Chemical Carcinogenesis

Current Cancer Research

For other titles published in this series, go to www.springer.com/series/7892

wwwwwwwwwwwwwwwww

Trevor M. Penning Editor

Chemical Carcinogenesis

Editor Trevor M. Penning Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, PA USA [email protected]

ISBN 978-1-61737-994-9 e-ISBN 978-1-61737-995-6 DOI 10.1007/978-1-61737-995-6 Springer New York Heidelberg London Dordrecht Library of Congress Control Number: 2011920809 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or ­dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

This monograph would not have been possible without the contributions of numerous scientists, too many to name individually, which have led to our understanding on how chemicals and environmental insults cause cancer. The monograph is also dedicated to my grandson “Trevor” who lights up my life with his smile.

wwwwwwwwwwwwwwwww

Preface

This volume will provide a contemporary account of advances in chemical carcinogenesis. It will promote the view that chemical alteration of DNA is the root cause of many cancers. This chemical alteration can result from exposure to biological reactive intermediates that may arise from the metabolism of endogenous and exogenous carcinogens. Furthermore, reactive metabolites can target 5¢-CpG’ islands and affect the epigenetic “footprinting” of tumor suppressor genes (leading to their silencing). Once the DNA is modified it is not predestined to cause mutations that may alter the function of critical growth control genes. Instead, eukaryotic cells have evolved sophisticated mechanisms for mutation avoidance using major DNA repair mechanisms including, but not limited to, nucleotide and base excision repair. If the ensuing DNA adducts escape repair, they can give rise to mutations during the replication phase. This often occurs as a result of a novel set of “by-pass” DNA polymerases, which can have low fidelity and processivity. It is faulty replication that leads to mutation of critical genes such as K-ras and p53. Knowledge of these events can identify critical genes and pathways involved in the carcinogenic process that can be exploited for cancer chemoprevention, intervention, and early diagnosis while an individual may be asymptomatic. This, in fact, is the driving force behind the field. The monograph starts with an historical overview of chemical carcinogenesis (Chap. 1 by Harvey) and describes how epidemiological data on migrant populations and exposure data identified major human carcinogens and their routes of activation. This is followed by a chapter (Chap. 2 by DiGiovanni) on chemical carcinogenesis as a multistage disease process using mouse skin as a model. Pertinent to the study of human carcinogenesis this chapter discusses that while we may study carcinogenesis as a sequential process, this may not be applicable to the human situation where there may be constant insult from initiators and promoters simultaneously. This is followed by an in-depth discussion of major classes of human carcinogens to which the general population is exposed: tobacco carcinogens (Chap. 3 by Hecht), estrogens (Chap. 4 by Bolton), heterocyclic amines in food (Chap. 5 by Turesky), and aflatoxins in contaminated food (Chap. 6. by Groopman). Often the goal of this work is to identify biomarkers of exposure and response for human biomonitoring and risk assessment. This approach requires sophisticated analytical chemistry and such approaches are discussed. Common mechanisms by vii

viii



which carcinogens are metabolized to biologically reactive intermediates and how this can be exploited in cancer chemoprevention studies are then discussed in Chaps. 7 and 8 by Penning and Kensler, respectively. Using polycyclic aromatic hydrocarbons as an example, Chap. 9 by Broyde and coworkers describes the importance of adduct stereochemistry and sequence context in dictating the mutations that may arise from diol epoxides. Three chapters (Chap. 10 by Dedon, Chap. 11 by Blair, and Chap. 12 by Sowers) deal with the damage of DNA by endogenous agents (e.g., reactive oxygen species and lipid peroxidation) as well as the modification of cytosine by deamination, oxidation, and chlorination. The repair of DNA damage and the tremendous advances made in the structural biology of DNA repair are discussed in depth with respect to the enzymes and associated proteins of nucleotide excision repair (Chap. 13 by van Houten), base-excision repair (Chap. 14 by Wilson), and repair of O6-methyl guanine by O6-alkylguanine– DNA alkyltransferase (Chap. 15 by Pegg). Mechanisms by which common DNA adducts are misread by “by-pass” DNA polymerases providing a “mutational code” are discussed in Chap. 16 by Guengerich and Chap. 17 by Basu. The final chapter is on mutation in critical protoncogenes and tumor suppressor genes (Chap. 18 by Field), which discusses the relevant importance of sequence context and biological selection in driving the mutational spectrum. The monograph does not contain detailed accounts of tumor promoters, tumor progression, or angiogenesis, since these are dealt with in other monographs in this series. Many contemporary texts on the biology of cancer now focus almost exclusively on the molecular and cell biology of the disease and do not cover the initiating (DNA-damaging) events of chemical carcinogenesis in depth. It is with this ­perceived gap in knowledge that world experts in their particular fields were asked and ­volunteered to contribute to this monograph. It is hoped that this monograph will be used as a reference source for students in training, postdoctoral scientists, and senior scientists who wish to gain an appreciation for this field. It is hoped that this monograph will be an invaluable reference source for years to come. Philadelphia PA, USA

Trevor M. Penning

Contents

  1 Historical Overview of Chemical Carcinogenesis................................. Ronald G. Harvey

1

  2 Multistage Carcinogenesis....................................................................... Erika L. Abel and John DiGiovanni

27

  3 Tobacco Smoke Carcinogens and Lung Cancer.................................... Stephen S. Hecht

53

  4 Mechanisms of Estrogen Carcinogenesis: Modulation by Botanical Natural Products............................................................... Judy L. Bolton   5 Heterocyclic Aromatic Amines: Potential Human Carcinogens......... Robert J. Turesky

75 95

  6 Aflatoxin and Hepatocellular Carcinoma.............................................. 113 John D. Groopman and Gerald N. Wogan   7 Metabolic Activation of Chemical Carcinogens.................................... 135 Trevor M. Penning   8 Detoxication of Chemical Carcinogens and Chemoprevention........... 159 Melinda S. Yates and Thomas W. Kensler   9 Covalent Polycyclic Aromatic Hydrocarbon–DNA Adducts: Carcinogenicity, Structure, and Function.............................................. 181 Suse Broyde, Lihua Wang, Yuqin Cai, Lei Jia, Robert Shapiro, Dinshaw J. Patel, and Nicholas E. Geacintov 10 Oxidation and Deamination of DNA by Endogenous Sources........................................................................... 209 Peter C. Dedon ix

x

Contents

11 Lipid Peroxide–DNA Adducts................................................................ 227 Seon Hwa Lee and Ian A. Blair 12 Chemical Carcinogenesis and Epigenetics............................................. 245 Agus Darwanto, Jonathan D. Van Ornam, Victoria Valinluck Lao, and Lawrence C. Sowers 13 Nucleotide Excision Repair from Bacteria to Humans: Structure–Function Studies.................................................................... 267 Ye Peng, Hong Wang, Lucas Santana-Santos, Caroline Kisker, and Bennett Van Houten 14 Base-Excision Repair: Role of DNA Polymerase b in Late-Stage Base Excision Repair............................................................................... 297 Kenjiro Asagoshi and Samuel H. Wilson 15 O6-Alkylguanine-DNA Alkyltransferase................................................ 321 Anthony E. Pegg, Sreenivas Kanugula, and Natalia A. Loktionova 16 Bypass DNA Polymerases........................................................................ 345 Jeong-Yun Choi, Robert L. Eoff, and F. Peter Guengerich 17 Mutagenesis: The Outcome of Faulty Replication of DNA.................. 375 Ashis K. Basu 18 p53 and Ras Mutations in Cancer and Experimental Carcinogenesis.......................................................................................... 401 Zahidur Abedin, Sushmita Sen, Elise Morocco, and Jeffrey Field Index.................................................................................................................. 423

Contributors

Zahidur Abedin Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA [email protected] Erika L. Abel Division of Pharmacology/Toxicology and Department of Nutritional Sciences , Colleges of Pharmacy and Natural Sciences, The University of Texas at Austin Austin, TX, USA [email protected] Kenjiro Asagoshi Laboratory of Structural Biology, NIEHS/National Institutes of Health, Research Triangle Park, NC, USA [email protected] Ashis K. Basu Department of Chemistry, University of Connecticut, Storrs, CT, USA [email protected] Ian A. Blair Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; Department of Pharmacology, Center of Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA [email protected] Judy L. Bolton Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago, IL, USA [email protected] xi

xii

Contributors

Suse Broyde Department of Biology, New York University, New York, NY 10003, USA [email protected] Yuqin Cai Department of Biology, New York University, New York, NY 10003, USA [email protected] Jeong-Yun Choi Department of Pharmacology, School of Medicine, Ehwa Womans University, Seoul, Republic of Korea [email protected] Agus Darwanto Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, CA, USA Peter C. Dedon Department of Biological Engineering and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA [email protected] John DiGiovanni Division of Pharmacology/Toxicology and Department of Nutritional Sciences, Colleges of Pharmacy and Natural Sciences, The University of Texas at Austin, Austin, TX, USA [email protected] Robert L. Eoff Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA; Department of Pharmacology, School of Medicine, Ehwa Womans University, Seoul, Republic of Korea [email protected] Jeffrey Field Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA [email protected] Nicholas E. Geacintov Department of Chemistry, New York University, New York, NY 10003, USA [email protected]

Contributors

John D. Groopman Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA [email protected] F. Peter Guengerich Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA [email protected] Ronald G. Harvey The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA [email protected] Stephen S. Hecht Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA [email protected] Lei Jia Pacific Biosciences Inc., Menlo Park, CA 94025, USA [email protected] Sreenivas Kanugula Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA, USA [email protected] Thomas E. Kensler Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA [email protected] Caroline Kisker Rudolf-Virchow-Center for Experimental Biomedicine, Wuerzburg, Germany [email protected] Victoria Valinluck Lao Department of Basic Science, Loma Linda University, Loma Linda, CA, USA Seon Hwa Lee Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Japan

xiii

xiv

Contributors

Natalia A. Loktionova Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA, USA [email protected] Elise Morocco Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA [email protected] Dinshaw J. Patel Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA [email protected] Anthony E. Pegg Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA, USA [email protected] Ye Peng Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA [email protected] Trevor M. Penning Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA [email protected] Lucas Santana-Santos Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA [email protected] Sushmita Sen Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA [email protected]

Contributors

Robert Shapiro Department of Chemistry, New York University, New York, NY 10003, USA [email protected] Lawrence C. Sowers Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, CA, USA [email protected] Robert J. Turesky Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA [email protected] Bennett Van Houten Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA [email protected] Jonathan D. Van Ornam Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, CA, USA Hong Wang Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; University of Pittsburgh Cancer Institute, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA [email protected] Lihua Wang Department of Biology, New York University, New York, NY 10003, USA Samuel H. Wilson Laboratory of Structural Biology, NIEHS/National Institutes of Health, Research Triangle Park, NC, USA [email protected] Gerald N. Wogan Department of Biological Enginearing, Massachusetts Institute of Technology, Cambridge, MA 02139, USA [email protected] Melinda S. Yates Department of Gynecologic Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA [email protected]

xv

wwwwwwwwwwwwwwwww

Chapter 1

Historical Overview of Chemical Carcinogenesis Ronald G. Harvey

Abstract  There is increasing evidence that carcinogens play a major role in causation of human cancer. This chapter reviews the advances in carcinogenesis research from a historical perspective. The classes of carcinogens surveyed include polycyclic aromatic hydrocarbons, aromatic amines and amides, nitroarenes, heterocyclic amines formed in cooking, N-nitroso compounds, aflatoxins, natural oils such as safrole, and other natural products, such as the pyrrolizidine alkaloids. For each of these categories, information is presented on historical developments, environmental occurrence, the identities of the active metabolites, the pathways of enzymatic activation, and the structures of the adducts formed with DNA. Despite the chemical and structural diversity of the carcinogens, the evidence indicates that their mechanisms of tumorigenesis are fundamentally similar. The active metabolites of most carcinogens are electrophiles (or reactive oxygen species) that react with DNA to induce mutations and/or other genotoxic changes.

1 Introduction 1.1 Chemical Carcinogenesis and Human Cancer Epidemiological and occupational studies support the importance of chemical carcinogens and other environmental factors as causative agents in human cancer (Doll and Peto 1981; Higginson et  al. 1992; Peto 2001). Differences in the incidences of specific types of cancer often vary widely from country to country. Five- to tenfold differences in the mortalities for cancers of the breast, stomach, colon, and liver are not uncommon, and these differences do not appear to be

R.G. Harvey (*) The Ben May Department for Cancer Research, The University of Chicago, Chicago, IL, USA e-mail: [email protected]

T.M. Penning (ed.), Chemical Carcinogenesis, Current Cancer Research, DOI 10.1007/978-1-61737-995-6_1, © Springer Science+Business Media, LLC 2011

1

2

R.G. Harvey

primarily related to genetic differences. The cancer risk patterns of migrant populations generally resemble those of the countries of origin, but the risk patterns of their descendants tend to change to match those of the adopted countries (Kmet 1970; Kolonel et al. 2004; Luch 2005). This suggests that most human cancer may derive from lifestyle and environmental factors rather than genetic differences. Indeed, the majority of cancer deaths in industrialized countries are associated with use of tobacco, diet, and environmental factors.

1.2 Early History of Carcinogenesis Research Research in chemical carcinogenesis dates back to the astute clinical observations of English physicians John Hill and Percival Pott in the latter half of the eighteenth century. Hill (1771) associated the occurrence of cancerous alterations in the nasal mucosa of snuff-users with their exposure to snuff. Pott (1775) observed a high incidence of scrotal cancer in chimney sweeps and proposed that it was due to exposure to soot. These reports had little impact at the time, but evidence began to accumulate relating the incidences of various cancers to exposure to chemicals in the workplace. Most notable was the report by German surgeon Ludwig Rehn of a high incidence of urinary bladder cancer in workers in the dye industry (Rehn 1895). Experimental research in carcinogenesis began with a report by Yamagawa and Ichikawa (1915) that repeated application of coal tar to the ears of rabbits produced malignant tumors at the site. Soon after, Tsutsui (1918) showed that skin tumors were produced by the application of coal tar to the skins of mice. Subsequently, the active carcinogenic component was isolated from coal tar pitch by British investigators who identified it as a polycyclic aromatic hydrocarbon (PAH), benzo[a] pyrene (BaP) (Fig. 1) (Cook et al. 1933).

bay region

K-region benzo[a]pyrene BaP

CH3

fjord region

K-region Dibenzo[def,p]chrysene DBC

CH3 7,12-dimethylbenz[a]anthracene DMBA

Fig.  1  Examples of carcinogenic PAHs. Dibenzo[def,p]chrysene (DBC) is the most potent in rodent tumor assays. Although BA is a weak tumor initiator, 7,12-dimethylbenz[a]anthracene (DMBA) ranks below only DBC in potency

1  Historical Overview of Chemical Carcinogenesis

3

2 Polycyclic Aromatic Hydrocarbons 2.1 Structure–Activity Relationships The discovery that some PAHs are carcinogens raised many questions. What are the structural requirements for activity? Are they directly active, or is metabolic activation needed? What are their cellar targets? Early investigators assumed that the PAHs acted directly because the urinary metabolites (phenols, dihydrodiols, quinones, and their conjugates) were inactive or exhibited only minimal activity as tumorigens. Extensive studies have been conducted on the structure–activity relations of PAHs and their heterocyclic analogs (review: Harvey 1991). Relatively up-to-date information on this topic may be found in the publications of the IARC (1976, 1983, 1987, 1993, 2002, 2004, 2007). Caution should be exercised in comparing data obtained using animals differing in strain, species, age, or sex, or employing different modes of administration (e.g., skin painting, oral feeding, subcutaneous injection, etc.), or whether promoters were used. Promoters are agents, such as 12-o-tetradecanoylphorobol-13-acetate (TPA), that are not carcinogens but which if administered to animals pretreated with a sub-effective dose of a carcinogen may elicit tumors. For example, benz[a]anthracene (BA) and chrysene are not carcinogenic on mouse skin, but both exhibit activity with TPA promotion. They are said to be initiators, but not complete carcinogens. The essential requirements for carcinogenic activity are possession of four to seven fused aromatic rings and a bay or fjord molecular region (Fig.  1). A “K-region,” typified by the 4,5-bond of BaP, is also usually present. Activity is also often dramatically enhanced by the substitution of methyl groups at key positions. BA is not a carcinogen, but it is transformed into one by the introduction of methyl groups into the 6-, 7-, 8-, or 12-positions (Huggins et al. 1967; Hecht et al. 1985). Indeed, 7,12-dimethyl-BA (DMBA, Fig.  1) is one of the most potent carcinogens known. In the series of 5-ring PAHs, BaP is a potent carcinogen, dibenz[a,h]anthracene, benzo[g]chrysene, and benzo[c]chrysene exhibit moderate activity, and other isomers are weak tumor initiators. BaP is classified by the IARC as a category 1 carcinogen (i.e., a confirmed human carcinogen). In the 6-ring series, dibenz[def,p] chrysene (DBC) is the most potent PAH carcinogen currently known (Cavalieri et al. 1991). With increasing size, the PAHs become more graphite-like, and the solubility diminishes imposing a practical upper size limit on activity. Polar substituents such as CO2H, NO2, and SO3H generally abolish activity.

2.2 Metabolism and Metabolic Activation Boyland (1950) hypothesized that arene oxides are the primary PAH metabolites that give rise to the phenols, dihydrodiols, and other oxidized metabolites. This idea

4

R.G. Harvey

is now widely accepted. He also proposed that K-region arene oxides, e.g., BaP 4,5-oxide (Figs. 1 and 2), were the active forms of the PAH carcinogens. The term “K-region” was introduced by Pullman and Pullman (1955) to designate electronrich aromatic bonds analogous to the 9,10-bond of phenanthrene. Metabolism takes place on the microsomes of the endoplastic reticulum mediated by P-450 enzymes. The pathways of metabolism of BaP are shown in Fig. 2. The arene oxides undergo transformation via three pathways: (1) rearrangement to phenols, (2) epoxide hydrolase-catalyzed hydration to trans-dihydrodiols (Oesch et  al. 1984), and (3) reaction with glutathione (Singer and Grunberger 1983). The least stable arene oxides, such as BaP 2,3-oxide, rearrange rapidly to phenols. Usually only one of the two possible phenol isomers is formed, and that isomer is predictable by molecular orbital theory (Fu et al. 1978). Thus, 3-HO-BaP is a major metabolite of BaP, but 2-HO-BaP is not formed (Yang et al. 1977). More stable arene oxides, e.g., BaP 7,8-oxide, survive long enough for partial conversion to dihydrodiols, and the most stable arene oxides, e.g., BaP 4,5-oxide, survive long enough for complete conversion to dihydrodiols. The phenols and dihydrodiols are transformed to water soluble conjugates and sulfate esters, and the glutathione conjugates are converted to mercapturic acid derivatives. Secondary oxidative metabolism affords quinones and other highly oxidized metabolites. Brooks and Lawley (1964) showed that application of 3H-labeled PAHs to the backs of mice gave adducts with nucleic acids and proteins. For binding to occur, oxidative metabolism of the PAHs by P-450 microsomal enzymes was required, and HO OH

HO 11,12-oxide

11,12-diol

HO HO

1,2-oxide O

O

1-phenol

O 9,10-diol O

9,10-oxide

OH 2,3-oxide

3-phenol

HO O

HO 7,8-diol

7,8-oxide

HO

O 4,5-oxide

OH OH 4,5-diol

8-phenol

Fig. 2  Metabolism of BaP proceeds via initial oxidation by cytochrome P-450 enzymes to form reactive arene oxide intermediates that undergo rearrangement to phenols and hydration to dihydrodiols

1  Historical Overview of Chemical Carcinogenesis

5

the extent of binding to DNA correlated with carcinogenic activity (Goshman and Heidelberger 1967; Grover and Sims 1968). These findings were consistent with K-oxides being the active metabolites. Syntheses of the K-oxides of the potent carcinogens BaP and DMBA (Fig. 2) were accomplished by Harvey (Harvey et al. 1975; Goh and Harvey 1978). Synthesis of BaP 4,5-oxide was also reported by Dansette and Jerina (1974). Using these compounds, Baird et al. (1973, 1975) showed that the chromatographic profiles of the hydrolysates of the products of the reactions of BaP 4,5-oxide and 7-methyl-BA 5,6-oxide with DNA were different than those of the adducts formed in vivo, thereby ruling out the K-oxides as active metabolites. 2.2.1 Diol Epoxide Pathway The DNA adduct formed by the metabolism of BaP in mouse cells was more polar chromatographically than the adduct formed by BaP 4,5-oxide. A clue to the identity of the DNA-bound metabolite was the finding by Borgen et al. (1973) that incubation of BaP 7,8-dihydrodiol with DNA and hamster liver microsomes gave a DNA-bound adduct. Sims et al. (1974) proposed that the active metabolite was the diol epoxide (Fig. 3). They oxidized 3H-BaP-7,8-dihydrodiol with m-chloroperbenzoic acid and incubated the product with DNA. The adduct formed was chromatographically identical with the adduct formed in mouse cells. Stereospecific syntheses of the anti- and syn-isomers of the BaP diol epoxides (anti- and syn-BaPDE) (Fig.  3) were reported by Beland and Harvey (1976) (who provided proof of the isomer assignments) and by Yagi et  al. (1975).

O

DNA

O

+

microsomes HO

HO

HO

OH

HO

HO

(±) syn-BPDE (minor isomer)

(±) anti-BPDE (major isomer)

(±) BaP 7,8-diol

d-ribose O N N

N

N

N NH

d-ribose HO

N

N

NH

NH +

HO HO

HO OH

anti-BPDE-dGua adduct

OH anti-BPDE-dAde adduct

Fig.  3  Metabolism of (±) BaP 7,8-dihydrodiol by CYP enzymes affords the anti- and syn-diolepoxide isomers. Reaction of (+)-anti-BaPDE takes place with DNA mainly at dGuo sites to furnish 2-NH2-dGuo adducts plus smaller amount of a 6-NH2-dAde adducts

6

R.G. Harvey

Anti-BaPDE has the benzylic OH group on the molecular face opposite the epoxide oxygen, while syn-BaPDE has these groups on the same face. Both isomers exist as pairs of (±)-enantiomers. The authentic anti- and syn-BaPDE were employed to assign the structures of the adducts formed with DNA in vivo (Jeffrey et al. 1976; King et  al. 1976; Jeffrey et  al. 1977; Meehan et  al. 1977; Nakanishi et  al. 1977; Weinstein et al. 1976). The principal DNA adduct (80–85%) was shown by NMR and mass spectral analysis to arise from reaction of (+)-anti-BaPDE on the 2-NH2group of dGuo (Fig. 3). A minor adduct formed by reaction of this BaPDE isomer on 6-NH2-dAde and several other minor adducts were also identified (Harvey and Geacintov 1988). Similar adducts were also shown to be formed by the metabolism of BaP in rodent, bovine, and human cells. Anti- and syn-BaPDE are strong mutagens in bacterial and mammalian cells (Huberman et al. 1976; Newbold and Brookes 1976). Anti-BaPDE generally exhibits higher activity than syn-BaPDE in mammalian cells, and (+)-anti-BaPDE is a more potent mutagen than (−)-anti-BaPDE. (±)-Anti-BaPDE is also more tumorigenic than (±)-syn-BaPDE on topical application to mouse skin (Levin et al. 1977; Slaga et  al. 1980). Syntheses of the diol epoxides of other PAH carcinogens (e.g., DMBA, DBC, 5-methylchrysene, benzo[g]chrysene) were also reported (Conney 1982; Harvey 1991). It is now generally accepted that the diol epoxide path plays an important role in PAH carcinogenesis. However, this does not rule out the involvement of other activation pathways. What is the molecular basis of the unique character of the bay and fjord region diol epoxide metabolites that accounts for their exceptional potency as carcinogens? According to the “bay region theory” proposed by Jerina and Daly (1977), bay region diol epoxides are predicted on the basis of molecular orbital calculations to be distinguished by their exceptional reactivity (Lehr et al. 1985). This appears counter-intuitive, since exceptional reactivity would likely result in the rapid destruction of these molecules by reactions with cellular nucleophiles prior to their migration from the endoplasmic reticulum to the nucleus where reaction with DNA takes place. The K-region oxides of BaP and DMBA (not considered in the theoretical treatment) were shown to react readily with nucleic acids to form adducts (Blobstein et al. 1975; Jennette et al. 1977). However, the evidence indicates that the arene oxide metabolites rapidly rearrange to phenols and/or undergo conversion to dihydrodiols (Wood et al. 1976; Oesch 1980; Gozukara et al. 1981) and do not form adducts with DNA in vivo. The author proposed (Harvey, 1991) that the unique feature of the bay and fjord region diol epoxides is their resistance to enzymatic detoxification that allows them to survive in a hostile environment. Survival is favored by the location of the epoxide function in a sterically restricted molecular region. Crowding interferes with enzyme-mediated transformation to water soluble conjugates. Consistent with this idea, bay region diol epoxides are relatively poor substrates for epoxide hydrolase (Wood et  al. 1976; Oesch 1980; Gozukara et  al. 1981). Also, bay-region epoxides are inherently more stable than arene oxides. The bay and fjord regions provide pockets of protection for the epoxide ring that allow

1  Historical Overview of Chemical Carcinogenesis

7

these compounds to survive long enough to be transported from the microsomes to the nucleus where they can cause DNA damage (Harvey 1991; Harvey and Geacintov 1988). 2.2.2 Radical-Cation Pathway This pathway is based on the finding that one-electron oxidation of PAHs yields short-lived PAH radical cations. Oxidation of BaP by Mn(OAc)3 in HOAc was shown by Cavalieri and Rogan (1985, 1995) to furnish the BaP radical cation which reacted with acetate to yield 6-AcO-BaP (Fig. 4). Oxidation of BaP and other PAHs by P-450 peroxidase was shown to furnish the same PAH radical cations, and these reacted with DNA to yield depurinating adducts. Cavalieri proposed that the ability of PAH radical cations to bind to cellular macromolecules depends upon their ease of formation as measured by PAH ionization potential (IP 60%) and oil of basil (24–70%) (Leung 1980). Methyleugenol is a component of the oils of basil, cinnamon, citronella, and pimento, and myristicin occurs in the oils of parsley (10–30%), nutmeg, and mace (4–8%).

16

R.G. Harvey H3CO H3CO

O

H3CO estragole

O H3CO

methyleugenol

myristicin

Fig. 11  Natural oils used as food flavorings that are implicated as carcinogens

O

O

O

O

O

O

safrole-2,3-epoxide

OH

1'-hydroxysafrole-2,3-epoxide

path C

path C O

O O

O

1'-hydroxysafrole

O2

H2O2

HO

O

1'-hydroxysafrole sulfate

4-allylcatechol

O2

O

O2

O

O e

adducts

OSO3H

path B HO

DNA

O

OH

path A safrole

O

+ ROS

DNA

damage

e semiquinone anion-radical

4-allylquinone

Fig. 12  Pathways of metabolic activation of safrole and other natural oils

The principal path of bioactivation of safrole (Fig. 12: path A) was shown by the Millers (Borchert et al. 1973) to entail P-450-mediated hydroxylation to 1¢-hydroxysafrole followed by sulfotransferase-mediated conversion to a sulfate ester. The ester decomposes to generate an allyl carbonium ion that reacts with DNA at dGuo sites to furnish the N2-linked adducts, N2-(trans-isosafrol-3-yl)-dGuo and N2(safrol-1-yl)-dGuo (Wiseman et al. 1985; Chung et al. 2008). A second activation pathway (B) entails initial O-dealkylation catalyzed by P-450-enzymes to form 4-allylcatechol which is readily oxidized to 4-allyl-o-quinone by a mechanism similar to that for formation of the PAH quinones (Bolton et al. 1994). In the process, ROS are generated that may lead to DNA damage. There is also evidence for a minor pathway (C) that involves epoxidation of 1¢-hydroxysafrole to form 1¢-hydroxysafrole-2¢,3¢-epoxide (Wiseman et  al. 1985). Safrole is classified as a group 2B carcinogen (IARC 1976), i.e., it is carcinogenic to rodents, but the evidence is not sufficient to conclude that it is carcinogenic to humans.

1  Historical Overview of Chemical Carcinogenesis

17

7.3 Pyrrolizidine Alkaloids The PAs are natural products, many of which are hepatotoxic and tumorigenic (IARC 1976). The most toxic PAs are produced by the Boraginaceae, Compositae, and Legumionsae plant families (Mattocks 1986). More than 660 PAs have been identified from these three families, and about half of them exhibit toxic properties (Stegelmeier et al. 1999). Pyrrolizidine-containing plants have a long history of use in folk medicine as herbal teas and medicinal herbs. Comfrey, a popular herbal tea, frequently contains PAs that can cause moderate to severe liver damage. Although the FDA took official action in 2001 to ban comfrey as a dietary supplement, comfrey in various forms is still readily available, and its supposed health benefits are widely touted on the Internet. Human exposure to PAs may also result from food contamination. Several examples of large-scale poisoning associated with consumption of bread made from wheat flour contaminated with PAs have been reported (Fu et al. 2001). Other modes of intake include consumption of milk from animals that graze on plants containing PAs, or eating honey obtained from bees that gather pollen from plants with significant levels of PAs. The structures of several important PAs are depicted in Fig.  13. The pyrrolizidine component possesses two fused five-membered rings having a shared nitrogen atom, a OH group in one ring, and a CH2OH group and a double bond in the other ring. PAs that lack a double bond are generally not toxic. In most cases, the rings are linked by a highly substituted diester group. Monoesterified PAs generally exhibit lower toxicity than PAs with a diester function, such as monocrotaline, riddelliine, and retrosine (Mattocks 1986; Fu et al. 2001). PAs exhibit a range of genotoxic effects that include binding to DNA, cross-linking DNA, cross-linking proteins to DNA, mutagenicity, and carcinogenicity (Fu et al. 2001). Mattocks (1968) showed that the PAs require metabolism to “pyrrole-like derivatives” to exert their hepatotoxic effects. The pathways for activation of riddelliine are shown in Fig. 13 (Fu et al. 2001). Path A entails hydrolytic de-esterification to yield a necine base (retronecine). Path B involves dehydrogenation of the unsaturated ring via hydroxylation at an allylic position to a 3- or 8-hydroxynecine derivative followed by dehydration to dehydro-riddelliine, and hydrolysis to dehydroretronecine (DHR). Path C entails oxidation to riddelliine N-oxide. Current evidence suggests that path B (formation of DHR catalyzed by cytochrome P-450 enzymes) probably plays a key role. Metabolism of riddelliine by either rat or human liver microsomes in the presence of calf thymus DNA gave eight adducts that were identical to those obtained from reaction of DHR with calf thymus DNA (Yang et al. 2001; Xia et al. 2003). Two of the adducts were epimeric DHR-dGua monophosphate adducts with a covalent linkage between the 7-position of DHR and the 2-amino group of dGua (Fig.  13). They were presumed to be formed by covalent binding of dehydro-riddelliine to DNA and/or by initial formation of DHR followed by its reaction with DNA.

18

R.G. Harvey

Structures of typical tumorigenic pyrrolizidine alkaloids.

H

OH

H

CH3

H3C

O

H3C

HO HO CH3

CH(CH3)2

HO HO

H3C

H

O

O

CH2

N

Lycopsamine

O

CH2

H

O

O

CH3

H H

O CH2

N

N

Monocrotaline

Seneceonine

CH2OH

HO

H H3C

O

O O

CH3

HO

H

O

CH3

H

O

O

O CH2

H N

Retrosine

Pathways of metabolic activation of riddelliine. CH2OH

HO

H H3C O

CH2 O

H

O CH2

O O

B

CH2

O CH2

O

HO

Riddelliine

Dehydroriddelliine oxidation

A

hydrolysis

H

CH2OH

N

N

dehydration

C

Dehydroretronecine (DHR) DNA

CH2OH

HO

H H3 C

O

O O

O

N

CH2

N

H

N

O CH2

N

Retronecine

O

CH2OH

hydrolysis

hydroxylation

N

HO

CH2OH

HO

H H 3C

O

Riddelliine N-oxide

HO

O

O O P OH OH

NH N

NH

CH2OH

N

+ additional adducts

Fig. 13  Structures of typical tumorigenic pyrrolizidine alkaloids, and the pathways of metabolic activation of riddelliine

8 Occupational Carcinogens Many carcinogens were first identified through observation of high incidences of cancers in workers exposed to them in the workplace. The PAHs and the aromatic amines were discovered in this way. Other prominent examples include chlorinated solvents, polybromobiphenyls, chlorinated dioxins, pesticides, compounds of toxic metals (arsenic, nickel, chromium, beryllium, and cadmium), asbestos, and other mineral fibers (Searle 1984; Searle and Teale 1990). PAHs are major occupational carcinogens as well as major environmental carcinogens. High exposure to PAHs occurs in aluminum production, coal gasification, coke production, iron and steel production, tar distillation, road paving, roofing, extraction of shale oil, production of carbon black, and carbon electrode production (Boffetta et al. 1997). PAHs are also a significant hazard for transportation workers (truck and taxi drivers, mechanics),

1  Historical Overview of Chemical Carcinogenesis

19

operators of tractors and farm machinery, and workers in the oil and petrochemical industries.

