153,99 €
Mehr erfahren.
- Herausgeber: Wiley-VCH
- Kategorie: Wissenschaft und neue Technologien
- Serie: Methods and Principles in Medicinal Chemistry
- Sprache: Englisch
Closing a gap in the scientifi c literature, this first comprehensive introduction to the topic is based on current best practice in one of the largest
pharmaceutical companies worldwide. The first chapters trace the development of our understanding of drug metabolite toxicity, covering basic concepts and techniques in the process, while the second part details chemical toxicophores that are prone to reactive metabolite formation. This section also reviews the various drug-metabolizing enzymes that can participate in catalyzing reactive metabolite formation, including a discussion of the structure-toxicity relationships for drugs. Two chapters are dedicated to the currently hot topics of herbal constituents and IADRs.
The next part covers current strategies and approaches to evaluate the reactive metabolite potential of new drug candidates, both by predictive
and by bioanalytical methods. There then follows an in-depth analysis of the toxicological potential of the top 200 prescription drugs, illustrating
the power and the limits of the toxicophore concept, backed by numerous case studies. Finally, a risk-benefi t approach to managing the toxicity risk of reactive metabolite-prone drugs is presented.
Since the authors carefully develop the knowledge needed, from fundamental considerations to current industry standards, no degree in pharmacology is required to read this book, making it perfect for medicinal chemists without in-depth pharmacology training.
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 560
Veröffentlichungsjahr: 2012
Ähnliche
Contents
Cover
Methods and Principles in Medicinal Chemistry
Title Page
Copyright
Preface
References
A Personal Foreword
Chapter 1: Origin and Historical Perspective on Reactive Metabolites
Abbreviations
1.1 Mutagenesis and Carcinogenesis
1.2 Detection of Reactive Metabolites
1.3 Induction and Inhibition: Early Probes for Reactive Metabolites and Hepatotoxicants
1.4 Covalent Binding and Oxidative Stress: Possible Mechanisms of Reactive Metabolite Cytotoxicity
1.5 Activation and Deactivation: Intoxication and Detoxification
1.6 Genetic Influences on Reactive Metabolite Formation
1.7 Halothane: the Role of Reactive Metabolites in Immune-Mediated Toxicity
1.8 Formation of Reactive Metabolites, Amount Formed, and Removal of Liability
1.9 Antibodies: Possible Clues but Inconclusive
1.10 Parent Drug and Not Reactive Metabolites, Complications in Immune-Mediated Toxicity
1.11 Reversible Pharmacology Should not be Ignored as a Primary Cause of Side Effects
1.12 Conclusions: Key Points in the Introduction
References
Chapter 2: Role of Reactive Metabolites in Genotoxicity
Abbreviations
2.1 Introduction
2.2 Carcinogenicity of Aromatic and Heteroaromatic Amines
2.3 Carcinogenicity of Nitrosamines
2.4 Carcinogenicity of Quinones and Related Compounds
2.5 Carcinogenicity of Furan
2.6 Carcinogenicity of Vinyl Halides
2.7 Carcinogenicity of Ethyl Carbamate
2.8 Carcinogenicity of Dihaloalkanes
2.9 Assays to Detect Metabolism-Dependent Genotoxicity in Drug Discovery
2.10 Case Studies in Eliminating Metabolism-Based Mutagenicity in Drug Discovery Programs
References
Chapter 3: Bioactivation and Inactivation of Cytochrome P450 and Other Drug-Metabolizing Enzymes
Abbreviations
3.1 Introduction
3.2 Pharmacokinetic and Enzyme Kinetic Principles Underlying Mechanism-Based Inactivation and Drug–Drug Interactions
3.3 Mechanisms of Inactivation of Cytochrome P450 Enzymes
3.4 Examples of Drugs and Other Compounds that are Mechanism-Based Inactivators of Cytochrome P450 Enzymes
3.5 Mechanism-Based Inactivation of Other Drug-Metabolizing Enzymes
3.6 Concluding Remarks
References
Chapter 4: Role of Reactive Metabolites in Drug-Induced Toxicity – The Tale of Acetaminophen, Halothane, Hydralazine, and Tienilic Acid
Abbreviations
4.1 Introduction
4.2 Acetaminophen
4.3 Halothane
4.4 Hydralazine
4.5 Tienilic Acid
References
Chapter 5: Pathways of Reactive Metabolite Formation with Toxicophores/Structural Alerts
Abbreviations
5.1 Introduction
5.2 Intrinsically Reactive Toxicophores
5.3 Toxicophores that Require Bioactivation to Reactive Metabolites
5.4 Concluding Remarks
References
Chapter 6: Intrinsically Electrophilic Compounds as a Liability in Drug Discovery
Abbreviations
6.1 Introduction
6.2 Intrinsic Electrophilicity of β-Lactam Antibiotics as a Causative Factor in Toxicity
6.3 Intrinsically Electrophilic Compounds in Drug Discovery
6.4 Serendipitous Identification of Intrinsically Electrophilic Compounds in Drug Discovery
References
Chapter 7: Role of Reactive Metabolites in Pharmacological Action
Abbreviations
7.1 Introduction
7.2 Drugs Activated Nonenzymatically and by Oxidative Metabolism
7.3 Bioreductive Activation of Drugs
7.4 Concluding Remarks
References
Chapter 8: Retrospective Analysis of Structure–Toxicity Relationships of Drugs
Abbreviations
8.1 Introduction
8.2 Irreversible Secondary Pharmacology
8.3 Primary Pharmacology and Irreversible Secondary Pharmacology
8.4 Primary or Secondary Pharmacology and Reactive Metabolites: the Possibility for False Structure–Toxicity Relationships
8.5 Multifactorial Mechanisms as Causes of Toxicity
8.6 Clear Correlation between Protein Target and Reactive Metabolites
8.7 Conclusion – Validation of Reactive Metabolites as Causes of Toxicity
References
Chapter 9: Bioactivation and Natural Products
Abbreviations
9.1 Introduction
9.2 Well-Known Examples of Bioactivation of Compounds Present in Herbal Remedies
9.3 Well-Known Examples of Bioactivation of Compounds Present in Foods
9.4 Summary
References
Chapter 10: Experimental Approaches to Reactive Metabolite Detection
Abbreviations
10.1 Introduction
10.2 Identification of Structural Alerts and Avoiding them in Drug Design
10.3 Assays for the Detection of Reactive Metabolites
10.4 Other Studies that can Show the Existence of Reactive Metabolites
10.5 Conclusion
References
Chapter 11: Case Studies on Eliminating/Reducing Reactive Metabolite Formation in Drug Discovery
Abbreviations
11.1 Medicinal Chemistry Tactics to Eliminate Reactive Metabolite Formation
11.2 Eliminating Reactive Metabolite Formation on Heterocyclic Ring Systems
11.3 Medicinal Chemistry Strategies to Mitigate Bioactivation of Electron-Rich Aromatic Rings
11.4 Medicinal Chemistry Strategies to Mitigate Bioactivation on a Piperazine Ring System
11.5 4-Fluorofelbamate as a Potentially Safer Alternative to Felbamate
11.6 Concluding Remarks
References
Chapter 12: Structural Alert and Reactive Metabolite Analysis for the Top 200 Drugs in the US Market by Prescription
Abbreviations
12.1 Introduction
12.2 Structural Alert and Reactive Metabolite Analyses for the Top 20 Most Prescribed Drugs in the United States for the Year 2009
12.3 Insights Into the Excellent Safety Records for Reactive Metabolite–Positive Blockbuster Drugs
12.4 Structural Alert and Reactive Metabolite Analyses for the Remaining 180 Most Prescribed Drugs
12.5 Structure Toxicity Trends
References
Chapter 13: Mitigating Toxicity Risks with Affinity Labeling Drug Candidates
Abbreviations
13.1 Introduction
13.2 Designing Covalent Inhibitors
13.3 Optimization of Chemical Reactivity of the Warhead Moiety
13.4 Concluding Remarks
References
Chapter 14: Dealing with Reactive Metabolite–Positive Compounds in Drug Discovery
Abbreviations
14.1 Introduction
14.2 Avoiding the Use of Structural Alerts in Drug Design
14.3 Structural Alert and Reactive Metabolite Formation
14.4 Should Reactive Metabolite–Positive Compounds be Nominated as Drug Candidates?
14.5 The Multifactorial Nature of IADRs
14.6 Concluding Remarks
References
Chapter 15: Managing IADRs – a Risk–Benefit Analysis
Abbreviations
15.1 Risk–Benefit Analysis
15.2 How Common is Clinical Drug Toxicity?
15.3 Rules and Laws of Drug Toxicity
15.4 Difficulties in Defining Cause and Black Box Warnings
15.5 Labeling Changes, Contraindications, and Warnings: the Effectiveness of Side Effect Monitoring
15.6 Allele Association with Hypersensitivity Induced by Abacavir: Toward a Biomarker for Toxicity
15.7 More Questions than Answers: Benefit Risk for ADRs
References
Index
Methods and Principles in Medicinal Chemistry
Edited by R. Mannhold, H. Kubinyi, G. Folkers
Editorial Board
H. Buschmann, H. Timmerman, H. van de Waterbeemd, T. Wieland
Previous Volumes of this Series:
Brown, Nathan (Ed.)
