Studies on Experimental Models (eBook)

Preface 6
Contents 8
Contributors 12
Part I: 
22 
Role of Oxidative Stress and Targeted Antioxidant Therapies in Experimental Models of Diabetic Complications 23
1 Introduction 24
2 Diabetes-Associated Atherosclerosis 25
2.1 Experimental Models of Diabetes-Associated Atherosclerosis with an Emphasis on Oxidative Stress 27
2.1.1 The GPx1 Knockout Mouse 27
Glutathione Peroxidase-1 and Its Role in the Antioxidant Pathway 27
GPx1 / Mice Fed High Fat Diets as a Model to Study 
29 
ApoE/GPx1 Double-Knockout Mouse Model 29
Diabetic ApoE/GPx1 dKO Mice as a Model of Accelerated Diabetes-Associated Atherosclerosis 30
2.1.2 The NOX Knockout Mouse 31
2.1.3 The RAGE Knockout Mouse Model 33
3 Diabetic Nephropathy 34
3.1 Diabetic ApoE/GPx1 / dKO Mouse as a Model of DN 36
3.2 Experimental Models of NADPH Oxidase-Mediated Oxidative Stress in DN 36
4 Diabetic Cardiomyopathy 37
5 Targeted Antioxidant Therapies 40
5.1 The GPx1-Mimetic Ebselen 42
5.2 Ebselen in an Experimental Model of Diabetes-Associated Atherosclerosis 42
5.3 A Mechanistic Understanding of the Anti-atherogenic Action of Ebselen 43
5.4 Mitochondrially Targeted Antioxidants 44
5.5 NOX Inhibitors 45
6 Conclusions 47
References 48
Experimental Models of Oxidative Stress Related to Cardiovascular Diseases and Diabetes 59
1 Introduction 60
2 Animal Models of Cardiovascular Diseases 60
2.1 Rabbit Dietary-Induced Atherosclerosis 62
2.2 Murine Models of Atherosclerosis 64
2.3 Genetically Modified Animals 65
2.4 Other Experimental Models of Cardiovascular Diseases 66
3 Experimental Models of Diabetes 67
3.1 Streptozotocin and Alloxan Murine Diabetes Models 68
3.2 Neonatal Streptozotocin-Induced Diabetes 69
3.3 Streptozotocin-Spontaneously Hypertensive Rat 69
3.4 Fat-Fed/Streptozotocin-Induced Diabetic Rodents 69
3.5 Zucker Diabetic Fatty Rats 70
3.6 Genetically Engineered Diabetic Mice 71
3.6.1 Otsuka Long-Evans Tokushima Fatty Rats 72
3.6.2 Goto-Kakizaki Rats 72
3.6.3 db/db (C57BL/KsJ-db/db) Mice 73
3.6.4 ob/ob (C57BL/6J-ob/ob) Mice 74
References 75
Part II: 
81 
Arachidonic Acid Metabolism and Lipid Peroxidation in Stroke: Alpha-Tocotrienol as a Unique Therapeutic Agent 82
1 Introduction 82
2 Ischemic Stroke (87% of all stroke cases) 83
2.1 Patterns of Oxidative Stress in Ischemic Stroke 84
2.2 Experimental Models of Ischemic Stroke 85
3 Hemorrhagic Stroke (13% of all stroke cases) 88
3.1 Patterns of Oxidative Stress in Hemorrhagic Stroke 88
3.2 Experimental Models of Hemorrhagic Stroke 89
4 Brain Oxygen, Lipid Metabolism, and Oxidative Stress 90
4.1 Arachidonic Acid Metabolism in Brain 90
4.2 Phospholipase A2 91
4.3 Nonenzymatic Oxidative Lipid Metabolism 92
4.4 Enzymatic Oxidative Lipid Metabolism 93
4.4.1 Cyclooxygenase 94
4.4.2 Lipoxygenases 95
4.4.3 Cytochrome P450 96
5 Management of Oxidative Stress in Stroke 97
5.1 Therapeutic Window of Opportunity for Hyperbaric Oxygen Therapy in Acute Ischemic Stroke 98
5.2 Neuroprotective Properties of Natural Vitamin E, Alpha-Tocotrienol 98
6 Conclusion 99
References 100
Assessment of Oxidative Stress in the Brainof Spontaneously Hypertensive Rat and Stroke-Prone Spontaneously Hypertensive Rat 
 
