Mitochondria in Pathogenesis

by Lemasters, John J.; Nieminen, Anna-Liisa

• ISBN13: 9780306464331
• ISBN10: 0306464330
• Format: Hardcover
• Copyright: 2001-09-01
• Publisher: Plenum Pub Corp

Summary

Mitochondria are organelles in each cell outside the nucleus and are the energy source of all cells. As such, they are crucial to the healthy functioning of cells. Recent research has shown that mitochondrial dysfunction underlies a broad spectrum of disease, from maternally inherited genetic disorders to metabolism defects, aging, stroke, and neurodegenerative diseases such as Parkinson's, Alzheimer's, and Lou Gehrig's disease. This book brings together top researchers whose work in examining the pathophysiologic processes will lead to new strategies for prevention and treatment.

Table of Contents

Section 1. Evaluation of Mitrochondrial Function in Intact Cells
Flow Cytometric Analysis of Mitochondrial Function	3	(18)
Hagai Rottenberg
Introduction	3	(1)
Flow Cytometry	4	(1)
Tools for Measuring Plasmalemmal and Mitochondrial Membrane Potentials	4	(12)
Flow Cytometry Measurements	4	(1)
Rhodamine 123	5	(1)
DiOC6(3)	6	(9)
JC-1 as a Flow Cytometry Probe for ΔΨp	15	(1)
Estimation of ΔΨm with the Fixable Dye CMX Ros (Mitotracker Red)	15	(1)
FAD Fluorescence	16	(1)
Mitochondrial Generation of Reactive Oxygen Species	16	(2)
Mitochondrial Calcium Stores	18	(3)
References	19	(2)
Confocal Microscopy of Mitochondrial Function in Living Cells	21	(32)
John J. Lemasters
Ting Qian
Donna R. Trollinger
Wayne E. Cascio
Hisayuki Ohata
Anna-Liisa Nieminen
Introduction	21	(1)
Image Formation in Confocal Microscopy	22	(1)
Pinhole Principle	22	(1)
Axial Resolution of Confocal Microscopy	22	(1)
Two-Dimensional Image Formation	23	(1)
Specimen Preparation for Live Cell Imaging	23	(1)
Dealing with Photodamage and Photobleaching	24	(2)
Greater Requirement by Confocal Microscopy for Excitation Light	24	(1)
High Numerical Aperture Objective Lenses	24	(1)
Laser Attenuation	24	(1)
Pinhole Size, Zoom Magnification, and Previewing	25	(1)
Fluorophores	25	(1)
Multiparameter Fluorescence Microscopy	26	(1)
Membrane Potential Imaging	26	(2)
Nernstian Uptake of Cationic Dyes	26	(1)
Quantifying Electrical Potential in Confocal Images	27	(1)
Nonideal Behavior by Potential-Indicating Fluoreophores	28	(1)
Ion Imaging	28	(5)
Ratio Imaging	28	(1)
pH Imaging	29	(1)
Temperature Dependence of Ester Loading	29	(4)
The Cold-Loading/Warm-Incubation Protocol	33	(1)
Free Ca2+ Imaging	33	(10)
Nonratiometric Imaging	33	(1)
Line-Scanning Confocal Microscopy of Ca2+ Transients	33	(3)
Ratio Imaging	36	(1)
Cold Loading of Ca2+ Indicators Followed by Warm Incubation	37	(2)
Simulataneous Labeling of the Cytosol and Mitochondria with Different Ca2+-Indicating Fluorophores	39	(1)
Lysosomal Localization of Ca2+-Indicating Fluorophores	40	(3)
Reactive Oxygen Species	43	(1)
The Mitochondrial Permeability Transition	44	(2)
Conclusion	46	(7)
References	47	(6)
Section 2. Mitochondrial Disease and Aging
Primary Disorders of Mitochondrial DNA and the Pathophysiology of mtDNA-Related Disorders	53	(28)
Eric A. Schon
Salvatore DiMauro
Introduction	53	(2)
Diseases Associated with mtDNA Point Mutations	55	(9)
Ribosomal RNA Gene Mutations	55	(3)
Transfer RNA Gene Mutations	58	(3)
Polypeptide-Coding Gene Mutations	61	(3)
Diseases Associated with mtDNA Rearrangements	64	(2)
