The
-
Carcinogenic Effects Polycyclic Aromatic Hydrocarbons r
Imperial College Press
The
Carcinogenic Effects r
Polycyclic Aromatic Hydrocarbons
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The
Carcinogenic Effects r
Polycyclic Aromatic Hydrocarbons
Editor
Andreas Luch Massachusetts Institute of Technology, USA
-jffi
Imperial College Press
Published by Imperial College Press 57 Shelton Street Covent Garden London WC2H 9HE Distributed by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE
Library of Congress Cataloging-in-Publication The carcinogenic effects of polycyclic aromatic hydrocarbons / editor, Andreas Luch. p. cm. Includes bibliographical references and index. ISBN 1-86094-417-5 (alk. paper) 1. Polycyclic aromatic hydrocarbons-Carcinogenicity. 2. Polycyclic aromatic hydrocarbons-Toxicology. 3. Chemical carcinogenesis. 4. Genetic toxicology. J. Luch, Andreas. RC268.7.P64C374 2004 616.99'4071-dc22
2004056977
British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.
Copyright © 2005 by Imperial College Press All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.
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Typeset by Stallion Press Email:
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For my two beloved children,
fttina & (Kubin who were born amidst the work on this book
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Preface
When I agreed in August 2002 to work on a book entitled The Carcinogenic Effects of Polycyclic Aromatic Hydrocarbons I finally overcame a period of almost a year of thinking and hesitation. Besides arranging my move from Munich to Boston, I was not sure about whether it would be worthwhile to invest a great amount of time and effort into a project that most researchers these days would rather consider somewhat 'old-fashioned' and boring. Their credo, "Cancer is a disease of the genes", has developed over the past decades as a result of the rise of molecular genetics and the discovery of genetic traits underlying tumorigenesis. But is it really? The relative contribution of heritable genetic constitution vs. environmental factors in cancer causation has been a matter of debate ever since the discovery of 'oncogenes', and even long before. Evidence from epidemiological observations, working place or migration studies however, points to environmental factors as the major players. In the age of cancer genetics it therefore seems reasonable to recall the importance of chemical carcinogenesis and to outline our present knowledge on the molecular mode of action of a very important and ubiquitously present group of tumorigenic compounds, the polycyclic aromatic hydrocarbons. The scientific work on carcinogenic polycyclic aromatic hydrocarbons as a part of Environmental and Molecular Toxicology requires knowledge and input not only from experts in the field of 'molecular' biology or 'molecular' pathology, but primarily the analytical skills and achievements of chemists and biochemists well-educated and trained to think in dimensions of molecules and their three-dimensional occurrence and interactions vii
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(stereochemistry). Work on carcinogenic chemicals indeed is molecular toxicology in its strictest sense, not to be mixed up with macromolecular biology and genetics which an increasing number of toxicology departments are mainly focusing on these days. In the country where I come from, Molecular Toxicology will only survive when it saves its roots and keeps its interdisciplinary character as a highly interactive field of research stimulated and further strengthened by all natural sciences and human medicine. This interplay of many disciplines is what makes the work on biological effects of chemicals interesting and exciting but also rather difficult and complex. But to return to the main point: assessing the biological effects of chemicals is the central issue, the mainstay in toxicology! — and that's true per definitionem. Research that avoids the application of chemicals and the investigation of their conversion, reactivity and fate within the living organism has nothing to do with toxicology. There were essentially these two issues that inspired me to get into this project: to