Free Radicals in Ophthalmic Disorders

about the book… Free radicals are molecules with an unpaired electron in the outer shell or an electron that was damaged from either attack or from a poor splitting bond. After a free radical is formed it will continue to attack other molecules, which usually results in the damage of tissue or destruction of a healthy cell. Free radicals arise normally through metabolism. However, sometimes the body’s immune system will create them on purpose to neutralize viruses and bacteria. Free radicals are implicated in many ophthalmic disorders including uveitis, optic nerve damage, retinal ischemia, and macular degeneration. Free Radicals in Ophthalmic Disorders presents the most current knowledge pertaining to the role of free radicals/oxidants in ocular disorders, and the use of antioxidants in the prevention of these disorders. Written by today’s leading ocular scientists and clinicians Free Radicals in Ophthalmic Disorders • gives comprehensive coverage of the role of free radicals/oxidants in ocular disorders • covers the use of antioxidants to prevent oxidative stress and ocular tissue damage • examines external factors that may result in the stimulation and heightened occurrence of free radicals/oxidants about the editors... MANFRED ZIERHUT is Associate Professor of Ophthalmology, University Eye Hospital, Tubingen, Germany. Dr. Zierhut received his M.D. from the University of Hannover, Germany, and has published 102 articles, co-authored 24 books, and completed over 3000 surgeries in ophthalmology. ENRIQUE CADENAS is Professor of Pharmacology and Pharmaceutical Sciences and Associate Dean of Research Affairs at the University of Southern California School of Pharmacy, Los Angeles. He is also Professor of Biochemistry at the Keck School of Medicine, University of Southern California. Dr. Cadenas received his M.D. in Medicine and his Ph.D. in Biochemistry/ Biophysics from the University of Buenos Aires, Argentina, and his main focus of research, besides free radicals, covers oxidative stress, mitochondrial dysfunction, aging, and neurodegenerative diseases. He is the author of over 200 peer-reviewed papers.

Printed in the United States of America

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Zierhut • Cadenas • Rao

NARSING A. RAO is Professor of Ophthalmology and Pathology at the Keck School of Medicine, and the first chair holder of the Stieger Vision Research Endowed Chair of Doheny Eye Institute, University of Southern California, Los Angeles and Director of the Intraocular Inflammation/ Uveitis Service and the Director of the Ophthalmic Pathology Laboratories at the Doheny Eye Institute. Dr. Rao was awarded his M.D. from Osmania University and completed his internship at Osmania General Hospital, Hyderabad, India. Following a year of rotating internships in upstate New York, he completed two residencies in pathology and ophthalmology at Georgetown University, Washington, D.C. and a fellowship in ophthalmic pathology at the Armed Forces Institute of Pathology, Washington, D.C. Dr. Rao is involved in both research aspects and the clinical treatment of inflammatory ocular diseases affecting the uveal tract, vitreous, retina and sclera and immune disorders affecting the eye. Dr. Rao has published over 375 peer-reviewed articles in U.S. and international journals and has authored or edited four books.

Free Radicals in Ophthalmic Disorders

Ophthalmology

Free Radicals in Ophthalmic Disorders H202

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Edited by

Manfred Zierhut Enrique Cadenas Narsing A. Rao

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Free Radicals in Ophthalmic Disorders

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Free Radicals in Ophthalmic Disorders Edited by

Manfred Zierhut University Eye Hospital Tubingen, Germany

Enrique Cadenas

School of Pharmacy University of Southern California Los Angeles, California, USA

Narsing A. Rao

Keck School of Medicine University of Southern California Los Angeles, California, USA

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Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2008 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-4200-4433-8 (Hardcover) International Standard Book Number-13: 978-1-4200-4433-1 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. 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 Free radicals in ophthalmic disorders / edited by Manfred Zierhut, Enrique Cadenas, Narsing A. Rao. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-1-4200-4433-1 (hardcover : alk. paper) ISBN-10: 1-4200-4433-8 (hardcover : alk. paper) 1. Eye—Diseases. 2. Free radicals (Chemistry)—Physiological effect. 3. Free radicals (Chemistry)— Toxicology. I. Zierhut, Manfred. II. Cadenas, Enrique. III. Rao, Narsing A. [DNLM: 1. Eye Diseases—drug therapy. 2. Eye Diseases—etiology. 3. Antioxidants— therapeutic use. 4. Free Radicals—adverse effects. 5. Free Radicals—metabolism. 6. Oxidative Stress—physiology. WW 140 F853 2008] RE48.F736 2008 617.7—dc22

2007041964

For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 16th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

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Preface

Over the last few decades, free radical biology has evolved into a discipline addressing various pathologic processes at the molecular level. In the past, the reactive oxygen–derived radicals and nitric oxide have been extensively studied in ischemia-reperfusion, involving cardiac, neuronal, hepatic, pulmonary, gastrointestinal, and other organs. Recently, in the eye, the role of free radicals in pathogenesis of cataract has gained momentum in delineating the effect of oxidative stress and antioxidant depletion in cataract formation. In the lens, extensive studies were conducted in evaluating both offending radicals and protecting agents against such insult. Most noteworthy, in recent years, the free radical biology has extended significantly in addressing other ophthalmic disorders, including macular degeneration, retinal degeneration in glaucoma, diabetic retinal complications, and intraocular inflammation or uveitis. For the first time, the pathogenesis of these diseases is seen from the context of free radical generation. The workshop held in Ettal in 2005 provided a unique opportunity for a gathering of free radical biologists with interest in basic biochemical interactions and ophthalmic scientists devoted to the field of oxidative stress and ophthalmic diseases. At that event, we came up with the idea to summarize the current status of free radical biology in addressing various ophthalmic diseases in form of a book. Recent studies on free radical–related ophthalmic diseases are distributed in diverse ophthalmic and nonophthalmic journals, and no effort has been made to summarize our knowledge in a single periodical or a book. The current book, Free Radicals in Ophthalmic Disorders, summarizes recent advances in free radical insults leading to various ophthalmic diseases. The conditions that are addressed include cataract, macular degeneration, diabetic retinopathy, corneal diseases, retinal degeneration, glaucoma, retinal ischemia, and intraocular inflammation or uveitis. Each chapter addressing these diseases is

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orderly, presented with a brief introduction of the disease and followed by the role of free radicals in the pathogenesis of the disorder and the potential therapeutic intervention with antioxidants and/or other means. In cataract, the current understanding of the mechanism of cataract formation and the protection by specific antioxidants were discussed. The feasibility of treating the diabetic retinopathy with peroxynitrite-scavenging agents was introduced. In corneal disease progression, the evidence of involvement of oxidants was shown, and in macular degeneration, the role of oxidative stress was reinforced. Linking to oxidation, antioxidants were also discussed in cardiovascular diseases. In glaucoma, retinal ganglion cell and trabecular meshwork cell death was ascribed to oxidative stress. The reduction of oxidative stress by tyrosinase is proposed in retinal diseases, and that the free radical formation is an element in retinal ischemia was demonstrated. In uveitis, the early involvement of mitochondrial peroxynitrite was demonstrated. Aside from the direct link of oxidants to eye diseases, the general aspects of oxidative and nitrative stresses were also discussed. Therefore, this book, which spans every aspect of ocular diseases, might bring a unifying understanding of the involvement of free radicals in disease as well as in health. The editors of the volume are appreciative of the contribution of various authors, who succinctly presented the current material with background pertinent to their topics and who focused on biological changes in the ocular tissues resulting from free radicals. Manfred Zierhut Enrique Cadenas Narsing A. Rao

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Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii 1.

Free Radical Biology, Mitochondrial Functions, and Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Li-Peng Yap, Allen H. K. Chang, Derick Han, and Enrique Cadenas

1

.....

11

......

33

Modulation and Determination of Cellular Glutathione Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lars-Oliver Klotz

45

2.

Antioxidants and Modulation of Cardiovascular Disease Regine Heller

3.

Nitric Oxide—Related Oxidants in Health and Disease Cecilia Gonza´lez de Ordu~ na and Santiago Lamas

4.

5.

Oxidants in Corneal Diseases Anders Behndig

.........................

55

6.

Involvement of Oxidative Stress in the Pathogenesis of Glaucoma ....................................... Neville N. Osborne

71

7.

Oxidative Stress and Cataract ........................ Susanne Hippeli, Matthias Elstner, Harald Schempp, and Erich F. Elstner

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Contents

8.

Nitric Oxide in Experimental Autoimmune Uveoretinitis . . . . 107 Janet Liversidge, Sharon Gordon, Andrew Dick, Morag J. Robertson, and Ross Buchan

9.

TNF Activation and Nitric Oxide Production in EAU . . . . . . Claudia J. Calder, Lindsay B. Nicholson, Morag J. Robertson, and Andrew D. Dick

121

10.

Peroxynitrite and Ocular Inflammation Guey-Shuang Wu and Narsing A. Rao

................

131

11.

Melanin and Oxidative Reactions . . . . . . . . . . . . . . . . . . . . . Tadeusz Sarna, Grzegorz Szewczyk, and Andrzej Zadlo

147

12.

Are Antioxidants Useful in Diabetic Retinopathy? . . . . . . . . . Maria Miranda, Francisco Bosch-Morell, Maria Muriach, Jorge Barcia, Manuel Diaz-Llopis, Angel Messeguer, and Francisco J. Romero

159

13.

Macular Degeneration: The Role of Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael E. Boulton

167

14.

Retinal Ischemia and Oxidative Stress Neville N. Osborne

.................

177

15.

Reduction of Oxidative Stress in Retinal Disease . . . . . . . . . . Ulrich Schraermeyer, J€ urgen Kopitz, Petra Blitgen-Heinecke, Despina Kokkinou, and Tobias Schwarz

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Index

..............................................

209

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Contributors

Jorge Barcia Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain Anders Behndig Department of Clinical Sciences/Ophthalmology, Umea˚ University Hospital, Umea˚, Sweden Michael E. Boulton Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, U.S.A. Petra Blitgen-Heinecke Sektion f€ ur Experimentelle Vitreoretinale Chirurgie, Universit€ ats-Augenklinik T€ ubingen, T€ ubingen, Germany Francisco Bosch-Morell Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain Ross Buchan Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, U.S.A. Enrique Cadenas Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, U.S.A. Claudia J. Calder Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K. Allen H. K. Chang Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, U.S.A. Manuel Diaz-Llopis Department of Surgery, Universitat de Valencia, Hospital General Universitario, Valencia, Spain

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Andrew D. Dick Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K. Erich F. Elstner TU-M€ unchen, Institute of Phytopathology, FreisingWeihenstephan, Germany Matthias Elstner Munich, Germany

Department of Neurology, Ludwig-Maximilian University,

Sharon Gordon Human Resources Development and Training, University Office, King’s College, Aberdeen, U.K. Derick Han Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Regine Heller Department of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich-Schiller-University of Jena, Jena, Germany Susanne Hippeli TU-M€ unchen, Institute of Phytopathology, FreisingWeihenstephan, Germany Lars-Oliver Klotz Department of Molecular Aging Research, Institut f€ ur Umweltmedizinische Forschung (IUF) at Heinrich-Heine-University, D€ usseldorf, Germany Despina Kokkinou Sektion f€ ur Experimentelle Vitreoretinale Chirurgie, Universit€ats-Augenklinik T€ ubingen, T€ ubingen, Germany J€ urgen Kopitz Zentrum f€ ur Pathologie, Abt. Angewandte Tumorbiologie, Klinikum der Ruprecht-Karls-Universit€ at, Im Neuenheimer Heidelberg, Germany Santiago Lamas Spain

Centro de Investigaciones Biol ogicas (CIB-CSIC), Madrid,

Janet Liversidge Department of Ophthalmology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, U.K. Angel Messeguer Department of Biological Organic Chemistry, Centre d’Investigaci o i Desenvolupament (CID), CSIC Jordi Girona Salgado, Barcelona, Spain Maria Miranda Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain Maria Muriach Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain Lindsay B. Nicholson Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K.

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Contributors

ix

Cecilia Gonza´lez de Ordu~ na (CIB-CSIC), Madrid, Spain

Centro de Investigaciones Biol ogicas

Neville N. Osborne Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, U.K. Narsing A. Rao Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Morag J. Robertson Aberdeen, U.K.

Department of Ophthalmology, University of Aberdeen,

Francisco J. Romero Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain Tadeusz Sarna Krakow, Poland

Department of Biophysics, Jagiellonian University Krakow,

Harald Schempp TU-M€ unchen, Institute of Phytopathology, FreisingWeihenstephan, Germany Ulrich Schraermeyer Sektion f€ ur Experimentelle Vitreoretinale Chirurgie, Universit€ats-Augenklinik T€ ubingen, T€ ubingen, Germany Tobias Schwarz Sektion f€ ur Experimentelle Vitreoretinale Chirurgie, Universit€ ats-Augenklinik, T€ ubingen, T€ ubingen, Germany Grzegorz Szewczyk Department of Biophysics, Jagiellonian University Krakow, Krakow, Poland Guey-Shuang Wu Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A. Li-Peng Yap Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, U.S.A. Andrzej Zadlo Krakow, Poland

Department of Biophysics, Jagiellonian University Krakow,

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1 Free Radical Biology, Mitochondrial Functions, and Nitric Oxide Li-Peng Yap and Allen H. K. Chang Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, U.S.A.

Derick Han Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Enrique Cadenas Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, U.S.A.

INTRODUCTION Oxygen-derived free radicals are generated during metabolism and energy production in the body and are involved in countless processes such as the regulation of signal transduction and gene expression, activation of receptors and nuclear transcription factors, oxidative damage to cell components, the antimicrobial and cytotoxic action inherent in immune system cells, as well as in aging and agerelated degenerative diseases. Conversely, the cell convenes antioxidant mechanisms to counteract the effect of oxidants; these antioxidants may remove oxidants either in a highly specific manner as in the case of superoxide dismutases or in a less specific manner (for example, small molecules such as vitamin E, vitamin C, and glutathione). Oxidative stress is classically defined as an imbalance between

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oxidants and antioxidants.1,2 This concept of oxidative stress entails a global view of, for example, thiol/disulfide balance –a major determinant of the cell redox state– and fails to recognize discrete redox pathways. Based on this, Jones3 provided a new definition of oxidative stress as a disruption of redox signaling and control, in essence, a mechanistic concept. This is important, for redox regulation of cell signaling occurs in discreet cellular regions that respond differently to oxidative and/or nitrosative stress situations. More recently, Sies and Jones introduced a new definition of oxidative stress in the Encyclopedia of Stress as an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage.4 THE UNIVALENT REDUCTION OF OXYGEN AND OXIDATION OF NITROGEN Univalent reduction of oxygen (O2) to superoxide anion (O2 ) is accomplished by various mechanisms. However, the two most significant sources in vivo are the mitochondria and inflammatory cells. Mitochondria are recognized as the major cellular sources of O2 , largely originating from the autoxidation of ubisemiquinone — a mobile carrier that (a) transfers electrons from complex I and II to complex III of the mitochondrial respiratory chain and from (b) rotenone-sensitive complex I. Another major source of O2, during inflammatory conditions is the activity of NADPH oxidase, a multi-subunit flavoheme enzyme. The four steps encompassed in the univalent reduction of oxygen yields free radicals and oxidants as shown in Fig. 1: superoxide anion





Figure 1 (See color insert.) Univalent reduction of oxygen and univalent oxidation of nitric oxide.

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Free Radical Biology, Mitochondrial Functions, and Nitric Oxide

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(O2 ), hydrogen peroxide (H2O2), hydroxyl radical (HO ), and water (H2O), the latter being generated by the four-electron reduction of O2 at the cytochrome c oxidase (complex IV) of the respiratory chain. The generation of reactive nitrogen species –formation of nitric oxide ( NO)—requires the five electron oxidation of the guanidine group of L-arginine by the nitric oxide synthases (L-arginine þ NADPH þ O2 ? L-citrulline þ NADPþ þ NO). Subsequent one-electron oxidations yield, among others, nitrite (NO2), nitrogen dioxide (NO2 ), and nitrate (NO3). Hence, univalent reduction of oxygen and univalent oxidation of NO yield a variety of oxidants and free radicals that are involved in several aspects of cell function ranging from redox regulation of cell signaling to irreversible damage of cellular constituents. Of interest, the reaction of O2  and NO yields peroxynitrite (ONOO), an oxidant with a reduction potential of about þ1.0 volt and is involved in oxidation and nitration reactions. This nonenzymic reaction proceeds at diffusioncontrolled rates (O2  þ NO ? ONOO; k ¼ 1.9  1010 M1s1), slightly faster than the dismutation of O2  by the enzyme superoxide dismutase (O2  þ O2  þ 2Hþ ? H2O2 þ O2; k ¼ 2.3  109 M1s1).






































PROTEIN POST-TRANSLATIONAL MODIFICATIONS BY REACTIVE OXYGEN AND NITROGEN SPECIES H2O2, NO, and ONOO are distinctly involved in different steps of redox cell signaling through specific protein post-translational modifications. H2O2, essential for cell signaling,5 is produced by mammalian cells to mediate several physiological responses such as cell proliferation, differentiation, and migration6 through reductiveoxidative-based mechanisms.5 The signaling properties of H2O2 are exerted in the cytosol, where this oxidant increases protein phosphorylation largely upon inhibition of protein phosphatases;5 it is important to recognize that these H2O2-driven signaling pathways are in unique equilibrium with the activities of peroxiredoxins.5 NO exerts its effects on cell signaling via (a) guanylyl cyclase and cyclic GMP-dependent pathways and (b) cyclic GMP-independent pathways, the latter including post-translational modifications of proteins. Protein S-nitrosylation, a post-translational modification of thiol residues to form S-nitrosothiols, is a major mechanism of redox signaling by which NO alters cellular function through the modification of protein thiol residues.7,8 NO-mediated S-nitrosylation of proteins appears to be a reversible process and has been identified in a limited subset of proteins in in vitro and in vivo studies.8 Hogg7 lists four major mechanisms of S-nitrosylation that potentially occur in biological systems. As mentioned above, S-nitrosation appears to be a reversible process: the reversible transfer of the nitroso group from an S-nitrosothiol to a thiol (transnitrosation: RSNO þ R0 S $ RS þ R0 SNO).7 S-Nitrosylation has been compared with phosphorylation as a cellular signaling mechanism.9,10 An interesting concept is that S-nitrosylation is likely to promote S-glutathionylation, that is, the incorporation of glutathione into proteins via mixed disulfide bonds. S-glutathionylation is an important protein





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post-translational modification deeply involved in the regulation of protein function.11 Additionally, S-glutathionylation of proteins is increasingly viewed as a representative mechanism whereby the changes in the redox environment and increase generation of reactive oxygen and nitrogen species can be translated into a recognizable modality and subsequently translated into a functional response.11 The redox environment of a cell is governed by the redox couple composed of glutathione (GSH), the most abundant non-protein thiol and its reduced counterpart, glutathione disulfide (GSSG). As the concentration of GSH far exceeds any other redox couples present in the cell, the GSH/GSSG can be used to define the cellular redox environment. The redox environment of the cell is closely associated with its life cycle. As a cell progressed from proliferation to differentiation, to apoptosis and necrosis, its cellular redox state becomes increasingly oxidized.12 Work done in our laboratory by Antunes et al13 showed that at low concentrations of H2O2, where the redox status is less oxidized, cells undergo apoptosis; however, at higher concentrations of H2O2, the cellular redox status becomes more oxidized, shifting the mode of cell death from apoptosis to necrosis. Redox regulation of protein function has become increasingly important in understanding cellular adaptation to oxidative and nitrosative stress. The formation of ONOO can result in oxidative modifications of proteins including the formation of 3-nitrotyrosine;10,11 the limited efficiency of nitration reactions in biology as well as the significance of 3-nitrotyrosine formation have been discussed in detail by several authors;14 oxidation of cysteine thiols by ONOO leads to sulfenic, sulfinic, and sulfonic acid derivatives15 (Fig. 2).

Figure 2 Protein post-translational modification.

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Figure 3 Specific removal of reactive oxygen species.

SPECIFIC REMOVAL OF REACTIVE OXYGEN SPECIES The intermediates in the univalent reduction of oxygen sequence described above are of free radical or oxidant nature. Mitochondria are endowed with specific antioxidant systems aimed at removing O2  and H2O2 (Fig. 3). The former is specifically reduced to H2O2 by Mn-superoxide dismutase, present in the mitochondrial matrix at a concentration of 0.3  105 M16. Mitochondria also contain a Cu,Zn-superoxide dismutase in the intermembrane space,17 the activation of which seems to require an oxidative modification of its critical thiol groups.18 The presence of Mn-superoxide dismutase in the mitochondrial matrix allowed an estimation of a steady-state concentration of O2  of about 1010 M18, slightly higher than that in the cytosolic compartment (1011 M). H2O2 is specifically removed by glutathione peroxidase, with an assumed concentration in the mitochondrial matrix of 1.17  106 M: the steady-state level of H2O2 in the matrix is estimated at 0.5  108 M16.





MITOCHONDRIAL FEATURES AND CELL FUNCTION As mentioned above, mitochondria are energy-transducing organelles (the powerhouses of the cell) that generate metabolic energy for cell function and maintenance; mitochondria are major cellular sources of O2  and H2O2, and also of NO by a mitochondrial nitric oxide synthase.16,17 It appears that mitochondrial nitric oxide synthase is a voltage-dependent enzyme, responsible for NO diffusion to cytosol and modulated by the mitochondrial metabolic states.19,20 Another function of mtNOS, at least in brain mitochondria or synaptosome mitochondria, is – in coordination with Mn-superoxide dismutase – to maintain brain redox status and participate in the normal physiology of brain development.21 Hence, mitochondria generate O2 , H2O2, and NO. Formation of O2  during mitochondrial electron transfer along with that of NO by mitochondrial NOS sets the ground for the formation of ONOO, which seems to specifically




















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inhibit NADH-ubiquinone reductase (complex I) activity.22,23 It was reported that S-nitrosated proteins are abundantly localized to mitochondria and the peri-mitochondrial space: because of the role of mitochondria in oxidative and nitrosative stress, it was suggested that ONOO generated by mitochondria can act as a nitrosating agent.8 We have reported on the sites and mechanisms of aconitase inactivation by ONOO, a process regulated by substrate availability and glutathione: specifically, LC/MS/MS analyses revealed that ONOO treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption.24 The binding and inhibition of NO to cytochrome c oxidase (complex IV)25,26 has profound regulatory implications: first, it expands the classical concept of mitochondrial respiration in that energy demands drive respiration but it places the kinetic control of both respiration and energy supply on the availability of ADP to F1-ATPase and O2 and NO to cytochrome oxidase.27 Hence, NO, in addition to its role as intercellular messenger in diverse physiological processes is a mitochondrial regulatory metabolite. Increasing concentrations of NO are required to observe: reversible inhibition of cytochrome oxidase (0.05–0.1 mM), binding to the bc1 segment (complex III) of the respiratory chain (0.3 mM), and oxidation of ubiquinol (Fig. 4).28 The second effect is similar to that elicited by antimycin A and supports O2  and H2O2 formation.














MITOCHONDRIAL GENERATION OF SIGNALING MOLECULES Mitochondria are considered the major cellular site for H2O2 production, a process that is kinetically controlled by the availability of O2 and NO to cytochrome oxidase and of ADP to F1-ATPase. Han et al. demonstrated clearly that mitochondria are cytosolic sources of O2 , whereby O2 – formed in the cytosolic








Figure 4 NO gradients and sites of NO action on the mitochondrial respiratory chain.

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side of the inner membrane space is released into the cytosol through voltage dependent anion chanels (VDAC).29 The multi-site regulation of mitochondrial respiration and energy-transducing pathways support a critical regulatory role of mitochondrion in cell signaling pathways. Mitochondrial H2O2 was shown to regulate MAPK activity; H2O2 might act at multiple levels to activate, for example, JNK and p38 kinase: under normal conditions, thioredoxin is bound to and inhibits the activity of the apoptosis signal-regulating kinase-1 (ASK-1), a MAPKKK involved in both JNK and p38 activation. Oxidative stress dissociates the thioredoxin-ASK-1 complex leading to activation of p38.23 A similar mechanism may function at the level of JNK: under non-stressed conditions, glutathione transferase binds to JNK and inhibits its activation, but this interaction is disrupted by oxidative stress.30 Alternatively, JNK activation by H2O2 may occur in part through suppression of phosphatases involved in JNK inactivation.31,32 Likewise, NO diffusing from mitochondria can differentially regulate MAPK signaling: ERK1/2 are activated by NO through cGMP-dependent protein kinase and promote cell proliferation by enhancing matrix metalloproteinase-13 expression in endothelial cells33,34 (Figs. 5 and 6). The intracellular GSH levels determine the kinetics of NO-stimulated ERK1/2 activation in glial cells.35 NO decreased protein levels of MAP kinase phosphatase-3 by destabilizing its mRNA and inhibited tyrosine-specific phosphatases, presumably, through modification of their catalytic cysteine.36,37 Of particular importance to cellular signaling is the ability of mitochondria to release apoptotic signaling factors such as cytochrome c, a component of the respiratory chain. Release of cytochrome c from the inner membrane space of the





Figure 5 Mitochondrial generation of signaling molecules.




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Figure 6 Hydrogen peroxide, superoxide anion, and nitric oxide in cell signaling and gene expression.

mitochondria represents the initial step in the executioner phase of mitochondrion driven apoptosis. When released after membrane permeability transition, cytochrome c interacts with Apaf-1 to form the apoptosome and then can recruit and activate pro-caspase-9 in an ATP dependent process. Caspase-9 in turn activates caspase-3 and -7. These effector caspases are then responsible for the biochemical and morphological changes characteristic to apoptosis. It has been recently demonstrated that caspase-2 which is activated by genotoxic stress is directly involved in cytochrome c release. This is important as it represents an important link between DNA damage and mitochondrial apoptotic pathway that is directly engaged by caspase-2.38 CONCLUSION Mitochondria are the powerhouses of the cell as they do generate energy in the form of ATP to support cellular metabolic processes. During respiration, a fraction of oxygen is reduced univalently to O2  with subsequent dismutation to H2O2; mitochondria are recognized as major cellular sources of these species along with NO by virtue of a mitochondrial nitric oxide synthase, probably attached to the inner mitochondria membrane and in close proximity to complex IV, cytochrome c oxidase. Mitochondrion-generated free radicals are involved in the redox regulation of cell signaling, for they act as second messengers: H2O2 and NO can easily cross membranes and regulate cytosolic processes. Because of these and other properties, mitochondria became the harbinger of cell death upon the release of factors—most notably cytochrome c—that activate cytosolic apoptotic cascades.








REFERENCES 1. 2. 3. 4.

Sies H. Biochemistry of oxidative stress. Angew Chem Int Ed Engl 1986; 25:1058–1071. Sies H. Oxidative Stress. New York: Academic Press, 1985. Jones DP. Redefining oxidative stress. Antioxid Redox Signal 2006; 8:1865–1879. Sies H, Jones DP. In: Fink G, ed. Encyclopedia of Stress. 2nd ed. Academic Press, 2007:45–48.

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5. Rhee SG. Cell signaling. H2O2, a necessary evil for cell signaling. Science 2006; 312:1882–1883. 6. Rhee SG, Bae YS, Lee SR, et al. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci STKE 2000 Oct 10; 2000(53):PE1. 7. Kettenhofen NJ, Broniowska KA, Keszler A, et al. Proteomic methods for analysis of S-nitrosation. J Chromatogr B 2007; 851:152–159. 8. Handy DE, Loscalzo J. Nitric oxide and posttranslational modification of the vascular proteome: S-nitrosation of reactive thiols. Arterioscler Thromb Vasc Biol 2006; 26:1207–1214. 9. Mannick JB, Schonhoff CM. Nitrosylation: the next phosphorylation?. Arch Biochem Biophys 2002; 408:1–6. 10. Stamler JS, Lamas S, Fang FC. Nitrosylation: the prototypic redox-based signaling mechanism. Cell 2001; 106:675–683. 11. Klatt P, Lamas S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem 2000; 267:4928–4944. 12. 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. 13. Antunes F, Cadenas E. Cellular titration of apoptosis with steady state concentrations of H2O2: submicromolar levels of H2O2 induce apoptosis through Fenton chemistry independent of the cellular thiol state. Free Radic Biol Med 2001; 30:1008–1018. 14. Radi R. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci U S A 2004; 101:4003–4008. 15. Carballal S, Radi R, Kirk MC, et al. Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite. Biochemistry 2003; 42:9906–9914. 16. Boveris A, Cadenas E. Cellular sources and steady-state levels of reactive oxygen species. In: Clerch LB, Massaro DJ, eds. Oxygen, Gene Expression, and Cellular Function. New York: Marcel Dekker, 1997:1–25. 17. In˜arrea P. Purification and determination of activity of mitochondrial cyanide-sensitive superoxide dismutase in rat tissue extract. Methods Enzymol 2002; 349:106–114. 18. In˜arrea P, Moini H, Rettori D, et al. Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase. Biochem J 2005; 387:203–209. 19. Valdez LB, Zaobornyj T, Boveris A. Mitochondrial metabolic states and membrane potential modulate mtNOS activity. Biochim Biophys Acta 2006; 1757:166–172. 20. Valdez LB, Boveris A. Mitochondrial nitric oxide synthase, a voltage-dependent enzyme, is responsible for nitric oxide diffusion to cytosol. Front Biosci 2007; 12:1210–1219. 21. Riobo´ NA, Melani M, Sanjuan N, et al. The modulation of mitochondrial nitric-oxide synthase activity in rat brain development. J Biol Chem 2002; 277:42447–42455. 22. Riobo´ NA, Clementi E, Melani M, et al. Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation. Biochem J 2001; 359:139–145. 23. Valdez LB, Alvarez S, Arna´iz SL, et al. Reactions of peroxynitrite in the mitochondrial matrix. Free Radic Biol Med 2000; 29:349–356. 24. Han D, Canali R, Garcia J, et al. Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione. Biochemistry 2005; 44:11986–11996.

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25. Brown GC, Copper CE. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett 1994; 356:295–298. 26. Antunes F, Boveris A, Cadenas E. On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide. Proc Natl Acad Sci U S A 2004; 101:16774–16779. 27. Boveris A, Costa LE, Poderoso JJ, et al. Regulation of mitochondrial respiration by oxygen and nitric oxide. Ann NY Acad Sci 2000; 899:121–135. 28. Poderoso JJ, Carreras MC, Lisdero C, et al. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys 1996; 328:85–92. 29. Han D, Antunes F, Canali R, et al. Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. J Biol Chem 2003; 278:5557–5563. 30. Adler V, Yin Z, Fuchs SY, et al. Regulation of JNK signaling by GSTp. EMBO J 1999; 18:1321–1334. 31. Foley TD, Armstrong JJ, Kupchak BR. Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain. Biochem Biophys Res Commun 2004; 315:568–574. 32. Chen YR, Shrivastava A, Tan TH. Down-regulation of the c-Jun N-terminal kinase (JNK) phosphatase M3/6 and activation of JNK by hydrogen peroxide and pyrrolidine dithiocarbamate. Oncogene 2001; 20:367–374. 33. Parenti A, Morbidelli L, Cui XL, et al. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium. J Biol Chem 1998; 273:4220–4226. 34. Zaragoza C, Soria E, Lopez E, et al. Activation of the mitogen activated protein kinase extracellular signal-regulated kinase 1 and 2 by the nitric oxide-cGMPcGMP-dependent protein kinase axis regulates the expression of matrix metalloproteinase 13 in vascular endothelial cells. Mol Pharmacol 2002; 62:927–935. 35. Canals S, Casarejos MJ, de Bernardo S, et al. Selective and persistent activation of extracellular signal-regulated protein kinase by nitric oxide in glial cells induces neuronal degeneration in glutathione-depleted midbrain cultures. Mol Cell Neurosci 2003; 24:1012–1026. 36. Rossig L, Haendeler J, Hermann C, et al. Nitric oxide down-regulates MKP-3 mRNA levels: involvement in endothelial cell protection from apoptosis. J Biol Chem 2000; 275:25502–25507. 37. Callsen D, Pfeilschifter J, Brune B. Rapid and delayed p42/p44 mitogen-activated protein kinase activation by nitric oxide: the role of cyclic GMP and tyrosine phosphatase inhibition. J Immunol 1998; 161:4852–4858. 38. Gogvadze V, Orrenius S, Zhivotovsky B. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta 2006; 1757:639–647.

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2 Antioxidants and Modulation of Cardiovascular Disease Regine Heller Department of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich-Schiller-University of Jena, Jena, Germany

INTRODUCTION Growing evidence from experimental and animal studies as well as correlative data from human studies suggest that oxidative stress is implicated in a variety of chronic progressive diseases, such as atherosclerosis, neurodegenerative disorders and cancer.1–7 Since low levels of antioxidants were associated with an increased risk to develop oxidative stress related diseases8–12 antioxidants were suggested to modulate or even to prevent these diseases. The outcome of randomized clinical trials undertaken to prove this hypothesis remained however largely inconclusive.13,14 This review focuses on the current state of antioxidant modulation of cardiovascular disease. It briefly summarizes types and sources of oxidants as well as molecular processes through which oxidants contribute to atherosclerotic processes. Furthermore, a short overview about the antioxidant defence system, protective effects of antioxidant vitamins and results of antioxidant studies is given. Finally, potential reasons for the disparity of experimental, observational and clinical data and possible future strategies for a specific targeted antioxidant therapy are discussed.

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REACTIVE OXYGEN SPECIES Reactive oxygen species (ROS) are biologically important O2 derivatives which possess higher reactivity than molecular oxygen.3,15 They include free radicals or one-electron oxidants such as superoxide anion ( O2 ), hydroxyl radical ( OH) or nitric oxide ( NO) and nonradical two-electron oxidants, for example hydrogen peroxide (H2O2), hypochlorite/hypochlorous acid ( OCl/HOCl) and peroxynitrite (ONOO ). The sources of ROS in mammalian tissues are manifold and involve enzymatic and nonenzymatic intracellular pathways as well as the extracellular milieu. O2 may derive from aerobic respiration in the mitochondria16 or from enzymatic sources including phagocytic and vascular NAD (P)H oxidases,17–19 xanthine oxidase20 and uncoupled endothelial nitric oxide synthase (eNOS), i.e. eNOS deficient in its substrate arginine or its cofactor tetrahydrobiopterin.21,22 H2O2 is generated from dismutation of O2 , ONOO derives from the reaction of O2 with NO and HOCl is produced from Cl and H2O2 by the phagocyte-derived myeloperoxidase. The most reactive radical, OH, is produced by high energy irradiation or via the superoxide-driven Fenton reaction using traces of catalytic metal ions such as iron or copper. This radical is not counteracted by specific defence strategies and is probably the major representative of ROS-mediated cell damage.23 Traditionally, ROS were considered as potentially injurious by-products of normal oxidative metabolism or as tools through which phagocytes accomplish antimicrobial activity. Current evidence suggests, however, that ROS participate in cell signalling pathways leading to changes in gene transcription and cellular functions.15,24,25 Intracellular production of ROS is elicited in response to a host of stimuli including growth factors, cytokines, vasoactive substances and shear stress. Under physiological conditions ROS are produced in a controlled manner and contribute to the regulation of growth and tissue repair. Dependent on the magnitude of dose, the kinetics and duration of exposure and the type of cells ROS can also lead to transient or permanent growth arrest and finally to apoptotic or necrotic cell death. These responses are coordinated by a large number of signalling pathways including mitogen-activated protein kinases, phosphoinositide-3-kinase/Akt, phospholipase C-g1, Janus protein tyrosine kinases, p53, the transcription factors NFkB, AP-1 and HIF-1 as well as heat shock proteins. Molecular targets for ROS involve protein thiol groups, methionine residues, iron sulphur clusters and metals.15,24,25 The generation of ROS is usually in balance with antioxidant defence. In pathological settings an increase of ROS production or a reduction of antioxidant reserves may lead to an imbalance between oxidants and antioxidants in favour of the oxidants. This situation is defined as oxidative stress26 and may potentially cause oxidative damage if adaptive responses are not sufficient to compensate. Oxidative stress may involve uncontrolled activation of specific ROS signalling pathways and/or direct oxidation of DNA, lipids, and proteins. These processes have been suggested to contribute to a variety of diseases, including atherosclerosis, neurodegenerative disorders, 















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cancer, diabetes, and cataract although causal relationships have not been firmly established.1–7,27,28 ANTIOXIDANTS An antioxidant has been defined as a substance that when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate.29 Antioxidants may prevent the formation of primary oxidizing species or remove ROS after they have been generated or they may interact with secondary reactive species that arise from oxidative processes and attenuate or stop these processes after they have begun (chainbreaking antioxidants). Antioxidants involve metal-binding proteins, enzymes and low molecular mass compounds which function interactively and synergistically to neutralize ROS30 (Table 1). For example, ferritin, transferrin or ceruloplasmin sequester iron or copper and prevent the metal-catalysed formation of peroxyl or hydroxyl radicals.30,31 Antioxidant enzymes catalyse reactions that dismutate or divert ROS. Superoxide dismutase, for instance, removes O2 ,32,33 catalase reduces H2O2 to water,34 and glutathione peroxidase converts H2O2 and lipid hydroperoxides to water and lipid alcohols.34,35 In the latter reactions reduced glutathione is used as a cofactor and subsequently recycled by glutathione reductase. Thiol-disulfide oxidoreductases such as thioredoxin or glutaredoxin, and peroxiredoxins maintain the protein thiol state.36,37 Further enzymes participating in the antioxidant defence are glutathione-S-transferase, methionine sulfoxide reductase, heme oxygenase, g-glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, and glucose-6-phosphate dehydrogenase which provides NADPH as a reducing equivalent.38 

Table 1 The Antioxidant Defence System Protein antioxidants Enzymes (conversion of ROS) Superoxide dismutases Catalase Glutathione peroxidases Glutathione reductase Thiol-disulfide oxidoreductases Peroxiredoxins

Metal chelators (removal of catalytic metal ions) Ferritin Transferrin Ceruloplasmin

Small molecular weight antioxidants (scavenging of ROS) Water-soluble Lipid soluble Glutathione Vitamin E (a-tocopherol) Vitamin C (ascorbic acid) Ubiquinol Uric acid Carotenoids Bilirubin Polyphenols Lipoic acid Polyphenols Abbreviation: ROS, reactive oxygen species.

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Interestingly, some antioxidant enzymes underlie adaptive responses initiated by electrophiles or oxidative stress and mediated by the antioxidantresponse element (ARE)-nuclear factor-erythroid-2-related factor (Nrf2) signalling pathway.39,40 Nrf2 is sequestered in the cytoplasm by the Kelch-like ECH-associated protein 1 (Keap 1). Oxidation of cysteine thiol groups of Keap 1 results in a conformational change that renders Keap 1 unable to bind to Nrf2 which then translocates to the nucleus, activates ARE and leads to transcriptional regulation of target genes.41 This pathway has been described for drug metabolizing enzymes but also for heme oxgenase, thioredoxin, gastrointestinal glutathione peroxidase, the subunits of g-glutamate cysteine ligase, manganese superoxide dismutase and catalase.39,40 It has been speculated that this adaptive response may contribute to the beneficial health effects of exercise42 since this is known to cause low levels of lipid peroxidation and formation of electrophilic lipids.43 Dismutation and diversion of ROS by antioxidant enzymes is efficiently supported by small molecules (scavengers) which interact with primary ROS, such as O2 or with secondary reactive species such as lipid radicals. These low molecular weight antioxidants include water-soluble compounds (glutathione, ascorbic acid, uric acid, bilirubin, lipoic acid, polyphenols) and lipid-soluble compounds (vitamin E, ubiquinol, carotenoids, polyphenols) and are either of endogenous or dietary origin.30 Interestingly, when these compounds react with free radicals they are transformed into radicals themselves. Antioxidant radicals comprise lower reactivity but still need to be reduced or recycled to avoid damage. This implies an interaction with other antioxidants in a so-called antioxidant network.44 For instance, a-tocopherol, the most active form of vitamin E in human tissues, produces the a-tocopheroxyl radical which can be reduced back by ascorbate, ubiquinols or bilirubin.45–47 Furthermore, glutathione is maintained in a reduced state via reduction of the glutathione thiyl radical by ascorbate.48 Conversely, glutathione or lipoic acid are able to recycle dehydroascorbic acid back to ascorbic acid.49,50 Through these interactions antioxidants may also spare each other and elicit synergistic effects. 

ANTIOXIDANT VITAMINS Dietary antioxidants seem to play a major role in the antioxidant defence system and to be critical for optimal cellular and systemic health. The best investigated natural compounds are ascorbic acid (vitamin C) and a-tocopherol, others are carotenoids and polyphenols such as flavonoids.51–55 Ascorbic acid is one of the most important water-soluble antioxidants with almost ideal properties.56,57 Due to its low reduction potential it is able to react with virtually all physiologically relevant reactive oxygen and nitrogen species including nonradical oxidants such as HOCl and ONOO although the protection against these oxidants may not be complete.29,58–60 Furthermore, the ascorbyl radical formed from ascorbate in one-electron oxidations has a low reactivity and ascorbate can

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be readily regenerated from its oxidized forms by spontaneous chemical or by enzymatic reactions.58 a-Tocopherol, on the other hand, is the major natural lipid-soluble antioxidant.61,62 It belongs to the vitamin E group which consists of two classes of compounds, i.e. tocopherols and tocotrienols, with four structurally related isoforms in each class (a, b, g and d-forms). Due to a selective sorting in the liver by a specific a-tocopherol transfer protein a-tocopherol predominates in human blood and tissues.63 It is located in membranes and lipoproteins and its major antioxidant action is thought to be scavenging of lipid peroxyl radicals.64 In contrast, a-tocopherol does not seem to protect against HOCl or ONOO .65–67 Interestingly, a-tocopherol has been shown to modulate cellular signalling and transcriptional regulation independent of its antioxidative properties, partially via inhibition of protein kinase C.68,69 Proteins downregulated by a-tocopherol are the scavenger receptors SRA and CD36, interleukins 1b and 4, as well as the adhesion molecules VCAM-1 and CD11b/CD18. Ascorbic acid has also activities in addition to oxidant scavenging which are, however, related to its electron donor abilities. Ascorbate acts as a cofactor for several enzymes engaged in hydroxylation reactions, for example enzymes involved in the biosynthesis of collagen or carnitin70,71 and it has also been shown to affect the expression of extracellular matrix proteins and to upregulate antioxidant enzymes.72 Dietary antioxidants have garnered considerable interest during the last years which is mainly based on the observation that diets rich in antioxidants seem to be associated with a lower risk to develop oxidative stress related diseases.10–12,73,74 In addition, antioxidant vitamins were generally thought to have few adverse side effects and to be safe in therapeutical trials. In this context, effects of natural antioxidants, especially vitamin C and vitamin E, on cardiovascular diseases were intensively investigated. OXIDATIVE STRESS AND ATHEROSCLEROSIS Cardiovascular disease and the underlying pathology of atherosclerosis have been shown to represent a state of increased oxidative stress in the vascular wall.3,4 Moreover, oxidative stress is thought to be a unifying mechanism for many risk factors of atherosclerosis, such as smoking, obesity, diabetes and hypertension.27,75–79 One of the hypotheses of atherogenesis, the oxidative modification hypothesis, proposes that oxidation of LDL converts the native lipoprotein into a particle with proatherogenic activities which is responsible for the formation and development of atherosclerotic lesions.3,80–82 LDL modification may be mediated by radicals which lead to lipid oxidation or by twoelectron oxidants such as HOCl or ONOO which primarily modify apoprotein B.3 Oxidized LDL is susceptible to macrophage uptake via scavenger receptors leading to foam cell formation, and stimulates processes known to be involved in lesion formation. These include monocyte chemotaxis (via direct effects or via

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induction of monocyte chemotactic protein-1), endothelial adhesion molecule expression, recruitment of inflammatory cells, smooth muscle cell proliferation, and apoptosis of several cell types.3,83 In support of the oxidation theory oxidized lipids and proteins have been found in atherosclerotic lesions, and oxidized LDL as well as autoantibodies to oxidized LDL have been detected in the plasma of patients.3,84 Furthermore, increased levels of urinary and circulating F2 isoprostanes (chemically stable free-radical-catalysed products of arachidonic acid) have been found in patients with atherosclerosis or with risk factors for atherosclerosis indicating oxidative stress in vivo.75 Oxidative processes, either directly or via oxidation of LDL, may also contribute to endothelial dysfunction, i.e. to a loss of NO bioavailability, which is thought to be an early step in atherogenesis.85–88 NO is produced in endothelial cells and is known to be a central regulator of vascular homeostasis with vasorelaxing and antiatherogenic properties including inhibition of platelet aggregation, monocyte and leukocyte adhesion to the endothelium and smooth muscle cell proliferation.89 ROS may affect NO bioavailability in several ways. O2 , for example, has been shown to scavenge and inactivate NO directly whereas ONOO is thought to inhibit NO biosynthesis via oxidation of the Znthiolate cluster of eNOS or via oxidation of tetrahydrobiopterin.21,22 Tetrahydrobiopterin is a reducing cofactor of eNOS.90,91 It is responsible for coupling oxygen reduction to arginine oxidation and prevents O2 formation by eNOS. Upon reaction with oxidants tetrahydrobiopterin forms a neutral trihydrobiopterin radical which further disproportionates to the quinonoid 6,7-[8H]dihydrobiopterin. Both compounds can either be recycled or irreversibly oxidized. The latter leads to tetrahydrobiopterin depletion and, as a consequence, not only less NO is formed but eNOS is uncoupled, i.e. converted into a O2 generating enzyme.21,22,91,92 

















ANTIOXIDANT VITAMINS AND ATHEROSCLEROSIS Experimental and Animal Studies Based on the observation that oxidative stress is associated with atherogenesis and that plasma levels of ascorbic acid and a-tocopherol are inversely correlated to the mortality from coronary heart disease, antioxidant vitamins were suggested to protect from cardiovascular disease. This assumption has been encouraged by the majority of in vitro and cell culture studies demonstrating inhibitory effects of ascorbic acid or a-tocopherol on key events of atherogenesis.93,94 It has been clearly demonstrated that a-tocopherol acts as a chain-breaking antioxidant by scavenging highly reactive lipid peroxyl and alkoxyl radicals and stopping the propagation of lipid peroxidation and thus LDL oxidation.95 Ascorbate supports a-tocopherol by regenerating the a-tocopheroxyl radical and by scavenging oxidants that may initiate lipid peroxidation in the aqueous milieu.45,96 Ascorbate and a-tocopherol have both been shown to decrease

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adhesion molecule expression on endothelial cells and to reduce leukocyte adhesion either by antioxidant mechanisms or, in the case of a-tocopherol, by inhibition of protein kinase C.97–101 Finally, both, ascorbic acid and a-tocopherol have been reported to improve endothelial dysfunction via several mechanisms.88,102 Ascorbate in high concentrations ( 10 mM) protects NO from inactivation by scavenging O2 .103 Furthermore, ascorbate is able to reduce the trihydrobiopterin radical as well as the quinonoid 6,7-[8H]-dihydrobiopterin and thus, to regenerate oxidized tetrahydrobiopterin and to prevent eNOS uncoupling.104–107 In contrast, the beneficial effect of a-tocopherol on endothelial dysfunction is mainly attributed to its ability to counteract adverse effects of oxidized LDL on NO formation.108 In addition, a-tocopherol has been shown to promote activation of eNOS via effects on eNOS phosphorylation.109 The vasoprotective effects of natural antioxidants described in vitro have been confirmed in animal models of atherogenesis and atherosclerosis regression although results have not been uniformly positive. 94,110,111 



Epidemiological and Clinical Studies The effect of ascorbic acid and a-tocopherol on cardiovascular disease in humans has been investigated in various epidemiological and clinical studies. As a first approach, several large prospective observational studies were performed which compared the development of cardiovascular disease as measured by defined endpoints (for example myocardial infarction or mortality from coronary heart disease) in subjects with a different estimated intake of antioxidant vitamins (dietary and supplemental). Many but not all of these studies suggested an inverse association of vitamin E or C intake and cardiovascular disease.9,93,111–113 Subsequently, large-scale randomized clinical trials were carried out to prove a causal relationship between the increased intake of natural antioxidants and the reduced risk for cardiovascular disease.13,14,111,114–117 Most of these trials were conducted on patients with established atherosclerosis or with high risk for cardiovascular disease. In most cases vitamin E alone or in combination with other antioxidants was investigated while vitamin C alone was not tested. Vitamin E was used at different doses and pharmaceutical formulations (natural or synthetic a-tocopherol preparations) and for different periods. The majority of controlled interventional trials was not able to demonstrate beneficial effects of antioxidant supplementation despite the fact that observational studies strongly suggested this benefit. Protective effects of vitamin E supplements on the progression of cardiovascular disease have only been documented in subgroups or in some smaller studies.118–120 On the other hand, there is some evidence of potentially adverse effects of vitamin E supplements including an increase of overall mortality.121 In addition to interventional trials with endpoint measurements, a large number of studies has examined the effect of natural antioxidants on several clinical markers of cardiovascular disease including flow-mediated vasodilation,

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carotid artery intima-media ratio as well as C-reactive protein levels, soluble adhesion molecules, antibodies against oxidized LDL and plasma levels of F2 isoprostanes.110 The results of these studies appear to be more promising. For example, beneficial effects of a-tocopherol in combination with vitamin C on the progression of the intima-media thickness have been reported in the Antioxidant Supplementation in Atherosclerosis Prevention study (ASAP)122 and the Intravascular Ultrasonography study (IVUS).123 Furthermore, with only a few exceptions, many studies have documented that ascorbic acid can reverse endothelial dysfunction in patients with atherosclerosis and several conditions that predispose to atherosclerosis.124,125 Endothelial function was determined as flow-mediated or acetylcholine-induced vasodilation and positive effects were seen in peripheral or coronary arteries, and with both ascorbic acid infusion and oral supplementation. a-Tocopherol has also been shown to improve endothelial dysfunction in some but not all studies and seems to be more effective when combined with vitamin C.126 ANTIOXIDANTS—DISAPPOINTMENT OR CHALLENGE? The ineffectiveness of antioxidants in reducing cardiovascular death and morbidity in controlled interventional trials has questioned the importance of oxidative stress in human atherosclerosis and the general belief that antioxidant supplementation may prevent cardiovascular disease. Consequently, many investigators have tried to explain the discrepancies between the protective role of antioxidants observed in most experimental and several human studies and the negative outcome of most randomized clinical trials. Generally, it has been argued that the large-scale trials suffer from inadequate dosage and type of the antioxidant, from inappropriate selection of patients suitable to test the hypothesis and from poor monitoring of the study.2,62 Furthermore, it becomes increasingly clear that a better understanding of the nature of oxidation involved in the disease process is necessary and that the complex chemistry and biochemistry of oxidative stress and antioxidants need to be considered to develop efficient therapeutic approaches (Table 2).