9 Importance of Chemical Carcinogens in Human Cancer Almost a century has passed since the beginning of experimental research in carcinogenesis. Progress was slow prior to Watson and Crick’s determination of the structure of DNA and the elucidation of its role as the primary carrier of genetic information. These discoveries provided the essential basis for understanding carcinogenesis at the molecular-genetic level, and the pace of discovery has rapidly accelerated. Principal advances include (1) identification of the major classes of environmental carcinogens; (2) appreciation of the role of lifestyle factors in human cancer; (3) recognition of the role of bioactivation; and (4) increased awareness of differences between individuals in genetic susceptibility.

9.1 Identification of the Major Classes of Environmental Carcinogens Since the discovery of the PAH carcinogens in the 1930s, the list of known carcinogens has expanded to include the classes of carcinogens reviewed in this chapter, as well as others (chloromethyl ethers, epoxides, vinyl chloride, chlorinated pesticides, polybromobiphenyls, chlorinated dioxins, steroid hormones, aldehydes, compounds of arsenic, nickel, chromium, beryllium, and cadmium, and asbestos). A complete list of substances evaluated as carcinogens by the IARC and the NTP is available through the Internet.

9.2 Appreciation of the Role of Lifestyle Factors Discoveries of new classes of chemical carcinogens have been primarily through their association with occupation or lifestyle factors. In the first half of the century, the occupational factor was dominant, probably because the association between exposure to a carcinogen and the incidence of cancer was more obvious in a workplace setting. Recognition of the role of lifestyle factors was inherently more difficult because of the long time between exposure to a carcinogen and the appearance of cancer. The time interval between the beginning of smoking and the diagnosis of lung cancer was often 20 or more years. Prior to World War I lung cancer was relatively rare in the American population. American men serving in the military adopted the habit of smoking cigarettes during the war, and after a time lag of 20+ years, the incidence of lung cancer in males rose to exceed that of other forms of cancer. Recognition of the role of lifestyle was enhanced by the report by the Surgeon General (1964) that lung cancer was associated with smoking tobacco.

20

R.G. Harvey

PAHs are major atmospheric pollutants that originate mainly from combustion processes. Specific sources include the burning of coal and oil for heating, emissions from power plants and other industrial sources, engine exhaust (auto, truck, aircraft), burning of garbage, forest fires, and volcanic emissions. The annual global emission of B[a]P in 1979 was estimated to be ~5,000 tons, the greatest contribution coming from combustion of coal (Grimmer 1983). The USA accounted for about a quarter of the total ~1,260 tons. Nitroarenes are formed from PAHs in the atmosphere by their reaction with oxides of nitrogen, and they are also produced in the combustion of fossil fuels. 1-Nitropyrene accounts for ~25% of the mutagenic activity of diesel emissions (Grimmer 1983). PAHs and the nitro-PAHs formed from them are major environmental carcinogens as well as important occupational carcinogens. It is worthy of note that noncarcinogenic PAHs, such as pyrene, may give rise to carcinogenic nitroarenes. Diet and food preparation are also important lifestyle factors that contribute to the cancer burden (National Research Council 1996; Jeffrey and Williams 2005). Approximately 35% of cancer mortality in the USA has been attributed to diet (Doll and Peto 1981). Carcinogens and procarcinogens in the diet derive from multiple sources (Grasso 1984; National Research Council 1996). They include naturally occurring carcinogens (caffeic acid, urethane), various food additives and flavorings (e.g., safrole), dyes, preservatives (e.g., nitrites), contaminants (pesticides, aflatoxins, and PAs), and carcinogens formed in cooking. Another important dietary factor is the quantity and quality of the food consumed. Epidemiological studies indicate that diets high in fat and caloric intake increase the risk of certain cancers. There is also considerable evidence that anticarcinogens are also present in foods (National Research Council 1996). Numerous studies have demonstrated that consumption of fruits and vegetables is associated with reduced rates of cancers of the stomach, lungs, breast, and colon. Members of the allium, cruciferous, and tea families are effective in preventing cancers of the esophagus, colon, lung, breast, and skin in rodents. These effects are thought to be due partially to the presence of flavonoid compounds (e.g., apigenin, myricitin, quercitin, rutin) in these types of plants. Animal studies support the usefulness of these types of compounds as anticancer agents.

9.3 Recognition of the Role of Bioactivation One of the most important advances in carcinogenesis research has been the acceptance of the concept that most carcinogens are not directly active but require enzymatic conversion to active forms in order to exert their genotoxic effects. At first glance, the chemical and structural diversity of the known chemical carcinogens appear too complex for their activities to be explicable in terms of a single mechanistic concept. However, investigations of the metabolic pathways have revealed underlying fundamental similarities. Miller and Miller (1966) proposed, based on their studies with AAF and other carcinogens, that the ultimate active

1  Historical Overview of Chemical Carcinogenesis

21

metabolites of most carcinogens are reactive electrophiles that form adducts with cellular macromolecules. The likely targets were assumed to be proteins, RNA, and/or DNA. A key piece of evidence was the finding by Brooks and Lawley (1964), who measured the extents of binding of the active metabolites of several 3H-labeled PAHs to proteins, RNA, and DNA in mouse skin. They observed good correlation between the extents of binding to DNA but no correlation with binding to RNA or proteins. This observation is consistent with the unique role of DNA as the primary carrier of genetic information. The Millers proposed “that the initiation step of chemical carcinogenesis is a mutagenic event that is caused by alteration of DNA by the ultimate carcinogens (Miller and Miller 1981).” This hypothesis has gained wide acceptance. As discussed in preceding paragraphs, the identities of the active metabolites of most of the major categories of carcinogens have been established (Fig. 14). There is strong evidence that formation of PAH diol epoxide metabolites is a major pathway of PAH activation. The structures of the principal DNA-bound adducts formed by the isomeric diol epoxide metabolites of BaP were determined by NMR and mass spectral analysis and shown to be the products of reaction of (+)-anti-BaPDE

Active Metabolites of Chemical Carcinogens Carcinogen PAH*

Active metabolite

Reactive species

diol epoxide

carbonium ion

quinone

ROS

Arylamine, arylamide or nitroarene

N-hydroxy ester

nitrenium ion

N-Nitroso compd

hydroxyN-nitroso compd

carbonium ion + aldehyde

Aflatoxin

epoxide

carbonium ion

dehydroretronecine

carbonium ion

hydroxysulfate ester

carbonium ion

Pyrrolizidine alkaloid Safrole* Alkylating agent *

direct acting

Addit ional mechanisms may also play a role.

Fig. 14  Active metabolites of chemical carcinogens

carbonium ion

22

R.G. Harvey

on the 2-amino group of dGuo (Jeffrey et al. 1976; King et al. 1976; Weinstein et al. 1976; Nakanishi et al. 1977). However, there is increasing evidence that the redoxactive quinone pathway may also make an important contribution (Penning et al. 1999; Park et al. 2005, 2006). Recently, it has been demonstrated that this pathway also functions in human lung A549 cells (Park et al. 2008). Similar redox-active quinone pathways are thought to be involved in estrogen-induced carcinogenesis (Chap. 4) and in activation of safrole and other natural oils (Bolton et  al. 1994) (Fig. 11).

10 Future Directions While there have been major advances in carcinogenesis research, much still remains unknown. An important question is what is the basis of the differences between individuals in their susceptibilities to the oncogenic effects of carcinogens? Not all cigarette smokers succumb to lung cancer. Inter-individual differences are dependent upon the balance between competing activation and detoxification pathways (Harris 1989). Formation of DNA adducts by chemical carcinogens may lead to mutations that activate protooncogenes and inactivate tumor suppressor genes in replicating cells. The steady-state levels of these adducts will depend upon the amounts of the active metabolites produced, their rates of loss by secondary reactions and detoxification, and the efficiency of the DNA repair processes. Many of the enzymes involved in these processes are highly polymorphic. Genetic variants in these enzymes contribute to determine the individual susceptibilities to carcinogen exposures. Acknowledgments  The author’s investigations were supported by NIH Grants (P01 CA 92537, R01 CA 039504, R01 ES 015857, and P30 ES 013508).

References Baird WM, Dipple A, Grover PL, Sims P, Brookes P (1973) Cancer Res. 33:2386–2392. Baird WM, Harvey RG, Brookes P (1975) Cancer Res. 35:54–57. Beland FA, Harvey RG (1976) J. Chem. Soc. Chem. Commun. 84–85. Beland FA, Kadlubar FF (1990) In Chemical Carcinogenesis and Mutagenesis I, Cooper CS, Grover PI (eds), Springer-Verlag, Berlin, pp. 267–325. Bennett JW, Klich M (2003) Clin. Microbiol. Rev. 16:497–516. Blobstein SH, Weinstein IB, Grunberger D, Weisgras J, Harvey RG (1975) Biochemistry 14:3451–3458. Boffetta P, Jourenkova N, Gustavasson P (1997) Cancer Causes Control 8:444–472. Bolton JL, Acay NM, Vukomanovic V (1994) Chem. Res. Toxicol. 7:443–450. Borchert P, Miller JA, Miller EC, Shires TK (1973) Cancer Res. 33:590–600. Borgen A, Darvey H, Castagnoli N, Crocker TT, Rasmussen RE, Wang IY (1973) J. Med. Chem. 16:502–506.

1  Historical Overview of Chemical Carcinogenesis

23

Boyland E (1950) Biochem. Soc. Symp. 5:40–54. Brooks P, Lawley PD (1964) Nature 202:781–784. Busby WF, Wogan GN (1984) In Chemical Carcinogens, Searle CE (ed), Vol. 2, American Chemical Society, Washington, DC, pp. 945–1169 (ACS Monograph 182). Cavalieri EL, Rogan EG, Roth RW, Saugier RK, Hakam K (1983) Chem. Biol. Interact. 47:87–109. Cavalieri E, Rogan E (1985) In Polycyclic Hydrocarbons and Carcinogenesis, Harvey RG (ed), ACS. Symp. Series 283, American Chemical Society, Washington, DC, pp. 289–305. Cavalieri EL, Higginbotham S, RamaKrishna NSV, Devanesan PD, Todorovic R, Rogan EG, Salmasi S (1991) Carcinogenesis 12:1939–1944. Cavalieri E, Rogan E (1995) Xenobiotica 25:677–688. Cavalieri EL, Rogan EG, Li K-M, Todorovic R, Ariese F, Jankowiak R, Grubor N, Small GJ (2005) Chem. Res. Toxicol. 18:976–983. Chen L, Devanesan PD, Higginbotham S, Ariese F, Jankowiak R, Small GJ, Rogan EG, Cavalieri EL (1996) Chem. Res. Toxicol. 9:897–903. Cheng KC, Cahill DS, Kasai H, Nishimura S, Loeb LA (1992) J. Biol. Chem. 267:166–172. Chung Y-T, Chen C-L, Wu C-C, Chan S-A, Chi C-W, Liu T-Y (2008) Toxicol. Lett. 183:21–27. Conney A (1982) Cancer Res. 42:4875–4917. Cook JW, Hewett CL, Hieger I (1933) J. Chem. Soc. 395–405. Cramer JW, Miller JA, Miller EC (1960) J. Biol. Chem. 235:885–888. Dai Q, Ran C, Harvey RG (2005) Org. Lett. 5:999–1002. Dai Q, Xu D, Lim K, Harvey RG (2007) J. Org. Chem. 72:4856–4863. Dansette P, Jerina DM (1974) J. Am. Chem. Soc. 96:1224–1225. Devanesan PD, RamaKrishna NVS, Todorovic R, Rogan EG, Cavalieri EL, Jeong H, Jankowiak R, Small GJ (1992) Chem. Res. Toxicol. 5:302–309. Diechmann WBJ, Radomski J, Glass E, Coplan M, Woods F (1965) Ind. Med. Surg. 34:640–649. Doll R, Peto R (1981) J. Natl. Cancer Inst. 66:1191–1308. Eaton DL, Gallagher, EP (1994) Ann. Rev. Pharmacol. Toxicol. 34:135–172. Ender F, Favre G, Helgebostad A, Koppang N, Madsen R, Ceh I (1964) Naturwissenschaften 51:637–638. Freundenthal RI, Stephens E, Anderson DP (1999) Int. J. Toxicol. 18:353–359. Fu PP, Harvey, RG, Beland FA (1978) Tetrahedron 34:857–866. Fu PP, Chou MW, Xia Q, Yang Y-C, Yan J, Doerge DR, Chan PC (2001) Environ. Carcinog. Ecotoxicol. Rev. C19:363–385. Fujita J, Sirvastava SK, Kraus MH, Rhim JS, Tronik SR, Aaronson SA (1985) Proc. Natl. Acad. Sci. USA 82:3849–3853. Goh SH, Harvey RG (1978) J. Am. Chem. Soc. 95:242–243. Goshman LM, Heidelberger C (1967) Cancer Res. 27:1678–1688. Gozukara EH, Belvedere, G, Robinson RC, Deutsch J, Guengerich FP, Gelboin HV (1981) Mol. Pharmacol. 19:153–161. Grasso P (1984) In Chemical Carcinogens, Searle CE (ed), Vol. 2, American Chemical Society, Washington, DC, pp. 1205–1239 (ACS Monograph 182). Grimmer G (1983) Environmental carcinogens: polycyclic aromatic hydrocarbons, chemistry, occurrence, biochemistry, carcinogenicity, CRC Press, Boca Raton, FL. Grover PL, Sims P (1968) Biochem. J. 110:159–160. Guengerich FP, Johnson WW, Shimada T, Ueng Y-F, Yamazaki H, Langouët S (1998) Mutat. Res. 402:121–128. Harris C (1989) Carcinogenesis 10:1563–1566. Harvey R (1991) In Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity. Cambridge Monographs on Cancer Research, Coombs MM (ed), Cambridge University Press, Cambridge, UK. Harvey RG, Dai Q, Ran C, Lim KP, Blair, IA, Penning TM (2005) Polycycl. Aromat. Compd. 25:371–391.

24

R.G. Harvey

Harvey RG, Geacintov NE (1988) Acc. Chem. Res. 21:66–73. Harvey RG, Goh SH, Cortez C (1975) J. Am. Chem. Soc. 97:3468–3469. Hatanaka N, Yamazaki H, Oda Y, Guengerich FP, Nakajima M, Yokoi T (2001) Mutat. Res. 497:223–233. Hecht SS (1998) Chem. Res. Toxicol. 11:559–603. Hecht SS (2008) Chem. Res. Toxicol. 21:160–171. Hecht SS, Amin S, Melikian AA, LaVoie EJ, Hoffmann D (1985) In Polycyclic Hydrocarbons and Carcinogenesis, Harvey RG (ed), ACS Symp. Series No. 283, American Chemical Society, Washington, DC, 41–163. Higginson J, Muir CS, Munoz, N (1992) Human Cancer: Epidemiology and Environmental Causes. Cambridge Monographs on Cancer Research, Coombs MM (ed), Cambridge University Press, Cambridge, UK. Hill J (1761) Cautions against the immoderate use of snuff founded on the known qualities of the tobacco plant and the effects it must produce when in this Way Taken into the Body; and enforced by instances of persons Who have Perished Miserably of Disease, Occassioned, or Rendered Incurable by its Use. Baldwin and Jackson, London UK. Huberman E, Sachs L, Yang SK, Gelboin HV (1976) Proc. Natl. Acad. Sci. USA. 73:607–611. Huggins CB, Pataki J, Harvey RG (1967) Proc. Natl. Acad. Sci. USA. 58:2253–2260. IARC (1976) IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans: Some naturally occurring substances, Vol. 10, IARC, Lyon, France. IARC (1983) IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans: Polynuclear aromatic compounds, Vol. 32, IARC, Lyon, France. IARC (1987) Overall evaluations of carcinogenicity. IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans, Supplement 7, IARC, Lyon, France. IARC (1993) IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans: Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins, Vol. 56, IARC, Lyon, France. IARC (2002) IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans: Traditional herbal medicines, some mycotoxins, naphthalene and styrene, Vol. 82, IARC, Lyon, France. IARC (2004) IARC Monographs on the evaluation of carcinogenic risks to humans: Tobacco smoke and involuntary smoking, Vol. 83, IARC, Lyon, France. IARC (2007) IARC Monographs on the evaluation of carcinogenic risks to humans: Smokeless tobacco smoke, Vol. 89, IARC, Lyon, France. Iyer R, Coles B, Raney KD, Thier R, Guengerich FP, Harris TM (1994) J. Am. Chem. Soc. 116:1603–1609. Jager J (1978) J. Chromatogr. 152:573–578. Jeffrey AM, Jennette K, Blobstein SH, Weinstein IB, Beland FA, Harvey RG, Kasai H, Miura I, Nakanishi K (1976) J. Am. Chem. Soc. 98:5714–5716. Jeffrey AM, Weinstein IB, Jennette K, Grzeskowiak K, Nakanishi K, Harvey RG, Autrup H, Harris C (1977) Nature (London) 269:348–350. Jeffrey AM, Williams GM (2005) Toxicol. Appl. Pharmacol. 207:S628–S635. Jennette KW, Jeffrey AM, Blobstein SH, Beland FA, Harvey RG, Weinstein IB (1977) Biochemistry 16:932–938. Jerina DM, Daly JW (1977) In Drug Metabolism – From Microbe to Man, Parke DV, Smith RI (eds), Taylor & Francis, London. Keefer LE, Roller PP (1973) Science 181:1245–1247. King CM, Glowinski IB (1983) Environ. Health Perspect. 49:43–50. King HWS, Osborne MR, Beland FA, Harvey RG, Brookes P (1976) Proc. Natl. Acad. Sci. USA 73:2679–2681. Kinosita R (1936) Gann 30:423–426. Kmet J (1970) J. Chronic Dis. 23:305–315. Kolonel LN, Altshuler D, Henderson BE (2004) Nat. Rev. Cancer 4:519–527. Lai C-C, Miller JA, Miller EC, Liem A (1985) Carcinogenesis 6:1037–1045.

1  Historical Overview of Chemical Carcinogenesis

25

Lawley PD (1990) In Handbook of Experimental Pharmacology, Cooper CS, Grover PI (eds), Vol. 94/1, Springer-Verlag, Berlin, pp. 409–469. Lehr R, Kumar S, Levin W, Wood AW, Chang RL, Conney A, Yagi H, Sayer JM, Jerina DM (1985) In Polycyclic Hydrocarbons and Carcinogenesis, Harvey RG (ed), American Chemical Society, Washington, DC, pp. 63–84 (ACS Monograph 283). Leung AY (1980) Encyclopedia of common natural ingredients used in foods, drugs, and natural cosmetics, Wiley, New York. Levin W, Wood AW, Chang RL, Slaga, TJ, Yagi H, Jerina DM, Conney AH (1977) Cancer Res. 37:2721–2725. Lijinsky W (1999) Mutat. Res. 443:129–138. Lindahl T, Nyberg B (1972) Biochemistry 11:3610–3616. Loeb LA, Harris C (2008) Cancer Res. 68:6863–6872. Löfroth G, Hefner E, Alfeim I, Møeller M (1980) Science 209:1037–1039. Luch A (2005) Nat. Rev. Cancer 5:113–125. Magee PN, Barnes JM (1956) Br. J. Cancer 10:114–122. Magee PN, Farber E (1962) Biochem. J. 83:114–124. Magee PN, Hultin T (1962) Biochem. J. 83:106–114. Mangino M, Scanlan RA, O’Brien TJ (1981) In N-Nitroso Compounds, Scanlan RA, Tannenbaum SR (eds), ACS Symp. Series No. 174, American Chemical Society, Washington, DC, pp. 229–245. Mattocks AR (1968) Nature (London) 217:723–728. Mattocks AR (1986) Chemistry and toxicology of pyrrolizidine alkaloids, Academic Press, London. McCoull KD, Rindgen D, Blair IA, Penning TM (1999) Chem. Res. Toxicol. 12:237–246. Meehan T, Straub K, Calvin M (1977) Nature (London) 269:725–727. Melendez-Colon V, Luch A, Seidel A, Baird W (1999) Carcinogenesis 20:1885–91. Miller EC, Miller JA (1966) Pharmacol. Rev., Part II 18:805–838. Miller EC, Miller JA (1981) Cancer 47:2327–2345. Miller JA, Miller EC, Hartmann HA (1961) Cancer Res. 21:815–824. Nakanishi K, Kasai H, Cho H, Harvey RG, Jeffrey AM, Jennette KW, Weinstein IB (1977) J. Am. Chem. Soc. 99:258–260. National Research Council (1996) Carcinogens and anticarcinogens in the human diet, National Academy Press, Washington, DC. National Toxicology Program (1996) Toxicology Publ. No. 34, U.S. DHHS, Washington, DC. Newbold RF, Brookes P (1976) Nature 261:52–54. Oesch F (1980) In Microsomal Epoxide Hydrolase. Enzymatic Basis of Detoxification. II, Jacoby WB (ed), Academic Press, New York, pp. 277–290. Oesch F, Timms CW, Walker CH, Guenther TM, Sparrow A, Watabe T, Wolf CR (1984) Carcinogenesis 5:7–9. Park J-H, Gopishetty S, Szewczuk LM, Troxel AB, Harvey RG, Penning TM (2005) Chem. Res. Toxicol. 18:1026–1037. Park J-H, Troxel AB, Harvey RG, Penning TM (2006) Chem. Res.Toxicol. 19:719–728. Park J-H, Mangal D, Tacka KA, Quinn AM, Harvey RG, Blair IA, Penning TM (2008) Proc. Natl. Acad. Sci. USA 105:6845–6851. Penning TM, Burczyski ME, Hung C-F, McCoull KD, Palackal NT, Tsuruda, LS (1999) Chem. Res. Toxicol. 12:1–18. Peto J (2001) Nature 411:390–395. Pitts JN Jr, van Cauwenberghe KA, Grosjean D, Schmidt JP, Fitz DR, Belser WL Jr, Knudson GB, Hynds PM (1978) Science 202:515–519. Pott P (1775) Chirugical observations relative to the cancer of the scrotum, Hawes, Clarke, and Collins, London. Reprinted in Natl. Cancer Inst. Monograph 10:7–13 (1963). Poupko JM, Hearn WL, Radomski JL (1979) Toxicol. Appl. Pharm. 50:479–484. Poirier MC, Hunt JM, True B, Laishes BA, Young JF, Beland FA (1988) Cancer Res. 5:1591–1596. Preussmann R, Eisenbrand G (1984) In Chemical Carcinogens, Searle CE (ed), Vol. 2. American Chemical Society, Washington DC, pp. 829–868 (ACS Monograph 182).

26

R.G. Harvey

Preussmann R, Stewart BW (1984) In, Chemical Carcinogens, Searle CE (ed), Vol. 2, American Chemical Society, Washington DC, pp. 643–828 (ACS Monograph 182). Pullman A, Pullman B (1955) Adv. Cancer Res. 3:117–169. Purohit V, Basu AK (2000) Chem. Res. Toxicol. 13:673–692. Ran C, Dai Q, Ruan Q, Penning TM, Blair IA, Harvey RG (2008) J. Org. Chem. 73:992–1003. Rehn L (1895) Arch. Klin. Chir. 50:588–600. Rosenkranz HS, McCoy EC, Sanders DR, Butler M, Kiriazides DK, Mermelstein R (1980) Science 209:1039–1043. Searle CE (1984) Chemical Carcinogens, Vol. 1&2, American Chemical Society, Washington, DC (ACS Monograph No. 182). Searle CE, Teale OJ (1990) In Chemical Carcinogenesis and Mutagenesis I, Cooper CS, Grover PI (eds), Springer-Verlag, Berlin, pp. 103–151. Shou M, Harvey RG, Penning TM (1993) Carcinogenesis 14:2707–2715. Sims P, Grover PL, Swaisland A, Pal K, Hewer A (1974) Nature 252:326–328. Singer B, Grunberger D (1989) Molecular biology of mutagens and carcinogens, Plenum Press, New York. Slaga TJ, Gleason GL, Mills G, Ewald L, Fu PP, Lee HM, Harvey RG (1980) Cancer Res. 40:1981–1984. Smithgall TE, Harvey RG, Penning TM (1986) J. Biol. Chem. 261:6184–6186. Smithgall TE, Harvey RG, Penning TM (1988) J. Biol. Chem. 263:1814–1820. Spiegelhalder B, Eisenbrand G, Preussmann R (1980) IARC Scientific publication No. 31, pp. 467–470. Snyderwine EG, Turteltaub KW (2000) In Food Borne Carcinogens, Nagao M, Sugimura T (eds), Wiley, New York. Stegelmeier BL, Edgar JA, Colegate SM, Gardener DR, Schoch TK, Coulombe RA Jr (1999) J. Nat. Toxins 8:95–116. Sugimura T, Nagao M, Wakabayashi K (2000) Mutat. Res. 447:15–25. Sugimura T, Wakabayashi K, Nakagama H, Nagao M (2004) Cancer Sci. 95:290–299. Surgeon General (1964) Smoking and Health. Report of the Advisory Committee to the Surgeon General of the Public Health Service. Publication No. 1103, pp. 149–196, U.S. Govt. Printing Office, Washington, DC. Tokiwa H, Ohnishi Y (1986) CRC Crit. Rev. Toxicol. 17:23–60. Tsutsui H (1918) Gann 12:21. Turesky RJ, Rossi SC, Welti D, Lay JO Jr, Kadlubar FF (1992) Chem. Res. Toxicol. 5:479–490. Weinstein IB, Jeffrey AM, Jennette K, Blobstein S, Harvey RG, Harris C, Autrup H, Kasai H, Nakanishi K (1976) Science 193:592–595. Wilson RH, DeEds F, Cox AJ (1941) Cancer Res. 1:595–608. Wiseman RW, Fennell TR, Miller JA, Miller EC (1985) Cancer Res. 45:3096–3105. Wood AW, Levin W, Lu AYH, Yagi H, Hernandez O, Jerina DM, Conney AH (1976) J. Biol. Chem. 251:4882–4890. Xia Q, Chou MW, Kadlubar FF, Chan PC, Fu PP (2003) Chem. Res. Toxicol. 16:66–73. Yagi H, Hernandez O, Jerina DM (1975) J. Am. Chem. Soc. 97:6881–6883. Yamagawa K, Ichikawa K (1915) Mitt. Med. Fak. Kaiserl. Univ. Tokyo 15:295–344. Yang SK, Roller PP, Fu PP, Harvey RG, Gelboin HV (1977) Biochem. Biophys. Res. Commun. 77:1176–1182. Yang YC, Yan J, Doerge DR, Chan PC, Fu PP, Chou MW (2001) Chem. Res. Toxicol. 14:101–109. Yoshida T (1933) Trans. Jpn. Pathol. Soc. 23:636–638.

Chapter 2

Multistage Carcinogenesis Erika L. Abel and John DiGiovanni

Abstract  The major stages of chemical carcinogenesis have been deduced over the past ~50 years, primarily from animal model studies (and particularly from studies using the mouse skin model); these stages are termed initiation, promotion, and progression. Tumor initiation begins when DNA in a cell or population of target cells is damaged by exposure to exogenous or endogenous carcinogens leading to mutations in critical target genes. The responsiveness of initiated cells to their microenvironment gives them a growth advantage relative to normal cells under certain conditions. In the classic two-stage chemical carcinogenesis system in the mouse skin, a low dose of a carcinogen such as 7,12-dimethylbenz(a)anthracene induces a mutation in Hras1 that does not give rise to tumors over the lifespan of the mouse unless a tumor promoter, such as TPA, is repeatedly applied. The tumor promotion stage is characterized by selective clonal expansion of the initiated cells, a result of the altered expression of genes whose products are associated with hyperproliferation, tissue remodeling, and inflammation. During tumor progression, preneoplastic cells undergo malignant transformation through a process of selection that is facilitated by progressive genomic instability and altered gene expression. While the processes involved in each stage of experimental chemical carcinogenesis also appear to be involved in human carcinogenesis, the temporal nature of initiation, promotion, and progression events is more complex. In addition, multiple mutational events are involved in the formation of human tumors. Genetic background and nutritional status can dramatically affect susceptibility to a carcinogenic exposure in both experimental animals and humans. An understanding of the multistage nature of carcinogenesis has led to the discovery of mechanismbased inhibitors that target events associated with specific stages. Further study of the cellular, biochemical, and molecular mechanisms associated carcinogenesis induced by chemicals and other types of carcinogens will lead to identification of effective strategies for cancer prevention.

J. DiGiovanni (*) Division of Pharmacology/Toxicology and Department of Nutritional Sciences, Colleges of Pharmacy and Natural Sciences, The University of Texas at Austin, Austin, TX, USA e-mail: [email protected]

T.M. Penning (ed.), Chemical Carcinogenesis, Current Cancer Research, DOI 10.1007/978-1-61737-995-6_2, © Springer Science+Business Media, LLC 2011

27

28

E.L. Abel and J. DiGiovanni

1 Introduction The concept of multistage carcinogenesis dates back over 70  years to studies by Deelman (reviewed in Vulimiri and DiGiovanni 1999), who found that wounding led to skin tumors in mice that had first been treated with a carcinogenic tar. These, and other early studies (reviewed in Boutwell 1964; DiGiovanni 1992), suggested that multiple steps were required for chemical induction of tumors and that cell proliferation and hyperplasia play critical roles in tumor development during skin carcinogenesis. The concept of multistage carcinogenesis is relevant in terms of human cancers for several reasons. First, human exposures to chemical carcinogens typically occur at low dose levels that, alone, are insufficient to produce cancer. Second, there is considerable evidence from both human epidemiologic as well as experimental animal studies that certain human carcinogens exhibit a strong tumor promoting activity [e.g., tobacco smoke, ultraviolet (UV) light]. Finally, many components of the human diet appear to influence cancer in humans through a tumor promotion effect.

2 Multistage Carcinogenesis in Mouse Skin Since much of what we know about multistage carcinogenesis has been learned from the mouse skin model, this chapter will draw heavily on studies in this system (see Fig. 1). Several other excellent reviews have also been written on this subject

Fig. 1  Multistage carcinogenesis in mouse skin

2  Multistage Carcinogenesis

29

(Boutwell 1964, 1974; Slaga et al. 1982; DiGiovanni 1992; Slaga et al. 1996; Yuspa 1998, 2000; Kemp 2005). From a historical standpoint, skin carcinogenesis can be accomplished using either a complete carcinogenesis protocol or a multistage protocol (Boutwell 1964, 1974). A complete carcinogenesis protocol involves applying a single large dose of a skin carcinogen or repeated applications of smaller doses of a skin carcinogen to the backs of mice to elicit skin tumors. Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a] anthracene (DMBA) are typical complete carcinogens. Mouse skin tumors can also be elicited using an operational protocol where a small sub-carcinogenic dose (initiating dose) of a skin carcinogen such as DMBA is applied once followed 1–2 weeks later by prolonged and repeated application of a tumor promoting agent such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Research applications of this model and current protocols on its use can also be found in a very recent review (Abel et al. 2009). The multistage model of mouse skin carcinogenesis has been and still remains an excellent model for the study of mechanisms associated with the process of multistage epithelial carcinogenesis in humans. In addition, this model has been extremely useful for identification, as well as, mechanistic studies of chemopreventive agents. In the multistage model of mouse skin carcinogenesis, three mechanistic stages: initiation, promotion, and progression can be defined (Boutwell 1964; Slaga 1984; DiGiovanni 1992; Conti 1994; Yuspa 1994). In the initiation stage, mutations occur in critical genes, such as Hras1 (reviewed in DiGiovanni 1992; Kemp 2005), which control epidermal proliferation and/or differentiation. This genetic alteration confers a selective growth advantage on skin epidermal cells such that these cells undergo clonal expansion during the early phase of promotion. The initiation stage is irreversible and phenotypically silent and the initiated skin behaves as normal skin unless challenged with a promoting stimulus (reviewed in DiGiovanni 1992; Kemp 2005). The promotion stage of mouse skin carcinogenesis occurs as a result of exposure of the initiated skin to a repetitive promoting stimulus (Slaga 1984; DiGiovanni 1992; Yuspa 1994). Most tumor promoters are not genotoxic but cause altered expression of genes whose products are associated with hyperproliferation, tissue remodeling, and inflammation. The endpoint of the promotion stage in the mouse skin model is the formation of squamous papillomas, which are exophytic, premalignant lesions consisting of hyperplastic epidermis folded over a core of stroma (Klein-Szanto 1989; Conti 1994). Tumor promoting stimuli are very diverse in this model system and include various chemicals such as phorbol esters (e.g., TPA), organic peroxides (e.g., benzoyl peroxide [BzPo]), anthrones such as chrysarobin (Chry), and okadaic acid (OA) (DiGiovanni 1992; Abel et al. 2009). In addition, UV light, repeated abrasion, full thickness skin wounding, and certain silica fibers when rubbed on the skin all function as skin tumor promoting stimuli (DiGiovanni 1992; Abel et  al. 2009). The process of tumor progression occurs when papillomas convert into squamous cell carcinomas (SCCs). Most, if not all, SCCs that appear during a multistage carcinogenesis protocol in mouse skin arise from preexisting papillomas (Klein-Szanto 1989; Conti 1994). The SCCs that develop in this model are histologically similar to human SCCs (Klein-Szanto 1989).

