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Cytochromes P450
Role in the Metabolism and Toxicity of Drugs and other Xenobiotics
Issues in Toxicology

Series Editors

Professor Diana Anderson, University of Bradford, UK

Dr Michael D Waters, Integrated Laboratory Systems, Inc, N. Carolina, USA
Dr Timothy C Marrs, Edentox Associates, Kent, UK

This Series is devoted to coverage of modern toxicology and assessment of risk and is
responding to the resurgence in interest in these areas of scientific investigation.

Ideal as a reference and guide to investigations in the biomedical, biochemical and

pharmaceutical sciences at the graduate and post graduate level.

Titles in the Series:

Cytochromes P450: Role in the Metabolism and Toxicity of Drugs and other Xenobiotics
Edited by Costas Ioannides, University of Surrey, Guildford, UK

Hair in Toxicology: An Important Bio-Monitor

Edited by Desmond John Tobin, University of Bradford, Bradford, UK

Male-mediated Developmental Toxicity

Edited by Diana Anderson and Martin H Brinkworth, University of Bradford,
Bradford, UK

Visit our website at www.rsc.org/issuesintoxicology

For further information please contact:

Sales and Customer Care, Royal Society of Chemistry, Thomas Graham House,
Science Park, Milton Road, Cambridge, CB4 0WF, UK
Telephone þ44 (0)1223 432360, Fax: þ44 (0)1223 426017, Email: [email protected]
Cytochromes P450
Role in the Metabolism and Toxicity of
Drugs and other Xenobiotics

Edited by

Costas Ioannides
Molecular Toxicology Group, School of Biomedical and Molecular Sciences,
University of Surrey, Guildford, UK
ISBN: 978-0-85404-274-6

A catalogue record for this book is available from the British Library

r Royal Society of Chemistry, 2008

All rights reserved

Apart from fair dealing for the purposes of research for non-commercial purposes or for
private study, criticism or review, as permitted under the Copyright, Designs and Patents
Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not
be reproduced, stored or transmitted, in any form or by any means, without the prior
permission in writing of The Royal Society of Chemistry or the copyright owner, or in the
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Licensing Agency in the UK, or in accordance with the terms of the licences issued by
the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning
reproduction outside the terms stated here should be sent to The Royal Society of
Chemistry at the address printed on this page.

Published by The Royal Society of Chemistry,

Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 0WF, UK

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For further information see our web site at www.rsc.org

Preface

From uniqueness to multiplicity

As an introduction to the fifteen chapters of this new survey on the latest
developments in the field of cytochrome P450, a short glimpse on the course
of events during the last fifty years may be appropriate.
During half a century, cytochrome P450 in its original uniqueness as an op-
tically ‘‘wrong’’ cytochrome has attracted many investigators, who have con-
tributed to the unveiling of a bewildering multiplicity of biologically important
functions of the by now very large superfamily of cytochrome P450 enzymes.
The various enzymes and the specificities of the multitude of the iron por-
phyrins are reflected in this book.
As a principal biological function of cytochrome P450 is the oxidative me-
tabolism of drugs and other xenobiotic substances, a backward look into the
‘‘Detoxication Mechanisms’’ (2nd edition in 1959) by R. T. Williams (often
adoringly nicknamed as the ‘Beilstein-Williams’) is worthwhile. Page 4 contains
the following statement: ‘The mechanism of these ‘oxidation’ reactions, however,
is unknown except in a few cases’. Thereafter, however, with its discovery in 1958
and with the advent of more refined spectroscopic methodologies, through the
double wavelength spectrophotometry, the mysterious enzyme system began to
reveal its secrets in a swift stream of investigative successes. They could super-
ficially and very incompletely be summarised in the following terms:

 Binding of substrates as photometrically characterised by different binding

spectra of either type I or type II.
 Binding of oxygen as well as of NADPH and the creation of the oxidative
reaction cycle.
 The induction of the enzyme protein by phenobarbital or 3-
methylcholanthrene (cytochrome P448).
 The description of oxidative metabolism pathways began after the dis-
covery of cytochrome P450, e.g. the O-demethylation of model substrates

