Redox-Genome Interactions in Health and Disease (Oxidative Stress and Disease)

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Redox-Genome Inttractionr in Health and e d i t e d b y

Jiirgen Fuchs

Maurizio Podda J. M? Goethe University Frankfurt, Germany

Lester Packer University of Southern California School of Pharmacy LQSAngeles, California, U.S.A.

MARCEL

MARCELDEKKER, INC. D EK K ER

NEWYORK BASEL

Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide speciﬁc advice or recommendations for any speciﬁc situation. Trademark notice: product or corporate names may be trademarks or registered trademarks and are used only for identiﬁcation and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-4048-3 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher oﬀers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above.

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All Rights Reserved.

Neither this book nor any part may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopying, microﬁlming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

OXIDATIVE STRESS AND DISEASE Series Editors

LESTERPACKER, PH.D. ENRIQUE CADENAS, M.D., PH.D. University of Southern California School of Pharmacy Los Angeles, California

1. Oxidative Stress in Cancer, AIDS, and Neurodegenerative Diseases, edited by Luc Montagnier, Rene Olivier, and Catherine Pasquier 2. Understanding the Process of Aging: The Roles of Mitochondria, Free Radicals, and Antioxidants, edited by Enrique Cadenas and Lester Packer 3. Redox Regulation of Cell Signaling and Its Clinical Application, edited by Lester Packer and Junji Yodoi 4. Antioxidants in Diabetes Management, edited by Lester Packer, Peter Rosen, Hans J. Tritschler, George L. King, and Angelo Azzi 5. Free Radicals in Brain Pathophysiology, edited by Giuseppe Poli, Enrique Cadenas, and Lester Packer 6. Nutraceuticals in Health and Disease Prevention, edited by Klaus Kramer, Peter-Paul Hoppe, and Lester Packer 7. Environmental Stressors in Health and Disease, edited by Jurgen Fuchs and Lester Packer 8. Handbook of Antioxidants: Second Edition, Revised and Expandled, edited by Enrique Cadenas and Lester Packer 9. Flavonoids in Health and Disease: Second Edition, Revised arid Expanded, edited by Catherine A. Rice-€vans and Lester Packer 10. Redox-Genome Interactions in Health and Disease, edited by Jiirgen Fuchs, Maurizio Podda, and Lester Packer

Related Volumes

Vitamin E in Health and Disease: Biochemistry and Clinical Applications, edited by Lester Packer and Jiirgen Fuchs Vitamin A in Health and Disease, edited by Rune Blornhoff Free Radicals and Oxidation Phenomena in Biological Systems, edited by Marcel Roberfroid and Pedro Buc Calderon Biothiols in Health and Disease, edited by Lester Packer and Enrique Cadenas Handbook of Antioxidants, edited by Enrique Cadenas and Lester Packer Handbook of Synthetic Antioxidants, edited by Lester Packer and Enn'que Cadenas

Vitamin C in Health and Disease, edited by Lester Packer and Jurgen Fuchs

Lipoic Acid in Health and Disease, edited by Jurgen Fuchs, Lester Packer, and Guido Zimmer Flavonoids in Health and Disease, edited by Catherine Rice-Evans and Lester Packer Additional Volumes in Preparation

Series Introduction

Oxygen is a dangerous friend. Overwhelming evidence indicates that oxidative stress can lead to cell and tissue injury. However, the same free radicals that are generated during oxidative stress are produced during normal metabolism and thus are involved in both human health and disease. Free radicals are molecules with an odd number of electrons. The odd, or unpaired, electron is highly reactive as it seeks to pair with another free electron. Free radicals are generated during oxidative metabolism and energy production in the body. Free radicals are involved in: Enzyme-catalyzed reactions Electron transport in mitochondria Signal transduction and gene expression Activation of nuclear transcription factors Oxidative damage to molecules, cells, and tissues Antimicrobial action of neutrophils and macrophages Aging and disease Normal metabolism is dependent on oxygen, a free radical. Through evolution, oxygen was chosen as the terminal electron acceptor for respiration. The two unpaired electrons of oxygen spin in the same direction; thus, oxygen is a biradical, but is not a very dangerous free radical. Other oxygen-derived free radical species, such as superoxide or hydroxyl radicals, formed during metabolism or by ionizing radiation are stronger oxidants and are therefore more dangerous. In addition to research on the biological eﬀects of these reactive oxygen species, research on reactive nitrogen species has been gathering momentum. NO, or nitrogen monoxide (nitric oxide), is a free radical generated by NO synthase (NOS). This enzyme modulates physiological responses such as vasodilation or signaling in the brain. However, during inﬂammation, synthesis of NOS (iNOS) is induced. This iNOS can result in the overproduction of NO, causing damage. More worrisome, however, is the fact that excess NO can react with superoxide to produce the very toxic product iii

iv

Series Introduction

peroxynitrite. Oxidation of lipids, proteins, and DNA can result, thereby increasing the likelihood of tissue injury. Both reactive oxygen and nitrogen species are involved in normal cell regulation in which oxidants and redox status are important in signal transduction. Oxidative stress is increasingly seen as a major upstream component in the signaling cascade involved in inﬂammatory responses, stimulating adhesion molecule and chemoattractant production. Hydrogen peroxide, which breaks down to produce hydroxyl radicals, can also activate NF-nB, a transcription factor involved in stimulating inﬂammatory responses. Excess production of these reactive species is toxic, exerting cytostatic eﬀects, causing membrane damage, and activating pathways of cell death (apoptosis and/or necrosis). Virtually all diseases thus far examined involve free radicals. In most cases, free radicals are secondary to the disease process, but in some instances free radicals are causal. Thus, there is a delicate balance between oxidants in health and disease. Their proper balance is essential for ensuring healthy aging. The term oxidative stress indicates that the antioxidant status of cells and tissues is altered by exposure to oxidants. The redox status is thus dependent on the degree to which a cell’s components are in the oxidized state. In general, the reducing environment inside cells helps to prevent oxidative damage. In this reducing environment, disulﬁde bonds (S–S) do not spontaneously form because sulfhydryl groups kept in the reduced state (SH) prevent protein misfolding or aggregation. This reducing environment is maintained by oxidative metabolism and by the action of antioxidant enzymes and substances, such as glutathione, thioredoxin, vitamins E and C, and enzymes such as superoxide dismutase (SOD), catalase, and the selenium-dependent glutathione and thioredoxin hydroperoxidases, which serve to remove reactive oxygen species. Changes in the redox status and depletion of antioxidants occur during oxidative stress. The thiol redox status is a useful index of oxidative stress mainly because metabolism and NADPH-dependent enzymes maintain cell glutathione (GSH) almost completely in its reduced state. Oxidized glutathione (glutathione disulﬁde, GSSG) accumulates under conditions of oxidant exposure, and this changes the ratio of oxidized to reduced glutathione; an increased ratio indicates oxidative stress. Many tissues contain large amounts of glutathione, 2–4 mM in erythrocytes or neural tissues and up to 8 mM in hepatic tissues. Reactive oxygen and nitrogen species can directly react with glutathione to lower the levels of this substance, the cell’s primary preventative antioxidant. Current hypotheses favor the idea that lowering oxidative stress can have a clinical beneﬁt. Free radicals can be overproduced or the natural antioxidant system defenses weakened, ﬁrst resulting in oxidative stress, and then leading to oxidative injury and disease. Examples of this process include heart disease and cancer. Oxidation of human low-density lipoproteins is considered the ﬁrst step in the progression and eventual development of atherosclerosis, leading to cardiovascular disease. Oxidative DNA damage initiates carcinogenesis. Compelling support for the involvement of free radicals in disease development comes from epidemiological studies showing that an enhanced antioxidant status is associated with reduced risk of several diseases. Vitamin E and prevention of cardiovascular disease is a notable example. Elevated antioxidant status is also associated with a lower incidence of cataracts and cancer, and some recent reports have suggested an inverse correlation between antioxidant status and occurrence of rheumatoid

Series Introduction

v

arthritis and diabetes mellitus. Indeed, the number of indications in which antioxidants may be useful in the prevention and/or the treatment of disease is increasing. Oxidative stress, rather than being the primary cause of disease, is more often a secondary complication in many disorders. Oxidative stress diseases include inﬂammatory bowel diseases, retinal ischemia, cardiovascular disease and restenosis, AIDS, ARDS, and neurodegenerative diseases such as stroke, Parkinson’s disease, and Alzheimer’s disease. Such indications may prove amenable to antioxidant treatment because there is a clear involvement of oxidative injury in these disorders. In this new series of books, the importance of oxidative stress in diseases associated with organ systems of the body will be highlighted by exploring the scientiﬁc evidence and the medical applications of this knowledge. The series will also highlight the major natural antioxidant enzymes and antioxidant substances such as vitamins E, A, and C, ﬂavonoids, polyphenols, carotenoids, lipoic acid, and other nutrients present in food and beverages. Oxidative stress is an underlying factor in health and disease. More and more evidence indicates that a proper balance between oxidants and antioxidants is involved in maintaining health and longevity and that altering this balance in favor of oxidants may result in pathological responses causing functional disorders and disease. This series is intended for researchers in the basic biomedical sciences and clinicians. The potential for healthy aging and disease prevention necessitates gaining further knowledge about how oxidants and antioxidants aﬀect biological systems. Oxidative stress and the ensuing redox modulation of cell signaling and gene expression may play a signiﬁcant role in the pathophysiology of disease. Small changes in the cellular redox status elicit a profound impact on cellular homeostasis and pathophysiology. This book highlights new developments in redox biology and molecular genetics relevant to human disease. Lester Packer Enrique Cadenas

Preface

The last decade has witnessed rapid growth in the identiﬁcation of genes inﬂuenced by redox changes and formation of reactive oxygen and nitrogen species. Molecular genetics is changing our understanding of cell biology and providing new insights into the pathophysiology of human disease. New information obtained in the ﬁeld of genomics is already revolutionizing our understanding of several diseases and will improve the management of patients who exhibit diﬃcult-to-treat conditions. Accordingly, increased interest in redox modulation of cell function and gene expression is evolving, along with mounting experimental and clinical evidence that oxidative injury and redox modulations are involved in all diseases thus far examined. In most cases, oxidative stress and redox modulation, rather than being the primary cause of disease, are secondary complications, but may play a signiﬁcant role in their pathophysiology. Thus small modulations of the cellular redox status may have a large impact on health and disease. This comprehensive and unique book bridges diﬀerent but connected research areas and creates a new global perspective in biomedicine. It will be useful in basic research as well as in clinical applications, such as treatment of inﬂammatory diseases, cancer, or gene therapy. With contributions by leading researchers and clinicians, this book brings together information that has emerged in the last few years in the ﬁelds of redox biology and molecular genetics. Part I focuses on the basic aspects of the cellular redox state, signaling, identiﬁcation, and functional analysis of responding genes and downstream events such as mitogenic responses, apoptosis, and senescence. Part II includes chapters on the role of oxidative stress in the pathogenesis of mono- and polygenic diseases aﬀecting the lung, eye, skin, and neuromuscular and cardiovascular systems, as well as cancer. This volume is an important and timely contribution to the biomedical community. Ju¨rgen Fuchs Maurizio Podda Lester Packer

vii

Contents

Series Introduction (Lester Packer and Enrique Cadenas) Preface Contributors

I.

iii vii xiii

The Cellular Redox Status and Gene Regulation System

1. Redox–Genome Interactions: Evolution of a Concept Ju¨rgen Fuchs, Maurizio Podda, R. Kaufmann, and Lester Packer 2. Measurements of Biological Reducing Power by Voltammetric Methods Ron Kohen, Eitan Moor, and Miriam Oron

1

13

3. Composition and Regulation of Thiol-Disulﬁde Redox State Yvonne S. Nkabyo, Thomas R. Ziegler, and Dean P. Jones

43

4. Thiols and Thioredoxin in Cellular Redox Control Hajime Nakamura, Norihiko Kondo, Kiichi Hirota, Hiroshi Masutani, and Junji Yodoi

61

5. Role of Free Radicals and Cellular Redox Status in Signal Transduction and Gene Expression Wulf Dro¨ge and Wulf Hildebrandt 6. Redox Regulation of Apoptosis Marie-Ve´ronique Cle´ment and Shazib Pervaiz

79

101 ix

x

Contents

7. Mitochondria, Aging, and Disease: A Genomic Perspective Stefano Salvioli, Massimiliano Bonafe`, Cristiana Barbi, Miriam Capri, Daniela Monti, and Claudio Franceschi 8. The Human Genome as a Target of Oxidative Modiﬁcation: Damage to Nucleic Acids Jean Cadet, Thierry Douki, and Jean-Luc Ravanat 9. Redox Modulation in Tumor Initiation, Promotion, and Progression Margaret Hanausek, Zbigniew Walaszek, and Thomas J. Slaga 10. The Potential Impact of Polymorphism on Oxidative Stress Status Louise Lyrena¨s, Emma Wincent, Lena Forsberg, Ulf de Faire, and Ralf Morgenstern

II.

123

145 193 217

Oxidative Stress in Genetic Diseases

11. Mitochondrial Dysfunction in Genetic Diseases Immo E. Scheﬄer 12. Oxidative and Nitrosative Stress in Cystic Fibrosis: Signiﬁcance or Triviality? Brian M. Morrissey, Albert van der Vliet, Jason Eiserich, and Carroll E. Cross 13. Oxyradicals in Iron Overload Syndromes: Hemochromatosis Shinya Toyokuni 14. Oxidative Imbalance in Hereditary Hemoglobinopathies: The Role of Reactive Oxygen Species in the Pathophysiology of Sickle Cell Anemia and Thalassemia Ju¨rgen Fuchs, Maurizio Podda, and Eliezer A. Rachmilewitz

235

263

287

305

15. Association of Oxidative Stress with Cataractogenesis Marjorie F. Lou

325

16. Oxidative Stress and Age-Related Macular Degeneration Paul S. Bernstein, Jerusha L. Nelson, Jessica L. Burrows, and E. Wayne Askew

351

17. Retinitis Pigmentosa: A Potential Role for Reactive Oxygen and Nitrogen Species During Photoreceptor Apoptosis Maryanne Donavan and Thomas G. Cotter

377

18. The Role of Oxidative Imbalance in the Pathogenesis of Down Syndrome Ting-Ting Huang, Sailaja Mantha, and Charles J. Epstein

409

Contents

19. Metal Homeostasis and Its Relation to Oxidative Stress in Alzheimer’s Disease Adam D. Cash, Ravi Srinivas, Catherine A. Rottkamp, Xiongwei Zhu, Marta A. Taddeo, Gjumrakch Aliev, Hisashi Fujioka, Craig S. Atwood, Lawrence M. Sayre, Rudolph J. Castellani, Mark A. Smith, George Perry, and Akihiko Nunomura

xi

425

20. Oxidative Stress and Huntington’s Disease C. Turner and Anthony H. V. Schapira

439

21. Oxidative Stress in Familial Amyotrophic Lateral Sclerosis Diane E. Cabelli

473

22. Oxidants and Antioxidants in the Pathology of Colon Cancer William L. Stone, K. Krishnan, and Andreas M. Papas

497

23. The Role of Oxidative Imbalance in Diabetes Mellitus Dominique Bonnefont-Rousselot, Alain Legrand, and Jacques Delattre

513

24. The Role of Reactive Oxygen Species, the Renin-Angiotensin System, and Endothelin in the Development of Essential Hypertension Jane F. Reckelhoﬀ, Laurent Juillard, and J. Carlos Romero 25. Oxidative Stress in Cardiovascular Disease: Role of Oxidized Lipoproteins in Macrophage Foam Cell Formation and Atherosclerosis Michael Aviram and Mira Rosenblat 26. Free Radicals in Rheumatoid Arthritis: Mediators and Modulators Tulin Bodamyali, Janos M. Kanczler, Tim M. Millar, Cliﬀ R. Stevens, and David R. Blake Index

541

557

591

611

Contributors

Gjumrakch Aliev, M.D., Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. E. Wayne Askew, Ph.D. U.S.A.

University of Utah College of Health, Salt Lake City, Utah,

Craig S. Atwood, Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Michael Aviram, D.Sc. Lipid Research Laboratory, Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel Cristiana Barbi

University of Bologna, Bologna, Italy

Paul S. Bernstein, M.D. Ph.D. John A. Moran Eye Center, University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. David R. Blake Department of Medical Sciences, University of Bath and Royal National Hospital for Rheumatic Diseases, Bath, England Tulin Bodamyali, Ph.D. England Massimiliano Bonafe`

Department of Medical Sciences, University of Bath, Bath,

University of Bologna, Bologna, Italy

Dominique Bonnefont-Rousselot, Ph.D. macy, Paris, France Jessica L. Burrows, M.S. Utah, U.S.A.

Biochemistry Laboratory, Faculty of Phar-

University of Utah College of Health, Salt Lake City, xiii

xiv

Contributors

Diane E. Cabelli, Ph.D. Chemistry Department, Brookhaven National Laboratory, Upton, New York, U.S.A. Jean Cadet, Ph.D. De´partement de Recherche Fondamentale sur la Matie`re Condense´e, Commissariat a` l’Energie Atomique Grenoble, Grenoble, France Miriam Capri

University of Bologna, Bologna, Italy

Adam D. Cash Ohio, U.S.A.

Institute of Pathology, Case Western Reserve University, Cleveland,

Rudolph J. Castellani, M.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Marie-Ve´ronique Cle´ment, Ph.D. of Singapore, Singapore

Department of Biochemistry, National University

Department of Biochemistry, University College Cork, County

Thomas G. Cotter Cork, Ireland

Carroll E. Cross Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California, Davis, School of Medicine, Sacramento, California, U.S.A. Ulf de Faire, M.D., Ph.D. tutet, Stockholm, Sweden Jacques Delattre

Institute of Environmental Medicine, Karolinska Insti-

Biochemistry Laboratory, Faculty of Pharmacy, Paris, France

Maryanne Donavan Cork, Ireland

Department of Biochemistry, University College Cork, County

Thierry Douki De´partement de Recherche Fondamentale sur la Matie`re Condense´e, Commissariat a` l’Energie Atomique Grenoble, Grenoble, France Wulf Dro¨ge

Deutsches Krebsforschungszentrum, Heidelberg, Germany

Jason Eiserich Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California, Davis, School of Medicine, Sacramento, California, U.S.A. Charles J. Epstein Department of Pediatrics, University of California, San Francisco, San Francisco, California, U.S.A. Lena Forsberg, Ph.D. Stockholm, Sweden

Institute of Environmental Medicine, Karolinska Institutet,

Claudio Franceschi University of Bologna, Bologna, and Italian National Research Center on Aging, Ancona, Italy

Contributors

xv

Ju¨rgen Fuchs, Ph.D., M.D. Frankfurt, Germany

Department of Dermatology, J. W. Goethe University,

Hisashi Fujioka, Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Margaret Hanausek, Ph.D. Cancer Causation and Prevention, AMC Cancer Research Center, Denver, Colorado, U.S.A. Wulf Hildebrandt

Deutsches Krebsforschungszentrum, Heidelberg, Germany

Kiichi Hirota Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, Ikeda, Japan Ting-Ting Huang, Ph.D. Stanford University and GRECC, Palo Alto VA Health System, Palo Alto, California, U.S.A. Dean P. Jones, Ph.D. Georgia, U.S.A.

Department of Biochemistry, Emory University, Atlanta,

Laurent Juillard, M.D., Ph.D. Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota, U.S.A. Janos M. Kanczler, Ph.D. Bath, England R. Kaufmann, M.D. furt, Germany

Department of Medical Sciences, University of Bath,

Department of Dermatology, J. W. Goethe University, Frank-

Ron Kohen, Ph.D. Department of Pharmaceutics, The Hebrew University of Jerusalem, Jerusalem, Israel Norihiko Kondo Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan K. Krishnan, M.D., F.R.C.P. Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee, U.S.A. Alain Legrand, Ph.D.

Biochemistry Laboratory, Faculty of Pharmacy, Paris, France

Marjorie F. Lou Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska, U.S.A. Louise Lyrena¨s, M.Sc. Stockholm, Sweden

Institute of Environmental Medicine, Karolinska Institutet,

Sailaja Mantha Department of Neurology and Neurological Sciences, Stanford University, Stanford, California, U.S.A.

xvi

Contributors

Hiroshi Masutani Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, and Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, Ikeda, Japan Tim M. Millar, Ph.D. England Daniela Monti

Department of Medical Sciences, University of Bath, Bath,

University of Florence, Florence, Italy

Eitan Moor, Ph.D. Department of Pharmaceutics, The Hebrew University of Jerusalem, Jerusalem, Israel Ralf Morgenstern, Ph.D. tet, Stockholm, Sweden

Institute of Environmental Medicine, Karolinska Institu-

Brian M. Morrissey Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California, Davis, School of Medicine, Sacramento, California, U.S.A. Hajime Nakamura, M.D., Ph.D. Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan Jerusha L. Nelson, M.S., Ph.D. Utah, U.S.A.

University of Utah College of Health, Salt Lake City,

Yvonne S. Nkabyo, M.Sc. Department of Molecular and Systems Pharmacology, Emory University, Atlanta, Georgia, U.S.A. Akihiko Nunomura, M.D., Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A., and Department of Psychiatry and Neurology, Asahikawa Medical College, Asahikawa, Japan Miriam Oron, Ph.D. Department of Pharmaceutics and Molecular Biology, The Hebrew University of Jerusalem, Jerusalem, Israel Lester Packer, Ph.D. Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles, California, U.S.A. Andreas M. Papas, Ph.D.

YASOO Health, Inc., Johnson City, Tennessee, U.S.A.

George Perry, Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Shazib Pervaiz, M.B.B.S., Ph.D. Singapore, Singapore Maurizio Podda, M.D. Frankfurt, Germany

Department of Physiology, National University of

Department of Dermatology, J.W. Goethe University,

Contributors

xvii

Eliezer A. Rachmilewitz, M.D. cal Center, Holon, Israel

Department of Hematology, The E. Wolfson Medi-

Jean-Luc Ravanat De´partement de Recherche Fondamentale sur la Matie`re Condense´e, Commissariat a` l’Energie Atomique Grenoble, Grenoble, France Jane F. Reckelhoﬀ, Ph.D. Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi, U.S.A. J. Carlos Romero, M.D. Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota, U.S.A. Mira Rosenblat Lipid Research Laboratory, Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel Catherine A. Rottkamp Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Stefano Salvioli

University of Bologna, Bologna, Italy

Lawrence M. Sayre, Ph.D. Cleveland, Ohio, U.S.A.

Institute of Pathology, Case Western Reserve University,

Anthony H. V. Schapira, D.Sc., M.D., F.R.C.P. Department of Clinical Neurosciences, Royal Free and University College Medical School, University College London, London, England Immo E. Scheﬄer Division of Biological Sciences, University of California, San Diego, La Jolla, California, U.S.A. Thomas J. Slaga, Ph.D. Cancer Causation and Prevention, AMC Cancer Research Center, Denver, Colorado, U.S.A. Mark A. Smith, Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Ravi Srinivas Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Cliﬀ R. Stevens, Ph.D. Department of Medical Sciences, University of Bath and Royal National Hospital for Rheumatic Diseases, Bath, England William L. Stone, Ph.D. Department of Pediatric Research, East Tennessee State University, Johnson City, Tennessee, U.S.A. Marta A. Taddeo Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A.

xviii

Contributors

Shinya Toyokuni, M.D., Ph.D. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan C. Turner, B.Sc. Department of Clinical Neurosciences, Royal Free and University College Medical School, University College London, London, England Albert van der Vliet Vermont, U.S.A.

University of Vermont College of Medicine, Burlington,

Zbigniew Walaszek, Ph.D. Cancer Causation and Prevention, AMC Cancer Research Center, Denver, Colorado, U.S.A. Emma Wincent, M.Sc. Stockholm, Sweden

Institute of Environmental Medicine, Karolinska Institutet,

Junji Yodoi, M.D., Ph.D. Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, and Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, Ikeda, Japan Xiongwei Zhu, Ph.D. Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, U.S.A. Thomas R. Ziegler, M.D. Georgia, U.S.A.

Department of Medicine, Emory University, Atlanta,

1 Redox–Genome Interactions: Evolution of a Concept ¨ RGEN FUCHS, MAURIZIO PODDA, and R. KAUFMANN JU J.W. Goethe University, Frankfurt, Germany LESTER PACKER University of Southern California School of Pharmacy, Los Angeles, California, U.S.A.

Exciting new information in the ﬁeld of genomics and proteomics is currently revolutionizing our understanding of cell biology and is providing new insights into the pathophysiology of human diseases. At the same time, an exponentially increasing interest in redox modulation of cell function, including gene regulation and gene expression, is evolving. There is a rapid if not explosive growth in the identiﬁcation of signal transduction processes and regulatory elements found to be inﬂuenced by redox changes. The developments in these diﬀerent but connected research areas create a new global perspective of redox-genome biology. An introduction to and overview of this exciting ﬁeld of redox biology follows.

I.

FREE RADICALS AS BIOCHEMICAL ENTITIES AND DAMAGING SPECIES

Two recent reviews present excellent overviews of past and present free radical research in biology and medicine [1,2]. The ﬁrst discovery of solution phase reactive oxygen chemistry is attributed to Fenton, who studied iron/hydrogen peroxide chemistry [3], whereas Gomberg in 1900 demonstrated the existence of an organic free radical. Radiation chemistry studies, beginning in the early twentieth century, led in 1954 to the discovery that free radicals are the molecular basis for oxygen toxicity [4]. The work of Weiss in the 1930s on metal-catalyzed peroxide decomposition, particularly with 1

2

Fuchs et al.

Haber [5,6], and discoveries in radiation chemistry research had implications far beyond traditional free radical chemistry. Free radicals were accepted as chemical entities by biological scientists in the 1950s. Early studies focused mainly on free radicals bound to macromolecules, such as melanin radicals and protein-bound ﬂavin semiquinone radicals. Stanford Moore and William Stein were awarded the Nobel Prize in chemistry in 1972 for their contribution to the understanding of the connection between chemical structure and catalytic activity of the active free radical center of ribonuclease, which catalyzes production of deoxyribonucleotides for DNA synthesis. It is interesting to note that several decades were required before solution phase oxyradicals were widely accepted as credible biochemical entities [1]. The measurement of free radicals in biological samples was hampered for a long time due to restrictions in bioanalytical methodologies, thus the development of the electron paramagnetic resonance (EPR) spectrometer by Zavoisky in 1945 was a technological breakthrough [7]. EPR allowed detection of free radicals in complex biological specimens with high speciﬁcity and good sensitivity. In the 1950s pioneering researchers applied this technology to the study of melanin-bound radicals in biological material [8]. The development of spin traps in the late 1960s [9] allowed detection of short-lived free radical species, such as the hydroxyl and the superoxide anion radical, in biological samples. In the early nineteenth century, oxygen was recognized as the agent causing rancidiﬁation of polyunsaturated natural oils Following the discovery of vitamin E in 1922 [10], it was found that vitamin E inhibited autoxidation of fats in stored food [11]. Following this there was an increasing demand for prepacked food, leading researchers to focus on the role of lipid peroxidation in food racidiﬁcation. Radical chain reactions and antioxidants as radical chain–breaking agents had already attracted the interest of polymer scientists in the 1940s in their endeavor to prepare synthetic rubber and other polymers. In 1956 Harman postulated his theory on the role of free radicals in the aging process [12], and in 1958 the ﬁrst studies were originated addressing cellular redox biochemistry [13]. In 1969 superoxide dismutase (SOD) was discovered as an enzyme whose role was to detoxify superoxide anion radicals [14]. At this time free radicals were considered primarily as damaging species that attack lipids, proteins, and nucleic acids, disrupting the cellular homeostasis, ﬁnally causing disease. Initially researchers focused on lipid peroxidation (see, e.g., Ref. 15), measuring free radical damage of lipids by analyzing thiobarbituric acid–reactive substances, mainly because other methods for measurement of oxidative molecular damage in biological samples were not yet available. During the late 1980s the interest in oxidative damage in biological systems shifted to studies of protein and nucleic acids oxidation. DNA is an important target of oxidative damage, since it carries the genetic information and mutations will be carried on to future generations or will fundamentally change the behavior of the cells. Reactive oxidants can generate a variety of DNA lesions, including modiﬁed bases, abasic sites, single as well as double strand breaks, DNAprotein cross-links, deletions, and duplications, and if left unrepaired such damage represent potentially mutagenic lesions. II.

FREE RADICALS AS A CAUSE OR CONSEQUENCE OF CELL DAMAGE

It was recognized early that free radicals not only cause cell injury, but may also be a consequence of cell damage [16]. Most human diseases were suggested to be accompanied by oxidative stress [17], and this concept is still valid today [1,18]. In most cases,

Concept of Redox–Genome Interactions

3

oxidative stress, rather than being the primary cause of disease, is a secondary complication, but in some cases it has a signiﬁcant role in the pathophysiology. For instance, oxidative stress arising from exposure to environmental stressors such as irradiation or chemicals can be a major source of pathophysiological change leading to disease initiation and progression. Examples are diseases of the lung, eye, and skin where signiﬁcant involvement of free radical–mediated tissue injury is indicated. Such environmental diseases comprise, e.g., UV-induced cataract, toxic dermatitis caused by oxidizing chemicals, asbestosis, silicosis, and tobacco smoke– and diesel exhaust particle–associated health problems [19]. It is clearly evident that many genetic diseases, such as thalassemias, hereditary hemochromatosis, and cystic ﬁbrosis, are also accompanied by an imbalance in the cellular redox state (oxidative stress), which may have an inﬂuence on the disease phenotype. Research in this area is still in its infancy and mostly observational. III.

FREE RADICALS AS REGULATING SPECIES

As molecular and cell biology evolved from biochemistry, microbiology and molecular genetics in the 1980s to become a leading new discipline, an increasing interest in redox Table 1

Free Radicals as Chemical and Biological Entities

Discovery

Year

Ref.

Prediction and demonstration of oxygen toxicity

1775 1785 1822

Lavosier Priestly Saussurer

1876 1900 1922 1931 1934 1941 1945 1954

3 Gomberg 10 5 6 11 7 4

1954

8

1956 1958 1968 1969

12 13 10 15

1969

14

1985 1987

37 20

1990 1991

21 24

Oxygen is recognized as the agent causing rancidiﬁcation of polyunsaturated natural oils Discovery of solution phase ROS chemistry First report of a free radical (triphenylmethyl) Discovery of vitamin E Suggestion of the existence of hydroxyl radicals Vitamin E inhibits fat autoxidation Development of the EPR spectrometer Oxygen free radicals mediate the toxic eﬀects of ionizing radiation Detection of melanin radicals in biological material by EPR spectroscopy Free radical theory of aging Studies on cellular redox biochemistry Free radical spin traps Discovery of SOD1 as an enzyme that detoxiﬁes superoxide anion radicals Concept of biological rancidiﬁcation suggesting that lipid peroxidation is involved in key pathophysiological processes Concept of oxidative stress Discovery of NO and its identiﬁcation as the endothelium-derived relaxing factor Discovery of OxyR and SoxyR Reactive oxidants activate transcription factor NF-nB

4

Fuchs et al.

modulation of signal transduction and gene expression was developed. Several independent discoveries contributed to the evolution of this new area. For instance, nitric oxide (NO) was identiﬁed as the endothelium-derived relaxing factor in 1987 [20], and it was recognized that the free radical gas NO was a key mediator in control of vascular function. In 1998 Furchgott, Ignarro, and Murad were awarded the Nobel Prize for the discovery of NO as a signaling molecule. In the late 1980s SoxR and OxyR were identiﬁed as redox-responsive transcription regulators in Escherichia coli [21], which now serves as a model of redox-operated genetic switches [22,23]. This was followed by the discovery of NF-nB as a redox-sensitive transcription factor in mammalian cells [24]. In the 1980s Fischer and Krebs discovered that phosphorylation and dephosphorylation reactions of proteins at tyrosine and serine/threonine mediate signal transduction from the cell surface to the nucleus; their work was awarded a Nobel Prize in 1992. It was later recognized that several kinases and phosphatases are redox sensitive (reviewed in Refs. 25, 26). These developments contributed to a paradigm shift in free radical research in biomedicine. A dualistic concept evolved postulating that reactive oxidants produced at high levels are damaging species but, when produced at low levels, they act as second messengers in numerous signal transduction pathways and gene expression systems. In contrast, the genome regulates antioxidant defense systems and their interactions and the intrinsic or constitutive level of reactive oxidants. It is now believed that redox-based regulation of signal transduction pathways and gene expression represents a fundamental cellular regulatory mechanism (see, e.g., Refs. 25–33). Table 1 presents a chronological list of important discoveries in free radical research. IV.

OXIDATIVE STRESS AND THE REDOX STATE

Oxidative stress, a term coined by Helmut Sies in the 1980s, is deﬁned as a disturbance in the prooxidant-antioxidant balance in favor of the former [34]. Oxidative stress can result from one- or two-electron processes, and both reactions can trigger redox cascades. Although changes in the redox state and oxidative stress are closely interconnected, one- or two-electron transfer reactions may occur without any net redox change. It should be pointed out that most redox reactions in biology are two-electron processes, and oxidative stress can result from nonradical processes. According to a deﬁnition given by Schafer and Buettner, the cellular redox state is determined by the half-cell reduction potential (voltage of the relevant redox couples) and the reducing capacity (concentration of the reduced species of the relevant redox couples) of all redox-linked couples present in the compartment [35]. Since it is diﬃcult to identify and quantitate all linked redox couples present in a compartment of interest, a representative redox couple is often used as indicator of the redox state. The redox systems PSSP/PSH (protein thiols) or PSSG/PSH (mixed thiols), NADP+/NADPH, GSSG/GSH (glutathione disulﬁde/glutathione), TrxSS/Trx(SH)2 (thioredoxin), and ascorbate/dehydroascorbate represent the most important interlinked redox couples of the cell. The concentration of PSH in cells is much greater than that of GSH, intracellular GSH concentrations vary from 1 to 10 mM, while NADPH is around 0.1– 0.5 mM, Trx(SH)2 levels in the range of 1–10 AM, and ascorbate levels are about 0.1–2 mM. The GSH/GSSG and the NADPH/NADP+ couple are well-deﬁned pools of the cellular reducing equivalents and are frequently used as surrogate markers of the redox state. The exact measurement of the overall redox state in biological samples is diﬃcult

Concept of Redox–Genome Interactions

5

to achieve, and many diﬀerent approaches to its evaluation have been developed [36,37]. Cyclic voltammetry, which is based on the polographic measurement of electron transfer between molecules, provides information concerning the overall behavior of the antioxidant activity derived from low molecular weight water- and lipidsoluble antioxidants [38]. It is interesting to note that Jaroslav Heyrovsky was awarded the Nobel Prize in chemistry in 1959 for his discovery and development of the polarographic methods of analysis. V.

QUANTITATION, SPECIFICITY, AND COMPARTMENTATION

The facts that (1) an enzyme with a free radical in its active center is intimately involved in DNA biosynthesis, (2) free radicals can damage DNA and cause potentially mutagenic lesions, (3) DNA repair responds to regulation by redox reactions [39], and (4) free radicals and the redox state are involved in physiological regulation of gene expression may give rise to speculations about the overall signiﬁcance of free radicalredox-genome interactions. The quantitative importance of redox regulation is unknown, and as yet there are no examples of regulation exclusively by oxidative stress. Also, the response to oxidative stress is variable, can be upregulated or downregulated, and diﬀerent responses to dose or magnitude of oxidative stress can be demonstrated [40]. Furthermore, the relative contribution of individual redox-sensitive signaling proteins to redox-regulated processes in vivo is presently unclear [26]. Cell proliferation, diﬀerentiation, senescence, apoptosis, and necrosis can be modulated by the redox status, depending on oxidant concentration and cell type. It was recently shown that changes of the half-cell reduction potential E(hc) of the GSSG/GSH couple appear to correlate with the biological status of the cell: proliferation E(hc) approximately 240 mV, diﬀerentiation E(hc) approximately 200 mV, or apoptosis E(hc) approximately 170 mV [38]. Cells are not homogeneous, and various redox compartments exist in the nucleus, mitochondria, peroxisomes, and endoplasmic reticulum. Compartmentation of redox changes is a key principle in the control of cell signaling [41]. VI.

DEVELOPMENTS IN MOLECULAR GENETICS

The chronology of the developments in molecular genetics reveals a fascinating story of scientiﬁc discoveries, many of which were recognized by Nobel Prizes in medicine or physiology or chemistry. In the 1950s experiments showed that bacterial recombination (splicing ‘‘foreign’’ DNA into ‘‘own’’ DNA) is possible in nature, and researchers began to perform similar recombination experiments in the laboratory. Gradually this led to development of recombinant DNA technology, pioneered by Arber, Smith, and Nathan’s discovery of restriction enzymes and their application to molecular genetics. After recombinant DNA technology was established in the mid- and late 1970s as a standard molecular tool, the DNA molecule was transformed from the most diﬃcult to the easiest to study. In 1983 the ﬁrst transgenic mouse, carrying the gene for rat growth hormone in its cells, was generated. This was soon followed in 1988 by the generation of a mouse with severe combined immunodeﬁciency and a cancer-prone transgenic mouse in 1987 yeast artiﬁcial chromosomes were introduced by Olson, and in 1992 megaYAC (unusually large chromosomes) was introduced by Cohen. Thus it became possible to clone large segments of genomic DNA, which signiﬁcantly im-

6

Fuchs et al.

Table 2

Milestones in Molecular Genetics

Discovery Discovery that DNA is the genetic material Determination of the DNA structure Identiﬁcation of the ﬁrst human chromosome abnormality (Down syndrome) Discovery that DNA replication is mediated by DNA polymerases Identiﬁcation of the ﬁrst of the 64 three-letter genetic codes for proteins Foundation of recombinant DNA technology Discovery of restriction enzymes and their application in DNA sequence analysis and DNA cloning Southern blot Construction of recombinant DNA molecules Fast DNA sequencing methods Development of genetic ﬁngerprinting using DNA to positively identify individuals Discovery of the PCR reaction Transgenic and knockout mice Yeast artiﬁcial chromosomes (YAC) Invention of the ﬁrst automatic DNA sequencer Human genome project is launched megaYAC Researchers at George Washington University clone the ﬁrst human embryos and nurture them in vitro for several days Development of DNA and protein arrays First cloned sheep ‘‘Dolly’’ is born Human genome sequenced

Year

Reference

1944 1953 1959

42 43

1960

44

1961 1960s–1970s 1975

45–46 47–48

1975 1977 1977 1984

49 50 51–52

1986 1983, 1988 1987 1986 1990 1992 1993

53 54–56 57

58

1990s 1996 2003

proved human gene mapping. In 1986 the discovery of the polymerase chain reaction (PCR) by Mullis and colleaques allowed researchers to produce a billion identical copies of a single strand of DNA in a few hours, and newer technologies undoubtedly will allow reduction of the ampliﬁcation time to the order of a few minutes. Tables 2 and 3 list selected historical milestones and Nobel Prizes awarded for discoveries in molecular genetics and related ﬁelds. VII.

THE MOLECULAR GENETIC TOOLBOX AND REDOX BIOLOGY

The tools for studying oxidative stress responses are now expanding as a result of the human genome development, which led to completion of the sequencing of the human genome in 2003. In the 1990s development of DNA microarrays and protein chips made large-scale genomic and proteomic investigations possible. DNA microarrays can measure the expression of thousands of genes simultaneously, providing extensive information on gene interactions and cell function. Thus, microarrays can monitor the global proﬁle of gene expression in response to food, dietary supplements, and phar-

Concept of Redox–Genome Interactions

Table 3

7

Selected Nobel Prices in Medicine and Physiology, Chemistry

Discovery Chromosomes as carriers of genetic information Genes are eﬀective via regulation of biochemical process Mechanism of RNA and DNA biosynthesis Discovery of the molecular structure of DNA Discovery of the genetic control of enzymes and viruses Interpretation of the genetic code and its function for protein synthesis Genetic structure of viruses and mechanism of virus replication Mechanism of interaction of oncogenic viruses and the host genome Discovery of restriction enzymes and their application in molecular genetics Hybrid DNA technology Fast nucleic acid sequencing Discovery of mobile genetic elements (controlling elements) Catalytic properties of RNA Discovery of the polymerase chain reaction Genetic regulation of organ development and apoptosis

Year of award

Nobel prize recipients

1933

Morgan

1957

Beadle + Tatum

1959

Kornberg + Ochoa

1962

Crick + Watson + Wilkins Jacob + Lwoﬀ + Monod

1966 1968 1969 1975 1978 1980 1980 1983 1989 1993 2002

Nirnberg + Khorana + Holley Delbru¨ck + Hershey + Luria Baltimore + Dulbecco + Temin Arber + Nathans + Smith Berg Gilbert + Sanger McClintock Altman + Cech Mullis Brenner + Horvitz + Sulston

macological agents. This technology is improving our understanding of the complex interactions of micronutrients, xenobiotics, and antioxidant networks and their eﬀects on gene expression. DNA microarrays have been successfully used to identify redoxsensitive elements controlling key cellular biochemical pathways [59]. The genetic control elements that coordinate cell responses to oxidative stress and regulate adaptation to externally induced changes in the redox state are currently under investigation [60,61]. Genetic variants of relevance to oxidative stress are studied by a molecular epidemiological approach [62], and large-scale studies using genomic techniques will help to evaluate the impact of natural genetic variation on disease variability. Recombinant DNA technology has already turned out to be important in the analysis of antioxidant gentopye/phenotype relationship. Numerous gene knockout and overexpression mouse models have been generated. Transgenic and knockout mice over-expressing or totally lacking certain antioxidants reveal functional abnormalities [63,64]. This has led to the understanding that there is a highly sophisticated interrelationship between dietary, constitutive, and inducible antioxidant systems in the body that are under genetic control. The complex interactions of the cellular antioxidant network under environmental and genetic control partially explains why many clinical trials and intervention studies using antioxidants as a ‘‘magic bullet’’

8

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against free radical–associated diseases are often inconclusive, presumably because the body will adapt to maintain redox balance, or because of problems to selectively modulate cell redox microenvironments. A better understanding of cellular redox regulatory mechanisms, the interconnection of spatially diﬀerent redox microenvironments, and their genetic control may help to reveal new approaches to selectively manipulate the redox state at target sites. In the postgenomic area, proteomic techniques are also rapidly expanding our knowledge and understanding of cellular redox regulation. Formation of glutathionylated proteins and the reversible reduction of protein disulﬁde bonds are important mechanisms through which protein functions can be regulated by the redox status. Such post-translational modiﬁcations of proteins can readily be studied by proteomic techniques [65–67]. With access to whole human genome sequences and availibity of new and emerging genomic and proteomic technologies, the entire process of discovery in redox biology is poised to change. New strategies promise to revolutionize how we approach and ﬁnally understand the complex biochemical circuitry responsible for controlling physiological redox homeostasis and disease processes. Hopefully these developments will lead to a comprehensive mechanistic understanding of normal and abnormal human redox physiology at the molecular level and result ﬁnally in improvements in patient management.

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16. Dormandy TL. Free radical reactions in biological systems. Ann R Coll Surg Engl 1980; 62:188–194. 17. Halliwell B, Gutterridge JMC. Lipid peroxidation, oxygen radicals, cell damage and anti-oxidant therapy. Lancet 1984; 1396–1398. 18. Halliwell B. Free radical reactions in human disease. In: Fuchs J, Packer L, eds. Environmental Stressors in Health and Disease. New York: Marcel Dekker Inc., 2001:1–16. 19. Fuchs J, Packer L, eds. Environmental Stressors in Health and Disease. New York: Marcel Dekker, 2001. 20. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327:524–526. 21. Storz G, Tartaglia LA, Ames BN. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science 1990; 248:189–194. 22. Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol 2001; 19:109–114. 23. Kim SO, Merchant K, Nudelman R, Beyer WF Jr, Keng T, DeAngelo J, Hausladen A, Stamler JS. OxyR: a molecular code for redox related signaling. Cell 2002; 109:383–396. 24. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 1991; 10:2247–2258. 25. Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radic Biol Med 2000; 28:463–499. 26. Dro¨ge W. Free radicals in the physiological control of cell function. Physiol Rev 2002; 82:47–95. 27. Dalton TPD, Shertzer HG, Puga A. Regulation of gene expression by reactive oxygen. Ann Rev Pharmacol Toxicol 1999; 39:67–101. 28. Arrigo AP. Gene expression and the thiol redox state. Free Radic Biol Med 1999; 27:936–944. 29. Kamata H, Hirata H. Redox regulation of cellular signaling. Cell Signal 1999; 11:1–14. 30. Marshall HE, Merchant K, Stamler JS. Nitrosation and oxidation in the regulation of gene expression. FASEB J 2000; 14:1889–1900. 31. Pfeilschifter J, Eberhardt W, Beck KF. Regulation of gene expression by nitric oxide. Pﬂu¨gers Arch Eur J Physiol 2001; 442:479–486. 32. Moran LK, Gutteridge JM, Quinlan GJ. Thiols in cellular redox signaling and control. Curr Med Chem 2001; 8:763–772. 33. Peterson RM. Redox signal transduction. J Lab Clin Med 2002; 140:73–78. 34. Sies H. Oxidative Stress: Introductory remarks. In: Sies H, ed. Oxidative Stress. London: Academic Press, 1985:1–8. 35. Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulﬁde/glutathione couple. Free Radic Biol Med 2001; 30:1191– 1212. 36. Halliwell B, Gutteridge JCM. The deﬁnition and measurement of antioxidants in biological systems. Free Radic Biol Med 1995; 18:125–126. 37. Pryor RL, Cao G. In vivo total antioxidant capacity: comparison of diﬀerent analytical methods. Free Radic Biol Med 1999; 27:1173–1181. 38. Kohen R, Vellaichmay E, Hrbac J, Gati I, Tirosh O. Quantiﬁcation of the overall ROS scavenging capacity of biological ﬂuids and tissues. Free Radic Biol Med 2000; 28:871– 879. 39. Lunec J, Holloway K, Cooke M, Faux S, Griﬃths H, Evans M. Urinary 8-oxo-2Vdeoxyguanosine: redox regulation of DNA repair in vivo? Free Radic Biol Med 2002; 33:875–881. 40. Poulsen HE, Jensen BR, Weimann A, Jensen SA, Sorensen M, Loft S. Antioxidants, DNA damage and gene expression. Free Radic Res 2000; 33(suppl):S33–S39.

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41. Pani G, Bedogni B, Colavitti R, Anzevino R, Borrello S, Galeotti T. Cell compartmentalization in redox signaling. IUBMB 2001; 52:7–16. 42. Avery OT, Mac Leod CM, McCarthy M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. J Exp Med 1944; 79:137–158. 43. Watson JD, Crick DHC. Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 1953; 171:737–738. 44. Kornberg A. The biologic synthesis of deoxyribonucleic acid. Science 1960; 131:1503– 1508. 45. Arber W, Linn S. DNA modiﬁcation and restriction. Ann Rev Biochem 1969; 38:467–500. 46. Boyer HW. DNA restriction and modiﬁcation mechanisms in bacteria. Ann Rev Microbiol 1971; 25:153–176. 47. Nathans D, Smith HO. Restriction endonucleases in the analysis and restructuring of DNA molecules. Ann Rev Biochem 1975; 44:273–293. 48. Cohen SN. The manipulation of genes. Sci Am 1975; 233:24–33. 49. Southern EM. Detection of speciﬁc sequences among DNA fragments seperated by gel electrophoresis. J Mol Biol 1975; 98:503–517. 50. Villarreal LP, Berg P. Hybridization in situ of SV40 plaques: detection of recombinant SV40 virus carrying speciﬁc sequences of nonviral DNA. Science 1977; 196:183–185. 51. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467. 52. Maxam AM, Gilbert WA. A new method for sequencing DNA. Proc Natl Acad Sci USA 1977; 74:560–564. 53. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically ampliﬁed beta-globin and HLA-DQ alpha DNA with allele-speciﬁc oligonucleotide probes. Nature 1986; 324:163–166. 54. Palmiter RD, Norstedt G, Gelinas RE, Hammer RE, Brinster RL. Metallothioneinhuman GH fusion genes stimulate growth of mice. Science 1983; 222:809–814. 55. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, Weissman IL. The SCID-hu mouse: murine model for the analysis of human hematolymphoid diﬀerentiation and function. Science 1988; 241:1632–1639. 56. Schmidt EV, Pattengale PK, Weir L, Leder P. Transgenic mice bearing the human c-myc gene activated by an immunoglobulin enhancer: a pre-B-cell lymphoma model. Proc Natl Acad Sci USA 1998; 85:6047–6051. 57. Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artiﬁcial chromosome vectors. Science 1987; 236:806–812. 58. Dausset J, Ougen P, Abderrahim H, Billault A, Sambucy JL, Cohen D, Le Paslier D. The CEPH YAC library. Behring Inst Mitt 1992; 91:13–20. 59. Voehringer DW, Hirschberg DL, Xiao J, Lu Q, Roederer M, Lock CB, Herzenberg LA, Steinman L, Herzenberg LA. Gene microarray identiﬁcation of redox and mitochondrial elements that control resistance or sensitivity to apoptosis. Proc Natl Acad Sci USA 2000; 97:2680–2685. 60. Wang XL, Rainwater DL, VandeBerg JF, Mitchell BD, Mahaney MC. Genetic contributions to plasma total antioxidant activity. Arterioscler Thromb Vasc Biol 2001; 121:1190–1195. 61. Dusinska M, Ficek A, Horska A, Raslova K, Petrovska H, Vallova B, Drlickova M, Wood SG, Stupakova A, Gasparovic J, Bobek P, Nagyova A, Kovacikova Z, Blazicek P, Liegebel U, Collins AR. Glutathione S-transferase polymorphisms inﬂuence the level of oxidative DNA damage and antioxidant protection in humans. Mutat Res 2001; 482:47–55. 62. Forsberg L, deFaire U, Morgenstern R. Oxidative stress, human genetic variation, and disease. Arch Biochem Biophys 2001; 389:84–93.

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63. Tsan MF. Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice. Int J Mol Med 2001; 7:13–19. 64. Huang TT, Carlson EJ, Raineri I, Gillespie AM, Kozy H, Epstein CJ. The use of transgenic and mutant mice to study oxygen free radical metabolism. Ann NY Acad Sci 1999; 893:95–112. 65. Fratelli M, Demol H, Puype M, Casagrande S, Eberini I, Salmona M, Bonetto V, Mengozzi M, Duﬃeux F, Miclet E, Bachi A, Vandekerckhove J, Gianazza E, Ghezzi P. Identiﬁcation of redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci USA 2002; 99:3505–3510. 66. MacCoss MJ, McDonald WH, Saraf A, Sadygov R, Clark JM, Tasto JJ, Gould KL, Wolters D, Washburn M, Weiss A, Clark JI, Yates JR III. Shotgun identiﬁcation of protein modiﬁcations from protein complexes and lens tissue. Proc Natl Acad Sci USA 2002; 99:7900–7905. 67. Yano H, Kuroda S, Buchanan BB. Disulﬁde proteome in the analysis of protein function and structure. Proteomics 2002; 2:1090–1096.

2 Measurements of Biological Reducing Power by Voltammetric Methods RON KOHEN, EITAN MOOR, and MIRIAM ORON The Hebrew University of Jerusalem, Jerusalem, Israel

I.

BACKGROUND AND DEFINITIONS

Redox is the balance between reducing equivalents and oxidants, which is extremely important to the living cell. It is kept under tight regulation and is responsible for controlling many biochemical pathways and cellular events [1]. Reducing equivalents are deﬁned by their ability to donate electrons. Reducing agents or reductants donate electrons to the acceptor molecule, the oxidant. In a reduction process of a chemical, electrons are added to another chemical compound. In an oxidation reaction there is a loss of electrons. An oxidant is a compound that accepts electrons and causes the other compound to be oxidized and, thus, to serve as a reductant. An oxidation process is impossible without a reduction process in the system [2,3]. These compounds— reductants and oxidants—play a major role in many biochemical pathways, cellular chemistry and biosynthesis, and in cellular regulation. They are also important players in the cellular defense mechanism [4]. While the terms reductant and oxidant are used in the chemical sciences, their biological equivalents have diﬀerent names [2]. The term antioxidant has the same meaning as reducing agent or reductant, and the term prooxidant replaces the chemical term oxidant. Halliwell [5] suggested a deﬁnition for antioxidant which states that this agent signiﬁcantly prevents or delays oxidation when present in low concentrations. Prior and Cao [2] suggested that a prooxidant can be deﬁned as a toxic substance that can cause oxidative damage to lipids, proteins, and nucleic acids. Chemically, a prooxidant is an oxidant of pathological importance. There are many reducing agents in the cell, all contributing to the reducing power of the cell [6]. The reducing power is a parameter representing the overall ability of the cell or biological ﬂuids and tissue to donate electrons to chemicals (radicals) or to 13

14

Kohen et al.

biological components. The reducing power is composed of two parameters: the ability (capacity) to donate electrons and the overall concentration of the reducing equivalents responsible for this quality [7]. The redox potential is a thermodynamic parameter (see below) and cannot be determined in biological environments. The reducing power or biological reducing power (BRP) parameter, on the other hand, is not a thermodynamic parameter [4]. Nevertheless, it can supply valuable information concerning cellular response to oxidative stress. The redox potential exists only in equilibrium and in a reversible system [1,3,4]: k aRed1 þ bOx2 ! cOx1 þ dRed2 c

d

a

k ¼ ½Ox1 ½Red2 =½Red1 ½Ox2

ð1Þ

b

ð2Þ c

d

a

E ¼ E RT=nFlnk ¼ E RT=nFln½Ox1 ½Red2 =½Red1 ½Ox2 o

o

b

ð3Þ

where R (gas constant) = 8.314 JK1 mol1 T = temperature in Kelvin F (Faraday constant) = 9.648 104 cmol1 As mentioned above, the redox potential can be deﬁned as the ratio between oxidant and reductant [Eqs. (1)–(3)] and can be calculated according to the Nernst equation [Eq. (3)] [8]. Its value can be calculated only under appropriate conditions in reversible systems where all the factors aﬀecting the system are known and can be controlled. Therefore, in biological systems, where equilibrium does not exist and the systems are not fully reversible and the various factors involved are diﬃcult to control, the redox potential does not ﬁt its ‘‘classical’’ deﬁnition but rather is a steady-state approximation. For this reasons, the terms ‘‘redox status’’ or ‘‘redox state’’ are preferred over the term ‘‘biological redox potential’’ [1,9,–11]. Biological sites contain a large variety of electrochemical couples, all contributing to the biological redox status as well as to the BRP. Therefore, in quantifying biological redox values, the existence of multiple compounds must be considered [3]. The redox status can be described as an intensive quantity characterizing the system in terms of a balance between the oxidized and reduced forms of the redox active species present in the system. The corresponding extensive quantity is composed of the concentrations of those species in the system. An alternative term describing redox in biological locations is ‘‘redox environment,’’ as suggested by Schafer and Buettner [3]. They suggested that this parameter is a total of the products of the reduction potential and reducing capacity of all the redox couples found in biological ﬂuids, organelle, cell, or tissue. The redox state of a biological system is kept within a narrow range under normal conditions. However, under pathological conditions the redox state can be changed towards lower (redosis) or higher (oxidosis) values [1]. A 30 mV shift in redox status means a 10-fold change in the ratio between reductants and oxidants. A.

Redox Potential Versus Reducing Power

Measurement of the redox state in the biological environment may be extremely diﬃcult to achieve. Although the importance of this parameter in controlling biochemical events (e.g., apoptosis [12] cell cycle [13], signal transduction pathways [14] gene expression [12,15,16]) is well known, the determination of its exact value is diﬃcult to obtain. There are many diﬀerent approaches for its evaluation, including

Measurements of Biological Reducing Power by Voltammetric Methods

15

direct measurement by redox electrodes [17] as well as indirect methods based on ﬂuorescence of speciﬁc dyes [18], determination of the exact concentration of cellular electrochemical couples (e.g., GSH/GSSG, NAD+/NADH) and introduction of their values into the Nernst equation [3,19]. However, no satisfactory method has been developed to indicate the biological redox status within the biological site. The reasons for this are, in part, due to the nonthermodynamic conditions present in biological sites, as mentioned above, and the complexity and heterogeneity of biological electrochemical couples. Furthermore, the measurements usually reﬂect only the dominant electrochemical couple surrounding the measurement site and not necessarily the real redox value of the entire biological location. The action of the dominant redox couple is often termed ‘‘redox buﬀering.’’ In contrast, the biological reducing power is a parameter that can be determined in ‘‘normal’’ biological environments that are not in equilibrium and not reversible. This parameter is composed of a variety of reducing agents all contributing to the overall reducing power. This parameter also undergoes dynamic changes due to the biosynthesis, accumulation, mobilization, and degradation of these compounds and their interaction with other cellular components [19,20]. Schafer and Buettner [3] distinguished between the reduction potential and reducing capacity (reducing power) of cells by describing the reduction potential as voltage and the reducing capacity as the total charge stored. Measurement of the reducing power can be achieved by determination of all the diﬀerent reducing compounds in the cell using advanced laboratory techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) or using chemical methods (e.g., dichlorphenolindophenol) or electrochemical methods (e.g., voltammetric measurements). We have found voltammetry to be a reliable and reproducible method for evaluating the overall BRP and determining the two parameters (the nature of the reducing equivalents and their overall concentration) responsible for the cellular reducing power. In the following sections [2–4] we discuss in detail the advantages of the voltammetric measurements. B.

Compounds Contributing to the Overall Biological Reducing Power

Living cells possess a strong reducing capacity that originates from a variety of reducing agents including high-molecular-weight compounds and low-molecular-weight compounds. Athough there are numerous compounds possessing reducing properties, not all of them are responsible for the reducing power measured. Compounds present in low concentrations below 10 nm are not considered here as compounds responsible for the reducing power in the cellular environment, although they may play a role in speciﬁc microcellular environments. The major players in creating reducing power in cellular and biological environments are summarized in Table 1 [1]. Various electrochemical couples possess their own characteristic redox potential and may be responsible for direct activity in many cellular functions. In addition, they all contribute to the total cellular ability to donate electrons to their surrounding. C.

Low-Molecular-Weight Antioxidants Are Major Constituents of the BRP

As described above, a reducing agent by deﬁnition is a compound capable of donating electrons to an acceptor molecule. A group of compounds in the living cell that possess

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Table 1 Standard Redox Potentials (Ej) for Some Redox Systems of Biological Interest Redox system H2O/0.5 O2 Cyt c (Fe+2/Fe+3) Hemoglobin/methemoglobin Ubiquinone red/ox Ascorbate/dehydroascorbate Lactate/pyruvate Cysteine/0.5 cystine GSH/GSSG Lipoate red/oxide NADH + H+/NAD+

Ej (volts) 0.82 0.22 0.17 0.10 0.08 0.21 0.22 0.24 0.29 0.32

this ability is the group of low molecular weight antioxidants (LMWA) [20–23]. This is a subgroup of the overall antioxidant defense system (enzymes and LMWA). The LMWA contain a large number of compounds capable of preventing oxidative damage by direct interaction with reactive oxygen species (ROS). These agents, also called scavengers, share a similar chemical trait that allows them to donate an electron to the reactive oxygen radical [21–23]. This scavenging mechanism can proceed only if the concentration of the scavenger is suﬃciently high to compete with the biolog-

Table 2

Low-Molecular-Weight Antioxidants in Biological Environments

Antioxidant Ascorbic acid Uric acid Tocopherols Carnosine, anserine, homocarnosine Glutathione NADH NAD(P)H Lipoic acid (ox) Lipoic acid (red) Carotenoids Thiol proteins Ubiquinol 10 Lycopene Carotene

Concentration in human blood plasma (AM) 30–150 160–450 15–40 V20,000 (in skeletal muscle, in brain) 0.9–2.8 0.002 (Amol in 1 mL of red blood cells) 0.032 (Amol in 1 mL of red blood cells) 0.005– 0.12 0.16–0.7 0.3–0.6 400–600 0.4–1.0 0.5–1.0 0.3–0.6

Comments WS, DS WS, WP LS, DS WS, ES (family of histidine dipeptides) WS, ES WS, ES WS, ES LS, DS WS, ES LS, DS WS LS LS LS

WS, water soluble; DS, dietary sources; WP, waste product; LS, lipid soluble; ES, endogenous source.

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17

ical target for the deleterious species. Table 2 shows a few examples of biological scavengers. Theoretically, every reducing agent in the cell is potentially an antioxidant [4,24]. However, in biological environments this is not always the case [2]. It can be stated that every LMWA that reacts directly with the ROS is a reductant.

II.

METHODS FOR EVALUATION OF BIOLOGICAL REDUCING POWER

Due to the diﬃculties in measuring each reducing antioxidant component separately and the interactions among diﬀerent antioxidant components, several methods have been developed to assess the total reducing capacity of biological tissues and ﬂuids. These methods consist of two general categories: direct and indirect approaches [2]. Direct methods for measuring reducing capacity in biological tissues and ﬂuids are those that use an external probe as a measure of the reducing or oxidizing capacity of a system. An example of a direct method is an electrode at which the current is proportional to the concentrations of the species present. Indirect methods are those that measure consequential factors of redox capacity, such as oxidation products formed, or concentrations of major redox couples in the biological environment, such as glutathione/glutathione disulﬁde. Within this category, a few techniques are available whereby free radicals are produced at a known rate, and their removal by scavenger, which is monitored by an endpoint [2,25]. It must be understood that each method measures diﬀerent factors using various technologies; therefore, diﬀerent results for identical samples should be expected. The results are then given as a measure of the speciﬁc factor of a certain method and not as a deﬁnite intrinsic parameter. A.

Indirect Methods for Measuring Reducing Power in Biological Tissue and Fluids

Indirect methods include those that measure the concentration of a speciﬁc redox couple in the biological tissue/ﬂuid using ﬂuorescent or spectrophotometric techniques. In this approach it is assumed that a biological redox buﬀer exists in the form of a redox couple that is sensitive to changes in the redox environment and thus reﬂects changes in reducing power. The concentration of a redox couple (its reduced and oxidized forms) reﬂects the reducing capacity of the sample but is not, by deﬁnition, the reducing power, which is measured by other methods, such as voltammetry. Other methods for measuring reducing power are inhibition methods that consist of adding a radical species to the sample together with a scavenger that can be detected due to its optical properties. 1.

NADP/NADPH

The redox state of the cell was deﬁned by the concentration ratio of free NAD+/ NADH2 [3]. Due to the fact that classical enzymatic techniques fail to distinguish between free and protein-bound nucleotides, this ratio cannot be obtained by direct measurement of free NAD+ and NADH2 in tissue [26]. The NAD+/NADH2 ratio is calculated using the concentrations of other linked redox couples that interact with the NAD+/NADH2 at a known equilibrium constant, such a lactate/pyruvate [26].

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Kohen et al.

Lactate/Pyruvate

The widest clinically used method for measuring tissue redox state is the lactate/ pyruvate redox sytem [1]. Analyses of lactate and pyruvate are carried out by extraction and spectrophotometry [27]. In human blood, concentrations of the lactate/pyruvate couple are 300–1300 and 30–70 mM, respectively [1]. Due to its ability to permeate cell walls, coronary venous lactate/pyruvate was postulated to reﬂect the NAD/NADH ratio in cells, but this is not always the case [28,29]. The lactate/pyruvate ratio remains extraordinarily constant, even when both lactate and pyruvate are raised above normal levels [29]. For this reason this redox couple was considered a redox buﬀer in blood and to some extent in tissue. Though unaﬀected by nutritional factors, the lactate/pyruvate ratio varies as a result of muscular work (excess lactate) [29]. 3.

Glutathione/Glutathione Disulfide

Glutathione (GSH) is the major free thiol in most living cells and participates in major biological processes. Oxidation of GSH results in glutathione disulﬁde (GSSG). Intracellular GSH is eﬀectively maintained in the reduced state by GSSG reductase. GSH and GSSG have been considered as the redox buﬀer of the living cell and tissue, and their absolute and relative concentrations a measure of the system’s redox state [1,19]. There are many methods for the measurement of GSH. The most common consists of complexation of a substance with GSH, which results in a product/complex that can be detected by spectrophotometry or ﬂuorescence. Examples of complexating agents are methylglyoxal [30] and ortho-phthaldialdehyde [30]. Some methods involve redox reaction with a reactant so that it or its product can be detected by spectrophotometry or ﬂuorescence. Examples are NADPH in presence of GSSG reductase [30] or NADPH and DTNB [5,5V-dithiobis (2-nitrobenzoic acid)] [30,31]. For HPLC analysis of GSH and GSSG, derivatization of thiol compounds with ﬂuorescent labeling agents such as monobromobimane [30] or 2,4-dinitroﬂuorobenzene [32] is necessary, although direct detection using an electrochemical detector is possible. 4.

Ascorbic Acid/Ascorbate in Blood Plasma

Table 2 shows the major antioxidants found in blood plasma. Since the concentration of ascorbic acid in human blood plasma is relatively high compared to other antioxidants, it has been considered a potential blood buﬀer, and a variety of assays for blood ascorbic acid exist [33]. The most widely used method for measuring both uric acid and ascorbic acid is paired ion, reversed phase HPLC with an electrochemical detector [34]. Most other antioxidants in blood plasma are measured using HPLC as well, with either spectrophotometric, ﬂuorescent, or electrochemical detectors. However, in contrast to GSH or NADH, ascorbic acid is an antioxidant supplied by a dietary source. Therefore, the BRP in blood, as measured by this method, will be inﬂuenced by dietary factors as well as by changes in redox active components. 5.

Trolox Equivalent Antioxidant Capacity

Trolox equivalent antioxidant capacity (TEAC) [34,35] is a measure of 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E mimic) antioxidant equivalents. The sample is placed in a solution with 2,2V-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H2O2, which generate ABTS

Measurements of Biological Reducing Power by Voltammetric Methods

19

cation radicals, in the presence of metmyoglobin as a peroxidase. The decay in ABTS cation radicals absorbance as a result of quenching by antioxidants in the sample is monitored in the absence and the presence of Trolox. Only a lag phase or inhibition percentage at a ﬁxed time point can be quantiﬁed as the result. The direct interaction of an added sample of antioxidants with H2O2 cannot be excluded as the molar ratios of H2O2:metmioglobin:ABTS:Trolox are 10.2:0.25:25:1. Direct interaction between H2O2 and ABTS cation radical should also be taken into account. The TEAC results for blood plasma are not linear with concentration of sample [36]. 6.

Total Radical Trapping Potential

The total radical trapping potential (TRAP) [2] has been widely used during the last decade and is based on O2 measurement with an O2 electrode. 2,2V-Azobis(2amidinopropane) dihydrochloride (AAPH) that undergoes spontaneous decomposition and produces peroxyl radicals at a temperature-dependent rate [37], is added to the sample, and the O2 consumed is monitored both in the absence and the presence of Trolox. This method is based on the assumption that all damage resulting from the attack of the peroxyl radicals involves O2 consumption. As this is not always the case, the TRAP assay result cannot be considered as a measure of the BRP. Furthermore, the use of the O2 electrode, which is not suﬃciently stable during prolonged measurement, sheds doubt on the value of this particular measurement of peroxyl radicals damage control [35]. 7.

Chemiluminescence

The chemiluminescence method [2,38] is based on the reaction of AAPH radicals with Luminol to produce a cation radical, which is monitored by chemiluminescence. Like previous methods discussed in this category, the sample capacity to inhibit the luminescence of luminol is measured relative to Trolox. The lag time is proportional to the total antioxidants present in the sample. 8.

Oxygen Radical Absorbance Capacity

Oxygen radical absorbance capacity (ORAC) [2] is based on the ﬂuorescence properties of phycoerythrin (PE). The ﬂuorescence of PE is highly sensitive to the conformational and chemical integrity of the protein. The loss of PE ﬂuorescence under appropriate conditions is indicative of oxidative damage. The ﬂuorescene decay of PE in the presence of radicals relates a lag phase or rate to antioxidant capacity of an added sample. The results are compared to those in the presence of Trolox. Unlike TEAC and TRAP, this method takes the reactive species to complete reaction and uses the area under the curve technique for quantiﬁcation, thus combining both the inhibition time and inhibition percentage of the reactive species action by antioxidants into a single quantity. B.

Direct Methods for Measuring Reducing Power in Biological Tissue and Fluids

1.

Chemical Methods

Chemical methods for measuring reducing capacity consist of introducing a redox compound whose reduced and oxidized states have diﬀerent physical properties, which can be measured as a function of concentration.

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The ferric reducing/antioxidant power (FRAP) assay [39] is based on the reaction of the redox couple Fe(III)/Fe(II) with antioxidants in the sample, resulting in an intense blue color as the ferrous tripyridyltriazine Fe(II)-TPTZ complex (Abs at 593 nm) is formed. The FRAP assay is suitable mainly for biological ﬂuids and gives good reproducibility and sensitivity (up to 2 AM). In contrast to other chemical methods (indirect methods), this assay does not have a lag time and pretreatment of the sample is not required. The result of this assay reﬂects the total capacity of reductants in the sample to reduce Fe(III)-TPTZ to Fe(II)-TPTZ. Thus, compounds possessing an oxidation potential above the oxidation potential of the complex cannot be detected using this technique. A modiﬁed version of this assay, known as the ferric reducing/antioxidant power and ascorbic acid concentration (FRASC) assay [39], supplies three indices of antioxidant status: (1) the total reducing power of the sample; (2) the absolute ascorbic acid concentration; and (3) the particular contribution of ascorbic acid to the total reducing power. This is performed by taking two identical samples and adding ascorbate oxidase to one of them, which selectively destroys ascorbic acid in the sample. 2.

Electrochemical Methods

Electrochemical methods include a wide range of techniques. In some methods the potential of a sample is directly measured by an electrode. In others the current is monitored as a function of the applied potential upon the electrode. The current (or potential) is a result of an electron transfer between the working electrode and one or many electroactive species in the medium. Potentiometry. Potentiometric methods are those that measure the potential of a sample or solution following electron transfer between the electrode and the electroactive species in the solution. The current that develops upon the working electrode is proportional to the concentration of the electroactive species. This is the most straightforward method to measure the actual redox state or BRP of the tissue/ ﬂuid [4]. Some redox couples exhibit sluggish electron exchange with the electrode. In some systems the electrode is ‘‘poisoned’’ by proteins and sulfur-containing species that adsorb on the surface of the electrode. Both cases lead to inconsistent results and variation in time [4]. Electrochemical Titration Methods. Titration methods used to measure BRP are mainly potentiometric and coulometric titrations [4]. A potentiometric titration involves measurement of a suitable indicator electrode as a function of titrant volume. This technique provides information about the concentrations of the oxidizable or reducible species in the sample and allows the formal redox potential of the sample to be determined. In coulometric titrations the titrant is generated in situ electrochemically from its reduced form on the electrode surface [40]. Voltammetry—Sweep Techniques. Voltammetry [41] is an established electrochemical technique to measure and detect electron transfer between an electroactive species and an electrode. These measurements are usually carried out for isolated compounds under speciﬁc conditions in homogeneous solutions. All the electrochemical methods mentioned above provide thermodynamic and analytical information concerning the species in question. Voltammertric methods can in ad-

Measurements of Biological Reducing Power by Voltammetric Methods

21

dition provide kinetic information regarding the electron transfer step and in many cases about coupled homogeneous steps and mechanisms [41]. A potentiostat with a three-electrode system is required for voltammetric experiments (Fig. 1A). An external potential is linearly applied upon the working electrode relative to the reference electrode. The current developing on the working electrode is recorded as a function of the potential applied on it (Fig. 1B). This potential is aimed to oxidize or reduce the electroactive species in solution. The cyclic voltammetry (CV) experiment is carried out in highly conductive solutions, with high concentrations of electrolytes so that at any given potential (up to a certain point) the current is kept constant and very close to zero. Only when the potential is raised suﬃciently to permit an electron transfer between the electrode and an electroactivespecies in solution does the current rise. The peak current is an expression of this electron transfer and is proportional to the bulk concentration of the electroactivespecies. The key feature of CV is the ability to reoxidize (or rereduce) the product created on the forward scan on reverse scan. This gives information concerning the product resulting from the electron transfer. An important parameter in CV is the scan rate ranging from a few mV/s up to several thousand depending on the equipment and electrodes used. The mechanism of reaction can be deducted and rate constants can be calculated [41] based on the separation between forward and reverse waves, their shapes and ratio of their heights, and the change in them as a function of the scan rate. The concentrations of the reduced and oxidized species in the double layer of the electrode are governed by the potential applied upon the working electrode and by the

Figure 1 Cyclic voltammeter cell and a typical voltammogram. (A) Voltammeter cell. Three electrodes are introduced into the cell. The minimal volume of the samples under investigation is 200 AL. Nitrogen (99.999%) is bubbled through the sample in order to remove oxygen when needed. (B) A cyclic voltammogram of potassium ferricyanide (1 mM) in PBS. Two waves can be detected. The anodic wave (indicates the oxidation potential) is detected at peak potential of 284 mV and the reversible cathodic wave at peak potential of 172 mV. The tracing was carried out starting towards the negative potential at a scan rate of 100 mV/s. The peak potential (Ep(a)) or Ep(a)1/2 is calculated from the x-axis and the current (proportional to the concentration of the analyte) is calculated from the y-axis.

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standard Eo of the couple. This relationship is represented by the Nernst equation [see Eqs. (1)–(3)]. The peak current, Ip, in a voltammetric experiment is given by the Randles-Sevcik equation: Ip ¼ 2:69 105 n3=2 AD1=2 Cr1=2

ð4Þ

where Ip = peak current (amperes) n = number of electrons transferred per molecule A = electrode surface area (cm2) D = diﬀusion coeﬃcient (cm2/s) C = concentration (mol/cm3) r = scan rate (V/s) A method has been developed for measuring reducing power in biological samples, physiological ﬂuids, lipophilic and hydrophilic extracts, tissue, and organ homogenates [4,7,24]. These samples contain LMWA that act directly with ROS as scavengers. These compounds can be measured by voltammetry since they are reducing compounds, the precise quality that makes them ROS scavengers. Thus, it is assumed that an evaluation of the overall reducing power (the sum of the contribution of the various reducing antioxidants, or scavengers, present in a speciﬁc environment) of a biological sample would reﬂect its overall antioxidant ability. These LMWA include ascorbic acid, uric acid, and a-tocopherol (see Fig. 2). The reducing process is monitored as the current associated with the potential sweep. The position of the oxidation/reduction wave on the potential axis is a function of the formal redox potential. The peak current is thus proportional to the concentration of the electroactive species in solution. The biological sample diﬀers in many ways from a single component in a solution. As far as voltammetric measurements are concerned, the solution medium is not a highly pure solvent with an electrolyte salt, but a complex solution containing many components. There is no single electroactive species in a biological sample, but a few (see examples below) that may interact with each other, altering the voltammetric peak current. Therefore, the voltammogram cannot provide speciﬁc information on the exact nature of individual LMWA, but it can suply information concerning the reducing power or reducing proﬁle of the sample. The area under the peak can be calculated for this purpose and has been found to be highly reliable and reproducible and therefore qualiﬁed for comparison studies [7]. The voltammogram may be complicated if some of the species in the reaction adsorb onto the electrode surface. The redox wave of adsorbed species, in biological samples, generally possesses diﬀerent potentials and wave shapes than that of the compound itself. Diﬀerential Pulse Voltammetry. Diﬀerential pulse voltammetry (DPV) [42,43] was developed to increase the sensitivity of voltammetric methods by eliminating capacitative background current. The exclusion of this nonfaradaic current from the voltammetric peak current allows the detection of small faradaic currents resulting from electron transfer. The exclusion of the background current is achieved by a special potential program imposed upon the working electrode. In this technique, a potential pulse is applied at linearly increasing potentials. The scan rate is usually 10 mV/s, the pulse height is in the order of 10 mV, the pulse duration about 25 ms, and the time between the pulses is hundreds of milliseconds. The current is sampled before and after the pulse and the diﬀerence plotted against the potential applied. The resulting

Measurements of Biological Reducing Power by Voltammetric Methods

23

Figure 2 Cyclic voltammograms of various LMWA. The measurements were conducted at scan rate of 100 mV/s starting towards positive potential using a glassy carbon electrode as a working electrode for the measurement in the positive zone and a mercury film electrode (Hg/Au) for measurements in the negative zone. An Ag/AgCl/KCl (3.0 M) electrode was used as a reference electrode and a platinum wire as an auxiliary electrode. Prior to measurements in the negative potential, nitrogen (99.999%) was bubbled into the sample and flushed over the sample during the measurement. (A) Voltammogram of ascorbic acid (1 mM); (B) voltammogram of uric acid (0.1 mM); (C) voltammogram of a combination of ascorbic acid (500 AM) and uric acid (500 AM); (D) voltammogram of Nacetylcysteine (1 mM) measured in the negative potential using a Pt/Hg electrode in the absence of oxygen; (E) voltammogram of glutathione (1 mM) measured in the negative potential using a mercury film electrode (Pt/Hg) in the absence of oxygen; (F) voltammogram of a-tocopherol (1 mM) (measurement conducted in ethanol using tetrabutylammonium perchlorate (1%) as a supporting electrolyte).

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voltammogram has a peak-shaped form (see Figs. 3,4). It can be used in various samples with an up to 108 M detection limit. This technique was successfully employed for monitoring the redox-active species and providing a unique reducing power proﬁle of biological ﬂuids (see Fig. 3) and various tissue homogenates (see Fig. 4). The voltammetric method, CV and DPV, is the only one that allows diﬀerentiation between groups of LMWA in biological samples according to their reducing potentials.

Figure 3 Voltammograms of various biological fluids obtained with cyclic voltammetry at a scan rate of 100 mV/s starting towards positive potential and differential pulse voltammetry (DPV) at scan rate 5 mV/s, pulse amplitude 50 mV, pulse width 50 ms; pulse period 200 ms using a carbon working electrode and an Ag/AgCl//KCl (3.0 M) reference electrode. The fluids were diluted with 0.3 mL PBS buffer before measurements.

Measurements of Biological Reducing Power by Voltammetric Methods

25

Figure 4 CV at a scan rate of 100 mV/s starting towards positive potential and DPV at scan rate 5 mV/s, pulse amplitude 50 mV, pulse width 50 ms, pulse period 200 ms of various rat tissues. Tissue homogenates were prepared in PBS at 4jC using a manual homogenizer, as described above. The samples were analyzed immediately after homogenization or following period of storage (2 weeks) in 70jC. The left column shows the cyclic voltammograms of the various tissues. The right column shows the DPV voltammograms.

3.

Cyclic Voltammetry—Sensitivity

In contrast to electrochemical detectors for HPLC analysis, where the potential applied is constant and stable and therefore the sensitivity is high (up to fM), the sensitivity of the CV measurements is much lower. In cyclic voltammetry the limit of detection of LMWA is about 1–10 AM, while in DPV measurements the detection limit could approach 10–100 nM. Although the method is not extremely sensitive, it is suitable for biological quantiﬁcation and physiological concentration of LMWA,

26

Figure 4

Kohen et al.

Continued.

which are present in the range of 10 AM to millimolar. The limited sensitivity of the voltammetric method is an advantage, since low concentrations of redox-active compounds, such as some brain neurotransmitters, cannot be detected using cyclic voltammetry and therefore will not interfere with the measurement (low concentrations of neurotransmitters can be detected using the in vivo micro voltammetry technique—see below). It should be noted that increasing the scan rate of CV measurements increases the sensitivity by the square root of the scanning rate [Randles-Sevcik equation, Eq. (4)] of components that have fast diﬀusion and electrode kinetics. Components possessing very slow electrode kinetics will not be visible at all in fast scan rates. We have found that 100 mV/s is an optimal scanning rate for detecting LMWA in biological samples.

Measurements of Biological Reducing Power by Voltammetric Methods

Figure 4

4.

27

Continued.

Cyclic Voltammetry—Methodology

Instrumental Set-Up. There are many types of cyclic voltammeters. We have used a cyclic voltammeter Model CV1-B apparatus (BAS, West Lafayette, IN; for cyclic voltammetry measurements only) and an electrochemical working station CV50W (BAS, West Lafayette, IN) for evaluating the reducing power by applying cyclic voltammetry, linear sweep voltammetry (LSV), and diﬀerential pulsed voltammetry (CV, LSV, and DPV, respectively). LSV is a suitable method for biological samples in which the reverse scan does not provide additional information. DPV was used in order to increase the sensitivity when normal LSV failed to show anodic waves [4]. The sample under investigation (e.g., puriﬁed compounds in electrolyte solution, biological ﬂuids, tissue, or cell homogenates) was placed in the voltammetry cell (Fig. 1A). Three diﬀerent electrodes were used throughout the experiments. The working electrode was a glassy carbon disk electrode 3.3 mm in diameter, the reference electrode used was an Ag/AgCl, and the auxiliary electrode was a platinum wire. The volume of the sample was between 200 AL and 1 mL. When the measurements were carried out in the negative potential, a diﬀerent working electrode was used (e.g., a mercury ﬁlm electrode such as Pt/Hg or Au/Hg). In order to avoid oxygen interference, nitrogen gas (99.99%) or argon was bubbled for 20 min prior to the measurement, ﬂushing it above the sample throughout the experiments when scanning the negative potential. The potential was applied to the working electrode at a constant rate, usually 100 mv/s. Before each set of samples a standard of 1 mM of potassium ferrocyanide in 0.1 M

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phosphate-buﬀered saline (PBS) was run in order to check the electrodes and the apparatus setup. Figure 1B shows a typical voltammogram of such sample. From such measurement the active area of the electrode surface can be determined [44]. Cyclic Voltammetry of LMWA. Figure 2 shows typical cyclic voltammograms of various LMWA [4,7,24,44]. These compounds show anodic waves, indicating their ability to donate electron(s). Each compound possesses its own characteristic proﬁle. Figure 2A shows the anodic wave of ascorbic acid in PBS and Figure 2B shows the voltammogram of uric acid. A combination of these two compounds results in two close anodic waves, as shown in Figure 2C [24,44]. It must be stressed that when protein is present in the sample (e.g., albumin), the two anodic waves of uric acid and ascorbic acid appear as one wave containing the two compounds. Therefore, when measuring biological samples, each anodic wave is composed from many diﬀerent reducing equivalents that share close or similar oxidation potentials. Figure 2D and E shows voltammograms of N-acetylcysteine and glutathione (7). These measurements were carried out in the negative potential using Hg/Au electrode. Figure 2F shows a voltammogram of a-tocopherol. This measurement was carried out in lipophilic buﬀer (methanol:chloroform) using 1% tert-butylammonium perchlorate as a supporting electrolyte in the solution. III.

EVALUATING THE REDUCING POWER OF BIOLOGICAL FLUIDS AND TISSUES

Biological ﬂuids such as saliva, seminal ﬂuid, cerebrospinal ﬂuid (CSF), sweat, urine, plasma, serum, gastric juice, and tears all possess reducing power that can easily be measured using voltammetric techniques. Following collection of the samples, they are diluted 1:1 or 1:0.5 with PBS in order to keep the pH constant and ensure suﬃcient levels of electrolytes in solution [24,44,45]. The samples can be kept under nitrogen for 3 months at 70jC before analysis. When measuring biological samples that contain high levels of proteins and other molecules, which can be absorbed onto the electrode surface, the electrode must be polished before each measurement to ensure the same electrode surface between measurements. The polishing procedure is as described elsewhere [4] using a polishing kit (BAS, Bioanalytical Systems, West Lafayette, IN) and alumina for the glassy carbon electrode and diamond paste for the platinum or gold electrodes before creating a new mercury ﬁlm [7,44,45]. A.

CV and DPV Measurements of Some Biological Fluids

In Figure 3, human CSF (A) shows one major anodic wave at peak potentials of 383 mV, indicating one group of LMWA in human CSF. The saliva (collected in the morning following overnight fast) voltammograms demonstrate one major anodic wave at a peak potential of 517 mV. Figure 3 clearly shows that the plasma, when separated carefully, contained only one major anodic wave at a peak potential of around 400mV. Several studies [46,47] claimed that plasma contains two groups of reducing antioxidants and tried to relate the second wave to compounds such as oxidized lipoic acid. However, laboratory work revealed that the second wave is due to contamination of the sample with reducing equivalents originating in the red blood cells [e.g., NAD(P)H] during the separation of plasma from the cells. It is unlikely that lipoic acid in its oxidized form is a component of this wave as claimed [46], since the

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oxidation potential of oxidized lipoic acid is above 950 mV, and it is present in low concentrations in plasma, below the detection level (Table 2). Extreme care must be taken when relating reducing compounds to the biological anodic waves. Only a prudent evaluation using a biochemical approach (e.g., the use of uricase and ascorbate oxidase [44]) to the components composing the wave or appropriate analytical methods such as HPLC equipped with electrochemical detector or GCMS will be adequate. In contrast, a lipophilic extraction of human plasma showed four diﬀerent anodic waves at diﬀerent peak potentials indicating four diﬀerent LMWA groups [49]. The presence of only one anodic wave in plasma conducted in hydrophilic media is in line with the occurrence of a limited number of oxidation waves in biological ﬂuids in general (saliva, semen, CSF, sweat), all demonstrating only one to two hydrophilic anodic waves. B.

CV and DPV of Water- and Lipid-Soluble LMWA in Tissue

1.

Preparation of Tissue for Voltammetric Measurements

Tissues (30–500 mg) were deep-frozen in liquid air or liquid nitrogen immediately after removal from the animal. Tissues can be kept in 70jC for up to 6 months before homogenization or can be immediately homogenized and analyzed [24,44]. The homogenization is carried out in 1 mL PBS on ice using a mechanical homogenizer. In order to remove large insoluble particles, the homogenates were centrifuged at 1000 g for 10 minutes at 40jC. The pH of the homogenates is adjusted when needed to 7.2– 7.4. In order to extract the lipid-soluble components for the CV measurements, the tissue homogenates (or biological ﬂuids; 3–4 mL) are combined 1:4 (v/v) with a mixture of ethanol:hexane (1:5) (or other suitable combinations of extraction solvents). The mixtures are shaken roughly (5 min on vortex) and then centrifuged at 4jC at 1000 g for 15 minutes. The upper layer is separated from the lower layer and the lower layer is extracted again. The organic solvents of the combined upper layers are removed by evaporation. The residue is dissolved in an acetonitrile solution containing 1% tert-butylammonium perchlorate as a supporting electrolyte. The samples should be protected from light and kept on ice. A 30–50 AL sample should be separated from the PBS homogenates before the extraction procedure for protein determination. 2.

Characterization of Compounds Composing the Waves

Three diﬀerent approaches were taken in order to analyze the compounds composing the various waves. An HPLC instrument equipped with an electrochemical detector was used to ‘‘ﬁsh out’’ and characterize speciﬁc LMWA. The potential was set according to the diﬀerent peak potentials detected by the voltammetry measurements, and conditions of the chromatography were adjusted according to the literature for the diﬀerent compounds. Many LMWA were detected using the HPLC-ECD technique. For example, uric acid and ascorbic acid were found to be major components composing the ﬁrst anodic wave in plasma and in many other biological tissues. In a diﬀerent approach to quantifying the compounds responsible for the anodic waves, speciﬁc enzymes have been used. For example, the contribution of uric acid to the ﬁrst anodic wave of the hydrophilic measurements was assessed with uricase (E.C. 1.7.3.3), and ascorbic acid was analyzed using the enzyme ascorbate oxidase (E.C. 1.10.3.3). The voltammetry measurements were conducted twice prior to the incubation with the enzyme and following the incubation. The changes in the anodic current were

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attributed to the decomposition of the LMWA by the enzyme used. Another approach was to add various LMWA into the sample (spike) in order to quantify their peak potential and to correlate it to the anodic waves detected in the biological samples [4,24,44,48,49]. 3.

Examples of CV and DPV of Biological Tissues

Figure 4 shows a variety of biological reducing proﬁles of water-soluble LMWA in tissue extracts. It can be seen that each tissue possesses its own characteristic voltammogram that is characteristic of the tissue and may serve as a ‘‘biological tissue print.’’ When scanning diﬀerent samples of the same kind of tissue, the proﬁle is unique and the peak potentials are identical for a speciﬁc tissue. The anodic current of diﬀerent samples of a tissue changes dramatically as a function of the LMWA concentrations. The DPV measurements can signiﬁcantly enhance the sensitivity of the CV measurements and conﬁrm the existence of an anodic wave (group of LMWA), even when the CV measurements are not conclusive. Figure 4A shows CV (left) and DPV (right) rat heart tracings, respectively. Two anodic waves can be seen indicating two groups of LMWA. Similarly, Figure 4B–G show CV and DPV tracings of a variety of biological tissues, including lung, liver, kidney, spleen, muscle, small intestine muscle, and small intestine mucosae, respectively. In general, most biological ﬂuids demonstrate one anodic wave, indicating one major group of LMWA. Most tissues demonstrate two anodic waves, indicating two groups of LMWA. However, skin and some parts of the gastrointestinal tract and speciﬁc sites in mouse brain demonstrate three anodic waves, indicating three diﬀerent groups of LMWA and, possibly, the ability of these tissues to cope with deleterious ROS [4,7,44,45]. C.

Cyclic Voltammetry and DPV of Tissue Culture from Cell Lines

This method is not sensitive enough (see above) to obtain a reducing proﬁle of cells in culture or of a single cell. However, we were able to measure a reducing proﬁle of cultured skin cells. Figure 5 shows CV (A) and DPV (B) tracings of a HaCaT cell line [50] in PBS. The sample consists of 300 million cells broken by glass beads. BRP can also be measured for single cell voltammetry. Chronoamperometry was carried out on Chinese hamster ovarian cancer cells using a carbon ﬁber microdisk electrode to monitor doxorubicin (down to 1 nM) eﬄux from single preloaded cancer cells [51]. Amperometry with a platinized ultramicroelectrode was used for detection of H2O2 in single cell human ﬁbroblasts [52]. D.

Application of Voltammetric Measurements for Studying Reducing Power Profile In Vivo and In Vitro

Since this methodology for quantiﬁcation of the overall LMWA was ﬁrst introduced by us [4,7,22,44,45,48,49,53–55], it has been used in a variety of clinical situations and pathological disorders, including diabetes [56,57], inﬂammatory bowel disease [58], brain degenerative diseases and head trauma [57,59–63], skin status and pathologies [6,22–24,45,64], irradiation therapy [54], and in the study of the aging process [6] and the embryonic stage [65–67]. The results obtained have been reliable and reproducible. The use of this technique assists in clarifying several hypotheses about the role of antioxidants and

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Figure 5 Voltammogam made with CV (A) at 100 mV/s starting towards positive potential, and DPV (B) at scan rate 5mV/s, pulse amplitude 50 mV, pulse width 50 ms, pulse period 200 ms of HaCaT cells in PBS, using a carbon working electrode and an Ag/AgCl// KCl (3.0 M) reference electrode. Samples consisted of about 300 million cells broken by glass beads. their regulation in biological environments. For example, the existence of so many diﬀerent LMWA and their wide distribution among various organs led us to the hypothesis that the living cell needs a certain level of antioxidant activity and the speciﬁc identity of LMWA that contribute to this activity is irrelevant. In other words, whenever there is a decrease in the level of a speciﬁc antioxidant molecule, the cell copes by increasing the level of another antioxidant possessing a suitable oxidation potential. Using voltammetric measurements we were able to demonstrate that in many cases the overall reducing power proﬁle is not aﬀected, even though speciﬁc changes in the concentration of speciﬁc LMWA are detected. However, in speciﬁc pathological cases and in deﬁned clinical situations, the overall proﬁle is altered, as can be easily detected by the voltammetric determination, and may imply a possible intervention using LMWA. Some of these cases are summarized below. 1.

Reducing Power Profile Along the Aging Process

Among the many theories concerning the aging process, the free radical theory of aging is a dominant one [68,69]. One of the major claims in this theory is that there is a decrease in antioxidant activity during the aging process. However, many reports contradict this assumption and many others support it. One of the reasons for this contradiction is that in many studies the investigators evaluated the levels of several LMWA, ignoring the presence of many other antioxidants and the total antioxidant activity. In order to address this point, we determined the overall LMWA capacity in various rat organs and human skin during the aging process using the above-described voltammetric techique. It was found that the overall reducing power has a bell-shaped curve. The same age-related pattern was found in most of the organs tested, including liver, lungs, and kidney. Although we found an increase in reducing power up to the age of 2 months, these ﬁndings are in accord with ﬁndings concerning a decay in the concentration of LMWA in old age. This pattern of behavior was similar for the diﬀerent anodic waves, indicating a decrease in the total concentration of LMWA with age in all of the antioxidants present in diﬀerent tissues [6,54]. In several rat tissues a diﬀerent pattern was detected in which no change in the overall concentration was detected. Human saliva taken from donors at various ages

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exhibited a diﬀerent phenomenon [55]. In saliva from older donors the biological oxidation potentials detected were 400 mV higher than the anodic potentials found in saliva obtained from young donors (800 mV vs. 400 mV). The change in the antioxidants composing the various anodic waves (groups of LMWA) as measured by the change in the reducing proﬁle does not allow for the determination of concentrations, since the nature of each LMWA is diﬀerent. Skin tissue possesses an elevated antioxidant activity as reﬂected by the unique voltammetry proﬁle showing the existence of three anodic waves [6,22,45]. When the voltammetric measurements were conducted in whole skin removed from the anesthetized rat back or rat abdomen and immediately deep-frozen in liquid nitrogen [6] a signiﬁcant decrease was found in the concentration (anodic current) of the three waves along the aging process. No change in the type of antioxidant was found, as indicated by the fact that the oxidation potentials of the three waves remained similar. Similarly, in a noninvasive procedure where the surface skin antioxidant was evaluated using voltammetric and HPLC analysis, a decrease in the surface antioxidant was found in older human subjects as compared to young human subjects. These ﬁndings support the general claim in the free radical theory of aging of a decrease in antioxidant capacity in old age [45,68,69]. 2.

The Reducing Power Profile as Measured by CV in Rat Embryos

Reactive oxygen species were suggested to be involved in the mechanisms of action of several teratogens. Therefore, the antioxidant defense system of the embryo is of major importance in preventing embryo abnormalities. We studied the development of the antioxidant system in rat embryo and rat yolk sac in vivo and in vitro using the cyclic voltammetry technique [65–67]. We found a reducing power at peak potential of 0.56 V at embryonic day 9.5. On embryonic day 12.5 an additional group of LMWA appeared at a peak potential of 0.95 V. There was a gradual increase in the concentration of the LMWA with an increase in embryonic age. 3.

Voltammetric Measurements Following Head Injury

The involvement of ROS in brain injury has been demonstrated [59]. Head injury triggers a cascade of cytotoxic processes, including the release of ROS, cytokines, free fatty acids, and excitatory amino acids. In an experimental rat and mouse model of closed head injury (CHI), we studied the overall reducing power of rat brain following head injury as an indicator of the overall changes in the LMWA capacity of brain following injury. Such changes reﬂect the consumption and/or recruitment of these molecules. In the CV tracings of brain extracts of water-soluble LMWA, two anodic waves were found representing two classes of reducing antioxidants at peak potentials of 350 F 50 and 750 F 50 mV. The ﬁrst group, at peak potentials of 350 F 50 mV, consists of ascorbate and urate and other yet unknown scavengers. The second group, at peak potentials of 750 F 50 mV, probably includes imidazole-containing molecules such as NADH, histidine, carnosine, and other histidine-related compounds. Following CHI, the nature of the LMWA was not altered, as the two anodic peak potentials remained basically unchanged. The anodic current, Ia, which represents the concentration of the LMWA, was markedly aﬀected. Five minutes, 1 hour, and 24 hours following cerebral head injury, a signiﬁcant change was observed. An initial decrease of 40% was detected after the ﬁrst 5 minutes. A transient increase was observed at the second

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phase (1–4 h) with a second drop (a decrease of 60% in the anodic current) at 24 hours. The basal level was reached only 48 hours later and was maintained for 7 days [59]. The ﬁndings from cyclic voltammetry were supported by HPLC analysis. The reducing power proﬁle was also examined in a rat heat-acclimated model. When closed head injury was induced in heat-acclimated rats, they exhibited faster and better recovery of motor functions as compared with that of normothermic rats subjected to a similar severity of injury [59,60]. A CV study of water-soluble LMWA revealed that the acclimated rats displayed two anodic waves similar to the waves observed in the normothermic rats (similar chemical characteristics of the reducing antioxidants). The anodic current in the two groups of the acclimated rats was lower in 60% than the anodic current found in normothermic rats. Following trauma, the pattern of changes in the reducing power proﬁle was diﬀerent. No drop in the LMWA concentration was found at 5 minutes, and the increase in the level of the LMWA (increase in anodic currents) was 40–50% higher than in nontraumatized rats. Determination of the changes and the ratio between the anodic current at each time point after trauma to the anodic current before the trauma led to the suggestion that heat acclimation potentiates a systemic mechanism that enables the rats to cope more eﬃciently with the additional acute stress of closed head trauma. We have also investigated the role of ApoE deﬁciency on the reducing proﬁle after closed head injury. ApoE is a 36 kDa plasma glycolipoprotein that plays a role in lipoprotein metabolism and in lipid redistribution during the normal development of the nervous system as well as a role in the regulation of lipid metabolism following peripheral injury [70–72]. It has been found that in the absence of ApoE, the recovery of the cognitive and neurological functions of mice after closed head injury was slower than in control mice containing the ApoE protein [73,74]. We demonstrated using cyclic voltammetry that although in nontraumatized mice both control and apoEdeﬁcient have similar levels of reducing equivalents, they diﬀer markedly in their response to closed head injury. It was found that the deﬁcient mice were unable to mobilize LMWA to the same extent as the controls, thus explaining their impaired ability to recover from brain injury. 4.

Voltammetric Measurements Along the Gastrointestinal Tract

The LMWA group of compounds is extremely important in the defense mechanism of the gastrointestinal (GI) tract to oxidants. This tissue is exposed to ROS both from the serosic side and from the lumen. The GI mucosa is constantly exposed to oxidants from dietary sources, including oxidized food debris, a high level of iron ions, saliva oxidants, bacteria, and bile acids [58,75,76]. It is well documented that this high exposure might lead to a variety of pathological disorders, especially in the colon, where residence time of luminal contents is prolonged. The occurrence of ROS-related diseases, especially in the colon and its anaerobic surroundings (e.g., colon cancer, colitis), led us to hypothesize that this part of the GI tract is more susceptible to oxidative stress, which may be due to a lower LMWA capacity. Therefore, the overall reducing proﬁles of the mucosa/submucosa and muscularis/serosa of various sections along the small intestine and colon of the rat were evaluated using voltammetric assessment. It was found that the reducing power in the mucosa/submucosa of the small intestine was higher as compared to the mucosa/submucosa of the colon. Diﬀerences were also observed in the reducing power in the muscularis/serosa of the rat small intestine as compared to the colon [58]. In another study [76] the relationship

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Figure 6 CV voltammograms of lipophilic extraction from rat liver at 100 mV/s starting towards positive potential. Homogenization and preparation of the sample was carried out as described above and in Ref. 56. Voltammogram of lipophilic rat liver homogenate: (A) control and (B) diabetic rat. Diabetes was induced by injection of streptozotocine 4 weeks prior to the measurements.

between total tissue LMWA proﬁle and inﬂammation severity in experimental colitis was assessed. It was demonstrated that while mild colonic inﬂammation caused an increase in the overall reducing power of the water-soluble LMWA as detected by an increase in the anodic current (increase in concentration), a severe inﬂammation caused a reduction in the tissue LMWA-reducing capacity [76]. 5.

CV Study of Tissues from Diabetic Rats

Oxidative stress plays a major role in diabetic complications. However, the importance of the antioxidant system in protecting biological sites from deleterious processes induced by diabetic conditions is not fully understood. In order to clear up some of the inconsistency in the scientiﬁc literature, the overall LMWA capacity was evaluated using cyclic voltammetry. It was found that the levels of both water- and lipid-soluble LMWA progressively decreased in the diabetic plasma, kidney, heart, and brain. However, a signiﬁcant elevation in the antioxidant capacity of the liver was detected 2, 3, and 4 weeks after induction of the diabetes, as reﬂected by an increase in the anodic current of the lipophilic extraction of the rat liver [53] (Fig. 6). Several other studies were performed in order to determine the reducing power proﬁle in diabetic rats under additional stress (e.g., brain injury) [54] and development of abnormalities in the embryo [65–67]. IV.

IN VIVO VOLTAMMETRY

In vivo voltammetry (IVV) was developed in the 1970s for measuring catecholamine neurotransmitters and their metabolites in the functioning brain. It was developed as an analytical tool for monitoring speciﬁc compounds in a complex mixture, and, therefore, the major concern about its use has been the issue of chemical selectivity. The catecholamine neurotransmitters dopamine (DA) and norepinephrine (NE) and the main DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) are oxidized at similar potentials to ascorbic acid (AA). AA is present in the brain extracellular ﬂuid (ECF) at concentrations higher by several orders of magnitude than the neuro-

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transmitters. Voltammograms obtained with standard methods contain waves composed of oxidation currents from several compounds (composite peaks), and therefore they are considered ‘‘of little or no analytical use’’ [43]. Diﬀerent electrode materials, electrode surface modiﬁcations, applied potential procedures, and mathematical models for data analysis have been tried in the pursuit of improved separation of the small neurotransmitter peaks from the large AA and metabolite peaks. Despite diﬃculties, measurement of speciﬁc compounds in the ECF has proved feasible, and IVV has generated a large volume of information on the release and metabolism of biogenic amines in human brain. Moreover, the search for better chemicals, as well as temporal and spacial resolution, has contributed to the development of the ﬁeld of voltammetry and generated a variety of new applications. The aim of this section is to brieﬂy describe IVV and its application for measuring redox phenomena in the ECF. (For a more detailed discussion of the subject, readers are referred to Refs. 43, 77,78.) A.

Electrode Types and Arrangement

The classical three-electrode conformation has been used in the majority of IVV studies. However, when ultra-microelectrodes (5–100 Am in diameter) are used, the currents that pass through the working electrode are so low (picoamperes) that the auxillary electrode can be omitted [79]. The working and reference electrodes are implanted in the desired brain area of an anesthetized animal (most commonly rat) through holes drilled in the skull. One of the stainless steel screws used for anchoring the electrodes to the skull also serves as an auxillary electrode. The reference electrode is an Ag/AgCl wire sealed in a pulled glass capillary with saturated NaCl, but a reliable reference electrode is also obtained by implanting an Ag/AgCl wire directly into the brain tissue because the concentration of Cl ions in the ECF is kept within narrow margins. Carbon paste was among the ﬁrst electrode materials used in IVV [80]. However, the most widely used material is carbon ﬁber. A single ﬁber (6–12 Am) or several ﬁbers are sealed in a glass capillary. The ﬁber can be either cut so that it protrudes 0.1–0.5 mm beyond the seal to attain a cylindrical electrode shape (Fig. 7A) or cut and polished to obtain an ultra-microdisk electrode (Fig. 7B). Platinum electrodes are used as a base for some enzyme-modiﬁed electrode surfaces. The separation of DA, DOPAC, and AA peaks requires modiﬁcation of the electrode surface [81,82]. It was found that pretreatment of the electrode with waveshaped potential in a phosphate-buﬀered saline selectively shifts the AA peak to a more negative potential. Another approach utilizes the opposite charges of DA (positive) vs. AA and DOPAC (negative) by coating the surface of the electrode with an anion-repellent polymer such as NaﬁonR [83]. An increasing variety of electrode coating materials have become available, some of which are pretreated electrically [84]. Yet another approach consists of the incorporation of one or several enzymes, mostly hydrogen peroxide-producing oxidases, onto the electrode surface or in the electrode coating. With this approach it is possible to measure nonelectroactive compounds with great selectivity. However, because the ECF is a hostile environment to exogenous proteins, only a few enzyme-modiﬁed electrodes have been tried with IVV, e.g., as biosensors for measuring glucose [85], glutamate [86], lactate [87], choline [88], and nitrogen monoxide [89].

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Figure 7 Photographs of cylindrically shaped working microelectrode (A) and diskshaped microelectrode (B) made from a single carbon fiber (8 Am diameter) sealed in a pulled glass capillary. The connection between the carbon fiber and the electrode connector was made with conductive epoxy.

B.

Methods of Applied Potential in IVV

Numerous methods of applied potential have been used in IVV to obtain better sensitivity, peak separation, and reproducibility. The need for better temporal resolution has also aﬀected the development of voltammetric methods for IVV. Of the linear ramp methods, fast cyclic voltammetry (FCV) is most commonly used. At scan rates of roughly 300 V/s, the oxidized form of a chemical is reduced before it can react any further, and the oxidation/reduction proﬁle is characteristic for the analyte. Scan rate and potential range are chosen to maximize the contribution of a speciﬁc molecule. FCV oﬀers excellent time resolution, the typical cycle duration being 10–20 ms, with intervals between subsequent cycles being approximately 10 times longer, allowing for real-time, subsecond detection of biogenic amines [90,91]. The pulsed potential methods most frequently used in IVV are normal pulse voltammetry (NPV), diﬀerential pulse voltammetry (DPV), diﬀerential normal pulse voltammetry (DNPV), and diﬀerential pulse amperometry (DPA) [43]. In general, these methods provide better sensitivity and peak separation because the pulse amplitude, duration, and interval can be adjusted for optimal resolution. Furthermore, the diﬀerence in current is measured over a short interval and the contribution of initial, faster charging, and other background currents is minimized. Constant potential amperometry (CPA) has no chemical selectivity at carbon electrodes but is suited for use with some enzyme-modiﬁed electrodes, where it provides continuous information on substrate levels. C.

Example of Reducing Power Measured with IVV

Although the major ﬁeld of application of IVV is measuring temporal changes in the levels of DA, NA 5-HT, and their metabolites in the brain, IVV can also be used to attain a general proﬁle of the reducing power by oxidation of the major LMWA in ECF. Recent results from our laboratory demonstrate the feasibility of recording antioxidant (or reducing) proﬁles from the rat brain in vivo. In brief, rats were implanted chronically with either single ﬁber or multiﬁber carbon electrodes (Fig. 7) in the ventral hippocampus. Following a recovery period of several days, rats were anesthetized and connected to the voltammetry station (CV-50W; BAS, West Lafayette, IN). Voltammograms obtained with DPV show two separate peaks, of which the ﬁrst, larger peak clearly corresponds to AA (Fig. 8). AA is the principal antioxidant in the brain ECF, but it acts as a neuromodulator and is released into the extracellular

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Figure 8 Voltammograms obtained with electrically pretreated microelectrodes similar to the electrode shown in Figure 7A, but made with multiple carbon fibers. (A) The result of DPV scan (scan rate 20 mV/s, pulse amplitude 50 mV, pulse width 50 ms, pulse period 200 ms) of ascorbic acid and uric acid solution (both 100 AM) in phosphate-buffered saline. (B) Two consecutive voltammograms recorded in vivo in the hippocampus of an anesthetized rat. In the second scan, which was performed 2 minutes following the first scan, the peak containing uric acid was diminished, indicating that uric acid was depleted around the probe. The peak returned to the initial size following a 20-minute interval.

space under a variety of physiological and pathological conditions [92]. A second, large peak appearing between 300 and 400 mV (Fig. 8) can be, in large part, attributed to uric acid [93]. Uric acid is synthesized in the brain by the enzyme (hypo)xanthine oxidase, and it has been suggested that it acts as an important antioxidant [94]. In addition, similar proﬁles can be obtained with IVV from the ECF of soft tissues like skin, skeletal muscle, and adipose tissue (Kohen, unpublished results). It should be noted that factors such as continuing changes in the electrode surface properties with use and the complex, dynamic diﬀusion characteristics in living tissue complicate the interpretation of IVV results.

ACKNOWLEDGMENTS This work was supported by a grant from the Yedidut Foundation Mexico and from The Israel Science Foundation. E. M. is supported by a grant from the Golda Meir Fellowship fund. R. K. is aﬃliated with the David R. Bloom Center of Pharmacy. We thank E. Kanevsky for her assistance in the CV measurements and M. Segev for editorial assistance. REFERENCES 1. 2.

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3 Composition and Regulation of Thiol-Disulfide Redox State YVONNE S. NKABYO, THOMAS R. ZIEGLER, and DEAN P. JONES Emory University, Atlanta, Georgia, U.S.A.

I.

INTRODUCTION

Sulfur is an essential element for life, providing catalytic diversity to enzymes and structural diversity to proteins and other biomolecules. Of special importance, sulfur exists stably in multiple oxidation states under biological conditions, and the interconversion of these oxidation states, especially involving thiols and disulﬁdes, provides both a mechanism to switch between active and nonactive states in redox signaling and a site of vulnerability to oxidative stress. The present consideration of the composition and regulation of thiol-disulﬁde redox provides a brief review of the major thiol and disulﬁde components in mammalian systems, the principles for use of redox state calculations in redox signaling and control, and recent studies of thioldisulﬁde redox in cells, tissues, and extracellular ﬂuids. II.

BIOLOGICAL THIOLS AND DISULFIDES

A.

Thiols

A list of some of the more abundant thiols in mammalian systems is given in Table 1. The total protein thiol content in tissues and plasma exceeds the total of low molecular weight thiols under most physiological conditions; tissues can have 20– 40 mM protein thiol while plasma has 0.4 –0.6 mM. However, protein thiols vary considerably in their reactivities, and individual proteins are rarely at concentrations greater than 1 AM. As discussed below, thiols undergo reversible oxidations to disulﬁdes under prevailing conditions in tissues because the reductant system NADPH/NADP+ is suﬃcient to 43

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Table 1 Representative Plasma and Cellular Concentration of Low-Molecular-Weight Thiols Thiol or disulﬁde Cys CySS GSH GSSG CySSG Cys-Gly Cys-bis-gly g-Glutamyl cysteine Homocysteine Taurine Methionine Cys-Sulﬁnic acid

Plasma concentration, AM (Ref.) 10 (4) 40 (4) 3 (4) 0.14 (4) 2 (4) 1.5 (4) 2.5 (4) 3.8 (82) 11 (82) 66 (82) 31 (82) 0.79 (82)

Cellular concentration, AM (Ref.) 3 Caco-2 (81) 2200 HT29 (55) 60 HT29 (55) 12 RBC (82) 100 HT29 3.9 RBC 142 RBC 27 RBC

(52) (82) (82) (82)

reduce most disulﬁdes, while the oxidant systems provided by H2O2/H2O or O2/H2O are suﬃcient to oxidize most thiols. Steady-state concentrations of individual components are determined by the ongoing kinetics of these overall reductive and oxidative reactions in combination with thiol-disulﬁde exchange reactions among the various thiols and disulﬁdes. Some of these exchange reactions are catalyzed by members of the thioredoxin superfamily, while others are chemical reactions involving the major low-molecular-weight thiols and disulﬁdes. 1.

Glutathione

Glutathione (GSH) is the major low-molecular-weight thiol in cells. It is composed of three amino acids—glutamate, cysteine, and glycine—with the glutamate linked to the cysteine through the g-carboxylate, rather than the usual a-carboxylate linkage that is present in proteins. This unique linkage renders GSH (g-glutamyl-cysteinyl-glycine) resistant to proteases and peptidases that function in protein degradation. Synthesis. GSH synthesis occurs independently of ribosomal protein synthesis and occurs in two distinct steps involving glutamate:cysteine ligase (GCL; formerly known as g-glutamylcysteine synthetase) and glutathione synthetase, both ATPdependent enzymes present in the cytosol of all mammalian cells. The intermediate, g-glutamylcysteine, is present in all cells at low micromolar concentrations. Degradation. Degradation of GSH occurs by a unique enzyme, g-glutamyltransferase (g-glutamyltranspeptidase, g-GT). This enzyme removes glutamate to yield cysteinylglycine (present in AM concentrations in plasma), which is degraded by at least three enzymes with dipeptidase activity, eventually yielding cysteine. g-GT is present mostly as an ectoenzyme associated with the plasma membrane of epithelial cells, thus GSH degradation occurs mostly in the extracellular compartment. Function. The sulfur of the cysteine provides the functional portion of GSH. The thiol in GSH is less reactive than that of free cysteine because of the substitution on the a-amino group. In cysteine, the protonated amine withdraws electrons from the thiol group on the h-carbon, lowering the pK and making the thiol more reactive.

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45

Thus, in addition to providing a structure resistant to degradation by proteases, the unique g-glutamyl linkage in GSH also functions to reduce the reactivity of the thiol group so that relatively high concentrations can be maintained in cells. This decreased reactivity compared to cysteine suggests that a primary driving force for the evolution of the GSH system was to serve as a short-term reservoir for cysteine. Interorgan GSH/Cysteine Cycle. An interorgan GSH/cysteine cycle allows use of GSH as a short-term reservoir for cysteine (Fig. 1). In this cycle, GSH is released from cells at a rate that is both dependent upon cellular concentration and modulated by hormones and other homeostatic mechanisms [1]. Although liver releases GSH at a rapid rate via transporters such as the MRP (multidrug resistance-associated proteins) and Oatp (organic anion-transporting polypeptide) [2], all mammalian cells release GSH, and rats following hepatectomy still have circulating GSH concentrations that are about 50% of normal [3]. GSH is rapidly cleared from the circulation so that steady-state concentrations are in the low micromolar range, while the end products of GSH—hydrolysis, glutamate, cysteine, and glycine—are present at higher concentrations. Thus, by this mechanism cellular GSH pools serve as sources of free amino acids for the entire body. The human liver content of Cys (f1 g)

Figure 1

Interorgan GSH/Cys cycle. Met and Cys are supplied from digestion of protein in the diet. Met is converted to Cys in the liver so that dietary Cys and Met both are precursors for GSH synthesis. The liver serves as the major GSH reservoir, providing constant supply of Cys despite meal-associated intake. The mechanism involves GSH release from the liver into the blood and transfer to the kidneys, where it is degraded to release Cys. Cys released from the kidneys is transported to the tissues, where it is utilized for both protein synthesis and GSH synthesis. In this way, the liver and kidneys function to maintain a constant supply of Cys to the tissues. Under conditions of malnutrition or starvation, the relative contribution of other tissues increases due to the contribution of extrahepatic GSH export into the blood. This is also returned to the kidney, where it is degraded to release Cys. Cys released by the kidneys is returned to the tissues for protein synthesis. Thus, under conditions of malnutrition, tissue GSH serves as a reservoir for Cys.

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in GSH approximates the daily sulfur amino acid requirement; this pool is used to provide a stable Cys supply despite intermittent Met and Cys consumption, and there is an associated diurnal variation in hepatic GSH. The total amount of cysteine present in GSH in the body is about 3 g, roughly equal to the sulfur amino acid requirement for 3 days. 2.

Cysteine

Cysteine is the major low-molecular-weight thiol in plasma, being present at concentrations two to ﬁve times higher than that of GSH. Cysteine concentrations are maintained by the continuous supply from GSH but also through bidirectional plasma membrane transport systems that balance cysteine availability among tissues and between the intracellular and plasma spaces. As indicated above, cysteine is more reactive than GSH, the thiol being oxidized in the presence of molecular oxygen to produce reactive oxygen species (ROS). High levels of cysteine are, therefore, toxic. In addition to its utilization for protein synthesis and GSH synthesis, cysteine is used for the synthesis of taurine and sulfate. These uses necessitate a constant nutritional intake of cysteine or its precursor, the essential amino acid methionine. Methionine is converted to the thiol homocysteine through the action of two enzymes: S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Homocysteine, together with serine, yields cystathionine. Cystathionine can be cleaved by g-cystathionase to yield cysteine and a-ketoglutarate. Homocysteine is present at low micromolar concentrations both in plasma and tissues. Total plasma homocysteine, which consists principally of disulﬁde forms, appears to be a useful indicator of cardiovascular disease risk, but the mechanistic basis for this remains unknown. B.

Disulfides

All of the thiols can exist in disulﬁde forms, the nature of which is determined by the relative abundances of the thiol forms, the stability of the disulﬁde, and the kinetics of formation and removal. The major low-molecular-weight disulﬁde in mammals is cysteine, which is present at 50– 100 AM in plasma [4]. Disulﬁdes of cysteine with GSH, cysteinylglycine, and homocysteine are present in plasma. Glutathione disulﬁde (GSSG) and homocysteine are typically at concentrations below 200 nM but are increased during oxidative stress and the genetic disease homocystinurea, respectively. In contrast to the very low concentration in plasma, GSSG concentration in tissues is in the low micromolar range, with values as high as 50 AM being found in some tissues and cell types. Thus, GSSG appears to be the major low-molecular-weight disulﬁde in cells. Cystine content would appear to be variable in cells due to the variation in expression of the cystine uptake system [5], but little information is available on this. Disulﬁdes of coenzyme A are also present, especially during oxidative stress [6]. Cysteine, cysteinylglycine, homocysteine, and GSH occur extensively in human plasma as mixed disulﬁdes with serum albumin.

III.

ENZYME SYSTEMS FOR CONTROLLING THIOL-DISULFIDE REDOX STATE

Control of thiol-disulﬁde balance in proteins involves oxidation of thiols by oxidases and peroxidases, reduction of disulﬁdes by reductases, and thiol-disulﬁde exchange

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47

reactions with thiol transferases and protein disulﬁde isomerases (Fig. 2). The activities of many proteins are sensitive to modiﬁcations induced on thiols by redox changes, but whether there are speciﬁc mechanisms for control of individual proteins is not known. In particular, thiol oxidase and peroxidase activities are poorly characterized. A principal limitation for such studies is the lack of an understanding concerning the nature of spontaneous auto-oxidation reactions. A common perception is that an inadvertent loss of activity of thiol-dependent systems is merely a cost of aerobic life. An alternative possibility is that such auto-oxidation reactions are an evolved and purposeful mechanism for control of protein function. Thiols with a high reactivity toward either oxygen or peroxides could spontaneously convert a protein to a ‘‘resting’’ form under prevailing conditions in tissues. Activation of a reductase or protein disulﬁde isomerase system could then convert the protein to an alternate reduced form. A.

GSH-Dependent Systems

The two principal reductase systems utilize GSH and thioredoxin (Trx) as central electron carriers. The GSH pool is oxidized to GSSG during its function as an antioxidant, for both nonenzymatic (e.g., dehydroascorbate reduction) and enzymatic reactions (e.g., glutathione peroxidase). GSSG is recycled to GSH by an NADPH-

Figure 2

Systems controlling thiol/disulfide redox. The intracellular and extracellular thiol/disufide redox pools are separated by cell membranes, and because the components cannot easily diffuse across the membrane, these two pools are separate and not in equilibrium. The cellular pool is more reduced and is maintained principally by the balance between oxidation and reduction processes. The extracellular pool, which is more oxidized, is maintained by transport of the components across the membrane and thiol/disulfide exchange reactions. GR, Glutathione reductase; TR, thioredoxin reductase; Grx, glutaredoxin; Prx, peroxiredoxin; GPx, glutathione peroxidase; Trx-2SH, reduced thioredoxin; Trx-SS, oxidized thioredoxin; PrSH, reduced protein thiol; PrSS, protein disulfide; ROH, lipid hydroxide; ROOH, lipid hydroperoxide.

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dependent GSSG reductase [7]. GSH reacts with most protein disulﬁdes only at a slow rate; glutaredoxin (Grx), a member of the Trx superfamily, contains a conserved active site sequence Cys-Pro-Tyr-Cys and catalyzes GSH-dependent reduction of proteins. Grx reduces protein disulﬁdes directly via its active site dithiol, which is then converted to a disulﬁde. GSH reduces Grx and in the process is oxidized to GSSG, which is recycled back to GSH by GSSG reductase. NADPH is the electron donor used to recycle both Trx and Grx. B.

Thioredoxin-Dependent Systems

Trx is a multifunctional protein with a redox-active disulﬁde/dithiol in the active site: Cys-Gly-Pro-Cys. It operates together with NADPH and Trx reductase as a general protein disulﬁde-reducing system [8], functioning both intracellularly and extracellularly. Human Trx is secreted by lymphocytes and other cell types, e.g., hepatocytes and ﬁbroblasts, through a unique leaderless pathway [9,10]. Normal hepatocytes can secrete Trx, and oxidative stress can enhance the secretion of Trx [11]. Mammalian cells contain two distinct forms of Trx; Trx1 is found in the cytoplasm and nuclei, while Trx2 (mtTrx) is found in the mitochondria [12]. By its general protein disulﬁde reductase activity, Trx1 can regulate enzymes and transcription factors by thiol redox control. This is based on the reversible formation of a disulﬁde involving the sulfur of a critical cysteine -SH group and another -SH group within the protein or a small-molecular-weight thiol such as GSH. Alternatively, certain thiols appear to be oxidized reversibly to sulfenic acids, which are stable due to the speciﬁc microenvironment in the protein. Oxidation of speciﬁc thiols to sulfenic acids is thought to inhibit DNA binding of transcription factors such as c-Fos and c-Jun. Disulﬁde formation generally leads to loss of function for intracellular proteins with -SH groups, whereas disulﬁde reduction inactivates certain proteins. In the process of reducing disulﬁdes or sulfenic acids, thioredoxin becomes oxidized and is recycled by the NADPH-dependent thioredoxin reductase. Both Trx and Grx have roles for the reductive cleavage of GSH-protein mixed disulﬁdes [13]. Both Trx1 and Trx2 have been found to protect against oxidant-induced apoptosis [12].

IV.

QUANTITATIVE DEFINITION OF REDOX STATE

The interactions between redox active components are determined both by their tendency to accept or donate electrons and the kinetics of their interactions. As indicated above, enzyme systems are present to catalyze many of these reactions. However, growing evidence has indicated that cellular and extracellular redox environments diﬀer and that the major thiol-disulﬁde systems are not in redox equilibrium. The reducing force available from an electron donor/acceptor couple is conveniently expressed using the Nernst equation: Eh ¼ Eo þ RT=nF lnð½electron acceptor=½electron donorÞ where R is the gas constant, T is the absolute temperature, F is Faradays constant, and Eo is the standard potential relative to a standard hydrogen electrode. Comparison of redox states of multiple redox couples provides a means to understand the direction of electron ﬂow; a more negative redox state within a cell or

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49

tissue indicates a more potent reducing force. The greater the diﬀerence between two couples, the greater the free energy available from the electron transfer (DG = nFDEh). The NADPH/NADP+ reductant system for both Trx and GSH systems has an Eh value of about 400 mV in rat liver [14,15]. In principle, in the absence of oxidative stress, the GSH/GSSG couple would be expected to be equilibrated with this redox state due to the GSSG reductase reaction. However, the GSSG reductase has a very high Km for GSSG, and the reaction is probably far from equilibration under most conditions [16]. For instance, the GSSG concentration would be nanomolar or less in cells if the reaction were equilibrated with the NADPH/NADP+ pool, whereas values of 20– 50 AM are common in tissues. Although most studies of GSH and GSSG during oxidative stress have used the ratio GSH/GSSG as a measure of the extent of oxidation, this parameter is most useful for discussions of S-glutathionylation of proteins and not very useful as a measure of the extent of oxidation. The major diﬃculty for the later application concerns the stoichiometry of oxidation in which 2 GSH are oxidized to form GSSG. The Nernst equation correctly takes into account the stoichiometry and allows comparison of the redox state of the GSH/GSSG couple with other redox active components. In a simpliﬁed form of the Nernst equation for pH 7.0, given in millivolts—Eh = 240 + 30 log([GSSG]/[GSH]2)—the concentrations of GSH and GSSG are given in molar units. Eo is pH dependent, shifting 59 mV per pH unit [17], so that the equation must be adjusted for conditions where pH is not 7.0. V.

REDOX, GROWTH CONTROL, AND SIGNALING

Growing evidence indicates that both extracellular and intracellular oxidation-reduction (redox) status regulates various aspects of cellular function. Thiols, especially GSH and its precursors, stimulate cell proliferation in vivo and in vitro [18 – 20]. Depending on experimental circumstances, oxidative states can stimulate cell proliferation or activation in response to growth factors [18,19,21,22], as well as negative responses such as growth inhibition or cell death [20]. For example, growth-related signaling of a number of growth factors, such as platelet-derived growth factor (PDGF), appears to require intracellular generation of ROS, including superoxide [23] or H2O2 production [24]. The extracellular environment also inﬂuences cell proliferation. Cell density in a variety of cell lines was a function of the redox potential of the culture medium, with redox potential being predominantly determined by medium thiol content [25]. Supporting this is the accumulating evidence indicating that extracellular thiol depletion inhibits cell growth [21], while thiol addition enhances growth in growth-inhibited cells [26]. Cysteine/cystine are the predominant thiols in plasma and therefore represent the major extracellular thiol pool. The extracellular redox can be varied over a wide range of reducing to oxidizing states in vitro by altering extracellular cysteine relative to the cystine concentrations in cell medium. Under these conditions, proliferation of human intestinal epithelial cells was higher under more reducing conditions (150 mV); in contrast, a proliferative response to growth factors such as epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) was stimulated only under more oxidizing conditions (0 to 80 mV) [21]. Taken together, this evidence indicates the importance and complexity of the cellular and the extracellular redox milieu in regulation of cellular growth control and signal transduction pathways. Data are

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now becoming available to distinguish thiol-disulﬁde redox pools and thereby gain a better understanding of redox control and signaling mechanisms. VI.

CHARACTERIZATION OF SPECIFIC REDOX COMPARTMENTS

A.

Plasma Redox

Literature values for GSH concentrations in human plasma vary over a 10-fold range, despite the use of apparently valid analytical procedures (Fig. 3). A large portion of this variation appears to be due to sample collection procedures. Even 0.1% hemolysis can result in a 50% increase in GSH in samples from some individuals. A short delay in processing can result in a substantial underestimation of GSH due to the reaction of GSH with the relatively high concentration of cystine in the plasma [27]. To minimize errors in factors such as sample collection, processing, and storage that contribute to these variations, a sampling procedure was developed in which blood is collected by a

Figure 3 GSH and Cys pools in the plasma. The steady-state GSH/GSSG pool at 138 mV is considerably more reduced than the Cys/CySS pool at 80 mV. These two pools are therefore not in redox equilibrium. The GSH/GSSG redox in plasma is considerably more oxidized than in cells, indicating that GSH released from cells is rapidly oxidized or broken down via the action of plasma membrane enzymes g-glutamyltranspeptidase (g-GT) and dipeptidases (DP) to release cysteine, leading to lower exracellular GSH concentration and consequent oxidation of the pool. This maintains the extracellular GSH pool at a more oxidized state than the intracellular pool. It is not known whether enzymes are present in the plasma membrane to directly oxidize thiols or reduce disulfides.

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syringe and butterﬂy needle to avoid hemolysis and transferred immediately to a preservation solution [27]. This solution contains serine borate, a complex that inhibits degradation of GSH by plasma g-GT. The borate also functions as a buﬀer (pH 8.6) to facilitate reaction of thiols with iodoacetic acid, also present in the preservation solution. When collections and centrifugations are performed at room temperature, derivatization of thiols is essentially complete by the end of the centrifugation to separate plasma. Subsequent derivatization with dansyl chloride provides samples in which GSH, GSSG, cysteine, and cystine concentrations can be determined by HPLC. The redox state (Eh) is calculated using the Nernst equation, with Eo = 264 mV for GSH and GSSG and Eo = 250 mV for cystine and cyteine [27]. The GSH/GSSG Eh in plasma in healthy adults (137 F 9 mV) is considerably more oxidized than in cells and tissues, indicating rapid oxidation of GSH upon release into the plasma. This value, however, does not diﬀer markedly between young, healthy individuals, suggesting that the rates of reduction and oxidation in the plasma are closely balanced to maintain this redox state. The cysteine and cystine pool, at 80 F 9 mV, is 57 mV more oxidized. Thus, the GSH/GSSG and cysteine/cystine pools are not in redox equilibrium in the plasma. Because the cystine pool is very large, it appears to be a major oxidant for the GSH pool. A growing number of conditions have been found to oxidize the GSH pool. A comparison between young and elderly subjects indicates that this pool becomes about 40 mV more oxidized with age, due to a signiﬁcant decline in GSH concentration and an increase in the GSSG pool. Age-related pathological conditions such as age-related macular degeneration and type II diabetes further exacerbate this oxidation, with the pool becoming signiﬁcantly more oxidized in the diabetics (20 mV more oxidized than age-matched controls) [28]. Oxidizing conditions such as chemotherapy also oxidize this pool. In young to middle-age adults with hematological malignancies undergoing autologous or allogeneic bone marrow transplantation (BMT), plasma GSH/GSSG Eh became increasingly oxidized over time following high-dose conditioning chemotherapy (from 116 mV prior to chemotherapy to 106 mV 14 days post-chemotherapy) [29]. These observations indicate that Eh in plasma varies according to physiology, disease, and toxicity. The mechanism to control the extracellular GSH redox state in plasma appears to involve the release of GSH from tissue, principally the liver [30]. Generalized protein-energy malnutrition or speciﬁc limitation of amino acid substrate for GSH synthesis (methionine and cysteine) has each been shown to decrease whole blood GSH synthesis and turnover, with varying eﬀects on whole blood GSH levels [31,32]. Eﬀects on plasma GSH/GSSG Eh under these conditions of nutrient substrate depletion are unknown. Human Trx, another important antioxidant, has been identiﬁed in human plasma/serum [33] and can be measured by a sensitive sandwich ELISA method [34]. Plasma levels of Trx are indicative of the inﬂammatory response against oxidative stress. For instance, plasma levels of Trx are signiﬁcantly elevated in HIV-infected individuals suﬀering from oxidative stress [34]. Trx in plasma/serum may play an important role against oxidative stress, although the origin and role of plasma/serum Trx are still to be clariﬁed. Trx and Grx have been reported as possible electron donors to plasma glutathione peroxidase [35], suggesting that Trx in plasma may act as an antioxidant together with plasma glutathione peroxidase. Exogenous Trx can protect cells from anti-Fas antibody-induced apoptosis and cytotoxicity by TNF-a [36] or H2O2. The mechanism of cytoprotective eﬀects of

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exogenous Trx remains to be clariﬁed. One unique eﬀect of exogenous Trx is to promote uptake of cystine into cells and upregulate the intracellular levels of glutathione [37]. Red blood cells, leukocytes, and platelets contain Trx, which they may release in response to oxidative stress. The endothelium and liver are also possible sources of plasma/serum Trx. B.

Redox in the Intestinal Lumen

Thiols in the intestinal lumen are supplied by a variety of sources. GSH and other thiols such as cysteinyl-glycine and cysteine are supplied to the intestinal lumen via bile [2,38], diet [39], and release from the small intestinal mucosal epithelial cells [40 –42], with cysteine comprising a major portion of the luminal thiol pool (Fig. 4). Cysteine functions as a reductant in the absorption of iron [43] and selenium [44]. Both GSH and cysteine can function as reductants in the maintenance of mucus ﬂuidity [45] and in the protection of enzymes and transport systems that contain critical thiol groups [16]. These functions are likely to depend on the redox potential of the respective thioldisulﬁde couples. The small intestine has the capacity to reduce intraluminal disulﬁdes and therefore the ability to adjust the luminal thiol-disulﬁde redox. Thiols in foods are often oxidized to disulﬁdes upon processing, storage, and preparation [46]. Dietary GSSG is partially reduced to GSH in the upper small intestine [47] by a mechanism

Figure 4

Luminal redox in the intestine is maintained by a mechanism dependent upon cellular release of cysteine. Available evidence suggests that a cysteine (Cys)cystine(CySS) shuttle maintains luminal thiol-disufide redox state. Cys is released either directly or as a degradation product of glutathione (GSH) from the intestinal epithelium into the lumen. Luminal Cys reduces disulfides, forming CySS, with intermediate formation of Cys-GSH disulfide. CySS is transported back into the epithelium and is reduced intracellularly by GSH, forming glutathione disulfide. Glutathione disulfide is reduced to GSH by the NADPH-dependent GSSG reductase. If this system functions to reduce luminal disulfides and maintain luminal redox, then it is likely that cysteine uptake (during protein digestion) is inhibited in concert with activation of the cysteine efflux. Alternatively, GSH release followed by degradation could be the source of luminal thiol.

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using cysteine directly released from the epithelium or derived from breakdown of GSH released from epithelium [48]. This is likely an important homeostatic response given the potential importance of the luminal GSH system for detoxiﬁcation, mucus ﬂuidity, and nutrient transport [49]. Unlike the plasma, the Eh of the cysteine/cystine pool is more reduced than the GSH/GSSG pool [50]. This is unusual in that the data indicate cysteine as the principal reductant to maintain luminal redox rather than GSH, which appears to serve this function at other sites in the body. C.

Redox in Cells

Little data are available on Eh values in tissues. In rat intestine, GSH/GSSG redox is maintained at about 203, 209, and 220 mV in the jejunal, ileal, and colonic mucosa, respectively, but becomes signiﬁcantly oxidized in each tissue with malnutrition [20]. Of interest, administration of keratinocyte growth factor (KGF) prevented the malnutrition-induced decrease in gut mucosal GSH-reducing capacity in malnourished animals [20]. In normal mice, liver and lung GSH/GSSG Eh were about 222 and 207 mV, respectively [51]. High-dose chemotherapy/irradiation in murine allogeneic BMT did not alter liver GSH/GSSG redox but oxidized lung GSH/GSSG redox to about 195 mV. KGF treatment increased liver GSH Eh and attenuated the oxidation of the GSH redox pool induced by BMT + chemotherapy/ irradiation [51]. The Eh values of cells in culture under diﬀerent conditions have been determined, and the narrow range of the values suggests that the redox states of these pools are tightly regulated. Eh values in cells tend to vary according to the cell growth conditions, with rapidly proliferating cells being most reduced, apoptotic cells most oxidized, and diﬀerentiating cells between the two extremes. Eh of the GSSG/2GSH pool is 260 mV in undiﬀerentiated HT29 cells, and substantial oxidation (200 mV) is observed when these cells are induced to diﬀerentiate with sodium butyrate [52]. This oxidation is as a result of a rapid and substantial decline in the GSH pool and a concurrent increase in the GSSG pool. Cells induced to undergo apoptosis are further oxidized to a range of about 164 to 140 mV. This extensive oxidation is observed after cytochrome c is released from the mitochondria. The GSH redox in HL60 cells is oxidized to 165 mV during apoptosis [53], and HT29 cells undergoing terminal diﬀerentiation and apoptosis are oxidized to 170 mV [52]. Apoptotic cells are therefore more oxidized than diﬀerentiating cells, which in turn are more oxidized than rapidly proliferating cells. This indicates a natural progression in the redox state from reducing during proliferation, to oxidizing during apoptosis, and intermediate during diﬀerentiation. The redox changes observed in HT29 cells using a chemical model of diﬀerentiation were conﬁrmed and extended in Caco-2 cells. When grown to conﬂuence under standard culture conditions, Caco-2 cells cease division and spontaneously diﬀerentiate in a manner similar to normal enterocytes [54]. Using this model, we demonstrated that as cells progress from proliferation to spontaneous diﬀerentiation, they become more oxidized (by about 40 mV). The redox state of Trx did not change under the same conditions, although the expression of Trx protein increased. This demonstrates that in contrast to changes in the GSH/GSSG pool with diﬀerentiation, the Trx pool increases rather than decreases and does not undergo oxidation in association with diﬀerentiation [55].

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Trx plays an important role in maintaining the redox environment of the cell. The intracellular expression of Trx is dependent on the cell cycle, suggesting its possible involvement in redox regulation of the cell cycle [56]. Trx and its reductase are overexpressed in many human cancers, further supporting an important role for Trx in cell growth. This may be due to the function of Trx as a reductant for deoxyribonucleotide reductase, which is required for DNA synthesis. Another important intracellular role for Trx is in redox regulation of transcription factors. Trx maintains a critical thiol of the DNA-binding region of glucocorticoid receptor [57], activates a redox-sensitive nuclear factor Ref-1/APEX, thus enhancing the binding of Jun/Fos to the AP-1 site [58], and reduces a cysteine residue that is important for the binding of NF-nB to DNA [59]. Trx also functions as a redox-regulated binding factor for apoptosis signal-regulating kinase-1 (ASK-1) in which oxidation causes release from an inhibited complex and allows kinase-mediated activation of apoptosis [60]. Under most conditions in cells, Trx appears to be principally in the reduced form. However, Trx becomes oxidized in parallel with GSH following treatment with tert-butylhydroperoxide [61] and is extensively oxidized during growth stimulation by EGF [62]. However, during the natural progression of Caco-2 cells from proliferation to diﬀerentiation, there was no change in redox state of Trx despite the change in redox of GSH/GSSG [55]. Thus, the results indicate that the redox state of Trx is likely to be important in control of fundamental processes such as cell growth and apoptosis and is subject to oxidation during oxidative stress. However, the results also indicate that Trx is maintained by control mechanisms that are distinct from those for GSH and therefore indicate that these key redox systems must be considered independently in studies of the circuitry of redox signaling and control systems. D.

Redox in Subcellular Compartments

1.

Secretory Pathway

In the endoplasmic reticulum (ER), disulﬁde bonds are simultaneously formed in nascent proteins and removed from incorrectly folded molecules destined for the secretory pathway. A central role is played by ER-resident oxidoreductases, such as protein disulﬁde isomerases and ERp57. These proteins react directly and selectively with diﬀerent substrate proteins and, therefore, participate in distinct oxidative pathways [63,64]. Optimum concentrations of thiol and disulﬁde are required for rapid and complete refolding of many proteins. The principal redox buﬀer in the endoplasmic reticulum is GSH, and the GSH redox state of the secretory pathway is more oxidized (170 to 185 mV) than that of the cytosol (221 to 226 mV) [65]. Since enzymes catalyzing synthesis of glutathione are found only in the cytosol [66], GSH and Cys must be transported into the microsomal lumen from the cytosol. The preferential transport of GSSG relative to GSH is one mechanism that generates a more oxidized state in the secretory pathway [65], although ﬂavin-dependent oxidases are also present [67]. 2.

Mitochondria

Numerous studies have implicated GSH, Trx, and coenzyme A (CoASH) as molecules that all play a role in protecting the mitochondria from oxidative stress. Mitochondria in liver cells contain 15– 20% of the total cellular GSH [68], and this

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pool is solely derived from the activity of mitochondrial transporters that translocate GSH for the cytosol into the matrix [69]. Chen et al. [70] have shown that GSH uptake occurs by the 2-oxoglutarate and dicarboxylate carriers. The mitochondrial GSH/GSSG redox state, calculated from mitochondrial concentrations of GSH and GSSG and assuming a matrix pH of 7.6, is 236 mV, a value similar to the cytosolic GSH/GSSG redox state which is at 239 mV under the same conditions [53]. GSH depletion has been shown to enforce mitochondrial permeability transition and cause cell death in cells overexpressing Bcl-2 [71]. CoASH is compartmentalized preferentially in the mitochondria, and it can form mixed disulﬁdes with GSH, resulting in CoASSG [6]. O’Donovan et al. [72] demonstrated that the CoASH/CoASSG can be used as criteria for oxidant stress in the mitochondria. CoASH/CoASSG ratios were lower in animals exposed to hyperoxia. Trx overexpression in osteoblastoma cells protected the cells against death induced by tert-butylhydroperoxide, suggesting that the mtTrx system complements the GSH system to protect against oxidant stress in the mitochondria [12]. 3.

Nuclei

GSH and GSSG are diﬃcult to measure in isolated nuclei because nuclear membranes contain a nuclear pore complex that allows diﬀusion of small compounds like GSH and GSSG during standard subcellular fractionation protocols [73,74]. Attempts have been made to circumvent this limitation by modifying the standard protocols to include nonaqueous buﬀers in which GSH is not soluble [75,76] or by using GSHspeciﬁc ﬂuorescent probes in intact cells [77,78]. The results indicate that the GSH concentration in nuclei is similar to that in cytoplasm. Of particular interest, nuclear GSH has been found to vary as a function of the expression of the antiapoptotic protein Bcl2 [78]. However, the use of ﬂuorescent probes to address the question of nuclear compartmentalization has been seriously questioned [79]. GSSG levels in nuclei have not been reported, and the ﬂuorescent probes used for GSH measurement do not react with GSSG. Thus, there is no information available on thiol-disulﬁde redox based upon GSH. Examination of Trx redox suggests that the redox state in the nuclear compartment is similar to that in the cytoplasmic compartment [80]. Additional studies are needed on nuclear thiol content and redox state because DNA synthesis and function of many transcription factors are redox-sensitive.

VII.

SUMMARY AND CONCLUSION

Redox state analysis of thiol-disulﬁde couples provides a useful means to quantitatively evaluate function of speciﬁc small molecule and protein components in redox signaling and control. The two major low-molecular-weight thiol/disulﬁde systems, GSH/GSSG in cells and cysteine/cystine in plasma, are not in redox equilibrium and therefore can have diﬀerent functions in redox control and signaling. Similarly, redox of cellular Trx is controlled independently of cellular GSH. Together, the results suggest that the redox circuitry controlling oxidation-reduction of thiol motifs in speciﬁc proteins is likely to be complex and regulated within the context of local redox environments. If so, this represents a major paradigm shift from common oxidative stress models wherein altered oxidant generation is viewed as a global insult and nonspeciﬁc antioxidants are viewed as useful interventions.

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ACKNOWLEDGMENTS The research in the laboratories of the authors was supported by NIH grants ES09047, ES011195, and DK55850.

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4 Thiols and Thioredoxin in Cellular Redox Control HAJIME NAKAMURA and NORIHIKO KONDO Institute for Virus Research, Kyoto University, Kyoto, Japan KIICHI HIROTA National Institute of Advanced Industrial Science and Technology, Ikeda, Japan HIROSHI MASUTANI and JUNJI YODOI Institute for Virus Research, Kyoto University, Kyoto, and National Institute of Advanced Industrial Science and Technology, Ikeda, Japan

I.

INTRODUCTION

Recent studies have shown that reactive oxygen species (ROS) generated by a variety of oxidative stresses are not only harmful to the cells but also quite important in signal transduction from cell surface to nucleus. Cellular redox (reduction/oxidation) status is balanced by generated ROS and endogenous antioxidants. The redox status of protein thiols plays a crucial role in the regulation of signal transduction in biological responses. The thioredoxin (TRX) system, composed of TRX reductase, TRX, and peroxiredoxin, regulates intracellular and extracellular redox balance as well as the glutathione (GSH) system. TRX was originally identiﬁed in Escherichia coli as a hydrogen donor for ribonucleotide reductase, an essential enzyme for DNA synthesis [1]. We cloned human TRX as a cytokine-like factor, adult T-cell leukemia (ATL)–derived factor, produced by human T-cell leukemia virus type-I (HTLV-I)– transformed ATL cells [2,3]. TRX is found ubiquitously from prokaryotes to eukaryotes and has two cysteine residues in the well-conserved active site: -Cys-Gly61

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Pro-Cys- [1]. Reduced TRX has dithiols, and oxidized TRX has a disulﬁde bond in this active site. Reduced TRX is a powerful protein-disulﬁde reductase, and oxidized TRX is reduced by NADPH and thioredoxin reductase. TRX scavenges singlet oxygen and hydroxyl radical by itself [4] and hydrogen peroxide in cooperation with peroxiredoxin [5]. There is growing evidence that redox regulation by the TRX system plays a crucial role in biological responses against oxidative stresses [6]. Human TRX is a 12 kDa protein consisting of 105, amino acids and exists with or without N-terminal methionine. In addition to the two cysteine residues in the active site, mammalian TRX has three additional structural cysteines at positions 62, 69, and 73 (numbering based on human TRX), which bacterial TRX does not have. The C-terminal Cys73 is involved in dimerization when mammalian TRX is suﬀering from oxidative stress, and the dimer of TRX shows no reducing activity [7]. TRX is induced by various stresses and secreted from cells. Moreover, human TRX is cleaved at the site of Lys80 or Lys84 to become truncated TRX [8]. Truncated TRX shows no reducing activity and more cytokine-like functions [9]. Evidence shows that mammalian or human TRX has many biological properties. This review will focus on the biological roles of TRX in cellular redox control. II.

TRX INDUCTION BY OXIDATIVE STRESS

TRX is induced by a variety of oxidative stresses, including viral infection, hydrogen peroxide, ultraviolet light or x-ray irradiation, and chemical carcinogens. The promoter region of human TRX contains oxidative responsive elements (ORE), xenobiotic responsive elements (XRE), antioxidant responsive elements (ARE), cyclic AMP responsive elements (CRE), and SP-1–binding sites [10,11]. There is no evidence that heat shock can induce TRX. Therefore, TRX is a stress-inducible protein. TRX is induced in peripheral blood mononuclear cells (PBMC) by mixed lymphocyte reaction, phytohemagglutinin (PHA), ConA, or OKT3 monoclonal antibody (mAb), which is suppressed by the immunosuppressant FK506. Cyclosporin A also inhibits TRX expression in OKT3 mAb–stimulated PBMC, although rapamycin fails to aﬀect it in spite of exhibiting growth inhibition. In addition, exogenous IL-2 does not increase TRX production in FK506-treated PBMC or in PHA blasts [12]. In the erythroleukemic cell line K562, TRX is induced by hemin (ferriprotoporphyrin X). Previously, heat shock factor-2 was indicated to be responsible for this activation [13]. We recently showed that hemin activates TRX gene expression through the antioxidant responsive element (ARE) and that TRX gene induction is regulated via ARE by the binding of NF-E2 p45/small Maf in the unstimulated condition, of Nrf2/small Maf in hemin stimulation, and of Jun/Fos families of proteins in PMA stimulation [10]. This is a demonstration of a novel molecular mechanism through the ARE/EpRE (electrophile-responsive element) by a switch of binding factors including CNC-bZIP (Cap ‘n’ Collar/basic leucine zipper)/small Maf transcription factors and Jun/Fos families of proteins, depending on diﬀerent stimuli. III.