30

E.L. Abel and J. DiGiovanni

Since this model has been well characterized and bears relevance to certain human epithelial cancers (including SCC of the skin), it provides an excellent paradigm for studying the molecular mechanisms of epithelial cell tumor formation.

3 Mechanisms of Skin Tumor Initiation During the first (or initiation) stage of chemically induced skin carcinogenesis (see Fig. 1), key genes in epidermal keratinocytes acquire mutations as a result of topical exposure to a mutagenic carcinogen. Currently, the most frequently utilized initiating agent is DMBA, but additional agents can serve as chemical initiators. DMBA, B[a]P, and N-methyl-N-nitrosourea (MNU) may be used to initiate skin carcinogenesis but these “pro-carcinogens” require metabolic activation to mutagenic metabolites referred to as “ultimate carcinogens.” In contrast, N-methyl-N¢-nitro-Nnitrosoguanidine (MNNG) and UV light are mutagenic, direct-acting carcinogens that may serve as initiating agents in this model (Abel et al. 2009). To initiate carcinogenesis, a subcarcinogenic dose of the carcinogen is applied to the shaved dorsal skin of the mouse. Activating mutations in a critical target gene, such as Hras1, can be detected in the epidermis as early as 3–4 weeks following treatment with DMBA (Nelson et  al. 1992) and are observed in the majority of papillomas that develop initially following tumor promoter treatment (Balmain et  al. 1984). While DMBA predominantly induces an A (182) ® T transversion mutation in codon 61 of the Hras1 gene, various other initiating agents each produce a unique spectrum of activating Hras1 mutations (Brown et  al. 1990). Furthermore, mutations in Kras have been demonstrated in lesions initiated with DMBA and MNNG, and Nras mutations have been observed in lesions initiated with UV light (Pierceall et al. 1992; Rehman et al. 2000). Confirmation that mutations in Hras1 represent an initiating event in mouse skin carcinogenesis comes from several lines of evidence including (1) different mutation spectra depending on the initiating carcinogen and the types of DNA adducts they produce (reviewed in DiGiovanni 1992); (2) transgenic mice expressing a mutant Ha-ras gene develop skin tumors at sites of skin irritation (Bailleul et al. 1990); (3) knockout of Hras1 significantly reduced susceptibility to skin tumor development in mice exposed to a two-stage carcinogenesis protocol (Ise et  al. 2000); and finally (4) deletion of Stat3 specifically in bulge-region keratinocyte stem cells leads to loss of bulgeregion keratinocyte stem cells (KSCs) with Hras1 mutations and dramatically reduces skin tumor initiation by DMBA (Kim et al. 2009). From early studies it became clear that the initiation stage is both irreversible and cumulative (Boutwell 1964; Slaga et al. 1982; DiGiovanni 1992; Vulimiri and DiGiovanni 1999). That is, the dose required for initiation can be divided and applied in portions over time or applied in a single dose with essentially the same result. Additionally, commencement of the promotion phase can be delayed since the DNA mutations induced by the initiating agent are permanent. The critical mutations (e.g., mutations in Hras1) for tumor initiation are believed to occur in epidermal

2  Multistage Carcinogenesis

31

multipotent stem/progenitor cells, which may reside in specific compartments of the hair follicle as well as in the basal layer of the epidermis (Morris et al. 1985; Gerdes and Yuspa 2005; Kangsamaksin et al. 2007; Trempus et al. 2007). While no phenotypically abnormal cells are apparent in this “initiated” skin, small populations of epidermal cells can be identified as early as 1 week after initiation that contain signature mutations of the Hras1 allele, which are characteristic of tumors initiated with DMBA (Nelson et al. 1992). Furthermore, the frequency of detection of mutant Hras1 alleles increases when initiated skin is treated with a tumor promoter indicating that clonal expansion of the initiated cells occurred (Finch et al. 1996). Additional work from our laboratory has shown that these mutations can be detected in hair follicle bulge-region keratinocyte stem cells as early as 1  day and 10  days after initiation with DMBA (Kim et al. 2009) further supporting the hypothesis that keratinocyte stem/progenitor cells are the targets for initiation in this model system. Activating mutations in Hras1 are believed to confer, at least in part, resistance to terminal differentiation of keratinocytes during tumor promoter treatment (Glick and Yuspa 2005) thus conferring a selective growth advantage to these cells allowing further clonal expansion during long-term tumor promoter treatment. However, activating mutations in the Hras1 gene lead to alterations in many downstream effectors, and it is likely that mechanisms regulating proliferation, differentiation, and cellular survival as well as altered cell–cell interaction all play a role during clonal expansion of initiated cells depending on the type of promoting stimulus used (Glick and Yuspa 2005; Kemp 2005; Kim et al. 2007).

4 Mechanisms of Tumor Promotion While the initiation stage of mouse multistage skin carcinogenesis is relatively well understood in terms of molecular targets, the mechanisms underlying the promotion stage (see Fig. 1) are complex and less defined. Furthermore, genetic control of the development of tumors in this model appears to reside primarily with the tumor promotion stage (reviewed in Angel and DiGiovanni 1999). The process of tumor promotion is a whole-organ event, evidenced by the development of a dramatic increase in epidermal cell proliferation (thought to originate in the bulge region of the hair follicle and possibly other stem/progenitor cell niches) and by significant dermal changes characterized by inflammation (Boutwell 1976; Slaga 1984; Miller et al. 1993; Yuspa 1994; Fischer 1997). Molecular processes altered during these events include increased DNA synthesis, elevated ornithine decarboxylase activity, elevated growth factor and cytokine production, altered redox status, and increased eicosanoid synthesis (reviewed in Slaga et al. 1982; DiGiovanni 1992; Fischer and DiGiovanni 1995; Fischer 1997; Yuspa 1998, 2000). The effects of tumor promoters are primarily the result of promoter-induced alterations in gene expression and activation of cellsignaling molecules (Boutwell 1976; Slaga et  al. 1982; Slaga 1984; Slaga and DiGiovanni 1984; Naito and DiGiovanni 1989a, b; DiGiovanni 1992; Conti 1994; DiGiovanni 1994; Yuspa 1994; Fischer and DiGiovanni 1995; Mukhtar et al. 1995;

32

E.L. Abel and J. DiGiovanni

Slaga et al. 1996; Fischer 1997; Yuspa 1998; Yuspa 2000; Kemp 2005; Abel et al. 2009). In general, the activities of many genes that encode growth regulatory molecules are up-regulated (at the mRNA, protein, or enzymatic level) in response to exposure of mouse skin to tumor promoting stimuli. These changes are thought to stimulate a cascade of cell signaling events that alter cell proliferation and/or differentiation. Some of these changes include protein kinase C (PKC), epidermal growth factor receptor (EGFR), transforming growth factor alpha (TGFa), transforming growth factor beta 1 (TGFb), glucocorticoid receptor (GR); cytokines such as interleukin 1 (IL-1), avian erythroblastosis oncogene B 2 (ERBB2), Rous sarcoma oncogene (Src); eicosanoids such as the prostaglandins, and many others. Downstream mediators of the effects of these growth regulatory proteins and molecules include but are not limited to c-myc proto-oncogene (Myc), FBJ osteosarcoma oncogene (c-Fos), E2F transcription factor 1 (E2F-1), signal transducer and activator of transcription 3 (STAT3), transformation related protein 63 (p63), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and cyclin D1 (Ccnd1). It is clear that growth factor signaling plays an important role during the tumor promotion stage of skin carcinogenesis in this model system. One such growth factor receptor system, the erbB family and in particular the EGFR (erbB1) has been shown to play a significant role in epithelial carcinogenesis in multiple tissues (Yates et al. 1991; Nicholson et al. 2001; Yarden and Sliwkowski 2001). Elevated expression of EGFR and/or its ligands are common in many types of epithelial cancer, and such changes are an important component for maintaining the proliferative capacity of the tumor cells. The erbB family includes erbB1 (EGFR), erbB2, erbB3, and erbB4. Although all the erbB family members share similarities in primary structure, receptor activation mechanisms, and signal transduction patterns, they bind to different ligands, and ligand-dependent activation of erbB family receptors can lead to both homodimerization and heterodimerization (Pinkas-Kramarski et al. 1996). The EGFR (or erbB1) was the first member of the erbB family to be cloned and showed considerable homology to the avian erythroblastosis virus transforming protein, v-erbB (Ullrich et  al. 1984). ErbB2 is the human homolog of the neu oncogene that was initially isolated from chemically induced rat neuroblastomas (Shih et  al. 1981) and shares close homology with the EGFR (Bargmann et  al. 1986). To date, no ligand has been identified for erbB2; it can only act as part of a heterodimer with a ligand-bound receptor, often EGFR or erbB3 (Karunagaran et al. 1996; Graus-Porta et al. 1997). In contrast, erbB3 cannot generate signals in isolation because the kinase function of this receptor is impaired, thus relying on interaction with erbB2 for subsequent downstream signaling events (Citri et  al. 2003). The expression of erbB family members except erbB4 has been reported in mouse keratinocytes and human skin and keratinocytes (Xian et  al. 1997; Stoll et  al. 2001). As noted, the level of erbB4 expression appears to be very low or absent in mouse epidermis and in cultured mouse keratinocytes (Xian et al. 1997; Panchal et al. 2007). Recently, Prickett and colleagues identified erbB4 mutations in cutaneous metastatic melanoma resulting in increased kinase activity and transformation activity (Prickett et al. 2009).

2  Multistage Carcinogenesis

33

Since erbB family signaling pathways are central to regulating epithelial cell growth, it is not surprising that they are dysregulated during mouse skin carcinogenesis. Multiple EGFR ligands (e.g., TGFa, amphiregulin, and HB-EGF) are coordinately upregulated during skin tumor promotion, leading to EGFR activation (Imamoto et al. 1991; Kiguchi et al. 1995, 1998). Previous studies from our laboratory demonstrated that activation of the EGFR is a common response in mouse epidermis following treatment with diverse skin tumor promoters including TPA, OA, and Chry (Xian et al. 1995, 1997). Moreover, the EGFR is overexpressed and constitutively activated in skin tumors (papillomas and SCCs) generated via the two-stage skin carcinogenesis protocol (Rho et al. 1994). Transgenic mice overexpressing TGFa or erbB2 have epidermal hyperplasia and are highly sensitive to two-stage skin carcinogenesis (Vassar et  al. 1992; Dominey et  al. 1993; Jhappan et  al. 1994; Wang et  al. 1994; Kiguchi et al. 2000). In addition to the EGFR, both erbB2 and c-src (see below) are activated in tumor promoter treated mouse epidermis (Xian et al. 1997). In contrast, blockade of EGFR kinase inhibited TPA-mediated epidermal hyperproliferation (Xian et  al. 1997), and transgenic mice expressing a dominant negative EGFR showed resistance to two-stage skin carcinogenesis (Casanova et al. 2002). The dual EGFR/erbB2 inhibitor, GW2974 (200 ppm in the diet), inhibited skin tumor promotion in both BK5.erbB2 transgenic mice and nontransgenic mice during a two-stage skin carcinogenesis protocol (Kiguchi et al. 2010). Furthermore, increasing evidence exists demonstrating that signaling through the EGFR and/or erbB2 is rapidly activated in response to UV irradiation leading to increased keratinocyte proliferation and epidermal hyperplasia (El-Abaseri et al. 2006; Madson et  al. 2006; Han et  al. 2008). UV-induced tumorigenesis in mouse skin was blocked by topical treatment of an EGFR tyrosine kinase inhibitor and an erbB2 inhibitor, suggesting an important role of erbB family members including EGFR and erbB2 during epithelial carcinogenesis and, in particular, during tumor promotion by UV irradiation (El-Abaseri et al. 2006; Madson et al. 2009). Multiple signaling pathways downstream of growth factor receptors (such as the EGFR) play a role in skin tumor promotion. Two of these pathways will be discussed here in more detail. First, one member of the signal transducers and activators of transcription (Stats) family, Stat3, was found to be activated in mouse epidermis following treatment with different classes of tumor promoters, including TPA, OA, and Chry (Chan et al. 2004). Studies using epidermal-specific Stat3-deficient mice showed that Stat3 is required for both the initiation and promotion stages of epithelial carcinogenesis (Chan et  al. 2004). In terms of its role in tumor promotion, deletion of Stat3 significantly reduced epidermal hyperproliferation induced by TPA (Chan et al. 2004). Stat3 deletion also reduced the levels of cyclin D1, cyclin E, and c-myc, which are required to support epidermal proliferation during the early stages of tumor promotion and clonal expansion of initiated cells. Further studies using inducible Stat3-deficient mice (K5.Cre-ERT2 × Stat3fl/fl) confirmed its critical roles in tumor development during both the initiation and promotion stages of carcinogenesis (Kataoka et al. 2008). Second, several lines of evidence point to an important role for Akt signaling in skin tumor promotion. In this regard, overexpression of IGF-1 in the epidermis of

34

E.L. Abel and J. DiGiovanni

transgenic mice induces epidermal hyperplasia, enhances susceptibility to two-stage skin carcinogenesis, and leads to spontaneous skin tumor formation (DiGiovanni et al. 2000; Wilker et al. 2005). Biochemical alterations in the epidermis of these transgenic mice included elevated levels of PI3K, Akt, and cell cycle regulatory proteins (e.g., cyclin-D1). Topical application of LY294002, a specific PI3K inhibitor, not only directly inhibited these constitutive epidermal biochemical changes, but also inhibited IGF-1-mediated skin tumor promotion in a dose-dependent manner. Segrelles et al. (2002) reported sustained activation of epidermal Akt throughout two-stage carcinogenesis in mouse skin. Recent data by the same group (Segrelles et  al. 2006) and others (Affara et  al. 2004, 2006) have further confirmed the involvement of Akt-mediated cellular proliferation in mouse skin tumorigenesis. Transgenic mice overexpressing either Akt1wt or Akt1myr were developed to further evaluate the effects of elevated Akt activation on multistage epithelial carcinogenesis in mouse skin. Both BK5.Akt1wt and BK5.Akt1myr mice exhibited significantly enhanced susceptibility to two-stage skin carcinogenesis (Segrelles et  al. 2007). Of note, the BK5.Akt1myr mice were generated on a C57BL/6 genetic background. C57BL/6 mice are generally resistant to two-stage skin carcinogenesis due to a dramatic resistance to tumor promotion (Angel and DiGiovanni 1999). However, overexpression of Akt1myr was a sufficient stimulus to overcome the resistance of C57BL/6 mice to skin tumor promotion. Collectively, the data from two-stage carcinogenesis experiments using both IGF-1 and Akt transgenic mice further support the hypothesis that elevated Akt signaling plays an important role in skin carcinogenesis, especially during the tumor promotion stage. The underlying mechanisms involved in Akt-mediated enhanced susceptibility to chemically induced carcinogenesis and its role in tumor promotion remain to be fully established, although studies performed in BK5.Akt1wt and BK5.Akt1myr mice identified potential molecular targets through which Akt exerts its effects on tumorigenesis. Overexpression or constitutive activation of Akt led to enhanced epidermal proliferation that correlated with significant elevations of G1 to S phase cell cycle proteins, including cyclin D1 (Segrelles et al. 2007). In conjunction with these changes, a marked increase in signaling downstream of mTORC1 was observed suggesting that protein translation was also upregulated. In addition, GSK3b phosphorylation was significantly elevated, as were b-catenin levels. These changes, possibly in concert with alterations in survival pathways (e.g., p-Bad, p-Foxo3a), may drive both spontaneous tumor development in Akt transgenic mice as well as the increased sensitivity to skin tumor promotion observed. Activation of both the mTORC1 and GSK3b signaling pathways may be particularly important during the early stages of epithelial carcinogenesis and tumor promotion. A number of other Akt downstream pathways may also contribute to this process (Luo et al. 2003; Engelman et al. 2006; Shaw and Cantley 2006). These pathways include the survival pathway involving Bad phosphorylation, although to date elevated phosphorylation of Bad in keratinocytes has not been shown to dramatically affect keratinocyte survival during tumor initiation or promotion. Furthermore, although Foxo3a is phosphorylated in keratinocytes with elevated Akt activity, which leads to reductions in p27 levels, p27 KO mice do not display a dramatic increase in

2  Multistage Carcinogenesis

35

sensitivity to two-stage skin carcinogenesis (Philipp et al. 1999). The NFkB signaling pathway is also known to play a role during tumor promotion and skin tumor development (Budunova et al. 1999; Cooper and Bowden 2007) and is downstream of Akt. Thus, these and potentially other signaling pathways may contribute either directly or indirectly to Akt-mediated effects on tumor promotion and epithelial carcinogenesis. Considerable evidence now supports the hypothesis that oxidative stress plays an important role in TPA-mediated skin tumor promotion (reviewed in Perchellet et  al. 1995). For example, levels of reactive oxygen species (ROS) such as hydroperoxides, and lipid peroxides increased while the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GpX) decreased after TPA treatment (Solanki et al. 1981; Perchellet et  al. 1985, 1987; Perchellet and Perchellet 1989; Reiners et  al. 1991; Wei and Frenkel 1993; Jang and Pezzuto 1998; Bilodeau and Mirault 1999; Zhao et  al. 1999; Alam et al. 2002). Several studies have reported correlations between strain sensitivity to TPA skin tumor promotion and the extent of oxidant response following treatment (SSIN > SENCAR > C57BL/6) (Fischer et  al. 1986; Perchellet and Perchellet 1989; Wei et al. 1993). In addition, treatment of mice with antioxidants suppressed TPA promotion of papilloma formation (Perchellet et al. 1987; Jang and Pezzuto 1998; Alam et al. 2002; Zhaorigetu et al. 2003). Furthermore, overexpression of antioxidant enzymes in transgenic mice decreased sensitivity to TPA skin tumor promotion (Bilodeau and Mirault 1999; Zhao et al. 2001). The idea that lipid peroxidation is involved in skin tumor promotion is supported by the observation that free radical generating compounds such as BzPo and lauroyl peroxide can promote skin tumor formation in the multistage skin carcinogenesis model (Slaga et al. 1981; Klein-Szanto and Slaga 1982; Zhao et al. 2000). In addition, a single TPA treatment of SENCAR mice resulted in a significant increase in lipid peroxidation in the epidermis compared with mice treated with acetone alone (Zhao et  al. 1999). Treatment of mice with silymarin, an antioxidant compound, prior to TPA or BzPo treatment resulted in a highly significant reduction in epidermal levels of lipid peroxidation (Zhao et  al. 1999, 2000). The observations that silymarin also exerts a protective effect against UV light, TPA, OA, and BzPo skin tumor promotion (Katiyar et al. 1997; Zi et al. 1997; Lahiri-Chatterjee et al. 1999; Zhao et al. 2000) suggests that lipid peroxidation plays an important role in skin tumor promotion by diverse promoting agents. A similar correlation of reduced epidermal lipid peroxidation levels and skin tumor promotion in Swiss albino mice treated with the antioxidant Vitis vinifera extract prior to TPA treatment (Alam et al. 2002) supports this hypothesis. 4-Hydroxy-2-nonenal (4-HNE), a highly reactive but stable a,b-unsaturated aldehyde, is an end product of lipid peroxidation (Esterbauer et  al. 1991). Zhaorigetu et al. reported a correlation of 4-HNE levels and the magnitude of TPA skin tumor promotion response in ICR mice (Zhaorigetu et al. 2003). In these studies, DMBA-initiated mice co-treated with TPA and the antioxidant protein, sericin, had a reduced tumor response and reduced 4-HNE levels relative to mice treated with TPA alone suggesting that lipid peroxidation and subsequent production of 4-HNE

36

E.L. Abel and J. DiGiovanni

may be a general mechanism involved in tumor promotion. Recent studies have associated 4-HNE with signal transduction, differentiation, cell proliferation, cell cycle, and apoptosis (Paradisi et  al. 1985; Rossi et  al. 1990; Ullrich et  al. 1996; Hammer et al. 1997; Zhou et al. 1997; Ruef et al. 1998; Cheng et al. 1999; Dianzani et al. 1999; Uchida et al. 1999; Camandola et al. 2000; Liu et al. 2000; Rinaldi et al. 2000; Soh et  al. 2000; Song et  al. 2001; Negre-Salvayre et  al. 2003). Therefore, intracellular levels of 4-HNE may need to be tightly controlled. A major pathway regulating 4-HNE levels is metabolism via glutathione conjugation catalyzed by the glutathione S-transferase, Gsta4 (Alin et al. 1985; Esterbauer et al. 1991; Zimniak et al. 1994; Hubatsch et al. 1998). Following conjugation, 4-HNE can be transported across membranes via transport proteins, negating its chemical reactivity. While additional studies are required to determine if epidermal 4-HNE levels correlate with susceptibility to skin tumor promotion, these initial observations suggest that Gsta4 may play a role in tumor promotion through regulation of 4-HNE levels in the epidermis. Very recently, we have found that Gsta4, which resides on mouse distal chromosome 9, is a TPA promotion susceptibility gene (Abel et al. 2010). In addition, several polymorphisms in this gene were found to be risk alleles for human nonmelanoma skin cancer (NMSC) (see below for further discussion of Gsta4 and its potential role in skin tumor promotion).

5 Mechanisms Associated with Tumor Progression Papillomas generated during two-stage skin carcinogenesis protocols may progress to invasive SCCs (see again Fig. 1). Histopathologically, SCCs can be distinguished from papillomas by downward growth as well as loss of ordered differentiation of epidermal keratinocytes. The frequency of malignant conversion of papillomas to SCCs is dependent on genetic background (Hennings et al. 1993; Stern et al. 2002; Woodworth et al. 2004). Further progression can lead to the formation of spindle cell carcinomas, although this is a relatively rare event. During tumor progression, progressive chromosomal abnormalities occur resulting in aneuploidy after 30–40 weeks of tumor promotion (Conti et al. 1986; Aldaz et al. 1987). In the mouse skin model, the conversion of papillomas to SCCs is associated with trisomies of chromosomes 6 and 7 as well as mutations in p53 (Aldaz et al. 1989; Ruggeri et al. 1991). SCCs are highly vascularized and downward invading lesions. Characteristic changes in gene expression such as elevation in gamma-glutamyltranspeptidase, a6b4 integrin, and keratin 13 expression as well as loss of E-cadherin expression are commonly associated with tumor progression in the two-stage skin carcinogenesis model (Navarro et al. 1991; DiGiovanni 1992; Caulin et al. 1993; Chan et al. 2008). During tumor progression, signaling events that recapitulate the developmental process, epithelial-mesynchymal transition (EMT), are activated (reviewed in Thiery 2002; Kang and Massague 2004). EMT, wherein polarized epithelial cells convert to motile cells, involves a number of signaling pathways through which cell–cell adhesion is lost, the cytoskeleton is remodeled, and a migratory phenotype

2  Multistage Carcinogenesis

37

is attained. A hallmark of EMT during development and tumor progression is loss of E-cadherin expression at sites of cell–cell adhesion. Transcription factors such as Twist, Snai1 (also known as Snail), and Snai2 (also known as Slug) have been shown to repress E-cadherin expression (reviewed in Kang and Massague 2004; Yang et al. 2006; Peinado et al. 2007). Analysis of keratinocyte cell lines derived from different stages of skin carcinogenesis reveals that E-cadherin expression is negatively correlated with tumorigenicity in this model (Navarro et  al. 1991; Llorens et al. 1998). In mouse epidermal cell lines, Snail has been shown to be a strong repressor of E-cadherin expression, and stable transfection of Snail appeared to induce EMT as well as enhanced migration and invasion capacity (Cano et al. 2000). Expression of Snail was noted in invasive cell lines and tumors that had loss of E-cadherin expression. Silencing of Snail inhibited invasion in  vitro, elevated expression of E-cadherin, and reduced tumor growth in a xenograft model (Olmeda et al. 2007). Thus, regulation of cadherin-mediated cell–cell adhesion may play a role in tumor progression during two-stage skin carcinogenesis in the mouse. Another aspect of tumor progression, involves stromal invasion, which is mediated, in part, by the expression of matrix metalloproteinases (MMPs). MMPs are enzymes that function to degrade extracellular matrix (ECM) proteins and adhesion molecules to disrupt surrounding tissue architecture and cellular contacts (reviewed in Munshi and Stack 2006). MMPs have been suggested to play a role in tumor progression in the two-stage skin carcinogenesis model since a positive correlation was noted between MMP-9 expression and malignancy of mouse skin tumor derived cell lines (Papathoma et  al. 2001). Additionally, PACE4, an activator of membrane-type MMPs, has been shown to enhance progression of mouse skin tumors (Bassi et al. 2005). In addition to these signaling pathways, dysregulation of p63 isoforms, TGF-beta1, Smad3, Stat3, PTEN, c-fos, and IKKalpha has also been shown to alter progression of mouse skin cancers (Saez et  al. 1995; Koster et al. 2006; Yao et al. 2006; Park et al. 2007).

6 Role of KSCs in Multistage Skin Carcinogenesis Evidence has accumulated that KSCs are the targets for chemical carcinogenesis in mouse skin (reviewed in Kangsamaksin et al. 2007). Cells with properties of KSCs are primarily found at the base of epidermal proliferative units (EPUs) in the interfollicular epidermis and in the bulge region of the hair follicles (Cotsarelis 2006). These properties include slow cycling, label retaining properties (e.g., with 3HTdr or BrdU) (the latter referred to as label retaining cells or LRCs), and high proliferative capacity (Bickenbach and Mackenzie 1984; Morris et al. 1986). Furthermore, bulge region KSCs were found to express the hematopoietic stem cell marker, CD34 (Trempus et al. 2003). Characterization of CD34/a6 integrin positive cells from the bulge region confirmed that these cells were slow cycling, co-localized with LRCs, and had high proliferative capacity in culture (Trempus et  al. 2003; Blanpain et al. 2004). Morris et al. showed that LRCs, not pulse-labeled cells, can

38

E.L. Abel and J. DiGiovanni

undergo mitosis and remain in the basal layer (Morris et al. 1985). This implies that LRCs may have an ability for clonal expansion during skin tumor promotion. In addition, Morris et  al. demonstrated that LRCs could retain carcinogen-DNA adducts (Morris et al. 1986). Recently, Trempus et al. reported that CD34 expression in KSCs is required for TPA-induced hair follicle stem cell activation and tumor formation via the two-stage carcinogenesis protocol (Trempus et al. 2007). Collectively, these data suggest that KSCs are the primary targets for chemical carcinogenesis in mouse skin. The question of which specific stem cell populations are the primary targets remains open and is currently under active investigation. As noted above, recent studies where Stat3 was deleted specifically in bulge-region KSCs (Kim et al. 2009) have provided evidence that this population may represent the major target cell population for skin carcinogenesis.

7 Multistage Carcinogenesis and Human Cancer The applicability of multistage carcinogenesis concepts to human cancer is supported by a number of observations (Hanahan and Weinberg 2000; Lippman and Hong 2002; Braakhuis et al. 2003; Brabletz et al. 2005). Human environmental carcinogen exposure outside of occupational settings usually occurs in low doses repeatedly delivered over the course of months or years. Each individual dose alone is likely insufficient to produce cancer. Additionally, it is unlikely that a single dose of an agent is the cause of most human cancers. Evidence from both human epidemiologic as well as experimental animal studies also indicates that certain human carcinogens such as tobacco smoke and UV light exhibit a strong tumor promoting activity. Furthermore, many components of the human diet appear to influence cancer in humans through a tumor promotion type of effect. Finally, histochemical and molecular examination of tumors at various stages indicates that human cancers develop via multiple steps. It has been postulated that human cancers require as many as 4–6 sequential genetic events for their development (Hahn and Weinberg 2002). In the case of colon cancer, the accumulation of numerous genetic lesions in an increasingly aberrant subset of tumor cells reflects the multiple steps required for epithelial carcinogenesis, and these genetic changes are reflected in progressive histopathological changes from hyperplasia to adenoma to true carcinomas (Kinzler and Vogelstein 1996; Wistuba et al. 1999; Segditsas et al. 2009). The two-stage skin carcinogenesis model in mice recapitulates the features of multistage carcinogenesis in humans. As noted above, mounting evidence suggests that activating mutations within stem cell niches in hair follicle and possibly interfollicular epidermis is the first step in a cascade of events leading to tumor formation (Morris 2004; Trempus et al. 2007). Likewise, the two-stage skin carcinogenesis protocol is a good model for human cancers because humans are typically exposed to multiple low doses of both carcinogens and promoting agents (Rundhaug et al. 1997). The long latency associated with most human cancers also strongly supports a promotional component for tumor development (Pitot and Dragan 1991; Klein 2005).

2  Multistage Carcinogenesis

39

Therefore, this extensively characterized model can be utilized to study the mechanistic basis of human epithelial cancers. Numerous other human cancers, particularly those of epithelial origin, also appear to develop in a multistage progression. For instance, regions of dysplasia and carcinoma in situ appear to precede invasive carcinoma when melanoma, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, and cervical cancer lesions are examined (Clark et  al. 1969; Lazo 1999; Perez-Ordonez et  al. 2006; Koorstra et  al. 2008). Supporting the multistage nature of cancer development, genetic alterations have been shown to accumulate during tumorigenesis in these lesions. For example, during colorectal carcinogenesis, mutations in the adenomatous polyposis coli (APC) gene appear to initiate tumorigenesis (Kinzler and Vogelstein 1996). A portion of the resulting dysplastic foci further accumulates mutations in the K-ras oncogene as well as other oncogene and tumor suppressor genes, and progresses from adenomas to invasive carcinomas (Pino and Chung 2010). A similar pattern of accumulation of molecular abnormalities has been noted for squamous cell lung carcinoma and pancreatic ductal adenocarcinoma. As the severity of the histopathologic appearance of these lesions increases, the frequency of loss of heterozygosity events also increases (Wistuba et al. 1999; Koorstra et al. 2008).

