
ND
No
NR NR
0.53
Ethoate-methyl
116-01-8
OP
ND
Etrimfos
38260-54-7
OP
23.65
ND
No
Famphur
52-85-7
OP
0.45
1
No
33
Fenchlorphos (Ronnel)
299-84-3
OP
12.23
3
No
NR
Fensulfothion
115-90-2
OP
0.13
1
No
5
3.86
2
No
NR
'
Fonofos
944-22-9
OP
Formothion
2540-82-1
OP
ND
3
No
NR
Fosmethilan
83733-82-8
OP
ND
ND
No
NR
Fosthiazate
98886-44-3
OP
1.47
2
No
NR
3.04
3
No
NR
ND
No
NR
2
No
1
23560-59-0
OP
IPSP
5827-05-4
OP
Isazofos
42509-80-8
OP
Heptenophos
ND 0.51
675
Use rank, 2001 e
(continues)
676
S E C T I O N V l l . A q u a t i c Life & W i l d l i f e
TABLE 1.
Compound
CAS No.
OP or CM
Isocarbophos
24353-61-5
OP
Isothioate
36614-38-7
OP
HD5 a
0.26
(continued) U.S. EPA category of toxicity b
Current U.S. registration? c
No. of U.S. incidents d
1
No
NR
ND
ND
No
NR
Isoxathion
18854-01-8
OP
ND
ND
No
NR
Jodfenphos
18181-70-9
OP
ND
ND
No
NR
Leptophos
21609-90-5
OP
3
No
NR
Kayaphos
7292-16-2
OP
ND
ND
No
NR
Mazidox
7219-78-5
OP
ND
ND
No
NR
Mecarbam
2595-54-2
OP
ND
ND
No
NR
Mephosfolan (Cytrolane)
950-10-7
OP
1
No
NR
Mevinphos
7786-34-7
OP
0.70
1
No
Monocrotophos
6923-22-4
OP
0.42
1
No
3 4
ND
No
NR
2
0.09
0.14
Morphothion
144-41-2
OP
Omethoate
1113-02-6
OP
No
NR
Oxydeprofos
2674-91-1
OP
ND
ND
No
NR
Oxydisulfoton
2497-07-6
OP
ND
ND
No
NR
Phenkapton
2275-14-1
OP
ND
ND
No
NR
Phenthoate
2597-7-3
OP
23.17
3
No
NR
Phosalone
2310-17-0
OP
106.27
5
No
NR
Phosfolan
947-02-4
OP
0.69
1
No
NR
Phosphamidon
297-99-4
OP
1.08
1
No
1
Phoxim
14816-18-3
OP
1.71
2
No
NR
Pirimiphos-ethyl
23505-41-1
OP
1.90
1
No
NR
Propaphos
7292-16-2
OP
0.18
1
No
NR
Prothidathion
20276-83-9
OP
ND
ND
No
NR NR
ND 4.14
Prothiofos
34643-46-4
OP
13.65
3
No
Prothoate
2275-18-5
OP
5.52
3
No
NR
Pyraclofos
77458-01-6
OP
ND
No
NR
No
NR
ND
Pyridaphenthion
119-12-0
OP
7.94
3
Quinalphos
13593-03-8
OP
0.42
2
No
NR
Schradan
152-16-9
OP
2.02
2
No
NR
Sulfotep
3689-24-5 ~
OP
50.63
3
No
NR
Sulprofos
35400-43-2
OP
6.85
2
No
NR
TEPP
107-49-3
OP
Thiometon
640-15-3
OP
ND 5.07
1
No
NR
3
No
NR
1
No
NR
ND
No
NR
Thionazin
297-97-2
OP
Triamiphos
1031-47-6
OP
Triazophos
24017-47-8
OP
1.68
1
No
NR
Trichloronat
327-98-0
OP
0.73
2
No
NR
2
No
NR
ND
No
NR
1.02 ND
Vamidothion
2275-23-2
OP
Alanycarb
83130-01-2
CM
Allyxycarb
6392-46-7
CM
3.37
2
No
NR
Aminocarb
2032-59-9
CM
6.59
2
No
NR
3.72 ND
Bendiocarb
22781-23-3
CM
0.72
2
No
11
Benfuracarb
82560-54-1
CM
4.23
3
No
NR
Bufencarb
8065-36-9
CM
3.09
2
No
NR
Butacarb
2655-19-8
CM
ND
No
NR
ND
Use rank, 2001 e
(continues)
CHAPTER 4 6
TABLE 1.
Compound
9Wildlife Toxicity of OPs and CMs
(continued)
U.S. EPA category of toxicity b
Current U.S. registration? c
No. of U.S. incidents d
6.17
3
No
NR
18.58
3
No
NR
9.52
3
No
NR
CM
0.43
ND
No
NR
CM
25.32
3
No
NR
87130-20-9
CM
234.13
5
No
NR
644-64-4
CM
0.92
2
No
NR NR
CAS No.
OP or CM
Butocarboxim
34681-10-2
CM
Butoxycarboxim
34681-23-7
CM
Carbosulfan
55285-14-8
CM
Cloethocarb
51487-69-5
Crufomate
299-86- 5
Diethofencarb Dimetilan
HD5 a
677
Dioxicarb
6988-21-2
CM
3.36
3
No
Ethiofencarb
29973-13-5
CM
14.96
1
No
NR
Fenobucarb
3766-81-2
CM
31.12
3
No
NR
Formetanate
22259- 30-9
CM
8.77
2
No
NR
Furathiocarb
65907-30-4
CM
2.41
2
No
NR
Isoprocarb
2631-40-5
CM
14.23
ND
No
NR
Metolcarb
1129-41-5
CM
ND
ND
No
NR
Mexacarbate
315-18-4
CM
1.39
1
No
NR
Promecarb
2631-37-0
CM
0.94
2
No
NR
Thiofanox
39196-18-4
CM
0.12
1
No
NR
Triazamate
112143-82-5
CM
0.93
1
No
NR
Trimethacarb XMC
12407-86-2 2655-14-3
CM CM
16.28 ND
3 ND
No No
NR NR
Xylylcarb
2425-10-7
CM
6.20
ND
No
NR
Use rank, 2001 e
*The views expressed are those of the author and do not necessarily reflect the views of syngenta Crop Protection, Inc. aSee Mineau et al. (2001) for details. bCategories 1-5 represent a median LDs0 value (from Mineau et al., 2001) that is < 10, 10-50, >50-500, > 500-2000, or >2000 mg ai/kg body weight, respectively (see EPA, 1994). CThe following sources were used to determine the current U.S. registration status of each OP and CM: EPA/OPP Pesticide-Related Database Queries (www.cdpr.ca.gov/docs/epa/epamenu.htm), Tomlin (2000), and the Crop Protection Handbook (2004). aNumber of U.S. incidents in the AIMS database (www.abcbirds.org/aims) as of April 2005. eTop 10 most commonlyused OP insecticide active ingredients in all market sectors of the United States in 2001 (Kiely et al., 2004). fNR, not reported. gND, not determined.
III. DATA EVALUATION Several interesting observations can be made from Table 1. First, there are an estimated 116 OP and 38 CM insecticides used worldwide. There are also an estimated 33 OPs and 10 CMs registered in the United States. Second, not surprisingly, there appears to be a negative relationship between HD5 or categorization of toxicity and avian incidents, which suggests that the more toxic OPs and CMs are responsible for the majority of the avian incidents. This is in agreement with an earlier review of bird-kill incidents (Grue et al., 1983) that indicated that most incidents could be explained by pesticide toxicity and use. Third, approximately 60% of the avian incidents involving OPs and > 9 0 % of the avian incidents involving CMs in the United States (reported in the AIMS database by April 2005) were
associated with diazinon and carbofuran, respectively. A word of caution is in order here. It would be easy to jump to the conclusion that simply removing diazinon and carbofuran from the market would necessarily reduce the number of avian incidents. However, the records in the AIMS database are a compilation of incidents from 1968 to 2004. Many of the incidents occurred prior to current restrictions for these products (i.e., restrictions to limit application rates, the types of crops on which the products can be applied, and market sectors). It will be interesting to determine if the same trend continues after all of the restrictions are in force. Also, it is important to perform a risk-risk analysis (i.e., weigh the risk of using a particular product against the risk of not having a particular product available) prior to making decisions regarding the cancellation of a pesticide. In some cases, the risk of having a more toxic
678
SECTION V l I . A q u a t i c
Life & Wildlife
TABLE 2. Comparison of the Toxicity of Methyl and Ethyl Analogs of the OP Insecticides
Compound Azinphos-methyl Azinphos-ethyl Bromophos Bromophos-ethyl Methyl chlorpyrifos Chlorpyrifos Demeton-methyl Demeton Pirimiphos-methyl Pirimiphos
HD5
Toxicity category
2.28 1.53 491.14 12.88 25.32 3.76 7.24 1.04 13.51 1.90
2 1 5 3 4 2 2 1 3 1
alternative outweighs the risk of not having it. This risk-risk analysis could vary from country to country and even state to state. Fourth, it is interesting to note that the methyl analogs of the OPs are less toxic to birds than the ethyl analogs, as shown in Table 2. The data in Table 2 agree well with results from an experiment by Clothier et al. (1981), who showed that bovine red blood cell AChE inhibited with dimethoxysubstituted compounds spontaneously reactivated and aged more rapidly than AChE inhibited by the diethoxysubstituted compounds. Importantly, however, the rate of spontaneous reactivation of the dimethoxy-substituted compounds was approximately an order of magnitude more rapid than the rate of aging.
IV. F I N A L T H O U G H T S Perhaps Ecobichon (1991) described the paradox of pesticide use best: "All pesticides possess an inherent degree of toxicity to some living organism; otherwise they would be of no practical use" and "There is no such thing as a 'completely safe' pesticide." Pesticides are unique among chemicals in that they are registered specifically to kill their intended target. The question is how can we most effectively reduce nontarget incidents? There are times when the correct choice is to discontinue the use of a pesticide, as was the case with the use of monocrotophos in Argentina (Hooper et al., 2003). This was a very focused and collaborative effort that involved a number of government agencies, university personnel, industry, nongovernmental
organizations, and many others. Perhaps the easiest and most effective first step that we as individuals can take is to read and follow pesticide labels because many of the avian incidents are a direct result of product misuse.
Acknowledgments The author wishes to thanks Dr. Joh Akins and Jeff Stabnau for their help finding referenced materials and Alan Hosmer for his review of an earlier version of this chapter.
References Clothier, B., Johnson, M. K., and Reiner, E. (1981). Interaction of some trialkyl phosphorothiolates with acetylcholinesterase: Charaterization of inhibition, aging, and reactivation. Biochem. Biophys. Acta 660, 306-316. Crop Protection Handbook, Vol. 90. (2004). Meister Media Worldwide, Willoughby, OH. Ecobichon, D. J. (1991). Toxic effects of pesticides. In Casarett and Doull's Toxicolgy: The Basic Science of Poisons (M. O. Amdur, J. Doull, and C. D. Klaassen, Eds.), 4th ed., pp. 565-622. Pergamon, New York. Grue, C. E., Flemming, W. J., Busby, D. G., and Hill, E. E (1983). Assessing hazards of organophosphate pesticides to wildlife. Trans. N. Am. Wildl. Nat. Res. Conf. 48, 200-220. Hooper, M. J., Mineau, P., Zaccagnini, M. E., and Woodbridge, B. (2003). Pesticides in international migratory bird conservation. In Handbook of Ecotoxicology (D. J. Hoffman, B. A. Rattner, G. A. Burton, Jr., and J. Cairns, Jr., Eds.), 2nd ed., pp. 737-754. CRC Press, Boca Raton, FL. Kiely, T., Donaldson, D., and Grube, A. (2004). Pesticides Industry Sales and Usage: 2000 and 2001 Market Estimates. Biological and Economic Analysis Division, Office of Pesticide Programs, Office of Prevention, Pesticides, and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC. Mineau, P., Jobin, B., and Baril, A. (1994). A critique of the avian 5-day dietary test (LCs0) as the basis of the avian risk assessment, Technical Report No. 215. Canadian Wildlife Service Headquarters, Hull, Quebec, Canada. Mineau, P., Baril, A., Collins, B. T., Duffe, J., Joerman, G., and Luttik, R. (2001). Pesticide acute toxicity reference values for birds. Rev. Environ. Contam. Toxicol. 170, 13-74. Smith, G. J. (1987). Pesticide Use and Toxicology in Relation to Wildlife: Organophosphorus and Carbamate Compounds, Resource Publication 170. U.S. Department of the Interior, Fish and Wildlife Service, Washington, DC. Tomlin, C. D. S. (2000). The Pesticide Manual 12th ed. British Crop Protection Council, Farnham, Surrey, UK. U.S. Environmental Protection Agency (1994). Pesticide reregistration rejection rate analysis: Ecological effects, EPA 738-R-94-035. U.S. Environmental Protection Agency, Washington, DC.
Analytical & Biomarkers
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CHAPTER 4
7
Analysis of Organophosphate and Carbamate Pesticides and Anticholinesterase Therapeutic Agents ANANT V. lAIN University of Georgia, Athens, Georgia
of the sample extract, (iii) concentration of purified extract, (iv) separation from impurities and detection, and (v) confirmation. In this chapter, the various steps are discussed briefly, and recent trends for each step are discussed.
I. I N T R O D U C T I O N Organophosphate (OP) and carbamate (CM) pesticides are an integral part of modern agriculture. The publication of Silent Spring by Rachel Carson led to the withdrawal of many organochlorine (OC) pesticides due to their persistence in the environment. At this time, the use of OPs and CMs soared due to their availability and quick degradation in the environment. Although OPs and CMs degrade quickly and are much less persistent in the environment, they are much more toxic to mammals than OC pesticides. Because of their adverse health affects, various governmental agencies set limits on allowable levels of pesticide residues in foods, animal feeds, and the environment. In order to enforce these allowable levels, pesticides are monitored in various types of samples. Also, humans and animals may be poisoned accidentally or maliciously. Thus, there is a need for analytical methods to determine OPs and CMs in food and feedstuffs as well as biological specimens. By nature, analytical results are variable. The science of trace analysis (analysis at parts per million or below levels) is not as precise as most layman and many scientists view it to be (Rogers, 1986). Once devised, analytical methods are like life-forms, subject to evolution. Natural selection is mediated by analytical chemists, which ensures that only the fittest analytical methods survive. Therefore, an analytical method must be fit for purpose. In order to be able to determine trace levels of OPs and CMs in the environment and biological, food, and feed samples, it is necessary to follow a series of operations. Note that most of the advancements and improvements in the analytical methods have occurred by making necessary changes and improvements in various steps used in analytical methods. The papers by Sawyer (1988) and Seiber (1988) clearly demonstrate this point. The various steps in an analytical method are (i) extraction of the sample, (ii) cleanup and purification Toxicology of Organophosphate and Carbamate Compounds
II. S A M P L E E X T R A C T I O N It is essential to separate the target pesticide(s) from the sample matrix. Traditionally, this has been done by blending a homogeneous ground sample with an organic solvent. Since OPs and CMs are nonpolar to polar compounds, the polarity of extraction solvents varies accordingly. There are three general methods for extracting pesticides from solids with organic solvents: soxhlet extraction, homogenization with a solvent, and ultrasonication of the ground sample with an organic solvent. Homogenization is the preferred method for the extraction of pesticides with solvents in the methods described in the Pesticide Analytical Manual (PAM) of the U.S. Food and Drug Administration (FDA, 1994). Handling of the dry samples usually requires the addition of water to moisten the sample, along with the organic solvents. Sonication of the blended mixture expedites the extraction of target compounds. However, it is not recommended for OPs [SW-846 method 8141A; U.S. Environmental Protection Agency (EPA, 1994)]. Acetone, methylene chloride, hexane, petroleum ether, acetonitrile, and methanol are some of the solvents used for extraction of pesticides depending on the sample matrix and the nature of the pesticide(s). Liquid samples are extracted directly from the sample with organic solvents. Water samples are extracted with organic solvents; these methods use solvents that are immiscible with the sample phase. Ideally, one selectively extracts the target compounds by using a solvent whose polarity is similar to that of the target compound. Volatile 681
Copyright 2006, Elsevier, Inc. All rights of reproduction in any form reserved.
682
SECTION Vlll 9Analytical & Biomarkers
solvents, such as ether, ethyl acetate, and methylene chloride, are usually used for the extractions of OPs and CMs. For air samples, a certain amount of air is pumped through a trap tube (OSV-2) that contains an adsorbent such as XAD-2, which efficiently traps OPs and CMs, and then the trap is eluted with organic solvents in the case of OPs and buffer in the case of CMs [National Institute for Occupational Safety and Health (NIOSH), 1994].
A. Current Trends in Sample Extraction Current trends have been to accelerate the extraction of pesticides from the sample matrix by accelerated solvent extraction (ASE) or pressurized solvent extraction (PSE), microwave-assisted solvent extraction (MASE), and super critical fluid extraction (SFE). 1. ACCELERATEDSOLVENT EXTRACTION OR
PRESSURIZEDSOLVENTEXTRACTION ASE or PSE performs extractions at elevated solvent temperatures and pressures to achieve higher extraction efficacy. The process may be compared to pressure cooking. After loading a sample into the extraction cell, the cell is filled with solvent, heated and pressurized, and held at the pressure and temperature for a predetermined time. The clean solvent is pumped into the sample cell, and the sample cell is purged with nitrogen gas. This extract is collected into a collection vial. ASE has been used for the determination of OP and N-methyl CM pesticides in foods (Obana et al., 1997; Okihashi et al., 1997; Richter et al., 2001). ASE has also been used for the extraction of OPs and other pesticides from soils, clays, sediments, sludge, and waste solids (SW-846, method 3545A; EPA, 1994). 2. MICROWAVE-ASSISTEDSOLVENT EXTRACTION This method is similar to ASE, except that heating power is supplied by microwaves. MASE has been used for the extraction of pesticides from soil (de Andrea. et al., 2001). Sun and Lee (2003) report a method for the optimization of MASE as well as supercritical fluid extraction of CM pesticides from soil. Lopez-Avila et al. (1998) studied the stability of OCs and OPs when extracted from solid matrixes with microwave energy. 3. SUPERCRITICALFLUID EXTRACTION In this method, CO2 gas at the critical temperature and pressure is used to extract pesticides and other chemicals from solid samples or liquid samples soaked in an inert powder. CO2 and other gases become fluid (neither gas nor liquid) when temperature and pressure reach a critical point called the supercritical phase. In SFE, this fluid is used to extract target chemicals (OPs and CMs) from solid sampies. Instruments connecting the SFE apparatus with online gas chromatography (GC) have been developed in which the series of steps from extraction to analysis have been
automated. The sample is placed in an extraction chamber through which the supercritical fluid is forced, and the target substances are extracted from the samples and trapped in vials by a small amount of methanol or adsorbent such as florisil or ODS resin. The most common gas used in SFE is CO2. However, supercritical CO2 is very nonpolar, so modifiers are added to improve extraction efficiency for polar compounds. Modifiers for CO2 include methanol, dichloromethane, acetonitrile, and water. Modifiers are mixed with the CO2 using a pump or added directly into the samples. The application and effectiveness of SFE for the extraction of pesticides from various types of samples for pesticide analysis have been demonstrated (Hopper, 1997; Hopper et al., 1995; Kim et al., 1998; Lehotay, 2002; Lehotay and Eller, 1995; Lehotay and Valverdegarcia, 1997; Lehotay et al., 1995). A method for the SFE extraction of methamidophos residue from vegetables has been described (Valverdegarcia et al., 1995). A limitation of SFE is that extraction of fatty foods and animal tissues with SFE extracts the fat along with pesticides. A systemic approach to optimization of SFE of pesticides is presented by Juhler (1997). A review of the usefulness of SFE for pesticide determination is presented by Camel (1998). ASE, MASE, and SFC, and their uses and potential pitfalls, have been discussed in a review (Camel, 2001).
III. C L E A N U P A N D P U R I F I C A T I O N Sample extracts for pesticide analysis from food, feed, biological, and environmental samples are usually complicated mixtures. The other chemical components in the sample are extracted along with the pesticide residue. Usually, the residues are present at the parts per million (ppm) level in the sample, whereas other components are present in very large amounts (Rogers, 1986). The other component chemicals in the sample extract can interfere with GC and highperformance liquid chromatography (HPLC) analysis due to coelution of the impurities with target compounds, and affecting the separation capacity of the analytical column due to overloading by impurities. Furthermore, large amounts of nonvolatile or polar compounds can contaminate GC injection ports and columns. It is therefore necessary to clean up or remove nontarget compounds as much as possible. For OPs and CMs, the usual techniques for purification are liquid-liquid partitioning and column chromatography. For cleanup of OPs and CMs, the adsorption columns used are Florisil, charcoal/Celite, and ODS, in which an octadecyl function is bonded to silica particles (FDA, 1994).
A. Current Trends in Cleanup and Purification 1. SOLIDPHASE EXTRACTION This technique came in vogue with the development of HPLC techniques. This technique is a form of partition
CHAPTER 47 9Analysis of OPs and CMs chromatography and works according to the partition equilibrium between a solid phase and a solvent. If the solid phase is polar, the solvent used is nonpolar. On the other hand, if the solid phase is nonpolar, such as ODS, the solvent used is polar. The advantage of this system is that it is much faster than the traditional column chromatography and uses much less solvent. Solid phases are even available in the form of disks, which can be used to separate OPs and CMs from water (Chiron and Barcelo, 1993; Mersie et al., 2002). The application of SPE has been described for the extraction of pesticides from vegetables (Casanova, 1996), oranges (Yamazaki and Ninomiya, 1999), water (Van-Hoof et al., 2002), and beef muscle (Kuivinen and Bengtsson, 2002). In the case of water, the samples are percolated through the column or filtered through the disk; the pesticides are retained on the column or disk. The pesticides are eluted with appropriate solvents to be used for determination. In the method of Kuivinen and Bengtsson, the bovine muscle is homogenized with ethyl acetate (EtOAc); the homogenate is centrifuged and filtered through anhydrous sodium sulfate. The fat in the filtered extract is precipitated in methanol by cooling, and the extract is diluted with water and passed through an SPE column (isoelute ENV +). After elution with EtOAc, evaporation, and redissolution, the sample is injected into a GC fitted with a capillary column (DB-1701) and detected with a flame photometric detector. In a variation of SPE, honey samples were mixed with Florisil and anhydrous sodium sulfate and loaded in a column, and the column was eluted with hexane-ethyl acetate (90/10 V/V) (Sanchez-Brunete et al., 2002). The column eluate was analyzed by GC and nitrogen phosphorus detector (NPD), as well as GC and mass spectrometry (MS). 2. GEL PERMEATION CHROMATOGRAPHY Gel permeation chromatography (GPC) separates molecules by size. Compounds are separated when sample mixtures are passed through a column packed with material of known pore size. Larger molecules that cannot penetrate the pore size of the packed material (usually known as a gel) elute faster. GPC is used as a general separation method for semivolatile compounds. The separation ability is poorer than that of other forms of chromatography. Thus, GPC is generally used to remove lipids, proteins, and natural resins from samples. It is a very good technique for removing fat in the analysis of fatty samples for pesticides. The GPC technique for pesticide residue determination has been used since 1972. An automated GPC instrument was introduced by Ault et al. (1979) for chromatographic preparation of vegetable, fruits, and crops for OP residue determination utilizing GC with flame photometric detection (FPD). Sannino et al. (1995) described a multiresidue method for the quantitative determination of 39 OPs in seven fatty foods using GPC as a cleanup technique. A report of a cooperative trial to validate the use of GPC
683
cleanup for the isolation of pesticide residues from fats and oils was published by the Committee for Analytical Methods (Anonymous, 1992). A multiresidue screen for quantitative determination of 43 OPs, 17 OCs, and 11 CMs in plant and animal tissues, including ingesta, was reported by Holstege et al. (1994). Ali (1989) applied GPC cleanup and HPLC (with postcolumn derivatization) for the determination of CMs in animal tissues. 3. IMMUNOEXTRACTIONS Immunoaffinity sorbents (ISs) are more selective compared to ODS sorbents. The first commercial ISs were introduced for the cleanup of samples for the determination of aflatoxins (Groopman and Donahue, 1988). ISs have been synthesized for a limited number of pesticides and pesticide classes. Immunosorbents are formed by covalently bonding antibodies to an appropriate sorbent. Immunosorbent is packed into a solid phase extraction cartridge or precolumn as a classical extraction sorbent (Bouzige and Pichon, 1998). There is much interest in developing ISs for single analytes, which are particularly difficult to analyze at the trace level because of a lack of available extraction methods, such as acephate and methamidophs. 4. SOLID PHASE MICROEXTRACTION The solid phase microextraction (SPME) technique is used in many areas. SPME combines the use of solid phase extraction and concentration of the eluate from SPE in one step. SPME probes are silica fibers coated with organic polymers (DB 1, DB 1701, etc.). These fibers capture and concentrate pesticides and, of course, some other chemicals. The fibers are sealed in a syringe. The sample can be injected directly into a GC by using an accessory. The needle of the syringe is lowered into the injection port and then fiber is introduced in the injection port. Pesticides are thermally desorbed from the fiber and then separated on the analytical column and detected. A review of the application of SPME to the analysis of pesticides has been published (Boyd Boland et al., 1996). Methods for the analysis of pesticides using the SPME technique coupled with GC or HPLC have been described for water (Sng et al., 1997; Beltran et al., 1998; Aguilar et al., 1998) and in a food plant (Chen et al., 1998), biological samples (Musshoff et al., 1999), and soil samples (Ng etal., 1999; Moder etal., 1999). SPME fibers are also finding use in the analysis of chemical warfare agents (CWAs) in water (Lasko and Ng, 1997).
IV. C O N C E N T R A T I O N The cleaned up sample extract has to be concentrated in order to inject microliter amounts of the sample extract in GC or HPLC systems connected to high-sensitivity detectors used in the determinative step. Large volumes of solvents in the sample extracts overload the analytical column, particularly in GC. Several types
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S E CT I 0 N V I I I 9 A n a l y t i c a l & B i o m a r k e r s
of concentrators, such as Kuderna-Danish (K-D), Rotory Evaporator (RE), Turbovap, and N-EVAP, are available for sample concentration. The use of K-D, RE, and Turbovap is described in the Pesticide A n a l y t i c a l M a n u a l (FDA, 1994). Information about the use of N-EVAP nitrogen evaporators for sample concentration is available from Organomation Associates.
V. SEPARATION FROM IMPURITIES, D E T E C T I O N , AND D E T E R M I N A T I O N The cleaned up sample extract still contains many impurities; thus, these impurities have to be separated from the analyte before it is determined. The detection and determination of OPs is usually with GC coupled with selective detectors, such as electron capture detector, NPD, FPD, and mass spectrometric detector (MSD or MS). Large varieties of packed as well as megabore and capillary columns are used with these detectors. CMs are determined with GC coupled to NPD or FPD (in S mode). CMs are effectively determined with HPLC/photo diode array or HPLC/fluorescence. For a trace amount, it is essential to do a postcolumn derivatization (Ali, 1989).
A. Chromatography Chromatographic methods in general are the most common methods used at the determinative step in pesticide analysis. Thus, basic understanding of chromatographic procedures and the detection devices is important for pesticide analysis. According to the International Union of Pure and Applied Chemistry, chromatography is the method used primarily for the separation of components of a sample in which the components are distributed between two phases, one of which is stationary while the other moves. The stationary phase may be a solid, a liquid supported on a solid, or a gel. The stationary phase may be packed in a column, spread as a layer, or distributed as a film, etc. The mobile phase may be gaseous or liquid. Thus, there are two movements in the chromatographic system: the mobile phase movement, which is usually at a constant rate, and the movement of the components of the sample. The movement of the components depends on their relative distribution between the stationary phase and the mobile phase. This results in the separation of the components. This separation process coupled with the detection and measuring device completes the chromatographic system.
1. GAS CHROMATOGRAPHY In GC, the basic instrument consists of a sample introduction port (injector), an analytical column in an oven (to separate the components of the mixture), a detector (which can detect the presence of chemicals eluting from the analytical column), and a recording or data handling device. The injector,
oven, and detectors are heated zones; the temperature regulation of these zones depends on the application. Initially, packed columns were used in GC, but these columns had only limited resolving power. With the advent of capillary and megabore columns in which the liquid phase is crosslinked to pure silica capillary tubes, the resolving power of the analytical column increased severalfold. Lawrence (1987) showed the separation of 38 OPs on a DB-17 capillary column used for GC with FPD.
2. HIGH-PERFORMANCELIQUID CHROMATOGRAPHY HPLC is a variant of traditional liquid chromatography. The resolution and separation are improved by using small, uniform particle size columns. These columns require the use of a high-pressure pump that can force the liquid mobile phase through the columns, presumably at a constant rate. The basic HPLC instrument consists of an injector, an analytical column (which may or may not be temperature controlled), and a detector. With the development of hardware for pumps, the performance of liquid chromatography has increased manyfolds. The greatest impediment to the use of HPLC in residue analysis has been the absence of selective detectors, as are available for GC. However, the use of HPLC coupled with the MSD in trace analysis of pesticides is increasing, particularly for polar and thermally labile compounds. The technique of postcolumn derivatization (attaching a fluorophore) and fluorometric analysis has proved very useful for the determination of N-methyl CMs in foods (Holstege et al., 1994) and tissues (Ali, 1989; Ali et al., 1993a,b).
B. Detectors 1. ELECTRONCAPTURE DETECTOR The modem ECD contains a 63Ni source. This source emits high-energy beta particles. When these particles collide with the carrier gas molecules to produce low-energy electrons, the electrons continually collect at the cell anode by applying voltage pulses to the cell electrode. Cell current thus produced is measured and the pulse interval (frequency) adjusted to maintain constant cell current. A standing pulse frequency describes the equilibrium condition that exists when only carrier gas is passing through the cell. When molecules of an electrophilic substance enter the detector, electrons are "captured" to a degree depending on the electron affinity of the substance. The electron supply in the cell decreases, and pulse frequency increases to generate constant current. Change in the frequency required to keep the cell current constant is converted to voltage and is sent to the recording device as the detectors response to the analyte.
2. FLAME PHOTOMETRICDETECTOR When GC column effluent is burned in a hydrogen/air flame, compounds containing phosphorus and/or sulfur
CHAPTER 47 9Analysis of OPs and CMs produce characteristic emissions. A narrowband pass (interference) filter of appropriate wavelength isolates emissions produced by either phosphorus or sulfur. These emissions can be viewed by a conventional photomultiplier tube; a filter with maximum transmittance at 526 nm permits detection of phosphorus compounds, whereas one with maximum transmittance of 304 nm detects sulfur compounds. A single optical filter and photomultiplier tube may be used, or two filters and photomultiplier tubes can be assembled to permit response to both phosphorus and sulfur compounds simultaneously. A variation of FPD known as the pulsed flame photometric (PFPD) has been introduced. The PFPD uses the time-delayed chemiluminescence produced by the heteroatom in a molecule. It is claimed that PFPD is much more sensitive than FPD. Moreover, several other heteroatoms, such as S, P, N, and Se, can be detected by PFPD (Jing and Amirav, 1998). PFPD has been used simultaneously with MS to enhance pesticide detection capabilities (Amirav and Jing, 1998) 3. NITROGEN PHOSPHORUS DETECTOR The NPD is selective to residues containing nitrogen and/or phosphorus atoms. Modem NPD detectors evolved from the potassium chloride thermionic detector. GC effluent impinges onto the surface of an electrically heated and polarized alkali metal salt source (usually rubidium) in the presence of air/hydrogen plasma; ionization occurs and the flow of ions between plasma and an ion collector is amplified and recorded. Detector response to analytes results from the increased ionization that occurs when compounds containing nitrogen or phosphorus elute from the column. At gas flow rates used for NPD operation, the degree of ionization of compounds containing nitrogen or phosphorus is 10,000 times greater than for hydrocarbons. The mechanisms of enhanced response to nitrogen and phosphorus are not completely clear. However, both gas phase and surface ionization processes have been proposed. ECD, FPD, and NPD are discussed in detail in Chapter 5 of PAM (FDA, 1994).
