Advances in Bioactivation Research (eBook)

Dr. Elfarra received his B.S. degree in Pharmacy at Cairo University, Cairo, Egypt, in 1975 and his Ph.D. degree in Medicinal Chemistry at the University of Minnesota, Minneapolis, MN, in 1983. In 1985, Dr. Elfarra joined Shell Development Company in Houston, Texas as an associate research toxicologist after he completed his postdoctoral training in Pharmacology at the University of Rochester in Rochester, NY. Dr. Elfarra joined the University of Wisconsin-Madison as assistant professor in 1986. He was promoted to associate and full professor in 1992 and 1996, respectively. He served as the chair of the campus Biosafety Committee and has trained a large number of undergraduate students, graduate students, and postdoctoral students in his laboratory. He is the current director of the institutional training grant from NIEHS. Dr. Elfarra has served on the executive committee for the Division of Toxicology of the American Society for Pharmacology and Experimental Therapeutics, and has organized and participated in many national and international symposia focused on topics related to his research. He has served as consultant for many organizations, such as NIH, the National Research Council Board on Environmental Studies and Toxicology, the Health Effects Institute, Health Canada, and the International Agency for Research on Cancer.

Advances in Bioactivation Research 2
Contents 6
Preface 8
Part 1: General Concepts and Basic Mechanisms 9
Metabolic Concerns in Drug Design 10
1.1 Introduction 10
1.2 Overview of Computational Modeling of ADMET 11
1.3 Predicting Inhibition/Affinities: Drug-Drug Interactions and Drug Design 13
1.3.1 Cytochrome P450 Inhibition 13
1.3.2 Predicting Metabolites: Efficient Redesign and Avoiding Bioactivation Pathways 16
1.3.3 Predicting Rates of P450-Mediated Reactions 26
1.4 Summary 27
References 28
Role of Bioactivation in Idiosyncratic Drug Toxicity: Structure-Toxicity Relationships 34
2.1 Adverse Drug Reactions 34
2.2 Link Between Drug Metabolism and Type B ADRs 35
2.3 Assays to Monitor Reactive Metabolites in Drug Discovery 38
2.3.1 Covalent Binding 38
2.3.2 Reactive Metabolite Characterization as Stable Sulfydryl, Amino, and/or Cyano Conjugates 39
2.3.3 Enzyme Inactivation Studies 40
2.3.4 Metabolite Identification 40
2.4 Strategies to Abrogate Reactive Metabolite Formation: Structure-Activity Relationship Studies 40
2.4.1 Removal of Structural Alerts 43
2.4.2 Blocking Sites of Bioactivation 45
2.4.3 Introduction of Alternate Metabolic Soft Spots 46
2.4.4 Modulation of Biochemical Reactivity via Steric Hindrance 50
2.4.5 Modulation of Biochemical Reactivity via Changes in the Electronic Properties 51
2.5 Factors That Mitigate IADR Risks Associated with Drug Candidates Containing Structural Alerts 52
2.6 Exploring Biochemical Mechanisms of Toxicity Other Than (or in Addition to) Bioactivation 55
2.7 Concluding Remarks 57
References 58
Michael Addition-Elimination Reactions: Roles in Toxicity and Potential New Therapeutic Applications 63
3.1 Introduction 63
3.2 Formation of Michael Acceptors During Metabolism of Halogenated Hydrocarbons 64
3.3 Stabilities and Chemical Reactivities of DCVCS and TCVCS 65
3.4 Nephrotoxicity of DCVCS and TCVCS 68
3.5 Addition-Elimination Reactions and the Bioactivation of Anticancer Thiopurine Prodrugs 69