8 Factors Affecting Susceptibility to Multistage Skin Carcinogenesis 8.1 Genetic Background Epidemiologic data indicate that tumor susceptibility genes have an important role in determining the risk of development of most sporadic human cancers (Peto 1980; Ponder 1990). They are high frequency, low penetrance genes that modify the response of individuals to carcinogen exposure and are involved in DNA repair, immune response, carcinogen metabolism, and cellular proliferation, differentiation, and death. Variants of tumor susceptibility genes, while not directly responsible for transformation, may increase the probability of genetic alterations in oncogenes and tumor suppressor genes which are directly involved in carcinogenesis or affect the probability of a genetically altered cell clonally expanding into a clinically relevant tumor through epigenetic mechanisms. The mapping and isolation of such low penetrance genes in humans is complicated by the multiplicity of unlinked loci involved. This, together with the absence of clear-cut familial inheritance patterns, necessitates the development of more sophisticated analytical techniques to detect linkage (Knudson 1993). Animal models of genetic susceptibility for tumor development are useful experimental tools for identifying and characterizing such genetic factors. The development of cancer in mice, as in humans, is controlled by multiple genes that modify tumor susceptibility (Ponder 1990; Demant 1992). Modifier loci that contribute to interstrain variance in the development of carcinogen-induced lung,

40

E.L. Abel and J. DiGiovanni

liver, colon, mammary, kidney, and hematopoietic cancers have been mapped by analyzing tumor development in segregating crosses of sensitive versus resistant mouse or rat strains (Hanigan et al. 1988; Moser et al. 1990; Moen et al. 1992; Su et al. 1992; Walker et al. 1992; Angel et al. 1993; Bennett et al. 1993; Dietrich et al. 1993; Gariboldi et al. 1993; Gilbert et al. 1993; Mock et al. 1993; Jacoby et al. 1994; Yeung et al. 1994; Lee et al. 1995; Fijneman et al. 1996; Moen et al. 1996; van Wezel et al. 1996; Angel et al. 2000; Angel and Richie 2002; Richie et al. 2002). The complexity of genetic control of cancer susceptibility has been demonstrated by studies of hepatocarcinogenesis in the mouse. Susceptibility in this model is determined by loci that increase or decrease sensitivity to carcinogen exposure (Bennett et al. 1993; Gariboldi et al. 1993; Lee and Drinkwater 1995). Lee and Drinkwater (1995) suggested that the highly susceptible phenotype may be determined by a combined effect of these two classes of genes. Demant and colleagues have shown that cancer susceptibility is further complicated by genic interactions (Fijneman et  al. 1996; van Wezel et  al. 1996). In one study, the effects of two colon tumor susceptibility loci (Scc4 and Scc5) had no obvious independent effects but the two loci interacted with the effect of either locus being dependent upon the genotype of the other locus (van Wezel et al. 1996). While the identification of specific genes that underlie these modifier loci has been difficult, genes that underlie several modifier loci, including Mom1 (Pla2g2a), Pctr1 (Cdkn2a), Skts13 (Aurka), Skts14 (Tgfb1), Ter (Dnd1), and Mtes1 (Sipa1), have recently been identified (MacPhee et al. 1995; Cormier et al. 1997; Zhang et al. 2001; Ewart-Toland et al. 2003; Zhang et al. 2003; Park et al. 2005; Youngren et al. 2005; Mao et al. 2006), and variants that affect both function and expression have been reported. Genetic differences in susceptibility to multistage skin carcinogenesis have been known for many years (Slaga 1984; Naito and DiGiovanni 1989a, b; DiGiovanni 1992; Angel and DiGiovanni 1999). Early work by Boutwell showed that the response of the skin to multistage carcinogenesis is strongly influenced by the genetic background of the host (Boutwell 1964). For example, C57BL/6 mice are quite refractory to initiation–promotion protocols using phorbol ester tumor promoters while SENCAR mice are highly sensitive even with low doses of initiators and promoters (Reiners et al. 1984; Naito et al. 1988; DiGiovanni et al. 1992). Although the existence of genetic differences in susceptibility to mouse skin carcinogenesis has been clearly documented, the mechanisms involved in these differences are not fully understood. Early studies focused on differences in the metabolism of carcinogens and DNA binding as a possible source of variability among mouse strains (Reiners et al. 1984; Naito et al. 1988; DiGiovanni et al. 1992). However, studies carried out by our laboratory as well as others showed that although some differences among strains in susceptibility to skin carcinogenesis can be attributed to differences in initiation events, the major contribution to susceptibility appears to be at the level of tumor promotion (Fischer et al. 1987b; Naito et al. 1988; Naito and DiGiovanni 1989a, b; Gimenez-Conti et al. 1992; Nagase et al. 1995; Stern et al. 1995; Angel et al. 1997; reviewed in DiGiovanni 1997; Mock et al. 1998; Angel and DiGiovanni 1999; Nagase et al. 1999; Coghlan et al. 2000; Angel et al. 2001; Stern et al. 2002; Angel et al. 2003). Furthermore, the observation that the inbred SENCAR line, SSIN,

2  Multistage Carcinogenesis

41

is highly susceptible to TPA tumor promotion but refractory to progression (Gimenez-Conti et al. 1992) while other inbred SENCAR lines are highly susceptible to both promotion and progression (Hennings et al. 1997; Coghlan et al. 2000) demonstrates that different subsets of genes modify the response to TPA promotion and tumor progression in this model. A locus that modifies skin tumor progression has been mapped to chr 14 in genetic crosses of SSIN with the progression sensitive inbred SENCAR line, SENCAR B/Pt (Stern et al. 2002). Numerous research groups have expended a significant amount of effort in an attempt to understand the biochemical basis of differential sensitivity to skin tumor promotion among various mouse stocks and strains (Wheldrake et al. 1982; Garte et al. 1985; Fischer et al. 1986; Lewis and Adams 1986; Fischer et al. 1987a; Cope et  al. 1988; Fischer et  al. 1988; Hirabayashi et  al. 1988; DiGiovanni 1989; Furstenberger et al. 1989; Mills and Smart 1989; Hirabayashi et al. 1990; Imamoto et al. 1992, 1993; Wei et al. 1993; DiGiovanni 1997; Kiguchi et al. 1997; Angel and DiGiovanni 1999; Guo et al. 1999). The results from many of these studies have been contradictory or inconclusive and the mechanistic basis for these genetic differences in response to skin tumor promotion by TPA remains to be determined. More recently, our laboratory, as well as others, has used a genetic approach to identify genes that modify the responsiveness to TPA. These studies have shown that susceptibility to skin tumor promotion is a multigenic trait and loci have been mapped to more than ten chromosomal regions affecting latency, tumor number, tumor size, and survivability using genetic crosses of BALB/cANPt with SENCARA/ Pt (Mock et al. 1998), NIH/Ola with Mus spretus (Nagase et al. 1995, 1999), PWK with FVB (Fujiwara et  al. 2007), Car-R with Car-S (Peissel et  al. 2001), and C57BL/6 with DBA/2 mice (Angel et al. 1997; Angel and DiGiovanni 1999; Angel et al. 2001, 2003). We mapped TPA promotion susceptibility loci to chr 1 (Psl3), 2 (Psl2), 9 (Psl1), and 19 (Psl4) in genetic crosses of C57BL/6 with DBA/2 mice (Angel et al. 2003). Additional susceptibility loci have been tentatively mapped to other genetic loci (Angel et al. 2003). Analysis of interval specific congenic mouse strains suggested that at least two genes that modify TPA promotion susceptibility map to distal chr 9 (Psl1). These loci have been designated as Psl1.1 and Psl1.2 (Abel et al. 2010). In further studies from our laboratory, Gsta4 has been identified as a candidate tumor promotion susceptibility gene underlying the effect of Psl1 on skin tumor promotion. Global gene expression analyses revealed that at least 44 genes were differentially expressed in the epidermis of C57BL/6 versus DBA/2 mice following topical application of 3.4  nmol TPA using a multiple treatment regimen (Riggs et al. 2005). Of these genes, Gsta4, which maps to Psl1.2, showed the most dramatic difference in expression between C57BL/6 and DBA/2 (Riggs et al. 2005). Gsta4 deficient mice were subsequently analyzed for susceptibility to skin tumor promotion by TPA and were found to be more sensitive than wild-type C57BL/6 mice (Abel et al. 2010). In addition, single nucleotide polymorphisms (SNPs) in GSTA4 from individuals in a case-control study of NMSC were analyzed. Inheritance of polymorphisms in GSTA4 was associated with risk of NMSC in humans (Abel et al. 2010). Thus, Gsta4/GSTA4 appears to be a novel susceptibility gene for NMSC that affects

42

E.L. Abel and J. DiGiovanni

risk of skin tumor development in both mice and humans. Data from the mouse studies indicate that this gene plays a role primarily during the promotion stage of skin tumor development.

8.2 Diet/Nutritional Status Dietary energy balance refers to the balance between caloric intake and energy expenditure (Patel et  al. 2004). Epidemiological studies suggest chronic positive energy balance, which can lead to obesity, heightens the risk of developing multiple cancers, as well as increases the risk of death from a range of cancer types (Calle et  al. 2003; Hedley et  al. 2004). Despite the increasing prevalence of obesity (Hedley et  al. 2004) and the established obesity–cancer link, the mechanisms underlying this relationship are poorly understood. Animal models previously utilized to examine the effect of obesity on type-2 diabetes and several other chronic diseases have been more recently used to examine the impact of obesity on carcinogenesis (Yakar et al. 2006). In contrast to the poorly understood obesity– cancer link, calorie restriction (CR), or negative energy balance, is arguably the most potent dietary-based intervention for preventing carcinogenesis in animal models (Hursting et al. 2003). CR has been shown to inhibit formation of spontaneous neoplasias in experimental model systems, including tumors arising in several models with alterations in the p53 or Wnt pathways (Patel et al. 2004; Hursting et al. 2005). Furthermore, CR suppresses chemically induced carcinogenesis in rodents, including the mouse two-stage skin carcinogenesis model (Boutwell 1964; Stewart et al. 2005). A number of studies have examined the effect of CR on tumorigenesis using the two-stage skin carcinogenesis model, which allows for examination of the effect of diet manipulation on both the initiation and promotion stages. As already mentioned, CR has consistently been shown to reduce skin tumorigenesis. Specifically, CR during promotion leads to a significant reduction in tumor incidence, multiplicity, and papilloma size (Boutwell 1964; Birt et al. 1991, 1993). Birt et al. (Stewart et al. 2005), as well as Pashko and Schwartz (1992) reported that adrenalectomy reversed most of the inhibition of skin carcinogenesis associated with 40% CR, while restoration of circulating corticosterone levels in adrenalectomized mice partially restored the CR inhibition. Thompson and colleagues showed similar effects in a rat mammary tumor model, as well as direct cytostatic effects of corticosterone in  vitro (Zhu et  al. 1998; Jiang et  al. 2002). However, the majority of these CR studies used fairly severe degrees of calorie restriction (i.e., 40%). In recent studies (Moore et al. 2008a), we examined the impact of dietary energy balance manipulation on steady-state signaling in multiple epithelial tissues in the mouse, with a focus on the Akt/mTOR pathway. For these experiments, male FVB/N and C57BL/6, and female ICR mice (all commonly used strains in cancer studies) were maintained on either a control (10  Kcal% fat) diet, a diet induced obesity (DIO: 60  Kcal% fat) regimen, or a 30% CR regimen for 17  weeks.

2  Multistage Carcinogenesis

43

As expected, all mice maintained on the DIO regimen exhibited a significantly increased level of circulating IGF-1, while CR mice exhibited a significant reduction in levels of circulating IGF-1, as compared to the control. Western blot analyses were performed to determine the effect of dietary manipulation on Akt and mTOR activation in the epidermis, liver, and dorsolateral prostate. The DIO diet enhanced, while CR inhibited, activation of Akt and mTOR, regardless of epithelial tissue or  genetic background. Further analyses demonstrated that activation of AMPactivated protein kinase (AMPK) was modulated by dietary energy balance manipulation in the liver but not in either the epidermis or dorsolateral prostate, suggesting the response of AMPK to positive or negative energy balance conditions may be tissue-dependent. Western blot analyses of epidermal extracts taken from these mice revealed reduced activation of both the IGF-1 and epidermal growth factor (EGF) receptors in CR mice, compared to control mice or mice maintained on the DIO diet. Taken together, these findings suggested that dietary energy balance manipulation modulates growth factor signaling in mouse epithelial tissues, and especially, the Akt/mTOR signaling pathway. As noted above, these signaling pathways play an important role during the tumor promotion stage. In additional studies, liver IGF-1 deficient (LID) mice, which have a 75% reduction in serum IGF-1, were utilized to evaluate the effect of reduced circulating IGF-1 on multistage skin carcinogenesis and tumor promotion (Moore et  al. 2008b). LID mice were subjected to the standard two-stage skin carcinogenesis protocol utilizing DMBA as the initiator and TPA as the promoter. A significant reduction in epidermal hyperplasia and labeling index was observed in LID mice treated with either vehicle or TPA. Furthermore, a significant decrease in both tumor incidence and tumor multiplicity was also observed in LID mice undergoing two-stage skin carcinogenesis relative to wild-type littermates. Western blot analyses of epidermal extracts revealed reduced activation of both the EGF and IGF-1 receptors in response to TPA treatment in LID mice. In addition, reduced activation of both Akt and mTOR was observed in LID mice following TPA treatment relative to wild-type controls. Signaling downstream of mTOR was also reduced. These data suggest a possible mechanism whereby reduced circulating IGF-1 leads to attenuated activation of the Akt and mTOR signaling pathways, and thus diminished epidermal response to tumor promotion and ultimately reduced susceptibility to two-stage skin carcinogenesis. The current data also suggest that reduced circulating IGF-1 levels that occur as a result of CR may lead to inhibition of skin tumorigenesis, at least in part, by a similar mechanism.

9 Multistage Carcinogenesis and Cancer Prevention The ultimate goal of carcinogenesis research in animal models and human tissues is to elucidate the processes involved in the induction of human cancer so that interventions may be developed to prevent the disease, either in the general population or in  susceptible subpopulations. Understanding the multistage nature of chemically

44

E.L. Abel and J. DiGiovanni

induced cancer has led to the discovery of stage-specific and mechanism-based interventions. Table 1 lists a number of agents that target specific stages of the carcinogenic process. Given the long time frame and reversibility of the tumor promotion process many agents that target processes involved in this stage of carcinogenesis are currently under investigation. Nevertheless, promising agents that target the tumor initiation step have also been identified. As shown in Table 1, possible ways of interfering with tumor initiation events include (1) modifying carcinogen activation by inhibiting enzymes responsible for that activation or by direct scavenging of DNA-reactive electrophiles and free radicals; (2) enhancing carcinogen detoxification processes by altering the activity of the detoxifying enzymes; and (3) modulating certain DNA repair processes. Possible ways of blocking the processes involved in the promotion and progression Table 1  Examples of dietary factors and chemopreventive agents that target specific stages of the carcinogenic process Carcinogenesis stage Prevention strategy Preventive agents Initiation Inhibit activation Coumarins, ellagic acid, epigallocatechin gallate (EGCG), genistein, indole-3-carbinol, phenyl-isothiocyanate (PEITC), resveratrol, selenium Scavenge electrophiles EGCG, ellagic acid Enhance carcinogen CDDO, diallyl sulfide, EGCG, detoxification N-acetylcysteine, oltipraz, PEITC, resveratrol Enhance DNA repair Calorie restriction (CR), EGCG, pathways selenium Antioxidants (e.g., a-tocopherol, Scavenge reactive Promotion/progression oxygen species ascorbic acid, EGCG), CR, selenium Alter gene expression CR, dehydroepiandrosterone (DHEA), fluasterone, genistein, monoterpenes (i.e. d-limonene), retinoids (all-trans retinoic acid, fenretinide), Decrease inflammation Antihistamines, CR, DHEA, fluasterone, nonsteroidal antiinflammatory drugs (e.g., suldinac, aspirin), resveratrol, selective COX-2 inhibitors (e.g., celecoxib) Inhibit proliferation CR, DHEA, difluoromethyl-ornithine, erlotinib, finasteride, fluasterone, genistein, GW2974, LY29004, perilyl alcohol, RAD-001, repamycin, retinoids, selenium, tamoxifen Induce differentiation Calcium, retinoids, sodium butyrate Induce apoptosis DHEA, fenretinide, fluasterone, HDAC inhibitors, sodium butyrate

2  Multistage Carcinogenesis

45

stages of carcinogenesis include (1) scavenging of ROS; (2) altering the expression of genes; (3) decreasing inflammation; (4) blocking specific intracellular signaling pathways; (5) inducing differentiation; and (6) inducing apoptosis. Some of the most promising agents either have or are currently undergoing clinical trials (Kelloff et al. 2006; Lippman and Hawk 2009; William et al. 2009).

10 Conclusions and Perspectives The major stages of carcinogenesis have been deduced over the past 50  years, primarily from animal model studies (particularly in the mouse skin); these stages are termed initiation, promotion, and progression and are shown in Fig. 1. Tumor initiation begins when DNA in a cell or population of target cells is damaged by exposure to exogenous or endogenous carcinogens. If this damage is not repaired, it can lead to mutations in a critical target gene. The responsiveness of initiated cells to their microenvironment gives them a growth advantage relative to normal cells under certain conditions. In the classic two-stage carcinogenesis system in the mouse skin, a low dose of DMBA induces a mutation in Hras1 that does not give rise to tumors over the lifespan of the mouse unless a tumor promoter, such as TPA, is repeatedly applied. The tumor promotion stage is characterized by selective clonal expansion of the initiated cells, a result of the altered expression of genes whose products are associated with hyperproliferation, tissue remodeling, and inflammation. During tumor progression, preneoplastic cells undergo malignant transformation through a process of selection that is facilitated by progressive genomic instability and altered gene expression. The classic view of experimental carcinogenesis, in which tumor initiation is followed by tumor promotion and progression in a sequential fashion, remains conceptually important to experimental carcinogenesis research. However, while the processes involved in each stage of experimental carcinogenesis also appear to be involved in human carcinogenesis, the temporal nature of initiation, promotion, and progression events is more complex. For instance, multiple mutational events are involved in the formation of human tumors (Fearon and Vogelstein 1990; Sugimura 1992; Hahn and Weinberg 2002). Humans are generally exposed to mixtures of agents that can simultaneously act at different stages of the carcinogenesis process, and it has become clear that promotional events, which frequently increase cellular proliferation or decrease apoptosis, can influence subsequent initiation events. It is also increasingly apparent that an individual’s genetic background can dramatically affect his or her susceptibility to a carcinogenic exposure. Although much work has focused on genes involved in the initiation process, studies in animal models suggest that the major modifier genes are likely to be those that affect promotion and progression-related events. Thus, human cancer rather than occurring in three discrete stages in a predictable order is best characterized as an accumulation of alterations in genes regulating cellular homeostasis, such as oncogenes, tumor suppressor genes, apoptosis genes, and DNA repair genes.

46

E.L. Abel and J. DiGiovanni

An understanding of the multistage nature of carcinogenesis has led to the discovery of mechanism-based inhibitors that target events associated with specific stages (see again Table  1). Further study of the cellular, biochemical, and molecular mechanisms associated carcinogenesis induced by chemicals as well as other types of carcinogens will lead to further identification of effective strategies for cancer prevention.

References Abel, E.L., Angel, J.M, et al. (2009). Nat Protoc 4: 1350–1362. Abel, E.L., Angel, J.M, et al. (2010). J Natl Cancer Inst 102: 1663–1675. Affara, N.I., Schanbacher, B.L., et al. (2004). Anticancer Res 24: 2773–2781. Affara, N.I., Trempus, C.S., et al. (2006). Anticancer Res 26: 2805–2820. Alam, A., Khan, N., et al. (2002). Pharmacol Res 46: 557–564. Aldaz, C.M. and Conti, C.J. (1989). Carcinog Compr Surv 11: 227–242. Aldaz, C.M., Conti, C.J., et al. (1987). Proc Natl Acad Sci USA 84: 2029–2032. Aldaz, C., Trono, M.D., et al. (1989). Mol Carcinog 2: 22–26. Alin, P., Danielson, U.H., et al. (1985). FEBS Lett 179: 267–270. Angel, J.M. and DiGiovanni, J. (1999). Prog Exp Tumor Res 35: 143–157. Angel, J.M. and Richie, E.R. (2002). Mol Carcinog 33: 105–112. Angel, J.M., Morizot, D.C., et al. (1993). Mol Carcinog 7: 151–156. Angel, J.M., Beltran, L., et al. (1997). Mol Carcinog 20: 162–167. Angel, J.M., Popova, N., et al. (2000). Mol Carcinog 27: 47–54. Angel, J.M., Caballero, M., et al. (2001). Mol Carcinog 32: 169–175. Angel, J.M., Caballero, M., et al. (2003). Cancer Res 63: 2747–2751. Bailleul, B., Surani, M.A., et al. (1990). Cell 62: 697–708. Balmain, A., Ramsden, M., et al. (1984). Nature 307: 658–660. Bargmann, C., Hung, M., et al. (1986). Nature 319: 226–230. Bassi, D.E., Lopez De Cicco, R., et al. (2005). Cancer Res 65: 7310–7319. Bennett, L.M., Winkler, M.L., et al. (1993). Proc Am Assoc Cancer Res 34: 144. Bickenbach, J.R. and Mackenzie, I.C. (1984). J Invest Dermatol 82: 618–622. Bilodeau, J.F. and Mirault, M.E. (1999). Int J Cancer 80: 863–867. Birt, D.F., Pelling, J.C., et al. (1991). Cancer Res 51: 1851–1854. Birt, D.F., Pinch, H.J., et al. (1993). Cancer Res 53: 27–31. Blanpain, C., Lowry, W.E., et al. (2004). Cell 118: 635–648. Boutwell, R. (1964). Prog Exp Tumor Res 4: 207–250. Boutwell, R. (1974). CRC Crit Rev Toxicol 2: 419–443. Boutwell, R. (1976). Cancer Res 36: 2631–2635. Braakhuis, B.J., Tabor, M.P., et al. (2003). Cancer Res 63: 1727–1730. Brabletz, T., Jung, A., et al. (2005). Nat Rev Cancer 5: 744–749. Brown, K., Buchmann, A., et al. (1990). Proc Natl Acad Sci USA 87: 538–542. Budunova, I.V., Perez, P., et al. (1999). Oncogene 18: 7423–7431. Calle, E.E., Rodriguez, C., et al. (2003). N Engl J Med 348: 1625–1638. Camandola, S., Poli, G., et al. (2000). J Neurochem 74: 159–168. Cano, A., Perez-Moreno, M.A., et al. (2000). Nat Cell Biol 2: 76–83. Casanova, M.L., Larcher, F., et al. (2002). Cancer Res 62: 3402–3407. Caulin, C., Bauluz, C., et al. (1993). Exp Cell Res 204: 11–21. Chan, K.S., Sano, S., et al. (2004). J Clin Invest 114: 720–728. Chan, K.S., Sano, S., et al. (2008). Oncogene 27: 1087–1094. Cheng, J.Z., Singhal, S.S., et al. (1999). Arch Biochem Biophys 372: 29–36.

2  Multistage Carcinogenesis

47

Citri, A., Skaria, K.B., et al. (2003). Exp Cell Res 284: 54–65. Clark, W.H., Jr., From, L., et al. (1969). Cancer Res 29: 705–727. Coghlan, L.G., Gimenez-Conti, I., et al. (2000). Carcinogenesis 21: 641–646. Conti, C.J. (1994). The mouse skin as a model for chemical carcinogenesis. In: J.P. Sundberg (ed) Handbook of Mouse Mutations with Skin and Hair Abnormalities. Boca Raton, FL, CRC Press: 39–55. Conti, C.J., Aldaz, C.M., et al. (1986). Carcinogenesis 7: 1845–1848. Cooper, S.J. and Bowden, G.T. (2007). Curr Cancer Drug Targets 7: 325–334. Cope, F., Wagner, F., et al. (1988). Mol Carcinog 1: 116–124. Cormier, R.T., Hong, K.H., et al. (1997). Nat Genet 17: 88–91. Cotsarelis, G. (2006). J Invest Dermatol 126: 1459–1468. Demant, P. (1992). Semin Cancer Biol 3: 159–166. Dianzani, M.U., Barrera, G., et al. (1999). Acta Biochim Pol 46: 61–75. Dietrich, W.F., Lander, E.S., et al. (1993). Cell 75: 631–639. DiGiovanni, J. (1989). Metabolism of polycyclic aromatic bydrocarbons and phorbol esters by mouse skin: relevance to mechanism of action and trans-species/strain carcinogenesis. In: T.J. Slaga, D. Stevenson and A.J.P. Klein-Szanto et  al. (eds) Progress in Clinical and Biological Research Skin Carcinogenesis: Research Directions Mechanisms and Human Relevance. New York, Alan R. Liss. Vol. 298: 167–199. DiGiovanni, J. (1992). Pharmacol Ther 54: 63–128. DiGiovannni, J. (1994). Multistage skin carcinogenesis. In: J. Ward and M.P. Waalkes (eds) Carcinogenesis, Target Organ Toxicology Series. New York, Raven Press: 265–299. DiGiovanni, J. (1997). Genetic determinants of cancer susceptibility. In: G.T. Bowden and S.M. Fischer (eds) Comprehensive Toxicology, Carcinogens and Anticarcinogens. New York, Pergamon Press Vol. 10: 425–451. DiGiovanni, J., Imamoto, A., et al. (1992). Carcinogenesis 13: 525–531. DiGiovanni, J., Bol, D.K., et al. (2000). Cancer Res 60: 1561–1570. Dominey, A.M., Wang, X.J., et al. (1993). Cell Growth Differ 4: 1071–1082. El-Abaseri, T.B., Putta, S., et al. (2006). Carcinogenesis 27: 225–231. Engelman, J.A., Luo, J., et al. (2006). Nat Rev Genet 7: 606–619. Esterbauer, H., Schaur, R.J., et al. (1991). Free Radic Biol Med 11: 81–128. Ewart-Toland, A., Briassouli, P., et al. (2003). Nat Genet 34: 403–412. Fearon, E.R. and Vogelstein, B. (1990). Cell 61: 759–767. Fijneman, R.J., de Vries, S.S., et al. (1996). Nat Genet 14: 465–467. Finch, J.S., Albino, H.E., et al. (1996). Carcinogenesis 17: 2551–2557. Fischer, S.M. (1997). Cellular and molecular mechanisms of tumor promotion. In: G.T. Bowden and S.M. Fischer (eds) Comprehensive Toxicology. New York, Elsevier Science Ltd. Vol. 12: 349–381. Fischer, S.M. and DiGiovanni, J. (1995). Cancer Bull 47: 456–463. Fischer, S.M., Baldwin, J.K., et al. (1986). Carcinogenesis 7: 915–918. Fischer, S., Baldwin, J., et al. (1987a). Carcinogenesis 8: 1521–1524. Fischer, S.M., O’Connell, J.F., et al. (1987b). Carcinogenesis 8: 421–424. Fischer, S., Baldwin, J., et al. (1988). Cancer Res 48: 658–664. Fujiwara, K., Igarashi, J., et al. (2007). BMC Genet 8: 39. Furstenberger, G., Rogers, M., et al. (1989). Int J Cancer 43: 915–921. Gariboldi, M., Manenti, G., et al. (1993). Cancer Res 53: 209–211. Garte, S., Edinger, F., et al. (1985). Cancer Lett 29: 215–221. Gerdes, M.J. and Yuspa, S.H. (2005). Stem Cell Rev 1: 225–231. Gilbert, D.J., Neumann, P.E., et al. (1993). J Virol 67: 2083–2090. Gimenez-Conti, I.B., Bianchi, A.B., et al. (1992). Cancer Res 52: 3432–3435. Glick, A.B. and Yuspa, S.H. (2005). Semin Cancer Biol 15: 75–83. Graus-Porta, D., Beerli, R.R., et al. (1997). EMBO J 16: 1647–1655. Guo, Y., Zhao, J., et al. (1999). Mol Carcinog 26: 32–36. Hahn, W.C. and Weinberg, R.A. (2002). Nat Rev Cancer 2: 331–341.

48

E.L. Abel and J. DiGiovanni

Hammer, A., Ferro, M., et al. (1997). Free Radic Biol Med 23: 26–33. Han, C.Y., Lim, S.C., et al. (2008). Cancer Sci 99: 502–509. Hanahan, D. and Weinberg, R.A. (2000). Cell 100: 57–70. Hanigan, M.H., Kemp, C.J. et al. (1988). Carcinogenesis 9: 885–890. Hedley, A.A., Ogden, C.L., et al. (2004). JAMA 291: 2847–2850. Hennings, H., Glick, A.B., et al. (1993). Carcinogenesis 14: 2353–2358. Hennings, H., Lowry, D.T., et al. (1997). Mol Carcinog 20: 143–150. Hirabayashi, N., Naito, M., et al. (1988). Carcinogenesis 9: 2215–2220. Hirabayashi, N., Warren, B., et al. (1990). Mol Carcinog 3: 171–180. Hubatsch, I., Ridderstrom, M., et al. (1998). Biochem J 330: 175–179. Hursting, S.D., Lavigne, J.A., et al. (2003). Annu Rev Med 54: 131–152. Hursting, S.D., Nunez, N.P., et al. (2005). Mutat Res 576: 80–92. Imamoto, A., Beltran, L.M., et al. (1991). Mol Carcinog 4: 52–60. Imamoto, A., Beltran, L.M., et al. (1992). Carcinogenesis 13: 177–182. Imamoto, A., Wang, X.-J., et al. (1993). Carcinogenesis 14: 719–724. Ise, K., Nakamura, K., et al. (2000). Oncogene 19: 2951–2956. Jacoby, R.F., Hohman, C., et al. (1994). Genomics 22: 381–387. Jang, M. and Pezzuto, J.M. (1998). Cancer Lett 134: 81–89. Jhappan, C., Takayama, H., et al. (1994). Cell Growth Differ 5: 385–394. Jiang, W., Zhu, Z., et al. (2002). Cancer Res 62: 5280–5287. Kang, Y. and Massague, J. (2004). Cell 118: 277–279. Kangsamaksin, T., Park, H.J., et al. (2007). Mol Carcinog 46: 579–584. Karunagaran, D., Tzahar, E., et al. (1996). EMBO J 15: 254–264. Kataoka, K., Kim, D.J., et al. (2008). Carcinogenesis 29: 1108–1114. Katiyar, S.K., Korman, N.J., et al. (1997). J Natl Cancer Inst 89: 556–566. Kelloff, G.J., Lippman, S.M., et al. (2006). Clin Cancer Res 12: 3661–3697. Kemp, C.J. (2005). Semin Cancer Biol 15: 460–473. Kiguchi, K., Beltran, L.M., et al. (1995). Mol Carcinog 12: 225–235. Kiguchi, K., Beltran, L., et al. (1997). J Invest Dermatol 108: 784–791. Kiguchi, K., Beltran, L., et al. (1998). Mol Carcinog 22: 73–83. Kiguchi, K., Bol, D., et al. (2000). Oncogene 19: 4243–4254. Kiguchi, K., Kitamura, T., et al. (2010). Cancer Prev Res 3: 940–952. Kim, D.J., Chan, K.S., et al. (2007). Mol Carcinog 46: 725–731. Kim, D.J., Kataoka, K., et al. (2009). Cancer Res 69: 7587–7594. Kinzler, K.W. and Vogelstein, B. (1996). Cell 87: 159–170. Klein, E.A. (2005). Nat Clin Pract Urol 2: 24–31. Klein-Szanto, A.J.P. (1989). Pathology of human and experimental skin tumors. In: C.J. Conti, T.J. Slaga and A.J.P. Klein-Szanto (eds) Skin Tumors: Experimental and Clinical Aspects. New York City, Raven Press: 19–53. Klein-Szanto, A.J. and Slaga, T.J. (1982). J Invest Dermatol 79: 30–34. Knudson, A.G. (1993). Proc Natl Acad Sci USA 90: 10914–10921. Koorstra, J.B., Hustinx, S.R., et al. (2008). Pancreatology 8: 110–125. Koster, M.I., Lu, S.L., et al. (2006). Cancer Res 66: 3981–3986. Lahiri-Chatterjee, M., Katiyar, S.K., et al. (1999). Cancer Res 59: 622–632. Lazo, P.A. (1999). Br J Cancer 80: 2008–2018. Lee, G.H. and Drinkwater, N.R. (1995). Carcinogenesis 16: 1993–1996. Lee, G.H., Bennett, L.M., et al. (1995). Genetics 139: 387–395. Lewis, J.G. and Adams, D.O. (1986). Cancer Res 46: 5696–5700. Lippman, S.M. and Hawk, E.T. (2009). Cancer Res 69: 5269–5284. Lippman, S.M. and Hong, W.K. (2002). Cancer Res 62: 5119–5125. Liu, W., Kato, M., et al. (2000). J Cell Sci 113: 635–641. Llorens, A., Rodrigo, I., et al. (1998). Lab Invest 78: 1131–1142. Luo, J., Manning, B.D., et al. (2003). Cancer Cell 4: 257–262.