4. MASS SPECTROMETRIC DETECTOR A mass spectrometer is an instrument that can separate charged atoms or molecules according to their massto-charge ratio. Relative molecular masses of organic compounds and biopolymers can be measured in this way, and the instrument is also capable of generating structural information. The sample is introduced into the mass spectrometer, which is generally kept under high vacuum (5-10 mbar). Compounds are converted into gas phase molecules either before or during the charging or ionization process, which takes place in the ion source. Many types of ionization mode are available: The type of compound to be analyzed and the specific information required determine which ionization mode is the most suitable. Once ionized, the molecule ion may fragment, producing ions of lower mass than the original precursor molecule. These fragment ions are dependent
685
on the structure of the original molecule. The ions produced are repelled out of the ion source and accelerated toward the analyzer region. Although both positive and negative ions may be generated at the same time, one polarity is chosen and either positive or negative ions are analyzed and recorded. Molecules that do not ionize (i.e., remain neutral) are pumped away and will not be detected. There are various types of ionization techniques, such as electron impact ionization (EI), chemical ionization, fast atom bombardment/ liquid secondary ionization, matrix-assisted laser desorption ionization, and electrospray ionization. Recently, tandem mass spectrometers (MS-MS) have been introduced. In a tandem mass spectrometer, two mass spectrometers are coupled together separated by a collision cell. By coupling together two analyzers, separated by a collision cell, additional information can be obtained. The first analyzer is used to select the ion of interest. This ion is then passed into the collision cell, which usually will have been pressurized with an inert gas such as argon. Collision of the ion with the atoms in the cell can induce dissociation of the ion. This is known as collision-induced dissociation: The original ion is referred to as the "precursor" ion and the dissociated ions are known as "product" ions. Product ions are then analyzed in the second mass spectrometer, thus generating a product ion mass spectrum of the original precursor ion. The tandem mass spectrometer provides much more structural information about the analyte molecule and thus is an excellent confirmation technique. The greatest impediment for the use of mass spectrometers in common laboratories has been their high price. A direct sample introduction device has been used for the GC/MS-MS determination and confirmation of pesticides (Lehotay, 2002). It has been claimed that the sample extracts need not be cleaned up before determination. A method for the deterruination of 37 OPs in human tissue using GC/MS has been described (Russo et al., 2002). A HPLC/MS method for the determination of eight different types of CM pesticides in serum has been reported (Kawasaki et al., 1993). In this method, 1.5 ml of serum is mixed with 1.5 ml of 0.2 M phosphate buffer. The mixture is applied to an Extrelute column and then eluted with 15 ml of methylene chloride. The eluate is evaporated and the residue is dissolved in mobile phase. Carbofuran has been determined in stomach/rumen contents by GC/MS. Stomach/rumen contents are extracted with methylene chloride and cleaned up with GPC, and then the sample is analyzed by GC/MS (Osheim et al., 1985). Detection of pesticides in various types of samples without any cleanup has also been reported (Smith and Lewis, 1988). In this method, the samples are shaken with a suitable solvent and then analyzed by GC/MS. This method is qualitative in nature. 5. ION MOBILITY SPECTROMETRY Ion mobility spectrometry (IMS) has been known for more than 25 years. In IMS, the analyte and impurity molecules
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SECTION VllI 9Analytical & Biomarkers
are ionized by a 63Ni source, which is housed in a shielded chamber. As discussed previously, the 63Ni source gives off high-energy electrons, which collide with sample molecules to produce ions. The aspirated air or any other gas used to introduce the sample in the reaction chamber also produces ions. The ions move through an electric field in the drift tube. Smaller ions move faster compared to larger ions. Thus, this technique is based on the size-to-charge ratio of ions. This technique differs from mass spectrometry, in which the separation is on the basis of mass-to-charge ratio of ions. Also, in IMS the ions are formed at atmospheric pressure, whereas in mass spectrometry ions are formed at very low pressures (5-10mbar). A walk-through portal based on IMS has already been installed at the Statue of Liberty for detecting traces of explosives and other illegal chemicals. Compact units are in use at many airports for the detection of traces of explosives and illicit drugs. The application of IMS for monitoring mevinphos (phosdrin), an OP, has already been demonstrated (Tuovinen et al., 2001). Fematoscan (1999), a company involved in the manufacture of IMS-based detectors, has demonstrated the application of a handheld detector, which is a combination of GC and IMS for pesticide detection, including some OPs.
VI. CONFIRMATION Mass spectrometry is the technique of choice for confirmation of pesticides. In the case of OPs, the sample extract can be run on different columns. Relative retention times (RRt) of a given peak can be compared with a table of RR t for different columns (FDA, 1994). For CMs, the alkylated CMs can be chromatographed using GC with NPD detector. The retention time of peaks can be compared with the retention time of the standard compound. If enough of the residue compound is present, then thin-layer chromatography can be used to confirm the presence of the pesticide (FDA, 1982).
VII. SOURCES OF A N A L Y T I C A L METHODS Several excellent sources of analytical methods for OPs and CMs are available. These sources also contain methods for other classes of pesticides:
1. Pesticide Analytical Manual, Vol. 1 (FDA, 1994) 2. Pesticide Analytical Manual, Vol. 2 (FDA, 1991) 3. Manual of Analytical Methods for the Analysis of Pesticides in Humans and Environmental Samples (EPA, 1980)
4. NIOSH Manual of Analytical Methods (NIOSH, 1994) 5. Test Methods for Evaluating Solid Wastes (EPA, 1994) 6. Manual of Pesticide Residue Analysis, Vol. 1 [Deutsche Forschungsgemeinschaft (DFG), 1987]
7. Manual of Pesticide Residue Analysis, Vol. 2 (DFG, 1992)
8. A World Compendium. The Pesticide Manual (Tomlin, 1997)
9. Official Methods of Analysis (OMA) of AOAC International [Association of Analytical Communities (AOAC), 2003a]
10. ATSDR Pesticide Profiles [Agency for Toxic Substances and Disease Registry (ATSDR), 1994]
11. Residue Analytical Methods (RAM) (EPA, 2003) 12. Environmental Chemistry Methods (ECM) (EPA, 2004) 13. Scientific reviews
A. Pesticide Analytical Manual, Vol. 1 (FDA, 1994) PAM, Vol. 1, is the most exhaustive and complete compendium of analytical methods for the multiresidue method (MRM) analysis of pesticides in food stuffs. There are six chapters in PAM. The first chapter deals with regulatory operations, which describes what pesticide tolerance is and under which laws and statutes the pesticide tolerances are regulated. It also describes preparation of analytical sample and method application. Chapter 2 provides information about general analytical operations and discusses basic analytical techniques such as column chromatography; solvent selection and evaporation; equipment and procedures for comminuting samples; and procedures for specific commodities, such as crabs, eggs, fish, hay, straw, food, and feed ingredients. Also discussed in this chapter are special reagent preparation, tests and purification for applicable reagents, reference standards for pesticides, quality assurance and quality control, preparation of standard operating procedures, hazardous waste disposal, and safety issues. Chapter 3 discusses the need for MRMs of analysis. When the target analyte (residue compound) is not known, it is essential to use a method that can detect the maximum number of residues. For this purpose, multiclass MRMs are used. This chapter describes multiclass MRMs, their capabilities, and their limitations. Three MRMs are described in detail for nonfatty foods as well as fatty foods. Methods 302 and 303 are for nonfatty foods, and method 304 is for fatty foods. Each method is composed of various modules, such as for extraction, cleanup, identification, and determination. The extraction, cleanup, and detection modules are selected based on the nature of the sample. For example, for method 302 for nonfatty foods, there are 7 extraction modules, 6 cleanup modules, and 19 determinative step modules. Thus, method 302 is not a single method but a combination of various analyrical methods. The validation references for each module are also provided. The methods described in PAM are all validated methods. The extraction modules are described as El, E2, E3, etc.; the cleanup modules are described as C1, C2, etc.; and those for the determinative step are described with a prefix D. The complete description of the determinative step
CHAPTER 4 7
is DG or DL followed by a number. The DG modules refer to GC methods and DLs refer to HPLC methods. It should be noted that the extraction module E1 and cleanup module C1 of method 302 are not same as described for method 303 or any other method. Thus, in order to define a method of analysis, the method number, such as 302, 303, or 304, as well as the extraction module and cleanup module should be specified. The determinative step modules, such as DG1, DG2, or DL, are independent of method number and can be used with sample extract prepared by method 302, 303, or 304. Chapter 4 of PAM discusses methods 401-404. Method 401 is for N-methylCMs, method 402 is for acids and phe-
TABLE 1.
Method
PAM 302
PAM 303
Matrix
Nonfatty, high-moisture (>75%) commodities
Fruits and vegetables, moisture >75 %, sugar 10 kPa (75 mmHg). If these measures fail,
9Management of OP Pesticide Poisoning
717
the patient should be intubated and mechanical ventilation (with positive end expiratory pressure) should be instituted. In severely poisoned patients who are hypotensive, it may be necessary not only to expand plasma volume but also to use a vasopressor (e.g., dopamine titrated to a systolic pressure >90 mmHg) or an inotrope (e.g., dobutamine 2.5-10 txg/kg/min to maintain cardiac output). Cardiac arrhythmias should be treated conventionally, and hypoxia must be considered as a possible etiology. The management of convulsions and muscle fasciculation with diazepam is discussed later. Careful attention must be given to fluid and electrolyte balance and adjustments to infusion fluids made as necessary. Heart rate, blood pressure, electrocardiogram, and arterial blood gases should be monitored routinely.
B. Minimizing Further Absorption of the OP Pesticide If exposure is dermal, thorough skin decontamination should be carried out, without caregivers themselves being contaminated, after resuscitation and stabilization. All contaminated clothing should be removed and affected skin should be washed thoroughly with soap and cold water, including exposed areas (e.g., hands, arms, face, neck, and hair). Gastric lavage may be considered in all potentially serious cases if ingestion has occurred less than 1 hr previously, although its value is unproven. Lavage should be performed with care and with an endotracheal tube in situ if the level of consciousness is depressed because hydrocarbons are present in many OP insecticide formulations. Syrup of ipecacuanha should be avoided because emesis is dangerous in a patient whose level of consciousness may deteriorate because of either the OP or the solvent in the insecticide formulation; aspiration pneumonia is a wellrecognized complication in these circumstances. Moreover, aspiration can also occur in conscious patients given syrup of ipecacuanha and is more likely to happen if hydrocarbons are present in the pesticide mixture. The capacity of activated charcoal to adsorb most OP compounds has not been demonstrated. On theoretical grounds, a single dose of activated charcoal (50-100 g) may be beneficial if administered less than 1 hr after OP insecticide ingestion. No cathartic should be administered with the charcoal because this may exacerbate OP-induced gastroenteritis.
V. APPROPRIATE USE OF ATROPINE, OXIMES, AND DIAZEPAM The development of specific therapies effective in poisoning by OP pesticides was undertaken in a number of institutions, including chemical defense establishments and academic laboratories. The first development was the discovery of the usefulness of anticholinergic drugs such as atropine, which was soon followed in the 1950s by the
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Measures
demonstration of oxime-induced cholinesterase reactivation (Wilson and Ginsburg, 1955). A later development was the addition of anticonvulsants to the atropine/oxime combination. Atropine competes with ACh and other muscarinic agonists for a common binding site on the muscarinic receptor, thus effectively antagonizing the actions of ACh at muscarinic receptor sites, which leads to increased tracheobronchial and salivary secretions, bronchoconstriction, and bradycardia. The usefulness of atropine is virtually undisputed. Clinically, the main benefit of oximes is to reverse cholinergic effects at peripheral nicotinic sites so that, for example, muscle strength may improve. Oximes are much less effective than atropine at peripheral muscarinic sites, and their effects on CNS-mediated symptoms and signs may not be clinically significant. Although the therapeutic combination of oxime and atropine is well established in the treatment of OP pesticide poisoning, there is still no international consensus on the choice of oxime and on aspects of dosing; indeed, some have doubted the worth of oxime therapy altogether. For these reasons, the discussion on the oximes is relatively long. Benzodiazepines (most often diazepam and sometimes midazolam) may also be of benefit by reducing anxiety and restlessness, reducing muscle fasciculation, arresting seizures, and possibly reducing morbidity and mortality when used in conjunction with atropine and pralidoxime.
A. Atropine Atropine competes with ACh and other muscarinic agonists for a common binding site on the muscarinic receptor, thus effectively antagonizing the actions of ACh at muscarinic receptor sites. However, the peripheral antimuscarinic effects of atropine may not be the only antidotal property of the drug in OP poisoning. Atropine may also be of value in treating acute dystonic reactions occasionally observed in acute OP poisoning (Joubert et al., 1984; Joubert and Joubert, 1988; Smith, 1977; Wedin, 1988). |. PHARMACOKINETICS After intravenous dosing, atropine distributes rapidly, with only 5% remaining in the blood compartment after 5 min (Berghem et al., 1980). The apparent volume of distribution (Vd) is 1-1.7 liters/kg. Atropine is partly metabolized in the liver by microsomal monooxygenases to noratropine, tropine, atropine-N-oxide, and tropic acid (Van der Meer et al., 1983) and partly excreted unchanged in the urine. Elimination kinetics can be fitted to a two-compartment model with a clearance of 5.9-6.8 ml&g/min and a half-life of 2.6-4.3 hr in the elimination phase (Aaltonen et al., 1984; Kanto et al., 1981; Virtanen et al., 1982). Since the renal plasma clearance ( 6 5 6 _ 18 ml/min) was found to approach the renal plasma flow (712 _+ 38 ml/min), tubular
excretion may occur. Thus, both liver and renal disease can be expected to influence the kinetics of atropine (Hinderling etal., 1985). The elimination half-life of atropine is longer in children younger than 2 years of age and in the elderly. In the neonate, the half-life is between 5 and 10 hr due to an increased volume of distribution. In the elderly (70 years or older), the half-life may be prolonged to 30 hr due to reduced clearance. 2. EFFICACY Sanderson (1961) studied the effect of intraperitoneally administered atropine 17.4 mg/kg given alone or combined with oximes on the survival of rats poisoned orally by 10 different OPs, excluding dimethoate. Atropine alone prevented the development of toxicity. Although the numbers of rats in each group were small (n = 6) and statistical analysis was not performed, this study demonstrated that atropine treatment alone did reduce mortality. In calves poisoned with intravenous dichlorvos, atropine was shown to reverse the respiratory effects of the OP (Lekeux et al., 1986). The OP-induced reduction in dynamic lung compliance, arterial oxygen tension, increase in total pulmonary resistance, work of breathing, and alveolar arterial oxygen gradient were reversed by atropine. Atropine may therefore reverse changes in ventilation-perfusion inequalities resulting from uneven distribution of ventilation caused by ACh-mediated airway constriction (Slocombe and Robinson, 1981). Although the clinical efficacy of atropine in OP poisoning is well established (Bardin et al., 1987; DuToit et al., 1981 ; Namba et al., 1971; Senanayake and Karalliedde, 1987; Zilker and Hibler, 1996), no controlled studies have been published. Finkelstein et al. (1989) performed a noncontrolled prospective study of severe OP poisoning. In this study of 53 adult patients, atropine 2 mg by intravenous bolus, followed by the same dose at intervals of 10 min or more, was administered and the dose was adjusted as necessary to control tracheobronchial secretions and bronchospasm. All 53 patients were mechanically ventilated and obidoxime was also given. Although it is not possible to quantify any beneficial effect from atropine administration alone, atropine appeared to counteract the muscarinic features.
3. DOSAGEREGIMEN Atropine sulfate 2 mg (0.02-0.1 mg/kg in a child) intravenously should be given as soon as possible to patients who have increased secretions or bronchorrhea. Repeated injections of atropine, every few minutes if necessary, may be required during the first few hours of therapy; the dose should be titrated to control peripheral muscarinic signs, notably bronchorrhea and bronchospasm. In very severe cases, 100 mg or more per day may be required to control symptoms.
CHAPTER 49 The degree of atropinization may be assessed by dryness of the mouth and the magnitude of the tracheobroncheal secretions, pupil size, and heart rate. Tachycardia and mydriasis can be unreliable indicators, however, since both may also result from nicotinic stimulation in severely poisoned patients or, in the case of mydriasis, be a direct local effect of exposure. Tachycardia may also reflect hypoxia. The most sensitive and useful measure of adequate atropinization is the repeated evaluation of the quantity of secretions produced (Bardin et al., 1987; DuToit et al., 1981). B. Oximes
Early work on cholinesterase reactivators has been reviewed (Hobbiger, 1963). The earliest reactivators of AChE studied were hydroxylamine, choline, and hydroxamic acids. Pralidoxime (PAM), a much faster reactivator than hydroxylamine, was introduced by Wilson and Ginsburg (1955). The next major advance was the introduction of obidoxime by Ltittringhaus and Hagedorn (1964) and von Erdmann and Engelhart (1964). Large numbers of pyridinium oximes, both mono- and bis-pyridinium compounds, have been studied in OP poisoning, but only PAM salts and obidoxime have achieved widespread use (Table 2). Other oximes, particularly the Hagedorn oximes, have been studied for use in nerve agent poisoning, especially poisoningby soman (Dawson, 1994), and one (HI-6) has been the subject of a large case series in former Yugoslavia (Kusic et al., 1991), where it was employed to treat OP pesticide poisoning. This discussion focuses predominantly on PAM because this oxime is clinically used most frequently. 1. ACTION OF OXIMES The fundamental action o f the pyridinium oximes is to reactivate AChE inhibited by OPs. The pyridinium oximes also reactivate BuChE (Hobbiger, 1963; Worek etal.,
TABLE 2. Oxime
Pralidoxime
Obidoxime Asoxime
9Management of OP Pesticide Poisoning
1999), but dimethyl BuChE is reactivated more slowly than dimethyl ACHE. The action on BuChE is probably not clinically significant in the action of the pyridinium oximes, although it has been suggested that BuChE acts as a sink for anticholinesterases, in which case reactivation of the enzyme would presumably restore the capacity of the sink. The reactivation of AChE allows ACh to be hydrolyzed in the normal way, and therefore normal cholinergic neurotransmission will resume. It is usually considered that the beneficial effects of oximes in OP poisoning are confined to peripheral nicotinic sites and that CNS effects are clinically insignificant (Bismuth et al., 1992), although there is evidence that PAM can cross the blood-brain barrier (Sakurada etal., 2003). This means that the beneficial effects will mainly be on neuromuscular transmission, and that there will be little action on parasympathetic effects, such as bronchorrhea, bronchoconstriction, and rhinorrhea, or on CNS effects. 2. FACTORS LIMITING OXIME EFFICACY Three main factors limit the success of oxime therapy: the continuing presence of high concentrations of inhibitor in the plasma (Willems et al., 1993; Worek et al., 1997), aging, and the formation of phosphoryl oximes. The first depends on the dose and pharmacokinetics of the inhibitor: Fat-soluble OPs may form a deep compartment. Aging results from monodealkylation of the dialkoxyphosphorylated AChE (Karalliedde and Szinicz, 2001). Aged enzyme reactivates neither spontaneously nor under the influence of oximes so that recovery of enzymic activity depends on synthesis de novo of more enzyme. Although the tl/2 for aging of inhibited human AChE by soman is on the order of a few minutes, it is clear that for most OP pesticides the tl/2's for aging are much longer; for dimethoxyphosphoryl ACHE, it is 2-32 hr and for diethoxyphosphoryl AChE > 3 6 hr (Mason etal., 2000; Wilson etal., 1992; Worek et al., 1999). Similar differences exist in aging rates of dimethoxy- and diethoxy-inhibited BuChE (Mason et al.,
Oximes That Have Been Used in Human OP Pesticide Poisoning Salt
Iodide Chloride Mesilate, methanesulfonate Methylsulfate Chloride Chloride
719
Abbreviation a
2-PAMI 2-PAMC1 P2S 2-PAM methylsulfate
Proprietary name
Protopam
Contrathion Toxogonin
HI-6
aThere is some discrepancy in the use of the abbreviation2-PAM, so PAM is used for pralidoximein general in this chapter and the abbreviations as above for the salts. Many papers fail to state which salt was used. Note that the mesilate and methylsulfate are not the same salt.
720
SECTION I X . Therapeutic M e a s u r e s
1993), and these differences are important when considering BuChE activity after exposure to OPs and during recovery from their effects. With the typical dimethoxy and diethoxy structure of pesticides, aging rates are such that the phenomenon only becomes a problem when treatment is delayed and/or exposure prolonged. It is commonly, but erroneously, believed that 1 day after intoxication with an OP insecticide virtually all the phosphorylated enzyme will be in the aged form so that oxime therapy will be ineffective after this time. However, this interpretation derives from in vitro studies in which AChE is rapidly inhibited and is maintained fully inhibited thereafter by the presence of an excess of inhibitor and in the absence of oxime throughout the experiment. Such experiments do not represent the case in vivo and should not be used as a reason to abandon the use of oxime therapy after 24 hr. There is evidence from case reports and series that with dimethoxy pesticides, the efficacy of oximes may be reduced by aging. Because of concerns that dimethylphosphoryl enzyme might age sufficiently fast to interfere with the efficacy of oximes, studies have been carried out in vitro to investigate this (Ganendran and Balabaskaran, 1976; Skrinjaric-Spoljar et al., 1973). Less is known about structures other than the typical dimethyl and dethylphosphoryl enzyme and the effects of oximes. With AChE inhibited by profenofos (Glickman et al., 1984; Wing et al., 1984), an O-ethyl, S-propyl structure, aging occurs rapidly and oximes would be expected to be ineffective. With phosphoramidothioates, such as methamidophos, spontaneous and oxime-induced reactivation occurs and aging does not seem to be a problem: These data should not be extrapolated to all phosphoramidothioates, however, because fairly rapid aging occurs with the dithio analog of methamidophos (de Jong etal., 1982; Langenberg etal., 1988). Indeed, human plasma cholinesterase inhibited by propetamphos and crufomate, the former a phosphoramidothioate and the latter a phosphoramidate, appears not to undergo spontaneous reactivation (Mason et al., 1993). A third factor that may interfere with the reactivating capability of oximes is the formation of phosphoryl oximes in the AChE reactivating reaction because phosphylated oximes can act as AChE inhibitors (de Jong and Ceulen, 1978; Nenner, 1974). Fortunately, those produced by reactivation of AChE inhibited by pesticides appear not to be very stable in the plasma because they are hydrolyzed rapidly (Kiderlen et al., 2000; Leader et al., 1999). 3. ACTIONS OF OXIMES OTHER THAN REACTIVATION OF CHOLINEsTERASES
In a study on guinea pigs performed by Inns and Leadbeater (1983) involving pretreatment with pyridostgmine in both treatment regimens, the administration of bis-pyridinium compounds that are not oximes with atropine and diazepam was more effective than atropine and diazepam alone in
poisoning by certain OP nerve agents. This suggested that bis-pyridinium compounds did not depend entirely on the oxime moiety for their antidotal effects. There is evidence that oximes may react directly with OPs and such effects have been reviewed (Van Helden et al., 1996). Oximes may have direct activating and inhibitory effects on AChE (Alkondon et al., 1988; Kuhnen, 1971). It has been shown that both 2-PAMI and HI-6 interact with the nicotinic receptor-ion channel complex (Alkondon etal., 1988). It is unclear how much these actions contribute to the efficacy of oximes. Kusic et al. (1991) suggested that a general improvement in the clinical condition of poisoned patients, which was more rapid than the rise of AChE activity with HI-6, indicated direct beneficial pharmacological effects. In a study on the rat to evaluate noncholinesterase reactivating effects of the Hagedorn oximes, Van Helden et al. (1994) treated atropinized rats iv with three times the LD50 of crotylsarin, O-(2-butenyl) methylphosphonofluoridate. This compound produces an inhibited AChE that ages instantly, thus precluding any beneficial reactivating effects of the oxime. After 5 min, the poisoned rats were treated with saline or an oxime (obidoxime, HI-6, HL6-7, HGG-12, or HGG-42). The oximes significantly prolonged survival time compared to the saline-treated rats and the authors suggested that this effect must be by action other than cholinesterase reactivation. However, because the prolongation of survival time did not correlate with differences in pharmacokinetics of the oximes, potency in restoring neuromuscular transmission, and affinity for muscarinic receptors, the authors also concluded that the effect must be mediated through an unknown mechanism.
C. Pralidoxime 1. PHARMACOKINETICS
The majority of kinetic studies on PAM have been performed in healthy, nonpoisoned subjects. There is evidence from animal (Green et al., 1985, 1986) and human (Jovanovic, 1989) studies that OP compounds can alter PAM kinetics in a complex manner; this may be due to the cardiovascular changes and reduced blood flow seen in OP pesticide poisoning. Therefore, it may be inappropriate, to extrapolate the results of volunteer studies to severely poisoned patients. Because they are quaternary amines, PAM salts and obidoxime are not well absorbed after oral administration, although PAM chloride tablets remain commercially available. Ethanol does not appear to interfere with the absorption of PAM (Calesnick et al., 1967). The distribution of PAM is largely determined by its small molecular size and quaternary amine structure. It is thus widely distributed in most body fluids and is not significantly bound to plasma proteins (Sidell etal., 1972b). PAM penetrates the erythrocyte membrane by
CHAPTER 49 9Management of OP Pesticide Poisoning simple diffusion and does not bind to either red blood cell stroma or hemoglobin (Ellin et al., 1974). PAM does not pass readily into the CNS because of its quaternary nitrogen structure (Uehara et al., 1993). The in vivo rat brain microdialysis technique with high-performance liquid chromatography/ultraviolet was used by Sakurada et al., (2003) to determine whether 2-PAMI penetrated the blood-brain barrier. After intravenous injection of 2-PAMI at doses of 10, 50, or 100 mg/kg, PAM appeared in the dialysate and concentrations were dose dependent; the striatal extracellular:blood concentration ratio at 1 hr after a 50 mg/kg dose was 0.093 _+ 0.053 (mean _+ SEM). Neural uptake of PAM was sodium dependent. These results suggest that PAM may have some capability in reactivating CNS ACHE. Furthermore, Ligtenstein and Kossen (1983) found some entry of HI-6 into the CNS in the rat, as did Klimmek and Eyer (1986) in the dog. The values for the apparent volume of distribution in the central compartment (V1), peripheral compartment (V2), and at steady state [(Vd)ss] in volunteer studies are given in Table 3; the values at steady state are 0.6-0.8 liters/kg. However, in poisoned patients treated with methylsupfate and chloride salts, respectively, the mean (_+SD) volumes of distribution were found to be 2.77 +_ 1.45 liters/kg (Willems and Belpaire, 1992) and 2.8 + 2.21iters/kg (Jovanovic, 1989). For children treated for OP poisoning with a loading dose and then continuous infusion of PAM, the volume of distribution was higher in those severely poisoned (Schexnayder et al., 1998). PAM is metabolized only to a minor extent in humans (Gibbon et al., 1979). PAM is excreted rapidly in urine (Calesnick et al., 1967; Jager and Stagg, 1958; Loomis, 1963; Rivero Gonz~ilez et al., 2001; Sidell et al., 1972a,b; Sidell and Groff, 1971; Swartz and Sidell, 1973; Vojvodic and Maksimovic, 1972). Most studies, whether performed on humans or animals, appear to demonstrate first-order disappearance of PAM from the plasma (Jager and Stagg, 1958; Kondritzer et al., 1968; Sidell e t a l . , 1972b). Because urine clearance of PAM exceeds simultaneously measured creatinine clear-
TABLE 3.
ance, it is probable that PAM is secreted by the renal tubules, at least in part, by secreting mechanisms shared by several other strong bases (Swartz and Sidell, 1973). In several volunteer studies, the PAM half-life varied from 67 to 84 min (Table 3) after intravenous dosing of PAM 5-10mg/kg body weight. In poisoned patients treated with the methylsulpfate and chloride salts, respectively, the mean (_+ SD) elimination half-lives were found to be 3.44 +_ 0.9 hr (Willems and Belpaire, 1992) and 2.9 +_ 1.18 hr (Jovanovic, 1989). Josselson and Sidell (1978) investigated the effect of intravenous thiamine hydrochloride on the elimination of pralidoxime chloride 5 mg/kg body weight administered intravenously. The addition of thiamine lengthened the elimination half-life and the oxime concentration increased, whereas the intercompartment clearances and rate constant for elimination of oxime declined. The authors suggested that either thiamine and PAM compete for a common secretory mechanism or thiamine alters the membrane transport of PAM. PAM is excreted preferentially in acid urine (Berglund et al., 1962) and intravenous sodium bicarbonate markedly reduces PAM excretion. Swartz and Sidell (1973) observed that in six volunteers given 2-PAMC1 5 mg/kg body weight (but not atropine), exercise alone and exercise and heat stress significantly (p < 0.05) increased the PAM elimination half-life [71.2 _+ 7.4 min (control) vs 87.7 + 14.7 and 86.2 +_ 13.5 min, respectively). This suggests that exercise and heat reduce the renal elimination of PAM. Because oximes are quaternary ammonium compounds, they will be fully ionized in aqueous solution. Unless the accompanying anion has pharmacological properties of its own, there would thus be no expectation that the PAM salts would differ in activity, if used on molar equivalent bases, either qualitatively or quantitatively. Sidell e t a l . (1972b) compared the pharmacokinetics of 2-PAMC1 and P2S after intravenous administration to human volunteers. The two PAM salts at the same (mass) dose (5 mg/kg) produced virtually identical plasma concentration-time curves. The
Pralidoxime Kinetics after Intravenous Dosing in Volunteers Vd (liters/kg) a
Dose and salt
5 mg/kg chloride 5 mg/kg chloride 5 mg/kg chloride 5 mg/kg P2S 10 mg/kg chloride
721
tl/2 [~ (min)
V1
V2
(Vd)ss
Reference
78 71 67 84 79
0.27 0.37 0.18 0.20 0.30
0.54 0.39 0.42 0.58 0.46
0.82 0.76 0.60 0.78 0.76
Sidell et al. (1972b) Swartz and Sidell (1973) Josselson and Sidell (1978) Sidell et al. (1972b) Sidell and Groff (1971)
agl, central compartment; V2,peripheral compartment;(Vd)ss,volumeof distribution at steady state.
722
SECTION IX. T h e r a p e u t i c
Measures
tl/2's were similar, being 1.31 and 1.41 for 2-PAMC1 and P2S, respectively. The major difference was in Vd, which at steady state was 815 ___ 105 ml/kg for 2-PAMC1 and 775 ___204 ml/kg for P2S (mean ___SD).
