Polycyclic Hydrocarbons and Carcinogenesis 9780841209244, 9780841211131, 0-8412-0924-3

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ACS

SYMPOSIUM

SERIES

Polycyclic Hydrocarbons and Carcinogenesis Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.fw001

Ronald G. Harvey, EDITOR University of Chicago

Developed from a symposium sponsored by the Division of Organic Chemistry at the 188th Meeting of the American Chemical Society, Philadelphia, Pennsylvania, August 26-31, 1984

American Chemical Society, Washington, D.C. 1985 In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

283

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.fw001

Library of Congress Cataloging in Publication Data Polycyclic hydrocarbons and carcinogenesis. (ACS symposium series, ISSN 0097-6156; 283) "Developed from a symposium sponsored by the Division of Organic Chemistry at the 188th Meeting of the American Chemical Society, Philadelphia, Pennsylvania, August 26-31, 1984." Includes bibliographies and index. 1. Polycyclic aromatic hydrocarbons—Physiological effect—Congresses. 2. Polycyclic aromatic hydrocarbons—Congresses. 3. Carcinogenesis— Congresses. I. Harvey, Ronald G., 1927. II. American Chemical Society. Division of Organic Chemistry. III. American Chemical Society. Meeting (188th: 1984: Philadelphia, Pa.) IV. Series. RC268.7.P64P65 1985 616.99'4071 85-13384 ISBN 0-8412-0924-3

Copyright © 1985 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

ACS Symposium Series

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.fw001

M . Joan Comstock, Series Editor Advisory Board Robert Baker U.S. Geological Survey Martin L . Gorbaty Exxon Research and Engineering Co.

Robert Ory USDA, Southern Regional Research Center Geoffrey D. Parfitt Carnegie-Mellon University

Roland F. Hirsch U.S. Department of Energy

James C. Randall Phillips Petroleum Company

Herbert D. Kaesz University of California—Los Angeles

Charles N . Satterfield Massachusetts Institute of Technology

Rudolph J. Marcus Office of Naval Research

W. D. Shults Oak Ridge National Laboratory

Vincent D. McGinniss Battelle Columbus Laboratories

Charles S. Tuesday General Motors Research Laboratory

Donald E . Moreland USDA, Agricultural Research Service

Douglas B. Walters National Institute of Environmental Health

W. H . Norton J. T. Baker Chemical Company

C. Grant Willson IBM Research Department

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.fw001

FOREWORD The ACS S Y M P O S I U M S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S IN C H E M I S T R Y S E R I E S except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

PREFACE

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.pr001

THE

E N V I R O N M E N T A L O R I G I N O F M A N Y H U M A N C A N C E R S is gaining increasing acceptance. Significant levels of cancer-causing agents are commonly present in polluted air, automobile exhaust, tobacco smoke, and many common foods. Direct evidence concerning the role of chemical carcinogens in the etiology of human cancer is difficult to obtain due to necessary restrictions on human experimentation. However, there is a mounting volume of indirect evidence that supports the importance of chemical agents as causative factors. Epidemiological studies show wide variation in the incidences of various types of cancers in different populations. These geographic differences are often dramatic. For example, the incidence of female breast cancer is particularly high in the United States and relatively low in Japan. On the other hand, the occurrence of stomach cancer in these two countries is approximately reversed, being exceptionally high in Japan and relatively low in the United States. These differences appear to be related more to diet and lifestyle factors than to inherent genetic differences. Thus, the cancer profile of the Japanese population in Hawaii shows a marked shift in the relative incidence of breast and stomach cancer away from the levels in Japan toward those in the United States. In general the cancer patterns of migrant populations tend to shift toward those patterns characteristic of the new environment. Carcinogenesis research has demonstrated the tumorigenic activities of a large number of chemical substances in experimental animals. These include molecules of diverse chemical classes, organic and inorganiC., and natural products as well as compounds synthesized in the laboratory or produced by industry. Man has served as the unintentional guinea pig for the identification of some major classes of carcinogens. These include the polycyclic aromatic hydrocarbons (PAH), or polyarenes, which have been identified as the active components of soot, which was recognized by the London surgeon Percivall Pott two centuries ago as responsible for cancer of the scrotum in chimney sweeps. Subsequently, polycyclic hydrocarbons have been implicated as agents responsible for skin cancer in other occupations such as shale oil distillation and mule spinning in the cotton industry. The carcinogenicity of aromatic amines, such as benzidine and 2-naphthylamine, was first recognized by Rehn in the 1890s as an occupational hazard in the German dyestuffs industry. Compounds in this class induce

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.pr001

tumors in man predominantly in the urinary bladder. The nitrosamines, another major class of carcinogens, were first recognized as such by their induction of tumors in the livers of workmen in the chemical industry who were using N-nitrosodimethylamine as a solvent. Polycyclic aromatic hydrocarbons hold a unique place in carcinogenesis research. The first pure compounds recognized as carcinogens more than 50 years ago were the PAH benzo[a]pyrene and dibenz[tf,/z]anthracene. Because only certain polyarenes exhibit tumorigenic activity and the level of activity is highly dependent upon molecular structure (e.g., number of fused rings, molecular shape, and presence of methyl or other groups in particular molecular regions), the PAH are ideally suited to studies of structure-activity relationships. The polycyclic hydrocarbons are also exceptional in their ability to induce various types of tumors selectively, dependent upon their mode of administration and other experimental conditions. Thus, oral administration of 10 mg of 7,12-dimethylbenz[tf]anthracene to female Sprague-Dawley rats was shown by Huggins to elicit mammary tumors with 100% incidence. Intravenous injection of a lipid emulsion of the same hydrocarbon to male or female Long-Evans rats selectively induced leukemia with similarly high incidence. In contrast, intramuscular injection of this hydrocarbon into the legs of Long-Evans rats gave predominantly local sarcomas at the site of injection. Other malignancies may be induced by PAH under appropriate experimental conditions. PAH-induced tumors are widely employed as standards in experimental oncology. Polycyclic aromatic hydrocarbons' potential importance in human cancer is strongly suggested by their environmental occurrence and their exceptional carcinogenic potency; PAH as a class rank second only to the potent hepatocarcinogenic aflatoxins. The environmental prevalence of PAH is largely a consequence of PAH formation as products of combustion of fossil fuels and other organic matter. Although human populations are chronically exposed to low levels of polyarenes, individual levels of exposure may vary widely, determined by lifestyle, particularly cigarette smoking, diet, and occupation. Research in PAH carcinogenesis has made major advances in the past decade. Most notable has been identification of diol epoxide metabolites as the active forms of benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and other carcinogenic PAH. This finding has stimulated enormous research activity and opened the way to determination of the detailed molecular mechanism of action of this important class of carcinogenic molecules. The symposium upon which this book is based brought together leading investigators concerned with the mechanisms of carcinogenesis of PAH at the molecular level. The individual chapters in this book are not merely verbatim reports of the symposium proceedings but rather critical reviews of symposium topics with extensive references to investigations in other laboratories. Since the pertinent literature references are scattered in journals viii

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.pr001

in diverse fields ranging from synthetic and theoretical chemistry to oncology and molecular biology, it has been difficult for nonspecialists to keep abreast of recent advances. This book provides a convenient summary of current developments that cuts across these diverse academic disciplines. Several additional chapters on relevant topics that could not be included in the symposium due to time limitations are also included in this volume. Chapter 10 presents a unified theoretical treatment of the covalent binding to DNA of the reactive diol epoxide metabolite of benzo[tf]pyrene implicated as the active form of this hydrocarbon. Chapter 15 on the in vitro metabolic activation of nitro polycyclic aromatic hydrocarbons is the first review of this very active area of investigation. Interest in this topic has been stimulated by the discovery that nitration of pyrene and other PAH by oxides of nitrogen occurs in the atmosphere to form nitro-PAH derivatives which are often highly mutagenic. Chapter 14 on the carcinogenic metabolites of arylamines and arylamides reviews the large body of literature on the mechanism of carcinogenesis of this important class of PAH compounds. Since nitro-PAH are reducible by bacterial enzymes to polycyclic arylamines and the fused aromatic ring systems of both these classes of PAH compounds may undergo activation to diol epoxide derivatives, multiple overlapping mechanistic pathways exist for the metabolic activation of unsubstituted PAH and their nitro- and amino-substituted derivatives, compounding the mechanistic complexity. This book is expected to be of interest to investigators active in all aspects of carcinogenesis research as well as to graduate students, educators, and others seeking an introduction to this important field of research. It is hoped that this volume will contribute toward the ultimate elucidation of the molecular mechanism of induction of cancer by PAH. This knowledge will provide a rational basis for the design of approaches for the prevention and cure of this dread disease. Additional support for the symposium was provided by the U.S. Department of Energy. I particularly thank Walter Trahanovsky of the American Chemical Society for his personal contribution toward making this project a success and my wife Helene for her support and understanding throughout this project. Any opinions, findings, conclusions, or recommendations expressed herein are those of the authors and do not necessarily reflect the views of the U.S. Department of Energy. R O N A L D G.

HARVEY

University of Chicago Chicago, Illinois March 22, 1985

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1 Polycyclic Aromatic Hydrocarbon Carcinogenesis An Introduction ANTHONY DIPPLE

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

LBI-Basic Research Program, Chemical and Physical Carcinogenesis Laboratory, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701

Since polycyclic aromatic hydrocarbons are widely distributed throughout the atmosphere and water sources of the world, it i s essentially impossible to avoid exposure to nanogram quantities of these substances on a daily basis. It i s particularly important, therefore, that the mechanism of action of these carcinogens be understood. This introduc­ tion provides a general background on experimental carcinogenesis and structure-activity relationships for hydrocarbons. It also traces the key steps involved i n the discovery that polycyclic aromatic hydrocarbons are the carcinogenic components of complex mixtures such as soots and tars and the more recent discovery that bay region dihydrodiol epoxides are probably the metabolites of hydrocar­ bons that initiate the carcinogenic process. Although there are earlier reports of lifestyle-associated can­ cers in the scientific literature, the 1775 observation of Percival Pott, surgeon to St. Bartholomew's Hospital i n London, that scrotal cancer in chimney sweepers originates from their occupational exposure to soot (1) represents the key historical development i n the fields of chemical carcinogenesis in general and polycyclic aromatic hydrocarbon carcinogenesis in particular. This observa­ tion was followed a century later by von Volkmann's reports of occupational skin cancers i n workers i n the coal tar industry i n Germany (_2), and by the early 1900 s it was widely recognized that soot [produced by the inefficient combustion of coal and containing up to 40% coal tar (3)], coal tar [produced by the destructive d i s t i l l a t i o n of coal], and pitch [the residue after d i s t i l l i n g coal tar] are a l l carcinogenic for man. At that time,itwas conceivable that a single carcinogenic substance might be respon­ sible for a l l the known occupational cancers (j4), but attempts to characterize the carcinogens in these complex combustion and pyrolysis products had to await the development of an experimental system for determining carcinogenic activity. It was not u n t i l f

0097-6156/85/ 0283-0001 $06.00/0 © 1985 American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

1915 t h a t such a system was developed by Yamagiwa and Ichikawa (5) who succeeded i n p r o d u c i n g m a l i g n a n t s k i n tumors i n r a b b i t s by the p e r s i s t e n t t o p i c a l a p p l i c a t i o n of c o a l t a r . Three y e a r s l a t e r T s u t s u i (6) produced tumors i n mice by r e p e a t e d a p p l i c a t i o n o f t a r s t o the s k i n , and t h i s p a r t i c u l a r assay, w h i c h was q u i c k l y adopted i n o t h e r l a b o r a t o r i e s , has proved t o be of l a s t i n g v a l u e and i s s t i l l used f r e q u e n t l y today. The s e r i e s of events w h i c h then l e d t o the i d e n t i f i c a t i o n of c e r t a i n p o l y c y c l i c a r o m a t i c hydrocarbons as the f i r s t pure c h e m i c a l c a r c i n o g e n s has been d e s c r i b e d i n depth by S i r E r n e s t Kennaway (_7), a key p a r t i c i p a n t i n the d i s c o v e r y , and o n l y a b r i e f o u t l i n e of t h i s e x c i t i n g s t o r y i s i n c l u d e d h e r e i n . I n 1921, B l o c h and D r e i f u s s (8) i n S w i t z e r l a n d had e s t a b l i s h e d t h a t the c a r c i n o g e n i n c o a l t a r was of h i g h b o i l i n g p o i n t , was f r e e of n i t r o g e n and s u l ­ phur, formed a s t a b l e p i c r a t e , and was p r o b a b l y a complex h y d r o c a r ­ bon. Kennaway pursued these l e a d s a f t e r j o i n i n g what i s now the I n s t i t u t e of Cancer R e s e a r c h i n London i n 1922, and, amongst a number of n o t a b l e s t u d i e s on c a r c i n o g e n i c t a r s , he demonstrated t h a t p y r o l y s e s of i s o p r e n e or a c e t y l e n e i n an atmosphere of h y d r o ­ gen gave r i s e t o c a r c i n o g e n i c d i s t i l l a t e s , thereby p r o v i n g t h a t c a r c i n o g e n i c a c t i v i t y r e s i d e d i n some compound c o n t a i n i n g o n l y carbon and hydrogen ( 9 ) . A second v i t a l o b s e r v a t i o n was made when Mayneord, a p h y s i c i s t , j o i n e d i n the r e s e a r c h e f f o r t and d e c i d e d t o examine the c o n s p i c u ­ ous f l u o r e s c e n c e of the many c a r c i n o g e n i c d i s t i l l a t e s p r e s e n t i n Kennaway s l a b o r a t o r y . He found t h a t most of the c a r c i n o g e n i c t a r s e x h i b i t e d a common f l u o r e s c e n c e spectrum (A 400, 418 and 440 nm) but, i n subsequent s t u d i e s w i t h H i e g e r , none of the hydrocarbons a v a i l a b l e a t t h a t time e x h i b i t e d these s p e c t r a l c h a r ­ a c t e r i s t i c s (_7). The spectrum of b e n z [ a j a n t h r a c e n e was found t o be s i m i l a r t o , but of l o n g e r wavelength t h a n , t h a t of the c a r c i n ­ ogenic p r e p a r a t i o n s but t h i s s i m i l a r i t y d i r e c t e d Kennaway s a t t e n ­ t i o n t o C l a r ' s r e p o r t of the s y n t h e s i s of dibenz[a_,h]anthracene ( 1 0 ) . Tumors were o b t a i n e d when t h i s h y d r o c a r b o n was r e p e a t e d l y p a i n t e d on t o mice and thus i t was e s t a b l i s h e d t h a t the p r o p e r t i e s n e c e s s a r y t o e l i c i t tumors i n animals were c o n t a i n e d w i t h i n the s t r u c t u r e of a s i n g l e pure c h e m i c a l compound ( 1 1 ) . S i n c e the spectrum of dibenz [a_,hjanthracene was not i d e n t i c a l t o t h a t of the c a r c i n o g e n i c t a r s , c a r c i n o g e n i c a c t i v i t y o b v i o u s l y was not c o n f i n e d t o a unique c h e m i c a l s t r u c t u r e and i n d e e d Cook, who had j o i n e d t h i s r e s e a r c h e f f o r t i n 1929, soon s y n t h e s i z e d a number of new h y d r o c a r b o n s , and many of these were c a r c i n o g e n i c . The o u t s t a n d i n g problem of the c a r c i n o g e n i n the t a r s was u l t i m a t e ­ l y r e s o l v e d by a massive experiment b e g i n n i n g w i t h the d i s t i l l a t i o n of 2 tons of gas-works p i t c h . T h e r e a f t e r , by f r a c t i o n a l d i s t i l l a ­ t i o n , d i f f e r e n t i a l e x t r a c t i o n s , f r a c t i o n a l c r y s t a l l i z a t i o n and by f o l l o w i n g the f l u o r e s c e n c e spectrum and c a r c i n o g e n i c a c t i v i t y of the v a r i o u s f r a c t i o n s , Cook, Hewett and H i e g e r (12) were a b l e t o o b t a i n gram q u a n t i t i e s of a c r y s t a l l i n e m a t e r i a l w h i c h was c a r c i n o ­ g e n i c and e x h i b i t e d the f l u o r e s c e n c e w h i c h had been a s s o c i a t e d w i t h c a r c i n o g e n i c a c t i v i t y . T h i s m a t e r i a l was r e s o l v e d i n t o t h r e e s e p a r a t e s u b s t a n c e s , p e r y l e n e and two unknown isomers of p e r y l e n e w h i c h were i d e n t i f i e d by s t r u c t u r e - d e t e r m i n i n g s y n t h e s e s as benzof

m a x

f

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

1.

DIPPLE

Polycyclic Aromatic Hydrocarbon Carcinogenesis

3

[ a j p y r e n e and b e n z o [ e j p y r e n e . Benzo[ajpyrene was t h e source o f b o t h c a r c i n o g e n i c a c t i v i t y and t h e c h a r a c t e r i s t i c f l u o r e s c e n c e o f the c a r c i n o g e n i c t a r s . T h i s f i n d i n g completed a remarkable episode of r e s e a r c h d u r i n g which t h e c a r c i n o g e n i c a c t i v i t y ( o r , a t l e a s t , some o f t h e o f t h e c a r c i n o g e n i c a c t i v i t y ) a s s o c i a t e d w i t h complex m a t e r i a l s l i k e s o o t s and c o a l t a r s came t o be a t t r i b u t e d t o a s p e c i f i c c h e m i c a l component, b e n z o [ a j p y r e n e .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

Human Exposure The c a r c i n o g e n i c a c t i v i t y o f s o o t s , t a r s and o i l s i n man i s beyond d i s p u t e (13-16) and, i n a d d i t i o n t o t h e s k i n c a n c e r s which were n o t e d i n i t i a l l y , t h e r e have a l s o been s e v e r a l r e p o r t s i n d i c a t i n g t h a t h i g h e r i n c i d e n c e s o f r e s p i r a t o r y t r a c t and upper g a s t r o i n t e s ­ t i n a l t r a c t tumors a r e a s s o c i a t e d w i t h o c c u p a t i o n a l exposures t o these c a r c i n o g e n s (summarized i n r e f s . 13-16). W h i l e p r e s e n t day w o r k i n g c o n d i t i o n s a r e d r a s t i c a l l y improved over those o f t h e 1 9 t h c e n t u r y , contemporary s t u d i e s [ f o r example on D a n i s h chimney sweeps ( 1 7 ) ] c o n t i n u e t o f i n d i n c r e a s e d c a n c e r r i s k s a s s o c i a t e d w i t h exposure t o these p o l y c y c l i c a r o m a t i c h y d r o c a r b o n - c o n t a i n i n g m a t e r i a l s . N e v e r t h e l e s s , because any g i v e n hydrocarbon i s b u t one component o f these complex o c c u p a t i o n a l c a r c i n o g e n s , t h e p o s i ­ t i o n t a k e n by Working Groups o f t h e I n t e r n a t i o n a l Agency f o r Research on Cancer over t h e l a s t t e n y e a r s (13-16) i s t h a t i n d i v i ­ d u a l h y d r o c a r b o n s , such as b e n z o [ a j p y r e n e , have n o t been proven t o be c a r c i n o g e n s f o r man. T h i s l a c k o f d i r e c t p r o o f o f t h e i r c a r c i n ­ o g e n i c i t y f o r man s h o u l d not be o v e r i n t e r p r e t e d . I t does not i m p l y t h a t t h e human r a c e I s r e s i s t a n t t o these p o t e n t e x p e r i m e n t a l c a r c i n o g e n s and most c a r c i n o g e n e s i s r e s e a r c h e r s adopt t h e v i e w - p o i n t t h a t , u n t i l p r o o f t o t h e c o n t r a r y i s o b t a i n e d , c h e m i c a l s found t o be c a r c i n o g e n s i n a n i m a l s s h o u l d be regarded as p r o b a b l e c a r c i n o g e n s f o r man. The p o l y c y c l i c a r o m a t i c hydrocarbons have t o be r e g a r d e d i n t h i s way s i n c e some o f them a r e v e r y p o t e n t e x p e r i m e n t a l c a r c i n o g e n s and t h e metabolism and DNA b i n d i n g p r o d u c t s o f benzo[a] pyrene i n human c e l l s and t i s s u e s a r e v e r y s i m i l a r t o those seen i n s u s c e p t i b l e e x p e r i m e n t a l a n i m a l s ( r e v i e w e d i n 16). Some i n v e s ­ t i g a t o r s b e l i e v e t h a t t h e hydrocarbons a r e proven s k i n c a r c i n o g e n s and p r o b a b l e r e s p i r a t o r y t r a c t c a r c i n o g e n s f o r man ( 1 8 ) . The presence o f p o l y c y c l i c a r o m a t i c hydrocarbons i n t h e environment i s o f obvious c o n c e r n and, a p a r t from s p e c i f i c occupa­ t i o n a l environments, human exposure t o these compounds d e r i v e s from combustion p r o d u c t s r e l e a s e d i n t o t h e atmosphere. E s t i m a t e s of t h e t o t a l a n n u a l benzo[a]pyrene e m i s s i o n s i n t h e U n i t e d S t a t e s range from 900 tons (19) t o about 1300 tons ( 2 0 ) . These t o t a l s a r e d e r i v e d from heat and power g e n e r a t i o n (37-38%), open-refuse b u r n i n g (42-46%), coke p r o d u c t i o n (15-19%) and motor v e h i c l e e m i s s i o n s (1-1.5%) (19,20). S i n c e t h e v a s t m a j o r i t y o f these e m i s s i o n s a r e from s t a t i o n a r y s o u r c e s , l o c a l l e v e l s o f a i r p o l ­ l u t i o n o b v i o u s l y v a r y . Benzo[ajpyrene l e v e l s o f l e s s than 1 ug/1,000 m^ c o r r e s p o n d t o c l e a n a i r ( 2 0 ) . A t t h i s l e v e l , i t can be e s t i m a t e d t h a t t h e average person would i n h a l e about 0.02 u g o f b e n z o [ a j p y r e n e p e r day, and t h i s c o u l d i n c r e a s e t o 1.5 pg/day i n p o l l u t e d a i r ( 2 1 ) .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

P o l y c y c l i c p a r t i c u l a t e s r e l e a s e d i n t o t h e a i r can be washed out by r a i n o r s e t t l e out under g r a v i t y , thereby c o n t a m i n a t i n g w a t e r and s o i l and p r o v i d i n g o t h e r p o s s i b l e r o u t e s f o r human exposure. I n f a c t , hydrocarbons a r e widespread through t h e w o r l d ' s waters ( 2 2 ) and c a n e n t e r our food c h a i n by b e i n g t a k e n up by p l a n k t o n , by f i l t e r - f e e d i n g m o l l u s k s , and by f i s h ( 2 3 ) . The average l e v e l o f benzo[a]pyrene i n d r i n k i n g water i s about 0.01 pg/1 (19) so t h a t d a i l y i n t a k e from d r i n k i n g w a t e r i s o f a s i m i l a r o r d e r t o t h a t from b r e a t h i n g r e a s o n a b l y c l e a n air. These i n t a k e s , t o g e t h e r w i t h t h e hydrocarbons p r e s e n t i n our uncooked foods ( 2 4 ) , a r e p r o b a b l y l a r g e l y u n a v o i d a b l e and because o f t h i s , and t h e h i g h c a r c i n o g e n i c potency e x h i b i t e d by t h e hydrocarbons, i t i s important t h a t t h e mechanism o f a c t i o n o f these u b i q u i t o u s c a r c i n o g e n s be i n v e s t i g a t e d i n o r d e r t o determine how t o m i n i m i z e t h e i r t h r e a t t o man. Experimental

Carcinogenesis

The p r o c e s s of c a r c i n o g e n e s i s remains p o o r l y understood and t h i s i s perhaps n o t s u r p r i s i n g g i v e n t h a t i t o c c u r s over a p e r i o d o f many months i n e x p e r i m e n t a l animals and many y e a r s i n man. W h i l e mouse s k i n remains a c o n v e n i e n t and p o p u l a r system f o r m o n i t o r i n g t h e c a r c i n o g e n i c a c t i v i t y o f p o l y c y c l i c a r o m a t i c hydrocarbons, i t i s by no means t h e o n l y t i s s u e s e n s i ­ t i v e t o t h i s c l a s s o f c a r c i n o g e n s . To a l a r g e e x t e n t , the t i s s u e a f f e c t e d i s determined by the r o u t e of a d m i n i s t r a t i o n o f t h e c a r c i n o g e n and the a n i m a l s p e c i e s under i n v e s t i g a t i o n . For example, 7 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a c e n e Is a particularly p o t e n t c a r c i n o g e n f o r t h e mammary g l a n d o f young female SpragueDawley r a t s a f t e r o r a l o r i n t r a v e n o u s a d m i n i s t r a t i o n (25,26), d i e t a r y b e n z o [ a j p y r e n e l e a d s t o l e u k e m i a , l u n g adenoma and stomach tumors i n mice ( 2 7 ) , and e i t h e r o f these hydrocarbons can induce hepatomas i n male mice when i n j e c t e d on t h e f i r s t day o f l i f e ( 2 8 ) . N e v e r t h e l e s s , the mouse s k i n system has proved t o be p a r t i c u l a r l y v a l u a b l e because o f t h e r a p i d i t y of tumor i n d u c t i o n , t h e ease o f d e t e c t i o n o f tumors and because the m u l t i - s t a g e n a t u r e o f t h e c a r c i n o g e n i c p r o c e s s was e x p e r i ­ m e n t a l l y e s t a b l i s h e d i n t h i s system. Tumors a r e induced i n mouse s k i n e i t h e r by t h e r e p e a t e d ap­ p l i c a t i o n of s m a l l doses o f p o l y c y c l i c hydrocarbons, by a s i n g l e a p p l i c a t i o n o f a l a r g e dose, o r by t h e s i n g l e a p p l i c a t i o n of a s u b - c a r c i n o g e n i c dose o f hydrocarbon ( i n i t i a t i o n ) f o l l o w e d by r e p e a t e d a p p l i c a t i o n s o f a n o n c a r c i n o g e n i c agent such as croton o i l or i t s active constituent l2-0-tetradecanoyl-phorbol-l3a c e t a t e (promotion) ( 2 9 ) . The c h a r a c t e r i s t i c s o f t h e l a t t e r i n i t i a t i o n - p r o m o t i o n system i n d i c a t e t h a t i n i t i a t i o n i s e s s e n t i a l l y i r r e v e r s i b l e . T h i s f o l l o w s from the f a c t t h a t even when treatment w i t h promoters i s begun s e v e r a l months a f t e r i n i t i a t i o n w i t h a hydrocarbon, a h i g h y i e l d o f tumors i s s t i l l u l t i m a t e l y o b t a i n e d . I n c o n t r a s t , promotion i s r e v e r s i b l e t o some e x t e n t , and i t i s o n l y e f f e c t i v e f o l l o w i n g , not p r e c e d i n g , t h e i n i t i a t i n g e v e n t s . These d i s c o v e r i e s suggest t h a t o n l y t h e i n i t i a t i o n s t a g e of c a r c i n ­ o g e n e s i s has an a b s o l u t e requirement f o r t h e c h e m i c a l c a r c i n o g e n .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

1.

DIPPLE

Polycyclic Aromatic Hydrocarbon Carcinogenesis

5

More d e t a i l e d r e v i e w s o f the complex i n i t i a t i o n - p r o m o t i o n l i t e r a t u r e s h o u l d be c o n s u l t e d f o r a f u l l a p p r e c i a t i o n of t h i s t o p i c (30,31). W h i l e s e v e r a l g e n e r a l mechanisms through w h i c h c h e m i c a l s might i n i t i a t e the c a r c i n o g e n i c p r o c e s s have been c o n c e i v e d , the weight o f e v i d e n c e a t p r e s e n t suggests t h a t the p o l y c y c l i c a r o m a t i c h y d r o ­ carbons i n i t i a t e c a r c i n o g e n e s i s through a mutagenic mechanism. T h i s i n v o l v e s an i n i t i a l c o v a l e n t i n t e r a c t i o n between a m e t a b o l i t e of the hydrocarbon and the DNA of the t a r g e t t i s s u e . W h i l e these i n t e r a c t i o n s are becoming f a i r l y c l e a r l y u n d e r s t o o d ( 3 2 ) , i t i s i m p o r t a n t to remember t h a t t h i s c h e m i c a l i n t e r a c t i o n i n i t s e l f does not c o n s t i t u t e the process o f i n i t i a t i o n . T h i s f o l l o w s because i n i t i a t i o n i s o p e r a t i o n a l l y d e f i n e d as an i r r e v e r s i b l e process and s e v e r a l s t u d i e s o f the t r a n s f o r m a t i o n of mammalian c e l l s i n v i t r o show t h a t u n t i l c e l l s exposed t o a c a r c i n o g e n have been p e r m i t t e d t o undergo c e l l d i v i s i o n , the t r a n s f o r m i n g e f f e c t s o f c a r c i n o g e n s can be r e v e r s e d (33,34). Thus, even the i n i t i a t i o n stage o f c a r c i n o g e n e s i s i s complex, and i s dependent not o n l y on the p r o p e r t i e s o f the h y d r o c a r b o n a d m i n i s t e r e d , but i t a l s o r e ­ q u i r e s complex c o n t r i b u t i o n s from the a n i m a l t i s s u e i n v o l v e d , f i r s t i n m e t a b o l i z i n g the hydrocarbon t o a n a p p r o p r i a t e c a r c i n o ­ g e n i c m e t a b o l i t e and secondly i n making permanent the p o t e n t i a l l y I n i t i a t i n g damage generated by t h i s m e t a b o l i t e . In order that tumors s h o u l d e v e n t u a l l y a r i s e f o l l o w i n g the i n i t i a t i o n s t a g e , a p p r o p r i a t e p r o m o t i o n a l s t i m u l i are a l s o r e q u i r e d and promotion i t s e l f has been r e s o l v e d i n t o s e v e r a l stages (29,35). Given t h i s degree o f c o m p l e x i t y , i t would be g r o s s l y o p t i m i s t i c t o expect any o b v i o u s r e l a t i o n s h i p between the s t r u c t u r e o f p o l y c y c l i c a r o m a t i c hydrocarbons and t h e i r c a r c i n o g e n i c a c t i v i t i e s . However, a t the o u t s e t o f i n v e s t i g a t i o n s i n t o the c a r c i n o g e n i c h y d r o c a r b o n s , t h e s t r u c t u r e - a c t i v i t y approach was the o n l y means a v a i l a b l e t o attempt t o determine the mechanism of a c t i o n of these compounds. W h i l e i t d i d not p r o v i d e the key e v i d e n c e l e a d i n g t o our p r e s e n t l e v e l o f u n d e r s t a n d i n g , i t has c o n t r i b u t e d c o n s i d e r a b l y t o the l a t t e r , and, w i t h the b e n e f i t o f h i n d s i g h t , i t i s c l e a r t h a t the success o f the s t r u c t u r e - a c t i v i t y approach was l i m i t e d p r i m a r i l y by our i n a b i l i t y t o i n t e r p r e t the i n f o r m a t i o n i t y i e l d e d , r a t h e r than by the i n f o r ­ mation i t s e l f . S t r u c t u r e and

Activity

Immediately a f t e r the f i r s t few c a r c i n o g e n i c hydrocarbons were i d e n t i f i e d , s c i e n t i s t s p u z z l e d over the d e v e l o p i n g s t r u c t u r e - a c t i v ­ i t y r e l a t i o n s h i p s and t r i e d t o i d e n t i f y the s t r u c t u r a l f e a t u r e s o f the hydrocarbons which are a s s o c i a t e d w i t h t h e i r c a r c i n o g e n i c activity. For u n s u b s t i t u t e d p o l y c y c l i c aromatic hydrocarbons (see examples on page 6 ) , i t seems t h a t a minimum o f f o u r benzene r i n g s i s r e q u i r e d f o r , but does not guarantee, c a r c i n o g e n i c a c t i v i t y . Thus, o n l y two of the s i x p o s s i b l e arrangements o f f o u r benzene r i n g s r e p r e s e n t compounds w i t h d e f i n i t e a l t h o u g h weak c a r c i n o g e n i c a c t i v i t y . These are benzo[c]phenanthrene and b e n z [ a ] a n t h r a c e n e w h i c h , as Hewett (36) n o t e d i n 1940, a r e both phenanthrene d e r i v a t i v e s . I n c o n t r a s t , l i n e a r s t r u c t u r e s such as naphthacene are not a s s o c i a t e d

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Unsubstituted

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

S t r u c t u r e s o f C a r c i n o g e n i c and N o n c a r c i n o g e n i c P o l y c y c l i c Aromatic Hydrocarbons

These compounds show t u m o r - i n i t i a t i n g

activity.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

1.

DIPPLE

Polycyclic Aromatic Hydrocarbon Carcinogenesis

with carcinogenic a c t i v i t y . S t r u c t u r e s such as t r i p h e n y l e n e and d i b e n z o [f£,op_]naphthacene, which behave c h e m i c a l l y l i k e condensed p o l y p h e n y l s , a r e a l s o i n a c t i v e as c a r c i n o g e n s . W h i l e pyrene i t ­ s e l f , does not e x h i b i t any c a r c i n o g e n i c a c t i v i t y , the more p o t e n t c a r c i n o g e n s amongst the u n s u b s t i t u t e d hydrocarbons a r e b e n z o [ a j ­ pyrene and s e v e r a l compounds which can be c o n s i d e r e d t o be benzod e r i v a t i v e s o f b e n z o [ a j p y r e n e . The presence o f a b e n z o [ a j p y r e n e s t r u c t u r e w i t h i n a more complex molecule does not guarantee c a r c i n ­ o g e n i c a c t i v i t y however, because anthanthrene i s not a c a r c i n o g e n . I n a d d i t i o n t o the benzenoid p o l y c y c l i c s , s t r u c t u r e s c o n t a i n i n g five-membered r i n g s a r e a l s o p r e s e n t i n the environment and some of these a r e a l s o c a r c i n o g e n i c . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s become even more complex when the e f f e c t o f v a r i o u s s u b s t i t u e n t s on c a r c i n o g e n i c a c t i v i t y i s c o n ­ s i d e r e d ( 3 7 ) . W h i l e benz [ a j a n t h r a c e n e i s c o n s i d e r e d t o be a f a i r l y weak c a r c i n o g e n , 7 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a c e n e i s one o f the most p o t e n t o f the hydrocarbon c a r c i n o g e n s . A number o f o t h e r d i m e t h y l b e n z [ a j a n t h r a c e n e s , some t r i m e t h y l b e n z [ a j a n t h r a c e n e s and a l l o f the t w e l v e p o s s i b l e methylbenz [ a j a n t h r a c e n e s have been s y n t h e s i z e d and t h e i r c a r c i n o g e n i c a c t i v i t i e s e v a l u a t e d . These f i n d i n g s have been reviewed many times (37-40) and though t h e r e a r e s l i g h t d i f f e r e n c e s from one study t o a n o t h e r the o v e r a l l con­ c l u s i o n s are f a i r l y c l e a r ( T a b l e I ) . Thus, f o r the me t h y l b e n z [ a j a n t h r a c e n e s , the most p o t e n t c a r c i n o g e n i s 7 - m e t h y l b e n z [ a j a n t h r a cene but s u b s t i t u t i o n a t p o s i t i o n s 6, 8 o r 12 i s a l s o a s s o c i a t e d with substantial a c t i v i t y . None o f the o t h e r m e t h y l b e n z [ a j a n t h r a cenes have e x h i b i t e d s u b s t a n t i a l c a r c i n o g e n i c a c t i v i t y , w i t h the p o s s i b l e e x c e p t i o n of 5-methylbenz[ajanthracene ( 4 1 ) , but i n the e a r l i e r t e s t s on mouse s k i n (38) s u b s t i t u t i o n i n the a n g u l a r r i n g i . e . , on the 1-, 2-, 3- o r 4 - p o s i t i o n s , was found t o y i e l d t o t a l l y i n a c t i v e compounds w h i l e the r e m a i n i n g isomers i . e . the 9-, 10-, and 1 1 - m e t h y l b e n z [ a j a n t h r a c e n e s a l l e x h i b i t e d some s l i g h t a c t i v i t y . The concept t h a t s u b s t i t u t i o n i n the a n g u l a r r i n g was i n v e r s e l y a s s o c i a t e d w i t h c a r c i n o g e n i c a c t i v i t y was s t r e n g t h e n e d by t h e f i n d i n g s w i t h d i m e t h y l b e n z [ a j a n t h r a c e n e s . Even though both 7and 1 2 - m e t h y l b e n z [ a j a n t h r a c e n e a r e potent c a r c i n o g e n s , the 1,7-, 1,12-, 4,7-, and 4 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a c e n e s a r e a l l i n a c t i v e (37) . S i m i l a r l y , the p o t e n t a c t i v i t y o f 7 , 1 2 - d i m e t h y l b e n z [ a j ­ anthracene can be d e s t r o y e d by the presence of m e t h y l groups on the 2-or 3- p o s i t i o n s but the 4-methyl d e r i v a t i v e remains a c t i v e . The w e a l t h o f s t r u c t u r e - a c t i v i t y d a t a f o r the h y d r o c a r b o n s , t o ­ g e t h e r w i t h the f a c t t h a t they were the f i r s t pure c h e m i c a l s r e c o g ­ n i z e d t o e x h i b i t c a r c i n o g e n i c potency, a t t r a c t e d a g r e a t d e a l o f a t t e n t i o n o v e r the y e a r s , w i t h numerous attempts b e i n g made t o d e f i n e the s t r u c t u r a l f e a t u r e s a s s o c i a t e d w i t h c a r c i n o g e n i c a c t i v ­ ity. A t t e n t i o n was d i r e c t e d l a r g e l y towards the presence o f a phenanthrene s t r u c t u r e w i t h i n most o f the c a r c i n o g e n i c hydrocarbons (38) and then s u b s e q u e n t l y , t o the presence o f an a r o m a t i c bond w i t h a h i g h degree o f double bond c h a r a c t e r analogous t o t h a t o f the 9,10-bond i n phenanthrene. W h i l e many i n v e s t i g a t o r s c o n t r i b u ­ t e d t o the development o f s o - c a l l e d e l e c t r o n i c t h e o r i e s o f c a r c i n o ­ g e n e s i s , the most w i d e l y a p p l i c a b l e d e s c r i p t i o n o f a hydrocarbon c a r c i n o g e n i n t h e s e terms was developed by the Pullmans ( 4 2 ) .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

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They found t h a t , w i t h few e x c e p t i o n s , the c a r c i n o g e n s and noncarc i n o g e n s among the u n s u b s t i t u t e d hydrocarbons c o u l d be d i s t i n ­ g u i s h e d by i n d i c e s d e s c r i b i n g the r e a c t i v i t y of K - r e g i o n s and L - r e g i o n s towards a d d i t i o n r e a c t i o n s ( F i g u r e 1 ) . Carcinogens were c h a r a c t e r i z e d by a h i g h r e a c t i v i t y a t the K - r e g i o n t o g e t h e r w i t h a low r e a c t i v i t y a t the L - r e g i o n (where such a s t r u c t u r a l f e a t u r e was p r e s e n t ) . The i n t e r p r e t a t i o n of t h i s c o r r e l a t i o n i m p l i e d t h a t some i n t e r a c t i o n between a K - r e g i o n and some c e l l u l a r c o n s t i t u e n t was r e s p o n s i b l e f o r i n i t i a t i n g the c a r c i n o g e n i c p r o c e s s , w h i l e some a l t e r n a t i v e i n t e r a c t i o n a t a r e a c t i v e L - r e g i o n c o u l d d e s t r o y the c a r c i n o g e n i c p r o p e r t i e s of an o t h e r w i s e p o t e n t i a l carcinogen. These i d e a s had a major i n f l u e n c e on t h i n k i n g about the mechanisms of a c t i o n of the hydrocarbon c a r c i n o g e n s from the e a r l y 1 9 4 0 s t o the e a r l y 1 9 7 0 s . W h i l e i t i s no l o n g e r thought t h a t the K - r e g i o n p l a y s a major r o l e i n the a c t i v a t i o n of c a r c i n o ­ gens, i t remains p o s s i b l e t h a t i n a c t i v a t i n g r e a c t i o n s may occur at L-regions. S p e c i f i c e x c e p t i o n s t o the K- and L - r e g i o n h y p o t h e s i s were a n t h a n t h r e n e w h i c h was e x p e c t e d t o be a c a r c i n o g e n but i s n o t , and the o v e r a l l e f f e c t of methyl groups i n the a n g u l a r 1,2,3, 4 - r i n g of b e n z [ a ] a n t h r a c e n e d e r i v a t i v e s i n r e d u c i n g c a r c i n o g e n i c activity. Once i t was a p p r e c i a t e d t h a t a v i c i n a l d i h y d r o d i o l epoxide might be the m e t a b o l i t e of benzo[a]pyrene r e s p o n s i b l e f o r c a r c i n o ­ g e n i c a c t i v i t y ( 4 3 ) , J e r i n a and Daly (44) were a b l e t o suggest t h a t a "bay r e g i o n " was the s t r u c t u r a l f e a t u r e r e q u i r e d f o r c a r c i n ­ o g e n i c a c t i v i t y and t h a t the a c t i v e m e t a b o l i t e s f o r many h y d r o c a r ­ bons would be found t o be bay r e g i o n d i h y d r o d i o l e p o x i d e s i . e . v i c i n a l d i h y d r o d i o l epoxides w h e r e i n the epoxide r i n g i s a d j a c e n t t o a bay r e g i o n ( F i g u r e 2 ) . (The term bay r e g i o n i s used t o d e s c r i b e a concave a r e a of the p e r i p h e r y of a r o m a t i c hydrocarbons and was i n i t i a l l y i n t r o d u c e d because p r o t o n s i n such a r e g i o n i . e . at 1 and 12 i n 7-methylbenz[ajanthracene or 10 and 11 i n b e n z o [ a j p y r e n e ( F i g u r e 2) e x h i b i t d i s t i n c t i v e nmr p r o p e r t i e s ) . T h i s s u g g e s t i o n n e a t l y accounted f o r the e x c e p t i o n s t o the K - r e ­ g i o n h y p o t h e s i s , above, s i n c e anthanthrene does not c o n t a i n a bay r e g i o n and s u b s t i t u t i o n s i n the a n g u l a r r i n g of b e n z [ a j a n t h r a c e n e and i t s homologues might be expected t o i n t e r f e r e w i t h metabolic a c t i v a t i o n t o a bay r e g i o n d i h y d r o d i o l epoxide. In a d d i t i o n , the presence of a phenanthrene s t r u c t u r e w i t h i n a more complex h y d r o ­ c a r b o n i s necessary f o r a bay r e g i o n t o be p r e s e n t as w e l l as f o r a K - r e g i o n t o be p r e s e n t . I t appears, then, t h a t the e x c e p t i o n s t o the e a r l i e r s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s were s i g n a l l i n g the c u r r e n t u n d e r s t a n d i n g of the s t r u c t u r a l f e a t u r e s r e q u i r e d f o r c a r c i n o g e n i c a c t i v i t y but, as d e t a i l e d i n the f o l l o w i n g s e c t i o n , e x p e r i m e n t a l advances i n u n d e r s t a n d i n g the mechanism of m e t a b o l i c a c t i v a t i o n of p o l y c y c l i c hydrocarbons had t o be made b e f o r e these s i g n a l s c o u l d be i n t e r p r e t e d .

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f

f

M e t a b o l i c A c t i v a t i o n of Hydrocarbon C a r c i n o g e n s I n e a r l y s t u d i e s of the metabolism of h y d r o c a r b o n s , i t was noted t h a t v i c i n a l t r a n s d i h y d r o d i o l s were f r e q u e n t l y found as h y d r o c a r ­ bon m e t a b o l i t e s and, because of t h e i r t r a n s c o n f i g u r a t i o n , Boyland

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Table I . E f f e c t o f a M e t h y l Group a t a S i n g l e S t a r r e d P o s i t i o n on C a r c i n o g e n i c A c t i v i t y o f Benz[a]anthracene D e r i v a t i v e s Active

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Inactive

F i g u r e 1. Benz[a]anthracene w i t h r e g i o n s o f low bond l o c a l i z a t i o n energy ( K - r e g i o n ) and low para l o c a l i z a t i o n energy ( L - r e g i o n ) indicated.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

(45) suggested t h a t these d i o l s p r o b a b l y a r o s e from an i n t e r m e d i a t e epoxide. Moreover, he argued t h a t such epoxides might be t h e metabolites responsible f o r i n i t i a t i n g the carcinogenic process. The s t r u c t u r e - a c t i v i t y c o n s i d e r a t i o n s a t t h a t time n a t u r a l l y enough f o c u s s e d i n t e r e s t on epoxides formed a t t h e K - r e g i o n s o f t h e c a r ­ c i n o g e n i c hydrocarbons ( F i g u r e 3 ) , but i t was n o t u n t i l 1964 t h a t the s y n t h e s i s o f such p u t a t i v e m e t a b o l i t e s was a c h i e v e d ( 4 6 ) . The M i l l e r s ' p i o n e e r i n g work on m e t a b o l i c a c t i v a t i o n i n t h e a r o m a t i c amine f i e l d (47) had e s t a b l i s h e d t h e r o l e o f s p e c i f i c metabolites i n the carcinogenic a c t i o n of N-2-fluorenylacetamide by d e m o n s t r a t i n g t h a t t h e N-hydroxy m e t a b o l i t e was o v e r a l l a more potent c a r c i n o g e n than t h e parent compound. Analogous experiments w i t h the K - r e g i o n epoxides o f s e v e r a l hydrocarbon c a r c i n o g e n s , however, i n d i c a t e d t h a t these arene epoxides were e i t h e r v e r y weak o r t o t a l l y i n a c t i v e as c h e m i c a l c a r c i n o g e n s ( r e v i e w e d i n 4 8 ) . W h i l e these f i n d i n g s d i d not support t h e h y p o t h e s i s t h a t h y d r o c a r ­ bons e x p r e s s e d t h e i r c a r c i n o g e n i c a c t i v i t y through t h e i n t e r m e d i a c y of K - r e g i o n e p o x i d e s , t h e f a c t t h a t such m e t a b o l i t e s were c h e m i c a l ­ l y r e a c t i v e and p o t e n t i a l l y s u b j e c t t o a v a r i e t y o f i n a c t i v a t i n g r e a c t i o n s d u r i n g t h e course o f a p p l i c a t i o n t o e x p e r i m e n t a l a n i m a l s l e d many workers t o f e e l t h a t t h e n e g a t i v e f i n d i n g s were not con­ c l u s i v e . T h i s f e e l i n g was s t r e n g t h e n e d by t h e b i o l o g i c a l a c t i v i ­ t i e s e x h i b i t e d by the K - r e g i o n epoxides i n v a r i o u s In v i t r o systems, w h i c h showed them t o be toxiC., mutageniC., and e f f e c t i v e i n d u c e r s o f t r a n s f o r m a t i o n in v i t r o ( 4 8 ) . Thus, by t h e e a r l y 1970s, t h e w e a l t h o f i n f o r m a t i o n on b i o l o g i c a l a c t i v i t i e s i n v a r i o u s systems and t h e l a c k o f an a c c e p t a b l e a l t e r n a t i v e h y p o t h e s i s was l e a d i n g t o a growing acceptance o f K - r e g i o n epoxides as t h e m e t a b o l i t e s through w h i c h the hydrocarbons e x e r t t h e i r c a r c i n o g e n i c p o t e n t i a l , despite t h e i r lack of carcinogenic a c t i v i t y . The developments w h i c h l e d t o t h e p r e s e n t day concepts o f t h e m e t a b o l i c a c t i v a t i o n o f hydrocarbons d i d n o t a r i s e from t h e c l a s s i ­ c a l approach o f i d e n t i f y i n g m e t a b o l i t e s o f g r e a t e r b i o l o g i c a l potency than t h e parent compound, but from an approach dependent upon t h e assumption ( o r presumption) t h a t t h e i n t e r a c t i o n o f c a r ­ cinogens w i t h DNA i s a key event i n t h e i n i t i a t i o n o f t h e c a r c i n o ­ g e n i c p r o c e s s . Brookes and Lawley (49) found i n 1964 t h a t when r a d i o a c t i v e hydrocarbons a r e a p p l i e d t o t h e s k i n o f mice, they become c o v a l e n t l y bound t o t h e DNA o f t h e s k i n . Moreover, t h e e x t e n t s o f b i n d i n g t o DNA f o r v a r i o u s hydrocarbons f o l l o w e d f a i r l y closely their r e l a t i v e carcinogenic a c t i v i t i e s . T h e r e a f t e r , Brookes sought t o i d e n t i f y t h e m e t a b o l i t e s i n v o l v e d i n b i n d i n g t o DNA assuming t h a t these same m e t a b o l i t e s were i n v o l v e d i n t h e c a r c i n o g e n i c p r o c e s s (50-53). Since the b i n d i n g of hydro­ carbon t o DNA i n c e l l u l a r systems does not generate enough m a t e r i a l t o examine d i r e c t l y , DNA i s o l a t e d from c e l l s exposed t o r a d i o a c t i v e hydrocarbons was e n z y m i c a l l y degraded t o d e o x y r i b o n u c l e o s i d e s and the r a d i o a c t i v e h y d r o c a r b o n - d e o x y r i b o n u c l e o s i d e adducts were compared c h r o m a t o g r a p h i c a l l y w i t h those o b t a i n e d from some p u t a t i v e r e a c t i v e m e t a b o l i t e . I n 1973 ( 5 3 ) , t h i s approach c l e a r l y showed t h a t i n mouse s k i n o r mouse embryo c e l l s i n c u l t u r e , t h e c a r c i n o g e n 7-methylbenz[a]anthracene d i d n o t b i n d t o DNA through a K - r e g i o n epoxide i n t e r m e d i a t e and, u n l i k e t h e c o r r e l a t i v e b i o l o g i c a l a c t i v i -

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Bay

Region

CH

3

Bay Region

OH

F i g u r e 2. 7-Methylbenz[a]anthracene and benzo[a]pyrene i n d i c a t i n g those r e g i o n s d e f i n e d as bay r e g i o n s and the s t r u c t u r e s o f the c o r r e s p o n d i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s .

F i g u r e 3. The K - r e g i o n epoxides o f 7-methylbenz[a]anthracene and benzo[ajpyrene.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

t y d a t a , t h i s d i r e c t measurement o f events o c c u r r i n g w i t h i n t h e b i o l o g i c a l system c o u l d not be c i r c u m v e n t e d . The s e a r c h f o r an a l t e r n a t i v e r e a c t i v e m e t a b o l i t e f o r t h e p o l y ­ c y c l i c h y d r o c a r b o n c a r c i n o g e n s was soon s u c c e s s f u l . A l s o I n 1973, Borgen e t a l . (54) r e p o r t e d t h a t , i n t h e presence o f a microsomal system from hamster l i v e r , t r a n s 7 , 8 - d i h y d r o - 7 , 8 - d i h y d r o x y b e n z o [ a j ­ pyrene ( a m e t a b o l i t e o f benzo[a]pyrene) was bound t o DNA i n v i t r o some t e n times more e x t e n s i v e l y than was benzo[a]pyrene i t s e l f . They c o n c l u d e d t h a t t h i s t r a n s 7 , 8 - d i h y d r o d i o l " i s f u r t h e r metabo­ l i z e d t o an a c t i v e a l k y l a t i n g agent", though they made no s p e c i f i c s u g g e s t i o n as t o i t s s t r u c t u r e . Sims and h i s c o l l e a g u e s , who had l o n g been proponents o f t h e r o l e of h y d r o c a r b o n e p o x i d e s i n c a r c i n ­ ogenesis, r a p i d l y r e a l i z e d that the a l k y l a t i n g a c t i v i t y could a r i s e from e p o x i d a t i o n o f t h e nonaromatic 9,10-double bond i n the t r a n s 7 , 8 - d i h y d r o d i o l ( F i g u r e 4 ) . They s y n t h e s i z e d a s m a l l amount o f t h i s d i h y d r o d i o l e p o x i d e , and were a b l e t o show t h a t i t s p r o d u c t s o f r e a c t i o n w i t h DNA i n v i t r o were c h r o m a t o g r a p h i c a l l y i n ­ d i s t i n g u i s h a b l e from those o b t a i n e d when benzo[a]pyrene i t s e l f was bound t o DNA i n c e l l u l a r systems through m e t a b o l i c a c t i v a t i o n (43). T h i s g e n e r a l sequence o f m e t a b o l i c s t e p s through which h y d r o ­ carbons become bound t o DNA has s u b s e q u e n t l y been found t o a p p l y t o s e v e r a l o t h e r p o l y c y c l i c a r o m a t i c hydrocarbon c a r c i n o g e n s and t h i s s u p p o r t s t h e bay r e g i o n d i h y d r o d i o l epoxide g e n e r a l i z a t i o n o f J e r i n a and D a l y , d i s c u s s e d e a r l i e r . Moreover, s e v e r a l s t u d i e s i n w h i c h m e t a b o l i t e s i n v o l v e d i n t h e d i h y d r o d i o l epoxide pathway have been t e s t e d f o r c a r c i n o g e n i c a c t i v i t y a r e l a r g e l y s u p p o r t i v e of t h e i d e a t h a t t h i s r o u t e o f m e t a b o l i c a c t i v a t i o n i s a l s o i n v o l v e d i n t h e c a r c i n o g e n i c a c t i o n o f hydrocarbon c a r c i n o g e n s . The most thorough s t u d i e s have been done i n t h e case o f b e n z o [ a ] p y r e n e , so i t i s c o n v e n i e n t t o summarize these as r e p r e s e n t a t i v e o f t h e most e x t e n s i v e developments i n t h e a r e a i n g e n e r a l . F o r t h i s c a r c i n o ­ gen, a l l o f t h e p o s s i b l e s t e r e o i s o m e r s and enantiomers o f t h e bay r e g i o n d i h y d r o d i o l epoxide m e t a b o l i t e s have been s y n t h e s i z e d and t h e i r b i o l o g i c a l a c t i v i t i e s e v a l u a t e d ( T a b l e I I ) (55-58 and r e f e r ­ ences c i t e d t h e r e i n ) . O v e r a l l , f i n d i n g s on t h e t u m o r i g e n i c i t y o f t h e compounds l i s t e d i n T a b l e I I i n d i c a t e s t h a t benzo[a]pyrene e x p r e s s e s i t s c a r c i n o g e n i c p o t e n t i a l through m e t a b o l i c c o n v e r s i o n t o a bay r e g i o n d i h y d r o d i o l e p o x i d e . The (+)-enantiomer o f benzo[a]pyrene-7,8e p o x i d e i s a more p o t e n t c a r c i n o g e n than t h e (-)-enantiomer but n e i t h e r o f t h e s e has demonstrated g r e a t e r a c t i v i t y t h a n b e n z o [ a j ­ pyrene i t s e l f . S i m i l a r l y , t h e d i h y d r o d i o l and a n t i and syn d i h y ­ d r o d i o l e p o x i d e s d e r i v e d from t h e (+) 7,8-epoxide a r e a l l more p o t e n t c a r c i n o g e n s than t h e i r enantiomers d e r i v e d from t h e (-) 7,8e p o x i d e . However, i n comparison w i t h t h e c a r c i n o g e n i c a c t i v i t y o f b e n z o [ a ] p y r e n e , t h e s i t u a t i o n i s l e s s c l e a r , w i t h t h e (-) 7,8-dihy­ d r o d i o l b e i n g t h e o n l y m e t a b o l i t e which c o n s i s t e n t l y e x h i b i t s an a c t i v i t y e q u a l t o o r g r e a t e r t h a n t h a t o f t h e p a r e n t hydrocarbon. I n t h e newborn mouse system ( T a b l e I I ) , t h e (+) a n t i d i h y d r o d i o l epoxide i s c l e a r l y more e f f e c t i v e than benzo[a]pyrene but t h i s i s not t h e case i n i n i t i a t i o n - p r o m o t i o n s t u d i e s o r i n complete c a r c i n ­ o g e n e s i s s t u d i e s on mouse s k i n . N e v e r t h e l e s s , t h e r e i s always

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Carcinogenesis

7,8-dihydrodiol 9,10-epoxide

7,8-dihydrodiol

F i g u r e 4. The bay r e g i o n d i h y d r o d i o l epoxide r o u t e o f metabolism of benzo[a]pyrene.

Table I I . C a r c i n o g e n i c A c t i v i t i e s

o f Benzo[a]pyrene

Tumor I n i t i a t i o n i n Mouse S k i n M e t a b o l i t e A c t i v i t y / A c t i v i t y of BP a t Same Dose

Compound

% Mice Dose w i t h Av. Tumors Qxmol) Tumors per Mouse

Lung Adenoma I n d u c t i o n i n Newborn Mice M e t a b o l i t e A c t i v i t y / A c t i v i t y o f BP a t Same Dose % Mice Dose w i t h Lung Adenoma (/nmol) Adenoma p e r Mouse

( + ) BP[7R,8S]-epoxide

0.1

a

38/68

0.76/2.1

0.7*

OH (-) BP[7R,8R]-dihydrodiol

0.1

77/77

3.8/2.6

0.14

b

(BP) M e t a b o l i t e s

d

71*

2.14*

88*

9.18*

C o n t i n u e d on next page.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Table I I . Continued Tumor Initiation in Mouse Skin Metabolite Activity/Activity of BP at Same Dose

Compound

Dose (^mol)

% Mice with tumors

Av. tumors per mouse

Lung Adenoma Induction in Newborn Mice Metabolite Activity/Activity of BP at Same Dose % Mice with lung Adenoma Dose adenoma per mouse (^mol)

0.014

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e

OH (-) BP[7R,8S]-dihydrodiol [9R,10S]-epoxide

0.1

0.014

e

C

11/68

= OH ( + ) BP[7S,8S]-dihydrodiol

0.11/2.1

0.78

0.44/2.6

0.14

HO

d

HO OH (-) BP[7S,8R]-dihydrodiol [9R,10SJ-epoxide

( + ) BP[7S,8R]-dihydrodiol [9S,10R]-epoxide

0.014

0.1

6

C

0.17/2.1

0.014*

" A c t i v i t i e s f o r benzo[a]pyrene under the same c o n d i t i o n s were not reported. epoxide

d a t a from Ref. 59; benzo[a]pyrene data from Ref. 56.

From Ref. 55. "From Ref. 56. d

From Ref. 57.

"From Ref. 58.

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some difficulty i n the interpretation of carcinogenesis tests with highly reactive compounds and thus, the high carcinogenic activity of the 7,8-dihydrodiol (as well as the high activity of analogous diols from other hydrocarbons) strongly indicates that the dihydro­ diol epoxide route of activation i s involved i n polycyclic aromatic hydrocarbon carcinogenesis. Acknowledgments Research sponsored by the National Cancer Institute, DHHS, under Contract No. NO1-CO-23909 with Litton Bionetics, Inc.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch001

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Pott, P. "Chirurgical Observations" (1775), Reprinted in National Cancer Inst. Monogr, 1963, 10, 7-13. von Volkmann, R. Beiträge zur ChirurgiëLeipzig, 1875. Ross, P. Br. Med. J. 1948, 2, 369-74. Haddow, A. Persp. i n Biol. Med. 1974, 17, 543-88. Yamagiwa, K.; Ichikawa, K. Mitt. Med. Fak. Tokyo 1915, 15, 295-344. Tsutsui, H. Gann 1918, 12, 17-21. Kennaway, E.L. Br. Med. J. 1955, 2, 749-52. Bloch, B.; Dreifuss, W. Schweiz. Med. Wochenschr. 1921, 51, 1035-7. Kennaway, E.L. Br. Med. J. 1925, 2, 1-4. Clar, E. Ber. Dtsch. Chem. Ges. 1929, 62, 350-9. Kennaway, E.L.; Hieger, I. Br. Med. J. 1930, 1, 1044-6. Cook, J.W.; Hewett, C.L.; Hieger, I. J. Chem. Soc. 1933, 395405. "Evaluation of Carcinogenic Risk" International Agency for Research on on Cancer Monographs, Vol. 3, 1972. "Chemicals and Human Cancer" International Agency for Research on Cancer Monographs, Suppl. 1, 1979. "Chemicals and Industries Associated with Human Cancer" Inter­ national Agency for Research on Cancer Monographs, Suppl. 4, 1982. "Evaluation of Carcinogenic Risk" International Agency for Research on Cancer Monographs, Vol. 32, 1983. Hansen, E.S. Am. J. Epidemiol. 1983, 117, 160-4. Schmähl, D.; Habs, M. In "Environmental Carcinogens: Poly­ cyclic Aromatic Hydrocarbons", Grimmer, G., Ed., CRC: Boca Raton, 1983; p. 237. Baum, E.J. In "Polycyclic Hydrocarbons and Cancer"; Gelboin, H.V.; Ts'O, P.O.P., Eds.; Academic: New York, 1978; Vol. 1, p 45. "Particulate Polycyclic Organic Matter," National Academy of Sciences 1972. Woo, Y.T.; Arcos, J.C. In "Carcinogens i n Industry and the Environment"; Sontag, J.M., Ed.; Dekker: New York, 1981; p. 167. Brown, R.A.; Huffman, H.L. Science 1976, 191, 847-9.

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23. Shabad, L.M. J. Natl. Cancer Inst. 1980, 64, 405-10. 24. Grasso, P.; O'Hare, C. In "Chemical Carcinogens" Searle, C.E., Ed., American Chemical Society, Washington, D.C., 1976; p. 701. 25. Huggins,C.; Briziarelli,G.; Sutton,H. J. Exp. Med. 1959, 109, 25-42. 26 Huggins,C.; Grand,L.C.; Brillantes, F.P. Nature 1961, 189, 204-7. 27. Rigdon, R.H.; Neal,J. Proc. Soc. Exp. Biol. Med. 1969, 130, 146-8. 28. Roe, F.J.C.; Waters, M.A. Nature 1967, 214, 299-330. 29. Boutwell, R.K. Progr. Exp. Tumor Res. 1964, 4, 207-50. 30. Slaga, T.J. "Mechanisms of Tumor Promotion, Vol. I I " ; CRC Press: Boca Raton, Florida, 1984. 31. Iversen, O.H.; Astrup, E.G., Cancer Investign. 1984, 2, 51-60. 32. Grunberger, D.; Weinstein, I.B. In "Chemical Carcinogens and DNA" Grover, P.L., Ed.; CRC Press, Boca Raton, 1979, Vol. II p. 59. 33. Borek, C.; Sachs, L. Proc. Natl. Acad. Sci., U.S.A. 1967, 57, 1522-7. 34. Kakunaga, T. Cancer Res. 1975, 35, 1637-42. 35. Slaga, T.J.; Fischer, S.M.; Nelson, K.; Gleason, G.L. Proc. Natl. Acad. Sc., U.S.A. 1980, 77, 2251-54. 36. Hewett, C.L. J. Chem. Soc. 1940, 293-303. 37. Dipple, A. In "Chemical Carcinogens" Searle, C.E., Ed.; ACS Monogr., 173 American Chemical Society, Washington, D.C., 1976, pp. 245-314. 38. Badger, G.M. Br. J. Cancer 1948, 2, 309-50. 39. Arcos, J.C.; Argus, M.F. "Chemical Induction of Cancer, Vol. IIA", Academic, New York, 1974. 40. Dipple, A.; Lawley, P.D.; Brookes, P. Eur. J. Cancer 1968, 4, 493-506. 41. Lacassagne, A.; Zajdela, F.; Buu-Hoi, N.P.; Chalvet, O. Bull. Cancer 1962, 49, 312-7. 42. Pullman, A.; Pullman, B. Adv. Cancer Res. 1955, 3,117-69. 43. Sims, P.; Grover, P.L.; Swaisland, A.; Pal, K.; Hewer, A. Nature 1974, 252, 326-8. 44. Jerina, D.M.; Daly, J.W. In "Drug Metabolism: Parke, D.V., Smith, R.L., Eds., Taylor and Francis, London, 1976, pp. 13-32. 45. Boyland, E., Biochem. Soc. Symp. 1950, 5, 40-54. 46. Newman, M.S.; Blum, S. J. Am. Chem. Soc. 1964, 86, 5598-600. 47. Miller, E.C.; Miller, J.A. Pharmacol. Rev. 1966, 18, 805-38. 48. Sims, P.; Grover, P.L. Adv. Cancer Res. 1974, 20, 165-274. 49. Brookes, P.; Lawley, P.D. Nature 1964, 202, 781-4. 50. Brookes, P.; Heidelberger, C. Cancer Res. 1969, 29, 157-65. 51. Dipple, A.; Brookes, P.; Mackintosh, D.S.; Rayman, M.P. Bio­ chemistry 1971, 10, 4323-30. 52. Baird, W.M.; Brookes, P. Cancer Res. 1973, 33, 2378-23. 53. Baird, W.M.; Dipple, A.; Grover, P.L.; Sims, P.; Brookes, P. Cancer Res. 1973, 33, 2386-92.

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54. 55. 56. 57. 58.

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Borgen, A.; Darvey, H.; Castagnoli, N.; Crocker, T.C.; Rasmussen, R.E.; Yang, I.Y. J. Med. Chem. 1973, 16, 502-6. Levin, W.; Wood, A.W.; Chang, R.L.; Slaga, T.J.; Yagi, H.; Jerina, D.M.; Conney, A.H. Cancer Res. 1977, 37, 2721-25. Slaga, T.J.; Bracken, W.J.; Gleason, G.; Levin, W.; Yagi, H.; Jerina, D.M.; Conney, A.H. Cancer Res. 1979, 39, 67-71. Kapitulnik, J.; Wislocki, P.G.; Levin, W.; Yagi, H.; Thakker, D.R.; Akagi, H.; Koreeda, M.; Jerina, D.M.; Conney, A.H. Cancer Res. 1978, 38, 2661-65. Buening, M.K.; Wislocki, P.G.; Levin, W.; Yagi, H.; Thakker, D.R.; Akagi, H.; Koreeda, M.; Jerina D.M.; Conney, A.H. Proc. Natl. Acad. Sci., U.S.A. 1978, 75, 5358-61. Levin, W.; Buening, A.; Wood, A.; Chang, R.L.; Kedzierski, B.; Thakker, D.H.; Boyd, D.R.; Gadaginamath, G.S.; Armstrong, R.N.; Yagi, H.; Karle, J.M.; Slaga, T.J.; Jerina, D.M.; Conney, A.H. J. Biol. Chem. 1980, 255, 9067-74.