Table 2 Strategies for Future Antioxidative Therapies Characterization of specific oxidants involved in disease aetiology Assessment of oxidative stress and antioxidant action via sensitive and specific biomarkers Inhibition of disease-related ROS formation Maintenance of physiological ROS signalling Antioxidant targeting to subcellular compartments Employment of antioxidant combinations Combination of antioxidative and anti-inflammatory approaches Abbreviation: ROS, reactive oxygen species.

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Requirement of a Basal Oxidant Tone Recent data on regulatory and signalling effects of ROS suggest that a basal or tonal concentration of ROS is essential for maintaining cellular functions.15,24,25 This is especially important at the level of the mitochondria where electron transport to molecular oxygen is coupled to production of ATP (oxidative phosphorylation). ROS are formed in different compartments and concentration gradients are highly important. Some degree of localized oxidation seems to play a role in protein folding in the endoplasmic reticulum to permit disulfide formation,127 in growth factor signalling,128 in activation of several gene transcription factors or in mediating adaptive responses and upregulation of protective systems that render the cells more resistant to a subsequent insult (antioxidant enzymes, ferritin, heat shock proteins).39,129 Low quantities of ROS are known to stimulate cell proliferation. H2O2, for instance, has been shown to inactivate protein tyrosine phosphatases via oxidation of a critical cysteine residue which may be essential for tyrosine phosphorylation of growth factor receptors.130,131 A similar mechanism may play a role in insulin signalling and thus in the regulation of insulin sensitivity.132 ROS are also involved in the regulation of protein degradation and apoptosis.133 Oxidation of cysteine sulfhydryl groups of thioredoxin, for example, leads to the release of the apoptosis signal-regulating kinase from its complex with thioredoxin and subsequently to stimulation of stress-activated protein kinases and apoptosis.134 The requirement of a basal ROS tone for cell signalling may help to explain why many antioxidant-based therapies failed. Abolishment of ROS by vigorous use of antioxidants may not always be beneficial. Antioxidants may inhibit cell proliferation, prevent adaptation to oxidative stress or even accelerate oxidative damage. Furthermore, inhibition of ROS-induced apoptosis may lead to increased necrotic cell death with release of cell contents such as transition metals that could amplify oxidative processes. Thus, a more subtle approach of antioxidant therapy appears to be required which should consider the type and location of ROS generation. A recent meta-analysis of clinical studies demonstrating that high-dosage vitamin E supplementation (>400 IU/day) increased all-cause mortality seems to support the concept that global suppression of oxidation may eliminate some beneficial processes121 although this analysis was not uniformly accepted.135,136,137 In contrast, anti-inflammatory activities of a-tocopherol (inhibition of pro-inflammatory cytokine release, reduction of monocyte adhesion to endothelial cells, decrease of C-reactive protein levels) which are increasingly thought to be implicated in its vasoprotective effects have been shown to require high doses (600–800 IU/day).69 Clearly, more data on dose-effect relationships of antioxidants are needed. Characterization of Oxidative Events as a Cause of Disease Antioxidants tested in intervention studies so far were selected according to their ability to inhibit free radical-induced LDL oxidation and may not have

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sufficiently targeted other relevant oxidants. There is growing evidence that twoelectron oxidants, in particular H2O2, HOCl or ONOO are involved in atherosclerosis.3 HOCl and ONOO have already been shown to modify LDL67,138 and other proteins, and ONOO seems to be a major oxidant reacting with tetrahydrobiopterin.139 Furthermore, H2O2 as a component of cell signalling is increasingly thought to mediate augmented proliferative processes which have been implicated in lesion formation.140 Importantly, lipid-soluble antioxidants such as vitamin E are not able to affect nonradical oxidants65–67 and as a consequence, processes due to uncontrolled generation of H2O2, HOCl or ONOO may not have been altered in intervention studies with vitamin E supplementation. In the future, a more complete understanding of the oxidative events promoting atherosclerosis will allow a more specific selection of appropriate antioxidants for therapeutic strategies. Additionally, it will be necessary to characterize the stage of disease which is mainly promoted by oxidative stress. It is possible, for example, that ROS generation is more relevant to the initiation of lesion formation and that antioxidant protection is needed at an early age. Accordingly, the beneficial effects seen in dietary studies may reflect a life-long support with dietary antioxidants. In contrast, most antioxidant interventional trials were performed in patients with advanced atherosclerosis.13,14 One must also consider the possibility that oxidative events represent rather a consequence than a cause of cardiovascular disease. Indeed, atherosclerotic lesion formation can also be dissociated from the occurrence of lipid peroxidation.141,142 It may be possible that oxidative events are a result of vascular inflammation and not strictly required for the progression of atherosclerosis (oxidative response to inflammation hypothesis of atherosclerosis3). In this case, antioxidant treatment may not have a major impact on the development of disease since it would not affect the link between inflammation and atherosclerosis. Moreover, antioxidants may even attenuate the healing response to inflammation which may be promoted by ROS at low levels, and, as a consequence, worsen lesion formation. Thus, a clear distinction and characterization of oxidative events as cause of atherosclerosis is requisite for antioxidant strategies. Antioxidant Supplementation Versus Diet The antioxidant intake recorded in dietary studies and shown to be inversely associated with cardiovascular disease may be a marker for some other dietary or lifestyle factor that is providing cardiovascular benefit. It is plausible to suppose that persons who select a diet rich in antioxidants have also other health habits that may lower their risk for cardiovascular disease. Furthermore, dietary compounds may act as Nrf-2-Keap1-ARE activators and improve the defence system provided by antioxidant enzymes.143,144 These components include sulforaphane, a metabolite of the glucosinolate glucoraphanin which is found in crucifers (particularly in broccoli),145 diallyl sulphide from allium vegetables146 as well as

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the flavonoids kaempferol, epigallocatechin-3-gallate and curcumin.147–149 It may also be that the combination of antioxidants (vitamin C and E, carotenoids, flavonoids and other polyphenols) provided with diet is more efficient than the supplementation of a single compound. In plants, for example, knockout of a single antioxidant may cause a serious injury to the cells despite the presence of many other antioxidants.150 It is known that antioxidants need to recharge each other after they have reacted with free radicals and have been converted into radicals themselves. The function of a-tocopherol as a chain-breaking, for example, requires the presence of a coantioxidant to reduce the a-tocopheroxyl radical which otherwise would mediate further formation of lipid radicals.151 Thus, supplementation with a combination of antioxidants may reduce the potential for a paradoxical increase in oxidant generation. According to their structural features antioxidants may also protect different intracellular compartments, i.e. membrane or cytoplasm, and react with different radical and/or nonradical oxidants. As a consequence, they may exhibit distinct protective effects. Data from our group, for instance, demonstrate that ascorbate and a-tocopherol affect endothelial NO synthesis independently from each other via different mechanisms, i.e. ascorbic acid but not a-tocopherol regenerates oxidized tetrahydrobiopterin and a-tocopherol but not ascorbate promotes eNOS phosphorylation at serine 1177. Additionally, we were able to show that interactions between the two compounds take place, i.e. ascorbate is able to potentiate the effect of a-tocopherol, most probably by recycling oxidized a-tocopherol.106,109 

Selection of Patients A detailed knowledge about the nature of oxidative processes which trigger atherosclerotic lesion formation is not only important for the selection of specific antioxidants but will also allow selection and monitoring of a population that may respond to antioxidant treatment. It is possible that patients included in previous intervention trials were inappropriate to test the therapeutic efficacy of antioxidants since they were not selected according to a biochemical evidence for elevated ROS formation. It will be important to identify novel biomarkers which indicate increased HOCl or ONOO generation in addition to the known markers of lipid peroxidation.152–154 Indeed, it has been shown that F2 isoprostanes were only linked to some (smoking, obesity, diabetes) but not all risk factors of atherosclerosis.77 Oxidative events other than LDL oxidation, for example a loss of NO bioavailability which can be measured as endothelialdependent vasodilation may be used to identify patients at risk and to monitor antioxidant action.155–158 Furthermore, evaluation of the endogenous antioxidant defence system and of oxidant enzymes will be important to characterize the risk to develop oxidative stress as a cause of disease.38,159 In this context, genetic factors involved in oxidative processes and antioxidant defence will help to identify patients that may respond to an antioxidant treatment.87,160 

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Selection of Antioxidants Future studies may better define targets for antioxidant therapy other than LDL oxidation. For example, restoring endothelial function has become an attractive therapeutic approach86,156 and may be realized by preventing tetrahydrobiopterin oxidation and eNOS uncoupling.21,22,91,92 Furthermore, it may be important to target specific ROS populations or compartments of ROS generation. Leakage from the mitochondrial electron transport chain, for example, is a significant source of O2 16 and antioxidants which unlike vitamin C and E preferentially accumulate in the mitochondria may be more effective in ameliorating oxidative stress-mediated disease.161,162 Mitochondrial targeting is based on biophysical properties of the mitochondria (high negative internal potential promoting accumulation of lipophilic cations) and on the unique mitochondrial localization of enzymes and transporters.163–165 The mitochondrially targeted compounds described so far have shown promising results in a range of in-vitro systems.162 Finally, the best antioxidants may be those that interfere with the production of ROS. Drugs that influence the expression and activity of NAD(P)H oxidases such as statins, angiotensin-converting enzyme inhibitors or ligands of peroxisome proliferator-activated receptor-gamma have already been shown to attenuate cardiovascular oxidative stress.166–169 The development of specific inhibitors that interfere with the assembly of NAD(P)H oxidase components170 or compounds that target the myeloperoxidase pathway171–172 may represent novel antioxidant strategies. 

CONCLUSION Although atherosclerosis represents a state of increased oxidative stress in the vasculature antioxidant strategies have not been proven to limit cardiovascular events based on atherosclerotic processes. It seems, however, to be premature to conclude that the oxidation hypothesis of disease causality has to be rejected and antioxidant modulation of disease is not effective (Table 2). Pharmacological intervention with antioxidants requires a better understanding of ROS signalling pathways and ROS localization as well as a clear definition of oxidants which are involved in disease aetiology. Antioxidants should target the dysregulation rather than interfere with physiological signalling of ROS. An important prerequisite for antioxidant strategies is the development of sensitive and specific biomarkers that can be used to assess the oxidative stress phenotype which underlies a certain vascular pathology and to monitor antioxidant action. Identification of patients at risk may include the characterization of genetic variants of oxidant and antioxidant enzymes. Future antioxidative acting drugs should target specific intracellular compartments of ROS production such as mitochondria or oxidant enzymes such as NAD(P)H oxidase. Furthermore, combinations of different antioxidants or of antioxidative and anti-inflammatory treatments may help in early intervention. In conclusion, the challenge of future

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research will be to develop specific antioxidant approaches for specific oxidant phenotypes of patients that are likely to develop atherosclerosis or other oxidative stress-related diseases.

REFERENCES 1. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: Part I: basic mechanisms and in vivo monitoring of ROS. Circulation 2003; 108:1912– 1916. 2. Griendling KK, FitzGerald GA. Oxidative stress and cardiovascular injury: Part II: animal and human studies. Circulation 2003; 108:2034–2040. 3. Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev 2004; 84:1381–1478. 4. Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 2005; 25:29–38. 5. Barnham KJ, Masters CL, Bush AI. Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov 2004; 3:205–214. 6. Andersen JK. Oxidative stress in neurodegeneration: cause or consequence? Nat Med 2004; 10(suppl):S18–S25. 7. Poulsen HE. Oxidative DNA modifications. Exp Toxicol Pathol 2005; 57 (suppl 1):161–169. 8. Gey KF, Puska P, Jordan P, et al. Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am J Clin Nutr 1991; 53(suppl 1):326S–334S. 9. Carr AC, Frei B. Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am J Clin Nutr 1999; 69: 1086–1107. 10. Berman K, Brodaty H. Tocopherol (vitamin E) in Alzheimer’s disease and other neurodegenerative disorders. CNS Drugs 2004; 18:807–825. 11. Frank B, Gupta S. A review of antioxidants and Alzheimer’s disease. Ann Clin Psychiatry 2005; 17:269–286. 12. Borek C. Dietary antioxidants and human cancer. Integr Cancer Ther 2004; 3: 333–341. 13. Riley SJ, Stouffer GA. Cardiology Grand Rounds from the University of North Carolina at Chapel Hill. The antioxidant vitamins and coronary heart disease: Part II. Randomized clinical trials. Am J Med Sci 2003; 325:15–19. 14. Kris-Etherton PM, Lichtenstein AH, Howard BV, et al. Antioxidant vitamin supplements and cardiovascular disease. Circulation 2004; 110:637–641. 15. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000; 279:L1005–L1028. 16. Andreyev AY, Kushnareva YE, Starkov AA. Mitochondrial metabolism of reactive oxygen species. Biochemistry (Mosc) 2005; 70:200–214. 17. Griendling KK. Novel NAD(P)H oxidases in the cardiovascular system. Heart 2004; 90:491–493. 18. Cai H. NAD(P)H oxidase-dependent self-propagation of hydrogen peroxide and vascular disease. Circ Res 2005; 96:818–822.

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24

Heller

19. Sumimoto H, Miyano K, Takeya R. Molecular composition and regulation of the Nox family NAD(P)H oxidases. Biochem Biophys Res Commun 2005; 338: 677–686. 20. Berry CE, Hare JM. Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications. J Physiol 2004; 555:589–606. 21. Fo¨rstermann U, Mu¨nzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 2006; 113:1708–1714. 22. Fo¨rstermann U. Endothelial NO synthase as a source of NO and superoxide. Eur J Clin Pharmacol 2006; 62(suppl 13):5–12. 23. Kehrer JP. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 2000; 149:43–50. 24. Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 2002; 192:1–15. 25. Boonstra J, Post JA. Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 2004; 337:1–13. 26. Sies H, ed. Oxidative stress: oxidants and antioxidants. London: Academic Press, 1991. 27. Evans JL, Goldfine ID, Maddux BA, et al. Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 2002; 23:599–622. 28. Spector A. Oxidative stress-induced cataract: mechanism of action. FASEB J 1995; 9:1173–1182. 29. Halliwell B. Free radicals, proteins and DNA: oxidative damage versus redox regulation. Biochem Soc Trans 1996; 24:1023–1027. 30. Halliwell B, Gutteridge JMC. Free Radicals in biology and medicine. New York: Oxford University Press, 1999. 31. Halliwell B, Gutteridge JM. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990; 280:1–8. 32. Faraci FM, Didion SP. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol 2004; 24:1367–1373. 33. Johnson F, Giulivi C. Superoxide dismutases and their impact upon human health. Mol Aspects Med 2005; 26:340–352. 34. Rhee SG, Yang KS, Kang SW, et al. Controlled elimination of intracellular H2O2: regulation of peroxiredoxin, catalase, and glutathione peroxidase via posttranslational modification. Antioxid Redox Signal 2005; 7:619–626. 35. Arthur JR. The glutathione peroxidases. Cell Mol Life Sci 2000; 57:1825–1835. 36. Holmgren A, Johansson C, Berndt C, et al. Thiol redox control via thioredoxin and glutaredoxin systems. Biochem Soc Trans 2005; 33:1375–1377. 37. Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 2005; 38:1543–1552. 38. Leopold JA, Loscalzo J. Oxidative enzymopathies and vascular disease. Arterioscler Thromb Vasc Biol 2005; 25:1332–1340. 39. Holtzclaw WD, Dinkova-Kostova AT, Talalay P. Protection against electrophile and oxidative stress by induction of phase 2 genes: the quest for the elusive sensor that responds to inducers. Adv Enzyme Regul 2004; 44:335–367. 40. Lee JS, Surh YJ. Nrf2 as a novel molecular target for chemoprevention. Cancer Lett 2005; 224:171–184.

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Antioxidants and Modulation of Cardiovascular Disease

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41. Kang KW, Lee SJ, Kim SG. Molecular mechanism of nrf2 activation by oxidative stress. Antioxid Redox Signal 2005; 7:1664–1673. 42. Leeuwenburgh C, Heinecke JW. Oxidative stress and antioxidants in exercise. Curr Med Chem 2001; 8:829–838. 43. Parthasarathy S, Santanam N, Ramachandran S, et al. Potential role of oxidized lipids and lipoproteins in antioxidant defense. Free Radic Res 2000; 33:197–215. 44. Thiele JJ, Schroeter C, Hsieh SN, et al. The antioxidant network of the stratum corneum. Curr Probl Dermatol 2001; 29:26–42. 45. May JM, Qu ZC, Mendiratta S. Protection and recycling of alpha-tocopherol in human erythrocytes by intracellular ascorbic acid. Arch Biochem Biophys 1998; 349:281–289. 46. Mukai K, Itoh S, Morimoto H. Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution. J Biol Chem 1992; 267:22277–22281. 47. Neuzil J, Stocker R. Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. J Biol Chem 1994; 269:16712–16719. 48. Tamba M, Simone G, Quintiliani M. Interactions of thiyl free radicals with oxygen: a pulse radiolysis study. Int J Radiat Biol Relat Stud Phys Chem Med 1986; 50:595–600. 49. May JM, Qu ZC, Cobb CE. Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid. J Biol Chem 2004; 279:14975–14982. 50. Kagan VE, Shvedova A, Serbinova E, et al. Dihydrolipoic acid - a universal antioxidant both in the membrane and in the aqueous phase. Reduction of peroxyl, ascorbyl and chromanoxyl radicals. Biochem Pharmacol 1992; 44:1637–1649. 51. Packer L, Traber MG, Kraemer K, et al., eds. The antioxidant vitamins C and E. Champaign, IL: AOCS Press, 2002. 52. Kelly F, Meydani M, Packer L, eds. Vitamin E and health. Annals of the New York Academy of Sciences, vol. 1031. New York: New York Academy of Sciences, 2004. 53. Stahl W, Sies H. Bioactivity and protective effects of natural carotenoids. Biochim Biophys Acta 2005; 1740:101–107. 54. Tapiero H, Tew KD, Ba GN, et al. Polyphenols: do they play a role in the prevention of human pathologies? Biomed Pharmacother 2002; 56:200–207. 55. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu Rev Nutr 2002; 22:19–34. 56. Frei B, England L, Ames BN. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci U S A 1989; 86:6377–6381. 57. Frei B. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage. Am J Clin Nutr 1991; 54(suppl):1113S–1118S. 58. Buettner GR. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch Biochem Biophys 1993; 300: 535–543. 59. Carr AC, Tijerina T, Frei B. Vitamin C protects against and reverses specific hypochlorous acid- and chloramine-dependent modifications of low-density lipoprotein. Biochem J 2000; 346:491–499. 60. Bartlett D, Church DF, Bounds PL, et al. The kinetics of the oxidation of L-ascorbic acid by peroxynitrite. Free Radic Biol Med 1995; 18:85–92.

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26

Heller

61. Brigelius-Flohe´ R, Traber MG. Vitamin E: function and metabolism. FASEB J 1999; 13:1145–1155. 62. Brigelius-Flohe´ R, Kelly FJ, Salonen JT, et al. The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr 2002; 76:703–716. 63. Stocker A, Azzi A. Tocopherol-binding proteins: their function and physiological significance. Antioxid Redox Signal 2000; 2:397–404. 64. Wang X, Quinn PJ. The location and function of vitamin E in membranes. Mol Membr Biol 2000; 17:143–156. 65. Hazell LJ, Stocker R. Alpha-tocopherol does not inhibit hypochlorite-induced oxidation of apolipoprotein B-100 of low-density lipoprotein. FEBS Lett 1997; 414:541–544. 66. Pattison DI, Hawkins CL, Davies MJ. Hypochlorous acid-mediated oxidation of lipid components and antioxidants present in low-density lipoproteins: absolute rate constants, product analysis, and computational modeling. Chem Res Toxicol 2003; 16:439–449. 67. Thomas SR, Davies MJ, Stocker R. Oxidation and antioxidation of human lowdensity lipoprotein and plasma exposed to 3-morpholinosydnonimine and reagent peroxynitrite. Chem Res Toxicol 1998; 11:484–494. 68. Zingg JM, Azzi A. Non-antioxidant activities of vitamin E. Curr Med Chem 2004; 11:1113–1133. 69. Singh U, Devaraj S, Jialal I. Vitamin E, oxidative stress, and inflammation. Annu Rev Nutr 2005; 25:151–174. 70. Ronchetti IP, Quaglino D Jr., Bergamini G. Ascorbic acid and connective tissue. Subcell Biochem 1996; 25:249–264. 71. Rebouche CJ. Ascorbic acid and carnitine biosynthesis. Am J Clin Nutr 1991; 54 (suppl):1147S–1152S. 72. Arrigoni O, De Tulli MC. Ascorbic acid: much more than just an antioxidant. Biochim Biophys Acta 2002; 1569:1–9. 73. Jacob RA, Sotoudeh G. Vitamin C function and status in chronic disease. Nutr Clin Care 2002; 5:66–74. 74. Rimm EB, Stampfer MJ. Antioxidants and chronic disease: evidence from observational epidemiology. Bibl Nutr Dieta 2001; 55:80–91. 75. Morrow JD. Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans. Arterioscler Thromb Vasc Biol 2005; 25:279–286. 76. Ambrose JA, Barua RS. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J Am Coll Cardiol 2004; 43:1731–1737. 77. Keaney JF Jr., Larson MG, Vasan RS, et al. Obesity and systemic oxidative stress: clinical correlates of oxidative stress in the Framingham Study. Arterioscler Thromb Vasc Biol 2003; 23:434–439. 78. Son SM, Whalin MK, Harrison DG, et al. Oxidative stress and diabetic vascular complications. Curr Diab Rep 2004; 4:247–252. 79. Lassegue B, Griendling KK. Reactive oxygen species in hypertension: an update. Am J Hypertens 2004; 17:852–860. 80. Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989; 320:915–924. 81. Chisolm GM, Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic Biol Med 2000; 28:1815–1826.

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Antioxidants and Modulation of Cardiovascular Disease

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82. Witztum JL, Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans? Trends Cardiovasc Med 2001; 11:93–102. 83. Steinberg D. Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 1997; 272:20963–20966. 84. Tsimikas S. Oxidized low-density lipoprotein biomarkers in atherosclerosis. Curr Atheroscler Rep 2006; 8:55–61. 85. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87:840–844. 86. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation 2004; 109(23 suppl 1):III27–III32. 87. Loscalzo J, Voetsch B, Liao R, et al. Genetic determinants of vascular oxidant stress and endothelial dysfunction. Congest Heart Fail 2005; 11:73–79. 88. Heller R, Werner-Felmayer G, Werner ER. Antioxidants and endothelial nitric oxide synthesis. Eur J Clin Pharmacol 2006; 62(suppl 13):21–28. 89. Dudzinski DM, Igarashi J, Greif D, et al. The regulation and pharmacology of endothelial nitric oxide synthase. Annu Rev Pharmacol Toxicol 2006; 46:235–276. 90. Werner ER, Gorren AC, Heller R, et al. Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects. Exp Biol Med (Maywood) 2003; 228:1291–1302. 91. Channon KM. Tetrahydrobiopterin: regulator of endothelial nitric oxide synthase in vascular disease. Trends Cardiovasc Med 2004; 14:323–327. 92. Kawashima S, Yokoyama M. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler Thromb Vasc Biol 2004; 24:998–1005. 93. Diaz MN, Frei B, Vita JA, et al. Antioxidants and atherosclerotic heart disease. N Engl J Med 1997; 337:408–416. 94. Carr AC, Zhu BZ, Frei B. Potential antiatherogenic mechanisms of ascorbate (vitamin C) and alpha-tocopherol (vitamin E). Circ Res 2000; 87:349–354. 95. Esterbauer H, Dieber-Rotheneder M, Striegl G, et al. Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr 1991; 53 (suppl):314S–321S. 96. Carr A, Frei B. Does vitamin C act as a pro-oxidant under physiological conditions? FASEB J 1999; 13:1007–1024. 97. Weber C, Erl W, Weber K, et al. Increased adhesiveness of isolated monocytes to endothelium is prevented by vitamin C intake in smokers. Circulation 1996; 93:1488–1492. 98. Woollard KJ, Loryman CJ, Meredith E, et al. Effects of oral vitamin C on monocyte: endothelial cell adhesion in healthy subjects. Biochem Biophys Res Commun 2002; 294:1161–1168. 99. Devaraj S, Li D, Jialal I. The effects of alpha tocopherol supplementation on monocyte function. Decreased lipid oxidation, interleukin 1 beta secretion, and monocyte adhesion to endothelium. J Clin Invest 1996; 98:756–763. 100. Martin A, Foxall T, Blumberg JB, et al. Vitamin E inhibits low-density lipoproteininduced adhesion of monocytes to human aortic endothelial cells in vitro. Arterioscler Thromb Vasc Biol 1997; 17:429–436. 101. Yoshida N, Yoshikawa T, Manabe H, et al. Vitamin E protects against polymorphonuclear leukocyte-dependent adhesion to endothelial cells. J Leukoc Biol 1999; 65:757–763.

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102. Tomasian D, Keaney JF Jr., Vita JA. Antioxidants and the bioactivity of endothelium-derived nitric oxide. Cardiovasc Res 2000; 47:426–435. 103. Jackson TS, Xu A, Vita JA, et al. Ascorbate prevents the interaction of superoxide and nitric oxide only at very high physiological concentrations. Circ Res 1998; 83:916–922. 104. Patel KB, Stratford MR, Wardman P, et al. Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate. Free Radic Biol Med 2002; 32:203–211. 105. Toth M, Kukor Z, Valent S. Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid: contribution to placental endothelial nitric oxide synthase activity. Mol Hum Reprod 2002; 8:271–280. 106. Heller R, Unbehaun A, Schellenberg B, et al. L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem 2001; 276:40–47. 107. d’Uscio LV, Milstien S, Richardson D, et al. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res 2003; 92:88–95. 108. Keaney JF Jr., Guo Y, Cunningham D, et al. Vascular incorporation of alphatocopherol prevents endothelial dysfunction due to oxidized LDL by inhibiting protein kinase C stimulation. J Clin Invest 1996; 98:386–394. 109. Heller R, Hecker M, Stahmann N, et al. Alpha-tocopherol amplifies phosphorylation of endothelial nitric oxide synthase at serine 1177 and its short-chain derivative trolox stabilizes tetrahydrobiopterin. Free Radic Biol Med 2004; 37: 620–631. 110. Meydani M. Vitamin E modulation of cardiovascular disease. Ann N Y Acad Sci 2004; 1031:271–279. 111. Kaliora AC, Dedoussis GV, Schmidt H. Dietary antioxidants in preventing atherogenesis. Atherosclerosis 2006; 187:1–17. 112. Riley SJ, Stouffer GA. Cardiology Grand Rounds from the University of North Carolina at Chapel Hill. The antioxidant vitamins and coronary heart disease: Part I. Basic science background and clinical observational studies. Am J Med Sci 2002; 324:314–320. 113. Gaziano JM. Vitamin E and cardiovascular disease: observational studies. Ann N Y Acad Sci 2004; 1031:280–291. 114. Upston JM, Kritharides L, Stocker R. The role of vitamin E in atherosclerosis. Prog Lipid Res 2003; 42:405–422. 115. Morris CD, Carson S. Routine vitamin supplementation to prevent cardiovascular disease: a summary of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med 2003; 139:56–70. 116. Heinecke JW. Clinical trials of vitamin E in coronary artery disease: is it time to reconsider the low-density lipoprotein oxidation hypothesis? Curr Atheroscler Rep 2003; 5:83–87. 117. Vivekananthan DP, Penn MS, Sapp SK, et al. Use of antioxidant vitamins for the prevention of cardiovascular disease: meta-analysis of randomised trials. Lancet 2003; 361:2017–2023. 118. Stephens NG, Parsons A, Schofield PM, et al. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 1996; 347:781–786.

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119. Rapola JM, Virtamo J, Ripatti S, et al. Randomised trial of alpha-tocopherol and beta-carotene supplements on incidence of major coronary events in men with previous myocardial infarction. Lancet 1997; 349:1715–1720. 120. Boaz M, Smetana S, Weinstein T, et al. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): randomised placebocontrolled trial. Lancet 2000; 356:1213–1218. 121. Miller ER III, Pastor-Barriuso R, Dalal D, et al. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005; 142: 37–46. 122. Salonen JT, Nyyssonen K, Salonen R, et al. Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study: a randomized trial of the effect of vitamins E and C on 3-year progression of carotid atherosclerosis. J Intern Med 2000; 248:377–386. 123. Fang JC, Kinlay S, Beltrame J, et al. Effect of vitamins C and E on progression of transplant-associated arteriosclerosis: a randomised trial. Lancet 2002; 359: 1108–1113. 124. Heller R, Werner ER. Ascorbic acid and endothelial NO synthesis. In: Packer L, Traber MG, Kraemer K, et al., eds. The Antioxidant Vitamins C and E. Champaign, IL: AOCS Press, 2002:66–88. 125. Hornig B. Vitamins, antioxidants and endothelial function in coronary artery disease. Cardiovasc Drugs Ther 2002; 16:401–409. 126. Heller R, Werner-Felmayer G, Werner ER. Alpha-tocopherol and endothelial nitric oxide synthesis. Ann N Y Acad Sci 2004; 1031:74–85. 127. Tu BP, Weissman JS. Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol 2004; 164:341–346. 128. Chiarugi P, Giannoni E. Anchorage-dependent cell growth: tyrosine kinases and phosphatases meet redox regulation. Antioxid Redox Signal 2005; 7:578–592. 129. Ceaser EK, Moellering DR, Shiva S, et al. Mechanisms of signal transduction mediated by oxidized lipids: the role of the electrophile-responsive proteome. Biochem Soc Trans 2004; 32:151–155. 130. Xu D, Rovira II, Finkel T. Oxidants painting the cysteine chapel: redox regulation of PTPs. Dev Cell 2002; 2:251–252. 131. Rhee SG, Kang SW, Jeong W, et al. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr Opin Cell Biol 2005; 17: 183–189. 132. Droge W. Oxidative enhancement of insulin receptor signaling: experimental findings and clinical implications. Antioxid Redox Signal 2005; 7:1071–1077. 133. Kern JC, Kehrer JP. Free radicals and apoptosis: relationships with glutathione, thioredoxin, and the BCL family of proteins. Front Biosci 2005; 10:1727–1738. 134. Saitoh M, Nishitoh H, Fujii M, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J 1998; 17:2596–2606. 135. Shekelle PG, Morton SC, Jungvig LK, et al. Effect of supplemental vitamin E for the prevention and treatment of cardiovascular disease. J Gen Intern Med 2004; 19:380–389. 136. Jialal I, Devaraj S. High-dosage vitamin E supplementation and all-cause mortality. Ann Intern Med 2005; 143:155. 137. Meydani SN, Lau J, Dallal GE, et al. High-dosage vitamin E supplementation and all-cause mortality. Ann Intern Med 2005; 143:153.

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138. Hazell LJ, Stocker R. Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages. Biochem J 1993; 290:165–172. 139. Kuzkaya N, Weissmann N, Harrison DG, et al. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem 2003; 278:22546–22554. 140. Ardanaz N, Pagano PJ. Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction. Exp Biol Med (Maywood) 2006; 231:237–251. 141. Upston JM, Niu X, Brown AJ, et al. Disease stage-dependent accumulation of lipid and protein oxidation products in human atherosclerosis. Am J Pathol 2002; 160:701–710. 142. Choudhury RP, Rong JX, Trogan E, et al. High-density lipoproteins retard the progression of atherosclerosis and favorably remodel lesions without suppressing indices of inflammation or oxidation. Arterioscler Thromb Vasc Biol 2004; 24:1904–1909. 143. Blomhoff R. Dietary antioxidants and cardiovascular disease. Curr Opin Lipidol 2005; 16:47–54. 144. Moskaug JO, Carlsen H, Myhrstad MC, et al. Polyphenols and glutathione synthesis regulation. Am J Clin Nutr 2005; 81(suppl):277S–283S. 145. Fahey JW, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci U S A 1997; 94:10367–10372. 146. Chen C, Pung D, Leong V, et al. Induction of detoxifying enzymes by garlic organosulfur compounds through transcription factor Nrf2: effect of chemical structure and stress signals. Free Radic Biol Med 2004; 37:1578–1590. 147. Uda Y, Price KR, Williamson G, et al. Induction of the anticarcinogenic marker enzyme, quinone reductase, in murine hepatoma cells in vitro by flavonoids. Cancer Lett 1997; 120:213–216. 148. Balogun E, Hoque M, Gong P, et al. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J 2003; 371:887–895. 149. Andreadi CK, Howells LM, Atherfold PA, et al. Involvement of Nrf2, p38, B-Raf, and nuclear factor-kappaB, but not phosphatidylinositol 3-kinase, in induction of hemeoxygenase-1 by dietary polyphenols. Mol Pharmacol 2006; 69:1033–1040. 150. Demmig-Adams B, Adams W III. Antioxidants in photosynthesis and human nutrition. Science 2002; 298:2149–2153. 151. Upston JM, Terentis AC, Stocker R. Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J 1999; 13:977–994. 152. Mohiuddin I, Chai H, Lin PH, et al. Nitrotyrosine and chlorotyrosine: clinical significance and biological functions in the vascular system. J Surg Res 2006; 133:143–149. 153. Shishehbor MH, Hazen SL. Inflammatory and oxidative markers in atherosclerosis: relationship to outcome. Curr Atheroscler Rep 2004; 6:243–250. 154. Dalle-Donne I, Rossi R, Colombo R, et al. Biomarkers of oxidative damage in human disease. Clin Chem 2006; 52:601–623.

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155. Verma S, Buchanan MR, Anderson TJ. Endothelial function testing as a biomarker of vascular disease. Circulation 2003; 108:2054–2059. 156. Landmesser U, Hornig B, Drexler H. Endothelial function: a critical determinant in atherosclerosis? Circulation 2004; 109(21 suppl 1):II27–II33. 157. Deanfield J, Donald A, Ferri C, et al. Endothelial function and dysfunction. Part I: Methodological issues for assessment in the different vascular beds: a statement by the Working Group on Endothelin and Endothelial Factors of the European Society of Hypertension. J Hypertens 2005; 23:7–17. 158. Brunner H, Cockcroft JR, Deanfield J, et al. Endothelial function and dysfunction. Part II: Association with cardiovascular risk factors and diseases. A statement by the Working Group on Endothelins and Endothelial Factors of the European Society of Hypertension. J Hypertens 2005; 23:233–246. 159. Wassmann S, Wassmann K, Nickenig G. Modulation of oxidant and antioxidant enzyme expression and function in vascular cells. Hypertension 2004; 44:381–386. 160. Madamanchi NR, Tchivilev I, Runge M. Genetic markers of oxidative stress and coronary atherosclerosis. Curr Atheroscler Rep 2006; 8:177–183. 161. Weissig V, Cheng SM, D’Souza GG. Mitochondrial pharmaceutics. Mitochondrion 2004; 3:229–244. 162. Sheu SS, Nauduri D, Anders MW. Targeting antioxidants to mitochondria: a new therapeutic direction. Biochim Biophys Acta 2006; 1762:256–265. 163. Coulter CV, Kelso GF, Lin TK, et al. Mitochondrially targeted antioxidants and thiol reagents. Free Radic Biol Med 2000; 28:1547–1554. 164. Muratovska A, Lightowlers RN, Taylor RW, et al. Targeting large molecules to mitochondria. Adv Drug Deliv Rev 2001; 49:189–198. 165. D’Souza GG, Weissig V. Approaches to mitochondrial gene therapy. Curr Gene Ther 2004; 4:317–328. 166. Cai H, Griendling KK, Harrison DG. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 2003; 24:471–478. 167. Endres M, Laufs U. Effects of statins on endothelium and signaling mechanisms. Stroke 2004; 35(suppl 1):2708–2711. 168. Brosnan J. Vascular NAD(P)H oxidase as a novel therapeutic target in vascular disease. Drug News Perspect 2004; 17:429–434. 169. Hwang J, Kleinhenz DJ, Lassegue B, et al. Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production. Am J Physiol Cell Physiol 2005; 288:C899–C905. 170. Cifuentes ME, Pagano PJ. Targeting reactive oxygen species in hypertension. Curr Opin Nephrol Hypertens 2006; 15:179–186. 171. Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol 2005; 25:1102–1111. 172. Sies H, Schewe T, Heiss C, et al. Cocoa polyphenols and inflammatory mediators. Am J Clin Nutr 2005; 81(suppl 1):304S–312S.

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3 Nitric Oxide—Related Oxidants in Health and Disease Cecilia Gonza´lez de Ordun˜a and Santiago Lamas Centro de Investigaciones Biolo´gicas (CIB-CSIC), Madrid, Spain

INTRODUCTION Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are molecules produced in all aerobic cells,1 and are implicated in numerous signaling pathways. When produced in excess, a condition called oxidative stress, they become potentially hazardous and may be in part responsible for the pathogenesis of many pathological conditions. WHAT ARE ROS AND RNS? This family of reactive species is characterized by their capacity to produce diverse modifications in biological macromolecules, including membrane lipids, DNA, and proteins. One of the most important ROS is the free radical superoxide anion, which is produced from different sources. This free radical undergoes selective chemical reactions with other cell components, leading to the formation of other ROS such as hydrogen peroxide or hydroxyl radicals. The principal molecule responsible for the generation of RNS is nitric oxide, which is produced by the nitric oxide synthases. RNS have oxidant properties and interact with biological systems in specific ways to produce postranslational protein modifications such

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as nitration, S-gluthathionylation or S-nitrosation. These modifications can have functional consequences. From a chemical viewpoint these reactive species can be divided into two main groups: those that possess an unpaired electron, called free radicals, and those that are not free radicals but have oxidizing effects. Free radicals include the superoxide anion, the hydroxyl radical, nitric oxide and lipid radicals; non free radicals include hydrogen peroxide, peroxynitrite and hypochlorous acid. Superoxide Anion The superoxide anion is produced by the reduction of one electron from molecular oxygen, yielding a negatively charged free radical. This oxygen species is very unstable and reacts with other species to produce other ROS. However, cells have a detoxifying system to control increased and deleterious levels of superoxide anion. The principal enzymes implicated in this detoxifying action are superoxide dismutases (SODs), which transform the superoxide anion into hydrogen peroxide and molecular oxygen. The presence of SOD ensures that superoxide anion concentrations do not exceed the picomolar range. When the enzyme is absent the levels of O2 can reach the nanomolar range, favoring the formation of other ROS. It can also react with NO giving rise to the production of peroxynitrite. This reaction is non enzymatic but occurs at a very fast rate which actually exceeds by 3-fold the capacity of SOD to reduce O2 . Peroxynitrite also reduces the availability of NO, with potential consequences for its physiological actions2; and ONOO is also involved in the formation of hydroxyl radicals by promoting the release of iron. 



Hydroxyl Radical This free radical appears to have a much greater potential for catalyzing reactions that could be involved in signaling processes than does the superoxide anion. It is formed via the Fenton reaction, which consists of the reaction of hydrogen peroxide with ferrous iron.3 It can react with thiols and lipids, generating vasoactive isoprostanes and lipid peroxidation products.4 Nitric Oxide This labile radical can interact with ferrous heme groups, certain other metal sites, thiol groups, and free radical species. The most potent actions of NO occur above the nanomolar range, and cells can produce this concentration under pathological conditions associated with inflammatory processes, neurotoxicity and ischemia. When it interacts with the superoxide anion, nitrogen dioxide (NO2) may be formed in addition to ONOO . NO2 can be formed from nitrite,

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which is a decomposition product of NO, or directly by the reaction of molecular oxygen with NO. Lipid Radicals (LO AND LOO ) These are able to react with NO to produce LONO and LOONO5,6 Hydrogen Peroxide This is a relatively stable species compared with the free radicals. It can be formed from the reaction of SOD with superoxide anion or by the action of certain oxidases through a two electron reduction of molecular oxygen. Because of its structural similarities it has comparable diffusion properties to water. Hence it may move freely into the cell and produce alterations such as activation of gluthathione redox cycles,7 oxidation of intracellular sulfhydryls,8 or DNA damage.9 The main enzymes which account for its metabolism are catalase, glutathione peroxidase and the cyclooxygenases Cox 1 and Cox 2. Hypochlorous Acid This species is less diffusible than hydrogen peroxide and thus interacts mainly with membrane components. It has toxic properties such as oxidative bleaching of heme groups and iron-sulfur centers,10 and chlorination of amines and unsaturated lipids. Peroxynitrite 

Because O2 and NO are both free radicals and contain unpaired electrons they undergo an extremely rapid reaction, leading to the formation of peroxynitrite, a much stronger oxidant than O2 . The most important effect of ONOO appears to be thiol modification, but it also causes the nitration of tyrosine residues on proteins. The formation of ONOO is associated with the inhibition of several antioxidant systems, such as catalase,11 GSH peroxidase,12 and mitochondrial SOD.13 At high concentrations ONOO promotes formation of NO donors via the modification of alcohols and sugars to nitrated species which release NO in the presence of thiols.14 

HOW DO THEY FORM? To better understand the effects of oxidant stress it is key to identify the sources of ROS and when they are produced. In the vascular context, in particular in endothelial cells, ROS can be derived from several systems such as mitochondrial respiration, enzymes of the arachidonic acid pathways, cytochrome

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p450, peroxidases, xanthine oxidases, NADH/NAD(P)H oxidases, nitric oxide synthase, and other hemoproteins. But the most important sources of ROS that have been studied in the cardiovascular system are xanthine oxidase, NAD(P)H oxidase and the nitric oxide synthases (Fig. 1). Xanthine oxidase is a molybdoenzyme capable of catalyzing the oxidation of hypoxanthine and xanthine in the process of purine metabolism.15 There are two possible forms of the enzyme, determined by conformational changes: the xanthine dehydrogenase and the xanthine oxidase. Xanthine oxidase can reduce molecular oxygen via one electron or two electrons to form superoxide anion and hydrogen peroxide, respectively.16 The absolute amount of xanthine oxidase is important and the ratio with the reduced form is critical in modulating cellular ROS generation. This enzyme has been implicated in diverse pathophysiological states in the cardiovascular system. Another source of ROS is NAD(P)H oxidase, a multi-subunit protein complex. The complex is formed by a membrane integrated cytochrome, which is itself composed of two subunits (gp91phox or its NOX analogues plus p22phox), and at least three cytosolic proteins (p47phox, p67phox and p21rac).17 This enzyme utilizes NADH and NADPH as substrates to produce superoxide anion.