Sporadic Rearrangements	64	(1)
Maternally Inherited Rearrangements	65	(1)
Diseases Associated with Nuclear DNA Mutations	66	(15)
Integrity Errors	66	(1)
Importation Errors	67	(1)
Mature Polypeptide Errors	68	(1)
Cofactor and Coenzyme Errors	69	(1)
Other Disorders	69	(1)
References	70	(11)
Transmission and Segregation of Mammalian Mitochondrial DNA	81	(14)
Eric A. Shoubridge
Introduction	81	(1)
Mitochondrial DNA: Structure and Replication	82	(1)
mtDNA Transmission: The Bottleneck Hypothesis	82	(2)
Testing the Bottleneck Hypothesis	84	(1)
Mechanisms of Paternal mtDNA Exclusion	85	(1)
Male Transmission of mtDNA in Interspecific Matings	86	(1)
Segregation of mtDNA during Embryogenesis and Fetal Life	87	(1)
Segregation of mtDNA after Birth	88	(1)
Genetic Counseling	89	(1)
Summary and Future Perspectives	89	(6)
References	91	(4)
Cardiac Reperfusion Injury: Aging, Lipid Peroxidation, and Mitochondrial Function	95	(20)
Luke I. Szweda
David T. Lucas
Kenneth M. Humphries
Pamela A. Szweda
Background and Significance	95	(4)
Cardiac Reperfusion and Free Radical Formation	95	(1)
Evidence Implicating Free Radicals in Cardiac Reperfusion Injury	96	(1)
Mitochondria as a Source of Free Radicals during Reperfusion	96	(1)
Aging and Mitochondrial Dysfunction during Ischemia and Reperfusion	97	(1)
Potential Mechanisms of Free Radical-Mediated Mitochondrial Dysfunction	97	(1)
Hypothesis and Experimental Design	98	(1)
Results	99	(6)
Cardiac Ischemia/Reperfusion	99	(3)
In Vitro Treatment of Intact Rat Heart Mitochondria with 4-Hydroxy-2-Nonenal	102	(3)
Discussion	105	(10)
Potential Mechanism of Reperfusion-Induced Mitochondrial Dysfunction	105	(1)
Does 4-Hydroxy-2-Nonenal Mediate Free Radical Damage during Cardiac Reperfusion?	106	(1)
Ischemic Conditions Prime Mitochondria for Free Radical Damage during Reperfusion	106	(1)
Effects of Age on Reperfusion-Induced Mitochondrial Dysfunction	107	(1)
Implications for Antioxidant Interventions	107	(1)
References	108	(7)
Section 3. Mitochondrial Ion Homeostasis and Necrotic Cell Death
Ca2+-Induced Transition in Mitochondria: A Cellular Catastrophe?	115	(10)
Robert A. Haworth
Douglas R. Hunter
Introduction	115	(1)
Properties of the Ca2+-Induced Transition	116	(2)
Transition as a Death Mechanism	118	(2)
Transition Induced by Specific Removal of Protection	120	(1)
Role of Spontaneous Transitions	121	(4)
References	122	(3)
Physiology of the Permeability Transition Pore	125	(28)
Mario Zoratti
Francesco Tombola
Introduction	125	(1)
Localization	126	(1)
Intrinsic Characteristics of the Permeability Transition Pore	127	(4)
Size and Selectivity	127	(3)
Voltage Dependence	130	(1)
Induction/Modulation	131	(10)
Ca2+	132	(1)
Matrix pH	133	(1)
Redox Events	134	(3)
Cyclophylin D	137	(3)
Adenine Nucleotides	140	(1)
Protease Action	140	(1)
Molecular Identity	141	(5)
Adenine Nucleotide Translocator	141	(1)
The Permeability Transition Pore as a Supramolecular Complex	142	(1)
Bax	143	(1)
Permeabilization of Yeast Mitochondria	144	(1)
Electrophysiological Observations	144	(2)
Perspectives	146	(7)
References	146	(7)
Control of Mitochondrial Metabolism by Calcium-Dependent Hormones	153	(24)
Paul Burnett
Lawrence D. Gaspers
Andrew P. Thomas
Introduction	153	(3)
InsP3-Mediated Ca2+ Signaling	156	(1)
Mitochondrial Ca2+ Transport Systems	157	(2)
The Ca2+ Uniporter	157	(1)