RECEIVED

March 5, 1985

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

2 Stereoselective Metabolism and Activations of Polycyclic Aromatic Hydrocarbons SHEN K. YANG, MOHAMMAD MUSHTAQ, and PEI-LU CHIU

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch002

Department of Pharmacology, F. Edward Hébert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799

Current understandings on the stereoselective metabo­ lism and activation pathways of the weak carcinogen benz[a]anthracene and two of the potent carcinogens, benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene, are reviewed. Different stereoselective pathways of metabolism occur i n the formations of the procarcinogenic dihydrodiols, the bay-region dihydrodiolepoxides, and the K-region dihydrodiols by rat liver microsomal enzymes. Recent evidence suggests that a methyl substituent at the C-12 position of benz[a]anthracene enhances the carcinogenicity of the methylated hydrocarbon and also changes the stereo­ selective metabolism in the formation and hydration of the K-region 5,6-epoxide as well as the procarcinogenic 3,4-epoxide. Polycyclic aromatic hydrocarbons (PAHs) are common particulate envi­ ronmental pollutants and may be responsible for some cancer induc­ tion i n man. The biological properties of PAHs, such as mutageni­ city, carcinogenicity, and covalent binding to cellular macromolecules, require metabolic activation by the cytochrome P-450 contain­ ing drug-metabolizing enzyme systems. The metabolism of PAHs has been studied intensively in the past thirty years and the recent rapid progress i n the understanding of their activation pathways i s largely due to the recognition of benzo[ajpyrene 7,8-dihydrodiol9,10-epoxide as the major carcinogenic and mutagenic metabolite of benzo[ajpyrene (BaP) (Figure 1; for reviews, see 1-4 and references therein)• BaP i s metabolically activated predominantly to the 7R,8S-dihydrodiol-9S,10R-epoxide (anti form) and to a minor extent to 7R,8Sdihydrodiol-9R,10S-epoxide (syn form) via 7R,8S-epoxide and 7R,8Rdihydrodiol (Figure 1). Evidence for the formation of the 7S,8Sdihydrodiol and the 7S,8R-dihydrodiol-9R,10S-epoxide from the meta­ bolism of BaP in vivo on mouse skin has been reported (5). BaP i s also stereoselectively metabolized to the 4R,5R-dihydrodiol via the 4S,5R-epoxide and to the 9R,10R-dihydrodiol via the 9S,10R-epoxide (Figure 1). This chapter not subject to U.S. copyright. Published 1985, American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

F i g u r e 1. The major pathways i n the m e t a b o l i s m o f BaP t o BaP e p o x i d e s , d i h y d r o d i o l , and 7 , 8 - d i h y d r o d i o l - 9 , 1 0 - e p o x i d e s . The abso­ l u t e c o n f i g u r a t i o n s a r e as shown. The p o s i t i o n o f t r a n s - a d d i t i o n o f water i s shown by an arrow. The o p t i c a l p u r i t y o f t h e 4,5-epoxide formed i n BaP m e t a b o l i s m i s dependent on the cytochrome P-450 isozymes p r e s e n t i n t h e m i c r o s o m a l enzyme system. EH = e p o x i d e hydrolase.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch002

2.

YANG ET AL.

Stereoselective Metabolism and Activations

21

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch002

Absolute Configurations of the Dihydrodiol Metabolites of Benzo[ajpyrene The configuration of the 4R,5R-dihydrodiol was established by a p p l i cation of the exciton c h i r a l i t y method (6). To minimize undesired interactions between the e l e c t r i c t r a n s i t i o n dipoles of the two j>N,N-dimethylaminobenzoate chromophores and the dihydrodiol chromophore, a 4,5-dihydrodiol enantiomer was f i r s t reduced to l,2,3,3a,4,5,7,8,9,10-decahydro and 4,5,7,8,9,10,11,12-octahydro derivatives (6). We found that i t i s not necessary to reduce the chrysene chromophore of a BaP 4,5-dihydrodiol enantiomer (Figure 2). S i m i l a r l y , the absolute configurations of the K-region dihydrodiol enantiomers of BA (7}> 7-bromo-BA (8), 7-fluoro-BA (9), 7-methyl-BA (10), and 7,12-dimethyl-BA (DMBA) (7) can also be determined by the exciton c h i r a l i t y method without further reduction. The absolute configuration of the 7,8-dihydrodiol metabolite was also established to be 7R,8R by the exciton c h i r a l i t y method (11.12). Our result (Figure 2) i s i n agreement with those reported e a r l i e r (11,12.). The absolute configuration of the 9,10-dihydrodiol metabolite was established to be 9R,10R both by nuclear magnetic resonance spectroscopy and by the structures of the hydrolysis products formed from the syn and a n t i 9,10-dihydrodiol-7,8-epoxides which were synthesized from the same 9,10-dihydrodiol enantiomer (13). The absolute configuration of a BaP trans-9.10-dihydrodiol enantiomer, after conversion to a tetrahydro product, can also be determined by the exciton c h i r a l i t y method (Figure 2) (19.20). Optical Purity of the Dihydrodiol Metabolites

of Benzo[ajpyrene

The o p t i c a l p u r i t i e s of the dihydrodiol metabolites of BaP have been determined by three methods; (i) d e r i v a t i z a t i o n of the dihydrodiol metabolites with either (-)menthoxyacetyl chloride (15-16) or (-)-amethoxy-a-trifluoromethylphenylacetyl chloride (17). ( i i ) c i r c u l a r dichroism spectra (14,16,18). and ( i i i ) direct separation of enantiomers by c h i r a l stationary phase HPLC (19.20). Method i. requires the a v a i l a b i l i t y of r e l a t i v e l y large amounts of racemic dihydrodiol standards and i s very time-consuming. Method i i . requires microgram quantities of dihydrodiol metabolites and the a v a i l a b i l i t y of a spectropolarimeter. Method i i i can analyze sub-microgram quantity of unlabeled dihydrodiol metabolites and sub-nanogram quantity of radiolabeled dihydrodiol metabolites. If the enantiomers of a dihydrodiol can be resolved by the c h i r a l stationary phase HPLC., method i i i has the advantages of speed and s e n s i t i v i t y . The enantiomers of the non-K-region dihydrodiols of BaP can either be analyzed as the dihydrodiol or as the tetrahydrodiol (Figure 3). The o p t i c a l p u r i t i e s of the dihydrodiol metabolites formed i n BaP metabolism by l i v e r microsomes from Sprague-Dawley rats (1,14,15) are higher than those from l i v e r microsomes from rats of Long-Evans s t r a i n (17). Repeated experiments i n our laboratory using both rat strains indicate that small differences indeed exist (Table I). However, the percentages of R,R enantiomers are consistently higher than those reported by another laboratory (17).

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch002

22

250

300

350

Wavelength (nm) F i g u r e 2, The e x c i t o n c h i r a l i t y CD s p e c t r a o f b i s - p - N , N - d i m e t h y l a m i n o b e n z o y l d e r i v a t i v e s o f BaP 7,8,9,10-tetrahydro-tranjB-7,8-diol (1.0 A / ^ / m l , d e r i v e d f r o m BaP t r a n s - 7 , 8 - d i h y d r o d i o l m e t a b o l i t e ) , BaP 7 , 8 , 9 , 1 0 - t e t r a h v d r o - t r a n s - 9 . 1 0 - d i o l (1.0 A A / m l , d e r i v e d f r o m BaP t r a n s - 9 , 1 0 - d i h y d r o d i o l m e t a b o l i t e ) , and BaP t r a n s - 4 , 5 - d i h y d r o d i o l m e t a b o l i t e (1.0 A yQ/ml). CD s p e c t r a a r e expressed by e l l i p t i c i t y a t t h e i n d i c a t e d c o n c e n t r a t i o n as d e s c r i b e d (9.50). A r = p-N,Ndimethylaminobenzoyl.
?7% of BaP 4,5-epoxide metabolic a l l y formed from the metabolism of BaP i n a reconstituted enzyme system containing p u r i f i e d cytochrome P-450c (P-448) i s the 4S,5R enantiomer (24). The epoxide was determined by formation, separation and quantification of the diastereomeric trans-addition products of glutathione. Recently a BaP 4,5-epoxide was isolated from a metabol i t e mixture obtained from the metabolism of BaP by l i v e r microsomes from 3-methylcholanthrene-treated Sprague-Dawley rats i n the presence of the epoxide hydrolase i n h i b i t o r 3,3,3-trichloropropylene oxide, and was found to contain a 4S,5R/4R,5S enantiomer r a t i o of 94:6 (Chiu et a l . unpublished results). However, the content of the 4S,5R enantiomer was p r i o r t o c y c l i z a t i o n a f f o r d e d 8, i n somewhat h i g h e r o v e r a l l y i e l d . Attempted i n t r o d u c t i o n o f the o l e f i n i c bond i n t o 8, w i t h excess DDQ f a i l e d , d e s p i t e t h e f a c t t h a t s i m i l a r r e a c t i o n o f t h e analogous t e t r a h y d r o d i o l d i b e n z o a t e o f BP proceeded smoothly. T h i s d i f f e r e n c e i n r e a c t i v i t y r e s u l t s from t h e f a c t t h a t the d i b e n z o a t e groups o f £ a r e f o r c e d t o adopt the d i a x i a l c o n f o r m a t i o n due t o s t e r i c i n t e r a c t i o n w i t h the a d j a c e n t aromatic r i n g . C o n s e q u e n t l y , h y d r i d e a b s t r a c t i o n from the u n s u b s t i t u t e d ben­ z y l i c p o s i t i o n o f 8, i s e f f e c t i v e l y b l o c k e d . The NMR spectrum o f g, c o n f i r m s t h e d i a x i a l o r i e n t a t i o n o f t h e b e n z y l i c groups ( J ^ I Q " 3.5Hz). C o n v e r s i o n o f 8 t o % was r e a d i l y a c h i e v e d by the 'Bromination-dihydrobromination method. Attempted s y n t h e s i s o f £ by r e d u c t i o n o f BeP 9,10-dione w i t h NaBH^ i n e t h a n o l i n t h e presence o f a i r gave o n l y t h e c o r r e s p o n d i n g c a t e c h o l , 1,2-dihydroxy-BeP ( 2 2 ) . S i m i l a r r e a c t i o n conducted under oxygen f o r a week f u r n i s h e d 9, i n moderate y i e l d ( 3 5 % ) . Epoxidation of w i t h m-chloroperbenzoic a c i d afforded a mix­ t u r e o f the a n t i and syn d i o l epoxides ( a n t i - a n d syn-BePDE). I n our i n i t i a l s t u d i e s o n l y t h e a n t i isomer was i s o l a t e d (48). Subse­ q u e n t l y , i t was found by Y a g i e t a l . (50) t h a t b o t h d i a s t e r e o m e r s are formed. I n o u r e x p e r i e n c e , t h e r e l a t i v e r a t i o o f isomers i s dependent upon e x p e r i m e n t a l c o n d i t i o n s . T h i s i s a n o t h e r example o f lack of s t e r e o s p e c i f i c i t y of epoxidation of a d i a x i a l dihydrodiol. =

Triphenylene T h i s PAH i s a common e n v i r o n m e n t a l c o n t a m i n a n t . However, i t i s i n a c t i v e as a c a r c i n o g e n i n animal t e s t s ( 5 1 ) . The t r a n s - l , 2 - d i ­ h y d r o d i o l o f t r i p h e n y l e n e has been s y n t h e s i z e d from phenanthrene by a r o u t e analogous t o t h a t employed f o r the p r e p a r a t i o n o f BeP 9,10d i h y d r o d i o l ( 4 8 ) . L i k e the l a t t e r compound, e p o x i d a t i o n w i t h p e r ­ a c i d a f f o r d s a m i x t u r e o f the a n t i and s y n d i o l e p o x i d e s ( F i g u r e 9) (48,50).

Phenanthrene (Method I V ) A l t h o u g h phenanthrene i s noncarcinogeniC., some o f i t s m e t h y l a t e d d e r i v a t i v e s e x h i b i t s i g n i f i c a n t a c t i v i t y as mutagens (52,53). The 1,2- and 3 , 4 - d i h y d r o d i o l s o f phenanthrene were f i r s t s y n t h e s i z e d by J e r i n a e t a l . (54) by a method i n v o l v i n g r e d u c t i o n o f t h e c o r ­ r e s p o n d i n g quinones w i t h L i A l H . However, the y i e l d s i n t h e r e In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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4

5i

5b

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F i g u r e 7. S t r u c t u r e s o f t h e DBA a n t i 3 , 4 - d i o l - l , 2 - e p o x i d e ( 4 ) , anti-1,2-diol-3,4-epoxide ( 5 a ) , and syn-1,2-diol-3,4-epoxide (5b).

ANTj-BePDE

SvN-BePDE

F i g u r e 8. S y n t h e s i s o f the 9 , 1 0 - d i h y d r o d i o l o f BeP (41,42). Re agents: ( i ) s u c c i n i c a n h y d r i d e , A1C1~; (ii)H NNH«, KOH; ( i i i ) HF; ( i v ) NaBH, ; ( v ) H ; ( v i ) AgOBz, T ; ( v i i ) DDf/; ( v i i i ) NBS; ( i x ) DBN; ( x ) NaOMe. 2

F i g u r e 9. E p o x i d a t i o n o f the 1 , 2 - d i h y d r o d i o l y i e l d s b o t h a n t i and s y n d i o l epoxide isomers.

of triphenylene

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Dihydrodiol and Diol Epoxide Metabolites

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d u c t i o n s t e p were o n l y 4% and 1%, r e s p e c t i v e l y . S u b s e q u e n t l y , these d i h y d r o d i o l s were s y n t h e s i z e d by Lehr e t a l . (30) by Method I from the c o r r e s p o n d i n g k e t o n e s , 1-oxo-and 4 - o x o - l , 2 , 3 , 4 - t e t r a h y d r o p h e n a n t h r e n e . Subsequent r e i n v e s t i g a t i o n of the quinone r e d u c t i o n r o u t e i n o u r l a b o r a t o r i e s l e d t o development o f an improved procedure which a f f o r d e d s u b s t a n t i a l l y improved y i e l d s ( 5 5 ) . R e d u c t i o n o f the phenanthrene 1,2-and 3,4-diones w i t h L i A l H , by t h i s method gave the 1,2- and 3 , 4 - d i h y d r o d i o l s i n y i e l d s o f 4 6 % and 27%, r e s p e c t i v e l y ( F i g u r e 10). F u r t h e r enhancement o f these y i e l d s was a c h i e v e d by c a r r y i n g out these r e d u c t i o n s w i t h NaBH^ i n e t h a n o l under a i r ( 2 2 ) . I t i s b e l i e v e d t h a t oxygen s e r v e s t o r e o x i d i z e c a t e c h o l b y p r o d u c t s t o quinones. The phenanthrene 1,2- and 3,4-diones are s y n t h e t i c a l l y a c c e s ­ s i b l e from the r e l a t e d 3 - p h e n o l s . O x i d a t i o n o f 2-phenanthrol w i t h e i t h e r Fremy's s a l t ((KSO^^NO) o r p h e n y l s e l e n i n i c anhydride gave phenanthrene 1,2-dione d i r e c t l y ( 5 5 ) . U n e x p e c t e d l y , o x i d a t i o n o f 3p h e n a n t h r o l w i t h (KSO^^NO y i e l d e d 2,2-dihydroxybenz(e)indan-l,3-dione ( F i g u r e 10). However, phenanthrene 3,4-dione was r e a d i l y ob­ t a i n e d from 3-phenanthrol by F i e s e r ' s method e n t a i l i n g d i a z o n i u m c o u p l i n g , r e d u c t i o n , and o x i d a t i o n o f the r e s u l t i n g 4-amino-3-phena n t h r o l w i t h chromic a c i d ( 5 6 ) . The development o f s a t i s f a c t o r y methods f o r the s t e r e o s e l e c t i v e r e d u c t i o n o f t e r m i n a l r i n g PAH quinones t o t r a n s - d i h y d r o d i o l s r e ­ p r e s e n t e d a s i g n i f i c a n t advance. Combined w i t h methods f o r t h e o x i d a t i o n o f 8-phenols t o o r t h o - q u i n o n e s i t p r o v i d e d the b a s i s o f what i s p r o b a b l y the most g e n e r a l s y n t h e t i c r o u t e t o n o n - K - r e g i o n dihydrodiols. T h i s s y n t h e t i c approach i s d e s i g n a t e d Method I V . W h i l e r e d u c t i o n s w i t h L i A l H ^ appear t o be g e n e r a l l y s t e r e o s p e c i f i C . , the r e a c t i o n s w i t h NaBH^ a f f o r d somewhat b e t t e r y i e l d s , b u t show lower s t e r e o s e l e c t i v i t y , y i e l d i n g v a r i a b l e amounts o f c i s - a s w e l l as trans-dihydrodiols. S y n t h e s i s o f t h e a n t i and s y n isomers o f t h e 1 , 2 - d i o l - 3 , 4 epoxide o f phenanthrene by e p o x i d a t i o n o f t h e 1 , 2 - d i h y d r o d i o l has been r e p o r t e d by Whalen e t a l . ( 5 7 ) . Anthracene Anthracene i s n o n c a r c i n o g e n i c and i s s t r u c t u r a l l y i n c a p a b l e o f f o r ­ ming a bay r e g i o n d i o l e p o x i d e . Anthracene 1 , 2 - d i h y d r o d i o l i s most c o n v e n i e n t l y s y n t h e s i z e d from 2 - a n t h r a n o l by o x i d a t i o n w i t h p h e n y l ­ s e l e n i n i c anhydride t o anthracene 1,2-dione (55) f o l l o w e d by r e ­ d u c t i o n w i t h NaBH^ i n e t h a n o l (22) o r L i A l H ^ ( 5 5 ) . Anthracene 1,2d i h y d r o d i o l has a l s o been s y n t h e s i z e d v i a the P r o v o s t r e a c t i o n r o u t e (30). Chrysene Chrysene i s a weak tumor i n i t i a t o r and i s i n a c t i v e as a complete carcinogen (38). The 1 , 2 - d i h y d r o d i o l i s more a c t i v e as a mutagen than the 3,4- o r the 5 , 6 - d i h y d r o d i o l s . The b i o l o g i c a l d a t a support the h y p o t h e s i s t h a t the p r i n c i p a l a c t i v e m e t a b o l i t e o f chrysene i s the bay r e g i o n a n t i - 1 , 2 - d i o l - 3 , 4 - e p o x i d e ( 5 8 ) . Two s y n t h e t i c approaches t o the 1,2- and 3 , 4 - d i h y d r o d i o l s o f chrysene (10 and 1J.) have been r e p o r t e d (41,59), and an a d d i t i o n a l n o v e l methoa w i l l ^ E e d e s c r i b e d h e r e i n ( 6 0 ) . "Syntheses o f 10 and 11

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from naphthalene (61) and phenanthrene (62) v i a 1-oxo- and 4-oxo1 , 2 , 3 , 4 - t e t r a h y d r o c h r y s e n e by Method I was d e s c r i b e d by K a r l e e t a l , ( 4 1 ) . A more c o n v e n i e n t s y n t h e t i c r o u t e t o these d i h y d r o d i o l s d i ­ r e c t l y from chrysene by Method I I I has been r e p o r t e d by Fu and Harvey (59) ( F i g u r e 1 1 ) . H y d r o g e n a t i o n o f chrysene over a p a l l a d i u m c a t a ­ l y s t a f f o r d e d r e g i o s p e c i f i c a l l y 5,6-dihydrochrysene, w h i l e s i m i l a r r e a c t i o n over PtO« gave 1 , 2 , 3 , 4 - t e t r a h y d r o c h r y s e n e , and hydrogena­ t i o n over a mixecT Pd-Pt c a t a l y s t f u r n i s h e d 1,2,3,4,5,6-hexahydrochrysene (37,59,63). Dehydrogenation o f the hexahydrochrysene de­ r i v a t i v e w i t h DDQ took p l a c e r e g i o s e l e c t i v e l y t o y i e l d 3,4,5,6-tetrahydrochrysene. T h i s o l e f i n underwent smooth t r a n s f o r m a t i o n t o the 1 , 2 - d i h y d r o d i o l (10) v i a the P r e v o s t r e a c t i o n , d e h y d r o g e n a t i o n , and m e t h a n o l y s i s . A l t h o u g h d e h y d r o g e n a t i o n o f 1 , 2 , 3 , 4 - t e t r a h y d r o ­ chrysene c o u l d n o t be stopped a t the d i h y d r o s t a g e , p a r t i a l de­ h y d r o g e n a t i o n was r e a d i l y a c h i e v e d by the b r o m i n a t i o n - d e h y d r o b r o m i n a t i o n method t o y i e l d a m i x t u r e o f 1,2-and 3,4-dihydrochrysene from which the 3 , 4 - d i h y d r o d i o l ( J J ) was s y n t h e s i z e d v i a the u s u a l PreVost r e a c t i o n r o u t e . E p o x i d a t i o n o f 10 w i t h m - c h l o r o p e r b e n z o i c a c i d y i e l d e d the c h r ­ ysene a n t i - 1 , 2 - d i o l - 3 , 4 - e p o x i d e , whereas s i m i l a r r e a c t i o n o f 11 gave a m i x t u r e o f the c o r r e s p o n d i n g a n t i and s y n d i o l epoxides i n a 5:3 r a t i o (57,59). These f i n d i n g s a r e i n a c c o r d w i t h p r e v i o u s o b s e r v a ­ t i o n s t h a t d i h y d r o d i o l s f r e e t o adopt the d i e q u a t o r i a l c o n f o r m a t i o n undergo a n t i s t e r e o s p e c i f i c e p o x i d a t i o n , whereas bay r e g i o n d i a x i a l d i h y d r o d i o l s y i e l d m i x t u r e s o f a n t i and s y n d i a s t e r e o m e r s . The s y n 1 , 2 - d i o l - 3 , 4 - e p o x i d e d i a s t e r e o m e r o f chrysene was s y n t h e s i z e d from 10 v i a b a s e - c a t a l y z e d c y c l i z a t i o n o f the b r o m o t r i o l i n t e r m e d i a t e (57,60). The o p t i c a l l y pure (+) and (-) enantiomers o f b o t h the a n t i and syn chrysene 1 , 2 - d i o l - 3 , 4 - e p o x i d e s have a l s o been p r e p a r e d (64). An a l t e r n a t i v e new s y n t h e t i c approach t o chrysene 1,2-dihydro­ d i o l based on Method IV has r e c e n t l y been developed ( 6 0 ) . This method ( F i g u r e 12) e n t a i l s s y n t h e s i s o f 2 - c h r y s e n o l v i a a l k y l a t i o n of l - l i t h i o - 2 , 5 - d i m e t h o x y - l , 4 - c y c l o h e x a d i e n e w i t h 2 - ( l - n a p h t h y l ) et h y l bromide f o l l o w e d by m i l d a c i d treatment t o ge n e r a t e t h e d i ketone 12. A c i d - c a t a l y z e d c y c l i z a t i o n o f 12 gave the u n s a t u r a t e d t e t r a c y c l i c ketone 13 which was t r a n s f o r m e d t o 2 - c h r y s e n o l v i a de­ h y d r o g e n a t i o n o f i t s e n o l a c e t a t e w i t h o - c h l o r a n i l f o l l o w e d by h y ­ d r o l y s i s . O x i d a t i o n o f 2 - c h r y s e n o l w i t h Fremy's s a l t gave chrysene 1,2-dione which underwent r e d u c t i o n w i t h NaBH^ i n the presence o f oxygen t o y i e l d 11. T h i s method i s r e a d i l y a d a p t a b l e t o s y n t h e s i s on any s c a l e . Benzo(c)phenanthrene Benzo(c)phenanthrene (BeP) i s e x c e p t i o n a l l y weak o r i n a c t i v e as a c a r c i n o g e n i n e x p e r i m e n t a l a n i m a l s ( 5 1 ) . On the o t h e r hand, the bay r e g i o n a n t i d i o l epoxide o f BeP (14) e x h i b i t s h i g h tumor i n i t i a t i n g a c t i v i t y on mouse s k i n ( 6 5 ) . *~ The 3 , 4 - d i h y d r o d i o l o f BeP was s y n t h e s i z e d from 4-oxo-l,2,3,4t e t r a h y d r o - B c P (15) by Method I ( 6 6 ) . The ketone L§ was i t s e l f p r e p a r e d from 4 - o x o - l , 2 , 3 , 4 - t e t r a h y d r o p h e n a n t h r e n e v i a a m u l t i s t e p sequence e n t a i l i n g Reformatsky r e a c t i o n w i t h m e t h y l bromocrotonate, d e h y d r a t i o n o f the r e s u l t i n g a l c o h o l , i s o m e r i z a t i o n t o the a r y l b u t y r i c a c i d , and c y c l i z a t i o n o f i t s a c i d c h l o r i d e w i t h S n C l * . F u l l

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch003

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F i g u r e 10. S y n t h e s i s o f the 1,2- and 3 , 4 - d i h y d r o d i o l s o f phen­ anthrene from the r e l a t e d phenols by Method I V ( 4 7 ) . Reagents: ( i ) ( K S 0 ) NO o r (PhSeO) 0; ( i i ) L i A l H ^ o r NaBH,,0,; ( i i i ) d i azonium s a l t o f s u l f a n i l i c a c i d ; ( i v ) N a 0 « S ; ( v ; fJrO~. 3

2

9

9

F i g u r e 11. S y n t h e s i s o f the chrysene 1,2- and 3 , 4 - d i h y d r o d i o l s and the c o r r e s p o n d i n g d i o l epoxide d e r i v a t i v e s from chrysene by Method I I I ( 5 1 ) . Reagents: ( i ) H ,Pd; ( i i ) H , P t ; ( i i i ) DDQ; ( i v ) A g O B z , I ; ( v ) NBSsA^fc}|P§ftS ^ ^ | ^ § f M e J ( v i i i ) 5L- 2

2

C P B A

2

Society Library 1155 1Grh St. N.

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e x p e r i m e n t a l d e t a i l s have n o t been p u b l i s h e d . 4-0xo-l,2,3,4-tetrahydrophenanthrene i s s y n t h e t i c a l l y a c c e s s i b l e from naphthalene v i a the Haworth s y n t h e s i s ( 6 7 ) . More c o n v e n i e n t s y n t h e t i c access t o 15 i s p r o v i d e d by t h e s e ­ quence i n F i g u r e 13 ( 6 8 ) . A l k y l a t i o n o f t h e p o t a s s i u m s a l t o f 2,6d i m e t h o x y - 1 , 4 - c y c l o h e x a d i e n e w i t h 2 - ( 2 - n a p h t h y l ) e t h y l bromide i n l i q u i d ammonia f o l l o w e d by m i l d a c i d i c h y d r o l y s i s generated t h e d i ketone ( 1 6 ) . C y c l i z a t i o n o f 16 i n p o l y p h o s p h o r i c a c i d took p l a c e smoothly i n the d e s i r e d d i r e c t i o n t o a f f o r d t h e p a r t i a l l y s a t u r a t e d ketone which underwent d e h y d r o g e n a t i o n w i t h DDQ t o 15. S y n t h e s i s o f t h e 1 , 2 - d i h y d r o d i o l o f BeP by c o n v e n t i o n a l methods was b l o c k e d by t h e f a i l u r e o f attempts t o s y n t h e s i z e i t s p o t e n t i a l s y n t h e t i c precursors 1-keto-l,2,3,4-tetrahydro-BcP and 1,2-dihydroBcP ( 6 6 ) . However, BeP 1 , 2 - d i h y d r o d i o l was o b t a i n e d i n low y i e l d ( ~ 1 % ) by o x i d a t i o n o f BeP w i t h a s c o r b i c a c i d - f e r r o u s s u l f a t e ( 6 6 ) . Dibenzo(a,i)pyrene

and D i b e n z o ( a , h ) p y r e n e

These h e x a c y c l i c hydrocarbons a r e g e n e r a l l y r e c o g n i z e d as two o f t h e most potent u n s u b s t i t u t e d c a r c i n o g e n i c PAH ( 3 8 ) . The 3 , 4 - d i h y d r o ­ d i o l o f d i b e n z o ( a , i ) p y r e n e (17) and t h e 1 , 2 - d i h y d r o d i o l o f d i b e n z o (a,h) pyrene (j^§) have been s y n t h e s i z e d from 4 - o x o - l , 2 , 3 , 4 - t e t r a h y d r o d i b e n z o ( a , l ) p y r e n e and 1-oxo-l,2,3,4-tetrahydrodibenzo(a,h)pyr e n e , r e s p e c t i v e l y , by Method I . ( 6 9 ) . Treatment o f these d i h y d r o ­ d i o l s w i t h m - c h l o r o p e r b e n z o i c a c i d gave t h e c o r r e s p o n d i n g a n t i d i o l epoxides ( 6 6 ) . 7-Methylbenz(a)anthracene W h i l e BA i s e s s e n t i a l l y i n a c t i v e as a complete c a r c i n o g e n , 7-met h y l b e n z ( a ) a n t h r a c e n e (MBA) e x h i b i t s r e l a t i v e l y p o t e n t a c t i v i t y i n t h i s r e s p e c t (27,38). This d i f f e r e n c e t y p i f i e s the o f t e n dramatic e f f e c t o f m e t h y l s u b s t i t u t i o n on t h e b i o l o g i c a l a c t i v i t y o f PAH compounds ( 7 0 ) . The m o l e c u l a r b a s i s o f a l k y l sub s t i t u t i o n e f f e c t s i s one o f t h e most i n t r i g u i n g problems i n c u r r e n t c a r c i n o g e n e s i s r e s e a r c h . However, much l e s s p r o g r e s s has been made i n e l u c i d a t i n g the d e t a i l s o f t h e m e t a b o l i c a c t i v a t i o n and DNA b i n d i n g o f m e t h y l s u b s t i t u t e d than u n s u b s t i t u t e d PAH because o f the g r e a t e r c o m p l e x i t y o f t h e i r m e t a b o l i s m and t h e g r e a t e r d i f f i c u l t y o f t h e s y n t h e s i s o f their active metabolites. B i o l o g i c a l s t u d i e s have i m p l i c a t e d t h e a n t i 3 , 4 - d i o l - l , 2 - e p o x i d e o f MBA as i t s u l t i m a t e c a r c i n o g e n i c me­ t a b o l i t e (71-73). Syntheses o f t h e 1,2- and t h e 3 , 4 - d i h y d r o d i o l s o f MBA v i a Me­ thods I I and I V have been d e s c r i b e d ( 7 4 ) . The 1,2- and 3 , 4 - d i o l d i b e n z o a t e s o f 1,2,3,4-tetrahydro-MBA prepared from MBA v i a the L i / NH^ r e d u c t i o n r o u t e were r e a d i l y s e p a r a b l e by c r y s t a l l i z a t i o n . I n ­ t r o d u c t i o n o f t h e o l e f i n i c bond i n t o t h e 1 , 2 - p o s i t i o n o f t h e 3,4d i o l d i b e n z o a t e by t h e u s u a l b r o m i n a t i o n - d e h y d r o b r o m i n a t i o n proce­ dure was c o m p l i c a t e d by the g r e a t e r f a c i l i t y o f b r o m i n a t i o n by NBS on the m e t h y l group than t h e 1 - p o s i t i o n . T h i s problem was s o l v e d ( F i g ­ ure 14) by a l l o w i n g b r o m i n a t i o n t o proceed t o t h e dibromo s t a g e , f o l l o w e d by s e l e c t i v e r e d u c t i o n o f t h e bromomethyl group w i t h NaBH^ i n diglyme. The monobromo d e r i v a t i v e underwent d e h y d r o b r o m i n a t i o n i t h an amine base t o f u r n i s h t h e 3 , 4 - d i h y d r o d i o l d i b e n z o a t e e s t e r 19b), which on treatment w i t h NaOMe i n methanol y i e l d e d t h e f r e e

r

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch003

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F i g u r e 12. S y n t h e s i s o f chrysene 1 , 2 - d i h y d r o d i o l by Method I V (52). Reagents: ( i ) H ; ( i i ) i s o p r o p e n y l a c e t a t e ; ( i i i ) DDQ; ( i v ) ( K S 0 ) NO; ( v ) NaBH ,0 . 3

4

2

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F i g u r e 13. S y n t h e s i s o f 4 - o x o - l , 2 , 3 , 4 - t e t r a h y d r o b e n z o ( c ) p h e n a n t h r e n e (1J) ( 5 9 ) .

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3,4-dihydrodiol (19a). A s i m i l a r sequence o f o p e r a t i o n s on t h e 1,2,3,4-tetrahydro-MBA 1 , 2 - d i o l d i b e n z o a t e f u r n i s h e d t h e i s o m e r i c 1 , 2 - d i h y d r o d i o l o f MBA. The 3 , 4 - d i h y d r o d i o l was a l s o s y n t h e s i z e d v i a Method I V ( 7 4 ) . O x i d a t i o n o f 3-hydroxy-MBA w i t h Fremy's s a l t gave t h e 3,4-quinone which underwent r e d u c t i o n w i t h L i A l H ^ t o g i v e 19a. The y i e l d i n t h e r e d u c t i o n s t e p was o n l y 15%, b u t i t i s l i k e l y t h a t t h i s c o u l d be s u b s t a n t i a l l y improved by t h e use o f t h e NaBH^/0 system (18) d e ­ v e l o p e d a f t e r these s t u d i e s were completed. The 1 0 , 1 1 - d i h y d r o d i o l o f MBA was s y n t h e s i z e d from MBA by Method I I I ( 1 2 ) . H y d r o g e n a t i o n o f MBA over a p l a t i n u m c a t a l y s t took p l a c e r e g i o s p e c i f i c a l l y i n the t e r m i n a l r i n g t o provide 8,9,10,11-tetrahydro-MBA ( 7 5 ) . Treatment o f t h e l a t t e r w i t h DDQ f u r n i s h e d 8,9dihydro-MBA which underwent c o n v e r s i o n t o the 1 0 , 1 1 - d i h y d r o d i o l by the u s u a l p r o c e d u r e s . O x i d a t i o n o f MBA w i t h a s c o r b i c a c i d - f e r r o u s s u l f a t e t o a f f o r d low y i e l d s ( 8

7

6

Benzo[c]phenanthrene BcPh weak care.

6

BenzOaJanthracene BA weak care. Triphenylene Tp noncarc. 12

1

11

.794

.4BB Benzo Dajoyrene BaP strong care. 7

6

5

Benzo &1 pyrene BeP noncarc. .500

' 8

7

5

.S45

6

Dibenzo&,hJ pyrene DBahP strong care.

9

8

7

DibenzoCa,0pyrene DBaiP strong care.

F i g u r e 1. S t r u c t u r e s , w i t h numbering and c a l c u l a t e d A £ v a l u e s a t b e n z y l i c p o s i t i o n s o f the t e t r a h y d r o b e n z o - r i n g t i v e s o f PAH d i s c u s s e d i n t h i s c h a p t e r .

/3 deriva­

d e l o c

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

68

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

along w i t h c a l c u l a t e d values of A E ^ ^ / 3 , which r e f e r to the v a l u e s c a l c u l a t e d a t t h e b e n z y l i c p o s i t i o n on t h e t e t r a h y d r o b e n z o r i n g d e r i v a t i v e o f t h e PAH. A l s o i n d i c a t e d i n t h i s F i g u r e a r e t h e a b b r e v i a t i o n s t o be used f o r t h e PAH i n t h i s a r t i c l e , and t h e numbering o f t h e PAH. The h i g h e r c a l c u l a t e d v a l u e s o f A E ^ ^ / 3 a t the b a y - r e g i o n p o s i t i o n s o f each PAH a r e e v i d e n t , and i t i s a p p r o ­ p r i a t e t o note t h a t e x p e r i m e n t a l r e s u l t s w i t h a l l the carcinogens i n F i g u r e 1 a r e c o n s i s t e n t w i t h m e t a b o l i c a c t i v a t i o n t o bay r e g i o n d i o l e p o x i d e s as a major r o u t e o f a c t i v a t i o n o f t h e PAH. Indeed, f o r t h e more than t e n e v e n - a l t e r n a n t PAH so f a r s t u d i e d i n some depth, the e x p e r i m e n t a l r e s u l t s s t r o n g l y support "bay-region" a c t i ­ v a t i o n . Recent r e v i e w s (12-14) have summarized t h e s e r e s u l t s , w h i c h came f r o m d e t a i l e d s t u d i e s o f t h e m e t a b o l i s m o f PAH and t h e i r d i h y d r o d i o l d e r i v a t i v e s , s t u d i e s o f t h e m u t a g e n i c i t y o f PAH and t h e i r d e r i v a t i v e s b o t h w i t h and w i t h o u t m e t a b o l i c a c t i v a t i o n , and s t u d i e s o f t h e t u m o r i g e n i c i t y o f PAH and t h e i r d e r i v a t i v e s . This m a n u s c r i p t w i l l f o c u s p r i m a r i l y upon t h e " u l t i m a t e " mutagens and c a r c i n o g e n s , t h e d i o l e p o x i d e s , as w e l l a s t h e r e l a t e d t e t r a h y d r o ­ epoxides, w i t h a view to e x p l o r i n g the s t r u c t u r e - a c t i v i t y r e l a t i o n ­ s h i p s t h a t g o v e r n t h e i r c h e m i c a l and b i o l o g i c a l b e h a v i o r , and t h e e x t e n t t o w h i c h quantum c h e m i c a l c a l c u l a t i o n s are u s e f u l i n e x ­ p l a i n i n g those r e l a t i o n s h i p s .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch004

E

Q C

T e t r a h y d r o e p o x i d e s as models. S i n c e the quantum c h e m i c a l c a l c u l a ­ t i o n s a p p l y most r i g o r o u s l y t o t h e s i m p l e b e n z o - r i n g t e t r a h y d r o ­ e p o x i d e s and s i n c e t h e c a l c u l a t i o n s n e g l e c t i n f l u e n c e s o f t h e h y ­ d r o x y l groups i n t h e d i o l e p o x i d e s , i t i s i n s t r u c t i v e f i r s t t o examine the b e n z o - r i n g t e t r a h y d r o e p o x i d e s as s i m p l i f i e d models f o r the r e a c t i v e s i t e i n t h e d i o l e p o x i d e s . Most o f t h e i n f o r m a t i o n about t e t r a h y d r o e p o x i d e r e a c t i v i t y d e r i v e s f r o m s t u d i e s o f t h e k i n e t i c s o f t h e i r h y d r o l y s i s r e a c t i o n s , i n w h i c h c i s - and t r a n s d i o l s , as w e l l as t e t r a h y d r o k e t o n e s can be formed ( E q u a t i o n 5).

OH

OH

The r e a c t i o n f o l l o w s t h e r a t e law: k i ^R^"*" o» ^ — ^ v a l u e s o f k f o r e i g h t PAH t e t r a h y d r o e p o x i d e s a r e p l o t t e d i n F i g u r e 2 against the AE^eloc^ values at the b e n z y l i c p o s i t i o n bearing the epoxide. S i n c e cne e x p e r i m e n t a l c o n d i t i o n s used f o r t h e naph­ t h a l e n e (Np) and phenanthrene (Ph) s t u d i e s d i f f e r e d f r o m those f o r the s t u d i e s o f BA, t r i p h e n y l e n e (Tp), benzo[e]pyrene (BeP) and BaP, a d i r e c t c o m p a r i s o n o f a l l compounds i s d i f f i c u l t . Interestingly, f o r a s e r i e s o f t e t r a h y d r o e p o x i d e s whose h y d r a t i o n p r o d u c t s have been s t u d i e d , t h e percentage o f c i s - h y d r a t i o n d u r i n g a c i d i c hy­ d r o l y s i s i n c r e a s e s as t h e c a l c u l a t e d ease o f f o r m a t i o n o f t h e c a r b o c a t l o n i n c r e a s e s , a r e s u l t c o n s i s t e n t w i t h l o n g e r - l i v e d (more s t a b l e ) c a r b o c a t i o n s as one proceeds a l o n g t h e s e r i e s (16). =

+

k

a

0 D S


X H

o

m

X

L/1

SO

thyl and Fluorine Substitutic

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985. 85 70 10 5 60 35 0 0 0

100 33 100 33 100 33 100 33

-

5-MeC-l,2-diol

5-MeC-7,8-diol

Anti-DE-I

Anti-DE-II

10

0 5

80 65

75 30

100 85

90 80

0

0 0

1.8 0.4

0.1 0.1

4.3 2.4

1.2 1.1

0.1

0 0.1

4.4 1.3

1.3 0.3

12.7 9.9

5.2 3.9

a

Weeks o f treatment

with

tetradecanoyl phorbol

acetate.

Note: Groups o f 20 female CD-1 mice (age 50-55 days) were shaved and t r e a t e d w i t h a s i n g l e dose o f each compound i n 0.1 ml acetone. Ten days l a t e r , each group was t r e a t e d 3 times weekly w i t h 2 .5 ug of t e t r a d e c a n o y l p h o r b o l a c e t a t e i n 0.1 ml acetone f o r 25 weeks.

Acetone

50 45

3

Tumors P e r Animal 15 weeks 25 weeks

o f 5-MeC M e t a b o l i t e s on Mouse S k i n

P e r c e n t Tumor Bearing Animals 15 w e e k s 25 weeks

100 33

Dose (nmol)

Tumor I n i t i a t i n g A c t i v i t y

5-MeC

Table I I .

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5.

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Effects of Methyl and Fluorine Substitution

95

DE-n-dG F i g u r e 5. S t r u c t u r e s o f the major adducts formed upon r e a c t i o n o f ( A ) a n t i DE-I and ( B ) a n t i - D E - I I w i t h DNA i n v i t r o .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

bon 4 o f a n t i - D E - I . A s t r u c t u r a l l y s i m i l a r major adduct i s formed from a n t i - D E - I I ( 2 6 ) . However, a n t i - D E - I adducts exceed a n t i - D E - I I adducts by 2-3 f o l d i n mouse s k i n , 4-48 h r a f t e r t r e a t m e n t w i t h [ % ] 5-MeC ( 2 7 ) . The predominance o f a n t i - D E - I adducts over a n t i - D E - I I adducts i n mouse s k i n i s n o t due t o d i f f e r e n c e s i n e x t e n t s o f forma­ t i o n of 5 - M e C - l , 2 - d i o l and 5-MeC-7,8-diol s i n c e t h e l e v e l s o f t h e s e m e t a b o l i t e s i n mouse e p i d e r m i s a r e t h e same from 0.33-4 h r a f t e r t r e a t m e n t w i t h [ H]5-MeC ( 2 7 ) . The r a t e s o f h y d r o l y s i s and b i n d i n g t o DNA o f a n t i - D E - I , syn-DEI , a n t i - D E - I I , s y n - D E - I I , and a n t i - 1 , 2 - d i h y d r o x y - 3 , 4 - e p o x y - l , 2 , 3 , 4 t e t r a h y d r o c h r y s e n e ( a n t i - c h r y s e n e - D E ) were s t u d i e d i n o r d e r t o r e l a t e the c h e m i c a l r e a c t i v i t y o f t h e s e d i h y d r o d i o l e p o x i d e s t o t h e i r b i o ­ logical activities. The h a l f - l i v e s o f t h e d i h y d r o d i o l e p o x i d e s i n c a c o d y l a t e b u f f e r a t pH 7.0 and 37°C a r e summarized i n T a b l e I I I and t h e i r r e l a t i v e e x t e n t s o f b i n d i n g t o DNA i n T a b l e I V . I t i s c l e a r t h a t t h e r a t e s o f h y d r o l y s i s o f t h e d i h y d r o d i o l e p o x i d e s do n o t c o r ­ r e l a t e w i t h t h e i r DNA b i n d i n g p r o p e r t i e s .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch005

3

Table I I I . H a l f - L i v e s o f D i h y d r o d i o l Epoxides o f 5-MeC and Chrysene at pH 7.0 and 37 °C i n the Absence and Presence o f N a t i v e and Denatured C a l f Thymus DNA t^/2 Compound

Buffer Solution Only

(minutes) Denatured DNA

Native DNA

Anti-DE-I

59

24

Syn-DE-I

62

48

22

Anti-DE-II

17.5

9

2

4.9

2.8

Syn-DE-II Anti-chrysene-DE

5.4 104

77

3.5

21

When t h e h y d r o l y s e s were c a r r i e d o u t i n t h e p r e s e n c e o f denatured DNA, a r a t e enhancement o f 1.1 t o 2.5 f o l d was o b s e r v e d w h i l e i n t h e p r e s e n c e o f n a t i v e DNA t h e enhancement was 2 t o 17 f o l d ( s e e T a b l e III). The r a t i o s o f t h e r a t e s o f h y d r o l y s i s i n t h e presence o f n a t i v e DNA t o t h e r a t e s o f h y d r o l y s i s i n t h e presence o f denatured DNA d i d c o r r e l a t e w i t h t h e e x t e n t s o f b i n d i n g t o DNA as i l l u s t r a t e d i n F i g u r e 6. Our i n t e r p r e t a t i o n o f t h e s e r e s u l t s i s t h a t i n t e r c a l a ­ t i o n o f t h e d i h y d r o d i o l e p o x i d e i n DNA precedes r e a c t i o n , as has been observed w i t h b e n z o [ a ] p y r e n e - 7 , 8 - d i h y d r o d i o l - 9 , 1 0 - e p o x i d e s (28-30). T h i s may be t h e k e y f a c t o r i n d e t e r m i n i n g e x t e n t s o f b i n d i n g o f d i h y ­ d r o d i o l e p o x i d e s t o DNA i n v i t r o . I t i s of i n t e r e s t that the g r e a t e s t r a t e enhancement and h i g h e s t e x t e n t o f b i n d i n g were o b s e r v e d f o r a n t i - D E - I , w h i c h a l s o b i n d s t o DNA t o a g r e a t e r e x t e n t t h a n a n t i DE-II i n v i v o and i s t h e most t u m o r i g e n i c o f t h e m e t h y l a t e d bay r e g i o n d i h y d r o d i o l e p o x i d e s t e s t e d . W h i l e t h e s e r e s u l t s suggest t h a t

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Effects of Methyl and Fluorine Substitution

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Table IV. R e l a t i v e E x t e n t s o f B i n d i n g o f D i h y d r o d i o l Epoxides o f 5-MeC and Chrysene t o N a t i v e Calf-Thymus DNA a t pH 7.0 and 37 °C

Compound

R e l a t i v e Extents of Binding

Anti-DE-I

4.9

Syn-DE-I

2.0

Anti-DE-II

2.6

Syn-DE-II

1

Anti-chrysene-DE

2.4

e x t e n t s o f DNA b i n d i n g o f t h e d i h y d r o d i o l e p o x i d e s a r e a d e t e r m i n a n t of t u m o r i g e n i c a c t i v i t y , t h e e x c e p t i o n a l t u m o r i g e n i c i t y o f a n t i - D E - I can p r o b a b l y n o t be e x p l a i n e d on t h i s b a s i s a l o n e . The r e s u l t s o f t h e s e s t u d i e s demonstrate t h a t t h e enhancing e f f e c t o f a bay r e g i o n m e t h y l group on t u m o r i g e n i c i t y , as i n 5-MeC., i s due t o t h e e x c e p t i o n a l t u m o r i g e n i c i t y and r e l a t i v e l y h i g h r e a c ­ t i v i t y w i t h DNA o f a d i h y d r o d i o l e p o x i d e m e t a b o l i t e , anti-DE-I, h a v i n g a m e t h y l group and a n e p o x i d e r i n g i n t h e same bay r e g i o n . Among t h e monoraethylchrysenes, o n l y 5-MeC c a n form such a metabo­ lite. T h i s p a r t i a l l y e x p l a i n s i t s unique a c t i v i t y . S t u d i e s on t h e metabolic a c t i v a t i o n of 15,16-dihydro-ll-methylcyclopenta[a]phenanthrene-17-one and 7 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a c e n e have shown t h a t bay r e g i o n d i h y d r o d i o l e p o x i d e s a r e l i k e l y u l t i m a t e carcinogens (31,32). I t appears l i k e l y t h a t t h e enhancing e f f e c t o f a bay r e g i o n m e t h y l group on t u m o r i g e n i c i t y i n these systems i s a l s o a r e s u l t o f the e x c e p t i o n a l t u m o r i g e n i c i t y o f t h e s e d i h y d r o d i o l e p o x i d e metabo­ l i t e s h a v i n g a m e t h y l group and epoxide r i n g i n t h e same bay r e g i o n . I n t h e case o f 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e , i t has been proposed t h a t t h e bay r e g i o n s y n - d i h y d r o d i o l e p o x i d e may be i m p o r t a n t i n i t s metabolic a c t i v a t i o n (33). The l o w t u m o r i g e n i c i t y o f syn-DE-I sug­ g e s t s , however, t h a t t h i s may n o t be t h e case and i n d i c a t e s t h e importance of s t e r i c f a c t o r s i n determining d i h y d r o d i o l epoxide t u m o r i g e n i c i t y , a s r e p o r t e d f o r u n s u b s t i t u t e d PAH. An i m p o r t a n t s t r u c t u r a l f e a t u r e o f the m e t h y l a t e d PAH w i t h a bay r e g i o n m e t h y l group i s t h e i r n o n - p l a n a r i t y . S t e r i c h i n d r a n c e between the m e t h y l group and t h e a d j a c e n t bay r e g i o n hydrogen causes d i s ­ t o r t i o n and d e v i a t i o n from p l a n a r i t y i n 7 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a ­ cene and 5-MeC (34,35). I t has been s u g g e s t e d t h a t n o n - p l a n a r i t y may p l a y a r o l e i n t h e h i g h t u m o r i g e n i c i t y o f t h e s e compounds. I n view of t h e r e s u l t s d i s c u s s e d above, i t would appear t h a t t h i s e f f e c t would have t o o p e r a t e a t t h e l e v e l o f t h e d i h y d r o d i o l e p o x i d e metabo­ lites. I t would be u s e f u l t o determine t h e x - r a y c r y s t a l s t r u c t u r e s of a n t i - D E - I and a n t i - D E - I I i n o r d e r t o e s t a b l i s h whether d i f f e r e n c e s i n p l a n a r i t y o f t h e s e m e t a b o l i t e s , i f any, c o u l d c o n t r i b u t e t o t h e i r d i f f e r i n g e x t e n t s o f r e a c t i o n w i t h DNA.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

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98

0

1

2

3

4

5

RELATIVE E X T E N T OF BINDING O F DIHYDRODIOL EPOXIDES TO DNA

F i g u r e 6, P l o t of the r a t i o s of the h a l f - l i v e s of d i h y d r o d i o l epoxides i n the p r e s e n c e of d e n a t u r e d DNA t o t h o s e i n t h e presence o f n a t i v e DNA v s . e x t e n t s o f DNA b i n d i n g i n v i t r o .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

5.

HECHT ET AL.

Effects of Methyl and Fluorine Substitution

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch005

M e c h a n i s t i c B a s i s f o r the I n h i b i t o r y E f f e c t P e r i M e t h y l Group

99

on T u m o r i g e n i c i t y o f a

Unhindered a n g u l a r r i n g t r a n s - d i h y d r o d i o l s such as t r a n s - 7 , 8 - d i h y d r o 7,8-dihydroxybenzo[a]pyrene e x i s t p r e f e r e n t i a l l y i n the pseudo d i e q u a t o r i a l c o n f o r m a t i o n and can u s u a l l y be o x i d i z e d e n z y m a t i c a l l y t o the c o r r e s p o n d i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s . However, a p e r i m e t h y l group causes crowding and as a r e s u l t the c o n f o r m a t i o n o f a t r a n s - d i h y d r o d i o l i n the a d j a c e n t a n g u l a r r i n g w i l l be p r e f e r e n t i a l l y diaxial. T h i s phenomenon has been n o t e d by s e v e r a l groups and h a s been p r e v i o u s l y r e v i e w e d (36, 3 7 ) . I t has been suggested t h a t the i n h i b i t o r y e f f e c t o f a p e r i m e t h y l group on t u m o r i g e n i c i t y i s due t o the r e l a t i v e d i f f i c u l t y o f enzymatic c o n v e r s i o n o f d i a x i a l d i h y d r o ­ d i o l s t o t h e i r corresponding d i h y d r o d i o l epoxides (36,37). Some experimental evidence supports t h i s suggestion although d e t a i l e d m e t a b o l i c s t u d i e s on compounds such a s 6-methylbenzo[ajpyrene, 3,11d i m e t h y l c h o l a n t h r e n e , and 5 , 7 , 1 2 - t r i m e t h y l b e n z [ a ] a n t h r a c e n e have not been r e p o r t e d . Whereas p e r i m e t h y l s u b s t i t u t i o n does n o t b l o c k d i h y d r o d i o l f o r m a t i o n i n t h e a d j a c e n t r i n g i n t h e b e n z [ a j a n t h r a c e n e system ( 3 8 , 3 9 ) , i t a p p a r e n t l y does so i n the c h r y s e n e system. 7,8-Dihydro7 , 8 - d i h y d r o x y - 5 , 1 2 - d i m e t h y l c h r y s e n e was a major m e t a b o l i t e o f 5,12d i m e t h y l c h r y s e n e i n r a t and mouse h e p a t i c 9000 x g s u p e r n a t a n t , b u t 1,2-dihydro-l,2-dihydroxy-5,12-diraethylchrysene c o u l d n o t be d e ­ tected. S i m i l a r l y , the r a t i o o f 7-hydroxy-5,12-dimethylchrysene t o 1-hydroxy-5,12-dimethylchrysene was about 100 t o 1 i n l i v e r s u p e r n a t a n t s from 3 - m e t h y l c h o l a n t h r e n e p r e t r e a t e d mice and r a t s . In c o n t r a s t , 1 , 2 - d i h y d r o - 1 , 2 - d i h y d r o x y - 5 , 1 1 - d i m e t h y l c h r y s e n e was a major m e t a b o l i t e of 5,11-dimethylchrysene ( 4 0 ) . These r e s u l t s suggest t h a t the low t u m o r i g e n i c i t y o f 5,12-dimethylchrysene i s due t o i n h i b i t i o n of f o r m a t i o n o f i t s l i k e l y major p r o x i m a t e c a r c i n o g e n , 1,2-dihydro1,2-dihydroxy-5,12-dimethylchrysene• E f f e c t s of F l u o r i n e S u b s t i t u t i o n on the T u m o r i g e n i c i t y o f PAH T a b l e V summarizes l i t e r a t u r e on the t u m o r i g e n i c a c t i v i t i e s of f l u o r ­ i n a t e d PAH, t e s t e d e i t h e r as tumor i n i t i a t o r s o r complete c a r c i n o g e n s on mouse s k i n . I n g e n e r a l , the r e s u l t s a r e c o n s i s t e n t w i t h the h y ­ pothesis that f l u o r i n e s u b s t i t u t i o n could block the formation of a n g u l a r r i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s . Thus, d e c r e a s e d tumor­ i g e n i c i t y was o b s e r v e d upon s u b s t i t u t i o n o f f l u o r i n e i n t h e a n g u l a r r i n g s o f 7-methylbenz[ajanthracene, 7,12-dimethylbenz[ajanthracene, b e n z o [ a ] p y r e n e , d i b e n z o [ a , 1 ] p y r e n e , and d i b e n z o [ a , h ] p y r e n e . I n the case o f 5-methylchrysene and 5-hydroxymethylchrysene w h i c h each have 2 a n g u l a r r i n g s and 2 bay r e g i o n s , d e c r e a s e d t u m o r i g e n i c i t y was observed o n l y upon f l u o r i n e s u b s t i t u t i o n i n the 1-4 r i n g which i s t h e major s i t e o f m e t a b o l i c a c t i v a t i o n a s d i s c u s s e d above. Decreased t u m o r i g e n i c i t y was a l s o observed upon f l u o r i n e s u b s t i t u t i o n a t t h e p e r i - p o s i t i o n s a d j a c e n t t o the a n g u l a r r i n g s i n v o l v e d i n m e t a b o l i c a c t i v a t i o n , as seen w i t h the m e t h y l a t e d PAH. Increases i n tumorigen­ i c i t y were observed upon s u b s t i t u t i o n o f f l u o r i n e a t t h e 7- and 1 2 - p o s i t i o n s of b e n z [ a j a n t h r a c e n e d e r i v a t i v e s , a l t h o u g h the i n c r e a s e s were much l e s s t h a n observed upon s u b s t i t u t i o n o f m e t h y l groups a t those p o s i t i o n s . Whereas the e f f e c t s o f f l u o r i n e s u b s t i t u t i o n i n t h e a n g u l a r r i n g s and p e r i - p o s i t i o n s a r e r e a s o n a b l y w e l l u n d e r s t o o d as

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100

T a b l e V.

T u m o r i g e n i c i t y o f F l u o r i n a t e d PAH on Mouse +

Benz[ajanthracene

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7-methylbenz[a]anthracene

b

7,12-dimethylbenz[ajanthracene

4F (41) 5F

12F (_16)

6F (41) 9F 10F

3F (16,41-43) 4F 5F 5F (16)

11F (17,44)

6F (45)

5-hydroxymethylchrysene

a

-

7F (16) 0

5-methylchrysene

benzo[a]pyrene

NC

7F (16) 12F 7,12diF

12-methylbenz[ajanthracene

Skin

IF (17,44) 2F 5F

6F (45,46) 7F 9F 11F

IF (45,46) 3F 12F

7F (24)

3F (24)

d

6F (47-49) 7F 8F 9F 10F

dibenzo[a,i]pyrene

e

2F (50,_51) 3F 2,10-diF

dibenzo[a,h]pyrene

3,10-diF (52)

a

+; more a c t i v e

b

When t e s t e d by s.c i n j e c t i o n i n r a t s , 6F was + (41,53); 2F, 3F, 5F, 9F, 10F were (22^41-43). when t e s t e d by s . c . i n j e c t i o n i n mice, 3F, 6F were + (41_); 5F, 9F, 10F were - (41-43).

c

When t e s t e d (22,53).

d

When t e s t e d f o r i n d u c t i o n o f l u n g adenomas i n mice o r by s . c . i n j e c t i o n i n r a t s , 6F was NC C49).

e

When t e s t e d by s . c . i n j e c t i o n

t h a n p a r e n t hydrocarbon; NC., no change; -, l e s s

by s . c . i n j e c t i o n

i n rats

I F , 2F, 4F were

-

active.

and 8F, 11F were

i n mice 3F and 2,10-diF were - ( 5 4 ) .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

NC

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Effects of Methyl and Fluorine Substitution

d e s c r i b e d below, t h e reasons f o r t h e i n c r e a s e s i n t u m o r i g e n i c i t y upon s u b s t i t u t i o n a t t h e 7- and 1 2 - p o s i t i o n s o f b e n z [ a j a n t h r a c e n e a r e obscure.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch005

E f f e c t s o f F l o r i n e S u b s t i t u t i o n on t h e M e t a b o l i c A c t i v a t i o n o f PAH M e t a b o l i s m s t u d i e s on f l u o r i n a t e d d e r i v a t i v e s o f 5-MeC., 5-hydroxym e t h y l c h r y s e n e , b e n z o [ a j p y r e n e and d i b e n z o [ a , i j p y r e n e have a l l shown t h a t f l u o r i n e e f f e c t i v e l y b l o c k s o x i d a t i o n o f a PAH a t t h e f o r m a l double bond t o w h i c h t h e f l u o r i n e i s a t t a c h e d (24,45,47,50). Only one example o f m e t a b o l i c l o s s o f f l u o r i n e has been r e p o r t e d ; 6f l u o r o b e n z o [ a ] p y r e n e was p a r t i a l l y c o n v e r t e d t o t h e 1,6- and 3,6quinones ( 4 8 ) . S i n c e d i h y d r o d i o l epoxide f o r m a t i o n r e q u i r e s s u c c e s ­ s i v e o x i d a t i o n i n t h e same r i n g , a s i n g l e f l u o r i n e atom i n a n a n g u l a r r i n g w i l l i n h i b i t t h i s pathway o f m e t a b o l i c a c t i v a t i o n . Thus, t h e DNA b i n d i n g ( 5 5 ) and c a r c i n o g e n i c i t y o f t h e a n g u l a r r i n g f l u o r i n a t e d compounds a r e lower t h a n those o f t h e c o r r e s p o n d i n g hydrocarbons (24,45,47,50). The e f f e c t s o f f l u o r i n e s u b s t i t u t i o n a t t h e p e r i - p o s i t i o n s a d j a c e n t t o a n g u l a r r i n g s o f PAH a r e n o t a s s t r a i g h t f o r w a r d . I t has been shown t h a t t h e t r a n s - d i h y d r o d i o l s a d j a c e n t t o p e r i f l u o r i n e subs t i t u e n t s adopt t h e p s e u d o - d i a x i a l c o n f o r m a t i o n , a s i n t h e case o f peri-methyl substitution. Thus, NMR e x p e r i m e n t s demonstrated t h a t the 5,6- and 8 , 9 - d i h y d r o d i o l s o f 7 - f l u o r o b e n z [ a j a n t h r a c e n e and t h e 7,8-dihydrodiol of 6-fluorobenzo[a]pyrene e x i s t i n the p s e u d o - d i a x i a l c o n f o r m a t i o n ( 4 8 , 5 6 ) . I t was suggested t h a t t h e i n h i b i t o r y e f f e c t o f a p e r i - f l u o r i n e s u b s t i t u e n t on t u m o r i g e n i c i t y , as observed f o r 5F-7methylbenz[ajanthracene, 5F-12-methylbenz[ajanthracene, 5F-7,12-di­ methylbenz [a] a n t h r a c e n e , 6F-benzo[a]pyrene, and 12F-5-methylchrysene, might be due e i t h e r t o a low r a t e o f c o n v e r s i o n o f t h e d i a x i a l d i h y ­ d r o d i o l s t o t h e c o r r e s p o n d i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s o r t o the i n h e r e n t l y lower t u m o r i g e n i c i t y o f t h e d i h y d r o d i o l e p o x i d e metab­ o l i t e (56). F o r 6F-benzo[a]pyrene, t h i s e x p l a n a t i o n appears t o be c o r r e c t ( 4 8 ) . Thus, t h e 7 , 8 - d i h y d r o d i o l i s formed m e t a b o l i c a l l y a t s i m i l a r r a t e s from b o t h 6F-benzo[ajpyrene and b e n z o [ a ] p y r e n e . Both d i h y d r o d i o l s have t h e same a b s o l u t e c o n f i g u r a t i o n , b u t t h e 7,8-dihy­ d r o d i o l o f 6F-benzo[a]pyrene i s d i a x i a l and i s n o t a p p r e c i a b l y muta­ g e n i c toward C h i n e s e hamster V79 c e l l s , i n c o n t r a s t t o b e n z o [ a ] pyrene-7,8-dihydrodiol. I n c o n t r a s t , t h e f l u o r i n e atom a t t h e p e r i - p o s i t i o n o f 12F-5m e t h y l c h r y s e n e i n f l u e n c e s d i h y d r o d i o l f o r m a t i o n i n t h e a d j a c e n t angu­ lar ring. Whereas t h e r a t i o o f 5-MeC-7,8-diol t o 5 - M e C - l , 2 - d i o l i n mouse e p i d e r m i s was 1:1, 2 h r a f t e r t o p i c a l a p p l i c a t i o n o f [ H]5-MeC., the r a t i o o f 1 2 F - 5 - m e t h y l c h r y s e n e - 7 , 8 - d i o l t o 12F-5-methylchrysene1 , 2 - d i o l was 68:1. I n c o n t r a s t t o 5-MeC., t h e m e t a b o l i t e s formed from 12F-5-methylchrysene i n mouse s k i n r e s u l t e d a l m o s t e x c l u s i v e l y from o x i d a t i o n a t t h e 7,8-bond ( 5 7 ) . Thus, m e t a b o l i c s w i t c h i n g t o t h e l e s s t u m o r i g e n i c 7 , 8 - d i h y d r o d i o l appears t o be t h e b a s i s f o r t h e lower t u m o r i g e n i c i t y o f 12F-5-methylchrysene compared t o 5-MeC. 3

Prospects f o r F u r t h e r Research W i t h some e x c e p t i o n s , t h e r e l a t i o n s h i p between s t r u c t u r e and a c t i v i t y of v a r i o u s m e t h y l a t e d and f l u o r i n a t e d PAH i s now r e a s o n a b l y w e l l understood. Important e x c e p t i o n s a r e 6-,7-, and 8 - m e t h y l b e n z [ a ] -

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

anthracene and 7,12-dimethylbenz[ajanthracene. There i s presently no satisfactory explanation for the potent tumorigenicity of these com­ pounds compared to other monomethyl or dimethylbenz[a]anthracenes. Nevertheless, current knowledge of structure-tumorigenicity relation­ ships should allow accurate prediction of the tumorigenic activities on mouse skin of untested methylated PAH. Extension of methylated PAH testing to other bioassay systems would be desirable. L i t t l e i s known, for example, about the carcinogenicity of methylated PAH administered by inhalation or i n the diet. In addition, the carcino­ genic activities of mixtures of PAH should be more extensively investigated since human exposure i s always to mixtures. Whereas bay region dihydrodiol epoxides appear to be major u l t i ­ mate carcinogens of a number of methylated PAH, the stereochemical aspects of dihydrodiol epoxide reactions with DNA and tumorigenicity require further investigation. Among the unsubstituted PAH, i t is known that the absolute configuration of dihydrodiol epoxide metabo­ l i t e s i s a key feature in their tumorigenic a c t i v i t i e s . Whether such stereochemical subtleties operate for the bay region dihydrodiol epoxides having a methyl group and epoxide ring i n the same bay region i s unknown, but appears likely based on the differences i n tumorigenicity between anti-DE-I and syn-DE-I. Since the general features of methylated PAH metabolic activa­ tion are known, i t should now be possible to design effective chemopreventive strategies. A key to this approach i s a better under­ standing of the a b i l i t y of the organism to detoxify dihydrodiol epox­ ide metabolites by conjugation with glutathione. If glutathione con­ jugates of methylated PAH dihydrodiol epoxides are formed, i t may be possible to enhance their rates of formation by various pretreatments. In addition, i t w i l l be important to identify naturally occurring or synthetic compounds that can prevent dihydrodiol epoxide formation or reaction with DNA i n vivo. Finally, i t i s important that further research be carried out on the identification and mechanism of action of environmental cocarcinogens and tumor promoters which can enhance the carcinogenicity of PAH. Human exposure to at least trace amounts of PAH i s unavoida­ ble. The probability of eventual tumor development may be controlled primarily by repeated exposure to cocarcinogens or promoters. Acknowle dgment s Our research on methylated PAH i s supported by Grant CA-32242 from The National Cancer Institute. Literature Cited 1. 2.