Figure 1 Sources of ROS and RNS.

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Nitric oxide synthases18 are hemeproteins that catalyze the oxidation of L- arginine to L-citrulline and nitric oxide. There exist three isoforms of the enzyme in mammals: a constitutive neuronal NOS19 (nNOS or NOS I); an endotoxin- and cytokine-inducible NOS20 (iNOS or NOS II); and a constitutive endothelial NOS21 (eNOS or NOS III). NOS contain four redox active prosthetic groups – FAD, FMN, iron protoporphyrin IX (heme), and tetrahydrobiopterin BH4 – and catalyze the flavin mediated electron transport from the donor, NADPH, to the heme group. In the absence of the cofactor BH4 or the substrate L-arginine, the enzyme can produce superoxide anion and hydrogen peroxide, a phenomenon known as NOS uncoupling. In this uncoupled state the electrons that normally flow from the reductase domain of one subunit to the oxygenase domain of the other are driven to the molecular oxygen rather than to L-arginine, giving rise to the formation of superoxide rather than NO.22 There are several mechanisms whereby NOS can became uncoupled. One of them is the inactivation of the cofactor BH4 by its oxidation with peroxynitrite. BH4 is essential for enzyme activity because it stabilizes the NOS dimer and facilitates its formation, but it also increases the affinity of NOS for L-arginine and affects the spin state of the heme iron, thereby playing an important role in oxygen activation.23 Peroxynitrite is capable of rapidly oxidizing the cofactor BH4, with superoxide formation as the inevitable result. Another mechanism of uncoupling is the absence of L-arginine or mutations in GTP cyclohydrolase I, the enzyme that catalyzes the first step in the biosynthesis of BH4.24 PROTEIN MODIFICATIONS PRODUCED BY ROS AND RNS When ROS and RNS are produced, the cell needs to sense the changed environment and activate diverse pathways to respond to it. There are several mechanisms for this regulation, including protein-protein interactions, allosteric changes induced by the ligand binding and proteolytic processing. One of the best characterized is postranslational modifications of proteins (Fig. 2). For a protein modification to be physiologically relevant to the modulation of protein function, it must be specific, preferable reversible, and its formation must occur within a physiological concentration range and time frame, (Table 1). S-Glutathionylation This protein modification is a reversible covalent addition of glutathione (GSH) to a cysteine residue of a protein, through the formation of a mixed disulfide. The reduced form of the tripeptide GSH is one of the most important antioxidant molecules in mammalian cells and is present in cells at concentrations between 1 and 10 mM.25 GSH provides reducing equivalents for enzymes involved in the metabolism of ROS and RNS, and thus exerts its antioxidant actions by

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Figure 2 Species implicated on postranslational modifications.

Table 1 Postranslational modifications related to ROS and RNS Post-Translational Modification

Proteins

Physiological Relevance

S-glutathionylation

c-Jun

Inhibition of the DNA binding activity of the transcription factor

Thioredoxin Tyrosine hydroxylase Glyceraldehyde-3-Phosphate Dehydrogenase S-nitros(yl)ation

p21RAS NF-kB Zinc Finger Transcription Factors MMP-9 Hsp90 HIF-1

Activation of NF-kB alters p50-p65 dimmer formation Inhibition of the DNA binding activity of the Transcription factor Direct activation Inhibition of its activity Stabilization of a subunit

Tyrosine nitration

MnSOD PGI2

Loss of enzyme activity

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scavenging NO and oxidants.26 The availability of GSH in oxidative situations is ensured by GSH recycling and biosynthetic pathways.27 Apart from providing a reducing environment, GSH plays a role in the regulation of protein function through the formation of mixed disulfides between the protein cysteine residues and GSH.28 This process is called S-glutathionylation or S-glutathiolation; and this modification is implicated in the protection of proteins against irreversible oxidation of critical cysteine residues. Because this modification is reversible, dethiolation of S-glutathionylated proteins occurs, and can take place either by a non-enzymatic reduction or by an enzymatic cleavage of the disulfide bond, involving the action of thioredoxins and glutaredoxins.29 Therefore this modification fulfils the criteria of physiological relevance and S-glutathionylation may confer specificity and regulatory potential to the posttranslational control of protein function. Nitric Oxide can induce protein S-glutathionylation. This was first proposed in 1988 by J.W. Park.30 Then in 1997 it was demonstrated that GSNO could form a mixed disulfide with aldose reductase;31 and in the following year the role of NO as a mediator of this modification was highlighted by experiments in endothelial cells demonstrating that exogenous NO leads to S-glutathionylation of a number of proteins.32 NO may target the incorporation of GSH into some proteins in the following way. Exposure of cells to NO and other RNS leads to the formation of GSSG by the oxidation of GSH33 and its conversion to GSNO.34 This GSNO may be a source of GSSG through its reaction with superoxide35 or thiols,36 or by the breakdown of nitrosothiol.37 Therefore RNS causes S-glutathionylation indirectly, by forming GSSG.38 S-Nitros(yl)ation This modification is one of the most extensively studied protein modifications induced by reactive species because it is implicated in all classes of cell signalling, ranging from the regulation of ion channels and G-protein coupled reactions to receptor stimulation and activation of nuclear regulatory proteins.39 S-nitrosylated proteins are formed when a cysteine thiol reacts with NO in the presence of an electron acceptor to form an S-NO bond. In fact the direct reaction of an NO radical with a thiol does not yield nitrosylation:40 previous reaction with molecular oxygen via the formation of higher nitrogen oxides is thought to be necessary.41 However, transnitrosylation can occur, involving the transfer of NO between a nitrosothiol and another thiol.42 S-nitrosylation is a very labile covalent modification under physiological conditions, which makes it difficult to study. The bond can be cleaved by reaction with transition metals, or by transnitrosation, but it is also very sensitive to ultraviolet light. Several enzymes have also been described that help in the

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breakdown of nitrosothiols.43 Another problem in the study of S-nitrosylation is the low intracellular concentrations of the nitrosylated proteins, which makes it difficult to detect using current methodologies. The methods to date available for the detection of nitrosylation include electrospray ionization mass spectrometry (ESI-MS44 or ozone chemiluminescence, which can measure NO released from nitrosothiols when the S-NO is broken by photolytic cleavage.45 Nitrosylated proteins can also be identified by the biotin-switch method, which can be combined with immunoprecipitation.46 With the combined use of all these techniques new proteins have been identified as nitrosylated, such as Hsp90, b-actin and anexin II.47 S-nitrosylation reactions cause specific physiological or pathophysiological activities by modifying protein function. S-nitrosylation can promote an increase in protein activity as in the case of p21ras or thioredoxin,48,49 but it can also inhibit the activity of proteins such as caspases, methionine adenosyl transferase, or Hsp90.50,51 Tyrosine Nitration Protein tyrosine nitration is a covalent protein modification resulting from the addition of a nitro group to one of the carbons of the aromatic ring of a tyrosine residue.52 It is mediated by reactive nitrogen species such as the peroxynitrite anion; and the presence of nitrotyrosine has been used as a marker of oxidative stress and pathology. The nitration of proteins has been proposed to play a role in diseases such as amyotrophic lateral sclerosis,53 Alzheimer’s disease,54 Parkinson’s disease,55 cancer,56 atherosclerosis,57 and myocardial contractile failure.58 Tyrosine nitration appears to be catalyzed primarily by metalloproteins. Enzymes such as myeloperoxidases or cytochrome P-450 catalyze the oxidation of nitrite to nitrogen dioxide, which is able to nitrate tyrosine residues.59 Other metalloproteins such as manganese superoxide dismutase can catalyze their own nitration from peroxynitrite.60 Other reactive species capable of nitrating tyrosines are the intermediates of the reaction between peroxynitrite with carbon dioxide and the acidification of nitrite to form nitrous acid.61 The level of protein nitration is low: under inflammatory conditions between one and five 3-nitrotyrosine residues per 10,000 tyrosine residues are detected59 This fraction of nitrated protein is very small in the context of total tissue protein and raises questions about its possible biological relevance. Given that the molecular species participating in nitration have short diffusion distances, nitration may be site-specific, resulting in localized foci of nitration in a particular cell or tissue compartment. This would clearly limit the number of proteins that are available as targets for nitration; and in addition to this, only a few specific tyrosines in any particular protein can be nitrated.

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When a protein is nitrated its function can be altered in two ways: it can undergo a loss of function or a gain of function. To be biologically significant, a loss of function must affect a large fraction of a specific protein, but for gain of function only a small fraction needs to be nitrated to elicit a substantive biological signal.62 In addition to its direct effects on protein structure and function, tyrosine nitration can also have an significant impact on cell function by altering the availability of tyrosine residues for phosphorylation.63 CONCLUSION All these posttranslational modifications are biological processes associated with nitric oxide and reactive oxygen biochemistry and biology. The physiological relevance of these processes has begun to emerge, but much more remains to be discovered and understood. The confinement of modifications to restricted subcellar locations will probably prove to be important for greater understanding of their biological relevance, as will their specificity and reversibility. In the future, in vivo experimental models will be required to demonstrate the involvement of these modifications in specific physiological and disease processes. REFERENCES 1. Gille G, Sigler K. Oxidative stress and living cells. Folia Microbiol (Praha) 1995; 40:131–152. 2. Furchgott RF, Jothianandan D, Khan MT. Comparison of nitric oxide, S-nitrosocysteine and EDRF as relaxants of rabbit aorta. Jpn J Pharmacol 1992; 58(suppl 2):185–191. 3. McCord JM, Day ED Jr. Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett 1978; 86:139–142. 4. Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol 2000; 20:1430–1442. 5. O’Donnell VB, Chumley PH, Hogg N, et al. Nitric oxide inhibition of lipid peroxidation: kinetics of reaction with lipid peroxyl radicals and comparison with alpha-tocopherol. Biochemistry 1997; 36:15216–15223. 6. Baker PR, Lin Y, Schopfer FJ, et al. Fatty acid transduction of nitric oxide signaling: identification of unsaturated fatty acid nitro derivatives and PPAR receptor-dependent signaling activity. J Biol Chem 2005; 280:42464–42475. 7. Hyslop PA, Hinshaw DB, Schraufstatter IU, et al. Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells. J Cell Physiol 1986; 129:356–366. 8. Harlan JM, Levine JD, Callahan KS, et al. Glutathione redox cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide. J Clin Invest 1984; 73:706–713. 9. Schraufstatter IU, Hinshaw DB, Hyslop PA, et al. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide. J Clin Invest 1986; 77:1312–1320.

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10. Schraufstatter IU, Browne K, Harris A, et al. Mechanisms of hypochlorite injury of target cells. J Clin Invest 1990; 85:554–562. 11. Wolin MS, Davidson CA, Kaminski PM, et al. Oxidant-nitric oxide signalling mechanisms in vascular tissue. Biochemistry (Mosc) 1998; 63:810–816. 12. Asahi M, Fujii J, Suzuki K, et al. Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity. J Biol Chem 1995; 270:21035–21039. 13. Ischiropoulos H, Zhu L, Chen J, et al. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys 1992; 298:431–437. 14. Moro MA, Darley-Usmar VM, Lizasoain I, et al. The formation of nitric oxide donors from peroxynitrite. Br J Pharmacol 1995; 116:1999–2004. 15. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87:840–844. 16. Harrison R. Structure and function of xanthine oxidoreductase: where are we now? Free Radic Biol Med 2002; 33:774–797. 17. Cai H. NAD(P)H oxidase-dependent self-propagation of hydrogen peroxide and vascular disease. Circ Res 2005; 96:818–822. 18. Marsden PA, Heng HH, Scherer SW, et al. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993; 268:17478–17488. 19. Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 1990; 347:768–770. 20. Hevel JM, White KA, Marletta MA. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. J Biol Chem 1991; 266:22789–22791. 21. Pollock JS, Forstermann U, Mitchell JA, et al. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A 1991; 88:10480–10484. 22. Vasquez-Vivar J, Kalyanaraman B, Martasek P, et al. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998; 95:9220–9225. 23. Panda K, Rosenfeld RJ, Ghosh S, et al. Distinct dimer interaction and regulation in nitric-oxide synthase types I, II, and III. J Biol Chem 2002; 277:31020–31030. 24. Canevari L, Land JM, Clark JB, et al. Stimulation of the brain NO/cyclic GMP pathway by peripheral administration of tetrahydrobiopterin in the hph-1 mouse. J Neurochem 1999; 73:2563–2568. 25. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969; 27:502–522. 26. Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 1999; 31:273–300. 27. Griffith OW. Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic Biol Med 1999; 27:922–935. 28. Cotgreave IA, Gerdes RG. Recent trends in glutathione biochemistry—glutathioneprotein interactions: a molecular link between oxidative stress and cell proliferation? Biochem Biophys Res Commun 1998; 242:1–9.

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29. Jung CH, Thomas JA. S-glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione. Arch Biochem Biophys 1996; 335:61–72. 30. Park JW. Reaction of S-nitrosoglutathione with sulfhydryl groups in protein. Biochem Biophys Res Commun 1988; 152:916–920. 31. Chandra A, Srivastava S, Petrash JM, et al. Modification of aldose reductase by S-nitrosoglutathione. Biochemistry 1997; 36:15801–15809. 32. Padgett CM, Whorton AR. Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation. Arch Biochem Biophys 1998; 358:232–242. 33. Luperchio S, Tamir S, Tannenbaum SR. NO-induced oxidative stress and glutathione metabolism in rodent and human cells. Free Radic Biol Med 1996; 21: 513–519. 34. Gaston B, Reilly J, Drazen JM, et al. Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proc Natl Acad Sci U S A 1993; 90: 10957–10961. 35. Jourd’heuil D, Mai CT, Laroux FS, et al. The reaction of S-nitrosoglutathione with superoxide. Biochem Biophys Res Commun 1998; 244:525–530. 36. Wong PS, Hyun J, Fukuto JM, et al. Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry. Biochemistry 1998; 37:5362–5371. 37. Gorren AC, Schrammel A, Schmidt K, et al. Decomposition of S-nitrosoglutathione in the presence of copper ions and glutathione. Arch Biochem Biophys 1996; 330:219–228. 38. Klatt P, Lamas S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem 2000; 267:4928–4944. 39. Gaston BM, Carver J, Doctor A, et al. S-nitrosylation signaling in cell biology. Mol Interv 2003; 3:253–263. 40. Wink DA, Nims RW, Darbyshire JF, et al. Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction. Chem Res Toxicol 1994; 7:519–525. 41. Hogg N. The biochemistry and physiology of S-nitrosothiols. Annu Rev Pharmacol Toxicol 2002; 42:585–600. 42. Liu Z, Rudd MA, Freedman JE, et al. S-transnitrosation reactions are involved in the metabolic fate and biological actions of nitric oxide. J Pharmacol Exp Ther 1998; 284:526–534. 43. Gaston B. Nitric oxide and thiol groups. Biochim Biophys Acta 1999; 1411: 323–333. 44. Mirza UA, Chait BT, Lander HM. Monitoring reactions of nitric oxide with peptides and proteins by electrospray ionization-mass spectrometry. J Biol Chem 1995; 270:17185–17188. 45. Welch GN, Upchurch GR Jr., Loscalzo J. S-nitrosothiol detection. Methods Enzymol 1996; 268:293–298. 46. Jaffrey SR, Snyder SH. The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001; 2001:PL1. 47. Martinez-Ruiz A, Lamas S. Detection and proteomic identification of S-nitrosylated proteins in endothelial cells. Arch Biochem Biophys 2004; 423:192–199.

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48. Haendeler J, Hoffmann J, Tischler V, et al. Redox regulatory and anti-apoptotic functions of thioredoxin depend on S-nitrosylation at cysteine 69. Nat Cell Biol 2002; 4:743–749. 49. Perez-Mato I, Castro C, Ruiz FA, et al. Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol. J Biol Chem 1999; 274:17075–17079. 50. Martinez-Ruiz A, Villanueva L, Gonzalez de Orduna C, et al. S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities. Proc Natl Acad Sci U S A 2005; 102:8525–8530. 51. Mannick JB, Hausladen A, Liu L, et al. Fas-induced caspase denitrosylation. Science 1999; 284:651–654. 52. Gow AJ, Farkouh CR, Munson DA, et al. Biological significance of nitric oxidemediated protein modifications. Am J Physiol Lung Cell Mol Physiol 2004; 287: L262–L268. 53. Beckman JS, Carson M, Smith CD, et al. ALS, SOD and peroxynitrite. Nature 1993; 364:584. 54. Smith MA, Richey-Harris PL, Sayre LM, et al. Widespread peroxynitrite-mediated damage in Alzheimer’s disease. J Neurosci 1997; 17:2653–2657. 55. Good PF, Hsu A, Werner P, et al. Protein nitration in Parkinson’s disease. J Neuropathol Exp Neurol 1998; 57:338–342. 56. Goldstein SR, Yang GY, Chen X, et al. Studies of iron deposits, inducible nitric oxide synthase and nitrotyrosine in a rat model for esophageal adenocarcinoma. Carcinogenesis 1998; 19:1445–1449. 57. Beckmann JS, Ye YZ, Anderson PG, et al. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler 1994; 375:81–88. 58. Ferdinandy P, Danial H, Ambrus I, et al. Peroxynitrite is a major contributor to cytokine-induced myocardial contractile failure. Circ Res 2000; 87:241–247. 59. Brennan ML, Wu W, Fu X, et al. A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species. J Biol Chem 2002; 277:17415–17427. 60. MacMillan-Crow LA, Crow JP, Kerby JD, et al. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci U S A 1996; 93:11853–11858. 61. Gow A, Duran D, Thom SR, et al. Carbon dioxide enhancement of peroxynitritemediated protein tyrosine nitration. Arch Biochem Biophys 1996; 333:42–48. 62. Radi R. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci U S A 2004; 101:4003–4008. 63. Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998; 356:1–11.

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4 Modulation and Determination of Cellular Glutathione Concentrations Lars-Oliver Klotz Department of Molecular Aging Research, Institut fu¨r Umweltmedizinische Forschung (IUF) at Heinrich-Heine-University, Du¨sseldorf, Germany

INTRODUCTION Exposure of mammalian cells to light results in the photochemical generation of reactive oxygen species, such as singlet oxygen1 or superoxide,2 with the potential of causing oxidative damage. Several cellular lines of defense exist to cope with this challenge, but the tripeptide glutathione appears to play a prominent role in the cellular response to a stressful stimulus with an oxidative component; for example, there is ample evidence that age-related nuclear cataract is linked to oxidative processes and apparently affected by cellular glutathione levels.3,4 GLUTATHIONE Glutathione, or g-glutamylcysteinylglycine (GSH), is the major thiol of low molecular mass present in mammalian cells, with concentrations usually in the millimolar region. It is involved in the cellular antioxidant defense as part of a network of enzymes (Figure 1) that use GSH as the supplier of electrons for the reduction of peroxides (glutathione peroxidases), that keep glutathione in its reduced state (glutathione reductase) and that covalently couple GSH to various electrophilic compounds in phase II drug metabolism (glutathione S-transferases). Due to its high intracellular concentrations, the cellular redox state is governed to a large extent by the glutathione redox status.

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Figure 1 Structure of glutathione (GSH) and involvement in peroxide (ROOH) reduction by glutathione peroxidases (GPx). Glutathione disulfide (GSSG) is formed during reduction of peroxides and is reduced back to GSH at the expense of NADPH by glutathione reductase (GR). Cellular sources of NADPH include the pentose phosphate pathway, as well as reactions catalyzed by malic enzyme (malate dehydrogenase, decarboxylating) or NADP+-dependent isocitrate dehydrogenase.

As a thiol (R-SH, or thiolate, R-S
), GSH is readily oxidized under physiological conditions, forming sulfenic acid (R-SOH, or sulfenate, R-SO
) or disulfides (R-S-S-R’). This oxidation may occur both enzymatically (Figure 1) or nonenzymatically by interaction with reactive oxygen species. Even higher glutathione oxidation states, sulfinic acid (R-SO2H) and sulfonic acid (R-SO3H), were observed, but they are usually not reduced under physiological conditions, although the reduction of sulfinates has recently been shown to be feasible in some cases.5 The balance between glutathione (GSH) and glutathione disulfide (GSSG) concentrations is believed to be a determinant in the cellular capability to cope with an oxidative stressful stimulus. In addition to serving as an electron donor in the cellular antioxidative defense, glutathione serves a regulatory role in affecting enzyme activities by glutathiolation, i.e., by the formation of mixed disulfides between GSH and a protein thiol.1,6 In order to analyse a possible role of glutathione in a cellular process of interest, it will have to be tested whether an elevation and/or lowering of cellular glutathione levels affects the investigated process. Thus, a brief introduction to experimental means of modulating cellular glutathione levels and to methods for the determination of glutathione concentrations will be given in this chapter. EXPERIMENTAL MODULATION OF CELLULAR GLUTATHIONE CONCENTRATIONS In order to experimentally elevate cellular GSH levels, glutathione precursors or derivatives need to be applied because glutathione is not taken up by cells to a significant extent. N-acetyl cysteine, a cell permeant derivative of cysteine, may

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Figure 2 Reaction of diazenedicarboxylic acid bis(N,N-dimethylamide), or diamide, with glutathione. The reactive agent is the glutathione thiolate, which reacts with diamide to form a sulfenyl hydrazine and, upon reaction with a second glutathione thiolate to release glutathione disulfide (GSSG), the corresponding hydrazine.

be used to feed cellular GSH synthesis by supplying cysteine which is coupled to glutamate by g-glutamylcysteine synthetase (see below), but due to feedbackinhibition of GSH biosynthesis this approach is not always successful. Membrane permeant glutathione esters that are hydrolysed to GSH intracellularly are frequently employed instead.7 Several compounds exist that deplete GSH, including (i) electrophilic compounds nonenzymatically reacting with thiols (or thiolates), such as diamide, (ii) compounds that are coupled to GSH enzymatically, such as diethyl maleate, or (iii) inhibitors of glutathione biosynthesis. Diamide, diazenedicarboxylic acid bis(N, N-dimethylamide), has been employed for decades to deplete cellular GSH following the reaction depicted in Figure 2.8 Although GSH was found to be more reactive towards diamide than other non-protein thiols, the reaction is not specific for GSH, and diamide will thus also deplete several other cellular thiols, including protein-bound cysteinyl residues, to form diamide-SR adducts and/or (mixed) disulfides.8 However, as GSH is the major non-protein thiol in mammalian cells, diamide will usually preferentially react with GSH. The reaction of diamide with GSH is nonenzymatic. Employing the cellular machinery of specifically coupling GSH to electrophiles, the glutathione S-transferases (GSTs), would thus result in an enhanced specificity in terms of depleting GSH rather than other available thiols. Diethyl maleate (DEM) is an example of a compound that is recognized as a substrate by GSTs and by being coupled to GSH (see Figure 3) causes the depletion of cellular GSH. Buthionine sulfoximine (BSO) was identified as a specific inhibitor of g-glutamylcysteine synthetase,9 the initial step in GSH biosynthesis. Application of BSO will cause a loss of cellular GSH by preventing its resynthesis when cellular stores are depleted by export or by normal cellular metabolism, e.g. by

Figure 3 Reaction of diethyl maleate with glutathione as catalyzed by glutathione S-transferases (GST).

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Figure 4 Glutathione (GSH) is synthesised de novo in two steps from glutamate via g-glutamylcysteine in reactions catalysed by g-glutamylcysteine synthetase (GCS) and glutathione synthetase (GS). GCS is inhibited by the transition state analogue buthionine sulfoximine (BSO).

GST-dependent coupling of GSH or by peroxide reduction (Figure 1) with the production and subsequent export of GSSG. Of all possible enantiomers, L-buthionine (S)-sulfoximine was demonstrated to be the effective form (Figure 4).10 METHODS FOR THE DETERMINATION OF CELLULAR GLUTATHIONE CONCENTRATIONS In order to experimentally assess cellular GSH levels, essentially the same types of reaction can be exploited that were discussed above as being applied for the more or less specific depletion of GSH. Thiol-reactive substances are used that, upon reaction with GSH, form a product that is detectable photometrically or fluorimetrically. A widely applied reagent is Ellman’s reagent (5,5’-dithiobis-2nitrobenzoic acid, DTNB),11 a disulfide that reacts with thiols to form mixed disulfides and thionitrobenzoate (TNB), a dianion with an absorption maximum around 412 nm (see below and Figure 6): DTNB þ RS ! TNB-SR ðTNB=RS-mixed disulfideÞ þ TNB TNB-SR þ R0 S ! TNB þ R0 SSR Thiol concentrations can be estimated by either comparing absorptions with those of a standard curve established by reacting DTNB with different thiol concentrations or by calculating concentrations using the published TNB absorption coefficient at 412 nm, which was recently reevaluated to be 14.15 mM
1cm
1 and 13.8 mM
1cm
1 at 258C and 378C, respectively (pH 7.4).12 A second group of widely employed thiol-reactive compounds is that of bimane (1,5-diazabicyclo[3.3.0]octadienedione) derivatives, most notably the bromobimanes.13 The nonfluorescent monobromobimane (mBBr), upon reaction with a thiol, forms a fluorescent adduct (Figure 5) the concentration of which can be estimated directly by fluorimetry using an appropriate standard. As with DTNB, mBBr does not exclusively react with GSH, and various non-protein thiols as well as protein-bound cysteines can also be labeled.

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Figure 5 Monobromobimane (mBBr) reacts with glutathione to form a fluorescent adduct.

Specificity for GSH of these simple assays is enhanced in both cases by employing cell extracts depleted of proteins by treatment with acid, such as sulfosalicylic acid or metaphosphoric acid: as GSH is the major non-protein thiol in most cells, the TNB absorption or bimane derivative fluorescence measured under these conditions will be largely due to GSH. However, specificity may be further enhanced by introducing another selection criterion. This can be either by adding a second analytical step to the GSH determination procedure, or by making the whole reaction enzymedependent and thus most specific. The first approach is found in the literature for GSH analysis with mBBr:13 the mixture of fluorescent thiol-bimane adducts from the reaction of mBBr with cells or cell extracts is further analysed by HPLC, yielding information specifically on the presence and concentration of the bimane-glutathione adduct. The second approach is frequently applied for GSH analysis employing DTNB (Figure 6). According to the reaction sequence of thiols with DTNB described above, glutathione disulfide will result from the reaction of two GSH molecules with one molecule of DTNB. GSSG, in turn, is a substrate of glutathione reductase (GR, see Figure 1). GSH can thus be recycled from GSSG in the presence of GR and NADPH, resulting in a steady depletion of

Figure 6 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) recycling assay for the determination of glutathione and glutathione disulfide (GSSG) concentrations. Both DTNB and TNB are depicted in their fully protonated forms. GR, glutathione reductase. See text for details.

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Figure 7 1-Chloro-2,4-dinitrobenzene (CDNB) is coupled to glutathione in a nucleophilic substitution catalyzed by glutathione S-transferases (GST). The formation of the adduct can be followed photometrically at 340 nm.

DTNB and increase in absorbance at 412 nm due to formation of TNB. The slope of TNB formation directly correlates with glutathione concentration. As GSSG is continuously recycled and thus introduced into the assay, glutathione concentrations determined actually comprise GSH plus GSSG levels which can be analysed separately with this assay only after derivatization of GSH, e.g., with 2-vinylpyridine.14 Glutathione S-transferases (GSTs) were mentioned before as another group of enzymes specifically recognising GSH. Employing GST and a substrate, 1-chloro-2,4-dinitrobenzoic acid (CDNB), GSH concentrations are determined according to the reaction depicted in Figure 7.15 Different from the DTNB/GR assay, GSH is determined directly. A comparison of the mentioned glutathione assays with the same array of different GSH concentrations revealed that assay sensitivities are in the following order:16 DTNB/GR & CDNB/GST > DTNB (nonenzymatic) > mBBr/ HPLC. In summary, the two enzymatic assays not only appear to be more specific but also more sensitive than the assays solely based on the direct interaction between reagent (DTNB or mBBr) and thiol. AN EXAMPLE: MENADIONE AND CELLULAR GLUTATHIONE LEVELS Menadione (2-methyl-1,4-naphthoquinone, vitamin K3) is a known redox cycler and alkylating agent17 that causes the production of reactive oxygen species (Figure 8) and the depletion of thiols (Figure 9) in cells exposed to the quinone. Intracellularly, menadione is reduced to the corresponding semi- or hydroquinone by one- and two-electron reduction, respectively (Figure 8). The semiquinone is oxidized back to the quinone form by molecular oxygen (which is present in physiological systems in high micromolar concentrations) under concomitant generation of superoxide. Similarly, the hydroquinone may be oxidized by oxygen unless it is deactivated in phase II reactions and exported. Superoxide will dismutate both spontaneously and catalysed by superoxide dismutases to form hydrogen peroxide, which in turn is reduced to water at the expense of GSH by glutathione peroxidases (see Figure 1). Hence, menadione

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Figure 8 Redox cycling of menadione (2-methyl-1,4-naphthoquinone). Menadione is reduced intracellularly by one-electron reductases or in a two electron-reduction catalyzed by NAD(P)H:quinone oxidoreductase-1 (NQOR, DT-diaphorase). The resulting semiquinone and, to a lesser extent, also the corresponding hydroquinone, may be oxidized by molecular oxygen which is thereby reduced to superoxide.

affects the cellular balance between GSH and GSSG. Menadione also causes direct depletion of GSH by arylation, i.e. through a Michael-type addition of thiolates at C-3 (Figure 9). To analyse the effect of menadione and a known glutathione depletor, DEM (see above), on cellular glutathione levels, rat liver epithelial cells were exposed to these agents as described in Figure 10. As expected from the mechanisms outlined above, both menadione and, more so, DEM deplete total glutathione. While total glutathione levels are lowered by approximately 30%, concentrations of GSSG are significantly enhanced and those of GSH strongly diminished in cells exposed to menadione. These data are in line with GSH being lost in at least two ways under the influence of menadione, i.e. by oxidation of GSH to GSSG and most probably by direct interaction with menadione (arylation).

Figure 9 Arylation of thiols by menadione, i.e., Michael addition of thiols/thiolates to menadione.

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Figure 10 Glutathione levels in cells exposed to menadione or diethyl maleate (DEM). Rat liver epithelial cells were exposed to menadione (50 mM), DEM (1 mM) or DMSO ("–") as vehicle control for 15 min. Cells were washed and lysed in 10 mM HCl, protein was precipitated from the lysates with 5-sulfosalicylic acid, followed by analysis of glutathione in the protein-free fraction employing the DTNB/GR assay: total glutathione (GSH and GSSG) was analyzed from the acidic lysates, for identification of GSSG thiols were blocked by 2-vinylpyridine prior to the assay. GSH levels were calculated from total glutathione and GSSG concentrations. Data are given as means of 3 independent measurements ± SD (modified and recalculated from Abdelmohsen et al.18).

Different from menadione, the changes in total glutathione levels seen in cells treated with DEM are not due to changes in GSSG concentrations but to a loss of GSH (see Figures 3 and 10). SUMMARY Glutathione is an essential component in the cellular line of antioxidative defense. Changes in glutathione concentrations and in glutathione redox state are important parameters for the evaluation of potential susceptibility of cells to oxidative damage. Methods for the experimental analysis of GSH and GSSG concentrations as well as tools for the modulation of cellular glutathione levels were described.

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ACKNOWLEDGMENT Research in the author’s laboratory is funded by Deutsche Forschungsgemeinschaft, Bonn, Germany (SFB 728/B3, SFB 575/B4, GRK 1033), and the Forschungskommission der Medizinischen Fakulta¨t at Heinrich-Heine-University, Du¨sseldorf. Dedicated to my mother, Mrs. Eva-Marie Klotz, on the occasion of her 60th birthday. REFERENCES 1. Klotz LO, Kro¨ncke KD, Sies H. Singlet oxygen-induced signaling effects in mammalian cells. Photochem Photobiol Sci 2003; 2:88–94. 2. Mahns A, Melchheier I, Suschek CV, et al. Irradiation of cells with ultraviolet-A (320–400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide. Free Radic Res 2003; 37:391–397. 3. Pau H, Graf P, Sies H. Glutathione levels in human lens: regional distribution in different forms of cataract. Exp Eye Res 1990; 50:17–20. 4. Truscott RJ. Age-related nuclear cataract-oxidation is the key. Exp Eye Res 2005; 80:709–725. 5. Chang TS, Jeong W, Woo HA, et al. Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine. J Biol Chem 2004; 279:50994–51001. 6. Klatt P, Lamas S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem 2000; 267:4928–4944. 7. Anderson ME. Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact 1998; 111–112:1–14. 8. Kosower NS, Kosower EM. Diamide: an oxidant probe for thiols. Methods Enzymol 1995; 251:123–133. 9. Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 1979; 254:7558–7560. 10. Campbell EB, Hayward ML, Griffith OW. Analytical and preparative separation of the diastereomers of L-buthionine (SR)-sulfoximine, a potent inhibitor of glutathione biosynthesis. Anal Biochem 1991; 194:268–277. 11. Ellman GL. A colorimetric method for determining low concentrations of mercaptans. Arch Biochem Biophys 1958; 74:443–450. 12. Eyer P, Worek F, Kiderlen D, et al. Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal Biochem 2003; 312:224–227. 13. Kosower EM, Kosower NS. Bromobimane probes for thiols. Methods Enzymol 1995; 251:133–148. 14. Anderson ME. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 1985; 113:548–555. 15. Brigelius R, Muckel C, Akerboom TPM, et al. Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide. Biochem Pharmacol 1983; 32:2529–2534. 16. Do¨ll M. Evaluation von Methoden zur Bestimmung des Glutathiongehaltes menschlicher Zellen nach Behandlung mit unterschiedlichen Noxen. [Evaluation of

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methods for the determination of glutathione in human cells exposed to various stressful stimuli]. MD thesis, Heinrich-Heine-Universita¨t Du¨sseldorf, Du¨sseldorf, Germany, 2004. 17. Abdelmohsen K, Patak P, von Montfort C, et al. Signaling effects of menadione: from tyrosine phosphatase inactivation to connexin phosphorylation. Methods Enzymol 2004; 378:258–272. 18. Abdelmohsen K, Gerber PA, von Montfort C, et al. Epidermal growth factor receptor is a common mediator of quinone-induced signaling leading to phosphorylation of connexin 43: role of glutathione and tyrosine phosphatases. J Biol Chem 2003; 278:38360–38367.

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5 Oxidants in Corneal Diseases Anders Behndig Department of Clinical Sciences/Ophthalmology, Umea˚ University Hospital, Umea˚, Sweden

INTRODUCTION The cornea is generally considered to be intensely exposed to reactive oxygen species (ROS), and also to have special problems dealing with these reactive species.1 There are many reasons to believe this is true: First, to enable vision, the cornea is by necessity intensely exposed to light, with a high risk for photochemical reactions. In addition, with the exception of the epithelium, the corneal tissues have slow turnover rates, which means that compounds damaged by oxidative processes are likely to be present in the corneal tissue for long periods of time. Furthermore, the cornea has optical demands requiring a macroscopically and microscopically perfect tissue organization, with demands by far exceeding those put upon most tissues and organs of the body. Last, like the lens and the vitreous body, the cornea is avascular, which also reduces its possibility to ‘‘export’’ compounds damaged by oxidation. The main roles of the cornea are to offer mechanical protection and stability to the anterior surface of the eye, but it also provides about 2/3 of the refractive power of the eye’s optical system.2 Ideally, virtually all visible wavelengths of light should pass through the cornea unaffected (which will require a very exact tissue organization) but, almost equally important, the cornea should absorb most of the UV-light entering the eye,3 to protect the retina and lens from these highly energetic wavelengths (which will result a considerable oxidative stress in the superficial cornea). The cornea is avascular, and

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Figure 1 Histological section of the cornea, showing the epithelium (top), the stroma (middle) and the monolayer endothelium (bottom). See text for details.

most of its nutrients are derived from the aqueous humor. On the contrary, the major part of the cornea’s oxygen supply is directly derived from the air. This rather odd route of oxygenation is more or less unique to the cornea, and is naturally associated with the avascularity of the cornea. As a practical consequence of its oxygenation route, the oxygen tension in the superficial cornea is higher when the eye is open, but is significantly reduced when the eye is closed during sleep (the local oxygen tension of the superficial cornea may in fact vary as much as three-fold over a 24-hour period).4 The cornea consists of three layers, separated by basal laminae and acellular layers: an epithelium, a stroma, and a monolayer endothelium (Fig. 1). These layers are separately described below. The Corneal Epithelium The epithelium of the cornea is a squamos epithelium with 4–6 cell layers, which makes up about 10% of the total corneal thickness. The germinative capacity of the epithelium is found in the columnar basal cells. Bowman’s layer is a 10mm thick amorphous layer, which separates the epithelium from the stroma. The epithelial cells continuously regenerate, and they move gradually from Bowman’s layer towards the surface, while undergoing a transition to squamous superficial cells, which in turn undergo continuous apoptosis, cellular disintegration and desquamation, not unlike the superficial cells of the skin. Simultaneously, the epithelial cells move from the periphery of the cornea towards the corneal center.5,6 The stem cells of the corneal epithelium are located in deep crypts at the corneoscleral transition (the limbus),7 and the regenerative capacity of the corneal epithelium is virtually unlimited under normal conditions. These cells are capable

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of creating a whole new corneal epithelium after larger injuries or surgery.8 The flat superficial epithelial cells form a watertight layer that prevents water from entering the stroma, which is essential for corneal transparency.2 Also, it provides a smooth, even refractive surface, and a mechanical protection against invading microorganisms. The epithelial thickness and morphology may be influenced by environmental factors, and interestingly from an oxidative perspective, the epithelial thickness increases in response to light exposure.9 ROS can be generated within the corneal epithelium by multiple mechanisms, including photochemical and inflammatory processes.10 Also, xanthine oxidase, an enzyme known to generate ROS, is present in the corneal epithelium.11 There are many examples of situations where the influence of ROS affects the integrity and normal function of the corneal epithelium. For example, the process of epithelial wound healing is slower when oxidative processes are involved, such as in diabetes mellitus. Healing of corneal epithelial wounds can be accelerated by addition of antioxidants, such as trolox,12 Vitamin E,13,14 Vitamin C (ascorbic acid)15 or superoxide dismutase derivates.10,16,17 The Corneal Stroma The corneal stroma constitutes 90% of the corneal thickness in humans and is mainly made of stacked lamellae of collagen fibrils. Especially in the posterior stroma, these lamellae are arranged in a highly precise and regular manner. Between the lamellae are the keratocytes, cells which maintain the stoma by synthesizing collagen and an extracellular matrix of glycosaminoglycans (GAGs), mainly keratan sulphate (KS) and chondroitin sulphate/dermatan sulphate (DS).2 Oxygen tension apparently has a role in regulating the synthesis of the GAGs.18 Accordingly, the keratocytes synthesize more DS and less KS in the anterior stroma, where the oxygen tension is higher.4,19 The polyanionic GAGs are essential to keep the lamellae in the regular arrangement with a constant distance between them, which, in turn, is essential for corneal transparency. The alterations in the composition of GAGs with a decreased KS/DS ratio and appearance of other GAGs like heparan sulphate in corneal scar tissue20–22 and in corneal healing processes23 may contribute to the reduced transparency of a corneal scar. Also in deeper wounds, involving the corneal stroma, beneficial effects on the healing can be seen with antioxidants13 and superoxide dismutase derivates.10,16,17 The Corneal Endothelium The corneal endothelium is a 5 mm thick monolayer of flat, uniform, hexagonal cells covering the entire inside of the cornea,2,24–26 the endothelium rests on a 5–10 mm thick basement membrane, the Descemet’s membrane, which in turn is loosely attached to the stroma. The hexagon is the ‘‘roundest’’ of the three

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geometrical figures that can cover a plane, which explains the hexagonal shape of the endothelial cells. Uniform hexagonal cells means that the endothelium is ‘‘at rest’’, and in a state of minimal stress.27 The endothelial cells dehydrate the corneal stroma by pumping fluid continuously from the stroma.2,28,29 Keeping the stroma dehydrated is essential for preservation of the stromal lamellar geometry, and thereby for the visual function; a loss of corneal endothelial pump function will immediately cause corneal swelling, opacification of the stroma and loss of visual function. The corneal endothelial cells increase in size, in humans from 200–250 mm2 at birth to 400–700 mm2 in adulthood. From a few years of age this is mainly explained by a continuous loss of corneal endothelial cells.2,24 Loss of endothelial cells in humans is exclusively compensated for by sliding and thinning of adjacent cells to cover the defect.2,30–32 Mitosis may also play a role in lower mammals,25,31,33,34 but corneal endothelial cells are essentially amitotic under resting conditions.35 Therefore, a gradual enlargement of cells is seen with age in many species.36 The normal enlargement of cells can accelerate under different stress conditions, such as intraocular surgery,25,37,38 endothelial wounds,29,39 ocular40–42 and systemic diseases such as diabetes.43 Oxidative stress has been shown to cause corneal endothelial cell death by apoptosis or necrosis,44,45 and ROS generated from ultrasonic energy may be a major mechanism behind corneal endothelial cell damage in phacoemulsification cataract surgery46 (see below). EXAMPLES OF IMPORTANT CORNEAL ANTIOXIDANTS The Superoxide Dismutases Superoxide dismutases (SOD) generally catalyze the reaction 2 O2  + 2 H+ O2 + H2O2. SOD comprises the main enzymatic system for O2  scavenging, and is present in all higher organisms and most aerobic bacteria. There are three specific superoxide dismutases in higher organisms, each confined to its own compartment in cells and tissues: the cytosolic Copper-Zinc-containing SOD (SOD1),47 the mitochondrial Manganese-containing SOD (SOD2),48 and the Extracellular SOD (SOD3).49 The cornea contains unusually large amounts of SOD3, among the highest levels measured in the human body, and close to that of SOD1. The cornea also has a relatively high SOD2 activity, just below that of the other two isoenzymes. SOD3 shows an uneven distribution within the human cornea, with significantly lower contents in the central cornea than in the periphery, and immunohistochemically lower contents in the anterior, than in the posterior stroma (Fig. 2A). The corneal epithelium is rich in SOD3, which indicates a high synthesis of SOD3 in the epithelial cells, given the high turnover rate of these cells (Fig. 2B). The epithelium is also rich in SOD1, localized in the cytosol of the epithelial cells (Fig. 2C).

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Figure 2 (See color insert.) Immunohistochemical staining for SOD3 in the human cornea. A: Note a pronounced staining of the cell borders in the epithelium, and a stromal staining which is interleaved between the stromal collagen lamellae. The stromal staining is slightly weaker in the anterior, than in the posterior stroma. B: Detail of immunohistochemical staining for SOD3 in the human corneal epithelium. Note intense staining of the cell borders and intercellular space. C: Staining for SOD1 in the human corneal epithelium. Note the staining of the cytosol and nuclei.

Ascorbic Acid Ascorbic acid (Vitamin C) is hydrophilic and acts as a reducing agent, which may sometimes be of benefit and sometimes not. It reacts rapidly with ROS, such as O2  and OH , to give the less reactive semidehydroascorbic acid, but oxidation of ascorbic acid in the presence of certain transition metal ions, especially Cu2+ can also produce both H2O2 and OH .50 Ascorbic acid has long been known to exert special protective functions in certain tissues and fluids. It is accumulated in very high concentrations (10–100x the concentrations in serum9) in, for example, the lens, the aqueous humor, and the cornea of the eye. There is at least indirect evidence to support that the role of ascorbate in the anterior part of the eye has to do with protection from light-induced damage, and that ascorbate acts as a filter for ultraviolet light in the eye.9,51,52 In diurnal species, including humans, which are exposed to high levels of light, there are high concentrations of ascorbic acid present in these tissues, as opposed to in nocturnal species.52–54 The distribution of ascorbic acid in the cornea is just as interesting as that of SOD3. The concentrations in the corneal endothelium and stroma approximately equal those in the aqueous humor and the lens, but the concentrations in the corneal epithelium are about 6-fold higher.9,52,55 The epithelial concentrations also vary with the degree of light exposure in the same species,9 and within the epithelium in the same individual, with higher concentrations in the centre, over the pupil area.55 When evaluating the radical protective properties of ascorbate it is important to remember that they may be situation-dependent (as mentioned, ascorbate can have opposite effects under certain conditions), and also that ascorbate is consumed (oxidized) when scavenging radicals, and needs to be regenerated. In the anterior part of the eye, however, this should be a minor problem, since the aqueous humor has a rather high turnover rate, with an exchange of several percent of its volume each minute.

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In analogy with the potential antioxidant and pro-oxidant properties of ascorbic acid, the literature offers indications of both positive and negative effects of ascorbic acid in the cornea. Ascorbic acid or analogs promote corneal healing after alkali injury, an injury known to involve inflammatory and oxidative components.15 Although the concentrations of ascorbic acid in the eye’s anterior segment are the result of active secretion mechanisms, they can be affected by dietary intake.56 Noticeably, though, the benefit of increasing the ascorbic acid concentrations in, for example, the aqueous humor, is controversial, and dietary restriction of ascorbic acid has even been shown to reduce the development of cataract in a mouse model.57 EXAMPLES OF CORNEAL DISEASES WHERE OXIDATIVE MECHANISMS CONTRIBUTE ROS and oxidative stress have been proposed as contributing mechanisms behind many corneal disorders. The following section exemplifies a few such disorders and conditions. Keratoconus Keratoconus (KC) is characterized as a non-inflammatory corneal thinning disorder with an incidence of about 1 in 2000 in the general population. KC is characterized by a central or paracentral corneal thinning, resulting in mechanical instability of the cornea. This instability, in turn, results in a protruding corneal cone with induction of high myopia and irregular astigmatism2 (Fig. 3). Around this cone, ferritin accumulates within the basal corneal epithelium, which is clinically known as Fleischer’s ring. The treatment options involve (with increasing severity of the disease) spectacles, stable contact lenses and various surgical procedures, including corneal transplantation. KC usually starts in early adulthood, and its progression rate decreases with time, meaning that the condition is usually more or less stable after the age of 30.58 There is a familiar appearance of KC, which has become even more evident with the development of computerized corneal topography, slit-scan tomography, and related anterior segment imaging devices,59,60 revealing subclinical cases

Figure 3 A cornea with advanced keratoconus. Note the protruding cone.