REDOX CONTROL BY TRX

Glutathione peroxidase is a powerful scavenger for hydrogen peroxide. Intracellular amounts of GSH are of the mM order and of TRX of the AM order. Therefore, TRX is a minor component as an intracellular antioxidant pool. However, TRX knockout

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mice are embryonically lethal [14]. Therefore, TRX plays a pivotal role in cells not only as an antioxidant but also as a redox regulator. A variety of biological functions of TRX are shown in Figure 1. There is accumulating evidence that the DNA-binding activities of several transcription factors are regulated by TRX in nucleus and transcription factors such as AP-1 and NF-nB, now called ‘‘redox-sensitive transcription factors.’’ In the case of NF-nB, oxidative stress induces the nuclear translocation of NF-nB and TRX. Intranuclear DNA binding of NF-nB is promoted by the reduction of the key cysteine residue of NF-nB by TRX [15,16]. In the case of AP-1, redox factor-1 (Ref-1) and TRX enhance the DNA binding of AP-1 [17]. Besides AP-1, p53 activation is also regulated by TRX and Ref-1 [18]. Even in the cytoplasm, the activities of some kinases are regulated by TRX. The activation of p38 mitogen– activated (MAP) kinase is suppressed by the overexpression of TRX [19]. Saitoh et al. identiﬁed apoptosis-regulating kinase-1 (ASK-1), a MAP kinase kinase kinase, as a TRX-binding protein using a yeast two-hybrid system [20]. Reduced TRX binds with ASK-1 to suppress the activation. When TRX is oxidized, ASK-1 is dissociated from TRX and dimerized to transduce the signal for apoptosis. By the yeast two-hybrid method, we also identiﬁed two proteins as TRX-binding proteins (TBP). TBP-1 was phox40, a component of NADPH oxidase [21]; TBP-2 was a 45 kDa protein previously reported as vitamin D3–upregulated protein-1 (VDUP-1) [22]. TBP-2/VDUP-1 suppresses the expression and reducing activity of TRX. We have found that TBP-2/ VDUP-1 is downregulated in IL-2–independent ATL cells, suggesting that TBP-2/ VDUP-1 may be involved in some step of the carcinogenesis of ATL. Quite recently it

Figure 1

Biological functions of TRX.

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was reported that mutation in TBP-2/VDUP-1 gene is associated with familial combined hyperlipidemia in mice [23]. IV.

TRX-FAMILY

Proteins sharing the active site -Cys-Xxx-Yyy-Cys- and a three-dimensional structure similar to TRX are now called the ‘‘TRX (super) family.’’ The number of TRX family members is growing quite rapidly. Each subcellular compartment has a speciﬁc TRX family; for example, mitochondria has thioredoxin-2 (TRX-2) [24] and glutaredoxin-2 [25] and the nucleus have nucleoredoxin [26,27]. The TRX family and TRX-related molecules are schematically shown in Figure 2. A.

TRX-2

TRX-2 was identiﬁed as a mitochondria-speciﬁc TRX by Spyrou et al. [24]. TRX-2 was cloned from a rat heart library and found to encode a 18 kDa protein with 166 amino acid residues that had a conserved TRX catalytic site but that lacked the other structural cysteine residues found in mammalian TRX. A 60-amino-acid N-terminal extension of TRX-2 exhibited characteristics consistent with a mitochondrial translocation signal, and Western blotting has conﬁrmed the mitochondrial localization of TRX-2. We recently cloned chicken TRX-2 and developed TRX-2 knockout cells using DT40 cells. TRX-2 knockout cells easily fall into apoptosis [28]. The radical-

Figure 2 Subcellular localization of TRX family. TRX: thioredoxin; GRX: glutaredoxin; PDI: protein disulfide isomerase; CaBP: calcium-binding protein; TMX: TRX-related transmembrane protein.

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scavenging activity in TRX-2 lacking cells is low, and intracellular ROS generation is increased. However, antioxidants cannot rescue TRX-2–deﬁcient cells completely. Release of cytochrome c from mitochondria into cytoplasm and activation of caspases occurs in TRX-2–deﬁcient cells. In vitro study shows that TRX-2 binds with cytochrome c in mitochondria, suggesting that TRX-2 may be involved in the regulation of mitochondria-mediated apoptosis signal pathway. V.

EXTRACELLULAR TRX

Besides ADF and 3B6-IL-1, other reports showed that other cytokine-like factors were also related or identical to human TRX. Human TRX was a component of early pregnancy factor (EPF), which was originally reported as an immunosuppressive factor of rosette formation (lymphocyte binding to heterologous red blood cells) in the serum of pregnant animals or women [29]. Eosinophil cytotoxicity enhancing factor (ECEF) was a 1-80 or 1-84 truncated form of TRX [8]. A B-cell growth factor derived from T-T cell hybridoma, MP-6, was demonstrated to an isoform of human TRX with truncated mRNA [30,31]. A sensitive enzyme-linked immunosorbent assay (ELISA) system using anti-human TRX monoclonal antibodies has been developed to measure TRX levels [32]. Serum or plasma levels of TRX in healthy individuals are roughly 20– 30 ng/mL. TRX is measurable in a variety of body ﬂuids, and serum/plasma TRX levels are good markers for oxidative stress.

Figure 3 Exogenous TRX protects endothelial cells. Recombinant human TRX (wild) attenuated the cytotoxicity of endothelial cells caused by activated neutrophils or hydrogen peroxide in a dose-dependent manner, whereas mutant TRX (C32S) did not. (Modified from Ref. 39.)

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Cytokine-Like Effect

Human TRX itself has growth-promoting eﬀects on leukemic cells and hepatocellular carcinoma cells [33,34]. Schenk et al. reported that human TRX enhances cytokine productions [35]. In contrast, TRX from Actinobacillus actinomycetemcomitans was identiﬁed as an immunosuppressive factor with an inhibitory eﬀect on cytokine actions [36]. Truncated TRX shows a more mitogenic eﬀect on lymphocytes and induces cytokine production from monocytes [8,37]. B.

Chemokine-Like Effect

Hori et al. showed that TRX is chemotactic for eosinophils from patients with hypereosinophilia [38]. Bertini et al. reported that TRX is chemotactic for neutrophils, monocytes, and T lymphocytes [39]. Because TRX does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin, the chemotactic action of TRX seems to diﬀer from that of known chemokines, which use G protein–coupled receptor system. A series of experiments using recombinant TRX with the mutation

Figure 4 Intravenous injection of TRX suppresses neutrophil extravasation in mouse air pouch model. Mouse air pouch was created by injecting 4 mL air subcutaneously at days-7 and day-3. LPS 0.1 Ag in 1 mL sterile pyrogen-free PBS or PBS alone was injected in the pouch. The exudate in the pouch was collected 4 hours later with 5 mL PBS and analyzed by FACS. When 40 Ag TRX was injected intravenously just prior to LPS injection into the air pouch, neutrophil extravasation induced by LPS was dramatically suppressed. The half-life of TRX in mouse blood was roughly 1 hour. (Modified from Ref. 41.)

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of the active site residues to serines resulted in loss of chemotactic activity, suggesting that the redox property is required. C.

Anti-Inflammatory Effect

We previously reported that exogenous recombinant human TRX (wild) but not mutant TRX (C32S) with no reducing activity suppresses endothelial cell injury caused by hydrogen peroxide or activated neutrophils, as shown in Figure 3 [40]. Based on this eﬀect, Fukuse et al. showed that intravenous administration of recombinant human TRX attenuates ischemia-reperfusion lung injury in animal models [41]. Recently, we revealed that TRX suppresses the activation, adhesion on endothelial cells, and extravasation of neutrophils [42]. The phosphorylation of p38 MAP kinase is detectable in lipopolysaccharide (LPS)–activated neutrophils. Pretreatment with recombinant TRX (wild) but not mutant TRX (C32S/C35S) that has no oxidoreduction activity suppresses LPS-induced phosphorylation of p38 MAPK. When LPS-stimulated human neutrophils are cultured on the monolayer of human umbilical endothelial cells, TRX (wild) but not mutant TRX (C32S/C35S) inhibits the adhesion of neutrophils on endothelial cells. Moreover, in mouse air pouch chemotaxis model, LPS induces extravasation of neutrophils into the air pouch. Intravenous injection of recombinant TRX from mouse tail vein suppresses the extravasation of neutrophils in this model. Moreover, in a mouse air pouch chemotaxis model, intravenously injected recombinant TRX from mouse tail vein suppressed LPS-induced extravasation of neutrophils into the pouch. The half-life of injected recombinant human TRX is roughly one hour. Even in TRX Tg mice, human TRX levels in the blood is elevated by injection of LPS into the air pouch and neutrophil extravasation is signiﬁcantly suppressed (Fig. 4). Together, intravenous injection of recombinant TRX can

Figure 5

Circulating TRX suppresses activation, adhesion on endothelial cells, and extravasation of neutrophils.

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Figure 6

Possible mechanism of signal transduction via extracellular TRX.

attenuate neutrophil inﬁltration into the inﬂammatory site and may be a novel tool for the regulation of inﬂammation (Fig. 5). The signal transduction mechanism of extracellular TRX is now under investigation. Since TRX is a 12 kDa protein, it is unlikely that TRX itself goes through the cell membrane. Our data showing that the redox-inactive mutant TRX does not show the extracellular cytoprotective or antiinﬂammatory eﬀect suggest that the active site of TRX is important for the signal transduction of extracellular eﬀect of TRX. The disulﬁde-dithiol exchange between extracellular TRX and possible target protein on the cell surface may be involved in the signal transduction. The study is ongoing to identify the possible target molecules for extracellular TRX (Fig. 6).

VI.

TRX IN INFECTION AND INFLAMMATION

A.

HTLV-I Infection

ATL is an endemic leukemia ﬁrst reported in Japan by Yodoi et al. [43,44]. HTLV-I was later discovered as a retrovirus causative of ATL [45,46]. ATL cells overexpress IL-2 receptor/alpha chain (CD25) [47]. Yodoi et al. puriﬁed a factor that has a CD25inducing potential on YT cells from the culture supernatant of HTLV-I–transformed ATL cell line ATL-2 cells and called it the ATL-derived factor (ADF) [48,49]. Gene cloning revealed that ADF is identical to human TRX [2]. HTLV-I–transformed cells overexpress and secrete TRX. TRX may be involved in the growth of ATL cells and a possible target for therapeutic approach to ATL. B.

Human Immunodeficiency Virus Infection

We analyzed the expression of TRX in human immunodeﬁciency virus (HIV)–infected cells. After acute infection of HIV in cell line cells, the expression of TRX as well as Bcl2 is transiently downregulated, and infected cells fall into apoptosis [50]. In persistent infection, intracellular TRX and Bcl-2 levels are restored. In HIV-infected individuals, TRX high-positive cells diappear in lymph nodes [51] and plasma levels of TRX ar elevated [31]. Plasma TRX levels are inversely correlated with intracellular GSH levels in lymphocytes, suggesting that systemic oxidative stress caused by inﬂammatory

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cytokines promotes the depletion of intracellular GSH levels and secretion of TRX. Moreover, immunocompromised HIV-infected individuals (AIDS patients) with elevated plasma TRX levels die more quickly than those with normal plasma TRX levels [52]. Anti-inﬂammatory eﬀects of circulating TRX may worsen the prognosis of such immunocompromised hosts. The possible roles of TRX in HIV infection are schematically shown in Figure 7. In vitro studies have shown that truncated TRX promotes the viral replication of HIV and the full length of TRX inhibits the viral replication and viral entry into CD4+ T cells, suggesting the possibility of clinical application of TRX for AIDS therapy. C.

Epstein-Barr Virus Infection

Around the same period when we cloned human TRX as ADF, human TRX was identiﬁed by several groups under several diﬀerent names. One of them was IL-1–like autocrine growth factor (3B6-IL-1) produced by Epstein-Barr virus (EBV)–transformed B cells [53,54]. TRX is induced and secreted from EBV-infected cells. Interestingly, exogenous TRX augments the proliferation of some EBV-transformed cells [32] but inhibits the mitogen-stimulated viral replication in other EBV-infected cells [55]. Other reducing agents such as N-acetylcysteine downmodulate the expression of CD21, a receptor for EBV entry, on the cell surface, suggesting that redox regulation may play a crucial role in the regulation of EBV infection [56]. D.

Hepatitis C Virus Infection

In Japan and other Asian countries, hepatitis C virus (HCV) infection is a serious problem, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. In

Figure 7

Possible roles of TRX in HIV infection.

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HCV infection, iron deposition in the liver induces oxidative stress and in some cases serum ferritin levels and serum TRX levels are elevated. Interestingly, cases with elevated serum ferritin and TRX levels are resistant to interferon therapy to reduce viral loads [57–59]. Therefore, serum TRX levels may reﬂect the redox status in the liver and be a good indicator for interferon therapy. E.

Rheumatoid Arthritis

In rheumatoid arthritis (RA), oxidative stress caused by inﬂammatory cytokines in the synovial ﬂuids leads to decreases of intracellular GSH levels in intrasynovial T cells and elevation of intrasynovial TRX levels [60]. Local oxidative stress in the joint with RA may cause a hyporesponsiveness of T cells (Fig. 8). In the synovial epithelial cells, macrophages are highly expressive of TRX [61]. Serum TRX levels also reﬂect the disease progress and inﬂammation in cases of RA [62,63]. Interesingly, one chemical compound with an anti-RA eﬀect suppresses the secretion of TRX from macrophages [64]. F.

Autoimmune Myocarditis

Autoimmune myocarditis sometimes precedes the development of dilated cardiomyopathy and heart failure. The myosin immunization model in rats mimics human fulminant myocarditis in the acute phase and human dilated cardiomyopathy in the chronic phase. In this rat myocarditis model, TRX expression is upregulated in the acute phase but not in the chronic phase and the expression is correlated with the

Figure 8

Possible roles of TRX in rheumatoid arthritis.

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severity of the disease [65]. Damaged myocytes are strongly immunostained for 8OHdG and NF-nB. Thus, TRX may be speciﬁcally induced by acute inﬂammatory stimuli and the development of acute immune-mediated myocarditis may be regulated by the cellular redox status via TRX.

VII.

TRX KNOCKOUT MICE

We attempted to develop TRX knockout mice. A part of the mouse TRX gene including the translation start codon was deleted by homologous recombination in embryonic stem cells. Heterozygotes of mutant allele are viable, fertile, and appear normal. In contrast, homozygous mutants die shortly after implantation at the egg cylinder formation stage. By day 6.5 of gestation, the embryos proper were not found in the deciduas and the tissues could not be recovered for genotyping [14]. Interestingly, Ref-1–deﬁcient mice also die shortly after implantation, at day 5.5 of gestation [66]. Because Ref-1 and TRX operate coordinately in the redox-sensitive activation of transcription factors such as AP-1, Ref-1 and TRX may be essential for the critical stage of early embryonic development.

VIII.

TRX TRANSGENIC MICE

Human TRX gene was inserted between the human beta-actin promoter and human beta-actin terminator in C57BL/6 mice and used to generate the transgenic mice. The phenotype and behavior of TRX-transgenic (TRX Tg) mice are apparently normal. TRX Tg mice contain severalfold larger amounts of human TRX protein in most organs compared with endogenous mouse TRX protein levels. TRX Tg are more resistant to a variety of oxidative stresses compared with wild-type C57BL/6 mice. Twenty-four hours after focal cerebral ischemia is induced by occlusion of the middle cerebral artery, the infarcted areas and volume are signiﬁcantly smaller in TRX Tg mice than in wild-type C57BL/6 mice [67]. Bone marrow cells from TRX Tg mice are more resistant to ultraviolet light exposure-induced cytocide than those of wild-type C57BL/6 mice. Interestingly, TRX Tg mice survive longer than wildtype C57BL/6 mice [68]. A.

Influenza Viral Infection

Transnasal administration of inﬂuenza virus induces severe pneumonia and body weight loss in mice. The median lethal dose (LD50) of inﬂuenza virus was 106 dilution in wild-type C57BL/6 mice and 105 dilution in TRX Tg mice, respectively. Therefore, TRX Tg mice are more resistant to inﬂuenza virus infection. Seven days after administration of sublethal dose inﬂuenza virus, body weight loss of wild-type C57BL/6 mice was roughly 20%, whereas that of TRX Tg mice not signiﬁcantly changed. There was no diﬀerence of anti-inﬂuenza hemagglutinin (HA) IgG antibody in serum and anti-HA IgA in the lung between wild-type C57BL/6 and TRX Tg mice. Histological examination showed that alveolar or bronchiolar destruction was prominent in wild-type C57BL/6 mice, whereas it was mild in TRX Tg mice, suggesting that the inﬂammatory response against inﬂuenza virus infection was apparently attenuated in TRX Tg mice [69].

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Interstitial Pneumonia

Intraperitoneal injection of bleomycin, an anticancer agent, induces leukocyte inﬁltration in the interstitial space of the lung in mice. Seven days after 2 mg bleomycin intraperitoneal injection, wild-type C57BL/6 mice exhibited severe interstitial pneumonia, whereas TRX Tg mice showed mild interstitial pneumonia. Daily intraperitoneal injection of IL-2 and IL-18 also induced severe interstitial pneumonia in mice. Wild-type C57BL/6 mice were all dead within 4 weeks of severe interstitial pneumonia, whereas 6 of 8 (75%) TRX Tg mice were survived. Inﬁltration of leukocytes in the interstitial space of the lung was dramatically attenuated in TRX Tg mice. IX.

CLINICAL APPLICATIONS OF TRX

As mentioned above, intravenous injection of recombinant human TRX attenuates ischemia-reperfusion injury in animal models [41]. In addition, local administration (intravitreous injection) of recombinant human TRX attenuates retinal cell damage caused by light exposure [70]. Moreover, intraperitoneal injection of recombinant human TRX also rescues mice from bleomycin- or cytokine-induced interstitial pneumonia. These results prompted us to develop GMP (good manufacturing practice) production of recombinant human TRX for clinical application. Geranylgeranylacetone (GGA), an acyclic polyisoprenoid developed in Japan, is widely used as an ulcer drug. In addition to a protective eﬀect on gastric mucosal cells, GGA also has antiapoptotic eﬀects against ischemia and reperfusion injury in hepatocytes and other cells. We have found that GGA induces TRX in hepatocytes and gastric mucosal cells and inhibits ethanol-induced cytotoxicity [71,72] GGA induces protein expression and secretion of TRX. GGA also induces TRX in PC12 cells and attenuates MPP-induced cytotoxicity, suggesting the use of GGA and/or TRX in the clinical treatment of Parkinson’s disease [73]. In addition to GGA, estrogen and prostaglandin E1 are also TRX inducers [74,75]. TRX induction by GGA and other TRX inducers may be beneﬁcial for oxidative stress–induced disorders.

ACKNOWLEDGMENTS This work was supported by a grant-in-aid for Scientiﬁc Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a grant-in-aid for Research for the Future from the Japan Society for the Promotion of Science. REFERENCES 1. 2.

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5 Role of Free Radicals and Cellular Redox Status in Signal Transduction and Gene Expression ¨ GE and WULF HILDEBRANDT WULF DRO Deutsches Krebsforschungszentrum, Heidelberg, Germany

I. A.

INTRODUCTION AND HISTORICAL BACKGROUND Role of Free Radicals in Biological Regulation

Free radicals in biological materials were originally viewed as byproducts of enzymatic reactions and as a source of cellular damage, mutagenesis, cancer, and aging-related degenerative processes [58]. Today we know, however, that nitric oxide and superoxide are generated by various isoforms of nitric oxide synthase and NAD(P)H oxidase, respectively, and that both radicals as well as the superoxide-derived reactive oxygen species (ROS) play an important role in biological regulation. In 1977 Mittal and Murad [117] reported the ﬁrst suggestive evidence that superoxide-derived hydroxyl radical may stimulate the activation of guanylate cyclase, which produces the ‘‘second messenger’’ cGMP. Ignarro and Kadowitz [69] and Moncada and colleagues [134] discovered independently the role of nitric oxide as a key regulatory agent in the regulation of vascular tone and in the inhibition of platelet adhesion in 1985 and 1987, respectively. Roth and Dro¨ge reported in 1987 that the superoxide radical or low micromolar concentrations of its derivative hydrogen peroxide can upregulate in activated T cells the production of the T-cell growth factor interleukin-2 (IL-2), i.e., the expression of an immunologically important protein [136]. Tyrrell and colleagues showed in a series of papers (reviewed in Ref. 83) that the superoxide derivative hydrogen peroxide induces the expression of the heme oxygenase (HO-1) gene. Storz and colleagues [159] found that hydrogen peroxide induces in bacteria the expression of various genes engaged in protective responses against oxidative stress, and Schreck 79

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and Baeuerle, ﬁnally, have shown that the activation of the transcription factor nuclear factor-nB (NF-nB) is enhanced by hydrogen peroxide in mammalian cells [147]. Not unexpectedly, many redox-responsive regulatory mechanisms in bacteria and higher organisms play a role in the induction of protective responses against oxidative stress. The relatively large number of nitric oxide synthase and NAD(P)H oxidase isoforms and their role in physiological functions illustrate, however, that nature has ‘‘learned’’ to use free radicals to her advantage for processes not directly related to protection against oxidative stress. The term ‘‘redox signaling’’ is generally used to describe a regulatory process in which the signal is delivered through redox chemistry. Nitric oxide either interacts with iron-containing proteins such as guanylate cyclase or aconitase [27,70], or it nitrosylates tyrosine or cysteine residues as in the case of the Ras protein [95]. S-Nitrosoglutathione, one of the important nitric oxide derivatives in vivo [47,78], was found to cause both S-nitrosylation (S-NO) and S-glutathiolation (S-SG) [77,187]. One of the typical reactions of hydrogen peroxide with proteins is the conversion of a cysteine thiol group into a sulfenic acid moiety (S-OH) (see, e.g., Ref. 87). The highly reactive sulfenic acid group may interact rapidly with small molecular weight thiol compounds and thereby form a mixed protein-cysteine or protein-glutathione disulﬁde. If such a redox-reactive protein happens to be a signaling protein, its derivatization may activate or suppress the corresponding signaling pathway. In the case of the bacterial OxyR transcription factor, it was shown that the various thiol derivatives S-NO, SOH, and S-SG occur in vivo as stable posttranslational modiﬁcations and diﬀer in functional properties [84]. B.

The Response of Signaling Cascades to Changes in the Intracellular Thiol/Disulfide Redox Status

Because many of the redox-sensitive signaling proteins function through redox-sensitive cysteine residues, they often respond not only to the chemical attack of ROS but also to changes in the intracellular redox status (REDST). In 1994, the group of Dro¨ge and colleagues showed that even moderate changes in the intracellular glutathione REDST may lead to a strong activation of NF-nB and the transcription factor activator protein-1 (AP-1) [46]. Independently, Kuge et al. reported the activation of the yAP-1 transcription factor of Saccharomyces cerevisiae by changes in REDST [92]. Subsequently, changes in REDST were also shown to modulate the K+ channel activity in the carotid body [6], the insulin receptor tyrosine kinase activity [145], the functional activity of the bacterial OxyR protein [11], the catalytic activity of protein tyrosine phosphatases [17] and Src family kinases [62], the activation of JNK and p38 MAPK signaling pathways and immunological functions [62], and the signaling of replicative senescence [154]. Numerous disease conditions appear to be mediated either by excessive ROS production or by an oxidative shift in REDST. A discussion of oxidative conditions in disease and aging is not within the scope of this chapter. II.

REDOX SIGNALING IN PROTECTIVE RESPONSES AGAINST OXIDATIVE STRESS

A.

Redox Signaling in Bacteria

The bacterial OxyR and SoxR proteins induce the expression of various genes that serve to protect the cells against oxidative stress, i.e., against the very agents that

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induce the regulatory response (reviewed in Ref. 190). The OxyR protein controls protective responses against normally lethal doses of hydrogen peroxide or against killing by heat. It is negatively regulated by its own expression. At least nine proteins synthesized in Escherichia coli after exposure to hydrogen peroxide, including catalase, hydroperoxidase I, glutathione reductase, and glutaredoxin 1, are under control of the oxyR locus [18,29,35]. Hydrogen peroxide converts the reduced form of OxyR into its oxidized and active form. Initially Cys-199 is converted into a sulfenic acid, which then forms an intramolecular disulﬁde bond with Cys-208 [18,191]. At the normal intracellular REDST of the bacteria (between 260 and 280 mV) OxyR is in the reduced form and can be activated either through direct oxidation by hydrogen peroxide or by an oxidative shift in REDST [11]. Both oxidized and reduced OxyR proteins bind to the oxyR/oxyS promotor region but have diﬀerent binding characteristics [171]. The transcription factor is inactivated through reduction of the disulﬁde bonds by glutaredoxin-1 or thioredoxin [191]. Glutathione reductase and glutaredoxin-1 are both regulated by OxyR and are therefore part of an autoregulatory circuit. The bacterial sox locus controls the protective response against superoxide and regulates the induction of various proteins, including Mn-SOD (reviewed in Ref. 18). The SoxR protein exists in solution as a homodimer containing two stable iron-sulfur centers that are anchored to four cysteine residues near the carboxy-terminal end [22,64,181]. Under normal physiological conditions, these iron-sulfur centers are in the reduced state but are reversibly oxidized under oxidative stress [38]. Only the oxidized form of SoxR stimulates transcription of soxS [22,38,48]. Additional examples of redox-responsive regulatory mechanisms in prokaryotic cells have been reviewed by Bauer et al. [18]. B.

Redox Signaling in Yeast

The oxidative stress response of the budding yeast S. cerevisiae after exposure to hydrogen peroxide involves the transcription factor yAP-1 [91]. The transcription factor binds speciﬁcally to the AP-1 site of the eukaryotic AP-1 family of transcription factors. A similar activation is seen under conditions associated with an oxidative shift in the intracellular glutathione REDST. The yAP-1 transcription factor induces the expression of thioredoxin from the TRX2 gene and other oxidative stress response genes. Oxidative activation of yAP-1 operates at the posttranslational level and involves the translocation of yAP-1 from the cytoplasm to the nucleus. Three conserved cysteine residues in the COOH terminus of yAP-1 are believed to be important in sensing the change in REDST and in mediating the translocation [92]. C.

Oxidative Stress Responses in Mammalian Cells

Similar redox-responsive signaling cascades are involved in oxidative stress responses of mammalian cells. Because activated macrophages and neutrophils in inﬂamed tissues of higher organisms generate large amounts of ROS to kill environmental pathogens, the host must protect its own cells against this oxidative burst. The oxidoreductase thioredoxin is one of the proteins that are inducibly expressed in lymphocytes and other cells by hydrogen peroxide and other conditions of oxidative stress [111,138,166]. Together with the glutathione system, thioredoxin plays a key role in the maintenance of a reducing intracellular REDST in higher organisms. The

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5V-upstream sequence of the human thioredoxin gene contains putative binding sites for the redox-responsive transcription factors AP-1 and NF-nB [125,126]. Exposure of macrophages to low levels of ROS or other conditions of oxidative stress also induces the expression of peroxiredoxin I (i.e., a thioredoxin peroxidase), heme oxygenase-1 (HO-1), and the cystine transporter xC [72,140]. In addition, ROS and other mediators of oxidative stress were found to enhance MnSOD mRNA levels in several cell types [152]. The HO-1 gene is one of the best studied models of redox regulation in higher organisms. HO-1 induction is believed to serve as an inducible defense pathway which removes heme liberated by oxygen. HO-1 protein and mRNA are strongly induced by ROS and other inducers of oxidative stress, including nitric oxide [60,82,172,173]. The sustained induction of HO-1 mRNA in conditions of oxidative stress and its inducibility in many tissues and diﬀerent mammalian species has rendered HO-1 mRNA a useful marker of cellular oxidative stress at the mRNA level. In murine macrophages, HO-1 expression was shown to be induced by hydrogen peroxide via the transcription factor AP-1 [8,24,25]. Activation of the mitogen-activated protein kinases (MAPKs), early-response kinase (ERK), and p38 was implicated in HO-1 expression in chicken hepatoma cells [43]. In view of the toxicity of ‘‘free’’ heme, HO-1 appears to be a redox-responsive product with protective activity against oxidative stress (reviewed in Ref. 7). More recently, however, it was found that certain products of HO-1 such as carbon monoxide (CO), biliverdin, and bilirubin may also mediate physiological functions, including vasorelaxation, induction of vascular endothelial growth factor (VEGF) expression, neurotransmission, and anti-inﬂammatory eﬀects [7,41,97]. D.

Protective Responses Against Oxidative Stress in Plants

Plants can also generate superoxide and hydrogen peroxide in response to environmental pathogens and other stress conditions [94,188]. These ROS induce certain defense genes or drive cells into apoptosis [75,94,99]. Even a local oxidative burst can lead to a state of systemic acquired resistance (SAR) [9], which is associated with the expression of several gene families [174]. Exposure of Arabidopsis to hydrogen peroxide leads to the activation of several MAPKs through the MAPK kinase ANP1 [89]. III.

REGULATED PRODUCTION OF FREE RADICALS

The HO-1 system has illustrated that responses to altered redox conditions may play a role not only in protective responses against oxidative stress but also in the regulation of other physiological functions. The numerous examples discovered during the last two decades strongly suggest that higher organisms would not exist without the signiﬁcant contribution of ‘‘redox regulation.’’ Most of the cases involve the regulated production of nitric oxide or superoxide by one of the nitric oxide synthase (NOS) or NAD(P)H oxidase isoforms, respectively. A.

Regulated Production of Nitric Oxide by Nitric Oxide Synthase

Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed but functionally regulated by the intracellular calcium concentration. The inducible isoform iNOS, in contrast, is expressed in macrophages and some other cell types

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after stimulation by cytokines or other immunologically relevant agents (reviewed in Ref. 20). Expression of iNOS is regulated at the transcriptional and posttranscriptional level by signaling pathways that involve the redox-responsive transcription factor NF-nB (see below) or MAPKs [107]. B.

The Regulated Production of Superoxide by NAD(P)H Oxidase Isoforms

The NAD(P)H oxidase of phagocytes plays a pivotal role in the host defense against microbial infection. It catalyzes the reduction of oxygen to superoxide by NAD(P)H. The enzyme is a heme-containing protein complex which is formed by the cytosolic proteins p67PHOX, p47PHOX, p40PHOX, and the small GTPase Rac together with the membrane-bound cytochrome b558 proteins gp91PHOX and p22PHOX. Activation of phagocytic NAD(P)H oxidase is typically regulated by Rac and induced by translocation of the cytosolic proteins to the membrane-bound cytochrome b558 complex [4,5,21,33,98]. The assembled activated oxidase complex is deactivated by interaction with a GTPase-activating protein (GAP) or by GDP [119]. The production of ROS by nonphagocytic NAD(P)H oxidase isoforms plays a role in the regulation of intracellular signaling cascades in various nonphagocytic cell types, including ﬁbroblasts, endothelial cells, vascular smooth mucle cells, cardiomyocytes, sperm, and thyroid tissue [10,12,42,52,54,79–81,101,113,161,163,165,168, 169,192]. Non-phagocytic cells appear to utilize structural homologues of gp91PHOX. Several such homologues have recently been identiﬁed. Muscle cells and ﬁbroblasts are the main source of superoxide in the normal vessel wall. The cardiovascular NAD(P)H oxidase isoforms are induced by hormones, hemodynamic forces, or local metabolic changes [54]. Angiotensin II stimulates NAD(P)H-dependent superoxide production in vascular smooth muscle cells and ﬁbroblasts. Thrombin, platelet-derived growth factor (PDGF), and tumor necrosis factor-a (TNF-a) stimulate NAD(P)H-dependent superoxide production in vascular smooth muscle cells. Interleukin (IL)-1, TNF-a, and platelet-activating factor (PAF) stimulate NAD(P)H-dependent superoxide production in ﬁbroblasts (reviewed in Ref. 54), and minimally oxidized low-density lipoprotein (LDL) was shown to activate NADPH oxidase in vascular endothelial cells [158]. The increased vascular superoxide formation under conditions of chronic hyperglycemia appears to result from the fact that high glucose increases mRNA expression of the oxidase subunit p22PHOX [28]. In most of these cases, Rac1 is involved in the induction of NAD(P)H oxidase activity [80,81,192]. Redox factor-1/APE was found to suppress oxidative stress in hepatocytes by inhibiting Rac1 GTPase [130]. There is a strong possibility that Rac-like proteins are also involved in the induction of NAD(P)H oxidase-like enzymes in plants [4,167].

IV.

REDOX REGULATION OF RECEPTOR-MEDIATED SIGNALING PATHWAYS

There is an increasing body of evidence that various receptors of growth factors, cytokines, or other ligands can stimulate ROS production and that the resulting ROS, in turn, can mediate a positive feedback eﬀect by enhancing the intracellular signaling from these receptors [12,15,19,31,83,101,102,103,106,114,153,162–164,169,176,182].

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This form of redox regulation also facilitates the synergistic interaction between diﬀerent types of membrane receptors, because one type of receptor may respond to the increased ROS production induced by another receptor. At least in some cases these membrane receptors or certain proteins in their signaling cascades respond not only to ROS, but also to physiological or pathological changes in REDST. A.

Enhancement of Signaling Cascades by Oxidative Inhibition of Protein Tyrosine Phosphatases

High concentrations of hydrogen peroxide in the order of 1 mM or strong prooxidative changes in the intracellular REDST lead typically to a strong increase in tyrosine phosphorylation of many cellular proteins [57,124,142–144,156]. The EGF receptor, for example, is typically dephosphorylated at all tyrosine residues less than one minute after ligand-induced autophosphorylation. This dephosphorylation is retarded by high concentrations of hydrogen peroxide or other inducers of oxidative stress [87]. These eﬀects of hydrogen peroxide are to some extent (but not exclusively) the consequence of the oxidative inhibition of protein tyrosine phosphatases. Phosphatases counteract the eﬀect of protein tyrosine kinases and reset membrane receptors after ligand-induced autophosphorylation. All protein tyrosine phosphatases share a common sequence motif with a catalytically essential cysteine residue in the active center [16]. Millimolar concentrations of hydrogen peroxide convert this cysteine residue into cysteine sulfenic acid (see Sec. I) and suppress thereby the catalytic activity of the enzyme [36]. Cysteine sulfenic acids are highly reactive and may be rapidly converted into a mixed proteinglutathione disulﬁde under the condition of the relatively high intracellular glutathione concentration. This disulﬁde derivative (i.e., the glutathiolated protein) also lacks catalytic activity [17]. Essentially the same oxidative inactivation of the phosphatase can also occur if glutathiolation is mediated by an oxidative shift in REDST [17]. Taken together, these ﬁndings indicate that protein tyrosine phosphatases are redox-responsive proteins that enhance signaling cascades through a loss of function. In view of the relatively high concentrations of hydrogen peroxide, typically used in these experiments, the physiological relevance of the oxidative inhibition of tyrosine phosphatases is still controversial. B.

Signaling by Angiotensin II

There is growing evidence that angiotensin II increases vascular oxidative stress [53,63,155]. In studies on glomerular mesengial cells as well as adult ventricular cardiomyocytes, angiotensin II–induced signaling cascades were found to be inhibited by diphenyleniodonium chloride (DPI), a speciﬁc inhibitor of NAD(P)H oxidase and other ﬂavoprotein-containing enzymes [51,180]. Moreover, the induction of p38 MAP kinase by angiotensin II in cardiomyocytes was shown to be abrogated by transfection with antisense oligonucleotides directed against phox 22 and nox, two distinct components of smooth muscle NAD(P)H oxidase [180]. Stimulation of superoxide production by angiotensin II was shown to also induce the dominant negative helixloop-helix protein Id3, which depresses the expression of p21WAF1/Cip1, p27KIP1, and p53 proteins. Hydroxyl radicals, in contrast, lead to the induction of the transcription factor gut-enriched Kru¨ppel-like factor (GKLF), which induces p21WAF1/Cip1, p27KIP1, and p53 [127].

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Signaling from the Epidermal Growth Factor Receptor and Related Membrane Receptors

A role of ROS has also been demonstrated in signaling processes stimulated by epidermal growth factor (EGF) in human epidermoid carcinoma cells [12], vascular endothelial growth factor (VEGF) in endothelial cells [3], platelet-derived growth factor (PDGF) [13,61], nerve growth factor (NGF) in neuronal cells [164], and ﬁnally in the a1-adrenoceptor signaling in adult rat cardiac myocytes [185]. Stimulation by the said growth factors induces transient ROS production through the signaling protein Rac1. Catalase was shown to inhibit EGF- and NGF-induced tyrosine phosphorylation of various cellular proteins, including the growth factor receptor itself. That the redox dependency of the signal transduction process may facilitate synergistic interactions between diﬀerent types of membrane receptors is exempliﬁed by the interaction between the angiotensin II type-1 receptor and the EGF receptor or PDGF-h receptor [54,61,135,177]. There is also suggestive evidence that the angiotensin II–mediated transactivation of the EGF receptor may involve the intermittent activation of the Src family kinase p60c-src by hydrogen peroxide [175]. D.

Role of ROS in the Regulation of Insulin Receptor Tyrosine Kinase Activity

Signaling by insulin involves autophosphorylation of the insulin receptor kinase domain at Tyr1158, Tyr1162, and Tyr1163. In line with the oxidative inhibition of tyrosine phosphatases by (approximately millimolar concentrations of) hydrogen peroxide or other strong oxidants (see previous section), it was found that similar strongly oxidative conditions enhance the tyrosine phosphorylation of the insulin receptor h-chain in an insulin-independent fashion. Lower and physiologically relevant concentrations (<100 AM) of hydrogen peroxide, in contrast, do not induce phosphorylation in the absence of insulin but enhance the response to insulin in a synergistic fashion [146]. More recent experiments have shown that the stimulatory eﬀect of hydrogen peroxide can be demonstrated on highly puriﬁed preparations of the insulin receptor kinase domain, indicating that the insulin receptor kinase domain serves by itself as a redox-sensitive signaling compound (W. Dro¨ge, unpublished observation). Because hydrogen peroxide production can be induced by insulin [90,113,121], the redox eﬀect appears again to be part of a positive feedback regulation that is reminiscent of the redox control of other membrane receptor signaling pathways (see previous sections). As in the case of several other signaling processes, the insulin-dependent insulin receptor kinase activity is enhanced not only by ROS, but also by a pro-oxidative shift in the intracellular glutathione REDST [145]. Antioxidants such as butylated hydroxyanisol or the glutathione precursor N-acetylcysteine inhibit the induction of insulin receptor kinase activity [145]. Higher concentrations of hydrogen peroxide, however, and prolonged exposure to moderate concentrations of hydrogen peroxide tend to inhibit insulin receptor signaling [56,170]. E.

Redox Regulation of Cytoplasmic Protein Kinases

High (approximately millimolar) concentrations of hydrogen peroxide or other strongly oxidative agents lead to the activation of protein tyrosine kinases and to

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the phosphorylation of numerous proteins. Exposure of T cells to 0.15–0.5 mM hydrogen peroxide, in contrast, was found to yield only a single major phosphorylated protein, the protein tyrosine kinase p56lck, and the induction of its catalytic activity [124]. Moderate oxidative conditions appear to also activate the related tyrosine kinase p59fyn as suggested by the ﬁnding that hydrogen peroxide causes the activation of JAK2 and Ras in normal murine ﬁbroblasts and p60src-deﬁcient ﬁbroblasts, but not in p59fyn-deﬁcient ﬁbroblasts [2]. Moreover, the Src family kinases p59fyn and p56lck were found to be strongly activated by a mild oxidative shift of the intracellular REDST in human T-lineage cells, and the kinase activity of isolated p59fyn protein was activated by low micromolar concentrations of glutathione disulﬁde [62]. The protein tyrosine kinase p60src, ﬁnally, was found to be critically involved in the activation of JNK by V100 AM hydrogen peroxide in red vascular smooth muscle cells and ﬁbroblasts [189]. Whether the redox responsiveness of this protein tyrosine kinase family can be conﬁrmed in studies of puriﬁed kinase preparations remains to be determined. F.

Oxidative Activation of Protein Kinase C Isoforms

Protein kinase C-a (PKC-a) and certain other PKC isoforms are activated by hydrogen peroxide in a phospholipid-independent process that involves tyrosine phosphorylation within the catalytic domain [50,88]. Oxidative activation of PKC-a is enhanced by vitamin A and certain derivatives such as 14-hydroxy-retroretinol, which binds to a zinc ﬁnger domain in the conserved cysteine-rich region of the regulatory domain [67]. A similar retinol-dependent activation has been demonstrated for the serine/threonine kinase cRaf [67,71]. G.

Oxidative Activation of Transcription Factor AP-1

The transcription factor AP-1 is typically composed of c-Fos and c-Jun proteins and generally implicated in diﬀerentiation processes. In T lymphocytes, AP-1 regulates the expression of the IL-2 gene and other immunologically relevant genes [110,160]. The mRNAs of c-fos and c-jun are induced by relatively small amounts of hydrogen peroxide, superoxide, nitric oxide, and other inducers of oxidative stress [32,39,76,115, 118,120,128,151]. Activation of AP-1 and its upstream signaling cascades can also be enhanced by a change in the intracellular REDST [46,62]. A mild oxidative shift induced by any of three diﬀerent oxidants, including hydrogen peroxide, was found to result in a strong enhancement of JNK and p38 MAPK activity [62]. H.

Oxidative Activation of Transcription Factor NF-n nB

The transcription factor NF-nB is implicated in inﬂammatory reactions, growth control, and apoptosis [14,15] and is involved in the induced expression of the immunologically important IL-2 gene. It was the ﬁrst eukaryotic transcription factor shown to respond directly to oxidative stress in certain types of cells [147]. A large body of evidence suggests that ROS comodulate the activation of NF-nB in combination with other inducing stimuli (reviewed in Ref. 39). NF-nB activation is also enhanced by a moderate prooxidative shift in the glutathione REDST [46,62] and inhibited by antioxidants including cysteine [115,116,141,148,157].

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Oxidative Induction of the Cell Cycle Inhibitor p21

The protein p21 (WAF1, CIP1, sdi1) inhibits the cell cycle by interacting with multiple targets, including cycline-dependent kinases [59,186]. ROS were shown to induce p21 gene expression by an unknown mechanism that may involve p53 [34,108,129]. Therefore, whereas oxidative conditions may induce or enhance cellular responses (see previous sections), they tend to inhibit the proliferative phase of the response. J.

Signal Amplification by a Late Shift to Reducing Conditions

Oxidative conditions are not only inhibitory for the cell cycle by enhancing p21 activity (see previous section) but also inhibit the DNA-binding activity of transcription factors such as AP-1 and NF-nB (reviewed in Ref. 39). Physiologically relevant concentrations of glutathione disulﬁde were found to inhibit DNA binding of NFnB even in the presence of a severalfold excess of low-molecular-weight thiols. DNA binding was restored by thiols such as 2-mercaptoethanol and dithiotreitol and by physiologically relevant concentrations of reduced thioredoxin [46,163]. NMR studies have demonstrated the binding of a thioredoxin peptide to the DNA-binding domain of NF-nB [133]. Oxidation of a conserved cysteine residue in the DNA-binding region of AP-1 was also shown to impair its DNA binding [183]. Binding activity was restored by thioredoxin and the nuclear signaling protein redox factor-1 (Ref-1) [1,183,184] and markedly enhanced by transient overexpression of thioredoxin in vivo [141]. Taken together, the available evidence indicates that the induction of signaling cascades leading to the activation of AP-1 and NF-nB is typically enhanced by relatively oxidative conditions, whereas the ﬁnal execution of these signaling processes is favored by more reducing conditions. K.

Regulation of T-Cell Proliferation by cAMP-Mediated Redox Pathways

Elevation of intracellular cyclic adenosine monophosphate (cAMP) in T lymphocytes typically inhibits the cell surface receptor–mediated stimulation of T-cell proliferation and other T-cell functions. This inhibition is associated with a decreased level of the phosphorylated (activated) MAPKs, ERK-1, and ERK-2. Recent studies of helper T cells of type 1 (Th1) have shown that the elevation of intracellular cAMP levels creates an oxidative environment that oxidizes and inactivates p56lck by a hydrogen peroxide– dependent mechanism [30]. In view of other reports on the oxidative activation of p56lck kinase activity (described above), there is a strong possibility that the redox regulation of this and other kinases of the Src family may be complex and may involve activating and inactivating redox eﬀects. L.

Redox-Mediated Amplification of Immune Responses

Immune responses against environmental pathogens typically involve the lymphocyte receptors for antigen, receptors for costimulatory signals, and various cytokine receptors [149,150]. In addition, the immune response is subject to regulation by redox processes due to the various redox-sensitive signaling pathways discussed in the previous section. The activation of T-cell functions is strongly enhanced by ROS and/or by an oxidative shift in the intracellular glutathione REDST [62,106]. Superoxide and/or physiologically relevant concentrations of hydrogen peroxide were

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shown to augment the production of IL-2 by antigenically or mitogenically stimulated T lymphocytes in various experimental systems [105,136]. In addition, low micromolar concentrations of hydrogen peroxide were found to induce the expression of the IL-2 receptor in mouse T-lineage cells [105]. The amplifying eﬀect of ROS has long been ignored by immunologists because T-cell functions, such as IL-2 production, can be readily induced by suﬃciently high concentrations of antigen or antibodies that bind to the T-cell receptors and the costimulatory receptor CD28 in the absence of additional ROS [100]. In T cells, strong activation of the co-stimulatory receptor CD28 causes a signiﬁcant decrease in intracellular glutathione levels and the endogenous production of hydrogen peroxide [37,105]. In B lymphocytes, ROS production was shown to be induced by ligation of the CD40 receptor [96]. In a typical immune response against environmental pathogens in vivo, however, high ligand concentrations for the various lymphocyte receptors may be the exception rather than the rule, because the antigen concentrations are relatively small in the initial phase of the infection. ROS production by activated macrophages or neutrophils in the inﬂammatory environment may therefore be decisive for the survival of the infected host, because it ampliﬁes the signaling cascades and decreases thereby the antigen thresholds required for lymphocyte activation. Importantly, physiologically relevant concentrations of ROS or other inducers of moderate oxidative stress were found to amplify the signaling cascades after relatively weak stimulation of the antigen receptor but did not replace the signal from the CD28 costimulatory receptor. To reconcile the seemingly conﬂicting redox requirements for the induction and execution of signaling processes (see previous section), the immune system requires, on the average, a delicately balanced intermediate redox state [40]. A study on healthy human subjects showed that persons with intermediate intracellular glutathione levels (20–30 nM/mg protein) had higher CD4+ and higher CD8+ T-cell numbers than persons with either lower or higher glutathione levels [85]. However, lymphocytes are extremely mobile and may migrate from an oxidative inﬂammatory environment into a more reducing environment in the course of an immune response. Experiments with cultured T cells have shown that activation of AP-1 and NF-nB transcription factors is strongly enhanced when cells are stimulated under relatively oxidative conditions and shifted after 1 hour to more reducing conditions [46]. This eﬀect of the shift in redox condition may be viewed as an additional fail-safe mechanism against the undesirable activation of immune reactions, for example, against autoantigens. In line with the diﬀerential redox requirement in the induction and execution phase of the signaling cascade, it was found that simultaneous injection of glutathione suppressed the in vivo immunization by small numbers of stimulator cells (i.e., small amounts of antigen) but enhanced the in vivo immunization with relatively large numbers of stimulator cells [137]. M.

Different Immunological Functions of Macrophages Associated with Differences in Intracellular REDST

Macrophages vary strongly in their release of nitric oxide, prostaglandins, and cytokines, such as IL-6 and IL-12, depending on their intracellular glutathione content. The balance between ‘‘reductive’’ and ‘‘oxidative’’ macrophages, which express high and low intracellular glutathione concentrations, respectively, was found

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to regulate the ratio of Th1 versus Th2 cells [122,123,131]. Exposure of macrophages to interferon-g (IFN-g) favors the reductive phenotype, and exposure to IL-4 favors the oxidative phenotype of macrophages. In view of the aging-related change in the oxidative shift in the plasma REDST, it is reasonable to assume that this change in REDST also accounts for the age-related shift in the Th1/Th2 ratio towards a Th2 phenotype of helper T cells. V.

PHYSIOLOGICAL RESPONSES TO CHANGES IN OXYGEN CONCENTRATION

A.

Transcription Factor Hypoxia-Inducible Factor 1

The adequate supply of oxygen to cells and tissues of higher organisms is regulated by a number of oxygen-sensing mechanisms, which control the red blood cell mass, the hypoxic ventilatory response, and other physiological functions. The red blood cell mass is tightly regulated by the hormone erythropoietin (Epo), which is produced mainly in kidney and liver cells. The expression of Epo and numerous other proteins, including the vascular endothelial growth factor (VEGF), is controlled by the transcription factor hypoxia-inducible factor-1 (HIF-1), a heterodimeric protein composed of the subunits HIF-1a and HIF-1h [178]. The level of HIF-1a is downregulated under high O2 concentrations through ubiquitination and proteasomal degradation [44, 68], which are initiated by the hydroxylation of HIF-1a at two speciﬁc prolyl residues and subsequent binding of the von Hippel-Lindau tumor suppressor protein (VHL) to the hydroxylated HIF-1a subunit [112]. Because molecular oxygen is a ratelimiting substrate in this process, the respective prolyl hydroxylase, a 2-oxoglutarate– dependent dioxygenase, functions as the oxygen sensor [44,73,74,109]. Earlier studies suggested that the regulation of Epo production and degradation of HIF-1a under normoxic conditions may involve the intermittent formation of superoxide or other ROS [26,68], but the more recent studies on the prolyl hydroxylation of HIF-1a have provided no evidence for a role of ROS. In vivo studies on human subjects have shown, however, that Epo production is enhanced after treatment with N-acetylcysteine, i.e., after a reductive shift in the plasma REDST and/or increase in plasma thiol concentrations [65]. This phenomenon is possibly explained by the fact that the 5V-ﬂanking region of the Epo gene contains several binding sites for the redox-sensitive NF-nB transcription factor (discussed above) and that activation of NF-nB leads to the inhibition of Epo mRNA expression and Epo secretion [93]. In addition, HIF-1–transactivating activity is increased by the redox-regulatory protein Ref-1 and by thioredoxin-1, the oxydoreductase known to reduce Ref-1 [45,179]. Overexpression of thioredoxin was found to increase the expression of HIF-1a protein and the production of VEGF in human breast cancer cells, colon cancer cells, and mouse lymphoma cells. It thus appears that the HIF-1– transactivating activity is modulated not only by the oxygen supply but also by the intracellular REDST. B.

Control of Ventilation

The carotid body is a sensory organ that controls (to a large extent) the ventilatory response to changes in arterial blood oxygen (hypoxic ventilatory response), as indicated by the fact that responses to hypoxemia are negligible in animals and humans

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with denervated or resected carotid bodies [66]. The mechanism of this response is not entirely clear but has been suggested to involve several diﬀerent ROS-producing proteins, including a b-type cytochrome with properties similar to those of the cytochrome b558 of the phagocytic NAD(P)H oxidase complex (reviewed in Ref. 5). Other studies suggest that the changes in oxygen tension may be sensed by the carotid body through changes in the rate of mitochondrial ROS production [23, 132]. It is widely believed that the transduction of the sinus nerve signal in response to changes in oxygen tension may involve changes in the conductivity of the K+ channel in type I cells [104], the h-subunit of which resembles the structure of NAD(P)H oxidase [55]. The evidence for a role of ROS in the regulation of the hypoxic ventilatory response, however, is largely indirect and still controversial (see also Ref. 49). This applies also to the role of the extracellular or intracellular REDST. Recently, it was also found that the ventilatory response to chronic hypoxia was impaired in mice partially deﬁcient for HIF-1a [86]. Acker and Xue [6] implicated ROS-mediated changes in the glutathione REDST in the control of the K+ eﬄux and Ca2+ inﬂux in the carotid body, but SanzAlfayate and colleagues [139] failed to see a signiﬁcant eﬀect of N-acetylcysteine on the normoxic or hypoxic release of 3H-catecholamines from rabbit and calf carotid bodies in vitro. In normal healthy subjects in vivo, in contrast, the hypoxic ventilatory response was shown to be signiﬁcantly enhanced by treatment with N-acetylcysteine, indicating that it is inﬂuenced by the plasma thiol concentration and/or the plasma REDST [65]. It remains to be determined whether this eﬀect acts directly on the carotid body chemoreceptor. Lipton and colleagues reported that hypoxia also causes hemoglobin to release S-nitrosothiols, which may increase ventilation by directly stimulating solitary tract neurons [101]. This mechanism was proposed to modulate the response of the peripheral chemoreceptors [52]. There is clearly the possibility that the amounts of nitrosothiols generated under hypoxic conditions are signiﬁcantly modulated by the plasma thiol concentration or plasma REDST [65].

GENERAL CONCLUSIONS Signaling pathways that are activated by free radicals or radical-derived reactive oxygen species (ROS) and that induce protective responses against these agents are found in higher organisms as well as bacteria. Higher organisms, however, have also evolved the use of free radicals and radical-derived ROS in the signaling of other physiological functions. The redox-responsive target proteins in many cases expose redox-sensitive cysteine residues that are oxidized not only by the said radicals or radical-derived ROS, but also by changes in the thiol/disulﬁde redox status (REDST).

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174. Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J. Acquired resistance in Arabidopsis. Plant Cell 1992; 4: 645–656. 175. Ushio-Fukai M, Griendling KK, Becker PL, Alexander RW. Role of reactive oxygen species in angiotensin II-induced transactivation of epidemal growth factor receptor in vascular smooth muscle cells. Circulation 1999; 100(suppl):I-263. 176. Wang CY, Mayo MW, Baldwin ASJ. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NFnB. Science 1996; 274:784–787. 177. Wang D, Yu X, Cohen RA, Brecher P. Distinct eﬀects of N-acetylcysteine and nitric oxide on angiotensin II-induced epidermal growth factor receptor phosphorylation and intracellular Ca2+ levels. J Biol Chem 2000; 275:12223–12230. 178. Wang GL, Jiang B-H, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic helixloop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995; 92:5510–5514. 179. Welsh SJ, Bellamy WT, Briehl MM, Powis G. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1a protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis. Cancer Res 2002; 62:5089–5095. 180. Wenzel S, Taimor G, Piper HM, Schlu¨ter K-D. Redox-sensitive intermediates mediate angiotensin II-induced p38 MAP kinase activation, AP-1 binding activity, and TGF-h expression in adult ventricular cardiomyocytes. FASEB J 2001; 15:2291–2293. 181. Wu J, Dunahm WR, Weiss B. Overproduction and physical characterization of SoxR, a [2Fe-2S] protein that governs an oxidative response regulon in Escherichia coli. J Biol Chem 1995; 270:10323–10327. 182. Wu M, Lee H, Bellas RE, Schauer SL, Arsura M, Katz D, FitzGerald MJ, Rothstein TL, Sherr DH, Sonenshein GE. Inhibition of NFnB B/Re1 induces apoptosis of murine B cells. EMBO J 1996; 15:4682–4690. 183. Xanthoudakis S, Curran T. Identiﬁcation and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA binding activity. EMBO J 1992; 11:653–665. 184. Xanthoudakis S, Miao G, Wang F, Pan YC, Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J 1992; 11:3323– 3335. 185. Xiao L, Pimentel DR, Wang J, Singh K, Colucci WS, Sawyer DB. Role of reactive oxygen species and NAD(P)H oxidase in a1-adrenoceptor signaling in adult rat cardiac myocytes. Am J Physiol Cell Physiol 2002; 282:C926–C934. 186. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993; 366:701–704. 187. Yanbin J, Akerboom TPM, Sies H, Thomas JA. S-Nitrosylation and S-glutathiolation of protein sulfhydryls by S-nitroso glutathione. Arch Biochem Biophys 1999; 362:67–78. 188. Yang T, Poovaiah BW. Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin. Proc Natl Acad Sci USA 2002; 99:4097–4102. 189. Yoshizumi M, Abe J, Haendeler J, Huang Q, Berk BC. Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem 2000; 275:11706–11712. 190. Zheng M, Storz G. Redox sensing by prokaryotic transcription factors. Biochem Pharmacol 2000; 59:1–6. 191. Zheng M, A˚slund F, Storz G. Activation of the OxyR transcription factor by reversible disulﬁde bond formation. Science 1998; 279:1718–1721. 192. Zweier JL, Broderick R, Kuppusamy P, Thompson-Gorman S, Lutty GA. Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation. J Biol Chem 1994; 269:24156–24162.

6 Redox Regulation of Apoptosis MARIE-VE´RONIQUE CLE´MENT and SHAZIB PERVAIZ National University of Singapore, Singapore

I.

INTRODUCTION

The cellular redox state is deﬁned as the balance between intracellular production of reactive oxygen species (ROS) and the antioxidant defense systems. An increase in the intracellular generation of ROS or a defect in the antioxidant scavenger enzymes renders the cells oxidatively stressed. Oxidative stress has often been referred to as a state harmful for cell survival, and consistent with that there is an abundance of literature directly or indirectly implicating ROS in cell and tissue damage [1]. However, under conditions where the intracellular production of ROS is not excessive, a mildly prooxidant state ensues, which could directly stimulate cell division and activation of transcription and at the same time inhibit the execution of the cell death signal [2,3]. These ﬁndings seem to tie up well with the observations that ROS generated upon receptor ligation can function as second messengers to signal proliferation and diﬀerentiation and that in certain cell types, in particular tumor cells, constitutively higher levels of ROS function as autocrine growth stimulation signals [4]. Thus, at doses that are not harmful to the cells, ROS have physiological roles ranging from induction of proliferation to modulation of cell death. This chapter is aimed at ﬁrst familiarizing the reader with the basic biology and intracellular regulation of ROS and then, keeping in view our work over the last few years and that of many other groups, discussing the role and/or eﬀect(s) of intracellular ROS on cell survival and death signals. 101

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II.

REACTIVE OXYGEN SPECIES

A.

Definition

Reactive oxygen species is a collective term that includes not only oxygen radicals (superoxide and hydroxyl) but also some nonradical derivatives of molecular oxygen (O2) such as hydrogen peroxide (H2O2) [5]. Indeed, the term ‘‘reactive’’ here is relative, as two of the principal ROS involved in cell signaling, i.e., superoxide (O2 ) and H2O2, are only moderately reactive with other biological molecules, but can sometimes give rise to the highly reactive hydroxyl radical (. OH), which might be directly responsible for much of the oxidative damage attributed to ROS in biological systems [5].

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B.

Intracellular Sources of ROS

ROS are produced in all mammalian cells, partly as a result of normal cellular metabolism and partly due to activation of membrane-bound enzyme systems, such as the NADPH oxidase complex, in response to exogenous stimuli. 1.

The Mitochondrial Electron Transport Chain

In many aerobic cells, electron transport chains are probably the most important in vivo source of ROS, in particular O2 . In the eukaryotic system these electron transport chains are speciﬁcally located in the mitochondria and endoplasmic reticulum. The mitochondrial electron transport chain is a multicomponent system involved in a series of oxidation-reduction reactions between redox couples or pairs—transfer of electrons from a suitable donor (reductant) to a suitable electron acceptor (oxidant). These oxidation-reduction reactions involve either the transport of electrons only, as in the case of the cytochromes, or electrons and protons together, as occurs between NADH and FAD. The part of the electron-transport chain that actually uses O2 is the terminal oxidase enzyme cytochrome oxidase [5]. Cytochrome oxidase releases no detectable oxygen radicals into free solution, but during the transfer of electrons through earlier components of the transport chain a few electrons do leak out directly on to O2, resulting in the generation of O2. It is estimated that under physiological O2 concentrations, only 1–3% of the O2 reduced in the mitochondria may form O2. This low rate of leakage is probably due to the relatively low intramitochondrial O2 and to the arrangement of electron carriers into complexes that facilitate movement of electrons to the next component of the chain rather than their escape to O2. It follows that damage to mitochondrial organization that severely aﬀects the smooth ﬂow of electrons through the electron transport chain could favor leakage of electrons and increase O2 production [5]. The supposition that mitochondria are the principal source of ROS during oxidative tissue injury derives from the observations that isolated mitochondria produce O2 through either autooxidation of the ﬂavin component of complex I (NADH hydrogenase) and/or by autooxidation of the ubisemiquinone at complex III.

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2.

NADH/NADPH Oxidase

A second potent source of intracellular ROS, best characterized in neutrophils and cells of the phagocytic lineage, is the plasma membrane–bound multisubunit NADPH enzyme complex. Indeed, the production of ROS by the NADPH oxidase

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system represents the major microbicidal mechanism of professional phagocytes [6,7]. The enzyme complex consists of a membrane heterodimeric ﬂavocytochrome (cytochrome b559) consisting of two subunits, gp91phox and p22phox, and four cytosolic proteins, p47phox, p67phox, p40phox [8], and the small guanosine triphosphate (GTP)–binding protein Rac (1 and 2) [9,10]. The primordial oxygen radical generated by this complex is O2 , produced by the NADPH-derived one-electron reduction of O2 [8]. Elicitation of O2 production in vivo involves the stimulus-dependent translocation of some or all cytosolic components of this complex to the plasma membrane and their assembly with cytochrome b559. In nonphagocytic cells, including tumor cells, the proteins responsible for producing O2 and other ROS are less well deﬁned, but could well function in a manner similar to the phagocyte NADPH-oxidase complex. Indeed, it has been shown that similar to the phagocyte oxidant generating system, the production of ROS in nonphagocytic cells is regulated by the small GTPbinding protein Rac1 [11–13].

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C.

Cell Antioxidant Defenses

Historically, ROS production has been thought to be harmful to the cell [1,14,15]. This idea is supported by the fact that levels of ROS are tightly regulated by multiple defense mechanisms involving small antioxidant molecules, which often contain sulfhydryl groups, and ROS-scavenging enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase [5]. Antioxidant enzymes are induced by exposure of cells to ROS and signaling molecules, such as cytokines. Deﬁciencies or alterations in the cell’s inherent antioxidant defenses modulate the fate of ROS in the cell, hence its redox status. In this regard, it is important to note that the composition of antioxidant enzyme systems varies from tissue to tissue and even from one cell to another in a given tissue, a factor that could explain the varied responses of diﬀerent cell types to oxidant challenge. 1.

Scavenging O2.

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In eukaryotic cells the intracellular concentration of O2 is tightly regulated by the activities of the two principal scavenger enzymes, Cu/Zn SOD and Mn SOD [16]. The discovery of SOD enzymes provides much of the basis for our current understanding of antioxidant defense systems. Cu/Zn SOD. The Cu/Zn SOD enzyme, found in virtually all eukaryotic cells, has a relative molecular mass of about 32 kDa and contains two protein subunits, each of which bears an active site containing one Cu and one Zn cation. Primarily located in the cytosol, but also present in lysosomes, nucleus, and the mitochondrial intermembranous space, Cu/Zn SOD catalyzes the dismutation of O2 to H2O2 and O2 [5].

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O.2 þ O.2 þ 2Hþ ! H2 O2 þ O2 ðground stateÞ MnSOD. First isolated from Escherichia coli, Mn SOD in eukaryotic cells is expressed mainly in the mitochondria. Unlike Cu/Zn SOD, Mn SOD is not inhibited by cyanide or diethyldithiocarbamate, has a relative mass of 40 kDa, is destroyed by treatment with chloroform plus ethanol (and hence does not survive the typical puriﬁcation methods for Cu/Zn SOD), and contains manganese at its active site.

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Despite these diﬀerences, Mn SOD catalyzes essentially the same reaction as Cu/Zn SOD [5]. 2.

Scavenging H2O2

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As mentioned previously, dismutation of O2 generates H2O2. H2O2 is usually removed in cells by two types of enzymes—catalase and peroxidases. Catalase directly catalyzes the decomposition of H2O2 to ground state O2 and is unique in that H2O2 serves as both a donor and acceptor, while the peroxidases remove H2O2 by using it to oxidize other substrates, such as NADH and GSH. Catalase. Catalase consists of four subunits, each of which contains a ferric heme group bound to its active site. Catalase activity in cells is largely found in subcellular organelles bound to a single membrane and known as peroxisomes. Mitochondria and the endoplasmic reticulum (ER) contain little, if any, catalase. Hence, any H2O2 produced by these organelles in vivo cannot be disposed of by catalase unless H2O2 diﬀuses to the peroxisomes. The catalase reaction is essentially a dismutation reaction similar to SOD; one H2O2 is reduced to H2O and the other oxidized to O2. Catalase FeðIIIÞ þ H2 O2 ! compound I þ H2 O Compound I þ H2 O2 ! catalase FeðIIIÞ þ H2 O þ O2 The exact structure of compound I is uncertain [5]. Glutathione Peroxidase. Glutathione peroxidase (GPX) consists of four protein subunits, each of which contains one atom of the element selenium at its active site [5]. GPX removes H2O2 by coupling its reduction to H2O with the oxidation (GSSG) of reduced glutathione (GSH) [17]. H2 O2 þ 2GSH ! GSSG þ 2H2 O Most cells contain substantial activities of both GPX and catalase [17]. Usually, subcellular compartmentalization inﬂuences H2O2 removal. H2O2 produced by peroxisomal enzymes such as glycolate oxidase and urate oxidase is largely disposed of by catalase, whereas H2O2 arising from the mitochondria, the ER, or by the action of cytosolic CuZn SOD is acted upon by GPX [17].

III.

ROS AND CELL DEATH

Despite the abundant literature demonstrating cellular toxicity of ROS, convincing evidence is now emerging to strongly suggest that ROS, in particular O2 and H2O2, constitute a novel class of cellular second messengers that appear to be involved in mitogenic stimulation of cultured cells [2,11,12,18]. Moreover, a cellular prooxidant state where the intracellular levels of activated forms of oxygen are increased due to either overproduction or deﬁcient antioxidant defenses can modulate expression of early growth–related genes like c-fos and c-jun [2], leading in some cases to aberrant expansion of rapidly growing clones and formation of tumors. Moroever, as an increase in cell survival could be due to either induction of proliferation or a reciprocal defect in cell elimination, it is logical that the survival advantage provided by a prooxidant state could also be through creating an intracellular environment inhib-

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itory to pathways required for eﬃciently carrying out the cells’ death execution program. Thus, at concentrations that are not harmful to the cells, ROS may have an important physiological role, such as inhibition of cell death. A.

Two Modes of Cell Death

Based on morphological and pathological criteria, cell death is currently subdivided into two categories: necrosis (accidental cell death) and apoptosis (programmed cell death) [19,20]. Oxidative stress has been implicated in both necrotic and apoptotic forms of death. For example, in mammalian cells, adding millimolar amounts of H2O2 often causes death by necrosis, whereas lower concentrations of the same can eﬀectively trigger the apoptotic pathway [21,22]. On the other hand, we and others have demonstrated the ability of a mild sustainable increase in intracellular ROS (levels that do not directly aﬀect cell survival) to inhibit death signaling, in particular the apoptotic pathway [3,23–27]. 1.

Necrosis

Necrosis is characterized by early cell and organelle swelling, loss of integrity of mitochondria, plasma membrane, peroxisomes and lysosomal and eventual breakdown of the cells, leading to release of its contents into surrounding area. Necrosis appears to be the result of acute cellular dysfunction in response to severe stress or after exposure to toxic agents. The role of ROS in necrotic cell death is attributed to the highly reactive intermediates, such as . OH , as a result of excessive O2 or H2O2 production [19].

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2.

Apoptosis

Contrary to necrotic cell death, apoptosis or programmed cell death represents a highly controlled form of cell death in which single cells are selectively eliminated without release of cellular debris and perturbation of neighboring tissues [20,28]. Apoptosis regulates several key physiological functions. For example, developing lymphocytes undergo extensive cell death during selection of the immune repertoire. Understanding the fundamental mechanism(s) and regulation of apoptosis is crucial for the development of therapeutic strategies to selectively target undesirable cells, as would be the case with cancer cells, and for ﬁnding ways to prevent unwanted death of normal cells as occurs in Alzheimer’s disease and HIV/AIDS. The Hallmarks of Apoptosis. Much of our knowledge on the mechanism of apoptosis comes from studies of the nematode worm Caenorhabditis elegans. During development of the hermaphrodite worm, speciﬁc cells undergo apoptosis. This programmed suicide is divided into three stages: execution, engulfment, and degradation. The execution stage involves ced-3 and ced-4 gene products and is inhibited by the product of ced-9 [29,30]. Mammalian counterparts of ced-9 and ced-3 genes have been identiﬁed. Ced-9 is homologous to a family of mammalian proteins of which the prototype is the Bcl-2 oncogene [31], while Ced-3 was ﬁrst identiﬁed as a homolog of the mammalian protein encoding the interleukin-1h– converting enzyme (ICE) [32]. To date, 14 ICE/Ced-3 homologs of human origin have been identiﬁed, all of which are cysteine proteases that cleave their substrates after aspartic acid. Members of this family of proteases are called caspases (c for

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cysteine proteases and aspase for their ability to cleave after aspartic acid [33–36]. Each of these enzymes is synthesized as a pro-enzyme that is proteolytically activated to form a heterodimeric catalytic domain. Overexpression of each of these proteases in various cell types results in apoptosis. Moreover, speciﬁc inhibition of the caspases both in vitro and in vivo using the cowpox virus gene product crmA [37,38], the recombinant baculovirus p35, and speciﬁc inhibitors such as the peptide substrates YVAD or DEVD has outlined the key role of caspases as the common execution apparatus of apoptotic cell death [39,40]. Caspases Activation During Apoptotic Signaling. At least three models for caspase activation have been proposed. Apoptosis induced by ligation of cell surface receptors like CD95/Fas or TNF-R, also known as death receptors, represents a pathway almost exclusively controlled by caspases. Indeed, ligand binding to a death receptor causes the assembly of a series of proteins called the DISC (death-inducing signaling complex), which then activates an initiator caspase, pro-caspase 8 [41]. Activated caspase 8 will then induce activation of the main executioner caspase, caspase 3. Caspase 3 can then activate other caspases and ultimately cleaves various cellular substrates, leading to the disassembly of the cell. Alternatively, numerous agents such as anticancer drugs trigger apoptosis without involving cell surface receptors. The essential powerhouse of this pathway is the mitochondria. During this form of apoptotic execution, mitochondrial dysfunction such as changes in the mitochondrial permeability, drop in the mitochondrial membrane potential, and cytosolic release of pro-apoptotic factors such as cytochrome c ampliﬁes the caspase cascade. In the cytosol, cytochome c binds to Apaf-1 (apoptotic protease-activating factor 1) and in the presence of dATP forms an oligomeric complex with other Apaf 1 molecules and pro-caspase 9. This functional complex is referred to as the apoptosome and, once formed, triggers the maturation (processing/activation) of pro-caspase 9 to mature (active) caspase 9, which can then activate executioner caspases, such as caspase 3 and 7 [42,43]. A third pathway that can activate the caspase cascade is initiated by cytotoxic T cells. In these cells, perforin and granzyme B cooperate to induce apoptosis in tumor cells and in cells infected by exogenous pathogens. Perforin permeabilizes cells, allowing granzyme B into the cytosol where it activates caspase 3 [44]. Thus, regardless of the mechanism, activation of the caspase cascade leads to the cleavage of numerous cellular proteins and cell death.

B.

ROS and Apoptosis

Current opinion holds that ROS participate in the induction of apoptosis [1]. Indeed, numerous agents that induce apoptosis stimulate intracellular production of ROS, leading most frequently to an accumulation of H2O2. Moreover, many inhibitors of apoptosis have antioxidant properties or enhance the cellular antioxidant defense mechanism. These observations have led to the suggestion that ROS are eﬀectors for a variety of triggers of apoptosis, including TNF-a, C2 ceramide, anti-IgM antibody, dexamethasone, irradiation, and numerous anticancer drugs [1]. On the other hand, the observation that hypoxia can induce apoptosis supports the argument that ROS are not necessary for mediating apoptotic cell death [45,46]. In addition, we and others have shown that an increase in intracellular ROS, in particular O2 and H2O2, can inhibit apoptotic signaling in tumor cells, irrespective of the trigger [3,22,23,25–

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27,47–49]. This divergent eﬀect of ROS on cell death pathways has stimulated enormous interest to decipher underlying mechanism(s) that allow ROS to function as prolife in one setting and pro-death in another. 1.

O2. and Apoptosis

Intracellular Production of O2. Inhibits Apoptosis. Compared to the highly reactive . OH, O2 seems innocuous in chemical terms [5]. Indeed, many in vitro studies that showed the ability of O2 generating systems to damage biomolecules and kill cells found that protection was achieved by adding not only SOD, but also catalase and . OH scavengers [50], thereby questioning the direct involvement of O2 in cellular damage and death. In agreement with these ﬁndings, an increase in the intracellular O2 concentration in the absence of cytotoxic production of H2O2 does not kill cells but, contrarily, inhibits activation of the apoptotic pathway. We demonstrated this eﬀect of elevated intracellular O2 on apoptosis triggered either through the cell surface receptor CD95/Fas or anticancer drugs [3,26]. In our system, an increased intracellular level of O2 was achieved either by incubation of cells with diethyldithiocarbamate (DDC), an inhibitor of Cu/Zn SOD, or by overexpressing or repressing the expression of cytoplasmic Cu/Zn SOD protein in vivo using a tetracycline-inducible expression system. Overexpression of Cu/Zn SOD in the human melanoma cell line M14 induced a signiﬁcant decrease in intracellular level of O2 that correlated with an increase in cell sensitivity to anticancer drugs such as etoposide, daunorubicin, and pMC540. Conversely, an increase in intracellular O2 due to repression of Cu/Zn SOD protein inhibited apoptosis induced by the same anticancer drugs [26]. Similarly, preincubation of M14 cells expressing a chimeric TNF-CD95 receptor with 1 mM DDC for one hour signiﬁcantly increased intracellular O2 and strongly inhibited CD95-mediated apoptosis [3]. Similar results have been reported by Lin et al. with virus-induced apoptosis; elevated intracellular O2 served as a survival signal and strategies to lower O2 favored execution of death [51]. These results demonstrate that the role of intracellular O2 in regulating cell sensitivity to apoptosis appears to be common to a variety of apoptotic stimuli such as receptors, drugs, and even viruses. Thus, depending upon the intracellular level of O2, the apoptotic signal can be either facilitated or inhibited [25,49].

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Intracellular Production of O2. Inhibits Caspase Activation. The evidence is deﬁnitely in support of a critical role of intracellular O2 in the regulation of cellular response to death signaling but the intracellular target(s) of O2 are not clearly understood. At least in the tetracycline-inducible system to overexpress Cu/Zn SOD we were able to correlate the inhibitory activity of O2 on the cell death pathway with a direct or indirect eﬀect on the main executioner caspase, caspase 3 activation/activity. A decrease in intracellular O2 facilitated activation of the caspase 3, whereas an increase in O2 levels lowered caspase 3 activity. These results suggested that the regulation of tumor cell sensitivity to apoptotic triggers by intracellular O2 could be via its eﬀect on intracellular caspase activation. A more physiological example of this is the NADPH oxidase–derived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells [52]. Human neutrophils have a short half-life and are believed to die by apoptosis both in vivo an in vitro. It has been observed that caspases are activated in time-dependent manner in neutrophils undergoing

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spontaneous apoptosis. However, in cells treated with the potent neutrophil activator phorbol 12–myristate 13–acetate (PMA), caspase activity was only evident after pharmacological inhibition of O2 production by the NADPH oxidase. Similarly, inhibition of the NADPH oxidase in constitutive as well as CD95/Fas-triggered apoptosis resulted in increased rather than suppressed level of caspase activity, suggesting that ROS may prevent caspases from functioning optimally in these cells. These redox-sensitive death-executing enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate neutrophil clearance under these conditions [52]. Taken together, these observations support the hypothesis that manipulation of cellular-redox state serves as a productive strategy to modulate caspase-mediated death. Indeed, the catalytic domain shared by the proteases from the caspase family (QACXG) contains an oxidation-sensitive cysteine residue. Under oxidative conditions in vitro, the thiol group of the active site cysteine becomes oxidized to form a mixed disulﬁde with glutathione. Oxidation of the caspases proteolytic site completely blocks their enzymatic activity [25,35,36, 39,49,53]. This result suggests that changes in the intracellular oxidative environment may inhibit apoptosis by intercepting caspase function, irrespective of the trigger [54].

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2.

H2O2 and Apoptosis

H2O2 is a weak oxidizing and reducing agent and is generally poorly reactive at physiological concentrations [5]. Despite its poor reactivity, exposure of cells to exogenous H2O2 could be harmful to cell survival. Depending on the concentration of H2O2, cells undergo necrosis ( > 0.5 mM) or apoptosis (<0.5 mM) [21,22]. Interestingly, exposure of cells to apoptotic concentrations of H2O2 also induces a decrease in intracellular O2 [21]. Moreover, at these concentrations of H2O2, the intracellular pH (pHi) drops and GSH/GSSG ratio increases signiﬁcantly prior to the activation of caspase proteases. In contrast, at higher concentrations of H2O2 (0.5–2 mM) the cells undergo necrosis with a concomitant increase in pHi and a drop in the GSH/GSSG ratio, consistent with the deﬁnition of oxidative stress. In the light of these data it appears that concentrations of H2O2 that activate the apoptotic cell death pathway induce a signiﬁcant decrease in the intracellular concentration of O2 and cytosolic acidiﬁcation, thereby favoring reduction of the intracellular milieu. On the contrary, necrotic concentrations of H2O2 generate intracellular oxidative stress. Moreover, Hampton and Orrenius demonstrated that at necrotic concentration, H2O2 suppresses both the activation and activity of caspases, possibly through the oxidation of cysteine residues in caspases [22]. In a more physiological scenario, exposure of tumor cells to apoptosis-inducing agents also results in increased intracellular production of H2O2. This increase in intracellular H2O2 is predominantly a function of the mitochondrial respiratory chain [55]. Due to the abundance of Mn SOD in the mitochondria, O2 generated by the electron transport chain is readily converted to H2O2, which could either diﬀuse out into the cytoplasm or induce peroxidative damage to mitochondrial membranes, thus facilitating the release of pro-apoptotic factors such as cytochrome c, AIF, and Smac/ DIABLO, or even mitochondrial caspases 3 and 9. There is evidence to show that H2O2 can trigger activation of caspases 3 and 9, but it is not clear whether this activation is a direct result of H2O2 or mediated through H2O2-induced release of

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mitochondrial death factors. The latter scenario appears more plausible considering the observation that the antiapoptotic activity of mitochondrial protein Bcl-2 is linked to its ability to inhibit mitochondrial H2O2 production [47,56]. C.

Cytosolic pH as the Critical Determinant of ROS-Mediated Apoptotic Signaling

Activation of cytosolic caspases by cytochrome c in vitro is minimal at neutral pH, but maximal at acidic pH [57]. As mentioned previously, similar to apoptosis induced by hypoxia, H2O2-induced apoptotic cell death results from a sustained decrease in the intracellular O2 concentration and acidiﬁcation of the intracellular milieu. These observations suggest that a decrease in the O2 concentration and augmentation of the reducing state of the intracellular milieu (decrease in intracellular pH) may, therefore, represent a common mechanism for triggering apoptosis by stimuli that induce H2O2 production or directly inhibit intracellular O2 production. Consistent with these results, we recently showed that intracellular acidiﬁcation triggered by mitochondrial-derived H2O2 is an eﬀector mechanism for drug-induced apoptosis in tumor cells [55]. These data lead us to hypothesize that dysregulation of intracellular pH is a critical component for cellular response to apoptotic cell death. Interestingly, in 1988 Shibanuma et al. showed that O2 was a major signal for activation of one of the regulators of pHi, namely the Na+/H+ antiporter [58]. Their results demonstrated that an increase in intracellular O2 resulted in a signiﬁcant increase in cytosolic pH. Hence, we propose that intracellular O2 could promote survival by maintaining cytosolic pH in the alkaline range, which inhibits apoptotic signaling. Conversely, a decrease in intracellular O2 achieved by inhibition of O2 production, either directly or through intracellular production of H2O2, induces cytosolic acidiﬁcation, thus creating an environment permissive for activation and execution of the apoptotic signal. Interestingly, anticancer drugs that activate the apoptotic pathway via production of intracellular H2O2 also trigger an early acidiﬁcation of the intracellular milieu, which could be reversed upon scavenging H2O2 [55]. Intracellular acidiﬁcation induced by H2O2 also has been linked to the inhibition of the membrane Na+/H+ exchanger in a variety of cell types, which appears to be mediated by the activation of the nuclear repair enzyme poly(ADP)-ribose polymerase (PARP) [59–62]. H2O2mediated PARP activation could rapidly decrease cellular NAD+ and, subsequently, ATP levels, which could then inhibit the ATP-dependent Na+/H+ exchanger and induce intracellular acidiﬁcation. Indeed, prior inhibition of PARP by 3-aminobenzamine or phenanthridinone completely inhibits H2O2-induced acidiﬁcation and the concomitant decrease in intracellular O2 [63]. In the light of these ﬁndings, we believe that the apoptotic activity of ROS is tightly linked to the intracellular level of H2O2 and its downstream eﬀect on cytosolic pH. Here it needs to be emphasized that at higher concentrations of H2O2 the cellular stores of ATP are completely depleted, leading to an inhibitory eﬀect on the execution of the apoptotic signal. At these concentrations the oxidative stress can manipulate the mechanism of cell death, diverting it away from apoptosis to necrosis. Similar eﬀects of H2O2 on the apoptotic pathway have been reported with quinone compounds such as menadione that induce oxidative stress and inhibit caspase activity [64]. Metabolism of menadione leads to generation of ROS and to oxidation of GSH to GSSG. Preincubation of apoptotic

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Figure 1 Survival or death can be determined by a critical balance between intracellular superoxide and hydrogen peroxide concentrations. This model proposes that survival is favored by a mild sustainable increase in intracellular O2. that maintains cytosolic pH in the alkaline range and inhibits caspase activation/activity. Apoptosis, on the other hand, is a function of intracellular H2O2 production accompanied by acidification of the intracellular milieu leading to a permissive environment for caspase activation/activity. At the extreme end of the spectrum, an overwhelming production of O2. and H2O2 induces an oxidative stress responsible for necrotic cell death.

cells with catalase but not Cu/Zn SOD reduces the ROS levels and reverses the inhibitory eﬀect of menadione on caspase activity. These data directly implicate H2O2 in the caspase inhibitory eﬀect of menadione, rather than a direct eﬀect of menadione on caspase thiol group(s). Taken together, these and many other reports highlight the multiple roles of H2O2 in cellular physiology, ranging from activation of proliferation to induction of caspase-dependent apoptosis to inhibition of caspases and acquisition of the necrotic phenotype. Thus, the response of a particular cell type may vary depending upon the relative concentration of H2O2 and, perhaps more importantly, on the intricate balance between intracellular O2, H2O2 and cytosolic pH [25] (Fig. 1).

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IV.

REGULATION OF APOPTOSIS BY ROS IN TUMOR CELLS

A.

Activation of the RacGTPase, Rac1 Inhibits Apoptosis in Human Tumor Cells

There is increasing evidence that ROS are involved in development of cancer, not only by direct eﬀects on DNA but also by aﬀecting signal transduction, cell proliferation,

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and cell death. Indeed, one characteristic of tumor cells with respect to normal cells is an increase in metabolic rate, ROS generation, and decreased elimination of ROS that provide them with a survival advantage over their normal counterparts [4]. Racl, one of the main subunits of the NADPH oxidase in nonphagocytic cells, is also one of the downstream targets of the oncogene ras, overexpression and mutations of which have been described in about 30% of all human tumors [65,66]. Interestingly, mitogenic signal triggered by Ras in a ﬁbroblast cell line was shown to be mediated by Rac-dependent intracellular production of O2 , thereby lending support to the hypothesis that O2 acts as an important proliferative signal in tumor cells [11,12]. However, our data demonstrating the apoptosis inhibitory activity of O2 led us to hypothesize that in addition to triggering proliferation, activation of the Ras protein may also result in inhibition of apoptosis through Rac-mediated O2 production in tumor cells. Indeed, expression of a constitutively active form of Racl in the human melanoma cells M14 signiﬁcantly inhibited tumor cell response to apoptotic triggers such as staurosporine and etoposide. Moreover, results of experiments performed with M14 and NIH3T3 cells transiently transfected with the lossof-function mutants of Rac in an activated RacV12 background further provided impetus to our hypothesis that the inhibitory eﬀect of activated Rac on apoptotic signaling was mediated by the interaction of Rac with intracellular oxidase and the subsequent production of O2. Inhibition of Rac resulted in a decrease in intracellular O2 and a signiﬁcant increase in the sensitivity of cells to apoptotic triggers. These ﬁndings demonstrate the existence of a Rac-dependent survival pathway mediated by intracellular O2 production in tumor cells [27].

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Inhibition of Apoptosis by Bcl-2: A Pro- or an Antioxidant Activity?

The antiapoptotic gene bcl-2 is one of the best-studied survival genes in the inhibition of apoptosis. bcl-2 is the ﬁrst member of a family of genes consisting of pro- and antiapoptotic members that participate in the control of apoptosis [67]. bcl-2 was discovered as a result of its translocation into the immunoglobulin locus in follicular B cell lymphomas [68] and has been shown to prevent apoptosis in a variety of situations and to promote tumor development in vivo [69]. In growth factor–dependent cell lines bcl-2 expression prevents apoptosis in response to withdrawal of growth factors like IL-3, IL-4, NGF, BDNF, NT-3, and GM-CSF [70–72]. Bcl-2 also abrogates cell death induced by c-myc overexpression in serum-starved ﬁbroblasts [73]. All members of the Bcl-2 family of proteins regulate apoptosis: some members are proapoptotic whereas others are antiapoptotic. These protein share homology clustered within four conserved regions, namely Bcl-2 homology (BHI-4) domains, which control the ability of these proteins to dimerize and function as regulators of apoptosis. It is believed that these proteins serve as checkpoints in the apoptosis cascade, upstream of caspases [74]. Expression of Bcl-2 appears to be localized to mitochondrial and nuclear envelope as well as the ER, all of which are sites of intracellular ROS generation [75]. In 1993, Hockenberry et al. [47] observed that antioxidants that scavenge peroxides, N-acetylcysteine and glutathione peroxidase, countered apoptotic death whereas Mn SOD did not. Similarly, Bcl-2 protected cells from H2O2- and menadione-induced oxidative deaths. Following an apoptotic signal, cells sustained progressive lipid peroxidation. Overexpression of Bcl-2 suppressed lipid peroxidation completely. Based on these results it was proposed that Bcl-2 regulates an antioxidant

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pathway at sites of free radical generation [47]. This proposal, however, was soon challenged by work showing that Bcl-2 is actually a prooxidant that poses oxidant insult to the cells and thus results in the upregulation of endogenous antioxidant defenses. Indeed, expression of Bcl-2 in SOD-deﬁcient Escherichia coli resulted in increased transcription of the KatG catalase-peroxidase, a 13-fold increase in KatG activity, and a 100-fold increase in resistance to H2O2. In addition, the mutation rate was increased threefold, and KatG and oxyR, a transcriptional regulator of KatG induction, were required for aerobic survival [76]. Hence, opinions diﬀer as to whether the inhibition of apoptosis by Bcl-2 is via a direct antioxidant activity or conversely through induction of oxidant pathways, which in turn activates the intracellular antioxidant machinery. Recent results from our laboratory may help to shed some light on these apparent conﬂicting results. Using a model cell line CEM, which is dependent upon mitochondrial death factors for apoptotic execution and its Bcl-2 overexpressing variant, we found that overexpression of Bcl-2 can inhibit death signaling upon CD95 ligation or drug exposure via distinct pathways [77]. In the case of CD95-induced cell death, the inhibitory eﬀect of Bcl-2 was found to be due to its prooxidant activity that inhibits caspase 8 activation and downstream acidiﬁcation, whereas during drug-induced apoptosis, Bcl-2 overexpression blocks mitochondrial H2O2 production and subsequent acidiﬁcation. The ability of Bcl-2 to inhibit drug-induced H2O2 production from the mitochondria was found to be due to the inhibitory eﬀect of Bcl-2 on mitochondrial ROS generation, which could be linked to the well-documented mitochondrial protective activity of Bcl-2, such as inhibition of peroxide-mediated damage to mitochondrial membranes that could allow pro-apoptotic factors such as cytochrome c to leak out from the mitochondria [47,78]. On the contrary, the inhibitory eﬀect of Bcl-2 overexpression in CD95-induced apoptosis was due to its ability to create a prooxidant intracellular milieu, speciﬁcally by increasing intracellular O2 , which in turn inhibited caspase 8 activation and thereby blocked receptor-mediated acidiﬁcation and cell death signaling. The prooxidant activity of Bcl-2 appeared speciﬁc for intracellular O2 as was evidenced by the ability of NADPH oxidase inhibitor DPI or by transient transfection with the dominant negative form of Rac1 to decrease intracellular O2 in Bcl-2 overexpressing cells and enhance their sensitivity to apoptosis without the involvement of the mitochondria. In addition, preincubation of CEM/Bcl-2 cells to catalase had no eﬀect on the inhibitory activity of Bcl-2 on caspase 8 activation and acidiﬁcation following CD95 ligation, thus ruling out a role for H2O2 in this pathway. These ﬁndings provide further support for our previous reports that a slight increase in intracellular O2 is inhibitory to apoptotic signaling irrespective of the trigger [3,26,27,49]. Moreover, our results provide evidence that inhibition of apoptosis by Bcl-2 could be due to either the inhibition of ROS production from the mitochondria (antioxidant activity) or a slight increase in cytosolic levels of O2 (prooxidant activity), suﬃcient to prevent caspase 8 activation and reinforce the cells’ antioxidant capacity [77]. In light of these results, we propose that the antioxidant activity could be the natural function of Bcl-2, but upon overexpression (seen in tumor cells such as lymphomas) the inhibition of ROS production in the mitochondria could be reinforced by the inhibition of caspase 8 due to Bcl-2mediated increase in intracellular O2 level. This prevents caspase 8–dependent recruitment of mitochondria and inhibition of the death signal. The mechanism by which Bcl-2 induces intracellular O2 production has not yet been identiﬁed.

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MODULATION OF APOPTOSIS BY NITRIC OXIDE

So far we have focused our attention on the role of ROS in the regulation of apoptotic cell death. However, a review of the role of reactive species in the regulation of apoptosis would be incomplete without discussing the role of reactive species whose free radical origin is not oxygen (O2) but nitric oxide (NO ). NO was the ﬁrst gas known to act as a messenger in mammalian cells. NO has an impaired electron on the nitrogen atom, making it highly reactive. Its reaction with redox modulators yields many reactive species. Chemically, NO can exist in three forms: nitrosonium cation (NOþ ), nitric oxide (. NO), and nitroxyl anion (NO). In the presence of O2 , NO combines to form peroxinitrite (ONOO), a strong prooxidant species [79]. In aqueous aerobic solutions, NO. predominantly forms nitrite (NO2). In the presence of oxyhemoglobin, NO is completely oxidized to nitrate (NO3) [79]. As opposed to reactive species whose free radical origin is O2, referred to as ROS, these diﬀerent forms of NO are referred to as reactive nitrogen species (RNS).

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NO. and Cell Death

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NO. Is Toxic

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NO is generated from guanido nitrogen of L-arginine by at least three distinct isoforms of NO synthase (NOS) encoded by three diﬀerent genes [80]. Similar to ROS, RNS have been shown to mediate various cell signaling functions or be cytotoxic depending on the level of RNS produced. For example, NO synthesized by neuronal constitutive NOS acts as a neurotransmitter [81]. Similarly, NO produced by an endothelial NOS regulates vasodilatation [82] and platelet aggregation [83]. In contrast, high levels of NO produced by an inducible NO synthase are utilized as a defense mechanism against pathogens [84] and tumor cells [85,86]. Although the mechanisms of NO - mediated toxicity are still controversial, several possibilities have been proposed. First, NO species inactivate several mitochondrial iron sulfur enzymes involved in ATP synthesis [87]. Second, NO may directly damage chromatin by deamination and cross-linking of DNA, which increases mutagenesis [88]. Third, generation of ONOO- may play a role in the cytotoxicity process [89]. Fourth, NO may inactivate catalase, glutathione peroxidase, and superoxide dismutase [90,91]. Finally, NO has also been shown to induce apoptosis by increasing ceramide generation through caspace 3 activation, induction of mitochondrial permeability transition, and activation of the CD95/Fas system [92].

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NO. Inhibits Apoptosis

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Despite evidence supporting cytotoxic activity of RNS, exposure to NO or activation of the inducible NOS have also been reported to inhibit apoptosis in several cell types [93,94]. NO - mediated inhibition of apoptosis is in most cases due to direct inhibition of caspase activity through S-nitrosylation of the active site cysteine conserved in all caspase, where S-nitrosylation refers to a transfer of NO group to a cysteine sulfhydryl to form an R-S-NO [95]. Inhibition of caspase by NO can be rescued by the reducing agent dithiothreitol, which is consistent with direct S-nitrosylation of the caspase catalytic cysteine residue [95,96]. In this regard it has been recently reported that pro-caspase 3 was S-nitrosylated on its catalytic site cysteine (Cyst-163) in unstimulated human cell lines, but was denitrosylated upon apoptotic signaling

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through the CD95/Fas pathway [97]. These results seem to suggest that nitrosylation/ denitrosylation could serve as a regulatory mechanism, analogous to phosphorylation/dephosphorylation, during apoptotic cell death. VI.

THIOL REGULATION OF APOPTOSIS

A.

The Thiol-Dependent Activation of Apoptosis

In addition to the balance between the intracellular generation and scavenging of reactive species, modulation of cellular thiols is another important regulator of the cellular redox status. Given the requirement of cysteine reduction for the enzymatic activity of caspase proteases during apoptosis, modulation of cellular thiols could play a critical regulatory role in cellular response to apoptosis. To that eﬀect, one recent focus has been to ﬁnd clinically relevant modulators of cellular thiol status to manipulate cellular response to apoptotic triggers. One such compound is a-lipoic acid, present in trace amounts in vegetables. Alpha-lipoic acid is promptly taken up by cells and enzymatically reduced to dihydrolipoic acid [98,99]. Dihydrolipoic acid is a potent stimulator of cellular GSH synthesis and enhances the overall cellular reduced thiol status [100,101]. Lipoic acid remarkably potentiates CD95/fas-mediated cell death in leukemic Jurkat cells, but not in healthy peripheral blood lymphocytes [102]. This observation is consistent with the role of a-lipoic acid as a potent thiol reducer that could increase caspase activity. Since dihydrolipoic acid is also known to quench O2 , these data further support our observations that a decrease in intracellular O2 facilitates CD95/fas-mediated apoptosis [103]. Thus, a pharmacological decrease in intracellular O2 associated with reduction of the cysteines at the caspases’ catalytic sites may represent a promising avenue to manipulate cellular sensitivity to apoptotic triggers.

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B.

The Thioredoxin Paradox

In vivo the cellular reducing environment is maintained by a family of small proteins of a relative mass of f12 kDa, found in both prokaryotes and eukaryotes, known as thioredoxins. Thioredoxin and related molecules maintain the cellular reducing environment, working in concert with the glutathione system. Thioredoxin contains two redox-active cysteine residues in an active center (Cys-Gly-Pro-Cys-) and operates together with NADPH and thioredoxin reductase as an eﬃcient reducing system of exposed protein disulﬁdes. There are two known thioredoxins in mammalians cells, thioredoxin-1 and thioredoxin-2. Thioredoxin-1 is predominantly a cytosolic protein but is also found on the outer aspect of the cell membrane, and although it has no known nuclear localization sequence, it has been detected in the nucleus of many cell types. Thioredoxin-2, on the other hand, is a mitochondrial protein. Expression of thioredoxin-1 is induced by a variety of stress signals including viral infection, mitogens, PMA, x-rays and UV irradiation, H2O2, and ischemic reperfusion (for review on thioredoxin, see Ref. 104). Supporting the contention that reductants may facilitate caspase activity, Baker et al. recently showed that various recombinant human thioredoxins directly activate caspase 3 [105]; EC50 (caspase 3 activation) for reduced thioredoxin-1, reduced glutathione, and dithiothreitol were 2.5, 1, and 3.5 mM, respectively. In addition, caspase activation was shown to correlate with the number of reduced cysteine residues in the thioredoxins. Paradoxically, thioredoxin-1 expres-

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sion seems to protect rather than increase apoptosis as one would have expected due to its thiol-reducing activity. This antiapoptotic eﬀect of thioredoxin-1 has been reported with diverse apoptotic triggers such as TNF-a, anti-CD95/fas antibody, H2O2, activated neutrophils, ischemia-reperfusion injury, and anticancer drugs [104]. Although the mechanism of this protective eﬀect of thioredoxin-1 is unknown, in light of our ﬁndings on the role of ROS in the regulation of apoptosis, one could put forward a few attractive hypotheses vis-a`-vis the apoptosis inhibitory eﬀect of thioredoxin-1. The ﬁrst plausible scenario could be a direct scavenging or removal of H2O2 by thioredoxin-1 [106] as thioredoxin-1 appears to exert most of its antioxidant eﬀect through thioredoxin peroxidase. The thioredoxin peroxidase belongs to a conserved family of antioxidant proteins, the peroxiredoxins, which use thyl groups as reducing equivalents to scavenge oxidants. The reduced form of thioredoxin peroxidase scavenges oxidant species such as H2O2 and alkyl peroxides and in the process homo- or heterodimerizes with other family members through disulﬁde bonds formed between conserved cystein residues [107,108]. Thioredoxin reduces the oxidized thioredoxin peroxidase to the monomeric form. The second hypothesis stems from a recent ﬁnding by Nishiyama et al. [109]. Upon screening a cDNA library from a B-cell population of Epstein-Barr virus–transformed human peripheral blood lymphocytes for thioredoxin-binding proteins by the yeast two-hybrid system, a plasmid containing an insert sharing homology to the human p40phox, a cytosolic component of phagocyte oxidase, was found. This insert sequence extended from the base +181 to the stop codon of p40phox. The entire coding region of p40phox was shown to interact with thioredoxin both in assays of histidine prototrophy and h-galactosidase activity. In contrast, no interaction was observed with substituted mutant thioredoxin (C32S/C35S), which lacks reducing activity. These results showed that p40phox interacts with thioredoxin and indicates the possibility of thioredoxindependent regulation of phagocyte oxidase activity. These data strongly suggest that thioredoxin, in addition to scavenging H2O2, may also regulate intracellular production of O2 . Although, it still remains to be seen if thioredoxin could induce intracellular production of O2 , in the eventuality that it happens to do so, we propose that the antiapoptotic activity of thioredoxin may be associated with the induction of an intracellular prooxidant state. Thus, the combination of an H2O2-scavenging activity and induction of intracellular O2 production may overcome the thiolreducing activity of thioredoxin and result in inhibition rather than induction of the apoptotic pathway. Here again, and as proposed above, deregulation of the balance between H2O2 and O2 may explain the anti-apoptotic activity of thioredoxin.

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VII.

SUMMARY AND CONCLUSION

In order to survive, all cells depend on a constant repression of their intrinsic suicide program by signals from surrounding cells and the extracellular matrix [110]. We believe that an important eﬀect of extracellular survival signals may be to maintain a critical cellular redox equilibrium, based, at least in part, on adequate intracellular ROS production. Thus, an oxidative shift in the cellular redox state may be critical in directly inducing or regulating cellular response to apoptotic stimuli. Whereas a high concentration of intracellular ROS (oxidative stress) provides a direct eﬀector mechanism for necrotic cell death, a mild increase in ROS, either H2O2 or O2 (prooxidant state), could provide protection against apoptosis. By contrast, a H2O2/O2 ratio that

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favors reduction of the intracellular milieu (reduced state) will sensitize cells to apoptotic triggers that could eventually lead to spontaneous apoptosis. Thus, the divergent signaling by ROS is a function of their absolute intracellular concentrations and the critical balance between O2 and H2O2. In addition, the critical determinant between survival and apoptotic or necrotic cell death may be the cytosolic pH downstream of ROS production. According to our model (Fig. 1), survival is favored with a mild sustainable increase in intracellular O2 that maintains cytosolic pH in the alkaline range and inhibits caspase activation/activity. Apoptosis, on the other hand, is a function of intracellular H2O2 production accompanied by reduction of the intracellular milieu and, more importantly, a decrease in O2 level and cytosolic acidiﬁcation leading to a permissive environment for caspase activation/activity. At the extreme end of the spectrum, an overwhelming production of O2 and H2O2 induces an oxidative stress that can lead to inhibition of caspases and necrotic cell death. It is, therefore, imperative to make a clear distinction when attributing a particular eﬀect on the apoptotic pathway to ROS (in general) as the downstream eﬀect could be extremely varied depending upon the nature of the ROS in question. Thus, in order to diﬀerentiate necrotic from apoptotic stress, we refer to the mechanism of apoptosis induced by an increase in ROS production as reductive stress as opposed to the term oxidative stress, which invariably involves ROS-induced necrosis. Understanding the precise mechanism(s) by which cells regulate the critical ratio between O2 and H2O2 to inﬂuence their intracellular redox state will surely represent the next challenge in the ﬁelds of free radical and apoptosis research.

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7 Mitochondria, Aging, and Disease: A Genomic Perspective STEFANO SALVIOLI, MASSIMILIANO BONAFE`, CRISTIANA BARBI, and MIRIAM CAPRI University of Bologna, Bologna, Italy DANIELA MONTI University of Florence, Florence, Italy CLAUDIO FRANCESCHI University of Bologna, Bologna, and Italian National Research Center on Aging, Ancona, Italy

I.

INTRODUCTION

A new perspective is emerging indicating that mitochondria play a critical role in aging not only because they are the major source and the most proximal target of reactive oxygen species (ROS), but also because they regulate stress response and apoptosis. Moreover, they are involved in the pathogenesis of a variety of age-related diseases that will be brieﬂy summarized in this chapter. In this scenario, the importance of the cross-talk between nuclear and mitochondrial genomes is emerging. Indeed, a growing number of molecules are known to mediate mitochondria–nucleus interaction and to regulate apoptosis. This relationship is now considered a hot topic of great potential relevance for the physiopathology of eukariots, including humans. In particular, this interaction appears to be crucial in the process of aging. All of the processes involved in cell growth and metabolism require an input of energy, most of which is supplied by ATP molecules. The production of ATP occurs at the mitochondrial level by means of oxidation of pyruvate or fatty acid to CO2, coupled to the reduction of NAD+ and FAD molecules. Further transfer of electrons 123

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from NADH and FADH2 to oxygen through the subunits of the electron transport chain generates an electrochemical gradient across the inner mitochondrial membrane. This gradient, also called DA, is utilized by F0-F1 ATPase to synthesize ATP molecules. This metabolism has as a side eﬀect the production of most of the ROS of the cell. Indeed, the incomplete reduction of O2 to H2O yelds harmful oxidative species that can damage membranes, proteins, and DNA. For this reason, mitochondria have been considered important since Harman proposed his theory of aging based upon the role of ROS [1]. Mitochondria are unique in that they are the only organelles that possess a small DNA molecule with a peculiar genetic code diﬀerent from that used by nuclear DNA. Mitochondrial DNA, a closed circular, double-stranded molecule of 16,569 base pairs, has been completely sequenced. One strand is G-rich and is thus designated H (heavy), while the other is C-rich and designated L (light). Transcription and replication start in a noding region of about 100 bp called the displacement loop (D-loop). Each strand has a unique promoter, which means that the whole sequence is copied in two transcripts that undergo further maturation. mtDNA encodes two rRNAs (16S and 12S) and 22 tRNAs used to translate mitochondrial mRNAs. Moreover, it contains 13 sequences encoding proteins for NADH-CoQ oxidoreductase (7 subunits), CoQH-cytochrome c oxidoreductase (1 subunit), cytochrome c oxidase (3 subunits), and F0-F1 ATPase (2 subunits). mtDNA is located in the mitochondrial matrix, often anchored to the inner membrane. Each organelle has multiple copies of mtDNA. II.

THE SEARCH FOR GENETIC DETERMINANTS OF AGING

The ongoing search for genetic determinants of aging and longevity has been one of the main features of recent biomedical research. This search starts from diﬀerent theories proposed to explain why aging occurs. Among them, the ‘‘network theory’’ and ‘‘remodeling theory’’ of aging received particular attention in recent years [2]. The ﬁrst theory suggests that aging is indirectly controlled by a network of cellular and molecular defense mechanisms that protect cells from a variety of internal and external stressors potentially dangerous for the maintenance of cell functional integrity [3]. Such stressors can be extremely diverse and include diﬀerent physical (UV and gamma radiation, heat), chemical (components of the body and products of metabolism, e.g., oxygen free radicals and reducing sugars), and biological agents (viruses). The mechanisms that may play a critical role in the aging process at the cellular level can be summarized as follows: In the course of evolution, a number of mechanisms have emerged that allow the cell to cope with a variety of potentially harmful agents. These mechanisms include DNA repair mechanisms, antioxidant defense systems, either enzymatic or nonenzymatic, production of heat shock proteins, and activation of poly(ADP-ribose) polymerase (PARP), among others; These mechanisms are in fact interconnected and integrated and constitute a network of cellular defense systems; they have to be considered altogether and not one by one. These considerations may explain the failure of all attempts to correlate aging and/or longevity with the eﬃciency of a single defense mechanism (e.g., DNA repair or antioxidants and senescence or maximum life span).

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A failure of these mechanisms does not allow the cell to maintain its homeostasis, and this fact coincides with cell senescence (aging at the cellular level). The consequences of this failure are particularly important as far as two of the major programs of the cell–cell proliferation and cell death—are concerned. The second theory, the remodeling theory of aging, was originally proposed in order to conceptualize the results emerging from studies on human immunosenescence and the new experimental model of healthy centenarians [4,5]. The main question was how the immune system contributes to longevity. In fact, several unexpected ﬁndings have emerged and constitute the basis of the remodeling theory of immunosenescence: 1.

2.

A variety of immune responses and parameters were unexpectedly well conserved in centenarians (e.g., immunoglobulin and cytokine production, responsiveness of CD34+ cells to hemopoietic growth factors, number of CD4+ and CD8+ cells with ‘‘virgin’’ phenotype, etc.) (reviewed in Ref. 2). Diﬀerent immune responses are aﬀected diﬀerently by the aging process, and some decline while others remain unchanged or increase [6–8].

In general, the hypothesis of remodeling suggests that immunosenescence is the result of the continuous adaptation of the body to the deteriorative changes occurring over time. According to this hypothesis, body resources are continuously optimized, and immunosenescence must be considered a very dynamic process that includes both loss and gain. The keyword is adaptation, and consequently the major prediction is that people who survived to the extreme limits of life, e.g., healthy centenarians, are those who have the better capacity to adapt to damaging agents and, in particular, to immunological stressors. Finally, we would like to stress that the remodeling capability is likely to be genetically controlled in humans and other species. According to these theories, a large body of experimental evidence demonstrates the existence of genes that can modulate aging and longevity. The problem is, however, that experimental evidence and theory tell us that aging and longevity may be profoundly aﬀected by either a single or multiple gene mutations. Indeed, mutations at a single gene, such as Age1, Ras, or p66sch, are by themselves able to prolong the life span of species over the entire evolutionary scale, from yeast and Caenorhabditis elegans to mammals. On the other hand, in lower organisms mutations in multiple genes have been demonstrated to signiﬁcantly increase life span, alone or in combinations [9]. Together with the phenomenon of caloric restriction (CR), the best known intervention for extending life span in animals, these data are in apparent paradox with the ﬁndings that mutations at a single gene can deeply aﬀect aging rate and longevity. This apparent paradox can be explained assuming that in the ﬁrst case we are dealing with master genes capable of inducing signiﬁcant modiﬁcations in the network of cellular defense mechanisms because of their hierarchical role in the metabolic and biochemical pathways of the network, while the second case, the manipulation is capable of modifying several basic targets among the components of the network. In other words, either the above-mentioned genes or CR likely exert their action through the network by markedly modifying and modulating it [10]. In particular, it seems that CR deeply aﬀects the insulin pathway by which energetic metabolism is regulated. Accordingly, insulin signaling regulates metabolism, reproduction, radical production, and eventually life span in animal models such as C. elegans and Drosophila melanogaster, and genes such as Daf-2, belonging to the insulin pathway, are known to

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be longevity genes in those models [11]. It is also known that CR can avoid the increase of production of proinﬂammatory molecules, such as interleukin (IL)-1h, IL-6, tumor necrosis factor (TNF)-a, cyclooxygenase-2, and inducible nitric oxide (NO) synthase [12]. This should protect individuals from damage due to a chronic proinﬂammatory state, which is related to higher morbidity and mortality [13]. Mitochondria are deeply involved in this process, as will be further discussed. Indeed, these organelles are also known to play a crucial role in the aging process, which will be the main topic of this chapter. Mitochondria are the key organelles of the so-called free radical theory of aging and of its further extension, the mitochondrial theory of aging, which will be discussed in the following sections. III.

MITOCHONDRIA AS KEY ORGANELLES OF THE AGING PROCESS

Progressive loss of mitochondrial function in several tissues is a common feature of aging. In general, it has been proposed that mitochondria can be considered as a sort of biological clock for cell timing and aging. According to the hypothesis originally proposed by Harman, lifelong production of ROS as by-products of the oxidative metabolism leads to the accumulation of DNA and protein damage at many cellular and tissue levels. This eventually induces the appearance of the aged phenotype at both the cellular and organismal levels [1,14,15]. In line with this theory, experimental evidence indicates that mitochondria are a major target of the aging process. In particular: An accumulation of large deletions and point mutations in mitochondrial DNA (mtDNA) have been described in aged individuals [16,17], and a decrease in the number of mtDNA copies in some tissues has been reported [18]. A decrease with age of the electron transport chain enzyme activity has been described in lymphocytes [19], skeletal muscle cells [18], and cardiomyocytes [20]. As a consequence of such gradual impairment of the respiratory function, increased ROS production has been reported in a variety of tissues [21]. Even if the eﬀect of oxidative stress on mitochondria may have been overestimated [22], progressive peroxidation has been described in membranes [23] and in both mitochondrial and nuclear DNA [24,25] of aged subjects. Changes in mitochondrial morphology and a decreased mitochondrial membrane potential (MMP), the driving force for ATP synthesis, in aged tissues have been reported [26]. It is thus well established that mitochondrial function undergoes deep changes during aging, and these changes have to be considered causal events of the aged phenotype, rather than consequences, as suggested by the following observations: Cells from young rats undergo rapid senescence and degeneration when microinjected with mitochondria extracted from ﬁbroblasts of old rats [27,28]. ROS production induced by V21Ras transfection in human ﬁbroblasts induces replicative senescence [29]. An inverse relationship exists between the rate of mitochondrial hydroperoxide production and the maximum life span of species [30,31].

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The administration of antioxidant compounds such as N-acetylcysteine [32] glutathione (GSH) [33], and vitamin C [34] is able to eliminate the mitochondria-induced oxidative stress and provokes an increase of mean and maximum life span. The administration of acetyl-L-carnitine also partially restores some metabolic parameters in old rats, such as cardiolipin content in mitochondrial membranes and metabolic activity [35]. Mitochondria are involved in the aging process because of their life-long production of ROS, which in turn aﬀects the integrity of mtDNA (accumulation of mutations) and, as a consequence, leads to a reduced functionality of the organelle. Moreover, recent evidence indicates that there is likely a role also for mtDNA haplogroups in aging and senescence. IV.

ROS PRODUCTION AND AGING

It is known that ROS production is increased in advanced age [36,37] and that ROS can induce mtDNA mutations [38]. This would lead, in turn, to a loss of mitochondrial function and eventually to the appearance of the aged phenotype. Nevertheless, it is still a matter of debate if mutated mitochondria produce more ROS than normal ones. Indeed, it seems that mitochondria with mutated mtDNA produce higher amounts of superoxide radical (O2), but do not produce harmful HO2 because of their reduced or inexistent proton gradient [39]. It is known that ROS can induce apoptosis [40]. Nevertheless, there is a lack of evidence that cell death is massively present in patients suﬀering from mitochondrial disorders. Hence, increased ROS production is not necessarily associated with cell death phenomena [41,42]. Furthermore, it is also known that susceptibility to apoptosis is decreased in the elderly [43,44]. Thus, the aged phenotype may not be caused by apoptotic or necrotic phenomena induced by ROS. To date, it has been reported that cells from mitochondrial encephalomyopathies exhibit MnSOD expression, suggesting that cells adapt to cope with free radical generation [45]. This suggests that ROS production might not be the primary cause of the appearance of the aged phenotype, but rather a sort of second step, or even a side eﬀect. V.

DECLINE IN MITOCHONDRIAL FUNCTION AND AGING

Taking these data into account, a possible link between an accumulation of mtDNA mutations and the appearance of aged phenotype could be the progressive decline in mitochondrial function and ATP production [46,47]. Indeed, the idea that genetically damaged mitochondria which accumulate with time are causally responsible for the aging phenotype via a disturbed energy budget is at the core of the mitochondrial theory of aging [48]. It is not clear why such accumulation is not avoided by selection within the mitochondrial population. Nevertheless, several studies have shown that muscle ﬁbers with abnormalities of the electron transport system are apparently taken over by mitochondria of a single mutant mtDNA genotype [49,50]. This suggests that defective mitochondria can somehow overgrow the wild type and accumulate during age. It has been proposed by de Grey [15] that this accumulation is due to a slower rate of degradation of damaged mitochondria. This hypothesis states that damaged mitochondria have a replication disadvantage, but, according to their lower HO2

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production, they have slower membrane damage and thus a degradation rate slower than that of intact mitochondria. This eventually leads to their accumulation in postmitotic cells. VI.

MITOCHONDRIAL DNA VARIANTS AND AGING

Mitochondrial ATP production via oxidative phosphorylation is essential for normal function and maintenance of human organ systems, and numerous mutations of mtDNA are known to cause severe maternally transmitted diseases. A common feature in this group of inborn diseases is the involvement of some enzymes in the pathway of aerobic energy production. Thus, these syndromes are characterized by defects in organs and functions where energy supply is of more importance such as muscles, heart, eye, and the central nervous system. Besides these pathogenic mutations, other maternally inherited variants of mtDNA are present in the population. The study of such variants of mtDNA led to the identiﬁcation of several mtDNA haplogroups (groups of haplotypes derived from the same mtDNA ancestor). Those present in Europe are called H, I, J, K, T, U1, U2, U3, U4, U5, U6, v, W, X, and others. It is now emerging that such variants have to be considered as ‘‘natural’’ but not necessary ‘‘neutral’’ from a phenotypic point of view. Indeed, we recently showed that some mtDNA haplogroups are more frequent in centenarians that in younger control subjects and likely favor longevity [51,52]. Moreover, in Japanese people, three associated mtDNA germline mutations have been found at higher frequency in centenarians than in controls [53]. On the other hand, it has been reported that patients suﬀering by multiple sclerosis or DIDMOAD, a rare human disease characterized by diabetes insipidus and mellitus, optic atrophy and deafness, show the prevalence of mitochondrial haplogroup T [54,55]. Thus, it appears evident that diﬀerences in mitochondrial function can be important in the process of aging. A relationship between mtDNA haplogroups and mitochondrial function has also been demonstrated. In particular, it has been shown that mtDNA haplogroup T is correlated with less eﬃcient oxidative phosphorylation in human spermatozoa [56]. By extension, it can be posited that mtDNA haplogroups having diﬀerent properties related to oxidative phosphorylation or other mitochondrial functions (such as control of apoptosis) may be associated with physiological or pathological conditions allowing the subject to reach extreme longevity. Hence, it is possible to hypothesize a more general relationship among mtDNA haplogroups, mitochondrial function, and aging or longevity. Indeed, mitochondrial germline variability has been suggested to reduce the rate of age-associated accumulation of mitochondrial mutations in somatic cells, adding further evidence to the general hypothesis that speciﬁc mitochondrial inherited variability can contribute to longevity by conferring resistance to age-associated diseases such as diabetes and atherosclerosis [57]. It appears that mtDNA haplogroups can modulate the penetrance of variants of nuclear genes. In particular, it has been found that mtDNA haplogroups and alleles of the H-Ras1 3V variable number of tandem repeats do not independently aﬀect longevity. Indeed, the distribution of mtDNA haplogroups in centenarians is aﬀected by the genotype at the H-Ras1 locus, while they are randomly associated with H-Ras1 genotypes in young people [58]. This led to the supposition that a nucleus-mitochondrion interaction plays a role in human longevity similar to that which occurs in the

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yeast. Another example of this interaction was found in Alzheimer’s disease. Indeed, it has been found that some mtDNA haplogroups seem to neutralize the harmful eﬀect of the APOE q4 allele, lowering the q4 odds ratio from statistically signiﬁcant to nonsigniﬁcant values in Alzheimer’s patients (see below). One example that best validates the scenario that ‘‘natural’’ mtDNA variation is not necessarily ‘‘neutral’’ is the mtDNA disease Leber hereditary optic neuropathy (LHON). Having classiﬁed virtually all European mtDNAs into haplogroups, we and others have shown that the western Eurasian–speciﬁc haplogroup J (7–10% in European populations) is preferentially associated with two of the primary LHON mutations (11778 in ND4 and 14484 in ND6). It was hypothesized that the combination of three ancient polymorphisms that characterize all haplogroup J mtDNAs plays a role in disease expression by increasing the penetrance of the primary LHON mutations. VII.

MITOCHONDRIA AND AGE-RELATED DISEASES

A possible role of mtDNA variation has been recently postulated not only for aging but also for many common age-associated diseases, including type 2 diabetes, chronic inﬂammation and atherosclerosis, cancer, and Alzheimer’s disease, in which a clearcut pattern of inheritance is often lacking. A.

Type 2 Diabetes Mellitus

Type II non–insulin-dependent diabetes (T2DM) is one of the most common ageassociated diseases (diabetes of the elderly) and is characterized by a variety of severe complications which can be responsible for disability and eventually death. Diabetic patients not only live shorter lives but also have longer periods of disability. The pathogenesis of T2DM is complex, and a large literature indicates that inﬂammation and oxidative stress play an important role. In particular, TNF-a and its polymorphic variants as well as IL-6 are associated with an increased risk of developing the disease. The genetics of type 2 diabetes is still unclear, although in most cases several nuclear genes appear to be involved (complex genetic disease). Recent data suggest that oxidative stress can play an important role in the pathogenesis of T2DM. Indeed, increased production of ROS and decreased levels of antioxidant enzymes and vitamins could explain, at least in part, the damage observed in a number of organs and tissues in patients aﬀected by T2DM [59]. A number of experimental and clinical observations suggest an involvement of mitochondria and mtDNA mutations in the pathogenesis of T2DM. Indeed, in some patients aﬀected by T2DM, the disease is associated with mtDNA mutations. It is commonly assumed that in most cases a number of nuclear genes are involved (polygenic disease). However, recent data show that mitochondrial genomes can play an important role in the pathogenesis of this disease. First, a small percentage of diabetes cases that show either type 2 or type 2 phenotype is monogenic, maternally inherited, and associated with deafness (maternally inherited diabetes and deafness, MIDD) [60,61]. This form of diabetes is associated with a mutation at mtDNA np 3243, which appears to play a role in decreased insulin secretion and to be involved in the microvascular complications observed in the late stage of the disease.

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Another diabetes subtype is associated with MELAS, a syndrome associated with the same mtDNA mutation and characterized by miopathy, encephalopathy, lactic acidosis, and stroke [60,61]. Moreover, in rho(0) cells depleted of mtDNA, intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were found to be decreased [62]. Thus, quantitative alterations of mtDNA appear to profoundly aﬀect the expression of nuclear genes coding for glucose transporters and the enzymes involved in glucose metabolism. On the whole, the above reported data indicated that an alteration in the interactive circuitry between mitochondrial and nuclear genomes, which play a major role in the response to oxidative stress and apoptosis [63], can occur in diabetes and could be part of the complex pathogenesis of T2DM. Such cross-talk is an age-old mechanism responsible for the survival of model systems such as yeast, and it is of crucial importance for very important biological phenomena, such as the capability of cells to cope with a variety of stressors, including oxidative stress, and to undergo apoptosis (see below). B.

Atherosclerosis and Chronic Inflammation

Inﬂammation has recently been proposed as the pathophysiological ‘‘common soil’’ of many age-related diseases such as atherosclerosis and Alzheimer’s disease, which are major causes of mortality and morbidity among the elderly [64]. The inﬂammatory status observed in such diseases not only lasts for several decades (chronic) but also shows characteristics of acute inﬂammation (increased plasma levels of acute phase proteins) [65]. A similar scenario, although less pronounced, is found in the physiological aging and in centenarians [2]. We suggested that aging is associated with a peculiar inﬂammatory status we called ‘‘inﬂamm-aging’’ [66], which is the result of lifelong chronic stimulation due to antigenic challenge as well as other stimuli, including oxidative stress [2]. Accordingly, people who are genetically predisposed to secrete high levels of the proinﬂammatory cytokine IL-6 are disadvantaged for longevity [67]. As discussed, ROS are produced mainly in mitochondria. Thus, it is likely that lifelong ROS production can cause oxidation of lipoproteins such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cholesterol, which can accumulate in endothelial cells, macrophages, and smooth muscle cells within the artery wall. This would lead to the depletion of the cell’s antioxidant defenses and to the activation of redox-sensitive kinase cascades and transcription factors, such as NFnB and AP-1. Cells undergoing oxidative stress may in turn die by apoptosis and/or necrosis, and an increased rate of cell death contributes to the formation of a necrotic core within the atherosclerotic plaque. Such a necrotic core is the hallmark of an advanced, unstable lesion [68]. Moreover, it is known that macrophage death in advanced atherosclerotic lesions leads to lesional necrosis and possibly plaque rupture and acute vascular occlusion. Accumulation of free cholesterol is among the likely causes of lesional macrophage death. It has been reported that such a death process involves a decrease of mitochondrial membrane potential which is independent of GSH levels [69]. Thus, mitochondria can play a crucial role in the initiation, progression, and destabilization of atherosclerotic lesions, either as ROS producers or as apoptotic eﬀectors of cells trapped within the lesions.

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Cancer

Since Warburg’s initial hypothesis [70], the possible role of mitochondria in tumorigenesis and tumor progression has been investigated [71,72]. The importance of mitochondria in cancer is demonstrated by the following observations: Anti apoptotic molecules such as Bcl-2 and other Bcl-2 family members, originally identiﬁed in tumor cells, are constitutively and/or inducibly located in outer mitochondrial membranes [73]. Mitochondrial dysfunction in cancer cells is often related to the appearance of a more aggressive and malignant phenotype [72]. A variety of mtDNA mutations have been reported in a number of human tumors [74–78]. It is unclear whether these mutations are simply the consequence of increased oxidative stress, known to occur during tumor progression, or whether they have a direct role in the progression of cancer. It is, however, reasonable to conceive that accumulation of mutations in mtDNA may have a role in tumor formation. It has been demonstrated that mitochondrial stress (due to mtDNA depletion) can induce in vitro an invasive phenotype in otherwise noninvasive tumor cells [79]. In particular, this mitochondrial stress can increase cytoplasmic Ca2+ concentration, which in turn is responsible for the induction of genes such as cathepsin L and TGF-h. This may account, at least in part, for the invasive phenotype [79]. Cancer is often characterized by alterations in the apoptotic process, and apoptosis is strictly connected to mitochondrial function (see below). Moreover, the propensity of cells to undergo apoptosis varies with age, and an individual variability exists regarding the susceptibility of normal and cancer cells to undergo apoptosis, but the mechanism(s) and the possible genetic basis of these phenomena are poorly understood. The possible relationship between cancer and mitochondrial function is far from being elucidated, and it likely lies within not only the mitochondrial control of apoptosis, but also other mitochondrial parameters, such as mtDNA germline variability. The mitochondrial genome is dependent upon the nuclear genome for transcription, translation, replication, and repair, but the precise mechanisms involved in how the two genomes interact and integrate with each other are still poorly understood (see below). It can be speculated that individual mtDNA variants (haplogroups, see above) may inﬂuence the penetrance of other nuclear genes, which likely aﬀect interindividual susceptibility to develop apoptosis or cancer [80] as well as other diseases like Alzheimer’s disease (see below). Thus, mtDNA germline variants may play an important—at present mainly unexplored—role in the pathogenesis of cancer in the elderly. It is also known that apoptosis, in vitro aging, and neoplastic transformation involve overlapping sets of oncogenes and oncosuppressor genes, such as H-Ras1 and p53, whose germline variability can modulate individual susceptibility to develop cancer. Since these proteins can have a mitochondrial localization, it appears that mitochondrial function can be important for the eﬃcient translocation of such proteins (see below). Studies are in progress to elucidate the mechanism(s) by which such proteins are relocalized to the mitochondria and how this can be aﬀected by individual germline variability of mtDNA and by the age of the subject.

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Alzheimer’s Disease

It is more than a decade since the genes responsible for Alzheimer’s disease (AD) were identiﬁed [81]. These data suggest that familial AD (FAD) can be caused by autosomal dominant mutations to three genes: amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2). Nevertheless, FAD accounts for only 4–8% of all AD cases, almost all occurring before age 60, with mutations at PS1 and PS2 genes being the most and the less frequent, respectively. Thus, a major challenge is to identify possible genetic factors involved in the most common form of the disease, i.e., sporadic AD (about 90%), especially those occurring later in life (late-onset AD). The most important genetic factor identiﬁed in sporadic AD is apolipoprotein E (ApoE). ApoE is involved in cholesterol metabolism, transport, and storage as well as in neurite outgrowth, h-amyloid (h-A) deposition, and microglial expression of several inﬂammatory mediators, although the mechanism(s) is (are) still unclear [82]. A polymorphic site involving three alleles (ApoE2, E3, E4) has been identiﬁed and thoroughly investigated. The frequency of these alleles varies in diﬀerent populations, with ApoE3 being the most common. Since the ﬁrst report, an enormous number of AD patients of diﬀerent ethnicities and nationalities have been studied. The results are unexpectedly concordant and indicate that individual heterozygotes and homozygotes for the ApoE4 allele are signiﬁcantly more highly represented among AD patients (45–60%) when compared to the rest of the population, suggesting that ApoE4 is a universal risk factor for AD [83]. Genetic studies showed that an interaction between ApoE alleles and mitochondrial DNA inherited variants (mtDNA haplogroups) occurs and indicate that in ApoE4+ subjects, the presence of haplogroup U decreases the risk of AD (odds ratio change from 3.77 to 1.12), while the presence of haplogroup J increases the risk of AD (odds ratio change from 3.77 to 9.7). Such an interaction between the ApoE4 allele and mtDNA haplogroups was not observed in age-matched controls or nondemented centenarians [84]. Within the framework of a possible mitochondrial involvement in AD [85], it has been postulated that mtDNA haplogroups can modulate the harmful eﬀects of the ApoE4 allele by different oxidative phosphorylation (OXPHOS) performance and production of ROS of mitochondria carrying diﬀerent mtDNA variants [84]. It is possible that the low antioxidant eﬀect of the E4 isoform compared to E2 and E3 is compensated by a moderate ROS production by mtDNA types included in haplogroup U. Thus, a possible link between ApoE and another genetic marker of AD risk correlated with inﬂammation is emerging. VIII.

NUCLEAR–MITOCHONDRIAL CROSS-TALK

An important point to take into account when the relationship between mitochondria, aging, and longevity is considered is the cross-talk between the nucleus and mitochondria. We surmise that not only the energy requirement of the cell, but also many other stimuli are conveyed to mitochondria, which in turn can adjust their metabolism and functions in response to this information ﬂow. Accordingly, nuclear gene expression must be regulated on the basis of the status of the cell, including energy availability. Thus, mitochondria are likely involved in the control of the aging process not only because they are the ‘‘power plant’’ of the cell and the major source of ROS,

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but also because they may regulate nuclear gene expression. In other words, it has to be expected that a reverse information ﬂow exists from the mitochondria to the nucleus as a consequence of changes in the status of mitochondria. It has been found that a number of proteins with a regulatory or adaptation role are primarily located in these organelles or migrate there when activated. This is the case with Nur77/TR3 [85], p53 [86], PKCy [87], JNK/SAPK [88], some caspases, and cytoplasmic members of Bcl2 family, such as Bid, Bax, Bim [89–91]. On the other hand, a variety of mitochondrial products should be considered as signal molecules and directly or indirectly induce biochemical pathways with cytoplasmic or nuclear targets. ATP [92], ROS [93], and mitochondrial Ca2+ ions [94,79] might be considered as messenger molecules. In particular, ROS can activate the ASK1-mediated apoptotic pathway [95], the p70(S6k)-mediated progression of cell cycle [96], and the induction of various transcription factors (AP-1, AP-2, NFnB) via the antioxidant response element (ARE) [93]. Ca2+ ions are also well-known second messengers in a variety of transduction pathways. Recently it has been hypothesized that mitochondria-to-nucleus stress signaling occurs through cytosolic Ca2+ concentration changes, which are likely to be due to reduced ATP and mitochondrial Ca2+ eﬄux [94] (Fig. 1; Table 1).

Figure 1 The possible mechanism(s) of nuclear–mitochondria interaction. A variety of signaling pathways originating from the mitochondrion are directed to cytoplasmic and nuclear targets, and, in turn, many nuclear gene products converge at the mitochondrion, directly or upon stimulation.

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Table 1 Factors Involved in the Traffic of Information Between Nucleus and Mitochondria Location, cell type Mitochondria JNK/SAPK Ras Bax, Bid, Bim PKCy p53

TR3/Nur77 Caspases Mediators ATP

ROS ASK1 p70(S6k) ARE AP-1, AP-2, NFnB Nucleus and cytoplasm Ras-Rtg pathway DAF-2-like pathway Clk-1/Coq7 Proapoptotic molecules

Characteristics c-Jun NH2-terminal kinase, or stress-activated protein kinase, which can inactivate the antiapoptotic protein Bcl-XL A family of GTP-binding proteins, which move in/out of mitochondria in response to various stressors Cytoplasmatic members of Bcl-2 family that modulate the apoptotic cascade together with other Bcl-2 family members A protein kinase Cy with a proapoptotic activity A transcription factor that induces cell cycle block and multiple pathways of apoptosis; one of these pathways is mediated by Bax; p53 itself can localize into mitochondria An intracellular steroid receptor with a proapoptotic activity Proteases involved in apoptosis; some of them (e.g., caspase 7) can act at the mitochondrial level The levels of ATP are crucial for many aspects of cell life; ATP drops can directly induce cell death or induction of stress response genes Ros are involved in the induction of gene expression Apoptosis signal-regulating kinase 1, a MAP kinase able to activate the JNK pathway Growth factor–regulated protein kinase (S6 kinase), which induces cell cycling Antioxidant response element Nuclear transcription factors that are involved in gene expression during apoptosis Controls stress resistance and life span in S. cerevisiae Controls stress resistance and life span in C. elegans A mitochondrial protein involved in ubiquinon synthesis Cytochrome c, AIF, procaspases, Smac/Diablo, the release of which induces apoptosis

More complicated mitochondria-to-nucleus cross-talk pathways include the action of many signal proteins found in animal models such as Saccharomyces cerevisiae and C. elegans. The pathway identiﬁed in S. cerevisiae is called the ‘‘retrograde response’’ and informs the cell as to the organelle’s status. The retrograde response involves several nuclear-encoded proteins located in the mitochondrion and in the cytoplasm. Multiple longevity genes have been isolated, some of which are involved in metabolic regulation, and have mitochondrial localization (PHB1 and PHB2, RAS1 and RAS2) or interact in the retrograde regulatory pathway (Rtg 1, 2, and 3) [97]. Similarly, in C. elegans the clk-1 gene encodes a protein with mitochondrial localization that is highly conserved among eukaryotes and determines the timing of embryonic and postembryonic development, adult worm behavior, reproduction, and aging [98,99]. Mutations in clk-1 determine the so-called clk-1 phenotype, charac-

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terized by a slower metabolic rate and prolonged life span. Clk-1 shares a close homology with the yeast gene coq7, which is a regulator of mitochondrial function important for the change from fermentative to respiratory metabolism in yeast [100]. It seems that clk-1 in C. elegans is somehow involved in notifying the nucleus when energy production slows down. As a result, a pattern of gene expression is implemented that renders the cell able to cope with a low energetic availability. This will in turn result in slow physiological rates that prolong life span. Thus, it can be predicted that coq7, in analogy to clk-1, would act as a longevity gene, slowing the metabolic rate of the yeast from respiration (fast) to fermentation (slow). Intriguingly, the extension of life by caloric restriction also relies on mitochondrial function, but it seems to involve pathways distinct from retrograde response [101]. IX.

MITOCHONDRIAL CONTROL OF APOPTOSIS

Apoptosis has an important role in the aging process [3,102–104], and mitochondria are now considered as key orgenelles in the control of this phenomenon [105–107]. This control is often deranged in aged cells, where an increased susceptibility to ROS production has been reported. In fact, ROS lower MMP, enable the triggering of permeability transition (PT) reactions and the lowering of the threshold for calciuminduced PT activation. This sequence of events results in apoptosis in lymphocytes, liver, and brain from aged mice [108]. In other cases, an increased resistance to apoptosis has been observed in resting lymphocytes from aged people and centenarians with respect to young people and in ﬁbroblasts that underwent in vitro senescence [43,44]. This behavior is likely dependent on the cell type and status (activated or resting, proliferating or postmitotic cells), on the experimental model (in vivo or in vitro), and on the apoptotic stimuli used. It can also be hypothesized that cells from aged people are less prone to apoptosis because their mitochondria have become ineﬃcient or more refractory in generating signals that induce apoptosis. This phenomenon could be considered as the consequence of the adaptation of cells to the detrimental eﬀects of life-long exposure to oxidative stress. In fact, the survival to a long-term exposure to stressful conditions is likely dependent upon the induction of protective mechanisms, such as heat shock proteins, which in turn allow cell recovery after stress and impede apoptosis [109]. This phenomenon would lead, on the one hand, to the survival of the cells and thus to longevity of the organism and, on the other hand, to the accumulation of damaged cells and thus to the appearance of the aged phenotype. X.

A POSSIBLE MITOCHONDRIAL SIGNALOSOME?

An emerging body of evidence indicates a role of mitochondria in signal integration and ampliﬁcation. Indeed, as described above, a number of signal and adaptor proteins are found in mitochondria (Fig. 1; Table 1). Most of them have physical interactions with other proteins present in mitochondria [110,111], as in the case of the p53, which is associated with mt hsp70, the major mitochondrial import protein [86]. Moreover, some of these proteins, such as Ras [110], Raf [112], Grb10 [113], and Bag-1 [114], interact with each other [114,115] and may form a multimolecular complex that could act as a ‘‘mitochondrial signalosome.’’ The localization of such a complex is at present unknown, but it may be the same as Bcl-2, i.e. the membrane compartment,

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since some of its components physically interact with Bcl-2. Alternatively, it could be placed on the outer face of the outer membrane. Indeed, recent data suggest that this is the more likely localization for mitochondrial p53 [116], and, by analogy, it could be the same for the mitochondrial signalosome. The complex would be capable of integrating the signals introduced by each of its components. Taking into account that some of the components of mitochondrial signalosome associate with Bcl-2, it can be hypothesized that a major function of this complex is to modulate the inner mitochondrial membrane permeability to ions. When this permeability is lost, the mitochondrial matrix swells, inner membrane cristae become ﬂat, and the outer membrane will eventually break, leading to the release in the cytoplasm of proapoptotic molecules normally sequestered in the intermembrane space, such as cytochrome c [117], apoptosis inducing factor (AIF) [118], Smac [119], and procaspases [120]. Another part of the regulation of the apoptotic pathway driven by the mitochondrial signalosome may occur at the level of Bax/Bcl-xL interaction. It is known that Bax can alter mitochondrial membrane permeability and induce apoptosis by forming homodimeric Bax/Bax or heterodimeric Bax/porin channels and that this activity is blocked by the interaction of Bax with Bcl-xL [121,122]. Bcl-xL can be blocked by the antiapoptotic Bad, which in turn can be inactivated by Raf, thought to be part of the mitochondrial signalosome. Thus, the apoptotic control of the signalosome may extend over the PT complex. Another candidate component of the mitochondrial signalosome is p66shc, a member of the shc adaptor protein family. It has been shown that targeted mutation of the mouse p66shc gene prolongs life span, likely modulating the signal transduction pathway that regulates stress apoptotic response to H2O2 and increasing resistance to paraquat [123]. Another role has been proposed for p66shc in apoptosis [124,125], namely, that it can act as an adaptor protein to stop mitosis and initiate apoptosis after phosphorylation at speciﬁc amino acid sites. According to this theory, the phosphorylation of p66shc would be triggered mainly by ROS. When the burden of ROS damage is dramatically increased, as in the case of aged individuals, the apoptotic cascade would predominate and determine the death of the whole organism. This phenomenon has been called ‘‘phenoptosis’’ [126]. Accordingly, it can be hypothesized that p66shc function would have a positive eﬀect in young individuals by purifying tissues from ROS-overproducing cells, while on the other hand it would impair the survival of old individuals, thus reducing life span. If this idea is correct, the activity of p66shc would be a perfect example of antagonistic pleiotropy (a factor that may exert beneﬁcial eﬀects at early ages and detrimental eﬀects later on). Should p66shc mitochondrial location be conﬁrmed, additional evidence of the primary role of mitochondria in aging and longevity would be obtained. Recent data indicate that mitochondria-controlled apoptosis can be modulated by antioxidants, such as vitamin E [127], which likely contribute to longevity, with the value of this antioxidant being signiﬁcantly higher in centenarians than in randomly selected old people [128]. XI.

CONCLUSIONS

As discussed in this chapter, mitochondria play a crucial role in the aging process and in the pathogenesis of many age-associated diseases. The involvement of such organelles occurs at diﬀerent levels, the most important being the control of apoptosis and the interaction of mtDNA with other nuclear genes.

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In the control of apoptosis, mitochondria seem to function as a ﬁnely tuned checkpoint for many, if not all, the apoptotic pathways, where apoptotic and antiapoptotic signals converge and integrate, leading to the decision to die or to survive and sending a variety of messages to nuclear and cytoplasmic targets. Such an important decision would be taken not by the nucleus, as the cell’s ‘‘brain,’’ but by the mitochondrion, until now considered a mere executioner. Mitochondria may aﬀect the aging process in their capacity to eﬃciently eliminate unwanted or damaged cells. When this capacity is impaired, as in the case of aged cells, senescence of the organism eventually occurs. Moreover, it is conceivable that a mitochondrial signalosome is involved in cell homeostasis, being responsible for a two-way (nucleus ! mitochondrion and mitochondrion ! nucleus information) exchange. It is becoming clear that natural mtDNA variants must be considered as ‘‘not neutral’’ from a phenotypic point of view. Indeed, some haplogroups are linked to diﬀerent OXPHOS performance and are found at diﬀerent frequencies in healthy centenarians or AD patients. Moreover, it appears that mtDNA haplogroups can modulate the penetrance of nuclear genes, as demonstrated by the interaction of mtDNA and APOE alleles. This is a relatively new research ﬁeld, and the amount of experimental data is rapidly growing. Such nuclear–mitochondria interactions will be likely clariﬁed in the near future and their role and importance in aging and disease will become better understood. REFERENCES 1. 2.

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8 The Human Genome as a Target of Oxidative Modification: Damage to Nucleic Acids JEAN CADET, THIERRY DOUKI, and JEAN-LUC RAVANAT Commissariat a` l’Energie Atomique Grenoble, Grenoble, France

I.

INTRODUCTION

Oxidation reactions are ubiquitous within cells as the result, for example, of aerobic metabolism that may lead to the leakage of reactive oxygen species from mitochondria and endoplasmic reticulum [1]. Exogenous chemical and physical agents such as environmental carcinogens, ionizing radiation, and UV-A light may also contribute to the induction of oxidative processes that have been shown to be implicated in aging, several types of cancer, and various other diseases such as arteriosclerosis, arthritis, cataracts, and diabetes [2–5]. DNA, among other key biomolecules including membrane lipids and proteins, appears to be a critical cellular target. It is now well documented that oxidation reactions mediated by one-electron oxidants together with oxygen ( OH radical, H2O2, 1O2, O3, HOCI)- and nitrogen (NO , OONO)-reactive species give rise to a wide array of DNA modiﬁcations (for early reviews, see Refs. 6–9). Thus, purine and pyrimidine base lesions, DNA single and double strand breaks, and true and oxidized abasic sites constitute major classes of oxidative DNA damage that have received much attention during the last two decades. Another type of relevant oxidative lesion consists of DNA-protein cross-links, about which there is still a paucity of both mechanistic and structural information. Increasing interest is now being given to the formation of adducts between amino-substituted nucleobases and reactive aldehydes such as malonaldehyde (MDA) and 4-hydroxy2-nonenal (HNE) that may arise from the breakdown of initially generated unstable lipid peroxides [10] and/or 2-deoxyribose oxidation. The assessment of the

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steady-state level of oxidative damage to DNA that, for the most part, is produced by oxygen metabolism associated with mitochondrial respiration burst has been hampered until recently by the absence of accurate methods of measurement. As will be discussed later, drawbacks implicated in the widely used gas chromatography–mass spectrometry method led for more than 15 years to an overestimation of the level of oxidized bases within cellular DNA (for reviews, see Refs 11, 12). In the present chapter, emphasis is placed on several recent achievements that allow an accurate measurement of oxidative damage within cellular DNA upon exposure to hydroxyl radical ( OH), singlet oxygen (1O2), or one-electron oxidation agents. This has beneﬁted from major technical improvements including optimized DNA extraction methods and the recent availability of the powerful high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/ MS) technique. Less speciﬁc methods that involve the use of DNA repair enzymes in association with either single cell electrophoresis (‘‘comet’’) assay or the alkaline elution technique have also become available. Interestingly, application of the latter assays has allowed the measurement of very small amounts of oxidative base damage within cellular DNA upon exposure to low doses of ionizing and UVA radiation. It should be mentioned that the strategy used for the assessment of oxidative damage formation within cellular DNA involves the previous determination of targeted lesions in model system studies. Therefore the ﬁrst part of the chapter focuses on the main oxidation reactions induced by OH radical, 1O2, and one-electron oxidants with the four predominant pyrimidine and purine DNA bases: thymine, cytosine, guanine, and adenine.

II.