Bioisosteres in Medicinal Chemistry
2012
ISBN: 978-3-527-33015-7
Vol. 54
Gohlke, Holger (Ed.)
Protein-Ligand Interactions
2012
ISBN: 978-3-527-32966-3
Vol. 53
Kappe, C. Oliver / Stadler, Alexander / Dallinger, Doris
Microwaves in Organic and Medicinal Chemistry
Second, Completely Revised and Enlarged Edition
2012
ISBN: 978-3-527-33185-7
Vol. 52
Smith, Dennis A. / Allerton, Charlotte / Kalgutkar, Amit S. / van de Waterbeemd, Han / Walker, Don K.
Pharmacokinetics and Metabolism in Drug Design
Third, Revised and Updated Edition
2012
ISBN: 978-3-527-32954-0
Vol. 51
De Clercq, Erik (Ed.)
Antiviral Drug Strategies
2011
ISBN: 978-3-527-32696-9
Vol. 50
Klebl, Bert / Müller, Gerhard / Hamacher, Michael (Eds.)
Protein Kinases as Drug Targets
2011
ISBN: 978-3-527-31790-5
Vol. 49
Sotriffer, Christoph (Ed.)
Virtual Screening
Principles, Challenges, and Practical Guidelines
2011
ISBN: 978-3-527-32636-5
Vol. 48
Rautio, Jarkko (Ed.)
Prodrugs and Targeted Delivery
Towards Better ADME Properties
2011
ISBN: 978-3-527-32603-7
Vol. 47
Smit, Martine J. / Lira, Sergio A. / Leurs, Rob (Eds.)
Chemokine Receptors as Drug Targets
2011
ISBN: 978-3-527-32118-6
Vol. 46
Ghosh, Arun K. (Ed.)
Aspartic Acid Proteases as Therapeutic Targets
2010
ISBN: 978-3-527-31811-7
Vol. 45
All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.
© 2012 Wiley-VCH Verlag & Co. KGaA,
Boschstr. 12, 69469 Weinheim, Germany
All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Composition Thomson Digital, Noida, India
Cover Design Schulz Grafik-Design, Fußgönheim
Print ISBN: 978-3-527-33085-0
ePDF ISBN: 978-3-527-65577-9
ePub ISBN: 978-3-527-65576-2
mobi ISBN: 978-3-527-65575-5
oBook ISBN: 978-3-527-65574-8
Preface
The most common reasons for clinical failure and the withdrawal of already marketed drugs are insufficient efficacy and unexpected human toxicity [1, 2]. To some extent both reasons are inherently related: low doses generate fewer side effects but often lack activity, whereas higher doses are effective but may be toxic. Target-related side effects, like hERG channel inhibition, were the reason for several drug failures in past decades. Nowadays, such antitarget activities are already discovered during preclinica profiling. More critical is idiosyncratic toxicity, not detectable in clinical phase II, sometimes not even in phase III studies, because it occurs only in a very minor percentage of the population. Most often such rare toxic events are not related to the drug itself but to the generation of reactive metabolites. Correspondingly, it is of utmost importance to know the structural features that are responsible for the production of such toxic metabolites and to understand the underlying mechanisms of action. By eliminating or modifying these partial structures, the risk of idiosyncratic toxicity can be avoided or at least reduced. However, as discussed in this book, about 50% out of the most prescribed drugs show a structural alert for potential toxicity; at least most of them must be considered to be “false positives”. Sometimes very minor differences determine whether a compound forms a toxic metabolite or not.
A lesson that is told to every young medicinal chemist is to stay away from irreversible inhibitors. Whereas this indeed is good advice, we have to accept that some of the most important drugs are either irreversible inhibitors themselves, like acetylsalicylic acid, the penicillins, cephalosporins and related lactam antibiotics, or produce reactive metabolites, like omeprazole, clopidogrel, isoniazid and several others. However, these drugs or their reactive metabolites have either sufficient specificity for their therapeutic target or react with it directly at their site of generation. As a consequence, these drugs are sufficiently safe for human use. Despite the many examples of successful covalent drugs, interest in their role as drug candidates has emerged only recently.
The book by Amit Kalgutkar and his colleagues Deepak Dalvie, Scott Obach, and Dennis Smith discusses all relevant aspects of reactive drug metabolites, especially under the perspective of their relevance for drug toxicity. Numerous examples illustrate their generation and the various mechanisms of toxic action. Introductory chapters present a historical perspective of reactive drug metabolites, their role in genotoxicity and the role of various drug-metabolizing enzymes, especially the cytochrome P450 enzymes, as well as the role of reactive metabolites in drug-induced toxicity. The following two chapters describe structural alerts that indicate the potential formation of reactive metabolites. The next two chapters are dedicated to the role of reactive metabolites in pharmacological action and to the analysis of structure-toxicity relationships. The common misunderstanding that natural products per se are nontoxic is discussed in a chapter on their bioactivation, followed by a chapter on experimental approaches for the detection of reactive metabolite formation. Highly valuable for medicinal chemists is a chapter on case studies that describe the elimination or reduction of reactive metabolite formation in lead optimization. Structural alerts give a warning, but almost all of the most-prescribed drugs have a structural toxicity alert, in contrast to the excellent safety records of (most of) these drugs, as discussed in the following chapter; especially the dose range is responsible, whether toxic effects are observed. The last three chapters discuss reversible and irreversible inhibitors as drug candidates and the important issue of risk-benefit analysis, ending in a section on “more questions than answers”.
The series editors would like to thank Amit Kalgutkar and his colleagues for their enthusiasm to write this book, which is another important contribution to our series “Methods and Principles in Medicinal Chemistry”. Due to its content it will be of utmost importance for all scientists being involved in drug research, medicinal chemists as well as biochemists and biologists. Last but not least we thank the publisher Wiley-VCH, in particular Frank Weinreich and Heike Nöthe, for their engagement in this project and their ongoing support of the entire book series.
September 2012
Düsseldorf
Weisenheim am Sand
Zürich
Raimund MannholdHugo KubinyiGerd Folkers
References
1. Kennedy, T. (1997) Managing the drug discovery/development interface. Drug Discovery Today, 3, 436–444.
2. Schuster, D., Laggner, C., and Langer, T. (2005) Why drugs fail – a study on side effects in new chemical entities. Current Pharmaceutical Design, 11, 3545–3559.
A Personal Foreword
Throughout our careers in drug research, we have tried to understand the role of metabolites in drug efficacy and safety. Oxidative processes invariably yield reactive intermediates that are part of the process of drug metabolism. In most cases, these processes are extremely rapid and confined to the catalytic active site of the drug-metabolizing enzyme (e.g., cytochrome P450) concerned with metabolism; the resultant stable metabolite is the only evidence that such a process has occurred. Occasionally, some reactive species are quasi-stable and escape this environment to interact with other enzymes, tissue macromolecules, and/or DNA. The possible outcomes of this escape have consumed the past 50 years of dedicated research.