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among family members of KC patients. The exact genetic mechanisms underlying the disease are current under investigation,61 but may vary among cases.58 KC corneas show a decreased KS/DS ratio (see above),22,62,63 but this may be secondary to the thinning or the scarring of advanced forms of the disease. There is also a degradation of the extracellular matrix of the superficial stroma with elevated degradative enzymes64,65 and wound-healing and stress-related proteins66,67 as well as altered proteinase inhibitors67–69 in KC. These biochemical changes may be initiated by keratocyte apoptosis mediated by the interleukin-1 system.70–72 Indeed, apoptosis of anterior stromal keratocytes and basal epithelial cells is found in KC.71 Various types of stress, including mechanical and oxidative stress, to the superficial cornea can induce apoptosis of these cells.71,73,74 Kenney et al. have suggested a working hypothesis for KC pathogenesis, with formation of peroxynitrite (ONOO ) from O2  and nitric oxide (NO)72 as an initiating factor causing the keratocytes to undergo apoptosis, and subsequent studies have provided further support for oxidative stress as a causative factor behind KC.1,75,76 KC corneas show immunohistochemical staining for both nitrotyrosine and malondialdehyde, markers of ONOO and lipid peroxidation, respectively,1 an up-regulation of catalase,76 and increased degradative enzymes.76 Indeed, a spatial relationship is seen between nitrotyrosine, the nitric oxide synthetase NOS III, and fibrosis, which is interpreted as an insufficient superoxide radical processing capacity, resulting in ONOO formation, in the KC cornea .1,72 NO has a variety of functions in the eye,77 and is synthesized by keratocytes under stress conditions.78,79 The chain reaction thereafter, eventually resulting in resorption of collagen with stromal thinning, may reflect an unspecific corneal reaction pattern under such circumstances, analogous to the local resorption of corneal stroma seen clinically after, for example, corneal trauma and infectious processes. Our group has demonstrated that the levels of Extracellular Superoxide Dismutase (SOD3) in KC are significantly reduced, to about half of the levels in normal central cornea, whereas the other two SOD isoenzymes, SOD1 and SOD2, are unaltered.80,81 It is striking that the earliest changes in KC occur in the anterior stroma, where the levels of SOD3 are the lowest, also in the normal cornea (Fig. 2A). Subsequently, Kenney’s group have demonstrated that the basal expression of SOD3 on the mRNA level in the KC cornea does not differ from that in the normal cornea,76 but alterations in the corneal SOD3 expression pattern globally or locally, or in response to cytokines, oxidative stress or trauma, may still be altered in the KC cornea. In conclusion, there is growing evidence that the KC cornea is unable to handle superoxide radicals in a normal manner,76,81 and that oxidative stress is an important factor in KC pathogenesis. Bullous Keratopathy/Fuchs Endothelial Dystrophy These two conditions have resemblances, but distinctly different pathogenetic backgrounds. The common feature of these two corneal diseases is the corneal

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Figure 4 Specular microscopy photographs of the human corneal endothelium in beginning bullous keratopathy (A). Note the enlarged endothelial cells. In B, a normal endothelium is shown, for comparison. C: Fuchs endothelial dystrophy. Note the fibrous patches, appearing dark in the picture, clinically known as guttae. Between the guttae, the endothelial morphology may appear rather normal.

edema, which occurs because of a failure of the corneal endothelial cells to remove fluid from the stroma. Corneal edema is painful and sight threatening, and together, these two diagnoses comprise the majority of corneal transplantation cases. In bullous keratopathy, the endothelium is damaged by external forces (mainly surgical procedures), whereas in Fuchs endothelial dystrophy, the etiology is largely unknown. Sometimes, there may have been a mild, subclinical form of Fuchs prior to a surgical procedure, which has contributed to the subsequent development of edema. In other words, mixed forms of the two diseases may occur, and there is likely some uncertainty regarding the clinical diagnosis in a portion of the cases. In typical cases, however, the endothelial morphology differs between the two diseases (Fig. 4A–B), which indicates that the conditions may also differ in pathogenesis and/or biochemical changes. The density of corneal endothelial cells decreases continuously with time due to cell loss.2,24 In man, the cell densities decrease from 3500–4000 cells mm 2 at birth to 1400–2500 cells mm 2 in adulthood. In man, and other higher mammals, the loss of endothelial cells is compensated for only by sliding and thinning of adjacent cells to cover the defect,2,30–32 but mitosis may also play a role in lower mammals25,31,33–35 Even so, a gradual enlargement of cells is seen with age is seen also in lower mammals.36 The normal enlargement of corneal endothelial cells seen with time can be accelerated in various stress conditions, and then often in combination with a deviation from the uniform hexagonal cellular pattern normally seen.24–26 Examples of such stress conditions include endothelial wounds,29,39 systemic43 or ocular diseases,40 including uveitis40–42 and intraocular surgery.25,37,38 There is much evidence to support that oxidative stress is a major factor behind corneal endothelial cell loss.

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Figure 5 The corneal endothelium of SOD3 null mice shows an accelerated spontaneous cell loss with age (A), which is further accentuated after an LPS-induced uveitis (B).

Corneal endothelial cell death can be induced by hydrogen peroxide perfusion of the anterior chamber in rabbits.45 In Fuchs’ endothelial dystrophy, an accelerated age-dependent endothelial cell loss with increased apoptosis of the cells is seen.82 Photooxidative injury with formation of ROS has been shown to induce corneal endothelial cell apoptosis in animal models,83,84 and may also be a mechanism underlying cell loss in Fuchs’dystrophy. Our group has demonstrated that mice lacking SOD3 have an accelerated loss of corneal endothelial cells with an otherwise largely preserved morphology in normal ageing (Fig. 5A), a finding which strongly indicates that superoxide radicals and oxidative stress contributes to the age-dependent corneal endothelial cell loss. Reduced scavenging of O2  generated by photooxidation with subsequently increased apoptosis of endothelial cells may be a mechanism behind the increased cell loss seen in the SOD3 null mouse strain. Interestingly, recent investigations have demonstrated that the formation of ROS and the oxidative tissue injury differs between Fuchs’ endothelial dystrophy and bullous keratopathy. Bullous keratopathy corneas predominantly accumulate byproducts of lipid peroxidation, whereas in Fuchs’ dystrophy corneas, signs of peroxynitrite formation dominate.1 These findings strongly suggest that these two diseases differ from a pathogenetic and oxidative point of view. Loss of Corneal Endothelial Cells in Inflammatory Eye Diseases In an acute or chronic inflammation of the anterior segment of the eye, the corneal endothelium always suffers some degree of injury. As opposed to in normal ageing, altered cell morphology, cell elongation and pleomorphism are more pronounced features in inflammations.25,29,31,37,39 A prominent feature of a uveitis is the invasion of polymorphonuclear leucocytes, known to generate both O2  and NO.85,86 Endotoxin-induced uveitis, with administration of lipopolysaccaride (LPS) systemically87 or intravitreally88 is often employed in models to study endothelial viability and regenerative capacity in vivo in inflammatory

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processes.89 Oxygen free radicals85 and their reaction products with nitric oxide86 are involved in the formation of endotoxin-induced uveitis, and scavengers of oxygen free radicals have also been shown to reduce the harmful effects of endotoxin-induced uveitis and related inflammatory processes in several models. For example, ROS formation with lipid peroxidation has been demonstrated to induce endothelial cell damage in experimental uveitis,85 and inhibitors of NO synthetases have been shown to protect the corneal endothelium from inflammatory injury.89 In addition, our group has demonstrated that mice lacking SOD3 are more susceptible to endotoxin-induced corneal endothelial damage,90 which indicates a role for SOD3 in preserving the corneal endothelial viability in inflammatory processes (Fig. 5B). Loss of Corneal Endothelial Cells After Phacoemulsification Cataract Surgery In routine phacoemulsification cataract surgery, the ultrasonic energy delivered to emulsify the lens generates ROS.46,91 Some degree of corneal endothelial cell

Figure 6 The relationship between central corneal swelling the day after routine phacoemulsification surgery, and central corneal endothelial cell loss. There is much evidence to support that ROS are responsible for much of the endothelial cell loss seen after phacoemulsification cataract surgery.

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loss is almost invariably seen after phacoemulsification, and many investigations indicate that ROS generation is involved in this process, as opposed to in the previous extracapsular technique, where the endothelial damage may have been more mechanical.92 With the phacoemulsification technique, there is a strong correlation between the reversible decrease in corneal endothelial cell function the day after surgery, and the irreversible cell loss seen,93 a finding which aligns well with an oxidative mode of endothelial injury (Fig. 6). The endothelial damage induced by ultrasonic energy in phacoemulsification cataract surgery can be diminished by the addition of SOD91 or hyaluronate, acting in this concept as a ROS scavenger.46 In addition, Rubowitz et al have elegantly demonstrated that addition of ascorbic acid to the irrigation solution can reduce the corneal endothelial cell loss in a rabbit model of phacoemulsification surgery with as much as 70%,94 a finding which indicates that oxidative mechanisms likely play the main role in this particular cell damage. REFERENCES 1. Buddi R, Lin B, Atilano SR, et al. Evidence of oxidative stress in human corneal diseases. J Histochem Cytochem 2002; 50:341–351. 2. Klyce S, Beuerman R. Structure and function of the cornea. In: Kaufman H, Barron B, McDonald M, eds. The Cornea. 2nd ed. Boston: Butterworth-Heinemann, 1998:3–50. 3. Ringvold A. Cornea and ultraviolet radiation. Acta Ophthalmol (Copenh) 1980; 58:63–68. 4. Fatt I, Bieber MT. The steady-state distribution of oxygen and carbon dioxide in the in vivo cornea. I. The open eye in air and the closed eye. Exp Eye Res 1968; 7:103–112. 5. Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance [letter]. Invest Ophthalmol Vis Sci 1983; 24:1442–1443. 6. Buck RC. Measurement of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci 1985; 26:1296–1299. 7. Tseng SC. Regulation and clinical implications of corneal epithelial stem cells. Mol Biol Rep 1996; 23:47–58. 8. Coster D. Surgical procedures to restore the corneal epithelium. Kaufman HE, Baron BA, McDonald MB, eds. The Cornea. 2nd ed. Boston: Butterworth-Heinemann, 1998:715–726. 9. Ringvold A, Anderssen E, Kjonniksen I. Impact of the environment on the mammalian corneal epithelium. Invest Ophthalmol Vis Sci 2003; 44:10–15. 10. Ando E, Ando Y, Inoue M, et al. Inhibition of corneal inflammation by an acylated superoxide dismutase derivative. Invest Ophthalmol Vis Sci 1990; 31:1963–1967. 11. Cejkova J, Ardan T, Filipec M, et al. Xanthine oxidoreductase and xanthine oxidase in human cornea. Histol Histopathol 2002; 17:755–760. 12. Hallberg CK, Trocme SD, Ansari NH. Acceleration of corneal wound healing in diabetic rats by the antioxidant trolox. Res Commun Mol Pathol Pharmacol 1996; 93:3–12. 13. Vetrugno M, Maino A, Cardia G, et al. A randomised, double masked, clinical trial of high dose vitamin A and vitamin E supplementation after photorefractive keratectomy. Br J Ophthalmol 2001; 85:537–539.

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14. Bilgihan K, Ozdek S, Ozogul C, et al. Topical vitamin E and hydrocortisone acetate treatment after photorefractive keratectomy. Eye 2000; 14(Pt 2):231–237. 15. Saika S, Uenoyama K, Hiroi K, et al. Ascorbic acid phosphate ester and wound healing in rabbit corneal alkali burns: epithelial basement membrane and stroma. Graefes Arch Clin Exp Ophthalmol 1993; 231:221–227. 16. Alio JL, Artola A, Serra A, et al. Effect of topical antioxidant therapy on experimental infectious keratitis. Cornea 1995; 14:175–179. 17. Matsumoto K, Shimmura S, Goto E, et al. Lecithin-bound superoxide dismutase in the prevention of neutrophil-induced damage of corneal tissue. Invest Ophthalmol Vis Sci 1998; 39:30–35. 18. Scott JE, Haigh M. Keratan sulphate and the ultrastructure of cornea and cartilage: a ‘stand-in’ for chondroitin sulphate in conditions of oxygen lack? J Anat 1988; 158:95–108. 19. Kwan M, Niinikoski J, Hunt TK. In vivo measurements of oxygen tension in the cornea, aqueous humor, and anterior lens of the open eye. Invest Ophthalmol 1972; 11:108–114. 20. Cintron C, Covington HI, Kublin CL. Morphologic analyses of proteoglycans in rabbit corneal scars. Invest Ophthalmol Vis Sci 1990; 31:1789–1798. 21. Funderburgh JL, Cintron C, Covington HI, et al. Immunoanalysis of keratan sulfate proteoglycan from corneal scars. Invest Ophthalmol Vis Sci 1988; 29:1116–1124. 22. Funderburgh JL, Chandler JW. Proteoglycans of rabbit corneas with nonperforating wounds. Invest Ophthalmol Vis Sci 1989; 30:435–442. 23. Goodman WM, SundarRaj N, Garone M, et al. Unique parameters in the healing of linear partial thickness penetrating corneal incisions in rabbit: immunohistochemical evaluation. Curr Eye Res 1989; 8:305–316. 24. Laing RA, Sanstrom MM, Berrospi AR, et al. Changes in the corneal endothelium as a function of age. Exp Eye Res 1976; 22:587–594. 25. Glasser DB, Matsuda M, Gager WE, et al. Corneal endothelial morphology after anterior chamber lens implantation. Arch Ophthalmol 1985; 103:1347–1349. 26. Yee RW, Edelhauser HF, Stern ME. Specular microscopy of vertebrate corneal endothelium: a comparative study. Exp Eye Res 1987; 44:703–714. 27. Collin HB, Grabsch BE. The effect of ophthalmic preservatives on the shape of corneal endothelial cells. Acta Ophthalmol (Copenh) 1982; 60:93–105. 28. Stiemke MM, Edelhauser HF, Geroski DH. The developing corneal endothelium: correlation of morphology, hydration and Na/K ATPase pump site density. Curr Eye Res 1991; 10:145–156. 29. Yee RW, Geroski DH, Matsuda M, et al. Correlation of corneal endothelial pump site density, barrier function, and morphology in wound repair. Invest Ophthalmol Vis Sci 1985; 26:1191–1201. 30. Chung JH, Fagerholm P. Corneal alkali wound healing in the monkey. Acta Ophthalmol (Copenh) 1989; 67:685–693. 31. Van Horn DL, Sendele DD, Seideman S, et al. Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci 1977; 16:597–613. 32. Gwin RM, Lerner I, Warren JK, et al. Decrease in canine corneal endothelial cell density and increase in corneal thickness as functions of age. Invest Ophthalmol Vis Sci 1982; 22:267–271.

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33. Chung JH, Fagerholm P. Endothelial healing in rabbit corneal alkali wounds. Acta Ophthalmol (Copenh) 1987; 65:648–656. 34. Olsen EG, Davanger M. The healing of rabbit corneal endothelium. Acta Ophthalmol (Copenh) 1984; 62:796–807. 35. Gordon SR, Rothstein H, Harding CV. Studies on corneal endothelial growth and repair. IV. Changes in the surface during cell division as revealed by scanning electron microscopy. Eur J Cell Biol 1983; 31:26–33. 36. Fitch KL, Nadakavukaren MJ. Age-related changes in the corneal endothelium of the mouse. Exp Gerontol 1986; 21:31–35. 37. Matsuda M, Suda T, Manabe R. Serial alterations in endothelial cell shape and pattern after intraocular surgery. Am J Ophthalmol 1984; 98:313–319. 38. Olsen T. Variations in endothelial morphology of normal corneas and after cataract extraction. A specular microscopic study. Acta Ophthalmol (Copenh) 1979; 57:1014–1019. 39. Landshman N, Solomon A, Belkin M. Cell division in the healing of the corneal endothelium of cats. Arch Ophthalmo 1989; 107:1804–1808. 40. Olsen T. Changes in the corneal endothelium after acute anterior uveitis as seen with the specular microscope. Acta Ophthalmol (Copenh) 1980; 58:250–256. 41. Setala K. Corneal endothelial cell density in iridocyclitis. Acta Ophthalmol (Copenh) 1979; 57:277–286. 42. Brooks AM, Gillies WE. Fluorescein angiography of the iris and specular microscopy of the corneal endothelium in some cases of glaucoma secondary to chronic cyclitis. Ophthalmology 1988; 95:1624–1630. 43. Schultz RO, Matsuda M, Yee RW, et al. Corneal endothelial changes in type I and type II diabetes mellitus. Am J Ophthalmol 1984; 98:401–410. 44. Cho KS, Lee EH, Choi JS, et al. Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 1999; 40:911–919. 45. Hull DS, Green K. Oxygen free radicals and corneal endothelium. Lens Eye Toxic Res 1989; 6:87–91. 46. Takahashi H. Free radical development in phacoemulsification cataract surgery. J Nippon Med Sch 2005; 72:4–12. 47. McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244:6049–6055. 48. Weisiger RA, Fridovich I. Mitochondrial superoxide simutase: site of synthesis and intramitochondrial localization. J Biol Chem 1973; 248:4793–4796. 49. Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A 1982; 79:7634–7638. 50. Wolff SP, Wang GM, Spector A. Pro-oxidant activation of ocular reductants. 1. Copper and riboflavin stimulate ascorbate oxidation causing lens epithelial cytotoxicity in vitro. Exp Eye Res 1987; 45:777–789. 51. Ringvold A. Corneal epithelium and UV-protection of the eye. Acta Ophthalmol Scand 1998; 76:149–153. 52. Ringvold A, Anderssen E, Kjonniksen I. Ascorbate in the corneal epithelium of diurnal and nocturnal species. Invest Ophthalmol Vis Sci 1998; 39: 2774–2777.

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53. Reddy VN, Giblin FJ, Lin LR, et al. The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium. Invest Ophthalmol Vis Sci 1998; 39:344–350. 54. Reiss GR, Werness PG, Zollman PE, et al. Ascorbic acid levels in the aqueous humor of nocturnal and diurnal mammals. Arch Ophthalmol 1986; 104: 753–755. 55. Ringvold A, Anderssen E, Kjonniksen I. Distribution of ascorbate in the anterior bovine eye. Invest Ophthalmol Vis Sci 2000; 41:20–23. 56. Taylor A, Jacques PF, Nadler D, et al. Relationship in humans between ascorbic acid consumption and levels of total and reduced ascorbic acid in lens, aqueous humor, and plasma. Curr Eye Res 1991; 10:751–759. 57. Taylor A, Jahngen-Hodge J, Smith DE, et al. Dietary restriction delays cataract and reduces ascorbate levels in Emory mice. Exp Eye Res 1995; 61:55–62. 58. Marguire L. Ectatic corneal degenerations. In: Kaufman H, Barron BA, McDonald MB, eds. The Cornea. 2nd ed. Boston: Butterworth-Heinemann, 1998:525–538. 59. Rao SN, Raviv T, Majmudar PA, et al. Role of Orbscan II in screening keratoconus suspects before refractive corneal surgery. Ophthalmology 2002; 109: 1642–1646. 60. Auffarth GU, Wang L, Volcker HE. Keratoconus evaluation using the Orbscan Topography System. J Cataract Refract Surg 2000; 26:222–228. 61. Rabinowitz YS, Dong L, Wistow G. Gene expression profile studies of human keratoconus cornea for NEIBank: a novel cornea-expressed gene and the absence of transcripts for aquaporin 5. Invest Ophthalmol Vis Sci 2005; 46:1239–1246. 62. Funderburgh JL, Funderburgh ML, Rodrigues MM, et al. Altered antigenicity of keratan sulfate proteoglycan in selected corneal diseases. Invest Ophthalmol Vis Sci 1990; 31:419–428. 63. Sawaguchi S, Yue BY, Chang I, et al. Proteoglycan molecules in keratoconus corneas. Invest Ophthalmol Vis Sci 1991; 32:1846–1853. 64. Kenney MC, Nesburn AB, Burgeson RE, et al. Abnormalities of the extracellular matrix in keratoconus corneas. Cornea 1997; 16:345–351. 65. Sawaguchi S, Yue BY, Sugar J, et al. Lysosomal enzyme abnormalities in keratoconus. Arch Ophthalmol 1989; 107:1507–1510. 66. Zhou L, Yue BY, Twining SS, et al. Expression of wound healing and stress-related proteins in keratoconus corneas. Curr Eye Res 1996; 15:1124–1131. 67. Kenney MC, Chwa M, Alba A, et al. Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas. Curr Eye Res 1998; 17:238–246. 68. Sawaguchi S, Twining SS, Yue BY, et al. Alpha-1 proteinase inhibitor levels in keratoconus. Exp Eye Res 1990; 50:549–554. 69. Whitelock RB, Fukuchi T, Zhou L, et al. Cathepsin G, acid phosphatase, and alpha 1-proteinase inhibitor messenger RNA levels in keratoconus corneas. Invest Ophthalmol Vis Sci 1997; 38:529–534. 70. Wilson SE, He YG, Weng J, et al. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996; 62:325–327. 71. Kim WJ, Helena MC, Mohan RR, et al. Changes in corneal morphology associated with chronic epithelial injury. Invest Ophthalmol Vis Sci 1999; 40:35–42.

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72. Connon CJ, Meek KM, Newton RH, et al. Hyaluronidase treatment, collagen fibril packing, and normal transparency in rabbit corneas. J Refract Surg 2000; 16:448–455. 73. Trinkaus-Randall V, Leibowitz HM, Ryan WJ, et al. Quantification of stromal destruction in the inflamed cornea. Invest Ophthalmol Vis Sci 1991; 32:603–609. 74. Helena MC, Baerveldt F, Kim WJ, et al. Keratocyte apoptosis after corneal surgery. Invest Ophthalmol Vis Sci 1998; 39:276–283. 75. Brown DJ, Lin B, Chwa M, et al. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis 2004; 10:281–288. 76. Kenney MC, Chwa M, Atilano SR, et al. Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: evidence that oxidative stress plays a role in this disorder. Invest Ophthalmol Vis Sci 2005; 46:823–832. 77. Becquet F, Courtois Y, Goureau O. Nitric oxide in the eye: multifaceted roles and diverse outcomes. Surv Ophthalmol 1997; 42:71–82. 78. Dighiero P, Behar-Cohen F, Courtois Y, et al. Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation. Invest Ophthalmol Vis Sci 1997; 38:2045–2052. 79. Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-II is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci 1999; 40:2773–2779. 80. Behndig A, Svensson B, Marklund SL, et al. Superoxide dismutase isoenzymes in the human eye. Invest Ophthalmol Vis Sci 1998; 39:471–475. 81. Behndig A, Karlsson K, Johansson BO, et al. Superoxide dismutase isoenzymes in the normal and diseased human cornea. Invest Ophthalmol Vis Sci 2001; 42:2293–2296. 82. Borderie VM, Baudrimont M, Vallee A, et al. Corneal endothelial cell apoptosis in patients with Fuchs’ dystrophy. Invest Ophthalmol Vis Sci 2000; 41:2501–2505. 83. Ashok BT, Ali R. The aging paradox: free radical theory of aging. Exp Gerontol 1999; 34:293–303. 84. Podskochy A, Gan L, Fagerholm P. Apoptosis in UV-exposed rabbit corneas. Cornea 2000; 19:99–103. 85. Ishimoto S, Wu GS, Hayashi S, et al. Free radical tissue damages in the anterior segment of the eye in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 1996; 37:630–636. 86. Parks DJ, Cheung MK, Chan CC, et al. The role of nitric oxide in uveitis. Arch Ophthalmol 1994; 112:544–546. 87. Rosenbaum JT, McDevitt HO, Guss RB, et al. Endotoxin-induced uveitis in rats as a model for human disease. Nature 1980; 286:611–613. 88. Ohta K, Norose K, Wang XC, et al. Apoptosis-related fas antigen on memory T cells in aqueous humor of uveitis patients. Curr Eye Res 1996; 15:299–306. 89. Behar-Cohen FF, Savoldelli M, Parel JM, et al. Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis. Invest Ophthalmol Vis Sci 1998; 39:897–904. 90. Behndig A, Karlsson K, Brannstrom T, et al. Corneal endothelial integrity in mice lacking extracellular superoxide dismutase. Invest Ophthalmol Vis Sci 2001; 42:2784–2788.

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91. Holst A, Rolfsen W, Svensson B, et al. Formation of free radicals during phacoemulsification. Curr Eye Res 1993; 12:359–365. 92. Bourne RR, Minassian DC, Dart JK, et al. Effect of cataract surgery on the corneal endothelium: modern phacoemulsification compared with extracapsular cataract surgery. Ophthalmology 2004; 111:679–685. 93. Lundberg B, Jonsson M, Behndig A. Postoperative corneal swelling correlates strongly to corneal endothelial cell loss after phacoemulsification cataract surgery. Am J Ophthalmol 2005; 139:1035–1041. 94. Rubowitz A, Assia EI, Rosner M, et al. Antioxidant protection against corneal damage by free radicals during phacoemulsification. Invest Ophthalmol Vis Sci 2003; 44:1866–1870.

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6 Involvement of Oxidative Stress in the Pathogenesis of Glaucoma Neville N. Osborne Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, U.K.

INTRODUCTION Oxidative stress can be defined as an increase over physiological values in the intracellular concentrations of Reactive Oxygen Species (ROS). ROS include molecules such as superoxide anion (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (OH), nitric oxide (NO), peroxyl radical (ROO) and singlet oxygen (1O2). This situation can occur when there are changes in the endogenous activity of antioxidant enzymes (e.g. catalase, glutathione, superoxide dismutase, metallothionein) and/or concentrations of vitamins (A,D,E) (Figure 1). Substantial evidence exists to suggest that oxidative stress plays a major part in the pathogeneses of glaucoma.1 Glaucoma, or glaucomatous optic neuropathy, is a chronic neurodegenerative disease characterised by a progressive loss of retinal ganglion cells. The disease is associated with a specific remodelling of the optic nerve head. Primary open-angle glaucoma (POAG) constitutes the majority of all forms of glaucoma where the iris position is not affected. Traditionally, glaucoma has been viewed as a disease of elevated intraocular pressure (IOP). Excessive elevation of IOP can cause compression of retinal ganglion cell axons at the optic nerve head to affect axonal transport and alter the appropriate nutritional requirements for ganglion cell survival. Blood flow in the optic nerve head is also reduced because of compression of blood vessels and/or

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Figure 1 Oxidative stress is imposed on cells because of an increase in oxidant generation (i.e reactive oxygen species or ROS), a decrease in endogenous antioxidant protection or a failure to repair oxidant damage.

altered perfusion occurring when IOP is moderately elevated. Compelling evidence therefore exists to show that raised IOP can be the cause of visual loss in glaucoma. This is supported by the finding that the lowering IOP is often linked with the prevention of visual loss. On the other hand a substantial number of glaucoma patients do not have raised IOP and often lowering of elevated IOP does not result in the prevention of visual loss. Moreover, not all ocular hypertensive patients have glaucoma. POSSIBLE CAUSES FOR GANGLION CELL DEATH IN GLAUCOMA It is now clear that the cause of ganglion cell death in glaucoma is not solely due to raised IOP and it has been hypothesised that a number of risk factors (one of which includes raised IOP) induce glaucoma or loss of ganglion cell function.2 The common aspect associated with all the putative risk factors is that they are proposed to cause an inadequate blood delivery to the components in the optic nerve head region.3 The following have been suggested to be risk factors in glaucoma: fluctuation of IOP, ageing, family history, severe myopia, central cornea thickness, hypertension, hypotension, vasospasm, hemorheology, immune system, diabetes mellitus, sleep disturbances, family history and light (Figure 2). It is likely that a combination of these risk factors is necessary to cause glaucoma. This might also explain why not all ocular hypertensive patients have glaucoma. Inadequate blood delivery to the optic nerve head region will result in ischemic/hypoxic insults being delivered to the components in the region. These will include retinal ganglion cell axons, astrocytes, microglia and the lamina cribosa. Since oxidative stress is intricately associated with ischemia4 it follows that oxidative stress is likely to play a major role in the pathogenesis of glaucoma.

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Figure 2 Likely causes for the initiation for loss of vision in different glaucoma patients. Any insult alone or in combination with other insults all result in a similar pathogenesis of ganglion cell death initiated by common influences on the optic nerve head components.

We have hypothesised5,6 that the initial ischemic/hypoxic insults to the ganglion cell axons does not result in the neurones dying but rather forces them to survive at a lower energetic status and in the process making them more susceptible to any additional insults. We have also suggested that this will ultimately occur because of altered glial function (astrocytes, Mu¨ller cells, microglia), originating from ischemia to the optic nerve head region. This is based partly on experimental studies which have shown that a variety of toxic substances (glutamate, TNF-a, serine, nitric oxide, potassium) become elevated in the extracellular retinal spaces when retinal glial cell function is affected. Elevation of such substances will particularly affect the survival of retinal ganglion cells because they are energetically compromised. Moreover, they will affect ganglion cells differentially depending partly on the nature of their receptors. As a consequence, ganglion cell death will occur at varying times as it does in glaucoma. Recent observations have made it necessary to modify this hypothesis because of the realisation that ganglion cell axons within the globe contain many mitochondria7 and that light can interact with mitochondrial enzymes to generate ROS.8 It is established that light can act on the mitochondrial photosensitizers, cytochrome and flavin-containing oxidases to generate ROS.8 It is therefore proposed that the secondary insults to initiate apoptosis to energetically compromised ganglion cells in glaucoma can also be mediated by light effects upon their many axonal mitochondria (Figure 3). It should be emphasised that light is probably not a risk factor to healthy ganglion cells where their mitochondria are likely to be able to scavenge all ROS produced in metabolism or because of light. However, in glaucoma the ganglion cells are proposed to exist initially at a compromised energetic state and only at this stage become prone to elevation of ROS caused by light.

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Figure 3 Hypothesis for differential ganglion cell apoptosis caused by variable or sustained changes in the normal blood supply to the optic nerve head.

RETINAL GANGLION CELL AXONS, MITOCHONDRIA, AND OXIDATIVE STRESS Mitochondria are the seat of a number of important cellular functions, including essential pathways of intermediate metabolism, amino acid biosynthesis, fatty acid oxidation, steroid metabolism, and apoptosis. Of key importance is the role of mitochondria in energy metabolism. Oxidative phosphorylation generates most of the cell’s ATP, and any impairment of the organelle’s ability to produce energy can have catastrophic consequences, not only due to primary loss of ATP, but also due to impairment of ‘‘downstream’’ functions, such as maintenance of organelle and cellular calcium homeostasis. Moreover, deficient mitochondrial metabolism may generate ROS that can wreak havoc in the cell because of oxidative stress. It is for such reasons that it is believed that mitochondrial dysfunction leads to apoptosis. Retinal ganglion cell axons within the globe are laden with mitochondria.7 The abundance of mitochondria is thought to satisfy the high energy requirements for nerve transmission within unmyelinated axons, compared with the lower amount required for salutatory conduction in the myelinated axons of the optic nerve, including the laminar and prelaminar portions of the optic nerve head.7 The abundance of mitochondria within the intraretinal retinal ganglion cell axons makes them particularly vulnerable to ischemic/hypoxic insults and to the light that constantly impinging upon them. It is now established that light can act on the mitochondrial photosensitizers, cytochrome and flavin-containing

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oxidases to generate ROS.8 The probability for mitochondrial dysfunction and oxidative stress being a primary cause for retinal ganglion cell death in glaucoma is therefore extremely high. ANALYSIS OF PLASMA FOR THE INVOLVEMENT OF OXIDATIVE STRESS IN POAG Yildirin et al9 compared the plasma level of the malondialdehyde and the enzymes myeloperoxidase and catalase in 40 POAG patients with 60 healthy controls, and concluded that malondialdehyde was elevated in the glaucoma subjects. Malondialdehyde is a product generated during oxidative stress. Such data suggest that there is a reduced systemic capacity for POAG patients to oxidative stress. This is supported by a recent study which demonstrated a reduced plasma level of glutathione (GSH) in newly diagnosed POAG patients when compared to age matched controls.10 Circulating GSH is a very important enzyme involved in counteracting oxidative stress and might be reduced either by reduction in synthesis or by increased consumption due to oxidative stress. Altered metabolism of GSH could also be the cause as indicated by the work of Izzotti and collaborators.1,11 These authors analysed the glutathione S-transferase isoenzymes (GSTM1 and GSTM2) involved in the synthesis of GSH and found that the GSTM1-null genotype was more common in POAG patients. These studies therefore suggest that there is an association between low systemic antioxidative capacity and POAG. OXIDATIVE STRESS AND RAISED IOP Aqueous humour contains several active oxidative agents such as hydrogen peroxide and superoxide anion12 and a rise in their levels could affect, for example trabecular cell function. Indeed, laboratory studies have shown that trabecular cells are susceptible to hydrogen peroxide which alters their adhesion properties and compromises their cellular integrity.13 Moreover, studies on the isolated perfused eye show that hydrogen peroxide affects the drainage of aqueous so causing a raise in IOP.14 These experimental studies are consistent with the hypothesis that trabecular cell malfunction might be caused by oxidative stress in glaucoma patients.15,16 Further support for this idea comes from studies which reveal that oxidative DNA damage17 and the expression of endothelialleukocyte adhesion molecule (ELAM-1)18 are significantly elevated in trabecular cells of glaucoma patients compared with unaffected controls. Both trabecular cells and aqueous humour contain a number of oxidative stress markers and an alteration in any of these can cause oxidative stress to the cells. There is some evidence that these oxidative stress markers are altered in the aqueous humour of glaucoma patients. For example, Ferreira et al19 found that the total antioxidant potential value in the aqueous humour of glaucoma patients is 64% less than that of a cataract group of patients. Moreover, aqueous

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humour superoxide dismutase and glutathione activities increased by 57% and 300% respectively, in the glaucoma group compared with the cataract group. However, the catalase activity was similar in both groups. There is therefore good reason to suggest that raised IOP in some glaucoma patients originates from trabecular cell malfunction caused by oxidative stress. For more detailed information see Izzotti et al.1 OXIDATIVE STRESS INVOLVEMENT IN GANGLION CELL APOPTOSIS A body of experimental evidence now exists, and supported by pathological studies on glaucoma eyes, that a cascade of mechanisms occurs to cause ganglion cells to die at differential rates. A hypothesis to summaries what may occur is shown in Figure 3 where it is difficult not to exclude the involvement ROS in every aspect (Figure 6). For example, an increase in extracellular glutamate would alter cystine transport into ganglion cells so causing reduced intracellular glutathione and oxidative stress (Figure 4). Overactivation of ganglion cell excitatory amino acid receptors4 will result in an intracellular stimulation of ROS (Figure 5). ROS generation is also a component of TNF-a signalling20 which is believed to play a major part in retinal ganglion cell apoptosis.21 The influence of light on retinal ganglion cell axon mitochondria will also result in a generation

Figure 4 Increasesd extracellular glutamate causes oxidative stress.

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Figure 5 (See color insert.) A rise in extracellular glutamate and overactivation of glutamate ionotropic receptors leads to generation of ROS and cell death.

of ROS.6 Ischemia/hypoxia to the astrocytes is also likely to generate ROS to potentially influence ganglion cell function.22 It would appear therefore that there are very good reasons to suggest that increased extracellular and intracellular levels of ROS initiated by ischemia/ hypoxia to the optic nerve head region causes ganglion cells to die at a differential rate (Figure 6). Increased retinal ROS also affects glial function and possibly activates immune responses. Detailed molecular mechanisms of the real impact of oxidative stress on the development and progression of glaucomatous neurodegeneration, however, remains to be elucidated. A greater understanding may offer unique opportunities for neuroprotective intervention with appropriate antioxidants. CONCLUSION Good evidence exists to support the tenant that oxidative stress to the trabecular cells and retinal cells are instrumental in the eventual cause for ganglion cells dying in glaucoma (Figure 6). Logic therefore suggests that adjunct treatment of glaucoma patients with IOP lowering agents and suitable antioxidants would be worthy of consideration. Oral intake of powerful antioxidants like a-lipoic acid and/or vitamin E, which are well tolerated and will reach the retina, are possible candidates.

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Figure 6 Oxidative stress may be involved in a number of events that are associated with the pathogenesis of glaucoma?

REFERENCES 1. Izzotti A, Bagnis A, Sacca SC. The role of oxidative stress in glaucoma. Mutat Res 2006; 612:105–114. 2. Pache M, Flammer J. A sick eye in a sick body? Systemic findings in patients with primary open-angle glaucoma. Surv Ophthalmol 2006; 51:179–212. 3. Osborne NN, Ugarte M, Chao M, et al. Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv Ophthalmol 1999; 43 (suppl 1):S102–S128. 4. Osborne NN, Casson RJ, Wood JP, et al. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog Retin Eye Res 2004; 23:91–147. 5. Osborne NN, Ugarte M, Chao M, et al. Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv Ophthalmol 1999; 43 (Suppl 1):S102–S128. 6. Osborne NN, Lascaratos G, Bron AJ, et al. A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies. Br J Ophthalmol 2006; 90:237–241. 7. Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 2004; 23:53–89. 8. Godley BF, Shamsi FA, Liang FQ, et al. Blue light induces mitochondrial DNA damage and free radical production in epithelial cells. J Biol Chem 2005; 280:21061–21066. 9. Yildirim O, Ates NA, Ercan B, et al. Role of oxidative stress enzymes in open-angle glaucoma. Eye 2005; 19:580–583. 10. Gherghel D, Griffiths H, Hilton E, et al. Systemic reduction in glutathione levels occurs in patients suffering from primary open-angle glaucoma. Invest Ophthalmol Vis Sci 2005; 46:877–883.

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11. Izzotti A, Sacca SC, Cartiglia S, et al. Oxidative deoxyribonucleic acid damage in eyes of glaucoma patients. Am J Med 2003; 114:638–646. 12. Spector A, Garner WH. Hydrogen peroxide and human cataract. Exp Eye Res 1981; 33:673–681. 13. Zhou L, Li Y, Yue BY. Oxidative stress affects cytoskeletal structure and cellmatrix interactions in cells from an ocular tissue: the trabecular meshwork. J Cell Physiol 1999; 180:182–189. 14. Kahn MG, Giblin FJ, Epstein DL. Glutathione in calf trabecular meshwork and its relation to aqueous humor outflow facility. Invest Ophthalmol Vis Sci 1983; 24:1283–1287. 15. Alvarado J, Murphy C, Polansky J, et al. Age-related changes in trabecular meshwork cellularity. Invest Ophthalmol Vis Res 1981; 21:714–727. 16. Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary openangle glaucoma and non-glaucomatous normals. Ophthalmology 1984; 91:564–579. 17. Sacca SC, Pascotto A, Camicione P, et al. Oxidative DNA damage in human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma. Arch Ophthalmol 2005; 123:458–463. 18. Wang N, Chintala SK, Fini ME, et al. Activation of a tissue specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype. Nat Med 2001; 7:304–309. 19. Ferreira SM, Lerner SF, Brunzini R, et al. Oxidative stress markers in aqueous humor of glaucoma patients. Am J Ophthalmol 2004; 137:62–69. 20. Xu YC, Wu RF, Gu Y, et al. Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase. J Biol Chem 2002; 277:28051–28057. 21. Tezel G, Yang X, Yang J, et al. Role of tumor necrosis factor receptor-1 in the death of retinal ganglion cells following optic nerve crush injury in mice. Brain Res 2004; 996:202–212. 22. Liu B, Neufeld AH. Expression of nitric oxide synthase-2 (NOS-2) in reactive astrocytes of the human glaucomatous optic nerve head. Glia 2000; 30:178–186.

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7 Oxidative Stress and Cataract Susanne Hippeli, Harald Schempp, and Erich F. Elstner TU-Mu¨nchen, Institute of Phytopathology, Freising-Weihenstephan, Germany

Matthias Elstner Department of Neurology, Ludwig-Maximilian University, Munich, Germany

INTRODUCTION Most inflammatory and degenerative processes include oxygen activating processes where reactive oxygen species, ROS, are produced. Intrinsic radical scavenging systems or compounds administered with food such as Vitamin C and E, carotenoids and polyphenols, warrant metabolic control within certain limits. Many of these are free radical scavengers or quenchers of activated states and operate additively or synergistically. In this review mechanisms of cataract formation and also of protection from oxidative damage by antioxidants, present in many plant extracts used as natural drugs, are summarized. For this purpose, principles of oxygen activation during cataract induction and protective actions of antioxidants are outlined in short. NATURAL HISTORY About 3.5 billion years ago, the first light—utilizing organisms only had one photosystem (‘‘cyclic photosystem I’’). Thus, for the purpose of carbon dioxide fixation (which is a reductive process), they had to use exogenous electron

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donors (reductants) such as hydrogen sulfide or hydroxylamine. The first ‘‘energy crisis’’ arose when these reduced compounds were exhausted (oxidized) in their aqueous environments. The solution of the problem for the ancestors of cyanobacteria was ‘‘inventing’’ photosystem II, i.e. a second photosystem containing a chlorophyll modification with an E0 o as high as þ830 mV thus allowing them to utilize water as an inexhaustible electron source for free. This novel photosystem produced oxygen, protons and electrons in a light-dependent reaction involving manganese as catalytic redox converter as an electron-trap with water as electron donor. This strategy was so efficient that it allowed to assemble high densities of these organisms, accumulating as pure carbon, geologically designated as graphite (coal is approximately 2.5–3 billion years younger!). All this actually happened in Bavaria: In the community of Hauzenberg, approximately 30 km northeast of the city of Passau, the only graphite mine in Middle Europe is still being exploited and worth wile visiting (‘‘graphite museum’’). THE BENEFITS AND THE PROBLEM From this time other unicellular organisms, devoid of chlorophyll (heterotrophes), took advantage of these novel ‘‘energy-unlimited’’ cells, using them as food source or even incorporating them as cellular organelles in a sense of photovoltaic elements: coevolution started and multicellular, higher organisms could develop. The trade–in of water-splitting by photosystem II was ‘‘oxygen toxicity’’, however. The worst case is, when light and oxygen are operative at the same place. Thus, it is not astonishing, that photosystem II, where a light–dependent oxygen liberation from water is achieved, is perfectly protected against oxygen toxicity by a wealth of cooperative systems involving ‘‘electron idling’’ as well as antioxidants such as tocopherol, carotenoids as well as a set of enzyme systems.1 Actually our eyes have to envisage similar problems as the photosystems in plants: they only operate in the light exposed to high oxygen tensions; therefore it is also not astonishing, that the solutions to the problem of ROS toxicity might ask for similar solutions. In the first couple of hundred million of years the problem for oxygen evolving cells was not that dramatic since most oxygen was bound and sedimented by the process of iron IIþ oxidation. An intermediate period might have allowed to re-reduce oxidized nitrogen (‘‘nitrate-respiration’’) thus supporting the original ‘‘one-photosystem’’ organisms as well as primitive heterotrophes living without oxygen. Finally, when land-plants developed and oxygen accumulated in the atmosphere, nitrate respiration was substituted by the more efficient oxygen respiration utilizing the ‘‘counterpart’’ of water-splitting, namely water formation via oxygen reduction by cytochrome a/a3. This system involved both iron and copper as redox converters in analogy to the manganeous system in photosystem I, now functioning as oxygen trap. Water splitting and water formation, i.e. oxygen formation from water and oxygen reduction to water are four-electron steps. Thus, another problem

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arose: four electrons could not be transferred simultaneously, but step by step thus involving superoxide, hydrogen peroxide and OH-radical as intermediates on the way of oxygen to water, according to: O2 þ 4e þ 4Hþ ! 2H2 O Since these intermediates are of great chemical reactivity, the mechanism of their production had to be ‘‘cryptic’’, i.e. with an excellent isolation towards its surroundings, similar to an atomic power plant. Other redox systems in internal metabolism were also thermodynamically able to reduce oxygen potentially producing the above toxic species. All aerobic organisms therefore had to develop antioxidative strategies and synthesize antioxidants in order to survive. In the following, both phototrophic (algae, higher plants) and heterotrophic organism (bacteria, fungi, animals) developed cooperative and adaptive strategies for detoxification: Again, the heterotrophes took advantage of the much better synthesizing capacities of the plants: they just ‘‘forgot’’ to build bioenergetically ‘‘expensive’’ (ATP-consuming) molecules such as aromats— with some exceptions. The ROS–detoxifying systems were developed synergistically and allowed both plants and animals to utilize oxygen activation as defence systems (‘‘respiratory burst’’) exhibiting homologous external and internal battle fields such as the apoplasts of the plant and the phagosome of the animals. Traditional and modern medicine use microbial and higher plant’s products i.e. their antioxidants as drugs, preventive therapies and food additives.2–4 CATARACT What Is Cataract Chemically? Cataract, the turbidity of the eye lens, is due to protein cross linking via sulfhydryl oxidation and protein glycation, dependent on the individual patterns of pathometabolism.5 In most cataractogenic reactions oxygen seems to be involved. This seems to be clearly supported by the recent finding, that ‘‘vitrectomy surgery increases oxygen exposure to the lens’’ with the risk of nuclear cataract formation.6 Fundamentally, extremely different influences may govern cataractogenic processes, measurable at different sites in the lens as outlined in the following Table 1 and Figure 1. Principally, there are initiating processes triggered by radiation of different qualities, and others initiated by certain metabolites operating also in the dark via reductive oxygen activation, as outlined in ref.5 and below. Principle Effects of Light As demonstrated below there are several compounds, such as riboflavine or tryptophan derivatives, which are absorbing light quanta, transiently forming an activated state (P*) which can transfer this activity onto molecular oxygen thus yielding highly reactive ‘‘Singlet oxygen’’, 1O2. This reaction is called ‘‘Type II’’

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Table 1 Possible Modes of Cataract Induction Nutritional disturbance Lack of certain amino acids Lack of vitamins Disturbance of the essential compounds present in aqueous humor Endocrinic disturbances Chemical influences Physical influences Changes in enzyme patterns Accumulation of toxic products

photodynamic reaction. Singlet oxygen in turn can spontaneously react with unsaturated fatty acid, since this reaction is not spin forbidden, forming hydroperoxides, which in turn can cause further destructions. In ‘‘Type I’’ reactions P* undergoes charge separation initiating oxidations involving superoxide (Table 2). Both types of reaction may be induced cooperatively, dependent on the individual surrounding, i.e. the presence of suitable electron donors and substrates. As shown in Table 3, there is a vast amount of compounds used as drugs, which may work as photodynamics potentially operative in the above outlined processes.