FORMATION OF SINGLE BASE LESIONS AND TANDEM DAMAGE IN ISOLATED DNA AND MODEL SYSTEMS

The main reactive oxygen and nitrogen species involved in oxidation reactions with DNA together with the conversion pathways of nucleobase radical cations, the reactive intermediates generated by one-electron processes, are listed in Table 1. It

Table 1

Reactive Species and Radicals Involved in Oxidative Stress

Species/radicals

Reactivity with DNA

Ferryl ion Hydrogen peroxide (H2O2) Hydroperoxide radical (HO2) Hydroxyl radical ( OH) Hypochlorous acid (HOCl) Peroxinitrite (ONOO) Oxyl (RO ) and peroxyl (ROO ) radicals Ozone (O3) Purine and pyrimidine radical cations Singlet oxygen (1O2) Superoxide radical (O2)

Oxidizes bases and sugar moieties Oxidizes adenine Not detectable reactivity Oxidizes bases and sugar moieties Chlorination of nucleobases Oxidizes bases and sugar moieties Oxidize sugar moieties Oxidizes pyrimidine and purine bases Hydration and deprotonation Oxidizes guanine No detectable reactivity (reduction of ROO radicals)

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appears that OH radical and/or related reactive species that may arise from a Fentontype reaction according to the Haber-Weiss cycle inside the cell are powerful oxidants. This clearly indicates that OH reacts at the site where it is produced, whereas O2 , H2O2, and, to a lesser extent, 1O2 are able to diﬀuse within the cell before being involved in oxidation reactions with either DNA or other cellular targets. Emphasis is placed in this chapter on the description of the main oxidative processes mediated by OH, one-electron oxidants, and 1O2 to the four main pyrimidine and purine bases, giving rise to a major class of DNA damage. Interestingly, information on radical oxidation of 5-methylcytosine, a minor but biologically relevant DNA base, has recently become available [13,14]. Peroxynitrite, a nitrogen-reactive species that may be generated through the reaction of nitric oxide with superoxide radical [15], has been shown to oxidize both purine and pyrimidine bases of nucleosides and DNA [16–18]. Other reactions of peroxynitrite involve homolytic addition to the guanine moiety through the 4,5 double bond [19], on the one hand, and formation of 8-nitroguanine [20–28], on the other hand. It was recently shown that HOCl, a reactive oxygen species generated during the inﬂammation process, is able to promote chlorination of cytosine and purine bases at C5 and C8 [28], respectively, together with oxidation of the guanine base [29]. Another major class of oxidative damage to DNA is represented by single strand breaks that are mostly generated by initial OH-mediated hydrogen abstraction at C3, C4, C5, and to a lesser extent at C2 of the 2-deoxyribose moiety [30–32]. The formation of double strand breaks, which can be thought of in terms of two single nicks within 10 base pairs, requires two independent hits that may include OH radical, ionization or excitation, typically provided by ionizing radiation. Hydrogen abstraction at C1, which may be induced by either OH radical or deprotonation of cytosine or guanine radical cation, was found to lead to the formation of 2-deoxyribonolactone [7,33–35]. The latter oxidized abasic site has been shown to be highly unstable, even at neutral pH [35–37], and to be a trap for DNA repair enzymes [37,38]. Normal abasic sites are usually formed as intermediates in base excision DNA repair processes and/or through hydrolytic cleavage of weakened N-glycosidic bond of oxidized bases such as ureids. Evidence was found that abasic sites are generated within cellular DNA. However, the assessment of the steady-state level of the latter lesions [39,40], which should be close to that of strand breaks, remains a matter of debate due to their apparent overestimation [41]. Other oxidative lesions that are currently receiving major attention deal with exocyclic adducts that arise from the reaction of a-h-unsaturated aldehydes with amino-substituted nucleobases, including adenine, guanine, and cytosine. Reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) may be generated in cells as breakdown products of lipid peroxidation [10] and/or as radical degradation compounds of the 2-deoxyribose moiety of DNA [42]. Thus, the reaction of 4-HNE and 2-phosphoglycolaldehyde, another 2-deoxyribose oxidation product, with the exocyclic amino group of nucleobases gives rise to several adducts that can be measured speciﬁcally and in a sensitive manner by HPLC-ESI-MS/MS and gas chromatography. This is the case for 1,N2-etheno-2V-deoxyadenosine, 1,N2-etheno2V-deoxyguanosine, N2,3-etheno-2V-deoxyguanosine, 3,N4-etheno-2V-deoxycytidine, and 1,N2-glyoxal adduct to 2V-deoxyguanosine [42–50]. In addition, oxopropenylation of guanine residues within DNA by MDA, as its enol tautomer, and base propenals has been found to generate the related pyrimidopurinone adduct [51,52].

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Oxidation Reactions of the Thymine Base Mediated by OH Radical and One-Electron Oxidants

An almost complete description of both the OH radical–mediated and one-electron oxidation reactions of the thymine moiety of DNA and related model compounds is now possible. The main reaction of OH with thymine is the addition across the 5,6-pyrimidine bond, whereas hydrogen abstraction on the methyl group is a minor process, at least, in free base and nucleoside. The main oxidation products of thymidine (1) arising from the reaction of OH radical in aerated aqueous solution have been isolated and fully characterized (see below). About 50% of the overall decomposition products are represented by nine hydroperoxides that were assigned as the cis and trans diastereomers of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine (5) and 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine (6) [53] together with 5-(hydroxyperoxymethyl)-2V-deoxyuridine (7). Interestingly, the structure of the thymidine (Thd) hydroperoxides 5–7 was conﬁrmed by chemical synthesis [54]. More recently, the x-ray crystal structure of cis-5-hydroperoxy-6-hydroxy-5,6-dihydrothymine was solved and electronic properties were gained from early theoretical investigations [55]. The mixture of the thymidine hydroperoxides was completely resolved by reversephase high-performance liquid chromatography (RP-HPLC), and each of the peroxides was individually detected using a sensitive postcolumn reaction method [56]. Interestingly the presence of 5-(hydroperoxymethyl)-2V-deoxyuridine (7) in the enzymatic digest of oxidized DNA was assessed using a sensitive assay that involved the association of HPLC analysis with tandem mass spectrometry detection technique (J.-L. Ravanat and J. Cadet, unpublished). The bulk of the stable Thd oxidation products have been also identiﬁed on the basis of extensive 1H and 13C NMR, mass spectrometry, and x-ray diﬀraction measurements [57–63]. Thus, the modiﬁed nucleosides that are ranged in their decreasing order of quantitative importance are the following: N-(2-deoxy-h-D-erythro-pentofuranosyl) formamide (8) > the four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (9) > the (5R*)- and (5S*)-forms of 1-(2-deoxy-h-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (10) > the (5R*)- and (5S*)-diastereomers of 1-(2-deoxy-h-D-erythro-pentofuranosyl)-5-hydroxy-5-methylbarbituric acid (11) > 5-(hydroxymethyl)-2V-deoxyuridine (12) > 5-formyl-2V-deoxyuridine (13). Relevant information on the structure and the redox properties of the transient pyrimidine radicals was gained from pulse radiolysis measurements combined with the redox titration technique [64,65]. Based on the product analysis (see above), a comprehensive mechanism for the OH-mediated oxidation of the pyrimidine moiety of thymidine (1) can be proposed (Fig. 1). The predominant reaction of the OH radical within the thymine base is the addition (60%) at carbon C5 [30,64] giving rise to the reducing C6-centered radical 2. The formation of the oxidizing C5 radical 3 that arises from the addition of the OH radical at C6 occurs with a lower yield (35%). The third reaction, which is a minor process (5%), at least, for the free nucleoside, involves the abstraction of a hydrogen atom from the methyl group, generating the aromatic radical 4. Then, the pyrimidine radicals 2–4 are converted into the corresponding peroxyl intermediates through a fast reaction with oxygen that is controlled by diﬀusion [66]. It is estimated that about half of the latter peroxyl radicals are transformed into the hydroperoxides 5–7, through a reduction step involving superoxide radicals [67]. The hydrolytic decomposition of the hydroperoxides whose lifetimes vary from several days to one week at 37jC is quite speciﬁc. It was shown that the trans and cis diastereomers of

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Figure 1

149

Main OH radical–mediated degradation pathways of the thymine moiety.

6-hydroperoxide 5 are predominantly converted into 11. On the other hand, the major decomposition products of 6 have been identiﬁed as the (5R*)- and (5S*)-diastereomers of 10 [54]. The rest of the peroxyl pyrimidine radicals (f50%) may undergo a competitive dismutation reaction, giving rise to highly reactive oxyl radicals [30]. In particular, the oxyl radicals thus generated are expected to be involved in a hydrogen abstraction reaction leading to the formation of the diol 9. Another reaction to be considered for an oxyl radical is the h scission process. This may lead to the cleavage of the 5,6-pyrimidine bond with subsequent loss of a pyruvyl group and ring contraction to provide rearrangement products such as the (5R*)- and (5S*)diastereomers of 1-(2-deoxy--D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydan-

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toin (10). This decomposition pathway applies to the OH radical oxidation to the thymine moiety in double-stranded DNA. This is inferred from several studies aimed at isolating and characterizing oxidized bases or nucleosides after suitable hydrolysis of oxidized DNA. Thus, in early attempts, 5,6-dihydroxy-5,6-dihydrothymine and 5-hydroxy-5-methylhydantoin were found to be generated within [14CH3-thymine] DNA upon exposure to OH radicals in aqueous solution [68]. Later on, evidence was provided for the hydroxyl radical–mediated formation of 5formyl-2V-deoxyuridine (13) in DNA [69]. As will be discussed later, the measurement of oxidized nucleosides including thymidine glycols, 5-(hydroxymethyl-2V-deoxyuridine, and 5-formyl-2V-deoxyuridine within DNA [70] can be now achieved using the highly resolutive and sensitive HPLC-tandem MS assay [71,72]. Photoexcited 2-methyl-1,4-naphthoquinone (menadione) has been used to eﬃciently generate the pyrimidine radical cation of thymidine through one-electron oxidation [73,74]. Two main degradation pathways were inferred from the isolation and characterization of the main oxidized nucleosides. In that respect, the exclusive formation of the four cis and trans diastereomers of 5-hydroperoxy-6-hydroxy-5,6dihydrothymidine (5) may be thought of in terms of the speciﬁc generation of the oxidizing pyrimidine radical 3 through hydration of the thymidine radical cation at C6 [73–76]. The competitive deprotonation reaction of the latter intermediate is likely to explain the formation of 5-(hydroperoxymethyl)-2V-deoxyuridine (7), which represents about 40% of total thymidine hydroperoxides. Interestingly, the ability of three type I photosensitizers, including menadione, riboﬂavin, and benzophenone, to oxidize the pyrimidine and purine bases was assessed on the basis of accurate HPLC-GC-MS measurements [77]. It was found that formyl–2Vdeoxyuridine (13) is generated with relatively good yield with respect to that of predominant 8-oxo-7,8-dihydro-2V-deoxyguanosine (35) (8-oxodGuo), despite the fact that guanine has the lowest ionization potential among the four DNA bases. However, the ratio is lower than that observed for OH radical–mediated DNA oxidation, whereas photoexcited benzophenone and riboﬂavin are even less eﬃcient in generating 13.

B.

Radical Oxidation Reactions of the Cytosine Base Mediated by OH Radical and One-Electron Oxidants

Hydroxyl radical is able to add to the 5,6-ethylenic bond of cytosine with a preference for the C5 carbon [30] in manner analogous to that observed for thymine. The four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydro-2V-deoxyuridine (21) that arise from the fast deamination of the initially generated cytosine glycols 19 (see below) are generated in the reaction of 2V-deoxycytidine (14) (dCyd) with OH in aerated aqueous solutions [78]. A reasonable mechanism for the formation of dCyd glycols 19 (Fig. 2) recently isolated and characterized [73] implies the fast addition of molecular oxygen to 5-hydroxy- and 6-hydroxy-5,6-dihydrocytosyl radicals (15,16) initially generated (74). The resulting peroxyl radicals are expected to behave like the related thymine hydroxyperoxyl radicals, being involved in dismutation reactions and/or reduction processes that lead to the formation of hydroperoxides 17,18. In addition, N-(2-deoxy-h-D-erythro-pentofuranosyl)-formamide (8) and the (5R*)- and (5S*)-diastereomers of N-(2-deoxy-h-D-erythro-pentofuranosyl)-5-hydroxyhydantoin (23) are generated (Fig. 2) through the rearrangement of the pyrimidine ring, as already observed for Thd 1. However, the dCyd hydroperoxides 17,18 have not been yet isolated, probably because they are too unstable. It may be added that the

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151

OH radical–mediated oxidation of cytosine: the 5-yl decomposition pathway.

expected predominant 6-hydroperoxides (18) have been shown to undergo intramolecular cyclization (see below). Interestingly, speciﬁc degradation products of the cytosine moiety of 14 were isolated and characterized. These include 5-hydroxy-2Vdeoxycytidine (20), 5-hydroxy-2V-deoxyuridine (22), the two trans diastereomers of N(2-deoxy-h-D-erythro-pentofuranosyl)-1-carbamoyl-4,5-dihydroxy-imidazolidin-2one (26), N1-(2-deoxy-h-D-erythro-pentofuranosyl)-N4-ureidocarboxylic acid (27), and the a and h anomers of N-(2-deoxy-D-erythro-pentosyl)biuret (25) [74,78]. A reasonable mechanism for the formation of the latter three classes of modiﬁed nucleosides (Fig. 3) involves the generation of endoperoxide 24 through intramolecular cyclization of 6-hydroperoxy-5-hydroxy-5,6-dihydro-2V-deoxycytidine (18) as a common initial pathway. The occurrence of such transposition reactions of the pyrimidine ring has been demonstrated by isotopic labeling experiments involving,

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Figure 3

OH-mediated oxidation of cytosine: the 6-yl decomposition pathway.

for example, the incorporation of an 18O atom into the carbomoyl group of the diastereomers of the 4,5-dihydroxyimidazolidin-2-one derivatives (26) [78]. A similar set of modiﬁed nucleosides but with a diﬀerent relative distribution was shown to be generated through photoexcited menadione-mediated one-electron oxidation of the cytosine moiety [74]. In addition, the competitive deprotonation of the pyrimidine radical cation of dCyd, the reactive intermediate of the latter reaction, that occurs at N3 and C1V gives rise to 2V-deoxyuridine and the release of cytosine, respectively. The cleavage of the N-glycosidic bond may be accounted for by oxidation of the sugar residue on the anomeric carbon that leads to the formation of 2-deoxyribonolactone [80]. The formation of 5-hydroxy-2V-deoxycytidine (20) and 5-hydroxy-2V-deoxyuridine (22) that may arise from dehydration of dCyd glycols 19 and related dUrd derivatives 21, respectively, was assessed by HPLC-electrochemical detection within calf thymus DNA upon exposure to ionizing radiation, photoexcited menadione, or H2O2 in the presence of either Fe3+ or Cu2+ and subsequent enzymatic hydrolysis [81]. The two latter oxidized nucleosides were also shown to be generated within isolated DNA by two other photoexcited sensitizers, including riboﬂavin and to a lesser extent benzophenone [77], using an accurate HPLC-GC-MS assay. C.

Oxidation Reactions of the Guanine Base

As already mentioned, guanine is a preferential DNA target for several oxidants: it shows the lowest ionization potential among the diﬀerent purine and pyrimidine nucleobases and it is the only nucleic acid component that exhibits signiﬁcant reactivity at neutral pH toward singlet oxygen (1O2).

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Decomposition Pathways of Guanine Involving OH Radical and One-Electron Oxidation

The two overwhelming oxidation products of the purine moiety of 2V-deoxyguanosine (28) resulting from either the reaction with OH radical or the transformation of the guanine radical cation (one-electron oxidation) were isolated and identiﬁed as 2,2diamino-4-[(2-deoxy-h-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (34) and its precursor 2-amino-5-[(2-deoxy-h-D-erythro-pentofuranosyl)amino]-4H-imidazol4-one (33) [82,83]. The mechanism of their production (Fig. 4) may be rationalized in terms of transient formation of the oxidizing guanilyl radical 30, which may arise from either dehydration of the OH adduct at C4 29 (k = 6 103 s1) or deprotonation of the guanine radical cation 31 [84]. The resulting neutral radical dGuo(H) 30, which may exist in several tautomeric forms, is implicated in a rather complicated decomposition pathway. Thus, addition of O2 or more likely of O2, whose rate constant has been estimated to be 3 109 M1 s1 [84], to tautomeric C5 carbon-centered radical leads to the transient formation of a peroxyl radical that is followed by a nucleophilic addition of a water molecule across the 7,8-ethylenic bond. Rearrangement that is accompanied by the release of formamide [85] leads to the formation of the oxazolone 34 through the quantitative hydrolysis of unstable imidazolone (33) (half-life = 10 h in aqueous solution at 20jC) [82,83]. However, side-oxidation of 8-oxodGuo (35) by the highly oxidizing oxyl radical 30 may also contribute to the formation of the oxazolone 34 and imidazolone 33 compounds (see below). More recently, it was shown that both 34 [86] and 33 [87] are strongly alkali-labile compounds, whereas 8-oxodGuo (35) under similar hot piperidine treatment is stable in terms of induction of DNA strand breaks [85,88]. Interestingly, the nucleophilic addition of a water molecule in the sequence of events giving rise to 34 represents a relevant model system for investigating the mechanism of the generation of DNA-protein cross-links under radical-mediated oxidative conditions [89,90]. Thus, it was shown that a lysine residue tethered to a dGuo molecule via the 5V-hydroxyl group is able to participate in an intramolecular cyclization reaction with the purine base at C8, subsequent to oneelectron oxidation. A second major radical-induced decomposition pathway of dGuo (28) involved the reducing 8-hydroxy-7,8-dihydroguanyl radical (32) [91] that arises through either addition of OH at C8 (17% yield) or hydration of the guanine radical cation 31 (Fig. 4). Two major competitive reactions were identiﬁed in the conversion of reducing 8-hydroxy-7,8-dihydroguanyl radical (32) thus generated, the relative ratio strongly depending on the redox status of the environment and in particular on the presence or not of oxygen. Therefore, oxidation of the latter radical gives rise to 8-dGuo, whereas its one-electron reduction leads to the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (36) (FapyGua). This involves the scission of the C8–N9 bond of the reducing radical precursor with an eﬃcient rate (k = 2 105 s1) inferred from pulse radiolysis measurements [84]. However, the formation of 8-oxodGuo (35) is a minor process when 2V-deoxyguanosine (28) is exposed to OH radical in aerated aqueous solution. This may be mostly explained by the fact that 8-oxodGuo (35), produced from the 8-hydroxy-7,8-dihydroguanyl radical (32) precursor, is gradually consumed in the reaction with the overwhelming oxidizing oxyl radical (30). In addition, the formation of FapyGua (36) is not detectable under these conditions, whereas the oxazolone (34) and its imidazolone (33) precursor are the predominant dGuo oxidation products. The situation is diﬀerent within isolated DNA since the formation

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Figure 4 moiety.

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OH radical and one-electron oxidation decomposition pathways of the guanine

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of 8-oxodGuo (35) is predominant [77]. Interestingly, FapyGua (36) is also generated, although in about a fourfold lower yield than 8-oxodGuo (35), probably as the result of a more reducing environment within DNA, likely due to the presence of bound transition metals. It should be noted that exposure of dGuo (28) to either Fenton or Underfriend reagents gives rise to a completely diﬀerent product distribution with the overwhelming formation of 8-oxodGuo (35) at the expense of the imidazolone (33) and oxazolone (34) compounds [92]. This may be rationalized in terms of eﬃcient reduction of the oxidizing radicals (29,30) by Fe2+, a reaction that is likely to protect 8-oxodGuo (35) against further oxidation (see below). In addition, the presence of FapyGua (36) is also noted under these somewhat reducing conditions. Interestingly, the relevant chemical and biochemical features of FapyGua (36) and its adenine homologue (54) have recently become available [93–96]. The formation of 8-oxodGuo (35) upon exposure of dGuo (28) and singlestranded DNA fragments to one-electron oxidants is only a minor process. This may be accounted for by an eﬃcient deprotonation of the guanine radical cation at neutral pH [97] since its pKa is 3.9. In contrast, hydration of the guanine radical cation (31), which is likely to be stabilized within native DNA through base stacking and base pairing, was found to lead to the predominant formation of 8-oxodGuo (35) (98) together with small amounts of FapyGua (36) both through the intermediacy of the transient 8-hydroxy-7,8-dihydro-2V-deoxyguanosyl radical (32) [98]. Various physical processes including direct eﬀect of ionizing radiation [99] and bi-photonic highintensity UV lasers [100–102] together with type 1 photosensitizers [77,103], are able to promote the formation of the guanine radical cation (31) precursor of 8-oxodGuo (35) in isolated DNA. This is also the case for chemical agents that include Co(II) ion in the presence of benzoyl peroxide [104], peroxyl and oxyl radicals [105], and several radicals such as CO3 [106] Br 2 , (SCN) 2 [107] TI2+, or SO 4 [108]. Evidence has been accumulated during the last decade indicating that charge transfer is able to occur within double-stranded DNA from initially generated radicals in the nucleobases and 2-deoxyribose units. As a consequence, oxidative damage at a distance from the initial hit that is predominantly located at guanine and, to a lesser extent, at adenine is produced. This may be partly explained by the fact that guanine and, less eﬃciently, adenine are sinks for hole transfer. Interestingly, it was found that positive hole migration occurs within double-stranded DNA to a rather relatively long distance, depending on the sequence, through various mechanisms such as phononassisted polaron-like hopping, hopping mechanism, or/and coherent super-exchange [109–112]. It was recently shown using both experimental and theoretical approaches that 193 nm laser ionization of double-stranded DNA gives rise to guanine modiﬁcations predominantly via intra- and not interstrand charge migration [102]. DNA duplex stability is a key parameter to ensure eﬃcient charge transfer from radical cations created on the diﬀerent purine and pyrimidine bases. Interestingly, it was shown that formation of 8-oxodGuo (35) increased at the expense of several oxidation products arising from the conversion of radical cations of thymine and adenine within UV laser-irradiated double-stranded DNA upon increasing the ionic strength of the solution [101]. Relevant kinetic information on two competitive reactions of the guanine radical cation, namely hydration and hole transfer to another guanine residue, has become available [113]. Thus, the pseudo-order rate for hydration of the guanine radical cation (31) has been estimated to be 6 104 s1. This is lower by about two

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orders of magnitude than the rate of hole migration between two single guanines that are separated by two AT base pairs [113]. 2.

Singlet Oxygen Oxidation of the Guanine Moiety

As already mentioned, guanine is the only normal nucleic acid base that signiﬁcantly reacts with 1O2 in the 1Dg state (E = 22.4 kcal) at neutral pH. The main reaction was found to be a Diels-Alder [4 + 2] cycloaddition of 1O2 across the 4,8-bond of the imidazole ring of the guanine producing unstable 4,8-endoperoxides (36) (Fig. 5). Support for the occurrence of the latter mechanism was provided by the NMR characterization at low temperature in organic solvent of the endoperoxide arising from type II photosensitization of the 2V,3V,5V-O-tert-butyldimethylsilyl derivative of 8methylguanosine [114]. The two main decomposition products of 1O2-mediated oxidation of 2V-deoxyguanosine (28) were initially tentatively identiﬁed as the (4R*)- and (4S*)-diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2V-deoxyguanosine (37) [115]. In

O N

HN H2N

N

N 28

1O2 O N

HN H2N

O N

HN

N O

O O

36 O

N

H2N

N

H

N OH

37

N

HN H2N

OOH N

N

39 Reduction O

O O NH

HN HN Figure 5

NH N

O 38

H N

HN H2N

O N

N 35

Singlet oxygen–mediated oxidation reaction of the guanine moiety.

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fact, the structure of the ribonucleoside derivatives of 37 that were generated as the main 1O2 oxidation products of guanosine was recently revisited; they were assigned as to the two diastereomers of spiroiminodihydantoin nucleosides (38). Work is in progress in order to further establish the mechanism of formation of 38 that should involve 5-hydroxy-8-oxo-7,8-dihydroguanine (45) as a reaction intermediate. A relatively minor product of the 1O2 oxidation of free dGuo (28) has been found to be 8-oxodGuo (35). Interestingly, the formation of (35) becomes predominant at the expense of the spiroiminodihydantoin nucleoside (38) in the presence of reducing agents such as thiols or Fe2+ ions. A similar situation is found in double-stranded DNA since only the formation of 8-oxodGuo (35) has been detected [116,117]. It has been proposed that initially generated 4,8-endoperoxides (36) are able to rearrange into 8-hydroperoxy-2V-deoxyguanosine (39) [118] prior to be reduced into 8-oxodGuo. It may be added that attempts to search for the formation of FapyGua (36) within isolated DNA upon exposure to a chemical source of 1O2 were unsuccessful. This rules out the possibility for 1O2 to act by charge transfer reaction. It was also found, and this contrasts with previous reports, that 1O2 is unable to generate direct strand breaks within both isolated and cellular DNA. 3.

Secondary Oxidation of 8-Oxo-7,8-Dihydroguanine Components

Interestingly, it was shown that 8-oxodGuo (35), an ubiquitous marker of exposure of DNA to most of the oxidizing agents, is a much better substrate than dGuo to further oxidation and particularly that mediated by 1O2 [119]. Thus, it was found that the rate of reaction of 1O2 with 8-oxodGuo (35) is about two orders of magnitude higher than with dGuo (28) [120,121]. It is likely that 1O2 adds across the 4,5-ethylenic bond of 8-oxodGuo (35) to generate a transient dioxetane (40) that decomposes according to two main pathways. This leads to the formation of the predominant cyanuric acid [122] together with 2,2,4-triamino-5-(2H)-oxazolone and the erroneously assigned diastereomers of 37 [104] that have been shown to be spiroiminodihydantoin (38) derivatives [123–125]. The reaction of 1O2 with an 8-oxo-7,8-dihydroguanine residue, site-speciﬁcally inserted within a single-stranded oligonucleotide, was found to be more speciﬁc (Fig. 5). Thus, a predominant oxidation product that was identiﬁed as oxaluric acid (44) was found to be generated [126]. A likely mechanism for the formation of the ureid derivative (44) involves initial formation of the dioxetane (40) by 1O2 addition across the 4,5-ethylenic bond [119] that, upon rearrangement, is converted to the unstable 5-hydroperoxide 41. Cleavage of the 5,6-bond of 41 and subsequent decarboxylation give rise to a dehydro-guanidinohydantoin derivative (42). Further decomposition of the latter nucleoside leads, in two successive hydrolytic steps, to the formation of oxaluric acid (44) through the parabanic acid (43) precursor. Interestingly, the dehydro-guanidohydantoin compound (42) that can be isolated was also generated by two-electron oxidation of the guanine moiety of d(GpT) using the Mn-TMPyp/KHSO5 oxidizing system [127]. There is a growing body of evidence showing that 8-oxodGuo (35), whose oxidation potential is about 0.5 eV lower than that of dGuo (28) [121], is a preferential target for numerous one-electron oxidizing agents. These include Na2IrCl6 [128,129], K3Fe(CN)6, CoCl2/KHSO5 [130], high-valent chromium complex [131], peroxyl radicals [132], triplet ketones, oxyl radicals [124], ionizing radiation through the direct eﬀect [133], and riboﬂavin as a type I photosensitizer [134]. Interestingly, the two (R*)and (S*)-diastereomers of (Sp) nucleosides (38) were found to be the predominant

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one-electron oxidation products of 8-oxodGuo (35) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) at neutral pH. The formation of the latter oxidized nucleosides was rationalized in terms of transient generation of 5-hydroxy-8-oxo-7,8-dihydroguanine derivatives (45) that rearrange via an acyl shift (Fig. 6). The latter precursors were found to undergo a diﬀerent decomposition pathway under slightly acidic conditions; this involves the opening of the 5,6-pyrimidine ring that is followed by a decarboxylation reaction with the subsequent formation of the two diastereomers of guanidinohydantoin (Gh) derivatives (46). It was suggested that the latter Gh nucleosides (46) exist in a dynamic equilibrium with a pair of diastereomeric iminoallantoin compounds (47) [129,134]. The oxazolone nucleoside (34) together with its imidazolone (33) precursor were also found to be one-electron oxidation products of 8-oxodGuo, although generated in lower yields than (Sp) (38) and (Gh) (46) nucleosides [134]. Peroxynitrite is able to oxidize 8-oxoGuo through the intermediacy of CO3 radical that is generated in bicarbonate-buﬀered aqueous solutions. This was found to lead to the predominant formation of (Sp) ribonucleosides in the presence of thiols [123]. Several secondary ONOO–-mediated oxidation products of 8-oxoGua that include oxazolone (34), oxaluric acid (44), and cyanuric acid were characterized within oligonucleotides [135]. The two diastereomers of the (Sp) 2V-deoxyribonucleosides (38)

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Figure 6

One-electron and singlet oxygen oxidation reactions of 8-oxoGua.

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were found to be the main reaction products of 8-oxodGuo (35) with either HOCl or a myeloperoxidase–H2O2-Cl system. The formation of the (Sp) nucleosides (38) was accounted for by either initial Cl+ addition or two-electron oxidation of the base moiety of 35 [136]. Insights were recently gained into the kinetic features of one-electron oxidation of 8-oxodGuo (35) by various inorganic and organic radicals including Br 2, N3 , SO 4 , CH3O2 , CCl3O2 , TyrO, Trp+, and dGuo(H) (30) [137]. The rate of the reaction of the oxyl radical (30) that may arise either from deprotonation of the guanine radical cation 31 or via dehydration of the OH radical adduct at C4 of guanine (29) with 8oxodGuo 35 was found to be 0.46 109 M1 s1 at pH 7.0. This fast reaction, together with the fact that the rate of the O2 addition to the oxyl guanine radical (30) is likely to be very low (k < 106 M1 s1), would explain why 8-oxodGuo (35) is barely detectable upon steady-state gamma radiolysis of an aerated aqueous solution of dGuo 28. In fact, 8-oxodGuo (35) is consumed as soon as it is produced through an oxidation reaction with dGuo(H) (30). Interestingly, the reduction of the oxidizing radical (30) that may be easily achieved, for example, by the Fe2+ ions of Fenton reagents, leads to a pronounced change in the dGuo oxidation product distribution. Thus, a predominant formation of 8-oxodGuo (35) is observed at the expense of the oxazolone (34) and imidazolone (33) derivatives. The reactivity of 8-oxoGua residue towards various oxidizing agents was investigated in deﬁned DNA fragments that show diﬀerent sequence contexts [138]. Interestingly, it was shown that the 8-oxodGuo (35) residue when stacked in a duplex with a 3V neighboring guanine, was more susceptible to oneelectron oxidation than any other possible doublet with the three other normal bases. This, which parallels the previously higher observed reactivity of the 5V guanine in a GG doublet toward one-electron oxidants, is in agreement with earlier calculations of the ionization potential of nucleobases. It should be pointed out that in contrast to one-electron oxidation, the reaction of 1O2 with 8-oxodGuo (35) (see above) does not show any marked sequence selectivity. Interesting insights into the one-electron oxidation at a distance of an 8-oxoGua residue in a duplex DNA were gained from a time-resolved photolysis study. This has involved the detection of two purine radicals that derive from 8-oxoGua upon UVB excitation of a 2-aminopurine (AP) residue [139]. The rate constant of electron transfer between the deprotonated product of the radical cation of AP and 8-oxodGuo (35) was found to be strongly dependent on the distance between the two targeted residues. The value was estimated to be (3.8 F 0.5) 104 s1 when the AP and 8-oxodGuo 35 were separated by two molecules of thymidine (1). Interestingly, the rate was found to decrease by more than 10-fold upon insertion of four intervening thymidines [139].

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D.

Oxidation Reactions of the Adenine Base

The main OH radical–mediated oxidation product of the base moiety of 2V-deoxyadenosine (48) (dAdo) in aerated aqueous solution was assigned as to 8-oxo-7,8-dihydro-2V-deoxyadenosine (53) (8-oxodAdo) [140]. A reasonable mechanism (Fig. 7) for the formation of 8-oxodAdo (53) involves oxidation of the initially generated 8-hydroxy-7,8-dihydro-2V-deoxyadenosyl radical (49) in a similar way to the pathway proposed for the production of 8-oxodGuo (35) (Fig. 3). However, as a main diﬀerence from the radical chemistry of dGuo (35), 8-oxodAdo (58), which exhibits a higher ionization potential than 8-oxodGuo (35), is only partly involved in secondary reac-

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Main OH radical and one-electron degradation pathways of the adenine moiety.

tions with the oxidizing adenine radicals, namely the OH radical adduct at C4 (50) and/or its related dehydrated product, the 6-aminyl radical 51 (Fig. 7). However, attempts to isolate 2V-deoxyinosine (56), which is a likely ﬁnal decomposition product of the 6-aminyl radical (51) (see below), were unsuccessful, raising the question of the chemical reactions in which 51 may be involved. It should be noted that like FapyGua (36), 4,6-diamino-5-formamidopyrimidine (54) (FapyAde), whose formation requires the reduction of the 8-hydroxy-7,8-dihydropurinyl radical (49) [91], is formed in a very low yield in aerated aqueous solutions [141,142]. Attempts were recently made to assess the formation of 2-hydroxy-2V-deoxyadenosine (55) (2-OHdAdo), which was suggested to be a major OH radical decomposition product of dAdo (48). In fact,

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using the accurate and highly sensitive HPLC-ESI-MS/MS assay, it was found that 2-OHdAdo (55) is produced in very poor yields, with an even lower eﬃciency than minor FapyAde (54). The presence of Fe2+ does not signiﬁcantly aﬀect the yield of formation of 8-oxodAdo (53) and 2-OHdAdo (55), whereas a slight increase in the induction of FapyAde (54) was noted. Interestingly, a signiﬁcant increase in the level of 2-OHdAdo (55) that occurs at the expense of 8-oxodAdo (53) was observed when the aerated aqueous solution of dAdo (48) was g-irradiated in the presence of the copper(1)-ortho-phenantroline complex [142]. Information is now available on the chemical reactions that are induced by OH with the adenine moiety of isolated DNA. Similar to the observation made for free dAdo (48), 8-oxodAdo (53) and to a lesser extent FapyAde (54) were found to be the main decomposition products of the adenine moiety within DNA [77]. The formaton of 2-OHdAdo (55), in contrast to recent claims [143,144], was not found to be privileged under Fenton chemistry reaction conditions [142]. A rather intriguing observation that has not yet been explained is the apparent deﬁcit in the extent of OH radical–induced decomposition of the adenine moieties within DNA; indeed, 8-oxodAdo (53) and FapyAde (54), the two main identiﬁed decomposition products, are generated with a 10-fold lower eﬃciency than their dGuo analogs. One-electron oxidation of the adenine moiety of DNA is likely to generate the related purine radical cation, which may either undergo hydration to generate the 8hydroxy-7,8-dihydroadenyl radical (49) or deprotonate to give rise to the 6-aminyl radical 51. The formation of 8-oxodAdo (53) and FapyAde (54) is likely to be explained in terms of oxidation and reduction of the 8-hydroxy-7,8-dihydroadenyl radical (49), respectively. Another modiﬁed nucleoside that was found to be generated upon type I–mediated one-electron oxidation by photoexcited riboﬂavin and menadione is 2V-deoxyinosine (56). The latter nucleoside is likely to arise from the deamination of the 6-aminyl radical (51). Overall, the yield of formation of 8-oxodAdo (53) and FapyAde (54) in one-electron oxidized DNA is about 10-fold lower than that of 8-oxodGuo (35) and FapyGua (36) as observed for OH radical–mediated reactions. Interestingly, it was recently shown that the one-electron oxidation reaction promoted by photoexcited menadione (MQ) gives rise to N6-formyladenine (57) and N6-acetyladenine (58) residues (Fig. 7) in several dinucleoside monophosphates, including d(ApA), d(CpA), and d(ApC) [145]. In the absence of reduced transition metals, H2O2 and peracids were found to be able to react speciﬁcally with adenine to generate adenine N1-oxide [146]. However, recent attempts to look for the formation of adenine N1-oxide within cellular DNA treated by either H2O2 or UVA radiation were not successful using a highly speciﬁc and sensitive immunoassay.

E.

Formation of Tandem Lesions

The concept of the formation of clustered oxidative damage to DNA was proposed more than 15 years ago as a speciﬁc contribution of ionizing radiation [147]. Thus, interaction of high-energy photons (E > 100 keV) with nuclear DNA is expected to generate along the radiation track several ionization and excitation events as the result of energy deposition. This gives rise within cellular DNA to multiply damaged lesions [147], which except for double strand breaks have not yet been characterized. It was suggested that they consist of several base lesions, abasic sites, and/or strand breaks

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within a few DNA helical turns. Indirect evidence to support the radiation-induced formation of clustered lesions involving at least one modiﬁed base in addition to a single strand break was recently provided. This was inferred from the observation of an increased yield of double strand breaks upon incubation of extracted DNA from girradiated cells with DNA N-glycosylase repair enzymes [148]. It was also recently shown that endogenous enzymes acting through the base excision repair pathway contributed to the formation of additional radiation-induced DNA double strand breaks in E. coli cells during postirradiation incubation [149]. In addition, theoretical calculations have provided clues as to the structure of radiation-induced clustered damage whose complexity and frequency would increase with the linear energy transfer of the photon or the particle [150]. In contrast, detailed structural and mechanistic information is available on tandem oxidative lesions that arise from one radical hit. This subclass of oxidative DNA damage consists of either two vicinal oxidized bases or cyclonucleosides through radical intramolecular cyclization between the 2-deoxyribose moiety and the base. Emphasis will be placed in the following sections on recent aspects of the chemistry and biochemistry of tandem oxidative damage (for a comprehensive review of previous studies, see Ref. 151). Other tandem lesions whose mechanism of formation involves the addition of 5-(uracilyl)-methyl radical to purine bases have been characterized [152–154]. 1.

Tandem Base Modifications Involving Formylamine and 8-Oxo-7,8-Dihydroguanine

Box and collaborators showed in their pioneering work [151] that among several tandem oxidized bases, formylamine (Fo), a radical degradation product of either of the two pyrimidine bases, was generated either 3V or 5V to a 8-oxoGua residue upon exposure of short oligonucleotides to OH radical in aerated aqueous solutions. New insights into the formation of 8-oxoGua/Fo and the opposite sequence isomer (Fo/8oxoGua) tandem lesions within DNA were recently gained from detailed studies involving site-speciﬁc insertion of the modiﬁed nucleosides into deﬁned sequence oligonucleotides [155,156]. Interestingly, the formation of both sequence isomers, namely 8-oxoGua/Fo and Fo/8-oxoGua (Fig. 8), was assessed as the related dinucleoside monophosphates using a recently designed HPLC-MS/MS assay [155]. It was found that the two tandem lesions are generated linearly with the dose within a low dose range (5–100 Gy), providing further support for the fact that only one radical hit is involved in their formation. Thus, the radiolytic yield for the induction of 8-oxoGua/ Fo, Fo/8-oxoGua, and 8-oxoGua was found to be 0.0001, 0.0013, and 0.0130 Amol/J, respectively. A reasonable mechanism to explain the formation of the two tandem lesions involves an initial addition of a OH radical at either C5 or C6 of either cytosine or thymine. This is followed by a fast reaction of molecular oxygen with the pyrimidyl radical generated, leading to the formation of the corresponding peroxyl radical. Evidence for the occurrence of the intramolecular addition of the peroxyl radical to the C8 position of vicinal guanine was gained from 18O-labeled experiments [157]. Subsequently, the adduct would rearrange, giving rise to 8-oxoGua on the one hand and an oxyl type pyrimidine radical on the other. In the ﬁnal step, the latter radical is expected, through a h-scission mechanism, to lead to the generation of the formylamine remnant. Interestingly, the Michaelis-Menton kinetic parameters (kcat and Km) of speciﬁc excision of the oxidized bases by bacterial DNA N-glycosylases, namely 8-oxoGua by

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Figure 8 Tandem DNA damage including (5VR)-5V,8-cyclo-2V-deoxyguanosine, (5VS),5V,8cyclo-2V-deoxyadenosine, 8-oxoGua/formylamine, and formylamine/8-oxoGua.

Fpg and formylamine by endo III, have been determined using speciﬁcally modiﬁed oligonucleotides. It was found that the excision ability of the enzymes was only slightly aﬀected by the presence of a vicinal oxidized base [155]. However, the situation may be diﬀerent when two modiﬁed bases, including 8-oxoGua, 5,6-dihydrothymine, or an abasic site, are located on the opposite DNA strands [158,159]. Thus, the eﬃciency of 8-oxoGua removal by Fpg protein was reduced by two orders of magnitude when either an abasic site or a strand break was present one base 3V to the site opposite the guanine lesion. In contrast, 5,6-dihydrothymine when inserted on the same strand as 8-oxoGua has only a slight eﬀect on the Fpg-mediated excision of the latter damage. However, the decrease in eﬃciency of 8-oxoGua removal is higher when 5,6-dihydrothymine is substituted by a second 8-oxoGua residue. Similar eﬀects were observed when the clustered lesions were processed by yOgg1, the yeast functional analog of bacterial Fpg protein [159]. The mutagenic potential of the 8-oxoGua/Fo tandem lesion has been evaluated using a single-stranded DNA shuttle vector in which a dedicated lesion was site-speciﬁcally inserted prior to be transfected into Simian COS7 cells [160]. The mutations induced after replication in mammalian cells were screened in bacteria. 8-OxoGua alone was not found to aﬀect survival (70% bypass), whereas formylamine was shown to code mostly for adenine. As a result, Fo exhibits high

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mutagenicity when it arises from a cytosine residue. The coding properties of the tandem 8-oxoGua/Fo damage are a combination of both individual 8-oxoGua and Fo with a high frequency of adenine insertion in front of the pyrimidine lesions [160]. 2.

Tandem Lesions Involving the Formation of a Covalent Bond Between the Sugar Moiety and Purine Bases

Another interesting class of tandem DNA lesions whose OH radical–mediated formation was established more than 20 years ago is represented by 5V,8-cyclopurine nucleosides [161]. Insight into the mechanism of the generation of a covalent bridge between the sugar moiety at C5V and the purine base at C8 was gained from model studies involving the photolysis of 8-bromo-2V-deoxyadenosine [162,163; M. Berger and J. Cadet, unpublished results]. It was proposed that the aden-8-yl radical thus generated is able to abstract one hydrogen atom from the sugar moiety at C5V, which subsequently may react with the reconstituted adenine base at C8. Attempts are currently made to measure both the (5VR)- and (5VS)-diastereomers of 5V,8-cyclo-2Vdeoxyadenosine (5V,8-cyclodAdo) within cellular DNA. Interestingly, the latter bulky lesions that are not removed by the base excision repair (BER) pathway may be involved in neurological disorders associated with deﬁciency in nucleotide excision repair (NER) processes such as in xeroderma pigmentosum patients. In that respect, HPLC coupled with either mass spectrometry (HPLC-MS) [164,165] or tandem mass spectrometry (HPLC-MS/MS) (J.-L. Ravanat and J. Cadet, unpublished data) was able to detect the four purine cyclonucleosides in isolated DNA upon exposure to gamma radiation with a level of sensitivity close to 1 fmol for the latter method. Interestingly, a sensitive 32P-postlabeling assay, which allows the measurement of the purine cyclonucleosides as dinucleotides, has the required sensitivity to search for the eventual accumulation of the latter lesions in NER-deﬁcient cell lines [166]. It should be added that both the (5VR)- and (5VS)-diastereomers of 5V,8-cyclonucleosides of 2Vdeoxyadenosine and 2V-deoxyguanosine (Fig. 8) have been site-speciﬁcally inserted by chemical synthesis into deﬁned sequence oligonucleotides [167,168]. The availability of such probes has allowed several types of biochemically orientated studies. It was shown that both diastereomers of 5V,8-cyclodAdo that are not substrates for bacterial DNA N-glycosylases, including E. coli endo III and Fpg proteins, are removed from speciﬁcally modiﬁed oligonucleotides by NER enzymes that are present in human cellular extracts [169,170]. In a subsequent study it was found that the (5VR)diastereomer of cyclodAdo was not a stop for human DNA polymerase D, which is implicated in translesional synthesis. In contrast, the elongation of the DNA chain was stopped after incorporation of either dAMP or dTMP opposite the (5VS)-diastereomer. This is highly suggestive of a higher cytotoxicity for the latter cyclonucleoside compared to the (5VR)-form [171]. III.

MEASUREMENT OF OXIDATIVE DNA BASE DAMAGE

The measurement of oxidative base damage in cellular DNA, which has been the subject of intensive research during the last two decades, still remains a challenging analytical issue [12]. This may be due, in part, to the high level of sensitivity required since the threshold of detection has to be close to one single lesion per 106–107 normal bases in a sample size of DNA higher than 30 Ag for chromatographic methods (see below). In addition, the multiplicity and the lability of the lesions together with the

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presence of cellular background due to the occurrence of oxidative metabolism must be considered. This explains why highly accurate methods are required to deal with the small variations in the steady-state level of oxidative damage to DNA expected from exposure to pro- or antioxidants. Spurious oxidation is still of major concern in most assays requiring DNA extraction and its subsequent work-up (see below), and obviously this must be prevented or at least minimized. The situation is not as critical for a second class of assays that operate on isolated cells through in situ release of DNA and subsequent enzymatic detection of the lesions, usually as sensitive sites to DNA repair proteins (for early reviews on the methods initially developed for monitoring oxidative base damage, see Refs. 172–175). A.

Specific Methods Aimed at Singling Out Oxidative Lesions to Cellular DNA

Most of the available assays require initial DNA extraction followed by its chemical hydrolysis or enzymatic digestion (Table 2). One major requirement is that the targeted damage has to be quantitatively released from DNA. Then, the mixture of nucleobases, nucleosides, nucleotides, or dinucleoside monophosphates must be resolved mostly by chromatographic methods either in the liquid or in the gas phase (nucleobases). Finally, the compounds of interest, which are present in very low amounts, have to be detected quantitatively and accurately at the output of the columns. Before focusing on the widely applied chromatographic methods, the other available assays, including immunoapproaches and the ligation mediated–polymerase chain reaction (LM-PCR) technique, are critically surveyed. 1.

Nonchromatographic Methods

Immunodetection that requires high speciﬁc mono- or polyclonal antibodies can be achieved either with whole DNA or its components upon chemical or enzymic hydrolysis. Most of the attempts aimed at measuring oxidative base damage after DNA extraction have failed due to the occurrence of signiﬁcant cross-reactivity in the detection of the targeted lesion with the overwhelming related normal nucleobase or nucleoside. This was the case for several available mono- and polyclonal antibodies raised against 8-oxodGuo (35) [176–181]. Unfortunately, inappropriate use of polyclonal antibody directed against 8-oxodGuo has led to a huge overestimation of the

Table 2

Methodologies for Measuring Oxidative DNA Damage DNA

Sensitivitya

Amount of DNA (Ag)

Hydrolyzed Hydrolyzed Hydrolyzed Hydrolyzed Intact or hydrolyzed Intact

1 105 105–106 1 105 1 107 104–105 1 107

25–50 30–40 50–100 1–5 2–10 1

104

<1

Methods HPLC/electrochemistry HPLC-MS/MS CG/SIM-MS HPLC-32P postlabeling Immunology (RIA-ELISA) DNA-glycosylases (alkaline elution) Ligation-mediated PCR a

Intact

Sensitivity is indicated with respect to normal bases.

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level of the latter oxidized purine lesion in the DNA of cells exposed to 0.1 M H2O2. Thus, the level of 8-oxodGuo (35) was estimaed to be 1 lesion per 16 guanine bases as inferred from application of an immunoslot blot (ISB) assay [182]. This is at least 5 orders of magnitude higher than the values of 8-oxodGuo (35) determined using accurate assays (see below). Other questionable measurements performed using the ISB method deal with reported values of 28 and 282 8-oxodGuo (35) lesions per 105 dGuo residues in the DNA of normal and breast tissues, respectively [183]. Another example of high cross-reactivity was provided by the polyclonal antibodies against 5hydroxycytosine [180], whereas those raised against adenine N1-oxide show a higher speciﬁcity [184]. Interestingly, a highly sensitive assay that associates capillary electrophoresis with laser-induced ﬂuorescence detection has been designed for monitoring the presence of thymine glycols within cellular DNA [185]. An improved version of the assay that takes into account the alkaline lability of the oxidized thymine lesions has recently become available [186]. However, the speciﬁcity of the monoclonal antibody [187] used in the assay remains to be established. Immunostaining has been also applied to the detection of 8-oxodGuo 35 within isolated cells and tissues using monoclonal antibodies. Thus, monoclonal IF7 was used to visualize the formation of 8-oxodGuo in single cells and in tissues upon exposure to aﬂatoxin B1 [188]. A second monoclonal antibody, N45.1, is also available for immunohistochemical dectection of 8-oxodGuo (35) [189]. The successful applications of the assay include the induction of 8-oxodGuo–sensitive sites in UVB-irradiated epidermal cells [190] and in ferric nitrilotriacetate–treated renal cells [191]. LM-PCR was initially successfully applied to map the sites and frequency of bulky damage in DNA at nucleotide resolution along a sequencing gel autoradiogram. Typical targeted lesions included cyclobutadipyrimidines, pyrimidine (6–4) pyrimidone photoproducts, and carcinogenic adducts (for an early review, see Ref. 192) as well as methylated base adducts [193]. Usually lesions are converted into strand breaks that should exhibit a 5V-phosphate group. The speciﬁc cleavage of DNA is achieved by either a hot piperidine treatment for alkaline-labile damage or enzymatic digestion with DNA repair proteins. In a subsequent step, the DNA fragments that contain these ligatable breaks are ampliﬁed in a single-sided, ligation-mediated PCR reaction. Interestingly, attempts have been made to extend the application of the latter method to the measurement of oxidative damage [194]. Thus, direct strand breaks together with endonuclease III (endo III)– and formamidopyrimidine DNA N-glycosylase (Fpg)–sensitive sites were detected in peroxyl radical–treated human ﬁbroblast DNA [195] and mitochondrial DNA from cultured rat cells [196]. The main limitation of the assay is that a relatively high frequency of damage—at least 1 lesion per 104 normal bases—is required. This frequency—about 3 orders of magnitude higher than the steady-state level of the main oxidized bases—is likely to allow only applications of the LM-PCR assay to severe exposure conditions of oxidative stress [197]. 2.

HPLC-32P Postlabeling Technique

The 32P postlabeling technique was designed about two decades ago to monitor the formation of bulky DNA adducts within cellular DNA for the purpose of epidemiological studies [198,199]. The method involves enzymatic digestion of extracted DNA followed by controlled digestion of targets into nucleoside 3V-monophosphates or dinucleoside monophosphates, depending on the chemical structure of the modiﬁcations. Then the enzymatic hydrolysate is subjected to T4 polynucleotide kinase–

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mediated phosphorylation of the 5V-OH, while the phosphate at C3V is removed by nuclease P1. Finally, the lesion is isolated, usually by two-dimensional thin-layer chromatography analysis, prior to being visualized by autoradiography and quantiﬁed. Overall, the method is highly sensitive, since in most cases it allows the detection of at least 1 modiﬁcation per 107 normal nucleotides in a sample size of a few Ag of DNA. The main drawbacks of the assay concern both the quantitative and qualitative aspects of the measurement. Usually no information is provided as to the eﬃciency of the three enzymatic reactions, including the release of the selected damage, its phosphorylation, and the hydrolysis of the cold phosphate. In addition, the assignment of the damage is, at best, only tentative. Several attempts were made to extend the application of the 32P postlabeling technique to the measurement of oxidative damage to DNA. This initially concerned 5-(hydroxymethyl)uracil [200] and adenine N1-oxide [146], which are oxidation products of thymine and adenine, respectively. Later studies included 5,6-dihydrothymine and 8-oxodGuo. A suitable strategy was designed in order to allow semi-quantitative measurement of the selected lesions. The eﬃciency of the key enzymatic step, including DNA digestion, phosphorylation of the oxidized nucleoside 3V-monophosphate, and dephosphorylation of the nucleoside 3,5V-diphosphate, was optimized. An external standard was used, and the ﬁnal searched damage was eﬃciently puriﬁed by HPLC analysis on two diﬀerent columns. In addition, the structure of the lesion received further support from a microreaction test [146]. The HPLC prepuriﬁcation step was found to be a requisite for the improvement of the 32P postlabeling eﬃciency by removing competitive molecules present in large excess. The other major advantage provided by the removal of other nucleotides is the prevention of self-radiolysis processes mediated by the radiolabeled compounds. Omission of this important step was found to increase by at least 40-fold the level of 8-oxodGuo (35) in the DNA of breast cancer cells [201] and neonatal rat liver [202]. Preconcentration of the 3V-phosphoric ester of 8-oxodGuo 35 by HPLC [204–206] or TLC [203] was found to reduce signiﬁcantly the level of oxidized dGuo, even at a value lower than that provided by HPLCelectrochemical detection. This is likely to be explained by the presence of traces of nucleotides, including trailing 2V-deoxyguanosine 3V-monophosphate, in that aﬀect the yield of phosphorylation of the targeted modiﬁed nucleotide. This diﬃculty in achieving quantitative measurement of the lesion together with the tedious experimental protocol are the reasons why other methods such as HPLC-ECD and HPLCMS/MS are preferentially used for monitoring relatively abundant oxidative damage like the ubiquitous 8-oxodGuo (35). However, the 32P postlabeling assay may represent a more suitable alternative for measuring lesions generated in very low yields such as the purine cyclonucleosides [166]. 3.

Gas Chromatography–Mass Spectrometry Assays

Gas chromatography associated with electron impact–mass spectrometry (GC-EI/ MS) detection and HPLC-ECD were the ﬁrst analytical methods to become available in the mid-1980s for the measurement of oxidative base damage to cellular DNA [207]. This was followed by attempts to measure another oxidized base, thymine glycol, in the form of either methyl glycerol or acetol chemically released from cellular DNA after reduction or alkali treatment [208,209]. The main drawback of the assays was a huge background in the level of thymine glycol due to radiolysis processes associated with the use of radiolabeled substrates [210]. The GC-MS assay is a versatile method,

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since it can be applied to a wide array of modiﬁed bases. However, one of the limitations rarely considered until recently is the instability of several lesions during the derivatization step (see below). Indeed, silylation of bases is required to make the analytes volatile [211–213]. The accuracy of the measurements was at one time in doubt due to the lack of isotopically labeled internal standards, introduced for the ﬁrst time in 1993 [214]. It rapidly appeared that the values provided by GC-MS were on the average 2 orders of magnitude higher than those obtained by HPLC-ECD [215]. The origin of this major drawback has been found to be the derivatization reaction [216,217]. Indeed, heating of the acidic DNA hydrolysate at 140jC for 30 minutes has been shown to lead to the spurious oxidation of the overwhelming normal nucleobases. Thus, under the latter derivatization conditions, 8-oxodGua is formed with a frequency up to 104 from guanine, whereas artifactual oxidation of thymine, cytosine, and adenine was found to generate 5-(hydroxymethyl)uracil, 5-formyluracil, 5-hydroxycytosine, and 8-oxo-7,8-dihydroadenine [12,218]. One eﬃcient way to prevent spurious oxidation of the normal constituents is to prepurify the selected lesions prior to the derivatization. This may be achieved by HPLC [216–218] or eventually by immunoaﬃnity chromatography [219], both methods requiring the use of internal standards for isotope dilution measurement purposes. Another major caveat is the instability of several base lesions, including thymine glycols, FapyAde (54) and FapyGua (36), during the hot formic acid treatment aimed at releasing the base residues from the DNA chain [220–222]. A mild hydrolytic protocol that involved ﬁrst the enzymatic digestion of FapyAde (54) and FapyGua (36) as nucleotides followed by formic acid–mediated cleavage of the labilized N-glycosidic bond of the latter compounds at room temperature is now available [223]. Altogether, the optimized HPLC-GC-MS assay is very accurate, although tedious due to the requirement of the time-consuming prepuriﬁcation step. It is now clear that the bulk of GC-MS measurements of oxidative base damage in cellular DNA are questionable. Therefore, the conclusions of biochemical studies based on the latter data have to be considered with caution. A typical example is illustrated by the claim that supplemented vitamin C may exhibit pro-oxidant eﬀects, as inferred from the increased level of 8-oxoAde in lymphocytes DNA [224]. However, the reported values of 8-oxoAde and 8-oxoGua were about 2 orders of magnitude higher than those assessed by HPLC-ECD in the DNA of similar biological samples [225] and further conﬁrmed by recent measurements [226,227]. This is also the case for the GC-MS determination of high background levels of oxidative lesions in the DNA of AG101111 normal human ﬁbroblasts: thus, levels of 121 FapyGua, 16.5 FapyAde, and 27.8 5-hydroxycytosine (5-OHCyt) per 106 bases were assessed [228]. Huge values of base damage were measured in the DNA of lymphoma L5178S cells upon exposure to 400 Gy of gamma radiation [229]. The reported level of FapyGua (0.5 lesion per 102 bases) is at least 3 orders of magnitude higher than the yield of the same base damage measured in human monocytes by HPLC-GCMS [230]. Several attempts were made to devise amended versions of the conventional GCMS method without prepuriﬁcation of the targeted lesions. It was proposed that the use of lower temperature for the silylation reaction [216] or the addition of antioxidants, either ethanethiol [231] or N-phenyl-1-naphthylamine [232], should prevent artifactual oxidation from occurring. This appears unconvincing in light of the reported high values of steady-state level of oxidized bases in cellular DNA in various samples. Thus, 5 FapyAde, 2.1 5-(hydroxymethyl)uracil (5-HmUra), 2.0 5-OHCyt,

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and 1.6 8-oxoGua per 103 nucleosides were estimated in the DNA primary hepatocytes treated with ferric nitrilotriacetate [233]. Other examples of high values of oxidative lesions measured by GC-MS after derivatization at low temperature (60jC) were recently reported [234,235]. They indicate that the steady-state levels of 8-oxoGua and FapyGua (36) were within the range of 2 to 3 lesions per 103 guanine bases in the DNA of human prostate tissue [234] and hind leg of BALB/c mouse [235]. The values of 8oxoAde and FapyAde (54) were between 0.4 and 0.8 lesions per 103 adenines in the same DNA. These high values question the validity of the conclusions as to the correlation between level of oxidized bases and incidence of prostate cancer, on the one hand [234], and the protective eﬀect of N-acetylcysteine on the background level of oxidative base damage to DNA [235]. A ﬁnal example of inconsistencies is borrowed from experiments involving derivatization at low temperature in the presence of ethanethiol [236]. Again, the background levels of the main purine lesions in the DNA of human cells were elevated, as shown by the following values with respect to 105 normal bases: 3.5 8oxoGua, 5 FapyGua, 9 8-oxoAde, and 8 FapyAde [236]. It is rather intriguing that apparently no attempts were made to validate these modiﬁed GC-MS assays. Recently, the use of Fpg-mediated release of 8-oxoGua was proposed as an alternative to formic acid hydrolysis [237]. Using this speciﬁc approach since no normal bases are released, the level of 8-oxoGua was found to be similar to that provided by HPLC-ECD measurement, about 2 orders of magnitude lower than the values obtained using the conventional GC-MS method. 4.

HPLC–Electrochemical Detection

The ﬁrst identiﬁcation of 8-oxodGuo (35) as an oxidation product of dGuo upon incubation with heated sugar derivatives [238] provided a strong impetus for the wide use of this oxidized purine component as a relevant biomarker of oxidative stress [239]. The development at the same time by Floyd et al. [240] of a reliable and simple analytical assay associating HPLC to speciﬁc and sensitive electrochemical detection was of major importance. 8-OxodGuo (35) rather than 8-oxoGua is usually the targeted lesion quantitatively released from DNA by enzymatic digestion and then eﬃciently separated on octadecylsilyl silicagel columns. This method, in contrast to GC-MS, has no major drawbacks if artifactual oxidation during DNA extraction and subsequent work-up is achieved (see below). However, until recently this aspect has been only occasionally considered, which explains why variations in the reported values of 8oxodGuo (35) were observed. Optimization of DNA extraction conditions, including the use of NaI for DNA precipitation in the so-called chaotropic technique [241] together with the use of desferal as a metal chelator [226], has led to a signiﬁcant decrease in the steady-state level of 8-oxodGuo (35) in cellular DNA [226]. Several improvements in the HPLC-ECD assay have been achieved during the last decade. Coulometric detectors more sensitive by at least a factor of 4 than the initially available amperometric apparatus have gradually appeared. The detection of 8-oxodGuo (35), performed in the oxidation mode, has been extended to the measurement of other oxidized bases with low ionization potential, including 8-oxoAde, 5-hydroxyuracil, 5OHCyt, and their related nucleosides [81,242,243]. It may be added that FapyGua (36) and FapyAde (54) may also be measured by electrochemical detection. However, the diﬃculty in achieving eﬃcient separation of these polar compounds has limited this application. Calibration of the measurement of 8-oxoGua was provided by the design of 2,6-diamino-8-oxopurine as an electroactive internal standard [244]. Incubation of

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DNA with Fpg protein leading to the release of 8-oxoGua was found to be a relevant alternative to traditional nuclease P1 digestion. Faster work-up and reduced risks of artefactual oxidation are the two main advantages of this new protocol [245]. 5.

HPLC-MS and HPLC-MS/MS

The potential of the HPLC-MS/MS method for monitoring oxidative base damage to cellular DNA was ﬁrst illustrated with the measurement of 8-oxodGuo (35) [71,246]. Ionization of 8-oxodGuo is performed using an electrospray source at atmospheric pressure that operates in the positive mode. The most sensitive technique of detection is achieved using the multiple reaction monitoring (MRM) technique. Typically, the pseudo-molecular ion is selected on the ﬁrst quadripole before undergoing fragmentation on a collision cell. The main fragmentation for 8-oxodGuo (35) is the cleavage of the N-glycosidic bond [72,246–249]. This allows detection of the lesion on the basis of the transition between the nucleoside and the base on the third quadripole. Interestingly, a threshold of detection of a few fmol was estimated for 8-oxodGuo (35), making the assay at least 5- and 10-fold more sensitive than the HPLC-ECD and HPLC-GC-MS assays, respectively. The use of [M+3] to [M+5] internal standards labeled with stable isotopes including 13C, 15N, and 18O provides highly accurate measurements. Application of this versatile method, still in the MRM mode, has been extended after appropriate optimization and selection of a suitable transition to the measurement of 8-oxodAdo, the four diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine (9) (ThdGly), 5-OHdUrd (22), 5-FordUrd (13), and 5-HMdUrd (12) together with FapyAde (54) and FapyGua (36) [72,248,250,251]. Interestingly, 2OHdAdo (55) [142] and several oxidation products of dGuo [125,252] were also included in the list of oxidized nucleosides that can be detected accurately by HPLCMS/MS. 8-OxodGuo (35), 8-oxodAdo (53), FapyGua (36), and FapyAde (54) were better detected in the positive ionization mode. In contrast, ionization in the negative mode was found to be more appropriate for the tandem MS analysis of the oxidized pyrimidine 2V-deoxyribonucleosides. In addition, several tandem base lesions including the Fo/8-oxoGua and 8-oxoGua/Fo double damage [156] together with thymine adducts to adenine and guanine [154] were detected by HPLC-MS/MS as dinucleoside monophosphates in the negative ionization mode. It is likely that further applications of this powerful tool to the detection of other oxidized nucleosides, including thymidine hydroperoxides, will be reported shortly. The use of HPLC-MS/MS has made possible a comparative study aimed at evaluating and optimizing conditions of DNA extraction and subsequent hydrolysis for monitoring the formation of 8oxodGuo (35) [227]. For this purpose, molecules of [18O]-labeled 8-oxodGuo (35) were speciﬁcally generated within cellular DNA [253]. The preparation of this internal standard was achieved using a chemical source of [18O]-singlet oxygen [254]. It was found that the chaotropic method that involves the use of NaI together with desferal was the best approach to minimize spurious oxidation of DNA during extraction and subsequent work-up [227]. It may be added that NaI is not able to oxidize 8-oxodGuo (35), in contrast to recent claims [255]. Therefore, it appears that the background level of 8-oxodGuo in lymphocyte cell lines under these optimized conditions of extraction that require at least 20 Ag of DNA is about 0.5 lesions per 106 normal bases [227]. This value is similar to that found using similar protocols of extraction by two other groups [223,256] using the HPLC-ECD assay. A comparison of the MRM and SIM modes of detection of 8-oxodGuo provided by HPLC-MS/MS analysis and HPLC-MS measurement, respectively, has been car-

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ried out [71]. The background level was found to be much higher in the SIM-MS analysis, which appears to be 50-fold less sensitive than the tandem MS measurement. In addition, the accuracy of the measurement provided by the MRM mode is much better than of the SIM detection technique, since measurement of transition in the former analysis may be used as a relevant structural parameter. Despite these apparent limitations, several applications of HPLC-MS analysis of oxidized nucleosides have been reported recently. Thus, assays were developed for monitoring the formation of 8-oxodGuo (35) [257], 8-oxodAdo (53) [258], 8V,5V-cyclodAdo [164], and 8,5V-cyclodGuo [165]. Using the latter assay it was found that the background levels of the two (5VR)- and (5VS)-diastereomers of 8,5V-cyclodGuo in three diﬀerent types of cultured human cells were about 2 and 10 lesions, respectively, per 106 nucleosides. This is much higher than the values that may be extrapolated from HPLC-MS/MS measurements by at least 2 orders of magnitude. A similar situation is observed in the measurement of radiation-induced formation of 8-oxodGuo and 8-oxodAdo performed on the DNA from various Cockayne syndrome Group B–transfected cell lines. It was found that the rates of formation per Gy of 8-oxodGuo and 8-oxodAdo were 2 and 0.3 lesions per 106 in three of the transfected cells tested [259,260]. This contrasts with the much lower values assessed by HPLC-MS/MS in the DNA of g-irradiated human monocytes, which are, respectively, 0.039 8-oxodGuo and 0.003 8-oxodAdo per 106 bases [261]. B.

Enymatic Measurement of Oxidative Base Damage to DNA in Single Cells

An alternative approach to the chromatographic measurements of oxidative base damage is represented by the modiﬁed versions of the alkaline single cell gel electrophoresis or ‘‘comet assay’’ [262], the alkaline elution technique [263], and the alkaline unwinding method [264]. The comet assay, like the other methods, is applicable to isolated cells. This is a highly sensitive ﬂuorescence-based detection method that allows, for example, the assessment of radiation-induced damage within human blood cells upon exposure to doses of ionizing radiation as low as 0.1 Gy [265]. However, the three assays in their basic version suﬀer from a lack of speciﬁcity in the measurement of the lesions they provide. Thus, the potential damage that may be monitored is very broad, consisting of single and double frank strand breaks, true and oxidized abasic sites, together with alkali-labile lesions. It should be also pointed out that transient strand breaks that may occur during DNA repair processes are also accessible by the three methods. The strategy to make the latter assays more speciﬁc has involved incubating the released DNA with several DNA repair enzymes prior to the analysis of the strand breaks. Optimized protocols have become available during the last decade for the modiﬁed enzymatic version of the comet assay [266–268], alkaline elution technique [269,270], and alkaline unwinding technique [264]. Two main base excision repair enzymes that, in addition to their ability to cleave the N-glycosidic bond of oxidized bases, show an AP-lyase activity are used. This leads to strand cleavage of DNA at the site of the lesion. Thus, the result of the enzymatic treatment is the induction of additional strand breaks that could be assessed by subtracting the level of strand breaks generated in the enzymatic treatment from the overall number of nicks. Bacterial Fpg was used to reveal 8-oxoGua together with FapyGua and FapyAde, whereas E. coli endo III is able to convert several oxidized pyrimidine lesions into strand breaks.

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The three methods show comparable results. However, values of Fpg-sensitive sites were until recently lower that the sum of individual lesions measured by chromatographic methods, consisting mostly of 8-oxodGuo (35) and FapyGua (36). Optimization of the DNA extraction conditions has led to a signiﬁcant reduction in the level of background level of 8-oxodGuo (35) as assessed by HPLC-ECD or HPLCMS/MS (see above). Work is in progress to calibrate the comet assay in a more direct way than using the reference number of strand breaks generated by 1 Gy of ionizing radiation. Again, cells that contain in their DNA a given number of 8-oxodGuo (35) residues [253] generated by a clean source of singlet oxygen [254] will be used to estimate the eﬃciency of the Fpg protein to cleave DNA and to calibrate the number of strand breaks thus generated. The three methods exhibit a very small hydrodynamic range of applicability in term of damage extent, which for ionizing radiation corresponds to doses of between 0 and 10 Gy. Interestingly, these highly sensitive methods are particularly suitable for investigating low-dose eﬀects and when only low amounts of DNA are available (f1 Ag). This is a major advantage with respect to chromatographic methods due to the almost complete lack of the occurrence of DNA artifactual oxidation on the agarose gel or during the elution technique analysis. It was possible using an optimized version of the comet assay to detect Fpg- and endoIII-sensitive sites in the DNA of human monocytes after exposure to a dose of g-rays as low as 0.2 Gy [271]. However, the enzymatic methods are less speciﬁc and quantitative than the HPLC-ECD and HPLC-MS/MS assays.

IV.

OXIDATIVE BASE DAMAGE IN CELLULAR DNA AND BIOLOGICAL FLUIDS

Numerous attempts have been made to assess the level of oxidized bases in cells under diﬀerent pro-oxidant or antioxidant conditions with respect to control. In addition, eﬀorts have been made to assess indirect damage to nucleic acids through the indirect measurement of oxidized bases and nucleosides in biological ﬂuids such as urine and plasma. A.

Oxidative Base Damage in Cellular DNA

As discussed in the previous sections, the measurement of oxidative damage to cellular DNA is still a challenging issue which, during the last 15 years, has led in many cases to an overestimation of the reported values by factors ranging from 10 to 1000, depending on the method used. Most of the drawbacks associated with the application of assays involving DNA extraction are now known (see above). However, the use of the optimized chaotropic method of DNA extraction together with either HPLCECD or HPLC-MS/MS now allows measurement of several oxidized bases when the increase in the level is higher than a few modiﬁcations per 106 bases. This is the case with recent HPLC-MS/MS measurement of several radiation-induced base lesions within the DNA of human monocytes [230,261]. Thus, a signiﬁcant increase in the levels of ThdGly (9) (8.3 lesions per 106 normal bases) and 8-oxodGuo (35) (2.6 lesions per 106 normal bases) was observed in the DNA of human monocytes upon exposure to a dose of 90 Gy of gamma rays. On the other hand, the levels of 8-oxodAdo (53) and 5-OHdUrd (22) were similar to those of the control. Another example of the suitability of the HPLC-MS/MS method was provided by the assessment of the background level

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of several oxidized nucleosides, including ThdGly (9) and 8-oxodGuo (35), in the liver and kidney of young and old mice [227]. The basal level of 8-oxodGuo (35), which is 0.8 lesion in the DNA liver of young mice (<3 months), was found to increase up to 1.8 modiﬁcations with age (>13 months). The association of bacterial DNA repair enzymes including the formamidopyrimidine DNA glycosylase (Fpg) and the endonuclease III (endo III) proteins with the single cell gel electrophoretic assay has allowed the measurement of three main classes of radiation-induced damage in the DNA of human monocytes at a dose as low as 2 Gy [230]. In a recent study attempts were made to calibrate the modiﬁed comet assay by singling out the main nucleobase decomposition products that correspond to the Fpgsensitive sites in cellular DNA exposed to relatively high doses of either gamma rays or UVA radiation [230,261,268]. This was achieved by HPLC-ECD for the measurement of 8-oxodGuo (35), whereas the search for FapyAde (54) and FapyGua (36) was made by HPLC-GC/MS. Interestingly, it was found that 8-oxodGuo (35) and FapyGua (36) (relative ratio 29:71) were generated in a linear relationship with the applied doses of ionizing radiation within the dose ranges 30–80 and 250–1000 Gy, respectively. It may be added that the formation of FapyAde (54) was not detected in the DNA of monocytes exposed to gamma rays. This is in agreement with the fact that FapyAde (54) is generated in an approximate 10-fold lower yield than FapyGua (36) in isolated DNA upon exposure to either OH radical or one-electron oxidation process. The chaotropic DNA extraction method, which minimizes artifactual oxidation of DNA, does not allow the chromatographic measurement of 8-oxodGuo (35) at doses of ionizing radiation lower than 20 Gy. In contrast, an increase in both Fpg-and endo III–sensitive sites was observed following exposure of the monocyte cells to a dose of ionizing radiation as low as 0.2 Gy [271]. Thus, estimation of the levels of several classes of cellular background and radiation-induced damage together with the yields of targeted base damage including 8-oxodGuo (35) and FapyGua (36) can be made. This makes possible to gain relevant information on the mechanistic aspects of the radiation-induced decomposition of cellular DNA. We note the relative high value of the ratio FapyGua/8-oxodGuo in cellular DNA (f2.5), which is, at least, four times higher than within isolated DNA in aerated aqueous solution. This is indicative of the higher occurrence of reducing processes and/or a lower oxygen concentration in cells since 8-oxodGuo (35) and FapyGua (36) are generated from a common guanine radical precursor (Fig. 4) through oxidation and reduction, respectively. Ionizing radiation produces a low amount of DNA damage, since 4 Gy are required to double the background level of 8-oxodGuo (35). This clearly demonstrates that more complex lesions, such as DNA strand breaks associated with base damage, are likely to be involved in the deleterious eﬀects of ionizing radiation. The molecular eﬀects of UVA radiation were also investigated using chromatographic methods together with the modiﬁed comet assay. The formation of 8oxodGuo (35) was found to increase linearly with the applied doses of UVA radiation within the dose range 0–150 kJ.m2 [230,272]. However, it was not possible under these conditions of irradiation to detect the formation of FapyGua (36) and FapyAde (54) in cellular DNA. This, together with the relatively low induction of both DNA strand breaks and endo III–sensitive sites, leads to the conclusion that the UVA-mediated formation of 8-oxodGuo (35) is mostly accounted for by singlet oxygen. Similar conclusions were inferred from the results provided by the modiﬁed version of the alkaline elution technique [273,274]. Information was also gained from the applica-

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tion of the latter method to the measurement of the background level of classes of oxidative damage in the DNA of mice organs. Thus, the number of Fpg-sensitive sites, estimated to be 0.18/106 bp in the liver DNA of wild-type mice, was found to increase by 1.4-fold in OGG1-deﬁcient animals [275]. The modiﬁed comet assay has received interesting applications with the delineation of the protective eﬀects of antioxidants on the steady-state level of oxidative damage to DNA in lymphocytes [276]. B.

Oxidative Base Damage in Biological Fluids

Evaluation of the molecular eﬀects of pro- or antioxidants on genomes in humans and animals requires noninvasive assays. Attempts were made more than 20 years ago to develop methods aimed at measuring the release of oxidized bases and nucleosides in urine and plasma as an index of DNA damage [277,278]. The working hypothesis was that oxidized bases and nucleosides are likely to be excreted in biological ﬂuids, such as urine and plasma, once excised from nucleic acids by base and nucleotide excision repair enzymes. Practically, HPLC-EC, in association with either solid phase extraction preconcentration [279] or immunoaﬃnity chromatography [177,219], has been used to monitor the presence of 8-oxoGua and related nucleosides in urine. An ELISA kit assay based on monoclonal antibody N.45.1 [191] is available for monitoring the level of urinary 8-oxodGuo 35 [280,281]. Another approach applies a column switching device prior to ECD measurement [282,283]. More recently, the analytical capacity of the assay was improved by the use of a highly hydrophobic column as an alternative to the cation exchange column [284]. In order to extend the measurement of urinary oxidative DNA damage components to nonelectroactive compounds, a CG-MS assay was designed [285]. This requires a somewhat tedious HPLC prepuriﬁcation step of the targeted lesions prior to GC-MS analysis, aided by the use of isotopically labeled internal standards. Thus, 8-oxo-7,8-dihydroguanine, 5-(hydroxymethyl)uracil, and their corresponding nucleosides (12,35) [286–288] were measured. More recently, application of the assay was extended, and therefore ﬁve lesions were measured in human urine [289]. The importance of the levels of oxidized bases and nucleosides, expressed in pmol/mL of urine, was found to decrease in the following order: 8oxoGua (893 F 376) > 5-(hydroxymethyl)uracil (121 F 56) > 5-hydroxyuracil (58 F 23) > 8-oxodGuo (35) (30 F 15) > 8-oxodAdo (53) (7 F 4). Several studies attempted to assess the modulating eﬀects on oxidative stress agents such as on the levels of oxidized bases and nucleosides and particularly 8-oxodGuo (35) and 8-oxoGua [279,287,288,290–292]. Attempts were also made to correlate dietary factors and lifestyles with cancer risk through the measurement of 8-oxodGuo (35) [5,283,293,294]. However, no convincing conclusions can be gained from the bulk of the available results. In most cases the eﬀects, when signiﬁcant, were of low amplitude and sometimes contradictory, as in the case of smoking [280,288,292,294,295]. This is likely due to the lack of accuracy in most of the measurements, the apparent high variability of the reported values of oxidized bases and nucleosides in human urine, the absence of biological validation of the assay, and the putative modulation eﬀect of diet. It was recently reported that the levels of both 8-oxoGua and 8-oxodGuo (35) in human urine are not dependent on diet [296], in contrast to a previous ﬁnding that showed a modulating eﬀect of the latter parameter on the urinary level of 8-oxoGua in rat [177]. The origin of the oxidized lesions found in urine has yet to be established, since in addition to the likely implication of the BER and NER enzymes, other possibilities

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should be considered. These include the MTH repair enzyme [297] that detoxiﬁes the nucleotide pools from the presence of 8-oxodGTP, contribution of dead cells, and the putative occurrence of metabolism pathways. Further studies are required to address these issues in order to validate the biochemical feature of the approach. The accurate measurement of several oxidized bases and nucleosides in the urine of human or animals subject to dedicated oxidative stress should be of major help. The availability of the HPLC-MS/MS method, which has been shown to be particularly appropriate for the determination of the level of 8-oxodGuo (35) [71], 8-oxodAdo (53), 8-oxoGua, and the related ribonucleoside [249,298], should facilitate the measurement of a wide spectrum of oxidized DNA constituents in biological ﬂuids, such as plasma and urine. V.

CONCLUSIONS

Major eﬀorts have been devoted to determining the predominant oxidation reactions involved in the degradation of isolated DNA and model compounds that may have biological relevance. Although the main oxidation products of the guanine moiety have been isolated and characterized, there is still a deﬁcit of information on their mechanism of formation. This applies also to adenine, which seems to have the lowest sensitivity among nucleobases to the damaging eﬀects of strong oxidizing agents like the OH radical. Progress has been made in the optimization and validation of analytical methods aimed at sensitive and accurate measurement of oxidative base damage to DNA. (See, for example, the publication from the European Standards Committee on Oxidative Damage, which has also involved non-European research groups [299].) Thus, estimation of the level of several oxidized nucleosides, including 8oxodGuo, has been made in the DNA of isolated cells or tissues following exposure to oxidizing agents such as solar UVA radiation and gamma rays. It may be added that the background level of the ubiquitous 8-oxodGuo lesion (35) in the DNA of lymphocytes and mouse organs is in the range of a few lesions per 107 normal bases. Special attention should be given to the development and wider use of two main complementary powerful tools—the highly accurate HPLC-MS/MS and the ultrasensitive but more global enzymatic methods. In particular, the application of chromatographic techniques should be extended to the measurement of new lesions, whereas better calibration of the modiﬁed comet assay and alkaline elution method should be achieved.

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296. Gackowski D, Rozalski R, Roszkowski K, Jawien A, Foksinski M, Olinski R. 8-Oxo7,8-dihydroguanine and 8-oxo-7,8-dihydro-2V-deoxyguanosine levels in human urine do not depend on diet. Free Radic Res 2001; 31:825–832. 297. Hayakawa H, Taketomi A, Sakumi K, Kuwano M, Sekiguchi M. Generation and elimination of 8-oxo-7,8-dihydro-2V-deoxyguanosine 5V-triphosphate, a mutagenic substrate for DNA synthesis, in human cells. Biochemistry 1995; 34:89–95. 298. Weimann A, Belling D, Poulsen HE. Quantiﬁcation of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry. Nucleic Acids Res 2002; 30:E7. 299. Collins AR, Gedik C, Wood S, et al. Comparison of diﬀerent methods of measuring 8oxoguanine as a marker of oxidative DNA damage. ESCODD (European Standards Committee on Oxidative DNA Damage). Free Radic Res 2000; 32:333–341.

9 Redox Modulation in Tumor Initiation, Promotion, and Progression MARGARET HANAUSEK, ZBIGNIEW WALASZEK and THOMAS J. SLAGA AMC Cancer Research Center, Denver, Colorado, U.S.A.

I.

INTRODUCTION

The concept that carcinogenesis is a multistep and multifunctional phenomenon is now widely supported by researchers. With the early experimental design [1], which employs a two-step procedure for the treatment of mouse skin, a useful animal model became available to study the multistep nature of carcinogenesis. In fact, some early investigators began to analyze the process, and they deﬁned the concepts of tumor initiation and promotion as well as co-carcinogenesis in operational terms [2]. For much of the time during which initiation-promotion studies in mouse skin were being pursued by a number of researchers [3], little experimental evidence existed, other than hormone-dependent tumors, that similar processes occurred at other organ sites. Later, however, these sequential events were also found to occur in liver, urinary bladder, breast, cheek pouch, esophagus, colon, stomach, lung, and prostate [4,5]. The generality of the sequential nature of biological events in carcinogenesis is especially true in those tumors induced by exogenous chemical agents. In addition to extensive studies in the mouse skin model, several approaches to the sequential evolution of liver tumors by multistep protocols have been studied [6,7]. The greatest understanding of the important biological and cellular events involved in tumor initiation, promotion, and progression has been provided by studies in the skin and liver models [5]. Understanding the mechanism(s) by which an agent induces, promotes, or enhances cancer is important in overall risk assessment. Although one can question the relevance to humans of tumor promotion in experimental animals, it is important to emphasize that tumor promoters, in general, induce the 193

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cell proliferation, which is critically involved in selectively expanding initiated cells into tumors. Cancer is a disease in which there is an uncontrolled proliferation of cells that express varying degrees of ﬁdelity to their precursor cell of origin [8]. Most human tumors have a long history of pathological development during which they pass through several preneoplastic and premalignant stages before they become malignant. The clinical stages of tumor development have been correlated with speciﬁc genetic alterations such as activation of proto-oncogenes and deletion of tumor suppressor genes. By providing conﬁrmation of molecular defects, human cancer genetics strongly, though indirectly, supports the concept of multistage carcinogenesis. The implication here is that malignant neoplasia is the result of multiple genetic defects successively accumulating over a period of time [8]. However, only animal models can provide direct information on the underlying mechanisms and enable a ﬁnal proof of the multistage concept. As indicated above, the animal models of multistage carcinogenesis that are presently fairly well deﬁned are skin cancer in mice and liver cancer in rats [5]. Both models allow a clear-cut distinction and mechanistic insight of individual stages of carcinogenesis. The results from the skin model, in particular, appear to be of relevance for a more in-depth understanding of the human epithelial cancers including colorectal cancer (reviewed in Ref. 8). Thus, the induction of a neoplasm is a multistage process that occurs over a long period of time, i.e., decades in humans. These stages have been deﬁned experimentally as initiation, promotion, and progression. Initiation involves mutation of cellular DNA resulting in the activation of oncogenes and the inactivation of tumor suppressor genes. Initiation is thought to be irreversible and consist of a single gene mutation that is caused in most cases by environmental genotoxic agents such as chemicals, radiation, and viruses. Oncogenes can also be activated by chromosomal translocations and gene ampliﬁcations. Studies in the human colon indicate that the carcinogenic process involves multiple genetic alterations in a staged fashion [9]. Promotion follows initiation and involves the process of gene activation such that the latent phenotype of the initiated cell becomes expressed through cellular selection and clonal expansion. This can occur through a variety of mechanisms, including toxicity, terminal diﬀerentiation or mitoinhibition of the noninitiated cells, and mitogenesis of the initiated cells [3,4]. While promotion occurs over a long period of time, it is reversible in its early stages. Proof that promotion is reversible in humans is supported by the observation that the rate of lung cancer induction in individuals who quit smoking approaches that of nonsmokers [10]. The breadth of the available data as well as the multistage nature of tumor promotion suggests that this process, which is now thought to occur in most tissues where cancer can be induced or where it occurs spontaneously, may involve the interaction of a number of endogenous factors as well as environmental factors such as chemicals, radiation, viruses, and diet and nutrition, thus unifying all current areas of cancer research [11]. In human cancer, smoking, environmental factors such as asbestos, hydrocarbons, radiation, hormones, alcoholic beverages as well as diet and nutrition, to mention a few, are now thought to have more of a promotional inﬂuence in the multistage carcinogenesis process [11]. The last step leading to cancer is called progression. Progression involves genetic damage that results in the conversion of

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benign tumors into malignant neoplasms capable of invading adjacent tissues and metastasizing to distant sites. The additional genetic alterations thought to be required for neoplastic progression often occur faster that would be expected form the statistics of accidental geneotoxic insults due to so-called genetic instability. The concept of genetic instability implies that while environmental genotoxic agents generally cause cancer initiation, the additional mutations required for neoplastic progression may be attributed to endogenous reaction and factors such as detoxiﬁcation and removal of damaged cells by programmed cell death. Genetic instability may happen due to the errors in DNA replication, spontaneous hydrolytic alterations of DNA such as depurination and deamination, in combination with an impaired ability of premalignant cells to repair DNA damage or due to oxidative DNA damage [12]. Oxidative DNA damage occurs through an overactivation or disregulation of metabolic reactions, which in turn give rise to reactive oxygen species (ROS) such as hydroxyl and superoxide anion radicals, singlet oxygen, hydrogen peroxides, and nitrogen oxide, as well as to free radicals and peroxides. Growing evidence implicates both oxygen and organic free radical intermediates in the biomolecular interactions, which contribute to each of the multistep stages of carcinogenesis (reviewed in Refs. 13,14). Many exogenous chemicals implicated in initiation, promotion, and progression can be activated to radical intermediates, which can serve as electrophiles or participate in reactive oxygen generating redox cycling processes. Similarly, the increased and/or continuous elaboration of reactive oxygen species by endogenous sources can create an increased oxidative state in cells and organs contributing to the promotion and progression of cancer [13,14]. Oxidative damage inﬂicted by reactive oxygen species has been called oxidative stress [15]. Biological systems contain powerful enzymatic and nonenzymatic antioxidant systems, and oxidative stress denotes a shift in the prooxidant/antioxidant balance in favor of the former. A steadily increasing body of evidence supports the concept implying that oxidative stress plays a critical role in cancer development. Evidence is also accumulating that suggests that free radicals are important in all stages of chemical carcinogenesis [13,14]. Several antioxidants have been found to inhibit all stages of carcinogenesis, whereas others are more eﬀective against tumor initiation, promotion, or progression [13]. The purpose of this chapter is to discuss mechanisms by which both intracellular and extracellular ROS derived from endogenous and exogenous sources can modify diﬀerent stages of the carcinogenic process. II.

OXYGEN FREE RADICAL DAMAGE AND MULTISTAGE CARCINOGENESIS

There has been signiﬁcant progress in the understanding of the multistage nature of carcinogenesis [16–20]. The mouse skin model that represents one of the best understood experimental models of multistage carcinogenesis has permitted the resolution of three distinct stages: initiation, promotion, and progression [16,17,19,20]. It is now apparent that the cellular evolution to malignancy involves the sequential alteration of proto-oncogenes [21] and/or tumor suppressor genes [22], whose gene products participate in critical pathways for the transduction of signals and/or regulation of gene expression. Extensive data have revealed a good correlation between carcinogenicity of many chemical carcinogens and their mutagenic activities [16]. Most tumor-

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initiating agents either generate or are metabolically converted to electrophilic reactants, which bind covalently to cellular DNA [16,20]. Oxygen free radicals and DNA bases modiﬁed by oxygen free radicals have also been strongly implicated in the tumor initiation and/or tumor promotion stages as well as carcinogenesis in general [23–37]. The majority of organic peroxides that have been tested for carcinogenic activity in various animal models have been found to have no carcinogenic activity [38–47]. However, several investigators have reported that certain peroxides have some carcinogenic activity [48–51]. For example, in evaluating toxicity, carcinogenicity, and genotoxicity data on hydrogen peroxide, it can be concluded that hydrogen peroxide does not appear to have signiﬁcant carcinogenic activity in mice, hamsters, and rats even when administered at near toxic levels [46,52,53]. Still, hydrogen peroxide had weak carcinogenic activity when male and female mice were combined for analysis, while there was no signiﬁcant carcinogenic eﬀect when the male and female mice were analyzed separately [49,50]. The weak carcinogenic activity of several peroxides may be due to the fact that certain peroxides have been shown to have skin tumor–promoting activity [39,51,54,55]. Inspite of the fact that tumor promoters are considered noncarcinogenic, some tumor promoters produce a weak carcinogenic eﬀect in some but not in all experiments, possibly due to the presence of preinitiated cells [17,39]. Although peroxide tumor promoters give a dose-related tumor response when tested as promoters, they do not have any tumor-initiating activity when tested as tumor initiators [38,39]. Modiﬁed DNA bases, especially 8-hydroxy-2V-deoxyguanosine, produced by oxygen free radicals have been implicated in the genesis of cancer [26–35]. Many carcinogens and tumor promoters have been shown to produce oxygen free radicals and in some cases modiﬁed DNA bases [26–35]. Certain peroxide tumor promoters such as benzoyl peroxide produce modiﬁed DNA bases under certain conditions [34,35]. In determining the importance of oxygen free radicals in toxicity and carcinogenesis, several investigators have developed methods to measure the type and level of free radicals as well as damage from these radicals [23–25,27–29]. Oxygen free radicals are now known to play an important role in many diseases including cancer [19,56]. The importance of free radicals in radiation carcinogenesis and oxygen free radicals and electrophiles in chemical carcinogenesis is also well recognized. Free radicals and reactive oxygen species are continuously produced in vivo. Consequently, organisms have evolved that possess not only antioxidant and electrophile defense systems to protect against them, but also repair systems that prevent the accumulation of oxidatively damaged molecules [56]. III.

ROS AS FACTORS MODIFYING TUMOR INITIATION

A number of genes have been found to be frequently altered by translocation or mutation in human or animal tumors [21]. Most genes in this category are members of the ras family [57], comprising Harvey (Ha-ras), Kirsten (Ki-ras), and N-ras, although several other genes have also been identiﬁed [58]. The capacity of many carcinogens to cause point mutations in DNA together with irreversible nature of skin tumor initiation [16,17,19,20,59] led to the hypothesis that initiation involves the induction of point mutations in a gene(s) that confers some selective growth advantage to the target epidermal cell(s). Strong evidence from several laboratories indicates that

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activation of the Ha-ras gene occurs early in the process of mouse skin carcinogenesis and perhaps is equivalent to the initiation event. The presence of an activated c-Ha-ras gene in mouse skin papillomas and carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) was found to be associated with a high frequency of A-to-T transversions at codon 61 [60]. Subsequent studies demonstrated that the type of mutation was dependent upon the chemical initiator and independent of the promoter, suggesting a direct eﬀect of the initiator on c-Ha-ras [61,62]. Furthermore, infection of mouse skin by a virally activated Ha-ras gene (v-Ha-ras) can serve as the initiating event in two-stage mouse skin carcinogenesis [63]. Thus, although many questions remain to be answered about proto-oncogene activation and chemical carcinogenesis, the collective data using the mouse skin model suggest that activation of ras is an initiating event in the tissue and that, in general, there is a good correlation between carcinogen-speciﬁc DNA damage and induction of mutations in this target gene [20]. Although skin tumor initiators such as polycylic aromatic hydrocarbons (PAH) have been shown to bind covalently to a wide variety of cellular macromolecules, including DNA, RNA, and protein, their interation with DNA correlates most closely with skin tumor–initiating activity (reviewed in Refs. 16,19,20). Substantial eﬀorts have been made to characterize covalent interactions between activated carcinogen metabolites (primarily PAH) and the DNA of epidermal cells, the target cells for tumor initiation in mouse skin. With regard to other types of skin tumor initiators, such as the direct-acting alkylating agents, ultraviolet (UV) light, ionizing radiation, psolarens, and other chemicals, much less is known about their interaction with mouse epidermal DNA in vivo. However, some of these agents have been studied in detail in other model systems, and speciﬁc DNA modiﬁcations have been correlated with their carcinogenic properties (reviewed in Ref. 20). Speciﬁcally, both UV light and ionizing radiation have been shown to initiate skin tumors in mice [64–68]. Recent results indicate [69] that brief exposure of the skin of SKH-1 mice to UV light causes permanent cellular changes that do not result in skin tumors unless the mice are treated with a tumor promoter for several weeks. These observations indicate that UV can function as an initiator of tumorigenesis in mouse skin. Exposure of cultured cells or hairless mice to UV causes mutations in a number of genes. The exact molecular mechanisms of UV-induced skin sunburn lesions and skin tumor initiation still remain unknown. The more often mutagenized gene in skin cancers is the tumor suppressor gene p53. Recently, p53 mutations were shown [69] to be early events in skin carcinogenesis induced by chronic UVB irradiation in SKH-1 mice. It is believed that point mutations on p53 may be an early event in skin carcinogenesis since one can ﬁnd them in pretumoral lesions such as keratoacanthomas and actinic keratosis [69]. Studies over the last several decades have demonstrated that ROS as mediators can be involved in all three stages of carcinogenesis. ROS can directly cause damage to genomic DNA, leading to mutation, activation of proto-oncogenes, inactivation of tumor suppressor genes, and consequently to tumor initiation. ROS induce many forms of DNA damage including base modiﬁcation, deoxyribose oxidation, base-free sites (apurinic/apyrimidinic sites), strand breakage, and DNA-protein crosslinks. One of the most abundant and studied oxidative modiﬁcations of DNA bases involves the C-8 hydroxylation of guanine, frequently estimated as the oxidized deoxynucleoside, 8-oxo-7,8-dihydro-2V-deoxyguanosine (8-oxo-dG or 8-OH-dG) [70]. Diﬀerent ROS species oxidatively modify DNA bases in diﬀerent ways. For example, superoxide (O 2 ) and H2O2 do not react with DNA bases.

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Hydroxyl radical (OH) generates a multiplicity of products from all four bases. In contrast, singlet oxygen (1O2) selectively attacks guanine, resulting in the formation of 8-oxo-dG [71]. On the other hand, DNA damage by ROS produces variety of mutagenic alterations. Mutations arising from selective modiﬁcations at G:C sites appear to be the ﬁngerprint of oxidative attack on DNA by ROS [72,73]. It appears that ROS-induced DNA mutations are not randomly distributed and that some hot spots are evident [72,73]. Several mechanisms have been suggested to be responsible for the ROS-induced DNA mutations [73]. The mutations that occur in a proto-oncogene or a tumor suppressor gene may cause activation of the protooncogene or inactivation of the tumor suppressor gene leading to tumor initiation. It has been demonstrated [74] that ROS could induce activation of mutations in human c-Ha-ras-1 proto-oncogene. When the mutations responsible for the c-Ha-ras-1 gene activation were examined in 11 transformed foci, G-to-T mutations at the second base of codon 12 were found in two transformed foci, A-to-T transversions at the second base of codon 61 in ﬁve foci, and G-to-T mutations at the third position of codon 61 in 4 transformed foci [74]. The above mutations are commonly found in human skin cancers, which are known to be closely related to UV radiation. Both ROS and UVB light have been shown to induce mutations in the p53 tumor suppressor gene in vitro and in in vivo animal models [69]. The mutations include C-to-T and C-to-A single base pair changes at dipyrimidine sequences and tandem double CC-to-TT mutations [75,76]. These mutations in p53 gene have also been observed in 50–60% of nonmelanoma skin cancer in humans [75]. Recently, p53 gene mutations were shown [69] to be very early, possibly initiating events in mouse skin carcinogenesis induced by UVB. It has been postulated that the initiation phase of mouse skin tumorigenesis is the result of persistent lesions which are not adequately removed by DNA repair system prior to DNA replication [77,78]. Alternatively, such lesions may be repaired in errorprone fashion, thus introducing mutagenic lesions in the DNA [77,78]. It is generally believed that rodent cells are less eﬃcient at DNA repair than human cells. This could help explain why rodents (e.g., mice) are in general quite susceptible to chemical carcinogenesis (reviewed in Ref. 20). At present, little is known about genetic diﬀerences in DNA repair between mouse strains and their role in modifying tumor initiation and multistage carcinogenesis in mouse skin [20]. In summary, DNA damage arising from exposure to chemical agents or radiation gives rise to the mutations responsible for initiating the carcinogenic process. Mutations can arise from replication bypass of unrepaired base damage or as a consequence of large-scale genetic events involving deletions and translocations associated with unrepaired strand breaks. Translocations have been implicated in the initiation of many human neoplasms through the activation of oncogenes. The most prominent example of this involves the activation of myc oncogene in Burkitt’s lymphoma by a translocation between chromosomes 2 and 8. Deletions and translocations of various other oncogenes (e.g., N- and Ha-ras, myb, sis, abl) have also been associated with the initiation of certain cancers such as neuroblastoma, various leukemias, and breast carcinoma. Inactivation of tumor suppressor genes may also be caused by small deletion such as the deletion of chromosome 13 found in retinoblastoma. Like ionizing radiation, ultraviolet light can damage cellular DNA directly or indirectly (reviewed in Ref. 79). In contrast to the direct induction of DNA damage by UVC and UVB light, UVA light produces damage indirectly through highly

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reactive chemical intermediates. Similar to ionizing radiation, UVA radiation generates oxygen and hydroxyl radicals that in turn react with DNA to form monomeric photoproducts, such as cytosine and thymine photohydrates. It also causes DNA strand breaks and DNA-protein crosslinks [79]. Although UV and chemical carcinogens induce DNA damage that may initiate mutagenesis and carcinogenesis, normal mammalian cells have the capacity to remove the DNA damage eﬃciently. Cells utilize diﬀerent processes to repair DNA damage, and this depends on the structure of the damage and its location in genomic DNA (reviewed in Ref. 79). Cells that lack this repair processes are more susceptible to DNA-damaging agent–induced cell death and mutation and, in consequence, are more susceptible to malignant transformation as a result of DNA damage formation. In conclusion, ROS are able to directly damage genomic DNA, resulting in mutations, activation of proto-oncogenes, and/or inactivation of tumor suppressor genes, potentially leading to tumor initiation. On the other hand, a large body of evidence suggests that through redox modulation of transcriptional factors or activators and/or oxidatively modulating protein kinase cascades, ROS may also dramatically interfere with normal cell signaling, resulting in altered gene expression. These events might contribute to tumor promotion/progression stages of carcinogenesis. IV.

ROS AS FACTORS MODIFYING TUMOR PROMOTION

A variety of chemical agents are known to act as tumor promoters in various systems. However, much of our knowledge of the cellular and molecular mechanisms of tumor promotion has come from studies in which the phorbol ester and peroxide tumor promoters in the mouse skin model and in various cell culture systems have been used [19]. Although the phorbol esters, especially 12-O-tetradecanoyl-phorbol-13-acetate (TPA), have been the most extensively studied skin tumor promoters to date, many other chemical compounds have been used successfully as skin tumor promoters (reviewed in Refs. 3,17,19,20). In addition to chemical promoting agents, a number of other types of stimuli can act as promoters of skin tumors in this model system [19,20]. Thus, UV light has a strong promoting action in mouse skin, and physical trauma of suﬃcient enormity has long been known to promote the formation of skin tumors in previously initiated mice. Also, full thickness skin wounding is a very strong promotion stimulus for epidermal tumorigenesis. Current information suggests that skin tumor promoters do not bind covalently to DNA and are not mutagenic but result in a number of important epigenetic changes [80]. The observed phorbol ester–induced eﬀects on the skin, like the induction of epidermal cell proliferation, ornithine decarboxylase (ODC), and subsequently polyamines, prostaglandins, and dark basal keratinocytes, have the best correlation with promoting activity [81]. Other important epigenetic changes in the skin include membrane and diﬀerentiation alterations, an increase in protease activity, cAMP independent protein kinase activity, and phospholipid synthesis. Promoters that interact with a phospholipid, calcium-dependent kinase, called protein kinase C, include TPA and related phorbol esters, teleocidin, and its analogs as well as aplysiatoxins [81]. Other promoters such as benzoyl peroxide, lauroyl peroxide, hydrogen peroxide, anthralin, palytoxin, and chrysarobin do not interact with protein kinase C. Their promotional activity is probably due to some other mechanism, possibly their ability to generate free radicals [19]. Many chemical

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skin tumor promoters, including TPA, do not appear to require metabolic activation for their eﬀects [20]. However, some classes of tumor promoters such as the organic peroxides, anthrones, hydrocarbons, and quinones, do require conversion, either spontaneously or enzymatically, in order to form reactive intermediates for their promoting action. Studies of the skin tumor promoter butylated hydroxytoluene hydroperoxide (BHTOOH) suggest that one or more reactive intermediates, including phenoxyl, peroxyl, quinoxyl or quinone methide, may be involved in its promoting activity [82,83]. Anthrone derivatives such as anthralin and chrysarobin autooxidize to a variety of reactive intermediates including anthranyl, peroxyl, and semiquinone [84,85]. Anthrones, in addition, generate superoxide anion (O2) during their autooxidation [85]. The available evidence indicates that one or more of these free radical intermediates are responsible for the promoting activity of anthrones (reviewed in Refs. 20,84). Thus, evidence is emerging that the generation of free radicals (especially activated forms of oxygen) may be involved in the skin tumor– promoting actions of several classes of promoting agents, including the phorbol esters. TPA was also shown to stimulate production of O2 and possibly other free radicals by polymorphonuclear leukocytes [86]. There is a large body of direct evidence for the involvement of free radicals in tumor promotion coming from studies with free radical–generating compounds such as organic peroxides. Thus, benzoyl peroxide and other organic peroxides are eﬀective skin tumor promoters in mice sensitive to carcinogenesis [39,87]. In addition, TPA, benzoyl peroxide, and anthralin have also been shown to decrease the activities of superoxide dismutase (SOD) and catalase (CAT) in mouse epidermis shortly after their application [88–90]. A wide variety of tumor promoters have also been shown to decrease the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in mouse epidermal cells [91]. These changes presumably reﬂect the induction of a prooxidant state in the epidermal cells by TPA and other types of tumor promoters. Although some early studies [59] ﬁrst suggested that the tumor-promotion phase of mouse skin carcinogenesis had two operationally distinct stages, only subsequent extensive studies on mouse skin tumor promotion [92], conﬁrmed later by another group [93], led to further development of this two-stage concept. Currently, the standard two-stage promotion protocol involves initiation followed by one to four applications of TPA (stage I) and then by multiple applications of a weak papilloma-promoting agent, such as mezerein or 12-O-retinylphorbol-13-acetate (RPA) (stage II) [80]. Thus, tumor promotion in mouse skin appears to include at least two stages, and there is evidence for the involvement of active oxygen in both [88]. Hydrogen peroxide and benzoyl peroxide induce dark basal keratinocytes, which represent a reliable marker for stage I promotion [94]. On the other hand, mezerein, which elicits a strong oxidative burst in human polymorphonuclear leukocytes, is a speciﬁc stage II promoter [80]. While 12-O-retinoylphorbol-13-acetate is a stage II promoter [80], the antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are stage II promotion inhibitors [95]. In addition, the induction by TPA of ornithine decarboxylase, associated with stage II promotion, is suppressed by SOD and CAT in mouse epidermal cells, suggesting the intermediacy of active oxygen in the induction process. Tumor promoters, especially the free radical–generating promoters such as benzoyl peroxide and hydrogen peroxide, are highly cytotoxic and may promote tumors by causin a regenerative hyperplasia in the skin similar to the response observed after wounding [19]. Phorbol esters also induce skin inﬂammation and

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epidermal hyperproliferation resembling a wound response [8]. The inﬂammatory process in the tumor-promotion stage of skin carcinogenesis plays an important role. It was shown that tumor promoters and UV light induce one of the isozymes of cyclooxygenase (COX-2) and that this enzyme, which synthesizes prostaglandins, is constitutively overexpressed in tumors. Injection of wound growth factors, such as transforming growth factor a (TGFa), in combination with TGFh, can replace wounding or phorbol ester treatment for skin tumor promotion [96]. Interestingly, mice deﬁcient in tumor necrosis factor (TNF) were found to be resistant to TPApromoted skin carcinogenesis [97]. Tumor-promoting eﬀect of chronic chemical and/ or mechanical irritation has also been found in other tissues [98]. For example, epidemiological evidence and animal experiments indicate that secondary bile acids, such as deoxycholate, promote intestinal tumor development [99,100]. In fact, these agents evoke quite similar responses in the intestinal epithelium as the phorbol ester tumor promoters do in skin, i.e., through chemical initiation they activate protein kinase C, cause ROS formation, and induce inﬂammation and epithelial hyperproliferation [100,101]. The exact biochemical and molecular mechanism(s) whereby certain reactive oxygen species might lead to the process of tumor promotion are still under investigation. Both genetic and epigenetic mechanisms have been postulated (reviewed in Refs. 16,19,88,101,102). The induction of a prooxidant state leading to altered gene expression through the activation of poly-ADP-ribose synthetase was proposed [88] to result from oxidant-induced DNA strand breaks and increased levels of oxidized pyridine nucleotides. A variety of cellular proteins and/or enzymatic pathways could also be changed because of the reactions of unsaturated and sulfur-containing molecules with free radicals, leading to altered phenotypic characteristics of a cell [101,103]. Protein kinase C (PKC) may be regulated to a certain extent by direct oxidation. In fact, mild oxidation of the regulatory domain of PKC may eliminate the requirement for Ca2+ and phospholipid for its activation; also, H2O2 has been reported to alter the distribution of PKC in JB6 cells and benzoyl peroxide to alter PKC distribution in mouse epidermis (reviewed in Ref. 20). The activities of other proteins may also be regulated to certain extent directly by redox reactions including c-fos, c-jun, tyrosine kinase located in endoplasmic reticulum, GSSG reductase, and Mg2+-dependent, Na+/K+-stimulated ATPase (reviewed in Ref. 20). During oxidative stress, most cells suﬀer from compromised energy homeostasis due to uncoupling of oxidative phosphorylation, decreased levels of GSH, and decreased levels of NADPH as a result of its utilization by the GSH-peroxidase redox cycle leading to the subsequent release of intracellular Ca2+ stores and a cascade of biochemical pathways. Interestingly, PKC and Ca2+ are believed to act synergistically in stimulating various cellular responses (reviewed in Ref. 20). In view of the role of PKCmediated signal transduction in cell proliferation, diﬀerentiation and oncogenic transformation, oxidative manipulation of PKC activity by ROS may indeed represent an important pathway in tumor promotion [14]. The redox regulation of protein tyrosine phosphorylation may also signiﬁcantly aﬀect growth signaling in cells [104]. Recently, mitogen-activated protein kinases (MAPKs), a group of serine/threoninespeciﬁc, proline-directed kinases, were shown to play a critical go-between role in mediating signal transduction from the membrane to the nucleus [105]. The three mammalian MAPK subgroups include extracellular signal–regulated kinase (ERK), c-jun–N-terminal kinase (JNK), and p38 MAP kinase. Interestingly, the tumor-

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promoting agent butylated hydroxytoluene hydroperoxide (BHTOOH) was also found to be capable of stimulating a rapid and potent activation of ERK in vivo and in cultured mouse keratinocytes [106]. Both ERK activation and induction of cjun by BHTOOH were inhibited by expression of dominant-negative Ras-N-17 in PC12 cells [106]. This suggests that Ras activation is also an upstream signal for ERK activation and the subsequent induction of c-jun by organic peroxides. Despite substantial evidence indicating that various protein kinase/phosphatase-mediated signal transduction events can be altered by ROS insults, the role of such oxidatively altered cell signaling in in vivo carcinogenesis needs to be further investigated [14]. An increasing body of evidence suggests that some cytokines and growth factors are capable of producing ROS in target cells and that the ROS further mediate the eﬀects of the cytokines and growth factors (reviewed in Ref. 14). Thus, the ability of TNF to induce NF-nB activation and IL-6 gene expression in L929 cells was shown to be abrogated by deletion of the mitochondrial electron transport chain components known as necessary for a TNF-mediated increase of cellular ROS [107]. A rapid intracellular formation of ROS was reported [108] following the activation of 5lipoxygenase, required for the activation of NF-nB/CD28-responsive complex and IL-2 expression. The induction of c-fos gene expression by TNF and bFGF in chondrocytes was also shown [109] to be mediated by cellular ROS. Also, MnSOD overexpression was demonstrated [110] to signiﬁcantly decrease the constitutive expression of IL-1 and its induction by TNF in human ﬁbrosarcoma cells. This indicates that superoxide anion may be a signal molecule involved in the constitutive and inducible expression of IL-1. ROS are also signal molecules involved in apoptotic cell death caused by certain agents [111]. The above observations support the concept of ROS being cellular messengers involved in cell growth regulation and diﬀerentiation. In summary, a number of cell signal transduction pathways appear to be targets of ROSmediated damage. Modifying cell signaling by ROS might result in dysregulated cell growth, diﬀerentiation, and apoptosis, together with DNA mutations, which ultimately leads to tumor progression and the development of cancer [14]. V.

ROS AS FACTORS MODIFYING TUMOR PROGRESSION

Although reactive oxygen species appear to play a role mostly in the tumor-promotion phase, during which gene expression of initiated cells is modulated by aﬀecting genes that regulate cell diﬀerentiation and growth, active oxygen, by inducing chromosomal aberrations, could also play a role in progression [88]. During progression, mostly benign neoplasms are stimulated to more rapid growth and malignancy. While the epigenetic eﬀects of the tumor promoters are reversible and thus may be more important in the earlier stages of promotion, the genetic eﬀects of the tumor promoters may be responsible for the irreversible portion of the late stage of skin tumor promotion and for tumor progression [112]. Free radicals may be the candidates for the many genetic eﬀects. Some tumor promoters such as benzoyl peroxide spontaneously give rise to free radicals, whereas others such as phorbol ester and teleocidin type promoters may give rise to free radicals by their clastogenic eﬀect. Oxidized lipids and oxygen radicals are likely candidates induced by the clastogenic eﬀect of TPA, which could have a direct eﬀect on the genetic material. The processing of indirectly induced oxidative genetic damage by repair and constitutive and damage-induced mechanisms of replication and recombination can

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lead to permanent alterations in DNA sequences, such as mutations, ampliﬁcation of certain sequences, or intra- and interchromosomal rearrangements of blocks of sequences [88]. Since DNA-damaging agents that operate by indirect action are strong clastogens but weak mutagens, they may preferentially induce sequence rearrangements of oncogenes, previously mutated by an initiator, leading to tumor progression. Several events that appeared during skin tumor promotion are continued or even exaggerated during tumor progression, such as an increase in dark cells, loss of glucocorticoid receptors, and increase in polyamines and prostaglandins [113]. The papillomas that initially develop during mouse skin initiation-promotion protocols are considered by many researchers to be heterogeneous. It is reﬂected in the fact that some will persist, some will disappear or regress, and only relatively few (5–7%) will progress to an invasive squamous cell carcinomas (SCC) during the time frame of initiation-promotion experiments in which phorbol esters were used as promoters (reviewed in Ref. 20). This malignant progression, concomitant with the appearance of additional genetic alterations [112,114,116], occurs spontaneously, even in the absence of any further treatment. It may reﬂect the genetic instability induced and eventually constitutively expressed in the course of tumor promotion. On the other hand, if mice with papillomas are treated respectively with N-methyl-NV-nitro-Nnitrososoguanidine (MNNG), a signiﬁcant increase in the conversion of papillomas to carcinomas takes place [117]. Similar results have been found with limited treatment of MNNG as well as with ethylnitrosourea (ENU), benzoyl peroxide, and hydrogen peroxide [118–120]. This type of treatment (initiation-promotion-initiation) produces a carcinoma response similar to complete carcinogenesis (i.e., the repetitive application of a carcinogen such as DMBA or MNNG). Such treatment probably supplies both initiating and promoting inﬂuences continuously. Why diﬀerent types of promoters such as benzoyl peroxide [119] and other free radical–generating compounds [120] can increase conversion of papillomas to carcinomas is still under investigation. The possible role of free radicals during progression has also been investigated by utilizing free radical scavengers and antioxidants to inhibit skin tumor progression [121]. Thus, free radical scavengers like glutathione and disulﬁram inhibited malignant progression, while the glutathione-depleting agent diethylmaleate reportedly enhanced tumor progression [122]. These studies suggest that certain types of free radical–generating tumor promoters may enhance conversion of papillomas to carcinomas and that it is possible to identify eﬀective inhibitors of malignant progression in this model system [123]. Cancer directly aﬀects at least one third of the human population, but the inherited genetic determinants of cancer risk remain largely unknown. Mouse models of human cancer are helping us to understand this disease as a complex genetic trait [114] and thus to identify the multiple genetic mechanisms involved in pathways that aﬀect individual cancer susceptibility. It was suggested [8] that among these mechanisms, an overactivation of arachidonic acid metabolism resulting in or at least contributing to oxidative stress might play a critical role. Recent studies on cancer prevention using nonsteroidal anti-inﬂammatory drugs (NSAIDs) have led to remarkable insights into the molecular mechanisms of tumor promotion/progression in animals and humans. Thus, colorectal tumorigenesis in humans as well as experimental carcinogenesis in a variety of organs including mouse skin and rat colon have been shown to be inhibited by a variety of NSAIDs [8]. They were shown to suppress prostanoid synthesis by inhibiting the corresponding cyclooxygenases.

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COXs and their products may be involved in diﬀerent stages of carcinogenesis. Thus, prostaglandin PGE2 speciﬁcally exhibits strong immunosuppressive eﬀects, which may favor tumor progression [124]. In addition, chemical carcinogens are known to become co-oxidized to genotoxic ultimate carcinogens by the peroxidase component of COX [8]. Both mechanisms may contribute to tumor promotion/progression. The COX expression is the source not only of physiologically highly active signaling compounds, but also of genotoxic products such as malondialdehyde (a breakdown product of prostaglandin endoperoxide) and reactive oxygen species (reviewed in Ref. 8). Genotoxic byproducts are also generated along the various lipooxygenase (LOX)catalyzed pathways of arachidonic acid metabolism in mouse skin [125–127] as well as in human prostate cancer [128] and human colorectal cancer tissues [129]. Thus, both COX and LOX pathways of arachidonic acid metabolism may lead to an endogenous genotoxic potential that, together with an impairment of DNA repair, is required for the spontaneous malignant progression of tumors, i.e., genetic instability (reviewed in Ref. 8). Indeed, oxidant-induced DNA damage may be one mechanism underlying the genomic instability associated with tumor development. An alternate hypothesis for the action of reactive oxygen species or their precursors as progressor agents relates to their high degree of cytotoxicity [112]. If one takes this model for tumor progression into account, highly cytotoxic agents may selectively or nonselectively kill cells within a tumor, allowing the growth of more malignant cells, and/ or kill normal cells, reducing the constraints against expansion along the border between normal and tumor tissue. Both alternatives assume that cells capable of invasion either preexist within the benign tumor or that expansion of tumor clones increases the chances of the natural progression of cells towards malignancy [112]. VI.

INHIBITION OF DIFFERENT STAGES OF CARCINOGENESIS BY ANTIOXIDANTS

Free radicals are known to play an important role in many diseases, including cancer. Since free radicals and reactive oxygen species are continuously produced in vivo, organisms have evolved that possess not only antioxidant and electrophile defense systems to protect against them, but also repair systems that prevent the accumulation of oxidatively damaged molecules [56]. Antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, are preventive antioxidants because they eliminate species involved in the initiation of free radical chain reactions while small molecule antioxidants, such as ascorbate, the tocopherols, glutathione, and reduced coenzyme Q10, can repair oxidizing radicals directly and therefore are chainbraking antioxidants. Since the induction of cancer is a multistage process resulting from the prolonged accumulation of genetic and epigenetic damage, including oxidative damage, there is an opportunity for preventive intervention. In fact, many different natural and synthetic antioxidants have been shown to inhibit the induction of cancer by a wide variety of chemical carcinogens and/or radiation at many target sites in mice, rats, hamsters, and humans [13]. Although the detailed mechanisms of the antagonistic eﬀects of antioxidants are not known, a number of theories have been advanced to explain their eﬀects. Possible mechanisms include direct interaction of the carcinogen or one of its activated metabolites with the antioxidant, decreased activities (or alteration) of enzyme pathways responsible for carcinogenic activation, and increased activities of enzyme pathways responsible for detoxifying carcinogens. A

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number of potent inhibitors of tumor initiation appear to be eﬀective because they either prevent the formation of the ultimate carcinogen and/or scavenge the ultimate carcinogen. The phenolic antioxidants BHT and BHA have been studied extensively, primarily because of their use as food preservatives. However, compounds such as disulﬁram, ethoxyquin, and selenium also received considerable attention. When BHA, BHT, and ethoxyquin were added to diet, they antagonized the carcinogenic action of DMBA and benzo[a]pyrene (B[a]P) on the forestomach of mice and mammary glands of rats. Moreover, BHA incorporated into the diet protected against pulmonary neoplasms produced by acute exposure to DMBA, B[a]P, urethane, and uracil mustard (reviewed in Ref. 13). BHA also inhibited the carcinogenic eﬀects of other polycyclic aromatic hydrocarbons. Other studies using the two-stage system of mouse skin tumorigenesis have demonstrated that BHA and BHT eﬀectively inhibited tumor initiation by DMBA (reviewed in Refs. 13, 20). Thus, it was reported that BHA and BHT inhibited the covalent binding of DMBA and B[a]P to epidermal DNA. This eﬀect on PAH activation to binding products could account for the anticarcinogenic activity of these compounds. BHA and BHT also inhibited the carcinogenic action in various animal model systems of several other carcinogens like diethylnitrosoamine (DEN), bracken fern, 4-nitroquinoline-N-oxide (4-NQO), 2-acetylaminoﬂuorene (AAF), N-OH-AAF, and 1,2-dimethylhydrazine (DMH). In addition to the alterations in oxidative metabolism, the phenolic antioxidants have also been shown to increase the detoxiﬁcation pathways for many chemical carcinogens (reviewed in Ref. 13). The indoles, aromatic isothiocyanates, coumarins, ﬂavones, di- and triterpenoids, dithiothiones, organosulﬁdes, and D-glucarates [131] have a potent eﬀect on the metabolism of carcinogens [13,95,131]. In general, they appear to have a major eﬀect on the detoxiﬁcation of carcinogens. Ellagic acid and 2,6-dithiopurine have also been shown to be highly potent in scavenging the ultimate (reactive) carcinogenic form of carcinogens [95]. The majority of these chemicals have properties like phenolic antioxidants, such as BHA and BHT, known to inhibit tumor initiation [132,133]. They have both antioxidizing activity and inﬂuence the metabolism of carcinogens, especially detoxiﬁcation. Also, disulﬁram, vitamin C, and vitamin E appear to inhibit chemical carcinogenesis in a manner similar to the phenolic antioxidants by their eﬀect on the metabolism of the carcinogen, their antioxidizing activity, and preventing the formation of ultimate carcinogens. The mechanism by which selenium inhibits chemically induced tumors may be related to its eﬀect on glutathione peroxidase (GSHP) since it is a cofactor for this enzyme [13]. Although the antioxidants have been shown to dramatically inhibit complete carcinogenesis and tumor initiation, they also are even more eﬀective inhibitors of tumor promotion and progression [13,20,95]. For example, BHA and BHT inhibited both TPA and benzoyl peroxide promotion in mouse skin. In addition, disulﬁram and 4-p-hydroxyanisole were eﬀective inhibitors of TPA promotion [20,95]. Although the mechanism by which antioxidants inhibit tumor promotion is not clearly understood, they may be scavenging radicals generated directly in the case of benzoyl peroxide or indirectly by TPA. The fact that benzoyl peroxide and other free radical–generating compounds such as lauroyl peroxide and chloroperbenzoic acid are eﬀective skin tumor promoters suggests that free radicals may be important in tumor promotion [56,134]. An analogous situation would be the phorbol ester tumor promoters, which can stimulate superoxide anion production in polymorphonuclear leukocytes, and the anti–tumor promoters such as dexamethasone and antioxidants

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can counteract this eﬀect [19,134]. Selenium was found to be an eﬀective inhibitor of skin tumor promotion by croton oil. Selenium is a necessary cofactor for the enzyme glutathione peroxidase (GSHP) that detoxiﬁes hydrogen peroxide and hydroperoxides within the cell. The possibility exists that Se-dependent GSPH lowers the level of potentially dangerous and damaging peroxide radicals generated from various cocarcinogenic and promoting chemicals. a-Tocopherol and L-ascorbic acid are also known to signiﬁcantly reduce tumor formation induced by DMBA and croton oil. a-Tocopherol also reduced the number of ﬁbrosarcomas induced by 3-methylcholanthrene (MCA) and mammary gland adenocarcinomas induced by DMBA (reviewed in Ref. 13). L-Ascorbic acid was found to inhibit transformation of C3H/10T1/2 cells by MCA. Since the inhibitory eﬀect of L-ascorbic acid was observed in some cases long after the carcinogen was given, this observation indicates it was active during the promotional stage or possibly during progression [13]. In recent years, several novel antioxidants such as proanthocyanidins and ursolic acid have also been found to inhibit chemical carcinogenesis and mouse skin tumor promotion [13,95]. Caventol was found to inhibit skin tumor promotion through inhibition of TNFa release and protein isoprenylation. Several polyphenolic antioxidants in green tea have been shown to inhibit chemical carcinogenesis and mouse skin tumor promotion [13,95]. A number of other antioxidants have also been found to inhibit tumor promotion and/or progression in mouse skin. Although their mechanisms of action are not deﬁnitely known, evidence points to several possibilities: they scavenge various radicals generated directly or indirectly by tumor promoters, they increase levels of enzymes that are important in detoxifying cellular radicals, or they have other speciﬁc functions [13]. Certain agents that raise glutathione levels appear to be capable of inhibiting TPA promotion, including glutathione (GSH) itself. These agents may be eﬀective inhibitors by increasing the available pool of GSH for scavenging tumor promoter–induced hydroperoxides [21]. For example, glutathione, ethyl ester of glutathione, and N-acetylcysteine were found to inhibit skin tumor promotion and progression, while diethylmaleate, a chemical that reduces glutathione levels, was found to be an eﬀective enhancer of tumor progression. Moreover, overexpression of g-glutamyltranspeptidase, which leads to a reduction in cellular glutathione levels, also enhanced tumor progression [135]. These studies suggest that glutathione is very important in both skin tumor promotion and progression. On the other hand, acyl dehydroalanine derivatives appeared to be very eﬀective inhibitors of skin tumor progression but did not have any signiﬁcant eﬀect on skin tumor promotion [136]. Several antioxidants, such as vitamins C and E, vitamin E and selenium, BHA and vitamin E, have been shown to have synergistic activities (reviewed in Ref. 13). When nordihydroguaiaretic acid (NDGA) was assessed as an antipromoting agent in mouse skin, it was found to be a very potent inhibitor. The possible mechanism of action may be due to its eﬀect as scavenger of free radicals and/or its inhibitory eﬀect on arachidonic acid metabolism [137]. Compounds such as the antiinﬂammatory steroids and dibromoacetophenone inhibit early in the pathway and consequently inhibit the formation of all the important end products of arachidonic acid metabolism [121]. The next most consistent inhibitors of tumor promotion/ progression in this general category are phenidone and 5,8,11,14-eicosatetranoic acid (ETA); these compounds inhibit both the cyclooxygenase and lipoxygenase pathways. This mode of inhibition also leads to a decrease in all the important end products of

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arachidonic acid metabolism (reviewed in Ref. 121). Indomethacin, a potent inhibitor of the cyclooxygenase pathway, has been found to inhibit and enhance skin tumor promotion [121]. Flurbiprofen, another cyclooxygenase inhibitor, enhances and inhibits skin tumor promotion. The reason for the dual eﬀects of the cyclooxygenase inhibitors is not quite clear, but the inhibitory eﬀect may be related to the toxicity of these compounds. The enhancing eﬀect of the cyclooxygenase inhibitors on promotion may occur because more arachidonic acid is being metabolized by the lipoxygenase pathway, while the cyclooxygenase pathway is inhibited. This hypothesis suggests that the lipoxygenase pathway is important in skin tumor promotion/progression [121]. Recent ﬁndings (reviewed in Refs. 8, 121) strongly indicate that both COX and LOX may represent novel targets of cancer chemoprevention. It remains to be shown to what extent arachidonic acid metabolism contributes to oxidative stress and whether or not chemopreventive properties of antioxidants such as are found in vegetables, green tea, and certain spices can, at least partially, be explained by an inhibition of COX- and LOX-catalyzed arachidonic acid metabolism. Although the protease inhibitors reportedly have little eﬀect on most events thought to be important in skin tumor promotion [121], it has been postulated that protease inhibitors may act through suppression of the formation of reactive oxygen species. Protease inhibitors are known to prevent the formation of superoxide anion and hydrogen peroxide by polymorphonuclear leukocytes that are activated by TPA (reviewed in Ref. 121). Recent studies demonstrate that plants are rich in compounds such as avicins, triterpenoid saponins that inhibit oxidative stress and induce programmed cell death of premalignant and malignant cells [138–140]. These studies indicate that avicins could develop as important chemopreventive agents in many conditions where chronic inﬂammation and oxidative and nitrosative stress may lead to tumorigenicity [138–140]. VII.

CONCLUDING REMARKS

Carcinogenesis is a multistage process, with the experimentally deﬁned stages of initiation, promotion, and progression. These stages can be targeted or inﬂuenced by many physical and chemical carcinogenic agents, including reactive oxygen species. A growing body of evidence indicates that ROS can directly cause oxidative DNA damage resulting in DNA mutations, activation of proto-oncogenes, and inactivation of tumor suppressor genes; ROS can also inactivate certain DNA-repairing enzymes, which may, in turn, increase the mutations, consequently leading to tumor initiation. ROS can also interfere with normal cell signaling through modifying transcriptional factors and protein kinase cascades. Oxidative modiﬁcation of cell signal transduction by ROS can directly cause dysregulated cell growth, diﬀerentiation, and cell death, together with additional DNA mutations, leading to tumor promotion/progression and ultimately to the development of cancer. Understanding the relationship of ROS to all three stages of carcinogenesis could lead to the development of mechanistically designed chemoprotective strategies, which can utilize antioxidants or other radical detoxiﬁers to prevent the development of cancer. Scores of epidemiological studies have noted a lower risk of cancer among persons whose diet includes a relatively large amount of vegetables, fruits, and other natural products of plant origin [130]. A popular explanation, both within the scientiﬁc community and among members of

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the public, is that diﬀerent vitamins and other micronutrients in vegetables and fruits as well as other plant products prevent carcinogenesis by interfering with detrimental actions of mutagens, carcinogens, and tumor promoters, including reactive oxygen species and their precursors. These natural inhibitors of carcinogenesis are of particular importance because they are nontoxic or markedly less toxic than synthetic chemopreventive agents. Many of the antioxidants and related compounds such as phenolic antioxidants and vitamins C and E appear to be eﬀective in counteracting the tumor-initiating phase of carcinogenesis. This appears to be related to their antioxidant activity and their eﬀect on carcinogen metabolism. Many of them are also potent inhibitors of the tumor promotion phase of carcinogenesis. Their eﬀect on the free radical defense mechanisms, their antioxidizing activity, and their eﬀect on many critical events in tumor promotion, such as arachidonic acid metabolism, may explain why antioxidants are potent inhibitors of tumor promotion. In some cases antioxidants interact synergistically to inhibit carcinogenesis. A number of antioxidants such as glutathione and some derivatives, cysteine and its derivatives; and N-acyl dehydroalanine derivatives have been shown to inhibit tumor progression. These ﬁndings suggest that many antioxidants and related compounds are eﬀective inhibitors of tumor initiation, promotion, and/or progression. In a number of cases the mechanism(s) of action are related to their abilities to prevent critical carcinogen metabolism and to increase detoxiﬁcation pathways for carcinogens and free radicals as well as their antioxidizing activity. Obviously, some antioxidants have both antiinitiating and anti–tumor promoting activity, but they usually inhibit the initiation and promotion/progression stages to a diﬀerent degree. In conclusion, a combination(s) of various antioxidants and related compounds with diﬀerent mechanisms of action will most likely prove to be more eﬀective in inhibiting the development of cancer compared to one antioxidant alone. REFERENCES 1. 2.

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10 The Potential Impact of Polymorphism on Oxidative Stress Status ¨ S, EMMA WINCENT, LENA FORSBERG, ULF DE FAIRE, LOUISE LYRENA and RALF MORGENSTERN Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

I.

HUMAN GENETIC VARIATION AND DISEASE

Recently, through coordinated eﬀorts, human genetic variants have been identiﬁed and stored in databases [1,2] at an unprecedented rate. The NCBI database (dbSNP) now contains some 4,116,037 entries (4/2-02), mostly in the form of single nucleotide polymorphisms (SNPs). It has been estimated that some 50% of existing, amino acid– altering SNPs are already in databases [3]. A given human gene most often contains several genetic variants. It should be realized, however, that only some of the genetic variants listed in databases have been experimentally veriﬁed, as many were derived from in silico approaches. Consequently, uncharacterized SNPs of potential interest need to be experimentally veriﬁed. As a rule, when exploring a particular gene, databases should be screened for in parallel with the literature, as several genes have recently been globally characterized experimentally in many subjects (for examples, see Refs. 4,5). Here we discuss SNPs, but the principal arguments apply to other genetic variations (insertions and deletions) as well. Types of SNPs include those situated in noncoding regions such as introns or the 5V- and 3V-untranslated ends of the mRNA, which might have little impact on gene function. These variants could, however, aﬀect splicing or mRNA stability. Variants in the coding region might be silent or result in a conservative or nonconservative exchange of an amino acid. The perceived functionality of nonconservative change is evident from a negative selection bias compared to other SNPs [6], but one must not assume that silent or nonconservative changes are without consequence. Finally, nucleotide changes in the regulatory regions of genes are not uncommon and have 217

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in several cases been shown to aﬀect gene expression [7,8]. It is generally held that SNPs that aﬀect gene function are preferable in association studies, although genetic variants have also been used as ‘‘surrogate markers’’ under the (often valid) assumption that they are in linkage disequilibrium to nearby genetic variants (in the same or neighboring genes). Implicit in the collection of an exhaustive map of genetic variants is their potential use for identifying disease-associated genes (as markers in linkage analyis) and deﬁning the roles of candidate genes in disease (by association studies). The information on disease etiology can lead to the identiﬁcation of drug targets and prevention strategies. Certainly, the merit of linkage analysis in rare monogenic disease has been amply demonstrated, but complex common diseases with a polygenic origin have proven more resilient in terms of ﬁnding reproducible associations to candidate genes. Most diseases in which oxidative stress has been implicated fall into the latter category. If we hypothesize that a certain disease is caused to some degree by oxidative stress, it would also be reasonable to assume that genetic variants in genes resulting in increased stress would indeed aﬀect the disease outcome (i.e., earlier onset, more severe symptoms). Armed with this hypothesis, we must ﬁrst ﬁnd suitable genetic variants and well-deﬁned populations for study. Experience from association studies in cardiovascular disease (coronary heart disease, stroke, blood pressure, lipids, and inﬂammation), cancer (metabolic capacity for carcinogens), and Alzheimer’s disease (oxidative stress, amyloid processing, lipoprotein proﬁle) tells us that only very few of many initially positively correlated genetic variants are reproducibly correlated to disease (e.g., Ref. 9). The reasons that these positive ﬁndings are often quickly refuted can be many: (1) population stratiﬁcation (diﬀerence in ethnic composition in cases and controls) is possible; (2) when the number of studies increases and small numbers of individuals are examined, the chances for signiﬁcant associations increase; (3) publication bias (where the ﬁrst positive study is published although many negative studies are stored in other labs). In complex diseases etiology might also vary between study groups, explaining quite valid signiﬁcance in certain cases. However, lacking knowledge of the complex mechanisms (actually these are exactly what one hopes to deﬁne), it is diﬃcult to determine which studies are valid. There are several ways to improve the situation, including a strict matching of cases and controls, familial approaches [10], computational approaches [11] haplotype analysis [12], and, last but not least, increasing the size of study populations. (For a more exhaustive review of these issues, see Ref. 13.) Returning to the choice of candidate genes that are related to oxidative stress, it is easy to make a list of the core protective enzymes [14]. In addition, many malfunctioning systems can be envisioned to lead to oxidative stress, such as mitochondrial respiratory complexes [15], vitamin uptake systems, iron storage [16], inﬂammatory reactions, and nitric oxide generation, etc. Many SNPs have been catalogued [14] in core protective enzymes and related genes, and doubtless the list will become more comprehensive during the coming years. We aim to characterize the consequences of genotypic variation on phenotype. Therefore, amino acid changes are studied on the enzyme function and stability level, whereas gene regulatory region variants are characterized by gene reporter and electrophoretic mobility shift assays. In silico tools that predict the potential functional consequence of amino acid changes have been developed [17] that can guide the characterization eﬀort. Once a functional polymorphism has been identiﬁed at the molecular level, attempts are made to ascertain that the genotype also corresponds to an altered phenotype in humans. At

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this point it is important to put in context the contribution of genotype to normal variation. Although this is a tedious process, it is hoped that one can draw conclusions from the variations in each gene. For instance, 20 human genes for glutathione transferases have been characterized, and null alleles are present for two superfamily members. The lack of a single form of these enzymes can contribute to cancer risk (albeit marginally [18]). Lack of the gene for mitochondrial superoxide dismutase in mice is, however, not tolerated (mice die young [19]). In contrast, a knock-out of extracellular superoxide dismutase in mice was not coupled to negative symptoms unless the mice were challenged with increased oxygen [20]. These observations agree with the ﬁnding that the latter enzyme displays a redistribution phenotype in humans (tissue to plasma) that appears unlinked to disease so far [21]. When low in ﬂies catalase does not shorten life span [22], and human acatalasemia appears to be well tolerated. Clearly, redundant systems and large gene families make it possible to harbor more extreme variants (catalase, glutathione peroxidase, glutathione transferase), whereas speciﬁc protection of mitochondria by Mn superoxide dismutase cannot be dispensed with (in mice). It is interesting, therefore, that targeting of human superoxide dismutase to mitochondria is polymorphic with the corresponding SNP deﬁned and used as a tool to study oxidative stress in disease. To make things more complex, many oxidative stress protection enzymes are inducible and the lack of one speciﬁc function can therefore be compensated. Taken together, the choice of suitable candidate genes and SNPs for the study of oxidative stress in disease is based on the perceived functionality of the polymorphism, and initially this appears to be a viable strategy. It follows that considerable eﬀort remains to comprehensively catalogue and characterize the variants. The impact of the genetic variants on disease can then be determined in well-controlled epidemiological studies (see above) but, importantly, also on in vivo markers of oxidative stress such as oxidized DNA and oxidized lipid [23]. For instance, a promising but experimentally diﬃcult marker of oxidative stress is 8-hydroxy-2-deoxyguanosine. One enzyme responsible for repair of this DNA lesion is 8-hydroxy-2-deoxyguanosine DNA glycosylase/apurinic lyase, but tissue levels were not related to the level of damage [24]. Rare polymorphisms in this gene have yet to be linked to repair capacity. With a clear goal of deﬁning the role of oxidative stress in disease, molecular epidemiology may provide information. Hopefully, a few gene variants will turn out to have an impact on human markers of oxidative stress and disease, and thereby reveal underlying mechanisms. The polygenic and complex nature of oxidative stress makes these studies particularly demanding. Large study populations together with careful marker evaluation will be required, especially if we are to rely on negative results to rule out oxidative stress in the etiology of certain diseases. Glutathione transferases (GST) are involved in the metabolism of reactive compounds, but their glutathione peroxidase activity also provides documented protection from oxidative stress in cellular systems [25]. A role in oxidative stress protection is also supported by a correlation between the GSTM null genotype and increased urinary 8-hydroxy-2-deoxyguanosine [26]. In fact, a link between several GST polymorphisms and commonly measured oxidative stress biomarkers has been observed [27]. The reader is referred to recent reviews on these enzymes [28], and the insightful discussions by Richard C. Strange are highly recommended [29,30]. We recently reviewed the status of genetic variants in key oxidative stress–related genes [14]. Here we have made a survey of these and new gene variants that have been subject to association studies.

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II.

SURVEY OF ASSOCIATION STUDIES

A.

Catalase

Catalase is an ubiquitous antioxidant enzyme that can be found in all known organisms. It is most abundant in liver, kidney, and erythrocytes [31]. Several rare polymorphisms have been detected in the catalase gene, among them those associated with catalase deﬁciency, acatalasemia [32,33]. Acatalasemia was ﬁrst described by Takahara and Miyamoto in 1948 in Japanese individuals [32]. It is an autosomal, recessive trait characterized by catalase erythrocyte levels 0.2–4% of normal levels [34]. Cases of acatalasemia have been reported from nine countries, with most cases in Japan (91 patients in 46 families) and Switzerland (11 patients in 3 families) [35]. The Japanese type is due to a defect in the synthesis of the protein, i.e., an abnormal splicing of catalase mRNA due to a guanine to adenine (G/A) transition at the ﬁfth position of intron 4 [32,35]. The Swiss type is caused by a point alteration, resulting in an amino acid substitution [36,37]. Association studies with Hungarian individuals have shown signiﬁcantly higher incidence of diabetes mellitus in subjects with familial catalase deﬁciency than in the general Hungarian population [38]. However, a Finnish study found no association between diabetes mellitus and the A/T –21 variant* in the catalase gene [21]. Several variants of the catalase gene are found in nonacatalasemic persons, among those a common promoter C to T exchange 262 bp from the translation start site that inﬂuences the levels of erythrocyte catalase [7]. There is no known association between catalase polymorphisms and familial amyotrophic lateral sclerosis (FALS) [39]. B.

Superoxide Dismutases

1.

Superoxide Dismutase 1

Cu/Zn Superoxide dismutase (SOD1) is a small cytosolic protein that is functionally active as a homodimer. It is found at the highest levels in the liver, erythrocytes, brain, and neurons. The principal function of SOD1 is to convert the superoxide anion to hydrogen peroxide. There are over 60 known variants in the coding region of the SOD1 gene [40]. They are all very rare and associated with the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). About 10% of the ALS cases are familial [41]. About 20% of familial ALS (FALS) cases are caused by variants in the Cu/Zn SOD gene [42,43]. Association to cardiovascular disease in type 2 diabetes mellitus [21] or to the neurological disorder motor neuron disease [21,44] has not been detected. No common polymorphism in SOD1 has been detected so far [45]. 2.

Superoxide Dismutase 2

The manganese-containing SOD (SOD2) has a function similar to that of SOD1 but within the mitochondria. In mammals the highest levels of the enzyme are expressed in heart, brain, kidney and liver [46]. Several polymorphisms have been studied in the MnSOD gene, among those Ala16Val (also called Ala9Val), which results in altered intracellular traﬃcking [47],

* Nucleotide numbering refers to translation start sites if not otherwise indicated.

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and Ile58Thr, which results in diminished enzyme activity [48]. Association studies show that the Ala variant is associated with an increased risk of breast cancer in both pre- and postmenopausal women [49,50] as well as macular degeneration [51]. The Ala16Val polymorphism in the MnSOD gene has also been studied in association with Parkinson’s disease (PD), rheumatoid arthritis (RA), schizophrenia, schizophrenics with or without tardive dyskinesia (TD), and FALS. The Val allele was found to be associated with Parkinson’s disease in Japanese patients [52] but not in Germans [53]. The Val allele was found to be associated with RA in Japan but not in the United States [54]. There was no association between MnSOD variants and schizophrenia. However, there was a signiﬁcant association with TD in schizophrenics [54,55], where it seems as though the Ala variant protects against TD. There was no association with FALS [56] and no association of the SOD2-targeting polymorphism in PD. 3.

Superoxide Dismutase 3

Extracellular SOD (EC-SOD/SOD3) is a secretory, tetrameric glycoprotein containing both copper and zinc. EC-SOD is the principal enzyme protecting against superoxide radicals in the extracellular space [57,58]. A single base substitution resulting in a amino acid change, Arg213Gly, aﬀects the heparin-binding domain of EC-SOD. The decreased aﬃnity for heparin probably leads to the elevated plasma levels of EC-SOD seen in studies in Japanese [59,60], Swedish [61], and American [62] individuals. There was no association to vascular disease in type 2 diabetes mellitus [21]. C.

Homocysteine

Homocysteine levels have been correlated to vascular disease [63]. Whereas the inﬂuence of common variants in genes involved in homocysteine metabolism was modest, levels of extracellular SOD were correlated to total homocysteine in blood [64]. In fact, it appears that homocysteine can aﬀect the expression and extracellular binding/ accumulation of EC-SOD [65]. It would, therefore, be interesting to determine the inﬂuence of homocysteine on the levels of the polymorphic (rare) variant of EC-SOD displaying high plasma levels [61]. D.

Glutathione Peroxidases

Only one of the four human glutathione peroxidases, the GPX1 gene, has been genotyped in association studies. A Pro198Leu substitution has been associated with lung cancer [66] but not to blood levels [67]. There was no association to myocardial infarction/stroke in hypertensive non–insulin-dependent diabetes mellitus patients [68]. The more recently characterized peroxiredoxins function as peroxidases and have been shown to protect cells from oxidative stress [69]. Genetic variants have not been characterized in humans but would certainly be of interest. E.

Quinone Reductases

1.

Quinone Reductase Type 1

Quinone reductase 1 (NQO1) is a cytoplasmic enzyme, also previously named DT diaphorase or NAD(P)H:quinone oxidoreductase, that is ubiquitously present in all tissue types. It consists of two identical subunits, each containing one ﬂavin adenine dinucleotide (FAD) prosthetic group noncovalently attached to the protein [70]. NQO1 catalyzes two-electron reductions of a number of quinones and quinoid com-

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pounds to hydroquinones (using either NADH or NADPH as electron donors), thereby protecting the cells against the deleterious eﬀects of redox cycling of quinones [71]. A C/T variant at position 609 of the NQO1 gene, resulting in a proline-to-serine substitution at amino acid position 187 [72], yields an enzyme with about 2% of the wild-type activity. The consequences of benzene poisoning were signiﬁcantly aggravated for individuals homozygous for the serine variant [73,74], conferring an elevated risk of contracting benzene-induced leukemia [73]. In the general case of leukemias, the serine variant was signiﬁcantly associated with increased risk [75–77]. Signiﬁcant associations were also seen between the serine variant and kidney stone formation [78] and renal cell and urothelial carcinoma [79]. The risk for colorectal carcinoma (CRC) was not associated to the Pro187Ser polymorphism in one study [80], while another study showed a signiﬁcant association between the serine variant and CRC [81]. In the case of lung cancer, some studies show no overall association between NQO1 gene variants and lung cancer [82–84], while others show signiﬁcant associations between the Pro/Pro genotype and increased risk for lung cancer in general [70,85] and adenocarcinoma in particular [84]. An additional study shows an association between the serine variant and increased lung cancer susceptibility for current and ex-smokers [82]. Finally, studies on the susceptibility to basal cell carcinomas [86], adult glioma [87], PD [88], and malignant and benign pancreatic diseases [89] show no signiﬁcant association to the frequency of the serine variant. A T/C substitution at nt 464 results in a tryptophan replacement of arginine. The resulting eﬀects have not yet been completely characterized in mammalian cells [90]. There was no association between infant leukemias and the variant alleles [75]. 2.

Quinone Reductase Type 2

Quinone reductase type 2 (NQO2) has a high nucleotide sequence identity to NQO1. The in vivo role of NQO2 is not well characterized, although NQO2 was found to be expressed selectively in kidney, skeletal muscles, liver, heart, and lung, suggesting tissue-speciﬁc action of the enzyme [91]. Neither the Phe47Leu, 747A/G, 938A/C, or 16427A/G polymorphisms of the NQO2 gene show any signiﬁcant association to the development of PD [4,88]. However, a deletion of 29 base pairs (bp) in the promoter region of NQO2 was signiﬁcantly over-represented in patients with PD [88]. F.

Paraoxonase

Paraoxonase (PON1) is a high-density lipoprotein (HDL)–associated enzyme that prevents low-density lipoprotein (LDL) oxidation, known to be involved in formation of atherosclerotic plaque. Three common polymorphisms, Met55Leu [92], Gln192Arg [93], and C/T-108 [8], are all known to inﬂuence the PON1 activity levels [94], and Met55Leu also inﬂuences urinary oxidized lipid levels [23]. These variants have been extensively genotyped in individuals suﬀering cardiovascular disease, with conﬂicting results. Individuals homozygous for Leu55 exhibit a higher risk of developing carotid artery atherosclerotic disease [95], macular degeneration [96], artery wall atherosclerosis [97], and PD [98]. Individuals homozygous for Arg192 exhibit a higher risk of developing macular degeneration [96], cerebrovascular stenosis [99], and coronary

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artery disease [100]. The promoter polymorphism (C/T-108) has also been associated with coronary heart disease [101]. The eﬀect of PON1 polymorphism in many of these studies is often modulated by other risk factors like diabetes type 2, smoking, or age, whereas negative correlations are often observed when comparing all subjects in a population. To improve the results of these association studies, it has been suggested that individual PON1 serum concentrations should be determined in addition to the analysis of genetic polymorphism [102]. G.

Heme Oxygenase-1

Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, is considered to be an antioxidant enzyme, since the bilirubin produced functions as an eﬃcient scavenger of reactive oxygen. The HO1 promoter region contains a (GT)n repeat polymorphism [103]. Yamada et al. [104] have shown that the length of the repeat correlates to the promoter activity and is associated with chronic pulmonary emphysema. The polymorphism has also been associated with postdilation restenosis [105]. Even though several lines of evidence suggest that HO-1 may be involved in the pathogenesis of neurodegenerative disease [106], no (GT)n allele was associated with PD or Alzheimer’s disease [103]. H.

UDP-Glucuronysyltransferase

Bilirubin UDP-glucuronysyltransferase 1A1 contains promoter polymorphisms that modulate transcriptional activity. As this enzyme is involved in the conjugation of serum bilirubin, which can serve as an antioxidant, it has been suggested as a potential genetic marker for oxidative stress–protective status [107]. I.