VI. E F F I C A C Y O F O X I M E S A N D A T R O P I N E De Silva etal. (1992) concluded that nothing is to be gained in cases of severe acute OP insecticide poisoning by the addition of oximes to the standard regimen of atropine plus mechanical ventilation. This conclusion was based on a study in which 21 patients received atropine alone and 24 patients received atropine plus 2-PAMC1 (median doses, 4 g in the first 24 hr and 1 g daily thereafter). The mortality in both groups was 29%, which is not dissimilar to that reported from other centers managing severe cases of OP insecticide poisoning. Thus, the need for more effective treatment for OP poisoning is undeniable. However, in all probability the supposed failure of oxime therapy in this study does not indicate ineffectiveness of the drug employed nor, necessarily, does it indicate delay in administration but, rather, inadequate oxime dosing. The value of oximes has also been challenged on the basis of a systematic review of clinical trials (Eddleston et al., 2002). What, then, is the role of oximes?
A. Pharmacodynamic Studies in Animals Many of the relevant pharmacodynamic studies on pesticides are in the proprietary literature supporting registration of pesticides. These data can be accessed through the Joint Expert Meeting on Pesticide Residues monographs or through national pesticide registration authorities. Note that in these studies, the oxime therapy is usually given a few minutes after the OP challenge. This is not particularly representative of the clinical situation, in which the patient reaches the hospital some hours after poisoning: in such circumstances, these studies may give an unrealistic idea of the likely efficacy of oxime therapy, particularly with dimethoxy OPs, where aging may occur if oxime therapy is delayed. Examples of such studies are those with mevinphos, a dimethoxy OP (Kassa and Fusek, 1997), and methidathion, also a dimethoxy OP. In a rat study, atropine sulfate, PAM, obidoxime, and a combination of atropine sulfate and obidoxime were all effective when given at the first sign of poisoning with methidathion [Food and Agriculture Organization/World Health Organization (FAO/WHO), 1993]. A number of diethoxy OPs have been studied in relation to oximes, including diazinon, phosalone, pyrazophos, triazophos, and terbufos. In the study with diazinon, antidotal therapy comprised atropine and 2-PAMC1 and studies were undertaken in both rats and rabbits (FAO/WHO, 1994). In the latter, although there was enzyme reactivation, recrudescence of
poisoning was seen at 2 hr and the authors suggested that this indicated the need for repeated oxime dosing. In three studies reviewed by FAO/WHO (1994) on phosalone and 2-PAM methylsulfate in mice, P2S in rats, and obidoxime in mice, all appeared effective, although various aspects of the design of the studies were not optimal. Oximes (PAM or obidoxime) in combination with atropine were successful in rats experimentally poisoned with pyrazophos, and there was some indication that repeated dosing was required for optimal antidotal efficacy (FAO/WHO, 1993). In a rat study of experimental terbufos poisoning, little benefit was observed from PAM and atropine (FAO/WHO, 1991). In rat studies on triazophos, combinations of atropine sulfate and 2-PAMI or atropine sulfate and obidoxime were successful as experimental therapies (FAOAVHO, 1994). The effects of oximes in profenofospoisoned chicks and mice were reported to be limited, as expected, although atropine was effective (FAOAVHO, 1991). An interesting study was undertaken in rats by Jokanovic and Maksimovic (1995) in which the efficacy of PAM, obidoxime, HI-6, and trimedoxime, given with atropine and diazepam 1 min after poisoning, was tested in the treatment of poisoning with two LDs0's of 25 different OP insecticides and one OP fungicide (pyrazophos). It was shown that the oximes were potent antidotes in poisoning with phosphate insecticides. Obidoxime, PAM, and HI-6 had low effectiveness in the treatment of poisoning with the phosphonate trichlorfon. However, none of the oximes were effective antidotes in poisoning with dimethoate and pyridaphenthion. The reasons for some of these differences are unclear. Trimedoxime was the most effective oxime in the treatment of insecticide poisoning, being especially efficacious at the lowest tested doses. In animal toxicology and pharmacodynamic studies, "CNS AChE activity" is often reported, usually measured on brain homogenate. Studying the effect of the nerve agents tabun, satin, and VX on ACHE, Gupta et al. (1987, 1991) found that there were major differences in the degree and time course of AChE inhibition between different brain regions (and between brain and muscles). The use of whole brain homogenates could clearly obscure regional changes in AChE activity (see review by Gupta, 2004). In interpreting brain and erythrocyte AChE and plasma BuChE data in animal studies, the same considerations should be borne in mind as would be for erythrocyte AChE and plasma BuChE data in humans. That is, red blood cells cannot synthesize AChE de novo; BuChE is a different gene product from ACHE, with its own kinetics of inhibition, reactivation, and aging; and the enzyme one is really interested in is AChE in the central and peripheral nervous systems. It is important not to overinterpret such data, which are influenced by numerous pharmacokinetic factors. Additionally, it is sometimes necessary to examine the methodology of enzyme activity measurement in detail because BuChE
CHAPTER 49 9Management of OP Pesticide Poisoning can hydrolyze acetylcholine and therefore some "ACHE" activity may in fact be due to BuChE: Specific inhibitors are available for use in assays (St. Omer and Rottinghaus, 1992). A further consideration is that recovery of activity of brain enzyme cannot be assumed to be due to reactivation of the enzyme because synthesis de novo of AChE in brain may be quite rapid. The literature on atropine, oximes, and nerve agents is enormous and some of it is of relevance to the management of pesticide poisoning. However, some caution is necessary because pharmacodynamic studies on nerve agents and oximes frequently employ pretreatment with carbamate drugs. Moreover, much of the literature is primarily concerned with treatment of soman poisoning, in which the inhibited enzyme rapidly ages; here, beneficial actions of oximes are presumably due to their noncholinesterase reactivating properties. Rapidly aging pesticide-inhibited AChE only occurs with one or two pesticides, such as profenofos, and is therefore not a widespread problem. The lessons of the pharmacodynamic studies on OPs, with relevance to pesticides, can be (cautiously) summarized as follows: 1. Typical inhibited cholinesterases produced by dimethoxy and diethoxy OPs can be reactivated by oximes. 2. Delay in the treatment of OP poisoning produced by dimethoxy OPs may result in a significant proportion of the inhibited cholinesterase undergoing the aging reaction and becoming refractory to oxime-induced reactivation. 3. Prolonged therapy may be of value in poisoning by diethoxy OPs. 4. Nonreactivating effects of oximes may produce therapeutic benefit in poisoning by those few pesticides for which aging is a problem.
B. Efficacy: Case Reports and Case Series in Humans In a case series, Willems et al. (1993) reported that ethyl parathion and methyl parathion poisoning could be treated effectively with 2-PAM methylsulfate (plasma concentrations approximately 4 mg/liter) and atropine provided that the pesticide plasma concentrations were low. In more severe cases, when the plasma pesticide concentrations were >30 ixg/liter, even plasma PAM concentrations of 14.6 mg/liter did not produce improvement. In dimethoate poisoning, omethoate-inhibited enzyme could not be reactivated at PAM concentrations of 6.3 mg/liter (Willems et al., 1993). In a case report, continuous PAM infusion (with atropine sulfate) was successful in treating chlorpyrifos poisoning, and the nicotinic signs and symptoms were controlled (Tush and Anstead, 1997). In a case series, Thiermann et al. (1997) reported that in parathion poisoning, obidoxime
723
(250 mg iv as a bolus followed by 750 mg/day by infusion) was effective, but that in severe poisoning, reactivation did not occur until the concentration of inhibitor in the plasma had declined. Obidoxime (250 mg iv as a bolus followed by 750 mg/day by infusion) was ineffective with oxydemeton methyl when oxime therapy was delayed more than 1 day after poisoning. In another case series, Thiermann et al. (1999) reported that in parathion poisoning, reactivation was possible 7 days after poisoning, whereas with oxydemeton methyl, response was only seen when obidoxime therapy was instituted soon after poisoning. Similarly, Zilker et al. (1997) reported that obidoxime (750 mg/day by infusion) drastically reduced the need for atropine in parathion poisoning, but that demeton-S-methyl poisoning only responded to obidoxime if therapy was instituted soon after intoxication. Human experience of oximes, other than PAM salts and obidoxime, is scanty. An exception is a case series reported by Kusic et al. (1991). The oxime HI-6, administered four times daily as a single intramuscular injection of 500 mg, accompanied by atropine and diazepam therapy, was studied in OP pesticide poisoning. Oxime treatment was started on admission and continued for a minimum of 48 hr and a maximum of 7 days. Most patients were treated with HI-6, but nine patients severely poisoned with quinalphos were treated with 2-PAMC1 (1000 mg four times daily). HI-6 rapidly reactivated human red blood cell AChE inhibited by diethoxy OPs (phorate, pyridaphenthion, and quinalphos) as well as that inhibited by dichlorvos. With the exception of dichlorvos, enzyme inhibited by dimethoxy OPs (dimethoate and phosphamidon) was reported to be generally resistant to treatment by HI-6, whereas reactivation with malathion was poor. Both HI-6 and 2-PAMC1 reactivated erythrocyte AChE in quinalphos-poisoned subjects, but reactivation was much more rapid following the use of HI-6. The general improvement of poisoned patients, which was sometimes more rapid than the increase in AChE activity with HI-6, suggested that direct pharmacological effects were occurring. However, this may be an overinterpretation of the data because the clinical status of patients is determined by the degree of inhibition of neuronal ACHE, which may not be mirrored by the inhibition of red blood cell cholinesterase. No adverse side effects were noted when plasma concentrations of HI-6 were maintained above therapeutic concentrations for up to 7 days.
C. The Choice of Oxime Possibly for historical reasons, as discussed previously, in English-speaking countries together with France, the use of PAM salts is standard, whereas in German-speaking countries, obidoxime is used. Thus, it is of interest to consider whether one or the other oxime is better. Based on studies using OP pesticide-inhibited human AChE in vitro, Worek
724
SECTION I X .
Therapeutic Measures
et al. (1996) concluded that obidoxime was superior to 2-PAMI in reactivation capability. The pesticides and pesticide oxons used were chlorfenvinphos, dichlorvos, dicrotophos, heptenophos, mevinphos, monocrotophos, paraoxon, phosphamidon, trichlorfon, malaoxon, omethoate, oxydemeton-methyl, and methamidophos, and the inhibition time was 30 min. The Hagedorn oxime HI-6 appears to work in pesticides poisoning, although there are data in vitro using human AChE suggesting that HI-6 would be less effective in poisoning by dimethoxy pesticides than either PAM or obidoxime (Worek et al., 1999). Another Hagedorn oxime, HL6-7, has a broad spectrum of activity in nerve agent poisoning, being capable of reactivating human erythrocytic AChE inhibited by the nerve agent tabun (de Jong et al., 1989). However, there is no evidence that the use of HL6-7 would produce any benefit compared to existing oximes in pesticide poisoning.
D. O x i m e D o s i n g R e g i m e n Oxime administration must produce clear, irreversible, clinical improvement and achieve rapid reactivation of ACHE. This must be achieved without producing major adverse effects and the need to monitor oxime concentrations. Reactivation of inhibited AChE and clinical improvement depend on the chemical form of inhibited ACHE, the plasma oxime concentration, the duration of oxime therapy, and the plasma OP concentration. Early experiments on anesthetized cats (possibly seven animals) given lethal doses of intravenous sarin and P2S 10 mg/kg intramuscularly, but not atropine, established that plasma PAM concentrations higher than 4 mg/liter were required to counteract neuromuscular block in vitro and bradycardia, hypotension, and respiratory failure in vivo (Sundwall, 1961). Crook e t a l . (1962) gave dogs oral P2S and 2-PAM lactate, 30-115 mg/kg body weight, 1-5 hr before exposure to sarin vapor. Atropine 5 mg/kg body weight was administered 1 min after the dogs were exposed
TABLE 4.
to sarin. The authors extrapolated from this study of dogs to humans and concluded that a plasma PAM concentration of at least 3 mg/liter would be required "for reasonably protective attenuation of the toxic effects of OP anfi-ChEs." The relationship between plasma oxime concentrations after dosing with PAM and obidoxime and protection against sarin poisoning has been investigated by Shiloff and Clement (1987) in rats (Table 4) and by Bokonjic et al. (1987) in quinalphos-poisoned rats (Table 5). A study on poisoned patients showed that reactivation of dimethoateinhibited enzyme was not achieved with a plasma PAM concentration of 6.37 mg/liter (Willems et al., 1993). In the same study, it was shown that reactivation of inhibited enzyme did not occur, even in the presence of a plasma PAM concentration of 14.6 mg/liter, when plasma ethyl and methyl parathion concentrations were >30 t~g/liter. Thus, the modest doses of PAM that have often been recommended in the past (to achieve plasma oxime concentrations of approximately 4 mg/liter) will be insufficient to produce not only reactivation of phosphorylated enzyme but also a lasting clinical improvement, unless the patient is only mildly poisoned. Moreover, clinically effective PAM concentrations need to be maintained as long as inhibitory oxons (active OP metabolites) are circulating. Data from case reports suggest that PAM concentrations of 40 rag/liter may be required to produce reactivation of inhibited AChE in some cases (Casey et al., 1995a,b). How may such plasma PAM concentrations be achieved and maintained if reactivation of inhibited AChE is to occur and clinical improvement is to ensue in severe cases? Sidell and Groff (1971) administered 2-PAMC1 at a dose of 10.0 mg/kg intravenously to volunteers: PAM concentrations > 10 mg/liter were maintained for approximately 30 rain. Medicis et al. (1996) administered 16 mg/kg 2-PAMC1 over 30 min or 4 mg/kg over 15 min, followed by 3.2 mg/kg/hr for 3 hr 45 min to human volunteers, using a randomized crossover design. Concentrations in plasma > 10 mg/liter were maintained for approximately 60 min after bolus infusion, but were not achieved following
Relationship between Plasma Oxime Concentrations and Mortality in Sarin-Poisoned Rats Also Given Atropine 17.4 mg/kg a
Oxime
n
Mean (_+ SD) oxime concentration (mg/liter)
Pralidoxime Pralidoxime Pralidoxime Obidoxime Obidoxime Obidoxime
5 4 5 10 8 5
0.7 _+ 0.1 2.0 _+ 0.4 3.3 _+ 2.3 3.6 + 0.2 9.2 _+ 0.6 19.7 _+ 3.7
aAfter Shiloff and Clement(1987).
% Mortality 100 80 20 90 62.5 0
CHAPTER 49
9Management of OP Pesticide Poisoning
725
TABLE 5. Relationship between Plasma Pralidoxime Concentrations and LD50 in Quinalphos-Poisoned Rats Also Given Atropine 10 mg/kg and Diazepam 2.5 mg/kg a Mean (--. SEM) plasma PAM concentration (mg/liter) 0 0.8 1.5 + 0.6 2.9 _+ 0.7
Mean (-4--SEM) LDs0 (mg/kg)
Protective index
10.5 _+ 3.8 353.6 + 33.6 457.9 _+ 121.6 498.7 +_ 137.9
33.7 43.6 47.5
aAfter Bokonjic et al. (1987).
the continuous infusion, when concentrations of approximately 6 mg/liter were observed. After a single intramuscular injection of 2-PAMC1 30 mg/kg, PAM concentrations >10 mg/liter were maintained for approximately 2 hr in volunteers (Calesnick et al., 1967). Also, after an intravenous infusion of 2-PAMC1 30 mg/kg, PAM concentrations > 10 mg/liter were maintained for approximately 30 min (Calesnick et al., 1967). In children with OP poisoning, Schexnayder et al. (1998) found that mean (_+SD) PAM steady-state concentrations were 22.2 mg/liter after a loading dose of 2-PAM C1 25-50mg/kg, followed by a continuous infusion of 10-20 mg/kg/hr. Green et al. (1985) investigated the impact of poisoning on PAM concentrations. Guinea pigs were administered 2-PAMC1 25 mg/kg and atropine 16 mg/kg intramuscularly 1 min after subcutaneous sarin and soman at various multiples of the LDs0. The PAM concentrations remained above 25 mg/liter for the duration of observation (10 min). Generally, the higher the LDs0, the higher the PAM concentration (>30 mg/liter). The plasma PAM concentrations were measured both in volunteers and in poisoned patients after the administration of 2-PAMC1 1000 mg intramuscularly (Jovanovic, 1989). Mean plasma PAM concentrations were almost one and half times higher in patients compared to volunteers. Based on these data (Casey et al., 1995a,b; Willems et al., 1993; Willems and Belpaire, 1992), we propose that 2-PAMC1 or P2S 30 mg/kg should be administered by intravenous injection as soon as possible after exposure. Repeat doses at 4- to 6-hr intervals or, preferably, an intravenous infusion of 8-10 mg/kg/hr should then be given. Administration of PAM should continue for as long as atropine is r e q u i r e d - that is, until clear, irreversible clinical improvement is achieved, which may take many days while residual insecticide is cleared from the body stores. E. A d v e r s e Effects of O x i m e s The toxicology of oximes has been reviewed (Dawson, 1994; Marrs, 1991). With the first oximes used in OP poisoning, such as diacetylmonoxime, it was suggested that
some toxic effects could be due to cyanogenesis. In the case of P2S, this seems unlikely to be important in toxicity (Ballantyne et al., 1975; Enander et al., 1961). In the context of the treatment of acute OP pesticide poisoning, the acute toxicity is the only consideration of importance. The acute toxicity of PAM salts and of obidoxime is of the same order of magnitude. Impaired liver function has been observed after treatment of human parathion poisoning with obidoxime (Barckow et al., 1969; Wirth, 1968), and reversible fatty change has been reported in the liver of rats treated with that oxime (Bisa et al., 1964). However, Boelcke and Gaaz (1970) did not find evidence of hepatotoxicity in mice in terms of enzyme elevation, and bilirubin clearance and bromsulfthalein retention were unaffected in the rat (Boelcke and Kamphenkel, 1970). Therefore, some or all of the hepatotoxicity of obidoxime may be attributable to other factors, such as the OP or solvents. In human volunteer studies, side effects have been seen with oximes. Thus, Jager and Stagg (1958) found that medical students given 2-PAMI developed dizziness, blurred vision, diplopia, impairment of accommodation, and headache. Dizziness, blurred vision, and, occasionally, diplopia have been reported in human volunteer studies after intravenous or intramuscular administration of 2-PAMC1 (Sidell and Groff, 1971). In a study on obidoxime, male volunteers were given tablets in quantifies ranging from 1.84 to 3.58 g as a single dose or 7.36 g divided into four equal doses. More than half the subjects complained of one or more side effects: pallor, nausea, burning sensation, headache, generalized weakness, sore throat, and paresthesia of the face. Activities of blood cholinesterase, alanine, and aspartate aminotranferases, as well as hematocrit values, heart rate, and blood pressure, were not affected (Simon and Picketing, 1976).
VII. A N T I C O N V U L s A N T S A large number of anticonvulsants have been studied in animals or used in OP poisoning (Sellstr6m, 1992). Many candidate anticonvulsants have been investigated in attempts
726
SECTION IX- T h e r a p e u t i c
Measures
to improve the treatment of OP nerve agent poisoning, such as the water-soluble diazepam prodrug avizafone (Lallement et al., 2000); other benzodiazepines, such as clonazepam (Lipp, 1974) and midazolam (Pieri etal., 1981); and anticonvulsants of other types, such as barbiturates and phenytoin. Other drugs that have been studied include tiagabine (a GABA uptake inhibitor) and glutamate receptor antagonists (Shih and McDonough, 1999). Only diazepam and midazolam have achieved widespread use in the treatment of OP pesticide poisoning. Anticonvulsants including benzodiazepines, especially diazepam, were originally studied in OP poisoning for the symptomatic relief of OP-induced convulsions. Benzodiazepines are CNS depressants, anxiolytics, and muscle relaxants (Diamantis and Kletzkin, 1966). The main site of action of benzodiazepines is the 7-aminobutyric acid A (GABAA) receptor. The GABAA receptor is a ligand-gated chloride ion channel (Ortells and Lunt, 1995), the GABAergic system being the major inhibitory neurotransmission system in the mammalian CNS. Benzodiazepines including diazepam alter GABA binding at the GABAA receptor in an allosteric manner, but these drugs do not activate the GABAA receptor by direct action (Charney et al., 2001). Nevertheless, the overall effect is to increase the inhibitory action of the GABAergic system. Data from experimental nerve agent poisoning (Anderson et al., 1997; Hayward et al., 1990) suggest that benzodiazepines, such as diazepam and midazolam, ameliorate or prevent the development of pathological changes in the CNS.
A. Diazepam Diazepam is the anticonvulsant that has been most studied for use in OP pesticide poisoning. In this context, the most likely mode of administration is by intravenous injection, but other modes of administration have been considered for self-administration or when administration by those not trained in intravenous injection is required. In OP-induced convulsions, diazepam would be given intravenously. 1. PHARMACOKINETICS In healthy human subjects, a peak mean serum concentration of 1607 ~g/liter was found 15 rain after single bolus intravenous injections of 20 mg (Hillestad et al., 1974). A two-compartment open model has been used to describe elimination kinetics of diazepam in humans after single intravenous injections were reported (Andreasen etal., 1976; Klotz et al., 1975, 1976). A two-compartment open model has also been used to describe elimination kinetics of diazepam in experimental animals; however, there were major interspecies differences in parameters such as tl/2 and Vd (Klotz et al., 1976), which indicated caution in the interpretation of animal studies. In human volunteers, the plasma protein binding of diazepam was greater than 95% (Klotz et al., 1976). The tl/2 of diazepam appears
to increase when liver damage is present and with age (Andreasen etal., 1976; Herman and Wilkinson, 1996; Klotz et al., 1975). It appears that intramuscularly administered diazepam produces lower peak blood levels in humans than does either intravenous injection or oral administration (Hillestad et al., 1974), whereas absorption following oral administration is almost complete with bioavailability close to 1 (Mandelli et al., 1978). 2. PHARMACODYNAMICS In a variety of pharmacodynamic studies on experimental animals, it can be concluded that diazepam adds to the effects of atropine and of the classical combination of atropine and pyridinium oximes (Bokonjic etal., 1987; Gupta, 1984; Kassa and Bajgar, 1994; Kleinrok and Jagiello-Wojtowicz, 1977; Krutak-Krol and Domino, 1985; Rump and Grudzinska, 1974). There seems to be little doubt that in experimental animal models, diazepam can prevent, stop, and/or ameliorate convulsions due to OP pesticides and render less severe or even prevent structural changes in the brain, but the effects on lethality are less clear (Marrs, 2003).
3. EFFICACY: CASE REPORTSAND CASE SERIES In the scientific literature, there are many case reports and series of the apparently successful treatment of OP insecticide poisoning with diazepam as an adjunctive but widely accepted therapy (Karalliedde and Szinicz, 2001). Examples include Barckow et al. (1969), Vale and Scott (1974), Yacoub etal. (1981), Merrill and Mihm (1982), Martf etal. (1985), LeBlanc etal. (1986), de Kort etal. (1988), Jovanovic etal. (1990), Kusic etal. (1991), and Weissmann-Brenner et al. (2002). In most cases, the indication for the use of diazepam was convulsions, but diazepam has also been used to control muscle fasciculation and agitation. In the case of convulsions, the adult dose is 10-20 mg iv, whereas that for children is 0.2 or 0.3 mg/kg iv. The elderly should receive half the adult dose. In the absence of convulsions, diazepam administration at doses of 5-10 mg intravenously has also been recommended in cases of OP poisoning accompanied by anxiety and restlessness (Johnson and Vale, 1992). If large doses of diazepam are required to suppress seizure activity, phenytoin should be considered as an alternative (Johnson and Vale, 1992).
VIII. O T H E R M E T H O D S OF A N T I D O T A L TREATMENT A number of novel approaches to the antidotal treatment of OPs have been studied, often using prophylactic protocols and mostly in relation to nerve agent poisoning, but some may be applicable, at least in principle, to OP pesticide poisoning.
CHAPTER 49 9Management of OP Pesticide Poisoning
A. Enzymes 1. CHOLINESTERASE Both AChE (Maxwell e t a l . , 1999; Wolfe, 1992; Wolfe et al., 1987) and BuChE (Broomfield etal., 1999) have been studied as scavengers for nerve agents.
2. PHOSPHOTRIESTERASE McGuinn et al. (1993) described a study in which squid DFP-hydrolyzing enzyme (DFPase) was entrapped within mouse erythrocytes. These red blood cells were shown to be capable of hydrolyzing DFP. This approach would presumably be effective against other OP esters that are hydrolyzed by DFPase. In a similar approach, Pei et al. (1995) reported that resealed murine erythrocyte cells containing recombinant phosphotriesterase protected against the lethal effect of paraoxon in mice. Also, when these carrier cells were administered in combination with 2-PAM and/or atropine, synergy was reported.
B. Calcium Channel Blockers Calcium channel blockers have been studied in OP poisoning (e.g., nimodipine) (Dretchen et al., 1992; Karlsson et al., 1994).
C. Adenosine Receptor Agonists Adenosine receptor agonists were reported to prevent clinical signs and increase survival in soman, satin, and DFP poisoning (Harrison et al., 2003; Tuovinen, 2004; Van Helden et al., 1998).
D. N-Methyl-D-Aspartate Receptor Antagonists Although the initial stimulus for seizures appears to be cholinergic overactivity, as the seizures develop, other excitatory neurotransmission systems become involved, including the glutamatergic system. The N-methyl-Daspartate (NMDA) receptor is a subtype of glutamatergic receptor: antagonists at this receptor, dizocilpine and 3-((R, S)-2-carboxypiperazin-4-yl)-propyl- 1-phosphonic avid, were found in mice experimentally poisoned with chlorfenvinphos to block seizures (Dekundy et al., 2001). It has been suggested that the beneficial activity of caramiphen, an anticholinergic drug, in soman poisoning may be modulated through activity at the NMDA receptor (Raveh et al., 1999, 2003).
E. Blockade of Acetylcholine Synthesis or Uptake An obvious therapeutic measure in AChE decrease the synthesis of ACh. Sterling et al. that administration of acetylsecocholinium to soman enhanced the protective effects of
would be to (1988) found 30 min prior atropine and
727
2-PAMC1 in the rat, whereas administration of N-hydroxyethylnaphthylvinylpyridine a few minutes before soman reduced mortality due to soman. Acetylsecocholinium is an inhibitor of high-affinity choline uptake and a choline acetyltransferase (CHAT) inhibitor, and N-hydroxyethylnaphthylvinylpyridine is a ChAT inhibitor. N-allylquinuclinidol, another inhibitor of high-affinity choline uptake, reduced mortality. Gray et al. (1988)studied naphthylvinylpyridine derivatives as antidotes for nerve agent poisoning and concluded that their beneficial experimental action in mice and guinea pigs was not related to ChAT inhibition.
IX. PREVENTION AND TREATMENT OF INTERMEDIATE SYNDROME Assuming that intermediate syndrome (IMS) is a consequence of ACh accumulation at the neuromuscular junction, oxime cholinesterase reactivators should protect against the development of IMS. It is noteworthy, and possibly relevant, that 2-PAM methylsulfate was reported to protect against the development of muscle fiber necrosis consequent on exposure to OP pesticides (Cavaliere et al., 1998). Johnson et al. (1996) reported experience with two treatment regimes of PAM in the treatment of patients with OP poisoning in a prospective trial. Seventy-two adults presenting with a history of consumption of OP compounds and requiting intensive care were entered into the trial. Patients were randomized using a block randomization to receive either a single bolus dose of 1 g PAM at admission followed by placebo infusion during the next 4 days or a single placebo bolus at admission followed by 12 g PAM as a continuous infusion during the next 4 days. A higher prevalence of IMS was observed in the latter group. Analysis of patients who received at least 1 g PAM within 12 hr of ingestion of the OP poison and those who received PAM after 12 hr suggested that the time of administration of PAM was important because the first group was less likely to develop IMS. Other case reports have suggested that a high dose of oxime may not avert IMS. Thus, in a case of severe malathion self-poisoning, 1 g PAM was administered iv approximately 7 hr after poisoning and at 12 hr an infusion of 400 mg/hr PAM was administered iv. The patient developed IMS on hospital day 3 (Sudakin et al., 2000).
X. PREVENTION AND TREATMENT OF ORGANOPHOSPHATE-INDUCED DELAYED POLYNEUROPATHY There is no recognized antidotal treatment for OPIDR Numerous substances, including certain carbamates, phenyl methane sulfonyl fluoride, n-butane-sulfonyl fluoride, and some phosphinates, have been shown to prevent the development of OPIDP when given before neuropathic OPs to
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SECTION I X .
Therapeutic Measures
sensitive species such as hens (Johnson and Vale, 1992). Furthermore, Petrovic et al. (2000) found that elements of the conventional treatment used in humans (atropine, trimedoxime, and midazolam), if injected into hens prophylactically, ameliorated the subsequent development of OPIDP induced by D F E Jokanovic et al. (2001) found that a combination of trimedoxime, atropine, and methylprednisolone given 20 min before experimental poisoning of hens with DFP reduced the severity of the OPIDP that developed subsequently. The effect was less if the treatment was given after the D F E None of the previously mentioned studies provide any support for the efficacy of any postexposure antidotal therapy, so the treatment of OPIDP is essentially symptomatic.
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Gibbon, S. L., Tong, H., Miranda, E M. S., and Way, J. L. (1979). Metabolism of pralidoxime (2-PAM) in man. J. Anal. Toxicol. 3, 14-17. Glickman, A. H., Wing, K. D., and Casida, J. E. (1984). Profenofos ' insecticide bioactivation in relation to antidote action and the stereospecificity of acetylcholinesterase inhibition, reactivation, and aging. Toxicol. Appl. Pharmacol. 73, 16-22. Gray, L. E., Jr., Ostby, J., Sigmon, R., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J., and Laskey, J. (1988). The development of a protocol to assess reproductive effects of toxicants in the rat. Reprod. Toxicol. 2, 281-287. Green, M. D., Jones, D. E., and Hilmas, D. E. (1985). Satin intoxication elevates plasma pralidoxime. Toxicol. Lett. 28, 17-21. Green, M. D., Talbot, B. G., and Clark, C. R. (1986). Pharmacokinetics of pralidoxime chloride in the rat. Life Sci. 39, 2263-2269. Gupta, R. C. (1984). Acute malathion toxicosis and related enzymatic alterations in Bubalus bubalis: Antidotal treatment with atropine, 2-PAM, and diazepam. J. Toxicol. Environ. Health 14, 291-303. Gupta, R. C. (2004). Brain regional heterogeneity and toxicological mechanisms of organophosphates and carbamates. Toxicol. Mech. Methods 14, 103-143. Gupta, R. C., Patterson, G. T., and Dettbam, W.-D. (1987). Acute tabun toxicity; Biochemical and histochemical consequences in brain and skeletal muscles of rat. Toxicology 46, 329-341. Gupta, R. C., Patterson, G. T., and Dettbarn, W.-D. (1991). Comparison of cholinergic and neuromuscular toxicity following acute exposure to sarin and VX in rat. Fundam. Appl. Toxicol. 16, 449-458. Hansen, M. E., and Wilson, B. W. (1999). Oxime reactivation of RBC acetylcholinesterases for biomonitoring. Arch. Environ. Contam. Toxicol. 37, 283-289. Harrison, E K., Bueters, T. J. H., Ijzerman, A. E, Van Helden, H. E M., and Tattersall, J. E. H. (2003). Partial adenosine A1 receptor agonists inhibit satin-induced epileptiform activity in the hippocampal slice. Eur. J. Pharmacol. 471, 97"-104. Hayward, I. J., Wall, H. G., Jaax, N. K., Wade, J. V., Marlow, D. D., and Nold, J. B. (1990). Decreased brain pathology in organophosphate-exposed rhesus monkeys following benzodiazepine therapy. J. Neurol. Sci. 98, 99-106. Herman, R. J., and Wilkinson, G. R. (1996). Disposition of diazepam in young and elderly subjects after acute and chronic dosing. Br. J. Clin. Pharmacol. 42, 147-155. Hillestad, L., Hansen, T., Melsom, H., and Drivenes, A. (1974). Diazepam metabolism in normal man. I. Serum concentrations and clinical effects after intravenous, intramuscular, and oral administration. Clin. Pharmacol. Ther. 16, 479--484. Hinderling, E H., Gundert-Remy, U., and Schmidlin, O. (1985). Integrated pharmacokinetics and pharmacodynamics of atropine in healthy humans. I: Pharmacokinetics. J. Pharm. Sci. 74, 703-710. Hobbiger, E (1963). Reactivation of phosphorylated ACHE. Handbuch Exp. Pharmakol. 15, 921-988. Inns, R. H., and Leadbeater, L. (1983). The efficacy of bispyridinium derivatives in the treatment of organophosphonate poisoning in the guinea-pig. J. Pharm. Pharmacol. 35, 427-433.