3.

International Agency for Research on Cancer. "IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volume 32"; IARC: Lyon, France, 1983; pp. 33-53. Committee on Pyrene and Selected Analogues, Board on Toxicology and Environmental Health Hazards, National Research Council. "Polycyclic Aromatic Hydrocarbons: Evaluation of Sources and Effects"; National Academy Press; Washington, D.C., 1983. Dipple, A. In "Chemical Carcinogens"; Searle, C.E., Ed.; American Chemical Society: Washington, D.C., 1976, pp. 245-314.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch005

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103

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30. MacLeod, M.C.; Selkirk, J.K. Carcinogenesis 1982, 3, 287-92. 31. Coombs, M.M.; Kissonerghis, A.M.; Allen, A.J.; Vose, C.W. Cancer Res. 1979, 39, 4160-5. 32. Moschel, R.C.; Baird, W.M.; Dipple, A. Biochem. Biophys Res. Commun. 1977, 76, 1092-8. 33. Sawicki, J.T.; Moschel, R.C.; Dipple, A. Cancer Res. 1983, 43, 3212-8. 34. Iball, J . Nature 1964, 201, 916-7. 35. Kashino, S.; Zacharias, D.E.; Prout, C.K.; Carrell, H.L.; Glusker, J.P.; Hecht, S.S.; Harvey, R.G. Acta Cryst. C., 1984. In press. 36. Yang, S.K.; Chou, M.W.; Fu, P.P. In "Carcinogenesis: Fundamen­ t a l Mechanisms and Environmental Effects"; Pullmann, B.; Ts'o, P.O.P.; Gelboin, H., Eds.; D. Reidel: London, 1980; pp. 143-56. 37. Slaga, T.J.; Iyer, R.P.; Lyga, W.; Secrist, A., III; Daub, G.H.; Harvey, R.G. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjorseth, A.; Dennis, A.J., Eds.; Batelle Press: Columbus, Ohio, 1980, pp. 753-69. 38. Yang, S.K.; Chou, M.W.; Fu, P.P. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjorseth, A.; Dennis, A.J., Eds.; Batelle Press: Columbus, Ohio, 1980, pp. 645-62. 39. Yang, S.K.; Chou, M.W.; Fu, P.P. In "Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate"; Cooke, M.; Dennis, A.J., Eds.; Batelle Press: Columbus, Ohio, 1981, pp. 253-64. 40. Amin, S.; Camanzo, J.; Hecht, S.S. Carcinogenesis 1982, 3, 1159-63. 41. Miller, J.A.; Miller, E.C. Cancer Res. 1963, 23, 229-39. 42. M i l l e r , E.C.; Miller, J.A. Cancer Res. 1960, 20, 133-7. 43. Newman, M.S. In "Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism and Carcinogenesis"; Freudenthal, R.; Jones, P.W., Eds.; Raven Press: New York, 1976, pp. 203-7. 44. Huberman, E.; Slaga, T.J. Cancer Res. 1979, 39, 411-14. 45. Hecht, S.S.; LaVoie, E.; Mazzarese, R.; Hirota, N.; Ohmori, T.; Hoffmann, D. J . Natl. Cancer Inst. 1979, 63, 855-61. 46. Hecht, S.S.; Hirota, N.; Loy, M.; Hoffmann, D. Cancer Res. 1978, 38, 1694-98. 47. Buhler, D.R.; Unlu, F.; Thakker, D.R.; Slaga, T.J.; Newman, M.S.; Levin, W.; Conney, A.H., Jerina, D.M. Cancer Res. 1982, 42, 4779-83. 48. Buhler, D.R.; Unlu, F.; Thakker, D.R.; Slaga, T.J.; Conney, A.H.; Wood, A.W.; Chang, R.L.; Levin, W.; Jerina, D.M. Cancer Res. 1983, 43, 1541-9. 49. Buening, M.K.; Levin, W.; Wood, A.W.; Chang, R.L.; Agranat, I.; Rabinovitz, M.; Buhler, D.R.; Mah, H.D.; Hernandez,O.;Simpson, R.B.; Jerina, D.M.; Conney, A.H.; M i l l e r , E.C.; Miller, J.A. J. Natl. Cancer Inst. 1983, 71, 309-15. 50. Hecht, S.S.; LaVoie, E.J.; Bedenko, V.; Pingaro, L.; Katayama, S.; Hoffmann, D.; Sardella, D.J.; Boger, E.; Lehr, R.E. Cancer Res. 1981, 41, 4341-45. 51. Chang, R.L., Levin, W.; Wood, A.W.; Lehr, R.E.; Kumar, S.; Yagi, H.; Jerina, D.M.; Conney, A.H. Cancer Res. 1982, 42, 25-9.

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Effects of Methyl and Fluorine Substitution

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Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch005

RECEIVED

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

6 Mechanisms of Interaction of Polycyclic Aromatic Diol Epoxides with DNA and Structures of the Adducts NICHOLAS E. GEACINTOV

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch006

Chemistry Department, New York University, New York, NY 10003

Spectroscopic studies on complexes derived from the binding of benzo(a)pyrene-7,8-diol-9,10-epoxide (BaPDE) to DNA indicate that the conformations of the adducts can be broadly classified into two types: site I which displays most of the properties of intercalative adducts, and site II which i s characterized by an orientation of the planar aromatic residues t i l t e d closer to the axis of the helix. Both the syn and the anti diastereomers of BaPDE form unstable type I physical intercalation complexes and undergo speci­ f i c and general acid catalysis to form tetraols (>90%) and covalent adducts (J>.) 5 i« - |AA|0CA. e

Two types o f DNA b i n d i n g s i t e s . Two d i f f e r e n t s p e c t r o s c o p i c a l l y d i s t i n c t types o f b i n d i n g s i t e s have been i d e n t i f i e d utilizing a b s o r p t i o n , f l u o r e s c e n c e and l i n e a r d i c h r o i s m d a t a on n o n - c o v a l e n t (6), and c o v a l e n t ( 7 ) p y r e n e - l i k e m e t a b o l i t e model compound-DNA complexes• S i t e I i s c h a r a c t e r i z e d by a r e l a t i v e l y l a r g e r e d shift of ~10 nm i n t h e a b s o r p t i o n maxima ( r e l a t i v e t o t h e aqueous s o l u t i o n s p e c t r a ) , e x h i b i t i n g maxima a t ^ 3 3 7 and ~ 3 5 4 nm, and a n e g a t i v e A A 8pectrum; a l l o f these p r o p e r t i e s a r e c o n s i s t e n t w i t h an i n t e r ­ c a l a t i o n - c o m p l e x geometry i n w h i c h the p l a n a r pyrene r i n g - s y s t e m i s n e a r l y p a r a l l e l t o the p l a n e s o f the DNA b a s e s . S i t e I I i s c h a r a c t e r i z e d by a r e l a t i v e l y s m a l l 2-3 nm r e d s h i f t i n t h e a b s o r p t i o n spectrum and a p o s i t i v e A A spectrum. In this c o n f o r m a t i o n , the planes o f the pyrene m o e i t i e s tend t o a l i g n p a r a ­ l l e l r a t h e r t h a n p e r p e n d i c u l a r t o the a x i s o f the DNA h e l i x . T h i s c l a s s i f i c a t i o n has r e c e n t l y been adopted by o t h e r wor­ k e r s as w e l l ( 8 - 1 0 ) .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch006

F o r m a t i o n o f P h y s i c a l I n t e r c a l a t i v e D i o l Epoxide-DNA Complexes Stopped f l o w k i n e t i c measurements i n d i c a t e t h a t when two aqueous s o l u t i o n s , one c o n t a i n i n g BaPDE and the o t h e r DNA, a r e mixed r a p i d ­ l y , a n o n - c o v a l e n t s i t e I - t y p e complex i s formed w i t h i n 5 ms o r l e s s (11). The l i n e a r d i c h r o i s m s p e c t r a o f such n o n - c o v a l e n t complexes can be measured by the f l o w o r i e n t a t i o n method, and t y p i c a l A A s p e c t r a o b t a i n e d w i t h r a c e m i c a n t i - and syn-BaPDE a r e shown i n F i g u r e 2. These e x p e r i m e n t s were c a r r i e d out a t pH 9.2 i n o r d e r t o m i n i m i z e the d e g r a d a t i o n o f the d i o l e p o x i d e m o l e c u l e s d u r i n g the measurement ( l e s s than 2 minutes). The s t r u c t u r e s and the n e g a t i v e s i g n o f the A A s p e c t r a suggest t h a t b o t h compounds form i n t e r c a l a t i v e p h y s i c a l complexes. The A A s p e c t r a o f the c o v a l e n t adducts a r e a l s o shown f o r comparison ( a f t e r a l l o w i n g the r e a c t i o n s t o go t o c o m p l e t i o n and e x t r a c t i n g the t e t r a o l h y d r o l y s i s p r o d u c t s o f BaPDE w i t h e t h e r ) ; f o r anti-BaPDE A A i s p o s i t i v e , as found p r e v i o u s l y C 5 ) , but f o r the syn adducts AA i s n e g a t i v e , as shown a l r e a d y by Undeman e t a l ( 1 0 ) . Thus, c o n s i d e r a b l e r e - o r i e n t a t i o n o f the p y r e n y l m o e i t y i s o c c u r r i n g i n the case o f (+) anti-BaPDE as a r e s u l t o f the c o v a l e n t b i n d i n g r e a c t i o n , w h i l e w i t h (+) syn-BaPDE the c o n f o r m a t i o n a l changes, i f any, appear t o be m i n o r . The k i n e t i c s o f such l i n e a r d i c h r o i s m changes have r e c e n t l y been s t u d i e d u t i l i z i n g the enantiomers (+) and (-) anti-BaPDE ( 1 2 ) . Analogous r e s u l t s have been r e c e n t l y o b t a i n e d w i t h t r a n s - l , 2 dihydroxy-anti-3,4-epoxy-l,2,3,4-tetrahydro-5-methyl chrysene (13) and the epoxide 1 - o x y r a n y l p y r e n e ( 1 4 ) . Thus, the f o r m a t i o n o f nonc o v a l e n t i n t e r c a l a t i v e s i t e I complexes appears t o be a g e n e r a l phenomenon w h i c h governs the i n t e r a c t i o n o f p o l y c y c l i c a r o m a t i c epo­ x i d e s w i t h DNA (15-17). R e a c t i o n Pathways o f BaPDE i n Aqueous DNA

Solutions

The e x p e r i m e n t a l l y observed p s e u d o - f i r s t o r d e r r a t e c o n s t a n t k i s i n c r e a s e d i n the presence o f DNA (18,19). T h i s enhanced r e a c t i v i t y i s a r e s u l t o f the f o r m a t i o n o f p h y s i c a l BaPDE-DNA complexes; the dependence o f k on DNA c o n c e n t r a t i o n c o i n c i d e s w i t h the b i n d i n g i s o t h e r m f o r the f o r m a t i o n o f s i t e I p h y s i c a l i n t e r c a l a t i v e com­ plexes (20). T y p i c a l l y , over ^ 9 0 % o f the BaPDE m o l e c u l e s a r e c o n v e r t e d t o t e t r a o l s , w h i l e o n l y a minor f r a c t i o n b i n d c o v a l e n t l y t o the DNA bases (18,21-23). The dependence o f k on temperature ( 2 1 , 2 4 ) , pH (21,23-25), s a l t c o n c e n t r a t i o n (16*20,21,25), and con­ c e n t r a t i o n o f d i f f e r e n t b u f f e r s (23) has been i n v e s t i g a t e d . I n 5 mM sodium c a c o d y l a t e b u f f e r s o l u t i o n s the f o r m a t i o n o f t e t r a o l s and c o v a l e n t adducts appear t o be p a r a l l e l p s e u d o - f i r s t o r d e r r e a c t i o n s c h a r a c t e r i z e d by the same r a t e c o n s t a n t k, but d i f f e r e n t r a t i o s o f p r o d u c t s (21,24). S i m i l a r r e s u l t s are o b t a i n e d w i t h o t h e r b u f f e r s ( 2 3 ) . The f o r m a t i o n o f carbonium i o n s by s p e c i f i c and g e n e r a l a c i d c a t a l y s i s has been assumed t o be the r a t e - d e t e r m i n i n g s t e p f o r b o t h t e t r a o l and c o v a l e n t adduct f o r m a t i o n (21,24). The e x p e r i m e n t a l o b s e r v a t i o n s i n c a c o d y l a t e b u f f e r s o l u t i o n s a r e c o n s i s t e n t w i t h a mechanism i n v o l v i n g a k i n e t i c a l l y common i n t e r m e d i a t e a c c o r d i n g t o the f o l l o w i n g r e a c t i o n scheme:

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

GEACINTOV

Interaction of Polycyclic Aromatic Diol Epoxides with DNA

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch006

6.

F i g u r e 2. T y p i c a l l i n e a r d i c h r o i s m s p e c t r a o f n o n - c o v a l e n t ( s o l i d l i n e s ) and c o v a l e n t (dashed l i n e s ) DNA complexes ( d a t a o f M. Shahbaz).

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

111

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

BaPDE + DNA ^

>

+

[BaPDE.. .DNA]

>• [BaPDE .. .DNA]

i.

(2)

i k

Tetraols

Tetraols

Covalent

k

t

n

e

adducts

As l o n g as t h e r a t e c o n s t a n t s k ^ j l ^ ^ 3> pseudo-first r a t e c o n s t a n t k f o r t h e r e a c t i o n o f BaPDE i s (20,21); k = (1-X )k Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch006

b

h

+ k X , 3

X

b

b

=

C

order

K[DNA]/( 1 + K[DNA] ) ( 3 )

where X i s t h e f r a c t i o n o f d i o l epoxide m o l e c u l e s bound t o DNA, and K = k^/k^ i s t h e e q u i l i b r i u m b i n d i n g c o n s t a n t . The two terms i n equation (3) represent weighted c o n t r i b u t i o n s o f the r e a c t i o n s o f f r e e BaPDE m o l e c u l e s , and o f m o l e c u l e s complexed w i t h DNA, and t h i s e q u a t i o n p r o v i d e s an adequate f i t t o t h e e x p e r i m e n t a l d a t a w i t h K = 12,000 M i n 5 mM sodium c a c o d y l a t e s o l u t i o n a t pH 7, 25°C(20). The f r a c t i o n o f d i o l e p o x i d e m o l e c u l e s w h i c h b i n d c o v a l e n t l y t o DNA ( f ) i s (21): b

C Q V

f

c

o

v

- [k /(k +k )][k X /k] c

c

T

3

(4)

b

The f i r s t term on t h e r i g h t - h a n d s i d e i s t h e f r a c t i o n o f c a r b o nium i o n s w h i c h decay by f o r m i n g c o v a l e n t bonds, w h i l e t h e second term denotes t h e f r a c t i o n o f a l l d i o l e p o x i d e m o l e c u l e s w h i c h r e a c t w h i l e complexed p h y s i c a l l y t o DNA, r a t h e r than as f r e e m o l e c u l e s i n solution. E q u a t i o n ( 4 ) demonstrates t h a t t h e r e l a t i o n s h i p between t h e a s s o c i a t i o n c o n s t a n t K, w h i c h i s s e n s i t i v e t o t h e i o n i c s t r e n g t h (16,17,21,25), and t h e l e v e l o f c o v a l e n t b i n d i n g , f v ' * P^ one. I t i s known t h a t f d e c r e a s e s upon t h e a d d i t i o n o f N a C l o r M g C ^ f and t h i s e f f e c t has been t a k e n as e v i d e n c e t h a t p h y s i c a l i n t e r c a l a t i o n complexes p l a y a r o l e i n t h e c o v a l e n t b i n d i n g r e a c t i o n (17,22,26). W h i l e t h i s c o n c l u s i o n may s t i l l be c o r r e c t , such e v i d e ­ nce i s i n s u f f i c i e n t s i n c e i t has been shown t h a t n o t o n l y K, b u t a l s o k ( 2 1 , 2 5 ) , and t h e b r a n c h i n g r a t i o k / k (21) i n E q u a t i o n ( 4 ) depend on t h e s a l t c o n c e n t r a t i o n . 8 a

c o m

e x

C O

c

o

v

3

c

P h y s i c a l I n t e r c a l a t i o n Complexes, C o v a l e n t

T

B i n d i n g and H y d r o l y s i s

The p o s s i b l e e x i s t e n c e o f two types o f b i n d i n g s i t e s f o r p h y s i c a l BaPDE-DNA complexes has s t i m u l a t e d v a r i o u s p r o p o s a l s r e g a r d i n g t h e r e l a t i v e importance o f each i n t h e two r e a c t i o n pathways o f t e t r a o l f o r m a t i o n and c o v a l e n t b i n d i n g . I n order t o f a c i l i t a t e the f o l ­ l o w i n g d i s c u s s i o n , t h e v a r i o u s p o s s i b l e r e a c t i o n pathways a r e summa­ r i z e d i n F i g u r e 3. I n a d d i t i o n , we have added t h e p o s s i b i l i t y t h a t t h e r e may be a r a p i d exchange between p h y s i c a l b i n d i n g s i t e s ( k g , k„£)» a f a c t o r w h i c h seems t o have been n e g l e c t e d up t i l l now. P h y s i c a l b i n d i n g s t u d i e s (&*9.*2T) suggest t h a t p h y s i c a l complex f o r m a t i o n w i t h DNA by i n t e r c a l a t i o n appears t o be s e q u e n c e - s p e c i f i c . Thus, BaPT and pyrene i n t e r c a l a t e much more s t r o n g l y i n p o l y ( d A -

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

6.

Interaction of Polycyclic Aromatic Diol Epoxides with DNA

GEACINTOV

dT):poly(dA-dT) than i n poly(dG-dC):poly(dG-dC) and i n t h e homopolymers p o l y ( d G ) : p o l y ( d C ) and p o l y ( d A ) : p o l y ( d T ) . This preference f o r dA-dT sequences has prompted Chen t o suggest t h a t t h e c o v a l e n t b i n d i n g o f BaPDE t o guanine proceeds a t e x t e r n a l s i t e I I p h y s i c a l complexes, r a t h e r than a t i n t e r c a l a t i v e s i t e I complexes, w h i l e BaPDE m o l e c u l e s i n t e r c a l a t e d a t dA:dT r i c h sequences p r e f e r e n t i a l l y undergo h y d r o l y s i s ( 8 ) . I n f i g u r e 3, Chen's h y p o t h e s i s c o r r e s p o n d s t o k ( T ) » k ( T ) , and k ( C ) » k ( C ) . Meehan and Bond (23) on t h e o t h e r hand, have t a k e n an o p p o s i t e v i e w , namely t h a t k ( C ) « k ( C ) , w h i l e k ( T ) » k ( T ) . Thus, i n t h i s view, the h y d r o l y s i s occurs a t e x t e r n a l b i n d i n g s i t e s , w h i l e covalent b i n d i n g occurs a t i n t e r c a l a t i o n s i t e s . F u r t h e r m o r e , they r e j e c t t h e common i n t e r m e d i a t e model ( E q u a t i o n 2) on t h e b a s i s o f t h e i r b e l i e f t h a t t h e r a t e s o f r e a c t i o n f o r t e t r a o l f o r m a t i o n and adduct f o r m a t i o n and t h e r a t i o o f t h e p r o d u c t s s h o u l d be t h e same i n such a model. W h i l e these r a t e s o f r e a c t i o n a r e t h e same and t h e p r o d u c t r a t i o s a r e observed t o be d i f f e r e n t , t h i s i s f u l l y c o n s i s ­ tent f o r a s e t o f p a r a l l e l p s e u d o - f i r s t order r e a c t i o n s i n v o l v i n g a common i n t e r m e d i a t e (29) as p o i n t e d o u t above. Thus, t h e d a t a o f Meehan and Bond does n o t demonstrate t h e v a l i d i t y o f t h e two-domain model ( 2 3 ) . The r e a c t i o n schemes o f Chen ( 8 ) and o f Meehan and Bond ( 2 3 ) , n e g l e c t t h e p o s s i b i l i t y t h a t exchange between t h e two p h y s i c a l b i n d i n g s i t e s ( F i g u r e 3) may be o c c u r r i n g on time s c a l e s w h i c h a r e much f a s t e r than those c h a r a c t e r i z i n g t h e c h e m i c a l r e a c t i o n pathways o f BaPDE. Thus, w h i l e i t i s s t i l l p o s s i b l e t h a t t h e r e a c t i o n s may be o c c u r r i n g a t p h y s i c a l l y d i f f e r e n t b i n d i n g s i t e s , k i n e t i c a l l y o n l y one common p r e c u r s o r f o r these r e a c t i o n s may be d i s t i n g u i s h a b l e (k ,k ,k ,k. » k i n Figure 3 ) . U t i l i z i n g a k i n e t i c flow d i ­ c h r o i s m method, we have e s t a b l i s h e d t h a t t h e r e i s a d i s t i n c t kine­ t i c r e l a t i o n s h i p between t h e d i s a p p e a r a n c e o f p h y s i c a l l y bound ( + ) anti-BaPDE m o l e l c u l e s a t type I i n t e r c a l a t i v e b i n d i n g s i t e s , and t h e appearance o f c o v a l e n t adducts a t s i t e s I I . However, because o f t h e f o r e g o i n g arguments i n v o l v i n g t h e r a p i d exchange o f p h y s i c a l l y bound m o l e c u l e s between s i t e s I and s i t e s I I , t h e e x a c t n a t u r e o f t h e microcomplexes w h i c h a r e i n v o l v e d i n t h e c o v a l e n t and h y d r o l y s i s r e a c t i o n s remains t o be e l u c i d a t e d . I

I I

3

I I

3

I

3

3

I 1

3

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1

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E

E

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I I

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I

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3

S t r u c t u r e o f the Covalent

Adducts

There i s c o n s i d e r a b l e disagreement between d i f f e r e n t r e s e a r c h e r s on the c o n f o r m a t i o n s o f c o v a l e n t adducts d e r i v e d from t h e b i n d i n g o f BaPDE t o DNA. I t has been r e p o r t e d t h a t t h e c o v a l e n t b i n d i n g o f BaPDE t o c l o s e d c i r c u l a r DNA r e s u l t s i n t h e u n w i n d i n g o f t h e DNA h e l i x (26,29). S i n c e analogous unwinding e f f e c t s a r e produced by n o n - c o v a l e n t l y i n t e r c a l a t e d a c r i d i n e d y e s , these e f f e c t s have been a t t r i b u t e d t o t h e f o r m a t i o n o f c o v a l e n t i n t e r c a l a t i v e BaPDE com­ plexes (29). However, these c o n c l u s i o n s c a n be c r i t i c i z e d s i n c e o t h e r types o f c o n f o r m a t i o n s , o r e f f e c t s o t h e r than c o v a l e n t - i n t e r c a l a t i v e b i n d i n g o f BaPDE may g i v e r i s e t o t h e unwinding o f t h e d o u b l e h e l i x (26,30). Furthermore, the a b s o r p t i o n , l i n e a r d i ­ c h r o i s m , and f l u o r e s c e n c e p r o p e r t i e s o f t h e c o v a l e n t (+)-anti-BaPDEDNA complexes a r e n o t c o n s i s t e n t w i t h those o f c l a s s i c a l i n t e r c a l a ­ t i o n complexes, as i s shown below.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch006

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Linear Dichroism. The A A s p e c t r a o f c o v a l e n t a d d u c t s d e r i v e d from the b i n d i n g o f r a c e m i c anti-BaPDE and o f t h e enantiomer ( + ) - a n t i BaPDE t o DNA a r e p o s i t i v e i n s i g n and s i m i l a r i n shape ( 5 , 3 1 ) ; t h i s i s e x p e c t e d s i n c e t h e (+) enantiomer b i n d s more e x t e n s i v e l y t o DNA t h a n t h e (-) enantiomer ( 1 5 ) . These c o v a l e n t a d d u c t s a r e t h e r e f o r e of the s i t e I I type. Subsequent s t u d i e s o f a d d u c t s d e r i v e d from t h e c o v a l e n t b i n d i n g o f o t h e r e p o x i d e model compounds w i t h p y r e n y l chromophores ( F i g u r e 4) t o DNA (7,14,32-33) show t h a t t h e o c c u r r e n c e o f p o s i t i v e AA spectra i s t h e e x c e p t i o n r a t h e r than t h e r u l e . A d e t a i l e d a n a l y s i s of the l i n e a r d i c h r o i s m s p e c t r a o f covalent adducts obtained w i t h the compounds shown i n F i g u r e 4, demonstrates t h a t t h e r e i s a marked h e t e r o g e n e i t y o f a d d u c t s . Q u a l i t a t i v e l y t h e l i n e a r d i c h r o i s m spec­ t r a c a n be accounted f o r i n terms o f s u p e r p o s i t i o n s o f p o s i t i v e A A s p e c t r a due t o s i t e I I b i n d i n g , and n e g a t i v e A A s p e c t r a due t o s i t e I b i n d i n g , w i t h t h e l a t t e r d o m i n a t i n g i n 9,10-BaPE, 7,8-BaPE (unpub­ l i s h e d ) , BePDE, BePE and 1-OP c o v a l e n t DNA a d d u c t s . Other s t u d i e s on a d d u c t s d e r i v e d from t h e b i n d i n g o f b e n z o ( a ) p y r e n e - 9 , 1 0 - d i o l - 7 , 8 o x i d e t o DNA (22) suggest t h a t s i t e I complexes a l s o dominate i n t h i s case. The s i t e I adducts a r e c h a r a c t e r i z e d by a n e a r - p a r a l l e l ( w i t h i n 25°) average o r i e n t a t i o n o f t h e p l a n a r pyrene r e s i d u e w i t h t h e p l a n e s o f t h e DNA b a s e s , and a r e l a t i v e l y s t r o n g i n t e r a c t i o n between the T T - e l e c t r o n s o f t h e pyrene r e s i d u e s and t h e DNA b a s e s . Hogan e t a l ( 3 4 ) , who s t u d i e d t h e e l e c t r i c l i n e a r d i c h r o i s m o f (+) anti-BaPDE bound c o v a l e n t l y t o s m a l l DNA fragments (~145 base p a i r s ) , showed t h a t t h e l i n e a r d i c h r o i s m w i t h i n t h e DNA a b s o r p t i o n band d e c r e a s e d w i t h an i n c r e a s i n g l e v e l o f b i n d i n g . These r e s u l t s i n d i c a t e t h a t k i n k s a r e produced i n t h e DNA m o l e c u l e upon i n t e r a c ­ t i o n w i t h the d i o l epoxide molecules. They suggested t h a t t h e c o v a l e n t l y bound BaPDE m o e i t i e s r e s i d e a t t h e s e k i n k s i n wedgeshaped i n t e r c a l a t i o n complexes. T h i s model i s r e a s o n a b l e ; however, such a s t r u c t u r e i s more c o n s i s t e n t w i t h t h e n e g a t i v e , r e d - s h i f t e d l i n e a r d i c h r o i s m s p e c t r a o f s i t e I b i n d i n g s i t e s ( 3 1 ) than w i t h t h e major s i t e I I type o f b i n d i n g s i t e o b s e r v e d w i t h t h e c o v a l e n t ( + ) anti-BaPDE-DNA adducts s t u d i e d by Hogan e t a l . The r e d - s h i f t o f o n l y 2-3 nm d i s p l a y e d by t h e c o v a l e n t l y bound r e s i d u e s appears t o be too s m a l l f o r an i n t e r c a l a t i v e geometry; t h e l a r g e r r e d s h i f t , and the n e g a t i v e l i n e a r d i c h r o i s m d i s p l a y e d by a d d u c t s bound a t s i t e s I , appear more c o n s i s t e n t w i t h t h i s model. W h i l e i t i s r e a s o n a b l e t o assume t h a t k i n k s a r e formed a t t h e s i t e o f t h e c o v a l e n t b i n d i n g o f BaPDE ( 3 4 ) , i t i s a l s o p o s s i b l e t h a t such bends a r i s e e l s e w h e r e on t h e double h e l i x , due t o t h e known f o r m a t i o n o f n i c k s and s i n g l e - s t r a n d b r e a k s ( 3 5 , 3 6 ) . T h e r e f o r e , t h e e x i s t e n c e o f k i n k s i n t h e DNA h e l i x a t s i t e I o r s i t e I I b i n d i n g s i t e s s h o u l d be f u r t h e r i n v e s t i g a t e d , b e f o r e such a model c a n be adopted d e f i n i t i v e l y . I t i s i n t e r e s t i n g t o n o t e t h a t t h e absence o f t h e two OH groups a t t h e 7 and 8 p o s i t i o n s o f 9,10-BaPE (7), and t h e 7,8-carbon atoms i n 1-OP ( 1 4 ) , l e a d t o a p a r t i a l l o s s o f s t e r e o s e l e c t i v e e f f e c t s i n the c o v a l e n t b i n d i n g o f t h e s e m o l e c u l e s t o DNA; i n t h e adducts de­ r i v e d from t h e s e two m o l e c u l e s , s i t e I adducts dominate. I n con­ t r a s t , i n t h e case o f t h e d i o l e p o x i d e (+)anti-BaPDE, s i t e I I com­ p l e x e s account f o r o v e r 9 0 % o f t h e b i n d i n g ( 3 1 ) .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

6.

GEACINTOV

Interaction of Polycyclic Aromatic Diol Epoxides with DNA TIME SCALES ™*0) p o s t u l a t e d a k i n k e d r e c e p t o r s i t e w i t h an i n t e r c a l a t i v e c o v a l e n t l y bound BPDE l ( + ) i n c o n t r a s t t o G e a c i n t o v and coworkers (kj) who p o s t u l a t e e x t e r n a l binding. A l t e r n a t e i n t e r p r e t a t i o n s o f t h i s d a t a may i n c l u d e l o c a l d e n a t u r a t i o n o f t h e duplex o r some form o f DNA damage. However, i f t h e DNA remains double s t r a n d e d a t t h e s i t e o f c o v a l e n t adduct f o r ­ m a t i o n , then a model f o r t h e s t e r e o s e l e c t i v i t y o f t h e BPDE can be presented. T h e o r e t i c a l l y determined r e c e p t o r s i t e s f o r k i n k e d DNA were generated by T a y l o r e t a l . (kk) t o model t h i s t y p e o f b i n d i n g . The k i n k e d DNA accommodates t h e c o v a l e n t b o n d i n g r e g i o n w i t h t e t r a h e d r a l and t r i g o n a l h y b r i d c o n f i g u r a t i o n s about t h e CIO o f BPDE l ( + ) and N2(G), r e s p e c t i v e l y . However, t h e l o n g a x i s o f t h e pyrene i s n e a r l y p e r p e n d i c u l a r t o t h e h e l i c a l a x i s , i . e . , ot(BPDE) > 80° i n a l l s t r u c t u r e s examined. An a l t e r n a t i v e model p r e s e n t e d i n t h i s paper shows t h a t t h e d a t a o f Hogan e t a l . (50) and G e a c i n t o v e t a l . (kj) a r e c o n s i s t e n t w i t h an e x t e r n a l l y bound adduct t o a deformed DNA k i n k e d t o accommodate t h e pyrene i n t h e minor groove. None t h e l e s s t h e r e c e p t o r s i t e k i n k e d t o accommodate an i n t e r c a l a t i v e c o v a l e n t bound adduct y i e l d s s t e r e o s e l e c t i v i t y f o r t h e l ( + ) d i a s t e r e o i s o m e r ( o f a l l f o u r s t u d i e d ) (37) i n an i n t e r m e d i a t e s t e p which has n o t been i d e n t i f i e d e x p e r i m e n t a l l y . Because t h e l i n e a r dichroism r e s u l t s f o r t h e o r i e n t a t i o n o f t h e pyrene m o i e t y i n t h e BPDE l ( + ) DNA adduct a r e v e r y s i m i l a r i n e x p e r i m e n t s performed by t h e e l e c t r i c f i e l d and f l o w t e c h n i q u e s (51) i t i s t e m p t i n g t o assume t h a t t h e angles r e f l e c t measurements r e l a t i v e t o t h e "average" DNA a x i s . Thus, t h e r e a r e two i n t e r p r e t a t i o n s t o t h e o r i e n t a t i o n o f 35°-^3° for t h e pyrene moiety: a p u r e l y e x t e r n a l l y bound adduct and an i n t e r c a l a t i v e c o v a l e n t l y bound a d d u c t . I n o u r proposed mechanism f o r l(+)-N2(G) a d d u c t s , t h e i n t e r c a l a t i v e c o v a l e n t l y bound form i n a k i n k e d r e c e p t o r s i t e has an o r i e n t a t i o n a(BPDE) > 80°, whereas t h e e x t e r n a l l y bound forms i n a r e l a x e d B-DNA t y p e s t r u c t u r e have v a l u e s o f ot(BPDE) « 15° f o r t h e G base i n t h e a n t i c o n f o r m a t i o n and a(BPDE) « 50° f o r G i n t h e s y n c o n f o r m a t i o n . The n o n - c o v a l e n t l y bound BPDEs t o DNA formed i n i t i a l l y appear t o be i n t e r c a l a t i o n complexes (U6,52-55). Meehan e t a l . (k§) r e p o r t t h a t t h e BPDE i n t e r c a l a t e s i n t o DNA on a m i l l i s e c o n d time s c a l e w h i l e t h e BPDE a l k y l a t e s DNA on a time, s c a l e o f m i n u t e s . Most o f t h e BPDE i s h y d r o l y z e d t o t e t r o l s (53-56). G e a c i n t o v e t a l . (5k) have shown w i t h l i n e a r d i c h r o i s m s p e c t r a l measurements t h a t t h e d i s ­ appearance o f i n t e r c a l a t e d BPDE l ( + ) i s d i r e c t l y p r o p o r t i o n a l t o t h e r a t e o f appearance o f c o v a l e n t a d d u c t s . These r e s u l t s suggest t h a t e i t h e r t h e r e may be a c o m p e t i t i o n between t h e p h y s i c a l l y nonc o v a l e n t l y bound BPDE l ( + ) and an e x t e r n a l l y bound adduct o r as sug­ g e s t e d by t h e mechanism i n t h e p r e s e n t p a p e r , an i n t e r c a l a t i v e c o ­ v a l e n t s t e p f o l l o w e d by a r e l a x a t i o n o f t h e DNA t o y i e l d an e x ­ t e r n a l l y bound a d d u c t . T h e i r r e s u l t s f o r t h e BPDE i ( - ) e x h i b i t b o t h i n t e r c a l a t i v e and e x t e r n a l l y bound a d d u c t s . The l i n e a r dichroism measurements do n o t d i s t i n g u i s h between p h y s i c a l l y bound and c o ­ v a l e n t bound forms w h i c h a r e i n t e r c a l a t i v e i n n a t u r e . Hence t h e a s ­ sumption t h a t a s u p e r p o s i t i o n o f i n t e r n a l and e x t e r n a l s i t e s o c c u r s f o r t h i s isomer.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

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The b i n d i n g o f i ( - ) t o base atoms a s s o c i a t e d w i t h t h e minor g r o o v e , s p e c i f i c a l l y N6(A) (38), 06(G) and N*+(C), have been observed (13-22,3*4,38). We propose t h a t t h e DNA can be k i n k e d t o a l l o w i n ­ t e r c a l a t i v e c o v a l e n t b i n d i n g t o t h e s e atoms, b u t t h a t t h e f i n a l o r i e n t a t i o n may be i n such a s i t e o r e x t e r n a l l y bound t o t h e DNA and t h a t t h i s f i n a l s i t e may a l s o be k i n k e d . G e a c i n t o v and coworkers (51,57) have s t u d i e d t h e o r i e n t a t i o n o f t h e pyrene moiety i n BPDE l ( + ) and i ( - ) adducts w i t h DNA and Undeman e t a l . (58) have s t u d i e d t h e o r i e n t a t i o n o f BPDE l(±) and Il(±) t o DNA. They i n t e r p r e t t h e i r e l e c t r i c l i n e a r d i c h o r i s m measurements i n terms o f a s u p e r p o s i t i o n o f two t y p e s o f b i n d i n g s i t e s which t h e y l a b e l s i t e I and s i t e I I . To a v o i d c o n f u s i o n w i t h t h e n o t a t i o n f o r our i n t e r c a l a t i o n s i t e s , t h e i r s i t e s w i l l be r e f e r r e d t o as IQ and IIX. I n s i t e IQ t h e complex has t h e pyrene m o i e t y o r i e n t e d w i t h i n 30° o f t h e DNA b a s e s , i . e . , 60°-90° r e l a t i v e t o t h e h e l i x a x i s . They c a l l t h i s q u a s i i n t e r c a l a t i o n . We r e f e r t o i t as i n t e r c a l a t i v e c o v a l e n t b i n d i n g . I t r e s u l t s a f t e r i n t e r c a l a t i o n and k i n k i n g o f t h e DNA and c o v a l e n t bond f o r m a t i o n . I n s i t e I I X t h e complex has t h e l o n g a x i s o f t h e t h e pyrene moiety o r i e n t e d 15°-27° f o r BPDE l ( + ) and 37°-*45° f o r BPDE i ( - ) r e l a t i v e t o t h e h e l i x a x i s . They i n t e r ­ p r e t t h i s as e x t e r n a l b i n d i n g . They a t t r i b u t e s i t e I I X b i n d i n g t o N2(G) and S i t e IQ b i n d i n g t o 06(G) and N6(A) i n agreement w i t h t h e i n t e r p r e t a t i o n o f Brookes e t a l . (38), t h a t t h e d i f f e r e n c e i n mu­ t a g e n i c i t y between t h e l ( + ) and i ( - ) isomers r e s u l t s from d i f f e r ­ ences i n s p a t i a l o r i e n t a t i o n . S p e c i f i c a l l y , t h e l ( + ) isomer ex­ h i b i t s s i t e I I X b i n d i n g w i t h 88-9*+% bound. The i ( - ) isomer e x h i b i t s s i t e I I X b i n d i n g w i t h 50-60% bound, t h e r e m a i n i n g being a t t r i b u t e d t o s i t e IQ. The i n t e r p r e t a t i o n o f e x p e r i m e n t a l d a t a and p r e s e n t a t i o n o f a d e t a i l e d m o l e c u l a r model can be a c c o m p l i s h e d o n l y a f t e r c e r t a i n a s ­ sumptions have been made about t h e a l i g n m e n t o f t h e DNA by an e l e c ­ t r i c f i e l d and i n f l o w t e c h n i q u e s , ( l ) The h e l i x a x i s o f t h e DNA i s o r i e n t e d a l o n g t h e e l e c t r i c f i e l d and f l o w a x i s i n e x a c t l y t h e same manner. F o r DNA w i t h s u p e r t u r n s t h i s assumption i m p l i e s o n l y t h e "average" h e l i x a x i s . (2) The c o n f o r m a t i o n a l change t h r o u g h t h e r e ­ c e p t o r s i t e i s smooth. That i s , t h e r e a r e no sharp bends which may o r i e n t t h e pyrene m o i e t y a l o n g t h e e l e c t r i c f i e l d o r f l o w a x i s w h i l e i t i s q u a s i i n t e r c a l a t e d , i m p l y i n g t h a t i t i s e x t e r n a l l y bound. (3) The average h e l i c a l a x i s t h r o u g h t h e r e c e p t o r s i t e l i e s a l o n g t h e e l e c t r i c f i e l d o r f l o w a x i s so t h a t t h e b e n d i n g i s s y m m e t r i c a l about the kink. I n F i g u r e 2, an i d e a l i z e d o r i e n t a t i o n o f t h e BPDEs i n b o t h bound forms i s i l l u s t r a t e d w i t h one s p e c i f i c example. F o r t r i g o n a l h y b r i d i z a t i o n about N2(G) and t e t r a h e d r a l h y b r i d i z a t i o n about CIO(BPDE), v a l u e s o f t h e k i n k , a , y(DNA) and a(BPDE) a r e i n ­ dicated. However, i n p r a c t i c e t h e pyrene m o i e t y i s n o t p a r a l l e l t o t h e a d j a c e n t base p a i r , and i t s l o n g a x i s does n o t l i e i n t h e plane o f t h e l o c a l h e l i c a l a x e s , t and X', and t h e average h e l i c a l axis, Thus, a(BPDE) > 6*5°, t h e i d e a l i z e d v a l u e . Similarly for t h e o u t s i d e form, a(BPDE) >20°. I n t h i s p a p e r , a m o l e c u l a r model i s p r e s e n t e d i n which a(BPDE) « 80° f o r t h e i n t e r c a l a t i v e c o v a l e n t l y bound form, and a(BPDE) « 15° and y(DNA) * 30° f o r t h e e x t e r n a l l y bound form. Both forms a r e bound t o DNA w i t h d i f f e r e n t k i n k c o n f o r ­ m a t i o n s . I n summary, a k i n k i n t h e DNA need not i m p l y i n t e r c a l a t i v e x

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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F i g u r e 2. Geometry f o r i d e a l i z e d o r i e n t a t i o n s o f t h e pyrene m o i e t y ( ) i n (a) an i n t e r c a l a t i v e c o v a l e n t l y bound form and i n (b) an e x t e r n a l l y bound form. The average h e l i x a x i s i s assumed t o l i e along The l o c a l segments o f DNA l i e a l o n g t and X'. They a r e o r i e n t e d by y(DNA) r e l a t i v e t o £ and t h e l o n g a x i s o f t h e pyrene m o i e t y i s o r i e n t e d by 6'0). (b) L e a s t squares t e c h n i q u e s a r e used t o r e ­ l a x a DNA fragment u n t i l c o n s t r a i n t s f o r t h e g e o m e t r i c a l c o n d i t i o n s o f p r o p e r bond l e n g t h s , bond a n g l e s w i t h a s p e c i f i c placement o f bases a r e s a t i s f i e d (6l,62). (c) S y s t e m a t i c adjustment o f bases

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249

t h r o u g h p a r a l l e l , k i n k e d o r any o t h e r o r i e n t a t i o n o f i n t e r e s t t o s e a r c h f o r backbone c o m p l e t i o n (63-66). A l a r g e number o f duplex DNA r e c e p t o r s i t e s have been o b t a i n e d f o r t h e i n t e r c a l a t i o n p r o c e s s , s e v e r a l d i n u c l e o s i d e monophosphate r e c e p t o r s i t e s have been used f o r modeling BPDE-DNA i n t e r a c t i o n s , and o n l y one s e t o f k i n k e d DNA s i t e s a r e a v a i l a b l e (36,66). The q u e s t i o n a r i s e s : which receptor s i t e s s h o u l d be used i n the modeling o f t h e b i n d i n g o f molecules w i t h DNA? In p r i n c i p a l , i t cannot be answered w i t h o u t t r y i n g a l l o f t h e them. In p r a c t i c e , c a l c u l a t i o n s w i t h t h e o r e t i c a l l y determined i n t e r c a l a ­ t i o n s i t e s (67-71), c a l c u l a t i o n s u s i n g e x p e r i m e n t a l d a t a (72,73), d i n u c l e o s i d e t r i p h o s p h a t e u n i t s (7**, 75) t e t r a m e r duplexes w h i c h f i t i n t o B-DNA (61,62,66-68), and t e t r a m e r - d u p l e x e s (76) w i t h the same and w i t h mixed sugar puckers y i e l d r e s u l t s w h i c h demonstrate f o r s i m p l e systems t h a t the optimum b i n d i n g o r i e n t a t i o n s a r e i n agree­ ment w i t h e x p e r i m e n t a l d a t a and t h a t the t r e n d s i n b i n d i n g e n e r g i e s f o r a l l p o s s i b l e sequences remain a p p r o x i m a t e l y the same. The t e t r a m e r duplex r e c e p t o r s i t e s used i n t h i s study have t h e f o l l o w i n g characteristics: ( l ) t h e y f i t i n t o B-DNA, (2) t h e y r e p r e s e n t con­ f o r m a t i o n s which possess s p e c i f i c f e a t u r e s , i . e . , i n t e r c a l a t i o n , k i n k s or DNA d i s t o r t e d from the B-DNA form. T h e r e f o r e , we proceed w i t h the assumption t h a t the i n t e r c a l a t i o n p r o c e s s w i l l r e v e a l the manner i n which the r e a c t i v e atoms approach each o t h e r t o p e r m i t i n ­ t e r c a l a t i v e c o v a l e n t b i n d i n g as a f i r s t s t e p f o l l o w e d by an ad­ j u s t m e n t o f the base p a i r s t o non-planar o r i e n t a t i o n s ( k i n k ) t o a c ­ commodate the p r o p e r h y b r i d c o n f i g u r a t i o n s about CIO on BPDE and N2(G), N6(A), 06(G) and NU(C) f o r c o v a l e n t bond f o r m a t i o n . For b i n d i n g , two o r i e n t a t i o n s a l o n g the DNA must be c o n s i d e r e d : 3' and 5" b i n d i n g . They a r e i l l u s t r a t e d w i t h the BPDEs bound t o a p u r i n e (pu) and t o a p y r i m i d i n e ( p y ) .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

5

The arrow denotes the 5" 3' d i r e c t i o n a l o n g t h e backbone, i . e . , 5'(sugar)3' o r e q u i v a l e n t l y 3'(p)5'. The diagram corresponds t o v i e w i n g the DNA i n t o the minor groove as seen i n most f i g u r e s i n t h i s paper. T h e r e f o r e , t h e energy o f opening t h e DNA to sites f a v o r i n g 3' o r 5" b i n d i n g p r o v i d e s t h e c o n t r i b u t i o n o f t h e DNA t o the process. Once the s i t e i s c r e a t e d , the a b i l i t y o f each BPDE t o f i t i n t o t h e r e c e p t o r i s examined. Two p r o c e s s e s a r e c o n s i d e r e d : intercala­ t i o n and c o v a l e n t i n t e r c a l a t i v e b i n d i n g r e p r e s e n t e d by

A BP? BPl

_R

+ BPDE

AE

B P D E

^

A B PBPo I A 1

I

2

BPDE BP!

I

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

250

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

The energy

AE

J

B P D E - BPDE

+

AC

BPDE

r e p r e s e n t s t h e energy f o r i n s e r t i o n o f BPDE i n t o s i t e R. c a l c u l a t e d w i t h a Coulomb p o t e n t i a l

( 2 )

It is

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

(3)

a 6-lU p o t e n t i a l

and a t o r s i o n a l p o t e n t i a l

T = J ( V / 2 ) [ 1 + c o s i^] b b

(5)

f o r i n t e r a c t i n g atoms i and j and f o r r o t a t i o n s about each bond b . A s s o c i a t e d w i t h t h e atoms a r e t h e n e t a t o m i c charges q^ and q j , and t h e van d e r Waals r a d i i and p j . The d i s t a n c e between atoms i and j i s r-j^ , t h e sum o f van d e r Waals r a d i i i s p i j = P i + P i > t h e r e ­ duced d i s t a n c e i s s-y = r i j / p i j , A±j = 1 . 5 a i c y I j l j / C l i + I j ; where a i , a j and I i , I j a r e t h e a t o m i c p o l a r i z a b i l i t i e s and i o n i z a t i o n p o t e n ­ t i a l s respectively, i s t h e b a r r i e r t o i n t e r n a l r o t a t i o n and l\> i s t h e p e r i o d i c i t y f o r a complete r o t a t i o n about bond b . The i n t e r m o l e c u l a r energy Igp^g c o n s i s t s o f a sum Q+U. The summation i n t h e two-body i n t e r a c t i o n s proceeds o v e r a l l atoms i i n t h e BPDE and j i n t h e DNA. The i n t r a m o l e c u l a r i n t e r a c t i o n s between non-bonded atoms i n BPDE as w e l l as t h e t o r s i o n a l energy a r e g i v e n by t h e change i n c o n f o r m a t i o n a l energy, A C ^ ^ g , measured relative t o t h e g l o b a l minimum f o r t h e f r e e m o l e c u l e . I t c o n s i s t s o f a sum o f terms Q + U + T. I n t h i s case t h e summation proceeds o v e r nonbonded atoms i and J i n d i f f e r e n t fragments o f BPDE, i . e . , fragments s e p a r a t e d by r o t a t a b l e bonds. The c o n f o r m a t i o n a l energy o f t h e benzo r i n g i s a l s o i n c l u d e d . For t h i s contribution the r e s t r i c t i o n s a r e made t h a t i > j and t h a t atoms i and j a r e non-bonded. The d e f i ­ n i t i o n s and t h e d e t a i l s o f t h e energy terms and t h e parameters a r e p r e s e n t e d elsewhere (69)• The t o t a l energy change o f t h e complex i s g i v e n by

A

W

AE

- DNA

+

A

4>DE

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

( 6 )

10.

MILLER ET AL.

For c o n v e n i e n c e , minimum as

t h e energy w i l l be r e p o r t e d r e l a t i v e t o

0G

BPDE

=

e

BPDE "

U e

the g l o b a l

;

BPDE min

F o r t h e c o v a l e n t l y bound adduct t h e b i n d i n g energy must be added t o t o AEgp^g. ^ For tj^e DNA, o n l y a c o n f o r m a t i o n a l change occurs and ^t)NA ^DNA ^ g l ° l minimum i s assumed t o be f o r B-DNA. By c a l c u l a t i n g t h e energy changes f o r t h e i n t e r c a l a t i o n p r o ­ c e s s , t h e p r e f e r e n c e f o r o r i e n t a t i o n o f t h e epoxide i n t h e major o r minor groove can be determined. This step provides a r a t i o n a l e f o r t h e f i r s t s t e p i n c o v a l e n t b i n d i n g by examining whether t h e r e a c t i n g atoms a r e i n p r o x i m i t y t o each o t h e r . The i m p o r t a n t s t e p i n s t e r e o ­ s e l e c t i v i t y i s t h e a b i l i t y o f t h e BPDEs t o f i t i n t o t h e s i t e once a c o v a l e n t bond has formed. The c o n f o r m a t i o n s o f t h e benzo r i n g a r e i m p o r t a n t i n t h e f i t o f each o f t h e d i a s t e r e o i s o m e r s t o t h e k i n k s i t e d u r i n g adduct f o r m a t i o n . They m o d i f y t h e o r i e n t a t i o n o f t h e d o u b l e bond o f t h e benzo r i n g and hence t h e e n t i r e pyrene m o i e t y . A

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

251

Binding of Benzo[a]pyrene Diol Epoxides to DNA

=

A(

w t i e r e

Mechanism f o r BPDE-DNA Adduct

e

Da

Formation

F o r m a t i o n o f t h e BPDE-DNA adduct r e q u i r e s a study o f ( l ) Benzo r i n g c o n f o r m a t i o n s o f BPDEs and a d d u c t s ; (2) The r e h y b r i d i z a t i o n o f amino groups on the benzo f o r t h e C10-N bond f o r m a t i o n ; (3) The r e ­ c e p t o r s i t e s r e s u l t i n g from a c o n f o r m a t i o n a l adjustment o f DNA t o accommodate an i n t e r c a l a t e d and f i n a l l y an i n t e r c a l a t i v e c o v a l e n t l y bound BPDE, and t h e base sequence s p e c i f i c i t y i n t h e f o r m a t i o n o f t h e r e c e p t o r s i t e ; (k) C l a s s i c a l i n t e r c a l a t i o n and the o r i e n t a t i o n o f CIO o f the BPDEs toward t h e r e a c t i v e N2(G), N6(A), 06(G) and Nl+(C) base atoms; (5) The s t e r e o s e l e c t i v i t y o f t h e BPDEs d u r i n g i n ­ t e r c a l a t i v e c o v a l e n t b i n d i n g i n k i n k e d DNA; and (6) The p o s s i b l e r e ­ o r i e n t a t i o n o f t h e complex t o y i e l d an e x t e r n a l l y bound a d d u c t . The e n e r g e t i c s f o r each o f t h e s e p r o c e s s e s w i l l be p r e s e n t e d t o i d e n t i f y the important steps t h a t i n f l u e n c e the b i n d i n g of s p e c i f i c isomers. I t w i l l be shown t h a t t h e o r i e n t a t i o n o f each d i a s t e r e o i s o m e r o f BPDE about s p e c i f i c base atoms i n k i n k e d r e c e p t o r s i t e s i n t h e du­ p l e x DNA d u r i n g c o v a l e n t bond f o r m a t i o n i s t h e d e t e r m i n i n g f a c t o r i n stereoselectivity• The parameters which d e f i n e t h e o r i e n t a t i o n o f t h e BPDE adduct t o N2 on guanine a r e g i v e n i n F i g u r e 3 i n terms o f t h e r e a c t i o n c o ­ o r d i n a t e s R, a , $, Y> 5l) t o DNA. The major p r o d u c t i n v o l v i n g t h e t r a n s l(+)-N2(G) adduct suggests t h a t a(BPDE) and y ( ) r e l a t e d t o the t h e o r e t i ­ c a l s t r u c t u r e s . The measured unwinding a n g l e s (1+2,1+5) i n v o l v e o t h e r a d d u c t s and i n t e r c a l a t i v e c o v a l e n t l y bound forms. B o t h o f t h e s e s t r u c t u r e s can a r i s e a f t e r a d e n a u t r a t i o n and r e n a t u r a t i o n o f t h e DNA. There i s e x p e r i m e n t a l e v i d e n c e f o r t h e dynamic p r o c e s s o f opening and c l o s i n g o f t h e DNA. I n hydrogen ex­ change s t u d i e s o f the amino and, i n Watson-Crick base pairing, b u r i e d imide g r o u p s , T e i t e l b a u m and Englander (105,106) c o n c l u d e t h a t t h e G»C and A»T base p a i r s a r e open about 1% o f t h e t i m e , and t h a t t h e opening r a t e c o n s t a n t i s about 0.01+ t o 0.06 s e c " i n both c a s e s . A study o f i n t e r c a l a t i o n by Gabbay e t a l . (107) o f two mole­ c u l e s , a 1,8-naphthylimide w i t h one b u l k y s u b s t i t u e n t and a 1,8,1+, 5n a p h t h y l i m i d e w i t h b u l k y s u b s t i t u e n t s on each end o f t h e m o l e c u l e t o d n a

x

z

D N A

a

r

e

1

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

278

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Table

K+:), K->, I I ( +)

XIII.

E x t e r n a l l y Bound Adducts t o N2(G) and N6(A)

Adduct

base

anti l ( + )-N2(G)*> l(+)-N2(G)

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

Il(-)-N6(A)

c

sugars

syn

a

o r i e n t a t i o n of

and I I ( -)

trans

a

z

y (DNA)

a(BPDE)

5e

BPDE

a f f e c t e d base

6.6 9.6

syn syn

s_ s^

0 +10

36.0 36.1

0 5

51 h9

syn

s_

0

36.0

0

51

10

10 11+1.7 60.8

3

a n t i o r i e n t a t i o n o f a l l bases l(+)-N2(G) l(+)-N2(G) l(+)-N2(G)

anti anti anti

s_ s^ s^

0 -20 -1+0

36.0 22.5 22.0

0 10 20

50 33

l(+)-N2(G) l(+)-N2(G) l(+)-N2(G) l(+)-N2(G)

anti anti anti

a a a

-30

26.0 19.2 7.3 1-11+

15 26 35 29

29 16 15 15-^3

63.7 5.7 -10.1

36.1 38.0 38.6

5 10 15

1+2 38

377. k 1+1+.6 7.5

l(-)-N6(A) l(-)-N6(A) K-)-N6(A) l(-)-N6(A)

b

d

anti anti anti

a

b

c

d

s_ s_ js

10 20 30

e

Il(+)-N2(G) Il(+)-N2(G) Il(+)-N2(G) Il(±)-N2(G) Il(-)-N6(A) II(-)-N6(A) II(-)-N6(A)

-50 -70

anti anti anti

a a a

-30

-50 -70

26.0 19.2 7.3

15 25 35

f

C

anti anti anti

s_ s_ s^

0 20 30

36.0 38.0 38.6

0 10 15

30

31

37-^5

5

exp

exp

5 7 15 >65

215.7 -5.3 -13.8

1+6 39

3xl0 67.2 12.7

31

f

3

The s u g a r s a r e C(2')-endo t o C(2')-endo (s_) and C(2')-endo t o C(3^)-endo (a) i n t h e 5 " ( p ) 3 ' o r 3 ' ( s u g a r ) 5 ' d i r e c t i o n . Base sequences: +G-C,OG,BPT,G«C,OG+ and +G«C,T«A,BPT,A«T,C«G+. E n e r g i e s f o r t h e BPDE-N2(G) and -N6(A) a d d u c t s a r e measured r e l a t i v e t o t h e 5 ' - o r i e n t a t i o n o f t h e c o r r e s p o n d i n g isomers i n T a b l e s V I I I and I X , r e s p e c t i v e l y . See F i g u r e 11. See F i g u r e 13. E x p e r i m e n t a l r e s u l t s (50,51,58). a = 36° - u n w i n d i n g angle f r o m (1+2,1+5). E x p e r i m e n t a l r e s u l t s (51.). N6(A) adduct f o r m a t i o n i s assumed. E x p e r i m e n t a l r e s u l t s a r e f o r t h e r a c e m i c m i x t u r e (j>8_). z

e

exp

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

10.

MILLER ET AL.

Binding of Benzo[a]pyrene Diol Epoxides to DNA

279

prevent d i r e c t i n s e r t i o n into an i n t e r c a l a t i o n s i t e , also supports a dynamical structure of DNA. Their k i n e t i c studies show that DNA complexes form i n ca. 100 msec for these two compounds, respectively. The f i r s t compound can be inserted into DNA d i r e c t l y from one groove; however, f o r the second naphthylimide to be i n t e r ­ calated, the substituents must l i e i n opposite grooves. The slower time needed for complex formation suggests a denaturation and renaturation of the DNA t o accommodate each substituent i n each groove. Their r e s u l t s suggest that there are two modes a v a i l a b l e for i n t e r c a l a t i o n : rapid (involving opening, RO) and slow ( i n v o l ­ ving denaturation, SD) equilibrium processes, and conversely, that these two modes are a v a i l a b l e for the reverse process. For a co­ v a l e n t l y bound adduct which r e s u l t s during i n t e r c a l a t i o n , the RO process w i l l not permit the pyrene moiety to be dislodged from the i n t e r c a l a t i o n s i t e , whereas the SD process w i l l . Therefore, the f i n a l state w i l l depend on the d i r e c t i o n of the equilibrium pro­ cess. The a n t i ->» syn r e o r i e n t a t i o n of G, and A i s i l l u s t r a t e d i n Figure 12. This transformation takes N2(G) from i t s p o s i t i o n i n the minor groove and places i t i n the major groove and quite f a r outside the h e l i x . Watson-Crick p a i r i n g i s l o s t . There are no poor s t e r i c contacts i n t h i s model, and energy can be recovered by hydrogen bonding with water. An a n t i syn r o t a t i o n of A displaces N6(A) only s l i g h t l y farther into the major groove and s i m i l a r l y for 06(G). As already shown, the a n t i o r i e n t a t i o n of G y i e l d s the most favor­ able f i t and the pyrene orientations are i n agreement with experi­ mental r e s u l t s f o r the l(+)-N2(G) adducts. Similar calculations were performed f o r the BPDE l ( - ) - and Il(-)-N6(A) adducts. The stereoselected Cda conformation of the BPDE i ( - ) and I l ( - ) adducts to N6(A) were chosen f o r study i n a reoriented complex with an e x t e r n a l l y bound pyrene moiety. In Figure 13, the adduct i s shown i n i t s optimum o r i e n t a t i o n i n B-DNA with adenine a f t e r an anti syn transformation for which the non-bonded contacts are poor, and with the normal a n t i base o r i e n t a t i o n with favorable con­ t a c t s . The f i t improves f o r the a n t i base as ct 30°. The o r i e n ­ t a t i o n of the pyrene moiety i s a(BPDE) =31° and the l o c a l h e l i c a l a x i s of the DNA i s oriented at y(DNA) = 15°. Calculations were not performed with e x t e r n a l l y bound BPDE-DNA adducts to 06(G) and NU(C). Calculations of e x t e r n a l l y bound BPDE l(-)-N6(A) adducts with kinked DNA with a 30° y i e l d s an o r i e n t a t i o n a(BPDE) = 31° i n good agree­ ment with experimental r e s u l t s for the e x t e r n a l l y bound component (51). The energies reported i n Table XIII f o r the e x t e r n a l l y bound forms are measured r e l a t i v e to that f o r the i n t e r c a l a t i v e covalently bound form. Thus, the trans BPDE l(+)-N2(G) adduct i s 10.1 k c a l / mole more stable and the trans BPDE Il(-)-N6(A) adduct i s 12.7 kcal/mole l e s s stable i n the e x t e r n a l l y bound form. S i m i l a r i l y , the trans BPDE Il(+)-N2(G) adduct i s -13.8 kcal/mole more stable and the trans BPDE l(-)-N6(A) adduct i s 7*5 kcal/mole l e s s stable. There­ f o r e , s i t e IQ ( i n t e r c a l a t i v e covalent) which i s favored by the i ( - ) isomer (5l) may be due to N6(A) and NMc) adduct formation, s p e c i ­ f i c a l l y trans a d d i t i o n . The d i s t r i b u t i o n of BPDE i ( - ) adducts observed by Brookes et a l . (38) as 59% N2(G), 21% 06(G), lQ% N6(A) and 2% other must be ad­ dressed. Although the y i e l d of N2(G) adduct i s 59% compared t o x

x

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

280

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

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t h a t w i t h o t h e r base atoms, i t i s a c t u a l l y 0.1 o f t h e l(+)-N2(G) a d ­ duct. The b i n d i n g t o N2(G) and t h e r e s u l t i n g form ( e x t e r n a l l y o r i n t e r n a l l y bound) w i l l predominant. I n t r a n s a d d i t i o n t h e i ( - ) i s o ­ mer i s u n f a v o r e d i n t h e i n t e r c a l a t i v e c o v a l e n t s t e p ; however, c i s a d d i t i o n i s p o s s i b l e as d i s c u s s e d i n t h e p r e v i o u s s e c t i o n . The c i s adduct s h o u l d y i e l d a s t a b l e complex analogous t o t h e r e s u l t s f o r

F i g u r e 12. A n t i ( - ) and s y n ( ) r e o r i e n t a t i o n o f G and A about t h e i r g l y c o s i d i c bonds (Cl'-O) i n B-DNA.

the l ( + ) adduct i n T a b l e X I I I . The c o n t r i b u t i o n t o b o t h IQ ( i n t e r ­ n a l ) and I I X ( e x t e r n a l ) b i n d i n g s i t e s by t h e i ( - ) isomer may be due t o t h e s m a l l amount o f c i s adduct which b i n d s t o N2(G) ( f o r IQ) and l o w r e a c t i v i t y toward N6(A) ( f o r s i t e I Q ) . Two o t h e r models which o r i e n t t h e pyrene m o i e t y e x t e r n a l l y have been proposed. Aggarwal e t a l . (108) s u c c e s s f u l l y f i t b o t h t h e l ( + ) and i ( - ) i n t o A-DNA w h i c h c a n a r i s e from a l o c a l d i s t o r t i o n o f BDNA. I n t h e l ( + ) a d d u c t , t h e chromophore i s d i r e c t e d out o f t h e minor g r o o v e , whereas f o r t h e i ( - ) i t f i t s s n u g l y i n t o t h e g r o o v e . The a n g l e subtended by t h e l o n g a x i s o f BP w i t h r e s p e c t t o t h e h e l i x a x i s i s 67° f o r l ( + ) and 63° f o r i ( - ) . H i n g e r t y and Broyde (109) have o p t i m i z e d t h e c o n f o r m a t i o n o f t h e dCpdG-BPDE l ( + ) a d d u c t . They f i n d a pyrene base s t a c k e d c o n f o r m a t i o n w i t h C and o r i e n t e d by a(BPDE) = 25°-30°. I f t h e i r complex can be i n c o r p o r a t e d i n t o a d o u b l e h e l i x , i t may r e p r e s e n t a l o c a l d e f o r m a t i o n o f an e x t e r n a l l y bound form w i t h t h e pyrene o r i e n t e d i n t h e minor g r o o v e . Thus, a l l t h r e e models f o r t h e " f i n a l " e x t e r n a l l y bound BPDE l ( + ) t o N2(G) i n the minor groove e x h i b i t s i m i l a r p h y s i c a l c h a r a c t e r i s t i c s . At the t i m e o f w r i t i n g t h i s m a n u s c r i p t , s t e r e o s e l e c t i v i t y has not been demonstrated w i t h t h e s e l a t t e r two models (108,109).

