Advances in Environmental Control Technology: Health and Toxicology (Advances in Environmental Control Technology)

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Author: Paul Cheremisinoff

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HEALTH and TOXICOLOGY

m/«A^

Advances in Environmental Control Technology Series

ENCYCLOPEDIA Ol^ ENVIRONMENTAL CONTROL TECHNOLOGY Volume 1: Thermal Treatment of Hazardous Wastes Volume 2: Air Pollution Control Volume 3: Wastewater Treatment Technology Volume 4: Hazardous Waste Containment and Treatment Volume 5: Waste Minimization and Recycling Volume 6: Pollution Reduction and Contaminant Control Volume 7: High-Hazard Pollutants Volume 8: Work Area Hazards Volume 9: Geotechnical Applications • Leak Detection • Treatment Options

ADVANCES IN ENVIRONMENTAL CONTROL TECHNOLOGY Storage Tanks Ecological Issues and Environmental Impact Assessment Health and Toxicology

Gulf Publishing Company Houston, Texas



Paul N. Cheremisinoff, Editor Nicholas P. Cheremisinoff, Associate Editor in collaboration M. Bacis P. Beggs F. Y. Bois M. Charretton P. Chiang B. Cole P. Curson P. Dingle J. W. Dobrowolski J. L. Domingo T. Driscoll

I. Ebihara L. Gerhardsson L. Giusti M. Hirata N. Hisanaga P. Hrelia J. Indulski Y. Ishikawa A. Karjalainen A. Leclerc P. Leghissa

with—

J. Leigh K. P. Li D. Luce W. Lutz M. A. McGeehin G. Mosconi A. Oskarsson J. S. Reif K. Sakai C. Sala E. Shibata

D.T.H.M. Sijm A. Sisko S. Skerfving R. Song M. A. Subramanian J. Tolls A. Trevisan G. Varadaraj R. Vincent S. C. Yang S. P. Yang



Copyright © 1997 by Gulf Publishing Company, Houston, Texas. All rights reserved. Printed in the United States of America. This book, or parts thereof, may not be reproduced in any form without permission of the publisher. Gulf Publishing Company Book Division P.O. Box 2608 D Houston, Texas 77252-2608 10

9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data

Health and toxicology / Paul N. Cheremisinoff, editor ; in collaboration with M. Bacis . . . [et al.]. V. < > ; p. cm. — (Advances in environmental control technology) Includes bibliographical references and index. ISBN 0-88415-386-X (alk. paper) 1. Environmental toxicology. I. Cheremisinoff, Paul N. II. Bacis, M. III. Series. RA1226.H43 1997 615.9'02—dc21 96-40506 CIP ISBN 0-87201-238-7 (Series) Printed on Acid-Free Paper (oo) IV

CONTENTS CONTRIBUTORS TO THIS VOLUME

vii

(For a note about the editor, please see page x) PREFACE 1. Environmental Risk Factors on Cancer and Their Primary Prevention I W. Dobrowolski 2. Respiratory Function Changes from Inhalation of Polluted Air Shieh-Ching Yang and Sze-Piao Yang

xi 1 47

3. Biological Markers of Early Health Effects in the Assessment of the Risk of Cancer in People Exposed to Environmental Carcinogens .. 53 JanuszA, Indulski and Waldemar Lutz 4. Sick Building Syndrome Peter Dingle

67

5. Respiratory Findings of Construction Workers Exposed to Asbestos Dust 93 Isamu Ebihara, Mamoru Hirata, Naomi Hisanaga, Eiji Shibata and Kiyoshi Sakai 6. Asbestos Exposure and the Risk of Lung Cancer in Urban Populations Anti Karjalainen andAnttila Sisko

127

7. Emphysema and Lung Mineral Content in Coalworkers J. Leigh, T. Driscoll, and B. Cole

137

8. Sinonasal Cancer and Wood Dust Exposure A. Leclerc and D. Luce

171

9. Chromium Accumulation of Chromate Workers' Bronchi Yuichi Ishikawa and Eiju Tsuchiya

191

10. Benzene Toxicokinetics in Humans Frederic Yves Bois 11. Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons in Biological Systems Kuang-Pang Li, Ping Chiang, and Raxia Song

207

219

12. Occupational Exposure to Organic Solvents During Paint Stripping and Painting Marc Charretton and Raymond Vincent

251

13. Occupational Exposure to Metallic Cobalt G. Mosconi, M. Bacis, P. Leghissa, and C. Sala

307

14. Climate and Chronic Respiratory Disease Paul Beggs and Peter Curson

329

15. Effects of Acid Precipitation on the Environment and on Human Health Lars Gerhardsson, Staff an Skerfving andAgneta Oskarsson

355

16. Toxic Effect of Tannery Effluent on the Biochemical Constituents in Organisms G. Varadaraj and M. A. Subramanian

365

17. Bladder Cancer and Water Disinfection Methods Michael A. McGeehin, John S. Reif

377

18. Metal-induced Development Toxicity in Mammals Jose L. Domingo

395

19. Genetic Evaluation of Pesticides in Different Short-Term Tests Patrizia Hrelia

415

20. Pesticide Residues in Food Peter Dingle

433

21. Biological Monitoring by Means of Urinary Samples and Problems Concerning Concentration-Dilution of Spot Urine 453 Andrea Trevisan 22. Causes of Artifacts in Sorption Studies with Trace Elements Lorenzino Giusti

461

23. Bioaccumulation of Surfactants J Tolls and D. T H. M. Sijm

493

INDEX

501

VI

CONTRIBUTORS TO THIS VOLUME M. BACIS, Department of Occupational Medicine, Ospedali Riuniti Bergamo, Bergamo, Italy PAUL BEGGS, Climate Impacts Centre, School of Earth Sciences, Macquarie University, New South Wales 2109, AustraUa FREDERIC YVES BOIS, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 MARC CHARRETTON, Caisse Regionale d'Assurance Maladie Rhone-Alpes, Laboratoire de Chimie du Val Rosay, F-69370 St Didier au Mont d'Or, France PING CHIANG, Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854 B. COLE, Worksafe Australia, National Occupational Health and Safety Commission, GPO Box 58, Sydney, New South Wales 2001, Australia PETER CURSON, Climate Impacts Centre, School of Earth Sciences, Macquarie University, New South Wales 2109, AustraUa PETER DINGLE, Murdoch University, Murdoch, Western Australia 6150 J. W. DOBROWOLSKI, Committee for Prot. Public Health, Inst. Manage, and Prot. Environ., PoHsh Acad. Sci., Mickiewicza 30, Paw, C 4, Pok, 117 30 059 Krakow, Poland JOSE L. DOMINGO, School of Medicine, Rovira i VirgiU University, 43201 Reus, Spain T. DRISCOLL, Worksafe Australia, National Occupational Health and Safety Commission, GPO Box 58, Sydney, New South Wales 2001, AustraUa IS AMU EBIHARA, The Institute of Science of Labour, Kawasaki, Japan LARS GERHARDSSON, Department of Environmental and Occupational Medicine, Lund University Hospital, S-221 85 Lund, Sweden LORENZINO GIUSTI, School of the Environment, Sunderland University, Sunderland, SR2 7BW, UK MAMORU HIRATA, Osaka Prefectural Institute of Public Health, Osaka, Japan NAOMI HISANAGA, National Institute of Industrial Health, Kawasaki, Japan PATRIZIA HRELIA, Department of Pharmacology, University of Bologna, Bologna, Italy

YUICHI ISHIKAWA, The Cancer Institute, Department of Pathology, Toshimaku, Tokyo, Japan JANUSZ A. INDULSKI, The Nofer Institute of Occupational Medicine, Lodz, Poland ANTI KARJALAINEN, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, Helsinki, Finland A. LECLERC, Unite 88 INSERM-HNSM, 14 rue du Val d'Osne, 94410 SaintMaurice, France P. LEGHISSA, Department of Occupational Medicine, Ospedali Riuniti Bergamo, Bergamo, Italy J. LEIGH, Worksafe Australia, National Occupational Health and Safety Commission, GPO Box 58, Sydney, New South Wales 2001, Austraha WALDEMAR LUTZ, The Nofer Institute of Occupational Medicine, Lodz, Poland KUANG-PANG LI, Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854 D. LUCE, Unite 88 INSERM-HNSM, 14 rue du Val d'Osne, 94410 Saint-Maurice, France MICHAEL A. MCGEEHIN, Centers for Disease Control, Atianta, GA 30341-3724 G. MOSCONI, Department of Occupational Medicine, OspedaU Riuniti Bergamo, Bergamo, Italy AGNETA OSKARSSON, Department of Food Hygiene, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden JOHN S. REIF, Colorado State University, Boulder, CO KIYOSHI SAKAI, Nagoya City Public Health Research Institute, Nagoya, Japan C. SALA, Department of Safety and Hygiene, Chemical Division USL 16, Lecco, Italy FIJI SHIBATA, Nagoya University School of Medicine, Nagoya, Japan D.T.H.M. SUM, Environmental Chemistry Group, Research Institute of Toxicology, NL-3508 TB Utrecht, The Netherlands ANTTILA SISKO, Finnish Institute of Occupational Health, TopeHuksenkatu 41 aA, Helsinki, Finland STAFFAN SKERFVING, Department of Environmental and Occupational Medicine, Lund University Hospital, S-221 85, Lund, Sweden

RAXIA SONG, Department of Physics, Beijing University, Beijing, People's Republic of China M. A. SUBRAMANIAN, P.G. & Research, Department of Zoology, Chikkaiah Naicker College, Tamil Nadu, India J. TOLLS, Environmental Chemistry Group, Research Institute of Toxicology, NL3508 TB Utrecht, The Netherlands ANDREA TREVISAN, Istituto di Medicina del Lavoro, Universita di Padova, Padova, Italy G. VARADARAJ, P.G. & Research, Department of Zoology, Chikkaiah Naicker College, Tamil Nadu, India RAYMOND VINCENT, INRS, Institut National de Recherche et de Securite, Service Evaluation et Prevention du Risque Chimique, BP 27, F-54501 Vanoeuvre Cedex, France SHIEH-CHING YANG, National Taiwan University Hospital, No. 7, Chung-Shan S. Rd., Taipei, Taiwan, Republic of China SZE-PIAO YANG, National Taiwan University Hospital, No. 7, Chung-Shan S. Rd., Taipei, Taiwan, Republic of China

IX

ABOUT THE EDITOR The late Paul N. Cheremisinoff, P.E., was a professor of civil and environmental engineering at the New Jersey Institute of Technology. Professor Cheremisinoff had more than 40 years of experience in research, design, and consulting for a wide range of government and industrial organizations. He was author and co-author of numerous papers and books on energy, resources, and the environment, and was a licensed professional engineer. He was a member of Sigma Xi and Tau Beta Pi, and a Diplomate of the American Academy of Environmental Engineers.

PREFACE This volume, entitled Health and Toxicology, contains twenty-three chapters prepared by an international group of scientists and engineers. The volume is designed to provide an extensive overview and reference on human physiological responses to various forms of pollution. Extensive discussions can be found on asbestos, carcinogenic and mutagenic poisons, poison inhalation hazards, and a wide range of industrial poisons. The volume is not organized as a collection of clinical data and information, but rather as a general working reference for engineers, scientists, industrial hygienists, and toxicologists, as well as environmental health and safety managers. With the advent of ISO 14000 and EMAS standards, the industrial community is taking on a more aggressive role in dealing with environmental and health and safety issues, fueled by the concept of Responsible Care. Industry managers and technologists must be technically versed in the health-care issues associated with industry pollution and human exposure. The information presented in this volume has been compiled and presented by many experts. These individuals should be congratulated for their efforts. A heartfelt thanks is also extended to Gulf Publishing Company for their fine production of this series. During the course of the production of the volume Ecological Planning Issues and Health and Toxicology, the editor, Paul N. Cheremisinoff, P.E., passed away. It was his vision and diligence that made this important series a reality. This volume represents in many ways an epitaph to a respected scientist and engineer who devoted his career to environmental problems. Nicholas P. Cheremisinoff, Ph.D. Associate Editor

This Page Intentionally Left Blank

CHAPTER 1 ENVIRONMENTAL RISK FACTORS ON CANCER AND THEIR PRIMARY PREVENTION J. W. Dobrowolski Committee for Prof. Public Health Inst. Manage, and Prof. Environ. Polish Acad. Sci. Mickiewicza 30, Paw C 4, Pok, 117 30 059 Krakow, Poland

CONTENTS LASER MICROANALYSIS AS A NEW TOOL FOR PRIMARY PREVENTION, 11 ELECTROCHEMICAL POTENTIAL AND BIOLUMINESCENCE FOR DIAGNOSIS, 12 CHEMILUMINESCENCE AND PHOTON EMISSION AS METHODS FOR EARLY DETECTION OF CANCER RISK, 15 PHAGOCYTIC ACTIVITY, 16 SMOKING, 17 AIR POLLUTION AND OTHER RISK FACTORS, 17 SEAFOOD, 18 MERCURY, 18 SALTS AND MINERALS, 22 CONCLUSIONS, 23 Short Review of the Environmental Risk Factors of Cancer Incidence, 24 Emotional Stress as a Risk Factor, 29 STAGES OF CARCINOGENSIS, 36 APPENDIX 1: CANCER RISK FACTORS AND PREVENTION, 40 APPENDIX 2: FIGURES, 42 REFERENCES, 44 To evaluate the influence of the quality of human environment on public health (including cancer incidence), it is imperative to perform interdisciplinary case studies involving epidemiological surveys supported by the application of new, highly sensitive methods of multi-factorial analyses of the natural environment, including the food chain. Both field and experimental studies are required for better understanding of the long-term effects of low concentrations of various pollutants, including their synergistic effects. Ecotoxicology and medical elementology (knowledge about the role of elements in health and diseases) are new and very promising sciences for the environmentally oriented prevention of diseases. With this background, special attention should be paid to the complex studies, carried out

2

Health and Toxicology

in recent years, on environmental deterioration and risk factors for the incidence of cancer, congenital malformations, and so-called "diseases of civilization," which are incurable at present. According to experts of the WHO/IARCAVEEC, over 80% of proliferative diseases are caused by environmental factors. There are two basic fields of study. The first one deals with the assessment of health hazards of long-term employment under the influence of pathogenic factors. One of the case studies in this area is team research carried out by Samal et al., who evolved laboratory tests for determining the relation between the time spent while working under hazardous conditions (with a minimal radiation load) and an increase in the number of thrombocytes in the whole blood accompanied with an increase in the activity of serum lactate dehydrogenase. The second field of study is connected with the relationship between the geographic pattern of the incidence of some selected diseases—e.g., cancer and the quality of the local living environment. It is necessary to take into consideration both geochemical and other natural human-made predisposing factors. Geochemical Environment and Disease Patterns is useful for the interpretation of spatial differentiation of the rate of cancer incidence and for nutritional prevention of neoplastic diseases. Epidemiological and ecological investigations are related to the areas under comparison, justifying their description in quantitative terms. Jansson applied the Monte Carlo technique for verification of geographical clusters characterized by increased mortality rates in cases of all selected malignant neoplasms. He defined "cluster" as an area consisting of one or more subareas, in which it is possible to move from every point to any other point without leaving the area. Jansson considered this method to be effective for the evaluation of spatial clustering of various types of cancer. Besides, it is an objective way to confirm or reject subjective assumptions of the significance of higher incidence of these diseases in a particular area. Following the principles of ecotoxicology, model studies were conducted with particular attention to the analysis of elements in a trophic chain (soil, water, plants, animal feed, cattle blood, cow milk, and human blood) in various regions of Poland, taking into consideration control areas comparable with "clusters of neoplasmatic diseases" with respect to similar climatic and other natural conditions [3, 6, 5]. This can be illustrated with results of an interdisciplinary pilot project from a control village-R and a neighboring administrative unit in rural region village-P as a "cluster" with high incidence of various types of human neoplasms and lymphatic leukemia in cattle. Different analytical methods (e.g., AAS, NA, PIXE) were used in this study. By application of the scanning electron microscope and electron microbeam, the quantity of oligo and trace elements was measured in various fractions of human and animal blood cells. Significant differences were observed in the levels of some elements. The model study showed the influence of an excess or a deficiency of these elements in soil, water, cultivated plants, animals, and human food on the elements' concentration in the human body. Some of the results are shown in Table 1.

Environmental Risk Factors on Cancer and Their Primary Prevention

3

It will be seen that: (a) Greatest differences were found in relation to the levels of Mg in drinking water and Co in cow milk; the values were significantly lower in cluster areas. (b) Elevated amounts of Zn, Pb, Cu, Mn, Ni, Hg, Br, and Rb were detected in the natural environment and local food in cluster areas. The most distinct differences were noted in the concentration of elements in bran. The latter can, therefore, be recommended as a good indicator of disturbances in elemental levels of the trophic chain. (c) Mean amounts of Mg were found to be lower and that of Zn higher in both cattle and human cells (lymphocytes and erythrocytes) in the cluster areas. According to some studies about regional differences in biogeochemical properties and patterns of distribution of neoplastic and cardio-vascular diseases related to some elements, the amount of water soluble oligo and trace elements is of special importance. Increasing numbers of scientific reports are now supporting previous concepts about the health implications of environmental geochemistry [6]. Haviland, as early as 1869, reported a higher incidence of tumors in magnesium-deficient regions of France [1]. Many studies conducted in various parts of the world indicate a correlation between geochemical environment and the frequency of neoplasms, sclerosis, myocardial infarction, hypertension, apoplexy, urolithiasis, and some other diseases. From a practical point of view and for prophylactic purposes, studies about the concentration of elements in drinking water in relation to the health status, seem to be particularly interesting. Progress of knowledge about the biological function of trace and ultra trace elements is followed by research developing studies on nutritional prevention of some kinds of carcinoma. The role of selenium in primary prevention of cancer and leukemias is of special importance in this respect [38]. A reverse correlation between selenium intake and the incidence of cancer and leukemias is well documented [1, 6, 38]. Against this background, it is interesting to note that in "clusters" of human cancers and leukemia enzootica, the levels of selenium in drinking water were found to be below 0.000 ppm. A protective effect of sub-toxic doses of selenium administered in drinking water has been reported [6]. This author did not find any increase in the levels of selenium in lymphocytes (deficient in this element) of leukemic patients even after administration of 300g of sodium selenate daily for 10 days [6]. It is well known that selenium has preventive properties against neoplasms in early stages of carcinogenesis. The element acts not only as an anti-oxidant and a free radical scavenger, but also as an immunostimulative factor. This is indicated by increased titer values of antibodies against some bacteria and toxicogenic mold antigens in response to supplementation of experimental diet with sodium selenate [1]. Radio-protective properties of selenium were discovered by Badilo and co-workers in organic selenium compounds, and by Aleksandrowicz and CzyzewskaWazewska in inorganic selenium compounds. These facts may be of special interest

Health and Toxicology Table 1 Comparative epidemiological and elementological studies in control and cluster areas [4]

Study Number 1 1. 2.

3.

4.

5.

Parameters of study II Mean index for the incidence of lymphatic leukemia in cattle (per 100,000 animals) Drinking water: 2.1. Magnesium (ppm) 2.2. Manganese (ppm) 2.3. Iron (ppm) 2.4. Copper (ppm) 2.5. Nickel (ppm) 2.6. Lead (ppm) 2.7. Zinc (ppm) 2.8. Rubidium (ppm) 2.9. Strontium (ppm) 2.10. Nitrite (mgN/I) 2.11. Nitrate (mgN/I) Soil: 3.1. Manganese (ppm) 3.2. Iron (ppm) 3.3. Copper (ppm) 3.4. Nickel (ppm) 3.5. Lead (ppm) 3.6. Zinc (ppm) 3.7. Rubidium (ppm) 3.8. Zirconium (ppm) 3.9. Ytterium (ppm) 2.10. Nitrite (mgN/I) Cattle feed: 4.1. Manganese (ppm) 4.2. Iron (ppm) 4.3. Copper (ppm) 4.4. Nickel (ppm) 4.5. Lead (ppm) 4.6. Zinc (ppm) Cow milk: 5.1. Manganese (ppm) 5.2. Iron (ppm) 5.3. Copper (ppm) 5.4. Nickel (ppm) 5.5. Lead (ppm)

Control area village-R III

43.5 46.50 0.06 20.28 0.26 0.02 0.03 0.71 0.02 0.47 N.D. 0.04 204.34 193,019.21 11.76 3.16 23.90 29.57 94.91 N.D. 161.60 2,326.47 2.46 0.37 0.50 132.43 0.157 2.522 0.552 0.045 0.112

Cluster area village-R IV

781.3 6.90 0.08 0.17 0.32 0.12 0.21 0.90 0.03 0.51 0.0005 0.76 502.96 14,310.04 16.55 15.27 43.22 42.89 67.92 198.29 16.93 760.57 103.53 12.89 5.96 8.58 ' 202.97 0.065 1.882 1.893 0.205 0.762


6.

7.

8.

9.

10.

5.6. Zinc (ppm) 5.7. Selenium (ppm) 5.8. Rubidium (ppm) 5.9. Strontium (ppm) Cattle blood: 6.1. Manganese (ppm) 6.2. Iron (ppm) 6.3. Copper (ppm) 6.4. Nickel (ppm) 6.5. Lead (ppm) 6.6. Zinc (ppm) 6.7. Rubidium (ppm) 6.8. Selenium (ppm) 6.9. Strontium (ppm) Catde lymphocytes: 7.1. Magnesium (wt%) 7.2. Calcium (wt%) 7.3. Zinc (wt%) Human blood: 8.1. Manganese (ppm) 8.2. Iron (ppm) 8.3. Cobalt (ppm) 8.4. Copper (ppm) 8.5. Nickel (ppm) 8.6. Zinc (ppm) 8.7. Selenium (ppm) 8.8. Rubidium (ppm) 8.9. Strontium (ppm) Human lymphocytes: 9.1. Magnesium (wt%) 9.2. Calcium (wt%) 9.3. Zinc (wt%) Human erythrocytes: 10.1. Magnesium (wt%) 10.2. Calcium (wt%) 10.3. Zinc (wt%)

5.655 0.020 1.013 1.673

8.055 N.D. 1.680 1.003

1.150 709.000 1.500 0.103 0.318 4.600 0.600 0.107 0.310

2.983 606.667 3.483 0.653 2.383 14.817 0.800 N.D. 0.803

0.295 0.860 0.197

01168 0.445 0.265

0.790 319.700 0.063 1.113 0.060 2.860 0.147 0.857 0.360

1.000 210.233 N.D. 2.317 0.083 3.393 N.D. 0.230 0.430

0.270 0.827 0.110

0.220 0.393 0.180

0.280 0.843 0.137

0.207 0.627 0.203

Figures represent mean values for the elements estimated by various methods of analyses (including X-ray microanalyses in dry blood cells). Note different ratios of antagonistic elements. N.D. = non-detectable.

because ionizing radiation is recognized as a well-known leukosogen and carcinogen. Selenium is a non-specific protective agent involving inhibitory effects against di-methyl-benzene-anthracene (DMBA)-induced mammary tumorigenesis, as well as growth of transplanted leukemic cells. A negative correlation has been observed between the level of selenium in human blood and cancer mortality in various populations. This kind of reverse cor-

6


relation is particularly seen in epidemiological data about leukemia, cancers of breast, anus, kidneys, prostate gland, pancreas, and lungs [38]. It is significant to note that in the USA and New Zealand, the mean values for human blood Se levels ranged between 0.08 to 0.2 ppm, and at the same time a high incidence of leukemia (7.0 and 6.6 per 100,000) with a very high mortality from breast cancer (23.3 and 16.9 per 100,000) was reported among the inhabitants of these countries. In countries having soil rich in selenium (e.g., Venezuela), elevated levels of this element were reported in human blood (0.81 ppm) with a low incidence of leukemia (2.8 per 100,000) and low mortality rate from breast cancers (2.6 per 100,000) [38]. Excellent reviews about links between selenium and cancer, and about the relation of this element to general health are available. According to these reports, decreased concentrations of selenium were found in the blood of almost all investigated cancer patients. Further selenopurines were shown to be highly effective for treating lymphomas. Shamburger discovered that anti-oxidants (e.g., sodium selenite, vitamin E, and vitamin C) inhibit DMBA-phorbital and DMBA-croton oilinduced papillomas in mice. Protective effects of selenium in relation to butter-yellow carcinogens (liver cancer caused by N9-methyl-p-dimethyl-aminobenzene) were also found. Similar results were obtained with respect to the relation between nutritional intake of selenium and prevention of liver cancer. Some mycotoxins (e.g., aflatoxins) are well-known hepatocarcinogens. In many regions of the world characterized by seasonal humidity, toxicogenic molds and intoxication of food with mycotoxins are very common. Therefore, it may be interesting to note that statistically significant differences were observed in the degree of contamination with toxicogenic molds (e.g., Aspergillus flavus and Penicillum meleagrinum) in the living rooms of people suffering from leukemias and those of other patients. These workers had much higher titer of antibodies against A. flavus antigen in peripheral blood of leukemic patients (suffering from chronic lymphatic leukemia, granulocytic leukemia, and acute leukemia) as compared to the values among their healthy relations. In some clusters of human cancer and bovine leukemia, a higher occurrence of the above-mentioned molds was reported in houses where leukemic patients lived and in "leukemic" breeding centers of cattle, compared with the houses of a control group. A similar correlation was later reported in a "leukemic cluster" of children in the USA. Some recent studies may be helpful in decreasing the risk of cancer due to indoor pollution with the toxicogenic molds and other pathogenic microorganisms. Good mycostatic and bacteriostatic effects were observed by fortification of paint with 150^/g sodium selenate or even 15mg/g sodium selenite. Such additives of the emulsion paint, covering rearing boxes, did not elicit any symptoms of chronic toxicity in mice, rats, or hamsters. Protective effects of selenium were also detected against the effects of in vitro cultures of human lymphocytes contaminated with mycotoxins. The aflatoxin Bl (0.5g/ml culture medium) decreased transformation of phytohaemoaglutine (PHA)-treated lymphocytes to 71.4% [6].


7

Selenium-caused neutralization of the inhibitory effect of this cancerogenic mycotoxin on blastic transformation is one more proof of the protective effects of Se on the immunological system. Our studies reveal neutralization of aflatoxin B1 (5g/ml) by sodium selenate (the same concentration) in respect to its embryotoxic and teratogenic effects. The element protects lysosomal enzymes of granulocytes against benzeneinduced inhibition of their activity [1]. Supplementation of diet with 0.2, 0.5, 1.0, and 2.0 ppm of Se proportionally reduced the incidence of DMBA-induced breast cancer by 44, 70, 76, and 85%, respectively. The protective effect was observed only during the period of initiation of mammary tumorigenesis by DMBA [38, 39]. Basic mechanisms involved in protective effects of selenium against chemical carcinogens seem to be: (a) competition with these carcinogens for protein binding sites, (b) enhancement of the immune response, and (c) normalization of DNA activity in neoplastic cells. Experimental data thus support a working hypothesis about an inverse relationship between the geographic pattern of neoplasms and regional deficiencies of selenium in soil, water, and food. Antagonistic elements give a real chance for the nutritional prevention of cancer. In a study about the ecotoxicology of cancer/leukemic clusters, pollution of the trophic chain with mercury was detected [6]. The protective effect of selenium against methyl mercury toxicity is well documented [38]. Coincidence of pollution of the natural and living environment with toxicogenic molds and mercury in feed, combined with the deficiency of selenium in human and animal food, may markedly increase the risk factor connected with these pathogens. Proper proportion between antagonistic elements is also very important [6]. In areas described as neoplastic clusters, this proportion was quite different from that in control regions (e.g., a deficiency of selenium and iron and the excess of zinc). Neutron activation analysis indicated a decreasing concentration of rubidium with an increased concentration of selenium in experimental mice, especially under acute intoxication with selenium. Many studies indicate a strong chemical competition between selenium and zinc [38]. Disturbance of the optimal ratio of antagonistic elements in the diet can aggravate the biological effects of a deficiency of essential trace elements. This aspect should be taken into consideration, but usually it is overlooked. Some reports give estimates of daily selenium intakes by US adults to be 60-160g. These data are not accompanied by information about average intakes of antagonistic elements (e.g., Zn, As). A similar situation exists in relation to the recommended daily intake of selenium in different age groups (Table 2). Ratios between selenium and other elements are not included in the assessment of human exposure to inhalatory and ingestion pathways of environmental seleni-

8

Health and Toxicology Table 2 Recommended daily intake of selenium after Schrauzer, 1978 [6, 38] Age (years)

RDA of Se (g)

0.0-0.5 0.5-1.0 1.0-3.0 4.0-6.0 7.0-adult

10-40 20-60 20-80 30-120 50-200

.

um [1, 6, 38]. State-of-the-art studies on ecological prophylaxis recommend increased intake of dietary selenium in many regions of the world. Nutritional risk factors involved in this respect are connected not only with the natural geochemical, climatic, and other conditions, but also with contemporary agriculture, food industry, and some new trends in nutrition and food habits (e.g., higher consumption of fat, meat, and purified sugar, and lower consumption of seafood, fiber cereals, and other traditional food in modem industrialized societies). Computerized Pattern Recognition is a new tool in cancer study and nutritional prevention in relation to geochemistry and food. Because of the multifactorial etiology of many diseases, including cancer, application of computerized pattern recognition seems to be very promising for the future [6]. This new method was used for studying changes in elements, antagonistic to selenium, for the detection of lung cancer. Based on the results of the analysis of the hair samples, it has been concluded that there were three distinguishable areas corresponding to elemental patterns characteristic for healthy people and groups with early lung cancer. The rate of accuracy of prediction of the early lung cancer was estimated as 86%. Such studies are of special importance in working environments with high health hazards. There are new perspectives of the personal approach to prevention based on elemental Analysis in Normal and Pathological Cells by the application of X-Ray microanalysis under SEM. Some other methods may also be useful for early recognition of risk factors. Observation of human blood cells under scanning microscopy connected with an X-ray microanalyzer made possible comparative studies on the quantity of elements present within the normal and pathological cells. This new technique was developed and patented by the Polish Academy of Science under Polish patent number 199102 [6]. A modification of the method relating to the preparation of blood cells for measurement of oligo and trace elements was recently introduced. The method of X-ray microanalysis—for elements from atomic number 4 (beryllium) to atomic number 92 (uranium)—has relative detectability of elements to be as high as 10-14 to 10-15 (higher detectability is for heavier elements). Application of this method of cell monitoring revealed significant changes in the amount of some elements in blood cells of leukemic patients (Table 3).


9

Table 3 Elemental changes in lymphocytes of leukemic patients after Dobrowolski et al. 1989,1990 [6] Elemental Content (wt %) Group

Mg

Zn

Ca

Healthy subjects Patients with chronic lymphatic leukemia Patients with acute lymphatic leukemia

0.23 0.94 0.20

0.13 0.02 0.10

0.91 0.30 1.55

Table 3 shows that the level of magnesium is increased in human blood cells in patients suffering from chronic lymphatic leukemia (CLL) with concurrent decrease in levels of zinc and calcium in these cells. Not only was the absolute amount of these elements drastically changed, but also the ratio between calcium and magnesium was recognized as very symptomatic for neoplastic diseases. This was confirmed by our investigations. Application of scanning microscopy coupled with energy dispersive X-ray microanalysis (SEMQ) has permitted detection of alterations in Mg/Ca ratios in groups exhibiting increased incidences of sudden coronary death syndrome, cardiac arrhythmias, and other chronic diseases. Under SEMQ, it became possible to discover crystal-like structures (corresponding to the diameter of red cells) observed only in peripheral blood of leukemic patients, especially those suffering from acute leukemia [6]. They are characterized by a high amount of sulphur and a very high concentration of calcium (more than 20%). Coincidence was observed between the degree of pathological changes in erythrocytes and an increasing level of calcium. Hypercalcaemia is well known in cancer and leukemic patients as evidenced from the findings of conventional analyses of blood samples. This study provided a proof of correlation between morphological and chemical alterations. A significantly elevated content of iron was detected by the same method in lympho-reticular cells of one patient with sideroblastic anemia. It was retrospectively analyzed as a preleukemic case [6]. Correlation between some morphological differences of B cells, T cells, and intermediate forms in peripheral blood of newborn children and phosphate marker of T cells was reported. Age-dependent changes in the concentration of nine elements, particularly in blood cells of premature and mature newborn children, were also reported. Therefore, it is absolutely essential that the groups of healthy subjects and patients under comparison are of similar age. Quantitative investigations (using standard cytochemical techniques including radionucleides in autoradiography) for some essential elements in blood cells (e.g., lymphocytes) and activity of selected enzymes, which are activated by these elements, may be very promising in the future. Another interesting use of the method of cell monitoring of these elements is that it gives an idea about the biogeochemical and ecotoxicological back-

10


ground. A correlation between the concentration of selected elements in the natural environment (and the trophic chain in particular) on their content in the blood cells of healthy persons and cattle was described. Such correlation was not found in neoplastic (e.g., leukemic) cattle. Differences between the amounts of some selected elements in the blood cells of healthy and leukemic cattle living in the same living center were found to be higher than those between normal animals from different regions. The average content of magnesium in cattle lymphocytes was 0.29 wt % in a magnesium-rich area. The values in a low magnesium area were 0.16 wt % in healthy cattle and 0.37 wt % in leukemic stock [6]. Similar changes were found in relation to calcium, viz: in the region having Ca-rich soil, mean Ca content in cattle lymphocytes was 0.86 wt %. In low Ca areas, the corresponding mean values were found to be 0.45 wt % in healthy catde and 0.66 wt % in leukemic animals. Significantly increased amounts of magnesium in lymphatic cells of both humans and livestock suffering from chronic lymphatic leukemia seems to be rather surprising because epidemiological data indicate a tendency for a higher incidence of leukemia in regions having lower concentrations of magnesium in soil. Such correlation is particularly evident in cases of lymphatic leukemia in cattle as indicated by Karaczkiewicz's study in Poland. Deficiency of magnesium in animal feed leads to hypomagnesemia and increased morbidity because of concurrence of this disease in livestock. This trend may be interpreted against the background of studies pertaining to the influence of a magnesium-deficient diet on lymphatic glands (particularly the thymus gland) of experimental animals [1, 3]. A tendency toward accumulation of magnesium and calcium in non-physiological amounts, in the lymphatic cells of human subjects and cattle with chronic lymphatic leukemia, might be related to pathological adaptation of neoplastic cells in endoecological and ecotoxicological circumstances. It might be interpreted in light of multifactorial etiology of leukemia and biocybemetic models [5]. It is worthwhile to mention that in samples of the whole blood, analyzed by the PIXE method, deficiency of selenium and excess of antagonistic trace element rubidium was detected in lymphatic cells of cattie suffering from leukemia enzootica bovina (e.g., level of Se in healthy cattle was 0.11 ppm while it was below 0.00 ppm in leukemic animals; levels of Rb in normal and leukemic groups were 0.60 ppm and 0.81 ppm, respectively). An even wider range of mean concentrations was observed for strontium in analogous cells (healthy animals: 0.31 ppm Sr; leukemic cattle: 0.80 ppm Sr). In addition to the deficiency of selenium in blood cells of leukemic subjects, its assimilability is also very poor. Administration of optional physiological doses of selenium was not found to increase the amount of this element in peripheral cells [6]. Deficiency of zinc was also detected in some neoplastic cells, but application of Zn may stimulate the growth of cancer. Magnesium compounds may, however, be used for supplementation of Mg pool in deficiency states. It is effective for treating even advanced forms of some tumors in animals but in cases of leukemia, Mg supplementation is effective only to induce remission at the very early phase of the


11

disease [IJ.This is another reason which emphasizes the importance of early detection of risk factors of neoplasms and ecologically oriented preventive activity, with particular reference to the amount of essential elements in the diet.

LASER MICROANALYSIS AS A NEW TOOL FOR PRIMARY PREVENTION One of the methods useful for quantitative analysis of essential elements (e.g, Ca and Mg) in small samples of biological material, as well as for the estimation of enzymes by these elements, is laser microanalysis introduced by R. Kozik. This method was also applied for early detection of risk related to low levels of some chemical pollutants in water [3, 6]. The method allowed simultaneous analysis of selected elements along with the enzymes activated by them. Large-scale screening studies may be necessary because a reverse relationship was observed between the level of Se in selected populations and the frequency of incidence of some neoplasms. Lower mean levels for this element were reported in whole blood of patients suffering from early stages of fibroblastic diseases and breast cancer as compared to those in healthy women both in Japan and the USA [1, 37, 38]. Decreased concentrations of Se and Zn along with increased levels of Mn and Cu were observed in blood of patients having stomach cancer [6, 37]. Similar assessment, like the one described for Se, may be recommended for ecological prevention of elemental disturbances due to human exposure by ingestion and inhalatory pathways. Evaluation for such exposures was carried out for representative values of environmental Se in air, soil, diet, and human organism. For estimation of environmental risk factors (due to excess or deficiency of trace elements), computer simulation of concentrations in the natural environment may be useful. Inhibitory effects of selenium compounds were found against many mutagens (including MNH) and chromosomal aberrations. Therefore, deficiency of Se in the areas of cancer clusters, accompanied by the excess of Hg in the trophic chain, seems to be important as it might increase the risk factor of chromosomal breakage by this heavy metal. A new cytochemical technique for the study of oxidative enzymes has recently been introduced as a highly sensitive indicator of intoxication with Hg and other harmful elements. Using this technique, Niwelinski and coworkers (unpublished work) found a correlation between the amount of total Hg in placenta and decrease of activity of these enzymes, as well as frequency of congenital malformations in some regions of South Poland [6]. A positive correlation (r = 0.637) was found between the incidence of human leukemias in rural regions and the intensity of utilization of seed dressings containing mercury. Mean concentration of this element was significantly higher in the hair samples of patients suffering from acute leukemia than in their healthy relations Hving in the same houses [6]. A positive, but weaker correlation (r = 0.345) was observed between the incidence of lymphatic leukemia in cattle and the amount of similar fungicides used in various rural areas in Cracow region.

12


ELECTROCHEMICAL POTENTIAL AND BIOLUMINESCENCE FOR DIAGNOSIS Application of different methods of high sensitivity could increase the possibility of early detection of cancer and other diseases. One of the newest methods introduced by Tomassi and improved by Janczarski uses powder electrodes for the measurement of electrochemical potential in blood samples [6]. The mean ratios of potential observed between blood plasma and blood cells were as follows: healthy subjects: 0.83; chronic lymphatic leukemia: 1.01; acute lymphatic leukemia: 1.06; and hemolytic anemia: 1.10. Changes in electrophoretic properties of lymphoblastic cells of patients with chronic lymphoblastic leukemia were reported. Differences were also observed in Verdet's coefficient (reflecting the magnetic rotation of polarized light by serum globulins), as well as in fluorescence, Raman dispersion, and optical activity in the infrared light. In the case of Zn deficiency, characteristic for lymphatic cells in chronic lymphatic leukemia patients, irradiation with lowintensity He-Ne laser light enhanced the intensity of bioluminescence much more than that seen in lymphocytes containing a normal amount of Zn and other essential elements. The results are presented in Table 4. Laser biostimulation could be used for nutritional prevention by stimulation of accumulation of some essential trace elements—e.g., selenium, iron, zinc [6] and Dobrowolski et al., in press). The pathological cells are more sensitive to red light. Irradiation has been suggested of both trace element-deficient solutions and the blood cells flowing out of the organism, in separate apparatus using laser light corresponding to the wavelength of

Table 4 Effect of trace element content of saline and laser stimulation on bioluminescence of lymphocyte suspensions after Dobrowolski et al. 1989,1990 [6]

Groups and sub-groups

Bioluminescence (mean number of impulses in 40 seconds)

1. Normal lymphocytes 1.1 Before irradiation 1.2 After irradiation (a) Cell suspension in trace element-deficient saline (b) Cell suspension in trace element-rich saHne 2. Lymphocytes from patients of bone aplasia 2.1 Before irradiation (a) Cell suspension in trace element-deficient saline (b) Cell suspension in trace element-rich saline 2.2 After irradiation

96,000 189,000 109,000 35,000 38,000 94,000


13

the spectrum of elements for possible increase in the activity of element-dependent enzymes [6]. Short exposure to He-Ne laser or 25 mW intensity was found to be specially effective for increasing the number of erythrocytes and hemoglobin level in rats suffering from Sarcoma-45 and Roux Sarcoma. Optimal activity of blood serum of these animals after laser irradiation became similar to that of normal serum under infrared light. It was accompanied by higher levels of albumin and globulins. Irradiation of pig ovary in vitro, with red light of 2.8 mW laser for 60 seconds, was found to significantly increase the activity of delta-5, 3-beta-hydroxy-steroid-dehydrogenase (HSD) as well as estrogen and progesterone levels. In control cultures of granulosa cells from porcine ovary, estrogen and progesterone concentrations were highest on the first day. In laser-stimulated cultures, peak progesterone levels were observed during 2-3 days when the estrogen levels dropped. In non-irradiated cultures, activity of delta 5, beta 3 HSD was gradually reduced, while in laser beamirradiated cultures, it rose in a manner similar to the progesterone levels. Hyperplasia and epithelization of granulosa cells was detected only in the experimental cultures. Stimulation of ovarian function, induced by 25 mW laser irradiation, was also put to gynecological application. There are new perspectives of application of laser light biostimulation for normalization of female endocrinological activity and elimination of some of risk factors of common neoplasms. Adequate use of low intensity laser beams could inhibit the growth rate of some species of toxicogenic molds, producers of well-known carcinogens. In relation to the nutritional aspects of medical elementology, it is interesting to note that treatment of seeds in two varieties of tomato increased bio-accumulation of selenium in the fruits. The results, which were particularly significant after supplementation of the standard soil with non-toxic doses of sodium selenate, are presented in Table 5. An increase in Se levels was accompanied with the decrease in Rb and Zn concentrations owing to an antagonistic relation between Se, Rb, and Zn. Laser photostimulation could be also a new method for higher accumulation of iron in the potato bulbs (Dobrowolski et al., unpublished report, 1995).

Table 5 Effect of soil treatment on selenium content of tomato fruits after Dobrowolski et al., 1989,1990 [6] Selenium Content (Mean values mg/kg dry weight) Groups

Untreated soil Soil treated with sodium selenate

Subjects: Venture tomato

Open-air tomato

0.0 2.6

0.1 2.3

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The results of comparative studies of normal and cancerous (leukemic) cells showed changes in bioluminescent, electrochemical, and metabolic properties. On this background, the concept of oncogenesis created by Klimek is a new trial of interpretation [32, 33]. Taking into consideration thermodynamic aspects of tumor origin on the presumption of the appearance of dissipative structures in biological systems, one could have a quite new starting point of neoplastic transformation of cells. Amaya (in press report) further developed the Polish concept of multifactorial, environmental carcinogenesis and thermodynamics of the irreversible processes. Amaya's hypothesis is based on decreasing dormant gene information during the transformation of a normal gene to a malignant one. The cell is recognized as a very complicated feedback system of genotype enzymes and their products. Mutated genes control synthesis of new isoenzymes having properties that differ from the original (e.g., in relation to optimal conditions for maximum activity, sensitivity to environmental factors as also to controlling effectors, etc.). The molecular anvil model of enzymes was recommended by Amaya for explaining changes in chemical composition of cytoplasm in transformed cells. Changes in response of cells to environmental factors can cause marginal oscillations of the material system, including genes and final products resulting from enzymatic reactions. Changes in cell metabolism involve heating effects necessitating high-sensitivity equipment for quick detection of neoplasmatic transformation of some cells in early stages. Recently, some new methods were recommended for early diagnosis of common diseases, including cancer. Good results were reported by the application of a method based on Vincent's research. A computer program was developed for data pertaining to examination and interpretation of pH, redox, and conductivity characteristics of saliva, urine, and blood. The importance of immunological examination (using complex parameters) was suggested for the detection of risk factors of neoplasms. Immunoelectrophoresis and scanning electron-microscopy, as well as cytochemical studies of immunologically active blood cells, especially subfractions of B- and T-lymphocytes, became useful. Blood sedimentation velocities of some elements (e.g., Se, Zn, Fe, and Cu) were recognized as markers of cancer activity. A simple technique of holistic blood diagnosis was reported by Aurus-Blank and Blank. According to the authors, the application of the test enabled the detection of cancer, its stage of development, metabolic disturbances, disorders of various organs, and stress conditions of the body, as well as measurement of the control brought about by the therapeutic measures. Auroscopy was recognized by experts as more convenient than other conventional diagnostic methods for better detection of early stages of cancer. Free radicals are unstable and their recombination is associated with chemiluminescence. Intensity of this process is proportional to the concentration of superoxides in the examined biological material. Ottolengi described the catalytic function of Fe2+ in the origin of superoxides in biological membranes. Oxidation of bivalent iron is stimulating these processes. A feedback relation exists between the consid-


15

eration of SH-groups, Fe^"^, Fe^"^ and lipid superoxides in living cells and the enzymes involved in regulation of ROOH-levels [6]. CHEMILUMINESCENCE AND PHOTON EMISSION AS METHODS FOR EARLY DETECTION OF CANCER RISK (ASSOCIATED EXCESS OF FREE RADICALS) Recombination of free radicals in the presence of O2 and Fe^"^ may be associated with photon emission. Bivalent iron has a key role in three stages of lipid oxidation: namely, in the initiation of the process, its multiplication, and its inhibition. The last one is connected with reactions between iron and slow radicals. Excess of iron was detected in pre-leukemic state. The similarity between hemoglobin (containing iron) and chlorophyll (containing magnesium) was described by Polish scientists Marchlewski and Nenchi. Experimental studies revealed that when the temperature of chloroplasts was increased, bivalent cations of magnesium were released. This process is accompanied by photon emission. Ultraweak luminescence has been detected in all investigated higher plants and animals. Photon emission is more intense in higher plants and animals. Photon emission is more intense in higher organisms than in simpler ones. Living cells exhibit marked increases in photon emission before mitosis and amplify a secondary radiation. There are great differences between living cells in spectral range of photon emission, as well as in the intensity of this ultraweak radiation. A new concept of biologically useful information is based on photon storage in DNA and a control system of photon emission of a different wavelength than the control cells [6]. It is interesting to note that a decrease in free radical scavengers—e.g., selenium—in the human body is associated with an increased risk of incidence of breast cancer. Deficiency of some elements may be compensated by pathological mechanisms leading to an excess of these elements in particular cells, e.g., during cancer development. A higher level of anti-oxidants in neoplastic cells seems to be related to a higher intensity of chemiluminescence according to the following equation: ImH -H RO2

k2

In + ROOH

where ImH = anti-oxidant In = mild inhibitor of free radicals. Concentration of anti-oxidants is especially high during early stages of cancerogenesis. Therefore, the measurement of photon emission is seen mainly in the lower part of the spectrum (blue and green light). The chemiluminescence method also allows indirect estimation of excited molecules by a single oxygen measurement of longer red light emission. Exposure of experimental animals to leucosogens results in a significant increase in the amount of free radicals in the spleen and liver. First

16


pathological changes in the body, like increases in weight of the spleen and number of leucocytes in peripheral blood, are observed only when the highest concentration of free radicals is reached. By application of the single photon-counting technique, a much higher photon emission was reported in the leucocytes of leukemic patients than in ones among healthy subjects. The highest intensity of chemiluminescence is characteristic of people with acute leukemia. In a logarithmic scale, opposite trends were found referring to changes of chemiluminescence with time, in relation to lymphocytes and blood serum of patients with lymphatic leukemia and control subjects [6]. Leukemic and cancer clusters exhibit deficiency of cobalt in the trophic chain [6]. After supplementation of suspension of lymphocytes and the blood serum with trace amounts of cobalt chloride, the above-mentioned differences in chemiluminescence rapidly decreased. A quite opposite situation exists with respect to the application of divalent cations. The differences between photon-emission of the tested material collected from healthy and leukemic persons are substantially increased under the influence of bivalent iron, H2O2 and ultraviolet (UV) light. These factors, UV light in particular, multiplied the chemiluminescence of control blood serum and lymphocytes to a much higher degree than the comparable leukemic materials. As a result of this stimulation, the intensity of photon emission from control blood serum became much lower than that from serum of leukemic patients. The same stimulant factors slightly changed the lower intensity of photon emission from healthy lymphocytes into the much higher intensity of photon emission in leukemic lymphocytes. Simulated chemiluminescence, therefore, appears to be a very useful screening test for early detection of the incidence of leukemia or cancer. Chemiluminescence may also be helpful in estimating risk factors, e.g., sensitivity to ionizing radiation. Concentration of singlet molecular oxygen is related to the oxygen-dependent radical damage to cells. Low-level chemiluminescence emitted from rat hepatocytes (wavelength beyond 600 nm), may serve as a monitoring tool for continuous measurement of simulative effects. PHAGOCYTIC ACTIVITY Another important parameter of homeostatic ability is phagocytic activity. In the presence of anti-Escherichia coli antibodies, guinea pig alveolar macrophages released more H2O2 than in their absence. The polymorphnuclear leucocytes have a high activity of myeloperoxidase, opposite to the alveolar macrophages. As it is known that the efficiency of delay chemiluminescence from polymorphnuclear granulocytes reflects their bactericidal activity, this method was applied for comparative study of blood cells from healthy persons and some patients. A significantly higher mean value of chemiluminescence was observed in the control group compared to patients suffering from acute lymphoblastic leukemia [6]. It was observed after 45 minutes of stimulation with latex particles. Phagocytic ability of neurophils may be


17

increased by optimization of concentration of some bivalent ions, e.g., Ca^"^ and Mg^"^ at 10"^m were stimulatory while Ba^"^ exhibited inhibitory effects at all concentrations. Levels higher than 10"^m inhibited phagocytic activity. SMOKING One of the well-documented risk factors of respiratory tract cancers is smoking. A positive correlation was found between the number of cigarettes smoked and mutagenicity of Salmonella typhimurium in urine. Even passive smoking (equivalent to two cigarettes) caused an increase in urinary excretion of mutagens in Wistar rats within 24 hours of exposure. These results were obtained by mammaUan microsome tests. In supplementation to the application of measurements of changes in chemiluminescence of the expired air smokers, a highly sensitive single-photon counter was introduced for estimation of ultraweak light emission in blood samples of smokers and non-smokers. Significantly higher photon emission in the blood plasma of smokers was reported. Heavy smokers showed increased levels of some harmful elements (e.g., Cd and Pb), especially if the tobacco plant was cultivated in polluted areas [6]. AIR POLLUTION AND OTHER RISK FACTORS A new chemiluminescence method is also very useful for indirect estimation of other risk factors to public health. The excited molecules causing the luminescence are associated with soot particles emanating from the exhaust of a combustion engine-driven power generator. The long lifetime of these particles and their ability to transfer energy seems to increase the activity of potentially carcinogenic compounds. Enzymatic processes in the human body could increase cancer risk by hydroxylation of weak carcinogens and mutagens, mainly in respect of higher concentration of K-region epoxides. Benzene extracts of these soot particles could produce chemiluminescence and fluorescence studies. This fraction contains epoxides of poly cyclic hydrocarbons produced during the combustion process. The mutation rates in these extracts were three times greater than expected by considering only the concentration of mutagenic poly cyclic hydrocarbons. The highest dose-dependent increase in mutagenicity by the Ames system was described for the 4-7 ring polycyclic hydrocarbons among all fractions of the petrol engine exhaust condensate of a passenger car. Traffic input on the air pollution is related also to increased concentration of N-oxides. A simple colorimetric method developed by Amaya for analysis of personal exposure to NO2 is recommended for field study about the time and spatial differentiation of this risk factor. More sensitive methods—e.g., laser and chemiluminescence studies—may be applied for measurement of N-oxides and Pb to study their impact on blood levels and human health. Interdisciplinary studies about the levels of Pb in air, soil, water, the food chain, and human blood samples from inhabitants of different regions were carried

18


out by the Monitoring and Assessment Research Center, London. An excellent review relating to the impact of air lead and other pathogenic pollutants on blood lead in man and a methodological report about quality control in the analysis of Pb, Cd, and other ecotoxins in human blood are periodically released by this center. For ecologically oriented prevention of diseases, multifactorial analysis about the interactions between metals and carcinogens seems to be of special importance. Metal oxide particles were shown to modify carcinogenic effects of benzo(a)-pyrene and nitrosamines. Enhancement of nickel and asbestos carcinogens by polycyclic hydrocarbons was reported. Opposite/inhibitory effects were described for zinc and cadmium, or nickel and manganese, or selenium and zinc, as well as arsenic. SEAFOOD The application of natural food supplements' reach in deficient elements is widely recommended for nutritional prevention of health hazards. Seafood is a common source of necessary elements. The anti-carcinogenic effects of an edible seaweed, Laminaria angustata, which is popular in Japan, may be related to its elemental content. Antimutagenic effect of these extracts was observed against a breast carcinogen on Ames/mammalian microsome system. Anti-tumor activity of various seaweed extracts tested on experimental animals was recendy reviewed. A correlation between anti-tumor effects of various fractions of Semicarpus sp. and concentration of some trace elements was reported. Application of selenium-rich sea animal food exhibited better protective effects against acute and sub-acute methyl mercury poisoning in rodents than observed after the use of similar amounts of total selenium administered as sodium selenite. Sea bass—Lateolabrax japonicum—a common fish found in the seas surrounding Japan, has 2.5 times more seleniumconcentrating ability than mercury, especially in the liver and muscles. Thus, this species may be recommended for lowering the retention rate of mercury in the human body [6]. Some other products are also good for this purpose, e.g., crustaceans, muscle meat, some varieties of corn-like yellow IV for direct use by people, and halophytes as an animal feed in some regions. Some mutants of yeast concentrate high levels of selenium, and these are widely used in the form of pills in the USA [38]. A good natural source of selenium is pollen from plants growing in areas rich in this element [6]. MERCURY Mercury contamination of the natural environment is a high health hazard. Different kinds of food additives for supplementation of diet with selenium can increase human resistance against environmental pathogens, including some carcinogens and teratogens like mercury. Intoxication with mercury is a risk factor for higher incidence of leukemia and Minamata disease. The first recognized case of


19

this civilization disease was reported in Minamata (Japan) by Dr. Hosokawa in 1953 [6]. Nine years later, similar disease was reported from Nigata, and then reports from countries such as Iraq and Canada followed. Nutritional contamination with organic mercury is highly neurotoxic, especially for young children and prenatal development. The conditions were described in detail by Harada. During early stages of poisoning with organic mercury, the highest concentration of this metal was found in the kidney, and at later stages in the liver and brain. Four to six weeks after the affliction, mercury content decreased significantly in the kidney and liver, although it still remained high in the brain. Introduced by Harada, neurological tests are, therefore, useful for detection of early symptoms of Minamata disease, associated also with increased risk of neoplastic changes of some blood cells. According to Janicki, contamination of rural environments with pesticides containing mercury is associated with elevated frequency of adult leukemia [6]. Patients suffering with acute leukemia have a higher mean concentration of mercury in the body (as it was found by Dobrowolski, Janicki, Krasnicki) than their healthy relatives living in the same places. Mercury accumulation for prolonged periods is pathogenic for phagocytes, microghal cells, neurons, and epithelial cells of Henle's loop. Monitoring of Hg and Se in these cells (micro- and macro-phagocytes in particular) appears to be promising for the prevention of acute intoxication. Epithelial cell linings from fetal lung tissue of golden hamsters and also from human trachea and bronchi are recommended for testing chemical toxicity. This material is useful for establishing more accurate correlations between in vitro and in vivo data. Present in the cell membranes, sulphydryl groups have high affinity for mercury and its compounds. Cell membranes, therefore, are the first site of damage by Hg and other heavy metals. Exposure to Hg compounds may raise the concentration of metalothioneine-like protein in kidneys. Referring to the cases of coincidence of environmental contamination with mercury and cadmium, it should be borne in mind during interpretation that protein binding is enhanced by the presence of cadmium in these organs. Despite the stated lack of critical enzymes for mercury intoxication, decreased histochemical activity of some enzymes (e.g., NADPH-dehydrogenase and cytochrome oxidase) was observed with increased placental levels of Hg. Experimental poisoning of pregnant rats with methyl mercury chloride induced hyperplasia of labyrinth in placenta and reduced activity of some lysosomal enzymes. This intoxication was, however, associated with an increased number of arthritic follicles in the ovaries and subsequently increased activity of some lyposomal enzymes. This indicates that histochemical reactions are organ-dependent. According to an epidemiological study, the highest frequency of dysarthria in the Cracow region was observed in the westem part (1.38%), which corresponds to the highest mean amount of Hg in the placenta (8 ppm). The lowest incidence of such congenital malformations was detected in the northern part of the regions (0.61%,) corresponding with lowest level of Hg in the placenta (4 ppm). The highest incidence of all kinds of congenital malformations

20


(5.33%) was reported in the town of Oswiecim, which has a large chemical plant using mercury. The corresponding value for Cracow City was 1.04%, and for the non-polluted town of Myslenice 0.84%. Epidemiological differences are associated with differences in mean levels of mercury in the human placenta. In view of the protective role of selenium against mercury intoxication, adequate control of concentration of both these trace elements is desirable. Umbilical cord Se levels among the inhabitants of the Minamata region were almost constant for 39 years despite tremendous increase in Hg concentration [6]. The amount of these and other trace elements varies considerably in human food including cow and human milk from various regions. Experimental studies using radionuclides indicate that heavy metals can penetrate the placenta. As a result of bio-accumulation, higher Hg, Cd, and Fe concentrations were observed in the cord blood of newborn children as compared to the blood levels in their mothers. This increases the risk, particularly because of the higher sensitivity of fetuses and infants compared to the adults. In the region of the metallurgical center at Miasteczko Slaskie, increased levels of Pb and Cd (more than 30 g/dl) were reported in 15% of mothers and 24% of newborn children, in contrast to only 1.2% in control rural areas. Increased concentration of these metals in the placenta is accompanied by the tendency for higher incidence of gestoses and premature births as well as higher mortality of the newborn. Newborn children contained more Hg in blood than their mothers. It is surprising to note that some of such children from Greenland had more than 170 g/1 Hg, which is equivalent to the blood levels of victims with congenital Minamata disease. The relationship between total Hg in the child and the mother is approximately linear. This can be expressed by the equation: Hg in child (g/1) = 6.35+1.46 Hg in mother (g/1). Blood Se relationship between the child and mother is, however, non-linear. Mean level of the Se exceeded that of Hg on a molecular basis [6]. This seems to be important for preventing Hg intoxication. The main source of Se is marine food, which is subject to seasonal variation in chemical composition. Average amount of Se in cord blood was 100 g/1 in summer and 300 g/1 in winter. Differences in the concentration of Se and Hg depended on factors like dietary intake and age. Both blood and hair samples are good indicators of Hg level in the body. Seasonal variation in placental Hg content has also been reported by Niwelinski et al. from some rural areas in Poland [6]. Methyl-mercury aggravates toxicity of some pesticides. Nutritional intoxication with organic mercury followed by exposure to phosphamides increases chances of teratogenic effects in experimental rodents. It is, therefore, necessary to take into consideration the interaction of many pollutants and other factors to plan measures for preventive action. Some environmental pollutants are risk factors of both higher rates of cancer and congenital malformations incidence. One example of this coincidence is contamination of the human food chain with mercury [6]. Therefore, primary prevention has to be related to both aspects of the health hazard [3]. Exposure to lead is indirectly involved in higher cancer incidence. Nowadays, no region in the world is free from health hazards [6]. Lead levels in blood samples


21

from inhabitants of Greenland were found to be similar to those of people from industrial regions. Experimental studies in rodents have shown that radioactive lead crosses the placental barrier in significant amounts even if blood Pb level in the pregnant female is low. Calcium cations lower the concentration of Pb in erythrocytes followed a linear-dose response pattern. These cations also reduce the rate of Pb transport between blood plasma and bones. Calcium may usefully be applied for dietary supplementation in children from similar uptake kinetics in adults. One of the important aspects of a global UNEPAVHO project on assessment of exposure to Pb and Cd through biological monitoring is quality control in the analysis of these trace elements in blood. A significant correlation exists between the increase in amount of lead in erythrocytes (above 40g/dl) and decrease in activity of Na/K ATP-ase in cell membranes. Elevated Pb levels in these human cells is recognized as symptomatic for anemia as a result of Pb hemotoxicity. Activity of erythrocyte delta-aminolevulic and dehydrase and its change by heat treatment was suggested as an index of Pb exposure. Field studies conducted in the regions of big metallurgical centers revealed coincidence of high Pb and Cd levels in blood among the inhabitants of some areas. The influence of pollution of the living areas was found to be more important than that of the working places. The data for whole blood correlated with those for single lymphocytes. Increased amounts of Pb and other heavy metals in these blood cells is associated with immunosuppresive effects as risk factors of manifestation clinical symptoms of cancer. Bioindicators are necessary for early detection of risk factors. By the application of X-ray microanalysis, a correlation was detected between Se and Pb in the air, and Se or Pb levels in blood cells—e.g., red cells investigated under scanning electron microscope from blood samples collected from humans of low exposition and from industrial workers. Such investigation appears to be particularly useful while selecting proper blood donors under elevated risk of environmentally born contamination—e.g., by heavy metals. Another highly sensitive bioindicator is human placenta. Wide variations in concentration of some trace elements were observed even in a protective zone of one metallurgical center in Poland viz., 0.01-0.38ppm Pb, 0.015-0.055ppm Cd, 5.1-11.7 ppm Zn. For the prevention of nutritional intoxication (e.g., with heavy metals), analytical studies about phyto- and zooindicators are also recommended. Dog hair analyzed for Pb, Cd, and Zn was found a useful zooindicator for determining spatial differences in mean amount of trace elements. In samples collected 2 km from a mining and metallurgy center, the average amount in air samples was 13-15 ppm Pb and 1.47 ppm Cd. In the next zone 8 km away, the mean levels were 7.32 ppm Pb and 0.67 ppm Cd. Zinc levels in these samples did not show significant differences. Phyto- and zooindicators seem to be very useful in estimating the degree of environmental pollution in the area and the production of uncontaminated food. In the most polluted area, the frequency of cattle intoxication was 13 times higher than that in the control area. This was associated

22


with a higher incidence of metabolic diseases (3.5-fold) and reproductive disorders (5.9-fold). Some precautions are, therefore, necessary for breeding of animals in highly polluted regions. Strict control over the quality of food is absolutely essential for nutritional prevention of diseases. In any case animal breeding in such areas is not economical. An eminent expert in ecotoxicology—Rene Truhaut from the French Academy of Sciences and Descartes University in Paris—recommends analysis of essential oligo and trace elements as complex in the human trophic chain. These studies should be supported by field and laboratory experiments. Application of human and other mammalian tissue cultures seems to be of special importance for evaluating biological effects of environmental pollutants, including their carcinogenic, cytotoxic, and immunosuppressive effects in vitro (on animals) and in vivo, as well. In this context, valuable experimental data is available about the influence of elements at cellular level, including chemiluminescence studies in human blood cells after treatment with industrial dusts [9]. SALTS AND MINERALS Mineral waters and salts are traditional and important food additives. Proper amounts of essential oligo and trace elements in the human diet is important not only for physiological functions of the body, but also for reducing bioaccumulation of toxic elements. Soft water may increase health hazards due to pollution of drinking water with heavy metals. Deficiency of essential minerals in drinking water is associated with higher risk of heart attack and other cardiovascular disorders. Therefore, supplementation studies were carried out, using water rich in Mg and other essential elements, in a group of people with a tendency toward deficiency of these elements in their bodies. After long-term application of Mg-rich mineral water instead of tap water, a statistically significant increase was observed in mean levels of Mg in the whole blood and single blood cells of steel workers [6]. This mineral water was also used for production of bread and special cakes for persons working at high temperatures, with good results. Referring to observations of Hawiland and others, optimalization of magnesium amounts in the human body could also be useful for nutritional prevention of neoplasms. Some animal experiments and later clinical studies were undertaken to assess effects of application of rock salt rich in essential elements, instead of commonly used purified table salt containing NaCl only. After using this natural salt from a Wieliczka mine (in a normal dose of 2.5 g per day), a significant increase in serum immunoglobin level (from 113 to 240.4 mg%) was observed after two weeks of application of the same salt. X-ray microanalysis revealed increased concentrations of oligo and trace elements in single blood cells of the patients under this experiment. The amount of the following elements increased in human lymphocytes: Fe from 0.15 to 0.26%, Zn from 0.17 to 0.25%, and Mg from 0.18 to 0.27%. A similar


23

tendency was detected in cattle blood cells under this experiment. The amount of the following elements increased in cattle lymphocytes: Fe from 0.15 to 0.26%, and Zn from 0.18 to 0.27%. The rock salt added to animal feed caused retarded frequency of spontaneous leukemic symptoms in AKR mice and decreased aflatoxin B-1, reducing mortality of these mice by 35%. This beneficial Wieliczka salt is rich in Fe and contains elements such as Mn, Se, Co, Cr, and Li. Deficiency of Fe, Se, and Co observed in the food chain of "cancer cluster" could be a starting point for searching about natural food additives as sources of these elements. Another Indian way is widescale fortification of table salt with iron compounds and vitamin C for better availability of Fe. This is recommended to prevent Fe-deficiency anemia. Supplementation of the human diet with high amounts of vitamin C was recommended by American Nobel Prize winner Louis Pauling. CONCLUSIONS 1. A systems approach to the relation between the quality of the whole human environment and pattern of incidence of selected diseases seems to be necessary for assessing the influence of complex external factors (with special reference to the excess or deficiency of particular elements in different regions) on the public health. 2. Interdisciplinary field studies should be supplemented with experimental research on various species of mammals. These should include investigations on synergistic or antagonistic relations of elements. 3. In "clusters" of neoplastic diseases, higher levels of Ni, Pb, Hg, Mn, Cu, Zn, and Rb, and lower levels of Mg, Fe, Se, and Co were observed in the food chain. Ecotoxicological studies were followed by the measurement of the quantity of these elements in biological samples (including a cellular level). 4. Risk of health hazards related to the deterioration of the environment is increased by the deficiency of essential elements. Higher incidence of cancer was associated with concurrent pollution of residences with toxicogenic molds and selenium deficiency in food. 5. Studies on the interdependence of different elements (e.g.. As, Cd, Hg, Se, Zn, Mg, Ca, and Pb) and other factors affecting pathogenesis of common diseases have received considerable attention in recent years. The deficiency of selenium in the body is associated not only with a higher risk of cancer, but also of myocardial infarction and Keshan cardiomyopathy, as well as with higher sensitivity to mycotoxins, benzenes, ionizing radiation, and some heavy metals. 6. The application of newer methods of analysis enables the measurement of trace amounts of both necessary and harmful elements in health and disease (even in single cells by use of X-ray microanalysis or PIXE analysis together with SEM observations of these cells) and also early detection of health hazards due to the deterioration of the human environment.