3.6 cis-AVTP and trans-AVTG are More Cytotoxic in Tumor Cells Than 6-MP or 6-TG 71
3.7 cis-AVTP and trans-AVTG Exhibited Reduced In Vivo Toxicity Than 6-TG 72
3.8 Conclusions 73
References 73
Induction of Drug-Metabolizing Enzymes: Contrasting Roles in Detoxification and Bioactivation of Drugs and Xenobiotics 75
4.1 Introduction 75
4.2 Enzyme Induction 76
4.3 Overview of Enzyme Induction Pathways 77
4.3.1 Aryl Hydrocarbon Receptor (Aromatic Hydrocarbon/ CYP1 Inducers) 77
4.3.2 Pregnane X Receptor (CYP3A Inducers) 78
4.3.3 Constitutive Androstane Receptor (Phenobarbital-Like Inducers) 80
4.3.4 Other Nuclear Receptors: FXR (Bile Acids), LXR (Sterols), PPAR (Fibrates), VDR (Vitamin D) 81
4.3.5 Nrf2/Keap1 (ARE Inducers) 82
4.3.6 Ethanol-Type Induction (CYP2E1 Inducers) 84
4.4 Techniques in Enzyme Induction Research 85
4.4.1 Measuring Induction Potential of New Chemical Entities 85
4.4.1.1 Primary Cultures of Human Hepatocytes 85
4.4.1.2 Reporter Gene Assays 86
4.4.1.3 Immortalized Hepatocytes and Minimally Derived Hepatocyte Lines 87
4.4.1.4 Ligand-Binding or Coactivator Recruitment Assays 87
4.4.1.5 In Silico Models 88
4.4.2 Use of Gene Arrays to Find Novel Target Genes and Elucidate Wider Role of Nuclear Receptor Activation 88
4.4.3 Use of Transgenic and Knockout Mice in Induction Rresearch 89
4.5 Consequences of Enzyme Induction - Examples of Detoxification and/or Bioactivation 90
4.5.1 Consequences of AhR Activation 90
4.5.2 Enzyme Induction in Detoxification of Supraphysiological Concentrations of Endogenous Compounds 92
4.5.3 ARE Inducers and Nrf2 Pathway in Detoxification and Chemoprevention 93
4.5.4 Consequences of Enzyme Induction on the Bioactivation of Acetaminophen 94
4.5.5 Consequences of CYP3A Induction by Troglitazone 96
4.5.6 Enhancement of Cocaine Bioactivation by Phenobarbital-Like Inducers 98
4.6 Conclusions 99
References 99
Mechanism-Based Inactivation of Cytochrome P450 2A and 2B Enzymes 109
5.1 Introduction 109
5.2 Characterization of Mechanism-Based Inactivation 112
5.2.1 MBI as Tools 114
5.2.2 MBI and Drug Metabolism 115
5.3 Inactivation of P450 2B6 116
5.3.1 17alpha-Ethynylestradiol (17EE) 118
5.3.2 Efavirenz 119
5.3.3 N,N’,N’’-Triethylenethiophosphoramide (tTEPA) 120
5.3.4 Grapefruit Juice and Bergamottin 121
5.3.5 Phencyclidine 122
5.4 Inactivation of Cytochrome P450 2A Enzymes 123
5.4.1 8-Methoxypsoralen 125
5.4.2 Benzylisothiocyanate and Phenethylisothiocyanate 126
5.4.3 Nicotine and Nicotine Delta5’(1’)iminium ion 127
References 129
CYP2E1 - Biochemical and Toxicological Aspects and Role in Alcohol-Induced Liver Injury 138
6.1 Cytochrome P450 and Oxidative Stress 138
6.2 Alcohol, Oxidative Stress and Cell Injury 140
6.3 CYP2E1 and the Microsomal Ethanol-Oxidizing System 142
6.3.1 CYP2E1 Localization 143
6.3.2 CYP2E1 Substrates 143
6.3.3 CYP2E1 Polymorphisms 147
6.3.4 Induction of CYP2E1 148
6.3.5 Regulation of CYP2E1 149
6.3.6 CYP2E1 and the Proteasome Complex 152
6.4 The CYP2E1 Knockout Mouse 153
6.5 CYP2E1 and Alcohol-Induced Liver Injury 153
6.6 Biochemical and Toxicological Properties of CYP2E1 in HepG2 Cells 156
6.7 Future Perspectives 160
References 162
One- and Two-Electron-Mediated Reduction of Quinones: Enzymology and Toxicological Implications 174
7.1 Enzymology of Quinone Reduction 174
7.1.7 Cytochrome P450 Reductase 174
7.1.7 Cytochrome b5 Reductase 175
7.1.7 Xanthine Oxidoreductase (Xanthine Dehydrogenase, Xanthine Oxidase) 176