The science of reactive intermediates formed as metabolites of drugs developed from the pioneering work on the carcinogenicity of polycyclic aromatic hydrocarbons and other planar heterocyclic aromatic compounds. This early work demonstrated how epidemiological studies (e.g., high rates of cancer in chimney sweeps and shoe manufacturers) could be biochemically linked to the irreversible reaction of small organic molecules with macromolecules such as DNA. These important advances also contributed to the understanding of oxidative enzymes present in liver and other organs. Detoxification and activation processes were seen as a vital balance to the health of individuals. The association of some oxidative processes leading to reactive metabolites rather than the more abundant stable forms was embraced in the search to understand drug toxicity. By the 1970s, a strong body of opinion had formed that cellular necrosis, hypersensitivity, and blood dyscrasias induced by drugs could result from the formation of reactive metabolites. The hepatotoxicity of certain drugs such as acetaminophen could be linked to an overdose, leading to the formation of an overwhelming amount of a reactive metabolite. However, the field was vastly more complicated since many drugs showed these toxicities in only a small percentage of patients. Clues to an immunological component through drugs such as halothane and tienilic acid that had circulating antibodies to drug or modified proteins moved the science into new fields. The metabolism of drugs, such as the sulfonamide antibiotics, which cause severe skin toxicities (Stevens–Johnson syndrome), to metabolites that produced specific T cell responses is a more recent finding in this context.
With this long history, the question could be asked why now publish a work dedicated to the field? The answer lies in the multiple directions the research has taken us and in the increasing focus on drug safety. An area that was researched mainly in academic centers (of undoubted excellence), drug toxicity is now being researched, in varying degrees, in every drug discovery and development organization. There is no recipe, no standard protocol, and no regulatory mandates. Best practice is a distillation of all the knowledge and an application to each institutions research and disease area interests. This volume tries to provide that knowledge.
The research on reactive metabolites already has major influences on drug discovery, development, and prescribing to the patient population. For instance, certain functional groups are associated with a high risk of reactive metabolite formation and their inclusion should always be subject to scrutiny. Despite earlier findings, there is emerging evidence that toxicities produced by reactive metabolites show a dose–response relationship. This directly influences the design of future agents in terms of their pharmacological potency, intrinsic clearance, and so on. The possibility to predict which patients will undergo toxicity is becoming more practical. Human leukocyte antigen (HLA) class I alleles process reactive metabolite adducts. The protein adduct is attached to specific HLA molecules on the antigen-presenting cells and recognized by effector T cells via the T cell receptor to cause T cell activation. HLA-B∗5701 is carried by 100% of patients who are patch test positive for abacavir hypersensitivity and screening for this HLA is highly predictive of the toxicity. Similar diagnostics are gradually emerging for other toxicities. Drugs may in future include this information and testing as part of individualized medicine.
In this book, we have tried to distill the knowledge gained during our careers and our passion for this subject into a single volume that takes the reader through all aspects of reactive metabolites and drug toxicity: the history, the chemistry, the way to search for reactive metabolites, which structures and which drugs form them, why natural products may not be as benign as their devotees think, and also the central debate on benefit risk and the imprecise sources of information on which judgments are made. The authors hope that this volume will contribute to safer and better drugs; if so, their efforts will be repaid many folds.
Amit S. KalgutkarDeepak DalvieR. Scott ObachDennis A. Smith
Chapter 1
Origin and Historical Perspective on Reactive Metabolites
Abbreviations
AFB1Aflatoxin B1BQBenzoquinoneBSABovine serum albuminCYPCytochrome P450GSHGlutathioneGSSGGlutathione disulfideNAPQIN-Acetyl-p-benzoquinoneimineTCPO1,2-Epoxy-3,3,3-trichloropropaneTFATrifluoroacetic acidTFACTrifluoroacetyl chloride1.1 Mutagenesis and Carcinogenesis
The concept that chemicals, including drugs, could exert harmful effects on living organisms by their conversion into reactive metabolites probably dates back to the 1950s. The most compelling evidence began to be drawn in the 1960s from the area of carcinogenicity and studies looking at processing by metabolism of compounds to unstable, highly reactive metabolites. These studies drew from the human occupational exposure and animal experiments, which linked polycyclic aromatic hydrocarbons and certain other planar heterocyclic aromatic compounds, containing one or more nitrogen, sulfur, or oxygen atoms, to carcinogenic effects in humans. Human exposure was via coal tars, soot, pitch, dyes, adhesives, oil products, and tobacco smoke. Detailed examination showed that these compounds required bioactivation to electrophilic metabolites to exert their mutagenic or carcinogenic effects [1, 2]. In most cases, oxidation by cytochrome P450 (CYP) enzymes was seen as the initial step in the activation process to produce the reactive electrophilic species. Among the metabolic pathways identified for polycyclic aromatic hydrocarbons was the bay-region dihydrodiol epoxide pathway [3]. It involves three enzyme-mediated reactions: first, oxidation of a double bond catalyzed by CYP enzymes to unstable arene oxides; second, hydrolysis of the arene oxides by microsomal epoxide hydrolase to dihydrodiols; and, finally, a second CYP-catalyzed oxidation at the double bond adjacent to the diol function to generate a vicinal diol epoxide [3]. The vicinal diol epoxide was formed in the sterically hindered bay region. The bay-region diol epoxides are electrophiles capable of covalently binding to DNA. The formation of bay-region diol epoxides has been demonstrated with several polycyclic aromatics such as benzo[a]pyrene, chrysene, 5-methylchrysene, benzo[c]phenanthrene, benz[a]anthracene, and phenanthrene (Figure 1.1). Other potentially reactive metabolites with possible mutagenic and carcinogenic activity were being discovered at the same time by studies of known carcinogens (e.g., N-hydroxylation of 2-acetylaminoflourene) [4].
Figure 1.1 Conversion of phenanthrene (a) to its diol (b) by cytochrome P450 and epoxide hydrolase and its ultimate carcinogen/mutagen (c) bay-region diol epoxide by cytochrome P450.
Functional in vitro tests for mutagens (and ultimately carcinogens) were pioneered in the 1970s by Ames and coworkers (see Chapter 2 for utility of the Ames test in drug discovery). Their work examined a set of carcinogens, including aflatoxin B1 (AFB1), benzo[a]pyrene, acetylaminofluorene, benzidine, and N,N-dimethylamino-trans-stilbene, and used a rat or human liver homogenate to form reactive metabolites and colonies of Salmonella histidine mutants for mutagen detection. These experiments demonstrated that for the set of carcinogens there was a ring system sufficiently planar for a stacking interaction with DNA base pairs and a chemical functionality capable of being metabolized to a reactive species [5].
The work on mutagenesis and carcinogenesis has evolved continuously so that the effects of DNA binding are far more understood. For instance, AFB1 is present in certain foodstuffs, particularly in developing mold, and is recognized as a major contributor to liver cancer in parts of the developing world (also see Chapter 9) (Figure 1.2).
Figure 1.2 Structure of aflatoxin (AFB1).
AFB1 forms an epoxide (the exo isomer is ~1000 times more genotoxic than the endo form) that reacts with DNA to form a guanine–AFB1 DNA conjugate [6]. This difference in DNA reactivity and toxicity is believed to be due to DNA intercalation (affinity) and reactivity via an SN2 pathway. The exo epoxide has an aqueous half-life of 1 s but is still stable enough to migrate into the cell nucleus and modify DNA [6]. Different CYPs produce different amounts of the exo and endo forms, but the major human CYP isozyme, that is, CYP3A4, produces exclusively the exo form. The damage caused to DNA is specific and the effects on the ultimate protein coded for have been researched. The activation of proto-oncogenes and inactivation of tumor suppressor genes in cells are considered as major events in the multistep process of carcinogenesis. p53 is a tumor suppressor gene, which is mutated in about half of all human cancers. About 80% of these mutations are missense mutations that lead to amino acid substitution and alter the protein conformation and stability of p53. These changes can also alter the sequence-specific DNA binding and transcription factor activity of p53. Thus, the role of p53 in DNA repair, cell cycle control, and programmed cell death can be substantially altered. AFB1 exposure is correlated with a G:C to T:A transversion that leads to a serine substitution at residue 249 of p53, resultant altered activity, and ultimately hepatocellular carcinoma [7].