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7. Nutritional prevention of diseases (including diseases of civilization) require constant control of elemental composition of trophic chain and modification of the human diet with widescale application of natural food additives (e.g., essential element-rich rock salts, plants, bee products, pollen, etc.). 8. Area characteristics, by higher incidence of human cancer and cattle leukemia, have elevated levels of different carcinogens, including precursors of nitrosoamines in drinking water. Therefore, there is recommended feedback between permanent control of quality of water and food (as a part of environmental and ecotoxicological monitoring) and development of technical activity focused on primary prevention of cancer risk factors (e.g., adequate use of efficient wastewater treatment, improvement of sanitary conditions, etc.), as well as education toward environmental health (nutritional prevention in particular). Short Review of the Environmental Risk Factors of Cancer Incidence The general recommendation for primary prevention is a reduction of the rate of emission of the carcinogens at source, minimizing exposure as well as biological effects of the risk factors Risk factor

I. Ionizing radiation [1].

II. Non-ionizing radiation [1].

Ultraviolet radiation [1] from sunlight as well as for therapeutic uses sometimes concomitant with medicine, e.g., furocoumarins [16]. UV A, B, C [7, 16, 28].

III. Radon-222 and manmade fibers (glasswool, glass filaments, rockwool, ceramic fibers) as indoor pollutants [19].

Preventive measures

Fortification of food with radioprotectors, free-radical scavengers, etc. Supplementation of diet with additives stimulating activity of immunological system, antioxidants, etc. Use of protective glasses, and creams. Irradiation of the skin with the photoreactivating light [2].

Prevention of ozone layer depletion by air protection (including cutting down traffic output of NOx, etc.). Effective system of ventilation, selection of proper construction materials [35].


IV. Silica, wollastonite, attapulgite, sepiolite, talc, erionite [18]. V. Asbestos fibers [9]. VI. Beryllium and beryllium compounds as well as cadmium and Cd compounds are carcinogenic to humans; methyl mercury is possibly carcinogenic [5, 30]. VII. Carbon black particles and tar-pitch aerosol [10]. VIII. Chromium (VI) is carcinogenic, nickel [24] and lead [6] are possibly carcinogenic. IX. Occupational exposure in petroleum refining is probably carcinogenic; gasoline and residual fuel oils are possibly carcinogenic [21]. X. Exposure to gasoline engine exhaust and to diesel engine exhaust is probably carcinogenic; respirable particles are risk factors of lung or bladder tumors and also childhood cancers [24]. XI. Exposure to polynuclear aromatic compounds in coal gasification, coke production, steel founding, and aluminum production is associated with elevated risk of cancer [13]. XII. Occupational exposure to some organic solvents and pigments is possibly carcinogenic to adults, and parental exposure increases risk of leukemia and for child cancer [23]. Tobacco smoking, including passive XIII. exposure, is a major cause of cancer of respiratory system [15]. XIV. Chewing of betel and tobacco leaf, and areca-nut is possibly carcinogenic, depending on concentration of N-nitroso compounds [14]. XV. Chlorophenoxy herbicides and chlorophenols pesticide are possibly

as above. as above. Hermetization of production, substitution of materials, etc.

as above.

as above.

25

26


XVI.

XVII.

XVIII.

XIX.

XX.

XXI.

cancerogenic [17]; a fungicide captafol is probably carcinogenic; some insecticides (e.g., DDT, atrazine, dichlorvos, heptachlor) are possibly carcinogenic, and workers exposed to them are under higher risk of lung cancer, multiple myeloma, and tumors of B-cell origin [27]. Exposure to N-nitroso compounds, and aromatic amines in the chemical and rubber industries are risk factors of bladder cancer [11]. Mycotoxins (e.g., aflatoxins, ochratoxin A) are carcinogenic [4, 29]. Chinese-style salted fish [29] and pickled vegetable concentrations after fermentation elevate risk for esophageal and gastric cancer [ibid]. Halogenated by-products of chlorination of drinking water are potentially carcinogenic [26]. Coffee is possibly carcinogenic to the urinary bladder [25] and coincidence of occupational exposure to Nnitro compounds could increase chance of incidence of this cancer. Drinking hot beverages is probably associated with higher incidence of esophageal and oral tumors [2]. Alcohol overconsumption is a risk factor of cancers of the oral cavity, pharynx, larynx, esophagus, and liver [20]. Application of estrogens to adult women increases rate of endometrial cancer [12].

Coincidence of some environmental risk factors (e.g., mycotoxins and nitrosoamines, or polycyclic aromatic hydrocarbons and aromatic amines, as well as some metals) could increase risk factors of incidence of some tumors. Another point for common action for primary prevention is elimination of exposure to carcinogenic factors both in working places and residences as well as in the outdoor


27

environment and in food (e.g., contact with mycotoxins is possible in buildings contaminated with toxicogenic molds as well as by food imported from countries of high temperature and humidity, and contact with aromatic amines could be in working places and through food additives). Some behavioral patterns may also contribute to higher incidence of particular types of cancer. Synergistic effects of tobacco smoking and occupational exposure is of special importance in this respect. Knowledge about related risk factors seems to be important for engineers, teachers, and decision makers. There are some new data about environmental etiology of some neoplastic diseases. Correlations between rate of incidence of the following carcinoma and factors have been established: • leukemia and ionizing radiation • skin cancer and exposure to UV (from sunlight) • lung, pleura cancer and asbestos • urinary bladder cancer and aromatic amines • liver cancer and aflatoxins • esophagus, pharynx, oral cavity cancer and alcohol • lung, larynx cancer and cigarettes (After Kacki E., Stemczynska J. [31]) The following information is a starting point for a data bank about the system approach to environmentally oriented primary prevention of neoplastic diseases based on risk factors of human leukemia (after Aleksandrowicz et al. 1982 [1]). A. Risk factors and markers of genetic-racial-family and ecological origin: 1. Enhanced development of leukemia in offspring of older mothers. 2. Chromosomal aberrations in somatic cells in Down's syndrome and Fanconi's anemia chromosomal abnormalities. 3. Sex chromosomes in Klinefelter, Turner, Louis-Barr, Kostmann's, and Bruton syndromes. B. Risk factors and markers connected with viruses: 1. Latent infection with oncorna virus. 2. Oncorna virus reservoirs. 3. Consumption of food infected with oncorna virus (milk and meat of leukemic cattle and poultry products). 4. Vaccine made from infected animals. C. Risk factors and markers of immunological origin: 1. The early deaths of siblings among children suffering from frequent upperrespiratory tract infection. 2. Impairment of delayed hypersensitivity reaction to common antigens. 3. The positive hypersensitivity reaction to application of human leukemia antigen. 4. Occurrence of specific antibodies to human leukemia antigen.

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5. Elastic transformation of lymphocytes in vitro upon exposure to human leukemia antigen. 6. Increased level oi Aspergillus flavus and Penicillum meleagrinum antibodies. 7. Alpha-feto-protein. 8. CEA—carcino-embryonic antigen. 9. Low level of E-rosette forming cells. 10. Impairment of lymphocyte response to PHA and Con A. 11. Risk factors and markers of enzymatic origin: a. decreased activity of alkaline phosphatase in granulocytes of chronic granulocytes leukemia. b. increased activity of muramidase—in chronic granulocytes leukemia. c. decreased activity of muramidase—in acute myelogenous and chronic lymphatic leukemia. d. elevation of IgM levels in polyclonal and monoclonal gammopathies. e. decreased activity of terminal deoxynucleoitidyl transferase in acute myelogenous and chronic granulocytic leukemias and high TdT values in most cases of acute lymphoblastic leukemia. f. decreased activity of serum RNA-se. g. decreased activity of antineoplastons. h. decreased 5' nucleotidase activity in chronic lymphocytic leukemia lymphocytes, i. reverse transcriptase level as risk factor. D. Risk factors and markers connected with the level of essential elements in blood and tissues: 1. Copper level increased in blood cells and serum in acute myelogenous leukemia. 2. Calcium level increased in blood cells and serum in acute myelogenous leukemia. 3. Zinc level subnormal in serum of leukemia and lymphoma patients and markedly decreased in peripheral leukocytes of chronic granulocytic leukemia and other neoplastic diseases. 4. Magnesium level decreased in serum and increased in leukocytes in leukemia selenium levels decreased in serum. E. Risk factors and markers connected with the level of vitamins in blood and tissues: 1. Vitamin C level decreased in blood cells. 2. Vitamin E level decreased. 3. Vitamin B-12 level increased. F. Environmental risk factors. Physical, chemical and biological immunosuppressors as leukemia risk factors: 1. Ionizing and non-ionizing radiation: a) Ionizing radiation—there are significant differences between wooden buildings and buildings constructed from prefabricates (reinforced concrete) with regard to the extent of emitted radiation.


29

b) Non-ionizing radiation—non-ionizing radiation in the range of 10-100 W/m^ may induce a significant hazard with respect to leukemic morbidity. c) Electromagnetic field and microwaves exceeding values of 1.0 mW/cm^. 2. Chemical leukemogens. 3. Biological leukemogens. G. Electronic risk markers: 1. The lowering of intracellular lymphocyte potential. H. Clinical risk factors: 1. Fewer unknown causative agents. 2. Therapy-resistant anemia and granulocytopenia. 3. Primary polycythemia. 4. Hemochromatosis. 5. Herpes zoster. 6. Urticaria. 7. Meningitis. 8. Decreased viability of granulocytes. 9. Decrease of phagocytic activity of granulocytes. 10. Decrease migrational capacity of granulocytes. Emotional Stress as a Risk Factor [1] Nowadays it is believed that the vast majority of neoplasms is caused by environmental risk factors. At the same time, an increase of cancer incidence has been observed, especially in heavily contaminated areas. Circumstances mentioned above cause common interest in the prospects of more efficient primary prevention of neoplasmatic diseases [1, 3, 5]. Etiology of neoplasms is multifactorial. Thus, to learn about environmental risk factors of cancer, it is not enough to investigate single factors and their biological effects. There is a need to create data banks of different scientific disciplines that can be fundamental to permanent updating of our knowledge. This synthesis makes a foundation for the development of related interdisciplinary case studies [4, 6]. They should provide not only deeper knowledge on actual risk of neoplasms for the population and help to predict future trends, but first of all define conditions necessary for the efficient, environmentally oriented prevention of cancer. This disease makes a potential threat for all. This implies the need for active involvement of everybody in the common action. That demands the popularization of the knowledge, both of environmental risk factors as well as various practical opportunities of permanent preventive action toward individuals as well as institutions, including the local, national, and international levels. The knowledge and skills necessary for this action can be used better if regarded as a necessary element for all the programs of formal and informal education. For moral reasons, the orientation for environmental risk factors of cancer and their primary prevention is a necessary part of training of decision makers (includ-

30


ing post-graduate courses). Different forms of open universities and distance education (TV satellite broadcasts) can make partnership between politicians and local communities easier. This partnership is to be focused on cancer prevention. This particularly refers to the consolidation and integration of local environmentally oriented efforts with solving global problems indirectly connected with environmental risk factors or cancer (e.g., prevention of ozone layer depletion or ecotoxicological causes of food contamination, including transboundary pollution). Updating and promotion of the knowledge provides motivation for common action. To make it effective, it seems necessary to create an efficient system of permanent exchange of scientific and practical information (international system of email and satellite communication), considering also the experience connected with socio-technical methods such as legal regulations, economic instruments, and cooperation between decision makers and the public on a regional and international scale. This refers to the links between primary prevention of mutations and cancer incidence with the sustainable development related not only to the promotion of environmentally sound technologies and system of management, but also the prevention of the introduction into less-developed countries technologies and equipment that are outdated and harmful to the environment or exporting wastes to these countries. Such wastes can be a source of contamination of the natural environment and the human food chain with carcinogens or co-carcinogens. That refers to, e.g., the export of outdated cars to less developed countries, where they are widely used and not only cause enormous waste of petrol resources, but also contaminated environment with strongly carcinogenic poly-aromatic carbohydrates. An essential element of primary prevention of cancer is proenvironmental education of the society by encyclopedias, textbooks, and mass media, particularly in countries of lower levels of environmental and hygienic awareness. Nowadays the use of effective means of visual propaganda for the increase of cigarette consumption, which is high anyway, becomes a bigger and bigger problem in Central and East European countries. The societies of rich countries, because of the increase of knowledge on the high risk of neoplasms in the respiratory system, caused by smoking, reduced cigarette consumption radically. Thus, tobacco companies intensify their activities in poorer countries. Their land is usually more contaminated by traditional high energy-consuming technologies (with carcinogens as well). Association of pollution from industry and transport with mass addition to tobacco gives a synergistic effect, i.e., increases the incidence of deaths caused by lung cancer and similar neoplastic diseases. International preventive actions such as "Europe against cancer" are developed mainly in rich countries. In less-developed countries, the lack of information on actual environmental risk factors is widespread. This refers also to persons with elevated risk levels such as radiologists and nuclear physicists. Usually the estimation of risk factors is limited to the measurements of exposure on ionizing radiation only. But according to the founder of ecotoxicology—Rene Truhaut, expert of WHO, FAO, EC, etc.—the ability of keeping homeostasis in an organism, including the risk of cancer incidence, is influenced by a synergistic


31

effect of physical, chemical, biological, and other factors that directly or indirectly cause neoplastic diseases. Thus, at the same working exposure the risk can be different depending on total exposure to all carcinogens, co-carcinogens, immunosuppressors, and promoters of carcinogenesis out of work. That refers not only to the content of harmful ingredients in food (potentially pathogenic), but also the content of protective ingredients in the diet of a particular person as well as individual biological susceptibility, which is defined genetically. These rules imply both the necessity of the development of interdisciplinary studies combined with the development of multifactorial risk analyses based on estimation of personal exposure. There are differences both in total exposure to complex environmental carcinogens as well as individual biological susceptibility. Thus, there is a trend to combine the assessment of the exposure on external factors with the biological monitoring of many different physiological, biochemical, biophysical, and other parameters characterized by an individual reaction. To minimize the risk of the occurrence of irreversible morphophysiological changes, it is necessary to develop interdisciplinary studies in larger and larger teams and combine them with the promotion of the methods of early detection of risk factors of cancer incidence. Unfortunately, present scientific policy usually prefers financing fragmentary research that does not provide adequate premises to estimate the risk. This has a negative influence on the effectiveness of the primary prevention of cancer. The future trend of cooperation in this field seems to be also the integration of basic and applied studies, and extensive scientific cooperation of the scientists and the whole society. The present state of knowledge resulting from the synthesis of epidemiological data referring to spatial differentiation of cancer incidence and the results of environmental monitoring (including multifactorial emission measurements) and ecotoxicological control of food contamination (together with experimental data) already provides some scientific premise for the active participation of everybody in the primary prevention of tumors. The basic trend in contemporary environmental management and reduction of related health hazards is the elimination of pollution at the source. Thus under democratic conditions, the knowledge of the society on environmental risk factors of cancer can generate adequate decisions in policy referring to better environment and life quality. That refers to the preferences in the modernization of technologies or the construction of vehicles or other equipment so that the emission of pollution induces changes in DNA and stimulates the increase of cancer incidence. Because the majority of environmental mutagens also have carcinogenic properties, such action is to protect the health of both present and future generations. The necessary condition of the effective primary prevention of environmentally born health hazards is a system approach to the elimination of all risk factors related to environmental deterioration. That demands the integration of present activities on the field of legal regulations, administrative management, education of the whole society, and the propagation of model-comprehensive solutions. For example, the achievements of American scientists are very valuable. They dis-

32


covered that increased incidence of some neoplasms could be linked with the deficiency of selenium in some populations and they suggested supplementing diet with selenium by using selenium-rich natural products like mutants of yeast, which were introduced by G. N. Schrauzer from California University [38]. There is the need for the development of more complex methods of primary prevention of cancer and their promotion on the international scale. Interdisciplinary research developing studies in this field were initiated in Poland by Julian Aleksandrowicz from the Copernicus University of Medicine in Cracow. Related case studies were done in small areas of very high incidence of leukemia and other neoplastic diseases called "cancer houses" and "cancer clusters" in villages. Statistically significant higher incidence of some species of toxicogenic molds in indoor environments of cancer patients was discovered. At the same time, the deficiency of a protective factor (i.e., selenium) in the natural environment and food chain in cancer clusters was reported. During the experimental studies, our team found preventive effects of selenium in relation to human lymphocytes of in vitro cultures under immunosuppressive effects of one mycotoxin—aflatoxin Bl produced by the above-mentioned mold was found. Complementary results of this research indicate the cause-result relation and imply practical recommendation for preventive measures. Thus, we also conducted a team study with Bolestaw Smyk from the University of Agriculture in Cracow and his team. The study referred to the opportunities to prevent contamination of indoor environments by adding nontoxic mycostatic agents to the paints. My team initiated research on the influence of laser light stimulation of yeast and cultivated plants for increasing accumulation of selenium and some of their essential elements and vitamins, which are deficient in the food chain when there is higher incidence of cancer [4, 5]. Our interdisciplinary research can find a feedback system between the application of new research techniques for early detection of environmental risk factors on basic studies, including synergistic effects of different pathogens, and research studies for a system approach to primary prevention of environmental risk factors of cancer (leukemia). The common base (submolecular level) for many environmental carcinogens is the elevation of the concentration of free radicals. Thus, we investigated the shape of the curve and intensity of photon emission from neoplastic cells and related normal cells of in vitro culture, finding some differences. There is a growing recognition of the role of free radicals in pathology. Nutritional prevention is based on the supplementation of the human diet with rich "free radicals scavengers"—natural additives (including some selenium compounds and vitamins). Premature delivery of otherwise normal neonates is a problem in both developed and developing countries. What is more, there are common thermodynamic roots of cervical cancer and premature delivery. The results of treatment of cervical carcinoma from 1950 to 1986, obtained from all over the world, show no practical improvement in the 5-year survival rate for stages I (75%-79%), II (53%-58%), and III (28%-33%). Still, the survival rates depend on the stage of cancer at the


33

moment of its detection, not on the method of treatment. It means that, therefore, therapeutic methods have reached their apogee and indicate that medicine has exhausted its possibilities. Medicine, as no other field of human activity, requires from gynecologists the specific responsibility for conscientious constant acquisition of the general knowledge, which always has determined man's approach to cancer. The decisive improvements on it can be achieved only by simultaneous medical and psychological cooperation throughout childhood and youth, since adult cervical cancer is called the "cancer of premature sexual activity." That is why researchers should play a more important part in conquering "cancer of mothers" and preventing "cancer of early sexual life." Cancer can be promoted by many non-specific factors, including such gynecological ones as: 1. Faulty prediction and determination of birth date; 2. Instrumental instead of possible natural labor; 3. Reduction rather than prolongation of lactation; 4. Acceptance of early sexual life; 5. Wrong hormonal therapy; 6. Long-lasting prescription of pills, especially the same ones; 7. Infrequent diagnosis and incorrect therapy of hypothalmic-conditioned abortions and premature deliveries; 8. Clinical, coloposcopic, and cytologic false negative diagnosis of cervical cancer; 9. Eradication of neoplastic lesion without normalization of its near-environment; and 10. Too radical and aggressive treatment of precancerous states. For example, the unexpected diagnosis of cervical intraepithelial neoplasia (CIN) in women seeking professional help due exclusively to infertility is a major emotional trauma, additionally intensified by direct medical manipulation of the uterine cervix. Eradication of cervical pathological changes, particularly cervical conization or amputation, additionally decreases fertility. In order to minimize this jeopardy, it is possible to introduce medical treatment by immunotherapy in such cases. Cryosurgery, CO-laser therapy, and radical electrocautery are all effective in eradicating CIN, but not the cancerogenic. This local ablative therapy must be followed by medical restoration of the body's defense mechanism to prevent the recurrence of the disease. Only immunotherapy as a means of treating the whole body can alone prevent and cure the dissipathogenic states, as a final cause of cancerogenesis of any part of the body, not only the uterine cervix. Neoplasms are biological dissipative structures, and their self-organization in the body is an alternative to the death of its subcellular structures, cells, and tissues whose equilibrium state has been disturbed for too long. This concept not only explains the previous difficulties in the elucidation of both the nature of oncogene-

34


sis and the course of neoplastic diseases but also unifies in thermodynamic terms all known theories of neoplasia. It demonstrates their compatibility with our present knowledge of neogenesis as well as with the description of prerequisites for carcinogenesis put forward by sciences such as physics, chemistry, biology, medicine, sociology, and ecology. For many scientists, such terms as "cancer," "neoplasm," and "tumor" seem interchangeable and carry the same meaning as for the general public. Similarly, for many years physicists were not able to determine unmistakably states far-fromequilibrium as proved by the history of final development of the second principle of thermodynamics by Prigogine. The principle has allowed us to comprehend the origin of so-called dissipative structures as self-organizing formations in states farfrom-equilibrium. The equilibrium state among all systems ensures proper functioning of the body as a unit. This is known to biologists as physiological equilibrium. One sees readily how difficult it is not only to delineate borders between these structures within the organism, but even to imagine them since collectively they constitute one entity, being a higher biological dynamic-spatial organization. Similarly, any of these structures consists of basic microsystems, which are subcellular formations. In turn, each fundamental microsystem is made up of atoms and molecules which together demonstrate biological properties and, like any system, it has characteristic internal features and proficiency of external work. As existence itself, the body's growth and development depends on the equilibrium between particular systems. Therefore, each system, including individual cells, is affected by the balance between its component microsystems. From a thermodynamic point of view, all systems ranging from a fundamental microsystem to a social community—i.e., a system consisting of human beings— are subject to the principles of thermodynamics. Their interrelationships may attain the following three states: equilibrium, near-equilibrium, or far-from-equilibrium. The concept of equilibrium is a state of dynamic balance in which two or more opposing processes simultaneously cancel each other's effects. This state is also known as physiological equilibrium, dynamic equilibrium or simply, steady state. The claim, whereby equilibrium structures (systems) remain stable and resistant to minor disturbances, is fundamental for our line of reasoning. Also, near-equilibrium systems spontaneously tend to attain equilibrium, while the formation of a new structure is not possible. In the two cases, both the entire organism and its elementary microsystems are characterized by the same feature, i.e., they must exist themselves and be capable of performing work directed outside for the benefit of their environment. In equilibrium (health) or near-equilibrium (any disease except cancer), the system remains capable to perform outside work and to respond to the state and requirements of its milieu. If the system attains a state far from equilibrium with the environment, then its capability of external work becomes, for various reasons, limited or virtually nonexistent. The very survival of the system and the preservation of its internal fea-


35

tures are of greater importance. In persistent far-from-equilibrium states, the system is either destroyed or it may evolve into another formation. This is known as a dissipative structure. To exist, it takes from the environment excessive amounts of energy and matter. Cancer constitutes such a biological dissipative structure. Any disease, to a greater or lesser extent, brings the organism to the bifurcation point at the thermodynamic branch of life, which is individually determined for each man. That branch is the finite set of states in which a given human lives and functions. Beyond that point is death, or a violent attainment of thermodynamic balance with the environment, i.e., achieving the same temperature, humidity, or partial pressure of mass as the world. Apart from death, far-from-equilibrium states may lead to a spontaneous organization of dissipative structures. Man as an entity may in this manner change only his personality if the state concerns exclusively psychic and/or emotional processes. A body part which is far from equilibrium in a dissipathogenic state (the bifucation point at the thermodynamic branch) may survive by means of either returning to near-equilibrium or equilibrium, or self-organization into a neoplasm (cancer). Otherwise it must perish (death). Cancer is firstly a local process and it may be restricted or destroyed by a still-efficient regulatory and defense mechanism of the body. In other cases it becomes a generalized disease, and only this stage of its development is commonly known, affecting our idea of neoplasms. Starting at dissipative subcellular structures, the organism is able to suppress any threat to its internal equilibrium. This happens continuously in our body and is thus one of the significant inherent features of life. The precancerous states evolve into cancer more frequently than unaltered tissue. One cannot claim, however, that the currently observed precancerous lesion will in fact develop into cancer. Currently such conditions may be observed in situ, without their removal from the body employing magnetic resonance of atomic nuclei. The conditions are known as dissipathogenic changes. If the organism as a unit does not efficaciously respond to the most primary, though universal signal, manifested as a locally increased requirement for dissipation of matter and energy, the tumor will continue to develop. Its development may still be arrested. Not infrequently, cancer may remain at this stage of development for several years without giviiig rise to any symptoms and complaints. As it disturbs the physiological equilibrium within the body, it is not able to suppress the cancer single-handedly. Medical intervention is required and the cancer's efficacy is nearly complete at this stage. The latency period in the formation of neoplasms is well known. The concept is founded essentially upon the idea that a carcinogen need not act throughout the entire period of carcinogenesis. What we encounter here is a disorder in body homeostasis to which particular cells respond with their whole range of potential states for survival (cellular thermodynamic branch) including a neoplasm. Exposure to radiation may produce mutations in the cellular genome. In case of persistent cellular mutation, the process is irreversible.

36


On its own, however, it does not produce or incite cancer until the cell attains the dissipathogenic state, far-from-equilibrium, as the ultimate cause of carcinogenesis. The course of neoplastic disease depends on the reaction of the organism to the formation and development of the neoplasm as an individual biological system capable of existence, and further growth only if it remains in a state far from the internal equilibrium of the body. Accordingly, the suppression of such a state alone is sufficient to achieve a cure. The sooner the removal from the body of the neoplasm as the source supporting the state far from the equilibrium of its environment by means of taking up and dissipating matter and energy, the faster and easier the cure. The phenomena of molecular fluctuation play a decisive role in nature, which is ruled by the second principle of thermodynamics augmented by these nonlinear events. Far from equilibrium, a system assumes a statistical character, being dependent on the probability of occurrence and the extent of fluctuation. Unlike stable states, i.e., the ones that return to normal after sUght perturbations, fluctuations are not suppressed in unstable states or processes, and so they may considerably increase in an area far from equilibrium. The system may then experience various considerable changes associated with the fluctuation, which has further unsettled the unstable state of the system. The system may also attain a stable stationary state of different dynamic-spatial structure. In the latter case, dissipative structures with a higher degree of spatial and/or temporal organization are formed. They become stable stationary states of the systems whose entropy decreases while the very existence ensures irreversible excessive dissipation of matter and energy. They are part of a complex comprising an open system and its environment, together determined by the second principle of thermodynamics. STAGES OF CARCINOGENESIS Stage 1-a: Intracellular Dissipative Structures When subcellular dissipative structures evolve within a cell, there is an internal cause of neoplastic transformation. Survival may give rise to a tumor as a clone of a single cell. In unicellular organisms, such alterations are known as mutations. Cancer is not observed in these organisms as it requires a multicellular system to thrive. Tissues require cell transformation to increase their chances of surviving those cells capable of self-organization or immortal cells. Stage 2-b: Dysplasia Differentiation of the daughter cells may be incomplete, attaining merely the G state. Accumulation of such cells in the tissue produces a disturbance, mostly within the tissue structure manifested as a varying degree of dysplasia. Simultaneous neoplastic transformation of distant cells occurs when dissipathogenic conditions coexist due to cellular transformation by external forces. The ulti-


37

mate picture in Stage 2 is similar, corresponding to the concept of tissue dysplasia in contrast to dysplastic changes affecting a single cell. Stage 3-c: Neoplastic Tissue (Tumor) In this stage, the tissue (tumor) is made of transformed cells separated from the rest of the body by systemic barriers. The process involves the formation of extracellular substances as well as new vascular and neural networks. Unlike changes in the first two stages, at this level of organization cancer persists in 80 to 90% of developed dissipative structures. Stage 3-d: The Stochastic Phenotype of a Cancer Cell A cell, being an open system, comprises a number of states that make up its thermodynamic branch ranging from thermodynamic equilibrium with the body environment to a far-from-equilibrium state. Beyond the bifurcation point, it may exist only in its new cancerous form. The most likely state in given conditions starts a new thermodynamic branch of a newly formed cell. This is the essential difference between a neoplastic cell and surrounding cells in its environment, although in its structure the former may resemble the latter to a varying degree. A cell's dissipathogenic condition does not constitute a neoplasm but corresponds to the bifurcation point at its thermodynamic branch. A cell within this thermodynamic branch but in a case of dissipathogenic state may return to equilibrium, perish (death), or change into a potential dissipative structure, thus starting an entirely new thermodynamic branch. The last possibility demonstrates that a neoplasm (cancer) is directly in line with a biological system finding itself in a dissipathogenic condition. When the boundary of stability found at a certain critical distance from the state of equilibrium is crossed, any open system may, and under certain circumstances must, become a new dissipative system. In its earliest stage, cancer represents an additional and easily received universal signal to the remaining organism by scattering matter and energy. More specialized means of contact, including via highly differentiated hormones, is possible though less commonly and efficiendy decoded. It involves mobilizing the entire organism to suppress dissipathogenic conditions when cancer formation preserves a state far from the thermodynamic equilibrium in a tissue or organ. A system whose birth, growth, or development has been affected by unfavorable disorders is infrequently capable of removal, suppression, or repair by means of natural homeostatic mechanisms. Such disorders either facilitate or actually lead to deviations from a state of physiological equilibrium and thus promulgate initiation of dissipathogenic conditions. The evolving neoplasm further accelerates unfavorable body entropy and precipitates death of the entire organism, acting therefore, according to the principle of suppression of evolutionarily adverse intrasystemic changes, by means of destroying the organism.

38


Initiation is the self-organization of biological subcellular structures, cells, and tissues into a new dissipative structure. As shown above, this is a long-term process. The onset of initiation facilitates processes of reversal or suppression. The final effect depends on the preservation of the dissipathogenic condition that induces carcinogenesis. Induction means above all the ability to alter the state of a system by changing the fields in which the system is found. The dissipathogenic condition is produced and determined by many factors. Further removal from the initial state of equilibrium may only have two alternative outcomes, (1) destruction, or death, and (2) self-organization, or cancer. Initiation of the latter process is effected through induction at the end of the present thermodynamic branch of the system. Promotion means aiding cancer to preserve in its environment the conditions of the far-from-equilibrium state at the start of its own new thermodynamic branch. Even natural hormones, which support tissue growth, or hyperplasia, may act as promoters. Certain tumors grow as long as there is such hormonal stimulation. Others disappear and are known as hormonally conditioned, but throughout a certain point growth ceases to be affected by hormones without perceptible cellular changes. Due to a different history, i.e., different origin and course of progression, any neoplasm is a separate biological entity unlike other neoplasms. In thermodynamic terms, progression refers to that stage in neoplasm development when acting as an independent biological system it may, unaided, remain in a state far from the dynamic equilibrium of its biological environment, such as whole or part of the organism. The rate at which its cells grow and differentiate is affected by environment and the cellular division cycle at the site where changes develop. There are two extreme situations in the cell cycle. A rapid growth of undifferentiated cells, and slow growth of well-differentiated cells. The points of growth arrested by promotion factors remain a challenge. In the latter instance, however, initiating damage merely affects the capability of terminal differentiation, while promotion begins to operate at the point of growth and differentiation integration, thus covering all points of cell self-regulation. Cancer seems to be a regulator of the lifespan of individual organisms. It is a mechanism involved in species survival, which includes the elimination of organisms poorly adjusted to prevailing environmental conditions. That final phase of its action is seen as neoplastic disease with a full range of consequences. A neoplasm as such is the first important signal informing the body of the necessity of self-defense at a purely biological level, versus so-called "apoptosis" (programmed death of body). Powerful protective and repair mechanisms are active in the organism at the subcellular level, cells, tissues, organs, and systems (immune, nervous, endocrine and humoral). Such structures, by means of additional dissipation of matter and energy, automatically become a warning signal launching distant mechanisms of defense and


39

repair. In a multicellular organism, structures are continually killed, or destroyed and subsequently removed at subcellular and cellular levels, precisely to maintain the life of tissues and hence the whole body. Thousands of cells perish every day in our body, including newly developed neoplastic cells. Additionally, the very formation of dissipative structures in cell organelles leads to their destruction together with the cells in which they are contained, so that the latter do not manage to evolve into overtly cancerous cells. Thus, through neoplasia nature hastens the death of our abnormal cells, allowing us to live longer. We can benefit from this generous stage in the biology of neoplasia, mainly, by avoiding that which weakens the body's defense. The cigarette smoker, alcohol or drug abuser, the unhygienic, etc., will respond poorly to these first neoplastic warning signals. Between the ages of 20 and 65, the incidence of cancer rises a hundred-fold, but the same increase in human mortality is observed a decade earlier, meaning that with age the body's powers of self-defense decrease. Intentional psychic attitude is another major form of self-defense against neoplasms. Knowledge of the cancer's essence frees one from the fear of its mysterious character, incurability, and possibilities of contagiousness and inheritance, offering a unique chance of purely human self-help when the neoplasm has already developed. Before a neoplasm defeats the body's defensive mechanisms, it has been restricted to an area long enough to be diagnosed and removed, although it might be subjectively asymptomatic. This is the task for health education, properly trained medical and nursing personnel, well-equipped health facilities, and rehabilitation centers. Social community self-defense is of utmost importance for controlling cancer. Improvement and widespread introduction of appropriate diagnostic methods of early stages in biological development of neoplasms should be instituted. The most important thing is to eradicate the disease prior to the beginning of pathological symptoms and signs. It is necessary to continue research into the body's natural defense mechanism and to pursue more recent modalities of therapy, such as: interferon, monoclonal antibodies, immunochemotherapy, and computerized techniques of hormone therapy. The community must protect the natural and indoor environment, limiting the conditions that favor the formation and development of social evils such as dependence on alcohol and drugs, tobacco smoking, industrial pollution, undernutrition, etc. In addition, it is necessary to set up community facilities to provide adequate care to people suffering from neoplastic diseases, with stress on emotional, as well as physical support. Review of environmental risk factors of neoplasms could be a starting point for primary prevention focused on elimination of related health hazards at the sources, as well as on fortification of diet with protective additives, etc.

40


APPENDIX 1: CANCER RISK FACTORS AND PREVENTION Causes and Secondary Factors

Time Scale

Genetic predisposition or susceptibility Exposure to mutagens; malnutrition

Years before birth Conception and pregnancy

Prevention

Genetic counseling; Diet adequate in free radical scavengers Perinatal examination; minimalization of exposure; Supplementation of nutrition with protective factors—e.g., antioxidants, radioprotectors, free-radical scavengers, etc.

Childhood UV-light; Carcinogens; Virus infection Lack of hygiene; Smoking, drugs. Alcoholism

Prenatal supervision; Education Adolescence Adulthood Old age

Improvement of living standard; Regular self-inspection; Early treatment; No smoking; Proper diet; Regular medical examinations (including NMR use) Avoidance of fats, sugar, excessive meat, alcohol, etc.

After G. N. Schrauzer, [38] modified

Active prevention involves the cooperation of scientists with persons dealing with education, agriculture, farming, the food industry, and various areas of technology (including civil engineering technology to protect the indoor environment). This refers to the elimination of potential risk factors from the whole human environment and the optimization of the composition of drinking water and food. In these activities everyone can participate as a consumer. The particular role, however, belongs to engineers, farmers, and decision makers. These professional groups have the strongest influence on the natural environment. Thus, as far as the elimination of the sources of contamination with carcinogens is concerned, their significance can be greater than the one of physicians or biologists, although biomedical studies are basic, both in relation to motivation as well as verification of effectiveness of common action for the elimination of environmental cancer risk factors. These activities are the main reasons for the management of environ-


41

ments suitable for healthy work, living conditions and rest, the promotion of environmentally friendly technologies of production, transport, and consumption models (including diet). Knowing synergism of many environmental risk factors of cancer, one must conclude the need of integrated action for the improvement of the whole human environment. This refers both to individual decisions (modifying habits, e.g., giving up smoking, modifying diet regarding increase of the consumption of pollutant-free fruit and vegetables rich in antioxidants, fiber, whole complex of essential elements, vitamins, etc.) as well as the cooperation of institutions on local and global scale. Apart from this action, there is prevention related to environmental carcinogenesis including chemical, radioactive, biological, and other contamination. Both primary and secondary sources of this contamination should be considered in land reclamation, proper management of heavily contaminated areas, and the monitoring of food on a large scale. Another direction is the fortification of soils and diet with protective substances that diminish the input (permeability) of carcinogens into the food chain and human organisms. One more area of primary prevention is creating conditions for proper interpersonal relationships, because psychological factors play an important role in the distortions of neurohormonal function and lowering the effectiveness of some protective mechanisms against carcinogenic process [9]. The interference of physical carcinogenic factors with the state of immunosuppression and the distortion of homeostasis in the organism can be regarded as frequent circumstances increasing the risk of the appearance of cancer symptoms. This can be confirmed in the contemporary interpretation of a carcinogenic process as a whole-organism phenomenon seen from the point of view of information theory [6] and thermodynamics [31, 32]. Cell-type specific gene mutation could be induced by some pollutants, e.g., 7,12-dimethylbenz (a) anthracene (DMBA). The type of tumors/transformed cells depends on the mode of administration of carcinogens in experimental mice, including transplacental exposure [36]. There are also some data about the interaction of genotoxic and non-genotoxic mechanisms in multistage carcinogenesis [39]. In addition to the accumulation of genetic changes during this process, some non-genotoxic effects could indirectly generate genetic changes, e.g., by active oxygen species, cytosine methylation, and stimulation of mitosis [39]. Discovery of the inhibition of gap junctional intercellular communication by different tumor-promoting agents is a new field of study about the contribution of non-genetic changes in tumor progression [34]. Disruption of feedback system and homeostatic properties seems to be related to disturbances in the exchange of information between some elements involved in spatial-temporal order within integrated biological systems from cellular to organismal level of organization [5]. Carcinogenesis can be interpreted as the result of so-called "environmental noises" that can induce as very important mutations in genetic code, but also disturb the transfer of information necessary for the synthesis of both structural

42


elements and enzymes, neurotransmitters, and hormones [8, 32, 33]. Changes appearing in single cells can be interpreted as the appearance of biological dissipative structures. However, when their range transgresses certain threshold value, irreversible pathological changes including possible death can take place. This happens with the transgression of a certain value of the distortion of information transfer and disturbance of bioenergetic processes, which causes the increase of entropy in the system [32, 33]. Closer discussion of both theoretical premises for this interpretation and some examples of interdisciplinary case studies could be achieved with more active participation of experts from different disciplines focused on primary prevention of cancer. There is a certain analogy from the biocybernetic point of view, between the integration of different levels of structure and function of human organism starting from an individual cell [6], primary prevention of cancer starting from the level of ecosystem, and ending on the level of biosphere [4]. For the effective prophylaxis it seems to be necessary to integrate individual and common efforts including both sphere of social and technical activity for an environmentally friendly model of civilization. APPENDIX 2 PAPER BY DOBROWOLSKI AND KLIMEK ANNEX

Model of the influence of external and internal risk factors for leukemia development Output (leukemic effect)

Input (risk factors)

innmunological j reactivity

Xl

U2

genetic factors

X2

U3

oncorna vinjses

X3

-•y

Basic data for mathematical approach to environmental leukemogenesis. (After Kwiatkowski/from Aleksandrowicz et al.)


43

In Vitro Assay Template Correct Substrates: dATP + [a-32p] dTTP Incorrect Substrate:[3H] dGTP DNA Polymerase Mg2+ - Exogenous Agents (Metal Cations)

Known Carcinogens and/or Mutagens Ag Be Cd Co Cr Cu Mn Ni Pb As* Se* *Not Tested

Increased Misincorporation

No Change in Fidelity

Ag Be Cd Co Cr Cu Mn Ni Pb

Al Ba Ca Fe K Rb Mg Na Sr Zn

Fidelity assay screen for mutagens and/or carcinogens. (After Loeb et al., from Schrauzer, 1978 [37])

(After M. and R. Klimek gestational calculator, from Klimek, 1992 [33])

44


REFERENCES 1. Aleksandrowicz, J. and Czyzewska-Wazewska, M., "Selenium as radioprotective substance," Haematologica, 17: 61-62, 1976. 2. Aleksandrowicz J., Skotnicki, A. B. et al.. Leukemia ecology, Natl. Library Med., Natl. Sci. Foundation, Washington, D.C., NCSTEI, Warsaw 1982. 3. Amaya, K., "Report on cancer origin," International Conference on Primary Prevention, Krakow, 1995. 4. Badiello, R., Trenta, A., Matti, M., Marettini, M., "Azione radioprotective dei seleno-derivati. Affesto della selenourea in v/vo," Med. Nucleare Radiobiol Latina, 10: 1-12, 1967. 5. Dobrowolski, J., "Studies on photoreactivation," Acta Biol Cracoviensa, ser. zool. 18:211-220, 1975. 6. Dobrowolski J. W., "New aspects of environmental protection against developmental malformations and the cancer incidents," Scientists for better environment, pp. 517-527, Fukushima, Y. et al., eds., Sci. Council of Japan, HESQ Asahi Press, Tokyo, 1976. 7. Dobrowolski, J. W., Smyk, B., "Environmental risk factors of cancer and their primary prevention,"/. Env. Pathol. Toxicol. Oncol, 12(1): 55-57, 1993. 8. Dobrowolski, J. W., Tadeusiewicz, R., "Interpretation of cancerogenesis from the point of view of information theory," (in Polish), Gin. Pol, 57(12): 111-111, 1986. 9. Dobrowolski, J. W., Vohora, S. B., eds., "Ecologism in health protection," (in Polish), Ossolineum, Wroceaw, Krakow, 1989. 10. Dobrowolski-unpublished 11. Ferenczi, L. Z., Hill, G. B., Scrimger, J. W., "Ultraviolet radiation and the incidence of cancer of the skin in Alberta," Medecine/Biologie/Environment, September-December, pp. 48-53, 1982. 12. Gupta, D., WoUmann, H. A., Fedor-Freybergh, P. G., eds., Pathofisiology ofimmune-neuroendocrine communication circuit.. Mattes Verlag, Heidelberg, 1994. 13. Harada, M., "Minamata disease," Report, Kumamoto University, 1976. 14. Heinrich, U., "Carcinogenic effects of solid particles," Toxic and carcinogenic effects of solid particles in the respiratory tract, Mohr U. et al., eds., pp. 69-73 ILSI Press, Washington, D.C., 1994. 15. Heinrich, U. et al., "The carcinogenic effects of carbon black particles and tar-pitch concentration aerosol after inhalation exposure of rats." Ann. occup. Hig. (38) suppl. 1: 351-356, 1994. 16.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 4, 1974. 17. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 21, 1979. 18. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 34, 1984. 19. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 37, 1985.


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20. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 38, 1986. ll.IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 40, 1986. 22. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 41, 1986. 23.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 42, 1987. 24. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 43, 1988. 25. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 44, 1988. 26. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 45, 1989. 27. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 46, 1989. 28. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 47, 1989. 29. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 49, 1990. 30. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 51, 1991. 31. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 52, 1991. 32. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 53, 1991. 33.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 55, 1992. 34. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 56, 1993. 35.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 58, 1993. 36. Janczarski, I. Personal communication, 1980. 37. Janicki, K., Dobrowolski, J. W., Krashicki, K., "Correlation between contamination of the rural environment with mercury and occurrence of leukemia in men and cattle," Chemosphere 16: 253-257, 1987. 38. Jansson, N. K., "Topographical study in cancer in Denmark," Ninth International Cancer Congress Proceedings, Abstract 1017, Tokyo, 1966. 39. Kacki, E., Stempczynska, J., "Correlational research into the environmental etiology of cancer." pp. 607-611, European Centre for Pollution Research, London, 1993. 40. Karaczkiewicz, M., "Remarks concerning the geographical distribution of bovine leukemia in Poland," Przegl Let, 11: 391-394, 1970. 42. Klimek, R., "Cause, Predisposing Factors and Host-defense," (in Polish, English summary), PWN, Warszawa, 1985.

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43. Klimek, R., Peri-natal Psycho-Medicine, DReAM, Cracow 1992. 44. Kozik, R., Personal communication, 1975. 45. Krasnicki, K., Personal communication, 1986. 46. Krutovskikh, V. A. et al., "Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with Aberrant Localization of Connexin Proteins," Laboratory Investigation, 72 (5), 571-577, 1995. 47. Marchlewski, Necki. 48. Niwelinski, J., Zamorska, L. "Report on contamination of human placenta and cytochemical changes of some enzymes activity," International Conference on Elements in Health and Disease, Adana, 1990. 49. NNAS, RNAE, CIOM, Indoor pollutants, National Academy Press, Washington, D.C., 1981. 50. Otto, H., "Vergleich der Krankheltsblider der menschlichen und tierischen leukosen, identische und differente symptoma," Z Arztl. Fortbil, 57: 461, 1963. 51. Samal, J. et al.. Report on environmental risk factors of cancer at International Conference on Ecology and Cancer, Brussels, 1982. 52. Sasaki, K. et al., "Cell-type-specific rat mutations but no microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells," Cancer Res. 55, 3,513-3,516, 1995. 53. Schrauzer, G. N., ed., "Inorganic and nutritional aspects oicdLWcox'' Advances in experimental medicine and biology. Plenum Press, New York, London, 1978. 54. Schrauzer, G. N., "Trace elements in cancer diagnosis and therapy. A review," Trace Element-Analytical Chemistry in Medicine and Biology, Vol. 4, P. Bratter, P. Schramel, eds., pp. 403^17, Walter de Gruyter Co., Berlin, New York, 1987. 55. Shamberger, R. J., "Relationship of selenium to cancer. Inhibitory effects of selenium on cancerogenesis," Journal of National Cancer Institution., 44: 931-936, 1970. 56. Smyk, B., Aleksandrowicz, J., "The association of neoplastic diseases and mycotoxins in the environment," Tx. Rep. Biol. Med., 31: 715-726, 1973. 57. Tomassi, J., Personal communication, 1977. 58. Truhaut, R., Dobrowolski, J. W., "Ecotoxicology and primary prevention," Introduction to the Human Environment, Team textbook, J. W. Dobrowolski, B. Bhatia, D. Banerjee, New Delhi Press. 59. Vincente, de R., Personal communication, 1986. 60. Vohora, S. B., Dobrowolski, J. W., eds.. New horizons of health aspects of elements, Hamdard University, New Delhi, 1990. 61. Yamasaki, H., "Non-genotoxic mechanisms of carcinogenesis: studies of cell transformation and gap junctional intercellular communication," Toxicology Letters 77:55-61, 1995.

CHAPTER 2 RESPIRATORY FUNCTION CHANGES FROM INHALATION OF POLLUTED AIR Shieh-Ching Yang and Sze-Piao Yang Pulmonary Function Laboratory National Taiwan University Hospital No. 7, Chung-Shan S. Rd. Taipei, Taiwan, Repubhc of China

CONTENTS FORMS OF AIR POLLUTANTS, 47 Effects of SO2 and NO2 on Lung Function, 48 URBAN CONCENTRATIONS OF AIR POLLUTANTS, 48 PULMONARY FUNCTION PARAMETERS, 49 BRONCHIAL PROVOCATION TEST, 50 PULMONARY EFFECTS OF AIR POLLUTANTS IN ASTHMATICS, 51 CONCLUSION, 51 REFERENCES, 51 Because of civilization and increased industrial activities, the effects of air pollution on health and diseases have attracted much attention in recent years. Air pollution means a state resulting from manmade wastes produced so rapidly that they accumulate in concentrations that cannot be dispersed by the normal selfcleansing propensities of the atmosphere. Air pollution is thought to be one of the most important risk factors for respiratory diseases, particularly for bronchial asthma and chronic obstructive pulmonary disease (COPD). However, a direct causal relationship is not easy to prove because air pollutants do not occur as individual entities but in combination. In addition, the concentration and duration of exposure to air pollutants required for inducing an adverse pulmonary effect have not yet been determined. FORMS OF AIR POLLUTANTS Components of air pollution are usually inhomogeneous and may include suspended particulates, metallic contaminants (lead), chemicals, and irritating or toxic gases such as sulfur dioxide (SO2), nitrogen dioxide (NO2), ozone and carbon monoxide (CO). Particulate pollutants may appear in the forms of fog, smoke, or dust, according to their size and physical properties. Small-sized particles (less than 1 ^m) have catalytic effects on other air pollutants, particularly the oxidation of SO2, so as to produce irritation to the lower respiratory tract. Dust, like carbon or silica, are large particles that can produce pneumoconiosis if heavily exposed. 47

48


Polycyclic aromatic hydrocarbons (PAHs) are chemicals generated mainly by automobile fuel combustion, and they are well-known carcinogens. Effects of SO2 and NO2 on Lung Function The adverse effects of SO2 and NO2 on respiratory function have been investigated. Inhalation of high concentrations of SO2 (4-6 ppm) induced bronchoconstriction and caused an acute reduction in maximal expiratory flow (MEF) in normal subjects. This response can be blocked by treatment with atropine, suggesting that it is mediated by parasympathetic reflex pathways. Sulfur dioxide itself appears to cause only mildly irritating effects. The oxidation of SO2 leads to a much more prominent injurious effect in the small airways. At higher concentrations (i.e., 10-15 ppm), SO2 can inhibit mucociliary clearance of the respiratory tract. Such a concentration, however, is 300-fold higher than those observed in an urban polluted atmosphere. In a clinical setting, it was reported that there was a threefold increase in acute asthmatic episodes of adults on days characterized by high, compared to low, sulfate content in the residential environment. A significant change in pulmonary function of COPD patients after exposure for 2 hr to 0.8 ppm or less of SO2 was also found. Studies showed that asthmatic patients were sensitive to SO2 at concentrations as low as 0.5-1 ppm. Therefore, persons with bronchial hyper-reactivity are more likely to develop bronchoconstriction as a result of inhaling concentrations of SO2 that are well tolerated by normal subjects. Nitrogen oxides are generated mainly by power plants and automobile fuel combustion. The effects of NO2 on pulmonary function is incompletely understood. Nitrogen dioxide in high doses (40 ppm) is a prominent airway irritant. Results of studies on the effects of exposure to low levels ( 0 . 1 ^ ppm) of NO2, however, have been controversial. It was found that exposure to 4 ppm NO2 for 75 min., accompanied by intermittent exercise, had no effect on pulmonary function. On the contrary, exposure of asthmatics to only 0.1 ppm NO2 for 1 hr resulted in increased airway reactivity. Increased airway reactivity to carbachol challenge in normal humans was also observed after exposure to 1.5 ppm NO2 for 3 hrs. Pulmonary epithelial injury associated with mast cell influx and/or mediator release were probably the underlying mechanisms. Moreover, increased capillary permeability might occur, which leads to focal pulmonary edema. URBAN CONCENTRATIONS OF AIR POLLUTANTS The polluted air is collected for a specific site within the city and compressed into 40-L sample bags. The concentrations of air pollutants in the sample are determined by various methods. For example, total suspended particulates (TSP) and size-fractionated particulate samples are collected by using high-volume samplers equipped with acid-washed quartz-fiber filters. Particle-size can be viewed directly

Respiratory Function Changes from Inhalation of Polluted Air

49

with a scanning electron microscope. Analysis of atmospheric SO2 is performed with the colorimetric method. The NO2 concentration can be measured with a spectrophotometer and a midget-fritted bubbler used with the absorbing reagent. The reagent is a mixture of sulfanilic acid, N-(l-naphthyl)-ethylenediamine dihydrochloride, and acetic acid. The carbon monoxide (CO) concentration is usually determined with an infrared CO analyzer or a gas chromatograph. Urban concentrations of SO2 and NO2 are usually in the range of 0-1,000 ppb and 100-1,000 ppb, respectively. The concentrations of air pollutants may be influenced by time and site of sample collection, and by meteorologic factors such as humidity, wind velocity, temperature, and barometric pressure. Tunnel air collected during traffic jams in rush hours has much higher levels of pollutants than the average urban air. Typical air pollutant concentrations of a sample collected from a large city are shown in Table 1. Table 1 Components of air pollution and their concentrations* Pollutant

Concentration

S02,ppb 124.0 NO2, ppb 502.0 CO, ppm 3.6 TSP, [ig/M^ 227.0 ^Sample collected from Lin-Shan South Road Tunnel of Taipei, Taiwan, in August, 1993. PULMONARY FUNCTION PARAMETERS More than fifty parameters are concerned in a complete pulmonary function testing. For epidemiologic studies and for determining the adverse effects of air pollutants on respiratory function, however, it is possible to employ less than 10 parameters. Of these, some are volume parameters, e.g., forced vital capacity (FVC) and functional residual capacity (FRC); some are flow parameters, e.g., forced expiratory volume in one second (FEVl) and peak expiratory flow rate (PEFR); the remaining may be a resistance parameter (airway resistance. Raw) or a diffusion parameter (diffusing capacity of the lung for carbon monoxide, DLCO). Most flow parameters and some of the volume parameters, such as FVC, can be simply determined with a maximal expiratory flow volume maneuver. An automated spirometer is often used for this purpose. The instrument requires periodical calibrations, and its accuracy for volume and flow measurements should be within ±3-5%, or less than 150 ml. Determination of FRC and Raw requires an integrated multifunctional lung function analyzer or a body-plethysmograph. Helium, nitrogen, and carbon monoxide are used as the reference or test gases.

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The standard procedures for a flow-volume determination are described briefly as follows: Subjects are seated and wear nose-clips. Once adjusted to breathing through the mouthpiece, the subject exhales to residual volume (RV) and then inspires to total lung capacity (TLC), and then rapidly expires to RV again with maximal effort. Time-volume and flow-volume curves are recorded simultaneously. Up to three trials are performed, and the average of two technically acceptable tests is reported. The tests have to agree within 5% of each other to be considered acceptable. BRONCHIAL PROVOCATION TEST The assessment of airway hyper-reactivity is an increasingly popular but difficult task for a pulmonary function laboratory. The test is often called a "challenge" and uses several kinds of stimulants such as methacholine, histamine, carbachol, cold exposure, and specific antigens like pollens and distilled water, to provoke airway changes. A number of techniques exist for using each stimulant, but no agreement has been reached on which stimulant or technique best evaluates bronchial hyperreactivity. The major indications for a bronchial challenge are to diagnose asthma and document the severity of bronchial hyper-reactivity. Methacholine and histamine are the two most commonly used stimulants for a bronchial provocation test. The results of challenges with methacholine and histamine have good correlation and reproducibility. It is generally agreed that equal concentrations of methacholine and histamine produce similar results in asthmatics. However, histamine is associated with more side effects, e.g., flushing, throat irritation, and headache, especially at higher doses. Consequently, methacholine has become the agent of choice in clinical practice. Response to bronchial provocation tests is measured as change or lack of change in pulmonary function. Simple spirometry produces FEVl; the body plethysmograph produces Raw and specific conductance by panting or quiet breathing. Although it is recommended that FEVl be included in all bronchial challenge tests, FEVl may not be the most sensitive measurement. Furthermore, repeated expiratory efforts during simple spirometry can cause bronchoconstriction. Apparently, the sensitivities among various lung function parameters to detect a change after bronchial challenge are unequal. Airway resistance and specific conductance are more sensitive parameters than FEVl. This is why, in some cases, Raw and specific conductance are measured with a body box or newer devices. The "Astograph" is a new tidal breathing method for the bronchial provocation test. Bronchial hyper-reactivity is examined by directly writing the dose-response curve of respiratory resistance (Rrs) during the continuous inhalation of methacholine in stepwise incremental concentrations. Respiratory resistance is measured by the forced oscillation method. The concentrations of methacholine tested are from 0.01 to 25 mg/ml. The instrumentation also consists of 12 nebulizers capable of generating aerosols with a particle size of less than 5 |Lim. Aerosols are delivered

Respiratory Function Changes from Inhalation of Polluted Air

51

from each nebulizer for 1 min in sequence and inhaled by the subject until Rrs reach twice the baseline values. In that case, challenge is terminated and a bronchodilator is administered immediately. The test results are thereby considered positive. The cumulative dose of methacholine at the point where Rrs starts to increase prominently is calculated and expressed as methacholine units, i.e., 1 unit equals 1 min inhalation of 1 mg/ml methacholine. This oscillation technique is characterized by its advantage to proceed the challenge during quiet tidal breathing and to complete the test during a short period of time. The cumulative dose of methacholine instead of PD20FEV1 (the provocative dose that causes a 20% fall in FEVl) is calculated. PULMONARY EFFECTS OF AIR POLLUTANTS IN ASTHMATICS Many pertinent studies concerning the pulmonary effects in asthmatic subjects after acute inhalation of low concentrations of air pollutants had been done in the 1980-1990s. It is difficult to make direct comparisons among these studies because of differences in clinical characteristics of the subjects, type and concentrations of pollutants, exposure time and conditions, and lung function measured. Polluted tunnel air is inhomogeneous and has higher component concentrations than that of ambient air. In addition, subjects with a hypersensitive airway may exhibit an exaggerated response to short-term exposure of such low levels of air pollutants. Moreover, bronchial challenge aggravates the pulmonary functional response to air pollutants. It is not a surprise, therefore, that by using polluted tunnel air and bronchial provocation with methacholine, it is possible to demonstrate directly the adverse effects of polluted urban air to subjects with airway hypersensitivity. CONCLUSION Although a cross-sectional study using short-term exposures to low levels of air pollutants may be insufficient to cause a fall in lung function of the general population, exposure to polluted tunnel air may trigger respiratory symptoms in certain subjects such as those who have underlying bronchial hyper-reactivity while riding motorcycles. Careful selection of subjects and lung function parameters are essential to a successful demonstration of the unfavorable effects of polluted air. REFERENCES Brunekreef, B., Kinney, P. L., Ware, J. H., et al. "Sensitive subgroups and normal variation in pulmonary function." Health Perspect., 1991; 90:189-93. Chai, H., Farr, R. S., Froehlich, L. A., et al. "Standardization of bronchial inhalation procedures." /. Allergy Clin. Immunol^ 1975; 56:323-27.

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Frampton, M. W., Morrow, P. E., Cox, C , Gibb, F. R., Speers, D. M., Utell, M. J. "Effects of nitrogen dioxide exposure on pulmonary function and airway reactivity in normal humans." Am. Rev. Respir. Dis., 1991; 143:522-27. Koenig, J. Q., Pierson, W. E., Horike, M. "The effects of inhaled sulfuric acid on pulmonary function in adolescent asthmatics." Am. Rev. Respir. Dis., 1983; 128:221-25. Koenig, J. Q., Covert, D. S., Marshall, S. G., Belle, G. V., Pierson, W. E. "The effects of ozone and nitrogen dioxide on pulmonary function in healthy and asthmatic adolescents." Am. Rev. Respir. Dis., 1987; 136:1,152-1,157. Linn, W. S., Solomon, L. C , Trim, S. C , et al. "Effects of exposure to 4 ppm nitrogen dioxide in healthy and asthmatic volunteers." Arch. Environ. Health, 1991; 46:296-99. Orehek, J., Massari, J. P., Gayrard, P., Grimaud, C , Charpin, J. "Effect of shortterm, low-level nitrogen dioxide exposure on bronchial sensitivity of asthmatic patients." J. Clin. Invest., 1976; 57:301-07. Takishima, T., Hida, W., Sasaki, H., Suzuki, S., Sasaki, T. "Direct-writing recorder of the dose-response curves of the airway to methacholine: clinical application." Chest, 1981;80:600-06. Xu, X. P., Dockery, D. W., Wang, L. H. "Effects of air pollution on adult pulmonary function." Arc/i. Environ. Health, 1991; 46:198-206. Yamaguchi, S., Kano, K., Shimojo, N., et al. "Risk factors in chronic obstructive pulmonary malfunction and 'chronic bronchitis' symptoms in Beijing district: a joint study between Japan and China." J. Epidemiol. Comm. Health, 1988; 43:1-6. Yang, S. C , Yang, S. P. "Respiratory function changes from inhalation of polluted dk:' Arch. Environ. Health, 1994; 49: 182-87. Zeidberg, L. D., Prindle, R. A., Landau, E. "The Nashville air pollution study: L Sulfur dioxide and bronchial asthma. A preliminary report." Am. Rev. Respir. Dis., 1961;84:489-503.

CHAPTER 3 BIOLOGICAL MARKERS OF EARLY HEALTH EFFECTS IN THE ASSESSMENT OF THE RISK OF CANCER IN PEOPLE EXPOSED TO ENVIRONMENTAL CARCINOGENS Janusz A. Indulski and Waldemar Lutz The Nofer Institute of Occupational Medicine Lodz, Poland The continuing chemicalization of human life associated with the development of civilization is responsible for a steady increase in health risk due to chemical pollution of the environment. Large quantities of industrial and household chemical wastes are deposited (often in an uncontrolled manner) in the vicinity of human habitation places. Toxic chemicals permeating the soil, water, or air pose a real threat to the people living in the polluted areas. In the majority of cases, the effects of exposure to environmental pollutants take the form of prolonged exposure to such concentrations of the pollutants that do not result in acute forms of diseases. The majority of these effects, however, lead to some biological changes in the organisms of the exposed people and often contribute to increased incidence of chronic disease. As no overt clinical manifestations are evident, the only way to detect minute pathological changes resulting from prolonged exposure to environmental chemicals is by detecting them on the cellular level, or in the cellular metabolism products present in the biological fluids available for testing. The idea of biomarkers [1,2,3,4,5] has become very useful in detecting the adverse effects of environmental pollution on the human organism and taking suitable preventive measures. The concept of a biomarker is associated with laboratory testing of physiological fluids or cells from a person exposed to toxic chemicals; the result of the test, expressed in the biochemical or cellular terms, points either to the presence or content of the toxic chemical agent, or to adverse health effects caused by the chemical agent in the human organism. The concept of the biomarkers also covers an estimate of the individual susceptibility to harmful agents [3]. By integrating the concepts of exposure, health effect, and individual susceptibility, enhanced analysis of the complex problem of health risk in a person exposed to environmental toxic factors has become possible. The measurements performed directly on the material taken from exposed people makes it possible to eliminate speculation from the estimations of the associated health risks. This has been made possible because the effects of toxic chemicals are not related to their concentrations in the polluted environment but to the quantities of the chemicals that have permeated the relevant tissues and organs, causing specified toxic effects. The concept of biomarkers also made it possible to eliminate errors resulting from separately assessing the effects of occupational and communal environment.

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as well as to the lifestyle factors, such as tobacco smoking, alcohol drinking, and dietetic habits. The concept of biomarkers tends to highlight the assessment of health condition, or possible risks to health condition, rather than the assessment of the conditions of external chemical pollution [1]. The biomarkers measure individual hazard from exposure to environmental toxicants. They are indices of abnormalities within different biological systems (at the level of the whole body or of particular organs, tissues, and cells) that may be induced by hazardous agents of various chemical or biological natures. The biomarkers make it possible to monitor the processes within the organism from the moment of toxic agent penetration to the development of clinical symptoms. Biomarkers can be compared to windows which allow us to look into a "black box" and track the fate of the toxic agent and the effects it produces in the exposed organism. It is worth pointing out, however, that the very notion of biomarker refers not only to the data on the particular laboratory test results—e.g., toxic agent concentration or enzymatic activity—but also to the fact that it may provide tentative information about the possible concentration of the agent or the enzymatic activity within those tissues which are inaccessible for testing. Biomarkers can supply measurable data on the rate of absorption of the environmental contaminant and answer whether the critical dose leading to eventual molecular, subcellular, and cellular changes has reached the critical organ. The nature of these abnormalities is determined both by the type of the toxic agent and the quantity that penetrates the target organ as well as the actual duration of exposure [6]. At present it seems that the assessment of chemical exposure of the individual people living in the specified environment is more essential than the assessment of the chemical contamination present in that environment. The assessment can be performed either by estimating the quantity of the absorbed toxic agent with the aid of exposure biomarkers, or by assessing biochemical changes occurring in the cells of the exposed organism with the aid of health effect biomarkers. In view of the fact that we cannot carry out a complete analysis of environmental chemicals entering the system or determine their toxic potential, it seems reasonable to undertake measurement of the resulting health effects in exposed individuals along with routine evaluation of exposure based on measuring the toxic agent concentration. Complicated and costly as it is, the performance of health effect assessment seems highly justified from the point of view of social and economic needs. In many a case, the excessive expenditure connected with the relocation of inhabitants of the hazardous territories can thus be avoided. This may also help to prevent panic among the population of contaminated areas. Moreover, the health effects may form the grounds for undertaking relevant preventive measures. The latter, in turn, increases the probability that the health effects of exposure, when detected early, will still be reversible and the discontinuation of exposure and the appropriate treatment will inhibit their transformation into permanent clinical effects [7]. Health effect biomarkers are indicators that provide information on the changes in the organism which took place as a result of exposure to an environmental toxic

Biological Markers of Early Health Effects in Environmental Carcinogenesis

55

agent. This biomarker group includes, in a measurable way, these phenomena which constitute either the individual stages of the pathogenic process or the manifestations of the disease itself. The biological response of the organism to environmental toxic agents includes a relatively wide range of changes, from temporary and reversible (which do not produce any noticeable health effects) to permanent changes associated with increased disease incidence and mortality. This wide spectrum of changes may be arbitrarily divided into several categories: a) Metabolic disturbances which remain within the physiological limits; b) Physiological dysfunctions not accompanied by any detectable morphological changes in tissues; c) Pathological changes in tissues, but not accompanied by overt manifestations of disease; d) Clinically overt pathology. Environmental medicine and environmental toxicology are interested primarily in these biomarkers that enable the detection of changes occurring in the organism which are classified to the first three categories. These biomarkers are referred to as pre-clinical or early health effect biomarkers [3]. In evaluating the adverse effects of a chemically polluted environment on the human organism, most research concentrates on finding biomarkers of the carcinogenic process. The research refers both to the search for the exposure and the health effect biomarkers. In the latter case, particular attention is paid to the biomarkers of early stages of carcinogenesis. It is intended to find such biomarkers that would make it possible to detect those stages of carcinogenesis which occur many years in advance of clinically overt cancer forms [8]. The principal stages of the carcinogenic process have already been studied in some detail, and it is known that the process involves many stages, including both cellular genetic as well as epigenetic changes [9]. The following stages can be distinguished in the carcinogenic process: initiation, promotion, conversion, and progression. During the first (initiation) stage, DNA is damaged as a result of exposing a cell to carcinogenic agents. This change is hereditary and it is transferred to descendant cells. In the next (promotion) stage, numerous agents are involved, which do not necessarily lead to further direct changes in the genetic material of the cell, but by acting rather as gene activators and changing the phenotype of the initiated cells, cause selective and clonal cell expansion. The initiation process itself seldom leads to the formation of cancer cells when not followed by the promotion stage. On the other hand, in order for the promotion stage to occur in the process of carcinogenesis, it must be preceded by the initiation process. The promoting agents can be described as those that display no, or very little, carcinogenic activity when they occur alone. When they are, however, combined with agents causing genetic changes in the cells (initiating agents), they remarkably enhance their mutagenic properties. During the stage of the carcinogenesis conversion process, the benign forms of cancer are transformed into malignant ones. Now it seems that this stage

56


requires some phenomena to occur which cause changes in DNA. Thus, both the initiation and the third stage of the carcinogenesis process require participation of chemical (or physical) agents that produce changes in DNA structure. Progression, the final stage of the carcinogenesis process, leads to the development of the clinically detectable form of cancer. Each of the above stages of the carcinogenesis process can be monitored with the aid of suitable biomarkers (Figure 1). The initiation stage, although short-lasting, can be relatively easily monitored by employing currendy available biochemical (e.g., carcinogen adducts with DNA or proteins) or cytogenetic (chromosome aberrations, sister chromatid exchange, presence of micronuclei) techniques. For example, the formation of the carcinogen/DNA adducts is detectable with the sensitivity of 1 adduct per lO'^ to 10^^ DNA nucleotides [10]. The new possibilities in detecting DNA structure changes attributable to the carcinogenesis initiation process have been opened up by the PCR (Polymerase Chain Reaction) techniques. The information supplied by the biomarkers in evaluating the process of initiation, unfortunately, plays a limited role in cancer prevention. It demonstrates that carcinogenes genotoxically affect the cells of the exposed people in the chemically polluted environment, but because the initiation process is very short-lasting and practically irreversible, detection of the initiation process does not offer any opportunity for effective intervention [11]. The biomarkers of early health effects are essential in the prevention of the neoplastic disease induced by environmental carcinogenic agents. They provide information supplementary to that obtained by using the exposure biomarkers. From the data obtained by assessment of these biomarkers, it can be concluded whether the exposure to environmental carcinogenic factors has triggered cellular mechanisms capable of causing neoplastic transformation. The biomarkers of early health effects represent the intermediate stages of the carcinogenesis process, between the initiation and the conversion, and the clinically overt stages. Thus, the cellular process capable of being indicated by the biomarkers of early health effects corresponds to the promotion stage. Detecting those processes is extremely important, as the promotion process lasts many years and seems to be reversible. Therefore, some preventive action can be taken to stop development of cancer [12]. The biomarkers of early health effects, from this point on referred to as promotion biomarkers, can be used not only to detect the early stages of the carcinogenesis process, but also to determine the efficiency of preventive steps taken [12]. The cellular changes, which involve activation of protooncogenes and their transformation into oncogenes, as well as deactivation of the suppressor genes, seem to play a dominant role in the promotion process. The knowledge of the influence of environmental carcinogenes on the cellular genome is, in fact, limited only to the process of protooncogene activation, and litde is known about their influence on the deactivation of suppressor genes. Nevertheless, the data collected thus far help us understand the mechanism that may lead to the neoplastic transformation of cells initiated by environmental carcinogenes. It seems likely that activation of

METABOLISM

ENVIRONMENTAL GENOTOMC CHEMICAL

m cn. 0 c

MALIGNANT TUMOR

2 0

(CLINICAL DISEASE)

FURTHER GENETIC CHANGES

Metabolder m body *id and excreia (INTERNAL DOSQ

-

MUTATIONS, MUTATIONS, Mtpkficalion, Gene Mtpkficalion, tramlocatwn, &tic &hc loss, deletion, CEA, TPA deletion, (EARLY BIOLOGICAL EFFECT ON STRUCTURE AND FUNCTION LEVEL)

I EXPOSURE BlOMARKERS

DNA andprotein adduce TWOLOGICALLY EFFECTIVE DOSG A

MU TATIONS Glycophorm, r(ls HPRT, HLA, Glycophorin, oncogenes,p 53 gene, onkoprotcm onkoprotcins SCE's, micronuclei mrcronuclez (EARLY BIOLOGICAL EFFECT ONMOLECUUR LEVEL)

f

I

-.3

CELL PROLIFER4 T I 0 CLONAL EXPANSION

EARLY HEALTH EFFECT BIOMARKERS

Figure 1. A simplified diagram of the cascade of events from exposure to environmental gentoxic chemicals to malignant tumor (clinical disease), showing the relationship among exposure markers and early health effect biomarkers.