110 
1 Introduction 111
2 L-Band ESR/Nitroxyl Spin Probe Methods 112
3 SHR and SHRSP 113
4 Measurement of Oxidative Stress in SHRand SHRSP Brain Using ESR 113
5 Clinical Significance of the Measurement of OxidativeStress in an Animal Model of Stroke Using ESR 114
6 Conclusion 117
References 120
Small Heat Shock Proteinsand Doxorubicin-Induced Oxidative 
123 
1 Introduction 124
2 Differential Mechanisms of Action of Doxorubicin:The Oxidative Stress in the Heart 124
3 Enhanced Heat Shock Proteins Expressionin Dox-Treated Hearts 127
4 Transcription of Hsps in Dox-Treated Hearts:The Role of Heat Shock Factors 128
5 Mechanism of Heat Shock Protein CardioprotectionAgainst Dox Toxicity 130
6 Doxorubicin-Activated MAP Kinase and Small Hsps Phosphorylation 137
7 Effect of Dox-Induced Small Heat Shock Proteinson Antioxidant Enzymes 138
8 Future Perspective 139
References 141
Oxidative Stress in Cardiovascular Disease: Potential Biomarkers and Their Measurements 149
1 Introduction 150
2 Production of Free Radicals 150
3 Oxidative Stress in Health and Disease 152
3.1 ROS in Vascular Disease 154
3.2 Atherosclerosis 154
3.3 Ischemia–Reperfusion Injury 156
3.4 Diabetes Mellitus 157
4 Potential Biomarkers of Oxidative Stress 158
5 Measurement of Oxidative Stress 159
5.1 Detection of Malonaldehyde for Oxidative Stress Measurement 159
5.2 Measurement of Oxidative Stress by Measuring Exhaled Volatile Alkanes 161
5.3 Measurement of Oxidative Stress by Measuring Isoprostanes 161
5.4 Detection of Oxidative DNA Damage by Measuring 8-Hydroxyguanosine Formation 162
5.5 Measurement of ROS Generation by Dihydroethidium 163
5.6 Measuring of Oxidative Stress by Using Dichlorofluorescein (DCFH) Diacetate 163
5.7 Measuring Cytochrome c Reduction 164
5.8 Measurement of Oxidative Stress by Reduced Glutathione Method 165
5.9 Measurement of Oxidative Stress by Using Electron Spin Resonance 166
5.10 Measurement of Oxidative Stress by Measuring Hydroxyl Free Radical Formation 167
References 167
In vivo Imaging of Antioxidant Effects on NF-kB Activity in Reporter Mice 175
1 Introduction 175
1.1 Inflammation in Cancer and Cardiovascular Diseases 176
2 Nuclear Factor Kappa B 177
2.1 The NF-kB Family of Transcription Factors 177
2.2 NF-kB in Disease 179
3 Effects of Antioxidants on NF-kB Activation 181
3.1 Extracts of Dietary Plants Are Efficient Modulatorsof Nuclear Factor Kappa B In Vitro [67] 181
3.2 The Transgenic NF-kB Reporter Mice 182
3.3 Extract of Oregano, Coffee, Thyme, Clove and Walnuts Inhibits Nuclear Factor Kappa B in Transgenic Reporter Mice [79] 
183 
3.4 Apple, Cherry and Blackcurrant Increases Nuclear Factor Kappa B Activation in Liver of Transgenic Mice [80] 183
3.5 Degree of Roasting Is the Main Determinant of the Effects of Coffee on NF-kB and Nrf2/EpRE [81] 185
4 Discussion of Results 186
4.1 The Luciferase Reporter 186
4.2 Dietary Plants as Modulators of NF-kB Activity 187
4.3 Organ-Specific Effects 189
4.4 Redox Regulation of NF-kB Activation 190
4.5 Preconditioning, Hormesis and Antioxidants 192
5 Conclusion 195
References 195