1.2 Detection of Reactive Metabolites
Reactive metabolites exist in aqueous solution for a short finite time. Their detection per se is difficult. The work in carcinogenicity established ways through which the problem could be tackled. Urinary products may reflect the presence of a reactive metabolite in downstream products (see Section 1.5) with stable metabolites formed by conjugation, hydration, or rearrangement of the reactive species. Sometimes, the conjugates include hydrolysis products of proteins (or genetic material), which are the target for covalent modification by the reactive metabolite. Often, in vitro systems such as liver microsomes with exogenously added nucleophilic trapping agents such as the endogenous antioxidant glutathione (GSH) provide further substantive chemical clues. Some form of functional tests (such as the Salmonella Ames test in Section 1.1) is invaluable, but broader toxicity findings are usually unavailable. Benzene is a toxin carcinogen that also causes certain blood dyscrasias including acute myeloid leukemia and aplastic anemia. It is thus carcinogenic and also myelotoxic. The principal site of toxicity is the bone marrow. The toxicity of the chemical was recognized by epidemiology and associated with, among others, the shoe industry, where it was a major constituent of glues used to bond the soles to the shoe upper. While metabolism of benzene to phenol was known before the nineteenth century, detailed investigations into the bioactivation mechanism revealed the existence of the electrophilic benzene oxide as the putative carcinogenic entity (similar to examples of polycyclic hydrocarbons in Section 1.1). The identification of the epoxide was through the isolation of the stable dihydrodiol metabolite of benzene [8]. It would be tempting to conclude that the toxicity could be explained with this finding.
In vivo studies [9] sampling urine have over the years revealed different evidence for reactive metabolite formation. These include
Detailed in vitro studies [8] have indicated that the complex pathway derives from a single enzymatic step involving the formation of benzene oxide primarily by CYP2E1 in the liver. Benzene oxide equilibrates spontaneously with the corresponding oxepine valence tautomer, which can open the ring to yield a reactive α,β-unsaturated aldehyde, trans,trans-muconaldehyde. Reduction or oxidation of trans,trans-muconaldehyde gives rise to 6-hydroxy-trans,trans-2,4-hexadienal and trans,trans-muconic acid. Both trans,trans-muconaldehyde and the hexadienal metabolites are myelotoxic in animal models. Alternatively, benzene oxide can undergo conjugation with GSH, resulting in the eventual formation and urinary excretion of S-phenylmercapturic acid. Benzene oxide is also a substrate for epoxide hydrolase, which catalyzes the formation of benzene dihydrodiol, itself a substrate for dihydrodiol dehydrogenase, producing catechol. Finally, benzene oxide spontaneously rearranges to form phenol, which subsequently undergoes conjugation (glucuronic acid or sulfate) or oxidation to hydroquinone and 1,2,4-trihydroxybenzene. The two diphenolic metabolites of benzene, catechol and hydroxyquinone, undergo further oxidation to the corresponding ortho-(1,2)- or para-(1,4)-benzoquinones (BQs) that can by myelotoxic. The 1,2- and 1,4-BQs are highly electrophilic and capable of reacting with DNA.
Benzene oxide is surprisingly stable and has a half-life of around 8 min when added to rat blood and 34 min in aqueous buffer. The metabolite therefore can perfuse out the liver to reach all the organs of the body. The toxicity of benzene in bone marrow may be due to benzene oxide formed in the liver being further oxidized in the bone marrow [8]. Benzene illustrated the complex nature of many investigations, revealing multiple possible chemical alternatives that may contribute to the toxicity in different ways.
1.3 Induction and Inhibition: Early Probes for Reactive Metabolites and Hepatotoxicants
Undoubtedly this focus on chemically reactive metabolites and cancer helped scientists to postulate and experimentally test if drug toxicity could also be initiated by such metabolites. Gillette [10] in the 1970s, among others, suggested cellular necrosis, hypersensitivity, blood dyscrasias, and fetotoxicities could be the result of reactive metabolites. Much of the early work focused on rodent toxicants at high doses and used metabolic inducers and inhibitors. Thus, bromobenzene was shown to be metabolized via a reactive metabolite pathway (epoxide) and was recovered as mercapturic acid (GSH conjugate) and dihyrodiol derivatives in the excreta. In studies utilizing identical doses of bromobenzene, pretreatment of rats with phenobarbital, which induced the metabolism of bromobenzene, increased liver cell necrosis, while SKF525A, which inhibited bromobenzene metabolism, decreased liver cell necrosis. Similar studies implicated reactive metabolites in the hepatotoxicity of acetaminophen and furosemide. Acetaminophen research helped define a number of important areas of reactive metabolite research as detailed below. Acetaminophen has remained a focus because the drug when given or ingested in large doses causes hepatotoxicity in all species, albeit with different sensitivities (in humans the toxic dose is around 20–40 g), although some changes in hepatic function have been observed in daily doses as low as 2 g. The toxicity is related to dose size, and in vitro and in vivo models can be readily established.
1.4 Covalent Binding and Oxidative Stress: Possible Mechanisms of Reactive Metabolite Cytotoxicity
Early investigations lacked many of the physicochemical measures available today, but the 1980s were critical in the identification of covalent binding per se and/or redox recycling and oxidative stress as possible toxic outcomes of reactive metabolite formation. Identifying the reactive species actually binding was problematic. Different target proteins were often added to incubations and synthetic metabolites used. Acetaminophen was a major focus. The compound when radiolabeled bound to hepatic microsomes from phenobarbital-pretreated mice [11]. Cysteine and GSH inhibited this binding, whereas several non-thiol amino acids did not. Bovine serum albumin (BSA), when used as an alternative target protein, inhibited covalent binding to microsomal protein in a concentration-dependent manner. BSA has a single thiol group and the binding now occurred to BSA. When α-s1-casein was substituted (a nonfree protein), little binding to the protein occurred. In duplicate experiments synthetic N-acetyl-p-benzoquinoneimine (NAPQI) (the two-electron oxidation product of acetaminophen) reproduced the results, identifying it as the reactive arylating metabolite of acetaminophen and suggesting that downstream effects of interactions with thiol groups may be responsible for the toxicity.
The reaction with thiol groups may be important but other aspects of reactive metabolite formation began to be examined [12]. The reaction of NAPQI with GSH in aqueous solution forms an acetaminophen–GSH conjugate and acetaminophen and glutathione disulfide (GSSG). Similar reactions occur in hepatocytes, but the GSSG is rapidly converted back to GSH by the NADPH-dependent glutathione reductase and a rapid oxidation of NADPH. Inhibitors of glutathione reductase prevent this and enhance cytotoxicity without changing the extent of covalent binding. Dithiothreitol added to isolated hepatocytes after maximal covalent binding of NAPQI, but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may not result simply from covalent binding but from its oxidative effects on cellular proteins.
1.5 Activation and Deactivation: Intoxication and Detoxification
As work on reactive metabolites has progressed, so the balance between competing pathways, detoxifying reactions (such as GSH conjugate formation), and actual inherent species or individual sensitivity has become progressively more important. Without this knowledge it is tempting to conclude that it is the formation rate and amount of a particular metabolite that is important.
Acetaminophen showed varying toxicity when tested in different species. The drug and its toxic reactive metabolite, NAPQI, were, therefore, investigated in hepatocytes from different species [13]. Clear conclusions were drawn from these early studies in which acetaminophen triggered cell blebbing and loss of viability in the cells from mouse and hamster in contrast to human and rat hepatocytes, which were much more resistant to these effects. When NAPQI itself was tested, there were no significant differences in the sensitivity of the cells, from any species, to the toxic effects. The conclusion reached in these studies was that species differences in sensitivity to the hepatotoxicity of acetaminophen were due to differences in the rate of formation of NAPQI and not due to any intrinsic differences in sensitivity or any difference in the fate of NAPQI once formed. Later studies [14] have seen some correlations but without such clear-cut conclusions, observations often concluded that the actual test system, or conditions, produced significant variation. In these studies all the metabolites were quantified. These were separated into those downstream of NAPQI (GSH, cysteinylglycine, cysteine, and mercapturate conjugates) and alternative pathways (metabolites, such as the glucuronide and sulfate conjugates). The ratio of downstream NAPQI/alternative pathway metabolites excreted was 2.2, 1.0, 0.25, 0.1, and 0.08 for hamsters, mice, rabbits, rats, and guinea pigs, respectively, and inversely related to the hepatotoxic dose reported for these species. This is supportive of species sensitivity being determined by the balance between toxification and detoxification metabolic pathways [15].