8

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some protooncogenes and related qualitative and quantitative changes in oncoproteins, involved in the control of growth and division of cells, is a basic mechanism used by environmental carcinogenes to evoke this transformation process [1,12,13]. The possibility of observing the trend of those changes through assessment of protein products of the oncogenes and suppressor genes in the easily obtainable biological material (blood, serum, urine) has proved to be extremely useful from the point of view of occupational medicine and environmental health. It provides new diagnostic methods in the assessment of carcinogenic effects of the occupational environment and human habitation places. Knowledge of the fact that oncoproteins can appear in biological fluids long before (many months or even years) clinical symptoms of cancer occur provides new opportunities for taking more effective preventive measures [14,15,16]. The measurements of oncoproteins in plasma or urine in order to assess the risk of cancer under conditions of occupational or environmental exposure to chemical carcinogenes have some limitation associated with the high cost of one measurement that requires highly specific monoclonal antibodies. The measurement of oncoproteins in very easily available saliva-material has been carried out in persons occupationally exposed to asbestos and in smokers, in other words, in groups with a high risk of lung cancer. The level of ras oncoprotein p21 was measured in saliva cells. In some persons the p21 test was positive; however, they did not show any clinical symptoms of the cancer [14]. Very interesting studies on the measurement of oncoproteins in blood serum as a biomarker of early cancer changes were carried out by Brandt-Rauf et al. [17]. The authors examined 18 workers employed in a foundry and exposed to polycyclic aromatic hydrocarbons (PAHs)—well-known occupational and environmental carcinogenes, in view of the presence of oncoproteins in blood serum. A high level of PAH adducts from DNA had been earlier found in the peripheral blood lymphocytes of those persons. This confirmed that those workers belonged to the group of a particularly high risk of malignant disease. The studies showed, among others, the presence of fes oncoproteins in the blood serum of a worker employed in the system of an eight-hour shift under condition of exposure to benzo(a)pyrene in concentration in ambient air exceeding 0.2 |ig/m^. In the serum of another worker also employed in a foundry and exposed to slightly higher concentrations of benzo(a)pyrene (0.05-0.2 iig/m^), fes and ras oncoproteins were detected. The level of adducts in lymphocytes of these two workers was doubled in comparison with workers whose blood serum was free from oncoproteins. It should be stressed that according to data presented by the authors, none of those workers showed clinical symptoms of neoplasm of the respiratory pathways or other organs. Another study was performed in persons working in fire brigades and involved in anti-fire campaigns in chemical plants. Out of 33 persons covered by the study, oncoproteins (corresponding in their properties with B-transforming growth factor) were found in the blood serum of 14 workers. It is worth mentioning that in none of


59

the controls (non-occupationally exposed to chemical carcinogenes) was the presence of these oncoproteins detected [14]. A similarly high percent of persons with oncoproteins present in their blood serum was found in those exposed to polychlorinated biphenyles (PCBs) [18]. This group was consisted of 16 workers involved in purification of transformer oil. The presence of fes oncoproteins was detected in six persons. It should be emphasized that all of them were smokers. Ras oncoproteins and sis oncoproteins were found separately in two workers. Moreover, in two workers free from fes oncoproteins, the presence of ras oncoproteins was detected. The highest blood level of PCBs was found in the worker with fes and ras oncoproteins detected in his blood serum. Data presented by Brandt-Rauf [14] on the significance of oncoprotein expression in the prognosis of malignant disease proved to be interesting. Studies were performed on samples of blood serum collected from 46 workers exposed to asbestos and silica dusts. The samples were kept in a blood bank. Fourteen workers developed cancer from the time of blood collection (including nine cases of the respiratory neoplasm). Usually, there was a period of 14 months between the blood collection and the development of cancer. In the group of nine persons with lung cancer, in as many as seven the presence of at least one oncoprotein was detected in blood serum collected earlier. One can draw a conclusion that the detection of increased expression of oncogene or its mutation through the measurement of oncoproteins in blood serum can be a useful biomarker in assessing the risk of cancer in populations exposed to occupational and environmental carcinogenes. The changes that lead to protooncogene activation and suppressor gene deactivation are not the only changes that lead to the neoplastic transformation. It seems, however, that the former correspond to the earliest stage of the promotion [19, 20]. Inhibition of intracellular communication by inhibiting specific intercellular junctions seems to be extremely important in the complex mechanism of the transformation of a normal cell into a neoplastic one [21]. Studies have indicated that the ability of forbol esters to promote carcinogenesis (cell canceration) is revealed by interaction with proteins responsible for the formation of intercellular junctions and cell adhesion [22]. It seems interesting to note here that the carcinoembryonic antigen (CEA), a neoplastic marker known in oncology for many years, acts a protein responsible for intercellular adhesion [23]. CEA belongs to the immunoglobulin super-family that includes also the adhesive protein of nerve cells. It is thought that the changes in the number of the sialic acid included in the saccharic component of the glycoproteins (to which CEA and neuronal adhesive protein belong) are correlated with the loss of the adhesive properties of those proteins [24]. When evaluating the role of CEA as a biomarker of the promotion stage within the neoplastic process, attention should be paid to the fact that the genes for coding that protein are located in chromosome 19, in the vicinity of the genes that code the betagrowth transforming agent, or the protein kinase [25]. The possible relationship between elevated CEA concentration in the serum and the carcinogenic process is supported not only by observations in the oncology clinics, but also by the data

60


from studies on populations that do not display clinically overt signs of cancer process. In the latter case, elevated CEA concentrations in blood serum are found in people characterized by the occurrence of the agents which are known to increase the risk of neoplastic disease, such as age, tobacco smoking, alcohol drinking, and occupational exposure to pollutants [26]. Studies published in 1986 by Pluygers et al. [27], which involved a large population of 2,000 people said to be clinically healthy, have supported those suggestions. The authors have demonstrated that during a 5-year observation, the incidence of cancer in the people in whom elevated values of CEA concentration in blood serum were found during the screening tests was ten times higher than among the people with normal CEA values. A number of studies have confirmed that staying in an environment containing toxic chemical substances (not necessarily considered to be carcinogenic) contributes to increasing the frequency of occurrence of elevated CEA concentrations in the blood serum of the exposed people. This refers both to chemical pollutants present in the communal and in the occupational environments. Thus, Schilpkorter et al. [28] observed higher values of CEA concentrations in non-smoking males residing close to industrial plants as compared with those non-smoking males who resided some distance from the industrial region. Pluygers et al. [29] presented data on the behavior of CEA concentrations (simultaneously, TPA concentrations were also determined) in people residing in close vicinity to refuse dumps. According to the authors, 10.3% of the tested population had elevated values of CEA concentration. Page et al. [cit. from 30] were some of the first authors who attempted using the assessments of CEA in blood serum to assess the risk of occupational cancer. They reported that in as much as 48% of the studied population working under conditions of occupational exposure to vinyl chloride, increased values of CEA concentration had been detected. During the study, overt clinical symptoms of neoplastic disease were not detected in any of the studied people. Those observations were then confirmed by Anderson et al. [31], who performed a similar study on a group of 1,115 factory workers exposed to vinyl chloride in a vinyl chloride polymerization plant. Elevated values of CEA concentration were also found to occur in people occupationally exposed to asbestos dust [32]. During the XXII International Congress on Occupational Medicine in the Chemical Industry held in 1987 in Sydney, Australia, Pluygers et al. [33] presented interesting reports on the application of tumor marker assessments, including CEA, to determine the risk of neoplastic disease in employees (subdivided into non-smokers and smokers) of three chemical plants (the type of the chemical exposure was not specified). Evidently the highest percentage of people with CEA concentration above the cutoff (53.3%) was detected in the group of exposed smokers. Therefore, it can be concluded that tobacco smoking under conditions of occupational exposure to potentially carcinogenic chemicals increases the risk of incidence of neoplastic disease by many times. In 1991, Plugers et al. [12] presented data suggesting that the assessment of CEA concentration in blood serum, in combination with several other tumor markers


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(TPA, ferritin, hyalouronic acid), may be used for evaluating the risk of neoplastic disease in people occupationally exposed to asbestos dusts. The authors report that assessment of CEA concentration (and of other markers quoted above) can be helpful in evaluating the effectiveness of preventive procedures aimed at reducing the risk of incidence of asbestos-induced cancer. The observations made heretofore indicate, however, that the use of assessment of CEA concentration in blood serum as an indicator of the risk of neoplastic disease cannot serve as a universal test for various exposures to chemical carcinogenes and for various cancer types. It is thought that the assessment of CEA concentration should be accompanied by the assessment of another tumor marker, tissue polypeptide antigen (TPA), known for some years already in clinical oncology [34]. TPA, a protein of the cellular cytoskeleton, is recognized by antibodies to cytokeratins and is a constituent of protein intercellular junctions. Functionally, TPA is related to the family of the adhesive proteins known as cadherins, participating in cell-cell intercellular interactions. According to Pluygers et al. [12], assessment of blood serum TPA concentration as a marker of neoplastic disease risk is particularly useful under the conditions of exposure to these carcinogenic chemicals that display their genotoxic and mutagenic properties by forming highly active free radicals. Studies by Lutz and Krajewska [35] and Lutz et al. [36] have revealed that under conditions of exposure to carcinogenic aromatic amines, assessments of TPA concentration in blood serum performed to evaluate the risk of bladder cancer are more useful than that of CEA. Somewhat earlier, Kumar et al. [37] demonstrated that TPA can be used as a biomarker of early bladder epithelium cell changes, which can but do not necessarily lead to neoplastic transformation. The two discussed tumor markers, CEA and TPA, are not the only markers used for early detection of neoplastic disease risk. Nevertheless, at the current state of our knowledge on tumor markers, it can be said that CEA and TPA are universal biomarkers that complement each other and should be assessed simultaneously. Assessment of TPA and CEA concentration in blood serum can be supplemented by assessment of those tumor markers specific to a given exposure to chemical carcinogenes or to the type of neoplastic disease they can induce. For example, under conditions of exposure to asbestos dusts, assessment of ferritin hyaluoronic acid is recommended in addition to assessing CEA and TPA concentration [12]. Assessment of some tumor markers (TPA, CEA, or oncoproteins) in easily accessible biological material (blood, serum, or urine) opens up new diagnostic possibilities for early detection of the risk of neoplastic disease under conditions of environmental and occupational exposure to carcinogenic chemicals. The introduction of biomarkers able to record the very early health effects produced by environmental carcinogenes make it possible to assess the risk of cancer incidence at a very early stage in its development. Of course, there are some limits to this assessment. It is known that exposure biomarkers—e.g., DNA or protein adducts (also call precancerogenic biomarkers)—occur more frequently under conditions of exposure to carcinogenes, and that the number of people in whom those adducts are detected is

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much greater than the number of people who may develop the neoplastic disease. Not every one of these people in whom DNA adducts have been detected will develop cancer. On the other hand, the failure to detect adducts does not mean that the person in question will not develop cancer. However, in many instances the likelihood of correct assessment of increased cancer risk on the basis of DNA or protein adduct determination can be augmented by the simultaneous determination of other biomarkers (for example, the presence of oncogenes) or their protein products (oncoproteins) in the cells or the biological fluids. A greater number of positive biomarker determination results shows significant increase in the risk of disease. However, the determination of a large number of biomarkers noticeably increases the cost of the research; this may be essential in epidemiological studies conducted on large populations. Therefore, before selecting such a biomarker set, the biomarkers should be carefully examined, and those which supply similar information, or those which are not related to the investigated pathologic process or harmful factor, should be eliminated. Biomarker-based biological monitoring is the most efficient measure capable of assessing the risk to an individual organism resulting from exposure to external harmful agents. According to the biomarker concept, environmental effects on the human organism can be assessed by the determination of exposure, health effect, and individual susceptibility, and enhanced analysis of the health risk in people exposed to environmental toxic agents has become possible. The assessments performed directly in the organism contribute to the elimination of uncertainties inherent in epidemiological studies.

REFERENCES 1. "Biomarkers and Risk Assessment: Concepts and Principles." Environmental Health Criteria 155. Geneva: World Health Organization, 1993. 2. Fowle, J. R. and Sexton, K. "EPA Priorities for Biologic Markers Research in Environmental Health." Environmental Health Prospectives, Vol. 98, 1992, pp. 235-241. 3. Gunn, P. M., Devra, L. and Perera, F. "Biological Markers in Environmental Epidemiology: Constrains and Opportunities," in Methods for Assessing Exposure of Human and Non-Human Biota, Chichester: John Wiley and Sons,Ltd, 1991, pp. 152-174. 4. Hanke, J., Indulski, J. A. and Lutz, W. "Biomarkers—A New Way of Evaluating the Effects of Environment on the Human Organism." Medycyna Pracy, Vol. 43, 1991, pp. 63-71 (in Polish). 5. US National Research Council. "Biological Markers in Environmental Health Research." Environmental Health Perspectives, Vol. 74, 1987, pp. 1-191. 6. Henderson, R. F. et al. "The Use of Biological Markers in Toxicology." Critical Reviews in Toxicology, Vol. 20, 1989, pp. 65-82.


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7. Gaylor, D. W., Kadluber, F. F. and Beland, F. A. "Application of Biomarkers to Risk Assessment." Environmental Health Perspectives, Vol. 98, 1992, pp. 139-141. 8. Indulski, J. A. and Lutz, W. "Biological Monitoring of Risk of Bladder Cancer in Persons Occupationally Exposed to Aromatic Amines." Polish Journal of Occupational Medicine and Environmental Health, Vol. 6, 1992, pp. 143-151. 9. Weinstein, B. J. "The Origin of Human Cancer: Molecular Mechanisms of Carcinogenesis and Their Implications for Cancer Prevention and Treatment." Twenty-seventh GAA Clowes Memorial Award Lecture. Cancer Research Vol. 48, 1988, pp. 4,135^,143. 10. Lutz, W. and Baranski, B. "The Role of Testing DNA and Protein Adducts in Cancer Risk Monitoring." Medycyna Pracy, Vol. 42, 1991, pp. 67-75 (in Polish). 11. Autrup, H. "Human Exposure to Genotoxic Carcinogens: Methods and Their Limitations." Journal of Cancer Research and Clinical Oncology, Vol. 117, 1991, pp. 6-12. 12. Pluygers, E. P. et al. "Biomarker Assessments in Asbestos Exposed Workers as Indicators for the Selective Prevention of Mesothelioma or Bronchogenic Carcinoma: Rationale and Practical Implementations (Parts I and II)." European Journal of Cancer Prevention, Vol. 1, 1991, pp. 57-68 and Vol. 2, 1922, 129-138. 13. Balmain, A. and Brown, K. "Oncogene Activation in Chemical Carcinogenesis." A^vflEfic^^" in Cancer Research, Vol. 51, 1988, pp. 147-182. 14. Brand-Rauf, P. W. "The Molecular Epidemiology of Oncoproteins." Scandinavian Journal of Work, Environment and Health, Vol. 18, 1992, pp. 4 6 ^ 9 . 15. Cooper, G. M. "Oncogenes as Markers for Early Detection of Cancer." Journal of Cellular Biochemistry, Suppl. 16G, 1922, pp. 131-136. 16. Niman, H. L. et al. "Anti-Peptide Antibodies Detect Oncogene-Related Proteins in Urine." Proceedings of the National Academy of Sciences, Vol. 82, 1985, pp. 7,924-7,928. 17. Brand-Rauf, P. W. et al. "Serum Oncogene Proteins in Foundry Workers." Journal of Social and Occupational Medicine, Vol. 40, 1990, pp. 11-14. 18. Brand-Rauf, P. W. and Niman, W. "Serum Screening for Oncogene Proteins in Workers exposed to PCBs." British Journal of Industrial Medicine, Vol. 45, 1988, pp. 689-695. 19. Brand-Rauf, P. W. "New Markers for Monitoring Occupational Cancer: The Example of Oncogene Proteins." Journal of Occupational Medicine, Vol. 30, 1988, pp. 399-404. 20. Travis, C. C. and Belefant, H. "Promotion as a Factor in Carcinogenesis." Toxicology Letters, Vol. 60, 1922, pp. 1-9. 21. Yamasaki, H. "Gap Junctional Intercellular Communication and Carcinogenesis." Carcinogenesis, Vol. 11, 1990, pp. 1,051-1,058.

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22. Trosko, J. E., Chang, C. and Medcalf, A. "Mechanisms of Tumor Promotion. Potential Role of Intermolecular Communications." Cancer Investigation, Vol. 1,1983, pp. 511-526. 23. Benchimol, S. et al. "Carcinoembryonic Antigen a Human Tumor Marker, Functions as an Intercellular Adhesion Molecule." Cell, Vol. 57, 1989, pp. 327-334. 24. Rutischauser, U. et al. "The Neural Cell Interactions." Science, Vol. 240, 1988, pp. 53-57. 25. Hosier, J. C. "Genes for Tumor Markers are Clustered with Cellular ProtoOncogenes on Human Chromosomes." Cancer Letters, Vol. 36,1987, pp. 235-245. 26. Herbeth, B. and Bargel, A. A. "A Study of Factors Influencing Plasma CEA Levels in an Unselected Population." Oncodevelopment and Biological Medicine, Vol. 1, 1980, pp. 191-198. 27. Pluygers, E. P. et al. "Tumor Markers for Cancer Detection." Cancer Detection and Prevention, Vol. 9, 1986, pp. 495-504 and 505-509. 28. Schilpkoter, H. W., Baginski, B. and Kramer, U. "The Carcinoembryonic Antigen (CEA) in Urban Populations: Epidemiological Studies." Zentralblatt fur Bakteriologie und Hygiene, Vol. 166, 1978, pp. 136-143. 29. Pluygers, E. P. et al. "Evaluation of the Cancer Risk Associated with Activities of the Chemical Industry in Production, Utilization and Environmental Pollution," in Occupational Health in the Chemical Industry. Copenhagen: Medichem and WHO, 1992, pp. 220-228. 30. Bernard, A. and Lauwerys, R. "Determination of Tumor Markers in Biological Fluids," in Indicators for Assessing Exposure and Biological Effects of Genotoxic Chemicals. A. Aitio et al. (Eds.), Brussels-Luxemburg: Office for Official Publications of the European Communities, 1988, pp. 153-169. 31. Anderson, H. A. et al. "CEA Levels in Workers Exposed to Vinyl Chloride Monomer," in The Abstract Book of the Medichem 4th International Conference, September 7-10, Haifa: 1976, pp. 47. 32. Jarvisallo, J. O. and Stemman, U. H. "Monitoring Human Exposure to Carcinogenic and Mutagenic Agents." IRAC Scientific Publications, Vol. 59, 1984, pp. 403-409. 33. Pluygers, E. P. et al. "Evaluating Carcinogenic Risk Among Workers in the Chemical Industry by Using Tumor Marker Assessments in Serum," in Occupational Health in the Chemical Industry. Medichem and WHO, Copenhagen, 1988, pp. 136-143. 34. Fischer, L. "TPA: Tracing Cancer in Serum and Tissue." International Clinical Products, Vol. 7/8, 1985, pp. 20-24. 35. Lutz, W. and Krajewska, B. "The Concentration of the Tissue Polipeptyde Antigen (TPA) and Carcinoembryonic Antigen (CEA) in the Serum of Workers Exposed to Aromatic Amines." Medycyna Pracy, Vol. 43,1992, pp. 297-301.


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36. Lutz, W., Indulski, J. A. and Krajewska, B. "Tissue Polypeptyde Antigen and Acetylation Phenotype," in Occupational Health in the Chemical Industry. Selected papers from the XX Medichem Congress, 6-9 October 1992, Copenhagen: World Health Organization Regional Office for Europe. 1993, pp. 127-131. 37. Kumar, S. et al. "Frequent Evaluation of Tissue Polipeptyde Antigen in the Sera of Workers Exposed to Bladder Carcinogens." International Journal of Cancer, Vol. 22, 1978, pp. 542-545.


CHAPTER 4 SICK BUILDING SYNDROME Peter Dingle Environmental Science Murdoch University Murdoch Western AustraUa 6150

CONTENTS INTRODUCTION, 67 THE NATURE OF THE SYNDROME, 68 The Symptoms of SBS, 69 The Costs of SBS, 70 Causal Mechanisms, 70 ENVIRONMENTAL CONTAMINANTS ASSOCIATED WITH THE SYNDROME, 70 Indoor Air Pollution, 70 Physical Factors, 74 Biological Contaminants, 76 CAUSAL FACTORS AND SOURCES ASSOCIATED WITH THE SYNDROME, 79 Psychological Factors, 79 Psychosocial Phenomena, 80 The Building and Environmental Control Systems, 81 Office Materials, Equipment, and Furnishings, 82 Combination of Causes, 84 CURRENT RESEARCH, 85 SOLUTIONS TO SBS PROBLEMS, 85 CONCLUSIONS, 86 REFERENCES, 86 INTRODUCTION In a substantial number of large modem office buildings, office personnel have complained of a similar set of symptoms now commonly known as sick building syndrome (SBS). SBS was first reported as a problem in buildings more than thirty years ago in the U.S.A. and Scandinavia. The problem began to reach epidemic proportions when the oil crisis of the early seventies required Western countries to reconsider their use of energy [1]. The major strategy used to reduce energy consumption was to construct buildings more air tight. This was based on observations that buildings accounted for more than a third of America's fuel consumption [2] and with 75% of energy used on environmental control systems [3]. 67

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The syndrome is mostly associated with buildings used for non-industrial purposes, especially office buildings. It has been estimated that 30% of new and remodeled office buildings show signs of SBS and that 10-30% of occupants are affected [4]. While current evidence suggests that general SBS symptoms and complaints may cause no lasting damage to the long-term health of occupants, health complaints related to buildings can be a significant disruption to the people who experience them on a regular basis [5].

THE NATURE OF THE SYNDROME To date, the SBS has not been concisely defined and has been loosely applied to various situations. This has resulted in the lack of a systematic approach to studying the syndrome, which has caused some controversy and confusion. There is now a clear distinction between the syndrome and other building-related health problems such as "Building Related Illnesses" (BRI). This is based on the observation that a BRI is a clinically defined illness and can be diagnostically attributed to an environmental exposure. The most serious BRI is the occasionally fatal Legionnaires' Disease. Other well-known BRIs include Humidifier Fever and Pontiac Fever. In order to define the SBS, it is important to understand that it is not a clinically defined disorder but is a set of general symptoms with no known single underlying cause in all buildings [6]. Synonyms of the SBS have included "building illness syndrome," "ill buildings," "stuffy offices," and "tight office syndrome" [7]. The World Health Organization (WHO) and other researchers have defined SBS with the following observations: • An increase in the frequency of building occupant-reported complaints; • Acute non-specific symptoms; • Occurs in non-industrial environments; • No single causal substance or agent has been identified; and • The symptoms improve when the effected persons are away from the building [4, 8]. The WHO has also made a distinction between "temporary sick buildings," where onset is acute and usually declines within one year of occupancy, and "permanently sick buildings," where symptoms are non-specific and ongoing. The former are often caused by off-gassing of new fabrics, building materials, and furnishings, and can generally be treated [9]. The latter are invariably sealed, air-conditioned buildings designed to be energy-efficient [10]. The WHO has also suggested a more technical definition of SBS is when more than 20% of a building's occupants "often experience" a specific set of symptoms. However, while there is general agreement on the set of symptoms, what is defined as "often experienced" is not clear.

Sick Building Syndrome

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The Symptoms of SBS The symptomology of SBS is varied, but five symptom complexes are regularly encountered and may occur singularly or in combination [8]. The primary symptoms of SBS include: • Mucous membrane irritation; • Neurotoxic symptoms; • Odor complaints; • Skin irritation; and • Asthma-like symptoms [11, 12, 13, 14]. These symptoms disappear or become considerably less pronounced minutes or hours after leaving the building [15]. However, some people do not get better until they have been away from the building for several days, or in some cases such as skin symptoms, several weeks [8, 15]. Table 1 shows a broader list of symptoms. Table 1 General glasses of symptoms Related to the sick building syndrome with examples of symptoms belonging to each glass

1. Sensory irritation in eyes, nose, and throat: • dryness • stinging, smarting, irritating sensation • hoarseness, changed voice 2. Skin irritations: • reddening of the skin • stinging, smarting, itching sensations • dry skin 3. Neurotoxic symptoms: • mental fatigue • reduced memory • lethargy, drowsiness • reduced power of concentration • headache • dizziness, intoxication • nausea • tiredness 4. Unspecific reactions: • running nose and eyes • asthma-like symptoms in non-asthmatic persons • respiratory sounds 5. Odor and taste complaints: • changed sensitivity • unpleasant smell or taste Source: M0lhave [12].

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The Costs of SBS Apart from the adverse health effects an individual may experience, SBS symptoms have been shown to impact on productivity and absenteeism [5]. SBS may also affect unofficial time off work, reduce overtime, increase staff turnover, and can make substantial demands on management to spend time on resolving problems [5]. In some extreme cases, buildings have been closed for a period while mitigation activities are carried out, with the potential to incur considerable costs. It is reasonable to assume that if workers are unhappy and irritated they are unlikely to be working at maximum capacity [16]. Some studies estimate that a reduction in efficiency by 20% may occur (self-assessed) [1]. Causal Mechanisms SBS symptoms are non-specific in character and have many potential causes [17], which are generally classified as physical, chemical, biological, or psychological [18]. Although a large number of causes of SBS have been hypothesized, only fragmentary evidence exists supporting any one hypothesis [5, 18]. The nature and time pattern of SBS symptoms suggests that it could result from a combination of allergic, irritant, and toxic reactions to environmental contamination [13]. One etiological hypothesis for SBS suggested by M0lhave and co-workers was developed from the observation that low-level exposure to volatile organic compounds can lead to symptoms similar to those typical of SBS [13, 19]. A plausible biologic hypothesis proposed by Bergland et al. [20] suggests that SBS symptoms may arise from multi-sensory adaptation to indoor air pollution. This is achieved by either exhaustive stimulation, or by a sensory deprivation of the variety of signal required to maintain normal sensory variation. A result may be sensory confusion, which becomes a strain on the particular system attempting to interpret the sensory signals. This hypothesis has been supported by cases where individuals no longer working in or visiting the original building concerned will continue to experience symptoms, or have an increased sensitivity to environmental exposures such as tobacco smoke, new paint odor, etc. [15]. This is also supported by the observation that SBS is predominantly stimulation of the olfactory and irritant receptors of the upper airways, and while the symptoms may not be severe, these receptors are peoples' warning systems. ENVIRONMENTAL CONTAMINANTS ASSOCIATED WITH THE SYNDROME Indoor Air Pollution Sources of indoor air pollutants include the occupants and their activities, building materials, microorganisms, and infiltration of contaminants from the exterior


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[21]. The indoor air quality of a building can be impacted by potentially hundreds of chemicals [22]. The concentration of an indoor air pollutant is a complex interaction between: • The source strength; • The location of the source; • The transportation and mixing of pollutants; • Indoor climatic factors; • Synergistic reactions with other pollutants; • Dilution with outside air; and • Pollutant removal mechanisms [22]. Major contaminants of indoor air are human bioeffluents, volatile organic compounds, formaldehyde, suspended particulate matter, environmental tobacco smoke, and outdoor air pollution. Human Bioeffluents Contaminants generated by the human body are described as bioeffluents and historically are a major indoor air quality (lAQ) concern of both odor and (dis)comfort. Carbon dioxide (CO2) is a bioeffluent produced by human beings as a byproduct of metabolism [19]. It is now well established that CO2 concentrations in the range of 600 ppm to 1,000 ppm and higher are associated with SBS [8, 19]. The concentrations normally observed in buildings are not associated with any symptoms, except the sensation of stale and stuffy air [8]. Other bioeffluents reported from building investigations include acetone, acetaldehyde, acetic acid, alkyl alcohol, amyl alcohol, butyric acid, diethyl ketone, athylacetate, ethyl alcohol, methyl alcohol, phenol, and toluene [23]. Volatile Organic Compounds (VOCs) Volatile organic compounds (VOCs) are one of the main pollutants inside sick new buildings. They are regarded as producing SBS effects even in low doses. Organic compounds include all chemicals containing carbon and hydrogen. VOCs are easily vaporized and are rapidly absorbed via the lungs, skin, and intestinal tract. It is generally accepted that VOCs include those organic compounds with a boiling point in the range of 50 to 250°C. Of the compounds in this range, over 900 compounds have been identified in indoor air. The number of VOCs present in any one building can vary considerably from as few as 20 to several hundred compounds [15]. VOCs are emitted from a wide variety of sources in buildings such as building materials, furnishings, consumer products, building maintenance materials, human bioeffluents, office equipment, tobac-

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CO smoke [8, 24], carpets, cleaning products, smoke, adhesives, caulking, paints, and solvents [25]. However, evidence of the role of VOCs in SBS has not been convincing [8]. This may be due in part to a lack of standard measuring methods. Different methods will yield different results and do not allow for the direct comparison of results from studies using different measurement techniques [15]. Formaldehyde Formaldehyde is a ubiquitous contaminant of indoor spaces [26] and is a known sensory irritant [27], an irritant of the respiratory tract [9, 28], an apparent neurotoxic substance [29], and may be responsible for allergic disorders including asthma [30]. However, its presence in indoor air is frequently attributed to mucous membrane irritation [19]. Major sources of formaldehyde in office and non-industrial buildings include: • Furniture made from pressed woods such as plywood, particle board, and medium density fiberboard (MDF) [26, 31]; • Storage cabinets, countertops, and workstations made from particle board; • Room office dividers with particle board or MDF cores; • Acid cured finishes on wood furniture [26]; • Office equipment and decomposition of VOCs [32]; • Urea formaldehyde foam insulation (UFFI); • A variety of products used for disinfection, cleaning, and painting [8, 26]; and • Fabrics, glues, and carbonless copy paper. Except in a few cases, formaldehyde concentrations reported in buildings have been too low to be responsible for SBS symptoms. Consequently, it has rarely been identified as a causal factor in problem buildings [8]. On the whole, it is unlikely that formaldehyde by itself is responsible for SBS symptoms, but it may be a contributor to SBS symptoms by potentiation of other factors [8]. Suspended Particulate Matter Airborne particulate matter is ubiquitous and exists in a wide range of particle sizes and chemical characteristics [33]. Dust in the indoor air consists of organic and inorganic particles, many of which can be classified as fibers [8]. The particulate size range of concern to human health is the range in aerodynamic diameters between 0.1 and 10 micrometers. This corresponds to the respirable suspended particulate matter (RSP) and the total suspended particulate matter (TSP) ranges [33]. Particles less than 3.0 mm in diameter (RSP) are the most biologically significant, as they can reach the thoracic or lower regions in the respiratory tract. Exposure to these particulates is suggested responsible for most of the adverse health effects of SBS [14, 26, 34].


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Typical sources of suspended particulate matter in buildings include: • Smoke, street dust, paper, duct insulation, water residue, tobacco smoke, carpets, heating, ventilation, air, and cooling (HVAC) filters; • Chemical disinfectants, corrosion or rust from humidification equipment; damaged insulation materials; and paper shredding; • Skin cells, dust mite feces, domestic animal dander on clothing, clothing fabrics, and biological activity [15]; and • Combustion by-products. Environmental Tobacco Smoke (ETS) The problems with environmental tobacco smoke (ETS), and sidestream smoke (SS) are extensively documented. This lead to the finding in the late eighties that the smoking of tobacco products indoors is the most important source of chemical pollution in indoor air [8, 35]. ETS is the mixture of smoke from the burning end of a cigarette, pipe, or cigar, and the smoke exhaled by a smoker [36]. The sidestream smoke of ETS is known to be responsible for mucous membrane irritation and has been found to be far more irritating than mainstream smoke [8]. Cigarettes contain a great number of chemicals, in the order of 3,800. Some of the more noxious chemicals include formaldehyde, carbon monoxide, nitrogen oxides, ammonia, nicotine, and an assortment of heavy metals and pesticides. Short-term studies have revealed that ETS can add significantly to indoor formaldehyde levels [32]. In buildings where smoking is allowed, environmental tobacco smoke (ETS) may account for 40% of the indoor pollution. Outdoor Air Because healthy indoor air quality requires the intake of outdoor air, and because of phenomena such as the thermal stack effect, pollution in the outdoor air is a potential problem. The most likely outdoor air pollutants are from adjacent roadways, parking lots, and loading bays. Indoor air pollution entering from outside may be from combustion by-products and may include carbon monoxide, carbon dioxide, nitrogen dioxide, sulphur dioxide, and VOCs, all of which can contribute to SBS. Oxides from combustion products are often respiratory irritants and can result in adverse health if long-term chronic exposure occurs. Indoor Surface Pollution There is growing evidence of an important role of surface dust or indoor surface pollution (ISP) in office buildings [14]. This is partly due to the observation that individuals create their own dust cloud when settled dust is stirred up during normal work activities and that concentrations in this dust cloud are significantly

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greater than ambient levels [5]. Exposure to settled dust can result in direct dermal contact, and possible transportation to eyes causing irritation, and disturbed or resuspended dust can result in potential respiratory, ocular, and dermal exposure [14]. The potential toxic or irritation effects of ISP may also be increased as a result of the adsorption of gases and vapors to particulates [5]. Initial studies by Skov et al. [37] found significant associations between floor dust levels and mucous membrane irritation symptoms. Intervention studies have found that steam-cleaning carpets and soft furnishings, wet wiping of hard surfaces, and high efficiency vacuum cleaning can significantly reduce symptoms, particularly when actions remove dust deposits from carpets and soft furnishings [5, 38, 39, 40]. Physical Factors Physical factors contributing to indoor environmental conditions with the potential to affect human health and comfort include temperature, relative humidity, air movement, ventilation, light intensity and flicker, noise, vibration, air ions, and electrostatic, electric, and magnetic phenomenon [41, 42]. Thermal Comfort Thermal comfort is described as a condition of mind, an expression of satisfaction [43], and is dependent on the level of work performed, the heat of metabolism, evaporative losses, radiant and convective heat transfers [19], individual physiology, and age [45]. Dissatisfaction with the thermal environment may be due to general bodily discomfort due to warm or cool ambient conditions, or to unwanted heating or cooling of a localized part of the body—local thermal discomfort. Unsatisfactory thermal conditions can act indirectly to produce irritable workers, which may increase the likelihood of SBS complaints arising. Temperature alone has been suggested as the most important indoor parameter affecting SBS. Thermal discomfort can also lead to synergistic effects with other parameters such as increased skin irritancy by particulates [44]. Relative Humidity There is no agreement on what constitutes the ideal range of relative humidity for indoor air [8]. An optimum range of relative humidity of 45-55% is now generally accepted, and is a much narrower range than previously thought [46]. A wider range may be acceptable for thermal comfort, but encourages bacterial, fungal, and viral growth. What is generally agreed upon is that raising humidity has a similar effect to raising the temperature [45]. Direct effects include discomfort at excessively high (above 70%) or low (below 20%) relative humidities [8, 45]. Humidities below 20% can evoke drying of mucous membranes or skin, and a particular dermatitis has been associated with


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dry, warm air and high rates of air movement [47, 48]. Indirect effects of humidity include phenomena such as significant increases in formaldehyde concentrations by a factor of 1% for every 1% rise in relative humidity. Lighting Some evidence exists that illumination could play a role in the SBS [49]. Artificial light only consists of some wavelengths in the spectrum, whereas natural light is a combination of all colors of the spectrum. Research has shown that people can suffer, to varying degrees, a syndrome known as seasonal affective disorder (SAD), which is a result of deprivation from full-spectrum lighting. Poor lighting conditions can cause eye strain and fatigue and appear to be associated with headache, dizziness, fatigue, nausea, and eye irritation [50, 51]. Excessive luminosity has been reported to be a major cause of complaints in some SBS investigations and has been suggested as a major causal factor in 20% of buildings with SBS [52]. Noise and Acoustic Conditions Acoustic conditions in a building depend on sound transmitted from the outside, from other rooms, the ventilation system, machines and persons in the room, and on structural conditions that provide attenuation or modification (structure-borne vibrations) [15]. However, few investigators have evaluated the significance of noise or vibrations for SBS [49]. Concern over the potential impact of noise is based on the notion that some sound vibrations have frequencies similar to the resonant frequencies of certain body parts, such as the eyes. Electromagnetic Radiation Offices full of electrical equipment are likely to have severely disturbed electromagnetic fields. In 1987, WHO endorsed a report that visual display unit (VDU) workers should not work within one meter of the rear or sides of other terminals unless they had been tested to emit only low levels of non-ionizing radiation (NIR). The dominant source of extremely low frequency (ELF) electromagnetic fields measured in buildings is now known to be all office equipment and not just the VDUs [53]. Research has shown that women who sit in front of a VDU for more than six hours a day are 40% more likely to have a miscarriage. Airborne Ions The indoor office environment has far more positive ions and far fewer negative ions than most outdoor environments. Electrical equipment, VDUs, synthetic fabric and carpets, and HVAC systems, which recirculate air through metal ducts, all contribute to ion loss. Some research has shown that negative ion generators can facili-

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tate removal of particulates from air by electrostatic precipitation. Godish [31] has concluded that the collective weight of the various studies indicates that air ions can have significant biological effects and that such effects can occur in humans. However, there is considerable debate occurring on the health impacts of air ions, and in particular their role in SBS. Biological Contaminants Biological contaminants come from many sources and include bacteria, molds, mildew, viruses, animal dander and cat saliva, house dust mites, cockroaches, pollen [36], yeasts, amoebae, and nematodes [54]. Some of the allergic reactions triggered by biological contaminants include hypersensitivity pneumonitis, allergic rhinitis, and some types of asthma [36]. A variety of biogenic contaminants have been suggested as risk factors for SBS symptoms regardless of atopic history. The major contaminants include: • Aerosols of both viable and nonviable organisms and their antigens; • Microbial products such as glucans, endotoxins, mycotoxins, and VOCs; and • Macro-molecular organic dust (MOD) found in particulate matter deposited on floor and indoor surfaces. Bioaerosols Biological aerosols are naturally occurring and have always been a factor in human health [55]; however, this does not mean they are benign. Water spray systems, humidifiers, filters packed with organic dust, high humidity, reduced ventilation, tighter buildings, and moisture damage to building materials all provide conditions for the growth of microorganisms. In particular, indoor air biological or microbial contamination is often the result of a fault in the building's HVAC system or maintenance procedures where microorganisms are provided with conditions to grow and proliferate [49, 56]. Airborne Infectious Disease Many of the typical symptoms for SBS are similar to those occurring in common airway infections [49]. Infectious illnesses such as influenza, measles, and chicken pox viruses are generally transmitted through the air [36] and close human contact. Many of these common airway infections may induce temporary bronchial hyperactivity [57], which could further act to make an individual more sensitive to irritants in the indoor air [49]. LaForce [58] and Brundage et al. [59] provide evidence that air conditioning systems may influence the spread of airborne infections.


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However, the pattern of SBS occurrence suggests that it most Ukely results from a combination of allergic, irritant, and toxic reactions to environmental contamination and not to infectious illness [14]. Bacteria While the concentrations of other biological contaminants in indoor air, such as mold spores, varies primarily as a function of outdoor concentrations, the concentration of bacteria in indoor air varies mainly with human occupancy [15]. Exposures to bacteria and microbial metabolites have rarely been studied as potential causal factors of SBS. Most cross-sectional epidemiological studies have failed to show any relationship between viable bacteria levels and SBS symptoms. Endotoxin Endotoxin is the name given to a class of biological molecules that have certain characteristic toxic effects. It is now recognized that the lipid portion of lipopolysaccharide (Lipid A) of Gram-negative bacteria is chemically distinct from all other lipids in biological membranes and is responsible for the molecules' characteristic toxicity [60]. Airborne endotoxin is ubiquitous in nature, as are the Gramnegative bacteria that produce endotoxins [61]. The most important type of reservoir for Gram-negative bacteria is recirculated water-based fluids that produce aerosols, such as the mechanism found in many humidifier systems [60]. Endotoxins are known to cause fever and malaise, changes in white blood cell counts, respiratory distress, shock, and even death when in the bloodstream in microgram quantities [60]. Endotoxins are also known to stimulate the immune system, which can have beneficial effects such as anti-tumor properties [62] and result in reduction of lung cancer risk [63, 64, 65]. Fungi Fungal spores are ubiquitous in outdoor air, but are considered a contaminant in indoor air [66]. However, very little baseline data exist on indoor air concentrations apart from case studies performed following occupant complaints or a diagnosis of illness due to environmental exposure [66]. Environmental factors that influence indoor fungus concentrations include outdoor air concentrations, type and rate of ventilation, and indoor moisture levels [67]. During the growing season, indoor fungus spore levels are typically 10-25% of outdoor levels [68]. Only in cases of extreme indoor contamination do indoor spore levels exceed outdoor levels [55]. A study on seven New York buildings by Morey and Jenkins [68] found indoor air levels exceeded outdoor levels by a factor of 1.5 to 13 during periods when the furniture and HVAC system were disturbed.

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Indoor environments exposed to contaminants such as quantities of bird or bat droppings are at contamination risk from strains of fungi such as Aspergillus fumigatus, Histoplasma capsulatum, and other fungi known to cause certain diseases. Exposure to airborne fungal spores, hyphal fragments, or metaboHtes is known to cause a variety of respiratory diseases. The full range of health effects due to fungal exposure include: • Allergic diseases including allergic rhinitis, asthma, and hypersensitivity pneumonitis; • Infectious diseases such as histoplasmosis, blastomycosis, and aspergillosis; • Acute toxicosis from exposure to mycotoxins; and • Cancer, also ascribed to exposure to mycotoxins [66]. Free-living Amoebae The impact of protozoa on human health in indoor environments is generally restricted to free-living amoebae. These can have direct effects as pathogens and allergens, or they can interact with bacteria and amplify bacterial pathogens [69]. Unlike bacterial contamination of reservoirs such as humidifier water, the aerosolization of living amoebae appears to pose a minimal threat. However, aerosolization of amoebic antigens may be harmful [69]. Of even greater potential impact on human health is the biological symbiosis between free-living amoebae and bacterial pathogens. It appears that free-living amoebae can harbor and or amplify known bacterial pathogens such as Mycobacterium, Listeria, and Legionella. This can result in the release of these bacteria and possible human exposure. However, the interactions between protozoa and potential human bacterial and viral pathogens and their impact on human health in the indoor environment are not fully understood. Allergens Potential allergens found in indoor environments include dust mite fecal wastes, mold spores, fungal hyphal fragments, and macromolecular organic dust (MOD). Exposure to allergens in the fecal wastes of dust mites appears to be the single most important cause of asthma and chronic allergic rhinitis in residential environments [70]. Allergens from domesticated animals can occur in buildings regardless of whether animals are kept on the premises. These allergens can be carried on clothing from places with domesticated animals, or may remain for many years after previous habitation by animals [15]. Surfaces of fabric-covered office panels can further act as reservoirs for cat dander allergen and fungal spores brought into the building by occupants [71].


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Volatile Organic Compounds as Bioejfluents Microorganisms such as bacteria, actinomycete, and fungi produce a variety of VOCs and semi-VOCs during metabolism [15]. VOC metabolites such as higher alcohols, ketones, and organic acids are responsible for odor problems associated with microbial growth. Exposure to these VOCs is suggested as a potential contributing factor to SBS symptoms, although only few studies have attempted to evaluate their potential health effects. CAUSAL FACTORS AND SOURCES ASSOCIATED WITH THE SYNDROME Psychological Factors There is litde direct evidence for the role of individual psychological factors in problem buildings [72]. The major role of psychosocial factors is most likely as modifiers of individuals' and organizations' responses to biological, chemical, and physical exposures in the office environment [72]. Some of the evidence from the research suggests some individuals have a predisposition to SBS [73]. It has also been found that the degree of control an individual has over the climatic conditions of an office affects the severity of reported complaints indirectly, via its effects on the workers' satisfaction with the prevailing environmental conditions and also their awareness of those conditions. Individual Sensitivity Individual sensitivity to exposures depends on factors such as a variety of personal characteristics and include: • Gender; •Age; • Marital status; • Atopic status; • A variety of psychosocial factors such as smoking, alcohol consumption, coffee consumption, regular exercise, and use of contact lenses; • Personal factors such as childhood exposures, residential factors, and industrial exposure to irritants [49]; • Skin type; • Previous exposures (sensitization); • Medication; and • Activity-related factors [6].

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Atopy Atopy is the genetic predisposition to allergic manifestation of exposure to common allergens such as dust mites, mold, pollen, and animal dander. Significant relationships have been shown between people with a history of atopy and the prevalence of SBS symptoms [6, 31]. Female Gender Female gender is generally associated with reporting higher levels of SBS symptoms [74, 75, 76, 78]. The differences can be as high as 3 to 1 with females consistently reporting higher rates of symptoms than males. These findings have been repeated by other researchers [73, 77]. PSYCHOSOCIAL PHENOMENA Psychosocial phenomena include mass psychogenic illness (MPI), job (dis)satisfaction, occupant density, satisfaction with the physical environment, and seasonal affective disorder (SAD). Job Satisfaction Physical discomfort, repetitive or boring work, under-utilization and career frustration, and poor relationships with co-workers or superiors are all factors that can add to job stress and result in reduced tolerance for substandard indoor air quality. Managers and professional or technical staff are also known to report fewer symptoms than clerical staff. Professional staff often have enhanced accommodation and have been identified as having a greater degree of control over their job with much greater ability to change their environmental conditions [56]. External Stressors Little research has been done on the effects of the domestic environment on the symptoms of SBS. External factors of life stress, such as home and family relations, are particularly important in influencing occupational stress [78]. One study found that smoking, high population-density flats, terraced houses, and building moisture or mold at home were related to a higher prevalence of headache or mucosal irritation [79]. Norback [49] found that childhood exposure to tobacco smoke, childhood and current urban residency, and preschool children at home were found to be related to SBS, with personal factors such as atopy, hyperactivity, nickel allergy, and infection-proneness also related to SBS.


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The Building and Environmental Control Systems Four key elements interact in a building to yield the conditions of the indoor environment. They are the building shell, the HVAC system and its condition, the outdoor environment, and the building's occupants and their activities [54]. Most modern buildings now have a mechanically controlled indoor environment that is provided by a central HVAC system. Environmental Control Systems Environmental control systems are generally considered the major causal factor contributing to occupant health complaints and dissatisfaction with indoor air quality. HVAC systems are designed to provide thermal comfort, to distribute outdoor air to occupants, to remove odors and contaminants through the use of exhaust fans or by dilution, and to control air pressure relationships between rooms [45]. However, these systems represent a considerable risk factor for building-related problems as they can serve to transmit contaminants from their source(s) to areas in a building where they may produce air quality problems. Outdoor Air Ventilation Rates Ventilation systems are of two basic types: naturally ventilated or mechanically controlled. Natural ventilation refers to the intentional displacement of air through openings such as doors and windows. Mechanical ventilation refers to the intentional use of air movers (fans) to bring in outdoor air or to exhaust indoor air [33]. The term ventilation efficiency is used to describe the ability of the ventilation system to distribute supply air and remove internally generated pollutants [45]. The effectiveness of the ventilation with outdoor air can be judged when comparing the elevation of carbon dioxide concentrations indoors to the average ambient outdoor concentration of approximately 325 ppm [19]. However, the focus on CO2 levels also disregards indoor air pollution emitted from sources other than human beings. The first attempt to regulate the indoor air of buildings was to develop a ventilation standard to control body odor and effluents. As a result of the energy conservation movement in the 1970s, the ventilation requirements in nonsmoking areas were reduced to minimum values [6]. In response to more recent interest in building hygiene, revised ventilation requirements have recommended minimum values for office spaces of 10 liters/sec/person by ASHRAE and 11 liters/sec/person by the Nordic Countries [6]. However, Fanger and co-workers proposed that in buildings with a large sensory pollution load (such as new or temporary sick buildings), the ventilation requirements may be as high as 50 liters/sec/person [43]. However, rarely has research investigated the ventilation requirements for providing and maintaining healthy indoor environments [6].

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Many outbreaks of SBS are "cured" by increasing the air exchange and the air flow. The main problem faced by health authorities regarding ventilation is that no one really knows what level of air exchange is necessary to stop SBS complaints from occurring [32]. Meeting current ventilation standards does not necessarily guarantee good indoor air quality and thereby reduce the symptoms of sick building syndrome. Inadequate Maintenance Problems with inadequate maintenance can result in poor operational procedures. These include: • Failure to keep air intakes clean; • Dirty filters not replaced or serviced adequately; • Fouled and contaminated heating and cooling coils; and • Disconnected components such as damper linkages, exhaust fans, and automatic controls [45]. A common problem of poor maintenance are fan motors with worn bearings, or fan belts that overheat and generate detectable odors [54]. Another typical example is the changing of HVAC filters while the air-handling unit fans remain operating. This can result in a concentrated release of particulates into the supply ductwork [54]. Maintenance of local fan coil units and other ventilation system components is also often difficult or impossible to achieve due to poor design and access [19]. Microbes, particulates, insects, rats, dead birds, animal feces, and many other unhygienic objects have been found in badly maintained HVAC systems [80]. Office Materials, Equipment, and Furnishings Investigations of complaint buildings and epidemiological studies have shown the health-affecting or contaminant-generating potential of carbonless copy paper (CCP), other papers, copying machines, VDUs and computer equipment. These products are considered an important source for VOCs, especially when products or materials are new [24]. Some furnishings, such as fabric-covered surfaces, can also act as a sink for VOCs and biological contaminants such as dust mites and molds [81]. Carbonless Copy Paper A variety of studies have implicated handling carbonless copy paper (CCP) or some component of CCP as a causal factor of skin and/or mucous membrane irritation [82], hoarseness, coughing, skin irritation, headache, fatigue [83], and contact dermatitis [84]. Controlled human exposures to the paper have also confirmed


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responses such as upper respiratory congestion, upper airway obstruction, contact urticaria, and acute systemic reactions. Ojfice Machinery Photocopying, the use of laser printers, and working with VDUs are all factors that influence the prevalence of SBS [15]. A major source of ozone, a known irritant, in indoor air is office machinery such as electrical equipment, computer terminals (or VDUs), laser printers, and dry-process photocopiers [81]. Wet-Process Photocopy Wet-process photocopy (WPC) machines have been reported to be a significant source of VOCs, and copier vapor has been reported to account for 90% of total VOC concentrations in a number of buildings [85]. This "older" type of copy process generally produces higher levels of VOCs than the dry-process type copiers. VOC emissions typically arise when a small amount of the solvent is released into the air each time a copy is made [81]. Kerr and Sauer [86] found that approximately 0.332 ml of solvent was released with each copy made. Dry-Process Photocopy In recent years, many office managers have replaced wet-process photocopiers with dry-process machines to reduce the emissions and servicing problems associated with toner and dispersant liquids [81]. However, emissions testing on dryprocess copiers has shown that this kind of equipment can be a significant indoor source of ozone [87, 88, 89, 90]. The dry-copy process typically requires 5 to 10 kilovolts to produce electrical charges and discharges of photoconductive material. This process has been reported as responsible for emissions of ozone [91], selenium, cadmium sulfide, zinc oxide, and organic polymers from the photo-conductive material, and carbon black particulate matter from toner powder in machine exhaust streams [92, 93]. It has been found that photocopying more than 25 sheets per week is a significant risk factor for mucous membrane symptoms [94]. Visual Display Units (VDU) Health and safety concerns associated with using VDUs in office work environments are controversial and represent a number of health concerns. There is general agreement that working with VDUs causes discomfort problems. A number of studies indicating that increasing hours of VDU usage are associated with higher SBS symptom reporting rates [95, 96, 97].

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Floor Coverings Floor coverings are a potentially significant risk factor for building-related health complaints and a source of indoor contaminants [49]. Several factors regarding floor coverings give rise to potentially significant effects on lAQ and human health comfort. These factors include: • The large quantities of materials that are not used; • The large surface-to-volume ratio of materials such as carpets; • The use of composite products that give them their physical and chemical properties; • They require physical or chemical fastening to substrates; and • They require periodic cleaning. Carpets are considered one of the major sources of VOCs indoors [98] and are a risk factor for increased symptom severity among asthmatics. Floor coverings can affect occupant health directly by releasing toxic contaminants into the indoor air, and can act indirecdy by releasing toxic substances from bonding agents, shampoos, and waxes, and by acting as a reservoir or a contaminant sink for VOCs and immunogenic macromolecular dust (MOD). Floor coverings are also known to act as a medium for microbiological contamination and growth. Combination of Causes SBS can no longer be dealt with in terms of simple dose-response relationships, as if it were a case of chemical exposure. A multi-factorial view of SBS is needed and may account for past failure to identify specific causal agents. The most likely underlying cause of SBS is a combination of environmental exposures with direct, indirect, and additive effects. For example, temperature added to humidity has not been shown to cause SBS directly [99], whereas increased temperature and low humidity has been shown to increase out-gassing of chemicals from indoor materials [100]. Osbom et al. [101] found that by raising indoor temperature from 23°C to 40°C, formaldehyde concentrations released from simulated urea formaldehyde foam insulation (UFFI) wall panels could be increased 13-fold. Similar results have been found with VOC emissions [100]. The synergistic and antagonistic relationships between contaminants and other factors are also important. It is now known that even when formaldehyde levels are extremely low, they can still be dangerous by reacting with one or more volatile organic compounds [102]. If activities such as smoking are allowed in buildings, then the entire indoor environment is changed by the many new toxins being introduced. Lercher et al. [41] found a strong correlation between low-level formaldehyde concentrations, unfavorable physical conditions, and SBS.


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CURRENT RESEARCH There are many gaps in the research and problems with design, collation, and interpretation of results. Hedge et al. [78] points out that none of the research from the USA has a theoretical framework, and neither does much of the European work [103]. Until recently, there has been no research on turnover intentions (the reasons people left their workplaces). Few studies have used multivariate statistical analysis, and none has surveyed a broad range of offices. The range of survey questionnaires used makes comparing data complex, and the inherent bias of self-administered surveys has left some researchers skeptical of others' findings. Many surveys have not used comparably designed "healthy" buildings as a baseline. SOLUTIONS TO SBS PROBLEMS Chemicals are ubiquitous in the workplace, and since the data on toxicological and sensory effects of VOCs are incomplete, overall reduction to exposure is the goal. This can be achieved through a variety of strategies including: • Selection of or replacement with low-emitting building materials and furnishings; • Continuous ventilation during and after installation to minimize the occurrence of temporary sick buildings; • A building bake-out method, increasing the temperature and ventilation for several days to speed up the off-gassing of chemicals; • Darkened glass or shading to minimize direct sun entry and thereby reduce episodic elevation of VOCs; • Use of non-toxic glues, pens, correction fluid, and other office materials; • Storing all chemicals, especially cleaning chemicals, in well-ventilated places with the lids secured; and • Dedicated outside exhaust fans near sources of indoor contaminants such as large office equipment. Air cleaning, filtration, and purification of contaminants from indoor air is now viable; however, technological limitations remain, such as the impracticality of removing organic chemicals [104]. Maintenance and repairs should also be done with the least disturbance to the building environment to avoid release of accumulated dust and fibers into the indoor air. Extensive work will require a ventilation strategy for the affected areas, otherwise the whole building may have to be temporarily closed to avoid occupant exposure to contaminants. Recent research is now showing the benefits of proper cleaning practices in reducing SBS. Studies by Raw [14] and Kemp and Dingle [34] have shown the positive benefits of dust mite reduction measures and high-performance cleaning practices applied to carpets and all other fabric-covered surfaces such as chairs and partitions. In particular, the research is showing that normal bag-type vacuum

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cleaners do not remove the smaller fraction of dust (RSP), which is the most biologically important. The appropriate cleaning of hard surfaces has also shown positive benefits in reducing SBS [39]. CONCLUSIONS Problems with indoor air quality arise from many sources in the office environment and contribute to many complaints concerning comfort, health, and well-being. These stressors interact in combination to produce more significant effects. The wide range of potential exposures in indoor environments is likely to cause a correspondingly wide variation in symptoms known as sick building syndrome (SBS). An ideal work environment has been suggested as one with no large changes in temperature or humidity and with no odors. However, ideal conditions have proven to be far more difficult to achieve than engineers and building designers would hope for. Satisfactory indoor air quality is of vital importance for health, comfort, work, and productivity, and is subsequently an important economic consideration. "Healthy" buildings also include comfort and welfare. Ensuring that people are safe at work may be an initial design goal, but the consideration of health should not end there. Once safety is ensured, avoidance of the potential allergenic, irritative, and toxic effects of indoor environments should also be a primary aim in the design, construction, and use of non-industrial buildings. Education as a solution to SBS is the most prudent path to follow. If people understand even the main causative factors of SBS, and are given the means of monitoring, controlling, and alleviating these factors, top-down action on management should not be necessary. The solutions to SBS also involve education on all the factors associated with the building process. REFERENCES 1. Sykes, J. M. "Sick Building Syndrome." Building Services Engineering Research and Technology, 10(1), pp. 1-11. 1989. 2. Spengler, J. D. and Sexton, K. "Indoor Pollution—A Public Perspective." Science, Vol. 221, No. 4605, pp. 9-16. 1983. 3. Turiel, I. et al. "The Effects of Reduced Ventilation on Indoor Air Quality in an Office Building." In: Atmospheric Environment, Vol. 17, No. 1, pp. 51-64. 1983. 4. WHO. Indoor Air Quality Research. EURO Reports and Studies, No. 103, 1986. 5. Raw, G. J. "Sick Building Syndrome." Building Research Establishment, September, EP230, BRE/109/1/3. 1993. 6. Sundell, J. "On the Association Between Building Ventilation Characteristics, Some Indoor Environmental Exposures, Some Allergic Manifestations and Subjective Symptom Reports."//PzJoorA/r. Supplement No. 2/94:9^2. 1994.


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74. Hedge, A. R. et al. "Psyhosocial Correlates of SBS." In: Proceedings of the Sixth International Conference on Indoor Air Quality and Climate. Helsinki. Vol. 1, pp. 345-350. 1993. 75. Lenvik, K. "Sick Building Syndrome Symptoms; Different Prevalences Between Males and Females." Environmental International. Vol. 8, pp. 11-17.1992. 76. Mendell, M. J. "Non-Specific Symptoms in Office Workers: A Review and Summary of the Epidemiological Literature." Indoor Air, Vol. 3, pp. 227-236. 1993. 77. Taylor, P. R. et al. "Illness in an Office Building with Limited Fresh Air Access." /. Environ. Health. 47:24-7. 1984. 78. Hedge, A. et al. "Work-Related Illness in Offices: A Proposed Model of the Sick Building Syndrome." Environ. Int., 15:143-158. 1989. 79. Valbjom, O. and Kousgard, N. "Headache and Mucous Membrane Irritation: An Epidemiological Study." In: Berglund, B., Lindvall, T., and Sundell, J. (Eds). Indoor Air, Vol. 2. Radon, Passive Smoking, Particulates and Housing Epidemiology. Stockholm: Swedish Council for Building Research, D17:249-54. 1984. 80. Robertson, A. and Burge, S. "Building Sickness—All in the Mind?" Occup. Health, pp. 78-81. March 1986. 81. Etkin, D. S. Office Furnishings, Equipment and lAQ: Health Impacts, Prevention and Mitigation. Cutter Information Corp. Arlington. 1992. 82. Morgan, M. S. and Camp, J. S. "Upper Respiratory Tract Irritation from Controlled Exposure to Vapour from Carbonless Copy Forms." Journal of Occupational Medicine, 28:41-49. 1986. 83. Marks, J. G. et al. "Contact Urticaria and Airway Obstruction with Carbonless Copy Paper." JAMA. 262:1,038-1,040. 1984. 84. Marks, J. G. "Allergic Contact Dermatitis from Carbonless Copy Paper." JAMA. 245:2331. 1981. 85. Tsuchiya, Y. and Stewart, J. B. "Volatile Organic Compounds in the Air of Canadian Buildings with Special Reference to Wet Process Photocopying Machines." Proceedings of Fifth International Conference on Indoor Air Quality and Climate. Vol. 2. Canada Mortgage and Housing Corporation, Ottawa, pp. 633-638; and Stewart, 1990. 86. Kerr, G. and Sauer, P. "Control Strategies for Liquid Process Photocopier Emissions." In: Indoor Air '90—The Fifth International Conference on Indoor Air Quality and Climate. Toronto, Canada. 3:759-764. 1990. 87. Sutton et al. "Predicting Ozone Concentrations in Residential Structures." ASHRAE Journal, pp. 21-26, Sept. 1976. 88. Allen, R. J. et al. "Characterisation of Potential Indoor Sources of Ozone." Am. Ind. Hyg. Assoc. J 39:456-466. 1978. 89. Selway, M. D. et al. "Ozone Production from Photocopying Machines." Am. Ind. Hyg. Assoc. J 41:455-459. 1980.

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90. Wadden, R. A. and Scheff, P. A. Indoor Air Pollution: Characterization, Prediction and Control New York, New York: John Wiley and Sons, p. 213. 1983. 91. Braun-Hansen, T. B. and Andersen, B. "Ozone and Other Air Pollutants from Photocopying Machines." Am. Ind. Hyg. Assoc. J. 47:659-665. 1986. 92. Markin, J. M. et al. "Elevation of Selenium Levels in Air by Xerography." Nature. 259:204-205. 1976. 93. Parent, R. A. "Elevation of Selenium Levels in Air by Xerography." Nature. 263:708. 1976. 94. Skov, P. et al. "Influence of Indoor Climate on the Sick Building Syndrome in an Office Environment." Scand. J. Work. Environ. Health. 16:363-71. 1990. 95. Zweers, T. et al. "Health and Indoor Climate Complaints of 7043 Office Workers in 61 Buildings in the Netherlands." Indoor Air. Vol. 2, pp. 127-136. 1992. 96. Nelson, C. J. et al. "EPA's Indoor Air Quality and Work Environment Survey: Relationships of Employee's Self-Reported Health Symptoms with Direct Indoor Air Quality Measurements." Proceedings of Healthy Buildings. Washington, D.C. pp. 22-32. Adanta: ASHRAE. 1991. 97. Wallach, C. "Video Display Health Hazard Safeguards." In: Proceedings of the Third International Conference on Indoor Air Quality and Climate. Stockholm. Vol. 3. 1991. 98. Levin, H. "Building Materials and Indoor Air Quality." Occupational Medicine: State of the Art Reviews. 4:667-93. 1989. 99. Morris, L. and Hawkins, L. "The Role of Stress in Sick Building Syndrome." In: Indoor Air '87: Proceedings of the Fourth International Conference on Indoor Air Quality and Climate. Berlin. Institute for Water, Soil and Air Hygiene. 1987. 100. Volkl, S., Gebfiigi, I. and Korte, F. "Emission of VOC from Coatings into Indoor Air." In: Indoor Air '90: Proceedings of the Fifth International Conference on Indoor Air Quality and Climate. Ottawa. Institute for Water, Soil and Air Hygiene. 1990. 101. Osbom, S. W. et al. "Urea-Formaldehyde Foam Insulation Study in Indoor Air Quality." CRC Press, 5th Edition, Boca Raton, 1987. 102. Berglund, B. and Lindvall, T. "Sensory Reactions to Sick Buildings." Eviron.Int. 12:147-159. 1986. 103. WHO. Indoor Air Pollutants: Exposure and Health Effects. EURO Reports and Studies, 78:23-26. 1983. 104. Hedge, A. R. et al. "Breathing Zone Filtration Effects on Indoor Air Quality and Sick Building Syndrome Complaints." In: Proceedings of lAQ '91: Healthy Buildings. ASHRAE. Atlanta, pp. 351-355. 1991.