2  Multistage Carcinogenesis

49

MacPhee, M., Chepenik, K.P., et al. (1995). Cell 81: 957–966. Madson, J.G., Lynch, D.T., et al. (2006). Am J Pathol 169: 1402–1414. Madson, J.G., Lynch, D.T., et al. (2009). Am J Pathol 174: 2357–2366. Mao, J.H., Saunier, E.F., et al. (2006). Proc Natl Acad Sci USA 103: 8125–8130. Miller, S.J., Wei, Z.G., et al. (1993). J Invest Dermatol 101: 591–594. Mills, K. and Smart, R. (1989). Carcinogenesis 10: 833–838. Mock, B.A., Krall, M.M., et al. (1993). Proc Natl Acad Sci USA 90: 9499–9503. Mock, B.A., Lowry, D.T., et al. (1998). Carcinogenesis 19: 1109–1115. Moen, C.J., Snoek, M., et al. (1992). Oncogene 7: 563–566. Moen, C.J., Groot, P.C., et al. (1996). Proc Natl Acad Sci USA 93: 1082–1086. Moore, T., Beltran, L., et al. (2008a). Cancer Prev Res 1: 65–76. Moore, T., Carbajal, S., et al. (2008b). Cancer Res 68: 3680–3688. Morris, R.J. (2004). Differentiation 72: 381–386. Morris, R.J., Fischer, S.M., et al. (1985). J Invest Dermatol 84: 277–281. Morris, R.J., Fischer, S.M., et al. (1986). Cancer Res 46: 3061–3066. Moser, A.R., Pitot, H.C., et al. (1990). Science 247: 322–324. Mukhtar, H., Mercurio, M.G., Agarwal, R. (1995). Murine skin carcinogenesis: relevance to humans. In: H. Mukhtar (ed) Skin Cancer: Mechanisms and Human Relevance. Boca Raton, FL, CRC Press. Munshi, H.G. and Stack, M.S. (2006). Cancer Metastasis Rev 25: 45–56. Nagase, H., Bryson, S., et al. (1995). Nat Genet 10: 424–429. Nagase, H., Mao, J.H., et al. (1999). Proc Natl Acad Sci USA 96: 15032–15037. Naito, M. and DiGiovanni, J. (1989a). Carcinog Compr Surv 11: 187–212. Naito, M. and DiGiovanni, J. (1989b). Genetic background and development of skin tumors. In: C.J. Conti, A.J.P. Klein-Szanto, and T.J. Slaga (eds) Carcinogenesis, Skin Tumors, Experimental and Clinical Aspects. New York, NY, Raven Press: 187–212. Naito, M., Chenicek, K.J., et al. (1988). Carcinogenesis 9: 639–645. Navarro, P., Gomez, M., et al. (1991). J Cell Biol 115: 517–533. Negre-Salvayre, A., Vieira, O., et al. (2003). Mol Aspects Med 24: 251–261. Nelson, M.A., Futscher, B.W., et al. (1992). Proc Natl Acad Sci USA 89: 6398–6402. Nicholson, R.I., Hutcheson, I.R., et al. (2001). Endocr Relat Cancer 8: 175–182. Olmeda, D., Jorda, M., et al. (2007). Oncogene 26: 1862–1874. Panchal, H., Wansbury, O., et al. (2007). BMC Dev Biol 7: 105. Papathoma, A.S., Zoumpourlis, V., et al. (2001). Mol Carcinog 31: 74–82. Paradisi, L., Panagini, C., et al. (1985). Chem Biol Interact 53: 209–217. Park, Y.G., Zhao, X., et al. (2005). Nat Genet 37: 1055–1062. Park, E., Zhu, F., et al. (2007). Cancer Res 67: 9158–9168. Pashko, L.L. and Schwartz, A.G. (1992). Carcinogenesis 13: 1925–1928. Patel, A.C., Nunez, N.P., et al. (2004). J Nutr 134: 3394S–3398S. Peinado, H., Olmeda, D., et al. (2007). Nat Rev Cancer 7: 415–428. Peissel, B., Zaffaroni, D., et al. (2001). Mamm Genome 12: 291–294. Perchellet, E.M. and Perchellet, J.P. (1989). Cancer Res 49: 6193–6201. Perchellet, J.P., Perchellet, E.M., et al. (1985). Cancer Lett 26: 283–293. Perchellet, J.P., Abney, N.L., et al. (1987). Cancer Res 47: 6302–6309. Perchellet, J.-P. and Perchellet, E., et al. (1995). Oxidant stress and multistage skin carcinogenesis. In: H. Mukhtar (ed) Skin Cancer: Mechanisms and Human Relevance. Boca Raton, FL, CRC Press: 145–196. Perez-Ordonez, B., Beauchemin, M., et al. (2006). J Clin Pathol 59: 445–453. Peto, J. (1980). Predisposition to cancer. In: J. Cairns, J.L. Lyon and M. Skolnick (eds) Cancer Incidence in Defined Populations (Banbury Report 4). Cold Spring Harbor, Cold Spring Harbor Laboratory Press: 203–213. Philipp, J., Vo, K., et al. (1999). Oncogene 18: 4689–4698. Pierceall, W.E., Kripke, M.L., et al. (1992). Cancer Res 52: 3946–3951.

50

E.L. Abel and J. DiGiovanni

Pinkas-Kramarski, R., Soussan, L., et al. (1996). EMBO J 15: 2452–2467. Pino, M.S. and Chung, D.C. (2010). Gastroenterology 138: 2059–2072. Pitot, H.C. and Dragan, Y.P. (1991). FASEB J 5: 2280–2286. Ponder, B.A. (1990). Trends Genet 6: 213–218. Prickett, T.D., Agrawal, N.S., et al. (2009). Nat Genet 41: 1127–1132. Rehman, I., Lowry, D.T., et al. (2000). Mol Carcinog 27: 298–307. Reiners, J.J., Jr., Nesnow, S., et al. (1984). Carcinogenesis 5: 301–307. Reiners, J.J., Jr., Thai, G., et al. (1991). Carcinogenesis 12: 2337–2343. Rho, O., Beltran, L.M., et al. (1994). Mol Carcinog 11: 19–28. Richie, E.R., Schumacher, A., et al. (2002). Oncogene 21: 299–306. Riggs, P.K., Angel, J.M., et al. (2005). Mol Carcinog 44: 122–136. Rinaldi, M., Barrera, G., et al. (2000). Biochem Biophys Res Commun 272: 75–80. Rossi, M.A., Fidale, F., et al. (1990). Biochem Pharmacol 39: 1715–1719. Ruef, J., Rao, G.N., et al. (1998). Circulation 97: 1071–1078. Ruggeri, B., Caamano, J., et al. (1991). Cancer Res 51: 6615–6621. Rundhaug, J.E., Fuscher, S.M., Bowden, G.T. (1997). Tumor promoters and models of promotion. In: G.T. Bowden and S.M. Fischer (eds) Comprehensive Toxicology. Oxford, UK, Pergamon. Vol. 12. Saez, E., Rutberg, S.E., et al. (1995). Cell 82: 721–732. Segditsas, S., Rowan, A.J., et al. (2009). Oncogene 28: 146–155. Segrelles, C., Ruiz, S., et al. (2002). Oncogene 21: 53–64. Segrelles, C., Moral, M., et al. (2006). Oncogene 25: 1174–1185. Segrelles, C., Lu, J., et al. (2007). Cancer Res 67: 10879–10888. Shaw, R.J. and Cantley, L.C. (2006). Nature 441: 424–430. Shih, C., Padhy, L., et al. (1981). Nature 290: 261–264. Slaga, T.J. (1984). Mechanisms involved in two-stage carcinogenesis in mouse skin. In: T.J. Slaga (ed) Mechanisms of Tumor Promotion. Boca Raton, FL, CRC Press. Vol. II: 1–16. Slaga, T.J. and DiGiovanni, J. (1984). Inhibition of carcinogenesis. In: C.E. Searle (ed) Chemical Carcinogens. ACS Monograph. Vol. II: 1279–1321. Slaga, T.J., Klein-Szanto, A.J., et al. (1981). Science 213: 1023–1025. Slaga, T.J., Fischer, S.M., et al. (1982). J Cell Biochem 18: 99–119. Slaga, T.J., Budunova, I.V., et al. (1996). J Investig Dermatol Symp Proc 1(2): 151–156. Soh, Y., Jeong, K.S., et al. (2000). Mol Pharmacol 58: 535–541. Solanki, V., Rana, R.S., et al. (1981). Carcinogenesis 2: 1141-1146. Song, B.J., Soh, Y., et al. (2001). Chem Biol Interact 130–132: 943–954. Stern, M.C., Gimenez-Conti, I.B., et al. (1995). Carcinogenesis 16: 1947–1953. Stern, M.C., Benavides, F., et al. (2002). Mol Carcinog 35: 13–20. Stewart, J.W., Koehler, K., et al. (2005). Carcinogenesis 26: 1077–1084. Stoll, S.W., Kansra, S., et al. (2001). Neoplasia 3: 339–350. Su, L.K., Kinzler, K.W., et al. (1992). Science 256: 668–670. Sugimura, T. (1992). Science 258: 603–607. Thiery, J.P. (2002). Nat Rev Cancer 2: 442–454. Trempus, C.S., Morris, R.J., et al. (2003). J Invest Dermatol 120: 501–511. Trempus, C.S., Morris, R.J., et al. (2007). Cancer Res 67: 4173–4181. Uchida, K., Shiraishi, M., et al. (1999). J Biol Chem 274: 2234–2242. Ullrich, A., Coussens, L., et al. (1984). Nature 309: 418–424. Ullrich, O., Siems, W.G., et al. (1996). Biochem J 315: 705–708. van Wezel, T., Stassen, A.P., et al. (1996). Nat Genet 14: 468–470. Vassar, R., Hutton, M., et al. (1992). Mol Cell Biol 12: 4643–4653. Vulimiri, S.V. and DiGiovanni, J. (1999). Carcinogenesis. In: R.E. Pollack (ed) Manual of Clinical Oncology. New York, Wiley-Liss: 19–43. Walker, C., Goldsworthy, T.L., et al. (1992). Science 255: 1693–1695. Wang, X.J., Greenhalgh, D.A., et al. (1994). Mol Carcinog 10: 15–22. Wei, H. and Frenkel, K., (1993). Carcinogenesis 14: 1195–1201.

2  Multistage Carcinogenesis Wei, L., Wei, H., et al. (1993). Carcinogenesis 14: 841–847. Wheldrake, J., Marshall, J., et al. (1982). Carcinogenesis 3: 805–807. Wilker, E., Lu, J., et al. (2005). Mol Carcinog 44: 137–145. William, W.N., Jr., Heymach, J.V., et al. (2009). Nat Rev Drug Discov 8: 213–225. Wistuba, II., Behrens, C., et al. (1999). Oncogene 18: 643–650. Woodworth, C.D., Michael, E., et al. (2004). Carcinogenesis 25: 1771–1778. Xian, W., Kiguchi, K., et al. (1995). Cell Growth Differ 6: 1447–1455. Xian, W., Rosenberg, M.P., et al. (1997). Oncogene 14: 1435–1444. Yakar, S., Nunez, N.P., et al. (2006). Endocrinology 147: 5826–5834. Yang, J., Mani, S.A., et al. (2006). Cancer Res 66: 4549–4552. Yao, D., Alexander, C.L., et al. (2006). Cancer Res 66: 1302–1312. Yarden, Y. and Sliwkowski, M.X. (2001). Nat Rev Mol Cell Biol 2: 127–137. Yates, R.A., Nanney, L.B., et al. (1991). Int J Dermatol 30: 687–694. Yeung, R.S., Xiao, G.H., et al. (1994). Proc Natl Acad Sci USA 91: 11413–11416. Youngren, K.K., Coveney, D., et al. (2005). Nature 435: 360–364. Yuspa, S.H. (1994). Cancer Res 54: 1178–1189. Yuspa, S.H. (1998). J Dermatol Sci 17: 1–7. Yuspa, S.H. (2000). Carcinogenesis 21: 341–344. Zhang, S.L., DuBois, W., et al. (2001). Mol Cell Biol 21: 310–318. Zhang, S., Qian, X., et al. (2003). Oncogene 22: 2285–2295. Zhao, J., Sharma, Y., et al. (1999). Mol Carcinog 26: 321–333. Zhao, J., Lahiri-Chatterjee, M., et al. (2000). Carcinogenesis 21: 811–816. Zhao, Y., Xue, Y., et al. (2001). Cancer Res 61: 6082–6088. Zhaorigetu, S., Yanaka, N., et al. (2003). Oncol Rep 10: 537–543. Zhou, Q., Zhao, J., et al. (1997). J Biol Chem 272: 18240–18244. Zhu, Z., Jiang, W., et al. (1998). Carcinogenesis 19: 2101–2106. Zi, X., Mukhtar, H., et al. (1997). Biochem Biophys Res Commun 239: 334–339. Zimniak, P., Singhal, S.S., et al. (1994). J Biol Chem 269: 992–1000.

51

wwwwwwwwwwwwwwwww

Chapter 3

Tobacco Smoke Carcinogens and Lung Cancer Stephen S. Hecht

Abstract  Cigarette smoking is the major cause of lung cancer, the largest cancer killer in the world. This chapter discusses the role of cigarette smoke carcinogens as causes of lung cancer. A general mechanistic framework is presented, in which cigarette smoke carcinogens and their metabolically activated forms cause mutations in critical growth control genes, along with other effects. Evidence and unresolved issues for the role of various groups of carcinogens, such as polycyclic aromatic hydrocarbons, nitrosamines, volatile organic compounds, and metals as causes of lung cancer are discussed. An overview of inhalation studies of cigarette smoke in laboratory animals is also presented. Collectively, the massive studies on carcinogenesis by cigarette smoke and its constituents provide a firm base for understanding the mechanisms of human lung carcinogenesis.

1 Introduction Among lifestyle factors definitely related to cancer, tobacco use arguably entails the largest human exposure to diverse chemical carcinogens. Tobacco products cause about one in five cancer deaths in the world, or about 1.4 million deaths per year (Mackay et al. 2006). The greatest impact is on lung cancer, which kills approximately 3,000 people per day in the world. Smoking causes 80% of the global lung cancer death toll in men and about 50% in women (Mackay et al. 2006). Tobacco smoking is also a cause of cancers of the oral cavity, pharynx, larynx, esophagus, pancreas, bladder, nasal cavity, stomach, liver, kidney, ureter, cervix, and myeloid leukemia (International Agency for Research on Cancer 2004a). This chapter focuses on tobacco smoke carcinogens and lung cancer. A mechanistic framework for understanding the relationship between cigarette smoking and lung cancer will be presented. The roles of different classes of chemical

S.S. Hecht (*) Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA e-mail: [email protected] T.M. Penning (ed.), Chemical Carcinogenesis, Current Cancer Research, DOI 10.1007/978-1-61737-995-6_3, © Springer Science+Business Media, LLC 2011

53

54

S.S. Hecht

carcinogens as causes of lung cancer will be discussed, noting evidence and unresolved issues. Inhalation experiments with cigarette smoke will be summarized.

2 Mechanistic Framework for Understanding Smoking and Lung Cancer This framework is presented in Fig. 1 (Hecht 1999, 2003). The major established pathway is represented by the horizontal arrows of the central track. Most people begin smoking when they are teen-agers, and become addicted to nicotine (Prokhorov et  al. 2003). Although many reports have described adverse cellular effects of nicotine, particularly with respect to uncontrolled growth, nicotine is not a carcinogen (Hecht 2003; Schuller 2009). A mixture of carcinogens accompanies nicotine in each puff of each cigarette. There are over 60 carcinogens in cigarette smoke that have been evaluated by the International Agency for Research on Cancer (IARC) as having sufficient evidence for carcinogenicity in either laboratory animals or humans (International Agency for Research on Cancer 2004a), and many of these are lung carcinogens, as discussed below. Convincing studies demonstrate the uptake of carcinogens by smokers, and confirm the expected higher levels of their metabolites in urine and blood of smokers than nonsmokers, or find a decrease in metabolites upon smoking cessation (Carmella et  al. 2009). There are large differences in carcinogen exposure among smokers because of the number and types of cigarettes that they smoke and the ways in which they smoke them. These differences can be monitored in part by biomarkers of exposure such as urinary metabolites of carcinogens (Hecht 2002). In one notable series of recent studies, polymorphisms in nicotinic receptor genes were associated with increased lung cancer risk due to increased uptake of nicotine and consequent increased exposure to carcinogens (Amos et al. 2008; Hung et al. 2008; Thorgeirsson et al. 2008; Le Marchand et al. 2008). The carcinogens inhaled by smokers can be metabolically activated, usually by cytochrome P450 enzymes, to intermediates that covalently bind to DNA, or in some cases the carcinogens may bind to DNA directly. The resulting DNA adducts

Fig. 1  Mechanistic framework for understanding mechanisms of tobacco carcinogenesis

3  Tobacco Smoke Carcinogens and Lung Cancer

55

are central to chemical carcinogenesis because they can cause miscoding and permanent mutations, as discussed elsewhere in this monograph. If these mutations occur in critical regions of important growth control genes, the result can be loss of normal cellular growth control mechanisms, genomic instability, and cancer. There is massive evidence, particularly from studies which use relatively nonspecific DNA adduct measurement methods such as 32P-postlabelling and immunoassays, that DNA adduct levels in the lung and other tissues of smokers are higher than in nonsmokers, and some epidemiologic data link these higher adduct levels to increased cancer risk (International Agency for Research on Cancer 2004b; Veglia et al. 2008). Evidence for DNA adducts in human lung is more limited when only structure-specific methods are considered (Pfeifer et  al. 2002; Boysen and Hecht 2003; Beland et al. 2005; Zhang et al. 2006). The chronic formation of DNA adducts from cigarette smoke carcinogens, over years of smoking, is consistent with the multiple genetic changes observed in tumors. A recent study again validated this premise by finding multiple mutations in critical growth control genes in lung adenocarcinomas (Ding et  al. 2008a). Thus, Ding and co-workers examined 188 human lung adenocarcinomas and sequenced 623 genes with known or potential relationships to cancer. They found more than 1,000 somatic mutations in critical growth control genes, with the tumor suppressor gene p53 and the oncogene KRAS being the most frequently mutated, consistent with previous studies (Ahrendt et al. 2001; Pfeifer et al. 2002; Ding et al. 2008a). Other frequently mutated genes were the tumor suppressors CDKN2A and STK11 and the oncogenes N-RAS and EGFR. The cancer causing role of mutations in genes such as K-RAS and p53 has been firmly established in animal studies (Lubet et al. 2000; Johnson et al. 2001). The vertical arrows of the central track represent protective mechanisms. There are well-established detoxification pathways for virtually all carcinogens in cigarette smoke. These are catalyzed by cytochrome P450s as well as phase II enzymes such as glutathione-S-transferases, UDP-glucuronosyl transferases, and sulfotransferases. Cellular DNA repair systems can excise DNA adducts and restore normal DNA structure, thus opposing the mutational consequences described above. These complex systems include direct base repair by alkyltransferases, removal of DNA damage by base and nucleotide excision repair, mismatch repair, and double strand repair (Christmann et  al. 2003). There are polymorphisms in genes coding for some DNA repair enzymes. If these variants lead to deficient DNA repair, the probability of cancer development can increase (Liu et al. 2005). Apoptosis, or programmed cell death, is another protective process, and can remove cells which have DNA damage, thus serving as a counterbalance to the mutational events. The balance between apoptotic mechanisms and those suppressing apoptosis will have a major impact on tumor growth (Bode and Dong 2005). While the central track of Fig. 1 is the major necessary pathway by which cigarette smoke carcinogens cause cancer, other mechanisms also contribute, as indicated in the top and bottom tracks (Schuller 2002; Hecht 2003). Nicotine and tobacco-specific nitrosamines bind to nicotinic and other cellular receptors resulting in activation of Akt (also known as protein kinase B), protein kinase A,

56

S.S. Hecht

and other changes, resulting in decreased apoptosis, increased angiogenesis, and increased transformation (Heeschen et al. 2001; West et al. 2003; Schuller 2009). Although nicotine is not carcinogenic, it may enhance carcinogenicity in as yet incompletely defined ways. Cigarette smoke activates the epidermal growth factor receptor and cyclooxygenase-2 (Moraitis et al. 2005), and contains well-established oxidants, co-carcinogens, tumor promoting fractions, and inflammatory agents. Many studies demonstrate the co-carcinogenic effects of catechol, an important constituent of cigarette smoke (Van Duuren and Goldschmidt 1976; Hecht et  al. 1981; Melikian et  al. 1989). Cigarette smoke downregulates the FHIT tumor suppressor gene (Tseng et al. 1999; D’Agostini et al. 2006). An epigenetic pathway frequently observed in tobacco-induced cancers is enzymatic methylation of promoter regions of genes, resulting in gene silencing. When this occurs in tumor suppressor genes, the result can be unregulated proliferation (Belinsky 2005). Furthermore, inflammation due to smoking is associated with tumor promotion and cancer development (Smith et al. 2006; Lee et al. 2008).

3 Lung Carcinogens in Cigarette Smoke 3.1 Polycyclic Aromatic Hydrocarbons 3.1.1 Evidence Beginning with the classic studies of Kennaway, Cook, and others more than 80 years ago, polycyclic aromatic hydrocarbons (PAHs) were characterized by fractionation, spectroscopy, and synthesis as carcinogenic constituents of coal tar, see Chap. 1 (Phillips 1983) This research was followed by synthetic and structure–activity studies which defined their carcinogenic properties. Tumor induction on mouse skin was used as a bioassay in many of these studies. Since PAHs are products of incomplete combustion, it was natural to suspect their role in tobacco carcinogenesis. Following the demonstration that cigarette smoke condensate caused tumors on mouse skin (Wynder et al. 1953), extensive fractionation studies showed that PAHs were indeed carcinogenic constituents of cigarette smoke condensate. Fractions enriched in PAH were tumor initiators on mouse skin and induced tumors when implanted in the rat lung (Stanton et al. 1972; Hoffmann et al. 1978). PAHs always occur in cigarette smoke and other products of combustion as mixtures and it can be misleading, unless properly qualified, to consider only single compounds (as is often done with benzo[a]pyrene [BaP]) when discussing them. Over 500 PAHs have been completely or partially identified in tobacco smoke, but only a few are routinely quantified (Rodgman and Perfetti 2009). Structures of some commonly measured PAH in cigarette smoke are shown in Fig. 2, and typical amounts in cigarette mainstream smoke, based on recent studies, are summarized in Table 1 (Ding et al. 2005, 2007).

3  Tobacco Smoke Carcinogens and Lung Cancer

Fig. 2  Structures of some PAH identified in cigarette smoke

57

58

S.S. Hecht Table  1  Representative levels of PAH in cigarette mainstream smoke (ng/cigarette)a PAH Naphthalene 350.3 Acenaphthalene 116.9 Acenaphthene 84.8 Fluorene 217.5 Phenanthrene 134.8 Anthracene 74.9 Fluoranthene 74.4 Pyrene 48.6 Benz[a]anthracene 13.4 Chrysene 15.7 Benzo[b]fluoranthene 9.4 Benzo[k]fluoranthene 1.5 Benzo[j]fluoranthene 18.5 Benzo[e]pyrene 2.9 Benzo[a]pyrene 10.3 Indeno[1,2,3-c,d ]pyrene 9.3 Dibenz[a,h]anthracene 4.8 Dibenzo[a,e]pyrene 2.4 Dibenzo[a,i]pyrene 1.1 5-Methylchrysene 2.5b From Ding et al. 2005, 2007 a  Values for benzo[j ]fluoranthene, indeno[1,2,3-c,d ]pyrene, dibenz[a,h]anthracene, dibenzo[a,e]pyrene, dibenzo[a,i] pyrene, and 5-methylchrysene are from a commercial cigarette. Others are from a reference cigarette. ISO machine smoking conditions were used (35 ml puff volume, 2 s puff duration, 60 s puff interval) b  May contain other methylchrysene isomers

PAHs act locally in most carcinogenesis bioassays, inducing tumors at the site of application, such as mouse skin when applied as a solution, or rat lung when instilled in a vehicle such as beeswax (Stanton et al. 1972; Deutsch-Wenzel et al. 1983). Extensive structure–activity studies of PAH tumorigenicity have been carried out (Dipple et al. 1984; Harvey 1991), and a detailed recapitulation of these would be beyond the scope of this chapter. With respect to the induction of lung tumors, and considering those commonly measured, convincing evidence has been presented for BaP, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, dibenzo[a,i]pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, and 5-methylchrysene (Hecht 1999). Based on the values in Table 1, the total of these PAHs in mainstream smoke is about 50–60 ng/cigarette. Mouse skin studies demonstrate that most PAHs with two to four rings are inactive (Dipple et al. 1984; Harvey 1991). Chrysene and benz[a]anthracene are inactive or only weakly active as complete carcinogens but do show some tumor initiating

3  Tobacco Smoke Carcinogens and Lung Cancer

59

activity on mouse skin (Hecht et al. 1974; Dipple et al. 1984; Harvey 1991). Among the five ring compounds in Fig. 2, BaP, dibenz[a,h]anthracene, and the benzofluoranthenes are carcinogenic, as mentioned above, while benzo[e]pyrene is generally considered inactive (Harvey 1991). Mixed results have been obtained in studies of hexacyclic PAH in various bioassay systems (Harvey 1991). Cigarette smoking induces cytochrome P450s 1A1 and 1B1 through interactions of its components with the AH receptor, and this inducibility, frequently measured as “aryl hydrocarbon hydroxylase,” (AHH) activity, has been associated with a higher risk for lung cancer in some studies (Nebert et al. 2004). Cytochrome P450s 1A1 and 1B1 are involved in both the metabolic activation and detoxification of PAH, but some studies show that induction is associated with increased metabolic activation of BaP. Thus, lung tissue from recent smokers with elevated AHH activity converted the proximate carcinogen BaP-7,8-diol to tetraols (resulting from hydrolysis of BaP diol epoxides) to a greater extent than lung tissue from nonsmokers or ex-smokers, and BaP diol epoxide DNA adduct levels were also associated with AHH activity in the same samples (Rojas et al. 1992; Alexandrov et al. 1992). The distribution of DNA adducts in the p53 gene has been mapped for diol epoxides of BaP and other PAH using either enzymatic or mass spectrometric techniques (Smith et  al. 2000; Tretyakova et  al. 2002; Matter et  al. 2004). Both approaches produced similar results, demonstrating frequent adduct formation at codons 157, 158, 245, 248, and 273. These positions of preferential adduct formation are also major mutational hot spots in human lung cancer, possibly providing further support for an important role of PAH in lung cancer induced by cigarette smoking. The potential role of PAH quinones, oxidative damage, and biological selection as a basis for this phenomenon has also been discussed (Shen et al. 2006). The pattern of mutations in codon 12 of the K-RAS gene in lung tumors induced by PAH in mice is also consistent with that observed in human lung adenocarcinomas (Nesnow et al. 1998). Collectively, there is strong evidence based on their occurrence and carcinogen­ icity, and from biochemical and molecular biological studies, that PAHs are involved in lung cancer induction by cigarette smoke. BaP is considered “carcinogenic to humans” by the IARC (Straif et al. 2005). 3.1.2 Unresolved Issues Although subfractions of cigarette smoke condensate enriched in PAH were identified as carcinogenic, testing on mouse skin for complete carcinogenicity of a synthetic mixture of 17 PAH in this fraction, in the concentrations in which they occur or even in double their concentrations, did not produce tumors (Hoffmann et al. 1978). Other studies concluded that the PAH accounted for only a few percent of the observed complete carcinogenic activity of cigarette smoke condensate on mouse skin (Hoffmann and Wynder 1971). However, addition of a mixture of the 17 PAH to the condensate resulted in significant increases in complete carcinogenicity (Hoffmann et al. 1978). Collectively, these results and others indicated that

60

S.S. Hecht

PAHs in cigarette smoke condensate are tumor initiators and that the condensate also contains co-carcinogens and tumor promoters, which are important in the expression of its activity on mouse skin. These components are found partially in the weakly acidic fraction of the condensate (Hoffmann and Wynder 1971). Catechol, a strong co-carcinogen on mouse skin when tested with BaP, is certainly one of these, but there are other co-carcinogens and tumor promoters that are as yet unidentified and may be very important (Van Duuren and Goldschmidt 1976; Hecht et al. 1981). Related to this issue is the fact that PAH are potent inhibitors of the cytochrome P450 enzymes that metabolize them, and that they can act both as inhibitors and enhancers of tumorigenicity (Rubin 2001; Shimada and Guengerich 2006). It could be that there are highly carcinogenic PAH in the active subfractions that had not been identified at the time the work described above was performed. A prime candidate is dibenzo[a,l]pyrene (Fig. 2). This PAH has tumorigenic activity on mouse skin far greater than that of BaP and is considered to be the most carcinogenic PAH ever tested (Cavalieri et al. 1991). It also induces lung tumors in mice (Platt et al. 2004; Castro et al. 2008). There are only limited data on dibenzo[a,l]pyrene in cigarette smoke condensate, and it is not routinely analyzed because of its extremely low concentration. One study provided qualitative evidence for its presence (Snook et al. 1977), while a second study indicated that its levels were about 0.1 ng/cigarette (Seidel et al. 2004). Another group of related carcinogens is the azaarenes. Two of these, dibenz[a,h]acridine and 7H-dibenzo[c,g]carbazole, have been reported in cigarette smoke and are lung carcinogens, but have not been routinely identified or analyzed (Hecht 1999; Rodgman and Perfetti 2009). A recent study demonstrated that acrolein, which occurs in cigarette smoke at levels up to 10,000 times greater than that of BaP, produces adducts at the same codons of the p53 gene as do PAH diol epoxides, indicating that the observed mutations in the p53 gene could be due to acrolein, and not due to PAH (Feng et  al. 2006). Acrolein is highly toxic, but not generally considered carcinogenic. This observation also highlights the fact that the concentrations of carcinogenic PAH in cigarette smoke are actually quite low, with the commonly measured ones amounting to only 50–60 ng/cigarette.

3.2 Nitrosamines 3.2.1 Evidence In 1956, Magee and Barnes reported that N-nitrosodimethylamine, a simple water soluble compound with only 11 atoms, caused liver tumors in rats (Magee and Barnes 1956). This was remarkable at the time because the physical properties of the widely investigated lipophilic PAH carcinogens were completely different from those of N-nitrosodimethylamine. The Magee and Barnes paper initiated a blizzard of research on the carcinogenic properties of nitrosamines, which were easily synthesized by nitrosation of secondary amines (Druckrey et al. 1967; Lijinsky 1992).