Figure 1 Different forms of cataract.

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Table 2 Inductions of Cataract Formation by Light Primary reactions P P*  +P + D  P + O2 P + A

light ! ! ! ! !

P + D+ P + O2  P + A

activation charge separation photooxidation activation of oxygen photoreduction

Secondary reactions 2O2  + 2H+ D+ D+ + S D+ + A

! ! ! !

H2O2 + O2 decay D + S+ D-A

peroxide formation charge separation photooxidation cross-linking

P*

 +P 

Table 3 Photodynamic Drugs Drug

Application

Clinical Observation

Sulfonamides

chemotherapy, antibacterial Agent antidiabetic diuretic, antihypertensive

phototoxic, photoallergic relations phototoxic papillic and edematous eruptions and plaques hyperpigmentation, hypersensitivity to sun exposure, hypersensitivity to sun exposure erythema, phototoxic and photoallergic reactions erythema, hyperpigmentation phototoxic reactions

Sulfonic urea Chlorothiazines phenothiazines

tranquilizer, antihistaminic antiseptic

antibiotics (tetracyclines) griseofulvin

antimycotic

furocumarines

psoriasis treatment

estrogens and progesterones chlorodiazepoxides triacetylphenolisatin

contraceptive tranquilizer cathartic

eczema eczema like photoallergic reaction

In cataract formation the ‘‘oxidation-sensitive’’ amino acid tryptophan has been shown to act as one predominant precursor for two compounds operating as photodynamic enhancers: 3-hydroxy kynurenine-glucoside and xanthurenic acid 8-0-b-D-glucoside (Figure 2). Both substances stem from oxidative splitting of tryptophan via N-formylkynurenine and subsequent deformylation

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Figure 2 Formation of xanthurenic acid.

and glycosylation. Xanthurenic acid undergoes some more transformations, including N-heterocyclic ring formation and additional hydroxylation. Xanthurenic acid accumulates as non-diabetic brunescent colour in cataractic eyes and acts as endogenous chromophore/fluorophore and UV region sensitizer with an excitation at 338nm and an emission at 440nm, efficiently generating singlet oxygen.7 Singlet oxygen again produces long living peroxides thus promoting and extending the initial damage8 Reductive Events Since the redox potential of the pair O2/O2  is 330mV, many electronegative compounds may represent potential candidates as initiators of monovalent oxygen reduction. Some of them were designed for this purpose, i.e. anti-cancer drugs such as adriamycin. Generally, benzo-, naphtho- and anthraquinones are well known as redox cyclers in biochemistry, as shown for a naphthoquinone in Figure 3. Figure 4 represents compounds (besides the mentioned naphthoquinones like juglone, and the anthraquinone rein), pyrroloquinolin quinones (PQQ), quat salts such as paraquat and nitroaromats such as nitrofuran which may act as redox cyclers, being ‘‘unspecifically’’ reduced by flavoprotein (FP)-oxidoreductases (diaphorases) inducing ROS-production and thus oxidative destruction.

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Figure 3 Redox cycle of 2-methyl naphthoquinone producing superoxide.

Figure 4 Redox substrates of NAD(P)H oxidoreductases (diaphorases).

Protein Glycosylation: Diabetic Events Aldehyde groups of sugars can react with amino groups forming Schiff bases (aldimines). These Schiff bases undergo so-called Amadori rearrangements finally forming enediols. Enediols are compounds that may form complexes with transition metals such as copper or iron (mostly as chelates) which easily

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Figure 5 Model reaction for protein glycation and transition metal catalyzed oxidations.

autooxidize forming superoxide and the well known follow-up ROS, hydrogen peroxide and OH-radical thus again starting the well known initiation of destructive events. This cascade is represented in Figure 5. In the experiment in vitro, Amadori products i.e. the ‘‘pure’’ mechanism of ene-diol oxidation, can be substituted by dihydroxyfumaric acid, HOOC-C(OH) =C(OH)-COOH (DHF): in the presence of iron-ADP-complexes, DHF transfers ‘‘two times one’’ electron onto oxygen yielding diketosuccinate (DKS) and again superoxide, H2O2 and OH-radical (Table 4). Protein glycosylation can also be simulated in vitro by incubation of lens proteins with ascorbic acid, which also contains such an enediol configuration acting as prooxidant in this situation. Thus situations may occur where ‘‘ascorbylations’’ represent glycation models producing superoxide and so on, as outlined Table 4 Autoxidation of Dihydroxyfumaric Acid (DHF) DHF + O2 O2  + H+ + DHF DHF + O2 2O2  + 2H+ O2  + Fe3+ADP H2O2 + Fe2+ADP

! ! ! ! ! !

DHF + O2  DHF + H2O2 DKS + O2  + H+ H2O2 + O2 O2 + Fe2+ADP Fe3+ADP + OH + OH

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Table 5 Experimental Cataract Induction Ø Ø Ø Ø

Naphtalin, Selenite, UV, Smoking, Transition metals, Redox cyclers (PMS) Diabetes: sugars via aldose reductase and/or Amadori reaction Vitrectomy: increase of oxygen tension in the eye genetic and/or ethnic

by Linetsky et al.9 These models are very important to learn about basic reaction mechanisms; whether these situations are of clinical significance, however, is a matter of ongoing debate around the problem: when are antioxidants prooxidative? Altogether, there are certain possibilities to explore mechanisms of cataractogenesis by a wealth of models, mimicking physiological aspects thus allowing to test for possible amendments of procataractic events. Although we have little influence on the genetic basis yet, genetically favoured cataract formation (see below and),10 for example in the (Emory)-mouse model also greatly contributed to the field. Experimentally by naphthalin induced cataract seems to be mediated via its hydroxylation to 1,2-hydroquinone and following superoxide formation, as shown by the protection by SOD.11 Other examples are selenite (via interaction with sulfhydryl groups) or the redox cycler phenazonium methosulfate (PMS, c.f. ref. 12) representing valuable tools in cataract research. Some well known conditions for cataract induction, including experimental models, are summarized in Table 5. First Biochemical Signs of Cataract Formation Before cataract becomes evident as measurable or visible lens opafication, certain biochemical processes can be measured in advance (Table 6), some of which were already mentioned above. Three points shell be especially addressed here: Ø

Formation of protein-bound dihydroxyphenylalanin (DOPA) by high energy radiation as one more ene-diol mediated, transition metal-catalysed ROS generator.13

Table 6 Early Events in Cataract Formation Ø

Ø

Ø Ø Ø Ø

Increase of methionine sulfoxide and cystin (electron donors for riboflavin-type I photooxidation); Increase of oxidation products of tryptophan as singlet oxygen generators and thus amplificators (3-hydroxy-kynurenine and its cyclic derivates xanthurenic acid and xanthurenic-8-glycoside); Increase of peroxides (lipid-OOH, Tyr-OOH) and hydroxynonenal; Induction of DT-Diaphorases; Decrease of GSH and ATP-ases; Protein binding of DOPA as amplificator

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Induction of the formation of so-called DT-diaphorases which reduce pquinones to the corresponding hydroquinones. By subsequent catalysis of SOD, semiquinones and superoxide, produced by the above mentioned autoxidation, are detoxified in a ‘‘hetero-dismutation’’. Peroxides14 and aldehydes such as hydroxynonenal and other aldehydes15 have to be under strict metabolic (enzymatic) control.

Intrinsic Light Reactions As demonstrated in Figure 6, lens homogenates from calf eyes drive time- and light-dependent ethene formation from a-keto-S-methyl-butyric acid (KMB), a sensitive indicator for ROS.16 Thus, an intrinsic photodynamic activity in these preparations is indicated. Prevention of Cataract in Model Reactions In Vitro and Ex Vivo One of the dominating late processes in cataract formation is protein agglomeration by S-S bridge formation and other condensations producing high molecular weight (HMW)-aggregates. This process can be followed by means of protein electrophoresis or FPLC chromatography.17 In the experiment, lens homogenates are illuminated in the presence of mM concentrations of riboflavin and FPLC chromatograms are developed after illumination in the presence or absence of iodide (KJ) as ‘‘quenching’’ electron donor (Figure 7).

Figure 6 Ethene formation from KMB by lens homogenates after illumination as indicator for intrinsic photodynamic activities and ROS formation.

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Figure 7 FPLC chromatogramme of lens homogenate (LH) illuminated for 120 min with 50mM riboflavin (Rib) in the absence or presence of 10mM iodide (KJ).

As shown in Figure 7 high molecular weight (HMW)-protein with a retention time around 15 min increases after illumination in the presence of the photodynamic activator, riboflavin. This process is partially reversed in the presence of KJ as electron donor. It should be mentioned here that KJ was in use as topical anticataractic in the 1970s–1980s. With the same method it could be shown that photodynamic formation of several HMW-aggregates can be prevented by antioxidants such as (dihydro)thioctic acid,6,18 which is also in use as drug for the prevention of certain neurological disorders (Figure 8). Models for Investigating Topical Penetration Rates Enucleated rabbit eye bulbs were used in a ‘‘droplet apparatus’’ (Figure 9) to investigate on potential penetration rates of drugs in the interior of the eye, i.e., vitreous humor and lens tissue.19 In this apparatus, tear flow and eye lid movements are simulated by a paper strip on top of a copper net, thus connecting the bulb surface with an electrolyte reservoir. After the corresponding droplet applications, the increase of concentration of drug can be analyzed timedependently in the individual compartments of the eye (compared to the electrolyte reservoir), by means of inhibition of light-dependent, riboflavin-driven ethene formation from KMB, as demonstrated in Figure 6.

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Figure 8 Prevention of photodynamic HWM-aggregate formation by reduced thioctic acid (Lip(SH)2) LH: lens homogenate, FPLC-separated crystalline proteins with a molecular weight (MG) of more than 300 kD and of ca. 45 kD respectively; riboflavin 2,5 mM; illumination: 15 min with 30 klux.

As shown in Figure 10, potassium iodide (KI) as potential anticataractic is present in all the compartments under investigation. It also became clear that in a realistic time of 2 min. no KI was found in the anterior lens cortex. Further incubation for 20 min. increased this rate considerably, especially in the aqueous humor.

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Figure 9 Droplet apparatus for the determination of penetration rates.

Figure 10 Concentrations of KI in different compartments of the eye after droplet application. KI: potassium iodide; FW: fresh weight; numbers shown at top of columns: mmol KI/ml (aqueous humor and vitreous humor), mmol KI/mg FG (anterior lens cortex).

If we now compare the rates of KI movement, either through the cornea into the aqueous or via paper strip downwards to the electrolyte, an approximate 3:5 ratio was measured, respectively. This situation is depicted in Figure 11.

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Figure 11 Corneal penetration of KI as compared to lateral transport.

PROTECTION BY ANTIOXIDATIVE STRATEGIES Protection from Oxygen Stress Ocular diseases in respect to ROS have been addressed in recent reviews: ‘‘Oxygen free radicals in ocular diseases’’ have dealt with by Schempp and Elstner20 and by Varma et al.21 ROS have to be continuously under strict control of integral detoxification processes, detoxificating enzymes and organic antioxidants. One principle way to deal with oxygen toxicity is ‘‘avoidance’’, i.e. circumventing one or two electron donating processes towards oxygen. This can be achieved by ‘‘tight’’ coupling of electron transport chains operating at the electronegative region of oxygen activation or by stoichiometric coupling of oxygen activating processes with utilization of activated oxygen. Another possibility is the inhibition or inactivation of oxygen activating processes or enzymes. This has been shown for xanthine oxidase, lipoxygenases, prostaglandine cyclase, NAD(P)H oxidases and other enzymes by a wealth of compounds used in medicine. The so-called NSAIDs (non-steroidal antiinflammatory drugs) and several flavonoids are good examples for this principle. Detoxifying Enzymes Detoxification by enzymatic processes is only possible, if the reactivity of the respective oxygen species is reasonably low under physiological conditions so that the enzymatic reactions allow k-values of at least 2–3 orders of magnitude between the reaction under enzyme catalysis and the non-catalyzed, spontaneous reaction between the oxygen species and any reaction partner in its ‘‘molecular’’

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neighbourhood. Therefore, the reactions of OH ,1O2, RO , ROO and HOO are not under enzymatic control; their reaction constants with potential reaction partners in their typical ‘‘environments’’ is too fast (generally k>>108) for enzyme catalysis. Thus, the reactions of biomolecules with these oxygen species have to be ‘‘amended’’ after damage. In order not to ‘‘flood’’ these repair processes the above mentioned antioxidative molecules serve as scavengers and quenchers of activated states. Enzyme-catalyzed detoxifications thus mainly concern superoxide, peroxides, semiquinones and epoxides (produced by cytochrome-P450-activities) as more or less ‘‘stable’’ reduced oxygen species. In most aerobic cells catalase (CAT), superoxide dismutases (SODs), monoor dehydro-ascorbate reductase, glutathione peroxidase (GSH-POD), glutathione reductases, DT-diaphorases and different peroxidases (PODs) either individually or cooperatively remove stable reactive oxygen species. Different individual physiological parameters or ‘‘stresses’’ may induce different enzymatic patterns. Microperoxidase, a ferriheme undecapeptide, derived from cytochromes, has been shown to degrade peroxides (similar to a-keto-acids; see below) was suggested as protective against oxidative stress in the lens.22 One early event in cataract induction is the appearance of organic peroxides (c.f. Table 5) partially of lipophilic character, which experimentally is reflected by tert-butyl-hydroperoxide (TBOOH). Exposure towards TBOOH induces resistance towards hydrogen peroxide in immortal murine lens epithelial cells probably via a whole set of defence enzymes: out of more than 12.000 gene expressions tested, 16 genes were found to account for protection including glutathione-S-transferases, SOD, zeta-crystallin, NADPH-quinone reductase, toxic lipoprotein degradation, control of iron metabolism and aldehyde detoxification.10 Thus it seem evident that the fate of cataract development is clearly under genetic control. If this control fails, some other potentials seem to be available: Nutritional low molecular weight supplements promise to support possibly failing intrinsic defence lines, sometimes with extremely doubtful prerequisites, however. External Helpers: Phenolic Derivatives Protect from Oxidative Stress Phenolic compounds play an important role in this context acting as antioxidants, inducers of enzymes, transition metal chelators thus avoiding Haber-Weiss(Fenton)-chemistry and cofactors of regulation of enzymatic activities. Detoxification in a wider sense thus also concerns the replacement of damaged molecules such as DNA, proteins and membrane lipids by a complex ‘‘crew’’ of integrated repair enzymes and replacement processes. A continuous involvement of these repair processes, however, would render them inactive since they also continuously function as targets of these reactive oxidants. Therefore, another batch of first aid molecules such as phenolics is biologically more than logic.

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The only ‘‘help’’ for the final repair teams are small molecules with ‘‘Kamikazee-type’’ properties, representing antioxidants with or without chance to be metabolically repaired themselves. Phenolic redox reactions are fundamentally involved in stress metabolism both in plants and in animals comprising redox processes and antioxidative functions including the formation of phenoxyl radicals, semiquinone radicals and o- or p-quinones undergoing electron donating reactions towards reactive radicals. Dependent on the neighbourhood the formed phenolic radical may be rather stable awaiting reduction by available electron donors such as ascorbate and a-tocopherol. In a ‘‘pecking order’’23 of these two important antioxidants, radical states in biomembranes are quenched where ascorbate or thiols such as reduced glutathione or lipoic acid (thioctic acid) regenerate the reduced state of phenolics such as tocopherol or ubiquinol in the interphase between lipophilic and hydrophilic plasmatic phases.24,25 With certain initiator radicals phenolics may be converted into alkoxyl radicals (RO ) or semiquinones thus acting as prooxidants depending on the substituents in the neighbourhood of the phenoxyl radical group; tocopherols acting as prooxidants are good examples for this process. In the presence of ubiquinol, however, the prooxidative activity of vitamin E is converted into an antioxidative function as shown for LDL-oxidation.26 Thus cooperative effects of diverse phenolics are indicated where the over-all antioxidative effect is due to ‘‘total phenolics’’ and not a single substance where additive, synergistic and supplementory effects are observed. In the case of transition metal catalysis (Fenton- or Haber-Weiss-chemistry), phenolics may act as chelators for iron- or copper-ions. In this respect they both may stimulate or inhibit oxidative reactions, strongly dependent on the model reaction or the type of damage looked at. Phenolics may simply act as radical scavengers or radical-chain breakers thus extinguishing strongly oxidative free radicals such as OH.; they also may react with non-radical species such as hypochlorous acid or peroxinitrite yielding products with much lower oxidative capacities as compared to the parent compounds.27–29 Some molecules such as quercetin seem to have more than just one function: a strong antioxidative (scavenger) function as well as iron chelating and enzyme-inhibitor properties. Very recently, Fiorani et al30 reported on the prevention of dehydroascorbate (DHA)-dependent GSH depletion in red blood cells due to the presence of quercetin. The mechanism was not simply a chemical interaction of quercetin with DHA or GSSG, but an activation of enzymatic GSSG-reduction downstream to this primary redox events. Cooperative Effects of Antioxidants LDL Oxidation LDL oxidation is supposed to be one initiating factor in atherogenesis and seem to be a good model reaction to study lipid peroxidation in vitro. There have been

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Figure 12 Cooperative detoxification of alkoxyl (LO ) and peroxyl (LOO ) radicals. DHLA: dihydrolipoic acid; LA: lipoic acid; Qred: reduced quinone; Qox: oxidized quinone

numerous publications in the past 10–15 years reporting on prevention of LDL oxidation by a ‘‘pecking order’’-principle23,26 by food supplements such as genistein,31 cocoa,32 grape seed powder33 and many others. Besides the well known cooperative ‘‘repair teams’’, tocopherol-ascorbate and tocopherolbiquinole-ihydrolipoic acid.26 and refs therein In this case, the oxidized quinones are re-reduced by a-keto acids via the diaphorase-thiocitic acid (dihydrolipoic (DHLA) and lipoic acid (LA)) pathway as shown schematically in Figure 12. a-keto acids such as ketoglutarate, KMB (see also above) or pyruvate34 react chemically with peroxides in an ionic process thus acting antioxidatively per se, in addition to act as potential electron donors in the above electron transport system. It has been mentioned that antioxidants such as vitamin C may act prooxidatively. This can be shown in the case of LDL oxidation using the copper model where 1mM ascorbate accelerates the lag phase of dienconjugation of intrinsic linolenic acid. In the presence of the flavonoid rutin, however, this prooxidative effect is reversed and ascorbate and rutin work cooperatively in protection35 (Figure 13). Jet other ‘‘lipid protecting teams’’ in LDL may be operating, involving intrinsic carotenoids (b-carotene, lycopene, lutein): carotenoid oxidation was strongly delayed by the lemon oil terpene, g-terpinene,36,37 in a similar manner as tocopherol by ubiquinole as shown above. Herbal Extracts Neuronal disorders such as Parkinson’s disease or Alzheimer’s disease38 and eye diseases such as AMD (age related macular degeneration) and cataract gain increasing importance due to increasing age of our population. Herbal extracts are in use against mental and generally neuronal disorders since the old times and envisage dramatic revitalization in our days. Prominent examples are Ginkgo biloba extracts,39–41 Ayurvedic medications42 and extracts from St. John’s wort, Hypericum perforatum.43,44 Since the onset of atherosclerosis

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Figure 13 Cooperative effects of rutin and ascorbate in LDL protection.

has some basic similarities to cataract induction, i.e. glycations and formation of organic peroxides, medication of both may be indicated. Formation of glycation end products may be prevented or ameliorated by drugs called ‘‘amadorins’’ such as aminoguanidine and pyridoxamine (‘‘Pyridorin’’).45,46 Furthermore, additional uptake of both vitamins C and E together with moderate physical exercise have been shown to strengthen the antioxidative defense system in an animal model.47 Neuronal hypoxia as quite ‘‘normal’’ age-related anatomic change, varying from mild deficits to massive neuropathological events, implies pharmacological benefits for GBE by means of its antioxidant flavonoids. GBE have been proven in many studies to be advantageous for the amelioration of the blood vessel system thus protecting cells from oxidative damage in connection with inflammatory processes. Since neurological (cerebral) disorders are based on inflammatory processes and limitations in blood circulation (ischemic situations), an attenuation by antioxidants is indicated especially if accompanied by other activities. Animal experiments with rats after occlusion of the carotid arteries showed that pre-ischemic administration of GBE (150 mg/kg p.o.) protected against postischemic injury measured as malondialdehyde (MDA), glutathion (GSH)-status,

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phospholipid levels as well as superoxide dismutase (SOD) and lactate dehydrogenase (LDH) activities48 and other metabolic activities, as recently outlined.49 Oyama et al50 studied the metabolism of brain neurons in resting and calcium-loaded cells and the effects of myricetin and quercetin as GBEconstituents on oxidative events by means of increased fluorescence after 20 ,70 - dichlorofluorescein oxidation: 3 nM myricetin and 10 nM quercetin reduced oxidation significantly indicating that these ingredients of GBE may be partly responsible for the observed beneficial effects in the cells after ischemic events. Lipid peroxidation during experimental spinal cord injury (‘‘paraplegic animals’’) was measured (MDA-test) by Koc et al51 either in the absence or presence of GBE, methylprednisolon (MP) or thyrotropin-releasing hormone (TRH). Both MP and GBE exhibit protective effects due to their antioxidative properties. Subarachnoid hemorrhage, where NO-levels in serum are decreased but increased in the brain, are followed by cerebral vasospasms and neuronal damage. GBE antagonizes these effects thus reversing pathological NO-alteration and relieving cerebral vasospasms.52 Glutamate-induced cytotoxicity in neuronal (HAT-4) cells is associated with glutathion depletion and thus oxidative stress. GBE and also maritime pine bark extracts (‘‘Pycnogenol’’) were able to protect against glutamate-induced damage.39 On the other hand AMPA- and NMDA-receptors are antagonized by 6-hydroxykynurenic acid (6-HKA) and kynurenic acid, which can be extracted from Ginkgo leaves. Therefore 6-HKA is suggested as a useful tool for the analysis of glutamate-mediated synaptic responses.53 Staurosporine (ST)-induced neuronal apoptosis was inhibited by GBE and some of its components: After treatment with ST (200nM) for 24 h, 74% apoptosis in chick neurons was observed. This was reduced to 24%, 62% and 31% by GBE (100mg/l), ginkgolide J (100mM) and ginkgolide B (10mM), respectively.54 Age-related problems in terms of nutritive aspects are addressed by Riedel et al.55 They recommend that supplementations with antioxidants and cofactors like folate, b-carotene and tocopherole, caffeine (in low doses) and GBE are beneficial for enhancing cognitive functions in elderly people. GBE, due to its ability to improve peripheral blood flow to the eye and general neuroprotection, may thus be also advantageous for the treatment of glaucoma.56 Glaucoma, an eye disease with increased intraocular pressure, is normally treated with b-blockers and calcium channel inhibitors. Selenite-induced cataract (see above) is prevented by propolis, diclofenac, vitamin C and quercetin by 70, 60, 58, and 40%, respectively, whereas GBE has surprisingly no effect in this study.57 Likewise, death of glioma cells was prevented if apoptosis was induced by hydrogen peroxide but not if it was induced by the lipid-lowering drug, simvastatin, indicating different signaling pathways of these different apoptosis inducers.58

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Age-related shortcomings such as ultrastructural changes of mitochondria in Muller (retinal) glial cells, accompanied with an increase of intrinsic glutathione, can be attenuated by GBE-feeding of the experimental animals.59 Other beneficial effects of GBE reported very recently are the protection from radiation-induced cataract60 or combined carrageenan-gamma radiation and acute inflammation61 or LPS-induced inflammation both in vitro and in vivo.62 As already mentioned above, amelioration of glutamate-induced neurotoxicity by GBE can also be shown in cultured retinal neurons thus definitely extending its importance63 for ‘‘extensions of the brain.’’ An overview on natural therapies on ocular disorders was presented by Head64 where especially regulatory functions of GBE were emphasized and by Christen and Maixent.65 The conclusion was that increased circulation to the optic nerve and antioxidative functions help to prevent, and potentially also to cure, cataracts and glaucoma. Ischemic organs such as hearts after reperfusion showed much better performance if the animals were fed with 50 or 100 mg GBE before the experiment: especially the contractile function after global ischemia was strongly improved.66,67 Another study on cardioprotective effects, where GBE was compared with ginkgolides A and B and also bilobalide used hemodynamic properties and EPR spectroscopy as analytical tools. Anti-ischemic effects were observed after repeated feeding of either GBE (15 d 60mg/kg orally) or ginkgolide A (15 d 4mg/kg orally) as compared to placebo. CONCLUSION The goal, as in atherosclerosis and heart diseases, is to combine ‘‘safe’’ drugs (herbal extracts) with supplemented nutrition (‘‘novel food’’, ‘‘nutriceuticals’’, ‘‘functional food’’) in order to yield preventive protection. Two books addressing and perfectly summarizing these subjects should be mentioned in this context: the book comprising aspects of parmacognosy by Bruneton68 and that on functional food by Wildman.69 Both treatises discuss their respective fields exhaustively, not avoiding critical aspects. In a recent review70 another very important new field is addressed: ‘‘Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants’’ open new visions and promise interesting future research areas. Recent developments of this rapidly growing research area of utmost medical and commercial importance are critically discussed, with respect to environmental concerns. One of the authors principle issues is that ‘‘plant derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals’’. May be in many, or most cases! There is almost nothing to add: this issue is close to our own concern and research field if it is carefully integrated into classical procedures and treatments.

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REFERENCES 1. Schempp H, Hippeli S, Elstner EF. Plant stress: avoidance, adaptation, defense. In: Hock B, Elstner EF, eds. Plant Toxicology. 4th ed. New York: Marcel Dekker, 2005:87–129. 2. Sies H, ed. Oxidative Stress—Oxidants and Antioxidants. London: Academic Press, 1991. 3. Sies H, ed. Oxidative stress. Biol Chem 2002; 383:343–715. 4. Augustin AJ, ed. Nutrition and the Eye. Basic and Clinical Research. In: W. Behrens-Baumann, series ed. Developments in Ophthalmology, vol. 38, Basel: Karger, 2005. 5. Schempp H, Elstner EF. Induction of cataract formation by redox processes. In: Eyer P, ed. Metabolic Aspects of Cell Toxicity. Pharmacology and Toxicology, vol 4. Mannheim: BI-Wissenschaftsverlag, 1994:31–50. 6. Holekamp NM, Shui YB, Beebe DC. Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation. Am J Ophthalmol 2005; 139:302–310. 7. Thiagarajan G, Shirao E, Ando K, et al. Role of xanthurenic acid 8-O-beta-Dglucoside, a novel fluorophore that accumulates in the brunescent human eye lens. Photochem Photobiol 2002; 76:368–372. 8. Wright A, Bubb WA, Hawkins CL, et al. Singlet oxygen-mediated protein oxidation: evidence for the formation of reactive side chain peroxides on tyrosine residues. Photochem Photobiol 2002; 76:35–46. 9. Linetsky M, James HL, Ortwerth BJ. Spontaneous generation of superoxide anion by human lens proteins and by calf lens proteins ascorbylated in vitro. Exp Eye Res 1999; 69:239–248. 10. Ma W, Li D, Sun F, et al. The effect of stress withdrawal on gene expression and certain biochemical and cell biological properties of peroxide-conditioned cell lines. FASEB J 2004; 18:480–488. 11. Martynkina LP, Qian W, Shichi H. Naphthoquinone cataract in mice: mitochondrial change and protection by superoxide dismutase. J Ocul Pharmacol Ther 2002; 18:231–239. 12. Kise K, Kosaka H, Nakabayashi M, et al. Reactive oxygen species involved in phenazine-methosulfate-induced rat lens opacification. An experimental model of cataract. Ophthalmic Res 1994; 26:41–50. 13. Jain R, Freund HG, Budzinsky E, et al. Radiation-induced formation of 3,4-dihydroxyphenylalanine in tyrosine-containing peptides and proteins as a function of X-irradiation dose. Bioconjug Chem 1997; 8:173–178. 14. Babizhayev MA, Costa EB. Lipid peroxide and reactive oxygen species generating systems of the crystalline lens. Biochim Biophys Acta 1994; 1225:326–337. 15. Choudhary S, Xiao T, Vergara LA, et al. Role of aldehyde dehydrogenase in the defense of rat lens and human lens epithelial cells against oxidative stress. Invest Ophthal Vis Sci 2005; 46:259–267. 16. Heinisch HH, Hippeli S, Elstner EF. Biochemical test reactions for the evaluation of the potential anticataractic function of iodide. In: Hockwin O, Sasaki K, Leske MC, eds. Risk Factors for Cataract Development. Developments in Ophthalmol, vol. 17. Basel: Karger, 1989:132–137.

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17. Schempp H, Elstner EF. Oxidative damage of eye tissue and protection by thioctic acid. In: Pleyer U, Schmidt KH, Thiel H-J, eds. Cell and Tissue Protection in Ophthalmology. Stuttgart: Hippokrates Verlag, 1995:101–114. 18. Elstner EF, Adamczyk R, Furch A, et al. Biochemical model reactions for cataract research. Ophthalmic Res 1985; 17:302–307. 19. Kro¨ner R, Elstner EF. Model systems for testing anticataractic activities in rabbit eyes. In: Hockwin O, Sasaki K, Leske MC, eds. Risk Factors for Cataract Development. Developments in Ophthalmol, vol. 17. Basel: Karger, 1989:138–144. 20. Schempp H, Elstner EF. Free radicals in the pathogenesis of ocular diseases. In: Haefliger IO, Flammer J, eds. Nitric Oxide and Endothelin in the Pathogenesis of Glaucoma. Philadelphia: Lippincott-Raven Publishers, 1998:112–135. 21. Varma SD, Devamanoharan PS, Ali AH. Oxygen Radicals in the pathogenesis of cataracts—possibilities for therapeutic intervention. In: Taylor A, ed. Nutritional and Environmental Influences on the Eye. Boca Raton: CRC Press, 1999:53–93. 22. Spector A, Ma W, Wang RR, et al. Microperoxidases catalytically degrade reactive oxygen species and may be anti-cataract agents. Exp Eye Res 1997; 65: 457–470. 23. Buettner GR. The pecking order of free radicals and antioxidants: lipid peroxidation, a-tocopherol and ascorbate. Arch Biochem Biophys 1993; 300:535–543. 24. Kontush A, Hubner C, Finckh B, et al. Antioxidative activity of ubiquinol-10 at physiologic concentrations in human low density lipoprotein. Biochim Biophys Acta 1995; 1258:177–187. 25. Schneider D, Elstner EF. Coenzyme Q10, vitamin E, and dihydrothioctic acid cooperatively prevent diene conjugation in isolated low-density lipoprotein. Antioxid Redox Signal 2000; 2:327–333. 26. Thomas SR, Neuzil J, Stocker R. Cosupplementation with coenzyme Q prevents the prooxidant effect of a-tocopherol and increases the resistance of LDL to transition metal-dependent oxidation initiation. Arterioscler Thromb Vasc Biol 1996; 16: 687–696. 27. Decker EA. The role of phenolics, conjugated linoleic acid, carnosine and pyrroloquinoline quinone as nonessential dietary antioxidants. Nutr Rev 1995; 53:49–58. 28. Stadler RH, Markovic J, Turesky RJ. In vitro anti- and pro-oxidative effects of natural polyphenols. Biol Trace Elem Res 1995; 47:299–305. 29. Gotoh N, Noguchi N, Tsuchiya J, et al. Inhibition of oxidation of low density lipoprotein by vitamin E and related compounds. Free Radic Res 1996; 24:123–124. 30. Fiorani M, De Sanctis R, Menghinello P, et al. Quercetin prevents glutathion depletion by dehydroascorbic acid in rabbit red blood cells. Free Radic Res 2001; 34:639–648. 31. Exner M, Hermann M, Hofbauer R, et al. Genistein prevents the glucose autoxidation mediated atherogenic modification of low density lipoprotein. Free Radic Res 2001; 34:101–112. 32. Osakabe N, Baba S, Yasuda A, et al. Daily cocoa intake reduces the susceptibility of low-density lipoprotein to oxidation as demonstrated in healthy human volunteers. Free Radical Res 2001; 34:93–99. 33. Fuhrman B, Volkova N, Coleman R, et al. Grape powder polyphenols attenuate atherosclerosis development in apolipoprotein E deficient (E0) mice and reduce macrophage atherogenicity. J Nutr 2005; 135:722–728.

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34. Zhao W, Devamanoharan PS, Varma SD. Fructose induced deactivation of antioxidant enzymes: preventive effect of pyruvate. Free Radic Res 2000; 33:23–30. 35. Janisch KM, Milde J, Schempp H, et al. Vitamin C, vitamin E and flavonoids. In: Augustin AJ, ed. Nutrition and the Eye. Basic and Clinical Research. Developments in Ophthalmology, vol. 38. Basel: Karger, 2005:59–69. 36. Grassmann J, Hippeli S, Elstner EF. Plant’s defence and its benefits for animal and medicine: role of phenolics and terpenoids in avoiding oxygen stress. Plant Physiol Biochem 2002; 40:471–478. 37. Grassmann J, Schneider D, Weiser D, et al. Antioxidative effects of lemon oil and its components on copper induced oxidation of low density lipoprotein. Drug Res 2001; 51:799–805. 38. Behl C. Apoptosis and Alzheimer’s disease. J Neural Transm 2000; 107:1325–1344. 39. Kobayashi MS, Han D, Packer L. Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity. Free Radic Res 2000; 32: 115–124. 40. Diamond BJ, Shiflett SC, Feiwel N, et al. Ginkgo biloba extract: mechanisms and clinical indications. Arch Phys Med Rehabil 2000; 81:668–678. 41. Thiagarajan G, Chandani S, Hrinarayana Rao S, et al. Molecular and cellular assessment of ginkgo biloba extract as possible ophthalmic drug. Exp Eye Res 2002; 75:421–430. 42. Thiagarajan G, Venu T, Balasubramanian D. Approaches to relieve the burden of cararact blindness through natural antioxidants: use of Ashwagandha (Withania somnifera). Curr Sci India 2003; 85:1065–1069. 43. Denke A, Schneider W, Elstner EF. Biochemical activities of extracts from Hypericum perforatum L—2nd Communication: inhibition of metenkephaline- and tyrosine-dimerization. Drug Res 1999; 49:109–114. 44. Denke A, Schempp H, Weiser D, et al. Biochemical activities of extracts from Hypericum perforatum L.—5th communication: dopamine-ß-hydroxylase-product quantification by HPLC and inhibition by hypericins and flavonoids. Drug Res 2000; 50:415–419. 45. Mene P, Festuccia F, Pugliese F. Clinical potential of advanced glycation endproduct inhibitors in diabetes mellitus. Am J Cardiovas Drugs 2003; 3:315–320. 46. Khalifah RG, Baynes JW, Hudson BG. Amadorins: novel post-Amadori inhibitors of advanced glycation reactions. Biochem Biophys Res Commun 1999; 257: 251–258. 47. Kutlu M, Naziroglu M, Simsek H, et al. Moderate exercise combined with dietary vitamins C and E counteracts oxidative stress in the kidney and lens of streptozotocin-induced diabetic rat. Int J Vitam Nutr Res 2005; 75:71–80. 48. Seif El Nasr M, El Fattah AA. Lipid peroxide, phospholipids, glutathione levels and superoxide dismutase activity in rat brain after ischaemia: effect of ginkgo biloba extract. Pharmacol Res 1995; 32:273–278. 49. Logani S, Chen MC, Tran T, et al. Actions of Ginkgo biloba related to potential utility for the treatment of conditions involving cerebral hypoxia. Life Sci 2000; 67:1389–1396. 50. Oyama Y, Fuchs PA, Katayama N, et al. Myricetin and quercetin, the flavonoid constituents of Ginkgo biloba extract, greatly reduce oxidative metabolism in both resting and Ca2+-loaded brain neurons. Brain Res 1994; 635:125–129.

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51. Koc RK, Akdemir H, Kurtsoy A, et al. Lipid peroxidation in experimental spinal cord injury. Comparison of treatment with Ginkgo biloba, TRH and methylprednisolone. Res Exp Med 1995; 195:117–123. 52. Sun BL, Xia ZL, Yang MF, et al. Effects of Ginkgo biloba extract on somatosensory evoked potential, nitric oxide levels in serum and brain tissue in rats with cerebral vasospasm after subarachnoid hemorrhage. Clin Hemorheol Microcirc 2000; 23:139–144. 53. Weber M, Dietrich D, Grasel I, et al. 6-Hydroxykynurenic acid and kynurenic acid differently antagonise AMPA and NMDA receptors in hippocampal neurones. J Neurochem 2001; 77:1108–1115. 54. Ahlemeyer B, Mowes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents. Eur J Pharmacol 1999; 367:423–430. 55. Riedel WJ, Jorissen BL. Nutrients, age and cognitive function. Curr Opin Clin Nutr Metab Care 1998; 1:579–585. 56. Ritch R. Potential role for Ginkgo biloba extract in the treatment of glaucoma. Med Hypotheses 2000; 54:221–235. 57. Orhan H, Marol S, Hepsen IF, et al. Effects of some probable antioxidants on selenite-induced cataract formation and oxidative stress-related parameters in rats. Toxicology 1999; 139:219–232. 58. Altiok N, Ersoz M, Karpuz V, et al. Ginkgo biloba extract regulates differentially the cell death induced by hydrogen peroxide and simvastatin. Neurotoxicology 2006; 27:158–163. 59. Paasche G, Gartner U, Germer A, et al. Mitochondria of retinal Muller (glial) cells: the effects of aging and of application of free radical scavengers. Ophthalmic Res 2000; 32:229–236. 60. Ertekin MV, Kocer I, Karslioglu I, et al. Effects of oral Ginkgo biloba supplementation on cataract formation and oxidative stress occurring in lenses of rats exposed to total cranium radiotherapy. Jpn J Ophthalmol 2004; 48:499–502. 61. Hedayat I, Salam OM, Baioumy AR. Effect of Ginkgo biloba extract on carrageenan-induced acute local inflammation in gamma irradiated rats. Pharmazie 2005; 60:614–619. 62. Ilieva I, Ohgami K, Shiratori K, et al. The effect of Ginkgo biloba extract on lipopolysaccharide-induced inflammation in vitro and in vivo. Exp Eye Res 2004; 79:181–187. 63. Wang YS, Xu L, Ma K, et al. Protective effects of Ginkgo biloba extract 761 against glutamate-induced neurotoxicity in cultured retinal neuron. Chin Med J (Engl) 2005; 118:948–952. 64. Head KA. Natural therapies for ocular disorders, part two: cataracts and glaucoma. Altern Med Rev 2001; 6:141–166. 65. Christen Y, Maixent JM. What is Ginkgo biloba extract EGb 761? An overview— from molecular biology to clinical medicine. Cell Mol Biol 2002; 48:601–611. 66. Tosaki A, Engelman DT, Pali T, et al. Gingko biloba extract (EGb 761) improves postischemic function in isolated preconditioned working rat hearts. Coron Artery Dis 1994; 5:443–450. 67. Tosaki A, Pali T, Droy-Lefaix MT. Effects of Ginkgo biloba extract and preconditioning on the diabetic rat myocardium. Diabetologia 1996; 39:1255–1262.

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68. Bruneton J. Pharmacognosy, Phytochemistry, Medicinal Plants. Paris: InterceptLavoisier, 1999. 69. Wildman REC, ed. Handbook of Nutraceuticals and Functional Foods. Boca Raton: CRC Press, 2001. 70. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 2001; 6:219–226.

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8 Nitric Oxide in Experimental Autoimmune Uveoretinitis Janet Liversidge Department of Ophthalmology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, U.K.

Sharon Gordon Human Resources Development and Training, University Office, King’s College, Aberdeen, U.K.

Andrew D. Dick Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K.

Morag J. Robertson Department of Ophthalmology, University of Aberdeen, Aberdeen, U.K.

Ross Buchan Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, U.S.A.