recall the importance of chemically-induced carcinogenesis in human cancer etiology, and to demonstrate that the work on biologically active compounds may still be interesting and, to use a more modern word, 'en vogue'. It opens the doors to a highly interdisciphnary field of research and provides fascinating insights into biological (and pathological) processes at a truly molecular level. Carcinogenic polycyclic aromatic hydrocarbons are among the most interesting and important compounds in environmental toxicology. The main goal of this book is to outline and to communicate the progress that has been achieved during the past decade in this field. I deeply hope that it will find its place and acceptance in the scientific community. This book is dedicated to Anthony Dipple. I decided to ask some of his long-term friends and colleagues to write a paragraph to honor him as an outstanding researcher and an amiable human being. To my knowledge, this book is the first monograph on carcinogenic polycyclic aromatic hydrocarbons that has been published since his premature death in 1999. Although I saw Tony only occasionally during scientific meetings, he left a huge impression on me. I was equally fascinated by his scientific achievements and personality. Since he committed his lifetime work to the investigation of carcinogenic hydrocarbons, this book may be an appropriate place
Preface •
ix
to remember him and to recall the importance of his contributions to our research field. Finally I want to thank all my co-authors for their great work and the time they invested to nicely cover a specilc topic in thefieldof carcinogenic polycyclic aromatic hydrocarbons. On my personal side, most credit goes to my lovely wife, Ina, who never complained about the additional burden and strain during a time when everything changed dramatically due to the arrival of two new souls to our small family. To help to make this book project possible despite the culminating workload far away from our home town and families, that surely is her invaluable merit. Andreas Luch Cambridge, MA, Summer 2004
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In Memoriam:
Anthony Dipple (1940-1999)
GS£,
thony (Tony) Dipple dedicated most of his si Minn better understanding of chemical carcinogenesis and ~| mechanisms underlying the actions of polyci. hydrocarbons. Over the span of his career, he displ.iuinterests ranging from mechanistic organic chemist! \ molecular biology. Tony was born on February 9, 1940 Mansfield, England. After finishing Queen Elizabeih Grammar School for Boys in Mansfield, where he was- .i chess captain, a member of the football (soccer) team and a prefect in his senior year, Tony was awarded .i university scholarship. In 1961 he received his B.Sc. in I*' Chemistry at the University of Birmingham. In 1964. J Tony submitted his Ph.D. thesis work, "Studies on Chemical Degradation of Ribonucleic Acids", performed under the supervision of Dr. A. S. Jones, and a week after receiving his Ph.D. in Biological Chemistry from the University of Birmingham, Tony and his wife, Hilary, were on their way to the United States on the Queen Mary for Tony's postdoctoral studies. From 1964 -1966, as a Damon Runyon Cancer Research Fellow, Tony worked on synthesis of fluoropyrimidine nucleosides as potential anti-cancer drugs in the McArdle Laboratory, University of Wisconsin, Madison. During this time, his daughter Joanne was born in 1965. The small family returned to England in 1966, when Tony started as a lecturer at the Institute of Cancer Research, Chester Beatty Research Institute in London, where he stayed until 1975. Here, Tony began research on polycyclic aromatic hydrocarbon carcinogen interactions with DNA, a field of interest he continued to pursue until his death. He synthesized reactive derivatives of polycyclic aromatic hydrocarbons such as 7-bromome thy lbenzfa] anthracene, and found that they reacted specifically with the amino groups of the purine DNA bases. These DNA-reactive model compounds were made available by Tony to many colleagues for studies of mutagenesis, toxicity and cell transformation. During the years in London, Tony's family grew, with the births of Geoffrey and Christopher in 1967 and 1969.