Microsomal Epoxide Hydrolase

In humans, microsomal epoxide hydrolase (mEH) is expressed in all tissues, with the highest levels in the liver, kidneys, and testis. Within the cell mEH is principally located in the endoplasmatic reticulum. [108]. mEH is a critical biotransformation enzyme that catalyzes the conversion of a broad array of xenobiotic epoxide substrates to more polar diol metabolites [109]. A number of polymorphisms in the mEH gene have been described, two of which cause amino acid substitutions [110]. A T/C variant results in the substitution of tyrosine for histidine at amino acid position 113 and has been shown to result in reduced enzyme activity [110]. In the case of lung cancer, some studies show signiﬁcant associations between the His variant and a decreased risk of cancer [111–113], while others show an increased risk [83,114] or no signiﬁcant association [115]. Studies also show an association between the His variant and colorectal adenomas. This risk is increased by smoking and high intake of cooked meat [116]. Another study showed signiﬁcant association between the Tyr variant and an increased risk of colorectal adenomas [117]. In the case of ovarian cancer, one study shows a higher frequency of the Tyr variant among the cases [118], while another study shows that the same variant may decrease the risk [119]. The risk of preeclampsia [120], oral/pharynx cancer, and larynx cancer [121] was signiﬁcantly increased with increasing mEH activity. The His variant was signiﬁcantly overrepresented in hepatocellular carcinoma [122,123], colon cancer [124], chronic obstructive pulmonary disease and emphysema groups

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(smokers) [115], suggesting a positive association between this genotype and the risk of attracting these diseases. Males carrying the His variant have a lower risk of developing lymphoma [125]. Finally, no signiﬁcant associations were found with PD [126], asthma [115], and oral clefting [127]. An A/G variant results in the substitution of histidine for arginine at amino acid position 139 and has been shown to yield increased enzyme activity (25% higher activity [110]. No signiﬁcant associations were found between His139Arg polymorphism and preeclampsia [120], colorectal adenomas [116,117], colon cancer [124], lung cancer [111], Hodgkins and non-Hodgkins lymphoma [125], and oral, pharynx, and larynx cancers [121]. In the case of lung cancer, one study showed that smokers homozygous for the Arg variant have a signiﬁcantly decreased risk of developing the disease [112], while the opposite was shown in a study where Mexican Americans homo- or heterozygous for the same variant were associated with an elevated risk [114]. Reduced birth weight was signiﬁcantly associated with occurrence of the His139Arg polymorphism (in the mother) [128]. J.

Soluble Epoxide Hydrolase

The soluble form of epoxide hydrolase (sEH) is often involved in the metabolism of endogenous substances, e.g., epoxides of steroids and arachidonic acid derivatives, but also participates in xenobiotic metabolism, with a preference for trans-substituted epoxides. sEH is conﬁned mainly to the cytoplasm [129]. A number of polymorphisms in the sEH gene have been characterized, including two causing amino acid substitutions: Arg287Gln [130] and Ser407Ile [5]. The substitution of arginine for glutamine at amino acid position 287 has been shown to result in an enzyme with reduced activity [130]. A study of the eﬀect of this polymorphism in regard to PD showed no signiﬁcant association [126]. K.

Receptor of Advanced Glycation End Products

The receptor of advanced glycation end products (RAGE) responds by free radical generation, and polymorphisms have been linked to diabetes and microvascular dermatoses. Signiﬁcant associations to antioxidant carotenoids and a-tocopherol were observed [131,132]. In addition, promoter polymorphism that aﬀects transcriptional activity and transcription factor binding was associated to diabetic retinopathy [133] and non–small cell lung cancer [134]. L.

Nitric Oxide Synthase

Nitric oxide synthase is represented by three forms (neuronal, endothelial, and inducible) that participate in various physiological processes such as immune response and maintenance of vascular tone. The nitric oxide produced can react readily with superoxide, yielding the very reactive peroxynitrite potentially resulting in oxidative stress. Genetic polymorphisms have sometimes been linked to vascular disease and impaired endothelial function (reviewed in Ref. 135). M.

Myeloperoxidase

Myeloperoxidase is responsible for the production of hypochlorous acid, a potent and reactive antimicrobial agent. The enzyme has been implicated in cardiovascular disease, and levels in blood were associated with one promoter polymorphism but not

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four others that have been identiﬁed [136]. Several molecular epidemiological studies have found association with disease [136].

III.

CONCLUSIONS

An increasing number of genetic markers that can be employed for oxidative stress studies are emerging. Deﬁnition of phenotype in terms of expression levels, function, and stability is also being performed at an increasing pace. The most important phenotypes are those of the glutathione transferase M1-1 and T1-1 null alleles, the lowactivity (Pro187Ser) variant of quinone reductase 1, followed by several others that display altered activity/expression. Studying the correlation of these genetic markers to indices of oxidative stress damage will yield insight into the relative importance/ redundancy of protection mechanisms. Armed with this knowledge, the possible impact of our natural genetic variation on disease can be studied. Although we cite here a wealth of positive association studies linking genetic variants to diseases with a suggested oxidative stress component, it is also evident that conﬂicting results in association studies are often obtained. Careful considerations of the relative importance of total genetic inﬂuences on phenotypic variation (assessed by calculating heretabilities [137]) before starting studies may overcome some of the problems regarding lack of sensitivity in association studies. The primary goal of research in this area is to ﬁnd general utility markers to assess the possible impact of oxidative stress in human disease.

ACKNOWLEDGMENTS Work from the authors’ laboratories was supported by the Swedish Cancer Society, the Swedish Medical Research Council (05933), King Gustav V and Queen Victorias Foundation, The Swedish Heart and Lung Foundation, and Funds from Karolinska Institutet.

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11 Mitochondrial Dysfunction in Genetic Diseases IMMO E. SCHEFFLER University of California, San Diego, La Jolla, California, U.S.A.

I.

INTRODUCTION

Mitochondrial diseases as a distinct category did not exist until almost 1987. Symptoms that are now associated with mitochondrial diseases had been described before, and heritable diseases resulting from mutations in genes encoding mitochondrial proteins had been well characterized prior to this date. One example is the defect in ornithine transcarbamylase resulting in a defective urea cycle. Since mitochondria contain an estimated 1000–3000 proteins, many of which are associated with wellknown biochemical pathways, one can indeed predict the potential occurrence of many unifactorial genetic disorders due to deﬁciencies in speciﬁc mitochondrial functions. What distinguishes a mitochondrial disease, and why the ﬂurry of excitement during the past decade? The physiological signiﬁcance of mitochondria was ﬁrst appreciated in the 1950s when their role as the ‘‘powerhouse of the cell’’ was identiﬁed. They were recognized to be the organelle responsible for respiration and oxidative phosphorylation, fatty acid degradation, nitric oxide metabolism, and portions of a number of other important metabolic pathways such as pyrimidine biosynthesis, heme synthesis, the urea cycle, and the biosynthesis of quinones, steroids, and many other important biological compounds (for reviews, see Refs. 1–3). Many patients with defects in various pathways were diagnosed and characterized over the years, but it was probably an unstated assumption that a defect in respiration or oxidative phosphorylation would be lethal and therefore rarely if at all encountered among humans. Clearly, there were microorganisms that could live under anaerobic conditions, some of them even being obligate anaerobes, and mutations were soon discovered in 235

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populations of Saccharomyces cerevisiae and Neurospora crassa that knocked out the electron transport chain and/or oxidative phosphorylation [4–7]. In the presence of abundant glucose, such mutants can satisfy their energy requirements from glycolysis alone. Respiration-deﬁcient mammalian cells in tissue culture were ﬁrst described by this author in 1974 [8–12], but the ability of mammalian cells to survive exclusively by glycolysis was believed to be a special property of ﬁbroblasts in culture medium rich in glucose. ‘‘Mitochondrial diseases’’ in humans made headlines as a novel class of disease because of the following characteristics: (1) there was a partial defect in respiration and/or oxidative phosphorylation, i.e., an energy deﬁciency; (2) the partial defect was due to mutations in the mitochondrial genome (mtDNA), and hence there was a deﬁnite genetic basis, but inheritance was non-Mendelian and exclusively through the maternal lineage; (3) the study of many apparently diverse neuropathies and myopathies suddenly received attention and reached a new level of understanding. From the beginning it should have been clear that nuclear mutations could cause similar symptoms, and such patients have indeed been found during the past decade. Mitochondrial diseases due to nuclear mutations are therefore included in the present review. However, as will become clear from the discussion below, there are some fundamental diﬀerences in diagnosis, prognosis, and heritability between mitochondrial diseases arisng from mitochondrial vs. nuclear mutations. This chapter will not attempt to present a comprehensive discussion of all the medical and physiological manifestations of mitochondrial diseases. Instead, it represents an attempt to illustrate a number of basic concepts and principles that must be understood in order to comprehend the complexities of the biochemical and genetic aspects of this group of diseases.

II.

THE GENETIC BASIS FOR MITOCHONDRIAL DISEASES

A.

Nuclear and Mitochondrial Genes

Some 1000–3000 genes are thought to encode proteins found in mitochondria. The structure and morphology of mitochondria is suﬃciently understood to allow these proteins to be categorized into matrix proteins, inner and outer membrane proteins, and proteins occupying the intermembrane space. All but 13 of these proteins are encoded by nuclear genes, synthesized in the cytosol, and imported into mitochondria by targeting and translocation mechanisms of considerable intricacy [13–17]. Thus, a substantial number of mitochondrial proteins are dedicated to the import and proper localization of mitochondrial proteins. The organelle cannot originate de novo, but existing mitochondria grow in size and eventually divide by ﬁssion. The assembly of many of these proteins into larger multisubunit complexes requires additional proteins (chaperones, scaﬀolding proteins). Prosthetic groups, cofactors, and metal ions become associated as part of the maturation of these complexes to a fully functional form. The discovery of mitochondrial DNA (mtDNA) in the early 1960s was another milestone in the history of the study of mitochondria. Hypotheses about the origin of mitochondria as prokaryotes and symbionts during the earliest history of the evolution of eukaryotic cells received a major boost from this discovery (for reviews see Refs. 1, 18–20). The subsequent sequence comparisons and examinations of phy-

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logenetic relationships between the encoded proteins and ribosomal RNAs have solidiﬁed our view of a monophyletic origin of mitochondria in all living eukaryotes. Thirteen proteins are encoded by the mitochondrial genome in metazoans (with some exceptions) [21–24]. This number is similar in most invertebrates, fungi, and plants; a few exceptional organisms with a higher number of functional genes on mtDNA have provided interesting evolutionary clues. The 13 proteins are constituents of complexes of the oxidative phophorylation system, as described in further detail below. Mammalian (metazoan) mtDNAs also encode two ribosomal RNAs for small and large ribosomes and 22 tRNAs. A distinct apparatus for protein synthesis is found in the mitochondrial matrix to synthesize 13 subunits for complexes of the oxidative phosphorylation system [21,23,25]. Ribosomal proteins, initiation factors, and elongation factors are all encoded by nuclear genes and imported from the cytoplasm. The replication of the mtDNA and the transcription of this genome have been elucidated and described in numerous reviews [26–28]. However, only recently there has been signiﬁcant progress in characterizing mtDNA repair mechanisms [29–31], while evidence about recombination between mitochondrial genomes in mammals is still controversial [32–35]. DNA and RNA polymerases, ligases, RNases, and numerous other factors are required for the maintenance and expression of the mitochondrialgenome. It is essential to emphasize at this point that a mammalian cell has many mitochondria, and each organelle contains more than one mtDNA. Since mitochondria are engaged in a continuous dynamic process of fusion and ﬁssion (and they may even form a continuous reticulum in some circumstances), it makes less sense to count mitochondria per cell, but it is useful to know the number of mtDNAs per cell, i.e., the copy number relative to the diploid nuclear genome. An average number of f1000 per cell is easy to remember, but it should be stated that precise determinations have not been made in many cells types, and this number is likely to be variable in diﬀerent tissues and cells. It may be that the number of mitochondrial genomes and/or the rate at which they are transcribed is reﬂected in the density of mitochondrial cristae, that is, the total surface area of the inner mitochondrial membrane. B.

The Electron Transport Chain

The 13 proteins encoded by the mtDNA and made in the mitochondrial matrix are distributed among the complexes of the electron transport chain (with the exception of complex II) and ATP synthase (complex V). It is relevant here to discuss each of these complexes brieﬂy to understand the consequences of diverse mutations on oxidative phosphorylation. Numerous reviews have covered this material in the past [1,36]. 1.

NADH–Ubiquinone Oxidoreductase (Complex I)

Complex I consists of f43 diﬀerent subunits, seven of which are encoded by the mitochondrial genome [37]. In mammals these are designated ND1, ND2, ND3, ND4, ND4L, ND5, and ND6. Together with seven subunits (75 kDa, 51 kDa, 49 kDa, 30 kDa, 24 kDa, TYKY, PSST)* encoded by nuclear genes, they were thought to *The nomenclature varies from organism to organism. In mammals the designation of the subunits en-

coded by mtDNA is uniformly accepted. The other subunits are frequently referred to by the molecular mass or by listing the sequence of the ﬁrst four amino acids from the N-terminus.

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constitute a functional ‘‘core,’’ while 29 additional subunits have been referred to as ‘‘accessory’’ in the past. The nomenclature is derived from the observation that 14 homologous proteins in prokaryotes make up a functionally similar complex. The mitochondrial complex catalyzes the oxidation of NADH in the matrix and reduces the mobile electron carrier ubiquinone to ubiquinol; in the process four protons are pumped from the matrix to the intermembrane space [38]. It has become clear from genetic studies with fungi and mammalian mutant cells that some of the ‘‘accessory’’ proteins are essential, but the role of most of these is still unknown [6,39–41]. There is also evidence that speciﬁc factors (chaperones, scaﬀolding proteins) may be transiently involved in the assembly of the complex but are not found in the complex I puriﬁed by a variety of methods [40]. Complex I is the one for which a crystal structure is not yet available. From lowangle x-ray scattering the overall shape has been deduced to be that of a boot or the letter L. One major subcomplex contains integral membrane proteins (including ND1, 2, 3, 4, 4L, 5, and 6), and another is a subcomplex of peripheral proteins; these two are connected by a narrower neck or hinge region. Functionally the subdomain in the matrix is an NADH dehydrogenase, and the connecting fragment is related to multisubunit hydrogenases [42]. Certain combinations of detergents and fractionation techniques appear to cleave the complex into three subcomplexes that may reﬂect this functional diﬀerentiation. The most mysterious integral membrane subdomain is likely to be involved in proton translocation (or two of the four protons). Two major subcomplexes appear to be assembled independently in the matrix and in the inner membrane, respectively, until they are joined to form the complete functional complex (in Neurospora crassa [40]). The seven mtDNA-encoded subunits are required for assembly of the membrane subcomplex, and in the absence of one or more of these subunits no functional complex I can be formed. 2.

Succinate–Ubiquinone Oxidoreductase (Complex II)

Complex II is the exception, with no mitochondrially encoded subunits. A peripheral subcomplex made up of two subunits (a ﬂavoprotein, Fp, encoded by the SDHA gene, and the iron-protein, Ip, encoded by the SDHB gene) is attached to two integral membrane proteins encoded by the SDHC and SDHD genes, respectively (see Refs. 43, 44 for reviews). The integral membrane subunits are associated with a b-type heme, whose function is still controversial. The enzyme catalyzes one of the reactions in the Krebs cycle and thus couples the Krebs cycle directly to the electron transport chain via the mobile carrier ubiquinone. The crystal structure of the closely related complex fumarate reductase from E. coli has been determined [45]. It greatly facilitates molecular modeling of the eukaryotic (mammalian) complex II, and an actual structure for this complex can be expected in the near future. 3.

Ubiquinone–Cytochrome c Oxidoreductase (Complex III)

The mammalian complex III has 11 subunits, one of which (cytochrome b with two heme groups) is encoded by mtDNA. Two other subunits, cytochrome c1 and the Rieske iron-sulfur protein, together with cytochrome b can also be considered to constitute the essential core, since they are also found in the corresponding prokaryotic complex. The role of the other 8 subunits is still unclear: they may be required for assembly, stability, or regulation, and in other organisms such as plants. Their large

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domains in the matrix appear to play a role in protein import and processing. The enzyme oxidizes ubiquinol (QH2) and in the process transfers two electrons in succession to the mobile carrier cytochrome c. Four protons are transferred from the matrix to the intermembrane space by a mechanism referred to as the Q-cycle [46,47]. The structure of the complex has been resolved by x-ray crystallography, conﬁrming the two Q-binding sites and the sites for the well-known inhibitors antimycin and myxothiazol [48]. 4.

Cytochrome c Oxidase (Complex IV)

The mammalian complex contains 13 subunits; the three largest subunits (COXI, COXII, and COXIII) are encoded on mtDNA. It contains cytochromes a and a3 and two copper centers. Four electrons from the oxidation of reduced cytochrome c are transferred successively to oxygen (O2) to form two water molecules. Several of the additional subunits with no cytochromes or metal centers have unknown functions, possibly including a regulatory function involving adenine nucleotide binding. Although the crystal structure of the complex has been deduced at high resolution [49–51], conﬁrming the path of the electrons through the complex, the mechanism of proton translocation (2 per 2 electrons) is still unclear. Another debate continues to center around the question as to whether small molecules like ATP or ADP can act as ligands in the allosteric control of complex IV activity [52,53]. The complex IV is the only complex for which tissue-speciﬁc isozymes/subunits have been identiﬁed [54], but the full signiﬁcance of this ﬁnding remains to be explored. A common assumption is that the ratio of protons pumped to electrons transferred to oxygen is an integral number. That is to say, there is a stoichiometry familiar to us from chemical reactions. However, biophysical principles do not exclude the possibility of a noninteger. This phenomenon can be described as ‘‘slippage,’’ and speculations are that this slippage may even be a means of control. If true, this would represent another mechanism for regulating the coupling of electron ﬂow and proton translocations across the inner membrane. 5.

ATP Synthase (Complex V)

The elucidation of the structure and function of complex V is one of the triumphs of biochemistry, bioenergetics, molecular genetics, and structural biology/x-ray crystallography [55–65]. It is a small molecular rotary engine that can use a proton and electrochemical gradient to drive the synthesis of ATP from ADP and inorganic phosphate. The chemiosmotic hypothesis of P. Mitchell should be familiar to most readers: an electrochemical (DC) and proton gradient (DpH) across the inner mitochondrial membrane transiently stores the free energy derived from the oxidation of NADH (and succinate, and the ﬁrst step in h-oxidation of fatty acids). Electron transport through the electron transport chain to oxygen and ATP synthesis occur in distinct complexes physically separated in the plane of the inner mitochondrial membrane. The two reactions are nevertheless ‘‘coupled,’’ not by mechanical, protein–protein interactions, but by the need for proton circuit, that is, protons pumped out of the matrix by the ETC have to be returned through the ATP synthase (complex V). Thus, in well-coupled mitochondria, ADP in the matrix can become rate limiting for electron transport. This ‘‘acceptor control’’ arises from the need to dissipate the proton gradient established by the ETC by the transfer of a phosphate group to the acceptor ADP on complex V [66–69].

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Supercomplexes

A question that was raised some time ago addressed the relative abundance of the individual complexes in the inner mitochondrial membrane. This problem has been revisited periodically, and a consensus is emerging, although it should perhaps be stressed that the experimental data are from a very restricted range of tissues, typically bovine heart. A ratio for complexes I:II:III:IV:V of 1:2:3:6–7:3–5 in bovine heart mitochondria was published in 1985 and again by a diﬀerent lab in 1988. Some recent measurements have narrowed the uncertainty [70]. Recently published pioneering studies by several laboratories have raised the issue of the formation of supercomplexes (‘‘respirasome’’), and speciﬁcally they have suggested the existence of large and small supercomplexes [71]. All of complex I is bound to complex III (in the large supercomplex), but some complex III is found associated with IV but not with I. Complex II and complex V have not yet been found in these supercomplexes (but complex V may function as a dimer). The ratio of complexes in relation to the ratio of supercomplexes raises further interesting questions related to the quinone pool and the possibility of quinone substrate channeling. Complex II could interact with a distinct subset of complexes III and IV, etc. The real physiological meaning of these considerations is yet to be established, but clearly, if the formation of supercomplexes is physiologically meaningful (for control and speed), then mtDNA mutations that aﬀect the level of individual complexes may cause serious perturbations by yet another, novel mechanism. C.

Coupling and Transport

Well-coupled mitochondria are mitochondria in which the return of protons to the matrix occurs largely through complex V. Other mechanisms for the return of protons through the inner membrane include the pathway through the uncoupling proteins (UCP1–6) [72–77], through various symporters and antiporters used in the transport of other ions in and out of mitochondria, through artiﬁcial uncouplers used in experiments (dinitrophenol was at one time promoted as a drug for weight control) and under pathological conditions (apoptosis) through the megapore associated with the mitochondrial permeability transition (MPT) [1,78,79]. A second important aspect of oxidative phosphorylation and of the mechanism of coupling is the import of ADP and inorganic phosphate and the export of ATP. ADP/ATP exchange occurs though the adenine nucleotide translocator (ANT), an antiporter that has been studied extensively [72,73,75,80–82]. Phosphate enters in symport with protons through a separate membrane transporter. An adult human uses more than his or her body weight in ATP per day (and more when engaged in strenuous activity), but clearly this is not accumulated ATP, but ATP constantly being recycled through ADP and inorganic phosphate in the performance of biological work. D.

Flux and Flux Control in Oxidative Phosphorylation

Enzymes typically do not function in isolation, but are catalysts for biochemical reactions that are organized in metabolic pathways. An important property of a pathway is the ﬂux through the pathway, measured by the rate at which starting material is consumed and ﬁnal product is produced. In the case of oxidative phosphorylation,

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one can speak about the ﬂux of electrons through the electron transport chain, measured ultimately by oxygen consumption (respiration). This ﬂux may be controlled on one side by the availability of NADH. On the other side, oxygen is likely to be limiting only under extreme, pathological conditions. However, as explained above, respiration is also controlled by the availability of ADP and the rate of ATP synthesis in normal, well-coupled mitochondria. Both NADH and ADP are not so much consumed as turned over; NADH/NAD+ turnover is the result of metabolic reactions, while ATP/ADP turnover results from metabolism as well as from numerous and diverse types of biological work. In a narrower sense one can consider mitochondrial diseases to arise from reductions in the ﬂow of electrons through the electron transport chain, but one should broaden the deﬁnition to include the activity of complex V, the activity of the adenine nucleotide transporter, and possibly other mitochondrial functions. At steady state the ﬂux through a pathway is also the ﬂux at an individual step in the pathway. The ﬂux is under constant control and adjusted to the physiological demands of a cell or tissue. One can distinguish long-term and short-term control. Long-term control might include the diﬀerentiation of diﬀerent cells in diﬀerentiated tissues and the adjustment of the total capacity for oxidative phosphorylation. The mechanism is likely to involve the control of gene expression at a transcriptional or posttranscriptional level. How many mitochondria are present and what is the density of the cristae? Since the mechanisms are still poorly understood, it has not been possible to identify patients with perturbations in any of these mechanisms. Short-term control, in general, includes allosteric mechanisms, feedback by products, and covalent modiﬁcations of individual enzymes to activate or deactivate them. These mechanisms are rapid and allow almost instant responses to cellular needs. Conﬁning the discussion to bioenergetics, there are several well-understood examples of the control of speciﬁc enzymes such as glycogen phosphorylase, phosphofructokinase (PFK-1) in glycolysis, enzymes in the Krebs cycle, etc. Enzymes such as phosphofructokinase are frequently referred to as rate-controlling enzymes and thought of as a ‘‘bottleneck’’ in the pathway, i.e., they control the ﬂux. The past decade has seen the development of a more sophisticated view of the control of metabolic ﬂux in which ﬂux control is distributed over the entire pathway, albeit unevenly [83–91]. Each step is associated with a ﬂux control coeﬃcient. In a simpliﬁed view, this coeﬃcient is a measure of how much control the enzyme catalyzing that step exerts over the entire pathway. A coeﬃcient of 0 means that changing the enzyme concentration by x% has zero eﬀect on the ﬂux, that is, the enzyme may be present in excess. A coeﬃcient of 1.0 implies that a change in the enzyme level by a factor y changes the ﬂux through the pathway by the same factor, i.e., this enzyme is truly limiting and rate controlling. Typically, ﬂux control coeﬃcients are somewhere in between these extremes. The ‘‘bottleneck’’ enzymes in the old terminology have greater ﬂux control coeﬃcients than the ‘‘nonregulatory’’ enzymes. These considerations are particularly relevant for the discussion of the electron transport chain in the context of mitochondrial diseases. Between NADH and oxygen there are three complexes (I, III, IV) and two mobile carriers and hence multiple steps of electron transfer. Is there a rate-limiting step, for example, from NADH to ubiquinone (complex I), or from cytochrome c to oxygen (complex IV)? In other words, what are the ﬂux control coeﬃcients for the various steps? Such questions have provoked much experimentation and controversy. The issue is acute, because mito-

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chondrial diseases arise from partial deﬁciencies in the activities of one or more complexes (see below). When a missense mutation in a subunit reduces the activity of complex I by, say, 50%, it would have no consequence if complex I activity was present in large excess. If the mutation in a single structural gene for a subunit is totally incapacitating, at what level of heteroplasmy (% mutant mtDNA) would one expect to see pathological consequences? The situation is even more complicated when one has heteroplasmy and mutant mtDNA with a large deletion. Speciﬁc structural genes for one complex might be present at lower copy number, but the level of all complexes might be aﬀected by a reduced rate of protein synthesis in the matrix due to a shortage of tRNAs. It could be that the capacity for mitochondrial protein synthesis is higher than the demand, and the expression of the mitochondrial genome is controlled exclusively by RNA polymerase, transcription factor(s) such as mtTFA, and processing of the polycistronic RNA. It could also be that under normal conditions the capacity for electron transport is not limiting, but respiration is controlled by NADH/NAD+ and ATP/ADP turnover or recycling. Natural control mechanisms could be operative such that they could compensate for a partial loss of activity due to mutations. Defects in the electron transport chain would then become serious and pathological only when a lower threshold is crossed. Anticipating the discussion of speciﬁc mitochondrial diseases and the genetic studies performed on representative patients, one must consider all of the above possibilities. Most characteristically for mitochondrial diseases, the organs aﬀected are the nervous system (brain and highly specialized portions of the nervous system such as the optic nerve), cardiac muscle, and skeletal muscle. These are the tissues with the highest energy demands, where under some conditions the capacity of the electron transport chain is exhausted. The total capacity may even change with age, and hence mitochondrial diseases have frequently been found to have an age-dependent onset (the ‘‘threshold hypothesis’’ of Wallace) [92–94]. Clearly, individuals can be near normal for the ﬁrst one or two decades of their lives before neuropathies and myopathies become apparent. Does this mean that most other tissues/organs can tolerate a signiﬁcant loss of OXPHOS capacity without pathological consequences? An alternative explanation is that the observed heteroplasmy in a muscle biopsy is not the same as in another tissue, and this has clearly been established in some patients. Large deletions in mitochondrial DNA have been found in muscle, while they were rare or even undetectable in blood cells [95]. Even more remarkably, mtDNA in muscle progenitor satellite cells was found to be largely normal, while surrounding myotubes contained mtDNA with deletions [96]. Age-dependent changes can result either from ﬂuctuating heteroplasmies or from a steady accumulation of random mutations in mtDNA as a consequence of the action of reactive oxygen species. The eﬀects of these random mutations would become superimposed on those of a speciﬁc mutation. E.

Partial OXPHOS Deficiency

1.

Homoplasmy and Heteroplasmy

It has been self-evident that mammalian organisms cannot live under anaerobic conditions. It follows that severe mutations aﬀecting any component of the electron transport chain, the ATP synthase, or the adenine nucleotide transporter would be lethal. They may account for a fraction of the spontaneous abortions that occur at high

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frequency during human pregnancies, and patients with a severe OXPHOS deﬁciency would not have been expected to show up at a clinic. The breakthrough came when patients with partial OXPHOS deﬁciencies were discovered and it was recognized that such deﬁciencies could be found even in adolescents/adults. Symptoms could range from mild to severe, they could be sporadic, and they often manifested themselves with a delayed onset, followed by a progressive deterioration. Not surprisingly, the major organs aﬀected were the nervous system and skeletal and cardiac muscles. A more detailed description will be presented later. The cause for a partial OXPHOS deﬁciency in some of the earliest patients to be characterized at the biochemical and genetic level was also interesting and revealing. First, the mutations were in the mitochondrial DNA. Proof was derived from direct DNA sequence comparisons and from pedigree analyses demonstrating the nonMendelian, maternal inheritance of the mutations. To ﬁnd mutations in mitochondrial DNA would a priori not have been so surprising. Mutagens (reactive oxygen species?) and errors in replication and/or repair should have been expected to leave their mark. But a major theoretical obstacle to be overcome and explained was to account for the consequence of a mutation in one mtDNA molecule when there are of the order of 1000 such molecules per cell. The basic facts were quickly established by the pioneering studies of several laboratories [97–100]. Aﬀected individuals were found to be heteroplasmic: aﬀected (sampled) tissues were found to contain mixed populations of mtDNA, one fraction being normal and another fraction having a unique point mutation or deletion (however, see below). The severity of the symptoms was related roughly to the proportion of the mutated mtDNA. Therefore, a partial deﬁciency in the capacity for OXPHOS could be rationalized by the observed heteroplasmy. A fraction of the normal mtDNAs could serve to express normal proteins at a proportionately lower level, yielding fewer active complexes or mixture of active and less active (inactive) complexes. Depending on the position of the mutation, a single complex could be partially defective, or all complexes containing proteins encoded by mtDNA could be aﬀected. At one end of the spectrum of mutations is a large deletion of mtDNA. Wild-type (wt) mtDNA and deleted mtDNA molecules coexist in a single cell and even in a single mitochondrion (subject to further mixing by fusions and ﬁssions; see below). The speciﬁc deletion is ‘‘covered’’ (complemented) by the wt mtDNA, but the reduced gene dose (% heteroplasmy) determines the level of normal gene products still being produced. The deletions may extend over several thousand base pairs (out of 16.6 kb of mtDNA), and because of the dense packing of genes in the mitochondrial genome such deletions will invariably include one or more structural genes (genes encoding the 13 proteins made in the matrix) and several tRNA genes. A priori the levels of proteins produced in the matrix could be aﬀected in two ways: (1) a reduced level of mRNAs from the deleted genes may cause a deﬁciency of a subset of speciﬁc proteins; (2) a deﬁency of speciﬁc tRNAs could aﬀect the translational eﬃciency as a whole, and therefore even proteins whose genes are not deleted may be made at reduced levels. At the other end of the spectrum of mutations are single point mutations in either a structural gene or a tRNA or rRNA gene. Single point mutations in structural genes can cause amino acid substitutions (missense mutations) or chain termination. Some missense mutations will not completely destroy the function of the protein. Thus, in

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extreme cases aﬀected individuals in a pedigree were found to be homoplasmic for the mutation, that is, all mtDNAs carried the point mutation. In that case the mitochondria contain a homogeneous population of a partially active complex, and the residual activity is determined by the nature of the amino acid substitution and not by the gene dose (heteroplasmy). Point mutations in tRNA genes have also been deﬁned. Their eﬀect is diﬃcult to predict from ﬁrst principles, but two possibilities can be distinguished. A nucleotide change may aﬀect the rate at which the tRNA can be charged with an amino acid, or it may aﬀect the eﬃciency with which it participates in peptide synthesis on mitochondrial ribosomes. An alternative possibility arises from the mechanism by which tRNAs are made in mitochondria. MtDNA is transcribed into large polycistronic transcripts, which then have to be processed; tRNAs are distributed as spacers between the protein-coding mRNAs. Hence, their splicing and maturation to functional amino acyl acceptors may also be inﬂuenced by speciﬁc, strategically placed nucleotide changes. Evidence is derived from in vivo studies, but unfortunately it is technically still diﬃcult if not impossible to distinguish between these possibilities by in vitro protein synthesis systems with isolated, mitochondria-derived components. From the above considerations it is quite clear that depending on the speciﬁc nature of the mutation, and on the % heteroplasmy, the reduction in capacity for oxidative phosphorylation and hence the severity of the symptoms may be extremely variable. This variability is one of the hallmarks of mitochondrial diseases. 2.

Random Mutations in mtDNA

It is generally agreed that the mutation rate for mtDNA is an order of magnitude greater than that for nuclear DNA. Two reasons may be cited: (1) although the evidence for the presence of mitochondrial DNA repair mechanisms is growing, repair mechanisms involving recombination and mismatch repair or excision of pyrimidine dimers appear to be absent; (2) mtDNA may be more exposed to the mutagenic action of reactive oxygen species (ROS) compared to nuclear DNA. One would therefore predict that over a lifetime a large number of mtDNA mutations would accumulate, and this idea is in fact central to popular current hypotheses on aging, as discussed elsewhere in this volume. If point mutations are generated, these will be distributed randomly and at diﬀerent positions in each of the many mtDNAs in a cell. It would require the accumulation of a substantial number of such mutations before a signiﬁcant eﬀect on electron transport and oxidative phosphorylation could be observed. In addition to the spontaneously occurring errors in mtDNA replication, one can also consider the superimposed formation of mutations due to external, environmental insults. Two examples can be cited for attempts to use mtDNA as a biomarker of cumulative exposure to mutagens. Several laboratories have examined mtDNA in skin as a target for ultraviolet radiation [101–104]. For technical reasons, PCR-based methods are most easily employed, and such methods can readily detect signiﬁcant deletions in mtDNA, but not point mutations or very small deletions. Signiﬁcantly, increasing the UVR exposure of skin caused an increase in the number of deletions, and speciﬁcally an increase in the 4977 bp ‘‘common deletion.’’ What remains to be explained in detail is the pathway that leads from thymine dimers induced by UVR to the formation of such large (and relatively speciﬁc?) deletions [104]. It is known that short sequence repeats play a role, and slippage during mtDNA replication may be

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enhanced by the presence of photoproducts, but recombinational mechanisms cannot be excluded. In another investigation, mtDNA mutations as the result of smoking were looked for in human hair follicles [105]. A signiﬁcantly increased incidence of the 4977 bp deletion was found in smokers, especially in older, heavier smokers. The observed fraction was even more dramatically raised in patients with lung cancer. Thus, there appeared to be a correlation of mtDNA damage in hair follicles with the occurrence of smoking-associated cancers, but it is more likely to be collateral damage. MtDNAs in easily accessible tissue therefore can serve as biosensors for exposure to either external (radiation) or internal (xenobiotics from smoking) carcinogens. Whether they preserve a long-term or short-term record may depend on the rates of cellular turnover in the tissues examined. 3.

Mutations in Nuclear Genes

The majority of the protein subunits for the electron transport chain and associated functions are encoded by nuclear genes. Mutations in such genes can also give rise to mitochondrial diseases [106–111]. The genetic picture for mutations in nuclear genes is simpler. Recessive mutations (including null mutations and deletions) must be homozygous (/), but could be heteroallelic, unless X-linked genes are involved. Heterozygous patients (+/) suﬀering the disease as a consequence of a reduced (50%) gene dose have not been described so far. There have also been no reports of dominant-negative mutations that could be phenotypically expressed in heterozygotes. For reasons explained above, homozygous mutant individuals (/) must have some residual activity to be born alive, and therefore at least one of the alleles must represent a missense mutation. There is also the possibility of mutations in regulatory elements of a gene. Depending on the severity of the mutation, the life expectancy of live-born children may be very limited (see below). A question is whether nuclear mutations also cause a delayed onset of a mitochondrial disease. In contrast to heteroplasmy, one would expect cells and tissues to be genotypically stable. Theoretically one could have an age-dependent mutational load in mtDNAs superimposed on a nuclear mutation to lower OXPHOS capacity below a critical threshold. A second mechanism to be considered for nuclear mutations in heterozygous individuals is loss of heterozygosity (LOH), frequently found in tumorigenesis. As an example one could cite autosomal dominant paraganglioma resulting from mutations in the SDHC gene [111]. While this tumor does not present as a typical mitochondrial disease, it is caused by a defective complex II in the electron transport chain [111a]. F.

Inheritance of Mitochondrial Diseases

1.

Nuclear Mutations

When mutations in nuclear genes are involved, the analysis of pedigrees would be expected to be straightforward. However, a limited life expectancy and reduced reproductive ﬁtness of aﬀected individuals may result in small pedigrees. Since the mutations are also rare, available pedigrees have not yet been exploited for extensive gene mapping. In some cases the aﬀected gene/protein has been identiﬁed, and in combination with information from the Human Genome Initiative, the gene is characterized and mapped. Examples are the mutations in patients with complex II deﬁciencies [112–115] and some of the patients with complex I deﬁciencies [116]. An

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unexplained observation from a cohort of patients diagnosed with a complex I deﬁciency in The Netherlands is that a large majority of them are male, but X-linkage has not been deﬁnitively proved; the responsible X-linked gene has not been identiﬁed. The single, known X-linked gene (NDUFAI ) encoding the MWFE protein of complex I was found to be normal in all the patients. It should be considered that mitochondrial diseases due to nuclear mutations are complex/multifactorial in the sense that speciﬁc alleles at other loci may inﬂuence the penetrance and expressivity of the primary mutation. Polymorphisms in mtDNA may also play a role. This is particularly true for the genes/proteins under discussion here, since none of the gene products are functional in isolation. They are invariably subunits of larger, heteropolymeric complexes with many protein-protein interactions. 2.

Non-Mendelian Inheritance

There are now many studies with a very diverse group of metazoan organisms including humans demonstrating that mitochondrial genomes are inherited exclusively through the female lineage. Conformity with this pattern of non-Mendelian inheritance constituted one of the earliest convincing proofs that mitochondrial mutations were responsible for the observed phenotype (disease). Modern methods based on the polymerase chain reaction (PCR) make it relatively routine to clone and sequence the entire mitochondrial genome from a small biopsy sample. When samples from an extended pedigree are available, the genotyping of all individuals is feasible and maternal inheritance can be proved. Similar methodology also makes it possible to establish heteroplasmy and the relative proportions of wild-type and mutant mtDNA. In this context, however, a potentially major issue must be introduced. The percentage of heteroplasmy is not necessarily the same in every tissue or even in every cell of the same tissue. Some tissues are easier to obtain by biopsy than others. Blood cells (lymphocytes) are most readily available, skeletal muscle biopsies require a more serious invasion, and brain biopsies are generally not available but may be obtainable after death. The observed heteroplasmy in a given tissue has also been found to change with age [117]. The picture that emerges is therefore potentially complicated, since heteroplasmy in a skeletal muscle specimen may not reﬂect the situation in cardiac muscle or the brain, and hence it is not trivial to correlate very precisely the severity of symptoms with the degree of observable heteroplasmy. Similarly, it may become diﬃcult to make long-range predictions for a given individual. The following scenario must be considered. A zygote (fertilized egg) with an estimated 100,000–500,000 mtDNAs [118,119] may start with a certain fraction of mutated mtDNAs. During subsequent zygotic divisions, these mtDNAs are distributed to daughter cells, and at some stage in development (see below) mtDNA replication and segregation will accompany cell division and multiplication. The possibility exists that some mtDNA molecules are preferentially replicated, and as the mature organism develops, heteroplasmy becomes variable in diﬀerent tissues. The reasons for selective replication are not always clear. Among the earliest examples of mitochondrial diseases that were deﬁnitely related to a mutation in mtDNA were cases with large deletions. These deletions were relatively homogeneous in the tissues examined (e.g., muscle) but were not found in other tissues (e.g., blood cells). Furthermore, it was concluded from pedigree analysis that these deletions were not maternally inherited, but were likely the result of prob-

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lems in mtDNA replication and segregation in early development. Mutations arising spontaneously in an early embryo will lead to the formation of a mosaic with regard to genotype. If, in addition, the mutated mtDNA has a replicative advantage over normal mtDNA, for example, when it is signiﬁcantly shorter, a further biased evolution of heteroplasmy can be imagined. This is the simplest scenario, and undoubtedly the situation may be more complicated. For example, it is not obvious to rationalize a replicative advantage for a mtDNA with a point mutation in a tRNA gene, especially when one must consider that a mixture of wild-type and mutated mtDNAs exists within a single (transient) mitochondrion. It is evident that this problem cannot be approached experimentally in humans. Until very recently no animal model was available. However, successes in making mice with heteroplasmies have opened the path for a study of the processes that determine the changing (?) distribution of heteroplasmic mtDNA populations in diﬀerent tissues. Extrapolations of conclusions to humans should probably be made with caution. 3.

Mitochondrial Heteroplasmies in Maternal Transmission

Pedigree analysis in families with mitochondrial diseases not only established maternal transmission of the trait, but it also revealed another challenging problem [120]. When the degree of heteroplasmy in the mother was compared with that in her siblings, a wide variation was found, which was also correlated with the severity of the symptoms. Mothers with hardly any notable symptoms but with a measurable heteroplasmy were found to have severely aﬀected children as well as almost normal children. This result could to some extent have been anticipated from preceding observations on mitochondrial inheritance in cows, the only other mammal in which heteroplasmy had been observed at that time [121,122]. A ‘‘bottleneck theory’’ was advanced at the time that in its simplest form postulates that only a very small subset of mtDNAs is utilized and replicated to populate the oocyte and to be passed to future generations. Statistical calculations showed that the selection of a small number (f100–200) of mtDNAs from a mixed (heteroplasmic) population could lead to stochastic ﬂuctuations in heteroplasmy of the magnitude observed in the eggs and zygotes of mice [123]. It become a major challenge to determine where this ‘‘bottleneck’’ occurred in oogenesis and to deﬁne the basis for the selection of a subset of mtDNAs to be passed to future generations. One may distinguish between an ‘‘active’’ and a ‘‘passive’’ mechanism in a detailed consideration of oogenesis and embryogenesis. The mammalian oocyte is one of the largest cells in the organism, and the number of mtDNAs per mature oocyte has been estimated to be in the range of 100,000– 500,000 (mouse or humans). After fertilization there are a series of zygotic cell divisions, and there are indications that during that period there is no replication of the existing mtDNAs. It may be relevant here that there is no respiration in the early embryo, and mouse embryos with a homozygous knock-out of the cytochrome c gene are viable up to midgestation [124]. Therefore, the mtDNAs present from the oocyte are distributed into 2, 4, 8, 10, etc. cells. After 10 cell divisions and starting with 100,000 mtDNAs, one would have f100 mtDNAs per cell, a number that would be consistent with statistical models proposed to explain the bottleneck. With the higher starting number, only 12 cell divisions would be needed to reach the same level. In order to examine this model experimentally, one would have to determine when mitochondrial DNA replication begins in the embryo relative to the major stages (morula, blastocyst, gastrulation,

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etc.). At some early stage the future ovary and germ cells are set aside, and it would be predicted that they contain a very small number of mtDNAs [125,126]. From semiquantitative evaluations of electron micrographs an estimate of the number of mitochondria in the human primordial germ cell has made to be less than 10, and furthermore it has been proposed that there may be only one mitochondrial genome per organelle [119]. The physiological signiﬁcance of such a bottleneck is that a sampling of such a small number may represent a mechanism of quality control. Those resulting oocytes with a substantial fraction of defective mtDNAs may be unable to produce viable embryos and hence prolonged pregnancies are avoided [125]. Jansen and De Boer [125] have argued, based on experimental and theoretical work of Chao et al. [127], that this mechanism represents a reversal of Muller’s ratchet: ‘‘the inexorable force that degrades a genome with time’’ by deleterious random mutations. An alternative hypothesis focuses on the events of oogenesis in the ovary. The primordial ovary has a subpopulation of cells, the oogonia, which undergo a rapid series of mitotic cell divisions before their premeiotic S-phase. Meiosis I and Meiosis II lead to the formation of primary and secondary oocytes that eventually mature into eggs. If the oogonia have a number of mtDNAs that is in considerable excess of 100– 200, the interpretation of some limited experimental observations in the mouse requires that only a subset of mtDNAs become engaged in replication during the mitotic divisions of oogonial cells in order to explain the observed stochastic variations in heteroplasmy in the eggs [123]. Again, insights gained from the study of oogenesis and embryogenesis in the mouse should be extrapolated to humans with caution, because the number of cells involved and the timing of landmark events may diﬀer. The major conclusion remains true, however: heteroplasmic females will have oﬀspring in which the proportion of mutated mtDNAs can be signiﬁcantly higher or lower in a way that is diﬃcult to predict for an individual pregnancy. A debate has recently been reopened about the possible contribution of genetic information from the sperm mitochondria. Cytological evidence shows that sperm mitochondria are introduced in the fertilized egg. What is their fate? There are some indications that the morphologically distinguishable sperm mitochondria may be ubiquitinated and targeted for destruction by proteasomes or lysosomes during the earliest embryonic cleavages [128]. On the other hand, in a series of interspecies crosses of mice and cattle with suitable mitochondrial markers, biparental inheritance has been demonstrated [128–130]. To reconcile these observations, it has been argued that the destruction of paternal mitochondria in intraspecies crosses fails in interspecies crosses [128,129]. How well do they persist and contribute to the population of mtDNAs in the developing organism? One should note that an oocyte has an estimated 100,000 mtDNAs, while the number of genomes contributed from the sperm is signiﬁcantly lower (a few hundred?). Highly sensitive detection methods must be used to identify mtDNA contributed by the sperm. It may take several generations before they can be expected to make a contribution to the phenotype of the descendents, unless there is a clear replicative advantage or an ‘‘ampliﬁcation’’ by a stochastic process described above. There is no doubt that over a few generations inheritance is maternal. The question is whether over an evolutionary time scale some contribution from a male could have been made. The expanding practice of cloning animals by fusing nucleated somatic cells with enucleated oocytes introduces a potential complication that was ignored in the case of the sheep Dolly. The somatic cells clearly contribute mitochondria (and mtDNA), and

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if such mtDNAs carry polymorphisms or even subtle mutations, they could aﬀect energy metabolism in the cloned oﬀspring and make it less than a perfect clone. Similar considerations apply to the practice of cloning rare animals by fusing their somatic cells to an enucleated oocyte of a more common, closely related species. The result will not be a perfect clone; it remains to be seen how much ‘‘species variation’’ can be contributed by the mt genome alone [131]. These considerations also have potentially serious implications for the practice of in vitro fertilization using sperm microinjection and oocyte cytoplasm ‘‘donation therapy’’ [132]. Oﬀspring with mtDNA from two mothers have been reported. Another unsettled issue is whether there can be recombination between mtDNAs. First, evidence has been presented that mammalian mitochondria have the enzymatic machinery for recombination [133]. This ﬁnding triggered thoughts about the possibility of recombination between (1) the maternal and the paternal mtDNA and (2) recombination between mtDNA in heteroplasmic individuals. An experimental resolution will require heteroplasmic animals with multiple mitochondrial sequence polymorphisms.

III.

CLINICAL SYNDROMES

A.

General Aspects

From a broader perspective, it is now recognized that mitochondria play a central role in the cell that goes far beyond energy metabolism. Thus, mitochondrial dysfunction can be the cause of liver and kidney disease, diabetes, cancer, and infertility. Agerelated neurodegenerative diseases including Parkinson disease and Alzheimer dementia may have a mitochondrial connection. Finally, attempts to explain the natural process of senescence itself have included provocative hypotheses about mitochondrial degeneration being the root cause. A discussion of these subjects is beyond the scope of this article. This review will adopt a narrower focus on diseases resulting from a defective energy metabolism. Many examples of clinical descriptions could be cited here. Having determined that the underlying defect is a deﬁciency in respiration and/or oxidative phosphorylation, does this insight account for a common set of clinical manifestations? It is a general observation that the aﬀected individuals have many neurological problems (neuropathies) and problems associated with abnormal heart (cardiomyopathy) and skeletal muscle performance (exercise intolerance, myopathies); in combination, they may be referred to as encephalomyopathies. More speciﬁcally, symptoms may include blindness, deafness, migraine headaches, and stroke. A common problem detectable biochemically is lactic acidosis. Frequently the onset of the disease is delayed, and the course of the disease may be progressive. The point to be made is that it would have been diﬃcult to predict a priori the range of symptoms, the severity of symptoms, and in some cases the speciﬁc cell/tissue(s) aﬀected by a given mutation. Mitochondrial diseases constitute a broad spectrum of diseases. Because of overlapping symptoms they are not always unambiguously categorized. The same mutation may give rise to diseases with diﬀerent names, depending on the degree of heteroplasmy, or completely diﬀerent mutations can give rise to a very speciﬁc disease such as LHON (see below). Our theoretical understanding of the molecular-genetic defects and the mechanisms of their expression can be used to rationalize many of the clinical ﬁndings, but as

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will become clear from the discussion of speciﬁc diseases, many new challenges have arisen. The reader who is interested in a comprehensive and up-to-date listing of mitochondrial diseases, their genetic basis, and references to original articles is referred to a database maitained at Emory University (http://inﬁnity.gen.emory.edu/ mitomap.html). B.

Specific Mitochondrial Diseases

Kearns-Sayre syndrome (KSS) is deﬁned by a combination of abnormalities, including ophthalmoplegia, ataxia, retinitis pigmentosa, cardiac conduction defect, and elevated cerebrospinal ﬂuid protein. It is caused by single, large mtDNA deletions in the range of thousands of base pairs. A ‘‘common deletion’’ of f4.9 kb has been found repeatedly in a substantial fraction of KSS patients, and a novel junction was mapped between base pairs 8482 and 13460. A closer examination of this and many other deletions has established that directly repeated sequences of 4 to 13 bp are found at each breakpoint in a large majority of mtDNA deletions, supporting speculations about the mechanism of formation of such deletions. It is hypothesized that mtDNA dimers generated by DNA replication can be resolved by recombination events between such direct repeats, but proof for such a mechanism is still elusive. It should be noted that with some notable exceptions, mtDNA deletions do not cover the two origins of mtDNA replication and the promoter regions for transcription in the D-loop of mtDNA. Such molecules would clearly be doomed by their failure to be replicated. Because the deletions are large, one or more coding regions as well as several tRNA genes may generally be absent. Clearly, such mtDNAs can only exist in the presence of wild-type mtDNA or in a rare (still hypothetical) case of two mtDNA populations with nonoverlapping deletions, i.e., in heteroplasmic individuals. The deletions were established to be the cause of mitochondrial encephalomyopathies (MEM) in landmark papers by Holt et al. [98,134] and DiMauro and colleagues [135,136], but interestingly, the characterization of this mitochondrial disease could not be based on criteria of maternal inheritance in a pedigree. Almost all of these mutations were sporadic and absent in the mothers of such patients. Their creation must have been a postzygotic event, leading to the development of chimeric individuals with regard to heteroplasmy in diﬀerent tissues. It is unclear at what stage after fertilization the deletion occurred, but it must have been a unique event, since almost all deletions are identical in an aﬀected individual. If it occurred relatively late in development, a large proportion of cells and tissues should still be homoplasmic for wild-type mtDNA. In agreement with such a hypothesis, it is found that such individuals have tissues, such as blood, in which the deleted mtDNA is absent or heteroplasmy is very low. Since aﬀected individuals have often a large percentage of deleted mtDNAs in biopsied tissue(s), one has to ask how a rare deleted mtDNA at the time of its generation expanded during subsequent somatic cell proliferation and diﬀerentiation. What was the selective advantage? A shorter replication cycle? Heteroplasmy depends on the tissue; it is high in muscle, low in blood cells, and it would be very interesting to be able to determine it in many more tissues and organs. Access to such tissues from patients, however, may be limited. Heteroplasmy may change with age, but again it is impractical (and may be restricted by medical ethics) to make measurements over a required time span. The disease starts with a delay but

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before 20 years of age, and it is progressive, leading to death between the ages of 30 and 50. Identical deletions can be found in the Pearson syndrome (sideroblastic anemia, exocrine pancreas deﬁciency, gastrointestinal dysmotility, and renal tubulopathy). One of the most widely studied mitochondrial diseases is Leber’s hereditary optic neuropathy (LHON) (OMIM 535000). LHON is characterized by bilateral central vision loss that occurs with a delayed onset, is predominant in males (but no X-linked genes have been implicated so far), and may be followed by a recovery after many months. Although a description of the disease had been in the medical literature for over 100 years, the molecular-genetic characterization of LHON became a milestone in the history of mitochondrial diseases when Wallace and colleagues identiﬁed a point mutation in the mitochondrial ND4 gene encoding a subunit of complex I of the electron transport chain. Many examples have been described since then. Ninety percent of all cases involve mutations in mitochondrial complex I genes (ND4, ND1, ND6; 3 primary mutations at basepairs 11778, 3460, and 14484), but mutations aﬀecting complex III, complex IV, and complex V have also been reported. A precise genotyping of aﬀected individuals is important for a prognosis of recovery, ranging from 4% of np 11778 patients showing recovery an average of 36 months after onset to 37% of np 14484 patients recovering after 16 months [137]. Homoplasmy with respect to the LHON mutation is observed in the majority of cases, but this is the exception rather than the rule for mitochondrial diseases. Such a homoplasmic mutant condition is possible, because the mutations are in coding regions causing missense substitutions of amino acids. A homogeneous population of complexes with reduced activity is present. An alternate explanation could be that the assembly of the complex is less eﬃcient. In other words, there could be a reduction in speciﬁc activity as well as a reduction in the amount of complex. The observed homoplasmy raises another issue. A delayed onset of a disease could in principle be explained by an increase in the fraction of mutated mtDNAs in certain somatic cells with age. When homoplasmy is present from birth, other changes with age must be invoked. What are they? One of the most puzzling and provocative aspects of LHON is the observation that in most LHON patients’ vision loss is the primary and often the only clinical manifestation of the disease. Other LHON pedigrees have been described with individuals suﬀering from one or more of a variety of more or less severe symptoms, such as cardiac conduction defects, minor neurological problems, including altered reﬂexes, ataxia, and sensory neuropathy, as well as skeletal abnormalities, dysarthria, deafness, ataxia, tremor, posterior column dysfunction, corticospinal trait dysfunction, and more (OMIM 535000). When OXPHOS activity (respiration measured by polarography) or complex I activity were measured in accessible tissues, severe reductions (50–80%) were observed in muscle tissue, lymphoblasts, or platelets. Why does the pathological eﬀect predominate in the optic nerve? Histopathological investigations have demonstrated selective but extensive losses of central axons in the optic nerve ﬁbers. This selective loss of smaller ﬁbers can provide some explanation of the clinical features, but it does not explain why these neurons are particularly vulnerable to cell death (apoptosis) induced by complex I dysfunction. It is perhaps signiﬁcant that these neurons have some of the highest densities of mitochondria observed of any tissue. Mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes, or MELAS (OMIM 540000) has a delayed onset, and symptoms include migraine head-

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aches, vomiting, seizures, mental retardation, strokes, hearing loss, exercise intolerance, and others. The most common mutation is the A3243G mutation in the tRNALeu gene, but mutations in other tRNA genes (Val, Ser, Phe) and in the COX3 gene can lead to the same symptoms. The nucleotide substitution is in the DHU loop of tRNALeu. Earlier speculations had suggested problems in transcription termination (the factor mTERF binds in this region on mtDNA) or a partially defective processing step from the observation of an incompletely processed transcript including the tRNALeu. However, recent results [138] have indicated that the mutation causes an amino acylation deﬁciency and hence a slow-down in the initiation of translation. At a suﬃciently high level of heteroplasmy, one would expect a reduction in all of the 13 proteins encoded by mtDNA and a reduction in the levels of complexes I, III, IV, and V. The decreased respiration and compensatory increase in glycolysis can account for lactic acidosis. Myoclonic epilepsy, ragged red ﬁbers (MERRF) (OMIM 545000) has been found in heteroplasmic individuals with mutations in tRNALys, tRNASer, and tRNALeu (C3256T). Not surprisingly, the clinical picture shows overlaps with the MELAS syndrome. In vitro studies have shown that mutations have severe eﬀects on mitochondrial protein synthesis with a corresponding reduction in the activity of the electron transport chain and the ATP synthase. Variable heteroplasmy is clearly responsible for the variable severity of the symptoms. However, the variation exists not only between diﬀerent patients, but apparently between cells and tissues in a single patient. When skeletal muscle ﬁbers are analyzed by histochemical methods, cytochrome oxidase activity is found to be distributed nonrandomly. Subpopulations of ﬁbers diﬀer in the fractions of mutated mtDNA, and further variation can be anticipated with time (age). NARP (neuropathy, ataxia, retinitis pigmentosa; OMIM 551500) includes symptoms such as sensory neuropathy, ataxia, retinitis pigmentosa, dementia, seizures, and developmental defects. A point mutation (T8993G/C) in the ATPase 6 subunit is responsible when present in 70–80% of the mtDNAs. This subunit (subunit ‘a’ in biochemical terminology) forms part of the Fo subcomplex of the ATP synthase. The mutation replaces a highly conserved leucine with an arginine, and it is speculated that this new charge interferes with the function of the proton channel of complex V. Lower levels of heteroplasmy are usually observed in NARP, while higher levels (often >60–80%) are typically associated with the Leigh syndrome presentation. Mild symptoms may appear as night blindness. The disease is generally progressive, and depending on the heteroplasmy, symptoms may appear shortly after birth or signiﬁcantly later in life. In one of the earliest families described there was no histological evidence of muscle myopathy, and muscle weakness was assumed to be neurogenic. Although mitochondrial DNA replication is relaxed, i.e., loosely coupled to nuclear DNA replication, the copy number of mtDNA molecules per cell must be controlled by a mechanism that may be diﬀerent in dividing and nondividing cells. This mtDNA maintenance must depend on a number of enzymes encoded by nuclear genes. Although rare, human patients have been found during the past decade in which this mechanism is disturbed. They have been classiﬁed into two major groups: disorders resulting from mtDNA depletions and disorders with multiple mitochondrial deletions [139]. They can be discussed under one heading, if one considers both maintenance of copy number and maintenance of mtDNA integrity. In the deletion syndromes, multiple large-scale deletions of mtDNA can be detected on Southern blots with DNA from selected tissue such as muscle. It has been

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suggested that such deletions arise as somatic mutations that accumulate in nondividing tissues, since they are absent in cultured cells derived from the patients, and they are also not found in leukocytes. Two syndromes have been characterized based on typical characteristics. Autosomal dominant progressive external ophthalmoplegia (adPEO) is frequently associated with a range of problems (exercise intolerance, ataxia, major depression, hearing loss, cardiomyopathy, Wolfram syndrome, etc.). The symptoms appear between 18 to 40 years when the proportion of deleted mtDNA reaches 60% in basal ganglia and cerebral cortex and >40% in skeletal and ocular muscles. Linkage analyses have implicated several loci in diﬀerent families, and the most complete analysis suggests that mutations in the adenine nucleotide translocator 1 (ANT1 at the adPEO locus on chromosome 4) are responsible. A second autosomal recessively inherited disorder is mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), with an age of onset ranging from 5 months to greater than 40 years. It is caused by mutations in the thymidine phosphorylase (TP) gene, a conclusion that is supported by the ﬁnding of 16 diﬀerent mutations in this gene in MNGIE pedigrees from diverse ethnic groups [140–142]. The most plausible explanation of the origin of the observed deletions is to suggest errors in mtDNA replication due to deﬁciencies in mitochondrial dNTP pools [139], but the precise role of the ANT1 in establishment of these pools is not yet completely clear. The genetic and biochemical mechanisms leading to the mtDNA depletion syndrome are still more mysterious. Children are born healthy but develop severe hepatopathy, myopathy, nephropathy, or encephalopathy from a tissue-speciﬁc loss of mtDNA down to levels of f10% of controls. Diﬀerent members of the same family may have diﬀerential tissue involvement [139]. Such an observation would suggest a mtDNA mutation, but none have been found, and no cases of maternal inheritance have been reported. Parents have all been normal, consistent with an autosomal recessive mutation. Additional pedigrees must be found to strengthen this conclusion. The treatment of HIV-infected patients with drugs such as zidovudine (nucleoside analogues) has been shown to lead to mtDNA depletion in various tissues. The mitochondrial DNA polymerase g is a known target for such drugs, but the inhibition is reversible and the mtDNA population recovers after withdrawal of the drug. The genes for polymerase g as well as genes for other proteins involved in replication (mtTFA, endonuclease G, mitochondrial single-strand binding protein) have been examined in one family without yielding a clue about the cause. C.

Testing of Mitochondrial Mutations in Cybrids

A potential complication that has challenged investigators in the biochemical analysis of cells os tissues from individuals with mitochondrial diseases has been the consideration of the diverse genetic background contributed by nuclear genes. To compare mitochondrial activities from normal and mutant mitochondria a constant nuclear background would be desirable, and this can in principle be achieved by the formation of cybrids from enucleated cells contributing the normal or mutant mitochondria and human Uj cells that have no mtDNA. The latter can be obtained by prolonged treatment of cells with sublethal doses of ethidium bromide or some other inhibitor of mitochondrial DNA replication [143–145]. Instead of enucleating the mitochondrial donor cells, one can in principle also use platelets or synaptosomes (e.g., Ref. 146). The latter may not be easily available from live donors.

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The approach is not entirely without pitfalls. Depending on the methods used for enucleation, the reconstituted cybrids may have a variable number of mitochondria. The Uj (mtDNA-) cells are frequently heteroploid established cell lines, and individual cybrid clones derived from them may not have a constant nuclear genetic constitution. Nevertheless, such experiments have proved to be very useful, and it should perhaps be pointed out that experiments of this kind were the ﬁrst to provide experimental proof for ‘‘cytoplasmic mutations,’’ i.e., the existence of a phenotype that could be transmitted by means of the cytoplasm (mitochondria). In the case of cybrids derived with mitochondria from a patient, such experiments are proof that the mitochondrial point mutation is indeed responsible for a reduced OXPHOS capacity, independently of a demonstration of maternal transmission in a pedigree. D.

Mouse Models for Mitochondrial Disease

As can be expected, this is a growth industry. Two types of models should be distinguished both in methodology and in the expected expression of a disease. Knockout technology starting with embryonic stem cells is well established and almost routine. The challenge is to knock out interesting genes and to get novel and informative results. It is possible that defects become apparent already in heterozygotes (+/) if the gene dose is critical, but in most examples the homozygous mutants (/) have to be produced. In the latter case it may not be surprising to ﬁnd that the mutations are lethal in the embryo, as exempliﬁed by the cytochrome c knock-outs [124]. The more noteworthy fact is that these mouse embryos develop in utero up to mid-gestation, and viable cell lines can be derived from them. Thus, oxidative phosphorylation may not be essential for early embryonic development, but it remains to be seen whether the block occurs at the same stage in similar knock-outs. Interesting complications and loss of predictability will arise when multiple isoforms of a protein exist. For example, Ant1* (/) mice are born alive but develop a variety of symptoms typical of mitochondrial diseases [147,148]. ANT2, 3, etc. may take over the essential role of ADP/ATP exchange in mitochondria in many tissues. Knock-outs of the mtTFA are lethal as expected, but conditional, tissue-speciﬁc inactivation of the Tfam gene gave variable results that were perhaps not very surprising (e.g., cardiomyopathy) [149,150]. The role of the uncoupling proteins (UCP) is of great interest. There are multiple isozymes, and Ucp1, Ucp2, and Ucp3 had to be inactivated to observe physiological eﬀects, for example, the dependence of ROS production on DC [154]. Such mice promise to be good model systems to study the role of ROS in senescence. Similarly, mice in which the scavenging enzymes glutathione peroxidase (Gpx1) and Mn–superoxide dismutase 2 (Sod2) have been knocked out have revealed the importance of ROS in aging and accompanying degenerative diseases. Many more examples could be mentioned, and many can be expected in the future. It is appropriate here to reemphasize that many mitochondrial diseases arising from nuclear mutations result from missense mutations and hence partially active proteins. Therefore, for many nuclear genes such as those for complex I, it will be necessary to identify such mutations ﬁrst (in tissue culture?), and then mice can be produced by the related ‘‘knock-in’’ technology, i.e., the replacement of the appropriate wild-type exon with a mutated version of the same exon. *The nomenclature for human and mouse genes is generally similar, except that capital letters are used to

designate human genes, while for mouse (wild type) genes only the ﬁrst letter is capitalized.

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Unless knock-outs are made that can be controlled with respect to timing (after birth) and tissue, one should consider that all tissues will be aﬀected in the same manner as in humans with nuclear complex I mutations, for example. Nevertheless, one may ﬁnd diﬀerences between mouse and human model systems. One of the most important puzzles and challenges in mitochondrial diseases is to understand the population genetics and dynamics of mtDNAs in diverse somatic tissues during development and aging. This challenge includes the study of the ﬂuctuations in heteroplasmy observed from one generation to another. Clearly, a mouse model would permit experiments and analyses that are impossible in humans. Since most laboratory mouse strains had been bred to be genetically homogeneous, few mutations and polymorphisms on mtDNA were available and heteroplasmic mice were not available. This situation has dramatically changed recently by the successful introduction of technology allowing the production of heteroplasmic mice [151–154]. Two general approaches have been found to be successful: (1) to fuse enucleated cytoplasts with mutant mtDNA to female stem cells and injection of the stem cell cybrids into blastocysts and (2) to fuse such cytoplasts directly to single-cell embryos (zygotes). The ﬁrst results on transmission of the mutated mtDNA to descendents are being published [154], and a full exploration of these interesting mice can be expected in the near future.

IV.

DIAGNOSIS AND INTERVENTION

It has become clear that an increasingly large and diverse number of symptoms of variable severity aﬀecting one or more organs, but primarily the nervous system and muscles can now be ‘‘explained’’ on the basis of a deﬁciency in the capacity for oxidative phosphorylation. Such symptoms should alert physicians to examine the state of mitochondria and mitochondrial DNA. Large deletions in mtDNA can be found eﬃciently, but biochemical assays of the activities of the individual complexes may be required in most instances to establish the precise defect and the location of the mutation. Both nuclear and mitochondrial genes should be considered; in optimal circumstances information from a pedigree will help distinguish between maternal inheritance and autosomal recessive mutations. As the diagnosis and establishment of the speciﬁc cause for the symptoms become more deﬁnitive, better prognoses can be made about the progression and management of the disease, and in many instances genetic counselors will be able to give more reliable estimates of risk in future pregnancies. It may ultimately become feasible to ameliorate diseases with pharmacological interventions or even gene therapy (for nuclear genes?). However, at this time treatments with ubiquinone, creatine, and other metabolites have not been notably successful.

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12 Oxidative and Nitrosative Stress in Cystic Fibrosis: Significance or Triviality? BRIAN M. MORRISSEY, JASON EISERICH, and CARROLL E. CROSS University of California, Davis, School of Medicine, Sacramento, California, U.S.A. ALBERT VAN DER VLIET University of Vermont College of Medicine, Burlington, Vermont, U.S.A.

I.

INTRODUCTION

Cystic ﬁbrosis (CF), one of the most common of the lethal autosomal recessive diseases in Caucasian populations, occurs with varying frequency in all ethnic groups and geographic locations. Its highest incidence is in individuals of northern European heritage, aﬄicting approximately 1 in every 2500 Caucasians in the United States [1]. CF is a generalized multi-organ system disease arising from a biochemical abnormality in a protein (the cystic ﬁbrosis transmembrane conductance regulator, CFTR) that is encoded by a 250 kb gene located on chromosome 7 (band q 31–32). Over 900 diseasecausing mutations of CFTR have been described,* but the most common is the deletion of phenylalanine at position 508 of the 1480-residue protein. CFTR appears to couple ATP hydrolysis with transmembrane chloride conductance across apical epithelial surfaces under control of cAMP-dependent protein kinase A regulation [2], but likely has numerous other physiological functions that remain to be fully described

* See the CFTR mutation database at http://www.genet.sickkids.on.ca/cftr/.

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[3–5]. These include its conductance of other anions (e.g., ATP [6]), its regulation of other distinct ion channels [7], and its probable complex interactions with various anchor and scaﬀold proteins [8] and with its own membrane traﬃcking machinery [9]. The disease aﬀects primarily epithelial tissues. The defect in CFTR impairs the normal movement of water and electrolytes across these various epithelial surfaces, most clinically signiﬁcant in the respiratory tract (RT) [10], but also in the pancreatohepatobiliary system, the gastrointestinal tract, the genital tract, and the sweat gland excretion system. The precise roles that CFTR plays in abnormalities of epithelial mucin (glyco-conjugate) secretion [11,12], the hypersusceptibility to respiratory tract bacterial colonization and infection [13–18], and excessive inﬂammatory response to colonization and infection are still to be clariﬁed. RT abnormalities dominate the clinical manifestations of the defective CFTR. There is decreased chloride secretion and increased sodium absorption, leading to an inadequate hydration of the RT secretions [1,10]. This ‘‘underhydration’’ of the airways leads to a thickened mucus, impaired mucociliary clearance, and chronic lung infection. Another factor leading to the increased susceptibility of CF airways to infection is the possible increased salt concentration of the underhydrated CF airway surface ﬂuid. This limits the activation of certain antimicrobial defenses that are only active when airway salt concentration is low [19,20]. Both the underhydration and the possible increased salt concentration contribute to the disordered bacterial clearance, persistent infection, and inﬂammatory–immune system overexuberance. These result in a chronic neutrophil-dominant inﬂammation and lung destruction that is characteristic of CF [1,21]. The disease is punctuated by acute episodes of increased airway sepsis exacerbating the RT disease, ultimately leading to severe airway and surrounding lung parenchymal damage, respiratory insuﬃciency, and death. Especially in the early phases of the disease, the CF host inﬂammatory response appears ‘‘hyperactive’’ in relationship to the burden of the RT pathogens. The clinical evolution of CF appears closely related to a conﬁned state of airway inﬂammation [13,22]. This dysregulation of inﬂammatory and immune response also appears to be present both in CF cells and in the mouse model of CF [23,24]. It is still speculative whether aberrantly hyperactive pro-inﬂammatory cytokine responses play a key trigger role in energizing the overly exuberant inﬂammatory processes [13,21]. It has been hypothesized that decreases in hydrophilic surfactant proteins contribute to the relative ineﬀectiveness of cellular inﬂammatory responses to kill invading bacteria and the dysregulation of the inﬂammatory response in children with CF [18]. Since the discovery of the CF gene in 1989, scientiﬁc eﬀorts in CF have included corrections of the basic defect in CFTR expression in human cell lines by CFTR gene transfer [25], the development of mouse models for CF [26,27], gene transfer to transgenic mouse models [25] and in vivo gene transfer to the human nasal [28] and tracheobronchial epithelia [29,30]. However, there are still many obstacles to overcome before gene transfer therapy becomes a routine treatment in CF [30–32]. Although genetic treatments could be expected to restore deranged RT water and electrolyte homeostasis and prevent the sequence of airway colonization, infection, and injury, gene therapy is unlikely to reverse lung injury and disease progression in patients who already have extensive lung damage. The chronic infection and resulting inﬂammatory-immune processes in the lungs of CF patients cause both severe oxidant [33–37] and protease [38–41] stress; these latter stresses occur presumably because of imbalances in the protective oxidant/antioxidant and protease/antiprotease RT defense systems. Thus, both anti-

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oxidant [42,43] and antiprotease [40,44,45] approaches have been proposed for therapy in CF patients. In this chapter, we will discuss the evidence that oxidative stress occurs in CF and assess whether or not oxidative stress, and interrelated nitrosative stress, contribute to the disease pathology. Finally, we will speculate on whether or not therapeutic strategies directed at these stresses would be helpful in treatments of the disease. II.

THE ROLE OF OXYGEN-DERIVED SPECIES

The biomedical literature contains a multitude of claims that oxygen free radicals (e.g., O2. and OH.) and other oxygen-derived species (e.g., H2O2 and HOCl/OCl) are involved in a wide spectrum of human diseases [46]. Oxidative and more recently nitrosative stress have been implicated in many pathophysiological conditions ranging from cataracts to arthritis and from AIDS to age-related diseases such as cancer and neurodegenerative diseases. This wide range of implicated involvement strongly suggests that oxidative stress is common to most, if not all, human diseases characterized by cell injury and repair including activation of inﬂammatory–immune system processes. As illustrated in Figure 1, tissue injury increases free radical formation by such mechanisms as injury of mitochondria (so that they ‘‘leak’’ more single electrons to O2 to form O2), release of ‘‘catalytic’’ iron ions, release of heme proteins, and induction or activation of radical-generating systems (e.g., xanthine oxidase, lipoxygenases, cyclooxygenases, and nitric oxide synthase). This is accompanied by activation of complex interacting components of inﬂammatory-immune processes, including the releases of proinﬂammatory cytokines and chemokines. Phagocytes recruited and activated to the site of tissue injury represent an additional major source of oxidant (e.g., NADPH oxidase and myeloperoxidase)- and nitrosant (NO synthase)-generating systems. Although the occurrence and documentation of oxidative (or nitrosative) stress can

Figure 1

Generation of oxidants and (nitrosants) in response to tissue injury. The resulting oxidants (nitrosants) can induce biomolecular damage causing further tissue injury.

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often be easily demonstrated, it is in most cases unclear whether or not it causes or contributes signiﬁcantly to the tissue injury. In some diseases oxidative and/or nitrosative stress may be a major mechanism of injury, amenable to therapy. In other diseases, it may make no signiﬁcant contribution to disease pathology. Of course, exactly the same is true of other putative mediators of injury, such as prostaglandins, leukotrienes, interleukins, other cytokines, and chemokines. III.

EVIDENCE OF OXIDATIVE STRESS IN CF

The lungs are particularly prone to oxidative insult because the RT is exposed to the highest O2 tension of any body tissue and presents a very large surface area of contact with the environment and perfusing blood. They are continuously exposed to the oxidants of inhaled air and to bloodborne toxins. Additionally, oxidant products are generated by activated phagocytic cells, which are normally resident in the lung (e.g., pulmonary alveolar macrophages), and phagocytic cells recruited following lung injury (e.g., inﬁltrated neutrophils in cigarette smokers) [47]. Table 1 lists some of the lung disorders in which oxidative stress is believed to play a role. Several abnormalities predispose patients with CF to enhanced susceptibility to oxidative stress. These include altered gastrointestinal absorption of fat-soluble vitamins and micronutrients and increased activity of inﬂammatory processes. A list of the processes that could contribute to oxidative stress in CF is shown in Table 2. Once reactive oxidants (or nitrosants) are present, they can have numerous eﬀects on a variety of biomolecular species, as schematically shown in Figure 2. Numerous reports support the presence of RT or systemic oxidative stress in patients with CF. Evidence of oxidative stress in CF includes alleged increases in lipid, protein, and DNA oxidation, reduced antioxidant status, increased susceptibility of plasma lipoproteins to peroxidation, susceptibility of red blood cells to oxidation, and evidence of RT adaptive responses to oxidative stress (Table 3).

Table 1

Lung Conditions in Which Oxidative Stress Could Be Involved

Oxygen toxicity Asthma Adult respiratory distress syndrome Infant respiratory distress syndrome/bronchopulmonary dysplasia Cigarette smoke exposure Lung cancer Emphysema and/or chronic bronchitis (especially during exacerbations) Idiopathic pulmonary ﬁbrosis Immune complex–mediated lung injury HIV-associated lung disease Acid aspiration Environmental exposure to gaseous oxidant pollutants (e.g., O3, NO2, SO2, N2O5) Ischemia-reperfusion injury (e.g., as in post–lung transplant pulmonary edema) Mineral dust and ﬁber exposures, including asbestos and industrial and urban particulates Drug toxicity (e.g., bleomycin, paraquat, BCNU) Radiation toxicity Lung transplantation (especially during infections, rejection) Cystic ﬁbrosis

Oxidative and Nitrosative Stress in CF

Table 2

267

Potential Contributors to Systemic Oxidative Stress in Cystic Fibrosis

Increased metabolic rate (particularly in later stages of the disease) Increased cytochrome P-450 oxidative reactions Phagocyte hyperresponsiveness and altered neutrophil oxidative activity Chronic infection, inﬂammation, and increased immune responses Increased amounts of TNF-a and IL-1, which can themselves induce intracellular oxidative stress Fat malabsorption leading to decreased levels of fat-soluble antioxidant micronutrients (e.g., vitamin E and carotenoids) Probable increased turnover of antioxidant micronutrients Disordered iron metabolism (especially in the lung) Altered respiratory epithelial GSH transport

Some of these eﬀects, such as the increased susceptibility of plasma lipoproteins to peroxidation [76] and the increased susceptibility of red blood cells to peroxideinduced hemolysis in vitro [64,69], were partially reversible by dietary supplementation with vitamin E [64]. Interestingly, red blood cell glutathione (GSH), superoxide dismutase (SOD), and catalase levels are all reportedly increased in CF [90,91] although a more recent study has reported normal red blood cell SOD levels [69]. Several groups have reported decreased red blood cell GSH peroxidase levels, presumably secondary to selenium deﬁciency [35,67–69]. These ﬁndings need further validation and characterization in so far as they may contribute to the mild hemolysis known to occur in CF patients [69]. Other forms of evidence for a role of oxidative stress in CF include (1) elevated breath pentane levels [78,79], an arguable ‘‘marker’’ of increased lipid peroxidation [80,81], (2) increased exhaled and/or plasma 8-isoprostane levels [82,83], an excellent ‘‘marker’’ of lipid peroxidation (although a challenging analytical technology) [92,93], and (3) decreased GSH levels in bronchoalveolar lavage ﬂuid [43] and lymphocytes [66] but not in red blood cells [94]. Observed increased levels of the enzyme myeloperoxidase (MPO) in RT secretions [51–54] are presumably secondary to the large numbers of neutrophils present in the RT of CF patients [49]. MPO uses H2O2 to oxidize Cl- and Br- to generate cytotoxic hypohalous acids that can inactivate a1-antiprotease [39,40]. Hypochlorous acid (HOCI/OCI), a powerful oxidizing and chlorinating agent, depletes ascorbate and —SH- containing compounds [95,96], presumably including those on cell surfaces, causing cellular damage. The increased activity of MPO in CF

Figure 2

Spectrum of biological effects induced by oxidative (or nitrosative) stress.

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Table 3

Morrissey et al. Reported Indices of Increased Oxidative Stress in CF

Activated oxidative/peroxidative processes Increased neutrophil and monocyte numbers and oxidase activities [48–50] Increases in myeloperoxidase in RT sputum [51–54] Elevated RT RNS levels during exacerbations [55] Increased neutrophil-derived long-lived oxidants in sputum [56] Increased ‘‘free’’ iron, catalytic for free radical reactions, in RT sputum [57–59] Increased concentrations of iron and isoferrins in lower respiratory tract [60] Abnormal GSH transport in airway epithelia [61,62] Increased amounts of pro-oxidant cytokines (e.g., TNFa, IL-1, IL-6) [13,50,63] Indices of antioxidant depletion Increased susceptibility of RBC to peroxide-induced hemolysis [64] Decreased GSH levels in bronchoalveolar lavage [43] Decreased S-nitrosothiol levels in bronchoalveolar lavage [65] Altered concentrations of lymphocyte GSH [66] Decreases in plasma selenium (and/or plasma and RBC GSH peroxidase) levels [35,67–69] Decreases in plasma ascorbic acid and a-tocopherol levels in some patients [34,36,68–70] Decreased plasma h-carotene levels in many patients [34,35,69,70]a Indices of lipid peroxidation Elevated plasma TBARS level [35,69]b Elevated levels of plasma malondialdehyde [36,74,75] Elevated levels of plasma organic hydroperoxides [35] Elevated levels of antibodies against oxidized low-density lipoproteins [69] Increased susceptibility of lipoproteins to peroxidation [76] Increased plasma and tissue 9–11 dioenoic acid levels [33]c Elevated breath pentane and/or ethane levels [78,79]d Increased isoprostane levels [82,83] Indices of protein modiﬁcation/oxidation Decreased plasma protein sulfhydryl levels [36] Increased levels of plasma carbonyls [36] Evidence of protein oxidation in sputum [40] Increased total amount of nitrated protein in sputum and serum [54,84,85] Increased protein tyrosine oxidation and chlorination [54] Indices of DNA oxidation Increased oxidative damage to DNA [86] Indices of oxidative responses to oxidant/nitrosants Increased carbon monoxide in exhaled air [87,88]e Increased heme oxygenase-1 in lungs with advanced CF [89] a It must not be assumed that any beneﬁcial eﬀects or carotenoids in CF are necessarily related to antioxidant eﬀects [71]. b Often measured by nonspeciﬁc assays [72,73]. c Putative product of FR attack on lipids, but can often result from bacterial metabolism and/or diet [77]. d Most early assays of pentane exhalation in human breath have been performed inaccurately [80], it seems that the measurement of both pentane and ethane would be more desirable [81]. e Increased carbon monoxide presumed to be a result of oxidant-induced increases in heme oxygenase activity in RT cells.

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sputum also coincides with increased levels of chloramines (formed by reaction of HOCI/OCI- with amino compounds such as taurine) [56] and 3-chlorotyrosine, a stable end product of MPO-dependent protein chlorination [54]. H2O2 does not appear to be increased and may be lower in breath condensate of CF patients [53,97], probably because of the increases in H2O2-metabolizing pathways (e.g., myeloperoxidase, catalase) present in RT secretions of CF patients [51–54,97]. Inactivation of a1-antiprotease by MPO-generated hypohalous acids likely leads to increased protease activity. Among its damaging eﬀects, the augmented protease activity could degrade various iron-containing proteins present in CF lung secretions, including the endogenous transferrin and secreted lactoferrin [58,59]. Bacterial siderophores also contribute to iron sequestration within RT secretions of CF patients [57]. Most important are probably the siderophores of Pseudomonas aeruginosa, the pathogen most frequently present in adults with CF. These siderophores are part of their iron-acquisition system and may directly relate to their virulence [98,99]. At sites of microbial accumulation, bacterial products, neutrophil proteases, and oxidant species could be expected to break down endogenous and exogenous iron chelators [100,101]. This would increase catalytically active iron that is capable of reducing H2O2 to highly reactiveOH [58]. The increased levels of ferritin in CF sputum may contribute to this reduction [102]. In addition, CF patients have been shown to have increases of pro-inﬂammatory and pro-oxidant cytokines, such as TNF-a, 1L-1, and IL-8, in RT secretions and, to a lesser extent, in blood [13,63]. NFnB, an important transcription regulator of the pro-inﬂammatory cytokines, may cause their up-regulation [103]. These cytokines can stimulate phagocyte oxidant production (e.g., by ‘‘priming’’ their NADPH oxidase–related mechanisms responsible for oxidant generation) [104]. Recruitment and activation of phagocytes (largely neutrophils but also eosinophils) play important pathogenic roles in the course of inﬂammatory airway diseases such as chronic obstructive pulmonary disease (COPD), bronchial asthma, and CF [105]. A provocative ﬁnding in CF patients is the report that their monocyte-derived macrophages appear to have an increased rate of TNF-a gene transcription [104]. TNF-a plays a fundamental role in the control of neutrophil movement by virtue of eﬀects on the regulation of chemokine expression, which itself can induce lung inﬂammation [105]. This could be one mechanism accounting for the overexuberance of the RT inﬂammatory response in CF [106]. A pathological regulation of the release of arachidonic acid has been described in CF [107]. This, too, could contribute to the enhanced inﬂammatory-immune state seen in CF airways. Neutrophil antimicrobial mechanisms may be compromised in CF [108,109]. As neutrophils appear to express CFTR message [110], it is possible that abnormalities in this protein aﬀect neutrophil function. Indeed, neutrophils of CF homozygotes and heterozygotes have elevated intracellular MPO activity, based on limited luminal oxidation [16,111]. Furthermore, inhibition of Na+/H+ exchange by amiloride corrects this abnormality, suggesting that alterations in cytosolic or phagosomal pH aﬀect neutrophil function [16]. In select patients with CF, decreases in ascorbic acid and vitamin E have been reported [34,68,76]. The decreases in a-tocopherol are of special interest because many investigators have shown that decreased levels of vitamin E potentiate lung inﬂammatory and injury [112], including several models of oxidant-induced damage [113]. Of relevance, vitamin E appears to downregulate O2. production in human phagocytes by impairing activation of their NADPH oxidase [114].

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Table 4 Levels of Selected Plasma Lipophilic Antioxidants in CF Patients and Controls

a-Tocopherol (vitamin E) h-Carotene a-Carotene Lutein Lycopene Retinol (vitamin A)

CF patients (AM)

Controls (AM)

15 0.04 0.01 0.08 0.08 0.97

18 0.20 0.03 0.38 0.80 1.42

Values listed are means and not corrected for LDL, cholesterol, or lipid levels. Source: Adapted from Ref. 70.

Finally, markedly decreased amounts of h-carotene are present in almost all CF patients with active disease not taking supplements (Table 4) [34,35]. This deﬁciency appears to extend to several diﬀerent plasma carotenoids and appears more severe than the deﬁciencies in vitamins A and E [70]. The deﬁciency in h-carotene is particularly intriguing as it can exert antioxidant eﬀects in lipid systems under certain well-deﬁned conditions [71,115] and its supplementation in patients with CF seems to decrease their levels of lipid peroxidation as reﬂected by plasma malondialdehyde concentrations [74]. The decreased h-carotene levels seen in CF cannot easily be explained by decreased intake or decreased fat absorption alone [34] and may also relate to an increased consumption and turnover of carotenoids, possibly due to their reaction with O2-derived species (analogous to the situation that has been proposed for cigarette smokers) [116]. Future studies are needed to address the pharmacokinetics of h-carotene in CF patients, its possible role in retinol metabolism (especially in the RT [117]), and the eﬃcacy of its supplementation. IV.

OXIDATIVE STRESS AT THE CF AIRWAY SURFACE

A major part of the pulmonary pathophysiology of CF takes place at the airway surface. There is chronic infection and inﬂammation: large numbers of bacteria (especially Pseudomonas spp.), 1000-fold increased numbers of neutrophils, and altered respiratory tract lining ﬂuids (RTLFs) [49]. Many of the neutrophils are activated, releasing proteases and a range of reactive oxygen-derived and nitrogenderived species, including O2., H2O2, HOCI/OCI, NO, NO2, and ONOO.* Of particular relevance to CF pathobiology, the conversion of Pseudomonas species from nonmucoid to mucoid strains may be inﬂuenced by local concentrations of hydrogen peroxide [118]. If a signiﬁcant part of the RT damage in CF is indeed mediated by oxidative and/ or nitrosative stress and related phagocyte-induced injury, then augmentation of RT antioxidants could be used as a treatment [42,50]. Various eﬀorts to characterize the antioxidant composition of RT surface ﬂuids of normal healthy individuals have

* At physiological pH, peroxynitrite (ONOO-) coexists with its conjugated acid peroxynitrous acid (ONOOH), which may be responsible for many of the oxidative properties of peroxynitrite. Although the term ONOO- is used throughout this paper, either form of peroxynitrite may be implicated.

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Table 5

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Antioxidant Species in Respiratory Tract Lining Fluids

Low-molecular-mass antioxidants (e.g., uric acid, GSH, ascorbic acid, tocopherols, carotenoids) Metal-binding proteins (e.g., lactoferrin) Extracellular antioxidant proteins (e.g., extracellular superoxide dismutase and extracellular glutathione peroxidase) Small quantities of antioxidant enzymes ‘‘leaking’’ from RTECs and inﬂammatory cells (e.g., intracellular catalase, glutathione peroxidase, superoxide dismutase) ‘‘Sacriﬁcial’’ reactive proteins and unsaturated lipids Antioxidant proteins diﬀusing from plasma (e.g., albumin, transferrin, ceruloplasm) Mucus including various species of secreted glycoconjugates (esp. mucin)

indicated that local extracellular antioxidant levels [119,120] are, in general, similar to those present in other extracellular ﬂuids [121,122] (Table 5). However, there are several other signiﬁcant diﬀerences. For example, considerably more GSH is present when one compares levels present in distal RTLFs (also called epithelial lining ﬂuids, ELF) to levels present in plasma (Table 6). Such a comparison is somewhat diﬃcult, because performing back-calculations of levels measured in airway or bronchoalveolar lining ﬂuids to those actually present in the RTLFs in vivo makes several assumptions, the most important of which involve the uncertainties of the various methods used to calculate the resident RTLF volume [119,123]. Nonetheless, it is clear that most antioxidants, including protein antioxidants (such as protein-SH), are present in much smaller amounts in distal RTLFs than in plasma. There are few if any accurate estimates of the full spectrum of antioxidant concentrations in upper RTLFs. Another major diﬀerence between plasma and upper RTLFs is the presence of mucus, which may be an important antioxidant in both the respiratory and gastrointestinal tracts [124,125]. Several mucus constituents (e.g., carbohydrates) are powerful scavengers of oxygen-derived species, such as .OH. Mucus contains an abundance of protein thiols and disulﬁde bonds [126] both of which would be excellent scavengers of .OH, ONOO, and HOCI/OCI [127]. Other features of RTLFs include the presence of uric acid, an important antioxidant, which may be secreted with mucus in the upper airways [128]. The presence of GSH in lower RTLFs at levels exceeding those of plasma suggests the possibility of active RT antioxidant secretory or regeneratory mechanisms by lower RTECs. Also important is the presence of high

Table 6

Antioxidant Species in Plasma and in Lower RTLFs

Antioxidant Ascorbic acid Glutathione Uric acid a-Tocopherol Albumin-SH

Plasma (AM) 67 F 25 1.0 F 0.7 387 F 132 16 F 5 500

Source: Adapted from Ref. 123.

Lower RTLFs (AM) 40 109 207 0.7

F 18 F 64 F 167 F 0.3 70

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concentrations of the iron-binding protein lactoferrin in the upper RTLFs, which, like uric acid, is actively secreted by some populations of upper RTECs cells [129]. Iron species bound to lactoferrin are incapable of catalyzing free radical reactions [127] but may be a source of catalytic iron in CF because of oxidant/proteolytic degradation (see earlier discussion). Ceruloplasmin and transferrin are present in RTLFs, but at much lower concentrations than in plasma [130]. Interestingly, the concentrations of GSH in CF RTLFs are greatly reduced [43], perhaps due to altered cellular transport [131]. All of these may worsen the oxidative stress that results from the intense inﬂammatory– immune cell activations that occur in the CF lung (see below). V.

POTENTIAL ROLE OF NITRIC OXIDE AND RELATED REACTIVE NITROGEN SPECIES IN CF

Nitric oxide (NO) plays an important role in biological systems as a cell-signaling molecule, anti-infective agent, and, as most recently recognized, an antioxidant [132]. The metabolic fate of NO gives rise to a further series of compounds, collectively known as reactive nitrogen species (RNS) and including the species responsible for the nitration (addition of NO2) and nitrosylation (addition of NO) of most classes of biomolecules. NO is produced constitutively in the lung, as evidenced by its detection in the expired air of healthy humans [133]. It is believed to play an important role in several aspects of normal lung biology, most notably in pulmonary vascular responses and host defense mechanisms against infection [134]. NO is produced by the action on Larginine of nitric oxide synthase (NOS), which has three isoforms. NOS-1 and NOS-3 are calcium-dependent, whereas NOS-2 is calcium independent and is produced primarily within neutrophils, macrophages, and epithelial cells following stimulation within an inﬂammatory milieu of microorganisms and cytokines. Increased NO is present in exhaled breath in several RT diseases where active inﬂammatory processes are present [133]. For example, increased levels of NO in the expired breath have been reported in patients suﬀering from asthma, chronic bronchitis, bronchiectasis, COPD, sepsis, and in normal subjects with upper RT infections [133,135–140]. In striking contrast to these observations, NO levels in the expired breath of patients with stable CF are reduced [141], as are NO levels in nasal airways of CF patients [142]. A related ﬁnding is that levels of RT S-nitrosothiol, a naturally occurring bronchodilator representing a potential longer-term reservoir of RT NO, are also decreased in CF [65]. As CF airways are characterized by increased numbers of bacteria and an active inﬂammatory-immune response, it would be expected that NOS-2 expression in epithelial cells would be upregulated, presumably to protect the lung [143]. However, this has recently been shown not to be the case [144–146]. It is now known from in vitro studies that loss of human epithelial cell CFTR activity reduces NOS-2 messenger RNA expression and reduces overall epithelial cell NO production [147]. That NO metabolites (e.g., NO2, NO3) in sputum appear to increase during CF exacerbations [55] suggests that inﬂammatory cell NO production may be intact in CF. However, other workers have not been able to conﬁrm that sputum NO metabolites represent a useful marker of the degree of airway infection/inﬂammation in CF patients [148]. Indirect evidence suggests that metabolism of NO within airway ﬂuids could explain at least some of the decrease in the expired NO seen in CF patients [85]. The degree to which abnormalities in CF RT NO metabolism aﬀect airway physiology remains uncertain.

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As emphasized elsewhere [149], the lack of epithelial NOS-2 in CF airways can be expected to compromise defenses against microbial pathogens at the RT surface [150]. NO appears to reduce the sequestration of activated neutrophils into the lung and to diminish lung macrophage inﬂammatory cytokine production [151], possibly in part by downregulation of the redox-sensitive transcription factor NF-nB [152]. The absence of epithelial NO may underlie the excessive transmigration and traﬃcking of neutrophils into the CF airway lumen in response to the presence of airway pathogens. However, there is an unexplained possible paradox in this rationale, as it is often claimed that overproduction of NO and other RNS may augment the inﬂammatory processes in numerous RT inﬂammatory diseases. This is mostly supported by the detection of nitrated protein at the site of pathobiology, which is indicative of pro-oxidant (and perhaps pro-inﬂammatory) actions of NO [153]. Nitrated protein has also been found to be present in the RT of CF patients [54,84,154]. Of relevance, the increased levels of neutrophil myeloperoxidase [51–54] and eosinophil peroxidase [52,155], known to be present at high levels within CF airway lumens, represent strong oxidizing and nitrating systems and are known to interact with active inﬂammatory–immune cell local productions of NO [156–159]. An interesting observation is that inhalation and infusion of L-arginine appear to increase exhaled NO concentrations in CF patients [160,161]. Evidence that NO plays a role in CFTR expression [162], airway glycoconjugate secretion [163], and neutrophil recruitment [164,165] suggests NO’s relevance in CF. NO has many helpful physiological properties [134] and may be protective towards RTLF lipids and RTEC membranes by acting as an inhibitor of radical chain propagation reactions [166]. However, excesses are generally thought to be harmful [167]. Of note, it has been observed that inhaled NO at either physiological levels present in normal individuals or at levels used therapeutically to treat pulmonary hypertension has no eﬀect on bronchomotor tone in patients with CF [168]. One model of immune complex–mediated lung injury is ameliorated by inhibition of NO formation [169]. This is signiﬁcant because immune complex depositions are present in airways of CF patients [170]. The pro-oxidant eﬀects of NO are usually ascribed to its conversion to more reactive metabolites by (auto)oxidation or by reactions with O2.to form peroxynitrite (ONOO). ONOO is a powerful oxidant that also nitrates proteins [171–173]. Its ability to nitrate tyrosine residues and yield nitrotyrosine has received much recent attention. This stable protein modiﬁcation is often monitored as an indicator of NOmediated oxidative/nitrosative stress. However, other mechanisms besides ONOO formation, including peroxidase-dependent pathways, can contribute to tyrosine nitration. This argument is strengthened by the ﬁnding of high levels of nitrotyrosine in a mouse model of asthma, which was markedly reduced in asthmatic mice with eosinophil peroxidase deﬁciency [174] and may be of relevance in CF because of the increased degranulation of peroxidases in this condition [155]. Thus, nitrotyrosine should be regarded as a collective biomarker of reactions involving RNS [153] but, importantly, may or may not be involved in the pathobiology of the disease. We have recently detected levels of 3-nitrotyrosine, 3-chlorotyrosine, and dityrosine in expectorated sputum of adult CF patients [54] (Fig. 3). These protein modiﬁcations can be brought about by multiple (inﬂammatory) oxidants and nitrosants. However, the presence of dityrosine, especially chlorotyrosine, strongly implicates oxidative reactions involving heme peroxidases such as MPO. Dityrosine is a

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Figure 3 Activation of RT epithelial or inflammatory-immune cells by bacterial infection generates various oxidizing, nitrating, and chlorinating oxidants. Characteristic tyrosine modification products can be monitored as selective ‘‘biomarkers’’ of these oxidants.

common product of (myelo)peroxidase/H2O2 reactions with tyrosine [175,176]. HOCI/OCI produced by activated neutrophils typically chlorinates tyrosine residues [177,178]. Presumably, such (myelo)peroxidase-dependent mechanisms are also involved in nitration in CF. All three of these modiﬁed tyrosine products can be used as ‘‘markers’’ or ‘‘dosimeters’’ for the production of reactive nitrogen and reactive O2 species in CF airways. As such, these could determine the eﬀectiveness of antioxidants and other therapeutic strategies in ameliorating the oxidative component of tissue injury in CF. VI.

OTHER CONSIDERATIONS

Several other characteristics and respiratory tract components warrant consideration when discussing antioxidant and nitrosative stress. First is that of the RTLFs. RTLFs appear to contain higher concentrations of GSH than plasma. RTLF volumes are relatively small and, as is the case for all other RTLF antioxidants, little is known about GSH turnover [119,120]. The capacity of RTLF antioxidants to protect RTECs against inﬂammatory, oxidative, or nitrosative stresses remains uncertain. A further consideration is the degree to which cellular adhesion mechanisms may bring neutrophils (and bacteria) in direct apposition to RTECs. Close cellular adhesion would minimize the eﬀect of RTLF antioxidants to protect RTECs from phagocyte oxidants. This raises in importance the contributing role of lipophilic membrane antioxidants, such as vitamin E.

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A third consideration is that inﬂammation and its mediators activate RT neurohumoral mechanisms. These result in the entry of increasing amounts of plasma constituents, including plasma antioxidant proteins, into the RTLFs [179]. While this could limit airway gas conductance, it would clearly increase the extracellular antioxidants present in both airway lumens and airway walls. Finally, and particularly in the presence of airway inﬂammation, intracellular antioxidants released from shed RTECs or from the recruited airway inﬂammatory cells may well augment the measured RTLF antioxidant capabilities. This may include the intracellular leakage of superoxide dismutase, catalase, and glutathione peroxidase. These would augment the antioxidant capacities of extracellular superoxide dismutase and glutathione peroxidase, already known to be present at RT surfaces. VII.

THE POTENTIAL FOR ANTIOXIDANT THERAPY

Although administration of antioxidants to CF patients will not cure the patient, it may be beneﬁcial. Should aerosolized antioxidants, including genes encoding for antioxidant or anti-inﬂammatory defenses [180], be administered in conjunction with bronchodilators, aerosolized DNase, and other anti-inﬂammatory agents? Should CF patients be treated with pharmacological doses of antioxidants? Given the current limited eﬃciency of pharmacological CFTR channel–‘‘optimizing’’ agents, such as aerosolized uridine triphosphate and CFTR gene [28–31,181], low molecular weight antioxidants may have a more favorable cost-beneﬁt ratio. It is prudent to suggest that vitamins E and C carotenoids be administered orally in amounts that normalize or nearly normalize plasma levels in CF patients. The evidence is strongest for vitamin E. Experimental deﬁciencies of this antioxidant predispose the lung to oxidant injury [112]. However, we are not convinced that supernormal doses of such compounds should be administered, as eﬀects are still unknown and, at least for vitamin A and possibly h-carotene, could be potentially harmful [182]. It is unknown whether oral supplementation of these antioxidants will substantially increase their levels in RTLFs or RTECs, let alone what eﬀects they may have on pulmonary infection, inﬂammation, or function. For example, if antioxidants are to be aerosolized directly into the respiratory tract, how would Pseudomonas be aﬀected? Mucoid Pseudomonas phenotypes already secrete an antioxidant, their slimy alginate, which presumably protects them from neutrophil-derived oxidants [183]. Would aerosolized antioxidants protect the bacteria even more against the oxidant-generating antimicrobial capabilities of phagocytes? Administration of augmented amounts of iron chelator substances presents a similar enigma—would the augmented iron chelators, engineered so as to be resistant to proteolytic and oxidant destruction, be inhibitory toward bacterial growth or merely stimulate additional bacterial siderophore production and virulence [98]? Since oxidants play an important role in antimicrobial host defense, the augmenting of RTLF antioxidants might not strengthen overall RT defense capabilities. VIII.

THE DILEMMA OF ADMINISTERING AEROSOLIZED THIOLS OR ASCORBIC ACID

If ‘‘free’’ iron (e.g., Fe3+ and/or Fe2+) represents an important potential pro-oxidant constituent of CF RTLFs (see earlier discussion), then thiols and ascorbic acid could

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potentiate iron toxicity by keeping it reduced in the Fe2+ state. This would promote the highly pro-oxidant Fenton reaction, which generates the powerful oxidizing hydroxyl radical [46,184–186] from the abundant ROS being produced by activated neutrophils [187]. Reducing thiols such as GSH represent a second important risk. Certain free radicals may convert them to thiyl radicals, which either react with proteins or form reactive peroxyl radicals after reaction with O2 [188]. Finally, since disulﬁde bonds play an important role in the conformational integrity, stability, and functional capabilities of receptor and plasma membrane proteins, ‘‘reductive stress’’ may play a role as important as ‘‘oxidative stress’’ at the boundary of RTLFs and RTECs. Thus, some degree of caution should be exercised before recommending administration of large amounts of reducing antioxidants to patients with CF, particularly if via the inhaled route and in the presence of airway sepsis. The recognition that RT GSH levels are reduced in CF [43] has given impetus to therapeutic strategies designed to increase airway GSH levels. Although aerosolized GSH is known to induce bronchospasm in some individuals [189] and theoretical constructs of just how it may be beneﬁcial are incomplete, it is apparent that clinical trials of these strategies are needed, if for no other reason than they are being touted and used by some of the CF patient population.* Mention should be made of antibiotics. Aminoglycosides (frequently administered to CF patients) may act as radical scavengers [190], while other antibiotics appear to suppress important transcriptional regulators of pro-inﬂammatory cytokines [191,192]. It is not certain if these could also cause signiﬁcant augmentations of RTLF antioxidants or decreases in RTLF oxidant-producing capabilities. Although there is controversial evidence presented with regards to h-carotene supplementation, there is increasing evidence that dietary vitamin E and h-carotene may provide some protection against cancer and cardiovascular disease [182]. There is no a priori reason to suspect they cannot be helpful to patients with CF. In fact, the nutritional guidelines available for caregivers of patients with CF recognize the contributions that micronutrient antioxidants might make to patient management [48]. However, as emphasized in a sobering review by McCall and Frei [193], the scientiﬁc evidence that supplementation of humans with vitamins C, E, or other antioxidant micronutrients lowers in vivo oxidative damage to lipids, proteins, or DNA is based on the measurement of biomarkers, not disease outcome. They call attention to the fact that, with the possible exception of certain cardiovascular diseases, there is insuﬃcient evidence to conclude that antioxidant supplementation (above ‘‘normal’’ levels) materially reduces oxidative damage and disease progression in humans [193]. IX.

SUMMARY

In this chapter we have focused on the possible contributions of oxidative and nitrosative stress to the pathophysiology of CF. These oxidative processes relate to

* A group called the Utah Valley Institute of Cystic Fibrosis has posted their review of the GSH literature as it relates to CF on their Web site: http://members.tripod.com/uvicf/index/htm. This institute has recently gathered information about 24 CF patients who used to inhale GSH. In this uncontrolled ‘‘trial,’’ patients were instructed as to how to use inhaled GSH and asked to report their perceived beneﬁts or side eﬀects on The Institute’s Web site.

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the intense activation of RT inﬂammatory-immune processes and the deﬁciencies of lipophilic antioxidant micronutrients in CF. We discussed some of the emerging ideas of how the balance between ROS and RNS may interact with biomolecular constituents at RT surfaces. An important area not addressed is how these events occurring at the CF airway surfaces might aﬀect the molecular events that control RTEC signaling and gene expression [194,195]. Further understanding of these processes will play an important role in future innovative treatments of CF airway pathology.

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13 Oxyradicals in Iron Overload Syndromes: Hemochromatosis SHINYA TOYOKUNI Graduate School of Medicine, Kyoto University, Kyoto, Japan

Hereditary hemochromatosis (HHC) is a late-onset, autosomal recessive disorder leading to continuous deposition of iron in parenchymal cells of many organs causing a chronic oxygen radical overload syndrome. Though the disease can be treated easily, symptoms are nonspeciﬁc, and onset and severity are inﬂuenced by environmental factors. Therefore, HHC can remain undetected until decades of iron overload lead to irreversible damage in a variety of organs, such as the liver, heart, and pancreas, which may result in their failure and eventually lead to diabetes mellitus, cardiomyopathy, hepatic cirrhosis, and liver carcinoma, among others. Mutations in the HFE gene located at 6p21.3 accounted for about 80–90% of the hemochromatosis cases. The HFE protein is a 343 residue type I transmembrane protein that is associated with class I light chain h2-microglobulin. This product binds to the transferrin receptor and reduces its aﬃnity for iron loaded transferrin by 5- to 10-fold, thus regulating iron absorption, which is the key control element in iron homeostatis in humans. Genetic heterogeneity of hemochromatosis is shown by multiple mutations within the HFE gene as well as by other genes, particularly in speciﬁc populations, leading to a phenotypically speciﬁc iron overload syndrome [1,2]. HHC is classiﬁed into HFEassociated hereditary hemochromatosis and non–HFE-associated hemochromatosis. HFE-associated hereditary hemochromatosis occurs predominantly in individuals of northern European origin. Thirty-seven allelic variants of the HFE gene have been described to date, C282Y and H63D mutations being the most important. C282Y occurs in 85–90% of Anglo-Celtic populations but is either absent or has low allele frequency in non-Caucasian populations. Expression of the disease in those homozygous for the C282Y mutation is very variable (20–70%), depending on the various 287

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features of the population studied (race, age, sex, and diet) [3]. At least 50% of male and 25% of female persons homozygous for the C282Y mutation are likely to develop potentially life-threatening complications of the disease if untreated, especially in countries with high dietary intake of iron. Approximately 20% of the C282Y/H63D compound heterozygotes developed a HHC phenotype identical to that observed in C282Y homozygotes [3,4] while H63D homozygotes only rarely expressed the HHC phenotype [5]. In the absence of other cofactors or an additional rare mutation, C282Y heterozygotes do usually not develop signiﬁcant disease. However, some heterozygotes for C282Y develop a hemochromatosis phenotype that is indistinguishable from that of the homozygotes. This phenomenon was suggested to be at least partly due to the candidate modiﬁer genes that may inﬂuence the course of hemochromatosis [6], but could also be caused by unknown second mutations in the HFE gene.

I.

INTRODUCTION

‘‘Redox cycling’’ is a characteristic of transition metals such as iron. Iron is an essential metal involved in oxygen transport by hemoglobin in mammals and in the activity of many enzymes, including catalase and cytochromes [7]. Both deﬁciency and overload may cause such pathological conditions in humans as microcytic anemia and hemochromatosis, respectively. Therefore, iron metabolism should be and indeed is ﬁnely regulated. Recently, our understanding of iron metabolism has been enormously expanded by several new ﬁndings such as the cloning of iron transporters, messenger RNA–based regulation of ‘‘iron metabolism–associated’’ genes, and the discovery of the hemochromatosis HFE gene. Furthermore, there is a growing body of evidence that suggests a role of iron in carcinogenesis [8]. This has been extensively investigated in animal models, and the precise molecular mechanisms are now being elucidated. In addition, new studies will reveal the molecular mechanism of hemochromatosis.

II.

FENTON CHEMISTRY AND ‘‘CATALYTIC’’ IRON

Iron present in heme or iron-sulfur clusters or closely associated with proteins plays an important role in a variety of fundamental cellular functions such as oxygen transport, energy metabolism, electron transport, and modulation of H2O2 levels. On the other hand, non–protein-bound ‘‘free’’ or ‘‘catalytic’’ iron works quite diﬀerently. The Nobel laureate Christian deDuve hypothesized that iron was an essential element in the origin of life on earth, stating [9]: ‘‘Thanks to the UV-supported photooxidation of Fe(II), CO2 and other inorganic precursors were reduced to prebiotic building blocks with the consumption of protons. Oxidation of the synthesized materials takes place with Fe(III) ions as electron acceptors and is coupled to thioesterdependent substrate-level phosphorylations, capable, in turn, of supporting work. Thanks to the iron cycle, UV-light energy is made to support vital work.’’ This hypothesis clearly describes the important characteristic of iron, ‘‘redox cycling.’’ Iron is the most abundant transition metal in the human body (approximately 2– 6 g) [7]. Redox cycling of iron is closely associated with the production of reactive oxygen species (ROS). Fenton reported as early as 1894 that ferrous sulfate and H2O2 cause the oxidation of tartaric acid, resulting in a beautiful violet color on the addition

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of caustic alkali [10]. This was the basis for the discovery of the Fenton reaction, which produces hydroxyl radicals (OH): FeðIIÞ þ H2 O2 ! FeðIIIÞ þ OH þ OH

ð1Þ

In order to understand the involvement of this chemical reaction in biological systems, the concept of ‘‘catalytic’’ or ‘‘free’’ iron proposed by Gutteridge et al. [11] is important. The original detection of ‘‘catalytic’’ iron was made by the use of thiobarbituric acid in an in vitro system that contained a biological sample, calf thymus DNA, bleomycin, ascorbic acid, and H2O2. Bleomycin in the presence of Fe(II) degrades DNA to form thiobarbituric acid–reactive substances. The chemistry of this system was precisely analyzed by mass spectrometry [12]. ‘‘Catalytic’’ iron has the following two characteristics: redox activity and diﬀusibility. In biological environments at neutral pH, the reduction potential of Fe(III) is +722 mV, close to that of the water/oxygen couple, which is +818 mV [13]. However, Fe(III) dissolves in water at a very low concentration (1017 M) at neutral pH. Most Fe(III) precipitates as iron hydroxides at neutral pH [14]. On the other hand, iron chelated with citrate, ADP, ATP, or GTP can remain as ‘‘catalytic’’ iron at neutral pH [15]. In these iron chelates, at least one of the six ligands of iron is left free to maintain catalytic activity [16]. It was suggested that the fewer the number of ligands involved in chelation, the higher the preservation of catalytic activity for ROS production [17]. This is further related to the redox potential of the iron chelate; in the redox potential between +460 and 160 mV, the ferrous state gives a Fenton reaction, whereas the ferric state can be reduced by O2 [18].

III.

‘‘CATALYTIC’’ IRON IN THE BIOLOGICAL ENVIRONMENT

Only a limited amount of data is currently available concerning the localization of ‘‘catalytic’’ iron in the cytoplasm or nucleus of cells due to a deﬁciency of appropriate methods. It has been believed that there exists a minute cellular ‘‘labile’’ pool of iron that is solubilized via chelation to low molecular weight biomolecules such as citrate and adenine nucleotides [19,20]. This pool of iron is considered to be at least partly responsible for the pathological free radical reactions. On the other hand, more data are available regarding extracellular ‘‘free’’ iron. The clinical signiﬁcance of ‘‘nontransferrin plasma iron’’ (‘‘catalytic’’ iron) has been well discussed [21]. Plasma transferrin acts as a considerable reserve for coping with increasing amounts of incoming iron. However, in acute iron poisoning, ‘‘catalytic’’ iron concentrations ranging from 128 to over 800 Amol/L have been documented, exceeding by several times the total binding capacity of transferrin [22]. Similarly, in severe idiopathic hemochromatosis and Bantu siderosis, acute episodes of abdominal pain and shock have been observed in individuals with extremely high serum iron measurements exceeding 2000 Amol/L [23]. Another important concept regarding iron-dependent oxidative damage is that of a site-speciﬁc mechanism. Fe(III) ions that are loosely bound to biological molecules such as DNA and proteins can undergo cyclic reduction and oxidation. This concept is diﬀerent from that of ‘‘catalytic’’ iron in that the iron is not diﬀusible, and it explains the funneling of free radical damage to speciﬁc sites and possible ‘‘multihit’’ eﬀects on the molecules at such site [24].

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SIGNIFICANCE OF SUPEROXIDE IN IRON-DEPENDENT OXIDATIVE DAMAGE

Whereas the reactivity of superoxide is relatively low, superoxide can reduce Fe(III), as shown in Eq. (2) [18]. The sum of Eqs. (1) and (2) is Eq. (3): FeðIIIÞ þ O 2 ! FeðIIÞ þ O2 H2 O 2 þ O 2 ! OH þ OH þ O2

ð2Þ ð3Þ

Equation (3) is called the Haber–Weiss reaction, ﬁrst postulated by Haber and Weiss in 1934 [25]. Furthermore, superoxide has the potential to release iron from lactoferrin [26], saturated transferrin [27], ferritin [28,29], or hemosiderin [29] in a catalytically active form. These reactions are thought to be important in situations that increase superoxide generation, such as inﬂammation [30] or ischemia-reperfusion [31]. V.

IRON TRANSPORTERS

How iron crosses the cellular membrane is a critical issue for the absorption and redistribution of the iron. There has been until recently little molecular information available on the mechanisms by which metal ions are taken up by mammalian cells. In the presence of oxygen, Fe(III) is more stable than Fe(II). However, there are situations in which Fe(II) is required in organisms. The uptake and transport of iron under physiological conditions require special mechanisms since Fe(III) has very low solubility at neutral pH, as described previously [14]. Thus, the reduction of iron has been considered necessary for iron absorption. While the process of transferrin receptor–mediated endocytosis has been well studied [32–34], this is not the pathway by which iron is taken up into the circulation in the duodenum. In 1997 mouse Nramp2 (natural resistance-associated macrophage protein 2)/ DMT1 (divalent metal transporter 2; DCT1, divalent cation transporter 1) was identiﬁed as an iron transporter by studying mk mice, which have microcytic anemia, via a genetic approach. The anemia of mk mice is unresponsive to increased dietary iron, and iron injections do not reverse the anemia, suggesting a block of iron entry into red blood cell precursors as well [35]. It was interesting that Nramp2 is a homologue of Nramp1, which mediates natural resistance to infection with intracellular parasites, aﬀecting the capacity of macrophages to destroy ingested intracellular parasites early during infection [36]. Independently, this gene was identiﬁed by the use of an expression cloning technique from a duodenal cDNA library prepared from rats fed a low-iron diet. This insightful approach was based on the idea that mRNA for iron transporter would be overexpressed in such a situation. DMT1 was isolated by screening this library using a radiotracer assay of Fe(II) uptake in Xenopus oocytes. These experiments further revealed that DMT1 transports not only Fe(II) but also Zn(II), Mn(II), Cu(II), Co(II), Cd(II), and Pb(II). Furthermore, this transporter was expressed in other organs such as kidney, liver, brain, heart, lung, and testis, although to a lesser extent [37] (Fig. 1). As described above, Nramp1 was cloned from functionally impaired macrophages [36]. Localization studies showed that Nramp1 protein is present in the lysosomal compartment of macrophages and in phagosomal membranes during phagocytosis. Nramp1 may play a role in resistance to infection by depleting the phagosomes of Fe(II) and other essential divalent metal cations [38,39]. It was reported that

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Figure 1 Current understanding of iron absorption and transport in mammals. DMT1, divalent metal transporter 1; NRAMP2, natural resistance–associated macrophage protein 2; IREG1, iron-regulated transporter 1.

in West Africans each of four Nramp1 polymorphisms was signiﬁcantly associated with tuberculosis infection. Subjects who were heterozygous for two Nramp1 polymorphisms in intron 4 and the 3V untranslated region (UTR) of the gene were particularly overrepresented among subjects with tuberculosis, as compared with subjects with the most common Nramp1 genotype [40]. Now we will return to our consideration of the mechanism of iron absorption in the duodenum. Intestinal epithelial cells have two diﬀerent iron transporters, one in the apical and one in the basolateral membrane, as indicated by the ﬁndings that sla mice show normal uptake of iron into the villus cells, via a process mediated by DMT1/ Nramp2, but show impaired release of iron into the bloodstream (Fig. 1). Recent attempts to clone the basolateral membrane transporter suggest that it consists of at least two subunits, one for Fe(II) transport and the other for oxidation of Fe(II) back to Fe(III) [41]. Finally, in 2000 a novel duodenal iron-regulated transporter, Ireg 1, implicated in the basolateral transfer of iron to the portal vein, was cloned [42]. This transporter was reported independently from two other laboratories as ferroportin 1 (FP1) [43] and metal transporter prtein 1 (MTP1) [44]. In contrast to Nramp2/DMT1, Ireg1 (FP1, MTP1) was expressed in the duodenum, reticuloendothelial system, pregnant uterus, and embryonic muscle and central nervous system cells [44]. It was reported that the nuclei of rat liver take up iron from ferric citrate by a process that is dependent on ATP [45]. However, there has been no demonstration yet of an iron transporter in the nuclear membrane. In addition to these iron transporters, frataxin, which is defective in the mitochondria of patients with Friedreich’s ataxia, has been isolated and shown to mediate an iron transport exit mechanism for mitochondria. Defects of frataxin lead to

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iron accumulation in the myocardium of patients [46,47]. The ﬁnding of mitochondrial iron overload suggests that the use of speciﬁc iron chelators that can permeate the mitochondrion may have potential for the treatment of this disease [48,49]. Thus, I believe that, based on these major breakthroughs, new ﬁndings about a variety of pathological conditions related to iron metabolism can be expected in the next few years. VI.

POSTTRANSCRIPTIONAL REGULATION OF IRON METABOLISM

The expression of proteins that modulate the iron metabolism of mammalian cells is controlled by the intracellular level of iron. It was shown that this regulation is mediated at a posttranscriptional level, namely by speciﬁc mRNA-protein interactions in the cytoplasm. Particular hairpin structures, called iron-responsive elements (IREs), in the respective mRNA are recognized by the transacting proteins, called ironregulatory proteins (IRPs), that can control the eﬃciency of mRNA translation and stability. IREs are present not only in the 5V untranslated region (UTR) of the ferritin H- and L-chains [50,51], but also in the 3V UTR of transferrin receptor (TfR) mRNA [52] (Fig. 2). The predicted interaction of IRP-1 with the TfR IRE was convincingly demonstrated in cells treated with iron chelator to remove iron, and a clear correlation was shown between iron deprivation and the induction of TfR mRNA and protein [53]. It has now been established that IRP-1 plays a dual role as an IRE-binding form without a [4Fe-4S] cluster in iron deﬁciency and as a cytoplasmic aconitase with a [4Fe-4S] cluster in iron suﬃciency. In iron deﬁciency, while translation of ferritin is blocked by

Figure 2

Posttranscriptional regulation of iron metabolism by iron-regulatory proteins. Binding of iron-regulatory proteins to iron-responsive element in messenger RNA plays a key role in the posttranscriptional regulation of iron-associated proteins. ORF, open reading frame. Desferal is a chelator that inactivates iron.

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the interaction of IRE and IRP-1 in the 5V UTR region, the mRNA of TfR is stabilized by the same interaction in the 3V UTR region. Furthermore, it was discovered that signals other than the iron level may regulate IRP-1 and IRP-2 and modulate iron metabolism. Nitric oxide or oxidative stress transforms an inactive form of IRP-1 with a [4Fe-4S] cluster to an active form without a [4Fe-4S] cluster, although the time for the transformation is diﬀerent (f15 h vs.<1 h, respectively). The molecular mechanism of this activation has been studied in detail [54,55] (Fig. 2). VII.

HEMOCHROMATOSIS

Hereditary hemochromatosis is an iron overload disorder that in the past could not be diagnosed until the progressive accumulation of iron, mainly in the form of ferritin (soluble form of iron-protein complex) and hemosiderin (insoluble form of ironprotein complex), caused organ injury, particularly to the liver, heart, and endocrine pancreas (especially insulin-secreting h-cells). The disease has been diagnosed on the basis of a classic triad: (1) a micronodular pigment liver cirrhosis in all cases (Fig. 3), (2) diabetes mellitus in about 75–80% of the cases, and (3) skin pigmentation in about 75–85% of the cases. However, the disease can now be discovered much earlier by

Figure 3

Liver of hemochromatosis obtained by autopsy. (A) Macroscopic view: micronodular surface is noted. (B) Hematoxylin-eosin staining. (C) Azan staining (40): there is a prominent damage in the hepatic parenchyma that lead to micronodular cirrhosis. (D) Perls’ iron staining (200): hepatocytes contain numerous hemosiderin granules.

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biochemical studies of the blood before cirrhosis and other organ injuries have developed [56]. Hemochromatosis subsequent to hemosiderosis may occur due to a secondary excessive iron deposition with functional deterioration of any aﬀected organ that is induced by a nongenetic etiology such as thalassemia major or repeated transfusion. Recently, HFE, the gene responsible for hereditary hemochromatosis, was identiﬁed by a positional cloning approach. HFE is related to major histocompatibility complex class I proteins and is mutated in hereditary hemochromatosis [57]. Two point mutations, C282Y and H63D, have been linked to the majority of disease cases [2]. Hereditary hemochromatosis is usually inherited with an autosomal-recessive pattern. Interestingly, it was reported that a mutation in the gene encoding ferroportin is associated with autosomal-dominant hemochromatosis [58,59]. The structure of the protein was analyzed by x-ray crystallography [60] and it was found that HFE binds to the transferrin receptor (TfR), a receptor by which cells acquire iron-loaded transferrin, and that in cases of hemochromatosis this interaction is disrupted [61]. The 2.8 A˚ crystal structure of a complex between the extracellular portions of HFE and TfR showed that two HFE molecules grasp each side of a twofold symmetrical TfR dimer [62]. Intestinal crypt cells express HFE and TfR, whereas mature villus cells express Nramp2/DMT1, but not HFE. These results lead to the speculation that HFE and TfR together sense serum iron in crypt cells. It was proposed that HFE and TfR in crypt cells regulate the expression of the proteins involved in iron absorption in villus cells, including Nramp2/DMT1, via the IRE/IRP system [63]. Recently, data have been accumulated about the status of iron transporters in patients with hemochromatosis. DMT1, ferroportin 1 (FP1), messenger RNA (mRNA), and protein expression were analyzed in duodenal biopsy specimens from such patients. DMT1 and FP1 mRNA levels were positively correlated with each other in all patient groups. Moreover, DMT1 and FP1 mRNA levels were signiﬁcantly increased in the patients with iron deﬁciency, HFE, and non-HFE hemochromatosis, whereas they were unchanged in patients with secondary iron overload. Alterations in DMT1 and FP1 mRNA levels were paralleled by comparable changes in the duodenal expression of the proteins. In the patients with normal iron status or iron deﬁciency, signiﬁcant negative correlations between DMT1, FP1 mRNA, and serum iron parameters were observed, but such correlations were absent in the subjects with hereditary hemochromatosis [64,65]. Thus, DMT1 and FP1 are centrally involved in iron uptake/ transfer in the duodenum and in the adaptive changes of iron homeostasis to iron deﬁciency and overload. It is of note that the major cause of death today in hereditary hemochromatosis is hepatocellular carcinoma [56]. Indeed, 219, 240, or 92.9 times greater risk was shown for primary hepatocellular carcinoma in hemochromatosis patients than in the agematched control population in three independent studies [66–68]. In general, hepatocellular carcinoma is preceded by cirrhosis. A high incidence of cancers originating from other organs (esophageal cancer, skin melanoma, etc.) has also been reported [68–70]. Furthermore, cases of hepatocellular carcinoma in the absence of cirrhosis [71,72] and after reversal of cirrhosis with therapy [73] have been reported. These facts suggest that irreversible genetic alteration may have occurred early in the course of the disease. It has been suggested that consistent lipid peroxidation is one of the important factors in the etiology of the high incidence of hepatocarcinogenesis in hereditary

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hemochromatosis. The suggested mechanisms include increased lysosomal membrane fragility and peroxidative damage of organelles such as microsomes and mitochondria [74]. It was shown that the serum ‘‘catalytic’’ form of iron in advanced hemochromatosis patients exists largely as complexes with citrate [75]. It was shown that ferric citrate eﬃciently induces oxidative single- and double-strand breaks in plasmid DNA in vitro [76]. VIII.

IRON AND CARCINOGENESIS IN HUMANS

Iron deﬁciency is an important nutritional problem in developing countries because it reduces work performance by inducing microcytic anemia and reducing the activity of iron-containing respiratory enzymes in mitochondria required for muscle performance. In meat-eating countries, however, iron excess may be more of a problem than iron deﬁciency [77,78]. It has been hypothesized that increased body iron stores are associated with an increased risk of cancer and with overall death rates. Two lines of evidence provide biological rationales for this hypothesis. First, iron can catalyze the production of reactive oxygen species, and these species may be proximate carcinogens. Second, iron may increase the chances that cancer cells will survive and ﬂourish [79]. Several human epidemiological studies support this hypothesis [80,81]. It has been hypothesized by Nelson that intestinal exposure to ingested iron may be a principal determinant of human colorectal cancer risk [82]. Nelson et al. thereafter demonstrated a dose-response relationship between serum ferritin level and colon adenoma risk [83]. On the other hand, Kato et al. suggested that luminal exposure to excessive iron may increase the risk of colorectal cancer in combination with a high-fat diet [84]. Indeed, large amounts of unabsorbed iron reach the colonic lumen, especially in individuals who consume processed foods adulterated with inorganic iron and vitamin C supplements. This issue has been reviewed [85–87]. Occupational or nonoccupational exposure to asbestos is considered to pose an increased risk for the development of lung cancer (i.e., diﬀuse malignant mesothelioma, bronchogenic carcinoma). Approximately 30% of asbestos ﬁber by weight is made of iron. There is a close association between the incidence of tumors and the iron content of inhaled asbestos ﬁber. Chrysotile and crocidolite are the two major asbestos ﬁber types that contain high amounts of iron [88]. Furthermore, increased body iron stores (as estimated by serum ferritin level, transferrin concentration, or transferrin saturation) are associated with poor prognosis of several human malignant neoplasms (neuroblastoma, childhood Hodgkin’s disease, acute lymphocytic leukemia) [89–91]. IX.

IRON AND CARCINOGENESIS IN ANIMALS

In 1959 Richmond ﬁrst reported that an iron compound, iron dextran, induced malignant tumors (sarcomas) at the injection site of animals [92]. In 1982 Okada and Midorikawa showed for the ﬁrst time that an iron compound induces malignant tumors at sites diﬀerent from those of the injections [93]. Repeated intraperitoneal injections of an iron chelate, ferric nitrilotriacetate (Fe-NTA), induced a high incidence (60–92%) of renal cell carcinoma in rats [94] and mice [95] (Fig. 4). Nitrilotriacetic acid (NTA) is a synthetic aminotricarboxylic acid that eﬃciently forms water-soluble chelate complexes with several metal cations at neutral pH and has been used as a

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Figure 4

Ferric nitrilotriacetate–induced renal cell carcinoma in rats. (A) Macroscopic view: renal cell carcinoma is invading the whole peritoneal cavity. (B) Histology of renal cell carcinoma (100): atypical cells forming glandular structure are invading the renal parenchyma, leaving one normal glomerulus in the center.

substitute for polyphosphates in detergents for household and hospital use in the United States, Canada, and Europe [96]. It is of note that this model was originally developed as a model of hemochromatosis [97], based on the fact that Fe-NTA can be used to load iron to apotransferrin [98]. Fe-NTA works as an eﬃcient ‘‘catalytic’’ iron in vitro at neutral pH [99]. This animal model is characterized by (1) induction of only carcinoma, not sarcoma, (2) highly malignant potential, as shown by pulmonary metastasis or peritoneal invasion [100], (3) marked increase in oxidatively modiﬁed biological molecules such as 8oxoguanine [101], thymine-tyrosine cross-link [102], 4-hydroxy-2-nonenal and its modiﬁed proteins [103,104], especially at an early stage, and (4) signiﬁcant reduction of mortality and cancer incidence by vitamin E pretreatment [105,106]. X.

TARGET GENES OF IRON OVERLOAD–INDUCED CARCINOGENESIS

We now know that iron overload is associated with carcinogenesis. But why? After more than 20 years of studies of the basis of carcinogenesis, it is well established that cancer is a disease or abnormality of genomic DNA. Reactive oxygen species may cause DNA damage, leading to an error of the genomic information. This is deﬁned as a mutation, a genetic change, and includes point mutations, deletions, and additions as well as chromosomal translocations. These events may cause activation of oncogenes (proliferation-associated genes) and inactivation of tumor suppressor genes, which are basically classiﬁed into two categories: caretakers (DNA repair genes) and gatekeepers (cell cycle inhibitors) [107]. The caretakers usually take good care of our genomic DNA and maintain its integrity. It is estimated that roughly 130,000 damage events may occur per day per genome in that rat [108]. However, in the case of excessive oxidative stress such as in the situation of iron overload, accidents or mistakes may occur in the processes of repair or replication of DNA. In contrast to the relatively selective antigen-antibody interactions in immunological reactions, it is considered that free radical reactions reveal no such speciﬁcity, at

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least in vitro. Thus, it may be hypothesized that the genome is randomly injured and that there is no speciﬁc target gene in iron overload–induced cancer. We doubted this hypothesis and used a genetic strategy to examine whether there are any target gene in Fe-NTA–induced renal cancer. We used polymerase chain reaction (PCR) in an attempt to scan the whole genome of Fe-NTA–induced renal cell carcinoma cells in F1 hybrid rats in search of allelic loss or loss of heterozygosity with microsatellite polymorphic markers. A preliminary experiment revealed a signiﬁcantly elevated frequency of allelic loss (>30%) on rat chromosomes 5 and 8. We then focused on chromosome 5 and collected data on all the cases of renal cell carcinoma registered. Microsatellite markers in the chromosomal areas around rat chromosome 5q32 showed allelic loss in more than 40% of tumors [109]. Common allelic loss suggests the presence of a target tumor suppressor gene, according to Knudson’s ‘‘two-hit theory’’ [110] that both the alleles need to be mutated to inactivate the tumor suppressor gene. Allelic loss is one of the main pathways of this inactivation. We then searched for candidate genes from the map position. p15INK4B (p15) and p16INK4A (p16) tumor suppressor genes were the only candidate genes reported thus far at the map position indicated. We then evaluated whether these two genes are among the targets for carcinogenesis by ﬁne molecular techniques such as Southern blot analysis, PCR/single strand conformation polymorphism analysis, and Northern blot analysis, as well as methylation-speciﬁc PCR analysis. We found that 44% of the renal cell carcinomas showed allelic loss of p15 or p16 (p16 only, 38%); in 38% of the tumors p15 or p16 was inactivated; there was no preference in respect to the allele for loss; tumors with high-grade pathology had a high inactivation frequency. This is the ﬁrst report that showed the presence of any target gene in an iron overlod– or oxidative stress–induced cancer model [109]. The biological signiﬁcance of this ﬁnding is immense, since p16 is associated not only with the retinoblastoma protein pathway as a cyclin-dependent kinase 4 and 6 inhibitor, but also with the p53 pathway via p19 ARF and MDM2 [111,112] (Fig. 5). Thus, allelic loss of p16 is a key event in our iron overload–induced carcinogenesis model. The next question was when the allelic loss occurs. To answer this question, we studied the alteration of the number of alleles of p16 in the renal proximal tubules. Fluorescent in situ hybridization experiments were performed at the singlecell level by the use of touch preparations of excised kidney. We clearly demonstrated that allelic loss of p16 occurs quite early in carcinogenesis (1–3 weeks after the start of the experiment) and is gene speciﬁc [113]. This fact is probably related to which sites in the genome are susceptible to the attack of reactive oxygen species and the timing of the DNA replication of each of these genes, which might diﬀer depending on the kind of cell and the conditions under which they are placed. Two studies on cancers induced in hereditary hemochromatosis were performed quite recently. The mutation spectrum of the p53 tumor suppressor gene was studied using cases of hemochromatosis-associated hepatocellular carcinoma. In a British study, 60% A:T to G:C and 40% A:T to T:A mutations were observed [114], whereas 45% G:C to C:G, 33% A:T to C:G, 11% G:C to A:T at CpG, and 11% G:C to T:A mutations were observed in an American study [115]. These mutation spectra suggest that etheno-deoxyguanine or -deoxyadenine DNA adducts may be responsible for the DNA damage. These DNA modiﬁcations are produced by the reaction with lipid

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Figure 5 Two distinct functions of p16INK4A tumor suppressor gene by alternative splicing. INK4, inhibitor of cyclin-dependent kinase 4; RB, retinoblastoma protein; RB-p, phosphorylated retinoblastoma protein; E2F, adenovirus E2 promoter-binding factor; ARF, alternative reading frame; MDM, mouse double minute. peroxidation products, and the increased formation of such modiﬁcations is observed in the livers of hereditary hemochromatosis patients [116]. XI.

CONCLUSION

Iron plays an important role in free radical–induced tissue damage and carcinogenesis. Clinical features of iron overload are seen in hemochromatosis. In the past few years, our understanding of iron metabolism, the molecular mechanism of hemochromatosis and iron–induced carcinogenesis, has been enormously expanded. Iron is associated with quite fundamental phenomena in life. Hereditary hemochromatosis can now be treated by phlebotomy if the disease is recognized early in life. Modulation of iron intake and iron metabolism might be helpful for the prevention of aging and carcinogenesis.

ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan, a grant from the program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN), and Eiko Yasuhara Memorial Fund.

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14 Oxidative Imbalance in Hereditary Hemoglobinopathies: The Role of Reactive Oxygen Species in the Pathophysiology of Sickle Cell Anemia and Thalassemia ¨ RGEN FUCHS and MAURIZIO PODDA JU J.W. Goethe University, Frankfurt, Germany ELIEZER A. RACHMILEWITZ The E. Wolfson Medical Center, Holon, Israel

I.

INTRODUCTION

Disorders of hemoglobin (Hb) structure and synthesis constitute the most frequent group of genetic diseases in the world. It has been estimated that 7% of the world population are carriers for diﬀerent inherited disorders of Hb [1]. They are most common in areas of the world where malaria has been and still is endemic. For instance, in Southeast Asia it is estmated that about 110 million out of a population of 512 million carry at least one or more genetic mutations resulting in thalassamia [1]. It has been postulated that the malaria parasite penetrates and multiplies more readily in normal red blood cells (RBC) compared to pathological RBC such as sickle cells and thalassemic RBC, which are the hallmark of the two major hemoglobinopathies, sickle cell anemia and thalassemia. Consequently, the heterozygotes for these diseases have an advantage over normals regarding the severity and the magnitude of the infection induced by the malaria parasites [2]. As a result of population migration, these diseases have become widespread, occurring throughout northern Europe and North and South America [3]. Structural variants, particularly sickle cell disease and the thalassemias, are a major cause of morbidity and mortality worldwide and were the ﬁrst genetic diseases to be charac305

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terized at the molecular level. The experimental strategies and methods developed to study hemoglobinopathies became a paradigm for the molecular analysis of other human diseases [4,5]. As a consequence of the presence of abnormal Hb and/or the relative excess in the amount of either a or h protein chains which make HbA (a2h2), which happens in thalassemia, the RBC become unstable and eventually disintegrate prematurely, as will be discussed below, in the bone marrow, resulting in ineﬀective erythropoiesis. The RBC was very early the focus of investigators studying autoxidation of Hb [6–8], and there is now overwhelming evidence that hereditary hemoglobinopathies are associated with signiﬁcant redox imbalance [9,10]. The increased oxidative stress contributes to a depletion of the available antioxidants in patients with hereditary hemoglobinopathies and is relevant in the pathophysiology of these diseases. In this chapter we review the evidence that redox imbalance occurs in hemoglobinopathies, focusing on sickle cell disease and various forms of thalassamia, and discuss the eﬀects of oxidative stress on the signal transduction processes leading to RBC membrane pathology. Finally, we discuss whether or not therapeutic strategies directed at oxidative stress would be helpful as adjuvant treatments of these diseases. II.

Hb VARIANTS

Hb is a tetrameric molecule composed of two diﬀerent globin chains with diﬀerent numbers and compositions of amino acids. Each globin chain contains a heme group. Each of the two major globin chains is produced in parallel in equal amounts, maintaining a balance in their production during development. The genes encoding the a- and h-like globin chains occur in clusters; the a-globin gene cluster is located on chromosome 16, and the h-globin gene cluster, including g and y globins, on chromosome 11. During embryonic and fetal development, there is sequential expression of diﬀerent globin chains. In healthy adults, 95% of the Hb is HbA (a2h2), with small amounts (<3, 5%) of HbA2 (a2y2) and fetal Hb HbF (a2g2). The a- and h-globin chains consist of 141 and 146 amino acids residues, respectively. There are now more than 1000 known human globin gene mutations, 90% of which are single amino acid substitutions in the a, h, g, or y chains. Many point mutations have no clinical signiﬁcance, depending on their location within the tertiary structure of the protein moiety. The remaining 10% result in formation of abnormal Hb, with one or more substitutions in the globin chains, insertions, or deletions. More than 70% of the mutations are located in the a or h chains. The most important Hb variants are HbS, HbC, and HbE. The heterozygous state for all three are in general symptomless, while the homozygous state results in moderate to severe clinical manifestations. Sickle cell anemia is the most important of the sickling disorders. It is manifested by a chronic hemolytic anemia associated with a variety of infections and vasoocclusive episodes, which can result in life-threatening crises. Further clinical manifestations include excruciating pain crises, acute chest syndrome (a life-threatening pneumonia-like illness), cerebrovascular accidents, and splenic and renal dysfunction [11]. The underlying abnormality in this disease is the appearance of a genetically modiﬁed Hb (HbS) in the RBC which is due to a single amino acid substitution of valine for glutamic acid at the 6 position of the h-globin chain, which under low oxygen saturation results in sickling of the RBC.

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The basic abnormality in sickle cell disease was ﬁrst postulated by Linus Pauling in 1949 [12] and later demonstrated by Vernon Ingram [13]. Despite its genetic homogeneity, the phenotype of sickle cell disease is heterogeneous [14]. Patients who are homozogous for HbS can present with remarkably diﬀerent clinical courses, varying from death in childhood, to recurrent painful vaso-occlusive crises and multiple organ damage in adults, to being relatively well even until old age. Several environmental factors inﬂuence expression of sickle cell disease, including socioeconomic and the nutritional status [15], but the major risk factor for disease severity seems to be genotype. Increasing numbers of genetic loci within the h-globin gene cluster as well as on genes on diﬀerent chromosomes have now been identiﬁed that can modulate sickle cell disease phenotype [16]. A small fraction of individuals with the sickle cell gene may also carry genes for other Hb variants or defects in Hb synthesis [17]. Sickle cell disease results from the presence of homozygous state for the sickle cell gene (hShS) or, in varying degress of clinical severity, from the double heterozygous state with other variants, e.g., HbD Punjab (hShD-Punjab) and HbO Arab (hShO-Arab), which are associated with severe disease, while HbC (hShC) usually results in a mild disease [11,18]. HbC results from an alteration at the same codon 6, which is modiﬁed in sickle cell anemia, changing GAG to AAG, and resulting in the synthesis of a lysine at this position. This is also a relatively common h-globin chain mutation. In addition, there is a frequent association between sickle cell anemia and thalassemia, resulting in mild to severe phenotypes depending on the type of thalassemia hShO or hSh+, where in the former no h chains are synthesized in one of the chromosomes (Table 1). Sickle cell carriers very rarely experience clinical problems resulting from the presence of HbS, although their RBC contain both HbA and HbS, with distribution favoring HbA. However, one study found that the sickle cell carrier state may be a risk factor for sudden death during physical training [19]. HbE, which arises from a point mutation in codon 26 of the h-globin gene (GAG to AAG), is an example of a hemoglobinopathy representing a combination of a qualitative and quantitative abnormality. Individuals with this mutation produce only about 60% of the normal amount of h-globin from this mutant allele. HbE is a common variant in up to 30% of the population in some parts of Southeast Asia, mainly in combination with hthalassemia. While until recently most of the organ damage induced in sickle cell anemia was thought to be due the sickling phenomenon in relation to reduced oxygen tension, in the last few years more information has been accumulated indicating that

Table 1

Classification of Sickle Cell Disease

Genotype h h hShA hShC hSh+; hShO hShS hShO-Arab, hShD-Punjab A A

Phenotype

Clinical condition

Normal Normal Mild Mild-severe Moderate-severe Moderate-severe

Healthy individual Sickle cell carrier HbSC disease Sickle cell h thalassemia Sickle cell anemia Sickle cell disease

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HbS is an unstable Hb with all the characteristic consequences of hemolytic anemia, including thrombophilia (see below). III.

PATHOPHYSIOLOGY OF SICKLE CELL DISEASE

A.

The Effect of Sickling

The pathophysiology of sickle cell disease has been reviewed extensively in recent years [20]. Deoxygenated HbS polymerizes and deforms RBC, making them relatively inﬂexible and rendering them unable to traverse the ﬁne capillary beds. After repeated cycles of oxygenation and deoxygenation, some cells become irreversibly sickled. The most important mechanism of sickling seems to be cellular dehydration, which is a consequence of abnormalities in potassium-chloride cotransport. The result of the RBC sickling is plugging of the microcirculation, especially in areas of low oxygen concentration. The bones, heart, lungs, and kidney are particularly aﬀected; cerebrovascular injuries are another serious complication. The vascular pathology is possibly caused by the sticky surface of sickle cells, which favors abnormal interaction between these cells and the endothelium. Destruction of the RBC causes hemolysis and anemia, which contributes to the cardiac ‘‘stress’’ and in addition may contribute to the hypercoagulable state and thrombophilia. B.

HbS—An Unstable Hb

Reactive oxygen species are believed to be at least partially involved in vasoocclusion in sickle cell disease. Approximately, 3% of oxyHb autoxidizes daily to metHb with subsequent formation of reactive oxygen species (ROS), such as superoxide anion radicals [8,21–23]. Therefore, normal RBC are equipped with large amounts of the reducing enzyme superoxide dismutase (SOD). To restore Hb function, metHb must be reduced by the RBC NADH-cytochrome b5 reductase to Hb. In comparison to HbA, HbS exhibits accelerated autoxidation and denaturation [24–26], and increased formation of ROS has been found in sickle RBC [24]. In contrast to the stable metHbA, metHbS is very unstable, resulting in formation of hemichrome (see below) and heme loss, with resultant denaturation and precipitation as Heinz bodies [27]. Degradation of unstable metHbs, such as metHbS, is signiﬁcantly accelerated by external factors such as exposure to oxidative substances [28]. The degradation of the unstable Hbs is associated with marked oxidative membrane damage, such as crosslinking of membrane proteins, membrane lipid peroxidation, and increased permeability to potassium ions [29–31]. The potassium-chloride cotransport in RBC is a redox-controlled membrane transport system, modulated by a cascade of kinases and phosphatases yet to be deﬁned at the molecular level [32]. Recently it was shown that oxidation-induced hemolysis in human RBC modulates the C1--dependent cation conductance of the RBC [33]. Prevention of sickle cell dehydration by use of speciﬁc blockers of ion transport pathways mediating potassium loss from sickle cells has been shown to be a feasible strategy in vitro in transgenic sickle mice and in patients [34]. Several membrane abnormalities have been described in RBC from patients with sickle cell disease, including iron decompartmentalization and abnormal Hb-membrane interactions [35,36]. The increased accumulation of free iron in the sickle cell membrane and membrane-associated denatured Hb (hemichromes) may serve as a catalyst for oxido-reductive reactions [37], leading to protein thiol oxidation and lipid

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peroxidation [37,38]. Sickle cells contain several more oxysterols in comparison to normal RBC, which may be an eﬀect of the increased oxidative stress [39]. The membranes of RBC and sickle RBC from patients with homozygous sickle cell disease are very susceptible to endogenous free radical–mediated damage, and this increased oxidative susceptibility correlates with the proportion of irreversibly sickled cells [40]. The abnormal adherence of sickle RBC (even the oxygenated ones) to endothelial cells has been thought to contribute to vascular occlusion [41], a major cause of morbidity in sickle cell disease. Studies have shown that ROS cause increased adhesion of monocytes and neutrophils to the endothelium. In human umbilical vein endothelial cells (HUVECs) oxidative stress caused a twofold increase in the transendothelial migration of monocyte-like HL-60 cells and a ﬁvefold increase in platelet endothelial cell adhesion molecule-1 (PECAM-1) phosphorylation. This suggests that oxidation triggers cellular signaling pathways, leading to the transendothelial migration of polymorphonuclear leukocytes, a crucial event in the diapedesis of leukocytes during pathophysiology of vascular diseases [42]. Sultana determined that the interaction of sickle RBC with cultured endothelial cells induced cellular oxidant stress that would culminate in the expression of cell adhesion molecules (CAMs) involved in the adhesion and diapedesis of monocytes and the adherence of sickle reticulocytes. The oxidant stress–induced signaling resulted in an increased surface expression of a subset of CAMs, ICAM-1, E-selectin, and VCAM-1 in cultured human umbilical vein endothelial cells [43]. Further experiments suggested that the adherence/contact of sickle RBC to endothelial cells in a large vessel can generate enhanced oxidant stress leading to increased adhesion and diapedesis of monocytes, as well as heightened adherence of sickle reticulocytes, indicating that injury/activation of endothelium can contribute to vasoocclusion in sickle cell disease [43]. Although RBC are well equipped with a wide array of antioxidant defense systems [44], they may become very susceptible to free radical–mediated injury in the case of hereditary hemoglobinopathies [45]. Sickle cells contain lower antioxidant enzyme activities (glutathione peroxidase, catalase) compared with normal RBC, while their SOD activity was found to be signiﬁcantly increased [38]. Observations on the main plasma parameters of oxidative stress in homozygous sickle cell disease showed decreased serum vitamin E, and in addition altered RBC redox status (high GSSG/GSH ratio) was found in homozygous sickle cell disease patients [46,47]. It was recently shown that vitamin E levels were signiﬁcantly lower in transfused sickle cell disease patients in comparison with nontranfused patients [48]. This indicates that transfusional iron overload in sickle cell disease may increase the potential for oxidative stress. Furthermore, plasma low-density lipoproteins (LDL) from patients with sickle cell disease are more susceptible to oxidation than LDL from control subjects [49]. In adults, the acute chest syndrome is the leading cause of death in patients with sickle cell disease, and it was found that increased oxidative stress, measured by generation of F2 isoprostanes, occurs during acute chest syndrome [50]. Nitric oxide (NO) metabolism is altered during the acute chest syndrome, and Hb derivates are known to readily bind NO [51], thus lowering the concentration of free NO, which functions as the endothelial-derived relaxing factor. This scenario might play a role in the pathology of sickle cell disease. ROS, produced in increased amounts in sickle RBC, may be involved in hemolysis and organ damage but could also be innocent bystanders. Since protection from hemolysis and tissue injury can be provided by treatment with antioxidants, it is

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possible that reactive oxidants are responsible, at least in part, for some of the damage associated with sickle cell disease. Antioxidant thiols such as N-acetylcysteine were shown to protect sickle cells from oxidative stress [52], and N-acetylcysteine diminished in vitro the formation of irreversibly sickled cells at high concentrations [53]. In homozygous sickle cell patients, oral vitamin E supplementation decreased the amount of irreversibly sickled RBCs signiﬁcantly [54]. A cocktail containing garlic extract and high-dose vitamins C and E was shown to improve the clinical status of homozygous sickle cell patients [55]. An antioxidant mixture containing vitamins E and C, zinc, and soybean oil decreased irreversibly sickled cells in homozygous sickle cell disease patients, however, without aﬀecting Hb concentrations, RBC age, or number of vasoocclusive crisis [56]. IV.