Issues in Toxicology
Cytochromes P450: Role in the Metabolism and Toxicity of Drugs and other Xenobiotics
Edited by Costas Ioannides
r Royal Society of Chemistry, 2008

v
vi Preface
such as nitroanisole, which by optically measuring the formation of ‘yel-
low’ nitrophenol allowed for the characterisation of the enzyme kinetics
even in turbid microsomal suspensions.1 Ring hydroxylation and the
structural preconditions for specific points of attack on polycyclic aro-
matic hydrocarbons (bay region), N-oxidation etc. were most important
further subjects of investigational interest.
 Attempts at the elucidation of the reaction mechanism by the use of in-
hibitors e.g. carbon monoxide (the action spectrum), SKF 525-A and
metyrapone revealed more of the nature of cytochrome P450. Inhibition
kinetics suggested inhibitor binding at a location different from the active
centre itself. This has led to the concept of physical changes in the protein
configuration during the reaction cycle by invoking a‘hinge’ mechanism as
modelled in a ‘mouse trap’.2
 Understanding the function of the haem iron was achieved by determining
its spin states.
 Finally, after numerous, long and difficult attempts, the purification of
cytochrome P450 was achieved, and opened the way to the amino acid
sequence and further realisation of its diversity. Observation of species
differences pointed to genetic and evolutionary variations in drug metab-
olism. The role of cytochrome P450 in the oxidative steroid metabolism
became essential in explaining sex differences.

In order to gain the ultimate insight into a natural phenomenon, the two gen-
erally possible pathways for a scientific approach have been used (sometimes
certainly inadvertently) also by the investigators of cytochrome P450 functions,
namely a hypothesis-driven experimentation as well as the random explorative
method. Both processes are intimately intertwined, and have proven to be suc-
cessful. The shrewd and extensive utilisation of the molecular biology method-
ology very rapidly led to a vast body of enzymes calling for a classification of the
plethora of different cytochromes P450, the superfamily, into families and sub-
families.3 This is aptly exemplified by the ten chapters in Part B of this book,
dealing with ten subfamilies and two families of cytochrome P450.
A short internet search has revealed a very intense as well as an almost uniform
distribution of interest into the cytochrome families discussed here: the sub-
families of CYP1 and CYP2 have shown hits between 45 000 and 14 000 times,
with the exception of CYP1B and CYP2F, which showed only 2 000 and 800 hits,
respectively. The CYP3 and CYP4 families generated about 28 000 hits. The
interpretation of these figures will be left to the readers of this monograph.
Part A of this monograph introduces the reader to the current knowledge of
the evolutionary development of cytochrome P450 structure and function.
Furthermore, it deals with the role of this enzyme in the formation of reactive

1
K. J. Netter, Naunyn-Schmiedeberg’s Arch exp Pathol Pharmakol., 1960, 238, 292.
2
K. J. Netter, G.F. Kahl, M.P. Magnussen, Naunyn-Schmiedebergs Arch Pharmacol., 1969, 265, 205.
3
D. W. Nebert, M. Adesnik, M.J. Coon, R.W. Estabrook, F.J.Gonzalez, F.P. Guengerich, I.C.
Gunsalus, E.F. Johnson, B. Kemper, W. Levin, I.R. Phillips, R. Sato, and M.R. Waterman, DNA,
1987, 6, 1.
Preface vii

intermediates. Figuratively speaking, Part C offers an insight into another as-

pect of cytochrome P450 research, namely its regulation through receptor-
mediated stimuli – as opposed to enzyme induction or inhibition. Equally
important are the recently recognised variations in the pharmacokinetics of
drugs emanating from the ingestion of phytochemicals, which can act as in-
ducers and as inhibitors of the drug metabolising enzymes. In an attempt to
‘translate’ the current data on this one of several drug metabolising systems
into clinical application, the closing chapter highlights the role of cytochromes
P450 in the treatment of neoplastic growth.
This most recent member of the large ‘family’ of previous monographs de-
voted to cytochromes P450 and drug metabolism, indirectly could provide a
basis for a more rational approach in the discovery and development of new
drugs and therapeutic procedures. The knowledge on this major drug meta-
bolising system assembled in this book may possibly be of help in better
understanding why certain drugs in humans do not fulfill the expectations re-
sulting from successful preclinical experiments. Recently this has led to costly
disappointments in establishing new effective tumour therapies in humans,
because obviously human and experimental mammalian organisms in them-
selves are far more complex and complicated interacting systems than e.g.
cultured cells etc. A promising drug candidate acted on the ErbB epidermal
growth factor receptor on the outside cell wall, which then is believed to be
internalised in an inactive state and, therefore, to downregulate the replication
pathway; but obviously frequently occurring mutant receptors had a low in-
ternalisation rate, and hence the drug could not be effective. This experience has
refined drug design and development by introducing another biological variant
into the already multifaceted interplay between organisms and drugs, which
requires novel approaches to correctly predict drug actions from preclincical
testing regimes. Consequently, these have to be adapted to ever more diversified
biological conditions. Understanding and coping with the multitude of inter-
relations has been provisionally labeled as ‘Systems Biology’. Whether it will
survive as a fact-filled new era in drug discovery and regulation remains to be
seen. But ‘Systems Biology’ begins to become a new concept-or just a terminus
technicus-describing a more subtle testing or precalculating of desired effects by
taking into account as many biological features as possible.4
It is to be hoped that the combined knowledge and expertise of the authors of
this monograph might be able to contribute to an eventual success of Systems
Biology in developing new biologically – and therapeutically-active xenobiotics.