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SECTION I X . T h e r a p e u t i c
Measures
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Index
Page references in bold type refer to tables.
A (atypical) variant, butyrylcholinesterase, 189-194, 204, 216 Abortions, spontaneous, 474 Absorption anticholinesterases, interspecies variation, 148 body temperature and, 558 dermal, s e e Dermal absorption/toxicity PBPK models, 108-114 percutaneous, 569-570; s e e a l s o Dermal absorption/toxicity sweating and, 562-563 transappendageal, 411 Accelerated solvent extraction, 682 Acceptable Daily Intakes pesticide mixtures, 608 in risk assessment, 543 WHO/FAO guidelines, 644-645,646-647, 648-649
Accidental poisonings, in children, 602 Acephate degradation products, 693 homeowner use, cancellation/phase out, 630 Acetylation, 130 carbamates metabolism, 137-138 xenobiotics metabolism, 130 Acetylcholine, 3 accumulation, 703 and adenosine receptor agonists, 260 and muscarinic receptor downregulation, 236 and myopathy, 511-512 active site, 90 Alzheimer's disease, levels, 26, 30, 42 autoregulation of, 259 cholinergic crisis, 451 cholinergic receptor binding, 145, 209 cholinesterase hydrolysis of, 199, 211-213 developmental chlorpyrifos exposure, 298-300 glutamate and, 513 hyperthermic response, 551 regulation, 271,275-276, 282
retinal, 435 role in placenta, 472-473 in senile plaques, 29 synthesis, 275 tissue distribution, 451 vascular smooth muscle effects, 29, 381-382 Acetylcholine receptors carbamates and, 5 overstimulation, 703 Acetylcholinesterase active site, 104, 213 activity measurements, 199-205 aged, 104, 152, 215-216, 716 and oxime efficacy, 719 binding sites, 175-179 active center gorge, 174-176, 210 acyl pocket, 177-179, 209, 210, 214 choline, 176, 177-178, 179-180 oxyanion hole, 162, 211 peripheral, 212 serine residue, 165-168, 213 stereoselectivity, 210, 214 as biomarker, carbofuran exposure, 666 brain, and core temperature, 555-556 carbamylation of, 151-152 catalytic turnover rates, 104, 175, 182, 213 control of cholinergic neurotransmission, 276 distribution in eye, 423-424 erythrocyte activity measurements, 577-579, 716-717 organophosphate-binding, 704 genes, 168, 172-173, 194, 206 genetic variants, 204-206, 216 knockout mice, 260-261,703 morphogenic activity, 240-241 nerve agent antidote, 727 organophosphate deactivation, 150 pharmacogenetics, 194-195 phosphorylation, 152, 214-216 placental, 472-473 prophylaxis, soman, 195
735
reactivation, s e e Acetylcholinesterase inhibition, reactivation reactivators, 719-722; s e e a l s o Oximes role of, 209 structure interspecies variation, 152-153 primary, 161-162, 163-167, 168, 169-171 quaternary, 181 secondary/tertiary, 168, 174-181 variants, 181-182 substrate specificity, 209 tissue distribution, 161, 187 tolerance development and, 263 Acetylcholinesterase inhibition carbamates, 90 structure-activity relationships, 214 carbofuran-induced, 660 interspecies variation, 151-152 irreversible, 703 kinetic modeling, 211-213 and muscarinic receptor down-regulation, 236 organophosphates, 90 neurotoxicity, 234-235 structure-activity relationships, 213-214 and oxidative stress biomarkers, 519 reactivation, 152 and behavioral recovery, 350-351 carbamates, 103, 104 d e n o v o synthesis, 263 organophosphates, 103, 104 oxime-induced, 214-215, 716 spontaneous, 716 time course, 199-200 tolerance, s e e Tolerance development WHO/FAO activity guidelines, 646 Acetylcholinesterase inhibitors for activity measurements, 201 in Alzheimer's disease, 17, 22, 27-30 analytical methods, 694-695 and dementia with Lewy bodies, 29-30
736
Index
Acetylcholinesterase inhibitors (continued) effects on amyloid/3 peptide, 28-29 on cerebrovascular parameters, 29 excitotoxic, 511 on tau protein, 29 first generation, 17 glaucoma and, 22 history of, 3-4 immune system and~ 495 indications, 389 irreversible, 4; see also Organophosphates memantine and, 39-42 myasthenia gravis and, 22 in organophosphate nerve agent poisoning, 23 oxidative stress, 511-515 and Parkinson's disease dementia, 29-30 reversible, 4; see also Carbamates second generation, 17 urine voiding dysfunction and, 22 uses, 3 in veterinary medicine, 23 Acetylcholine synthesis, blocking, 727 Acetyl coenzyme A, 275 Acetylesterases, 129 Acetylpeptide hydrolase, 707 Acetylsalycylic acid, developmental neurotoxicity, 638 Acetylsecocholinium, 727 Acetylthiocholine, 112-113, 182, 199 Acetyltransferases, role of, 130 ACHE, 194-195,206 Acid phosphatase, 453-454 Acrylamide, developmental neurotoxicity, 634 Actin-myosin cross-bridges, 382 Action potential effect of pesticides, 339-340 organophosphate-induced cardiotoxicity, 383-386 Activated charcoal, 717 Active transport, placental toxicity, 465 Acute akinetic rigidity syndrome, 280 Acute Emergency Guideline Levels, 399 Acute Exposure Guideline Levels, 48, 57-62 Acyl peptide hydrolase, 281 Acyl pocket binding site acetylcholinesterase, 177-179, 209, 210,214 butyrylcholinesterase, 176, 214 carboxylesterases, 224 cholinesterases, 175-176, 177-179, 209, 210,214 AD-DX 384, 258-259 Addictive behaviors, cholinergic modulation of, 272 Adenosine, 260 Adenosine receptor, tolerance development, 260
Adenosine receptor agonists, and acetylcholine accumulation, 260 Adenosine receptor antagonists, 727 Adenylyl cyclase signaling, 704 chlorpyrifos-induced alterations, 239, 295, 296-297, 301 inhibition, 236, 237, 240 Adipose tissue, accumulation of organophosphates, 536 Adolescents, acute poisoning incidences, 572 Adrenocorticotrophic hormone, 498 Adulticides, 601 Adverse drug reactions, pharmacogenetics and, 187 Adverse-observed effect level, agricultural pesticides, 585 Aedes, pesticide resistance, 601 Aedes aegypti, 600, 601 Aerodynamic diameter, inhaled particles, 401 Aerosols inhalation exposure, 401-402, 570 respiratory absorption of, 404 A-esterases, 128-129 activity, interspecies variation, 150 detoxification potential, 52 in organophosphate metabolism, 106, 109-110, 133 modeling, 109-118 role of, 247 Affective function cholinergic modulation of, 274 and chronic pesticide exposure, 351,352 Africa, vector-b0rne disease, 599-600 Age acute pesticide exposure, neurobehavioral effects, 354-356 and anticholinesterase sensitivity, 154-155 and butyrylcholinesterase activity, 204 and dermal absorption, 416 OPIDN and, 362 organophosphate sensitivity and, 91, 118-119 and paraoxonase activity, 251 pesticide sensitivity and, 448 Aggregate risk, defined, 618 Aggregate risk assessment, 618-620 Aggression, cholinergic modulation of, 274 Aging acetylcholinesterase, 104, 152, 215-216, 716 and oxime efficacy, 719 cholinesterases, 215-216 nerve agents, 51, 83 neuropathy target esterase, 361,662 of organophosphates, 183 push-pull mechanism, 215 Agitation, and Alzheimer's disease, 26 Agriculture workers children of, 604-605 neurobehavioral effects in, 351,352
Agrochemicals and male reproduction, 451 and Parkinson's disease, 281 AIDS, dementia associated with, 39 Airborne exposure guidelines, 581-582, 585 Air samples extraction methods, 682 nerve agent analysis, 694 Airway hyperreactivity, 237-239, 574, 707 Airway resistance, 391 Albumin, organophosphate binding, 148, 706, 707, 708 Aldicarb age-related sensitivity, 355 developmental neurotoxicity, 639 endocrine disruption, 485 history of, 3, 134 immune system effects, 501,503 -induced myopathy, 512 interaction with memantine, 41 metabolism, 137 metabolites, 607 mixtures, 610 poisoning incidences, 4 sulfone, 607 toxicity, 4 Aldicarb sulfoxide, 607 Algae, carbofuran toxicity, 659 Aliesterases, 148, 150, 574 Alimentary tract, and ocular absorption, 431-432 Alkali thermionic detector, 691 Alkylating agents, cholinesterase inhibitors as, 498 Alkylphenols, estrogen receptor binding, 452, 458 Alkyl phosphates analysis, 691 excretion, 691 occupational exposure, 490 Alkyl pyrophosphates, lethal inhalation toxicity, 405 Alkyltins, developmental neurotoxicity, 638 Allergic sensitization, pesticides, 504 a-fl hydrolase fold carboxylesterases, 224 cholinesterases, 174 Alveoli, pulmonary toxicity, 394 Alzheimer's disease acetylcholine levels in, 42 amyloid hypothesis, 28-29, 30 cholinergic hypothesis, 25-26 cognitive reserve, 28 dementia, 26, 36-38 functional decline in, 26 nicotinic agonists and, 260 pathogenesis, 25, 28-29, 30, 35 prevalence, 25, 42 risk factors, 28 seizures and, 279
Index
symptoms, 26, 42 treatment acetylcholinesterase inhibitors, 3, 4, 17, 22, 27-30 acetylcholinesterase variants and, 194 approaches to, 41 cholinesterase inhibitors, 283 galantamine, 28 memantine, 26, 36-38 memantine/acetylcholinesterase inhibitor combination therapy, 41-42 donepezil, 27 rivastigmine, 27-28 tacrine, 27 "use it or lose it" hypothesis, 28 visual symptoms, 437-438 Alzheimer's Disease Assessment Scale-Cognitive Subscale, 26, 37 Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory, 36 Amantadine, 35, 36-38, 280 Ambenonium analysis, 693,694 in myasthenia gravis, 22 Ambient temperature, and anticholinesteraseinduced hypothermia, 554, 555-557 American Association of Poison Control Centers, 94, 571 Ameridine, 17 Amerindians, butyrylcholinesterase genotypes, 191 Ames assay method, 662 Aminocarb, immune system effects, 503 Aminopyrine, induction of carboxylesterases, 227 Ammonium polyphosphate, 406 Amnesia, scopolamine-induced, 27 Amphibians, anticholinesterases sensitivity, 148, 150, 152 Amygdala, cholinergic innervation, 271 Amyloid/3 peptide, effects of acetylcholinesterase inhibitors, 28-29 Amyloid precursor protein, 28-29 Amyotrophic lateral sclerosis, in Gulf War veterans, 74-75 Anabas testudineus, carbofuran toxicity, 659 Analgesia, cholinergic regulation of, 275 Analytical methods acetylcholinesterase inhibitor therapeutic agents, 694-695 cleanup/purification, 682-683 detectors, 684-686 HORRAT value, 696 immunoassays, 690-691 impurities separation, 684 metabolites, 691-693 nerve agents, 693-694 pesticide confirmation, 686 pesticide screens, 691 QuEChERS method, 690
sample concentration, 683-684 sample extraction, 681-682 sources for, 686-690 Androgen production, site of, 449 Androgen receptor, pesticide binding, 452-453,481,485-487 Angiotensin-converting enzyme inhibitors, 219 Anhedonia, 300 Anopheles, 600 Anthelmintics, 23 Anthrax vaccine, 69 Antiandrogenic compounds, 481, 491 Anticholinesterase eye drops, and accomodative capacity, 433 Anticholinesterase poisoning/intoxication biomarkers, 91-93 blood-brain barrier, 278-279, 283 chronic exposure, 281-282 clinical aspects, 91-93 CNS symptoms, 145 diazepam for, 93,277, 386, 394 extrapyramidal effects, 280-281 fatality rates, 91 global epidemiology, 93-98 Gulf War illnesses, 279-280 hypothermia and, 549, 551-554 interspecies variation age and, 154-155 gender and, 154-155 LDs0, 145-148 pharmacodynamics, 151-155 pharmacokinetics, 148-151 mechanism of, 90 muscarinic symptoms, 145 neuropathy, 278-279, 283 nicotinic symptoms, 145 respiratory effects, 277 seizures, 278-279, 283 signs/symptoms, 89, 276-277, 283 treatment, 93 Anticholinesterases. see Cholinesterase inhibitors Anticonvulsants, 726-727 Antidotes anticonvulsants, 725-726 atropine, 584, 718-719 atropine/oxime, 722-725 diazepam, 93,277, 386, 394 HI-6, 584-585 obidoxime, 584-585 oximes, 200, 584-585, 719-722 pralidoxime, 584-585 Antioxidants, see also specific antioxidant endogenous, 512 pesticide-induced alterations, 456 Antitumor drugs, carboxylesterase metabolism of, 220 Anxiety, 260 and acute pesticide exposure, 350 Bhopal accident-associated, 84
737
chlorpyrifos-associated, 354 and organophosphate exposure, 574 AOAC, validation methods, 695-606 AP5, 341 Apathy, and Alzheimer's disease, 26 Aphids, acetylcholinesterase genes, 168, 172-173 Apnea, scoline, 188, 189 Apoptosis neuronal stem cells, 318 organophosphate-induced, 235,454-456 AP prolongation, organophosphate-induced, 383-386 Aquatic ecosystem biomagnification of pesticides, 658 metals in, 666 pesticide accumulation, 657 pollutants, 664 Aquatic toxicity, methods, 658-659 Aqueous humor fluorescein flare, 425 and intraocular pressure, 426-427 ophthalmic toxicity, 434 Arachidonic acid, 514 Arecoline, 502 Areflexia, 376 Aricept, 27 Arousal, behavioral, 273-274 Arsenicals, Lewisite, 49 Arylalkylphosphatase/paraoxonase, 263 Arylesterases, 128-129, 150; see also A-esterases Arylformamidase, 707 Aryl hydrocarbon hydroxylase, 664 Aryl hydrocarbon receptor, 545 Asphyxia, organophosphate-induced, 394-395 Aspiration pneumonia, 717 Asthma irritant receptor activation, 391 organophosphate exposure and, 237, 395,574 Astrocyte-endothelial interactions, organophosphate effects, 331-332 Astrocytes, 315-316 and blood-brain barrier integrity, 331-332 function of, 330 Astroglia, 330 Asulam, acetylation of, 138 Atlantic salmon, DDT absorption, 658 Atmosphere closed facilities, 585 inhalable fraction, 401 respirable fraction, 401 sampling, 402 ATP carbamate-induced changes, 517 organophosphate-induced changes, 517-518 and oxidative stress, 513 Atresia, carbofuran-induced, 659
738
Index
Atropine blocking of hypothermic response, 554-555 clinical efficacy, 718 dosage regimen, 718-719 hyperthermic effect, 561 mechanism of action, 718 and methyl isocyanate toxicity, 82 muscarinic receptor blocking, 281 organophosphate poisoning/intoxication, 93,584 cardiotoxicity, 386 ocular exposure, 433 pulmonary toxicity, 394, 395 oxidative stress prevention, 519-521 and oximes, efficacy of, 722-725 pharmacokinetics, 718 ATSDR Pesticide Profiles, 689-690 Attention deficit disorder, 633 Attention/vigilance, 274, 350 Australia, pesticide poisoning in, 93-94 Autism, 633 Autoimmune myasthenia gravis, 376 Autonomic nervous system disorders, in Gulf War veterans, 75 Autoradiography, cholinergic pathways, 271 Avian acute oral toxicity test, EPA, 673 Avian toxicity, 673,674-677, 677-678 Avizafone, 726 Axonopathies, promotion of, 365-366 Axons degeneration, 234, 362-363,703 myelination, 330 outgrowth, effects of organophosphates on, 241 5-Azacytidine, developmental neurotoxicity, 638 Bacillus thuringiensis, 601
Balance, chronic pesticide exposure and, 353 Bambuterol, butyrylcholinesterase binding, 161 Barbiturates, 726 Basal forebrain, 26, 35, 271-272, 274 Basal ganglia, cholinergic neurons, 272-273 Basudin, 543 Bayer Crop Sciences, 630-631 Baygon, endocrine disruption, 485 BCHE, 190-194, 204-206 de novo amplification of, 195 variants, 204 Behavioral toxicity, 573 acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 Benchmark dose, 618, 619, 627, 628 Bendiocarb, endocrine disruption, 485 Benfuracarb, metabolites, 693 Benomyl, 134 inhalation toxicity, 403 metabolites, 693
in mixtures, 607-608 reproductive toxicity, 454 Benzodiazepines, 726 Benzoylcholine, 176, 201,202 Bergmann glia, 330 B-esterases, 129 activity, interspecies variation, 150 in organophosphate metabolism, 106, 109-110 modeling, 109-118 role of, 247 Beta-adrenoreceptor agonists, and developmental neurotoxicity, 302 BeWo cells, 469 Bhopal accident, 4, 79-80, 568 clinical toxicity of methyl isocyanate, 83-85 methyl isocyanate toxicity, 81-83 Bicyclophosphorus esters, neurotoxicity, 407 Bidrin, 538 Binding globulins, 452 Biochemical studies, WHO/FAO guidelines, 645 Bioconcentration pesticides, 481 placental, 471-472 Biological exposure index, 579, 581 Biomagnification, of pesticides in aquatic ecosystems, 658 Biomarkers muscle injury, 512, 515-516 organophosphate intoxication cholinesterase, 91-92 egasyn, 92-93 oxidative stress carbamate-induced changes, 516-517 organophosphate-induced changes, 517-519 parathion poisoning, 92 Biomonitoring pesticide exposure, 576 workplace, 581-582 Bioscavengers, 708 Biotransformation carbamates, 106 organophosphates, 104-106, 715 in vitro models, 332-333 Birds anticholinesterases sensitivity, 147-148, 150, 152 insecticide toxicity, 673, 674--677, 677-678 paraoxonase activity, 249 pesticide metabolism, 661 thermoregulation, 550 Birth defects, 84, 605 Bispyridinium SAD-128, 341 Bitot spots, 83 Blepharitis, eye irritation tests, 425 Blepharospasm, 431
Blood human, measuring acetylcholinesterase activity, 199-204 measuring anticholinesterase metabolites, 679-680 sample collection/storage, 200 Blood-brain barrier and anticholinesterase intoxication, 278-279, 283 artificial, 332 cell culture models, 331-332 factors effecting permeability, 279-280 and organophosphates neurotoxicity, 149 pralidoxime and, 721 role of, 278 Blue gill sunfish, carbofuran toxicity, 659 Body temperature core, 551 effects of cholinesterase inhibitors on, 551-554 Boll Weevil Eradication Program, 602 Botulinum toxin, pretreatment, and myopathy prevention, 512 Botulinum toxin poisoning, 376 Botulinum toxoid vaccine, 69 Bovine adrenal chromaffin cells, 329 Bovine microvascular endothelial cells, 332 Bradycardia, 703 Brain cholinergic pathways, 271,273,282-282 developmental neurotoxicity long-term outcomes, 298-301 mechanisms, 294-298 vulnerable subpopulations, 301-303 excitotoxicity in, 521-527 muscarinic receptors, and tolerance development, 258-259 sex differentiation, 299 Brain morphometry, effects of developmental organophosphate exposure, 241 Brain slice model, soman, 341 Brain stem, upper, cholinergic neurons, 273 Breathing pattern, and properties of inhaled materials, 404 Bromophos, metabolism of, 715 Bronchi, pulmonary toxicity, 393-394 Bronchioles, pulmonary toxicity, 394 Bronchoconstriction methacholine-induced, 237 organophosphate-induced, 393, 574 vagally-induced, 237, 574, 707 Bronchospasm, 574 Brownian diffusion, inhaled particles, 401 BSA, 693 n-Butane-sulfonyl fluoride, OPIDN prevention, 727 Buthionine sulfoximine, 514 Butylate, 17
Index Butyrylcholinesterase A (atypical) variant, 189-194, 204, 216 activity decreases in, 188 measurements, 199-204 aged, 215-216 in Alzheimer's disease, 17, 25 binding sites, 175-179 active gorge, 174-175 acyl pocket, 176, 209, 214 choline, 176 oxyanion hole, 211 as biomarker of organophosphate exposure, 187 C5 variant, 190 catalytic turnover rates, 175, 182 deficiencies, 187 mode of inheritance, 188-193 dibucaine-resistant variant, 192 distribution in eye, 423-424 fluoride-resistant variant, 192, 204 function, 703-704 gene for, 190-194, 204-206 genetic variants, 204-206, 216 H (Hammersmith) variant, 189-194, 204 J (James) variant, 189-194 kinetic modeling, 211-213 K (Kalow) variant, 189-194, 204, 216 nerve agent antidote, 727 in organophosphate metabolism, 106, 109-110, 150, 707, 708 modeling, 109-118 oxime reactivation of, 719 pharmacogenetics clinical aspects, 187-188 genetic variants, 189-194, 204, 216 inhibition studies, 188-189 phenotypes, 188-189, 204 phenotyping and succinyldicholine sensitivity, 201,202, 216 placental, 472-473 plasma activity measurements, 577-579, 716-717 organophosphate-binding, 704 Sc (scoline) variant, 194 S (silent) variant, 189-194, 204, 216 structure primary, 161-168 quaternary, 181 secondary/tertiary, 168, 174-181 variants, 181-182 substrate specificity, 209 tissue distribution, 161, 187 tolerance development and, 263 U (usual) variant, 189-194, 204, 216 Butyrylcholinesterase inhibitors for activity measurements, 201 immune system and, 495
Butyrylthiocholine, 112-113 butyrylcholinesterase binding sites, 176 BW286c51, cholinesterase binding site, 176, 179 BW2845c51, acetylcholinesterase inhibition, activity measurements, 201 C5 variant, butyrylcholinesterase, 190, 204 C6 rat glioma cell line, 330-331, 541-542 Ca2+-ATPase, pesticide inhibition of, 665 Ca2+/cAMP-response element binding protein, 240, 331,363 Ca2+/cAMP response element binding protein, organophosphate activation of, 239-240 Calabar bean, 4, 48, 599 Calcium/calmodulin-dependent protein kinase II, organophosphate activation of, 234, 239 Calcium channel blockers, 727 Calcium ions homeostasis, in OPIDN pathogenesis, 363 myopathy and, 512 and organophosphate-induced cardiotoxicity, 383-386 pesticide disruption of, 453 and smooth muscle regulation, 382 California Health Services Department, 578 Canal of Schlemm, 434 Cancer, childhood, 605 Cannabis, developmental neurotoxicity, 634 Capecitabine, 220 Capillary electrophoresis, 694 Caramiphen, 727 Carbachol, and down-regulation of muscarinic receptors, 258 Carbamate compounds acetylcholinesterase inhibition, 90, 214 allergic sensitization, 504 androgen receptor binding, 481 behavioral toxicity, 347-356 bioaccumulation, 658 biodegradation, 658 biotransformation, 106 body temperature and, 553, 554 carcinogenesis, 544-545 chemistry, 17, 18--21, 103 CNS effects, 451 crop pest control, 602 cumulative effects, 607-613 dermal absorption/toxicity, 411-4 19 ectoparasiticides, 23 embryocidal/fetocidal effects, 474-475 endocrine disruption, 481-491 EPA subgroups, 620 estrogenic effects, 447, 485-487 extraction solvents, 682 and gene expression, 545 and Gulf War syndrome, 69-75 history of, 3-4
739
in human medicine, 17, 22-23 immunomodulation by, 498-503 inhalation pharmacology/toxicology, 399-407 mechanism of action, 3, 103-104, 211, 339-344 memantine interactions, 40-41 metabolism in v i t r o studies using human tissues, 138-140 phase I reactions, 134-137 phase II reactions, 137-138 mixtures, 610 mosquito resistance, 601 neuropathy target esterase inhibition, 362 neurotoxicity, 276-283, 339-344 occupational exposure, s e e Occupational toxicology ophthalmic toxicity, 423-438 OPIDN prevention, 727 peripheral neuropathy, 364 pharmacokinetics, 104 placental toxicity, 463-475 pulmonary toxicity, 393 reproductive toxicity, 447-459 residue persistence, 4 resistance, 4 routes of exposure, 104 selectivity, 4 specific metabolites, analysis, 693 thermolabile, 406 thermoregulatory effects, 549-564 tolerance, s e e Tolerance development toxicity interspecies variation, 145-155 potentiation in mixtures, 610 uses, 4, 5, 17, 22-23,567 U.S. registered, 673,674-677 U.S. use of, 602 vector-borne disease control, 599-602 Carbamate poisoning/intoxication behavioral toxicity acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 blood cholinesterase measurements, 576-579 children's exposures, 602-605 interspecies variation age and, 154-155 gender and, 154-155 LDs0s, 145-148 pharmacodynamics, 151-155 pharmacokinetics, 148-151 medical surveillance, 572-576 neurotoxicity, 276-283, 339-344 signs/symptoms, 276-277, 348 treatment, 584-585 urine/blood metabolite measurement, 579-580
740
Index
Carbamic acid, 134 Carbamylcholine, 341, 512 Carbaryl absorption/elimination of, 148 aryl hydrocarbon receptor and, 545 cholinesterase inhibition, interspecies variation, 152 cognitive effects, 350 dermal absorption, 416-417 developmental neurotoxicity, 639 ectoparasiticide, 23 endocrine disruption, 485,489-490 history of, 4 immune system effects, 503 metabolism, 106, 135 CYP isoforms in, 139 interspecies variation, 139 metabolites, 580 mixtures, 610, 611 nitric oxide synthase inhibition, 455 PBPK/PD model for, 631 peripheral neuropathy, 364 placental toxicity, 475 Carbendazim, 693 Carbofuran and cholesterol, 665 CNS effects, 451 and creatine kinase activity, 516 developmental neurotoxicity, 639 effect on blood-brain barrier permeability, 279 endocrine disruption, 489-490 half-life, 663 immune system effects, 503 -induced myopathy, 512 interaction with memantine, 41 mechanism of action, 661 metabolism, 135, 662-664 CYP isoforms in, 139 interspecies variation, 139 metabolites, 580, 662, 693 biomarkers, 666 mixtures, 610 nerve agent protective effects, 611 peripheral neuropathy, 364 and phospholipids, 665 reactive oxygen species formation, 455 reproductive toxicity, 454 residue persistence, 659 restricted entry level, 568 toxicity, 4 in fish, 659-661 and route of exposure, 659 signs/symptoms, 659 uses, 658 Carbofuran phenol, 693 Carbon dioxide, 682 Carbon tetrachloride, protein kinase C modulation, 541 Carbosulfan, metabolites, 693
Carboxylesterase inhibitors, and tolerance development, 261-262 Carboxylesterases activity, interspecies variation, 150 acyl pocket binding site, 224 catalytic triad, 224 CES 1 isozyme family, 221-222 genes, 228 induction of, 227-228 structure-activity relationships, 222 CES2 isozyme family, 221-222 genes, 228-229 induction of, 227-228 structure-activity relationships, 222 CES3 isozyme family, 221-222 CES4 isozyme family, 221-222 detoxification potential, 52 gene structure, 228-229 human gene for, 224 induction of, 227-228 inhibition, and organophosphate potentiation, 610 isozymes as biomarkers, 92-93 structure and catalytic mechanism, 223-227 in organophosphate metabolism, 106, 109-110, 150, 706, 707 modeling, 109-118 oxyanion hole, 224 regulation, 228-229 role of, 133 scavenger function, 263 structure, 3-D, 224 substrate specificity, 219, 227, 229 tissue distribution, 219 tolerance development, 261-263 -UGT interaction, 220 Carboxylic ester hydrolases, 129 Carcinogenicity carbamates, 544-545 methyl isocyanate, 84 organophosphates, 542-544, 662 rat mammary tumor model, 543-544 WHO/FAO guidelines, 647 Cardiac arrest, 703 Cardiolipin, 455 Cardiomyopathy, anticholinesterase exposure and, 574 Cardiotoxicity organophosphate-induced effect on contractile tissue, 381-382 in silico approaches, 382-386 treatment, 386 Cardiovascular disease, role of paraoxonase, 247, 248-249 Carson, Rachel, 681 Caspase-3,455-456 Cat, as OPIDN model, 153 Catalase, 456
Catalytic triad carboxylesterases, 224 cholinesterases, 162, 165-168 organophosphate binding, 706 Cataractogenesis, 434-435 Cathepsin, 665 Caucasians butyrylcholinesterase genotypes, 191,204 paraoxonase polymorphisms, 247 Caudate putamen, cholinergic innervation, 273 CBDP, 262 Cell cultures, see also specific cell line fish hepatocytes, 666-667 gonadotoxicity, 457 neurotoxicant screening, 303-304 neurotoxicity testing, 317-318, 319-327, 328-329 placental, 469 Cell damage, see Cytotoxicity Cell signaling, adenylyl cyclase, 704 developmental chlorpyrifos exposure and, 239, 295,296-297, 301 inhibition, 236, 237,240 Cell swelling, organophosphate-induced, 383-386 Centers for Disease Control and Prevention, National Health and Nutrition Examination Survey, 603 Centers for Disease Control and Prevention, no adverse effects limit, 47 Central America, acute pesticide poisonings, 570-571 Central nervous system anticholinesterase intoxication blood-brain barrier, alterations, 278-279, 283 chronic exposure, 281-282 extrapyramidal effects, 280-281 Gulf War illnesses, 279-280 neuropathy, 277-278, 283 respiratory effects, 277 seizures, 277-278, 283 signs/symptoms, 276-277, 283 sites of action, 145 brain excitotoxicity, 521-527 cell types, 315-316 cholinergic failure, 26, 35 cholinergic neurons, 271-273,282-283 cholinergic neurotransmission, 275-276 functions associated with, 273-275 and thermoregulation, 549 cholinergic nuclei, 271 cholinesterase inhibitors, effects, 451 GABAergic transmission, 51-52 inhaled cholinesterase inhibitors, systemic effects, 404-405 muscarinic pathways, and hypothermic response, 554-555 muscarinic receptors, 236, 707 nerve agents and, 51-52
Index
OPIDN effects, 234 interspecies variation, 153-154 organophosphate toxicity, 91 chronic, 234-235 delayed, 662; see also Organophosphate-induced delayed neuropathy respiratory centers organophosphate-induced effects, 391,394 pulmonary toxicity and, 394 thermoregulation, 549, 551,554-555 Cerebral cortex alterations in Alzheimer's disease, 26 cholinergic innervation, 271 Cerebrovascular disease, dementia and, 29 CES1, 228 CES2, 228-229 C-esterases, 129 Channa punctatus, carbofuran toxicity, 659 Chemical Industry Institute for Toxicology Centers for Health Research, 630 Chemical Materials Agency, 47-48, 62 Chemical/physical properties, nerve agents, 50 Chemical Stockpile Emergency Preparedness Program, 47, 62 Chemical warfare agents, see also specific agents; Matsumoto incident; Nerve agents; Tokyo subway attack analytical methods, 693-694 at Khamisiyah, 73-74 decontamination/cleanup, 48 emergency preparedness plans, 47-48, 62 exposure guidelines, estimated reference doses, 48, 55-57 history of, 48-49 non-stockpile material, 48 organophosphate-induced delayed neuropathy, 54-55 properties, 49-52 response to release of, 48, 55 stockpile destruction, 332-333 stress and, 71, 73 toxicity, 52-55 mechanisms of, 51-52 Chemical weapons of mass destruction, 3 Chemiluminescence, time-delayed, 685 Chemiluminescence assay, 535-536 Chemotaxis, 496 Chest tightness, organophosphate exposure and, 392 Chick eggs, developmental neurotoxicity screening, 305 Chickens, as OPIDN model, 153 Children anticholinesterase poisoning/intoxication, signs of, 276-277 breathing zones, 604 and chlorpyrifos exposure, 293