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F i g u r e 13. An e x t e r n a l l y bound BPDE I l ( - ) - N 6 ( A ) adduct w i t h t h e pyrene m o i e t y p l a c e d i n t h e major g r o o v e . The r e c e p t o r s i t e s a r e (upper) B-DNA except f o r an a n t i s y n r o t a t i o n b y 180° about t h e g l y c o s i d i c bond o f A, and ( l o w e r ) DNA w i t h an a = 30° k i n k . x

D i s c u s s i o n and C o n c l u s i o n T h e o r e t i c a l r e s u l t s have been p r e s e n t e d w i t h a n a l y t i c a l d a t a and s t e r e o g r a p h i c p r o j e c t i o n s t o support a mechanism g i v e n i n F i g u r e ll+ i n which s t e r e o s e l e c t i v i t y occurs during i n t e r c a l a t i v e covalent binding. We propose a m o l e c u l a r model and demonstrate t h a t each step plays the f o l l o w i n g roles: The i n t e r c a l a t i o n s t e p o r i e n t s t h e e p o x i d e toward t h e major o r minor groove w i t h t h e r e a c t i v e CIO atom i n t h e groove a d j a c e n t t o t h e a p p r o p r i a t e base atoms: N 2 ( G ) , N 6 ( A ) , 06(G) and NU(c). A proton catalyzed nucleophilic SJJ2 r e a c t i o n i s favored d u r i n g i n t e r c a l a t i o n because p o s i t i v e i o n s , e s p e c i a l l y H , r e s i d e i n t h e grooves and t h e i r p r e s e n c e a s s i s t s i n t h e a c t i v a t i o n o f t h e BPDEs. However, d u r i n g c o v a l e n t bond f o r m a t i o n , t h e DNA must k i n k t o o r i e n t t h e pyrene m o i e t y w i t h i n t h e k i n k e d s i t e f o r proper b o n d i n g between CIO o f t h e BPDEs and t h e r e a c t i v e base atoms, i . e . , t o a c h i e v e a bond l e n g t h o f a p p r o x i m a t e l y 1.5 A and t e t r a h e d r a l h y ­ b r i d i z a t i o n on CIO o f t h e BPDE and t r i g o n a l h y b r i d i z a t i o n on t h e r e ­ a c t i v e base atoms. In t h i s s t e p t h e base p a i r s a r e deformed from the p a r a l l e l o r i e n t a t i o n o f a c l a s s i c a l i n t e r c a l a t i o n s i t e while the BPDE remains i n s e r t e d . The DNA k i n k s o r bends w i t h a wedge o p e n i n g i n t o t h e major o r minor g r o o v e , r e s p e c t i v e l y , w h i l e t h e a d j a c e n t base p a i r s remain s e p a r a t e d t o accommodate t h e q u a s i i n t e r c a l a t e d BPDE as i t undergoes i n t e r c a l a t i v e c o v a l e n t b i n d i n g t o DNA. I t was +

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shown t h a t s t e r e o s e l e c t i v i t y o c c u r s d u r i n g t h i s s t e p . Specifically, BPDE l ( + ) and I l ( + ) a r e t h e o n l y isomers w h i c h f i t i n a k i n k e d s i t e (+39 ) when bound t o N2 on g u a n i n e . I n c o n t r a s t , BPDE i ( - ) and Il(-) a r e t h e o n l y isomers w h i c h f i t i n a k i n k e d s i t e (-30°) when bound t o atoms N 6 ( A ) , 06(G) and i t appears t h a t b i n d i n g t o Hk(c) may not be s t e r e o s e l e c t i v e . The l e s s f a v o r e d p r o t o n a d d i t i o n v i a an Sjji r e a c t i o n forms t h e carbonium i o n o f BPDE. This permits both trans and c i s a d d i t i o n , and minor p r o d u c t s . For c i s a d d i t i o n , the mirror images a r e s t e r e o s e l e c t e d , namely, i ( - ) and I I ( - ) by N2(G), l ( + ) and I l ( + ) by N6(A) and 06(G) and I l ( - ) and l ( + ) by N U ( C ) . I f both trans and c i s a d d i t i o n occurred t o an e q u a l extent, s t e r e o s e l e c t i v i t y would n o t be o b s e r v e d . T h e r e f o r e , t h e f a v o r e d t r a n s a d d i t i o n as w e l l as t h e s t e r i c f i t o f s p e c i f i c s t e r e o i s o m e r s during i n t e r c a l a ­ t i v e covalent binding contribute t o s t e r e o s e l e c t i v i t y . Possible r e ­ arrangements o f t h e DNA t o y i e l d o u t s i d e b i n d i n g c a n o c c u r i n two ways: F i r s t , an a n t i s y n r o t a t i o n about t h e g l y c o s i d i c bond o f t h e a f f e c t e d bases a l l o w s t h e r e m a i n i n g p o r t i o n o f t h e DNA t o resume i t s normal B-DNA c o n f o r m a t i o n w i t h an e x t e r n a l l y bound adduct t h a t f i t s w e l l i n t h e c a s e o f BPDE l ( + ) and I l ( + ) bound t o N2(G), b u t n o t w e l l f o r BPDE i ( - ) and I l ( - ) bound t o N 6 ( A ) . Second, a denatura­ t i o n , rearrangement o f t h e adduct and r e n a t u r a t i o n o f t h e DNA a l l o w s t h e adduct t o l i e i n a groove i n a s l i g h t l y k i n k e d DNA.

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P

H+ BPT+

BPDE

BPDE

B-DNA

Activation

Intercalation

H+ Relaxation

I n t e r c a l a t i v e covalent binding; stereoselectivity; final predominant s t r u c t u r e f o r t r a n s a d d i t i o n t o N 6 ( A ) and 06(A1 o f BPDE I ( - ) and I l ( - ) .

Figure lU.

Outside b i n d i n g ; f i n a l predominant structure for trans a d d i t i o n t o N2(G) o f BPDE l ( + ) and I l ( + ) .

Mechanism f o r BPDE-DNA

adducts.

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The main f e a t u r e s o f t h i s proposed mechanism a r e ( l ) t h e s t e r e o s e l e c t i v i t y o f t h e BPDEs by t h e DNA d u r i n g i n t e r c a l a t i v e c o ­ v a l e n t b i n d i n g and (2) t h e f i n a l o r i e n t a t i o n o f t h e bound pyrene w h i c h may be o r i e n t e d i n t e r n a l l y ( i n t e r c a l a t i v e c o v a l e n t ) o r ex­ t e r n a l l y (outside the h e l i x ) . The s t e r e o s e l e c t i v i t y o c c u r s d u r i n g c o v a l e n t bond f o r m a t i o n and a f t e r i n t e r c a l a t i o n . Relaxation of the DNA a l l o w s t h e adduct t o a d j u s t t o i t s f i n a l o r i e n t a t i o n . I f the e x p e r i m e n t a l measurements a r e assumed t o be made on t h e DNA-adducts a f t e r t h e f i n a l o r i e n t a t i o n has been a c h i e v e d , t h e n t h e f o l l o w i n g i n t e r p r e t a t i o n s can be made. The l ( + ) and I l ( + ) isomers a r e s t e r e o s e l e c t e d by N2(G), whereas t h e i ( - ) and I l ( - ) isomers a r e s t e r e o s e l e c t e d by t h e N6(A) and 06(G) during i n t e r c a l a t i v e covalent steps w i t h t r a n s a d d i t i o n . The l ( + ) and I l ( + ) - N 2 ( G ) adducts a r e r e a r r a n g e d t o an e x t e r n a l l y bound form w i t h t h e pyrene i n t h e minor g r o o v e , b u t t h e l ( - ) - N 6 ( A ) and I l ( - ) - 0 6 ( G ) adducts remain q u a s i i n t e r c a l a t e d . T h i s i s determined by t h e r e l a t i v e energy change between t h e two forms as we see from Table X I I I . However, t h e r e i s a s u p e r p o s i t i o n o f t h e two t y p e s o f s i t e s , IQ and I I X (51,57,58), and BPDE i ( - ) DNA adducts e x h i b i t b o t h types o f binding. By symmetry, t h e c i s BPDE l ( - ) - N 2 ( G ) adduct i s p r e d i c t e d t o behave s i m i l a r i l y t o t h e t r a n s l(+)-N2(G) adduct. I t s h o u l d be e x t e r n a l l y bound. The N2(G) adducts a r e more s t a b l e t h a n t h e N6(A) and 06(G) and Nl*(c) a d d u c t s . Because c i s a d d i t i o n p r o d u c t s a r e p r e s e n t , minor amounts o f t h e o t h e r adducts a r e found. I f o n l y c i s a d d i t i o n o c c u r ­ r e d , t h e n t h e i ( - ) and I l ( - ) isomers would be s t e r e o s e l e c t e d by N2(G), and t h e l ( + ) and I l ( + ) isomers would be s t e r e o s e l e c t e d by N6(A) and 06(G). A l t h o u g h we d i d n o t p e r f o r m c a l c u l a t i o n s on t h e c i s a d d u c t s , i t can be seen from t h e s t e r e o g r a p h i c p r o j e c t i o n s t h a t t h e change accompanied b y a r e f l e c t i o n o f o n l y t h e BPDE atoms t h r o u g h t h e p l a n e o f t h e pyrene changes t h e ( + ) i n t o (-) i s o m e r s . Thus, t h e r u l e s o f s t e r e o s e l e c t i v i t y a r e r e v e r s e d . However, t h e s m a l l amount o f c i s adduct y i e l d s t h e s e minor components; t h e l ( - ) - N 2 ( G ) adduct i s most p r e v a l e n t (38) f o r r e a c t i o n s o f BPDE i ( - ) w i t h DNA and we assume t h a t t h i s a r i s e s from t h e c i s a d d i t i o n . I f b o t h t r a n s and c i s a d d i t i o n o c c u r r e d e q u a l l y , we p r e d i c t t h a t s t e r e o s e l e c t i v i t y would not be o b s e r v e d . Based on t h e r e s u l t s i n t h i s p a p e r , t h e f o l l o w i n g e x p e r i m e n t a l d a t a s h o u l d be o b t a i n e d f o r each o f t h e d i a s t e r e o i s o m e r s . ( l ) The r e l a t i v e y i e l d s o f t r a n s and c i s a d d i t i o n p r o d u c t s s h o u l d be d e t e r ­ mined f o r adduct f o r m a t i o n t o each base atom. (2) A l t e r n a t i n g and n o n - a l t e r n a t i n g homopolymers s h o u l d be used t o e v a l u a t e t h e base s e ­ quence s p e c i f i c i t y . (3) B i n d i n g t o s i t e s IQ and I I X s h o u l d be c o r ­ r e l a t e d t o t r a n s and c i s adducts and t o s t e r e o s e l e c t i v i t y .

Acknowledgments The a u t h o r s acknowledge t h e g r a n t o f computer t i m e from R e n s s e l a e r P o l y t e c h n i c I n s t i t u t e and support by t h e N a t i o n a l I n s t i t u t e s o f H e a l t h under Grant CA-28921+. A l s o , t h e a u t h o r s w i s h t o thank C h r i s Bonesteel f o r her t e c h n i c a l assistance with the preparation of t h i s manuscript•

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CHEMISTRY AND BIOCHEMISTRY Vol. 13; Jerusalem, Israel; 1980; pp. 565-579. Hogan, M. E.; Dattagupta, N.; Whitlock, Jr., J. P. J. Biol. Chem. 1981, 256, 4505-4513. Geacintov, N. E.; Ibanez, V.; Gagliano, A. G.; Jacobs, S. A.; Harvey, R. G. J. Biol. Struct. and Dynam. 1984, 1, l473-l484. Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1981, 100, 1569-1577. Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Harvey, R. G. Biochem. 1982, 21, 1864-1869. Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Jacobs, S. A.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1984, 122, 3339. MacLeod, M. C. & Selkirk, J. K. Carcinogenesis 1982, 3, 287292. Geacintov, N. E.; Ibanez, V.; Gagliano, A. G.; Yoshida, H.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1980, 92, 13351342.

57. Geacintov, N. E.; Gagliano, A. G.; Ibanez, V.; Harvey, R. G. Carcinogenesis 1982, 3, 247-253. 58. Undeman, O.; Lycksell, P.-O.; Gräslund, A.; Astlind, T.; Ehrenberg, A.; Jerström, B; Tjerneld, F.; Norden, B. Cancer Research 1983, 43, 1851-1860. 59. Olson, W. Macromolecules 1975, 8, 272-275. 60. Smith, P. J. C.; Arnott, S. Acta Cryst. 1978, A34, 3-11. 61. Alden, C. J.; Arnott, S. Nucleic Acids Res. 1975, 2, 17011717. 62. Alden, C. J.; Arnott, S. Nucleic Acids Res. 1977, 4, 38553861. 63. Tumanyan, V. G.; Esipova, N. G. Biopolymers 1975, 14, 22312246. 64. Zhurkin, V. B.; Lysov, Yu. P.; Ivanov, V. I. Biopolymers 1978, 17, 377-412. 65. Miller, K. J. Biopolymers 1979, 18, 959-980. 66. Taylor, E. R.; Miller, K. J. Biopolymers 1984, 23, 2853-2878. 67. Miller, K. J. Proceedings of the Second SUNYA Conversation in the Discipline Biomolecular Stereodynamics; R. H. Sarma, ed.; New York, 1981; Vol. II, pp. 469-486. 68. Miller, K. J.; Pycior, J. F. Biopolymers 1979, 18, 2683-2719. 69. Miller, K. J.; Brodzinsky, R.; Hall, S. Biopolymers 1980, 19, 2091-2122. 70. Newlin, D. D.; Miller, K. J.; Pilch, D. F. Biopolymers 1984, 23, 139-158. 71. Taylor, E. R.; Olson, W. K. Biopolymers 1983, 22, 2667-2702. 72. Pack, G. R.; Loew, G. Biochim. Biophys. Acta 1978, 519, 163-172. 73. Nuss, M. E.; Marsh, F. J.; Kollman, P. A. J. Am. Chem. Soc. 1979, 101, 825-833. 74. Ornstein, R. L.; Rein, R. Biopolymers 1979, 18, 2821-2847. 75. Ornstein, R. L.; Rein, R. Biopolymers 1979, 18, 1277-1291. 76. Berman, H. M.; Neidle, S. In "Stereodynamics of Molecular Systems"; Sarma, R. H., Ed.; Pergamon: New York; 1979, pp. 367-382.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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77. For example, Bingham, R. C.; Dewar, M. J. S.; Lo, D. H. J. Am. Chem. Soc. 1975, 97, 1285-1293 and subsequent papers. 78. Miller, K. J.; Pycior, J. F.; Moschner, K. F. QCPE BULLETIN 1981, 1, 67-70. 79. Neidle, S.; Subbiah, A.; Cooper, C. S.; Riberio, O. Carcinogenesis 1980, 1, 249-254. 80. Zacharias, D. E.; Glusker, J. P.; Fu, P. P.; Harvey, R. G. J. Am. Chem. Soc. 1979, 101, 4043-4051. 81. Silverman, B. D. Cancer Biochem. Biophys. 1983, 6, 131-136. 82. Lavery, R.; Pullman, B. Int. J. Quant. Chem. 1979, XV, 271280. 83. Kikuchi, O.; Pearlstein, R.; Hopfinger, A. J.; Bickers, D. R. J. Pharm. Sci. 1983, 72, 800-808. 84. Politzer, P.; Daiker, K. C.; Estes, V. M. Int. J. Quant. Chem. Quant. Biol. Symp. 1979, 6, 47-53. 85. Klopman, G.; Grinberg, H.; Hopfinger, A. J. J. Theor. Biol. 1979, 79, 355-366. 86. Zielinski, T. J.; Breen, D. L.; Rein, R. J. Am. Chem. Soc. 1978, 100, 6266-6267. 87. Klopman, G.; Andreozzi, P.; Hopfinger, A. J.; Kikuchi, O. J. Am. Chem. Soc. 1978, 100, 6267-6268. 88. Neidle, S.; Subbiah, A.; Kuroda, R.; Cooper, C. S. Cancer Res. 1982, 42, 3766-3768. 89. Politzer, P.; Daiker, K. C.; Estes, V. M.; Baughman, M. Int. J. Quant. Chem.: Quant. Biol. Symp. 1978, 5, 291-299. 90. Loew, G, H.; Pudzianowski, A. T.; Czerwinski, A.; Ferrell, Jr., J. E. Intl. J. Quant. Chem.: Quant. Biol. Symp. 1980, 7, 223244. 91. Malhotra, D.; Hopfinger, A. J. Nucleic Acids Res. 1980, 8, 5289-5304. 92. Subbiah, A.; Islam, S. A.; Neidle, S. Carcinogensis 1983, 4, 211-215. 93. Miller, K. J.; Macrea, J.; Pycior, J. F. Biopolymers 1980, 19, 2067-2089. 94. Pullman, B. J. Biomolec. Struct, and Dynam. 1983, 1, 773-794. 95. Clementi, E.; Corongiu, G. J. Biol. Phys. 1983, 11, 33-42. 96. Whalen, D. L.; Ross, A. M.; Montemarano, J. A.; Thakker, D. R.; Yagi, H.; Jerina, D. M. J. Am. Chem. Soc. 1979, 101, 50865088. 97. Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1981, 100, 1569-1577. 98. Geacintov, N. E.; Hibshoosh, H.; Ibanez, V.; Benjamin, M. J.; Harvey, R. G. Biophys. Chem. 1984, 20, 121-133. 99. Michaud, D. P.; Gupta, S. C.; Whalen, D. L.; Sayer, J. M.; Jerina, D. M. Chem.-Biol. Interactions 1983, 44, 41-52. 100. Kootstra, A.; Haas, B. L.; Slaga, T. J. Biochem. Biophys. Res. Commun. 1980, 94, 1432-1438. 101. Lavery, R.; Pullman, B. Intl. J. Quant. Chem. 1979, XVI, 175188. 102. Lavery, R.; Pullman, A.; Pullman, B. Int. J. Quant. Chem.: Quant. Biol. Symp. 1978, 5, 21-34. 103. Lin, J.-h.; LeBreton, P. R.; Shipman, L. L. J. Phys. Chem. 1980, 84, 642-649.

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104. Nakata, Y.; Malhotra, D.; Hopfinger, A. J.; Bickers, D. R. J. Pharm. Sci. 1983, 72, 809-811. 105. Teitelbaum, H.; Englander, S. W. J. Mol. Biol. 1975, 92, 5578. 106. Teitelbaum, H.; Englander, S. W. J. Mol. Biol. 1975, 92, 7992. 107. Gabbay, E. J.; DeStefano, R.; Baxter, C. S. Biochem. Biophys. Res. Commun. 1973, 51, 1083-1089.

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108. Aggarwal, A. K.; Islam, S. A.; Neidle, S. J. Biomol. Struct. and Dynam. 1983, 1, 873-881. 109. Hingerty, B.; Broyde, S. Biomol. Struct. and Dynam. 1983, 1, 905-912. RECEIVED April 18, 1985

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

11 One-Electron Oxidation in Aromatic Hydrocarbon Carcinogenesis ERCOLE L. CAVALIERI and ELEANOR G. ROGAN

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68105

Two main pathways are involved in the carcinogenic activation of polycyclic aromatic hydrocarbons (PAH): one-electron oxidation and monooxygenation. One-electron oxidation produces PAH radical cations, which can react with cellular nucleophiles. Biochemical and biological data indicate that only PAH with relatively low ionization potentials (below ca. 7.35 eV) can be activated by one-electron oxidation. Furthermore, a carcinogenic PAH must have a relatively high charge localization in its radical cation to react effectively with target cellular macromolecules. Binding of benzo[a]pyrene (BP) to DNA in vitro and in vivo occurs predominantly at C-6, the position of highest charge density in the BP radical cation, and binding of 6-methylBP to mouse skin DNA yields a major adduct in which the 6-methyl is bound to the 2-amino of deoxyguanosine. PAH radical cations are also involved in the metabolic conversion of PAH to PAH diones. Carcinogenicity studies of PAH in rat mammary gland indicate that only PAH with ionization potential low enough for activation by one-electron oxidation induce tumors in this target organ. These results and others indicate that one-electron oxidation of PAH is involved in their tumor initiation process. C o v a l e n t b i n d i n g o f chemical c a r c i n o g e n s t o c e l l u l a r m a c r o m o l e c u l e s , DNA, RNA and p r o t e i n , i s w e l 1 - a c c e p t e d t o be t h e f i r s t s t e p i n t h e tumor i n i t i a t i o n p r o c e s s (_1,_2). Most c a r c i n o g e n s , i n c l u d i n g p o l y c y c l i c a r o m a t i c hydrocarbons (PAH), r e q u i r e m e t a b o l i c a c t i v a t i o n t o produce t h e u l t i m a t e e l e c t r o p h i 1 i c s p e c i e s which r e a c t w i t h c e l l u l a r macromolecules. U n d e r s t a n d i n g t h e mechanisms o f a c t i v a t i o n and t h e enzymes which c a t a l y z e them i s c r i t i c a l t o e l u c i d a t i n g t h e tumor i n i t i a t i o n process. H i s t o r i c a l l y t h e p r o c e s s o f a c t i v a t i o n has almost e x c l u s i v e l y been s t u d i e d by m e t a b o l i z i n g compounds w i t h l i v e r p r e p a r a t i o n s , l e a d i n g most i n v e s t i g a t o r s i n chemical c a r c i n o g e n e s i s t o t h i n k t h a t

0097-6156/85/0283-0289S06.00/0 © 1985 American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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o x y g e n a t i o n i s t h e c r i t i c a l s t e p t o produce p r o x i m a t e a n d / o r u l t i mate c a r c i n o g e n s . T h i s emphasis has i n d e e d been predominant f o r PAH, i n which f o r m a t i o n o f b a y - r e g i o n v i c i n a l d i o l e p o x i d e s has been d e s c r i b e d t o be t h e most i m p o r t a n t , i f not e x c l u s i v e , pathway o f a c t i v a t i o n (2-5.)* At p r e s e n t a v a r i e t y o f s t u d i e s w i t h PAH, as w e l l as o t h e r c h e m i c a l s , s u g g e s t t h a t m e t a b o l i c a c t i v a t i o n i n t a r g e t t i s s u e s can o c c u r by o n e - e l e c t r o n o x i d a t i o n (6^,7). The e l e c t r o p h i l i c i n t e r m e d i a t e r a d i c a l c a t i o n s g e n e r a t e d by t h T s mechanism can r e a c t d i r e c t l y with various c e l l u l a r n u c l e o p h i l e s . In t h i s p a p e r , we w i l l d i s c u s s c h e m i c a l , b i o c h e m i c a l and b i o l o g i c a l e v i d e n c e which i n d i c a t e s t h a t o n e - e l e c t r o n o x i d a t i o n p l a y s an i m p o r t a n t r o l e i n t h e m e t a b o l i c a c t i v a t i o n o f PAH.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

Chemical

Properties of

PAH R a d i c a l

Cations

N u c l e o p h i l i c Trapping of Radical Cations. To i n v e s t i g a t e some o f the p r o p e r t i e s o f PAH r a d i c a l c a t i o n s t h e s e i n t e r m e d i a t e s have been g e n e r a t e d i n two o n e - e l e c t r o n o x i d a n t s y s t e m s . The f i r s t c o n t a i n s i o d i n e as o x i d a n t and p y r i d i n e as n u c l e o p h i l e and s o l v e n t ( 8 - 1 0 ) , w h i l e t h e second c o n t a i n s M n ( 0 A c ) i n a c e t i c a c i d ( 1 0 , 1 1 ) . Studies w i t h a number o f PAH i n d i c a t e t h a t t h e f o r m a t i o n o f p y n d i n i u m - P A H o r acetoxy-PAH by o n e - e l e c t r o n o x i d a t i o n w i t h M n ( 0 A c ) o r i o d i n e , r e s p e c t i v e l y , i s r e l a t e d t o t h e i o n i z a t i o n p o t e n t i a l (IP) o f t h e PAH. For PAH w i t h r e l a t i v e l y h i g h IP, such as p h e n a n t h r e n e , c h r y s e n e , 5 - m e t h y l c h r y s e n e and d i b e n z [ a , h ] a n t h r a c e n e , no r e a c t i o n o c c u r s w i t h t h e s e two o x i d a n t s y s t e m s . Another i m p o r t a n t f a c t o r i n f l u e n c i n g t h e s p e c i f i c r e a c t i v i t y o f PAH r a d i c a l c a t i o n s w i t h n u c l e o p h i l e s i s l o c a l i z a t i o n o f t h e p o s i t i v e c h a r g e at one o r a few carbon atoms i n t h e r a d i c a l c a t i o n . 3

3

For u n s u b s t i t u t e d PAH, such as b e n z o [ a ] p y r e n e ( B P ) , p y r i d i n i u m o r a c e t o x y d e r i v a t i v e s a r e formed by d i r e c t a t t a c k o f p y r i d i n e o r a c e t a t e i o n , r e s p e c t i v e l y , on t h e r a d i c a l c a t i o n at C - 6 , t h e p o s i t i o n o f maximum c h a r g e d e n s i t y (Scheme 1 ) . T h i s i s f o l l o w e d by a second o n e - e l e c t r o n o x i d a t i o n o f t h e r e s u l t i n g r a d i c a l and l o s s o f a proton t o y i e l d the 6 - s u b s t i t u t e d d e r i v a t i v e . For m e t h y l - s u b s t i t u t e d PAH i n which t h e maximum c h a r g e d e n s i t y o f t h e r a d i c a l c a t i o n a d j a c e n t t o t h e methyl group i s a p p r e c i a b l e , as i n 6 - m e t h y l b e n z o [ a ] pyrene (6-methylBP) (Scheme 2 ) , l o s s o f a methyl p r o t o n y i e l d s a benzylic T h i s r e a c t i v e s p e c i e s i s r a p i d l y o x i d i z e d by i o d i n e o r Mn t o a b e n z y l i c carbonium i o n w i t h subsequent t r a p p i n g by p y r i d i n e o r a c e t a t e i o n , r e s p e c t i v e l y . For a c t i v a t i o n by o n e - e l e c t r o n o x i d a t i o n , t h e s e p r o p e r t i e s o f PAH r a d i c a l c a t i o n s e n a b l e us t o p r e d i c t t h e p o s i t i o n ( s ) at which c o v a l e n t b i n d i n g o f PAH t o c e l l u l a r t a r g e t s may o c c u r .

radical.

S y n t h e s i s o f R a d i c a l C a t i o n P e r c h l o r a t e s and Subsequent C o u p l i n g with NucleophilesT Syntheses o f t h e r a d i c a l c a t i o n p e r c h l o r a t e s o f BP and 6-methylBP (12) were a c c o m p l i s h e d by t h e method r e p o r t e d e a r l i e r f o r the preparation of the perylene r a d i c a l c a t i o n (13,14). More r e c e n t l y we have a l s o s y n t h e s i z e d t h e r a d i c a l c a t i o n p e r c h l o r ate of 6-fluoroBP (15). O x i d a t i o n o f t h e PAH w i t h i o d i n e i n benzene i n t h e p r e s e n c e o f AgClO. i n s t a n t a n e o u s l y produces a b l a c k p r e c i p i t a t e c o n t a i n i n g t h e r a d i c a l c a t i o n p e r c h l o r a t e adsorbed on A g l w i t h

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291

One-Electron Oxidation

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11. CAVALIER I AND ROGAN

Scheme

2.

Stepwise

sequent t r a p p i n g

one-electron

oxidation

of

6-methylBP

and

by a n u c l e o p h i l e (Nu).

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

sub-

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y i e . l d s _ o f 28, 28 and 39% f o r BP*C10~ 6 - m e t h y l B P » C 1 OT and 6 - f l u o r o BP'CIOT, r e s p e c t i v e l y . The BP and 8-methylBP r a d i c a l c a t i o n s have been c h a r a c t e r i z e d by e l e c t r o n s p i n resonance s p e c t r o s c o p y (12) and by t r a p p i n g w i t h s t r o n g n u c l e o p h i l e s . R e a c t i o n o f t h e BP r a d i c a l c a t i o n w i t h t h e two s t r o n g n u c l e o p h i l e s NaSCN and NaN0 y i e l d s 6 - t h i o c y a n o - and 6 - n i t r o B P , but a l s o d e r i v a t i v e s at C - i . Incident a l l y , i n t h e BP r a d i c a l c a t i o n , C-6 i s t h e p o s i t i o n o f h i g h e s t c h a r g e d e n s i t y , f o l l o w e d by C - l and C - 3 . When t h e 6-methylBP and 6 - f l u o r o B P r a d i c a l c a t i o n s r e a c t w i t h NaNOp and NaSCN, o n l y d e r i v a t i v e s at t h e 1 a n d / o r 3 - p o s i t i o n a r e o b t a i n e d . Neither s u b s t i t u t i o n at t h e 6-methyl group nor d i s p l a c e m e n t o f t h e f l u o r i n e atom i s o b served. These r e s u l t s g e n e r a l l y i n d i c a t e t h a t s t r o n g n u c l e o p h i l e s d i s p l a y low s e l e c t i v i t y toward t h e p o s i t i o n i n which the p o s i t i v e c h a r g e i s b e t t e r l o c a l i z e d . R e a c t i o n o f BP and 6 - f l u o r o B P r a d i c a l c a t i o n s w i t h t h e weak n u c l e o p h i l e H 0 a f f o r d s a m i x t u r e o f B P - 1 , 6 - , - 3 , 6 - and - 6 , 1 2 - d i o n e . These p r o d u c t s a r e t h e r e s u l t o f an i n i t i a l a t t a c k o f FLO at C - 6 . 2

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

?

+

BP*C107

When t h e weak n u c l e o p h i l e a c e t a t e i o n i n water i s u s e d , y i e l d s s p e c i f i c a l l y 6-acetoxyBP and t h e t h r e e d i o n e s , which a r e t h e r e s u l t o f R e a c t i o n o f t h e r a d i c a l c a t i o n w i t h FLO. In t h e c a s e o f 6-fluoroBP#C10T, BP d i o n e s a r e t h e predominant p r o d u c t s , whereas o n l y t r a c e s o f 6-acetoxyBP a r e o b t a i n e d . This i n d i c a t e s that the a t t a c k by t h e a c e t a t e i o n i s s t e r i c a l l y h i n d e r e d at t h e 6 - p o s i t i o n in the 6-fluoroBP*ClOT. The o v e r a l l c o n c l u s i o n from t h e r e a c t i o n o f BP and 6 - s u b s t i t u t e d BP r a d i c a l c a t i o n s w i t h n u c l e o p h i l e s o f v a r i o u s s t r e n g t h s i s t h a t weak n u c l e o p h i l e s d i s p l a y h i g h e r s e l e c t i v i t y toward t h e p o s i t i o n o f highest charge l o c a l i z a t i o n . Thus a n o t h e r i m p o r t a n t f a c t o r i n t h e chemical r e a c t i v i t y o f r a d i c a l c a t i o n s i s r e p r e s e n t e d by t h e strength of the n u c l e o p h i l e . Ionization Cations

Potential

of

PAH and Charge L o c a l i z a t i o n i n

Radical

From knowledge p r e s e n t l y a v a i l a b l e , t h e a b i l i t y o f PAH t o b i n d c o v a l e n t l y t o c e l l u l a r macromolecules appears t o depend m a i n l y on two factors: t h e ease o f f o r m a t i o n o f PAH r a d i c a l c a t i o n s , which i s measured by t h e i r IP, and l o c a l i z a t i o n o f p o s i t i v e c h a r g e i n t h e radical cation. The IP o f numerous PAH have been d e t e r m i n e d and compared t o a q u a l i t a t i v e measure o f t h e i r c a r c i n o g e n i c i t y ( 1 6 ) . Some o f t h e most r e p r e s e n t a t i v e PAH w i t h h i g h and low IP a r e p r e s e n t e d i n T a b l e I. Only PAH w i t h r e l a t i v e l y low IP (below c a . 7.35 eV) can be b i o l o g i c a l l y a c t i v a t e d by o n e - e l e c t r o n o x i d a t i o n ( 1 6 ) . T h i s has been o b s e r v e d i n s t u d i e s o f r a t mammary g l a n d c a r c i n o g e n e s i s ( 1 0 , 1 7 , 1 8 ) , i n which t h e r e s u l t s from d i r e c t a p p l i c a t i o n o f PAH i n d i c a t e t h a t o n l y PAH w i t h low IP i n d u c e tumors i n t h i s t a r g e t organ (see b e l o w ) . In a d d i t i o n when t h e b i n d i n g o f PAH t o DNA i s s t u d i e d u s i n g h o r s e r a d i s h p e r o x i d a s e / H 0 , a system which c a t a l y z e s o n e - e l e c t r o n o x i d a t i o n o f a v a r i e t y o f c n e m i c a l s , o n l y t h o s e PAH w i t h IP < ca 7.35 eV a r e s i g n i f i c a n t l y bound ( 1 6 ) . The c a r c i n o g e n i c i t y o f PAH w i t h r e l a t i v e l y h i g h IP, such as benzo[c]phenanthrene, benz[a]anthracene, chrysene, 5-methylchrysene and d i b e n z [ a , h ] a n t h r a c e n e ( T a b l e I ) , can be r e l a t e d t o t h e f o r m a t i o n o f b a y - r e g i o n d i o l e p o x i d e s c a t a l y z e d by monooxygenase enzymes (j>). However, t h e most p o t e n t c a r c i n o g e n i c PAH have IP < c a . 7.35 eV. 2

2

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

11.

CAVALIERI AND ROGAN

Table

I.

Structure,

Compound

One-Electron Oxidation

Ionization Potential, S e l e c t e d PAH

Structure

and C a r c i n o g e n i c i t y

Ionization potential ieVj

of

8

. Carcinogenicity 1

Phenanthrene

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

Benzo[c]phenanthrene

Chrysene

5-Methylchrysene

Benzo[e]pyrene

Dibenz[a,h]anthracene

Benz[a]anthracene

Pyrene

Anthracene

7-Methylbenz[a]anthracene

C o n t i n u e d on next

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

page.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

T a b l e I.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

Compound

Continued. Ionization* potential (eV)

Carcinogenicity

Dibenzo[a,e]pyrene

7.35

+++

Dibenzo[a,l]pyrene

7.26

+++

D1benzo[a,i]pyrene

7.25

++ + +

Benzo[a]pyrene

7.23

++ + +

6-FluorobenzoLajpyrene

7.23

-I- +

7,12-D1methy1benz[a]anthracene

7.22

- H - + +

3-Methylcholanthrene

7.12

+ ++ +

6-Methylbenzo:a]pyrene

7.08

Peryle

+

+ ++

7.06

C o n t i n u e d on next

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

page.

11. CAVALIERI AND ROGAN

T a b l e I.

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295

One-Electron Oxidation

Continued. Ionization P o t e n t i a l (eV) C a r c i n o g e n i c i t y 9

Compound

Structure

Dibenzo[a.hjpyrene

6.97

+++ +

Anthanthrene

6.96

+

Determined from a b s o r p t i o n maximum o f t h e c h a r g e - t r a n s f e r complex o f each compound w i t h c h l o r a n i l , w i t h t h e e x c e p t i o n o f d i b e n z [ a , h ] a n t h r a c e n e d e t e r m i n e d by p o l a r o g r a p h i c o x i d a t i o n ( 2 4 ) . E x t r e m e l y a c t i v e , +++++; v e r y a c t i v e , ++++; a c t i v e , +++; m o d e r a t e l y a c t i v e , ++; weakly a c t i v e , +; v e r y weakly a c t i v e , +.; and i n a c t i v e , — .

b

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

296

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

T h i s l i s t i n c l u d e s BP, 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e , 3 - m e t h y l c h o l a n t h r e n e , d i b e n z o [ a , i ] p y r e n e and d i b e n z o [ a , h ] p y r e n e . These PAH can be a c t i v a t e d both by o n e - e l e c t r o n o x i d a t i o n a n d / o r monooxygenation. There a r e a few PAH w i t h low IP which a r e i n a c t i v e ( T a b l e I ) , such as p e r y l e n e , o r weakly a c t i v e , such as a n t h a n t h r e n e . This i n d i c a t e s t h a t low IP i s a n e c e s s a r y , but not s u f f i c i e n t f a c t o r f o r d e t e r m i n i n g c a r c i n o g e n i c a c t i v i t y by o n e - e l e c t r o n o x i d a t i o n . These i n a c t i v e o r weakly a c t i v e PAH have t h e h i g h e s t d e n s i t y o f p o s i t i v e charge d e l o c a l i z e d o v e r s e v e r a l a r o m a t i c carbon atoms i n t h e i r r a d i c a l c a t i o n s , whereas t h e a c t i v e PAH w i t h low IP have c h a r g e m a i n l y l o c a l i z e d on one o r a few carbon atoms i n t h e i r r a d i c a l c a t i o n s . These o b s e r v a t i o n s l e a d us t o suggest t h a t t h e second c r i t i c a l f a c t o r i n b i n d i n g o f PAH r a d i c a l c a t i o n s i s t h a t t h e c a r c i n o g e n i c PAH must have r e l a t i v e l y h i g h c h a r g e l o c a l i z a t i o n i n t h e i r r a d i c a l c a t i o n s t o g i v e them s u f f i c i e n t r e a c t i v i t y t o b i n d w i t h c e l l u l a r n u c l e o p h i l e s (6.,_7)E v i d e n c e on t h i s p o i n t has been o b t a i n e d by o n e - e l e c t r o n o x i d a t i o n o f PAH w i t h i o d i n e (8-10) and Mn(0Ac)~ ( 1 0 , 1 1 ) , a l t h o u g h t h i s concept o f charge locaTTzation r e q u i r e s f u r t h e r s t u d y by more q u a n t i t a t i v e a p p r o a c h e s . Metabolic o f BP

Formation o f Quinones by an I n i t i a l

One-Electron

Oxidation

Metabolism o f BP mediated by t h e cytochrome P-450 monooxygenase system forms t h r e e c l a s s e s o f p r o d u c t s : p h e n o l s , d i h y d r o d i o l s and quinones. Formation o f p h e n o l s and d i h y d r o d i o l s i s o b t a i n e d by an i n i t i a l e l e c t r o p h i l i c a t t a c k o f an enzyme-generated oxygen atom. The same pathway o f a c t i v a t i o n has been p o s t u l a t e d i n t h e f o r m a t i o n o f q u i n o n e s , a l t h o u g h t h e p u t a t i v e 6-hydroxyBP p r e c u r s o r has never been i s o l a t e d ( 1 9 , 2 0 ) . In t h i s mechanism, f o r m a t i o n o f quinones would proceed by a u t o x i d a t i o n o f 6-hydroxyBP ( 2 0 ) . However, s u b s t a n t i a l evidence i n d i c a t e s that the f i r s t step in formation of quinones does not i n v o l v e t h e t y p i c a l a t t a c k o f t h e e l e c t r o p h i l i c a c t i v e oxygen t o y i e l d 6-hydroxyBP, but i n s t e a d c o n s i s t s o f t h e l o s s o f one e l e c t r o n from BP t o produce t h e r a d i c a l c a t i o n . The f i r s t l i n e o f e v i d e n c e d e r i v e s from t h e predominant f o r m a t i o n o f quinones when metabolism o f BP i s conducted under p e r o x i d a s e c o n d i t i o n s , namely by p r o s t a g l a n d i n H s y n t h a s e (21) or by cytochrome P-450 w i t h cumene h y d r o p e r o x i d e as c o f a c t o r ~ T 2 2 ) • Under these metabolic c o n d i t i o n s o n e - e l e c t r o n o x i d a t i o n i s the prepond e r a n t mechanism o f a c t i v a t i o n . Second, metabolism o f 6 - f l u o r o B P by r a t l i v e r microsomes y i e l d s the same BP quinones o b t a i n e d i n t h e metabolism o f BP ( 2 3 ) . T h i s s u g g e s t s t h a t t h e s e p r o d u c t s a r e formed by an i n i t i a l a t t a c k o f a n u c l e o p h i l i c oxygen atom at C-6 i n t h e 6 - f l u o r o B P r a d i c a l c a t i o n w i t h d i s p l a c e m e n t o f t h e f l u o r o atom. In f a c t , when 6 - f l u o r o B P i s t r e a t e d w i t h t h e o n e - e l e c t r o n o x i d a n t M n ( 0 A c ) , t h e major p r o d u c t s o b t a i n e d are 6-acetoxyBP and a m i x t u r e o f 1,6- and 3 , 6 - d i a c e t o x y B P ( 1 5 ) , i n d i c a t i n g t h a t r e a c t i o n o c c u r s v i a an i n i t i a l a t t a c k o f a c e t a t e i o n at C-6 o f t h e 6 - f l u o r o B P r a d i c a l c a t i o n . On t h e o t h e r hand e l e c t r o p h i l i c s u b s t i t u t i o n o f 6 - f l u o r o B P w i t h bromine o r d e u t e r i u m i o n shows no d i s p l a c e m e n t o f f l u o r i n e at C - 6 , a l t h o u g h i n both c a s e s s u b s t i t u t i o n o c c u r s at C - l a n d / o r C - 3 . These r e s u l t s i n d i c a t e t h a t 3

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

11.

CAVALIERI AND ROGAN

297

One-Electron Oxidation

t h e o n l y p l a u s i b l e c h e m i s t r y i n t h e m e t a b o l i c f o r m a t i o n o f quinones from 6 - f l u o r o B P i s c o n s i s t e n t w i t h a n i n i t i a l o n e - e l e c t r o n o x i d a t i o n o f t h e compound t o form 6 - f l u o r o B P » . F i n a l l y , we have s t u d i e d t h e metabolism o f a s e r i e s o f PAH w i t h d e c r e a s i n g IP. In t h e s e m e t a b o l i c s t u d i e s w i t h A r o c l o r - i n d u c e d r a t l i v e r microsomes, t h e f o r m a t i o n o f quinones was measured i n t h e p r e sence o f NADPH o r cumene h y d r o p e r o x i d e as c o f a c t o r . As p r e s e n t e d i n T a b l e II, no quinones a r e o b t a i n e d w i t h NADPH f o r d i b e n z [ a , h ] a n t h r a c e n e and b e n z [ a ] a n t h r a c e n e , whereas w i t h cumene h y d r o p e r o x i d e a t r a c e amount o f b e n z [ a ] a n t h r a c e n e quinone i s o b served. For t h e PAH w i t h low IP, quinones a r e formed i n t h e p r e sence o f both c o f a c t o r s . The r e l a t i o n s h i p between IP and f o r m a t i o n o f quinones c o n s t i t u t e s f u r t h e r e v i d e n c e t h a t t h e s e m e t a b o l i t e s a r e o b t a i n e d by an i n i t i a l o n e - e l e c t r o n o x i d a t i o n o f t h e PAH w i t h f o r m a tion of i t s radical cation.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

+

Table

II.

M e t a b o l i c Formation o f Quinones Various I o n i z a t i o n P o t e n t i a l s

Ionization Potential

(eV)*

Compound

f o r PAH o f

Formation o f Quinone by A r o c l o r - i n d u c e d Rat L i v e r Microsomes w i t h Cumene Hydroperoxide NADPH

Dibenz[a,h]anthracene

7.57

-

Benz[a]anthracene

7.54

-

Benzo[a]pyrene

7.23

+

+

Dibenzo[a,i]pyrene

7.20

+

+

Dibenzo[a,h]pyrene

6.97

+

+

Anthanthrene

6.96

+

+

-

Determined from a b s o r p t i o n maximum o f t h e c h a r g e - t r a n s f e r complex o f each compound w i t h c h l o r a n i l , w i t h t h e e x c e p t i o n o f d i b e n z [ a . h ) ] a n t h r a c e n e . which was d e t e r m i n e d by p o l a r o graphic oxidation (24). +_ i n d i c a t e s

f o r m a t i o n o f a t r a c e amount o f q u i n o n e .

We propose t h a t t h e f i r s t s t e p i n t h e f o r m a t i o n o f q u i n o n e s , as shown i n Scheme 3 f o r BP, i n v o l v e s an e l e c t r o n t r a n s f e r from t h e hydrocarbon t o t h e a c t i v a t e d cytochrome P - 4 5 0 - i r o n - o x y g e n complex. The g e n e r a t e ^ n u c l e o p h i l i c oxygen atom o f t h i s complex would r e a c t at C-6 o f BP* i n which t h e p o s i t i v e c h a r g e i s a p p r e c i a b l y l o c a l i z e d . The 6-oxy-BP r a d i c a l formed would then d i s s o c i a t e t o l e a v e t h e i r o n o f cytochrome P-450 i n t h e normal f e r r i c s t a t e . Autoxidation of the 6-oxy-BP r a d i c a l i n which t h e s p i n d e n s i t y i s l o c a l i z e d m a i n l y on t h e o x y g e n , C - l , C-3 and C-12 U 9 , 2 0 ) would produce t h e t h r e e BP diones.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

298

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

CAVALIERI AND ROGAN

One-Electron Oxidation

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

11.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

299

300

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

Binding

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS of

PAH t o

DNA i n v i t r o and i n

vivo

While most r e s e a r c h on t h e e n z y m a t i c a c t i v a t i o n o f c h e m i c a l c a r c i n o gens has f o c u s e d on monooxygenation by cytochrome P-450, i t has become i n c r e a s i n g l y c l e a r t h a t a c t i v a t i o n by c e l l u l a r p e r o x i d a s e s , i n c l u d i n g t h e p r o s t a g l a n d i n H s y n t h a s e c o m p l e x , p l a y s an important r o l e i n t h e a c t i v a t i o n o f many c a r c i n o g e n s ( 2 5 ) . The model h o r s e r a d i s h peroxidase/HgOp system has been found t o m e t a b o l i z e N-hyd r o x y - 2 - a c e t y l a m i n o f l u o r e n e ( 2 6 , 2 7 ) , d i e t h y l s t i l b e s t r o l ( 2 8 ) , phenol ( 2 9 ) , a m i n o p y r i n e ( 3 0 ) , benzicTTne and d e r i v a t i v e s (3U, ^ 2 7 7 t e t r a methyl h y d r a z i n e (33T"~and BP (34) by o n e - e l e c t r o n o x i d a t i o n . Mamm a l i a n p e r o x i d a s e s a l s o f o l l o w t h i s mechanism: f o r example, mouse u t e r i n e p e r o x i d a s e and r a t bone marrow p e r o x i d a s e w i t h d i e t h y l s t i l b e s t r o l (28) and phenol ( 2 9 ) , r e s p e c t i v e l y . Furthermore p r o s t a g l a n d i n H s y n t h a s e has been proposed t o a c t i v a t e b e n z i d i n e i n k i d n e y c a r c i n o g e n e s i s (35, 3 6 ) , N - h y d r o x y - 2 - a c e t y l a m i n o f l u o r e n e i n mammary c e l l s ( 3 7 ) , t e t r a m e t h y l h y d r a z i n e (38) and d i e t h y l s t i l b e s t r o l (39), a p p a r e n t l y by o n e - e l e c t r o n o x i d a t i o n . Both h o r s e r a d i s h p e r o x i d a s e and p r o s t a g l a n d i n H s y n t h a s e e f f i c i e n t l y c a t a l y z e t h e b i n d i n g o f BP t o DNA in_ v i t r o , y i e l d i n g 89 +^ 5 and 310 + 64 y m o l e BP bound/mole DNA-P, r e s p e c t i v e l y . Horseradish p e r o x i d a s e has a l r e a d y been seen t o b i n d o t h e r PAH w i t h r e l a t i v e l y low IP t o DNA ( 1 6 ) . For both BP (34) and 6-methyl BP ( 4 0 ) , we have o b t a i n e d c l e a r e v i d e n c e c o n f i r m i n g o n e - e l e c t r o n o x i d a t i o n as t h e mechanism o f a c t i v a t i o n . In t h e c a s e o f 6-methylBP we have i d e n t i f i e d a DNA adduct i n which t h e 6-methyl group i s c o v a l e n t l y bound t o the 2-amino group o f deoxyguanosine ( 4 0 ) . T h i s DNA adduct i s a l s o p r e s e n t i n mouse s k i n t r e a t e d w i t h racTTolabeled 6-methyl BP, p r o v i d i n g t h e f i r s t e v i d e n c e f o r a c t i v a t i o n o f a PAH i n a t a r g e t t i s s u e by o n e - e l e c t r o n o x i d a t i o n ( 4 0 ) . We have begun t o examine BP-DNA adducts formed i n mouse s k i n u s i n g h i g h p r e s s u r e l i q u i d c h r o m a t o g r a phy a f t e r enzymic d i g e s t i o n o f t h e p u r i f i e d DNA t o m o n o n u c l e o s i d e s . In a d d i t i o n t o BP d i o l e p o x i d e a d d u c t ( s ) , we o b s e r v e an adduct p r o f i l e which i s q u a l i t i a t i v e l y s i m i l a r t o t h e adduct p r o f i l e s o b t a i n e d from DNA w i t h BP bound by i n c u b a t i o n w i t h h o r s e r a d i s h p e r o x i d a s e / H p 0 and from BP r a d i c a l c a t i o n bound t o d e o x y g u a n o s i n e . We are c u r r e n t l y i d e n t i f y i n g t h e s t r u c t u r e o f t h e common adducts o b t a i n e d on t h e ^ k i n , w i t h h o r s e r a d i s h p e r o x i d a s e a c t i v a t i o n and by r e a c t i o n o f BP w i t h d e o x y g u a n o s i n e . I d e n t i f i c a t i o n o f DNA adducts formed by o n e - e l e c t r o n o x i d a t i o n can p r o v i d e e v i d e n c e t h a t t h i s mechanism o f a c t i v a t i o n i s o p e r a t i v e i n t a r g e t t i s s u e s , a l t h o u g h t h i s does not prove t h a t i t i s r e s p o n s i b l e f o r i n i t i a t i n g t h e tumor process. 2

Carcinogenicity

Studies

i n Two T a r g e t

Organs

The c a r c i n o g e n i c i t y o f a s e r i e s o f PAH i n t h e mammary g l a n d has been examined i n 5 0 - d a y - o l d f e m a l e Sprague-Dawley r a t s u s i n g d i r e c t a p p l i c a t i o n o f t h e compound t o t h e mammary t i s s u e ( 1 0 , 17, 1 8 ) . The r e s u l t s o f t h e s e e x p e r i m e n t s , p r e s e n t e d i n T a b l e III, a r e compared t o t h e c a r c i n o g e n i c i t y r e s u l t s i n mouse s k i n from r e p e a t e d a p p l i c a t i o n o b t a i n e d i n our l a b o r a t o r y and o t h e r s . PAH were s e l e c t e d b e cause t h e y were o r were not e x p e c t e d t o be a c t i v a t e d by o n e - e l e c t r o n o x i d a t i o n , based on t h e h y p o t h e s i s t h a t compounds w i t h r e l a t i v e l y h i g h IP cannot be a c t i v a t e d by t h i s mechanism. F u r t h e r m o r e , some

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

11.

Table

III.

Comparative

C a r c i n o g e n i c i t y o f PAH i n Mouse Skin and Rat Mammary Gland

Ionization Potential (eV)

Compound

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Cyclopenta[cd]pyrene Benzo[a]pyrene dihydrodiol

301

One-Electron Oxidation

CAVALIER I AND ROGAN

7,8-

3

Carcinogenicity in: Rat Mammary Gland Mouse Skin

++

-

+ + ++

-

+ ++

Di b e n z [ a , h ] a n t h r a c e n e

7.57

+ ++

Benz[a]anthracene

7.54

+

-

7-Methylbenz[a]anthracene

7.37

+ ++

+

Benzo[a]pyrene

7.23

+ + ++

+ ++

7,12-Dimethylbenz[a]anthracene

7.22

+ + + ++

+ + ++

10-F1uoro-3-methylcholanthrene

7.17

N.T.

++

1,3-Dimethylcholanthrene

7.15

8-F1uoro-3-methylcholanthrene

7.14

N.T.

++

2,3-Dimethylcholanthrene

7.13

N.T.

++

3-Methylcholanthrene

7.12

+ + ++

+ + ++

6-Methylbenzo[a]pyrene

7.08

+ ++

+

5-Methylchrysene

ca.

7.7

C

-

++

Determined from a b s o r p t i o n maximum o f t h e c h a r g e - t r a n s f e r complex o f each compound w i t h c h l o r a n i l , with t h e e x c e p t i o n o f dibenzC&J}.]a n t h r a c e n e determined by p o l a r o g r a p h i c o x i d a t i o n ( 2 4 ) . Extremely a c t i v e , + + + + + ; v e r y a c t i v e , + + + +; a c t i v e , + + +, m o d e r a t e l y a c t i v e , + +; weakly a c t i v e , +; v e r y weakly a c t i v e , HH; inactive, C

N.T.

= not t e s t e d .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PAH were chosen i n which a c t i v a t i o n by monooxygenation o r o n e - e l e c t r o n o x i d a t i o n was b l o c k e d . Compounds which have low IP and s u f f i c i e n t c h a r g e l o c a l i z a t i o n i n t h e r a d i c a l c a t i o n , namely 7 - m e t h y l b e n z [ a ] a n t h r a c e n e , BP, 7,12-dimethylbenz[a]anthracene, 10-f1uoro-3-methylcholanthrene, 8-fluoro-3-methylcholanthrene, 2,3-dimethylcholanthrene, 3-methylc h o l a n t h r e n e , and 6-methylBP, a r e g e n e r a l l y c a r c i n o g e n i c , both i n mouse s k i n and r a t mammary g l a n d . However, 1 , 3 - d i m e t h y l c h o l a n t h r e n e , which has a low IP, i s i n a c t i v e i n r a t mammary g l a n d and a c t i v e i n mouse s k i n . T h i s i s presumably due t o s t e r i c h i n d r a n c e at C - l , the p o s i t i o n of n u c l e o p h i l i c s u b s t i t u t i o n in the 3-methylcholanthrene r a d i c a l c a t i o n . Its c a r c i n o g e n i c a c t i v i t y i n mouse s k i n can be a t t r i b u t e d t o a c t i v a t i o n by monooxygenation. In c o n t r a s t 2 , 3 - d i m e t h y l c h o l a n t h r e n e , i n which t h e methyl s u b s t i t u e n t at C-2 does not p r e v e n t n u c l e o p h i l i c s u b s t i t u t i o n at C - l i n t h e r a d i c a l cation, is carcinogenic. PAH w i t h r e l a t i v e l y h i g h IP, such as d i b e n z [ a , h ] a n t h r a c e n e and 5 - m e t h y l c h r y s e n e , are not a c t i v e when d i r e c t l y a p p l i e d t o t h e mammary g l a n d . The c a r c i n o g e n i c i t y o f 5 - m e t h y l c h r y s e n e i n mouse s k i n has been demonstrated t o o c c u r v i a a d i o l e p o x i d e mechanism ( 4 1 ) , and t h e p o t e n t a c t i v i t y o f d i b e n z [ a , h ] a n t h r a c e n e i s presumably induced by t h e same mechanism ( 5 ) . The i n a c t i v i t y o f t h e s e two s k i n c a r c i n o g e n s s u g g e s t s t h a t dTol e p o x i d e s are not formed i n t h e mammary g l a n d . No c a r c i n o g e n i c a c t i v i t y i s o b s e r v e d i n t h i s t a r g e t organ f o r t h e two mouse s k i n c a r c i n o g e n s BP 7 , 8 - d i h y d r o d i o l (5^) and c y c l o p e n t a [ c d ] p y r e n e ( 4 2 ) , both o f which r e q u i r e a s i m p l e e p o x i d a t i o n t o become a c t i v e . From t h e s e experiments we can draw t h r e e main c o n c l u s i o n s : 1) o x y g e n a t i o n o f PAH by cytochrome P-450 monooxygenase enzymes does not seem t o p l a y a r o l e i n e l i c i t i n g c a r c i n o g e n i c i t y i n r a t mammary g l a n d ; 2) t h e r e s u l t s i n t h e mammary e x p e r i m e n t s s u p p o r t t h e h y p o t h e s i s t h a t o n e - e l e c t r o n o x i d a t i o n might be t h e predominant mechanism o f a c t i v a t i o n i n t h i s t a r g e t o r g a n ; and 3) m u l t i p l e mechanisms o f a c t i v a t i o n appear t o o c c u r i n mouse s k i n , a l t h o u g h t h e s e e x p e r i ments do not p r o v i d e d i r e c t e v i d e n c e on t h i s p o i n t . Conclusions Based on p r e s e n t knowledge t h e c a r c i n o g e n i c i t y o f PAH i s best u n d e r s t o o d i n terms o f two major mechanisms o f a c t i v a t i o n : one-electron o x i d a t i o n and monooxygenation. The b a y - r e g i o n d i o l e p o x i d e s can be c o n s i d e r e d major u l t i m a t e c a r c i n o g e n i c i n t e r m e d i a t e s when a c t i v a t i o n o c c u r s by monooxygenation (2-5.) • O n e - e l e c t r o n o x i d a t i o n o f PAH w i t h f o r m a t i o n o f r a d i c a l c a t i o n s can o n l y p l a y a r o l e i n b i o l o g i c a l systems when PAH have an IP below c a . 7.35 eV ( T a b l e I) (6,_7). Thus c a r c i n o g e n i c i t y o f compounds w i t h r e l a t i v e l y h i g h IP (TabTe I ) , such as b e n z o [ c ] p h e n a n t h r e n e , c h r y s e n e , 5 - m e t h y l c h r y s e n e and d i b e n z [ a , h ] a n t h r a c e n e , can be a t t r i b u t e d t o monooxygenation w i t h f o r m a t i o n o f bay-region diol epoxides. Most o f t h e p o t e n t PAH, however, have IP below c a . 7.35 eV. T h i s l i s t i n c l u d e s BP, 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e , 3 - m e t h y l c h o l a n t h r e n e , d i b e n z o [ a , i ] p y r e n e and d i b e n z o [ a , h j pyrene. These PAH can be a c t i v a t e d by both o n e - e l e c t r o n o x i d a t i o n and monooxygenation, depending on t h e enzymes p r e s e n t i n t h e t a r g e t o r g a n . The u b i q u i t y o f p e r o x i d a s e s , i n p a r t i c u l a r p r o s t a g l a n d i n H s y n t h a s e , i n e x t r a h e p a t i c t i s s u e s which are r e s p o n s i v e t o PAH l e a d s us t o s u g g e s t t h a t o n e - e l e c t r o n o x i d a t i o n may be a major pathway o f

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

11. CAVALIERI AND ROGAN

One-Electron Oxidation

303

a c t i v a t i o n i n most t a r g e t t i s s u e s . Combined s t u d i e s o f enzymology, c a r c i n o g e n i c i t y and b i n d i n g t o c e l l u l a r macromolecules s h o u l d p r o vide the information necessary to determine the r o l e of the d i f f e r e n t mechanisms o f PAH a c t i v a t i o n r e s p o n s i b l e f o r i n i t i a t i o n o f the cancer process in a c e r t a i n target organ. Acknowl edgments We a p p r e c i a t e t h e v a l u a b l e c o l l a b o r a t i o n o f D r s . C. Warner, P. Cremonesi and A. Wong and o f Mr. S. T i b b e l s . We a r e a l s o g r a t e f u l t o Ms. M. Susman f o r e x c e l l e n t e d i t o r i a l a s s i s t a n c e . F i n a l l y we thank t h e N a t i o n a l I n s t i t u t e s o f H e a l t h f o r s u p p o r t i n g t h i s r e s e a r c h t h r o u g h g r a n t s R01 CA25176, R01 CA32376 and R01 ES02145.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

Literature Cited 1. Miller, J.A. Cancer Res. 1970, 30, 559-76. 2. Miller, E.C.; Miller J.A. Cancer, 1981, 47, 2327-45. 3. Nordqvist, M.; Thakker, D.R.; Yagi, H.; Lehr, R.E.; Wood, A.W.; Levin, W.; Conney, A.H.; Jerina, D.M. In "Molecular Basis of Environmental Toxicity"; Bhatnager, R.S., Ed.; Ann Arbor Science Publishers: Ann Arbor, Mich., 1979; pp. 329-357. 4. Sims, P.; Grover, P.L. In "Polycyclic Hydrocarbons and Cancer"; Gelboin, H.V.; Ts'o, P.O.P, Ed.; Academic: New York, 1978; Vol. 1, pp. 117-81. 5. Conney, A.H. Cancer Res. 1982, 42, 4875-917. 6. Cavalieri, E.L.; Rogan, E.G. In "Free Radicals in Biology"; Pryor, W.A., Ed.; Academic: New York, 1984; Vol. VI, pp. 323-369. 7. Cavalieri, E.; Rogan, E. In "Chemical Induction of Cancer"; by Woo, Y.-T; Lai, D.Y., Arcos, J.C.; Argus, M.F.; ; Academic: New York, 1984; in press. 8. Cavalieri, E.; Roth, R. J. Org. Chem., 1976, 41, 2679-84. 9. Cavalieri, E.; Roth, R.; Rogan, E.G. In "Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism and Carcinogenesis"; Freudenthal, R.I.; Jones, P.W., Eds.; Raven: New York, 1976; Vol. 1, pp. 181-190. 10. Cavalieri, E.; Rogan, E. In "Polynuclear Aromatic Hydrocarbons: Formation, Metabolism and Measurement"; Cooke, M.; Dennis, A.J., Eds.; Battelle Press, Columbus, Ohio, 1983; pp. 1-26. 11. Rogan, E.G.; Roth, R.; Cavalieri, E. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjørseth, A.; Dennis, A.J., Eds.; Battelle Press: Columbus, Ohio, 1980; pp. 259-265. 12. Cavalieri, E.; Rogan, E.; Warner, C.; Bobst, A. In "Polynuclear Aromatic Hydrocarbons: Mechanisms, Methods and Metabolism"; Cooke, M.; Dennis, A.J., Eds.; Battelle Press, Columbus, Ohio, in press. 13. Sato, Y.; Kinoshita, M.; Sano, M.; Akamatu, H. Bull. Chem. Soc. Jap., 1969, 42, 3051-5. 14. Ristagno, C.V.; Shine, H.J. J. Org. Chem., 1971, 36, 4050-5. 15. Cavalieri, E.; Cremonesi, P.; Warner, C.; Tibbels, S.; Rogan, E. Proc. Am. Assoc. Cancer Res., 1984, 25, 124. 16. Cavalieri, E.L.; Rogan, E.G.; Roth, R.W.; Saugier, R.K.; Hakam, A. Chem.-Biol. Interact. 1983, 47, 87-109.

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17. Cavalieri, E.; Sinha, D.; Rogan, E. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjørseth, A.; Dennis, A.J., Eds.; Battelle Press: Columbus, Ohio, 1980; pp. 215-231. 18. Cavalieri, E.; Rogan, E. In "Polynuclear Aromatic Hydrocarbons: Physical and Biological Chemistry"; Cooke, M.; Dennis, A.J.; Fisher, G.L., Eds.; Battelle Press: Columbus, Ohio, 1982; pp. 145-155. 19. Nagata, C.; Kodama, M.; Ioki, Y.; Kimura, T. In "Free Radicals and Cancer"; Floyd, R.A., Ed.; Marcel Dekker: New York, 1982; pp. 1-62. 20. Lorentzen, R.J.: Caspary, W.J.; Lesko, S.A.; Ts'o, P.O.P. Biochemistry, 1975, 14, 3970-7. 21. Marnett, L.J.; Reed, G.A. Biochemistry, 1979, 18, 2923-9. 22. Renneberg, R.; Capdevila, J.; Chacos, H.; Estabrook, R.W.; Prough, R.A. Biochem. Pharmacol., 1981, 30, 843-8. 23. Buhler, D.R.; Unlü, F.; Thakker, D.R.; Slaga, T.J.; Conney, A.H.; Wood, A.W.; Chang, R.L.; Levin, W.; Jerina, D.M. Cancer Res., 1983, 43, 1541-9. 24. Pish, E.S.; Yang, N.C. J. Am. Chem.Soc.,1963, 85, 2124-30. 25. Eling, T.; Boyd, J.; Reed, G.; Mason, R.; Sivarajah, K. Drug Metab. Rev., 1983, 14, 1023-53. 26. Bartsch, H.; Hecker, E. Biochim. Biophys. Acta, 1971, 237, 567-78. 27. Floyd, R.A.; Soong, L.M.; Culver, P.L. Cancer Res., 1976, 36, 1510-9. 28. Metzler, M.; McLachlan, J.A. Biochem. Biophys. Res. Comm., 1978, 85, 874-88. 29. Sawahata, T.; Neal, R.A. Biochem. Biophys. Res. Comm., 1982, 109, 988-94. 30. Griffith, B.W.; Ting, P.L. Biochemistry, 1978, 17, 2206-11. 31. Josephy, P.D.; Eling, T.; Mason, R.P. J. Biol. Chem., 1982, 257, 3669-75. 32. Josephy, P.D.; Mason, R.P.; Eling, T. Carcinogenesis, 1982, 3, 1227-30. 33. Kalyanaraman, B.; Mason, R.P. Biochem. Biophys. Res. Comm., 1982, 105, 217-24. 34. Rogan, E.G.; Katomski, P.A.; Roth, R.W.; Cavalieri, E.L. J. Biol. Chem., 1979, 254, 7055-9. 35. Mattammal, M.B.; Zenser, T.V.; Davis, B.B. Cancer Res., 1981, 41, 4961-6. 36. Josephy, P.D.; Eling, T.E.; Mason, R.P. J. Biol. Chem., 1983, 258, 5561-9. 37. Wong, P.K.; Hampton, M.J.; Floyd, R.A. In "Prostaglandins and Cancer: First International Conference", Powles, T.J.; Bockman, R.S.; Honn, K.V.; Ramwell, P., Eds.; Alan R. Liss: New York, 1982; pp. 167-179. 38. Kalyanaraman, B.; Sivarajah, K.; Eling, T.E.; Mason, R.P. Carcinogenesis, 1983, 4, 1341-3. 39. Degen, G.H.; Eling, T.E.; McLachlan, J.A. Cancer Res., 1982, 42, 919-23. 40. Rogan, E.G.; Hakam, A.; Cavalieri, E.L. Chem.-Biol. Interact., 1983, 47, 111-22.