INTRODUCTION During inflammation, and depending upon cytokine microenvironment, tissue resident and infiltrating macrophages can undergo polarisation towards a classically activated phenotype (IFN-g, TNF, or LPS) or towards an alternatively activated phenotype (IL-4, IL-10, TGF-b or PGE2). Classically activated macrophages drive increased intensity of inflammation associated with Th1 driven cellular responses

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and nitric oxide driven tissue damage whilst alternatively activated macrophages mediate Th2 cell differentiation, tolerance induction, down regulation of inflammation and healing. These opposing functional effects are controlled by cytokine or other polarising factors. Driving the balance towards an alternatively activated, healing phenotype is crucial to re-establish tissue homeostasis and disease remission. We have used rodent models to explore the role of macrophages in experimental autoimmune uveoretinitis (EAU), and how their function might be manipulated to limit retinal damage. In the normal CNS and retina, tissue resident macrophages and myeloid cells appear to be polarised towards alternatively activated phenotype, and this polarisation appears irreversible, providing a regulatory mechanism within the tissue that is over-ridden during autoimmune inflammation. Infiltrating classically activated monocyte-macrophages are essential for full expression of disease and our histological and trafficking experiments indicate that they are amongst the first cells to infiltrate the retina and may be the key cells initiating blood retina barrier breakdown. Infiltrating macrophages that are reactivated locally by T cell derived cytokines are also primary effectors of photoreceptor damage through nitric oxide and super-oxide generation but show greater resistance to apoptosis during EAU than would otherwise expected under normal inflammatory conditions, due to expression of a caspase 8 inhibitory molecule, FLIP. Down regulation of these classically activated macrophages through altering the cytokine microenvironment is key to controlling inflammation. PATHOLOGY OF NITRIC OXIDE IN EAU Effects of Nitric Oxide on Immune Function It is now understood that all known isoforms of NO synthase catalyse the same reaction and all operate within the immune system. Neuronal NO synthase (nNOS or NOS1) and endothelial NO synthase (eNOS or NOS3) are constitutively expressed and regulated by Ca2+ flux and need not necessarily be expressed only by neurons and endothelium within the retina, but it is inducible NO synthase (iNOS or NOS2) that is frequently implicated in the inflammatory immune response.1,2 A hypothesis emerged that the constitutive forms of NO synthase were critical to normal physiology and their inhibition caused damage whist, induction of inducible NO synthase could be harmful (Figure 1). The generation of NOS2 deficient mice was supposed to provide an insight into the role of NOS2 in normal physiology and inflammation, but conflicting or contradictory results in various models raised even more questions than answers.3–5 In addition to well-described toxic effects, NOS2 has subsequently been shown to have multiple biological effects, including normal healing, regulation of T cell proliferation and differentiation.6 Considering that many of the targets of NO are themselves regulatory molecules (for example, transcription factors and components of various signalling cascades) it is evident that NO frequently exerts diverse phenotypic effects.7 NO mediatedstress will alter gene expression patterns, and the number of genes known to be involved is increasing. In addition, NO can act as powerful inducer of apoptosis or necrosis in some cells, it may also provide equally powerful protection from cell

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Figure 1 Biological Effects of NO molecules. (i) NO may bind directly to the iron of a protein heme group in a reversible manner and exert signalling function. (ii) At higher concentrations, NO will react with O2 forming reactive nitrogen oxide intermediates that may S-nitrosate thiols. Under conditions of simultaneous oxidative and NO-mediated stress, NO may react with O2  to yield the unstable strong oxidant peroxynitrite anion, which can nitrate tyrosine residues. The stable NO oxidation product nitrite in the presence of peroxidases and H2O2 also leads to tyrosine nitration (adapted from8).

death in other situations. These effects may be in part due to differences in a cells capacity to cope with the stress of NO exposure.8 Within the immune system, many cells are capable of generating NO. Relevant to the eye these include microglia, dendritic cells, monocyte macrophages, granulocytes including mast cells, neutrophils and eosinophils.9 The expression of NOS2 is also regulated by cytokines often immune system derived. Cytokines such as IL-1, IFN-g and TNF-a activate the NOS2 gene promoter via transcription factors such as NF-kB and AP-1,10,11 but equally, type 1 interferons can inhibit NOS2 transcription.12 TGF-b post-transcriptionally regulates the production of NOS2 through enhanced degradation13 and IL-4 inhibits gene expression and NO production via a different pathway.14 Another factor that determines NOS activity is the availability of its substrate arginine, and that is regulated enzymatically by production of arginase. In macrophages and dendritic cells, Th2 cytokines and TGF–b strongly increase arginase activity thus limiting availability of arginine,14,15 and preventing the induction of NOS2 by subsequent exposure to IFN-g plus TNF-a. Regulation of NOS2 can also be mediated by cell-cell contact, and uptake of apoptotic (but not necrotic) lymphocytes by macrophages down regulates expression and at the same time shifts arginine metabolism towards the arginase pathway.16 Approximately 200 genes, including genes related to inflammation, infection and apoptosis are subject to regulation by NO,17 illustrating the complexity of NO induction and regulation. Protective and toxic effects frequently seen in parallel are reviewed by Bogdan,9 and are summarized in Table 1. POTENTIAL CELL SOURCES OF NITRIC OXIDE IN EAU Together with other inflammatory mediators, NO is known to be involved in the induction of ocular inflammation.18–20 In experimental autoimmune uveoretinitis (EAU), the inflammation is characterised by a breakdown of the blood retina

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Table 1 No Effects on Immune Function l l

Non specific cytotoxicity towards microbes Immunopathology*apoptosis, extracellular matrix effects Inflammation Necrosis or fibrosis of parenchyma Anti-inflammatory/immunosuppressive T and B lymphocyte proliferation *apoptosis Antibodies*disruption of signalling Leukocyte recruitment *adhesion, chemokines Immune regulation Cytokine, chemokine and growth factor modulation. (signalling cascades, transcription factors, mRNA stability) T helper cell deviation Regulation of Th1/Th2 immune responses. IL-12 regulation? ~

l

!

l

~

!

l

~

!

barrier, primarily at the post-capillary venules21 and at the retinal pigment epithelium (RPE).22,23 The disease is induced in animal models by immunisation with various retinal antigens.24,25 In acute disease an increase in vascular permeability together with fibrin exudation is associated with polymorphonuclear neutrophils and affects the anterior as well as the posterior chamber of the eye, in addition to the monocyte macrophages and T lymphocytes that characterise the delayed type hypersensitivity response in the retina in more moderate disease.22,25,26 The cytokine response associated with the inflammation represents mainly an elevation of Th1 type cytokines, such as IFN-g, TNF-a and IL-2 and other generally proinflammatory cytokines such as IL-1b and IL-6.27,28 Although NOS2 is not expressed within the normal retina and choroid, it is not surprising therefore that NOS2 is induced within the eye during inflammation.29–31 Potential cell sources of NO in EAU are tissue resident cells and inflammatory cells. Tissue resident cells include the photoreceptors and these have recently been reported to express NOS2 very early in disease and before inflammatory infiltrates are evident.32 The possible implications of this are discussed more fully in another Chapter of this volume. Expression of NOS2 in vivo by microglia and Mu¨ller glia (astroglia) is well described.33 In Mu¨ller cells NOS2 is associated with neurotoxicity,34 whilst microglia expressed NOS2 may be regulatory.20,35,36 Vascular endothelium forms the inner blood retina barrier and together with perivascular pericytes also expresses NOS early in disease37 but whether this plays any role in leukocyte recruitment to the CNS is less clear.38,39 In contrast, the RPE that forms the outer blood retina barrier does not appear to express NOS2 in vivo.30,31 In common with the cell types mentioned above, cultured RPE cells do express NOS2 and produce high levels of nitrite when stimulated with cytokines,40–44 but there is no clear evidence that they can

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Figure 2 A. The RPE layer in vivo (arrow head) is NOS2 negative, whilst infiltrate (arrowed) is NOS2 positive. B. RT-PCR for cytokine expression by RPE cultured in vitro with activated T lymphocytes.

produce NO in vivo. Indeed the evidence is that RPE may, in fact be protective as co-culture with T cells or cross-linking of CD2 ligands on RPE cells induces PGE2 secretion and TGF-b expression (Figure 2).40,45,46 Inflammatory cells appear to be the major sources of NOS2 within the eye in EAU. In hyperacute uveitis models such as endotoxin induced uveitis (EIU), neutrophils as well as monocytes express high levels of NOS2,47–49 but in the posterior chamber in EAU, infiltrating monocytes are the principal inflammatory cell expressing NOS2 and the major cause of tissue damage.30,31,36,50 The role of NOS2 expression by leukocytes in EAU is discussed more fully in the following section. PATHOGENESIS OF NITRIC OXIDE INDUCTION IN EAU Nitric oxide production is an important aspect of the innate response to microbial or parasitic infection,51,52 and deleterious effects of NOS2 expression in inflammatory settings involving endotoxin induced shock or haemorrhage and resuscitation are well recognised.4 However in macrophage driven inflammation, including autoimmunity, there is evidence for both beneficial, and deleterious effects. This is highlighted by the contradictory evidence from EAU models using NOS2 KO mice or inhibitors of nitric oxide synthase. Protective effects could be demonstrated using NOS2 deficient mice53,54 or with a NOS2 inhibitor.55 On the other hand, also using a NOS2 KO model, NO donors or inhibitors of NOS, a pathogenic role for NO could also be demonstrated by others.29,30,56–58 The molecular basis for these contradictory results has been extensively reviewed.1,8,9 The most relevant mechanisms for autoimmune inflammation such as EAU would appear to involve cytotoxic effects leading to apoptosis or necrosis of local tissue cells as NO is a key stimulus for DNA damage and p53 activation. This occurs particularly in the presence of superoxide that drives formation of the strong oxidant peroxynitrite anion that nitrates tyrosine residues. In addition, although stable, nitrite can also lead to tyrosine nitration in the presence of peroxidases and H2O2 (Figure 1). On the other hand, in smaller quantities, NO appears to be regulatory, particularly with respect to T cell growth and differentiation. The effect of NO can be profound, suppressing T cell

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proliferation in response to mitogens as well as inhibiting antigen specific T cell expansion.40,59 Nitric oxide production is regulated by two enzymes. Nitric oxide synthase generates NO, and arginase, an enzyme that limits the availability of arginine, a substrate for NO production as well as T cell growth. NOS2 can also interfere with T cell growth through blocking the phosphorylation of signalling molecules required for IL-2 receptor signalling.60 Cytokines that induce NOS2 are produced predominantly by Th1 cells, therefore NO can also control Th1 cell responses by providing a negative feedback regulator of autoimmune Th1 driven autoimmune responses.5 Equally, Th2 cytokines up-regulate arginase, limiting the availability of arginine, the substrate for NO production, thus reducing NO production. Where NO levels are damaging, as in early EAU, then this will clearly be protective. When both enzymes are produced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T cell apoptosis providing an additional regulatory mechanism during inflammation.61–64 Further regulation of immune responses driven by NO release is through the immunosuppressive cytokine TGF-b. This is affected via three routes, decreased stability and translation of NOS2 mRNA, and increased degradation of NOS2 protein.65 Macrophage cytotoxic activity is also reduced by T helper 2 cytokines IL-10 and IL-4 that can synergise with TGF-b to limit tissue damage.66 Thus in Th1 driven organ specific autoimmune diseases such uveitis, NO production is part of a natural, negative feedback mechanism designed to limit inflammatory damage and promote healing. Such a complex role for this molecule explains much of the conflicting data found in models eliminating NOS activity, either by gene manipulation of specific inhibitors, or models providing NO donors. In the retina, nitric oxide clearly has physiological functions.67 Neuronal NOS1 may be responsible for producing NO in photoreceptors and bipolar cells and may be required for stimulus of guanylate cyclase in photoreceptor rod cells increasing calcium channel currents as inhibition of NOS is known to impair phototransduction. Endothelial NOS3 is required to maintain vascular tone and inducible NOS2 in RPE and Mu¨ller cells may be required for phagocytosis of ROS. In EAU, the NOS inhibitors aminoguanidine and L-NAME cause enhanced rolling of leukocytes on vascular endothelium, but decreases firm adhesion and inhibits overall leukocyte infiltration, indicating that during inflammation, NOS may contribute to the pro-inflammatory response.39 We have also found that L-NAME is protective in EAU.30,68 L-arginine was found to enhance IFN-g and exacerbate retinal inflammation, whereas L-NAME significantly reduced NOS2 expression and severity of tissue damage via an IFN-g dependent mechanism.30 The principle source of NOS2 expression was found to be infiltrating monocytes in the target organ, whilst tissue resident macrophages in the choroid, and RPE cells did not express the enzyme. It was also evident that monocyte NOS2 expression peaked early in the inflammatory process, subsiding after peak disease despite increasing infiltrations of monocytes in later stages of the inflammation (Figure 3).

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Figure 3 Infiltration of NOS2+ monocytes coincides with peak disease, but NOS2 monocytes continue to accumulate within tissue in later stages. Serial sections of rat retina were immuno-stained and then percentage of retina positive for NOS2 or ED1 monocyte marker were determined using computer assisted densitometric scanning, n–6.

Further analysis of the mechanisms of NO tissue damage revealed that tissue damage correlated with peroxynitrite formation within monocytes in the outer retina, together with extensive photoreceptor apoptosis and apoptosis of Fas+ T cells within the retina. However the monocytes, despite showing evidence of lipid peroxydation remained resistant to apoptosis. The protective effect of L-NAME could be attributed to dramatically reduced photoreceptor damage, absence of nitrotyrosine formation and overall reduced NOS2 protein expression. However, as T cell apoptosis was also reduced, accumulations of these cells was increased despite continued expression of FAS and Fas ligand indicating that normal regulation of T cells within the inflammatory lesion via activation induced cell death was compromised.57 THERAPEUTIC STRATEGIES TO REDUCE NITRIC OXIDE INDUCED TISSUE DAMAGE From the preceding sections it is clear that inhibiting NO production during retinal inflammation may not have purely beneficial effects. Our work, and that of others show that inhibition of NOS can inhibit or exacerbate inflammation depending upon the model. Even specific inhibitors of NOS2 were not protective.55 Other approaches to target monocyte cytotoxicity may perhaps be more effective. Nitric oxide is a product of classically activated monocytes. The cytokines IFN-g and TNF-a induce NOS2 activity in monocytes, but targeting IFN-g, a stimulator of classical activation, has not proved effective in controlling EAU.54,69 It is now known that IFN-g (and IL-4) inhibit IL-23 dependent IL-17 production,70 and as IL-23 is the major cytokine driving neural inflammation in EAE71 this result is perhaps less surprising. Targeting TNF-a has been more successful,20,50 and is now used therapeutically to control intractable uveitis in the clinic with some success.72,73

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We have also looked at the mechanisms controlling monocyte/macrophage resistance to apoptosis within the inflamed retina. Monocyte/macrophages within the retina appear to exert a double role in inflammation, producing tissue damaging NO during early stages, but also having a role in resolution of inflammation and tissue healing.50,78 If monocyte apoptosis could be induced early in disease it may be possible to prevent early photoreceptor damage. Using flow cytometry we isolated monocytes from uveitic retina and found that up to 30% of monocytes expressed the FLICE-inhibitory protein (FLIP). This protein inhibits caspase 8 activation and prevents caspase 3 cleavage that leads to apoptosis.74 To test whether FLIP expression in monocytes could be targeted therapeutically, we challenged cultured monocytes with IFN-g and TNF-a and treated them with either L-NAME, PKC inhibitors or an anti-TNF receptor fusion protein.50 Whilst L-NAME had no effect on FLIP expression, both the PKC inhibitor and the TNF-fusion protein reduced FLIP expression by up to 50%. This approach has yet to be tested in vivo, but may provide a clue to the efficacy of anti-TNF therapies. CONCLUSION In EAU we may hypothesise that early infiltrates of monocytes are classically activated by IFN-g and TNF-a produced by T cells being reactivated by retinal antigen by local or infiltrating APC.25,75 This induces NOS2 in infiltrating monocytes as well as susceptible tissue resident cells and the production of reactive oxygen species leads to activation induced cell death of the T cells. Uptake of apoptotic T cells by APC is known to induce IL-10 production that will in turn have an immuno-regulatory effect on the immune response reducing inflammation and driving alternative activation of monocytes towards a healing phenotype.76–78 As IL-10 and IL-4 produced by alternatively activated macrophages synergise with TGF-b, known to be present within ocular tissues, NOS2 expression is rapidly down regulated as the inflammation progresses. Thus down regulation of these classically activated monocyte/macrophages through altering the cytokine balance, or through manipulation of specific receptor agonists or antagonists will be the key to controlling ocular inflammation. REFERENCES 1. Kroncke KD, Fehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities. Biol Chem Hoppe Seyler 1995; 376:327–343. 2. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43:109–142. 3. MacMicking JD, Nathan C, Hom G, et al. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 1995; 81:641–650.

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4. Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest 1997; 100:2417–2423. 5. Bogdan C. The multiplex function of nitric oxide in (auto)immunity. J Exp Med 1998; 187:1361–1365. 6. Kubes P. Inducible nitric oxide synthase: a little bit of good in all of us. Gut 2000; 47:6–9. 7. Kroncke KD, Fehsel K, Kolb-Bachofen V. Nitric oxide: cytotoxicity versus cytoprotection—how, why, when, and where? Nitric Oxide 1997; 1:107–120. 8. Kroncke KD, Fehsel K, Suschek C, et al. Inducible nitric oxide synthase-derived nitric oxide in gene regulation, cell death and cell survival. Int Immunopharmacol 2001; 1:1407–1420. 9. Bogdan C. Nitric oxide and the immune response. Nat Immunol 2001; 2:907–916. 10. MacMicking JD, North RJ, LaCourse R, et al. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci U S A 1997; 94:5243–5248. 11. Kleinert H, Wallerath T, Fritz G, et al. Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-kappaB-signaling pathways. Br J Pharmacol 1998; 125:193–201. 12. Faure V, Courtois Y, Goureau O. Inhibition of inducible nitric oxide synthase expression by interferons alpha and beta in bovine retinal pigmented epithelial cells. J Biol Chem 1997; 272:32169–32175. 13. Mitani T, Terashima M, Yoshimura H, et al. TGF-beta1 enhances degradation of IFN-gamma-induced iNOS protein via proteasomes in RAW 264.7 cells. Nitric Oxide 2005; 13:78–87. 14. Bogdan C, Vodovotz Y, Paik J, et al. Mechanism of suppression of nitric oxide synthase expression by interleukin-4 in primary mouse macrophages. J Leukoc Biol 1994; 55:227–233. 15. Gotoh T, Mori M, Arginase II downregulates nitric oxide (NO) production and prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells. J Cell Biol 1999; 144:427–434. 16. Freire-de-Lima CG, Nascimento DO, Soares MB, et al. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature 2000; 403:199–203. 17. Zamora R, Vodovotz Y, Aulak KS, et al. A DNA microarray study of nitric oxideinduced genes in mouse hepatocytes: implications for hepatic heme oxygenase-1 expression in ischemia/reperfusion. Nitric Oxide 2002; 7:165–186. 18. Smith JR, Hart PH, Williams KA. Basic pathogenic mechanisms operating in experimental models of acute anterior uveitis. Immunol Cell Biol 1998; 76: 497–512. 19. Forrester JV. Uveitis: pathogenesis. Lancet 1991; 338:1498–1501. 20. Dick AD, Carter D, Robertson M, et al. Control of myeloid activity during retinal inflammation. J Leukoc Biol 2003; 74:161–166. 21. Xu H, Forrester JV, Liversidge J, et al. Leukocyte trafficking in experimental autoimmune uveitis: breakdown of blood-retinal barrier and upregulation of cellular adhesion molecules. Invest Ophthalmol Vis Sci 2003; 44:226–234. 22. Liversidge J, Forrester JV. Experimental autoimmune uveitis (EAU): immunophenotypic analysis of inflammatory cells in chorioretinal lesions. Curr Eye Res 1988; 7:1231–1241.

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23. Bamforth SD, Lightman S, Greenwood J. The effect of TNF-alpha and IL-6 on the permeability of the rat blood-retinal barrier in vivo. Acta Neuropathol (Berl) 1996; 91:624–632. 24. Nussenblatt RB, Gery I. Experimental autoimmune uveitis and its relationship to clinical ocular inflammatory disease. J Autoimmun 1996; 9:575–585. 25. Caspi RR. Immune mechanisms in uveitis. Springer Semin Immunopathol 1999; 21:113–124. 26. Kuppner MC, Liversidge J, McKillop-Smith S, et al. Adhesion molecule expression in acute and fibrotic sympathetic ophthalmia. Curr Eye Res 1993; 12:923–934. 27. de Vos AF, Hoekzema R, Kijlstra A. Cytokines and uveitis, a review. Curr Eye Res 1992; 11:581–597. 28. de Vos AF, Klaren VN, Kijlstra A. Expression of multiple cytokines and IL-1RA in the uvea and retina during endotoxin-induced uveitis in the rat. Invest Ophthalmol Vis Sci 1994; 35:3873–3883. 29. Goureau O, Bellot J, Thillaye B, et al. Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase. J Immunol 1995; 154:6518–6523. 30. Hoey S, Grabowski PS, Ralston SH, et al. Nitric oxide accelerates the onset and increases the severity of experimental autoimmune uveoretinitis through an IFN-gamma-dependent mechanism. J Immunol 1997; 159:5132–5142. 31. Zhang J, Wu LY, Wu GS, et al. Differential expression of nitric oxide synthase in experimental uveoretinitis. Invest Ophthalmol Vis Sci 1999; 40:1899–1905. 32. Wu GS, Lee TD, Moore RE, et al. Photoreceptor mitochondrial tyrosine nitration in experimental uveitis. Invest Ophthalmol Vis Sci 2005; 46:2271–2281. 33. Goureau O, Hicks D, Courtois Y, et al. Induction and regulation of nitric oxide synthase in retinal Muller glial cells. J Neurochem 1994; 63:310–317. 34. Goureau O, Regnier-Ricard F, Courtois Y. Requirement for nitric oxide in retinal neuronal cell death induced by activated Muller glial cells. J Neurochem 1999; 72:2506–2515. 35. Broderick C, Duncan L, Taylor N, et al. IFN-gamma and LPS-mediated IL-10dependent suppression of retinal microglial activation. Invest Ophthalmol Vis Sci 2000; 41:2613–2622. 36. Robertson MJ, Erwig LP, Liversidge J, et al. Retinal micro-environment determines both resident and infiltrating macrophage function during experimental autoimmune uveoretinitis (EAU). Invest Ophthalmol Vis Sci 2002; 43:2250–2257. 37. Chakravarthy U, Stitt AW, McNally J, et al. Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes. Curr Eye Res 1995; 14:285–294. 38. Hickey MJ, Granger DN, Kubes P. Inducible nitric oxide synthase (iNOS) and regulation of leucocyte/endothelial cell interactions: studies in iNOS-deficient mice. Acta Physiol Scand 2001; 173:119–126. 39. Baatz H, Pleyer U. Modulation of leukocyte-endothelium interaction by nitric oxide synthase inhibitors: effects on leukocyte adhesion in endotoxin-induced uveitis. Inflamm Res 2001; 50:534–543. 40. Liversidge J, Grabowski P, Ralston S, et al. Human retinal pigment epithelial cells produce nitric oxide in the presence of activated T cells. Moncada S, Feelish M, Busse R, Higgs EA, eds. The Biology of Nitric Oxide, vol 4. Cambridge: Portland Press, 1994:378–383.

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41. Liversidge J, Grabowski P, Ralston S, et al. Rat retinal pigment epithelial cells express an inducible form of nitric oxide synthase and produce nitric oxide in response to inflammatory cytokines and activated T cells. Immunology 1994; 83:404–409. 42. Goureau O, Hicks D, Courtois Y. Human retinal pigmented epithelial cells produce nitric oxide in response to cytokines. Biochem Biophys Res Commun 1994; 198:120–126. 43. Goureau O, Amiot F, Dautry F, et al. Control of nitric oxide production by endogenous TNF-alpha in mouse retinal pigmented epithelial and Muller glial cells. Biochem Biophys Res Commun 1997; 240:132–135. 44. Faure V, Courtois Y, Goureau O. Differential regulation of nitric oxide synthase-II mRNA by growth factors in rat, bovine, and human retinal pigmented epithelial cells. Ocul Immunol Inflamm 1999; 7:27–34. 45. Liversidge J, Sewell HF, Forrester JV. Interactions between lymphocytes and cells of the blood-retina barrier: mechanisms of T lymphocyte adhesion to human retinal capillary endothelial cells and retinal pigment epithelial cells in vitro. Immunology 1990; 71:390–396. 46. Liversidge J, McKay D, Mullen G, et al. Retinal pigment epithelial cells modulate lymphocyte function at the blood-retina barrier by autocrine PGE2 and membranebound mechanisms. Cell Immunol 1993; 149:315–330. 47. McMenamin PG, Crewe J. Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body. Invest Ophthalmol Vis Sci 1995; 36:1949–1959. 48. McMenamin PG, Crewe JM. Cellular localisation and dynamics of nitric oxide synthase expression in the rat anterior segment during endotoxin-induced uveitis. Exp Eye Res 1997; 65:157–164. 49. Marie O, Thillaye-Goldenberg B, Naud MC, et al. Inhibition of endotoxin-induced uveitis and potentiation of local TNF-alpha and interleukin-6 mRNA expression by interleukin-13. Invest Ophthalmol Vis Sci 1999; 40:2275–2282. 50. Robertson M, Liversidge J, Forrester JV, et al. Neutralizing tumor necrosis factoralpha activity suppresses activation of infiltrating macrophages in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 2003; 44:3034–3041. 51. Bogdan C, Rollinghoff M, Diefenbach A. The role of nitric oxide in innate immunity. Immunol Rev 2000; 173:17–26. 52. Liew FY, Wei XQ, Proudfoot L. Cytokines and nitric oxide as effector molecules against parasitic infections. Philos Trans R Soc Lond B Biol Sci 1997; 352: 1311–1315. 53. Silver PB, Tarrant TK, Chan CC, et al. Mice deficient in inducible nitric oxide synthase are susceptible to experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 1999; 40:1280–1284. 54. Tarrant TK, Silver PB, Wahlsten JL, et al. Interleukin 12 protects from a T helper type 1-mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon gamma, nitric oxide, and apoptosis. J Exp Med 1999; 189:219–230. 55. Charlotte F, Ito S, Wu G, et al. Highly selective inhibitor of inducible nitric oxide synthase enhances S-antigen-induced uveitis. Curr Eye Res 2003; 26:1–7.

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56. Thillaye-Goldenberg B, Goureau O, Naud MC, et al. Delayed onset and decreased severity of experimental autoimmune uveoretinitis in mice lacking nitric oxide synthase type 2. J Neuroimmunol 2000; 110:31–44. 57. Liversidge J, Dick A, Gordon S. Nitric oxide mediates apoptosis through formation of peroxynitrite and fas/fas-ligand interactions in experimental autoimmune uveitis. Am J Path 2002; 160:905–916. 58. Kwak HJ, Yang YS, Pae HO, et al. Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1dependent immune response. Mol Cells 2001; 12:178–184. 59. Albina JE, Abate JA, Henry W Jr. Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway. J Immunol 1991; 147:144–148. 60. Bronte V, Serafini P, Mazzoni A, et al. L-arginine metabolism in myeloid cells controls T-lymphocyte functions. Trends Immunol 2003; 24:302–306. 61. Munder M, Eichmann K, Moran JM, et al. Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells. J Immunol 1999; 163:3771–3777. 62. Munder M, Eichmann K, Modolell M. Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype. J Immunol 1998; 160:5347–5354. 63. Mills CD. Macrophage arginine metabolism to ornithine/urea or nitric oxide/citrulline: a life or death issue. Crit Rev Immunol 2001; 21:399–425. 64. Mills CD, Kincaid K, Alt JM, et al. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 2000; 164:6166–6173. 65. Vodovotz Y, Bogdan C, Paik J, et al. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta. J Exp Med 1993; 178: 605–613. 66. Oswald IP, Gazzinelli RT, Sher A, et al. IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity. J Immunol 1992; 148:3578–3582. 67. Goldstein IM, Ostwald P, Roth S. Nitric oxide: a review of its role in retinal function and disease. Vision Res 1996; 36:2979–2994. 68. Liversidge J, Dick A, Gordon S. Nitric oxide mediates apoptosis through formation of peroxynitrite and Fas/Fas-ligand interactions in experimental autoimmune uveitis. Am J Pathol 2002; 160:905–916. 69. Jones LS, Rizzo LV, Agarwal RK, et al. IFN-gamma-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response. J Immunol 1997; 158:5997–6005. 70. McKenzie BS, Kastelein RA, Cua DJ. Understanding the IL-23-IL-17 immune pathway. Trends Immunol 2006; 27:17–23. 71. Cua DJ, Sherlock J, Chen Y, et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003; 421: 744–748. 72. Dick AD, Forrester JV, Liversidge J, et al. The role of tumor necrosis factor (TNFalpha) in experimental autoimmune uveoretinitis (EAU). Prog Retin Eye Res 2004; 23:617–637.

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73. Murphy CC, Greiner K, Plskova J, et al. Neutralizing tumor necrosis factor activity leads to remission in patients with refractory noninfectious posterior uveitis. Arch Ophthalmol 2004; 122:845–851. 74. Pope RM. Apoptosis as a therapeutic tool in rheumatoid arthritis. Nat Rev Immunol 2002; 2:527–535. 75. Shimizu K, Wu GS, Sultana C, et al. Stimulation of macrophages by retinal proteins: production of reactive nitrogen and oxygen metabolites. Invest Ophthalmol Vis Sci 1999; 40:3215–3223. 76. Rizzo LV, Xu H, Chan CC, et al. IL-10 has a protective role in experimental autoimmune uveoretinitis. Int Immunol 1998; 10:807–814. 77. Stumpo R, Kauer M, Martin S, et al. Alternative activation of macrophage by IL-10. Pathobiology 1999; 67:245–248. 78. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23–35.

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9 TNF Activation and Nitric Oxide Production in EAU Claudia J. Calder and Lindsay B. Nicholson Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K.

Morag J. Robertson Department of Ophthalmology, University of Aberdeen, Aberdeen, U.K.

Andrew D. Dick Department of Clinical Sciences South Bristol, University of Bristol, Bristol Eye Hospital, Bristol, U.K.

ABSTRACT Retinal destruction during inflammatory responses are mediated by non-specific infiltration of mononuclear cells and polymorphonuclear cells. In particular macrophages which are predominant in the retinal cell infiltrate during disease course of experimental models of automimmune retinal inflammation are adaptable in their behaviour. Cytokine conditioning of macrophage behaviour is well recognised, for example when maximal retinal destruction occurs during experimental autoimmune uveoretinitis, macrophages generate nitrite. Nitrite production is dependent upon operational Tumour Necrosis Factor-alpha (TNFa) p55 receptor signalling following interferon-gamma activation of macrophage. INTRODUCTION Experimental Autoimmune Uveoretinitis (EAU) is an animal model providing an established paradigm for clinical inflammatory disorders affecting the retina and choroid, including sympathetic ophthalmia.1,2 EAU is a CD4þ Th1 mediated 121

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disease, producing pro-inflammatory cytokines. Following T cell infiltration into the eye, antigen specific recognition leads to a cytokine cascade including IFNg and IL-2, which activates resident microglia as well as infiltrating macrophages. Tissue damage is predominantly mediated via reactive oxygen species (ROS) and lipid peroxidation of cell membranes secondary to nitrotyrosine formation.3 More specifically, during autoimmune inflammation of the retina, the driving systemic CD4þ T cell response activates the production of TNFa from macrophages4 which in turn is required for complete classical macrophage activation and subsequent nitric oxide (NO) production.5 To elucidate mechanisms we will describe a series of experiments utilising the EAU model and describing pathways of macrophage activation and NO production. Materials and Methods Animals and Induction of EAU EAU was induced in C57Bl/6 and TNFRp55/ mice or Lewis rats as previously described5,6 (respectively). Briefly, mice were immunised with Interphotoreceptor retinoid-binding protein (IRBP) peptide 1–20 ((GPTHLFQPSLVLDMAKVLLD) (500mg/mouse)) in CFA (v/v) with additional intraperitoneal injection of 1.5mg of Bordetella pertussis toxin (PTX).7 Rats were immunised with retinal extract (RE; 5mg/ml) in CFA (v/v) with additional i.p. injection of 1mg of PTX. Animals were maintained in accordance with Home Office Regulations for Animal Experimentation, UK. Immunohistochemistry Eyes were enucleated for histological grading8 at time points indicated. Tissues were snap frozen, and fixed in acetone for 5 to 10 minutes and air-dried. Mouse sections were single stained for F4/80 and CD45 and visualised using VectorTM DAB. Stained slides were counterstained with haematoxylin and mounted in Histomount. Rat sections were dual fluorescent stained for ED1-FITC and NOS2 visualised with Texas Red. Generation of Bone Marrow-Derived Macrophages (BM-MF) and Retinal Myeloid Cell Isolation Bone marrow cells were cultured as previously described9 in hydrophobic TeflonTM bags in M-CSF supplemented media. Retinal myeloid cells were isolated as previously described5,6 using a graduated density gradient (PercollTM). Cytokine Stimulation of Macrophage Cultures Macrophages were seeded at 5  105/ml/well in 24 well plates and stimulated with cytokines, IFNg (20U/ml), TNF-a (20U/ml; Peprotech EC, UK), and TGF-b (10ng/ml; R&D systems, UK), alone or administered sequentially in combination,

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with the administration of each cytokine separated by a 4-hour period. Macrophage function was assessed 24 hours after addition of the first cytokine. IFN-g, followed 4 hours later by TNF-a (IFNg/TNFa), was used as a positive control for NO production, confirming previous in vitro studies.10 Quantification of NO Synthesis and Cytokine Analysis NO generation was measured after 24 hours by assaying culture supernatants for the stable reaction product of nitric oxide (NO2; nitrite) using the Greiss reagent (0.5% sulphanilamide, 0.05% N-(1-napthyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid), the optical densities were measured at 540nm, with a reference filter of 630nm. Cytokines, IL-2, IFNg, IL-10, IL-12p40 and TNFa production were assayed by capture ELISA. Statistical Analysis Statistical analysis was performed by two-tailed unpaired t tests (GraphPad Instant software) amongst the groups and p values equal or less than 0.05 were considered significant, unless otherwise stated. Results are expressed as mean  SEM. Disease incidence was compared using Fisher’s exact test (StatsDirect). RESULTS Retinal Microenvironment Controls Resident and Infiltrating Macrophage Function During EAU During EAU, macrophages show behavioural characteristics of cytokine conditioning at various phases of EAU.6 In summary, macrophages isolated from normal rat retina (consisting of perivascular ED2þ macrophages and microglia) generated little NO spontaneously, and furthermore, they remained unresponsive to further cytokine stimulation as there was no increase in NO production following stimulation with IFNg (Figure 1). The apparently stable state of the microglia, which has been termed a tonically deactivated state, may be secondary to either TGFb, found abundantly in the eye11 or via the negative signal received by the macrophage from CD200 receptor upon ligation with neuronal CD200;12 both mechanisms would render the cells unresponsive to IFNg-induced classical activation. Macrophages isolated from inflamed uveitic eyes at peak disease (corresponding to maximal macrophage infiltration in the retina) spontaneously produced significant amounts of NO (Figure 1). At this stage the population consists predominantly of infiltrating monocytes and not resident microglia. Evidence of nitrite production was supported by the increase in NOS2 expression just prior to maximal disease and nitrotyrosine expression on immunohistochemistry

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Figure 1 Quantification of NO production by infiltrating macrophages during EAU.

Figure 2 (See color insert.) Immunohistochemical analysis of macrophage NOS2 expression during EAU. Two-colour immunofluorescence, with ED1 (FITC; green; arrow) and NOS2 (Texas Red; arrow head). A: An increased number of ED1þ NOS2þ macrophages were found during prepeak phase EAU only. B: NOS2 expression was absent in ED1þ macrophages during peak phase EAU.

(Figure 2). Furthermore, during the post-peak (days 13–15) and resolution (days 15–17) phases, when infiltrating monocyte/macrophage numbers are comparable to peak disease, the macrophages produced little NO and remained unresponsive to IFNg and TNFa stimulation (Figure 1). In summary these set of experiments showed that resident retinal myeloidderived cells (predominantly microglia) are conditioned or tonically deactivated and thus remain resistant to further cytokine stimulation, at least in vitro. This was similar to the response seen in the larger number of macrophages isolated during EAU recovery. It was evident though that during peak inflammation, infiltrating macrophages adapt to the Th1 T cell response (IFNg/TNFa), inducing classical activation of cells and generating NO. Extrapolating the in vitro data

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which represents responses of cells isolated directly ex vivo, the results infer in vivo programming of macrophages within the retina.6 Neutralising TNFa Activity Suppresses Activation of Infiltrating Macrophages in EAU Previous experiments have shown that treatment of rats or mice with sTNFr-Ig after immunisation suppresses EAU.13–15 During the course of EAU there is no selective inhibition of myeloid cell infiltration into the retina after treatment with sTNFr-Ig (Figure 3a), although macrophages were delayed in entering the retina (day 11 and day 13, control and sTNFr-Ig-treated animals, respectively). Despite myeloid cell infiltration in sTNFr-Ig treated animals, histological disease scores were significantly lower, both at the height and the resolution of disease, compared with controls (Figure 3b). As we have discussed, infiltrating macrophages within the retina generate NO only during peak disease, at which time they remain unresponsive to further cytokine stimulation, in particular deactivation following TGFb exposure. Therefore, we sought to determine whether interrupting classical activation via sTNFr-Ig therapy would inhibit NO production in vivo. Subsequently data showed that retinal macrophages isolated from peak phase of disease from animals treated with sTNFr-Ig showed significantly suppressed NO production. Control animals maintained the capacity to generate NO, and in both groups, macrophages remained unresponsive to further cytokine stimulation (Figure 4). The data shows that sTNFr-Ig successfully suppresses retinal damage and impairs macrophage activation but not trafficking during EAU. Additionally, sTNFr-Ig mediated suppression of NO production results in reduced levels of apoptosis of inflammatory cells and reduction in photoreceptor damage.16

Figure 3 sTNFr-Ig therapy suppressed target organ destruction without impairing retinal myeloid cell infiltrate. a: percentage of CD11bþ macrophages infiltrating the retina, despite sTNFr-Ig therapy. b: Histological scoring of structural changes showing marked reduction in structural damage in the retina at the height of disease.

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TNFRP55/ has a Selective Role in Autocrine Signalling Following IFNg Stimulation in EAU sTNFr-Ig, which binds free TNFa and inhibits even low levels of TNF mediated signalling, can impair in vivo macrophage activation (Figure 4). Taking this observation further we sought to examine the apparent TNF-dependency of NO production in macrophages utilising naı¨e bone marrow derived macrophage responses in vitro and TNFRp55 knock out animals. Following IFNg stimulation, TNFRp55/ bone marrow derived macrophages (BM-MF) failed to produce NO compared with wild-type (WT) BM-MF. Furthermore, supporting a dependency of TNF, experiments showed that pre-treating BM-MF with sTNFr-Ig converted WT BM-MF behaviour and responses and suppressed NO production (Figure 5). To confirm the effect in vivo, EAU was induced in TNFRp55/ and WT animals.

Figure 4 sTNFr-Ig treatment suppressed generation of nitrite by infiltrating macrophages during height of disease. At peak disease there was a significant suppression of nitrite production in sTNFr-Ig-treated animals.

Figure 5 Pre-incubation of sTNFr-Ig prior to IFNg stimulation abrogates NO production and results in WT BM-MF displaying a similar response to TNFRp55/ BM-MF, TNF activity is neutralised.

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Figure 6 TNFRp55/ mice have display reduced incidence and severity of EAU.

Figure 6 shows that TNFRp55/ animals had significantly reduced histological scores at peak disease (day 18) with concomitant suppression of splenocyte proliferation, IL-2 and IFNg production (data not shown). At day 10, however, before onset of disease, TNFRp55/ splenocyte proliferative and IL-2 responses were reduced but associated with a significant increase in IFNg production, indicating normal T cell priming in these mice (data not shown; see ref.5). Although T cell priming is relatively unaffected, macrophages lacking the TNFp55 receptor fail to produce NO following IFNg activation, because of a requirement for autocrine TNFa signalling through the TNFp55 receptor.5 DISCUSSION Selected aspects of IFNg (adaptive immune system) activation are controlled by autocrine secretion of TNFa (Figure 7). NO production and MHC-class II upregulation are both critically dependent on autocrine secretion of TNFa, but only NO secretion requires signals from the TNFp55 receptor.5 However, data from TNFRp55/ macrophages demonstrate that other signals, notably from the innate immune system, via pathogen-associated molecular pattern (PAMP) recognising receptors such as the Toll family of receptors (e.g. TLR4) can induce NO production independent of signals through the TNFp55 receptor.5 This raises a question in autoimmune disease, when pathogens need not necessarily be present (for example sympathetic ophthalmia), of whether endogenous ligands for PAMP receptors contribute to the inflammatory milieu that promulgates disease. If this is the case it might provide an alternative therapeutic target for intervention.

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Figure 7 Selective autocrine signalling from p55 and p75 induced by IFN-g.

REFERENCES 1. Forrester JV, Liversidge J, Dua, HS, et al. Comparison of clinical and experimental uveitis. Curr Eye Res 1990; 9(supp1):75–84. 2. Forrester JV, Dick AD, McMenamin PG, et al. The Eye: Basic Sciences in Practice. Edinburgh: W B Saunders, 2002. 3. Liversidge J, Dick AD, Gordon S. Nitric oxide mediates apoptosis through formation of peroxynitrite and Fas/Fas-ligand interactions in experimental autoimmune uveitis. Am J Pathol 2002; 160:905–916. 4. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23–35. 5. Calder CJ, Nicholson LB, Dick AD. A selective role for the TNF p55 receptor in autocrine signalling following IFN-gamma stimulation in experimental autoimmune uveoretinitis (EAU). J Immunol 2005; 175:6286–6293. 6. Robertson MJ, Erwig LP, Liversidge J, et al. Retinal microenvironment controls resident and infiltrating macrophage function during uveoretinitis. Invest Ophthalmol Vis Sci 2002; 43:2250–2257. 7. Avichezer D, Silver PB, Chan CC, et al. Identification of a new epitope of human IRBP that induces autoimmune uveoretinitis in mice of the H-2b haplotype. Invest Ophthalmol Vis Sci 2000; 41:127–131. 8. Dick AD, Cheng YF, Liversidge J, et al. Immunomodulation of experimental autoimmune uveoretinitis: a model of tolerance induction with retinal antigens. Eye 1994; 8:52–59. 9. Munder M, Eichmann K, Modolell M. Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4þ T cells correlates with Th1/Th2 phenotype. J Immunol 1998; 160:5347–5354. 10. Erwig LP, Kluth DC, Walsh GM, et al. Initial cytokine exposure determines function of macrophages and renders them unresponsive to other cytokines. J Immunol 1998; 161:1983–1988.

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11. Streilein JW, Ksander BR, Taylor AW. Immune deviation in relation to ocular immune privilege. J Immunol 1997; 158:3557–3560. 12. Broderick C, Hoek RM, Forrester JV, et al. Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol 2002; 161:1669–1677. 13. Dick AD, McMenamin PG, Korner H, et al. Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina. Eur J Immunol 1996; 26:1018–1025. 14. Dick AD, Duncan L, Hale G, et al. Neutralizing TNF-alpha activity modulates T-cell phenotype and function in experimental autoimmune uveoretinitis. J Autoimmun 1998; 11:255–264. 15. Hankey DJ, Lightman SL, Baker D. Interphotoreceptor retinoid binding protein peptide-induced uveitis in B10.RIII mice: characterization of disease parameters and immunomodulation. Exp Eye Res 2001; 72:341–350. 16. Robertson MJ, Liversidge J, Forrester JV, et al. Neutralizing tumor necrosis factoralpha activity suppresses activation of infiltrating macrophages in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 2003; 44:3034–3041.

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10 Peroxynitrite and Ocular Inflammation Guey-Shuang Wu Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

Narsing A. Rao Department of Ophthalmology and Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, U.S.A.

INTRODUCTION In humans, uveitis is a complex, inflammation that primarily involves intraocular structures such as iris, ciliary body, choroid, and retina. An animal model of this inflammatory disease, experimental autoimmune uveitis (EAU), can be produced by immunizing Lewis rats with the retinal soluble protein, S-antigen.1 In EAU, the most direct cause of retinal damage is the various cytotoxic agents and free radicals that are released by the infiltrating macrophages and polymorphonuclear leukocytes.1–4 These reactive free radical species can amplify the local inflammatory processes and cause photoreceptor cell damage. Superoxide and nitric oxide are among the most important primary species generated by the macrophages. Further, at the peak of inflammation, on day 14 postimmunization (p.i.), the oxidative damage inflicted by these reactive species is concentrated in the photoreceptors, as indicated by the localization of hydroperoxide-derived cellular carbonyls,5,6 due to an unusually high concentration of docosahexaenoic acid (22:6) in the photoreceptor outer segments. Cellular protein modification by tyrosine nitration occurs at the same time, mainly in the photoreceptor layer, with only minor lesions seen in the retinal blood vessels.5,6 Contrary to these earlier observations and the dogma that tissue damage is initiated by activated macrophages, the present study revealed that retinal

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nitration damage occurs earlier, on day 5 p.i. before any histologic or immunohistochemical evidence of macrophage infiltration. Therefore, the retinal damage of EAU appears to be derived from an alternative mechanism; and this mechanism operates apart from the effects of macrophages, especially in the release of reactive nitrogen species and reactive oxygen species. These early events that set the destructive pathway in EAU have not been elucidated. SUPEROXIDE AND NITRIC OXIDE IN EAU At physiological pH, peroxynitrite formed in vivo can directly nitrate phenolic rings to form 3-nitrotyrosine from tyrosine residues. In recent years, although other metabolites of nitric oxide have also emerged as biological oxidants, it is generally agreed that peroxynitrite is generally considered the most plausible entity for causing biological nitration and oxidation.7 Peroxynitrite has been implicated in the pathogenesis of a series of diseases, including acute and chronic inflammatory processes, sepsis, ischemic-reperfusion and a variety of neurodegenerative and retinal disorders.8 The presence of nitric oxide synthase (NOS) has recently been shown in mammalian mitochondria.9,10 Thus, with an abundance of substrate,10 nitric oxide is continuously produced in the mitochondria. Mitochondria are also a copious source of superoxide, which is generated at the sites of complexes I and III of the electron transport chain.11 In tissues and in mitochondria, peroxynitrite forms from a facile reaction of superoxide and nitric oxide concomitantly generated in close proximity. These facts suggest that mitochondria are continuously challenged by peroxynitrite formed within the organelles themselves. In the past, nitration of Mn superoxide dismutase, mitochondrial aconitase, the voltage-dependent anion channel, mitochondrial ATPase and cytochrome c have been detected in animals undergoing inflammatory processes.12,13 Photoreceptor cells are known to have the highest rates of glycolysis and respiration among all retinal cells.14 For these reasons, inner segments of photoreceptor are densely packed with mitochondria.15 Our study was designed to determine the primary nitration target(s) of peroxynitrite and to detect the onset of this post-translational modification in EAU. The retina contains numerous proteins that complement its complex visual functions. Three of these proteins, all of which are essential for mitochondrial energetics and metabolism functions, were found to be selective prime targets of peroxynitrite nitration. Moreover, the protein nitration was found to commence early in the inflammatory process, far before the entry of inflammatory cells known to release superoxide and nitric oxide in the retina. In experimental uveitis, the insult that initiates the spiral of degenerative processes in the photoreceptors has not been defined in the past. Nitration of Retinal Proteins in Uveitis Experimental uveitis was induced by a hind foot-pad injection of 60 mg of bovine S-antigen in Freund’s complete adjuvant containing 4 mg/ml of heat killed Mycobacterium tuberculosis H37 RA (Difco, Detroit, MI). To investigate the

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early phase of the disease, animals were sacrificed on days 5 and 10 p.i., with the normal peak of inflammation being at day 14 p.i. In ultraviolet/visible (UV/VIS) absorption and Western blot analyses, in vitro nitrated bovine serum albumin (BSA) was used as a model protein to establish the sensitivity and specificity of 3-nitrotyrosine absorption embedded in proteins.16 Bovine serum albumin containing 19 tyrosine residues/molecule was nitrated in good yield in vitro by the peroxynitrite donor 3-morpholinosydnonimine (SIN-1; Sigma, St Louis, MO) to give 354 nm absorption at pH 7 (see insert in Fig. 1). The tyrosine residues were nitrated at a much higher level in BSA compared with the level of nitrotyrosine formed in SIN-1-reacted retina and in inflamed retina nitrated in inflammation. Since the molar absorption coefficient for nitrotyrosine is only 4400/M/cm,17 the maximal obtainable intensity for 354 nm, the pH 7 band for the SIN-1-reacted retinal proteins is small (Fig. 1A). In these spectra, the tyrosine-nitrated proteins (spectrum 4, Fig. 1A) revealed 360 nm nitrotyrosine chromophore after subtracting spectrum 3, sum of controls, spectra 1 and 2 (Fig. 1A). Similarly, in the inflamed retina (Fig. 2A), subtraction of spectrum 1, non-immunized control retina from spectrum 2, EAU day 5 retina revealed an absorption peak centered at 350 nm, indicative of nitrotyrosine chromophore and an absorption for cytochrome c at 407 nm (Fig. 2A).18 Although the absorption bands were not completely resolved, as was commonly seen in the in vivo samples, they clearly demonstrated the presence of nitrotyrosine chromophore and released cytochrome c. These observations are consistent with other reported tissue studies in which tyrosine-nitration can also be detected by immunohistochemistry,5 by electrochemically monitored HPLC,19 or by Western blotting20; but no one method alone will totally ascertain the formation of nitrotyrosine in vivo. In this study, to assay and confirm the protein tyrosine-nitration in both in vitro and in vivo samples, we used UV/VIS absorption for initial screening, Western blot in conjunction with mass spectrometry for confirmation, and immunohistochemical staining for subsequent localization in the retina. Identification of Nitrated Retinal Protein Exposure of naı¨ve retina to the peroxynitrite donor SIN-1 resulted in seven tyrosine-nitrated proteins, as revealed by Western blot analysis (Fig. 1C, lane 1). The molecular masses of these proteins are 68, 52, 50, 41, 39, 35, and 29 kDa, as estimated by the relative mobility (Rf value) of these proteins compared with that of the protein standards. The relative intensities of these nitrated bands in Western blot appeared to follow closely the intensities seen in the total retinal protein profile (Fig. 1B, lane 1). In this system, major proteins were all nitrated as compared with the controls (Fig. 1C, lane 2). The EAU eyes were obtained from the animals on days 0, 5, 10, 12, and 14 p.i. Day 0 denotes non-immunized control animals. In the EAU retina, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel)

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Figure 1 UV/VIS absorption and Western blot of retinal proteins nitrated by SIN-1. A. Analysis of UV/VIS spectra of nitrated retinal proteins at pH 7. Spectrum 1: retinal protein end absorption; spectrum 2: degradation products of SIN-1 following reaction; spectrum 3: sum of spectra 1 and 2; spectrum 4: nitrated retinal proteins. Subtraction of spectrum 3 from 4 resulted in an absorption centered near 360 nm, indicative of nitrotyrosine chromophore. The insert shows the absorption of nitrated BSA. B. Coomassie Blue staining of retinal proteins incubated with (lane 1) and without (lane 2) SIN-1. C. Western blot probed with anti-nitrotyrosine. Lane 1: retinaþSIN-1; lane 2: retina-SIN-1. Note that all major proteins in the retina are equally nitrated.