The Ca2+ Efflux Mechanisms	158	(1)
Role of Mitochondria in [Ca2+]c Homeostasis	159	(5)
Quantitation of Mitochondrial Matrix [Ca2+]	160	(3)
Modulation of [Ca2+]c Signals by Mitochondria	163	(1)
Control of Mitochondrial Metabolism by Ca2+	164	(5)
Regulation of the Respiratory Chain	164	(2)
Regulation of the Mitochondrial Ca2+-Sensitive Dehydrogenases by Intrinsic Factors and Ca2+	166	(1)
Regulation of Mitochondrial Metabolism by Ca2+ in Intact Cells	167	(2)
Conclusions	169	(8)
References	170	(7)
The Permeability Transition Pore in Myocardial Ischemia and Reperfusion	177	(24)
Andrew P. Halestrap
Paul M. Kerr
Sabzali Javadov
M-Saadah Suleiman
Introduction	177	(1)
The Mitochondrial Permeability Transition Pore and Reperfusion Injury	178	(12)
Intracellular Conditions during Reperfusion Favor Pore Opening	178	(1)
Methods for Measuring Pore Opening in Isolated Heart Cells and in Perfused Heart	179	(3)
Pore Opening Occurs upon Reperfusion, but Not Ischemia	182	(1)
Pore Closure Follows Opening in Hearts that Recover during Reperfusion	183	(2)
Transition Inhibitors Protect Hearts from Reperfusion Injury	185	(5)
The Permeability Transition and Apoptosis in Reperfusion Heart Injury	190	(2)
Conclusions	192	(9)
References	192	(9)
Mitochondrial Calcium Dysregulation during Hypoxic Injury to Cardiac Myocytes	201	(14)
Elinor J. Griffiths
Introduction	201	(1)
Mitochondrial Ca2+ Transport under Normal Conditions	202	(2)
Ca2+ Transport Studies in Isolated Mitochondria	202	(1)
Mitochondrial Ca2+ Transport in Intact Cells	203	(1)
Mitochondrial Ca2+ Transport during Hypoxia and Reoxygenation	204	(7)
Importance of [Ca2+]m during Hypoxia/Reoxygenation Injury	204	(1)
Single-Cell Model of Hypoxia/Reoxygenation	204	(1)
Myocyte Morphological Changes	205	(1)
Changes in [Ca2+]m during Hypoxia and Reoxygenation	205	(2)
Route of Ca2+ Entry into Mitochondria during Hypoxia and Reoxygenation	207	(2)
Role of Ca2+ Transporters during Hypoxia	209	(1)
Activity of Mitochondrial Ca2+ Transporters upon Reoxygenation	210	(1)
Role of the Mitochondrial Permeability Transition Pore?	210	(1)
Conclusions	211	(4)
References	211	(4)
Mitochondrial Implication in Cell Death	215	(32)
Patrice X. Petit
Introduction	215	(2)
Reactive Oxygen Species as Mediators of Programmed Cell Death	217	(5)
Programmed-Cell-Death-Mediating ROS Are Produced Mainly by Mitochondria	218	(2)
Reactive Oxygen Species: Effects and Signaling	220	(2)
Mitochondrial Membrane Potential, Permeability Transition, and Early Apoptosis	222	(2)
Membrane-Potential Decrease: Universal Apoptotic Event	222	(1)
Kinetic Data Implicating Mitochondria in Apoptosis	222	(2)
Permeability-Transition Modulation and Apoptosis	224	(1)
Permeability Transition and Apoptosis-Inducing Factor	224	(3)
Cytochrome c Release and Induction of Caspases	227	(1)
Permeability Perturbation and Cytochrome c Release	228	(7)
Cytochrome c: Execution-Phase Caspase-Activation Regulator	232	(1)
Apoptosis-Inducing Factor and Mitochondrial Cell Death	233	(2)
The Bc12 Family: Counteracting Mitochondria Pro-Apoptotic Signals	235	(1)
Toward an Endosymbiotic Theory of Apoptosis Origin and Evolution	236	(3)
Conclusions	239	(8)
References	240	(7)
Role of Mitochondria in Apoptosis Induced by Tumor Necrosis Factor-α	247	(18)
Cynthia A. Bradham
Ting Qian
Konrad Streetz
Christian Trautwein
David A. Brenner
John J. Lemasters