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305

41. Hecht, S.S.; Mazzarese, R.; Amin, S.; LaVoie, E.; Hoffmann, D. In "Polynuclear Aromatic Hydrocarbons. Third International Symposium on Chemistry and Biology—Carcinogenesis and Mutagenesis"; Ann Arbor Science Publishers: Ann Arbor, Mich., 1979; pp. 733-52. 42. Cavalieri, E.; Rogan, E.; Toth, B.; Munhall, A. Carcinogenesis, 1981, 2, 277-81.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

RECEIVED March 5, 1985

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

12 Hydroperoxide-Dependent Oxygenation of Polycyclic Aromatic Hydrocarbons and Their Metabolites LAWRENCE J. MARNETT

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

Department of Chemistry, Wayne State University, Detroit, MI 48202

Fatty acid hydroperoxides in the presence of heme complexes and heme proteins oxidize benzo(a)pyrene and 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to quinones and diol epoxides, respectively. The oxidizing agent is a peroxyl radical derived from the fatty acid hydroperoxide but not a higher oxidation state of a mammalian peroxidase. The stereochemistry of (±)-BP-dihydrodiol epoxidation is distinct from that catalyzed by mixed-function oxidases, which provides a convenient method for discriminating the contributions of the two systems to BP-7,8-dihydrodiol metabolism in cell homogenates, cell or organ culture. Using this method, epoxidation of BP-7,8-dihydrodiol has been detected during prostaglandin biosynthesis, l i p i d peroxidation, and xenobiotic oxygenation. Fatty acid hydroperoxide-dependent oxidation constitutes a novel pathway for metabolic activation of polycyclic hydrocarbons and other carcinogens which has widespread potential in vivo significance. Oxidation is intimately linked to the activation of polycyclic aromatic hydrocarbons (PAH) to carcinogens (1-3?. Oxidation of PAH in animals and man is enzyme-catalyzed and is a response to the introduction of foreign compounds into the cellular environment. The most intensively studied enzyme of PAH oxidation is cytochrome P-450, which is a mixed-function oxidase that receives its electrons from NADPH via a one or two component electron transport chain (_1) . Some forms of this enzyme play a major role in systemic metabolism of PAH (4). However, there are numerous examples of carcinogens that require metabolic activation, including PAH, that induce cancer in tissues with low mixed-function oxidase activity (_5) . In order to comprehensively evaluate the metabolic activation of PAH, one must consider a l l cellular pathways for their oxidative activation. Peroxidases have been implicated in carcinogenesis by PAH, aromatic amines, and estrogens inter alia (6-9). These enzymes catalyze the reduction of hydrogen peroxide and organic 0097-6156/85/0283-0307S06.00/0 © 1985 American Chemical Society

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

h y d r o p e r o x i d e s and u s e a wide v a r i e t y o f compounds a s r e d u c i n g a g e n t s ( E q u a t i o n 1 ) . Important o b s e r v a t i o n s on t h e o x i d a t i o n o f PAH

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

ROOH + DH

2

+ ROH + D + H 0 2

(1)

by p e r o x i d a s e s have been made by C a v a l i e r i and Rogan and a r e d e s c r i b e d i n t h e i r c h a p t e r i n t h i s volume and elsewhere (6^) . T e n y e a r s ago we r e p o r t e d t h a t b e n z o ( a ) p y r e n e (BP) i s o x i d i z e d d u r i n g the o x y g e n a t i o n o f a r a c h i d o n i c a c i d by p r o s t a g l a n d i n H (PGH) s y n t h a s e (10). PGH s y n t h a s e i s a w i d e l y d i s t r i b u t e d enzyme o f p o l y u n s a t u r a t e d f a t t y a c i d m e t a b o l i s m t h a t p o s s e s s e s a p e r o x i d a s e a c t i v i t y and g e n e r a t e s h y d r o p e r o x y e n d o p e r o x i d e s as i n i t i a l p r o d u c t s o f f a t t y a c i d o x y g e n a t i o n (11-13). Its principal function i s to biosynthesize PGH2i t h e e n d o p e r o x i d e i n t e r m e d i a t e o f p r o s t a g l a n d i n and thromboxane b i o s y n t h e s i s ( F i g u r e 1) (11,14). The o t h e r enzyme o f u n s a t u r a t e d f a t t y a c i d o x y g e n a t i o n i s l i p o x y g e n a s e (15^ . I t oxygenates u n s a t u r a t e d f a t t y a c i d s t o hydroperoxides t h a t a r e reduced t o a l c o h o l s o r c o n v e r t e d t o l e u k o t r i e n e s ( F i g u r e 1) ( 1 6 ) . These two enzymes, PGH s y n t h a s e and l i p o x y g e n a s e , r e p r e s e n t t h e p r i n c i p a l s o u r c e s o f o r g a n i c h y d r o p e r o x i d e s i n mammalian t i s s u e ( 1 7 ) . Our i n v e s t i g a t i o n s o f t h e o x i d a t i o n o f PAH by t h e h y d r o p e r o x i d e p r o d u c t s o f PGH s y n t h a s e and l i p o x y g e n a s e c a t a l y s i s i n d i c a t e t h a t t h i s pathway c a n g e n e r a t e u l t i m a t e c a r c i n o g e n i c forms o f PAH and t h a t t h e mechanisms o f o x i d a t i o n a r e d i s t i n c t from t h o s e o f c l a s s i c p e r o x i d a s e - c a t a l y z e d o x i d a tion. F a t t y a c i d hydroperoxide-dependent o x i d a t i o n , t h e r e f o r e , r e p r e s e n t s a n o v e l pathway f o r t h e m e t a b o l i c a c t i v a t i o n o f PAH. Benzo(a)pyrene

Oxidation

I n c u b a t i o n o f BP w i t h a r a c h i d o n i c a c i d and ram s e m i n a l v e s i c l e m i c r o somes, a r i c h s o u r c e o f PGH s y n t h a s e , p r o d u c e s 1,6-, 3,6-, and 6,12q u i n o n e s as t h e e x c l u s i v e p r o d u c t s o f o x i d a t i o n ( F i g u r e 2) ( 1 8 ) . These a r e t h e same quinones t h a t a r e formed when 6-hydroxy-BP i s o x i d i z e d by a i r o r microsomes ( 1 9 ) . However, t h e r e i s no d e f i n i t i v e e v i d e n c e t h a t 6-hydroxy-BP i s an i n t e r m e d i a t e i n t h e i r f o r m a t i o n by PGH s y n t h a s e . Among a l l o f t h e s t a b l e m e t a b o l i t e s o f BP, t h e q u i n o n e s a r e d i s t i n c t i v e because, u n l i k e p h e n o l s and d i h y d r o d i o l s , t h e y a r e n o t d e r i v e d from arene o x i d e s . Thus, arene o x i d e s do n o t appear t o be p r o d u c t s o f BP o x i d a t i o n by PGH s y n t h a s e (19,20). P o t e n t i n h i b i t i o n o f PGH synthase-dependent BP o x i d a t i o n by a n t i o x i dants suggests t h a t t h e quinones are p r o d u c t s o f f r e e r a d i c a l r e a c t i o n s (18) . A d d i t i o n o f RNA o r DNA p r i o r t o o x i d a t i o n o f BP by PGH s y n t h a s e r e s u l t s i n s u b s t a n t i a l n u c l e i c a c i d b i n d i n g (17,21). Addition of RNA f i v e m i n u t e s a f t e r i n i t i a t i o n o f o x i d a t i o n l e a d s t o no c o v a l e n t b i n d i n g ( 1 7 ) . T h i s i m p l i e s t h a t t h e quinones do n o t b i n d t o n u c l e i c a c i d b u t r a t h e r a s h o r t - l i v e d i n t e r m e d i a t e i n t h e i r format i o n does. A r a c h i d o n i c a c i d o x y g e n a t i o n i n ram s e m i n a l v e s i c l e microsomes i s complete w i t h i n two min, which s u g g e s t s t h a t t h e r e a c t i v e i n t e r m e d i a t e i s g e n e r a t e d c o n c u r r e n t l y w i t h PGH2« The s t r u c t u r e s o f t h e n u c l e i c a c i d a d d u c t s have n o t been e l u c i d a t e d so the i d e n t i t y o f t h e r e a c t i v e i n t e r m e d i a t e i s unknown. Despite the high l e v e l o f nucleic a c i d binding that i s evident, no mutagenic s p e c i e s c a n be d e t e c t e d when BP i s i n c u b a t e d w i t h ram

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

12.

MARNETT

Hydroperoxide-Dependent

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Oxygenation

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Hydroxy Acids

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

PGH2

Malondialdthydo

Prostacyclin F i g u r e 1.

Thromtoxano

Pathways o f o x y g e n a t i o n o f u n s a t u r a t e d f a t t y a c i d s i n animal t i s s u e .

6,12F i g u r e 2.

P r o d u c t s o f BP o x i d a t i o n b y a r a c h i d o n i c a c i d s e m i n a l v e s i c l e microsomes.

and ram

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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310

POLYCYCLIC HYDROCARBONS AND

CARCINOGENESIS

s e m i n a l v e s i c l e microsomes and a r a c h i d o n i c a c i d i n t h e p r e s e n c e o f Salmonella typhimurium strains. The p r e s e n c e o f t h e Salmonella s t r a i n s and n u t r i e n t b r o t h i n t h e i n c u b a t i o n s does n o t i n h i b i t quinone f o r m a t i o n . F u r t h e r m o r e , one o f t h e s t r a i n s employed, TA98, has been r e p o r t e d t o d e t e c t 6-hydroxy-BP as a mutagen (23_) . I t may be t h a t t h e i n t e r m e d i a t e r e s p o n s i b l e f o r n u c l e i c a c i d b i n d i n g i s t o o u n s t a b l e t o s u r v i v e t r a n s i t a c r o s s t h e b a c t e r i a l c e l l w a l l and membrane. A l t e r n a t i v e l y , t h e i n t e r m e d i a t e may b i n d t o DNA b u t n o t induce mutation. T h i s i s u n l i k e l y because t h e g e n e r a t i o n o f b u l k y a d d u c t s on a DNA m o l e c u l e u s u a l l y r e s u l t s i n m u t a t i o n . Although some adducts o f p o l y c y c l i c h y d r o c a r b o n s t o DNA appear t o be more mutagenic than o t h e r s , t h e d i f f e r e n c e s a r e n o t g r e a t e r than an o r d e r o f magnitude (24,25). T h u s , i t i s u n l i k e l y t h a t i f adducts a r e formed they a r e n o t m u t a g e n i c . Two o t h e r p o l y c y c l i c h y d r o c a r b o n s , 3 - m e t h y l c h o l a n t h r e n e and 7,12-dimethylbenzanthracene are oxidized during arachidonate metabolism (21,26). Hydroxymethyl compounds t h a t do n o t a r i s e from arene o x i d e s appear t o be t h e p r o d u c t s formed from 7 , 1 2 - d i m e t h y l benzanthracene . 7,8-Dihydroxy-7,8-Dihydrobenzo(a)pyrene

Oxidation

In c o n t r a s t t o t h e r e s u l t s w i t h BP, i n c u b a t i o n o f B P - 7 , 8 - d i h y d r o d i o l w i t h ram s e m i n a l v e s i c l e microsomes and a r a c h i d o n a t e g e n e r a t e s a s p e c i e s t h a t i s s t r o n g l y mutagenic t o Salmonella s t r a i n s TA98 and TA100 ( F i g u r e 3) ( 2 2 ) . F o r m a t i o n o f t h e mutagen i s i n h i b i t e d by i n d o m e t h a c i n i n d i c a t i n g t h e i n v o l v e m e n t o f PGH s y n t h a s e . Similar e x p e r i m e n t s w i t h B P - 4 , 5 - d i h y d r o d i o l and B P - 9 , 1 0 - d i h y d r o d i o l do n o t g e n e r a t e p o t e n t mutagens, which s u g g e s t s t h a t a c t i v a t i o n i s s p e c i f i c f o r t h e p r e c u r s o r o f t h e b a y - r e g i o n d i o l e p o x i d e (22). The o b v i o u s i n t e r p r e t a t i o n o f t h e s e e x p e r i m e n t s i s t h a t PGH s y n t h a s e c a t a l y z e s the e p o x i d a t i o n o f B P - 7 , 8 - d i h y d r o d i o l t o the u l t i m a t e c a r c i n o g e n BP-diol epoxide. To c o n f i r m t h i s we i d e n t i f i e d t h e p r o d u c t s o f BP7 , 8 - d i h y d r o d i o l o x i d a t i o n (27,28). T h e o r e t i c a l l y , t h e epoxide oxygen c a n be i n t r o d u c e d from e i t h e r s i d e o f t h e m o l e c u l e g i v i n g r i s e to syn- o r a n t i - d i o l epoxides. Each epoxide h y d r o l y z e s r a p i d l y t o a m i x t u r e o f c i s and t r a n s t e t r a h y d r o t e t r a o l s ( F i g u r e 4 ) . When i n c u b a t i o n s o f B P - 7 , 8 - d i h y d r o d i o l and PGH s y n t h a s e a r e a l l o w e d t o p r o c e e d f o r 15 min, two p r o d u c t s a r e o b t a i n e d t h a t we i d e n t i f i e d as t h e c i s and t r a n s t e t r a o l s d e r i v e d from t h e a n t i - d i o l e p o x i d e (27,28,29). H y d r o l y s i s p r o d u c t s o f t h e s y n - d i o l e p o x i d e were n o t d e t e c t e d . When i n c u b a t i o n s were t e r m i n a t e d a f t e r 3 min, a new p r o d u c t was d e t e c t e d t h a t we i d e n t i f i e d as a m e t h y l e t h e r t h a t i s formed by m e t h a n o l y s i s o f t h e a n t i - d i o l e p o x i d e ( E q u a t i o n 2) ( 2 9 ) , T h i s r e a c t i o n can o n l y have o c c u r r e d a f t e r t e r m i n a t i o n o f t h e r e a c t i o n because t h e r e was no methanol i n t h e i n c u b a t i o n m i x t u r e . A d d i t i o n a l experiments confirmed t h a t methanolysis occurs d u r i n g chromatography ( r e v e r s e p h a s e , methanol-water g r a d i e n t s ) . The d e t e c t i o n o f t h e m e t h y l e t h e r i s i m p o r t a n t because i t c o n f i r m s t h a t a d i o l e p o x i d e i s g e n e r a t e d , s u r v i v e s s o l v e n t e x t r a c t i o n , and t h e n undergoes s o l v o l y s i s on t h e HPLC column. T h i s p r o v i d e s d i r e c t e v i d e n c e f o r t h e f o r m a t i o n o f t h e a n t i - d i o l e p o x i d e as a p r o d u c t o f PGH synthase-dependent c o o x i d a t i o n o f B P - 7 , 8 - d i h y d r o d i o l . The c o r r e l a t i o n o f the rate o f BP-7,8-dihydrodiol o x i d a t i o n , a n t i - d i o l e p o x i d e f o r m a t i o n , and mutagen g e n e r a t i o n a r e shown i n F i g u r e 5 (30).

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

12.

Hydroperoxide-Dependent

MARNETT

311

Oxygenation

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

600-

1BP7,8-DI0U /tM

F i g u r e 3.

F i g u r e 4.

Induction of mutation i n typhimurium TA98 by BP-7,8d i h y d r o d i o l , a r a c h i d o n i c a c i d , and ram s e m i n a l v e s i c l e microsomes. C o n c e n t r a t i o n dependence on B P - 7 , 8 - d i h y d r o ­ diol. (Reproduced w i t h p e r m i s s i o n from Ref. 22. C o p y r i g h t 1978 Academic.)

D i o l epoxide products o f BP-7,8-dihydrodiol t h e i r hydrolysis products.

o x i d a t i o n and

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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312

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

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Mutagenicity

10

15

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Time (min) Figure 5 .

Comparison o f the time course o f PGH2 b i o s y n t h e s i s , BP7 , 8 - d i h y d r o d i o l metabolism, and g e n e r a t i o n o f a mutagen from B P - 7 , 8 - d i h y d r o d i o l by RSVM. (Reproduced w i t h p e r m i s s i o n from Ref. 30. C o p y r i g h t 1982 M a r c e l Dekker.)

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

12.

MARNETT

Hydroperoxide-Dependent

313

Oxygenation

Further support f o r e p o x i d a t i o n o f BP-7,8-dihydrodiol t o the a n t i - d i o l e p o x i d e i s t h e i d e n t i f i c a t i o n o f RNA and DNA a d d u c t s formed as a r e s u l t o f i n c u b a t i o n o f B P - 7 , 8 - d i h y d r o d i o l , PGH s y n t h a s e , and p o l y g u a n y l i c a c i d o r DNA (30,31). Following digestion of the nucleic a c i d , t h e major g u a n o s i n e and deoxyguanosine a d d u c t s were i d e n t i f i e d as a r i s i n g by a d d i t i o n o f t h e e x o c y c l i c amino group o f g u a n o s i n e t o the b e n z y l i c c a r b o n o f t h e a n t i - d i o l e p o x i d e ( T a b l e I) (.31) . These experiments a l s o d e f i n e d the s t e r e o c h e m i s t r y o f e p o x i d a t i o n . Both enantiomers o f B P - 7 , 8 - d i h y d r o d i o l are e p o x i d i z e d a t equal r a t e s t o e n a n t i o m e r s o f t h e a n t i - d i o l e p o x i d e . The d i r e c t i o n o f oxygen i n t r o d u c t i o n i s from t h e same s i d e o f t h e m o l e c u l e as t h e h y d r o x y l group a t carbon-8 o f B P - 7 , 8 - d i h y d r o d i o l .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

Table I .

R e l a t i v e Y i e l d s o f D i a s t e r e o m e r i c A d d u c t s From A n t i - d i o l E p o x i d e P l u s P o l y g u a n y l i c A c i d Compared t o A d d u c t s Genera t e d D u r i n g M e t a b o l i s m o f B P - 7 , 8 - d i h y d r o d i o l by Ram S e m i n a l V e s i c l e s i n the Presence o f A r a c h i d o n i c A c i d

Incubation 1 4

% Radioactivity as ( - ) - c i s and (-)-trans diastereomers

% Radioactivity as ( + ) - c i s diastereomer

% Radioactivity as ( + ) - t r a n s diastereomer

[ c]-anti-

50

12

38

d i o l epoxide [3H]-BP-7,8dihydrodiol

51

7

42

When t h e 7 , 8 - h y d r o x y l groups a r e m i s s i n g , e p o x i d e i n t r o d u c t i o n o c c u r s from b o t h s i d e s o f t h e p y r e n e r i n g . Thus 7 , 8 - d i h y d r o b e n z o ( a j pyrene i s c o o x i d i z e d by PGH s y n t h a s e t o a p o t e n t mutagen t h a t i s i d e n t i f i e d by p r o d u c t and n u c l e i c a c i d b i n d i n g s t u d i e s as 9,10-epoxy7 , 8 , 9 , 1 0 - t e t r a h y d r o b e n z o ( a ) p y r e n e ( E q u a t i o n 3) (32J . The s t r u c t u r e s o f t h e g u a n o s i n e a d d u c t s formed i n i n c u b a t i o n s c o n t a i n i n g p o l y g u a n y l i c a c i d i n d i c a t e t h a t e q u a l amounts o f e p o x i d e a r e formed by i n t r o d u c t i o n o f oxygen from above and below t h e p l a n e o f t h e p y r e n e r i n g (Equation 3 ) . These f i n d i n g s i n d i c a t e t h a t PGH s y n t h a s e i n t h e p r e s e n c e o f a r a c h i d o n a t e c a n c a t a l y z e t h e t e r m i n a l a c t i v a t i o n s t e p i n BP c a r c i n o g e n e s i s and t h a t t h e r e a c t i o n may be g e n e r a l f o r d i h y d r o d i o l metabol i t e s o f p o l y c y c l i c hydrocarbons. G u t h r i e e_t. a l . have shown t h a t PGH s y n t h a s e c a t a l y z e s t h e a c t i v a t i o n o f c h r y s e n e and b e n z a n t h r a c e n e d i h y d r o d i o l s t o p o t e n t mutagens ( 3 3 ) . As i n t h e c a s e w i t h BP, o n l y the d i h y d r o d i o l t h a t i s a p r e c u r s o r t o bay r e g i o n d i o l e p o x i d e s i s activated. We have r e c e n t l y shown t h a t 3 , 4 - d i h y d r o x y - 3 , 4 - d i h y d r o b e n z o ( a ) a n t h r a c e n e i s o x i d i z e d by PGH s y n t h a s e t o t e t r a h y d r o t e t r a o l s d e r i v e d from t h e a n t i - d i o l e p o x i d e ( E q u a t i o n 4) ( 3 4 ) . N a t u r e o f O x i d a n t s G e n e r a t e d From F a t t y A c i d

Hydroperoxides

PGH s y n t h a s e c o n t a i n s two h e m e - r e q u i r i n g a c t i v i t i e s ( 1 3 ) . The c y c l o oxygenase component oxygenates a r a c h i d o n i c a c i d t o t h e h y d r o p e r o x y e n d o p e r o x i d e , PGG2, and t h e p e r o x i d a s e component r e d u c e s PGG t o t h e hydroxy e n d o p e r o x i d e , PGH2. The c y c l o o x y g e n a s e i s i n h i b i t e d by nons t e r o i d a l a n t i i n f l a m m a t o r y agents such as a s p i r i n and i n d o m e t h a c i n , 2

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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but t h e p e r o x i d a s e i s n o t (35,36) . Both components a r e c o n t a i n e d on the same 70,000 D a l t o n p r o t e i n ( 1 3 ) . The p r e s e n c e o f a p e r o x i dase as an i n t e g r a l component o f PGH s y n t h a s e i m p l i e s t h a t h y d r o p e r o x i d e - d e p e n d e n t o x i d a t i o n s are c a t a l y z e d by t h i s component (37_) . As a f i r s t a p p r o x i m a t i o n one m i g h t e x p e c t t h a t the mechanisms o f t h e s e o x i d a t i o n s would be analogous t o t h o s e o f o t h e r heme p e r o x i dases. E x t e n s i v e s t u d i e s have e s t a b l i s h e d t h a t the c a t a l y t i c c y c l e f o r the r e d u c t i o n o f h y d r o p e r o x i d e s by h o r s e r a d i s h p e r o x i d a s e i s t h e one d e p i c t e d i n F i g u r e 6 (38) . The r e s t i n g enzyme i n t e r a c t s w i t h t h e p e r o x i d e t o form an e n z y m e - s u b s t r a t e complex t h a t decomposes t o a l c o h o l and an i r o n - o x o complex t h a t i s two o x i d i z i n g e q u i v a l e n t s above the r e s t i n g s t a t e o f t h e enzyme. F o r c a t a l y t i c t u r n o v e r t o o c c u r the i r o n - o x o complex must be r e d u c e d . The two e l e c t r o n s a r e f u r n i s h e d by r e d u c i n g s u b s t r a t e s e i t h e r by e l e c t r o n t r a n s f e r from s u b s t r a t e t o enzyme o r by oxygen t r a n s f e r from enzyme t o s u b s t r a t e . S u b s t r a t e o x i d a t i o n by the i r o n - o x o complex s u p p o r t s c o n t i n u o u s h y d r o p e r o x i d e r e d u c t i o n . When e i t h e r r e d u c i n g s u b s t r a t e o r h y d r o peroxide i s exhausted, the c a t a l y t i c c y c l e stops. We have d e v e l o p e d an a s s a y t o i d e n t i f y p e r o x i d a s e r e d u c i n g subs t r a t e s based on t h e i r a b i l i t y t o s t i m u l a t e r e d u c t i o n o f 1-hydrop e r o x y - 5 - p h e n y l — 4 - p e n t e n e ( E q u a t i o n 5) ( 3 9 ) . The h y d r o p e r o x i d e i s i n c u b a t e d w i t h l i m i t i n g c o n c e n t r a t i o n s o f p e r o x i d a s e i n the p r e s e n c e o r absence o f a p o t e n t i a l r e d u c i n g s u b s t r a t e . I n the absence o f r e d u c t a n t c a t a l y t i c r e d u c t i o n cannot o c c u r and n e g l i g i b l e q u a n t i t i e s o f a l c o h o l a r e p r o d u c e d (the h y d r o p e r o x i d e and a l c o h o l are q u a n t i t a t e d a f t e r s e p a r a t i o n by HPLC). I n the p r e s e n c e o f a good r e d u c i n g s u b s t r a t e c a t a l y t i c t u r n o v e r o c c u r s and q u a n t i t i e s o f a l c o h o l a r e produced t h a t are s t o i c h i o m e t r i c w i t h reducing s u b s t r a t e o x i d i z e d . The a s s a y appears t o be g e n e r a l f o r a l l p l a n t and a n i m a l , heme and non-heme, p e r o x i d a s e s . One can rank the r e l a t i v e e f f i c a c y o f reducing substrates u s i n g t h i s assay. A r o m a t i c amines, p h e n o l s , c a t e c h o l s , 8 - d i c a r b o n y l s , n i t r o g e n h e t e r o c y c l e s , and a r o m a t i c s u l f i d e s a r e good t o e x c e l l e n t r e d u c i n g s u b s t r a t e s (39^) . In c o n t r a s t , p o l y c y c l i c h y d r o c a r b o n s and d i h y d r o d i o l m e t a b o l i t e s o f PAH a r e v e r y poor t o n o n - r e d u c i n g compounds. Because BP and B P - 7 , 8 - d i h y d r o d i o l do not s t i m u l a t e h y d r o p e r o x i d e r e d u c t i o n they cannot be o x i d i z e d by h i g h e r o x i d a t i o n s t a t e s o f the p e r o x i d a s e ( i r o n - o x o complexes) The c o n c e n t r a t i o n s o f h y d r o p e r o x i d e , PGH s y n t h a s e , and BP o r BP-7,8d i h y d r o d i o l a r e analogous t o t h o s e i n which BP o r B P - 7 , 8 - d i h y d r o d i o l o x i d a t i o n can be d e t e c t e d i n ram s e m i n a l v e s i c l e microsomes. T h e r e f o r e , we c o n c l u d e t h a t t h e o x i d i z i n g agent t h a t c o n v e r t s BP t o q u i n o n e s o r B P - 7 , 8 - d i h y d r o d i o l t o d i o l e p o x i d e s i s n o t an i r o n - o x o intermediate of peroxidase turnover. S u p p o r t f o r t h i s c o n c l u s i o n i s p r o v i d e d by the h y d r o p e r o x i d e s p e c i f i c i t y o f BP o x i d a t i o n . The scheme p r e s e n t e d i n F i g u r e 6 r e q u i r e s t h a t the same o x i d i z i n g agent i s g e n e r a t e d by r e a c t i o n o f 2 ° 2 r perox> a c i d s , o r a l k y l h y d r o p e r o x i d e s w i t h t h e p e r o x i d a s e . O x i d a t i o n o f any compound by the i r o n - o x o i n t e r m e d i a t e s s h o u l d be s u p p o r t e d by any h y d r o p e r o x i d e t h a t i s r e d u c e d by the p e r o x i d a s e . T h i s i s c l e a r l y not the case f o r o x i d a t i o n o f BP by ram s e m i n a l v e s i c l e microsomes as t h e d a t a i n F i g u r e 7 i l l u s t r a t e . Quinone f o r m a t i o n i s s u p p o r t e d by f a t t y a c i d h y d r o p e r o x i d e s b u t v e r y p o o r l y o r not a t a l l by s i m p l e a l k y l h y d r o p e r o x i d e s o r H 0 2 . The f a c t t h a t H

2

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

12.

MARNETT

Hydroperoxide-Dependent

Oxygenation

F i g u r e 6. C a t a l y t i c c y c l e o f h o r s e r a d i s h

peroxidase.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

PGG

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

2

somes on t h e c o n c e n t r a t i o n o f d i f f e r e n t h y d r o p e r o x i d e s . A b b r e v i a t i o n s used a r e 20:4, a r a c h i d o n i c a c i d ; 15-HPEA, 1 5 - h y d r o p e r o x y - e i c o s a t e t r a e n o i c a c i d ; t-BuOOH, t - b u t y l hydroperoxide. The s t r u c t u r e i s PGG i s g i v e n i n F i g u r e 1. 2

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

12.

MARNETT

Hydroperoxide-Dependent

Oxygenation

317

H2O2 does n o t s u p p o r t o x i d a t i o n i s e s p e c i a l l y s i g n i f i c a n t because the same c o n c e n t r a t i o n s o f H 0 2 s u p p o r t v i g o r o u s o x i d a t i o n o f r e d u c i n g s u b s t r a t e s such as a r o m a t i c amines and p h e n y l b u t a z o n e . T h e r e f o r e , we c o n c l u d e t h a t BP and B P - 7 , 8 - d i h y d r o d i o l a r e o x i d i z e d by a s p e c i e s t h a t i s n o t a f u n c t i o n a l i n t e r m e d i a t e o f p e r o x i d a s e catalysis. The o x i d i z i n g agent t h a t i s r e s p o n s i b l e f o r t h e o x y g e n a t i o n o f BP and B P - 7 , 8 - d i h y d r o d i o l appears t o be a f r e e r a d i c a l . Reaction o f f a t t y a c i d h y d r o p e r o x i d e s w i t h m e t a l complexes g e n e r a t e s a l k o x y l and p e r o x y l r a d i c a l s t h a t can o x i d i z e o r g a n i c m o l e c u l e s (40-43). Incub a t i o n o f f a t t y a c i d hydroperoxides with c e r t a i n hemeproteins o r t h e i r p r o s t h e t i c group, h e m a t i n , c a u s e s o x i d a t i o n o f BP t o q u i n o n e s and B P - 7 , 8 - d i h y d r o d i o l t o d i o l e p o x i d e s (17,40). I n the case o f BP7 , 8 - d i h y d r o d i o l e p o x i d a t i o n , t h e s o u r c e o f t h e e p o x i d e oxygen i s m o l e c u l a r oxygen; e p o x i d a t i o n i s p o t e n t l y i n h i b i t e d by a n t i o x i d a n t s , and e p o x i d a t i o n i s s u p p o r t e d by u n s a t u r a t e d b u t n o t s a t u r a t e d f a t t y a c i d hydroperoxides (Table I I ) ( 4 0 , 4 4 ) . These o b s e r v a t i o n s a r e analogous t o t h e r e s u l t s o f m i c r o s o m a l i n c u b a t i o n s and a r e c o n s i s t e n t w i t h a f r e e r a d i c a l mechanism o f h y d r o p e r o x i d e - d e p e n d e n t e p o x i dation. BP o x i d a t i o n t o q u i n o n e s o c c u r s d u r i n g a u t o x i d a t i o n o f l i p i d s i n i t i a t e d by enzymes o r y - i a d i a t i o n (45,46) .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

2

r r

In t h e case o f t h e h e m a t i n - c a t a l y z e d r e a c t i o n we have p r o p o s e d t h a t p e r o x y l r a d i c a l s a r e t h e e p o x i d i z i n g a g e n t s ( 4 0 ) . The mechanism i s i l l u s t r a t e d i n F i g u r e 8. Hematin r e d u c e s t h e h y d r o p e r o x i d e t o an a l k o x y l r a d i c a l t h a t c y c l i z e s t o t h e a d j a c e n t d o u b l e bond. The i n c i p i e n t c a r b o n - c e n t e r e d r a d i c a l c o u p l e s w i t h O2 t o form a p e r o x y l r a d i c a l t h a t we p r o p o s e e p o x i d i z e s B P - 7 , 8 - d i h y d r o d i o l . P e r o x y l r a d i c a l s a r e well-known i n c h e m i c a l systems t o e p o x i d i z e i s o l a t e d d o u b l e bonds such as t h e 9 , 1 0 - d o u b l e bond o f B P - 7 , 8 - d i h y d r o d i o l ( E q u a t i o n 6) ( 4 7 ) . However, t h e y have been l a r g e l y i g n o r e d as p o t e n t i a l o x i d i z i n g a g e n t s i n b i o c h e m i c a l systems a l t h o u g h t h e i r h a l f - l i v e s ( 0 . 1 - 1 0 sec) s u g g e s t they c a n s e r v e as d i f f u s i b l e , s e l e c t i v e o x i d a n t s (48) . The mechanism o u t l i n e d i n F i g u r e 8 i s c o n s i s t e n t w i t h a l l o f t h e e x p e r i m e n t a l o b s e r v a t i o n s and e x p l a i n s t h e r e q u i r e m e n t f o r a d o u b l e bond i n t h e v i c i n i t y o f t h e h y d r o p e r o x i d e (Table I I ) . The a b i l i t y o f p e r o x y l r a d i c a l s t o e p o x i d i z e d o u b l e bonds appears t o depend upon t h e a b i l i t y o f t h e p e r o x y l r a d i c a l o l e f i n adduct t o s t a b i l i z e t h e c a r b o n - c e n t e r e d r a d i c a l . Thus, 3,4dihydroxy-3,4-dihydrobenzo(a)anthracene i s o x i d i z e d t o 1/6 t h e e x t e n t o f B P - 7 , 8 - d i h y d r o d i o l and a f l a t o x i n B i i s e p o x i d i z e d t o o n l y a s l i g h t e x t e n t (34,49). Peroxyl r a d i c a l s are the species t h a t propagate a u t o x i d a t i o n o f the u n s a t u r a t e d f a t t y a c i d r e s i d u e s o f p h o s p h o l i p i d s (50). In addit i o n , p e r o x y l r a d i c a l s a r e i n t e r m e d i a t e s i n the metabolism o f c e r t a i n d r u g s s u c h as p h e n y l b u t a z o n e ( 5 1 ) . E p o x i d a t i o n o f BP-7,8d i h y d r o d i o l has been d e t e c t e d d u r i n g l i p i d p e r o x i d a t i o n i n d u c e d i n r a t l i v e r microsomes by a s c o r b a t e o r NADPH and d u r i n g t h e p e r o x i d a t i c o x i d a t i o n o f p h e n y l b u t a z o n e (52,53) . These f i n d i n g s suggest t h a t peroxyl radical-mediated epoxidation o f BP-7,8-dihydrodiol i s general and may s e r v e as t h e p r o t o t y p e f o r s i m i l a r e p o x i d a t i o n s o f o t h e r o l e f i n s i n a v a r i e t y o f b i o c h e m i c a l systems. In addition, peroxyl radical-dependent epoxidation of BP-7,8-dihydrodiol e x h i b i t s the same s t e r e o c h e m i s t r y as t h e a r a c h i d o n i c a c i d - s t i m u l a t e d e p o x i d a t i o n by ram s e m i n a l v e s i c l e microsomes. T h i s n o t o n l y p r o v i d e s a d d i t i o n a l

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Table I I ,

E p o x i d a t i o n o f U n s a t u r a t e d and S a t u r a t e d F a t t y A c i d Hydroperoxides 0

HYDROPEROXIDE

2

UPTAKE

(*M)

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

n

" l6 33 C

H

7 , 8 - D I O L OXIDATION

VI (ttM/ain.)

0.1610.02

0 0 H

A ^ X C O O H 6 I ±3

6.510.6

160116

12.310.9

160113

1211.3

16015

1211.6

00H

00H

H00

/=V=W

C 0 0 H

\=A7 vv 00 H / V ^ / V

/

C

0

0

C

3

M

y v v w 00H

/

N

^ ^ C 0 0 C H

3

6511

7.011.1

240115

1611.6

00H

/ \

/

^ s / V

/

C

0

0

C

H

3

0OH

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

MARNETT

Hydroperoxide-Dependent

Oxygenation

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

12.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

319

320

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

e v i d e n c e t h a t the o x i d i z i n g agent i n the enzymatic r e a c t i o n i s a p e r o x y l r a d i c a l b u t a l s o s u g g e s t s t h a t the s t e r e o c h e m i s t r y o f BP7 , 8 - d i h y d r o d i o l o x i d a t i o n i s an i m p o r t a n t and g e n e r a l d i a g n o s t i c probe t o d i f f e r e n t i a t e e p o x i d a t i o n by m i x e d - f u n c t i o n o x i d a s e s and by peroxyl r a d i c a l s .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

S i g n i f i c a n c e o f F a t t y A c i d Hydroperoxide-Dependent PAH

Oxidation

What i s the s i g n i f i c a n c e o f a r a c h i d o n i c a c i d - d e p e n d e n t x e n o b i o t i c metabolism? E x p e r i m e n t s d e s c r i b e d above f i r m l y e s t a b l i s h t h a t i t can cause m e t a b o l i c a c t i v a t i o n in vitro, Dihydrodiol metabolites of p o l y c y c l i c hydrocarbons are o x i d i z e d t o d i o l epoxides t h a t r e p r e s e n t the u l t i m a t e c a r c i n o g e n i c forms o f the p a r e n t h y d r o c a r b o n s . Intere s t i n g l y , o n l y d i h y d r o d i o l s t h a t form bay r e g i o n d i o l e p o x i d e s a r e a c t i v a t e d by PGH s y n t h a s e ; no a c t i v a t i o n o f o t h e r PAH d i h y d r o d i o l s occurs. A r a c h i d o n a t e - d e p e n d e n t c o o x i d a t i o n i s e s s e n t i a l l y an a c t i v a t i o n pathway s p e c i f i c f o r g e n e r a t i o n o f b a y - r e g i o n d i o l e p o x i d e s . Work d e s c r i b e d e l s e w h e r e i n d i c a t e s t h a t a r o m a t i c amines can a l s o be o x i d i z e d t o mutagenic d e r i v a t i v e s a l t h o u g h the i d e n t i t y o f the mutag e n i c d e r i v a t i v e i s , a t p r e s e n t , u n c e r t a i n (54). Is i t p o s s i b l e t c q u a n t i t a t e the r e l a t i v e c o n t r i b u t i o n o f h y d r o p e r o x i d e - d e p e n d e n t and m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t o x i d a t i o n o f PAH in vitro, i n c e l l s and o r g a n s , and in vivo? Adding a r a c h i d o n i c a c i d o r NADPH t o s u p p o r t o x i d a t i o n i i i vitro g i v e s a good estimate of o x i d a t i v e p o t e n t i a l but i t s r e l a t i o n to c e l l u l a r o x i d a t i o n i s n o t s t r a i g h t f o r w a r d . L i k e w i s e , " s p e c i f i c " i n h i b i t o r s can be h e l p f u l i n in vitro e x p e r i m e n t s but t h e i r use can be compromised i n c e l l u l a r , o r g a n i s m a l , o r in vivo e x p e r i m e n t s by o v e r l a p p i n g s p e c i f i c i t i e s o r a l t e r e d p o t e n c i e s . F o r example, many compounds t h a t i n h i b i t l i p o x y g e n a s e a c t i v i t y a t low c o n c e n t r a t i o n i n m i c r o s o m a l o r c y t o p l a s m i c f r a c t i o n s a r e i n e f f e c t i v e when t h e y a r e employed i n c e l l u l a r experiments. The r e a s o n f o r the d i f f e r e n t i a l e f f e c t i s unc l e a r b u t the i m p l i c a t i o n f o r the use o f such compounds as in vivo i n h i b i t o r s i s obvious. A p o t e n t i a l l y p o w e r f u l probe f o r s o r t i n g o u t the c o n t r i b u t i o n of h y d r o p e r o x i d e - d e p e n d e n t and m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t p o l y c y c l i c hydrocarbon o x i d a t i o n i s stereochemistry. F i g u r e 9 summ a r i z e s the s t e r e o c h e m i c a l d i f f e r e n c e s i n e p o x i d a t i o n o f (±)-BP-7,8d i h y d r o d i o l by h y d r o p e r o x i d e - d e p e n d e n t and m i x e d - f u n c t i o n o x i d a s e dependent pathways (31,55,56). The (-)-enantiomer o f BP-7,8d i h y d r o d i o l i s c o n v e r t e d p r i m a r i l y t o the ( + ) - a n t i - d i o l epoxide by b o t h pathways whereas the (+)-enantiomer o f B P - 7 , 8 - d i h y d r o d i o l i s c o n v e r t e d p r i m a r i l y t o the ( - ) - a n t i - d i o l e p o x i d e by h y d r o p e r o x i d e dependent o x i d a t i o n and t o the ( + ) - s y n - d i o l e p o x i d e by m i x e d - f u n c t i o n oxidases. The s t e r e o c h e m i c a l c o u r s e o f o x i d a t i o n by cytochrome P-450 isoenzymes was f i r s t e l u c i d a t e d f o r t h e m e t h y c h o l a n t h r e n e i n d u c i b l e form but we have d e t e c t e d the same s t e r e o c h e m i c a l p r o f i l e u s i n g r a t l i v e r microsomes from c o n t r o l , p h e n o b a r b i t a l - , o r m e t h y l c h o l a n t h r e n e - i n d u c e d a n i m a l s (32). The o n l y d i f f e r e n c e between the microsomal p r e p a r a t i o n s i s the r a t e of o x i d a t i o n . The f i n d i n g s summarized i n F i g u r e 9 p r o v i d e a p r a c t i c a l d i a g n o s t i c t o o l f o r d i s t i n g u i s h i n g the two r o u t e s o f o x i d a t i o n . R e a c t i o n s can be p e r f o r m e d w i t h c e l l u l a r o r s u b c e l l u l a r p r e p a r a t i o n s and ( ± ) - o r ( + ) - B P - 7 , 8 - d i h y d r o d i o l and the t e t r a o l h y d r o l y s i s

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

12.

MARNETT

Hydroperoxide-Dependent

Oxygenation

321

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

(•>-ANTI

(•>-8YN

-ANTI

F i g u r e 9.

©

-

MIXED-FUNCTION 0XIDA8E DEPENDENT

(D

- P E R O X I D E - M E T A L DEPENDENT

S t e r e o c h e m i c a l d i f f e r e n c e s between f a t t y a c i d h y d r o p e r o x i d e - and m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t o x i d a t i o n of (±)-BP-7,8-dihydrodiol.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch012

322

POLYCYCLIC HYDROCARBONS AND

CARCINOGENESIS

p r o d u c t s o f the d i o l e p o x i d e s s e p a r a t e d by HPLC and q u a n t i t a t e d (57). When the s u b s t r a t e i s (±)-BP-7,8-dihydrodiol an a n t i / s y n r a t i o i n e x c e s s o f 2.5 i s seen f o r p e r o x i d e - d e p e n d e n t o x i d a t i o n and an a n t i / s y n r a t i o o f 1 f o r m i x e d - f u n c t i o n o x i d a s e - d e p e n d e n t o x i d a tion. When the s u b s t r a t e i s ( + ) - B P - 7 , 8 - d i h y d r o d i o l the a n t i / s y n r a t i o f o r the mixed-function oxidase-dependent r e a c t i o n decreases t o MD.3. The t e n f o l d d i f f e r e n c e i n the a n t i / s y n r a t i o between p e r o x i d e - and cytochrome P-450-dependent e p o x i d a t i o n makes i t an e x t r e m e l y s e n s i t i v e i n d i c a t o r o f the pathway o f o x i d a t i o n . We have e x p l o i t e d i t t o demonstrate t h a t l i p i d p e r o x i d a t i o n i n r a t l i v e r microsomes c a u s e s e p o x i d a t i o n ( 5 2 ) . By u s i n g (+)-BP-7,8-dihydrodiol, we have been a b l e t o d i s t i n g u i s h e p o x i d a t i o n caused by NADPHdependent l i p i d p e r o x i d a t i o n i n m e t h y l c h o l a n t h r e n e - i n d u c e d r a t l i v e r microsomes (52). These microsomes c o n t a i n an e x t r e m e l y a c t i v e c y t o chrome P-450 toward B P - 7 , 8 - d i h y d r o d i o l but i t i s p o s s i b l e to d i f f e r e n t i a t e the c o n t r i b u t i o n o f l i p i d p e r o x i d a t i o n t o e p o x i d a t i o n by d e t e r m i n i n g the y i e l d o f t e t r a o l s from the ( - ) - a n t i - d i o l e p o x i d e . A l t h o u g h i t has been s u s p e c t e d f o r some time t h a t l i p i d p e r o x i d a t i o n c o u l d cause x e n o b i o t i c o x i d a t i o n i n t h e p r e s e n c e o f an a c t i v e c y t o chrome P-450, our s t u d i e s o f B P - 7 , 8 - d i h y d r o d i o l oxidation provided the f i r s t c l e a r c u t d e m o n s t r a t i o n o f i t . S t e r e o c h e m i s t r y has a l s o been employed t o d e t e c t a r a c h i d o n i c a c i d - d e p e n d e n t B P - 7 , 8 - d i h y d r o d i o l e p o x i d a t i o n i n c u l t u r e d hamster t r a c h e a (58). These examples i l l u s t r a t e the power o f such s t e r e o c h e m i c a l p r o b e s . PGH s y n t h a s e and t h e r e l a t e d enzyme l i p o x y g e n a s e occupy a p o s i t i o n a t t h e i n t e r f a c e o f p e r o x i d a s e c h e m i s t r y and f r e e r a d i c a l c h e m i s t r y and can c l e a r l y t r i g g e r m e t a b o l i c a c t i v a t i o n by b o t h mechanisms. The p e r o x i d a s e pathway a c t i v a t e s compounds such as d i e t h y l s t i l b e s t r o l and a r o m a t i c amines whereas the f r e e r a d i c a l pathway a c t i v a t e s p o l y c y c l i c h y d r o c a r b o n s (59). B o t h pathways r e q u i r e s y n t h e s i s of hydroperoxide i n order to t r i g g e r o x i d a t i o n . The r a t e - l i m i t i n g s t e p i n h y d r o p e r o x i d e s y n t h e s i s i s r e l e a s e o f a r a c h i d o n i c a c i d from p h o s p h o l i p i d s t o r a g e (60,61). Release i s c a t a l y z e d by p h o s p h o l i p a s e s and i s s t i m u l a t e d by a q e n t s t h a t a c t a t the c e l l s u r f a c e such as hormones, i o n o p h o r e s , tumor p r o m o t e r s , e t c (62). Arachidonic acid-dependent c o o x i d a t i o n i s , t h e r e f o r e , a pathway t h a t l i n k s e v e n t s a t t h e c e l l s u r f a c e t o i n t r a c e l l u l a r o x i dation of xenobiotics. I t i s a l s o a model f o r o x i d a t i o n o f xenob i o t i c s by o t h e r p e r o x i d a s e s and by f r e e r a d i c a l s . T h e r e a r e few r e p o r t s o f x e n o b i o t i c m e t a b o l i s m by p e r o x y l o r a l k o x y l f r e e r a d i c a l s b u t the p o t e n t i a l i s enormous. U n s a t u r a t e d f a t t y a c i d s are p r e s e n t i n a l l c e l l s t o some e x t e n t and, i n f a c t , a r e q u i t e abundant i n most cells. F o r example, v e n t r i c u l a r m y o c a r d i a l muscle c o n t a i n s 14.1 ymol l i n o l e i c and a r a c h i d o n i c a c i d s p e r gram wet w e i g h t t i s s u e (63). B o t h f a t t y a c i d s a r e q u i t e s u s c e p t i b l e t o l i p i d p e r o x i d a t i o n which generates peroxvl r a d i c a l s capable of o x i d i z i n g c e r t a i n x e n o b i o t i c s , e.g., B P - 7 , 8 - d i h y d r o d i o l . I n h i b i t i o n of l i p i d peroxidation i s o b v i o u s l y a t a s k t h a t must be c o n s t a n t l y p e r f o r m e d by c e l l s t o p r e v e n t t i s s u e d e s t r u c t i o n and x e n o b i o t i c m e t a b o l i s m . The t u r n o v e r o f o n l y 0.1% o f the u n s a t u r a t e d f a t t y a c i d r e s i d u e s o f c e l l s c o u l d g e n e r a t e a v e r y s i g n i f i c a n t amount o f p e r o x y l r a d i c a l s i n s i d e membrane r e g i o n s o f c e l l s where many x e n o b i o t i c s arp d i s s o l v e d . M e t a b o l i s m o f a r o m a t i c amines and B P - 7 , 8 - d i h y d r o d i o l has been d e t e c t e d d u r i n g a r a c h i d o n a t e o x y g e n a t i o n i n i n t a c t c e l l s and i n c u l t u r e d t r a c h e a (64,58). Exogenous a r a c h i d o n a t e was added t o

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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MARNETT

Hydroperoxide-Dependent

Oxygenation

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s t i m u l a t e h y d r o p e r o x i d e s y n t h e s i s and c o o x y g e n a t i o n i n most o f t h e studies. R e c e n t l y , though, Amstad and C e r u t t i r e p o r t e d t h a t t h e l e v e l s o f a f l a t o x i n B^-DNA a d d u c t s formed i n C3H i O l S f i b r o b l a s t s were d e c r e a s e d by t r e a t m e n t o f t h e c e l l s w i t h i n d o m e t h a c i n o r e i c o s a t e t r a y n o i c a c i d , i n h i b i t o r s o f a r a c h i d o n a t e o x y g e n a t i o n ( 6 5 ) . They concluded that a s i g n i f i c a n t f r a c t i o n o f t o t a l a f l a t o x i n e p o x i d a t i o n by i O T ^ c e l l s o c c u r s as a r e s u l t o f a r a c h i d o n a t e - d e p e n d e n t cooxygenation. T h i s i m p l i e s t h a t c o o x y g e n a t i o n t a k e s p l a c e i n c e l l s and t h a t i t i s t r i g g e r e d by r e l e a s e o f a r a c h i d o n a t e from endogenous s t o r e s . To what e x t e n t does c o o x y g e n a t i o n o c c u r in vivo and i s i t important i n chemical c a r c i n o g e n e s i s ? This i s a very d i f f i c u l t q u e s t i o n t o answer a t t h e p r e s e n t t i m e . Recent r e s u l t s demonstrate t h a t a r o m a t i c amines and d i a m i n e s c a n be c o o x i d i z e d in vivo (66,67). In t h e case o f 3-napthylamine i t i s e s t i m a t e d t h a t 30% o f t h e a d d u c t s t h a t form t o DNA i n t h e dog b l a d d e r , a t a r g e t o r g a n f o r n a p t h y l a m i n e c a r c i n o g e n e s i s , a r i s e as a r e s u l t o f a r a c h i d o n a t e - d e p e n d e n t c o o x i d a t i o n ( 6 6 ) . T h i s c o n c l u s i o n i s based on t h e d e t e c t i o n o f u n i q u e p e r o x i d a s e a d d u c t s t c DNA t h a t a r e s t r u c t u r a l l y d i s t i n c t from mixedf u n c t i o n oxidase-generated adducts. In contrast, pretreatment o f A/HeJ mice w i t h a s p i r i n o r i n d o m e t h a c i n does n o t lower t h e l e v e l s o f DNA a d d u c t s formed from BP i n l u n g n o r does i t reduce t h e i n c i d e n c e o f l u n g neoplasms i n d u c e d by BP ( 6 8 ) . C o n t r o l e x p e r i m e n t s i n d i c a t e t h a t a s p i r i n t r e a t m e n t a b o l i s h e s PGH s y n t h a s e a c t i v i t y in vivo (68). T h i s s u g g e s t s t h a t PGH synthase-dependent c o o x i d a t i o n does n o t p l a y a r o l e i n l u n g t u m o r i g e n e s i s by b e n z o ( a ) p y r e n e i n t h e adenoma model. T h i s may be r e l a t e d t o t h e h i g h l e v e l s o f t h e endogenous a n t i o x i d a n t , v i t a m i n E , i n r o d e n t l u n g ( 6 9 ) . However, a d m i n i s t r a t i o n o f a s p i r i n t o g u i n e a p i g s does n o t lower t h e l e v e l s o f p r o t e i n o r DNA a d d u c t s formed from BP i n s e v e r a l d i f f e r e n t t i s s u e s , so t h e l e v e l s o f v i t a m i n E may n o t be a d e t e r m i n a n t o f BP c o o x i d a t i o n ( 7 0 ) . The t i s s u e d i s t r i b u t i o n o f PGH s y n t h a s e s u g g e s t s t h a t i t does n o t p l a y a major r o l e i n s y s t e m i c d r u g m e t a b o l i s m because most o f t h e t i s s u e s where i t i s p r e s e n t i n h i g h c o n c e n t r a t i o n do n o t r e c e i v e a s i g n i f i c a n t p r o p o r t i o n o f c a r d i a c o u t p u t (12) . However, s e v e r a l o f t h e s e t i s s u e s , e.g., k i d n e y and u t e r u s , a r e t a r g e t o r g a n s f o r carcinogens that require metabolic a c t i v a t i o n . In oraer t o detect arachidonate-dependent metabolic a c t i v a t i o n i n these t i s s u e s , i t w i l l be n e c e s s a r y t o d e v e l o p u n i q u e and s p e c i f i c p r o b e s . I f s y s t e m i c m e t a b o l i s m o f a g i v e n compound p r o c e e d s w i t h a unique p a t t e r n o f s t e r e o c h e m i s t r y (e.g., BP-7,8-dihydrodiol) o r produces u n i q u e DNA a d d u c t s (3-napthylamine) t h e n i t s h o u l d be p o s s i b l e t o q u a n t i t a t e t h e e x t e n t t o which u n i q u e s t e r e o i s o m e r s o r DNA a d d u c t s a r e formed. In vitro studies d e f i n e these d i s t i n c t i v e features o f c o o x i d a t i v e m e t a b o l i s m and g u i d e t h e i n t e l l i g e n t d e s i g n o f c r i t i c a l experiments. H o p e f u l l y , by u s i n g s u c h d i a g n o s t i c p r o b e s i t w i l l be p o s s i b l e t o p r o v i d e q u a n t i t a t i v e answers t o q u e s t i o n s about t h e e x t e n t t o which c o o x i d a t i o n o f p o l y c y c l i c h y d r o c a r b o n s and o t h e r c a r c i n o g e n s o c c u r s in vivo. Acknowledgments T h i s r e s e a r c h h a s been g e n e r o u s l y s u p p o r t e d by g r a n t s from t h e American Cancer S o c i e t y (BC244) and t h e N a t i o n a l I n s t i t u t e s o f H e a l t h (GM23642). LJM i s a r e c i p i e n t o f an American Cancer S o c i e t y F a c u l t y R e s e a r c h Award (FRA243).

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13 The Mutational Consequences of DNA Damage Induced by Benzo[a]pyrene ERIC EISENSTADT

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Department of Cancer Biology and Laboratory of Toxicology, Harvard School of Public Health, Boston, MA 02115 Induced mutagenesis i n Escherichia c o l i i s an active process involving proteins with DNA replication, re­ pair, and recombination functions. The available evi­ dence suggests that mutations are generated at sites where DNA has been damaged and that they arise v i a an error-prone repair activity. In an attempt to under­ stand what specific contributions to mutagenesis are made by DNA lesions, we have studied the mutational specificity of some carcinogens, such as benzo[a]pyrene and aflatoxin B1, whose chemical reactions with DNA are well-studied. Our results, obtained by monitoring the distribution of lacI nonsense mutations i n E. coli, suggest that the major mutational events induced by benzo[a]pyrene and aflatoxin B are base substitutions. The base substitutions are primarily transversions at G:C base pairs and the available evidence suggests that these mutations are induced by apurinic sites which are generated as secondary consequences of the initial al­ kylation event. The significance of these results i n the context of carcinogenesis i s briefly considered. 1

The h i g h f i d e l i t y w i t h w h i c h genomes a r e r e p l i c a t e d in v i v o and passed on t o daughter c e l l s i s a c h i e v e d by a r e p e r t o i r e o f a c t i v i t i e s which f u n c t i o n d u r i n g r e p l i c a t i o n , r e p a i r , and r e c o m b i n a t i o n (1^,2). These a c t i v i t i e s , which c o l l e c t i v e l y m a i n t a i n the s t r u c t u r a l and i n ­ f o r m a t i o n a l i n t e g r i t y o f the DNA m o l e c u l e , a r e s e v e r e l y t e s t e d when the DNA t e m p l a t e i s damaged and becomes n o n - r e p l i c a b l e . Under these c i r c u m s t a n c e s , which o b t a i n , f o r example, when c e l l s a r e exposed t o such human c a r c i n o g e n s as U V - l i g h t (3) o r p o l y c y c l i c a r o m a t i c h y d r o ­ carbons ( 4 ) , i t i s commonly observed t h a t the f r e q u e n c y o f m u t a t i o n i s enhanced by many o r d e r s o f magnitude. The c o r r e l a t i o n between t h e mutagenic and c a r c i n o g e n i c a c t i v i t y o f many p h y s i c a l and c h e m i c a l agents has been well-documented (_5). Recent o b s e r v a t i o n s even sug­ g e s t the p o s s i b i l i t y t h a t one s t e p i n t u m o r i g e n e s i s might l i t e r a l l y i n v o l v e the m u t a t i o n a l a l t e r a t i o n o f s p e c i f i c chromosomal genes (6-8). 0097-6156/ 85/ 0283-0327506.00/ 0 © 1985 American Chemical Society

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I n t h i s c h a p t e r I w i l l r e v i e w some a s p e c t s o f mutagenesis mecha­ nisms and t h e m u t a t i o n a l consequences o f DNA damage g e n e r a t e d by ben­ zo [a] p y r e n e . The f o c u s w i l l be on knowledge d e r i v e d from i n v e s t i g a ­ t i o n s i n v o l v i n g the bacterium E s c h e r i c h i a c o l i .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Mutagenesis i s an a c t i v e p r o c e s s 12. c o l i and e u k a r y o t i c c e l l s can respond t o DNA damage by i n d u c i n g the s y n t h e s i s o f s p e c i f i c gene p r o d u c t s ( 9 - 1 2 ) . The phenomenon o f gene i n d u c t i o n by DNA damage has been most t h o r o u g h l y d e s c r i b e d f o r 12. c o l i and has r e c e n t l y been reviewed by Walker ( 9 ) . Among t h e i n ­ d u c i b l e r e s p o n s e s t o DNA damage i s the mutagenic r e p a i r p r o c e s s , whose e x i s t e n c e was f i r s t suggested o v e r 30 y e a r s ago by t h e e x p e r i ­ ments o f W e i g l e ( 1 3 ) . W e i g l e showed t h a t U V - l i g h t was mutagenic t o b a c t e r i o p h a g e lamb­ da o n l y i f t h e U V - i r r a d i a t e d lambda were grown on b a c t e r i a w h i c h had a l s o been i r r a d i a t e d w i t h U V - l i g h t . I n o t h e r words, t h e UV treatment was n o t mutagenic p e r s e . F u r t h e r m o r e , he demonstrated t h a t i r r a d i ­ ated lambda phage c o u l d be r e a c t i v a t e d by growing t h e phage on p r e i r r a d i a t e d b a c t e r i a . H i s r e s u l t s suggested t h e p o s s i b i l i t y t h a t bac­ t e r i a had an i n d u c i b l e system f o r DNA r e p a i r and mutagenesis w h i c h a c t e d on U V - i r r a d i a t e d lambda phage. The g e n e t i c s o f what i s now c a l l e d W e i g l e o r W - r e a c t i v a t i o n and W-mutagenesis i s now v e r y w e l l understood. Some twenty genes i n J2. c o l i — known c o l l e c t i v e l y as d i n genes (damage i n d u c i b l e ; 9,14) a r e c o o r d i n a t e l y r e g u l a t e d by t h e p r o d u c t s o f t h e genes recA and l e x A . The LexA p r o t e i n r e p r e s s e s d i n gene e x ­ p r e s s i o n by b i n d i n g t o t h e o p e r a t o r r e g i o n o f each gene and p r e v e n t ­ i n g i t s t r a n s c r i p t i o n i n t o RNA by RNA polymerase. Treatments w h i c h damage t h e c e l l ' s DNA o r o t h e r w i s e i n t e r f e r e w i t h DNA s y n t h e s i s , a c t ­ i v a t e t h e RecA p r o t e i n ; a c t i v a t e d RecA p r o t e i n t h e n promotes t h e p r o ­ t e o l y t i c i n a c t i v a t i o n o f LexA r e p r e s s o r ( 1 5 ) . Genes whose t r a n s c r i p ­ t i o n had been r e p r e s s e d by LexA p r o t e i n c a n now be t r a n s c r i b e d and new p r o t e i n s c a n be s y n t h e s i z e d . The o v e r a l l response o f 12. c o l i t o DNA damage, w h i c h i s g e n e t i c a l l y r e g u l a t e d by t h e r e c A and l e x A l o c i , i s known as t h e SOS-response ( 1 6 , 1 7 ) . M u t a t i o n s i n e i t h e r recA o r l e x A c a n a b o l i s h t h e SOS-response and e l i m i n a t e b o t h W - r e a c t i v a t i o n and W-mutagenesis. These m u t a t i o n s a l s o e l i m i n a t e t h e m u t a b i l i t y o f t h e b a c t e r i a by U V - i r r a d i a t i o n ( 1 6 ) . The o b s e r v a t i o n t h a t UV mutagenesis depended on t h e SOS-response e s ­ t a b l i s h e d t h a t m u t a t i o n s were n o t i n e v i t a b l e outcomes o f DNA damage and t h a t DNA damage r e q u i r e d p r o c e s s i n g by c e l l u l a r mechanisms i n o r ­ der f o r m u t a t i o n s t o be r e c o v e r e d . What s p e c i f i c p r o c e s s e s r e g u l a t e d by t h e SOS-response a r e r e s p o n s i b l e f o r mutagenesis? A major c o n t r i b u t i o n towards answering t h i s q u e s t i o n was made by the i s o l a t i o n o f m u t a t i o n s w h i c h s p e c i f i c a l l y e l i m i n a t e d t h e m u t a b i l ­ i t y o f 12. c o l i w i t h o u t a f f e c t i n g any o f t h e o t h e r components o f t h e SOS-response. M u t a t i o n s a t t h e umuDC l o c u s were i n d e p e n d e n t l y d i s ­ covered by Kato and S h i n o u r a (18) and S t e i n b o r n (19) t o a b o l i s h t h e m u t a t i o n a l a f f e c t s o f DNA damage by U V - i r r a d i a t i o n . These mutants were a l s o shown t o be d e f e c t i v e i n W-mutagenesis (18,20) and W-react­ i v a t i o n (18,20). The b i o c h e m i c a l n a t u r e o f t h e a c t i v i t y performed by the umuDC gene p r o d u c t s i s n o t known. However, s e v e r a l o b s e r v a t i o n s suggest t h a t t h e f u n c t i o n o f t h e umuDC p r o t e i n s i s t o enhance some mode o f DNA r e p a i r , e i t h e r d i r e c t l y o r i n d i r e c t l y : 1) J2. c o l i c a r r y -

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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i n g t h e umuC36 a l l e l e i s more s e n s i t i v e t o t h e l e t h a l e f f e c t s o f UVi r r a d i a t i o n (18) and a n g e l i c i n p l u s near-UV ( 2 1 ) ; 2) P l a s m i d borne a n a l o g s o f the umuDC l o c u s (mucAB; 12) enhance W - r e a c t i v a t i o n and t h e resistance of b a c t e r i a to the l e t h a l e f f e c t s of U V - i r r a d i a t i o n (23); 3) As p r e v i o u s l y n o t e d , W - r e a c t i v a t i o n i n U v r b a c t e r i a i s e l i m i n a t e d by m u t a t i o n s a t the umuDC l o c u s ( 1 8 ) . Of t h e a p p r o x i m a t e l y twenty genes induced by DNA damage, the umuDC genes and t h e i r p r o d u c t s a r e the b e s t c a n d i d a t e s f o r d i r e c t p a r t i c i p a n t s i n the b i o c h e m i c a l p r o ­ c e s s i n g o f DNA l e s i o n s t o m u t a t i o n s . The p r o c e s s i n g o f DNA damage i n °li umuDC gene p r o d u c t s and t h e a s s o c i a t e d p r o t e i n s r e g u ­ l a t e d by t h e SOS-response i s c a l l e d SOS-processing (9) o r , sometimes, e r r o r - p r o n e r e p a i r ( 1 6 , 1 7 ) . Mutagenesis i n IS. c o l i , t h e r e f o r e , a p ­ pears t o be a g e n e t i c a l l y and b i o c h e m i c a l l y a c t i v e p r o c e s s r e q u i r i n g the p a r t i c i p a t i o n o f i n d u c i b l e p r o t e i n s . Not a l l mutagenesis i n IS. c o l l i s dependent on S O S - p r o c e s s i n g . M u t a t i o n s may a r i s e q u i t e s i m p l y d u r i n g DNA r e p l i c a t i o n i f a base i s s u b s t i t u t e d by o r c o n v e r t e d t o a n o t h e r , i n c o r r e c t , base. Consider the consequence o f o x i d a t i v e d e a m i n a t i o n o f t h e base 5 - m e t h y l c y t o s i n e to thymine. R e p l i c a t i o n f o l l o w e d by daughter s t r a n d s e g r e g a t i o n w i l l r e s u l t i n a G:C base p a i r h a v i n g been mutated t o an A:T base p a i r . S i t e s c o n t a i n i n g 5 - m e t h y l c y t o s i n e a r e h o t s p o t s f o r G:C t o A:T t r a n s i ­ t i o n s i n E. c o l i ( 2 4 ) . A l k y l a t i o n o f some bases a t the e x o c y c l i c oxygen atoms c a n l e a d to c h e m i c a l l y s t a b l e a l t e r a t i o n s i n t h e base p a i r i n g p r o p e r t i e s o f a base and, t h e r e b y , d i r e c t l y induce base m i s - p a i r i n g by DNA polymer­ a s e . A w e l l - s t u d i e d example o f t h i s i s t h e consequence o f a l k y l a t i n g guanine a t the 0-6 p o s i t i o n ( 2 5 - 2 7 ) . T h i s has t h e e f f e c t o f f r e e z i n g guanine i n i t s ( r a r e ) e n o l tautomer p e r m i t t i n g the G:T mismatch t o form i n p l a c e o f the u s u a l G:C base p a i r . A subsequent round o f DNA r e p l i c a t i o n l e a d s t o the g e n e r a t i o n o f a G:C t o A:T t r a n s i t i o n muta­ t i o n . These e x c e p t i o n s n o t w i t h s t a n d i n g , most DNA damaging agents i n ­ duce mutations i n IS. c o l i v i a SOS-processing.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

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How u n i v e r s a l i s t h e SOS-processing

system o f E. c o l i ?