Part III: 
203 
MPTP and Oxidative Stress: It’s Complicated! 204
1 Introduction 204
2 PD and Oxidative Stress 205
3 Other Toxin PD Models 206
4 Why MPTP? 208
5 MAO-B and MPTP 208
6 DA Neurons, DA Terminals and MPTP 210
7 Cytokines and MPTP 211
8 Superoxide and MPTP 214
9 Nitric Oxide and MPTP 216
10 Peroxynitrite and MPTP 217
11 Mitochondria, MPTP and Oxidative Stress 218
12 Conclusion 219
References 219
Oxidative Stress in Alzheimer’s Disease:A Critical Appraisal of the Causes 
227 
1 Introduction 227
2 AD Neuropathology 228
3 Oxidative Stress in AD 229
4 Sources of Oxidative Stress Defined 231
4.1 Mitochondrial Dysfunction in AD 231
4.2 The Role of Metals in Oxidative Stress Production and AD 231
5 Conclusion 232
References 232
Retinal Disturbances in Patients and Animal Models with Huntington’s, Parkinson’s and Alzheimer’s Disease 237
1 Introduction 238
1.1 Retinal Organization 238
1.2 Transmission of Visual Information 240
2 Huntington’s, Parkinson’s and Alzheimer’s Diseases 242
2.1 Etiology of Huntington’s, Parkinson’s and Alzheimer’s Diseases 242
2.2 Pathologic Mechanisms 244
3 Retinal Disturbances in Patients with Huntington’s, Parkinson’s and Alzheimer’s Diseases 245
3.1 Visual Disturbances and Huntington’s Disease 245
3.2 Visual Disturbances and Parkinson’s Disease 245
3.3 Visual Disturbances and Alzheimer’s Disease 247
3.3.1 Role of Melatonin in the Retina of Patients with Alzheimer’s Disease 248
3.3.2 Retinal Pathologies and Alzheimer’s Disease 248
4 Retinal Impairments in Animal Models of Huntington’s, Parkinson’s and Alzheimer’s Diseases 249
4.1 Retinal Alterations in Animal Models with Huntington’s Disease 249
4.2 Retinal Alterations in Animal Models with Parkinson’s Disease 252
4.2.1 6-Hydroxydopamine Model 252
4.2.2 1-Methyl-4-phenyl-1, 2, 3, 6-Tetrahydropyridine Model 253
4.2.3 Rotenone Model 255
4.3 Retinal Alterations in Animal Models with Alzheimer’s Disease 256
5 Conclusion 258
References 259
Stress Gene Deregulation in Alzheimer Peripheral Blood Mononuclear Cells 267
1 Alzheimer’s Disease and Peripheral Blood Mononuclear Cells 267
2 Recruitment of Participants and Experimental Designs 269
3 Functional Annotations Associated with Oxidative Stress 271
4 Working Model for Alzheimer’s Disease 275
References 277
The Use of Gpx4 Knockout Mice and Transgenic Mice to Study the Roles of Lipid Peroxidation in Diseases and Aging 280
1 Introduction 281
2 Glutathione Peroxidase 4 281
3 Isoforms of Gpx4 282
4 Gpx4 Knockout Mice 283
5 Gpx4 Transgenic Mice 284
6 The Use of Gpx4 Knockout and Transgenic Mice in Studying the In Vivo Role of Lipid Peroxidation in Aging and Disease 285
6.1 The Use of Gpx4 Knockout and Transgenic Mice to Study Lipid Peroxidation in Aging 285
6.2 The Use of Gpx4 Knockout Mice and Transgenic Mice to Study Lipid Peroxidation in Alzheimer’s Disease 286
6.3 The Use of Gpx4 Knockout and Transgenic Mice to Study the Role of Lipid Peroxidation in Cardiovascular Disease 287
7 Conclusion 288
References 289
An Experimental Model of Myocardial and Cerebral Global Ischemia and Reperfusion 294
1 Introduction 294