CHAPTER 5 RESPIRATORY FINDINGS OF CONSTRUCTION WORKERS EXPOSED TO ASBESTOS DUST Isamu Ebihara The Institute of Science of Labour Kawasaki, Japan Mamoru Hirata Osaka Prefectural Institute of Public Health Osaka, Japan Naomi Hisanaga National Institute of Industrial Health Kawasaki, Japan Eiji Shibata Nagoya University School of Medicine Nagoya, Japan Kiyoshi Sakai Nagoya City Public Health Research Institute Nagoya, Japan

CONTENTS ASBESTOS UTILIZATION IN THE CONSTRUCTION INDUSTRY, 94 Asbestos-Containing Construction Materials, 94 Levels of Asbestos Exposure, 94 Delay of Measures of Occupational Safety and Health in the Construction Industry, 98 REGULATION OF ASBESTOS, 102 Asbestos Regulations in Different Countries, 102 ILO Convention No. 162 and Recommendation No. 172, 103 RESPIRATORY DISORDERS DUE TO ASBESTOS AMONG CONSTRUCTION WORKERS, 105 Asbestos Burden in Lung Tissues of Construction Workers, 105 Subjective Symptoms, 108 Pleural Plaques and Asbestosis Among Construction Workers, 108 Cancers of the Trachea, Bronchus, and Lung, 111 Malignant Mesothelioma, 113

93

94


MEASURES AGAINST RESPIRATORY DISORDERS DUE TO ASBESTOS EXPOSURE, 118 Prevention of Asbestos Exposure, 118 Health Control, 120 REFERENCES, 123 ASBESTOS UTILIZATION IN THE CONSTRUCTION INDUSTRY Asbestos-containing Construction Materials Chrysotile and amphiboles (including crosidlite, amosite, and ansophylite) have traditionally been used in asbestos-containing construction materials. Today, however, due to regulations restricting their use, chrysotile is mainly used. The regulations include prohibition of crosidlite and often amosite, also. There is even a total ban of the utilization of asbestos in some European countries. Asbestos exposure from construction materials results from the use of cementreinforcement materials and insulatory materials against sound, heat, and fire (e.g., asbestos-containing materials are required by law for use as fireproof construction materials by the Building Standards Law in Japan). After asbestos control regulations came into effect, such materials began being replaced by man-made mineral fibers (MMMF), including fiberglass and rock wool, but asbestos continues to be used in cement-reinforcement and fireproofing materials, many of which are asbestos cement-made materials. In 1995, about 90% of asbestos consumption was accounted for by asbestos cement (A/C) products (asbestos slate, asbestos perlite, roofing tiles, etc.) [1]. For example, about 42,000 tons of asbestos was consumed as asbestos slate in Japan in 1994 (21% of total consumption of about 199,800 tons) [2]. Fireproof construction materials certified by the Ministry of Construction of Japan from 1989 to 1994 showed a decrease in the content of asbestos, which ranged from 2% to 20%. Also, the number of asbestos-containing materials certified by the Ministry of Construction of Japan has decreased: two types in 1994 and 1992, three in 1993, compared with 23 types in 1987, 16 in 1986, and 17 in 1988 (Table 1) [3]. Accordingly, the risk of asbestos exposure among construction workers in the 1990s should have been lower than in the 1980s. However, the total amount of asbestos utilization did not decrease, indicating that old types of asbestos-containing construction materials might still be in use with or without transformation. Levels of Asbestos Exposure Table 2 shows the available data of airborne asbestos concentrations associated with construction work [4-13]. In home construction, the asbestos exposure of workers ranged widely from 0.04 to 787 fibers/ml depending on the type of work and the ventilatory conditions. Indoor sawing with an electric circular saw is considered to be one of the most hazardous operations [6] (Figures 1 and 2).

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

95

Table 1 Asbestos-containing fire-proof construction materials certified by the ministry of construction of Japan from 1989 to 1994, and asbestos contents Number of materials

Item

Fiber-mixed cement board Fiber-mixed calcium silicate board Fiber-mixed asbestos cement board Fiber-mixed slug cement board Fiber-mixed slug plaster board Fiber-mixed cement calcium silicate board Asbestos cement calcium silicate board Asbestos cement board Asbestos perlite slug plaster board Asbestos slate Fly ash mixed slug cement board Calcium silicate board

J.

Total

28

Range of content

4-5% 14-22.5 17 0.5 4.5 4.5 17 20.5 5 5 4.5 5

2

Source: Department of Construction Leading, Bureau of Housing, Ministry of Construction of Japan, Handbook of fireproof structure and materials, 1995 [3].

Table 2 Airborne concentrations of asbestos fibers in construction work

Reference

Application

Number of samples

Concentration (Mathematical mean, fibers/cm^) (range)

10 3 2 11 2 2 2 3 2

10.0(1.2-19.3) 8.6(3.5-19.8) 4.8 (0.7-8.8) 5.3(1.3-16.9) 2.3(2.1-2.5) 4.3(1.5-7.1) 47.2.(35.4-59.0) 5.8(0.5-13.1) 2.6(2.1-3.1)

1 1

41.4 26.4

Drywall construction

Fischbein et al. (1979) USA [4]

- pole sanding (3-5 ft.) background (8 ft.) same room background (25 ft.) adjacent room - hand sanding (3-5 ft.) background (8 ft.) same room background (15 ft.) adjacent room - dry mixing (3-5 ft.) background (10-20 ft.) same room background (16-35 ft.) adjacent room - sweeping floor (10-50 ft.) 15 minutes after sweeping 35 minutes after sweeping

(table continued on next page)

96


Table 2 (Continued)

Reference

Number of samples

Application

Concentration (Mathematical mean, fibers/cm^) (range)

Drywall tapping process Verma& Middleton (1980) Canada [5]

- application - mixing (dry powder) - mixing (pre-mix) - mixing area (pre-mix) - hand sanding - pole sanding - sanding area - sweeping

10 3 7 7 22 52 10 10

3.2 (0.3-7.0) 15.1 (4.0-26.5)

4 3 8

214(125-787) 245 (103-630) 11.0(1.3-1319)

7 8

5.4(0.9-48.1) 2.0(0.3-14.1)

15 1 2

1.3(0.1-4.6) 12.1 0.08(0.04-0.12)

5

0.3(0.1-0.5)

12 8

6.67*1

3

1.10*1

2 2

0.19*1 3.07*1

22

8.5(0.1-24.3)

45

6.4(0.1-61.6)

0.9(0.4-1.3) 11.2(9.0-12.4) 2.4(1.2-3.2) 2.0(1.2-2.7) 11.5(2.1-24.2) 4.9(1.2-10.1)

Home construction work sitek Hisanaga etal. (1991) Japan [6]

- Sawing construction boards - 1.5-2 m from the above work - Screwing, drilling, or nailing (partly including sawing) - 1-10 m from the above work - Screwing, drilling, or nailing (not including sawing) - 1-4 m from the above work - Cutting and filing - 5-30 m from the work operating board - Finishing or cleaning (1 to 7 days after the work) Home construction work sitek

Sakai etal. (1993) Japan [7]

- Sawing - Screwing, drilling, nailing, cutting. or filing (partly including sawing) - Screwing, drilling, or nailing (not including sawing) - Cutting or filing (not including sawing) - Boarding

leY^

Insulation Balzer & Cooper (1968) USA [8]

- Prefabrication: precut and shaped materials using hands or power tools - Application: fitted, hammered, or curved and attached materials to surface by wiring or gluing

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

- Finishing: coating materials with asbestos-containing cements, resins, asbestos or cotton cloth, or petroleumbased sealers - Tearing out: removal of old or unusable materials in the process of insulating or reinsulating - Mixing: mixing mineral wool, asbestos, fibrous glass, and cements or glues - General: cleaning up of old insulation, transporting of materials (1 to 7 days after the work using board)

31

2.7 (0.1-24.4)

17

8.9 (0.2-26.3)

22

2.6 (0.2-10.7)

16

4.8 (0.1-22.9)

97

Renovation and demolition of asbestos-cement (A/C)-clad buildings

Brown (1987) Australia [9]

- Cleaning A/C roofing with water jets - Painting weathered A/C sheeting • by roller • by airless spray - Replacing weathered A/C roofing - Removing weathered A/C roofing - Demolishing A/C warehouses

2

0.08-0.10

3 6 27 20 40

0.12-0.22 0.01-0.14 0.03-0.27 50%). There was good agreement shown between the two observers for separate readings of 400 lungs using this method [32]. Figures 1-8 illustrate different levels of emphysema. Classification of pneumoconiosis was made on the basis of macroscopic and microscopic examination of lung tissue and an examination of whole lung sections. Macules/nodules were classed as sparse (equivalent to ILO 1/0 or less), moderate (less than ILO 2/2), or profuse (ILO 2/2 or greater), and massive lesions documented. Distinction was made between coal only (macules), mixed coal/silica (nodules, palpable), and siHca only changes. One observer made all classifications. Pneumoconiosis classification was available for 259 (98%) subjects. Figures 9-12 illustrate typical histological appearances. (text continued on page 150)

Figure 1. Large lung sections from inflated post-mortem coalworkers' lungs showing normal lung (E score 1).

^~/-&&7^

Figure 2. 0.55). The distribution of emphysema score was positively skewed, suggesting that a log transformation might be appropriate when using regression techniques. However, results from the regression equations were similar with and without transformation. A slightly better fit was obtained when the log transformation of emphysema score was used. Residuals were appropriately distributed whether the emphysema score or its logarithm was used as the outcome variable. For clarity, only the analyses involving the untransformed emphysema score are presented. The correlation matrix for variables of interest is shown in Table 4. Emphysema score (E score) increased significantly with lung coal content, age, and amount of smoking (Equation 1). Equation 1 E score = -1.73 +0.22 (coal g/1) +0.07 (age years) +0.10 (smoking oz/week) (Adjusted R^ = 0.23)

t = 3.12 t = 2.05 t = 8.18 t = 2.50

p < 0.002 p < 0.05 p < 0.0001 p < 0.02

Table 2 Distribution of pneumoconiotic changes (frequency [n] and percent)

Pneumoconiosis severity Sparse n % Pneumoconiosis Type Coal Mixed Silica Sparse Total

123 (47) 123 (47)

Moderate n %

Profuse % n

72 29 4 105

10 11 21

(28) (11) (2) (40)

(4) (4) (8)

IVlassive n %

4 6 10

(2) (2) (4)

n

Total %

86 (33) 46 (18) 4 (2) 123 (47) 259 (100)*

^Subjects did not have data regarding pneumoconiotic change; errors in total percentages are due to rounding.

158

Health and Toxicology Table 3 Mean coal and silica content by type of pneumoconoisis Lung content (g/l predicted vital capacity) Coal* Pneumoconiosis type

Sparse (n = 123) Coal (n = 86) (moderate, profuse, massive) Mixed (n = 46) (moderate , profuse, massive)

Silica**

Mean

SD

Mean

SD

0.53 1.15

(0.41) (0.84)

0.03 0.05

(0.02) (0.03)

1.41

(1.11)

0.07

(0.06)

^Mean lung coal content significantly different for each group (F = 31.8, p < 0.0001), using Duncan's multiple range test. ^^Mean lung silica content significantly different for each group (F = 30.6, p < 0.0001), using Duncan's multiple range test.

Table 4 Correlation matrix of main variables* (Pearson correlation coefficient (r)) Emphysema n=264 Emphysema (Score) Coal (g/l) Silica (g/l) Age (years) Smoking (oz/week) Face (years) FEVi%

1.00 0.20*** QJ9*** 0.46* 0.04 0.24* -0.44*

Coal n=264

Silica n=264

Age n=264

Smoking n=264

Face FEVi% n=264 n=183

1.00 0.54* 1.00 0.23** 0.26* 1.00 —0 15**** -0.25* -0.17*** 1.00 0.26* 0.45* -0 19*** 0.27* 1.00 0.05 -0.31* -0.02 -0.11 -0.01

1.00

*/7 < 0.0001 **/7 < 0.001 ***/? < 0.005 ****/? < 0.02

Lung silica content did not contribute significantly to the model. Addition of a coal-smoking interaction term improved the model slightly, with coal and smoking having a negative interaction (Equation 2). Note that the effect of lung coal content on E score must be obtained by using a combination of the coal-only and coal x smoking regression coefficients, as the effect of coal depends on the level of smoking.

Emphysema and Lung Mineral Content in Coalworkers

159

Equation 2

E score = -1.79 +0.62 (coal g/1) +0.06 (age years) +0.21 (smoking oz/week) -0.17 (coal X smoking) (Adjusted R^ = 0.25)

t = 3.28 t = 3.64 t = 7.76 t = 3.91 t = 2.99

p< p< p< p< p
It!*

proportion of lung dry weight t value p > Itl*

absolute amount per subject t value p > it!*

3.64

0.0003

3.53

0.0005

3.79

0.0002

7.76 3.91 -2.99

0.0003 0.0001 0.004 0.25

7.86 3.28 -1.89

0.0001 0.002 0.06 0.26

7.87 4.02 -3.15

0.0001 0.0001 0.002 0.26

^probability of obtaining an absolute value oft as great or greater

160


Emphysema score had a highly significant negative relationship with FEVi% for the 184 subjects who had available FEVi% measurements (Equation 4). Almost identical results were found when lung coal content was added to the regression model (P E score = -7.39, t = -6.73, p < 0.0001), with coal not making a significant contribution to the model (|3coal = 2.86, t = 1.00, p > 0.32). This relationship appeared to be similar for non-smokers, with the low numbers of non-smokers with available FEVi data (28) probably responsible for the borderline statistical significance of this relationship (Equation 5). Equation 4 FEVi%= 99.2 -7.24 (E score)

t = 28.22 t = 6.66

p < 0.0001 p < 0.0001

t= 10.24 t = 1.76

p 0.17). Discussion The results from this study show clearly that lung coal content is quantitatively related to the development of emphysema, taking into account age and smoking. This is true whether the effect of smoking is controlled using regression methods or by examining only non-smokers. The results indicate that a non-smoking miner with about the average lung coal content would have emphysema involving an extra 7 to 10% of the whole lung mass compared to a non-smoking person of the same age who was not exposed to coal. The influence of coal appears less marked in smokers, possibly due to a negative interaction between smoking and coal. Although other possible causes of emphysema in coalworkers, apart from coal and smoking, have been suggested [44], the very high R^ term in the regression equation for non-smokers strongly suggests that coal exposure and age are the main determinants of emphysema in non-smoking coalworkers. Biologically plausible mechanisms for a pathological effect of coal on lungs to produce emphysema are now well-documented [45, 46]. The analytical technique used in this study measures total coal and silica content of the lung, which includes both dust in the bronchi and in the parenchyma. This measurement is used as a proxy for the true measure of interest, parenchymal dust, as it is in the parenchyma that any pathological effects that might result in emphysema presumably occur. Therefore, any process (such as dust being trapped in the increased mucus produced in bronchitis) that might alter the proportion of total lung dust in the parenchyma could affect the observed relationship between total lung dust and emphysema. Unfortunately, it is not known how much variation in this proportion might occur due to pathological processes or differences between individuals, because it is too difficult to dissect out the bronchial tree from the parenchyma to allow separate measurements of dust content in each. However, known particle deposition distributions would suggest that the parenchymal component would be much larger than the bronchial component, except in very unusual circumstances. An alternative explanation for the observed coal-emphysema association might be that emphysema somehow impairs clearance of dust from the lungs. Greater emphysema would therefore be associated with dust in the lung because of decreased clearance rather than because the dust was causing the emphysema. The effect of emphysema on clearance is uncertain, and there is animal and human evidence of both decreased and increased clearance in emphysematous lungs [47^9].

162


The strong positive association between age and emphysema found in this study is well-documented elsewhere [50]. An association between smoking and emphysema as found in this study has been well-documented in the general community [51], coal miners [52], and goldminers [34, 35]. The negative interaction between coal and smoking does not have a clear explanation. Perhaps it is due to the fact that smokers have more bronchitis [51], and that the increased mucus production traps more cqal in the bronchi before it reaches the lung parenchyma. Therefore, for a given amount of measured dust in the lung, more smoking may result in a smaller proportion of dust reaching the parenchyma and correspondingly less emphysema than would otherwise be expected. Alternatively, the negative interaction may be due to a form of competitive inhibition, with the individual effects of coal and smoking having a final common pathway in which the presence of one interferes with the effect of the other. This is plausible, as macrophage or polymorphonuclear leucocyte damage or activation, with subsequent release of elastase or other proteases, is a probable mechanism for the development of emphysema in both smokers [52] and coal miners [45, 46]. Finally, the observed relationship might simply be a result of statistical associations inherent in this data set rather than a reflection of a true biological phenomenon. Silica content in the lung was not found to be significantly associated with emphysema in this study. Previous investigators have found conflicting results. One group investigating British coalworkers found a negative effect of silica on emphysema prevalence in coalworkers [30]. This was true for lung silica content for subjects with progressive massive fibrosis; and for percentage silica of lung dust or percentage silica in lung for subjects with any fibrotic lung disease. They postulated that silica-induced fibrosis might make emphysematous change less likely. In goldminers exposed to "silica dust," the prevalence of centriacinar emphysema was found to be related to dust exposure level and to the presence of silicosis in one study [35]. In contrast, an earlier study of goldminers found a positive association between the presence of any emphysema and dust exposure levels, but no significant effect of silicosis [34]. It is possible that the relationship between emphysema and "silica dust" in these two studies was due to some other component of the dust apart from silica, although the dust is known to have had a high silica content [35]. A very recent study of lifelong non-smoking white South African goldminers found only low levels of emphysema, mainly panacinar, unrelated to dust exposure, pathological silicosis, or loss of respiratory function [53]. The lack of an emphysema-silica association in the present study may be due to relatively low silica exposures and correspondingly less silica-related fibrotic lung disease, even though there was a strong relationship between the presence of fibrotic lung disease (mixed coal-silica or silica-only disease) and lung content of silica. The subjects included in this study were mosdy recent miners who had a relatively low prevalence of fibrotic lung disease. Of the 259 subjects who had data available on lung histology, 19% had mixed coal-silica or silica-only nodules, with only 34% of these (6% of the total) showing profuse nodularity or massive fibrosis (ILO 2/2 or


163

greater). In comparison, in a study of British coalworkers that showed a negative emphysema-siUca association, 79% of the subjects had some fibrotic lung disease and 33% were said to have progressive massive fibrosis [30]. A study of an earlier group of miners from the same mining regions as the subjects used in this study, but exposed to a higher concentration of respirable dust, had a much higher prevalence of fibrotic lung disease, with 48% having mixed nodules and 54% of these (26% of the total) having ILO 2/2 or greater [33]. In the latter group, there was a significantly lower prevalence of emphysema in those subjects with mixed nodules compared to those with coal-only macules. This difference was especially marked in those with profuse mixed nodules, supporting the suggestion that there may be a negative association between silica and development of emphysema. Unfortunately, the lungs of those subjects were not available for the mineral analysis used in this study. Lung silica content of subjects in this study was also lower than in other studies, both on an absolute and a percentage basis. This study showed a mean lung silica content of 0.11% of dry lung weight, compared to 0.35% in the above study of British coalworkers [30]. The comparable absolute amounts were 0.18 g compared with 0.30 g per subject. Percentage lung coal content could not be directly compared between the two studies because of differences in the data presentation, but the mean absolute amount of coal per subject found in this study (3.9 g) was lower than that found in the British study (4.9 g). Lung coal content was an order of magnitude higher than lung silica content in this study (mean coal g/1: mean silica g/1 = 22:1), similar to the British study, which found a corresponding ratio of about 16:1. Emphysema score had a highly significant negative relationship with FEVi% in this study. This result has been found previously in a larger group of coalworkers that included the subjects from this study [32] and in other studies of coalworkers [29, 30]. The emphysema-FEVi% relationship was independent of lung coal content, suggesting that the association was not a consequence of dust exposure independently causing emphysema and a decrease in FEVi%. The regression coefficient suggests that the emphysema found in coalworkers is of some functional relevance, with an increase of emphysema of 10% leading to a predicted decrease of FEVi% of about 7%. This same negative relationship between FEVi% and emphysema was suggested in the analysis, which included only non-smokers, but the small number of subjects with available FEVj measurements makes firm conclusions difficult. Recent evidence suggests that occupational exposure to coal dust alone might produce clinically important airways dysfunction [54-56], although this is not accepted by all researchers [57, 58]. In this study, as the severity of pneumoconiosis increased the severity of emphysema increased. This relationship appeared to be independent of lung dust content and suggests a direct pathological connection between pneumoconiosis and emphysema. FEVi% showed a non-significant decline with increased severity of pneumoconiosis. The lack of statistical significance of this relationship was possibly due to the limited number of subjects with available FEVi measurements.

164


Lung content of coal and silica were both highly correlated with each other, as would be expected because the majority of coalworkers were exposed to mixed coal/silica dust. Lung contents of coal and silica were also both significantly correlated with years worked at the face, although the correlation coefficients of 0.26 and 0.27 suggest that years worked at the face gives a more qualitative estimate of the lung burden from dust exposure. Years at the face was highly significantly correlated with emphysema score, but once age was taken into account, years at the face no longer had a significant relationship with emphysema score, presumably because years at the face was acting as a proxy measure of age in the univariate correlations. A number of sources of bias are possible in a study of this type. However, for reasons outlined below, bias is in fact not thought to have been a significant problem. Most of the emphysema found in previous studies of coalworkers was reported to be of the centriacinar type [28, 29] with relatively little panacinar emphysema, and this was also the case in the present study. In advanced cases, centriacinar emphysema was seen to extend to involve whole lobules and to coalesce between lobules, ultimately involving the whole lung. In accordance with current thinking [59-61], we regarded the two types of emphysema as part of a spectrum and hence made no attempt to separately quantify panacinar emphysema. Tissue preparation methods were the same for all lung specimens used, and emphysema measurements were made by either of two observers using standard techniques, with a high degree of inter-observer agreement, blinded to the other variables of interest. Assessment of emphysema using macroscopic measurements has been criticized [59], but the technique is widely used [9] and, in this study, emphysema score had a highly significant negative relationship with FEVi%, as would be expected clinically. The measurements of overall emphysema are therefore believed to be appropriate and reasonably accurate. Dust analysis was performed using minor modifications of established techniques, with good agreement between duplicate samples, and analysis was done without knowledge of the emphysema measurements made on each specimen. Lung content of coal and silica can be regarded as the best quantitative estimate of exposure as, once clearance mechanisms are saturated, accumulation of dust in the lungs is proportional to further exposure [62]. Lung coal and silica contents were adjusted using predicted vital capacity, which was used as an estimate of lung volume, thereby providing a measure of the concentration of coal and silica in the lung. However, this adjustment introduces some (presumably non-differential) error. Expressing lung coal and silica content as a proportion of dry lung weight would appear to give an even better measurement of the concentration of dust in the lung. Unfortunately, this measurement can be criticized on the basis that more emphysematous lungs probably contain less total lung tissue. Thus, for a given amount of total lung dust, more emphysematous lungs would appear to have a higher concentration of dust per dry weight of lung, thereby potentially introducing a spurious positive relationship between dust content and


165

amount of emphysema. Conversely, more fibrotic lungs might contain more lung tissue and so introduce an apparent negative relationship. Nevertheless, a number of authors have expressed lung dust content on a per dry weight of lung basis [30, 63, 64], and in this study analyses using this measurement for lung coal and silica content produced similar results to the main analyses performed using measures adjusted by predicted vital capacity. Using the absolute mass of coal or silica (unadjusted) does not give a measure of the concentration of dust in the lung as it does not take into account the fact that bigger lungs will contain more dust than smaller lungs. Therefore, this measure is likely to contain significant non-differential error, although analyses using these unadjusted measures gave similar results to the primary analysis. The 264 subjects included in this study are a subset of a continuous series of 1,086 post-mortem examinations on coalworkers in New South Wales from 1949 to 1987. Lung tissue obtained prior to 1966 was no longer available for mineral analysis. Subjects who had a post-mortem during the study period 1966-1983 but were not included in this study had similar values for the main known variables of interest compared to subjects who were included in the study. Since the main reason for exclusion of subjects at this stage was inadequate lung specimens, it is unlikely that significant bias was introduced because of this exclusion. It is likely that those miners who reached post-mortem had a higher prevalence of lung disease than the overall mining population of the same age during the study period. Post-mortem proven lung disease would be more likely to attract compensation for the families of the miners than lung disease that was not confirmed pathologically, so families of miners who suspected that they had lung disease are more likely to have agreed to the conduct of a post-mortem examination. However, there is no reason to believe that selection would have been influenced by emphysema severity and lung dust content in such a way as to artificially produce the results found in this study. In conclusion, the results from this study provide strong support for the hypothesis that emphysema in coalworkers is quantitatively and causally related to coal content in the lung and thus to exposure to coal in life. In this regard, it is of interest that emphysema has recently become a prescribed disease for underground coal miners in the United Kingdom [65]. The importance of age and smoking in emphysema severity is also confirmed. The results do not support a relationship between silica exposure and the development of emphysema. This review is based on the previously published papers [66, 67] and material in Reference [68]. REFERENCES 1. Miller, W. S. The Lung. 2nd edition. C. C. Thomas, Springfield, 1947. 2. Reid, L. "The secondary lobule in the adult human lung, with special reference to its appearance in bronchograms." Thorax, 1958; 13: pp. 110-115.

166


3. Ciba Guest Symposium. "Terminology, definitions and classification of chronic pulmonary emphysema and related conditions." Thorax, 1959; 14: pp. 286-299. 4. Thurlbeck, W. M., Henderson, J. A., Fraser, R. G., Bates, D. V. "Chronic obstructive lung disease. A comparison between clinical, roentgenologic, functional and morphologic criteria in chronic bronchitis, emphysema, asthma, and bronchiectasis." Medicine, 1970; 49: pp. 81-145. 5. Snider, G. L., Kleinerman, J., Thurlbeck, W. M., Bengali, Z.H. "The definition of emphysema." Report of a National Heart, Lung, and Blood Institute, Division of Lung Diseases Workshop. Am Rev Resp Dis, 1985; 132: pp. 182-185. 6. Weibel, E. R. Morphometry of the Human Lung. Heidelberg: Springer. 1963. 7. Glazier, J. B., Hughes, J. M. B., Maloney, J. E., West, J. B. "Vertical gradient of alveolar size in lungs of dogs frozen intact." J Appl Physiol, 1967; 23: pp. 694-705. 8. Dunnill, M. S. "The contribution of morphology to the study of chronic obstructive lung disease." Am J Med, 1974; 57: pp. 506-519. 9. Thurlbeck, W.M. Chronic Airflow Obstruction in Lung Disease. Philadelphia: W. B. Saunders, 1976. 10. Heard, B. E. "Further observations on the pathology of pulmonary emphysema in chronic bronchitis." Thorax, 1959; 14: pp. 58-70. 11. Snider, G. L., Brody, J. S., Doctor, L. "Subclinical pulmonary emphysema. Incidence and anatomic patterns." Am Rev Resp Dis, 1962; 85: pp. 66-83. 12. Thurlbeck, W. M. "The incidence of pulmonary emphysema with observations on the relative incidence and spatial distribution of various types of emphysema." Am Rev Resp Dis, 1963; 87: pp. 206-215. 13. Gough, J. "The pathology of emphysema." Postgrad Med J, 1965; 41: pp. 392-400. 14. Leopold, J. G., Gough, J. "The centrilobular form of hypertrophic emphysema and its relation to chronic bronchitis." Thorax, 1957; 12: pp. 219-235. 15. Heppleston, A. G., Leopold, J. G. "Chronic pulmonary emphysema: anatomy and pathogenesis. Am J Med, 1961; 31: pp. 279-291. 16. Gough, J. "Pneumoconiosis in coal trimmers." J Path Bacteriol, 1940; 51: pp. 277-285. 17. Heppleston, A. G., Leopold, J. G. "Chronic pulmonary emphysema: anatomy and pathogenesis." Am 7 MeJ, 1961; 31: pp. 279-291. 18. Heppleston, A. G. "The pathological anatomy of simple pneumoconiosis in coal workers." 7 PRE>Other, (ii) RE>RC>Others, (iii) RC>Other or RE>Other, (iv) Other>RC and Other>RE.

198


The results were: (i) 50%, (ii) 30%, (iii) 9%, and (iv)ll%. The most remarkable finding was the higher Cr concentrations at most (80%) of the bifurcations than at the other portions [(i) + (ii)]. Within the ridge of many bifurcations, concentrations at the centers were higher than at the edges. Whereas the concentrations were relatively constant in other portions, they varied considerably from site to site at the centers of bifurcation ridges (Table 2). This was particularly the case in more peripheral regions, where very high values were found almost exclusively at the ridge centers. In portions other than the centers of bifurcation ridges, Cr levels were generally higher in the peripheral than in the central bronchi in both lungs. Those at the trachea and tracheal bifurcations were the lowest, in agreement with the scarcity of cancers arising at these sites. Mean Cr concentration ratios between portions at ridge centers and at other portions for each generation of tracheobronchial branching, calculated from Table 2, are shown in Figure 4. For this purpose we propose the term "accumulation ratios at ridge centers." In all the generations of tracheobronchial branching from tracheal to segmental bifurcations, the mean accumulation ratios were more than 1. Interestingly, the mean accumulation ratios increased with generation of tracheobronchial branching, although the high values of 8.4 (n = 4) and 10.9 (n = 3) were largely due to two particularly high ratios of 25.4 (14.20/0.56) and 25.2 (38.54/1.53), respectively, both of which were found in Case 9. Using all the ratios (n = 44) from both sides of the lungs of all the cases, the correlation coefficient (R) regarding the accumulation ratios and branching generation was calculated to be significantly positive, at 0.06 < R < 0.58 (p < 0.05). DISCUSSION The present investigations revealed 80% of bifurcations to show higher concentrations of Cr at ridges than in other nearby portions. Furthermore, all mean accumulation ratios at ridge centers proved to be more than 1, with values increasing significantly with increasing generations of bronchial branching. These findings imply that the bifurcation ridges, and in particular the ridge centers, are hot spots of persisting Cr deposits even more than 10 years after cessation of exposure. This study thus produced solid evidence for the deposition hot spot concept of long-term retention. There are two main aspects, physical and pathophysiological, that should be taken into account in determining the factors responsible for formation of deposi-

Table 2 Cr concentrations of former chromate workers’ bronchi Tracheal Case

RC

RE

Main Other

Left lung and trachea 1

RE

Other

RC

RE

Segmental Other

RC

RE

Other 0

-

2 3

1.1

4 6

7

RC

Lobar

0.6

0.4

8 (table continued on next page)

1.7 2.6

-

0.8 2.2

0.2 0.7 1.9

0.2

4.4 1.3

1.8 2.0

2.7 1.1

0.4

13.4

6.4

1.1

4.7 3.0

1.1 7.4

1.o 2.9

12.3 0.4

3.9 0.8

zz.

0.7 0.4

1.0 8.0 3.8 8.4 14.4 14.6

1.4 4.3 3.1 4.0 4.1 6.5

1.9 2.6 1.4 1.4 2.6 3.3

2.1 2.2 2.3 3.3 1.5 4.2 9.2

3.4 1.8 2.6 4.8 1.6 2.8 4.5

1.1 1.3 1.8 1.9 1.3 1.1 2.2

C

3 D

9.2

5.8

1.8

0 0

C

3

E

$ !

2 s

3 a3

2 (D

z

% v?

m

a

3 0

z A

(0 (0

Table 2 (Continued) Cr concentrations of former chromate workers' bronchi Main Case

RC

RE

Intermediate Other

RC

RE

Other

Lobar RC

RE

pl

Segmental Other

RC

RE

Subsegmental

Other

RC

RE

Other

d

+ .!0

0 0

9

Right lung 1

1.2

1.2

1.9

2 4 5

2.2 1.6

2.0 1.1

0.8

6 7 9

1.1

0.3

0.4 4.6

0.4 2.3

0.5 1.2

0.8 0.04

1.2 1.2

0.7

-

1.1 2.4 2.2

1.8 2.3

1.1 1.9

8.0 3.7 12.6 0.6 1.9 1.5 6.7 6.4

3.9 0.5 3.4 0.2 3.0 5.7 -

2.3 3.1 3.3

18.0 12.3 14.7 2.5 1.6 1.7

4.9 3.9 1.9

2.5 2.1 9.7 0.7

1.9

14.2 16.5

0.6

1.9 4.2 38.5 7.8

0.8 1.5

-

-

1.0 1.0 1.5 0.9

Centers of bifurcation ridges (RC), the edges of bifurcation ridges (RE), and other (i.e., more central or more peripheral) parts near the bifurcation (Other). Measured by neutron activation analysis, at 15 years on average after cessation of exposure. Unit of Cr concentration is x l @"g/pm tissue thickness/mm2.

Chromium Accumulation of Chromate Workers' Bronchi

201

Mean Cr accumulation ratios at ridge centers of bifurcations 4

6

8

10

12

Tracheal bif. (n=1)

L Main br. bif. (n=7) L Lobar br. bif. (n=15) L. Seg. br. bif. (n=1)

R. Main. br. bif. (n=3) R. Interm. tr. bif. (n=5) R. Lobar, br. bif. (n=7) R. Seg. br. bif. (n=4) R. Sub-seg. br. bif. (n=3) Figure 4. Mean ratios of Cr concentrations between the ridge centers of bifurcations and other portions (mean Cr accumulation ratios) for each generation of bronchial branching. Note that all the mean Cr accumulation ratios are more than 1 and that the ratio increases with increasing generation of tracheobronchial branching. L., left; R., right; bif., bifurcation; br., bronchus or bronchial; Interm. tr., intermediate trunk; Seg., segmental; n, number of bifurcations available for calculation of the accumulation ratios.

tion hot spots at bifurcations. Physically, there are four ways in which solid particles can be deposited in the lung: sedimentation, inertial impaction, interception, and diffusion [28]. Among these, the former two are of primary importance in the enhanced deposition at bronchial bifurcations of the chromate workers because the other two may concern particles of irregular shape and very small particles. In fact, microscopic measurements in lung tissues obtained at autopsy showed that most of the chromate particles were 1-3 jim in diameter [29], with an approximately spherical shape. (Actual assessment of particle size distribution in the workplace is now impossible because the factory in which the workers were employed terminated production of Cr compounds in 1975.) Considering the size and shape of particles and airway generations of interest (main to subsegmental bronchi), inertial impaction and sedimentation may be chiefly responsible for the enhanced deposition in the workers' bronchi [28].

202


However, the results obtained by this approach should be interpreted in the light of physiological considerations, where possible, using animal models for example, because deposited particles are largely cleared by mucociliary streaming. It is known that the ciliary streaming of mucus and the lymphatic flow tend to slow down and stagnate around the bifurcation [14, 30]. Other factors that may facilitate augmented deposition are related to pathology in human beings. Prolonged exposure to toxic inhalants such as tobacco smoke causes loss of cilia and/or squamous metaplasia occurring preferentially at or near bifurcations. This would further delay the mucociliary removal and hence accelerate the formation of hot spots. Additionally, we should consider the question as to whether Cr sticks to bronchial tissues for more than 10 years or whether steady removal of Cr from alveolar regions is responsible for the enhanced concentration in bronchial bifurcations. If the former possibility is true, most of the Cr detected in bronchial tissues must be deposited in bronchial stroma rather than in epithelium, considering that cell renewal of the epithelium is estimated to be an order of 1 year or less for adults in our age range [31, 32]. On the other hand, the latter possibility would imply that most of the Cr measured is localized to the epithelium. Further fine topographical determination of Cr within bronchial tissue may answer this question. Another finding requiring discussion is the fact that higher enhancement was evident in the more peripheral bifurcations. This may be a clue to the question on how the deposition hot spots are formed, because one of the main causes is the stagnation of mucus stream at the bifurcations mentioned above. The mucus flow must carry considerable amounts of Cr compounds deposited in alveolar regions, which retain mainly particles with diameters of more than sub-|im and less than 4 [xm [33], comparable with typical chromate particles [29]. In other words, the fact that at the more peripheral bifurcations a more marked deposition was evident may imply that the primary mechanism of forming hot spots at bifurcations is the stagnation of mucus flow at such sites. Further, it is very interesting that the distribution of Cr is comparable with that of bronchial cancer which is known to arise most frequently at segmental bronchi, not at more central bronchi [3]. In this study we successfully examined up to subsegmental bifurcations, but could not analyze more peripheral bifurcations using the same methods because of their very small dimensions. Chemical forms may be crucial for metal carcinogenesis. As is well known, 6valence Cr is more harmful than the 3-valence form. However, actual measurements showed that most of the Cr deposited in pulmonary tissues was 3-valence like Cr203 [34]. We agree with Dr. T. Sano, a pioneer of occupational disease pathology in Japan, who proposed that 6-valence Cr is so strong an irritant that it can induce severe inflammation and subsequently is reduced to 3-valence, which can exist as an insoluble form and might persist to cause cancer [29]. It is known that carcinomas and atypical lesions preferentially occur at bronchial bifurcations in smokers [4]. This is also the case for ex-chromate workers [26]. Hence, the results of this study would indicate a direct relationship between the concentration of Cr deposits and neoplasia. Additionally, it should be noted that the


203

Cr concentrations at ridge edges were not found to be low. In fact, among the 44 bifurcations where the values at the ridge center, ridge edge, and another portion were available, 13 (30%) showed the highest level at the edge as mentioned above. Cigarette smoking may have been a factor in the induction of carcinomas in the bronchi of the population of ex-chromate workers used in this study. Actually, most of the cases were smokers with an average pack-year of 32.7 (Table 1). However, an epidemiological study of the same population of ex-chromate workers suggested that the exposure to Cr compounds played the main role in causing cancer development [20]. Summarizing briefly, the lung cancer morbidity rate for the ex-chromate workers with 9 years or more of exposure was 21.6 times higher than that for the general population. The mortality rate for lung cancers in non-chromate smokers, adjusted to the ex-chromate worker population by age of starting smoking and by number of cigarettes per day, is generally 3-4.7 times higher than for non-smokers in Japan. Unless cigarette smoking had an extraordinarily synergistic effect on cancer induction, or super-multiplicative synergism with Cr inhalation, we can therefore conclude that the Cr exposure was primarily responsible, always bearing in mind that there is a minor difference between morbidity and mortality. ACKNOWLEDGMENTS We wish to thank Drs. T. Kitagawa and H. Sugano, Cancer Institute, for their support; Drs. K. Nakagawa, Cancer Institute Hospital; T. Hirano, Hirano Kameido Himawari Clinic; and Prof. T. Tamai, Kyoto University, for collaboration. Y. I. is grateful to Professor Emeritus S. Hatakeyama for his encouragement. This study was supported financially by the Visiting Researchers' Program, Research Reactor Institute, Kyoto University, Osaka, Japan. REFERENCES 1. Kreyberg, L. Aetiology of lung cancer—a morphological, epidemiological and experimental analysis, Oslo: Oslo Universitets Forlaget, 1969, pp. 1-90. 2. National Academy of Sciences. Health Risks of Radon and Other Internally Deposited Alpha-emitters, BEIR IV Report, Washington, D.C.: National Academy Press, 1988, pp. 1-602. 3. Marsh, B. et al. "Bronchoscopic localization of radiologically occult cancer," in Recent Results in Cancer Research, Vol. 82, Early Detection and Localization of Lung Tumours in High Risk Groups. R. R. Band (Ed.), Heidelberg: Springer-Verlag, 1982. pp. 87-89. 4. Auerbach, O. et al. "Changes in bronchial epithelium in relation to cigarette smoking and in relation to lung cancer." New England Journal of Medicine, Vol.265, 1961, pp. 253-267.

204


5. Hofmann, W. and Balashazy, I. "Particle deposition patterns within airway bifurcations—solution of the 3D Navier-Stokes equation." Radiation Protection Dosimetry, Vol. 38, 1991, pp. 57-63. 6. Kinsara, A. A. et al. "Computational flow and aerosol concentration profiles in lung bifurcations." Health Physics, Vol. 64, 1993, pp. 13-22. 7. Martell, E. A. "Alpha-radiation dose at bronchial bifurcations of smokers from indoor exposure to radon progeny." Proceedings of National Academy of Science of USA, Vol. 80, 1983, pp. 1,285-1,289. 8. Martonen, T. B. et al. "Cigarette smoke and lung cancer." Health Physics, Vol.52, 1987, pp. 213-217. 9. Ermala, P. and Holsti, L. R. "Distribution and absorption of tobacco tar in the organs of the respiratory tract." Cancer, Vol. 8, 1955, pp. 673-678. 10. Martin, D. and Jacobi, W. "Diffusion deposition of small-sized particles in the bronchial trees." Health Physics, Vol. 23, 1972, pp. 23-29. 11. Sussman, R. G. et al. "Asbestos fiber deposition in a human tracheobronchial cast. I. Experimental, and II. Empirical model." Inhalation Toxicology, Vol. 3, 1991, pp. 145-179. 12. Schlesinger, R. B. and Lippmann, M. "Selective particle deposition and bronchogenic carcinoma." Environmental Research, Vol. 15, 1978, pp. 424^31. 13. Gore, D. J. and Patrick, G. "The distribution and clearance of inhaled UO2 particles on the first bifurcation and trachea of rats." Physics of Medicine and Biology, Vol. 23, 1978, pp. 730-737. 14. Svartengren, M. et al. "Retention of particles on the first bifurcation and the trachea of rabbits." Bull Eur. Physiopathol Respir., Vol. 17, 1981, pp. 87-91. 15. Brody, A. R., et al. "Chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages." American Review of Respiratory Disease, Vol. 124, 1981, pp. 670-679. 16. Brody, A. R. and Ore, M. W. "Deposition pattern of inorganic particles at the alveolar level in the lungs of rats and mice." American Review of Respiratory Disease, Vol. 128, 1984, pp. 724-729. 17. Little, J. B. et al. "Distribution of polonium-210 in pulmonary tissues of cigarette smokers." New England Journal of Medicine, Vol. 273, 1965, pp. 1,343-1,351. 18. Mancuso, T. F. and Hueper, W. C. "Occupational cancer and other health hazards in a chromate plant: a medical appraisal. I. Lung cancers in chromate workers." Industrial Medicine and Surgery, Vol. 20, 1951, pp. 358-363. 19. Abe, S. et al. "Chromate lung cancer with special reference to its cell type and relation to the manufacturing process." Cancer, Vol. 49, 1982, pp. 783-787. 20. Nakagawa, K. et al. "Surveillance study of a group of chromate workers— early detection and high incidence of lung cancer." (in Japanese) Japanese Journal of Lung Cancer, Vol. 24, 1984, pp. 301-310.


205

21. Langard, S. and Norseth, T. "A cohort study of bronchial carcinomas in workers producing chromate pigments." British Journal of Industrial Medicine, Vol. 32, 1975, pp. 62-65. 22. Machle, W. and Gregorius, F. "Cancer of the respiratory system in the United States chromate-producing industry." Public Health Report, Vol. 63, 1948, pp. 1,114-1,127. 23. Newman, D. "A case of adeno-carcinoma of the left inferior turbinate body and perforation of the nasal septum in the person of a worker in chrome pigments." Glasgow MedicalJournal, Vol. 33, 1890, pp. 469-470. 24. Bidstrup, P. L. "Carcinoma of the lung in chromate workers." British Journal of Industrial Medicine, Vol. 8, 1951, pp. 302-305. 25. Baetjer, A. M. "Pulmonary carcinoma in chromate workers I. A review of the literature and report of cases." AMA Archives of Industrial Hygiene and Occupational Medicine, Vol. 2, 1950, pp. 487-504. 26. Ishikawa, Y. et al. "Characteristics of chromate workers' cancers, chromium lung deposition and precancerous bronchial lesions: an autopsy study." British Journal of Cancer, Vol. 70, 1994, pp. 160-166. 27. Ishikawa, Y. et al. "Quantitative determination of elements in paraffinembedded specimens by activation analysis—an example of Thorotrastinjected patients." (in Japanese) Journal of Clinical and Experimental Medicine (Igaku-no-ayumi), Vol. 141, 1987, pp. 959-960. 28. Parks, W. R. Occupational Lung Disorders, 2nd ed. London: Butterworth & Co., 1982, pp. 1-529. 29. Sano. T. "Occupational cancers in chromate workers." in Pneumoconiosis and Public by Dust in Japan, 2nd ed. (in Japanese) T. Sano (Ed), Kawasaki: Institute for Science for Labour, 1983, pp. 315-325. 30. Hilding, A. C. "Ciliary streaming in the bronchial tree and the time element in carcinogenesis." New England Journal of Medicine, Vol. 256, 1957, pp. 634-640. 31. Fawcett, D. W. A Textbook of Histology, Philadelphia: W. B. Saunders Co., 1986, p. 737. 32. Creagh, T., and Krausz, T. "13. The respiratory system, 13.1 Normal structure and function." in Oxford Textbook of Pathology, Vol. 2a, J. O'D. McGee et al. (Eds.), Oxford: Oxford University Press, 1992, pp. 943-947. 33. Lippmann, M. and Albert, R. E. "The effect of particle size on the regional deposition of inhaled aerosols in the human respiratory tract." American Industrial Hygiene Association Journal, Vol. 30, 1969, pp. 257-275. 34. Kudo, H. et al. "The concentration and X-ray microanalysis of chromium in the lung tissues of a chromate worker with multicentric lung cancer." (in Japanese) Japanese Journal of Lung Cancer, Vol. 19, 1979, pp. 385-392.


CHAPTER 10 BENZENE TOXICOKINETICS IN HUMANS Frederic Yves Bois Indoor Environment Program Energy and Environment Division Lawrence Berkeley National Laboratory Berkeley, CA 94720

CONTENTS INTRODUCTION, ABSORPTION, DISTRIBUTION, ELIMINATION, BENZENE SECONDARY METABOLISM, MODELING OF BENZENE TOXICOKINETICS, REFERENCES, INTRODUCTION Benzene is an ubiquitous chemical, present in some natural foodstuffs and raw petroleum minerals, but mostly encountered in industry as a basis for plastic monomers and as a solvent [1]. It is also found in refined petroleum products such as gasoline. Benzene is volatile at usual temperatures and slightly miscible with water (0.8 parts by weight in 1,000 parts of water at 20°C). Overall, it has been estimated that 10 kg of benzene is lost per ton of benzene produced during transfer and storage, with approximately 94% lost as air emissions and 6% as water effluents [1]. It is therefore found in ambient air, water, and soil. For example, benzene has been detected in U.S. water supplies [2]. Concentrations between 0.3 to 2.4 ppm in the air near gas stations for benzene have been reported, compared to 0.017 ppb for rural areas [1]. The main exposure for smokers is from mainstream cigarette smoke: This represents 50% of the population exposure burden [3]. The highest exposure levels occur for workers exposed when using it as a solvent. Benzene is a known human carcinogen [4, 5], most likely through its metabolites. It has been long recognized that chronic exposure to benzene at high levels in the workplace can lead to bone marrow depression and aplastic anemia [6]. Benzene is also an etiologic agent of acute myelogenous leukemia and some of its variants [7]. Humans appear to be more susceptible to the leukemogenic potential of benzene than most animal species studied. Yet, most animals exposed to benzene develop bone marrow hypoplasia and cancers at various sites. At very high concentrations, unhkely to be encountered nowadays except in case of accident, it induces acute toxicity, particularly to the central nervous system [7]. Understanding the toxicokinetics of benzene in humans is important to achieve better monitoring of workers (through the choice of sensitive measurement endpoints and sampling 207

208


times), and in risk assessment of low-level exposures (through a better characterization of effective exposure). ABSORPTION, DISTRIBUTION, ELIMINATION Data have been collected for the last hundred years on benzene toxicokinetics. Those experiments are usually summarized by a few characteristics (e.g., fraction absorbed, terminal half-life) whose interpretation requires some understanding of some basic concepts related to benzene absorption, distribution, and elimination. Absorption Benzene can enter the body by inhalation, ingestion, or dermal absorption. The respiratory route is believed to be the major source of human exposure to benzene from the workplace, gasoline vapors, automobile emissions, and tobacco smoke. Yet, the use of benzene for hand cleaning, by rotogravure workers for example, can lead to significant (but unquantified) exposures [1]. Skin absorption represents less than 5% of that absorbed through the lungs if exposure is only to the vapor phase [8]. There is apparently no human data on benzene absorption through ingestion, but it is likely that most if not all benzene ingested passes the intestinal wall because of its lipophilicity. The fraction absorbed by inhalation is difficult to measure and often to interpret because it is time and dose dependent, based on benzene blood concentration; therefore, penetration decreases when exposure length increases. At steady-state (achieved after about 60 hr of constant exposure, bur reasonably approximated after 10 hr), it can be measured by the difference between inhaled and exhaled air concentration and is the complement of the fraction metabolized. Yet, this fraction itself tends to decrease with the exposure concentration because benzene metabolism eventually saturates. Interindividual variability further complicate the picture. It should also be realized that in a closed-exposure setting, all benzene would eventually be absorbed and metabolized by the subjects. With all these caveats, at steady-state, for exposure levels of the order of 10 ppm or below, the fraction absorbed is about 50% [9, 10]. A potentially more useful quantity is the blood over air partition coefficient (i.e., the ratio of blood over air concentration at equilibrium). This partition coefficient, when measured in vitro, is close to 7 [8, 11, 12]. In vivo, values of about 14 (range 5-40) and 9.6 (SD 0.8) can be inferred from the data of Brugnone et al. [13], Pekari et al. [10], and Bois et al. [14] data, respectively. Lower in vitro values could be due to the effect of uninhibited metabolism, other losses, or inter-subject variability. Distribution Once in the body, benzene is dispersed mostly by blood through the various organs. It has a particular affinity, characteristic of apolar solvents, for fat tissues.

Benzene Toxicokinetics in Humans

209

where it accumulates. The affinity of benzene for the various tissues or organs can be measured by the organ-over-air-partition coefficients (Table 1). These coefficients can be combined to give tissue-to-tissue ratios: For example, the in vivo fatover-blood partition coefficient can be obtained as 183/9.6 = 19, indicating that at equilibrium the concentration of benzene in fat is about 20 times higher than its concentration in blood. Bone marrow, a target organ for toxicity, has a high partition coefficient and accumulates benzene more than most other tissues. It has been suggested that benzene could bind to blood proteins [15], but this claim is based on little evidence and has not been confirmed experimentally. Table 1 Benzene tissue-over-air-partition coefficients (mean ± SE)

in vitro [11] in vivo [10, 14]^^)

Liver

Bone marrow

Fat

Muscle

Viscera

425 ± 50 183 ±55

16 ±2.5 16 ±3.2

12±4 23 ± 4.5 — 9.7 ± 2.4 17 ±7.6 74 ±30

Blood 6.4 ± 0.6 9.6 ±0.8

('^^Population averages determined using a physiologically-based pharmacokinetic model.

It was noted experimentally that the contribution of smoking to blood and exhaled air benzene can be a confounder of occupational exposures, but this problem can be overcome if the subjects refrain from smoking on the day of sample collection, or if the collection takes place one hour after a high (>5 ppm) exposure [10]. Elimination Benzene is eliminated out of the body by exhalation and by metabolism. Very little benzene is found in the urine (less than 1%) [12]. Post exposure, benzene blood concentration-vs.-time curves are at least triphasic [16]. The terminal component of the concentration curve is controlled by the balance of exhalation, metabolism, and slow release from fat storage, processes which vary from individual to individual. Early studies give terminal half-Hves ranging from 16 hr to 75 hr [17-19]. It has been shown, however, that these studies lead to unreconcilable estimates of pharmacokinetic parameters [20]. The variability seen above could therefore be due to artifacts. Recently, the average terminal half-life was found to be 10 hr for 3 subjects in a controlled experiment [14]. Caution should be exercised in interpreting the results of some studies [16, 21, 22] with follow-up too short to observe the terminal phase. The population average of the fraction metabolized was estimated to be 57% ± 6% in a recent pharmacokinetic analysis of controlled exposure data [10, 14]. Benzene metabolism occurs in several organs, but mostly in the liver (at least for rodents [1, 7]), and is mediated by cytochrome P450 2E1 [7]. A single pathway.

210


from benzene to benzene oxide is involved. Benzene oxide is itself further transformed (see secondary metabolism below). The high metabolic clearance of benzene points to the possibility of a perfusion limitation of benzene metabolism (particularly at exposure levels below 10 ppm) [14]. This would imply that benzene rate of metabolism is essentially controlled by its rate of delivery to the liver, and that it is linear for constant exposure levels below 10 ppm. The high hepatic metabolism of benzene would also induce a potential difference between the inhalation and the ingestion route. Upon ingestion, benzene is transported through the portal vein to the liver where P450 oxidizes it and only the unmetabolized fraction escapes to the general circulation. After inhalation, benzene is directly available to the other tissues. The impact of this "first pass" effect is unknown because all experimental data on humans are from inhalation exposures. Simultaneous treatment of animals with both benzene and toluene increases the excretion of unchanged benzene [1]. Toluene appears to inhibit benzene metabolism, thereby increasing its respiratory elimination. A report that this was not observed in humans [22] is inconclusive because the kinetics were not followed enough in time to make meaningful inference about altered metabolism. While the majority of the experimental evidence suggests that the liver is the major organ involved in benzene metabolism, the bone marrow is the target site for benzene toxicity. Hepatic metabolites are carried via the bloodstream to the bone marrow, where they may accumulate and undergo further transformations [7], but this has not been shown in humans. In situ bone marrow metabolism can also contribute to benzene toxicity [23]. These two possible mechanisms of bone marrow exposure to metabolites would lead to different patterns of metabolites accumulation, but no human data is available on this question. This complicates tremendously the identification of the actual active species responsible for bone marrow toxicity, and hence benzene risk assessments. BENZENE SECONDARY METABOLISM Benzene is actually not likely to be directly carcinogenic; rather, its metabolites are involved [1]. Most of the evidence relating to the reactions involved comes from animal experiments, but the same metabolites are found in humans, and it is reasonable to assume that similar pathways are followed. A brief summary is given here (Figure 1). A first step involves P450 oxidation of benzene, leading to benzene oxide. Three pathways then lead to mercapturic acids, ring-opened metabolites, or ring-hydroxylated metabolites. Premercapturic acid is formed by glutathione conjugation of benzene oxide. It leads to S-phenylmercapturic acid, which has been found in the urine of exposed workers and proposed as a biomarker of benzene exposure [24]. This pathway gives nontoxic metabolites but is quantitatively minor (representing less than 1% of all metabolites).

Benzene Toxicokinetics in Humans

H HOOC

0 =

I

H

I

C

I

0 =

C

I

H

COOH

H

H2C— O H — COOH

trans,trans-mucon\c acid

H OHO—C= C

I

H

211

t

I 0 =

I

I

S

benzene H C

OHO

I

N H — COCH3

P450

H

phenylmercapturic acid

frans, frans-m ucona Ide hyde epoxide hydrolase^

GSH benzene oxide

premercapturic acid

benzene-/ra/is-dihydrodioi

P450 phenol

OH

hydroquinone

catechol OH

I

A o

OH

1,2,4-benzenetrlol

o-benzoquinone

T o

p-benzoquinone Figure 1. IVIajor pathways of benzene metabolism in humans. Unlabeled arrows can represent several reactions.

212


Ring opening of benzene oxide (which can be achieved by free radical reactions) leads to trans,trans-muconMthyde. Muconaldehyde is cytotoxic and could be involved in benzene toxicity. It is further oxidized in trans,trans-muconic acid, found in urine of benzene-exposed individuals. Muconic acid amounts to about 10% of all urinary benzene metabolites in humans. Several other minor muconic derivatives can also be formed, at least in vitro [7]. The major metabolites are ring-hydroxylated compounds. Phenol is formed by non-enzymatic rearrangement of benzene oxide. Direct oxidation of benzene by hydroxyl radicals could also be an important mechanism in micromolar concentrations (which is the range encountered in the body after occupational exposures) [7]. Phenol is detoxified by sulfo- and glucurono-conjugation. Phenol (free and conjugated) is the main metabolite found in urine (where it accounts for about 70% of total metabolites) [25]. Hydroquinone, catechol, and 1,2,4-benzenetriol are formed by further P450 mediated hydroxylation of phenol. Hydroquinone, catechol sulfate, and glucuronic acid conjugates can be detected in the urine of benzene-exposed individuals, where each represents about 10% of excreted metabolites. An alternative pathway for the formation of catechol involves metabolism of benzene oxide by epoxide hydrolase to benzene-rran^-dihydrodiol, converted to catechol by the action of dehydrogenase. Hydroquinone and catechol can be further oxidized into p- and o-benzoquinones. The hydroxylated metabolites are very likely to be involved in the toxicity of benzene [1, 7]. Little is known of the kinetics of these compounds in humans, and even less in the bone marrow. MODELING OF BENZENE TOXICOKINETICS Several models have been developed to describe the toxicokinetics of benzene in humans. Early work used classical pharmacokinetic models with three compartments to describe the various phases of benzene elimination from blood [16]. These models were only moderately helpful because they did not describe in any detail inhalation or metabolism and could not account for first-pass effects. They also did not provide insight on the fraction of benzene dose delivered to the bone marrow. For these reasons (and for the hypothetical possibility of extrapolating results from animals) physiologically based pharmacokinetic (PBPK) models have recently been developed [14, 20, 26-30]. A typical design for these models is given in Figure 2. The equations used to describe this type of models can be found in the literature [26, 30]. Problems remain with the calibration of those models. For example, Travis et al. [27], used visual fitting to model several sets of data [12, 17-19, 21, 31] and give no measures of uncertainty for the model parameters or predictions. Yet, many of these parameters, typically those controlling metabolism, are not known with precision. Proper statistical inference about their value is therefore necessary, while conserving the strong prior information conferred by their physiological definition. Bayesian statistics provide a natural way to merge a priori knowledge, gained by implementing a physiological model, with evidence from experimental data [32]. In

Benzene Toxicokinetics in H u m a n s

ALV ^ ^

213

Exhaled Air

r y^ VPR^'^Pba

ô 11 CD 1 o 1

WELL PERFUSED

>a>

Vwp, Pwp

c 1 1

^Fwp

1^

FAT

4:"

^

Vf,Pf

POORLY PERFUSED

^Fpp

Vpp, Ppp

^

BONE MARROW

^Fbm

\^

Vbm, Pbm

LIVER VI, PI VmaxI, Kmi

Vmaxbm, Kmbm

\ fKf -^

1

Fl

L^-J ^

9

1 METABOLITES

^

Ke, Fu

URINARY PHENOL OTHERS

*L_. 1-fp

Figure 2 . Schematic representation of a 5 - c o m p a r t m e n t physiological model used to simulate t h e distribution a n d m e t a b o l i s m of b e n z e n e in h u m a n s [14]. S y m b o l s are given in T a b l e 2.

addition, scientific interest generally resides in inference about benzene metabolism in humans—i.e., in a diverse population—rather than in any one individual studied in published experiments. It is therefore preferable to design a statistical model describing the relationships between individual and population physiological parameters to estimate population variability [33-35]. Once properly parameterized, PBPK models can be powerful simulation tools. Figures 3 and 4 show the fit obtained to data from one subject exposed for 4 hr to 1.7 ppm and 10 ppm benzene by inhalation, using the model depicted in Figure 2. Distributions for the parameter values of this model are given in Table 2. On the basis of this model, it was established that no significant dose-rate effect occur for benzene [20], that benzene primary metabolism is essentially linear below 10 ppm exposure levels, and that the fraction metabolized at those levels is close to 55% (Figure 5) [14, 27]. Many other endpoints of interest for benzene toxicology or risk

214


H 1 1 1 1 1 1 1 1—j 1 1 1 1 1 I I I I I I I I 1 I I I I I I I I I I I I I I I I t I I I

500

1000

500

1500

Time (min)

2500

Time (min)

Figure 3. Simulated and observed time course of tlie venous blood and exhaled air concentrations of benzene for subject 1 of Pekari et al. [10]. Exposures were to 1.7 ppm (A and C) and 10 ppm (B and D) for 4 hours. The data points are bracketed by ±2 estimated SDs. Results are similar for the other two subjects studied.

30H 25H

c o c o

o

"o c .

v'^'"^ B

1 9"

A

/

>y^

y'''''!^--''''***'*^^^.

\

o

i

60

l-i

o 30 -jirvv ^"V'Â u •

390

^^^

440

490

540

590

Wavelength (nm)

Figure 9. Emission spectra of BaP measured at 30 s after mixing with increasing amounts of cells, (a) BaP in buffer with 0 |jL (A), 10 |jL (B), 20 pL (C), and 30 |JL (D) of mouse liver cells; (b) BaP in buffer with 0 |JL (A), 10 |JL (B), 20 |JL (C), and 30 ML (D) of pig kidney cells; (c) BaP in buffer with 0 ML (A), 10 |JL (B), 20 |JL (C), and 30 ML (D) of monkey kidney cells; (d) BaP in buffer with 0 pL (A), 10 |JL (B), 20 |JL (C), and 30 ML (D) of human cancer cells. Cell counts are approximately 100/|JL. The rate equations can be written as: drnj dt dni2

"dT dt

= -kimi

(2)

= kiiTii - (k2 + k4) m2 + k3m3

(3)

= k2m2 - k3m3

(4)

Solution of these equations yield: i^2 A -kt T^ bt f /C ,\sinat — ^ = Ae ^^+Be^^Jcosat+ —+ b kimo [ \B / a

(5)

s-b (s - b)^ + a^ -k(t-u)U^-ku^g^bu

•+ •

C/B + b

(s - b)^ + a '

/ C , \ sin au cos au + — + b IB

) a

.du

(6)

120

a

390

440

490

S90

540

Waveloopb (om)

Wavsloapb bn)

n

b

d

80 x

'z

60

H 9

40

8 20

0'

J

390

uo

490 Wwclcngtb (om)

540

590

I 390

uo

490

540

S90

Wavelength (nm)

Figure 10. Emission spectra of BaP measured at different incubation times in the presence of (a) mouse liver cells, (b) pig kidney cells, (c) monkey kidney cells, and (d) human liver cells. Incubation times: (A) 0 hr, (B) 1 hr, (C) 2 hr, and (D) 3 hr.

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons C^^FQI^ J]l'

239

heme group of P-450

Iic4

membrane

BaP

microcrystals BaP(m) m3 membrane

Figure 11. Simplified sketch of the theoretical model.

where:

hz^^_

A

(7)

kik2 + kik3 + kik4 - kj - k3k4

^'-^'

B

,

(8)

kik2 + kik3 + kik4 - kj - k3k4

Q^

^3 (^2 + k3 - ki) kik2 + kik3 + kik4 - kj^ ~ k3k4

^^^mn^

(10)

and: a2 = k3k4-b2

(11)

The rate constant k^ can be obtained from the rate of dissolution of BaP microcrystals [90] or that of BaP uptake by the membrane [86]. Rate constants k2 and k3 can be obtained, at least in principle, from kinetic measurements of BaP distribution in phospholipid bilayers, but have not been reported. Because of this, the rate constant of metabolism (or more accurately, the rate constant of association with P450) k4 can only be estimated indirectly.

240


From the structure of the phospholipid bilayer, it is conceivable that k2 should have a larger value than k3 and both of them should be greater than, or at least comparable to, k4. This is because the inward diffusion of BaP is a transport process from an orderly environment, the glycerol backbone, to a much more random domain, the membrane midplane. It is also because the membrane midplane is more hydrophobic than the membrane surface, making the inward diffusion of BaP energetically more favorable than the reversed transport process. Based on the assumption that k4 is rate determining, one would expect that the midplane BaP concentration m3 would rise, pass through a maximum, and eventually die away as BaP microcrystals completely disappear. The incubation time needed for m3 to approach zero level, of course, depends on the value of k4. The larger k4 is, the shorter the incubation will be. A similation of the k4 effect on m3 is given in Figure 12 and temporal emission profiles of BaP in different cell cultures is given in Figure 13. Comparison of these profiles with the similated ones reveals that the mouse liver cells have the largest and pig kidney the smallest k4.

0.25

2: 0.15

0.05

Time (hour)

Figure 12. Effect of k4 on m^ of BaP is similated using Equation 5. The value of k4 used for similation is (A) 0.04 hr^; (B) 0.4 hr^; (C) 1.0 hr^ and (D) 5.0 hr\ respectively.

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons

0.6

-

/

0.5

A

0.4

/

0.3

/

-

^ ^

^-^

•

1

B

/ A

c

"/

oJ

-

-0.1 -0.2

^

/

0.2 0.1

-

/

241

1

^^ „.

-v"^^^ ^ s ; ; ; ,

1 ,

J

^^

1 24

TIME (hour) Figure 13. Corrected temporal profiles of BaP monomer emission. BaP emission at zero incubation time was used as background for correction. BaP in (A) human liver cancer cells, (B) monkey kidney cells, (C) mouse liver cells, and (D) pig kidney cells.

CONCLUSION PAH carcinogenesis is a long, multiple-stage process. The crucial step leading to the ultimate carcinogen is conceived to be the metabolic activation of the hydrocarbon by the enzyme cytochrome P450 to form an arene oxide at a critical position, e.g., in the bay region. Oxygenation at other positions most likely gives rise to nontoxic metabolites.The substrate binding site of the enzyme is capacious enough to accommodate some molecular reorientations but restrictive enough to permit stereoselectivity. In other words, the heme-0-BaP complex could have only a few configurations. If these configurations were stable enough to permit characterization and differentiation, biochemical modifications to inhibit activation or to enhance detoxication could be designed. Many P450 isozymes have been expressed and cloned. Production of large quantities of isoforms with specific structures for commercial or industrial uses in environmental control is not out of reach. Analytical methods capable of monitoring and differentiating the configurations of the heme-substrate complexes can be very helpful in the construction and production of these specific isoforms. Among these methods, fluorescence, particularly the time-resolved technique, is probably the most appropriate one and should be further explored.