The dependence o f m u t a t i o n on f u n c t i o n s i n v o l v i n g DNA r e p a i r seems t o be widespread among organisms. Many p r o k a r y o t i c s p e c i e s a r e i n h e r ­ e n t l y non-mutable by U V - l i g h t b u t become mutable when p l a s m i d s encod­ i n g f o r f u n c t i o n s analogous t o the umuDC f u n c t i o n s a r e i n t r o d u c e d (e.g. 2 8 ) . Non-mutable mutants o f the y e a s t Saccharomyces c e r e v i s i a e have been i s o l a t e d and shown t o possess d e f e c t s which i m p l i c a t e DNA r e p a i r and r e c o m b i n a t i o n p r o c e s s e s ( s e e 29^ f o r a r e c e n t r e v i e w ) . F u r t h e r m o r e , t h e r e a r e many examples o f DNA r e p a i r s t r a t e g i e s which a r e common t o p r o k a r y o t i c and e u k a r y o t i c organisms ( n u c l e o t i d e e x c i ­ s i o n r e p a i r , DNA g l y c o s y l a s e s , a p u r i n i c / a p y r i m i d i n i c e n d o n u c l e a s e s , 0 -methylguanine-DNA-methyl t r a n s f e r a s e ; 2,30). P u r i f i e d DNA p o l y ­ merases from mammalian c e l l s and v i r u s e s behave s i m i l a r l y _in v i t r o t o IS. c o l i DNA polymerase when DNA damage i s encountered — r e p l i c a ­ t i o n ceases a t t h e s i t e o f t h e l e s i o n ( 3 1 ) . Of c o u r s e , even i f ana­ logues o f SOS-processing a r e i d e n t i f i e d i n e u k a r y o t e s , t h e r e g u l a t i o n of these a c t i v i t i e s might d i f f e r i n d e t a i l from the scheme which ob­ t a i n s i n IS. c o l i (e.£. analogous f u n c t i o n s may be c o n s t i t u t i v e l y ex­ p r e s s e d ) . N o n e t h e l e s s , Ruby and S z o s t a k (10) have demonstrated t h e e x i s t e n c e o f DNA damage i n d u c i b l e l o c i i n j>. c e r e v i s i a e and e s t i m a t e

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t h a t t h e r e may e x i s t as many as 80 such genes ( 1 1 ) . Shorpp e t a l . (12) have r e c e n t l y r e p o r t e d t h a t UV l i g h t enhances t h e s y n t h e s i s o f a t l e a s t e i g h t p r o t e i n s i n human f i b r o b l a s t c e l l s .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Are m u t a t i o n s d i s t r i b u t e d a t s i t e s o f DNA damage? The dependence o f mutagenesis on SOS p r o c e s s i n g r a i s e d q u e s t i o n s about t h e r o l e ( s ) p l a y e d by DNA l e s i o n s i n mutagenesis. Do DNA l e ­ s i o n s s i m p l y t r i g g e r the SOS response by i n t e r f e r i n g w i t h DNA r e p l i ­ c a t i o n t h e r e b y g e n e r a t i n g m u t a t i o n s i n d i r e c t l y v i a an e r r o r - p r o n e form o f DNA r e p l i c a t i o n ? Or do m u t a t i o n s a r i s e d i r e c t l y a t t h e s i t e s i n DNA where damage has been generated? The o b s e r v a t i o n s t h a t m u t a t i o n f r e q u e n c i e s a r e e l e v a t e d s e v e r a l f o l d above normal l e v e l s i n mutants w h i c h c o n s t i t u t i v e l y e x p r e s s t h e i r S O S - f u n c t i o n s (32) and t h a t the m u t a t i o n f r e q u e n c y o f u n i r r a d i ­ ated phage i s e l e v a t e d by growing them on i r r a d i a t e d ( i . ^ e . SOS-in­ duced) b a c t e r i a ( 3 3 ) , have been i n v o k e d t o argue f o r the n o t i o n t h a t mutagenesis v i a SOS-processing may be i n d i r e c t . On the o t h e r hand, the o b s e r v a t i o n t h a t 95% o f the UV induced base s u b s t i t u t i o n m u t a t i o n s a r o s e a t the v e r y s i t e s ( p y r i m i d i n e - p y r i midine sequences) where the major f r a c t i o n o f UV damage i s d e p o s i t ­ ed suggested t h a t a t l e a s t the UV i n d u c e d m u t a t i o n s were t a r g e t e d (24). Drake and B a l t z (34) and W i t k i n and Wermundsen (35) p r e s e n t e d arguments i n f a v o r o f the n o t i o n t h a t , f o r the most p a r t , SOS muta­ g e n e s i s was o c c u r r i n g a t s i t e s o f DNA damage. More r e c e n t e v i d e n c e , based on a n a l y z i n g the d i s t r i b u t i o n o f m u t a t i o n s w i t h i n the l a d gene of _E. c o l i , s t r o n g l y s u g g e s t s t h a t m u t a t i o n s a r i s i n g v i a SOS-process­ i n g a r e o c c u r r i n g a t the s i t e s o f DNA damage ( 3 6 , 3 7 ) . B r i e f l y , when one examines t h e spectrum o f m u t a t i o n s i n d u c e d by a v a r i e t y o f muta­ gens whose a c t i v i t y i s dependent on S O S - p r o c e s s i n g , one f i n d s t h a t both where the m u t a t i o n s a r e i n d u c e d and w h i c h m u t a t i o n s a r e induced depends on the mutagen. The observed d i f f e r e n c e s among mutagens a p ­ p l y b o t h t o the m u t a t i o n a l e v e n t s t h a t a r e d i s t r i b u t e d non-randomly at o n l y a few s i t e s ( h o t s p o t s ) and t o e v e n t s t h a t a r e d i s t r i b u t e d randomly a t many d i f f e r e n t s i t e s w i t h i n t h e gene ( l o w f r e q u e n c y o c ­ c u r r e n c e s o r LFO e v e n t s ) ( 3 6 ) . S i n c e each mutagenic t r e a t m e n t l e a v e s behind i t s own c h a r a c t e r i s t i c d i s t r i b u t i o n o f m u t a t i o n s w i t h i n t h e gene ( 3 7 ) , m u t a t i o n s g e n e r a t e d b y SOS-processing o f damaged DNA must be o c c u r r i n g a t the s i t e s o f damage. F u r t h e r m o r e , t h e r e c e n t s t u d y by M i l l e r and Low (38) on the d i s t r i b u t i o n o f m u t a t i o n s g e n e r a t e d by t u r n i n g on the SOS-response w i t h o u t DNA-damaging t r e a t m e n t s shows t h a t even these m u t a t i o n s a r e g e n e r a t e d a t s p e c i f i c s i t e s i n a gene as i f t h e y arose a t s i t e s where s p o n t a n e o u s l y g e n e r a t e d l e s i o n s o c c u r with a h i g h frequency. The w e l l c h a r a c t e r i z e d r e a c t i o n s o f c a r c i n o g e n s such as benzo[a]pyrene and a f l a t o x i n B^ w i t h DNA (39-50) suggested t o us t h a t an a n a l y s i s o f the k i n d s o f m u t a t i o n s t h e s e agents i n d u c e d c o u l d shed l i g h t on the c o n t r i b u t i o n o f s p e c i f i c DNA l e s i o n s t o mutagenesis. Such an a n a l y s i s c o u l d , i n t u r n , p r o v i d e c l u e s as t o w h i c h s p e c i f i c DNA l e s i o n s g e n e r a t e d by t h e s e agents were mutagenic. I would l i k e t o d e s c r i b e o u r i n v e s t i g a t i o n s , performed i n c o l ­ l a b o r a t i o n w i t h J e f f r e y M i l l e r . To b e g i n , I w i l l b r i e f l y o u t l i n e t h e g e n e t i c system d e v e l o p e d by M i l l e r w h i c h p e r m i t s a r a p i d , r i g o r o u s d e t e r m i n a t i o n o f t h e p o s i t i o n and k i n d s o f mutants i n d u c e d i n a p a r ­ t i c u l a r gene by DNA. damaging a g e n t s *

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

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l a d system f o r a n a l y z i n g nonsense m u t a t i o n s I n E. c o l i

331

The l a d system has been d e s c r i b e d i n d e t a i l by M i l l e r ( 5 1 ) . The l a d gene product i s the r e p r e s s o r o f t h e l a c operon. C e l l s w h i c h have normal r e p r e s s o r a c t i v i t y a r e r e p r e s s e d f o r t h e s y n t h e s i s o f the l a c Z gene p r o d u c t , 3 - g a l a c t o s i d a s e , and t h e o t h e r p r o d u c t s o f t h e l a c operon. C e l l s w h i c h c a r r y m u t a t i o n s i n l a d w h i c h l e a d t o s y n t h e s i s of a d e f e c t i v e r e p r e s s o r p r o t e i n w i l l c o n s t i t u t i v e l y s y n t h e s i z e 3g a l a c t o s i d a s e . Such mutants can be s e l e c t e d by demanding growth o f b a c t e r i a on a medium c o n t a i n i n g a g a l a c t o s i d e a n a l o g such a s p h e n y l 0 - D - g a l a c t o s i d e ( P - g a l ) . P - g a l i s n o t i t s e l f an i n d u c e r o f t h e l a c operon. Thus, i t i s a s i m p l e m a t t e r t o t r e a t a p o p u l a t i o n o f b a c t e r ­ i a l c e l l s w i t h a DNA damaging a g e n t , grow them o u t n o n - s e l e c t i v e l y t o p e r m i t p r o c e s s i n g o f DNA damage and p h e n o t y p i c e x p r e s s i o n , and t h e n p l a t e them on P - g a l t o s e l e c t f o r c e l l s c a r r y i n g m u t a t i o n s i n l a c l . A l a r g e c l a s s o f base s u b s t i t u t i o n mutants can be a n a l y z e d d i ­ r e c t l y by s c r e e n i n g f o r s u p p r e s s i b l e m u t a t i o n s among t h e c o l l e c t i o n of l a c l mutants. The s u p p r e s s i b l e m u t a t i o n s a r e due t o w i l d - t y p e codons h a v i n g been mutated t o TAA, TAG, o r TGA. These nonsense c o dons a r e n o r m a l l y s i g n a l s f o r t h e t e r m i n a t i o n o f p r o t e i n s y n t h e s i s by ribosomes and c a n a r i s e v i a a l l s i n g l e base p a i r s u b s t i t u t i o n muta­ t i o n s w i t h t h e e x c e p t i o n o f t h e A:T t o G:C t r a n s i t i o n . Thus, a l l base p a i r s u b s t i t u t i o n s , e x c e p t f o r t h e one t r a n s i t i o n , can be moni­ t o r e d by c o l l e c t i n g nonsense m u t a t i o n s i n l a c l . There a r e o v e r 60 s i t e s i n l a c l a t w h i c h a s i n g l e base p a i r s u b s t i t u t i o n w i l l g e n e r a t e a nonsense codon. L a c l nonsense mutants can be i d e n t i f i e d u s i n g c l a s s i c a l b a c t e r ­ i a l g e n e t i c methods. The e n t i r e gene has been sequenced ( 5 2 ) . The s i t e , and t h e r e f o r e , t h e base p a i r w h i c h has been mutated c a n be in­ dent i f l e d s i m p l y by mapping t h e p o s i t i o n o f t h e nonsense m u t a t i o n . T h i s can be a c c o m p l i s h e d by u s i n g an e x t e n s i v e s e t o f l a c l d e l e t i o n mutants ( 5 3 ) . Mapping t h e m u t a t i o n a l l o w s one t o determine w h i c h base p a i r s u b s t i t u t i o n has been g e n e r a t e d by a p a r t i c u l a r t r e a t m e n t . Thus, by i d e n t i f y i n g many nonsense m u t a t i o n s induced by a mutagen, a p i c t u r e emerges o f b o t h t h e mutagens s i t e s p e c i f i c i t y (where, w i t h i n the gene t h e m u t a t i o n s a r i s e ) and i t s mutagenic s p e c i f i c i t y (which p a r t i c u l a r base s u b s t i t u t i o n s a r e g e n e r a t e d ) . To d e t e r m i n e c l a s s e s of m u t a t i o n o t h e r than base p a i r s u b s t i t u t i o n s , i t i s p o s s i b l e t o g e n e t i c a l l y c r o s s a g i v e n l a c l a l l e l e o n t o s m a l l p l a s m i d o r phage m o l e c u l e s and d e t e r m i n e t h e sequence o f t h e mutant a l l e l e (54,55)• We have a p p l i e d t h e g e n e t i c system f o r a n a l y z i n g l a c l nonsense mutants t o t h e i n v e s t i g a t i o n o f t h e mutagenic s p e c i f i c i t y o f benzo[a]pyrene (56) and a f l a t o x i n B. ( 5 7 ) . The r e s u l t s o f our s t u d i e s have p r o v i d e d some i m p o r t a n t c l u e s as t o t h e c h e m i c a l n a t u r e o f t h e mutagenic l e s i o n s induced by b e n z o [ a ] p y r e n e . B e f o r e I d i s c u s s these r e s u l t s , I w i l l b r i e f l y summarize p r e v i o u s i n v e s t i g a t i o n s on the mu­ t a g e n i c i t y o f BPDE. The m u t a g e n i c i t y inves t igat ions

of benzo[a]pyrene d i o l e p o x i d e —

previous

C a r c i n o g e n s f i r s t began t o be e v a l u a t e d d i r e c t l y f o r mutagenic a c -

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

332

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

t i v i t y a g a i n s t microorganisms 35 y e a r s ago by B a r r e t t and Tatum ( 5 8 ) . However, the s y s t e m a t i c use of s e n s i t i v e m i c r o b i a l m u t a t i o n a s s a y s t o m o n i t o r the b i o l o g i c a l a c t i v i t y o f c a r c i n o g e n s was not a c h i e v e d u n t i l the r e a l i z a t i o n t h a t m e t a b o l i c a c t i v a t i o n o f c a r c i n o g e n s was e s s e n ­ t i a l ( r e v i e w e d i n 5 9 ) . By the use of s u b - c e l l u l a r f r a c t i o n s d e r i v e d from l i v e r homogenates, i t became p o s s i b l e t o d e t e c t the mutagenic a c t i v i t y of benzo[a]pyrene and many o t h e r p o l y c y c l i c a r o m a t i c h y d r o ­ carbons • The m u t a g e n i c i t y of benzo[a]pyrene f o r b a c t e r i a was demonstrated by Ames e t a l . ( 6 0 ) . They found t h a t i n the presence o f r a t l i v e r homogenates benzo[a]pyrene i n d u c e d b o t h f r a m e s h i f t and b a s e - p a i r sub­ s t i t u t i o n m u t a t i o n s . When the c h e m i s t r y of benzo[a]pyrene a c t i v a t i o n had been worked out and t h e u l t i m a t e c a r c i n o g e n i c form i d e n t i f i e d as a d i o l e p o x i d e , BPDE ( r e v i e w e d i n 61-62), s e v e r a l i n v e s t i g a t o r s ( 6 3 66) showed t h a t BPDE was an e x t r e m e l y p o t e n t mutagen, a l s o c a p a b l e o f i n d u c i n g b o t h f r a m e s h i f t and b a s e - p a i r s u b s t i t u t i o n m u t a t i o n s . McCann e t a l . (67) had shown t h a t benzo[a]pyrene was mutagenic f o r J5. typhimurium o n l y i f the b a c t e r i a c a r r i e d the m u t a t i o n enhancing p l a s mid pKMlOl whose a c t i v i t y was l a t e r shown by Walker (23) t o be en­ t i r e l y dependent on b a c t e r i a l r e c A and l e x A c o n t r o l l e d f u n c t i o n s . T h i s p r o v i d e d e a r l y e v i d e n c e t h a t the m u t a g e n i c i t y o f c a r c i n o g e n s such as b e n z o [ a ] p y r e n e was dependent on S O S - r e p a i r . L a t e r , I v a n o v i c and W e i n s t e i n (68) d i r e c t l y showed t h a t benzo[a]pyrene was mutagenic +

f o r JE. c o l i o n l y i f the b a c t e r i a were b o t h r e c A and l e x A * . Two q u e s t i o n s t h a t a r e r a i s e d by t h e s e o b s e r v a t i o n s a r e : 1) what i s the mutagenic s p e c i f i c i t y o f BPDE, i_.je. what k i n d s o f m u t a t i o n s are i n d u c e d by t r e a t i n g c e l l s w i t h BPDE? 2) what i s ( a r e ) the p r e - m u t a t i o n a l l e s i o n ( s ) g e n e r a t e d by BPDE which i s ( a r e ) r e s p o n s i b l e f o r mutations? The mutagenic

s p e c i f i c i t y o f BPDE

We have o b t a i n e d i m p o r t a n t c l u e s t o t h e s e q u e s t i o n s by d e t e r m i n i n g the spectrum of 185 nonsense m u t a t i o n s induced i n the l a c l gene o f IS. c o l i by BPDE. The r e s u l t s o f t h i s i n v e s t i g a t i o n (56) a r e summarized i n T a b l e s I t o I I I and i n F i g u r e 1. The r e s u l t s were s t r i k i n g . They showed t h a t t r a n s v e r s i o n m u t a t i o n s a t G:C base p a i r s were the domi­ nant i n d u c e d e v e n t , a l t h o u g h , o t h e r s u b s t i t u t i o n s , i n p a r t i c u l a r A:T to T:A t r a n s v e r s i o n were c l e a r l y i n d u c e d , but a t l o w e r f r e q u e n c i e s . The s p e c i f i c i t y of i n d u c t i o n o f G:C t o T:A was most c l e a r l y seen by examining the m u t a t i o n s o c c u r r i n g a t the TAC codons f o r t y r o s i n e ( T a ­ ble I I I ) . A t t h e s e s i t e s , b o t h G:C t o T:A m u t a t i o n s ( y i e l d i n g TAA, ochre nonsense mutants) and G:C t o C:G ( y i e l d i n g TAG, amber nonsense mutants) t r a n s v e r s i o n s are m o n i t o r a b l e . T a b l e I I I c l e a r l y shows t h a t at the two t y r o s i n e codons where m u t a t i o n s were w e l l - i n d u c e d t h e r e i s a s t r i k i n g p r e f e r e n c e f o r one m u t a t i o n a l event over the o t h e r . We have not d i r e c t l y determined t h e r e l a t i v e f r e q u e n c i e s o f f r a m e s h i f t m u t a t i o n s and o t h e r m u t a t i o n a l e v e n t s i n comparison t o the base s u b s t i t u t i o n m u t a t i o n s . However, based on the h i g h f r e q u e n c y o f nonsense m u t a t i o n s (11%) among a l l l a c l mutants induced by BPDE and because nonsense m u t a t i o n s a r e m o n i t o r a b l e a t l e s s than o n e - f i f t h o f the l a c l codons and, even t h e n , o n l y v i a c e r t a i n base p a i r s u b s t i t u ­ t i o n s , we b e l i e v e t h a t base s u b s t i t u t i o n s account f o r a major f r a c ­ t i o n o f m u t a t i o n s induced by BPDE.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13.

EISENSTADT

Mutational Consequences of DNA Damage

333

T a b l e I . Summary of Base S u b s t i t u t i o n Events Generated by BPDE

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Substitution

No. o f Available Sites

No. o f Sites Found

T o t a l No. o f Occurrences

% of Analyzed Mutations

G:C t o A:T

26

12

22

12

G:C t o T:A

23

21

123

66

A:T t o T:A

15

33

18

9

A:T t o C:G

5

3

4

2

G:C t o C:G

3

2

3

2

Total

72

47

185

Amber

36

26

96

Ochre

36

21

89

What i s ( a r e ) the p r e - m u t a t i o n a l

l e s i o n ( s ) i n d u c e d by BPDE?

BPDE r e a c t s a t s e v e r a l d i f f e r e n t s i t e s on DNA t o g e n e r a t e s e v e r a l k i n d s o f l e s i o n s a t t h e N2 (43-45) and N7 (46,47) p o s i t i o n s o f guan­ i n e , a p u r i n i c s i t e s ( 4 8 , 4 9 ) , and s t r a n d b r e a k s ( 5 0 ) . Which o f t h e s e l e s i o n s a r e r e s p o n s i b l e f o r t h e t r a n s v e r s i o n m u t a t i o n s a t G:C s i t e s ? E v i d e n c e d e r i v e d from a number o f e x p e r i m e n t s s u g g e s t s t h e h y p o t h e s i s t h a t a p u r i n i c s i t e s g e n e r a t e d by BPDE r e a c t i o n s w i t h DNA a r e r e s p o n ­ s i b l e f o r the transversion mutations: 1. When we examined t h e mutagenic s p e c i f i c i t y a f l a t o x i n B^, a c a r c i n o g e n w h i c h s p e c i f i c a l l y r e a c t s w i t h t h e N7 atom o f guanine ( 3 9 4 2 ) , we found v i r t u a l l y o n l y G:C t o T:A t r a n s v e r s i o n s were i n d u c e d ( 5 7 ) ; N7 p u r i n e adducts can i n d u c e d e p u r i n a t i o n by d e s t a b i l i z i n g t h e N - g l y c o s y l i c bond ( 6 9 ) . 2. The work o f Loeb and K u n k e l and t h e i r c o l l e a g u e s (70-72) h a s c l e a r l y e s t a b l i s h e d t h a t a p u r i n i c s i t e s i n DNA a r e mutageniC.; t h e y s p e c i f i c a l l y cause t r a n s v e r s i o n m u t a t i o n s , due t o a s t r o n g p r e f e r e n c e f o r t h e i n c o r p o r a t i o n o f adenine r e s i d u e s d u r i n g bypass o f a p u r i n i c s i t e s i n t e m p l a t e DNA. Thus, A:T t o T:A and G:C t o T:A t r a n s v e r s i o n s a r e t h e major mutagenic outcome g e n e r a t e d by d e p u r i n a t i o n o f DNA. 3. R e c e n t l y , Sage and H a s e l t i n e (49) have q u a n t i t a t i v e l y d e t e r ­ mined t h e spectrum o f DNA l e s i o n s i n d u c e d by r e a c t i o n s o f BPDE w i t h DNA. They found t h a t a l k a l i - l a b i l e l e s i o n s account f o r about 40% o f the DNA a d d u c t s . There was a s t r i k i n g c o r r e l a t i o n between t h e muta­ t i o n f r e q u e n c i e s induced by BPDE i n l a c l and t h e f r e q u e n c i e s o f a l k a ­ l i s e n s i t i v e l e s i o n s a t G, A, and C r e s i d u e s . Apurinic/apyrimidinic s i t e s a r e common a l k a l i - s e n s i t i v e l e s i o n s . E a r l i e r work by D r i n k ­ w a t e r e t a l . ( 4 8 ) had a l s o shown t h a t t r e a t m e n t o f DNA w i t h BPDE gen­ erated a p u r i n i c / a p y r i m i d i n i c s i t e s .

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

334

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS Table I I .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Base Substitutions

D i s t r i b u t i o n o f l a c l Nonsense M u t a t i o n s Induced by BPDE

Site

No. Independent Occurrences

Site

No. Independent Occurrences 0 0 0 0 0 3 3 2 1 1 0 0

G:C - A:T

A5 *A6 A9 *A15 A16 A19 A21 A23 A24 A26 A31 A33 *A34 A35

0 5 0 0 0 1 2 0 1 0 1 1 1 0

09 010 Oil 013 017 021 024 027 028 029 034 035

G:C - T:A„ G:C - C:G

A2 A7 A10

7 5 5

A12 A13 A17 A20 A25 A27 A28

18 5 5 5 4 5 3

03,A1 06 07 „ 08, A8 014 015 019 020 025 026 030,A29 031 032 036

#

12,1 0 0 4,0 2 7 4 12 0 4 1,2 2 1 12

#

(13)

(4)

(3)

2 5 0 (0) 0*>A3^ 05,A4 2 (2) 0 012 6 0 018,A14 1 (1) 4 A 023,A22^ 0 (0) 033,A30 1 (1) S i t e s a t w h i c h nonsense m u t a t i o n s a r e d e t e c t e d a r e i d e n t i f i e d by t h e i r amber (A) o r o c h r e (0) a l l e l e s ( C o u l o n d r e and M i l l e r , 1977). The 8 t y r o s i n e codons i n l a c l each have two nonsense a l l e l e s , one amber and one o c h r e . The amber a l l e l e s a t t h e s e s i t e s a r e marked by the symbols # and @. * S i t e s c o n t a i n i n g 5 - m e t h y l c y t o s i n e s (CCAGG). These a r e spontaneous l a c l hotspots.

A:T - T:A A:T - C:G

0

All A18 A32 A36

2 4 1 8

01

&

0 2

L

0 1 6

L

0

2

2

L

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

13.

EISENSTADT

Table I I I .

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Site

BPDE-Induced M u t a t i o n s a t the Three TAC T y r o s i n e Codons i n the l a c l Gene

coding p o s i t i o n

^,03

tyr 7

A8,08 A29,030 Total

335

Mutational Consequences of DNA Damage

# o f independent o c c u r r e n c e s o f : TAC - TAA TAC - TAA (GC - CG) (GC - TA)

12

1

t y r 47

4

0

tyr

1

2

17

3

273

Both amber and o c h r e m u t a t i o n s can be g e n e r a t e d a t t h e s e s i t e s , a l l o w i n g b o t h G:C t o T:A and G:C t o C:G t r a n s v e r s i o n s t o be monitored.

Thus, w h i l e BPDE and a f l a t o x i n might g e n e r a t e G:C t o T:A t r a n s v e r s i o n s v i a d i f f e r e n t pathways, i t i s r e a s o n a b l e t o c o n s i d e r the h y p o t h e s i s t h a t t h e r e i s a common mechanism by w h i c h t h e y i n d u c e t h i s m u t a t i o n and t h a t , t h e r e f o r e the t r a n s v e r s i o n m u t a t i o n s induced by BPDE r e s u l t , not from the major adduct t o the N2 atom o f guanine but from the g e n e r a t i o n o f a p u r i n i c s i t e s i n DNA. These secondary l e s i o n s might be g e n e r a t e d s p o n t a n e o u s l y o r v i a the a c t i v i t y of DNA g l y c o s y l a s e s (2_, 3 0 ) . An a l t e r n a t i v e h y p o t h e s i s i s t h a t b u l k y l e ­ s i o n s i n g e n e r a l , the N2 adduct among them, may be n o n i n f o r m a t i o n a l s i t e s o p p o s i t e w h i c h adenines a r e p r e f e r e n t i a l l y i n s e r t e d d u r i n g r e p ­ l i c a t i o n a f t e r DNA damage. Other m o l e c u l a r g e n e t i c s t u d i e s on the m u t a g e n i c i t y o f BPDE The g e n e t i c system we used t o s t u d y the mutagenic s p e c i f i c i t y o f BPDE l i m i t s one t o a n a l y z i n g base s u b s t i t u t i o n m u t a t i o n s . What i s known about the a b i l i t y o f BPDE t o i n d u c e o t h e r c a t e g o r i e s o f m u t a t i o n ? As mentioned above, r e s u l t s from the Ames t e s t r e v e a l e d t h a t b e n z o [ a ] p y ­ rene and i t s d i o l e p o x i d e were c a p a b l e o f i n d u c i n g f r a m e s h i f t muta­ t i o n s (60,63,64). More r e c e n t l y , Mizusawa and co-workers (73-76) have i n v e s t i g a t e d the m u t a t i o n a l consequences o f m o d i f y i n g p l a s m i d DNA i n v i t r o w i t h BPDE. I n a s e r i e s o f s t u d i e s t h e y have shown t h a t : _ 1. p l a s m i d m o l e c u l e s a r e i n a c t i v a t e d (become n o n - r e p l i c a b l e ) i n Uvr b a c t e r i a by 1 c o v a l e n t adduct (73,76) per m o l e c u l e ; t h i s r e s u l t i s i n p e r f e c t agreement w i t h an e a r l i e r r e p o r t by Hsu eit a l . (77) w h i c h had demonstrated t h a t one m o l e c u l e o f bound BPDE was s u f f i c i e n t

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

GC-^AT

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

10-,

I

I

20p

GC-^TA

10 +

A

I II I ,l I ll GC-^CG

AT-^TA

IX AT-^CG

10r

oL 100

200

300

AMINO ACID RESIDUE F i g u r e 1. The f r e q u e n c i e s o f amber m u t a t i o n s i n t h e l a c l gene i n d u c e d by BPDE. S o l i d b a r s , i n d i v i d u a l s i t e s a t w h i c h we d e t e c t e d m u t a t i o n s ; open b a r s , s i t e s a t w h i c h we d i d not detect mutations; a s t e r i s k s ( i n a ) , s i t e s a t which the t a r g e t codon c o n t a i n s 5 - m e t h y l c y t o s i n e .

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to i n h i b i t t h e r e p l i c a t i o n o f s i n g l e m o l e c u l e o f t h e s i n g l e s t r a n d e d b a c t e r i o p h a g e 0X174. 2. BPDE-induced m u t a t i o n s i n p l a s m i d borne genes c a n be depen­ dent on umuC ( 7 5 ) ; 3. m u t a t i o n s induced by BPDE i n c l u d e t r a n s v e r s i o n , t r a n s i t i o n , and f r a m e s h i f t m u t a t i o n s ( 7 4 , 7 6 ) . Wei e t a l . ( 7 8 ) c h a r a c t e r i z e d m u t a t i o n s r e s u l t i n g from a l k y l a ­ t i o n o f a 10-base p a i r o l i g o n u c l e o t i d e w i t h BPDE. D e l e t i o n m u t a t i o n s were t h e major m u t a t i o n a l event d e t e c t e d . The number o f m u t a t i o n s a n a l y z e d i n each o f t h e s e i n v e s t i g a t i o n s was t o o s m a l l ( o n l y 7 t o 8) t o p e r m i t drawing f i r m c o n c l u s i o n s about m u t a t i o n a l and s i t e s p e c i f i c i t i e s . However, t h e r e s u l t s suggest t h a t , under some c i r c u m s t a n c e s , BPDE c a n i n d u c e many d i f f e r e n t k i n d s of m u t a t i o n s . F u t u r e s t u d i e s on t h e g e n e t i c e f f e c t s o f BPDE To r i g o u r o u s l y e s t a b l i s h t h e g e n e t i c consequences r e s u l t i n g from BP adduct t o t h e N2 p o s i t i o n o f g u a n i n e , t h e approach t a k e n by Essigman and h i s c o l l e a g u e s (79,80) w i l l be r e q u i r e d . They have been d e v e l o p ­ i n g t e c h n i q u e s f o r p l a c i n g d e f i n e d c h e m i c a l l e s i o n s i n t o p l a s m i d DNA at pre-determined s i t e s a t which m u t a t i o n s c a n be m o n i t o r e d . If a BP adduct can be " b u i l t " i n t o DNA a t t h e N2 o f g u a n i n e , i t s b i o l o ­ g i c a l and g e n e t i c e f f e c t s can be d e t e r m i n e d . I t would be i n t e r e s t i n g t o know i f t h e m u t a t i o n a l consequences of DNA l e s i o n s i n mammalian c e l l s were t h e same as t h o s e w h i c h o b t a i n i n b a c t e r i a . Methods f o r r e t r i e v i n g and sequencing m u t a t i o n s i n mam­ m a l i a n c e l l s and t h e i r v i r u s e s a r e now b e i n g developed ( 8 1 - 8 3 ) . I f y e a s t , a e u k a r y o t i c m i c r o o r g a n i s m , c a n be c o n s i d e r e d r e p r e s e n t a t i v e of h i g h e r e u k a r y o t e s , then j u d g i n g from t h e o b s e r v a t i o n s t h a t t h e m u t a t i o n a l s p e c t r a f o r U V - i r r a d i a t i o n and 4 - n i t r o q u i n o l i n e - l - o x i d e t r e a t m e n t a r e i d e n t i c a l f o r y e a s t (84) and b a c t e r i a ( 8 5 ) , t h e spec­ trum o f m u t a t i o n s induced by BPDE i n mammalian c e l l s c o u l d w e l l r e ­ semble those induced i n IS. c o l i . I s t h e m u t a g e n i c i t y o f BPDE d i r e c t l y r e s p o n s i b l e f o r i t s carcinogenicity? Though one i s f a r from b e i n g a b l e t o make a d e f i n i t i v e s t a t e m e n t , t h e r e a r e some i n d i c a t i o n s t h a t t h e answer t o t h i s q u e s t i o n might be no. Recent experiments w i t h mammalian c e l l c u l t u r e systems, w h i c h a l l o w one t o s t u d y t h e p r o g r e s s i o n o f c e l l s from a s t a t e where t h e i r growth i s n o r m a l l y r e g u l a t e d t o a t u m o r i g e n i c s t a t e have r e v e a l e d t h a t t h e t r a n s f o r m a t i o n p r o c e s s r e q u i r e s a t l e a s t two s t e p s ( 8 6 - 8 8 ) • The f i r s t s t e p , which i s induced f o l l o w i n g exposure t o a c a r c i n o g e n ( X - r a y s : 86,88; 3 - m e t h y l c h o l a n t h r e n e : 8 7 ) , o c c u r s w i t h a v e r y h i g h f r e q u e n c y and s e e m i n g l y a f f e c t s e v e r y exposed c e l l i n t h e t r e a t e d p o p u l a t i o n . The f r e q u e n c y o f t h e i n i t i a l event i m p l i e s t h a t i t i s not a m u t a t i o n a l event b u t r a t h e r an e p i g e n e t i c one r e l a t e d , perhaps, to t h e responses i n d u c e d by DNA damage i n IS. c o l i and S* c e r e v i s i a e . The second event i s a v e r y r a r e event c o n s i s t e n t w i t h t h e p o s s i b i l i t y t h a t i t might be m u t a t i o n a l i n n a t u r e . However, s i n c e t h e second event o c c u r s many c e l l g e n e r a t i o n s a f t e r t h e exposure t o DNA damaging a g e n t s , i t seems h i g h l y improbable t h a t i t o c c u r s as a d i r e c t conse­ quence o f r e p a i r i n g t h e i n i t i a l DNA damage. Thus, t h e l i n k between

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c a r c i n o g e n e s i s and mutagenesis may s i m p l y be t h a t t h e two p r o c e s s e s o r i g i n a t e from t h e same s t a r t i n g p o i n t , namely DNA damage. Acknowledgments Work i n my l a b o r a t o r y has been supported by g r a n t s from t h e NIH. I am i n d e b t e d t o many o f my p r e s e n t and former c o l l e a g u e s , i n p a r t i c u ­ l a r t o D r s . A . J . Warren and P.L. F o s t e r f o r t h e i r work on the muta­ g e n i c s p e c i f i c i t y o f c h e m i c a l c a r c i n o g e n s and t o D r . J.H. M i l l e r f o r h i s c o l l a b o r a t i v e e f f o r t i n s t u d y i n g t h e mutagenic s p e c i f i c i t y o f b e n z o [ a ] p y r e n e and a f l a t o x i n B^.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

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14 Chemical Properties of Ultimate Carcinogenic Metabolites of Arylamines and Arylamides FRED F. KADLUBAR and FREDERICK A. BELAND

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch014

National Center for Toxicological Research, Jefferson, AR 72079

A number of arylamines and arylamides are carcinogenic in a variety of tissues of several species including the urinary bladder of man. These compounds undergo metabolic activation to ultimate carcinogens through a number of enzymatic and nonenzymatic pathways. In this review, these activation mechanisms are considered in detail and their relative con­ tribution to the observed carcinogenicity of these compounds is discussed. The metabolism o f c a r c i n o g e n i c a r y l a m i n e s and a r y l a m i d e s r e s u l t s i n a broad spectrum o f r e a c t i v e , e l e c t r o p h i l i c m e t a b o l i t e s t h a t form c o v a l e n t adducts w i t h c e l l u l a r c o n s t i t u e n t s . These a c t i v a t i o n p a t h ­ ways a r e summarized i n F i g u r e I . Arylamides and p r i m a r y a r y l a m i n e s are r e a d i l y i n t e r c o n v e r t e d by N - a c e t y l t r a n s f e r a s e s and N-deacetyl a s e s (reviewed i n 1) and they a r e i n i t i a l l y a c t i v a t e d by cytochrome P-450- and f l a v i n - c o n t a i n i n g monooxygenases t o form N-hydroxy a r y l ­ amides and N-hydroxy a r y l a m i n e s , r e s p e c t i v e l y (reviewed i n 2_). These N-hydroxy m e t a b o l i t e s , which can a l s o be i n t e r c o n v e r t e d by enzymatic N - d e a c e t y l a t i o n / N - a c e t y l a t i o n (1_), a r e proximate c a r c i n o g e n s s i n c e they a r e g e n e r a l l y more c a r c i n o g e n i c and mutagenic than t h e i r parent compounds. F u r t h e r enzymatic o r non-enzymatic p r o c e s s e s lead to u l t i m a t e c a r c i n o g e n s , which a r e u s u a l l y d e f i n e d by t h e i r e l e c t r o ­ p h i l i c r e a c t i v i t y w i t h n u c l e i c acids or p r o t e i n s 03). N-Hydroxy a r y l a m i d e s a r e c o n v e r t e d t o u l t i m a t e carcinogens through conjugation with s u l f u r i c , a c e t i c or g l u c u r o n i c acids (reviewed i n J ^ , 4 ) . S u l f u r i c a c i d c o n j u g a t i o n i s c a t a l y z e d by 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulf©trans­ f e r a s e s and y i e l d s N - s u l f o n y l o x y a r y l a m i d e s ( I ) ; w h i l e N-acetoxy a r y l a m i d e s ( I I ) a r e formed through nonenzymatic e s t e r i f i c a t i o n w i t h a c e t y l coenzyme A o r by a p e r o x i d a s e - m e d i a t e d , one-electron oxida­ t i o n and s u b s e q u e n t d i s m u t a t i o n o f a n i t r o x y l r a d i c a l (5^,6^). N - G l u c u r o n y l o x y a r y l a m i d e s ( i l l ) a r e a l s o formed by enzymatic con­ j u g a t i o n and they can undergo subsequent N - d e a c e t y l a t i o n t o N - g l u c u r o n y l o x y a r y l a m i n e s ( I V ) . An a d d i t i o n a l pathway by which N-hydroxy a r y l a m i d e s a r e a c t i v a t e d i s through an i n t r a m o l e c u l a r rearrangement to N-acetoxy arylamines ( V ) w h i c h i s c a t a l y z e d by c y t o s o l i c N,0-acyltransferases. This chapter not subject to U.S. copyright. Published 1985, American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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N-Hydroxy a r y l a m i n e s a r e a l s o c o n v e r t e d t o N-acetoxy a r y l a m i n e s ( V ) , b u t a p p a r e n t l y by an a c e t y l coenzyme A-dependent enzymatic O-esterification 0,8). S i m i l a r l y , N-sulfonyloxy arylamines ( V I ) are thought t o a r i s e by a PAPS-dependent enzymatic O - s u l f o n y l a t i o n o f N-hydroxy a r y l a m i n e s ( 9 , 1 0 ) ; w h i l e 0 - s e r y l o r 0 - p r o l y l e s t e r s ( V I I ) a r e formed by t h e i r c o r r e s p o n d i n g aminoacyl tRNA s y n t h e t a s e s i n a ATP-dependent r e a c t i o n ( 1 1 , 1 2 ) . N-Hydroxy a r y l a m i n e s r e a d i l y form g l u c u r o n i d e c o n j u g a t e s , b u t i n c o n t r a s t t o the N-hydroxy a r y l a m i d e s , these are N - g l u c u r o n i d e s which are u n r e a c t i v e and s t a b l e a t n e u t r a l pH. The N - g l u c u r o n i d e s a r e r e a d i l y t r a n s p o r t e d t o t h e lumens o f t h e u r i n a r y b l a d d e r and i n t e s ­ t i n e where they can be h y d r o l y z e d t o the f r e e N-hydroxy a r y l a m i n e s by m i l d l y a c i d i c u r i n e o r by i n t e s t i n a l b a c t e r i a l 3 - g l u c u r o n i d a s e s ( 1 3 , 1 4 ) . Non-enzymatic a c t i v a t i o n o f N-hydroxy a r y l a m i n e s c a n occur i n an a c i d i c environment by p r o t o n a t i o n (15,16) o f t h e N-hydroxy group ( V I I I ) as w e l l as by a i r o x i d a t i o n ( r e v i e w e d i n 17) t o a nitrosoarene (IX). A l t e r n a t i v e m e t a b o l i c p a t h w a y s i n v o l v e r i n g - o x i d a t i o n and peroxidation of arylamines. Although r i n g - o x i d a t i o n i s g e n e r a l l y c o n s i d e r e d a d e t o x i f i c a t i o n r e a c t i o n , an e l e c t r o p h i l i c iminoquinone (X) can be formed by a secondary o x i d a t i o n o f t h e aminophenol m e t a b o l i t e ( J ^ 8 , J _ 9 ) . L a s t l y , r e a c t i v e imines ( X I ) can be formed from the p r i m a r y a r y l a m i n e s by p e r o x i d a s e - c a t a l y z e d r e a c t i o n s t h a t i n v o l v e free r a d i c a l intermediates (reviewed i n 20). Only a l i m i t e d number o f a c t i v a t i o n pathways appear t o be a v a i l a b l e t o N-methyl a r y l a m i n e s . F o l l o w i n g enzymatic N - h y d r o x y l a t i o n t o secondary N-hydroxy a r y l a m i n e s (21,22), these compounds a r e c o n v e r t e d i n t o r e a c t i v e e l e c t r o p h i l e s through enzymatic e s t e r i f i c a t i o n (9) t o N - s u l f o n y l o x y - N - m e t h y l a r y l a m i n e s ( X I I ) o r by f u r t h e r oxidation to N-arylnitrones (XIII). I n t h i s r e v i e w , t h e c h e m i c a l p r o p e r t i e s o f these e l e c t r o p h i l i c m e t a b o l i t e s ( I - X I I I ) a r e d i s c u s s e d i n terms o f t h e i r m e t a b o l i c f o r m a t i o n and r e a c t i v i t y w i t h n u c l e o p h i l e s , s o l v o l y s i s and redox c h a r a c t e r i s t i c s , r e a c t i o n mechanisms, and t h e i r r o l e as u l t i m a t e carcinogenic metabolites. N-Sulfonyloxy Arylamides ( i ) The m e t a b o l i c f o r m a t i o n o f N - s u l f o n y l o x y - N - a c e t y l - 2 - a m i n o f l u o r e n e (N-sulfonyloxy-AAF) and i t s observed electrophilic reactivity, p r o v i d e d the f i r s t e v i d e n c e f o r t h e importance o f enzymatic con­ j u g a t i o n r e a c t i o n s i n chemical c a r c i n o g e n e s i s (23,24). This r e a c t i o n was shown t o be c a t a l y z e d by PAPS-dependent s u l f o t r a n s f e r a s e s t h a t a r e l o c a t e d p r e d o m i n a n t l y i n l i v e r c y t o s o l and has been s u b s e q u e n t l y demonstrated f o r N-hydroxy a r y l a m i d e m e t a b o l i t e s o f s e v e r a l other carcinogens, i n c l u d i n g N-acety1-4-aminobipheny1 (AABP), b e n z i d i n e , N-acetyl-2-aminophenanthrene and p h e n a c e t i n . Accordingly, thecontribution of this metabolic activation pathway t o t h e f o r m a t i o n o f c o v a l e n t l y - b o u n d adducts o f a r y l a m i d e s w i t h c e l l u l a r p r o t e i n s and n u c l e i c a c i d s has been t h e s u b j e c t o f numerous i n v e s t i g a t i o n s , and has been reviewed e x t e n s i v e l y by Mulder (25). From t h e s e and more r e c e n t d a t a (4,26,27) i t i s a p p a r e n t , p a r t i c u l a r l y i n t h e case o f N-hydroxy-AAF TN-OH-AAF), t h a t _in v i v o formation of reactive N-sulfonyloxy derivatives i s primarily

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r e s p o n s i b l e f o r the c a r c i n o g e n adducts w i t h h e p a t i c p r o t e i n , RNA, DNA and g l u t a t h i o n e (GSH) t h a t r e t a i n the N - a c e t y l group. With N-OH-AAF, f o r e x a m p l e , t h e s e N - a c e t y l a t e d a d d u c t s a c c o u n t f o r 70-80%, 60-80%, and 15-30% of the t o t a l b i n d i n g to r a t l i v e r p r o t e i n , RNA, and DNA, r e s p e c t i v e l y (28,30); and GSH-AAF adducts e x c r e t e d i n the b i l e account f o r about 10% of the dose g i v e n ( 2 6 ) . I n comparable s t u d i e s w i t h N-hydroxy-AABP (N-OH-AABP), N - a c e t y l a t e d adducts r e p r e s e n t about 10% and 20% of the RNA and DNA b i n d i n g , r e s p e c t i v e l y (3C0 ; and N - a c e t y l a t e d adducts d e r i v e d from 4 ' - f l u o r o N-OH-AABP and N , N ' - d i a c e t y l b e n z i d i n e amount t o 10-20% of the t o t a l DNA-bound p r o d u c t s (_3jL^,_32_). I n c o n t r a s t , o n l y d e a c e t y l a t e d adducts are d e t e c t a b l e i n r a t h e p a t i c DNA a f t e r a d m i n i s t r a t i o n of N - a c e t y l 4 - a m i n o s t i l b e n e (33) or N - a c e t y l - 7 - f l u o r o - 2 - a m i n o f l u o r e n e ( 3 4 ) , both of which induce tumors i n the l i v e r and o t h e r t i s s u e s . S i m i l a r l y , o n l y d e a c e t y l a t e d DNA adducts are found i n r a t l i v e r a f t e r treatment w i t h the e x t r a h e p a t i c a r y l a m i d e c a r c i n o g e n , N-acetyl-2-aminophenanthrene ( 3 5 ) , or w i t h the N - h y d r o x y - N - a c e t y l d e r i v a t i v e of the c o l o n carcinogen, 3,2'-dimethyl-4-aminobiphenyl (36). S t r u c t u r a l i d e n t i f i c a t i o n of the N - a c e t y l a t e d adducts found i n v i v o has shown t h a t b i n d i n g t o p r o t e i n or GSH i n v o l v e s p r e d o m i n a n t l y o r t h o - r i n g s u b s t i t u t i o n of the a r y l a m i d e w i t h the s u l f u r atom i n m e t h i o n i n e or c y s t e i n e , r e s p e c t i v e l y . I n c o n t r a s t , a r y l a m i d e b i n d i n g t o n u c l e i c a c i d s in. v i v o i n v o l v e s both ^ - s u b s t i t u t i o n at the C-8 p o s i t i o n of guanine and o r t h o - r i n g s u b s t i t u t i o n w i t h the e x o c y c l i c N atom of guanine (26,29-31,37,38). S e v e r a l s y n t h e t i c N - s u l f o n y l o x y a r y l a m i d e s have been prepared i n o r d e r t o compare t h e i r r e a c t i v i t y w i t h n u c l e o p h i l e s t o t h a t observed i n v i v o and i n i n v i t r o m e t a b o l i c systems. S y n t h e t i c N - s u l f o n y l o x y AAF r e a c t s a p p r e c i a b l y w i t h b o t h p r o t e i n or m e t h i o n i n e t o g i v e h i g h y i e l d s of ortho-methylmercapto d e r i v a t i v e s t h a t are i d e n t i c a l to those formed jLn v i v o . S i m i l a r l y , m e t h i o n i n e has been shown to t r a p 65-85% of N - s u l f o n y l o x y - A A F generated i n i n c u b a t i o n s c o n t a i n i n g PAPS, N-OH-AAF, and h e p a t i c c y t o s o l i c sulf©transferase ( 9 ) . NS u l f o n y l o x y - A A F a l s o r e a c t s w i t h GSH Ln v i t r o t o g i v e 1-, 3-, 4-, and 7-AAF r i n g - s u b s t i t u t e d g l u t a t h i o n - S - y l adducts ( 3 9 ) , of which two ( 1 - , 3-) are major b i l i a r y m e t a b o l i t e s ( 2 6 ) . N-(Guanosin-8-yl)AAF, a major i n v i v o adduct w i t h h e p a t i c RNA, can be prepared by r e a c t i o n of guanosine w i t h N - s u l f o n y l o x y - A A F or by _in v i t r o s u l f o t r a n s f e r a s e a c t i v a t i o n of N-OH-AAF i n the presence of RNA or guano­ s i n e (40_). R e a c t i o n of N - s u l f onyloxy-AAF w i t h DNA y i e l d s both N-(deoxyguanosin-8-yl)-AAF and 3 - ( d e o x y g u a n o s i n - N - y l ) - A A F , which are i d e n t i c a l t o the N - a c e t y l a t e d adducts found _in v i v o (30,41). However, a s i m i l a r r e a c t i o n w i t h deoxyguanosine i n an aqueous medium g i v e s o n l y the C 8 - s u b s t i t u t e d p r o d u c t ; w h i l e both C8- and ^ - s u b s t i ­ t u t e d adducts can be prepared by r e a c t i o n of N - s u l f o n y l o x y - A A F w i t h deoxyguanosine i n anhydrous d i m e t h y l s u l f o x i d e / t r i e t h y l a m i n e ( 4 1 ) . Though much l e s s r e a c t i v e than N - s u l f o n y l o x y - A A F , N-sulfonyloxy e s t e r s of N-OH-AABP and i t s 4 ' - f l u o r o d e r i v a t i v e have been prepared and shown t o r e a c t w i t h m e t h i o n i n e t o g i v e o r t h o - s u b s t i t u t e d m e t h y l mercapto a r y l a m i d e s and w i t h DNA t o g i v e C8- and N - s u b s t i t u t e d d e o x y g u a n o s i n e - a r y l a m i d e adducts (reviewed i n 4 2 ) . A g a i n , o n l y C8s u b s t i t u t e d guanine d e r i v a t i v e s are o b t a i n e d on r e a c t i o n of N - s u l fonyloxy-AABP w i t h deoxyguanosine, guanosine, or RNA. N-Sulfonyloxy-N-acetyl-2-aminophenanthrene has been prepared and shown to r e a c t t o a l i m i t e d e x t e n t w i t h m e t h i o n i n e , deoxyguanosine and deoxy2

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adenosine t o g i v e 1-methvlmercapto, N - ( d e o x y g u a n o s i n - 8 - y l ) , and l-(deoxyadenosin-N -yl) derivatives, respectively. Metabolic f o r m a t i o n o f N - s u l f o n y l o x y p h e n a c e t i n has a l s o been proposed s i n c e hepatic sulfotransferase-catalyzed activation o f N-hydroxy p h e n a c e t i n l e a d s t o t h e f o r m a t i o n o f adducts w i t h p r o t e i n , n u c l e i c a c i d s and GSH (25,43). From these s t u d i e s and those i n v o l v i n g N-acetoxy arylamides ( v i d e i n f r a ) , i t i s c l e a r t h a t any proposed r e a c t i o n mechanism must account f o r the a b i l i t y o f d i f f e r e n t n u c l e o p h i l e s t o d i r e c t sub­ s t i t u t i o n t o the _N-, o r t h o - and m e t a - r i n g p o s i t i o n s o f t h e a r y l a m i d e and should be c o n s i s t e n t w i t h r e a c t i o n k i n e t i c s and w i t h s o l v o l y s i s or rearrangement p r o d u c t s found i n t h e r e a c t i o n medium. In this r e g a r d , s t u d i e s w i t h model compounds such as m e t h a n e s u l f o n a t e e s t e r s of N-hydroxy a c e t a n i l i d e s (44,45) and N - s u l f o n y l o x y a c e t a n i l i d e s (46) have been p a r t i c u l a r l y u s e f u l . These data i n d i c a t e t h a t r e a c ­ t i v e N - s u l f o n y l o x y d e r i v a t i v e s undergo h e t e r o l y t i c cleavage o f t h e N-0 bond t o form an i n t i m a t e i o n p a i r c o n s i s t i n g o f a p a r t i a l l y d e l o c a l i z e d s i n g l e t n i t r e n i u m / c a r b e n i u m c a t i o n and t h e s u l f a t e a n i o n ( F i g u r e 2 ) , as o r i g i n a l l y proposed by S c r i b n e r e t a l . (47) and more r e c e n t l y supported by m o l e c u l a r o r b i t a l c a l c u l a t i o n s ( 4 8 ) . Collapse of t h e i o n p a i r by i n t e r n a l r e t u r n r e s u l t s i n an o - s u l f onyloxy a c e t a n i l i d e w h i l e reducing agents convert i t to the parent acetanilide. E v i d e n c e has a l s o been p r e s e n t e d t h a t h y d r o l y s i s o f the i o n p a i r may proceed through an imine i n t e r m e d i a t e which would account f o r met a- and p o s s i b l y ^ - s u b s t i t u t e d products (45,46). In a d d i t i o n , e a r l i e r s t u d i e s w i t h the m e t a b o l i c a l l y generated N - s u l fonyloxy ester of phenacetin (£-ethoxyacetanilide) i n d i c a t e t h a t N - a c e t y l benzoquinone imine i s formed as a r e a c t i v e i n t e r m e d i a t e (49). R e c e n t l y , t h e d e c o m p o s i t i o n o f N - s u l f o n y l o x y - A A F under aqueous c o n d i t i o n s has been f u r t h e r examined and appears t o be c o n s i s t e n t w i t h t h i s o v e r a l l mechanism ( 5 0 ) . That i s , t h e major products appear t o be 1- and 3 - s u l f onyloxy-AAF w i t h s m a l l amounts o f AAF, 4-hydroxy-AAF, and a dimer formed by a d d i t i o n o f t h e e l e c t r o p h i l e onto t h e aromatic r i n g o f another AAF m o l e c u l e ( 5 1 ) . Furthermore, the r e l a t i v e y i e l d s o f AAF c o u l d be i n c r e a s e d by a d d i t i o n o f t h e r e d u c i n g agent, a s c o r b i c a c i d ( 5 2 ) . The involvement o f t h e n i t r e n i u m / c a r b e n i u m c a t i o n - s u l f a t e anion p a i r as t h e major e l e c t r o p h i l i c r e a c t a n t from a r y l a m i d e carcinogens i s a l s o c o n s i s t e n t w i t h t h e nature o f the p r o d u c t s formed w i t h c e l l u l a r n u c l e o p h i l e s ( v i d e supra) and i s i n a c c o r d w i t h t h e P e a r s o n h a r d / s o f t acid-base concept o f e l e c t r o p h i l i c s u b s t i t u t i o n ( 5 3 ) . Thus, o r t h o - s u b s t i t u t i o n o f t h e a r y l a m i n e i s f a v o r e d by s o f t n u c l e o ­ p h i l e s (RSCH , RSH, RNH ) w h i c h tend t o advance i o n p a i r s e p a r a t i o n r e s u l t i n g i n g r e a t e r charge d e r e a l i z a t i o n i n t h e a r o m a t i c ring ( 4 8 ) ; w h i l e ^ - s u b s t i t u t i o n i s f a v o r e d w i t h hard n u c l e o p h i l e s t h a t a t t a c k a t i g h t i o n p a i r w i t h a p o s i t i v e n i t r o g e n c e n t e r ( 5 4 ) . Both types o f s u b s t i t u t i o n r e p r e s e n t an S I r e a c t i o n mechanism w h i c h i s determined by t h e s t r e n g t h o f t h e s u l f a t e l e a v i n g group, a l o n g w i t h f o r m a t i o n o f t h e i o n p a i r whose o v e r a l l r e a c t i v i t y o r s e l e c t i v i t y (N v s . o r t h o s u b s t i t u t i o n ) can be i n f l u e n c e d by changes i n r e a c t i o n medium and by t h e nature o f t h e n u c l e o p h i l e (41,42,55). Although metabolically-formed N-sulfonyloxy arylamides are s t r o n g e l e c t r o p h i l e s , b i n d t o c e l l u l a r macromolecules, and have l o n g been c o n s i d e r e d u l t i m a t e c a r c i n o g e n s , t h e i r p r e c i s e r o l e i n a r y l 6

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SR (-OH)

Figure

2.

R e a c t i o n Mechanism f o r N - S u l f o n y l o x y A r y l a m i d e s ( I ) . Ac, a c e t y l ; RSCH , m e t h i o n i n e ; RSH, g l u t a t h i o n e o r c y s t e i n e ; RNH , N - g u a n i n e and/or N - a d e n i n e - n u c l e o s i d e s , - n u c l e o ­ t i d e s , o r - n u c l e i c a c i d s ; RCH, C 8 - g u a n i n e - n u c l e o s i d e s , - n u c l e o t i d e s , o r - n u c l e i c a c i d s , o r C7-AAF. 3

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amide t u m o r i g e n e s i s i s n o t c e r t a i n . F o r example, N - s u l f o n y l o x y - A A F i s n o t a d i r e c t - a c t i n g , l o c a l c a r c i n o g e n ( 5 6 ) , even though i t i s highly toxic (57). I t i s mutagenic when r e a c t e d w i t h p u r i f i e d _B. s u b t i l i s t r a n s f o r m i n g DNA ( 5 8 ) , b u t does n o t serve as a d i r e c t - a c t ­ i n g mutagen i n the IS. typhimurium t e s t system (51,52,59,60) . D u r i n g c h r o n i c a d m i n i s t r a t i o n o f a c a r c i n o g e n i c dose o f AAF, h e p a t i c s u l f o t r a n s f e r a s e a c t i v i t y i s g r e a t l y d i m i n i s h e d ( 6 1 ) and d e a c e t y l a t e d arylamine-DNA adducts e v e n t u a l l y account f o r 97-100% o f t h e t o t a l adducts ( 6 2 ) . I n a d d i t i o n , e x t r a h e p a t i c t i s s u e s which have l i t t l e or no sulf©transferase a c t i v i t y and c o n t a i n o n l y d e a c e t y l a t e d a d ­ d u c t s , a r e a l s o s u s c e p t i b l e t o AAF o r N-OH-AAF c a r c i n o g e n e s i s (63,64). However, s e n s i t i v i t y t o h e p a t i c tumor i n d u c t i o n by AAF c o r r e l a t e s w e l l w i t h h e p a t i c s u l f a t e a v a i l a b i l i t y and w i t h s e x , s t r a i n , and s p e c i e s d i f f e r e n c e s i n h e p a t i c sulf©transferase l e v e l s (reviewed i n 4^,25^). Thus, i t has been proposed t h a t N - s u l f o n y l o x y a r y l a m i d e s may n o t be r e s p o n s i b l e f o r i n i t i a t i n g h e p a t i c tumorigenes i s , b u t may r a t h e r s e r v e t o promote f i x a t i o n o f an i n i t i a t i n g l e s i o n through a c y t o t o x i c response t h a t induces c e l l r e p l i c a t i o n ( 25^, 6 0 ) . N-Acetoxy A r y l a m i d e s ( I I ) N-Acetoxy a r y l a m i d e s have been w i d e l y used as s y n t h e t i c models t o study e l e c t r o p h i l i c r e a c t i v i t y w i t h c e l l u l a r c o n s t i t u e n t s and they y i e l d r e a c t i o n p r o d u c t s s i m i l a r t o those observed w i t h t h e N - s u l f o n y l o x y e s t e r s . F u r t h e r m o r e , s i n c e they a r e h i g h l y c a r c i n o g e n i c a t l o c a l s i t e s o f a p p l i c a t i o n (56,65,66) they have a l s o been regarded as u l t i m a t e c a r c i n o g e n s (47jT~ However, t h e N-acetoxy e s t e r s a r e g e n e r a l l y l e s s r e a c t i v e than t h e c o r r e s p o n d i n g s u l f o n y l o x y d e r i v a ­ t i v e s , they e x h i b i t much l o n g e r h a l f - l i v e s i n aqueous s o l u t i o n (41,55,57,67-71) and t h e i r r e a c t i o n mechanism i s d e c i d e d l y more complex. They r e a c t , a t l e a s t i n p a r t by an S ^ l mechanism i n v o l v i n g i o n p a i r f o r m a t i o n s i m i l a r t o t h a t shown i n F i g u r e 2. This i s supported by: a) t h e i r lower e l e c t r o p h i l i c r e a c t i v i t y and s e l e c t i v i ­ t y i n comparison t o N - s u l f onyloxy e s t e r s which i s due t o t h e d e c r e a s e d s t r e n g t h and h a r d n e s s o f t h e a c e t a t e l e a v i n g g r o u p (41,55,67,72); b) t h e i r thermal rearrangement t o o r t h o - a c e t o x y a r y l ­ amides ( 6 9 ) ; c ) t h e i r f a c i l e r e d u c t i o n t o t h e parent arylamide (68,73); ~d) t h e i r c o n v e r s i o n t o r e a c t i v e imines (45,74); and e) t h e i r r e a c t i v i t y w i t h n u c l e o p h i l e s t o g i v e N^-, o r t h o - and metas u b s t i t u t e d p r o d u c t s (42,75). Y e t h e t e r o l y t i c c l e a v a g e a t t h e N-0 bond must occur t o o n l y a minor e x t e n t because u n l i k e N - s u l f o n y l o x y a r y l a m i d e s (46,50), N-acetoxy a r y l a m i d e s have been shown t o undergo p r e f e r e n t i a l l y cleavage o f e s t e r l i n k a g e t o form a hydroxamate a n i o n and presumably an a c e t y l c a t i o n which would account f o r the observed a c e t y l a t i o n o f l y s i n e i n p r o t e i n s and r i b o s e i n n u c l e i c a c i d s (66,72,76,77). E v i d e n c e has a l s o been p r e s e n t e d that N-acyloxy a r y l a m i d e s may decompose h o m o l y t i c a l l y t o y i e l d f r e e r a d i c a l s t h a t c o u l d a r y l a m i d a t e DNA bases and a l s o r e s u l t i n DNA-protein c r o s s ­ l i n k s (78-81). However, i n v i e w o f t h e r e l a t i v e s t a b i l i t y o f Nacetoxy a r y l a m i d e s i n aqueous media, t h e i r r a p i d r e a c t i o n w i t h added n u c l e o p h i l e s , and t h e f a i l u r e t o d e t e c t r a c e m i z a t i o n t o N - [ ( ( ^ - 0 ) acetoxy]-arylamides on prolonged incubation of N-[(carbonyl- 0)a c e t o x y ] - a r y l a m i d e s i n t h e absence o f n u c l e o p h i l e s ( 8 2 ) , i t appears t h a t an S^2 r e a c t i o n i ^ y o l v i n ^ b | ^ ^ e c | ^ displacement o f a c e t a t e l 8

1 8

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or hydroxamate by t h e a t t a c k i n g n u c l e o p h i l e r e p r e s e n t s a more p r o b a b l e mechanism t o account f o r t h e major a r y l a m i d a t e d o r a c e t y l a t e d p r o d u c t s o b t a i n e d (27,75,76,82). The r o l e o f N - a c e t o x y a r y l a m i d e s as m e t a b o l i c a l l y f o r m e d u l t i m a t e c a r c i n o g e n s in v i v o a l s o appears t o be l i m i t e d . Their enzymatic f o r m a t i o n v i a p e r o x i d a t i o n o f N-hydroxy a r y l a m i d e s can be e x c l u d e d s i n c e t i s s u e s c o n t a i n i n g h i g h l e v e l s o f p e r o x i d a s e s such as the r a t mammary g l a n d (83) and t h e dog u r i n a r y b l a d d e r (84) do n o t form a c e t y l a t e d carcinogen-DNA adducts in v i v o ( 6 3 ) . T h e i r non­ enzymatic f o r m a t i o n by r e a c t i o n o f a c e t y l coenzyme A w i t h N-hydroxy a r y l a m i d e s (6^) cannot be e x c l u d e d ; however, even i f formed, t h e i r d i r e c t r e a c t i o n w i t h c e l l u l a r DNA appears u n l i k e l y as treatment o f c u l t u r e d c e l l s w i t h s y n t h e t i c N-acetoxy AAF (85,86) r e s u l t s p r i m a r i ­ l y i n d e a c e t y l a t e d arylamine-DNA a d d u c t s , a p p a r e n t l y due t o r a p i d N - d e a c e t y l a t i o n t o form t h e r e a c t i v e N-acetoxy a r y l a m i n e ( V ) . N-Glucuronyloxy

A r y l a m i d e s ( I I I ) and A r y l a m i n e s ( I V )

M e t a b o l i c c o n j u g a t i o n o f N-hydroxy a r y l a m i d e s t o form N - g l u c u r o n y l oxy e t h e r s ( i l l ) r e p r e s e n t s a major pathway f o r b i l i a r y and u r i n a r y e x c r e t i o n o f a r o m a t i c amine c a r c i n o g e n s (87,88). W h i l e these c o n j u ­ gates a r e g e n e r a l l y c o n s i d e r e d t o be s t a b l e d e t o x i f i c a t i o n p r o d u c t s , the N - g l u c u r o n y l o x y d e r i v a t i v e s o f AAF, N - a c e t y l - 4 - a m i n o s t i l b e n e , p h e n a c e t i n , b u t n o t o f AABP o r N-acetyl-2-aminophenanthrene, have been shown t o r e a c t s l o w l y e i t h e r w i t h p r o t e i n , n u c l e i c a c i d s , o r t h e i r c o n s t i t u e n t s (89-91). S i n c e r e a c t i o n o f N-glucuronyloxy-AAF w i t h m e t h i o n i n e and g u a n o s i n e y i e l d s o r t h o - m e t h y l m e r c a p t o and N - ( g u a n o s i n - 8 - y l ) d e r i v a t i v e s (89) , r e s p e c t i v e l y , a r e a c t i o n mechanism i n v o l v i n g f o r m a t i o n o f a n i t r e n i u m / c a r b e n i u m c a t i o n g l u c u r o n y l l a c t o n a t e a n i o n p a i r can be envisaged ( F i g u r e 3, path a ) . S t u d i e s on the mechanism o f d e c o m p o s i t i o n o f N - g l u c u r o n y l o x y phenac­ e t i n (92) a r e c o n s i s t e n t w i t h t h i s h y p o t h e s i s as o r t h o - g l u c u r o n y l o x y p h e n a c e t i n was the major rearrangement p r o d u c t , and evidence f o r an imine i n t e r m e d i a t e (45,92) l e a d i n g t o a m e t a - s u b s t i t u t e d d e r i v a t i v e and t o N - a c e t y l benzoquinone imine and i t s r e a c t i o n p r o d u c t s was obtained. The r e d u c t i o n p r o d u c t , p h e n a c e t i n , was a l s o o b t a i n e d a l t h o u g h i t s f o r m a t i o n was n o t i n c r e a s e d by a s c o r b a t e . However, an i n t e r n a l redox p r o c e s s y i e l d i n g p h e n a c e t i n and s a c c h a r i c a c i d i s plausible. C o n v e r s i o n o f N-glucuronyloxy-AAF t o an N - g l u c u r o n y l o x y a r y l ­ amine ( I V ) has a l s o been demonstrated ( F i g u r e 3, paths b and c ) . T h i s can occur s p o n t a n e o u s l y a t a l k a l i n e pH by m i g r a t i o n o f t h e N - a c e t y l group t o t h e 2 ' - h y d r o x y l o f t h e g l u c u r o n y l moiety (93) o r i n t i s s u e s by enzymatic N - d e a c e t y l a t i o n ( 9 4 ) . N-Glucuronyloxy-2a m i n o f l u o r e n e (AF) i s h i g h l y e l e c t r o p h i l i c , d i r e c t l y mutagenic, and r e a c t s w i t h n u c l e i c a c i d s and w i t h m e t h i o n i n e and guanosine (and 5 ' - g u a n y l i c a c i d ) t o g i v e t h e c o r r e s p o n d i n g ortho-methylmercapto and N-(guan-8-yl) d e r i v a t i v e s ($9_>95_,j>6_), presumably v i a an S 1 mecha­ nism. I n t e r e s t i n g l y , enzymatic f o r m a t i o n o f N - g l u c u r o n y l o x y a r y l a ­ mines by d i r e c t O - g l u c u r o n i d a t i o n o f N-hydroxy a r y l a m i n e s does n o t appear t o o c c u r , as o n l y s t a b l e N-hydroxy a r y l a m i n e N - g l u c u r o n i d e s are o b t a i n e d i n i n v i t r o h e p a t i c microsomal i n c u b a t i o n s ( 1 6 ) . N - G l u c u r o n y l o x y a r y l a m i d e s do n o t appear t o be i m p o r t a n t i n h e p a t o c a r c i n o g e n e s i s as t h e i r i n c r e a s e d m e t a b o l i c f o r m a t i o n does n o t result i n i n c r e a s e d h e p a t i c macromolecular binding (4,25). N

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch014

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F i g u r e 3. R e a c t i o n Mechanism f o r N - G l u c u r o n y l o x y A r y l a m i d e s ( I I I ) and A r y l a m i n e s ( I V ) . AC.; a c e t y l ; R S C H , m e t h i o n i n e ; RCH, C8-guanine-nucleosides, - n u c l e o t i d e s , o r - n u c l e i c a c i d s . Pathways a, b , and c are d i s c u s s e d i n the t e x t . 3

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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P r o l o n g e d r e s i d e n c e i n the i n t e s t i n e o r u r i n a r y b l a d d e r lumen c o u l d a l l o w time f o r s i g n i f i c a n t r e a c t i o n w i t h t i s s u e components; however, N-glucuronyloxy-AAF was o n l y weakly c a r c i n o g e n i c a t l o c a l subcu­ taneous s i t e s o f a p p l i c a t i o n ( 8 9 ) . Enzymatic d e a c e t y l a t i o n t o N - g l u c u r o n y l o x y - A F has been d e t e c t e d i n h e p a t i c t i s s u e but t h i s a c t i v i t y i n d i f f e r e n t s p e c i e s does n o t c o r r e l a t e w i t h t h e i r r e l a t i v e s u s c e p t i b i l i t y t o AAF h e p a t o c a r c i n o g e n e s i s ( 9 4 ) . On the o t h e r hand, the a l k a l i n e pH-induced c o n v e r s i o n t o a r e a c t i v e d e r i v a t i v e may p l a y an important r o l e i n u r i n a r y b l a d d e r c a r c i n o g e n e s i s (87) by AAF and o t h e r a r y l a m i d e s i n those s p e c i e s o r i n d i v i d u a l s where normal u r i n e pH i s a l k a l i n e ( e . g . normal r a b b i t u r i n e pH i s 8.5-9.0).