2 Methodology of Studies of Experimental Cardiac Arrest and Resuscitation Studies [3] 295
2.1 Analytical Methods 296
3 Review of Results 297
4 Conclusion 311
References 311
Part IV: 
315 
Neovascular Models of the Rabbit Eye Induced By Hydroperoxide 316
1 Introduction 317
2 Materials and Methods 318
2.1 Lipid Hydroperoxide Synthesis 318
2.2 Corneal Model 318
2.3 Retinal Model 318
2.4 Choroidal Model 319
2.5 Biomarkers 319
2.6 Histology 319
2.7 Electrophysiology 319
2.8 Tissue Culture 319
3 Clinical Results 320
3.1 Corneal Model 320
3.2 Retinal Model 321
3.3 Choroidal Model 323
3.4 Tissue Culture Models 324
4 Biochemical and Molecular Biology Results 324
5 Histological Results 326
6 Tissue Culture Results 326
7 Conclusion 327
References 328
Purinergic Signaling Involved in the Volume Regulation of Glial Cells in the Rat Retina: Alteration in Experimental Diabetes 331
1 Introduction 332
1.1 Retinal Edema in Diabetics 332
1.2 Retinal Fluid Clearance 333
2 Osmotic Swelling of Retinal Glial Cells 335
2.1 Diabetes Alters the Osmotic Swelling Properties of Retinal Glial Cells 335
2.2 Factors Involved in Evoking Glial Swelling 338
2.2.1 Decrease in Potassium Conductance 338
2.2.2 Oxidative Stress and Inflammatory Lipids 339
3 Purinergic Inhibition of Glial Swelling 340
3.1 Purinergic Receptor Cascade 340
3.2 Involvement of Ecto-Nucleotidases in Swelling Inhibition 342
3.3 Diabetes Alters Glial Expression of Ecto-Nucleotidases 343
4 Functional and Clinical Implications 344
4.1 Functional Implications 344
4.2 Clinical Implications 346
References 347
Part V: 
353 
Helicobacter pylori-Induced Oxidative Stress and Inflammation 354
1 Oxygen Radicals, NADPH Oxidase and Cag A 355
2 Nuclear Factor-k.B 358
3 Activator Protein-1 358
4 Mitogen-Activated Protein Kinases 359
5 Chemokines 360
6 Proinflammatory Enzyme-Inducible Nitric Oxide Synthase and Cyclooxygenase-2 361
7 NF-kB, iNOS, and Apoptosis 363
8 Protease-Activated Receptors 363
9 Integrins 365
10 Genetic Variability of H. pylori Strain 366
11 Experimental Models of H. pylori Infection 367
11.1 Mongolian Gerbil Model 368
11.2 Mouse Model 368
12 H. pylori as a Putative Promoter of Gastric Carcinogenesis 370
13 Concluding Remarks 372
References 372
Experimental Models for Ionizing 
382 
1 Introduction 384
2 Radiation Chemistry Basics 384
3 Radiation-Induced Damage: Molecular Target 385
4 Radiation-Induced Damage: Cell Killing 386
4.1 Clonogenic Assay 386
4.2 Apoptosis 388
4.3 Necrosis and Autophagy 388
4.4 Colorimetric Assays for Cytotoxicity Assessment 389
5 Radiation-Induced Damage: DNA Damage and Repair 390
5.1 DNA Repair 390
5.2 Nonhomologous End-Joining 391
5.3 Homologous Recombination 392
5.4 Measurement of DNA Damage Repair 392
6 Radiation-Induced Damage: GenomicInstability/Bystander Effects 394
6.1 Measurements of Nontargeted Effects 395
7 Radiation Responses Using In Vivo Models 399
8 Modifiers of the Radiation Response 401
8.1 Radiosensitizers 401
8.2 IR Protectors 403
References 403
Experimental Models to Study Cigarette Smoke-Induced Oxidative Stress In Vitroand In Vivo in Preclinical Models, and in Smokers and Patients with Airways Disease 