Apoptosis Signaling and TNFα	247	(3)
The TNFR Superfamily and Death Domains	248	(1)
Receptor Protein Complex	248	(1)
Caspase Cascade	249	(1)
Cytochrome c Release	250	(1)
The TNFα-Mediated Apoptosis Model in Primary Culture	250	(9)
Mitochondrial Permeability Transition during TNFα-Mediated Apoptosis	251	(2)
The Mitochondrial Permeability Transition Precedes and is Required for Cytochrome c Release and Caspase 3 Activation	253	(3)
The Mitochondrial Permeability Transition as a Component of the Signaling Cascade	256	(2)
Role of Bc12 Proteins	258	(1)
Conclusions	259	(6)
References	260	(5)
The ATP Switch in Apoptosis	265	(16)
David J. McConkey
Introduction	265	(1)
Molecular Regulation of Apoptosis	266	(1)
Triggers for Apoptosis	267	(1)
Mechanisms of Caspase Activation	268	(1)
Molecular Overlap of Apoptosis and Necrosis	269	(1)
The ATP Switch in Apoptosis	270	(1)
Other Biochemical Switches	271	(2)
Role of Calcium	271	(1)
Role of Reactive Oxygen Species	272	(1)
Role of Intracellular Thiols	273	(1)
Conclusions	273	(8)
References	274	(7)
Section 4. Mitochondria, Free Radicals, and Disease
Reactive Oxygen Generation by Mitochondria	281	(20)
Alicia J. Kowaltowski
Anibal E. Vercesi
Introduction	281	(1)
The Mitochondrial Electron Transport Chain and Superoxide Generation Sites	282	(1)
Different Reactive Oxygen Species Detectable in Mitochondria	283	(1)
Conditions that Increase or Decrease Mitochondrial Reactive Oxygen Species Generation	284	(2)
Antioxidant Defenses	286	(2)
Effects of Mitochondrial Reactive Oxygen Species under Oxidative Stress Conditions	288	(4)
Oxidative Damage to Mitochondrial Membrane Proteins	288	(1)
Mitochondrial Permeability Transition	288	(3)
Oxidative Damage to Mitochondrial Membrane Lipids	291	(1)
Oxidative Damage to Mitochondrial DNA	291	(1)
Beneficial Effects of Mitochondrial Oxidative Stress	292	(1)
Effects of Mitochondrial Oxidative Stress on Cell Integrity	292	(1)
Conclusions	293	(8)
References	294	(7)
Role of the Permeability Transition in Glutamate-Mediated Neuronal Injury	301	(16)
Ian J. Reynolds
Teresa G. Hastings
Introduction	301	(1)
Role of Mitochondria in Glutamate Toxicity	302	(3)
Mitochondria in Neuronal Ca2+ Homeostasis	302	(1)
Reactive Oxygen Species and Glutamate Toxicity	303	(2)
The Permeability Transition in Neuronal Mitochondria	305	(1)
Limitations of the Permeability Transition Hypothesis	306	(6)
Transition Measurement in Intact Cells	306	(2)
Limitations of Pharmacological Approaches	308	(2)
Limitations of Cell Culture Methodology	310	(1)
Acutely Isolated Mitochondria Preparations	310	(1)
Mitochondrial Heterogeneity	311	(1)
Conclusions	312	(5)
References	312	(5)
Mitochondrial Dysfunction in the Pathogenesis of Acute Neural Cell Death	317	(16)
Gary Fiskum
Introduction	317	(1)
Significance of Mitochondrial Injury	318	(2)
Mitochondria as Primary Targets in Excitotoxicity	318	(1)
Mitochondria as Storage Sites for Apoptogenic Proteins	318	(2)
Mechanisms of Mitochondrial Injury	320	(2)
Mitochondrial Ca2+ Overload	320	(1)
Oxidative Stress	321	(1)
Induction of Apoptosis via Release of Mitochondrial Proteins	322	(3)
Stimulation and Inhibition Signals for Release of Apoptogenic Mitochondrial Proteins	322	(1)
Release Mechanisms of Apoptogenic Mitochondrial Proteins	323	(2)
Conclusions	325	(8)
References	326	(7)
Varied Responses of Central Nervous System Mitochondria to Calcium	333	(8)