It is likely that species differences in acetaminophen toxicity are due to a combination of pharmacokinetic (metabolism) differences and other biological variations. There is a natural inclination to believe that metabolism differences explain different responses, but the evidence in many cases is lacking. This is particularly true when the products of metabolism differ along with species responses.
1.6 Genetic Influences on Reactive Metabolite Formation
Early focus, even in the 1980s, was to look for at-risk populations. Understanding of enzymology and genetic variation was at an early stage but some links were established. Isoniazid, an antitubercular drug, caused hepatotoxicity in around 1% of the population. Increased risk was observed in fast acetylators of the drug that formed comparatively more of the metabolite N-acetylisoniazid. This hepatotoxicity could also be observed in animal studies together with covalent binding of radioactivity in the liver. Radiolabeled () versions of the metabolite bound covalently only when the acetyl group was labeled and not when the was incorporated into the pyridine ring [16]. Pathways suggested involved the formation of an N-hydroxy derivative, which dehydrated to a diazene that could fragment in the presence of oxygen to radical species (Figure 1.3).
Figure 1.3 Pathway of isoniazid (a) metabolism forming N-acetylisoniazid (b) by acetylation, hydrolysis to isonicotinic acid (c) and N-acetylhydrazine (d), and subsequent N-hydroxylation (e).
1.7 Halothane: the Role of Reactive Metabolites in Immune-Mediated Toxicity
Halothane anesthesia may be followed by changes in liver function. For 25–30% of patients there is a minor degree of disturbance of liver function shown by increased serum transaminases or glutathione-S-transferase. With this mild change subsequent reexposure to halothane is not necessarily associated with evidence of liver damage. In 1 in 20 000 patients normally having past experience of halothane anesthesia, massive liver cell necrosis can occur, frequently leading to fulminant hepatic failure. This type of liver damage has clinical, serological, and immunological features strongly indicating an immune-mediated idiosyncratic reaction [17].
Halothane metabolism [18] produces three main excreted metabolites: trifluoroacetic acid (TFA), N-triflouroacetyl-2-aminoethanol, and, to a lesser extent, N-acetyl-S-(2-bromo-2-chloro-1,1-difluoroethyl)-l-cysteine. The last two metabolites are downstream products of reactive metabolites. They are formed by hydroxylation of halothane with spontaneous loss of hydrogen bromide to form trifluoroacetyl chloride (TFAC), which would form TFA by hydrolysis or N-triflouroacetyl-2-aminoethanol by reaction with intracellular products. TFAC is also known to acylate lysine residues on proteins. Dehydrofluorination to 2-bromo-2-chloro-1,1-difluroethylene is the likely route to the cysteine conjugate.
CYP2E1 is the major catalyst in conversion of halothane to the reactive metabolite TFAC [19] and formation of trifluoroacetylated proteins. Trifluoroacetylated CYP2E1 was detected immunochemically in livers of rats treated with halothane and high levels of autoantibodies that recognized purified rat CYP2E1 but not purified rat CYP3A were detected by enzyme-linked immunosorbent assay in 14 of 20 (70%) sera from patients with halothane hepatitis. In contrast, only very low levels of such antibodies were detected in sera from healthy controls, from patients anesthetized with halothane without developing hepatitis, or from patients with other liver diseases. The intracellular location of trifluoroacetyl adducts was predominantly in the endoplasmic reticulum and also, to a lesser extent, on the cell surface. Thus, halothane metabolism by CYP2E1 results in the cell surface expression of acetylated CYP2E1 that could be important as an antigen in halothane hepatotoxicity.
1.8 Formation of Reactive Metabolites, Amount Formed, and Removal of Liability
Other inhaled anesthetics such as isoflurane and desflurane [20] also have TFAC as a metabolic product as evidenced by recoveries of TFA. However, the degree of biotransformation of these anesthetics is much less than that of halothane. This lower degree of exposure to the reactive metabolite may be an important factor in a much lower immune response and hepatotoxic risk. A later gaseous anesthetic sevoflurane [20] is biotransformed to a lesser degree than halothane (3–5% versus 18–25%), and much less total mass of metabolites is formed. The primary organic metabolite of sevoflurane is hexafluoroisopropanol, not TFAC. This metabolite is not chemically reactive in comparison to TFAC. Hexafluoroisopropanol does not accumulate, being rapidly cleared by glucuronidation that in turn is rapidly excreted in the urine. This is in contrast to halothane, where TFA (from TFAC) is detectable in urine for up to 12 days after 75 min of anesthesia (Figure 1.4).
Figure 1.4 Structure of (I) halothane, its hydroxylated metabolite (II), the reactive trifluoroacetyl chloride (III, TFAC) formed by HBr loss, and the downstream product trifluoroacetic acid (IV, TFA). The metabolism of sevoflurane (V) is shown in comparison, where the principal stable metabolite is hexafluoroisopropanol (VI), rapidly converted to a glucuronic acid conjugate.
1.9 Antibodies: Possible Clues but Inconclusive
Similar to halothane, tienilic acid forms antibodies that are associated with hepatotoxicity. In this case the circulating antibodies recognize CYP2C9, the principal CYP isozyme in the metabolism of tienilic acid. Moreover, tienilic acid is a very potent mechanism-based inhibitor of CYP2C9. Again the haptenized protein appears on the surface of the hepatocyte [21]. Other covalently altered enzymes are apparently the hapten for antibody production. For instance, iproniazid, an irreversible monoamine oxidase-B inhibitor, causes antibodies to be formed against monoamine oxidase-B. While it is enticing to link outcome to an immunological mechanism, many patients have circulating antibodies with no sign of toxicity. Practolol, a β-adrenoceptor antagonist with an acetylated aniline template, was responsible for oculomucocutaneous syndrome. An antibody specific to a practolol-reactive metabolite, most probably formed by oxidation of the aniline nitrogen, was found in the plasma of practolol-treated patients with or without a history of adverse reaction to the drug. No antibodies were detected in patients treated with other β-blocking drugs. Titer of the antibody was highly variable [22].
1.10 Parent Drug and Not Reactive Metabolites, Complications in Immune-Mediated Toxicity
Sulfonamide antibacterials are one of the earliest examples of chemotherapy against infection and are inhibitors of tetrahydropteroic acid synthetase. The natural substrate for this enzyme is para-aminobenzoic acid. Sulfonamides mimic the natural substrate with the para-aminobenzene (aniline) being retained but with the carboxylic acid being replaced by the isostere sulfonamide. The drugs cause serious skin toxicities including erythema multiforme, Stevens–Johnson syndrome, and toxic epidermal necrolysis. The N-4-hydroxylamine metabolite of these drugs, which can be formed in the skin, was initially identified and a rationale of covalent binding to proteins and induction of specific adverse immune response was developed. Anilines can be oxidized to more than one reactive metabolite and the nitroso intermediate has been shown to be a potent antigenic determinant [23, 24]. Surprisingly and contrary to the theories on reactive metabolites, T cell responses against the parent drug have also been detected. These T cells are low in proportion and highly selective, being responsive only to the particular drug used in the treatment such as sulfamethoxazole. Thus, they do not react to related sulfonamide antibacterial agents such as sulfapyridine or sulfadiazine. In contrast, those generated from reactive metabolites of sulfamethoxazole can be stimulated by other structurally related drugs such as sulfapyridine and sulfadiazine.
1.11 Reversible Pharmacology Should not be Ignored as a Primary Cause of Side Effects
Phenytoin is responsible for “fetal hydantoin syndrome,” a defined set of side effects on the embryo, which include embryonic death, intrauterine growth retardation, mild central nervous system dysfunction, and craniofacial abnormalities. Original theories as causes included reactive metabolite formation. Phenytoin teratogenesis was postulated to result from epoxide formation [25] and covalent binding of the epoxide, the ultimate teratogen, to constituents of gestational tissue. Some experimental evidence was obtained in which Swiss mice were given teratogenic doses of phenytoin with and without a nonteratogenic dose of 1,2-epoxy-3,3,3-trichloropropane (TCPO), an epoxide hydrolase inhibitor. TCPO significantly increased the incidence of I-induced cleft lip and palate and enhanced the embryolethality twofold compared to phenytoin alone. The covalent binding of phenytoin-derived radioactivity in fetuses and placenta was enhanced by TCPO.