3  Tobacco Smoke Carcinogens and Lung Cancer

61

Ultimately, more than 200 nitrosamines were found to be carcinogenic, and many of these were extremely potent (Preussmann and Stewart 1984). The carcinogenic properties of nitrosamines are quite different from those of PAH. Nitrosamines are generally systemic carcinogens that are selective for particular organs, and they seldom exhibit local carcinogenicity, such as seen in mouse skin application studies of PAH. Studies on the nitrosation of nicotine, following the mechanistic principles established by Smith and Loeppky (Smith and Loeppky 1967), demonstrated that, in addition to N¢-nitrosonornicotine (NNN), which had been previously synthesized and tested by Boyland in 1964 (Boyland et  al. 1964), two other nitrosamines – 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)1-(3-pyridyl)butanal (NNAL) – were formed, along with a number of other products (Hecht et al. 1978b). As these and related nitrosamines were formed from nicotine and other tobacco alkaloids, they came to be known as “tobacco-specific nitrosamines.” Ultimately, seven tobacco-specific nitrosamines were identified in tobacco products (Hoffmann et al. 1994; Hecht 1998). Their structures are shown in Fig. 3. NNK, NNAL, and NNN are the most carcinogenic of the tobacco-specific nitrosamines (Hecht 1998). The organoselectivity of NNK for the lung is particularly remarkable. It induces lung tumors in rats, mice, hamsters, and ferrets independent of the route of administration (Hecht 1998). The tumors are adenomas and adenocarcinomas. The F-344 rat, which has a very low incidence of spontaneous lung tumors, is particularly sensitive to lung tumor induction by NNK. Dose–response studies demonstrate that a total dose of 6 mg/kg, and 1.8 mg/kg when considered as part of a dose–response trend, induce a significant incidence of lung tumors (Belinsky et al. 1990). Lung tumors have been induced by NNK in different mouse strains, whether or not they are normally considered susceptible or resistant (Hecht 1998). Various routes of administration have been used in carcinogenesis studies with NNK, and the lung is generally the main organ where tumors are observed, although tumors of the liver, nasal mucosa, and pancreas are also seen in rats (Hecht 1998). NNAL has lung carcinogenic activity similar to that of NNK in rats and mice (Hecht 1998). NNN, which causes tumors of the esophagus and nasal mucosa in rats, is not generally considered as a lung carcinogen, but lung tumors have been

Fig. 3  Tobacco-specific nitrosamines in tobacco products

62

S.S. Hecht

observed in a number of studies in mice treated with NNN, and tumors of the trachea are produced in hamsters (Hecht 1998). The only other nitrosamine lung carcinogen found in cigarette smoke is N-nitrosodiethylamine, which causes lung tumors in hamsters (Preussmann and Stewart 1984). Virtually all unburned commercial tobacco products contain NNN and NNK, and they always occur together (International Agency for Research on Cancer 2007). They are mainly formed during the curing of tobacco and are partially transferred into smoke. There is a great variation in levels of NNN and NNK in mainstream smoke of cigarettes. This is mainly due to differences in tobacco types used, agricultural practices, curing methods, and manufacturing processes. Factors that lead to relatively high levels of NNN and NNK in cured tobacco include the use of Burley tobacco, the use of midribs from air cured tobacco or lamina from flue cured tobacco, storage of tobacco leaves under humid conditions or in bales, processes that encourage bacterial growth thus leading to increased nitrite, and heating with propane during curing (International Agency for Research on Cancer 2007). Since the first reports of NNN and NNK in unburned tobacco (Hoffmann et al. 1974; Hecht et  al. 1978a), many studies have quantified their levels in various tobacco products. Extensive compilations of recent data may be found in the International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risks to Humans, volumes 83 and 87 (International Agency for Research on Cancer 2004b, 2007). Levels of NNN ranged from 20 to 58,000 ng/ cigarette and NNK from 19 to 10,745  ng/cigarette in tobacco from commercial cigarettes sold in different parts of the world; and from 4 to 2,830  ng/cigarette (NNN) and 3–1,749  ng/cigarette (NNK) in mainstream smoke of internationally available commercial cigarettes. In one recent study, levels of NNN and NNK were quantified in the smoke of research cigarettes made from different tobacco varieties. Levels of NNN and NNK were greatest in Burley tobacco smoke, with substantially lower amounts in the smoke of Oriental and Bright cigarettes. Nitrate content of the tobacco was significantly related to smoke NNK (but not NNN), and was inversely proportional to PAH levels (Ding et  al. 2008b). These results are completely consistent with earlier studies (International Agency for Research on Cancer 1986, 2007). Another recent study of multiple brands reported NNK levels of 54–101 ng/ cigarette (ISO method) and 110–212 ng/cigarette (Canadian intense method) while the corresponding levels of NNN were 25–168  ng/cigarette and 53–353  ng/ cigarette, respectively (Hammond and O’Connor 2008). Levels of N-nitrosodiethylamine in cigarette smoke have been reported as ranging from not detected to 25 ng/cigarette (International Agency for Research on Cancer 2004a). This compound is not routinely quantified in smoke, mainly because of its low levels. Extensive studies on the metabolism and DNA binding of NNK and NNN have been carried out in laboratory animals and humans. These have been reviewed and summarized (Hecht 1998; Jalas et al. 2005; International Agency for Research on Cancer 2007), and a detailed recapitulation is beyond the scope of this chapter. In summary, NNK and NNN metabolites have been identified in the blood and urine of smokers and smokeless tobacco users, and their adducts, measured as

3  Tobacco Smoke Carcinogens and Lung Cancer

63

4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts, have been positively identified and quantified in DNA and globin of tobacco users. There is a general concordance between metabolic pathways in rodents and humans, although there are some quantitative differences. On the basis of carcinogenicity studies in laboratory animals, exposure data, and biochemical studies in humans, NNK and NNN are considered to be carcinogenic to humans by the International Agency for Research on Cancer (2007). Two recent molecular epidemiology studies are consistent with this conclusion. In one study, urinary levels of NNAL plus its glucuronides (total NNAL), metabolites of NNK, were significantly associated with risk of lung cancer in a dose-dependent manner (Yuan et al. 2009). Relative to the lowest tertile, risks associated with the second and third tertiles of total NNAL were 1.43 (95% CI, 0.86–2.37) and 2.11 (95% CI, 1.25–3.54), respectively (P for trend = 0.005) after adjustment for smoking history and total cotinine. In a second study, similar results were obtained using prospective measurements of total NNAL in serum (Church et al. 2009). Collectively, there is strong evidence based on its occurrence and carcinogenicity, and from biochemical studies, that NNK is involved in lung cancer induction by cigarette smoke. The evidence for a role of NNN in lung cancer is less conclusive. 3.2.2 Unresolved Issues HPB-releasing DNA adducts of NNK and NNN were higher in lung tissue from smokers than nonsmokers in two studies, but a third study which obtained lung tissue from sudden death victims did not show a significant difference (Foiles et al. 1991; Hölzle et  al. 2007; Schlobe et  al. 2008). There was also no correlation between HPB-releasing DNA adducts and Hb adducts in one study (Hölzle et al. 2007), and no difference between smokers and nonsmokers in HPB-releasing DNA adducts in the lower esophagus and cardia in the second study (Schlobe et al. 2008). These results suggest that there could be other sources of HPB-releasing adducts. Nitrosation of myosmine has been proposed as one source, but studies in rats did not support that hypothesis (Hecht et al. 2007). The metabolism of NNK has been examined in human lung tissue in many studies [reviewed in (Richter et al. 2009)]. The results of these studies demonstrate extensive conversion to NNAL, but only small amounts of metabolic activation by the established a-hydroxylation pathway. The reasons for this are not clear, particularly since a-hydroxylation is required to form HPB-releasing DNA adducts, which have been detected in human lung, as described above. Furthermore, there is evidence based on the analysis of urinary metabolites that a-hydroxylation is the major metabolic pathway of NNK in laboratory animals and humans (Stepanov et al. 2008). There may be defects in the in vitro systems used for these metabolic studies with human lung tissue. Studies of mutations in the p53 gene have so far not provided evidence for a role of NNK, while results from studies of the K-RAS gene are inconclusive (Hecht 1999).

64

S.S. Hecht

However, there is also no evidence for mutational activation of these genes in rat lung tumors induced by NNK, indicating the involvement of other pathways. There are no reports in the open literature of lung tumor induction by NNK administered by inhalation. Although this would be expected to produce lung tumors, the absence of data prevents an analysis of the potential role of NNK as a cause of lung tumors produced in rodents by inhalation of tobacco smoke (Hutt et al. 2005). The dose of NNK in those tobacco smoke inhalation studies may be too low to explain the observed effects, but there could be co-carcinogenic or even inhibitory effects which remain unexplored.

3.3 Butadiene 3.3.1 Evidence Beginning in the mid-1980s, a series of studies carried out by the U.S. National Toxicology Program demonstrated that B6C3F1 mice exposed by inhalation to 6.25–1,250  ppm of 1,3-butadiene developed significant incidences of bronchiolar/ alveolar adenomas and carcinoma of the lung in addition to lymphoma, heart hemangiosarcoma, forestomach papillomas and carcinomas, and other tumors (Huff et al. 1985; International Agency for Research on Cancer 2008). There is no doubt that inhalation of 1,3-butadiene causes lung tumors in mice, but lung tumors were not observed in rats exposed to 1,000 or 8,000  ppm of 1,3-butadiene, nor were other tumors as common as in mice (International Agency for Research on Cancer 2008). Extensive data are available on levels of 1,3-butadiene in cigarette smoke, as it is a commonly measured constituent. In one study of 48 Philip Morris brands, levels of this carcinogen ranged from 6.4 to 54.1 mg/cigarette (mean 32.1 ± 12.7 mg/cigarette [ISO conditions]) (Counts et al. 2004). A second study of eight Philip Morris brands provided similar data: range 12.5–50.8 mg/cigarette (mean 31.5 ± 14.6 mg/cigarette) (Roemer et al. 2004). A study of Canadian brands reported an average of 43 mg/cigarette. Levels of 1,3-butadiene in cigarette smoke do not seem to have changed much in the past 20 years (International Agency for Research on Cancer 2004a). Like PAH and nitrosamines, 1,3-butadiene requires metabolic activation to exert its carcinogenic effects. It is metabolized to mutagenic epoxybutene, diepoxides, and a diol epoxide which form adducts with DNA and hemoglobin, and the latter have been quantified in humans (International Agency for Research on Cancer 2008). DNA cross-links have also been observed and quantified in exposed animals (Goggin et al. 2008, 2009). The epoxides are detoxified by conjugation with glutathione and the conjugates are metabolized and excreted as urinary mercapturic acids (International Agency for Research on Cancer 2008). The mercapturic acids derived from glutathione conjugation of epoxybutene have been identified in human urine and decreases tenfold upon smoking cessation (Carmella et al. 2009). Levels of 1,3-butadiene in the exhaled breath of smokers are also significantly higher than in nonsmokers (International Agency for Research on Cancer 2008).

3  Tobacco Smoke Carcinogens and Lung Cancer

65

Collectively, there is no doubt that smokers are exposed to substantial amounts of the mutagenic carcinogen 1,3-butadiene, and that these amounts decrease markedly upon smoking cessation. 3.3.2 Unresolved Issues 1,3-Butadiene is a strong pulmonary carcinogen in the mouse, but not in the rat. It is unclear which species is a better model for humans, but studies of the molecular dosimetry of diepoxybutane-induced DNA–DNA cross-links suggest that rats and humans are more similar than mice and humans with respect to this ultimate carcinogen of 1,3-butadiene (Goggin et al. 2009). Although genotoxicity data indicate that diepoxybutane is the most genotoxic epoxide metabolite of 1,3-butadiene, the relative contribution of all epoxide metabolites to its mutagenicity and carcinogenicity is not known (International Agency for Research on Cancer 2008). Epidemiologic studies have been carried out on workers in the 1,3-butadiene monomer industry and in the styrene – 1,3-butadiene rubber industry. The results of these studies provide evidence that 1,3-butadiene exposure causes leukemia and non-Hodgkin lymphoma, and it is considered a human carcinogen. There is no evidence that exposures in these industries cause lung cancer (International Agency for Research on Cancer 2008).

3.4 Ethylene Oxide 3.4.1 Evidence Inhalation studies demonstrate that ethylene oxide causes alveolar/bronchiolar adenomas and carcinomas of the lung in male and female B6C3F1 mice. Tumors of the Harderian gland, malignant lymphomas, uterine adenocarcinomas, and mammary gland carcinomas were also observed. Lung tumors were not observed in rats treated with ethylene oxide by inhalation or gavage (International Agency for Research on Cancer 2008). Ethylene oxide is not routinely measured in tobacco smoke: a value of 7 mg per cigarette has been given (International Agency for Research on Cancer 2004a). Levels of an ethylene oxide adduct with the terminal valine of hemoglobin are elevated in smokers, and correlate with numbers of cigarettes smoked (International Agency for Research on Cancer 2008). Levels of this hemoglobin adduct were higher in hemoglobin of newborns from smoking mothers compared to nonsmoking mothers (International Agency for Research on Cancer 2008). 7-(2-Hydroxyethyl)guanine, from reaction of ethylene oxide with DNA, has been reported in DNA samples from lung tissue and leukocytes of smokers (Zhao et al. 1999, 2000). Concentrations of an ethylene oxide-derived mercapturic acid in urine decrease about fivefold when smokers stop smoking cigarettes (Carmella et al. 2009).

66

S.S. Hecht

3.4.2 Unresolved Issues Ethylene oxide causes lung tumors in mice exposed by inhalation, but not in rats. Ethylene oxide is considered carcinogenic to humans by the IARC, based on a combination of epidemiological evidence for associations between occupational exposure to ethylene oxide and lymphatic and hematopoietic cancers, and consistent mechanistic data demonstrating its alkylating and mutagenic effects in various test systems and humans. However, occupational exposure to ethylene oxide has not been related to lung cancer (International Agency for Research on Cancer 2008).

3.5 Ethyl Carbamate (Urethane) 3.5.1 Evidence Ethyl carbamate causes lung tumors in various strains of mice treated by different routes of administration and doses (International Agency for Research on Cancer 1974; U.S. Department of Health and Human Services 2004). Tumors at other sites are also observed. Lung tumors are not induced in rats or hamsters treated with ethyl carbamate (International Agency for Research on Cancer 1974; U.S. Department of Health and Human Services 2004). Ethyl carbamate is the classic carcinogen for induction of lung tumors in A/J mice and is still used routinely for this purpose (Shimkin and Stoner 1975; O’Donnell et al. 2006). Ethyl carbamate is not routinely analyzed in tobacco smoke. Levels of 20–38 ng/cigarette have been reported (International Agency for Research on Cancer 2004a). 3.5.2 Unresolved Issues Although ethyl carbamate is demonstrably carcinogenic to the mouse lung, there are simply inadequate data on its levels in tobacco smoke to evaluate it further with respect to its possible contribution to lung carcinogenicity in smokers.

3.6 Inorganic Compounds 3.6.1 Evidence The same as any plant, tobacco contains metals, and some of these are transferred to smoke. Among these are some pulmonary carcinogens, including arsenic, cadmium, chromium, and nickel. Arsenic in drinking water causes cancers of the

3  Tobacco Smoke Carcinogens and Lung Cancer

67

urinary bladder, lung, and skin, and is evaluated as carcinogenic to humans (IARC Group 1) (International Agency for Research on Cancer 2004b). Cadmium salts produced local tumors in animals and exposure to cadmium has been associated with increased risks of prostatic and respiratory cancers (IARC Group 1) (International Agency for Research on Cancer 1993). Chromium (hexavalent) is considered carcinogenic to humans, inducing lung cancer (IARC Group 1) (International Agency for Research on Cancer 1990). Nickel sulfate and combinations of nickel sulfides and oxides as encountered in the nickel refining industry cause lung and nasal cancers (IARC Group 1) (International Agency for Research on Cancer 1990). Levels of these metals in cigarette smoke have been assessed in recent studies (Baker et  al. 2004; Counts et  al. 2004; Pappas et  al. 2006; Hammond and O’Connor 2008). Arsenic, chromium, and nickel were below the limits of detection of 15, 3, and 12 ng/cigarette, respectively (Baker et al. 2004; Counts et al. 2004). Levels of cadmium in the smoke of U.S. commercial brands ranged from 13.8 to 62.4 ng/cigarette (Pappas et al. 2006). The mean cadmium level in the smoke of Canadian brands was 57.6 ± 21.6  ng/cigarette (Hammond and O’Connor 2008). Large studies in Europe and North America demonstrated that urinary cadmium increased with age and smoking, while environmental and occupational exposure played only a minor role (International Agency for Research on Cancer 2004a). Serum and pulmonary cadmium have also been related to smoking (Stavrides 2006). There is sufficient evidence in animals but inadequate evidence in humans for the carcinogenicity of the radioelement 210Po. 210Po is a pure a-particle emitter and internalized radionuclides that emit a-particles are considered carcinogenic to humans (IARC Group 1) (International Agency for Research on Cancer 2001). The levels of 210Po in cigarette smoke are probably too low to be involved in lung cancer induction, based on data from uranium miner studies (Harley et al. 1980; Tso 1990). Hydrazine produces tumors at various sites, including the mouse lung, and is considered possibly carcinogenic to humans (IARC Group 2B) (International Agency for Research on Cancer 1999). Hydrazine levels in cigarette smoke are not routinely measured, but have been listed as 24–43  ng/cigarette (International Agency for Research on Cancer 2004a). 3.6.2 Unresolved Issues There are inadequate data on hydrazine to assess its role in lung carcinogenesis in smokers. Levels of several metals and 210Po in cigarette appear to be quite low and perhaps need further assessment. Specific mechanistic studies on the role of Cd in lung cancer induced by cigarette smoke need to be performed in order to better evaluate its potential role.

68

S.S. Hecht

4 Other Carcinogens and Agents Possibly Related to Lung Cancer Isoprene causes lung tumors in mice when administered by inhalation, but its activity is considerably less than that of the structurally related 1,3-butadiene. It does not affect the lung in rats treated by inhalation (International Agency for Research on Cancer 1994). Isoprene levels in mainstream cigarette smoke are typically about 300  mg/cigarette (Hammond and O’Connor 2008). Benzene caused tumors at multiple sites including the lung when administered to mice by gavage, and some lung tumors were also observed upon inhalation of benzene (Farris et al. 1993; U.S. Department of Health and Human Services 2004). Lung tumors were not observed in rats treated with benzene. Benzene is considered to be a cause of various types of leukemia in humans (International Agency for Research on Cancer 1987), but one study also found an increased risk for lung cancer upon exposure to benzene (Hayes et  al. 1996). Levels of benzene in mainstream cigarette smoke are typically about 45  mg/cigarette (Hammond and O’Connor 2008). Acetaldehyde and formaldehyde, while not lung carcinogens, do induce nasal carcinomas in rats exposed by inhalation, and are “reasonably anticipated to be human carcinogens” (U.S. Department of Health and Human Services 2004). Levels of acetaldehyde and formaldehyde in mainstream cigarette smoke are typically about 590 and 40–73  mg/cigarette, respectively (Hammond and O’Connor 2008). Leukocyte DNA adducts of acetaldehyde are marginally higher in smokers before cessation, while clear differences in formaldehyde–DNA adducts have been observed (Chen et  al. 2007; Wang et al. 2009). Cigarette smoke causes oxidative damage, possibly due to free radicals such as nitric oxide, mixtures of catechols, hydroquinones, semiquinones, and quinones which can induce redox cycling, and the involvement of redox cycling in PAH metabolism (Pryor et  al. 1998; Hecht 1999; Shen et  al. 2006; Park et  al. 2008). Smokers have lower levels of ascorbic acid, higher levels of oxidized lipids, and sometimes higher levels of oxidized DNA bases than nonsmokers but the role of oxidative damage as a cause of specific tobacco-induced cancers remains unclear (Hecht 1999; Dietrich et al. 2002; Phillips 2002). It is worth noting that inhalation studies to determine the possible carcinogenicity of ozone have consistently been negative, and that ozone had no enhancing effect on lung carcinogenesis by NNK (Boorman et al. 1994; Kim and Cho 2009). Pro-inflammatory changes have been observed in smokers’ lungs, and inflammation is closely associated with tumor promotion and activation of NFĸB (Fischer 1997; Malkinson 2005; Smith et al. 2006; Lee et al. 2008). Inflammation plays a role in COPD associated with smoking (Kim et al. 2007), and COPD (especially emphysema) in turn is a risk factor for lung cancer (Turner et al. 2007). The specific agents in cigarette smoke responsible for inflammation are poorly defined, but oxidants and reactive aldehydes such as acrolein may be involved (Kim et al. 2007; Thompson and Burcham 2008).

3  Tobacco Smoke Carcinogens and Lung Cancer

69

5 Insights from Inhalation Studies of Cigarette Smoke Inhalation studies of cigarette smoke have been carried out in hamsters, rats, mice, rabbits, dogs, and nonhuman primates. The model systems used in these studies have various problems, and none is able to duplicate accurately human smoking habits. These studies have been reviewed (International Agency for Research on Cancer 1986; Coggins 1998; Witschi 2000; Hecht 2005). In early studies, the most consistent results with respect to induction of cancer were observed in Syrian golden hamsters, in which whole cigarette smoke and its particulate phase induced malignant tumors and other lesions in the larynx. Dose to the larynx was considerably higher than to the lung. Tumors were not induced by cigarette smoke gas phase in the hamster experiments. Evidence was also obtained for tumor promotion by inhaled cigarette smoke. Some data however indicate that gas phase constituents contribute to tumor induction. Early studies in Snell’s mice demonstrated an increase in pulmonary adenocarcinoma in animals exposed to gas phase alone (International Agency for Research on Cancer 2004a). In an exposure model which uses 89% sidestream and 11% mainstream smoke, increased lung adenoma multiplicity is consistently observed in A/J mice exposed to smoke for 5 months, then allowed a 4-month resting period. Tumor response in this model is clearly due to the gas phase, as filtration has no effect on lung adenoma multiplicity (Witschi 2000, 2005; International Agency for Research on Cancer 2004b). The results of these studies indicate that a volatile carcinogen of cigarette smoke, possibly 1,3-butadiene, produces a tumorigenic response in the A/J mouse lung when smoke is administered by inhalation. These experiments were however complicated by lack of weight gain and the possible contribution of stress (Stinn et al. 2005). More recent studies demonstrate convincingly that mainstream cigarette smoke, administered by whole body inhalation to rats or mice for extended periods of time, induces benign and malignant tumors of the respiratory tract (Mauderly et al. 2004; Hutt et al. 2005). Male and female F344 rats were exposed 6  h per day, 5 days per week for up to 30 months to mainstream smoke from research cigarettes or to clean air. Cigarette smoke exposure significantly increased the incidence of nonneoplastic and neoplastic proliferative lung lesions in females. The combined incidence of bronchioloalveolar adenomas and carcinomas was 14% in the high exposure (250 mg/m3 particulate) group, 6% in the low exposure (100 mg/m3) group, and zero in controls. Both males and females had significant increases of nasal cavity neoplasia (Mauderly et al. 2004). Female B6C3F1 mice were exposed 6 h per day, 5 days per week for 925 days (250 mg/m3), or sham exposed. Significant incidences of lung adenoma (28%) and adenocarcinoma (20%) were observed (Hutt et al. 2005). Recent studies have also produced high incidences of lung tumors by the exposure of newborn mice to mainstream cigarette smoke (Balansky et al. 2007). Collectively, the results of these studies leave no doubt that inhalation of cigarette smoke causes lung and other respiratory tract tumors in laboratory animals.

70

S.S. Hecht

Consistent with the data presented above on individual carcinogens and fractions of condensate, both volatile and particulate phase components induce tumors, and evidence for tumor promotion has also been obtained.

6 Summary Decades of conclusive epidemiologic studies demonstrate that cigarette smoking causes lung cancer. Experimental studies lagged behind at first, with operational difficulties in inhalation experiments and possibly over-reliance on mouse skin studies. However, experimental data are now on an equal footing with epidemiologic studies. We have an excellent view of cigarette smoke carcinogens that are likely causes of lung cancer. These include volatiles such as 1,3-butadiene and possibly other compounds, and particulate phase constituents such as PAH and tobacco-specific nitrosamines. While the individual contributions of each carcinogen may never be known because of the complexity of the system, there can be little doubt that removal of all of these agents from cigarette smoke, if that were even possible, would decrease its horrible consequences. There are mountains of data, not summarized here but discussed in other chapters of this monograph, which support the central track of Fig. 1 as a basic mechanism of carcinogenesis by cigarette smoke. Where more research is needed is on the upper and lower tracks, particularly the roles of tumor promoters, co-carcinogens, and inflammatory agents. These are not nearly as well characterized as the carcinogens, either with respect to identity or mechanism. While there are still unresolved issues, our current state of knowledge allows us to develop reasonable and testable hypotheses concerning the susceptibility of individual smokers to tobacco-induced lung cancer. If methods were available to predict which smoker will get lung cancer, they might be effectively used for preventing it. Acknowledgments  Studies in the Hecht laboratory on cigarette smoking and cancer are supported by grants CA-81301 and CA-92025 from the National Cancer Institute and grant ES-11297 from the National Institute of Environmental Health Sciences.

References Ahrendt SA, Decker PA, Alawi EA et al (2001) Cancer 92:1525–1530 Alexandrov K, Rojas M, Geneste O et al (1992) Cancer Res 52:6248–6253 Amos CI, Wu X, Broderick P et al (2008) Nat Genet 40:616–622 Baker RR, Pereira DS, Jr., Smith G (2004) Food Chem Toxicol 42 Suppl:S3–S37 Balansky R, Ganchev G, Iltcheva M et al (2007) Carcinogenesis 28:2236–2243 Beland FA, Churchwell MI, Von Tungeln LS et al (2005) Chem Res Toxicol 18:1306–1315 Belinsky SA (2005) Carcinogenesis 26:1481–1487 Belinsky SA, Foley JF, White CM et al (1990) Cancer Res 50:3772–3780 Bode AM, Dong Z (2005) Prog Nucl Acid Res Mol Biol 79:237–297

3  Tobacco Smoke Carcinogens and Lung Cancer

71

Boorman GA, Hailey R, Grumbein S et al (1994) Toxicol Pathol 22:545–554 Boyland E, Roe FJC, Gorrod JW (1964) Nature 202:1126 Boysen G, Hecht SS (2003) Mutat Res 543:17–30 Carmella SG, Chen M, Han S et al (2009) Chem Res Toxicol 22:734–741 Castro DJ, Lohr CV, Fischer KA et al (2008) Toxicol Appl Pharmacol 233:454–458 Cavalieri EL, Higginbotham S, Rama Krishna NVS et al (1991) Carcinogenesis 12:1939–1944 Chen L, Wang M, Villalta PW et al (2007) Chem Res Toxicol 20:108–113 Christmann M, Tomicic MT, Roos WP et al (2003) Toxicology 193:3–34 Church TR, Anderson KE, Caporaso NE et  al (2009) Cancer Epidemiol Biomarkers Prev 18:260–266 Coggins CRE (1998) Toxicol Pathol 26:307–314 Counts ME, Hsu FS, Laffoon SW et al (2004) Regul Toxicol Pharmacol 39:111–134 D’Agostini F, Izzotti A, Balansky R et al (2006) Cancer Res 66:3936–3941 Deutsch-Wenzel RP, Brune H, Grimmer G et al (1983) J Natl Cancer Inst 71:539–543 Dietrich M, Block G, Hudes M et al (2002) Cancer Epidemiol Biomarkers Prev 11:7–13 Ding L, Getz G, Wheeler DA et al (2008a) Nature 455:1069–1075 Ding YS, Ashley DL, Watson CH (2007) J Agric Food Chem 55:5966–5973 Ding YS, Trommel JS, Yan XJ et al (2005) Environ Sci Technol 39:471–478 Ding YS, Zhang L, Jain RB et al (2008b) Cancer Epidemiol Biomarkers Prev 17:3366–3371 Dipple A, Moschel RC, Bigger CAH (1984) Polynuclear aromatic hydrocarbons. In: Searle CE (Ed.), Chemical Carcinogens, Second Edition, ACS Monograph 182, vol. 2. American Chemical Society, Washington, DC, pp. 41–163 Druckrey H, Preussmann R, Ivankovic S et al (1967) Z Krebsforsch Klin Onkol 69:103–201 Farris GM, Everitt JI, Irons RD et al (1993) Fundam Appl Toxicol 20:503–507 Feng Z, Hu W, Hu Y et al (2006) Proc Natl Acad Sci USA 103:15404–15409 Fischer SM (1997) Cellular and molecular mechanisms of tumor promotion. In: Bowden GT, Fischer SM (Eds.), Comprehensive Toxicology, vol. 12, Chemical Carcinogens and Anticarcinogens. Elsevier Science, New York, pp. 349–381 Foiles PG, Akerkar SA, Carmella SG et al (1991) Chem Res Toxicol 4:364–368 Goggin M, Anderson C, Park S et al (2008) Chem Res Toxicol 21:1163–1170 Goggin M, Swenberg JA, Walker VE et al (2009) Cancer Res 69:2479–2486 Hammond D, O’Connor RJ (2008) Tob Control 17 Suppl 1:i24–i31 Harley NB, Cohen BS, Tso TC (1980) Polonium-210: a questionable risk factor in smoking related carcinogenesis. In: Gori GB, Beck FG (Eds.), Banbury Report 3: A Safe Cigarette? Cold Spring Harbor Laboratory, New York, pp. 93–104 Harvey RG (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity. Cambridge University Press, Cambridge, England, pp. 26–49 Hayes RB, Yin SN, Dosemeci M et al (1996) Environ Health Perspect 104 Suppl 6:1349–1352 Hecht SS (1998) Chem Res Toxicol 11:559–603 Hecht SS (1999) J Natl Cancer Inst 91:1194–1210 Hecht SS (2002) Carcinogenesis 23:907–922 Hecht SS (2003) Nat Rev Cancer 3:733–744 Hecht SS (2005) Carcinogenesis 26:1488–1492 Hecht SS, Bondinell WE, Hoffmann D (1974) J Natl Cancer Inst 53:1121–1133 Hecht SS, Carmella S, Mori H et al (1981) J Natl Cancer Inst 66:163–169 Hecht SS, Chen CB, Hirota N et al (1978a) J Natl Cancer Inst 60:819–824 Hecht SS, Chen CB, Ornaf RM et al (1978b) J Org Chem 43:72–76 Hecht SS, Han S, Kenney PMJ et al (2007) Chem Res Toxicol 20:543–549 Heeschen C, Jang JJ, Weis M et al (2001) Nat Med 7:833–839 Hoffmann D, Brunnemann KD, Prokopczyk B et al (1994) J Toxicol Environ Health 41:1–52 Hoffmann D, Hecht SS, Ornaf RM et al (1974) Science 186:265–267 Hoffmann D, Schmeltz I, Hecht SS et  al (1978) Tobacco carcinogenesis. In: Gelboin H, Ts’o POP (Eds.), Polycyclic Hydrocarbons and Cancer. Academic Press, New York, pp. 85–117

72

S.S. Hecht

Hoffmann D, Wynder EL (1971) Cancer 27:848–864 Hölzle D, Schlöbe D, Tricker AR et al (2007) Toxicology 232:277–285 Huff JE, Melnick RL, Solleveld HA et al (1985) Science 227:548–549 Hung RJ, McKay JD, Gaborieau V et al (2008) Nature 452:633–637 Hutt JA, Vuillemenot BR, Barr EB et al (2005) Carcinogenesis 26:1999–2099 International Agency for Research on Cancer (1974) Some Anti-Thyroid and Related Substances, Nitrofurans and Industrial Chemicals. IARC Monographs on the Carcinogenic Risk of Chemicals to Man, vol. 7. IARC, Lyon, France International Agency for Research on Cancer (1986) Tobacco Smoking. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, vol. 38. IARC, Lyon, France International Agency for Research on Cancer (1987) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 7. International Agency for Research on Cancer, Lyon, France International Agency for Research on Cancer (1990) Chromium, Nickel, and Welding. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 49. IARC, Lyon, France International Agency for Research on Cancer (1993) Beryllium, Cadmium, Mercury, and Exposures in the Glass Manufacturing Industry. Monographs on the Carcinogenic Risk of Chemicals to Humans, vol. 58. IARC, Lyon, France International Agency for Research on Cancer (1994) Some Industrial Chemicals. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, vol. 60. IARC, Lyon, France International Agency for Research on Cancer (1999) Re-evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide (Part Three). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 71. IARC, Lyon, France International Agency for Research on Cancer (2001) Ionizing Radiation, Part 2: Some Internally Deposited Radionuclides. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 78. IARC, Lyon, France International Agency for Research on Cancer (2004a) Tobacco Smoke and Involuntary Smoking. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 84. IARC, Lyon, France International Agency for Research on Cancer (2004b) Some Drinking-Water Disinfectants and Contaminants, Including Arsenic. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 84. IARC, Lyon, France International Agency for Research on Cancer (2007) Smokeless Tobacco and Tobacco-Specific Nitrosamines. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 89. IARC, Lyon, France International Agency for Research on Cancer (2008) 1,3-Butadiene, Ethylene Oxide, and Vinyl Halides (Vinyl Fluoride, Vinyl Chloride, and Vinyl Bromide). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 97. IARC, Lyon, France Jalas J, Hecht SS, Murphy SE (2005) Chem Res Toxicol 18:95–110 Johnson L, Mercer K, Greenbaum D et al (2001) Nature 410:1111–1116 Kim MY, Cho MH (2009) Toxicol Ind Health 25:189–195 Kim V, Rogers TJ, Criner GJ (2007) Semin Thorac Cardiovasc Surg 19:135–141 Lee JM, Yanagawa J, Peebles KA et al (2008) Crit Rev Oncol Hematol 66:208–217 Le Marchand L, Derby KS, Murphy SE et al (2008) Cancer Res 68:9137–9140 Lijinsky W (1992) Chemistry and Biology of N-Nitroso Compounds. Cambridge University Press, Cambridge, England Liu G, Zhou W, Christiani DC (2005) Semin Respir Crit Care Med 26:265–272 Lubet RA, Zhang Z, Wiseman RW et al (2000) Exp Lung Res 26:581–593 Mackay J, Jemal A, Lee NC et al (2006) The Cancer Atlas. American Cancer Society, Atlanta, GA, pp. 30–31 Magee PN, Barnes JM (1956) Br J Cancer 10:114–122 Malkinson AM (2005) Exp Lung Res 31:57–82

3  Tobacco Smoke Carcinogens and Lung Cancer

73

Matter B, Wang G, Jones R et al (2004) Chem Res Toxicol 17:731–741 Mauderly JL, Gigliotti AP, Barr EB et al (2004) Toxicol Sci 81:280–292 Melikian AA, Jordan KG, Braley J et al (1989) Carcinogenesis 10:1897–1900 Moraitis D, Du B, De Lorenzo MS et al (2005) Cancer Res 65:664–670 Nebert DW, Dalton TP, Okey AB et al (2004) J Biol Chem 279:23847–23850 Nesnow S, Ross JA, Mass MJ et al (1998) Exp Lung Res 24:395–405 O’Donnell EP, Zerbe LK, Dwyer-Nield LD et al (2006) Cancer Lett 241:197–202 Pappas RS, Polzin GM, Zhang L et al (2006) Food Chem Toxicol 44:714–723 Park J-H, Mangal D, Tacka KA, Quinn AM et al (2008) Proc Natl Acad Sci USA 105:6846–8851 Pfeifer GP, Denissenko MF, Olivier M et al (2002) Oncogene 21:7435–7451 Phillips DH (1983) Nature 303:468–472 Phillips DH (2002) Carcinogenesis 23:1979–2004 Platt KL, Dienes HP, Tommasone M et al (2004) Chem Biol Interact 148:27–36 Preussmann R, Stewart BW (1984) N-nitroso carcinogens. In: Searle CE (Ed.), Chemical Carcinogens, Second Edition, ACS Monograph 182. American Chemical Society, Washington, DC, pp. 643–828 Prokhorov AV, Hudmon KS, Stancic N (2003) Paediatr Drugs 5:1–10 Pryor WA, Stone K, Zang LY et al (1998) Chem Res Toxicol 11:441–448 Richter E, Engl J, Friesenegger S et al (2009) Chem Res Toxicol 22:1008–1017 Rodgman A, Perfetti T (2009) The Chemical Components of Tobacco and Tobacco Smoke. CRC Press, Boca Raton, FL, pp. 1483–1784 Roemer E, Stabbert R, Rustemeier K et al (2004) Toxicology 195:31–52 Rojas M, Camus AM, Alexandrov K et al (1992) Carcinogenesis 13:929–933 Rubin H (2001) Carcinogenesis 22:1903–1930 Schlobe D, Holzle D, Hatz D et al (2008) Toxicology 245:154–161 Schuller HM (2002) Nat Rev Cancer 2:455–463 Schuller HM (2009) Nat Rev Cancer 9:195–205 Seidel A, Frank H, Behnke A et al (2004) Polycycl Aromat Compd 24:759–771 Shen YM, Troxel AB, Vedantam S et al (2006) Chem Res Toxicol 19:1441–1450 Shimada T, Guengerich FP (2006) Chem Res Toxicol 19:288–294 Shimkin MB, Stoner GD (1975) Adv Cancer Res 21:1–58 Smith CJ, Perfetti TA, King JA (2006) Inhal Toxicol 18:667–677 Smith LE, Denissenko MF, Bennett WP et al (2000) J Natl Cancer Inst 92:803–811 Smith PAS, Loeppky RN (1967) J Am Chem Soc 89:1148–1152 Snook ME, Severson RF, Arrendale RF et al (1977) Beitr Tabakforsch 9:79–101 Stanton MF, Miller E, Wrench C et al (1972) J Natl Cancer Inst 49:867–877 Stavrides JC (2006) Free Radic Biol Med 41:1017–1030 Stepanov I, Upadhyaya P, Feuer R et al (2008) Cancer Epidemiol Biomarkers Prev 17:1764–1773 Stinn W, Teredesai A, Kuhl P et al (2005) Inhal Toxicol 17:263–276 Straif K, Baan R, Grosse Y et al (2005) Lancet Oncol 6:931–932 Thompson CA, Burcham PC (2008) Chem Res Toxicol 21:2245–2256 Thorgeirsson TE, Geller F, Sulem P et al (2008) Nature 452:638–642 Tretyakova N, Matter B, Jones R et al (2002) Biochemistry 41:9535–9544 Tseng JE, Kemp BL, Khuri FR et al (1999) Cancer Res 59:4798–4803 Tso TC (1990) Production, Physiology, and Biochemistry of Tobacco Plant. Ideals, Bethesda, MD, pp. 369–378 Turner MC, Chen Y, Krewski D et al (2007) Am J Respir Crit Care Med 176:285–290 U.S. Department of Health and Human Services (2004) Report on Carcinogens, 11th Edition. Research Triangle Park, North Carolina Van Duuren BL, Goldschmidt BM (1976) J Natl Cancer Inst 56:1237–1242 Veglia F, Loft S, Matullo G et al (2008) Carcinogenesis 29:932–936 Wang M, Cheng G, Balbo S et al (2009) Cancer Res 69:7170–7174 West KA, Brognard J, Clark AS et al (2003) J Clin Invest 111:81–90

74 Witschi H (2000) Exp Lung Res 26:743–755 Witschi H (2005) Exp Lung Res 31:3–18 Wynder EL, Graham EA, Croninger AB (1953) Cancer Res 13:855–864 Yuan JM, Koh WP, Murphy SE et al (2009) Cancer Res 69:2990–2995 Zhang S, Villalta PW, Wang M et al (2006) Chem Res Toxicol 19:1386–1392 Zhao C, Kumar R, Hemminki K (2000) Biomarkers 3:327–334 Zhao C, Tyndyk M, Eide I et al (1999) Mutat Res 424:117–125

S.S. Hecht

Chapter 4

Mechanisms of Estrogen Carcinogenesis: Modulation by Botanical Natural Products Judy L. Bolton

Abstract  The longer women are exposed to estrogens, through early menarche and late menopause and/or through hormone replacement therapy (HRT), the higher is the risk of developing certain hormone-dependent cancers. It has become clear that there are likely multiple overlapping mechanisms of estrogen carcinogenesis. This review is focused on the chemical mechanism of estrogen carcinogenesis involving metabolism of estrogens to catechols mediated by cytochrome P450 and further oxidation of these catechols to estrogen o-quinones. These electrophilic/ redox active quinones can cause damage within cells by alkylation and/or oxidation of cellular proteins and DNA in many tissues. Finally, there is evidence to suggest that botanical dietary supplements have cytoprotective/cytotoxic properties that could modulate estrogen-dependent cancers in both pre- and postmenopausal women by blocking key critical steps in the estrogen genotoxicity pathway. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.