Nickolay Brustovetsky
Janet M. Dubinsky
Mitochondrial Dysfunction in Oxidative Stress, Excitotoxicity, and Apoptosis	341	(20)
Anna-Liisa Nieminen
Aaron M. Byrne
Kaisa M. Heiskanen
Introduction	341	(1)
Oxidative Stress	342	(1)
Mitochondrial Permeability Transition (MPT)	343	(1)
Visualization of the Transition In Situ during Oxidative Stress	344	(1)
Contribution of Pyridine Nucleotide Oxidation to the Permeability Transition	344	(2)
Contribution of Increased Mitochondrial Free Ca2+ to the Permeability Transition	346	(3)
Contribution of Mitochondrial Reactive Oxygen Species to the Permeability Transition	349	(2)
Contribution of the Permeability Transition to Excitotoxicity in Neurons	351	(1)
Role of Mitochondrial Permeability Transitions in Apoptosis	352	(9)
References	355	(6)
Mitochondria in Alcoholic Liver Disease	361	(18)
Jose C. Fernandez-Checa
Carmen Garcia-Ruiz
Anna Colell
Introduction	361	(1)
Oxidative Alcohol Metabolism	362	(1)
Alcohol and Mitochondrial Dysfunction	363	(2)
Alcohol, Mitochondrial Biogenesis, and Membrane Properties	365	(2)
Alcohol and Mitochondrial Oxidative Stress	367	(2)
Alcohol-Induced Cytokine Overproduction	369	(4)
Tumor Necrosis Factor and Mitochondria	369	(1)
Ceramide Interaction with Mitochondria	370	(2)
Mitochondrial GSH Role in Sensitization to Tumor Necrosis Factor	372	(1)
Treatment of Alcoholic Liver Disease and Mitochondrial GSH Restoration	373	(1)
Conclusion	374	(5)
References	374	(5)
Mitochondrial Changes after Acute Alcohol Ingestion	379	(14)
Hajimi Higuchi
Hiromasa Ishii
Introduction	379	(1)
Ethanol-Associated Oxidative Stress in Hepatocytes	380	(2)
Mitochondria as Targets of Oxidative Stress	382	(1)
Mitochondrial Membrane Permeability Transition and Depolarization	383	(2)
Effect of Ethanol on Intracellular Glutathione Content	385	(1)
Ethanol-Induced Mitochondrial Injury and Apoptosis	385	(3)
Conclusion	388	(5)
References	388	(5)
Mitochondrial Dysfunction in Chronic Fatigue Syndrome	393	(20)
Brad Chazotte
Introduction	393	(4)
Origins of Chronic Fatigue Syndrome	393	(1)
Symptoms and Case Definitions for Chronic Fatigue Syndrome	394	(1)
Role for Mitochondrial Dysfunction in Chronic Fatigue Syndrome	395	(1)
Cytokine Role in Chronic Fatigue Syndrome	396	(1)
Methods	397	(4)
Cell Isolation, Culture, and Labeling	397	(1)
Confocal Microscopy	398	(1)
Membrane-Potential Imaging Analysis	398	(3)
Criteria for Patient and Control Populations	401	(1)
Results	401	(3)
Confocal Imaging of Membrane Potential in TMRM-Labeled Human Cells	401	(1)
Patient and Control Populations in this Study	401	(1)
Comparison of Patient and Control Populations	402	(1)
Comparison of Male and Female Chronic Fatigue Syndrome Patients	402	(1)
Interferon-α Effects on Human Fibroblasts	402	(2)
Discussion	404	(3)
Membrane-Potential Evidence for a Mitochondrial Dysfunction Role in Chronic Fatigue Syndrome	404	(2)
Searching the Mitochondrial Pathways for Possible Defects	406	(1)
Cytokines Affect Mitochondrial Bioenergetics, Cellular Bioenergetics, or Both	406	(1)
Conclusions	407	(6)
References	407	(6)
Section 5. Chemical Toxicity
Bile Acid Toxicity	413	(12)
Gregory J. Gores
James r. Spivey
Ravi Botla
Joong-Won Park
Mark J. Lieser
Introduction	413	(2)
Cholestasis	413	(1)
Hepatocellular Injury and Cholestasis	414	(1)
Mitochondria and Cell Death	414	(1)