Further experiments looking at other causes have shown that the syndrome is unequivocally linked to phenytoin's reversible secondary pharmacology, namely, its blockade of the IKr delayed rectifier K+ channel. In rodents the expression of this channel is age-specific, making the fetal heart especially sensitive compared to the mature animal. IKr blockers (which include phenytoin) initiate concentration-dependent embryonic bradycardia/arrhythmia resulting in hypoxia, explaining embryonic death and growth retardation, and episodes of severe hypoxia, followed by generation of reactive oxygen species within the embryo during reoxygenation, causing orofacial clefts and distal digital reductions and alterations in embryonic blood flow and blood pressure, inducing cardiovascular defects [26].
1.12 Conclusions: Key Points in the Introduction
This historical review serves as a stepwise introduction to the book. The important topics that have been introduced in this chapter will be explored in much greater detail in subsequent chapters and include the following:
References
1. Miller, E.C. and Miller, J.A. (1966) Mechanism of chemical carcinogenesis: nature of proximate carcinogens and interactions with macromolecules. Pharmacology Reviews, 18 (1), 805–838.
2. Miller, E.C. and Miller, J.A. (1981) Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules. Cancer, 47 (10), 2327–2345.
3. Xue, W. and Warshawsky, D. (2005) Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage. Toxicology and Applied Pharmacology, 206 (1), 73–93.
4. Cramer, J.W., Miller, J.A., and Miller, E.C. (1960) N-Hydroxylation: a new metabolic reaction observed in the rat with the carcinogen 2-acetylaminofluorene. Journal of Biological Chemistry, 235 (3), 885–888.
5. Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.D. (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proceedings of the National Academy of Sciences of the United States of America, 70 (8), 2281–2285.
6.Guengerich, F.P. (2001) Forging the links between metabolism and carcinogenesis. Mutation Research, 488 (3), 195–209.
7. Hussain, S.P., Schwank, J., Staib, F., Wang, X.W., and Harris, C.C. (2007) P53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene, 26 (15), 2166–2176.
8. Monks, T.J., Butterworth, M., and Lau, S.S. (2010) The fate of benzene-oxide. Chemico-Biological Interactions, 184 (1–2), 201–206.
9. Medeiros, A.M., Bird, M.G., and Witz, G. (1997) Potential biomarkers of benzene exposure. Journal of Toxicology and Environmental Health, 51 (6), 519–539.
10. Gillette, J.R. (1974) A perspective on the role of chemically reactive metabolites of foreign compounds in toxicity. I. Correlation of changes in covalent binding of reactivity metabolites with changes in the incidence and severity of toxicity. Biochemical Pharmacology, 23 (21), 2785–2794.
11. Streeter, A.J., Dahlin, D.C., Nelson, S.D., and Baillie, T.A. (1984) The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro. Chemico-Biological Interactions, 48 (3), 349–366.
12. Albano, E., Rundgren, M., Harvison, P.J., Nelson, S.D., and Moldeus, P. (1985) Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity. Molecular Pharmacology, 28 (3), 306–311.
13. Tee, L.B., Davies, D.S., Seddon, C.E., and Boobis, A.R. (1987) Species differences in the hepatotoxicity of paracetamol are due to differences in the rate of conversion to its cytotoxic metabolite. Biochemical Pharmacology, 36 (7), 1041–1052.
14. Jemnitz, K., Veres, Z., Monostory, K., Kobori, L., and Vereczkey, L. (2008) Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes. Toxicology In Vitro, 22 (4), 961–967.
15. Gregus, Z., Madhu, C., and Klaassen, C.D. (1988) Species variation in toxication and detoxication of acetaminophen in vivo: a comparative study of biliary and urinary excretion of acetaminophen metabolites. Journal of Pharmacology and Experimental Therapeutics, 244 (1), 91–99.
16. Timbrell, J.A., Mitchell, J.R., Snodgrass, W.R., and Nelson, S.D. (1980) Isoniazid hepatotoxicity: the relationship between covalent binding and metabolism in vivo. Journal of Pharmacology and Experimental Therapeutics, 213 (20), 364–369.
17. Neuberger, J.M. (1990) Halothane and hepatitis. Incidence, predisposing factors and exposure guidelines. Drug Safety, 5 (1), 28–38.
18. Cohen, E.N., Trudell, J.R., Edmunds, H.N., and Watson, E. (1975) Urinary metabolites of halothane in man. Anesthesiology, 43 (4), 392–401.
19. Eliasson, E. and Kenna, J.G. (1996) Cytochrome P450 2E1 is a cell surface autoantigen in halothane hepatitis. Molecular Pharmacology, 50 (3), 573–582.
20. Frink, E.J. (1995) The hepatic effects of sevoflurane. Anesthesia and Analgesia, 81 (6S), S46–S50.
21. Dansette, P.M., Bonierbale, E., Minoletti, C., Beaune, P.H., Pessayre, D., and Mansuy, D. (1998) Drug-induced immunotoxicity. European Journal of Drug Metabolism and Pharmacokinetics, 23 (4), 443–451.
22. Amos, H.E., Lake, B.G., and Artis, J. (1978) Possible role of antibody specific for a practolol metabolite in the pathogenesis of oculomucocutaneous syndrome. British Medical Journal, 1 (6110), 402–404.
23. Sanderson, J.P., Naisbitt, D.J., Farrell, J., Ashby, C.A., Tucker, M.J., Rieder, M.J., Pirmohamed, M., Clarke, S.E., and Park, B.K. (2007) Sulfamethoxazole and its metabolite nitroso sulfamethoxazole stimulate dendritic cell costimulatory signaling. Journal of Immunology, 178 (9), 5533–5542.
24. Castrejon, J.L., Berry, N., El-Ghaiesh, S., Gerber, B., Pichler, W.J., Park, B.K., and Naisbitt, D.J. (2010) Stimulation of human T cells with sulfonamides and sulfonamide metabolites. Journal of Allergy and Clinical Immunology, 125 (2), 411–418.
25. Martz, F., Failinger, C., and Blake, D.A. (1977) Phenytoin teratogenesis: correlation between embryopathic effect and covalent binding of putative arene oxide metabolite in gestational tissue. Journal of Pharmacology and Experimental Therapeutics, 203 (1), 231–239.
26. Danielsson, B.R., Skold., A.C., and Azarbayjani, F. (2001) Class III antiarrhythmics and phenytoin: teratogenicity due to embryonic cardiac dysrhythmia and reoxygenation damage. Current Pharmaceutical Design, 7 (9), 787–802.
Chapter 2
Role of Reactive Metabolites in Genotoxicity
Abbreviations
CYPCytochrome P450GSHGlutathioneHPB4-Hydroxy-1-(3-pyridyl)-1-butanoneNATN-AcetyltransferaseNNAL4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanolNNK4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanoneNNNN′-NitrosonornicotineSULTSulfotransferases2.1 Introduction
The role of metabolism in the generation of reactive metabolites that are responsible for forming covalent bonds to nucleic acids and causing mutagenic lesions is now well established for many endogenous and exogenous xenobiotics. Approximately half of the chemicals listed as “known” human carcinogens and “probable” human carcinogens (Table 2.1) by the International Agency for Research on Cancer require metabolic activation to reactive species that are ultimately responsible for the carcinogenic activity of the parent compound. Virtually, any molecule that forms reactive metabolites possesses the propensity to modify DNA and elicit a genotoxic/carcinogenic response. However, there are certain commonalities in carcinogenic substances with respect to functional groups capable of generating DNA-reactive metabolites.
Table 2.1 Examples of carcinogenic substances that require metabolic activation to reactive intermediates for their carcinogenic activities.