THALASSEMIA

The thalassemias are characterized by a relative deﬁciency or total absence of one of the major globin chains that compose the Hb molecule. They are classiﬁed as a- or h(+ or 0) thalassemia on the basis of the particular globin chain(s) with impaired synthesis (+) or not synthesized at all (0). Each of these forms of thalassemia is extremely heterogeneous [17]. a-Thalassemia is caused by relative or complete deﬁciency of a-globin chains, while the production of h-globin chains remains unaltered. The clinical severity of the a-thalassemia is highly variable. A normal individual carries two functional a-globin genes (a1 and a2) per haploid genome (aa/aa). The a-thalassemias generally involve inactivation of anywhere from one to all four of these genes, therefore several types of a-thalassemia can be distinguished (Table 2). The a1- and a2-globin genes encode identical a-globin chains of 141 amino acid residues, but the a2-globin gene accounts for twice the amount of a-globin chains produced relative to the a1-globin gene. Consequently, a2 globin mutations generally are associated with more adverse eﬀects than the same mutations of the a1-globin chain [17]. More than 95% of a-thalassemias are caused by deletions, and two deletions are commonly encountered in speciﬁc ethnic groups, (a3,7) and (a4.2), while point mutations in the a-globin genes (ax) only rarely cause thalassemic syndromes. In a+thalassemias, one of the linked a-globin genes is lost by deletion (-a/aa) or by point mutation (axa/aa). In the a0-thalassemias, where both of the linked a-globin chains are lost, the heterozygous genotype is expressed as (– –/aa). In Southeast Asian individuals frequently both a-globin chains on one chromosome are defective, whereas both a-globin genes on the other chromosome are normal (– –/aa). Blacks,

Table 2

Classification of a-Thalassemia Syndromes

Genotype aa/aa (-a/aa), (axa/aa), (aax/aa) (-a/-a), (axa/-a), (aa/- -) (- -/-a), (axa/- -), (aax/- -) (- -/- -)

Phenotype

Clinical condition

Normal Normal Relatively asymptomatic Mild-severe Lethal

Healthy individuals a-Thalassemia silent carrier a-Thalassemia trait HbH disease Hb Bart’s

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however, usually have the alternative arrangement, in which one a-globin gene on each chromosome is functioning normally (–a/–a). Individuals who have a single a-globin chain inactivated either by deletion (–a) or point mutation (ax) usually have a normal blood count and are clinically well because the relative ‘‘mini’’ excess of the remaining h chains is not suﬃcient to cause any signiﬁcant pathological changes in the RBC. These carriers are deﬁned as a-thalassemia silent carriers. Individuals with two defective a-globin genes usually have slightly pathological RBC indices but are clinically asymptomatic and are classiﬁed to have the a-thalassemia trait. Patients with a loss or inactivation of three a-globin chains have moderate to severe anemia and may require transfusions [57], since the excess of h chains in this form of a-thalassemia are still capable of forming a homotetramer (h4), also known as HbH. However, this homotetramer is less stable than a2h2, which make HbA, and eventually precipitates around the half-life of 120 days of the mature RBC and forms inclusion bodies, also known as H bodies, which can be identiﬁed by regular and/or electronic microscopy (Fig. 1). In fetal life, the g chain is the counterpart of the a chain, and together they form fetal Hb a2g2. Around the ﬁrst 3 months after birth there is a switch from g chain synthesis to h chain synthesis by a mechanism that still has not been fully elucidated. If g chains continue to be synthesized after birth, they can also form a tetramer of g chains known as Hb Bart’s (g4). Both HbH and Hb Bart’s have a very high aﬃnity to oxygen and are ‘‘useless’’ oxygen carriers. In fact, babies who have a complete absence

Figure 1

Phase contrast micrograph showing the presence of at least one inclusion body (Heinz bodies) in every red cell ghost of h-thalassemia major.

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of a chains and have only Hb Bart’s cannot survive after birth due to a lack of oxygen delivery to the tissues. Such infants die in utero with a clinical picture known as ‘‘hydrops fetalis.’’ In h-thalassemia the h-globin chains are insuﬃciently synthesized, and therefore a-globin chains are in excess and are also capable of forming homotetramers. However, these tetramers are very unstable and disintegrate shortly after being formed and precipitate within the RBC, even in nucleated RBC and reticulocytes, leading to their premature destruction in the bone marrow and marked trapping in the spleen. The h-thalassemias result from a variety of molecular defects that either reduce the synthesis of h-globin (h-thalassemia) or completely abolish it (h0-thalassemias). Approximately 200 diﬀerent mutations have been described in patients with hthalassemia [58]. Each aﬀected population has a small number of common mutations as well as rarer ones. Eighty percent of the mutations are associated with only 20 diﬀerent haplotypes [59]. Hardison constructed a Web-accessible database of Hb variants and thalassemia mutations [60]. Most commonly, h-thalassemias result from point mutations of the h-globin gene and only rarely from gross gene deletions. Because of the wide diversity of mutations, most individuals with h-thalassemia major are in fact compound heterozygotes, having inherited a diﬀerent mutation from each parent. The h-globin gene can also be silenced by deletions involving the h-globin gene cluster that remove either the y and h genes or all the genes of the cluster. For example, hereditary persistence of fetal Hb (HPFH) and the yh- and qgyh thalassemias, which are quite rare, result from such gross gene deletions [1]. The h-thalassemias have extremely diverse clinical phenotypes. For clinical purposes, h-thalassemia is divided into thalassemia major (transfusion dependent), thalassemia intermedia (of intermediate severity), and thalassemia minor (asymptomatic or mild severity). The severity of h-thalassemia is related to the degree of globin chain imbalance. Patients with thalassemia major present in the ﬁrst year of life with severe anemia; they are unable to maintain a Hb level of f5 g/dL [61]. Heterozygotes for hthalassemia are known to have thalassemia minor and are asymptomatic (silent carriers). They carry one normal h-globin gene, while the other gene has a mutation that reduces partially or completely the synthesis of h globin. It is usually impossible to tell by looking at the h-globin protein levels in such a person whether the thalassemia allele carried is a h+ or a h0 mutation, because the normal chromosome is producing the vast majority of h globin in both situations. Individuals who carry mutations in both of their h-globin chains are signiﬁcantly anemic but usually do not require transfusions. One or both of these mutations is relatively mild (h+/h0) or (h+/h+), so that a signiﬁcant amount of h globin production still occurs. Clinically, they are deﬁned as having h-thalassemia intermedia. An alternative mechanism for thalassemia intermedia is for an individual to have a-thalassemia with triple a genes (aaa genes) along with h+-thalassemia with the consequent signiﬁcant excess of remaining a chains to cause anemia. If both h-globin chains are completely abolished (h0/h0), there will be no synthesis of HbA and consequently severe anemia, which is transfusion dependent, and encounter a variety of serious medical problems throughout the life of the aﬀected individuals. This clinical condition is deﬁned as h-thalassemia major (Table 3). The broad spectrum of h-thalassemia alleles can produce a wide variety of diﬀerent h-thalassemia phenotypes, but a wide phenotypic diversity exists even among individuals with the same h-thalassemia genotype. Some of this diversity can be

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Table 3

313

Classification of h-Thalassemia Syndromes

Genotype

Phenotype

Clinical condition

(h/h) (h/h+), (h/h0) (h+/h0), (h+/h+) (h0/h0)

Normal Normal Mild-severe Severe

Healthy individuals h-Thalassemia minor h-Thalassemia intermedia h-Thalassemia major

explained by secondary and tertiary modifying factors involving other genetic loci [62]. Co-inheritance of a-thalassemia may reduce the severity of the globin chain imbalance and consequently result in a milder form of the disease. Some carriers of h-thalassemia mutations have tripled or quadrupled a-globin genes with an increased imbalance of a and h globin synthesis [63]. The interaction of h-thalassemia with other structural Hb variants, such as HbS and HbE, are of global importance [64]. HbE, the most common Hb variant, is synthesized at a slightly reduced rate and has a homozygous phenotype similar to heterozygous h-thalassemia. Yet when it is inherited together with a hthalassemia allele, the resulting condition, HbE/h-thalassemia, the most frequent form of hemoglobinopathy in Southeast Asia, is sometimes characterized by a severe, transfusion-dependent thalassemia major [65]. A.

Pathophysiology of Thalassemia

The pathophysiology of the a-thalassemias has much in common with that of the hthalassemias, but there are also some distinct diﬀerences, resulting mainly from the properties of the globin that is produced in excess. Normal tetrameric Hb is a highly stable and very soluble protein, while the individual a chains which accumulate in h-thalassemia are not. The h4 and g4 tetrameters are soluble and relatively stable. The ineﬀective erythropoiesis and hemolysis characteristic of all forms of thalassemia is a consequence of the excess of the unstable individual a or h chains. As mentioned, excess a and h chains are relatively unstable, dissociate into monomers, and then experience a change in their tertiary structure; they are oxidized ﬁrst to metHb and then to hemichrome, which is unstable and precipitates with time. The basic structure of hemichromes results from the covalent binding of the distal histidine E7 to the sixth coordination position of the heme iron in a similar axial arrangement as that of the distal histidine F8 (Fig. 2). The steps in this cascade after the precipitation of hemichrome is heme loss, release of free ‘‘toxic’’ iron species, which are not bound to transferrin (NTBI), and precipitation of the protein moiety of the globin. The outcome of this chain of events is excess formation of oxygen free radicals catalyzed by free iron, with a variety of deleterious eﬀects on RBC membrane components (e.g., lipids, proteins) and induction of premature apoptosis and possibly also direct impairment of DNA replication and cell division [64,66–68]. The underlying cause of organ pathology in thalassemia is the premature destruction of RBC, both in the bone marrow and by the reticuloendothelial system. Iron overload of tissues, which will be fatal with or without transfusion if not adequately treated, is the most important complication of h-thalassemia and is a major focus of management [69]. Iron-induced liver and heart damage are the most serious and life-threatening long-term complications.

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The oxidation of either a- or h- globin subunits results in the ultimate formation of hemichromes. The rate of formation of hemichromes is a prime determinant for the severity of anemia.

Figure 2

Ineﬀective erythropoiesis results in a compensatory massive erythroid hyperplasia, causing bone deformities and pathological fractures as well as marked enlargement of the liver and spleen. The massive consumption of caloric and vitamin resources devoted to the useless process of ineﬀective erythropoiesis causes developmental disturbances and increased susceptibility to infections [5]. In thalassemia major, clinical signs become apparent during the ﬁrst year of life, after the switch from g to h chains has been completed. The mainstay of existing therapies for severe h-thalassemia is chronic transfusion therapy in order to overcome the severe anemia in addition to intensive iron chelation therapy in order to relieve the body of the excess iron delivered by repeated ongoing blood transfusions. There are additional supportive therapies for thalassemia, such as erythropoietin to increase the number of RBC and consequently the basic circulating Hb level [70]. Preliminary trials have been conducted with g chain inducers, such as hydroxyurea and butyrates, that will increase the levels of fetal Hb [71], and recently the addition of antioxidants has been implemented. Excess globin chains bind to diﬀerent membrane proteins and alter their structure and function. The pattern is diﬀerent in a- and h-thalassemia. In hthalassemia the insoluble a chains aggregate at the RBC membrane. The heme, hemichromes, and iron component of a globin presumably serve as foci for generation of ROS. This presumably causes partial oxidation of membrane protein (band 4.1) and a decrease in the spectrin/band 3 ratio in h-thalassemia [72]. In a-thalassemia the g and h chains produced in excess do not precipitate right away, but form the soluble tetramers g4 (Hb Bart’s) and h4 (HbH). HbH contains two reactive SH groups per h chain, while the h chains in HbA have only one ractive SH group in the oxy form. Thus, it might be less stable than HbA and have an increased susceptibility to oxidation. Indeed, the generation of HbH is associated with increased hemichrome formation [73], and HbH precipitates during the aging of the RBCs to form Heinz bodies. In a-thalassemia there is no evidence of band 4.1 oxidation, and the membranes are hyperstable (Table 4). However, the membranes of a-thalassemic cells presumably undergo oxidative damage by broadly similar mechanisms to those observed in h-thalassemia. It has been observed that h-globin chains lose heme eight times faster than a-globin chains, but that a chains are more likely to auto-oxidize.

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Table 4

Differences Between a- and h-Thalassemia

a-Thalassemia Relatively stable tetramers of g4 > h4 Slow rate of hemichrome formation and heme loss Protein band 4.1 not oxidized Moderate degree of iron overload

B.

315

h-Thalassemia Unstable tetramer of a4 Rapid rate of hemichrome formation and heme loss Protein band 4.1 oxidized Severe degree of iron overload

Redox Imbalance in Thalassemia

Destruction of h-thalassemic RBC is largely due to increased oxidative stress to these cells due to the instability of excess a globin chain interaction with the RBC membrane, which is presumably triggered by their increased binding [74–76]. Additional oxidative stress may be imposed by the higher metabolic turnover due to increased protein synthesis of the remaining globin chain. Therefore, HbH (h4) chains prossess higher peroxidative reactivity than the normal tetramer of HbA (a2h2). Under oxidative stress, HbA can trigger oxidation of low-density lipoproteins (LDL). It was shown that the order of relative oxidation of LDL protein and lipids was a chains > h chains > HbA, indicating that extracellular globin chains may be the trigger of lipoproteins alterations observed in h-thalassemia [77]. Furthermore, high intracellular and plasma concentrations of non–transferrin-bound iron (NTBI) most probably contributes to the generation of ROS. The antioxidant status in h-thalassemia patients is compromised, and lipid membrane peroxidation products are increased in the blood [78,79]. Inconsistent changes were also observed in the reducing enzymes in thalassemic RBC. A signiﬁcant increase in thiobarbituric acid–reactive substances and a compensatory increase in superoxide dismutase and glutathione peroxidase activities were found in h-thalassemia major patients [79]. In h-thalassemia intermedia patients, glutathione (GSH) content in RBC was remarkably decreased and catalase and glutathione reductase activity were normal. Additional antioxidant enzymes were found to have increased activity [80]. In other studies, patients with hthalassemia major had decreased plasma levels of selenium and glutathione peroxidase activity [81] and decreased ascorbic acid level of platelets [82]. The mechanism by which normal RBC are selected for removal from the circulation has not yet been fully elucidated, but it seems to be obvious that Hb denaturation by ROS plays an important role in RBC survival [83]. Membrane protein thiol oxidation seems to play a role, but is not an obligatory step in the sequence of events leading to premature sequestration in, e.g., xenobiotic-induced hemolysis [84].A summary of the pathophysiological changes occurring in thalassemic cells and possible sites for therapeutic intervention is depicted in Figure 3. 1.

The Role of Iron

Iron can be readily released from Hb in a number of conditions in which RBC are subject to oxidative stress. It was recently demonstrated that iron-dependent oxidative reactions in h-thalassemic RBC membranes are involved in premature cell removal and anemia [85,86]. The current understanding of the role of iron in the deleterious oxidation of biomolecules was recently reviewed [87]. The released iron could have a

Figure 3 Pathophysiological processes within and outside red blood cells and possible sites for therapeutic interventions in a- and h-thalassemia.

316 Fuchs et al.

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role in the oxidation of membrane proteins and the formation of senescent cell antigen formation, one of the major pathways for RBC removal from the peripheral blood [88]. A study investigating the iron and peroxidation status in regularly transfused hthalassemia major patients and in untransfused h-thalassemia intermedia patients found signiﬁcant diﬀerences in their oxidative stress parameters. Free and total malondialdehyde (MDA) and NTBI levels were higher in the regularly transfused h-thalassemia major patients compared with patients with untransfused h-thalassemia intermedia. In transfused patients, the free MDA levels correlated positively with serum iron, whereas the total MDA correlated positively with the NTBI levels. A negative correlation was observed between the total peroxyl radical–trapping activity and NTBI levels in patients with h-thalassemia intermedia. There was no signiﬁcant correlation between free or total MDA and total peroxyl radical–trapping activity or NTBI levels [89]. These results clearly highlight the oxidative imbalance in diﬀerent groups of h-thalassemia patients. 2.

The Role of Vitamin E

Severe h-thalassemia patients are frequently vitamin E [90–92] and vitamin C deﬁcient [92]. Several studies have investigated the eﬀect of vitamin E supplementation on RBC survival (see, e.g., Ref. 93) and peroxidative status. In patients with homozygous h-thalassemia, parenteral administration of vitamin E was more eﬀective than oral administration in improving RBC survival [94]. In h-thalassemia heterozygotes, high-dose oral vitamin E decreased RBC lipid peroxidation and increased RBC survival, but the hematological parameters remained unchanged [95]. Oral treatment of h-thalassemia intermedia patients with vitamin E improved the antioxidant/prooxidant balance in plasma [96]. Another study showed that the impaired osmotic fragility of RBC obtained from patients with h-thalassemia returned almost to normal following administration of vitamin E in a daily dose of 750 IU for 3–6 months [97]. The summary of all the studies pertaining to vitamin E in thalassemia indicates that the levels of the vitamin are decreased, most probably due to its increased consumption in order to neutralize the ROS formed in excess. Administration of vitamin E resulted in improvement in various pathological parameters of the aﬀected RBC. However, these positive changes were insuﬃcient to change signiﬁcantly the number of circulating RBC and increase the basic Hb level. One reason for this could be that vitamin E by itself is insuﬃcient to induce the required changes, and one has to consider administration of additional antioxidants to supplement the eﬀect of vitamin E. For instance, N-acetylcysteine, a protein antioxidant, has been shown to improve several parameters in oxidized sickle RBC [98]. In addition, the oral iron chelator LI (deferiprone) has been shown to penetrate the RBC membrane and chelate NTBI in both sickle cells and thalassemic RBC [99]. Consequently, one can design a protocol where all three and perhaps more antioxidants will be given together, and the combined eﬀect may induce more signiﬁcant positive changes in the number of RBC and Hb levels, which will yield a positive objective clinical response. Such a protocol is currently in progress. V.

HYPERCOAGULABLE STATE IN HEMOGLOBINOPATHIES

In both major hemoglobinopathies (i.e., sickle cell anemia and thalassemia) as well as in other congenital anemias such as hereditary dyserythropoietic anemia (HEMPAS)

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as well as in normal neonatal RBC, cumulative data suggest the existence of a hypercoagulable state, which may result in thrombophilia. In sickle cell anemia, thromboembolic phenomena were historically related to the sickling of the RBC and the vessel wall (see above), while in thalassemia, clinical manifestations of thrombophilia are not a common complication. However, recent data suggest that this is not exactly the case. In both sickle cell anemia and thalassemia, it has been shown that one of the consequences of lipid peroxidation of the RBC membrane is a ‘‘ﬂip-ﬂop’’ change resulting in the exposure of phosphatidylserine (PS) from the inner lipid membrane bilayer to the outer bilayer of the RBC [100,101]. PS exposure activated the prothrombinase complex and generates thrombin in vivo. In a recent report [102] using a speciﬁc ﬂuorescent technique to detect ROS by FACS in RBC grown in liquid culture, a direct correlation between PS exposure and ROS generation in normal stimulated and thalassemic RBC ws found. In fact, the magnitude of both PS and ROS was decreased following incubation of the cells with N-acetylcysteine [102]. In both sickle cell anemia and thalassemia there was a correlation between PS exposure of RBC and hemostatic markers of both thrombin generation and activation of ﬁbrinolysis [100,101]. PS exposure also occurs in platelets in the latter diseases. While a direct correlation was found between activated platelets and annexin V binding to PS in thalassemic RBC [101], there was no correlation between PS exposure of platelets with other hemostatic markers in sickle cell anemia [100]. RBC intercellular interactions, i.e., self-aggregation and adherence to endothelial cells, play important roles in microcirculation and result in thromboembolic phenomena. The RBC ﬂow properties are determined by cell membrane components, which are prone to damaging ROS produced in oxidative stress states. Several studies have been performed in sickle cell disease demonstrating the various interactions between the pathological RBC and endothelial cells. In h-thalassemia patients, increased RBC adherence to endothelial cell was found [103]. However, data about the results of the interaction between the thalassemic RBC and the vessel wall remain to be studied. The prevalence of clinical presentation of thrombophilia in hemoglobinopathies is not entirely clear. It is not easy to distinguish in sickle cell anemia the etiology of vascular events between the sickling phenomena and the hypercoagulability induced by ROS PS–exposed RBC. Only recently is more attention being paid to the clinical manifestations of the hypercoagulable state in thalassemia. More studies are indicated, particularly in the areas where treatment with regular blood transfusions is not available, in order to assess the magnitude of the problem and, consequently, to decide whether treatment, for instance with aspirin as an antiplatelet aggregant, is indicated. VI.

CONCLUSION

The data presented above demonstrate that diﬀerent genetic insults to the Hb molecule, which eventually result in the production of abnormal circulating RBC, ignite a chain of similar deleterious reactions in the cell. These reactions are mediated by ROS no matter the primary cause and will result in premature denaturation and precipitation of pathological RBC with consequent damage to almost all organs in the

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body. Prevention of or neutralizing the ill eﬀects of ROS may result in symptomatic improvement by ameliorating the damage to the aﬀected RBC. REFERENCES 1. 2. 3. 4. 5. 6. 7.

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15 Association of Oxidative Stress with Cataractogenesis MARJORIE F. LOU University of Nebraska, Lincoln, Nebraska, U.S.A.

I.

INTRODUCTION

The cataract is a complex eye disease. Just based on the anatomical location of the lens opacity, it can be classiﬁed into cortical, nuclear, posterior subcapsular, and mixed cataracts. Based on etiology, it can be divided into senile or the age-related cataract, congenital and juvenile cataract, traumatic cataract, or cataract associated with intraocular diseases (such as uveitis/inﬂammation, glaucoma, or retinal degeneration), cataract associated with systemic disease (such as diabetes, Lowe’s, homocystinuria, Marfan’s, or Wilson’s), or cataract caused by noxious agents (such as the ionizing radiation of ultraviolet light and x-ray, and drug induced such as steroids, naphthalene, lovastatin, and selenium). Of all these cataracts, the age-related type is most common in humans and covers all three types of cortical, nuclear, and posterior subcapsular cataracts, which diﬀer both in the location of the opacity where they originate and in the pathology underlying the opaciﬁcation. It often begins as a pure type of cataract and later matures into a mixed type of cataract. Cortical cataracts characteristically show opacity in the outer cortical region (about 25% of the lens) with vacuoles, water clefts, and spokes. The likely mechanism for such cataract formation may derive from an osmotic imbalance, which may originate from damage of the cell membrane where the normal permeability and active transport systems are destroyed and unable to maintain ionic and metabolic homeostasis, resulting in an inﬂux of water. Vacuoles or ‘‘lakes’’ containing this water have a low refractive index relative to the protein-rich cytoplasm in the ﬁber cells. Therefore, discontinuities are created, resulting in light scattering and cataract. The opacity of nuclear cataracts is located in the nucleus or the oldest, central region of the lens. The 325

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alteration appears to be an acceleration of processes that occur normally in aging, involving a large number of posttranslational modiﬁed proteins, likely from oxidation, leading to large protein aggregates that scatter light. This type of cataract, unlike the cortical type, can become very hard and less hydrated than age-matched normal lenses. Posterior subcapsular cataracts occur at the posterior region of the lens immediately beneath the capsule. It likely results from improper posterior suture formation and abnormal diﬀerentiation of the ﬁber cells. It can also be a result of radiation or longterm corticosteroid use or be a consequence of retina degeneration. Human congenital cataracts also display a wide phenotypic and genetic heterogeneity. At least eight genes have been associated with autosomal dominant congenital cataracts based on the currently identiﬁed genetic mutations. The examples of inherited cataract phenotypes include central anterior polar cataract, posterior polar cataract, nuclear cataract, pulverulent cataract with dust-like opacities, and cerulean cataract with blue-white pinhead and wedge-shaped opacities. Several candidate genes are connected to cataract formation. Mutations of lens structural protein (alpha, beta, and gamma crystallins) genes often result in cataract formation. The gamma-crystallin gene cluster at loci 2q33-q35 consists of several gamma subspecies. Missense mutations of gammaC and gammaD genes are considered to associate with Coppock-like (pulverulent) cataract and a progressive nuclear cataract, respectively. Mutation apparently drastically lowered the protein solubility. Such is the case in gammaD R36S or R58H mutation, in which crystallization of gammaD has been reported. The phenotype of R58H mutation is linked to the aculeiform cataract. Missense mutation of the crystallin betaB2 gene at loci 22q is known to result in a cerulean cataract. Mutation of the alpha-crystallin gene is also cataractogenic. A missense mutation in the crytallin alphaA gene has been identiﬁed in a family with zonular nuclear cataract. Connexins, the protein family that forms an integral part of the gap junction network of lens ﬁber membrane, are known to directly link to cataract when mutation occurs at connexin 46 gene on 13q. Mutation of the gene encoding the major intrinsic protein of the lens membrane (MIP) is found to associate with polymorphic and lamellar cataract in humans, while mutation within the homeobox gene, PITX3 (at 10q25), is shown to associate with the congenital cataract along with other anterior segment abnormalities. Human senile cataract is considered a multifactorial-induced disease. Several risk factors are deemed important in cataractogenesis. These include oxidative stress, hyperglycemia, ultraviolet light–exposure, cigarette smoking, genetic factors, and aging [1,2]. Oxidative stress is thought by many to be the most important risk factor in senile cataract formation [3–7]. This chapter reviews the current concepts and advances in the understanding of the biochemical mechanism of age-related cataracts from the viewpoint of oxidative stress. Previous reviews on this subject [3–7] are recommended for in-depth reading. II.

THE LENS AND CATARACTOGENESIS

The lens is an avascular organ whose major function is to maintain transparency so that light can be transmitted and focused on the retina. It increases in weight and thickness throughout life, with new lens ﬁbers being continuously derived from the active monolayer of epithelial cells. This process occurs at the equator of the lens. The elongating ﬁbers diﬀerentiate and divide, eventually losing their nuclei and other or-

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ganelles. This process allows the older cells to be continuously compressed toward the center of the lens, with newly formed ﬁbers building up layers on the outside. Therefore, the lens nucleus represents the oldest lens ﬁbers and is the oldest tissue of the animal body. The region between the epithelial monolayer and the nucleus is the cortex, which is relatively younger and, apart from the epithelium, the most metabolically active region of the lens [8]. Therefore, the lens is often considered a good model system for studies of aging and developmental biology. A unique feature of the lens is its high protein content. The whole lens may have a protein content equal to 35% of its weight; in the center region it can be 65–70%. Over 90% of the total are proteins characteristic to the lens, called crystallins. Alphacrystallin is the main protein, followed by beta- and gamma-crystallin, each with several isoforms. Delaye and Tardieu [9] proposed that these proteins may be packed in a short range spatial order so that a smooth gradient of refractive index from the periphery to the center of the lens is achieved, allowing the lens to be transparent and to have necessary refractive power. Another unique feature of the lens is its high content of a natural antioxidant glutathione (GSH), which averages 4–6 mM, depending on the animal species [10]. The lens maintains GSH in a reduced state and apparently uses it to counteract oxidative stress and to protect proteins from oxidative damage [11]. As the lens ages, it tends to lose transparency, causing impaired vision. This agerelated cataract (the senile cataract) is the leading cause of blindness in the world, accounting for about 45% of all blindness. More than half of the population over 65 years of age will develop a certain degree of lens opacity [1]. According to the World Health Organization (WHO), over 20 million people worldwide are blind due to cataracts. With increasing life expectancy, the number of cases of blindness from this disorder may double by the year 2010 [12]. Currently, surgery is the only means to restore vision lost due to cataracts. In the United States, cataract surgery is the most frequently performed surgical procedure among 30 million Medicare beneﬁciaries. Approximately 1.35 million cataract extractions per year are performed. It is the single largest cost in the Medicare budget, at $3.5 billion per year. As the age of our population increases, it becomes more and more costly to maintain a cataract-free population. Therefore, it is important to study the mechanism of cataract formation so that new therapeutic approaches can be developed. A delay in cataract progression of even 5–10 years would be beneﬁcial and could decrease the need for cataract extraction by 50%, saving billions of dollars in health care [13]. III.

OXIDATIVE STRESS

Oxidative stress can be deﬁned as the biochemical damage and morphological changes resulting directly or indirectly from oxidation. This chemical oxidation can be derived from cellular sources generated in situ or from sources in the environment. For the eye lens, such damage often has been associated with loss of transparency. The best example of a cellular oxidant is hydrogen peroxide (H2O2), which has been found in the aqueous humor of the eye [14]. The most commonly observed damage is to cytosolic and membrane proteins in which the thiol groups form disulﬁde bridges and high molecular weight (HMW) protein aggregates, which eventually become water insoluble and lead to light scattering [5,7]. The other contributor to oxidative stress is light, in particular, ultraviolet light. This photooxidative stress results from light

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absorption by the constituents of the lens. This aspect of photooxidation is beyond the scope of this review. Several excellent reviews on this subject [6,15] are recommended for further reading. IV.

SOURCES OF OXIDATIVE STRESS

Eukaryotic aerobic organisms, which require oxygen for their survival, also suﬀer simultaneously from the inherent danger of the reactive oxygen species (ROS) generated from molecular oxygen [16,17]. The ROS group includes superoxide anion radical (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH). Superoxide is formed in the mitochondrial electron transport chain and during cellular response to inﬂammation. Superoxide can be dismutated nonenzymatically or by superoxide dismutase enzymes to produce the relatively stable H2O2. Hydrogen peroxide can also be generated in a site-speciﬁc, metal-catalyzed reaction when a protein-bound transition metal ligand (Cu2+ or Fe3+) is being reduced by ascorbate (AH2) or other reducing agents [18]: 2 AH2 þ 2 Fe3þ þ 2O2 ! 2 Fe2þ þ H2 O2 þ 2 AH þ O2 H2O2 can readily form a very reactive radical, OH, by receiving one electron from Fe2+. This reaction, discovered by Fenton [19] is: Fe2þ þ H2 O2 ! 2 OH þ Fe3þ The OH radical has a very short half-life and can be quickly dissipated to water molecule. Superoxide reacts mainly with proteins that contain a transition metal, damages the amino acid proximal to the metal catalyst, and causes loss of certain biological function. Hydroxyl radical can react with all biomolecules and readily oxidizing proteins, lipids, carbohydrates, or nucleic acid [20,21]. The toxic eﬀect of H2O2 orignates both in its ability to form the damaging hydroxyl radicals and in its direct damage to biomolecules containing sulfhydryl groups or iron and copper moieties [17]. The stability of H2O2 and its ability to cross membranes easily enhances its capability to damage cells. H2O2 is probably the most common and harmful oxidant in biological systems, including the lens. Because of the anatomical locale, the lens can receive ROS from both its anterior and posterior sides. ROS generated from iris, ciliary body, or corneal endothelial cells can be collected in the anterior chamber and diﬀused into the lens [22], while the ROS (such as lipid peroxide) generated from the retina, collected in the vitreous body, can be diﬀused posteriorly into the lens [23]. The best example for a cellular oxidant is H2O2, which is the most stable species present in the aqueous humor [24]. Therefore, the lens is accessible and vulnerable to oxidative stress, and often cataract formation is a ﬁnal consequence. V.

OXIDATION DEFENSE AND DAMAGE REPAIR IN THE LENS

Like other organs, the lens has a well-designed system of defenses against oxidation [3]. It uses primary defenses to neutralize oxidative species and to repair, recover, or degrade molecules that do become damaged. The primary antioxidants include non-

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enzymatic (e.g., glutathione, vitamin C, vitamin E, and carotenoids) and enzymatic (e.g., superoxide dismutase, glutathione peroxidase, and catalase) systems. The repair systems include enzymes that can degrade the damaged proteins or repair damaged nucleic acids [5,6]. Another recently identiﬁed enzyme thioltransferase (also called glutaredoxin) [25,26] may be critical in maintaining the lens in a reduced state by cleaving protein-thiol mixed disulﬁde bonds formed on the oxidation of lens proteins. The NADPH-dependent thioredoxin/thioredoxin reductase system [27] is very eﬀective in reducing protein-protein disulﬁde bonds. This system is likely involved in maintaining the thiol/disulﬁde homeostasis in the lens since both thioredoxin [28,29] and thioredoxin reductase [30] have recently been found in the lens. Under oxidative stress some of these defense enzymes are upregulated as much as 50-fold, as in the case of catalase in mouse lens epithelial cells adapted to long-term exposure to H2O2 [31]. Raghavachari et al. [32] recently reported that thioltransferase could be transiently upregulated two- to threefold in the human lens epithelial cells when the cells were brieﬂy exposed to a bolus of low-level H2O2. The oxidation defense system in the lens is summarized in Figure 1. A.

Primary Defense

Of several small molecular antioxidants, glutathione (GSH) is perhaps the most important in the lens. This sulfhydryl-containing tripeptide, g-glutamyl-cysteinyl-glycine, is highly abundant in the lens (4–6 mM) [33]. GSH can be synthesized by the lens

Figure 1 The primary oxidative defense systems in the lens. H2O2, generated by the dismutation of superoxide anion or by the reaction between ascorbate and Fe3+, can be degraded by several pathways. These include catalase, glutathione peroxidase, and Fenton reaction. The decrease in the SH/S-S ratio by oxidation can be reversed by the glutathione reductase (GR)–pentose phosphate shunt cycle and by thioltransferase (TTase). These mechanisms protect the lens from oxidative damage. GR: glutathione reductase; GPx: glutathione peroxidase; SOD: superoxide dismutase; TTase: thioltransferase. (From Ref. 52.)

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[34], and its reduced form is believed to protect oxidation-sensitive-SH groups in enzymes and structural proteins [10]. Oxidized glutathione (GSSG) molecules, generated from GSH by oxidative stress, can be reduced back to GSH by glutathione reductase and NADPH. Thus, cellular GSH is always available and ready for a new round of oxidant attack. A more detailed review of this subject is available in previous publications [10,11]. Ascorbic acid (vitamin C), another water-soluble antioxidant in the lens, is present at a relatively high concentration (mM level) [35]. Most lenticular ascorbate originates from the diet. Ascorbate can be oxidized to dehydroascorbate, which in turn is reduced by GSH. Another important antioxidant is the lipid-soluble vitamin E, the main function of which is to protect membranes from oxidation. Epidemiological surveys showed that high lenticular concentrations and high dietary intake of both vitamins C and E correlated well with a lower incidence of cataracts [36]. Carotenoids (vitamin A precursors) are other lipid-soluble, lenticular antioxidants obtained from food intake [36]. Carotenoids may also contribute to lenticular defense against oxidants. The enzymatic antioxidant defense systems in the lens involve several enzymes, including catalase [37], glutathione peroxidase [38], and superoxide dismutase [39]. Superoxide dismutase detoxiﬁes superoxide radicals and produces H2O2 and O2 [16]. Both glutathione peroxidase [40] and catalase [41] detoxify H2O2 molecules by reducing them to H2O and O2. Glutathione peroxidase catalyzes the reaction of GSH and peroxides to form GSSG, which is recycled to GSH by glutathione reductase and NADPH. This process permits the continuous regeneration of GSH. These reactions illustrated the importance of GSH in the lens. Catalase is another enzyme that detoxiﬁes H2O2. In tissues, including the lens, catalase removes H2O2 in high concentrations, whereas glutathione peroxidase removes H2O2 at lower concentrations [42,43]. Catalase resides mainly in the membrane, whereas most glutathione peroxidases are located in the cytosol. Thus it has been suggested that glutathione peroxidase reduces cytoplasmic H2O2, whereas catalase reduces membrane lipid peroxides [44]. DT diaphorase (quinone reductase) [45], an antioxidant enzyme, reduces divalent quinones, such as quinonoid drugs and other environmental agents, before they can be conjugated and eliminated by other enzyme systems. B.

Repair Systems

Extensive studies in other tissues have revealed repair mechanisms in the cells that counteract oxidant-induced damage, provided the damage is not so severe that it is irreparable. For example, proteolytic enzymes recognize oxidatively damaged proteins and degrade and remove them, so that the cells can replace them with other de novo synthesized protein molecules. A series of in vitro experiments have shown that the lens also uses proteolysis to control the accumulation of oxidatively damaged proteins [46,47]. These proteolytic systems use ATP or ubiquitin for their reactions. Ubiquitin is a tag protein that is found in almost all tissues, including the lens [48]. It conjugates with a target protein (in this case, an oxidatively damaged protein) to signal the degradation process. Wagner and colleagues [49] ﬁrst demonstrated that bovine lens proteasome, a complex of proteolytic enzymes, can preferentially hydrolyze oxidatively

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modiﬁed glutamine synthetase but not the native protein. Taylor and associates [47] have shown that bovine epithelial cells can selectively degrade OH-oxidized acrystallin much faster than untreated proteins. They also showed that oxidative stress alters the proteolytic pathway, thus reducing cells’ clean-up mechanism [50]. Similarly, damaged DNA can be repaired by a series of direct and excision-repair mechanisms. Poly (ADP-ribose) polymerase is the key enzyme for repairing oxidatively damaged DNA in the lens [51]. Thioltransferase (TTase), a thiol-disulﬁde regulatory enzyme, may repair oxidative damage in the lens. Lou et al. [52] showed that TTase is very resistant to oxidative stress in comparison with other oxidative defense enzymes in cultured rabbit lens epithelial cells during H2O2 treatment (Fig. 2). Qiao et al. [25] found that lens TTase is structurally and functionally similar to the TTase present in other tissues (e.g., liver, placenta, red blood cells) [53], where it plays an essential role in maintaining the reduced state in the cells. These authors proposed that TTase can function as part of a primary defense system as well as to repair oxidized lens proteins by cleaving the disulﬁde bonds in protein-thiol mixed disulﬁdes, thus preventing proteins from aggregating with each other. Thioredoxin (TRx), another thiol-regulating and repair enzyme, is reported to be present in the human lens [28,29]. It was found that TRx-1 and TRx-2 (cytosolic and mitochondrial enzyme, respectively) gene expressions were downregulated with age and both TRx proteins were found in all regions of the lens [28]. Furthermore, the gene for the cytosolic TRx-1 was upregulated ﬁvefold in the lens of Emory mouse (a model for age-related human cataract) when the mouse was subjected to photochemical oxidative stress. The upregulation was lens-speciﬁc since the expression of TRx-1 in other organs of the mouse was not changed [54]. These authors proposed that upregulation of TRx-1 is a protective response of the lens to the altered redox status

Figure 2 Effect of bolus H2O2 (0.5 mM) on enzyme activities in cultured rabbit lens epithelial cells (N/N1003A). TTase: thioltransferase; G-3PD: glyceraldehyde-3-phosphate dehydrogenase; GPx: glutathione peroxidase; GR: glutathione reductase. (From Ref. 52.)

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induced by oxidative stress. Addition of E. coli TRx in the culture medium of the lens epithelial cell culture eﬀectively protected the cells from H2O2-induced damage [55]. The enzyme showed reduction of the disulﬁde bonds in the lens water-soluble proteins, restored the initial activity of H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase, and even restored the reducing potential for lens methionine sulfoxide peptide reductase and the membrane function. VI.

ASSOCIATION OF OXIDATION TO AGE-RELATED CATARACT FORMATION

As mentioned above, the lens can protect itself against oxidation quite eﬀectively by utilizing various protecting agents and enzyme systems. However, as the lens ages the de novo synthesis and recycling system for GSH become less eﬃcient [56], causing the net concentration of GSH to decline. The progressive loss of lens GSH during aging (Fig. 3A) has been shown in rats [57] and humans [58,59]. With aging, the mechanisms that protect and repair oxidative damage in the lens and other tissues slowly deteriorate. The sulfhydryl group is most susceptible to oxidation. Thus, as GSH is depleted, protein sulfhydryl groups form intramolecular and intermolecular crosslinks. Many biochemical and physiological alterations in human cataractous lenses result from oxidative damage. In these cataracts, more than 60% of the GSH is depleted [5,56] and in some isolated polypeptides, over 50% of the methionine and nearly all the cysteine moieties are oxidized [4]. In addition, many disulﬁde crosslinks are found in the water-insoluble (WI) protein fraction [3,4]. Most of these disulﬁdes are in HMW aggregates (>1000 kDa) [60]. Although HMW components are also found in the normal lens, they are formed by hydrophobic interactions [61,62]. The light scattering theory of Benedek [63] predicts that when the lens protein aggregates reach a size greater than 5107 daltons, increased light scattering and cataract occurs.

Figure 3

The levels of free GSH, PSSG and PSSC in normal human lenses as a function of aging: (A) Free glutathione (GSH); (B) protein-GSH mixed disulfide (PSSG); (C) proteincysteine mixed disulfide (PSSC). Individual values are expressed in lmol/g wet weight. PSSG was measured as GSO3H, and PSSC was measured as CSO3H. Linear regression lines are shown. (From Ref. 58.)

Association of Oxidative Stress with Cataractogenesis

VII.

333

CURRENT HYPOTHESIS FOR THE BIOCHEMICAL MECHANISM OF OXIDATION-INDUCED CATARACT

Several hypotheses for cataractogenesis have throughout the years been developed, including the role of advanced glycation [64] the loss in a-crystallin chaperone-like function [65], and the phase separation of the aqueous protein-rich and protein-poor phases in the lens [66]. However, the hypothesis with the most consensus support is oxidative stress, although opinions may vary on the initiation site of the oxidative damage in the lens. This may also depend on what type of cataract is formed, since there are diﬀerent cataracts in human lenses, based on where the opacity is observed, including nuclear, subcapsular, cortical, and mixed cataract (where both nuclear and cortical cataracts were found in the same lens). In all cataractous lenses, the most prominant changes are loss in GSH level and increase in disulﬁde bond–containing HMW aggregate formation. The mechanisms of HMW aggregate formation are not understood. Evidence indicates that posttranslational modiﬁcation of proteins and oxidation of membrane lipids precede HMW protein aggregation. Posttranslational modiﬁcation of a protein often exposes functional groups and leads to conformational change. Oxidation of membrane lipids also causes crosslinks between lens proteins and membranes and creates large aggregates. If these damaged proteins accumulate, eventual opaciﬁcation occurs. The nature of these posttranslational modiﬁcations has been the subject of intense study for many years. Glycation of protein by glucose, ascorbate, or other sugar molecules is one major factor [67,68]. Protein thiolation (S-modiﬁcation) through protein-thiol mixed disulﬁde formation has been proposed as a possible mechanism leading to protein disulﬁde crosslinking and high molecular weight aggregation [57]. Oxidative damage to membrane lipids may also initiate cataract formation [69,70]. Taylor and Davies [71] speculate that the inactivation of protease by oxidative stress is responsible for accumulation of highly damaged proteins. Additionaly, a sitespeciﬁc, metal-catalyzed oxidation of proteins has been proposed as the mechanism for cataract formation [18]. In recent years numerous studies have identiﬁed oxidative damage of DNA in lens epithelial cells and upregulation of the genes responsible for apoptosis as important contributing factors to cataractogenesis [72,73]. VIII.

EVIDENCE OF OXIDATIVE STRESS IN HUMAN AGE-RELATED CATARACTS

Much evidence supports the hypothesis that oxidation is the major factor in formation of age-related cataracts. A long list of oxidation-related biochemical changes in human cataractous lenses have been reported (Table 1). A.

Damage to the Membranes

Oxidation of the membrane lipids was observed in cataractous lenses proportional to the severity of the opacity [74]. Borchman and Yapper [75] reported that lipid oxidation increased linearly and uniformly throughout the human lens with age, as represented by the accumulation of malondialdehyde (MDA), a by-product of membrane damage (Fig. 4). MDA was also found to damage the lens by forming crosslinks with proteins, phospholipids, or nucleic acids [69,76–78]. The MDA-

334

Table 1

Lou Indications That Oxidative Stress Is Associated with Human Cataracts

Indication Decrease in protein -SH and increase in protein -SSProtein HMW aggregates Methionine sulfoxide and cysteic acid found in lens proteins Cytosol-membrane proteins with reducible linkage Loss of GSH Increase in protein-thiol mixed disulﬁde formation, including protein-SS-glutathione (PSSG), protein-SS-cysteine (PSSC), and protein-SS-gamma-glutamylcysteine (PSSGC) Lipid peroxidation (MDA) Elevated H2O2 in aqueous humor of cataract patients Modiﬁcation of membrane transport enzymes, Na/K ATPase, Ca2+ ATPase Advanced glycation in proteins DNA strand break Epidemiological studies

Ref. 3,60 61 4,83 4 4,5,10 58,85,93

69,70 23 4,5 68 72 142

Source: Ref. 6.

phospholipid adduct, phosphatidyl ethanolamine-MDA-phosphatidylserine, is highly elevated in cataractous human lenses [78]. Measurement of trans double bonds in the lipids conﬁrmed the earlier report that an age-dependent oxidation of membrane lipid occurs in the human lens [75]. Age-related changes in phospholipid composition of human lens membranes, such as increased sphingomyelin and decreased phosphotidylcholine, may augment the rigidity of ﬁber cell membranes [79,80]. These changes may inﬂuence the function of membrane-associated transport enzymes for ions, such as Na/K-ATPase [81] and Ca2+-ATPase [82].

Figure 4 Malondialdehyde in whole lens homogenates determined chemically. (- - -) 95% confidence interval. The solid line represents the best fit determined by linear regression analysis for all data points. (From Ref. 75.)

Association of Oxidative Stress with Cataractogenesis

B.

335

Damage to Lens Proteins

In normal young lens, no evidence of oxidation is found in cytosolic or membrane proteins. Most thiols in these proteins are buried in the macromolecular structure. In older lenses, however, some oxidation is detected in the membrane proteins [83]. Miesbauer et al. [84], using mass spectrometry, showed that there is little or no oxidation of thiols in water-soluble proteins except aA-crystallin. However, there was extensive oxidation in water-insoluble proteins in a normal lens [85]. With the complete sequence of aA-crystallin known, these authors established the sites of damage to be Cys-131 and Cys-142, which formed an intramolecular disulﬁde bond. Takemoto [86] showed that the amount of this intradisulﬁde bond gradually increased as the lens aged, and in lenses older than 60 years old, there was nearly twice the amount found in a 2-month-old lens (Fig. 5). Takemoto [87,88] subsequently showed that intradisulﬁde bonds were formed in hB2- and hA3/A1-crystallins during cataractogenesis of the human lens but not in the clear lenses. Disulﬁde bonding and methionine oxidation have been identiﬁed by mass spectrometry as a major posttranslational modiﬁcation in several g-crystallin species in aging human lens [89], especially in the water-insoluble portion. Interestingly, hB2-crystallin was most stable and unmodiﬁed in the watersoluble protein portion of an old lens and only showed a small amount of disulﬁde formation in the water-insoluble fraction [90]. Protein thiols conjugated with nonprotein thiol molecules form protein-thiol mixed disulﬁdes [57]. Lou and Dickerson [58] found a postive correlation between aging and elevated protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) (Fig. 3B,C). Interestingly, both these thiol modiﬁcations are intensiﬁed in lenses older than 50 years. In cataractous lenses, most thiols are exposed, and oxidation causes

Figure 5 Relative change in the amount of oxidized forms of cysteine-131 and cysteine142 in alpha-A cyrstallin from normal human lenses as a functin of aging. Each point is the average of three determinations plus or minus the standard deviation. The curve was fitted using second-order regression. (From Ref. 54.)

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them to form either protein-thiol mixed disulﬁde conjugates (PSSG and PSSC) [91], intermolecular, intramolecular protein-protein disulﬁdes, or both [92,93]. PSSG and PSSC formation is most pronounced in brunescent cataracts [91,94]. In advanced brunescent nuclear cataracts, the PSSG content can be four or ﬁve times higher than the GSH content of the same lens. A third protein-thiol species, protein-S-S-gglutamylcysteine (PSSGC) [94], which is barely detectable in a normal old lens, is present in the same magnitude as that of PSSG and PSSC in a cataractous lens. Lou and associates [95] reported a strong correlation of protein-thiol mixed disulﬁde level with the degree of opalescence and brunescence in the human lenses (Table 2). In some nuclear cataracts, the number of protein SH groups is less than 10% of that of a normal lens nucleus [96]. Thiols may be further oxidized to cysteic acid [83,97– 99]. Extensive oxidation of cysteine residues may be responsible for shifting the lens proteins from water-soluble to highly water-insoluble state. Takemoto [100] found intramolecular disulﬁde bonds in a-crystallin in all cataractous lenses. He speculated that such modiﬁcation might diminish the chaperone-like activity of a-crystallin [65] and thus increase the chances for lens proteins to aggregate. Indeed, Cherian and Abraham [101] demonstrated in vitro that modiﬁcation of the thiol groups in aA-crystallin decreased its chaperone-like activity. They also found that a-crystallin from an older lens (>70 years) had only half the chaperone-like activity of a lens younger than 18 years. The role of site-speciﬁc, metal-catalyzed oxidation in lens proteins was proposed to contribute to the age-related changes and cataractogenesis [18]. Metal ions such as copper and iron, which can catalyze oxidation, were also found to be elevated in the human cataractous lenses. However, it was only until recently that the ﬁrst speciﬁc evidence for the presence of HO -damaged proteins in cataractous lenses was provided by Fu et al. [102]. Hydroxyl radical generation is better produced in a diseased lenses, particularly in the nuclear region and closely associated with nuclear cataracts [103].

Table 2

Levels of Nuclear GSH and Protein-Thiol Mixed Disulfides in Human Cataractsa

Group

n

GSH

TPSSX

GSH/TPSSX

% protein thiolated

Control NO-1 NO-2 NO-3 NO-4 I (Y) II (DY) III (B) IV (DB)

3 7 15 37 14 19 27 15 13

1.43F0.59 0.80F0.19 0.58F0.08 0.52F0.05 0.20F0.01 0.73F0.13 0.68F0.07 0.33F0.06 0.13F0.03

0.32F0.08 0.31F0.07 0.84F0.13 0.91F0.17 1.37F0.18 0.47F0.10 0.61F0.06 1.29F0.13 1.55F0.22

4.47 2.58 0.69 0.57 0.15 1.55 1.11 0.26 0.08

2.3F0.5 2.1F0.1 5.6F0.8 7.0F1.1 7.9F1.2 3.1F0.7 4.1F0.4 8.6F0.8 10.1F1.5

Data expressed as lmol/g wet wt, meanFS.D. Control group represents clear, normal lenses. TPSSX: Total protein-thiol mixed disulﬁdes. NO-1 to NO-4 represents the degree of nuclear opacity. Groups I–IV (from yellow to dark brown) represent nuclear pigmentation. Source: Ref. 95. a

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337

Methionine is also prone to oxidative damage through conversion to methionine sulfoxide, which has been found in normal water-insoluble proteins [72], but moreso in cataractous water-insoluble proteins [72,97,104]. Other protein modiﬁcations including glycation, phosphorylation, deamination, C-terminus truncation (in the a-crystallin proteins) were all observed in both normal aged and cataractous lenses but will not be covered in this review. C.

Damage to Oxidation Defense Mechanisms

Two of the earliest changes in aging and cataractous lenses are the loss of the primary antioxidant molecules (GSH, ascorbate) and the decreased activity of antioxidant defense enzymes. GSH decreases by 50–60% in lenses older than 70 years and decreases further in cataractous lenses [58,59,105]. There is little or no GSSG accumulated in the same lens. GSSG can conjugate with protein thiols through thiol exchange to form PSSG [58,59,91–93,106]. PSSG content is 3–5% of the GSH level in the normal lens, but increases in lenses older than 50 years [58] and in all cataractous lenses [94]. Reduced ascorbate level has been demonstrated both in aging guinea pig lenses [107] and in human cataractous lenses [108]. The concentration of cysteine is about 5% of that of GSH in a normal lens and also decreases with aging [109]. The activity of glutathione reductase (GR) decreases with aging and to a greater extent in cataractous lenses [110–113]. The epithelium of cataractous lenses has little or no GR activity, but this activity can be restored by adding the cofactor FAD [113]. Both superoxide dismutase and glutathione peroxidase undergo loss of activity in the cataractous lens [114], but the activity of catalase is not changed in cataractous lenses [74]. IX.

ANIMAL MODELS

A.

Oxidative Stress-Induced Cataract Models

Cataracts have been successfully induced by oxidants both in vivo and in vitro. These models are listed in Table 3. Cataracts can be easily induced and monitored in vitro under organ culture conditions. For instance, medium containing 0.5 mM H2O2 (50– 100 times higher than the observed physiological concentration in the aqueous humor of humans) opaciﬁes rat lens cortex overnight. The lens becomes completely opaque if it is exposed to either a higher H2O2 concentration (>1 mM) or a longer exposure time (3 days) of a less concentrated H2O2 solution. This cataract model has been induced successfully in several mammalian lenses, including human lenses [56,57]. Other oxidants, such as naphthalene dihydrodiol [112], xanthine-xanthine oxidase [116], selenite [117], photo-oxidation [118], and lipid peroxides [119], all have been used to induce cataract. Most of these agents induce cortical cataracts; naphthalene dihydrodiol, however, induces a characteristic perinuclear cataract. It is more diﬃcult to develop oxidative cataract in a live animal. Over many years only a handful of such models have been achieved. Giblin and colleagues [120] used successive exposure to hyperbaric oxygen to induce nuclear cataract in older guinea pigs; the model is meant to mimic the nuclear cataract found in patients after receiving hyperbaric oxygen treatment for other illness [121]. Many biochemical alterations in this nuclear cataract model showed parallel changes as the human cataractous lenses. For instance, the early formation of PSSC and PSSG (Fig. 6) [117], the increased

338

Table 3

Lou Cataract Models Induced by Oxidants or Antioxidant Inhibitors

Models H2O2 (in vitro) Aminotriazol (in vivo) Naphthalene dihydrodiol (in vitro) Naphthalene (in vivo) Selenite (in vitro) Selenite (in vivo) Hyperbaric oxygen (in vitro, in vivo) Photo-oxidation (in vitro, in vivo) Lipid peroxides (in vitro) Lipid peroxides (in vivo) X-ray (in vivo, in vitro) Ultraviolet light (in vitro, in vivo) Xanthine-xanthine oxidase (in vitro) Diquat (in vitro, in vivo) Buthionine sulfoxamine (BSO) (in vivo) Emory mouse (in vivo)

Cataract type

Animal species

Refs.

Cortical Cortical Perinuclear Perinuclear Cortical Nuclear Nuclear Cortical Subcapsular

Most animals, human Rabbit Rat Rabbit, rat Rat, rabbit Neonatal rat Guinea pig, rabbit Rat, monkey Rat, rabbit

Cortical Cortical Cortical Cortical Cortical Cortical

Rabbit Mouse, rabbit, squirrel Rat Rat, rabbit Neonatal mouse Aging mouse

58,134 131 115 129 117 124 120 118 119 23 128 127 116 125 130 132

Source: Ref. 6.

disulﬁde bonds [122], and elevated lipid oxidation and hydrocarbon chain disorder [123] were all more visible in the nuclear area of the guinea pig lens, correlating with the increased lens nuclear opacity. Nuclear cataract can be induced in neonatal rats by subcutaneous injection of selenite [124]. Direct injection of an oxidant in the vicinity of the lens can be an eﬀective technique to induce a cataract. Yu et al. [125] reported that injection of diquat intravitreally into rabbit eyes induced cortical cataract within a few days. Injection of naphthoquinone also induced cortical cataract [126]. Intravitreal injection of docosahexenoic acid into rabbit eyes induced posterior subcapsular cataract within 24 hours. This model has been proposed for retinitis pigmentosa associated–cataract in humans [23]. Zigman and coworkers [127] successfully induced ultraviolet ray– associated cataracts after exposing squirrels to near-ultraviolet light. Studies of xray–induced cortical cataracts were successful with rabbits [128]. Feeding naphthalene to rats can induce perinuclear cataracts within one week [129]. Under conditions in which the oxidation defense systems are compromised, the lens spontaneously becomes opaciﬁed. For instance, Calvin et al. [130] observed that when a near-total depletion of GSH in lenses of young mice was achieved by using a glutathione synthase inhibitor, buthionine sulfoxamine (BSO), opaciﬁcation in the lens occurred in a few days and a mature cataract developed in 2 weeks. Aminotriazole, a speciﬁc inhibitor of catalase, has been found to be a potent cataractogenic agent [131]. Another model of age-related cataract, the Emory mouse [132], which forms cataract spontaneously during aging, mimics cataracts induced by oxidizing agents [133]. Detailed studies of the H2O2-induced cataract model showed that PSSG formation was one of the earliest events, preceding PSSP cross-linking and protein solubility loss [134]. Mass spectrometric studies [135] on the isolated gB-crystallin

Association of Oxidative Stress with Cataractogenesis

339

Figure 6

Concentrations of protein-bound glutathione (A) and protein-bound cysteine (B) in lens cortex and nucleus (total protein fractions) of guinea pigs receiving different numbers of treatments with hyperbaric O2. Ages of control and experimental animals were 19, 20, 24, and 30 months after 15, 30, 65, and 100 O2 treatments, respectively. Error bars indicate S.D. Numbers of lenses analyzed were 12–13, 9–10, 4–8, and 2–3 for 15, 30, 65, and 100 O2 treatments, respectively. Closed circle: nucleus, O2 treated; open circle: nucleus, control; closed triangle: cortex, O2-treated; open triangle: cortex, control. (From Ref. 200.)

from the H2O2-treated lenses showed that the number of protein thiols being thiolated (as PSSG) was dependent on the H2O2 concentration used. PSSG formation also changed the protein conformation, so much so that a previously buried thiol group was exposed and formed an intradisulﬁde bonding [135]. Additional studies indicated that PSSG formation was reversible and the degree of lens opacity was reduced when the oxidant was removed before PSSP formation, suggesting the action of an in situ repair mechanism such as thioltransferase [134]. Besides S-thiolation of lens proteins, methionine was also oxidized to methionine sulfoxide [135]. DNA single-strand breaks were also found in cataract formation in vitro [136].

340

B.

Lou

Gene Overexpression and Gene Knockout Models

The most important evidence to demonstrate the protective role of oxidation defense enzymes is the use of the technique of gene knockout in the mouse. Glutathione peroxidase-1 knockout mouse model has been the subject of studies for recent years. In this knockout mouse model, Spector et al. [137] did not observe any morphological changes over 2 years, but Reddy and associates [138] reported an increase in nuclear light scattering, extensive membrane damage, and cataract formation in the GPx-1 gene knockout mice as a function of age (Fig. 7). Another important antioxidant enzyme, superoxide dismutase (SOD), has also been studied. Reddy et al. [139] reported that when SOD (Mn) gene was overexpressed in human lens epithelial cell line cells (SRA 01/04), the cells were more resistant to the cytotoxic eﬀect of H2O2 with greater cell viability, while the SOD (Mn)–suppressed cells showed dramatic mitochondrial damage and DNA strand break. Behndig et al. [140] irradiated lenses from Cu-Zn SOD knockout mice for 1.5 hours by a daylight ﬂuorescent light (the lenses were preincubated with riboﬂavin) under organ culture conditions. These lenses showed more opaciﬁcation and hydration with less ability for the lens membrane to transport K+ in comparison to normal controls. Similarly, the overexpression of the human thioltransferase gene (TTase) showed stronger protection of the human lens epithelial cells against oxidative stress by H2O2 [141].

Figure 7 Percentage of lenses showing opacification in glutathione peroxidase-1 knockout and control mice by age group. Lenses showing lamellar ring opacify or those with entire lens opacity were compared in the various age groups. The increase in opacities in knockout (K) mice compared with control (C) animals in all groups after 5 months of age are highly significant. The number of lenses examined were: 0–4.9 m (C 32, K 22); 5–9.9 m (C 50, K 70); 10–14.9 m (C 8, K 18); >15 m (C 28, K 38). (From Ref. 138.)

Association of Oxidative Stress with Cataractogenesis

Table 4

341

Antioxidants That Can Protect Against Cataract in Model Systems

Cataract models In vivo Diquat Selenite BSO X-ray Emory mouse Ultraviolet light In vitro H2O2 Photo-oxidation Lipid peroxidation Xanthine-xanthine oxidase

Antioxidants Desferal-Mn (III) (143), Captopril (144) WR cpd (145), pantethine (145), pyruvate (146), ascorbate (147), GIE (YM737) (148) Ascorbate (149), GEE (149), lipoic acid (150), g-GCEE (151) WR cpd (152), GIE (YM737) (153) ALO5741 (154) Vitamin E (155) AL03823 A (156), Tempol H (157), AL05741 (158), AL05712 (158) AL03823 A (156), pyruvate (159) Vitamin E (160) Ascorbate (161)

WR cpd: Walter Reed compound; GEE: glutathione ethyl ester; AL03823 A: glutathione peroxidase mimic (Alcon Laboratories, Fort Worth, TX); AL05741 and its ester form, AL05712: thioltransferase mimic (Alcon Laboratories); Tempol H: tempol hydroxylamine; g-GCEE: gglutamylcysteinyl ethyl ester; GIE: glutathione isopropyl ester. Source: Ref. 6.

X.

PREVENTION OF CATARACT BY ANTIOXIDANTS AND CLINICAL SIGNIFICANCE

Several cataract models mentioned previously have been used to test the eﬃciency of antioxidants in decelerating or preventing cataract. Some natural or synthetic antioxidants or enzyme mimics were moderately eﬃcacious in slowing or preventing cataract. Such antioxidants/anticataract agents are summarized in Table 4. These results suggest that oxidative stress does induce cataract and that reactive oxygen species are the causative agents. Antioxidants may be of clinical value for anticataract use in the future, provided they can be delivered eﬃciently with little or no long-term side eﬀects. In recent years, Ansari and associates [162] have devised a noninvasive technique of dynamic light scattering that can detect quantitatively the lens structure on a molecular level by measuring the sizes of the predominant particles and mapping the three-dimensional topographic distribution of the light scattering macromolecules in an aging lens or cataractous lens. Preliminary studies showed that this powerful tool can provide early detection of cataracts and be useful in monitoring therapeutic treatment of cataract, for example, the anticataract eﬃcacy of pantetheine treatment of selenite cataract in rat lenses [163]. Such a device can be potentially of great value for clinical evaluation of anticataract therapy.

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22. 23.

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16 Oxidative Stress and Age-Related Macular Degeneration PAUL S. BERNSTEIN John A. Moran Eye Center, University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. JERUSHA L. NELSON, JESSICA L. BURROWS, and E. WAYNE ASKEW University of Utah College of Health, Salt Lake City, Utah, U.S.A.

I.

INTRODUCTION

Age-related macular degeneration (AMD) is a devastating disease that is the leading cause of irreversible blindness in the developed world. It is a disorder that results from the complex interaction of numerous inherited and acquired risk factors, including increasing age, smoking, family history, race, light exposure, and nutrition. Many new treatments have been introduced for patients with advanced cases of the wet form of AMD, such as photodynamic therapy and macular translocation surgery, but the vast majority of these patients will still progress to legal blindness. Thus, there is considerable interest among both patients and clinicians to identify individuals at high genetic risk for the disease at a young age so that they can begin to alter modiﬁable risk factors before signiﬁcant damage has begun. Nutritional interventions with antioxidant supplements have been widely touted in the United States for many years to individuals at risk for AMD, but high-quality scientiﬁc data to support the marketing claims have been notably lacking. The recent publication of the results of the Age-Related Eye Disease Study (AREDS) ﬁnally puts some of these nutritional claims on more solid scientiﬁc footing. This very large longterm prospective placebo-controlled study demonstrated that oral supplementation with antioxidant vitamins and minerals (zinc, vitamin C, vitamin E, and h-carotene) could slow the rate of progression to advanced stages of AMD with severe visual loss 351

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in patients with ‘‘moderate’’ AMD or with advanced AMD in the fellow eye [1]. The AREDS study was able to show for the ﬁrst time that AMD is likely to be in part a disorder related to excessive oxidative stress and that antioxidant nutritional supplements could alter its course. In this chapter, the relationship between genetic risk of AMD and oxidative stress will be examined. II.

MACULAR BIOLOGY AND PHYSIOLOGY

AMD is a disease of a part of the retina called the macula lutea, or ‘‘yellow spot,’’ a region with high endogenous levels of the yellow carotenoid pigments lutein and zeaxanthin [2]. It is a portion of the central retina 5–6 mm in diameter, less than 5% of the total area of the retina (Fig. 1). A high density of photoreceptors is present in the macula, and it provides for the central 15–20j of visual angle [3]. Its center (the umbo) lies approximately 4 mm temporal and 0.8 mm inferior to the center of the optic disc. The macula is subdivided anatomically into concentric circular regions with progressively decreasing densities of cone photoreceptors (Fig. 2): the foveola (diameter f0.35 mm), the fovea (diameter f2 mm), the parafovea (diameter f3 mm), and the perifovea (diameter f6 mm) [4]. A functional macula makes it possible for a person to drive, read, and recognize faces. Because the macula is primarily responsible for central vision, peripheral vision is generally not aﬀected in individuals with AMD. Cross-sectionally, the retina has a highly ordered structure (Fig. 3). Starting from the vitreous humor, through which incident light must pass, the retina contains several layers of neural cells including the ganglion cells responsible for transmission of visual signals to the central nervous system as well as the amacrine, bipolar, and horizontal cells responsible for intermediate stages of visual signal processing and integration. The adjacent Mu¨ller cells are retinal glial cells that provide structure and metabolic support for these inner retinal cells. In the foveola, these inner layer cells

Figure 1

Cross-sectional anatomy of the human eye.

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Figure 2

Macula of the human eye. The concentric circles mark the boundaries of the regions of the human macula: foveola, fovea, parafovea, and perifovea (smallest to largest).

are pushed concentrically toward the foveal crest (0.55 mm thick) so as to provide minimal impedance to incident light at the umbo (0.13 mm thick). Working outwards, beyond these inner retinal layers is the layer of photoreceptors, which consists of the rods and cones. There are four diﬀerent photoreceptor cells of the human retina: the rod cells and the red, green, and blue cone cells. Rods are predominant in the peripheral retina, while cones predominate in the foveal area. All

Figure 3

Cross-sectional anatomy of the human retina. (Courtesy of Helga Kolb, Ph.D.)

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of these photoreceptors consists of an inner segment containing nuclei, mitochondria, Golgi apparatus, and other cellular organelles connected by a modiﬁed cilium to the outer segment, a specialized structure packed with photoreceptor pigments and the various enzymes and channels responsible for visual transduction (3). Rod outer segments contain membrane discs distinct from the plasma membrane, while cone cells have deep invaginations of their plasma membranes. The predominant protein in the rod outer segment disc is the visual pigment rhodopsin with a peak absorption at f500 nm, while the three cone types contain distinct visual pigments with absorption maxima at 437, 533, and 564 nm, respectively [5]. Next, there is an interphotoreceptor space, which is followed by the retinal pigment epithelium (RPE), a single layer of hexagonal cells that are closely associated with the photoreceptors [6]. They contain the enzymatic machinery essential for the regeneration of rhodopsin’s 11-cis-retinal chromophore [7]. Each day the photoreceptor cells shed many discs from their outer segments, which are phagocytosed by the RPE [8]. RPE cells form tight junctions with each other, and they are partially responsible for sustaining the blood-retina barrier [9]. The basement membrane of the RPE rests on Bruch’s membrane. Bruch’s membrane is a condensation of part of the choricocapillaris, which is responsible for supplying the outer retina with nutrients and oxygen from the blood. Bruch’s membrane is quite porous and is essentially a layer of elastic tissue sandwiched between two layers of collagen [3]. III.

RETINAL CHANGES ASSOCIATED WITH EARLY AND ADVANCED AMD

Deﬁnite changes in the retina are observed in individuals with both early- and latestage AMD (Fig. 4). On a cellular level, a ﬂuorescent yellow-brown compound called lipofuscin accumulates in RPE cells with age [10]. Lipofuscin is thought to contain debris resulting from oxidative stress, and it ﬁlls some RPE cells to bulging since it is indigestible by the cell [10]. Its major constituent is A2E, a pyridinium bis-retinoid produced by the condensation and oxidation of two molecules of retinal with one molecule of ethanolamine [11,12]. Greater amounts of lipofuscin are thought to correlate with increased risk for developing AMD [10]. Another pigment present in the RPE is melanin. The amount of melanin in RPE cells decreases with age, and decreased amounts are associated with a greater risk of AMD [13]. For a detailed analysis of the potential roles of lipofuscin and melanin as endogenous light-induced free-radical generators in the eye, see a previous volume in this series [14]. Bruch’s membrane also changes as a result of AMD. It thickens signiﬁcantly with age, and it may begin to degenerate. This, along with signiﬁcant age-related RPE dysfunction, leads to the accumulation of cellular debris between the RPE and Bruch’s membrane called drusen, the most obvious clinical sign of early AMD [15]. Hard drusen are small (<125 Am, and usually<63 Am in diameter), ﬂat, yellowish-white deposits. They are thought to consist of oxidized lipids [16] and proteins [17]. While the presence of a few hard drusen are considered a normal part of the aging process, very large numbers of hard drusen may be associated increased risk of AMD [18]. Soft drusen are larger ( >63 Am in diameter) yellow or grayish deposits that are strongly associated with high risk of development of advanced AMD. Distinct soft drusen have a uniform density, sharp edges, and a thick, solid appearance, while indistinct drusen are of nonuniform density and have fuzzy edges. At this stage of the disease, some

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Figure 4 Aging of the retina/RPE organocomplex during the course of AMD. With increasing age, photoreceptors are lost, disc membrane lipids are oxidized, lipofuscin accumulates in the RPE, Bruch’s membrane thickens, drusen are deposited between the RPE and Bruch’s membrane, and choroidal neovascularization may occur.

patients begin to notice disturbances of central vision and loss of visual acuity, although many are asymptomatic. Eventually, the disruption of retinal and RPE structure and function can become so severe that substantial portions of the macula become atrophic. This advanced form of dry AMD takes many years to evolve and is known as geographic atrophy. Ultimately, these patients may become legally blind with visual acuities of 20/200 or worse. In other patients, the dry form of AMD progresses into the exudative or wet form of AMD. The integrity of Bruch’s membrane is disrupted, and blood vessels derived from the choroid invade the subretinal and sub-RPE space, where they bleed and leak ﬂuid and lipid, causing severe disruption of photoreceptor function. The progression of wet AMD may be so rapid that many lines of vision can be permanently lost within a matter of weeks. If a patient has developed wet AMD in one eye, there is a 5–10% risk per year that the fellow eye will also develop exudative changes [19]. Examples of wet and dry AMD are shown in Figure 5. Treatment options for advanced AMD are limited. If the choroidal neovascular membrane is small, well-deﬁned, and outside of the fovea, it can be treated with laser ablation [20] although the persistence and recurrence rate after treatment can be as high as 50% [21]. If the lesion extends under the fovea, laser ablation can still be performed, but the permanent laser-induced tissue damage is often unacceptably high for the physician and the patient [22]. Recently, photodynamic therapy using lightactivated photosensitizing dyes has been introduced as a less damaging alternative for the treatment of subfoveal neovascularization [23]. The majority of patients, however, are ineligible for any laser treatments due to the size or other angiographic characteristics of the lesion. Heroic surgical interventions such as subfoveal surgery or macular

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Figure 5 Examples of age-related macular degeneration: (A) extensive drusen; (B) choroidal neovascularization; (C) geographic atrophy; (D) disciform (fibrotic) scarring. translocation beneﬁt only a few patients, and other interventions such as external radiation and antiangiogenic compounds are still in clinical trials. Ultimately, the vast majority of patients with wet AMD will end up legally blind in the aﬀected eye. Treatment options for patients with geographic atrophy are even more limited since there are no proven interventions that can halt the progression of the disease. Restorative treatments that could beneﬁt these patients such as retinal and RPE transplantation, gene therapy, and various prosthetic devices are still in the very early research phases. IV.

RISK FACTORS ASSOCIATED EPIDEMIOLOGICALLY WITH AMD

The prevalence and incidence of early and advanced AMD rises dramatically with age, especially after age 70. Below age 50, virtually all patients with signiﬁcant macular pathologies resembling AMD actually have inherited macular dystrophies such as

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Stargardt’s disease, Best’s disease, Sorsby’s fundus dystrophy, dominant drusen, or the pattern dystrophies [24]. Within families, these diseases are highly concordant in clinical presentation, and it is often possible to establish dominant or recessive inheritance patterns. The prevalence of AMD progressively rises with age, such that approximately 30% of the U.S. population over age 75 exhibits at least the early signs of AMD, and 7% have advanced stages of the disease including geographic atrophy or exudative disease [25]. Exudative or wet AMD accounts for the majority of legal blindness (vision 20/200 or worse in both eyes) attributed to AMD [26]. As will be discussed in detail later, this increased prevalence with age is thought to be due in part to cumulative oxidative damage to ocular structures from environmental insults. Cigarette smoke is a reactive oxygen species (ROS) generator and has been identiﬁed as a risk factor for many diseases, including cancer, lung diseases, and cardiovascular disease [27]. The Eye Disease Case Control Study Group found that current and past smokers have a higher risk for AMD than those who have never smoked [28]. Another study of 21,157 physicians found that heavier smokers had a higher risk of AMD than those who smoked less [29]. Smoking may lead to the development of AMD by promoting neovascularization, by damaging the blood vessels of the choroid (by promoting hypoxia), and by increasing oxidative stress in the retina, leading to increased peroxidation [30]. Whites have signiﬁcantly higher amounts of lipofuscin than blacks or Hispanics, while blacks have signiﬁcantly higher amounts of choroidal melanin, which may help explain the increased risk of AMD in whites. Some researchers estimate that whites have up to a 40 times greater risk of having AMD than blacks [10]. The Beaver Dam Eye Study found an association between systemic hypertension and AMD [31], as did a study by Hyman et al. [32]. A family history of AMD increases the likelihood of having the disease [33], and higher blood lipid levels may also present an increased risk [32]. Having light- or medium-colored eyes, lens opacities, and hyperopia are all associated with increased risk [32,34]. Persons exposed to pollution [35] and those exposed to high amounts of sunlight also have increased risk of AMD [36,37]. In some studies, women have been found to have a higher risk for AMD [38], although this is controversial since the population of elderly women is so much larger than elderly men. Macular pigment density and nutrition are other important factors that will be discussed in detail below. V.

GENETICS OF AMD

Genetic susceptibility to visual loss from AMD has long been suspected, especially since many patients note that AMD seems to ‘‘run in the family.’’ This notion is supported by an epidemiological study that demonstrated an age-adjusted 2.4-fold elevated relative risk of AMD in ﬁrst-degree relatives of patients with AMD [33]. Deﬁnitive demonstration of an inherited risk factor for AMD has proven elusive, however. Human molecular genetics has had by far the most success in deﬁning the genetic etiology of ophthalmic diseases with a clear monogenic hereditary basis and well-deﬁned diagnostic criteria. Genetic studies on retinitis pigmentosa [39], retinoblastoma [40], and color blindness [41] are excellent examples of such successes. Determining genetic factors involved in late-onset common ophthalmic disorders with variable clinical presentations such as glaucoma and AMD has been a far more evanescent goal.

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A cornerstone methodology for studying the genetics of human diseases is the collection and characterization of multigenerational kindreds with the disorder. This approach is quite problematic for AMD, however. As a disease of the elderly, the aﬀected proband’s parents are usually dead, siblings are often dead or in widely scattered locations, and oﬀspring are typically too young to manifest symptoms. Even with the ascertainment of a large kindred with AMD, the researcher is faced with substantial challenges due to the high prevalence of the disease, its variable expressivity, and its apparent multifactorial etiology. Unlike a relatively uncommon disease, such as retinitis pigmentosa, in which aﬀected family members almost certainly suﬀer from identical genetic defects, the situation is not as simple for AMD. Interaction of multiple AMD-associated alleles in many genes may be needed for increased susceptibility within a particular family. Thus, it cannot be assumed that aﬀected siblings must have the same AMD-associated allele. The broad spectrum of clinical presentation of AMD ranging from exudative changes to geographic atrophy raises the question of whether AMD is truly one disorder or actually represents a multitude of diseases with diﬀerent genetic etiologies, and the variable presentation and progression of AMD often requires an arbitrary delineation of which individuals are or are not aﬀected. The interplay of nongenetic risk factors for AMD such as smoking history, nutritional status, and light exposure complicates genetic studies, because even if an individual has inherited a putative AMD susceptibility allele, the disease may not manifest if a protective lifestyle has been practiced. Also, age must be considered as a factor since a few soft and hard drusen in the macula of a 95-year-old patient may be ‘‘normal,’’ while the same ﬁndings in a 45-year-old patient might be considered the ﬁrst signs of AMD. Even if linkage is established to a chromosomal locus, it is often a long and arduous task to determine the actual genetic defect since the chromosomal locus may encompass dozens of genes. Signiﬁcant linkage has been reported only recently for one AMD family at locus 1q25–31 [42], and it is likely that further progress with this approach will continue to be slow [43]. The genetic investigation of AMD is amenable to the ‘‘candidate disease’’ approach. AMD shares phenotypic similarities to a number of hereditary diseases of the macula, and as the genetic bases for these diseases are ascertained, cohorts of AMD patients can then be screened to determine whether or not comparable mutations are involved in the pathogenesis of AMD [44]. Stargardt disease (STGD1) has proven to be the most promising candidate disease for AMD. This autosomal recessive disorder is the most common early-onset macular dystrophy encountered in clinical practice (estimated frequency 1 in 10,000) [45]. It is characterized by macular atrophy and drusen-like ﬂecks with associated central visual loss that typically occurs in the second or third decade of life but with earlier and later onsets well documented. Homozygous and compound heterozygous mutations in ABCR are responsible for STGD1 [46], some cases of recessive retinitis pigmentosa [47], and a substantial fraction of autosomal recessive cone-rod dystrophies [48]. The ABCR (ABCA4) gene codes for a retina-speciﬁc protein on the rims of photoreceptor outer segment discs that appears to facilitate the transport of retinoids during the visual cycle [49]. The RPE accumulates large enough amounts of lipofuscin to exhibit a dark choroid on ﬂuorescein angiography. Exudative complications are rare [44]. If mutations in both alleles of ABCR can lead to protein dysfunction severe enough to manifest as an early-onset macular dystrophy such as STGD1, is it possible

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that a mutation in one ABCR allele could lead to moderate dysfunction suﬃcient to cause a late-onset macular degeneration such as AMD? Our ﬁndings of an elevated frequency of amino acid–changing ABCR variants in AMD patients relative to agematched controls supports this hypothesis [50,51]. Also, AMD-aﬀected relatives of STGD1 patients are more likely to carry pathogenic ABCR alleles than predicted by chance alone [52]. Physiologically, this hypothesis is tenable. In STGD1 patients, severe ABCR dysfunction disrupts vitamin A transport pathways from the outer segment disks leading to formation of massive amounts of lipofuscin which accumulates in the RPE [49]. Less profound disruption of ABCR function in the heterozygous state acting over a prolonged time period could lead to a similar accumulation of lipofuscin, albeit at a much slower rate. Indeed, lipofuscin formation is strongly associated with the progression of AMD [53,54], and knock-out mouse studies have conﬁrmed that both homozygous and heterozygous mutations in ABCR are associated with increasing lipofuscin accumulation over time, especially when these animals are exposed to light [55–57]. VI.

DIETARY FAT AND RISK OF AMD

Recently it has been proposed that high intakes of certain polyunsaturated fatty acids (PUFA) are associated with a signiﬁcantly lower risk of AMD. This ﬁnding is particularly intriguing since the retina has an extraordinarily high concentration of highly polyunsaturated long-chain omega-3 fatty acids such as docosahexaenoic acid (DHA; C-22:6 N3) [58], which must be ingested preformed in the diet or synthesized from shorter-chain essential omega-3 fatty acid precursors such a-linolenic acid (C-18:2 N3) [59]. Furthermore, many patients with X-linked retinitis pigmentosa have also been found to have low plasma levels of DHA and impaired biosynthesis of DHA [60]. Dietary fat, both the type and amount of total fat, has been shown to be related to the incidence of AMD [61,62]. Speciﬁcally, dietary intake of linolenic acid has been positively associated with the risk of AMD, whereas DHA (usually of ﬁsh origin) was inversely related to AMD [62]. In another recent study with 3654 participants, a higher frequency of ﬁsh consumption (once per week vs. once per month) was associated with a decreased odds of late AMD [63]. Other investigators [64] have also reported that diets high in omega-3 fatty acids and ﬁsh were inversely associated with the risk for AMD but only when the intake of linoleic acid was low. Oxidation of long-chain PUFA may be pathogenically involved in AMD. Although there is less DHA in the macula compared to the overall composition in the retina [65,66], peroxidized DHA is a major peroxidized fatty acid identiﬁed in Bruch’s membrane that increases with age [67]. This could be interpreted to mean that DHA is particularly susceptible to oxidative stress and is reﬂective of tissue damage. Conversely, it could mean that DHA is the ﬁrst line of defense against free radical attack, and an increase in its peroxidized form might mean it is acting in a protective role, somewhat like a sacriﬁcial antioxidant. The results of these epidemiological and postmortem tissue analysis studies suggest that dietary fat may function to facilitate or prevent the onset of AMD. Diets high in cold water marine ﬁsh may provide certain omega-3 fatty acids that protect against AMD, whereas diets high in grains or other foodstuﬀs that contain saturated or omega-6 fatty acids may permit the earlier expression of AMD. Seddon et al. have

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proposed that high dietary intakes of speciﬁc types of fat rather than the total fat intake may alter the risk factors or odds ratio for AMD [64]. For example, neither omega-3 fatty acids nor ﬁsh intake was related to risk for AMD among people with high linoleic acid intake in Seddon’s study. The concentration of eicosanoid and docosanoid precursors in membranes and adipose tissue depends upon the dietary supply of these fatty acids and, to a certain extent, upon the subsequent desaturation-elongation of ingested precursor fatty acids [68]. The fatty acid composition of adipose tissue has been used in epidemiological studies as a marker for habitual dietary fatty acid intake [69–71]. For example, several investigators have shown that the presence of omega-3 fatty acids in adipose tissue reﬂects habitual ﬁsh intake patterns [71–73]. The fatty acid composition of adipose tissue triacylglycerol reﬂects, but is not identical to, the fatty acid composition of the habitual diet [70,74]. The array of fatty acids in adipose tissue could also arise through diﬀerences in the early metabolic processing or subsequent mobilization of individual dietary fatty acids, leading to accumulation of some fatty acids relative to others [75]. The characteristic fatty acid pattern of adipose tissue reﬂects the metabolic deposition, mobilization, and oxidation of diﬀerent fatty acids. Mobilization of fatty acids into the plasma is inﬂuenced by their molecular structure; as an example, eicosapentanoic acid (20:5 N3) and arachidonic acid (20:4 N6) seem to be preferentially mobilized into the plasma compared to saturated fatty acids [75]. Since the half-life of certain fatty acids in the adipose depot can be up to 2 years, the fatty acid composition of adipose tissue may be a good marker of habitual long-term fatty acid intake [70]. Serum long-chain fatty acid proﬁles, on the other hand, are reﬂective of short-term dietary intake of lipids in fed individuals [74] and perhaps best representative of adipose depot fatty acids during prolonged periods of food restriction. To obtain a complete dietary picture of food intake patterns and hence subsequent long-chain fatty acid availability to target tissues, dietary records (food frequency questionnaires) and evidence of both recent (serum) and long-term (adipose) dietary habits need to be examined. It was recently reported that a ﬁve base-pair deletion in the ELOVL4 gene is associated with dominantly inherited Stargardt-like macular dystrophy and pattern dystrophy [76]. All reported families with this disease carried the same mutation, presumably from a common founder eﬀect. The ELOVL4 gene product is highly homologous to yeast enzymes involved in long-chain polyunsaturated fatty acid synthesis, and it is likely that it performs a similar function in the human retina and brain. Since this is the ﬁrst fatty acid metabolism genetic defect reported to be associated with a macular dystrophy phenotype, these families could provide a key link in furthering our understanding of the role of fatty acid intake, metabolism, and oxidative stress in AMD. We have recently identiﬁed an unrelated Utah family (K4175) that has a novel noncontiguous two base-pair deletion in the same region of the ELOVL4 gene [77]. We have examined 25 family members and found 12 to be clinically aﬀected. All 12 aﬀected members carry the two base-pair deletion in ELOVL4, and the median age of onset is 13. Interestingly, we have also identiﬁed two clinically unaﬀected adults and one unaﬀected child who carry the family’s mutation despite having aﬀected siblings and/ or children. The presence of nonpenetrant adults raises the question of whether these individuals may be practicing some form of protective lifestyle that has delayed or prevented the onset of the disease. If this protective lifestyle can be identiﬁed, it may be

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possible to ameliorate visual loss in other members of these families, especially if they can be identiﬁed at an early age. Some of these recommendations may be applicable to the general population at risk for AMD.

VII.

OXIDATIVE STRESS AND AMD

The retina of the eye is particularly at risk of oxidative damage for several reasons. First, oxygen consumption in the retina is greater than in any other tissue [78]. Second, the retina is subject to high levels of radiation from short-wavelength visible light, also known as the ‘‘blue light hazard,’’ which can generate large amounts of ROS from endogenous retinal photosensitizers such as lipofuscin and melanin [14]. Furthermore, photoreceptor outer membranes are rich in PUFA, which are highly prone to oxidative stress [58,66]. Finally, immunostimulation within the retina also produces ROS locally, increasing the risk of oxidative damage to the tissue [79]. Interestingly, AMD-like characteristics have been demonstrated in animals fed diets deﬁcient in antioxidant nutrients. In a study conducted by Malinow et al., it was observed that macaque monkeys fed a diet deﬁcient in lutein and zeaxanthin had virtually no macular pigment, and they developed symptoms reminiscent of AMD [80]. Deﬁciencies of vitamin E have been shown to increase light-induced retinal damage in rats, while vitamin C supplementation has been shown to decrease light-induced retinal damage [81,82]. Further evidence for the oxidative stress theory is demonstrated by the fact that rats fed an antioxidant-deﬁcient diet had less accumulation of retinal lipofuscin [83].

VIII.

ANTIOXIDANTS IN THE RETINA

The retina of the eye does have some endogenous protection against oxidative damage. Selenium-dependent glutathione peroxidase, superoxide dismutase (SOD), catalase, and glutathione reductase all work to disable ROS and stop chain reactions in the retina [84]. These antioxidant systems may deteriorate with age, but results of recent studies are ambiguous. De La Paz et al. found that some enzyme activities decreased with age (e.g., hexokinase), while most did not (e.g., catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione reductase) [84]. They did ﬁnd, however, that the ratio of the diﬀerence between the activity of glutathione peroxidase in the macular and peripheral areas increased with age, and this may be signiﬁcant in the development of AMD, because it is the macular region that is aﬀected in the disease. Frank et al. found that there is an upregulation of heme oxygenase-1 and-2 lysosomal activities relative to the cytoplasm of the macular RPE cells of young individuals and that this degree of upregulation declined substantially with age, especially in AMD eyes [85]. This suggests that this endogenous antioxidant system is less functional with increasing age, placing the eye at greater risk of oxidative damage. Additionally, some antioxidant vitamins and minerals are involved in protecting the retina of the eye from oxidative damage. Ascorbate and a-tocopherol are present in the retina [86,87]. Because carotenoids can quench singlet oxygen and scavenge other ROS, the presence of lutein and zeaxanthin in the macula may be protective. These vitamins, minerals, and carotenoids function in the antioxidant capacities discussed above. Zinc-dependent enzymes in the eye include carbonic anhydrase, collagenase,

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superoxide dismutase, retinol dehydrogenase, and catalase [10]. Zinc’s major site of deposition in the retina appears to be melanin. A decrease in melanin leads to a decrease in zinc levels and zinc-dependent enzyme and protein function [10]. IX.

THE MACULAR CAROTENOID PIGMENT

Macular pigment (MP) was ﬁrst observed anatomically in the eighteenth century. Wald deduced in the 1940s that it was likely to consist of xanthophyll carotenoids based on biochemical and spectroscopic analysis [88], but it was not until 1985 that Bone et al. discovered that the macular pigment consisted of the carotenoids lutein and zeaxanthin [89]. In 1993, the stereochemical conﬁgurations of the macular carotenoids were determined by Bone et al. [90]. Macular pigment is entirely of dietary origin and the relationship between dietary intake of lutein and zeaxanthin to MP has been well researched. Lutein is present in green leafy vegetables such as kale, spinach, and broccoli, while zeaxanthin can be found in corn, peaches, citrus fruits, and some orange-ﬂeshed melons [91]. Malinow et al. found that the eyes of macaque monkeys fed a lutein- and zeaxanthin-free diet had no macular pigment and exhibited changes reminiscent of early human AMD [80]. Also of note is the fact that lutein and zeaxanthin were undetectable in the serum of the monkeys fed the deﬁcient diet, but they were present in normal concentrations in monkeys fed unmodiﬁed diets [80]. Hammond et al. [92] further examined the relationship between diet and lutein and zeaxanthin. In one of the ﬁrst studies on the topic, the macular pigment of 88 male and female subjects was measured, and the group attempted to correlate the serum levels of lutein and zeaxanthin with dietary intakes [92]. MP was found to be 38% greater in males than in females, and it was positively and signiﬁcantly ( p<0.002) correlated to dietary intake for males only. However, plasma lutein and zeaxanthin concentrations correlated positively and signiﬁcantly to macular pigment density ( p<0.01) for both genders. A review of the National Health and Nutritional Examination Survey (NHANES) showed that persons who eat more fruits and vegetables high in vitamin A (which also have high amounts of lutein and zeaxanthin) had lower risk of age-related maculopathy than those who eat less [93]. There is a 1:2.4 concentration of lutein:zeaxanthin in the central retina and a 2:1 concentration in the periphery of the retina [94,95], even though there is a 3:1 concentration of lutein:zeaxanthin in the serum [96]. More than one diastereoisomer of zeaxanthin is present in the retina. Not only is the dietary form, 3R,3VR-zeaxanthin, present, but in the fovea there is an equal amount of a nondietary form, 3R,3VS-mesozeaxanthin [90]. It is thought that dietary lutein is converted to 3R,3VS-meso-zeaxanthin in the retina via an as yet unidentiﬁed mechanism [90,97]. In a study conducted by Sujak et al., it was found that zeaxanthin was a better antioxidant when oxidative damage was induced by radiation; this may account for zeaxanthin’s greater central concentration [98]. Lutein and zeaxanthin have also been shown to be present in substantial amounts in the photoreceptor outer membranes [99]. This gives evidence that they play a role as ocular antioxidants, as they are in close enough proximity to photoreceptor outer membranes to be protective against possible oxidative damage. Another carotenoid in the eye, 3-hydroxy-h-q-carotene-3V-one, is an oxidation product of lutein at high amounts relative to serum levels, suggesting that lutein is in fact oxidized in the retina [97]. Additionally, there are other oxidation products of both

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lutein and zeaxanthin in the retina as well as an oxidation product of lycopene, although lycopene itself has not been identiﬁed in the retina [97]. Bernstein et al. [100] considered how these carotenoids concentrate in the retina given these relatively low serum concentrations. They initially found that retinal tubulin binds the carotenoids speciﬁc to the macula and that microtubules of the Henle ﬁber layer may serve as a site for the passive deposition of carotenoids in the macula [100]. In a further study by some of the members of the same group, Yemelyanov et al. reported that they have found evidence for the existence of a family of xanthophyllbinding proteins from membranes in human retinas and maculas that speciﬁcally bind lutein and zeaxanthin [101]. In a separate study, Bernstein et al. found through highpressure liquid chromatography (HPLC) analysis of human donor eyes that dietary lutein and zeaxanthin and their metabolites were present in almost all parts of the eye [102]. In addition to functioning as free radical quenchers, MP ﬁlters out high-energy blue light, which is known to cause free radical formation [103]. MP also reduces the eﬀects of light scattering and chromatic aberrations, thus contributing to increased visual acuity [104]. X.

MEASURING MACULAR PIGMENT

Macular pigment can be measured in living subjects and in postmortem eyes. In postmortem subjects, MP can be measured using chemical extraction and HPLC [105] and by microdensitometry, a spectroscopic method that calculates the diﬀerences in the MP’s ability to absorb blue light (460 nm) and green light (560 nm) [106]. The most common technique for measuring MP in live subjects is a psychophysical method called heterochromatic ﬂicker photometry (HFP), which estimates the perceived optical density of the MP at the foveal center [107]. In HFP, a stimulus of blue light close to the absorbance peak of MP (460 nm) is alternated with green light (560 nm), which is not absorbed by the macular pigment. The ﬂickering stimulus is focused on the foveal center where macular pigment reaches its maximum concentration and then on the parafovea where macular pigment does not appear to be detectable. The subject can adjust the luminance of one light source, and thus the ﬂicker can be eliminated or minimized. The diﬀerence between the foveal and parafoveal sensitivities to blue light is used as a measurement of MP density [107]. HFP yields reproducible results and shows acceptable test-retest reliability; however, some subjects ﬁnd the task of HFP diﬃcult to perform, and they must have good visual acuity and be free of signiﬁcant macular pathology [108]. Therefore, it is not an adequate screening tool for the elderly, who are at highest risk for developing AMD. Due to the subjective nature of HFP and its inadequacy for examining an older population, Bernstein et al. have developed a method for measuring macular pigment based on the principles of resonance Raman spectroscopy [109,110]. In the Raman method, 488 nm argon laser light is used to resonantly excite the electronic absorption of retinal carotenoids. The scattered light is then collected and analyzed by a Raman spectrometer. To test this method, Bernstein et al. measured the carotenoid levels of ﬂat-mounted human retinas and then analyzed them for carotenoids using HPLC. They found that resonance Raman scattering was a highly sensitive and speciﬁc quantitatively valid method for measuring human retinal macular pigment [109]. This method is noninvasive, and it only requires subjects to line up their eyes by looking

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into an aiming beam; thus it may be useful as a clinical instrument for those at high risk for AMD [111,112]. XI.

MACULAR PIGMENT AS PROTECTION AGAINST AMD

Several studies suggest that macular pigment is protective against the development of AMD. Bone et al. [113] looked at macular pigment of living subjects and in donor eyes. Nineteen subjects ﬁlled out questionnaires regarding food habits. Blood was analyzed for lutein and zeaxanthin, and the subjects also had their macular pigment density measured by heterochromatic ﬂicker photometry. The researchers found that 55% (r=0.74; p<0.001) of the variability in serum lutein and zeaxanthin and that 17% (r=0.42; p<0.01) of the variability in macular pigment density could be explained by dietary intakes of lutein and zeaxanthin. Forty-six human eyes were also examined postmortem along with blood samples from the tissue donors, and serum concentrations were found to correlate with macular carotenoids measured by HPLC. In a separate study by the same researchers, they found that autopsy eyes from doors with a known history of AMD had lower levels of lutein and zeaxanthin relative to control eyes without a known history of AMD [114]. Thus, these papers support the hypothesis that low concentrations of MP may be associated with increased risk of AMD. In a diﬀerent study conducted by Beatty et al., their group examined subjects from a northern European population (ages 21–81) and compared the healthy eyes of individuals with unilateral AMD to the healthy eyes of control individuals [115]. They found that eyes at risk for AMD (because of an aﬀected neighboring eye) had signiﬁcantly less macular pigment as measured by ﬂicker photometry than healthy eyes with no such risk ( p<0.015). More recently, Bernstein et al. used the resonance Raman method to compare MP levels objectively in AMD patients and in a normal population [116]. AMD patients had 32% lower levels of macular carotenoids relative to age-matched controls ( p=0.001). Interestingly, a subgroup of AMD patients who had begun taking highdose lutein supplements after their initial diagnosis of AMD had returned to ‘‘normal’’ age-adjusted levels, implying that carotenoid binding sites in the macula [101] are not saturated in AMD patients consuming an unsupplemented diet. In the same studies just mentioned, both Bernstein et al. and Beatty et al. also found that MP density declined signiﬁcantly with age in subjects with healthy eyes ( p<0.001) [115,116]. In a report of the Age-Related Eye Disease Study (AREDS), the group reported that age was signiﬁcantly correlated to the presence of intermediate drusen, large drusen, and both forms of late AMD [117]. Hammond et al. also found that visual sensitivity decreases with age and that photopic sensitivity was positively related to MP density in older subjects ( p<0.0003) [118]. Interestingly, the visual sensitivity of subjects over age 60 with high MP density was not signiﬁcantly diﬀerent from that of younger subjects [118]. These results suggest that MP protects the retina from age-related sensitivity losses and that it may even delay or prevent disease progression of AMD. Iris color has also been shown to be related to risk for AMD. In a study conducted by Hammond et al., the authors reported a signiﬁcant, positive relationship between macular pigment density and iris color. They found that despite similarities in diet and blood concentrations of carotenoids, people with lighter irises had a lower

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macular pigment density than those with darker irises [119]. The authors postulated that eyes with lighter irises might allow for greater transmission of light and, therefore, they may experience greater levels of oxidative stress [119]. Bernstein et al., however, could not conﬁrm any relation between iris color and macular carotenoid levels when resonance Raman spectroscopy was used as an objective test [116]. The Eye Disease Case Control Study (EDCCS) group obtained personal, medical, physiological, biochemical, and ocular data on 421 subjects with AMD and 615 subjects without the disease [120]. They found that groups with higher and medium levels of serum carotenoids had one-third and one-half, respectively, the risk for neovascular AMD as did the group with the lowest level of serum carotenoids. The group did not ﬁnd any statistically signiﬁcant eﬀect of vitamin C, vitamin E, or selenium alone; however, when these were grouped with the carotenoids to create an antioxidant index, a statistically signiﬁcant protective eﬀect was found ( p=0.005). In a case-control study conducted by Seddon et al., 356 subjects with late AMD and 520 control subjects were examined [121]. After controlling for other risk factors for AMD, the group found that those in the highest quintile of carotenoid intake had a 43% decrease in their risk for developing AMD compared to the lowest quintile. Intake of green leafy vegetables such as spinach and collard greens, which are high in lutein and zeaxanthin, were also associated with a signiﬁcantly lower risk for exudative AMD ( p<0.001). Intakes of vitamin A (as retinol), vitamin C, and vitamin E were not related to risk for AMD. After examining the serum and fundus photographs of participants in the Beaver Dam Eye Study, Mares-Perlman et al. [122] found that plasma levels of only lycopene were correlated with reduced risk for early ARM (age-related maculopathy, often a precursor to advanced AMD). Persons with serum lycopene levels in the highest quintile had half the risk of those in the lowest quintile [122]. Since lycopene is such a powerful quencher of singlet oxygen, this eﬀect could be due to the systemic removal of potentially destructive ROS. XII.

NONCAROTENOID ANTIOXIDANTS AND RISK OF AMD

Researchers in the Pathologies Oculaires Liees a l’Age (POLA) study sought to clarify the relationship between serum antioxidant status and AMD. They examined fundus photographs and serum antioxidants of 2584 subjects. Subjects in the highest quintile of serum a-tocopherol-to-lipid ratio had an 82% decrease in risk for late AMD compared to those in the lowest quintile, but this relationship was not signiﬁcant [123]. No associations were found with plasma vitamin A, vitamin C, or red blood cell glutathione levels. Obisesan et al. [124] examined data collected from the NHANES-1 study, which included 3072 adults age 45–74 with macular changes indicative of AMD. These researchers found that there was a statistically signiﬁcant, inverse correlation between wine consumption and AMD. This association persisted after controlling for the eﬀect of age, gender, income, history of congestive heart failure, and hypertension. Researchers propose that the reason for these ﬁndings is the high level of antioxidants present in wine [124]. The Blue Mountains Eye study examined the associations between dietary antioxidant intake and AMD in 3654 subjects in Australia. Food intake was assessed using a food frequency questionnaire, and researchers found no association between any

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dietary antioxidants and AMD [125]. Results did not change even when the dietary food source of the antioxidants was considered. It should be noted that blood samples were not taken, and carotenoids were not subdivided into individual carotenoids. XIII.

ANTIOXIDANT SUPPLEMENTATION TRIALS RELATED TO AMD

Due to the vast amount of epidemiological and circumstantial evidence, researchers have begun to investigate the eﬃcacy of antioxidant supplementation in modifying serum levels, tissue levels, and risk for AMD. Several studies have shown promising results to date. Christen et al. [126] reviewed data collected in the Physicians’ Health Study 1, which consisted of 21,120 male physicians who did not have AMD at baseline in 1982. After 12.5 years, researchers found that supplemental vitamin E and multivitamins slightly, though not signiﬁcantly, decreased risk for AMD. Supplementation with vitamin C was found to yield a slightly increased risk of AMD with an odds ratio of 1.03 (126). The authors did not consider dietary sources of antioxidants. Landrum et al. supplemented the diets of two subjects with lutein esters, equivalent to 30 mg of free lutein, for 140 days [127]. Macular pigment density and serum data were measured before, during, and after supplementation. Twenty to 40 days after supplementation, the MP density of both subjects increased linearly. Serum lutein levels were increased 10-fold and remained elevated 40–50 days after supplementation was discontinued. The investigators estimated that during the period of supplementation, the amount of blue light reaching tissues vulnerable to AMD was decreased by 30–40% and, therefore, possibly reduced the light-induced oxidative damage that took place over that period. In a study conducted by Hammond et al. [128], 11 subjects modiﬁed their diets by eating an extra 60 g of spinach per day. Ten subjects ate the spinach and 150 g of corn, and two other subjects were given only 150 g of corn. Together this supplementation regime provided 10.8 mg lutein, 0.6 mg zeaxanthin, and 5.0 mg of h-carotene daily. Subjects consumed the modiﬁed diets for 15 weeks [128]. Three types of responses to the dietary treatments were identiﬁed. Eight ‘‘retinal responders’’ had 33% increases in serum lutein concentrations and a 19% increase in MP density. Two ‘‘retinal nonresponders’’ experienced a 31% increase in serum lutein concentrations, but actually showed an 11% decrease in MP density. One subject did not exhibit any changes in serum lutein concentrations or MP density. Berendschot et al. gave 8 subjects 10 mg of supplemental lutein per day for 12 weeks [129]. After 4 weeks, mean serum levels of lutein had increased by 5 times the baseline value. This elevated level was maintained until 4 weeks after supplementation was terminated, when serum lutein levels were about 1.5 times what they were at baseline. MP density increased by 5.3% every 4 weeks ( p<0.001). In a study conducted by Carughi and Hooper [130], 11 subjects were fed a lowcarotenoid diet for 2 weeks, and they then consumed diets supplemented with hcarotene, a-carotene, and lycopene for 4 weeks. After the 2-week washout period, the subjects’ serum carotenoid levels were 60% of that of baseline, and after 4 weeks of supplementation, they were signiﬁcantly higher than the baseline value ( p<0.05). In a study conducted by Johnson et al., 7 subjects consumed extra spinach and corn in addition to their daily diets for 15 weeks [131]. At 0, 4, 8, and 15 weeks, and 2 months after the study, serum, buccal mucosa cells, and adipose tissue were analyzed

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for carotenoids and MP density was measured. Signiﬁcant inverse correlations were found between lutein concentrations in adipose tissue and MP in women, but not in men, implying that males and females may metabolize lutein diﬀerently. They also found that all measures of lutein had returned to baseline at 2 months postintervention, suggesting that the spinach consumption was indeed responsible for the increase in lutein measured in the diﬀerent tissues. Yeum et al. [132] determined the plasma carotenoid responses in 36 men and women before and after being fed diets of moderate fat content and high in carotenoid content for two 15-days periods. In addition, an extra 205 g/day of broccoli was provided in one of the two periods for each subject. Plasma concentrations of lycopene, a- and h-carotenes, cryptoxanthin, and lutein were all signiﬁcantly increased on days 6–16 of the study ( p<0.05). The provision of broccoli resulted in a further signiﬁcant increase in serum lutein concentrations relative to the high-carotenoid diet alone. Handelman et al. wished to determine the bioavailability of lutein and zeaxanthin found in egg yolks [133]. Eleven subjects were given two baseline diets, which contained 29–33% as fat, with 20% of the energy as either beef tallow or corn oil. These diets were then supplemented with 1.3 cooked egg yolks/day. Each subject consumed all four diets. Each diet was consumed for 4.5 weeks with a 2-week washout period between diets. Egg supplementation with the beef tallow diet increased plasma lutein by 28% ( p<0.05) and zeaxanthin by 142% ( p<0.001). Supplementation of the corn oil diet increased plasma lutein by 50% ( p<0.05) and zeaxanthin by 114% ( p<0.001). No changes in lycopene or h-carotene were detected. The investigators concluded the lutein and zeaxanthin were highly bioavailable from egg yolk, but the beneﬁts of introducing carotenoids in this manner may be counterbalanced by added dietary cholesterol and saturated fat. Thompson et al. investigated the eﬀects of additional antioxidants on oxidative stress indicators in the body [134]. In this study, 28 women increased their daily fruit and vegetable intakes from an average of 5.8 servings per day to 12 per day. Serum levels of 8-OHdG, urine malondialdehyde (MDA), and 8-EPG (8-epiprostaglandin F2 alpha), all indicators of oxidative stress, were measured before and after the intervention. Plasma levels of h-carotene, lutein, lycopene, and cryptoxanthin were also measured pre- and postintervention. Investigators found a signiﬁcant increase in all plasma carotenoids. Levels of 8-OHdG and 8-EPG were reduced, but urine MDA was not signiﬁcantly aﬀected. Of special note is that subjects with lower average plasma levels before supplementation showed the greatest reduction in 8-OHdG, suggesting they beneﬁted most from the intervention. This study is also important because it gives evidence that the antioxidants derived from supplemental intakes may actually have an eﬀect on oxidative damage. Fifty-four subjects underwent dietary modiﬁcations in a 4-week study by van het Hof et al. [135]. Twenty-two of the subjects ate a high-vegetable diet (490 g/day), 10 ate a low-vegetable diet (130 g/day) supplemented daily with 6 mg h-carotene and 9 mg lutein, and 22 ate a low-vegetable diet (130 g/day) with no supplementation. Plasma levels of vitamin C, lutein, zeaxanthin, lycopene, a- and h-carotene, and h-cryptoxanthin were measured pre- and postintervention. The low-vegetable with supplementation group showed signiﬁcant increases in h-carotene and lutein and signiﬁcantly higher increases in h-carotene and lutein than either of the other two groups. The highvegetable group showed a signiﬁcant increase in all serum vitamins measured. Plasma

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lycopene decreased signiﬁcantly in both the low-vegetable with supplementation group and the high-vegetable group, possibly because the high-vegetable diet had low lycopene content. Carroll et al. [136] supplemented the diets of 51 subjects who were all at least 65 years old. They supplemented with 13.3 mg lycopene, 11.9 mg a- and h-carotene mixture, or placebo for 12 weeks. The subjects’ plasma a- and h-carotene and lycopene concentrations were measured before and after intervention. The carotene mixture produced signiﬁcant increases in the plasma concentrations of both carotenes; lycopene supplementation signiﬁcantly increased serum lycopene levels. Zinc is widely promoted in supplements targeted toward the population at risk for AMD based largely on a clinical study by Newsome and colleagues, which reported signiﬁcant protection against the progression of AMD in individuals randomized to zinc supplementation [137]. Subsequent studies by other researchers have failed to conﬁrm these ﬁndings. It has been noted that the Newsome study did not measure visual acuity in a standardized manner and that the placebo group had unusually rapid progression of AMD [138]. In an eﬀort to sort out the often conﬂicting relationships seen in epidemiological studies and in small clinical studies, the National Eye Institute established the AgeRelated Eye Disease Study [1]. This comprehensive randomized, placebo-controlled clinical trial followed 3640 elderly subjects at risk for severe visual loss from AMD for an average of 6.3 years. Subjects were randomized equally to receive daily zinc supplementation (80 mg of zinc oxide and 2 mg of cupric oxide), daily antioxidant supplementation (500 mg of vitamin C, 400 IU of vitamin E, and 15 mg of h-carotene), both zinc and antioxidants, or placebo. Serum levels of h-carotene increased severalfold, and vitamin E nearly doubled in the subjects receiving supplements, but changes in vitamin C and zinc were modest. They found that the zinc plus antioxidant group had a 25% lower rate of progression to advanced AMD relative to the placebo group. Zinc alone and antioxidants alone groups had smaller protective eﬀects that did not reach statistical signiﬁcance. AREDS did not include lutein or zeaxanthin in the study formulations because they were not readily available in supplement form at the time the study was initiated, but there is general agreement that these two carotenoids will eventually need to be examined in a similar manner as an intervention against AMD [1,139], especially since h-carotene is essentially undetectable in the human macula while lutein and zeaxanthin and their oxidation products are abundant [102]. XIV. OUTLOOK FOR AMD We are currently at a pivotal time in our understanding and treatment of AMD. As the ‘‘baby boomer’’ generation ages and as general life expectancy increases, the population at risk for blindness due to AMD is enormous and still growing, creating a potentially major public health crisis. In the meantime, our knowledge of the pathogenic mechanisms responsible for AMD is still at an early stage, and our current treatments have signiﬁcant limitations such that the vast majority of patients suﬀer a progressive downhill course ultimately leading to legal blindness. Recent studies discussed above have shown that oxidative stress is an important contributor to AMD and that intervention to decrease oxidative stress by smoking cessation and by nutritional interventions can make a diﬀerence. No major gene responsible for AMD has been discovered yet, and it is unlikely that one will ever be found since AMD

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appears to be a complex multigenic trait inﬂuenced by a multitude of environmental modifying factors. We do know, however, that the defects in genes associated with early-onset macular dystrophies such as ABCR and ELOVL4 can have substantial eﬀects on retinal lipid metabolism that could in turn exert important inﬂuence on levels of macular oxidative stress. Since dietary intakes of nearly all antioxidant vitamins and minerals are well below the RDAs for many older adults [140], supplementing older adults with antioxidants (particularly vitamin E, vitamin C, zinc, and carotenoids) may be the best way to reduce the risk of AMD, especially if they have a known family history of the disease.

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17 Retinitis Pigmentosa: A Potential Role for Reactive Oxygen and Nitrogen Species During Photoreceptor Apoptosis MARYANNE DONAVAN and THOMAS G. COTTER University College Cork, County Cork, Ireland

I.

RETINITIS PIGMENTOSA

Retinitis pigmentosa (RP) is a group of hereditary disorders of the retina leading to progressive photoreceptor degeneration and visual impairment. This genetically diverse disease aﬀects 1.5 million people worldwide, with an incidence of approximately 1/4000, making it one of the most commonly encountered retinal dystrophies and the most signiﬁcant cause of registered blindness in working populations in nontropical countries [1]. RP is characterized by the early loss of rod photoreceptors followed by a slow, progressive degeneration of the cones. Nyctalopia (night-blindness), an initial symptom of the disease, is associated with rod photoreceptor degeneration. As the disease progresses, severe loss of peripheral and central ﬁelds occur due to the subsequent loss of cone photoreceptors. Pigmentory changes typically arise following photoreceptor degeneration and are a result of the release of pigment by cells of the adjacent retinal pigment epithelium (RPE). These pigmented deposits are ophthalmoscopically visible in advanced RP and are referred to as ‘‘bone-spicule formations’’ due to their branching pattern. 377

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Molecular Genetics of RP

RP is the most heterogeneous of retinal disorders, displaying autosomal dominant (adRP), autosomal recessive (arRP), and X-linked (xlRP) modes of inheritance. AdRP and arRP account for approximately 20% of RP cases, while xlRP, the most rapid and severe form of RP, has an incidence of approximately 10%. A digenic segregation pattern has also been identiﬁed (DRP) [2,3]. Digenic inheritance of the disease is rare and involves co-inheritance of mutations within the peripherin/RDS and ROM 1 genes, both photoreceptor structural components that function in formation and stabilization of photoreceptor outer segments. While the majority of cases of RP are inherited, isolated instances are also common, occurring in about 40% of RP cases. In addition, RP can be a component of several systemic diseases including Usher’s syndrome, Bardet-Biedl syndrome, and Kearns-Sayre syndrome. To date, more than 30 genes have been implicated in the etiology of RP, the majority of which encode photoreceptor-speciﬁc proteins that are components of the phototransduction cascade (Fig. 1). This G-protein–coupled enzyme cascade begins with the absorption of light by rhodopsin, the primary photoreactive pigment of the rod photoreceptor cells. Once activated, rhodopsin binds and activates transducin, a G protein composed of three subunits: a, h, and g.The a subunit of transducin activates

Figure 1 Schematic diagram of a rod photoreceptor cell and the phototransduction cascade. The phototransduction proteins are located within the outer segment, which is composed of flattened membrane sacs called discs. Following absorption of a photon, the activated rhodopsin (R*) repeatedly contacts molecules of the heterotrimeric G protein, transducin (T), catalyzing the exchange of GDP for GTP, producing the active form Ta. Two a subunits bind to the two inhibitory g subunits of the phosphodiesterase (PDE), thereby activating the corresponding a and h catalytic subunits, which rapidly hydrolyze cytoplasmic cGMP. Depletion of cGMP causes cGMP-gated cation channels to close, resulting in a decline in intracellular Na+ and Ca2+ levels, and hyperpolarization of the plasma membrane.

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phosphodiesterase (PDE), which rapidly hydrolyzes cytoplasmic cGMP. Depletion of cGMP causes cGMP-gated cation channels to close, resulting in a decline in intracellular calcium levels, hyperpolarization of the cell, and synaptic signaling. The cascade is terminated via several mechanisms. The serine/threonine kinase rhodopsin kinase works together with arrestin to shut oﬀ rhodopsin activity. Rhodopsin, once phosphorylated by rhodopsin kinase, binds to arrestin, preventing its interaction with transducin and ultimately resulting in decreased PDE activity and opening of the cGMP-gated channels. To date, genes encoding rhodopsin (RHO), the catalytic PDE subunits a (PDE6A) and h (PDE6B), the a subunit of the rod cGMP-gated channel (CNGA1), and arrestin (SAG) are known to be mutated in RP [4–11]. Mutations in the rhodopsin gene are the most common cause of RP. These mutations account for about 25% of autosomal dominant forms of RP and a proportion of autosomal recessive cases [12,13]. More than 100 mutations have been found in the rhodopsin gene of RP patients and appear to eﬀect protein folding, stability, or intracellular traﬃcking. Genes encoding proteins involved in the visual cycle (a series of events that result in visual pigment regeneration) are also mutated in RP. These include cellular retinaldehyde-binding protein (CRALBP), the ATP-binding cassette transporter of rods (ABCR), RPE65, and RPE-retinal G-protein–coupled receptor (RGR) [14–18]. Mutations in these genes aﬀect the processes involved in recycling the rhodopsin chromophore 11-cis-retinaldehyde. Structural components of photoreceptors have also been found to harbor mutations that cause RP. Peripherin/RDS encodes a 36 kDa glycoprotein located in the rims of rod and cone outer-segment (OS) discs and is thought to be critical to the formation and stabilization of photoreceptor outer segments [19]. This is supported by the demonstration that rds mice homozygous for a null mutation in the peripherin-2 gene (Prph2) fail to develop outer segments, and heterozygous rds mice form highly disorganized structures [20,21]. Over 40 mutations have been identiﬁed within the peripherin/RDS gene in patients with retinal dystrophies [22]. The majority of these mutations are associated with adRP. Peripherin/RDS has been demonstrated to associate with Rom 1, a 37.3 kDa protein with similar membrane topology and identical distribution in the OS as peripherin/RDS [23]. Recently, Rom 1 was shown to be required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors [24]. In this study, Rom 1-/- mice displayed highly disorganized outer segments and rod photoreceptors slowly degenerated. Clearly, RP is a complex genetic disorder. In addition to those genes mentioned above, genes that encode transcription factors, mitochondrial proteins, and several proteins of unknown function are also mutated in RP. Furthermore, despite the large number of genes identiﬁed to date, molecular defects in > 50% of patients with RP have yet to be identiﬁed. An extensive account of the genes involved in RP is outside the scope of this review. Several excellent reviews on the molecular genetics of RP have recently been published [25–27], and an updated list of mutations in RP is available on the Internet http://utsph.sph.uth.tmc.edu/www/utsph/RetNet/genes. htm). B.

Apoptosis in RP

Despite the diverse genetics underlying the pathology of RP, there is one feature common to both human cases and animal models: the apoptotic cell death of rod and

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cone photoreceptors [28,29]. Apoptosis is a tightly regulated form of cell death in which a cell eﬀectively participates in its own demise. The execution of the death program is characterized by morphological and biochemical changes. These include mitochondrial depolarization and alterations in phospholipid asymmetry, chromatin condensation, nuclear fragmentation, membrane blebbing, cell shrinkage, and the formation of membrane-bound vesicles termed apoptotic bodies [30]. The generation of reactive oxygen species as well as alterations in cellular redox state have also been observed in many systems following the induction of apoptosis by a range of stimuli [31,32]. Many of the morphological changes associated with apoptosis are orchestrated by activation of a cascade of proteases termed caspases [33,34]. The caspases are a family of cysteine proteases comprised of at least 14 members, all of which contain an active site thiol group necessary for activity (Fig. 2) [35]. The highly conserved cysteine

Figure 2 Procaspase and active enzyme. Caspases are synthesized as inactive proenzymes that require proteolytic cleavage between their three domains (N-terminal prodomain, large P20 and small P10 subunits) at aspartic acid residues. Caspases contain an active site thiol group, which is necessary for activity. The highly conserved cysteine residues within their active site render caspases highly susceptible to S-nitrosylation and oxidative modification by reactive oxygen species.

Reactive Oxygen Species in Retinitis Pigmentosa

381

residues within their active site render caspases highly susceptible to oxidative modiﬁcation by reactive oxygen species, such as H2O2, and they can be readily converted to disulﬁdes and mixed disulﬁdes under conditions of oxidative stress [36,37]. Two major caspase pathways have been described (Fig. 3). The receptormediated pathway entails ligand binding to cell surface receptors, receptor oligomerization and recruitment, and activation of caspase-8, while the mitochondrial pathway involves caspase activation from within the cell. This intrinsic pathway is initiated by release of cytochrome c from the mitochondrial intermembrane space. Cytochrome c associates with Apaf-1 in a dATP-dependent manner to promote the activation of caspase-9. Caspase-9 then cleaves and activates the executioner caspases-3 and -7. In turn, caspase-3, described as one of the key executioners of apoptosis, has been demonstrated to cleave at least four other caspases (-2, -6, -8, and -10) [38] and ICAD

Figure 3

The mitochondrial and receptor-mediated caspase pathways. The intrinsic mitochondria-mediated pathway involves the complexing of mitochondrially released cytochrome c and Apaf-1 to caspase-9, leading to its autolytic activation, which in turn activates caspase-3, while the death-receptor pathway involves caspase-3 activation by caspase-8. Activation of caspase-3 results in activation of downstream effector caspases -6, -2, and -10. The two pathways are not necessarily mutually exclusive. Caspase-8 can indirectly induce activation of caspase-9 through Bid, an intermediate protein that acts at the level of the mitochondria, while activation of caspase-6 through the mitochondrial pathway can result in activation of caspase-8.

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(inhibitor of caspase-activated DNase), resulting in the translocation of CAD (caspase activated DNase) to the nucleus and DNA fragmentation [39,40]. Photoreceptors have been shown to die via apoptosis in several animal models of RP, including Royal College of Surgeon (RCS) rats [41], the retinal degeneration (rd) mouse [42–44], the retinal degeneration slow (rds) mouse [28,44], albino rats and mice exposed to excessive levels of white light [45,46], rhodopsin knockout mice [47], and rhodopsin S334ter rats [28,44]. Apoptosis, therefore, represents a common ﬁnal pathway in the pathology of RP. As a genetically encoded program of cell destruction, apoptosis is under the control of a network of highly ordered and conserved components. Regulatory components of the apoptotic pathways in photoreceptors, therefore, represent possible therapeutic targets for the inhibition of apoptosis and the amelioration of blinding retinal disorders such as RP. Indeed, manipulation of apoptotic regulatory components has proven successful to the retardation or prevention of retinal degeneration in several animal models of RP (Table 1). In addition to

Table 1

Rescue of Photoreceptor Degeneration by Various Agents

Agent Gene therapy Bcl-2 overexpression

Bcl-2 and BAG-1 co-expression Absence of c-fos Absence of p53 Survival factors Ciliary neurotrophic factor (CNTF) Brain-derived neurotrophic factor (BDNF) Fibroblast growth factor (FGF) Nerve growth factor Midkine Interleukin-1h Antioxidants and NOS inhibitors Methylprednisolone Dimethylthiourea Ascorbic acid L-NAME 7-Nitroindazole (7NI) Calcium channel blockers Diltiazem Flunarizine Verapamil Caspase inhibitors P35 Ac-YVAD-CHO z-DEVD-fmk

Model

Ref.

Pdegtm1/Pdegtm1 rd mouse rds mouse Light-induced injury (mouse) S334ter rhodopsin mutant Light-induced injury (mouse) rds mouse

226 180 181 182 227 228 229

rds mouse Light-induced Light-induced RCS rat rd mouse Light-induced Light-induced

injury (rat) injury (rat)

230 231 231 232 233 234 231

injury injury injury injury injury

79 78 77 235 66

Light-induced Light-induced Light-induced Light-induced Light-induced

injury (rat) injury (rat)

(rat) (rat) (rat) (rat) (mouse)

rd mouse Drosophila Light-induced injury (rat) Drosophila

200 236 237 236

Drosophila RCS rat Rhodopsin S334ter rats

238 239 240

Reactive Oxygen Species in Retinitis Pigmentosa

383

antioxidants, other agents used to delay or prevent retinal degeneration in animal models to date include calcium channel blockers, peptide inhibitors of caspase activation, survival or growth factors such as ciliary neurotrophic factor (CNTF), brainderived neurotrophic factor (BDNF), ﬁbroblast growth factor (FGF), nerve growth factor (NGF), midkine and interleukin-1h. The introduction of the antiapoptotic transgene Bcl-2 can also delay the degeneration of photoreceptors in several established models of retinal degeneration as well as in a new autosomal-recessive murine model (Pdegtm1/Pdegtm1). Coexpression of Bcl-2 with BAG-1 in the S334ter rhodopsin mutant also protects against photoreceptor apoptosis. In addition, gene therapy targeting other apoptosis-related genes such as c-fos, p53, and p35 has also been demonstrated to improve photoreceptor cell survival. Interestingly, p35 has recently been attributed antioxidant properties [48], and emerging evidence supports possible antioxidant functions for Bcl-2 [49]. Oxidative stress, therefore, may be a common mediator of photoreceptor apoptosis in RP. II.

REACTIVE OXYGEN SPECIES

Reactive oxygen species (ROS) are constantly generated under normal conditions as a consequence of aerobic metabolism. ROS include free radicals such as the superoxide . anion (O2 ), hydroxyl radicals (. OH), and the nonradical hydrogen peroxide (H2O2). They are particularly transient species due to their high chemical reactivity and can react with DNA, proteins, carbohydrates, and lipids in a destructive manner. Polyunsaturated fatty acids (PUFA) are particularly susceptible to oxidation. The membranes of both photoreceptor rods and cones are enriched with PUFA (f50%). Docosahexaenoic acid (DHA) is the most highly polyunsaturated fatty acid occurring in nature and is the principal fatty acid in photoreceptor membranes, rendering the retina innately vulnerable to lipid peroxidation [50,51]. The cell is endowed with an extensive antioxidant defence system to combat ROS, either directly by interception or indirectly through reversal of oxidative damage. When ROS overcome the defense systems of the cell and redox homeostasis is altered, the result is oxidative stress. A role for oxidative stress in the induction of apoptosis is provided by studies where addition of low levels of ROS induces apoptosis and the observation that various antioxidants such as N-acetylcysteine (NAC) can inhibit cell death [52,53]. Additionally, ROS generation has been reported to occur in tumor necrosis factor (TNF) and Fas-mediated apoptosis [54,55] as well upon treatment of cells with various agents including ultraviolet (UV) irradiation and chemotherapeutic drugs [56,57]. In addition to RP, oxidative stress is implicated in the pathogenesis of age-related macular degeneration, another blinding degenerative disorder of the retina, as well as several other diseases including acquired immunodeﬁciency (AIDS), Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease. Evidently the brain is particularly vulnerable. This is not suprising as the central nervous system is highly aerobic and thus extremely susceptible to oxidative stress. Additionally, the brain’s antioxidant defenses are relatively low, having almost no catalase and very low levels of glutathione [58,59]. Reactive nitrogen intermediates (RNI) are now also recognized as important radicals. Nitric oxide (NO) is formed endogenously from the oxidation of L-arginine to L-citrulline by a family of NADPH-dependent enzymes, the NO synthases. NO

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exists in diﬀerent chemical forms (NO, NO , and NO+) and thus has a wide-ranging degree of chemical reactivity and functions in a variety of diﬀerent biological roles [60]. These include regulation of the cardiovascular system, smooth muscle relaxation, neurotransmission, coagulation, and immune regulation. In addition, NO is thought to play a role in the control of intraocular pressure [61,62] as well as phagocytosis of photoreceptor outer segments by RPE cells [63]. There is increasing evidence to support a role for NO as a mediator of physiological processes in the retina such as phototransduction and the modulation of synaptic transmission from photoreceptors to bipolar and horizontal cells [64]. Despite these beneﬁcial functions, the molecule has a pivotal role in cell death, having the ability to both induce and protect against apoptosis, as well as driving an apoptotic response into a necrotic one. NO can also . combine with O2 to form peroxynitrite (ONOO), which like NO can diﬀuse freely intra -and intercellularly and also acts as a powerful oxidant. NO has been shown to play a role in uveitis, retinal ischemia, glaucoma, and retinal vascular dysfunction seen in diabetes [65]. Moreover, this laboratory has recently reported a key role for NO in light-induced photoreceptor apoptosis, an animal model frequently employed to study retinal degenerations such as RP [66]. .

A.

Sources of ROS in the Retina

In the retina ROS are produced in the mitochondrion, during RPE phagocytosis of outer segments, and as a result of photosensitization reactions. The mitochondrion, which is the organelle in eukaryotes responsible for aerobic respiration, is the most common source of ROS during apoptosis (Fig. 4). In normal resting cells, 1–2% of electrons carried by the mitochondrial electron transport chain (ETC) leak from this pathway and form the superoxide free radical. Complex I, NADH–ubiquinone oxidoreductase, and complex III, ubiquinol–cytochrome c oxidoreductase, are the . two sites where superoxide is produced [67]. O2 generated by the cycling of ubiquinol in the inner mitochondrial membrane is produced primarily in the mitochondrial matrix [68]. High concentrations of the enzyme MnSOD in the mitochondrial matrix ensure that this basal level of superoxide production is neutralized before it can cause damage to the cell. Rod and cone photoreceptor cells are particularly vulnerable to the generation of ROS by mitochondria due to their high rate of respiration and high density of mitochondria in inner segments. As a result, oxygen consumption by the retina is much greater than any other tissue. ROS can also be generated during phagocytosis. Phagocytes, including macrophages and neutrophils, are capable of generating large quantities of RNI and ROS, respectively. These free radicals are important for phagocytic antimicrobial and tumoricidal immune responses. The NADPH oxidase complex found at the plasma membrane of phagocytes produces reactive oxygen species that kill bacteria and other invaders [69]. Evidence is mounting that a similar, NADPH-like oxidase is expressed in a variety of cells throughout the body. Indeed, activation of this enzyme and . subsequent O2 production is necessary for apoptosis in a variety of systems [70,71]. . Most of the O2 produced reacts with itself to form H2O2. A large number of highly reactive oxidants may then be formed, including hypochlorous acid (HOC1), OH, and ONOO. While the reactive oxidants mentioned above are manufactured for the purpose of killing invading microorganisms, they may also inﬂict damage on nearby tissues. The RPE, a single layer of postmitotic cells, which lies close to the outer segments of photoreceptor cells, is the most active phagocytic cell in the body [72]. It

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Figure 4 Schematic diagram illustrating the major subunits of the electron transport chain (ETC) and sites of O2. production. Electrons enter the ETC at either Complex I (NADH–ubiquinone oxidoreductase) or Complex II (succinate dehydrogenase) following the oxidation of NADH and succinate, respectively. Ubiquinone is a lipid-soluble electron carrier and carries the electrons from Complex I and Complex II to Complex III (ubiquinol– cytochrome c oxidoreductase). Superoxide (O2. ) can be produced at both Complex I and Complex III. It is believed that semiquinone formation at both Complex I and Complex III results in the production of O2. . The inhibitors Rotenone and Myxathiazol inhibit electron transport at Complex I and Complex III of the ETC, respectively. These inhibitors can be used to prevent increases in superoxide produced by the mitochondria.

has been demonstrated that in the rat, a single RPE cell ingests and degrades 25,000– 30,000 membrane outer segment discs every day. RPE cells possess NADPH oxidase and have been shown to generate of H2O2 during phagocytosis [73]. The increase in H2O2 is accompanied by an increase in the activity of the antioxidant enzyme catalase, an event that appears to be essential in protecting the RPE cell against ROS produced during phagocytosis [74]. The retina is also susceptible to production of ROS during photosensitization reactions. These reactions involve the absorption of light by a photosensitizing molecule or chromophore, resulting in the excitation of this molecule and the formation of singlet oxygen, a particularly destructive oxygen metabolite [75]. Retinal photosensitezers include rhodopsin, lipofusin, and melanin. A recent study has demonstrated that rhodopsin is essential for light-induced photoreceptor apoptosis [76]. The involvement of ROS in this rhodopsin-mediated light damage is supported by several studies that employ antioxidants to ameliorate light-induced retinal degeneration [77– 79]. Lipofusin, a major chromophore located within the RPE, has been shown to accumulate with age and thought to play a role in age-related macular degeneration (AMD) [80]. Several studies have shown that photoactivation of lipofusin results in the generation of superoxide ions, singlet oxygen, and hydrogen peroxide, leading to lipid

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peroxidation and RPE dysfunction [75,81–84]. Protoporphyrin IX is another example of a photosensitizing molecule found in the RPE. Blue light–dependent activation of this molecule has been shown to result in the production of O2. and singlet oxygen [85]. B.

Sources of RNI and Regulation of Apoptosis by NO

NO is synthesized endogenously by a family of enzymes called nitric oxide synthases (NOS). Three major isoforms of this enzyme exist. While neuronal NOS (nNOS, NOS1) and endothelial NOS (eNOS, NOS3) are Ca2+/calmodulin dependent and constitutively expressed in a wide variety of cells, inducible NOS (iNOS, NOS2) is Ca2+ independent and is expressed in cells of the immune system and other cells in response to various stimuli [86]. All three NOS isoforms—eNOS, nNOS, and iNOS— have been reported in the retina, with NOS activity mainly reported in rod inner segments [87–90]. The ability of some cells to regulate the expression of iNOS allows them to produce large amounts of nitric oxide (NO) on demand. While nNOS, eNOS, and iNOS are all present in the cytoplasm, the most recently discovered NOS, mitochondrial NOS (mtNOS), is present exclusively in the mitochondrion [91–93]. Co-stimulation of superoxide production and mtNOS can result in the formation of high concentrations of the highly reactive and damaging peroxynitrite [94,95]. All NOS enzymes function as homodimers. They generate NO by a two-step oxidation of the amino acid L-arginine to citrulline and NO via the intermediate compound Nhydroxy-L-arginine (NHA). Activation of NOS requires the binding of several cofactors, as reviewed by Groves and Wang [96]. NO is perhaps one of the most potent regulators of apoptosis, having the ability to both induce and block cell death. Antiapoptotic eﬀects of NO are associated with low levels (10 nM to 1 AM) of exposure from the activation of endogenous NOS and from the slow release of NO from NO. donors. In addition, inhibition of apoptosis by NO can switch apoptotic cell death to necrosis in systems where NO may be acting below the point of no return. In these systems, inactivation of the execution machinery is not suﬃcient to prevent cell death, and cells eventually die by necrosis. The outcome depends on several factors including cell type, the level of stress, redox state, the chemical form of NO, and the ability of the cell to scavenge and detoxify ROS. NO either delivered by NO donors or endogenously produced by NOS enzymes induces apoptosis both in vivo [97,98] and, in several cell types, in vitro (e.g., neuronal cells [99] macrophages [100], cardiac myocytes [101], endothelial cells [102], lymphocytes and thymocytes [103,104]). The mechanisms of NO-induced apoptosis are presently under intensive investigation, and several mechanisms underlying the eﬀects of NO on apoptosis have now been elucidated. These include activation of the death receptor Fas through upregulation of Fas ligand expression [105], generation of peroxynitrite [106], inhibition of mitochondrial ATP synthesis [107], and inactivation of several antioxidant enzymes [108,109]. NO activation of guanylate cyclase and resultant increases in intracellular calcium through cGMP-gated channels has also been demonstrated as a mechanism of NO-induced apoptosis in photoreceptor cells in vivo [97]. NO has been reported to have protective eﬀects against apoptosis in a variety of cell types including lymphocytes [110–112], hepatocytes [113], endothelial cells [114,115], neurons [116], and eosinophils [117]. Several mechanisms have been pro-

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posed to elucidate the ability of NO to confer protection against cell death. These can be divided into cGMP-dependent and cGMP-independent mechanisms (Fig. 5). NO can stimulate cGMP production through the activation of soluble guanylyl cyclase. NO-dependent generation of intracellular cGMP has been shown to protect against apoptosis in lymphocytes [118], eosinophils [119], embryonic motor neurons [116], PC12 cells [120], and ovarian follicles [121]. Exactly how cGMP protects against apoptosis is at present unclear. In serum-deprived PC12 cells it is thought to involve the activation of protein kinase G and the inhibition of caspase activation through the prevention of cytochrome c release [120]. In other studies, activation of the serine/ threonine kinase Akt is associated with cGMP signaling [122]. Activation of Akt can promote cell survival through phosphorylation of the proapoptotic protein Bad [123] and procaspase-9 [124] and by preventing cytochrome c release from mitochondria [125]. cGMP-independent mechanisms of NO protection of cell death include inhibition of lipid peroxidation [126] or peroxyl radical scavenging [127], prevention of Bcl-2 cleavage [128] and cytochrome c release, and the induction of protective proteins such as Hsp70 and Bcl-2 [129,130]. In addition, many of the protective actions of NO are mediated by S-nitrosylation of proteins. S-nitrosylation involves the transfer of a nitric group to cysteine sulfhydryls, leading to the formation of a nitrosothiol (RSNO). While the movement and activity of nitric oxide is often restricting due to its very short half-life, nitrosothiols in comparison can be very stable compounds. Caspases, as previously mentioned, contain a highly conserved cysteine residue within their active site and are therefore a target for S-nitrosylation. Evidence in

Figure 5 Schematic model of NO regulation of cell death. NO can regulate cell death through cGMP-dependent and cGMP-independent mechanisms. Activation of GC by NO results in the production of cGMP, which can regulate apoptosis through modulation of ion channels or activation of protein kinases such as Akt. cGMP-independent mechanisms are largely mediated through S-nitrosylation reactions. Proteins been reported to be S-nitrosylated include several members of the caspase family, tissue transglutaminase, the two cysteine transcription factors NF-nB and AP-1, the G protein Ras, several ion channels, including the NMDA receptor, and complex 1 of the electron transport chain.

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support of caspase inhibition via S-nitrosylation initially came from a study carried out on puriﬁed caspases. This study demonstrated reversible inhibition of seven members of the caspase family. Dithiothreitol (DTT), an agent that removes the NO group bound to the thiol group on proteins, reversed inhibition by NO, indicating direct Snitrosylation of the caspase catalytic cysteine residue by NO [131]. NO inhibition of caspase activity has also been observed in a number of cells where NO donors were employed to inhibit caspase activities [132,133]. Endogenous NO has also been shown to inhibit caspase-3–like activity. In a study by Mannick et al. caspase-3 was found to be nitrosylated intracellularly and denitrosylated on Fas/Fas ligand cross-linking [111]. Recently pro caspase-3 was found to be S-nitrosylated on its catalytic-site cysteine in vivo [134]. Finally, NO has been reported to protect against Fas-induced liver injury by inhibiting caspase activity [135]. This caspase inhibition is DTT reversible, suggesting that cysteine S-nitrosylation is the underlying mechanism of caspase regulation by NO in this study. In addition to S-nitrosylation of caspase-3, both caspase-1 and caspase-8 have also been reported to be nitrosylated in cells. NO produced by the NO donor SNAP or iNOS is suﬃcient to prevent TNF-induced apoptosis in hepatocytes. Both caspase-8 and caspase-3 were found to be reversibly inhibited in this system, and caspase-8 was found to be S-nitrosylated [136]. Caspase-1, demonstrated to be involved in both apoptosis and cytokine maturation, is also thought to be S-nitrosylated with the demonstration of the reversal of NO-induced inhibition of caspase-1 by DTT in macrophages [137]. It is evident that NO is a potent inhibitor of caspase activity both in vitro and in vivo and that this inhibition is largely due to S-nitrosylation of the catalytic site cysteine conserved in all caspases. This discovery has signiﬁcant implications for the regulation of apoptosis by NO. Under an oxidizing intracellular environment the caspases are inactivated, the cell, therefore, must maintain a reducing environment in order for the execution phase of apoptosis to occur. Evidence is accumulating that NO modulates the biological functions of many other intracellular signaling proteins by S-nitrosylation. These include tissue transglutaminase (Ttg) [112], the two cysteine-transcription factors NF-nB [138,139] and AP-1 [140], both implicated in the regulation of apoptosis, several ion channels [141– 143], and the protease calpain [144] (Fig. 4). In addition, NO has also been reported to impair p53 function, possibly through amino acid modiﬁcations such as S-nitrosylation [145]. The antiapoptotic activity of NO is now well established. Inhibition of caspases via S-nitrosylation is probably the best-characterized mechanism of inhibition and explains the extensive protective eﬀect of NO. However, additional targets for Snitrosylation containing critical cysteine residues are increasingly being identiﬁed. The transfer of a NO group to a free SH group is now gaining acceptance as a ubiquitous mode of regulation of protein function comparable to phosphorylation during apoptotic cell death. C.

NO and the Mitochondrion

Exposure of cells to NO concentrations of greater than 1 AM can result in inhibition of apoptosis. However, while apoptosis is inhibited, necrotic cell death ensues. This may be due to a drop in cellular levels of ATP due to the failure of mitochondrial energy

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production and the greater dependence on ATP by apoptosis compared to necrosis [146,147]. NO disrupts mitochondrial function by reversibly inactivating cytochrome c oxidase, the terminal electron acceptor in the respiratory chain, thus stimulating superoxide generation by the respiratory chain, production of peroxynitrite, and ATP depletion [148,149]. Complex I and complex II are also inactivated by NO. It is thought that NO-induced complex I inhibition may result from S-nitrosylation of critical thiols [150] while complex II is inactivated in vivo by mechanisms resembling ONOO–induced oxidative stress [149]. Another site of action of NO on the mitochondria is the permeability transition pore (PTP) [151,152]. There is now increasing evidence in support of redox regulation of cytochrome c release during apoptosis [153]. The PTP plays an essential role in apoptosis; once opened, cytochrome c is released, resulting in the formation of the apoptososme, activation of caspase-9, and execution of apoptosis. Recently, inhibition of mitochondrial PTP opening was identiﬁed as a novel site of action for NO signaling in apoptosis [154]. Inhibition of PTP opening would result in less cytochrome c available to initiate apoptosis. This study suggests that a ﬁne balance exists between the pro- and antiapoptotic properties of NO at the level of the mitochondrion. III.

ANTIOXIDANT DEFENSE MECHANISMS

Both ROS and RNI can be generated as a result of normal metabolic processes or from toxic insult. It is clear that they are key participants in cellular injury, causing oxidative damage to all of the major classes of macromolecules, including proteins, lipids, carbohydrates, and nucleic acids. Under normal conditions, antioxidant systems minimize the adverse eﬀects caused by ROS (Fig. 6). However, when ROS overwhelm the biological defenses of the cell, the result is oxidative stress. The importance of the antioxidant defense system is emphasized by antioxidant enzyme dysfunctions, which have been demonstrated to be associated with several neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and ALS [155,156]. Primary antioxidant defense functions to prevent oxidative damage directly by intercepting ROS before they can damage intracellular targets. Components of this system include superoxide dismutase (SOD), glutathione peroxidase (Gpx), and catalase, all of which are found in photoreceptors and in the RPE. The potential importance of these enzymes as antioxidant defenses in the retina is illustrated by their upregulation in the RPE after exposure to excessive levels of light [157]. Their upregulation may act as a compensatory mechanism to combat production of ROS during light exposure [66]. Four classes of SOD have been identiﬁed to date. These are Mn-SOD, Cu, ZnSOD, Ni-SOD, and extracellular SOD. All four SOD enzymes destroy the free radical superoxide by converting it to H2O2 as follows: O2

.

þ O2

.

SOD

þ 2Hþ ! H2 O2 þ O2

H2O2 is one of the major ROS in the cell. While low levels result in apoptosis, high levels can lead to necrosis or caspase-independent apoptosis [158,159]. The primary defense mechanisms against H2O2 are catalase [160] and GPx through the glutathione (GSH) redox cycle [161]. Catalase is one of the most eﬃcient enzymes known and cannot be saturated by H2O2 at any concentration [162]. It is present only

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Figure 6 Intracellular sources of ROS and principal defense mechanisms. Major sources of ROS production include the mitochondria, endoplasmic reticulum, plasma membrane, and cytosol. The mitochondria generate O2. during respiration, which is converted to H2O2 by MnSOD. In the cytosol, O2. is converted to H2O2 by CuZnSOD. The two major defense systems against H2O2 are the GSH redox cycle, present in both the cytosol and mitochondria, and catalase, present only in the peroxisome fraction. Other sources of O2. include the enzymes xanthine oxidase in the cytosol, NADPH oxidase in the membrane, and cytochrome p450 in the ER. Bcl-2 can function as an antioxidant in some apoptotic systems through inducing the relocalization of GSH to the nucleus. NO can be produced in the cytosol by nNOS, eNOS, and iNOS or in the mitochondria by mtNOS. Additionally, TNFa can induce NOS activation, resulting in the generation of nitric oxide. NO2 can react with membrane lipids and can also cause mutations in DNA. In addition, ONOO can induce lipid peroxidation. Vitamin E (g-tocopherol) is the main membrane antioxidant and is effective in inhibiting peroxynitrite oxidation of lipids and can also detoxify NO2 [224,225]. Photosensitization reactions in the retina involving melanin, rhodopsin, lipofusin, and protoporphyrin IX can result in the production of singlet oxygen 1O2. For more detail see text.

in the peroxisome fraction, whereas the GSH redox cycle exists in the cytosol and mitochondria. Catalase reacts with H2O2 to form water and molecular oxygen: 2 H2 O2

Catalase

!

2H2 O þ O2

Overexpression of catalase in cytosolic or mitochondrial compartments has been demonstrated to protect cells against oxidative injury [163]. In addition, catalaseoverexpressing thymocytes are resistant to apoptosis [164]. These studies not only demonstrate the cytotoxic eﬀects of H2O2, but also emphasize the importance of catalase in antioxidant defense.