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In Memoriam: Anthony Dipple (1940-1999)
In 1975, Tony moved back to the United States to join the newly created NCI-Frederick Cancer Research and Development Center in Frederick, Maryland. As head of the Molecular Aspects of Chemical Carcinogenesis Section and later also as director of the Chemistry of Carcinogenesis Laboratory, Tony continued his studies of mechanisms involved in the initiation of cancer by chemicals. Over the years, Tony mentored numerous postdoctoral fellows and assistants in his laboratory. The success of the laboratory benefited from contributions from his colleagues C. Anita Bigger, Robert C. Moschel and Karen H. Vousden in Frederick as well as from numerous collaborators including William M. Baird, Ronald G. Harvey, Shantu Amin, Donald M. Jerina and their associates. The laboratory was highly productive, and highlights of these investigations are described below. A major area of study involved the chemistry of alkylation of DNA by benzylating agents and styrene oxides. Simple alkylating agents were found to modify primarily the N-7 ring nitrogens and to a lesser extent the exocyclic oxygen atoms, whereas the benzylic alkylating agents derived from polycyclic aromatic hydrocarbons modified the exocyclic amino groups, often quite extensively. These investigations led to the conclusion that reactions on the exocyclic amino groups of the purine bases require a considerable degree of SN1 character, whereas reactions on the ring nitrogens (specifically N-7) are favored for agents which react via a pathway with greater SN2 character. Studies of a deoxyadenosine adduct generated by reaction with the directly alkylating 7-bromomethyl -12-methylbenz[a]anthracene led to the first crystal structure of a polycyclic aromatic hydrocarbon-nucleoside adduct. 7,12-Dimethylbenz[a]anthracene is one of the most potent tumor initiators among the polycyclic aromatic hydrocarbons. The finding in Tony's laboratory that there is poor repair of DNA damage caused by this carcinogen may be associated with its high potency. Since the highly hindered fjord-region 3,4-diol 1,2-epoxides of 7,12-dimethylbenz[a]anthracene and benzo[c]phenanthrene both react extensively with deoxyadenosine, it was proposed that the poor repair of the deoxyadenosine adducts was responsible for their high tumorigenicity. Less hindered bay-region diol epoxides derived from carcinogens such as benzo[a]pyrene, chrysene and benz[a]anthracene modify deoxyguanosine to a greater extent than deoxyadenosine and are less tumorigenic. To investigate the mutagenic properties of the reactive diol epoxide metabolites of the polycyclic aromatic hydrocarbons, molecular biology approaches were used. A shuttle vector system (pS189) was used and showed that the mutational spectra for diol epoxides from benzo[c]phenanthrene, 5-methylchrysene and 7-methylbenz[a]anthracene were not identical, although some similarities among the types of mutations were found. To examine further the correlation between adduct structure and mutations, site-specific mutagenesis was investigated with diol epoxide adducts derived from benzo[c]phenanthrene and benzo[a]pyrene in two different DNA sequence contexts using a bacteriophage (M13) as a vector in E. coli. Both sequence context and adduct structure had complex and interdependent effects on mutational frequencies and distributions. This complexity may have resulted from the involvement of multiple DNA polymerases in E. coli with different specificities for nucleotide incorporation opposite individual adducts. Tony's most recent area of research interest involved the effects of polycyclic aromatic hydrocarbon diol epoxides on the cell cycle. These potent carcinogens were found to damage DNA in human cells without inducing the expected cell cycle arrest in the Gl phase. Normally DNA damage triggers Gl cell cycle arrest to allow for DNA repair before the cell enters S (synthesis) phase during which DNA replication occurs. This lack of G l arrest is likely to enhance error-prone insertions opposite the unrepaired adducts in the S phase and lead to enhanced mutations compared to other types of DNA damage.