Human CarcinogensProbable Human CarcinogensAflatoxinsAcrylamidepara-Cresidine4-AminobiphenylAdriamycin (doxorubicin)DacarbazineAristolochic acidBischloroethyl nitrosourea2,4-Diaminoanisole sulfateBenzeneChloramphenicolDibenz[a,h]acridineBenzidine (and dyes metabolized to benzidine)1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosoureaEthylene dibromideBenzo[a]pyrene4-Chloro-ortho-toluidineDiepoxybutaneBis(chloromethyl)ether and chloromethyletherCyclopenta[cd]pyrene1,1-Dimethylhydrazine1,3-ButadieneDibenz[a,h]anthracene1,6-DinitropyreneChlorambucilDibenzo[a,l]pyreneFuranCyclophosphamideDiethyl sulfateHydrazobenzeneDiethylstilbestrol5-Methoxypsoralen5-MethylchryseneEthylene oxideN-Methyl-N-nitrosoureaMethyleugenolMelphalanNitrogen mustardMetronidazole8-MethoxypsoralenN-NitrosodiethylamineNitrobenzene2-Naphthylamine2-Nitrotoluene1-NitropyreneN′-NitrosonornicotineTrichloroethyleneOchratoxin A4-N-(Nitrosomethylamino)-1-(3-pyridyl)-1-butanone1,2,3-TrichloropropanePropylthiouracilPhenacetinVinylbromideSafroleSulfur mustardsVinylfluorideStyreneTamoxifen2-AcetylaminofluoreneThioureaThiotepa2-Aminoanthraquinoneortho-Toluidineortho-AminoazotolueneVinyl chloride2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)2-Amino-3,4-dimethylimidazo[4,5-f]quinoxaline (MeIQx)2-Amino-3-methylimidazo[4,5-f]quinoline (IQ)2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (phIP)ortho-AnisidineBenz[a]anthraceneBromodichloromethane4-Chloro-ortho-phenylenediamine2.2 Carcinogenicity of Aromatic and Heteroaromatic Amines
Several aromatic amines (e.g., benzidine, 2-naphthylamine, and 4-aminobiphenyl, Figure 2.1) are of industrial importance because of their applications in the dyestuff industry and as antioxidants in rubber and other commercial products. The carcinogenic properties of aromatic amines were evident in the late 1800s from epidemiological reports on the appearance of urinary bladder tumors among workers employed in the German aniline dyestuff industry in the production of “fuchsin” (magenta). This workplace was subsequently associated with frequent occurrence of cancer of the urinary tract, referred to as “aniline cancer” [1]. Although aniline and other aromatic amines failed to produce tumors in rabbits, Hueper et al. showed that 2-naphthylamine induced bladder tumors in dogs when administered to the stomach and skin [2]. Likewise, hepatocarcinogenicity of aminoazo dyes such as ortho-aminoazotoluene and N,N-dimethyl-4-azobenzene (“butter yellow”) was also demonstrated in rats [3]. In 1941, the first report on tumorigenesis in bladder, liver, kidney, pancreas, and lung of rats induced by 2-acetylaminofluorene, an aromatic amine intended to be used as a pesticide, was published [4]. Around the same time, Swedish chemist Widmark demonstrated that extracts of fried horse meat induced cancer when applied to mouse skin [5]. Sugimura and coworkers investigated the smoke produced by broiling fish and meat; they demonstrated that the smoke condensate and charred surfaces of broiled fish and meat were highly mutagenic in Salmonella typhimurium test systems [6]. Subsequently, heteroaromatic amine derivatives formed as a consequence of pyrolysis of amino acids or protein-containing foods were isolated, their structures were determined, and their biological effects were examined, specifically mutagenicity and carcinogenicity in animals [7, 8] (also see Chapter 9 for additional discussions on bioactivation pathways of natural products). The formation of heteroaromatic amines is the result of a Maillard reaction, which occurs when amino acids and reducing sugars are heated together [9]. More than 20 such compounds have been identified (see Figure 2.1). Heterocyclic arylamines have also been identified in cigarette smoke condensate and have been shown to be genotoxic [10]. Aromatic amines are also formed in commercial hair dyes, and their contributions to an increased risk of bladder, breast, colon, and lymphatic cancer have been investigated [11].
Figure 2.1 Examples of carcinogenic aromatic amines and heteroaromatic amines found in food.
The mechanism of carcinogenicity of aromatic and heteroaromatic amines involves bioactivation to reactive metabolites [12, 13]. The rate-limiting step is the N-oxidation of the amine nitrogen, which is mediated primarily by cytochrome P450 (CYP) enzymes, although flavin-containing monooxygenases and peroxidases are also known to play a role in the bioactivation pathway (Figure 2.2) [14–16]. Studies on aromatic amine oxidation go back to the 1940s, with early work on aminoazo dyes by Mueller and Miller [17]. N-Hydroxylation by CYP enzymes was first demonstrated with the acetamide derivative of 2-aminofluorene [18, 19], and further studies extended the work to unsubstituted aromatic amines [20]. CYP1A1 and 1A2 have been generally recognized to be the major isoforms involved in the bioactivation of aromatic and heterocyclic amines in human liver and lung microsomes [21, 22]. The findings with CYP1A2 have been confirmed in vivo in human studies, at least with the heteroaromatic amines PhIP and MeIQx (see Figure 2.1). The CYP1A2-selective inhibitor furafylline blocked most of the in vivo elimination of these compounds in studies in which the human volunteers consumed burned meat [23]. The N-hydroxy products of some heteroaromatic amines can be further oxidized to produce the nitroso intermediates (Figure 2.2). This reaction seems to be selective among the heteroaromatic amines substrates and may contribute to toxicity, through covalent binding to either proteins or DNA. The nitroso derivatives of heteroaromatic amines have been shown to react with DNA, proteins, as well as the endogenous antioxidant glutathione (GSH) [24].
Figure 2.2 Bioactivation of aromatic and heteroaromatic amines via phase I (oxidative) and phase II (conjugation) pathways.
N-Hydroxylamine metabolites are also prone to bioactivation via a phase II conjugation reaction to produce highly reactive ester derivatives that bind covalently to DNA (also see Chapter 9). Among the phase II enzyme systems responsible for the secondary activation step in mammals, N-acetyltransferases (NAT) and sulfotransferases (SULT) are most prominent and lead to the formation of reactive N-acetoxy and N-sulfonyloxy esters, respectively [25–27]. NAT-catalyzed acetylation of N-hydroxy aromatic and heteroaromatic amines enhances the genotoxic activity and DNA adduct levels via the generation of reactive N-acetoxyl esters [25–28]. In a similar way, the sulfur esters formed by the action of SULT enzymes are unstable and react readily with DNA [26]. Aromatic and heteroaromatic amines yield DNA adducts primarily through covalent adduction with guanine residues [29], reacting at the N2 and C8 atoms. The finding that photoactivated azide derivatives of IQ, MeIQx, and PhIP bind to DNA to form the same adducts as the N-acetoxy species indicates that the nitrenium ion may be a common intermediate for both reactive intermediates [30]. The N-hydroxy heteroaromatic amines can react directly with DNA, but the reaction is facilitated when reactive ester derivatives undergo heterocyclic cleavage to yield reactive aryl nitrenium ion species, which preferentially react to form DNA adducts (Figure 2.2).
2.3 Carcinogenicity of Nitrosamines
Tobacco-specific nitrosamines have emerged as one of the most important groups of carcinogens in tobacco products (also see Chapter 9 for additional discussions) [31]. Seven tobacco-specific nitrosamines have been identified in tobacco products, but two of these – 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) (Figure 2.3) – are most important because of their carcinogenic activities and their consistent presence in both unburned tobacco and its smoke, frequently in considerable amounts. NNK selectively induces lung tumors in all species tested and is a particularly potent carcinogen in the rat [32]. It also causes tumors of the pancreas, nasal mucosa, and liver [32]. NNN produces esophageal and nasal cavity tumors in rats and respiratory tract tumors in mice and hamsters [32]. Both NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer (Table 2.1).
Figure 2.3 Bioactivation pathways of nitrosamines leading to DNA-reactive metabolites.