developmental neurotoxicity, 633 FQPA 10x factor, 618, 619, 633,635,639 organophosphates sensitivity, 118-119 pesticide exposure, 490, 602-605 sensitivity to, 354-356 Chironomus adult emergence test, 658 Chlorfenvinphos, 23 chronic exposure, neurobehavioral effects, 353 CNS effects on respiration, 277 metabolism, interspecies variation, 139 pharmacokinetics, interspecies variation, 148-149 retinal effects, 436 seizure induction, 278 successive exposures, 612 Chlorothalonil, endocrine effects, 474 Chlorphoxim, mosquito resistance, 601 Chlorpyrifos, 23 adenylyl cyclase signaling and, 239, 295, 296-297, 301 age-related sensitivity, 354-355 airway hyperreactivity, 237-239, 395 antidotal treatment, 723 anxiogenic effects, 354 biotransformation, 104-106 cognition effects, 350 developmental neurotoxicity, 293-294, 639, 704 glial targeting, 541-542 long-term outcomes, 298-301 mechanisms, 294-298 vulnerable subpopulations, 301-303 and dioxalon binding, 259 DNA synthesis inhibition, 331,533 effects on Ca2+/cAMP response element binding protein, 240, 331 estrogenic activity, 485 gene expression of, 538-540 and Gulf War, 69 hyperthermia, delayed, 559, 560 hypothermic response to, 555 immune system effects, 498, 501 indoor use, 604, 630 induction of heat shock proteins, 538-540 inhibition of cAMP synthesis, 239 metabolism CYP isoforms in, 139, 140 interspecies variation, 139 metabolites, 579 mixtures, 612 neonate vs. adult toxicity, 293 neurobehavioral effects, chronic exposure, 352,353 neurotoxicity testing cell culture systems, 330 whole rat embryo, 329 nicotinic receptor inhibition, 259 oxidative stress and, 534 partitioning coefficient, 111
741
PBPK/PD model for, 108-121 potency, 131 prenatal exposure, 253,604 protein kinase C modulation, 541 and tolerance to acetylcholinesterase inhibition, 258-259 toxicity and paraoxon status, 249-250, 264 transendothelial permeability, 332 uses, 293, 533 Chlorpyrifos-methyl adrenal gland toxicity, 454 endocrine disruption, 488 Chlorpyrifos-oxon activation, 104-106 developmental neurotoxicity, 542 effects on Ca2+/cAMP response element binding protein, 240 hydrolysis of, 133 multidrug resistance-1 gene and, 540 neurotoxicity testing, cell culture systems, 330 noncompetitive muscarinic receptor binding, 237 protective effects of paraoxonase, 249 Choice behavior, effects of anticholinesterase poisoning, 350 Cholesterol, and carbofuran, 665 Choline, 275, 719 Choline acetyltransferase, 271 in Alzheimer's disease, 25-26 developmental chlorpyrifos exposure, 298 levels in mild cognitive impairment, 26 Choline binding site, cholinesterases, 175-176, 177-179, 179-180 Cholinergic agonists chronic exposure and muscarinic receptor decrease, 258 neurobehavioral teratology, 294 Cholinergic crisis, 145,209, 233 acetylcholine accumulation, 451 signs/symptoms, 552, 572-574 Cholinergic fibers, arborization, 272-273 Cholinergic hypothesis, Alzheimer's disease, 25-26 Cholinergic neurons, 271-273, 282-283 Cholinergic neurotransmission, 275-276 and cholinesterase inhibitors, 276-283 functions associated with, 273-275 Cholinergic nuclei, central nervous system, 271 Cholinergic receptors acetylcholine binding, 209 age-related pesticide sensitivity, 355 postsynaptic, 275 presynaptic, 276 tolerance and, 236 Cholinergic system distribution, 103 impairment of, 35 placental, 472-474
742
Index
Cholinesterase inhibitors as alkylating agents, 498 allergic sensitization, 504 autonomic thermoeffectors and, 556-557 behavioral thermoeffectors and, 557 behavioral toxicity acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 cardiotoxicity, 381-386 and cholinergic neurotransmission, 276-283 combustion toxicology, 406-407 and core temperature, 555-556 dermal absorption/toxicity, 411-4 19 endocrine disruption, 481-491 eye accommadative capacity and, 433 glaucoma treatment, 434 history of, 3-4 hypersensitivity to, 194-195 immunomodulation, 498-503 mechanism, 496-498 inhalation pharmacology/toxicology, 399-407 LDs0, interspecies variation, 145-148 lens opacities, 435 low doses, 704 mechanism of action, 51-52 apoptosis, 454-456 carbamates, 211 central nervous system effects, 451 endocrine -related effects, 452-453 hypothalamic-pituitary-gonadal axis, 452,457 metabolic effects, 453-454 organophosphates, 209-211 oxidative stress, 454-456 neurotoxicity, electrophysiological mechanisms, 339-344 occupational exposure, s e e Occupational toxicology ophthalmic toxicity, 423-438 placental toxicity, 463-475 potentiation, 574 progressive, irreversible, 183-184, 199-200 and pulmonary toxicity, 389-395 reproductive toxicity, 447-459 retinal effects, 436-438 reversible, 182-183 therapeutic uses, 283,495 thermoregulation, 551-564 tolerance, s e e Tolerance development uses, 567 vapor pressure, 399 Cholinesterases activity measurements, 716-717 expressing, 200-201 methods, 201-204 quality control, 204 sample collection, 200
standards, 204 substrates, 201 aged, 215-216 Alzheimer's disease and, 25-26 binding sites, 175-179, 199 active center gorge, 174-176, 179-180 acyl pocket, 175-176, 177-179, 209, 210,214 amino acid sequence, 165-168 catalytic triad, 162, 165-168 choline, 175-176, 177-179, 179-180 oxyanion hole, 162, 165-168, 175,211 peripheral, 175-176, 177-179, 209-211, 212,214 serine residue, 162, 165-168, 213 catalytic turnover rates, 104, 175, 213 characteristics of, 161 cysteine residues, 168, 169-171 disulfide loops, 168, 169-171 erythrocyte, 52; s e e a l s o Acetylcholinesterase as biomarker of organophosphate exposure, 91-92 eye, distribution in, 423-424 genetic variants, 204-206 inhibition as biomarker of organophosphate exposure, 91-92 and neurotoxicity, 233-236 oxime reactivation of, 21 4-215 pharmacodynamic models, 111-118 spontaneous reactivation, 200, 213 placental, 472-473 plasma, 52; s e e a l s o Butyrylcholinesterase activity measurements, 576-579 as biomarker of organophosphate exposure, 91-92 human, 112 rat, 112 structure primary, 161-168, 169-171,177-179 quaternary, 181 secondary/tertiary, 168, 174-181 variants, 181-182 substrate activation, 182 substrate hydrolysis, 182, 199-200 substrate inhibition, 182 tissue distribution, 161, 187, 423-424, 451 WHO/FAO activity guidelines, 646 Cholinomimetic agents, effect on motor activity, 274-275 Choreiform movements, 280 Choreoathetosis, 280 Chorioallantoic membrane, in eye irritation tests, 427-428 Choriocarcinoma cell line, 469 Chorionic gonadotropin, placental, 473-474 Chorionic somatomammotrophin, 474
Chromatography, 683,684 Chronic organophosphate-induced neuropsychiatric disorder, 573,662 Cilazapril, 219 Ciliary body, ophthalmic toxicity, 433-434 Citrulline and anticholinesterase-induced seizures, 278 carbamate-induced changes, 516-517 correlation with acetylcholinesterase inhibition, 519 organophosphate-induced changes, 517 pesticide-induced changes memantine/atropine pretreatment, 520 and PBN pretreatment, 525-527 and vitamin E pretreatment, 525-527 CL 18706, in mixtures, 609 Clara cells, 394, 403 Clinical Global Impression of Change, 36 Clinician's Interview-Based Impression of Change Plus Caregiver Input, 36 Clonazepam, 726 CNQX, 341 Cocaine butyrylcholinesterase hydrolysis of, 187 carboxylesterase metabolism of, 220, 222 developmental neurotoxicity, 634 Cockroaches, synaptic transmission, 343-344 Code of Federal Regulations, 40 CFR Part 158, 618, 634 Codex Alimentarius Commission, 644-645 Cognex, 27 Cognition cholinergic regulation of, 274 impaired in Alzheimer's disease, 26 Bhopal accident-associated, 84 measures of, 26-27, 36 organophosphate-induced deficits, 234-235, 282 pesticide exposure acute, 573 chronic, 351,353 Coho salmon, carbamates sensitivity, 342 Collaborative Behavioral Teratology Study, 634 Collision cell, 685 Collision-induced dissociation, 685 Colorimetric test, pesticides, 691 ColQ, 181 Combat stress, 70 Combustion toxicology, 406-407 Comet assay, 458, 537-538 Common mechanism group, 620, 627, 629 Compartmental pharmacokinetic models, 107-108 Compound muscle action potential, 371 Conditioned behaviors, 350-351 Confocal microscopy, corneal thickness, 426
Index
Conjunctivae absorption from, 431 inflammation of, 425 Contact dermatitis, organophosphateinduced, 504 Contractile tissue, effect of cholinesterase inhibitors, 381-382 Convulsions and anticholinesterase intoxication, 278-279, 283 role of nitric oxide, 522 treatment, 725-726 Cooperative Threat Reduction Program, 49 Copper, in aquatic ecosystems, 666 Coquillettidia, 600 Co-Ral, mixtures, 610 Comea cholinergic activity, 424-425 eye irritation tests, 425-428 isolated, cell preparations, 427 Coronary arterial disease, and butyrylcholinesterase activity, 204 Cortex, cholinergic innervation, 273,274 Corticotrophin-releasing factor, 474 Cortisol, immunomodulatory effects, 498 Coughing, 392, 393-394, 574 Coumaphos, 23 CPT- 11,220 Crack-and-crevice application, 604 Cranial nerve palsies, intermediate syndrome, 371 Crayfish, 659 Creatine kinase as biomarkers of muscle injury, 512, 515-516 isoenzymes, 512, 515-516 myonecrosis and, 512 pesticide-induced changes in, 527 Creatine phosphokinase, as biomarker, 580 o-Cresyl saligenin phosphate, and OPIDN syndrome, 7 Critical toxic effect, risk assessment, 618, 621-626
Crop yield, and pesticide usage, 602 Cross-resistance, insecticides, 601 Croton oil, 544 Crotylsarin, 720 Crufomate, 23 Crystal violet staining, eye irritation tests, 428 Cule tarsalis, 600 Culex, 600, 601 Culex pipiens quinquefasciatus, 600, 601 Culiseta, 600 Cumulative assessment group, 620, 627, 630 Cutaneous exposure modeling, 576 Cyanide poisoning, 80 Cyanophos, 716 carbamates interaction, 611 mixtures, 612
cyclicAMP, 704 chlorpyrifos interactions, 297, 301 organophosphate inhibition of, 239 cyclicAMP/PKA signaling pathway, organophosphate modulation of, 239 Cyclic nucleotidase, 498 Cycloate, 17 Cyclopentolate, 432, 433 N6-Cyclopentyladenosine, 260 Cycloplegia, 432 Cyclosarin, 62 chemistry, 7 history, 389 neuropathy induced by, 278 properties, 49-52 CYP 19-aromatase, 487 Cyprinus carpio communis, carbofuran toxicity, 659 Cysteine, cholinesterases, amino acid sequence, 168-171 Cythioate, 23 Cytochrome c oxidase, 455 fasciculations-induced changes, 515 loss of activity, 513 Cytochrome P450 activity, interspecies variation, 150 ancestral gene, 663 and inhalation toxicity, 403 inhibitory effects of pesticides, 140 isozymes CYP1A2, 663-664 CYP1A EROD activity, 666-667 in fish, 664-666 function, 663-664 induction of, 664 placenta and, 465,470, 474 polymorphisms, 139-140 selectivity, 139-140 organophosphate metabolism, 104-106, 111,707-708, 715 oxon activation, 211 pesticide metabolism, 663-664 role of, 128 Cytokines, 560 Cytoskeletal proteins, in OPIDN, 363 Cytotoxicity biomarkers creatine kinase, 512, 515-516 lactate dehydrogenase, 512, 516 nitric oxide and, 518 organophosphate-induced DNA single strand breaks, 537-538 reactive oxygen species-mediated, 533 tubular 538 i ' Cytotoxicity assays, eye irritation tests, 428 Cytotrophoblasts, 463,474 2,4-D, dermal absorption, 416
Daphnia magma life cycle test, 658
743
Data-call in notice, 619, 636 Data extrapolation, WHO/FAO guidelines, 650, 7647 DBCP, and male reproduction, 451 DDT developmental neurotoxicity, 634 endocrine disruption, 481 environmental persistence, 600 estrogenic effects, 447, 452 history of, 657 and male reproduction, 451 mosquito control, 600-602 Dealkylation carbamate metabolism, 136 organophosphates metabolism, 131 Dearylation, organophosphates metabolism, 131 Decamethonium, 168, 176, 179 de Clermont, Phillipe, 3 Decontamination, 584 DEET dermal absorption, regional variability, 416 and Gulf War, 69 interaction with pyridostigmine bromide, 71 Delayed neuropathy, nerve agents, 53-55 Delayed neurotoxicity, WHO/FAO guidelines, 645-646 Dementia and Alzheimer's disease, 26 and cerebrovascular disease, 29 Lewy bodies with, 29-30 memantine for, 36-38 mixed, 29 Parkinson's disease, 29-30 vascular, 37 Demeton, tolerance development and carboxylesterase, 261 Demyelination, organophosphate-induced, 234 Dengue, 599-601 Dengue hemorrhagic fever, 600, 601 Department of Veterans Affairs, Gulf War illness, 71 Depression and Alzheimer's disease, 26 Bhopal accident-associated, 84 Derivatizing agents, 691,693 Dermal absorption factor, 619 Dermal absorption/toxicity additives and, 417 dose and, 413 factors affecting, 416-418 formulation and, 417 mechanism, 411-413 models, 413-415 occlusion and, 418 organophosphates, 715 species differences, 415-416 vehicle effects, 417-418 Dermis, 411 Desferrioxamine, 538
744
Index
Desulfuration, organophosphates metabolism, 131 Detection paper, nerve agents, 694 Detection tickets, pesticides, 691 Detection tubes, nerve agents, 694 Detectors analytical, 684-686 nerve agents, 694 Detoxification age-related differences, 355 organophosphates, in vitro models, 332-333 Developmental neurotoxicity children, 633 chlorpyrifos, 704 glial targeting, 541-542 long-term outcomes, 298-301 vulnerable subpopulations, 301-303 chlorpyrifos-oxon, 542 comparative cholinesterase data, 638-639 diazinon, 542 EPA guidelines, 619 guideline, 634-635 mechanisms, 294-298 neurotoxicants screening, 303-305 organophosphate-induced, 235-236 pituitary-gonadal axis, 448 testing requirements, 635-638 WHO/FAO guidelines, 646 Dexamethasone, induction of carboxylesterases, 228 Di(2-ethylhexyl) phthalate, induction of carboxylesterases, 227 Diacetyl monoxime, 262, 263, 725 Diacylglycerol, 239, 666 Diacylglycerol lipase, 239 Dialkyl phosphates analysis, 691-692 in fruit juices, 603 in water, 692 Diazepam, 726 for anticholinesterases poisoning, 93,277, 386, 394 for convulsions, 585 Diazinon, 23 avian toxicity, 677 chronic exposure, central effects, 281-282 delayed hyperthermia, 560 developmental neurotoxicity, 303,542 DNA synthesis inhibition, 534 endocrine disruption, 487, 489 environmental persistence, 543 EPA classification, 543 homeowner use, cancellation/phase out, 630 immune system effects, 502 intermediate syndrome, 371-372, 377 metabolism of, 715 mixtures, 611 partitioning coefficient, 111 PBPK/PD model for, 108-118
retinal effects, 436 Saku disease, 430 Diazopentane, 691 Diazoxon, 133,248, 249-250 Dibromochloropropane, reproductive toxicity, 448 Dibucaine and butyrylcholinesterase phenotyping, 204 cholinesterase inhibition, 188,204 Dibucaine number, 189 Dibucaine-resistant variant, butyrylcholinesterase, 192 Dibutyryl cyclic AMP, 328 Dichlorodiphenyltrichloroethane. see DDT Dichlorvos, 23 absorption/elimination of, 148 blood-brain barrier permeability and, 279 CNS effects, 277, 281 extrapyramidal effects, 280, 281 immune system effects, 502 intermediate syndrome, 376 lipid peroxidation and, 534-535 memantine and, 41 mixtures, 611 noncompetitive muscarinic receptor binding, 236 Saku disease, 430 seizure induction, 278 tolerance development and carboxylesterase, 261 Dieldrin, estrogenic activity, 485 Diesel oil, 664 Diet, pesticide exposure, in children, 602-604 Diethiofencarb, 134 O,O-Diethyl dithiophosphate, 579, 691 O, O-Diethyl phosphate, 579, 691 Diethyl phosphorofluoridate, 3 Diethylsuccinase, 574 O,O-Diethyl thiophosphate, 106, 579, 691 Diethylumbelliferyl phosphate, 239 Diffusion cell model, dermal absorption/toxicity, 413-414 Diisopropyl fluorophosphatase, 727 Diisopropyl fluorophosphatase/somanase, 263 Diisopropyl fluorophosphate acetylcholinesterase knockout mice and, 234 central respiratory depression, 394 cholinesterase inhibition, interspecies variation, 152 chronic exposure central effects, 281 neurobehavioral effects, 353, 354 cognitive effects, 350 delayed hyperthermia, 560 hypothermic response to, 555 immune system effects, 496 -induced myopathy, 512 inhalation toxicity, 404 memantine and, 39-40 neuropathy target esterase inhibition, 362
neurotoxicity, 277-278 electrophysiological, 340, 341 nicotinic receptor effects, 260 PBPK model for, 108 reactive oxygen species formation, 455 structure of, 390 and tolerance to acetylcholinesterase inhibition, 258 toxicity, physostigmine protection, 611-612 N,N'-Diisopropylphosphorodiamidic fluoride, 7 Diisopropyl phosphorofluoridate, 3 and OPIDN syndrome, 7 Dimethoate birth defects and, 475 cancellation/phase out, 630 immune system effects, 502 intermediate syndrome, 371,372, 376 reproductive toxicity, 454 Dimethyl-4-phenylpiperazinium, 260 O, O-Dimethyl dithiophosphate, 579, 691 O, S-Dimethyl hydrogen phosphorothioate, 693 O, O-Dimethyl phosphate, 579, 580, 691 Dimethyl phosphorofluoridate, 3 O, O-Dimethyl S-(N-ethylcarbamoylmethyl) phosphorodithioate (CL 18706), 609 O, O-Dimethyl thiophosphate, 579, 691 Dioxalan binding, effect of organophosphates on, 259 Dioxins estrogenic effects, 447 and male reproduction, 451 Diplopia, oxime-related, 725 Dipterex, mixtures, 610 Diquat, 415 Disposition, PBPK models, 108-114 Distigmine bromide, in urine voiding dysfunction, 22 Distribution, anticholinesterases, interspecies variation, 148-149 Disulfide loops, cholinesterases, amino acid sequence, 168-171 Disulfiram, methylation of, 138 Disulfoton intermediate syndrome, 377 nicotinic receptor inhibition, 259 tolerance development and carboxylesterase, 261 and tolerance to acetylcholinesterase inhibition, 258 5,5'-Dithiobis-2-nitrobenzoic acid, 202 Diuron, endocrine disruption, 487 Dizziness, oxime-related, 725 DNA adducts, carbamates and, 137 organophosphate-induced damage, 537-538 single strand breaks, 537-538 DNA flow cytometry, 458 DNA stability assay, 458
Index
DNA synthesis inhibition cell cultures, 303-304 and developmental chlorpyrifos exposure, 297 DNQX, 341 Docosahexaenoic acid, 514 Donepezil for Alzheimer's disease, 27,283 analysis, 693,694 combination therapy with memantine, 40, 41-42 mechanism of action, 27, 28 Dopamine, 272-273 effects of developmental chlorpyrifos exposure, 300 retinal, 435 Dopaminergic fibers, arborization, 272-273 Dose-additive design, mixture studies, 608-609 Dosimetry, and PBPK/PD models, 108-118 Doxorubicin, 470 Draize injury scores, 427 Drift, sprays/dusts, 568 Drosophila melanogaster, cholinesterases 3-D structure, 168, 174-181 amino acid sequence, 162, 163-164 Drug metabolism, see also Xenobiotics metabolism butyrylcholinesterase, 187 carboxylesterases induction of, 227-228 isozymes, 219-220, 229 organophosphate enzyme-induction, 536 role of paraoxonase, 247 Drugs of abuse, developmental neurotoxicity, 634 Dugway Proving Grounds, 3 Dusts in closed environments, 585 dermal absorption, 411 drift, 568 inhalation exposure, 570 Dyskinesia, 280 Dyslexia, 633 Dyspnea nerve agent exposure and, 52, 59-61 organophosphate exposure and, 392, 393-394 Dystonia, 280 Eastern equine encephalitis, 600 Echothiophate effect on nicotinic receptors, 260 for glaucoma, 22, 283,432, 433 noncompetitive muscarinic receptor binding, 237 Ectoparasiticides, organophosphates, 23 Eel, freshwater, 168 Effect-additive design, mixture studies, 608-609
Egasyn, 92-93,223-224 Electrocardiogram, organophosphate-induced changes, 92, 383-386 Electroencephalogram, pesticide-related abnormalities, 573 Electromyography in intermediate syndrome, 373, 374, 375-376 for monitoring exposure, 581 OPIDN, 365,573 single-fiber, 376 Electron capture detector, 684 Electron impact ionization, 685 Electron spin resonance spectroscopy, 521 Electroolfactogram, 342 Electrophoresis microchips, 694 Electrophus electricus, 168 Electrophysiological mechanisms, insecticide neurotoxicity action potential conductance, 339-340 neuromuscular transmission, 340-341 synaptic transmission, 341-344 Electroretinography, 430, 435-438 Electrospray ionization, 685 Electrospray ionization mass spectrometry, 705 Elimination anticholinesterases, interspecies variation, 148 PBPK models, 108-114 ELISA, pesticide analysis, 690 Ellman method, cholinesterase activity measurements, 202-203 Embryolethality, pesticide-induced, 474-475 Embryotoxicity, pesticide-induced, 474-475 Emergency preparedness plans, chemical warfare agents, 47-48 Emotion, cholinergic modulation of, 274 Employee periodic medical examination, 574 Encephalitis, 600 Endocannabinoids, 707 Endocrine disruption, 448, 452-453 carbamates in vitro, 485-487 in vivo, 489-490 in humans, 490-491 organophosphates in vitro, 481-485 in vivo, 488-489 Endoplasmic reticulum KDEL receptor, 223,229 localization of carboxylesterase, 219, 220, 223-227 " retention tetrapeptide, 223,229 Endosulfan, estrogenic activity, 485 Endothelium-derived relaxing factor, 473 Endplate potential, 340 Endpoint assessment, WHO/FAO guidelines, 646-647 Endpoint methods, cholinesterase activities, 201-202
745
Enucleated eyes, eye irritation tests, 427 Environmental Chemistry Methods, 690
Environmental Genome Project, 248 Environmental Protection Agency avian acute oral toxicity test, 673 benchmark dose guidance document, 618 canceled/phased out pesticides, 630 carbamates subgroups, 620 chlorpyrifos restrictions, 293, 306 cumulative risk, defined, 620 developmental neurotoxicity guidelines, 633-639 Integrated Risk Information System, 619 mechanism of toxicity, defined, 620 ocular toxicity testing, 430-431 Office of Pesticide Programs, 617-632, 633 anticholinesterase toxicity guidelines, 52-53 data call-in, 619, 636 pesticide additives regulations, 417 pesticide docket, 617 pesticide exposures, 94 pesticide regulations, 618 pesticide reregistration, 635 pesticide use, 523 reference doses, 55-57, 618 registered pesticides, 617, 633, 643 residue guidelines, 690 website, 617 Enzyme activities, measuring, 201 Epichlorhydrin, and male reproduction, 451 Epidemics, mosquito-borne diseases, 600 Epidemiology, organophosphate intoxication, 93-98 Epidermis, 411-412 EPN, 609-610, 716 Eptastigmine in Alzheimer's disease, 17 analysis, 693,694 toxicity, 27 EPTC, methylation of, 138 Equilibrium dialysis, partitioning coefficient determination, 110-111 Erectile dysfunction, 447 Eserine. see Physostigmine Esotropia, 433 Esterases activity, interspecies variation, 150 and immune response, 496 role of, 128-129 Ester drugs/prodrugs, carboxylesterases metabolism of, 219-220, 227-229 Estimated reference doses, nerve agent exposure, 48, 55, 57 Estrogen hypothesis, reproductive toxicity, 447, 448, 452-453,458-459 Estrogen proliferative screening assay, 481 Estrogen receptor carbamate binding, 485-487 pesticide binding, 452-453,481-485
746
Index
Estrogen receptor competitive binding assay, 481 Estrogens, environmental, 447, 448, 452-453,458-459 Ethanol developmental neurotoxicity, 634, 638 hypothermic response, 558 Ethenoadenosine, 137 Ethion, 23,630 Ethopropazine, 161, 176, 201 Ethoxycoumarin-o-deethylase, 664 Ethoxyresorufin-o-deethylase, 664, 665-667 Ethyl carbamate carcinogenicity, 137, 544 immunotoxicity, 498 Ethyldichlorvos, aging of, 183 S-Ethyl dipropylthiocarbamate, 17 Ethylene dibromide, and male reproduction, 451 Ethyl methyl phosphonic acid, 693 O-Ethyl O-p-nitrophenyl phenylphosphonothionate, potentiation of malathion, 609-610 Ethyl parathion antidotal treatment, 723 intermediate syndrome, 376 retinal effects, 436 Ethylthiometon, 433-434, 438 Europeans, butyrylcholinesterase genotypes, 191 European Union, airborne exposure guidelines, 585 Excitotoxicity, 42, 521-527 glutamate, 513 mechanism, 511 neuromuscular junction, 511-512 and nitric oxide, 528 Executive Order No. 13045, 633 Exelon, 28 Exercise effect on pralidoxime, 721 and heat stress, 562-563 Exhaust methods, closed facilities, 585 Explant cultures, placental, 469 Explosives, IMS analysis, 686 Extracellular signal-regulated kinase signaling pathway, 239 Extraction methods, 681-684 Extraneous maximum residue limit, 650, 651 Extrapolation factors, risk assessment, 619 Extrapyramidal motor effects, 280, 281 Eye(s) absorption pathways, 431-432 accomodative capacity, 433-434 cholinergic activity, 429-430 distribution of cholinesterases, 423-424 enucleated, 427 methyl isocyanate toxicity, 83 occupational pesticide exposure protection, 583
as route of absorption, anticholinesterases, 423-430 as target organ, 423,430-431 Eye irritation tests, 424-426 crystal violet staining, 428 cytotoxicity assays, 428 fluorescein staining, 425 in vitro alternatives, 427-429 in vivo alternatives, 426-427 neutral red uptake, 428 ocular/periocular lesions, 425 photobacterium phosphoreum luminescence, 428 plasminogen activation assays, 428 slit-lamp biomicroscopy, 425,426 structure-activity relationships, 429 tiered scheme, 429 Eyelid twitching, 438 F2-isoprostanes, 514 carbamate-induced changes, 516 correlation with acetylcholinesterase inhibition, 519 pesticide-induced changes in, memantine/atropine pretreatment, 519 F4-neuroprostanes, 514 Facial nerve paralysis, 438 Famphur, 23 Farm workers children of, pesticide exposure, 604-605 occupational pesticide exposure, 571 neurobehavioral effects, 352 Fasciculations, evaluating, 581 Fasciculins, 168, 176, 182-183 Fas ligand, 464 Fast atom bombardment/liquid secondary ionization, 685 Fatality rates, organophosphate intoxication, 91 Fatty acid amide hydrolase, 707 Febrile response, anticholinesterases, 559-562 Fecal samples, metabolite analysis, 692 Federal Emergency Management Agency, 47, 62 Federal, Food, Drug, and Cosmetic Act, 618, 633 Federal Insecticide, Fungicide, and Rodenticide Act, 633,635 Feeding studies, WHO/FAO guidelines, 647 Fematoscan, 686 Fenarimol, 474, 485 Fenitrothion antiandrogenic activity, 485 carbamates interaction, 611 endocrine disruption, 488 formulations, 608 immune system effects, 502 mixtures, 612 partitioning coefficient, 110-111 retinal effects, 438
Saku disease, 430 steroid receptor binding, 453 Fenthion, 23 antiandrogenic activity, 485 carbamates interaction, 611 cholinergic crisis, clinical course, 372 endocrine disruption, 488 gene expression of, 538-540 induction of heat shock proteins, 538-540 intermediate syndrome, 371,372 mixtures, 610 nicotinic receptor inhibition, 259 oxidative stress and, 534 protein kinase C modulation, 541 retinal effects, 436-437 Saku disease, 430 uses, 533 Ferbam, 17 Fetotoxicity, pesticide-induced, 474-475 Fetus, pesticide sensitivity, 448 Fever, anticholinesterase-induced, 559-562 Fick's law of diffusion, 412 Filariasis, 599-600 Fire retardants, thermal decomposition products, 406-407 Fires, thermal decomposition products, 406 Fish anticholinesterases sensitivity, 148, 150, 152 carbofuran toxicity, 659-661 cholinesterases, amino acid sequence, 162, 163-164
CYP isozymes, 663-666 malathion toxicity, 489 organophosphate metabolism, 661 pesticides residues, 481 species used in toxicity testing, 658 xenobiotics metabolism, 664-666 Fish eggs, toxicity testing, 659 Flame photometric detector, 683,684-685 " Flame retardants chemistry, 7, 17 organophosphate, 5, 406 Flavin-containing monooxygenase, role of, 128 Flavin-containing oxygenases, 708 Flea collars, 70 Florisil, 682 Fluorescein staining, eye irritation tests, 425 Fluorescein uptake, blood-brain barrier integrity, 279 Fluorescent tracer methods, pesticide exposure, 576 Fluoride number, 189 Fluoride-resistant variant, butyrylcholinesterase, 192, 204 Fluorochromes, corneal permeability, 425,427 Flutamide, 488 Follicle-stimulating hormone, pesticideinduced alterations, 452