7.1.7 NAD(P)H:quinone Oxidoreductase 1 177
7.1.7 NRH:quinone Oxidoreductase 2 178
7.1.7 Mitochondrial Reductases 179
7.1.7 Carbonyl Reductases 180
7.1.7 Other Enzymes 180
7.2 Toxicological Implications of Quinone Reduction 181
7.2.1 One-Electron Reduction of Quinones and Redox Cycling 181
7.2.2 Two-Electron Reduction of Quinones 181
7.2.2.1 Detoxification of Benzene-Derived Quinones 182
7.2.2.2 Cellular Protection by Generation of Antioxidant Quinones via Two-Electron Reduction 182
7.2.2.3 Naphthoquinones: Toxification or Detoxification Depending on the Properties of the Hydroquinone 183
7.3 Bioreductive Activation of Antitumor Quinones 184
7.3.1 Mitomycins 185
7.3.2 Diazirdinyl 1,4-benzoquinones 186
7.3.3 Indolequinones 186
7.3.4 Streptonigrin 187
7.3.5 ß-Lapachone 187
7.3.6 Benzoquinone Hsp90 Inhibitors 188
7.3.7 The Case of Hypoxia-Activated Quinone Prodrugs: Therapeutic Exploitation of One-Electron Reduction of Quinines 190
7.4 Summary 190
References 191
Lipoxidation-Derived Electrophiles as Biological Reactive Intermediates 203
8.1 Introduction Generation of Lipoxidation-Derived Electrophiles 203
8.2 Chemistry of Protein and Polynucleotide Covalent Modification 206
8.2.1 Malondialdehyde 206
8.2.2 Glyoxal 208
8.2.3 2-Hydroxyalkanals 210
8.2.4 Levuglandins 211
8.2.5 Acrolein, 2-Alkenals, and 2,4-Dienals 212
8.2.6 HNE, ONE, HODA, KODA 216
8.2.7 EKODE 220
8.2.8 4,5-Epoxy-2-alkenals 221
8.2.9 Multicomponent LPO-Derived Protein Adducts 222
8.3 Functional Biological Consequences of Nonenzymatic LPO 222
8.4 Conclusions 224
References 224
Bioactivation and Protein Modification Reactions of Unsaturated Aldehydes 237
9.1 Processes that Produce Aldehydes 237
9.2 Metabolism of Aldehydes 238
9.3 The Chemistry of Aldehyde-Protein Adducts 240
9.3.1 Adducts Formed by Acrolein 241
9.3.2 Adducts Formed by HNE 243
9.3.3 Adducts Formed by 4-oxo-trans-2-nonenal 244
9.4 Reactivity of Nucleophilic Residues in Protein 245
9.5 Effect of Neighboring Nucleophilic Amino Acid Residues 247
9.6 Reversibility of Aldehyde-Protein Adduction Reactions 247
9.7 Detection and Characterization of Aldehyde-Protein Adducts 248
9.7.1 Top-Down Approaches to Identify Protein Modifications Using MS 249
9.7.2 Bottom-Up Approaches to Identify Protein Modification Using MS 249
9.7.3 Analysis of Unstable Aldehyde-Protein Adducts 250
9.7.4 Techniques for Aldehyde-Protein Adduction Detection by MS 250
9.7.5 Quantification of Protein and Adduct by MS 251
9.8 Conclusions and Summary 253
References 254
Part 2: Tissue-Specific Features and Risk Assessment Applications 258
Adaptive Responses and Signal Transduction Pathways in Chemically Induced Mitochondrial Dysfunction and Cell Death 259
10.1 Introduction 259
10.2 Role of Redox Status in Determining Mitochondrial Function and Adaptation 263
10.3 Mitochondrial MPT and Cell Death 267
10.4 Mitochondrial Sensors and Mediators of Adaptation 270
10.4.1 Bcl-2 Family Proteins 270
10.4.2 Heat Shock Proteins 271
10.4.3 Protein Kinases: PKB (Akt), PKC-alpha, PKC-epsi, PKD 273
10.4.4 MAPK Pathway 274
10.4.5 Nuclear Factor-Kappa B Pathway 275
10.4.6 Epidermal Growth Factor 276
10.4.7 Peroxisome Proliferator-Activated Receptor gamma Cofactor-1alpha and Mitochondrial Biogenesis 276
10.5 Physiological, Pathological and Toxicological States Affecting Mitochondrial Function 277
10.5.1 Aging 278