242


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246


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66. Kulisch, G. P. and Vilker, V. L. "Application of Pseudomanas putida PpG 786 Containing P450 Cytochrome Monooxygenase for Removal of Trace Naphthalene Concentrations," Biotechnol Prog 1991, 7, pp. 93-98. 67. Selkirk, J. K., Yang, S. K., and Gelboin, H. V. "Analysis of Benzo[a]pyrene Metabolism in Human Liver and Lymphocytes and Kinetic Analysis of Benzo[a]pyrene in Rat Liver Microsomes," in Carcinogenesis—A Comprehensive Survey, Vol. 1, R. Fredenthal and P.W. Jones (Eds.), New York, Raven Press, 1976, pp. 153-169. 68. Selkirk, J. K. "Comparison of epoxide and free-radical Mechanisms for Activation of BaP by Sprague-Dawley Rat Liver Microsomes," J Natl Cancer Inst. 1980,64,771-774. 69. Capdevila, J., Estabrook, R. W., and Prough, R. A. "The Microsomal Metabolism of BaP Phenols," Biochem Biophys Res Commun, 1978, 82, pp. 518-525. 70. Jernstrom, B., Orrenius, S., Undeman, O., Graslund, A., and Ehrenberg, A. "Fluorescence Study of DNA-binding Metabolites of BaP Formed in Hepatocytes Isolated from 3-Methylcholanthrene Treated Rats," Cancer Res, 1978, 38, pp. 2,600-2,607 71. Wiebel, F.J. "Metabolism of Monohydroxy-BaPs by Rat Liver Microsomes and Mammalian Cells in Culture," Arch Biochem Biophys, 1975, 168, pp. 609-621. 72. Yang, S. K., Selkirk, J. K., Plotkin, E. V., and Gelboin, H. V. "Kinetic Analysis of the Metabolism of BaP to Phenols, Dihydrodiols and Quinones by HPLC Compared to Analysis by Aryl Hydrocarbon Hydroxylase Assay, and the Effect of Enzyme Induction," Cancer Res, 1975, 35, pp. 3,642-3,650. 73. Ribeiro, O., Kirkby, C. A., Hirom, P. C , and Millburn, P. "Secondary Metabolites of BaP: 3-Hydroxy-rraw5'-7,8-dihydro-7,8-dihydroxy-BaP, a Biliary Metabolite of 3-Hydroxy-BaP in Rat," Carcinogenesis, 1985, 6. pp. 1,507-1,511. 74. Capdevila, J. Estabrook, R. W., and Prough, R. A. "The existence of BaP3,6-Quinone Reductase in Rat Liver Microsomal Fractions," Biochem Biophys Res Commun, 1978, 83, pp. 1,291-1,298. 75. Prough, R. A., Saeki, Y., and Capdevila, J. "The Metabolism of BaP Phenols by Rat Liver Microsomal Fractions," Arch Biochem Biophys, 1981, 212, pp. 136-146. 76. Keller, G. M., Turner, C. R., and Jefcoate, C. R. "Kinetic Determinants of BaP Metabolism to Dihydrodiol Epoxides by 3-methylcholanthrene Induced Rat Liver Microsomes," Mol Pharmacol 1982, 22, pp. 451^58. 77. Amir, D. and Haas, E. "Estimation of Intramolecular Distance Distributions in Bovine Pancreatic Trypsin Inhibitor by Site-Specific Labeling and Nonradiative Excitation Energy-Transfer Measurements," Biochemistry, 1987, 26, pp. 2,162-2,175.

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CHAPTER 12 OCCUPATIONAL EXPOSURE TO ORGANIC SOLVENTS DURING PAINT STRIPPING AND PAINTING Marc Charretton Caisse Regionale d'Assurance Maladie Rhone-Alpes Laboratoire de Chimie du Val Rosay F-69370 St Didier au Mont d'Or, France Raymond Vincent Institut National de Recherche et de Securite Service Evaluation et Prevention du Risque Chimique BP 27, F-54501 Vandoeuvre Cedex, France CONTENTS INTRODUCTION, 252 ASSESSMENT OF OCCUPATIONAL EXPOSURE TO ORGANIC SOLVENTS, 252 Evaluation of Exposure by Atmospheric Sampling, 253 Exposure Evaluation by Biological Sampling, 255 PAINT STRIPPING, 256 COMPOSITION OF CHEMICAL STRIPPING AGENTS, 258 TOXICITY OF DICHLOROMETHANE, 259 Metabolism, 259 Acute Toxicity, 260 Chronic Toxicity, 261 LIMIT VALUES FOR OCCUPATIONAL EXPOSURE TO DICHLOROMETHANE, 262 LEVEL OF EXPOSURE TO DICHLOROMETHANE DURING STRIPPING OPERATIONS, 262 CURRENT TRENDS IN PAINT-STRIPPING TECHNIQUES, 264 GENERAL OVERVIEW OF PAINTS, 265 THE CONSTITUENTS OF PAINTS, 269 Binders, 269 Pigments, 271 Extenders, 271 Additives, 271 Solvents, 272 TOXICITY OF SOLVENTS FOUND IN PAINT, 272 Pathways by w^hich Solvents can Enter into the Organism, 272 Effects of Solvents on the Organism, 273

251

252


DIFFERENT AREAS IN WHICH PAINTS ARE USED, 275 METHODS OF APPLICATION OF LIQUID PAINTS, 275 Application by Pressurized Spraying of Paint, 276 Application by Electrostatic Pulverization, 279 Application by Dipping in Paint Baths, 280 Other Processes for Application of Liquid Paints, 281 SECTORS OF USE AND APPLICATIONS OF PAINT, 283 Industry, 283 OCCUPATIONAL EXPOSURE TO PAINT SOLVENTS, 283 Epidemiological Studies, 283 EVOLUTION OF PAINT FORMULATIONS, 285 REFERENCES, 300 INTRODUCTION The use of paint dates back to the beginning of civilization. Employed throughout prehistory, paints were used to create ritual decorations, and to embellish monuments and dwellings. Since then, their use has expanded to include a protective role: to prevent damage to wood and construction materials, and to stop the corrosion of metals. Due to relatively recent chemical developments and the appearance of polymers, the manufacturing of paint has lost the empirical character that dominated it for millennia. In our industrial society, the production of objects made from different materials (wood, plastics, metals, concrete) is closely associated with the application of paint—an application that both protects and beautifies the object, be it an automobile, an airplane, furniture, a boat, or a building, etc. The majority of paints, varnishes and paint strippers (with the exception of coating powders) contains solvents. These volatile components allow the paint to be easily spread, or in the case of stripping agents, to participate in the destruction of the paint film. The use of paints and stripping agents thus leads to the emission of organic solvent vapors, to which different categories of people are exposed during the course of their occupational, or domestic, activities. ASSESSMENT OF OCCUPATIONAL EXPOSURE TO ORGANIC SOLVENTS Two methods are generally used to evaluate the degree of exposure of workers to organic solvents:

Occupational Exposure to Organic Solvents

253

1. the measurement of the concentration of pollutants in the atmosphere at the workplace; and 2. the measurement of the quantity of a toxic substance absorbed by the worker, accomplished by evaluating the levels of pollutant or metabolites in the biological fluids of the individual: urine, blood, exhaled air. Evaluation of Exposure by Atmospheric Sampling [1,2,3] This approach usually consists of two steps in industrial hygiene: 1. sampling of air polluted by organic solvents; and 2. determination of concentration of pollutants in the air. This type of approach can only be used to evaluate exposure caused by the inhalation of polluted air. Sampling In the classical approach, sampling is usually carried out with a sampling tube that contains some type of absorbent material that traps the solvent vapors. This tube, placed in a support, is attached to a battery-powered individual sampling pump (Figure 1) that assures a regular and constant air flow rate. The support is placed near the respiratory tract of the individual through the sampling period (Figure 2). The most commonly used sampling tubes contain activated charcoal for

Figure 1. Sampling pump and tubes.

254


Figure 2. Worker equipped with a sampling device.

non-polar solvents (e.g., hydrocarbons), and silica gel for polar components (e.g., alcohols). Porous polymers (e.g., TENAX, CHROMOSORB, etc.) containing variable quantities of absorbent material (on the order of tens to hundreds of mg) can also be used. The choice of which tube to use thus depends on the polarity of the solvent in question, its concentration (assumed to be low, high), sampling flow rate (on order of tens to hundreds of ml/min), and the sensitivity of the method of analysis for the pollutant(s) being sought. Sampling badges, or passive samplers that do not need a pump, can also be used. In this case, the absorbent material is contained in a pouch, covered by a grid that regulates the rate at which air diffuses across the absorbent material. The solvent vapors are trapped on the absorbent material in the same manner as for the sampling tubes (Figure 3) [4]. The workers wear the sampling device in question for periods representative of the exposure related to their activity (on the order of a few minutes to several hours). At the end of the sampling period, using a known flow rate (usually measured at the


255

Figure 3. Different sampling badges.

beginning and end of the sampling period), the volume of air sampled can also be calculated. The solvent vapors present in the sampled air are trapped in the tube and their respective concentrations quantified using different analytical methods. Analysis The first step of the analysis consists of desorbing the vapors trapped in the sampling tube or badge. The absorbent material is transferred to a glass flask to which 1 or 2 ml of solvent (carbon disulfide normally) are added. The vapors are desorbed by being solubihzed in the added solvent. It is the analysis of this desorption solution that will yield the amount of pollutants trapped in the sampler. This can be used in turn to estimate the atmospheric concentration using the estimate of the volume of air that was sampled. The analysis of the desorbed solution constitutes the second step of the analysis. The most widely used analytical technique is gas chromatography, often coupled with mass spectrometry to identify and quantify the pollutants. The results are generally expressed in terms of mg of pollutant per cubic metre (mg/m^), or in parts per million (ppm). The industrial hygienist then compares the results to the current occupational limit values (TLV-TWA, TLV-STEL, PEL, etc.) to evaluate the exposure to which the worker(s) was (were) exposed. Exposure Evaluation by Biological Sampling [5] The second method is based on the measurement of the concentration of the pollutant itself, or of its metabolites in a blood or urine sample taken at the beginning or end of the exposure period. It is also possible to measure the concentration of pollutant in the air exhaled by the individual at the end of the work shift. This method allows one to evaluate respiratory and cutaneous exposures, and, in certain cases, the amount of pollutant ingested by the subject. Additionally, this method

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also accounts for the level of physical activity since, in the case of heavy physical effort, respiration is accelerated and the quantity of polluted air inhaled increases. Given the kinetics of the elimination of toxins by the organism, the moment at which the biological samples are taken is very important in order to be able to evaluate the exposure levels of the individual with any precision. A wide range of analytical techniques are available to measure pollutant concentrations, and the one chosen will depend on the nature of pollutant or metabolite to be measured. Once the concentration of pollutant has been evaluated, it is compared to the value of the Biological Exposure Indices (BEI) proposed by different organizations such as the ACGIH. The ACGIH currently proposes BEI values for approximately sixty substances [6]. The BEI values for conmion substances used in paints and in stripping agents are listed in Table 1. PAINT STRIPPING It is necessary to periodically apply a new coat of paint or varnish to trains, airplanes, boats, outdoor furniture, and parts of buildings (e.g., doors, wooden or metal shutters and blinds) among other objects. In order to guarantee a maximum level of protection and aesthetic quality, it is necessary to remove the old layers of paint from the surfaces to be treated. The elimination of old paint can be accomplished using different techniques: • abrasion of paint film by sanding, milling, or projection of particles (sandblasting); • burning and abrasion; • stripping with laser light (used in restoring art treasures); or • chemical strippers. Table 1 BEI Values (ACGIH) recommended for certain organic solvents

Substance Acetone Methyl alcohol (Methanol) Ethylbenzene Ethylglycol Ethylglycol acetate Methylethylketone Methylisobutylketone Xylenes

Parameter

Fluid

BEI

Sampling time*

Acetone Methanol Formic acid Mondelic acid 2-ethoxyacetic acid

Urine Urine Urine Urine Urine

100 mg/1 15mg/l 80 mg/g creatinine 1,500 mg/g creatinine 100 mg/g creatinine

A A I D D

Methylethylketone Methylisobutylketone Methylhippuric acids

Urine Urine Urine

2 mg/1 2 mg/1 1,500 mg/g creatinine

A A A

Sampling time: A = end of shift; D = end of shift at week's end; I = before last shift of work week.


257

Chemical stripping agents are often used when it is necessary to protect the mechanical properties of the materials in question, for example in the case of airplanes, or in instances where mechanical stripping would aesthetically damage the surface of the object, e.g., furniture. Additionally, chemical stripping, when performed with a liquid product, can be applied to all parts of the object, even those where it would be difficult to strip the paint by abrasion or scraping. Two principal techniques are typically used for the stripping of painted surfaces. The technique to be used is generally chosen as a function of the size of the object to be stripped:

Figure 4. Hand stripping of furniture dipped in a stripping agent (Courtesy of Ets COMONT).

258


Figure 5. Industrial stripping of an airplane by spraying (Courtesy of Didier TOULORGE).

• soaking, widely used for the stripping of furniture, shutters, doors, etc. (Figure 4). • direct application of stripping agent with a brush or spray gun, followed by elimination of the paint by sanding or scraping. This type of procedure is generally used for large objects such as airplanes (Figure 5). Because chemical stripping agents always contain organic solvents, these types of operations lead to exposure to solvent vapors of the individuals performing the work. The intensity of the exposure will depend on the size of the surfaces to be treated, the premises, the procedures used, etc. COMPOSITION OF CHEMICAL STRIPPING AGENTS A chemical stripping agent is generally composed of: • a caustic product, the primary function of which is to soften the paint film. Potassium and sodium hydroxides are the most widely used products for the stripping of paint applied to wood and ferrous metals. For other metals, such as aluminum, copper, and tin and their alloys, phenol or organic acids are generally used to strip paint in order to avoid corrosion associated with the formation of metal hydroxides in the presence of sodium or potassium hydroxide.


259

• organic solvents to permeate the paint film. The action of these solvents, sometimes coupled with that of a caustic agent, also softens the paint film and causes it to become detached from the surface. Methylene chloride, or dichloromethane (CH2CI2), is by far the solvent most widely used in paint strippers—in fact this is the principal application of dichloromethane [7]. Methanol, or methyl alcohol, is also often present in these formulations. A study on the composition of paint strippers [8] showed that 67.9% of the 215 formulations studied contained dichloromethane at an average concentration of 73% (standard deviation ±12%), 45.6% contained methanol (average concentration 11%, standard deviation ±4%), and 19.5% contained phenol (average concentration 13%, standard deviation ±7%). Methylene chloride is widely used in this type of formulation because it is such a good solvent, but also because it is not flammable [7]. • thickeners such as waxes or methyl cellulose. (The methanol present in paint strippers swells the methyl cellulose.) The thickeners form a barrier at the surface of the stripper that limits the evaporation of the solvents and guarantees that they produce their maximum effect on the paint surface. • surfactants that improve the wetting capacity of the solution and increase the speed at which the stripper works at ambient temperature. Paint strippers are sold in different containers depending on their end use: in small bottles for domestic use, or in barrels or tanks for industrial applications. Given the composition of paint strippers, occupational exposures to organic solvents resulting from these products is generally related to exposure to dichloromethane. TOXICITY OF DICHLOROMETHANE Metabolism The biotransformation of dichloromethane can follow one of two paths, both of which lead to the production of electrophile reactants [9, 10, 11]. The first pathway, dependent on cytochrome P450, leads to the production of a carbon monoxide (CO) endogen that causes an increase in the levels of carboxyhemoglobin. The second path follows the conjugation of dichloromethane with glutathion (GSH), and leads to the formation of formaldehyde. The two different pathways are shown in Figure 6. In subjects that have been intensively exposed to dichloromethane vapors at levels high enough to make them lose consciousness, carboxyhaemoglobin levels can be as high as 50% [12]. Given this phenomenon of hypoxia, those suffering from cardiac problems can be considered to be particularly at risk if they inhale strong doses of dichloromethane vapors [13]. The second pathway of biotransformation that leads to the formation of formaldehyde could explain the mutagenic character of dichloromethane that has been

260


I CYTP-450'~|

H

o

O

II

C

H - O - C - CL

C^rLj v^Lo

NADPH I CYTOSOL + GSH

I

CL

|

\

:C = 0

H CL

\ HCL

HCL + GSH

h^HCL GS - CH2 - CL

D

^

^HCL GS -'CH2 - OH

//

G-S-C I "^H ^

CH2O + GSH

H

NAD+

>.^°

o

v&

CO9

GS-C

\ i

O

// GS-C \

o H

HCOOH + GSH GSH

CO,

CO,

Figure 6. Metabolism of dichloromethane as described by Andersen et al. [11].

observed by several authors [14-16]. Given the polymorphism of the enzyme involved in the formation of the formaldehyde (glutathion-S-transferase Theta; GSH), the actual quantity of formaldehyde produced is highly dependent on the level of GSH activity—an activity that can vary greatiy from one individual to the next. For this reason, certain subjects have a higher risk than others of genetic damage. Acute Toxicity Animal Studies The LD50 for oral administration in rats is approximately 3,000 mg/kg [17], and for inhalation, the LC50 is 52,000 mg/mV6h [18]. For mice, the LC50 is 49,100 mg/m3/6h [19]. Numerous studies carried out on different species of animals (rats, mice, guinea pigs, rabbits, dogs, and monkeys) have demonstrated that dichloromethane acts on the liver, kidneys, heart, and central nervous system after only short exposures (on the order of a few minutes to several hours) at atmospheric concentrations varying


261

from 3,600 mg/m^ (1,000 ppm) to 60,000 mg/m^ (approximately 17,000 ppm). Damage to the central nervous system is reflected by a decrease in overall activity levels, narcosis, and perturbation of the sleeping patterns [13]. Human Studies The exposure of humans to atmospheric concentrations of 3,600 mg/m^ (1,000 ppm) leads to the appearance of anesthetic effects. After 5 minutes of exposure to concentrations of up to 8,000 mg/m^ (2,300 ppm), symptoms including dizziness, and irritation of the eyes and of the respiratory tract can appear [20]. After two hours of exposure to atmospheric concentrations of 700 mg/m^ (200 ppm) neurobehavioral changes were observed, including problems of alertness and the alteration of overall performance levels in surveillance/pursuit tests [21]. The narcotic effect of its vapors can lead to a deep coma [12], or even death [22-25], whereas cutaneous contact with dichloromethane lasting on the order of a few minutes can cause severe bums [26, 27]. Chronic Toxicity Animal Studies Rats and mice are the studied animals that are the most sensitive to this substance. An increase in dose-dependent levels of mammary benign tumors (in both males and females) was noted in groups of rats exposed to 0, 500, and 3,500 ppm for 5 hours a day, 6 days a week for 2 years. Sarcomas in the region of the salivary glands were also observed, but only in males—this effect was statistically insignificant in females [28]. These results were confirmed by another study where groups of rats and mice were exposed in much the same conditions of frequency and duration, but to atmospheric concentrations of 1,000, 2,000, and 4,000 ppm. Other than the appearance of tumors in the mammary and salivary glands, the authors also demonstrated a dosedependent increase in the frequency of pulmonary (males and females) and hepatic (in males only, statistically insignificant in females) cancers [29]. Human Studies There are relatively few epidemiological studies on the risks associated with prolonged exposure to dichloromethane [30-32]. None of those studies available was able to demonstrate a particular cause (pulmonary and liver cancer, cardiac problems) of an increase in mortality rates associated with a occupational exposure to dichloromethane. A long-term study from 1964 to 1984 of a cohort of 1,013 workers exposed to an average atmospheric concentration of 26 ppm of dichloromethane per day showed

262


that there was an insignificant increase in the number of deaths due to pancreatic cancer—8 observed cases against 4.2 expected cases [32]. The prolongation of this cohort study until 1988 did not lead to the confirmation of this observation [33]. More recently, an examination of a group of workers exposed to average atmospheric concentrations on the order of 475 ppm for at least ten years did not reveal any significant cardiac or hepatic damage, nor any damage to the central nervous system [34]. The International Centre for Cancer Research (CIRC) [35] has classified dichloromethane in the category 2B: a substance suspected to have a possible carcinogenic effect on humans. Additionally, when discussing the toxicity of dichloromethane, it should be pointed out that the thermal degradation of this substance leads to the production of phosgene gas, a highly toxic compound that causes severe lesions to the pulmonary system. This phenomenon can arise when paint strippers are used near a source of heat such as a gas-burning radiator [36]. LIMIT VALUES FOR OCCUPATIONAL EXPOSURE TO DICHLOROMETHANE The 8-hour exposure value in the United States (TLV-TWA: Threshold limit value, time-weighted average) reconmiended by the American Conference of Governmental Industrial Hygienists (ACGIH) is currently 50 ppm (174 mg/m^). The ACGIH has also classified this substance in the category A2: substance suspected of having a carcinogenic effect in humans [6]. In 1993, the Occupational Safety and Health Administration (OSHA) reduced the Permissible Exposure Limit (PEL) from 500 ppm to 25 ppm [37]. In France, the Ministry of Labour recommends an average limiting exposure value (AEV) over eight hours of 50 ppm. The short-term limiting exposure value (LEV) for periods no longer than 15 minutes has been fixed at 100 ppm. Dichloromethane is classified in category C3: carcinogenic effects observed on animals [38]. In Germany, the 8-hour exposure value (the MAK value) has been fixed at 100 ppm, and dichloromethane has been classified in category IIIB: substance suspected of having a carcinogenic potential. The values of biological indicators of exposure to dichloromethane in Germany are a level of carboxyhaemoglobin of 5%, or 1 mg dichloromethane/1 in a blood sample taken at the end of the working day [39]. LEVEL OF EXPOSURE TO DICHLOROMETHANE DURING STRIPPING OPERATIONS Anundi et al. [40] studied the exposure of workers to organic solvents during the elimination of graffiti from subway stations. The exposure levels of dichloromethane varied from 18 to 1,188 mg/m^ (5-330 ppm) over the course of the workday, but varied from 6 to 5,315 mg/m^ (1.7 to 1,476 ppm) for short periods. The same authors also measured the atmospheric concentration of other sol-


263

vents contained in the stripping agents, but found that exposure to these substances, as well as their atmospheric concentration levels, were relatively low. They detected the presence of some glycol ethers, including dipropyleneglycolmonomethylether (DPGME), propyleneglycolmonobutylether (PGBE), and nmethyl-2-pyrolidone (nMP). The trimethylbenzenes present in the stripping agents were not detected in the air of the workplace. The concentrations of the different glycol ethers ranged from 3.4 to 11.8 mg/m^, and exposure to nMP was detected at 9.9 mg/m^, but only in one single sample. McCammon et al. [41] evaluated exposure of 12 workers from 5 different workshops involved in furniture stripping to dichloromethane with both atmospheric and biological samples. The atmospheric concentrations measured over the course of the workday varied from 54 to 1,317 mg/m^ (15-366 ppm). At the end of the workday, the concentration of dichloromethane in the air exhaled by the workers varied from 2.3 to 167 ppm, and the concentrations of carboxyhemoglobin varied from 2.1 to 13.5%. The concentration of dichloromethane in these same blood samples varied from 0.1 to 8.8 ppm (approximately 0.1 to 8.8 mg/1). The exposure values varied from one workshop to the next, and depended to a great extent on the procedures employed by the workers and on the means of protection provided (i.e., ventilation). The authors also point out that the concentrations of dichloromethane measured during the course of the study are probably the lowest observed during the year because their study was carried out during the summertime when all the doors of the different workshops were wide open, thereby offering the maximum level of ventilation possible. Hall et al. [42] also studied exposure to dichloromethane in a furniture stripping workshop to aid in the conception of a ventilation system designed to reduce exposure levels. Before modification, exposure levels varied from 230 to 2,200 ppm (828-7,920 mg/m^). After the ventilation system was installed, the exposure varied from 6 to 93 ppm (21.6-334.8 mg/m^). Hall and Rumach [43] gave questionnaires to workers from 21 furniture stripping workshops to determine how dichloromethane was used, the ventilation systems employed, methods of individual protection, and the physiological effects noticed by the individuals in question during the stripping operations. Half of the workers questioned stated that they suffered nausea, headaches, and dizziness during this type of work. Given the descriptions of symptoms provided by the workers, the authors deduced that the exposure level in these workshops was between 500 and 5,000 ppm for periods varying from a few minutes to several hours. Shusterman et al. [44] presented a case study of a worker in a furniture stripping shop exposed to between 100 and 200 ppm of dichloromethane, and whose carboxyhemoglobin level attained 10.4%. The authors also point out the lack of efficiency of the protective respiratory equipment using activated cartridges; the authors state that the worker reported being able to detect solvent odors while wearing the mask.

264


This phenomenon was studied experimentally by Moyer and Peterson [45]. Their results demonstrate that the duration of the trapping efficiency of an activated carbon cartridge varied as a function of the relative humidity and dichloromethane concentration, and was relatively short—approximately 30 minutes in the case of a dichloromethane concentration of 50 ppm at a relative humidity of 80%. This lack of dichloromethane capture efficiency in the cartridges of respiratory masks is linked to the physical characteristics of this solvent, especially the low molecular weight and low boiling point (high vapor pressure). Vincent et al. [8] measured individual exposure levels to dichloromethane in a door, shutter, and furniture stripping workshop, and found that they varied from 258 to 659 ppm (929-2,373 mg/m^). Concentration levels in the same workshop were measured by ambient sampling, and were between 69 and 683 ppm (250-2,460 mg/m^). In order to reduce exposure to dichloromethane in furniture stripping workshops, NIOSH published a good practices guide that suggests the use of appropriate and efficient ventilation systems [46]. The most crucial problem associated with such stripping operations concerns the treatment of large objects, for which it is difficult to install a ventilation system. This is especially true in the case of airplanes. When Vincent et al. [47] measured the exposure to dichloromethane and to phenol during the stripping of a Boeing B747 aircraft, they found that the total surface to be treated was on the order of 2,500 m^, and required the participation of a dozen workers for approximately 72 hours. The exposure of workers performing the actual paint stripping was on the order of 83 to 525 ppm (299-1,889 mg/m^), an exposure that corresponds to a daily level of between 24 to 344 ppm (86-1,240 mg/m^). Those workers who did not directly handle the paint stripper, but performed other tasks in the workshop, were exposed to concentrations between 88 and 212 ppm (317-762 mg/m^) during the stripping work carried out by the other workers. This corresponds to an 8-hour exposure of 27-48 ppm (97.2-174.6 mg/m^). Atmospheric phenol concentrations—phenol being present in the stripper—varied from 1.1 to 5.3 mg/m^ (0.3-1.4 ppm), which represents levels below the exposure levels generally recommended for this product (TLV-TWA, AGCIH = 5 ppm or 19 mg/m^). CURRENT TRENDS IN PAINT-STRIPPING TECHNIQUES Given the supposedly carcinogenic nature of dichloromethane, the decreasing limiting occupational exposure values, new laws on the emissions of VOCs in the atmosphere, the difficulty of installing efficient ventilation systems while at the same time respecting the environment, and the lack of efficient protective respiratory devices, industrial groups are looking for substitute chemical stripping processes that do not rely on the use of dichloromethane. Two routes are currently being


265

explored: Replace the dichloromethane by other solvents thought to be less toxic, or use mechanical stripping procedures based on the projection of different materials. In strippers for domestic use, dichloromethane has been replaced by different mixtures of ketones, alcohols, esters, etc. The principal problem associated with the implementation of these new stripping agents is higher cost, lower efficiency, and especially problems of flammability when used in confined spaces. Furthermore, the use of hot caustic sodium hydroxide solutions for the stripping of furniture is not possible because this product attacks wood. In the area of aeronautics, the International Air Transport Association (lATA) has created a working group to study different alternatives to paint strippers based on dichloromethane and/or phenol [48]. Several different processes are being considered. Also being considered are acidic or basic strippers containing benzylic alcohol. This product has a low toxicity, a relatively high boiling point (200 °C), and it acts 10 to 20 times more rapidly than dichloromethane-based stripping agents. Paint stripping through the projection of particles of ice, carbon ice, starch, and water under high pressure can also be envisaged. The use of a pulsed laser beam also seems to be a possible means for stripping airplanes, railway cars, etc. The principal obstacles to the use of such new processes is also their higher cost as compared to that of chemical processes using dichloromethane. The operating principal of a process using the projection of ice particles that can be used to eliminate exposure to toxic substances is illustrated in Figures 7 and 8. The efforts undertaken by different companies to commercialize new stripping processes should lead to a considerable reduction in the use of chemical stripping agents over the course of the next few years, and at the same time reduce occupational exposure to organic solvents. GENERAL OVERVIEW OF PAINTS A paint is a fluid preparation (liquid, paste, or solid) that can be spread in thin layers on all types of materials (called subjectiles), which, after a hardening period (referred to as drying), forms a resistant, adhesive thin coating or layer (film or sheet) that plays both a decorative and/or protective role. If the film is opaque (white, or colored), it is classified as a paint. It is the presence of pigments in the paint that gives it the ability to cover or mask the support on which it is applied. A varnish corresponds to a paint without pigment. The development of synthetic macromolecules has led to the creation of a wide diversity of products—a development that makes the task of formulating paints more complex, but that also enriches the possibilities of new formulations, better adapted to a given end use. Constraints related to hygiene and safety in the workplace and in the environment have also contributed to the evolution of the makeup of paints. The manufacturer of a paint must currently consider the following parameters when developing a new product:

266


Figure 7. Schematic diagram of the operating principle of a process using the projection of ice particles to strip paint (Courtesy of LINDE-CRYOCLIN, France).

• end use of the paint—whether it is an adherent, an anti-corrosive agent, an intermediate layer, or for finishing an object. • the nature of the support onto which it will be applied—whether this be wood, concrete, metal, aluminum, or a plastic material. • the desired properties of the paint—its hardness, flexibility, weather resistance, resistance to chemical agents, etc. • form in which the paint is sold. The different forms in which paint is found are summarized in Table 2. • is the chemical reaction necessary. Generally begun during the hardening phase of a paint, if the reaction is to occur at ambient temperature, it is necessary to

Figure 8. Stripping of a painted surface using the projection of ice particles to strip paint (Courtesy of LINDE-CRYOCLIN, France).

Table 2 Fornfis in which paints are found Presentation

Solid Aqueous Liquid • solution • emulsion Organic Liquid • solution • emulsion

Common name

Coating powders

Containers

1

Waterbome paints Water-thinnable paints

lor 2 lor 2

Solvent-based paints High solids paints Plastisols Organosols

lor 2 lor 2 1 1

mix the reactive constituents only at the instant of application. In this case, the paint is generally delivered in two separate containers. It can be seen from Table 2 that practically all types of liquid paints (aqueous or organic) can be conditioned in two separate containers, depending on their reactivity. • method of curing—summarized in Table 3.

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Health and Toxicology Table 3 Paint curing methods

Drying category

Physical curing

Chemical curing

Physico-chemical curing

Type of drying

Solvent evaporation Coalescence Fusion Oxidation Polycondensation Polyaddition Polymerization Each type of chemical curing method can be preceded by one of the three types of physical curing methods

Example of binding agents used

Acrylics, chlorinated rubbers, nitrocellulose Acrylics, vinyls Polyesters, acrylics Alkyds Alkyd-melamines Polyurethanes Unsaturated polyesters See above-mentioned binding agents.

Whenever a paint cures only by the simple evaporation of solvents, no chemical transformation of a binder occurs during the drying stage. The paint film therefore remains sensitive to solvents even when it is dry. In this case, the binders used are thermoplastic. Coalescence is the mechanism by which film forming occurs in emulsion paints when paint is applied. The evaporation of water causes the spreading, coming together, and finally the fusion of the emulsion droplets. It should be remembered that after the drying process, the film is insensitive to water. If a chemical reaction occurs in the film at the same time as the coalescence process, the film can equally become resistant to the majority of solvents (physico-chemical drying). In the opposite case, it can remain sensitive to solvents that dissolve the binder present in the paint. Fusion is the mechanism by which coating powders, or plastisols and organosols, form films. In the case of emulsion paints, if a chemical reaction occurs at the same time as fusion, the film becomes insensitive to attack by the majority of solvents. The binders used in paints that dry via a chemical process (or a physico-chemical process) are generally thermoset resin. During the drying process they undergo an irreversible transformation. This transformation can also cause the paint film to become insoluble in the majority of solvents. The chemical process can occur at ambient temperature (2 component paints), via the addition of a catalyst (oxidation catalysts: siccatives or acid catalysts), or be caused by radiation (e.g., infrared, ultraviolet, or beta-rays). The principal methods for applying paints, as well as their capacity for solvent emission (+-I-++ = high emissions, + = weak emission), are summarized in Table 4.


269

Table 4 Methods for applying paint

Category

Pulverization

Dipping Pouring Other processes

Nature

Pneumatic Airless Airmix HVLP Purely Electrostatic Assisted Electrostatic Conventional processes Electrodeposition Flowcoating Rolling machines Sray curtains Coil coating Can coating Brush, roller

Estimated level of emission of solvents

++++ + +++ ++ + ++ +++ + +++ ++ + + ++ +

The rheological properties of paints must be adapted to the way in which they are used, and the.manufacturer must account for this in the formulation of the paints. Other than the above-mentioned parameters, the manufacturer must also consider the toxicity of the substances used in the paints in order to satisfy the health and safety requirements for the workplace and environmental legislation, during both the manufacturing and use phases. The importance of the list of requirements shows that paints are highly technical products, and the technological evolutions in this area have led to the creation of highly specific formulations; each paint is formulated for a specific end use. THE CONSTITUENTS OF PAINTS The constituents necessary for paint formulation are listed in Table 5. The column corresponding to coating powders is mentioned for the sake of completeness, but this category of paint does not produce any solvent vapors and will therefore not be discussed. Binders The binder is the essential ingredient of a paint. It forms bonds between all of the components and assures that the paint film adheres to the surface to which it is applied. The principal characteristics of a paint depend on the binder, and paint is

270

Health and Toxicology Table 5 Composition of paints

Water-based paints Constituents Binders Pigments Extenders Solvents -Organic -Water -Coalescing agents -Cosolvents Additives -Conservation agents -Neutralizing amines

Coating powders

Waterborne Solventpaints based paints

Water-thinnable paints

+ + +

+ + +

+ + +

+ + +

+ -

+ + -

+ + + +

+ + + + -

(+) presence (-) absence

frequently named after which principal binder it contains. A paint can have one binder, or a combination of many different ones. The "principal binder" is the one whose chemical and physical characteristics define the general functional properties of the paint. The "complimentary binders" are those added to bring particular properties to the finished product. Binders are made up of macromolecules of natural (e.g., cellophane), artificial (nitro-cellulose, chlorinated rubbers), or synthetic origin (acrylics, epoxides, polyesters, polyurethanes, etc.). The principal characteristics of a binder include: • form—liquid or solid; • chemical nature—organic (natural and synthetic), organo-mineral (silicones), or mineral (silicates); • reactivity—non-reactive binders such as cellulose or rubber derivatives, reactive binders such as acryUcs, polyesters or epoxides, etc.; • molecular weight—low (oils), or medium to high (synthetic binders); • form in which they are used—solution, dispersion, powder; • solubility—in solvents or water (waterborne binders). It should be noted that the type of solvent used in a paint is dictated by the solubility-related properties of the binder that it contains.


271

Pigments As previously mentioned, pigments provide the opacity and color of the paint film. In addition, they can also have an impact on certain other properties such as corrosion resistance, hardness, impermeability, etc. Pigments are pulverized solids of submicron-sized particles, either white or colored, mineral or organic, and insoluble in the dispersion medium. Mineral pigments are metal derivatives. With the exception of titanium oxide (the white pigment found in the majority of paints), mineral-based pigments are being used less and less because of the danger of toxicity that they can pose (e.g., lead or zinc chromates). Mineral pigments provide high levels of opacity and have average tinting capacities. There is a wide variety of formulas for organic pigments, but the most commonly used are phthalocyanine, azoic, and quinacridone derivatives. Organic pigments offer poor degrees of opacity, but very high powers of coloration. Extenders Originally, extenders were added to paints to lower production costs. However, experience has shown that this practice can have an impact on the mechanical, chemical or rheological properties of the paint. Extenders are generally pulverized soHds, usually white, mineral-based, and insoluble in the dispersion medium of the paint. The most widely used include calcium carbonate, barium sulphate, and silica derivatives. Extenders have poor powers of opacity. Additives Additives can bring different properties throughout the life cycle of a paint: •Fabrication: aid in the dispersion of pulverizing agents, anti-foaming agents (surfactants); • Storage: "anti-skin forming" agents (cetoxime derivatives), anti-sedimentation agents, rheological agents; • Film formation: spreading agents (surfactants), agents that render the film matte (silica), anti-corrosion agents (nitrites), anti-bacterial agents or fungicides; • Curing: accelerating agents (siccatives, acids), coalescing agents (glycol derivatives). Additives are introduced at low concentrations (

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336


Similarly, respiratory disease deaths, such as those from chronic bronchitis, rise markedly during the colder winter months in Britain [29]. Seasonal influences [30, 31, 32] include gradual changes in temperature, and radiation period and intensity. Such changes regulate the growth of allergen-producing organisms, such as the house dust mite, molds, and plants. Wind Wind, a collective term for characteristics of air flow, including air speed, direction, and turbulence, has a marked influence over the composition of the atmosphere. It plays a major role in the dispersion and distribution of air pollution. Low wind velocity leads to an accumulation of pollutants, whereas high velocity and turbulence tend to disperse particulates and decrease their concentration [8]. Temperature inversions suppress vertical mixing and thus reduce the dispersion of pollutants. Outdoors, wind can trigger the release of pollen grains from the anthers of flowers and the distribution of mold/fungal spores. Onshore winds in coastal areas reduce the concentration of airborne allergens such as spores and pollen [33, 34], where as offshore winds may lead to increases in airborne allergen concentrations [33]. Winds such as katabatic (downslope or drainage) and anabatic (upslope) breezes are topographically generated. Land and sea breezes result from thermal circulation systems generated by contrasting thermal responses of land and water. Radiative heating of surfaces produces convective updrafts and mixing. Indoors, air movements can be generated by human activity. These air movements are the mechanism by which allergenic particles such as house dust mite feces and mold/fungal spores can be lifted to the level of the mouth or nose. Air Pollution The respiratory sensitivity of people suffering from diseases like asthma and emphysema makes them especially sensitive to episodes of atmospheric pollution. Ozone, sulphur dioxide, and nitrogen dioxide, as well as heavy loads of particulate matter, increase airway resistance and have an irritant effect on mucous membranes. All these elements are common byproducts of living in modem cities. The production of ozone (O3), a major constituent of photochemical smog, requires NOx (NOx = nitric oxide + nitrogen dioxide) and reactive organic compounds as precursors, and high temperature and ultraviolet radiation intensity for its formation [35]. Temperature inversions are perhaps the other major link between climate, air pollution, and CRD. In many places there is a strong seasonal character to air pollution episodes. Summer-type episodes often involve photochemical smog, while winter-type episodes more typically involve sulphur dioxide and suspended particulate matter [36]. Both have been associated with CRD [37]. Indeed, Bates and Sizto [11] have found respiratory admissions in Southern Ontario to be correlated with ozone, SO4, and SO2. They refer to this as the Acid Summer Haze Effect.

Climate and Chronic Respiratory Disease

337

Mold and Fungal Spores Many people suffering from chronic respiratory diseases are atopic with some degree of allergic status. Plants and animals provide the greatest store of allergens. Pollens, mold/fungal spores, and house dust mite feces are the allergen groups of most importance. CRDs such as asthma, which are affected by mold and fungal spores, are affected by climate indirectly via its effects on the growth of molds and the liberation of mold fungal spores. Moisture is required for mold growth, although requirements of different species vary widely [38]. Maximum growth for most fungi occurs at a relative humidity of 95-100% and growth declines or ceases in humidity of 80-85%, although a few fungi will grow at a relative humidity as low as 65% [39]. Reed [40] commented that mold growth would not occur unless the humidity consistendy exceeded 70%. The ability to grow on a given substrate is partially determined by the moisture content of the substrate. The liberation of mold/fungal spores into the air is also very much related to the humidity of the atmosphere. While the production of allergens may depend on moist conditions, their liberation into the air may be favored by dry air. For example, spores from certain of the Fungi Imperfecti (Deuteromycetes), Cladosporium and Alternaria, are shed into dry winds [8, 41]. Other spores, however, such as those from Ascomycetes and lower Basidiomycetes classes, are discharged during periods of high humidity (rainfall and dampness). As well as being sensitive to air humidity and substrate moisture levels, the growth of molds is responsive to temperature. All fungi have minimum temperatures below which they will not grow and maximum temperatures above which they are inactivated. They achieve their best growth at what are known as optimum temperatures [42, 43]. These temperatures can vary according to the stage of life of the fungus. Different molds are suited to different temperature regimes, so much so that they have been grouped accordingly: mesophilic, thermophilic, psychrophilic, and thermotolerant fungi growing in moderate, high, low, and wide temperature ranges, respectively. Various fungi can grow over a wide range of temperatures, though most (known as mesophilic fungi) grow at moderate temperatures within the range of 10-40°C [38], with optimal growth generally occurring between 15-40°C. Thermophilic fungi grow at higher temperatures with an optimum for growth of about 40°C, tolerating a maximum up to about 60°C and a minimum of 20°C [43]. Other fungi known as psychrophiles (e.g., certain Cladosporium species) grow at low temperatures, to a minimum of-5°C [42] with a maximum of 20°C and an optimum of 0-17°C [43, 44]. By contrast, thermotolerant fungi (e.g., certain Aspergillus species) tolerate a wide temperature range [41]. Aspergillus fumigatus, for example, grows between 12-55°C. The production or germination of spores often requires more specific conditions than does vegetative growth. Particles of any type of airborne allergen over 2|im in diameter are liable to be washed out of the atmosphere by rain [18, 28, 45]. Rain therefore decreases the atmospheric concentration of many types of spores, although some like Ascospores

338


are increased [8]. Experimental work conducted by Hirst and Stedman [46] has shown that large transient increases in the concentration of some dry airborne spores can be due to rapid air movement in advance of radially spreading raindrop splashes and vibration. High wind velocity has also produced decreased atmospheric concentrations of some spores such as Altemaria, Cladosporium, and Botrytis species, and some Basidiospores [8]. Plants and Pollen In a similar way, CRDs of an allergic nature are indirectly affected by climate via its effects on plant growth and airborne pollen concentrations (Figure 4). The growth and distribution of some significant asthma-triggering plants, such as Parietaria judaica, is partially determined by temperature and rainfall levels [47]. Pollen release is also closely related to daily fluctuations in temperature, and occurs predominantly in the morning. Flowering, and thus the production of pollen, of many plants is responsive to the relative amounts of light and dark sensed by the plant. This phenomenon is known as photoperiodism. Ragweed, for example, is a short-day plant, flowering when the light period is shorter than a critical length. There is evidence to suggest that the initial cause of asthma, or at least sensitization, may be closely related to climate through seasonal variation in flowering and pollen production. Exposure to high allergen levels in the first year of life may lead to the development of asthma. More specifically, the timing and type of the first high allergen exposure may be critical. For example, Bjorksten et al. [48] and Bjorksten [49] found an increased risk of immediate hypersensitivity to birch pollen in patients who were born in the months immediately prior to the birch flowering season. Humidity also effects the concentration of pollens in the atmosphere. Lopez and Salvaggio [8] comment that pollen is shed into dry winds. Bass and Bass [47] comment that pollen release from P. judaica is closely related to daily fluctuations in humidity, and occurs predominantly in the morning. Pollen release into the air would seem to be triggered by a fall in relative humidity [45]. Conversely, high humidity can lead to the accumulation of moisture on airborne particles and accelerate their deposition. Rain also decreases the atmospheric concentration of pollen [8]. Much of the wind-borne pollen produced globally is lost by fallout at or within several meters of its source [50]. For example, probably no more than 5% to 10% of short ragweed pollen achieves appreciable dispersion. House Dust Mite At the broad level, climate exerts a considerable influence on the levels of house dust mite allergen and mites (Figure 4). Lower levels of house dust mite allergen have been found at high altitudes compared to sea level [51], and inland locations tend to have fewer mites than locations on the coast [52, 53, 54]. However,


339

Figure 4. Scanning electron microscope photograph of a female house dust mite (Dermatophagoides pteronyssinus), upper right; mite feces, upper left; mite egg, lower left; and rye grass pollen, lower right. (Courtesy of Dr. Euan Tovey, University of Sydney.)

Tovey [54] has noted that "while regions with similar climates have similar mean levels of mite allergens, the level between houses in the same region may cover at least a hundredfold range ... for reasons which are not yet characterized." A wide range of allergen concentrations and mite numbers have been determined, ranging from negligible in locations such as Alice Springs in central Australia [54] and Briangon in the French Alps [51], to high on the east coast of Australia [54, 55] and Sao Paulo, Brazil [56]. It has become clear in recent times that the house dust mite and the allergen it produces is a major factor in asthma in many locations around the world. As such, considerable research has been conducted on its life history, and it has been determined that it has quite specific microclimatic requirements. Some of this research has focused on domestic design as a means of controlling house dust mite numbers and therefore improve asthma severity. For example, house dust mites such as Dermatophagoides farinae and D. pteronyssinus are very sensitive to the temperature and humidity of their micro-environment, thriving at moderate temperatures and high humidities. Tovey [54], who has examined mite allergen levels in Australia,

340


suggests that patterns of mite abundance are controlled by regional humidity. A relative humidity of 70-80% (at 25 °C) would seem optimal for the development of a population of house dust mites [57]. Specifically, it has been determined that the optimum conditions for mite development and reproduction are 25 °C and 75% relative humidity. Humidity greatly influences fecal production in mites. A reduction of humidity from 85% to 65% reduces feeding (and therefore production of allergen-containing feces) by more than tenfold, so although mites may not be killed by minor reductions in humidity, allergen production is severely curtailed. At a relative humidity of 60% or lower, the population stops growing and dies out. It has been found that very few house dust mites can develop if the internal relative humidity is less than 45% at an indoor temperature of 20-22°C [58]. House dust mite populations and allergen levels have been surveyed at many locations around the world (Table 2). Three dwelling sites have been studied: the subject's bed, bedroom floor, and the living-room floor. Of these three sites, beds have usually been found to contain the greatest numbers of mites or quantity of allergen, though a few studies have found higher numbers on the bedroom floor or living-room floor (e.g., Korsgaard [59] and Tovey [54], in Wellington NZ). This may reflect the microclimate and food supply generated by humans as they sleep. It has been demonstrated that in some locations such as Sydney, Australia, and South Manchester, United Kingdom, there is little fluctuation in the quantity of house dust mite allergen with season of the year [60, 61]. Studies at other locations such as in the United States of America [31, 62] have indicated seasonal variation in house dust mite allergen quantities and house dust mite body counts, which are related to seasonal variation in humidity. Table 2 House dust mite allergen quantities for selected locations Site* Reference

Location

Charpin et al. [51]

Brianôn, France Martigues (near Marseille), France Sydney, Australia Sao Paulo, Brazil

Green et al. [55] Naspitz et al. [56] Tovey [54]

Alice Springs, Australia

Sample (n)

Bed

Bedroom floor

Living-room floor

—

—

0.36 9 to 11 years old (211)

15.8

random (72-74)

22.42(18.3, 38.86 (95% c.i.** 31.8, 47.5) 27.5) 38.4 >4

13.68 (10.7, 17.6) >4

negligible

negligible

moderate to severe asthmatic and positive sldn test (20) general

negligible

* Quantities are geometric means expressed as ng{ofmite allergen Der p I)/g of fine dust. **Confidence interval.


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Viruses The seasonality of upper and lower respiratory tract infections, and their common association with exacerbations of asthma, chronic bronchitis, and emphysema, suggests that viral infections are both related to the climate and can provoke respiratory diseases [63, 64]. Derrick [18] has proposed a mechanism whereby an asthma attack is brought about by a sudden cold temperature change first inducing a respiratory infection. He cites Andrewes' [65] suggestion that cold temperatures might activate latent viruses, and that rhinoviruses are very prevalent among the cold infections associated with the first cold weather of autumn. It is likely that very low humidity can cause irritation of mucous membranes and may predispose the susceptible to respiratory tract infections. DWELLING CHARACTERISTICS, DAMP HOUSING, MOLD GROWTH, AND ASTHMA It has recendy become clear that certain characteristics of the home environment, such as the presence of damp, mold, and house dust mites, may be of the utmost importance in some CRDs like asthma. Older buildings, for example, can have problems of rising damp resulting from the disintegration of damp-proof building materials. The activities of humans in buildings can also gready effect the indoor climate. Activities involving clothes driers, showers, gas heaters, and cookers can inject large volumes of moisture into the air. Burr et al. [66], in their survey of respiratory disease in a South Wales town, found an association between those with a history of wheezing with breathlessness, and reported dampness and use of open coal fires throughout the year. Strachan and Elton [67], in a study of the relationship between respiratory morbidity in children and the home environment in Edinburgh, Scotland, found parental reports of wheeze, nocturnal cough and school absence owing to chest trouble were significantly more common among children from damp or moldy housing. They also found an association between coal fires and nocturnal cough. Brunekreef et al. [68], in a study of children living in the United States, also found a strong and consistent association between measures of home dampness and respiratory symptoms. However, only a weak relationship was found between home dampness and pulmonary function. Similar results have been found in a more recent North American study [69] where the odds ratio between symptoms and dampness was 1.62 (95% c.i. 1.48 to 1.78). Burr et al. [70, 71] found asthmatics reported mold on the inside walls of their homes in Cardiff more than twice as often as controls (Table 3). Piatt et al. [72], in a study of the relation between damp and mold growth and symptomatic ill health in the United Kingdom, found that both adults and children living in damp and moldy dwellings had a greater prevalence of respiratory symptoms such as breathlessness and wheeze compared with those living in dry dwellings. Although other studies have found such an association (e.g., Strachan [73]), it is possible that the simple awareness of dampness or mold in the home might be a determinant of parental reporting of respiratory symptoms.

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Health and Toxicology Table 3 Studies of reported and measured prevalence of mold in dwellings

Reported mold (%)

Reference

Location

Sample (n)

Brunekreef et al. [68]

Kingston, TN Steubenville, OH Watertown, MA St. Louis, MO Topeka, KS Portage, WI

eight- to 12-yr-old children (4,625)

38.1 27.9 20.9 26.9 33.0 35.4

Burr et al. [70] &Burretal. [71]

Cardiff

asthmatics (72) controls (72)

26.0 12.5

Piatt et al. [72]

Glasgow, Edinburgh and London

households (597)

Strachan & Elton

Edinburgh

children (165)

Actual mold

45.9%

21.0

m Finally, it has recently become clear that there are close relationships between damp housing and the prevalence of molds and house dust mite populations. A recent study in the Netherlands found home dampness was associated with increased sensitization to dust mite and molds [74]. SYNERGISTIC EFFECTS One of the major challenges that faces modem society is the need to understand the complex synergistic interactions involved in CRD. Experimental studies by Bethel et al. [75] and Sheppard et al. [76] suggest how one synergy operates. This study found that cold dry air potentiated S02-induced bronchoconstriction, and that exposure to 0.1 ppm SO2 potentiated the effect of cold and dry air. Further, Linn et al. [77] found that warm humid air ameliorated the response to SO2. EPIDEMICS The circumstances surrounding epidemics of respiratory diseases like asthma have been the focus of many investigations. Many asthma epidemics have been associated with thunderstorms, which are characterized by rapid temperature drops, wind speed and direction changes, and heavy rainfall. The actual mechanisms involved in such epidemics have often been difficult to unravel. A number of studies have identified a sudden increase in airborne mold spores, as well as the characteris-


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tic atmospheric changes, as the cause of the outbreak [78]. Recently, however, more complex mechanisms have been proposed, particularly that sulphuric acid particulates could be transported to the surface from high altitude by thunderstorms [79]. It would also seem that the rainfall associated with thunderstorms could trigger the release of allergenic starch grains from pollens [80, 81]. The passage of cold fronts, involving rapid temperature decreases, wind speed and direction changes, and pressure changes, have also been shown to be associated with asthma attacks [9, 23, 82]. In some circumstances, epidemics of asthma may result from a combination of meteorological and aerobiological factors. Asthma epidemics in Barcelona, for example, seem to have originated from plumes of soybean particles being produced when beans were unloaded into silos from ships in the harbor on days with high pressure and little wind, the city lying downwind on every occasion in which the air movement occurred. Although there is still much we do not know about the impacts of the atmospheric environment on CRDs, at least one attempt has been made recently to integrate the sum of knowledge gained through the numerous and varied studies conducted to date [83]. Given the apparently complex interrelated factors involved, these authors have constructed a schematic model showing the linkages and relationships between the human and biophysical environments and the asthmatic person. Such models are beneficial as they can clarify and enhance our understanding of complex disease processes. THE FUTURE: CLIMATE CHANGE AND CHRONIC RESPIRATORY DISEASE In the context of global warming, the relationships and interactions between climate and CRD assume a new significance. Any deterioration of the status of chronic respiratory diseases associated with future climate change will undoubtedly have significant social and economic consequences. To date, reviews of climate change and health with respect to asthma have largely considered the impacts of atmospheric triggers such as urban air pollution and, to a lesser extent, allergens and the direct impacts of climate and meteorological phenomena. There are a number of problems and difficulties associated with gauging the impacts of global climate change on asthma. These include difficulties in predicting regional and local patterns of climate change and in assessing human and architectural influences on asthma triggers, as well as the problems associated with clarifying the effects of individual factors in a multifactorial disease [84]. Further, Longstreth [85] recognized that there are such complexities involved in even assessing the role current weather plays in human disease that one could only speculate at the effects of global warming on human health. Climate change is generally expected to lead to increases in morbidity and mortality from respiratory diseases like asthma. Many of the effects of climate change on respiratory diseases will be indirect, or secondary, as well as direct, or primary

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[16, 86, 87]. As will be discussed below, climate change will affect asthma indirectly through its impacts on atmospheric irritants and allergens and infections, and directly through its influence on the climate (Figure 5). Air Quality (Irritants) Longstreth [85] recently suggested that "respiratory diseases brought about by increased air pollution," such as asthma, would be "one of the largest public health issues" associated with global warming and stratospheric ozone depletion. Many studies have discussed the effects of atmospheric pollutants on asthma and how their occurrence may vary with climate change. An acceleration of photochemical reaction rates among the precursors of photochemical smog would be associated with increased temperatures and ultraviolet-B (UV-B) radiation [87-92]. Thus, high levels of O3 would be achieved earlier in the day and maintained longer more frequently [85, 93]. Such increases in ozone would be significant, as this irritant has been linked to sunmier peaks in the incidence of asthma [16].

Figure 5 Change in January air temperature over the globe associated with an equivalent duubling uf uarbuH diUAide. Frum the Austrcilicin Buruciu uf Muttijurulugy Research Centre's General Circulation Model. (Courtesy of Dr. Bryant MoAvaney and Ann-Maree Hansen.)


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Future emissions of NO2 and SO2 will rely primarily on human decisions and actions, largely being dependent on trends in fossil fuel consumption and, to a lesser extent, on the balance between the need for heating and cooling [91]. For example, increased winter temperature would lessen the demand for energy production for heating and thus would reduce acid aerosol exposure and reduce asthma morbidity [89]. Regional-scale (synoptic) conditions such as high pressure systems (anticyclones) and local-scale conditions such as katabatic flows and sea breezes play a large role in air pollution production. Changes in the occurrence of such regional and local conditions will influence urban air quality [92]. Allergens There is a clear relationship between many allergen-producing organisms such as plants, mold, and house dust mites, and climatic factors such as humidity, temperature, rainfall, and sunshine. Increased levels of pollens and other biologic agents causing allergic respiratory system reactions in humans may be expected to result from higher temperatures coupled with elevated humidity. Last and Guidotti [94] and Last [95] have suggested there would be significant changes in the distribution of vegetation, including many allergens. Similarly, Longstreth [85] has suggested that "changes in the prevalence or intensity of asthma . . . episodes in affected individuals" could come about through likely "quantitative and/or qualitative changes in the airborne concentration of allergens" such as mold spores and pollens. Climate Wind plays a major role in the transport of pollens, dust and other irritants [96]. Curson [97] has suggested "drier winters and changed wind movements and cyclonic activity" may lead to asthma increases in Australia. Waves of extreme heat and cold impose stress on asthmatic individuals [98]. Ewan et al. [92, 99] indicated "heat stress combined with high humidity act as a trigger to attacks." Hennessy [100] has estimated the change in frequency of extreme cold events in southeast Australia (winter days when temperature falls below 0°C) associated with a 3°C warming. It is clear that such events may become far less frequent. It is likely that acute asthma symptoms triggered by sudden exposure to cold air will be reduced if such climate change occurs in this region. Similarly, Langford and Bentham [29] have suggested that there would be fewer winter deaths from respiratory disease such as chronic bronchitis associated with the warming predicted to occur with climate change. Infections Ewan et al. [92] have stated that "infections such as the common cold and influenza" could increase in southeast Australia and decrease in southwest Aus-

346


tralia due to predicted increases and decreases in rainfall, respectively. Such infections are commonly associated with asthma symptoms. These potential increases are significant because peaks in hospital admissions for asthma in autumn/winter have been related to viral infections [16, 101]. In the case of asthma, each asthmatic is unique in his sensitivity to, and the composition of, factors that provoke acute symptoms. As such, it is difficult to generalize about the overall impacts that climate change will have on the status of the disease. Asthmatics rarely, if ever, react or are reactive to only one trigger. While there is increasing evidence that triggers act synergistically or in combination, such complex mechanisms are not fully understood. Indeed, Goldstein and Reed [86], in their commentary on global atmospheric change and research needs in environmental health sciences, recognized the need to better understand both the individual and multiple effects of indoor and outdoor irritants and allergens on people with chronic respiratory diseases such as asthma. It is possible that a wide range of factors will be suited to a variety of environmental conditions. Just as the conditions close to the coast favor the house dust mite while those inland favor grass pollen production (reflected in the status of respective allergies present in the two populations), different molds grow over very different temperature ranges. As such, no matter what the change in climate, there may still be molds that can grow in that new climate. TECHNOLOGICAL WARNING SYSTEMS Because of the knowledge base that has been built with regard to the relationships between climate and weather and the occurrence of air pollutants and allergens, local and regional authorities are able to issue warnings and advise the public in advance of potentially threatening events. Warnings of elevated levels of air pollution are provided in many urban centers. Such warnings require an intimate knowledge of the local and regional setting for which the forecast is to be made. Factors that are often taken into consideration include the forecast wind direction and speed, the existence of temperature inversion, the day of the week, and whether rain is expected. As such, it is usually the local environmental protection authority and meteorological bureau that are best placed to issue such warnings. The need for warnings of elevated pollen and mold spore levels is perhaps more wideranging because it is not only urban areas that are affected by such events. Such warning systems have not been as well developed as those for air pollution, though the potential benefits could be equal to or greater than those derived from air pollution warnings. ENVIRONMENTAL CONTROL In addition to making forecasts of potentially harmful levels of air pollution and airborne allergens, attempts have been made to determine housing characteristics


347

that are best suited to the prevention and management of CRDs. Recently, adequate ventilation and dehumidification have been targeted as essential factors in the control of the house dust mite [102, 103]. In cold climates, ventilation systems have been designed to exchange cold and dry outdoor air for warm and moist indoor air. In order to further reduce the humidity of indoor air, and thus house dust mite populations, a heat exchange system warms the outdoor air (which is to replace the indoor air), thus reducing its relative humidity. Indoor climate can also be controlled by using moisture absorbers, extractor fans in rooms where large amounts of moisture are generated such as bathrooms, and by using flues on gas appliances to prevent moisture from gas combustion being added to the indoor atmosphere. Older dwellings also often have structural defects such as leaking roofs and deteriorated damp-proof materials. The renovation of such dwellings can greatly improve the indoor microclimate that is so important for house dust mites and molds. The control, prevention, and treatment of mold growth in dwellings have been well documented [38]. These, too, focus on moisture control and ventilation. CONCLUSIONS There is no doubt that climate is related to chronic respiratory disease in many ways. Climate and weather have been linked to major outbreaks of chronic respiratory disease, seasonal variations of such disease, and more subtle instantaneous effects on the respiratory system. This chapter has viewed the relationship between climate and chronic respiratory disease by first looking at the direct effects of climate and weather such as the well-documented effects of cold dry air on asthmatics, and second by examining the diversity of indirect effects. The latter include the effects of climate and weather on potent stimuli such as air pollutants, and allergens, including mold spores, pollens, and house dust mite. It has also been shown that both the outdoor and indoor environments are important, with some stimuli such as house dust mites exclusively occurring in the latter. Perhaps the major challenge is to understand the complex synergistic relationships between many environmental agents and chronic respiratory disease. Such relationships may prove to be the dominant mechanisms producing such disease. Though our understanding of chronic respiratory disease is far from complete, we must look to the future and see what it holds for these significant diseases. While climate change may in some ways be detrimental to diseases such as asthma, our growing understanding of the relationships between the natural biophysical environment and human disease enables us to predict changes in and improve our environment. REFERENCES 1. National Institute of Health (NIH). International Consensus Report on Diagnosis and Management of Asthma. U.S. Department of Health and Human Services, NIH publication 92-3091, 1992.

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CHAPTER 15 EFFECTS OF ACID PRECIPITATION ON THE ENVIRONMENT AND ON HUMAN HEALTH Lars Gerhardsson and Staffan Skerfving Department of Environmental and Occupational Medicine Lund University Hospital S-221 85 Lund, Sweden Agneta Oskarsson Department of Food Hygiene Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden CONTENTS INTRODUCTION, 355 AIR, 356 WATER AND SOIL, 357 Calcium, 358 Aluminum, 358 Cadmium, 359 Copper, 359 Lead, 359 Magnesium, 360 Mercury, 360 Metal Exposure in Farmers, 360 CONCLUSIONS, 361 REFERENCES, 362 INTRODUCTION Environmental acid precipitation is a global problem that may cause a number of adverse health effects in man. These effects can be of both primary and secondary nature. Primary effects in humans are mainly confined to the respiratory tract and are caused by inhalational exposure. These effects most often result in impairment of pulmonary function, and symptoms such as cough, dyspnea, and increased mucus secretions. Secondary effects are caused by increased solubilization and mobilization of toxic metals—such as aluminum, cadmium, copper, lead, and mercury—in lakes and waters, leading to groundwater pollution and uptake in plants [1]. Through the animal and vegetable food chain, ingestion of polluted sources, e.g., fish and vegetables, will increase the intake of toxic metals in man, which may lead to health implications [2]. For some elements, such as the essential element selenium, acid 355

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precipitation results in decreased mobility and decreased levels taken up in crops. The greatest problems are found in regions with a combination of high fallout of acid precipitation and a low buffering capacity of bedrock, water, and soil. AIR The atmospheric acidity consists of both gaseous and particulate phases, which are mainly formed from the oxidation of sulfur oxides (SOx, e.g., SO2), nitrogen oxides (NOx, e.g., NO2), and hydrocarbons from combustion sources [3]. The SO2 gas passes a series of photo-oxidation reactions in the atmosphere, leading to formation of sulfate particles. The principal forms are sulfuric acid (H2SO4) and ammonium sulfate [(NH4)2S04]. They are characterized by relatively long residence time and long-range atmospheric transport. Laryngeal and tracheobronchial deposition is expected to be greater when breathing H2SO4 as fog droplets as compared with humid haze particles. On the other hand, deposition of acid in the alveolar region seems to be greater via humid haze particles than via fog droplets [4]. Various NOx compounds are formed in combustion processes and emissions from motor vehicles. The principal component is NO2, which is hydrolyzed to HNO2 (nitrous acid) and HNO3 (nitric acid), occurring mainly in fog or cloud droplets. Most of the HNO3 is expected to be deposited in the nose and upper part of the respiratory tract when breathing either haze or fog. In the former case, the acid is inhaled and rapidly removed as a gas, whereas in the latter case it may be efficiently removed in large droplets by the extrathoracic airways [4]. Also, through photo-oxidation processes, NOx contributes to the formation of ozone (O3), which is implicated as a main human health hazard in photochemical pollution. Acid aerosols in ambient air are in the respirable size and will reach the alveolar region of the lung [3]. In animal experiments, acid aerosols have caused airway narrowing, changes in particle lung clearance, and changes in response to inflammation [3]. In humans, a relation between increased acid aerosol levels and total mortality has been reported [5]. Other adverse health effects connected to exposure to acid aerosols include increased hospital admissions due to mainly respiratory symptoms [6]. Exposure to acid aerosols has been associated with increased prevalence of bronchitis symptoms in children [7]. Concomitant exposure to acid aerosols and ozone have affected lung function in normal children exercising outdoors [3]. For example, decrements of forced expiratory volume (FEVl) and peak expiratory flow (PEP) have been associated with air pollution episodes [8]. In the latter study, a larger decrement was indicated for children with a positive response to metacholine challenge compared to their nonresponsive counterparts. In experimental studies in humans [9, 10], asthmatics show statistically significant dose-related increases in respiratory symptoms, as well as dose-related decrements in forced expiratory performance when exposed to sulfuric acid aerosols. An association between summer hospital admissions for respiratory diseases in Southern

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Ontario and daily concentrations of particularly SO4 has been reported [6]. Multiple regression analysis showed that SO4 accounted for about 3% of the variance. Pollution episodes are often worse in winter, when high concentrations of pollutants may be trapped in the bottom few hundred meters of the atmosphere [11]. High concentrations may lead to severe adverse health effects. One of the worst disasters took place in London in 1952 in which coal smoke containing sulfuric acid was implicated as the causing agent. It is estimated that more than 4,000 deaths, primarily in elderly people, occurred in London during that single episode [12]. During the 1950s and early 1960s, deaths connected to chronic bronchitis were higher in London than in other, less-polluted areas in the U.K. and considerably higher than in other countries in northern Europe [12]. Later investigations showed that the excess mortality in London was more closely associated with blackness of sampled particles (BS) than with SO2. However, BS is mainly a measure of the carbon content of the aerosol, and is thus unlikely as a causal factor. It seems more probable that another aerosol compound, e.g., the free hydrogen ion (H"^), was the causal factor [12]. Increases have been observed in the total number of deaths (8%), hospital admissions (15%), and ambulance use (30%) for respiratory and cardiovascular illnesses in subjects living in a polluted area during the January 1985 air-pollution event in western Europe, compared to a before-and-after interval, and with a control area [13]. However, the effects could not be attributed to acidic aerosols as such [14]. The observation of an association between fine particulate air pollution and health outcomes in regions where little aerosol acidity has been measured suggests that particulate acidity alone may not be the primary component causing fine particulate air pollution toxicity [15]. Thus, respirable particulate air pollution is probably an important contributing factor to respiratory disease (e.g., symptoms and decreased lung function), increased hospitalizations, and other health care visits for respiratory and cardiovascular disease [16, 17]. There are two important defense systems against inhalation of acid aerosols. One is the conversion of acid to the ammonium salts by respiratory ammonia, and the other is buffering by the mucous lining of the airways [18], especially by its glycoproteins. The mucous increases in viscosity, leading to decreased clearance, as well as increased airway resistance and reduced pulmonary gas exchange [19, 20]. Further, after saturating the buffering capacity, the underlying tissues will receive an increased dose of acid. WATER AND SOIL Environmental pollution increases the exposure to metals in two ways. They can either be deposited from the atmosphere or mobilized from soil and water [21]. The solubilized metals will cycle through the ecosystems. The primary exposure to man is mainly through food and water, and adverse health effects may occur if the intake exceeds critical levels.