409 
1 Introduction 410
2 In Vitro Cigarette Smoke Treatment Model 410
2.1 Preparation of Aqueous CSE 411
2.2 Preparation of CSC 411
2.3 Biophysical Comparison and Standardization of Different Research Grade Cigarettes Based on Their Total Particular Matter Concentration 
413 
2.4 Air–Liquid Interface System 413
3 In Vivo Models to Investigate Cigarette Smoke-Induced Oxidative Stress 414
3.1 Cigarette Smoke Exposure to Mice 415
3.2 Cigarette Smoke Exposure in Rats 416
3.3 Characteristics of Cigarette Smoke-Induced Lung Inflammationand Emphysema 417
4 Collection of Samples from Smokers and COPD Patients to Determine Oxidative Stress Markers 418
4.1 Exhaled Breath Condensate 418
4.2 Induced Sputum 419
4.3 Bronchoalveolar Lavage 420
4.4 Collection of Human Lung Tissue 420
5 Measurement of Oxidative Stress Biomarkers 421
5.1 Measurement of Superoxide Anion Releasein Bronchoaveolar Lavage Cells 421
5.2 Measurement of ROS by Flow Cytometry and Fluorescent Microscopy in Cells and Bronchoaveolar Lavage Cells 422
5.3 Myeloperoxidase Assay 422
5.4 Determination of Intracellular 4-Hydroxy-2-nonenal Levels and Its Adducts 423
5.5 Immunohistochemistry for 8-Hydroxy-2'-deoxyguanosine 
423 
5.6 Carbonylated Nuclear Erythroid-Related Factor 2: Protein Carbonyl Assay 424
5.7 Measurement of Intracellular Glutathione Levels 425
5.8 Electron Paramagnetic Resonance Measurement 425
6 Conclusions 426
References 426
Exhaled Breath Condensate Biomarkers of Airway Inflammation and Oxidative Stress in COPD 431
1 Introduction 431
2 Methodology of EBC Analysis 432
2.1 Experimental Setup 432
2.2 Analytical Techniques 434
2.3 Methodological Aspects of EBC Analysis 436
2.3.1 Origin(s) of Biomolecules in EBC 436
2.3.2 Flow-Dependence of Biomolecules in EBC and Effect of Respiratory Patterns 437
2.3.3 Time-Dependence of Biomolecules in EBC 438
2.3.4 Organ Specificity of EBC 438
2.3.5 Nasal and Saliva Contamination 439
2.3.6 Dilution Reference Indicators 439
2.3.7 Within-Day and Between-Day Variability of Biomolecules in EBC 439
3 Analysis of EBC in Patients with COPD and Healthy Smokers 440
3.1 Eicosanoids 440
3.1.1 Leukotrienes 440
3.1.2 Prostanoids 441
3.1.3 Isoprostanes 441
3.2 Hydrogen Peroxide 442
3.3 NO-Derived Products 443
3.4 pH 443
3.5 Aldehydes 444
3.6 Adenosine Triphosphate 444
3.7 Other Markers 444
4 Advantages and Limitations 445
5 Future Research 446
References 446
Induction of Oxidative Stress by Iron/Ascorbate in Isolated Mitochondria and by UV Irradiation in Human Skin 451
1 Introduction 452
1.1 Initiation of Oxidative Stress in Isolated Mitochondria 452
1.2 Initiation of Oxidative Stress by UV Irradiation 453
2 Materials and Methods 453
2.1 Isolation of Functionally Intact Mitochondria from Rat Liver, Brain and Heart 453
2.2 Protein Content 454
2.3 Incubation Conditions in the Presence of Iron/Ascorbate 454
2.4 Respiration and Membrane Potential 454
2.5 Phospholipid and Fatty Acid Analysis 454
2.6 Lipid Peroxidation Products 455
2.6.1 TBARS 455
2.6.2 4-Hydroxynonenal and Other Monofunctional Aldehydes 455
2.6.3 Lipohydroperoxides 455
2.6.4 Monohydroxyeicosatetraenoic Acids and F2-Isoprostanes 455