K. J. Netter
Department of Pharmacology & Toxicology
University of Marburg
Marburg
Germany

4
B. Borell, The Scientist, August 2007, 21, No. 8, 37.
Contents

Part A

Chapter 1 Cytochrome P450 Structure and Function: An Evolutionary

Perspective
David F.V. Lewis and Yuko Ito

1.1 Introduction 4
1.2 Evolutionary Aspects 7
1.3 Binding Functions of P450 11
1.4 Substrate Binding and Selectivity 15
1.5 P450 Catalysis 24
1.6 Structural Modelling of P450s 29
1.6.1 Molecular Modelling and Dynamics of P450s 30
1.6.2 Lipophilicity Relationships in P450 Substrate
Binding and Selectivity 35
1.7 Conclusions 37
Abbreviations 38
Acknowledgements 39
References 39

Chapter 2 Generation of Reactive Intermediates by Cytochromes P450

Hermann M. Bolt and Peter H. Roos

2.1 Introduction 47
2.2 Determinants of Biologically Reactive Metabolites 49
2.2.1 General Principles of Biological Reactivity 49
2.2.2 Application to Aliphatic Epoxides 50
2.2.3 Structure-Genotoxicity Relationship: Aliphatic
Olefins and Their Epoxides 52
Issues in Toxicology
Cytochromes P450: Role in the Metabolism and Toxicity of Drugs and other Xenobiotics
Edited by Costas Ioannides
r Royal Society of Chemistry, 2008

ix
x Contents
2.3 Suicide Substrates for CYP Enzymes 55
2.4 Formation of Reactive Intermediates:
Toxicological Determinants 57
2.4.1 Bioactivation, Organotropism and Species
Differences of N-Nitrosamines 57
2.4.2 Endogenous Compounds: Biological Activation
of Oestrogens 59
2.5 Application to Polycyclic Aromatic Hydrocarbons 63
2.5.1 Dihydrodiolepoxides (DDE) 66
2.5.2 Radical Cations 72
2.5.3 Quinone Pathway 74
2.5.4 ‘Polar Metabolites’ 75
2.5.5 Specific PAHs 78
2.5.6 Specific CYPs 82
2.5.7 Specific Sites: CYP Profiles, Subcellular
CYP-localisations 84
2.5.8 General Conclusions Regarding PAH-derived
Reactive Metabolites 85
2.6 Final Remarks 86
References 86

Part B

Chapter 3 The CYP1A Subfamily

Bhagavatula Moorthy

3.1 Introduction 98
3.2 Human CYP1A Enzymes and Their Role
in Drug Metabolism 99
3.3 Ontogenic Expression of CYP1A Enzymes 101
3.4 Role of CYP1A1 Enzymes in Human Carcinogenesis 102
3.4.1 Human Exposure to PAHs 102
3.4.2 Role of CYP1A Enzymes in PAH Metabolism 103
3.4.3 Genetic Polymorphisms of CYP1A and Human
Cancers 103
3.5 Molecular Mechanisms of CYP1A Induction 104
3.6 Persistent Induction of CYP1A1 106
3.6.1 AHR Contributes to Sustained Induction of
CYP1A1 by Persistent Chemicals 106
3.6.2 MC Elicits Sustained Induction of CYP1A1 by
Mechanisms Other than Persistence of the
Parent Compound 106
3.6.3 Persistent CYP1A1 Induction in Extrahepatic
Tissues 107
Contents xi