10.5.2 Diabetic Nephropathy and Chronic Kidney Disease 278
10.5.3 Compensatory Renal Cellular Hypertrophy 280
10.5.4 Cysteine Conjugate-Induced Nephrotoxicity 281
10.6 Summary and Conclusions 283
References 283
Hepatic Bioactivation and Drug-Induced Liver Injury 292
11.1 Introduction 292
11.2 Mechanisms of Drug-Induced Liver Injury 296
11.3 Enzymology of Hepatic Bioactivation 296
11.3.1 Phase I Reactions 297
11.3.2 Phase II Reactions 298
11.4 Structural Alerts for Hepatic Bioactivation 299
11.4.1 Epoxidation of Alkenes and Aromatic Rings 300
11.4.2 Michael Acceptors 302
11.4.3 Acetyl Halides 304
11.4.4 Hydroxylamines and Nitroso Compounds 304
11.4.5 Carbocations 306
11.4.6 Free Radicals 306
11.4.7 Acyl Glucuronides and Acyl-CoA thioesters 307
11.5 Molecular Targets of Reactive Metabolites in Liver 309
11.5.1 Acetaminophen 309
11.5.2 Diclofenac 311
11.6 Methods for Assessment of Reactive Metabolites 314
11.7 Conclusions 316
References 316
Role of Cysteine S-Conjugate beta-Lyases in the Bioactivation of Renal Toxicants 325
12.1 Introduction 325
12.2 The Mercapturate Pathway 326
12.3 Nephrotoxic Haloalkene Glutathione- and Cysteine S-Conjugates 328
12.4 The Cysteine S-Conjugate beta-Lyase Reaction 329
12.5 Identification of Cysteine S-Conjugate beta-Lyases 330
12.6 Nephrotoxicity of Haloalkene Cysteine S-Conjugates 331
12.7 beta-Lyase-Catalyzed Generation of Reactive Fragments from Nephrotoxic Halogenated Cysteine S-Conjugates 333
12.8 Mechanisms Contributing to the Nephrotoxicity of Haloalkene Cysteine S-Conjugates - Toxicant Channeling 337
12.9 Metabolism of Electrophiles Other than Haloalkenes via the Mercapturate/Cysteine S-Conjugate beta-Lyase Pathways 340
12.10 Conclusion 342
References 343
Bioactivation of Xenobiotics in Lung: Role of CYPs and FMOs 349
13.1 Introduction 349
13.2 Tools for the Study of Xenobiotic Bioactivation and Lung Toxicity in Humans 349
13.2.1 Animal Models 349
13.2.2 Human Lung Cell Lines 350
13.3 Xenobiotic-Metabolizing Enzymes in the Lung 350
13.3.1 Cytochrome P450s 351
13.3.1.1 The CYPs that are Selectively Expressed in the Human Lung are CYPs 2A13, 2F1, 2S1, and 4B1 352
13.3.2 Flavin-Containing Monooxygenases 353
13.3.3 Other Phase I Xenobiotic-Metabolizing Enzymes 354
13.3.4 Phase II Xenobiotic-Metabolizing Enzymes in Human Lung 354
13.3.5 Xenobiotic Transporters (Phase III Enzymes) 355
13.4 Genetic Polymorphisms in Drug-Metabolizing Enzymes and Susceptibility to Disease 356
13.5 Examples of Xenobiotic Bioactivation in Lung by POR (Paraquat), CYP (3-Methyindole), and FMO (alpha-Naphthylthiourea) 357
13.6 Future Directions 360
References 360
Generation of Reactive Metabolites and Associated DNA Adducts from Benzene, Butadiene, and PAH in Bone Marrow. Their Effects on Hematopoiesis and Impact on Human Health 376
14.1 Introduction 376
14.2 BM and Hematopoiesis 380
14.3 Chemical Disruption of Hematopoiesis 381
14.4 In Vitro and Ex Vivo Model System for BM Toxicity 382
14.5 Benzene Disruption of Hematopoiesis and Initiation of Cancer 383
14.6 Benzene Metabolism and Formation of Reactive Metabolites in BM 384
14.7 1,3-Butadiene and BM Toxicity 387
14.8 PAH Activation and BM Toxicity 388
14.9 Reactive PAH Metabolites and Formation of DNA Adducts 390
14.10 Enzymes Involved in Activation and Detoxification 391
14.11 Liver Metabolism of PAHs versus BM Metabolism 395
14.12 Disruption of Hematopoietic Maturation by PAHs 395