2.6.5 Oxidized Cardiolipin [(C-18:2)3 Monohydroxylinoleic acid-CL] 455
2.7 Antioxidants 456
2.7.1 Reduced and Oxidized Glutathione 456
2.7.2 a-Tocopherol and Ubiquinol 
456 
2.8 UVA Irradiation 456
2.8.1 UVA Exposure of Cells and Plasma In Vitro 456
2.8.2 Western Blotting for Detection of Oxidatively Modified Proteins 457
2.8.3 Total Antioxidant Status 457
2.8.4 Quantification of HETE Isomers and Enantiomer Separation of 5-HETE 457
2.9 UVB Irradiation and HaCaT Keratinocytes In Vitro 457
2.9.1 UVB Irradiation and Cell Culture 457
2.9.2 Determination of Cell Viability: Vital Staining and TUNEL-Staining 458
2.9.3 Determination of Biomarkers of Oxidative Stress and Inflammation 458
2.9.4 Mitochondria and Submitochondrial Membranes 458
2.9.5 Mitochondrial Electron Transfer Activities and Mitochondrial Nitric oxide Synthase (mtNOS) Activity 458
2.9.6 Quantification of Cardiolipin Species 459
2.9.7 Western Blot Analysis of MnSOD and CuZnSOD 459
2.10 UVB Irradiation and Microdialysis of Human Skin In Vivo 459
2.10.1 Control Persons 459
2.10.2 Induction of Skin Erythema by UVB Irradiation 459
2.10.3 Microdialysis Technique 460
2.10.4 Determination of Concentrations of F2-Isoprostanesand Prostaglandins 460
3 Results and Discussion 460
3.1 Iron/Ascorbate Induced Peroxidation in Mitochondria 460
3.1.1 Iron/Ascorbate Induced Generation of Lipid Peroxidation and Protein Oxidation 461
3.1.2 Iron/Ascorbate Mediated Impairment of Functional Intactnessof Mitochondria 462
3.1.3 Role of Endogenous and Exogenous Antioxidants During Iron/Ascorbate-Induced Oxidative Stress 463
3.2 UV Irradiation and Human Skin 465
3.2.1 UVA-Mediated Damage to Proteins and Lipid Mediators in Extracorporeal Photoimmunotherapy 465
3.2.2 Effects of UVB Irradiation on HaCaT Keratinocytes In Vitro and Microdialysates of Human Skin In Vivo 466
UVB Irradiation-Induced Damage of HaCaT Keratinocytes and Adaptive Responses to Oxidative Stress 466
UVB Irradiation-Induced Damage of Human Skin In VivoUsing Microdialysis Technique 469
References 470
Carbon Tetrachloride-Induced Hepatotoxicity: A Classic Model of Lipid Peroxidationand Oxidative Stress 476
1 Introduction 477
2 Isoprostane Formation and Release Following 
478 
3 Prostaglandin F2a Formation and Release FollowingCCl4 Challenge 
479 
4 Metabolism of Carbon Tetrachloride 479
5 Induction of Free Radical-Mediated Lipid Peroxidationand Oxidative Injury by Carbon Tetrachloride 480
6 Induction of Cyclooxygenase-Mediated Lipid Peroxidation by Carbon Tetrachloride 481
7 Methodological Protocol of Induction of Oxidative Stressby Carbon Tetrachloride 481
8 Role of Antioxidants in Carbon Tetrachloride-Induced Isoprostane and Prostaglandin Formation 484
9 Conclusion 485
References 486
Enhanced Urinary Excretion of Lipid Metabolites Following Exposure to Structurally Diverse Toxicants: A Unique Experimental Model for the Assessment of Oxidative Stress 
490 
1 Introduction 490
2 Toxicant-Induced Oxidative Stress 492
3 Urinary Lipid Metabolites 494
4 Experimental Models 495
5 Conclusion 499
References 499