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch014

N-Acetoxy A r y l a m i n e s (V) E a r l y s t u d i e s on t h e jLn v i t r o m e t a b o l i c a c t i v a t i o n o f c a r c i n o g e n i c N-hydroxy a r y l a m i n e s i n d i c a t e d t h a t N-acetoxy a r y l a m i n e s (V) a r e formed as h i g h l y r e a c t i v e i n t e r m e d i a t e s t h a t y i e l d adducts w i t h p r o t e i n s and n u c l e i c a c i d s (40,97). W i t h N-hydroxy a r y l a m i d e s as s u b s t r a t e s , an enzyme mechanism i n v o l v i n g i n t r a m o l e c u l a r N , 0 - a c e t y l t r a n s f e r was proposed ( 9 8 ) ; w h i l e an i n t e r m o l e c u l a r p r o c e s s c o u l d be demonstrated u s i n g N-hydroxy a r y l a m i n e s as s u b s t r a t e s and N-hydroxy a r y l a m i d e s as a c e t y l donors (^?). S i n c e t h a t time, t h i s a c y l t r a n s ­ f e r a s e has been e x t e n s i v e l y c h a r a c t e r i z e d (J_) and p u r i f i e d t o homo­ g e n e i t y from h e p a t i c and e x t r a h e p a t i c t i s s u e s o f s e v e r a l s p e c i e s (reviewed i n 100). More r e c e n t l y , Flammang e_t a l . (7,101) have shown t h a t a c e t y l coenzyme A can serve e f f e c t i v e l y as an a c e t y l donor f o r t h i s enzyme, c a t a l y z i n g the apparent d i r e c t O - a c e t y l a t i o n of s e v e r a l c a r c i n o g e n i c N-hydroxy a r y l a m i n e s . Because o f t h e i r i n s t a b i l i t y and h i g h r e a c t i v i t y , s y n t h e t i c N-acetoxy a r y l a m i n e s have never been i s o l a t e d (97,99). However, NMR s p e c t r a l evidence f o r the e x i s t e n c e o f N - a c e t o x y - 4 - a m i n o q u i n o l i n e 1-oxide has been o b t a i n e d (102,103); and N-acetoxy-4-aminoazobenzene (104), N-acetoxy-2-amino-6-methyldipyrido [l,2-a:3 ,2 -d]imidazole (N-acetoxy-Glu-P-1); r e f . 1 0 5 ) , and N-acetoxy-3-amino-l-methyl-5Hp y r i d o [ 4 , 3 - b ] i n d o l e (N-acetoxy-Trp-P-2; r e f . 106) have been prepared as i n t e r m e d i a t e s and then r e a c t e d w i t h n u c l e o s i d e s o r n u c l e i c a c i d s t o a f f o r d N-(guan-8-yl) p r o d u c t s . I n each o f these c a s e s , a more s t a b l e imino tautomer can e x i s t ( F i g u r e 4 ) . S i m i l a r attempts a t p r e p a r a t i o n o f N-acetoxy-4-aminobiphenyl have n o t been s u c c e s s f u l (107); however, N - a c e t o x y - N - t r i f l u o r o a c e t y l - 4 - a m i n o b i p h e n y l has been prepared and shown t o r e a c t r a p i d l y i n aqueous b u f f e r w i t h guanosine (or 5 - g u a n y l i c acid) to give N-(guan-8-yl)-4-aminobiphenyl d e r i v a t i v e s , a p p a r e n t l y by s e q u e n t i a l d e t r i f l u o r o a c e t y l a t i o n and g e n e r a t i o n o f an e l e c t r o p h i l i c N-acetoxy a r y l a m i n e (108). Evidence f o r t h e f o r m a t i o n o f o t h e r N-acetoxy a r y l a m i n e s _iri s i t u has been o b t a i n e d by treatment o f N-hydroxy a r y l a m i n e s w i t h a c e t i c anhydride i n b u f f e r e d aqueous s o l u t i o n s c o n t a i n i n g N - a c e t y l m e t h i o n i n e which yielded the corresponding ortho-methylmercapto a r y l a m i n e s ( 9 7 ) . W i t h i n v i t r o m e t a b o l i c a c t i v a t i o n systems, e n z y m a t i c a l l y generated N-acetoxy arylamines have a l s o b e e n shown t o r e a c t with N - a c e t y l m e t h i o n i n e o r 2-mercaptoethanol t o y i e l d ortho-aIkylmereapto a r y l a m i n e s (68,97,99) and w i t h n u c l e o s i d e s o r n u c l e i c a c i d s t o g i v e N - ( g u a n - 8 - y l ) - and o r t h o - ( g u a n - N - y 1 ) - a r y l a m i n e s (97,101,103,104). From t h e i r h i g h r e a c t i v i t y and n u c l e o p h i l i c s e l e c t i v i t y , i t seems l i k e l y t h a t N-acetoxy a r y l a m i n e s r e a d i l y undergo h e t e r o l y t i c f

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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4. R e a c t i o n M e c h a n i s m f o r N - A c e t o x y A r y l a m i n e s ( V ) . A c , a c e t y l ; RSCH m e t h i o n i n e ; RNH , N - g u a n i n e - n u c l e o s i d e s , - n u c l e o t i d e s , o r - n u c l e i c a c i d s ; RCH, C8-guanine-nucleosides, -nucleotides, or - n u c l e i c a c i d s . Pathways and h e t e r o l y t i c c l e a v a g e s a and b a r e d i s c u s s e d i n t h e t e x t . Dashed arrows i n d i c a t e proposed pathways. 2

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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c l e a v a g e t o form s i n g l e t n i t r e n i u m / c a r b e n i u m c a t i o n - a c e t a t e anion p a i r s ( F i g u r e 4, path a ) . N u c l e o p h i l i c a t t a c k by RSCH , RNH , o r RCH would then g i v e t h e observed o r t h o - and ^ - s u b s t i t u t e d p r o d u c t s . Under a c i d i c c o n d i t i o n s , h y d r o l y s i s o f N - a c e t o x y - 4 - a m i n o q u i n o l i n e 1-oxide t o 4 - h y d r o x y a m i n o q u i n o l i n e - l - o x i d e ( F i g u r e 4, path b) has a l s o been observed (102). A l t h o u g h t h e i d e n t i f i c a t i o n o f decompo­ s i t i o n p r o d u c t s o f c h e m i c a l l y o r e n z y m a t i c a l l y - g e n e r a t e d N-acetoxy a r y l a m i n e s i n n e u t r a l aqueous s o l u t i o n has n o t been r e p o r t e d , model s t u d i e s w i t h N-benzoyloxy-4-aminophenanthrene (109) suggest t h a t i n ­ t e r n a l rearrangement t o an o r t h o - a c e t o x y a r y l a m i n e and an N-hydroxy a r y l a c e t a m i d e should occur ( F i g u r e 4, dashed a r r o w s ) . The l a t t e r c o n v e r s i o n has i m p o r t a n t i m p l i c a t i o n s f o r enzyme mechanisms. Thus, f o r N-hydroxy a r y l a m i d e N , 0 - a c e t y l t r a n s f e r a s e , c o n v e r s i o n t o an N-acetoxy a r y l a m i n e and i n t e r n a l r e t u r n t o an N-hydroxy a r y l a c e t a ­ mide r e p r e s e n t s a c y c l i c p r o c e s s w h i c h would t e r m i n a t e upon a d d i t i o n of a n u c l e o p h i l e and may be r e s p o n s i b l e f o r t h e s u i c i d e i n a c t i v a t i o n of t h e enzyme ( 9 9 ) . F o r N-hydroxy a r y l a m i n e O - a c e t y l a s e , t h e rearrangement o f t h e i n i t i a l N-acetoxy a r y l a m i n e i n t e r m e d i a t e t o an N-hydroxy a r y l a c e t a m i d e product r e p r e s e n t s an o v e r a l l enzymatic N - a c e t y l a t i o n o f an N-hydroxy a r y l a m i n e , which i s a well-documented m e t a b o l i c pathway f o r a r o m a t i c amines (1_). An i m p o r t a n t r o l e f o r N-acetoxy a r y l a m i n e s as u l t i m a t e c h e m i c a l c a r c i n o g e n s seems l i k e l y i n v i e w o f t h e i r h i g h r e a c t i v i t y , t h e wide t i s s u e and s p e c i e s d i s t r i b u t i o n (7,98) o f enzyme(s) t h a t c a t a l y z e t h e i r f o r m a t i o n , and t h e p r e v a l e n c e o f n o n - a c e t y l a t e d arylamine-DNA adducts i n c a r c i n o g e n - t a r g e t t i s s u e s (110). In addition, synthetic N - a c e t o x y - 4 - a m i n o q u i n o l i n e - l - a c e t a t e , which generates t h e acetoxy a r y l a m i n e on r e a c t i o n w i t h t h i o l s (102,103), i s h i g h l y c a r c i n o g e n i c at s i t e s o f a p p l i c a t i o n ( 1 1 1 ) . R e c e n t l y , S a i t o et_ a l . ( 8 ) have shown t h a t t h e N-hydroxy m e t a b o l i t e s o f t h e mutagenic h e t e r o c y c l i c amines, Trp-P-2 and G l u - P - 1 , a r e m e t a b o l i c a l l y a c t i v a t e d t o u l t i m a t e mutagens by an a c e t y l coenzyme A-dependent enzyme p r e s e n t w i t h i n t h e t e s t b a c t e r i u m , as o r i g i n a l l y proposed by S a k a i £t a l . (112) and McCoy e_t a l . (113). Thus, m e t a b o l i c f o r m a t i o n o f N-acetoxy a r y l a m i n e s would appear a major pathway f o r both m u t a t i o n i n d u c t i o n and i n i t i a t i o n o f c a r c i n o g e n e s i s .

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3

2

N-Sulfonyloxy Arylamines ( V I ) F o r c e r t a i n c a r c i n o g e n i c p r i m a r y N-hydroxy a r y l a m i n e s , m e t a b o l i c 0- s u l f o n y l a t i o n t o a r e a c t i v e e s t e r has been demonstrated. With r a t h e p a t i c sulf©transferase p r e p a r a t i o n s , the PAPS-dependent a c t i v a t i o n of N-hydroxy d e r i v a t i v e s o f 4-aminobiphenyl, 4-aminoazobenzene, 1- naphthylamine, and 2-naphthylamine y i e l d e d e l e c t r o p h i l i c i n t e r ­ mediates t h a t formed adducts w i t h m e t h i o n i n e o r n u c l e i c a c i d s ; w h i l e N-hydroxy-4-aminostilbene, N-hydroxy-3,2'-dimethyl-4-aminobiphenyl, N-hydroxy-N -acetybenzidine and N-hydroxy-AF were n o t a c t i v a t e d i n t h i s i n v i t r o system (9,37,101,114). By comparison, mouse h e p a t i c sulf©transferase has r e c e n t l y been shown t o c a t a l y z e t h e a c t i v a t i o n of both N-hydroxy-AF (10) and N-hydroxy-4-aminoazobenzene (115) t o i n t e r m e d i a t e s t h a t r e a c t w i t h guanosine t o y i e l d N-(guan-8-yl) products. L i k e t h e N-acetoxy a r y l a m i n e s , a r e a c t i o n mechanism f o r N - s u l f o n y l o x y e s t e r s would be expected t o i n v o l v e f o r m a t i o n o f a n i t r e n i u m / c a r b e n i u m c a t i o n - s u l f a t e a n i o n p a i r which then r e a c t s w i t h 1

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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m e t h i o n i n e i n p r o t e i n s and guanine ( o r adenine) i n n u c l e i c a c i d s t o g i v e o r t h o - and N - s u b s t i t u t e d p r o d u c t s ( F i g u r e 5 ) . I n t e r e s t i n g l y , the s i m p l e e l e c t r o p h i l i c e s t e r , h y d r o x y l a m i n e - O - s u l f o n i c a c i d , a l s o r e a c t s w i t h guanosine under a c i d i c c o n d i t i o n s t o g i v e t h e C8-subs t i t u t e d araino-guanosine a p p a r e n t l y by a s i m i l a r mechanism ( 1 1 6 ) . Furthermore, t h i s mechanism i s c o n s i s t e n t w i t h d e c o m p o s i t i o n p r o ­ d u c t s i d e n t i f i e d from sulf©transferase i n c u b a t i o n s o f N-hydroxy-2naphthylamine (9) and w i t h rearrangement p r o d u c t s observed w i t h s y n t h e t i c a l l y - p r e p a r e d N - s u l f o n y l o x y a n i l i n e and N - s u l f o n y l o x y - 2 naphthylamine (117). That i s , t h e o r t h o - s u l f o n y l o x y a r y l a m i n e was a major product and t h i s c o u l d a r i s e by i n t e r n a l r e t u r n upon c o l l a p s e of t h e i o n p a i r ( o f . N - s u l f o n y l o x y a r y l a m i d e s ) . N-Hydroxy-N-sulfonyl-2-naphthylamine may a l s o be f o r m e d as an i n t e r m e d i a t e rearrangement p r o d u c t as i t has been r e p o r t e d (117,118) t o decompose to ortho-sulfonyloxy-2-naphthylamine and 2 - a m i n o - l - n a p h t h o l , t h e l a t t e r o f which was a l s o d e t e c t e d i n t h e sulf©transferase i n c u b a ­ t i o n w i t h N-hydroxy-2-naphthylamine ( 9 ) . The e l e c t r o p h i l i c i o n p a i r a l s o appeared t o undergo a f a c i l e r e d u c t i o n t o 2-naphthylamine. I n t h i s m e t a b o l i c a c t i v a t i o n system, t h i s proceeded a t t h e expense o f N-hydroxy-2-naphthylamine, w h i c h was o x i d i z e d t o 2,2'-azoxynaphthal e n e ; however, o t h e r r e d u c i n g agents may serve t h i s purpose Jjri v i v o and e f f e c t i v e l y d e t o x i f y t h e r e a c t i v e e s t e r . S i m i l a r redox p r o c e s ­ ses c o u l d occur w i t h N-acetoxy a r y l a m i n e s and o t h e r p r i m a r y a r y l ­ amine O-esters b u t t h i s has n o t y e t been i n v e s t i g a t e d . The r o l e o f N - s u l f o n y l o x y a r y l a m i n e s as u l t i m a t e c a r c i n o g e n s appears t o be l i m i t e d . F o r N-hydroxy-2-naphthylamine, c o n v e r s i o n by r a t h e p a t i c sulf©transferase t o a N - s u l f o n y l o x y m e t a b o l i t e r e s u l t s p r i m a r i l y i n d e c o m p o s i t i o n t o 2-amino-l-naphthol and 1 - s u l f o n y l o x y 2-naphthylamine which a r e a l s o major u r i n a r y m e t a b o l i t e s ; and r e a c ­ t i o n w i t h added n u c l e o p h i l e s i s v e r y low, which suggests an o v e r a l l d e t o x i f i c a t i o n process (9,17). However, f o r 4-aminoazobenzene and N-hydroxy-AAF, which a r e p o t e n t hepatocarcinogens i n t h e newborn mouse, evidence has been p r e s e n t e d t h a t s t r o n g l y i m p l i c a t e s t h e i r N - s u l f o n y l o x y a r y l a m i n e e s t e r s as u l t i m a t e h e p a t o c a r c i n o g e n s i n t h i s s p e c i e s (10,104). T h i s i n c l u d e s the i n h i b i t i o n o f arylamine-DNA adduct f o r m a t i o n and t u m o r i g e n e s i s by t h e s u l f o t r a n s f e r a s e i n h i b i t o r p e n t a c h l o r o p h e n o l , t h e reduced tumor i n c i d e n c e i n brachyraorphic mice t h a t a r e d e f i c i e n t i n PAPS b i o s y n t h e s i s (10,115), and t h e r e l a t i v e l y low O - a c e t y l t r a n s f e r a s e a c t i v i t y of mouse l i v e r f o r N-hydroxy-4aminoazobenzene and N-OH-AF (7,114,115). O-Seryl

( O - P r o l y l ) E s t e r s ( V I I ) o f N-Hydroxy

Arylamines

The f o r m a t i o n o f 0 - s e r y l o r 0 - p r o l y l e s t e r s ( F i g u r e 1) o f c e r t a i n Nhydroxy a r y l a m i n e s has been i n f e r r e d from the o b s e r v a t i o n s t h a t h i g h l y r e a c t i v e i n t e r m e d i a t e s can be generated _in v i t r o by i n c u b a ­ t i o n w i t h ATP, s e r i n e o r p r o l i n e , and t h e c o r r e s p o n d i n g aminoacyl tRNA s y n t h e t a s e s (11,12,119). F o r example, a c t i v a t i o n o f N-hydroxy4 - a m i n o q u i n o l i n e - l - o x i d e (119 ,120) , N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) t o n u c l e i c acid-bound p r o d u c t s was dem­ o n s t r a t e d u s i n g s e r y l - t R N A s y n t h e t a s e from y e a s t o r r a t a s c i t e s hepatoma c e l l s . More r e c e n t l y , h e p a t i c c y t o s o l i c p r o l y l - , b u t n o t s e r y l - , tRNA s y n t h e t a s e was shown t o a c t i v a t e N-hydroxy-Trp-P-2 ( 1 2 ) ; however, no a c t i v a t i o n was d e t e c t a b l e f o r t h e N-hydroxy metab­ o l i t e s o f AF, 3 , 2 - d i m e t h y l - 4 - a m i n o b i p h e n y l , or N -acetylbenzidine (122). ,

1

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

F i g u r e 5.

2

2

OH

R e a c t i o n Mechanism f o r N - S u l f o n y l o x y A r y l a m i n e s ( V I ) . R S C H , m e t h i o n i n e ; R N H » N - g u a n i n e - and -adeninen u c l e o s i d e s o r - n u c l e i c a c i d s ; RCH, C 8 - g u a n i n e - n u c l e o s i d e s or - n u c l e i c a c i d s . The dashed arrow i n d i c a t e s a proposed pathway. 3

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6

The i d e n t i f i c a t i o n o f C 8 - g u a n y l and N - a d e n y l a d d u c t s o f 4-aminoquinoline-l-oxide (102,103) i n DNA m o d i f i e d by t h e m e t a b o l i c a l l y - g e n e r a t e d 0 - s e r y l e s t e r and t h e s i m i l a r i t y o f t h e adduct p r o ­ f i l e w i t h t h a t o b t a i n e d on r e a c t i o n o f DNA w i t h N-acetoxy-4-arainoq u i n o l i n e - 1 - o x i d e s u g g e s t an e l e c t r o p h i l i c r e a c t i o n m e c h a n i s m s i m i l a r t o t h a t f o r t h e N-acetoxy o r N - s u l f o n y l o x y arylamines ( F i g u r e s 4 and 5 ) . However, N - s e r y l o x y o r N - p r o l y l o x y arylamines have n o t been s y n t h e s i z e d and t h e d e c o m p o s i t i o n products of the e s t e r s generated i n v i t r o have n o t y e t been s t u d i e d . A l t h o u g h aminoacyl-tRNA s y n t h e t a s e s a r e n e c e s s a r y f o r p r o t e i n s y n t h e s i s i n a l l t i s s u e s , t h e i r importance i n c h e m i c a l c a r c i n o ­ genesis i s d i f f i c u l t to assess. M u t a t i o n i n d u c t i o n by t h i s pathway has been s t u d i e d e x t e n s i v e l y ( 1 2 3 ) , y e t m e t a b o l i c a c t i v a t i o n i n a c a r c i n o g e n - t a r g e t t i s s u e has n o t been demonstrated. The o n l y e x c e p ­ t i o n i s h e p a t i c p r o l y l - t R N A s y n t h e t a s e a c t i v a t i o n o f N-hydroxy-TrpP-2; however, h e p a t i c O - a c e t y l a t i o n o f t h i s s u b s t r a t e a l s o o c c u r s t o an a p p r e c i a b l e e x t e n t ( 1 2 ) . F u r t h e r i n v e s t i g a t i o n s i n v o l v i n g the use o f s p e c i f i c enzyme i n h i b i t o r s would be h e l p f u l i n a d d r e s s i n g t h i s problem. P r o t o n a t i o n o f N-Hydroxy A r y l a m i n e s

(VIII)

The f o r m a t i o n o f O-protonated N-hydroxy a r y l a m i n e s ( F i g u r e 6) under a c i d i c c o n d i t i o n s has been w e l l documented as an i n t e r m e d i a t e s t e p i n t h e Bamberger rearrangement t o form aminophenols and o t h e r o r t h o or p a r a - s u b s t i t u t e d p r o d u c t s (124-128). From a b s o r p t i o n s p e c t r a l d a t a i n v o l v i n g p r o t o n a t i o n e q u i l i b r i a ( 1 2 8 ) , t h e exchange e x p e r i ­ ments o f [ 0]H20 i n t o p r o d u c t s o r s t a r t i n g m a t e r i a l (126,127), and from s t u d i e s o f r e a c t i o n k i n e t i c s (125,128), t h e p r o t o n a t e d h y d r o x y l a m i n e s appear t o be r e l a t i v e l y s t a b l e s p e c i e s whose r e a r r a n g e ­ ment proceeds by an S ^ l mechanism w i t h e l i m i n a t i o n o f water as t h e rate-determining step. The e l e c t r o p h i l i c n a t u r e o f t h i s i n t e r m e d i ­ a t e was i n i t i a l l y c o n s i d e r e d by H e l l e r e_t a l . ( 1 2 5 ) ; w h i l e K r i e k (15), who proposed t h a t e l i m i n a t i o n o f water r e s u l t e d i n a h i g h l y e l e c t r o p h i l i c a r y l n i t r e n i u m i o n , f i r s t demonstrated r e a c t i o n s w i t h biological nucleophiles. Since t h a t t i m e , t h e r e a c t i o n o f c a r ­ c i n o g e n i c N-hydroxy a r y l a m i n e s w i t h n u c l e i c a c i d s under m i l d l y a c i d i c c o n d i t i o n s has been shown t o be an e f f e c t i v e procedure f o r p r e p a r a t i o n and i d e n t i f i c a t i o n o f a r y l a m i n e - n u c l e o s i d e adducts and b o t h o r t h o - and N - s u b s t i t u t e d p r o d u c t s have been o b t a i n e d (reviewed i n 110). These i n c l u d e o_-(guan-N - y l ) , o_-(guan-0 - y l ) , o_-(aden-N y l ) , N-(guan-8-yl), and N - ( a d e n - 8 - y l ) a d d u c t s . Of t h e s e , t h e N( g u a n - 8 - y l ) d e r i v a t i v e s have u s u a l l y been t h e major r e a c t i o n product. I n c o n t r a s t t o the r e a c t i v i t y o f N - s u l f o n y l o x y and N-acetoxy e s t e r s o f a r y l a m i d e s and a r y l a m i n e s , t h e r e l a t i v e r e a c t i v i t y o f p r o ­ t o n a t e d N-hydroxy a r y l a m i n e s w i t h n u c l e o p h i l e s g e n e r a l l y d e c r e a s e s i n t h e o r d e r : DNA > d e n a t u r e d DNA > rRNA = p r o t e i n > tRNA » n u c l e o ­ t i d e s s n u c l e o s i d e s s m e t h i o n i n e s GSH (2,13-17,30,36,40,127,129, 130). F u r t h e r m o r e , t h e r a t e o f r e a c t i o n w i t h DNA was found t o be not o n l y f i r s t o r d e r w i t h r e s p e c t t o N-hydroxy a r y l a m i n e concen­ t r a t i o n , b u t a l s o f i r s t o r d e r w i t h r e s p e c t t o DNA c o n c e n t r a t i o n (127,129,131). These d a t a suggested t h a t t h e r e a c t i o n mechanism was e i t h e r S 2 o r S I w i t h t h e i n v o l v e m e n t o f an i n t e r m e d i a t e i n t h e N N XT

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r a t e - d e t e r m i n i n g step ( 1 3 2 ) . I n v i e w o f the r e l a t i v e l y slow e x ­ change ( 4 0 % / h r ) o f [ 0 ] H 0 i n t o N - h y d r o x y - l - n a p h t h y l a m i n e a t pH 5, the slower c o n v e r s i o n o f N - h y d r o x y - l - n a p h t h y l a m i n e t o aminonaphthols ( 1 % / h r ) , i t s r a p i d r e a c t i o n w i t h DNA. ( 2 5 % / h r ) , and t h e e s t a b l i s h e d S ^ l r e a c t i o n mechanism f o r the Bamberger r e a c t i o n , a p a r t i a l l y d e l o c a l i z e d h y d r a t e d n i t r e n i u m / c a r b e n i u m i o n i n t e r m e d i a t e ( F i g u r e 6) was proposed (127,132,133). T h i s i n t e r m e d i a t e i s analogous t o an i n t i m a t e i o n p a i r formed under n e u t r a l c o n d i t i o n s as d e s c r i b e d f o r the e l e c t r o p h i l i c O - e s t e r s o f N-hydroxy a r y l a m i d e s and a r y l a m i n e s (vide supra). Thus, the o v e r a l l r e a c t i v i t y and s e l e c t i v i t y ( N - v s . r i n g s u b s t i t u t i o n ) o f p r o t o n a t e d N-hydroxy a r y l a m i n e s should be d e t e r ­ mined by a b i l i t y o f the n u c l e o p h i l e t o d e s o l v a t e the h y d r a t e d i o n , t o d e l o c a l i z e f u r t h e r t h e p o s i t i v e c h a r g e , and t o r e s u l t i n product f o r m a t i o n (132,134). Such a mechanism i s c o n s i s t e n t w i t h the p r e f ­ e r e n t i a l formation o f N - s u b s t i t u t e d products from r e a c t i o n w i t h n u c l e i c a c i d s (110) and from s o l v o l y s i s o f N-hydroxy a r y l a m i n e s i n b e n z e n e / t r i f l u o r o a c e t i c a c i d (135); w h i l e a r y l r i n g - s u b s t i t u t e d products a r e p r e f e r e n t i a l l y o b t a i n e d on s o l v o l y s i s o f N-hydroxy arylamines (135) o r 1 - n a p h t h y l a z i d e (136) i n b e n z e n e / t r i f l u o r o m e t h a n e s u l f o n i c a c i d . A l t e r n a t i v e l y , upon d e s o l v a t i o n , a t r u e i o n p a i r c o u l d be formed between a n e g a t i v e l y charged n u c l e o p h i l e o r c a t a l y s t and t h e n i t r e n i u m / c a r b e n i u m c a t i o n , which c o u l d c o l l a p s e t o the product o r undergo i n t e r n a l rearrangement. However, t h e l a t t e r mechanism seems improbable s i n c e , u n l i k e t h e e l e c t r o p h i l i c O - e s t e r s , the r e a c t i v i t y o f p r o t o n a t e d N-hydroxy a r y l a m i n e s w i t h DNA i s u n ­ a f f e c t e d by r e d u c i n g agents and t h e i r r e a c t i o n w i t h s t r o n g , low molecular-weight nucleophiles such as 4 - ( _ p - n i t r o b e n z y l ) p y r i d i n e cannot be d e t e c t e d (127,129,131). The e x c e p t i o n a l r e a c t i v i t y o f DNA f o r p r o t o n a t e d N-hydroxy a r y l a m i n e s can be r a t i o n a l i z e d by a t l e a s t two mechanisms. First, i n t e r c a l a t i o n o f the e l e c t r o p h i l i c i n t e r m e d i a t e between DNA bases c o u l d s t e r i c a l l y a s s i s t i n d e s o l v a t i o n and i n d i r e c t i n g t h e e l e c ­ t r o p h i l i c c e n t e r o f the c a r c i n o g e n over the n u c l e o p h i l i c r e g i o n o f the DNA base. T h i s seems u n l i k e l y , however, as p r e t r e a t m e n t o f DNA w i t h c i s - P t , which decreased t h e DNA contour l e n g t h by 50%, f a i l e d t o reduce t h e r e a c t i v i t y o f N - h y d r o x y - l - n a p h t h y l a m i n e f o r t h e DNA (137). A second p o s s i b i l i t y i n v o l v e s an e l e c t r o s t a t i c a t t r a c t i o n between t h e e l e c t r o p h i l e and t h e phosphate backbone o f t h e DNA ( 7 7 ) . T h i s seems more p r o b a b l e s i n c e e i t h e r h i g h i o n i c s t r e n g t h o r s t o i ­ c h i o m e t r i c ( t o DNA-P) amounts o f Mg s t r o n g l y i n h i b i t DNA adduct f o r m a t i o n (77,137). I n a d d i t i o n , e v i d e n c e has been presented t h a t N-hydroxy arylamine-DNA/RNA p h o s p h o t r i e s t e r s may be formed which induce s t r a n d breaks (137,138) and c o u l d serve as a c a t a l y s t f o r d e s o l v a t i o n and subsequent adduct f o r m a t i o n . The importance o f p r o t o n a t e d N-hydroxy a r y l a m i n e s as u l t i m a t e c a r c i n o g e n s has been suggested f o r some time (28,40,139). From s t u d i e s on t h e i r r e a c t i v i t y w i t h n u c l e i c a c i d s a t d i f f e r e n t pH's (2,15,16,63,130,131), the pK f o r p r o t o n a t i o n o f t h e N-hydroxy group appears t o be between pH 5 and 6; t h u s , a s i g n i f i c a n t p r o p o r t i o n (1-10%) o f t h e N-hydroxy d e r i v a t i v e e x i s t s as t h e p r o t o n a t e d form even under n e u t r a l c o n d i t i o n s . T h i s would account f o r the s i g ­ n i f i c a n t l e v e l s o f c o v a l e n t m o d i f i c a t i o n o f DNA observed i n v i t r o by r e a c t i o n w i t h N-hydroxy a r y l a m i n e s a t n e u t r a l pH. C o n s e q u e n t l y , i t has been proposed t h a t in_ v i v o f o r m a t i o n o f n o n - a c e t y l a t e d aryl l 8

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2

+

a

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amine-DNA adducts may a r i s e , a t l e a s t i n p a r t , by t h e d i r e c t r e a c ­ t i o n with protonated N-hydroxy arylamines (2,28,139) . This h y p o t h e s i s i s f u r t h e r supported by t h e o b s e r v a t i o n t h a t s y n t h e t i c o r m e t a b o l i c a l l y - g e n e r a t e d N-OH-AF r e a c t s a p p r e c i a b l y w i t h DNA i n i s o ­ l a t e d l i v e r n u c l e i t o y i e l d d e t e c t a b l e l e v e l s o f N-(deoxyguanosin8-yl)-AF ( 2 ) . T h i s i s c o n s i s t e n t w i t h t h e h i g h c o n c e n t r a t i o n o f DNA w i t h i n c e l l n u c l e i ( c a . 50 mg/ml) and w i t h the f i r s t order r e l a t i o n between r e a c t i o n r a t e s and DNA c o n c e n t r a t i o n s . P r o t o n a t e d N-hydroxy a r y l a m i n e s have a l s o been proposed t o be u l t i m a t e c a r c i n o g e n s f o r t h e u r i n a r y b l a d d e r (16,17,140,141) s i n c e u r i n e pH i s s l i g h t l y a c i d i c i n a number o f s p e c i e s (14,142). F u r t h e r m o r e , p h a r m a c o k i n e t i c s t u d i e s have shown t h a t i n c r e a s e d u r i n e a c i d i t y and decreased frequency o f u r i n a t i o n a r e p r e d i c t i v e o f r e l a ­ t i v e s p e c i e s s u s c e p t i b i l i t y t o u r i n a r y b l a d d e r c a r c i n o g e n e s i s (142); and n e o p l a s t i c t r a n s f o r m a t i o n o f c u l t u r e d human f i b r o b l a s t s by N-hydroxy a r y l a m i n e s i s g r e a t l y enhanced by i n c u b a t i o n a t pH 5 as compared t o pH 7 ( 1 4 3 ) . Nitrosoarenes (IX) N i t r o s o a r e n e s a r e r e a d i l y f o r m e d by t h e o x i d a t i o n o f p r i m a r y N-hydroxy a r y l a m i n e s and s e v e r a l mechanisms appear t o be i n v o l v e d . These i n c l u d e : 1) t h e m e t a l - c a t a l y z e d o x i d a t i o n / r e d u c t i o n t o n i t r o ­ soarenes, azoxyarenes and a r y l a m i n e s (144); 2) t h e 0 - d e p e n d e n t , m e t a l - c a t a l y z e d o x i d a t i o n t o n i t r o s o a r e n e s (145); 3) t h e 0 - d e p e n d e n t , hemoglobin-mediated c o - o x i d a t i o n t o n i t r o s o a r e n e s and methem o g l o b i n (146); and 4) t h e 0 -dependent c o n v e r s i o n o f N-hydroxy arylamines to nitrosoarenes, nitrosophenols and n i t r o a r e n e s (147,148) . Each of these processes can i n v o l v e i n t e r m e d i a t e n i t r o x i d e r a d i c a l s , s u p e r o x i d e a n i o n r a d i c a l s , hydrogen p e r o x i d e and h y d r o x y l r a d i c a l s , a l l o f which have been observed i n model systems (149,151). Although these r a d i c a l s a r e e l e c t r o p h i l i c and have been suggested t o r e s u l t i n DNA damage (151,152), a c a u s a l r e l a t i o n s h i p has n o t y e t been e s t a b l i s h e d . N i t r o s o a r e n e s , on t h e o t h e r hand, a r e r e a d i l y formed i n i n v i t r o m e t a b o l i c i n c u b a t i o n s (2,153) and have been shown t o r e a c t c o v a l e n t l y w i t h l i p i d s (154), p r o t e i n s (28,155) and GSH (17,156-159). N i t r o s o a r e n e s a r e a l s o r e a d i l y reduced t o N-hydroxy a r y l a m i n e s by a s c o r b i c a c i d (17,160) and by reduced p y r i d i n e n u c l e o t i d e s (9,161) . The mechanism o f r e a c t i o n o f n i t r o s o a r e n e s w i t h GSH has been s t u d i e d e x t e n s i v e l y and i s known t o i n v o l v e an a d d i t i o n r e a c t i o n w i t h the t h i o l group t o form an N-hydroxy-N-(glutathion-S-yl)a r y l a m i n e adduct. T h i s i n t e r m e d i a t e can r e a r r a n g e t o an N - ( g l u t a t h i o n - S - y l ) - a r y l a m i n e S-oxide o r can be reduced t o an N-hydroxy a r y l a m i n e o r an N - ( g l u t a t h i o n - S - y l ) - a r y l a r a i n e ( F i g u r e 7 ) . I t i s i n t e r e s t i n g t o note t h a t 4-aminobiphenyl has r e c e n t l y been r e p o r t e d to form h i g h l e v e l s o f a hemoglobin adduct ( 5 % o f t h e dose) t h a t appears t o a r i s e by a d d i t i o n o f 4 - n i t r o s o b i p h e n y l t o a c y s t e i n y l s u l f h y d r y l group i n t h e p r o t e i n , forming an N-S l i n k a g e ( 1 6 2 ) . B i n d i n g o f n i t r o s o a r e n e s t o n u c l e i c a c i d s has been suggested ( 4 3 , 163), b u t n e g a t i v e r e s u l t s were o b t a i n e d i n subsequent s t u d i e s (40,159). Thus, t h e r o l e o f n i t r o s o a r e n e s as u l t i m a t e c a r c i n o g e n s per se seems u n l i k e l y , a l t h o u g h m o d i f i c a t i o n o f a c r i t i c a l c e l l u l a r p r o t e i n cannot be e x c l u d e d . A r o l e f o r n i t r o s o a r e n e s i n a r y l a m i n e c a r c i n o g e n e s i s has been 2

2

2

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OH

-

OH

/

2

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©

8 ^

N 8

(H O)

2

2

OH

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(ROH) —%

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F i g u r e 6. R e a c t i o n Mechanism and F o r m a t i o n o f P r o t o n a t e d N - H y d r o x y Arylamines ( V I I I ) . R N H , N - g u a n i n e - and N - a d e n i n e nucleic a c i d s ; ROH, 0 - g u a n i n e - n u c l e i c a c i d s ; RCH, C8-guanine- and C 8 - a d e n i n e - n u c l e i c a c i d s . 2

8

2

6

OH + RSH SR

+ 2e"

+ 2e

fe

H + RSH

I N—SR

i

OH

F i g u r e 7. R e a c t i o n Mechanism f o r N i t r o s o a r e n e s ( I X ) , RSH, g l u t a t h i o n e o r c y s t e i n e .

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t SR

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suggested t o be due t o t h e i r f a c i l e i n t e r c o n v e r s i o n w i t h N-hydroxy a r y l a m i n e s by o x i d a t i o n and r e d u c t i o n and t h e i r r a p i d d e t o x i f i c a t i o n by r e a c t i o n w i t h GSH ( 1 5 9 ) . C o n s e q u e n t l y , a d d i t i o n o f a s c o r b i c a c i d s i g n i f i c a n t l y increased 2-nitrosofluorene mutagenicity (160); whereas, a d d i t i o n o f GSH s t r o n g l y i n h i b i t e d mutagenic a c t i v i t y (164) and GSH d e p l e t i o n h a s r e s u l t e d i n i n c r e a s e d DNA damage i n h e p a t o c y t e s by N-OH-AF ( 1 6 5 ) .

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Iminoquinones (X) and D i i m i n e s ( X I ) The f o r m a t i o n o f iminoquinones (166,167) and d i i m i n e s (20,168) as i n t e r m e d i a t e s i n t h e o x i d a t i o n o f aminophenols and a r y l d i a m i n e s has been w e l l e s t a b l i s h e d . These i n t e r m e d i a t e s r e a d i l y undergo a d d i t i o n r e a c t i o n s w i t h n u c l e o p h i l e s t o y i e l d N-, o r t h o - , o r m e t a - s u b s t i t u t e d p r o d u c t s ( F i g u r e 8 ) . F o r example, 2 - a m i n o - l - n a p h t h o l , w h i c h has l o n g been suggested as a p r o x i m a t e carcinogenic metabolite of 2-naph t h y lamine ( 1 6 9 ) , i s r e a d i l y o x i d i z e d i n a i r o r by cytochrome c_ t o 2-imino-l-naphthoquinone. T h i s iminoquinone i s e l e c t r o p h i l i c and can b i n d c o v a l e n t l y t o p r o t e i n and DNA, can undergo r e a c t i o n w i t h aryl-NH2 groups t o g i v e m e t a - s u b s t i t u t e d p r o d u c t s , o r can h y d r o l y z e t o form 2-amino-l,4-naphthoquinone (19,166,167,170-172). In this r e g a r d , t h e major r e a c t i o n p r o d u c t o f 2-imino-l-naphthoquinone with DNA has been r e c e n t l y i d e n t i f i e d as 4-(deoxyguanosin-N - y l ) - 2 - a m i n o 1,4-naphthoquinoneimine ( 1 9 ) . D i i m i n e s a r e formed d i r e c t l y by p e r o x i d a t i v e m e t a b o l i s m o f aryldiamines. F o r example, 4 , 4 ' - d i i m i n o b i p h e n y l (or benzidined i m i n e ) , a p r o d u c t o f b e n z i d i n e p e r o x i d a t i o n whose f o r m a t i o n i n ­ v o l v e s a c a t i o n r a d i c a l i n t e r m e d i a t e (20,168), r e a d i l y b i n d s t o p r o t e i n and n u c l e i c a c i d (173,174). This diimine a l s o reacts with i t s e l f t o form, an azo d^mer (20) o r r e a c t s w i t h GSH t o g i v e an o r t h o - s u b s t i t u t e d g l u t a t h i o n - S - y l c o n j u g a t e ( 1 7 5 ) , w i t h phenols t o g i v e an N - s u b s t i t u t e d i n d o d y e ( 1 7 6 ) , and w i t h DNA t o g i v e N-(deoxyguanosin-8-yl)-benzidine (174). Other s i m i l a r l y r e a c t i v e i m i n e s and iminoquinones have been shown t o be formed i n b i o l o g i c a l systems, n o t a b l y N-acetyl-jr-benzoquinone i m i n e , w h i c h has been i d e n t i f i e d as t h e major h e p a t o t o x i c m e t a b o l i t e o f acetaminophen and phenacetin (reviewed i n 91). Over t h e l a s t few y e a r s , t h e s i g n i f i c a n c e o f these i n t e r m e d i a t e s as u l t i m a t e c a r c i n o g e n s has r e c e i v e d new impetus s i n c e p r o s t a g l a n d i n H s y n t h a s e , a mammalian p e r o x i d a s e which i s w i d e l y d i s t r i b u t e d i n e x t r a h e p a t i c t i s s u e s ( 1 7 7 ) , can mediate t h e c o o x i d a t i o n o f s e v e r a l carcinogenic arylamines t o intermediates that bind c o v a l e n t l y t o p r o t e i n and n u c l e i c a c i d (20,168,178,179). For the u r i n a r y bladder c a r c i n o g e n , 2-naphthy1amine, t h e f o r m a t i o n o f 2 - a r a i n o - l - n a p h t h o l and i t s subsequent o x i d a t i o n t o 2-imino-l-naphthoquinone have been shown t o be p r i m a r i l y r e s p o n s i b l e f o r DNA b i n d i n g i n t h e i i i v i t r o p e r o x i ­ dase system ( 1 8 0 ) . Furthermore, about 20-30% o f t h e 2 - n a p h t h y l amine-DNA adducts formed i n t h e dog u r i n a r y b l a d d e r , which c o n t a i n s h i g h l e v e l s o f p r o s t a g l a n d i n H synthase ( 8 4 ) , appears t o be d e r i v e d from the a d d i t i o n r e a c t i o n o f 2-imino-l-naphthoquinone (19). For b e n z i d i n e , another u r i n a r y b l a d d e r c a r c i n o g e n , t h e major b e n z i d i n e DNA adduct formed i n t h e p r o s t a g l a n d i n H synthase-mediated r e a c t i o n and i n t h e u r i n a r y b l a d d e r o f dogs g i v e n b e n z i d i n e was shown t o be i d e n t i c a l t o t h e N - ( g u a n - 8 - y l ) d e r i v a t i v e t h a t was p r e p a r e d by r e a c t i o n w i t h s y n t h e t i c 4,4'-diiminobiphenyl (174). Recently, the

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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F i g u r e 8.

R e a c t i o n Mechanisms f o r Iminoquinones (X) and I m i n e s ( X I ) . RNH , N - g u a n i n e - n u c l e i c acids or arylamines; RCH, C8guanine-nucleic a c i d s o r _ D _ - s u b s t i t u t e d p h e n o l s ; RSH, glutathione or cysteine. 2

2

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p e r o x i d a t i v e m e t a b o l i s m o f AF has been c a r e f u l l y s t u d i e d and found t o r e s u l t i n t h e f o r m a t i o n o f a " h e a d - t o - t a i l " dimer, 2-aminod i f l u o r e n y l a m i n e , whose f u r t h e r o x i d a t i o n t o a r e a c t i v e d i i m i n e may be r e s p o n s i b l e f o r macromolecular b i n d i n g (181-183). However, f o r each o f these c a r c i n o g e n s , t h e r e i s a l s o good evidence t h a t e l e c t r o ­ p h i l i c r a d i c a l c a t i o n s (20,150,168,182) can be produced and t h a t these may y i e l d c o v a l e n t adducts w i t h p r o t e i n and n u c l e i c a c i d s . Further s t u d i e s on t h e i d e n t i f i c a t i o n o f these adducts should p r o v i d e u s e f u l i n f o r m a t i o n on the r o l e o f r a d i c a l i n t e r m e d i a t e s i n arylamine carcinogenesis.

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N - S u l f o n y l o x y ( X I I ) and N - A r y l n i t r o n e N-Methyl A r y l a m i n e s

( X I I I ) D e r i v a t i v e s of

Although several N-methyl-substituted a r y l a m i n e s have been shown t o be c a r c i n o g e n i c (184-186), m e t a b o l i c a c t i v a t i o n pathways have been i n v e s t i g a t e d p r i m a r i l y f o r the h e p a t o c a r c i n o g e n i c aminoazo dyes, N-methyl-4-aminoazobenzene (MAB) and i t s 3'-methyl d e r i v a t i v e (9,21, 22,187,188). N-Hydroxy-N-methyl a r y l a m i n e s a r e g e n e r a l l y regarded as p r o x i m a t e c a r c i n o g e n i c m e t a b o l i t e s (22,187,189) and have been shown t o be c o n v e r t e d t o e l e c t r o p h i l i c N - s u l f o n y l o x y d e r i v a t i v e s by h e p a t i c s u l f o t r a n s f e r a s e s (9,187) o r t o r e a c t i v e N - a r y l n i t r o n e s by a i r oxidation (21). M e t a b o l i c a l l y - f o r m e d N-sulfonyloxy-MAB was found t o r e a c t w i t h m e t h i o n i n e , g u a n o s i n e , and GSH t o g i v e o r t h o - m e t h y l m e r c a p t o , g u a n - 8 - y l , and o r t h o - g l u t a t h i o n - S - y l p r o d u c t s (9,190); and these were t h e same major adducts found i n v i v o i n r a t h e p a t i c p r o t e i n , n u c l e i c a c i d , and b i l e , r e s p e c t i v e l y , a f t e r MAB a d m i n i s t r a t i o n (191-193) . T h e s e s t u d i e s were a i d e d by t h e a v a i l a b i l i t y o f s y n t h e t i c N-benzoyloxy e s t e r s which show s i m i l a r r e a c t i v i t y toward n u c l e o p h i l e s and a r e p o t e n t , d i r e c t - a c t i n g c a r c i n o g e n s and mutagens (57,194,195). A d d i t i o n a l experiments have shown t h a t s u b s t i t u t e d guan-N - y l and aden-N - y l d e r i v a t i v e s a r e a l s o formed i n DNA a f t e r r e a c t i o n Ln v i t r o w i t h N-benzoyloxy-MAB and a f t e r d o s i n g w i t h MAB i n vivo (196-198) , which suggests a similar reactivity for metabolically-formed N-sulfonyloxy esters. Thus, l i k e the N - s u l f o n y l o x y e s t e r s o f a r y l a m i d e s and o f p r i m a r y a r y l a m i n e s ( F i g u r e s 2 and 5 ) , a r e a c t i o n mechanism f o r N - s u l f o n y l oxy-N-methyl a r y l a m i n e s i s expected t o i n v o l v e f o r m a t i o n of a n i t r e n i u m / c a r b e n i u m c a t i o n - s u l f a t e a n i o n p a i r which r e a c t s t o g i v e b o t h N- o r r i n g - s u b s t i t u t e d p r o d u c t s , depending on t h e s o f t n e s s o r hardness o f t h e n u c l e o p h i l e ( F i g u r e 9 ) . R e c e n t l y , N-sulfonyloxy-MAB was prepared s y n t h e t i c a l l y and i t s s o l v o l y s i s and r e a c t i o n w i t h GSH was examined ( 1 9 9 ) . I n a d d i t i o n t o t h e expected r i n g - s u b s t i t u t e d g l u t a t h i o n - S - y l a d d u c t s , a g l u t a t h i o n - S - m e t h y l e n e c o n j u g a t e was obtained. T h i s suggests t h a t i n t e r n a l d e c o m p o s i t i o n o f t h e i n t i m a t e i o n p a i r i n v o l v e s l o s s o f s u l f u r i c a c i d and f o r m a t i o n o f a methimine ( F i g u r e 9 ) , w h i c h can h y d r o l y z e t o formaldehyde and the p r i m a r y a r y l a m i n e o r c a n r e a c t w i t h GSH v i a a Mannich c o n d e n s a t i o n t o y i e l d the g l u t a t h i o n - S - m e t h y l e n e p r o d u c t ( 2 0 0 ) . I n i n v i t r o N-hydroxy-MAB s u l f o t r a n s f e r a s e - a c t i v a t i n g systems, N-sulfonyloxy-MAB a l s o appears t o undergo r a p i d r e d u c t i o n t o MAB ( F i g u r e 9) w i t h t h e concomitant o x i d a t i o n o f N-hydroxy-MAB t o the N - a r y l n i t r o n e (9K The o x i d i z i n g p r o p e r t i e s o f t h e N - s u l f o n y l o x y MAB i o n p a i r i s c o n s i s t e n t w i t h r e s u l t s o b t a i n e d f o r t h e p r i m a r y 2

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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Nitro Polycyclic Aromatic

Hydrocarbons

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Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

a n a e r o b i c c o n d i t i o n s i n the presence o f FMN. T h i s suggests t h a t 1-nitronaphthalene i s l e s s mutagenic than 5-nitroacenaphthene because i t i s more r e s i s t a n t t o n i t r o r e d u c t i o n . I t i s also s i g n i f i c a n t t h a t when n i t r o r e d u c t i o n d i d o c c u r , N - h y d r o x y - l naphthylamine was n o t d e t e c t e d as a m e t a b o l i t e (125) s i n c e t h i s compound i s b o t h mutagenic (5,126-127) and c a r c i n o g e n i c (127-129). I t i s p o s s i b l e t h a t d u r i n g the S 9 - c a t a l y z e d r e d u c t i o n o f 1 - n i t r o ­ naphthalene o n l y t h e t e r m i n a l l y - r e d u c e d s p e c i e s , 1-naphthylamine, i s r e l e a s e d from the enzyme complex. Recent s t u d i e s have i n d i c a t e d t h a t 1-naphthylamine i s not o x i d i z e d by mixed f u n c t i o n o x i d a s e s t o i t s N-hydroxy a r y l a m i n e d e r i v a t i v e ( 1 3 0 ) . Taken t o g e t h e r , these d a t a h e l p e x p l a i n why N-hydroxy-l-naphthylamine c a n be a potent c a r c i n o g e n w h i l e 1 - n i t r o n a p h t h a l e n e and 1-naphthylamine a r e apparently noncarcinogenic. 9-Nitroanthracene, 7-nitrobenz[a]anthracene and 6 - n i t r o c h r y s e n e . Rat l i v e r microsomes o x i d i z e d 9 - n i t r o a n t h r a c e n e p r i m a r i l y t o 9-nitroanthracene-trans-3,4-dihydrodiol, with 9-nitroanthracenet r a n s - 1 , 2 - d i h y d r o d i o l and 9,10-anthraquinone b e i n g d e t e c t e d as minor m e t a b o l i t e s ( F i g u r e 3; 8 9 ) . The p r e f e r e n t i a l o x i d a t i o n o f the 3- and 4-carbons, as opposed t o carbons 1 and 2, suggests t h a t metabolism i s i n h i b i t e d i n r e g i o n s p e r i t o the n i t r o s u b s t i t u e n t . 9-Nitroanthracene and i t s two d i h y r o d i o l m e t a b o l i t e s were n o t d i r e c t - a c t i n g mutagens i n S^. typhimurium and were o n l y weakly mutagenic i n t h e presence o f an S 9 - a c t i v a t i n g system. When m i c r o s o m a l i n c u b a t i o n s were conducted under a n a e r o b i c c o n d i t i o n s , r e d u c t i o n t o 9-aminoanthracene was not observed. The p r i m a r y product formed d u r i n g the m i c r o s o m a l metabolism o f 7-nitrobenz[a]anthracene was 7-nitrobenz[a]anthracene-trans-3,4d i h y d r o d i o l ( F i g u r e 4; 131). A s m a l l amount o f 7 - n i t r o b e n z [ a ] a n t h r a c e n e - t r a n s - 8 , 9 - d i h y d r o d i o l was a l s o d e t e c t e d w h i c h i s c o n s i s ­ t e n t w i t h t h e p r e v i o u s o b s e r v a t i o n (89) t h a t n i t r o s u b s t i t u t i o n i n h i b i t s p e r i - r e g i o n o x i d a t i o n . Both d i h y d r o d i o l m e t a b o l i t e s were f o r m e d i n a s t e r e o s e l e c t i v e manner w i t h t h e R,R e n a n t i o m e r s predominating. S i n c e the same t r a n s - 3 , 4 - and 8,9-enantiomers a r e formed d u r i n g the m i c r o s o m a l metabolism o f b e n z [ a ] a n t h r a c e n e , i t appears t h a t n i t r o s u b s t i t u t i o n a f f e c t s the r e g i o s e l e c t i v i t y b u t not the s t e r e o s e l e c t i v i t y o f metabolism. Nitro substitution also a f f e c t s the c o n f o r m a t i o n o f the r e s u l t a n t d i h y d r o d i o l m e t a b o l i t e s . Thus, w h i l e the h y d r o x y l groups o f 7 - n i t r o b e n z [ a ] a n t h r a c e n e - t r a n s 3 , 4 - d i h y d r o d i o l p r e f e r e n t i a l l y adopt a q u a s i - d i e q u a t o r i a l conforma­ t i o n ( 1 3 1 ) , t h e 8,9-isomer has a s i g n i f i c a n t p o p u l a t i o n w i t h a q u a s i - d i a x i a l c o n f o r m a t i o n . The e f f e c t o f these c o n f o r m a t i o n s upon the f u r t h e r metabolism of 7 - n i t r o b e n z [ a ] a n t h r a c e n e i s p r e s e n t l y not known, a l t h o u g h w i t h o t h e r PAH d e r i v a t i v e s , q u a s i - d i a x i a l conforma­ t i o n s tend t o i n h i b i t metabolism t o d i o l epoxides ( 1 3 2 ) . As was observed w i t h 9 - n i t r o a n t h r a c e n e (89) , 7 - n i t r o b e n z [ a ] a n t h r a c e n e and i t s two d i h y d r o d i o l m e t a b o l i t e s were not d i r e c t - a c t i n g mutagens i n S^. typhimurium and were o n l y weakly mutagenic i n the presence o f S9 (96). El-Bayoumy and Hecht (95) have r e c e n t l y examined the metabo­ l i s m o f 6 - n i t r o c h r y s e n e by r a t l i v e r S9. 6 - N i t r o c h r y s e n e - t r a n s 1 , 2 - d i h y d r o d i o l was d e t e c t e d as the major m e t a b o l i t e and t h i s was f u r t h e r metabolized t o a product t e n t a t i v e l y i d e n t i f i e d as

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Figure 4. Microsomal metabolites of 7-nitrobenz[a]anthracene.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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1,2-dihydroxy-6-nitrochrysene ( F i g u r e 5 ) . O x i d a t i o n i n the r e g i o n p e r i t o the n i t r o f u n c t i o n ( i . e . , carbons 7 and 8) was not observed and r e d u c t i o n t o 6-aminochrysene o n l y o c c u r r e d when the 0^ concen­ t r a t i o n was d e c r e a s e d . I n S_. typhimurium TA100, 6 - n i t r o c h r y s e n e t r a n s - 1 , 2 - d i h y d r o d i o l was a b e t t e r d i r e c t - a c t i n g m u t a g e n t h a n 6-nitrochrysene o r t h e o t h e r two m e t a b o l i t e s . This latter o b s e r v a t i o n s u g g e s t s t h a t t h e d i h y d r o d i o l may be a p r o x i m a t e mutagenic form o f 6 - n i t r o c h r y s e n e and t h a t i t i s f u r t h e r a c t i v a t e d by b a c t e r i a l n i t r o r e d u c t i o n . I n t h e presence o f S9, a l l o f t h e m e t a b o l i t e s were mutagenic i n s t r a i n TA100, w i t h 6-aminochrysene b e i n g the most a c t i v e . 1-Nitropyrene. 1 - N i t r o p y r e n e i s the p r i n c i p a l n i t r o PAH found i n d i e s e l exhaust (40) and, t h e r e f o r e , has been the s u b j e c t o f i n t e n s e study. Nachtman and Wei (133) found t h a t under a n a e r o b i c c o n d i ­ t i o n s , 1 - n i t r o p y r e n e was r e d u c e d b y h e p a t i c S 9 , c y t o s o l o r microsomes t o p r i n c i p a l l y 1-aminopyrene. Only l i m i t e d r e d u c t i o n o c c u r r e d i n the absence o f c o f a c t o r s , w h i l e maximum m e t a b o l i s m was observed i n t h e presence o f b o t h FMN and NADPH. Although the microsomal f r a c t i o n had t h e g r e a t e s t s p e c i f i c a c t i v i t y toward 1 - n i t r o p y r e n e m e t a b o l i s m , t h e c y t o s o l had 30 times the t o t a l activity. S a i t o et_ a l . (134) found t h a t t h e c y t o s o l i c n i t r o r e d u c t a s e a c t i v i t y was due t o DT-diaphorase, aldehyde o x i d a s e , xanthine oxidase plus other u n i d e n t i f i e d n i t r o r e d u c t a s e s . As a n t i c i p a t e d , the microsomal r e d u c t i o n o f 1 - n i t r o p y r e n e was i n h i b i t e d by 0^ and s t i m u l a t e d by FMN w h i c h was a t t r i b u t e d t o t h i s c o f a c t o r a c t i n g as an e l e c t r o n s h u t t l e between NADPH-cytochrome P-450 r e d u c t a s e and cytochrome P-450. Carbon monoxide and type I I cytochrome P-450 i n h i b i t o r s d e c r e a s e d t h e r a t e o f n i t r o r e d u c t i o n w h i c h was c o n s i s t e n t w i t h the i n v o l v e m e n t o f cytochrome P-450. I n d u c t i o n o f cytochromes P-450 i n c r e a s e d r a t e s o f 1-aminopyrene f o r m a t i o n and n i t r o r e d u c t i o n was demonstrated i n a r e c o n s t i t u t e d cytochrome P-450 s y s t e m , w i t h i s o z y m e P - 4 4 8 - I I d c a t a l y z i n g t h e r e d u c t i o n most efficiently. El-Bayoumy and Hecht (91) were the f i r s t t o r e p o r t the o x i d a ­ t i v e metabolism o f 1 - n i t r o p y r e n e . U s i n g r a t l i v e r S9, they found 3-, 6-, and 8 - h y d r o x y - l - n i t r o p y r e n e and l - n i t r o p y r e n e - t r a n s - 4 , 5 - d i hydrodiol (Figure 6). A s m a l l amount o f 1-aminopyrene was a l s o formed and t h i s i n c r e a s e d i n c o n c e n t r a t i o n as the 0^ c o n c e n t r a t i o n was d e c r e a s e d . S i m i l a r m e t a b o l i t e s have r e c e n t l y been d e t e c t e d w i t h mouse l i v e r and l u n g S9 ( 1 0 3 ) . When assayed i n _S. typhimurium TA98 and TA100, 3- and 6 - h y d r o x y - l - n i t r o p y r e n e were found t o be b e t t e r d i r e c t - a c t i n g mutagens than 1 - n i t r o p y r e n e . Thus, as was found w i t h 6 - n i t r o c h r y s e n e (95) and 5 - n i t r o a c e n a p h t h e n e ( 8 4 - 8 6 ) , r i n g h y d r o x y l a t i o n does not n e c e s s a r i l y r e p r e s e n t a d e t o x i f i c a t i o n process. I n t e r e s t i n g l y , when the mutagenic a s s a y s were conducted i n the presence o f S9, 1 - n i t r o p y r e n e was more mutagenic t h a n i t s phenolic metabolites. The r e a s o n f o r t h i s apparent dichotomy i s not known. Bond (135) found t h a t S9 p r e p a r a t i o n s from r a t n a s a l t i s s u e have t w i c e t h e s p e c i f i c a c t i v i t y f o r the o x i d a t i v e m e t a b o l i s m o f 1 - n i t r o p y r e n e as l i v e r S9 and 10 times the a c t i v i t y o f l u n g S9. Each S9 p r e p a r a t i o n gave s i m i l a r m e t a b o l i c p r o f i l e s with the

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Figure 5. S9-catalyzed metabolites of 6-nitrochrysene.

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15.

F i g u r e 6. 1 - N i t r o p y r e n e tions.