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391

Glutathione and the GSH Redox Cycle

The GSH system is probably the most important cellular defense mechanism that exists in the cell. The tripeptide GSH (g-glu-cys-gly) not only acts as a ROS scavenger but also functions in the regulation of intracellular redox state. The system consists of GSH, glutathione peroxidase, and glutathione reductase. Glutathione peroxidase catalyzes the reduction of H2O2 and other peroxidases and converts GSH to its oxidized disulﬁde form (GSSG), as outlined below: GPX

ROOH þ 2GSH ! ROH þ GSSG þ H2 O GSSG is then reduced back to GSH by glutathione reductase. The cell’s ability to regenerate GSH (reduction by either of GSSG or new synthesis of GSH) is an important factor in the eﬃciency of a cell to manage oxidative stress. Under normal conditions, more than 95% of a cell’s GSH is reduced and so the intracellular environment is usually highly reducing. A depletion of intracellular GSH has been reported to occur with the onset of apoptosis in a number of studies [165,166]. Moreover, this laboratory has reported a depletion of intracellular GSH during photoreceptor apoptosis in an in vitro model of retinal degeneration, an event accompanied by a concomitant increase in ROS [167]. The drop in GSH levels in this study may be due to an increased rate of GSH eﬄux, as described in Fas-induced apoptosis in Jurkat T lymphocytes [168]. In this study, inhibitors of the GSH transporter prevented the eﬄux of GSH, suggesting that the selective opening of a GSH-speciﬁc membrane channel is responsible. A more recent study supports this explanation, demonstrating that two speciﬁc inhibitors of carrier mediated GSH extrusion decrease GSH eﬄux and apoptosis [169]. The mechanism of GSH depletion in photoreceptor cell apoptosis, however, remains to be elucidated and may to be due to an increased rate of GSH eﬄux, an inhibition of synthesis, or intracellular oxidation of the tripeptide. B.

Bcl-2 as an Antioxidant

The proto-oncogene Bcl-2 was initially characterized for its unique ability to block cell death triggered by a diverse array of agents. At least 15 Bcl-2 family members have now been identiﬁed, all possessing at least one of four conserved motifs known as Bcl-2 homology domains. The Bcl-2 family consists of both proapoptotic members such as Bax, Bad, Bid and Bik and antiapoptotic members including Bcl-2, Bcl-xL, Bcl-w, and Mcl-1. The mechanism by which Bcl-2 protects against apoptosis remains somewhat elusive, with many novel functions for Bcl-2 suggested [170,171]. The majority of research on Bcl-2 and inhibition of apoptosis focuses on its interaction with and regulation of mitochondrial function, particularly the mitochondrial permeability pore [172–175]. However, the localization of Bcl-2 to intracellular sites of oxygen free radical generation, including the cytoplasmic face of mitochondrial outer membrane, endoplasmic reticulum, and nuclear membranes, led Hockenbery and colleagues to study possible antioxidant properties of Bcl-2. This study demonstrated that Bcl-2 expression protects cells against oxidant stress and led to the suggestion that Bcl-2 is a death repressor molecule functioning in an antioxidant pathway. Further evidence in support of this suggestion came from the observation that Bcl-2 knockout mice expressed a phenotype consistent with that of mice exposed to chronic oxidative stress

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(polycystic kidney disease and follicular hypopigmentation) [176]. These early studies led to research into possible redox aspects of Bcl-2. A study carried out in two mouse lymphoma cell lines demonstrated that overexpression of Bcl-2 leads to an increase in intracellular GSH levels [177]. Conversely, downregulation of Bcl-2 expression is associated with depletion of cellular glutathione [178]. These studies indicate that Bcl-2 may modulate apoptosis through an intermediary pathway such as GSH metabolism. Further studies on the relationship between Bcl-2 and GSH demonstrated that Bcl-2 expression alters GSH compartmentalization [179]. Overexpression of Bcl-2 leads to a relocalization of GSH from the cytosol to the nucleus. It is possible, therefore, that Bcl-2 inhibits apoptosis through modulation of intracellular glutathione levels and its redistribution to the nucleus, where it may act as a transcriptional regulator by altering the nuclear redox environment. Overexpression of Bcl-2 has been demonstrated to have a protective eﬀect in several models of RP, including the rd mouse [180], the rds mouse [181], and the lightinduced injury model [182]. The exact mechanism by which Bcl-2 protects against photoreceptor apoptosis in these models is unclear. A role for oxidative stress in lightinduced photoreceptor apoptosis has been demonstrated directly with the detection of O2. generation during light exposure and indirectly with the ability of antioxidants to ameliorate retinal degeneration [66]. Clearly there is considerable evidence in support of the hypothesis that Bcl-2 can inhibit apoptosis through an antioxidant eﬀect. However, there is also evidence to suggest that Bcl-2 acts against nonoxidative injury. This is supported by Bcl-2’s ability to protect against apoptosis in systems where ROS generation is considered to be negligible [183,184]. These studies were carried out in near-anaerobic conditions under the assumption that cells do not produce any signiﬁcant ROS in such conditions. Measurements of ROS were not carried out in these studies, and since then Degli Esposti and McLennan have demonstrated that mitochondria and cells do produce ROS in virtual anaerobiosis [185]. Therefore, the debate as to the mechanism of Bcl-2 protection against apoptosis may be due at least in part to a lack of crucial measurements of ROS. IV.

MECHANISMS OF PHOTORECEPTOR DEGENERATION IN RP: A POTENTIAL ROLE FOR ROS

Although considerable progress has been made in identifying genes responsible for RP, the sequence of events by which genetic defects such as those listed above can lead to retinal degeneration remain unclear. Animal models that carry spontaneous or engineered mutations in multiple genes implicated in human RP have been employed to study such mechanisms. In addition, constant exposure of animals to white light results in photoreceptor apoptosis and is also employed to study mechanisms of retinal degeneration. According to the ‘‘light equivalent hypothesis,’’ the molecular defects observed in some forms of RP may be equivalent to continuous light exposure [186]. Mutations in rhodopsin, rhodopsin kinase, transducin, and arrestin could potentially lead to prolonged signal ﬂow. Certain mutations in rhodopsin can lead to constitutive activity of the visual pigment [187]. These include mutations of Gly90, Glu113, Ala292, and Lys296 and result in constitutive activation of the G protein transducin. Two of these mutations, Gly90 and Ala292, have been shown to cause complete night blindness, while the Lys296 mutation results in RP. A mutation in the a subunit of transducin, which is found in patients with the Nougaret form of

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congenital stationary night blindness, is thought to lead to prolonged transducin activity [188]. It is easy to imagine how mutations in the rhodopsin shut-of machinery could lead to unabated signaling. Phosphorylation of rhodopsin by rhodopsin kinase and subsequent binding of arrestin completely inactivate the visual pigment. Absence of arrestin has been demonstrated to result in defective rhodopsin inactivation and subsequent prolonged photoresponse [189]. Mutations that lead to nonfunctional arrestin and rhodopsin kinase have been identiﬁed in patients with Oguchi disease, a type of stationary night blindness [190,191], while null mutations in arrestin occur in patients with arRP [192]. It is evident that mutations in several genes involved in the phototransduction cascade could potentially mimic the eﬀects of constant light exposure, leading to unabated cascade activation. While it is unclear exactly how continuous activation of the cascade leads to photoreceptor degeneration, it has ben suggested that oxygen toxicity may play a role. Both continuous light exposure and constitutive activation of the visual cascade could potentially lead to increased oxygen partial pressure (pO2) values in the photoreceptor microenvironment, due to unloading of the Na+/K+-ATPase pumps and reduced oxygen consumption by inner segment mitochondria [193]. Support for the involvement of ROS and oxidative stress in photoreceptor apoptosis following light exposure comes from several studies in which antioxidants appear to retard or inhibit the degenerative pathology. These include methylprednisolone [79], dimethylthiourea [78,194–196], and ascorbic acid [77]. However, in contrast to the light injury model, a role for oxidative stress in models of RP with mutations that potentially mimic continuous light exposure has yet to be established. This laboratory has recently reported on the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice [66]. This study demonstrated signiﬁcant O2. generation during photoreceptor apoptosis, an event accompanied by an increase in intracellular calcium levels and mitochondrial membrane depolarization. Furthermore, we showed that light-induced retinal degeneration is accompanied by increased expression of neuronal nitric oxide synthase (nNOS) and that inhibition of nNOS by 7 nitroindazole (7NI) is suﬃcient to prevent retinal degeneration. Continuous light exposure has also been demonstrated to increase rat nNOS levels, as measured by the NADPH diaphorase technique [197]. Evidently, neuronal NO plays a key role in light-induced retinal degeneration, possibly by activating guanylate cyclase and eliciting a cGMP increase in this model (Fig. 7). This is supported by the demonstration that an inhibitor of guanylate cyclase completely inhibits photoreceptor apoptosis [66]. Indeed, there is considerable evidence from other animal studies supporting a role for increased intracellular cGMP in rod photoreceptors in retinal degeneration. Increased levels of cGMP have been demonstrated to precede photoreceptor degeneration in the rd mouse [198] and rod-cone dysplasia in Irish setter dogs [199]. In the rd mouse, photoreceptor cell death is triggered by a null mutation in the gene encoding the h subunit of cGMP PDE. It is hypothesized that cell death in rd retinas is a direct consequence of elevated cGMP concentrations. Accumulation of cGMP in the outer segment would result in an increase in the opening of the cGMP-gated channels and a subsequent increase in intracellular calcium, resulting in cell death. This hypothesis is supported by the potent antiapoptotic eﬀects of calcium channel blockers in the rd mouse [200]. It is possible that calcium mediates cell death through the generation of ROS in this model, as excessive calcium uptake by mitochondria can

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Figure 7 Schematic diagram of the events involved in light-induced retinal degeneration. Exposure to excessive levels of light results in activation of neuronal NOS and subsequent activation of NO-sensitive guanylate cyclase. Calcium may then enter the rod photoreceptor outer segment through cGMP-gated channels, and intracellular calcium levels escalate. Excess calcium is taken up by mitochondria, resulting in increased mitochondrial calcium, ROS production, and mitochondrial depolarization.

lead to deenergized mitochondria and trigger the mitochondrial production of ROS [201–204]. This is supported by the demonstration that increased O2. production occurs as a result of elevated calcium levels in the light-induced model of retinal degeneration [66,205]. However, while the potential for generation of ROS in the rd model (and indeed other models where cGMP levels increase and result in elevated intracellular calcium) is evident, ROS measurements have not been made nor have the eﬀects of antioxidants been examined in such models. Clearly further work is necessary to conﬁrm or refute the involvement of ROS in these models. It has been hypothesized that oxygen toxicity is the mechanism behind photoreceptor degeneration in the rds/rds mouse, a spontaneous mutant in which photoreceptor cell death is triggered by a null mutation in the peripherin/RDS gene [193]. This mutation results in the failure of photoreceptor outer segments to develop normally. The potential involvement of oxidative stress in this model is supported by the recent demonstration that daily administration of melatonin can delay photoreceptor degeneration in the rds/rds mouse [206]. Melatonin has been demonstrated to have several neuroprotective mechanisms, including scavenging free radicals, inhibition of NOS, increasing the activity of antioxidant defenses and modulating the expression of apoptosis related genes such as Bcl-2. While the mechanism of action of melatonin remains to be elucidated in rds/rds mice, it could potentially act as a potent antioxidant in retarding photoreceptor apoptosis. Further evidence in support of the involvement of ROS in RP comes from studies on neurogenic ataxia retinitis pigmentosa (NARP). NARP is a maternally inherited syndrome clinically characterized by peripheral neuropathy, ataxia, seizures, and dementia. The ocular abnormalities in the NARP syndrome are reported in most

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cases as typical RP [207–209]. NARP is associated with a thymine to guanine point mutation at position 8993 in mtDNA (T8993G) in the ATPase 6 subunit gene of the mitochondrial ATP synthase (F1F0-ATPase). ATP synthase is located in the inner mitochondrial membrane of eukaryotic cells and is the key enzyme involved in aerobic ATP production. The T8993G mutation has been shown to result in impaired ATP synthase assembly and activity and decreased respiration rates and ADP/oxygen ratios [210–213]. A recent study investigating oxidative stress resulting from the NARP mutation in the mitochondrial ATPase 6 gene reported a huge induction of SOD activity in ﬁbroblasts harboring >90% of mutant mitochondrial DNA. This study implicates the participation of superoxides in the pathology of NARP with the demonstration that oxidative stress (denoted by the high SOD activity) is associated with increased cell death via apoptosis and inhibited by perﬂuoro-tris-phenyl nitrone, an antioxidant spin-trap molecule [214]. It is possible, therefore, that the T8993G mutation in the retina results in photoreceptor degeneration as a result of oxidative stress. This laboratory has previously described an in vitro primary culture model for the study of rod photoreceptor apoptosis in retinal degenerations such as RP [215]. Photoreceptor apoptosis in this model occurs over 24 hours, allowing the analysis of biochemical events occurring during the death process, such as the generation of ROS and changes in cellular redox state. Studies on this model established a central role for ROS as mediators of photoreceptor apoptosis in vitro [167]. Intracellular peroxide and superoxide anion levels were analyzed using the cell-permeable ﬂuorescent probes DCFH/DA and DHE, respectively. Peroxide levels were found to increase early in the death process, while superoxide generation was demonstrated to be a late event. Furthermore, a depletion of GSH levels was also observed, an event that could facilitate the early and prolonged ROS generation during apoptosis in these cells. The antioxidants pyrrolidine dithiocarbamate (PDTC) and zinc chloride completely inhibit photoreceptor cell death, demonstrating a crucial role for ROS during in vitro photoreceptor apoptosis. ROS are thought to act as mediators of photoreceptor apoptosis in this model because ROS generation and GSH depletion occur prior to biochemical and morphological features of apoptosis, such as mitochondrial depolarization, cell shrinkage, and nuclear condensation. Oxidative stress has also been shown to result in caspase inhibition during in vitro photoreceptor apoptosis. This is likely due to oxidation of the cysteine residue in the active site as DTT, a thiol-reducing agent, restores caspase activity in retinal lysates [216]. There is considerable signiﬁcance to this result, as it establishes that photoreceptors are capable of caspaseindependent apoptosis, suggesting that caspases may not be suitable therapeutic targets. These in vitro studies not only establish a crucial role for ROS as mediators of photoreceptor apoptosis, but also suggest the potential therapeutic value of antioxidants for the treatment of retinal degenerations such as RP. Indeed, a wide variety of experimental antioxidant therapeutic regimens have been recommended, including h-carotene, zinc, lutein, zeaxanthin, vitamin A, and n-acetylcysteine [217]. Moreover, vitamin E, a very eﬀective free radical scavenger, has been employed in treatment trials for RP. While one study showed vitamin E to have a beneﬁcial eﬀect on the disease, another found vitamin E to have adverse eﬀects [218,219]. Further trials are clearly necessary to establish whether or not antioxidant treatments such as vitamin E have beneﬁcial eﬀects on RP.

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CONCLUSION

Apoptosis represents a ﬁnal common pathway in the pathology of RP. The involvement of ROS and cellular redox state in apoptosis in several systems, taken together with the susceptibility of the retina to oxidative stress, has led to the investigation of the potential role of ROS in photoreceptor apoptosis. The majority of the evidence for the involvement of ROS in RP has come from studies carried out in this laboratory on in vitro photoreceptor cell death as well as studies on the light-induced model of retinal degeneration. In both models, photoreceptors die by apoptosis as evidenced by terminal dUTP nick end labeling (TUNEL), retinal DNA laddering, cell shrinkage, and mitochondrial depolarization. The light-induced model of retinal degeneration is particularly relevant to the study of RP. Several animal studies have demonstrated that photoreceptors from retinal degeneration mutants are more susceptible to the damaging eﬀects of excessive light than normal photoreceptors [220–223]. The evidence from these animal models suggests that excessive light may enhance the progression and severity of some forms of human RP. There is no doubt that ROS and RNI play crucial roles in this model with the demonstration that both antioxidants and inhibitors of NOS block photoreceptor apoptosis. Oxidative stress could therefore play a role in enhancing RP following exposure to excessive light and could also be involved in RP models with mutations that potentially mimic continuous light exposure, as previously discussed. The evidence for the involvement of ROS in inherited models of RP, while biologically plausible, is less convincing and mainly comes from studies where proteins and agents with antioxidant properties (e.g., Bcl-2, p35, and melatonin) improve photoreceptor cell survival. However, the mechanism of action of these antiapoptotic molecules in photoreceptor apoptosis is unclear, as actual ROS measurements have not been made. Analysis of ROS production in inherited models of RP is potentially diﬃcult, due to the time course of photoreceptor degeneration. In contrast to the light-injury and in vitro models where photoreceptor apoptosis occurs over 24 hours, thereby allowing the analysis of dynamic biochemical events such as ROS generation, retinal degeneration in inherited models of RP occurs over weeks (rd) and even months (rds). Antioxidants, therefore, may be more eﬀective in determining the role of ROS in inherited models of RP. Undoubtedly there is substantial evidence supporting a role for ROS in photoreceptor apoptosis, but future work is necessary to resolve whether or not oxidative stress acts as common mediator of retinal degeneration in RP.

ACKNOWLEDGMENTS This work was supported by Fighting Blindness (Ireland), The Health Research Board of Ireland and Bausch and Lomb, Co., Waterford, Ireland. REFERENCES 1. 2.

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18 The Role of Oxidative Imbalance in the Pathogenesis of Down Syndrome TING-TING HUANG Stanford University and GRECC, Palo Alto VA Health System, Palo Alto, California, U.S.A. SAILAJA MANTHA Stanford University, Stanford, California, U.S.A. CHARLES J. EPSTEIN University of California, San Francisco, San Francisco, California, U.S.A.

I.

INTRODUCTION

Down syndrome (DS), a frequent cause of mental retardation and congenital abnormalities, is caused in most instances by trisomy 21, the presence in the aﬀected person’s genome of an extra copy of all of human chromosome 21. However, in a minority of cases DS may result from trisomy for just the long arm (21q) of the chromosome or, rarely, from a segmental trisomy of 21q. The prominent clinical features of DS include mental retardation, hypotonia, characteristic minor dysmorphic features aﬀecting the head, face, and extremities, and defects in T-lymphocyte– mediated cellular immunity. Congenital heart disease (most characteristically an atrioventricular canal) is present in about half of aﬀected individuals, and the frequency of childhood leukemia is increased. Of particular interest is the development of Alzheimer’s disease among DS patients in the fourth decade of their lives, which makes DS the only known condition, other than mutations of the amyloid precursor protein (APP) or of the presenilin (PS1, PS2) genes, that uniformly predisposes to the development of Alzheimer’s disease at an accelerated rate [1]. 409

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The sequence of human chromosome 21 was completed in 2000 [2], and a total of 127 known genes and 98 predicted genes were identiﬁed, numbers that are still in a state of ﬂux. Attempts have been made to assign the diﬀerent aspects of the DS phenotype to particular regions of chromosome 21 by examining the phenotypes exhibited by persons with segmental trisomies of chromosome 21. However, it is still not possible to attribute speciﬁc aspects of the phenotype to the increased expression of particular genes or sets of genes. Nevertheless, there has been a keen interest in the role of oxidative imbalance in the pathogenesis of DS ever since the gene SOD1, which encodes CuZn–superoxide dismutase (CuZnSOD), was mapped to chromosome 21. Indeed, the level of CuZnSOD activity is increased by 50% in tissues of persons with DS, an increase attributable to the extra copy of SOD1 [3].

II.

THE SINET HYPOTHESIS

The increase in CuZnSOD activity in DS is not always accompanied by a concomitant increase in the activities of catalase or glutathione peroxidase (GPx), both of which act to remove H2O2, the reactive product of the reaction of CuZnSOD with superoxide (O2). In 1982, Sinet hypothesized that the increased CuZnSOD activity in the tissues of DS patients leads to increased oxidative damage within cells that, in turn, could aﬀect the function of the nervous system and contribute to mental retardation and the development of Alzheimer’s disease [4]. This hypothesis was based on the observation that there is an increase in CuZnSOD activity and an accompanying increase in GPx and hexose monophosphate shunt (HMPS) activities in red cells of persons with DS. The increases in GPx and HMPS were thought to be a response to increased oxidative stress in the red cells. Whereas GPx acts directly downstream from SOD to remove H2O2 at the expense of reduced glutathione (GSH), the HMPS acts indirectly to maintain high levels of GSH. Further support of Sinet’s hypothesis was derived from the positive correlation between memory function and GPx levels in red blood cells (RBC) [5]. If the GPx levels in RBC are representative of GPx levels in the brain, these results suggest a relationship between an inability to upregulate GPx and the severity of memory impairment in DS (see also Ref. 6). A.

The Biochemical Reactions

CuZnSOD is one of three superoxide dismutases present in mammalian cells. It is a copper- and zinc-containing dimer that is expressed constitutively and localized in the cytoplasm and mitochondrial intermembrane space. MnSOD, encoded by the gene SOD2, is a highly inducible manganese-containing tetramer that localizes to the mitochondrial matrix, and EC (extracellular)-SOD, encoded by the gene SOD3, is a copper- and zinc-containing tetramer found primarily in extracellular spaces and adherent to the endothelium of blood vessels. Superoxide radicals (O2) are generated in all oxygen-utilizing organisms from normal metabolism and enzymatic reactions that use O2 as the ﬁnal electron acceptor. O2 is very unstable and is quickly converted into H2O2 by spontaneous dismutation [Eq. (1)], by enzymatic dismutation [Eq. (2)], or by reacting directly with the (4Fe-4S) cluster of (Fe-S)-containing enzymes [Eq. (3)]. O2 can also interact with NO to form highly reactive peroxynitrite [Eq. (4)], which can cause protein nitration. H2O2 is converted into H2O by catalase, glutathione peroxidase (GPx), and other peroxidases

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such as peroxiredoxin [Eq. (5)]. However, in the presence of Fe2+ or other transition metals, H2O2 is converted into the highly reactive hydroxyl radical (OH.) [Eq. (6)] that is able to initiate a chain reaction of hydroperoxide formation [Eq. (7)] and thereby to produce further cellular damage. Although H2O2 is a mild oxidant by itself, the damaging eﬀect of H2O2 lies in its ability to cross membranes and diﬀuse for long distances into nuclei, mitochondria, and other organelles. O2 þ 2 Hþ ! H2 O2 E 2 O2 þ 2 Hþ ! H2 O2 þ O2 O2

þ

þ 2 H þ ½4Fe 4S

O2 þ NO ! ONOO E 2 H2 O 2 ! 2 H2 O þ O 2 H2 O2 þ Fe

2þ .

.

2þ

ð1Þ E ¼ SOD

! H2 O2 þ Fe

2þ

ð2Þ þ

þ ½3Fe 4S

ð3Þ ð4Þ

E ¼ GPx; catalase; or peroxiredoxin

! OH þ OH þ Fe

3þ

.

R1 H þ OH þ O2 ! R1 OO þ H2

ð5Þ ð6Þ ð7Þ

R1 OO. þ R2 H ! R1 OOH þ R2. The Sinet hypothesis raises the following question: if the role of CuZnSOD is to protect tissues and cells against the harmful eﬀects of superoxide radicals, how could having more CuZnSOD in the system be injurious? Deleterious eﬀects associated with overproduction or administration of CuZnSOD have been observed in multiple experimental systems [7–11] (see also examples cited for SOD1-transgenic mice below). It was postulated that increased CuZnSOD would lead to increased H2O2 production, but direct evidence for this has been inconclusive until recent years. It has now been shown that a concomitant increase in catalase activity will oﬀset the deleterious eﬀects of SOD overproduction [12,13], and H2O2-sensitive ﬂuorescence probes have revealed the presence of higher levels of H2O2 [12,13]. It has also recently been shown that CuZnSOD can act as a superoxide reductase (SOR) or as a superoxide oxidase (SOO) in biological systems [14]. In the presence of a reductant, such as Fe(II), CuZnSOD can act as a SOR to reduce the active site Cu(II) and catalyze an O2-dependent Fe(II) oxidation: E O2 þ 2 Hþ þ Fe2þ ! H2 O2 þ Fe3þ E ¼ CuZnSOD; acting as SOR

ð8Þ

In this situation, one molecule of O2 is converted into one molecule of H2O2, eﬀectively doubling the amount of H2O2 that can be generated when CuZnSOD is acting as a SOD [Eq. (2)]. Based on reaction rate calculations, the ratio of SOR to SOD activities increases with increasing amounts of CuZnSOD when the level of Fe(II) and the steady-state level of O2 generation remain constant. Therefore, increasing CuZnSOD levels would favor a higher level of H2O2 production. On the other hand, increasing the O2 concentration would favor SOD activity, which helps to explain the protective eﬀect of CuZnSOD under conditions of acute oxidative stress [14]. In addition to acting as a SOR, CuZnSOD has been shown to enhance H2O2- and Cu(II)mediated DNA damage [15] and to catalyze peroxynitrite-mediated tyrosine nitration [16]. Therefore, CuZnSOD can act as a prooxidant and lead to oxidative damage under several diﬀerent conditions.

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STUDIES IN TRISOMIC HUMANS AND HUMAN TISSUES

In this and the following sections we shall review the evidence relating to whether there is increased oxidative damage in the ﬂuids and tissues of humans with DS and of the various animal models of DS that have been developed. A.

Human In Vivo Data

Studies of blood and urine of persons with DS and of brain tissues from trisomic fetuses suggest that there is a general trend toward increased oxidative stress and damage. For example, measurements of the levels of total hydroperoxide in the serum of children with DS with a mean age 4.5 years showed a 1.67-fold increase over those of age-matched diploid controls [17]. Conversely, the total serum antioxidant capacity in persons with DS, which is determined by the collective antioxidant capacity of uric acid, glutathione, thiol groups, vitamins, glutathione peroxidase, superoxide dismutase, catalase, etc., is only 74% that of diploid controls. Consistent with this, the mean level of thiol (sulfhydryl) groups in the serum of children with DS measured only 72% that of the control level. It is possible that the lowered antioxidant capacity is due to an increase in reactive oxygen species (ROS), which would consume antioxidants in the system. However, it is not clear if there is a reduction in the antioxidant capacity independent of the increased ROS. Increased levels of markers for oxidative damage have been demonstrated in persons with DS. Elevated levels of thiobarbituric acid–reactive products (TBARS) were detected in the RBC, but not in the plasma [18] of persons with DS. Urine levels of products of lipid peroxidation and oxidative DNA damage were also increased in children with DS as indicated, respectively, by elevated levels of F2-isoprostane and malondialdehyde (MDA), both biomarkers of lipid peroxidation, and by a 1.74-fold increase in 8-hydroxy-2V-deoxyguanosine (8-OHG) [19,20]. Analysis of brain tissues from fetuses with DS showed elevated levels of lipid peroxidation, protein oxidation, and glycation products [21,22]. Brain tissue obtained at autopsy from adults with DS showed an increase in 8-OHG immunoreactivity in the cerebral cortex that preceded the deposition of the amyloid peptide Ah. However, the degree of Ah42 deposition was inversely related to the 8-OHG levels in the brain of adults with DS [23]. This paradoxial ﬁnding may be the result of sampling bias of the available postmortem tissues. Studies of the mRNA levels of the DNA repair genes XRCC1 (x-ray repair complementation 1), ERCC2 (excision repair complementation 2), and ERCC3 (excision repair complementation 3) in diﬀerent brain regions of persons with DS showed a consistent increase in expression that could be caused by a persistent increase in oxidative DNA damage [24]. Additional in vivo evidence for increased oxidative stress in the brains of persons with DS comes from the observation of chronic overexpression of the gene DSCR1 (DS candidate region 1), which is located in the putative DS region of chromosome 21 [25]. The expression of DSCR1, which encodes a protein that inhibits calcinurin, can be induced by oxidative stress (H2O2) and, according to gene dosage, is expected to be expressed under basal conditions at 1.5 times the normal level. However, expression has been shown to be in the 2- to 3-fold range, exceeding the expected level and suggesting a possible response to increased oxidative stress in DS patients. Recent studies of the proﬁles of proteins from the brains of persons with DS showed signiﬁcant increases in the concentrations of peroxiredoxins (Prx) I and II and a reduction in peroxiredoxin III [26]. Peroxiredoxins are a group of newly identiﬁed

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peroxidases with conserved cysteine residues that scavenge reactive oxygen species using thioredoxin as the electron donor. Prx I and II reside in the cytoplasm, and Prx III is in the mitochondria. The presence of increased levels of Prx I and II suggests an adaptive response to increased H2O2 in the environment, whereas decreased Prx III suggests impaired mitochondria integrity. Taken together, alteration in the levels of Prx I, II, and III suggests an increased level of oxidative stress in tissues of persons with DS. B.

Human In Vitro Data

Neuronal cultures derived from brains of fetuses with DS have been shown to diﬀerentiate normally, but then, while normal neurons remain viable, to undergo apoptosis and degeneration. The DS neuronal cultures exhibit a 3- to 4-fold increase in intracellular ROS and elevated levels of lipid peroxidation preceding neuronal death, and their early degeneration can be reversed by addition of antioxidants such as PBN, vitamin E, N-acetylcysteine, and catalase to the culture medium [27]. However, addition of SOD had no eﬀect, suggesting that OH. and H2O2 are the oxygen species causing the early degeneration of the trisomic neurons. A recent report showed that astrocytes and neurons isolated from patients with DS have a decrease in the production of the a-secretase–processed APPs and Ahs (the secreted forms) and an increase in the h-secretase–processed APPi and Ahi (the intracellular forms). Diploid astrocytes treated with the uncoupler CCCP (carbonylcyanide m-chlorophenylhydrazone) to inhibit mitochondrial energy metabolism showed a similar eﬀect on APP processing. Therefore, it is possible that trisomic neurons and astrocytes have a defect in mitochondrial energy generation, which could lead to increased oxidative stress and abnormal APP processing [28]. Furthermore, defective repair of oxidative damage in the mitochondrial DNA of trisomic ﬁbroblasts has been reported. This observation suggests that there may be an increased vulnerability to ROS-induced damage [29].

IV.

MOUSE MODELS FOR DOWN SYNDROME

The mouse chromosomal regions orthologous to human chromosome 21 include a 1 cM region of chromosome 10 (41–42.1 cM), a 24 cM region of chromosome 16 (47.2– 71.2 cM), and a 5 cM region of chromosome 17 (13.2–18.8 cM). Because chromosome 16 includes the bulk of the orthologous region, the development of mouse models to mimic the genetic abnormality of humans with DS has focused primarily on the mouse chromosome 16 syntenic region. These models include full and segmental trisomy 16 (Ts16) models and single gene transgenic mouse strains with extra copies of human or mouse genes from human chromosome 21 or mouse chromosome 16. A.

Data from Transgenic Mouse Models

Several strains of transgenic mice have been generated to assess the potential contributions of individual genes to the pathogenesis of DS and the DS phenotype. These include mice with the following transgenes: SOD1, APP, Dyrk (or minibrain), Ets2, Sim2, S100b, and PFKL. With the exception of S100b and PFKL, which reside on mouse chromosome 10, all the other transgenes are from mouse chromosome 16 or human chromosome 21. The copy number of the transgenes range from one to several

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copies, and their expression patterns do not always parallel those of the endogenous genes. Furthermore, the levels of expression of the transgenes are often greater than the 1.5 times the levels present in diploid animals. Perhaps the most direct evidence for a possible relationship between oxidative imbalance and DS-like abnormalities comes from studies of SOD1-transgenic mice expressing 3- to 4-fold increased levels of CuZnSOD [30,31]. Groner and colleagues, as well as other investigators, have observed defects in the structure of neuromuscular junctions (NMJ) [32] and serotonin uptake by platelets [33], an early onset of thymic involution [34–37], increased ROS generation in the skeletal muscles [38], and a defect in long-term potentiation (LTP) in hippocampal slices [39]. The defects in the NMJ of transgenic tongue and hind limb muscles were characterized as withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membrane area is decreased, and the secondary folds are often complex and hyperplastic. The morphological changes observed in the NMJ of SOD1-transgenic mice are similar to those found in aged mouse and rat muscles and in the tongues of children with DS [32,40,41]. The data were interpreted as indicating an accelerated aging process in the NMJ of SOD1-transgenic mice and of children with DS. In addition to the NMJ changes, impairment of muscle function [38] and muscular degeneration [42] have been observed in SOD1-transgenic mice. A defect in catecholamine and serotonin uptake in cells expressing increased levels of CuZnSOD was demonstrated in SOD1-transfected PC12 (rat pheochromocytoma) cells, and the defect was attributed to a diminished pH gradient (DpH) in the chromaﬃn granules [43]. A similar result was obtained for serotonin uptake and accumulation in the dense granules of the platelets of SOD1-transgenic mice [33]. These ﬁndings suggest that overexpression of CuZnSOD aﬀects the dense granule transport system for neurotransmitter and help to explain the depressed level of blood serotonin found in patients with DS. Long-term potentiation is a measurement of activity-dependent plasticity in the hippocampal formation [44]. There is growing evidence that LTP may represent the cellular substrate of at least some kinds of learning and memory. Hippocampal slices taken from SOD1-transgenic mice were shown to have normal synaptic physiology, but they were impaired in the ability to express LTP [39]. The defect could be abrogated by treating the hippocampal slices with catalase, an H2O2 scavenger, or with the OH scavenger, N-t-butyl-phenylnitrone (BPN). These results suggest that increased levels of CuZnSOD might lead to impaired learning and memory and that the defect may be caused by increased amounts of H2O2 and OH. in the hippocampus. Recently, a hypoxia/ischemia injury study with neonatal SOD1-transgenic mice showed enhanced brain injury because of the accumulation of H2O2 in the transgenic brain [45]. Similarly, a study of prenatal ischemia/reperfusion injury showed an increased level of lipid peroxidation in the brains of SOD1-transgenic fetuses immediately after reperfusion [46]. Adult SOD1-transgenic mice that had been subjected to ischemia/reperfusion injury while in utero also had a poorer performance in motor and coordination skills, suggesting a long-term eﬀect from the in utero treatment. Contrary to the examples cited above, increased levels of CuZnSOD have been shown to provide protection in a large number of experimental acute oxidative stress paradigms (see review by Huang et al., Ref. 47). These include adult brain ischemia/

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reperfusion injury, myocardial ischemia/reperfusion injury, brain trauma, MPTPinduced degeneration of dopaminergic neurons, methylamphetamine-induced neurotoxicity, and paraquat toxicity. The dichotomy between these and the previously cited results points to the complexity of free radical generation and interactions, but the general trend seems to indicate deleterious eﬀects from chronic overexpression of CuZnSOD and protective eﬀects under conditions of acute oxidative insult. Increased oxidative stress has been reported in transgenic mice expressing high levels of APP with the Swedish mutation, a cause of familial Alzheimer disease. Using isoprostanes (iPs) as biomarkers for lipid peroxidation, the mutant APP-transgenic mice were shown to have elevated levels of iPs in the urine, plasma, and homogenates from cerebral cortex and hippocampus at 8 months of age [48], which precedes the increase in Ah levels and the appearance of Ah deposits. APP and APP-like proteins have been shown to bind to and inhibit heme oxygenase (HO) [49], and APP with the Swedish mutation is substantially more inhibitory to HO than is wild-type APP. HO catabolizes heme, thereby leading to the generation of bilirubin, an important antioxidant. Cortical neuronal cultures from Swedish mutation APP-transgenic mice have a lower level of bilirubin, which strongly suggests a direct link between increased inhibition of HO and a reduced concentration of bilirubin. Based on these ﬁndings it has been suggested that a consequence of increased production of APP, as in the brain of persons with DS, would be increased inhibition of HO by the extra amount of APP with a resulting increase in oxidative neurotoxicity. The existence of defects in mitochondrial energy metabolism has been well established in the brains of persons with DS and Alzheimer disease and of transgenic mice expressing high levels of APP with the Swedish mutation. It is not clear, at least in the case of DS, if defects in mitochondrial energy metabolism cause or are caused by the increased oxidative imbalance. However, what is clear is that defects in mitochondrial energy metabolism, once present, lead to increased levels of intracellular APP and Ah and decreased levels of secreted APP and Ah. The decreased level of extracellular Ah and accumulation of the intracellular Ah leads to a disruption of calcium homeostasis and a disruption of the mitochondrial membrane potential. This process results in an increased production of ROS and increased oxidative stress, which in turn cause further depletion of mitochondrial energy metabolism and, ultimately, cell death [50]. This loop reinforces the deleterious eﬀects of APP overproduction and increased oxidative stress in patients with DS. No other transgenic models for DS have as yet been shown to have a connection to oxidative imbalance. B.

Data from Trisomic Mouse Models

Full Ts16 mice are generated from the crosses between two diploid (2N) mice, one with a normal chromosome complement and the other with two Robertsonian (Rb) fusion chromosomes that involve chromosome 16. One frequently used breeding scheme is the cross between Rb32Lub/Rb2H (11;16 and 16;17 fusions, respectively) and 2N mice [51]. Roughly 15% of fetuses from such a cross are Ts16. However, with very few exceptions, Ts16 mice die in the late gestation, making it impossible to carry out postnatal studies and a systematic analysis of oxidative imbalance. Furthermore, since mice with Ts16 are also segmentally trisomic for regions orthologous to segments of human chromosomes 10 and 17, the results are sometimes diﬃcult to interpret. The

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Figure 1 Antioxidant enzyme levels and aconitase activities in 3-month-old Ts65Dn mice. Tissues from five each male Ts65Dn and 2N controls were collected, flash frozen in liquid nitrogen, and processed for enzyme assays. CuZnSOD, MnSOD, and the aconitases were analyzed according to the protocols outlined recently [73]; catalase and glutathione peroxidase were assayed according to previous procedures [74,75]. Assay results are analyzed with two-tailed t-test. *p < 0.05.

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Figure 1

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Continued.

generation of Ts16:2N chimeras and transplantation of Ts16 neurons into the brains of normal hosts have allowed some limited postnatal studies [52,53]. Several years ago, a segmental trisomy model was generated at the Jackson Laboratory by using radiation to induce chromosome translocations [54,55]. This approach yielded a mouse model, designated Ts(1716)65Dn, with an extra chromosome composed of the centromere of chromosome 17 and the distal part of chromosome 16. The break point on chromosome 16 lies just proximal to the amyloid precursor protein gene (App) [56]. Since Ts65Dn mice are viable and survive well into adulthood, this model allows for postnatal analysis and the examination of agerelated changes. Shortly after Ts65Dn became available, another segmental trisomy model, designated Ts1Cje, was created during the generation of a Sod1 knockout [57]. By an unknown mechanism, the insertion of the knockout construct resulted in a chromosome translocation between chromosomes 16 and 11. This created a segmental trisomy with normal number of chromosomes and a triplicated region from Sod1 to the telomere region, but with only two functional copies of Sod1. Similar to Ts65Dn mice, Ts1Cje mice are viable, and studies on postnatal and age-related changes are possible. From the genetic point of view, Ts65Dn and Ts1Cje are better models for DS than is Ts16, since they do not have extra copies of genes that are not orthologous to genes on human chromosome 21. Studies of the Ts16 models have focused principally on the pathogenesis of neuronal defects. Similar to the human results, the activity of GPx in fetal Ts16 brain was shown to be the same as in 2N controls, but, interestingly, the level of lipid peroxidation in the fetal trisomy 16 brains was decreased. Cultures of microglia from Ts16 fetuses produced 2.8- to 20-fold more superoxide when stimulated by opsonized zymosan (OPZ) or phorbol myristate acetate (PMA), suggesting the possibility of increased oxidative stress during inﬂammatory responses [58]. Furthermore, cultured

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neurons and astrocytes derived from Ts16 fetuses were shown to have increased levels of protein oxidation and an upregulation of metallothioneins (MT) I and II [59,60]. Cultured hippocampal neurons from Ts16 fetuses were shown to have a lower level of GSH and augmented cell death compared to the 2N cultures [61]. A follow-up study using the rate of hydroethidine (HEt) oxidation as the indicator for superoxide generation showed a > 50% increase in the rate of HEt oxidation in Ts16 hippocampal neuron cultures [62]. This study also showed that the increased basal level of superoxide generation is probably due to a defect in complex I of the mitochondrial electron transport chain [62]. Similar studies have not been done with neuronal cultures derived from the segmental Ts16 mouse models. Early thymic involution is another hallmark of DS phenotype, and increased oxidative stress has been thought to contribute to this process. Cultures of Ts65Dn thymocytes have been shown to have higher levels of ROS generation and to be more susceptible to programmed cell death induced by LPS and dexamethasone [63]. As was observed with SOD1-transgenic mice, Ts65Dn mice display abnormalities of LTP [64]. They also have structural abnormalities of dendritic spines [65] and develop a time-dependent atrophy and loss of function of basal forebrain cholinergic neurons [66], the latter reminiscent of changes observed in Alzheimer disease. However, it has not been shown that any of these abnormalities are in any way related to oxidative stress. In addition to structural and physiological abnormalities, Ts65Dn mice display abnormalities of learning in the Morris water maze [67]. They display an impaired ability to learn the location of a platform hidden under the surface of the water in the maze and show an even greater impairment in their ability to learn the platform’s location after it had been moved to another quadrant of the tank (the reverse hidden platform test). However, Ts1Cje mice also have impaired learning, particularly of the reverse hidden platform task. Therefore, it is not possible to attribute these deﬁcits, at least not in their entirety, to the presence of three copies of either the App or Sod1 genes, since Ts1Cje has only two App genes and two functional Sod1 genes. To evaluate the antioxidant proﬁle of Ts65Dn mice, we measured CuZnSOD, MnSOD, GPx, catalase, and cytoplasmic and mitochondrial aconitase activities in brain, liver, and kidney of 3-month-old Ts65Dn animals (T-T Huang et al., unpublished data). CuZnSOD activities were increased by 25–38%, somewhat less than the 50% increase expected, and increased MnSOD activities were observed in brain (40%z) and kidney (30%z). Whereas there was a 31% reduction in GPx activity in the liver, a 38% increase in GPx level was detected in the brain. No signiﬁcant changes in catalase or in cytoplasmic and mitochondrial aconitase activities were observed (Fig. 1). Overall, although the increased MnSOD activities in the brain and kidney are intriguing, the results do not show any changes indicative of a signiﬁcant degree of oxidative imbalance in 3-month-old Ts65Dn mice. V.

CONCLUSIONS

The ﬁndings in humans with DS and in several transgenic and trisomic mouse models can be brieﬂy summarized as follows: 1. There is both in vivo and in vitro evidence for increased oxidative stress associated with DS. Changes compatible with oxidative stress are present in

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a variety of ﬂuids and tissues, including, in particular, the brain and cultured neurons. 2. Transgenic mice with increased levels of CuZnSOD recapitulate some of the cellular phenotypes found in DS. 3. Although there is evidence for oxidative stress in mice with full Ts16, there is presently no convincing evidence for a signiﬁcant degree of oxidative stress in mice with segmental Ts16, which are better genetic models for DS than is full Ts16. Although not identical to the human situation, animal models recapitulate certain aspects of the defects observed in patients with DS. SOD1 is the only gene in the DS region that has a direct association with oxidative metabolism, and it is therefore not surprising that most of the work related to oxidative imbalance has been done in SOD1-transgenic mice. Remarkably, SOD1-transgenic mice do show certain phenotypic resemblances at the cellular level (NMJ, serotonin uptake, thymocyte degeneration) to persons with DS, suggesting a role of oxidative imbalance in the pathogenesis of at least some aspects of DS. However, the level of CuZnSOD in SOD1-transgenic mice signiﬁcantly exceeds the 1.5-fold increase observed in DS. Furthermore, it is not known whether Ts65Dn mice, which do have a 1.5-fold increase in CuZnSOD activity and have a genetic makeup that more closely resembles that of DS, show similar defects. This needs to be done, and if similar defects are found, the role of increased SOD activity needs to be established by subtracting out one copy of Sod1 from the trisomic mouse by mating Ts65Dn mice with mice carrying a Sod1knockout allele. Humans with segmental trisomy 21 that does not involve SOD1 or APP, similar to the situation in the Ts1Cje mouse, display many if not all of the features of DS, including mental retardation and hypotonia [68,69]. Therefore, increases in the expression of these genes are not required for the development of mental retardation. However, it certainly cannot be concluded that such increases, as occur in full Ts21, do not make any contribution to the pathogenesis of the cognitive deﬁcits. It is likely that they do. With regard to the pathogenesis of Alzheimer’s disease, it should be noted that there is one case report in the literature of a 78-year-old woman with a mild DS phenotype who had no pathological evidence of Alzheimer’s disease [70]. She was found to have a segmental Ts21 that did not include the region encompassing both APP and SOD1 (the breakpoint was distal to SON). This result, as limited as it is, has been taken to mean that increased levels of APP production may be in fact be required for the development of Alzheimer’s disease in DS. However, this is certainly not proven since many other genes were also present in just two copies. To assess fully the role of oxidative imbalance in the pathogenesis of DS, it will be important to take advantage of the existing animal models and to carry out systematic analyses in the prenatal, postnatal, and adult stages. It will be important to know if oxidative imbalance is the cause or the eﬀect of DS phenotype, and how much it actually contributes to the defects observed in patients with DS. Although mental retardation and Alzheimer’s disease have been regarded as the most important phenotypes to study in DS postnatally, other phenotypes such as premature thymic involution and hypotonia deserve attention as well. Current technology allows high throughput analyses on gene expression and protein proﬁling. Therefore, it should be possible to screen tissues from animal mod-

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els and patients with DS for dysregulation of a large set of genes involved in oxidative and energy metabolism and to assess the possible involvement of oxidative stress in the pathogenesis of DS. The development of methods for the accurate detection of biomarkers for oxidative damage will allow for a more deﬁnitive ‘‘real-time’’ determination of the state of oxidative imbalance in tissues and even in whole animals or people. In addition, large-scale RNA in situ hybridization on whole-mount mouse embryos and on tissue sections of newborn mice have just become available for the expression patterns of genes on orthologous to those on human chromosome 21 [71,72]. The information on the tissue-speciﬁc and temporal-speciﬁc expression of chromosome 21 genes should enhance our understanding of the pathogenesis of DS and assist in the design of better animal models for mechanistic studies in the future. Why is it important to know if oxidative imbalance or stress is involved in the pathogenesis of DS? The principal reason is that oxidative imbalance is amenable to pharmacological intervention. Speciﬁc scavengers of ROS can be designed and targeted to speciﬁc locations if the types of ROS generated and sites at which the oxidative imbalance occurs are known. If oxidative imbalance does indeed play an important role in the prenatal development of DS phenotype and in the age-dependent postnatal degenerative and functional changes, then the possibility of reducing the severity of defects generated during prenatal development and delaying or preventing the postnatal consequences by pharmacological intervention may prove to be realizable in the future.

ACKNOWLEDGMENTS The writing of this chapter and the unpublished experiments reported were supported by NIH grants AG-16998, HD-31498, and AG-16633.

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19 Metal Homeostasis and Its Relation to Oxidative Stress in Alzheimer’s Disease ADAM D. CASH, RAVI SRINIVAS, CATHERINE A. ROTTKAMP, XIONGWEI ZHU, MARTA A. TADDEO, GJUMRAKCH ALIEV, HISASHI FUJIOKA, CRAIG S. ATWOOD, LAWRENCE M. SAYRE, RUDOLPH J. CASTELLANI, MARK A. SMITH, and GEORGE PERRY Case Western Reserve University, Cleveland, Ohio, U.S.A. AKIHIKO NUNOMURA Case Western Reserve University, Cleveland, Ohio, U.S.A., and Asahikawa Medical College, Asahikawa, Japan

I.

INTRODUCTION

There is an emerging acceptance that Alzheimer’s disease (AD) is a genetically complex heterogeneous disorder with no single mode of inheritance. Research has recently provided information on four genes that account for incidence of AD. The gene encoding the amyloid h protein precursor (AhPP) has 11 diﬀerent pathogenic mutations, which although accounting for less than 0.1% of all AD cases, has virtually complete penetrance of AD in the fourth to seventh decades of life. Presenelin 1 (PS-1) has been controversially hypothesized to play a role in the liberation of Ah from AhPP. A majority of early-onset familial AD mutations reside in PS-1, and PS-2 mutations exhibit a later age of onset than either AhPP or PSF-1. The gene encoding apolipoprotein E (APOE) in an allelic variation known as q4 has been associated with increased risk of early-onset, sporadic AD. The least common allele, q2, has been noted to have a weak, yet signiﬁcant protective eﬀect against AD. While the mechanistic details of AD genetics remains unclear, these genetic factors have been implicated, and detailing the connection between genetics and disease has yet to be done. 425

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Individuals aﬄicted by AD show increased oxidative damage to every class of biological macromolecule. In this chapter we review the role of iron, copper, and other redox-active transition metals and some observations related to oxidative homeostasis in the pathogenesis of AD. Analysis of the location of oxidative damage can provide clues to the mechanistic source. Subcellular localization shows that oxidative damage in AD primarily involves reactive oxygen species (ROS) that are generated in the neuronal cytoplasm. This compartment also shows extensive mitochondrial abnormalities as well as redox metal accumulation, indicating a possible cause-and-eﬀect relationship between metal abnormalities and increased oxidative damage. All aerobic organisms produce free radicals, predominantly superoxide, a side product of the reduction of molecular oxygen by mitochondria, where it is estimated that 1% of respired molecular oxygen will form O2. The average cell utilizes 1013 O2 molecules per day; this equates to 1011 free radical species produced per cell per day, and even more for highly metabolic neurons. Most of the metabolically formed free radicals remain within the mitochondria. Age, metabolic demand, or disease process increases oxidative insult. With respect to neurodegenerative disease, it is notable that most diseases show increases in prevalence with age and that the brain, though only 2–3% of total body mass, utilizes 20% of basal oxygen consumption. Additionally, H2O2, or O2 produced by brain-speciﬁc oxidases such as monoamine oxidase and xanthine oxidase, can immensely add to this oxygen radical burden in compartments less protected from reactive oxygen than mitochondria, and the actual location of oxidative damage in AD. Of the various free radicals produced by cells, the hydroxyl radical is the most highly reactive with all categories of biomacromolecules. Most hydroxyl radicals arise as a consequence of the Fenton reaction between reduced transition metals [usually iron(II) or copper(I)] and H2O2. Importantly, reduction of the resulting oxidized transition metal ions [iron(III) or copper(II)] by vitamin C or other cellular reductants regenerates the ‘‘active’’ transition metal and leads to the catalytic production of free radicals by the process of redox cycling. Notably, cellular reductants are often altered during neurodegenerative diseases, suggesting their use in redox cycling as well as autooxidant defense. An alternate reactive oxygen product is peroxynitrite, the result of a combination of NO and O2 [1]. The role of this process must be taken with care, as metal-dependent peroxidative nitration [2] makes peroxynitrite-dependent nitration indistinguishable from metal-dependent processes. Therefore, chains of metalindependent oxidative damage must demonstrate that they can occur without catalytic transition metals. Protection against oxidant damage comes through a variety of enzymatic and nonenzymatic antioxidants. In the former regard, metal-containing enzymes such as cytosolic copper-zinc-superoxide dismutase (CuZnSOD) and mitochondrial manganese-superoxide dismutase (MnSOD) convert superoxide to O2 and H2O2. Since these protective enzymes contain metals, there is seemingly a delicate balance in place within cells regarding the distribution of prooxidant versus antioxidant metal ions, which is critical to cellular homeostasis. Here we review evidence that strongly implicates a pivotal role for redox-active metal ions in the pathogenesis of AD and other neurodegenerative diseases such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS).

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Finally, that pleiotropic oxidative and metabolic changes occur in a chronic state argues that they represent a new homeostatic balance. Induction of heme oxygenase-1 (HO-1), the pentose phosphate pathway, increased free sulfhydryls as well as induction of the MAP kinase cascade argue for a dynamic state that protects neurons from death in AD. With this idea in mind, we also argue that much of the ‘‘pathology’’ of the disease, speciﬁcally Ah and H deposits, is a reﬂection of the compensations necessary to protect neurons from death. II.

OXIDATIVE DAMAGE

Oxidative stress, deﬁned as a breaching of the oxidant defense system, is often demonstrated as increased damage to biological macromolecules. While many of the earliest studies of AD focused on malonyldialdehyde and lipofuscin formation [3], factors poorly linked to oxidative stress in vivo, it was not until the study showing twoto fourfold increase in reactive carbonyls [4] that oxidative damage was established as a feature of the disease. In rapid succession, carbonyls derived from sugar were identiﬁed in the speciﬁc lesions of AD [5] as well as in their component proteins, Ah [6] and H [7,8]. While these studies suggested that oxidative damage primarily involved the lesions, possibly due to low protein turnover [9], it is also the case that the glycoxidative chemistry underlying the observed carbonyl condensation requires active metal-centered ROS for formation [10]. Therefore, it was not a surprise when the lipid peroxidation adduction product of hydroxynonenal [11,12] and acrolein [13] were found in the lesions of AD. It was more surprising, however, that the dominant site of damage was not lesions but rather the cytoplasm of neurons vulnerable to death in AD [1,12]. Since the products of lipid peroxidation and glycation can yield crosslinked molecules, oxidative modiﬁcation by these pathways can make breakdown of molecules less likely. In model studies, crosslink formation not only makes proteins resistant to removal by the proteosome but also inhibits proteosome activity [14]. Oxidative inhibition of proteolytic activity underlies the accumulation of ubiquitin conjugates in AD [15,16] as well as the cytopathology noted below. To address the site of ROS formation, we focused our eﬀorts on ﬁnding a marker resulting from primary attack, rather than the result of complex reactions involving adduction of secondary metabolites. This necessitated examining damage to a cell constituent with a short half-life. When we analyzed protein-based reactive carbonyl and nitrotyrosine formation, we found that they were conﬁned primarily to the cytoplasm of vulnerable neurons with less evidence of their formation in Ah or H deposits. These ﬁndings point to the cytoplasm, not the lesions, as the source of ROS. Nevertheless, protein-based modiﬁcations are far from ideal since they are often associated with crosslinking that slows their turnover. Therefore, crosslink modiﬁcations of proteins, while useful to assess history, may reveal less of the current state of oxidative stress. Our observation that RNA, a rapidly turned over cell constituent, is a target for oxidative stress provided a possible marker, 8-hydroxyguanosine (8OHG), a nucleic acid modiﬁcation predominantly derived from .OH attack of guanidine 8OHG is greatly increased in cytoplasmic RNA in vulnerable neuronal populations [17]. Ultrastructural analysis shows that most 8OHG is present in the endoplasmic reticulum (Fig. 1).

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Figure 1

Most 8OHG is in the neuron endoplasmic reticulum rather than cytoplasmic membrane bound organelles such as mitochondria.

In quantitative analysis of the extent of 8OHG oxidation, we found that cases of AD with the most extensive Ah deposits show the lowest 8OHG levels, and neurons containing neuroﬁbrillary tangles (NFT) have about half the levels of 8OHG (i.e., current oxidative stress status) despite an obvious history of oxidative damage (i.e., advanced glycation end products or lipid peroxidation), suggesting that both Ah and neuroﬁbrillary tangles are associated with reduced oxidative stress. 8OHG is likely to form at the site of .OH production, a process dependent on redox-active metal-catalyzed reduction of H2O2 together with cellular reductants such as ascorbate or O2. Both iron and copper in their redox-competent states

Figure 2 The extent of oxidative damage (8OHG) is highly dependent on the degree of mitochondrial abnormalities for normal aging (r 2 = 0.87) but displays a nonlinear relationship among cases of AD.

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lysosome mitochondria

O2-

H2O2

Mered

O2ascorbate

Meoxd •OH

Figure 3 The hypothesis of how mitochondrial abnormalities promote oxidative damage is through release of redox-active metal (Me), most likely iron and copper, into the cytosol to catalyze .OH production through Fenton or Haber–Weiss reactions.

are bound to neuroﬁbrillary tangles and Ah deposits [18–20]. That 8OHG levels are inversely related to the extent of Ah deposits, yet are distant from the deposits, suggests a complex interplay between Ah redox metal activity that may be critical to metal dynamics within the neuronal cytoplasm. A possible key element to these dynamics is mitochondria in the cell body. While morphometry of the number of mitochondria in AD remains essentially unchanged, both mtDNA and the protein cytochrome oxidase 1 protein are increased severalfold in AD. The extent of mtDNA increase for individual cases of AD correlates highly with the extent of 8OHG levels during normal aging, but not as much for cases of AD (Fig. 2). Ultrastructural analysis of the subcellular localization of mtDNA showed the increase was restricted to the vacuolar portion of lipofuscin. These ﬁndings suggest a fundamental alteration in mitochondrial dynamics, possibly involving diﬀerence in proliferation, transport, or turnover related to deﬁciencies in microtubules [69] or degradative insuﬃciencies [16]. The increased release of redox active metal ions as mitochondria are catabolized is a possible reason for the increased oxidative damage observed in the cytoplasm (Fig. 3).

III.

COMPENSATORY RESPONSES

Intracellular oxidative balance is highly regulated, and, therefore, in the face of stress, an increase of compensatory mechanisms would be expected in AD. One of the ﬁrst reports of oxidative stress in AD came from the ﬁnding of pentose phosphate

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pathway induction in AD [21]. Reducing equivalents in the form of NADPH are essential to maintain oxidant defenses. Later ﬁndings of induction of the NADPHrequiring enzyme HO-1, the rate-limiting enzyme required for transforming prooxidant heme to antioxidant bilirubin, substantiated the view of active antioxidant defense in AD. Since HO-1 induction is synchronous with H accumulation in neurons [22,23], reduced oxidative damage in neurons with H accumulation may be a part of this antioxidant response. Another consequence of increased NADPH generation is increased glutathione and free sulfhydryls. The latter are speciﬁcally increased in vulnerable neurons [24] since sulfhydryls are a major component of the cellular antioxidant defenses to ROS and secondary metabolites. Additionally, vulnerable neurons show activation of the MAP kinases ERK, SAPK, and p38 [25–27], denoting a survival response consistent with the protracted nature of AD where neuronal death is an inconsistent feature [28]. Finally, the observed decrease in oxidative damage with Ah and H accumulation suggests that they may represent important survival responses [29].

IV.

METAL IMBALANCE IN NEURODEGENERATIVE DISEASE

The generation of oxygen free radicals and consequent tissue oxidative stress in vivo is thought to be mediated by redox transitions associated with ‘‘free’’ iron more than any other cellular transition metal. Indeed, alterations in free iron and iron homeostasis have been implicated in a number of neurodegenerative disorders including AD [18, 20,30]. While cellular iron homeostasis is mainly mediated by the transferrin receptor and ferritin, the lactotransferrin receptor [31], melanotransferrin, ceruloplasmin [32], and divalent cation transporter also play important roles. Therefore, alterations in any of these proteins could contribute to alterations in brain iron metabolism in AD, PD, ALS, and Huntington’s disease (HD) [33]. Control of these regulatory proteins and therefore overall regulation of cellular iron metabolism involves the action of two iron regulatory proteins, IRP-1 and IRP-2. IRP-1, but not IRP-2, is rapidly activated by extracellular H2O2, establishing a regulatory connection between the control of iron metabolism and response to reactive oxygen. Interestingly, IRP proteins show signiﬁcant alterations in AD [30], paralleling alterations in redox-active iron [18]. Tightly regulated homeostasis involving copper transport, storage proteins, and chaperonins has evolved of the necessity of copper transport to essential enzymes without engendering cellular toxicity arising from reactive oxygen generation by ‘‘free’’ copper [34]. Ceruloplasmin, a key copper storage protein, responds to oxidative stress. However, while ceruloplasmin is increased in brain tissue and cerebrospinal ﬂuid in AD, PD, and HD [32,35], neuronal levels of ceruloplasmin remain unchanged [32]. Therefore, while increased ceruloplasmin may indicate a compensatory response to increased oxidative stress in AD, its failure to do so in neurons may play an important role in potentiating metal-catalyzed reactive oxygen formation [32]. Prevention of toxic interaction between free radicals and reactive transition metals is a primary function of glutathione (GSH). Perturbations to GSH metabolism may play an important role in neurodegenerative disorders such as AD and PD as well as the prion diseases, conditions linked to abnormalities in copper homeostasis [36–38] and redox balance [24,32,39,40].

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METALLOENZYME DYSFUNCTION

The principal metalloenzymes implicated in cellular regulation of reactive oxygen are the Mn and CuZn superoxide dismutases (SOD-2 and SOD-1, respectively) that remove O2 and the enzymes catalase and peroxidase that remove H2O2. Usually SOD is considered the ﬁrst line of defense against buildup of reactive oxygen because it removes O2, the initial form of metabolically produced reactive oxygen. SOD has attracted considerable attention due to the linkage of about 5% of ALS cases to mutations in SOD. With the advent of transgenic mouse knockout and overexpression models, it has become possible to directly evaluate the extent to which alterations of SOD activity might aﬀect cell viability. If one or the other of the SOD enzymes serve a singular and noncompensable antioxidant function, then knockout animals should exhibit increased oxidative stress parameters. This oxidative stress may be global or may be localized to the cellular compartment normally protected by SOD. In support of this notion, while MnSOD knockout mice are embryonic lethal, the heterozygotes, suﬀering a 50% drop in mitochondrial SOD activity but no reduction of CuZnSOD or GSH peroxidase activity, were found to exhibit increased oxidative damage to mitochondria as evidenced by increased mitochondrial protein carbonyls and 8-hydroxydeoxyguanosine in mitochondrial protein and DNA, respectively [41]. In contrast, no damage to cytosolic proteins or to nuclear DNA was observed, indicating that mitochondrial oxidative imbalance is contained within the organelle. Analysis of homozygote knockouts showed mitochondrial degeneration so extensive that they could only be followed for the 2 weeks that these mice lived with administration of metal-centered antioxidants [42]. These results suggest that decreases in MnSOD activity in vivo can explain increased oxidative damage in mitochondria and alterations in essential mitochondrial function but not changes in the cytoplasm. Overexpression of human CuZnSOD in mice resulted in a 10-fold higher level of SOD protein and SOD activity in both myocytes and endothelial cells and was able to quench a burst of superoxide (EPR detection) and reduce functional damage following 30 minutes of global ischemia [43]. These results suggest that superoxide is an important mediator of postischemic injury, and it is therefore surprising that CuZnSOD knockout mice under basal conditions show little, if any, neurodegenerative phenotype [44] but do show abnormalities in injury repair [45]. Nonetheless, it is apparent that decreases in CuZnSOD activity can lead to a perturbation of cellular antioxidant defense mechanisms that increase sensitivity to prooxidant conditions. VI.

METALS AND A

Microparticle-induced x-ray emission revealed that zinc(II), iron(III), and copper(II) are signiﬁcantly increased in AD neuropil and are further highly concentrated within the core and peripheral areas of senile plaques [21]. Using an in situ iron-detection technique, we found a marked association of redox-active iron with both NFT and senile plaques in AD [18]. This association of iron with NFT may partly be related to iron binding to H [41,42]. Also, whereas the iron-regulatory protein IRP-1 was present at similar levels in both AD and control brain tissues, IRP-2 co-localized with redoxactive iron in NFT, senile plaques, and neuropil threads [30] suggests that alterations in IRP-2 might be directly linked to impaired iron homeostasis in AD.

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Recently we found that the redox activity of AD lesions can be detected directly and can be inhibited by initial exposure of the tissue sections to copper- and ironselective chelators. Redox activity can be reinstated following reexposure of the chelator-treated sections to either copper or iron salts [20]. These studies suggest that the lesions may play critical roles in metal homeostasis. Interest in the factors responsible for aggregation of Ah led to an obvious early focus on the possible involvement of trace metal ions. The ﬁrst systematic study reported that aluminum, iron, and zinc but not calcium, cobalt, manganese, copper, magnesium, sodium, or potassium accelerated aggregation of Ah [44]. However, more recent studies suggest that the aggregatory eﬀect of metals depends critically on the pH. Copper(II) induced aggregation of Ah (1-40) when the pH was lowered from 7.4 to 6.8,a phenomenon that was not common to other metals tested [45]. A mildly acidic environment together with increased zinc(II) and copper(II) are common features of inﬂammation, and this could explain the rapid deposition of A following head injury as well as inﬂammation associated with increased oxidative damage due to promotion of .OH-like activity from microglial-derived peroxynitrite. The association of copper(II), zinc(II), and iron(II) with Ah found in vitro could explain the enrichment of these metals with senile plaques in AD. Evidence that metal ions may actually be involved in pathological aggregation of Ah in vivo is provided by the recent study reporting that divalent metal ion chelators facilitate solubilization of Ah deposits from AD tissue, though eﬃcient extraction of Ah also required the presence of magnesium(II) and calcium(II) [46,47]. The ﬁnding that divalent metal ion-induced aggregation of Ah is aﬀected by apolipoprotein E, and diﬀerentially by the diﬀerent apolipoprotein E isoforms, was recently proposed to be consistent with a role for apolipoprotein E as an in vivo chaperone for Ah [48]. Although metal-induced aggregation of Ah may not reﬂect redox chemistry, the metals bound to soluble or aggregated forms of the peptide do seem to be redox active and thus capable of mediating ROS production [42,49–52]. Taken together, the studies by us and others indicating the presence of redox-active iron and copper in AD pathology suggest that these metal accumulations could be major contributors to metal homeostasis abnormalities. The recent interest in the possible contribution of transition metals bound to Ah in mediating redox chemistry and possibly oxidative stress is intimately tied to the ongoing studies directed at clarifying the association of Ah with free radical production [53]. Since the ‘‘peptide-only shear’’ mechanism, initially proposed to explain radical production, lacked chemical precedent, the consensus has been that increased oxidative stress associated with Ah must represent either peptide interactions with redox-active metal ions [54,55] or indirect mechanisms triggered by Ah ﬁbrils [56]. The probability that Ah-associated oxidative stress might arise at least in part from direct reactive oxygen production by Ah-bound transition metals has become the subject of detailed investigation over the past year. A role for bound copper in possibly mediating Ah peptide neurotoxicity is supported by the ﬁnding that added copper(II) markedly potentiates A toxicity in primary neuronal cultures to an extent that is greatest for Ah (1-42) HAh (1-40) >Ah (40-1) f rodent Ah (1-40), apparently via H2O2 production [53]. Recent studies with iron show that Ah toxicity for cells in culture can be potentiated by iron addition and greatly reduced by the iron chelator deferoxamine [55,57]. These ﬁndings suggest a critical role for both iron and copper in Ah activity. Also, Cu(II) binds to amyloid protein precursor (AhPP) and appears to be reduced to copper(I) concomitant with production of a disulﬁde linkage [58]. Sub-

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sequent exposure to H2O2 results in reoxidation of copper(I) and concomitant sitespeciﬁc cleavage of AhPP. Redox chemistry associated with AhPP-bound metals could thus, alongside that mediated by Ah-bound metals, contribute to a perturbation of oxygen radical homeostasis and resulting ROS-mediated neuronal toxicity in AD. In this regard, a recent study demonstrated that copper-dependent (but not iron- or zinc-dependent) toxicity in primary neuronal cultures is potentiated by AhPP, and in particular by the copper(II)-binding domain of AhPP [59]. These same workers showed that the levels of copper (but not iron or zinc) in certain brain regions determined by atomic absorption are increased in AhPP-knockout mice as well as knockout mice for the amyloid precursor-like protein 2, also known to bind copper and zinc [59]. This ﬁnding was proposed to suggest that perturbations to AhPP metabolism could initiate pathological changes associated with copper metabolism in AD. While in cell culture [55] and in transgenic mice [60], Ah is associated with oxidative damage, in AD there is no such association [17]. In fact, Ah deposition is marked by a reduction in the quantity of oxidative damage in AD, sporadic [61–63] and genetic cases [64], as well as in Down syndrome [65]. These ﬁndings suggest that a more complex interplay between homeostatic imbalance in metals, Ah and other metabolic features is critical to the quantity of oxidative damage observed. One of the most striking of these changes is a shift in redox balance signaled by induction of the pentose phosphate pathway, NQO1 induction, as well as increased sulfhydryls [24,39,40]. Increased availability of reducing equivalents will fundamentally alter the relationships between metals and redox balance and may explain why we note prooxidant eﬀects with normal cells, while in AD, Ah is associated with apparent antioxidant activity. VII.

CONCLUSIONS AND THERAPEUTIC POTENTIAL

This chapter has highlighted the substantial body of evidence for the fundamental role that metals play in oxidative balance in AD and other neurodegenerative diseases. Although most focus has been on direct redox roles of the transition metals iron and copper, non–redox-active metal ions, such as zinc or aluminum, can alter free radical homeostasis by displacing iron and/or copper from sites, e.g., storage proteins, where they are inactive in oxyradical production. In addition, dysregulation of metalloenzymes or operation of mutant forms adds to a neuron’s burden in its eﬀorts to maintain redox homeostasis. The stage is now set to critically examine the importance of these basic research ﬁndings as they are translated into therapeutic modalities, such as antioxidants and chelating agents, that are being used clinically. In these studies it is essential to understand the delicate homeostatic balance between metals, redox control, and the pathological lesions [66]. We think it is essential to understand the contribution of each before proposing to remove the ‘‘pathological’’ entity [67], which may further exacerbate the disease process. We are encouraged that use of antioxidant and iron chelators show clinical eﬃcacy for AD [68]. We think the greatest beneﬁts in metal-directed therapeutics will derive from treatments that sequester metals responsible for oxidative imbalance while leaving those metals that may have adaptive value in neuronal survival. Ultimately, the most eﬃcacious therapy will result from directing our eﬀorts to the fundamental abnormalities, displacing neurons from physiological homeostasis. Therapeutics based on the latter will require a detailed mechanistic understanding of the diseases. While the prominent increase in neuronal oxidative damage and mitochondrial abnormalities of AD would seem to mark an unstable state leading to cell death, instead, extensive compensatory

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mechanisms protect neurons from death. We suggest that the pleiotropic nature of changes in AD, broadly considered pathological, is instead the display of neuronal oxidant defenses.

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20 Oxidative Stress and Huntington’s Disease C. TURNER and ANTHONY H.V. SCHAPIRA Royal Free and University College Medical School, University College London, London, England

I.

INTRODUCTION: THE CLINICAL AND PATHOLOGICAL SPECTRUM OF HUNTINGTON’S DISEASE

In 1872 George Huntington ﬁrst described a devastating movement disorder clinically characterized by chorea, personality changes, and dementia. The disease now carries his name, Huntington’s chorea. The ﬁrst clinical symptoms of Huntington’s disease (HD) are personality changes, mood disturbances, and other psychiatric abnormalities in the fourth or ﬁfth decade of life. Involuntary choreiform movements develop over the next 20 years, and these are often associated with progressive cognitive decline. Death usually occurs due to the complications of immobility. There is no treatment for the progressive neurodegenerative process underlying HD, and management is purely symptomatic control of the movement disorder and psychiatric features. HD aﬀects approximately 4–8 per 100,000 persons throughout the world [1]. HD is an autosomal dominant condition caused by an expansion of a CAG repeat sequence within the IT15 gene on chromosome 4, which encodes a 349 kDa protein, huntingtin (htt) [2]. Huntingtin is associated with cytoplasmic granules, organelles involved in retrograde transport and protein degradation in some cortical and striatal neurons. The normal function of huntingtin is otherwise speculative [3,4]. The huntingtin gene is expressed in cells throughout most tissues. There is widespread neuropathology in the HD brain but the levels of gene expression do not correlate with the pathology. There is a 30% reduction in brain mass with a direct correlation between length of disease and brain weight [5]. The loss is severe in the basal ganglia with up to 60% reduction in mass in the caudate, putamen, and globus pallidus. The neu439

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ropathology is most severe in the caudate nucleus where there is marked neuronal loss and astrogliosis. The GABA-nergic medium spiny neurons are mostly aﬀected within the caudate. These neurons project to the globus pallidus and receive glutaminergic input from the cortex and dopaminergic input from the substantia nigra. The larger cholinergic and aspiny NADPH-diaphorase interneurons are relatively spared. The putamen and cerebral cortex are less aﬀected, and the cerebellum is only mildly aﬀected [6]. The cause of such speciﬁc regional and subpopulation neuronal loss and the absence of obvious pathology in other tissues remain uncertain. There has been increasing evidence over the past 20 years that there is a defect in energy metabolism in HD. This possibly leads or contributes to free radical generation, slow excitotoxicity, mitochondrial dysfunction, decreased mitochondrial membrane potential, a lower threshold for apoptosis, and alterations in the hemodynamic responsiveness of the cerebral vessels. Some or all of these processes could lead to neurodegeneration. The role of mutant htt in these processes is currently speculative. Recent evidence suggests that mutant htt binds preferentially to CREB-binding protein (CBP) and possibly other transcription factors [7]. This may lead to widespread cellular transcriptional dysfunction. Other evidence suggests that htt binds to key metabolic enzymes such as GADPH. There is conﬂicting evidence as to the pathological role of intranuclear inclusions that have been observed in neurons of in vitro and animal models of HD and postmortem brain tissue [8]. The intranuclear inclusions may sequester vital proteins such as transcription factors or have a direct physical disruptive eﬀect on nuclear processes. Alternatively, the inclusions may be protective by removing toxic mutant htt from its pathological role [9]. We will review the evidence for the role of oxidative stress in the pathogenesis of HD and discuss the putative sources of free radical generation. We will also discuss the interplay between oxidative stress, excitotoxicity, and mitochondrial dysfunction. II.

FREE RADICALS AND OXIDATIVE STRESS

Free radicals are independent and reactive species that contain one or more unpaired electrons. Oxidative stress can be deﬁned as a condition in which the production of free radicals and the damage they cause exceed the ability to scavenge the free radicals or repair their damage. Some of the most important free radicals in biological systems are oxygen-centered free radicals or reactive oxygen species (ROS) such as hydroxyl (OH), superoxide (O2), and nitric oxide (NO). Free radicals extract electrons from neighboring molecules to complete their own orbital. This leads to oxidation of the recipient molecule, including proteins, lipids, and carbohydrates. The hydroxyl radical is the most reactive oxygen species that acts in close proximity to its site of generation. Transition metals such as iron, copper, and zinc catalyze its formation, and any molecule that has one of these heavy metals as a constituent component is susceptible to oxidative damage. The superoxide radical is less reactive than the hydroxyl radical and is therefore capable of crossing membranes and acting at a distance from its site of generation. It is especially important in inactivating enzymes containing Fe-S clusters. Both of these radicals can react very rapidly with nitric oxide to form peroxynitrite. Peroxynitrite can diﬀuse rapidly across cell membranes and oxidize many types of cellular macromolecules.

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III.

441

THE PATHOGENIC ROLE OF FREE RADICALS IN NEURODEGENERATION

There are a number of intracellular generators of radicals, but current evidence suggests that mitochondria produce the majority of free radicals that are constantly being produced as a byproduct of aerobic metabolism. Mitochondrial production of free radicals increases when the electron transport chain is inhibited or acquires molecular defects [10,11]. Defects in energy metabolism caused by toxins or genetic defects can result in reduced levels of ATP and increased free radical generation. Reduced ATP leads to a number of deleterious consequences. One of these is a partial depolarization of the neuronal membrane due to a failure to maintain the ionic gradient across the plasma membrane. This can lead to a loss of the Mg2+-dependent block of the NMDA Ca2+ channel and enable ambient levels of glutamate to activate the receptor. This allows large inﬂuxes of calcium into the cell, resulting in excitotoxicity [12]. One consequence of elevated intracellular calcium levels is an increase in activation of nitric oxide synthase and generation of nitric oxide (NO). This may also

Figure 1

Putative mechanisms in the generation of free radicals in HD.

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result in peroxynitrite formation produced by the reaction of NO with superoxide. Ca2+ is also sequestered in mitochondria and in vitro experiments have demonstrated that Ca2+ concentrations similar to those seen with excitotoxic stimuli can induce excess ROS [11]. Redox-sensitive dyes, such as dihydrorhodamine, have demonstrated glutamate-induced increases in mitochondrial Ca2+ and free radical generation in vitro [10]. An adaptive cellular response to increased free radicals is to upregulate components of the antioxidant system such as superoxide dismutase (SOD), catalase, and reduced glutathione (GSH). When these systems become overwhelmed and fail to control macromolecular oxidation, the system undergoes oxidative stress. It has been suggested that mitochondria are particularly susceptible to oxidative injury because of the high levels of free radicals generated during aerobic metabolism. Mitochondrial DNA (mtDNA) is especially susceptible to oxidative damage due to its location in the mitochondrial matrix next to the respiratory chain, its lack of histones, and the paucity of mtDNA repair mechanisms [13]. The slow progressive nature of neurodegenerative diseases could be explained by a cycle of energy impairment and oxidative damage (see Fig. 1). IV.

CAUSES OF OXIDATIVE STRESS IN HD

There have been several mechanisms by which oxidative stress may be produced in HD. These include cellular energetic defects, nitric oxide dysregulation, excitotoxicity, an uncontrolled inﬂammatory response, and heavy metal accumulation. Although these processes are discussed individually, there is considerable overlap in the mechanisms underlying their eﬀect. This interplay is not fully understood, and any experimental data that connect these processes will be highlighted. A.

Defects in Energy Metabolism

There has been increasing evidence from imaging studies, animal models, and postmortem data over the past 20 years that there is a defect in energy metabolism and mitochondrial function in HD. This is potentially the primary mechanism leading to free radical generation in HD. It has not been proven whether mitochondrial dysfunction is the direct consequence of mutant htt or results from defects in other cellular pathways caused by mutant htt. This uncertainty is less important when considering putative therapies for HD as defects in energy metabolism are probably deleterious to cellular homeostasis irrespective of when and how they occur in the pathogenic cascade leading to neurodegeneration in HD. 1.

The Role of Imaging in Detecting Metabolic Defects

Positron Emission Tomography. During the 1980s positron emission tomography (PET) in conjunction with 18F-deoxyglucose were used extensively to investigate regional utilization of glucose in HD brain. Most studies demonstrated a decrease in striatal regional cerebral metabolic rates for glucose and oxygen (rCMRGlc and rCMRO2, respectively) in symptomatic HD patients [14,15]. More recent studies have demonstrated a defect in the cerebral cortex and striatum but not in the thalamus and cerebellum [16,17]. The degree of dementia and the duration of chorea correlated with the severity of the defects in most cortical regions and the caudate nucleus. The severity

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of the chorea correlated more speciﬁcally with the defect in the lentiform nucleus, whereas the degree of disability correlated with defects in most cortical regions except the occipital and superior frontal cortex. These initial studies provided evidence that there are widespread changes in HD brain, which involve cortical as well as subcortical structures. Further studies examined individuals at risk of HD. Before genetic testing became available in the late 1990s, it was suggested that striatal hypometabolism could be used as a method of presymptomatic diagnosis. Striatal defects have been described in presymptomatic/chorea-free/at-risk patients [18–21]. In one study [18], 31% of individuals at risk for HD had reduced caudate glucose metabolism bilaterally compared to an expected gene frequency in this group of 34%. As these studies were performed before genetic testing was available, precise correlation between genotype and PET data could not be made. The similar predicted frequency of HD in the at-risk populations and the frequency of hypometabolism in the striatum was suggestive that there was an association between presymptomatic HD and a striatal energy defect. All the presymptomatic patients with striatal hypometabolism had a normal CT brain scan, which suggested that the metabolic defect preceded gross tissue loss and dysfunction. In conclusion, PET studies are suggestive of a defect in energy utilization in HD striatum and cortex and imply that the dementia of HD is not purely subcortical. The PET studies do not distinguish between failure of energy utilization secondary to atrophy that cannot be detected on a CT scan, or a primary defect in energy metabolism within the neurons. There is the potential for using PET to monitor progression of disease and putative treatments in the future, although the advent of NMR spectroscopy has made it possible to study metabolic defects more accurately and in greater detail. Nuclear Magnetic Resonance Spectroscopy. An initial study using 1H nuclear magnetic resonance (NMR) spectroscopy in HD demonstrated elevated lactate levels in the occipital cortex and variable rises in the basal ganglia of symptomatic patients [22]. The level of occipital lactate also correlated with the duration of illness. Unfortunately, there was wide variability in the data from the striatum possibly because of spectroscopic artefacts but also due to relatively fewer living neurons in the striatum. The striatal levels of N-acetyl aspartate (NAA), a putative neuronal marker, were reduced, and levels of choline (Ch), a putative glial marker, were elevated. This suggested that there was striatal neurodegeneration and gliosis. Further studies conﬁrmed these ﬁndings [23,24]. Subsequently 1H MRS demonstrated increased lactate in the frontal cortex of approximately 50% of symptomatic HD patients and four presymptomatic patients [25]. A reduced N-acetyl aspartate/choline (NAA/Ch) ratio in the symptomatic patients was also found, and this was related to disease severity. The reduced ratio in HD suggested that there was selective neuronal degeneration. In the same study, there was no reduction in the NAA/Ch ratio in the presymptomatic HD patients, suggesting that defects in energy metabolism occur before gross neuronal loss and before phenotypic expression. A further study using 31P MRS demonstrated that there was a signiﬁcant decrease in the phosphocreatine–to–inorganic phosphate ratio in symptomatic HD patients’ muscle. This suggested a widespread defect in energy metabolism in HD that

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was not conﬁned to the nervous system. In the same study it was demonstrated that the oral administration of coenzyme Q10, an essential component of the mitochondrial respiratory chain, signiﬁcantly reduced occipital cortex lactate levels as measured by 1 H MRS in symptomatic HD patients. The levels of lactate returned to pretreatment values when the coenzyme Q10 was stopped supporting a treatment-related eﬀect [26]. A recent randomized placebo-controlled trial of coenzyme Q10 and remacemide [a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist] demonstrated no signiﬁcant beneﬁt in early HD. There was a trend towards slower disease progression with coenzyme Q10 but remacemide had no clinical beneﬁt. Unfortunately, the trial was designed to identify an approximate 35–40% slowing in functional decline, and so smaller beneﬁts would have been missed [27]. A larger cohort of symptomatic HD patients have been studied using 1H MRS, and an elevated lactate level in the occipital cortex was conﬁrmed as well as variable increases in the frontal and parietal cortices. They also demonstrated, using extremely small voxels (2–4 AL), highly elevated lactate levels in the striata of HD patients that showed a marked asymmetry towards higher levels in the left striatum. This has been related to asymmetrical activation of the striata, as all patients were right-handed. The lactate levels were independent of NAA and choline levels, suggesting a metabolic eﬀect as opposed to diﬀerences in neuronal and glial populations. They also demonstrated that CAG repeat length, especially when over 45, correlated with levels of striatal lactate. Three of the eight asymptomatic patients they studied demonstrated elevated striatal lactate, suggesting that energy-related defects occur before clinical diagnosis is possible [28]. A recent study examined in vivo muscle energy metabolism in HD using 31P MRS. At rest, there was reduced phosphocreatine: inorganic phosphate ratio in the symptomatic patients only. Muscle ATP/(PCr + inorganic P) was signiﬁcantly reduced in both symptomatic and presymptomatic patients. During recovery from exercise the maximum rate of ATP production (Vmax) in muscle was also signiﬁcantly reduced. The Vmax deﬁcit overage also correlated with CAG repeat length. These ﬁndings support a role for mitochondrial dysfunction in the pathogenesis of HD in particular, the possibility that mutant htt expression could aﬀect mitochondrial function directly, independent of any of the speciﬁc biochemical or pharmacological features of the striatum. It also suggests that 31P MRS may act as a surrogate marker by which to study disease progression and response to therapy [29]. Defects in creatine and NAA levels have been found in presymptomatic (30%) and symptomatic (60%) HD caudate on 1H MRS. The levels of creatine also correlated with CAG repeat length and performance in cognitive timed tests [30]. 1 H-MRS has been performed on the CSF of patients with HD. A slight but signiﬁcant reduction in lactate and citrate was found in HD patients but not PD patients. The signiﬁcance of these observations is uncertain but may relate to impaired glycolysis and Krebs cycle function or to gross neuronal loss [31]. Several studies have found an elevated lactate-to-pyruvate ratio in symptomatic HD CSF samples, which directly supports the data obtained from NMR of the brain but not CSF [26]. A more recent study using 1H and 31P MRS failed to demonstrate a signiﬁcant increase in putaminal or occipital cortical lactate levels or a decrease in the phosphocreatine or ATP levels. The authors suggested that contamination from surrounding CSF may explain apparent increases in HD brain lactate [32].

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There are alternative explanations for the elevated lactate levels observed in HD patients. Enhanced neuronal stimulation leads to elevated lactate levels due to increased anaerobic glycolysis. This explanation appears unlikely as one would then expect elevated glucose utilization in the cortex and striatum, but PET studies suggest that there is a reduction [14,15]. Mismatching of blood ﬂow and neural activity might also cause elevated lactate levels, but there are conﬂicting data on whether there are changes in cerebral blood ﬂow in HD [33]. Imaging in Animal Models of HD RODENTS. The development of the ‘‘MicroPET’’ has enabled functional in vivo studies in rodents. Rats with unilateral quinolinic acid–induced striatal lesions demonstrated decreased 2-deoxy-2-[18F]ﬂuoro-D-glucose uptake in the lesioned side one week after injection consistent with a defect in energy metabolism secondary to excitotoxic damage to the striatum [34]. The R6/2 mouse was the ﬁrst transgenic mouse model of HD. The transgene contains the ﬁrst exon of huntingtin with an expanded CAG repeat length between 141–157 [35]. NMR analysis of R6/2 mouse brains has demonstrated a large nonlinear drop in NAA levels commencing at 6 weeks of age coincident with the onset of symptoms and the presence of intranuclear inclusions but in the absence of neuronal death [36]. In vitro NMR analysis also revealed signiﬁcant increases in glutamine, taurine, cholines, and inositol and decreases in glutamate and succinate. It is speculated that the elevation in glutamine represents a profound metabolic defect associated with a decrease in activity of glutaminase. This is a mitochondrial enzyme in neurons responsible for the conversion of glutamine to glutamate and is probably involved in the glutamate/glutamine neuronal/glial cycling system. Further studies using the R6/2 mouse have demonstrated that dietary supplementation with creatine enhances brain creatine levels and reduces the drop in NAA with time. This suggests that enriching metabolic pathways with creatine may slow neuronal dysfunction [37]. Chronic systemic administration of 3-nitropropionic acid (3-NP), which is an inhibitor of succinate dehydrogenase (SDH) or complex II of the mitochondrial respiratory chain, reproduces most of the motor, cognitive, and histopathological features of HD in primates, rodents, and humans. The temporal and spatial evolution of lesions induced by 3-NP in rats has been studied using MRI, where diﬀusionweighted images were more sensitive at detecting lesions than T2-weighted images (3 h postdose versus 4.5 h), and lesions were observed in the striatum, hippocampus, and corpus callosum but not the cerebral cortex [38]. 1 PRIMATES. Serial H-NMR spectroscopy has been used to assess the striatal and occipital cortical concentrations of NAA, phosphocreatine/creatine, choline, and lactate every 2 weeks in 3-NP–treated baboons [39]. A region-selective increase in lactate was detected in the striatum in association with the formation of a lesion in the dorsolateral putamen on T2-weighted MRI. There was also a region-speciﬁc and progressive decrease in NAA, creatine, and choline occurring 3 weeks before the ﬁrst reduction in lactate. This suggested that NMR may be used to detect the early stages of brain metabolic impairment. The inhibition of SDH by 3-NP occurs throughout the brain, but there are selective decreases in NAA and creatine in the striatum, which suggests that there is preferential vulnerability of the striatum to impairment of mitochondrial function by 3-NP.

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The Mitochondrial Respiratory Chain in HD

Recently there has been much interest in the role of mitochondria in neurodegenerative diseases. This is based on the ﬁnding of several primary mitochondrial mutations associated with various neurological phenotypes, e.g., Leber’s hereditary optic neuropathy, as well as in vitro and in vivo evidence for mitochondrial respiratory chain dysfunction in several neurodegenerative conditions such as Parkinson’s disease, Friedreich’s ataxia, and Huntington’s disease [40]. It is postulated that a defect in mitochondrial function could lead to a defect in energy metabolism, which in turn produces depleted intracellular levels of ATP and an increased susceptibility to excitotoxic damage, increased free radical production, and apoptosis. Mitochondrial Cell Biology. The bacterial hypothesis of the origin of mitochondria suggests that alpha-purple bacteria were incorporated into eukaryotic cells, and, during evolution, these bacteria transferred many of their essential genes to the nuclear chromosomes [41]. The mitochondria still have remnants of their bacterial origin such as the use of N-formylmethionyl-tRNA as the initiator of protein synthesis [42,43]. The mitochondrial respiratory chain consists of ﬁve multisubunit protein complexes, which are located in the inner mitochondrial membrane, and two mobile electron carriers, cytochrome c and ubiquinone (Fig. 2). The respiratory chain produces ATP by the production of a proton gradient across the inner mitochondrial membrane, which drives ATP production by ATP synthase or complex V. The proton gradient is produced by the release of protons from complexes I, III, and IV into the intermembranous space. The energy for this process is yielded from the transfer of electrons from NADH/FADH2 through the protein complexes to the ﬁnal electron acceptor, oxygen. Complex V couples the reentry of protons to the mitochondrial matrix with the production of ATP. Complex III and to a lesser extent complex I are major sites of generation of potentially toxic ROS [44]. Each mitochondrion contains 2–10 molecules of circular double-stranded mitochondrial DNA (mtDNA), which encode 22 transfer RNAs, 2 ribosomal RNAs, and 13 proteins: 7 subunits of complex I, cytochrome b of complex III, cytochrome oxidase (CO) I, COII, and COIII of complex IV, and subunits 6 and 8 of complex V (ATPase) [45,46]. MtDNA is inherited through the maternal line. More than 100 mtDNA mutations have been associated with a wide spectrum of human disease [47]. Mitochondrial DNA displays heteroplasmy: this is where mutations coexist with normal wild-type molecules within the same cell type [48]. The proportion of the mutant form varies between tissues depending on segregation during mitosis. A high level of the mutant form is required to produce respiratory chain dysfunction, although this is partly determined by the level of dependence of the tissue on oxidative metabolism. Thus, tissues with a high level of dependence on oxidative respiration, such as neurons, require a high level of ATP production to restore ionic homeostasis following the controlled ﬂux of ions across the cell membrane during electrical signaling. Most of the ATP utilized by Na+/K+ ATPase and Ca2+ ATPase in maintaining ionic homeostasis is generated in mitochondria. The Mitochondrial Hypothesis in Neurodegeneration. The mitochondrial hypothesis in neurodegeneration suggests that defects in mitochondrial metabolism lead to a chronic depletion in cellular ATP and a lowered threshold for apoptosis. This may trigger the neuron to enter apoptosis, leading to neurodegeneration. Mitochondrial dysfunction may be precipitated by exogenous and endogenous oxidative phosphor-

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Figure 2 A schematic diagram of the mitochondrial respiratory chain. Cyt c = cytochrome c; Q = ubiquinone; ROS = reactive oxygen species.

ylation (OXPHOS) inhibitors or by mutations in nuclear or mitochondrial DNAencoded OXPHOS subunits or proteins involved in other aspects of mitochondrial function [49,50]. This hypothesis is supported by evidence that disorders of mitochondrial function caused by mutations in mtDNA or mitochondrial inhibitors have a role in a variety of neurodegenerative diseases. An extrapolation of this hypothesis would suggest that normal aging could be associated with a gradual reduction in mitochondrial function secondary to mtDNA mutations or other mitochondrial inhibitory processes. Mitochondrial Respiratory Chain Function in HD Tissues. Many defects in oxidative phosphorylation in HD postmortem tissue have been described over the past 30 years. The respiratory chain is illustrated in Figure 2. The early studies demonstrated a complex II defect [51,52]. One of the ﬁrst studies to examine regional mitochondrial respiratory activity in HD isolated mitochondrial membranes from caudate and cerebral cortex and found a reduction in caudate cytochome oxidase (COX) activity,

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cytochrome aa3 (complex IV subunit), whereas cytochromes c1 and b were normal. There were no abnormalities in cortical tissue. This study did not account for activities relative to a marker of mitochondrial number such as citrate synthase (CS) [53]. It was demonstrated subsequently that there was a 77% decrease in caudate II/III activity corrected for CS activities. Activities of complexes I and IV were similar to control values [54]. At the same time, a complex I defect was found in symptomatic HD patients’ platelet mitochondria. Complex II/III and IV were not statistically diﬀerent from controls and at-risk individuals. These results were corrected for protein content only and not for a mitochondrial protein such as CS [55], and this abnormality of complex I has not subsequently been reproduced. The direct measurement of respiratory chain activity in postmortem HD caudate nucleus homogenate subsequently demonstrated a severe deﬁciency of complex II and III (56%) and IV (33%) activity [56]. Platelet mitochondrial function was not found to be diﬀerent from controls in contrast to an earlier study [55]. One of the largest postmortem studies (18 HD patients and 29 controls) examined respiratory chain function in mitochondrial fractions from the frontal and parietal cortex, caudate, putamen, and cerebellum [57]. They found a signiﬁcant reduction in complex II/III activity in caudate and putamen but not in cortex or cerebellum. A defect in complex IV was also observed in putamen but not elsewhere. This pattern of mitochondrial defect parallels the neuronal cell loss in HD brain. Similar defects have not been described in Parkinson’s disease (PD) or multisystem atrophy (MSA), suggesting that it is not secondary to nonspeciﬁc cell loss. There were also increased levels of 8-hydroxydeoxyguanosine (OH8dG), a marker of free radical generation, in caudate but not putaminal nuclear DNA There was no eﬀect on caudate or putaminal SOD or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity [57]. A similar but less severe pattern was observed in HD putamen but not cortex, cerebellum or cultured HD ﬁbroblasts [58]. In agreement with Browne et al. [57], no defect in GADPH activity was found. Aconitase deﬁciency was found in HD caudate (92%), putamen (73%), and cortex (48%) but not cerebellum, and this deﬁciency closely follows the pathology in HD [58]. Aconitase is an iron-sulfur (FeS)–containing enzyme that is involved in the Krebs cycle and iron homeostasis. Its activity is especially susceptible to inhibition by O2 and by the reaction product of O2 with NO , ONOO, or peroxynitrate [59–61]. Complexes II and III are also FeS-containing compounds, and they are also susceptible to inhibition by these free radicals. Thus, the pattern of enzyme loss in HD suggests that free radicals and excitotoxicity have a role in the pathogenesis of the disease. In contrast to brain, skeletal muscle oﬀers a tissue that can be relatively easily biopsied and has many characteristics in common with neurons; they are postmitotic cells with a high dependence on oxidative metabolism. 31P MRS has demonstrated a defect of ATP synthesis in skeletal muscle in symptomatic and presymptomatic HD patients which correlates with the length of the CAG repeat. A complex I deﬁciency has also been described in three out of four muscle biopsies from symptomatic HD patients [62]. This supports a direct role for a mitochondrial defect secondary to an expanded CAG repeat in HD outside of the CNS and independent of excitotoxicity. We are currently studying respiratory chain activity in muscle from presymptomatic and symptomatic HD patients. If a mitochondrial defect were found presymptomatically, this would add further weight to the direct role of mitochon-

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drial dysfunction in HD [40,63]. HD lymphoblasts demonstrated an increased mitochondrial depolarization in response to cyanide, a complex IV inhibitor, but not in response to complex I, II, or III inhibitors. The degree of depolarization was correlated with the CAG repeat length [64]. Thus, a widespread peripheral as well as CNS defect in the mitochondrial respiratory chain seems to occur in HD. 3-Nitropropionic Acid and Malonate: Mitochondrial Inhibitors and Models of HD. The use of mitochondrial inhibitors to emulate HD have been used extensively in the past. Succinate dehydrogenase or complex II converts succinate to fumarate with the concomitant reduction of ﬂavin adenine dinucleotide and the passage of electrons to fumarate. The two most widely used complex II toxins are 3-nitropropionic acid (3-NP), an irreversible inhibitor and malonate, a reversible inhibitor. 3-NP is a widely distributed plant and fungal neurotoxin that causes damage to the basal ganglia, hippocampus, spinal tracts, and peripheral nerves in animals [65]. Reports from northern China have suggested that 3-NP can cause putaminal necrosis and delayed dystonia in children who have eaten mildewed sugar cane [66]. Accidental ingestion of 3-NP in humans leads to nausea, vomiting, encephalopathy, coma, and, when the subject survives, chorea and dystonia and basal ganglia degeneration [66]. EVIDENCE FOR 3-NP–INDUCED OXIDATIVE STRESS USING RODENT AND PRIMATE

The intrastriatal administration of 3-NP to rats causes a dose-dependent ATP depletion, increased lactate concentration, and neuronal loss in the striatum [67]. The neuronal loss is ameliorated by decortication of the rats. This suggests that the glutaminergic input from the corticostriatal pathway may be required to cause excitotoxic damage in 3-NP–mediated cell death. NMDA receptor antagonists also block the toxic eﬀects of 3-NP, indicating that mitochondrial respiratory chain dysfunction and impaired energy metabolism may predispose to excitotoxic damage [68]. The reversible SDH inhibitor malonate has also been injected intrastriatally into rats to produce similar but milder lesions than are seen with 3-NP. These lesions are also reduced by glutamate antagonists, suggesting an excitotoxic mechanism [69]. The chronic systemic administration of 3-NP in rats produces an animal model displaying lesions that closely resemble the neuropathological features of HD with selective loss of striatal medium spiny neurons. This suggests that the cell population that is most vulnerable in HD is sensitive to energy impairment. Younger animals display more resistance to the neurotoxic eﬀects of 3-NP, suggesting that cellular handling of 3-NP is increasingly impaired with age [67]. The Sprague-Dawley rat appears more susceptible to 3-NP than BALB/c ByJ mice. The rats also demonstrate striatal apoptosis as demonstrated by TUNEL staining, whereas this is not observed in the mice [70]. This suggests that there are diﬀerent mediating factors, such as genetic background, which confer susceptibility to 3-NP. The administration of 3-NP to rodents by some investigators induces an increase in catalase and SOD activities and a reduction in glutathione levels [71,72]. In contradiction to these ﬁndings, Segovia et al. have found that intrastriatal quinolinic acid (a NMDA agonist) injections into rodents produce reduced Cu/Zn SOD levels and in a model with 3-NP both Cu/Zn and Mn SOD were reduced [73]. 3-NP toxicity is accentuated in mice deﬁcient in cellular glutathione peroxidase [74] and attenuated in mice overexpressing Cu/Zn SOD [72]. 3-NP toxicity is attenuated in mice overexpressing bcl-2, which suggests that the mechanism involved

MODELS.

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may be apoptotic [75]. Bcl-2 has also been shown to inhibit necrotic neuronal cell death caused by glutathione depletion [76], to protect against peroxides in a dose-dependent manner [77,78], and protect cells against lipid peroxidation [79]. Further evidence of free radical involvement in 3-NP toxicity is seen in SODdeﬁcient knockout mice. The homozygous SOD (Mn) knockout mice die perinatally following myocardial injury and neurodegeneration [80]. Mice heterozygous for the same knockout display striatal lesions 50% larger than control mice and elevated striatal hydroxyl radical levels when treated with either 3-NP or malonate [81]. An increase in fatty acid release has been associated with 3-NP neurotoxicity. Lipid peroxidation activates phospholipase A2, which leads to cleavage of fatty acid esters and fatty acid release [82]. Free fatty acid production itself may contribute to free radical generation and increased oxidative stress [83]. Direct evidence of oxidized proteins has been found in animal models of 3NP toxicity. Electron paramagnetic resonance (EPR) has been used to detect conformational changes in synaptosomal membranes in both the striatum and cortex of rats treated with intraperitoneal 3-NP [84]. The W/S ratio, the relevant EPR parameter used to determine levels of protein oxidation of MAL-6–labeled striatal or cortical synaptosomal membranes, was signiﬁcantly lowered, suggesting increased oxidized proteins. These changes occurred prior to the appearance of morphological changes in the striatum and suggest that oxidative stress procedes cell death in 3-NP toxicity [84]. Increased levels of protein carbonyls, another marker of proteins under oxidative stress, have also been reported in rats treated with 3-NP. The levels of protein carbonyls were not attenuated by MK-801, suggesting that excitotoxicity does not play a role in the formation of mitochondrial protein carbonyls. Rodents injected with salicylate and 3-NP exhibit increased levels of 2,3-dihydrobenzoic acid (DHBA) and 2,5-DHBA, both of which are metabolic products of salicylate in the presence of hydroxyl radicals. Peroxynitrite-mediated stress may also occur as demonstrated by increased 3-nitrotyrosine following systemic 3-NP or intrastriatal malonate injections [85]. Increased levels of striatal 8-OHdG have also been found in rats following systemic administration of 3-NP, suggesting oxidative damage to DNA. The size of the lesions and the associated increases in oxidative damage markers were markedly attenuated by overexpression of the superoxide free radical scavenger Cu/Zn superoxide dismutase, suggesting that ROS were involved in lesion formation [86]. Lesions were also attenuated, for both malonate and 3-NP, with free radical spin traps and nitric oxide synthase inhibitors. Neuronal NO synthase knockout mice also display attenuated lesions with malonate. These models support nitric oxide–mediated induced cell damage and death as well as peroxynitrite and hydroxyl radical formation following energetic disruption by 3-NP [87]. The spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) has been reported to protect against 3-NP-induced striatal lesions [88]. The spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), a more stable phosphorylated analogue of DMPO, has also been shown to be protective and reduce levels of protein oxidation [89]. a-Phenyl-tertiary-butynitrone (PBN) a commonly used spin trap was demonstrated to increase 3-NP toxicity, probably by interfering with 3-NP degradation [90]. Elevation of glutathione levels by injection of the glutathione precursor N-acetylcysteine has also been shown to be protective and reduce levels of protein oxidation [89].

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Neuroprotection against 3-NP by caloric restriction has also been reported [91]. Caloric restriction is known to decrease basal levels of oxidative damage [92]. Creatine and acetyl-L-carnitine have also been reported to protect from 3-NP, possibly by enhancing energy production and availability [93,94]. Three to 6 weeks of systemic 3-NP administration in primates is suﬃcient to cause choreiform movements, foot and limb dystonia, and dyskinesia induced by apomorphine, a dopamine agonist. More prolonged exposure results in spontaneous dystonia and dyskinesia accompanied by lesions in the caudate and putamen on MRI. The histopathology is similar to HD with depletion of calbindin-positive neurons, gliosis, sparing of NADPH-diaphorase neurons, and growth-related proliferative changes in dendrites of spiny neurons. There is also preservation of the striosomal organisation of the striatum and nucleus accumbens which are features seen in HD [95]. EVIDENCE FOR 3-NP–INDUCED OXIDATIVE STRESS USING IN VITRO MODELS. The addition of 3-NP to neuronal cell cultures results in a dose-dependent increase in neuronal death after 48 hours. During such studies some neurons undergo rapid necrotic cell death, while others exhibit a slow necrotic cell death over 48 hours. This suggests that both these mechanisms can be induced by 3-NP. The rapid necrosis was inhibited by MK-801, an allosteric inhibitor of the NMDA receptors, whereas the delayed mechanism was not inhibited [96]. POSSIBLE SOURCES OF 3-NP–INDUCED OXIDATIVE DAMAGE. Excitotoxicity may be a signiﬁcant source of free radicals in 3-NP toxicity. MK-801 has been shown to protect against 3-NP–mediated neurotoxicity by some researchers but not by others [97,98]. In summary of the available data, it has been shown that NMDA receptor antagonists attenuate but do not prevent neuronal death by 3-NP. Part of the variability may be due to diﬀerences in species and strain used for these experiments [70]. Another possible source of oxidative damage in the 3-NP model is mitochondrial dysfunction, which is associated with increased free radical generation. The role of 3NP in causing mitochondrial dysfunction has already been discussed. 3-NP–induced oxidative stress may result from an excessive and chronic inﬂammatory response. 3-NP–induced striatal lesions are inﬁltrated with neutrophils and are associated with immunoreactivity to serum/immune complement factors C3b/ C4b4 [99]. 3-NP also leads to increased expression of inducible NOS [100]. Dopamine metabolism may play a role in 3-NP and malonate toxicity. Dopamine levels are elevated in rodents treated with malonate [101]. When dopamine is catabolized by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (DOPAC), hydrogen peroxide is produced, which can lead to generation of hydroxyl radicals by the Fenton reaction. Dopamine can also undergo autooxidation with molecular oxygen to form radical semiubiquinones and superoxide radicals. Striatal neurons have more dopaminergic input than any other area of the brain. In support of this model, both deprenyl and clorgyline (MAO-A and -B inhibitors) reduce striatal lesions caused by 3-NP and malonate [102]. Protein carbonyl levels are slightly reduced in rat striatum but not cortex following treatment with these MAO inhibitors. The modest level of protection suggests that dopamine metabolism may contribute only slightly to 3-NP–induced damage. 6-Hydroxydopamine (6-OHDA) pharmacologically depletes the dopaminergic input into the striatum from the substantia nigra. In rodents treated with 6-OHDA, malonate toxicity is attenuated and the generation of hydroxyl radicals is blocked

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[101]. Systemic or intrastriatal application of L-dopa or dopamine, respectively, reconstitutes malonate toxicity and the generation of ROS. D2 receptor agonists, such as lisuride, partially restored malonate-induced toxicity but not the generation of ROS in rodents lesioned with 6-OHDA. Blockage of the dopamine transporter does not reduce malonate-induced dopamine release but signiﬁcantly impairs the generation of hydroxyl radicals. The conclusions that can be drawn from these experiments are that there are two independent pathways mediating malonate-induced dopamine toxicity: (1) dopamine transporter uptake–dependent but dopamine receptor–independent generation of ROS and (2) excessive stimulation of dopamine receptors [103]. Antioxidants alone and in combination can be used to protect dopamineinduced cell death in dissociated cortical neurons, striatal cell culture, and neuroblastoma cells. In none of these experiments was the identity of the free radical found, but partial protection and reduced formation of protein-bound cysteinyl catechols after treatment with antioxidants ascorbic acid or glutathione have been reported. This suggests that dopamine-mediated toxicity may be mediated by oxidized dopamine products [104]. In conclusion, there is abundant evidence that 3-NP causes excessive free radical production and subsequent cell death in rodent and primate models as well as in in vitro cell culture systems. The mechanism linking these two phenomena is less uncertain but almost certainly involves disrupted energy metabolism, excitotoxicity, and subsequent free radical disruption of macromolecules. Mitochondrial Dysfunction and Oxidative Stress in HD Transgenic Mouse Models. Over the past 5 years, many transgenic mouse models of HD have been produced. The R6/2 transgenic mouse was the ﬁrst HD mouse model and exhibits progressive neurological disease from 2 months of age. The light microscopic and ultrastructural pathology are very similar to those changes observed in postmortem HD brain. These neuropathological features occur approximately 4 weeks prior to a progressive movement disorder and muscle wasting and 10 weeks before neuronal cell death in selected brain regions. This suggests, at least in this model, that neuronal dysfunction is responsible for the initial phenotype rather than cell death [105]. Several defects in the respiratory chain have recently been characterized in the R6/2 mouse model. A reduction in complex IV in the striatum and cerebral cortex and a reduction in aconitase in the striatum have recently been described [106], and these were associated with increased immunostaining for inducible nitric oxide synthase (iNOS) and nitrotyrosine (a marker of increased peroxynitrate generation) in the mouse brain. An increase in the lesion size produced by 3-nitropropionic acid in the R6/2 mice and increased striatal 3,4-dihydroxybenzoic acid (a marker of ROS) also support a role for mitochondrial dysfunction and free radical damage [107]. Recently, creatine has been found to have a protective eﬀect in the R6/2 mouse. Dietary creatine improved survival, slowed the development of brain atrophy, and delayed degeneration of striatal neurons and the formation of intranuclear inclusions. The onset of diabetes was also delayed. NMR in these mice demonstrated delayed decreases in NAA and elevated brain creatine concentrations [37]. R6/1 mouse model with 116 CAG repeats demonstrates increased Cu/Zn (cytosolic) SOD in 19-week-old striatal homogenates when the phenotype is mild but decreased levels in 35-week-old mice when the phenotype is severe. These results were explained by initial compensation to oxidative stress followed by decompensation when neuronal dysfunction is more severe [73].

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Further work by this group examined levels of lipid peroxidation in 24-week-old R6/1 mice striata. The levels were signiﬁcantly elevated and correlated with the neurological phenotype [108]. 3.

Weight Loss and Huntington’s Disease

It is a well recorded but unexplained observation that HD patients suﬀer extreme weight loss in spite of an adequate caloriﬁc intake. Further studies have found that a higher BMI at presentation is associated with slower disease progression [109]. These features have also been observed in the R6/2 transgenic mouse. Weight loss was initially found not to be related to the severity of chorea, but a recent study did ﬁnd a relationship between the severity of chorea and energy expenditure [110]. Patient’s sedentary energy expenditure is proportionately related to the severity of the movement disorder, but total energy expenditure was the same as in controls because HD patients tended to not take part in as much voluntary physical activity. Further studies will elucidate whether there is a widespread metabolic defect that can explain the weight loss often observed in HD. 4.

The Role of Glyceraldehyde-3-phosphate and HD

Glyceraldehyde-3-phosphate (GAPDH) has an essential role in glycolysis. GAPDH binds to normal and mutant huntingtin in vitro, and both GAPDH and huntingtin are present in the cytoplasm and nucleus. The intrastriatal administration of the GAPDH inhibitor iodoacetate produces striatal lesions that are attenuated by decortication, suggesting an excitotoxic mechanism [111]. Striatal murine cultures are more sensitive than cortical cultures to the GAPDH inhibitor a-monochlorohydrin, with selective sparing of NADPH diaphorase–positive neurons [112]. Cell death can be attenuated in these experiments by the addition of supraphysiological levels of pyruvate. Several studies have found normal GAPDH activity in HD postmortem brain tissue [57,58] or unstressed HD ﬁbroblasts [113]. However, a recent study examined the activity of GAPDH in skin ﬁbroblasts from patients with symptomatic HD and AD. They found impairment of GAPDH glycolytic function in subcellular fractions in spite of unchanged gene expression in AD and HD patients. In HD this was most prominent in the nuclear fraction and suggested that the inhibition was a posttranslational event. This suggests that preparation of whole cell or brain tissue destroys subcellular GAPDH interactions, which inhibit its glycolytic activity [114]. GAPDH activity has also been found to be decreased in metabolically stressed HD ﬁbroblasts [115]. Tissue transglutaminase may cross-link GAPDH with proteins produced from mutant huntingtin processing, resulting in reduced GAPDH activity [116]. The nuclear translocation of GAPDH has been linked to the induction of apoptosis, and the ﬁnding of markedly reduced activity in the nuclear fraction may indicate a change in catalytic function and an involvement in apoptosis. The changes in function may be induced by the nuclear localization of truncated mutant huntingtin. 5.

Impaired Energy Metabolism and Cell Death

Impaired energy metabolism reduces the threshold for glutamate toxicity and can lead to activation of excitotoxic mechanisms and increased production of reactive oxygen species. Energy depletion can result in partial depolarization of the outer membrane and releases the voltage-dependent Mg2+ ion block of the Ca2+ channel in the NMDA receptor. Na+ can also enter via the NMDA receptor, and under conditions of reduced ATP the Na+/K+ ATPase will less eﬀectively extrude Na+ and

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intracellular Na+ levels will rise. This will in turn reduce the eﬃcacy of the Na+/Ca2+ antiport system and also cause an accumulation of Ca2+. Mitochondria as well as the endoplasmic reticulum and Golgi apparatus are involved in Ca2+ homeostasis in neurons. Calcium entry into mitochondria is linked with opening of the mitochondrial permeability transition pore and cell death. [117]. This function is impaired if the inner mitochondrial membrane potential is more positive than it normally is because of impaired energy metabolism and a reduced ability to extrude protons [118]. B.

The Role of Nitric Oxide in Huntington’s Disease

There has been increasing interest in the role of NO and NO synthase (NOS) in many diseases over the past decade. There is also growing support for the involvement of the NO/NOS system in the pathogenesis of HD. 1.

A Biological Overview of NO and NOS

NO is a gas produced when the semi-essential amino acid l-arginine is converted to citrulline in a two-step process by the enzyme NOS [119]. When this reaction is uncoupled, the second part does not occur and superoxide is liberated [120]. There are three isoforms of NOS. Inducible NOS (iNOS) is associated with modulation of the inﬂammatory response and is independent of calmodulin levels [121]. Endothelial NOS (eNOS) is found in endothelial Golgi and is involved in vascular reactivity in the periphery and to a lesser extent the brain [122]. Neuronal NOS (nNOS) is found in the CNS, testes, and skeletal muscle and has diverse function including regulation of cerebral vascular tone [121]. The activities of eNOS and nNOS are calmodulindependent and are referred to as constitutive NOS (cNOS). 2.

The Link Between htt and NO

Huntingtin-associated protein (HAP-1) interacts with htt [123]. This complex binds to calmodulin (CAM). Mutant htt/HAP-1 complex has been reported to have higher aﬃnity for CAM than wild-type htt/HAP-1 [124]. The relevance of this interaction to NOS function is uncertain, but there is evidence to suggest that calmodulin-dependent NOS function is reduced by HAP-1/htt complex and therefore increased aﬃnity of mutant htt would lead to a reduction in constitutive NOS function. CBP (CREB-binding protein) is a transcription factor critically important in regulating nNOS transcription [125]. Recently it has been reported that mutant htt interacts with CBP and represses transcription [7]. Mutant htt may therefore be involved in dysregulation of nNOS at the transcriptional level. 3.

The In Vivo Link Between NO and HD

The level of nNOS mRNA has been found to be reduced in HD striatum but not in cerebral cortex. This was especially evident in clinically advanced HD cases. The loss of signal was most profound in the dorsal striatum where the earliest changes are seen in HD [126]. Both nNOS expression and activity are reduced in the cerebellum, striatum, and cortex of the R6/2 mouse model of HD. nNOS activity was inversely correlated with body weight, the ability of the mice to stay on a rotating rod, and abnormal clasping reﬂexes. A NOS inhibitor was also found to accelerate disease progression in these HD transgenic mice [127].

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The precursor of NO, L-arginine, when given via dietary supplements, accelerates the disease process in HD mice. Increased dietary arginine also increases resting cerebral blood ﬂow (CBF) in HD mice, which correlates with symptom progression. Conversely, diets low in l-arginine slow the onset of body weight loss in HD mice [128]. Western blotting demonstrates increased production of 3-NT in mouse HD brains and reduced expression of nNOS proteins [127]. There is also evidence of increased deposition of 3-NT in brain, choroid plexus, and blood vessels of HD mice [127,128]. 4.

Cerebral Blood Flow Abnormalities in HD

Changes in vascular activity represent an indirect measure of NO production in the endothelium [129]. In early-stage HD patients, a reduction in the blood ﬂow velocity has been observed using functional transcranial Doppler (f TCD) in the region of the anterior cerebral artery during a combined cognitive/motor activating task [130]. Increased ﬂow velocity was observed during an oral word ﬂuency test, which is a sensitive indicator of early deﬁcits in early-stage HD [131]. PET has demonstrated decreased CBF in the caudate nucleus of HD patients during a combined motor/cognitive activation test [132]. Functional MRI has also been used to detect a decrease in CBF in HD striatum during motor/cognitive activation [133]. In contrast to the classic paradigm of increased CBF during activation of neurons, it has been reported that CBF increases during rest in HD patients and HD mice [128,132]. 5.

NO, Excitotoxicity, and Energetic Dysfunction

3-Nitropropionic acid causes striatal lesions by inhibition of complex II of the respiratory chain. The lesions are associated with elevated NO and 3-nitrotyrosine– related products such as peroxynitrite [134,135]. Quinolinic acid causes stimulation of the NMDA receptors, resulting in striatal lesions [136]. This eﬀect can be attenuated by administration of NOS inhibitors. 6.

Reduced NO and Neurotoxicity in HD

Elevated NOS activity can theoretically be protective or harmful to vulnerable neurons in HD, depending on the stage of the disease, the isoform of NOS involved, and the balance between nitrosative and oxidative stress. Existing evidence suggests that NOS activity and protein expression are reduced in HD. There is also evidence that NO itself is potentially protective in HD. NADPH-diaphorase–positive cells are resistant to neurodegeneration in HD [137]. These cells also produce small amounts of htt [138]. The reason for these ﬁndings is unknown, but elevated NO production by these cells may be neuroprotective. NO has been found to be neuroprotective during hypoxia and glucose deprivation [139]. This may be related to the antioxidant properties associated with NO, although when superoxide is produced secondary to NOS uncoupling, the highly toxic peroxynitrite is formed [140]. There is further evidence that a reduction in NO could be detrimental in HD from studies of CBF. Imaging studies have suggested that there is reduced blood ﬂow in the territory of the anterior cerebral artery in HD patients during cognitive and motor tasks which may be due to reduced NO production. It is postulated that during times of increased metabolic demands (e.g., motor tasks), the HD cerebral vessels are less able to increase CBF due to a reduction in NO. This would lead to a mismatch between metabolic demand and perfusion leading to oxidative stress.

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Ischemic tolerance occurs when brief episodes of sublethal ischemia protect against future potentially lethal periods of ischemia [141]. When the NMDA receptor is activated, an inﬂux of calcium activates calmodulin and in turn activates NOS. This increases NO production and activates the Ras cell survival-signaling pathway and extracellular signal–regulated protein kinase. Blockade of NO production by LNAME inhibits ischemic tolerance, whereas the administration of a NO donor such as DETA/NO promotes it [141]. This suggests that the reduction in NO could result in a reduction in the capacity for HD brain to tolerate ischemia. It has been suggested that NO can be toxic or protective depending on its redox state. When NO is in a NO+ state, it confers neuroprotection by the S-nitrosylation of NMDA receptors. Similarly, NO may inﬂuence Ca2+ inﬂux through the NMDA receptor [142]. In conclusion, the experimental data in HD patients and HD mice suggest that a reduction in NOS activity and NO production are involved in the pathogenesis of HD. The exact role of NOS/NO in the sequence of events underlying neurodegeneration remains uncertain, although therapeutically there appears to be a role for redressing the imbalance of NO and NOS dysfunction. C.

Heavy Metals and HD

Multiple lines of evidence link redox-active transition metals such as copper and iron as mediators of oxidative stress in neurodegenerative diseases [143]. Huntingtin has recently been described as an iron-responsive protein, which may link the toxicity of mutant huntingtin to feedback control of iron stores [144]. To support this role, MRI studies have demonstrated that iron accumulates in HD basal ganglia in asymptomatic patients [145]. The presence of iron in the basal ganglia is also a risk factor for developing the disease in patients in whom gene analysis has not been performed. In rats, lesions in the globus pallidus result in an initial reduction in iron followed by iron accumulation [146]. Iron has been reported to damage synapses resulting in lower uptake of GABA and elevated uptake of dopamine [147]. Ceruloplasmin, as well as being involved with copper metabolism, is also involved in iron homeostasis [148]. A rare recessive condition of aceruloplasminemia gives rise to a neurological phenotype with basal ganglia iron deposition [149]. Elevated levels of ceruloplasmin have been described in HD [150]. The pathological signiﬁcance of these ﬁndings are uncertain but may relate to the iron accumulation within the HD basal ganglia. D.

Inflammatory Response

There are few published data on the putative role of the inﬂammatory response in HD. The regions of the brain most associated with neurodegeneration (e.g., the caudate nucleus), demonstrate the highest degree of astrogliosis. 3-NP-Induced oxidative stress has also been suggested to result from an inﬂammatory response. As stated previously, the striatal lesions are inﬁltrated with neutrophils and are associated with C3b/C4b4 [99]. 3-NP also leads to increased expression of inducible NOS, which is involved in modulating the inﬂammatory response [100]. E.

Dopamine and Oxidative Stress

The striatum receives the largest dopaminergic input into the brain. Dopamine has been found to be toxic to striatal neurons in vivo and in vitro [151,152]. There is very

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little evidence linking dopamine and oxidative stress in vivo in HD. However, in a recent study, baseline primary striatal cultures of R6/2 mice did not display more cell death than their wild-type littermates. However, following exposure to dopamine, R6/ 2 striatal neurons displayed increased cell death and a variety of structural and functional changes associated with stimulation of autophagy [153]. First, there was an induction of discrete cytoplasmic puncta, which contained high levels of oxyradicals. Dopamine induced more oxidative stress in the R6/2 than wild-type neurons, and the R6/2 neurons had a signiﬁcantly higher number of DCF-labeled free radical–containing puncta per neuron, demonstrating increased dopamine-triggered oxidative stress. Second, it has been observed that DCF puncta were often co-localized with vital autophagic/lysosomal labels but not with vital markers for ER or mitochondria. DCF-labeled puncta contained high levels of ubiquitin. Third, R6/2 neurons exposed to dopamine displayed classic autophagic granules, as well as distinctive lysosomal morphology, with an electron-dense luminal matrix indicative of stimulation of autophagic/lysosomal degradative pathways. F.

Excitotoxicity

During the 1980s and early 1990s, there were many animal studies suggesting that excitotoxicity was involved in the pathogenesis of HD. The striatal injection of NMDA agonists, such as quinolinic acid, in rats and primates produced lesions that closely followed the neurochemical, neuropathological, and behavioral changes seen in HD [154,155]. Fewer data have been produced in humans, but one of the most compelling studies demonstrated that in HD postmortem brain, striatal neurons with high levels of NMDA receptor expression had increased degeneration [156]. There has been much in vitro work to support the role of excitotoxicity in HD. More recently it has been demonstrated that in HEK 293 cells co-transfected with the glutamate receptor NR1A/NR2B and mutant full-length huntingtin, there is increased apoptotic cell death compared to cells transfected with the same glutamate receptor but wild-type huntingtin. The predominant receptor type on medium spiny neurons is NR1A/NR2B. N-Terminal huntingtin was also co-transfected, and a slight increase in cell death but not apoptosis was observed, which suggests that full-length huntingtin is required to mediate all the toxic eﬀects of mutant huntingtin [157]. Since the late 1990s, the intracellular signaling pathways involved in excitotoxicity in HD have been described. The most important elements of this cascade are the kainate and NMDA receptors, a protein called postsynaptic density protein 95 (PSDP-95), mixed lineage kinase (MLK), and c-Jun-N-terminal kinase (JNK). Glutamate receptor stimulation has previously been shown to be associated with MLK-1 activation [158]. JNK activation is also associated with apoptosis [159]. PSDP-95 is a scaﬀold protein that binds to several intracellular proteins and possesses guanylate cyclase activity. PSDP binds via several repeat units in its structure, termed PDZ domains, to the NMDA receptor. PSDP-95 plays a pivotal role in regulating synaptic plasticity and synaptogenesis [160]. Mutant huntingtin interferes with the binding of PSDP-95 to the NMDA and kainate receptors, causing both receptors to become hypersensitive [161,162]. This enables increased Ca2+ uptake and activation of MLK-2 [163], which causes activation of MAP kinase kinases 4 and 7 and stress signaling kinases, SEK-1, and consequently activates JNK-2 [164]. Activated JNK2 phosphorylates the N-terminal region of c-Jun, which is one half of the transcription factor AP-1. Activated JNK-2 also phosphorylates the C-terminal region of MLK-2.

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This is critical in triggering apoptosis. Co-transfection of a dominant negative MLK-2 blocks apoptotic cell death induced by mutant huntingtin [165]. The treatment of primary rat mixed spinal cord cultures with NMDA causes microglial proliferation and subsequently neuronal death. Administration of extra microglia on top of these cultures augments the cell death [166]. Minocycline, a tetracycline derivative with anti-inﬂammatory eﬀects, attenuated all the pathological responses to NMDA in this model. Minocycline also reduced the NMDA-dependent microglial proliferation. These results suggested that migroglia contribute to NMDAmediated excitotoxicity and that minocycline may represent a therapeutic agent in neurodegenerative diseases. In support of these results, minocycline has previously been demonstrated to attenuate the neurological phenotype in a mouse model of HD [167]. NF-nB is an important transcription factor in regulating apoptotic cell death. NF-nB is maintained inactive within the cytoplasm by being bound to unphosphorylated I-nBa. QA induces apoptosis partly, if not fully, by accelerating the degradation of I-nBa and thus enabling the nuclear translocation of NF-nB [168]. This is probably mediated by caspase-3 or a caspase-3–like protein. Using a quinolinic acid excitotoxic model of HD in rats, it has been found that the free radical scavenger, OPC-14117, attenuates the degradation of I-nBa. This suggests that free radicals are involved in the breakdown of I-nBa. OPC14117 has also been found to attenuate the activation of NF-nB and reduce the ensuing apoptotic cell death of rat striatal neurons [169]. The role of ROS in mediating some of the pathogenic eﬀects of elevated cytosolic calcium has been studied in primary striatal neuronal cultures. The antioxidant MnTBAP inhibits cell death induced by the calcium ionophore A23187. Cyclosporin attenuated cell death as well, which suggests that mitochondrial dysfunction may mediate cell death by the production of free radicals [170]. HD skin ﬁbroblasts demonstrate increased vulnerability to glutamate toxicity. This can be attenuated by antioxidants and further links the interplay between excitotoxicity and free radicals [171]. The cellular pathways underlying excitotoxicity are currently under intensive investigation. Some of these pathways may be free radical independent, although there is growing evidence that many of the eﬀects of NMDA receptor stimulation can be attenuated by free radical scavengers. This suggests that free radicals may be involved in the modulation of the excitotoxic response or directly associated with the downstream toxic eﬀects of macromolecular disruption.

V.

THE EFFECTS OF OXIDATIVE STRESS ON MACROMOLECULES

There is growing evidence for the presence of free radical–damaged macromolecules, such as DNA, lipid, and proteins, in HD. Their pathogenic role remains uncertain, and it is suggested that they may merely be byproducts of other toxic events and not the cause of macromolecular and subsequently cellular dysfunction. In spite of this, free radical–damaged macromolecules may represent a way of monitoring cellular dysfunction and response to therapy. A.

Proteins

Peroxynitrite is a highly reactive free radical that forms when superoxide reacts with NO. Peroxynitrite can react with Cu/Zn SOD to form nitronium ions that irreversibly

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nitrate tyrosine residues in proteins to form 3-nitrotyrosine (3-NT). This can aﬀect phosphorylation/dephosphorylation events at the tyrosine residue and modify the tertiary structure of the targeted protein [172]. The levels of 3-NT and heme oxygenase are elevated in postmortem HD striatum and cortex compared to age-matched controls. The extent of staining mirrored the severity of the disease. Vonsattel grades were associated with greater immunoreactivity and a gradient from enhanced dorsal to lesser caudal striatal staining. The extent of staining was reduced in the grade 3 and 4 cases. This may be due to loss of neurons in more severe cases [173]. One group has also described a slight reduction in Cu/Zn SOD activity in parietal cortex and cerebellum in HD postmortem brain. Mn SOD activity was normal [57]. Another mechanism of amino acid side chain modiﬁcation following oxidative stress is associated with products of lipid peroxidation. It involves the addition of various reactive aldehydes (e.g., malondialdehyde) to nucleophilic side chains of electron-rich Cys, His, or Lys residues. This results in the addition of an aldehyde carbonyl group to the peptide chain and alters the conformation and function of membrane proteins. Elevated protein carbonyls have been found in rat brain following administration of 3-NP [84]. B.

Lipids

Lipofuscin is a ﬂuorophore produced by the reaction of amino compounds with secondary aldehydic products of oxidative free radical–induced oxidation of macromolecules, especially lipid peroxidation. It accumulates in lysosomes of post-mitotic cells such as neurons and cardiac myocytes and is often called ‘‘age pigment,’’ as it increases with age [174]. The rate and extent of lipofuscin deposition increases under conditions of increased oxidative stress and metabolic rate [175]. Both striatal and cortical HD neurons contain more lipofuscin than their age-matched controls, and the level of lipofuscin is related to the severity of the neuropathology [173,176]. These results suggest that in regions aﬀected in HD there is enhanced lipid peroxidation. Isoprostanes are prostaglandin-like compounds which are not formed by cyclooxygenase (COX) but by free radical–catalyzed peroxidation of esteriﬁed arachidonic acid in membrane phospholipids. A 35% increase in CSF F2 isoprostanes has been described in 20 early HD patients. The overlap in the cases suggested that the oxidative damage did not occur in all early HD patients. There was no correlation between age or disease duration and F2 isoprostane levels. Isoprostanes may represent a novel group of molecules that can be used as a marker of disease progression and response to therapy [177]. Malondialdehyde (MDA) is a marker of oxidative damage to lipids. Elevated levels have been found in HD striatum and cortex compared with age-matched controls [173]. The most compelling evidence for the direct relationship between lipid peroxidation and HD phenotype is in HD mice, where striatal lipid peroxidation (measured by TBARS) parallels the neurological phenotype [108]. C.

DNA

Free radical oxidative damage to DNA can induce DNA strand breaks [178]. A number of studies show evidence of increased strand breaks in HD striatal neurons and have suggested the involvement of both apoptotic and necrotic mechanisms of cell

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death. A recent study suggests that the degree of DNA fragmentation in HD striatum is correlated with the length of the CAG repeat in huntingtin [179]. Using in situ end labeling techniques (ISEL), it has also been found that there are marked increased levels of DNA fragmentation in HD cortex and striatum and that this occurs in the cytoplasm and not the nucleus, suggesting that mitochondrial DNA is more susceptible to fragmentation [13]. Oxidative damage can also induce excessive oxidation of DNA bases such as deoxyguanosine to produce 8-hydroxydeoxyguanosine (8-OHdG). HPLC has been used to demonstrate increased levels of nuclear DNA 8-OHdG in grade 4 HD postmortem caudate but not in relatively spared regions such as frontal and parietal cortices and the cerebellum [57]. Immunocytochemical detection of 8-OHdG also demonstrates elevated levels in the striatum and the cerebral cortex [173]. More recently, increased levels of 8-OHdG in the parietal cortex but not the frontal cortex or cerebellum have been reported. This suggests against widespread DNA oxidation as a primary factor in the pathology of HD. 8-OHdG can also be caused by mechanisms other than oxidative stress and DNA strand breaks can be caused by apoptosis or DNA repair [180]. When peroxynitrite reacts with DNA poly(ADP-ribose) synthetase (PARP) can be activated. PARP is a nuclear enzyme involved in DNA repair, but excessive PARP activation may exhaust cellular energy supplies, promoting the induction of cell death cascades. This may be another mechanism by which peroxynitrite exerts a direct toxic eﬀect [181]. Most of the evidence presented so far has been highly suggestive of oxidative stress in HD. One lab has challenged the view of protein, lipid, and DNA damage in HD. They found no evidence for increased levels of protein carbonyls, 8-hydroxydeoxyguanosine, or TBARS in HD postmortem frontal cortex, caudate, or putamen compared to age-matched controls. They did not examine mtDNA separately from nuclear DNA and did not comment on the Vonsattel or clinical stage of disease in their subjects. The same methods of looking for oxidative damage have found oxidative stress in PD, AD, and CLBD. They found no changes in the level of antioxidant enzymes or in glutathione levels in HD brain [182]. VI.

THERAPEUTIC APPROACHES TO OXIDATIVE STRESS IN HD

The interplay between energy dysfunction, excitotoxicity, and ROS has led to several approaches to treat not only HD but also other neurodegenerative diseases. Coenzyme Q10, or ubiquinone, has been demonstrated to protect cultured neurons against glutamate toxicity and acts as an antioxidant [183]. Ubiquinone also protects against malonate and 3-NP–induced striatal lesions in a dose-dependent manner [184]. This is also associated with protection of ATP levels. In HD patients, oral administration of ubiquinone resulted in signiﬁcant decreases in occipital cortex lactate concentrations that reversed on withdrawal of therapy [26]. These led to the use of ubiquinone and remacemide (an NMDA receptor antagonist) in the ﬁrst large clinical therapeutic trial in HD. Unfortunately, there appeared to be no beneﬁt from this combination, although there were methodological problems in the trial such as overexpectant endpoints [27]. A recent study with two transgenic mouse models demonstrated a signiﬁcant beneﬁt from remacemide and ubiquinone on phenotype and pathology of the mice [185]. These compounds may still demonstrate clinical beneﬁt if given for

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longer periods and earlier in the disease. Creatine has been demonstrated, in toxininduced and transgenic animal models of HD, to improve neuronal survival [37,93]. OPC-14117 is a potent lipophilic synthetic free radical scavenger. The compound collects in rodent brain to levels ﬁve times those in plasma. OPC-14117 blocks ironinduced lipid peroxidation in rat brain synaptosomes in vitro. OPC-14117 is probably a better free radical scavenger than vitamin E. In a short 16-week trial of safety, there was no change in clinical scales measures, but there was a trend towards a reduction in markers of CSF protein oxidation. This would be consistent with a recent trial demonstrating a reduction in occipital lactate in HD with ubiquinone therapy but without any change in clinical outcome [186]. In quinolinic acid models of HD, BDNF NGF3 and NGF4/5 are protective. This opens the possibility of using growth factor–releasing implants as a putative treatment in HD [187]. VII.

CONCLUSIONS

The presence of defects in respiratory chain activity associated with excitotoxicity and increased free radical production is now well established in HD. The interaction between these factors and their contribution to the pathogenesis of HD are less well understood. Free radical metabolism is disturbed in HD, and there is evidence that there are many potential causes of free radical generation. Whether the free radicals cause macromolecular dysfunction or their damaged end products merely reﬂect underlying cellular abnormalities of minor physiological signiﬁcance has yet to be deﬁned. Free radical abnormalities may function as a marker of the disease process and its progression. Future therapeutic strategies will aim to target the primary causes of neurodegeneration in HD, and maintaining free radical equilibrium may represent one method of attenuating this process. ACKNOWLEDGMENTS The authors’ work described in this review was supported by the Medical Research Council and Wellcome Trust. REFERENCES 1. 2.

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Turner and Schapira mediated striatal neuron autophagy and degeneration. Hum Mol Genet 2001; 10(12): 1243–1254. Cull-Candy S, Brickley S, Farrant M. NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 2001; 11:327–335. Hantraye P, Riche D, Maziere M, Isacson O. A primate model of Huntington’s disease: behavioral and anatomical studies of unilateral excitotoxic lesions of the caudateputamen in the baboon. Exp Neurol 1990; 108(2):91–104. Albin RL, Young AB, Penney JB, Handelin B, Balfour R, Anderson KD, Markel DS, Tourtellotte WW, Reiner A. Abnormalities of striatal projection neurons and N-methylD-aspartate receptors in presymptomatic Huntington’s disease. N Engl J Med 1990; 322(18):1293–1298. Zeron MM, Chen N, Moshaver A, Lee AT, Wellington CL, Hayden MR, Raymond LA. Mutant huntingtin enhances excitotoxic cell death. Mol Cell Neurosci 2001; 17:41– 53. Schwarzschild MA, Cole RL, Hyman SE. Glutamate, but not dopamine, stimulates stress-activated protein kinase and AP-1-mediated transcription in striatal neurons. J Neurosci 1997; 17:3455–3466. Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, Rakic P, Flavell RA. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 1997; 389:865–870. Che YH, Tamatani M, Tohyama M. Changes in mRNA for post-synaptic density-95 (PSD-95) and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection. Brain Res Mol Brain Res 2000; 76:325–335. Sun Y, Savanenin A, Reddy PH, Liu YF. Polyglutamine-expanded huntingtin promotes sensitization of N-methyl-D-aspartate receptors via post-synaptic density protein 95. J Biol Chem 2001; 276:24713–24718. Savinainen A, Garcia EP, Dorow D, Marshall J, Liu YK. Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via post-synaptic density protein 95. J Biol Chem 2001; 276:11382–11386. Liu YF, Dorow D, Marshall J. Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin. J Biol Chem 2000; 275(25):19035–19040. Liu YF. Expression of polyglutamine-expanded Huntingtin activates the SEK1-JNK pathway and induces apoptosis in a hippocampal neuronal cell line. J Biol Chem 1998; 273:28873–28877. Phelan DR, Price G, Liu YF, Dorow DS. Activated JNK phosphorylates thec-terminal domain of MLK2 that is required for MLK2-induced apoptosis. J Biol Chem 2001; 276:10801–10810. Tikka TM, Koistinaho JE. Minocycline provides neuroprotection against N-metyl-Daspartate neurotoxicity by inhibiting microglia. J Immunol 2001; 166:7527–7533. Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, Bian J, Guo L, Farrell LA, Hersch SM, Hobbs W, Vonsattel JP, Cha JH, Friedlander RM. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med 2000; 6:797–801. Qin ZH, Chen RW, Wang Y, Nakai M, Chuang DM, Chase TN. Nuclear factor kappaB nuclear translocation upregulates c-Myc and p53 expression during NMDA receptor-mediated apoptosis in rat striatum. J Neurosci 1999; 19:4023–4033. Nakai M, Qin Z, Wang Y, Chase TN. NMDA and non-NMDA receptor-stimulated IkappaB-alpha degradation: diﬀerential eﬀects of the caspase-3 inhibitor DEVD.CHO, ethanol and free radical scavenger OPC-14117. Brain Res 2000; 859:207–216. Petersen A, Castilho RF, Hansson O, Wieloch T, Brundin P. Oxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187induced death of cultured striatal neurons. Brain Res 2000; 857:20–29.

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171. May PC, Gray PN. The mechanism of glutamate-induced degeneration of cultured Huntington’s disease and control ﬁbroblasts. J Neurol Sci 1985; 70:101–112. 172. Beckman JS. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol 1996; 9:836–844. 173. Ferrante RJ, Kowall NW, Hersch SM, Brown RH, Beal MF. Immunohistochemical localisation of markers of oxidative injury in Huntington’s Disease. Soc Neurosci Abstr 1996; 22:227. 174. Terman A, Brunk UT. Lipofuscin: mechanisms of formation and increase with age. APMIS 1998; 106:308–332. 175. Nakano M, Gotoh S. Accumulation of cardiac lipofuscin depends on metabolic rate of mammals. J Gerontol 1992; 47:B126–B129. 176. Tellez-Nagel I, Johnson AB, Terry RD. Studies on brain biopsies of patients with Huntington’s chorea. J Neuropathol Exp Neurol 1995; 33:308–332. 177. Greco A, Minghetti L, Levi G. Isoprostanes, novel markers of oxidative injury, help understanding the pathogenesis of neurodegenerative diseases. Neurochem Res 2000; 25:1357–1364. 178. Driggers WJ, Holmquist GP, LeDoux SP, Wilson GL. Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA. Nucleic Acids Res 1997; 25:4362–4369. 179. Butterworth NJ, Williams L, Bullock JY, Love DR, Faull RL, Dragunow M. Trinucleotide (CAG) repeat length is positively correlated with the degree of DNA fragmentation in Huntington’s disease striatum. Neuroscience 1998; 87:49–53. 180. Polidori MC, Mecocci P, Browne SE, Senin U, Beal MF. Oxidative damage to mitochondrial DNA in Huntington’s disease parietal cortex. Neurosci Lett 1999; 272:53–56. 181. Zhang J, Dawson VL, Dawson TM, Snyder SH. Nitric oxide activation of poly(ADPribose) synthetase in neurotoxicity. Science 1994; 263:687–689. 182. Alam ZI, Halliwell B, Jenner P. No evidence for increased oxidative damage to lipids, proteins, or DNA in Huntington’s disease. J Neurochem 2000; 75:840–846. 183. Favit A, Nicoletti F, Scapagnini U, Canonico PL. Ubiquinone protects cultured neurons against spontaneous and excitotoxin-induced degeneration. J Cerebr Blood Flow Metab 1992; 12:638–645. 184. Beal MF, Henshaw DR, Jenkins BG, Rosen BR, Schulz JB. Coenzyme Q10 and nicotinamide block striatal lesions produced by the mitochondrial toxin malonate. Ann Neurol 1994; 36:882–888. 185. Ferrante RJ, Andreassen OA, Dedeoglu A, Ferrante KL, Jenkins BG, Hersch SM, Beal MF. Therapeutic eﬀects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington’s disease. J Neurosci 2002; 22:1592–1599. 186. The Huntington Study Group. Safety and tolerability of the free-radical scavenger OPC-14117 in Huntington’s disease. Neurology 1998; 50:1366–1373. 187. Menei P, Pean JM, Nerriere-Daguin V, Jollivet C, Brachet P, Benoit JP. Intracerebral implantation of NGF-releasing biodegradable microspheres protects striatum against excitotoxic damage. Exp Neurol 2000; 161:259–272.

21 Oxidative Stress in Familial Amyotrophic Lateral Sclerosis DIANE E. CABELLI Brookhaven National Laboratory, Upton, New York, U.S.A.

I.

FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS: AN INTRODUCTION AND THE SOD LINK

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with the hallmark of paralysis to the limbs with retention of cognitive function. Death usually occurs 2–5 years after onset of symptoms. It is commonly known as Lou Gehrig’s disease after the New York Yankees baseball player whose life it claimed at age 37. It was suggested in 1993 [1] that approximately 20% of inherited or familial ALS (fALS) is linked to damage to the gene on chromosome 21 that is responsible for the expression of copper, zinc-superoxide dismutase (CuZnSOD). With the production of transgenic mouse models expressing the G93A, G37R, and the G86R fALS point mutations to their SOD and exhibiting the symptoms of ALS [2–4], these mutated forms of the enzyme, and by analogy the other fALS mutant SODs, were inextricably linked to the disease. This connection between transgenic animal models and fALS was recently strengthened with the production of rats carrying the human transgene for fALS mutant SODs G93A and H46R [5]. The familial version of the disease diﬀers from the sporadic form in that the age of onset is approximately 5 years earlier, in the mid-40s instead of the early 50s, and the severity seems to be generally greater [6]. There is, however, great variability in both severity and age of onset. An intriguing feature of fALS mutant SODs is that mutations at approximately 70 diﬀerent sites in the enzyme, leading to more than 90 diﬀerent mutant proteins, have been identiﬁed thus far in the SOD-linked inherited form of the disease. There is not yet a discernible correlation between the severity or age of onset of symptoms and any particular pattern in the mutations [7], although 473

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glimmers are beginning to be observed. Spurring the great interest in fALS as initiated by mutant SODs is the fact that the clinical symptoms of the familial version and the sporadic version of the disease are almost indistinguishable [8], suggesting that understanding the initiation of disease by fALS mutant SOD may help us understand how to treat the much more common sporadic ALS. A number of mechanisms have been suggested to initiate the deleterious eﬀects associated with the fALS mutant enzymes. These can be divided into two categories: those related to oxidative stress and those related to protein aggregation. In fact, it is not easy to distinguish between the two mechanisms, as aggregation can be viewed as potentially resulting from oxidative damage. Mechanisms that have been postulated to derive from oxidative stress/damage involve (1) enhanced production of the highly toxic OH radical [9,10], leading to OH-induced oxidative damage, (2) copper loss in the mutant enzyme with free copper–induced cellular damage [11], (3) changes in the metal-binding properties common to the diﬀerent mutated enzymes that are manifested at lower pH [12], (4) damage induced by the very eﬃcient reaction of superoxide with NO an in vivo neurotransmitter, to form peroxynitrite (ONOO), a putative agent of damage [13], and (5) protein aggregation [14]. The link between these processes and fALS mutant enzymes is strengthened by studies showing enhanced oxidative damage in ALS patients [15–17]. The protective eﬀects of penicillamine [18], a reductive copper chelator, observed in transgenic mouse studies also implies a connection between metals and the toxic eﬀects induced by the ALS mutant SOD. This chapter will cover the mechanisms of oxidative stress associated with fALS but not cover issues of protein aggregation as a causative factor. Several excellent reviews have been published [19,20] that discuss aggregation and ﬁbril formation in both human and animal systems. Mechanistic and structural features of superoxide and superoxide dismutase are reviewed ﬁrst. Then each of the proposed mechanisms for oxidative damage is discussed, primarily from the view of isolated chemical reactions and not in vivo models. Both cultured motor neurons and transgenic mouse models of the disease are powerful tools for allowing insight into the relationship between enzymatic studies and living systems. The evidence for these various mechanisms of disease as seen in both neuronal and mouse models is then reviewed. Finally, we will discuss some of the interesting results, seemingly unrelated but suggesting possible connections, speciﬁcally some recent studies on superoxide toxicity, calcium channels, and pH eﬀects.

II.

SUPEROXIDE AND SUPEROXIDE DISMUTASE

The sequential addition of electrons to oxygen leads initially to the formation of superoxide and then peroxide, OH radicals, and ﬁnally water (Fig. 1). The main source of excess electrons in solution comes from leakage of electrons in the process of oxidative phosphorylation in the mitochondria. In fact, it is estimated that roughly 1–2% of all electrons leak out in this process. Some biologically important reactive oxygen species (ROS) formed in this process are (1) the superoxide radical (O2 ), formed by the reaction of O2 with an electron [Eq. (1)], (2) the hydroxyl radical ( OH), presumably formed by a Fenton-type reaction of peroxide with a metal [Eq. (2)], (3) peroxide itself (H2O2), not a radical but a reactive species formed by the dismutation of superoxide radical [Eqs. (3) and (4)], (4) peroxy radicals (RO2 ), and (5) the most

Oxidative Stress in Familial ALS

475

SOD as a 1 e- peroxidase +e-

+e- / 2 H+ +e- / H + +e-/ H+ . O2 H2O2 OH, H2O 2H2O

O2

SOD as a dismutase

Figure 1 The four-electron reduction of oxygen to water involves both superoxide and hydroxyl radicals as intermediates. SOD functions as a dismutase to yield peroxide and oxygen but can also function as a one-electron peroxidase to generate OH radical and superoxide.

recent additions to this list, nitric oxide [21] (NO ), peroxynitrite [22] (ONOO), and other nitrogen oxide radicals (e.g., NO2 , NO+, NO).

eaq þ O2 ! O2

ð1Þ

Mnþ þ H2 O2 ! Mðnþ1Þþ þ OH þ OH

ð2Þ

HO2 þ HO2 ! H2 O2 þ O2

ð3Þ

HO þ O ðþH Þ ! H O þ O O þ O ! does not react pK ¼ 4:8 HO X O þ H 2

2 2

2 2 2

þ

2

2

ð4Þ ð5Þ

2

þ

ð6Þ

ROS have been implicated in a variety of normal and pathological processes that range from aging [23] to cancer [24] and, more recently, neurodegenerative disease such as ALS [1–3,25] and Alzheimer’s disease [26]. Enzymatic responses to these oxy radicals are superoxide dismutases, metalloenzymes that dismutate O2 into H2O2 and O2, and catalase/glutathione peroxidase, enzymes that eliminate peroxide by converting H2O2 into H2O/O2. Another cellular response to ROS is metal sequestration to eliminate the metal-mediated oxidation-reduction processes that lead to OH radical production. Superoxide radical itself is not particularly reactive in water [27] with the exception of its reactivity with radicals (NO , ascorbate radical, etc.) and metal cations. It can be a very eﬀective reductant for Fe3+ ions [28] (k7 = 1.5 108 M1/s1), thus driving the Fenton reaction [Eq. (8)] and leading to the production of very highly oxidizing OH radicals.