Index Food pesticide residues, 603 pesticide tolerances, 618 Food and Drug Administration acetylcholinesterase inhibitors approved for Alzheimer's disease, 27-28 food monitoring program, 617 Pesticide Analytical Manual, 681 pesticide guidelines, 643 Food Quality and Protection Act, 617, 618 public health pesticide uses, 601, 602, 604 10x factor, 618,619, 633,635,639 Formulations, pesticide, interaction of compounds, 608 Formulators, pesticide, 568 FP-biotin, 704 Free radicals and heat shock proteins, 538 and male infertility, 454-456 and pesticide-induced oxidative stress, 90 protein kinase C interaction, 541 Free radical scavenging system, 456 Fruit juices, dialkylphosphates in, 603 Fruits, maximum residue limits, 650-651 Fungicides, thiocarbamates, 17 Furathiocarb, metabolites, 693 Galantamine for Alzheimer's disease, 28,283 analysis, 693,694 mechanism of action, 28 in myasthenia gravis, 22 Gallamine, cholinesterase binding site, 176 Gamma-aminobutyric acid retinal, 435 VX-induction of, 51 Gamma aminobutyric acid receptor benzodiazepines and, 726 TMPP blocking of, 407 Gamma-glutamylcysteine synthetase, 512 Gamma-glutamyl transpeptidase, 456 Gammarus plexus, 660-661 Ganstigmine, in Alzheimer's disease, 17, 22 GAPs. see Good agricultural practices Gas chromatography, 684 Gastrointestinal tract, occupational pesticide exposure, 570 Gel permeation chromatography, 683 Gender and anticholinesterase sensitivity, 154-155 and butyrylcholinesterase activity, 204 Gene expression carbamates and, 545 organophosphates and, 538-540 Genotoxicity, organophosphates, 537-538, 542-544 Germany, nerve agent production, 49, 389
Gestation chlorpyrifos exposure during, 296 and placental choline acetyltransferase activity, 473 Gills, 663 Glaucoma cholinesterase inhibitors for, 3, 4, 22, 434 echothiophate for, 22, 283,432, 433 physostigmine for, 4, 283 Glia chlorpyrifos targeting, 297-298, 533-534, 541-542 organophosphate neurotoxic effects, 330-332 types of, 330 Globus pallidus, cholinergic innervation, 272,273 GLPs. see Good laboratory practices Glucagon, ocular absorption, 431 Glucocorticoids, pretreatment and methyl isocyanate toxicity, 82 Glucose-6-phosphate dehydrogenase, 456 Glucosidation, 129 Glucuronic acid, 129 /3-Glucuronidase, 92-93 Glucuronidases, 129-130 Glucuronidation, 129-130 Gluphosinate, toxicity, 5 Glutamate dementia and, 42 memantine and, 36 role at neuromuscular junction, 513 Glutamate receptor, nerve agents and, 51 Glutamate receptor antagonists, 726 Glutathione carbofuran plus/3-napthoflavone, 667 composition, 129 conjugation reactions, 137 protective effects, 512 Glutathione peroxidase, 456 Glutathione reductase, 456 Glutathione transferases, 129, 456, 708 Glyceraldehyde-3-phosphate dehydrogenase, 363 Glycerophosphocholine, 362 Glycine, retinal, 435 Glycogen synthase kinase-3, 29 Glycopyrrolate, 93, 377 Glycosolation, 129 Glyphosate, toxicity, 5 Goldfish, carbofuran toxicity, 659 Gonadotoxicity, assessing, 456-458 Gonads, male, 449-450 Good agricultural practices, 643,650, 653 Good laboratory practices, 643,653 gpl20, inhibition by memantine, 39 G protein/cAMP-dependent protein kinase A, pesticide disruption of, 453 G protein-coupled receptors, 259, 275-276 down-regulation, 258
747
G proteins, 258 chlorpyrifos interactions, 295,297, 301 Gravitational settlement, inhaled particles, 401 Greenhouse workers, pesticide exposure central effects, 282 endocrine disruption, 490 Growth, methyl isocyanate exposure and, 84 G series agents, see also Sarin; Soman; Tabun delayed neuropathy, 54-55 history, 3, 48-49, 389-390 properties, 49-52 Guinea pigs plasma carboxylesterase, 263 tolerance development to carboxylesterase inhibitors, 261-262 Gulf War environmental exposures, 69, 252 nerve gas warning systems, 73 products used during, 70 Gulf War syndrome animal studies, 72-73 anticholinesterase intoxication, long-term effects of, 281-282 carbamates and, 69-75 and cholinesterases variants, 195 cognitive dysfunction, 234 neurological diseases, 74-75 organophosphates and, 69-75 and paraoxonase status of veterans, 252 pyridostigmine and, 279-280 self-reported exposures, 70-71 "symptom belief", 283 symptoms, 69, 252 synergistic effects of multiple exposures, 71-73 Guthion, mixtures, 610 H7,665 Haber's rule, 392 Hagedorn oximes, 719, 720 efficacy, 722, 723,724 Half-maximal effective concentrations, 318 Half-maximal inhibitory concentrations, 318 Halothane, developmental neurotoxicity, 638 Haloxon anthelmintic use, 23 and OPIDN syndrome, 7 Hand rinses, 575 Hatch rate, 658 Hazard assessment, 618-619 Hazard identification, 618-619 HD5 method, 673, 674-677 Health care facilities, workplace, 587 Health and Safety Program, auditing, 583 Heart, organophosphate-induced toxicity, 381-386, 703 Heat, dissipation methods, 562 Heat loss pathways cholinergic stimulation, 551 rodents, 556-557
748
Index
Heat shock proteins overexpression of, 666 pesticide induction of, 538-540, 665 and protein kinase C, 667 Heat shock/stress genes, 538-540 Heat stress effect on pralidoxime, 721 and exercise, 562-563 and toxicity, 562-563 Heavy metals, hypothermic response, 558 Hematocrit, effects of organophosphate intoxication, 92 Hematopoietic system, organophosphate suppression of, 502 Hemicholinium-3,274, 298 Hemolymph calcium, 659 Hen, as OPIDN model, 361 Hen egg, chorioallantoic membrane, in eye irritation tests, 427-428 Hepatocytes, fish, 666-667 Herbicides, thiocarbamates, 17 Heroin butyrylcholinesterase metabolism of, 187 carboxylesterase metabolism of, 220, 222 Hestrin method, cholinesterase activities, 201-202 H (Hammersmith) variant, butyrylcholinesterase, 189-194, 204 HI-6, 215, 719, 724 efficacy, 722, 723 organophosphate poisoning/intoxication, 485-486 and pulmonary toxicity, 393-394 High-affinity choline uptake, 271,276 High-energy phosphates and anticholinesterase-induced seizures, 278 carbamate-induced changes, 517 correlation with acetylcholinesterase inhibition, 519 organophosphate-induced changes, 517-518 pesticide-induced changes, 522-523 memantine/atropine pretreatment, 520 and PBN pretreatment, 525-527 and vitamin E pretreatment, 525-527 High-performance liquid chromatography, 684 High-stepping gait, 365 Hippocampal slices, insecticide mechanism of action studies, 341 Hippocampus alterations in Alzheimer's disease, 26 cholinergic innervation, 272 developmental chlorpyrifos exposure, 298-299 HIV, and neuronal damage, 39 HLr-7, 724 Homogenization, 681 Hormones, pesticide-induced alterations, 452 HORRAT value, 696 Households, pesticide use, 602, 604 HPTE, steroid receptor binding, 452
Human chorionic gonadotropin, 458 Human placental endothelial cells, 469 Human placental perfusion method, 466-469 Humans anticholinesterase poisoning behavioral effects, 351-352 neurobehavioral effects, 348-351 sensitivity to, 148, 150, 152 signs of, 276-277 carbofuran toxicosis, 659 carboxylesterases genes, 228 plasma, 263 cholinesterases 3-D structure, 174-181 amino acid sequence, 162, 163-164 variants, 181-182 endocrine disruption, pesticide-induced, 490-491 fever/hyperthermia, anticholinesteraseinduced, 561-562 immunotoxicity and cholinesterase inhibitors, 498, 501 observations in, WHO/FAO guidelines, 646 OPIDN, clinical aspects, 364-365 organophosphate-binding proteins, 704-705 placenta, cholinergic system, 472-473 spermatogenesis, 450 Huperzines, 176, 201,386 Hydraulic fluids, 364 Hydrogen cyanide and Bhopal accident, 79-80 inhalation hazard, 400 Hydrogen peroxide, detoxification, 456 Hydroxamic acids, 3-4, 719 3-Hydroxycarbofuran, 666 Hydroxylamine, 3-4, 719 Hydroxylation, carbamate metabolism, 135-136 Hydroxyl radical, lipid peroxidation initiation, 522 3-Hydroxynitrosocarbofuran, 662 5-Hydroxytryptamine, 551 Hygiene occupational, 582-587 personal, 584 Hyperactive airway, 395 Hypersensitivity pneumonitis, 574 Hyperthermia, 559-562 Hypoglycemia, 92 Hypogonadism, 452 Hypokalemia, 92, 384 Hypomagnesaemia, 92 Hyporeflexia, 376 Hypothalamic-hypophysial-gonadal axis, 659 Hypothalamic-pituitary-adrenal axis, role in regulating immunity, 498 Hypothalamic-pituitary-gonadal axis, effects of pesticides on, 452, 457
Hypothalamic-pituitary-thyroid axis, 660 Hypothalamus, acetylcholine activity, 704 Hypothermia and anticholinesterase poisoning, 549, 551-554 benefits of, 558 development of, 550 Idolamine, retinal, 435 Imidapril, 219 Immune system adaptive, 496 allergic sensitization, 504 anticholinesterase pesticides and, 496-503 developing, 496 developmental chlorpyrifos exposure and, 301 innate, 496 Immunity cell-mediated, 496 humoral, 496 Immunoaffinity sorbents, 683 Immunochemical assays, metabolites, 580 Immunoextractions, 683 Immunoglobulins, 496 Immunomodulation, cholinesterase inhibitors, 496-498 Immunosuppression, organophosphates, 495,502 Immunotoxicity methyl isocyanate, 84 and phosphorylating properties of organophosphates, 498 Impaction, inhaled particles, 401 Indentation tonometry, 426 India acute pesticide poisonings, 571 Bhopal accident, 79-85,568 butyrylcholinesterase genotypes, 191 pesticide poisoning in, 93-94 Indian Council of Medical Research, 79-80 Industrial chemicals, and male reproduction, 451 Infants dietary pesticide exposure, 603 FQPA 10x factor, 618, 619, 633,635, 639 neurobehavioral disorders, 633 organophosphate sensitivity, 118-119 Ingestion, pesticides, 570 Inhalable fraction, 401 Inhalation toxicity, 392; see a l s o Pulmonary toxicity acute lethal toxicity, 399-401 exposure route, 399 exposure studies, 402 lethality, 405 material distribution, 401-402 material formulation, 399 respiratory tract effects, 402-403 study design, 399
Index
systemic effects, 403-406 vapor inhalation hazards, 399 Inhibition rate constant, 113 Inositol triphosphate, 237 Inotrope, 717 Insecticides, s e e a l s o Pesticides biotransformation, 104-106 carbamates, 339 cross-resistance, 601 mechanism of action, 339-344 organochlorine, 130 organophosphates, 339 vector resistance, 601-602 Insects acetylcholinesterase genes, 168, 172-173 cholinesterases, amino acid sequence, 162, 163-164 resistance development, 176, 183 Insulin, ocular absorption, 431 Integuments, 663 Interception, inhaled particles, 401 Interleukin-6, 560 Intermediate syndrome, 7 clinical aspects, 91,373-375 diagnostic criteria, 377 experimental animal studies, 372-373 future studies, 377-378 inhalation toxicity, cholinesterase inhibitors, 404 pathogenesis, 376-377 prevention, 727 signs/symptoms, 371-372, 373-374, 573, 661-662 treatment, 371,372, 377, 727 Intermolt phase, 659 International Programme on Chemical Safety, 634 International Union of Pure and Applied Chemistry, 684 Interspecies variation, anticholinesterase intoxication LDs0s, 145-150 pharmacodynamics, 151-155 pharmacokinetics, 148-151 Intracellular neurofibrillary tangle, 26 Intracellular signaling pathways, 239 Intracytoplasmic sperm injection, 447 Intraocular pressure, 426-427, 434 Inuits, butyrylcholinesterase genotypes, 191 Invertebrates, aquatic toxicity testing, 658 Ionization techniques, 685 Ion mobility spectrometry, 685-686 Iran acute pesticide poisonings, 571 butyrylcholinesterase genotypes, 191 Iris eye irritation tests, 425 ocular toxicity, anticholinesterases, 432-433 Iritis, 433 Irritant receptors, 391
Islands of Calleja, cholinergic innervation, 273 Isocyanates, 80 Isomalathion, 608 Isoniazid, 187 iso-OMPA butyrylcholinesterase inhibition, 161,176, 214 activity measurements, 201 lethal inhalation toxicity, 405 and tolerance development, 262 toxicity potentiation, 610 2-Iso-propoxyphenol, 693 Isopropyl methyl phosphonic acid, 693 Isoprostanes as biomarkers of oxidant injury, 514,516,517 correlation with acetylcholinesterase inhibition, 519 Isosafrol, 664 Japan butyrylcholinesterase genotypes, 192 nerve agent poisoning incidents, 49 pesticide poisoning in, 94-95 Japanese encephalitis, 600 Japanese medaka, carbofuran toxicity, 659 Jar cells, 469 Jeg3 cells, 469 J (James) variant, butyrylcholinesterase, 189-194
Joint FAO/WHO Expert Committee on Food Additives, 644 Joint WHO/FAO Meeting on Pesticide Residues in Food, 644-653 Juxtacapillary (J) receptors, 391 Kainic acid, 528 KDEL receptor, endoplasmic reticulum, 223,229 Kepone and male reproduction, 451 nitric oxide synthase inhibition, 455 Kerala (India), acute pesticide poisonings, 571 Keratinocytes, 411 Ketamine, 39 Ketanserin, 300 3-Ketocarbofuran, 666 Ketoconazole, 139 Khamisiyah, and chemical warfare agents exposure, 73-74 Kinetic disorders, anticholinergic agents for, 274-275 Kinetic modeling, acetylcholinesterase metabolism/inhibition, 211-213 K (Kalow) variant, butyrylcholinesterase, 189-194, 204, 216 Knox Out, 543 Kuderna-Danish, 684
749
Kuhn, Richard, 389 Kynurenic acid, 707 Kynurenine, plasma, 707 "Lab on a chip", 694 Lacrimation due to methyl isocyanate toxicity, 83 excess, ocular absorption and, 431 eye irritation tests, 425 LaCross encephalitis, 600 Lactate dehydrogenase as biomarker of muscle injury, 512, 516 isozymes, 512, 516 leakage by organophosphates, 536 myonecrosis and, 511-512 pesticide-induced changes in, 527 Lactation, developmental neurotoxicity guideline, 634, 637 Lambert-Eaton myasthenic syndrome, 376 Lange, Willy, 390 Lannate, reproductive toxicity, 453-454 Larvicides, 601 Laskin nebulizer, 402 Laterodorsal tegmental nucleus (Ch6) cholinergic innervation, 273 role in respiratory depression, 277 Lavage, gastric, 717 Lazaroid (U78517F), 514 Lead acetate, developmental neurotoxicity, 634 Learning, 36 cholinergic modulation of, 273,274 chronic pesticide exposure effects, 354 conditioned, 273 developmental chlorpyrifos exposure and, 299, 300 disabilities, 633 effects of memantine on, 39 measuring deterioration, 39 reward-based, 272 Leishmaniasis, 599 Lens isolated, 427 ophthalmic toxicity, 434-435 Leptophos, 330, 716 Leukocytes, 496 Leukocytosis, organophosphate intoxicationassociated, 92 Leutinizing hormone, 449, 450, 452 Lewisite, 49 Lewy bodies, 29-30 Leydig cells, 449-450 Libido, development of, 450 Ligand binding methods, insecticide mechanism of action, 339 Lindane dermal absorption, 416, 417 hormone alterations, 452 metabolism of, 665 Linear free energy relationships, 412-413 Linuron, 452, 487
750
Index
Lipid peroxidation biomarkers, carbamate-induced changes, 516 dichlorvos and, 534-535 initiators, 522 isoprostanes as indicators, 514, 516, 517 and muscle hyperactivity, 514-515 organophosphate-induced, 534-535 pesticide-induced changes, 522-523 pesticide-induced oxidative stress, 90 Lipids intercellular, and dermal absorption, 412 metabolism, role of paraoxonase, 247 Liquid chromatography/electrospray ionization-tandem mass spectrometry, 693 Liver disease, and butyrylcholinesterase activity, 204 Lou Gehrig's disease, in Gulf War veterans, 74-75 Lowest-observed-adverse-effect level anticholinesterase exposure, interspecies variation, 145-147 estimated reference doses, 55-57 risk assessment, 618-619, 621--626 Lowest-observed-effect level, anticholinesterase exposure, interspecies variation, 145-147 Lubricants, 364, 406 LtiH6, 215 Lungs control of ventilation, 391 methyl isocyanate toxicity, 82, 83 muscarinic receptors, 237-239 organophosphates effects, 237-239, 703 receptors, 391 respiratory physiology, 390-391 Lymphocyte activation, 496 Macrophages, 496 Madhya Pradesh (India), acute pesticide poisonings, 571 Malaoxon, inhibition of cAMP synthesis, 239 Malaria, 599-600 malathion epidemic poisoning, 608 reemergence, 600-601 vaccine research, 601 Malathion, 23 absorption/elimination of, 148 allergic response to, 504 boil weevil eradication, 602 carbamates interaction, 611 carboxylesterase hydrolysis of, 133 cholinesterase inhibition interspecies variation, 152 specificity, 183 decomposition products, 406 delayed hyperthermia, 559, 560 history of, 3 immune system effects, 502 intermediate syndrome, 372, 376
metabolites, 579 mixtures, 610, 611, 612 nitric oxide synthase inhibition, 455 oxidative stress induction, 454 poisoning incidences, 3 potentiation, 608 by EPN, 609-610 by tri-o-cresylphosphate, 609-610 rat mammary tumor model, 543-544 retinal effects, 436 Saku disease, 430 and thyroid activity, 489 toxicity to fish, 489 ultra-low volume spraying, 600 uses, 543 Male reproduction and antiandrogen compounds, 491 assessing gonadotoxicity, 456-458 pesticides and, 450-451 reproductive tract, 449-450 sperm quality and, 448 Malondialdehyde, 538 Mammals anticholinesterases sensitivity, 148, 152 paraoxonase activity, 249 pesticide metabolism, 661 thermoregulation, 549, 550-551 Mancozeb, 17 endocrine disruption, 487, 490 reproductive toxicity, 454 Maneb, 17 Manual of Analytical Methods for the Analysis of Pesticides in Humans and Environmental Samples, 688-689 Manual of Pesticide Residue Analysis, 689 Mass balance studies, dermal absorption/toxicity, 414 Mass spectrometer, 658 Matching accuracy, effects of anticholinesterase poisoning, 350 Material safety data sheets, 582 Maternal-placental-fetal unit, 471-472 Matrix-assisted laser desorption ionization, 685 Matrix-assisted laser desorption time-offlight, 705 Matsumoto incident, delayed hyperthermia, 561 Maximum residue limits, WHO/FAO guidelines, 644, 650-651 Meat, maximum residue limits, 650 Mecamylamine, 259, 278 Meconium, organophosphate metabolites in, 543 Medical surveillance, occupational pesticide exposure, 572-576 Medulla, respiratory centers, 391,394 Memantine adverse effects, 38 clinical use, 36-38 interaction with acetylcholinesterase inhibitors, 39-42
in vitro profile, 38-39 mechanism of action, 42-43 and pesticide-induced reactive oxygen species, 512 postmarketing surveillance studies, 38 prevention of acetylcholinesterase inhibitorinduced oxidative stress, 519-521 properties, 35-36 safety, 38 Membrane signaling, and stress, 665-666 Memory, 36 acute pesticide poisoning effects, 573 Ca2+/cAMP response element binding protein and, 240 cholinergic modulation of, 273, 274 chronic pesticide exposure and, 353 developmental chlorpyrifos exposure and, 299, 300 impairment, 236 loss of, in Alzheimer's disease, 26 Mental retardation, 633 Meperidine, carboxylesterase metabolism of, 220 Merphos, 5 Metabolism anticholinesterases, interspecies variation, 149-151 carbamates in vitro studies, 138-140 phase I reactions, 134-137 phase II reactions, 137-138 effects of pesticides on, 453--454 organophosphates in vitro studies, 138-140 phase I reactions, 131-133 phase II reactions, 134 PBPK models, 108-114 Metabolites carbamates, analytical methods, 693 maximum residue limits, 651 organophosphates, analytical methods, 691-693 Metals, in aquatic ecosystems, 666 Metam, methylation of, 138 Methadone, developmental neurotoxicity, 634 Metham, 17 Methamidophos, 716 age-related sensitivity to, 355 birth defects and, 475 cumulative risk assessment, 629 extraction of, 682 extrapyramidal effects, 280 metabolites, 693 neurotoxicity, 340, 353, 354 Methidathion, 722 Methiocarb, 485,487 Methomyl endocrine disruption, 474, 485 interaction with memantine, 41 seizure induction, 278
Index
Methoxychlor, 452, 481 Methyl-5-hydroxy-2benzaimidazolecarbamate, 693 Methyl amines, toxicity, animal models, 82-83 2-Methylaminochroman, 538 Methylation, carbamates metabolism, 137-138 Methyl azinphos, 610 Methylazomethanol, developmental neurotoxicity, 638 Methylcarbamates, anticholinesterase activity, 214 Methyl-carbamic acid, 106 Methyl chlorpyrifos, neurotoxicity, 234 Methyl isocyanate Bhopal accident, 4, 79-80, 568 conjugates, 81 degradation products, 81 physicochemical characteristics, 80-81 toxicity animal models, 81-83 clinical, 83-85 Methyl mercury, developmental neurotoxicity, 634, 638 Methyl parathion activation of, 150 antiandrogenic activity, 485 antidotal treatment, 723 cancellation/phase out, 630 demethylation of, 131 dermal absorption, 417 immune system effects, 501-502 memantine and, 41 mixtures, 610, 612 neurobehavioral effects, chronic exposure, 353 nicotinic receptor and, 259 protein synthesis and, 474 Saku disease, 430 and tolerance to acetylcholinesterase inhibition, 259 transplacental transfer of, 470 Methyl phosphonate alkyl esters, 693 Methyl phosphonic acid, 693 Methylprednisolone hemisuccinate, carboxylesterase metabolism of, 222, 228 Metolcarb, peripheral neuropathy, 364 Metrifonate in Alzheimer's disease, 17 chronic exposure, central effects, 281 interaction with memantine, 40 Mevinphos birth defects and, 475 IMS analysis, 686 retinal effects, 436 Mexico, acute pesticide poisonings, 571 Michaelis complexes, 210-214 Microarray technology, neurotoxicant screening, 304
Microchips electrophoresis, 694 single-channel, 694 Microelectrode studies, 581 Microglia, 330 Microwave-assisted solvent extraction, 682 Midazolam, 726 Mild cognitive impairment, 26 Milk, maximum residue limits, 650 Mimivirus, cholinesterases, amino acid sequence, 162-164 Miniature endplate currents, 340 Miniature endplate potential, 260, 340 Mini-Mental Status Examination, 26 Miosis nerve agent poisoning, 52-53, 58-61 organophosphate-induced, 392, 432 and sarin exposure, 58-59 Mipafox neurotoxicity testing, cell culture systems, 329, 330 OPIDN, 7, 316 Miscarriages, methyl isocyanate-associated, 83-84 Mission Oriented Protective Posture protection, 73 Mists, inhalation exposure, 570 Mitochondria acetylcholinesterase inhibitor-induced changes, 512 dysfunction, 522 nitric oxide and, 518 Mivacurium, butyrylcholinesterase hydrolysis of, 187 Mixtures administration sequence, 612 carbamates, 610 defined, 607-608 homergic, 608 more-than additive interactions, 610 organophosphates, 609-610 organophosphates and N-methyl carbamates, 611-612 study design, 608-609 time course of interactions, 611-612 WHO/FAO guidelines, 651 MK-801, 39, 341 Mobam, nerve agent protection, 611 Modifying factors, estimating reference doses, 56-57 Molinate, 17 endocrine disruption, 490 metabolism, interspecies variation, 139 toxicity in fish, 659 Monitoring, occupational, 575-576 Monkey, placental cholinergic system, 472 Monoamine systems, effects of developmental chlorpyrifos exposure, 300
751
Monocrotophos, 678 extrapyramidal effects, 280 intermediate syndrome, 371 Monoethylamine, 106 Mood, cholinergic modulation of, 274 Morbidity, organophosphate intoxication, 91 Morphine, carboxylesterase metabolism of, 222 Morris swim tank, 704 Mortality, organophosphate intoxication, 91 Mosquitoes acetylcholinesterase genes, 168, 172-173 disease vectors, 600-601 resistance, 600-602 Motivation, cholinergic modulation of, 274 Motor function, cholinergic modulation of, 273,274-275 Motor nerve conduction, evaluating, 581 Mouse acetylcholinesterase knockout, 234, 260-261 carboxylesterases genes, 228 cholinesterases 3-D structure, 174-181 amino acid sequence, 162, 163-164 neurotoxic target esterase knockout, 234 paraoxonase knockout, 249-250, 264 paraoxonase transgenic, 250 Mouse neuroblastoma cell lines, 328 MRLs. s e e Maximum residue limits MTBSTFA, 693 Muller, Paul, 657 Multidrug resistance- 1 gene, 540 Multidrug resistance-associated protein, 2, 220 Multiresidue method analysis, 686 Munitions binary, 48 unitary, 48, 49 Muscarinic effects nerve agents, 52 organophosphate intoxication, 91 Muscarinic receptors activation, 273 antagonists, 237, 259 binding assays, 258 chlorpyrifos effects, 294 cholinergic hyperstimulation, 145 cholinesterase-independent effects, 236-237 down-regulation, 236 inhibition of acetylcholine, 271 lung, direct effects of organophosphates, 237-239 nerve agents and, 51 organophosphate binding, 706-707 overstimulation of, 233,236 postsynaptic, 707 presynaptic, 707 respiration and, 274 subtypes, 236-239, 257-258 tolerance development and, 236, 257-259, 261
752
Index
Muscle fasciculations, acetylcholinesterase inhibitor-induced, 511-512 Muscle hyperactivity, and lipid peroxidation, 514-515 Muscle injury, biomarkers, 512, 515-516 Muscle weakness intermediate syndrome, 371,374 in OPIDN, 364-365 Mutagenicity, organophosphates, 662 Myasthenia gravis characteristics, 375 treatment acetylcholinesterase inhibitors, 3, 4, 22, 194 cholinesterase inhibitors, 283 galantamine, 28 genetic variability in, 194 neostigmine, 4 Mydriasis, 432 Myelin, organophosphate-induced degradation, 234 Myocardium, organophosphate-induced toxicity, 382-386 Myonecrosis, in intermediate syndrome, 372, 374 Myopathy acetylcholinesterase inhibitor-induced, 511-512 memantine/atropine and, 519-521 organophosphate-induced, role of nitric oxide, 518 prevention of, 512 Myopia, ethylthiometon-induced, 433-434 Myotoxicity, acetylcholinesterase inhibitorinduced, memantine and, 41 NADPH cytochrome P450 reductase, 128 Naled, 600 N-allylquinuclinidol, 727 Naphthalophos, 23 a-Naphthol, 106 a-Napthoflavone, 667 /3-Napthoflavone, 665,667 1-Napthol, 693 Narcotics, carboxylesterase metabolism of, 220 Nasal mucosa, absorption and, 431 Nasal passages, inhalation toxicity, 392-393 Nasolacrimal drainage system, 431 National Health and Nutrition Examination Survey, 603 National Home and Garden Pesticide Use Survey, 617 National Research Institute of Police Sciences, 94-95 Necrotizing myopathy, in intermediate syndrome, 372, 374 Neonates and anticholinesterase sensitivity, 154 chlorpyrifos neurotoxicity, 293-294, 296
paraoxonase activity, 252-253 pesticide sensitivity, 448 Neostigmine analysis, 693,694 in myasthenia gravis, 22 neurotoxicity, electrophysiological mechanisms, 340 respiratory effects, 393 in urine voiding dysfunction, 22 uses, 4 Neostriatum, cholinergic innervation, 272 Nephrotoxicity, anticholinesterase exposure and, 574 Nerve agents, see also specific agent; Chemical warfare agents aging of, 51, 183 analytical methods, 693-694 antidotes acetylcholine synthesis blockade, 727 adenosine receptor antagonist, 727 anticonvulsants, 726 cholinesterase, 727 blood flow alterations, 382 carbamate protection against, 611 cardiotoxicity, 382 chemical/physical properties, 50 CNS effects, 52, 277 degradation products, 693-694 delayed neuropathy, 53--55 destruction of, in vitro models, 332-333 dissemination, 389 exposure guidelines, estimated reference doses, 48, 55-57, 62 history of, 389-390 hypothermic response to, 555 -induced myopathy, 512 muscarinic effects, 52 nicotinic effects, 52 ocular toxicity, 430 OPIDN, 54-55 organophosphate, 5 history of, 3 prophylactic agents, 3, 4 protective effects of memantine, 41 properties of, 49-52 pulmonary toxicity, 277, 392-394, 395 pyridostigmine prophylaxis, 3, 23, 69, 71, 279-280, 365, 611 relative potency, 60-61 retrospective detection, 694 Russian stockpiles, 49 toxicity, 52-55 mechanisms of, 51-52 U.S. stock piles, 47--48, 49 Nerve conduction studies, 581 Nerve excitability, neurotoxicity and, 340 Neural cell cultures, neurotoxicant screening, 303 Neural tracts, interspecies variation, 154 Neural tube defects, 296, 605
Neurite growth, 241,297 Neurobehavioral disorders, infants, 633 Neuroblastoma cell lines, human (SY5Y), 328-329, 332-333 Neuroeffector junctions, acetylcholine accumulation, 209 Neuroendocrine dysfunction, in fish, carbofuran-induced, 659 Neurofibrillary tangles, tau and, 29 Neuromotor function, chronic pesticide exposure and, 353 Neuromuscular blockade, reversal of, galantamine, 28 Neuromuscular conduction techniques, 581 Neuromuscular junction cholinergic excitotoxicity, 511-512 nitric oxide at, 513 role of glutamate, 513 Neuromuscular transmission and acetylcholinesterase inhibition, 511-512 effect of pesticides, 340-341 glutamate and, 513 Neuromuscular transmission disorders, intermediate syndrome comparisons, 375-376 Neuronal stem cells, apoptosis, 318 Neurons, 315-316 cholinergic, see Cholinergic neurons effects of organophosphate neurotoxicants, 318, 328-330, 333 Neuropathology, and anticholinesterase intoxication, 278-279, 283 Neuropathy, delayed, see Delayed neuropathy; Organophosphate-induced delayed neuropathy Neuropathy target esterase, 7, 54, 316 aging of, 361,662 as biomarker, 580 placental, 473 tissue distribution, 362 WHO/FAO guidelines, 645-646 Neuropathy target esterase inhibition, 234, 703 DFP, 362 interspecies variation, 153-154 and neurotoxicity testing, 329-330 and OPIDN, 704 phenylmethane sulfonyl fluoride, 363 phenylpentyl phosphinate, 363 Neuropsychiatric disorder, organophosphateinduced, 89; see also Organophosphateinduced delayed neuropathy Neuropsychiatric Inventory, 26 Neurospora crassa, cholinesterases, amino acid sequence, 162-164 Neurotoxicity bicyclophosphorus esters, 407 carbamate-induced acetylcholinesterase inhibition, 276-283 electrophysiological mechanisms, 339-344
Index