Oxidative Stress in Animal Models with Special Reference to Experimental Porcine Endotoxemia 505
1 Introduction to Animal Models of Inflammation 506
1.1 The Validity of Animal Models in Inflammatory Research 506
1.2 The Choice of Animals 507
1.3 Ethical Considerations 508
2 Experimental Sepsis Models 508
2.1 Sepsis Models 508
2.2 SIRS and Sepsis Models 509
2.3 Endotoxins 511
2.4 Host–Endotoxin Interactions 512
2.5 Endotoxemic Models 512
2.6 Live Bacteria Models 513
2.7 Peritonitis Models 513
2.8 Appendix on Induction of Endotoxemia and Acute Inflammation 514
References 516
Models and Approaches for the Study of Reactive Oxygen Species Generation and Activities in Contracting Skeletal Muscle 519
1 Introduction 519
2 Measurements of Circulating Markers 520
3 Measurement of End Products of ROS Reactions in Muscle 520
4 Techniques to Study Extracellular ROS 521
5 Techniques to Study Intracellular ROS 523
6 Conclusion 524
References 525
Exercise as a Model to Study Interactions Between Oxidative Stress and Inflammation 528
1 Introduction 528
2 Acute Exercise, Muscle Damage and Antioxidative Vitamins 529
3 Exercise, Cytokines and Oxidative Vitamins 530
3.1 The Cytokine Response to Exercise 530
3.2 Exercise-Induced IL-6 Production Is Not Linked with Muscle Damage 530
3.3 Exercise-Induced IL-6 and Antioxidative Vitamins 531
4 Chronic Inflammation and Disease 532
5 Linking Inflammation with Disease 532
6 Training Studies and Antioxidative Vitamins 533
7 Conclusion 534
References 534
Exercise as a Model to Study Oxidative Stress 537
1 Free Radical Generation and Oxidative Stress in Exercise 537
2 Exercise and Antioxidants 539
3 Redox Control of Muscle Adaptation to Exercise 541
4 Hypoxia, Exercise and Oxidative Stress 543
5 Role of RONS in Muscle Health/Disease 543
6 Conclusion 544
References 544
Part VI: 
549 
Models of Mitochondrial Oxidative Stress 550
1 Mitochondrial Production of Superoxide Anion and Hydrogen Peroxide 551
2 Measurement of Mitochondrial Superoxide and Hydrogen Peroxide Production 551
3 Models of Mitochondrial Oxidative Stress 554
3.1 Formation of O2• /H2O2 by Complex I 555
3.2 Inhibition of Complex III: The Outer and Inner UQ Pools 556
3.3 Complex II 559
4 Cellular H2O2: Mitochondrial and Nonmitochondrial Sources 559
5 Conclusion 562
References 563
Protection of Oxidant-Induced Neuronal Cells Injury by a Unique Cruciferous Nutraceutical 568
1 Introduction 569
2 Materials and Methods 570
2.1 Chemicals and Materials 570
2.2 Cell Culture 570
2.3 Cell Extract Preparation 570
2.4 Assay for GSH Content 571
2.5 Assay for Activities of NQO1 and Other Antioxidant Enzymes 571
2.6 Assay for Cell Viability 571
2.7 Statistical Analysis 572
3 Results 572
3.1 Induction of Cellular Antioxidant GSH by D3T in SH-SY5Y Neuroblastoma Cells, Primary Human Neurons and Astrocytes 572
3.2 Induction of Cellular Phase-2 Enzyme NQO1 by D3T in SH-SY5Y Neuroblastoma Cells, Neurons and Astrocytes 572
3.3 D3T Pretreatment Protects SH-SY5Y Neuroblastoma Cells Against Acrolein, 4-Hydroxy-2-nonenal (HNE) or 6-OHDA-Induced Neurocytotoxicity 
574 
3.4 D3T Pretreatment Protects Human Primary Neurons Against HNE and 6-OHDA-Induced Neurocytotoxicity 574