Fe3þ þ O2 ! Fe2þ þ O2 Fe

2þ

þ H2 O2 ! Fe

3þ

þ OH þ OH

ð7Þ

ð8Þ

The importance of superoxide as the reductant in this mechanism was reconsidered [29] in light of the high cellular content of reducing agents such as ascorbate that can function very adequately as reductants for Fenton or Fenton-type reactions. The lack of reactivity of O2 with many cellular components (e.g., amino acids are unreactive

476

Cabelli

with O2 ) in aqueous medium can allow it to diﬀuse relatively large distances to potentially critical sites prior to involvement in Fenton-type reactions, and a sitespeciﬁc mechanism of toxicity was suggested [30] where a Fenton-type reaction occurs at speciﬁc cellular sites leading to greatly ampliﬁed damage. The dismutation mechanism for superoxide radical described by Eqs. (3)–(5) allows for the production of peroxide, the other reactant in the Fenton or Fenton-type reaction, again possibly in a very site-speciﬁc fashion. All aerobic organisms contain SODs [31], with the exception of Lactobacillus, which instead has high levels of manganese that presumably serves to carry out the same function as SOD [32]. SODs come with a variety of metal centers, and in all cases the metal centers are alternately reduced and oxidized to facilitate the dismutation of superoxide radicals in what has been referred to as a ping-pong mechanism:

Mðnþ1Þþ þ O2 ! Mnþ þ O2

ð9Þ

Mnþ þ O2 ðþ2Hþ Þ ! Mðnþ1Þþ þ H2 O2

ð10Þ

The SOD that is found in the cytosol is CuZnSOD, or SOD1. Mitochondrial SOD is a manganese-based enzyme, MnSOD or SOD2. Another CuZnSOD that is found extracellularly is called SOD3. It is a mutant form of SOD1 that has been linked to ALS. CuZnSOD is a dimeric enzyme that catalyzes the dismutation of superoxide (O2) to oxygen and hydrogen peroxide [31,33]. The extraordinary features of this enzyme are that (1) it carries out this catalysis with pH-independent (pH 5–9.5) rate constants that are virtually diﬀusion controlled [34,35]. (k11 = k12 c 2 109 M1/s1), and (2) catalysis is electrostatically facilitated by the charge on the amino acid residues at and near the active site channel [36–38].

SOD þ O ðþ2H

Cu2þ Zn2þ SOD þ O2 ! Cuþ Zn2þ SOD þ O2 þ

2þ

Cu Zn

2

þ

Þ ! Cu Zn SOD þ H2 O2 2þ

2þ

ð11Þ ð12Þ

Each monomer contains an active site that consists of one copper and one zinc bridged by an imidazole group of histidine 63. The copper is also bound by three histidines, His 46, His 48, and His 120, and, in the oxidized state, a water molecule. The zinc is bound by two histidines (His 71 and His 80) and an aspartate (Asp 83) as well as the bridging histidine (His 63). The structures of the oxidized and reduced enzymes are generally similar with the notable diﬀerence that reduced copper loses the bond to the bridging histidine and shifts position somewhat [39,40]. Copper is the redox-active metal, while zinc has been viewed as playing a structural role only. Recently the role of zinc has been scrutinized more closely in the ALS mutant CuZnSODs and will be discussed later. The active site channel plays an important role in electrostatically funneling the superoxide anion to the copper center. The channel is 24 A˚ wide at the top but gradually narrows until it is only 4 A˚ wide at the copper ion. The arginine (Arg 143 in the human enzyme) at the top lip of the active site channel is responsible for a great deal of the electrostatic guidance, with the rate constant for dismutation decreasing roughly an order of magnitude when going from 0.01 to 0.5 M salt. This electrostatic channeling is reﬂected in studies in which the arginine was selectively mutated to a lysine (reﬂecting the same positive charge but with a diﬀerent pH for protonation of

Oxidative Stress in Familial ALS

477

the amine), an isoleucine or alanine (neutral charge), or a glutamate or aspartate (negatively charged residues at pH 7 or greater when the carboxylate group is deprotonated) (Fig. 2). In these studies [41], a decrease in one order of magnitude for enzymatic activity was observed for each charge, underscoring the importance of this active site lip residue and the role of electrostatic guidance of the small anionic superoxide radical to the active site channel in maintaining the very fast rate constant for catalysis. Another catalytic cycle is operative in this enzyme. A peroxidative cycle is described by reactions of peroxide with both the reduced and oxidized enzyme (see Scheme I). Cu2þ Zn2þ SOD þ H2 O2 ! Cuþ Zn2þ SOD þ HO2 þ Hþ þ

ð13Þ

Cu Zn SOD þ HO2 ! Cu Zn SOD OH þ OH 2þ

H2 O2 ¼ HO2 þ H

þ

2þ

2þ

pK ¼ 11:9

ð14Þ ð15Þ

Equations (13) and (14) represent a cycle in which H2O2 is converted to HO2 and OH in the presence of CuZnSOD. In particular, Eq. (14) is a Fenton-type reaction where the reduced metal reacts with peroxide to yield OH radical. The HO2 produced in Eq. (13) is catalytically removed via Eqs. (11) and (12), leading to an overall cycle that generates O2 and OH radical and regenerates one quarter of the

human WTSOD Arg (+)

k calculated , M - 1sec - 1

Arg143Lys hSOD (+) 1

09

Arg143Ile hSOD (0)

1 08 Arg143Asp hSOD (-)

Arg143Glu hSOD (-) 1 07

5

6

7

8

9

10

11

12

pH

Figure 2 Rate constants as a function of pH for superoxide dismutation by wild-type CuZnSOD and a variety of point mutations to the arginine 143 that sits at the lip of the active site channel. The arginine has a positive charge at neutral/high pH. The point mutations are labeled with the effective charges.

478

Cabelli

H2O2. If the OH that is produced in Eq. (14) reacts with and deactivates the CuZnSOD, then this is clearly not a catalytic cycle. Insofar as the OH is captured by a substrate, diﬀuses away from the active site, or reacts at the active site with a residue that does not lead to deactivation, it remains a catalytic cycle. The actual site of OH radical attack in the enzyme has come under renewed attention [42] through the use of sophisticated mass spectrometry techniques, and studies suggest that the OH radical reactions are not as localized at a single amino acid, His 120, as previously thought. That this peroxidative cycle occurs in the wild-type enzyme is well documented [43– 45]. The observed bimolecular rate for reaction 13 is approximately 103 M1/s1 at pH 8. The rate constant for reaction 14 [46] is 2.6 103 M1/s1, but the reaction of the reduced enzyme is with HO2, not H2O2, and, as the acid dissociation constant, pK15, for peroxide is 11.9, the observed rates at pH 5–8 are very slow (0.001–1 M1/s1, respectively) (Fig. 3). In the wild-type enzyme, it is quite clear that bleaching of the copper [Eq. (13)] (reduction of the ‘‘blue’’ copper(II) absorbance at 680 nm) occurs at a much faster rate than deactivation [Eq. (14)]. The mechanism deﬁned Eqs. (13) and (14) describes a method for converting the more benign superoxide radical into the much more reactive OH radical, albeit at a very slow rate in the wild-type enzyme. Another point to note is that the HO2 anion is also a small, negatively charged species and, like O2 , is subject to the electrostatic eﬀects described above and illustrated in Figure 1. In particular, the rate constant for the rate-limiting step in this cycle, which is Eq. (14) in the wild-type enzyme, will also vary with ionic strength as well as charge at the active site channel.

103

k calc, M - 1sec- 1

102 101 100 1 0 -1 1 0 -2 1 0 -3 1 0 -4 6

8

10

12

14

pH A plot of the calculated rate constant for the reaction between HO2 and Cu SOD as a function of pH. The mechanism assumes that only the deprotonated form of peroxide reacts and was calculated [46] using the equation kcalc = k14K15/[H+]/(1+ K15/[H+]) and assuming that k14 = 2.6 103 M1/s1 and K15 = 1.26 1012 M or pK15 = 11.9.

Figure 3 +

Oxidative Stress in Familial ALS

III.

479

SUGGESTED MECHANISMS OF OXIDATIVE STRESS

The association of familial amyotrophic lateral sclerosis with mutant forms of CuZnSOD led initially to the proposal that increased or decreased enzymatic activity was the explanation for the observed motor neuron dysfunction associated with fALS mutant SODs [1,4]. Subsequent studies showed that activity was not altered and that toxicity was associated with a gain-of-function property [47]. Recently, activity studies for a wide range of fALS mutant SODs [48] demonstrated that the mutant enzymes may be grouped into two categories. By far the larger group is that for which activity is similar to that of the wild-type enzyme. The other category is fALS mutant enzymes with mutations of binding ligands or that directly aﬀect metal binding. This latter group includes such mutants as H46R, where the copperbinding histidine is replaced by an arginine, or D124N, where a ligand that has been shown previously [49] to control zinc loss from the active site has been modiﬁed. In general, this latter group of fALS mutant enzymes shows greatly lowered copper aﬃnity, lowered activity, or a loss of pH independence for the enzymatic activity. The retention of full activity in the majority of fALS mutant enzymes, however, making them very ‘‘wild-type-like,’’ led to a number of proposals for oxidative damage in ALS. A.

Enhanced OH Radical Production

One gain-of-function mechanism involves enhancement of the rate constant for OH radical production that occurs when the reduced copper in SOD reacts with H2O2 [Eq. (14)]. This mechanism postulates that the disease process is initiated by (1) escape of the OH radical into solution with its subsequent reactivity with cellular targets or (2) increased scavenging of the OH radical by the histidine at the active site [43,44], leading to destruction of the integrity of the active site and loss of copper to solution with subsequent deleterious copper-related oxidation/reduction processes. Experiments aimed at testing the hypothesis of enhanced OH radical production have led to two seemingly contradictory results. Using spin-traps such as DMPO (5V,5V-dimethylpyrroline-N-oxide) to trap the free radicals formed via Eqs. (13) and (14), the peroxidative cycle of CuZnSOD, has led to results that consistently show an elevated level of trapped free radicals [9,10] in fALS mutant SODs. The mechanism was hypothesized to involve more ﬂexibility at the active site, allowing either greater escape of the OH radical in solution or accessibility of the spin trap to the active site channel of CuZnSOD, normally excluded because of steric constraints. The crystal structure of the G37R mutant enzyme showed a greater ﬂexibility at the copper-binding site [50], adding support to this mechanistic hypothesis. These observed enhanced levels of OH radical generation were not reproduced, however, when enzyme deactivation was used as a measure of Eq. (14), i.e., the enzyme itself was used as the OH radical scavenger [51,52]. One would have expected enhanced levels of deactivation using the fALS mutant SODs because increased ﬂexibility would allow increased access of peroxide as well as a spin trap. The same argument for enhanced reactivity could be made, of course, for enzyme activity [Eqs. (9) and (10)] except that the catalytic rate constant is already virtually diﬀusion controlled and thus diﬃcult to accelerate, whereas the deactivation rate constant is quite slow.

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In the past few years, the role of bicarbonate in the reaction of reduced CuZnSOD with peroxide [Eq. (11)] has received scrutiny. Surprisingly, enhanced peroxidative activity, as determined in spin-trapping experiments, was absolutely predicated upon the presence of bicarbonate (HCO3) in the solution [53]. Because the spin trap experiments were carried out in bicarbonate buﬀer and the deactivation experiments were carried out with no added bicarbonate, this observation helps to reconcile the observed diﬀerences in OH radical generation in fALS mutant SODs. The involvement of bicarbonate is very signiﬁcant as the in vivo concentration of bicarbonate in the cytosol of cells is reported to be 12–30 mM. The involvement of bicarbonate has implications in proposed mechanisms of peroxynitrite damage as well (see below). The literature so far indicates that substantial levels of bicarbonate are necessary in order to experimentally measure the enhancement of peroxidase activity (greater than 1 mM bicarbonate in spin-trap experiments). Clearly bicarbonate, as a small anionic species, can enter the active site channel in the same fashion as O2 or HO2. Interestingly, phosphate, as a small inorganic species, can competitively replace bicarbonate. Experiments by Sankarapandi and Zweier [54] demonstrated that at 100–200 mM phosphate and 25 mM bicarbonate, the enhanced signal for spin-trapped radical was lost. The enhanced OH radical production by fALS mutant SODs is also observed in yeast cells expressing the human mutant enzymes. Use of POBN, a spin trap, demonstrated enhanced radical production for a series of fALS mutant SODs expressed within the yeast cells: G93A, G93C, A4V, and L38V [55]. This observed increase in spin-trap signal when hydrogen peroxide is mixed with fALS mutant SOD versus the wild-type enzyme that occurs only in the presence of substantial concentrations of bicarbonate has led to an increased interest in the bicarbonate dependence of peroxidase activity in CuZnSOD. A number of diﬀerent mechanisms have been proposed for the involvement of bicarbonate in the peroxidative cycle [53,54,56–61]. One mechanism involves bicarbonate entering the active site channel and scavenging the (bound) OH radical.

Cuþ Zn2þ SOD þ HO2 ! Cu2þ Zn2þ SOD : : : OH Cu2þ Zn2þ SOD : : : OH þ HCO ! Cu2þ Zn2þ SOD þ H2 O þ CO3

3

ð16Þ ð17Þ

The carbonate radical diﬀuses away and the longer lifetime of the carbonate radical relative to the OH radical leads to the oxidative reactions observed experimentally. In this system, bicarbonate (HCO3) is the OH radical scavenger (see below), but the carbonate radical (CO3) is the reactive species as the pK for the carbonate radical is very acidic [62]. An alternate mechanism has been proposed in which the observed increase in oxidative equivalents arises from an enhanced interaction of the peroxide anion, or possibly even a reaction of the protonated peroxide itself, with the carbonate that is bound at the active site. The eﬀects of protonation and ionic strength should not be underestimated or ignored here. A consideration of Figure 3 makes quite clear that a change in one pH unit will change the rate constant for OH radical production via a Fenton-type mechanism by one order of magnitude. This dwarfs the signals observed in the experiments described here. In addition, the change in rate constant with charge of the residue near the active site that is seen in Figure 1 for enzymatic activity must be viewed as applicable for OH radical generation. As mentioned previously, the active species is HO2, not H2O2, and therefore it is subject to the same eﬀects from ionic strength and electrostatic forces as O2 .

Oxidative Stress in Familial ALS

B.

481

Copper Trafficking: Copper–Chaperone Interactions

Copper traﬃcking in cells has received much attention recently. This is particularly relevant to fALS and CuZnSOD with the discovery and characterization of the copper protein responsible for loading copper into CuZnSOD, CCS (copper chaperone for SOD) in human cells and yCCS or Lys7 in yeast cells [63,64]. Studies in the copper transport pathways for loading copper in CuZnSOD, cytochrome c oxidase, and fet3p, a multicopper oxidase controlling iron uptake, have shown that copper transport is an exquisitely controlled process within cells. An experiment looking at cellular copper concentration led to an estimated upper limit of 10–18 M free copper or less than one atom per cell [65]. This was a very startling conclusion as it was thought previously that pools of free copper existed intracellularly and the binding aﬃnity for copper is what drove copper loading in enzymes. The mechanism for copper loading into CuZnSOD from the copper chaperone has been shown to be very complex [66]. The copper chaperone, CCS, consists of three domains. Domain 2 is very similar to a monomer of CuZnSOD, with the same copper-binding motif as the enzyme with the exception of one binding histidine, which is replaced by an aspartate. Upon insertion of copper into domain 2, no SOD activity is observed. However, if the aspartate is replaced by histidine via site-directed mutagenesis, SOD activity is acquired [67]. Domain 1 and domain 3 both have cysteine residues, and, in particular, domain 1 contains a very common motif (MTCXXC) that is found in the Menkes and Wilson’s proteins and other copper chaperone systems. An x-ray absorption spectral study showed that reduced copper is bound by these two domains in a bimolecular cluster [68]. Crystal structures of the copper chaperone CCS [69] and the yeast chaperone Lys7 [70] were published; the latter study postulated the formation of heterodimers between the chaperone and SOD. A beautiful crystal structure of the heterodimer between a monomer of CuZnSOD and domain 2 of CCS was determined recently [71]. A theory for the structure-function relationship of CCS is that the copper is transported by the cysteines as it is carried in the reduced state and cysteine is a powerful ligand for copper (I). The similarity of domain 2 with the active site of CuZnSOD allows a dimerization and a driving force for copper transport into the enzyme. That the copper chaperone, CCS, contains the binding site for copper but with the aspartate replacement of one of the histidine ligands probably allows the copper to migrate from the domain 1–domain 3 pincer of CCS into the CuZnSOD monomer preferentially and not domain 2 of CCS. It has been clearly demonstrated that copper insertion into CuZnSOD occurs via a copper chaperone for the wild-type system. That this same phenomenon holds true for fALS mutant SOD was demonstrated in a series of experiments involving A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T fALS mutant SODs [72]. These experiments, carried out in Saccharomyces cerevisiae, where Lys7 is the functioning chaperone but the human mutant enzyme was expressed, involved looking at expressing the fALS mutant enzymes in yeast cells both with and without the Lys7 copper chaperone system. Copper was found in mutant enzymes only in the presence of the chaperone. Gain-of-function theories for the deleterious eﬀect of the mutant enzyme have been postulated that involve the enzyme-chaperone interaction. It has been suggested that copper could be lost in the copper migration process because of unfavorable dimerization between the SOD monomer and CCS domain 2. The free copper could then initiate metal-induced redox processes that are toxic to motor neurons. The very

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low levels of free copper in cells argues towards the intrinsic toxicity of cellular free copper, making this a plausible mechanism to consider. In all of these as isolated enzymes, the concentration of zinc is signiﬁcantly higher than the concentration of copper. No zinc chaperone for CuZnSOD has been isolated, and there is speculation that zinc may have been inserted into domain 2 of CCS prior to copper loading in the domain 1–domain 3 pincer. Interestingly, all of the zinc-binding ligands of the active site in CuZnSOD are maintained in CCS. How CCS would then transfer the bound zinc to the SOD monomer is a matter of speculation. An alternate possibility is that a speciﬁc zinc chaperone may have already inserted zinc into the SOD monomer prior to the CCS-SOD interaction and copper transfer. The role that zinc plays in normal enzymatic function in CuZnSOD has been of continuous interest, and zinc as a mediator in fALS SOD–induced damage has come under increased scrutiny, as will be discussed later. The discovery of the copper chaperone led to immediate interest in whether mutations in CCS could also lead to ALS or another form of motor neuron disease. Thus far no mutation linked to ALS has been found in the CCS gene in a study of 20 sporadic ALS subjects [73]. In addition, no link between mutations in CCS and the ALS has been found in the investigation of two Italian families with fALS which is not linked to mutations in superoxide dismutase [74]. C.

Peroxynitrite

A mechanism was proposed involving damage induced by peroxynitrite (ONOO), formed by the very eﬃcient reaction of superoxide with NO, an in vivo neurotransmitter [13,75]. The rate constant:

NO þ O2 ! ONOO

ð18Þ

is k18 = 5 109 M1/s1 [76,77]. Peroxynitrite was shown to react with superoxide dismutase to form 3-nitrotyrosine [78], and this led [13] to a proposal of a gain-offunction mechanism involving greater ﬂexibility at the active site of fALS mutant SODs promoting the ability of peroxynitrite to nitrate exogenous proteins. Indeed, in studies of the transgenic mouse expressing G37R fALS mutant SOD, elevated levels of nitrotyrosine were observed [79]. There is, however, another step in the peroxynitrite mechanism. It was shown in 1995 [80] that peroxynitrite reacts rapidly with CO2 with a bimolecular rate constant of 2.9 104 M1/s1. The level of CO2 in cells is actually quite signiﬁcant as CO2 is in equilibrium with bicarbonate with the equilibrium lying in a physiologically relevant pH range. CO3 2 þ Hþ X HCO3

þ

HCO3 þ H X H2 CO3 H2 CO3 X CO2ðgÞ þ H2 O

pK19 ¼ 9:9

ð19Þ

pK20 ¼ 6:1

ð20Þ ð21Þ

ONOO þ CO2 ! ONOOCO2

ð22Þ

The carbon dioxide complex with peroxynitrite ultimately disappears with approximately 30% of the product being carbonate radical and NO2 radical and the remainder being nitrite and carbon dioxide. This is in contrast to the disappearance of the protonated peroxynitrite, where the products are approximately 30% diﬀusible

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free OH radical and NO2 radical and the remainder rearranging to nitrite. In both cases there is formation of an oxidizing free radical. Chemically, the reactivity of peroxynitrite with CO2 and the in vivo levels of CO2 suggest that peroxynitrite itself may have limited involvement in the system and that the reaction is mediated by the carbonate free radical. The connection between peroxynitrite and ALS, particularly the fALS mutant SOD, is, however, not obvious. The observed increase in nitrotyrosine levels associated with the transgenic mouse model of fALS is a tantalizing indication that there is some important association between nitrogen radicals and fALS that is yet undetermined. However, there are many caveats associated with nitrotyrosine production as an indicator of damage [81]. One study [82] showed that enhanced tyrosine nitration was associated with the zinc-free enzyme and not the ALS mutant. However, the authors also demonstrated that zinc binding in a number of fALS mutant SODs was lowered and that neuroﬁlament-L possessed zinc-binding aﬃnity suﬃcient to remove zinc from the fALS mutant SODs. A subsequent study from the same group demonstrated a further association between peroxynitrite toxicity and metal misplacement [75]. In particular, the authors found that it was the loss of zinc in both the mutant and wild-type forms of the SOD that was crucial for apoptosis in cultured neuronal cells. The common thread of toxicity as a result of metal misplacement will be discussed below. D.

Metal Binding

Metal binding is quite robust and speciﬁc in the wild-type enzyme. In the presence of a metal chelator such as ethylenediaminetetraacetic acid (EDTA), which binds copper with a stability constant of approximately 1018 M1, the enzyme will still retain the bound copper and zinc. As well as the very strong copper and zinc chelation at the active site of CuZnSOD, metal speciﬁcity is exquisitely controlled in the human wildtype enzyme. Remetallation of the wild-type apo enzyme invariably leads to an enzyme with copper in the copper-binding site and zinc in the zinc-binding site. However, studies have demonstrated that, upon remetallation, the Gly93Ala, Ala4Val, and Lys38Val fALS mutant enzymes remetallate with a loss of site speciﬁcity, with the extent of mismetallation progressing in the order given here [83]. Remarkably, the remetallated form of L38V has almost completely reversed the copper and zinc binding to where approximately 95% of the copper is now located in the zinc site, with the concomitant loss of activity associated with copper in a zinc-binding site. This leads to interesting questions as to the in vivo metallation of the enzyme and whether metal aﬃnity loss is common to fALS mutant SODs. A recent study of the activity of a large number of fALS mutant enzymes [48] demonstrated that the majority of the enzymes are fully active. It should be noted that these were isolated enzymes where the human enzyme was expressed in a baculovirus expression system. This suggests that the in vivo metallation of the fALS mutant enzymes occurs with correct placement of the metals; copper in the copper site and zinc in the zinc site. Interestingly, the copper content in these enzymes was low relative to both protein concentration and zinc concentration. It may be that the observed in vitro mismetallation of an apo enzyme does not indicate that mismetallation is an in vivo mechanism for oxidative damage but is rather a reﬂection of a much lowered binding aﬃnity and speciﬁcity for the copper and zinc sites in the mutant enzymes. Both in

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vitro studies demonstrating aberrant metal binding upon remetallation in the three fALS mutant human CuZnSODs discussed above [83] and the report of altered metal site structure in a diﬀerent fALS mutant enzyme [50] suggest that ﬂaws in metal binding and metal discrimination may be a common feature in the wide variety of mutations that have been identiﬁed thus far. This reduction of metal aﬃnity in the mutant SOD could lead to metal loss or misplacement during the in vivo lifetime of the enzyme. The role of the zinc-binding site in enzymatic activity has received much attention in the past few decades. Studies of CuapoSOD (enzyme containing only one equivalent of copper in each copper site, with the zinc-binding site remaining empty) showed that there is a pH-dependent migration of the copper from the copper-binding site in one subunit (monomer) to the other subunit, resulting in an SOD dimer without metal in one subunit and with two bound copper ions in the other subunit [84]. Later, it was demonstrated that, in the absence of zinc, Cu2+apoSOD is reduced in a pH invariant fashion (Cu2+apoSOD + O2 ! Cu+apoSOD) with a diﬀusion-controlled rate constant [85]. However, the reoxidation of the enzyme (Cu+apoSOD + O2 (+2H+) ! Cu2+apoSOD + H2O2) becomes pH dependent well below the pH where the copper becomes ﬂuxional. This suggests that the zinc plays a role in protonation and release of peroxide. These features highlight the importance of both the copper- and zinc-binding sites in the enzyme and points out some possible avenues for research vis-a-vis the diﬀerences between fALS mutant SODs and wild-type SOD. Changes in the zinc-binding site had been observed in yeast CuZnSOD below pH 5. In a very careful study [12] of the zinc-binding site in the yeast wild-type enzyme and a series of single amino acid mutants, including three fALS mutants, the authors took advantage of the fact that cobalt(II) can substitute for zinc and that the cobalt bound in the zinc site has a very characteristic spectral signal. They found that zincbinding is altered in all of these mutants and is dependent upon pH and copperbinding site occupancy. The modiﬁed zinc-binding aﬃnity observed in some fALS mutant SODs [82] prompted further investigation of the role of zinc in the peroxide-mediated production of oxidizing species (whether OH radical, bound OH radical, Cu(III), bound carbonate radical, or free carbonate radical). A study [86] of the deactivation of the bovine zinc-free enzyme by hydrogen peroxide indicated that zinc-binding aﬃnity was reduced much more quickly than enzyme activity. In addition, substrates that are known to protect the fully metallated enzyme in the presence of peroxide (e.g., ascorbate, urate) were shown to aﬀord no protection for the CuapoSOD. This, taken with the enhanced nitration by peroxynitrite observed in zinc-free enzyme (see above), suggests that zinc is involved in mechanisms of oxidative damage and certainly warrants further investigation in these systems.

IV.

IN VIVO EVIDENCE

A number of transgenic rodent models have been generated that can be used to test some of the mechanistic proposals of fALS mutant SOD toxicity. These include the (/) CuSOD mouse, the (/) MnSOD mouse, the (/) CCS mouse (CCS, as discussed previously, being the copper chaperone for CuZnSOD), and three fALS mutant SOD mouse models. The production of these three transgenic mouse models

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of ALS expressing three of the fALS mutant SODs (G37R, G85R, and G93A) and reproducing the phenotype of ALS with striking ﬁdelity lent enormous credence to the association of mutant SOD with fALS. Two of these mouse models, expressing G37R [87] and G93A [2], require overexpression of the mutant enzyme to generate the fALS phenotype and thus have higher SOD activity than normal (these being two of the ‘‘normal’’ fALS mutant SODs where activity assays have demonstrated wild-type-like activity). The disease phenotype, as mentioned earlier, is only observed in mice overexpressing fALS mutant SOD, not wild-type SOD, supporting both the decoupling of SOD activity from disease and a gain-of-function mechanism. The third of these mouse models, however, the G85R fALS SOD transgenic mouse [3], exhibits the characteristics of ALS without overexpression of the mutant enzyme. In this latter mouse model, disease was observed in mice that expressed 20–100% of the wild-type level with earlier onset for the higher expression levels [88]. In all cases, severity of the disease was great. However, a most interesting feature was that astrocyte inclusions were observed and these inclusions stained positive for the presence of mutant SOD and some ubiquitin. Very recently two transgenic rat models of ALS have been produced: the H46R fALS mutant SOD rat and the G93A fALS mutant SOD rat [5]. The H46R enzyme is very interesting as His 46 is a copper-binding histidine (see above) and the isolated enzyme is known to have very low observed activity as it does not retain much copper. In both of these transgenic rat models, disease is not observed until the enzyme is highly overexpressed, and the disease course is milder at comparable expression levels in the H46R transgenic rat. In accord with the rat and mouse models, the disease course in humans carrying the H46R fALS SOD mutation is known to be very mild, while humans carrying the G93A fALS mutant SOD, the fALS mutant SOD expressed in the both the transgenic mouse and rat models described above, exhibit a severe form of ALS. The CuZnSOD (/) (knockout) mouse does not show any phenotype similar to the motor impairment observed in the fALS mutant SOD mouse and, in fact, shows a phenotype of mild impairment of response to oxidative stress. In contrast, the MnSOD knockout mouse is severely impaired [89] and, depending upon what genetic background is used for expressing the knockout, survives for varying but short times [90]. This highlights the critical role for mitochondrial SOD2 versus cytosolic SOD1. These diﬀerences are not surprising as mitochondria are where the reactions involving oxidative phosphorylation occur and where leakage of electrons leads to relatively high concentrations of ROS. In analogy with the CuZnSOD knockout mice, mice that are null for CCS, the copper chaperone to SOD, also do not exhibit any form of motor neuron impairment. It was clearly demonstrated from the production of this CCS knockout mouse that CCS is essential for delivering copper to CuZnSOD [91]. Studies carried out with isotopically labeled copper reinforced this result in that they demonstrated that all of the other copper-delivery systems functioned as normal and only the delivery of copper to SOD1 was aﬀected. The transgenic CCS knockout mouse exhibited the phenotype of mice null in SOD1, with impaired fertility and paraquat sensitivity [91]. Some very recent experiments, however, have shown that mice that are null in CCS but that overexpress the fALS mutant SOD do indeed develop motor neuron disease [92]. The authors demonstrated that the SOD has a 20% or less residual activity in the various fALS mutant SOD transgenic mice and, in isotopic studies

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using 64-Cu, that almost no copper has been loaded into the SOD. This study reiterates the requirement for CCS to incorporate copper into SOD and suggests that copper is not associated with the gain of function in the fALS mutant SODs that leads to disease or that the role of copper in the process is more obscure. The role of metal, speciﬁcally copper, in the progression of motor neuron disease was addressed by treating the fALS SOD transgenic mouse with a variety of pharmaceutical agents in order to modify the disease course. Of interest here is a study in which penicillamine, a copper chelator, was shown to prolong the life of transgenic G93A fALS mutant SOD mice [18]. This is further supported by the observed beneﬁcial eﬀect of penicillamine on the viability of motor neurons in this mouse model. These data suggest that copper is indeed responsible for at least some of the symptoms of motor neuron disease. It is hard to reconcile the observations that in the fALS SOD transgenic mice removal of copper via ablation of CCS results in motor neuron disease, while removal of copper through the use of a chelator, penicillamine, leads to attenuation of the disease. The beneﬁcial eﬀect of copper chelators was further reinforced in a study of cultured cells expressing the H46R fALS mutant SOD. The cells exhibited an increased sensitivity to paraquat that was alleviated by the addition of penicillamine [11], again suggesting the occurrence of copper-mediated chemistry inducing oxidative stress. The observed beneﬁcial eﬀect on motor neurons expressing G93A fALS mutant SOD extends beyond penicillamine to a variety of other copper-speciﬁc chelators [93]. Elevated oxidative damage, OH radicals, and H2O2 observed in transgenic mice transfected with a fALS mutant SOD [94,95] also suggest that metal-mediated chemistry is linked to fALS mutant SOD. The oxidative damage and other phenomena in the latter studies are not necessarily speciﬁcally copper based but may represent CuZnSOD inducing some other metal toxicity. This quandary concerning the role of copper in motor neuron disease as evidenced by the role of CCS is more perplexing in light of results showing the beneﬁcial eﬀects of catalase on transgenic mice. Catalase is an enzyme that dismutates peroxide to water and oxygen and is suggested to remove the peroxide that results from the dismutation of superoxide by SOD. The authors in one study [96] prepared a catalase that was modiﬁed to cross the blood-brain barrier more eﬀectively and administered it to fALS SOD transgenic mice. A beneﬁcial eﬀect on the transgenic mice was observed, with both increased survival time and delay in onset of disease. The same eﬀect was seen with another modiﬁed catalase [97] and, recently, a synthetic catalase [98]. While these studies do not demonstrate the intermediacy of copper in the disease process, they strongly suggest the involvement of processes linked to oxidative damage. The theory of peroxynitrite or NO involvement was tested using the mouse model that expressed G93A fALS mutant SOD [99]. The authors found that only one of three neuroprotective agents increased survival time of the transgenic mice and that when the G93A mouse was crossed with a mouse in which the gene for neuronal nitric oxide synthase (NOS) was removed, the disease course was also unchanged. Another study [74] involving expression of fALS mutant SOD in cultured motor neurons demonstrated that, in the absence of zinc, both mutant and wild-type SODs were toxic to the motor neurons when in the presence of NO . These studies seems to preclude the direct involvement of neuronal NOS in initiating the disease

Oxidative Stress in Familial ALS

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process, and the latter work rather suggests the importance of zinc binding to proper enzyme function. Another variation on this theme involved crossing a fALS SOD mouse model with a mouse that was transgenic for overexpression of parvalbumin [100], a protein that binds calcium. It is known that there is excess intracellular calcium in ALS. Interestingly, the mice that overexpressed parvalbumin and fALS mutant SOD lived signiﬁcantly longer than those expressing just the mutant SOD. Delay of disease onset was also seen in these mice. Another piece of evidence to add to the importance of calcium in the process involves studies of the SOD knockout mouse where the authors observed a lowered level of calcium intercellularly in motor neurons [101]. A parallel phenomenon was not observed in ocular neurons [102], suggesting that calcium homeostasis is particularly important in motor neurons. There is a fascinating report that overexpression of human WT CuZnSOD in a mouse leads to some similar pathology to that observed in the G93A transgenic mouse, particularly in the mitochondrial eﬀects [103]. More strikingly, when the mouse overexpressing hWT SOD is crossed with the mouse expressing G93A fALS mutant SOD, the authors observed an earlier onset of symptoms than in the G93A mouse alone. This leads to some fascinating and perplexing questions about the role of SOD itself, particularly in light of the reports that overexpressing SOD in Caenorhabditis elegans and in Drosophila has reportedly a beneﬁcial eﬀect on overall life span [104]. This observation likely ties into the idea that motor neurons are particularly sensitive oxidative damage and, by analogy, to levels of SOD. There has been a theme of mutant SOD in mitochondria [105] (either because they leak there across the cell membrane or mutant SOD is actually expressed in the mitochondria) and being linked to motor neuron death. Mitochondria swell and then become vacuoles, and these vacuoles are a hallmark of the disease [106]. Two rather interesting eﬀects have been associated with mitochondria and fALS. One is that overexpression of MnSOD was shown to inhibit cell death in a cell line that expressed the G37R fALS mutant SOD [107]. Additionally, it was shown that in a cell line expressing G93A fALS mutant SOD, overexpression of MnSOD or pretreatment by DMSO, a spin trap for oxyradicals, inhibited cell death. As a further step, treatment of a transgenic mouse expressing G93A fALS mutant SOD with DMSO led to increased survival [108]. This then ties the mouse studies back to the earlier discussion of increased OH radical production in the in vitro assays using fALS mutant SOD and peroxide in the presence of biologically signiﬁcant concentrations of bicarbonate. One ﬁnal and very interesting transgenic mouse experiment involved expression of mutant SOD speciﬁcally in motor neurons of the mouse [109]. The mouse did not develop any form of motor neuron disease, suggesting that the disease process is initiated not in the motor neuron itself but rather in initial genetic expression of SOD in other sites such as the brain. Another animal model for fALS is the recently produced transgenic C. elegans that produces human mutant G93A, A4V, and G37R superoxide dismutase [110]. These transgenic nematodes were more sensitive to oxidative stress induced by paraquat exposure as measured by survival than their wild-type analogues. The authors measured oxidative damage and saw increased levels of lipid peroxidation (malondialdehyde assay) for the transgenic C. elegans. In addition, SOD degradation and

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removal was slowed in the transgenic C. elegans, leading to aggregation. The results shown here are analogous to the results for transgenic mice. V.

CONCLUSION

The relationship between oxidative damage and fALS is highly complex. As time passes and additional experiments are carried out, the topic becomes both more obscure and tantalizingly clearer. Initially, it seemed that an answer would be immediately forthcoming. After all, here was an enzyme that had been studied for years, decades even, with at least one established function and a direct connection with a disease process. Early experiments demonstrated ﬁrst a gain-of-function and then a potential mechanism. Finally, very robust mouse models were developed that mimicked the disease process in consummate detail. What is quite clear is that all is not so clear. The latest studies on the mechanism of neutrophils suggest that superoxide itself may not be the toxic entity and that cell killing by neutrophils may be associated with calcium channel activation and possibly pH change [111]. This places a diﬀerent slant on the role of superoxide dismutases— not in any way reducing their essential roles in the well-being of aerobic organisms, but suggesting that an additional function may be associated with their reactivity with superoxide. Superoxide radical may also serve to mediate in a process that may not involve direct oxidative damage but rather triggering of signaling pathways and SOD. More interestingly, the issue of pH and proton concentration in cells becomes important via this kind of mechanism. Mechanisms of oxidative damage (discussed above), such as production of OH radicals via a peroxidative reaction [see Eqs. (13) and (14)], certainly are extraordinarily proton dependent in that the reaction accelerates an order of magnitude for each increase in pH unit. The in vivo results are seemingly hard to reconcile. Animal studies seem to demonstrate a role for oxidative damage through the response to antioxidants and the use of penicillamine to ameliorate the damage to cultured motor neurons. However, the very striking demonstration of motor neuron disease in transgenic mice null in CCS, the copper transport chaperone for SOD, that express the mutant enzyme seems to eliminate the role of metals in the damage resulting from fALS mutant SOD. What seems readily apparent is that the results in the next few years will be very exciting and may say something about the link between calcium, superoxide, cellular signaling, and metals in the disease. Finally, there is evidence that the observed aggregates contain SOD [112] and the copper chaperone CCS [113] and are intimately related to motorneuron death. In addition, axonal transport is aﬀected very early in the processes leading to motor neuron death [114]. A very interesting new paper provides evidence that, in the absence of copper, a number of fALS mutant SODs exhibit greater thermal instability than the wild-type enzyme [115]. The relationship between protein aggregation and oxidative damage and at what point protein aggregation occurs in the pathway from initiation of damage to motor neuron death are clearly vital to understanding the disease process. ACKNOWLEDGMENTS I would like to acknowledge the very rewarding collaboration with Professor Joan Valentine (UCLA) and her group over the past few years. In addition, support

Oxidative Stress in Familial ALS

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from the ALS Association for many of the studies described here is gratefully acknowledged. This chapter was written at Brookhaven National Laboratory, which is supported by the U.S. Department of Energy.

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22 Oxidants and Antioxidants in the Pathology of Colon Cancer WILLIAM L. STONE and K. KRISHNAN East Tennessee State University, Johnson City, Tennessee, U.S.A. ANDREAS M. PAPAS YASOO Health, Inc., Johnson City, Tennessee, U.S.A.

I.

INTRODUCTION

The purpose of this chapter is to provide an oversight into the relationship between oxidants, antioxidants, and the etiology of colon cancer. Although considerable evidence supports the hypothesis that antioxidant factors protect against colon cancer and proxidant factors promote colon cancer, the overall picture is far from complete. Nevertheless, it is encouraging that a coherent and compelling argument can be made to support this hypothesis. It is encouraging because relatively simple and costeﬀective measures are available to maximize antioxidant factors and minimize proxidant factors and thereby provide a primary treatment paradigm that could potentially reduce the incidence of colon cancer.

II.

PHENOTYPIC AND GENETIC ASPECTS OF COLON CANCER

Colorectal cancer is a heterogeneous disease. Genetic and environmental factors play a variable role in the colorectal cancer. A genetic component is identiﬁable in only about 20% of colorectal cancer. Hence the majority of colorectal cancer cases are sporadic without a known deﬁned genetic basis. Other risk factors described in the literature include diet, especially excess animal fat, inﬂammatory bowel diseases, streptococcus, bovis bacteremia, and ureterosigmoidostomy. 497

498

A.

Stone et al.

Inherited Colorectal Cancer Syndromes

Inherited large-bowel cancers can be divided into polyposis syndromes (uncommon) and nonpolyposis (common) syndromes [1]. The polyposis syndromes include familial adenomatous polyposis (FAP), Gardner’s syndrome, Turcot syndrome, Peutz-Jeghers syndrome, and juvenile polyposis. Of the polyposis syndromes, malignant potential is highest with FAP, Gardner’s syndrome, and Turcot syndrome. All the syndromes have a variety of extraintestinal manifestations. B.

Familial Adenomatous Polyposis

FAP is an autosomal dominant familial colorectal cancer syndrome due to a germline mutation of the adenomatous polyposis coli (APC) gene [2,3]. The disease develops during the teenage years. The aﬄicted individuals develop hundreds of adenomas throughout the colon. All patients with FAP will develop colorectal cancer if colectomy is not performed. Colorectal cancer develops in the third or fourth decade of life. The APC gene is a tumor suppressor gene. It has 15 exons and encodes for a 2843amino-acid protein. The APC protein is important in cell adhesion, signal transduction, and transcriptional activation. Insertions, deletions, and nonsense mutations lead to the production of a truncated protein product. Mutations in the 5V and 3V ends of the APC gene lead to a less aggressive variant with fewer polyps called attenuated FAP, a later onset of colorectal cancer, and a variety of upper tract lesions [4]. A missense mutation in the APC gene, APC I1307K, is found in 6% of the general Ashkenazi Jewish population [5]. Individuals with this missense mutation are at increased lifetime risk of developing colorectal cancer. Genetic testing is available for this mutation. C.

Hereditary Nonpolyposis Colorectal Cancer Syndromes

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder accounting for about 5% of all colorectal cancer cases in the United States [6]. This inherited colorectal cancer syndrome is due to a mutation in one of the DNA mismatch repair genes. DNA repair genes are critical for the maintenance of DNA ﬁdelity during replication. The mismatch repair genes include hMSH2 on chromosome 2p16, hMLH1 on chromosome 3p21, and hPMS1 and hPMS2 on chromosomes 2q31 and 7q11. Germline mutations of hMSH2 can account for the majority of HNPCC mutations. Microsatellite instability is found in the colorectal cancer DNA from these individuals. Microsatellite instability is characterized by expansion or contraction of short DNA repeat sequences. Individuals with the HNPCC mutation carry a 70–80% risk of developing colorectal cancer in their lifetime. Clinically, colorectal cancers arising in HNPCC syndromes are predominantly right-sided. This is in contrast to sporadic colon cancer, which is predominantly left-sided. The precursor lesion for HNPCC is called the ‘‘ﬂat’’ adenoma. The survival of patients who develop colorectal cancer in HNPCC syndromes is better than for those with sporadic colorectal cancer. D.

Indications for Genetic Testing

Several organizations including the American Society of Clinical Oncology have published guidelines for the rational use of genetic testing to identify inherited colorectal

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cancer syndromes [7]. Genetic testing is oﬀered only when there is a strong family history of cancer or early age of onset of the disease, the test can be adequately interpreted, and the results will inﬂuence the treatment of the patient or family member. III.

OXIDATIVE STRESS AND COLON CANCER

It is well known that cancer of the colon is at least 30-fold higher than that of the small intestine [8]. In contrast to the small intestine, the colon is exposed to high levels of fecal bacteria [8]. Fecal bacteria account for about 50% of the dry weight of stools. The respiratory activity of the microﬂora in feces has been found to be a signiﬁcant source of superoxide radicals (O2 ) that, in the presence of iron, can generate hydroxyl radicals [9]. Babbs [9] has calculated that fecal bacteria can generate hydroxyl radicals ( OH ) at a rate equivalent to that produced by 10,000 rads of g-radiation per day. Most dietary iron is not absorbed and is concentrated in the feces at levels about 10-fold higher than in most tissues [9]. Bile pigments such as bilirubin and biliverdin are also concentrated in feces, and these pigments can chelate iron in a form capable of supporting the superoxide-driven Fenton reactions [Eqs. (1) and (2)] [9]. In addition to reactive oxygen species (ROS), nitric oxide ( NO ) is also generated in the colon, particularly in inﬂammatory bowel disease (see detailed discussion below). Nitric oxide can rapidly react with superoxide radicals to generate peroxynitrite, which is a powerful mutagenic reactive nitrogen oxide species (RNOS). These data support the view that the colon is uniquely exposed to a high level of oxidative stress. .

.

.

Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH . O2 þ Fe3þ ! Fe2þ þ O2

ð1Þ ð2Þ

.

NO þ O2 .

.

! ONOO

ð3Þ

The potential molecular mechanisms linking oxidative stress to colon cancer will also be reviewed here since an understanding of these mechanisms will undoubtedly be important in identifying potential targets for lifestyle, nutraceutical, pharmaceutical, and gene therapy interventions. Oxidative stress can result in damage to DNA and, in rapidly dividing cells, e.g., the epithelial cells of the colon, this damage can escape repair mechanisms resulting in somatic mutations. The age-related accumulation of somatic mutations in the epithelial cells of the intestinal mucosa gives rise to colon cancer. It is also of considerable interest (as will be discussed below) that some ROS, such as the superoxide radical, appear to play a key role in the signal transduction pathways that mediate cell growth. Of particular importance is the recent observation that the ras protein’s oncogenic message is relayed by O2 . In addition to discussing the antioxidant/proxidant factors that are operative in the colon, this review will also summarize the evidence suggesting that systemic oxidative stress, from cigarette smoking or excess alcohol consumption, could also contribute to colon cancer. The role of vitamin E in the etiology of colon cancer was reviewed in 1997, and this chapter will therefore focus only on the relevant developments since that date [10]. .

IV.

IMPACT OF COLON CANCER

Colorectal cancer is the fourth most commonly diagnosed form of cancer and the second most common cause of cancer deaths in the United States [11]. Colon cancer is

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primarily a disease of aging. Of the 140,000 cases of colon cancer reported in the United States each year, 110,000 cases are in people aged 65 or older. Cancer accounts for about 10% of all health care expenditures in the United States and colorectal cancer accounts for about 24% of these funds (i.e., colorectal cancer costs are about $24 billion annually) [12]. Surgical resection is the primary treatment for colon cancer, and early detection is critical in assuring a positive outcome. Despite the well-publicized need for early detection, compliance with screening programs remains poor. Primary prevention therapies that can prevent or inhibit the development of adenomatous polyps and their progression into colorectal carcinoma therefore aﬀord great promise. Even primary prevention therapies that are only 10% eﬀective in reducing colon cancer mortalities could save (on a yearly basis) about 5000 lives in the United States and potentially save some $2.4 billion in colon cancer costs.

V.

KNOWN RISK FACTORS AND THEIR RELATIONSHIP TO OXIDATIVE STRESS

A.

Environmental Factors

It generally accepted that the development of colon cancer results from an interplay between environmental and genetic factors. Lack of physical exercise [13–16], smoking [17,18], a western-style diet [19,20], and excess alcohol consumption [21,22] are environmental risk factors for colon cancer. Excess alcohol consumption has also been linked with oxidative stress [23]. Hartman et al. [24] recently found that even moderate alcohol consumption (30 g/day) for 8 weeks can cause a statistically signiﬁcant decrease in plasma a-tocopherol levels. In this review, redox–genome interactions, as modulated by other dietary factors, will be discussed in some detail. It is signiﬁcant, however, that even smoking and physical exercise are factors that modulate oxidative stress. Oxidative stress occurs when the production of oxidants outstrips antioxidant protection mechanisms. Smoking increases oxidative stress as measured by increased levels of isoprostanes in urine and plasma [25,26] as well as increased levels of breath alkanes [27]. Isoprostanes are nonenzymatically derived from arachidonate and active oxygen. Breath alkanes are thought to arise from the in vivo lipid peroxidation of polyunsaturated fatty acids. It is also interesting that breath levels of alkanes are also increased in inﬂammatory bowel disease [27], which is also associated with a dramatically increased risk of developing colon cancer (discussed below in more detail). There is convincing evidence that early smoking cessation is associated with a reduction in colon cancer risk [17]. Smoking cessation may, therefore, represent a very cost-eﬀective primary prevention measure that reduces an oxidative stress component contributing to the etiology of colon cancer. The interrelationships between exercise and oxidative stress and colon cancer have not yet been adequately studied. Based on observational studies, Thune et al. [13] suggest that there is an inverse dose-response between physical activity and colon cancer for both sexes. Lack of physical activity may contribute to colon cancer by inﬂuencing overall energy balance. Recent data support the notion that physical inactivity, high-energy intake, and large body mass strongly interact to increase the risk of developing colon cancer [28].

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The inﬂuence of exercise on modulating oxidative stress could, however, also be an important factor. During exercise there is a transient increase in oxidative stress that causes a marked increase in plasma levels of oxidized glutathione [29]. Nevertheless, subjects who undergo a physical exercise training program have an increased physical work capacity but experience less oxidative stress than their nonexercising counterparts [30]. It is likely that the transient oxidative stress caused by exercise triggers a long-lasting adaptive response that actually increases antioxidant defense mechanisms in the resting state, i.e., ‘‘the exercise oxidative stress paradox’’ [30,31]. Well-controlled studies with rats suggest that exercise training increases rat heart cytosolic glutathione levels by 131% compared to sedentary controls. B.

Genetic Factors

1.

Ras-p21

Studies of cancer in twins supports the notion that individuals may have a genetic predisposition to colon cancer and cancer in general [32]. This is a very active area of research, which has reasonably focused on the potential role of mutations in proteins that produce free radicals or provide antioxidant defense. The ﬁnding that O2 relays the ras protein’s oncogenic message is particularly interesting and will be reviewed in some detail due to its direct relevance to colon cancer. The ras gene is frequently mutated or overexpressed in colon cancer tissue. The proto-oncogene p21-ras is a 21 kDa guanine nucleotide-binding protein (a low molecular weight G-protein) with GTPase activity, and it plays a key role in regulating cell growth. P21-ras is in an active state (stimulating cell growth) when it complexes with GTP and is inactive when it binds GDP. The GTPase activity of ras provides this protein with the ability to automatically convert from the ‘‘on’’ to the ‘‘oﬀ’’ state. The active ras-GTP complex exerts its downstream signals by binding to eﬀector molecules that, in turn, eventually activate transcription factors that stimulate cell growth. This signal transduction pathway is very dependent upon the proper anchoring of the ras protein on the cytoplasmic face of the plasma membrane. The posttranslational modiﬁcation of ras precursors by farnesyl protein transferase plays a key role in this anchoring process by catalyzing the addition of a lipophilic farnesyl moiety. Oncogenic ras proteins have a mutation that inactivates the GTPase activity, thereby preventing the ras-GTP complex from switching to the ‘‘oﬀ ’’ state. The etiological role of mutated ras proteins in colon cancer has been convincingly demonstrated by the work of Sugiyama et al. [33]. These investigators showed that rat colonocytes could be transformed solely by introducing retroviruses containing the activated human K-ras oncogene. These transformed cells formed colonies in soft agar cultures and tumors in athymic nude mice [33]. There is also excellent evidence suggesting that expression of the ras oncogene is involved at the very early stages of colon cancer, i.e., the formation of aberrant crypt foci (ACF) [34,35]. The pioneering work of Irani et al. [36,37] has shown that O2 functions as a signal-transduction messenger that mediates the downstream eﬀects of ras and rac in nonphagocytic cells. In particular, this group demonstrated that NIH 3T3 ﬁbroblasts transformed with constitutively active forms of ras-p21 produce large amounts of O2 [36]. This O2 production could be suppressed by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, which is an inhibitor of NADPHoxidase complex [36]. It is also intriguing that the mitogenic activity of cells trans-

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formed with a constitutively active form of ras-p21 could be inhibited with N-acetylcysteine, which is a chemical antioxidant and precursor of glutathione. It is clear that ras mutations or factors that inﬂuence the expression of wild-type ras could be extremely important in the etiology of colon cancer. 2. 8-Hydroxyguanine Glycosylase A key enzyme in the repair of DNA is 8-hydroxyguanine (oh8Gua) glycosylase, which removes oh8Gua, which is an abundant and highly mutagenic oxidative DNA adduct [38]. In an excellent review, Halliwell [39] has critically evaluated the use of oh8Gua as a biomarker for oxidative DNA damage. oh8Gua, if not excised by repair enzymes, can result in G ! T and A ! C substitutions during cell division. Park et al. [40] found that oh8Gua levels were higher in colon cancer tumors compared to normal tissues. Surprisingly, the activity of oh8Gua glycosylase was found to be higher in the tumor tissues compared to the normal tissues [40]. Catalase and superoxide dismutase activity were, however, lower in the tumor tissues compared to normal tissue [40]. Park et al. [40] found no evidence that somatic mutation or polymorphism of oh8Gua glycosylase plays a role in colon cancer, but the sample size in this study was very small. 3. Unscheduled DNA Repair and ADP-Ribosyl Transferase Unscheduled DNA repair and ADP-ribosyl transferase activity (ADPRT) can also be used to estimate DNA repair. Pero et al. [41] have reported that both unscheduled DNA repair and ADPRT are sensitive to oxidative stress and are suppressed in patients with colon cancer. All of the published epidemiological studies relating DNA repair to human cancer were recently reviewed (for data prior to the year 2000) [42]. Cancer susceptibility does appear to be associated with defects in DNA repair capacity. The rapidly progressing ability to study polymorphisms in DNA repair genes (such as oh8Gua glycosylase) will undoubtedly increase our understanding of colon cancer etiology. 4. p53 Suppressor The p53 tumor suppressor gene functions to maintain the integrity of DNA, and somatic mutations in this gene are a major contributor to colon cancer and cancers in general. It is particularly important that damaged DNA be repaired before a cell enters the S phase of the cell cycle to prevent the duplication of the damaged DNA. The p53 protein is a transcription factor that increases in response to DNA damage and turns on the synthesis of p21 protein, which in turn blocks the progression of cells from the G1 phase to the S phase. The p53 protein is not an antioxidant protein, but the p53 gene may be an important locus of mutations induced by ROS and RNOS generated in chronic inﬂammatory diseases [43]. Ulcerative colitis is a chronic inﬂammatory disease of the colon, and individuals with this disease are at least 10 times more likely to develop colon cancer than the general public. It is highly signiﬁcant, therefore, that Hussain et al. [43] found higher p53 mutations in nontumorous colon tissue from ulcerative colitis subjects than normal adult controls. In addition, the levels of inducible nitric oxide synthase (iNOS) were higher in ulcerative colitis subjects compared to the controls.

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Inflammatory Bowel Diseases

It is well known that inﬂammatory bowel diseases dramatically increase the risk of developing colon cancer and decrease the age at which colon cancer occurs [44]. Considerable evidence supports the view that inﬂammation is associated with the excess production of ROS and RNOS [45,46]. In the case of inﬂammatory bowel diseases, there is direct evidence of oxidative damage to epithelial crypt proteins [47] as well as dramatic decreases in the levels of antioxidant defense mechanisms (i.e., ascorbate, urate, glutathione, ubiquinol-10, and total peroxyl radical-scavenging capacity) from inﬂamed versus noninﬂamed mucosal tissues [48,49]. There has been considerable progress in identifying the particular free radicals produced during inﬂammatory bowel disease. Kimura et al. [50] as well as Oudkerk Pool et al. [51] found that patients with ulcerative colitis or Crohn’s disease have elevated plasma levels of nitrite/nitrate. Kimura et al. [50] also found that serum nitrite levels positively correlated with the mucosal activity of iNOS. These data support a role for excess production of nitric oxide in inﬂammatory bowel disease. More recent work by this group [52] has shown that iNOS levels increased in parallel with the degree of inﬂammation and that peroxynitrite formation (as measured by nitrotyrosine staining) in the lamina propria was also associated with actively inﬂamed mucosa. Rachmilewitz et al. [53] have directly monitored nitric oxide production in the colon using a nitric oxide electrode. These investigators found that patients with ulcerative colitis or Crohn’s disease have an at least twofold higher level of colonic nitric oxide than control subjects [53]. In addition, colonic levels of NO positively correlated with the levels of NOS in mucosal biopsies as well as the clinical severity of the inﬂammatory bowel disease [53]. A direct role for NOS in the etiology of colitis has been provided in a rat model [54]. In these studies colitis was induced by intracolonic administration of trinitrobenzene, and some rats were simultaneously provided with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, in their drinking water. Rats provided with L-NAME had signiﬁcantly less colonic tissue damage than rats not provided with this nitric oxide synthase inhibitor [54]. D.

Nutritional Factors

There are considerable country-to-country variations in the incidence of colorectal cancer. For example, male colon cancer rates vary 19-fold from country to country, suggesting that environmental or nutritional factors could be a signiﬁcant factor [55]. Many studies have also supported the notion that the consumption of vegetables and fruits is inversely associated with the incidence of colon cancer. Both fruits and vegetables contain relatively large quantities of antioxidants that could prevent somatic mutations in colonic cells caused by the generation of highly reactive free radicals or by-products of lipid peroxidation. It is reasonable, therefore, to suggest that dietary levels of antioxidants, as well as dietary levels of proxidants (such as iron), could play a key role in accounting for the wide country-to-country variation in the incidences of colon cancer. The antioxidant status of humans is primarily evaluated in plasma and, to a much lesser extent, in tissues such as buccal epithelial cells or adipose cells. Papas [56] and Stone et al. [10] have argued that the antioxidant status of the digesta (a term

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deﬁning the total contents of the digestive tract, including food, drink, gut microﬂora, and endogenous secretions) could be particularly important in understanding the etiology of colon cancer. In this regard, antioxidants that are not eﬃciently absorbed may be as important as antioxidants that are eﬃciently absorbed. Nevertheless, this area of research has not yet received much emphasis and deserves considerable focus in the future. This chapter will focus on new information with regard to a few selected antioxidants and their relationship to colon cancer. Albanes and Hartman [57] have written an extensive review that covers the general relationship between antioxidants and cancer and provides an excellent and detailed summary of the current clinical trials addressing this issue. 1. Salicylates Recent work by Blacklock et al. [58] found that serum levels of salicylic acid (in subjects not taking aspirin) was higher in vegetarians then in nonvegetarians presumably due to the high salicylic acid content of fruits and vegetables. Salicylic acid is a nonsteroidal anti-inﬂammatory phenol that is thought to act as a chemotherapeutic agent against colon cancer by virtue of its ability to inhibit the synthesis of prostaglandins [59]. As detailed above, inﬂammatory reactions are also characterized by excess free radical production. Although salicylic acid is considered a weak antioxidant [60], it is capable of inhibiting low-density lipoprotein from oxidation induced by the simultaneous generation of superoxide and nitric oxide radicals [61]. Moreover, salicylate is able to inhibit the nitric oxide production and iNOS protein expression in cultured ﬁbroblasts [62,63] and RAW 264.7 macrophages [64,65]. It is likely, therefore, that salicylates in fruits and vegetables also play a role both as chemical antioxidants and as inhibitors of nitric oxide (and subsequent peroxynitrite formation) production in the colonic mucosa. 2. Flavonoids Other antioxidants in food may similarly be acting as anti-inﬂammatory agents. Quercetin is a common plant ﬂavonoid that generally occurs as a glycosidic derivative such as rutoside. In a rat model, Cruz et al. [66] found that oral administration of rutoside could ameliorate trinitrobenzenesulfonic acid (TNBS)–induced colitis and was also capable of inhibiting glutathione depletion in colonic tissues caused by TNBS. Flavonoids also may be acting to prevent cancer by nonantioxidant mechanisms [67]. Quercetin (10 AM) was recently found, for example, to inhibit the steadystate levels of ras-p21 (see discussion above) in both colon cancer cell lines and primary colorectal tumors [68]. Flavone is the core structure of ﬂavonoids, and the molecular basis of its anticancer activity has been investigated by Wenzel et al. [69]. These investigators found that ﬂavone induces apoptosis, diﬀerentiation, and growth inhibition in transformed colonocytes, and these properties may all contribute to the putative anticancer properties of ﬂavonoids [69]. 3. Vitamin E New Developments in Vitamin E Biochemistry. Vitamin E is not a single compound, and the term can be applied to any of the four (a-, h-, g-, y-) common tocopherols or any of the four (a-, h-, g-, y-) common tocotrienols. Tocopherols have a phytyl tail structure with all saturated bonds, whereas tocotrienols have a tail structure with three

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double bonds (a farnesyl group). a-Tocopherol (Fig. 1) is the primary form of vitamin E found in plasma, red blood cells, and the liver. It has, therefore, been the primary focus of most clinical, animal, and biochemical investigations. Nevertheless, the primary dietary form of vitamin E in the United States is g-tocopherol (Fig. 1), which is present in a typical diet at levels two- to threefold higher than that of atocopherol. The fate of dietary g-tocopherol has only been clearly understood in the last few years and will be reviewed here because this form of vitamin E may be uniquely important in colon cancer. All forms of dietary vitamin E are absorbed from the intestine and secreted into chylomicrons equally well. The liver rapidly takes up the secreted chylomicrons, and the hepatic vitamin E (from the chylomicrons) is then transferred into newly synthesized very low-density lipoproteins (VLDL), which are secreted into circulation. A major step in bio-discrimination between a-and g-tocopherol takes place during the transfer of hepatic tocopherol to the VLDL. A hepatic a-tocopherol transfer protein (a-TTP) selectively transfers a-tocopherol to VLDL. It has been suggested that the hepatic g-tocopherol not transferred to VLDL could be secreted from the liver into the intestine via bile [70]. This would suggest that g-tocopherol levels in the intestine could be very high. Nevertheless, when rats are fed diets with identical amounts of either atocopherol or g-tocopherol, the levels of g-tocopherol in feces, colonocytes, plasma, or liver (see Fig. 2) are always less than the levels of a-tocopherol [71]. These data suggest that g-tocopherol may be metabolized more rapidly than a-tocopherol rather than being selectively secreted into the intestine. The data in Figure 2 do suggest, however, that the g-tocopherol: a-tocopherol ratio found in plasma is not predictive of the ratio found in colonocytes or feces, where the ratio was two- to threefold higher. Most tissues are likely to have chemical distribution of tocopherol similar to that of plasma since most tissues take up vitamin E from circulating lipoproteins. The Data in ﬁgure 2 suggest, however, that feces and colonocytes could take up g-tocopherol directly from the digesta [71]. In humans it is

Figure 1

Structures of alpha- and gamma-tocopherols.

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The ratio of g-tocopherol for rats fed 0.156 mmol RRR-g-tocopherol/kg diet to a-tocopherol for rats fed 0.156 mmol RRR-a-tocopherol/kg diet in feces, colonocytes, plasma, and liver. Data indicate that the ratio of g-tocopherol to a-tocopherol in plasma is not a good predictor of tissue ratios. (From Ref. 64.)

Figure 2

likely that g-tocopherol makes a signiﬁcant contribution to the antioxidant defense mechanism of the colon since the dietary intake of g-tocopherol is about three fold higher than that of a-tocopherol. It is particularly interesting that rats fed diets containing a constant level of RRR-a-tocopherol and graded levels of RRR-gtocopherol show higher concentrations of a-tocopherol in all tissues compared to rats fed a control diet containing RRR-a-tocopherol alone [72]. These data suggest that the levels of g-tocopherol in food also may be an important variable inﬂuencing tissue levels of a-tocopherol. Yamashita et al. [73] have shown that the secretion of g-tocopherol (or atocopherol) into bile is not a signiﬁcant metabolic route, and this mechanism cannot, therefore, explain the lower plasma and tissue levels of g-tocopherol compared to that of a-tocopherol. Swanson et al. [74] have shown that the major pathway for the elimination of g-tocopherol is its catabolism to 2,7,8-trimethyl-2(h-carboxyethyl)-6hydroxychroman (g-CEHC) followed by glucuronide conjugation and urinary excretion. g-CEHC is a recently identiﬁed metabolite of g-tocopherol and is consistent with an omega-like oxidation of the phytyl trail to a water-soluble form [75,76]. Vitamin E and Colon Cancer. Shklar and Oh [77] reviewed the experimental basis for cancer prevention by vitamin E. In general, in vitro biochemical studies and animal studies support a protective eﬀect of vitamin E. Human observational studies are also generally supportive. For example it was demonstrated that vitamin E status, as measured by the ratio of plasma a-tocopherol to g-tocopherol, was associated with a decreased occurrence of large but not small adenomas [78]. Subjects in the highest versus the lowest quintile of a-tocopherol: g-tocopherol ratio had an odds ratio of 0.36 for large adenomas. In this study it was also suggested that measurement of dietary vitamin E intake might not provide an adequate index of vitamin E status [78]. It is interesting that supplementation with a-tocopherol results in a marked decrease in the levels of plasma g-tocopherol [79]. A high ratio of a-tocopherol to g-tocopherol is likely to be the result of taking a supplement containing only a-tocopherol.

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There are only a very few well-designed clinical intervention studies investigating the inﬂuence of vitamin E on colon cancer. Colon cancer is primarily a disease of aging that takes some 30–40 years to progress to the point of being an invasive cancer. This suggests that short-term trials in middle-aged adults may not be adequate to demonstrate clinical eﬃcacy. It may take large-scale, very long-term intervention (50 or so years) trials, starting in adolescents, to clearly test the clinical eﬃcacy of vitamin E or other antioxidants. Funding agencies do not, however, typically provide such long-term support. In the a-tocopherol, h-carotene (ATBC) cancer prevention study, long-term (5– 8 years) supplementation with 50 IU of all-rac-a-tocopheryl acetate was found to substantially reduce prostate cancer incidence and mortality in male smokers [80]. In this same study there was evidence suggesting that the vitamin E supplement showed some promise in reducing the colon cancer mortality [81]. In a randomized placebocontrolled study, Cascinu et al. [82] found that a daily dietary supplement for 12 months containing calcium and vitamins, including vitamin E (all-rac-a-tocopheryl acetate 70 mg) did not reduce cellular proliferation (as measured by the proliferating cell nuclear antigen or PCNA) in colonic mucosa of subjects who had previously been operated on for colon cancer. Is the Right Form of Vitamin E Being Used? Almost all clinical studies with vitamin E have used either synthetic a-tocopheryl acetate (all-racemic-a-tocopheryl acetate) or natural a-tocopheryl acetate (RRR-a-tocopheryl acetate). The acetate form of vitamin E is inactive until converted, by intestinal esterses, into the free unesteriﬁed form. Maximal protection of the colon (and its contents) from oxidative stress is more likely to be achieved with unesteriﬁed forms of vitamin E. g-Tocopherol may also play a unique role in preventing colon cancer or cancer in general. Moyad et al. [83], for example, compared the ability of RRR-g-tocopherol and synthetic vitamin E (all-raca-tocopherol) to control the growth of a human prostate cancer cell line. RRR-gTocopherol was found to be superior to a-tocopherol in inhibiting cell growth in vitro. Cooney et al. [84] found that g-tocopherol was a more potent inhibitor of neoplastic transformation in 3-methylcholanthrene–treated C3H/10T1/2 murine ﬁbroblasts than a-tocopherol. The diﬀerences in the ability of g- and a-tocopherol to prevent neoplastic transformation may be related to some of the striking diﬀerences in their chemistry [84,85]. g-Tocopherol shows a remarkable ability to reduce nitrogen dioxide (NO2) to nitric oxide that is not shared with a-tocopherol [84,85]. Nitrogen dioxide, if not reduced, can react with unsaturated fatty acid moieties to yield nitrite esters capable of nitrosating amines. In addition, Bittrich et al. [86] found that NO2 can induce single strand DNA breaks in V79 cells that is optimally inhibited by g-tocopherol in comparison to other lipid-soluble antioxidants. g-Tocopherol also appears to have a unique chemistry with peroxynitrite. Christen et al. [87] found that peroxynitriteinduced lipid peroxidation in liposomes was more eﬀectively inhibited by g-tocopherol than a-tocopherol. These authors also suggest that g-tocopherol is required to effectively remove peroxynitrite-derived nitrating species. g-Tocopherol has biological characteristics that also distinguish it from atocopherol. In vitro cell studies by Jiang et al. have shown that a-tocopherol is a more potent inhibitor of cyclooxygenase-2 (COX-2) than a-tocopherol [88]. This is particularly important with regards to colon cancer since COX-2 inhibitors have, in

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general, been found to be chemotherapeutic. It has also recently been found that gtocopherol, in contrast to a-tocopherol, can suppress the expression of ras-p21 in rat colonocytes [71]. The role of ras-p21 in colon cancer has been described above. VI.

CONCLUSION

The hypothesis that colon cancer risk is increased by in vivo oxidative stress is compelling but not yet convincingly supported by large-scale, long-term, placebo-controlled randomized clinical studies. Nevertheless, oxidative stress appears to be an underlying mechanism that provides insight into how lifestyles, genetic factors, inﬂammatory bowel disease, and nutritional antioxidants play a role in the etiology of colon cancer. REFERENCES 1. 2. 3. 4. 5. 6.

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23 The Role of Oxidative Imbalance in Diabetes Mellitus DOMINIQUE BONNEFONT-ROUSSELOT, ALAIN LEGRAND, and JACQUES DELATTRE Faculty of Pharmacy, Paris, France

I.

INTRODUCTION

Diabetes mellitus is a metabolic disorder characterized by a chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolism. It results from a deﬁciency of insulin secretion and/or of insulin activity. Classiﬁcation is based on the etiology of the disease and distinguishes between four types of diabetes: type 1, type 2, gestational, and other types (e.g., genetic alterations of pancreatic h cells, genetic deﬁciencies leading to a decrease in insulin activity, mitochondrial diabetes, and several diseases like endocrinopathies or pancreas disease). The majority of cases of diabetes are accounted for by type 1 and type 2, which account for approximately 15 and 80% of cases, respectively, the rest being constituted by rarer forms [e.g., maturityonset diabetes of the young (MODY)] that represent less than 5%. Type 2 diabetes is a heterogeneous disorder that develops in response to both genetic and environmental factors. It is characterized by a deﬁciency in insulin action as a result of a combination of insulin resistance and h-cell dysfunction that is manifest as inadequate insulin secretion in the face of insulin resistance and hyperglycemia. Obesity is an additional contributing factor to type 2 diabetes via its eﬀects on insulin sensitivity. Its rapidly increasing prevalence makes it a major cause of morbidity and mortality. Abundant evidence supports a genetic predisposition to type 2 diabetes and insulin resistance and/or insulin secretion. The results of linkage and association studies demonstrate that multiple genes are involved in the susceptibility to type 2 diabetes, each making a modest contribution to the overall risk. A locus has been mapped and potential causative variants identiﬁed on chromosome 2q, but the 513

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completion of the human genome project will likely result in the discovery of new genes. Type 1 diabetes constitutes more than 80% of diabetes in the young. It is a T-cell–mediated autoimmune disease characterized by the selective destruction of pancreatic h cells, leading to an absolute deﬁciency of insulin. Susceptibility to the disease is determined by a combination of genetic and environmental factors. The ﬁrst genetic markers of type 1 diabetes to be determined were genes of the major histocompatibility complex (MHC), especially genes of the HLA system that are located on the short arm of chromosome 6. The loci most associated with type 1 diabetes were HLA-B (class I genes) and HLA-DR,-DQ, and -DP (class II genes). Alleles of diabetes susceptibility have been discovered (DQA1, DQB1). Carrying a HLA haplotype of diabetes susceptibility multiplies by ﬁve or six times the risk of diabetes. HLA region (or IDDM1 region) accounts for about 30–50% of the familial risk of diabetes, whereas the insulin gene region on chromosome 11 (IDDM2) accounts for about 10%. Further research is necessary to determine the precise location and identify other diabetes-susceptibility genes. Gestational diabetes is deﬁned as a glucose intolerance leading to hyperglycemia that occurs during pregnancy. It probably results from an insulin resistance status present before pregnancy and associated with an inability to adapt insulin secretion. MODY is characterized by an onset of disease generally before the age of 25 without any insulin resistance. Patients exhibit an autosomal dominant pattern of inheritance and can be divided into at least ﬁve subtypes (MODY 1–5), with each subtype being caused by mutations in a speciﬁc gene. This type of diabetes is related to mutations on genes of glucokinase and several transcription factors. An oxidative imbalance, i.e., a disturbance in the balance between the production of reactive oxygen species (ROS) (especially free radicals) and antioxidant defenses, has already been described in diabetes mellitus [1,2]. This oxidative stress is thought to be involved in tissue damage [3] and could account for a new risk factor for coronary heart disease in type 2 diabetic patients. As will be developed below, oxidative stress can be demonstrated by several markers related to the oxidation of biological targets (i.e., lipids, proteins, and nucleic acids) and to lowered antioxidant defenses. The question can thus arise whether damage induced by reactive oxygen species is the cause or the consequence of diabetes. Indeed, hyperglycemia results by itself in oxidative stress status, which can be involved in some complications of diabetes (nephropathy, retinopathy, neuropathy) [4,5], especially via the production of advanced glycation end products (AGEs) and the oxidation of macromolecules such as the extracellular matrix. Conversely, oxidative stress could induce destruction of pancreatic h cells and thus be responsible for type 1 diabetes, but it also could result in impaired insulin production, release, or function, thereby inducing the insulin resistance observed in type 2 diabetes. II.

EVIDENCE FOR OXIDATIVE IMBALANCE IN DIABETES MELLITUS

Biological markers can be useful to assess increased oxidation of cellular targets and/ or lowered antioxidant defenses in diabetes. Three kinds of oxidative stress markers are classically used to evaluate oxidative damage: markers of lipid peroxidation, protein oxidation, and DNA oxidation [6]. Evidence for lowered antioxidant defenses can be assessed by determining the status of enzymatic and nonenzymatic antioxidant systems (Table 1).

Oxidative Imbalance in Diabetes Mellitus

Table 1

515

Some Markers of Oxidative Stress in Diabetes

Marker TBARS (thiobarbituric acid– reactive substances)

Hydroperoxides 8-epi-PGF2a (8-epi-Prostaglandin F2a) Oxysterols Protein carbonyls

8OHdG (8-Hydroxy-deoxyguanosine) Total antioxidant status

Vitamin E

Carotenoids, vitamin A Vitamin C

Cu,Zn-SOD (superoxide dismutase)

z: increased; ! unchanged; # decreased.

Variation (medium) z (plasma) z (plasma) z (plasma) z (plasma) z (plasma) z (erythrocytes) z (leukocytes) z (plasma) z (plasma) z (plasma) z (urine) z (plasma) z (urine) z (plasma) z (plasma) z (plasma) z (lens, vitreous humour) z (urine) z (lymphocytes) # (plasma) # (plasma) # (plasma) # (plasma) # (plasma) # (plasma) z (plasma) ! (plasma) ! (plasma) # (plasma) # (plasma) # (platelets) ! (platelets) # (plasma) ! (plasma) ! (plasma) # (plasma) z (serum) # (leukocytes) # (serum) !, # or z (erythrocytes) ! or z (erythrocytes) ! (erythrocytes) # (erythrocytes) # (erythrocytes) # (leukocytes)

Type of diabetes

Types

Types

Types

Types

Types

Types

Types

Types Types

Type Type Type Type Type Type Type Type Type Type 1 and Type Type Type Type Type Type Type 1 and Type Type Type 1 and Type Type Type Type 1 and Type Type Type 1 and Type Type Type 1 and Type Type 1 and Type Type Type 1 and Type 1 and

2 1 1 1 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 2 2 2 1 1 2 2 2 2 1 2 2 1 2 2 1 2 2 1 2 1 2 1 2

[185] [7] [11] [186] [187] [188] [189] [13] [12] [190] [16] [20] [20] [191] [23] [192] [193] [94] [194] [195] [12] [196] [57] [62] [9] [195] [196] [57] [51] [197] [52] [53] [51] [195] [196] [57] [56] [189] [8] [30] [30] [11,31] [187] [198] [199]

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A.

Evidence for Increased Oxidation of Cellular Targets

1.

Markers of Lipid Peroxidation

Although the assay lacks speciﬁcity, measurement of thiobarbituric acid–reactive substances (TBARS) is often used to quantify the eﬀects of oxidative damage on lipids containing polyunsaturated fatty acids (i.e., the eﬀects of lipid peroxidation). Several studies report elevated plasma TBARS levels in type 1 or type 2 diabetic patients compared to a normoglycemic population [7,8]. The question arose whether the diabetes type, the glycemic balance, or the presence of cardiovascular complications could modulate the TBARS level. Griesmacher et al. [7] showed that type 2 diabetic patients exhibited signiﬁcantly higher plasma TBARS levels than type 1 diabetic patients and that good glycemic control (HbA1c <6.5%) was in favor of lower plasma TBARS concentrations. This has been conﬁrmed in recent studies, which showed that concentrations of one TBARS, i.e., malondialdehyde (MDA), were increased in diabetic ketoacidosis and in poorly controlled diabetes [9,10]. In type 1 diabetic patients, plasma TBARS concentrations are higher in patients with vascular complications [11]. Finally, it has been reported that type 2 diabetic patients with angiopathy exhibited higher TBARS values than those without angiopathy [7]. Lipid hydroperoxides are earlier markers of lipid peroxidation than TBARS, whose values are elevated in both types of diabetes [12,13]. They could also constitute a marker associated with increased renal disease susceptibility, as recently shown in AfricanCaribbean patients with type 2 diabetes mellitus compared to Caucasian patients [14]. F2-isoprostanes have been proposed as alternative markers of lipid peroxidation in diabetic patients. They consist of a series of prostaglandin F2–like compounds formed during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Plasma levels of one of these, the 8-epi-PGF2a, are increased in subjects with non–insulin-dependent diabetes mellitus (NIDDM) [15]. Urinary levels of 8-epi-PGF2a are higher in type 1 and type 2 diabetic patients than in control subjects and are lowered by vitamin E supplementation [16]. As shown in streptozotocininduced diabetic rats, this increase in F2-isoprostanes may be partly responsible for the rise in renal TGF-h, a well-known mediator of diabetic nephropathy [17]; it may also be involved in maternal complications and fetal abnormalities associated with pregnancy complicated by diabetes [18]. Nevertheless, it seems too early to correlate an increased urinary excretion of 8-iso-PGF2a in diabetic patients with the occurrence of cardiovascular complications [19]. Another way to assess lipid peroxidation in diabetic patients is to assay plasma oxysterols, i.e., cholesterol oxidation products, which have been shown to be higher in patients with poorly controlled type 2 diabetes than in control subjects [20]. It seems that plasma levels of lipid peroxidation markers (e.g., TBARS, lipid hydroperoxides) are higher in long-standing type 1 diabetic subjects than in recently diagnosed patients, which could be in favor of a deterioration of the oxidative stress status as a function of diabetes development [21]. It would be interesting to take this into account to interpret the oxidative stress status in diabetic patients. With regard to lipid peroxidation, another target of interest is lipoproteins, especially low-density lipoproteins (LDLs), which are particularly susceptible to the oxidation process. This will be developed in the second part of this chapter, dealing with the oxidation of macromolecules; indeed, LDL susceptibility to oxidation cannot be considered a routine marker of lipid peroxidation due to the heaviness of the LDL isolation

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procedure, although new tests for LDL susceptibility to oxidation have recently been developed. 2.

Markers of Oxidative Protein Damage

The oxidative stress status demonstrated in diabetic patients also leads to oxidative protein damage. Carbonyl groups resulting from protein oxidation [22] can thus be found at high levels in the plasma and tissues of diabetic patients, with higher values in patients with complications [23,24]. A sandwich enzyme-linked immunosorbent assay has also been constructed to measure modiﬁcations of serum albumin induced by reaction with an aldehyde from lipid peroxidation, hydroxynonenal (HNE) [25]. The use of a monoclonal antibody against HNE-histidine–stable adducts showed that serum of type 2 diabetic patients exhibited signiﬁcantly higher levels of HNE-modiﬁed albumin than the matched nondiabetics. Nevertheless, and in opposition with what was observed with other markers like TBARS, no correlation was observed between the levels of HNE-modiﬁed albumin and glycated hemoglobin, diabetes duration, or complications. In the same way, Jakus et al. [26] studied oxidative stress in children with type 1 diabetes by measuring the speciﬁc ﬂuorescence due to MDA-protein adducts; this ﬂuorescence, which was high in these subjects, was correlated with an increased formation of AGEs. According to these authors, this increased oxidative stress could contribute to the development of complications. It is noteworthy that other protein modiﬁcations occur in diabetes, especially due to ‘‘glycoxidation’’ (i.e., AGE formation through an oxidative pathway), as will be explained in the third part of this chapter. Among the major carbonyl compounds, Nq-(carboxymethyl)lysine (CML) can accumulate in the plasma and tissues of diabetic patients [27]. 3.

Markers of DNA Oxidation

DNA is susceptible to free radical attack. As noted above for lipoprotein oxidation, evidence for DNA oxidative damage requires speciﬁc techniques not available for routine assessment [6]. B.

Evidence for Lowered Antioxidant Defenses

1.

Enzymatic Antioxidant Defenses

Tissue expression of enzymatic antioxidant defenses can usually be induced by reactive oxygen species, which constitute a defensive mechanism. Ceriello et al. [28] showed that exposure to high glucose concentrations, which signiﬁcantly increased lipid peroxidation in ﬁbroblasts from type 1 diabetic patients, induced a selective increase in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) mRNA and activity, whereas Mn-SOD did not change. These authors also demonstrated that type 1 diabetic patients with nephropathy had a failure in their defensive mechanism (especially catalase and glutathione peroxidase). However, discrepancies appeared in the literature data. Indeed, human extracellular superoxide dismutase (EC-SOD) can undergo glycation, which is associated with a decrease in its aﬃnity for heparin without aﬀecting its enzymatic activity. Nevertheless, Hartnett et al. [8] described a decreased serum SOD activity in type 1 and type 2 diabetic patients, without any correlation with either glucose or glycated hemoglobin levels. The primary glycation sites on ECSOD have been identiﬁed as two lysine residues present in the heparin-binding

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domain in the carboxy-terminal end. In diabetes, the proportion of glycated EC-SOD is markedly higher than in healthy subjects, so that the proportion of EC-SOD present on the surface of the endothelium (bound to heparan sulfate proteoglycan) may be decreased, enhancing the susceptibility of cells to superoxide radicals produced in the extracellular space [29]. Besides, erythrocyte Cu,Zn-SOD activity has been reported to be unchanged, elevated, or decreased in type 1 diabetic patients, whereas it was unchanged or lowered in type 2 diabetic subjects [30,31]. Increased glycated Cu,Zn-SOD has been reported in the erythrocytes of patients with type 1 or type 2 diabetes [32,33], and this enzyme is less active than the nonglycated fraction [32] due to the glycation of two lysine residues probably located in an active site liganding loop [34]. The glycation reaction further results in site-speciﬁc and random fragmentation of human Cu,Zn-SOD [35]. Plasma glutathione peroxidase (GSH-Px) activity has been reported to be elevated in both types of diabetes [30]. Erythrocyte GSH-Px activity has been found either unchanged or decreased in type 1 diabetes or enhanced in type 2 diabetes [30]. The rise in GSH-Px activity observed, for example, in diabetic patients with retinopathy may be a compensatory mechanism to prevent tissue damage [36]. Nevertheless, others have found a decreased GSH-Px activity in diabetic patients with retinopathy [8,37]. The site of glycation of GSH-Px has been identiﬁed in the bovine enzyme and is located at approximately 15 A˚ from the active site, selenocysteine [38]. Glycation of GSH-Px seems to be responsible for a decrease in aﬃnity of this enzyme, which could contribute to the hyperaggregability of the diabetic platelet due to the impairment of glutathione peroxidase [39]. 2.

Metals

Metals also play an important role in diabetes-associated oxidative stress, especially since some of them are involved in the function of antioxidant enzymes. Zinc deﬁciency has commonly been described [40], in association with hyperzincuria, although some authors reported no signiﬁcant diﬀerence in plasma zinc levels between diabetic patients and healthy controls [41,42]. It has been reported that a low groundwater content of zinc may be associated with later development of childhood-onset diabetes [43]. Zinc supplementation corrects zinc deﬁciency and decreases lipid peroxidation in insulin-dependent diabetes [44]. Manganese also seems involved in the oxidative stress status in diabetes. A manganese deﬁciency in streptozotocin-induced diabetes rats can lead to markedly decreased activities of Mn-SOD in kidney and heart and of Cu,Zn-SOD in kidney, in association with an increased lipid oxidizability of erythrocytes and a depletion of reduced glutathione in liver and kidney [45]. Urinary excretion of manganese is higher in diabetic patients than in control subjects; moreover, diabetic patients not treated with insulin or those with liver disorders excreted signiﬁcantly more manganese than diabetic patients without such disorders [46]. With regard to selenium, concentrations measured in serum or plasma of patients with diabetes mellitus were signiﬁcantly lower than those determined in healthy subjects [42,47], whereas urine selenium concentrations were not signiﬁcantly diﬀerent between both groups [47]. A negative correlation between the plasma contents of selenium and glycated hemoglobin has even been reported [42]. Selenium supplementation (and more eﬃciently selenium + vitamin E supplementation) could play a beneﬁcial role by controlling oxidative stress in experimental diabetes [48].

Oxidative Imbalance in Diabetes Mellitus

3.

519

Nonenzymatic Antioxidant Defenses

Among the nonenzymatic antioxidant defense systems, most studies have focused on vitamin E, a liposoluble antioxidant carried by lipoproteins in plasma. Investigations conducted in diabetic patients led to heterogeneous results, with either unchanged or elevated or decreased plasma vitamin E concentrations [49–51]. Such discrepancies could be explained by the fact that the circulating lipid level was not always taken into account to interpret the plasma vitamin E concentration. Heterogeneous results have also been reported in platelets and erythrocytes. Indeed, Kunisaki et al. [52] reported low vitamin E levels in platelets from type 1 diabetic subjects, whereas Caye-Vaugien et al. [53] found unchanged vitamin E concentrations in platelets and erythrocytes of type 1 and type 2 diabetic patients. Other lipophilic antioxidant compounds (i.e., carotenoids) have been reported to be useful indicators for a prediabetic condition, since they were detected in a Japanese population at lower levels in the plasma of newly detected diabetic subjects than in controls [54]. Similarly, Polidori et al. [51] reported in old type 2 diabetic patients low plasma levels of vitamin A and carotenoids (h-cryptoxanthine, h-carotene, lycopene) compared to age-matched controls. Vitamin C, which plays a major role in regenerating vitamin E from the a-tocopheroxyl radical, has been reported in diabetes either at unchanged plasma concentrations [50,55] or at elevated [56] or lowered [36,57] levels. Lower vitamin C concentrations could result from an impaired NADPH-dependent regeneration of vitamin C, as shown in diabetic rats [58]. Finally, cellular reduced glutathione level has been suggested to be lowered in animal diabetes models as well as in diabetic patients [59,60]. Similarly, intraplatelet content of reduced glutathione is signiﬁcantly lower in diabetic patients with high glycated hemoglobin than in those with low glycated hemoglobin [39]. 4.

Plasma Total Antioxidant Status

Methods have been developed to measure the total antioxidant status (which involves metal chelators, free radical scavengers, and several antioxidant enzymes) or the total peroxyl radical trapping antioxidant parameter in plasma [61]. Most studies conducted in type 1 or type 2 diabetic patients showed a signiﬁcant decrease of the plasma total antioxidant status [50,57,62], especially in poorly controlled diabetic patients and in cases of diabetic ketoacidosis [9]. This status is lowered during an oral glucose tolerance test in normal and non–insulin-dependent diabetic subjects, showing that even an acute hyperglycemia can induce an oxidative stress [63]. However, one study reported no decrease of plasma antioxidant activity in subjects at increased risk for type 1 diabetes (risk assessed by the presence of type 1 diabetes–associated autoantibodies) [64]. This study suggested that the clinical onset of diabetes was not preceded by signs of increased systemic oxidative stress as evaluated by plasma antioxidant capacity. It is of note that a new marker, called the ‘‘redox compensation index,’’ has been proposed to evaluate the oxidative stress in a group of patients with type 2 diabetes [65], by combining the values of plasma hydroperoxides and of a ferric reducing/ antioxidant power (FRAP) assay for total antioxidant potential of plasma. In these patients, a signiﬁcant inverse correlation was observed between levels of glycated hemoglobin and redox compensation values, in favor of the beneﬁt of good glycemic control. Another methodology, cyclic voltammetry, has been used to evaluate the

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antioxidant capacity of blood plasma in healthy and diabetic subjects and can be useful to monitor a response to treatment and/or antioxidant supplementation [66]. III.

OXIDATION OF MACROMOLECULES IN DIABETES

A.

Oxidation of Extracellular Matrix Molecules

Abnormalities of the extracellular matrix could be involved in the pathogenesis of diabetic complications. Indeed, increased permeability of the glomerular ﬁltration barrier may be attributed to an alteration of the matrix molecules [67]. Matrix glycation interferes with normal cell–matrix interactions and intracellular signaling that can potentially result in diﬀerential gene expression contributing to the changes seen in diabetic nephropathy [68]. In addition, glycation of extracellular matrix components may contribute to altered interaction of human mononuclear cells with collagen I, as shown in vitro by Menon and Sudhakaran [69]. Moreover, as will be described below, under oxidative conditions glucose can react with proteins to form AGEs. AGE-modiﬁed proteins bind to cell surface receptors and other AGE-binding proteins, several types of which have been identiﬁed [70]. Cell activation in response to AGE-modiﬁed proteins is associated with increased expression of extracellular matrix proteins, vascular adhesion molecules, cytokine, and growth factors [70]. In diabetic rats, the presence of AGEs has been shown by immunocytochemistry in the renal extracellular matrix and could thereby participate in the pathogenesis of renal complications [71]. It has been hypothesized that AGEs could induce crosslinking of collagen, as recently shown in vitro [72]. Another factor that plays a role in extracellular matrix regulation is the metabolism of ascorbic acid, which is known to be abnormal in diabetes. Ascorbic acid has a stimulatory eﬀect on the sulfate incorporation into cell and matrix, and glucose is able to inhibit this action [73]. This would be of particular importance in the pathophysiology of diabetes. In addition, glycoxidation of type I and type IV collagens (two major extracellular matrix collagens) considerably alters their ability to modulate polymorphonuclear leukocyte (PMN) migration and production of reactive oxygen species [74]. B.

Lipoprotein Oxidation

Diabetic patients have an increased risk of cardiovascular disease (two- to fourfold higher than nondiabetic subjects). Conventional risk factors (plasma lipids, lipoproteins, hypertension) only partly account for the excessive risk of developing cardiovascular disease in type 2 diabetic patients. Indeed, insulin resistance, hyperinsulinemia, and hyperglycemia may constitute other regulating factors for this cardiovascular risk [75]. Given the oxidative theory of atherosclerosis, the question of increased in vitro oxidizability of LDLs in diabetic patients has been addressed. In vitro, glycated LDLs (obtained by incubation of LDLs with glucose or glucose 6-phosphate) is more prone to copper-induced oxidation than native LDLs [76]. However, data related to the oxidizability of LDLs from diabetic patients show discrepancies, with either increased [77,78] or unchanged [79,80] or even decreased [81] oxidizability. Such discrepancies could be due to the heterogeneity of the diabetic populations. These studies should have taken into account the glycemic control and vascular complications. Jenkins et al. [79] have evaluated the LDL oxidizability of 15 diabetic patients whose type 1 diabetes

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was well controlled and who did not have any vascular complications. No change in the oxidizability of their LDL was observed, in comparison with LDL from normoglycemic subjects. An increased oxidizability of LDLs could appear associated with a severe hyperglycemia or vascular complications [82]. The inﬂuence of the severity of the hyperglycemia on the hyperoxidizability of LDLs and VLDLs has recently been conﬁrmed in diabetic subjects with frequent hyperketonemia [83]; indeed, it has been reported that ketosis (acetoacetate) could generate free radicals and thereby increase lipid peroxidation [84]. Glycemic control is thus very important to avoid an increased susceptibility of LDLs to oxidation in diabetic patients [85]. It has been reported that the amount of partially oxidized LDLs in plasma was signiﬁcantly correlated with insulin resistance and its metabolic consequences [86]. Several studies showed that glycation of apolipoproteins, especially of apolipoprotein B [87], was enhanced in diabetic subjects. Since glycation and lipoxidation are two closely related phenomena, the question arose whether glycation could inﬂuence the susceptibility to oxidation. In vitro incubation of LDLs with glucose leads to an increased oxidizability of these LDLs [88,89]. In nonhuman primates, diabetes-induced glycation of LDLs increases their binding to arterial proteoglycans compared to LDLs of nondiabetic animals [90]. This may result in an increased retention of LDLs in the arterial intima and be one mechanism for increased atherogenicity of diabetic LDLs. Moreover, LDL oxidation generates new epitopes and leads to the formation of antibodies raised against oxidized LDLs. The presence of antioxidized LDL antibodies in plasma has been proposed as a predictive factor for the progression of carotid atherosclerosis. Such antibodies have been found both in plasma and in atherosclerotic plaques of diabetic patients [91]. It has been reported that serum of type 1 diabetes patients contained both antibodies to oxidized LDLs and antigen-antibody complexes (oxidized LDL–containing immune complexes) [92]. The presence of these complexes may be a risk factor for the development of macrovascular disease in these patients. An enhancement of the plasma concentration of antibodies raised against oxidized LDLs has also been observed in type 2 diabetic subjects [93]. A recent study conducted in fairly controlled type 2 diabetic patients reported a higher in vitro oxidizability of high-density lipoproteins (HDLs) as compared to control subjects [80]. Nevertheless, the protective eﬀect of these HDLs against LDL oxidation was maintained in relation with a normal paraoxonase activity [80]. C.

DNA Oxidation

DNA oxidative damage can be assessed by measuring speciﬁc oxidation products such as 8-hydroxydeoxyguanosine (8OHdG) using high-performance liquid chromatography (HPLC). Leinonen et al. [94] reported higher levels of 8OHdG in the urine of type 2 diabetic patients than in matched controls and observed a correlation with the level of glycated hemoglobin. DNA breakage was more frequent in a diabetic population than in a control normoglycemic population [95]. Signiﬁcantly elevated levels of DNA damage were found in the neutrophils from type 1 diabetics as compared to controls, even with acceptable glycemic control [96]. Interestingly, in experimental diabetes in rats (streptozotocin diabetic and fructose-fed rats), it has been suggested that the oxidative stress could modulate the transcriptional activation of genes by decreasing the aﬃnity of some transcription factors, due to DNA damage [97]. It is noteworthy that high glucose concentrations can also induce mutations in mitochon-

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drial DNA in vitro [98]. Indeed, the latter is more susceptible to damage than nuclear DNA, given its absence of coating with histones and its lower repair activity. IV.

HYPERGLYCEMIA AND OXIDATIVE STRESS

Elevated extra-and intracellular glucose concentrations result in an oxidative stress. Endothelial cells from bovine aorta incubated with 30 mmol/L glucose thus lead to increased production of oxygenated free radicals and enhanced concentration of thiobarbituric acid–reactive susbtances (TBARS) in cells [99]. Moreover, the increased free radical production is associated with a concomitant increase in intracellular AGE formation [99]. Conversely, antioxidants such as a-tocopherol, desferroxamine, and dimethylsulfoxide inhibit both production of free radicals and AGE formation [99]. In addition, as already mentioned, high glucose concentrations can lead to an enhancement of both the activity and mRNA of antioxidant enzymes (Cu,Zn-superoxide dismutase, catalase, glutathione peroxidase) [28]. This overexpression of antioxidant enzymes could constitute a response to glucose-induced oxidative stress. Several mechanisms seem to be involved in the development of oxidative stress in the presence of elevated glucose concentrations, namely glucose autoxidation, protein glycation, AGE formation, and the polyol pathway (Fig. 1). A recent study showed that increased production of superoxide radicals by the mitochondrial electron transport chain could be involved in the pathways of hyperglycemic damage [100]. This could provide a basis for the development of new pharmacological agents to inhibit this overproduction or dismutate the superoxide produced [100]. Oxygen free radicals, antioxidant defenses, and cellular redox status could thus be considered as central

Figure 1 Relationship between hyperglycemia and oxidative stress.

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players in diabetes pathogenesis, and the role of glycemic equilibrium in prooxidant/ antioxidant balance deserves special attention. Indeed, metabolic disturbances and oxidative stress seem to be closely related, with improved glycemic control being associated with a lowering of the prooxidant status [101]. Nevertheless, total normalization of the parameters of oxidative stress appears not to be reached by glycemic control alone, indicating continued oxidant injury despite optimal control of the diabetes [102]. In type 2 diabetes it has been suggested that hyperinsulinemia induces an additional oxidative stress via the activation of membrane NADPH-oxidase and H2O2 production [103]. A.

Glucose Autoxidation

In the presence of transition metals, glucose leads to an ene-diol radical anion; the latter radical reduces molecular oxygen, resulting in the formation of superoxide anions (O.2 –), with a concomitant production of a carbonyl compound. Superoxide anion can disproportionate into hydrogen peroxide, which in the presence of transition metals produces extremely reactive hydroxyl radicals (.OH) [104]. B.

Protein Glycation and AGEs

Protein glycation results from covalent binding between the aldehydic glucose function and the free amino groups of proteins. In the presence of transition metals (e.g., copper or iron), glycated proteins can give an electron to molecular oxygen, leading to oxygenated free radicals. This was shown for the ﬁrst time by Gillery et al. [105] and has been further conﬁrmed by others [106,107], even in the absence of transition metal ions [108]. When the protein half-life is longer than 10 weeks, glycated proteins undergo irreversible modiﬁcations leading to Maillard products or AGEs [109]. As glycated proteins, AGEs are also able to produce oxygenated free radicals via complex biochemical mechanisms [110]. Interaction of AGEs with endothelial cells leads to oxidative stress by a receptor-mediated process. Binding of AGEs to their speciﬁc receptors (RAGE) results in intracellular oxidant stress and activation of NFnB in cultured endothelial cells [111]. Hyperglycemia activates NFnB and promotes leukocyte adhesion to the endothelium through upregulation of cell surface expression of vascular cell adhesion molecule-1 (VCAM-1) and other adhesion proteins [112]. NFnB activation is an early event in response to increases in glucose and may contribute to vascular complications [111]. Reactive oxygen species can in turn accelerate AGE formation. Thus, glycoxidation refers to AGE formation through an oxidative pathway. During these complex reactions, protein modiﬁcation is generally due to compounds with a carbonyl or a dicarbonyl function. Therefore, a ‘‘carbonyl stress’’ hypothesis has been proposed [113]. Among these carbonyl compounds, Nq-(carboxymethyl)lysine (CML) in proteins could bind redox active transition metal ions such as Cu2+, which could ease free radical production [114]. CML accumulation may thus result in a deleterious vicious cycle, since CML formation itself is catalyzed by lipoxidation and glycoxidation [114]. Increased levels of CML have been observed in serum from children and adolescents with type 1 diabetes, and interestingly this increase preceded the development of micro- and macrovascular complications [115]. Among the intermediates occurring in the cross-linking of proteins by glucose, special attention should be paid to the a-dicarbonyl compound methylglyoxal [116]. This compound, which is formed in early glycation, is a precursor of advanced glycation

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adducts. Short periods of hyperglycemia (as observed in impaired glucose tolerance) may be suﬃcient to increase methylglyoxal concentration in vivo [117]. Methylglyoxal can react with collagen, thereby interfering with crucial cell–matrix interactions, especially via the loss of speciﬁc arginine residues involved in integrin-mediated attachment [118]. Methylglyoxal is physiologically detoxiﬁed by the cytosolic glutathione-dependent glyoxalase system [119]. Aberrations in the expression of the human glyoxalase have been reported in diabetes [120]. Impairment of the detoxiﬁcation via glyoxalase contributes to cytotoxicity and to the chronic pathogenesis associated with diabetes mellitus [121]. It is noteworthy that degradation of oxidized proteins can also be impaired in diabetes, as shown by Portero-Otin et al. [122] in the cytosol of kidney and liver of rats with streptozotocin-induced diabetes. Nevertheless, a serine protease that preferentially degrades oxidized and glycated proteins has been characterized in human erythrocyte cytosol [123]. This enzyme is adherent to oxidized membranes and is responsible for the degradation of proteins modiﬁed by oxidation and glycation [123]. C.

The Polyol Pathway

Glucose present at abnormally high intracellular concentration is preferentially metabolized via the polyol pathway. Its reduction by the aldose reductase leads to sorbitol, which is oxidized to fructose by the sorbitol dehydrogenase. An increased formation of AGEs may elicit activation of the polyol pathway and amplify endothelial cell damage leading to diabetic microvascular dysfunction [124]. Since NADPH is required for the activity of aldose reductase, an enhancement of the polyol pathway results in an intracellular depletion of NADPH [125]. Antioxidant enzymes such as glutathione reductase, which regenerates reduced glutathione, need NADPH. By decreasing the activity of glutathione reductase, an intracellular depletion of this cofactor decreases the intracellular content of reduced glutathione, which constitutes an important factor for the protection against oxygenated free radical–induced damage. Glucose-6-phosphate dehydrogenase (G6PD) plays an essential role in the regulation of oxidative stress by primarily regulating NADPH, the main intracellular reductant. Asahina et al. [126] showed that human umbilical endothelial cells incubated with high glucose (33 mmol/L) and exposed to H2O2 exhibited an inhibition of the pentose phosphate pathway, which predisposes cells to oxidative damage. Zhang et al. [127] showed that increased glucose concentrations (10–25 mmol/L) caused G6PD inhibition, resulting in decreased NADPH levels in bovine aortic endothelial cells. In these cells, high glucose concentrations also stimulated increased cAMP (via increased activity of adenylate cyclase), which led to increased protein kinase A activity, phosphorylation of G6PD, and inhibition of G6PD activity [127]. Such inhibition of G6PD predisposed cells to death and thus could play an important role in high glucose–induced cell damage/death. It is interesting to note that the use of an aldose reductase inhibitor (sorbinil) in streptozotocin-diabetic rats corrected diabetes-induced sorbitol and fructose accumulation and partially corrected malondialdehyde accumulation, reduced glutathione depletion and the increase in oxidized glutathione/reduced glutathione ratio [128]. Similarly, epalrestat, an aldose reductase inhibitor, decreased the levels of CML protein adduct in erythrocytes from diabetic patients [129]. This strongly suggests that aldose reductase activity plays a substantial role in the intracellular

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formation of CML. Intracellular depletion of NADPH also leads to another deleterious eﬀect, decreased nitric oxide (.NO) synthesis, since NADPH is cofactor of NOS, which synthesizes .NO from L-arginine. Moreover, .NO metabolism can also be altered by an abnormal production of superoxide anions, which is a consequence of an elevated intracellular glucose concentration. Indeed, superoxide anions react with .NO to form peroxynitrite (OONO), which is a potential oxidizing agent. It has also been hypothesized that proteins such as collagen, which contain AGEs, could scavenge .NO in endothelium and thus limit its diﬀusion into smooth muscle cells [130]. An elevated intracellular glucose concentration could thus result in several abnormalities of .NO metabolism, which could be involved in some vascular complications of diabetes. V.

ROLE OF OXIDATIVE STRESS IN THE ORIGIN OF TYPE 1 DIABETES OR INSULIN RESISTANCE IN TYPE 2 DIABETES

It has been suggested that .NO is involved in autoimmune pancreatic h-cell destruction in insulin-dependent diabetes mellitus [131]. The overproduction of .NO by activated immunocompetent cells during the development of streptozotocin-induced diabetes is probably an important part of the complex autoimmune reaction which leads to the destruction of pancreatic h cells [132]. Modulation of streptozotocininduced diabetes could thus be achieved by treatment with NOS inhibitors [132]. Apoptosis of pancreatic h cells could also result from the eﬀects of aldose reductase (role of the polyol pathway in the redox imbalance), as shown in a pancreatic h-cell line transfected with rat aldose reductase cDNA [133]. Thioredoxin, a redox-active protein, has been shown to protect cells from oxidative stress and apoptosis and to lead to a lower incidence of diabetes in nonobese diabetic transgenic mice that overexpress thioredoxin [134]. Conversely, catalase deﬁciency, which might predispose to cumulative oxidant damage of pancreatic h cells, is associated with a higher frequency of diabetes [135]. Insulin resistance may also be associated with intracellular production of free radicals, which in turn could be responsible for a deterioration of insulin action, thus leading to a vicious circle [136]. Thus, 3T3-L1 adipocytes exposed to low micromolar hydrogen peroxide concentrations display impaired insulin-stimulated GLUT4 translocation from internal membrane pools to the plasma membrane [137]. The mechanism involved would be an impairment of the cellular redistribution of the normal insulin-stimulated insulin receptor substrate-1 and phosphatidylinositol 3-kinase between the cytosol and the internal membrane [138]. Hydrogen peroxide at low concentrations would be a potent inhibitor of insulin signaling and may thus be involved in the development of insulin resistance [139]. Moreover, lipid peroxidation products such as 4-hydroxynonenal have been shown to decrease glucose-induced insulin secretion in isolated rat pancreatic islets, thus impairing glucose utilization and glucose oxidation, especially by aﬀecting the glycolytic pathway [140]. VI.

ANTIOXIDANTS AND ANTI-AGEs AS COMPLEMENTARY TREATMENTS IN DIABETES

Given the involvement of oxidative stress in diabetes complications, supplementation with classical antioxidants could be of interest by allowing a delay in the appearance or

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in the development of vascular complications. Some information is available on the eﬀects of treatments with classical antioxidants such as vitamin E, lipoic acid, or Nacetylcysteine. Vitamin E supplementation in diabetic patients has been reported to result in an improvement in insulin eﬀect and better glycemic equilibrium [102], as shown by lowered glucose, hemoglobin A1c and fructosamine values, and by decreased plasma lipid peroxidation and LDL oxidizability [141]. In addition, vitamin E supplementation could normalize the blood ﬂow in diabetes [142]. However, others showed no clear protective eﬀect of vitamin E supplementation, especially on blood glycated hemoglobin [143], and one study even reported that vitamin E could deteriorate insulin action and worsen the hypoﬁbrinolysis in obese type 2 diabetic patients [144]. Results of these studies remain divergent, and the use of a combination of antioxidants could be of interest, as antioxidants act synergistically. In experimental diabetes, the use of vitamin E, vitamin C, and N-acetylcysteine in diabetic mice can preserve h-cell function [145]. In vitro studies [146] and trials in animals [147] reported beneﬁcial eﬀects of a-lipoic acid. Clinical investigation has been carried out to assess the eﬀects of lipoic acid in the treatment of neuropathy in diabetic subjects. Clinical studies in type 2 diabetic patients showed an improvement in some clinical features of neuropathy after treatment with a-lipoic acid [148,149]. Finally, a clinical study conducted on type 2 diabetic patients demonstrated that N-acetylcysteine might slow down the progression of vascular damage by decreasing plasma soluble VCAM-1 concentrations [150]. Since AGEs and oxidative stress could result in many metabolic disorders in diabetes, elaboration of molecules that can ﬁght against the eﬀects of AGEs could be of great interest (Fig. 2). They can act either by inhibiting AGE formation (trapping

Figure 2 Antioxidants and anti-AGEs as complementary treatments.

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of reactive carbonyl intermediates) or by preventing cellular action of AGEs (antagonists of AGE receptors). Inhibitors of AGE formation are generally nucleophilic compounds. Novel drugs are thus able to inhibit the conversion of Amadori compounds (ketoamins resulting from the rearrangement of Schiﬀ bases) to AGEs [151]. These drugs are called amadorins, and their ﬁrst member is pyridoxamine. Pyridoxine, pyridoxal phosphate, and pyridoxamine form the vitamin B6 group of compounds, which are interconvertible within the cell. A pyridoxine deﬁciency has been reported in both type 1 and type 2 diabetes [152]. Clinical trials have shown that supplementation with pyridoxine lowered glycated hemoglobin levels in type 2 diabetic patients [153] and had beneﬁcial eﬀects on the clinical symptoms of neuropathy and retinopathy [152,154]. It has been demonstrated that pyridoxamine inhibits formation of AGEs in vitro [155]. Moreover, pyridoxamine lowered hyperglycemia-induced lipid peroxidation and protein glycation in red blood cells by a mechanism involving an increase in Na+K+-ATPase activity, which might contribute to its ability to delay or inhibit the development of cellular damage in diabetic patients [156]. Because of its nucleophilic properties, pyridoxamine can also inhibit formation of advanced lipoxidation end products (ALEs), such as adducts of malondialdehyde (MDA) or 4-hydroxynonenal (4HNE), with lysine [157]. Moreover, other inhibitors of AGE formation have been tested in animals and delayed diabetes-induced complications. They are tenilsetam [3(2-thienyl)-2-piperazinone] [158], OPB-9195 (2-isopropylidene-hydrazono-4-oxothiazolidine-5-ylacetanilide) [159], and 2,3-diaminophenazine [160]. These are nucleophilic compounds that act by trapping reactive carbonyl intermediates involved in AGE formation. They also play a role by limiting the formation of ALEs. In addition, aminoguanidine (or pimagedine) is a nucleophilic hydrazine that reacts with early protein glycation products, such as Amadori products. It inhibits the formation of AGEs such as pentosidine [161], which leads to intra- and intermolecular binding in proteins with a long half-life, such as collagen. Moreover, aminoguanidine and h-resorcylene aminoguanidine inhibit malondialdehyde formation in erythrocytes incubated with hydrogen peroxide, and thus may have therapeutic potential [162]. In vivo, treatment with aminoguanidine in animals inhibits the development and progression of major complications of diabetes such as retinopathy, nephropathy, and neuropathy [163]. Renal AGE levels, as assessed by ﬂuorescence or by radioimmunoassay in rats, which were increased after 3 weeks of diabetes compared to nondiabetic control animals, were attenuated by aminoguanidine therapy [164]. In addition, it has been reported that aminoguanidine, which is structurally related to N-nitro-L-arginine, can prevent experimental diabetes-induced vascular changes that could be mediated by NO production [165]. More recently, speciﬁc antioxidant properties of aminoguanidine have been demonstrated. Philis-Tsimikas et al. [166] reported an antioxidant eﬀect of aminoguanidine at high concentrations on the LDL peroxidation initiated by copper ions, which has also been shown by others [167]. A scavenging eﬀect of aminoguanidine toward hydroxyl (.OH) and peroxyl (RO.2) radicals has been demonstrated [168,169]. Endogenous LDL vitamin E and h-carotene (lipophilic antioxidants) have also been shown to be protected by this antioxidant upon oxidation of LDLs by .OH/O.2– free radicals [170]. To investigate the in vivo eﬀect of aminoguanidine on lipid peroxidation in diabetes, aminoguanidine was given for 9 weeks to streptozotocin-induced diabetic rats and lipid peroxidation was measured in their plasma (lipid hydroperoxides) and red blood cells (membrane malondialdehyde and

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thiobarbituric acid–reactive substances) [171]. Lipid peroxidation was signiﬁcantly lower in aminoguanidine-treated rats than in untreated rats. The results of this study thus suggest that aminoguanidine may have an additional beneﬁcial eﬀect as an antioxidant against lipid peroxidation in a prevention trial for diabetic vascular complications. Since diabetes complications are partly related to oxidative stress and to AGEs, a molecule such as aminoguanidine would be of great interest. Separate multicenter clinical trials of aminoguanidine in types 1 and 2 diabetes are in progress to monitor the eﬀect of treatment on the amount of proteinuria, the progression of renal insuﬃciency, and the course of retinopathy. A randomized, double-blind, placebocontrolled trial comparing two dose levels of aminoguanidine with placebo on the progression of the diabetic nephropathy was conducted in 599 type 2 diabetic patients with renal disease from 84 centers in the United States and the Canada [172]. Followup was 2 years after the date of randomization of the ﬁnal enrolled patient, so that the trial ended in March 1998. The results of this study would contribute to determining whether aminoguanidine slowed the progression of diabetic renal disease. Speciﬁc antagonists of the AGE receptors (RAGE) are an interesting ﬁeld of research. The 35 kDa proteolytic fragment of the extracellular domain of RAGE is called soluble RAGE (sRAGE) and exhibits an aﬃnity towards AGE-modiﬁed proteins [70]. It constitutes a potential pharmacological agent to prevent vascular eﬀects of AGEs in experimental diabetes [173]. A third category of molecules is constituted by oral antidiabetic drugs exhibiting antioxidant properties (without any anti-AGE eﬀect). Among them, thiazolidinediones are of particular interest. As an example, troglitazone has a combined chemical structure of thiazolidinedione and a-tocopherol–like structure (chroman ring). Troglitazone is able to inhibit Cu2+-induced oxidation of LDLs and the subsequent uptake and degradation of these LDLs by macrophages by acting as an aqueous-phase antioxidant in addition to its eﬀect on glucose homeostasis [174]. The antioxidant activity of troglitazone seems also to be involved in the inhibition of the expression of adhesion molecules such as intercellular cell adhesion molecule-1 (ICAM-1), VCAM1, and E-selectin on human umbilical vein endothelial cells (HUVECs), induced for example by oxidized LDLs [175]. This reduction in expression is paralleled by a signiﬁcant fall in NFnB translocation [175]. Similarly, sulfonylureas can exhibit antioxidant activity. Thus, in addition to beneﬁcial vascular eﬀects on the hemorrheological abnormalities seen in diabetic vascular disease [176], gliclazide decreases LDL oxidation and monocyte adhesion to endothelial cells, suggesting a beneﬁcial eﬀect of this drug in the prevention of atherosclerosis associated with type 2 diabetes [177]. Treatment with gliclazide also induces a decrease in plasma lipid peroxides and an enhancement of erythrocyte Cu,Zn-SOD activity, this eﬀect resulting from a free radical–scavenging activity independent of glycemic control [178]. Gliclazide seems to possess antioxidant properties that produce measurable clinical eﬀects at therapeutic doses. Indeed, administration of gliclazide to type 2 diabetic patients resulted in a fall of 8-isoprostanes and in an increase in the total plasma antioxidant capacity, superoxide dismutase activity and thiol levels [179]. Finally, a prospective path of research is devoted to oral antidiabetic drugs exhibiting both antioxidant and anti-AGEs properties. One molecule of interest is metformin (dimethylbiguanide), which is a leading drug in the therapy of type 2 diabetes [180]. In high fructose–fed rats (a diet that leads to insulin resistance), metformin is thus able to improve erythrocyte antioxidant activities (Cu,Zn-SOD,

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GSH-Px) [181]. It also increases blood glutathione levels classically low in diabetic animals [181], independent of its eﬀect on insulin activity. Due to the fact that guanidino compounds can block dicarbonyl groups, the eﬀects of the diamino biguanide metformin have been investigated on methylglyoxal formation in type 2 diabetes. Indeed, metformin reduces the levels of methylglyoxal (mentioned above as a reactive a-dicarbonyl thought to contribute to diabetic complications as a precursor of AGEs) [182]. Therefore, metformin treatment can prevent diabetic complications not only by lowering plasma glucose, but also by inhibiting AGE formation. In addition, a 4-week treatment with metformin in newly diagnosed obese patients with type 2 diabetes has resulted in a reduction of malondialdehyde level in erythrocytes and plasma and in an increase in the erythrocyte activities of Cu,Zn-SOD, catalase, and in glutathione level [183]. In conclusion, possible sources of oxidative stress in diabetes include increased production of radical oxygen species, especially from glycation or lipoxidation processes, and decreased enzymatic or nonenzymatic antioxidant defense systems [184]. Improvement of glycemic control seems to be a beneﬁcial factor to decrease oxidative stress in diabetic patients. Prevention of AGE formation by drugs such as aminoguanidine may help to delay the development of diabetic complications (kidney, eye, blood vessel, and nerve damage). Apart from classical antioxidants (vitamin E, acid lipoic, N-acetylcysteine) used to decrease oxidative stress, oral antidiabetic agents themselves can exhibit antioxidant activity independent of their action on glycemic control, which confers on them a high therapeutic potential.

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24 The Role of Reactive Oxygen Species, the Renin-Angiotensin System, and Endothelin in the Development of Essential Hypertension JANE F. RECKELHOFF University of Mississippi Medical Center, Jackson, Mississippi, U.S.A. LAURENT JUILLARD and J. CARLOS ROMERO Mayo Clinic, Rochester, Minnesota, U.S.A.

I.

INTRODUCTION

This chapter examines what is known about the pathophysiology of essential hypertension, with special emphasis on the role of the renin-angiotensin system, its interaction with paracrine and autocrine renal factors (such as nitric oxide and superoxide), and the eﬀects of angiotensin II (Ang II) on oxidative stress and on the interaction with endothelin (ET). Furthermore, we examine the experimental evidence indicating that most, if not all, of the deleterious eﬀects of angiotensin are triggered when the plasma level of this substance is inappropriately elevated with respect to the extracellular ﬂuid volume. In essential hypertension, the functional mechanisms responsible for long-term maintenance of hypertension, in the presence of so-called ‘‘normal’’ levels of plasma renin activity, remain unexplained [1,2]. Plasma renin activity is deﬁned as the amount of Ang I generated in plasma during one hour of incubation under predeﬁned laboratory conditions (pH, peptidase inhibitors, 37jC) [3]. In most cases the level of circulating Ang II is determined by the activity of renin. Therefore, in 50–60% of essential hypertensive patients, the levels of plasma renin activity are not diﬀerent from those seen in normotensive individuals [1,2]. Furthermore, a subset of the population 541

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of hypertensives exists (25–35%) in whom the level of circulating Ang II is signiﬁcantly less than that detected in normotensives [1]. However, these levels of Ang II may contribute to the maintenance of hypertension, because blood pressure is markedly reduced by the administration of either converting enzyme inhibitors [4] or angiotensin antagonists [2]. These observations raise the question as to whether hypertension can be induced with normal levels of Ang II. After a short summary concerning the established involvement of genetics in essential hypertension, this latter issue will be better understood by examining the characteristics of fast and slow pressor responses of angiotensin. II.

GENETIC INVOLVEMENT IN ESSENTIAL HYPERTENSION

Studies have demonstrated that blood pressure analyzed as a quantitative trait is heritable. Two principal mechanisms lead to sustained elevation of arterial pressure: general vasoconstriction or enhanced plasma volume due to increased sodium retention. Three examples of the direct action of the genome on the development of hypertension [5,6] can be cited. The most relevant here are the polymorphisms in the renin-angiotensin system. Higher levels of angiotensinogen are demonstrated to to be associated with higher levels of angiotensin II. The M235T polymorphism of the angiotensinogen gene was demonstrated to inﬂuence angiotensin levels. Jeunemaitre et al. reported a signiﬁcant linkage between the allele T235 and hypertensive patients, especially those with severe hypertension [7]. In contrast, the gene polymorphism I/D of the ACE and the renin gene failed to demonstrate signiﬁcant association with essential hypertension [8]. The second example is the C825T polymorphism of G protein h3. G proteins are heterodimeric molecules that mediate stimuli from a large variety of receptors, and the 825T allele was shown to increase NHE1 activity in cultured cells. Siﬀert et al. [9] demonstrated an association between 825T and hypertension in a casecontrol design study including 427 hypertensive and 426 normotensive German subjects. The third example is the demonstration by Bianchi that the genetic defect in the Milan hypertensive rat model was extendable to humans. The polymorphism of the adducing protein, a heterodimeric cytoskeletal protein implicated in the regulation of signal transduction, inﬂuence Na+K+-ATPase and sodium reabsorption in the proximal tubule. This polymorphism was associated with hypertension and salt sensitivity [10]. These results could lead to important strategies in drug therapy especially targeted to the renin angiotensin system [6]. Unexpectedly, variants of genes encoding angiotensinogen and ACE, were found to be associated with diﬀerences in blood pressure reductions in response to dietary sodium restriction. In the C825T polymorphism, the declines in blood pressure in response to hydrochlorothiazide diﬀered signiﬁcantly among phenotypic groups, with the magnitude of the response increasing progressively with the number of T alleles. Variation in the gene encoding adducin was also associated with enhanced response to acute loop-acting diuretics and chronic thiazide diuretic delivery. Most of these genetic involvements in essential hypertension support the main hypothesis developed here about oxidative stress–mediated angiotensin eﬀect, since response to oxidative stress could be genetically transmitted. Lacy et al. [12] reported that normotensive subjects with a genetic risk of hypertension (i.e., a positive family

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history of hypertension) had signiﬁcantly greater hydrogen peroxide (a critical molecule in oxidative stress cascade) production than blood pressure–matched normotensives without a family history of hypertension. They demonstrated, in a family-based cohort study, that hydrogen peroxide production was heritable in family with history of hypertension [13]. These ﬁndings emphasize that genetic loci inﬂuencing hydrogen peroxide production may represent logical candidates in the transmission of essential hypertension. III.

FAST AND SLOW PRESSOR RESPONSE TO ANGIOTENSIN II

The fast pressor response is produced when a relatively high concentration of Ang II is administered as a bolus, thus triggering a rapid smooth muscle contraction through the phosphoinositide-Ca2+-protein kinase C eﬀector system. The response reaches a maximal pressor eﬀect in seconds and returns to normal levels in 2–3 minutes [14]. We believe that the fast responses to angiotensin are just a pharmacological curiosity, because the circulating levels of Ang II necessary to evoke such a rapid elevation of blood pressure are higher than 2500 pg/mL of plasma [15]. This concentration is never seen in any physiological (sodium deprivation) or pathological (severe renovascular hypertension, severe hemorrhage) circumstances. The ﬁrst demonstration of a slowly progressive rise of arterial pressure during constant small infusion of Ang II was by Dickinson and Lawrence in 1963 [16]. Using conscious rabbits, they showed that amounts of Ang II below the threshold of the direct vasoconstrictor eﬀect elicited a gradual increase in blood pressure. Two years later McCubbin et al. reported similar ﬁndings in dogs [17]. These studies were critical to the determination of the fast and slow pressor eﬀects of Ang II. Slow pressor responses have been demonstrated in studies conducted in diﬀerent species such as rabbits [16], dogs [17], rats [15], swine [18], and humans [19]. The important characteristics underlying this response are as follows: the slow pressor response (1) needs 5–10 hours to develop and reaches a maximal peak 3–5 days after the onset of the infusion [20]; (2) evolves at low plasma concentrations that are close to or below the threshold of steroidogenic, renal, and dipsogenic actions typical of bloodborne angiotensin and well below the plasma concentration required to produce an immediate elevation of blood pressure [15,18,19,21]; and (3) is also evoked by continuous infusion of norepinephrine [22,23]. However, a signiﬁcantly higher (18fold) plasma concentration of norepinephrine is required to produce a similar pressor response as Ang II and is confounded by its intrinsic h-adrenergic agonist eﬀect, which can stimulate renin release [15]. An important study on the development of slow responses to Ang II was performed by Brown et al. [15]. These investigators found that the continuous intravenous infusion of 20 ng/kg/min of Ang II to conscious rats failed to elicit any detectable pressor response during the ﬁrst hour of the infusion. However, the following day, MAP increased by 15 mmHg. On day 7 of infusion, MAP reached 153F6 mmHg (Table 1). The level of circulating Ang II in these hypertensive animals was 249F25 pg/mL of plasma, signiﬁcantly higher than the level recorded on day 7 in normotensive controls infused with dextrose (80F19 pg/mL). It was also found that the amount of angiotensin needed to produce, during 1 hour, an elevation of MAP to levels comparable to that seen in the chronically infused hypertensive animals was

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Table 1 Response to Intravenous Infusion of Dextrose and Angiotensin II

270,000 pg/kg/min. This infusion yielded a pressor response of 146F3 mmHg, while the level of angiotensin was approximately 2750 pg/mL. This ﬁnding proved that continuous infusion of subpressor doses of angiotensin, accompanied by small or undetectable increments of angiotensin in plasma (see later), is capable of producing the same increment in blood pressure as that induced by the fast infusion of a dose of angiotensin that is approximately 13–14 times higher than that needed to produce the slow responses. This observation led Dickinson and Lawrence [16] to state that ‘‘angiotensin may bring into action some secondary mechanism which sustains arterial pressure by means other than general arterial vasoconstriction due directly to the hormone.’’ It should be noted that speculations as to whether the hypertension elicited by slow responses to angiotensin could be attained with plasma levels similar to those detected in normotensive individuals is important to consider because it will support the concept that in people suﬀering from essential hypertension, the same pressor mechanism has been activated. An important study to deﬁne this problem was undertaken by Hu et al. [24], who investigated whether slow responses of angiotensin could also be evoked by the administration of small amounts of renin. In fact, these investigators showed that slow responses can be developed in the rat by the administration of either Ang II (3.5 ng/min) (Fig. 1a) or rat recombinant renin (0.6 ng/min) (Fig. 1b), compared to vehicle control (Fig. 1c). The results are slightly diﬀerent from those reported by Brown et al., in that the control levels of plasma angiotensin were statistically elevated from f4.0 to 13F5 ng/mL [15] for 10 days after the onset of the renin infusion [15]. However, on days 12–14, the concentrations of angiotensin in plasma were not statistically diﬀerent from those recorded during the control period (Fig. 2). These renin-infused animals experienced a slow and progressive increase in blood pressure, which was characterized by two stages. In the ﬁrst stage, blood pressure increased from 90 mmHg in the control period up to approximately 120 mmHg. Five to 6 days after this ﬁrst plateau, blood pressure increased to approximately 140 mmHg, where it stayed for 14 days, in spite of the fact that, as mentioned above, the peripheral levels of angiotensin decreased back to normal levels. The studies of Hu et al. [24] and Brown et al. [15] clearly show that the administration of subpressor doses of Ang II induced an increase in blood pressure which was sustained,

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Figure 1 Changes in mean arterial pressure (MAP) and heart rate (HR) during control (C1–C7) and during intravenous infusion of (a) angiotensin II at 3.5 ng/kg (E1–E14) (*p < 0.05 vs. control period); (b) rat recombinant renin at 0.6 ng/min (E1–E14) (*p < 0.05 vs. control period); and (c) saline vehicle (E1–E14) (*p < 0.05 vs. control period).

despite the fact that the concentration of Ang II in plasma was never increased by more than a small amount [15] or was only transiently increased [24]. The consistent delay of small subpressor doses of Ang II to produce an increase in blood pressure in a chronic fashion is indicative of a time requirement for the activation of additional vasoconstrictor processes, which can then trigger an autocatalytic reaction that accelerates or potentiates the vasoconstrictor eﬀect of Ang II [16]. Like fast pressor responses, slow pressor responses are triggered by the binding of Ang II to its receptor (AT1), which activates a G-protein that subsequently stimulates phospholipase C [25]. This enzyme induces the hydrolysis of phosphatidylinositol bisphosphate and thus liberates inositol triphosphate (IP3), which releases Ca2+ from intracellular stores and diacylglycerol, which activates protein kinase C [26,27].

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Figure 2 Changes in the plasma level of angiotensin in pg/mL in response to the intravenous infusion of recombinant renin (5) or angiotensin II (n) during control days (C1– C7), during experimental days (E1–E13), and during recovery days (R5–R7) (*p < 0.05 vs. control days).

Rasmussen and Barrett suggested that diﬀerent signaling pathways may be involved in the fast and slow pressor response mechanisms, mainly due to the length of the stimulation of the AT1 receptor [28]. Thus, stimulation of the IP3-Ca2+ system may account for the rapid smooth muscle contraction by releasing Ca2+ from intracellular stores. The sustained pressor eﬀect of Ang II could be through protein kinase C, which stimulates Ca2+ channels and perpetuates a contractile response (1) by facilitation of the entrance of extracellular Ca2+, (2) by stimulation of lipoxygenase production, or (3) by the release of other autocoids that may potentiate the vasoconstrictor response of Ang II [29]. However, there is additional evidence that Ang II could stimulate superoxide production through the activation of the NADH system [30]. This could produce pressor eﬀects through mechanisms that will be discussed later. IV.

FLUID VOLUMES AND LEVELS OF ANG II

The renin–angiotensin system accounts for the long-term regulation of arterial pressure through its eﬀect on the pressure natriuresis relationship. Under physiological conditions, the relationship between arterial pressure and sodium excretion is very steep, so that minimal changes in arterial pressure are necessary to maintain sodium balance. Thus, a high sodium intake that induces a proportional ﬂuid volume expansion correlates with low levels of plasma renin activity. This inverse relationship between ﬂuid volume and plasma renin is extremely critical to maintaining blood pressure within normal limits [31]. Studies conducted with speciﬁc receptor antagonists for Ang II or blockade with angiotensin-converting enzyme inhibitors have

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shown the quantitative importance of the endogenous renin–angiotensin system in modulating sodium excretion and pressure natriuresis [32,33]. It is now evident that the renin–angiotensin system does not exert its sodium regulatory action solely through the stimulation of aldosterone. The fact that aldosterone has a latent period of 60–120 minutes suggests that, contrary to what was once thought, its role becomes increasingly important under chronic physiological and pathophysiological conditions rather than in the acute setting. The intrarenal eﬀects of Ang II are the result of its eﬀects on renal hemodynamics and direct stimulation of sodium transport [32,33]. Ang II has been shown to decrease peritubular capillary pressure and inner medullary blood ﬂow [34]. Thus, alterations in Starling forces across the peritubular capillaries and an increased medullary interstitial tonicity provide renal hemodynamic mechanisms whereby the renin–angiotensin system aﬀects the tubular reabsorption of sodium. Experimental evidence also suggests a direct tubular action of Ang II [35] that can be completely inhibited by an Ang II receptor antagonist [36]. The increased proximal tubule transport, in response to physiological concentrations of Ang II, is mediated by changes in HCO3 coupled with Na+ transport [37]. This eﬀect of Ang II has been attributed to activation of Na+/H+ exchange. Studies in animal models have shown that if the levels of Ang II are driven above those that correspond to a given level of extracellular ﬂuid volume, the organism becomes susceptible to develop hypertension through slow responses to Ang II [29]. DeClue et al. [38] demonstrated that when the circulating levels of Ang II are maintained by a constant infusion of 5 ng/kg/min, blood pressure is strictly determined by the level of sodium intake. Ames et al. [19] also observed in volunteers that a continuous infusion of Ang II suﬃcient to raise and maintain blood pressure 10 mmHg above normal caused a marked sodium retention with weight gain, which was attributable to the antinatriuretic eﬀect of Ang II as well as the concurrent stimulation of aldosterone secretion. After 5 days weight gain, sodium retention leveled oﬀ. As the infusion continued, less and less Ang II was needed to maintain a slight rise in blood pressure. These investigators concluded that the increased sodium retention was mainly responsible for the potentiation of Ang II. The mechanisms underlying potentiation of the vasoconstrictor eﬀect are not well deﬁned. However, its recognition is important because it may explain the development of slow pressor responses, that is, hypertension with normal levels of Ang II. In an attempt to ﬁnd the pressor mechanism responsible for the slow pressor response to Ang II, Hu et al. [24] measured concomitant alterations in renal hemodynamics, electrolyte balance, and the renin system–related substances such as renin concentration, prorenin, and angiotensinogen. They found that the development of the slow pressor responses to renin or Ang II on day 12 of the infusion was accompanied by a signiﬁcant reduction (60%) in renal blood ﬂow while sodium balance shifted to the positive side on days 12 to 14, especially in the animals infused with renin. Furthermore, as mentioned above, in both groups of animals (infused with Ang II or renin), the concentration of angiotensin in plasma was exactly the same. The level of angiotensinogen was not altered, while the prorenin level, which is an indication of the amount of inactive renin released from the kidney, was signiﬁcantly decreased. In brief, this study showed that small amounts of angiotensin infused in normal animals was not enough to chronically increase circulating levels of Ang II in plasma, but was suﬃcient to produce a signiﬁcant elevation of renovascular resistance and sodium retention, which presumably yields a level of extracellular ﬂuid volume inappropriate

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to the activity of the renin–angiotensin system. The concept that the vasoconstrictor eﬀect of Ang II is not only deﬁned by a given plasma level, but rather by the extent to which the volume of extracellular ﬂuid is inappropriately high, is important. According to this, hypertension may be corrected not only by blocking the biological eﬀects of angiotensin, but also by reducing ﬂuid volume down to a level appropriate to the concentration of Ang II, as it occurs after the administration of diuretics [1]. Consistent with these observations are the studies of Hollenberg and colleagues [39], who were the ﬁrst to characterize the eﬀect of angiotensin on renal blood ﬂow and aldosterone secretion with high- and low-sodium diet. The administration of 1 or 10 ng/kg/min (doses of Ang II that neither increase blood pressure nor alter the level of Ang II in blood) produces a signiﬁcant decrease of renal blood ﬂow in humans fed with a high- but not low-sodium diet [39]. Conversely, on a low-sodium diet, adrenal release of aldosterone became very sensitive to Ang II, whereas this response was decreased on a high-sodium diet (Fig. 3). These very interesting studies suggested that patients with an inability to adjust the plasma levels of renin to the existing levels of plasma volume may be prone to develop hypertension. In fact, they found a number of patients with essential hypertension, called ‘‘nonmodulators,’’ who exhibited an enhanced sensitivity of the adrenal cortex to increased aldosterone secretion. These patients were also unable to suppress renin release during sodium overload or Ang II infusion, and it was

Figure 3 Changes in blood pressure (bp), plasma angiotensin II, mean renal blood flow, and plasma aldosterone in response to the intravenous infusion of angiotensin II at 1 and 10 ng/kg/min during high sodium intake (.) and low sodium intake (o).

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found that they had a strong family history of hypertension. These investigators showed later on that this group of essential hypertensives, with inappropriate levels of renin and excessive sodium retention, comprised 45–50% of the hypertensive population. Importantly, Hollenberg et al. [39] found that the hypertension in these patients, a result of their inability to modulate levels of Ang II, was corrected by the administration of a converting enzyme inhibitor. We have suggested that when volume expansion fails to reduce Ang II to levels proportionate to the existing level of ﬂuid volume, the actions of Ang II are not only limited to vasoconstriction and aldosterone stimulation, but can also increase endothelin (ET) expression and the expression of components of oxidative stress. Some of the oxidative stress–dependent eﬀects consist of a decrease in nitric oxide (NO) concentration and an increase in the production of isoprostanes [40]. V.

ANGIOTENSIN AND OXIDATIVE STRESS .

Ang II stimulates superoxide (O2) production through its AT1 receptor and activation of a membrane NADH/NAD(P)H oxidase [30]. Pryor and Squadrito have shown that oxygen free radicals (O2) are constantly being combined with NO to form peroxynitrite (OONO), which is in equilibrium with peroxynitrous acid [41]. Peroxynitrite is a potent oxidant that could oxidize arachidonic acid and thus release a potent renal vasoconstrictor, antinatriuretic substance, 8-iso-prostaglandin F2a (isoprostane). Superoxide can then increase blood pressure via at least two mechanisms: quenching NO and forming isoprostanes. An important observation that links superoxide production to an increased level of Ang II was described by Rajagopalan et al. [30]. This study showed that arteries isolated from rats rendered hypertensive by the administration of a pharmacological dose of Ang II (0.7 mg/kg/day for 5 days) exhibited an impaired relaxation to acetylcholine associated with an increased level of superoxide. These alterations were corrected by pretreating the rats with losartan (an Ang II antagonist) or by treatment of vessels with liposome-encapsulated superoxide dismutase. The role of oxidative stress in Ang II–induced hypertension is further supported by studies conducted with the administration of a superoxide dismutase mimetic (tempol) [43–45], which has been shown to scavenge superoxide anions in vitro [46]. In these studies, Ang II– infused rats had a 100% increase in vascular superoxide production, which was normalized by treatment with tempol. We have shown that oxidative stress can be stimulated by very low doses of Ang II in swine, because infusion of 10 ng/kg/min of Ang II for 28 days signiﬁcantly increases the plasma-free isoprostane F2a, which was proposed as a marker of oxidative stress [42,47]. This eﬀect was not seen in age-matched control animals that were not treated with Ang II. We also described similar ﬁndings in rats receiving 10 ng/ kg/min for 14 days [48]. However, further studies conducted by our laboratory showed that isoprostanes by themselves are probably not responsible for the slow pressor response to Ang II, since bosentan (a nonspeciﬁc endothelin ETA and ETB inhibitor) blocked Ang II–induced hypertension, but did not decrease isoprostanes. This is consistent with the observation that isoprostanes can stimulate production of ET [49]. In any case, these studies suggest that there might be an additional factor or an intermediate step in the development of Ang II hypertension and that one of the mediators may be ET [50]. .

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ANGIOTENSIN AND ENDOTHELIN

As mentioned above, there is strong evidence that ET is involved in the slow responses to angiotensin. However, the role played by ET in other forms of hypertension deserves to be discussed. In this respect, it will be important to bear in mind that the relationship between the renin angiotensin system and ET is reciprocal. That is to say, there are a number of ways through which Ang II and ET inﬂuence each other. These are detailed below. A.

Effect of the Renin–Angiotensin System on Endothelin

Angiotensin II can activate the transcription of preproendothelin by acting via the AT1 receptor in both cultured vascular smooth muscle [51] and endothelial cells [52,53]. This eﬀect may have a signiﬁcant clinical implication, because Ang II is a wellknown promoter of cardiac hypertrophy and ET-1 has been shown to contribute to the hypertrophic response of cardiomyocytes [54]. Furthermore, long-term treatment with ET-1 antagonists greatly improved the survival of rats with chronic heart failure by preventing the increase in left ventricular mass and cavity enlargement [55] as well as the increase in collagen deposition [56]. The possibility that ET is responsible for mediating the pressor response to Ang II was ﬁrst suggested by the studies of Balakrishnan et al. [57]. These investigators found that bosentan pretreatment blunted the Ang II–induced increase in blood pressure and the fall in cardiac output in conscious spontaneous hypertensive rats (SHR) and Wistar Kyoto rats (WKY). However, this hypotensive eﬀect was more pronounced in SHR. An important ﬁnding of this study was that the blunting eﬀect of ET antagonism on the pressor response to angiotensin was more pronounced in rats infused with low (2–10 ng/kg/min) than with high ( > 30 ng/kg/min) doses of Ang II. This preliminary observation suggested that ET plays an important role in potentiating mild increments of angiotensin in peripheral circulation but does not exert this eﬀect when angiotensin plasma level is very high. However, these data are controversial, since there are many studies in which ET antagonism blunts the eﬀects of Ang II even at higher doses. For example, studies by Moreau et al. [58] suggested that ET-1 could play an important role in the structural changes induced by Ang II infusion. The administration of a high dose (200 ng/kg/ min) of Ang II increased medial thickness, the medial-lumen ratio, and the crosssectional area of mesenteric and cerebral arterioles. Furthermore, Ang II doubled the content of ET-1 in mesenteric tissue through a mechanism most likely involving ETA receptors, because the ETA-speciﬁc antagonist LU135252 was capable of preventing the development of these eﬀects [58]. Similarly, a 5-day infusion of Ang II at 0.7 mg/kg/day to Sprague-Dawley rats enhanced immunostaining for ET-1 in vascular smooth muscle. This eﬀect was abolished by the administration of an ETA-selective antagonist, PD155080, as well as the AT1 receptor antagonist losartan [59]. The beneﬁcial eﬀect of bosentan in preventing the development of hypertension, the reduction in renal blood ﬂow, and the increase in albuminuria and heart weight during a 10day infusion of 200 ng/kg/min of Ang II has also been demonstrated by Herizi et al. [60]. Bosentan has also been found to exert a cumulative hypotensive eﬀect, with losartan, in a canine model of hypertension (kidney wrapping) [61] and other forms of renin-dependent hypertension [62]. These ﬁndings are consistent with studies reporting that in models of renovascular hypertension induced by clipping one renal artery,

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the administration of an orally active endothelin antagonist, Ro-00203, attenuated the increase in blood pressure without aﬀecting renin expression or secretion [63]. In a recent study conducted by Ortiz et al. [50], it was suggested that subpressor doses of Ang II enhance either the local formation or the activity of ET directly or stimulate oxidative stress, causing an increase in plasma isoprostane levels. With AT1 receptor blockade using losartan, Ang II–induced hypertension was abolished, isoprostanes were decreased, and urinary nitrite excretion increased (suggesting that oxidative stress fell and the bioavailability of NO was enhanced). As mentioned before, ET blockade with bosentan in this model prevented Ang II-induced hypertension without altering isoprostane levels and urinary nitrite excretion. Thus, ET antagonism did not decrease oxidative stress or shift the balance between NO and oxidative stress in favor of NO. Taken together, these data suggest that Ang II increases oxidative stress, causing a subsequent rise in plasma isoprostanes, which, in turn, may stimulate the local production of ET, inducing an ET-dependent hypertension. B.

Model with High Salt Intake

In contrast with the controversial data on the involvement of ET-1 in renindependent forms of hypertension, clear data exist regarding the role of this peptide in mineralocorticoid-dependent forms of hypertension [64]. Increased levels of ET-1 were found both in plasma and in the arterial wall of rats with deoxycorticosterone acetate (DOCA) salt–induced hypertension [49,65,66]. Elegant in situ hybridization studies by Day et al. [67] showed enhanced expression of the preproendothelin mRNA in the vessel wall in the same model. Treatment with bosentan was quite eﬀective in preventing vascular hypertrophy in this model. This ﬁnding further supports to the idea of a direct involvement of ET-1 in target organ damage in low-renin forms of hypertension [68]. The contention that ET-1 plays an important role in salt-dependent forms of hypertension is further supported by the ﬁndings of Barton et al., who reported that the ETA-speciﬁc antagonist, LU135252, partially prevented the development of hypertension and the structural and functional alterations caused by chronic salt administration in Dahl salt-sensitive rats [69]. A role for ET-1 in cardiovascular damage of salt-sensitive hypertension is suggested also by data obtained in humans [70]. C.

Effects of Endothelin on the Renin–Angiotensin System and Aldosterone

These ﬁndings are quite interesting because there are studies showing there is a signiﬁcant eﬀect of ET on the adrenal gland (this issue has been studied by Nussdorfer). It is known that there are abundant speciﬁc ETA and ETB receptors in human and animal adrenal zona glomerulosa [71–73]. ET appears to regulate the transcriptional statement of the aldosterone synthase gene in normal human adrenal cells. Furthermore, in dispersed normal human adrenal cortical zona glomerulosa cells, ET-1 was found to be equipotent with Ang II [74]. Thus, in vivo administration of ET is expected to produce, at low doses, sodium retention, stimulation of aldosterone, and suppression of renin release. It should be mentioned, however, that ET can suppress renin release directly, which has been shown to occur in vitro in juxtaglomerular cells as well as in vivo [75].

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SUMMARY

Most studies support a strong participation of ET in mediating or potentiating the vasoconstrictor eﬀect of angiotensin in a number of situations. These are secondary forms of hypertension, which are mediated through the renin-angiotensin system, such as renovascular and malignant forms of hypertension. An important animal model of hypertension obtained by the infusion of small doses of angiotensin, which evoke slow responses, also appears to be mediated by ET. The studies of Ortiz have been important in determining that angiotensin may stimulate oxidative stress, which per se or through the production of isoprostanes may stimulate the release of ET [50]. In fact, this sequence of events is supported by ﬁndings from the same investigator showing that blockade of angiotensin with an angiotensin antagonist, or oxidative stress with tempol, reduces blood pressure. Interestingly, blockade of ET with bosentan produces the same eﬀect on blood pressure, although this latter maneuver is not accompanied by any alterations in oxidative stress. These studies show that isoprostanes by themselves are not responsible for the slow responses to Ang II, but rather that ET may play a role as a cofactor in the sequence of events that triggers hypertension with low plasma levels of Ang II.