and cholinesterase inhibition, 233-236 organophosphate-induced, 661-662 acetylcholinesterase inhibition, 276-283 acetylcholinesterase knockout mice and, 234 blood-brain barrier, 149 chronic, 234-235 developmental, see Developmental neurotoxicity effects, 233-234 electrophysiological mechanisms, 339-344 intracellular targets, 239-241 molecular targets, 239-241 muscarinic receptor effects, 236-239 neurotoxic target esterase knockout mice and, 234 and paraoxonase status, 251-253 pathways, 316 studying, 315 symptoms, 234 trimethylolpropane phosphate, 406, 407 tri-o-cresyl phosphate, 406 WHO/FAO guidelines, 646 Neurotoxicity testing bioavailability and, 318 cell/tissue culture models, 317-318, 319-327, 328-329 developmental cell culture models, 303-304 nonmammalian models, 305 effects on glia, 330-332 effects on neurons, 318, 328-330, 333 exposure regimens, 318, 333 extrapolation of results, 333 future directions, 333-334 goals of, 315 in vitro vs. in vivo systems, 316-317 neural cell types, 315-316 neuronal morphology and differentiation, 330 neuron-like cell lines, 328-329 noncholinergic/nonantiesterase mechanisms, 329-330 primary cultures, 329 prophylactic organophosphate detoxification, 332-333 whole embryo cultures, 329 Neurotransmission, cholinergic, 161,275-276 Neurotransmitters developing brain, 294 retinal, 435-436 Neutral red uptake, eye irritation tests, 428 NEvap, 684 N-formyl-L-kynurenine, 707 NGF, 328 NGF receptor, 271 N-hydroxyethylnaphthylvinylpyridine, 727 Nicotinamide adenine dinucleotide, 474 Nicotine, 259, 302-303
Nicotinic acetylcholine receptors organophosphate binding, 707 prolonged stimulation, 511, 512 Nicotinic agonists, and Alzheimer's disease, 260 Nicotinic effects nerve agents, 52 organophosphate intoxication, 91 Nicotinic receptors acetylcholine and, 271 activation, 273 antagonists, 259 chlorpyrifos effects, 294 cholinergic hyperstimulation, 145,233, 236 direct effects of insecticides, 340, 343-344 nerve agents and, 51 organophosphate inhibition of, 574 respiration and, 274 and sodium influx, 276 tolerance development, 259-260 Nictitating membrane, inflammation of, 425 Nimodipine, 727 N I O S H Manual o f Analytical Methods, 689 Nitric oxide acetylcholine and, 29 at neuromuscular junctions, 513 biomarkers carbamate-induced changes, 516-517 organophosphate-induced changes, 517-518 developmental neurotoxicity, 638 and excitotoxicity, 528 male infertility and, 454--455 neurotoxic effects, 522 and organophosphate-induced cardiotoxicity, 381 pesticide-induced changes, 522-523 respiration regulation, 274 role in organophosphate-induced myopathy, 518 sleep regulation, 274 Nitric oxide synthase activation of, 235 biomarkers carbamate-induced changes, 516-517 organophosphate-induced changes, 517-518 effects of memantine pretreatment, 41 inhibition, 235, 274, 455 isoforms, 513 N~-Nitro-L-arginine, 274 Nitro-L-arginine methyl ester, 518 Nitrogen phosphorus detector, 685 7-Nitroindazole, 518 p-Nitrophenol, urinary, 92, 579 p-Nitrophenylacetate, carboxylesterases and, 228 Nitrosocarbofuran, 662 Nitrosopropoxur, mutagenic potential, 544 NK cells, 496
753
NMDA. see N-methyl-D aspartate N-methyl-D aspartate antagonists, analgesic properties, 39 N-methyl-D aspartate receptors, 35, 36 activation, 235 antagonists, 39, 727 blocking, 513 memantine inhibition of, 38-39 N-methyl carbamates, cumulative risk assessment, 620, 630 N-methyl hydroxylamine, 214 N-methyl scopolamine, 258 N,N-diethyl m-toluamide, see DEET N,N'-methylene bi-acrylamide, developmental neurotoxicity, 634 No-observed-adverse-effect level estimated reference doses, 55-57 risk assessment, 618-619, 621-626 WHO/FAO guidelines, 646, 647, 652-653 No-observed-effect level, anticholinesterase exposure, interspecies variation, 145-147 Noradrenergic pathway, thermoregulation, 555 Norepinephrine, 300 North Americans, butyrylcholinesterase genotypes, 191 Northern Europeans, paraoxonase polymorphisms, 247 Nucleic acid synthesis, 474 Nucleophilic agents, 3 Nucleus accumbens, cholinergic innervation, 272,273 Nucleus basalis of Meynert, 26, 272 Nurse cells, 450 Nutrition, and pesticide usage, 602 Obesity, and butyrylcholinesterase activity, 204 Obidoxime, 719, 720 adverse effects, 725 efficacy of, 722-723 organophosphate poisoning/intoxication, 584-585 Occlusion, and dermal absorption, 418 Occupational hygiene, 582-585 closed facilities, 585-586 open air, 586-587 Occupational Safety and Health Administration, exposure limits, 585 Occupational toxicology biomarkers, 580 blood cholinesterase measurements, 576-579 exposure, 423,424, 448, 567-570 behavioral deficits, 351-354 "carry home", 605 dermal absorption, 411 and endocrine disruption, 490-491 NIOSH guidelines, 689 routes of, 569-570 sources of, 567-569
754
Index
Occupational toxicology (continued) medical surveillance, 572-576 neurophysiological monitors, 581 urine/blood metabolite measurement, 579-580 workplace biomonitoring, 581-582 Oconne River, 661 Octamethyl pyrophosphortetramide, lethal inhalation toxicity, 405 ODS resin, 682 ODS sorbents, 683 Office of the Army Surgeon General, AEGLs, 48, 57-62 Office of Pesticide Programs, 617-632, 633 developmental neurotoxicity guidelines, 633-639 Official Methods o f Analysis (OMA) o f AOAC International, 689 Oikopleura dioica, cholinesterases, amino
acid sequence, 162, 163--164 Oleamide, 707 Olfactory bulb, cholinergic innervation, 271 Olfactory tubercle, cholinergic innervation, 273 Oligodendrocytes, 316 Oligodendroglia, 330 Onchocerciasis, 601 ONT (Osterrieder-Noma-Trautwein) formulation, 386 O, O,S-trimethyl phosphorothioate, immunotoxic effects, 502-503 Operation Desert Shield/Storm, 69 pyridostigmine bromide, prophylactic use, 23 Ophthalmic toxicity anticholinesterases intraocular effects, 430 ocular effects, 424-428 aqueous humor, 434 ciliary body, 433-434 exposure accidental, 423,424 deliberate (medicinal), 423,424 occupational, 423,424 extraocular muscles, 438 eye, cholinergic activity, 429-430 eye irritation tests, 424-426 glaucoma and, 434 intraocular pressure, 434 iris, 432-433 lens, 434-435 retina, 435-438 systemic effects extraocular absorption, 430-431 transocular absorption, 431-432 Ophthalmoscopy, retinal lesions, 435 OPIDN. see Organophosphate-induced delayed neuropathy Optic neuritis, 438 Oral studies, WHO/FAO guidelines, 647 Ordered Uni Bi Bi kinetic scheme, 212-213
Organization for Economic Co-operation and Development, pesticide guidelines, 643 Organochlorine pesticides, 130 environmental persistence, 600 estrogenic activity, 481 withdrawal of, 681 Organomation Associates, 684 Organophosphate compounds acetylcholinesterase inhibition, 90 structure-activity relationships, 213-214 activation, interspecies variation, 150 aging, 183 allergic sensitization, 504 androgen receptor binding, 481 anthelmintics, 23 asthma and, 395 behavioral toxicity, 347-356 biotransformation, 104-106 body temperature and, 553, 554 carcinogenicity, 542-544, 662 rat mammary tumor model, 543-544 cardiotoxicity, 381-386 chemistry/structure, 5-7, 8-16, 103, 130-131,390 children, sensitivity to, 118-119 classes of, 104 crop pest control, 602 cumulative effects, 607-613 deactivation, interspecies variation, 150-151 decline in use, 673 delayed neurotoxicity/neuropathyassociated, 7; see also Organophosphate-induced delayed neuropathy dermal absorption/toxicity, 411-419 developmental neurotoxicity chlorpyrifos, 293-306 and paraoxonase, 251 DNA damage, 537-538 ectoparasiticides, 23 embryocidal/fetocidal effects, 474-475 endocrine disruption, 481-491 estrogenic effects, 447, 485 extraction solvents, 682 flame retardants chemistry, 7, 17 neurotoxic, 406 gene expression, 538-540 genotoxicity, 537-538, 542-544 and Gulf War syndrome, 69-75 history of, 3, 89, 130, 599 in human medicine, 17, 22-23 immune system and, 495-504 impurities associated with, 502-503 inhalation pharmacology/toxicology, 399-407 intermediate syndrome-associated, 7 lung muscarinic receptors and, 237
mechanism of action, 3, 90, 103-104, 209-211,715-716 electrophysiological, 339-344 non-acetylcholinesterase inhibition, 706-708 memantine interactions, 40-41 metabolism, 130-131 in fish, 661 and genetic polymorphisms, 119-120 in vitro studies using human tissues, 138-140 phase I reactions, 131-133 phase II reactions, 134 metabolite analysis, 691-693 metabolites, blood/urinary, 579-580 mixtures, 609-610 more-than additive interactions, 610 mosquito resistance, 601 mutagenicity, 662 nerve agents chemistry, 7 prophylactic agents, 23 neurotoxicity electrophysiological mechanisms, 339-344 noncholinesterase mechanisms, 233-241 noncholinesterase interactions, 236 occupational exposure, see Occupational toxicology ophthalmic toxicity, 423-438 OPIDN associated, 573 paraoxonase activity, and chronic exposure, 252-253 pesticides, chemistry, 5-7, 8-16 pharmacokinetics, 104 compartmental models, 107-108 physiologically based models, 108-114 principles, 107 placental toxicity, 463-475 prenatal exposure, biomarkers, 543 prophylactic detoxification, in vitro testing, 332-333 protein binding in vitro, 706 in vivo, 704-705 protein kinase C modulation, 540-541 pulmonary toxicity, 389-395 reproductive toxicity, 447-459 residue persistence, see Pesticide residues resistance, 4 risk assessment, see Risk assessment routes of exposure, 104 selectivity, 4 sensitivity to, 118-119, 708 sites of action, 703-704 specific metabolites, analysis, 692-693 thermoregulatory effects, 549-564 tolerance, see Tolerance development
Index
toxicity biomarkers, 91-93 and butyrylcholinesterase variants, 195 characteristics of, 89 clinical aspects, 91-93 fatality rates, 91 global epidemiology, 93-98 interspecies variation, 145-155 mechanism of, 3, 90, 103-104, 706-708, 715-716 and paraoxonase status, 249-250, 251 tolerance, s e e Tolerance development types of, 5-7, 17, 89 uses, 4, 5, 7, 17, 567 U.S. registered, 673,674-677 U.S. use of, 602 vector-borne disease control, 599-602 Organophosphate-induced delayed neuropathy, 7, 316, 703 animal models, 365-366 characterization of, 234, 361 clinical aspects, 91,364-365 DFP-induced, 728 early events, 363 inhalation toxicity, cholinesterase inhibitors, 404 intermediate events, 363 interspecies variation, 153-154 late events, 363 mechanism, 361-362, 573 nerve agents, 54-55 nerve conduction, 340 organophosphates associated with, 573 prevention/treatment, 727-728 signs/symptoms, 573,662 Organophosphate poisoning/intoxication, 717 behavioral toxicity acute effects, 348-351 age-related sensitivity, 354-356 chronic effects, 351-354 biomarkers, 91-93 blood cholinesterase measurements, 576-579 and butyrylcholinesterase variants, 195 characteristics of, 89 children's exposures, 602-605 chronic, low level, 281-282 clinical aspects, 91-93 CNS effects, 91 diagnosis/assessment of, 716-717 fatality rates, 91 global epidemiology, 93-98 intermediate syndrome, 371-378 interspecies variation age and, 154-155 gender and, 154-155 LDs0s, 145-148 pharmacodynamics, 151-155 pharmacokinetics, 148-151 management of, 715
mechanism of, 90 medical surveillance, 572-576 muscarinic effects, 91 neurotoxicity, 276-283 nicotinic effects, 91 signs/symptoms, 90, 276-277, 348, 552 treatment, 93,584-585 acetylcholine synthesis blockade, 727 adenosine receptor agonists, 727 anticonvulsants, 726-727 atropine, 718-719 calcium channel blockers, 727 cholinesterases, 727 NMDA receptor antagonists, 727 oxime/atropine, 722-725 oximes, 200, 719-722 phosphotriesterase, 727 urine/blood metabolite measurement, 579-580 Organophosphate-specific PBPK/PD model, 108-114 O , S , S - t r i m e t h y l phosphorothioate immunotoxic effects, 502-503 and inhalation toxicity, 403 Oxamyl, 41,485 Oxidant injury, isoprostanes as indicators, 514,516,517 Oxidant tumor promotors, protein kinase C modulation, 541 Oxidation carbamates, 135-137 organophosphates metabolism, 131-132 Oxidative destruction, 692 Oxidative phosphorylation, inhibition of, 518 Oxidative stress acetylcholinesterase inhibitor-induced, 511-515 memantine/atropine, 519-521 antioxidant protection, 512 and ATP, 513 biomarkers carbamate-induced changes, 516-517 organophosphate-induced changes, 517-519 chlorpyrifos and, 534 delayed, 278 and developmental neurotoxicity, 302 fenthion and, 534 and heat shock proteins, 538-540 isoprostanes as indicators of, 514, 516,517 and memantine pretreatment, 41 organophosphate-induced, 662 DNA single strand breaks, 537-538 and male infertility, 454-456 protein kinase C activation, 540-541 pesticide-induced, 90 seizures and, 278, 522 skeletal muscle, 513
755
Oximes adverse effects, 725 antidotal effect, 340 choice of, 723-724 clinical efficacy, 719-720 dosing regimen, 724-725 efficacy as reactivators, 200, 715, 716 history of, 3-4 intermediate syndrome treatment, 377 mechanism of action, 719-720 electrophysiological, 341 myopathy prevention, 512 organophosphate poisoning/intoxication, 584-585 plus atropine, efficacy of, 722-725 reactivation of inhibited cholinesterases, 214-215 Oxonases, organophosphate deactivation, 150-151 Oxons anticholinesterase activity, 211 carboxylesterase detoxification of, 133,715 Oxotremorine, hypothermic response to, 551 Oxyanion hole carboxylesterases, 224 cholinesterases, 162, 165-168, 175, 211 Oxydemeton-methyl, birth defects and, 475 Pachymetry, 425,426 Pakistan, malathion epidemic poisoning, 608 2-PAM. s e e Pralidoxime Pancreas, anticholinesterase exposure and, 574 Paralysis, due to organophosphate intoxication, 91 Paraoxon, 247 aging of, 183 anticholinesterase activity, 213 axon growth and, 241 blood-brain barrier permeability and, 279 central effects, chronic exposure, 281 cholinergic crisis, clinical course, 372 -induced myopathy, 512 inhibition of cAMP synthesis, 239 neurobehavioral effects, 354 neurotoxicity, 278 electrophysiological, 340, 341 neurotoxicity testing cell culture systems, 329 exposure regimens, 318 noncompetitive muscarinic receptor binding, 236 and OPIDN, 7 potency, 131 respiratory effects, 393 sensitivity in paraoxonase knockout mice, 249-250 tolerance development and carboxylesterase, 261
756
Index
Paraoxonase, 133, 708; see also A-esterases activity, interspecies variation, 150-151 clinical relevance of paraoxonase status, 251-253 genetic polymorphisms, 119-120 genotype/phenotype, 248-249 levels in newborns, 154 mutations, 708 in organophosphate metabolism, 106, 109-110, 249-250, 251 modeling, 109-118 polymorphisms, 247-248 structure, 247 substrates, 247 type Q, 75 Paraquat, 95, 97, 415 Parasiticides carbamate, 5 organophosphate, 5 Parathion activation of, 150 antiandrogenic activity, 485 antidotal treatment, 723 biological exposure index, 581 cancellation/phase out, 630 cholinesterase inhibition specificity, 183 dermal absorption, 563,582 regional variability, 416 developmental neurotoxicity, 303, 634 and dioxalon binding, 259 endocrine disruption, 488-489 history of, 3 immune system effects, 502 inhibition of CYE 140 intermediate syndrome, 376 metabolism of, 715 metabolites, 579 mixtures, 610, 611, 612 nicotinic receptor inhibition, 259 and OPIDN, 7 poisoning, biomarkers of, 92 rat mammary tumor model, 543-544 retinal effects, 436 Saku disease, 430 steroid receptor binding, 453 tolerance development, 236, 264 and tolerance to acetylcholinesterase inhibition, 258 transplacental transfer of, 470 uses, 543 Parkinson's disease and agrochemical exposure, 281 dementia and acetylcholinesterase inhibitors, 29-30 Partition coefficient, 110-111,412 Passive diffusion and dermal absorption, 412 placental toxicity, 465 Patch clamp analyses, 342
Patch technique, pesticide exposure monitoring, 576 PC-12 cell line, 297,328 chlorpyrifos developmental neurotoxicity, 541-542 PCBs, estrogen receptor and, 452 Pebulate, 17 Pedunculopontine nucleus (Ch5), 274 cholinergic innervation, 273 innervation, 273 role in respiratory depression, 277 Peer-reviewed programs, AOAC, 695 Pentaerythritol, 406 Pentafluobenzylbromide, 691 Pentylenetetrazol, 277-278, 350 Performance-tested methods, AOAC, 695 Perfused skin preparations, dermal absorption/toxicity, 414 Peripheral binding site, cholinesterases, 175-176, 177-179, 209-211,212, 214 Peripheral nervous system anticholinesterases, sites of action, 145 cell types, 316 cholinergic neurotransmission, 275-276 chronic organophosphate neurotoxicity, 234-235 compensation/repair mechanisms, 365 muscarinic receptors, distribution, 236 OPIDN effects, 234, 662 Permeability coefficient, dermal absorption, 412 Permethrin, 70, 71 Peroxisome proliferators, induction of carboxylesterases, 227 Peroxynitrite radicals, 455, 513,522 Persian Gulf War and paraoxonase status of veterans, 252 pyridostigmine bromide, prophylactic use, 23 Perspiration, and toxicant absorption, 562-563 Pesticide Analytical Manual, 681 Pesticide Analytical Manual, Vol. 1,686-688 Pesticide Analytical Manual, Vol. 2, 688 Pesticide applicators acute poisoning, 571-572 chronic poisoning, central effects, 282, 35 l, 352 Pesticide exposure assessment, 448 Pesticide poisoning blood cholinesterase measurements, 576-579 in children, 602-605 diagnosis/assessment of, 716-717 exposure accidental, 423,424 deliberate (medicinal), 423,424 dermal, 411 inhalation, 570 occupational, 423,424 paraoccupational "take-home", 569
routes of, 567-569 sources of, 567-569 global incidence of, 4, 570-572 in homicides, 569 management of, 715, 717 medical surveillance, 572-576 self, 569 toxicological effects of, 4 treatment, 584-585 acetylcholine synthesis blockade, 727 adenosine receptor agonists, 727 anticonvulsants, 726-727 atropine, 718-719 calcium channel blockers, 727 cholinesterases, 727 NMDA receptor antagonists, 727 oxime/atropine, 722-725 oximes, 719-722 phosphotriesterase, 727 urine/blood metabolite measurement, 579-580 Pesticide residues analysis, sources for, 686-690 carbamates, 4 carbofuran, 659 fish, 481 in food/beverages, 603 organophosphates, 4 persistence, 448 water, 657-658 WHO/FAO guidelines, 644-645, 650-651 Pesticides, see also Insecticides additives, classification, 417 aerial drift, 568 age-related sensitivity to, 354-356 aging of, 183 allergic sensitization, 504 androgen receptor binding, 485-487 bioaccumulation, 481 bioconcentration, 481 carbamates, chemistry, 17, 18-21 detection tickets, 691 estrogenic effects, 447, 481-485 exposure pathways, 617 extraction methods, 681-682 formulators, 568 household use, 602 impurities, toxicity and, 608 inhibition of CYP, 140 labeling, 583 and male reproduction, 450-451 mechanism of action, 451-456 apoptosis, 454-456 central nervous system effects, 451 endocrine -related effects, 452-453 hypothalamic-pituitary-gonadal axis, 452, 457 metabolic effects, 453-454 oxidative stress, 454-456
Index
metabolism of, and cytochrome P450, 139, 663-664 mixtures, see Mixtures organophosphates, chemistry, 5-7, 8-17 progesterone receptor binding, 484-485 registration, 583 reentry restrictions, 587 reregistration, 635 resistance, see Resistance development storage, 602 transplacental transfer, 465-474 turf, 543 U.S. registered, 673,674-677 Pesticides in the Diets of infants and Children, 354
P-glycoproteins mammalian, 149 placental transfer, 470 Phagocytes, 496 Pharmacodynamic models cholinesterase inhibitors, 111-118 validation, 115-118 Pharmacodynamics, anticholinesterases, interspecies variation, 151-155 Pharmacogenetics acetylcholinesterase, 194-195 butyrylcholinesterase BCHE, 190-194, 204-206 clinical aspects, 187-188 genetic variants, 189-194, 204, 216 inhibition studies, 188-189 phenotypes, 188-189, 204 role of, 187 Pharmacokinetic models compartmental, 107-108 physiologically based, 108-121 Pharmacokinetics anticholinesterases, interspecies variation, 148-151 applications, 103, 107 role of, 103 Phase I metabolism, 219, 220 carbamates hydrolysis reactions, 134-135 oxidation reactions, 135-137 enzymes, 127-129 age and, 154 carboxylesterases, see Carboxylesterases placental, 465-466 organophosphates hydrolysis reactions, 132-133 oxidation reactions, 131-132 Phase II metabolism, 219, 220 carbamates, 137-138 enzymes, 128, 129-130 age and, 154 placental, 466 organophosphates, 134 Phencyclidine, developmental neurotoxicity, 634
Phenobarbital, induction of carboxylesterases, 227 Phenols, 693 Phenserine, in Alzheimer's disease, 17 Phenylmethane sulfonyl fluoride, 363 Phenyl methane sulfonyl fluoride, OPIDN prevention, 727 Phenyl-N-tert-butylnitrone
action of, 521 pretreatment with, 525-527 Phenylpentyl phosphinate, 363 Phenytoin, 726 for convulsions, 585 developmental neurotoxicity, 634 Pheochromocytoma cell line, 328 Phorbol esters, protein kinase C modulation, 541 Phosdrin, IMS analysis, 686 Phosmet, 23, 630 Phosphamidon, mixtures, 610 Phosphatidylcholine, deacylation, 362 Phosphatidyl inositol, pesticide disruption of, 453 Phosphatidyl inositol diphosphate, 666, 667 Phosphatidyl-inositol-phosphate, 667 Phosphinates, OPIDN prevention, 727 Phosphine, 406 3'-Phosphoadenosine 5'-phosphosulfate, 130 Phosphocreatine carbamate-induced changes, 517 organophosphate-induced changes, 517-518 Phosphoinositide-specific phospholipase C, 237 Phospholine, in glaucoma, 22 Phospholine iodide, 278-279 Phospholipase 2, 707 Phospholipase C, activation, 236 Phospholipids, and carbofuran, 665 Phosphonofluoridates, 7 lethal inhalation toxicity, 405 Phosphonothioates, 7 Phosphoric triester hydrolases, 129 and tolerance development, 263-264 Phosphoroamidothiolates, biotransformation, 104 Phosphorodithioates, biotransformation, 104 Phosphorofluoridates, lethal inhalation toxicity, 405 Phosphorothioates bioactivation, 715 and inhalation toxicity, 403 Phosphorothionates biotransformation, 104 desulfuration, 131 oxons, 131 S,S,S,-Phosphorotrithioate, hypothermic response to, 555 Phosphotriesterase, 727 Photobacterium phosphoreum luminescence, eye irritation tests, 428
757
Photophobia, due to methyl isocyanate toxicity, 83 Physiologically-based pharmacokinetic/dynamic models, and risk assessment, 630-631 Physiologically based pharmacokinetic models, 108-121 organophosphate-specific, 108-114 validation, 115-118 Physostigma venenosum, 599 Physostigmine central effects, chronic exposure, 281 cholinesterase inhibition, interspecies variation, 152 cognition and, 27 DFP toxicity and, 611-612 for glaucoma, 22, 283 history of, 48, 599 hypothermic response to, 555 inhalation toxicity, 403 mixtures, 611, 612 in myasthenia gravis, 22 nerve agent protection, 611 neuropathy induced by, 278 neurotoxicity, electrophysiological, 340, 341 rat mammary tumor model, 543-544 respiratory effects, 393 in urine voiding dysfunction, 22 uses, 4, 17, 35 Pig, as dermal absorption/toxicity model, 415 Pigeon chest, 84 Pilocarpine, 237 Ping Pong Bi Bi kinetic scheme, 211-213 Pirimicarb endocrine disruption, 474, 485, 487 metabolites, 580 Pirimiphos-methyl, 23 Pituitary-gonadal axis, developmental toxicity, 448 Pituitary hormone, pesticide-induced alterations, 452 Placenta as barrier, 470 bidiscoid, 463 bioconcentration of toxicants, 471-472 cholinergic system, 472-474 endotheliochorial, 463 function of, 463 hemochorial, 463 human, 463-465 perfusion method, 466-469 multicotyledonary, 463 pesticide metabolism, 470 syndesmochorial, 463 term, 464-465 xenobiotic-metabolizing enzymes, 465-466, 467 zonary, 463 Placental choline acetyltransferase, gestation age and, 473
758
Index
Placental toxicity abnormal pregnancy outcomes, 474-475 experimental methods, 466-469 placenta structure/function, 463-465 species differences, 463-464 toxicodynamics, cholinesterase inhibitors, 470-474 toxicokinetics, 465-466 cholinesterase inhibitors, 469-470 Plague, 599 Plaques, senile, 26, 29 Plasminogen activation assays, eye irritation tests, 428 Plasticizers, 364 Platelet-activating factor acetylhydrolase, 707 Point of departure, 618-620, 629 Poiseuille's law, 391 Poisonings, pesticide, see Pesticide poisoning Polychlorinated biphenyls developmental neurotoxicity, 634 estrogenic effects, 447 Polydactyly, 475 Polyurethane foams, thermal decomposition products, 406 Pons, respiratory centers, organophosphateinduced effects, 391,394 Pons medulla, cholinergic innervation, 273,274 Pontomesencephalic tegmentum, innervation, 273 Porton Down, 389 Posttraumatic stress disorder, 70 Potassium ions, and organophosphateinduced cardiotoxicity, 383-386 Potentiation anticholinesterases, 574 pesticide mixtures, 607-613 Potentiometric methods, cholinesterase activities, 202 Pralidoxime, 719 adverse effects, 725 history of, 3-4 intermediate syndrome treatment, 727 mechanism of action, electrophysiological, 341 organophosphate poisoning/intoxication, 93, 395, 584-585 pharmacokinetics, 720-722 and pulmonary toxicity, 393, 394 reactivation of inhibited cholinesterases, 214,215 Prazocin, 555 Precinorm S, 204 Precinorm U, 204 Precursor ions, 685 Preemployment medical examination, 575 Pregnancy abnormal outcomes, 474-475 and butyrylcholinesterase activity, 204 chlorpyrifos exposure and, 293,604
developmental neurotoxicity guideline, 634 nicotine, developmental neurotoxicity, 302-303 and paraoxonase activity, 252-253 Presenilin 1 and 2, 29 Pressurized solvent extraction, 682 Preterm labor, pharmacotherapy and developmental neurotoxicity, 302 PRIMA, 181 Primary cultures containing glia, 331 neurotoxicity testing, 329 Primates cholinergic neurons, 271-272 plasma carboxylesterase, 263 Procaine, 187 Prochloraz, estrogenic activity, 474, 485,487 Procymidone, steroid receptor binding, 452 Produce, pesticide residues, 603 Production facility, occupational exposure, 568 Product ions, 685 Profenofos, 716 Progesterone, toxic effects of methyl isocyanate, 82 Progesterone receptor, pesticide binding, 453,484--485 Propamocarb, estrogenic activity, 485, 487 Propanil, endocrine disruption, 487 Propham, 134, 135 Propidium, cholinesterase binding site, 176 Propoxur cognition effects, 350 ectoparasiticide, 23 immune system effects, 503 metabolites, 580, 693 mutagenic potential, 544-545 neurobehavioral effects, chronic exposure, 353 Protamine sulfhydryl, 458 Protective clothing, 583,586-587 Protein kinase C and developmental chlorpyrifos exposure, 297 and heat shock proteins, 667 organophosphates and, 239, 540-541 pesticide disruption of, 453 regulation of CYP1A, 665-666 role of, 540-541 Protein kinases, OPIDN and, 363 Protein phosphatase, 39 Protein synthesis, effects of anticholinesterases on, 474 Proxyfur, intermediate syndrome, 376 pS2 expression assay, 481 Pseudocholinesterase. see Butyrylcholinesterase Psorophora, 600 Psychic state, acute pesticide poisoning effects, 573
Psychomotor function, and chronic pesticide exposure, 351,353 Psychosis, and Alzheimer's disease, 26 Public health control of vector-borne disease, 599-602 pesticide exposure, children, 602-605 Pulmonary fibrosis, 574 Pulmonary stretch receptors, 391 Pulmonary toxicity alveoli, 394 bronchi, 393-394 bronchioles, 394 exposure Ct, 392 nasal airway, 392-393 organophosphate-induced, 395 and respiratory center, 394 signs/symptoms, 392 trachea, 393-394 Pulsed flame photometric detector, 685 Punarbhaba River, 657 Pupils, soman-induced constriction, 257 Putamen, cholinergic innervation, 272 Pyrazophos, antidotal treatment, 722 Pyrethrin, dermal absorption, regional variability, 416 Pyridinium oximes, 719 Pyridostigmine analysis, 693,694 blood-brain barrier permeability and, 279 blood flow alterations, 382 and Gulf War, 69, 71 hypersensitivity to, 194-195 -induced myopathy, 512 in myasthenia gravis, 22 neurotoxicity, electrophysiological, 341 potentiation by stress, 280 prophylactic use, 3, 23, 69, 71,279-280, 395, 611 respiratory effects, 393 QNB, 258-259 QT prolongation, organophosphate-induced, 383-386 Quadriplegia, in OPIDN, 365 Quantitative structure permeability relationships, 412-413 QuEChERS method, 690 Quinalphos antidotal treatment, 723 endocrine disruption, 488, 489 male infertility and, 457 steroid receptor binding, 453 Quinapril, 219 Quinidine, butyrylcholinesterase inhibition, activity measurements, 201 Quinolinic acid, 707 Rabbits, paraoxonase activity, 249 Radial glia, 330 Radiation, developmental neurotoxicity, 634
Index