Dialkylnitrosamines such as NNK require metabolic activation via CYP-catalyzed α-hydroxylation to exert their carcinogenic properties. Figure 2.3 illustrates the α-hydroxylation pathways of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), as well as the 2′-α-hydroxylation pathway for NNN. α-Hydroxylation of the NNK methyl group leads to the carbinolamine species 1, which spontaneously loses formaldehyde yielding 4-(3-pyridyl)-4-oxobutanediazohydroxide 2. The same intermediate is formed by 2′-hydroxylation of NNN. α-Hydroxylation at the methylene group of NNK produces the secondary alcohol metabolite 3, which spontaneously decomposes to methanediazohydroxide. Similar intermediates are produced by α-hydroxylation of NNAL. Methanediazohydroxide, formed in these reactions from NNK and NNAL, reacts with DNA to produce the well-known DNA adducts, O6-methyl-dGuo, 7-methyl-dGuo, and O4-methyl-dThd, which are common to many methylating carcinogens [32]. Subsequently, it was demonstrated that neutral thermal or acid hydrolysis of DNA from NNK-, NNN-, or NNAL-treated animals produced 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), confirming the pathway of DNA alkylation involving the diazo intermediate resulting in pyridyloxobutyl (POB)–DNA adducts [33, 34]. Released HPB could be quantified by mass spectrometry, and its presence was established in the lung DNA of smokers, as well as in NNK-treated rodents [35].
2.4 Carcinogenicity of Quinones and Related Compounds
Quinones are ubiquitous in nature and constitute an important class of naturally occurring compounds found in plants, fungi, and bacteria. Naturally occurring quinones are synthesized via the shikimate or polyketide pathways, which are absent in animals. Humans are exposed to endogenous quinones primarily via the oxidative metabolism of catecholamines and estrogens. Human exposure to quinones also occurs via the diet, clinically, or via airborne pollutants such as quinone derivatives of polycyclic aromatic hydrocarbons. From a toxicological perspective, quinones are oxidants and electrophiles. They possess the soft (α,β-unsaturated carbonyl) and hard (carbonyl) electrophilic centers capable of reacting with GSH in a classic 1,4-Michael fashion or with DNA bases in a 1,2- and/or 1,4-Michael fashion [36]. Since nucleophilic 1,4-Michael addition to quinone represents a formal two-electron reduction resulting in a catechol–nucleophile conjugate, their oxidant and electrophilic properties are intimately related.
The carcinogenic activity of estrogens used as replacement therapy is thought to be mediated through CYP-mediated metabolism to the corresponding electrophilic/redox-active quinones (via the intermediate catechol metabolites) [37]. Once formed, the catechol estrogen metabolites can be oxidized by virtually any oxidative enzyme or metal ions affording the reactive ortho-quinone metabolites. ortho-Quinones and quinone methides (the products of isomerization) are known estrogen metabolites, which cause alkylation and/or oxidative damage to cellular proteins and DNA with estradiol, as illustrated in Figure 2.4 [38]. ortho-Quinones are also redox-active compounds and can undergo redox cycling with the semiquinone radical, generating superoxide radicals mediated through CYP/CYP reductase (see Figure 2.4). The reaction of superoxide anion radicals with 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 hydroxyl radicals, which are powerful oxidants responsible for damage to essential macromolecules and DNA [39]. It is noteworthy to point out that the quinone-containing anticancer drugs such as mitomycin C, adriamycin, and daunorubicin (Figure 2.5) exert their pharmacological action via such a quinone–hydroquinone redox cycling, leading to reactive oxygen species [40].
Figure 2.4 Metabolism of estradiol into DNA-reactive ortho-quinone and quinone methide metabolites.
Figure 2.5 Structures of quinone-containing anticancer drugs.
An additional illustration on the carcinogenicity of quinones is evident with tamoxifen, which is used to treat hormone-dependent breast cancer. The increased risk of endometrial cancer associated with tamoxifen therapy has been associated with its bioactivation potential to reactive quinone species (Figure 2.6) [41, 42]. One of the suggested bioactivation pathways involves aromatic ring oxidation to 4-hydroxytamoxifen, which on a two-electron oxidation can generate the electrophilic quinone methide species. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. Interestingly, the quinone methide is unusually stable; its half-life under physiological conditions is ~3 min and its half-life in the presence of GSH is ~4 min [43]. In addition, tamoxifen can also be metabolized to a catechol intermediate followed by further two-electron oxidation to the ortho-quinone, which is capable of reacting with DNA [44]. Apart from bioactivation pathways arising from aromatic ring hydroxylation to quinones, Shibutani et al. [41, 42] have characterized tamoxifen–DNA adducts in the endometrium of women treated with tamoxifen, which presumably arise from the α-hydroxylation (on the pendant ethyl group) of a tamoxifen N-oxide metabolite. α-Hydroxytamoxifen undergoes sulfation and loss of sulfate group to a carbocation species, which reacts with 2′-deoxyguanosine (Figure 2.6).
Figure 2.6 Bioactivation pathways of tamoxifen leading to protein- and DNA-reactive metabolites.
The final example in this category is that of safrole, the main constituent of sassafras oil, which is also present in a number of herbs and spices, such as nutmeg, mace, cinnamon, anise, black pepper, and sweet basil. Sassafras oil is extracted from the root bark of the tree Sassafras albidum and was widely used as a natural diuretic, as well as a remedy against urinary tract disorders, until safrole was discovered to be hepatotoxic and weakly carcinogenic [45]. In 1960 the US Food and Drug Administration banned the use of sassafras oil as a food and flavoring additive because of the high content of safrole and its proven carcinogenic effects. Two bioactivation pathways of safrole to potentially hepatotoxic and carcinogenic intermediates have been reported (Figure 2.7) [46–48]. The first one involves CYP-catalyzed hydroxylation of the benzyl carbon producing 1′-hydroxysafrole and conjugation with sulfate generating a reactive sulfate ester. This ester undergoes an SN1 displacement reaction creating a highly reactive carbocation, which alkylates DNA [49]. The second pathway involves methylenedioxy ring scission leading to the formation of the catechol derivative, hydroxychavicol, which is a natural product found in betel leaf [50, 51]. Hydroxychavicol can easily be oxidized to the ortho-quinone, which isomerizes nonenzymatically to the more electrophilic para-quinone methide. Both pathways could explain the genotoxic effects of safrole, and DNA adducts consistent with the carbocation pathway have been identified in vitro and in vivo [52–54]. Recently, studies with betel quid chewers demonstrated that betel quid containing safrole-induced DNA adducts is highly associated with the development of oral squamous cell carcinoma in Taiwan [55].
Figure 2.7 Bioactivation pathways of safrole leading to DNA-reactive metabolites.
2.5 Carcinogenicity of Furan
Furan is an important industrial intermediate and solvent. It is also an environment contaminant, present in wood smoke, tobacco smoke, and car exhaust. Furan is a liver and kidney toxicant in laboratory animals [56]. It induces hepatocellular adenomas and carcinomas in rats and mice and hepatic cholangiocarcinomas in rats. Based on these results and the large potential for human exposure, furan has been listed as a possible human carcinogen (see Table 2.1) by the National Toxicology Program and the International Agency for Research on Cancer.
The rate-limiting step in furan toxicity appears to be its metabolism to reactive species. The metabolism of furan is initiated by a CYP-catalyzed oxidation to the reactive α,β-unsaturated dialdehyde cis-2-butene-1,4-dial presumably via the epoxide intermediate (Figure 2.8) [57, 58]. The dicarbonyl metabolite can be trapped either as a bis-semicarbazone derivative or as GSH conjugate [57–59]. cis-2-Butene-1,4-dial also possesses the propensity to alkylate cellular nucleophiles such as amino acids and DNA [60–64]. Products of these reactions have been observed in the urine of furan-treated rats including the mono-GSH reaction product, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinylglycine cyclic sulfide (4), and a downstream metabolite of this product, S-[1-(1,3-dicarboxypropyl)-1H-pyrrol-3-yl]methylthiol (5) [60, 65]. Three additional urinary metabolites of furan have also been identified as follows: (R)-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid (6), N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (7), and its sulfoxide (8) [65]. Metabolite 6 results from the reaction of the dicarbonyl metabolite with lysine, whereas metabolites 7 and 8 result from the cross-linking of cysteine and lysine by cis-2-butene-1,4-dial (intermediate 9).
Figure 2.8 Oxidative bioactivation of furan to the electrophilic cis-2-butene-1,4-dial metabolite and its downstream adduction with nucleophiles.
cis-2-Butene-1,4-dial reacts readily with 2′-deoxycytidine, 2′-deoxyadenosine, and 2′-deoxyguanosine to form diastereomeric oxadiazabicyclo(3.3.0)octaimine adducts (Figure 2.9