1 Risks of Estrogen Exposure Recent data have estimated that 192,370 women will develop breast cancer in 2009 in the USA and 40,170 will die from this disease (Jemal et al. 2009). Experimental and epidemiological data strongly associate excessive estrogen exposure to the development of hormone-dependent cancers, particularly breast and endometrial cancer (Chen 2008). The longer women are exposed to estrogens, through early menarche and late menopause and/or through hormone replacement therapy (HRT), the higher is the risk of developing these cancers. In the past, it was thought that the J.L. Bolton (*) Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago, IL, USA e-mail: [email protected]

T.M. Penning (ed.), Chemical Carcinogenesis, Current Cancer Research, DOI 10.1007/978-1-61737-995-6_4, © Springer Science+Business Media, LLC 2011

75

76

J.L. Bolton

purported benefits of HRT, which included relief of menopausal symptoms, decrease in coronary heart disease, osteoporosis, stroke, and Alzheimer’s disease, justified its long-term use. However, the release of the initial results from the Women’s Health Initiative Study in July 2002 cast serious doubt on this paradigm for the treatment of postmenopausal women (Rossouw et al. 2002). The estrogen plus progestin arm was halted 3 years early due to significant increases in breast cancer, coronary heart disease, stroke, and pulmonary embolism, with more recent data suggesting an increase in vascular disease in women over 65 on HRT (Shumaker et  al. 2003). In 2004, the estrogen only arm was halted because of increased incidence of stroke (Brass 2004). A recent analysis of data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) registries have shown that age-adjusted incidence rate of breast cancer fell sharply in 2003 and has been sustained through 2005, which seemed to be related to the drop in the use of HRT (Ravdin et al. 2007; Chlebowski et al. 2009). The reductions in breast cancer incidence were greatest among 50–60-year-old women (those most likely to be current users of HRT) and most pronounced for ER+ and PR+ cancers (most strongly related to HRT use) (Verkooijen et  al. 2009). Similar trends have since been reported in other industrialized countries (Kumle 2008). Finally, a reanalysis of nine prospective studies has shown that exposure to estrogens is associated with an increase in breast cancer risk with evidence of a dose–response relationship (Key et al. 2002). These troubling findings highlight the urgent need for a full understanding of all the deleterious effects of estrogens including their potential to initiate and/or promote the carcinogenic process. Most of the epidemiological studies on HRT and cancer risk, including the WHI study discussed above, have been conducted with either Premarin® or Prempro® (Premarin® plus the progestin, medroxyprogesterone acetate), both of which remain the estrogen replacement treatments of choice and widely prescribed drugs in North America (Wysowski and Governale 2005). Premarin® was approved by the Food and Drug Administration in the 1940s, yet very little is known about the metabolism and potential toxic metabolites that could be produced from the various equine estrogens, which make up approximately 50% of the estrogens in Premarin® (Purdy et al. 1982; Li et al. 1995; Sarabia et al. 1997; Bhavnani 1998; Zhang et al. 1999) (Fig. 1).

Fig. 1  Major estrogens present in Premarin

4  Mechanisms of Estrogen Carcinogenesis

77

It is known that treating hamsters for 9 months with either estrone or equilin + equilenin or sulfatase-treated Premarin® resulted in 100% kidney tumor incidences and abundant tumor foci (Li et al. 1995). Furthermore, in a small clinical trial of 596 postmenopausal women, a significant increase in endometrial hyperplasia was found in those women receiving a daily dose of 0.625  mg of Premarin® (Judd et al. 1996). Nevertheless, HRT is still the most effective remedy for relief of symptoms of menopause such as sleeplessness, hot flashes, and mood swings, and provides protection against early menopausal bone loss, and lowers the risk of colon cancer (Rossouw et al. 2002; Hays et al. 2003). For these reasons, women continue to use HRT (Hersh et  al. 2004) in spite of the well-recognized risks (Zumoff 1998). Although the sales of Premarin® and Prempro® prescriptions have plummeted since July 2002 when NHLBI terminated the clinical trial on the long-term risks and benefits of estrogen plus progestin therapy (Barbaglia et  al. 2009), both remain on the list of the “Top 200 prescribed drugs in North America” in 2008 (2008). This highlights the need to fully understand the deleterious effects of equine and endogenous estrogens in this large population of postmenopausal women exposed to HRT.

2 Mechanisms of Estrogen Carcinogenesis 2.1 Hormonal Mechanism The molecular mechanisms of estrogen carcinogenesis are still not well understood (Jefcoate et  al. 2000; Russo et  al. 2000; Cavalieri et  al. 2006; Russo and Russo 2006; Yager and Davidson 2006). Malignant phenotypes arise as a result of a series of mutations, most likely in genes associated with tumor suppression, oncogenesis, DNA repair, or endocrine functions (Henderson and Feigelson 2000). One major pathway considered to be important is the extensively studied hormonal pathway (Fig. 2, hormonal, using estradiol as an example) by which estrogen stimulates cell proliferation through nuclear ER-mediated signaling pathways, thus resulting in an increased risk of genomic mutations during DNA replication (Nandi et  al. 1995; Feigelson and Henderson 1996; Henderson and Feigelson 2000; Flototto et al. 2001). A similar “non-genomic pathway,” potentially involving newly discovered endoplasmic reticulum (GPR30) and/or membrane-associated ERs, also appears to regulate extranuclear estrogen signaling pathways (Revankar et  al. 2005; Song et  al. 2006; Levin 2009; Prossnitz and Maggiolini 2009). Recent studies have also shown the presence of ERa and ERb in the mitochondria of various cells and tissues, which may be involved in deregulation of mitochondrial bioenergetics, contributing to estrogen-related cancers (Chen et al. 2008). Cross-talk between these genomic and second-messenger pathways probably has important roles in estrogenic control of cell proliferation, inhibition of apoptosis, and induction of DNA mutation and damage.

78

J.L. Bolton

Fig.  2  Summary of potential carcinogenic mechanisms for estrogens using estradiol as an example. ER estrogen receptor, mER membrane-associated estrogen receptor, ERE estrogen response element, NF-kB nuclear factor kappa B, CRE cyclic AMP responsive element, AP-1 activator protein 1, Sp1 steroidogenic protein 1, ROS reactive oxygen species

2.2 Chemical Mechanism Estrogen metabolism, mediated by cytochrome P450, that generates reactive electrophilic estrogen o-quinones and reactive oxygen species (ROS) through redox cycling of these o-quinones may also contribute to estrogen carcinogenesis (Fig. 2, chemical). Studies have shown that constitutive and TCDD-inducible P450 isozymes, P4501B1 (Fig. 3, step 2) and P4501A1 (Fig. 3, step 3), selectively catalyze hydroxylation of estrone and 17b-estradiol (Spink et al. 1997, 1998; Shimada et al. 1999), suggesting that excessive exposure to environmental pollutants could lead to enhanced production of these catechols. P4501B1 metabolism could be particularly significant since only 4-hydroxyestrone/estradiol was found to be carcinogenic in the male Syrian golden hamster kidney tumor model, whereas 2-hydroxylated metabolites were without activity (Liehr et al. 1986; Li and Li 1987). Similarly, Newbold and Liehr have shown that 4-hydroxyestradiol induced uterine tumors in 66% of CD-1 mice, whereas mice treated with 2-hydroxyestradiol or 17b-estradiol had much lower uterine tumor incidence (Newbold and Liehr 2000). In women, significantly higher amounts of GSH conjugates resulting from reaction of GSH with the 4-OHE1/E2-o-quinones were detected in the nontumor tissue from women with breast cancer compared to women without the disease (Rogan et al. 2003). In addition, estrogen 4-hydroxylase

Fig. 3  Botanical modulation of chemical mechanisms of estradiol carcinogenesis

4  Mechanisms of Estrogen Carcinogenesis 79

80

J.L. Bolton

levels (P4501B1 and 1A1) had higher expression in breast tissue of women with breast cancer, whereas expression of protective enzymes (e.g., NQO1) was lower (Singh et  al. 2005). Finally, epidemiological studies have suggested a link between genetic polymorphism in estrogen 4-hydroxylases and a risk for developing breast cancer (Zheng et al. 2000; Kisselev et al. 2005), and recent studies have suggested that specific polymorphisms in these P450 genes may modify the effect of HRT use on breast cancer risk (Diergaarde et al. 2008). These data suggest that estrogen metabolites are obligate contributors to the development of cancer.

2.3 Estrogen Receptor as a Trojan Horse Estrogens that are potent ER agonists and are oxidized to electrophilic and redoxcycling metabolites have the potential to contribute to the initiation, promotion, and progression of hormone-sensitive cancers as dual-mechanism carcinogens (combined hormonal and chemical mechanisms discussed above, Fig. 2). If catechol estrogens represent good estrogenic ligands, the ER would be capable of translocating these genotoxins to the nucleus where oxidative DNA damage would be amplified, even at low concentrations. The ER would act as a Trojan horse, and ER-positive cells would be highly sensitive to DNA damage (Fig.  2). We have preliminary data that this mechanism may play a role in catechol estrogen-induced DNA damage (Chen et al. 2000; Liu et al. 2002). We have examined the effect of ER status on the relative ability of 4-OHEN and 4,17b-OHEN to induce DNA damage in ER-negative cells (MDA-MB-231), ERa-positive cells (S30), and ERbpositive cells (b41). The data showed that both 4-OHEN and 4,17b-OHEN induced concentration-dependent DNA single-strand cleavage in all three cell lines. However, cells containing ERs had significantly higher DNA damage. The endogenous catechol estrogen metabolite 4-hydroxyestrone was considerably less effective in inducing DNA damage in breast cancer cell lines as compared to 4-OHEN (Chen et al. 2000). Recently, we have shown that the rate of 4-OHENinduced DNA damage was significantly enhanced in ERa(+) cells, whereas ER status had no effect on the rate of menadione-induced DNA damage (where menadione is a nonestrogenic quinone) (Wang et al. 2009). Imaging of ROS induced by 4-OHEN showed selective accumulation in the nucleus of ERa(+) cells within 5 min, whereas in ER(−) cells or menadione-treated cells, ROS did not selectively increase in the nucleus. Our data suggest that the genotoxic effects of 4-OHEN could be related to its ability to induce DNA damage in hormone-sensitive cells in vivo, and these effects may be potentiated by the ER. The Trojan horse model could potentially apply to any nuclear receptor whose ligands are genotoxic, e.g., the binding and translocation of polycyclic aromatic hydrocarbon o-quinones to the nucleus by the aryl-hydrocarbon receptor (Burczynski and Penning 2000; Park et al. 2009a).

4  Mechanisms of Estrogen Carcinogenesis

81

3 DNA Damage 3.1 Oxidative DNA Damage Estrogen chemical carcinogenesis describes the capacity of biologically reactive intermediates derived from estrogen metabolism to cause DNA damage by electrophilic and oxidative reactions leading to genotoxicity (Liehr 2001). For example, estrogens (Figs. 2 and 3) o-quinones can undergo redox cycling with the semiquinone radical generating superoxide radicals mediated through cytochrome P450 NADPH oxidoreductase. The conversion of superoxide anion radicals to hydrogen peroxide, formed by the enzymatic or spontaneous dismutation of superoxide anion radical, in the presence of trace amounts of iron or other transition metals gives rise to hydroxyl radicals. The hydroxyl radicals are powerful oxidizing agents that may be responsible for damage to essential macromolecules. In support of this mechanism, various free radical toxicities have been reported in hamsters treated with 17b-estradiol including DNA single-strand breaks (Nutter et  al. 1991; Roy and Liehr 1999), 8-oxo-dG formation (Cavalieri et  al. 2000; Lavigne et  al. 2001; Rajapakse et al. 2005), and chromosomal abnormalities (Li et al. 1993; Banerjee et al. 1994; Russo and Russo 2006). It has also been shown that 4-hydroxyestradiol also induces oxidative stress and apoptosis in human mammary epithelial cells (MCF-10A), although the concentrations used in this study (25 mM) have questionable physiological relevance (Chen et al. 2005). Studies in MCF-7 cells required depletion of GSH levels prior to detection of significant increases in 8-oxo-dG after treating cells with 2-hydroxyestradiol or 4-hydroxyestradiol (10  mM) for 30  min (Mobley and Brueggemeier 2002). Similarly, pretreatment of MCF-7 cells with the P450 inducer dioxin as well as the COMT inhibitor Ro 41-0960 was necessary to detect increased 8-oxo-dG levels after treatment with 0.1  mM estradiol (Lavigne et al. 2001). Micromolar concentrations (20 mM) of 4-hydroxyestradiol in MCF-10A cells were also required to generate time-dependent increases in DCF-fluorescence staining, which is indicative of induced intracellular accumulation of ROS (Park et  al. 2009b). Given the limited number of reports of oxidative damage to DNA induced by catechol estrogens and the high concentrations necessary to achieve significant increase in these oxidized lesions, it is unclear if oxidative damage to DNA plays a significant role in the carcinogenesis mechanisms of endogenous estrogens. The equilenin catechol 4-OHEN, which is the major phase 1 metabolite of both equilin and equilenin (Fig. 1), is also capable of causing DNA single-strand breaks and oxidative damage to DNA bases both in  vitro and in  vivo (Chen et  al. 2000; Zhang et al. 2001; Liu et al. 2002; Okamoto et al. 2008). Injection of 4-OHEN into the mammary fat pads of Sprague Dawley rats resulted in a dose-dependent increase in single-strand breaks and oxidized bases as analyzed by the comet assay (Zhang et al. 2001). In addition, extraction of mammary tissue DNA, hydrolysis to deoxynucleosides, and analysis by LC-MS-MS showed the formation of 8-oxo-dG as well

82

J.L. Bolton

as 8-oxo-dA. In mice treated with equilenin, the levels of 8-oxo-dG were increased 1.5-fold in the uterus (Okamoto et al. 2008). In women, a recent study has evaluated the potential of HRT to induce DNA damage in peripheral blood leukocytes of postmenopausal women using the comet assay (Ozcagli et  al. 2005). Significant increases in DNA damage were observed among women receiving 0.625  mg/day conjugated equine estrogens or conjugated equine estrogens plus medroxyprogesterone acetate as compared to the control group that had never received HRT. Finally, the excessive production of ROS in breast cancer tissue has been linked to metastasis of tumors in women with breast cancer (Malins et  al. 1996; Malins et  al. 2006; Karihtala and Soini 2007; Benz and Yau 2008). These and other data provide evidence for a mechanism of estrogen-induced tumor initiation/promotion by redox cycling of estrogen metabolites generating ROS, which damage DNA.

3.2 Covalent DNA Adducts Estrogen quinoids can form covalent adducts with cellular DNA, leading to genotoxic effects (Liehr 2000; Chakravarti et al. 2001; Bolton et al. 2004; Li et al. 2004; Russo and Russo 2004; Prokai-Tatrai and Prokai 2005; Cavalieri et al. 2006; Bolton and Thatcher 2008; Gaikwad et al. 2008; Zhang and Gross 2008). Cavalieri’s group has reported that the major DNA adducts produced from 4-hydroxyestradiol-oquinone are depurinating N7-guanine and N3-adenine adducts resulting from 1,4-Michael addition both in vitro and in vivo (Fig. 3) (Stack et al. 1996; Cavalieri et al. 2000; Li et al. 2004; Cavalieri et al. 2006; Zahid et al. 2006; Saeed et al. 2007; Gaikwad et  al. 2008). Interestingly, they have recently concluded that only the N3-adenine adduct is likely to induce mutations, since this adduct depurinates instantaneously, whereas the N7-guanine adduct takes hours to hydrolyze (Saeed et al. 2005; Zahid et al. 2006). In contrast, the considerably more rapid isomerization of the 2-hydroxyestradiol-o-quinones to corresponding quinone methides results in 1,6-Michael addition products with the exocyclic amino groups of adenine and guanine (Fig. 4) (Stack et al. 1996; Debrauwer et al. 2003). Unlike the N3 and N7 purine DNA adducts, these adducts are stable, which may alter their rate of repair and relative mutagenicity in  vivo. A depurinating N3-adenine adduct of 2-hydroxyestradiol quinone methide has recently been reported in reactions with adenine and DNA (Fig. 4) (Zahid et al. 2006). The levels of this adduct were considerably lower than corresponding depurinating adducts observed with similar experiments with 4-hydroxyestradiol-o-quinone, which may explain why 2-hydroxylation is considered a benign metabolic pathway, whereas 4-hydroxylation results in carcinogenesis. Finally, this same study (Zahid et  al. 2006) suggested that depurinating DNA adducts of estrogen quinoids were formed in much greater abundance compared to stable bulky adducts, implying a causal role for these adducts in estrogen carcinogenesis; however, the depurinating adducts were analyzed by different methods (HPLC with electrochemical detection) as compared to the stable adducts (32P-postlabeling/TLC), making direct quantitative

4  Mechanisms of Estrogen Carcinogenesis

83

Fig. 4  Stable quinone methide DNA adducts formed from 2-hydroxyestradiol

comparisons problematic. The mutagenic properties of 2-hydroxyestrogen-­quinonemethide-derived stable DNA adducts have been evaluated using oligonucleotides containing site-specific adducts transfected into simian kidney (COS-7) cells where G → T and A → T mutations were observed (Terashima et al. 2001). It is important to mention that stable DNA adducts have been detected by 32P-postlabeling in Syrian hamster embryo cells treated with estradiol and its catechol metabolites (Hayashi et  al. 1996). The rank order of DNA adduct formation that correlated with cellular transformation was 4-hydroxyestradiol > 2-hydroxyestradiol > estradiol. Finally, stable bulky adducts of 4-hydroxyestrone and 4-hydroxyestradiol corresponding to alkylation of guanine have been detected in human breast tumor tissue (Embrechts et al. 2003). These data suggest that the relative importance of depurinating adducts versus stable DNA adducts in catechol estrogen carcinogenesis remains unclear. Recently, there have been efforts to correlate depurinating estrogen DNA adducts with breast cancer risk. Ratios of depurinating DNA adducts to their respective estrogen metabolites were significantly higher in high-risk women (12 subjects) and women with breast cancer (17 subjects) compared to healthy women (46 subjects) (Gaikwad et al. 2008). However, another much smaller study (six subjects total) did not have the precision to conclude if the levels of depurinating estrogen DNA adducts were elevated in breast tissue from cancer patients (Zhang et  al. 2008). More importantly, the levels of depurinating DNA adducts

84

J.L. Bolton

were close to the detection limits of the instrument (20–70 fmol/g tissue) and were two orders of magnitude less than that reported in an earlier Cavalieri study (Markushin et al. 2003). It is difficult to compare the results from the more recent Cavalieri study (Gaikwad et  al. 2008) with the gross results (Zhang et al. 2008), since only ratios of adducts were reported, instead of fmol/g tissue. As a result, it is still not clear if depurinating estrogen DNA adducts can be used as biomarkers for breast cancer risk. For the major equine estrogens (equilin and equilenin and 17b-ol derivatives), the data strongly suggest that the majority of DNA damage results from reactions of 4-hydroxyequilenin-o-quinone through a combination of oxidative damage (i.e., single-strand cleavage and oxidation of DNA bases), and through generation of apurinic sites, as well as through formation of stable bulky cyclic adducts (Fig. 5) (Bolton and Thatcher 2008). For example, a depurinating guanine adduct was detected in in vivo experiments with rats treated with 4-OHEN, following LC-MS-MS analysis of extracted mammary tissue (Zhang et  al. 2001). However, isolation of mammary tissue DNA, hydrolysis to deoxynucleosides, and analysis by LC-MS/MS also showed the formation of stable cyclic deoxyguanosine and deoxyadenosine adducts, as well as the above-mentioned oxidized bases and single-strand breaks. Interestingly, the ratio of the diasteriomeric adducts detected in vivo differs from in vitro experiments, suggesting that there are differences in the response of these stereoisomeric lesions to DNA replication and repair enzymes (Ding et al. 2003; Kolbanovskiy et al. 2005; Yasui et al. 2006; Ding et al. 2007). Finally, in a recent report, highly sensitive nano LC/MS-MS techniques have been used to analyze the DNA in five human breast

Fig. 5  Metabolism of equilenin benign and mutagenic metabolites forming stable DNA adducts

4  Mechanisms of Estrogen Carcinogenesis

85

tumor and five adjacent tissue samples, including samples from donors with a known history of Premarin-based HRT (Embrechts et al. 2003). While the sample size was small, and the history of the patients was not fully known, cyclic 4-hydroxyequilenin-dC, -dG, and -dA stable adducts were detected for the first time in four out of the ten samples. These results suggest that 4-hydroxyequilenin has the potential to be carcinogenic through the formation of a variety of DNA lesions in vivo.

4 Chemoprevention of Estrogen Carcinogenesis If catechol estrogen-induced DNA damage is a major mechanism contributing to estrogen carcinogenesis, it should be possible to lower the level of DNA damage, which may lead to a reduction in breast cancer risk. A number of protection mechanisms have been proposed including preventing the formation of estrone/estradiol with aromatase inhibitors (Fig. 3, step 1) (Castrellon and Gluck 2008). However, this strategy is not practical for healthy women, since it places women under chemical menopause and removes all benefits of estrogens including protection from osteoporosis. Another obvious strategy would, therefore, be the inhibition of CYP1B1 (Bruno and Njar 2007), which catalyzes 4-hydroxyestrogen formation (Fig.  3, step 2). Studies with CYP1B1 knockout mice demonstrated that animals lacking this gene developed normally and showed no noticeable deficiencies. Furthermore, CYP1B1 knockout mice showed strong resistance to 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumor formation (Gonzalez 2002). These studies provide evidence for the potential efficacy and safety of a chemopreventive agent for estrogen carcinogenesis that blocks CYP1B1 expression or activity. However, chemoprevention strategies based on inhibition of P450s are probably not practical due to the lack of isoform selectivity manifested by inhibitors. Alternatively, agents that control regulation of CYP1B1 may be a more persuasive approach to chemopreventive therapy. If it is not practical to prevent formation of the catechols/o-quinones, it may be possible to enhance their rate of detoxification. This could be achieved by COMT-catalyzed methylation of catechol estrogens (Fig.  3, step 4), reduction of estrogen quinones by quinone reductase (Fig.  3, step 5), scavenging of estrogen semiquinone radicals by antioxidants (Fig.  3, step 7), or conjugation of estrogen quinones with thiols such as GSH (Fig. 3, step 6) (Zahid et al. 2008). It has been shown that treatment of MCF-10F nontumorigenic breast epithelial cells with 4-hydroxyestradiol and the COMT inhibitor Ro41-0960 resulted in threefold to fourfold increases in the levels of depurinating N3Adenine and N7guanine adducts (Zahid et al. 2007). Similarly, knockdown of COMT expression increased neoplastic transformation of immortalized human endometrial glandular (EM) cells treated with 4-hydroxyestradiol (Salama et  al. 2008). As far as a link between genetic polymorphisms in COMT and risk of breast cancer are concerned, the data are equivocal (Bugano et al. 2008).

86

J.L. Bolton

It has been reported that induction of NQO1 activity protects against estrogeninduced oxidative DNA damage in vitro and in vivo (Montano et al. 2007). These correlative findings were supported by findings that NQO1 downregulation led to increased levels of estrogen quinone metabolites and enhanced estrogen-induced transformation in MCF10A nontumorigenic breast epithelial cells. Since epidemiological evidence indicates that genetically deficient NQO1 is a risk factor for development of cancer (Cornblatt et al. 2007), it is quite reasonable to hypothesize that NQO1 deficiency plays an important role in estrogen-dependent cancer etiology. A recent report has showed that 4-hydroxyestrone o-quinone was observed to be a substrate for NQO1; however, the acceleration of NADPH-dependent reduction by NQO1 over the nonenzymatic reaction was less than tenfold and the same at more relevant nanomolar concentrations of substrate was less than twofold (Chandrasena et al. 2008). These results indicate that a key role for NQO1 in direct detoxification of 4-hydroxy-estrogen quinones is problematic.

5 Botanical Modulation of Estrogen Carcinogenesis Women frequently use botanical dietary supplements for the alleviation of menopausal symptoms, especially for the reduction of hot flashes (Newton et al. 2002; Mahady et al. 2003). As botanical dietary supplements are considered to be safer than traditional HRT, many women prefer botanical alternatives. However, the efficacy of the alleviation of menopausal symptoms and safety of these botanical dietary supplements have not been established yet (The North American Menopause Society 2004). Previous studies have shown that botanical dietary supplements have a number of biological effects that could be related to efficacy in relieving menopausal symptoms including estrogenic (red clover, hops) (Liu et  al. 2001; Burdette et al. 2002; Overk et al. 2005; Overk et al. 2008), progestinic (red clover), and serotoninergic (black cohosh, dang gui) symptoms (Burdette et al. 2003; Deng et al. 2006; Powell et al. 2008). However, numerous clinical trials (Piersen et al. 2004; Booth et al. 2006; Geller et al. 2009; Palacio et al. 2009; Rees 2009) suggest that most botanicals have little effect on post menopausal hot flashes. In contrast, there is limited evidence to suggest that botanical dietary supplements have cytoprotective/cytotoxic properties that could modulate estrogen-dependent cancers in both pre- and postmenopausal women (Fig. 3) (Mandlekar et al. 2006; Mense et al. 2008b).

5.1 Hormonal Phytoestrogens are weak estrogenic compounds that can compete with endogenous estrogens for binding to both ERa and ERb. Several phytoestrogens have been identified in botanical dietary supplements, including the isoflavones genistein and diazein in red clover (Liu et al. 2001; Overk et al. 2005), 8-prenylnaringenin in hops

4  Mechanisms of Estrogen Carcinogenesis

87

Fig. 6  Structures of representative phytoestrogens and resveratrol in botanical dietary supplements

(Milligan et al. 2002; Overk et al. 2005), and liquiritigenin in licorice (Cvoro et al. 2007; Kupfer et al. 2008; Mersereau et al. 2008) (Fig. 6). Unlike estradiol, which binds ERa and ERb with similar affinity, many phytoestrogens have much higher affinity for ERb and may induce ERb-mediated antiproliferative effects (McDonnell 2004). Alternatively, some phytoestrogens have been found to act synergistically with estradiol to activate both ERa- and ERb-induced gene transcription (Harris et al. 2005) as well as potentiate the growth of ER-positive xenografts in nude mice (Ju et al. 2006). Therefore, phytoestrogens have the potential to reduce and/or enhance the hormonal-related carcinogenic effects of endogenous estrogens (Fig. 2, hormonal pathway). In addition to this pathway, it has been suggested that phytoestrogens can reduce cancer risk through other pathways, including their effect on estrogen metabolism and their antioxidant effects (Fig. 3) (Messina et al. 1994; Kurzer and Xu 1997; Horn-Ross et al. 2001).