3.6.4 Sustained CYP1A1 Induction in Humanised

Transgenic Mouse 108
3.7 Relevance of CYP1A1 Induction to Tumourigenesis 108
3.8 CYP1A, Ki-ras Mutations, and Lung
Tumourigenesis 109
3.9 Mechanisms of CYP1A2 Induction 110
3.10 CYP1A in Laboratory Animals 110
3.10.1 Species Differences 110
3.10.2 Tissue-specific Expression 111
3.11 The cyp1a Knockout Mouse Models 112
3.12 Studies with Humanised Mice 114
3.13 Role of CYP1A Enzymes in the Metabolism
of Endogenous Substances 114
3.13.1 Role of CYP1A Enzymes in ROS Metabolism 114
3.13.2 Modulation of ROS by CYP1A Enzymes 115
3.13.3 CYP-dependent Arachidonic Acid (AA)
Metabolism 115
3.13.4 AA is an Endogenous CYP1A Substrate
in Humans 116
3.13.5 Other Endogenous CYP1A Substrates 116
3.13.6 Role of CYP1A Enzymes in Hyperoxic
Lung Injury 118
3.13.7 F2 Isoprostanes, Isofurans, and Hyperoxic
Lung Injury 119
3.14 Protective Effects of CYP1A Induction 120
3.14.1 Mechanisms for Beneficial Effects of CYP1A
Enzymes 121
3.15 Conclusions 122
Acknowledgements 123
References 123

Chapter 4 The CYP1B Subfamily

Morag C.E. McFadyen and Graeme I. Murray

4.1 Introduction 137

4.2 Mechanisms of Regulation of CYP1B1 137
4.2.1 Transcriptional Regulation of CYP1B1 138
4.3 Tissue Specific Expression of CYP1B1 140
4.3.1 CYP1B1 and Disease 140
4.4 Role of CYP1B1 in Drug Metabolism 141
4.4.1 CYP1B1 Metabolism of Specific Drugs 143
4.4.2 CYP1B1 Activated Prodrugs in Development 143
4.5 Inhibitors of CYP1B1 144
4.6 Drug Metabolism and Pharmacogenetics of CYP1B1 144
4.7 Conclusions 145
References 146
xii Contents
Chapter 5 The CYP2A Subfamily
Hannu Raunio, Jukka Hakkola and Olavi Pelkonen

5.1 Introduction 151

5.2 Species and Tissue Distribution 151
5.3 Substrate Specificity 153
5.4 Developmental and Tissue Selective Regulation and
Effect of Physiological and Pathological Factors 154
5.4.1 Ontogenic Expression 154
5.4.2 Regulation of Tissue Expression 154
5.4.3 Regulation by Physiological Factors 155
5.4.4 Impact of Disease on CYP2A Liver Expression 156
5.5 Regulation by Xenobiotics 156
5.6 Structure of the Human CYP2A6 Enzyme 158
5.6.1 Molecular Modelling 158
5.6.2 Mutagenesis Studies 159
5.7 Pharmacogenetics of Human CYP2A Genes 159
5.8 Role in Drug Metabolism 160
5.8.1 Drug Substrates 160
5.8.2 Inhibitors 161
5.9 Role in Xenobiotic Toxicity 162
5.9.1 Toxic Substrates 162
5.9.2 Nicotine 162
5.9.3 N-Nitrosamines 165
5.10 CYP2A6 Polymorphism and Cancer 167
5.10.1 Lung Cancer 167
5.10.2 Other Cancers 168
5.11 Implications for Risk Assessment – Coumarin 168
5.12 Conclusions 170
References 171

Chapter 6 The CYP2B Subfamily

Laurent Corcos and François Berthou

6.1 Introduction 179

6.2 The CYP2B Gene Family 180
6.2.1 Substrates and Inhibitors 181
6.3 Regulation of CYP2B Gene Expression 184
6.3.1 Species and Tissue Sites in Mammals 184
6.3.2 Sex-dependent Regulation of CYP2B
Expression 185
6.3.3 Hormonal Regulation 185
6.3.4 The Influence of Pathology and Inflammation 186
6.3.5 Model Systems to Investigate Enzyme Activity
and Gene Regulation 186
6.4 Genetic Polymorphism and Splice Variants 188
Contents xiii