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In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

incuba­

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch015

386

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

predominant m e t a b o l i t e s b e i n g 3-, 6-, and 8 - h y d r o x y - l - n i t r o p y r e n e p l u s a t l e a s t t h r e e o t h e r u n i d e n t i f i e d p r o d u c t s . K i n g ej: a i l . (136) conducted s i m i l a r s t u d i e s u s i n g S9 from r a b b i t l i v e r . A t l e a s t 12 m e t a b o l i t e s were d e t e c t e d and, based upon cochromatography w i t h known s t a n d a r d s , the major compounds appeared t o be K - r e g i o n ( i . e . , 4,5- and 9,10-) d i h y d r o d i o l s , 1 0 - h y d r o x y - l - n i t r o p y r e n e and o t h e r phenols. Bond and Mauderly (137) a l s o r e p o r t e d t h e presence o f 1 0 - h y d r o x y - l - n i t r o p y r e n e as w e l l as N - a c e t y l - l - a m i n o p y r e n e i n perfused r a t lung preparations. Howard ejt a l . (138) have s t u d i e d t h e metabolism o f 1 - n i t r o ­ pyrene u s i n g r a t l i v e r microsomes. I n microsomes from c o n t r o l and p h e n o b a r b i t a l - p r e t r e a t e d r a t s , t h e p r i n c i p a l m e t a b o l i t e was 3 - h y d r o x y - l - n i t r o p y r e n e , whereas A r o c l o r p r e t r e a t m e n t r e s u l t e d i n 6- and 8 - h y d r o x y - l - n i t r o p y r e n e b e i n g t h e major p r o d u c t s . These d a t a suggest t h a t d i f f e r e n t cytochrome P-450 isozymes may be responsible f o r the formation of the i n d i v i d u a l phenolic metabolites. I n a d d i t i o n t o these hydroxylated d e r i v a t i v e s , 1 - n i t r o p y r e n e - t r a n s - 4 , 5 - d i h y d r o d i o l , 1 - a m i n o p y r e n e , a n d two a d d i t i o n a l m e t a b o l i t e s were d e t e c t e d . More r e c e n t l y , t h i s m i c r o s o m a l metabolism has been examined i n g r e a t e r d e t a i l and l - n i t r o p y r e n e - 4 , 5 - o x i d e , l - n i t r o p y r e n e - 9 , 1 0 - o x i d e and 1-hydroxypyrene were i d e n t i f i e d as m e t a b o l i t e s through s p e c t r a l a n a l y s i s and by comparison t o s y n t h e t i c s t a n d a r d s ( 1 3 9 ) . 1-Nitropyrene-trans9 , 1 0 - d i h y d r o d i o l was a l s o d e t e c t e d , w h i c h c o n f i r m e d t h e o r i g i n a l i s o l a t i o n o f t h i s d e r i v a t i v e by K i n g et_ a l . ( 1 3 6 ) . I n subsequent s t u d i e s , m i c r o s o m a l i n c u b a t i o n s were conducted i n t h e presence o f Chinese hamster ovary (CHO) c e l l s . Under a n a e r o b i c c o n d i t i o n s , t h e p r i n c i p a l m e t a b o l i t e was 1 - a m i n o p y r e n e a n d one m a j o r a d d u c t , N - ( d e o x y g u a n o s i n - 8 - y l ) - 1 - a m i n o p y r e n e , was d e t e c t e d i n t h e CHO c e l l genome ( 1 4 0 ) . I n c o n t r a s t , under o x i d a t i v e c o n d i t i o n s amine f o r m a t i o n was suppressed and two DNA adducts were found. One o f these adducts c o e l u t e d w i t h N-(deoxyguanosin-8-yl)-1-aminopyrene, w h i l e the o t h e r was more p o l a r . T h i s l a t t e r adduct may r e s u l t from l - n i t r o p y r e n e - 4 , 5 - o x i d e and suggests t h a t g e n o t o x i c damage c a n r e s u l t from the o x i d a t i v e metabolism o f 1 - n i t r o p y r e n e . 1,3-, 1,6- and 1 , 8 - D i n i t r o p y r e n e . A l t h o u g h d i n i t r o p y r e n e s account f o r o n l y a s m a l l amount o f t h e n i t r o PAHs found i n d i e s e l e x h a u s t , they make a s i g n i f i c a n t c o n t r i b u t i o n t o the m u t a g e n i c i t y a s s o c i a t e d w i t h d i e s e l p a r t i c u l a t e s ( 4 0 ) . As noted e a r l i e r , i n S. typhimurium these d i n i t r o p y r e n e s appear t o be m e t a b o l i c a l l y a c t i v a t e d through s e q u e n t i a l n i t r o r e d u c t i o n and O - a c e t y l a t i o n . T h i s i s i n c o n t r a s t t o t h e r e l a t e d , l e s s mutagenic 1 - n i t r o p y r e n e , w h i c h o n l y r e q u i r e s n i t r o r e d u c t i o n f o r i t s m u t a g e n i c i t y (70,73,118). A s i m i l a r pathway has r e c e n t l y b e e n f o u n d u s i n g mammalian n i t r o r e d u c t a s e s a n d acetylases (141). Incubation of rat l i v e r cytosol with 1 - n i t r o ­ pyrene o r 1,3-, 1,6- o r 1 , 8 - d i n i t r o p y r e n e r e s u l t e d i n t h e f o r m a t i o n of 1-aminopyrene and t h e r e s p e c t i v e a m i n o n i t r o p y r e n e s . When DNA was i n c l u d e d i n t h e i n c u b a t i o n s , o n l y a l o w l e v e l o f DNA b i n d i n g was d e t e c t e d . However, a d d i t i o n o f a c e t y l coenzyme A (AcCoA) i n c r e a s e d t h e b i n d i n g o f t h e d i n i t r o p y r e n e s t o DNA 20- t o 4 0 - f o l d w h i l e t h e b i n d i n g o f 1 - n i t r o p y r e n e was o n l y s l i g h t l y a f f e c t e d . T h i s i n c r e a s e i n b i n d i n g o f d i n i t r o p y r e n e s t o DNA i n t h e presence of AcCoA was n o t d e t e c t e d when u s i n g dog l i v e r c y t o s o l w h i c h i s

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known t o be d e f i c i e n t i n a c e t y l a s e s . These d a t a i n d i c a t e t h a t mammalian c y t o s o l i c n i t r o r e d u c t a s e s c a t a l y z e t h e f o r m a t i o n o f N-hydroxy a r y l a m i n e i n t e r m e d i a t e s which i n t h e case o f d i n i t r o ­ pyrenes a r e c o n v e r t e d t o r e a c t i v e N-acetoxy a r y l a m i n e s by c y t o s o l i c AcCoA-dependent a c e t y l a s e s . 6-Nitrobenzo[a]pyrene. 6-Nitro-BaP i s a l s o a component o f d i e s e l exhaust (16,40); however, i n c o n t r a s t t o t h e n i t r a t e d p y r e n e s , i t i s o n l y mutagenic i n t h e presence o f S9 ( 1 5 - 1 8 ) . T h i s suggests t h a t o x i d i z e d m e t a b o l i t e s o f 6 - n i t r o - B a P may be r e s p o n s i b l e f o r m u t a t i o n i n d u c t i o n , and t h e r e f o r e , t h e s e p r o d u c t s have been c h a r a c t e r i z e d . When i n c u b a t e d w i t h l i v e r microsomes from 3-methylc h o l a n t h r e n e - p r e t r e a t e d r a t s , 6 - n i t r o - B a P was c o n v e r t e d i n t o 1- and 3 - h y d r o x y - 6 - n i t r o - B a P , 6 - n i t r o - B a P - l , 9 - and -3,9-hydroquinone and BaP-3,6-quinone ( F i g u r e 7, 1 4 2 ) . The same m e t a b o l i t e s were found when c o n t r o l o r p h e n o b a r b i t a l - i n d u c e d microsomes were used, w i t h 1and 3-hydroxy-6-nitro-BaP b e i n g t h e predominant p r o d u c t s i n each instance (143). The l a t t e r two phenols were more mutagenic than 6 - n i t r o - B a P , b u t as was observed w i t h 6 - n i t r o - B a P , they r e q u i r e d S9 t o be a c t i v e ( 1 4 2 ) . I n c o n t r a s t , 6 - n i t r o - B a P - l , 9 - and -3,9-hydroquinones appear t o be d i r e c t - a c t i n g b a c t e r i a l mutagens ( 9 6 ) . S e v e r a l c o n c l u s i o n s c a n be drawn from these r e s u l t s . First, as has been noted w i t h 5-nitroacenaphthe n e and 6 - n i t r o c h r y s e n e , t h e r e appears t o be no p e r i - r e g i o n ( i . e . , 4,5- o r 7,8-) o x i d a t i o n . Second, a l t h o u g h r i n g o x i d a t i o n appears t o be an a c t i v a t i o n s t e p , S9 i s s t i l l r e q u i r e d f o r 1- and 3-hy d r o x y - 6 - n i t r o - B a P t o be mutagenic. I n a d d i t i o n , e s s e n t i a l l y t h e same mutagenic a c t i v i t y was found i n TA98 and TA100 and t h e i r r e s p e c t i v e n i t r o r e d u c t a s e deficient derivatives. Thus, i n c o n t r a s t t o t h e n i t r o PAHs c o n s i d e r e d p r e v i o u s l y , t h e m e t a b o l i c a c t i v a t i o n o f 6 - n i t r o - B a P may not i n v o l v e n i t r o r e d u c t i o n , but o n l y r i n g o x i d a t i o n . Third, since 6 - n i t r o - B a P - l , 9 - and -3,9-hydroquinones a r e d i r e c t - a c t i n g mutagens, t h e s e m e t a b o l i t e s may be t h e u l t i m a t e m u t a g e n i c f o r m s o f 6-nitro-BaP. R e c e n t l y , t h e mechanism o f 6 - n i t r o - B a P r i n g h y d r o x y l a t i o n h a s been e l u c i d a t e d by u s i n g 3 - d e u t e r o - 6 - n i t r o - B a P ( 1 4 4 ) . When i n c u ­ b a t e d w i t h 3 - m e t h y l c h o l a n t h r e n e - i n d u c e d r a t l i v e r microsomes, t h i s d e u t e r a t e d analogue y i e l d e d t h e same m e t a b o l i t e p r o f i l e p r e v i o u s l y observed w i t h 6 - n i t r o - B a P . S p e c t r o s c o p i c a n a l y s i s o f 3-hydroxy-6n i t r o - B a P and 6-nitro-BaP-3,9-hydroquinone i n d i c a t e d t h a t 30% o f the d e u t e r i u m l a b e l had m i g r a t e d t o carbon 2, presumably v i a a n NIH shift. T h e r e f o r e , i t appears t h a t 6 - n i t r o - B a P - 2 , 3 - o x i d e i s a common i n t e r m e d i a t e f o r these two m e t a b o l i t e s . 1- and 3 - N i t r o b e n z o [ a ] p y r e n e . 1- and 3-Nitro-BaP a l s o appear t o be components o f d i e s e l e x h a u s t , b u t u n l i k e 6 - n i t r o - B a P , these n i t r o PAHs a r e d i r e c t - a c t i n g b a c t e r i a l mutagens ( 1 5 - 1 8 ) . When 1- and 3 - n i t r o - B a P were i n c u b a t e d w i t h l i v e r microsomes from 3-methylcholanthrene-pretreated r a t s , 7 , 8 - t r a n s - d i h y d r o d i o l s , 9,10-transd i h y d r o d i o l s , and 7 , 8 , 9 , 1 0 - t e t r a h y d r o t e t r o l s were formed as major m e t a b o l i t e s ( F i g u r e 8; 145-146), w h i l e amine f o r m a t i o n was d e t e c t e d o n l y under a n a e r o b i c i n c u b a t i o n s ( 1 4 7 ) . These r e s u l t s a r e i n c o n t r a s t t o those found w i t h 6 - n i t r o - B a P ; w i t h 1- and 3 - n i t r o - B a P , a l l o f t h e o x i d a t i o n o c c u r r e d i n t h e t e r m i n a l benzo r i n g ( i . e . ,

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carbons 7, 8, 9, and 1 0 ) , w h i l e 6 - n i t r o - B a P metabolism initially o c c u r s a t carbons 1 and 3. Thus, the n i t r o group has a s i g n i f i c a n t e f f e c t upon t h e r e g i o s e l e c t i v i t y o f m e t a b o l i c o x i d a t i o n . The absence o f h y d r o x y l a t i o n a t carbon 3 i n 1 - n i t r o - B a P i s noteworthy because, i n a d d i t i o n t o b e i n g a major s i t e o f o x i d a t i o n i n 6 - n i t r o - B a P (142) , 3-hydroxy-BaP i s a predominant m i c r o s o m a l m e t a b o l i t e o f BaP ( 1 4 8 ) . Furthermore, t h e analogous carbon i n 1 - n i t r o p y r e n e i s a s i t e f o r e x t e n s i v e metabolism (91,138). The r e a s o n f o r t h i s marked r e g i o s e l e c t i v i t y i s not known. The c o n f o r m a t i o n s o f the 1- and 3 - n i t r o - B a P m e t a b o l i t e s were determined through a n a l y s i s o f t h e i r NMR s p e c t r a (145-146). Both 1- and 3 - n i t r o - B a P - t r a n s - 7 , 8 - d i h y d r o d i o l s e x i s t e d p r e d o m i n a n t l y i n q u a s i - d i e q u a t o r i a l c o n f o r m a t i o n s , w h i c h corresponds t o the pre­ f e r r e d c o n f o r m a t i o n of the proximate c a r c i n o g e n BaP-Jtrans-7 ,8-dihy­ d r o d i o l (149). T h i s suggests t h a t these d i h y d r o d i o l m e t a b o l i t e s may be c o n v e r t e d i n t o e l e c t r o p h i l i c d i o l epoxides and i n support o f t h i s c o n t e n t i o n , t h e s t e r e o c h e m i s t r i e s o f 1- and 3 - n i t r o - B a P 7 , 8 , 9 , 1 0 - t e t r o l s were i n d i c a t i v e o f t r a n s - 7 , 8 - d i h y d r o d i o l - a n t i 9,10-epoxide i n t e r m e d i a t e s . I t i s p o s s i b l e , however, t h a t a p r o p o r t i o n o f the t e t r o l m e t a b o l i t e s were formed from t r a n s - 9 , 1 0 dihydrodiol-anti-7,8-epoxides. The m u t a g e n i c i t i e s o f 3 - n i t r o - B a P , 3-amino-BaP, and 3 - n i t r o BaP-7,8- and - 9 , 1 0 - d i h y d r o d i o l s have r e c e n t l y been compared i n S. typhimurium s t r a i n s TA98, TA98NR, and TA98/1,8-DNP, ( 1 4 7 ) . I n the absence o f S9, 3 - n i t r o - B a P showed decreased a c t i v i t y i n TA98NR and s l i g h t l y h i g h e r m u t a g e n i c i t y i n TA98/1,8-DNP compared t o TA98, which i s c o n s i s t e n t w i t h n i t r o r e d u c t i o n , but not e s t e r i f i c a t i o n , being e s s e n t i a l for mutation i n d u c t i o n . This conclusion i s s u p p o r t e d by the o b s e r v a t i o n t h a t the d i r e c t - a c t i n g m u t a g e n i c i t y o f 3-amino-BaP was the same i n a l l t h r e e s t r a i n s , p r o b a b l y as a r e s u l t of i t b e i n g s p o n t a n e o u s l y o x i d i z e d t o N-hydroxy-3-amino-BaP. The d i h y d r o d i o l s behaved s i m i l a r l y t o one a n o t h e r : t h e i r d i r e c t - a c t i n g m u t a g e n i c i t y was s i m i l a r t o t h a t o f 3 - n i t r o - B a P i n TA98, decreased by a b o u t 5 0 % i n TA98NR, a n d was s u b s t a n t i a l l y d e c r e a s e d i n TA98/1,8-DNP^. These r e s u l t s suggest t h a t b o t h n i t r o r e d u c t i o n and e s t e r i f i c a t i o n a r e r e q u i r e d f o r the d i r e c t - a c t i n g m u t a g e n i c i t y o f these d i h y d r o d i o l m e t a b o l i t e s . 6

Conclusions The d a t a p r e s e n t e d i n t h i s r e v i e w show t h a t b o t h o x i d a t i v e and r e d u c t i v e pathways a r e i n v o l v e d i n t h e m e t a b o l i c a c t i v a t i o n o f n i t r o PAHs t o g e n o t o x i c agents jLn v i t r o . The p r e c i s e pathways depend upon the p a r t i c u l a r compound, but i n each i n s t a n c e the n i t r o group appears t o p l a y a c r i t i c a l r o l e i n the a c t i v a t i o n p r o c e s s . W i t h r e g a r d t o o x i d a t i v e metabolism, the l o c a t i o n of the n i t r o f u n c t i o n i n f l u e n c e s the r e g i o s e l e c t i v i t y o f r i n g o x i d a t i o n s i n c e metabolism i s generally inhibited i n peri regions. When p e r i r e g i o n o x i d a t i o n does o c c u r , i t i s q u i t e l i m i t e d i n e x t e n t and a s i g n i f i c a n t p r o p o r t i o n o f the d i h y d r o d i o l m e t a b o l i t e e x i s t s i n a q u a s i - d i a x i a l conformation. Thus, i n a d d i t i o n t o i n f l u e n c i n g t h e r e g i o s e l e c t i v i t y o f o x i d a t i o n , the n i t r o group can have a marked e f f e c t upon the c o n f o r m a t i o n o f the r e s u l t a n t m e t a b o l i t e w h i c h , i n t u r n , may i n f l u e n c e subsequent metabolism. The n i t r o f u n c t i o n may a l s o be d i r e c t l y i n v o l v e d i n the a c t i v a t i o n sequence v i a r e d u c t i o n

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to r e a c t i v e N-hydroxy a r y l a m i n e s . N-Hydroxy a r y l a m i n e s c a n r e a c t d i r e c t l y w i t h DNA, o r i n some i n s t a n c e s they may be f u r t h e r a c t i v a t e d by 0 - e s t e r i f i c a t i o n . A l t h o u g h t h e f a c t o r s t h a t a l l o w an N-hydroxy a r y l a m i n e t o undergo 0 - e s t e r i f i c a t i o n a r e n o t known, e s t e r f o r m a t i o n g e n e r a l l y r e s u l t s i n t h e p r o d u c t i o n o f a compound which i s more g e n o t o x i c than t h e N-hydroxy a r y l a m i n e p r e c u r s o r . F i n a l l y , some n i t r o PAHs a r e m e t a b o l i z e d t o u l t i m a t e mutagens by a combination o f o x i d a t i v e and r e d u c t i v e pathways. With these compounds, r i n g o x i d a t i o n g e n e r a l l y precedes n i t r o r e d u c t i o n and t h e net e f f e c t appears t o be t h e f o r m a t i o n o f an N-hydroxy a r y l a m i n e m e t a b o l i t e which can serve as a s u b s t r a t e f o r O ^ e s t e r i f i c a t i o n . The d a t a from these i n v i t r o s t u d i e s c a n p r o v i d e i n s i g h t i n t o the t u m o r i g e n i c i t y o f n i t r o PAHs. Thus, n i t r o d e r i v a t i v e s o f n o n c a r c i n o g e n i c PAHs, such as 2 - n i t r o n a p h t h a l e n e o r 4 - n i t r o b i ­ p h e n y l , a r e e x t e n s i v e l y reduced _in v i t r o which i s c o n s i s t e n t w i t h t h e i r showing t h e same t a r g e t s p e c i f i c i t y as t h e i r a r o m a t i c amine analogues. T h i s v i e w i s s t r e n g t h e n e d by t h e o b s e r v a t i o n t h a t i d e n t i c a l DNA adducts have been found i n t h e b l a d d e r e p i t h e l i u m o f dogs a d m i n i s t e r e d e i t h e r 4 - n i t r o b i p h e n y l o r 4-aminobiphenyl ( 1 1 6 ) . N i t r o d e r i v a t i v e s o f c a r c i n o g e n i c PAHs demonstrate v a r i e d t u m o r i ­ g e n i c r e s p o n s e s w h i c h may be a s s o c i a t e d w i t h d i f f e r e n c e s i n m e t a b o l i c pathways. F o r example, 6 - n i t r o c h r y s e n e i s t u m o r i g e n i c on mouse s k i n and undergoes e x t e n s i v e d i h y d r o d i o l f o r m a t i o n i n v i t r o w h i c h suggests t h a t i t i s m e t a b o l i z e d t o a r e a c t i v e d i o l e p o x i d e . I n c o n t r a s t , d i h y d r o d i o l m e t a b o l i t e s a r e a p p a r e n t l y n o t formed from 6 - n i t r o b e n z o [ a ] p y r e n e and t h i s compound g i v e s a n e g a t i v e t u m o r i ­ g e n i c response on mouse s k i n . I n a d d i t i o n , 6-nitrobenzo[a]pyrene has two p r o t o n s p e r i t o i t s n i t r o f u n c t i o n , which appears t o r e s t r i c t i t s a b i l i t y t o be e n z y m a t i c a l l y c o n v e r t e d t o a r e a c t i v e N-hydroxy a r y l a m i n e ( 1 5 0 ) . These i n v i t r o m e t a b o l i c s t u d i e s a l s o i n d i c a t e t h a t a c o m b i n a t i o n o f o x i d a t i v e and r e d u c t i v e pathways may be i n v o l v e d i n t h e t u m o r i g e n i c i t y o f c e r t a i n n i t r o PAHs. T h i s may be p a r t i c u l a r l y i m p o r t a n t w i t h t h e r i n g - o x i d i z e d m e t a b o l i t e s o f 1 - n i t r o p y r e n e , 5 - n i t r o a c e n a p h t h e n e and 3 - n i t r o b e n z o [ a ] p y r e n e , which a r e a t l e a s t as g e n o t o x i c as t h e i r parent n i t r o PAHs. These o b s e r v a t i o n s suggest t h a t i n o r d e r t o a s s e s s t h e human h e a l t h r i s k from n i t r o PAHs, t u m o r i g e n i c i t y assays s h o u l d be conducted n o t o n l y w i t h t h e p a r e n t compounds and t h e i r n i t r o r e d u c t i o n p r o d u c t s , b u t a l s o w i t h t h e r i n g - o x i d i z e d m e t a b o l i t e s t h a t have been d e t e c t e d i n i n v i t r o incubations. Acknowledgment We thank Ruth Y o r k f o r h e l p i n g prepare t h i s

review.

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142. Fu, P.P.; Chou, M.W.; Yang, S.K.; Beland, F.A.; Kadlubar, F.F.; Casciano, D.A.; Heflich, R.H.; Evans, F.E. Biochem. Biophys. Res. Commun. 1982, 105, 1037. 143. Fu, P.P.; Chou, M.W. In "Cytochrome P-450, Biochemistry, Biophysics and Environmental Implications"; Hietanen, E.; Laitinen, M.; Hanninen, O., Eds.; Elsevier: Amsterdam, 1982, p. 71. 144. Chou, M.W.; Evans, F.E.; Yang, S.K.; Fu, P.P. Carcinogenesis 1983, 4, 699. 145. Chou, M.W.; Fu, P.P. Biochem. Biophys. Res. Commun. 1983, 117, 541. 146. Chou, M.W.; Von Tungeln, L.S.; Unruh, L.E.; Fu, P.P. In "Eighth International Symposium on Polynuclear Aromatic Hydrocarbons"; Cooke, W.M.; Dennis, A.J., Eds.; Battelle: Columbus, OH, in press. 147. Chou, M.W.; Heflich, R.H.; Fu, P.P. submitted. 148. Holder, G.; Yagi, H.; Dansette, P.; Jerina, D.M.; Levin, W.; Lu, A.Y.H.; Conney, A.H. Proc. Natl. Acad. Sci. USA 1974, 71, 4356. 149. Jerina, D.M.; Selander, H.; Yagi, H.; Wells, M.C.; Davey, J.F.; Mahadevan, V.; Gibson, D.T. J. Amer. Chem. Soc. 1976, 98, 5988. 150. Fu, P.P.; Chou, M.W.; Miller, D.W.; White, G.L.; Heflich, R.H.; Beland, F.A. submitted. RECEIVED May 2, 1985

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix001

Author Index

Amin, S h a n t u , 85 B e l a n d , F r e d e r i c k A., 341,371 C a v a l i e r i , E r c o l e L . , 289 Chang, R i c h a r d L . , 63 C h i u , P e i - L u , 19 Conney, A l l a n H., 63 D i p p l e , Anthony, 1 Dommen, J o s e f , 239 E i s e n s t a d t , E r i c , 327 F u , P e t e r P., 371 G e a c i n t o v , N i c h o l a s E . , 107 G l u s k e r , Jenny P., 125 H a r v e y , Ronald G., 35 H e c h t , S t e p h e n S., 85 H e f l i c h , R o b e r t H., 371 Hoffmann, D i e t r i c h , 85 Howard, P a u l C , 371 J e f f r e y , A l a n M., 185

J e r i n a , Donald M., 63 K a d l u b a r , F r e d F., 341 Kumar, Subodh, 63 L a V o i e , Edmond J . , 85 L e B r e t o n , P. R., 209 L e h r , Roland E . , 63 L e v i n , Wayne, 63 M a r n e t t , Lawrence J . , 307 M e l i k i a n , A s s i e h A., 85 M i l l e r , Kenneth J . , 239 Mushtaq., Mohammad, 19 Rogan, E l e a n o r G., 289 S a y e r , Jane M., 63 T a y l o r , E r i c R., 239 Wood, A l e x a n d e r W., 63 Y a g i , H a r u h i k o , 63 Yang, Shen K., 19

Subject Index

A

N-Acetoxy arylamides c a r c i n o g e n i c i t y , 347 e l e c t r o p h i l i c r e a c t i v i t y , 347 p r o p e r t i e s , 347 r e a c t i o n mechanism, 347-48 r o l e as u l t i m a t e c a r c i n o g e n s , 347 N-Acetoxy arylamines i s o l a t i o n , 350 m e t a b o l i c f o r m a t i o n , 350,352 r e a c t i o n mechanism, 350-52 r o l e as u l t i m a t e c a r c i n o g e n s , 352 A c t i v a t e d carcinogen-DNA i n t e r a c t i o n s , computer m o d e l i n g , 170,176 Activated carcinogen-nucleotide interactions d i s c u s s i o n , 159,161,169 r i b o s e 0 atom-methyl group i n t e r a c t i o n , 170,172f s t a c k i n g o f p o l y c y c l i c aromatic h y d r o c a r b o n s and b a s e s , 169-70,171f-72f

Activated carcinogen-polypeptide i n t e r a c t i o n s , 157 Aflatoxin B c r y s t a l s t r u c t u r e , 131 mode o f a c t i o n , 131 Alkylated nucleotides, s t r u c t u r e s , 161,165f-68f,169-70,173f Alkylated tripeptide, s t r u c t u r e , 157-59 A l k y l a t i o n o f amino g r o u p s o f DNA b a s e s , 139,140f Angstrom u n i t s , d e f i n i t i o n , 128 A n t h r a c e n e , c a r c i n o g e n i c a c t i v i t y , 45 A n t h r a c e n e d i h y d r o d i o l s , s y n t h e s i s , 45 A r y l n i t r o n e s , r e a c t i o n mechanism, 363f

B

Bay r e g i o n d e f i n i t i o n , 8,128 structures, 8 , l l f

397

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

Author Index

Amin, S h a n t u , 85 B e l a n d , F r e d e r i c k A., 341,371 C a v a l i e r i , E r c o l e L . , 289 Chang, R i c h a r d L . , 63 C h i u , P e i - L u , 19 Conney, A l l a n H., 63 D i p p l e , Anthony, 1 Dommen, J o s e f , 239 E i s e n s t a d t , E r i c , 327 F u , P e t e r P., 371 G e a c i n t o v , N i c h o l a s E . , 107 G l u s k e r , Jenny P., 125 H a r v e y , Ronald G., 35 H e c h t , S t e p h e n S., 85 H e f l i c h , R o b e r t H., 371 Hoffmann, D i e t r i c h , 85 Howard, P a u l C , 371 J e f f r e y , A l a n M., 185

J e r i n a , Donald M., 63 K a d l u b a r , F r e d F., 341 Kumar, Subodh, 63 L a V o i e , Edmond J . , 85 L e B r e t o n , P. R., 209 L e h r , Roland E . , 63 L e v i n , Wayne, 63 M a r n e t t , Lawrence J . , 307 M e l i k i a n , A s s i e h A., 85 M i l l e r , Kenneth J . , 239 Mushtaq., Mohammad, 19 Rogan, E l e a n o r G., 289 S a y e r , Jane M., 63 T a y l o r , E r i c R., 239 Wood, A l e x a n d e r W., 63 Y a g i , H a r u h i k o , 63 Yang, Shen K., 19

Subject Index

A

N-Acetoxy arylamides c a r c i n o g e n i c i t y , 347 e l e c t r o p h i l i c r e a c t i v i t y , 347 p r o p e r t i e s , 347 r e a c t i o n mechanism, 347-48 r o l e as u l t i m a t e c a r c i n o g e n s , 347 N-Acetoxy arylamines i s o l a t i o n , 350 m e t a b o l i c f o r m a t i o n , 350,352 r e a c t i o n mechanism, 350-52 r o l e as u l t i m a t e c a r c i n o g e n s , 352 A c t i v a t e d carcinogen-DNA i n t e r a c t i o n s , computer m o d e l i n g , 170,176 Activated carcinogen-nucleotide interactions d i s c u s s i o n , 159,161,169 r i b o s e 0 atom-methyl group i n t e r a c t i o n , 170,172f s t a c k i n g o f p o l y c y c l i c aromatic h y d r o c a r b o n s and b a s e s , 169-70,171f-72f

Activated carcinogen-polypeptide i n t e r a c t i o n s , 157 Aflatoxin B c r y s t a l s t r u c t u r e , 131 mode o f a c t i o n , 131 Alkylated nucleotides, s t r u c t u r e s , 161,165f-68f,169-70,173f Alkylated tripeptide, s t r u c t u r e , 157-59 A l k y l a t i o n o f amino g r o u p s o f DNA b a s e s , 139,140f Angstrom u n i t s , d e f i n i t i o n , 128 A n t h r a c e n e , c a r c i n o g e n i c a c t i v i t y , 45 A n t h r a c e n e d i h y d r o d i o l s , s y n t h e s i s , 45 A r y l n i t r o n e s , r e a c t i o n mechanism, 363f

B

Bay r e g i o n d e f i n i t i o n , 8,128 structures, 8 , l l f

397

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

398

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Bay-region d i h y d r o d i o l epoxide generalization, d i s c u s s i o n , 8,12,15 Bay-region hypothesis, carcinogenic mechanism, 145 B a y - r e g i o n m e t h y l group enhanced e f f e c t on t u m o r i g e n i c i t y , m e c h a n i s t i c b a s i s , 91,96,97 Bay-region theory d i a g r a m , 65 d i s c u s s i o n , 64,65 t h e o r e t i c a l b a s i s , 65 Benz[a]acridine e f f e c t o f n i t r o g e n s u b s t i t u t i o n on t u m o r i g e n i c i t y , 78 s t r u c t u r e , 78 t u m o r i g e n i c i t y , 78 BenzfaJacridine tetraepoxide e f f e c t o f n i t r o g e n s u b s t i t u t i o n on m u t a g e n i c i t y , 79 m u t a g e n i c i t y , 79,80f Benz[a_] a c r i d i n e d i o l e p o x i d e e f f e c t o f n i t r o g e n s u b s t i t u t i o n on m u t a g e n i c i t y , 79 m u t a g e n i c i t y , 78,79,80f Benz[ c j a c r i d i n e e f f e c t o f n i t r o g e n s u b s t i t u t i o n on t u m o r i g e n i c i t y , 78 s t r u c t u r e , 78 t u m o r i g e n i c i t y , 78 Benz[cJacridine tetraepoxide e f f e c t o f n i t r o g e n s u b s t i t u t i o n on m u t a g e n i c i t y , 79 m u t a g e n i c i t y , 79,80f B e n z [ c J a c r i d i n e d i o l epoxide e f f e c t o f n i t r o g e n s u b s t i t u t i o n on m u t a g e n i c i t y , 79 m u t a g e n i c i t y , 78,79,80f Benzf a] a n t h r a c e n e s a c t i v i t y , 5,7,9t c a r c i n o g e n i c a c t i v i t y , 38

DNA adducts, 200-201 e f f e c t o f n i t r o g e n s u b s t i t u t i o n on t u m o r i g e n i c i t y , 78 m e t a b o l i c a c t i v a t i o n pathway, 25

m e t a b o l i t e adducts, 200-201 s t e r e o s e l e c t i v e m e t a b o l i s m a t the K r e g i o n , 27 s t r u c t u r e s , 6,78,126,138,140f Benz[a_] a n t h r a c e n e t e t r a e p o x i d e effect of nitrogen substitution on m u t a g e n i c i t y , 79 m u t a g e n i c i t y , 79,80f Benz[a_] a n t h r a c e n e d i h y d r o d i o l s enantiomeric c o m p o s i t i o n s , 25,28t,29t m a j o r e n a n t i o m e r , 27,28t m e t a b o l i c pathways, 25 o p t i c a l p u r i t y , 25

BenzfaJ a n t h r a c e n e d i h y d r o d i o l s — Continued s e p a r a t i o n , 25 s t e r e o s e l e c t i v e m e t a b o l i s m , 29 s y n t h e s i s v i a Methods I I and I I I , 40,41f s y n t h e s i s v i a quinone r e d u c t i o n , 40,42 Benz[ a_] a n t h r a c e n e d i o l s , s t r u c t u r e s , 145,146f ,147f B e n z [ a J a n t h r a c e n e d i o l epoxide e f f e c t o f n i t r o g e n s u b s t i t u t i o n on m u t a g e n i c i t y , 79 m u t a g e n i c i t y , 78,79,80f Benz[a_,hjanthracene, s p e c t r u m , 2 Benzene c r y s t a l s t r u c t u r e , 128 X - r a y d i f f r a c t i o n s t u d i e s , 128 Benzofluoranthenes, carcinogenic a c t i v i t y , 56 Benzofc]phenanthrenes c a r c i n o g e n i c a c t i v i t y , 5,46 s t r u c t u r e , 6,46,49 s y n t h e s i s v i a Method I , 46,48,49f Benzo[a]pyrenes bay r e g i o n , 8 , l l f b a y - r e g i o n d i h y d r o d i o l epoxide m e t a b o l i s m r o u t e , 12,13f bond l e n g t h s , i n t e r b o n d a n g l e s , and t o r s i o n a n g l e s , 128,129f c a r c i n o g e n i c a c t i v i t y , 7,12,13t,14t c o n f o r m a t i o n s , 389

DNA adducts, 198-200 levels i n a i r , 3 l e v e l s i n d r i n k i n g water, major a c t i v a t i o n pathways, 19,20f m e t a b o l i c a c t i v a t i o n , 387-89 m e t a b o l i s m , 64 m i c r o s o m a l m e t a b o l i t e s , 387,388f m u t a g e n i c i t y , 387-89 n o n r e a c t i v e m e t a b o l i t e s , 215 physical binding i n t e r a c t i o n s , 215-16 stereoselective m e t a b o l i s m , 19-21 s t r u c t u r e , 6,126 structure of bay-region d i h y d r o d i o l epoxide, 8,1If structure of K-region dihydrodiol epoxide, 1 0 , l l f s t r u c t u r e s o f m e t a b o l i t e s and m e t a b o l i c model compounds, 212,213f Benzo[a]pyrene epoxides s t r u c t u r e , 139,141f i s o l a t i o n , 23 s t e r e o c h e m i s t r y , 23,25 BenzofaJpyrene metabolism, c l a s s e s o f p r o d u c t s , 296

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

399

INDEX

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

BenzofaJpyrene metabolites a b s o l u t e c o n f i g u r a t i o n d e t e r m i n e d by X - r a y d i f f r a c t i o n , 145,148f c a r c i n o g e n i c a c t i v i t i e s , 12,13t,14t comparison of p h y s i c a l b i n d i n g p r o p e r t i e s , 227 p h y s i c a l b i n d i n g to s e c o n d a r y s i t e s on DNA, 225 s t r u c t u r e s , 12,13t,14t BenzofaJpyrene o x i d a t i o n a d d i t i o n of RNA o r DNA, 308 a d d i t i o n o f S a l m o n e l l a typhimurium s t r a i n s , 310 PGH s y n t h a s e , 308 quinone p r o d u c t s , 308,309f BenzofaJ p y r e n e d i h y d r o d i o l m e t a b o l i t e s a b s o l u t e c o n f i g u r a t i o n s , 21 e x c i t a t i o n c h i r a l i t y CD s p e c t r a , 21,22f o p t i c a l p u r i t y , 21,23t s t e r e o c h e m i s t r y , 23,25 B e n z o f a J p y r e n e d i o l s , s t r u c t u r e s , 143 Benzofajpyrenediol epoxides a s s o c i a t i o n c o n s t a n t s , 223-25 c o n f o r m a t i o n s , 152,153f-54f c o n f o r m a t i o n a l e n e r g i e s and a v e r a g e benzo r i n g c o n f o r m a t i o n s , 253-56 c o n f o r m a t i o n a l s t a b i l i z a t i o n by h y d r o g e n b o n d i n g , 256 d i a s t e r e o i s o m e r s , 240,252,253f i n t e r c a l a t i o n t o DNA, 223 p r e m u t a t i o n a l l e s i o n , 333,335 p r o p e r t i e s of aromatic chromophore, 109 s t e r e o i s o m e r s , 107,108f s t r u c t u r e s of a n a l o g u e s , 149,151f-53f s t r u c t u r e s of s t e r e o i s o m e r s , 149,150f s y n t h e s i s , 64 t o r s i o n a n g l e s , 152,155t UV a b s o r p t i o n b i n d i n g c o n s t a n t s , 225,226t B e n z o f a J p y r e n e d i o l epoxide-DNA a d d u c t s binding energies v i a trans a d d i t i o n , 267-73 b i n d i n g e n e r g y , 249-51 b i n d i n g p r o c e s s s t e p s , 248 e n e r g y change f o r l o s s o f e l e c t r o n p a i r c o n j u g a t i o n on NHR, 259-61 e x t e r n a l l y and i n t e r n a l l y bound

adducts, 275 f o r m a t i o n mechanism, 251 i n t e r c a l a t i o n optimum b i n d i n g o r i e n t a t i o n s , 263-65,266f i n t e r c a l a t i v e covalent

b i n d i n g , 267,268f-69f,272f,274 i n t e r m o l e c u l a r e n e r g y dependence f o r t r a n s a d d i t i o n , 266,267f

Benzofajpyrenediol

epoxide-DNA

adducts

Continued mechanism f o r

stereoselectivity,

246-48,281-83

possible reaction schemes, 112-13,115f r e a c t i o n c o o r d i n a t e s , 251,252f r e a c t i o n mechanism, 212,215 r e a c t i o n pathways, 110,112 r e h y b r i d i z a t i o n o f amino g r o u p s , relative intercalation e n e r g i e s , 264t,265

259

s t e r e o s e l e c t i v i t y , 266-69,272-75,276t s t r u c t u r e o f the

covalent

adducts, 110 t h e o r e t i c a l m o d e l i n g , 246 B e n z o f a j p y r e n e d i o l epoxide-DNA adduct r e a c t i o n pathways c o v a l e n t b i n d i n g mechanism, 110 f r a c t i o n of d i o l e p o x i d e m o l e c u l e s b i n d i n g to DNA, 112 i o n i c s t r e n g t h - l e v e l of c o v a l e n t b i n d i n g r e l a t i o n s h i p , 112 mechanism, 110,112 r a t e c o n s t a n t , 112 sequence s p e c i f i c i t y , 112-13 B e n z o f a j p y r e n e d i o l epoxide-DNA covalent adducts a b s o r b a n c e and l i n e a r d i c h r o i s m s p e c t r a o f e n a n t i o m e r s bound to DNA, 120,121f f l u o r e s c e n c e h e t e r o g e n e i t y , 116-18 f l u o r e s c e n c e p r o p e r t i e s , 116-18,119f f l u o r e s c e n c e q u e n c h i n g , 117-18 l i n e a r s p e c t r a d i c h r o i s m , 114 s o l v e n t a c c e s s i b i l i t y , 117 stereoselective covalent b i n d i n g , 120 s t r u c t u r e s o f model r e a c t a n t s t h a t g i v e s i t e I a d d u c t s , 114,115f B e n z o [ a j p y r e n e d i o l epoxide mutagenicity m o l e c u l a r g e n e t i c s t u d i e s , 335,337 p r e v i o u s i n v e s t i g a t i o n s , 331-32 s p e c i f i c i t y , 332 two-step t r a n s f o r m a t i o n p r o c e s s , 337-38 B e n z o f a j p y r e n e d i o l e p o x i d e mutagenic specificity base s u b s t i t u t i o n e v e n t s , 332,333t d i s t r i b u t i o n o f l a c l nonsense m u t a t i o n s , 332,334t i n d u c e d m u t a t i o n s i n the l a c l gene, 332,335t,336f B e n z o f a J p y r e n e d i o l epoxide-N2 (quanine) adducts benzo r i n g c o n f o r m a t i o n s and c o n f o r m a t i o n a l e n e r g i e s , 257,259t preferred conformations, 257

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

400

B e n z o [ a J p y r e n e d i o l epoxide-N2 adducts—Continued

(quanine)

r e l a t i v e energies of various c o n f o r m a t i o n s , 257,258t Benzo[e]pyrene

adduct f o r m a t i o n , 200

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

c a r c i n o g e n i c a c t i v i t y , A3 Benzo[ej p y r e n e d i h y d r o d i o l s e p o x i d a t i o n , A3 s y n t h e s i s , A3,AAf

.Carcinogenesis, process, A Carcinogenic a c t i v i t y dependence on b e n z o [ a ] p y r e n e , 3 determination, 2 mouse s k i n m o n i t o r i n g system, A route o f a d m i n i s t r a t i o n - t i s s u e affected relationship, A structure relationship, 2 C a r c i n o g e n i c a r y l a m i n e s and arylamides, metabolic a c t i v a t i o n pathways, 3 A l , 3 A 2 f Carcinogenic d i s t i l l a t e s , fluorescence, 2 Carcinogenic p o l y c y c l i c aromatic hydrocarbons b a y - r e g i o n geometry, 133,135f b u c k l i n g o f the m o l e c u l e , 133,136 c r y s t a l s t r u c t u r e s , 131 p l a n a r i t y d i s t o r t i o n s , 131,133 shapes and s i t e s o f a c t i v a t i o n , 131,132f s i t e o f a c t i o n , 139 t o r s i o n a n g l e s , 133 views o f d i s t o r t i o n s due t o s t e r i c e f f e c t s , 133,13Af-35f Carcinogens c a r c i n o g e n i c i t y f o r man, 3 c h a r a c t e r i z a t i o n , 1,2 examples, 1 o c c u p a t i o n a l exposure, 3 Chemical c a r c i n o g e n e s i s , m u l t i s t a g e p r o c e s s , 185-86 Chrysenes c a r c i n o g e n i c a c t i v i t y , A5

DNA

a d d u c t s , 201

C h r y s e n e d i h y d rod i o l s e p o x i d a t i o n , A6 s y n t h e s i s v i a Methods I I I and IV, A6,A7f,A9f C o c a r c i n o g e n s , d e f i n i t i o n , 2A1 Computer-modeling s t u d i e s d o c k i n g a base i n t o Z-DNA, 170,17Af-75f model o f q u a s i - i n t e r c a l a t e d p o l y c y c l i c aromatic h y d r o c a r b o n s , 176,177f

C o o x y g e n a t i o n , importance i n c h e m i c a l c a r c i n o g e n e s i s , 323 C r y s t a l s t r u c t u r e , d e f i n i t i o n , 127 Cytochrome P-A50 d e f i n i t i o n , 307 p r o p e r t i e s , 307 Cytochrome P-A50c s u b s t r a t e b i n d i n g s i t e model d e s c r i p t i o n , 29,31 s c h e m a t i c , 29,30f

D e o x y r i b o n u c l e i c a c i d — S e e DNA Dibenz[ £,c]anthracene, s t r u c t u r e , 88 Dibenz[£,h]anthracene c a r c i n o g e n i c a c t i v i t y , A2 spectrum, 2 s t r u c t u r e , 127 Dibenz[a_,Jh] a n t h r a c e n e d i o l e p o x i d e s e p o x i d a t i o n , A2 s t r u c t u r e s , A2,AAf s y n t h e s i s v i a Method I I , A2, Dibenzo[a_,eJ f l u o r a n t h e n e c a r c i n o g e n i c a c t i v i t y , 58 p r i n c i p a l m e t a b o l i t e s , 58 Dibenzof a_,Ji] p y r e n e c a r c i n o g e n i c a c t i v i t y , A8 s t r u c t u r e , A9 s y n t h e s i s v i a Method I , A8 Dibenzo[a_,JJ p y r e n e c a r c i n o g e n i c a c t i v i t y , A8 s t r u c t u r e , A9 s y n t h e s i s v i a Method I , A8 D i h y d r o d i o l epoxides c a r c i n o g e n i c i n i t i a t i o n , 10 structures, 10,llf Dihydroxy-7,8-dihydrobenzo[aJpyrene o x i d a t i o n c o r r e l a t i o n of the rate o f BP-7,8-dihydrodiol o x i d a t i o n , a n t i - d i o l epoxide f o r m a t i o n , and mutagen g e n e r a t i o n , 310,312f d i o l e p o x i d e p r o d u c t s , 310-15 mutation i n d u c t i o n i n Salmonella typhimurium, 310,311f PGH s y n t h a s e , 310 y i e l d s o f a d d u c t s from d i o l e p o x i d e v e r s u s those from d i h y d r o d i o l , 313t Diimines m e t a b o l i c f o r m a t i o n , 359 r e a c t i o n mechanism, 359,360f r o l e as u l t i m a t e c a r c i n o g e n s , 359,361

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

401

INDEX

7,12-Dimethylbenz[aJanthracene

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

bond l e n g t h s , i n t e r b o n d

angles,

and

t o r s i o n a n g l e s , 128,129f b r e a s t tumor i n d u c t i o n , 85,86 c a r c i n o g e n i c a c t i v i t y , 50 physical binding i n t e r a c t i o n s , 215-16 s t e r e o s e l e c t i v e m e t a b o l i s m a t the K r e g i o n , 28 s t r u c t u r e , 126,143 s t r u c t u r e s o f m e t a b o l i t e s and m e t a b o l i t i c model compounds, 212,214f v i e w o f 5 , 6 - c i s - d i o l , 143,144f v i e w o f K - r e g i o n o x i d e , 143,144f 7,12-Dimethylbenz[a]anthracene epoxide, s t r u c t u r e , 139,141f 7,12-Dime t h y l b e n z [ a_] a n t h r a c e n e m e t a b o l i t e s , comparison of p h y s i c a l b i n d i n g p r o p e r t i e s , 227 7,12-Dimethylbenz[a]anthracenedihydrodiols enantiomeric c o m p o s i t i o n s , 27,28t,29t e n a n t i o m e r i c r a t i o s , 28 metabolic a c t i v a t i o n pathways, 27,30f o p t i c a l p u r i t y , 27 p r o p e r t i e s , 27 s t e r e o s e l e c t i v e m e t a b o l i s m , 29 Dinitropyrenes, metabolic a c t i v a t i o n , 386 D i o l epoxides conformational p r e f e r e n c e s , 69,711,72 conformational-reactivity r e l a t i o n s h i p , 72,73 d i a s t e r e o m e r i c s e r i e s , 69 f o r m a t i o n , 127 log ^ v e r s u s AE, /p, 73,74f log r e l a t i v e mutagenicity versus AE^ /p, 75-77 m u t a g e n i c i t y , 73,75,77 r e a c t i o n mechanism, 65 r e a c t i v i t y , 73 t u m o r i g e n i c i t y , 77 t y p e s , 69 D i o l epoxide adducts conformational-biological activity r e l a t i o n s h i p , 120,122 f l u o r e s c e n c e q u e n c h i n g , 116-17 l i n e a r dichroism spectra, 1 1 0 , l l l f s o l v e n t a c c e s s i b i l i t y , 110 D i o l epoxide-DNA a d d u c t c o n f o r m a t i o n s , t h e o r e t i c a l m o d e l i n g , 118,120 DNA alignment, 245 a l k y l a t i o n by a d i o l e p o x i d e , 176,178f

DNA—Continued b i n d i n g c o n s t a n t s , 210,211t c r y s t a l s t r u c t u r e , 136 d i a g r a m s o f major c o n f o r m a t i o n s , 159,162f-63f i n t e r c a l a t i o n , 136 major c o n f o r m a t i o n s , 159,160t s i t e s a c c e s s i b l e to a l k y l a t i n g a g e n t s , 161,164f DNA a d d u c t s c o n f o r m a t i o n a l s t r u c t u r e o f the r e c e p t o r s i t e , 242-43 c o v a l e n t bond f o r m a t i o n , 242 d e t e c t i o n , 189 e x t e n t o f r e a c t i o n , 242 i n t e r c a l a t i o n complexes, 244-45 i n t e r c a l a t i v e c o v a l e n t b i n d i n g , 245 o r i e n t a t i o n o f the p y r e n e m o i e t y and the base p a i r s , 243 DNA adduct d e t e c t i o n d i r e c t methods, 189-90 f l u o r e s c e n c e measurements, 194-95 i m m u n o l o g i c a l methods, 192-94 i n d i r e c t methods, 189 p o s t l a b e l i n g t e c h n i q u e s , 188-90 DNA a t o m i c c o o r d i n a t e s , m a t h e m a t i c a l a p p r o a c h e s , 248-49 DNA b i n d i n g DMBA m e t a b o l i t e s , 27,30f e n e r g y f o r i n s e r t i o n , 250-51 m e t a b o l i c s t e p s , 12 metabolites, 10,12 r e c e p t o r s i t e s , 249-50 DNA b i n d i n g - c a r c i n o g e n i c i t y r e l a t i o n s h i p , 188-89 DNA b i n d i n g s i t e s d e f i n i t i o n s , 261t e n e r g y r e q u i r e d to a l t e r DNA t o r e c e p t o r s i t e s , 261t,262-63 k i n k s i t e s , 261t,262-63 p r o p e r t i e s , 109 t y p e s , 109 DNA r e p a i r s t r a t e g i e s m u t a g e n i c r e p a i r p r o c e s s , 328 SOS r e s p o n s e , 328 DNA s i t e a d d u c t s b i o l o g i c a l a c t i v i t y , 120,122 c h a r a c t e r i s t i c s , 114

E l e c t r o n e n e r g y parameter ( A E ^ ^ / ( 3 ) , e s t i m a t i o n of r e l a t i v e e

reactivity,

65,66,68

E l e c t r o n i c t h e o r i e s of carcinogenesis, 7

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Q c

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

402

Epoxide r i n g , averaged geometry, 139,142f E p o x i d e s , c o n t o u r s o f hydrogen-bond p o s i t i o n s , 139,142f,143 E x t e r n a l l y bound b e n z o j a j p y r e n e d i o l epoxide-DNA a d d u c t s adduct w i t h t h e p y r e n e m o i e t y i n t h e

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

major groove, 279-80,281f a n t i and syn r e o r i e n t a t i o n o f G and A, 279,280f d i s t r i b u t i o n , 279-80 i n t e r c a l a t i o n s , 277,279 o r i e n t a t i o n s , 275-76,277f o r i e n t a t i o n a n g l e s and e n e r g i e s , 276-79

F

F a t t y acid hydroperoxide o x i d a t i o n , pathways, F a t t y a c i d hydroperoxide aromatic hydrocarbon s i g n i f i c a n c e , 320 Fluoranthenes carcinogenic a c t i v i t y ,

dependent 308,309f polycyclic oxidation,

56

DNA a d d u c t s , 201 Fluoranthenedihydrodiols e p o x i d a t i o n , 56 s t r u c t u r e , 57 s y n t h e s i s , 56,57f N - 2 - F l u o r e n y l a e e t a m i d e , r o l e o f metabol i t e s i n c a r c i n o g e n i c a c t i o n , 10 F l u o r e s c e n c e quenching a s s o c i a t i o n constant f o r i n t e r c a l a t i o n , 216 f l u o r e s c e n c e decay p r o f i l e s , 218,221f f l u o r e s c e n c e l i f e t i m e s , 218 mechanism, 216 q u e n c h i n g c o n s t a n t s , 223-24 S t e r n - V o l m e r p l o t s , 218,219f-20f F l u o r i n e - p r o b e approach, d e s c r i p t i o n , 86

G

N-Glucuronyloxy arylamides r e a c t i o n mechanism, 348 role i n h e p a t o c a r c i n o g e n e s i s , 348,350 N-Glucuronyloxy arylamines, r e a c t i o n mechanism, 348,349f

H

Hydroperoxide-dependent e p o x i d a t i o n , unsaturated versus saturated f a t t y a c i d h y d r o p e r o x i d e s , 317,318t Hydroperoxide-dependent o x i d a t i o n s c a t a l y t i c cycle of horseradish p e r o x i d a s e , 314,315f h y d r o p e r o x i d e s p e c i f i c i t y o f BP o x i d a t i o n , 314 i d e n t i f i c a t i o n of peroxidaser e d u c i n g s u b s t r a t e s , 314-15 o x i d i z i n g a g e n t , 314,317 N-Hydroxy a r y l a m i d e s , r e a c t i o n s , 341 N-Hydroxy a r y l a m i d e s and N-hydroxy a r y l a m i n e s , c a r c i n o g e n i c i t y , 341 N-Hydroxy a r y l a m i n e s p r o t o n a t i o n , 355 r e a c t i o n s , 343 N-Hydroxy a r y l a m i n e 0 - s e r y l ( 0 - p r o l y l ) esters m e t a b o l i c f o r m a t i o n , 342f,353 r e a c t i o n mechanism, 355 r o l e i n c a r c i n o g e n e s i s , 355 Hydroxybenzo[ajpyrene a l t e r n a t e s y n t h e s i s , 38,41f c o n f o r m a t i o n a l and s t e r e o c h e m i c a l a s s i g n m e n t s , 38,39f h a l f - l i v e s , 38 p u r i f i c a t i o n , 38 r e a c t i v i t y , 38 s y n t h e s i s v i a Method I , 36,38,39f Hydroxy-3-methylcholanthrenes, s y n t h e s i s , 53,54f,56 Hydroxy-5-methylchrysene, s y n t h e s i s , 53,55f,56

I

I b a l l i n d e x , d e f i n i t i o n , 128,131 Iminoquinones m e t a b o l i c f o r m a t i o n , 359 r e a c t i o n mechanism, 359,360f r o l e as u l t i m a t e c a r c i n o g e n s , 359,361 Initiation i n t e r a c t i o n o f carcinogens with DNA, 10 irreversibility, 4 m u t a g e n i c mechanism, 5 reversibility, 4 I n i t i a t i o n - p r o m o t i o n system, characteristics, 4 I n t e r c a l a t i o n o f an a c r i d i n e nucleic

a c i d , views,

ina

136-38

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

403

INDEX K

K-

and L - r e g i o n h y p o t h e s i s description, 8 exceptions, 8 regions of benz[ajanthracene, K - r e g i o n , d e f i n i t i o n , 128 K-region epoxides b i o l o g i c a l a c t i v i t i e s , 10 m e t a b o l i c a c t i o n , 10

8,9f

L

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

lacl

system f o r a n a l y z i n g nonsense mutations i n E s c h e r i c h i a c o l i , d e s c r i p t i o n , 331 L i n e a r d i c h o i s m , d e s c r i p t i o n , 109

M

Metabolic a c t i v a t i o n , one-electron o x i d a t i o n , 290 Method I s y n t h e t i c a p p r o a c h , d e s c r i p t i o n , 36 Method I I s y n t h e t i c a p p r o a c h , d e s c r i p t i o n , AO Method I I I s y n t h e t i c a p p r o a c h , d e s c r i p t i o n , AO Method IV s y n t h e t i c a p p r o a c h , d e s c r i p t i o n , A5 l l - M e t h y l - 1 5 , 1 6 - d i h y d r o c y c l o p e n t a [ aj p h e n a n t h r e n e , s t r u c t u r e , 133 Methylated p o l y c y c l i c aromatic hydrocarbons examples, 85,86 h i g h l y t u m o r i g e n i c examples h a v i n g a m e t h y l a d j a c e n t to an a n g u l a r r i n g , 86,87f m e t a b o l i c a c t i v a t i o n , 88 o c c u r r e n c e , 85 s t r u c t u r a l requirements f a v o r i n g m u t a g e n i c i t y , 86,88 7-Methylbenz[ajanthracene, carcinogenic a c t i v i t y , A8 7-Methylbenz[aJ a n t h r a c e n e d i h y d r o d i o l e p o x i d a t i o n , 50,52 p e r a c i d e p o x i d a t i o n , 50 s y n t h e s i s v i a Methods I-IV, A8,50-52,5Af 3-Methylcholanthrene c a r c i n o g e n i c a c t i v i t y , 52 DNA a d d u c t s , 201 s t r u c t u r e , 126

3-Methylcholanthrenedihydrodiols s t r u c t u r e , 5A s y n t h e s i s v i a Method IV, 52 3-Methylcholanthrenediol epoxides, s t r u c t u r e s , 5A,55 3 - M e t h y l c h o l a n t h r e n e t r i o l epoxide, s t r u c t u r e s , 5A 5-Methylchrysene c a r c i n o g e n i c a c t i v i t y , 53 s t r u c t u r e , 136 5-Methylchrysene m e t a b o l i t e s h a l f - l i v e s of d i h y d r o d i o l e p o x i d e s , 96t h a l f - l i v e s of d i h y d r o d i o l epoxides v e r s u s e x t e n t s o f DNA b i n d i n g s , 96,98f r e l a t i v e extents of b i n d i n g of d i h y d r o d i o l e p o x i d e s t o DNA, 91 s t r u c t u r e s of bay-region d i h y d r o d i o l e p o x i d e s and d i h y d r o d i o l s , 91,92f s t r u c t u r e s o f major adducts formed upon D N A - m e t a b o l i t e r e a c t i o n , 91,95f,96 t u m o r i g e n i c i t y i n r a t s , 91,93t t u m o r - i n i t i a t i n g a c t i v i t y on mouse s k i n , 91,9At 5-Methylchrysenedihydrodiols e p o x i d a t i o n , 56 s y n t h e s i s v i a Method IV, 53,55f,56 ll-Methyl-15,16-dihydrocyclopenta[ji]p h e n a n t h r e n e , s t r u c t u r e , 133 Mouse s k i n system advantages, A tumor i n d u c t i o n , A Mutagenesis base m i s p a i r i n g , 328 p r o c e s s e s o f DNA damage, 328-29 r o l e s p l a y e d by DNA l e s i o n s , 330 SOS p r o c e s s i n g , 329 M u t a g e n i c mechanism, d e s c r i p t i o n , 5

N

Naphthacene activity, 7 structure, 6 N i t r o p o l y c y c l i c aromatic hydrocarbons b i o l o g i c a l e f f e c t s , 37A e n v i r o n m e n t a l o c c u r r e n c e , 372,37A mammalian m e t a b o l i s m , 378 m i c r o b i a l m e t a b o l i s m , 377 m u t a g e n i c i t y , 37A,375t p u r i f i c a t i o n , 372 s t r u c t u r e s , 372,373f s y n t h e s i s , 372 t u m o r i g e n i c i t y , 37A,376t,377

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

404

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

N i t r o p o l y c y c l i c aromatic hydrocarbon mammalian m e t a b o l i s m e f f e c t s o f n i t r o s u b s t i t u t i o n , 381 m i c r o s o m a l m e t a b o l i t e s , 381,382f o x i d a t i o n and n i t r o r e d u c t i o n , 379,381,383, 386-87,389 r e a s o n s f o r s t u d y , 378 S-9-catalyzed m e t a b o l i t e s , 379,380f,384f N i t r o p o l y c y c l i c aromatic hydrocarbon m i c r o b i a l metabolism o t h e r b a c t e r i a , 378 S a l m o n e l l a t y p h i m u r i u m , 377-78 1-Nitropyrene metabolites detected i n i n v i t r o i n c u b a t i o n s , 383,385f,386 m u t a g e n i c i t y , 383 o x i d a t i v e m e t a b o l i s m , 383 r e d u c t i o n , 383 Nitrosoarene m e t a b o l i c f o r m a t i o n , 357 r e a c t i o n mechanism, 357,358f r o l e i n c a r c i n o g e n e s i s , 357,359

0 Optical purity determination CSP-HPLC s e p a r a t i o n o f e n a n t i o m e r s , 23,24f h y d r a t i o n mechanism o f K - r e g i o n e p o x i d e e n a n t i o m e r s , 21,22f methods, 21 Oxidation a c t i v a t i o n o f p o l y c y c l i c aromatic h y d r o c a r b o n s t o c a r c i n o g e n s , 307 d e s c r i p t i o n , 307

P P e r i m e t h y l group i n h i b i t o r y e f f e c t on tumorigenicity, mechanistic b a s i s , 99 Peroxidases c a r c i n o g e n i c a c t i v a t o r s , 307 p r o p e r t i e s , 307-8 Peroxy r a d i c a l s e p o x i d a t i o n , 317,319-20 r e a c t i o n s , 317 Peroxy r a d i c a l g e n e r a t i o n , mechanism, 317,319f Perturbational molecular o r b i t a l c a l c u l a t i o n s , 65,66 Phenanthrene, c a r c i n o g e n i c a c t i v i t y , 43

Phenanthrene e p o x i d e , s t r u c t u r e , 139,141f Phenanthrenedihydrodiols, synthesis v i a Method I V , 45,47f Physical binding properties, s p e c t r o c o p i c p r o b e s , 216 P o l y c y c l i c aromatic hydrocarbons a d d i t i o n o f an epoxide g r o u p , 143 b i o l o g i c a l p r o p e r t i e s , 19 carcinogenic i d e n t i f i c a t i o n , 2 c a r c i n o g e n i c i t y , 126,292-96 c a r c i n o g e n i c i t y i n mouse s k i n v e r s u s t h a t i n r a t mammary g l a n d , 300-302 c h a r g e l o c a l i z a t i o n i n the r a d i c a l c a t i o n , 292,296 c l a s s i f i c a t i o n s o f m e t a b o l i t e s , 241 c o v a l e n t b i n d i n g f a c t o r s , 292 e n v i r o n m e n t a l e x p o s u r e , 3,4 examples, 198 i n t e r c a l a t i o n i n DNA, 138-39,140f i o n i z a t i o n p o t e n t i a l , 292,293t-95t n i t r a t i o n , 372 numbering, a b b r e v i a t i o n s , and A

/ p

v

a

l

u

e

s

6

6

6

8

^deloc » " r e a c t i v e m e t a b o l i t e s , 196 s t r u c t u r a l f o r m u l a s , 36,37f structure-activity relationship, 5 s t r u c t u r e s , 292,293t-95t s y n t h e s i s o f d i h y d r o d i o l and d i o l e p o x i d e d e r i v a t i v e s , 36 view o f c a r c i n o g e n i c molecules showing K and bay r e g i o n s , 128,130f X - r a y d i f f r a c t i o n s t u d i e s , 128 P o l y c y c l i c aromatic hydrocarbon d i o l epoxide-DNA i n t e r a c t i o n , p e r p e n d i c u l a r i t y , 152,156f,157 P o l y c y c l i c a r o m a t i c hydrocarbon-DNA adducts a c t i v a t i o n by c e l l u l a r p e r o x i d a s e s , 300 b i n d i n g c o n s t a n t s , 210,211t ,227,229 binding of hydrocarbon m e t a b o l i t e s , 210,212 e f f e c t s o f DNA on s o l u b i l i t y , 210 e l e c t r o n i c i n f l u e n c e on s t a c k i n g i n t e r a c t i o n s , 227,228f evidence f o r one-electron o x i d a t i o n a c t i v a t i o n , 300 metabolitic reactivityc a r c i n o g e n i c i t y r e l a t i o n s h i p , 212 p h o t o e l e c t r o n spectrum, 229,230f p h y s i c a l b i n d i n g , 210 s t r u c t u r e - a c t i v i t y s t u d i e s , 210 s t r u c t u r e s , 197-98 P o l y c y c l i c a r o m a t i c hydrocarbon-DNA adduct s t r u c t u r e s , d e t e r m i n a t i o n , 195-98

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

405

INDEX

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

P o l y c y c l i c a r o m a t i c hydrocarbon-DNA i n t e r c a l a t i o n , i n f l u e n c e o f DNA s t r u c t u r e and e n v i r o n m e n t , 229,231f,232 P o l y c y c l i c aromatic hydrocarbon metabolic a c t i v a t i o n , e f f e c t s of f l u o r i n e s u b s t i t u t i o n , 101 P o l y c y c l i c aromatic hydrocarbon o x i d a t i o n , h y d r o p e r o x i d e and mixed-function oxidase d i f f e r e n c e s , 320-23 P o l y c y c l i c aromatic hydrocarbon t u m o r i g e n i c i t y , e f f e c t of f l u o r i n e s u b s t i t u t i o n , 99-101 P r i m a r y c a r c i n o g e n s , d e f i n i t i o n , 241 Procarcinogen—See Secondary carcinogen P r o s t a g l a n d i n H synthase b i o s y n t h e s i s o f PGH2» 308,309f h e m e - r e q u i r i n g a c t i v i t i e s , 313-14 o x i d a t i o n of p o l y c y c l i c a r o m a t i c hydrocarbons, 308 p r o p e r t i e s , 308 Proteins, polypeptide chain f o l d i n g , 157 P r o t o n a t e d N-hydroxy a r y l a m i n e s r e a c t i o n mechanism and f o r m a t i o n , 355-56,358f r e a c t i v i t y and s e l e c t i v i t y , 355-56 r o l e as u l t i m a t e c a r c i n o g e n s , 356-57

Q

Quantum c h e m i c a l c a l c u l a t i o n s , 65 Quinones m e t a b o l i c f o r m a t i o n by an i n i t i a l one-electron o x i d a t i o n of b e n z o [ a ] p y r e n e , 296-99 metabolic formation for p o l y c y c l i c aromatic hydrocarbons of v a r i o u s i o n i z a t i o n p o t e n t i a l s , 297t

R

Radical cations c h e m i c a l p r o p e r t i e s , 290 nucleophilic trapping mechanism, 290-91 o n e - e l e c t r o n o x i d a t i o n and subsequent n u c l e o p h i l i c t r a p p i n g , 290-91 s p e c i f i c r e a c t i v i t y with n u c l e o p h i l e s , 290,292

Radical cation perchlorates, synthesis and subsequent n u c l e o p h i l i c c o u p l i n g , 290,292

S

S e c o n d a r y c a r c i n o g e n s , d e f i n i t i o n , 241 SOS m u t a g e n e s i s , o c c u r r e n c e a t s i t e s o f DNA damage, 330 SOS-processing system, d e s c r i p t i o n , 329-30 S p e c t r o s c o p i c probes e m i s s i o n s p e c t r a o f DNA, 216,217f f l u o r e s c e n c e quenching, 216 UV a b s o r p t i o n , 218 S t r u c t u r a l requirements f a v o r i n g mutagenicity b a y - r e g i o n m e t h y l g r o u p , 86,88,91 comparative t u m o r i g e n i c i t y of m e t h y l c h r y s e n e s , 88,90f f r e e p e r i p o s i t i o n , 86,88 i n h i b i t i o n o f t u m o r i g e n i c i t y by p e r l - m e t h y l s u b s t i t u t i o n , 88,89f n o n p l a n a r i t y , 97 u n s u b s t i t u t e d a n g u l a r r i n g , 86,88 Structure-activity relationships e f f e c t o f a m e t h y l g r o u p , 7,9t,48 substitution, 7 u n s u b s t i t u t l o n , 5,7 Sugar c o n f o r m a t i o n s , d i a g r a m s , 159,161,163f N-Sulfonyloxy arylamides decomposition, 345 e l e c t r o p h i l i c r e a c t i v i t y , 343 e l e c t r o p h i l i c s u b s t i t u t i o n , 345 m e t a b o l i c f o r m a t i o n , 343-45 r e a c t i o n mechanism, 345,346f role i n arylamide t u m o r i g e n e s i s , 345,347 N-Sulfonyloxy arylamines m e t a b o l i c f o r m a t i o n , 352 r e a c t i o n mechanism, 352-53,354f r o l e as u l t i m a t e c a r c i n o g e n , 353 N-(Sulfonyloxy)-N-methyl arylamines r e a c t i o n mechanism, 361-63 r e a c t i v i t y , 361 r o l e as u l t i m a t e c a r c i n o g e n s , 361

T Tetrahydroepoxides l o g kg v e r s u s A E ^ ^ /P p l o t , 68 l o g r e l a t i v e m u t a g e n i c i t y toward S. t y p h i m u r i u m TA 100, 69,70f ~~

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

406

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

Tetrahydroepoxides—Continued

Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ix002

models f o r d i o l e p o x i d e r e a c t i v e s i t e , 68 m u t a g e n i c i t y , 69 r e a c t i v i t y , 68 t u m o r i g e n i c i t y , 69 T e t r a o l s , f l u o r e s c e n c e , 116 T r i p e p t i d e s , s t r u c t u r e s , 157 Triphenylene activity, 7 c a r c i n o g e n i c a c t i v i t y , 43 structure, 6 Triphenylened ihydrod i o l s e p o x i d a t i o n , 43,44f s y n t h e s i s , 43

Unstructured p o l y c y c l i c aromatic h y d r o c a r b o n s , s t r u c t u r e s , 5,6 UV a b s o r p t i o n a b s o r p t i o n s p e c t r a , 218,222f a s s o c i a t i o n c o n s t a n t s , 223,224t

W

W e i g l e r e a c t i v a t i o n and m u t a g e n e s i s , d e s c r i p t i o n , 328

X

U

Ultimate carcinogen—See carcinogen

Primary

X-ray d i f f r a c t i o n analyses of c r y s t a l s , d e s c r i p t i o n , 127-28

Production by Meg Marshall Indexing by Deborah H. Steiner Jacket design by Pamela Lewis Elements typeset by Hot Type Ltd., Washington, D.C. Printed and bound by Maple Press Co., York, Pa.

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.