3.5 D3T Pretreatment Protects Human Primary Astrocytes Against 1-Methyl-4-phenylpyridinium (MPP+), HNE or 6-OHDA-Induced Neurocytotoxicity 
574 
4 Discussion 576
References 579
Oxidatively Generated Damage to DNA and Biomarkers 583
1 Introduction 584
2 Radical Reactions Involving •OH and One-Electron Oxidation Processes 585
2.1 Single-Base Lesions 586
2.1.1 Thymine 586
2.1.2 Guanine 589
2.1.3 Adenine 591
2.2 Clustered and Tandem Lesions 592
2.2.1 Cytosine Adducts Arising from •OH-Mediated-Hydrogen Abstraction at C4' 
593 
2.2.2 Formation of (5'R)-5',8-cyclo-2'-deoxyadenosine (33) through •OH-mediated hydrogen abstraction at C5' 
594 
3 Singlet Oxygen Oxidation of Guanine 596
4 Measurement of Oxidatively Generated DNA Damage 597
4.1 HPLC-Based Methods 597
4.2 Enzymatic Assays 599
5 Conclusion and Future Perspectives 600
References 601
Measuring Oxidative Stress in Cell Cultures, Animals and Humans: Analysis and Validation of Oxidatively Damaged DNA 609
1 Introduction 609
2 Markers for Oxidative Stress 611
2.1 Non-Cellular Markers 611
2.2 Markers for Intracellular ROS 612
2.3 Changes in Cellular Antioxidant Status 613
2.4 Oxidation of Lipids and Proteins 614
2.5 Oxidatively Damaged DNA 614
2.5.1 Analysis of Cellular Levels of 8-oxodG 615
2.5.2 The Comet Assay 617
2.5.3 Oxidatively Modified DNA Lesions in Urine 618
3 Validation of Oxidatively Damaged DNA 619
3.1 Results in ESCODD 619
3.2 Work in ECVAG 619
3.3 Work in ESCULA 620
4 Oxidative DNA Damage and Relation to Disease 621
5 Conclusion 621
References 622
The Roles of cAMP and G Protein Signalingin Oxidative Stress-Induced Cardiovascular Dysfunction 625
1 Cyclic AMP and G Protein Signaling 626
2 Oxidative Stress and Reactive Oxygen Species 627
3 Messenger Action of ROS 629
3.1 G Protein Signaling and Oxidative Stress in Hypertension 631
3.1.1 Role of superoxide anions 631
4 Role of Peroxynitrite in G-Protein Signaling 633
5 NPR-C and Oxidative Stress 634
6 G Protein Signaling and Oxidative Stress in Hyperglycemia 635
7 Conclusion 636
References 637
Cellular and Chemical Assays for Discovery of Novel Antioxidants in Marine Organisms 640
1 Introduction 641
1.1 CAA 642
1.2 CLPAA 644
1.3 The Comet Assay 645
1.4 ORAC 645
1.5 FRAP 646
2 Materials and Methods 646
2.1 Cell Culture, CAA and CLPAA Experimental Procedures 646
2.2 Extracting, Screening and Identifying Marine Natural Compounds 647
3 Results and Discussion 649
3.1 CAA 649
3.2 CLPAA 650
3.3 The Comet Assay 653
3.4 Comparison of Chemical and Cellular Assays: FRAP, ORAC, CAA and CLPAA 653
3.5 Testing Antioxidants in Animals and Humans 654
4 Conclusions 657
References 658
Arsenic-Induced Oxidative Stress: Evidence on In Vitro Models of Cardiovascular, Diabetes Mellitus Type 2 and Neurodegenerative 
661 
1 Introduction 662
2 Arsenic-Induced Oxidative Stress in the Cardiovascular System 663
2.1 Endothelium Dysfunction 664
2.2 Atherosclerosis 665
2.3 Hypertension 668
3 Arsenic-Induced Oxidative Stress in Diabetes Mellitus Type 2 670
4 Arsenic-Induced Oxidative Stress in CNS Degeneration 674
5 Conclusions and Perspectives 676
References 677
Index 683