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69. Barton M, D’Uscio LV, Shaw S, Meyer P, Moreau P, Luscher TF. ETA receptor blockade prevents increased tissue endothelin-1, vascular hypertrophy, and endothelial dysfunction in salt-sensitive hypertension. Hypertension 1998; 31:499–504. 70. Ferri C, Bellini C, Desideri G, Mazzocchi C, De Siati L, Santucci A. Elevated plasma and urinary endothelin-1 levels in human in salt-sensitive hypertension. Clin Sci (Colch) 1996; 93:35–41. 71. Koseki C, Imai M, Hirat Y, Yanagisawa M, Masaki T. Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide? Am J Physiol 1989; 256: R858–R866. 72. Imai T, Hirata Y, Eguchi S, Kanno K, Ohta K, Emori T, Sakamoto A, Yanagisawa M, Masaki T, Marumo F. Concomitant expression of receptor subtype and isopeptide of endothelin by human adrenal gland. Biochem Biophys Res Commun 1992; 182: 1115–1121. 73. Cozza EN, Gomez Sanchez CE, Foecking MF, Chiou S. Endothelin binding to cultured calf adrenal zona golmerulosa cells and stimulation of aldosterone secretion. J Clin Invest 1989; 84:1032–1035. 74. Rossi GP, Albertin G, Bova S, Belloni AS, Fallo F, Pagotto U, Trevisi L, Palu G, Pessina AC, Nussdorfer GG. Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca in human adrenocortical carcinoma. Endocrinology 1997; 138:4421–4426. 75. Rossi GP, Sacchetto A, Cesari M, Pessina A. Interactions between endothelin-1 and the renin-angiotensin-aldosterone system. Cardiovasc Res 1999; 43:300–307.

25 Oxidative Stress in Cardiovascular Disease: Role of Oxidized Lipoproteins in Macrophage Foam Cell Formation and Atherosclerosis MICHAEL AVIRAM and MIRA ROSENBLAT The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel

I. A.

OXIDATIVE STRESS AND ATHEROSCLEROSIS Risk Factors for Atherosclerosis

Atherosclerosis is the major cause of morbidity and mortality in the western world. It is a multifactorial disease, and it is also considered as a form of chronic inﬂammation resulting from complex interactions between modiﬁed lipoproteins, monocyte-derived macrophages, T cells, and cellular elements of the arterial wall [1–3]. This inﬂammatory process leads to the development of complex lesions, which grow suﬃciently large to block blood ﬂow. Plaque rupture and thrombosis results in acute clinical complications of myocardial infarction and stroke [3]. Recruitment of macrophages and their subsequent uptake of low-density lipoprotein (LDL)–derived cholesterol are the major cellular events contributing to fatty streak formation in early atherogenesis [1–4]. The events of atherosclerosis have been greatly clariﬁed by studies in animal models. Mice deﬁcient in apolipoprotein E (apo E) or in the LDL receptor develop advanced atherosclerotic lesions, which have many features in common with human lesions [5]. Epidemiological studies revealed numerous risk factors for atherosclerosis. They can be grouped into factors with an important genetic component and factors that are largely environmental. The genetic risk factors include elevated levels of LDL or very-low-density lipoprotein (VLDL) [6], reduced levels of high-density lipoprotein 557

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(HDL) [7], elevated levels of lipoprotein (a) [Lp(a)] [8], elevated levels of homocysteine [9] and increased levels of ﬁbrinogen, plasminogen activator inhibitor type 1, and platelet reactivity [6]. Hypertension [6,10], diabetes, obesity [6] gender [11], and rheumatoid arthritis [12] are also associated with accelerated atherosclerosis. If several risk factors exist, their eﬀect is not simply additive; for example, the eﬀects of hypertension on coronary heart disease (CHD) are considerably ampliﬁed if cholesterol levels are high [13]. When all known risk factors are controlled, family history remains a very signiﬁcant independent factor [14]. The environmental risk factors include high-fat diet [6], smoking [6], infectious agents [15], and lack of physical exercise [6]. Thus, the common forms of CHD result from the combination of an unhealthy environment, genetic susceptibility, and increased life span. B.

Atherosclerosis Heterogeneity and Genetic Background

Molecular cardiology is changing our concepts of atherosclerosis development, disease etiology, pathophysiology, and therapy [16]. The understanding of the genetic basis, or the molecular origin, of a disorder and the insight into the way genetic information is expressed or modulated by the environment will have a major impact on treatment and prevention of the disease. Multiple genes are involved in the genetic predisposition for atherosclerotic disease, but the role of only a few of these genes is known. Far less is known about gene–gene and gene–environment interactions. Once the trigger for atherosclerosis has initiated disease development, various genes are activated or silenced and contribute to lesion progression. Every stage of lesion development depends on a diﬀerent gene expression programe. In some families disease can be explained mostly by a single, major gene (monogenic). In other cases, one or several variations in minor genes (multigenic) contribute to atherosclerosis development. 1.

The LDL Receptor Gene

The gene coding for LDL receptor has been extensively characterized. Mutations in this gene cause an inherited disorder called familial hypercholesterolemia (FH). FH is one of the most frequent autosomal dominant inherited disorders. Diagnosis of FH can be conﬁrmed by demonstration of a mutation in the LDL receptor gene, and today more than 500 diﬀerent mutations, have been identiﬁed [16]. Because of a diminished number of active LDL receptors on the liver cell surface in the case of the heterozygous form, or a complete absence of LDL receptors in the homozygous form, the LDL in plasma is severely elevated. 2.

The Lp(a) Gene

The gene that codes for apolipoprotein A, the main protein constituent of this particle, is highly polymorphic. The gene contains a varying number of speciﬁc protein domains called Kringle IV repeats, and the number of repeats directly determines the levels of Lp(a): the more Kringle IV repeats, the lower the plasma level of Lp(a). Over a level of approximately 300 mg/L, Lp(a) is an independent risk factor for cardiovascular disease [8]. 3.

Homocysteine

Elevated levels of homocyteine in plasma have recently been found to be a risk factor for atherosclerosis [9]. Homocysteine is an amino acid that is intermediate in the conversion of methionine to cysteine. The plasma level of homocysteine is dependent

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on the dietary intake of methionine and on the activity of the methionine- and homocysteine-converting enzymes methylenetetrahydrofolate reductase and cystathionine-h-synthetase [16]. Mutations in the genes encoding for these enzymes have been shown to increase homocysteine levels [16]. The activity of these enzymes is regulated by the cofactors vitamin B6 and folic acid, and a deﬁciency of these cofactors consequently leads to elevated homocysteine levels in plasma [9,16]. 4.

The Angiotensin-Converting Enzyme Gene

The DD genotype of angiotensin-converting enzyme (ACE) was recently identiﬁed as an additional risk factor for atherosclerosis [1,4,16]. The ACE gene is positioned on the long arm of choromosome 17. In the ACE gene a deletion of 287 bp (DD) was found in intron 16, which appeared to be associated with increased risk for myocardial infarction and restenosis following precutaneous coronary angiography (PTCA) [16]. C.

Theories of Atherogenesis

Although it is well documented that elevated levels of LDL cause an increased rate of atherosclerosis, the molecular and cellular mechanisms for the pathobiological changes that lead to the disease are still poorly understood. The ‘‘response-to-injury’’ hypothesis proposes that the initial event in atherosclerosis is injury to the endothelium [17]. The injury may result from high concentrations of lipoprotein (LDL,VLDL) Lp(a), oxidized LDL (Ox-LDL), or from inﬂammation caused by bacteria or viruses (e.g., cytomegalovirus, Chlamydia). According to this theory, physiologically active substances such as platelet-derived growth factor (PDGF), macrophage colonystimulating factor (M-CSF), and monocyte-derived cytokines are released in response to endothelium injury, and these substances induce pathological reactions in the cells of the arterial wall. Most of the accumulated cholesterol in foam cells originates from plasma LDL, which is internalized into the cells via the LDL receptor. However, native LDL does not induce cellular cholesterol accumulation, since the LDL receptor activity is down-regulated by cellular cholesterol content [18,19]. Therefore, alternative mechanisms are needed to explain the accumulation of cholesterol in macrophage foam cells. The ‘‘response-to-retention’’ hypothesis proposes that when the levels of circulating LDL are raised, LDL diﬀuses passively through endothelial cell junctions, and its retention in the vessel wall seems to involve interactions between the LDL apolipoprotein B (apo B) and the extracellular matrix (ECM) proteoglycans [20,21]. This interaction can be a direct one, between glycosaminoglycans and apolipoproteins, or it may occur via lipoprotein lipase bridging [22]. Proteoglycan-bound LDL forms aggregates, and aggregation of LDL stimulates its uptake by macrophages, via phagocytosis, thus converting macrophages into foam cells [23,24]. Furthermore, the retained LDL is more susceptible to oxidation [25]. The oxidative modiﬁcation hypothesis of atherosclerosis proposes that LDL oxidation plays a pivotal role in early atherogenesis [26–34]. This hypothesis is supported by evidence that LDL oxidation occurs in vivo. D.

Evidence for the Existence of Ox-LDL In Vivo

Using speciﬁc antibodies against oxidized LDL (Ox-LDL), it was recently demonstrated that elevated levels of circulating Ox-LDL exist in human plasma of patients

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with cardiovascular diseases, and these levels show a positive relationship with the severity of acute coronary syndromes [35,36]. The presence of Ox-LDL was also demonstrated in atherosclerotic lesions from humans and animals [37–39]. Patients at high risk for atherosclerosis (e.g., hypercholesterolemic, hypertensive, diabetic, and renal failure patients) exhibit an increased susceptibility of their LDL to lipid peroxidation [40–43]. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in cholesterol synthesis. Hypocholesterolemic drug therapy, including HMG-CoA reductase inhibitors such as atorvastatin, ﬂuvastatin, lovastatin, and the hypotriglyceridemic drugs bezaﬁbrate and gemﬁbrozil, signiﬁcantly reduced the enhanced susceptibility to oxidation of the LDLs isolated from hyperlipidemic patients [44–48]. Recently, it was shown that the antioxidative properties of ﬂuvastatin also contribute to the prevention of atherosclerosis in cholesterol-fed rabbits [49]. In hypertensive patients the increased levels of angiotensin II (Ang-II) may be associated with increased LDL oxidation and atherosclerosis [50]; thus, ACE inhibitors or Ang-II receptor antagonist therapy may be beneﬁcial in these patients. Captopril and enalapril therapy indeed reduced the susceptibility of the patients’ LDL to copper ion–induced oxidation [41]. ACE inhibitor (captopril, ramipril, fosinopril) or Ang-II receptor antagonist (losartan) administration to apo E–deﬁcient mice reduced both LDL oxidation and the development of atherosclerotic lesions [51– 54], and a combination of ACE inhibitors and Ang-II receptor blockers acted synergistically [53]. Another approach to reduce oxidative modiﬁcation of LDL is the use of antioxidants. Epidemiological studies linking dietary antioxidant consumption with a reduction in cardiovascular disease showed an inverse association between the intake of various fruits and vegetables and the mortality from coronary heart diseases [55–57]. The increased LDL oxidation state and its propensity to oxidation in diabetic patients were signiﬁcantly reduced (to normal levels) following 3 weeks of supplementation with natural h-carotene [58]. Furthermore, in healthy subjects we have also demonstrated an inhibitory eﬀect of h-carotene on the susceptibility of LDL to oxidation [59,60]. In kidney transplant recipients, dietary selenium supplementation reduced the enhanced susceptibility to lipid peroxidation of their plasma and LDL [61]. Polyphenols and ﬂavonoids are extremely powerful antioxidants [56,62]. Oxidation of LDL was inhibited in vitro, as well as in vivo, in humans after dietary consumption of several nutrients rich in ﬂavonoids, including red wine [63], licorice root extract and its major polyphenol glabridin [64], olive oil [65], green tea [66], and pomegranate juice [67]. Supplementation with red wine or its ﬂavonoids quercetin or catechin [68], licorice or glabridin [63], ginger extract [69], and pomegranate juice [66,70] resulted in reduced susceptibility of LDL to oxidation, decreased basal LDL oxidative state, and attenuation in the development of atherosclerotic lesions in apo E–deﬁcient mice. E.

Atherogenic Properties of Ox-LDL

Ox-LDL has several atherogenic and pro-inﬂammatory properties [71–73]. Among the various atherogenic properties of oxidized LDL, stimulation of macrophage cholesterol accumulation is of major importance in the development of atherosclerosis. Ox-LDL receptors, unlike the native LDL receptor, are not regulated by cellular cholesterol levels, and therefore uptake of Ox-LDL via these receptors—scavenger

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receptor A (SRA) and CD-36—leads to cholesterol accumulation and foam cell formation [74,75]. Ox-LDL is also chemoattractant for monocytes, and minimally oxidized LDL increases the adherence and penetration of circulating monocytes from the blood stream to the subendothelial space by inducing endothelial cell production of the potent monocyte chemoattractant protein-1 (MCP-1) [76]. Moreover, Ox-LDL was demonstrated to induce the expression of the adhesion molecules ICAM-1 and VCAM-1 [77]. Minimally oxidized LDL can stimulate the release of M-CSF from endothelial cells, which facilitate monocyte diﬀerentiation into tissue macrophages [78]. In addition, Ox-LDL also inhibits the migration of tissue macrophages back to the circulation [79]. After binding to the macrophage scavenger receptors, Ox-LDL initiates a series of intracellular events that include the induction of inﬂammatory cytokines such as the interleukins (IL-8, IL-1, and IL-6) [80,81]. Ox-LDL also results in cell injury by necrotic and apoptotic pathways, both in vitro and in vivo [82–85]. Ox-LDL induces migration and proliferation of smooth muscle cells, impedes endothelial cell migration, intereferes with endothelium-mediated relaxation, and promotes procoagulant properties of vascular cells [86,87]. F.

Mechanisms for LDL Oxidation

Oxidation of LDL involves free radicals attack on the lipoprotein components, including its cholesterol, phospholipids, fatty acids, and apolipoprotein B-100 (apo B-100) moieties. LDL oxidation starts by the consumption of its antioxidants (mainly vitamin E and carotenoids) [89]. After depletion of LDL from its antioxidants, transition metal ions catalyze propagation reactions, breakdown of lipid hydroperoxides, and the formation of aldehydes, which are responsible for apolipoprotein B100 modiﬁcation. LDL cholesterol is converted during oxidation to oxysterols, such as 7-hydroxycholesterol and 7-ketocholesterol [84,90,91]. The polyunsaturated groups of esteriﬁed cholesterol and phospholipids are also major targets for oxidation [88,89], and the primary oxidation products formed are hydroperoxides, which can undergo subsequent reduction to hydroxides and aldehydes. Nonenzymatic peroxidation of arachidonic acid results in the formation of isoprostanes [92]. During LDL oxidation, the apo B-100 molecule is also modiﬁed, with the formation of oxidized amino acid side chains and fragmentation of the polypeptide backbone. In addition, short-chain aldehydes (e.g., malondialdehyde and hydroxynonenal) can form stable adducts with the amino acid residues of the LDL apo B-100 [89]. All these reactions result in changes in the LDL structure, and the formed Ox-LDL cannot bind to the LDL receptor, but rather interacts with the macrophage scavenger receptors, leading to macrophage accumulation of cholesterol and oxidized lipids and to foam cell formation. The process of LDL oxidation is unlikely to occur in plasma because of the high concentrations of antioxidants and metal ions chelators. LDL oxidation is more likely to occur within the artery wall, an environment depleted of antioxidants [93–95]. The identity of the cells responsible for LDL oxidation in the arterial wall is uncertain. Early studies focused on LDL oxidation by endothelial cell (EC) [96,97] and by smooth muscle cells (SMC) [96], which require the presence of transition metal ions. Both copper and iron ions were found in atherosclerotic lesion [98] and copper ions are more potent in inducing LDL oxidation [99]. Reduction of ceruloplasmin copper by cell-derived superoxide anions was found to be critical for LDL oxidation

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mediated by EC or SMC [100]. LDL oxidation by monocyte-derived macrophages [101–103] has a major role during the early stages of atherogenesis. These cells are prominent in early arterial lesions and generate reactive oxygen/nitrogen species [104,105]. Macrophages also express ceruloplasmin mRNA and protein [106] and activated macrophages are capable of oxidizing LDL even in media free of transition metal ions. II.

MACROPHAGE-MEDIATED OXIDATION OF LDL

LDL can be oxidized in vitro by isolated monocytes, monocyte-like cell lines, and macrophages [107]. LDL cholesterol is signiﬁcantly oxidized after about 5 hours of incubation with macrophages, forming cholesterol ester core aldehydes and oxysterols [108,109]. Using antibodies to the macrophage LDL receptor or to the LDL receptor–binding domains on the LDL apo B-100, we showed that binding of LDL to the LDL receptor is required for cell-mediated oxidation of LDL [110]. Other studies, however [111,112], suggested that lipoprotein–receptor interactions are not absolutely required, as macrophages from LDL receptor-deﬁcient mice were still able to oxidize LDL. Several biological and biochemical mechanisms were suggested to be involved in macrophage-mediated oxidation of LDL [113], including metal ions, superoxide anions, NADPH oxidase, lipoxygenases, myeloperoxidase, and reactive nitrogen species. A.

Role of Transition Metal Ions

Cell incubation with LDL requires the presence of iron or copper ions for LDL oxidation [114–116]. One mechanism for this is the ability of macrophages to reduce these transition metals [117,118] to Fe (II) or to Cu (I), which then rapidly react with lipid hydroperoxides, leading to the formation of reactive lipid radicals and to the conversion of the reduced metal back to its oxidized form. Recently, it was demonstrated that both thiol-dependent and thiol-independent pathways for transition metal reduction are enhanced by protein kinase C (PKC) activation [118]. Iron-containing proteins such as ferritin, transferrin, hemoglobin, myoglobin, or the hemin moiety itself, as well as copper-containing proteins such as ceruloplasmin, were suggested as ‘‘physiological’’ sources for iron and copper [119,120], inducing LDL oxidation [121– 123]. Monocytes-macrophages express ceruloplasmin mRNA and protein [106], and cell-derived ceruloplasmin was shown to contribute to LDL oxidation by these cells [124]. A neutralizing antibody to ceruloplasmin blocked LDL oxidation by activated U937 macrophage cell line, as did antisense targeted against segments of the ceruloplasmin mRNA [124]. B.

Role of NADPH Oxidase

The role of superoxide anion in macrophage-mediated LDL oxidation is under debate. Unstimulated cells produce small amounts of superoxide anions and contribute minimally to cell-mediated LDL oxidation. Activation of the cells with phorbol myristate acetate (PMA), however, results in an increase in both superoxide anion production and LDL oxidation [125–129]. Superoxide is required for the initiation of LDL oxidation, whereas propagation of LDL oxidation was shown to be superoxide independent [125]. It was speculated that superoxide dismutase (SOD) could be

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inhibitory by acting as a metal chelator in addition to its superoxide scavenging capability [130]. In macrophages, the predominant source of superoxide is the NADPH oxidase system [131]. This enzyme is composed of at least ﬁve subunits: two cytosolic components, p47phox and p67phox; two membrane-bound components, p22 phox and gp91phox (which together compose the cytochrome b558); and a G protein, rac-1 [132]. We have demonstrated that LDL in the presence of copper ions activates the NADPH oxidase complex, as PMA does in human monocyte–derived macrophages (HMDM), causing the translocation of p47phox from the cytosol to the macrophage plasma membrane, thus forming the active enzyme complex [128]. Using PKC inhibitors, its involvement in NADPH oxidase activation and macrophagemediated oxidation of LDL was demonstrated [127]. Further evidence for the role of superoxide anions in the oxidation of LDL by macrophages was obtained using cells from patients with chronic granulomatous disease (CGD), who lack active NADPH oxidase. PMA-stimulated cells from these patients neither generated superoxide anions nor oxidized LDL [128]. Recent studies using in vitro knockout of p47phox by antisense showed that both superoxide anion and LDL oxidation were inhibited [133]. Other studies, however, suggested that NADPH oxidase may not be essential for LDL oxidation by macrophages [134,135], as deﬁciency in the gp91 in mice macrophage membrane component failed to inhibit atherosclerosis progression [135]. However, there is no reason to assume that only one cell type or only one enzyme source of free radicals is important for the oxidation of lipids. In the case of severe hypercholesterolemia, as produced in cholesterol-fed mice or apo E–deﬁcient mice, it is quite likely that diﬀerent ROS/RNS, derived from several diﬀerent sources, can react with lipoproteins that accumulate in the vessel wall. It is possible that under normal conditions, superoxide (and other free radicals) derived from NADPH oxidase activation contribute to lipid peroxidation, but when the enzyme is absent, other sources, such as smooth muscle cells (which contains mox1 instead of gp91phox), are perfectly capable of producing ROS/RNS and of oxidizing LDL. C.

Role of Myeloperoxidase

Another oxygenase that may participate in phagocyte-dependent oxidation of LDL is myeloperoxidase (MPO). MPO is an abundant heme protein released by activated neutrophils and monocytes, and it is present in tissue macrophages such as those found in vascular lesions [136]. MPO may play a role in monocyte-macrophage oxidation of LDL [137] by a variety of distinct pathways [138–144]. MPO can act to amplify the oxidizing potential of H2O2 by using it as a co-substrate to generate a variety of oxidants, including diﬀusible reactive oxygen species (ROS) [144], reactive halogens [137,142], aldehydes [139,142,145–147], and nitrating agents [141,148,149]. MPO-mediated oxidation reactions occur in the absence of transition metal ions and are resistant to inhibition by chelators. Because of its high concentration in biological matrices, chloride is regarded as a major substrate for MPO [138,150–152]. MPO catalyzes the two-electron oxidation of chloride, forming the powerful oxidant, hypochlorous acid (HOCL). Exposure of LDL to HOCL resulted in chlorination and oxidation of protein and lipid constituents of LDL, induces LDL aggregation, and converts the lipoprotein into a high-uptake form by macrophages [137,138,151]. MPO can catalyze the one-electron oxidation of L-tyrosine, generating the tyrosyl radical.

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PMA-activated phagocytes produce tyrosyl radical and initiate LDL lipid peroxidation and dityrosine cross-linking of proteins [145]. Recent studies have identiﬁed another potential MPO-dependent pathway that involves the formation of reactive nitrogen species (RNS) [153]. MPO can use H2O2 and nitrite to generate a reactive intermediate capable of nitrating aromatic compounds and tyrosine residues [148,154]. Recent studies demonstrate that exposure of LDL to nitrite or to human monocytes resulted in LDL lipid peroxidation and in protein nitration [154]. Moreover, LDL modiﬁed by MPO-generated nitrating intermediates is taken up by macrophages at an increased rate, resulting in cholesteryl ester accumulation and foam cell formation [154]. D.

Role of Lipoxygenases

Lipoxygenases (LO) are nonheme iron–containing enzymes found in various cells, including reticulocytes, monocytes-macrophages, and certain endothelial cells [155, 156]. They catalyze the direct insertion of molecular oxygen into polyenoic fatty acids, forming hydroperoxides. There are a number of ways by which LO could participate in LDL oxidation. LO could oxidize cellular fatty acid, cholesteryl ester, or phospholipid substrates, and the hydroperoxide products could then be transferred to LDL, making the LDL more susceptible to oxidation [157]. If LO is brought in contact with LDL, it could use the LDL phospholipids or cholesteryl ester moieties as substrates [158], leading to lipid peroxidation. The 12/15-lipoxygenase, which is present in monocytes-macrophages, contributes to cell-mediated LDL oxidation [159– 162]. Incubation of LDL with 12/15-lipoxygenase led to a signiﬁcant oxidation of LDL [160,163], and lipoxygenase inhibitors can greatly diminish the ability of macrophages to oxidatively modify LDL [157,161,162]. It was recently demonstrated that 12/15-lipoxygenase overexpression in mouse macrophage-like J774A. 1 cells was involved in the oxidation of LDL, and secretion or leakage of the 12/15-lipoxygenase to the medium was ruled out [159]. In a recent study it was shown that the LDL receptor–related protein is required for macrophage-mediated oxidation of LDL by 12/15-lipoxygenase [164]. The involvement of 12/15-lipoxygenase in LDL oxidation in vivo was suggested because 15-lipoxygenase mRNA and protein colocalized with epitopes of Ox-LDL in macrophage-rich areas of the atherosclerotic lesions [165]. Furthermore, a disruption of the 12/15-lipoxygenase gene diminished lipid peroxidation and atherosclerosis in apo E–deﬁcient mice [166,167], whereas overexpression of 12/15-lipoxygenase facilitated atherosclerosis in the LDL receptor–deﬁcient mice [168]. E.

Role of Nitric Oxide

The role of nitric oxide (NO) and reactive nitrogen species derived from NO in LDL oxidation is an area of intense research. NO is a long-lived free radical [169] formed by multiple inﬂammatory and vascular cells including monocyte and macrophages [170,171], and the expression of nitric oxide synthase was demonstrated in human coronary atherosclerotic plaques [172]. Both pro- and antioxidant eﬀects of NO have been proposed [173–175]. NO has an unpaired electron in the highest orbital, and thus it behaves as a potential antioxidant agent by virtue of its ability to reduce other molecules [174]. Nitric oxide acts as a potent inhibitor of lipid peroxidation chain reaction by scavenging propagatory lipid peroxyl radicals. In addition, nitric oxide

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can also inhibit many potential initiators of lipid peroxidation, such as peroxidase enzymes [173–176]. Enhanced production of NO by monocytes-macrophages exerted an antioxidant eﬀect, attenuating the extent of cell-mediated oxidation of LDL [177]. However, when endogenous tissue rates of NO production are accelerated, or when tissue oxidant defenses become depleted, NO gives rise to secondary oxidizing species that can initiate membrane and lipoprotein lipid peroxidation [178]. NO reacts with superoxide to form peroxynitrite (ONOO-) [179,180], and apocynin, an NADPH oxidase inhibitor, inhibited the formation of both superoxide and peroxynitrite by macrophages [181]. Peroxynitrite is a potent agent to induce LDL oxidation [182–186], resulting in the consumption of LDL-carotenoids and tocopherol [183] and in the formation of oxysterols [184] and isoprostanes [185]. Peroxynitrite modiﬁcation of LDL leads to its recognition by the macrophage scavenger receptors [186,187], and there is evidence for the existence of peroxynitrite-modiﬁed LDL in human [188,189] and in rabbit atherosclerotic lesions [189]. F.

LDL Oxidation by Lipid Peroxide–Rich Cells (‘‘ ‘‘ ‘‘Oxidized Macrophages’’ ’’ ’’)

Under oxidative stress not only does lipoprotein lipid peroxidation take place, but peroxidation of cellular lipids and the formation of oxidized phospholipids, isoprostanes, and oxysterols in atherosclerotic lesions [190,191], including arterial macrophages [192], also occurs. Macrophage lipid peroxidation under oxidative stress can aﬀect their ability to oxidize LDL. It was indeed demonstrated that macrophages from atherosclerotic lesions, as well as macrophages that were lipid peroxidized in vitro (by incubation with iron ions or with Ang-II), oxidize LDL at enhanced rate, even in the absence of transition metal ions [193–196]. Recently, we demonstrated the role of macrophage oxysterols in cell-mediated oxidation of LDL [197]. Macrophages from apolipoprotein E–deﬁcient mice that contain speciﬁc oxysterols (e.g., 7-ketocholesterol, h-epoxycholesterol, 7h-hydroxycholesterol) or macrophages from control mice (C57BL6) that were enriched with these oxysterols (in vitro) demonstrated increased ability to oxidize LDL [197]. In these cells we found increased amounts of arachidonic acid (AA) and superoxide anions [197]. Macrophage oxysterols induce translocation of p47phox from the cytosol to the cell’s plasma membrane [197], and in the oxysterolrich macrophages increased activity of protein kinase C (PKC) was shown [197]. Because both PKC and AA participate in NADPH-oxidase activation [198,199], we proposed a mechanism for oxysterol-induced macrophage-mediated oxidation of LDL (Fig. 1). Macrophage’s oxysterols activate the enzyme phospholipase A2, which in turn acts on membranal phospholipids to release AA. AA stimulates PKC, and PKC phosphorylates p47phox and induces its translocation to the plasma membrane. The active NADPH-oxidase complex can then convert molecular oxygen into superoxide anion, which is further transformed to more reactive oxygen species, resulting in the oxidation of LDL and initiating atherosclerosis. G.

Role of Cellular Antioxidants

The extent of LDL oxidation by macrophages is determined by the balance between cellular oxygenases and antioxidants [4,200,201]. Reduced glutathione (GSH) is the major antioxidant in cells [202]. We found that GSH content and glutathione peroxidase (GPx) activity (which reduces lipid peroxides to alcohols) are both

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Figure 1

Proposed mechanism for oxysterol-induced NADPH oxidase activation and macrophage-mediated oxidation of LDL. Macrophage oxysterols activate phospholipase A2 (PLase A2) (a). PLase A2 acts on membrane phospholipids (b); releasing arachidonic acid (AA) (c). AA stimulates protein kinase C (PKC) (d). AA can also activate the cytosolic component of NADPH oxidase p47phox directly (e). PKC phosphorylates p47phox and activate it (f). Both processes (e and f) stimulate the translocation of p47phox to the macrophage plasma membrane, where it can form the active NADPH-oxidase (NADPH-Ox) complex (g). Active NADPH-Ox converts molecular oxygen into superoxide anion (h). Released superoxide anions are converted under oxidative stress to reactive oxygen species (ROS) and reactive nitrogen species (RNS) (i); and ROS/RNS can finally oxidize native LDL to form the atherogenic oxidatively modified LDL (Ox-LDL) (j).

inversely related to macrophage-mediated oxidation of LDL [203]. Upon incubation of J774A.1 macrophages with 0.05 mM of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, cellular GSH content and GPx activity were reduced by 89% and 50%, respectively, and this eﬀect was associated with a twofold elevation in macrophage-mediated oxidation of LDL [203]. The BSO-treated cells contained high levels of peroxides and released more superoxide anions than nontreated cells. In order to increase macrophage GSH content, we used L-2-oxothiazolidine-4carboxylic acid (OTC), which delivers cysteine residues to the cells for GSH synthesis. GSH content and GPx activity in J774A.1 macrophages were increased by 80% and 50%, respectively, following cell incubation with 2 mM of OTC for 20 hours, and this was paralleled by 47% inhibition in LDL oxidation by these cells [203]. Upon incubation of macrophages with selenomethionine (10 ng/mL) for 1 week, cellular GSH content and GPx activity were increased by about twofold as compared to control cells, and this eﬀect was associated with a 30% reduction in cell-mediated oxidation of LDL [203]. Dietary selenium supplementation (1 Ag/day/mouse) to apo E–deﬁcient mice for 3 months also increased GSH content and GPX activity in peritoneal macrophages (MPM), and this eﬀect was associated with a reduction in cell-

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mediated oxidation of LDL and with 30% reduction in atherosclerotic lesion size in these mice, compared to nontreated mice [203]. Recently we demonstrated that peritoneal macrophages from apo E–deﬁcient (E0) mice contain decreased GSH levels (by 58%) and fourfold increased lipid peroxide content compared to macrophages from C57BL6 control mice. The E0 MPM demonstrated an increased ability to release superoxide anions and to oxidize LDL [204]. OTC supplementation (500 mg/kg/d) to E0 mice resulted in a 26% increase in MPM GSH content, paralleled by a 25% reduction in cellular lipid peroxide content. Decrement by about 30% in superoxide anion release and LDL oxidation and also in the atherosclerotic lesion size were found in OTC-treated mice compared to the control untreated mice [204]. An additional way to increase cellular GSH was achieved by the use of dietary antioxidants. Vitamin E (40 mg/kg/d) or the isoﬂavan glabridin (25 Ag/kg/d) administration for 2 months to apo E–deﬁcient mice resulted in the accumulation of these antioxidants in their MPM and increased macrophage GSH content by 24% and 80%, respectively, followed by reduced cell-mediated oxidation of LDL [204]. In the above study [204], we have demonstrated that vitamin E reduced oxidized glutathione and GSH together with vitamin E act synergistically in the scavenging of free radicals. Furthermore, in vitamin E–enriched MPM from apo E–deﬁcient mice, reduced levels of oxysterols were found, and this resulted in the inhibition of NADPH oxidase activation and in cell-mediated oxidation of LDL [197,205]. In vitro, however, vitamin E supplementation to macrophages does not inﬂuence their ability to oxidize LDL [206]. The eﬀect of dietary carotenoids (such as h-carotene or lycopene) on MPM-mediated LDL oxidation was also studied [207,208]. In one study, h-carotene inhibited–HMDMmediated LDL oxidation [207], but in other studies both h-carotene and lycopene had no signiﬁcant eﬀect [59,208]. Flavonoids are an additional group of antioxidants that accumulate in the macrophages and aﬀect their capability to oxidize LDL [209]. Consumption of nutrients rich in ﬂavonoids, such as pomegranate juice [66] and ginger extract [69], or in puriﬁed ﬂavonoids, such as catechin, quercetin [68], or glabridin [210], by the apo E–deﬁcient mice resulted in a signiﬁcantly reduced capacity of their harvested macrophages to oxidize LDL. Glabridin that accumulates in the macrophage plasma membrane inhibited cell-mediated oxidation of LDL via the inhibition of superoxide anion release and attenuation of macrophage NADPH oxidase activation [210]. Glabridin decreased the activation of NADPH oxidase, secondary to its inhibitory eﬀect on the translocation of the cytosolic p47phox to the plasma membrane, and this eﬀect was also related to the inhibition of cellular PKC [210]. The glabridin hydroxyl groups were required to obtain these eﬀects, as methylation of these groups completely blocked the eﬀects of glabridin on NADPH oxidase and macrophage-mediated oxidation of LDL [210]. Similar to licorice-derived glabridin, pomegranate hydrolyzable tannins [66], red wine [211], or tea ﬂavonoids [212] were also shown to substantially inhibit macrophage-mediated oxidation of LDL.

III.

UPTAKE OF Ox-LDL BY MACROPHAGES

A.

Macrophage Scavenger Receptors

Oxidized LDL can be taken up by macrophages at an increased rate via their scavenger receptors, SR-A and CD36, leading to cellular cholesterol accumulation and foam cell formation [74,75]. Class A SR consist of SR-A types I and II, the nonfunctional splice

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variant type III, and a distant receptor called MARCO [213,214]. Ox-LDL–binding studies on a series of truncation and point mutant receptors show that the collagenous region of SR-A is required for ligand recognition, at least for binding of oxidized LDL [215]. In order to evaluate the role of SR-A, Suzuki and colleagues generated a mouse deﬁcient in both isoforms of this receptor [216]. Isolated peritoneal macrophages from these mice showed a signiﬁcant decrease in the endocytotic degradation of Ox-LDL by 30% and of acetylated LDL by 80% [216]. To study the contribution of SR-A to atherosclerosis, the above mice were crossed with apo E–deﬁcient mice. The double knockout mice had moderately increased serum cholesterol and signiﬁcant reduced lesion formation (by 60%) compared with the apo E–deﬁcient controls, suggesting that the SR-A receptor accelerates atherosclerosis [216]. Studies in other models, however, have raised doubts about the generality of this ﬁnding [217], and it was suggested that the SR-A receptor might function diﬀerently in animals with diﬀerent genetic background [218]. A second macrophage scavenger receptor, the CD36 receptor, belongs to the SR-B scavenger receptors and, like the SR-A receptor, also contributes to enhanced macrophage uptake of Ox-LDL [219,220]. In HMDM from CD36-deﬁcient subjects, reduced uptake of Ox-LDL was shown [220], and in human atherosclerotic lesion, lipid-laden macrophages showed a strong positive immunoreactivity towards CD36 [221]. Minimally oxidized LDL can upregulate the expression of scavenger receptors including SR-A and CD36 in resident mouse peritoneal macrophages by increasing the number of receptors and not their aﬃnity towards Ox-LDL [222]. Similarly, incubation of HMDM with Ox-LDL for 24 hours increased CD36 mRNA and protein levels by more than 50% [223]. Ox-LDL uptake by macrophages and the expression of CD36, SR-A I and SR-A II were increased during diﬀerentiation of monocytes to macrophages [224], and this phenomenon was signiﬁcantly enhanced in cells from hypercholesterolemic patients [224], whereas hypocholesterolemic therapy with atorvastatin decreased macrophage uptake of Ox-LDL. The role of the CD36 receptor in atherosclerosis was recently demonstrated in vivo, where targeted disruption of CD36 protected against atherosclerotic lesion development in mice [225]. As the sum of Ox-LDL binding to SR-As (assessed by competition with excess unlabeled acetylated LDL) and to CD36 (assessed by blockage with anti-CD36 antibodies) does not account for the total binding of Ox-LDL to macrophages [226], additional pathways for uptake of Ox-LDL by macrophages should exist. By using speciﬁc glcosaminoglycan (GAGs)-hydrolyzing enzymes, we have shown that macrophage plasma membrane GAGs also contribute to the cellular uptake of OxLDL and to cellular cholesterol accumulation [227]. Unlike Ox-LDL degradation, there was no role for macrophage surface GAGs in the catabolism of native LDL or of acetylated LDL. Removal of cell-surface GAGs can inhibit Ox-LDL catabolism by nearly 50%, whereas augmentation of macrophage-associated GAGs almost doubled the cellular catabolic rate of Ox-LDL [227]. There was a clear correlation between GAG cellular content and Ox-LDL cellular uptake. The inhibitory eﬀects of both heparinase and chondroitinase on Ox-LDL uptake and degradation by macrophages were dose-dependent, as well as additive, with a more potent eﬀect for chondroitinase, indicating that chondroitin sulfate is mostly involved in proteoglycan-mediated uptake of Ox-LDL by macrophages [227]. Recently, it was demonstrated that extracellular matrix (ECM)-bound Ox-LDL is taken up by PMA-activated macrophages [228]. This uptake was lipoprotein dose- and time-dependent and required

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preincubation of ECM with lipoprotein lipase, which acts as a bridge between the GAGs and the lipoprotein. ECM-bound Ox-LDL is taken up by macrophages together with the ECM-GAG [228], leading to cholesterol and oxysterol accumulation in arterial macrophages. The uptake of Ox-LDL by macrophages in vivo was recently shown, as monoclonal antibodies against Ox–LDL from apo E–deﬁcient mice blocked the uptake of Ox-LDL by peritoneal macrophages harvested from these mice [229,230]. Macrophage uptake of Ox-LDL resulted in lysosomal accumulation of unesteriﬁed cholesterol [231]. This latter phenomenon may be related to sphingomyeline binding to unesteriﬁed cholesterol avidly, leading to inhibition of cholesterol processing out of the lysosome [232]. B.

Ox-LDL Uptake by Lipid Peroxide–Rich Macrophages

Under oxidative stress, not only LDL lipids but also cellular lipids, are oxidized. Macrophages produce reactive oxygen and nitrogen species (including superoxide anion, hydrogen peroxide, peroxynitrite), which contribute to lipid peroxidation in lipoproteins and in cells and also modulate scavenger receptors gene expression [233]. In macrophages that were enriched with lipid peroxides by incubation with Ang-II, deoxycholic acid, or with iron ions, we found increased macrophage uptake of the lipoprotein, which was the result of an initial enhanced LDL receptor activity, followed by LDL oxidation and enhanced Ox-LDL cellular uptake [234]. Injection of Ang-II for 2 weeks to the apo E–deﬁcient mice (these mice are under oxidative stress and Ang-II further increases cellular oxidative state in the mice) signiﬁcantly increased Ox-LDL degradation and CD36 mRNA and protein expression by their harvested MPM by 28% and 53%, respectively [235]. The involvement of interleukin 6 (IL-6) in Ang-II–induced stimulation of macrophage uptake of Ox-LDL was suggested, as incubation of IL-6 with MPM or injection of IL-6 to mice resulted in a dose-dependent elevation in Ox-LDL degradation and in macrophage CD36 mRNA expression. Furthermore, injection of anti-IL-6 receptor antibodies to mice prior to Ang-II injection signiﬁcantly inhibited Ox-LDL degradation and CD36 mRNA expression in the MPM harvested from these mice [235]. In accordance with the above data, Ang-II injection of IL-6 deﬁcient mice did not aﬀect cellular Ox-LDL uptake and CD36 protein level [235]. Administration of the ACE inhibitors fosinopril (12.5 mg/ kg/d) or ramipril (5 mg/kg/d) [236] to the apo E–deﬁcient mice signiﬁcantly reduced Ox-LDL uptake by their MPM. Similarly, treatment of the apo E–deﬁcient mice with the Ang-II receptor antagonist losartan (12.5 mg/kg/d), followed by Ang-II injection, resulted in a signiﬁcant decrement in Ox-LDL degradation, in CD36 mRNA expression, and in serum IL-6 levels by 30, 48, and 76%, respectively, compared to mice that were injected only with Ang-II [235]. Losartan administration to eight hypercholesterolemic patients for 4 weeks also resulted in a signiﬁcant reduction (by 78%) in OxLDL uptake by monocytes-macrophages derived from these patients [237]. We have recently shown [238] that lipid peroxide content in MPM from apo E–deﬁcient mice progressively increased, by up to 4.6-fold, during mice aging, and this was accompanied by an age-dependent increase in the cellular uptake of Ox-LDL (by up to 90%) and in the expression of the CD36 macrophage receptor (by up to 41%). Inhibition of cellular oxidative stress by administration of dietary antioxidants such as vitamin E, licorice root–derived glabridin [238], or pomegranate juice [66,70] resulted

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in a signiﬁcant inhibition in Ox-LDL uptake by macrophages and in CD36 mRNA expression. Stimulation of cellular oxidative stress, on the other hand, by depletion of cellular GSH signiﬁcantly increased Ox-LDL uptake by macrophages [238].

IV.

PARAOXONASE AND LDL OXIDATION

The paraoxonase gene family in mammals includes at least three members: PON1, PON2, and PON3 [239,240]. These PON genes appear to have arisen by gene duplication of a common evolutionary precursor, because they share considerable structural homology and are located adjacently on chromosome 7 in humans and on chromosome 6 in mice. Within a given species, PON1, PON2, and PON3 share about 70% identity at the nucleotide level. Until recently there has been little insight into the role of the respective gene products in human physiology and pathology. However, emerging evidence from biochemical and genetic experiments is providing some clues about the possible role(s) of the products of these genes. Whereas PON1 and PON3 proteins are present in serum, PON2 protein was not found in serum, but only in tissues. A.

Serum PON1

Paraoxonase 1 (PON1) is a protein of 354 amino acids with a molecular mass of 43 kDa [240,241]. Human serum PON1 is an esterase, which is physically associated with HDL, and is also distributed in tissues such as liver, kidney, and intestine [241,242]. PON1 is bound to the HDL phospholipids via its retained N-terminal leader sequence [243]. PON1 can reversibly bind to organophosphate substrates, which it hydrolyzes, and thus PON1 can protect the nervous system against the neurotoxicity of organophosphates entering the circulation. It was in this context that it was ﬁrst discovered, as PON1 hydrolyzes paraoxon, a metabolite of the insecticide parathion, and this is reﬂected in its name. Activities of PON1, which are routinely measured, include hydrolysis of paraoxon and arylesters, such as phenyl acetate. Recently, the capability of PON1 to hydrolyze lactones and cyclic carbonate esters was also demonstrated [244]. Human serum paraoxonase activity was shown to be inversely related to the risk of cardiovascular disease [245–247], as shown in atherosclerotic, hypercholesterolemic, and diabetic patients [248–250]. PON1 activity is reduced in the atherosclerotic apo E–deﬁcient mice [251], and it is also decreased by a pro-atherogenic diet in rabbits [252]. Since organophosphates are not present in the human body, other possible substrates of physio/pathological relevance were studied. Oxidized cholesteryl esters, as well as oxidized phospholipids in oxidized lipoproteins [251,33] and in atherosclerotic lesion lipids [253], were shown to be substrates for PON1. PON1 exists in two major isoforms, Q and R, and the R allele frequency was signiﬁcantly higher in patients with coronary artery disease or with ischemic stroke in comparison to controls [254]. PON1 protects both LDL and HDL against lipid peroxidation [251,255–257] and was shown to hydrolyze lipid peroxides in human carotid and coronary atherosclerotic lesions [253], with the Q isoform being more potent that the R isoform in this respect [253,258]. Oxidized lipids were shown to inactivate PON1, and the Q isoform was less inactivated than the R isoform [259]. Furthermore, injection of oxidized 1-palmitoyl2-arachidonyl-sn-glycerol-3-phosphoryl choline (Ox-PAPC) into C57BL/6J mice resulted in a marked reduction in PON1 activity. Similarly, incubation of HepG2

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cells with Ox-PAPC decreased PON1 mRNA levels [260]. In accordance with these results, antioxidants were shown to preserve PON1 activity as they decrease the formation of lipid peroxides [66,68]. Inhibition of HDL oxidation by PON1 was shown to preserve the antiatherogenic eﬀects of HDL in reverse cholesterol transport [251]. The free sulfhydryl group on the PON1 cysteine-284 is required for its protection against lipid peroxidation in lipoproteins and in lesions, but not for its paraoxonase/arylesterase activities [258]. These eﬀects of PON1 may be relevant to its beneﬁcial properties against cardiovascular diseases. Using immunolocalization techniques, Mackness et al. have shown that PON1 accumulates in the arterial wall during the development of atherosclerosis [261] and hence it can protect the LDL from oxidation in the arterial wall. In addition, it was recently shown that mice lacking serum paraoxonase are becoming susceptible to organophosphate toxicity and atherosclerosis [262]. Combined serum paraoxonase knockout with apo E knockout mice exhibit increased lipoprotein oxidation and accelerated atherosclerosis [263]. We have recently questioned the eﬀect of human PON1 on oxidative state of macrophages and on their ability to oxidize LDL. In PON1 knockout mice on the background of C57BL6, serum arylesterase activity was reduced by 82%, as compared to the activity in control C57BL6 mice. Lipid peroxides content in their harvested MPM was signiﬁcantly increased (sixfold), and reduced glutathione (GSH) content was decreased compared to control MPM. Superoxide anion release from macrophages in response to PMA was increased twofold after 1 hour of incubation with MPM from PON1 knockout mice compared to the amount of superoxide anions released from control C57BL6 mice MPM. Cell-mediated oxidation of LDL (after 5 hours of incubation in the presence of copper ions) by MPM from PON1 knockout mice was increased by 41%, compared to LDL oxidation by macrophages from control C57BL6 mice. These results clearly demonstrate increased oxidative stress in macrophages from PON1 knockout mice. Similar results were obtained with the double knockout mice: PON1 knockout/apo E knockout. Interestingly, we observed a signiﬁcant twofold increment in cholesterol biosynthesis from [3H]-acetate in MPM harvested from PON1 knockout mice, as compared to macrophages from control mice. This may be related to the increased oxidative stress in the cell, as a result of cellular accumulation of oxidized lipids, which are substrates for PON1. Upon adding puriﬁed PON1 to MPM from the PON1 knockout mice, a signiﬁcant dose-dependent reduction in cholesterol biosynthesis was noted, further suggesting that PON1 has a role in inhibiting cellular cholesterol biosynthesis. Finally, intraperitoneal injection of puriﬁed human PON1 into apo E–deﬁcient mice resulted in a 40–65% decrement in lipid peroxides content in MPM harvested from these mice. This eﬀect was associated with a similar 30–40% reduced uptake of Ox-LDL and a decreased expression of CD36 mRNA [238]. B.

PON2 and PON3

The roles of PON2 and PON3 are less understood than are those for PON1. To date, all PON2 and PON3 cDNAs seguenced lack the three nucleotide residues of codon 106 present in PON1 [240,264]. By using PON3-speciﬁc peptide antibodies, human PON3 was detected as a 40 kDa protein, which like PON1 is also associated with serum HDL [265,266]. Human PON3 gene from HepG2 cells was cloned and characterized [265], and a rabbit serum lactonase distinct from PON1 was recently puriﬁed and charac-

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terized as PON3 [267]. In contrast to PON1, PON3 has very limited arylesterase activity and no paraoxonase activity, but it rapidly hydrolyzes lactones such as statin pro-drugs like lovastatin [267]. PON3 hydrolyzes aromatic lactones and 5- or 6member ring lactones with aliphatic substituents, but not simple lactones or those with polar substituents [267]. Rabbit serum PON3 is more eﬃcient than rabbit PON1 in protecting LDL from copper-induced oxidation [267]. Unlike PON1, PON3 mRNA expression was not inactivated in HepG2 cells by oxidized phospholipids and was also not inactivated in the livers of mice fed a high-fat atherogenic diet [265]. While the mRNA expression of PON1 and PON3 is restricted primarily to the liver, PON2 mRNA is more widely expressed and is found in a number of tissues, including brain, liver, kidney, testis, and also in white blood cells [268]. Furthermore, like PON1, PON2 polymorphism is associated with numerous pathophysiological conditions, including variations in plasma lipoprotein concentrations [269] and the risk of coronary disease [270,271], but the exact function of PON2 in humans is not known yet. The high similarity observed in amino acid sequence between the PON proteins suggests that PON2 may possess a biochemical function similar to that of PON1 and PON3. Unlike PON1 and PON3, PON2 protein is not detectable in HDL (by Western blot analysis using speciﬁc antibodies towards PON2) [272]. It was recently shown [272] that PON2 is constitutively expressed (mRNA and protein) in both primary and immortalized human endothelial cells and in human aortic smooth muscle cells. PON2 overexpression was recently shown to lower the intracellular oxidative state of cells that have been treated with either hydrogen peroxide or Ox-PAPC [272]. Furthermore, cells that overexpressed PON2 were able to reverse the oxidative modiﬁcation of minimally modiﬁed LDL (MM-LDL), and MM-LDL that has been incubated with cells that overexpressed PON2 showed lower levels of lipid hydroperoxides and was less able to induce monocyte chemotaxis than MM-LDL incubated with control cells [272]. These data suggest that PON2 may act as cellular antioxidant and may play an antiatherogenic role by reducing the cellular oxidative stress.

Figure 2 Lactonase action of PON1 and of PON3 on statins. PON1 and PON3 can hydrolyze lactone-containing drugs such as the HMG-CoA reductase inhibitors, mevastatin, lovastatin, and simvastatin, forming the prospective acids.

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Figure 3 Statins-paraoxonase interactions protect against oxidative stress and atherosclerosis. Some statin metabolites are powerful antioxidants and can directly inhibit the oxidation of LDL [1]. These metabolites can also increase paraoxonase activity [2], which can than act on OX-LDL, resulting in the hydrolysis of specific oxidized lipids, forming ‘‘neutralized Ox-LDL’’ [3]. On the other hand, paraoxonase possess lactonase activity, and can act on statins, forming statin metabolites [4]. Altogether, paraoxonase-statins interactions reduce Ox-LDL accumulation and hence attenuate atherosclerosis.

We have recently found mRNA for PON2 and PON3, but not for PON1, in macrophages: J774A.1 macrophages, mouse peritoneal macrophages (MPM), and human monocyte–derived macrophages. The cell sonicate possesses little arylesterase activity and no activity with paraoxon. However, we observed signiﬁcant macrophage lactonase activity (with dihydrocoumarin) and also signiﬁcant activity with lovastatin. Both PON1 and PON3 possess lactonase activity, and they are capable of hydrolyzing statins, the HMG-CoA reductase inhibitors (Fig. 2). On the other hand, there is a considerable interest in the potential pharmacological eﬀects on PONs activity. Of the lipid-lowering drugs, ﬁbric acid derivatives have been reported to raise serum PON1 activity in some studies [45,273], but this eﬀect was not found in other studies [274,275]. Statin therapy may also raise PON activity, because some statins possess antioxidant-like activity [45]. As paraoxonase is inactivated by oxidized lipids [259,260], statin therapy can reduce the level of oxidized lipids in serum of hypercholesterolemic patients which are under oxidative stress. Indeed, atorvastatin metabolites [45] or simvastatin or ﬂuvastatin therapies [276] increased serum PON1 activity. The interactions between statins and paraoxonase are summarized in Figure 3. V.

CONCLUSIONS

LDL is oxidized in vivo by arterial cells, and during early atherogenesis macrophages play a major role. The extent of LDL oxidation depends on its own oxidative status and on the balance between activities of cellular oxygenases and cellular

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antioxidants. Under oxidative stress not only are lipoproteins oxidized, but also arterial cells including macrophages. Macrophage lipids are also oxidized as demonstrated in vitro by several oxidants and in vivo in macrophages from atherosclerotic patients or mice. These lipid peroxide–rich macrophages easily oxidize LDL and take up oxidized LDL, leading to foam cell formation, the hallmark of early atherogenesis. As a result of the atherogenic properties of Ox-LDL, reduction in oxidative stress in general and that of lipoproteins in particular is needed. Strategies to reduce LDL oxidation and atherosclerosis may thus include: 1.

Reduce activities of cellular oxygenases (NADPH oxidase, myeloperoxidase, lipoxygenase)

Figure 4 Paraoxonase and macrophage foam cell formation. Under oxidative stress, both plasma LDL and blood monocytes invade the arterial wall. LDL binds to extracellular matrix proteoglycans (retention) and can be oxidized by arterial wall macrophages. The extent of LDL oxidation is determined by the balance between LDL-associated cholesteryl ester (CE), and LDL-associated vitamin E, h-carotene, lycopene, and some polyphenols. Macrophage oxidative state also determines the extent of LDL oxidation, and it is determined by the balance between cellular oxygenase activities (such as NADPH oxidase) and macrophage antioxidants (such as the GSH). Macrophage paraoxonases (PON2 and PON3) may also protect the cell from oxidation. Nutritional antioxidants such as flavonoids are not only associated with LDL, but can also accumulate in arterial cells, including macrophages, and can thus inhibit cell-mediated oxidation of LDL. Finally, if oxidized LDL is formed in the arterial wall under oxidative stress, HDL-associated paraoxonase (PON1 and PON3) can hydrolyze specific lipid peroxides in Ox-LDL. Antioxidants, which reduce OxLDL lipid peroxide formation, can also preserve paraoxonase activity. HDL-associated PON1 and PON3 as well as cellular PON2 can act on specific oxidized lipids in macrophages, resulting in the hydrolysis of cellular lipid peroxides, hence reducing their ability to oxidize LDL, to take up Ox-LDL, and to form foam cells. HDL-associated PON1 and PON3 also protect the HDL from oxidation and, hence, promote HDL-mediated cholesterol efflux from macrophage foam cells.

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4. 5.

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Increase cellular antioxidant activities (PON2, PON3, GSH) [277–279] Enrich LDL environment, as well as enrichment of arterial cells, including macrophages, with antioxidants (ﬂavonoids, vitamin E, h-carotene, lycopene, vitamin C) Use angiotensin II–converting enzyme inhibitors and angiotensin II antagonists Increase serum paraoxonases (PON1, PON3) activities [278]

The role of antioxidants and that of paraoxonases in preventing macrophage foam cell formation and atherosclerosis is summarized in Figure 4.

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26 Free Radicals in Rheumatoid Arthritis: Mediators and Modulators TULIN BODAMYALI, JANOS M. KANCZLER, and TIM M. MILLAR University of Bath, Bath, England CLIFF R. STEVENS and DAVID R. BLAKE University of Bath and Royal National Hospital for Rheumatic Diseases, Bath, England

I.

INTRODUCTION

Free radicals have long been accepted as bioactive molecular species that were originally attributed toxic roles. Their involvement in a wide range of pathologies has been extensively reviewed [1]. Our current opinions with respect to their role in pathology, however, based on evidence from relatively recent research, are somewhat diﬀerent from previous beliefs. We now know of their signiﬁcance with respect to numerous physiological processes that govern cellular and tissue responses. The role of free radicals in the physiological control of cell functions has recently been reviewed [2]. Within an altered microenvironment, disruption of the tight control that regulates these responses may be the basis for a pathological outcome. Our ongoing research indicates that the observed pathological features of rheumatoid arthritis are no exception. In the following sections, we review the current understanding of how free radicals function as mediators of acute inﬂammation, considered to be a physiological defense mechanism. We argue that, in the context of the pathology of rheumatoid arthritis, acute inﬂammatory response is an initial tissue reparative response, which is designed to reestablish homeostasis. In the subsequent sections, we describe how the microenvironment of the inﬂamed joint is conducive to the perpetuation of this response, imbalance in the intrinsic control mechanisms, and the development of chronic 591

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inﬂammation, leading to the observed pathology. The role of free radicals as modulators of chronic, unresolved, and pathological inﬂammation is discussed at this level. II.

FREE RADICALS AND THE ACUTE INFLAMMATORY RESPONSE

There is an increasing body of experimental evidence implicating partially reduced forms of oxygen in a wide variety of pathological states and xenobiotic metabolism and associated toxicity [1]. What is often dismissed, however, is the role of such species in physiology. Relatively recent evidence showing the signaling capacity of many forms of reactive oxygen species (ROS) has strengthened the view that they are not only associated with toxicity and pathology but have signiﬁcant controlling inﬂuences in many physiological processes such as the acute inﬂammatory response. Recently, the biological signaling function of nitric oxide, a reactive nitrogen species (RNS), has come to the fore. Figure 1 summarizes the multiple reactions and the cellular eﬀects of nitric oxide. In this section we summarize the characteristics of well known ROS and RNS, discuss the endogenous sources of such species in diﬀerent environmental settings, and describe how such species can bring about a physiological defense mechanism such as the acute inﬂammatory response. All free radicals, as well as reactive oxygen and nitrogen species, with their capacity as potent oxidants, are capable of oxidizing multiple targets within a cell. These targets include critical biomolecules such as DNA, RNA, proteins, and lipids, thus having altered functionality, which may lead to pathology. An increasing number of studies now support the modulating role of biological oxidants in inﬂammatory processes in which antigen-autoantibody reactions and immune complex pathogenesis may play an important role. Recent studies on the properties of IgG modiﬁed by hypochlorous acid (HOCl) and peroxynitrite (ONOO) have revealed the proinﬂam-

Figure 1

Biological effects of nitric oxide and its metabolites.

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matory properties of such oxidant-modiﬁed molecules [3]. Although such interactions of free radicals appear to be detrimental, in physiology they are tightly controlled by a variety of eﬃcient antioxidant and scavenger systems. The main antioxidants are listed in Table 1. This control ensures a slow, steady ﬂux of oxidants, which mediate an acute inﬂammatory response. The controlled functioning of this physiological defense mechanism results in the resolution of damage and the homeostasis is reestablished. The signiﬁcance of the net balance of oxidants and antioxidants/scavengers in preserving the inﬂammatory response as a physiological defense mechanism is thus clear. Under pathological conditions, however, where the microenvironment is altered, this control mechanism is somewhat compromised. In this setting, the levels of oxidants and antioxidants are variable and modulate the magnitude and the nature of responses exhibited by resident cells. These responses, discussed later in the text, can facilitate a futile cycle of inﬂammation, which fails to resolve and is recognized as chronic inﬂammation, a pathological condition. It is clear that a wide range of inﬂammatory mediators interact in order to achieve homeostasis. These include plasma enzyme cascades and inhibitors/acute phase proteins, arachidonic acid products (prostaglandins and leukotrienes), plateletderived mediators, mediators derived from mast cells, polymorphonuclear(PMN)leukocytes, monocytes/macrophages, and lymphocytes. One signiﬁcant feature of many of these inﬂammatory mediators, however, is that ROS may be involved in their mechanism of action. Many of the cell types regulating the inﬂammatory response can, under deﬁned microenvironmental conditions, be induced to generate ROS. Such capacity infers a potential for all cells to utilize ROS as a means of rapid bactericidal or antimicrobial defense and/or to employ them as eﬃcient signaling molecules in the communication and relay of messages. There is no doubt that aerobic cells utilize these species as a bactericidal defense system via the neutrophil respiratory burst during acute inﬂammation. In chronic and/or persistent inﬂammation, ROS may have an even more signiﬁcant role via their signaling characteristics. The following sections of this chapter discuss in detail the pivotal role of these species in the modulation of cellular responses to the altered redox environment. A.

Vascular Responses

It is now widely accepted that one of the earliest responses of tissues to inﬂammatory stimuli is the constriction/dilatation of its microvasculature. The role of peroxynitrite in mediating vascular responses is becoming more evident [4]. Peroxynitrite can also activate a variety of signaling pathways, which tend to be proinﬂammatory. Peroxynitrite promotes the inﬂammatory synthesis of prostaglandins. The conjugate acid of

Table 1

Biological Antioxidants and Antioxidant Systems

Reducing agents and/or scavengers Thioredoxin Glutathione Tocopherol Ferritin Biliverdin and bilirubin

Enzymes Superoxide dismutase Catalase Glutathione peroxidase Thioredoxin reductase Heme oxygenase

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peroxynitrite (ONOOH) plays an important role in vivo in the activation of cyclooxygenase by providing the peroxide tone necessary for enzyme activity. The nonheme iron in cyclo-oxygenase must initially be oxidized to an unstable and reactive ferryl state to be able to oxidize arachidonic acid. Peroxynitrite has been shown to be the major peroxide responsible for activating cyclo-oxygenase in vivo [5,6]. The product of cyclo-oxygenase, PGH2, is usually converted by prostacyclin synthatase to the vasodilatory and antithrombotic prostaglandin (PGl2). Accumulation of PGH2 can activate thromboxane receptors. Thus, the reaction of superoxide with nitric oxide in the vasculature can help subvert the principal vasodilating mechanisms into becoming strongly vasoconstricting. This rapid vascular response leads to alterations in the pO2 in the immediate milieu, resulting in a net redox change. A redox gradient naturally exists between the site of inﬂammation and the distant cells within the tissue. Cellular responses in such a microenvironment show a signiﬁcant variation and are depicted in Figure 2. B.

Necrosis and Apoptosis

Cells at the focus of the direct site of stimulus are most oxidatively stressed and often show necrotic responses, releasing tissue destructive products. One of the most readily observed cellular responses to such alteration within the microenvironment is the recruitment and activity of acute inﬂammatory cells, namely PMNs, which mainly function, through oxidant activity, to clear cellular debris. Around the periphery of this focal region, cells may die by more controlled apoptosis. It appears that relatively lower level of oxidants exert their eﬀects through their capacity to act as signaling molecules, modulating biologically signiﬁcant events such as phosphorylation, transcription factor activity, and gene transcription. Oxidants may inﬂuence apoptosis in a variety of ways, and such signals may be both extra- and

Figure 2

Redox gradient and cellular responses.

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intracellular. Intracellular sources include mitochondrial oxidation, the cytochrome P-450 system, and the plasma membrane NAD(P)H oxidases. Mitochondrial oxidants are particularly important in apopototic responses since cellular activation requires an increase in oxidative phosphorylation. In apoptotic death, the electron transport chain uncouples, resulting in the direct reduction (addition of electrons) of molecular oxygen. The level of oxidants generated during oxidative phosphorylation thereby predicts whether the cells within that locality will proliferate or apoptose. Regulation of mitochondrial or cytosolic oxidants is thought to be mediated by the bcl-2 gene product, which inhibits apoptosis possibly by interacting with mitochondrial superoxide dismutase (SOD). Various antioxidants, which raise intracellular thiol levels, can substitute for bcl-2 expression in preventing growth factor–deprived cells from apoptosis. These observations conﬁrm the role of oxidants in apoptosis [7,8]. Similar control mechanisms may be eﬀective at the ruﬄed border of the osteoclast, mediating osteoclastic bone resorption [9]. The level of involvement of oxidants in apoptotic responses is summarized in Figure 3, indicating H2O2 and bcl-2 as examples of interacting components. C.

Defensive—Respiratory Burst and Phagocytosis

In evolutionary terms, the major role of such oxidants is their microbicidal activity, phagocytes being the primary source. The main enzymes responsible for generating oxidants recognized primarily for their microbicidal activity are the neutrophil NAD(P)H oxidase(s). However, increasing evidence shows that most tissue-resident cells also have the capacity to generate free radicals as an intrinsic, ﬁrst-line defense mechanism against infectious and potentially pathogenic stimuli. Enzymes such as xanthine oxidoreductase, nitric oxide synthetase, and the cytochrome P-450 family are known to generate reactive oxygen and nitrogen species with such characteristics.

Figure 3 apoptosis.

Oxidants and apoptosis: possible interactions for oxidants and Bcl-2 in

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Combative Stress Protein Response

Subcytotoxic levels of oxidants lead to a cellular stress response. The expression of a group of proteins, referred to as stress proteins, is increased. These proteins function as a unit in cellular defense. Certain stress proteins (HSP-70, for example) function as molecular chaperones involved in various aspects of protein synthesis and maturation. The association of HSP-70 with nascent poplypeptides prevents their premature folding until synthesis is completed. The oxidative modiﬁcation of proteins, both extracellularly and intracellularly, is a feature of the inﬂammatory response. Proteins oxidized intracellularly associate with HSP-70, which, in turn, dissociates from the heat shock factors (HSFs), the transcriptional regulators of stress genes, allowing for a stress protein response [10]. Our recent observations suggest that in synovial microvessels, a select aspect of the stress protein response (HSP-32) is impaired [11]. Stress proteins have been directly linked to arthritis and autoimmunity. It has been argued that because stress proteins are immunodominant antigens of microorganisms, they may contribute to T-cell selection within the thymus producing a biased T- and B-cell repertoire, favoring recognition of stress proteins. The hypoxic and oxidative inﬂuences eﬀective within the inﬂamed joint lead to the induction of stress proteins [11]. T- and B-cell autoreactivity has also been found in patients with synovitis as well as normal individuals [12]. In this context, it is still far from clear whether an autoimmune response to stress proteins plays any part in the initiation or maintenance of synovitis in humans. In the later sections, we describe how both HSP response and T- and B-cell repertoire can be aﬀected by varying pO2 and subsequent local redox changes. E.

Combative and Reparative Gene Activation

Oxidants also mediate combative and reparative responses in surviving cells. Two redox-controlled transcription factors involved in such responses are the nuclear factor nB (NFnB) and the activating protein 1 (AP-1) [13,14]. NFnB is a multisubunit transcription factor that can rapidly activate the expression of genes encoding cytokines, cell surface receptors, cell adhesion molecules, acute phase proteins, and hemopoietic growth factors [15]. It is believed that NFnB functions as a signal transducer between cytoplasm and the nucleus and is an immediate-early activator of gene transcription. It is also argued that the activation of NFnB may be brought about via the oxidant-mediated cleavage of its inhibitory subunit InB [13]. The transcription factor AP-1 (a heterodimer of c-jun and c-Fos protooncoproteins) has also been described to be responsive to antioxidants as well as oxidants [14]. The coordinated function of both NFnB and AP-1 in response to redox changes may control the net eﬀect of oxidants on exposed cells. Oxidant stress– regulated activation of transcription factors such as NFnB and AP-1, and cross-talk between them, in turn, modulate the expression of a second wave of genes involved in reparative responses to tissue injury. One such response is angiogenesis, a process integral to the resolution of inﬂammation and physiological tissue perfusion. Indeed, the redox-sensitive transcription factors NFnB and AP-1 are known to cooperate with another transcription factor, c-Ets-1, for transcriptional activation of the collagenase, stromelysin, and uPA genes, which play a signiﬁcant role in angiogenesis [16,17]. In this context, cell proliferation can also be considered as a ‘‘combative’’ or ‘‘reparative’’ response to the changes in the tissue microenvironment. Transcriptional

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control of many growth competence genes is also modulated by oxidants. Two such examples are the oncogenes c-fos and c-jun (the products of which are components of the transcription factor AP-1) [18,19]. F.

Reactive Oxygen Species and Cell Proliferation

Although the processes that regulate mammalian cell proliferation are already known to be complex, several lines of investigation suggest that ROS may have an important regulatory role. Some of this evidence comes from studies on a-tocopherol levels in regenerating liver in relation to periods of DNA synthesis, levels of cellular lipid peroxidation in relation to cell proliferation, and the eﬀects of polyunsaturated fatty acids on growth. Experiments carried out in relation to the eﬀects of lipid peroxide breakdown products (such as IP3) on growth and speciﬁc intracellular signal transduction mechanisms [20] have also provided further evidence in this context. H2O2 at very low concentrations (108 M) was reported to be growth stimulatory [21]. Similarly, Garrett et al. [22] have shown that ROS, particularly O2. –, are intermediaries in the formation and activation of osteoclasts both in vitro and in vivo and that osteoclasts have the capacity to generate ROS themselves. Growth promotion by oxidants is observed with cultured human and mouse ﬁbroblasts, as well as epithelial cells. Indeed, oxidants are known to trigger (patho)physiological reactions, which resemble those induced by growth and diﬀerentiation factors. For example, ROS have been shown to cause S6-phosphorylation, activation, and translocation of protein kinase C, Ca2+-dependent DNA breakage, and transcriptional activation of the growth competence genes c-fos and c-myc [23]. The regulation of both NFnB and AP-1 by redox-mediated mechanisms has been reviewed recently [24]. Furthermore, two other redox-regulated transcription factors, speciﬁcity protein (Sp1) and peroxisome proliferator–activated receptors (PPARs), have also been discussed with relevance to inﬂammation, aging, and agerelated pathologies. Reactive oxygen species, however, are not the only species that bring about gene activation. Peroxynitrite, a potent oxidant and a reactive nitrogen species, also has signiﬁcant eﬀects on inﬂammatory activity. Peroxynitrite induces longer-lasting changes in the vasculature and the immune system by deactivating anti-inﬂammatory agents as well as activating other stress-related signaling cascades. Peroxynitrite is reported to be particularly eﬀective with inactivated tyrosine kinases, such as CD45 [4]. CD45 is a JAK phosphatase, which appears to have a central role in downregulating proinﬂammatory responses. In addition, endogenous formation of peroxynitrite in cultured endothelial cells has been reported to activate c-Jun-NH2-terminal kinase (JNK), which induces a variety of stress-related responses [25].

III.

REDOX MICROENVIRONMENT OF THE INFLAMED JOINT

The activity of a whole network of factors during acute inﬂammation often results in the establishment of homeostasis in most tissues and organs. The joint, however, is structurally and functionally unique in that movement renders this tissue susceptible to above atmospheric pressure, which in turn alters the oxygenation proﬁle. A change in the local pO2, hypoxia, initiates a plethora of free radical–driven responses that lead to pathology. There is a considerable body of evidence for the existence of hypoxia within the inﬂamed synovium.

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The hypoxic nature of rheumatoid synovium was originally suggested on the basis of measurements of oxygen tension within the inﬂamed cavity [26]. Other studies reporting raised CO2 tension, lactate, lowered glucose, and acidosis also supported these observations [27]. More indirect, but supportive evidence was also provided by studies of the synovial oxidative metabolism, revealing increased metabolic demand and shift to glycolytic metabolism [28]. These observations were conﬁrmed with our studies of the synovial membrane and ﬂuid from rheumatoid joints, utilizing nuclear magnetic resonance (NMR) spectroscopy [29]. The results of this sensitive technique displayed a proﬁle of low-molecular-weight metabolites consistent with hypoxic metabolism. Decreased synovial pH, raised lactate, 3-D-hydroxybutarate, acetate levels and ketone body formation conﬁrmed a progressive shift to glycolytic metabolism, in accordance with chronic hypoxia. Further evidence came from studies utilizing a polarographic needle electrode, which directly measured pO2 levels in diseased synovium and veriﬁed the hypoxic nature of inﬂamed synovia [30]. The most hypoxic regions were found to be the innermost synovium in contact with the synovial ﬂuid, irrespective of eﬀusions. Morphometric analysis of inﬂamed synovial tissues also supported these observations and revealed structural features that explain chronic hypoxia [31]. Although synovitis was long considered to be an angiogenesis-driven pathology, in our analyses the capillary density was calculated to be a third of that in normal synovium. In addition, the average distance of the capillaries from the joint cavity was found to increase in rheumatoid synovia. These measurements indicate inadequate perfusion due to failure of angiogenesis to vascularize innermost synovium. We interpret this as the failure of angiogenesis to keep pace with synovial thickening and have previously discussed the implications in the context of synovitis [32]. Indeed, the prevalence of such events in inﬂamed rheumatoid synovium is exempliﬁed by the presence of an avascular and predominantly hypoxic pannus tissue. We further argue that the loss of highly organized vascular structure causes relatively less uniform perfusion through the tissue. Subsequent poor blood ﬂow aggravates hypoxia by diminishing the oxygen gradient out of the vessels and reduces the capillary release rate in small, remote capillaries. With sustained hypoxia, the capillaries become ‘‘paralyzed’’ and lose their normal vasoactive responses. In addition to this hypoperfusion, mobility of the inﬂamed joint causes increased intra-articular pressure, further restricting synovial blood ﬂow [33]. Other hypoxia-induced factors prevail. The presence of saturable, high-aﬃnity vascular endothelin (ET-1)-binding sites within RA synovial sections and the localization of ET-1–like immunoreactivity to synovial microvascular endothelial cells is a further indication of a hypoxia-mediated contribution to the reduction in local synovial perfusion [34]. A.

Hypoxia and Subsequent Events Modulated by Free Radicals

1.

Immune Responses

The pathogenesis of rheumatoid arthritis (RA) is hypothesized to involve inappropriate triggering of the MHC-controlled immune surveillance, resulting in altered Tand B-cell proﬁles. Recently, the cytokine proﬁle of T-helper (Th) lymphocytes has been associated with the disease. The cytokine repertoire of inﬂamed synovia is categorized as that of a Th1 response. The characteristic Th1 response displays enhanced expression of select cytokines such as TGF-h, IFN-g, TNF-a, IL-1, and IL-2.

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In contrast, Th2 responses exhibit enhanced IL-4, IL-5, IL-10, and IL-13 (predominantly anti-inﬂammatory cytokines). While the levels of TNF-a and IL-1 are high within RA synovium [35], the level of IL-2 is paradoxically low [36]. This is atypical of a Th1 response, but may be accounted for by the overriding hypoxic condition of the RA synovium. Hypoxia transcriptionally upregulates TNF and IL-1 but downregulates IL-2 [37,38]. The predominance of a given cytokine in the microenvironment of the responding Th cell inﬂuences Th1/Th2 diﬀerentiation [39]. This phenomenon is believed to be regulated by phosphorylation of the relevant signal transducers and activators of transcription (STATs), following cytokine binding to their associated receptors. Free radicals are known to modulate both cytokine expression and phosphorylation events. This knowledge infers that within inﬂamed synovial tissues, hypoxia-driven redox uncoupling and free radical generation may underlie the functional polarization of the Th1/Th2 lymphocytes and the apparent Th2-to-Th1 switch in RA. The T-cell cytokine proﬁle of inﬂamed synovia may account for the development of an unorganized or abnormal inﬂammatory response. However, it does not convincingly explain the persistence of the existing inﬂammatory reaction or more distant events such as the development of pannus tissue and the erosion of cartilage and bone. Evidence of an ancillary mechanism to cytokine-induced inﬂammatory changes comes from a study of RA patients with HIV. In this situation, even when terminal loss of T cells occurs and all immune and cytokine activity ceases (patients in remission), destructive rheumatoid pathology still continues [40]. The apparent RA synovial T-cell hyporesponsiveness (or anergy) may also be related to hypoxia and free radical interactions. It is known that transient carbonylamino condensation, or Schiﬀ base formation, regulates T-cell and antigen presenting cell (APC) interactions [41]. Inhibition of T-cell responses can thus occur, depending on the species and concentration of Schiﬀ base–forming aldehydes. Such lowmolecular-weight species are generated during hypoxic metabolism. We have demonstrated the presence of several low molecular mass carbonyl compounds, notably acetone and acetoacetate, in RA synovial ﬂuid (SF) [42] that can potentially lead to the observed synovial T-cell anergy. Indeed, recent observations have revealed that the hyporesponsiveness of synovial ﬂuid T lymphocytes in rheumatoid arthritis is related to the displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations. This hyporesponsiveness has been restored after supplementation of the intracellular glutathione levels, supporting the role of oxidant stress in this phenomenon [43]. Other studies indicate that the cytokine and growth factor proﬁle of the Type A (macrophage) and Type B (ﬁbroblastic) synoviocytes in culture match more closely with that displayed by rheumatoid synovial tissues [44]. These observations reﬂect a dominant and persistent cytokine and growth factor inﬂuence by resident synovial cells rather than the T lymphocytes, in particular, macrophage or Type A–derived IL1, IL-6, TNF-a, and GM-CSF. The production of macrophage (Type A synoviocyte)– derived cytokines is also inﬂuenced by hypoxia and redox uncoupling [37]. 2.

Pannus Development

Vascular insuﬃciency, hypoxia, and inﬂammatory cell inﬁltration leads to synovial ﬁbroblastic hyperplasia. Free radicals have been reported to have direct eﬀects on the proliferative proﬁle of ﬁbroblasts [45], projecting these species as the modulators of the

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unscheduled proliferative response of the resident ﬁbroblastic cells. In this respect, free radicals are also known to induce the activation of a number of cellular oncogenes such as c-fos and c-jun, which not only constitute transcription factors but also function as growth competence genes. Macrophages contribute to this process by releasing potent cytokines such as TNF-a and TGF-h [37,46]. Hypoxia induces TGF-h through pathways common to hypoxia-sensing mechanism(s) described for erythropoietin [47]. Both hypoxia and TGF-h are known to induce ET-1 production from endothelial cells, although diﬀerential expression is observed in diﬀerent vascular beds [48,49]. Fibroblastic cells are then primed by ET-1 to produce factors that initiate extracellular matrix production and angiogenic factors, collagens, FGF, PDGF, and VEGF, further contributing to the development of the pannus tissue [50–52]. Signiﬁcant transcriptional upregulation of VEGF expression in rheumatoid synovial ﬁbroblastic lining cells has also been reported [53]. The genes for these factors are controlled by hypoxia/redox-sensitive transcription factors such as Hif-1, AP-1, and NFnB [54], such that their activation is modulated by free radicals. Moreover, peroxynitrite is also thought to promote intracellular release of acidic FGF, inducing vascular remodeling and ﬁbroblastic proliferation. These features parallel the structural changes that occur within the rheumatoid synovial tissues. Enhanced activation of a hypoxia/redox-controlled transcription factor, NFnB, and its predominant vascular localisation within the inﬂamed synovium further stresses the pivotal inﬂuence of free radicals in the disease [55]. NFnB controls the transcription of the integrins. Inﬂamed synovial endothelial cells show enhanced surface expression of a series of integrin molecules (ELAM-1/ICAM-1) [56]. Lymphocytes and neutrophils within inﬂamed synovial tissues also show enhanced expression of the associated integrins (LFA-1, VCAM-1, VLA-4, and CD11a/CD18, respectively) [57]. The enhanced expression of such surface molecules facilitates the recruitment of inﬂammatory cells, further exaggerating the inﬂammatory reaction. The pannus of chronic RA, which is adjacent to areas of predominant connective tissue destruction, contains a heterogeneous population of cells. In common with both normal and inﬂamed synovial membrane, pannus contains phagocytic type A and secretory type B synoviocytes. Lymphocytes (both T and B) and plasma cells constitute the tissue to variable extents, with activated T lymphocytes predominating. In vascularized regions, endothelial cells, attendant pericytes, and interstitial ﬁbroblasts are also present. Neutrophils have also been found at the cartilage-pannus junction, implicating some direction to their distribution in pannus. Thus, the cellular constituents of pannus confer a formidable armory for the destruction of cartilage. All of these cell types recruited to the pannus tissue have the capacity, once aﬀected by factors such as cytokines and redox imbalance within the local milieu, to generate a range of reactive oxygen and nitrogen species, broadly classiﬁed as free radicals. Oxidant-mediated or -modulated events within the inﬂamed rheumatoid joint are represented in Figure 4. 3.

Cartilage Breakdown and Bone Resorption

The proteoglycans are important structural constituents of connective tissue, particularly of cartilage, where their loss diminishes the rigidity and load-bearing properties of articular cartilage. Enzymes produced by PMNs are known to cleave the core protein of the proteoglycan subunit, in particular elastase and chymotrypsin. Activated neutrophils are also capable of producing the superoxide radical, which can

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Figure 4

601

Oxidants and the regulation of transcription factor and gene expression.

degrade proteoglycan in a similar manner as its eﬀect on hyaluronate. It is now clear that PMN are not the sole source of these oxidants and that other oxidant-generating enzyme systems are operative at cellular level, which inﬂuence both physiological and pathological function. Recent studies on the regulation of mitochondrial respiration and functions of articular chondrocytes by nitric oxide provide further evidence for oxidant involvement in the alterations of chondrocyte function under mimicked hypoxic conditions of the inﬂamed joint. This study shows that nitric oxide inhibits respiration and ATP and proteoglycan synthesis of cultured chrondrocytes, particularly under low oxygen concentrations. In addition, cellular levels of both HSP-70 and HSP-32 (heme oxygenase-1) were reported to be increased markedly under these conditions [59]. The bone microenvironment involves a complex sequence of cellular events occurring at speciﬁc regions. Site-speciﬁc signals that can promote cell diﬀerentiation, proliferation, and coupling [between osteoblasts (OB) and osteoclasts (OC)] must occur at the correct time in the sequence of bone remodelling. A multitude of mediators are implicated in regulating this mechanism. Such mediators include systemic hormones, cytokines, and growth factors (see Table 2). Reactive oxygen and nitrogen species, often regarded as free radicals, have also been recognized as mediators of bone remodeling. A number of growth factors and cytokines are known to induce free-radical generation from a variety of cell types, implicating that the mediators of the responses to eﬀector molecules such as growth factors and cytokines could still be free radicals [60]. Recent ﬁndings relating to the bone remodeling control systems have identiﬁed a signaling cascade that may be the basic mechanism through which both physiological and pathological bone remodeling

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is brought about. The signaling molecules and pathways involved downstream of the growth factor/cytokine-receptor binding facilitate the resultant eﬀect. It has been discovered that OBs play a signiﬁcant role in osteoclastogenesis via cell-cell contact with OC precursors. This interaction is mediated through a ligand on the OB surface [known as osteoprotegerin ligand (OPGL) or osteoclast diﬀerentiation factor (ODF), or receptor activator of NFnB ligand (RANKL)], with a receptor on the OC precursor [receptor activator of NFnB (RANK)] [61–63]. The interaction is also modulated by the presence of a soluble decoy receptor for OPGL, which is identiﬁed as the osteoprotegerin molecule [termed OPG or osteoclast inhibitory factor (OCIF)] [64]. The interaction of these eﬀector molecules and how this network can be modulated by oxidants such as H2O2 are summarized in Figure 5. RANKL expression in RA synovial ﬁbroblasts and the lining layer where synovium abuts the bone has recently been described [65]. The OPGL-RANK interaction is believed to lead to the activation of a range of transmembrane factors on the OC and/or OC precursors, namely, TNF receptor– associated factors (TRAF). Several have been identiﬁed ranging from TRAF-1 to TRAF-6. These factors then mediate the activation of NFnB and c-Jun N-terminal kinase, leading to diﬀerentiation, survival, and activation of OC [61–63]. Involvement of free radicals in modulating these systems is not limited to such recently identiﬁed factors but also at the level of a number of cellular oncogenes such as c-fos and c-src, which are established eﬀector molecules in bone remodeling [66,67]. We and others have previously reported that reactive oxygen species, such as hydrogen peroxide and nitric oxide/peroxynitrite, inﬂuence the bone resorption process [68–70]. It is now generally believed that hydrogen peroxide directly inﬂuences

Figure 5

OPG/RANKL interactions.

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the formation, diﬀerentiation, and activation of the OC. It is also known that hydrogen peroxide is involved in the cytokine-mediated activation of NFnB [13]. Such activation has also been reported speciﬁcally in bone cells [71]. In addition, another transcription factor involved in bone-remodeling processes, namely AP-1, is also modulated by oxidants [14]. It is clear that both NFnB and AP-1 play signiﬁcant roles in bone remodeling and their activation is modulated by oxidants such as hydrogen peroxide and nitric oxide/peroxynitrite. The involvement of NFnB in the OPGL-RANK interactions is evident. Recent reports have also revealed an important link between RANK signaling and the expression of AP-1 proteins (mainly Fos and Jun) in osteoclast diﬀerentiation [72]. We argue that although clear evidence is not yet available, a mediator or signaling role of oxidants in the OPGL-RANK interactions and thus bone remodeling is clearly justiﬁed. In this context, the eﬀects of free radicals on bone resorptive processes have been reported previously. One such example is the observation that cytokines stimulate cells from both synovium and cartilage to produce free radicals [69]. It is believed that these radicals can modulate the control of OB/OC coupling, thus mediating bone remodeling. Free radicals may be mediator/modulator molecules at several diﬀerent stages of bone remodeling. Superoxide radical is generated via NADPH oxidase in the ruﬄed border and released into the site of resorption. Superoxide dismutase can inhibit resorption at this level, implying that the degradation of the matrix itself may be enhanced by oxidative damage caused by superoxide or its derivative species. At the level of osteoclastic diﬀerentiation and development, hydrogen peroxide (although not a free radical but a highly reactive oxidant) can induce ostoclastogenesis and osteoclast motility in vitro [70]. Although the role of oxidants in bone remodeling is beginning to become clear, there is still scope for extensive and detailed investigations in order to elucidate the exact mechanisms by which oxidants mediate and/or modulate bone remodeling. B.

Xanthine Oxidoreductase: A Unique Source of Free Radicals Within the Environmental Conditions of the Inflamed Synovium

The relevance of this enzyme in hypoxia/redox-driven pathology has been the subject of much debate since xanthine was considered to be the essential substrate for the enzyme-mediated generation of ROS via reduction of molecular oxygen. It is now clear that the enzyme is also capable of using NADH as a substrate, which facilitates the reduction of molecular oxygen to ROS [73–75]. There is a large body of evidence for the accumulation of NADH in hypoxic tissues. In fact, the recycling of this molecule forms an essential part of the basic redox cycling events during oxidative phosphorylation, facilitating ATP generation. Under physiological conditions, the xanthine oxidoreductase is believed to metabolize xanthine/hypoxanthine to urate with the aid of NAD+ as the electron acceptor. It follows, therefore, that this redox-active enzyme, having three intact redox centers, is capable of maintaining the redox balance via alternately using NAD+ as an electron acceptor and generating NADH as well as using NADH as a substrate during hypoxia, leading to ROS generation. The reactions of the redox-active centers of the enzyme are indicated in Figure 6. The former mechanism feeds into the ‘‘salvage pathway’’ of purine metabolism, maintaining the nucleotide pool, whereas the latter

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Redox-active catalytic centers of the enzyme xanthine oxidoreductase.

mechanism may act as a signal transducer for redox changes due to hypoxia. We have described the presence and the localization of the enzyme to the inﬂamed synovial microvascular beds [75]. We have recently reported that under conditions of hypoxia, this enzyme also generates nitric oxide from nitrates and nitrites [76]. We previously showed the presence of high levels of nitrite in rheumatoid plasma and synovial ﬂuid [77]. In addition, we have also identiﬁed that the dual capacity of this enzyme to generate both nitric oxide and superoxide radical facilitates, under deﬁned oxygen tensions, the simultaneous generation of both of these species, which react together to form a more potent oxidant, peroxynitrite [78]. The reactions of this enzyme are such that in fully oxygenated settings, the predominant oxidant generated is the superoxide radical.

Figure 7 Nitric oxide and superoxide interactions generating peroxynitrite at varying oxygen concentrations.

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Table 2

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Mediators of Bone Remodeling

Mediators Parathyroid hormone 1,25-Dihydroxyvitamin D3 Glucocorticoids Calcitonin Interleukins

Tumor necrosis factor Colony stimulating factors

Interferon-g Receptor activator of NFnB ligand/osteoprotegerin ligand Osteoprotegerin Insulin-like growth factors Transforming growth factor Fibroblast growth factor Platelet-derived growth factor

Epidermal growth factor Bone morphogenic proteins Estrogen Heparin Thyroid hormones Bradykinin Thrombin Vitamin A Prostaglandin E2 Nitric oxide Superoxide Hydrogen peroxide Peroxynitrite

Abbreviation PTH 1,25Vit D CT IL-1 IL-2 IL-3 IL-4 IL-6 IL-8 IL-10 IL-11 IL-13 TNF-a GM-CSF M-CSF LIF SCF IFN-g RANKL/OPGL OPG IGF-1 IGF-2 TGF-h aFGF bFGF PDGF-AA PDGF-AB PDGF-BB EGF BMPs 1-8

NO O.2 H2O2 ONOO

Stimulators of bone resorption

Osteoclast diﬀerentiation/ formation

+ + +/ + +/ ? +/ + + + ? +/ +

+ + +/ + +/ + + + ? + + + + + +

+/ +/ +/ + + + + + + +/ + + + + + + +/ +/ + ?

+ + +/ + + + + + + +/ ? + + ? + + ? ? ? ?

+, positive eﬀects of bone resorption, diﬀerentiation, and activation; , negative eﬀects.

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Conversely, under conditions where oxygen is limited, the predominant oxidant is nitric oxide. Under conditions where the oxygen tension lies midway within this range, both oxidants are simultaneously generated and react together to form peroxynitrite. These reactions are diagrammatically represented in Figure 7. Recently we also provided indirect evidence for the generation of peroxynitrite within the inﬂamed rheumatoid synovium and discussed its signiﬁcance. In this study we showed the abundance and the distribution of 3-nitrotyrosine (3-NT) immunoreactivity, a marker for the reaction of peroxynitrite with protein tyrosine residues [79]. Considering the signiﬁcance of modiﬁed (phosphorylated and nitrated) amino acid residues in signaling and gene activation events, this particular function of the enzyme presents its potential for acting as a ‘‘sensor’’ for alterations in the local oxygen tension, with redox-mediated signaling capacity. The predominant endothelial localization of the enzyme indicates a strategic locality for it to act as a sensor of any pO2 changes as well as related metabolic alterations, such as NADH/NAD+ redox balance. The enzyme has speciﬁc high aﬃnity for sulfated glycosaminoglycans (GAGs) [80], integral structural components of extracellular matrix. The sulfation pattern of GAGs is particularly inﬂuenced by hypoxia [81]. In this context, we propose xanthine oxidoreductase as a putative sensor for environmental changes transduced via the extracellular matrix GAGs. The presence of high circulating levels of this enzyme in various conditions, including rheumatoid arthritis [82], may also indicate the GAG-mediated ‘‘trapping’’ or ‘‘recruitment’’ of this putative sensor where it is required to transduce the signal from an extracellular to intracellular environment via free radical or reactive oxygen species production. Its capacity to generate such species under nonphysiological conditions (particularly low pO2) in human tissues gives the enzyme its uniqueness in mediating oxidant-driven processes, which modulate cellular function. This, we believe, in evolutionary terms, is a ﬁrst-line defense and repair mechanism, which has the potential, under conditions where redox balance is compromised, to mediate pathological changes. REFERENCES 1. 2. 3. 4. 5.

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Index

AAF, 203 ABCR, 377 ABPP, 423 2-acetylaminoﬂuorene (AAF), 203 Acetylcysteine, 315 Aconitase, Huntington’s disease (HD), 446 Acquired immunodeﬁciency (AIDS), 381 Activating protein-1 (AP-1), 594 oxidative activation, 84 Acute chest syndrome, 307 Acute inﬂammatory response, apoptosis, 592–593 cell proliferation, 595 combative and reparative gene activation, 594–595 combative-stress protein response, 594 defensive-respiratory burst, 593–594 free radicals, 590–595 necrosis, 592–593 phagocytosis, 593 reactive oxygen species (ROS), 595 vascular responses, 591–592 Acute lymphocytic leukemia, 293 Adaptation, 123 Adenine base, oxidation reactions, 157–159 Adenomatous polyposis coli (APC) gene, 496 ADP-ribosyl transferase activity (ADPRT), 500 Advanced glycation end products (AGE), 512, 521–522 inhibitors, 525

Advanced lipoxidation end products (ALE), 525 A2E, 352 Aerosolized thiols, cystic ﬁbrosis (CF), 273–274 AGE, 512, 521–522 inhibitors, 525 Age-related cataract (see Senile cataract) Age-related disease, mitochondria, 127–130 Age-related macular degeneration (AMD), 354–355 antioxidant supplements, 364–366 Bruch’s membrane, 352–353 cigarette smoke, 355 dietary fat, 357–359 dry, 353 examples, 354 genetics, 355–357 iris color, 362–363 lutein, 364–366 lycopene, 364–366 macular pigment, 362–363 noncarotenoid antioxidants, 363–364 outlook, 366–367 oxidative stress, 349–367 polyunsaturated fatty acids (PUFA), 357–359 prevalence, 355 retina, 352–354 risk factors, 354–355 vitamin E, 364 zeaxanthin, 364–366 zinc, 366 611

612 Aging, colon cancer, 498 genetic determinants, 122–124 mitochondria, 124–125 mitochondrial decline, 125–126 mitochondrial DNA variants, 126–127 network theory, 122 reducing power proﬁle, 29–30 remodeling theory, 122–123 AIDS, 381 Aldosterone, endothelin (ET), 549 essential hypertension, 546 ALE, 525 Alkaline elution technique, 144, 169 Alkaline single cell gel electrophoresis, 169 Alkaline unwinding method, 169 Alpha-thalassemia, vs. beta-thalassemia, 313 classiﬁcation, 308 Alpha-tocopherol, 504 retina, 359–360 structure, 503 ALS, 218, 381, 428 Alzheimer’s disease, 127, 381, 413, 423–432, 428 aluminum, 430–431 antioxidants, therapeutic potential, 431–432 compensatory responses, 427–428 metals, 429–431 mitochondria, 130 oxidative damage, 425–427 Amadorins, 525 AMD (see Age-related macular degeneration) Aminoguanidine, 525 Aminotriazole, inducing cataracts, 336 Amyloid beta protein precursor (ABPP), 423 Amyotrophic lateral sclerosis (ALS), 218, 381, 428 Ang II (see Angiotensin II) Angiotensin-converting enzyme gene, 557 Angiotensin II (Ang II), 539 aldosterone, 547 blood pressure, 547 endothelin (ET), 547–549 ﬂuid volumes, 455–457 heart rate, 543 intrarenal eﬀects, 455–456 levels, 455–457 mean arterial pressure, 543

Index [Angiotensin II (Ang II)] oxidative stress, 547 plasma level, 543 pressor response, 541–544 renal blood ﬂow, 547 response, 542 signaling, 82 Angiotensinogen, 540 Anthrones, 198 Anti-AGEs, diabetes mellitus, antioxidants, 524–527 Antioxidants, carcinogenesis inhibition, 202–205 cataract protection, model systems, 339 defense mechanisms, retinitis pigmentosa (RP), 387–390 deﬁned, 11 depletion, 266 retina, 359–360 species, plasma, 269 respiratory tract lining ﬂuids, 269 supplements, age-related macular degeneration (AMD), 364–366 AP-1, 594 oxidative activation, 84 Apaf-1, 379 APC gene, 496 Apolipoprotein E (APOE), 423, 555–556 closed head injury (CHI), voltammetric measurements following, 31 Apoptosis, 103–104 acute inﬂammatory response, 592–593 Bcl-2, 109–110 caspases activation, 104 hallmarks, 103–104 hydrogen peroxide, 106–107 mitochondria, 133 nitric oxide (NO), 111–112, 384–385 oxygen, 105–106 Rac1, tumor cells, 108–109 RacGTPase, tumor cells, 108–109 reactive oxygen species (ROS), 104–107 mediated apoptotic signaling, 108– 112 redox regulation, 99–114 retinitis pigmentosa (RP), 377–381 thiol, 112–113 Applied potential, in vivo voltammetry (IVV), 34 Asbestos, lung cancer, 293

Index Ascorbate, glutathione/glutathione disulﬁde, 16 retina, 359–360 Ascorbic acid (vitamin C), cystic ﬁbrosis (CF), 267, 273–274 glutathione/glutathione disulﬁde, 16 lens, 328 Atherogenesis, theories, 557 Atherosclerosis, genetics, 556–557 mitochondria, 128 oxidative stress, 555–560 risk factors, 555–556 Atorvastatin, 566 ATP-binding cassette transporter of rods (ABCR), 377 ATP synthase (complex V), 237 Atrial natriuretic peptide, 540 Autoimmune myocarditis, thioredoxin (TRx), 68–69 Bacteria, redox signaling, 78–79 Bacterial infection, respiratory tract lining ﬂuids (RTLF), 272 Bacterial siderophores, iron sequestration, 267 Bcl-2, 389–390 apoptosis, 109–110 BDNF, 381 Benzoyl peroxide, 201 Best’s disease, 355 Beta-thalassemia, vs. alpha-thalassemia, 313 classiﬁcation, 311 glutathione (GSH), 313 Heinz bodies, 309 point mutations, 310 Bezaﬁbrate, 558 BHA, 198, 203 BHT, 198, 203 BHTOOH, 198 Bilirubin UDP-glucuronysyltransferase, 221 Biological eﬀects, oxidative stress, 265 Biological environment, catalytic iron, 287 Biological ﬂuids, cyclic voltammetry (CV), 26–27 diﬀerential pulse voltammetry (DPV), 26–27 oxidative base damage, 171–172 Biological reducing power (BRP), 12 chemical measurement, 17–18

613 [Biological reducing power (BRP)] chemiluminescence, 17 compounds contributing, 13 direct measurement, 17–22 electrochemical measurement, 18–22 evaluation, 15–26 glutathione/glutathione disulﬁde, 16 indirect measurement, 15–17 lactate pyruvate, 16 low-molecular-weight antioxidants (LMWA), 13–15 NADP/NADPH, 15 oxygen radical absorbance capacity (ORAC), 17 reducing power evaluation, 26–27 voltammetric measurements, 28–32 Biological regulation, free radicals, 77–78 Biological tissues, cyclic voltammetry (CV), 28 diﬀerential pulse voltammetry (DPV), 28 Bone remodeling, 599 mediators, 603 Bone resorption, rheumatoid arthritis, 598–601 Bottleneck enzymes, 239 Brain-derived neurotrophic factor (BDNF), 381 BRP (see Biological reducing power) Bruch’s membrane, age-related macular degeneration (AMD), 352–353 Buthionine sulfoxamine (BSO), inducing cataracts, 336 Butylated hydroxyanisole (BHA), 198, 203 Butylated hydroxytoluene (BHT), 198, 203 Butylated hydroxytoluene hydroperoxide (BHTOOH), 198 cAMP-mediated redox pathways, T-cell proliferation, 85 Cancer, mitochondria, 129 Candidate genes, 216 Captopril, 558 Carcinogenesis, inhibition, antioxidants, 202–205 free radicals, 202–205 iron, 293–294 Carotenoids, 513 lens, 328 retina, 359–360 Cartilage breakdown, rheumatoid arthritis, 598–601

614 Caspases, 378 Activation, apoptosis, 104 nitric oxide (NO), 385–386 pathways, 379 Catalase, 102, 198, 218 lens, 328 Catalytic iron, 286–287 biological environment, 287 Cataractogenesis, lens, 324–325 oxidative stress, 323–339 Cataracts, congenital, 324 Coppock-like, 324 cortical, 323–324 extractions, 325 nuclear, selenite induced, 336 CBP, 438 Cell antioxidant defenses, 101–102 Cell damage, free radicals, 2–3 Cell death, Huntington’s disease (HD), 451–452 modes, 103–104 nitric oxide (NO), 111 reactive oxygen species (ROS), 102–108 Cell electrophoresis (comet) assay, 144 Cellular antioxidants, low density lipoproteins, oxidation, 563–565 Cellular DNA, gas chromatography-mass spectrometry assays (GC-MS), 165–167 HPLC-electrochemical detection, 167 HPLC-MS/MS, 167–169 HPLC32-P postlabeling technique, 164–165 ligation mediated-polymerase chain reaction (LM-PCR), 163–164 oxidative base damage, 170–172 enzymatic measurement, 169–170 oxidative lesion, nonchromatographic methods, 163–164 singling out, 162–169 Cellular redox control, thiols, 59–70 thioredoxin (TRx), 59–70 Cellular retinaldehyde-binding protein (CRALBP), 377 Cerebral blood ﬂow abnormalities, Huntington’s disease (HD), 453 Ceruloplasmin, 428, 454 CF (see Cystic ﬁbrosis)

Index CFTR, 261–262 nitric oxide (NO), 271 CGD, 561 CGMP, nitric oxide (NO), 385 Chaperones, 234 Chaperonins, 428 Chemiluminescence, biological reducing power (BRP), 17 CHI, voltammetric measurements following, 30–31 Cholesterol, 557 Choroidal neovascularization, 354 Choroidal neovascular membrane, laser ablation, 353 Chromosome 21, 407 Chronic granulomatous disease (CGD), 561 Chronic inﬂammation, mitochondria, 128 Chrysotile, 293 Cigarette smoke, age-related macular degeneration (AMD), 355 Ciliary neurotrophic factor (CNTF), 381 Clinical syndromes, mitochondria, 247–253 Closed head injury (CHI), voltammetric measurements following, 30–31 CNTF, 381 Coenzyme Q10 (ubiquinone), Huntington’s disease (HD), 458 Colon cancer, 495–506 environmental factors, 498–499 ﬂavonoids, 502 genetic factors, 499–500 genetic testing, indications, 496–497 impact, 497–498 inﬂammatory bowel disease, 501 inherited syndromes, 496 iron, 293 nutritional factors, 501–506 oxidative stress, 497 phenotypic and genetic aspects, 495–497 risk factors, 498–506 salicylates, 502 smoking, 498 vitamin E, 502–506 Combative and reparative gene activation, acute inﬂammatory response, 594–595 Combative-stress protein response, acute inﬂammatory response, 594 Comet assay, 144, 169 Cones, 351 Congenital cataracts, 324

Index Connexins, 324 Constant potential amperometry (CPA), in vivo voltammetry (IVV), 34 Copper, 428 Alzheimer’s disease, 429–431 familial amyotrophic lateral sclerosis (FALS), 479–480, 481–482, 484 low-density lipoproteins (LDL) oxidation, 560 Copper-zinc-superoxide dismutase (CuZnSOD), 424, 515–516 deleterious eﬀects, 409 Down syndrome (DS), 408 enzyme, 101 overexpression, 429 Coppock-like cataracts, 324 Cortical cataracts, 323–324 COX-2, 199 CPA, in vivo voltammetry (IVV), 34 CRALBP, 377 Creatine, 459 CREB-binding protein (CBP), 438 Crocidolite, 293 CuZnSOD (see Copper-zinc-superoxide dismutase) CV (see Cyclic voltammetry) Cybrids, mitochondrial mutations, 251–252 Cyclic voltammetry (CV), 5, 19, 22, 23–24 biological ﬂuids, 26–27 biological tissues, 28 diabetic rats, 32 instrumental setup, 25–26 low-molecular-weight antioxidants (LMWA), 21, 26 tissue, 27–28 methodology, 25–26 rat cells, reducing power proﬁle, 30 sensitivity, 23–24 tissue culture from cell lines, 28 Cyclooxygenase (COX-2), 199 Cysteine, 44 Cystic ﬁbrosis (CF), 261–275 antioxidant therapy, 273 ascorbic acid, 267 gene, 262 neutrophil antimicrobial mechanisms, 267 nitric oxide, 270–272 oxidative stress, 264–268 airway surface, 268–270 evidence, 265–266 indices, 266

615 [Cystic ﬁbrosis (CF)] potential contributors, 265 oxygen-derived species, 263–264 plasma lipophilic antioxidants, 268 vitamin E, 267 Cystic ﬁbrosis transmembrane conductance regulator (CFTR), 261–262 nitric oxide (NO), 271 Cytochrome c, 379 Cytochrome c oxidase (complex IV), 237 Cytoplasmic protein kinases, redox regulation, 83–84 Cytosine base, OH radical, oxidation reactions, 148–150 Cytosolic pH, reactive oxygen species (ROS)-mediated apoptotic signaling, 107–108 DBMA, 195 DbSNP, 215 Decomposition pathway, 5-yl, 149 6-yl, 150 Defensive-respiratory burst, acute inﬂammatory response, 593–594 Deferiprone, 315 DEN, 203 DEPMPO, 448 Dextrose, response, 542 DHA, 381 DHBA, 448 Diabetes mellitus, Antioxidants, with anti-AGEs, 524–527 DNA, oxidation, 519–520 enzymatic antioxidant defenses, 515– 516 macromolecules, oxidation, 518–520 manganese, 516 metals, 516 nonenzymatic antioxidant defense, 517 oxidative imbalance, 511–527 cellular targets, 514–515 evidence, 512–518 markers, 513 plasma total antioxidant status, 517–518 selenium, 516 vitamin C, 517 vitamin E, 517, 524–527 zinc, 516 Diabetic rats, cyclic voltammetry (CV), 32 Dietary fat, age-related macular degeneration (AMD), 357–359

616 5–Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), 448 Diﬀerential pulse amperometry (DPA), in vivo voltammetry (IVV), 34 Diﬀerential pulse voltammetry (DPV), 25 biological ﬂuids, 26–27 biological reducing power (BRP), 20–22 biological tissues, 28 low-molecular-weight, antioxidants (LMWA), tissue, 27–28 tissue culture from cell lines, 28 in vivo voltammetry (IVV), 34 Digenic retinitis pigmentosa (DRP), 376 2,3-dihydrobenzoic acid (DHBA), 448 Dihydroguanine, secondary oxidation, 155–157 tandem base modiﬁcations, 160–161 Dihydrorhodamine, 440 3,4-dihydroxyphenylacetic acid (DOPAC), 33, 449 7,12-dimethylbenz-anthracene (DBMA), 195 Dimethylbiguanide, 526–527 Dimethylnitrosamine (DEN), 203 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 448 Disciform scarring, 354 Disulﬁdes, 44 Dithiothreitol (DTT), 386 DMPO, 448 DNA, cellular (see Cellular DNA) Huntington’s disease (HD), 457–458 microarrays, 7 mitochondrial, 126–127, 234, 242–243. (see Mitochondrial DNA) oxidation, 266 markers, 515 repair enzymes, 144 single base lesions, 144–162 Docosahexaenoic acid (DHA), 381 Docosanoid precursors, 358 Dominant drusen, 355 DOPAC, 33 Dopamine, 33 Huntington’s disease (HD), 454–455 3-nitropropionic acid, 449 Down syndrome, 407–418 copper/zinc SOD, 408 glutathione peroxidase (GSHP), 408 mouse models, 411–416

Index [Down syndrome] sinet hypothesis, 408 transgenic mouse models, 411–413 trisomic mouse models, 413–416 DPA, in vivo voltammetry (IVV), 34 DPV (see Diﬀerential pulse voltammetry) DRP, 376 Dry age-related macular degeneration (AMD), 353 DT diaphorase (quinone reductase), 328 DTT, 386 EC-SOD, 515–516 ECT, 235–238, 382, 383 EGFR, signaling, 83 Eicosanoid precursors, 358 Eicosatetraenoic acid (ETA), 204 8-epi-PGF2 alpha, 513 8-hydroxydeoxyguanosine (8-OHdG), 458, 513, 519–520 8-hydroxyguanosine (8-OHG), 425–427, 500 8-oxo-7,8-dihydroguanine, secondary oxidation, 155–157 tandem base modiﬁcations, 160–161 Electrochemical titration, biological reducing power (BRP), 18 Electrodes, in vivo voltammetry (IVV), 33–34 Electron paramagnetic resonance (EPR) spectrometer, 2, 448 Electron transport chain (ECT), 235–238, 382, 383 ELF, 269 Enalapril, 558 Endoplasmic reticulum (ER), 52 Endothelial NO synthase, 540 Endothelin, 539 aldosterone, 549 angiotensin II, 547–549 renin-angiotensin system, 548–549 Energy metabolism, Huntington’s disease, 440–452 ENU, 201 Environmental factors, colon cancer, 498–499 Enzymatic antioxidant defenses, 515– 516 Enzyme systems, thiol-disulﬁde redox state, 44–46 Epidermal growth factor receptor (EGFR), signaling, 83

Index 8-epi-PGF2 alpha, 513 Epithelial lining ﬂuids (ELF), 269 EPR spectrometer, 2, 448 Epstein-Barr infection, thioredoxin (TRx), 67 ER, 52 Essential hypertension, 539–550 genetics, 540–541 ETA, 204 Ethylnitrosourea (ENU), 201 Extensive drusen, 354 Extracellular matrix molecules, oxidation, diabetes mellitus, 518 Extracellular superoxide dismutase (EC-SOD), 515–516 Extracellular thioredoxin (TRx), 63–66 anti-inﬂammatory eﬀect, 65–66 chemokine-like eﬀect, 64–65 cytokine-like eﬀect, 64 Eye, anatomy, 350 FALS (see Familial amyotrophic lateral sclerosis) Familial adenomatous polyposis (FAP), 496 Familial amyotrophic lateral sclerosis (FALS), 218, 471–487 copper, 479–480, 481–482, 484 metal binding, 481–482 nitric oxide (NO), 484 OH radical, 477–478 oxidative stress, mechanisms, 477–482 peroxynitrite, 480–481, 484 superoxide, 471–476 superoxide dismutase (SOD), 471–476 in vivo evidence, 482–486 zinc, 481–482 FAP, 496 Fat-soluble vitamins, gastrointestinal absorption, 264 Fenton chemistry, 286–287 Ferric nitrilotriacetate, 294 Ferric reducing/antioxidant power and ascorbic acid concentration(FRASC) assay, 18 Ferric reducing/antioxidant power (FRAP) assay, 18, 517–518 Ferritin, 428 FGF, 381 Fibroblast growth factor (FGF), 381 5-yl decomposition pathway, 149 Flavonoids, 558 colon cancer, 502

617 Flurbiprofen, 205 Formylamine, tandem base modiﬁcations, 160–161 Fosinopril, 558 Foveola, 350 FRAP assay, 18, 517–518 FRASC assay, 18 Free radicals, 438 acute inﬂammatory response, 590–595 biochemical entities, 1–2 biological regulation, 77–78 carcinogenesis inhibition, 202–205 cell damage, 2–3 damaging species, 1–2 Huntington’s disease, generation, 439 regulated production, 80–81 regulating species, 3–4 rheumatoid arthritis, 589–605 GAG, 605 Gamma-tocopherol, 504 structure, 503 GAPDH, Huntington’s disease (HD), 451 Gas chromatography (GC), 13 Gas chromatography-mass spectrometry assays (GC-MS), 165–167 cellular DNA, 165–167 Gastrointestinal tract, voltammetric measurements along, 31–32 GC, 13 GC-MS, 165–167 cellular DNA, 165–167 Gemﬁbrozil, 558 Gene knockout mouse models, 7 gene overexpression, 338 Gene overexpression, gene knockout mouse models, 338 Genetic diseases, mitochondrial dysfunction, 233–253 Genetics, age-related macular degeneration (AMD), 355–357 atherosclerosis, 556–557 colon cancer, 499–500 essential hypertension, 540–541 Genetic testing, colon cancer, indications, 496–497 Geographic atrophy, 353, 354 Glial cells, retina, 350 Gliclazide, 526

618 Globin chains, excess, 312 Glucose, autoxidation, 521 Glutathione (GSH), 42–44, 198, 389, 428, 440 beta-thalassemia, 313 dependent systems, thiol-disulﬁde redox state, 45–46 redox cycle, 389 respiratory tract lining ﬂuids (RTLF), 272 Glutathione/glutathione disulﬁde, ascorbic acid/ascorbate, 16 total radical trapping potential (TRAP), 17 trolox equivalent antioxidant capacity (TEAC), 16–17 Glutathione peroxidase, 102, 204, 219, 516 Down syndrome (DS), 408 Glutathione transferases (GST), 217 Glyceraldehyde-3-phosphate (GAPDH), Huntington’s disease (HD), 451 Glycosaminoglycans (GAG), 605 G-protein-coupled enzyme cascade, 376 Growth control, 47–48 GSH (see Glutathione) GST, 217 Guanine, decomposition pathways, 151–154 oxidation reactions, 150–157 singlet oxygen oxidation, 154 Haber-Weiss cycle, 145 HbE, Southeast Asia, 305 HbS, 304–305, 306–307 Hb variants, 304–305 HDL, 519 Heavy metals, Huntington’s disease (HD), 454 Heinz bodies, beta-thalassemia, 309 Heme oxygenase (HO-1), 221 gene, 77 Hemichromes, formation, 312 Hemoglobin disorders, hypercoagulable state, 315–317 Southeast Asia, 303 Hemoglobinopathy, population migration, 303–304 HEMPAS, 315–316 Hepatitis C, thioredoxin (TRx), 68 Hepatocellular carcinoma, hereditary hemochromatosis (HHC), 292, 295–296

Index Hereditary dyserythropoietic anemia (HEMPAS), 315–316 Hereditary hemochromatosis (HHC), 285–296 hepatocellular carcinoma, 292, 295–296 liver, autopsy, 291 Hereditary nonpolyposis colorectal cancer (HNPCC), 496 Hereditary persistence of fetal Hb (HPFH), 310 Heterochromatic ﬂicker photometry (HFP), 361 Heteroplasmy, 240–242, 249–250 Hexose monophosphate shunt (HMPS), 408 HFP, 361 HHC (see Hereditary hemochromatosis) High density lipoproteins (HDL), 519 High-performance liquid chromatography (HPLC), 13 electrochemical detection, cellular DNA, 167 low -molecular-weight antioxidants (LMWA), 27–28 MS, cellular DNA, 167–169 postlabeling technique, cellular DNA, 164–165 HIV, thioredoxin (TRx), 65–66 HLA genes, 512 HMPS, 408 HNE, 515 4-HNE, 145 HNPCC, 496 HO-1, 221 gene, 77 HOC1, 382, 590 Hodgkin’s disease, 293 Homocysteine, 219, 556–557 Homoplasmy, 240–242, 249 HPFH, 310 HPLC, electrochemical detection, cellular DNA, 167 low-molecular-weight antioxidants (LMWA), 27–28 MS, cellular DNA, 167–169 postlabeling technique, cellular DNA, 164–165 HSP–32, 594 HSP-70, 594 HTLV-1, 59–60 infection, thioredoxin (TRx), 65

Index Human colon, carcinogenic process, 192 Human genome, oxidative modiﬁcation, 143–173 Human Genome Initiative, 243–244 Human globin gene mutations, 304 Human T-cell leukemia virus type-1 (HTLV-1), 59–60 Infection, thioredoxin (TRx), 65 Human umbilical vein endothelial cells (HUVEC), 307 Huntington’s disease, 381, 428, 437–459 aconitase, 446 animal models, imaging, 443–444 cell death, 451–452 cerebral blood ﬂow abnormalities, 453 coenzyme Q10 (ubiquinone), 458 dopamine, 454–455 excitotoxicity, 455–456 free radicals, generation, 439 glyceraldehyde-3-phosphate (GAPDH), 451 heavy metals, 454 inﬂammatory response, 454 iron, 454 macromolecules, oxidative stress, 456–459 malonate, 447 mitochondrial respiratory chain, 444–451, 445–447 nitric oxide (NO), 452–454, 453–454 3-nitropropionic acid, 447 nuclear magnetic resonance spectroscopy, 441–443 oxidative stress, causes, 440–456 positron emission tomography (PET), 440–441 remacemide, 458 therapy, 458–459 transgenic mouse models, 450–451 weight loss, 451 HUVEC, 307 Hydrogen peroxide, 201 apoptosis, 106–107 Hydroperoxides, 513 Hydrops fetalis, 310 8-hydroxy-1’-deoxyguanosine, 194 8-hydroxydeoxyguanosine (8-OHdG), 458, 513, 519–520 6-hydroxydopamine (6-OHDA), 449–450 8-hydroxyguanosine (8-OHG), 425–427, 500

619 Hydroxyl radical (OH), 196 familial amyotrophic lateral sclerosis (FALS), 477–478 Hydroxynonenal (HNE), 515 4-hydroxynonenal (4-HNE), 145 Hyperbaric oxygen, 337 Hypercoagulable state,hemoglobin disorders, 315–317 Hyperglycemia, oxidative stress, 520–524 polyol pathway, 522–523 Hypochlorous acid (HOC1), 382, 590 Hypoxia, rheumatoid arthritis, 596–601 Hypoxia/ischemia injury, neonatal SOD1-transgenic mice, 412 Immune responses, redox-mediated ampliﬁcation, 85–86 rheumatoid arthritis, 596–597 Indomethacin, 205 Ineﬀective erythropoiesis, 312 Inﬂammatory bowel disease, colon cancer, 501 Inﬂammatory process, increased activity, 264 Inﬂuenza, thioredoxin (TRx), 69–70 Inheritance, mitochondrial disease, 243–247 Inherited hemoglobinopathy, redox imbalance, 303–317 Insulin resistance, 523–524 Interleukin-1 beta, 381 Interorgan glutathione/cysteine cycle, 43–44 Interstitial pneumonia, thioredoxin (TRx), 70 Intestinal lumen, redox, 50–51 Intracellular REDST, macrophages, immunological functions, 86–87 Intracellular thiol/disulﬁde redox status, signaling cascades, 78 In vivo voltammetry (IVV), 32–35 applied potential, 34 electrodes, 33–34 reducing power measured, 34–35 Iris color, age-related macular degeneration (AMD), 362–363 Iron, 428 Alzheimer’s disease, 429–431 carcinogenesis, 293–294 colorectal cancer, 293 excess, meat-eating countries, 293

620 [Iron] Huntington’s disease (HD), 454 low-density lipoproteins (LDL) oxidation, 560 metabolism, posttranscriptional regulation, 290–291 redox cycling, 286 thalassemia, 313–315 transporters, 288–290 Iron chelator LI, 315 Iron-dependent oxidative damage, 288 Iron overload-induced carcinogenesis target genes, 294–296 Iron overload syndrome, oxyradicals, 285–296 Iron-regulatory proteins (IRP), 290–291 Iron-responsive elements (RE), 290–291 Iron sequestration, bacterial siderophores, 267 IRP, 290–291 Isoprostanes, 457 IVV, 32–35 applied potential, 34 electrodes, 33–34 reducing power measured, 34–35 Kallikrein, 540 Kearns-Sayre syndrome (KSS), 249 Knockout mice, thioredoxin (TRx), 69 Krebs cycle, 239 KSS, 249 Lactate pyruvate, biological reducing power (BRP), 16 Lactoferrin, 267 Laser ablation, choroidal neovascular membrane, 353 LDL (see Low-density lipoproteins) Leber’s hereditary optic neuropathy (LHON), 127, 249 Lens, ascorbic acid (vitamin C), 328 carotenoids, 328 catalase, 328 cataractogenesis, 324–325 oxidation defense and damage repair, 326–330 protein, 325 repair systems, 328–330 vitamin E, 328 LHON, 127, 249

Index Ligation mediated-polymerase chain reaction (LM-PCR), cellular DNA, 163–164 Light equivalent hypothesis, 390 Linear sweep voltammetry (LSV), 25 Lipid peroxidation, 266, 526 markers, 514–515 Lipid peroxide-rich macrophages, oxidized LDL (Ox-LDL), 567–568 Lipids, Huntington’s disease (HD), 457 Lipofuscin, 352, 355, 383, 457 Lipoproteins, oxidation, diabetes mellitus, 518–519 Lipoxygenase (LO), low-density lipoproteins (LDL), oxidation, 562 Liver, autopsy, hereditary hemochromatosis (HHC), 291 LM-PCR, cellular DNA, 163–164 LMWA (see Low-molecular-weight antioxidants) LO, low-density lipoproteins (LDL), oxidation, 562 Long-term potentiation (LTP), 412 Losartan, 558 Low-density lipoproteins (LDL), 514–515, 518–519, 555–556, 558 cellular antioxidants, oxidation, 563–565 HbA, 313 lipoxygenase (LO), oxidation, 562 myeloperoxidase (MPO), oxidation, 561–562 nitric oxide (NO), oxidation, 562 oxidation, copper, 560 iron, 560 macrophage-mediated, 560–565 mechanisms, 559–560 metals, 560 NADPH oxidase, 560–561 paraoxonase, 568–573 paraoxonase, oxidation, 568–573 peroxide, oxidation, 563 receptor gene, 556 very, 503, 555–556 Low-molecular-weight antioxidants (LMWA), 20 biological reducing power (BRP), 13–15 cyclic voltammetry (CV), 21, 26 tissue, 27–28 diﬀerential pulse voltammetry (DPV)

Index [Low-molecular-weight antioxidants (LMWA)] tissue, 27–28 high-performance liquid chromatography (HPLC), 27–28 reactive oxygen species (ROS), 14 Lp(a) gene, 556 LSV, 25 LTP, 412 Lung cancer, asbestos, 293 Lung conditions, oxidative stress, 264 Lutein, 360–361 age-related macular degeneration (AMD), 364–366 Lycopene, 363 age-related macular degeneration (AMD), 364–366 Macrophages, intracellular REDST, immunological functions, 86–87 oxidized LDL (Ox-LDL), 565–568 scavenger receptors, 565–567 Macula, anatomy, 350 physiology, 350–351 regions, 351 Macula lutea, 350 Macular carotenoid pigment, 360–361 Macular pigment, age-related macular degeneration (AMD), 362–363 measuring, 361–362 Magnesium SOD enzyme, 101–102 Major histocompatibility complex (MHC), 512 Major intrinsic protein (MIP), 324 Malaria parasite, 303 Malonaldehyde (MDA), 143, 145, 315, 525 Malonate, Huntington’s disease (HD), 447 Mammalian cells, oxidative stress responses, 79–80 Manganese, diabetes mellitus, 516 Manganese-superoxide dismutase (MnSOD), 424 MAO, 449 Maternally inherited diabetes and deafness (MIDD), 127–128 Maternal transmission, mitochondrial heteroplasmies, 245–247

621 Maturity-onset diabetes of the young (MODY), 511 MDA, 143, 145, 315, 525 Meat-eating countries, iron excess, 293 MEH, 221 Melanin, 352, 383 Melanotransferrin, 428 MELAS (myopathy, encephalopathy, lactic acidosis, stroke), 128, 250 MERRF (myoclonic epilepsy, ragged red ﬁbers), 250 Metalloenzyme dysfunction, 429 Metals, Alzheimer’s disease, 429–431 diabetes mellitus, 516 low density lipoproteins (LDL) oxidation, 560 Metformin (dimethylbiguanide), 526– 527 MHC, 512 Micronutrients, gastrointestinal absorption, 264 Microsomal epoxide hydrolase (mEH), 221 MIDD, 127–128 Midkine, 381 Minocycline, 456 MIP, 324 Mitochondria, 52–53, 121–135 age-related disease, 127–130 aging, 124–125 Alzheimer’s disease, 130 apoptosis, 133 atherosclerosis, 128 cancer, 129 cell biology, 444 chronic inﬂammation, 128 clinical syndromes, 247–253 coupling and transport, 238 decline, aging, 125–126 Mitochondrial disease, diagnosis, 253 inheritance, 243–247 mouse models, 252–253 non-Mendelian inheritance, 244–245 nuclear mutations, 243–244 Mitochondrial DNA (mtDNA), 234 random mutations, 242–243 variants, aging, 126–127 Mitochondrial dysfunction, genetic basis, 234–247 genetic diseases, 233–253

622 Mitochondrial electron transport chain, 100 Mitochondrial genes, 234–235 Mitochondrial heteroplasmies, maternal transmission, 245–247 Mitochondrial hypothesis, neurodegenerative disease, 444–445 Mitochondrial mutations, cybrids, 251–252 Mitochondrial permeability transition (MPT), 238 Mitochondrial respiratory chain, diagram, 445 Huntington’s disease (HD), 444–451, 445–447 Mitochondrial signalosome, 133–134 Mitochondrion, nitric oxide (NO), 386–387 MNNG, 201 MnSOD, 424 MODY, 511 Molecular genetics, developments, 5–6 retinitis pigmentosa (RP), 376–377 toolbox, redox biology, 7–8 Monoamine oxidase (MAO), 449 Mouse models, Down syndrome (DS), 411–416 MPO, 222–223 low-density lipoproteins (LDL), oxidation, 561–562 MPT, 238 MRM, 167–169 MtDNA, 234 random mutations, 242–243 variants, aging, 126–127 Muller cells, 350 Multiple reaction monitoring (MRM), 167–169 Multistage carcinogenesis, oxygen free radicals, 193–200 Myeloperoxidase (MPO), 222–223 low-density lipoproteins (LDL), oxidation, 561–562 Myoclonic epilepsy, ragged red ﬁbers, 250 Myopathy, encephalopathy, lactic acidosis, stroke, 128, 250 N-acetylcysteine, 315 NADH, oxidase, 100–101 ubiquinone oxidoreductase (complex I), 235–236

Index NADP, biological reducing power (BRP), 15 oxidase, 100–101 NADPH biological reducing power (BRP), 15 oxidase, low-density lipoproteins (LDL) oxidation, 560–561 NARP, 250, 392 Natural resistance-associated macrophage protein 2, 288, 289 NCBI database, 215 NDGA, 204 Necrosis, 103 acute inﬂammatory response, 592–593 Neonatal SOD1-transgenic mice, hypoxia/ ischemia injury, 412 Nerve growth factor (NGF), 381 Network theory, aging, 122 Neuroblastoma, 293 Neurodegenerative disease free radicals, pathogenic role, 439–440 metal imbalance, 428 mitochondrial hypothesis, 444–445 Neurogenic ataxia retinitis pigmentosa (NARP), 250, 392 Neuromuscular junctions (NMJ), 412 Neutrophil antimicrobial mechanisms, cystic ﬁbrosis (CF), 267 NF-jB, 4, 456, 594 oxidative activation, 84 NGF, 381 Nitric oxide (NO), 4 apoptosis, 111–112, 384–385 biological eﬀects, 590 biological overview, 452 caspases, 385–386 cell death, 111 CFTR, 271 cGMP, 385 cystic ﬁbrosis (CF), 270–272 familial amyotrophic lateral sclerosis (FALS), 484 htt, 452 Huntington’s disease (HD), 452–454, 453–454 low-density lipoproteins (LDL), oxidation, 562 mitochondrion, 386–387 regulated production, 80–81 retinitis pigmentosa (RP), 381–382 Nitric oxide synthase (NOS), 222, 384 biological overview, 452

Index 3-Nitropropionic acid, dopamine, 449 Huntington’s disease (HD), 447 malonate, 449 oxidative damage, sources, 449–450 in vitro models, 449 Nitrosants, generation, 263 N-methyl-N-nitro-N-nitrososoguanidine (MNNG), 201 NMJ, 412 Nobel-Prize winning discoveries, 6 Noncarotenoid antioxidants, age-related macular degeneration (AMD), 363–364 Non-Mendelian inheritance, mitochondrial disease, 244–245 Nonregulatory enzymes, 239 Nonsteroidal anti-inﬂammatory drugs (NSAID), 201 Nordihydroguaiaretic acid (NDGA), 204 Norepinephrine, 33 Normal pulse voltammetry (NPV), in vivo voltammetry (IVV), 34 NOS, 222, 384 biological overview, 452 Not bound to transferrin (NTBI), 311–312, 315 NPV, in vivo voltammetry (IVV), 34 Nramp2 (natural resistance-associated macrophage protein 2), 288, 289 NSAID, 201 NTBI, 311–312, 315 Nuclear cataracts, selenite induced, 336 Nuclear genes, 234–235 mutations, 243 Nuclear magnetic resonance spectroscopy, Huntington’s disease (HD), 441–443 Nuclear-mitochondrial cross-talk, 130–133 Nuclear mutations, mitochondrial disease, 243–244 Nuclei, 53 Obesity, type 2 diabetes mellitus, 511 ODF, 600 Oguchi disease, 391 8-OHdG, 458, 513, 519–520 8-OHG, 425–427, 500 Oncogenes, 192 Oogenesis, 246 OPC-14117, 459 OPGL, 600

623 ORAC, biological reducing power (BRP), 17 Osteoblasts, 599 Osteoclast diﬀerentiation factor (ODF), 600 Osteoclasts, 599 Osteoprotegerin ligand (OPGL), 600 Overexpression mouse models, 7 Oxidants, deﬁned, 11 generation, 263 oxidative responses, 266 Oxidation-induced cataract, biochemical mechanism, 331 Oxidative base damage, biological ﬂuids, 171–172 cellular DNA, 170–172 Oxidative DNA base damage, measurement, 162–170 Oxidative imbalance, diabetes mellitus, evidence, 512–518 markers, 513 Oxidative/peroxidative processes, 266 Oxidative phosphorylation (OXPHOS), 130 ﬂux, 238–240 inhibitors, 445 partial deﬁciency, 240–247 Oxidative protein damage, markers, 515 Oxidative stress, 438 age-related macular degeneration (AMD), 349–367 angiotensin II, 547 atherosclerosis, 555–560 biological eﬀects, 265 cataractogenesis, 323–339 colon cancer, 497 cystic ﬁbrosis (CF), airway surface, 268–270 evidence, 265–266 indices, 266 potential contributors, 265 familial amyotrophic lateral sclerosis (FALS), mechanisms, 477–482 Huntington’s disease, causes, 440–456 hyperglycemia, 520–524 lung conditions, 264 plants, protective responses, 80 polymorphism, 215–223 radicals, 144 reactive oxygen species (ROS), 144 redox signaling, 78–80 redox state, 4–5 retina, 359

624 [Oxidative stress] senile cataract, 331–335 sources, 326 thioredoxin (TRx), 60 type 1 diabetes mellitus, 523–524 type 2 diabetes mellitus, 523–524 Oxidative stress-induced cataract models, 335–337 Oxidative stress responses, mammalian cells, 79–80 Oxidized LDL (Ox-LDL), atherogenic properties, 558–559 evidence, 557–558 lipid peroxide-rich macrophages, 567–568 macrophages, 565–568 Oxidized macrophages, 563 Ox-LDL (see Oxidized LDL) 8-oxo-7,8-dihydroguanine, secondary oxidation, 155–157 tandem base modiﬁcations, 160–161 OXPHOS, 130 ﬂux, 238–240 inhibitors, 445 partial deﬁciency, 240–247 Oxygen, apoptosis, 105–106 concentration changes, physiological responses to, 87–88 Oxygen concentration, transcription factor hypoxia-inducible factor 1, 87 ventilation control, 87–88 Oxygen-derived species, cystic ﬁbrosis (CF), 263–264 Oxygen free radicals, multistage carcinogenesis, 193–200 Oxygen radical absorbance capacity (ORAC), biological reducing power (BRP), 17 Oxyradicals, iron overload syndrome, 285–296 Oxysterols, 513 sickle cells, 306 p21, oxidative activation, 85 PAH, 195 Pannus development, rheumatoid arthritis, 597–598 Paraoxonase, 220–221 low-density lipoproteins (LDL), oxidation, 568–573

Index Paraoxonase 1 (PON1), 568–569 Paraoxonase 2 (PON2), 569–571 Paraoxonase 3 (PON3), 569–571 Parkinson’s disease, 219, 381, 428 Pattern dystrophies, 355 PDE, 377 PDTC, 393 PE, 17 PECAM-1, 307 Permeability transition pore (PTP), 387 Peroxide, low-density lipoproteins (LDL), oxidation, 563 Peroxiredoxins, 410–411 Peroxynitrite, 145, 456–457, 590, 591–592, 602 familial amyotrophic lateral sclerosis (FALS), 480–481, 484 PFK-1, 239 PGF2 alpha, 513 Phagocytosis, acute inﬂammatory response, 593 Phenotypic and genetic aspects, colon cancer, 495–497 Phosphatidylserine (PS), 316 Phosphodiesterase (PDE), 377 Phosphofructokinase (PFK-1), 239 Photoreceptor degeneration, retinitis pigmentosa (RP), 390–393 Photoreceptors, 350, 351, 376, 380 degeneration rescue, 380 Photosensitization, retina, reactive oxygen species (ROS), 383 Phototransduction cascade, 376 Phycoerythrin (PE), 17 Physical training, sudden death, sickle cell carrier state, 305 p16INK4A, functions, 296 Plants, oxidative stress, protective responses, 80 Plaque, 555–556 Plasma, antioxidant species, 269 Plasma redox, 48–50 Platelet endothelial cell adhesion molecule-1 (PECAM-1), 307 Point mutations, alpha-thalassemia, 308 beta-thalassemia, 310 tRNA genes, 242 Polycyclic aromatic hydrocarbons (PAH), 195 Polymorphism, oxidative stress, 215–223

Index Polyphenols, 558 Polyunsaturated fatty acids (PUFA), 381 age-related macular degeneration (AMD), 357–359 PON1, 568–569 PON2, 569–571 PON3, 569–571 Population migration, hemoglobinopathy, 303–304 Posterior subcapsular cataracts, 324 Potentiometry, biological reducing power (BRP), 18 Potentiostat, 19 Pressor response, angiotensin II, 541–544 Primates, Huntington’s disease (HD), imaging, 443–444 Proanthocyanidins, skin carcinogenesis, 204 Procaspase, 378 Progressive nuclear cataract, 324 Prooxidant, deﬁned, 11 Protein carbonyls, 513 Protein chips, 7 Protein glycation, 521–522 Protein kinase C isoforms, oxidative activation, 84 Protein modiﬁcation/oxidation, 266 Proteins, Huntington’s disease (HD), 456–457 lens, 325 posttranslational modiﬁcations, 7–8 Protein tyrosine phosphatases, signaling cascades, oxidative inhibition, 82 PS, 316 PTP, 387 P53 tumor suppressor gene, 500 PUFA, 381 age-related macular degeneration (AMD), 357–359 Pyridoxal phosphate, 525 Pyridoxamine, 525 Pyridoxine, 525 Pyrrolidine dithiocarbamate (PDTC), 393 Quinone reductases, 219–220 Rac1, apoptosis, tumor cells, 108–109 RacGTPase, apoptosis, tumor cells, 108–109 Radicals, oxidative stress, 144 RAGE, 222, 522, 526

625 Ramipril, 558 Random mutations, mtDNA, 242–243 RANKL, 600 Ras-p21, 499–500 RBC (see Red blood cells) RE, 290–291 Reactive nitrogen intermediates (RNI), retinitis pigmentosa (RP), 381 sources, 384–385 Reactive nitrogen species (RNS), 270 retinitis pigmentosa (RP), 381–387 Reactive oxygen species (ROS), 100–102 acute inﬂammatory response, 595 aging, 125 apoptosis, 104–107 cell death, 102–108 deﬁned, 100 intracellular sources, 100–101, 388 low-molecular-weight antioxidants (LMWA), 14 oxidative stress, 144 retina, 382–384 tumor initiation, 194–196 tumor progression, 200–202 tumor promotion, 197–200 Reactive oxygen species (ROS)-mediated apoptotic signaling, Apoptosis, tumor cells, 108–112 cytosolic pH, 107–108 Receptor activator of NFKB ligand (RANKL), 600 Receptor-mediated signaling pathways, redox regulation, 81–82 Receptor of advanced glycation end products (RAGE), 222, 522, 526 Recombinant DNA technology, 5–6, 7 Red blood cells (RBC), hemoglobinopathy, 303–304 intercellular interactions, 316 membrane abnormalities, 306 Redox, 47–48 cells, 51–52 deﬁned, 11 intestinal lumen, 50–51 subcellular compartments, 52–53 Redox biology, molecular genetics toolbox, 7–8 Redox compartments, characterization, 48–53 Redox control, thioredoxin (TRx), 60–62 Redox cycling, iron, 286

626 Redox-genome interactions, 1–8 compartmentation, 5 quantitation, 5 speciﬁcity, 5 Redox imbalance, inherited hemoglobinopathy, 303–317 Redox-mediated ampliﬁcation, immune responses, 85–86 Redox modulation, tumor initiation, promotion, and progression, 191–206 Redox potential, vs. reducing power, 12–13 Redox regulation, receptor-mediated signaling pathways, 81–82 Redox signaling, 78 bacteria, 78–79 oxidative stress, 78–80 yeast, 79 Redox state, quantitative deﬁnition, 46–47 Redox status (REDST), 78 Reducing power, deﬁned, 12 vs. redox potential, 12–13 Remacemide, Huntington’s disease (HD), 458 Remodeling theory, aging, 122–123 Renin angiotensin system, 455, 540 endothelin (ET), 548–549 Resonance Raman method, 362 Respirasome, 238 Respiratory tract lining ﬂuids (RTLF), 268, 269 antioxidant species, 269 bacterial infection, 272 glutathione (GSH), 272 Retina, age-related macular degeneration (AMD), 352–354 alpha-tocopherol, 359–360 antioxidants, 359–360 ascorbate, 359–360 carotenoids, 359–360 cross-section, 350, 351 glial cells, 350 oxidative stress, 359 reactive oxygen species (ROS), 382–384 Retinal pigment epithelium (RPE), 352, 375 Retinitis pigmentosa (RP), 375–394 antioxidant defense mechanisms, 387–390 apoptosis, 377–381 molecular genetics, 376–377 nitric oxide (NO), 381–382

Index [Retinitis pigmentosa (RP)] photoreceptor degeneration, 390–393 reactive nitrogen intermediates (RNI), 381 reactive nitrogen species (RNS), 381–387 vitamin E, 393 Retrograde response, 132 Rheumatoid arthritis, 219 bone resorption, 598–601 cartilage breakdown, 598–601 free radicals, 589–605 hypoxia, 596–601 immune response, 596–597 pannus development, 597–598 redox microenvironment, 595–604 thioredoxin (TRx), 68 xanthine oxidoreductase, 601–604 Rheumatoid synovium, hypoxic nature, 596 Rhodopsin, 376, 383 RNA genes, point mutations, 242 RNI, retinitis pigmentosa (RP), 381 sources, 384–385 RNS, 270 retinitis pigmentosa (RP), 381–387 Rodents, Huntington’s disease (HD), imaging, 443 Rods, 351, 352 ROS (see Reactive oxygen species) RPE, 352, 375 RTLF, 268, 269 antioxidant species, 269 bacterial infection, 272 glutathione (GSH), 272 Salicylates, colon cancer, 502 Scaﬀolding proteins, 234 Scavenger receptors, macrophages, 565–567 Scavengers, 14–15 Scavenging hydrogen peroxide, 102 Scavenging oxygen, 101 SCC, 201 Schizophrenia, 219 SDH, 443 Secretory pathway, 52 SEH, 222 Selenite, inducing nuclear cataracts, 336 Selenium, 204 diabetes mellitus, 516 Senile cataract, 324, 325 oxidation, 330 oxidative stress, 331–335

Index [Senile cataract] lens proteins damage, 333–335 membrane damage, 331–332 oxidative defense damage, 335 Sickle cell anemia, 303–308 Sickle cell carrier state, sudden death, physical training, 305 Sickle cell disease, classiﬁcation, 305 pathophysiology, 306–308 Sickling, eﬀect, 306 Signal ampliﬁcation, reducing conditions, 85 Signaling cascades, 47–48 intracellular thiol/disulﬁde redox status, 78 protein tyrosine phosphatases, oxidative inhibition, 82 Single nucleotide polymorphisms (SNP), 215 Singlet oxygen oxidation, guanine, 154 6-yl decomposition pathway, 150 Skin carcinogenesis, proanthocyanidins, 204 transforming growth factor alpha (TGF alpha), 199 tumor necrosis factor (TNF), 199 ursolic acid, 204 Skin tumor promoters, 197 Smoking, colon cancer, 498 S-nitrosylation (S-NO), 78 S-NO, 78 SNP, 215 SOD (see Superoxide dismutase ) SOD1, 218 SOD2, 218–219 SOD3, 219 S-OH, 78 Soluble epoxide hydrolase (sEH), 222 Sorsby’s fundus dystrophy, 355 Southeast Asia, HbE, 305 hemoglobin disorders, 303 Squamous cell carcinoma (SCC), 201 Stargardt’s disease, 355, 356, 358 Statins-paraoxonase interactions, 571 Stress proteins, 594 Subfoveal neovascularization, photodynamic therapy, 353 Succinate dehydrogenase (SDH), 443 Succinate-ubiquinone oxidoreductase (complex II), 236

627 Sudden death, physical training, sickle cell carrier state, 305 Sugary moiety/purine base, covalent bond, tandem lesions, 161–162 Sulfenic acid moiety (S-OH), 78 Supercomplexes, 238 Superoxide, familial amyotrophic lateral sclerosis (FALS), 471–476 Superoxide dismutase (SOD), 198, 218–219, 440, 513, 560–561 discovery, 2 familial amyotrophic lateral sclerosis (FALS), 471–476 overexpression, 338 regulated production, 81 Superoxide dismutase 1 (SOD1), 218 Superoxide dismutase 2 (SOD2), 218–219 Superoxide dismutase 3 (SOD3), 219 Tandem lesions, formation, 159–162 Tardive dyskinesia, 219 Target genes, iron overload-induced carcinogenesis, 294–296 TBARS (thiobarbituric acid-reactive substances), 513, 514–515, 520 T-cell proliferation, cAMP-mediated redox pathways, 85 TEAC, glutathione/glutathione disulﬁde, 16–17 TfR, 292 TGF alpha, skin carcinogenesis, 199 Thalassemia, 308–317 vs. beta-thalassemia, 313 classiﬁcation, 308 glutathione (GSH), 313 Heinz bodies, 309 iron, 313–315 pathophysiology, 311–312 point mutations, 310 redox imbalance, 313–315 vitamin E, 315 Thiobarbituric acid-reactive substances, 513, 514–515, 520 Thiol(s), 41–44 apoptosis, 112–113 cellular redox control, 59–70 Thiol-disulﬁde redox state, 41–54 enzyme systems, 44–46 Thioltransferase (TTase), 49–50, 329 Thioredoxin (Trx), 329 autoimmune myocarditis, 68–69 biological functions, 61

628 [Thioredoxin (Trx)] cellular redox control, 59–70 clinical application, 70 dependent systems, 46 Epstein-Barr infection, 67 family, 62–63 subcellular localization, 62 hepatitis C, 68 HIV, 65–66 HTLV-1 infection, 65 inﬂuenza, 69–70 interstitial pneumonia, 70 knockout mice, 69 oxidative stress, 60 paradox, 112–113 redox control, 60–62 rheumatoid arthritis, 68 transgenic mice, 69–70 Thioredoxin-2 (Trx-2), 62–63 Threshold hypothesis, 240 Thymine base, OH radical, oxidation reactions, 146–148 Tissue, preparation for voltammetric measurements, 27 TNF, skin carcinogenesis, 199 Tocopherol, 504 retina, 359–360 structure, 503 Total radical trapping potential (TRAP), glutathione/glutathione disulﬁde, 17 Transcription factor hypoxia-inducible factor 1, 87 oxygen concentration, 87 Transducin, 376 Transferrin, 267 Transferrin receptor (TfR), 292 Transforming growth factor alpha (TGF alpha), skin carcinogenesis, 199 Transgenic mice, thioredoxin (TRx), 69–70 Transgenic mouse models, 7 Down syndrome (DS), 411–413 Huntington’s disease (HD), 450–451 TRAP, glutathione/glutathione disulﬁde, 17 Trisomic mouse models, Down syndrome (DS), 413–416 Trisomic studies, 409–411 human in vitro, 411 human in vivo, 410–411 tRNA genes, point mutations, 242

Index Trolox equivalent antioxidant capacity (TEAC), glutathione/glutathione disulﬁde, 16–17 TRx, 63–66 anti-inﬂammatory eﬀect, 65–66 chemokine-like eﬀect, 64–65 cytokine-like eﬀect, 64 Trx (see Thioredoxin) Trx-2, 62–63 TTase, 49–50, 329 Tumor cells, apoptosis, ROS-mediated apoptotic signaling, 108–112 Tumor initiation, redox modulation, 191–206 Tumor necrosis factor (TNF), skin carcinogenesis, 199 Tumor progression, reactive oxygen species (ROS), 200–202 redox modulation, 191–206 Tumor promotion, reactive oxygen species (ROS), 197– 200 redox modulation, 191–206 Type 1 diabetes mellitus, oxidative stress, 523–524 Type 2 diabetes mellitus, 511 mitochondria, 127–128 obesity, 511 oxidative stress, 523–524 Ubiquinone, Huntington’s disease (HD), 458 Ubiquinone-cytochrome c oxidoreductase (complex III), 236–238 UDP-glucuronysyltransferase, 221 Unscheduled DNA repair, 500 Ursolic acid, skin carcinogenesis, 204 Vascular endothelial growth factor (VEGF), 83 Vascular occlusions, sickle cells, 307 Vascular responses, acute inﬂammatory response, 591–592 VEGF, 83 Ventilation control, oxygen concentration, 87–88 Very low density lipoproteins (VLDL), 503, 555–556 Vitamin A, 513 Vitamin C, 513 cystic ﬁbrosis (CF), 267, 273–274 diabetes mellitus, 517

Index [Vitamin C] glutathione/glutathione disulﬁde, 16 lens, 328 Vitamin E, 513 age-related macular degeneration (AMD), 364 biochemistry, 502–504 colon cancer, 502–506 cystic ﬁbrosis (CF), 267, 273 diabetes mellitus, 517, 524–527 form, 505–506 lens, 328 retinitis pigmentosa (RP), 393 thalassemia, 315 VLDL, 503, 555–556 Voltammetric measurement, 11–35 biological reducing power, 11–35 Voltammetric measurements, biological reducing power (BRP), 28–32 Voltammetry-sweep measurement, biological reducing power (BRP), 18–20 Weight loss, Huntington’s disease (HD), 451

629 Xanthine oxidoreductase, rheumatoid arthritis, 601–604 Yeast, redox signaling, 79 Yellow spot, 350 5-yl decomposition pathway, 149 6-yl decomposition pathway, 150 Zeaxanthin, age-related macular degeneration (AMD), 360–361, 364–366 Zinc, age-related macular degeneration (AMD), 366 Alzheimer’s disease, 429–431 copper/zinc SOD, deleterious eﬀects, 409 Down syndrome (DS), 408 enzyme, 101 copper-zinc-superoxide dismutase (CuZnSOD), 424, 515–516 overexpression, 429 diabetes mellitus, 516 familial amyotrophic lateral sclerosis (FALS), 481–482 Zinc chloride, 393