In Memoriam: Anthony Dipple (1940-1999)
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As Executive Editor and co-founder in 1980 with Dr. Colin Garner of the journal Carcinogenesis, Tony was well-known within the scientific community. Dedicated efforts by the two editors were rewarded as Carcinogenesis became a highly respected, premier journal. He was a member of the editorial boards of Chemical Research in Toxicology, Woman and Cancer and of the Editorial Academy of the International Journal of Oncobgy. In addition, Tony served on NIH site visit panels and review boards, such as the NIH Chemical Pathology Study Section, the NIEHS Environmental Health Sciences Review Committee, the American Institute for Cancer Research Review Panel and the American Cancer Society Advisory Committee for Biochemistry and Carcinogenesis. In 1987, Tony was awarded a Doctor of Science in Biological Chemistry by the University of Birmingham for his research accomplishments and scientific excellence in his field. Tony is remembered by his colleagues as "a pleasure to work with on professional matters". He had an "ability to make a point without diminishing his opponent which resulted from clear thinking and remarkable personal graciousness". They "had great respect for Tony because of his outstanding and seminal contributions to our field, and for his qualities as a person", "a towering intellect". "As editor of Carcinogenesis Tony gained his greatest professional recognition... his forbearance was enormous..." "His thinking was focused on the problem in question. He was kind and compassionate and had a very pleasant demeanor. Even when he had to reject a paper submitted to Carcinogenesis he did so humanely. He was noted for writing rather long sentences some of which filled an entire paragraph that still retained absolute clarity." "I saw Tony as a positive, forward looking, matter-of-fact person." "Tony was a kind, gracious person, always there for those who needed advice, always patient, as if he had all the time in the world to listen." "All those who worked with him will miss his patience as a teacher, and the goodwill and support he gave to all." As the quotations above demonstrate, Tony was a warm and generous person who enjoyed life. Although his health did not allow challenging physical activities after his two serious health incidents (a heart attack in 1984 and a kidney failure and transplant in 1990), he found ways to exercise and at the same time enjoy the outdoors. He could spend hours biking on the Chesapeake and Ohio Canal tow path and traversed the entire 185 miles from Washington, DC to Cumberland, MD in bits and pieces. Most recently he began playing golf as a particularly enjoyable pastime. Another favorite recreation was camping on the beach at Assateague State Park, where he found peace and rest from the stress at work, and where the main worry would be what to prepare for dinner. He found cooking at the campsite over an open fire as much fun as preparing a feast for friends at home. There, he would delight in playing the organ and coax them into singing together after dinner. Tony is missed by his many friends and colleagues for his analytical mind and ability to dissect a problem, but most of all for the cheerful times they shared. Ingrid Ponten, 1 Jane M. Sayer2 and Donald M. Jerina 2 1
Safety Assessment, AstraZeneca R&D Sodertalje, S-15185 Sodertalje, Sweden Email:
[email protected] 2
Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda MD 20892, USA Emails:
[email protected] &
[email protected] Acknowledgement: Information for this article was obtained from Dipple A, Curriculum vitae; Dipple A, "Mechanisms of action of chemical carcinogens", D.Sc. thesis, 1987; Dipple A (1999) int. J. Oncol. 14:1019 -1020; and Bigger C A H {1999) Erewran. Moi. Mutagen. 34: 227 - 232.
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List of Contributors
Shantu Amin American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, NY, USA E-mail:
[email protected] William M. Baird Oregon State University, Department of Environmental and Molecular Toxicology, Corvallis, OR, USA E-mail: william. baird® orst. edu Ahmad Besaratinia Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, CA, USA E-mail:
[email protected] Karam El-Bayoumy American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, NY, USA E-mail:
[email protected] Nicholas E. Geacintov Chemistry Department, New York University, New York, NY, USA E-mail: nicholas. geacintov @ nyu. edu
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List of Contributors
Hansruedi Glatt German Institute of Human Nutrition, Department of Toxicology, Potsdam-Rehbriicke, Germany E-mail:
[email protected] Ari Hirvonen Laboratory of Biomonitoring, Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland E-mail:
[email protected] Andreas Luch Massachusetts Institute of Technology, Center for Cancer Research, Cambridge, MA, USA E-mail:
[email protected] Hanspeter Naegeli Institute of Pharmacology and Toxicology, University of ZUrich-Tierspital, Zurich, Switzerland E-mail: naegelih @ vetpharm. unizh. ch GerdP.Pfeifer Division of Biology, Beckman Research Institute of the City of Hope National Medical Center, Duarte, CA, USA E-mail:
[email protected] David H. Phillips Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK E-mail: david.phillips @ icr. ac. uk Albrecht Seidel Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gemot Grimmer-Foundation, Grosshansdorf, Germany E-mail:
[email protected] Pablo Steinberg Institute of Nutritional Science, University of Potsdam, Bergholz-Rehbriicke, Germany E-mail: steinber® rz. uni-potsdam. de
Contents
Preface
vii
In Memoriam: Anthony Dipple (1940-1999)
xi
List of Contributors
xv
1
Polycyclic Aromatic Hydrocarbon-Induced Carcinogenesis — An Introduction 1 Andreas Luch
2 Metabolic Activation and Detoxification of Polycyclic Aromatic Hydrocarbons Andreas Luch and William M. Baird
19
2.1
Introduction
19
2.2
Structure-Activity Relationships
21
2.3 Enzymatic Activation
22
2.3.1
Monooxygenation and Dihydrodiol Epoxide Pathway 2.3.2 Stereochemistry of Activation 2.3.3 One-Electron Oxidation 2.3.4 Formation of Quinones 2.3.5 'Bioalkylation' and Benzylic Ester Pathway
xvii
22 33 39 42 49
xviii
3
• Contents
2.4
Detoxification
53
2.5
Summary and Perspectives
57
Biomonitoring of Polycyclic Aromatic Hydrocarbons — Human Exposure Albrecht Seidel
97
3.1
Introduction
97
3.2
Studies at Work Places/Occupational Exposure to PAHs
4
99
3.3
Non-Occupational Exposure to PAHs
102
3.4
Metabolism and Excretion of PAHs
103
3.5 Biomonitoring of PAHs and Their Metabolites 3.5.1 Principal Considerations 3.5.2 1-Hydroxypyrene and Its Glucuronide 3.5.3 Enzyme Polymorphisms and Excretion Levels of 1-Hydroxypyrene 3.5.4 Phenanthrene Metabolites 3.5.5 Benzo[a]pyrene Metabolites
105 105 105
3.6
Summary
118
3.7
Conclusions
119
109 112 114
Macromolecular Adducts as Biomarkers of Human Exposure to Polycyclic Aromatic Hydrocarbons David H. Phillips
137
4.1
137
Introduction
4.2 Methods of Detection
138
4.3
Occupational Exposure to PAHs
140
4.3.1 Iron and Steel Production 4.3.2 Aluminum Production
140 141
Contents •
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4.3.3
Coke Ovens and Graphite Electrode Manufacture
142
4.3.4
Other Occupational Exposures
150
4.4
Environmental Exposure to PAHs
152
4.5
Coal Tar Therapy
154
4.6
Diet
155
4.7
Discussion and Summary
157
5 DNA Damage and Mutagenesis Induced by Polycyclic Aromatic Hydrocarbons Ahmad Besaratinia and Gerd P. Pfeifer
171
5.1
Introduction
171
5.2
Evolution of Research on PAHs
172
5.3
Chemistry and Biological Effects
173
5.4
Significance of Stable versus Unstable PAH-DNA Adducts
178
5.5
Mutagenicity of PAH-DNA Adducts
179
5.6
5.5.1 Site-Specific Mutagenicity of PAH-DNA Adducts 5.5.2 Translesional Synthesis Cancer Epidemiology and PAH-DNA Adducts
180 184 187
5.6.1 Mapping of PAH-DNA Adducts 188 5.6.2 Additional Evidence for the Etiological Relevance of PAHs in Human Carcinogenesis: The Exemplary Case of p53 Mutations in Lung Cancer 191 5.7 6