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Rainwater contains a variety of chemicals, including both strong and weaker acids. As strong acids such as sulfuric and nitric acids contribute up to 95% or more of the total acidity, the pH of rainwater can be used as an index of the acidity. Calcium Present acid rainfalls in the range pH 4.0-4.5 in many European countries will have a great impact on aquatic and terrestrial systems [22]. Generally, about 90% of the rain will pass through one or more soil layers before reaching a stream that feeds a lake. Water flowing over chalk and limestone will normally be completely neutralized. In more acidic soils, however, a cation exchange process may take place. In this process, hydrogen ions from the acid rain will be exchanged by soilderived elements, e.g., calcium. This gradually decreases the acidity of the water passing through the soil, and in parallel increases the calcium concentration in the water. In contrast, in soils naturally poor in exchangeable calcium, the soil may become progressively acidified, leading to increased leakage of, e.g., aluminum instead of calcium into the water [11]. The bedrock in, for example, Norway and Sweden has a low buffering capacity, which makes these countries more susceptible to acid downfalls. Afforestation may also worsen the effects of soil acidification. Growing trees take up calcium and other basic elements from the soil. The harvesting of timber removes this calcium, which otherwise in a natural ecosystem would be recycled [11]. Adequate concentrations of calcium seems to be as important for fish survival as is low acidity. The calcium/acidity ratio has been suggested as an index for fish survival as high values are associated with well-stocked lakes and low values with fishless lakes. High concentrations of calcium also seem to counteract harmful effects of toxic aluminum compounds as shown by experimental studies [11]. Liming of lakes has been practiced in Sweden for several years, showing good results. Usually, limestone has been added to the water itself. It would, however, probably be more efficient to lime the lake catchment instead [11]. Then it would be possible not only to increase the calcium supply to the soil, but also to prevent the release of aluminum into water. Aluminum Aluminum is an ubiquitous metal constituting about 8% of the earth's crust. It has generally been considered relatively non-toxic to humans. However, some studies have reported an increased incidence of Alzheimer's disease in regions with elevated aluminum concentrations in drinking water. Headwater streams exhibit episodic declines in pH and increases in dissolved aluminum leaching from soils during rainfall and snowmelt events. The elevated aluminum levels in source streams can directly affect the quality of domestic tap-

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water. Acid precipitation runoff episodes, thus, may give significant decreases in tapwater pH and increases in tapwater aluminum [23]. Cadmium Important exposure sources for the general population are diet and tobacco smoke. In many regions, the margin between present exposure levels and adverse health effects is small or non-existent. This is exemplified by the Cadmibel study in Belgium, where approximately 10% of the general population had an internal dose of cadmium sufficient to cause slight renal dysfunction [24]. The cadmium uptake in crops is considerably raised by acid precipitation, leading to increased human dietary intake. The long biological half-time (10-30 years) in the kidneys, the main storage pool in the body, means that virtually all absorbed cadmium is retained. More than two thirds of the increased concentrations of cadmium in winter wheat in Sweden seem to originate from increased soil concentrations. In this context, alterations of soil pH is probably of less importance. In the southern part of Sweden, cadmium concentrations in the grain of winter wheat close to or exceeding the maximum permissible level of 100 |Lig Cd/kg, as suggested by FAOAVHO, is often found. Thus, not much could be gained by Uming, because wheat is grown on the best soils with a near optimal pH [25]. Copper Copper is an essential element for man with a daily need of 1-1.2 mg in adults (0.3 mg in children up to one year of age). The safety margin between beneficial intake of copper and potentially harmful effects seems to be small [26]. The concentration of copper in drinking water originates from the piping system. Acidic water increases the corrosion processes and elevates the copper concentration. However, after flushing, the levels diminish rapidly. In a Swedish study of 600 children aged 9-21 months (Pettersson and Rasmusson, personal communication), most children had a daily intake of copper from water below 0.5 mg. Approximately 25% had an intake between 0.5-1 mg/day, and 10% exceeded 1 mg/day. Thus, copper in drinking water accounts for more than the daily need, for most children in this study. Lead Acid precipitation will increase the lead solubility, mobilizing lead from soils, especially in poorly buffered systems. The decreased pH will also solubiHze lead from pipes and lead soldered joints in cisterns. Acid deposition from the environment will also accelerate the weathering of lead-painted surfaces.

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Magnesium During the past few decades, there have been suspicions that acid deposition may severely damage growing trees. This issue attracted serious concern in the early 1980s when suddenly and unexpectedly a rapid decline of Norway spruce began in southern Germany. Recent studies suggest that soil magnesium deficiency is a main reason for this type of forest damage, probably due to a reduced magnesium supply [11]. Mercury Inorganic mercury is transformed to methylmercury in water. The methylation mostly takes place on sediments in fresh and ocean waters, but also in fresh and sea waters, and the process is enhanced by acidity. Through the food chain, the methylmercury concentration is increasing, giving levels exceeding 1.2 mg/kg in e.g. shark, swordfish, pike, and bass. Through this mechanism, long-term and high consumption of fish from contaminated waters will increase the risk of toxic effects on the central nervous system [21], as shown from the epidemic in Minamata. Historic discharges in Sweden have the greatest impact on present mercury concentrations in pike, followed by water acidity and airborne exposure of mercury emissions from other countries. The latter are of greatest importance in southern Sweden. A program has been implied, testing various remedial measures such as lake and wetland liming, potash and selenium treatment, and intensive fishing [27]. After proper liming, a 30% reduction of mercury concentrations in small perch have been observed in 2 years. The levels in 1-kg pikes can probably be reduced by 15^0% after another 2 years. No discernible differences between various sorts of lime were noted with respect to the reduction of mercury concentrations in small perch [25]. Intensive fishing caused a reduction of the biomass in the lake by about 25%, and reduced the mercury levels in 1-kg perch by 20-30% after 2 years. It is expected that the effect of intensive fishing would last about 6-8 years. When using selenium treatment, the dosing is very important, as the reproduction of perch was shown to decrease considerably. Also, other species of animals and plants may be sensitive to increased selenium levels in the water. On a long-term basis, however, the best solution is to minimize the mercury contamination of air, soil and water [25]. Metal Exposure in Farmers A Swedish study included 237 farmers with a high consumption of locally produced food and water from their private wells, showed different grades of acidity (range of pH 4.7-8.6). A significant correlation between the pH and the metal concentration in drinking water was observed [1]. The relation was negative for aluminum (median 0.07 jimol/L), cadmium (0.44 nmol/L), copper (0.24 jimol/L)


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and lead (1.9 nmol/L), and positive for calcium (0.62 mmol/L) and magnesium (0.21 mmol/L). However, no relation was observed between metal uptake in the subjects, as indicated by concentrations in blood and urine on the one hand, and pH in drinking water on the other (used as an acidity index). Other sources of metal exposure had a stronger impact on the concentrations in blood and urine than the acid precipitation had. Thus, the concentration of mercury in blood was related to fish consumption, cadmium and lead in blood to smoking, aluminum in urine to intake of antacids, lead in blood to rifle shooting and hunting, and mercury in blood to hunting [1]. In this study, the acid precipitation had an effect on the metal concentrations in drinking water, but not on the body retention of those elements. In another study in Scandinavia [28], concentrations of cadmium, nickel, zinc, and selenium were studied in spring wheat, potatoes, and carrots from commercial farming fields and correlated to pH and different soil factors. The selenium levels in potatoes decreased with decreasing pH. However, with decreasing pH in soil, there was an increasing concentration of cadmium, nickel, and zinc in potatoes and carrots, while in wheat only nickel and zinc levels were raised. In a multiple regression analysis, a combination of pH in soil, organic matter content, and cadmium concentration in soil explained 25, 68 and 41% of the total variation in cadmium concentration in potatoes, carrots, and spring wheat, respectively. Preliminary findings indicate that liming with CaC03 will lead to a decreased uptake of nickel and zinc in spring wheat and potatoes. The effect on cadmium, however, seems to be more complex, probably due to a competition between cadmium and calcium on the soil particles. Approximately 75% of the total dietary intake of cadmium originates from cereals, potatoes, and vegetables. It is thus probable that acid deposition causing decreases in pH of arable soils in combination with other acidifying processes in agriculture, e.g., fertilization and harvest of biomass in connection with insufficient liming, will lead to an increased cadmium intake via food as well as altered dietary exposure of other elements. CONCLUSIONS Air pollution with SOx and NOx causes exposure to acid pollutants and may result in irritant effects in the respiratory tract of man, causing symptoms such as cough and dyspnea. Impaired pulmonary functions have been reported at such exposures. Environmental acidification is a global problem that increases levels of toxic metals in water and food. Of these metals, aluminum, cadmium, lead, and methylmercury are of particular concern. Acid precipitation may increase the concentrations of aluminum, cadmium, copper, and lead in drinking water. On the other hand, levels of calcium and magnesium seem to be positively related to the pH. Furthermore, acid rain can cause an accumulation of methylmercury in fish in rivers and lakes.

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In parallel, acid precipitation may lead to increased metal concentrations in plants and vegetable foods, although, as shown for cadmium, the soil concentration seems to be more important. Accordingly, acid precipitation may have an impact on the exposure/uptake of metals. For several elements, e.g., cadmium, lead and mercury, there is a clear connection between environmental exposure and measured concentrations in biological indicator media such as blood and urine. The safety margin between current exposure and critical effect levels in humans is small for many toxic metals in some geographical areas. Moreover, in other regions around the world, these critical effect levels are indeed exceeded, causing effects on the health status of the population. One example is cadmium, which has been observed to affect the kidney function in a minor portion of the general population. For exposure to lead and methylmercury, the fetus has an increased susceptibility, and developmental effects in infants and children may occur at various exposure levels in pregnant women living in several regions. Up to now, no firm evidence linking acid precipitation to metal-related adverse health effects in man has been presented. However, there is a small or non-existent margin between the present intake of some metals, on the one hand, and toxic exposures, on the other. Further, for some essential metals, acidification may decrease the intake. This makes it necessary to follow the ongoing acid precipitation with greatest concern. To deal with this task, a global approach has to be adopted. It is important that accurate steps are taken on both national and international levels to enforce legislation that protects everyone. REFERENCES 1. Bensryd, I. et al. "Effect of acid precipitation pn retention and excretion of elements in man." Sci. Total Environ., Vol. 145, 1994, pp. 81-102. 2. NAPAP, National Acid Precipitation Assessment Program. "Indirect health effects associated with acidic deposition. National Acid Precipitation Assessment Program. Acid deposition: state of science and technology." NAPAP Report 23, Government Printing Office, Washington, D.C., 1990, p. 173. 3. American Thoracic Society. "Health effects of atmospheric acids and their precursors. Report of the ATS workshop on the health effects of atmospheric acids and their precursors." Am. Rev. Respir. Dis., Vol. 144,1991, pp. 464-467. 4. Larson, T. V. "The influence of chemical and physical forms of ambient air acids on airway doses." Environ. Health Perspect., Vol 79, 1989, pp. 7-13. 5. Thurston, G. D. et al. "Reexamination of London, England, mortality in relation to exposure to acidic aerosols during 1963-1972 winters." Environ. Health Perspect., Vol. 79, 1989, pp. 73-82. 6. Bates, D. V. and Sizto, R. "The Ontario air pollution study: identification of the causative agent." Environ. Health Perspect., Vol. 79, 1989, pp. 69-72.


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25. Gerhardsson, L., Oskarsson, A., and Skerfving, S. "Acid precipitation— effects on trace elements and human health." Sci. Total Environ., Vol. 153, 1994, pp. 237-245. 26. Pettersson, R. and Sandstrom, B. "Copper," in Risk Evaluation of Essential Trace Elements, Essential versus toxic levels of intake, A. Oskarsson (Ed.), Copenhagen, Denmark: Nord Series, Nordic Council of Ministers, 1995, pp. 149-167. 27. Hakansson, L. "Measures to reduce mercury in fish." Water Air Soil Pollut., Vol.55, 1991, pp. 193-216. 28. Obom, I., Jansson, G. and Johnsson, L. "Acidification of arable land—a field study on the influence of soil pH on trace element uptake in spring wheat, potatoes and carrots." Abstract No. 445 at "Acid Rain '95," Gothenburg, Sweden, 1995. To be published in Water Air Soil Pollut.

CHAPTER 16 TOXIC EFFECT OF TANNERY EFFLUENT ON THE BIOCHEMICAL CONSTITUENTS IN ORGANISMS G. Varadaraj and M. A. Subramanian P.G. & Research Department of Zoology Chikkaiah Naicker College Tamil Nadu, India CONTENTS INTRODUCTION, 365 OVERALL EFFECTS OF TANNERY EFFLUENT (TE), 366 EFFECTS OF TE ON BIOCHEMISTRY OF ORGANISMS, 367 In Plants, 367 In Animals, 368 REFERENCES, 372 INTRODUCTION The tannery industry is growing in diverse proportions at cottage-, medium-, and large-scale sectors, particularly in developing countries. It provides employment opportunities to several thousand people and earns considerable sums via foreign exchange, thereby contributing to the economy. Unfortunately, this industry is linked with water pollution. The quantity of water used is voluminous in the tannery unit; it discharges 3,000-3,200 liters of water per 100 kg of skins and hides processed. The spent water of the tanning industry is invariably let off as effluent, becoming either stagnant or, in most cases, confluent with the nearby water bodies. Thus, pollution due to tannery wastes and its control has gained international importance. Hides and skins are the principal raw materials in the tannery industry. Common salt, lime, sodium sulfate, ammonium sulfate, ammonium chloride, enzymatic products, sulfuric acid, sodium carbonate, dyes, and sulfated vegetable oils are the chemicals used in the industry. Small tannery units may use vegetable tanning materials like wattle bark extract, pungum oil, myrobalam, and kodukai, whereas big tanneries perform chrome tanning. The inherent nature of the tanning process leads to the discharge of wastewater having foul smell, highly putrescible matter and toxic materials. Generally, the effluent consists of both organic matter such as fatty acids, protein, other soluble tannin, and inorganic matter like chlorides, trivalent chromium, nitrates, phosphorus, sulfides, sulfates, etc.

365

366


OVERALL EFFECTS OF TANNERY EFFLUENT (TE) Considerable work has been carried out on the characterization of tannery wastes (Guruprasada Rao and Nandakumar, 1981; Manivasakam, 1987; Subramanian et al., 1988; Kadam, 1995). Available literature clearly indicates that TE contains a high concentration of total solids, high biochemical oxygen demand (BOD) and chemical oxygen demand (COD), chlorides, sulfides, chromium, oil, grease, tannin and lignin, and low dissolved oxygen (DO) (Table 1). The TE when discharged into water courses adversely affects aquatic life and thereby poses a threat to the aquatic ecosystem (Eye and Lawrence, 1971). Sastry and Madhavakrisnan (1984) have reported that TE affects physical, chemical and biological characteristics of water, and depletes DO from the water body. On land, tannery wastes are found to cause damage to the soil quality, crop pattern, vegetation, and yield (Miakhan and Raman, 1972; Guruprasada Rao and Nandakumar, 1981). Harihara Iyer et al. (1957), Yusuff and Ismail (1984) and Varadaraj and Subramanian (1995a) have noticed the seepage of effluents from tannery units into the surrounding irrigation as well as drinking wells. Moreover, dermatitis, bronchitis, acute pharyngitis and other respiratory diseases, and acid burns are associated with tannery workers (Backyavathy et al., 1986).

Table 1 Physicochemical characteristics of tannery effluent and tolerance limit (ISi, 1981)

Characteristics

Tannery effluent

Tolerance limit (ISI)

Color Odor Temperature pH Total solids BOD COD DO Chlorides Sulfides Sodium Chromium Oil and grease Tannin and lignin

Brown or black Disagreeable, foul 29°C-31°C 8-10 1,250-23,920 96-1,200 116-27,810 0.84-1.10 298-6,700 10-84.7 74 32-381 12-16 50-90

Without practicable color Without unpleasant odor Within 40°C 5.5-9.0 2,950 30 250 — 1,000 2 — 2 10 —

Values in mgA except temperature andpH.

Toxic Effect of Tannery Effluent on the Biochemical Constituents in Organisms

367

EFFECTS OF TE ON BIOCHEMISTRY OF ORGANISMS In Plants On Seed Germination The first process in the growth of a plant is germination, a resumption of metaboHc activity by the seed involving dehydration, utilization of nutrient reserves, and gradual development of biosynthetic systems, enabling the young plant to assume an autotrophic existence. All plants store proteins as reserve food in their seeds. Mobilization of these stored proteins and their rapid translocation from the cotyledons to the growing axes involves proteolytic activity. During germination, insoluble carbohydrates like starch are first hydrolyzed into maltose in the presence of amylase. Maltose is then converted into glucose, which is transported to the growing embryonic axes. TE has been reported to diminish the reducing and non-reducing sugar levels as well as proteolytic and amylolytic activity levels in cotyledons and embryonic axes of Vigna radiata (Figure 1) (Varadaraj and Subramanian, 1995b). Saxena et al. (1986) also have documented restricted growth of radicals and plumules and suppression of seed germination with raw TE in Vigna mungo, Cicer arietinum, and Pisum sativum. The alteration in seed germination could be due to the increased salinity condition of TE as noticed by Narale et al. (1969) in Oryza sativa, Gaur and Tomar (1975) in Helianthus annus, Ramana and Ramarao (1978) in Rhaphanus sativus, and Varma and Poonia (1979) in Pennisetum typhoides. This is further strengthened by the investigation of Ogra and Baijal (1982), who have shown decreased activity of amylase and acid protease in Sorghum sp. during seedling growth in increased salinity. On Chlorophyll Content In green plants, chlorophyll is involved in the formation of organic substances from simple inorganic ones. A marked decrease in the chlorophyll content has been noticed in the shoots of Cicer arietinum in concentrations of 25, 50, 75, and 100% TE (39.40%, 41.84%, 43.56%, and 58.58%, respectively) (Guruprasada Rao and Nandakumar, 1983). Similar decrement in the chlorophyll content has been recorded by Kadam (1995) in the leaves of C arietinum under the influence of 75% and 100% TE (34.25% and 25.30%), whereas the chlorophyll content has been shown to increase (13.10%) in 25% concentration of TE. This reduction in the chlorophyll content in plants could be due to the effect of chromium present in the effluent as envisaged by Arvind Bharati et al. (1979).

368


50 Concentration (%)

Figure 1. Effect of TE on sugar and activity levels of enzymes in y. radiata during germination on 5th day. (Varadaraj and Subramanian, 1995b) PC—Protease activity in cotyledon AC—Amylase activity in cotyledon PE—Protease activity in embryonic axis AE—Âmylase activity in embryonic axis NSC—Non sugar in cotyledons RSC—Reducing sugar In cotyledons NSE—Non reducing sugar in embryonic axis RSE—Reducing sugar in embryonic axis

In Animals Invertebrates Varadaraj et al. (1994) have reported decreased levels of body constituents like total free amino acids, total proteins, total free sugars, glycogen, and total lipids in various tissues of tannery effluent-treated Pila globosa (Table 2). They have shown that the effect of the effluent on the biochemical constituents of tissues is dosedependent. The influence of TE and its ingredients on the succinic dehydrogenase (SDH) activity level in the hepatopancreas of Pila globosa has been investigated by Guruprasada Rao and Nandakumar (1982) (Table 3). Total haemolymph proteins, free amino acids, and free sugars of the freshwater prawn, Macrobrachium idella, exhibit marked diminution (6.94-32.22%, 8.16-36.90%, and 10.12-41.21%, respectively) when exposed to different sublethal


369

Table 2 Dose-dependent changes in biochemical constituents in the tissues of P. globosa exposed to tannery effluent Ranges of % decrease over control Biochemical constituent

Total free amino acids Total proteins Total free sugars Glycogen Total lipids

Liver

Gill

Mantle

Foot

11.81-37.00 43.33-66.67 43.04-58.06 6.94-38.38 6.25-31.25

14.01-42.92 17.65-47.04 28.12-46.87 16.75-62.50 9.09-27.27

14.28-61.90 4.78-28.57 4.08-44.89 7.14-21.42 13.63-45.45

7.14-21.43 4.00-42.85 8.10-59.45 6.67-20.00 11.76-47.05

Varadaraj et al (1994).

Table 3 Effect of different media on SDH activity levels in the hepatopancreas oipila globosa after 10-day exposure Medium

Tannery effluent Potassium dichromate Tannin Sodium chloride

Concentration

% change in SDH activity

10% 2.5 ppm 20ppm 1,000 ppm

-16.43 (P< 0.005) +20.75 (P < 0.05) -19.76 (P< 0.05) -31.16 (P< 0.05)

- indicates % decrease over control. + indicates % increase over control. Guruprasada Rao and Nandakumar (1982).

concentrations of TE (5, 10, 15, 20, and 25%) for a period of 15 days (Subramanian and Varadaraj, 1993). After exposure to 25% tannery effluent for 15 days, the concentrations of total haemolymph proteins, free amino acids and free sugars in the larvae of dragonfly Macromia cingulata exhibit a reduction of about 20% (Varadaraj et al., 1993). Subramanian and Varadaraj (1994) have reported decreased total free amino acids in haemolymph (1.60% to 20.35%), cuticle (9.96% to 26.97%), fatbody (17.90% to 33.46%), and nervous tissue (2.90% to 21.16%) in the larvae of dragonfly Pantala flavescens under sublethal concentrations of TE (1.2, 1.4, 1.6, 1.8, and l.Wc). The effect of TE leading to mortality in the larvae of P. flavescens is found to be dose- and duration-dependent (Subramanian, 1994). In these animals, the tannery effluent reduces total proteins, total free sugars, glycogen, and total lipids, and

370


increases lactic acid and Ach content in vital tissues (Table 4). Activity levels of various enzymes such as protease, LDH, acid and alkaline phosphatases, and lipase increase in the tissues under the influence of sublethal concentrations of TE (1.2, 1.4, 1.6, 1.8, and 2.0%). At the same time, all the tissues exhibit a decreased rate of SDH and acetylcholine esterase (AchE) activities (Table 5).

Table 4 Ranges of percent changes of various biochemical constituents in the tissues of larvae of P. flavescens under sublethal concentrations of tannery effluent

Biochemical constituents/tissue

Haemolymph

Cuticle

Fat body

Nervous tissue

Total proteins* Total free sugars* Glycogen* Total lipids* Lactic acid** Ach*

20.52-52.33 19.57-52.72 — 6.37^3.58 8.04-24.90 9.26-50.00

17.32-53.15 20.00-51.33 23.08-69.23 5.33-50.30 14.28-61.90 13.04-95.65

26.03-67.64 24.48-56.51 10.69-66.63 12.13-36.38 2.41-54.25 25.00-187.50

31.50-65.35 18.92-70.27 9.41-59.22 16.67-47.22 1.38-61.29 13.13-71.05

* Ranges of % decrease over control. ** Ranges of% increase over control. Subramanian, 1994.

Table 5 Ranges of percent changes in the activity levels of enzymes in the tissues of larvae of P. flavescens under sublethal concentrations of tannery effluent

Activity levels of enzymes/tissue Protease** SDH* LDH** Acid phosphatase** Alkaline phosphatase** Lipase** AchE*

Haemolymph

Cuticle

Fat body

Nervous tissue

22.22-66.67 17.11-64.33 21.42-67.00

22.22-100.00 24.88-52.07 15.68-54.90

25.71-97.14 15.85-78.05 13.21-75.47

16.67-83.33 6.72-39.08 8.16-49.00

5.36-35.50

6.45-37.10

13.13-48.67

14.29-71.43

5.56-37.04 7.41-34.57 8.57-60.00

5.94-27.72 4.59-39.08 12.24-53.06

12.87-39.11 6.18-29.45 11.43-60.00

11.76-39.22 30.00-60.00 16.39-70.49

* Ranges of % decrease over control. ** Ranges of% increase over control. Subramanian, 1994.


371

Vertebrates A sublethal concentration of TE (0.2, 0.4, 0.6, 0.8, and 1.0%) is found to cause decreased levels of biochemical constituents in various tissues of the fingerlings of Cyprinus carpio (Varadaraj and Subramanian, 1995c). The percent decrease of total free amino acids ranges between 20.37-28.89, 17.70-48.03, 4.00-38.82, and 25.20-55.20 in gills, intestine, liver, and muscle, respectively; the values for total proteins are 0.46-14.16, 7.53-24.92, 0.52-21.28, and 2.72-15.40; for total sugars 7.78-54.86, 2.29-27.12, 19.14-33.33, and 9.43-40.57; for glycogen content 11.11-55.56, 10.53-36.84, 8.03-57.28, and 12.24-46.26; and for total lipids 16.84-37.76, 1.49-30.21, 0.49-24.27, and 3.72-23.09. Contamination of chromium compounds of TE is shown to cause a high degree of pollution in the waters of irrigation reservoirs (Nandakumar, 1990). Backyavathy and Nandakumar (1992) have recorded pronounced decrease in the activity levels of dehydrogenases, adenosine triphosphatase (ATPase) and AchE in various tissues of albino rats under chromium contaminated irrigation well water (Figure 2). The increase in percent inhibition of the activity levels of dehydrogenases could affect the oxidative metaboUc activity of citric acid, glycolytic pathways, and energy production. The impairment of oxidative phosphorylation due to chromate through inhibition of dehydrogenases and cytochrome system of mitochondria has also been reported by Sigova (1974). Decrement of the activity level of ATPase in the tissues could possibly impair oxidative phosphorylation from ADP to ATP (Berwin et al., 1977; Luciana et al., 1979; Marino et al., 1980). The chromium has been reported to deplete the activity level of AchE disrupting neural transmission in rats (Burmistrov, 1978) and in frogs (Zefirov et al., 1980). The perusal of literature clearly indicates that the toxicants in TE inflict stress on animals. To combat the stress, they have to allocate excessive energy. As a result, they are forced to utilize metabolic reserves, particularly glycogen. The TE causes a shift in the metabolism toward the anaerobic side, leading to the accumulation of lactic acid and the reduction of glycogen reserves and free sugars. At a point of time during stress, metabolism shifts to the utilization of lipid stores as well as tissue proteins, leading to the decrease of these biochemical constituents as well. These results explain the overall decrease in the biochemical parameters in animals, the increase in the levels of lactic acid and LDH activity, and the decrease in SDH activity. The change in the levels of Ach and AchE activity suggests spontaneous and inhibited nervous activity, which would cause fatigue and breakdown of the nervous system. These biochemical alterations would cause the death of animals under TE stress. Subramanian (1994) has recorded Juvenile Hormone Analogue (JHA) mimetic property of TE on dragonfly larvae, and therefore the effluent may be further investigated for application in the control of insects.

372


20

10020

B

10020

K

10020

L

ioo"

M

iiir

Mi -t-.U: '20 100 20

10020

B

K

SDH

10020'

L

20

10020

B

M

10020

K

10020

L

100

M

GDH

Concentration

20

10020

10020

B

MDH

100*

MtVc

:Mni

K

(%)

n-ni:

iod2o

L

AT Paso

100

M

rir i_ 20

B

T 1C020

r 10020

K

TJ|.!.i.>.M-i10020 100

L

M

AchE

C o n c e n t r a t i o n (%)

Figure 2, Enzyme activity levels in tissues of rat under chromium contaminated water. (Backyavathy and Nandakumar, 1992) B—Brain; K—Kldney; L—Liver; M—Muscle; SDH—Succinic dehydrogenase GDH—Guanine dehydrogenase LDH—Lactic dehydrogenase MDH—Malate dehydrogenase ATPase—Âdenosine triphosphatase AchE—Âcetylcholine esterase

REFERENCES Arvind Bharathi, Saxena, R. P., and Pandy, G. N. 1979. "Physiological imbalances due to hexavalent chromium in freshwater algae." Ind. J. Env. Hlth., 21: 243. Backyavathy, D. M., and Nandakumar, N. V. 1992. "Effects of industrial chromium on rat dehydrogenases, ATPase and AchE." Poll. Res., 11 (3): 139-150.


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Backyavathy, D. M., Nandakumar, N. V., and Bhaskar, 1986. "Epidemiological studies on tannery industrial workers exposed to occupational environment." Poll Res., 5 (2): 73-78. Berwin, Yip., Rudolph, P., and Frederick, B. 1977. "Effect of chromium ATP on the association of the reacting forms of yeast hexokinase." J. Biol. Chem., 252 (6): 1,844-1,846. Burmistrov, N. V., Sanitari, Sb. Tr. Nil., and Virieny. 1978. "Determination of the quantitative dose time dependence for hexavalent chromium as a pollutant of wastes of a circulating water supply." Gruz., 14:12-1 A. Eye, J. D., and Lawrence, L. 1971. "Treatment of waste from sole leather tannery." /. Wat. Pollut. Cont. Feb., 43: 2,291-2,303. Gaur, B. L., and Tomar, D. S. 1975. "Effect of salinity on germination of sunflower (Helianthus annuus L.) varieties." Sci. and Culture, 41: 429-430. Guruprasada Rao, M., and Nandakumar, N. V. 1981. "Physicochemical characteristics of tannery effluent-contaminant irrigation reservoir." Ind. J. Env. Hlth., 23.-239-241. Guruprasada Rao, M., and Nandakumar, N. V. 1982. "The tannery industrial effluent effect on succinate dehydrogenase activity pattern in a freshwater snail, Pila globosa." Proc. Indian Acad. Sci. (Anim. Sci.)., 91: 427-481. Guruprasada Rao, M., and Nandakumar, N. V. 1983. "Impact of tannery effluent on seed germinability and chlorophyll content in Cicer arietinum L." Poll. Res., 2 (1): 33-36. Harihara Iyer, C. R., Rajagopalan, R., and Pillay, S. C. 1957. Curr. Sci., 10: 187. ISI 1981. Indian Standards Institution, Indian standard tolerance limits for industrial effluents discharged into inland surface water. IS-2490 (Part I). Kadam, S. D. 1995. "A study on the effect of tannery effluent on the growth of Cicer arietinum." J. of Env. and Poll., 2 (2): 59-62. Luciani, S. Dal., Rebellta, A. M., and Levis, A. G. 1979. "Effect of chromium compounds on plasma membrane magnesium ATPase activity of B cells." Chem. Biol Interest., 27(1): 56-69. Manivasakam, N. 1987. Industrial effluents—origin, characteristics, effects, analysis, and treatment, Sakthi Publications, Coimbatore, TN., India. Marino, D., and Cheland, W. W. 1980. "Investigation of substrate specificity and reaction mechanism of several kinases using chromium (III) adenosine 5' triphosphate." ^/oc/z^m/^^ry, 19(7): 1,506-1,515. Miakhan, M., and Raman, A. 1973. Proc. Symp. on Envi. Pollution, NEERI, Nagpur, India. Nandakumar, N. V. 1990. "Tannery and chromate industrial effluent effect on soil, animals and plants." In: Soil Pollution and Soil Organism. (Ed. P. C. Mishra). Ashish PubHshing House, New Delhi, India, 81-105. Narale, R. P., Subramanyam, T. K., and Mukherjee, R. K. 1969. "Influence of salinity on germination, vegetable growth and grain yield of rice (Oryza sativa var. Dulas)." Agraw. /., 61: 341-344.

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Ogra, R. K., and Baijal, B. D. 1982. "Physiological studies on the effect of salinity on Sorghum. I. Changes in alpha-amylase and acid protease during seedling growth." Ind. J. PL Physiol, 25: 133-140. Ramana, K. V. R., and Ramarao, V. S. 1978. "Physiological studies on the influence of salinity and alkalinity. Changes in growth, respiration, carbohydrates and fats during seedling growth of radish (Raphanus sativus L)." Ind. J. PI Physiol, 21: 93-105. Sastry, C. A., and Madhavakrishnan, W. 1984. "Pollution problems in leather industries of India." Dept. of Environment, Govt, of India Public, 34-35. Saxena, R. M., Keral, P. F., Yadav, R. S., and Bhatnagar, A. K. 1986. "Impact of tannery effluents on some pulse crops." Indian J. Environ. Hlth., 28 (4): 345-348. Sigova, N. V. 1974. "Mechanism and means of rendering the toxic effects of chromium." Yopu. Exsp. Klin. Ter. Profil Prom. InasikatsL 110-115. Subramanian, M. A. 1994. "Studies on the impact of tannery effluent on ecophysiology and biochemistry of the nymphs of dragonfly, Pantala flavescens (Fabricius) (Libellulidae: Anisoptera)." Ph.D. Thesis, Bharathiar University, Coimbatore, TN., India. Subramanian, M. A., Viswanathan, S., and Varadaraj, G. 1988. "A report on the physicochemical parameters of the sewage and industrial effluents in and around Erode." The Indian Zool, 12 (1 & 2): 185-187. Subramanian, M. A., and Varadaraj, G. 1993. "The impact of tannery effluent on the biochemical constituents in the haemolymph of the freshwater prawn, Macrobrachium idella Heller," J. Environ. Biol, 14 (3): 255-259. Subramanian, M. A., and Varadaraj, G. 1994. "Impact of tannery effluent on tissue free amino acids in the larvae of dragonfly Pantala flavescens (Fabricius)." Fraseria, 1 (1): 19-21. Varadaraj, G., Subramanian, M. A., and Jayasuriya, S. 1993. "Sublethal effects of industrial effluents on the biochemical constituents of the haemolymph in the larvae of Macromia cingulata Rambur (Anisoptera: Corduliidae)." Odonatologica, 22 (1): 89-92. Varadaraj, G., Jayasuriya, S., and Subramanian, M. A. 1994. "Toxic effect of tannery effluent on the biochemical constituents in different tissues of Pila globosa." Environment & Ecology, 12 (2): 303-307. Varadaraj, G., and Subramanian, M. A. 1995a. "Studies on groundwater contamination at the vicinity of tanneries near Erode." (Unpublished). Varadaraj, G., and Subramanian, M. A. 1995b. "A study on the physicochemical properties of tannery effluent and its effect on the germination of Vigna radiata (L) Wilezek." (UnpubHshed.) Varadaraj, G., and Subramanian, M. A. 1995c. "Studies on food utilization, oxygen consumption and biochemistry in the fingerlings of common carp Cyprinus carpio on exposure to tannery effluent." (Unpublished.)

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Varma, S. R., and Poonia, S. R. 1979. "Germination and early seedling growth of pearl millet, Pennisetum typhoidis (L) as affected by salinity." Ind. J. PI. Physiol, 22 (2): 144-146. Yusuff, R., and Ismail, S. A. 1984. "Contamination of drinking water in wells by tannery effluents at Pammal (Pallavaram, Madras)." IV Annual Conference of STOX India, P.O. Institute of Basic Medical Sciences, University of Madras, Madras, India, Abs. No. C18. Zefirov, A. L., Poletaer, G. I., and Guselikova, G. G. 1980. "Effect of chromium ions on neuromuscular synapse function." Rifolizile., 25 (1): 115-119.


CHAPTER 17 BLADDER CANCER AND WATER DISINFECTION METHODS Michael A. McGeehin Centers for Disease Control Atlanta, GA 30341-3724 John S. Reif Colorado State University Boulder, CO

CONTENTS INTRODUCTION, 377 WATER CHEMISTRY, 378 Organic Compounds in Source Waters, 378 Disinfection Processes, 378 CARCINOGENICITY OF DISINFECTION BY-PRODUCTS, 381 Trihalomethanes, 381 Haloacetonitriles, 381 Chlorophenols, 382 Summary, 382 BLADDER CANCER EPIDEMIOLOGY, 382 Bladder Cancer Risk Factors, 382 REFERENCES, 390 INTRODUCTION Since early in this century, the chlorination of drinking water has been considered one of the most significant advances in public health by preventing the waterborne transmission of infectious disease. In 1974, it was discovered that chlorine combines with organic materials in water to form halogenated compounds [1]. Some of these compounds are possible human carcinogens [2]. Since this discovery, a number of epidemiologic studies have been conducted to investigate the possible association between cancer and exposure to chlorinated drinking water. Based on the results of early investigations, more recent studies have focused on bladder cancer as the health outcome of interest. Cancer of the urinary bladder accounted for 4.7% of all newly diagnosed cancers in the United States between 1983 and 1987 [3]. Approximately 50,000 cases of bladder cancer occurred in the United States in 1995, almost three-fourths in men [4]. Bladder cancer incidence has been increasing among white males at approximately one percent per year since 1973 and at a slightly lower rate among white females [3]. Although the incidence rate among blacks is lower than among whites, 377

378


it has been increasing faster among members of this racial group [3]. Bladder cancer will claim approximately 11,200 lives nationally in 1995 [4]. Certain risk factors for bladder cancer, such as smoking and occupation, have been studied extensively. The results of numerous investigations have indicated that cigarette smoking increases the risk of bladder cancer approximately two-fold [5]. Investigators conducting a nationwide case-control study of bladder cancer concluded that a moderately increased risk was found for several occupations, but that only 21% of bladder cancer incidence could be attributed to occupational exposure [6]. After accounting for the contribution of smoking and occupation, however, a large portion of bladder cancer cases have no known cause. One of the possible etiologic factors under investigation is exposure to chlorinated water. WATER CHEMISTRY The interest in possible adverse health effects associated with chlorinated drinking water centers on the by-products produced by reactions of chlorine with certain organic compounds present in raw water. Rook's [1] discoveries that chloroform and other trihalogenated methanes (THMs) are formed during the chlorination of drinking water first focused attention on this subject. In subsequent investigations, Trehy and Bieber [7, 8] identified dihaloacetonitriles (DHANs) as other by-products of the chlorination disinfection process with potential implications for human health. The results of these studies caused an acceleration in the development of analytical procedures to isolate, identify, and quantify microorganic drinking water contaminants. Organic Compounds in Source Waters The production of hazardous by-products during the chlorination of drinking water depends on the concentration of organic material in the source water [9]. The majority of organic material in natural waters comes from natural sources through decay of vegetation and animal tissues, photosynthetic by-products, animal excretion, and release of organic matter by plankton and aquatic microphytes [9]. These natural humic substances constitute most of the carbon found in typical surface waters [10, 11]. Disinfection Processes In addition to the natural humic material found in source waters, the production of potentially hazardous by-products during drinking water treatment requires the presence of free chlorine used in the disinfection process. When free chlorine combines with organic carbon precursors, trihalomethanes (THMs) are formed [1, 12]. The four THMs most commonly found in potable water are chloroform, bro-

Bladder Cancer and Water Disinfection Methods

379

modichloromethane, dibromochloromethane, and bromoform [13]. A fifth, dichloroiodomethane, is found less frequently [14]. The concentration of THMs found in treated water is heavily dependent on the concentration of humic material, reaction time, pH, temperature, chlorine dose, and residual chlorine [14-16]. Chlorination remains the most common water disinfection method used in the United States. Alternative methods, such as chloramination, are becoming more prevalent. The basic chemistry of these processes will be reviewed, along with the potential for formation of toxic by-products. Chlorination Chlorination has been the dominant method for disinfecting drinking water for more than 70 years. Chlorine is employed as a disinfectant principally because of its potent germicidal activity, but it is also unique in its ability to maintain a sustained, residual disinfecting action during distribution of the water after treatment. When chlorine gas is dissolved in water, the following chemical reactions occur [17]: CI2 + H2 = HOCl + H^ + CI-

(1)

HOCl = H^ + OCl-

(2)

At the pH normally found in water treatment. Equation 1 proceeds rapidly to the right with complete conversion to dissociated hypochloric acid within a few tenths of a second [17]. Hypochlorous acid (HOCl) is a weak acid and ionizes according to Equation 2. During chlorination, the relative amount of HOCl and OCl (-) (hypochlorite ion), together termed "free chlorine," is a function of the pH of the water [18]. HOCl, a more effective biocide than OCl (-), dissociates into OCl (-) between a pH of 7.0 and 8.0, the range in which most drinking water undergoes treatment [18]. Natural waters have a "chlorine demand" produced by inorganic molecules, organic molecules, and microbiota reacting with and consuming free chlorine ^19]. Addition of chlorine beyond the chlorine demand "breakpoint" produces a freechlorine residual, which forms the basis for determining required amounts of disinfectant [18]. The "breakpoint reaction" involves the reaction of HOCl with ammonia (NH3) to form products known collectively as chloramines [20]. The following reactions are classically used to characterize the formation of chloramines [21]: NH3 + HOCl = NH2 CI + H2O

(3)

NH2 CI + HOCl = NHCI2 + H2O

(4)

NHCI2 + HOCl = NCI3 + H2O

(5)

380


The product in Equation 3 is monochloramine, followed by dichloramine in Equation 4 and trichloramine in Equation 5. Although these chloramines are not as reactive nor as biocidal as hypochloric acid, they are more stable and provide persistent disinfectant activity throughout the distribution system [17]. The chlorination of water supplies involves two steps in the disinfection process. The first is primary disinfection where an agent of sufficient biocidal strength is used to kill any pathogen [17]. HOCl is the only agent formed in the chlorination process with the potency against all forms of pathogenic organisms to fulfill the requirements as a primary disinfectant. The second step is residual disinfection, which involves the maintenance of a small concentration of germicide throughout the distribution system to preserve the integrity of the water supply. Both chloramine and HOCl are suitable for this purpose [17]. Chloramination Chloramination is becoming more widely used as a drinking water disinfection process, primarily because it limits the concentration of trihalomethanes produced when compared to chlorination [22, 23]. Monochloramine produces chlorine substitution into humic material to yield an organic halogen that can be measured using the total organic halogen (TOX) method [24, 25]. The quantity of TOX produced by monochloramine is only 5% to 50% of that produced by an equivalent amount of free chlorine, but the concentration of monochloramine is higher because it is a less-effective disinfectant [25]. In practice, chloramination has been applied in three different ways. Each of these methods results in a water product of different bacteriological and chemical quality [26]. In the first method, marginal chlorination is practiced when chlorine is added to water containing ammonia to generate monochloramine as the primary disinfectant. However, as discussed above, monochloramine is a much poorer disinfectant than chlorine [27]. Marginal chlorination, therefore, may not provide the necessary biocidal activity to prevent bacterial growth in the system [26]. Much greater biocidal activity can be generated if sufficient chlorine is added beyond that needed to remove ammonia from the source water, producing a freechlorine residual [18]. Ammonia is then added to the water after a contact time sufficient to achieve primary disinfection. This process achieves maximum disinfection, but it also produces chlorinated by-products [18]. The third method of chloramination utilizes a solution of preformed monochloramine as both the primary and residual disinfectant [18]. Although the bactericidal quality of this method may be better than marginal chlorination, the poorer disinfection capacity of monochloramines may still pose a problem [28-30]. The reactions between chlorine and ammonia which result in the formation of monochloramine and dichloramine were represented in Equations 3 and 4. The difference in the methodologies is essentially the length of time during which the "free chlorine" is allowed to react with the waterbome pathogens prior to the addition of


381

ammonia. Because "free chlorine" has much greater biocidal activity compared to chloramines, this "free chlorine" reaction period greatly influences the disinfection efficacy of the process. This period, however, is also the time during which the chlorine reacts with organic compounds to form THMs and other potentially harmful halogenated products. CARCINOGENICITY OF DISINFECTION BY-PRODUCTS The reaction of free chlorine with naturally occurring organic substances produces thousands of organic compounds [31]. Approximately 90% of these compounds have yet to be identified and are present in very low concentrations [32]. The remaining 10% have been identified as volatile organic compounds (VOCs), of which trihalomethanes are the major constituent [32]. Meier et al. [33] investigated the mutagenicity of the mixture of compounds resulting from chlorination of surface water and found that more than 90% of the total mutagenicity was attributed to unidentified compounds. Because of the lack of appropriate toxicological information for large numbers of the by-products of water disinfection, this review will concentrate on three categories of volatile organic compounds, with particular emphasis on the trihalomethanes. Trihalomethanes The trihalomethanes are the most widely recognized by-products of the chlorination of drinking water. The most common forms include chloroform, dichlorobromomethane, dibromochloromethane, and bromoform [13]. Of the THMs, the toxicological effects of chloroform have been characterized most completely [34]. The evidence that trihalomethanes are carcinogenic is primarily based on data from bioassays of chloroform. It is clear that chloroform can produce renal tumors in one strain of rats and one strain of mice [35, 36]. When given in an oil-based vehicle, which adds significantly to the lipid intake of the subject, chloroform induced liver tumors in one strain of mice [37]. These results suggest that chloroform may be carcinogenic in humans [18]. In a 2-year rodent bioassay, dibromochloromethane was shown to increase the incidence of liver carcinomas, but not renal tumors, in male mice [38]. Haloacetonitriles The haloacetonitriles that have been identified in chlorinated drinking water have been the dihalogenated chlorinated and brominated derivatives [39]. The investigations into the carcinogenicity of this class of compounds have been limited to four compounds: dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), bromochloroacetonitrile (BCAN), and dibromoacetonitrile (DBAN). All four of these

382


compounds have been shown to be powerful mutagens [2]. However, evidence of carcinogenicity of these chemicals in experimental animals is very limited. Although the haloacetonitriles are the first group of by-products of chlorination other than THMs to show both mutagenic and carcinogenic properties [34], this class of compounds does not seem to represent a serious carcinogenic hazard to humans. There is no evidence available that clearly demonstrates the haloacetonitriles' ability to initiate cancer by an oral route of administration [34]. Chlorophenols Small quantities of chlorinated phenol derivatives result from the chlorination of drinking water [34]. The principal products formed from the reaction of chlorine with humic acid in source water include 4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol [40]. Carcinogenic testing has been done only for 2,4,6-trichlorophenol. High-dose rodent bioassays have shown a dose-related increase in leukemias and lymphomas in male rats and an increase in liver carcinomas in male and female mice [41]. The National Research Council has recommended that further toxicological data be developed for this class of compounds [18]. Summary The lack of certainty concerning disinfection by-products from water chlorination makes it difficult to estimate the overall carcinogenic hazard to humans. However, it is not necessary for each of the chemicals in a mixture of compounds to be identified and assayed before epidemiologic studies investigate the effects of the mixture on human health. There is sufficient toxicological evidence to suspect that by-products of drinking water chlorination may be carcinogenic in humans. Wellconducted epidemiological studies will assist in assessing the human cancer risk that these substances may represent, while the sciences of chemistry and toxicology attempt to assay the individual compounds and their possible interactions. BLADDER CANCER EPIDEMIOLOGY Bladder Cancer Risk Factors Smoking Much of the increase in bladder cancer incidence may be attributable to changing cigarette smoking habits [42]. Analysis of Connecticut incidence rates for bladder cancer shows a close correspondence of trends for bladder cancer with trends for cigarette smoking by birth cohort [43]. Although the increases are not as steep as those for lung cancer, the points trends are essentially the same in each gender [43].


383

The association between tobacco smoking and bladder cancer has been thoroughly investigated. A positive association has been found consistendy in descriptive, case-control, and cohort studies. Ecological studies have found positive correlations of incidence [44] and mortality [45] with cigarette consumption. Cohort analyses of incidence in the United States and Denmark [46] and mortality in England [46-48] showed an association with tobacco consumption in those countries. The epidemiological evidence strongly suggests that cigarette smoking is a risk factor for bladder cancer [49]. Although dose-response was not evaluated in the early investigations, in those studies in which the quantity of cigarettes or duration of smoking was characterized, a dose-response relationship was demonstrated [50, 51]. In general, ex-smokers have elevated risks compared to non-smokers, but the increases are lower than those of current smokers [51]. Cessation had to have been seven to fifteen years before the risk was reduced [49]. The results of these studies suggest that the latency period for bladder cancer with respect to smoking may be as long as 20 years [5]. Occupation Although the association between bladder cancer risk and occupation is one of the most investigated areas in occupational cancer epidemiology, the results remain unclear. Certain occupations have been consistently associated with increased risk; e.g., printers, dye workers, rubber workers [6]. However, numerous studies during the past 30 years have suggested approximately 40 occupations that may have an increased risk for bladder cancer. Most of these studies have been hampered by small numbers of exposed subjects and results that were not statistically significant. It is difficult to compare the various studies' outcomes because each used a different method to classify occupation and different analytical methods were employed. Artificial Sweeteners Collectively, epidemiologic studies have shown no increased risk of bladder cancer in relation to intake of artificial sweeteners. The one case-control study that did show an elevated risk for males showed a protective effect for females [52]. It appears that if the use of artificial sweeteners represents any risk of bladder cancer in humans, it is negligible. Coffee Drinking Epidemiologic investigations of coffee drinking and bladder cancer have produced mixed results. Most of the epidemiological studies have shown slight to moderate increased risk for coffee drinkers with the risks being inconsistent for various subgroups [53, 54]. While it appears that coffee may not play a causal role in the

384


etiology of bladder cancer, there is sufficient evidence to suggest that coffee should be investigated in any study of an association of bladder cancer and risk factors. Hair Dye There is presently no epidemiologic evidence of an association between bladder cancer and hair dye use. This possible risk factor, however, has not been extensively investigated. Water Disinfection Epidemiologic studies investigating the association between exposure to chlorinated surface water and risk of bladder cancer have had mixed results. The early investigations were ecologic studies using various measurements of water quality as surrogate exposure variables. In a review of these early studies. Cantor [55] reported that eight of the eleven ecologic studies showed positive and statistically significant associations between bladder cancer rates and some measure of chlorinated water contamination for white males. Case-control and cohort studies have also been conducted to investigate the association between bladder cancer and drinking water disinfection. Although the results of these studies were not completely uniform, those studies that used an interview to assess exposure and controlled for possible confounders all found an association between bladder cancer and drinking water chlorination. Most epidemiologic studies investigating an association between drinking water disinfection and bladder cancer have had important methodologic weaknesses [56]. The most serious of these is the lack of historical environmental measurements to assess individual past exposures to water contaminants. Ecologic Studies Early studies utilized an ecological design and failed to show a consistent relationship between chlorinated drinking water consumption and deaths from bladder cancer [57-60]. Cantor et al. [57] investigated the association of cancer mortality with trihalomethane levels by analyzing cancer mortality rates by county. Age-standardized cancer mortality rates by sites and sex in whites for the years 1968-1971 were adjusted for socioeconomic, industrial, and demographic factors. The analysis included counties with more than 50% of the population served by the sampled water supply. Positive correlations with THM levels measured by the Environmental Protection Agency were found for bladder and brain cancers among both males and females [57]. Non-Hodgkin's lymphoma and kidney cancer rates were associated with THM levels in males only. Bladder cancer mortality rates in both sexes showed the strongest correlation with THM exposure after controlling for differ-


385

ences in ethnic group, urbanization, region of the U.S., and industrialization of the county. This study provided the first evidence of a possible association between THM levels and surface water chlorination with bladder cancer. Hogan et al. [61] examined the association between chloroform levels in drinking water and cancer mortality data from 1950 to 1969 for all counties in the 48 contiguous states of the United States plus the District of Columbia. The researchers used 20-year average annual, age-adjusted, sex- and race-specific rates for 16 cancer sites. The analysis was restricted to whites only. County estimates from the 1960 census of family income, years of education, and percent of the population which was non-white were included in a regression model to attempt to control for socioeconomic factors. The investigators found associations between potable water chloroform levels and rates of cancer of the large intestine, rectum, and bladder for both sexes. Urinary tract cancer mortality was studied in Houston, Texas, populations exposed to heavily chlorinated and lightly chlorinated drinking water [58]. In 1954, construction of a reservoir changed the type of drinking water supplied to many Houston residents from ground water to surface water. Mortality rates were studied by gender, race, and age cohorts for the period 1940 to 1970. Deaths due to cancer of the urinary tract were compared to mortality rates from respiratory cancer, emphysema, and homicide. Census tracts were classified according to duration of use of surface water. By the 1970s, approximately 20 years following the change to surface water, white females showed an increase in urinary tract cancer mortality rates [58]. A similar effect was not seen among males, nor among the non-white males or females. The investigators had difficulty in classifying exposure because the water supply was changed for each of the study areas gradually. Further, the study could not control for smoking or occupational exposure. The follow-up period may not have been long enough for bladder cancer to become manifest because the maximum induction period allowed was less than 20 years. These ecological studies used existing data bases and evaluated the mortality experience of geographically defined population groups. Exposure at the level of the individual was not evaluated. Case-control Studies—Death Certificates Case-control studies conducted prior to 1980 relied on data obtained from death certificates [61-64]. Exposure to water in each of these studies was classified based on the individual's residence as listed on the death certificate. Excess urinary tract and gastrointestinal cancer deaths were investigated for an association with chlorinated water use in a case-control study of seven New York counties [61]. The study populations included 3,446 deaths due to these cancers from 1968 to 1970 and 3,444 individually matched control deaths from other causes for the same period. Exposure to surface water or ground water and chlorinated or nonchlorinated water was determined based on the residence listed on the death certificate. The

386


researchers attempted to control for occupation by using the occupation listed on the death certificate for a number of randomly chosen cases and controls. The odds ratio for bladder cancer for residence in a county served by chlorinated versus nonchlorinated water was 1.69 for both genders combined (p ^ .025) [61]. The main limitation of this study was that exposure was based entirely on the residence listed on the death certificate, not on a lifetime residential history. An analysis of data from the National Bladder Cancer Study has shown that determining exposure by death certificate residence depresses the odds ratio estimates compared to lifetime residential history [65]. The study was also not able to control for smoking status. A death-certificate-based case-control study design was used to investigate the association of various cancers with water supply among white women in Wisconsin [62]. Cases included deaths for various cancer sites between 1972-1977 in the 28 study counties of eastern Wisconsin. Non-cancer deaths were matched to each case on gender, race, year of death, county of residence, and age. Exposure was assigned on the basis of the water facility that served the population of the town, city, or village listed on the death certificate as the usual place of residence. More detailed information on water quality was obtained through a questionnaire mailed to each water utility superintendent. Logistic regression analysis was used to determine odds ratios for site-specific cancer deaths for high, medium, and low chlorine levels as compared to unchlorinated water exposure. There were no significantly elevated odds ratios for any chlorine dose category in an analysis of 460 bladder cancer deaths. However, classifying lifetime chlorination exposure by the residence listed on the death certificate may have resulted in misclassification of exposure in the Wisconsin study. Exposure misclassification in this study may have been nondifferential, which could have decreased the odds ratios [66]. An investigation in Massachusetts examined the relationship between site-specific cancers and drinking water disinfection methods [64]. Death certificates for approximately 50,000 fatal cancers were compared to those for approximately 215,000 controls who died from cardiovascular, cerebrovascular, pulmonary disease, or lymphatic cancer. Exposure was classified according to residence at death, and the water systems were categorized as chlorinated or chloraminated. After adjusting for age, a moderate increase in risk in exposure to chlorinated water systems was found among bladder cancer deaths compared to controls who died from lymphatic cancer (O.R. = 1.7; 95% CI = 1.3-2.2). No association was found for chlorinated water exposure among bladder cancer deaths when compared to all controls combined (O.R. = 1.1; 95% CI = 0.97-1.14) [64]. None of the death-certificate-based case-control studies utilized interviews to assist in an accurate determination of exposure. The researchers were unable to estimate duration or quantity of exposure. Control for smoking and other potential confounders was generally not possible, and control for possible occupational exposure was limited to county-wide census estimates. Most importantly, malignancies with good long-term survival, such as bladder cancer, may be under-represented in studies based on death certificate data.


387

Cantor has suggested that case-control studies based solely on death certificate information may underestimate the true exposure to carcinogenic by-products of the chlorination process [56]. In an analysis of Iowa data, investigators also found an underestimation of exposure to chlorinated water for bladder cancer cases based on death certificate residence compared to interviewing for lifetime exposure [65]. Case-control Studies—Interview-based More recent studies have improved on the limitations of the earlier analyses. Investigators analyzed data from the National Bladder Cancer Study, a large casecontrol study (2,982 cases and 5,782 controls) conducted in 10 geographic areas of the U.S. [67]. Exposure was determined by interview and based on lifetime residential information, which was combined with water utility data. Logistic regression analysis was used to control for possible confounding by age, smoking, gender, employment in a high-risk occupation, and geographical area. Duration and frequency of exposure were examined by stratification. The analysis was limited to white respondents (2,805 cases and 5,258 controls). The results showed that estimates of bladder cancer risk increased with total tap water consumption among respondents using chlorinated water sources. Study participants using nonchlorinated ground water did not show an increased risk for bladder cancer with tap water consumption. Increasing odds ratios for increasing duration of exposures to chlorinated water was found among women (p < .02) and nonsmokers of both sexes reporting higher-than-median tap water consumption (p = .01) (Table 1). For nonsmokers with an exposure duration of at least 60 years, the odds ratio was 3.1 (95% CI = 1.3-7.3). For female bladder cancer cases with an exposure of at least 60 years, the odds ratio was 3.2 (95% CI = 1.2-8.7) [67]. In a case-control study in Massachusetts, surrogates for 614 individuals who died of bladder cancer and 1,074 individuals who died of other causes were interviewed Table 1 Estimated risk of bladder cancer for nonsmokers with duration of residence with chlorinated surface water for respondents reporting tap water consumption above the median [67]*

# of years

Odds ratio

95% CI

0 1-19 20-39 40-59 >59

1.0 1.1 1.9 2.0 3.1

0.5-2.4 1.0-3.7 1.0-4.1 1.3-7.3

"Âdjusted for age, smoking habit, high-risk occupation, population size of usual residence, reporting center, and sex.

388


about residential and smoking information [68]. Exposure was defined according to duration of residence in communities using either chlorination or chloramination disinfection. Individuals were classified as having "usual exposure" to chlorinated by-products if they spent more than 50% of their lives in communities using chlorinated drinking water and "usual nonexposure" if they spent more than 50% of their lives in communities using chloraminated drinking water (Table 2). An odds ratio of 1.6 (95% CI = 1.2-2.1) was found for bladder cancer among individuals who resided only in communities supplied with chlorinated drinking water compared to those who resided only in communities served with chloraminated drinking water. Participants classified as "usual exposure" had an estimated risk of bladder cancer of 1.4 (95% CI = 1.1-1.8) compared to the "usual nonexposure" group. An historical cohort study was conducted on 31,000 persons in Washington County, Maryland [69]. Three cohorts, each distinguished by a different degree of exposure to chlorinated water, were examined for site-specific cancer incidence rates. Rates for cancer of the bladder in men and cancer of the liver in women were nearly two-fold higher in the group exposed to chlorinated water than in the nonchlorinated cohort. Neither rate elevation was statistically significant. Morris and his colleagues used meta-analytic methods to pool the results of epidemiologic studies and examine the association between chlorination of drinking water and cancer [70]. Relative risk estimates from pertinent case-control and cohort studies were abstracted and combined. The pooled relative risk estimate for consumption of chlorinated water and bladder cancer was 1.2 (95% CI = 1.09-1.34). A population-based case-control study of bladder cancer and drinking water disinfection methods was conducted during 1990-1991 in Colorado [71]. Surface water in Colorado has historically been disinfected with chlorination in some municipalities and chloramination in others. A total of 327 histologically verified bladder cancer cases were frequency matched by age and sex to 261 other-cancer controls. Subjects were interviewed about residential and water source histories. Table 2 Estimated risk of bladder cancer mortality in massachusetts associated with drinking water disinfection process [68]* Exposure duration

Lifetime: chlorine vs. chloramination Usual exposure: chlorine vs. chloramination

OR

95% CI

1.6

1.2-2.1

1.4

1.1-1.8

"Âdjusted for age, sex, cigarette pack-years, residence in community with high risk occupation.


389

This information was linked to data from water utility and Colorado Department of Health records to create a drinking water exposure profile. After adjustment for cigarette smoking, tap water and coffee consumption, and medical history factors by logistic regression, years of exposure to chlorinated surface water were significantly associated with risk for bladder cancer (p = 0.0007). The odds ratio for bladder cancer increased for longer durations of exposure to a level of 1.8 (95% CI = 1.1-2.9) for more than 30 years of exposure to chlorinated surface water compared with no exposure (Table 3). The increased bladder cancer risk was similar for males and females, and for nonsmokers and smokers. In summary, epidemiologic studies of the association between water disinfection and bladder cancer have been of four general types—ecological, case-control mortality, case-control incident, and cohort studies. Each of the studies used a different measure of exposure, and each looked at a different period of exposure. Only three studies were able to control for smoking. Only two studies controlled for high-risk occupational exposure and included interviews with the cases to determine lifetime exposure at the individual level. Despite differences in study design, eight of the epidemiologic investigations reviewed above showed some association between bladder cancer and chlorinated water. Similar findings from independently conducted studies in different populations and geographical locations by independent investigators strengthens the plausibility of the association [72]. All four of the studies that used interviews for lifetime exposure assessment found an association between bladder cancer and chlorinated drinking water. Although the epidemiologic evidence of the association between bladder cancer and chlorination of drinking water certainly does not warrant abandoning chlorination as a water disinfection method, efforts should be made to reduce chlorine concentration and levels of disinfection by-products by protecting water sources and using water filtration for all but the most pristine water sources.

Table 3 Estimated risk of bladder cancer for lifetinfie duration of exposure to chlorinated water in Colorado [71]*

# of years

OR

95% CI

0 1-10 11-20 21-30 >30

1.0 0.7 1.4 1.5 1.8

0.4-1.3 0.8-2.5 0.8-2.9 1.1-2.9

"Âdjusted for sex, age, smoking, tap water intake, family history of bladder cancer, and medical history of bladder infection.

390


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44. Morrison, A. S. "Geographic and time trends of coffee imports and bladder cmcQrr Eur J Cancer, 1978; 14: 51-54. 45. Fraumeni, J. F. "Cigarette smoking and cancers of the urinary tract: geographic variation in the United States." JNCI, 1968; 41: 1,205-1,211. 46. Hoover, R., and Cole, P. "Population trends in cigarette smoking and bladder cmctxr Am J Epidemiol, 1971; 94: 409-418. 47. Armstrong, B., and Doll, R. "Bladder cancer mortality in England and Wales in relation to cigarette smoking and saccharin consumption." Br J Prev Soc Med, 1974; 28: 233-240. 48. Stevenson, R. G., and Moolgavkar, S. H. "Estimation of relative risk from vital data: smoking and cancers of the lung and bladder." JNCI, 1979; 63: 1,351-1,357. 49. Wynder, E. L., and Stellman, S. D. "Artificial sweetener use and bladder cancer: a case-control study." Science 1980; 207: 1,214-1,216. 50. Wynder, E. L., and Goldsmith, R. "The epidemiology of bladder cancer—a second look." Cancer, 1977; 40: 1,246-1,268. 51. Howe, G. R., Burch, J. D., and Miller, A. B. "Artificial sweeteners and human bladder cancer." Lancet, 1977; 2: 578-581. 52. Howe, G. R., Burch, J. D., and Miller, A. B. "Tobacco use, occupation, coffee, various nutrients, and bladder cancer." JNCI, 1980; 64: 701-713. 53. Marrett, L. D., Walter, S. D., and Meigs, J. M. "Coffee drinking and bladder cancer in Connecticut." Am 7 £p/J^m/6>/, 1983; 117: 113-127. 54. Hartge, P., Hoover, R., West, D. W., and Lyon, J. L. "Coffee drinking and the risk of bladder cancer. 7A^C/,1983; 70: 1,021-1,026. 55. Cantor, K. P., and McCabe, L. J. "Epidemiologic studies on the health effects of waterbome carcinogens." In: Proceedings, 1978 annual conference. Denver: Am. Water Works Assoc, 1979. 56. Cantor, K. P. "Epidemiological evidence of carcinogenicity of chlorinated organics in drinking water." Environ Health Perspect, 1982; 46: 187-195. 57. Cantor, K. P., Hoover, R., Mason, T. J., and McCabe, L. J. "Associations of cancer mortality with halomethanes in drinking water." JNCI, 1978; 61:979-985. 58. Cech, I., Holguin, A. H., Littell, A. S., Henry, J. P., and O'Connell, J. "Health significance of chlorination byproducts in drinking water: The Houston experience." M / £/7/J^m/o/, 1987; 16: 198-207. 59. Carlo, G. L., and Mettlin, C. J. "Cancer incidence and trihalomethane concentrations in a public drinking water system." Am J Public Health, 1980; 70: 523-525. 60. Alvanja, M., Goldstein, I., and Susser, M. "Case control study of gastrointestinal and urinary cancer mortality and drinking water chlorination." In: Jolley, R. J., Gorchev, H., and Hamilton, D. H., Jr., eds. Water Chlorination: Environmental Impact and Health Effects. Vol. 1. Ann Arbor: Ann Arbor Science Publ. 1978: 395-409.

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61. Hogan, M. D., Chi, P. Y., Hoel, D. G., and Mitchell, T. B. "Association between chloroform levels in finished drinking water supplies and various site-specific cancer mortality rates." / Environ Pathol Toxicol, 1979; 3: 873-887. 62. Young, T. B., Kanarek, M. S., and Tsiatis, A. A. "Epidemiologic study of drinking water chlorination and Wisconsin female cancer mortality." JNCI, 1981; 67: 1,191-1,198. 63. Gottlieb, M. S., Carr, J. K., and Clarkson, J. R. "Drinking water and cancer in Louisiana: A retrospective mortality study." Am J Epidemiol, 1982; 116:652-667. 64. Zierler, S., Danley, R., and Feingold, L. "Type of disinfectant in drinking water and patterns of mortality in Massachusetts." Environ Health Perspect, 1986; 69: 275-279. 65. Lynch, C. F., Woolson, R. F., O'Gorman, T., and Cantor, K. P. "Chlorinated drinking water and bladder cancer: Effects of misclassification of risk estimates." Arch Environ Health, 1989; 44: 252-259. 66. Breslow, N. E., and Day, N. E. Statistical Methods in Cancer Research, Volume 1—The Analysis of Case-control Studies. Lyon: International Agency for Research on Cancer, 1980. 67. Cantor, K.P., Hoover, R., Hartge, P., Mason, T. J., and Silverman, D. T. "Bladder cancer, drinking water source, and tap water consumption: a casecontrol study." 7A^C/, 1987; 79: 1,269-1,279. 68. Zierler, S., Feingold, L., Danley, R. A., and Craun, G. "Bladder cancer in Massachusetts related to chlorinated and chloraminated drinking water: A case-control study." Arch Env Health, 1988; 43: 195-200. 69. Wilkins, J. R., and Comstock, G. W. "Source of drinking water at home and site-specific cancer incidence in Washington County, Maryland." Am J Epidemiol, 1981; 114: 178-190. 70. Morris, R. D., Audet, A. M., Angelillo, I. F., Chalmers, T. C , and Mosteller, F. "Chlorination and cancer: a meta-analysis." Am J Pub Health, 1992; 82: 955-963. 71. McGeehin, M. A., Reif, J. S., Becker, J. C , and Mangione, E. J. "Case-control study of bladder cancer and water disinfection methods." Am J Epidem, 1993;137:492-501. 72. Cantor, K. P., Kanarek, M. S., and Young, T. B. "Epidemiologic approaches to the assessment of carcinogens in drinking water." In: Ram, N. M., Calabrese, E. J., and Christman, R. F., eds. Organic carcinogens in drinking water. New York: John Wiley and Sons, 1986.