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Figure 2 Retinal protein nitration in the early phase of EAU. A. UV/VIS absorption spectra of control animal (D0) (spectrum 1) and early phase of EAU (spectrum 2). Subtraction of spectrum 1 from 2 reveals absorption of nitrated tyrosine at 350 nm and cytochrome c at 407 nm (arrow). B. Coomassie Blue staining of electrophoresed retinal proteins. Lane 1: D0; and lane 2: D5 p.i. C. Western blot of nitrated retinal proteins. Lanes 1 (D0), 2 (D5), 3 (D10), 5 (BSAþSIN-1), and 6 (BSA-SIN-1) were probed with antinitrotyrosine. Lane 4 (D5) was probed with preimmune serum. Band A: mitochondrial import stimulation factor; band B: phosphoglycerate mutase; and band C: cytochrome c.

revealed 10 major protein bands (Fig. 2B, lane 2); this profile was similar to that of the non-immunized control animals (Fig. 2B, lane 1). Western blots of EAU samples were then run in parallel with nitrated BSA (Fig. 2C, lane 5). The blots of EAU retinas indicated three relatively intense tyrosine-nitrated protein bands,

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Figure 3 Western blot analyses of nitrated cytochrome c in the early phase of EAU. Lane 1 (D0) and lane 2 (D5) were blotted with anti-rat cytochrome c, and lane 3 (D5) and lane 4 (D10) were blotted with anti-nitrotyrosine antibody. Band A: cytochrome c trimer; band B, cytochrome c and band C: nitrated cytochrome c.

located at 32, 29, and 16 kDa (Fig. 2C, lanes 2 and 3). Moreover, these three bands appeared early in the inflammation, on days 5 and 10 p.i., long before the peak of inflammation at day 14 p.i. The 32 kDa (upper) and 29 kDa (lower) bands were excised separately from an electrophoresed gel and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Sequest database search revealed that the upper band is mitochondrial import stimulation factor and the lower band, rat phosphoglycerate mutase. In both mitochondrial import stimulation factor and phosphoglycerate mutase, six peptides each were identified to match the known sequences. Using Western blot (15% gel) gel, the 14 kDa band from EAU days 5 and 10 p.i. (Fig. 2C, band C) was identified as cytochrome c. This sample was also blotted in parallel with both rat cytochrome c antibody (lanes 1 and 2, Fig 3) and nitrotyrosine antibody (lanes 3 and 4, Fig. 3). The identity of the cytochrome c band was also confirmed by LC-MS/MS, using cytochrome c from both whole retina and isolated mitochondria. Sequential studies covering days 0, 5, 10, 12, and 14 p.i. revealed that three Tyr-nitrated proteins, including mitochondrial import stimulation protein, phosphoglycerate mutase and cytochrome c, were at near maximal intensities on days 5 and 10 p.i., then leveled off gradually from day 10 to the peak of inflammation on day 14 (Fig. 4). During the period from days 0 to 10, the retinal morphology was well preserved. Day 12 signified the onset of disease, and the entrance of inflammatory cells was visible.

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Figure 4 (See color insert.) Retinal morphology and protein nitration during the course of EAU. Nitrated retinal proteins from days 0 (D0), 5 (D5), 10 (D10), 12 (D12), and 14 (D14) p.i. were immunoblotted with anti-nitrotyrosine (B), the relative intensities of nitrated proteins were quantified (C) and correlated with morphologic changes in EAU (A). Maximal intensities of tyrosine-nitration were seen in days 5 and 10, with well preserved retinal structures. Day 12 marked the onset of inflammation with arrival of inflammatory cells.

Localization of Nitrated Retinal Protein in the Early Phase of EAU To assess the cellular source of peroxynitrite in the early phase of EAU, tyrosinenitrated proteins in the retina were localized. Using immunohistochemical methods, positive nitrotyrosine staining was localized exclusively at the photoreceptor inner segments in day 5 p.i. retina (Fig. 5B). No nitrotyrosine staining

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Figure 5 (See color insert.) Localization of tyrosine-nitrated proteins in the retina. Polyclonal nitrotyrosine antibody and anti-rabbit IgG conjugated with biotin were used for the detection. A: non-immunized control animals and B: EAU day 5 p.i. Note the intense localization of nitrated proteins seen only in the photoreceptor inner segments (B).

was seen in the non-immunized controls (Fig. 5A). The specificity of primary antibody was established by (1) replacing the primary antibody with phosphatebuffered saline and (2) reacting the primary antibody with authentic nitrotyrosine before staining. Both procedures abolished the nitrotyrosine staining in the inflamed retinas. Displacement of Cytochrome C from Electron Transport Assembly The release of cytochrome c in EAU animals on days 5 and 10 p.i. was examined by isolating intact mitochondria.21 The retinal cytosol and intact mitochondria were separated initially. In cytosolic fractions, the presence of cytochrome c was not detected on days 5 and 10 p.i., indicating that cytochrome c was not released into the cytosol at the early phase of disease. When the isolated mitochondria were sonicated briefly to rupture the outer membranes,22 a substantial release of cytochrome c was observed on both days 5 and 10 (Fig. 6, band A in lanes 2 and 3). A functional cytochrome c binds to both mitochondrial respiratory complexes III and IV and is, therefore, stable to sonication but sensitive to detergents.23 No detergent was used in these isolation procedures. It appears that in this early phase of inflammation, although cytochrome c (more likely nitrated cytochrome c) was already displaced from its normal binding site in the electron transport chain, the mere separation of cytosol from intact mitochondria did not result in the significant release of cytochrome c. However, upon mechanical rupture of

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Figure 6 Nitration and release of cytochrome c in the early phase of EAU. The release of cytochrome c was not seen in the cytosolic fraction separated from the intact mitochondria. However, after mild sonication of mitochondria fraction to disrupt the outer membranes, substantial release of cytochrome c/nitrated cytochrome c was detected by Western blot probed with anti-rat cytochrome c. Lane 1: non-immunized control (D0); lane 2: D5 and lane 3: D10.

mitochondrial outer membranes, cytochrome c was released into the supernatant. No cytochrome c release was detected in the controls, even with sonication to disrupt the mitochondrial outer membranes (Fig. 6). ROLE OF PEROXYNITRITE IN EAU From total proteins in EAU retina, we have detected three mitochondria-related proteins that were specifically nitrated in the early phase of EAU, prior to any macrophage or other phagocytic infiltration (Figs. 2, 4). Using LC-MS/MS, we identified two nitrated proteins near 30 kDa as mitochondrial import stimulation factor and phosphoglycerate mutase. The third protein (14 kDa) was identified as cytochrome c (Fig. 3). Levels of tyrosine-nitration were also correlated with the extent of cellular infiltration and photoreceptor degeneration in the course of EAU (Fig. 4). In the early phase of EAU, the tyrosine-nitrated retinal proteins were localized exclusively in the photoreceptor inner segments, which are densely populated with mitochondria (Fig. 5). Further, in vivo nitrated cytochrome c was found to be displaced from its original binding site at the electron transport chain assembly. The in vitro nitration of naı¨ve retina was also carried out by peroxynitrite donor SIN-1 to result in nitration of all seven major proteins with similar intensities (Fig. 1). Therefore, in vitro nitration in the solution phase lacks the selectivity displayed by the in vivo nitration.

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Peroxynitrite has been implicated in the pathogenesis of a series of diseases. In these systems, peroxynitrite generation requires nitric oxide and superoxide.8 The concomitant generation of these two agents at a localized site results in the formation of peroxynitrite by a combination reaction, threefold faster than the rate of superoxide dismutation by superoxide dismutase.24 We and others have reported in the past that both superoxide and nitric oxide are among the most important primary oxidant species generated by macrophages in inflammatory diseases such as uveitis.3,4,25 However, our study showed that nitration of mitochondrial proteins occurred prior to the infiltration of macrophages (Fig. 4), indicating that the generation of reactive species and formation of peroxynitrite occurred within the retinal cells and was not from macrophages or other infiltrating inflammatory cells. Photoreceptor cells, which are responsible for all visual processes, have the highest rate of glycolysis and respiration, as revealed by the metabolic mapping of mammalian retina using H3-2-deoxyglucose autoradiography.14 Because of this high metabolic requirement, the inner segments in the photoreceptor cells are packed with mitochondria, with a density unseen in any other cells.15 Mitochondria are also an important cellular source of superoxide. It is estimated that 1–2% of the oxygen consumed undergoes partial reduction, generating superoxide.11 In recent years, mitochondrial production of nitric oxide by mitochondrial NOS was recognized.9,10 Nitric oxide produced by mitochondrial NOS and L-arginine is readily diffusible through cell membranes, whereas superoxide is not; therefore, it is conceivable that a charged combination product, such as peroxynitrite will be principally formed in the same compartment as superoxide, probably near the inner membranes.26 In the absence of macrophages, the actively respiring mitochondria in the inner segments of photoreceptor cells would be the early source of peroxynitrite, causing the nitration of cellular proteins at the proximity. Mitochondrial DNA contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides. The mitochondrial DNA-encoded polypeptides are all subunits of enzyme complexes of the oxidative phosphorylation system.27 Therefore, most of the proteins required for the mitochondrial functions are encoded by nuclear genes, synthesized by cytoplasmic ribosomes and imported to mitochondria post-translationally.28 Therefore, there are cytosolic protein factors that chaperone and target cytoplasmic precursor proteins to mitochondrial membrane receptors. Mitochondrial import stimulation factor serves these functions.29 Although mitochondrial import stimulation factor originates as a cytosolic factor, during the chaperone process, it sits on the mitochondrial membrane receptors to transfer preproteins; therefore, mitochondrial import stimulation factor is exposed to the peroxynitrite generated within mitochondria.29 Phosphoglycerate mutase catalyzes the interconversion of 2- and 3phosphoglycerate in the glycolytic/gluconeogenic pathways. These reactions are essential components in the metabolism of glucose and/or 2, 3-bisphosphoglycerate in all cells.30 Although this protein does not reside intramitochondrially, it

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is an essential enzyme in glycolysis, one of the major reactions in mitochondrial metabolism. Cytochrome c is a member of the mitochondrial respiratory chain assembly situated between complexes III and IV, and is an electron carrier in the electron transport process. Unlike other respiratory chain complexes, cytochrome c faces intermembrane space rather than matrix.31 Therefore, nitration of cytochrome c without nitration of complexes I through IV might indicate that the gradient of peroxynitrite produced in the mitochondria could be concentrated in the intermembrane space rather than in the matrix (Fig. 7). When intact mitochondria and cytosol were separated in EAU day 5 retina, only a trace of cytochrome c released was observed in the cytosolic fraction.

Figure 7 Location and function of cytochrome c in the mitochondria. The respiratory chain complexes are embedded in the mitochondrial inner membrane. This assembly includes four complexes (I to IV), coenzyme Q and cytochrome c. Electrons flow down the chain to complex IV where O2 is reduced to H2O. Cytochrome c, which carries electrons between complexes III and IV, is the only member facing the intermembrane space. The mitochondrial respiratory chain is also a copious producer of superoxide, which reacts with nitric oxide to form peroxynitrite. Mitochondrial nitric oxide synthase is previously shown to associate with inner membrane.31

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However, when mild sonication was applied to disrupt outer mitochondrial membranes, substantially more cytochrome c was detected. In the respiratory chain assembly, cytochrome c is bound to complex III and cytochrome oxidase by electrostatic interaction and is therefore stable to sonication but sensitive to most detergents.23 In the present study, no detergent was used in processing retina and mitochondria. In the previous reports, when cytochrome c was released from apoptotic or permeabilized mitochondria, it was often found that cytochrome c was already dissociated from the electron transport chain before pathologic membrane rupture.22 Therefore, it appears that the release of cytochrome c requires two simultaneous impairments: 1) rupture or permeabilization of mitochondrial outer membranes; and 2) detachment of cytochrome c from the respiratory chain complex. In this study, the integrity of mitochondrial outer membranes was still mostly intact on day 5; but cytochrome c was already displaced from its normal binding site in the respiratory chain due to tyrosinenitration in the molecule. The initial signal leading to upregulation of mitochondrial NOS in S-antigen induced EAU has not been dealt in the past. In an organ-specific autoimmune disease such as EAU, the CD4-positive T-cells are present in the retina early in the inflammation. For example, after adaptive transfer of S-antigen specific T-cells, these T-cells were seen in the retina within 24 hours, although loss of retinal stratification was not observed until after 120 hours.32 The local antigen presentation to these S-antigen autoreactive T-cells can result in the generation of tumor necrosis factor-a (TNF-a) by the antigen-presenting cells. Tumor necrosis factor-a, an inflammatory agonist, is known to upregulate NOS, and subsequently to produce reactive oxygen species.33 Tumor necrosis factor-a can also increase mitochondrial Ca2þ, a known stimulator of mitochondrial reactive oxygen species.34 In this process, TNF-a initially mobilizes Ca2þ from its endoplasmic storage to the mitochondria; Ca2þ then triggers mitochondrial NOS activity.35 CONCLUSION In the early phase of EAU, prior to leukocyte infiltration, we found three major nitrated retinal proteins and these were mitochondrial import stimulation factor, phosphoglycerate mutase and cytochrome c, all of which are mitochondriarelated proteins. Immunohistochemical staining revealed that these nitrated proteins are exclusively localized in the inner segments of photoreceptor cells, a layer known to be densely populated with mitochondria. These findings provide evidence for a rather selective tyrosine-nitration process that modifies specific proteins in vivo. In this early stage of inflammation, mitochondria are the major source of peroxynitrite and mitochondrial proteins the prime target for damage by the mitochondrial oxidative stress. Hence, for the first time, these findings implicate the photoreceptor damage at the molecular level by peroxynitrite generated in the mitochondria. Such oxidative damage may lead to microglial

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activation, recruitment of blood-born monocytes/macrophages and neutrophils in the amplification of retinal damage and clinical and histologic findings of amplified uveitis. ACKNOWLEDGMENT This study was supported in part by grants EY015714 and EY03040 from National Institutes of Health. REFERENCES 1. Rao NA. Role of oxygen free radicals in retinal damage associated with experimental uveitis. Trans Am Ophthalmol Soc 1990; 88:797–850. 2. Rao NA, Patchett R, Fernandez MA, et al. Treatment of experimental granulomatous uveitis by lipoxygenase and cyclo-oxygenase inhibitors. Arch Ophthalmol 1987; 105:413–415. 3. Zhang J, Wu GS, Rao NA. Role of nitric oxide in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 1993; 34(4):1000. 4. Zhang J, Wu LY, Wu GS, et al. Differential expression of nitric oxide synthase in experimental uveoretinitis. Invest Ophthalmol Vis Sci 1999; 40:1899–1905. 5. Wu GS, Zhang J, Rao NA. Peroxynitrite and oxidative damage in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 1997; 38:1333–1339. 6. Rao NA, Wu GS. Free radical mediated photoreceptor damage in uveitis. Prog Retin Eye Res 2000; 19:41–68. 7. Valdez LB, Alvarez S, Arnaiz SL, et al. Reactions of peroxynitrite in the mitochondrial matrix. Free Radic Biol Med 2000; 29:349–356. 8. Radi R. Peroxynitrite reactions and diffusion in biology. Chem Res Toxicol 1998; 11:720–721. 9. Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 1997; 418:291–296. 10. Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem 1998; 273:11038–11043. 11. Turrens JF, Alexandre A, Lehninger AL. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys 1985; 237:408–414. 12. MacMillan-Crow LA, Cruthirds DL, Ahki KM, et al. Mitochondrial tyrosine nitration precedes chronic allograft nephropathy. Free Radic Biol Med 2001; 31:1603–1608. 13. Aulak KS, Miyagi M, Yan L, et al. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc Natl Acad Sci U S A 2001; 98: 12056–12061. 14. Winkler BS, Pourcho RG, Starnes C, et al. Metabolic mapping in mammalian retina: a biochemical and 3H-2-deoxyglucose autoradiographic study. Exp Eye Res 2003; 77:327–337. 15. Tsacopoulos M, Poitry-Yamate CL, MacLeish PR, et al. Trafficking of molecules and metabolic signals in the retina. Prog Retin Eye Res 1998; 17:429–442.

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16. Spencer JP, Wong J, Jenner A, et al. Base modification and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholinosydnonimine. Chem Res Toxicol 1996; 9:1152–1158. 17. Crow JP, Ischiropoulos H. Detection and quantitation of nitrotyrosine residues in proteins: in vivo marker of peroxynitrite. Methods Enzymol 1996; 269:185–194. 18. Cassina AM, Hodara R, Souza JM, et al. Cytochrome C nitration by peroxynitrite. J Biol Chem 2000; 275:21409–21415. 19. Skinner KA, Crow JP, Skinner HB, et al. Free and protein-associated nitrotyrosine formation following rat liver preservation and transplantation. Arch Biochem Biophys 1997; 342:282–288. 20. MacMillan-Crow LA, Crow JP, Kerby JD, et al. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci U S A 1996; 93:11853–11858. 21. Netto LE, Kowaltowski AJ, Castilho RF, et al. Thiol enzymes protecting mitochondria against oxidative damage. Methods Enzymol 2002; 348:260–270. 22. Adachi S, Cross AR, Babior BM, et al. Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis. J Biol Chem 1997; 272:21878–21882. 23. Capaldi RA, Darley-Usmar V, Fuller S, et al. Structural and functional features of the interaction of cytochrome c with complex III and cytochrome c oxidase. FEBS Lett 1982; 138:1–7. 24. Crow JP, Beckman JS. The importance of superoxide in nitric oxide-dependent toxicity: evidence of peroxynitrite-mediated injury. Adv Exp Med Biol 1996; 387:147–161. 25. Liversidge J, Dick A, Gordon S. Nitric oxide mediates apoptosis through formation of peroxynitrite and Fas/Fas-ligand interactions in experimental autoimmune uveitis. Am J Pathol 2002; 160:905–916. 26. Alvarez MN, Trujillo M, Radi R. Peroxynitrite formation from biochemical and cellular fluxes of nitric oxide and superoxide. Methods Enzymol 2002; 359: 353–366. 27. Taanman JW. The mitochondrial genome: structure, transcription, translation and replication. Biochim Biophys Acta 1999; 1410:103–123. 28. Hartl FU, Pfanner N, Nicholson DW, et al. Mitochondrial protein import. Biochim Biophys Acta 1989; 988:1–45. 29. Omura T. Mitochondria-targeting sequence, a multi-role sorting sequence recognized at all steps of protein import into mitochondria. J Biochem 1998; 123: 1010–1016. 30. Fothergill-Gilmore LA, Watson HC. The phosphoglycerate mutases. Adv Enzymol Relat Areas Mol Biol 1989; 62:227–313. 31. Ghafourifar P. Characterization of mitochondrial nitric oxide synthase. Methods Enzymol 2002; 359:339–350. 32. Prendergast RA, Iliff CE, Coskuncan NM, et al. T-cell traffic and the inflammatory response in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 1998; 39:754–762. 33. Parthasarathi K, Ichimura H, Quadri S, et al. Mitochondrial reactive oxygen species regulate spatial profile of proinflammatory responses in lung venular capillaries. J Immunol 2002; 169:7078–7086.

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34. Borutaite V, Morkuniene R, Brown GC. Release of cytochrome C from heart mitochondria is induced by high Ca2þ and peroxynitrite and is responsible for Ca2þ-induced inhibition of substrate oxidation. Biochim Biophys Acta 1999; 1453:41–48. 35. Rizzuto R, Pinton P, Carrington W, et al. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2þ responses. Science 1998; 280:1763–1766.

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11 Melanin and Oxidative Reactions Tadeusz Sarna, Grzegorz Szewczyk, and Andrzej Zadlo Department of Biophysics, Jagiellonian University Krakow, Krakow, Poland

INTRODUCTION Melanins are a group of pigments with distinct physicochemical properties whose molecular structure and biological functions are only partially understood. Although melanin in the human skin and eyes is usually considered as a natural sunscreen and antioxidant that protects the pigmented tissue against adverse effects of solar radiation, some studies suggested that melanin could also act as a photosensitiser i.e., a system that utilizes energy of the absorbed photons to generate so-called reactive oxygen species. To explain these seemingly contradictory findings, this chapter briefly reviews basic physical and chemical properties of eumelanins and pheomelanins—the two main classes of melanin pigments found in human—that determine their antioxidant and, under special conditions, pro-oxidant action.

BIOSYNTHESIS OF MELANIN AND ITS PHYSICOCHEMICAL PROPERTIES RELEVANT FOR PHOTOPROTECTION In the human skin and eye, melanin biogenesis occurs in melanocytes, specialized cells that contain the necessary machinery for the pigment granule ensemble.1 The synthesis of melanin is controlled by several enzymes and

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involves the formation of highly reactive transient species that are potentially very toxic to the melanocyte.2 Therefore all key steps of melanogenesis take place in melanosomes—sub cellular organelles that limit the exposure of the cellular environment to melanogenic intermediates.3 As a result, melanin in melanocytes is present in the form of discernible units such as pigment granules whose size and geometry are determined by the phenotype of the melanosomes. Melanin granules in the human retinal pigment epithelium are typically elongated and relatively large (2–3 mm long and 1 mm wide), while such pigment granules in the human choroids are smaller and somewhat more spherical.4 In the human skin, the ultrastructure of melanosomes usually relates to the type of melanin they produce.5 Thus typical melanosomes that produce so-called eumelanin have ellipsoidal-lamellar structure with melanin deposited in a uniform pattern. On the other hand, melanosomes that form so-called pheomelanin, are usually round and granular with uneven deposition of pigment. Eumelanin originates from tyrosine or DOPA, and pheomelanin formation requires, in addition, the presence of cysteine or glutathione.6 It is believed that key intermediates in the biosynthetic pathway for eumelanin, are 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid, as well as their fully oxidized forms.7 In the biosynthetic pathway for pheomelanin, a similar role may be played by 1,4-bezothiazynylalanine, which is derived from cysteinyldopas.8 The understanding of the molecular structure of melanin has undergone a substantial evolution. Thus while previously melanin was viewed as very large molecule of hetero-polymeric structure,9 recent studies using advanced imaging techniques such as scanning electron, tunnelling and atomic force microscopies indicate that the actual building block of eumelanin is a relatively small planar oligomer with maximum dimension of 0.4
1.0 nm that is preferentially aggregated into fundamental aggregates of 3–4
-stocked oligomers.10–12 According to this view, the macroscopic morphology of eumelanin pigment granules is a result of hierarchical self-assembly, in which the building blocks of eumelanin assemble into hundred-nanometre structures, which then aggregate to form the final pigment granule.13,14 Although the exact nature of the forces that are involved in the assembly of nanoaggregates and of hundred-nanometer structures remain unknown, it can be speculated that Van der Waals,
–
and hydrophobic interactions play a key role. One of the most characteristic and unique features of melanin is its paramagnetism. Melanins are the only biological material that both in vivo and in vitro contain a significant amount of persistent free radical centres that are easily detected by electron paramagnetic resonance (EPR) spectroscopy.15,16 Importantly, the EPR signals of melanin are specific for the two main types of melanin pigments. At standard EPR frequency (X-band), eumelanins have a single slightly asymmetric line 0.4–0.6 mT wide with a g-factor close to 2.004. The EPR spectrum of pheomelanin typically consists of three spectral features with an overall width of about 3.0 mT and g = 2.005. It must be stressed that even though the EPR signal of melanin is very persistent, the free radicals in

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Figure 1.1 Univalent reduction of oxygen and univalent oxidation of nitric oxide (see page 2).

Figure 5.2 Immunohistochemical staining for SOD3 in the human cornea. A: Note a pronounced staining of the cell borders in the epithelium, and a stromal staining which is interleaved between the stromal collagen lamellae. The stromal staining is slightly weaker in the anterior, than in the posterior stroma. B: Detail of immunohistochemical staining for SOD3 in the human corneal epithelium. Note intense staining of the cell borders and intercellular space. C: Staining for SOD1 in the human corneal epithelium. Note the staining of the cytosol and nuclei (see page 59).

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Figure 6.5 A rise in extracellular glutamate and overactivation of glutamate ionotropic receptors leads to generation of ROS and cell death (see page 77).

Figure 9.2 Immunohistochemical analysis of macrophage NOS2 expression during EAU. Two-colour immunofluorescence, with ED1 (FITC; green; arrow) and NOS2 (Texas Red; arrow head). A: An increased number of ED1þ NOS2þ macrophages were found during prepeak phase EAU only. B: NOS2 expression was absent in ED1þ macrophages during peak phase EAU (see page 124).

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Figure 10.4 Retinal morphology and protein nitration during the course of EAU. Nitrated retinal proteins from days 0 (D0), 5 (D5), 10 (D10), 12 (D12), and 14 (D14) p.i. were immunoblotted with anti-nitrotyrosine (B), the relative intensities of nitrated proteins were quantified (C) and correlated with morphologic changes in EAU (A). Maximal intensities of tyrosine-nitration were seen in days 5 and 10, with well preserved retinal structures. Day 12 marked the onset of inflammation with arrival of inflammatory cells (see page 137 ).

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Figure 10.5 Localization of tyrosine-nitrated proteins in the retina. Polyclonal nitrotyrosine antibody and anti-rabbit IgG conjugated with biotin were used for the detection. A: non-immunized control animals and B: EAU day 5 p.i. Note the intense localization of nitrated proteins seen only in the photoreceptor inner segments (B) (see page 138).

Figure 15.2 For tyrosinase immunocytochemistry, the RPE monolayer was prepared and exposed to ROS for 4 hrs. The expression of tyrosinase was investigated before feeding with ROS, as well as 5 and 24 hours afterwards. 2A Five hours after feeding with ROS, no staining was visible with anti-tyrosine hydroxylase antibodies. 2B Without feeding with ROS no staining was found with anti-tyrosinase antibodies. 2C Five hours after feeding with ROS faint staining was observed with anti-tyrosinase antibodies corresponding to DOPA positive vesicles in Fig. 1B, 2D. Twenty-four hours after feeding with ROS intense staining of lysosome-like organelles (arrows) was found with anti-tyrosinase antibodies. These organelles correspond to those shown in Fig. 1G, 1B (see page 203).

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melanins are by no means chemically stable. Indeed it has been demonstrated that the concentration of melanin free radicals can be changed reversibly by almost two orders of magnitude.17 Among agents that can induce melanin free radicals are ultraviolet and visible radiation, high pH, redox systems and diamagnetic multivalent metal ions.18 It is believed that most of the changes in the free radicals induced by theses agents are due to changes in the so-called comproportionation equilibrium, i.e. the equilibrium between the fully reduced and oxidized melanin subunits, and their semi-reduced (semi-oxidized) states, as shown in the equation below: Q þ QH2 $ 2SQ þ 2Hþ The monomers are o-quinones, o-hyrdoquinones, and o-semiquinones in the case of eumelanin; corresponding units for pheomelanin are o-quinonimines, o-aminophenols, and o-semiquiononimines respectively. The effect of complexing of diamagnetic multivalent metal ions on the melanin EPR signal is an important diagnostic test that can be used to determine the molecular nature of the subunits and, hence, the type of melanin studied.19 Thus, EPR spectroscopy is a unique physical method that enables non-destructive analysis and characterization of melanin pigments with good sensitivity and high accuracy.20 It seems generally accepted that melanin in the skin and eye acts as a natural sunscreen that by absorbing and scattering, hence attenuating, solar radiation, particularly the energetic UV and short wavelength visible photons, protects the pigmented tissue against adverse photo-reactions. Indeed a distinct correlation between the resistance of the human skin to UV-induced erythema and sunburn, and constitutive pigmentation of the skin is usually observed.21 Epidemiological data also suggest that the incidence of solar radiation-related skin cancer is higher in individuals with genetically-determined poor ability of the skin to tan and low pigmentation.22 In addition, skin susceptibility to socalled photo-ageing may inversely correlate with pigmentation of the skin.23 In cultured melanocytes, melanin was shown to offer protection against induction of major DNA lesions by UVB24,25 and UVA-induced membrane damage.26 A significant inverse correlation between baseline skin pigmentation and the extent of UV-induced DNA damage was also reported by an independent study.27 Although the role of chronic exposure of the human retina to solar radiation in the ethiology of AMD remains controversial,28,29 it is of interest to note that AMD is more often found in individuals with lower content of the uveal melanin.30 The molecular and cellular mechanism of photoprotection offered by melanin is not fully understood. Of course, the ability of melanin pigments to absorb light with the efficiency that increses inversely with the light wavelength is intrinsically photoprotective, providing that the energy of the absorbed photons is rapidly and safely utilized in non-photochemical processes. Indeed recent studies confirm a very efficient nonradiative de-excitation of melanin, following

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the absorption of ultraviolet and visible photons.31–33 The studies clearly show that melanin is a system in which a very efficient thermal relaxation occurs, this is to say that energy absorbed by melanin photons is rapidly converted into heat via very fast internal conversion. As a result, the risk of potentially damaging photochemical reactions, mediated by melanin, is significantly reduced. Antioxidant Properties of Melanin There is another mode of photoprotective action of melanin. It is related to its ability to act as an antioxidant, i.e., an agent that protects other molecules by neutralizing oxidizing free radicals and other so-called ‘‘reactive oxygen species’’, being present at lower concentration than the oxidisable substrate molecules. While photochemical oxidising reactions are typically accompanied by the formation of reactive oxygen species, the presence of redox-active metal ions, such as iron and copper, is believed to elevate the oxidative damage via Fenton-type processes.34 That’s why antioxidant action may also depend on sequestration of redox-active metal ions. In model systems of different complexity, synthetic and natural melanins have been shown to act as efficient scavengers of reactive free radicals, quenchers of singlet oxygen and excited triplet states of certain photosensitising dye molecules, and inhibitors of lipid peroxidation.35–43 Using pulse radiolysis as a direct method for generation of selected free radicals and for monitoring their lifetime in the absence and presence of synthetic DOPA-melanin, apparent rate constants of the interaction of the radicals with melanin were obtained.41,44,45 The data, shown in Table 1, can be summarized as follows: this synthetic eumelanin exhibits reactivity with both oxidizing and reducing radicals. The observable reactivity increases with the absolute value of the one-electron reduction potential of the radicals studied and with their intrinsic lifetime. The reactivity also depends on the electric charge of the radicals being higher for the positively charged species and lower for the negatively charged radicals. As expected, melanin interacted most rapidly with OH (one of the most oxidising free radicals) and with hydrated electron (the most reducing species known). However, this eumelanin also interacted quite efficiently with superoxide anion, which is a poor oxidant and only a mild reductant.46 In addition, melanin interacted with reasonably high rate constants with peroxyl and carbon-centred radicals that may be involved in peroxidation of lipids. The interaction of melanin with oxidizing and reducing radicals can be explained by the hydroquinone and quinone nature of the melanin subunits, which can act as efficient electron donors and acceptors, respectively. Using steady-state photosensitised generation of singlet oxygen and EPRoximetry to monitor oxygen consumption, rate constant of the interaction of singlet oxygen with synthetic DOPA-melanin was measured.47 Again, the corresponding bimolecular rate constant was quite high (above 107 M1s1), indicating that melanin could be a good quencher of this important reactive oxygen species.

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Table 1 Second-order Rate Constants for the Interaction of DOPA-melanin with Free Radicals and Singlet Oxygen Radical

k(M1s1)  20%

OH SO4 N3 CCl3O2 NO2 Trp TyrO Asc RF O2 DQþ PQþ RB2 TriQþ TetraQþ NAD CH2OH CO2 eaq O2(1Dg)

1.5
109 107  108 1.8
108 1.2
108 1.2
106 1.4
107 0.8
107 8
104 <106 5
105 103 103 1.2
106 3
106 6
106 4.6
106 1.1
107 106  107 2.6
108 2
107



Redox Potential of R (V) 2.3 2.4 1.3 7 1.1 7 1.0 0.9 0.8 0.3 0.3 0.3 0.3 7 0.4 7 0.5 0.5 7 0.6 0.9 1.0 1.3 2.9 0.6 7

2.0 1.3

0.4 0.5 0.6

0.7

Note: Rates estimated for melanin monomers, assuming the molecular weight of a representative melanin monomer is 150.

Although the results of the reviewed studies are consistent with melanin being an efficient scavenger of reactive free radicals and a quencher of singlet oxygen, it seems rather unlikely that these properties of melanin play a critical role in the antioxidant action of this pigment. This is because of the limited lifetime of randomly generated reactive species. These species would interact with many constituents of the pigmented cell before having a chance to diffuse to the proximity of the melanosomes (or pigment granules), where they would then need to penetrate the melanosome surface and their membrane, in order to interact with the melanin active groups. Of course, the free radical scavenging and singlet oxygen quenching abilities of melanin may be of importance if the formation of such reactive species is ‘‘site-specific’’, i.e. their generation predominantly occurs within the melanin granule or in its proximity.48 It can be postulated that melanin principally exerts its antioxidant action by binding of redox-active metal ions and photosensitising dye molecules. It has been demonstrated that iron and copper ions that are bound to melanin are inefficient generators of free hydroxyl radicals.49,50 Even though melanin complexes with ferrous and cuprous ions are readily oxidized by molecular oxygen and hydrogen peroxide, very few OH radicals leak out of the melanin

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structure.50 Ferric and cupric ions, on the other hand, after binding to melanin, become significantly more difficult to reduce by mild reductants. Sequestration of iron ions has been identified as a major mechanism for the inhibitory effects of melanin on lipid peroxidation.51,52 Free radical and singlet oxygen-induced oxidation of lipids can schematically be written as: . . LH þ R ! L þ RH . . L þ O2 ! LOO . . LOO þ LH ! L þ LOOH . LOOH þ Fe2þ ! LO þ OH þ Fe3þ S þ hv ! 1S ! 3S; 3 S þ3 O2 ! S þ 1 O2 LH þ 1 O2 ! LOOH 





R is an initiating free radical; LH is unsaturated lipid; LOO , LO , LOOH are the lipid peroxyl and alkoxyl radical, and hydroperoxide, respectively; S, 1S, and 3S are a photosensitising dye molecule in its ground state, singlet and triplet excited states, respectively; 3O2, 1O2 are ground triplet and excited singlet states of molecular oxygen. The inhibitory effect of melanin on oxidation of lipids is described by the following reactions responsible for scavenging of radicals and binding of iron ions: . Mel þ R ! Mel  RðMelox þ RHÞ . Mel þ LOO ! Mel  LOOðMelox þ LOOHÞ Mel þ1O2 ! Melox Mel þ Fe2þ ! Mel  Fe2þ Mel  Fe2þ þ H2 O2 ! site specific formation and decay of hydroxyl radicals Mel þ Fe3þ ! Mel  Fe3þ ðdifficult to reduce by biological electron donorsÞ Mel; Melox are melanin in its initial and oxidised state; respectively Similar mechanism for antioxidant action of melanin can be considered in systems where photo-oxidation reactions are sensitised by positively charged dye molecules. Using laser flash photolysis and EPR-oximetry it has been shown that binding by melanin of two cationic porphyrins dramatically reduced the photosensitising efficiency of the dyes, i.e. their abilities to photo-generate singlet oxygen and free radicals.53 The mechanism of the quenching of excited sates of the dyes bound to melanin was recently determined by femto-second absorption and pico-second emission spectroscopies.54 It has been concluded that such a binding facilitates an ultrafast energy transfer from the excited porphyrin molecule to melanin. The excited energy is then rapidly converted into heat. Because of its speed, the process involves only singlet excited states. No triplet states are formed and, consequently, no photochemistry occurs: Mel þ Sþ ! Mel  Sþ Mel  Sþ þ hv ! 1ðMel  Sþ Þ ! Mel  Sþ þ heat þ S is the ground state of a positively charged photosensitising dye molecule:

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MELANIN PROPERTIES RELEVANT FOR ITS REPORTED PRO-OXIDANT AND PHOTOTOXIC ACTION There is ample literature indicating that melanotic systems may act as prooxidizing agents. Thus, it has been reported that: melanogenic intermediates photo-initiated DNA damage in vitro,55 in human melanocytes from dark skin, UVA induced 40 times more DNA single strand breaks than in melanocytes from light skin,56 UV-irradiated melanin, particularly pheomelanin, sensitised adjacent cells to capsase-3 independent apoptosis,57 and pheomelanin synthesis sensitised melanocytes to oxidative DNA damage by UVA.58 RPE melanin and synthetic DOPA melanin mediated photooxidation of ascorbic acid,59,60 synthetic eumelanin sensitised isolated DNA to induction of the oxidative DNA base damage by UVA irradiation61 and, under certain experimental conditions, isolated RPE melanosomes were apparently able to photosensitise peroxidation of lipids.62 In view of the reported data, it should be explained how melanin, which usually behaves as a good antioxidant, may become a pro-oxidant. It appears that certain experimental conditions can stimulate pro-oxidant properties of melanin. Thus in the presence of photosensitising dye molecules that do not bind to melanin53 or high concentration of electron donors,60 as a result of an overload with redox-active metal ions,40 and after aerobic exposure of melanin to high intensity UV-vis radiation, melanin may behave as an oxidizing system. It was postulated that melanotic system could act as a pro-oxidant if the pigment contained high percentage of the pheomelanin component or was present in a very low aggregation state.63 In addition, results of a recent study suggest that also in vivo melanin may loose its antioxidant efficiency and even become a pro-oxidant.64 As a redox-system, melanin can engage in a number of electron-transfer reactions. It has been demonstrated that melanin mediates aerobic oxidation of ascorbic acid and NADH, and that such electron-transfer reactions are greatly accelerated by irradiation with short-wavelength visible light.59,60 Even in the dark, melanin was shown to reduce ferric complexes with EDTA or other strong chelators.49 Under such conditions, by acting as an electron donor, melanin may drive the Fenton-reaction that generates highly damaging free hydroxyl radicals or other strongly oxidizing species. This is in a striking contrast to melanotic systems, in which the iron ions are tightly bound to the melanin. As discussed above, in the latter, due to site-specific formation and decay of reactive species, virtually no free hydroxyl radicals are generated and very little or no oxidation of other substrates occurs. It is important to realise that even though melanin very efficiently converts energy of the absorbed photons into heat, the residual aerobic photochemistry of melanin may lead to significant changes of the pigment granules that will lower their ability to sequester metal ions and scavenge reactive oxygen species; particularly, if melanin is exposed to high fluxes of intense UV or short-wavelength visible radiation. The aerobic photochemistry of melanin is responsible for generation of superoxide anion and hydrogen peroxide, which, in the presence of

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adventitious redox-active metal ions, will lead to the formation of hydroxyl radicals or other strongly oxidizing species. The mechanism of melanin-induced photo-formation of superoxide anion and hydrogen peroxide is believed to involve the interaction of transient radicals generated in melanin by light with molecular oxygen. Interestingly, simple orto-semiquinones, generated by pulse radiolysis, did not show any significant reactivity with molecular oxygen.65 The higher efficiency of the melanin ortho-semiquinone centres to interact with oxygen may be due to their longer lifetime compared to that of the free orto-semiquinones generated in solution. The light induced oxygen reduction by melanin can be described by the following reactions: MelfðmÞQ; ðnÞQH2 g þ hv ! Melfðm  kÞQ; ðn  kÞQH2 ; ð2kÞSQg Melfðm  kÞQ; ðn  kÞQH2 ; ð2kÞSQg þ O2 ! : Melfðm þ kÞQ; ðn  kÞQH2 g þ ð2kÞO2 Q, QH2 and SQ are the quinone, hydroquinone and semiquinone functional groups of eumelanin, and indices m, n, k denote the number of the corresponding groups in the melanin. For simplicity, in the notation used above, the intrinsic melanin free radicals are ignored and only the extrinsic, i.e. the inducible melanin radicals are indicated. Melanin of the human retinal pigment epithelium (RPE) is unique in that it is formed early during fetal development and serves its biological role(s) for the entire life of an individual.66 This is because melanin in these post-mitotic nondividing cells shows very little, if any, metabolic turnover.67 Using EPR spectroscopy for unambigous detection and quantification of melanin, we have recently shown that the amount of human RPE melanin decreases with age of the donors.64 While the exact molecular and cellular mechanisms of this phenomenon are not clearly understood, it can be postulated that the RPE melanin undergoes in situ photobleaching. Considering the exposure of the outer retina to high accumulative doses of visible light and oxygen concentration, this is not an impossible scenario, particularly if the normal antioxidant defense systems are compromised. It can further be postulated that the decreased amount of melanin in the ageing RPE may reduce the efficiency of melanin to protect the RPE, as well as the entire retina, against oxidative stress. Importantly, chronic oxidative stress to the retina has been suggested to be a contributing factor in the development of age-related macular degeneration.68 Interestingly, not only the amount of melanin decreases in the aging human RPE, also the photoreactivity of the remaining melanin granules increases with age.69 Using EPR-oximetry and EPR-spin trapping, we have shown that purified RPE melanosomes, isolated from human donors of different age, exhibit a distinct age-dependent increase in their efficiency to photo-consume oxygen and to photogenerate superoxide anion. It appears that melanin in the senescent human RPE becomes substantially modified and that this chemical modification increases its aerobic photoreactivity. It can be postulated that, in part, this is due

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to an accumulation of photosensitising component(s) in the aging RPE melanin granule. Cosidering that the aging RPE also accumulates high amounts of lipofuscin, a powerful photogenerator of reactive oxygen species,70 it can be speculated that the risk of oxidative stress in the human retinal is greatly elevated in senescence. ACKNOWLEDGMENT The authors thank the Ministry of Science and Information Technology (KBN), and NIH (R01 EY013722) for providing financial support. REFERENCES 1. Hearing VJ. Unraveling the melanocyte. Am J Hum Genet 1993; 52:1–7. 2. Prota G. Melanins and Melanogenesis. San Diego: Academic Press, 1992. 3. Seiji M. Melanosomes 1980. In: Seiji M, ed. Pigment Cell: Proceedings of the XIth International Pigment Cell Conference. Tokyo: University of Tokyo Press, 1981: 3–13. 4. Boulton M. Melanin and the retinal pigment epithelium. In: Marmor MF, Wolfensberger TJ, eds. The Retinal Pigment Epithelium. Function and Disease. New York: Oxford University, 1998:68–85. 5. Jimbow K, Miyake Y, Homma K, et al. Characterization of melanogenesis and morphogenesis of melanosomes by physicochemical properties of melanin and melanosomes in malignant melanoma. Cancer Res 1984; 44:1128–1134. 6. Prota G. Progress in the chemistry of melanins and related metabolites. Med Res Rev 1988; 8:525–556. 7. Hearing VJ, Tsukamoto K. Enzymatic control of pigmentation in mammals. FASEB J 1991; 5:2902–2909. 8. Ito S, Wakamatsu K. Melanin chemistry and melanin precursors in melanoma. J Invest Dermatol 1989; 92(suppl 5):261S–265S. 9. Nicolaus RA. Melanins. Paris: Hermann Press, 1968. 10. Zeise L, Murr BL, Chedekel MR. Melanin standard method: particle description. Pigment Cell Res 1992; 5:132–142. 11. Zajac GW, Gallas JM, Cheng J, et al. The fundamental unit of synthetic melanin: a verification by tunneling microscopy of X-ray scattering results. Biochim Biophys Acta 1994; 1199:271–278. 12. Gallas JM, Zajac GW, Sarna T, et al. Structural differences in unbleached and mildly-bleached synthetic tyrosine derived melanins identified by scanning probe microscopies. Pigment Cell Res 2000; 13:99–108. 13. Clancy CMR, Nofsinger JB, Hanks RK, et al. A hierarchical self-assembly of eumelanin. J Phys Chem B 2000; 104:7871–7873. 14. Clancy CMR, Simon JD. Ultrastructural organization of eumelanin from Sepia officinalis measured by atomic force microscopy. Biochemistry 2001; 40:13353–13360. 15. Sarna T, Swartz HM. The physical properties of melanin. In: Nordlund JJ, Boissy RE, Hearing JV, King RA, Ortonne JP, eds. The Pigmentary System. Physiology and Pathophysiology. New York: Oxford University, 1998:333–357.

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156

Sarna et al.

16. Sealy RC, Felix CC, Hyde JS, et al. Structure and reactivity of melanins: influence of free radicals and metal ions. In: Pryor WA, ed. Free Radicals in Biology. New York: Academic Press, 1980:209–259. 17. Sarna T, Korytowski W, Pasenkiewicz-Gierula M, et al. Ion-exchange studies in melanins. In: Seiji M, ed. Proceedings of the 11th International Pigment Cell Conference, Sendai, 1980. Tokyo: University of Tokyo Press, 1981:23–29. 18. Sarna T, Plonka P. Biophysical studies of melanin paramagnetic, ion-exchange and redox properties of mealnin pigments on their photoreactivity. In: Eaton SS, Eaton GR, Berliner LJ, eds. Biological Magnetic Resonance vol. 23 Part A. New York: Kluwer Academic/Plenum Publishers, 2004:125–146. 19. Sealy RC. Free radicals in melanin formation, structure and reactions. In: Armstrong D, Sohal RS, Cutler RG, Slater TF, eds. Free Radicals in Molecular Biology, Aging and Disease. New York: Raven Press, 1984:67–75. 20. Enochs WS, Nilges MJ, Swartz HM. A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy. Pigment Cell Res 1993; 6:91–99. 21. Rees JL. The genetics of sun sensitivity in humans. Am J Hum Genet 2004; 75: 739–751. 22. Armstrong BK, Kricker A. The epidemiology of UV induced skin cancer. J Photochem Photobiol B 2001; 63:8–18. 23. Wlaschek M, Tantcheva-Poor I, Naderi L, et al. Solar UV irradiation and dermal photoaging. J Photochem Photobiol B 2001; 63:41–51. 24. Barker D, Dixon K, Medrano EE. Comparison of the responses of human melanocytes with different melanin contents to ultraviolet B irradiation. Cancer Res 1995; 55:4041–4046. 25. Smit NP, Vink AA, Kolb RM. Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6–4 photoproducts by UVB in cultured human melanocytes. Photochem Photobiol 2001; 74:424–430. 26. Kvam E, Dahle J. Pigmented melanocytes are protected against ultraviolet-A-induced membrane damage. J Invest Dermatol 2003; 121:564–569. 27. Tadokoro T, Kobayashi N, Zmudzka BZ. UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin. FASEB J 2003; 17:1177–1179. 28. Young RW. Solar radiation and age-related macular degeneration. Surv Ophthalmol 1988; 32:252–269. 29. Boulton M, Rozanowska M, Rozanowski B. Retinal photodamage. J Photochem Photobiol B 2001; 64:144–161. 30. Nicolas CM, Robman LD, Tikellis G, et al. Iris colour, ethnic origin and progression of age-related macular degeneration. Clin Experiment Ophthalmol 2003; 31: 465–469. 31. Crippa PR, Martini F, Viappiani C. Direct evidence of electron–photon interaction in melanins. J Photochem Photobiol B 1991; 11:371–375. 32. Meredith P, Riesz J. Radiative relaxation quantum yields for synthetic eumelanin. Photochem Photobiol 2004; 79:211–216. 33. Forest SE, Lam WC, Millar DP, et al. A model for the activated energy transfer within eumelanin aggregates. J Phys Chem B 2000; 104:811–814. 34. Girotti AW. Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms. J Photochem Photobiol B 2001; 63:103–113.