Concluding Remarks
193
Mechanisms of Repair of Polycyclic Aromatic Hydrocarbon-Induced DNA Damage Hanspeter Naegeli and Nicholas E. Geacintov
211
6.1
211
Introduction
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• Contents
6.2
6.3
6.4
Nucleotide Excision Repair
213
6.2.1 6.2.2 6.2.3 6.2.4
213 215 217 219
Mammalian DNA Nucleotide Excision Repair . . . Subunits of the Human NER Machinery Transcription-Coupled DNA Repair Global NER Deficiency and Cancer
Repair of PAH-DNA Adducts
221
6.3.1 The In Vitro Oligonucleotide Excision Reaction .. 6.3.2 Base Pair Conformation-Dependent Excision of B[a]PDE-dG Adducts 6.3.3 Unrepaired Fjord-Region PAH-DNA Adducts in Ras Codon 61 Mutational Hotspots 6.3.4 Bipartite Recognition of PAH-DNA Adducts ..... 6.3.5 Modulation of Human NER Activity by 5-Methylcytosines 6.3.6 Antagonistic Interaction of NER Factors between Substrate and Decoy DNA Adducts 6.3.7 Mechanism of PAH Adduct Recognition by the Human NER Machinery
221
Conclusion
7 Aberrant Gene Expression and Cell Signalling/Epigenetic Effects Induced by Polycyclic Aromatic Hydrocarbons . . . . Pablo Steinberg
224 228 235 237 238 243 246
259
7.1
Introduction
259
7.2
Cancer-Related Genetic and Epigenetic Alterations Induced by PAHs
260
7.2.1 7.2.2 7.2.3 7.2.4 7.2.5
260 263 265 265 266
Ras Activation Effects onp53 Effects on Mitogen-Activated Protein Kinases ... Disruption of BRCA1 Expression Inhibition of Intercellular Communication
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7.3 Atherosclerosis-Related Alterations Induced by PAHs . . .
268
7.4 Apoptosis-Related Alterations Induced by PAHs
270
7.5
271
Summary
Indicator Assays for Polycyclic Aromatic Hydrocarbon-Induced Genotoxicity Hansruedi Glatt
283
8.1
Introduction
283
8.2
Bacterial Systems
284
8.2.1
In Vitro Mutagenicity Tests Using Bacterial Target Cells 8.2.2 Other Endpoints of Genotoxicity in Bacterial Target Cells 8.2.3 Host-Mediated Assays Using Microbial Target Cells
8.3
8.5
289 290
Mammalian Systems
291
8.3.1 8.3.2
291
Mutations in Mammalian Cells in Culture Other Endpoints of Genotoxicity Using Mammalian Cells in Culture 8.3.3 Genotoxicity in Mammalian Cells In Vivo
8.4
284
294 295
Characterization of DNA Sequence Changes Induced by PAHs
298
Summary
299
9 Tumorigenicity of Polycyclic Aromatic Hydrocarbons
315
Shantu Amin and Karam El-Bayoumy 9.1 Introduction
315
9.2
316
Environmental Genotoxic Agents
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9.3
PAHs as Representative Examples of Environmental Mammary Carcinogens 9.3.1 9.3.2
9.4
319 328
NC«2-PAHs as Representative Examples of Environmental Mammary Carcinogens 330 9.4.1 9.4.2
9.5
Levels and Carcinogenic Potency of PAHs Metabolic Activation of PAHs and Potential Biomarkers
319
Levels and Carcinogenic Potency of NO2-PAHS . 330 Metabolic Activation of NO2-PAHS and Potential Biomarkers 334
Summary and Future Recommendations
10 Genetic Susceptibility to Polycyclic Aromatic Hydrocarbon-Induced Carcinogenesis Ari Hirvonen
338
353
10.1 Introduction
353
10.2 Cytochrome P450-Dependent Monooxygenases
355
10.2.1 CYP1A1 10.2.2 CYP1B1 10.2.3 CYP2C9
355 357 357
10.3 Epoxide Hydrolase
358
10.4 Glutathiones-Transferases
359
10.5 NAD(P)H:Quinone Oxidoreductase
361
10.6 Myeloperoxidase
362
10.7 Combined Genotype Effects
362
10.8 Concluding Remarks
363
Contents •
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11 Polycyclic Aromatic Hydrocarbon-Induced Carcinogenesis — An Integrated View 379 Andreas Luch 11.1 Exposure and Risk
380
11.2 Incorporation and Biotransformation
383
11.3 Monitoring Human Exposure
386
11.4 Molecular Epidemiology: Individual's Susceptibility? .. 388 11.5 Molecular Mechanisms of DNA Damage
397
11.6 Reprise
414
List of Abbreviations
453
Index
457
1 Polycyclic Aromatic Hydrocarbon-Induced Carcinogenesis - An Introduction Andreas Luch Massachusetts Institute of Technology, Center for Cancer Research, Cambridge, MA, USA E-mail:
[email protected] The association of human cancer with the exposure to polycyclic aromatic hydrocarbons (PAHs) dates back to Percivall Pott's observation of chimney sweeps' cancer in 1775.J Pott (1714-1788), who was surgeon to St. Bartholomew's Hospital in London, described the occurrence of scrotal cancer in chimney sweeps, and traced it to the contamination of the skin by soot. The interest of this observation lay in the first proof of the environmental origin of one particular form of cancer. About 100 years later, Volkmann and Bell confirmed the early observation made by Pott by describing several cases of scrotal skin tumors among workers in the German and Scottish paraffin industry, respectively.2'3 In 1907, the following definition was included into the Workmen's Compensation Act of Great Britain: "scrotal epithelioma occurring in chimney sweeps and epitheliomatous cancer or ulceration of the skin occurring in the handling or use ofpitch, tar or tarry compounds".4 With this addendum it has been officially acknowledged for the first time that cancer of any cutaneous site could be caused by pitch, tar or tarry compounds. A few years later, bitumen, mineral oil and paraffin were also included into the Compensation Act. All of these regulations resulted from the knowledge that was gained in the second half of the nineteenth and the beginning of the twentieth century, when the skin carcinogenicity of soot, tar and related PAH-containing mixtures was confirmed under various work place conditions.4 Until then, physicians collected the unforeseen 1
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outcome of an undesigned and undesirable grand-scale 'natural experiment' based on the rise of industrialization. The imperative next step was that of systematic inquiry and reproduction of the diseases at will. After many failures to reproduce the human outcome in laboratory animals, success was finally achieved by Yamagiwa and Ichikawa in 1915. Their report on the production of malignant epithelial tumors by repetitive application of coal tar to the ear skin of rabbits5,6 marked the transition into the modern era of PAH-related experimental cancer research. Shortly thereafter, painting of the back of mice was introduced as a method of biological testing of carcinogenic tars,7 and subsequently ethereal extracts of soot were confirmed to be carcinogenic in this mouse model.8 After the first successful production of cancer under experimental conditions, the scientific interest naturally shifted to the identification of the nature of the chemical(s) responsible. It was in this field more than any other that the greatest strides have been made in the 1920s and 30s, in large measures as an outcome of the studies of Sir Ernest Kennaway and his co-workers at the Royal Cancer Hospital in London. When Kennaway started with his attempt to identify the cancer-producing compound(s) in coal tar, it was known from previous work by Bloch and Dreifuss that the active fraction was of high boiling-point, neutral, nitrogen- and sulphur-free, and capable of forming a picrate complex.9 In 1925, Kennaway reported on the generation of carcinogenic tars by heating of petroleum, isoprene and acetylene up to 700-900°C in a hydrogen-containing atmosphere.10 As already known at this time from initial work of Berthelot in 1866, 'pyrolytic' conditions such as those applied would cause molecular condensation and rearrangement reactions that ultimately lead to the generation of PAHs. The carcinogenic tars produced under these conditions as well as carcinogenic products derived from the incubation of tetralin (1,2,3,4-tetrahydronaphthalene) with aluminum chloride at moderate temperatures (30-40°C; 'Schroeter reaction')11 were found to exhibit strong and characteristic fluorescence spectra with several distinct bands in the blue and violet region (at 4000,4180 and 4400 A) — a quality that finally turned out to be "...the single thread that led all through the labyrinth" (Kennaway) towards identifying the carcinogenic species. It was the work of Mayneord and Hieger from the Cancer Hospital that confirmed the identity of the characteristic fluorescence bands ('cancer bands') found