Does Mitochondrial Dysfunction Occur in Cholestasis?	415	(1)
Do Toxic Bile Salts Directly Cause Mitochondrial Dysfunction?	415	(3)
Bile Salts and Mitochondrial Respiration	415	(2)
Bile Salts and Oxidative Stress	417	(1)
Do Bile Salts Lead to the Mitochondrial Membrane Permeability Transition?	418	(2)
Bile Salts and the Permeability Transition	418	(1)
Bile Salts and Cytochrome c Release	418	(1)
Bile Salts and Mitochondrial Depolarization In Situ	419	(1)
Do Adaptations Occur in Cholestasis to Inhibit Bile-Salt-Induced Transition?	420	(1)
Conclusion	421	(4)
References	422	(3)
Reye's Syndrome and Related Chemical Toxicity	425	(26)
Lawrence C. Trost
John J. Lenasters
Introduction	425	(1)
Reye's Syndrome, a Toxic Metabolic Crisis Caused by Mitochondrial Dysfunction	426	(6)
Aspirin and Salicylate Induce the Mitochondrial Permeability Transition	428	(2)
Salicylate MPT Induction Precedes Cell Death in Isolated Hepatocytes	430	(2)
Metabolic Deficiencies in Reye's Syndrome and Related Chemical Toxicity	432	(3)
Reye's-Related Chemical Toxicity	435	(5)
Valproic Acid	435	(2)
Jamaican Vomiting Sickness	437	(1)
Neem Oil	437	(1)
Adipic, Benzoic, 4-Pentenoic, and 3-Mercaptopropionic Acids	438	(2)
Mechanism of MPT Induction In Vitro by Salicylate and Agents Implicated in Reye's-Related Chemical Toxicity	440	(1)
Role of Calcium in Reye's Syndrome and Reye's-Related Chemical Toxicity	440	(4)
Fasting and Fatty Acid Mobilization in Reye's Syndrome and Reye's-Related Chemical Toxicity	444	(1)
Conclusion: Implications for Other Diseases	445	(6)
References	446	(5)
Purinergic Receptor-Mediated Cytotoxicity	451	(16)
J. Fred Nagelkerke
J. Paul Zoeteweij
Introduction	451	(1)
Purinergic Receptors	451	(1)
Functional Role of P2 Purinergic Receptors In Vivo	452	(1)
Mechanisms of ATP-Induced Cell Death	452	(9)
Intracellular Calcium	452	(2)
Determination of Mitochondrial Ca2+	454	(1)
Isolated Mitochondrial Ca2+-Induced Changes	454	(1)
Role of Ca2+-Induced Mitochondrial Damage in Cytotoxicity	455	(6)
Conclusion	461	(6)
References	462	(5)
Doxorubicin-Induced Mitochondrial Cardiomyopathy	467	(22)
Kendall B. Wallace
Drug-Induced Microvesicular Steatosis and Steatohepatitis	489	(30)
Dominique Pessayre
Bernard Fromenty
Abdellah Mansouri
Introduction	489	(1)
Mitochondria: Our Strength and Achilles' Heel	489	(2)
Microvesicular Steatosis	491	(2)
Morphology	491	(1)
Mechanism	491	(1)
Short-term Severity: Energy Crisis	492	(1)
Secondary Inhibition of β-Oxidation	493	(1)
Diversity of Mechanisms Impairing β-Oxidation	494	(6)
Mitochondrial DNA Lesions	494	(2)
Termination of mtDNA Replication: Dideoxynucleosides	496	(1)
Inhibition of mtDNA Replication: Fialuridine	497	(1)
Inhibition of mtDNA Transcription: Interferon-α	497	(1)
Sequestration of CoA and Opening of the Mitochondrial Permeability Transition Pore	497	(2)
Inhibition of β-Oxidation	499	(1)
Susceptibility Factors Affecting Drug Metabolism	500	(1)
Susceptibility Factors Affecting Mitochondrial Function	501	(4)
Reye's Syndrome	501	(2)
Acute Fatty Liver of Pregnancy	503	(2)
Possible Outcome of Prolonged Steatosis: Steatohepatitis	505	(4)
Features	505	(1)
Steatosis, Basal Formation of Reactive Oxygen Species, and Lipid Peroxidation	505	(1)
Increased Formation of Reactive Oxygen Species	506	(1)
Reactive Oxygen Species and Steatohepatitis	507	(2)
Conclusions	509	(10)
References	510	(9)
Index	519