14.13 Cooperation of PAH Metabolites and TNFalpha in BM Toxicity 398
14.14 Conclusions 399
References 400
Butadiene-Mediated Mutagenesis and Carcinogenesis 410
15.1 Exposure to Butadiene 410
15.2 Human Epidemiology and Carcinogenesis 411
15.3 Rodent Carcinogenesis 411
15.4 Metabolic Activation 412
15.5 DNA Adducts 412
15.5.1 Reactions of EB 412
15.5.2 Reactions of EBD and DEB 414
15.5.3 Crosslink Formation by DEB 415
15.5.4 Reactions of HMVK 416
15.6 Mutagenic Effects of BD 416
15.6.1 Introduction 416
15.6.2 Evidence that BD and its Epoxide Metabolites are Mutagens 416
15.6.3 Mutagenic Effects of BD in Human Studies 418
15.6.4 Modulation of Mutagenicity 419
15.6.4.1 Disruption of mEH 419
15.6.4.2 Disruption of DNA Repair 419
15.7 Structure-Function Analyses of Mutagenicity 420
15.7.1 Overall Experimental Strategy 420
15.7.2 Chemistry by which DNAs have been Prepared Containing Structurally Defined, Site-Specific BD Lesions 422
15.7.2.1 N6-dAdo, 1-Hydroxy-3-buten-2-yl Adducts 422
15.7.2.2 N6-dAdo, 2,3,4-Trihydroxybutyl-1-yl Adducts 422
15.7.2.3 N6-dAdo-N6-dAdo, 2,3-Dihydroxybutane-1,4-diyl Intrastrand Crosslinks of DEB 423
15.7.2.4 N1-dIno, 1-Hydroxy-3-buten-2-yl Adducts 424
15.7.2.5 N2-dGuo, 1-Hydroxy-3-buten-2-yl, and N2-dGuo, 2,3, 4-Trihydroxybut-1-yl Adducts 424
15.7.2.6 N2-dGuo-N2-dGuo, (2R,2R)- and (2S,3S)-2, 3-Dihydroxybutane-1,4-diyl Intrastrand Crosslinks 425
15.7.2.7 N3-dUri, 1-hydroxy-3-buten-2-yl adducts 425
15.7.3 Mutagenesis Conferred by Replication of DNAs Containing Specific BD Adducts 426
15.7.3.1 Vector Design and Mutagenesis Assay 426
15.7.3.2 Mutagenicity and Structural Analyses of N6-dAdo BD Adducts 426
15.7.3.3 Mutagenecity and Structural Analyses of N6-dAdo-N6-dAdo, 2,3-Dihydroxybutane-1,4-diyl Intrastrand Crosslinks 427
15.7.3.4 Mutagenicity and Structural Analyses of N1-dIno, 1-Hydroxy- 3-buten-2-yl Adducts 429
15.7.3.5 Mutagenicities of DNAs Containing N2 Guanine Adducts of R and S EB and R,R and S,S DEB 430
15.7.3.6 Mutagenicity of N2-dGuo-N2-dGuo, 2,3-Dihydroxybutane-1, 4-diyl Intrastrand Crosslinks 430
15.7.3.7 Mutagenicity of N3-dUri, 1-Hydroxy-3-buten-2-yl Adducts 431
References 432
Pharmacogenetics of Drug Bioactivation Pathways 441
16.1 Introduction 441
16.2 Cytochrome P450s 442
16.2.1 Bioactivation by the CYP1 Family 442
16.2.1.1 CYP1A1 442
16.2.1.2 CYP1A2 443
16.2.1.3 CYP1B1 444
16.2.2 Bioactivation by the CYP2 Family 445
16.2.2.1 CYP2E1 445
16.2.2.2 Other CYP2 Bioactivation Pathways 446
16.2.3 Bioactivation by the CYP3 Family 446
16.3 Other Oxidative Pathways 447
16.3.1 Flavin-Containing Monooxygenases 447
16.3.2 Peroxidases 448
16.4 Conjugation Reactions 448
16.4.1 N-Acetyltransferases 449
16.4.2 Sulfotransferases 449
16.5 Hydrolysis 450
16.5.1 Epoxide Hydrolases 450
16.6 Summary 450
References 451
Human Phenanthrene Metabolites as Probes for the Metabolic Activation and Detoxification of Carcinogenic Polycyclic Aromatic Hydrocarbons 461
17.1 Introduction 461
17.2 Metabolic Activation and Detoxification of PAH 462
17.3 Carcinogen Metabolite Phenotyping to Assess Individual Differences in PAH Metabolism 466
17.4 Development of Methods for Analysis of Phe Metabolites in Human Urine 467
17.4.1 Longitudinal Study of Urinary Phe Metabolite Ratios 469
17.4.2 Relationship of PheT: HOPhe Ratios to Genotyping Data 471
17.5 Future Directions in Phe Metabolite Phenotyping 476
References 476
Index 483