PAH-lnduced Carcinogenesis - An Introduction
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3
in the spectra of carcinogenic tars and in carcinogenic substances produced via the 'Schroeter reaction'. Among all available PAHs synthesized and characterized at this time, 1,2-benzanthracene (benz[a]anthracene, B[a]A, Figure 1.1) was found to give a fluorescence spectrum similar (but not identical) to that of the carcinogenic mixtures.12 About the same time, synthetic
5
s
4
Naphthalene
10
4
Anthracene
Benz[a]anthracene, B[a]A
3-Methylcholanthrene, 3-MC
("1,2-benzaiithraceiie")
("20-methyleholanthrene")
2
Benzo[e]pyrene, B[e]P ("1^-benzpyrene")
Benzo[a]pyrene, B[a]P ("3,4-benzpyrene")
2 1 14
Dibenz[a,/j]anthracene, DB[a,ft]A ("l,2;5,6-dibenzanthracene")
Dibenz[a,/]anthracene, T)B[a,j]A (" 1,2;7,8-dibenzanthracene")
Figure 1.1: Polycyclic aromatic hydrocarbons. (The older nomenclature system used prior to 1966 is written in brackets: see text for explanation of inconsistencies between older and IUPAC-based nomenclature.)
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preparations of higher molecular PAHs such as l,2;5,6-dibenzanthracene (dibenz[a,/i]anthracene, DB[a,/t]A) and l,2;7,8-dibenzanthracene (dibenz[a,j']anthracene, DB[a, j]A) (Figure 1.1) have been described;13 and subsequently these newly synthesized and pure compounds were tested for carcinogenic activity by Kennaway and Hieger.14,15 Since DB[a,h]A, DB[a, j]A and 3-methyl-DB[a,/t]A scored positive in the mouse skin bioassay, these pentacyclic hydrocarbons were actually the first pure compounds proved to act independently as real and strong carcinogens. In addition to their biological activity, the carcinogenic dibenzanthracenes displayed fluorescence spectra with similar (but again not identical) features as those of carcinogenic tars or carcinogenic 'Schroeter products'. Beginning in 1930, and with the help of the British Gas, Light and Coke Company, Hieger isolated about 7 g of a yellow powder out of two tons (!) of coal tar pitch by means of repetitive steps of fractional distillation, extraction and crystallization. The product showed both strong carcinogenic activity and high fluorescence in the spectral positions designated as the 'cancer bands'. 16,17 Further fractionation of the carcinogenic powder by Hewett and Cook afforded two pure crystalline products with melting points of 176 and 187°C. Both compounds were shown to be isomeric with the pentacyclic perylene (C20H12), but only the lower melting major component displayed the characteristic 'cancer bands' in its fluorescence spectrum. Subsequent synthetic preparation of '3,4-benzpyrene' (benzo[a]pyrene, B[a]P) and '1,2-benzpyrene' (benzo[e]pyrene, B[e]P) (Figure 1.1) unequivocally revealed that the major component of the crystals prepared from the pitch distillate was identical to B[a]P, which was also proven to be highly carcinogenic18 (see also Cook et alP). The synthetic B[e]P was identical with the minor and non-carcinogenic component of the crystallate melting at 187°C. In 1939, Kennaway, Cook, Hewett, Hieger and Mayneord were awarded with the first Anna Fuller Memorial Prize "... in recognition of their notable accomplishments in thefieldsof Cancer Research, and specificallyfor the isolation and synthesis of cancer-producing hydrocarbonsfrom coal tar, the identification by fluorescence spectroscopy, and for the study of the biological effects of these substances"?® At this point it should be noted that the nomenclature used in the classical literature mentioned above is based on older, sometimes confusing and
PAH-lnduced Carcinogenesis - An Introduction
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5
contradictory ring numbering systems, all of which have been replaced by the IUPAC rules established in 1966.21 For that reason, the old and now obsolete names for B[