5.2 Chemical Aromatase, CYP1A1, CYP1B1, COMT, and NQO1 have been identified to play critical roles in the proposed pathway of carcinogenic activation of estrogens to estrogen o-quinones, ultimate carcinogens (Fig. 3). These enzymes have polymorphic variants with altered enzyme activities that are known to contribute to the risk of breast cancer (Huang et al. 1999; Cheng et al. 2005; Modugno et al. 2005; Hu et al. 2007). The net effect of the polymorphic variants of these genes could be to unbalance estrogen homeostasis, thus favoring formation of estrogen o-quinones, their reaction with DNA, and generation of tumor-initiating mutations. Cavalieri’s group has shown that resveratrol can prevent estrogen DNA adduct formation and neoplastic transformation of MCF-10A cells through a variety of different mechanisms (Lu et  al. 2008). Resveratrol downregulates P4501B1 (step 2, Fig.  3),

88

J.L. Bolton

which decreases the levels of the putative carcinogen 4-hydroxyestradiol. Resveratrol upregulates the potentially potent detoxification enzyme NQO1, which could reduce the quinone back to the catechol and prevent DNA damage (step 5, Fig. 3). Resveratrol has also been shown to be a potent inhibitor of aromatase, resulting in significant decreases in estradiol levels (Wang et al. 2006). Finally, resveratrol is a potent antioxidant that could scavenge ROS and reduce oxidative DNA damage (step 7, Fig. 3). In contrast to the chemopreventive effects of resveratrol on estrogen carcinogenesis, the isoflavone genistein (phytoestrogen, red clover), despite numerous reports of chemopreventive properties (Banerjee et  al. 2008), seems to modulate estrogen metabolism, which would enhance estrogen carcinogenesis (Mense et al. 2008b). For example, genistein caused a significant reduction in both COMT mRNA levels and COMT activity, which would enhance the levels of 4-hydroxyestradiol (step 4, Fig. 3) (Lehmann et al. 2008). Genistein also inhibited the expression of P4501A1 (step 3, Fig. 3) and NQO1 (step 5, Fig. 3), which would also be expected to increase estrogen-induced DNA damage (Wagner et al. 2008). Other examples of natural products which potentially alter estrogen carcinogenesis include quercetin (Zhu and Liehr 1994; Mense et al. 2008a), polyphenolic components present in coffee and green tea (Goodin and Rosengren 2003; Zhu et al. 2009), and tocopherols (Lee et al. 2009). These preliminary studies suggest that it may be possible to reduce estrogen-dependent cancer risk by modulation of estrogen metabolism and detoxification of reactive intermediates. Even though millions of women are taking botanical extracts daily, there is little information on the effect of the most common botanical extracts on the enzyme targets involved in the estrogen carcinogenic process.

6 Conclusions and Future Directions Receptor-mediated responses to hormones are a plausible and probably necessary mechanism for hormonal carcinogenesis. The results of research over the past few years add considerable support for a direct genotoxic effect of hormones or their associated by-products such as ROS. Current knowledge does not provide a conclusion as to whether either of these mechanisms is the major determinant of hormonally induced cancer. It is entirely possible that both mechanisms contribute to and are necessary for carcinogenesis. In addition, it is quite feasible that popular botanicals can be used to modulate estrogen carcinogenesis by inhibiting/enhancing enzymatic pathways (steps 1–6, Fig.  3) and/or by scavenging ROS (step 7). Establishing the effects of popular extensively utilized botanicals on these key pathways will allow predictions on the risk/benefit of these supplements in hormone-dependent cancers as well as on other estrogen-associated biological effects crucial to women’s health. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that the factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.

4  Mechanisms of Estrogen Carcinogenesis

89

Acknowledgements  This work is supported by NIH Grants CA102590, CA79870, and CA73638.

References (2008) The top 200 drugs. http://www.rxlist.com/. Banerjee, S., Li, Y., Wang, Z., and Sarkar, F. H. (2008) Cancer Lett. 269, 226–242. Banerjee, S. K., Banerjee, S., Li, S. A., and Li, J. J. (1994) Mutat. Res. 311, 191–197. Barbaglia, G., Macia, F., Comas, M., Sala, M., del Mar Vernet, M., Casamitjana, M., and Castells, X. (2009) Menopause 16, 1061–1064. Benz, C. C. and Yau, C. (2008) Nat. Rev. Cancer 8, 875–879. Bhavnani, B. R. (1998) Proc. Soc. Exp. Biol. Med. 217, 6–16. Bolton, J. L. and Thatcher, G. R. (2008) Chem. Res. Toxicol. 21, 93–101. Bolton, J. L., Yu, L., and Thatcher, G. R. (2004) Methods Enzymol. 378, 110–123. Booth, N. L., Piersen, C. E., Banuvar, S., Geller, S. E., Shulman, L. P., and Farnsworth, N. R. (2006) Menopause 13, 251–264. Brass, L. M. (2004) Stroke 35, 2644–2647. Bruno, R. D. and Njar, V. C. (2007) Bioorg. Med. Chem. 15, 5047–5060. Bugano, D. D., Conforti-Froes, N., Yamaguchi, N. H., and Baracat, E. C. (2008) Eur. J. Gynaecol. Oncol. 29, 313–320. Burczynski, M. E. and Penning, T. M. (2000) Cancer Res. 60, 908–915. Burdette, J. E., Liu, J., Chen, S., Fabricant, D. S., Piersen, C. E., Barker, E. L., Pezzuto, J. M., Mesecar, A., van Breemen, R. B., Farnsworth, N. R., and Bolton, J. L. (2003) J. Agric. Food Chem. 51, 5661–5670. Burdette, J. E., Liu, J., Lantvit, D., Lim, E., Booth, N., Bhat, K. P., Hedayat, S., Van Breemen, R. B., Constantinou, A. I., Pezzuto, J. M., Farnsworth, N. R., and Bolton, J. L. (2002) J. Nutr. 132, 27–30. Castrellon, A. B. and Gluck, S. (2008) Expert Rev. Anticancer Ther. 8, 443–452. Cavalieri, E., Chakravarti, D., Guttenplan, J., Hart, E., Ingle, J., Jankowiak, R., Muti, P., Rogan, E., Russo, J., Santen, R., and Sutter, T. (2006) Biochim. Biophys. Acta 1766, 63–78. Cavalieri, E., Frenkel, K., Liehr, J. G., Rogan, E., and Roy, D. (2000) J. Natl Cancer Inst. Monogr. 27, 75–93. Chakravarti, D., Mailander, P. C., Li, K. M., Higginbotham, S., Zhang, H. L., Gross, M. L., Meza, J. L., Cavalieri, E. L., and Rogan, E. G. (2001) Oncogene 20, 7945–7953. Chandrasena, R. E., Edirisinghe, P. D., Bolton, J. L., and Thatcher, G. R. (2008) Chem. Res. Toxicol. 21, 1324–1329. Chen, J. Q., Brown, T. R., and Yager, J. D. (2008) Adv. Exp. Med. Biol. 630, 1–18. Chen, W. Y. (2008) Best Pract. Res. Clin. Endocrinol. Metab. 22, 573–585. Chen, Y., Liu, X., Pisha, E., Constantinou, A. I., Hua, Y., Shen, L., van Breemen, R. B., Elguindi, E. C., Blond, S. Y., Zhang, F., and Bolton, J. L. (2000) Chem. Res. Toxicol. 13, 342–350. Chen, Z. H., Na, H. K., Hurh, Y. J., and Surh, Y. J. (2005) Toxicol. Appl. Pharmacol. 208, 46–56. Cheng, T. C., Chen, S. T., Huang, C. S., Fu, Y. P., Yu, J. C., Cheng, C. W., Wu, P. E., and Shen, C. Y. (2005) Int. J. Cancer 113, 345–353. Chlebowski, R. T., Kuller, L. H., Prentice, R. L., Stefanick, M. L., Manson, J. E., Gass, M., Aragaki, A. K., Ockene, J. K., Lane, D. S., Sarto, G. E., Rajkovic, A., Schenken, R., Hendrix, S. L., Ravdin, P. M., Rohan, T. E., Yasmeen, S., and Anderson, G. (2009) N. Engl. J. Med. 360, 573–587. Cornblatt, B. S., Ye, L., Dinkova-Kostova, A. T., Erb, M., Fahey, J. W., Singh, N. K., Chen, M. S., Stierer, T., Garrett-Mayer, E., Argani, P., Davidson, N. E., Talalay, P., Kensler, T. W., and Visvanathan, K. (2007) Carcinogenesis 28, 1485–1490.

90

J.L. Bolton

Cvoro, A., Paruthiyil, S., Jones, J. O., Tzagarakis-Foster, C., Clegg, N. J., Tatomer, D., Medina, R. T., Tagliaferri, M., Schaufele, F., Scanlan, T. S., Diamond, M. I., Cohen, I., and Leitman, D. C. (2007) Endocrinology 148, 538–547. Debrauwer, L., Rathahao, E., Jouanin, I., Paris, A., Clodic, G., Molines, H., Convert, O., Fournier, F., and Tabet, J. C. (2003) J. Am. Soc. Mass Spectrom. 14, 364–372. Deng, S., Chen, S. N., Yao, P., Nikolic, D., van Breemen, R. B., Bolton, J. L., Fong, H. H., Farnsworth, N. R., and Pauli, G. F. (2006) J. Nat. Prod. 69, 536–541. Diergaarde, B., Potter, J. D., Jupe, E. R., Manjeshwar, S., Shimasaki, C. D., Pugh, T. W., Defreese, D. C., Gramling, B. A., Evans, I., and White, E. (2008) Cancer Epidemiol. Biomarkers Prev. 17, 1751–1759. Ding, S., Shapiro, R., Geacintov, N. E., and Broyde, S. (2003) Chem. Res. Toxicol. 16, 695–707. Ding, S., Shapiro, R., Geacintov, N. E., and Broyde, S. (2007) Biochemistry 46, 182–191. Embrechts, J., Lemiere, F., Dongen, W. V., Esmans, E. L., Buytaert, P., van Marck, E., Kockx, M., and Makar, A. (2003) J. Am. Soc. Mass Spectrom. 14, 482–491. Feigelson, H. S. and Henderson, B. E. (1996) Carcinogenesis 17, 2279–2284. Flototto, T., Djahansouzi, S., Glaser, M., Hanstein, B., Niederacher, D., Brumm, C., and Beckmann, M. W. (2001) Horm. Metab. Res. 33, 451–457. Gaikwad, N. W., Yang, L., Muti, P., Meza, J. L., Pruthi, S., Ingle, J. N., Rogan, E. G., and Cavalieri, E. L. (2008) Int. J. Cancer 122, 1949–1957. Geller, S. E., Shulman, L. P., van Breemen, R. B., Banuvar, S., Zhou, Y., Epstein, G., Hedayat, S., Nikolic, D., Krause, E. C., Piersen, C. E., Bolton, J. L., Pauli, G. F., and Farnsworth, N. R. (2009) Menopause 16, 1156–1166. Gonzalez, F. J. (2002) Toxicology 181–182, 237–239. Goodin, M. G. and Rosengren, R. J. (2003) Toxicol. Sci. 76, 262–270. Harris, D. M., Besselink, E., Henning, S. M., Go, V. L., and Heber, D. (2005) Exp. Biol. Med. 230, 558–568. Hayashi, N., Hasegawa, K., Barrett, J. C., and Tsutsui, T. (1996) Mol. Carcinog. 16, 149–156. Hays, J., Ockene, J. K., Brunner, R. L., Kotchen, J. M., Manson, J. E., Patterson, R. E., Aragaki, A. K., Shumaker, S. A., Brzyski, R. G., LaCroix, A. Z., Granek, I. A., and Valanis, B. G. (2003) N. Engl. J. Med. 348, 1839–1854. Henderson, B. E. and Feigelson, H. S. (2000) Carcinogenesis 21, 427–433. Hersh, A. L., Stefanick, M. L., and Stafford, R. S. (2004) JAMA 291, 47–53. Horn-Ross, P. L., John, E. M., Lee, M., Stewart, S. L., Koo, J., Sakoda, L. C., Shiau, A. C., Goldstein, J., Davis, P., and Perez-Stable, E. J. (2001) Am. J. Epidemiol. 154, 434–441. Hu, Z., Song, C. G., Lu, J. S., Luo, J. M., Shen, Z. Z., Huang, W., and Shao, Z. M. (2007) J. Cancer Res. Clin. Oncol. 133, 969–978. Huang, C. S., Chern, H. D., Chang, K. J., Cheng, C. W., Hsu, S. M., and Shen, C. Y. (1999) Cancer Res. 59, 4870–4875. Jefcoate, C. R., Liehr, J. G., Santen, R. J., Sutter, T. R., Yager, J. D., Yue, W., Santner, S. J., Tekmal, R., Demers, L., Pauley, R., Naftolin, F., Mor, G., and Berstein, L. (2000) J. Natl Cancer Inst. Monogr. 27, 95–112. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., and Thun, M. J. (2009) CA Cancer J. Clin. 59, 225–249. Ju, Y. H., Allred, K. F., Allred, C. D., and Helferich, W. G. (2006) Carcinogenesis 27, 1292–1299. Judd, H. L., Mebane-Sims, I., Legault, C., Wasilauskas, C., Johnson, S., Merino, M., BarrettConnor, B., and Trabal, J. (1996) JAMA 275, 370–375. Karihtala, P. and Soini, Y. (2007) APMIS 115, 81–103. Key, T., Appleby, P., Barnes, I., and Reeves, G. (2002) J. Natl Cancer Inst. 94, 606–616. Kisselev, P., Schunck, W. H., Roots, I., and Schwarz, D. (2005) Cancer Res. 65, 2972–2978. Kolbanovskiy, A., Kuzmin, V., Shastry, A., Kolbanovskaya, M., Chen, D., Chang, M., Bolton, J. L., and Geacintov, N. E. (2005) Chem. Res. Toxicol. 18, 1737–1747. Kumle, M. (2008) Lancet 372, 608–610. Kupfer, R., Swanson, L., Chow, S., Staub, R. E., Zhang, Y. L., Cohen, I., and Christians, U. (2008) Drug Metab. Dispos. 36, 2261–2269.

4  Mechanisms of Estrogen Carcinogenesis

91

Kurzer, M. S. and Xu, X. (1997) Annu. Rev. Nutr. 17, 353–381. Lavigne, J. A., Goodman, J. E., Fonong, T., Odwin, S., He, P., Roberts, D. W., and Yager, J. D. (2001) Cancer Res. 61, 7488–7494. Lee, E. J., Oh, S. Y., Kim, M. K., Ahn, S. H., Son, B. H., and Sung, M. K. (2009) Nutr. Res. Pract. 3, 185–191. Lehmann, L., Jiang, L., and Wagner, J. (2008) Carcinogenesis 29, 363–370. Levin, E. R. (2009) J. Physiol. 587, 5019–5023. Li, J. J., Gonzalez, A., Banerjee, S., Banerjee, S. K., and Li, S. A. (1993) Environ. Health Perspect. 5, 259–264. Li, J. J. and Li, S. A. (1987) Fed. Proc. 46, 1858–1863. Li, J. J., Li, S. A., Oberley, T. D., and Parsons, J. A. (1995) Cancer Res. 55, 4347–4351. Li, K. M., Todorovic, R., Devanesan, P., Higginbotham, S., Kofeler, H., Ramanathan, R., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (2004) Carcinogenesis 25, 289–297. Liehr, J. G. (2000) Regul. Toxicol. Pharmacol. 32, 276–282. Liehr, J. G. (2001) Hum. Reprod. Update 7, 273–281. Liehr, J. G., Fang, W. R., Sirbasku, D. A., and Ari-Ulubelen, A. (1986) J. Steroid Biochem. 24, 353–356. Liu, J., Burdette, J. E., Xu, H., Gu, C., van Breemen, R. B., Bhat, K. P., Booth, N., Constantinou, A. I., Pezzuto, J. M., Fong, H. H., Farnsworth, N. R., and Bolton, J. L. (2001) J. Agric. Food Chem. 49, 2472–2479. Liu, X., Yao, J., Pisha, E., Yang, Y., Hua, Y., van Breemen, R. B., and Bolton, J. L. (2002) Chem. Res. Toxicol. 15, 512–519. Lu, F., Zahid, M., Wang, C., Saeed, M., Cavalieri, E. L., and Rogan, E. G. (2008) Cancer Prev. Res. (Phila) 1, 135–145. Mahady, G. B., Parrot, J., Lee, C., Yuri, G. S., and Dan, A. (2003) Menopause 10, 65–72. Malins, D. C., Anderson, K. M., Jaruga, P., Ramsey, C. R., Gilman, N. K., Green, V. M., Rostad, S. W., Emerman, J. T., and Dizdaroglu, M. (2006) Cell Cycle 5, 1629–1632. Malins, D. C., Polissar, N. L., and Gunselman, S. J. (1996) Proc. Natl Acad. Sci. USA 93, 2557–2563. Mandlekar, S., Hong, J. L., and Kong, A. N. (2006) Curr. Drug Metab. 7, 661–675. Markushin, Y., Zhong, W., Cavalieri, E. L., Rogan, E. G., Small, G. J., Yeung, E. S., and Jankowiak, R. (2003) Chem. Res. Toxicol. 16, 1107–1117. McDonnell, D. P. (2004) Maturitas 48(Suppl 1), S7–S12. Mense, S. M., Chhabra, J., and Bhat, H. K. (2008a) J. Steroid Biochem. Mol. Biol. 110, 157–162. Mense, S. M., Hei, T. K., Ganju, R. K., and Bhat, H. K. (2008b) Environ. Health Perspect. 116, 426–433. Mersereau, J. E., Levy, N., Staub, R. E., Baggett, S., Zogovic, T., Chow, S., Ricke, W. A., Tagliaferri, M., Cohen, I., Bjeldanes, L. F., and Leitman, D. C. (2008) Mol. Cell. Endocrinol. 283, 49–57. Messina, M. J., Persky, V., Setchell, K. D., and Barnes, S. (1994) Nutr. Cancer 21, 113–131. Milligan, S., Kalita, J., Pocock, V., Heyerick, A., De Cooman, L., Rong, H., and De Keukeleire, D. (2002) Reproduction 123, 235–242. Mobley, J. A. and Brueggemeier, R. W. (2002) Toxicol. Appl. Pharmacol. 180, 219–226. Modugno, F., Zmuda, J. M., Potter, D., Cai, C., Ziv, E., Cummings, S. R., Stone, K. L., Morin, P. A., Greene, D., and Cauley, J. A. (2005) Breast Cancer Res. Treat. 93, 261–270. Montano, M. M., Chaplin, L. J., Deng, H., Mesia-Vela, S., Gaikwad, N., Zahid, M., and Rogan, E. (2007) Oncogene 26, 3587–3590. Nandi, S., Guzman, R. C., and Yang, J. (1995) Proc. Natl Acad. Sci. USA 92, 3650–3657. Newbold, R. R. and Liehr, J. G. (2000) Cancer Res. 60, 235–237. Newton, K. M., Buist, D. S. M., Keenan, N. L., Anderson, L. A., and LaCroix, A. Z. (2002) Obstet. Gynecol. 100, 18–25. Nutter, L. M., Ngo, E. O., and Abul-Hajj, Y. J. (1991) J. Biol. Chem. 266, 16380–16386. Okamoto, Y., Chou, P. H., Kim, S. Y., Suzuki, N., Laxmi, Y. R., Okamoto, K., Liu, X., Matsuda, T., and Shibutani, S. (2008) Chem. Res. Toxicol. 21, 1120–1124.

92

J.L. Bolton

Overk, C. R., Guo, J., Chadwick, L. R., Lantvit, D. D., Minassi, A., Appendino, G., Chen, S. N., Lankin, D. C., Farnsworth, N. R., Pauli, G. F., van Breemen, R. B., and Bolton, J. L. (2008) Chem. Biol. Interact. 176, 30–39. Overk, C. R., Yao, P., Chadwick, L. R., Nikolic, D., Sun, Y., Cuendet, M. A., Deng, Y., Hedayat, A. S., Pauli, G. F., Farnsworth, N. R., van Breemen, R. B., and Bolton, J. L. (2005) J. Agric. Food Chem. 53, 6246–6253. Ozcagli, E., Sardas, S., and Biri, A. (2005) Maturitas 51, 280–285. Palacio, C., Masri, G., and Mooradian, A. D. (2009) Drugs Aging 26, 23–36. Park, J. H., Mangal, D., Frey, A. J., Harvey, R. G., Blair, I. A., and Penning, T. M. (2009a) J. Biol. Chem. 284, 29725–29734. Park, S. A., Na, H. K., Kim, E. H., Cha, Y. N., and Surh, Y. J. (2009b) Cancer Res. 69, 2416–2424. Piersen, C. E., Booth, N. L., Sun, Y., Liang, W., Burdette, J. E., van Breemen, R. B., Geller, S. E., Gu, C., Banuvar, S., Shulman, L. P., Bolton, J. L., and Farnsworth, N. R. (2004) Curr. Med. Chem. 11, 1361–1374. Powell, S. L., Godecke, T., Nikolic, D., Chen, S. N., Ahn, S., Dietz, B., Farnsworth, N. R., van Breemen, R. B., Lankin, D. C., Pauli, G. F., and Bolton, J. L. (2008) J. Agric. Food Chem. 56, 11718–11726. Prokai-Tatrai, K. and Prokai, L. (2005) Ann. NY Acad. Sci. 1052, 243–257. Prossnitz, E. R. and Maggiolini, M. (2009) Mol. Cell. Endocrinol. 308, 32–38. Purdy, R. H., Moore, P. H., Williams, M. C., Goldzheher, H. W., and Paul, S. M. (1982) FEBS Lett. 138, 40–44. Rajapakse, N., Butterworth, M., and Kortenkamp, A. (2005) Environ. Mol. Mutagen. 45, 397–404. Ravdin, P. M., Cronin, K. A., Howlader, N., Berg, C. D., Chlebowski, R. T., Feuer, E. J., Edwards, B. K., and Berry, D. A. (2007) N. Engl. J. Med. 356, 1670–1674. Rees, M. (2009) Best Pract. Res. Clin. Obstet. Gynaecol. 23, 151–161. Revankar, C. M., Cimino, D. F., Sklar, L. A., Arterburn, J. B., and Prossnitz, E. R. (2005) Science 307, 1625–1630. Rogan, E. G., Badawi, A. F., Devanesan, P. D., Meza, J. L., Edney, J. A., West, W. W., Higginbotham, S. M., and Cavalieri, E. L. (2003) Carcinogenesis 24, 697–702. Rossouw, J. E., Anderson, G. L., Prentice, R. L., LaCroix, A. Z., Kooperberg, C., Stefanick, M. L., Jackson, R. D., Beresford, S. A., Howard, B. V., Johnson, K. C., Kotchen, J. M., and Ockene, J. (2002) JAMA 288, 321–333. Roy, D. and Liehr, J. G. (1999) Mutat. Res. 424, 107–115. Russo, J., Hu, Y. F., Yang, X., and Russo, I. H. (2000) J. Natl Cancer Inst. Monogr. 27, 17–37. Russo, J. and Russo, I. H. (2004) Trends Endocrinol. Metab. 15, 211–214. Russo, J. and Russo, I. H. (2006) J. Steroid Biochem. Mol. Biol. 102, 89–96. Saeed, M., Rogan, E., Fernandez, S. V., Sheriff, F., Russo, J., and Cavalieri, E. (2007) Int. J. Cancer 120, 1821–1824. Saeed, M., Zahid, M., Gunselman, S. J., Rogan, E., and Cavalieri, E. (2005) Steroids 70, 29–35. Salama, S. A., Kamel, M., Awad, M., Nasser, A. H., Al-Hendy, A., Botting, S., and Arrastia, C. (2008) Int. J. Cancer 123, 1246–1254. Sarabia, S. F., Zhu, B. T., Kurosawa, T., Tohma, M., and Liehr, J. G. (1997) Chem. Res. Toxicol. 10, 767–771. Shimada, T., Watanabe, J., Kawajiri, K., Sutter, T. R., Guengerich, F. P., Gillam, E. M., and Inoue, K. (1999) Carcinogenesis 20, 1607–1614. Shumaker, S. A., Legault, C., Rapp, S. R., Thal, L., Wallace, R. B., Ockene, J. K., Hendrix, S. L., Jones, B. N., Assaf, A. R., Jackson, R. D., Kotchen, J. M., Wassertheil-Smoller, S., and Wactawski-Wende, J. (2003) JAMA 289, 2651–2662. Singh, S., Chakravarti, D., Edney, J. A., Hollins, R. R., Johnson, P. J., West, W. W., Higginbotham, S. M., Cavalieri, E. L., and Rogan, E. G. (2005) Oncol. Rep. 14, 1091–1096. Song, R. X., Fan, P., Yue, W., Chen, Y., and Santen, R. J. (2006) Endocr. Relat. Cancer 13(Suppl 1), S3–S13.

4  Mechanisms of Estrogen Carcinogenesis

93

Spink, D. C., Spink, B. C., Cao, J. Q., Depasquale, J. A., Pentecost, B. T., Fasco, M. J., Li, Y., and Sutter, T. R. (1998) Carcinogenesis 19, 291–298. Spink, D. C., Spink, B. C., Cao, J. Q., Gierthy, J. F., Hayes, C. L., Li, Y., and Sutter, T. R. (1997) J. Steroid Biochem. Mol. Biol. 62, 223–232. Stack, D. E., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1996) Chem. Res. Toxicol. 9, 851–859. Terashima, I., Suzuki, N., and Shibutani, S. (2001) Biochemistry 40, 166–172. The North American Menopause Society. (2004) Menopause 11, 11–33. Verkooijen, H. M., Bouchardy, C., Vinh-Hung, V., Rapiti, E., and Hartman, M. (2009) Maturitas 64, 80–85. Wagner, J., Jiang, L., and Lehmann, L. (2008) Adv. Exp. Med. Biol. 617, 625–632. Wang, Y., Lee, K. W., Chan, F. L., Chen, S., and Leung, L. K. (2006) Toxicol. Sci. 92, 71–77. Wang, Z., Wijewickrama, G. T., Peng, K. W., Dietz, B. M., Yuan, L., van Breemen, R. B., Bolton, J. L., and Thatcher, G. R. (2009) J. Biol. Chem. 284, 8633–8642. Wysowski, D. K. and Governale, L. A. (2005) Pharmacoepidemiol. Drug Saf. 14, 171–176. Yager, J. D. and Davidson, N. E. (2006) N. Engl. J. Med. 354, 270–282. Yasui, M., Laxmi, Y. R., Ananthoju, S. R., Suzuki, N., Kim, S. Y., and Shibutani, S. (2006) Biochemistry 45, 6187–6194. Zahid, M., Gaikwad, N. W., Ali, M. F., Lu, F., Saeed, M., Yang, L., Rogan, E. G., and Cavalieri, E. L. (2008) Free Radic. Biol. Med. 45, 136–145. Zahid, M., Kohli, E., Saeed, M., Rogan, E., and Cavalieri, E. (2006) Chem. Res. Toxicol. 19, 164–172. Zahid, M., Saeed, M., Lu, F., Gaikwad, N., Rogan, E., and Cavalieri, E. (2007) Free Radical Biol. Med. 43, 1534–1540. Zhang, F., Chen, Y., Pisha, E., Shen, L., Xiong, Y., van Breemen, R. B., and Bolton, J. L. (1999) Chem. Res. Toxicol. 12, 204–213. Zhang, F., Swanson, S. M., van Breemen, R. B., Liu, X., Yang, Y., Gu, C., and Bolton, J. L. (2001) Chem. Res. Toxicol. 14, 1654–1659. Zhang, Q., Aft, R. L., and Gross, M. L. (2008) Chem. Res. Toxicol. 21, 1509–1513. Zhang, Q. and Gross, M. L. (2008) Chem. Res. Toxicol. 21, 1244–1252. Zheng, W., Xie, D. W., Jin, F., Cheng, J. R., Dai, Q., Wen, W. Q., Shu, X. O., and Gao, Y. T. (2000) Cancer Epidemiol. Biomarkers Prev. 9, 147–150. Zhu, B. T. and Liehr, J. G. (1994) Toxicol. Appl. Pharmacol. 125, 149–158. Zhu, B. T., Wang, P., Nagai, M., Wen, Y., and Bai, H. W. (2009) J. Steroid Biochem. Mol. Biol. 113, 65–74. Zumoff, B. (1998) Proc. Soc. Exp. Biol. Med. 217, 30–37.

wwwwwwwwwwwwwwwww

Chapter 5

Heterocyclic Aromatic Amines: Potential Human Carcinogens Robert J. Turesky

Abstract  Heterocyclic aromatic amines (HAAs) are formed at parts per billion concentrations during the cooking of meats, poultry, and fish. All of the HAAs tested thus far are carcinogenic in experimental animals and induce tumors in multiple organs. Because of the presence of HAAs in a wide range of food items, the exposure to them can be appreciable. Some epidemiological studies have linked an increased risk for cancer development of the colon, prostate, and female mammary gland with frequent consumption of well-done cooked meats containing HAAs. Therefore, much research has been devoted to determining the potential role of HAAs in the etiology of human cancer. This chapter highlights investigations on the biochemistry of metabolism of several prototypical HAAs, the formation of DNA adducts by these HAAs and the ensuing biological effects, and the analytical approaches that are employed for biomonitoring of these procarcinogens in humans. Abbreviations AaC 4,8-DiMeIQx 7,8-DiMeIQx 7,9-DiMeIgQx Glu-P-1 Glu-P-2 IQ IQx MeAaC MeIQ 7-MeIgQx

2-Amino-9H-pyrido[2,3-b]indole 2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline 2-Amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline 2-Amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline 2-Amino-6-methyldipyrido[1,2-a:3¢,2¢-d]imidazole 2-Aminodipyrido[1,2-a:3¢,2¢-d]imidazole 2-Amino-3-methylimidazo[4,5-f]quinoline 2-Amino-3-methylimidazo[4,5-f]quinoxaline 2-Amino-3-methyl-9H-pyrido[2,3-b]indole 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline 2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline

R.J. Turesky (*) Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA e-mail: [email protected]

T.M. Penning (ed.), Chemical Carcinogenesis, Current Cancer Research, DOI 10.1007/978-1-61737-995-6_5, © Springer Science+Business Media, LLC 2011

95

96

MeIQx PhIP Trp-P-1 Trp-P-2

R.J. Turesky

2-Amino-3,8-dimethylimidazo[4,5-f ]quinoxaline 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole 3-Amino-1-methyl-5H-pyrido[4,3-b]indole

1 Introduction More than 20 heterocyclic aromatic amines (HAAs) have been identified in cooked meats, fish, and poultry (Fig.  1) (Felton et  al. 2000; Sugimura et  al. 2004). Several HAAs are also known to occur in cigarette smoke condensate and diesel exhaust (Manabe et al. 1991). Many HAAs induce cancers in rodents in a number of organs that include the oral cavity, liver, stomach, lung, colorectum, prostate, and female mammary gland, during long-term feeding studies (Sugimura et al. 2004). The colon, prostate, and female mammary gland are common sites of human cancers in Western countries, and some epidemiological studies have linked frequent consumption of well-done cooked meats containing HAAs with the elevated risk for these cancers (Le Marchand 2002). The International Agency for Research on Cancer has classified eight HAAs, including 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), as possible human carcinogens (Group 2B), and one HAA, 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), as a probable human carcinogen (Group 2A) (IARC 1993). More recently, the Report on Carcinogens, 11th Edition, of the National Toxicology Program, has concluded that several prevalent HAAs are “reasonably anticipated” to be human carcinogens (National Toxicology Program 2005). Therefore, questions have been raised about the safety of foods containing HAAs, and considerable research has been devoted to understanding the health risk posed by these carcinogens.

Fig. 1  Chemical structures of prominent HAAs

5  Heterocyclic Aromatic Amines: Potential Human Carcinogens

97

2 HAA Formation and Levels in Cooked Meat One class of HAAs arises during the pyrolysis (>250°C) of individual amino acids such as tryptophan, glutamic acid, and phenylalanine, or during the high-temperature heating of proteins (Sugimura et  al. 2004). The high temperature catalyzes the formation of deaminated and decarboxylated amino acids, and reactive radical fragments, which combine to form heterocyclic ring structures (Fig. 1). The high temperature of burning cigarettes also catalyzes the formation of pyrolytic HAAs. 2-Amino-9H-pyrido[2,3-b]indole (AaC) and 2-amino-3-methyl-9H-pyrido[2,3-b] indole (MeAaC) are the two most abundant HAAs that arise in mainstream cigarette smoke (Yoshida and Matsumoto 1980), with levels reported at up to 258 and 37 ng/cigarette, respectively. These levels are considerably higher than those levels reported for many polycyclic aromatic hydrocarbons and aromatic amines, which are established human carcinogens (Hecht 2003). The second class of HAAs is formed in meats cooked at temperatures (150–250°C) that are commonly utilized in the household kitchen. Amino acids, sugars, and creatine are precursors to these HAAs. The 2-amino-N-methylimidazole portion of the HAA molecule is derived from creatine, and the remaining parts of the skeleton are assumed to arise from Strecker degradation products (for example, pyridines or pyrazines) formed in the Maillard reaction between hexoses and amino acids (Jagerstad et al. 1991). An aldol condensation is thought to link the two molecules through an aldehyde or related Schiff base to form 2-amino-Nmethyl-imidazoquinoline or 2-amino-N-methyl-imidazoquinoxaline ring structure. PhIP can form in a model system containing phenylalanine, creatinine, and glucose (Shioya et al. 1987), but this HAA can also form in the absence of sugar. The types and concentrations of HAAs formed in meats, poultry, and fish under various cooking conditions have been reported: The concentrations can typically range from