6.5 Suicide-based Strategies in Cancer Therapy Protocols 190

6.6 Biochemistry of CYP2B Enzymes 192
6.6.1 Structural Features of CYP2B Family Members 192
6.7 Conclusions and Future Prospects 194
Acknowledgements 195
References 195

Chapter 7 The CYP2C Subfamily

Stephen S. Ferguson, Karen Black and Jonathan P. Jackson

7.1 Introduction to the CYP2C Subfamily,

Importance and Scope 201
7.2 CYP2C Chromosomal Comparisons: Human,
Mouse and Rat 202
7.3 Contribution of CYP2C to Drug
and Xenobiotic Metabolism 205
7.3.1 Metabolism Catalysed by CYP2C8 205
7.3.2 Metabolism Catalysed by CYP2C9 208
7.3.3 Metabolism Catalysed by CYP2C19 210
7.4 Pharmacogenetics of CYP2C 211
7.4.1 CYP2C8 Pharmacogenetics 212
7.4.2 CYP2C9 Pharmacogenetics 212
7.4.3 CYP2C19 Pharmacogenetics 218
7.4.4 Perspective on Pharmacogenetics of CYP2Cs 219
7.5 Drug-Drug Interactions Involving CYP2C 220
7.5.1 Drug-Drug Interactions - Inhibition 220
7.5.2 Drug-Drug Interactions - Induction and
Suppression 226
7.6 Conclusions and Summary 233
References 233

Chapter 8 The CYP2D Subfamily

Ulrich M. Zanger

8.1 Introduction and Molecular Genetics

of the CYP2D Subfamily 242
8.2 Catalytic Properties and Structure
of CYP2D Enzymes 243
8.2.1 Role in Drug and Xenobiotic Metabolism 244
8.2.2 Potential for Drug-Drug Interactions 247
8.2.3 Role in Endogenous Biotransformation 247
8.3 Species Differences in CYP2D Molecular Genetics,
Expression and Function 248
8.3.1 CYP2D in Nonhuman Primates 248
8.3.2 CYP2D in Rat 249
8.3.3 CYP2D in Mouse 250
xiv Contents
8.3.4 CYP2D in Other Species 251
8.4 Hepatic and Extrahepatic Expression
of CYP2D Enzymes 252
8.4.1 Ontogeny, Induction and Regulation 252
8.4.2 Extrahepatic Expression and its Significance 253
8.5 Pharmacogenetics of Human CYP2D6 255
8.5.1 Definition and Determination of CYP2D6
Phenotype 255
8.5.2 Genetic Polymorphism and Interethnic
Variation 258
8.5.3 CYP2D6 Genotyping 262
8.5.4 Clinical Relevance of CYP2D6
Pharmacogenetics 262
Acknowledgement 264
References 264

Chapter 9 The CYP2E Subfamily

Lowell C. Overton, Alice Hudder and Raymond F. Novak

9.1 Introduction 277

9.2 CYP2E1 Tissue and Species Distribution 278
9.3 Age and Gender Differences in Expression 279
9.4 Role in Xenobiotic Metabolism and Toxicity 279
9.5 Substrate Specificity 282
9.6 Regulation by Endogenous and Exogenous Factors 282
9.6.1 Cytokine Regulation of CYP2E1 Expression 282
9.6.2 Hormonal Regulation of CYP2E1 Expression 283
9.6.3 Xenobiotic Regulation of CYP2E1 Gene
Expression 291
9.6.4 Metabolic Conditions, Disease States and
Oxidative Stress Alter CYP2E1 Expression 295
9.7 Pharmacogenetics and SNPs 297
9.8 Ontogenic Expression 300
References 301

Chapter 10 The CYP2F, CYP2G and CYP2J Subfamilies

Qing-Yu Zhang and Xinxin Ding

10.1 CYP2F 310

10.1.1 Introduction 310
10.1.2 Human CYP2F1 310
10.1.3 Mouse CYP2F2 314
10.1.4 Goat CYP2F3 318
10.1.5 Rat CYP2F4 319
10.1.6 CYP2F in Other Species 320
10.1.7 Perspective 320
10.2 CYP2G1 321
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