Radiometric methods, cholinesterase activities, 201 Radiotelemetry, 551 Rainwater, pesticides in, 608 Ramathibodi Poison Center, 96 Ramshorn snail, 658 Rapid-eye movement sleep, 274, 277 Rat adrenal pheochromocytoma cell line, 328 Rat brain endothelium-4 cell line, 332 Rat embryos, neurotoxicity testing, 329 Rating Scale for Geriatric Patients, 36 Rat mammary tumor model, 543-544 Rats anticholinesterases sensitivity, 148, 150, 152 inhalation toxicology, 401 neurobehavioral effects, anticholinesterase poisoning, 348-351,352-354 neuropathic response to organophosphates, 153 paraoxonase activity, 249 plasma carboxylesterase, 263 spermatogenesis, 450 toxicity testing, 457 tolerance development to carboxylesterase inhibitors, 261-262 Reactivation acetylcholinesterase, 152 and behavioral recovery, 350-351 nucleophilic agents, 3-4 cholinesterases by oximes, 214-215 time course, 200, 213 oxime-induced, 21 4-215, 716 resistance to, 216 spontaneous, 716 Reactivators, 214-215 Reactive airways dysfunction syndrome, 83 Reactive nitrogen species and excitotoxic injury, 511 male infertility and, 454-455 and pesticide-induced oxidative stress, 90 Reactive oxygen species chemiluminescence assay, 535-536 and cytochrome oxidase, 513, 515 and excitotoxic injury, 511 male fertility and, 454 organophosphate-induced, 534-535 peroxynitrite, 513 pesticide-induced, 90, 533 protein kinase C interaction, 541 role in organophosphate-induced myopathy, 512 spin trapping agents, 521 and xanthine dehydrogenase, 513, 515 Red nucleus, cholinergic innervation, 272 Reentry restrictions, pesticides, 587 Reference concentration, risk assessment, 618, 639
Reference dose nerve agents, 48, 55-57, 62 risk assessment, 618-620, 621--626, 639 WHO/FAO guidelines, 651-652 Reflexes, alterations in intermediate syndrome, 371,374 Registry of Toxic Effects of Chemical Substances, database, 147 Relative potency factor, 628-629 Relative retention times, 686 Relative standard deviation, 696 Remediation, organophosphates, in vitro models, 332-333 Reminyl, 28 Repeatability, 696 Reproducibility, 696 Reproductive toxicity assessing gonadotoxicity, 456-458 carbofuran, in fish, 659 estrogen hypothesis, 447, 448, 452-453, 458-459 male reproductive tract, 449-450 mechanisms, 451-456 methyl isocyanate, 83-84 Residue Analytical Methods, 690 Residues. see Pesticide residues Resistance development carbamates, 4 insects, acetylcholinesterase structural changes, 183 mosquito vectors, 600-602 organophosphates, 4 pesticides, cholinesterase binding sites and, 176 Resource Utilization in Dementia Scale, 38 Respirable fraction, 401 Respiration anticholinesterase intoxication, central effects, 277 cholinergic regulation of, 274 control of, 391,394 muscles of, 391 Respirators air-purifying, 583-584 atmospheric, 584 Respiratory system control of ventilation, 391 distress/paralysis, and intermediate syndrome, 371,372, 373-374 methyl isocyanate toxicity, 82, 83 organophosphate-induced failure, 394-395 physiology, 390-391 pulmonary toxicity, 392-394 Respiratory tract distribution of inhaled materials, 401-402 local pharmacological effects, 402-403 local toxicity, 403 occupational pesticide exposure, 570
759
Restricted entry interval, 587 Retention tetrapeptide, endoplasmic reticulum, 223,229 Rete testis, 449 Reticular activating system, cholinergic innervation, 273 Retina detachment of, 436 ophthalmic toxicity, 435438 Retinoic acid, 328 Reward, cholinergic modulation of, 272, 273,274 Rhabdomyolysis, anticholinesterase exposure and, 574 Rhinorrhea, 392-393 nerve agent exposure, 52-53, 58 Rice fields, pesticide-contaminated runoff, 660 Rigidity, 280 Risk assessment, see also World Health Organization/Food and Agriculture Organization acceptable daily intake, 543 cumulative common mechanism of toxicity, 620, 627, 629 cumulative assessment group, 620, 627 N-methyl carbamates, 620, 630 organophosphates, 620, 627-630 relative potency factor, 628-629 dermal absorption/toxicity, 414, 620 developmental neurotoxicity, 633-635 comparative cholinesterase data, 638-639 testing requirements, 635-638 dietary, 619, 620 gonadotoxicity, 456-458 inhalation, 620 occupational, 619 and paraoxonase status, 248 and PBPK/PD models, 630-631 pesticides, 448 pharmacokinetic approach, 630-632 pharmacokinetic modeling, 107-121 reference concentration, 639 reference dose, 639 residential, 619 single chemical, aggregate, 618-620 Risk-risk analysis, pesticide cancellation, 677-678 Rivastigmine for Alzheimer's disease, 17, 27-28, 283 analysis, 693,694 effects on tau levels, 29 mechanism of action, 28 memantine and, 40, 41-42 River blindness, 601 Ro 02-0683, and butyrylcholinesterase phenotyping, 204
760
Index
Rodents anticholinesterase-induced hypothermia, 551-559 anticholinesterase sensitivity, 148, 150, 152 anticholinesterases-induced hyperthermia, 559-560 heat loss pathways, 556-557 organophosphate binding proteins, 705 tolerance development to carboxylesterase inhibitors, 261-262 Ronnel, 23 RO number, 189 Root mean square, 174-175 Rotory Evaporator, 684 Runoff, 657, 660-661 Russia, nerve agent stockpiles, 389 Sacramento River, 660 Safety factors, data extrapolation, 647, 650 St. Louis encephalitis, 600 Saku disease, 430 Samples cleanup/purification, 682-683 concentration, 683-684 impurities separation, 684 Sampling atmosphere, 402 skin exposure, 575-576 Sarcomeres, 512 Sarin AEGLs, 58-62 analytical methods, 693-694 chemistry, 7 Ct, 392 estimated reference dose, 56-57 history, 3, 48, 389 hypothermic response to, 555 -induced myopathy, 512 inhalation toxicity, 405 long-term effects, 73-74 neuropathy induced by, 278 noncompetitive muscarinic receptor binding, 237 ocular toxicity, 433 paraoxonase hydrolysis of, 248 properties, 49-52 respiratory effects, 277, 393, 394-395 retinal effects, 436 structure of, 390 tolerance development and carboxylesterase, 261-263 toxicity and adenosine receptors, 260 delayed neuropathy, 54-55 effects, 52-55 mechanism of, 51-52 and paraoxonase status, 251 use in warfare, 3
Satin poisoning delayed hyperthermia, 561 Matsumoto incident, 49 oximes for, 724 Tokyo subway attacks, 3, 47, 49 paraoxonase status and, 251 postmortem findings, 97 Schradan, tolerance development and carboxylesterase, 261 Schrader, Gerhard, 3,389, 599, 657 Schwann cells, 316, 330 Scoline apnea, 188, 189 Scoline (Sc) variant, butyrylcholinesterase, 194 Scopolamine, blocking of hypothermic response, 554-555 Sea urchins, developmental neurotoxicity screening, 305 Seizures and anticholinesterase intoxication, 278-279, 283 anticonvulsants for, 585 organophosphate-induced, 235,662 and oxidative stress, 522 role of nitric oxide, 235,522 Semen, decreased quality, and environmental toxicants, 448 Seminiferous tubules, 449 Senile plaques, 26, 29 Sensory nerve conduction, evaluating, 581 September 11,2001 attacks, 3 Septohippocampal (Chl) pathway, 274 Serine residue acetylcholinesterase active site, 165-168, 213 cholinesterases, 162, 165-168, 213 organophosphate binding, 706 Serotonin chlorpyrifos interactions, 294, 300-301 heat loss pathways, 551 Sertoli cells, 450 Serum, organophosphate stability in, 580 Severe Impairment Battery, 36 Sexdifferentiation, developmental chlorpyrifos exposure and, 299 Sex steroid hormone-binding globulin, 452 Sheep dippers chronic pesticide exposure, neurobehavioral effects, 351-352 paraoxonase status and organophosphates exposure, 252 Signaling pathways and developmental chlorpyrifos exposure, 295,296-297, 301 pesticide disruption of, 453 Silent Spring, 681 Silicon microphysiometer, 428 Simulium damnosum, 601 Single-channel microchips, 694 Single fiber electromyography, 59 .Single laboratory validation, 695-696
Single nucleotide polymorphisms acetylcholinesterases, 181-182 butyrylcholinesterase variants, 191 paraoxonase, 248 Skeletal muscle, oxidative stress, 513 Skin acidity, 412 composition, 411 decontaminating, 717 monitoring pesticide exposure, 575-576 permeability humidity effects on, 418, 562-563 regional variation, 416 species differences, 415-416 pesticide absorption, 569-570 protection, 570, 583 sensitization, anticholinesterase-induced, 504 Sleep, cholinergic regulation of, 274 Slit-lamp biomicroscopy, eye irritation tests, 425,426 Slow-wave sleep, 274 SLUD, 354 Smoking, and developmental neurotoxicity, 302-303 Smooth muscle, vascular, effect of cholinesterase inhibitors, 381-382 SN-38, 220 Snake venom toxins, 168, 176, 182-183 S-(N-methylcarbamoyl) glutathione, 81, 83 Sodium ion, and organophosphate-induced cardiotoxicity, 383-386 Sodium salicylate, 560 Sodium thiosulfate, and Bhopal accident, 79-80, 82 Solid phase extraction, 682-683 Solid phase microextraction, 683 Solvents, extraction, 681 Soman acetylcholinesterase prophylaxis, 195 AEGLs, 59-62 aging of, 183 analytical methods, 693-694 blood-brain barrier permeability and, 279 brain slice model, 341 cardiotoxicity, 382, 383 chemistry, 7 estimated reference dose, 57 history of, 3, 48--49, 389 hypothermic response to, 555 -induced myopathy, 512 inhalation exposure, 403,404 memantine and, 40-41 miotic effect, 257 neuropathy induced by, 54-55,277-278 noncompetitive muscarinic receptor binding, 237 paraoxonase hydrolysis of, 248 pharmacokinetics, interspecies variation, 149 poisoning, oximes for, 719
Index
properties, 49-52 pyridostigmine pretreatment, 395 respiratory arrest, 394-395 respiratory effects, 277, 393 structure of, 390 tolerance development and carboxylesterase, 261-263 toxicity delayed neuropathy, 54-55 effects, 52-55 mechanism of, 51-52 Somanase activity, 263-264 Somatotrophin, 496 Sonication, 681 Soviet Union, nerve agent stockpile, 49 Spastic ataxia, 365 Spectrophotometric methods, cholinesterase activities, 201 Speech, acute pesticide poisoning effects, 573 Sperm, function, and oxidative stress, 454-456 Spermatogenesis, 450 and environmental antiandrogens, 491 parathion exposure and, 489 site of, 449 Sperm chromatin structure assay, 458 Spermination, 450 Sperm nuclear integrity assessment, 458 Spices, maximum residue limits, 651 Spinal cord, OPIDN-associated lesions, 365 Spin trapping agents, reactive oxygen species, 521 Spraying, indoor, children and, 604 Sprays drift, 568 pesticide, dermal absorption, 411 S (silent) variant, butyrylcholinesterase, 189-194, 204, 216 Status epilepticus, 585 Staurosporine, 665 Steroid hormones pesticide-induced alterations, 452 placental, 473-474 Steroidogenesis, disruption of, in vitro, 487-488 Steroid receptor, pesticide binding, 452 Stillbirths, 474 Strategic Diagnostics, 691 Stratum corneum, 411-4 13 species differences, 415-4 16 tape stripping, 414 Stress blood-brain barrier permeability and, 279-280 and membrane signaling, 665-666 and susceptibility to chemical warfare agents, 71, 73 Striatum acetylcholine, 704 cholinergic innervation, 272, 273 dopaminergic transmission, 272-273
Structure-activity relationships carbamates, 214 organophosphates, 213-214 Substantia nigra, cholinergic innervation, 272, 273 Substantia pars compacta, 272 Subthalamic nucleus, cholinergic innervation, 272, 273 Succinylcholine, 188 Succinylcholine sensitivity, and butyrylcholinesterase phenotyping, 181-182, 201,202, 216 Succinyldithiocholine, 202 Suicide by pesticide poisoning, 93,569 chronic pesticide exposure and, 352 with organophosphates, 91 Sulfation reaction, 130 Sulfones, 136 Sulfonyl fluorides, neuropathy target esterase inhibition, 362 Sulfotransferases, role of, 130 Sulfoxidation carbamate metabolism, 136-137 organophosphates metabolism, 132 Sulfoxides, 136 Sulfur compounds, as insecticides, 657 Sulfur mustard agent, 49 Sulphanilamide, acetylation of, 138 Sulphorhodamine B, 425 Supercritical fluid extraction, 682 Superoxide anion radical, 455,522 Superoxide dismutase, 456, 522 Supervised trials median residue level, 651 Surrogate skin techniques, 576 Suxamethonium, butyrylcholinesterase hydrolysis of, 187, 188 Sweating, 562-563 SY5Y human neuroblastoma cell line, 328-329, 332-333 Synapses acetylcholine accumulation, 145,209, 233 cholinergic, 275-276 transmission, effects of pesticides on, 341-344 Synaptogenesis, impaired, 328 Syncytiotrophoblasts, 463,474 Syndactyly, 84 Syrup of Ipecacuanha, 717 Tabun AEGLs, 59-62 analytical methods, 693-694 chemistry, 7 delayed neuropathy, 54-55 estimated reference dose, 57 history of, 3, 48-49, 389 -induced myopathy, 512 noncompetitive muscarinic receptor binding, 237
761
properties, 49-52 pulmonary toxicity, 277 respiratory arrest, 394-395 structure of, 390 toxicity, 278 and adenosine receptors, 260 effects, 52-55 electrophysiological mechanisms, 340 mechanism of, 51-52 Tachycardia, 703 Tacrine for Alzheimer's disease, 27, 35,283 analysis, 693,694 cholinesterase binding site, 176 combination therapy with memantine, 41-42 retinal effects, 437-438 toxicity, 17, 27 Tail, rat, skin temperature, 556-557 Taiwan, pesticide poisoning in, 95, 97 TAK-802, 22 Tamoxifen, 453 Tandem mass spectrometers, 685 Tape stripping, 414, 575 Tau protein effects of acetylcholinesterase inhibitors, 29 memantine and, 39 and OPIDN, 363 T cells, 496 Teleost fish carbofuran toxicity, 659 CYP1A in, 664-666 Temephos, mosquito resistance, 601 Temocapril, 219, 222, 227 Temperature gradient tube, 557 Temperature regulation, see Thermoregulation Teratogenesis, pesticide-induced, 474-475 Terbutaline, and developmental neurotoxicity, 302 Termiticide applicators, chronic pesticide exposure, neurobehavioral effects, 352 Terrorism, chemical, 47--48; see also Chemical warfare agents; Nerve agents Testicular cancer, 448 Testis composition, 449-450 dysfunction, and oxidative stress, 454-456 Test kits, cholinesterase activity measurements, 203 Test-mate field kit, 203 Test Methods for Evaluating Solid Wastes, 689 Testosterone effects, 449-450 effects of organophosphates on, 488-489 metabolism, pesticide inhibition of, 140 production, 449 Testosterone-estrogen-binding globulin, 452 Tetanic fade, 581 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 545
762
Index
Tetrachlorfenvinphos, 23 Tetraethyl pyrophosphate, 3 central respiratory depression, 394 extraocular effects, 438 noncompetitive muscarinic receptor binding, 236 and OPIDN syndrome, 7 structure of, 390 toxicity, 599 Tetrodotoxin, 512 Thailand, pesticide poisoning in, 95-98 Thalamus, cholinergic innervation, 273 Thermogenesis, 550 Thermoneutral zone, 550 Thermoregulation autonomic response, 556-557 behavioral response, 557 cholinergic pathways, 551 cholinesterase inhibitors and core temperature, 555-556 CNS control of, 551,554-555 developmental effects, 558 dopaminergic pathways, 551,555 exercise and heat stress, 562-563 fundamentals of, 550-551 hyperthermia, 559-562 hypothermia, 551-559 neurochemical studies, 551 noradrenergic pathways, 555 serotonergic pathways, 555 Thiamine, effect on pralidoxime, 721 Thin-layer chromatography, 692 5-Thio-2-nitrobenzoic acid, 202 Thiobarbituric acid-malondialdehyde complex, 514 Thiobarbituric acid reactive substances, 534 Thiocarbamates, 17, 138 Thiocholine, 202 Thionophosphorus organophosphate insecticides, PBPK/PD model for, 108-110 Thiram, 17 Threshold Limit Values, 399 Thymosin, 496 Thyroid gland, fish, carbofuran-induced abnormalities, 660 Thyroid hormone carbamate-related effects, 490 organophosphate-related effects, 489 Tiagabine, 726 Timolol, ocular absorption, 431 Titration methods, cholinesterase activities, 202 T-maze test, 39 TMB4, 215 Tobacco workers, organophosphate exposure, extrapyramidal effects, 280 Tokyo subway attack, 3, 47, 49 paraoxonase status and, 251 postmortem findings, 97
Tolclofosmethyl, estrogenic activity, 485 Tolerance development in acetylcholinesterase knockout mice, 260-261 and acetylcholinesterases, 263 adenosine receptors, 260 and butyrylcholinesterases, 263 carboxylesterases, 261-263 cholinesterase inhibitors, 257-264 muscarinic receptors and, 235, 257-259, 261 nicotinic receptors and, 259-260 parathion, 236, 258, 264 pathways, 257 phosphoric triester hydrolases and, 263-264 presynaptic changes, 260 Tolerance reassessment, 618 Toluene, developmental neurotoxicity, 634 Tonometry, indentation, 426 Torpedo californica cholinesterases, 3-D structure, 168, 174-181 cholinesterases, amino acid sequence, 162, 163-164 Torsade de Pointes, and hypokalemia, 384 Toxic Exposure Surveillance System, 571 Toxicity body temperature and, 558 heat stress and, 562-563 pesticide mixtures, 607-613 Toxicity studies LOAEL, 55-56 NOAEL, 55-56 WHO/FAO guidelines, 645,647 Trace analysis, 681 Trachea, 393-394 Transappendageal pathways, absorption, 411 Transcription factors, 239-240 Transport, and occupational exposures, 568 Transthyretin, 489 Trialkyl phosphates, thermal decomposition products, 406 Trialkyl phosphorothioates, and inhalation toxicity, 403 Triarylphosphates, 362, 364 Triazines, 691 Triazoles, cholinesterase inhibition, 176, 183 Triazophos, antidotal treatment, 722 S,S,S-Tributyl phosphorotrithioate, 5 S,S,S-Tributyl phosphorotrithioite, 5 Tributyrinase, 574 Trichlorfon, 23 in Alzheimer's disease, 17 anthelmintic use, 23 effect on action potential conductance, 340 intermediate syndrome, 376 mixtures, 610 Saku disease, 430 3,5,6-Trichloro-2-pyridinol, 106 in fetal/maternal samples, 604 Trichloroethane. see DDT
Trichloronat, 716 Trichloropyridinol, 117, 240 Triethyl tin, developmental neurotoxicity, 634 Trifluoroacetophenones, cholinesterase inhibition, 183 Trimedoxime, efficacy, 722 Trimethylopropane phosphate, 406, 407 Trimethyl phosphorodithioate, pulmonary toxicity, 394 Tri-o-cresyl phosphate, 7 neurotoxicity, 406, 703 neurotoxicity testing, cell culture systems, 330 OPIDN and, 316, 364-365 potentiation of malathion, 609-610, 612 potentiation of paraoxon toxicity, 148 pretreatment and tolerance development to carboxylesterase inhibitors, 262 Tri-o-tolyl phosphate, 503 Triphenyl phosphate antiandrogenic activity, 485 neurobehavioral effects, 353 thermal decomposition products, 406 Tris (1,3-dichloroisopropyl) phosphate, 7, 17 Tris (2-chloroethyl) phosphate, 7, 17 "Iris (2-chloropropyl) phosphate, 7, 17 Trophoblasts, in cell cultures, 469 Tropicamide, eye drops, 432 Trypsanomiasis, 599 Tryptophan, 707 d-Tubocurarine, 512, 514, 515 alpha-Tubulin, 363 Tumorigenesis, organophosphate modifying effects, 542-543 Tumor necrosis factor-a, 560 Turbovap, 684 TV3326, 17 T-wave abnormalities, 383-386 Twitch potentiation, 581 Typhus, louse-borne, 599 Tyrosine kinase, pesticide disruption of, 453 syn-TZ2PA6, 179 Ultrafiltration, partitioning coefficient determination, 110-111 Ultralow-volume aerial application, 600 Uncertainty factors, risk assessment, 56-57, 59, 62, 619, 621-626 Unconditioned behaviors, acute effects of anticholinesterases, 348-350 Union Carbide plant, 4, 79-85 United Kingdom, airborne exposure guidelines, 585 United States federal pesticide regulations, 617; see also Environmental Protection Agency nerve agent production, 49 pesticide poisoning in, 94, 528, 571 pesticide use, 599, 602 registered pesticides, 673,674-677
Index
U.S. Department of Agriculture, pesticide data program, 617 U.S. Environmental Protection Agency. see Environmental Protection Agency U.S. Federal Emergency Management Agency, 47, 62 U.S. Food and Drug Administration. see Food and Drug Administration U.S. Geological Survey, water monitoring, 617 Urethane carcinogenicity, 137 immunotoxicity, 498 Uridine diphosphate glucuronic acid, 129 Uridine diphosphate glucuronosyltransferase, 129, 220 Urine children, metabolites in, 603 collection, dermal absorption/toxicity, 414 metabolite analysis, 679-680, 692, 693 nerve agents analysis, 694 Urine voiding dysfunction, and acetylcholinesterase inhibitors, 22 U (usual) variant, butyrylcholinesterase, 189-194, 204, 216 Vaccines, vector-borne diseases, 601 Valproic acid, developmental neurotoxicity, 639 Vapor diffusion rate, 404 Vapor pressure cholinesterase inhibitors, 399 nerve agents, 693 Vapors, inhalation exposure, 401,404, 570 Vascular smooth muscle, effect of cholinesterase inhibitors, 381-382 Vasopressors, 717 Vector-borne disease, eradication of, 599-600; see also Public health Vegetables maximum residue limits, 650-651 pesticide residues, 603 Velnacrine, toxicity, 27 Ventilation assisted, 584, 717 control of, 391 Ventilation systems, closed facilities, 585 Ventral striatum, cholinergic innervation, 272 Ventral tegmental area, 272 Ventricular fibrillations, organophosphateinduced, 384 Vernolate, 17 Vertebrates, pesticide metabolism, 661 Vesicant (blister) agents, in unitary munitions, 49 Vestibular function, chronic pesticide exposure and, 353 Veterinary medicine, acetylcholinesterase inhibitors in, 23
Vial equilibrium method, partitioning coefficient determination, 110-111 Vinclozolin, androgen receptor binding, 452 Vineyard workers, endocrine disruption, 490 Vinyl carbamate epoxide, carcinogenicity, 137 Vision, blurred organophosphate-induced, 433 oxime-related, 725 Visual evoked response, 62 Vitamin E, pretreatment with, 525-527 Vitellogenin assay, 484 Volaton, 610 Voltage-dependent calcium channel, 329 Volume transmission, 272-273 von Krueger, Gerde, 390 VR, neuropathy induced by, 278 V series compounds, 3 VX acetylcholinesterase knockout mice and, 234 AEGLs, 59-62 analytical methods, 693-694 chemistry, 7 CNS effects on respiration, 277 dermal absorption, 563 estimated reference dose, 57 history of, 3,389 -induced myopathy, 512 muscarinic receptors, noncompetitive binding, 237 neurotoxicity testing, cell culture systems, 329 nicotinic receptors and, 260 properties, 49-52 respiratory arrest, 394-395 stereoselectivity of acetylcholinesterase toward, 210 structure of, 390 supralethal doses, 55 toxicity, 278 effects, 52-55 electrophysiological, 341,342 mechanism of, 51-52 Wasting syndrome, 533 Water dialkyl phosphates in, 692 pesticide residues, 657-658 Water samples, extraction of, 681 Western equine encephalitis, 600 West Nile virus, 600 organophosphate insecticides used for, 601 U.S emergence, 600 Wipe sampling, 575 Workers occupationally-exposed, withdrawal of, 578 protective clothing, 583, 586-587 supervision of, 583, 586
763
Workplace biomonitoring, 581-582 health care facilities, 587 NIOSH guidelines, 689 Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity, 634 A World Compendium. The Pesticide Manual, 689 World Health Organization acute pesticide poisonings, 570 eradication of malaria, 600 IPCS, 644 Neurobehavioral Core Test Battery, 282 pesticide classification, 643-644 pesticide poisoning, incidence of, 93,528 World Health Organization/Food and Agriculture Organization acceptable daily intakes, 644-645, 646-647,648-649 acute reference dose, 651-652 assessment of endpoints, 646-647 data extrapolation, 647, 650 maximum residue limit, 644, 650-651 mixtures guidelines, 651 pesticide residues, 644-645,650-651 toxicity assessment guidelines, 645-646 World War II, nerve agent stockpiles, 389 Worms, acetylcholinesterase genes, 168
Xanthine dehydrogenase, and reactive oxygen species, 513, 515 Xanthine oxidase, fasciculations-induced changes, 515 Xanthurenic acid, 707 Xenobiotic-metabolizing enzymes phase I, 127-129 phase II, 127, 129-130 placental, 465-466, 467 Xenobiotic responsive element of nucleus, 664 Xenobiotics estrogenic effects, 447 metabolism, see also Drug metabolism carboxylesterases, 219-220, 227-228, 229 in fish, 664-666 transplacental transfer, 465-474 Xenopus oocytes, 343 Y chromosome deletions, 447 Yellow fever, 599-600 YT blood group, 194 Zebra fish, 704 developmental neurotoxicity screening, 305 toxicity testing, 659 Zimbabwe, acute pesticide poisonings, 570
Index U.S. Page Department of Agriculture, pesticide This Intentionally Left Blank data program, 617 U.S. Environmental Protection Agency. see Environmental Protection Agency U.S. Federal Emergency Management Agency, 47, 62 U.S. Food and Drug Administration. see Food and Drug Administration U.S. Geological Survey, water monitoring, 617 Urethane carcinogenicity, 137 immunotoxicity, 498 Uridine diphosphate glucuronic acid, 129 Uridine diphosphate glucuronosyltransferase, 129, 220 Urine children, metabolites in, 603 collection, dermal absorption/toxicity, 414 metabolite analysis, 679-680, 692, 693 nerve agents analysis, 694 Urine voiding dysfunction, and acetylcholinesterase inhibitors, 22 U (usual) variant, butyrylcholinesterase, 189-194, 204, 216 Vaccines, vector-borne diseases, 601 Valproic acid, developmental neurotoxicity, 639 Vapor diffusion rate, 404 Vapor pressure cholinesterase inhibitors, 399 nerve agents, 693 Vapors, inhalation exposure, 401,404, 570 Vascular smooth muscle, effect of cholinesterase inhibitors, 381-382 Vasopressors, 717 Vector-borne disease, eradication of, 599-600; see also Public health Vegetables maximum residue limits, 650-651 pesticide residues, 603 Velnacrine, toxicity, 27 Ventilation assisted, 584, 717 control of, 391 Ventilation systems, closed facilities, 585 Ventral striatum, cholinergic innervation, 272 Ventral tegmental area, 272 Ventricular fibrillations, organophosphateinduced, 384 Vernolate, 17 Vertebrates, pesticide metabolism, 661 Vesicant (blister) agents, in unitary munitions, 49 Vestibular function, chronic pesticide exposure and, 353 Veterinary medicine, acetylcholinesterase inhibitors in, 23
Vial equilibrium method, partitioning coefficient determination, 110-111 Vinclozolin, androgen receptor binding, 452 Vineyard workers, endocrine disruption, 490 Vinyl carbamate epoxide, carcinogenicity, 137 Vision, blurred organophosphate-induced, 433 oxime-related, 725 Visual evoked response, 62 Vitamin E, pretreatment with, 525-527 Vitellogenin assay, 484 Volaton, 610 Voltage-dependent calcium channel, 329 Volume transmission, 272-273 von Krueger, Gerde, 390 VR, neuropathy induced by, 278 V series compounds, 3 VX acetylcholinesterase knockout mice and, 234 AEGLs, 59-62 analytical methods, 693-694 chemistry, 7 CNS effects on respiration, 277 dermal absorption, 563 estimated reference dose, 57 history of, 3,389 -induced myopathy, 512 muscarinic receptors, noncompetitive binding, 237 neurotoxicity testing, cell culture systems, 329 nicotinic receptors and, 260 properties, 49-52 respiratory arrest, 394-395 stereoselectivity of acetylcholinesterase toward, 210 structure of, 390 supralethal doses, 55 toxicity, 278 effects, 52-55 electrophysiological, 341,342 mechanism of, 51-52 Wasting syndrome, 533 Water dialkyl phosphates in, 692 pesticide residues, 657-658 Water samples, extraction of, 681 Western equine encephalitis, 600 West Nile virus, 600 organophosphate insecticides used for, 601 U.S emergence, 600 Wipe sampling, 575 Workers occupationally-exposed, withdrawal of, 578 protective clothing, 583, 586-587 supervision of, 583, 586
763
Workplace biomonitoring, 581-582 health care facilities, 587 NIOSH guidelines, 689 Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity, 634 A World Compendium. The Pesticide Manual, 689 World Health Organization acute pesticide poisonings, 570 eradication of malaria, 600 IPCS, 644 Neurobehavioral Core Test Battery, 282 pesticide classification, 643-644 pesticide poisoning, incidence of, 93,528 World Health Organization/Food and Agriculture Organization acceptable daily intakes, 644-645, 646-647,648-649 acute reference dose, 651-652 assessment of endpoints, 646-647 data extrapolation, 647, 650 maximum residue limit, 644, 650-651 mixtures guidelines, 651 pesticide residues, 644-645,650-651 toxicity assessment guidelines, 645-646 World War II, nerve agent stockpiles, 389 Worms, acetylcholinesterase genes, 168
Xanthine dehydrogenase, and reactive oxygen species, 513, 515 Xanthine oxidase, fasciculations-induced changes, 515 Xanthurenic acid, 707 Xenobiotic-metabolizing enzymes phase I, 127-129 phase II, 127, 129-130 placental, 465-466, 467 Xenobiotic responsive element of nucleus, 664 Xenobiotics estrogenic effects, 447 metabolism, see also Drug metabolism carboxylesterases, 219-220, 227-228, 229 in fish, 664-666 transplacental transfer, 465-474 Xenopus oocytes, 343 Y chromosome deletions, 447 Yellow fever, 599-600 YT blood group, 194 Zebra fish, 704 developmental neurotoxicity screening, 305 toxicity testing, 659 Zimbabwe, acute pesticide poisonings, 570