CHAPTER 18 METAL-INDUCED DEVELOPMENTAL TOXICITY IN MAMMALS Jose L. Domingo School of Medicine Rovira i Virgili University 43201 Reus, Spain

CONTENTS SUMMARY, 395 DEVELOPMENTAL TOXICITY OF ENVIRONMENTAL TOXIC ELEMENTS, 396 Arsenic, 396 Cadmium, 397 Lead, 398 Mercury, 399 Uranium, 400 Vanadium, 401 DEVELOPMENTAL TOXICITY OF INDUSTRIAL METALS, 402 Chromium, 403 Cobalt, 403 Manganese, 403 Zinc, 404 DEVELOPMENTAL TOXICITY OF THERAPEUTIC METALS, 405 Aluminum, 405 Gallium, 405 Lithium, 406 REFERENCES, 406 SUMMARY Metals are widely distributed in nature. Elements such as arsenic, cadmium, lead, and mercury are well-known environmental pollutants that have profound effects upon embryonic and fetal development, whereas other metals such as uranium or vanadium are developmental toxicants. Although several metals such as chromium, cobalt, manganese, and zinc are considered essential for mammals, these elements may also represent a serious health hazard as a result of high environmental concentrations derived from a number of industrial activities. On the other hand, metals such as aluminum, gallium, and lithium are examples of current therapeutic elements that also possess potential embryotoxicity and fetotoxicity. The developmental toxicity in mammals of various elements is herein reviewed.

395

396


DEVELOPMENTAL TOXICITY OF ENVIRONMENTAL TOXIC ELEMENTS Toxicities are summarized in Table 1 and are detailed in the text following. Arsenic Although exposure to arsenic (As) compounds may occur from sources such as herbicides, insecticides, rodenticides, paint pigments, and wood preservatives [1], environmental arsenic exposure has received attention primarily because of disease resulting from ingestion of food or water containing arsenic [2]. Most of environmental inorganic arsenic is present as arsenate (As^"^), but arsenite (As^"^) which is more toxic than arsenate, is also found in some cases [3]. Abundant evidence exists for the developmental toxicity of arsenic in the hamster, mouse, and rat [4, 5]. Arsenate teratogenicity has been demonstrated in golden hamsters [6] and rats [7] when given at high doses. In those species, malformations consisted mainly of exencephaly, eye defects, renal agenesis, and gonadal agenesis. Fetal anomalies resulting from intraperitoneal (ip) arsenate treatment of pregnant mice on one of gestation days 6-12, or from oral administration on one of gestation days 7-15, have also been reported [8-10]. It was found that oral dosing with arsenate had less effect on the conceptus than did treatment by ip injection, suggesting that fetal arsenic uptake is more rapid and extensive following ip than oral maternal exposure, with significantly greater peak levels attained [9, 10]. It was suggested

Table 1 Developmental toxicity in mammals of a number of environmental toxic elements: A summary of results

Element Arsenic Arsenate Arsenite Cadmium Lead Mercury Inorganic Methylmercury Uranium (uranyl ion) Vanadium Vanadyl Vanadate

Species

Route

Developmental effects

References

hamsters, mice, rats hamsters, mice, rats hamsters, mice. rats hamsters, rats

oral, ip oral, ip iv, diet, sc iv, ip

Teratogenicity Embryolethality, teratogenicity Embryotoxicity, teratogenicity Embryotoxicity, teratogenicity

[4-10] [4, 5, 12, 14] [22-32] [43, 44]

rats hamsters, cats, rats, mice mice

iv oral, ip

Teratogenicity Embryolethality, teratogenicity

[63] [52-59]

oral, sc

Embryolethality, fetotoxicity (including teratogenicity)

[69-75]

mice

oral

hamsters, rats, mice

oral, ip, iv

Embryolethality, fetotoxicity (including teratogenicity) Embryolethality, fetotoxicity (including teratogenicity)

[78] [79-84]

Metal-Induced Developmental Toxicity in Mammals

397

that the primary effect of arsenate might be to prevent the neural folds from approaching close enough for fusion to occur during critical development periods [11]. With regard to arsenite, the first report that it might adversely affect the conceptus was the study by Hood [12], which found that arsenite treatment in mice resulted in high rates of fetal deaths, and tended to decrease fetal weights when given at 10 or 12 mg/kg on one of days 7-12 of gestation. Exencephaly, micrognathia, open eye, tail defects, and skeletal anomalies of the ribs and vertebrae were the most common defects associated with arsenite treatment. More recently, the effects of orally and ip administered arsenic were compared in the mouse [1] and hamster [13], with oral dosing being significantly less effective. In pregnant mice, trivalent arsenic administered ip or by gavage on gestation day 18 was readily transferred via the placenta and entered the near-term fetuses [14]. Arsenic uptake was significantly greater following maternal ip vs. oral dosing, whereas methylation of the administered arsenic occurs to a considerable extent regardless of treatment made and is presumed to decrease the toxic effect [14]. Embryotoxic effects of arsenite and arsenate on the development of mouse embryos during early organogenesis were also found using the whole embryo culture technique [15]. Moreover, at least one case of human neonatal death has also been ascribed to maternal ingestion of arsenic [16]. With regard to the prevention of arsenic-induced developmental toxicity, subcutaneous (sc) treatment with 2,3-dimercaptopropanol (BAL) diminished the incidence of arsenate-induced gross malformations and growth retardation in mice [17], although sc administration of this chelator was not able to alleviate the embryotoxicity and teratogenicity of arsenite [18, 19]. However, 2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane-l-sulfonate (DMPS) were effective in the prevention of both arsenite- and arsenate-induced developmental toxicity in mice [19-21]. Cadmium In animal models, cadmium (Cd) has been found to be a developmental toxin that can induce defects in the development of the lung, brain, testes, eye, and palate, as well as intrauterine growth retardation and fetal death [22-25]. In rats, a single intravenous (iv) injection of 1.25 mg Cd^'^/kg between days 9 and 15 of gestation resulted in a high incidence of hydrocephalus together with other malformations in the fetuses, while 1.35 mg Cd^"^/kg killed all the embryos. The no-teratogenic-observed-effect level (NOEL) was 1.1 mg Cd^"^/kg [26]. Other threshold doses for embryotoxicity of Cd chloride in rats were 1,000 ppm in the diet and 10 mg/kg by oral gavage [27]. Fetal death in rats was produced by sc injection of CdCl2 (40 jimoles/kg) on day 18 of pregnancy. The high incidence of these deaths (74.9%) following maternal exposure to Cd would not be solely explained by the direct effect of the metal on the fetus, because the fetal toxicity may also be the result of some extra-fetal mechanisms such as maternal toxicity or the observed

398


placental necrosis [24]. Hydrocephalus was the most frequent abnormality when Cd was given between days 8 and 12 of gestation. Other malformations in rats included eye defects, gastroschiasis and umbilical hernia, and forelimb ectrodactyly [26, 28]. The placenta is another target organ for Cd toxicity [25]. In hamsters, the types of malformations depend upon the stage of organogenesis during which Cd exposure occurs, but consisted primarily of craniofacial and limb bud defects when the metal was injected iv [29, 30]. Growth retardation and malformations were also observed in mice [31, 32]. In general terms, the adverse effects of Cd are produced despite limited embryonic and fetal accumulation of the metal. A number of studies showed that the placenta serves as a barrier to the free movement of Cd^"^ to the fetus. It was suggested that many of the teratogenic effects of Cd^"^ might result from damage to the placenta and the disruption of the delivery of nutrients to the fetus [33-35]. The oral administration of alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) (50 mg/kg) protected hamsters against cadmium-induced malformations and embryonic death following a teratogenic dose of cadmium sulfate (2 mg/kg, iv, day 8 of gestation) [36]. The MFA protective mechanism against cadmium-induced teratogenesis in the hamster was mediated by the chelation of the cadmium ion rather than by metallothionein synthesis. A similar conclusion was obtained in a subsequent study, when the relative ability of four dithiocarbamate agents (DTC) to influence the developmental toxicity of parenteral cadmium in the hamster was examined [37]. Lead Early reports indicated that high doses of lead (Pb) could induce abortions in humans [38]. While Pb-induced malformations have been demonstrated in birds, fish and rodents [39], there is no evidence that Pb produces gross malformations in humans [40]. Although it would be possible that fetuses aborted by high-dose exposure to Pb were malformed, there is no evidence supporting this suggestion. The possibility that a number of adverse maternal health conditions can affect the transfer of Pb to the fetus and/or the retention of Pb by the mother or the fetus has received little systematic study [40]. In experimental investigations, negative findings were obtained in studies on Pb acetate for teratogenicity in rats and mice given oral doses of 3.9-390 mg Pb/kg/day during organogenesis [41], whereas Jacquet et al. [42] established that Pb added to the diet of pregnant mice from day 1 of gestation provoked a marked reduction in fertility as well as a retarded growth in utero. Studies by McClain and Becker [43] showed embryotoxic and fetotoxic effects of Pb in rats. The toxic effects depended on the day of gestation that Pb was administered. On day 9, teratogenic effects were observed with few resorptions, while Pb given on day 16 of pregnancy resulted in hydrocephalus and central nervous system hemorrhage. These effects were only observed when Pb was given iv. Very large doses of Pb given orally to rats produced few effects, whereas the ip injection of Pb on individ-


399

ual days of gestation produced up to 100% resorptions, although much fewer and nonspecific malformations were detected [43]. In hamsters, malformations induced by iv administration of Pb were primarily localized within the developing sacral and tail vertebrae, and were characterized by varying degrees of tail malformations ranging from stunting to complete absence of the tail [44]. In mice, ip injection of Pb acetate increased the postimplantation mortality and the rate of skeletal anomalies among the fetuses. These anomalies were restricted to the anterior part of the axial skeleton and consisted essentially of the fusion of two or more cervical vertebrae [45]. Cohen and Roe [46] reviewed 23 teratogenicity studies of Pb acetate or nitrate given orally to rats and mice. No evidence of teratogenic effects was found. The route of administration of Pb appears to play an important role in the specificity of malformations provoked by this metal. Although significant quantities of Pb are transferred into the fetus, the placenta might limit the passage of Pb, because large maternal-fetal concentration gradients exist [43]. An assessment of the teratogenicity and fetal toxicity of a 1:1 molar ratio of lead to four chelating agents—ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), and penicillamine (PEN)—was made in rats at three stages of gestation. The iv administration of lead nitrate with the chelating agents resulted in less embryotoxic and fetotoxic effects than those produced by lead nitrate alone. EDTA produced the greatest reduction, with PEN and NTA intermediate, and IDA resulting in the least overall protective effects [47]. Mercury Agriculture, consumption of fossil fuels, and industry are the main sources of pollution by mercury (Hg), which is discharged into the environment as metallic mercury, as inorganic mercury compounds, or as organic compounds. Inorganic mercury can be transformed into methylmercury by action of microorganisms, particularly in sediments, and this biotransformation in aquatic food chains is recognized as a serious environmental hazard [48]. Methylmercury is known as the main causative agent of Minamata disease, one of the most visible tragedies caused by methylmercury pollution. This was the first known incident in history where the natural bioaccumulation of a toxin (methylmercury) in the aquatic environment killed human beings who ate fish accumulating the contaminant. More than 50 people died, whereas over 700 people around Minamata Bay (Japan) were permanently paralyzed. A high rate of congenital abnormalities was also observed. They included high palates, auditory canal defects, syndactylia, congenital retinochoroidal colobamata, congenital heart abnormalities, congenital pupil malformations, microcornea, microcephalia, hydrocephaha, archynoid cysts, and so forth [49]. Although the placenta may represent a certain barrier to Hg, transfer of alkyl Hg is possible, being more important than that of aryl or inorganic Hg [48]. Methylmercury has been recognized to cause neurologic disturbances and death in

400


human adults and teratological anomalies in newborn infants [50, 51]. Methylmercury has been reported to be embryotoxic and teratogenic in golden hamsters [52, 53], cats [54], rats [53, 55, 56], and mice [55, 57-59]. A high incidence of resorptions and dead fetuses was observed, and cleft palate was the most frequent malformation. Generalized edema, brain lesions, wavy ribs, assymetrical stemebrae, and decreased ossification of parietal and occipital bones were other malformations and developmental variations seen in animals exposed to methylmercury [55, 58, 60]. The inorganic mercury detected in the fetal liver of hamsters after maternal exposure to methylmercury would probably be due to demethylation of methylmercury in the dam and transplacental transfer of inorganic mercury [61]. Mercuric mercury (Hg^"^) not only produces a range of toxic manifestations in adult male and non-gravid female animals [62], but is also fetotoxic, and at lower dose levels may affect embryonic development and cause fetal malformations in pregnant rodents [39]. Holt and Webb [63] suggested that the fetal abnormalities observed in rats given Hg^"^ iv resulted not from any direct action of Hg on the conceptus, but either from the effects of the metal on the transport of essential metabolites from the mother, or the maternal kidney disfunction. In mice exposed to low, non-toxic levels of methylmercury, the deposition of mercury in offspring due to transplacental transport was quantitatively more important than later deposition due to intestinal absorption of mercury from mother's milk [64]. Exposure of guinea pigs to inorganic mercury or methylmercury via breast milk resulted in significant accumulation of mercury in the offspring, the pattern being dependent on the chemical form of mercury given to the dams [65]. The chelating agents DMSA [58], DMPS [66], and 2-mercaptopropionylglycine [67] were effective in alleviating methylmercury-induced teratogenesis in mice, whereas the administration of penicillamine to pregnant rats poisoned with methylmercury prevented the production of morphological changes in the fetal brain [68]. In contrast, the methylmercury-induced teratogenicity was not influenced by the administration of trisodium nitriloacetate (NTA) [56], while the incidence of malformations produced by methylmercury was increased by treatment with N-acetylcysteine (NAC) [57]. Uranium The best known use of uranium (U) is as a fuel in nuclear power reactors and nuclear weapons. The increasing role of this metal in nuclear industry results in an increase in occupational exposure to uranium and puts the general population at risk for chronic exposure to low levels of uranium either by inhalation or by dietary intake. Although uranium can exist in the oxidation states of +3, +4, +5, or +6, in solution the uranyl ion (U02^"^) is the most stable species and the form in which this element is present in the mammalian body [69]. In recent years, it has been demonstrated that uranium is a developmental toxicant when given orally or sc to mice [4, 69]. Teratogenic effects of U were found in


401

mice when uranyl acetate dihydrate (UAD) was given by gavage at 5, 10, 25, and 50 mg/kg/day on gestational days 6-15 [70]. Fetal toxicity consisted primarily of reduced fetal body weight and body length as well as an increased incidence of malformations (cleft palate, bipartite sternebrae) and developmental variations (reduced ossification and unossified skeletal variations), while in contrast, there was no evidence of embryolethality at any dosage level. In a subsequent study, UAD was given orally to pregnant mice at daily doses of 0.05, 0.5, 5, and 50 mg/kg from day 13 of pregnancy until weaning of the litters on day 21 post-birth. Although U exposure had no significant effect on sex ratio, mean litter size, pup body weight, or pup body length throughout lactation, significant decreases in the mean litter size on postnatal day 21 and in viability and lactation indices were observed at the 50 mg/kg/day dose level [71]. No adverse changes on fertility of mice were found when UAD was tested for its effects on reproduction, gestation, and postnatal survival. Mature male mice were orally treated with UAD (5, 10, and 25 mg/kg/day) for 60 days prior to mating with mature virgin female mice treated orally for 14 days prior to mating with UAD at the same doses as those of males. Significant increases in the number of dead young per litter were seen at birth and at day 4 of lactation in the 25 mg/kg/day group, whereas the growth of the offspring was always significantly lower for the U-exposed animals [72]. The effects of multiple maternal sc injections of UAD (0.5, 1, and 2 mg/kg/day) from day 6 to 15 of gestation were also evaluated in mice [73]. Although not doserelated, embryotoxicity occurred in all U-exposed groups. Fetal body weight was significantly decreased at 1 and 2 mg/kg/day, whereas the number of total internal and skeletal defects showed dose-dependent increases at 0.5, 1, and 2 mg/kg/day. Most morphological defects were developmental variations, while malformations (cleft palate) were only detected at 1 and 2 mg/kg/day. Most of these anomalies were not resulting from maternal toxicity [74], and they were probably the primary effects of the developmental toxicity of U. Investigation of whether the day of exposure would modify the embryo/fetal toxicity of U in mice showed that gestational day 10 would be the most sensitive time for U-induced developmental toxicity [75]. When the ability of Tiron, an effective chelator in acute uranium poisoning, to protect the developing mouse fetus against uranium-induced developmental toxicity was assessed, the results offered only modest encouragement with regard to its possible therapeutic potential for pregnant women exposed to this heavy metal [76]. Vanadium Vanadium (V) occurs at relatively high concentrations in crude oils and coals. Combustion of these fuels constitutes the major source of vanadium emissions into the atmosphere. Other possible sources of vanadium contamination are effluent discharges from titanium and uranium processing plants. Consequently, in the non-

402


industrial community, vanadium exposure in humans arises primarily from its environmental presence. Vanadate (V^"^) is the most stable form of vanadium at neutral pH in the presence of oxygen. However, reducing environments readily convert vanadate to vanadyl (V^"^). Although vanadate is more toxic than vanadyl, oxidation states IV and V interconvert easily under physiological conditions. It has been demonstrated that both vanadate and vanadyl cross the placental barrier and reach the mammalian conceptus [77, 78]. Vanadate has been found to cause developmental toxicity in pregnant hamsters, rats, and mice, whereas vanadyl exposure causes embryotoxic and teratogenic effects in mice. The ip exposure to ammonium vanadate (0.47, 1.88, 3.75 mg/kg/day) of pregnant Syrian golden hamsters from gestation days 5 to 10 resulted in micrognathia, supernumerary ribs, and alterations in stemebral ossification, while embryolethality was not observed at any dosage level [79]. Also, the iv administration of 0.15 ml of V2O5 1 mM injected in mice on day 8 of pregnancy caused a low degree of skeletal ossification without increasing the number of nonviable implants [80]. On the other hand, the oral administration of vanadyl sulfate pentahydrate (37.5, 75, 150 mg/kg/day) to pregnant mice during organogenesis caused maternal toxicity, embryofetotoxicity, and teratogenicity (cleft palate, micrognathia) at all dose levels tested [78], while the NOEL for maternal toxicity and for developmental toxicity were, respectively, 7.5 and 15 mg/kg/day of sodium orthovanadate when this compound was given by gavage to mice on gestational days 6-15 at doses of 7.5, 15, and 30 mg/kg/day [81]. A significant increase in the number of resorptions and dead fetuses was also observed when sodium metavanadate (NaV03) was administered orally to rats (20 mg/kg/day) during organogenesis [82]. Fetotoxicity (decreases in fetal body weight) and teratogenicity (increases in the incidence of cleft palate) were also found when NaV03 was injected ip to Swiss mice during organogenesis [83], with gestational day 12 being the most sensitive time for NaV03-induced developmental toxicity in mice [84]. The chelating agent sodium 4,5-dihydroxybenzene-l,3-disulfonate (Tiron) has been reported to ameliorate the vanadate-induced developmental toxicity in mice [85]. There was a significant decrease in the number of resorbed fetuses, an increase in the mean fetal weight, and a reduction in the incidence of the skeletal variations (reduced ossification of supraoccipital, parietal, metacarpals, and metatarsals) caused by vanadate. DEVELOPMENTAL TOXICITY OF INDUSTRIAL METALS Toxicities are summarized in Table 2 and are detailed in the text following.


403

Table 2 Developmental toxicity in mammals of various industrial metals: a summary of results Element Chromium Cobalt Manganese Zinc

Species

Route


References

hamsters, mice hamsters, rats mice rats, sheep

oral, ip oral sc diet

Embryotoxicity, teratogenicity No remarkable embryo/fetal effects Embryotoxicity, fetotoxicity Embryotoxicity

[86-88] [90, 93] [98, 99] [103, 104]

Chromium The major toxic action of chromium (Cr) is due to its hexavalent form, which acts as a strong oxidant. Chromium (VI) crosses the placenta in humans, as evidenced by elevated concentrations in the newborns. In hamsters, chromium trioxide (Cr03) produced malformations in 64% of the pregnant animals receiving a parenteral dose of 15 mg/kg [86]. However, no malformations or developmental variations were observed in mice treated ip on day 9 of gestation with 15 mg Cr03/kg [57]. Embryonic and fetal developmental changes in mice receiving chromium (VI) in drinking water from conception to day 19 of gestation were also found [87], while gestational weight gain of dams, fetal weight and crown-rump length were decreased when female mice received 500 or 750 ppm chromium (VI) as potassium dichromate in drinking water on days 14 to 19 of pregnancy [88]. Cobalt Although cobalt (Co) is an essential trace element, excess dietary cobalt produces toxic effects in mammals [89]. Early teratogenesis tests of Co salts were negative in hamsters [90], whereas Co (II) chloride caused cleft palate and delayed ossification in mouse embryos [91]. When C0CI2 was administered in the drinking water of male rats for 60 days prior to mating, and female rats for 14 days [92], by gavage (25, 50 and 100 mg/kg/day) on days 6 to 15 of gestation [93], or from the 14th day of gestation through 21st day of lactation [94], no embryotoxic or fetotoxic effects were observed at doses as high as 100 mg/kg/day. However, cobalt affected the period of late gestation as well as the postnatal survival and development of the pups [92, 94]. Manganese IVIanganese (JMn) is also an essential nutrient. However, an excess of this element in an individual's environment can produce devastating results. For select popula-

404


tions of humans, in addition to CNS pathologies, Mn toxicity (acute and chronic) can influence lung, liver, kidney, pancreas, and reproductive function [95]. Developmental toxicity of Mn may also be a serious health hazard for pregnant women living in high-exposure environments. Experimental studies have shown that although administration of Mn during pregnancy did not result in structural malformations in rats, mice, and hamsters, ip exposure during the organogenic period in hamsters caused embryolethality [90, 96, 97]. Unlike the fetus, it has been reported that the neonate seems to be particularly vulnerable to high levels of manganese [97]. Recent investigations also showed embryotoxic and fetotoxic effects in mice following sc exposure to 2, 4, 8, and 16 mg/kg/day of manganese chloride tetrahydrate from gestation day 6 through 15. Embryotoxicity was evidenced by significant increases in the number of late resorptions as well as in the percentage of postimplantation loss. Fetotoxicity consisted primarily of reduced fetal body weight and an increased incidence of skeletal defects. No teratogenic effects were seen in any of the groups [98]. Although the mouse conceptus would be adversely affected by parenteral exposure to Mn in any of the gestational days, mice would be more susceptible to Mn-induced embryo/fetal toxicity on days 9 and 10 of gestation [99]. Zinc Zinc (Zn) is a common metal in the human environment and constitutes an important trace element intervening in many biological processes [100]. Postulated mechanisms for zinc deficiency-induced abnormalities include: reductions in protein and/or nucleic acid synthesis, abnormal microtubule polymerization, free radical damage, altered gene expression, and altered cell cycles with subsequent distortion in morphogenesis [101, 102]. Little is known on the potential developmental toxicity of Zn to human subjects, but it was reported that of four women dosed orally with 300 mg Zn/day during pregnancy, three gave birth prematurely and the child of a fourth was stillbom [103]. Zinc concentrations of up to 5,000 mg/kg in the diet of pregnant rats induced hypocuprosis in the fetuses and caused a high incidence of stillbirths and fetal resorptions [104], toxic effects which were also observed in pregnant sheep receiving 750 mg Zn/kg diet [103]. In mice, ip Zn administration (20 mg/kg) on days 8, 9, 10, or 11 of gestation produced a delay in development and some abnormalities in fetal ossifications [105]. However, iv administration of Zn sulfate to pregnant golden hamsters at 2 mg/kg on day 8 of gestation did not reveal a pattern of malformations in the embryos [90], whereas a slight increase in the frequency of hydrocephalus in rat embryos was found following excess Zn in the maternal diet [106].


405

Table 3 Developmental toxicity in mammals of some therapeutic metals: A summary of results Element/ compound

Species

Route


References

Aluminum hydroxide nitrate Gallium nitrate Lithium

rats, mice rats mice rats, mice

oral oral ip oral

No embryo/fetal effects Fetotoxicity Embryotoxicity, fetotoxicity Fetotoxicity

[108, 109] [112] [115] [117, 120]

DEVELOPMENTAL TOXICITY OF THERAPEUTIC METALS Toxicities are summarized in Table 3 and are detailed in the text following. Aluminum It is well established that aluminum (Al) is a developmental toxicant when administered parenterally [107]. However, until recently there was little concern about embryo/fetal consequences of aluminum ingestion because bioavailability was considered low. The importance of the route of exposure and the chemical form of the aluminum compound on the developmental toxicity of this element are now well established. Although no evidence of maternal and embryo/fetal toxicity was observed when high doses of aluminum hydroxide were given orally to pregnant rats and mice during organogenesis [108, 109], signs of maternal and developmental toxicity were found in mice when aluminum hydroxide was given concurrently with citric [110] or lactic acids [HI]. In contrast to those studies, oral administration of Al nitrate nonahydrate (13, 26, and 52 mg Al/kg/day) to pregnant rats on days 6-14 of gestation resulted in decreased fetal body weight and increased incidence and types of external, visceral and skeletal abnormalities in all the Al-treated groups [112]. On the other hand, studies in rabbits have shown that aluminum-induced behavioral toxicity is greater in adult and aged animals than in young adults [107, 113]. Maternal dietary exposure to excess Al during gestation and lactation, which did not produce maternal toxicity, would be capable of causing permanent neurobehavioral deficits in weanling mice and rats [113]. Gallium There are very few data concerning the developmental toxicity of gallium (Ga). When this element was injected iv to pregnant hamsters as Ga sulfate at 40 mg/kg

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on day 8 of gestation, only three embryos from Ga-treated mothers showed mild malformations consisting of one limb bud abnormality, one case of spina bifida, and one mild exencephaly [114]. In a subsequent study, maternal toxicity was observed in mice given ip gallium nitrate at 12.5, 25, 50 and 100 mg/kg/day on days 6, 8, 10, 12, and 14 of gestation. Embryo/fetal toxicity was also evidenced at all doses by a decrease in the number of viable implants, a reduction in fetal weight, and an increase in the number of skeletal variations (delayed ossification of parietal and occipital, dorsal hyperkiphosis, wavy ribs), whereas no significant teratogenic effects were observed. The NOEL for both maternal and developmental toxicity of Ga nitrate was < 12.5 mg/kg [115]. Lithium Lithium (Li) carbonate is used in the treatment of manic depressive psychosis and other psychiatric disorders. Hence, the knowledge of the teratogenic potential of this element has been important. Although lithium-related congenital abnormalities in rats were reported by Gralla and Mcllhenny [116], a number of anomalies were found by other investigators after administration of Li salts [117-119]. Reductions in number and weight of the litter, increase in the number of resorptions, wavy ribs, short and deformed bones of the limbs, or an increased incidence of incomplete ossifications of sternebrae, thoracic vertebrae, phalanges, and metatarsal and metacarpal bones were the most remarkable effects caused by Li when given orally to rats [117, 120]. Exencephaly, fused ribs, and defective vertebra were the morphologic defects found in mice after oral administration of Li carbonate during pregnancy [118]. There is also sufficient evidence that lithium, when administered for therapy, causes developmental toxicity in offspring of humans who have major affective disorders. Data from registries, prospective studies, and case histories of women who used lithium therapy during their pregnancies indicate that lithium causes developmental toxicity in some offspring [121, 122]. However, recent epidemiological data indicate that the teratogenic risks of first-trimester lithium exposure is lower than those previously suggested [123]. REFERENCES 1. Baxley, M. N. et al. "Prenatal toxicity of orally administered sodium arsenate in mice." Bulletin of Environmental Contamination and Toxicology, 26,1981,749-756. 2. Franzblau, A., and Lilis, R. "Acute arsenic intoxication from environmental arsenic exposure." Archives of Environmental Health, 44, 1989, 385-390. 3. Vahter, M. "Biotransformation of trivalent and pentavalent arsenic in mice and rats." Environmental Research, 25, 1981, 286-293.


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87. Trivedi, B. et al. "Embryotoxicity and fetotoxicity of orally administered hexavalent chromium in mice." Reproductive Toxicology, 3, 1989, 275-278. 88. Junaid, M. et al. "Chromium fetotoxicity in mice during late pregnancy." Veterinary and Human Toxicology, 37, 1995, 320-323. 89. Domingo, J. L. "Cobalt in the environment and its toxicological implications." Reviews of Environmental Contamination and Toxicology, 108, 1989, 105-132. 90. Perm, V. H. "The teratogenic effects of metals on mammalian embryos." Advances in Teratology, 6, 1972, 51-75. 91. Kasirsky, G. et al. "Inhibition of cortisone-induced cleft palate in mice by cobaltous chloride." Journal of Pharmaceutical Sciences, 56, 1967, 1,330-1,332. 92. Paternain, J. L. et al. "Estudios en ratas de los efectos producidos por la administracion oral de C0CI2 sobre la fertilidad, gestacion, parto y lactancia." Revista de Toxicologia, 2, 1985, 93-103. 93. Paternain, J. L. et al. "Developmental toxicity of cobalt in the rat." Journal of Toxicology and Environmental Health, 24, 1988, 193-200. 94. Domingo, J. L. et al. "Effects of cobalt on postnatal development and late gestation in rats upon oral administration." Revista Espanola de Fisiologia, 41,1985,293-298. 95. Keen, C. L., and Zidenberg-Cherr, S. "Manganese," in Present Knowledge in Nutrition, M. L. Brown (Ed.), Washington D.C.: ILSI, Nutrition Foundation, 1990, 279-286. 96. Laskey, J. W. et al. "Effects of chronic manganese (manganese oxide) exposure on selected reproductive parameters in rats." Journal of Toxicology and Environmental Health, 9, 1982, 677-687. 97. Webster, W. S., and Valois, A. O. "Reproductive toxicology of manganese in rodents, including exposure during the postnatal period." Neurotoxicology, 8, 1987, 437-444. 98. Sanchez, D. J. et al. "Maternal and developmental toxicity of manganese in the mouse." Toxicology Letters, 69, 1993, 45-52. 99. Colomina, M. T. et al. "Effect of the day of exposure on the developmental toxicity of manganese in mice." Veterinary and Human Toxicology, 38, 1996, 7-9. 100. Leonard, A. et al. "Mutagenicity, carcinogenicity and teratogenicity of zincr Mutation Research, 168, 1986, 343-353. 101. Keen, C. L., and Hurley, L. S. "Zinc and reproduction: Effects of deficiency on fetal and postnatal development," in Zinc in Human Biology, C. F. Mills (Ed.), New York: Springer-Verlag, 1989, 183-220. 102. Peters, J. M. et al. "Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos." Proceedings of the Society for Experimental Biology and Medicine, 198, 1991, 561-568.


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120. Hoberman, A. L. et al. "Developmental toxicity study of orally administered lithium hypochlorite in rats." Journal of the American College of Toxicology, 9, 1990, 367-379. 121. Weiner, M. L. "Overview of Lithium Toxicology," in Lithium in Biology and Medicine, G. N. Schrauzer (Ed.), New York: VCH, 1991, 83-99. 122. Moore, J. A. et al. "An assessment of lithium using the lEHR evaluative process for assessing human developmental and reproductive toxicity of 2igtni%r Reproductive Toxicology, 9, 1995, 175-210. 123. Cohen, L. S. et al. "A reevaluation of risk of in utero exposure to lithium." The Journal of the American Medical Association, 111, 1994, 146-150.

CHAPTER 19 GENETIC EVALUATION OF PESTICIDES IN DIFFERENT SHORT-TERM TESTS Patrizia Hrelia Department of Pharmacology University of Bologna Bologna, Italy

CONTENTS INTRODUCTION, 415 THE ASSESSMENT OF GENOTOXICITY OF PESTICIDES, 416 MUTAGENICITY TESTING OF PESTICIDES: EXPERIENCE WITH OLD COMPOUNDS, 417 NEW APPROACHES TO THE STUDY OF PESTICIDES, 422 Integration of Cytogenetic Assays with Biochemical and Metabolism Studies, 422 Potential to Detect Cytogenetic Damage by Using Molecular Cytogenetic Techniques, 427 CONCLUSIONS, 427 REFERENCES, 428 INTRODUCTION The high level of agricultural productivity that has been achieved during the last half-century is due, at least in part, to the availability of a wide range of pesticides, e.g., fungicides, herbicides, insecticides and other plant protection products. Moreover, pesticides are used domestically in wood preservation or as household insecticides. A principal goal in pesticide research and development is identifying the specificity of harmful action of an agent toward life forms. However, it has to be recognized that many pesticides may be toxic to organisms other than those they are intended to control, and as such may constitute a potential health hazard to livestock, domestic animals, wildlife, and man. The impact of pesticides on the health of agricultural workers and consumers is still largely unknown, even if pesticides are extensively studied from a toxicological point of view. Continued attention is focused on pesticides applied to crops or soil because of the large amount of these chemicals currently in use, the extent of field worker exposure and the possibility of direct entry of pesticides into the food chain. Clinical poisoning by residues seems to be extremely rare, and acute poisoning seems likely to be a very uncommon occurrence.

415

416


Cancer and the induction of hereditary damage represent the stochastic effects of agricultural chemicals and are regarded as the principal risk to health. Such effects are of special concern because of the generally irreversible nature of the processes and the long latency associated with their manifestation. Damage to germ cells may result in a genetic mutation expressed in a later generation; if pesticides induce lesions in critical genes of somatic cells, the result may be cancer induction in the exposed individual. Pesticide induced lesions in cells may be manifest at any level of organization of the genetic material, i.e., the chromosome, the gene, or the primary DNA level. Chromosome aberrations and gene mutations are both part of a complex and overlapping spectrum of genetic events, but have different genetic consequences and are induced in different proportions by different chemical agents. In some cancers, oncogenes are commonly activated by chromosome aberrations, whereas in others gene mutations are responsible [1]. Clear examples are now available of each of these mutational changes in different tumors, and these provide critical support for the somatic mutations theory of carcinogenesis [2, 3, 4]. These observations support the use of mutagenicity assays in the evaluation of carcinogenic risks of chemicals to humans and in the elucidation of mechanisms of action of known carcinogens. THE ASSESSMENT OF GENOTOXICITY OF PESTICIDES The use of short-term tests for mutagenicity is an important tool for identification of chemicals with the potential of posing a threat to human health. A large number of genotoxicity tests are presently available, and in order to assess adequately any expression of genotoxicity, a simplified systematic approach to the selection of these tests is required. In the early 1970s, Bruce Ames popularized that all mutagens were carcinogens [5, 6], but this optimistic relation between genotoxicity and carcinogenicity was clearly demonstrated to be incorrect. It was shown that mutagenicity is not always synonymous with carcinogenicity, and that in vitro conditions do not adequately mimic the metabolism and physiology of animal models or human exposure [7]. The activity of genotoxic carcinogens in the Ames test and in other tests for genotoxicity provide supports for discouraging reliance solely on the Salmonella assay. There is a genetic specificity of mutagenic action [8], and the induction of damage by chemicals can be specific or preferential for one or other genetic endpoints. As mentioned before, two basic categories of endpoints, gene mutations and chromosome alterations are believed responsible for the induction of somatic (including carcinogenics) as well as heritable mutations leading to genetic disorders in offspring. For a newly developed chemical, such as a pesticide, or for a hitherto unstudied "existing" chemical, the process of ascertaining its significant primary mutagenicity and potential genotoxic carcinogenicity usually involves the conduct of, at most, four or five genetic toxicity assays [9]. Because the reliability of risk evaluation

Genetic Evaluation of Pesticides in Different Short-Term Tests

417

mainly depends on the quality of testing, adherence in test protocols to the current state of the art is obligatory. In Europe, EEC has recently been revising its recommendations for design of studies [10]. The protocols usually to be followed are those of OECD [11, 12]. In the United States, the USEPA developed a set of guidelines [13, 14]. The harmonization of testing methods will ensure mutual acceptance of data in the international community. MUTAGENICITY TESTING OF PESTICIDES: EXPERIENCE WITH OLD COMPOUNDS In this very problematic field, regulatory authorities have to be concerned with the evaluation of safety of so called "old" compounds. Many of these compounds were not subjected to systematic toxicological investigations, and therefore risk assessment has to be made on the basis of incomplete data. Mutagenicity data are part of the weight-of-evidence approach for classifying potential human carcinogens [15]. The basis of the approach is to evaluate a pesticide on the basis of results from a range of functionally distinct in vitro assays (e.g., gene mutations, chromosome mutations, DNA damage), and then to assess individual activities in in vivo assays with the same genetic endpoint. Moreover, most inadequate information is published, and the evidence that does exist is mostly based on non-mammalian bioassays. In several studies, technical-grade chemicals were used, and therefore it is possible that the reported genotoxic activity may be related to the presence of contaminants. The observation of positive results in vivo is regarded as defining a potential hazard, while activity that is evident only in vitro is regarded with circumspection and may even be completely ignored [16, 17, 8]. Negative and dubiously positive results from insufficiently performed tests usually require reinvestigation. Results of such tests could only be considered if they are clearly positive. In recent studies, we investigated the genetic safety of a series of pesticides with a relevant economic importance and widespread agricultural use: cyanazine, cyhexatin, dicamba, and DNOC (see Table 1 for chemical structure). Insufficiently performed or poorly documented tests covering in vitro and in vivo toxicological assays were submitted, and therefore further tests were required to enable risk/benefit decision. As an example, the oncogenic potentials of cyanazine and dicamba were negative in a feeding study on rats and mice [18, 19], but no data are available on the carcinogenic potential of DNOC. Cyhexatin did not cause increases in tumor incidence in rats and mice [20, 21]. Nevertheless, criticisms exist about experimental protocols of these bioassays. Scanty and often inconclusive and conflicting results are available in the open literature on genetic activity (Table 2). Most of the data come from mutation assays with bacteria and yeasts, but very few results on induced chromosomal damage are available. We concentrated our efforts on genetic endpoints evaluated in the whole

418

Health and Toxicology Table 1 Chemical structure of pesticides tested

Chemical

Cyanazine (H)

CAS No.

21725-46-2

Structure CH

1

^W N

N

NH-CH,CHj

Cyhexatin (A)

13121-70-S

Dicamba (H)

1918-00-9

DNOC (I)

534-52-1

lO-l

SnOH

COOH

OH 02N^X,CH,

¥ NO2

Fenarimol (F)

60168-88-9

Vinclozolin (F)

50471-44-8

Metalaxyl (F)

57837-19-1

^

^

I ^N

y\J^zH=cH,

Cl

32809-16-8

23564-05-8

H, herbicide; A, acaricide; /, insecticide; F, fungicide

CHC0.OCH3

t r'"' 0

-f"'

^ "^H N >=/ V- ^ c - ,

Ct

Thiophanate-methyl (F)

1

CH3OCHJC0 CHj

Procymidone (F)

OH

fY

0

NHCS.NHCO.OCH

K. Jl NHCS.NHCO.OCH

419

Genetic Evaluation of Pesticides in Different Short-Term Tests Table 2 Summary of available results on the genotoxicity of pesticides tested Compound

Test organism

Genetic effect

Cyanazine

S. typhimurium

Gene mutations with mammalian activation Gene mutations with plant activation Gene mutations Chromosome aberrations

-

[24, 26] [24]

+

[25]

Cytological effects

+

[27]

S. cerevisiae Human peripheral lymphocytes in vitro Viciafaba and tradescandia

Result

Reference

[23] +

Cyhexatin

S. typhimurium

Gene mutations

-

[23]

Dicamba

S. typhimurium S. typhimurium

Gene mutations Gene mutations with plant activations

-

[28, 29]

+

-

[24] [24]

+ + +

[24] [29, 30] [29, 30]

-

[29] [28, 29]

Maize S. cerevisiae Bacillus subtilis rec A Escherichia coli pol A Drosophila melanogaster Human lung fibroblasts Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat liver in vivo DNOC

Drosophila melanogaster Proteus mirabilis Hela cells Mouse in vivo Human peripheral lymphocytes in vitro

Gene mutations with mammalian activation DNA damage DNA damage Sex-linked recessive lethal mutations UDS SCE UDS DNA strand breaks Sex-linked recessive lethal mutations DNA damage UDS DNA strand breaks Chromosome aberrations Chromosome aberrations

[31] -H -h

+ + +

+ +

[31] [31] [35] [36] [37] [37] [38] [38]

420


animal and in in vitro systems that utilize human cells. Chromosomal aberrations are direct indicators of genetic damage and serve as excellent markers to assess the genotoxic and clastogenic potential of pesticides. Description of each bioassay system, procurement of pesticides, purity, and qualitative aspects of pesticide data were reported previously [22]. Either the lowest effective dose or the highest dose tested that produced no effect was recorded for each test agent and bioassay system. For each negative result, the highest dose studied was termed the highest ineffective dose (HID). Similarly, for positive results, the lowest effective dose (LED) was recorded. The doses or concentrations for all in vitro tests are expressed as pig/ml, and these for in vivo tests were reported in mg/Kg body weight (Table 3). Cynazine was uniformly negative in three tests for genotoxicity: unscheduled DNA synthesis (UDS) and sister chromatid exchanges (SCE) in cultured human

Table 3 Results on the genotoxicity of pesticides tested

Compound (purity >99%) Cyanazine

Cyhexatin

Test organism Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat in vivo Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat in vivo

Dicamba

Mouse in vivo

DNOC

S. typhimurium TA98, TAIOO, TA97, TA102 Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat in vivo

Genetic effect

Results^ Metabolic activation with without

Dose*' LED/HID

UDS

100

SCE

100

Chromosome aberrations UDS

224 50

SCE

25

Chromosome aberrations Chromosome aberrations Reverse mutations

160 832 1,000

UDS

100

SCE

50

Chromosome aberrations

15

"+, positive; -, negative În vitro tests, \ig/ml; in vivo tests, mg/Kg b.w. LED: lowest effective dose; HID: highest ineffective dose.

Genetic Evaluation of Pesticides In Different Short-Term Tests

421

lymphocytes, and in vivo cytogenic assay in rat bone marrow. These results are in agreement with reported data on microorganisms [23, 24]. The reported clastogenic response in human lymphocytes based only on a doubling of break frequency at one dose level [25] is regarded with circumspection. Altogether this evidence establishes that cyanazine did not exert genotoxic activity in experimental systems. Nevertheless, clear evidence of a specificity of mutagenic action which depends upon plant activation was obtained in three studies [24, 26, 27]. Cyhexatin was reported to be non-mutagenic in Salmonella [23]. Negative results on the induction of UDS in human lymphocytes and of chromosome damage in rats endorse that the observed induction of SCEs (Table 3) cannot be regarded as providing evidence of mutagenicity for cyhexatin. An extensive number of in vitro data sets exist for dicamba. Conflicting results were obtained in microbial assay systems with mammalian or plant activation [28, 29, 24]. Dicamba was found positive in Bacillus subtilis rec A and in E. coli pol A assay [29, 30], in human lymphocytes in vitro by means of SCE stimulation and UDS activity [31], but not in human lung fibroblasts [28, 29]. Studies in rats have resulted in the production of DNA-damaging activity in the liver [31], but not of clastogenic activity in the bone marrow (Table 3). This discrepancy may imply that mutagenic species produced in the whole animal do not reach the bone marrow in sufficient concentration, if at all. Interaction of dicamba with germ cell DNA was excluded by Waters et al. [29] in Drosophila melanogaster sex-Hnked recessive lethal mutation assay. The genotoxic effects of DNOC in different experimental systems are outlined in Table 2. Clearly, the data from both in vitro systems and rodents establish that DNOC is genotoxic. In particular, the significant activity exhibited in vivo (Table 3) may be of potentially significant health concern. Shelby [32] and Shelby and Zeiger [33] have noted that the majority of human carcinogens are clastogenic to the rodent bone marrow, almost irrespective of the tissue in which these carcinogens induce cancer in rodents and man. The previously reported DNA-damaging activity in the liver of treated rats [34] provides strong confirmation of this potential risk. Interaction of DNOC with germ cell DNA was suggested by Muller and Haberzettl [35], who demonstrated sex-linked recessive mutations in Drosophila melanogaster, while no sperm abnormalities have been found following subchronic toxicity testing in mice [39]. Nevertheless, the identification of DNOC as a potential human mutagen requires other studies to verify an interaction with mammalian germ cells. Clearly, the data from experimental animals and cells in vitro estabhsh that pesticides tested may exert genotoxic activity as judged by the methodologies usually used to make these evaluations. Although the quantitative significance of these responses has not been determined, results on genotoxic activity provide additional information that may contribute to the design of legislative and regulatory instruments to minimize significant risk to humans from pesticides. Research into the etiology of cancer has provided us with a better understanding of the processes and agents that are crucial for the induction of tumors. This knowl-

422


edge provides support for the idea that minimization of exposure to carcinogens is a prudent objective and is consistent with actions taken to safeguard public health. If one accepts that pesticides may contribute to the develop of human cancer, a logical question is how tumor induction occurs and what the mechanisms of carcinogenicity and toxicity are. NEW APPROACHES TO THE STUDY OF PESTICIDES Integration of Cytogenetic Assays with Biochemical and Metabolism Studies As stated before, carcinogenesis in human and animals is a complex, multistage process [4]. Tumors are known to show a range of genetic disturbances, such as chromosomal aberrations, aneuploidy, and gene amplification, which may not be detected using only the Salmonella assay. As suggested by Ashby [8], definition of the genotoxicological character has to be done avoiding to conduct intrinsically insensitive or repetitive assays that add no new positive information, or those that have an insecure basis. Development of assays with a cytogenetic endpoint, either in vitro or in vivo, will leave essentially no genotoxins undetected. However, it is increasingly clear that the primary biological effect of many carcinogenic chemicals involves events other than direct DNA reactivity in many experimental rodent models as well as human cancers. At this time, it is predominantly the nongenotoxic carcinogens that present the greatest problems in detection and risk assessment [40]. Chemicals that act to promote the probability that an initiated cell will progress to malignancy may be important as risk determinants [41, 42]. Recently we proposed a new approach which combines cytogenetic data with a comprehensive examination of the mechanisms of toxicity down to the metabolic level as a possible surrogate measure for promotional and cocarcinogenic activity [43]. This approach permits one to study the pathogenic mechanisms of cellular and tissue lesions at various stages of carcinogenesis (initiation and promotion). Many carcinogens can alter cytochrome P450 enzyme expression (induction or inhibition), leading to metabolic differences that may be related to variations in kinetic factors and in tissue dosimetry [42]. Moreover, the promoting properties of cyt. P450 enzyme inducers (e.g., barbiturates, haliphatic halogenated hydrocarbons, alcohols, dioxins) are acknowledged [44, 45]. We focused our attention to fungicides because these agents have been stated to contribute nearly 60% of all estimated oncogenic risk from pesticides [46]. Our recent studies on fenarimol, vinclozolin, metalaxyl, and procymidone (see Table 1 for the chemical structures) demonstrate a means of identifying potential carcinogenic pesticides that can act through genotoxic and nongenotoxic mechanisms of carcinogenesis. Fenarimol is not carcinogenic in mice [47], and results on vinclozolin are doubtful [48]. Metalaxyl did not exert oncogenic effects in rodents [49], whereas procymidone was found to increase the incidence of testicular interstitial cell tumors [50]. The evidence in the literature on genotoxic effects of the

423


fungicides is conflicting and incomplete. In most cases, data consist of summary of results from mandatory toxicological assays submitted for registration and cited in USEPA or FAO-WHO reports (Table 4). Fungicides were assessed with two different cytogenetic assays, namely, frequency of chromosome aberrations in human peripheral lymphocytes and an assessment of the micronuclei frequency in mouse bone marrow. Description of each bioassay system and qualitative aspects of results were reported previously [56, 57].

Table 4 Sunnmary of available results on the genotoxicity of fungicides tested Compound

Test organism

Genetic effect

Fenarimol

S. typhimurium E. coli Mouse lymphoma cells Primary rat hepatocytes Mouse bone marrow

Gene mutations Gene mutations Gene mutations UDS Micronuclei (two repeated doses) Chromosome aberrations Dominant lethals Mitotic non-disjunction Gene mutations Gene mutations Gene mutations SCE Dominant lethals Mitotic recombination Mitotic non-disjunction Hemoglobin adducts Gene mutations Gene mutations Gene mutations Gene mutations Chromosome aberrations Host mediated assay with S. typhimurium Chromosome aberrations SCE UDS UDS Mitotic crossover Mitotic gene conversion Reverse mutations UDS Gene mutations Dominant lethals

Vinclozolin

Procymidone

Metalaxyl

Chinese Hamster in vivo Rat in vivo A. nidulans S. typhimurium S. pombe S. typhimurium CHO cells Mouse in vivo A. nidulans Rat in vivo S. typhimurium E. coli B. subtilis Mammalian cells CHO-Kl cells Mouse in vivo Mouse in vivo Mouse embryo cells Rat hepathocytes Epithelian human cells S. cerevisiae Primary rat hepatocytes Mouse lymphoma cells Mouse in vivo

Result

Reference

-

[47]

+ + + + + + — -

[51] [52] [48] [53, 54] [55] [50]

[49]

424


Treatment of human lymphocytes in vitro with different doses of fenarimol, vinclozolin, and procymidone (1-100 îgA), both in the presence and in the absence of exogenous microsomal activation (S9 fraction) did not induce any significant increase in chromosome aberrations (Figure 1). Dose-dependent enhancements in chromosomal damage, not associated with mitotic inhibition or cell death, were shown in metalaxyl-treated (300-1,000 fxg/l) lymphocytes. On the other hand, in in vivo experiments metalaxyl (75 to 300 mg/Kg b.w.), as well as procymidone (97.5 to 390 mg/Kg b.w.), had no effects on the frequency of micronuclei, detected in murine polychromatic erythrocytes (Figure 2). Significant increases in the incidence of micronucleated erythrocytes were found in fenarimol (75 to 300 mg/Kg b.w.) and vinclozolin-treated mice (312.5 to 1,250 mg/Kg b.w.). The discordant results from the in vitro and in vivo cytogenetic assays for the fungicides tested suggest that metalaxyl has an intrinsic genotoxic activity which is

Figure 1. Incidence of structural chromosome aberrations induced in cultured human peripheral blood lymphocytes treated with fungicides. Treatment dose: fenarimol, procymidone and vinclozolin, 100 pg/ml; metalaxyl, 1,000 pg/ml. • , without metabolic activation; 0 with metabolic activation. * p < 0.05, ** p < 0.01 with respect to controls pc^ test).

Genetic Evaluation of Pesticides In Different Short-Term Tests

425

Figure 2. Frequency of polychromatic erythrocytes with micronuclel (MN) In mice treated with fungicides. Data Indicate the percentage mean ± S.E. valued on 1,000 erythrocytes after an intraperitoneal dose of fenarlmol (300 mg/Kg), metalaxyl (300 mg/Kg), procymidone (390 mg/Kg), vinclozolin (1,250 mg/Kg). * p < 0.05, ** p < 0.01 with respect to controls (X^ test).

expressed only in vitro; metabolism in the whole animal provides detoxified products which do not react with DNA moieties in bone marrow erythrocytes. Bioavailability data in blood of metalaxyl [49] provide sufficient evidence of bone marrow exposure. Fenarimol and vinclozolin are clastogenic in vivo but non in vitro. This behavior may reflect a deficiency of the activating fraction in vitro, or effects detected may be due to interactions with other cellular structures than chromosomes resulting in mitotic anomalies. Procymidone was confirmed to be nongenotoxic; therefore, the induction of Leyding cell tumors evaluated in chronic and oncogenic studies [50] may be a species-specific nongenotoxic mechanism. Biochemical analysis, performed on purified microsomes from different organs of treated mice [58], indicated a toxic, co-toxic, co-carcinogenic, and "promoting" activity of fungicides tested through the induction of P450 function. Results shown in Figure 3, as activity profiles in liver of treated animals, pointed out a selective

426


1.000

pNFI (CYP 2E1) PROD (CYP 2B1) APND (CYP 3A) ECOD (CYPs)

Figure 3. Expression of carcinogen metabolizing enzymes in liver microsomes from fungicide treated mice. Intraperitoneal doses for 3 days: fenarlmol, 150 mg/Kg; metalaxyl, 200 mg/Kg; procymidone, 800 mg/Kg; vinclozolin, 750 mg/Kg.

increase of the expression of CYP 2B1 by fenarimol, CYP 2E1 by vinclozolin, and CYP 3A by metalaxyl, whereas procymidone was a non-selective substrate, inducing all the considered isoforms. These inductive capabilities were confirmed at a molecular level. There are two major reasons why it is important to establish whether or not a pesticide is an enzyme inducer of inhibitor. First, to determine if the pesticide is likely to cause chemical interactions. Secondly, to evaluate the possible toxicological consequences of perturbation of drug-metabolizing enzymes. A combined study in which critical genetic endpoints have been correlated with alterations in oxidative metabolism, and with urine metabolites, was carried out in mice treated with fenarimol and trichloroethylene as a probe carcinogen [59]. In this time-course study, we found that the enhanced effect of fenarimol is correlated with a significant induction of cyt. P4502B1 family of isozymes and the increased presence of activated trichloroethylene metabolites in vivo. Observed significant potentiation of clastogenic effects of trichloroethylene (about two-fold increase, p < 0.01) by pretreatment (1 h) with fenarimol was paralleled by a 20% increase in excretion of trichloethanol. However, concomitant exposure to trichloroethylene


427

and fenarimol decreased both the metabolism (by 51%) and the genetic effects of trichloroethylene (by 38%, p < 0.05), probably because of inactivation of the microsomal enzymes critical for its metabolism; both trichloroethylene and fenarimol are substrates of the same P450 enzymes. Potential to Detect Cytogenetic Damage by Using Molecular Cytogenetic Techniques Structural and numerical chromosome aberrations are associated with a range of clinical genetic disorders and diseases, such as spontaneous abortions, mental retardation, congenital malformations, and cancer [60, 61]. These same events are important in tumor initiation and progression [62, 63]. It is therefore important to develop methods for cytogenetic analysis that are applicable to both laboratory animals and humans. Recent advances in molecular biology led to facilitated analysis of cytogenetic abnormalities. One approach uses fluorescence in situ hybridization (FISH) with centromeric DNA probes to detect increased frequencies of hyperploidy in human lymphocytes that have been exposed to chemicals in vitro [64]. Initial studies performed on Thiophanate-methyl (see Table 1 for chemical structure), which has a well-documented aneugenic activity in lower eukaryotes, indicate that the fungicide (0.3 to 30 [ig/ml) induced dose-dependent increases in structural chromosome aberrations (up to eleven-fold, p < 0.01) and micronucleus frequency (up to two-fold, p < 0.01) in human lymphocytes in vitro [65]. Thiophanate-methyl was relatively more active in the induction of centromere-negative than centromere-positive micronuclei (73% vs. 27%). The two types of micronuclei were present in similar proportion in cells treated with bleomycin, used as positive control, indicating a relatively stronger clastogenic activity of the fungicide. CONCLUSIONS Evidence for genotoxicity of pesticides in different short-term tests has been and still is viewed as a useful diagnostic signal for potential human genetic diseases and cancer. Genotoxicity testing will continue to play an increasingly important role in distinguishing genotoxic and nongenotoxic compounds, which are treated differently in risk assessment models. Currendy, the Salmonella typhimurium test is the most widely used short-term test for the screening of mutagens and carcinogens. However, it may be only poorly correlated to carcinogenesis [66]. Because each test system detects some genotoxins than other might not, a battery of tests is liable to attain a greater number of hits and possibly a higher sensitivity. The vast majority of genotoxic pesticides will be alerted to by a consideration of chemical structure coupled to an assessment of mutagenic activity in the Salmonella assay and in a cytogenetic assay, either in vitro or in vivo, or perhaps both [8]. Evidence was pre-

428


sented to illustrate the challenge to scientists to evaluate the genetic safety of pesticides and improve the standard of genotoxicity assays despite their limitations. Although it is possible to prioritize cancer risk using only genetic data, additional information is needed for quantitative risk evaluation. The prediction of potential mutagenic and/or carcinogenic pesticides is mainly toxicological. One most challenging and useful next step forward for the development of testing strategies is to consider biochemical techniques or metabolism studies that can identify and characterize the cellular mechanisms capable of initiating and promoting the carcinogenic response. The addition of toxicokinetic studies with relevant carcinogens offers a pointer to investigate interactive effects between agents to which humans are exposed. Moreover, new techniques (e.g., molecular cytogenetic) provide a means to increase further the information obtained from routine studies and to answer questions that could not be answered before.

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45. Schulte-Hermann, R. "Tumor promoting in the liver." Arch. Toxicol, 57, 1985, 147-158. 46. National Research Council "Regulating pesticides in food." Washington, D.C.: National Academy Press, 1987. 47. U.S. EPA. Pesticide Fact Sheet "Fenarimol." Tox. Chem. N. 207AA, June 17, 1985. 48. U.S. EPA. Pesticide Fact Sheet "Vinclozolin." Tox. Chem. N. 323C, Sept. 7, 1983; April 23, 1986; April 24, 1987. 49. FAOAVHO "Pesticides residues in food—Metalaxyl." Joint Meeting on Pesticide Residues, Rome, 1982, pp. 259-270. 50. FAOAVHO "Pesticide residues in food—Procymidone." Eval. II-ToxicoL, 100(2), 1989, 161-181. 51. Bellicampi, D. et al. "Membrane-damaging agents cause mitotic nondisjunction in Aspergillus nidulans." Mutat. Res., 79, 1980, 169-172. 52. Chiesara, E. et al. "Detection of mutagenicity of Vinclozolin and its epoxide intermediate." Arc/z. Toxicol, suppl. 5, 1982, 345-348. 53. Georgopoulos, S. G., Sarris, M., and Ziogas, B. N. "Mitotic instability in Aspergillus nidulans caused by the fungicides iprodione, procymidone and vinclozolin." Pest. Scl, 10, 1979, 389-392. 54. Vallini, G., Pera, A., and de Bertoldi, M. "Genotoxic effects of some agricultural pesticides in vitro tested with Aspergillus nidulans." Environ. Poll, 30, 1983, 39-58. 55. Sabbioni, G., and Neumann, H.G. "Biomonitoring of arylamines: hemoglobin adducts of urea and carbammate pesticides." Carcinogenesis, 11, 1990, 111-115. 56. Hrelia, P. et al. "A battery of biomarkers for detecting carcinogenic risk from fungicides." Clin. Chem., 40 (7), 1994, 1,460-1,462. 57. Hrelia, P. et al. "Genetic and biochemical markers as predictors of carcinogenic effects of pesticides." Pharmacol Toxicol, 76 (suppl. 1), 1995, 18 (Abs.). 58. Paolini, M. et al. "Wide spectrum detection of precarcinogens by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes." Carcinogenesis, 12, 1991, 759-766. 59. Hrelia, P. et al. "Interactive effects between trichloroethylene and pesticides at metabolic and genetic level in mice." Environ. Hlth. Perspect. 102 (suppl. 9), 1994, 65-68. 60. Oshimura, M., and Barrett, J. C. "Chemically induced aneuploidy in mammalian cells: Mechanisms and biological significance in cancer." Environ. Mutagen., 8, 1986, 129-159. 61. Hecht, F., and Hecht, B. K. "Aneuploidy in humans: Dimensions, demography, and dangers of abnormal numbers of chromosomes," in: Aneuploidy, Part A: Incidence and Etiology, B. K. Vig and A. A. Sandherg (Eds.), New York: Alan R. Liss, 1987, pp. 9-49.

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62. Nowell, P. C. "Origins of human leukemia: An overview," in: Origins of Human Cancer, J. Brugge, T. Curran, E. Harlow, and F. McCormick (Eds.), Plainview, NY: Cold Spring Harbor Laboratory Press, 1991, pp. 513-520. 63. Knudson, A. "Genetic events in human carcinogenesis," in: Origins of Human Cancer, J. Brugge, T. Curran, E. Harlow, and F. McCormick (Eds.), Plainview, NY: Cold Spring Harbor Laboratory Press, 1991, pp. 17-25. 64. Becker, P., Scherthanh, H., and Zanki, H. "Use of centromere-specific DNA probe (p82H9) in non isotopic in situ hybridization for classification of micronuclei." Genes Chromosome Cancer, 2, 1990, 59-62. 65. Hrelia, P. et al. "A cytogenetic approach to the study of genotoxic effects of fungicides: an in vitro study in lymphocyte cultures with thiophanate methyl" AJLA, 24,1996,597-601. 66. Tennant, R. W. et al. "Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays." Sciences, 236, 1987, 933-944.

CHAPTER 20 PESTICIDE RESIDUES IN FOOD Peter Dingle Adrian Strahco and Peter Franklin Murdoch University Perth, Western AustraUa 6150 CONTENTS INTRODUCTION, 433 HISTORY, 434 RESIDUES AND FACTORS AFFECTING RESIDUE LEVELS, 435 PESTICIDE REGULATION IN THE DEVELOPED AND DEVELOPING WORLD, 437 TOXICOLOGICAL TESTING OF PESTICIDES, 438 Limitations, 440 ESTABLISHING STANDARDS, 441 MONITORING OF RESIDUES, 442 INTERNATIONAL TRADE AND PESTICIDE RESIDUES, 443 RESIDUE LEVELS, 444 HEALTH EFFECTS, 445 Acute Poisonings, 446 REFERENCES, 447 INTRODUCTION Pesticides are used to control insect pests, weeds, rodents, and diseases on food and other crops. They have also been used to control insect-borne diseases in humans (e.g., control of malaria in the tropics). The major groups of pesticides include insecticides, fungicides, herbicides, and rodenticides. The use of pesticides has developed principally because such chemicals are capable of destroying certain animal species perceived as economic threats. In no instance, however, has it been shown that these chemicals are only toxic to target species. It is well known that most pesticides are toxic to a wide range of other species, including, in many cases, humans. In spite of such knowledge, the use of pesticides remains a global pursuit, particularly in the agricultural industry, where the reduction of losses to pests through the use of pesticides has been the principal benefit. According to Melifrondes and Kashouli-Kouppari (1994) the benefits of pesticides are that their use "enhances and stabilizes crop yield, protects the nutritional integrity of food, facilitates storage to assure year round supplies, and provides for attractive and appealing food products" [1]. One of the major benefits of pesticides has been the reduction in food losses caused by insect pests, crop diseases, and 433

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weeds. It has been estimated that on a world scale there would be 30% more food, cotton and other crops if it were not for losses caused by insects, weeds, and disease [2]. International losses in agricultural production as a result of pest attack have been estimated at $74.9 U.S. billion annually [3]. One of the legacies of pesticide use, however, has been the adverse affect that pesticides can have on human health. The main source of non-occupational exposure to pesticides is through the diet [4, 5]. For example, it has been estimated that greater than 90% of DDT in human tissue came from food [4]. However, considering food pesticide residues in isolation may give a false sense of safety to the public, who may in fact conceive that food pesticide residues are the only significant source of contamination. Studies such as the Non-Occupational Pesticide Exposure Study (NOPES) carried out by the U.S. Environmental Protection Agency concluded that for "14 of the 25 pesticides tested, food appears to be the major contributor to total exposure, whereas air appears to be the dominant contributor for six of the other eleven compounds" [6]. Although a wide variety of pesticides finds its way into our food, debate has raged over whether pesticide residues in food can affect our health and what, if any, levels are safe. However, consumer surveys have shown that the majority of people consider pesticide residues in food to be a major concern. Consumers worldwide are increasingly concerned about the chemical residues in the food they eat and expect safeguards to ensure that chemical residues do not pose a health risk. This chapter will examine the history of pesticide use, factors that affect pesticide residue levels in food, regulations and standards, and exposure to pesticide residues and the associated health risks. HISTORY Pesticides have been used throughout history; however, it was not until the insecticidal potential of chlorinated organic compounds was first discovered during the Second World War, with the use of dichlorodiphenyl trichloroethane (DDT) and hexachlorocyclohexane (HCH) that pesticide use in agriculture proliferated [7, 8]. For example, in 1945 there were 15 chemicals (60 formulations) registered in the U.K., but by 1975 the number had grown to 200 (more than 800 formulations) [9]. In the U.S., agricultural pesticide use more than doubled from 150 million kg in 1964 to 400 million kg in 1980 [7]. World sales of agrochemicals amounted to $950 U.S. million in 1964; however, international sales of pesticides alone was $2.32 U.S. billion in 1971 [3]. In 1991, 2.53 million tons of pesticides, approximately $20 billion worth, was used worldwide [1]. The United States uses approximately one-fifth of this with an annual cost of $4.1 billion [10], and of the pesticides used in the United States, 69% are herbicides, 19% are insecticides, and 12% are fungicides [10].

Pesticide Residues in Food

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Equally, the concern about the ecological consequences and health effects of pesticide use has increased regulations relating to their use. For example, due to its known persistence and possible carcinogenicity, DDT was banned in Sweden in 1970 and the U.S. in 1973 [11]. Although its use was severely restricted, it was not totally banned in the U.K. and Australia until the late 1980s. Now, the use of 20 or more individual pesticides of major economic importance have been banned or restricted in one or more countries [12]. More recently, there seems to be an effort in many industrialized countries to reduce the reliance on pesticides in agriculture. Some Western European countries now have policies to reduce pesticide use over the next decade. These countries include The Netherlands, Sweden, Denmark, and Germany [13, 14]. In other countries, such as the U.K., it is felt that pesticide approval regulations, as well as more effective application techniques and the increased use of products that are more biologically active at lower dosage rates, will decrease pesticide use without the need to impose arbitrary reduction targets [15]. Although pesticide use has seemed to stabilize or even decrease in developed countries in recent years, world consumption is still growing. Indeed it has been estimated that pesticide use will increase at an annual rate of 12.5% [2]. The reason for this is the increasing amount of pesticides that are being applied in developing countries [2]. This includes substances that have been banned or severely restricted in developed countries. For example, organochlorines, which are banned in most developed countries, are being used at ever-increasing rates in Asian tropical agroecosystems and in southern Pacific islands [16]. RESIDUES AND FACTORS AFFECTING RESIDUE LEVELS A residue can be defined as something that is left behind. "A pesticide residue is any substance or mixture of substances in food for humans or animals resulting from the use of a pesticide and includes any specified derivatives, such as degradation products, metabolites, reaction products and impurities, which are considered to be of toxicological significance" [17]. Residues will originate from the normal use of agricultural chemicals, misuse of chemicals, and unintended exposure or environmental contamination. Normal use may result in residues, particularly if the chemical is applied to protect the produce on its way to the consumer. Chemicals are applied with the expectation that they will break down or disperse over time, and that the produce will have little or no residue on it by the time it gets to market. Misuse of chemicals includes: failure to observe the withholding period, the application of too much of the chemical, or the use of the chemical on a commodity for which there is not registered utility [18]. The most common form of incorrect use is the failure to have the correct withholding period between application of the pesticide and harvesting [18]. Cases also exist where illegal pesticides or excessive concentrations of legal pesticides have left residues that are a health risk [19, 20].

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The other sources of residues are the unintended exposures of crops or animals to pesticides, either prescribed or illegal. Environmental contamination may be the result of past pesticide use, and some has remained due to its persistence and nonbiodegradability, such as in the case of the organochlorines, or pesticide drift from nearby applications. Apart from residues acquired during growing, some pesticides are applied after harvesting has occurred. They are applied during storage and transport to ensure that they last. Fungicides are common to prevent mold and other storage diseases, and pests. For example, potatoes, leafy vegetables, and grains are treated [18]. A fungicide is added to a wax, and together they help keep the fruits' and vegetables' appearance intact and prevent mold. There was concern that the fungicide, not the wax, could have some health effects as a result of increasing the residue level present [22]. Waxes are used primarily to make foods look more attractive to the consumer and therefore increase produce marketability. A number of fungicides that are added to waxes have been shown to cause health problems [23]. Foods that are typically waxed include apples, citrus fruits, and tomatoes. A number of factors affect the residue levels in produce at harvest. Hayes [24] and Precheur et al. [25] noted the following as contributing factors: a) Dosage and number of applications—several small doses led to more residue than did one large dose. b) Time from application to harvest—shorter intervals between last dose and harvest resulted in elevated residues [25]. Reduction in residues involves two factors: 1) loss of pesticide through means, and 2) relative loss of residue through plant growth. c) Nature of the compound—where some compounds break down faster than others. d) Character of the formulation—dusts give rise to lower residues than liquid formulations. If the plant surface is wet, then the reverse is true. e) Character of the plant—waxy plants retain less residue than hairy plants. f) Migration from the soil—depended on soil type and crop, where sandy soils retain the most pesticide. Carrots were found to absorb more pesticide than any other crop. g) Weather—rain soon after application decreased residues. h) Volatilization—when some chemicals evaporated due to temperature, higher residues resulted, i) Method of harvest—handling during harvest decreased residues considerably, j) Possible intentional or unintentional post-harvest application—grains may have several applications during transportation, k) Method of storage—under different storage conditions, residues may or may not decrease to safe levels [24]. Even when taking all these factors into account, uncertainty still remains as to resultant residue levels.


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Many factors also affect residue levels between harvest and consumption. Eilrich (1991) traced vegetables treated with the fungicide chlorothalonil from picking to packing houses, grocery stores, and restaurants [26]. The residue levels fell steadily between test points. During processing of foods, most residues will decrease greatly while others may undergo as much as a ten-fold concentration, as in the processing of tomatoes [25]. The amount of reduction or concentration depends upon the properties of the individual pesticide, the food on which it is present, and the types of processing steps [27]. The National Food Processors Association (NFPA) in the U.S.A. showed that washing and peeling removed 99% of carbaryl and malathion residues from tomatoes. Benomyl residues were reduced 83% by washing and 98% by subsequent processing into tomato puree and sauce [28]. Processing may also lead to the formation of breakdown products that may be toxic. In the case of the ethylenebisdithiocarbamate (EBDC) fungicides such as maneb, zineb, mancozeb, and metiram, ethylenethieourea (ETU) is occasionally detected as a breakdown product. Toxicology studies have shown that ETU possesses carcinogenic, mutagenic, goiterogenic, and teratogenic properties [27]. In the U.S. there was much public concern when supplies of apple juice were recalled from the market after discovery that they were contaminated with unsymmetrical dimethylhydrazine (UDMH), a breakdown product from the pesticide daminozide (Alar). PESTICIDE REGULATION IN THE DEVELOPED AND DEVELOPING WORLD Most developed countries have registration requirements and regulatory systems for pesticides [11]. In the U.S., for example, all pesticides must be registered with the U.S. EPA, which also sets tolerances (the maximum amount of residue that is permitted in or on a food) [29]. The U.S. Department of Agriculture is responsible for setting tolerances for meat, poultry, and certain egg products, while the Food and Drug Administration (FDA) is charged with monitoring and enforcing residue levels [29]. In the European Community, each of the individual member states is able to have its own pesticide registrations and set individual tolerances; however, most legislate to take into account a European Community Directive which seeks to harmonize registration requirements throughout the Community [30]. This directive provides for a list of "Active Substances Authorized for Incorporation in Plant Protection Products"; however, authorization of products containing these active substances is at a member state level [30]. In most industrialized countries, for a pesticide to be registered it must comply with certain criteria regarding its effectiveness in controlling pests and diseases, persistence, toxicity, and residue tolerance [14]. Before approving the use of an individual pesticide, regulatory bodies may require the manufacturer to provide data from standard animal toxicity tests and from field studies [31].

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In contrast to the developed world, many developing countries do not have pesticide regulations. Indeed, 50% of developing countries have no legislation for pesticide availability and use (the proportion in Africa was 76%) [32]. As a case in point, over the past 10-15 years in Nigeria there has been a great upsurge in pesticide use; however, apart from a recent publication of guidelines and standards for environmental pollution control from the Federal Environmental Protection Authority, there is no legislation or control of pesticide use [33]. Where there is some form of regulation, it may well be inadequate or poorly implemented [34]. The situation in Third World countries is exacerbated by a lack of resources and expertise to monitor use, a growing population that needs feeding, and a lack of political will to deal with the situation [35]. A number of UN and other international agencies have collaborated in the development of national policies on pesticides. There are about 50 international organizations, approximately 15 of them within the UN system, with some involvement in pesticides [12]. A good review of the international structure is provided by Ekstrom and Akerblom (1990) [12]. Following is a brief outline of some of the areas the UN has contributed to the regulation of pesticides: • Food and Agriculture Organization (FAO), in cooperation with WHO, UNEP and others, has developed a code of conduct on the distribution and use of pesticides, outlining the potential hazards and appropriate actions for their prevention. It also defines the responsibilities of national regulatory bodies. • FAO has published guidelines for improved pesticide management including registration and control. • World Health Organization (WHO) has conducted a literature review of the public health impact of pesticides used in agriculture. • Data sheets about pesticides, where the information is available, are published jointly by FAO and WHO. • FAO and WHO have recommended restrictions on the availability of pesticide formulations, and the UN has published a list of pesticides that have been banned, withdrawn, or severely restricted by governments. The FAO and WHO through the Codex Alimenterius Commission recommend maximum residue levels (MRLs) for pesticides in foods. In most developed countries, the basic principles for assessing pesticides and setting tolerances follow those set by the joint committee of the FAO and WHO. TOXICOLOGICAL TESTING OF PESTICIDES Toxicological testing is done to assess acute and chronic toxic effects of pesticides. These include the potential to promote or initiate tumors, exhibit neural toxicity, alter genetic material, affect fetal development, affect reproductive capacity, or give rise to chronic effects such as allergies or environmental sensitivities [20].


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The cost of these toxicological and field trials is high, approximately $20 million per chemical. Acute toxicity is that which is due to short periods of exposure to high concentrations of the toxin, usually less than 96 hours. These tests have largely evolved from the concerns of the groups of people most at risk, such as through occupational exposure. Given the ubiquity of chemicals in our total environment, though, they are increasingly seen as a tool for investigating community concerns. The most common techniques used to measure acute toxicity determine the amount of a toxic substance required to kill 50 percent of target organisms in a given time. This is referred to as the LD50 and is a measure of the concentration of toxic substances ingested or absorbed through the skin. In contrast, the LC50 is a measure of the concentration of a toxic substance in the ambient environment of the organism, such as in the air or water. This technique is particularly relevant and more realistic for air pollutants that may be inhaled, but is more difficult to assess and more expensive to determine than the LD50. Subacute toxicity studies assess the toxic effects of substances at sublethal concentrations. Subacute toxicity is most commonly tested on rodents by route of intended exposure to three or four different concentrations. A high dose, just below the acute toxicity levels, is usually selected to highlight any subacute affects, and a very low dose is selected to identify the level at which no toxic effect is noticeable, referred to as the no observable effect level (NOEL). These levels are usually set in the testing protocols established by regulatory agencies such as the U.S. EPA. The remaining concentrations tested are intermediate to these two levels. Subacute tests are usually carried out over 90 days or for 10 to 15% of an organism's normal lifespan. Observations on test animals are usually numerous and diverse, and include changes in body weight, diet consumption, organ dysfunction, metabolic, physiological and somatic changes, and obvious behavioral changes. Chronic toxicity tests are designed to investigate the long-term effects of exposure to subacute concentrations of toxic substances and typically explore the genotoxic, mutagenic, carcinogenic, and/or teratogenic potential of the substance. Classical chronic toxicology studies consist of animal bioassays using surrogate species. Carcinogenic bioassays are usually performed on medium-sized small mammals, such as rats. During the course of the study, some of the experimental animals are sacrificed and pathological examinations are used to determine whether tumors have developed. The remaining animals are sacrificed for the same purpose at the end of the study. Teratogenic bioassays are designed to assess malformations induced during development from conception to birth. Teratogens are most effective during the first trimester of pregnancy, the period of organogenesis, so most studies expose the pregnant animals during this period. As a result of the high financial and animal costs and time (very important), there has been much interest shown in developing short-term tests (STT). The first attempt to identify carcinogens using STT was using a Esherichia coli mutagenesis

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assay. However, these earliest studies failed to take into account that most mutagens require metabolic activation before they become active genotoxins, thus few mutagens were identified. In the 1970s, liver extracts were first used to metabolize promutagens to mutagens. Limitations From the above it is clear that the tools used by toxicologists are important in assessing how toxic substances adversely affect living organisms. However, it is also apparent that these tools have inherent limitations that need to be considered when assessing the toxicology of a substance. Clearly there is a large gap between our present levels of understanding in toxicology and the knowledge needed to make confident judgments about chemicals in our environment. The limitations of our understanding must be recognized and taken into account so we do not develop a false sense of our knowledge. It must also be recognized that total understanding is not necessary for action to be taken. Some of the limitations include the fact that the toxicological tests carried out do not consider the possible synergies between chemicals, such as the ability of nicotine to enhance the ability of certain organophosphate pesticides to inhibit the enzyme acetylclolinesterase, causing chronic weakness, blurred vision, and in extreme cases respiratory failure [37]. Toxicological studies often only consider the effects of the active ingredient, even though toxic effects many be attributable to the so-called "inert" ingredients [38]. In addition, there are concerns that toxicological calculations do not adequately consider the different dietary habitats and susceptibility of particular groups in the population, such as infants [39]. Only about one-third of the active ingredients used in agricultural chemicals have even been assessed by the Codex Committees [40]. The toxicological effects of many chemicals in widespread use are unknown. A number of older chemicals that were previously thought to be safe, based on overseas assessment, were found to be carcinogenic in animals [20]. These include Caftan, Captafol, Folpet, a major metabolite of Chlordimeform, and alachlor. At present, as much as 80% of the registered chemicals have not been fully tested for health effects on humans, to say nothing of the environmental effects [41]. Despite the lengthy process to introduce chemicals into the market, there is no guarantee that a chemical is entirely safe for use. An example of this is where fraud occurred when a major independent laboratory. International Bio Test of Illinois, was found to have faked tests. As the cost of the tests is so high, the regulatory bodies cannot afford to duplicate many of the tests, so it is uncertain how often this fraud takes place. The problems of such fraud would be carried through when other companies developing new formulations use the fraudulent data as a referential standard. While the testing may be carried out to the best possible standard, there still remain many adverse health effects that the tests fail to acknowledge, especially in regard to hypersensitivity. At present the basis for hypersensitivity is not fully


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understood. Hypersensitivity includes such ailments as asthma, migraines, arthritis, and skin rashes. Nor is it fully understood how the chemicals affect such things as emotions, intelligence, memory, and other complex neural functions and thus the general quality of life [31]. It is hard to establish a causal link between increased exposure levels of chemicals and allergies, as no long-term research on chronic effects of pesticides on humans has been undertaken [42]. ESTABLISHING STANDARDS After completion of the toxicological tests, the data are used to develop standards. The no observable adverse effect level (NOAEL) is the highest dose at which there are no observable effects in the most sensitive test species in the most sensitive tests [31, 43]. From this the acceptable daily intake (ADI) is set. The ADI represents the amount of a chemical that you can consume every day over an entire lifetime "without appreciable risk" [31]. The ADI is usually set at one-hundredth the NOAEL of the most sensitive animal studied. The ADI is expressed in milligrams of the chemical, as it appears in the food, per kilogram of body weight (mg/kg) per day. In order to arrive at an ADI, the following information is desirable: • The chemical properties of the residue and its derivatives due to metabolization in the plant/animal tissues. • The toxicity of the chemicals forming the residues based on data from acute, long-term and short-term toxicity studies (usually in animals), and knowledge of metabolism, mechanism of action, and carcinogenicity of residue chemicals when consumed. • An adequate knowledge of the effects of the chemicals in people. The maximum residue limit (MRL) is defined to be the maximum concentration of a residue that is recommended to be legally permitted, or recognized as acceptable in, or on, that food at any stage of harvesting, transport, marketing, or preparation, up to the final consumption. Firstly, the MRL is set with regard to the ADI of the chemical concerned. The MRL is set so that the ADI is unlikely to be exceeded. This is done through assessment of the population's diet and also exposure through non-food sources. Secondly, field trials are conducted to determine the levels of a chemical and its metabolites expected to be present when the chemical is used according to officially sanctioned directions or good agricultural practice (GAP). Residue levels can vary depending on soil, climatic, and other factors even when GAP is used. MRLs need to accommodate countries where climatic conditions, pests, crop, and farming practices can cause GAP to vary considerably. Thus it is not appropriate for countries simply to adopt standards from other countries because differing agricultural conditions require different practices. For instance, hot dry conditions in Australia

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require high insecticide use, whereas cool damp conditions in Europe require use of fungicides. Trade and poUtical considerations also come into the setting of MRLs. ADIs for pesticides are reconmiended by the WHO/FAO Joint Meeting on Pesticide Residues (JMPR). International MRLs are set by FAO/WHO through the Codex Committees on Pesticide Residues (Codex). MONITORING OF RESIDUES A critical component of dietary pesticide risk assessment is the estimation of the levels of pesticides that are likely to be in foods [29]. In practice, the major approach to estimation of pesticide residues involves monitoring a sample of foods, selected to be representative of the normal diet of the population, and extrapolating this information to the broader food market. Monitoring programs also aim to detect illegal use of pesticides. Because it is not practical to analyze all commodities for all possible residues, the analytical program is based on risk profiles. Commodity-residue combinations of highest risk are included in most surveys. The following factors are taken into account in assessing risk profiles: • Toxicology of residue • Incentive for misuse • Persistence • Previous survey results • Trade considerations • Cost and availability of analytical method. The Food and Drug Administration (FDA) of the U.S. conducts an annual monitoring program, the Total Diet Study or Market Basket Survey. It is designed to estimate the total dietary intake of pesticide residues of 8 age/sex groups ranging from infants to senior citizens [45]. Each basket within the FDA study contains 234 foods compared to a maximum of 54 foods in the Market Basket Survey. These foods were purchased four times within the year to account for a greater variety of foods purchased under different climatic conditions. In total, the FDA study analyzes 19,798 foods for a total of 270 pesticides [45]. The FDA survey could not be considered as conclusive as not all the pesticides or foods used in America are tested, but in comparison to the other countries, it is more comprehensive. The FDA (1989) reported that approximately 40% of the foods monitored had detectable residues and that approximately 3% exceeded the tolerance levels with similar results being reported in other years [45]. While many countries conduct market basket surveys, they generally have a number of limitations and at best can only be used as a guide.


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For example, the surveys are limited because: • A small number of pesticides is questioned. • No account is taken of those pesticides not permitted to be used on certain crops but that may still be freely available to growers. • In many cases the environmental conversion products or metabolites are not sought. In addition, not all breakdown products or even contaminants of some pesticides are known. It is therefore not possible to test for these chemicals, let alone identify how toxic they may be. • The cumulative total or toxic equivalents of all pesticides present within a sample is also often not accounted for within the test procedures. Two or more of a particular group of pesticides in a sample must not exceed the MRL allowable for a single substance. • Only a limited number of foods can be monitored for pesticides. In Australia, a maximum of only 21 samples of a particular food were purchased. As little as 7 samples were collected for some foods. • Samples analyzed may be composites and, consequently, the values of the residues only represent average values, not the values that may be present within individuals. It is likely then that individual samples of foods with residue levels above the maximum allowable levels will be diluted by samples within the composite for which little to no residue levels exist. This fact alone renders the results of the residue monitoring of little use. INTERNATIONAL TRADE AND PESTICIDE RESIDUES Different registration requirements and regulatory systems for pesticides among countries has meant that compounds that may be banned in one country can still be used in another [11]. Countries may also differ in national tolerance levels for pesticides. The Codex Committee on Pesticide Residues (CCPR) was established through the Codex Alimenterius Commission to try to achieve international harmonization of national tolerances of pesticide residues in food [46] to protect public health and facilitate international trade. In 1989, approximately 160 pesticides were subject to Codex tolerances but very few countries adopted these unless they already complied with existing national levels [46]. Countries include the monitoring of imported foods in their sampling surveys. The U.S. puts a large emphasis on the monitoring of imported food with 60% of sampled food being imports [46]. Imported foods in the U.S. are subject to the same tolerance levels as domestically produced goods, and most violations occur through the discovery of pesticide residues of compounds not registered in the U.S. [46]. Foods that violate U.S. laws can be refused entry into the U.S..

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The General Agreement on Tariffs and Trade (GATT) proposes that Codex standards be used for international trade and to remove non-tariff barriers [13], and under new changes all restrictions, even those based on health, will no longer be possible. The new proposals for GATT would set worldwide standards for acceptable pesticide residue levels called harmonization. Under harmonization, 42% of current U.S. food residue standards would be changed to allow higher levels of pesticide residue. The changes in the EC and Australia are anticipated to be slightly less. The allowed food residues of pesticides such as the organochlorines, no longer used in agriculture in the U.S., EC and Australia, would be higher than present levels [13]. This can increase the limits for residues in food in some countries as the U.S. and EC often prescribe tighter controls on pesticide residues than does Codex [13]. The harmonization of health, safety, and environmental standards could mean, in practice, the lowest standards of the most poorly regulated member state could be adopted. RESIDUE LEVELS Human exposure to a wide variety of pesticides in foods is extensive. Since concerns were first raised about the environmental and health effects of organic pesticides, a number of have been banned or their use restricted in various countries [12]. Of these, the organochlorines have been one of the most tightly controlled. The bans on a number of the organochlorine pesticides, including DDT, heptachlor, aldrin, and dieldrin, have been reflected in the results of many monitoring surveys with marked decreases in residue levels since restrictions on their use [47, 48]. Leoni et al. (1995) reported that total dietary intake of chlorinated pesticides in the U.S. has dropped from about 100% of ADI in the early 1970s to 10% by the mid-1980s [49]. Despite these decreases in residue levels, detectable levels of organochlorine pesticides are still found in a high number of tested samples. Trotter and Dickenson (1993) found that 49.4% of milk supplies monitored throughout the U.S. had pesticide residues, and p,p'-DDE and dieldrin accounted for 84.4% of these [50]. In Spain, DDT was found in 83% of lamb samples, although at moderate levels [48]. There is also concern that pesticide residues in developing nations are likely to be higher than those experienced in the developed countries. For example, Dogheim et al. (1990) reported that most milk samples in Egypt had high residue levels of 15 pesticides included in the investigation [51]. Butter in Argentina contained detectable levels of Lindane in 93% of samples, heptachlor in 78%, aldrin in 55%, and DDT and dieldrin in 30% [52]. Kashyup et al. (1994) reported that the total dietary intake of DDT for the average Indian vegetarian adult was 19.24 |Lig/day with 50% of this being contributed by fatty foods [5]. Organophosphorous pesticides are used extensively as grain protectants and have therefore been found in a number of grains and grain products. Organophosphate pesticides are broken down rapidly, and this probably accounts for the low residue


445

levels reported from most [53]. Mean intakes of organophosphate (OP) pesticides reported from a number of different countries are generally below 1% of the respective ADIs [47]. In Italy, the sum of all the percentages of ADIs of nearly all organophosphates ingested was about 20 [49]. Smart (1987), in a review of OP residues in foods from EC countries, reported that only a small proportion of fruit and vegetables contain levels that exceeded FAOAVHO MRLs or EC Maximum Limits [53]. Violations of the recommended levels were generally below 1. It would seem that pesticide residues reported from most surveys are sufficiently below MRLs for the respective compounds. In the 1994 U.S. Market Basket Survey, only 3.4% of the samples violated U.S. levels [29]. However a high number of samples (in some cases 100%) still have detectable levels of pesticide residues. Much of the concern over residue levels, particularly for consumers, is whether current levels are safe rather than whether they are under the legal tolerance level [54]. Toxic impHcations of pesticide residues in humans are not completely understood, and they may be unsafe even at low daily intake [48]. HEALTH EFFECTS The possible effects of pesticides include acute or chronic poisonings, altered immune responses, allergic reactions, effects on the nervous system, and mutagenic, teratogenic, or oncogenic effects. Although acute poisoning is of great concern, it is fairly rare and easily detectable as the acute toxicity of many pesticides to humans is well documented. Less is known about the health effects of chronic exposure to pesticides at much lower doses, with findings in relation to such exposure often being debated. Whether or not an exposure of twenty to thirty years will cause cancer or nerve damage, for example, is a very real concern. Various studies have linked chronic pesticide exposure with oncogenetic, mutagenetic, fetotoxic, and teratogenic activity, as well as damage to primary metabolic body organs (kidneys and liver) and central nervous system degradation. It is difficult to detect the cause of some delayed effects or subtle effects. This is due to the fact that there are many possible causative agents to which an individual is exposed, and the amount of exposure is likely to be unknown. The International Agency for Research on Cancer reported sufficient evidence on carcinogenicity for eighteen pesticides and limited evidence on carcinogencity on another 15 pesticides based on animal studies [56]. Animal studies have shown the tumorigenic effects of organochlorine and dithiocarbamate pesticides, while the long-term effects of a range of other agricultural chemicals are unknown [3]. Many other chronic health effects are also being associated with pesticide use. The risk associated with pesticide residues in foods depends on the dosage of the chemical, the time of exposure, and the susceptibility of the individual human. The National Academy of Science (1987) estimates that nearly 60% of the carcinogenic risk from pesticides in foods comes from fungicides, 27% from herbicides, and

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13% from insecticides [58]. In addition, approximately 20% of the current cancer risk is associated with the consumption of processed foods; within this, fungicides account for approximately 75% of this risk. Linuron, a herbicide, accounts for more than 98% of the risk associated with herbicides, while the insecticides chlordimeform and pyrethrin account for more than 95% of the estimated dietary risk from insecticides [58]. The National Academy of Science (1987) also suggested 15 foods are associated with approximately 80% of the dietary cancer risks from pesticide residues. These include tomatoes, beef, potatoes, oranges, and lettuce. Tomatoes pose the highest estimated risk with a dietary carcinogenic risk as high as 15% of the total risk attributed to all pesticide residues as a result of the high use of fungicides [58]. The National Academy of Science (1987) concluded that the estimated additive cancer risk for the American public averages at no greater than 1 x 10"^ [58]. Archibald and Winter (1989) suggest a lower estimated cancer risk than the National Academy of Science [58]. However, Gots (1992) reports that it is impossible to directly relate any increase in cancers directly to pesticide residues [60]. The role of trace amounts of pesticide residues at the parts per million and parts per billion level are not understood and will continue to be debated. Some researchers suggest that the health risks associated from natural chemicals in foods are greater than those from pesticide residues in food [61, 62]. They claim that specific constituents of common fruits such as apples, bananas, grapefruit, orange juice, peaches, pineapples, and vegetables such as broccoli, cauliflower, cabbage, and celery are more toxic than chemical pesticide residues [64]. The extent of risk to human health associated with naturally occurring toxicants remains a scientifically contentious matter. There is an almost complete lack of data on the effects on human populations of long-term ingestion of natural toxicants in foods. At present, there is no firm evidence to demonstrate a link between common foods and any chronic human illness [64], and considerably less evidence supporting this than there is for the evidence that pesticide residues in food cause long-term health effects. Acute Poisonings There have been very few reported cases of acute pesticide poisoning from residues on food. In Singapore during December 1988, 110 people were treated for gastroenteritis after ingestion of a green leafy vegetable, known as gai-lan, that was contaminated with the organophosphates methamidophos and profenofos, and also dithiocarbamate. Based on the gastrointestinal systems of the patients and the depressed serum cholinesterase levels detected, methamidophos was assumed to be mainly responsible for the poisoning. Methamidophos is highly hazardous and has an LD50 in rats of 30 mg/kg body weight and its no-effect level (NEL) for cholinesterase activity in rates is 2 ppm. Profenophos is moderately hazardous with an LD50 of 350 mg/kg body weight. Its NEL is 0.38 ppm. In this case, human poisoning occurred after ingestion of approximately 0.56 mg of insecticide [65].


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In another case, California during July 1985 experienced the largest known outbreak of illness due to pesticide-contaminated food in the U.S.A. when the pesticide Aldicarb was illegally applied to watermelons. In that case, 1,370 illnesses were reported after the consumption of watermelons contaminated with the carbamate pesticide. Although no deaths occurred, some people were severely ill with possible life-threatening conditions: brachycardia, and hypotension. In addition, two stillbirths were reported in women who were pregnant at the time of their poisoning. Symptoms included vomiting, abdominal pain, blurred vision, loss of consciousness, seizures, and muscle twitching [19]. These cases illustrate the problems of assuring the safe use of potent pesticides. When inadvertent or illegal uses of a pesticide occur, there is no method of protecting the public from exposure. Mild symptoms are likely to be mistaken for those of unspecified gastroenteritis and the cause not investigated [19, 65]. The major problem in this area is establishing a scientifically proven link between pesticide exposure and the physical symptoms. Only where there has been relatively large-scale poisoning, requiring hospitalization of a large number of people, has a pesticide link been diagnosed. Lower-level exposures are unlikely to be detected and traced to pesticide exposure. It could be said that the vast majority of pesticide residue levels in food are relatively safe and are within legal limits. It is also true to say that some levels will continue to decrease, as people become aware of the adverse effects of certain pesticides and decline the usage of those chemicals. However, the effect of these low levels of pesticides is still poorly understood and may, for all we know, be contributing to long-term chronic health effects. REFERENCES 1. Melifrondes, I. D., and Kashouli-Kouppari, A. (1994). "Pesticide use and regulation in Cyprus." Reviews of Environmental Contamination and Toxicology, 134: 91-1,103. 2. British Medical Association (1990). Pesticides, Chemicals and Health. Report of the Board of Science and Education, October 1990. 3. Spynu, E. I. (1989). "Predicting pesticide residues to reduce crop contamination." Reviews of Environmental Contamination and Toxicology, 109: 89-107. 4. Barthel (1993). Pesticides and Cancer Risk, Z. Erkrank Atm-Org., 161: pp. 257-265. 5. Kashyap, R., Iyer, L. R., and Singh, M. (1994). "Evaluation of daily dietary intake of dichloro-diphenyl-trichloroethane (DDT) and benzene hexachloride (BHC) in India." Archives of Environmental Health, 49: 63-66. 6. United States Environmental Protection Agency (1990). Pesticide Exposure Study (NOPES), U.S. Environmental Protection Agency, Research Triangle Park, N.C.

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7. Lichtenberg, E., and Zilberman, D. (1986). "Problems of pesticide regulation: Health and environment versus food and fibre." In: Phipps, T. T., Crosson, P. R., and Price, K. A. (Eds). Agriculture and the Environment. Resources for the Future, Washington, D.C. pp. 123-145. 8. Hassall, K.A. (1990). The Biochemistry and Uses of Pesticides, 2nd edn, Macmillan Press Ltd, London. 9. Sly, J. M. (1977). "Changes in the use of pesticides since 1945." In: Perring, F. H., and Mellanby, K. (Eds.). Ecological Effects of Pesticides. Academic Press, London. 10. Pimental, D., McLaughlin, L., Zepp, A., Lakitan, B., Kraus, T., Kleinman, P., Vancini, P., Roach, W., Graap, E., Keeton, W., and Selig, G. (1991). "Environmental and economic impacts of reducing U.S. agricultural pesticide use." Handbook on Pest Management in Agriculture. Boca Raton, Florida: CRC Press, pp. 679-718. 11. Costa, L. G. (1986). "Toxicology of pesticides: A brief history." In: Costa, L. G., Galli, C. L., and Murphy, S. D. (Eds). Toxicology of Pesticides: Experimental, Clinical and Regulatory Perspectives. Springer-Verlag, Berlin, pp. 1-10. 12. Ekstrom, G., and Akerblom, M. (1990). "Pesticide management in food and water safety: International contributions and National approaches." Reviews of Environmental Contamination and Toxicology, 114: 23-55. 13. Beaumont, P. (1993). "GATT and pesticides in food." Chemistry and Industry, p. 424. 14. Jansma, J. E., van Keulen, H., and Zadoks, J. C. (1993). "Crop protection in the year 2000: a comparison of current policies towards agrochemical usage in four Western European countries." Crop Protection, 12: 483-489. 15. Lawson, H. M. (1994). "Changes in pesticide usage in the United Kingdom— policies, results and long term implications." Weed Technology, 8: 360-365. 16. Iwata, H., Tanabe, S., Sakai, N., Nishimura, A., and Tatsukawa, R. (1994). "Geographical distribution of persistent organochlorines in air, water and sediments from Asia and Oceania, and their implications for global redistribution from lower latitudes." fnv/ronm^nra/Pt^/to/ow, 85: 15-33. 17. Guidelines on Residue Trials (1981) Department of Primary Industry. Australian Government Publishing, Canberra. 18. BRR (1989). Report on National Residue Survey: 1987 and 1988 Results, Australian Government Publishing Service, Canberra. 19. Goldman L. R., Smith, D. F., Neutra, R. R., Saunders, L. D., Pond, E. M., Stratton, J., Walker, K., Jackson, R., and Kizer, K. (1990). "Pesticide food poisoning from contaminated watermelons in California 1985." Archives of Environmental Health, 45: 229-236. 20. AustraHan Science and Technology Council (ASTEC) (1989). Health, Politics and Trade. Controlling Chemical Residues in Agricultural Products. AGPS, Canberra.


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21. Americans For Safe Food (AFSF) (1988). The Wax Cover Up—What Consumers Aren 't Being Told about Pesticides in Fresh Produce. Center for Science in the Public Interest, Washington, D.C., U.S.A. 22. Americans For Safe Foods (1990). The Wax Cover Up. What Consumers Aren't Told about Pesticides on Fresh Produce. Washington, D.C. 23. Hayes, W. J. (1975). Toxicology of Pesticides. Waverley Press: Baltimore. 24. Precheur, R. J., Bennet, M. A., Ridel, R. M., Wiese, W. L., and Dudet, J. (1992). "Management of Fungicide Residues on Processing Tomatoes." Plant Diseases. 76: 700-702. 25. Eilrich, G. L. (1991). "Tracking the fate of residues from farm gate to the table." In: Tweedy, B. G., Dishburger, H. J., Ballantine, L. G., and McCarthy, J. (eds). "Pesticide Residues and Good Safety: a Harvest of Viewpoints." Am^ncaw Chemists' Society, Washington, D.C, pp. 202-212. 26. Winter, C. K. (1992). "Dietary pesticide risk assessment." Reviews of Environmental Contamination and Toxicology, 127: 23-67. 27. Elkins, E. R. (1989). "Effect of commercial processing on pesticide residues in selected fruit and vegetables." Journal of the Association of Official Analytical Chemists, 72: 533-535. 28. Food and Drug Administration (1994). Pesticide Program. Residue Monitoring 1994. 29. Chapman, P. J., and Mason, R. D. (1993). "British and European Community regulations and registration requirements for non-pesticidal co-formulants in pesticides and for adjuvants." Pesticide Science, 37: 167-171. 30. World Health Organization (WHO) (1990). "Principals for the Toxicological Assessment of Pesticide Residues in Food." (Publications of the World Health Organization: Finland.) 31. Loevinsohn, M. E. (1993). "Improving the pesticide regulation in the Third World: The role of the independent hazard auditor." Environmental Management, 17: 705-712. 32. Mbagwu, I. G., and Ita, E. O. (1994). "Pesticide use in the sub-humid zones of Nigeria: Implications for conservation of aquatic resources." Environmental Conservation, 21: 214-219. 33. Bajet, C. M., and Tejada, A. W. (1995). "Pesticide residues in the Philippines: an analytical perspective." Trends in analytical chemistry, 14: 430^34. 34. Pelfrene, A. F. (1986). "Pesticide use exposure and regulation in developed and developing countries." In. Costa, L. G., Galli, C. L., and Murphy, S. D. (Eds). Toxicology of Pesticides: Experimental, Clinical and Regulatory Perspectives. Springer-Verlag, Berlin, pp. 253-262. 35. Hayes, W. J. (1982). Pesticides Studied in Man. Baltimore, Williams and Wilkins, 1982. 36. Hallenbeck, W. H., and Cunningham-Burns, K. M. (1985). Pesticides and Human Health. (Springer-Verlag: New York.)

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37. Blair, D. (1989). "Uncertainties in Pesticide Risk Estimation and Consumer Concern." Nutrition Today, November/December 1989, pp. 13-19. 38. Martin, R. (1989). Health, Politics, Trade: Controlling Chemical Residues in Agricultural Products. Australian Government Publishing Service, Canberra. 39. Jerry, M. (1989) "Don't Downplay the Risk of Pesticides." Pest Control. 57 (10): 55. 40. The Parliament of the Commonwealth of Australia (POCA) (1990). Report of the Senate Select Committee on the Agricultural and Veterinary Chemicals in Australia. (Commonwealth government Printer: Canberra.) 41. Bureau of Rural Resources (BRR) (1992). Report on the National Residue Survey 1989-1990 Results (AGPS: Canberra). 42. Food and Drug Administration (1989). Residues in Food 1989. Food and Drug Administration Pesticide Program, Washington, D.C. 43. Wessel, J. R., and Yess, N. J. (1991). "Pesticide residues in foods imported into the United States." Reviews of Environmental Contamination and Toxicology, 120: 83-104. 44. Galal-Gorchev, H. (1991). "Dietary intake of pesticide residues, cadmium, mercury, and lead." Food Additives and Contaminants, 8: 793-806. 45. Herrera, A., Arino, A. A., Conchello, M. P., Lazaro, R., Bayarri, S., and Perez, C. (1994). "Organochlorine pesticide residues in Spanish meat of different species." Journal of Food Protection, 57: 441-444. 46. Leoni, V., Caricchia, A. M., Cremisini, C , Chiavarini, S., Fabiani, L., Morabito, R., Rodolico, S., and Vitali, M. (1995). "Levels of pesticide residues in food—evaluation of data from total diet studies in Italy." International Journal of Environmental Analytical Chemistry, 58: 411-422. 47. Trotter, W. J., and Dickerson, R. (1993). "Pesticide residues in composited milk collected through the United States pasteurised milk network." Journal of AOAC International, 76: 1,220-1,225. 48. Dogheim, S., Nasr, E., Almaz, M., and El-Tohamy, M. (1990). "Pesticide residues in milk and fish samples collected from two Egyptian govemorato." Jour. Assoc. Off. Anal. Chem., 73: 19-21. 49. Lenardon, A., Maitre de Hevia, M. I., and Enrique de Carbone, S. (1994). "Organochlorine pesticides in Argentinian butter." The Science of the Total Environment, 144: 273-277. 50. Smart, N. (1987). "Organophosphorous pesticide residues in fruits and vegetables in the United Kingdom and some other countries of the European Community since 1976." Reviews of Environmental Contamination and Toxicology, 98: 99-147. 51.Gianessi, L. P., and Greene, C. R. (1988). Vegetables and Specialties. Situation and Outlook Report. United States Department of Agriculture, pp. 27-41. 52. WHO/UNED (1989). Public health impact of pesticides used in agriculture. Geneva: World Health Organization/United Nations Environment Program.


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53. National Academy of Sciences (1987), Regulating Pesticides in Food, The Delaine Paradox, Washington, D.C., National Academy Press, 272 pp. 54. Gots, R. E. (1992). Toxic Risks: Science, Regulation and Perception. Lewis Publishers: Florida. 55. Ames, B. N., Magaw, R., Gold, L. S. (1987). "Ranking possible carcinogenic hazards." Science, 236: 271-280. 56. Ames, B. N., and Gold, L. S. (1989). "Pesticides, risks and applesauce." Science, 244: 755-757. 57. Culliney, T. W., Pimentel, D., Pimentel, M. H. (1992). "Pesticides and Natural Toxicants in Foods, Agriculture, Ecosystems and Environment," 41:297-320. 58. Goh, K. T., Yew, F. S., Ong, K. H., Tan, I. K. (1990). "Acute organophosphorus food poisoning caused by contaminated green leafy vegetables." Archives of Environmental Health, 45: 180-184.


CHAPTER 21 BIOLOGICAL MONITORING BY MEANS OF URINARY SAMPLES AND PROBLEMS CONCERNING CONCENTRATION-DILUTION OF SPOT URINE Andrea Trevisan Istituto di Medicina del Lavoro Universita di Padova Padova, Italy

CONTENTS INTRODUCTION, 453 UNADJUSTED URINE, 454 ADJUSTMENT FOR OSMOLALITY, 454 ADJUSTMENT FOR SPECIFIC GRAVITY, 455 ADJUSTMENT FOR CREATININE, 456 OTHER TYPES OF ADJUSTMENTS, 456 CONCLUDING REMARKS, 457 REFERENCES, 458 INTRODUCTION Urinary sampling is a method utilized to detect occupational xenobiotic exposure and to determine damages of the nephron caused by diseases, drugs, poisons, or industrial substances. Commonly, 24-hour collection of urine is of clinical use, but this type of collection is subjected to practical and scientific problems during biological monitoring in an industrial environment. The practical restriction is that a careful collection is often difficult even during hospitalization, and much more so on the job or during leisure time. The scientific restriction is that metabolites are frequently excreted during and immediately after the end of exposure to xenobiotics; thus, prolonged collection period intervals could cause a dilution of the samples with urine not containing the xenobiotic and therefore underestimate its true value. For these reasons, long time collection is not reliable during biological monitoring. Other timed collections (4, 8, 12 hours) show similar problems; in addition, a too-short period could cause problems related to an overestimation of the values. Another common difficulty is the precise control of the time-related volume. The type of urine collection that appears to be most suitable to monitor work exposure and possible effects of industrial substances on the kidney is urine spot sampling. Obviously, this method presents the problem of concentration-dilution of urine, and four practical solutions were suggested:

453

454


1) unadjusted 2) adjustment 3) adjustment 4) adjustment

urine; for osmolality; for specific gravity; for creatinine concentration excretion.

These, in addition to other types of adjustment requiring a short period of collection, are reviewed in this chapter. UNADJUSTED URINE Some authors support unadjustment of the urinary values during biological monitoring, justifying that a rough concentration factor, such as specific gravity, had been multiplied by a precise analytical measurement [1]. Other following studies [2-4], regarding creatinine adjustment also, agree against urine adjustment for concentration-dilution factors. The main argumentation supporting this choice is that no significant difference was observed among correlation between spot samples unadjusted, adjusted for specific gravity or creatinine, and values measured in 24-hour collected urine [3, 5]; thus, no practical advantage is offered by adjusted values in biological monitoring. ADJUSTMENT FOR OSMOLALITY Osmolality measures one of the colligative properties of water and depends on the number of particles of solutes in solution. Its unit of measure is mOsmoles for kg of water (mOsm/kg H2O): 1 mOsm/kg H2O means 6 x 10^^ particles of solutes are in one kg of water (1/1,000 of Avogadro's number). Sodium, the main extracellular cation, along with accompanying anions are the determinants of osmolality. In normal subjects, the value of osmolality in plasma is between 285-295 mOsm/kg H2O, with slight interindividual differences. On the contrary, depending on water consumption, urine osmolality shows high inter- and intraindividual differences, with values that range from less than 100 mOsm/kg H2O (high diluted urine), to urine that is four times the concentration of plasma with values higher than 1,000 mOsm/kg H2O (very concentrated urine). The maximum urine concentration capacity of the kidney is about 1,200 mOsm/kg H2O; this value is obtained when water consumption is limited to 0.5 1/day. Therefore, osmolality is a good parameter by which to measure concentration-dilution of urine, but, as with specific gravity (detailed in the next section), it is dependent on sodium and other solutes. Diseases that cause a variation of solute excretion, water restriction, or water loading, or an increase of sweat during work may modify osmolality. The use of osmolality adjustment is rare in biological monitoring and is used only for comparison with other methods of adjustment. In healthy subjects, an

Biological Monitoring by Means of Urinary Samples and Problems

455

almost perfect correlation between adjusted values for osmolality and for specific gravity was found [6]; this was not unexpected in that urinary excretion did not contain abnormal concentrations of proteins or sugar. The osmolality is measured by the freezing point depression method with an osmometer. ADJUSTMENT FOR SPECIFIC GRAVITY Specific gravity (relative density or density number) of a substance is the result of a ratio of its density to the density of a reference substance under conditions that must be stated for both. Commonly, the reference substance for liquids and sohds is water. Sodium chloride, urea, sulfate, and phosphate [7] are the main contributors to specific gravity. Urea contributes about 20%, and phosphate and sulfate account for another 20%. Wide variations of urinary specific gravity were observed in a healthy population (coefficient of variation ranging from 5.8% to 13%) submitted to 24-hour and consecutive urine collections [8]. According to these observations, a reference value for specific gravity is difficult to define. In the United Kingdom, a mean value of the standard population is 1.016, whereas in the United States this value is 1.024 [9]. These authors [9] observed that the urine's solid content is roughly proportional to the specific gravity of 1.024. In an Italian population of 333 subjects, the average specific gravity, using a 24-hour collection period, was 1.024, whereas in 1,079 spot specimens this value was 1.027 [10]. These differences agree with differences in salt, protein, and fluid intake and ambient temperature. As with osmolality, specific gravity may be influenced by dysfunction of kidney tubules with impairment of sodium, phosphate, and protein reabsorption. In these subjects, correction for specific gravity during biological monitoring seems inadequate. This is true particularly for workers exposed to substances that are toxic to the renal tubules. A standard specific gravity is needed before this type of adjustment can be made. Commonly, a reference specific gravity of 1.024 is often used, but this, or other choices such as 1.016, must always be specifically expressed with the results. According to Elkins et al. [11] the correction is made as follows: unadjusted value x 0.024 ^ Unit of measure (îg, mg, g or others)/liter (L) specific gravity of sample - 1.000 adjusted for a specific gravity standard of 1.024

It is common practice to reject over-diluted (specific gravity lower than 1.010) or over-concentrated (specific gravity higher than 1.030) urine samples. Recent research [10] supports a range in spot specimens between 1.010 and 1.035 because in 1,079 spot samples analyzed by the Laboratory of Industrial Toxicology of the Institute of Occupational Health of Padua University Medical School

456


(Italy), a high percentage (almost 40%) of urine samples showed specific gravity values higher than 1.030. If the acceptable range is increased to 1.035, only 6% of the values exceed the upper limit. In addition, specific gravity of 1.035 corresponds to a creatinine concentration of about 3 g/1, the suggested upper limit for this urinary parameter (detailed in the next section). Commonly, specific gravity is measured by means of a difractometer. ADJUSTMENT FOR CREATININE Folin was the first [12] to report that creatinine output during a 24-hour period was constant. On the contrary, following studies showed that daily excretion may change significantly not only in different subjects but in the same subject from day to day [13]. Creatinine excretion is related to body muscle mass and muscular activity; creatinine output is also affected by ingestion of cooked meats, but there are fewer dietary factors that affect excretion of creatinine compared to specific gravity. In 24-hour specimens, an average creatinine concentration of 1.25 g/1 was found, and the relationship with specific gravity of 1.024, calculated from the correlation equation, was 0.88 g/1 [10]. The choice of creatinine was related to its high concentration in urine and to the hypothesis that it was filtered by the glomerulus and not reabsorbed or secreted by the tubule. This conviction was corrected by the observation that a small portion of creatinine is added to the urine by active tubular secretion. Measurement of creatinine is based on Jaffe's alkaline-picrate reaction. Usually, unadjusted results are related to g (or nmioles) of creatinine; one gram of creatinine corresponds to 8.8 nmioles (molecular weight (WB) of creatinine is 113.07). Biological Threshold Limit Values (BTLVs) of substances and metabolites in urine are conventionally expressed adjusted for creatinine, as indicated in ACGIH publications [14] and by Lauwerys and Hoet [15]. As for specific gravity, urine with a creatinine concentration lower than 0.5 g/1 (4.4 mmoles/1) or higher than 3 g/1 (26.5 mmoles/1) should be discarded and the collection repeated. More than 95% of spot samples collected in our laboratory [10] show a creatinine concentration within this range. Furthermore, following observations [16] show that adjustment for creatinine is justified in a wide range of concentrations (too diluted or concentrated urine). OTHER TYPES OF ADJUSTMENTS Following early observation by Araki [17] that some urinary indices were influenced by urinary volume, an equation was introduced to eliminate the effects of urinary volume on urinary concentration [18]. This equation is as follows: U(V-i) = UixVl'


457

where:

U(v=i) = concentration adjusted to one ml urinary volume per minute; Ui = urinary concentration of the substance examined; Vi = urinary volume (ml/min); b = slope of regression coefficient of the relationship between urinary volume and urinary concentrations in each subject. This adjustment needs a timed collection of urine and a preliminary evaluation of "b" values for the measured substances, but the urinary volume-adjustment concentration is applicable to virtually all urinary substances within a wide variation in urinary volume [19]. In addition, concentration adjusted to urinary flow rate is independent by definition of urinary flow; therefore, this adjustment is applicable for highly diluted and highly concentrated urine samples without repetition of urine collection [20]. More recently, according to observations that creatinine elimination is not constant, adjustment for creatinine related to urinary flow was introduced [21]. The estimated slope (b) of the log-log dependence of creatinine excretion rate on urinary flow is 0.67 ± 0.07 standard error of the mean (SEM), and this value was used as an exponent for urinary volume to correct the measured concentrations to a standardized volume. Obviously, this type of adjustment required a timed, although short (60-90 minutes), collection. The following formula defines the urinary flow adjusted creatinine ratio: mg substance

1000 x substance concentration ^ x flow ^ ^^^

g creatinine

creatinine concentration ^^

Customarily, units are chosen as milligrams of substance per gram of creatinine (which requires multiplying the concentration ratio by 1,000). Substance concentrationM is the crude substance concentration (in mg/dl), and creatinine concentratiouM the measured creatinine concentration (in mg/dl). Flowî^^ is urinary flow (subscript M designates the measured volume) in ml/min, and superscript 0.67 is slope (b). CONCLUDING REMARKS Discussions about urinary values of xenobiotic substances measured in biological monitoring may often be academic. Omitting long-time collection for the known reasons, short-time collections are also difficult to obtain during or immediately after the work shift. Urinary volume adjustment or urinary flow-adjusted creatinine ratio are also difficult to apply, requiring timed collection and appHcation of complicated mathematical calculations.

458

Health and Toxicology Table 1 Intra-individual coefficients of variation of urinary values

Within day Between day

Creatinine

Specific gravity

Osmolality

Unadjusted

22.4 15^6

32.2 22^0

36.5 —

47.3 37.3

Results are drawn from [22].

A spot urine sample offers the only practical alternative in biological monitoring, and we agree with Pryde [22] that these are unacceptable without some adjustment for short-term concentration-dilution effects because of the wide variation in urine output from hour to hour. Creatinine correction reduces uncertainty of a spot urinary concentration, reflecting accurately the true substance excretion in an individual subject. Although the majority of researchers [4, 5, 16] do not find great differences in the relationship between values of unadjusted spot samples, samples adjusted for creatinine or specific gravity and 24-hour samples, adjustment for creatinine, and to a lesser extent for specific gravity, reduces scatter of the results and intraindividual coefficients of variation [23], as summarized in Table 1. Moreover, to evaluate individual exposure, unadjusted urine appears inadequate because in occupational medicine the aim is the safety of the workers and the reduction of the exposure within acceptable limits. Thus, a single unadjusted value, influenced by concentration-dilution of urine, could be underestimated or overestimated, and could cause failure of prevention. In conclusion, adjustment of urinary values in biological monitoring is needed for the explained reasons. Adjustment for creatinine appears more satisfactory in that it yields results in subjects with altered renal function, a situation that instead influences specific gravity and osmolality. Furthermore, adjustment for creatinine appears to offer less intraindividual variations. REFERENCES 1. R. J. Graul and R. L. Stanley, "Specific gravity adjustment of urine analysis results," Am. Ind. Hyg. Assoc. J. 43, 863 (1982). 2. C. N. Ong, B. L. Lee, S. C. Foo, H. Y. Ong, and L. H. Chua, "Specific gravity adjustment for urinary analysis of 6-aminolevulinic acid," Am. Ind. Hyg. A^ôc.y. 4(5, BIO (1985). 3. A. Berlin, L. Alessio, G. Sesana, A. Dell'Orto, and I. Ghezzi, "Problems concerning usefulness of adjustment of urinary cadmium for creatinine and specific gravity," Int. Arch. Occup. Environ. Health 55, 107 (1985).


459

4. A. DeirOrto, A. Berlin, F. Toffoletto, B. Losito, and L. Alessio, "Creatinine and specific gravity adjustment of ALA in urinary spot samples: Is there any need?," Am. Ind. Hyg. Assoc. J. 48, A331 (1987). 5. L. Alessio, A. Berlin, A. Dell'Orto, F. Toffoletto, and I. Ghezzi, "Reliability of urinary creatinine as a parameter used to adjust values of urinary biological indicators," Int. Arch. Occup. Environ. Health 55, 99 (1985). 6. T. E. Barber and G. Wallis, "Correction of urinary mercury concentration by specific gravity, osmolality, and creatinine," J. Occup. Med. 28, 354 (1986). 7. J. W. Price, M. Miller, and J. M. Hayman, "The relation of specific gravity to composition and total solids in normal human urine," J. Clin. Invest. 19, 537 (1940). 8. H. Buchwald, "The expression of urine analysis results-observations on the use of a specific gravity correction," An/i. Occup. Hyg. 7, 125 (1964). 9. L. Levine and J. P. Fahy, "Evaluation of urinary lead determinations. I. The significance of the specific gravity," J. Ind. Hyg. Toxicol. 27, 217 (1945). 10. A. Trevisan, "Concentration adjustment of spot samples in analysis of urinary xenobiotic metabolites," Am. /. Ind. Med. 17, 637 (1990). 11. H. B. Elkins, L. D. Pagnotto, and H. L. Smith, "Concentration adjustments in urinalysis," Am. Ind. Hyg. Assoc. J. 35, 559 (1974). 12. O. Folin, "Approximately complete analyses of thirty 'normal' urines," Am. J. Physiol. 13, 45 (1905). 13. G. Curtis and M. Fogel, "Creatinine excretion: Diurnal variation and variability of whole and part day measures," Psychosom. Med. 32, 337 (1970). 14. Threshold Limit Values and Biological Exposure Indices. ACGIH 1994-95. 15. R. R. Lauwerys and P. Hoet, Industrial Chemical Exposure. Guidelines for Biological Monitoring, 2nd ed., Lewis Publishers, 1993. 16. A. Trevisan, G. Nicoletto, S. Maso, G. Grandesso, A. Odynets, and L. Secondin, "Biological monitoring of cadmium exposure: Reliability of spot urine samples," Int. Arch. Occup. Environ. Health 65, 373 (1994). 17. S. Araki, "The effects of water restriction and water loading on urinary excretion of lead, 6-aminolevulinic acid and coproporphyrin," Br. J. Ind. M^J. 55, 312(1978). 18. S. Araki, "Effects of urinary volume on urinary concentrations of lead, 6aminolevulinic acid, coproporphyrin, creatinine, and total solutes," Br. J. Ind. Med. 37, 50 (1980). 19. S. Araki, H. Aono, and K. Murata, "Adjustment of urinary concentration to urinary volume in relation to erythrocyte and plasma concentrations: An evaluation of urinary heavy metals and organic substances," Arch. Environ. Health 41, 171(1986). 20. S. Araki, F. Sata, and K. Murata, "Adjustment for urinary flow rate: an improved approach to biological monitoring," Int. Arch. Occup. Environ. Health 62, 471(1990).

460


21. G. N. Greenberg and R. J. Levine, "Urinary creatinine excretion is not stable: A new method for assessing urinary toxic substance concentrations," /. Occup. Med. 31, 832 (1989). 22. D. E. Pryde, "Is it appropriate to adjust the results of urine analyses for concentration-dilution effects?," International Workshop on Biological Indicators of Cadmium Exposure: Diagnostic and Analytical Reliability, CEC-IUPAC, Luxembourg, 7-9 July 1982. 23. H. J. Mason and I. M. Calder, "The correction of urinary mercury concentrations in untimed, random urine samples," Occup. Environ. Health 51, 287 (1994).

CHAPTER 22 CAUSES OF ARTIFACTS IN SORPTION STUDIES WITH TRACE ELEMENTS Lorenzino Giusti School of the Environment, Sunderland University, Benedict Building, St. George's Square, Sunderland, SR2 7BW, UK CONTENTS INTRODUCTION, 461 SORPTION STUDIES: THEORETICAL BACKGROUND, 462 ARTIFACTS IN SORPTION STUDIES, 466 Artifacts caused by contamination, 466 Artifacts caused by separation techniques and type of sorbent material, 466 Artifacts associated with the use of voltammetric methods, 473 Artifacts caused by the use of radioactive tracers, 474 Artifacts caused by incorrect data manipulation, 475 SUMMARY, 481 REFERENCES, 485 INTRODUCTION The interactions between solid phases and trace elements dissolved in a liquid phase have been the object of investigation by scientists working in very diverse disciplines. Examples of fields in which sorption plays an important role include the following: the leaching of elements from the lithosphere and their transport and deposition in aquatic environments, pedogenetic processes, the transfer of nutrients from soil to vegetation, and more in general the uptake of substances by living organisms; transmembrane ion fluxes in patients with a number of physical and mental disorders. Environmental chemists, ecotoxicologists, and water engineers need to investigate the partitioning of trace elements between solution and particulate material as this will affect their bioavailability, toxicity, and the methods to be used in the treatment of water intended for domestic and industrial uses. Nuclear scientists are interested in the sorption mechanisms taking place between radionuclides and soil particles, sediments, suspended material in aquatic environments, living cells, humic substances, clays, and materials used to encapsulate nuclear waste. Waste disposal engineers have to ensure the efficient sequestration by natural or synthetic sorbents of potentially toxic pollutants that may contaminate groundwater. The reliability of the data obtained from sorption experiments and related computer simulations must be carefully assessed in order to rule out the possibility that

461

462


some of the results may be attributed to experimental artifacts. Some of these problems have been described in the literature [1-5]. The purpose of this chapter is to describe the main sources of these and to review the methods used for their detection and correction. SORPTION STUDIES: THEORETICAL BACKGROUND The aim of this section is to describe the basic principles relating to the interactions between particulate material and trace elements, and to summarize the terminology commonly used in sorption studies. Sorption processes such as adsorption, absorption, ion exchange, and surface precipitation are interactions between solutes (sorbates) and sorbing phases (sorbents). Figure 1 illustrates the main categories of sorption. The term adsorption refers to the reactions of a solute with surface functional groups of sorbing phases (sorbents) such as living or dead organic matter; humic and fulvic acids; aluminosilicates (especially clays); iron-, manganese-, and aluminium oxyhydroxides; and sulphides. In this process, the solute (sorbate) accumulates on or near the surface of the sorbent. Depending on the prevailing type of attractive forces [6], adsorption can be physical (involving polar molecules), chemical (chemisorption, arising from hydrogen and covalent bonds), and electrostatic (due to ion-ion and ion-dipole forces). The term absorption is used to describe the actual penetration of the solute into the sorbent. Ion exchange involves the replacement of an ion in a solid phase by another ion present in the solute. When the solute concentration near the surface of the sorbent is relatively high, the solute forms a precipitate onto the surface, i.e., a new solid phase; in this case the interaction between the different phases is called precipitation. The uptake of metal ions and other elements or compounds by living cells can be achieved in a number of ways depending on the level of specialization of the cell itself [7]. The terminology used by biochemists and clinical pharmacologists to describe the possible interactions of metals with living tissue is thus more complex. The term ligand refers to any substance which binds to a specific binding site, and the term binding site is normally used for any molecule of the cell membrane which interacts with the ligand. The behavior of trace elements in biogeochemical cycles depends on two main factors: (i) the chemical species in which they are present in the system, and (ii) the interactions of trace metals with dissolved complex formers and with particulate matter. Although metal complexation by organic compounds and inorganic ligands in natural water keeps a significant fraction of metals in solution [8-14], a number of studies has indicated that an important fraction of trace elements is associated with suspended particles and sediments [15-17]. According to Martin and Meybeck [18], more than 95% of heavy metals in freshwater is transported in the form of

Causes of Artifacts in Sorption Studies with Trace Elements ADSORPTION

463

(a)

Solid particlej

ABSORPTION

(b)

Figure 1. Simplified models representing three sorption mechanisms: (a) adsorption, in which the solute (Pb) reacts with the surface of the solid particle (sorbent); (b) absorption, characterized by the penetration of the solute Into the particle; and (c) ion exchange, which results in the replacement, both on the surface and inside the sorbent phase, of one ion (Zn in this case) by another (Pb).

particulate material. In aqueous environments, the distribution of metals between natural water and suspended particles (inorganic and organic) depends mostly on i) the affinity of metals for solute ligands and solid particles, ii) particle coagulation and sedimentation rates, iii) the seasonal composition of settling particles, iv) pH conditions, and v) the ratio of metal concentration to suspended particle concentration. Fe- and Mn-oxides are known to be important scavengers in lake water, sea water, and soils [19-21]. Organic matter, in the form of phytoplankton and humic substances, is a major carrier of metal ions [22-25]. Carbonates and aluminosilicates appear to be less efficient in removing metals from surface water [26].

464


Suspended and settling particles tend to agglomerate and form heterogeneous particles. In natural water, inorganic particles are normally coated with humic substances and other organic material [27, 28]. As there is no completely successful method for the determination of the metal fraction bound to each type of carrier phase, model substances (especially hydrous metal oxides) have been used in many laboratories to mimic components of the natural particles [29-33]. Useful information concerning the adsorption/desorption mechanisms of trace metals have been obtained from these experiments. The fewer studies carried out with natural particles [19, 22, 24, 34-37] have confirmed that the sorption models developed for artificial phases may be applicable to more complex natural mixtures of particles. However, longer metal equilibration times have been reported in natural settings than in experiments with pure metal oxides [37], the difference being ascribed to biological activity or considered to be a result of the use of more realistic particle concentration. Also, the metal binding capacity of cell surfaces has been found to be orders of magnitude larger than for metal oxides [22, 24]. The effect of the simultaneous presence of natural concentrations of mineral surfaces and humic substances has only rarely been investigated [36, 38, 39] despite the fact that humic-metal interactions may modify the pH dependency and kinetics of the metal adsorption. Many adsorption studies failed to take into account particle residence times. The reported conclusions may thus be valid only for systems where the particle residence time is long with respect to reaction half-life. Normally, adsorption of trace metals onto particulates increases from nearly 0% to almost 100% as the pH increases. By plotting the adsorption data as a function of pH, a typical S-shaped curve is obtained, usually referred to as the adsorption (or binding) curve. The pH range over which absorption increases from undetectable levels to almost 100% is called the "adsorption edge." The position of the adsorption edge on the pH scale is an indication of the degree of affinity of the metal for the binding sites on the surface of the particulate phase(s). Figure 2 summarizes the typical adsorption trends reported for some trace metals. The following generalizations can be drawn from the available information: (i) metal binding to particles increases with increasing pH, (ii) increased adsorption (at a given pH) is observed with increasing particle concentration, reaction time, and temperature, (iii) decreased adsorption occurs with increasing metal concentrations, and increasing competing cations. The latter decrease the available binding sites. Some anions (e.g., sulphate) appear to promote adsorption of metals [30]. In experiments with synthetic and natural samples (sorbents and sorbates), metal desorption processes can be detected, especially in media of relatively high pH.

Figure 2. Diagrams showing the general sorption trends of metals onto sorbent phases. The percent sorption onto a constant amount of particulate material increases with increasing pH (all diagrams), decreases with increasing metal concentration (a), and competing cations (b), and increases at higher temperature (c), longer reaction time (d). Assuming other parameters are constant, increasing particle concentration has the effect of increasing fractional sorption of the metal in solution (e).

466


ARTIFACTS IN SORPTION STUDIES Artifacts Caused by Contamination Contamination may affect both accuracy and precision of analytical results. It may occur during sampling, transport, storage, sample preparation, or during analysis in the laboratory. Sampling methods, sampling equipment, containers, labware, or dust may introduce contamination in trace and ultra-trace analysis. The addition of stabilizing reagents and buffers can also be responsible for significant levels of contamination. Routine quality controls and interlaboratory calibrations are a must to minimize potential errors. Proper selection and maintenance of sampling equipment and labware, clean-air facilities, and routine blank determinations (partialmethod and total-method blanks) are likely to result in a considerable reduction and correction of bias associated with contamination. In trace-element adsorption studies, particular care is necessary when physical separation of sorbent phases from the incubating medium is required. Contamination at this stage and in the subsequent steps before analysis is quite common given the small amounts of analyte normally involved and the contaminants that can be released by filtration apparatus. In addition to causing changes to the physico-chemical speciation of the trace elements, preconcentration procedures on natural samples are likely to result in high blanks. With the exception of cases of extremely polluted environments, the concentration of trace elements of environmental samples is usually quite low (often smaller than 10"^ to 10"^ mol). Similarly, synthetic solutions containing extremely low concentrations of metals are required in simulated sorption studies designed to mimic environmental conditions. Contamination and adsorption during storage and separation procedures must thus be minimized. Useful reviews of recommended techniques have been published [40-42]. Many workers avoided the problems associated with contamination by setting up experimental conditions in which the solute under investigation and/or the sorbent phase(s) were present in high or very high amounts. Consequently, contamination from lab apparatus and sorption onto containers and filters became unimportant. The main shortcoming of the data gathered from these experiments is their limited applicability to natural settings. Artifacts Caused by Separation Techniques and Type of Sorbent Material Filtration and Ultrafiltration Filtration and ultrafiltration (i.e., filtration with membranes of nominal pore size smaller than 15 nm) will inevitably affect the concentration and speciation of trace metals in filtered solutions. Important fractions of trace elements are normally associated with particulate and colloidal material so that the size discrimination of filters and ultrafilters will vary depending on pore size and material used. Filter membranes with different nominal pore size will give filtrate residues containing different amounts, size fractions, and variety of sorbing particles. Clogging of

Causes of Artifacts in Sorption Studies with Trace Elements

467

filters arid ultrafilters may cause adsorption of trace metals onto the material retained by the membranes [43]. Large fractions of the trace metals adsorbed onto suspended particles in aquatic systems are likely to be associated to colloidal-sized particles (< l|Lim) as a result of their large surface area. As colloids can pass through the pores (0.45|im, 0.2|Lim, 0.1 îm) of filter membranes conventionally used to separate the "dissolved" fractions of trace metals, binding experiments that make use of filtration may be affected by large errors, unless a strict experimental protocol including a number of control and blank determinations is followed [4]. Values of partition coefficients (K^, see below) have been found to decrease with increasingly high concentration of suspended soHds [44, 45]. One of the possible explanations of this trend is improper phase separation with membrane filters (e.g., increasing concentrations of colloidal particles passing through the filter pores that are accounted for as "dissolved" fraction). The lapse of time between collection of natural samples and execution of an experiment must be taken into consideration because coagulation of colloids into larger particles is bound to cause errors of size fractionation of the particles in suspension and thus bias the final partitioning of trace metals between "dissolved" and "particulate" fractions. An example of this problem is summarized in Table 1, which shows the variations in Fe concentration in natural water samples collected from the anoxic hypolimnion of a lake. These variations are a direct result of the filtration/centrifugation of the water, of the oxidation of the samples (Fe^"^ -^ Fe^"^) and a function of the time lag between sampling and analysis. The term "colloidal pumping" [46] describes the gradual accumulation of trace metals into increasingly larger particles forming as a result of aggregation of colloids in natural aquatic environments. Artifacts caused by changes of chemical/physical properties of natural particles prior to filtration can be minimized by carrying out the filtration immediately after Table 1 Changes in Fe concentration (MQ/L) in anoxic water samples from Esthwaite Water, Lake District, Cumbria, UK (n = 3) Sample treatment

Not filtered/Not centrifuged Filtered (0.45îm)/Not centrifuged Not filtered/Not centrifuged Filtered (0.45|Lim)/Not centrifuged Not filtered/Centrifuged Not filtered/Centrifuged Not filtered/Centrifuged Not filtered/Centrifuged

Fe concentration {\ig/L)

311.5 267.4 89.2 85.5 190.4 171.1 149.7 60.8

(5.7) (3.2) (4.3) (0.6) (1.2) (1.7) (2.5) (1.7)

Time of analysis

2h 2h 2d 2d 2h Id 2d 28d

after after after after after after after after

collection collection collection collection collection centrifugation centrifugation centrifugation

468


sampling. During filtration, a relatively low flow rate and stirring may reduce the problems of particle coagulation and sorption by the filters [47]. Coagulation and settling of particles during sorption experiments are also important problems that can be partially offset by gentle stirring and sonication. Filtration can thus cause a number of artifacts resulting in apparent increased or decreased metal concentrations. Figure 3 shows the concentration of Al detected in samples of surface water filtered with different cellulose membranes. In this case dialysis was more effective, though more time-consuming, in removing colloidal and larger particles. In general, the smaller the nominal pore size of the filter membrane the smaller the Al concentration in the "dissolved" fraction. Samples from sites 7 and 8 did not follow this trend, probably due to their high concentration in humic substances and other organic material. Living or dead cells in suspension may break during filtration, especially when excessive positive or negative pressure is applied, and release a number of elements and organic compounds that affect the behavior of trace metals. Filters, ultrafilters, and dialysis membranes should normally be decontaminated with diluted nitric acid and later rinsed in distilled deionized water. In order to prevent losses of trace elements, apparatus and filters may also need to be preconditioned with solutions such as NaN03 or Ca(N03)2. Figure 4 shows a reduction in Pb losses at pH 6.3 when a 0.005 M NaN03 solution is used instead of Milli-Q water.

300

z o p

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