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Melanin and Oxidative Reactions

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35. Stepien K, Zajdel A, Wilczok A, et al. Dopamine-melanin protects against tyrosine nitration, tryptophan oxidation and Ca(2þ)-ATPase inactivation induced by peroxynitrite. Biochim Biophys Acta 2000; 1523:189–195. 36. Ostrovsky MA, Sakina NL, Dontsov AE. An antioxidant role of ocular screening pigments. Vision Res 1987; 27:893–899. 37. Scalia M, Geremia E, Corsaro C, et al. Lipid peroxidation in pigmented and unpigmented liver tissues: protective role of melanin. Pigment Cell Res 1990; 3:115–119. 38. Porebska-Budny M, Sakina NL, Stepien KB, et al. Antioxidative activity of synthetic melanins. Cardiolipin liposome model. Biochim Biophys Acta 1992; 1116:11–16. 39. Reszka KJ, Matuszak Z, Chignell CF. Lactoperoxidase-catalyzed oxidation of melanin by reactive nitrogen species derived from nitrite (NO2): an EPR study. Free Radic Biol Med 1998; 25:208–216. 40. Krol ES, Liebler DC. Photoprotective actions of natural and synthetic melanins. Chem Res Toxicol 1998; 11:1434–1440. 41. Ro´zanowska M, Sarna T, Land EJ, et al. Free radical scavenging properties of melanin interaction of eu- and pheomelanin models with reducing and oxidising radicals. Free Radic Biol Med 1999; 26:518–525. 42. Zhang X, Erb C, Flammer J, et al. Absolute rate constants for the quenching of reactive excited states by melanin and related 5,6-dihydroxyindole metabolites: implications for their antioxidant activity. Photochem Photobiol 2000; 71:524–533. 43. Sichel G, Corsaro C, Scalia M, et al. In vitro scavenger activity of some flavonoids and melanins against O2. Free Radic Biol Med 1991; 11:1–8. 44. Sarna T, Pilas B, Land EJ, et al. Interaction of radicals from water radiolysis with melanin. Biochim Biophys Acta 1986; 883:162–167. 45. Dunford R, Land EJ, Rozanowska M, et al. Interaction of melanin with carbon- and oxygen-centered radicals from methanol and ethanol. Free Radic Biol Med 1995; 19:735–740. 46. Koppenol WH, Butler J. Energetics of interconversion reactions of oxyradicals. Adv Free Radic Biol Med 1985; 1:91–131. 47. Sealy RC, Sarna T, Wanner EJ, et al. Photosensitization of melanin: an electron spin resonance study of sensitized radical production and oxygen consumption. Photochem Photobiol 1984; 40:453–459. 48. Sarna T, Swartz HM. Interaction of melanin with oxygen (and related species). In: Scott G, ed. Atmospheric Oxidation and Antioxidants, vol. III. Amsterdam: Elsevier, 1993:129–169. 49. Pilas B, Sarna T, Kalyanaraman B, et al. The effect of melanin on iron associated decomposition of hydrogen peroxide. Free Radic Biol Med 1988; 4:285–293. 50. Korytowski W, Sarna T. Bleaching of melanin pigments. Role of copper ions and hydrogen peroxide in autooxidation and photooxidation of synthetic dopa-melanin. J Biol Chem 1990; 265:12410–12416. 51. Korytowski W, Sarna T, Zareba M. Antioxidant action of neuromelanin: the mechanism of inhibitory effect on lipid peroxidation. Arch Biochem Biophys 1995; 319:142–148. 52. Zareba M, Bober A, Korytowski W, et al. The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems. Biochim Biophys Acta 1995; 1271:343–348.

[pradeepr][D:/informa_Publishing/Zierhut_H4433_112023/z_production/ z_3B2_3D_files/978-1-4200-4433-1_CH0011.3d] [29/1/08/12:1:35] [147–158]

158

Sarna et al.

53. Bielec J, Pilas B, Sarna T, et al. Photochemical studies of porphyrin-melanin interactions. J Chem Soc Farad Trans 1986; 82:1469–1474. 54. Ye T, Simon JD, Sarna T. Ultrafast energy transfer from bound tetra(4-N,N,N,Ntrimethylanilinium) porphyrin to synthetic dopa and cysteinyldopa melanins. Photochem Photobiol 2003; 77:1–4. 55. Koch WH, Chedekel MR. Photoinitiated DNA damage by melanogenic intermediates in vitro. Photochem Photobiol 1986; 44:703–710. 56. Wenczl E, Van der Schans GP, Roza L, et al. (Pheo)melanin photosensitizes UVAinduced DNA damage in cultured human melanocytes. J Invest Dermatol 1998; 111:678–682. 57. Takeuchi S, Zhang W, Wakamatsu K, et al. Melanin acts as a potent UVB photosensitizer to cause an atypical mode of cell death in murine skin. Proc Natl Acad Sci U S A 2004; 101:15076–15081. 58. Kvam E, Dahle J. Melanin synthesis may sensitize melanocytes to oxidative DNA damage by ultraviolet A radiation and protect melanocytes from direct DNA damage by ultraviolet B radiation. Pigment Cell Res 2004; 17:549–550. 59. Glickman RD, Lam KW. Oxidation of ascorbic acid as an indicator of photooxidative stress in the eye. Photochem Photobiol 1992; 55:191–196. 60. Ro´zanowska M, Bober A, Burke JM, et al. The role of retinal pigment epithelium melanin in photoinduced oxidation of ascorbate. Photochem Photobiol 1997; 65:472–479. 61. Kvam E, Tyrrell RM. The role of melanin in the induction of oxidative DNA base damage by ultraviolet A irradiation of DNA or melanoma cells. J Invest Dermatol 1999; 113:209–213. 62. Dontsov AE, Glickman RD, Ostrovsky MA. Retinal pigment epithelium pigment granules stimulate the photo-oxidation of unsaturated fatty acids. Free Radic Biol Med 1999; 26:1436–1446. 63. Nofsinger JB, Liu Y, Simon JD. Aggregation of eumelanin mitigates photogeneration of reactive oxygen species. Free Radic Biol Med 2002; 32:720–730. 64. Sarna T, Burke JM, Korytowski W, et al. Loss of melanin from human RPE with aging: possible role of melanin photooxidation. Exp Eye Res 2003; 76:89–98. 65. Kalyanaraman B, Hintz P, Sealy RC. An electron spin resonance study of free radicals from catechol estrogens. Fed Proc 1986; 45:2477–2484. 66. Marmor MF. Structure, function and disease of the retinal pigment epithelium. In: Marmor MF, Wolfensberger TJ, eds. The Retinal Pigment Epithelium. Function and Disease. New York: Oxford University, 1998:3–9. 67. Boulton M. Melanin and the retinal pigment epithelium. In: Marmor MF, Wolfensberger TJ, eds. The Retinal Pigment Epithelium. Function and Disease. New York: Oxford University, 1998:68–85. 68. Beatty S, Koh H, Phil M, et al. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol 2000; 45:115–134. 69. Ro´zanowska M, Korytowski W, Ro´zanowski B, et al. Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin. Invest Ophthalmol Vis Sci 2002; 43:2088–2096. 70. Ro´zanowska M, Jarvis-Evans J, Korytowski W, et al. Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species. J Biol Chem 1995; 270:18825–18830.

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12 Are Antioxidants Useful in Diabetic Retinopathy? Maria Miranda, Francisco Bosch-Morell, Maria Muriach, Jorge Barcia, and Francisco J. Romero Department of Physiology, Pharmacology and Toxicology, Universidad CEU-Cardenal Herrera, Valencia, Spain

Manuel Diaz-Llopis Department of Surgery, Universitat de Vale`ncia, Hospital General Universitario, Valencia, Spain

Angel Messeguer Department of Biological Organic Chemistry, Centre d’Investigacio´ i Desenvolupament (CID), CSIC Jordi Girona Salgado, Barcelona, Spain

INTRODUCTION Diabetes mellitus is an endocrine disorder resulting primarily from inadequate insulin release (Type 1 insulin-dependent diabetes mellitus) or insulin insensitivity coupled with inadequate compensatory insulin release (Type 2 non-insulin dependent diabetes mellitus). Though strict glycemic control is desirable to prevent diabetes complications, this is not always achievable. Thus, adjuvant therapies are needed to help in preventing or delaying the onset of diabetic complications.

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It has been repeatedly suggested that oxidative stress may play an important role in the pathogenesis of late diabetic complications,1 though it is not clear yet whether increased oxidative stress has a primary role in the pathogenesis of diabetic complications, or if it is simply the consequence of the presence of complications.2 In diabetes, oxidative stress seems to be caused by both an increased production of free radicals and a sharp reduction in antioxidant defences.3 DIABETIC RETINOPATHY AND ANTIOXIDANTS Diabetic retinopathy is the major cause of adult blindness in developed countries. It has been recently reviewed that one third of the diabetic patients will have some degree of retinopathy within the first ten years after the onset of diabetes; twenty years after diabetes onset, 100% of patients with type 1 diabetes and 60% of patients with type 2, will develop diabetic retinopathy; 30% out of them will develop proliferative diabetic retinopathy.4 Retina is the neurosensorial tissue of the eye and is extremely rich in membranes with polyunsaturated lipids. This feature makes it especially sensitive to oxygen free radicals and to lipid peroxidation. There is substantial evidence from animal and clinical studies for both impaired antioxidant defences and increased oxidative damage in the retina of diabetic subjects that may be, in the case of animal studies, reversible with antioxidant supplementation. The antioxidants used in animal models of diabetic retinopathy, include: ascorbic acid, trolox, alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, selenium, vitamin C, alpha-lipoic acid, pycnogenol, green tea, zinc, ebselen, lutein, taurine, etc.5–14 In humans clinical trials with antioxidants have failed to reverse these effects or have shown contradictory results.15 Recently Ceriello16 suggested that new compounds which act as SOD or catalase mimics or superoxide scavengers, may be adequate tools in the prevention of diabetic vascular complications. This author suggested that the hyperglycemia-induced process of overproduction of superoxide seems to be the key event in the activation of all other pathways involved in the pathogenesis of diabetic complications. Peroxynitrite Scavengers The product of the reaction between nitric oxide (
NO), which is also increased in diabetes, and superoxide is peroxynitrite, a potent prooxidant. Therefore, we suggest that the use of peroxynitrite scavengers may also be a good option in the prevention of diabetic complications, and in particular of diabetic retinopathy. These changes in superoxide and peroxynitrite production and the subsequent damage to DNA can also occur in nervous cells. Neural cell apoptosis in human tissues has been demonstrated in diabetic retina17 and more recently in neuropathy18 and the hippocampus of type 1 diabetic rats.19 It seems that neurons begin to die soon after the onset of experimental diabetes in rats. Peroxynitrite has

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been suggested to be involved in apoptotic cell death. Cells that constitutively express
NO synthase, such as neurons, may be more vulnerable to peroxynitriteinduced cell death.20 Neurodegeneration should be considered a component of diabetic retinopathy. An increase in neural cell apoptosis could also contribute to microangiopathy because the apoptotic cells may include glia and if retinal glial cells die, this could lead to a loss of blood retina barrier properties.17 We have previously reported the biochemical (in terms of increase lipid peroxidation and decrease antioxidant defences), and functional changes in the retina of diabetic mice only one week after the induction of diabetes, and the ability of ebselen and lutein to reverse these effects.11,12 These two antioxidants share a common feature; both of them are peroxynitrite scavengers. In a new set of experiments we prolonged diabetes in mice to two and three weeks and decided to use a new antioxidant. CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) is a a-tocopherol analogue that has shown a potent inhibitory activity against lipid peroxidation in rat liver microsomes21 and can act as an efficient scavenger of nitric oxide and peroxynitrite.22 Recently, CR-6 has been shown to reduce apoptosis induced by sodium nitroprusside in retinal photoreceptor cells.23 Hyperglycaemia was obtained by means of the injection of Alloxan. Mice were identified as diabetic based on their blood glucose levels (greater than 16 mM at least four days after alloxan treatment). Animals were divided into subgroups as required by the experiment (control, controlþCR-6, diabetic, diabeticþCR-6). CR-6 was administered daily since day 4 after alloxan or citrate buffer injection until the end of the experiment (2 or 3 weeks after). CR-6 administration did not affect mice glycaemia (Table 1). GPx activity (Table 2), the key enzymatic activity metabolizing cytosolic and mitochondrial hydrogen peroxide, was assayed in eye homogenate without lens after two and three weeks of the induction of diabetes and it decreased in the diabetic condition. CR-6 restored GPx activity, having no effect on glycaemia, though the best results were obtained three weeks after the induction of diabetes. The longer we administered the treatment, the better results were achieved. It is known that MDA determination by high-pressure liquid chromatography is a good marker of oxidative stress involvement in a pathological process.24 Table 1 Glycaemia (mM) of the Different Groups of Mice after Two and Three Weeks of Experiment Glycaemia (mM) after 2 weeks Glycaemia (mM) after 3 weeks Control ControlþCR-6 Diabetic DiabeticþCR-6

7.7 9.1 45.98 33.31

   

Note: *p < 0.05 vs. control and controlþCR-6.

1.8 0.4 16.67* 9.62*

6.6 8.6 36.10 36.00

   

1.5 0.2 7.97* 0.25*

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Table 2 GPx Activity (%) in the Eyes of Different Groups of Mice Treated with CR-6 During Two or Three Weeks Compared with the Control Groups GPx activity (%) Control Diabetic 2 weeks Diabetic 3 weeks DiabeticþCR-6 2 weeks DiabeticþCR-6 3 weeks

100.00 51.49 48.64 69.30 120.26

% % % % %

Figure 1 MDA concentration (mM) in the different groups of mice (*p < 0,05 vs control and diabeticþcr6 two and three weeks; **p < 0,05 vs. control and diabeticþcr6 three weeks).

After two or three weeks of diabetes, MDA levels in retina of diabetic mice (Figure 1) are increased when compared to controls, confirming the role of lipid peroxidation in diabetes. CR-6 is able to prevent this effect. We demonstrate herein in an experimental model of diabetic retinopathy that an antioxidant, like CR-6 which is a peroxynitrite scavenger and that is also able to inhibit photoreceptor apoptosis,23 can reverse the biochemical changes induced by oxidative stress in diabetic retina. These results are similar to those reported by our group with lutein and ebselen11,12 and lead us to conclude that oxidative stress is an early event in diabetic retinopathy. Electroretinogram and Its Utility The electroretinogram (ERG) has been used for decades to study the mechanisms of retinal physiology. In diabetes electrophysiological changes have been shown to occur before clinical evident retinopathy.25,26 A reduction in the a-wave amplitude has been demonstrated in patients with type 1 diabetes25,27 and it has been repeatedly reported that b-wave has reduced amplitude in diabetes in humans and also in animal models of diabetes11,12,25,28 (Figure 2).

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Figure 2 Example of an electroretinogram (b-wave) recorded from a diabetic mice and from a diabetic mice treated with CR-6.

In this sense, the electroretinogram can help us to determine if antioxidant treatment is able not only to restore the biochemical markers of oxidative stress to control values in diabetic retina but also to restore retinal function. So, we recorded serial electroretinograms (ERG) of diabetic and CR-6 treated diabetic mice and measured b-wave amplitude, latency time and implicit time (Figure 3). The abnormalities in the b-wave of the electroretinogram have pointed out to a possible involvement of Muller cells, the site of generation of the b-wave.29 Muller cells possess two other features of certain relevance, they express nitric oxide synthase30 and produce molecules that confer barrier properties to vascular endothelium.31 Maximal electroretinogram amplitude (mostly b-wave) decreased in diabetic animals respect to controls. Latency time and implicit time were not affected by diabetes (data not shown). Abnormalities in the electroretinogram have been reported to precede vasculopathy.32 Indeed, we have reported that b-wave amplitude is affected

Figure 3 b-wave amplitude (mV) from the electroretinograms recorded to control, diabetic and diabetic treated with CR-6 mice. (*p < 0.05 vs control, controlþCR-6 and diabeticþCR-6 two and three weeks).

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already one week after the induction of diabetes11 in a similar animal model, and it is known that most diabetic rodent models develop changes such as pericyte loss, capillary dilation and increased basal membrane thickening, even after a year of diabetes.33 We agree with emergent theories which affirm that changes in neuronal and glial cell function often occur prior to vascular abnormalities, indeed it is possible that changes in retinal neurons and glia contribute to the development of the vascular changes. In Figure 3 we can observe the decreased amplitude of the b-wave of the electroretinogram of diabetic mice only after two and three weeks of the induction of diabetes. Treatment with antioxidants, such as CR-6, restored b-wave amplitude, as well the biochemical changes-related to oxidative stress previously reported, without modifying glucose levels in blood mice. Although we have not noticed any changes in latency times of the electroretinogram, other studies have reported alterations in latency but at later times.34 CONCLUSION Our data are consistent with those of Ceriello’s group, that in a recent review proposed antioxidant therapy for diabetic complications. We suggest antioxidants with peroxynitrite scavenger capacity such as CR-6, ebselen or lutein may also be adequate for the treatment of diabetic complications. CR-6 and ebselen are synthetic antioxidants but lutein35 is a natural antioxidant that can not be synthesized within the body and is provided by dietary intake. Other promising molecules are sulforaphanes.36 Sulforaphanes produce induction of phase 2 genes and result in the elevation of proteins that exert antioxidant activities, including the glutathione synthesis enzymes. We have also demonstrated that GSH metabolism is altered in diabetic retinopathy. Further studies on ebselen, lutein or CR-6 as adequate adjuvant diabetes therapies must be performed to confirm the exact mechanism of action of these antioxidants. ACKNOWLEDGMENTS This work was supported by projects PI03/1710 from the Fondo de Investigacio´n Sanitaria to FB-M, and PRUCH04/30 to FJR. REFERENCES 1. Baynes JW, Thorpe SR. The role of oxidative stress in diabetic complications. Curr Opin Endocrin Diab 1996; 3:277–284. 2. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications. A new perspective on an old paradigm. Diabetes 1999; 48:1–9. 3. Giugliano D, Ceriello A, Paolisso G. Diabetes mellitus, hypertension and cardiovascular disease: which role for oxidative stress? Metabolism 1995; 44:363–368.

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4. Aiello LP, Gardner TW, King GL, et al. Diabetic retinopathy. Diabetes Care 1998; 21:143–156. 5. Kowluru RA, Koppolu P, Chakrabarti S, et al. Diabetes-induced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants. Free Radic Res 2003; 37:1169–1180. 6. Dene BA, Maritim AC, Sanders RA, et al. Effects of antioxidant treatment on normal and diabetic rat retinal enzyme activities. J Ocul Pharmacol Ther 2005; 21:28–35. 7. Yatoh S, Mizutani M, Yokoo T, et al. Antioxidants and an inhibitor of advanced glycation ameliorate death of retinal microvascular cells in diabetic retinopathy. Diabetes Metab Res Rev 2006; 22:38–45. 8. Mustata GT, Rosca M, Biemel KM, et al. Paradoxical effects of green tea (Camellia sinensis) and antioxidant vitamins in diabetic rats: improved retinopathy and renal mitochondrial defects but deterioration of collagen matrix glycoxidation and crosslinking. Diabetes 2005; 54:517–526. 9. Kowluru RA, Odenbach S. Effect of long-term administration of alpha-lipoic acid on retinal capillary cell death and the development of retinopathy in diabetic rats. Diabetes 2004; 53:3233–3238. 10. Moustafa SA. Zinc might protect oxidative changes in the retina and pancreas at the early stage of diabetic rats. Toxicol Appl Pharmacol 2004; 201:149–155. 11. Miranda M, Muriach M, Johnsen S, et al. Oxidative stress in a model for experimental diabetic retinopathy: treatment with antioxidants. Arch Soc Esp Oftalmol 2004; 79:289–294. 12. Miranda M, Muriach M, Roma J, et al. Oxidative stress in a model for experimental diabetic retinopathy II: the utility of peroxynitrite scavengers. Arch Soc Esp Oftalmol 2006; 81:27–32. 13. Di Leo MA, Ghirlanda G, Gentiloni Silveri N, et al. Potential therapeutic effect of antioxidants in experimental diabetic retina: a comparison between chronic taurine and vitamin E plus selenium supplementations. Free Radic Res 2003; 37:323–330. 14. Abiko T, Abiko A, Clermont AC, et al. Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase-C activation. Diabetes 2003; 52:829–837. 15. Marchioli R, Schweiger C, Levantesi G, et al. Antioxidant vitamins and prevention of cardiovascular disease: epidemiological and clinical trial data. Lipids 2001; 36(suppl):S53–S63. 16. Ceriello A. New insights on oxidative stress and diabetic complications may lead to a ‘‘causal’’ antioxidant therapy. Diabetes Care 2003; 26:1589–1596. 17. Barber AJ, Lieth E, Khin SA, et al. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin. J Clin Invest 1998; 102:783–791. 18. Cellek S, Qu W, Schmidt AM, et al. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes. Diabetologia 2004; 47: 331–339. 19. Li ZG, Zhang W, Grunberger G, et al. Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 2002; 946:221–231. 20. Cowell RM, Russell JW. Nitrosative injury and antioxidant therapy in the management of diabetic neuropathy. J Investig Med 2004; 52:33–44.

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21. Irurre J Jr., Casas J, Ramos I, et al. Inhibition of rat liver microsomal lipid peroxidation elicited by 2,2-dimethylchromenes and chromans containing fluorinated moieties resistant to cytochrome P-450 metabolism. Bioorg Med Chem 1993; 1:219–225. 22. Montoliu C, Llansola M, Sa´ez R, et al. Prevention of glutamate neurotoxicity in cultured neurons by 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6), a scavenger of nitric oxide. Biochem Pharmacol 1999; 58:255–261. 23. Sanvicens N, Go´mez-Vicente V, Masip I, et al. Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6. J Biol Chem 2004; 279:39268–39278. 24. Halliwell B. Oxidative stress markers in human disease: application to diabetes and to evaluation of the effects of antioxidants. In: Packer L, Ro¨sen P, Tritschler HJ, King GL, Azzi A, eds. Antioxidants in Diabetes Management. New York: Marcel Dekker, 2000:33–62. 25. Juen S, Kieselbach GF. Electrophysiological changes in juvenile diabetics without retinopathy. Arch Ophthalmol 1990; 108:372–375. 26. Vadala M, Anastasi M, Lodato G, et al. Electroretinographic oscillatory potentials in insulin-dependent diabetes patients: a long-term follow-up. Acta Ophthalmol Scand 2002; 80:305–309. 27. Levin RD, Kwaan HC, Dobbie JG, et al. Studies of retinopathy and the plasma cofactor of platelet hyperaggregation in type 1 (insulin-dependent) diabetic children. Diabetologia 1982; 22:445–449. 28. Hancock HA, Kraft TW. Oscillatory potential analysis and ERGs of normal and diabetic rats. Invest Ophthalmol Vis Sci 2004; 45:1002–1008. 29. Mizutani M, Gerhardinger C, Lorenzi M. Muller cell changes in human diabetic retinopathy. Diabetes 1998; 47:445–449. 30. Newman E, Reichenbach A. The Muller cell: a functional element of the retina. Trends Neurosci 1996; 19:307–312. 31. Ikeda T, Puro DG. Regulation of retinal glial cell proliferation by antiproliferative molecules. Exp Eye Res 1995; 60:435–443. 32. Di Leo MA, Caputo S, Falsini B, et al. Presence and further development of retinal dysfunction after 3-year follow up in IDDM patients without angiographically documented vasculopathy. Diabetologia 1994; 37:911–916. 33. Su EN, Alder VA, Yu DY, et al. Continued progression of retinopathy despite spontaneous recovery to normoglycemia in a long-term study of streptozotocininduced diabetes in rats. Graefes Arch Clin Exp Ophthalmol 2000; 238:163–173. 34. Li Q, Zemel E, Miller B, et al. Early retinal damage in experimental diabetes: electroretinographical and morphological observations. Exp Eye Res 2002; 74: 615–625. 35. Davies NP, Morland AB. Macular pigments: their characteristics and putative role. Prog Retin Eye Res 2004; 23:533–559. 36. Gao X, Talalay P. Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage. Proc Natl Acad Sci U S A 2004; 101:10446–10451.

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13 Macular Degeneration: The Role of Reactive Oxygen Species Michael E. Boulton Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, U.S.A.

INTRODUCTION Age-related macular degeneration (AMD) is the leading cause of registerable blindness in the developed world and its prevalence is likely to increase as a result of increased longevity.1 AMD is currently estimated to affect 12.7 million people in Europe and the USA.2,3 Furthermore, the incidence of AMD within the population appears to be increasing and there appears to be a tendency for a decrease in the age of onset.4 AGE-RELATED MACULAR DEGENERATION AMD is associated with pathological changes in the macular region of the retina which in the later stages lead to loss of central vision. Early stage AMD (often referred to as age-related maculopathy) presents clinically as soft Drusen, choroidal or outer retinal hyperpigmentation and/or depigmentation of the retinal pigment epithelium (RPE). Late stage AMD is broadly subdivided into two pathologies; dry or atrophic AMD and wet or neovascular AMD.5 Dry AMD refers to geographic atrophy of the RPE leading to degeneration of the overlying photoreceptor cells which are dependent on the RPE for their maintenance and survival. In vivo imaging studies demonstrate fundus hypofluorescence, presumed to be derived from the age-pigment lipofuscin, at the edge of the areas of 167

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RPE atrophy.6,7 Wet AMD describes any of the following findings in the macular area: choroidal neovascularization (CNV), hard exudates, fibrous scar, RPE and sensory retinal detachment (with or without hemorrhage). Dry AMD is the most common form of AMD affecting around 85% of AMD sufferers but while wet AMD is less common (*15%) it is the most severe form of the condition and can result in an acute loss of central vision. Epidemiological studies to identify risk factors have been fundamental to our current understanding of the pathobiology of this debilitating condition. However, despite all the studies, the scientific community remains divided as to whether AMD is predominantly of environmental or genetic origin. It seems most likely that AMD is a multigenic condition in which variable combinations of gene defects make the individual more susceptible to environmental insults. Mutations associated with AMD have been located and include the bestrophin, fibulin-3, ELOVLA4, ABCR4, Ccl2, RPE65, superoxide dismutase, APOE, complement factor H genes and HTRA1.8,9 Epidemiological studies such as the Beaver Dam Eye Study, Blue Mountain Eye Study, Chesapeake Bay Study and Rotterdam Study have identified a number of risk factors.10,11 Of these the two most prominent are aging and smoking.12 Cumulative exposure to visible light is widely believed to contribute to AMD although the evidence is equivocal probably due to sampling technique. Other risk factors include pigmentation, diet, pharmacological agents and hypertension.12 PHOTOREACTIVITY OF THE RETINA The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its constant exposure to visible light. Furthermore, environmental risk factors such as diet (reduced intake of antioxidants, eg, carotenoids, vitamin E and vitamin C), smoking, photochemical reactions (e.g. light exposure of the retina) and mutations affecting the antioxidant status provide strong support that the generation of reactive oxygen species (ROS) plays a key role in cumulative oxidative damage to the retina.1,13 It is thought by many that AMD represents an extension of the normal aging process and when this passes a critical point some or all of the symptoms we associate with AMD are manifest. Susceptibility to ROS damage will be dependent on antioxidant potential, efficiency of repair processes and genetic susceptibility. These varied parameters and variations in gene penetrance help explain the variable age of onset of AMD ranging from 50 through to 100þ years. It has long been considered that exposure to visible light, in particular the more energetic ‘‘blue light’’ component of the visible spectrum, makes a major contribution to retinal ageing, RPE dysfunction and the pathogenesis of AMD.1,14,15 The retina contains numerous light absorbing species. These include (a) the visual pigment rhodopsin and its retinal metabolites which collectively absorb across the whole of the visible spectrum, (b) the macular pigment

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carotenoids (zeaxanthin and lutein) which strongly absorb between 400 and 530 nm, (c) flavins and flavoproteins which absorb blue light with maximum at about 430 nm, (d) hemoglobin and other proteins (e.g., mitochondrial enzymes such as cytochrome c) containing porphyrin which absorb strongly around 400 nm and (e) the broad band absorbers lipofuscin and melanin which have been implicated in blue light damage to the retina.16,17 It is these latter broad band absorbers which will be considered in detail in this review. PHOTOREACTIVITY OF LIPOFUSCIN Lipofuscin is predominantly a lipid aggregate that accumulates with increasing age within metabolically active, post mitotic cells in a variety of tissues throughout the body. Lipofuscin, or age pigment as it is often referred to, is generated within the lysosomal system and is considered to represent the incomplete degradation of intracellular or extracellular substrates. The common substrate for lipofuscin formation in most postmitotic tissues is autophagy of spent organelles such as mitochondria, Golgi bodies and endoplasmic reticulum.16,18,19 However, the accumulation of lipofuscin within the retinal pigment epithelium (RPE) is unique in that in this cell type the major substrate for lipofuscin appears to be photoreceptor outer segment tips.16 There is support for this from biochemical analyses of lipofuscin.16,20–22 The predominant chloroform-soluble lipofuscin fluorophores are di-retinal conjugate A2E and other retinal metabolites. However, protein may also represents a component of lipofuscin granules ranging from estimates of 0–75% protein per granule. Proteins have been identified and show diverse origin ranging from photoreceptors to intracellular organelles and enzymes. Furthermore, the proteins exhibit extensive protein modifications, advanced glycation end products and lipid-protein adducts. However, the composition reported to date has to be treated with caution as the lipofuscin preparations are contaminated with cellular debris and a recent study by our group indicates that lipofuscin contains minimal if not zero protein.23 Notwithstanding, lipofuscin is a broad band absorber and contains a variety of chromophores with potential for the generation of reactive oxygen species (ROS).16 RPE lipofuscin granules exhibit a strong broad band emission spectrum with a peak at 600 nm and subsidiary shoulders located at 470 and 550 nm.25 We and others have demonstrated that RPE lipofuscin granules photogenerate the superoxide anion, singlet oxygen and hydrogen peroxide and that production increases with age and at the shorter wavelengths of the visible spectrum.26 Lipofuscin-photosensitized lipid peroxidation was also confirmed by the generation of lipid hydroperoxides and malondialdehyde.26 The reactive oxygen species generated not only oxidise intragranular and extragranular lipids but can also oxidise proteins and carbohydrates plus cause damage to nucleic acids. Analysis of the blue light photoreactivity of isolated human RPE cells demonstrates that the rate of photo-inducible oxygen uptake increases with donor age, and that oxygen uptake is predominantly due to endogenous lipofuscin.27

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Furthermore, the photogeneration of ROS is strongly wavelength dependent with, for example, efficiency increasing with wavelength by a factor of 10 when excitations of 520 and 420 nm are compared.28 This suggests that lipofuscin may make a major contribution to the ‘‘blue light hazard’’ associated with retinal photodamage.29 The biological significance of the photogeneration of ROS by lipofuscin granules is confirmed by the oxidation of lipids and the inactivation of antioxidant (catalase) and lysosomal (acid phosphatase) enzymes.30 This suggests that RPE cells containing significant levels of lipofuscin and exposed to visible light could be compromised by oxidative damage to critical biomolecules. To confirm the phototoxic potential of lipofuscin in a biological system cultured RPE cells containing lipofuscin granules were exposed to ‘‘blue’’ (400–550 nm) and ‘‘amber’’ (550–800 nm) light with an irradiance of 2.8 mW/cm2 for periods up to 48 hours.31,32 Exposure of lipofuscin containing cells to ‘‘blue’’ light caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nononal), protein oxidation of integral proteins (protein carbonyl formation), an increase in lysosomal pH and loss of lysosomal integrity, cytoplasmic vacuolation and membrane blebbing culminating in cell death by 48 hours exposure. By contrast, cells exposed to amber light or maintained in the dark showed no adverse effect. Interestingly, exposure of RPE cells to blue light in the absence of lipofuscin resulted in mitochondrial DNA damage due to the generation of ROS within the mitochondrion which could be blocked by mitochondrial specific antioxidants.32 However, the exposure of RPE cells containing lipofuscin to blue light resulted in nuclear DNA damage in addition to the light-alone effect onmitochondrial DNA (mtDNA) suggesting that lipofuscin was able to generate longer life time lipid peroxides which are able to reach the nucleus.32 The most well characterized photosensitizer of RPE lipofuscin is A2E. Upon photoexcitation with blue light A2E is able to generate singlet oxygen and to promote an apoptotic form of cell death.33–35 However, A2E is only weakly photoreactive in comparison with the hydrophobic components of RPE lipofuscin.16 It is likely that photooxidative forms of A2E such as A2E epoxides are more photoreactive than A2E itself, and it is these which make a significant contribution to lipofuscin phototoxicity. However, it has recently been reported that a considerable proportion of lipofuscin photoreactivity is present at the interface between the chloroform and methanol phases of Folch extracted material (A2E is in the chloroform phase).36 It is an increase in this chloroform insoluble phase which is responsible for the increase photoreactivity of lipofuscin granules with increasing age. Thus the role of A2E within lipofuscin remains equivocal. However, since A2E is a lysosomotrophic agent it may cause RPE dysfunction by destabilizing the lysosomal proton pump and impairing proteolytic degradation.37,38 Thus it is possible that A2E is formed initially to detoxify the highly photoreactive trans-retinal generated during the phototransduction process and is then irreversibly sequestered within lipofuscin to protect the RPE from further damage.

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MELANOSOME PHOTOXICITY Melanosomes are prominent within the young RPE however their number per cell decreases with increasing age.39 Furthermore, with age there is an increase in melanosome/lipofuscin complexes. The role of melanosomes within the human RPE is equivocal but may include absorption of stray light, scavenging of ROS, sequestration of metal ions and the binding of xenobiotics.39,40 Notably the photophysical properties of melanosomes change with increasing age. Blue light photoexcitation of melanosomes results in an age-related increase in both oxygen uptake and the accumulation of superoxide anion spin adducts.41 We have recently confirmed the phototoxicity of aged human melanosomes in an vitro model.42 Cell viability of RPE cells containing aged melanosomes was reduced by 60% after exposure to blue light for 48 hours as compared to 10% for young human melanosomes. This confirmed that the ROS generated by aged melanosomes can contribute to RPE dysfunction and that reduced metal binding by aged melanosomes may also contribute to oxidative stress in RPE cells by making more iron available for the Fenton reaction. ANTIOXIDANTS Although the RPE is constantly exposed to oxidative stress it is able to reduce oxidative damage through an efficient antioxidant system and active repair of oxidatively damaged biomolecules. The RPE relies on both enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic, dietary (ascorbate, a-tocopherol, lutein, zeaxanthin) antioxidants to negate the action of ROS.13,43 While the antioxidant system functions efficiently in the young there appear to be a decreased overall antioxidant activity with increasing age. It would further appear that the RPE is able to reverse nuclear DNA (nDNA) damage, protein oxidation and lipid peroxidation through efficient repair and replacement mechanisms. However, mtDNA repair, as for most other cell types, is poor. This, together with an age-related increase in ROS generators, such as lipofuscin, in the elderly results in the RPE being exposed to ever increasing levels of oxidative stress. Since oxidative damage is thought to play a fundamental role in the aging process it is likely that oxidative stress in the retina will be a major contributor to retinal aging and the pathogenesis of AMD.1,13 SUSCEPTIBILITY AND ADAPTATION OF THE RPE TO REACTIVE OXYGEN SPECIES Recent studies from this laboratory indicate that the RPE has a greater resistance to a diverse range of ROS compared to other cell types localised to tissues similarly exposed to high levels of oxidative stress.44 For instance, the RPE demonstrated a significantly greater resistance to hydrogen peroxide, tertbutylhydroperoxide, Paraquat and sodium arsenite than did liver hepatocytes, alveolar cells or corneal fibroblasts. It would appear that this increased resistance of the RPE to oxidative damage is, at least in part, influenced by the local

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environment. Prior exposure of RPE cells to sublethal hydrogen peroxide demonstrated an adaptive response resulting in a greater resistance to subsequent toxic exposures compared to non-adapted RPE.45 This was associated with a greater catalase, CuZn-superoxide dismutase and glutathione peroxidase enzymatic activity and increased nuclear DNA protection. Interestingly, there was no adaptive benefit for mitochondrial DNA protection or repair in response to sublethal oxidative stress. This identifies the mitochondrion as a potential weak link in the otherwise efficient oxidative stress defenses of the RPE and that this may contribute to retinal aging and age-related disease. CONCLUSION The retina, due to its high oxygen environment, high concentration of unsaturated fatty acids and regular exposure to visible light is an ideal environment for the generation of ROS. Oxidative stress is further increased by the age-related accumulation of photoinducible-generators of ROS (e.g. lipofuscin). There is a wealth of literature supporting a relationship between oxidative stress and agerelated macular degeneration. While genetic and inflammatory components (e.g. complement factor H) are currently in vogue and clearly contribute to the pathobiology of AMD the recent findings that antioxidant therapy has a protective effect46 reinforces the importance of oxidative stress in the aetiology of AMD. Greater research is now required to determine the best dose regime in terms of combinations of antioxidants and their concentration. REFERENCES 1. Beatty S, Koh H, Phil M, et al. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol 2000; 45:115–134. 2. Klein R, Klein BE, Cruickshanks KJ. The prevalence of age-related maculopathy by geographic region and ethnicity. Prog Retin Eye Res 1999; 18:371–389. 3. Klein R, Wang Q, Klein BE, et al. The relationship of age-related maculopathy, cataract, and glaucoma to visual-acuity. Invest Ophthalmol Vis Sci 1995; 36:182–191. 4. Evans J, Wormald R. Is the incidence of registrable age-related macular degeneration increasing? Br J Ophthalmol 1996; 80:9–14. 5. Kanski J. Clinical Ophthalmology: A Systematic Approach. 5th ed. Oxford, UK: Butterworth-Heinemann, 2003. 6. Holz FG, Bellman C, Staudt S, et al. Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci 2001; 42:1051–1056. 7. Lois N, Owens SL, Coco R, et al. Fundus autofluorescence in patients with agerelated macular degeneration and high risk of visual loss. Am J Ophthalmol 2002; 133:341–349. 8. Klaver CC, Allikmets R. Genetics of macular dystrophies and implications for agerelated macular degeneration. Dev Ophthalmol 2003; 37:155–169.

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9. Abrera-Abeleda MA, Nishimura C, Smith JL, et al. Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease). J Med Genet 2006; 43:582–589. 10. Klein R, Peto T, Bird A, et al. The epidemiology of age-related macular degeneration. Am J Ophthalmol 2004; 137:486–495. 11. De Jong PT. Risk profiles for ageing macular disease. Ophthalmologica 2004; 218 (suppl 1):5–16. 12. van Leeuwen R, Klaver CC, Vingerling JR, et al. Epidemiology of age-related maculopathy: a review. Eur J Epidemiol 2003; 18:845–854. 13. Winkler BS, Boulton ME, Gottsch JD, et al. Oxidative damage and age-related macular degeneration. Mol Vis 1999; 5:32. 14. Margrain TH, Boulton M, Marshall J, et al. Do blue light filters confer protection against age-related macular degeneration? Prog Retin Eye Res 2004; 23:523–531. 15. Boulton M, Ro´zanowska M, Ro´zanowska B. Retinal photodamage. J Photochem Photobiol B 2001; 64:144–161. 16. Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal pathology. Exp Eye Res 2005; 80:595–606. 17. Boulton M, Rozanowska M, Wess T. Ageing of the retinal pigment epithelium: implications for transplantation. Graefes Arch Clin Exp Ophthalmol 2004; 242: 76–84. 18. Boulton M. Ageing of the retinal pigment epithelium. In: Osborne N, Chader G, eds. Progress in Retinal Research. Vol. 11. Oxford, New York: Pergamon Press, 1991:125–151. 19. Brizee KR, Ordy, JM. Cellular features, regional accommodation, and prospects of modification of age pigments in mammals. In: Sohal RS, ed. Age Pigments. Amsterdam: Elsevier/North Holland Biomedical Press, 1981:101–154. 20. Renganathan K, Ng K, Davies M, Gu X, Rozanowska M, Rayborn M, Salomon R, Hollyfield J, Boulton M, Crabb J. Does lipofuscin contain protein? Amino acid, protein and ultrastructural analysis of human lipofuscin. Invest Ophthalmol Vis Sci 2007; 48:5059. (ARVO E-abstract). 21. Schutt F, Ueberle B, Schnolzer M, et al. Proteome analysis of lipofuscin in human retinal pigment epithelial cells. FEBS Lett 2002; 528:217–221. 22. Warburton S, Southwick K, Hardman RM, et al. Examining the proteins of functional retinal lipofuscin using proteomic analysis as a guide for understanding its origin. Mol Vis 2005; 11:1122–1134. 23. Eldred GE, Katz ML. Fluorophores of the human retinal pigment epithelium: separation and spectral characterization. Exp Eye Res 1988; 47:71–86. 24. Schutt F, Bergmann M, Holz FG, et al. Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation and products in lipofuscin of human retinal pigment epithelium. Invest Ophthalmol Vis Sci 2003; 44:3663–3668. 25. Boulton M, Docchio F, Dayhaw-Barker P, et al. Age-related changes in the morphology, absorption and fluorescence of melanosomes and lipofuscin granules of the retinal pigment epithelium. Vision Res 1990; 30:1291–1303. 26. Boulton M, Rozanowska M, Rozanowski B, et al. The photoreactivity of ocular lipofuscin. Photochem Photobiol Sci 2004; 3:759–764.

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27. Rozanowska M, Jarvis-Evans J, Korytowski W, et al. Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species. J Biol Chem 1995; 270:18825–18830. 28. Rozanowska M, Wessels J, Boulton M, et al. Blue light-induced singlet oxygen generation by retinal lipofuscin in non-polar media. Free Radic Biol Med 1998; 24:1107–1112. 29. Ham WT Jr, Mueller HA, Ruffolo JJ Jr, et al. Basic mechanisms underlying the production of photochemical lesions in the mammalian retina. Curr Eye Res 1984; 3:165–174. 30. Wassell J, Davies S, Bardsley W, et al. The photoreactivity of the retinal age pigment lipofuscin. J Biol Chem 1999; 274:23828–23832. 31. Davies S, Elliott MH, Floor E, et al. Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells. Free Radic Biol Med 2001; 31:256–265. 32. Godley B, Shamsi F, Liang F-Q, et al. Blue light induces mitochondrial DNA damage and free radical production in epithelial cells. J Biol Chem 2005; 280:21061–21066. 33. Schutt F, Holz F, Kopitz J, et al. A2E, a retinoid component of lipofuscin, is damaging to RPE cells. Invest Ophthalmol Vis Sci 2000; 41:2303–2308. 34. Sparrow JR, Nakanishi K, Parish CA. The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 2000; 41:1981–1989. 35. Sparrow J, Cai B. Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2. Invest Ophthalmol Vis Sci 2001; 42:1356–1362. 36. Rozanowska M, Pawlak A, Rozanowski B, et al. Age-related change in the photoreactivity of retinal lipofuscin granules: role of chloroform-insoluble components. Invest Ophthalmol Vis Sci 2004; 45:1052–1060. 37. Holz FG, Schutt F, Kopitz J, et al. Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin. Invest Ophthalmol Vis Sci 1999; 40:737–743. 38. Bermann M, Schutt F, Holz FG, et al. Does A2E, a retinoid component of lipofuscin and inhibitor of lysosomal degradative functions, directly affect the activity of lysosomal hydrolases? Exp Eye Res 2001; 72:191–195. 39. Boulton ME. The role of melanin in the RPE. In: Marmor M, Wolfensberger T, eds. The Retinal Pigment Epithelium. Oxford University Press, 1998:68–85. 40. Sarna T. Properties and function of the ocular melanin. A photobiophysical view. J Photochem Photobiol B 1992; 12:215–258. 41. Rozanowska M, Korytowski W, Rozanowski B, et al. Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin. Invest Ophthalmol Vis Sci 2002; 43:2088–2096. 42. Rozanowski B, Cuenco J, Davies S, Shamsi F, Zadlo A, Dayhaw-Barker P, Rozanowska M, Sarna T, Boulton M. The phototoxicity of aged human retinal melanosomes. Photochem. Photobiol. 2007; 83 (In Press). 43. Boulton M, Dayhaw-Barker P. The role of retinal pigment epithelium: topographical variation and ageing changes. Eye 2001; 15:384–389. 44. Jarrett S, Albon J, Boulton M. The contribution of DNA repair and antioxidants in determining cell type-specific resistance to oxidative stress. Free Radic. Res. 2006; 40:1155–1165.

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45. Jarrett S, Boulton M. Antioxidant up-regulation and increased nuclear DNA protection play a key role in adaptation to oxidative stress in epithelial cells. Free Radical Biol Med 2005; 38:1382–1391. 46. Age-Related Eye Disease Study Research Group. A randomized, placebocontrolled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol 2001; 119:1417–1436.

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14 Retinal Ischemia and Oxidative Stress Neville N. Osborne Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, U.K.

INTRODUCTION Retinal ischemia, in its various guises, is a common clinical entity and, due to relatively ineffective treatment, remains a common cause of visual impairment and blindness in the industrialized world. In recent years, a considerable amount of data has accumulated on the subject of retinal ischemia, particularly regarding the contribution of glutamate-induced excitotoxicity to the ischemic injury, and although, to date, this laboratory-based research has had questionable clinical impact, researchers remain optimistic that a better understanding of the fundamental pathophysiology of retinal ischemia will lead to better management and an improved clinical outcome. In this article the role of oxidative stress in the pathogenesis of retinal ischemia will be focussed upon. ISCHEMIA The word ischemia was coined by Virchow, who combined the Greek iskho, meaning ‘‘I hold back’’, with ha´ima, meaning ‘‘blood’’. Hence, ischemia refers to a pathological situation involving an inadequacy (not necessarily a complete lack of) blood flow to a tissue, with failure to meet cellular energy demands. Ischemia should be distinguished from anoxia (a complete lack of oxygen) and hypoxia (a reduction in oxygen): ischemia always has a component of hypoxia/ anoxia, but hypoxia/anoxia does not imply ischemia (Figure 1). For example, the retina may become hypoxic at high altitudes, producing loss of vision, but it is

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Figure 1 Terminology.

Figure 2 Ocular diseases where retinal ischaemia is implicated (retina blood supplies: retinal, choroidal, optic nerve).

not ischemic. Similarly, anaemia (generally a reduction, rather than complete absence of haemoglobin) is always a component of ischemia, but not vice versa. Ischemia deprives a tissue of three requirements: oxygen, metabolic substrates, and removal of waste products. The loss of these requirements will initially lower homeostatic responses and with time will induce injury to the tissue. If withheld for a sufficiently long time the tissue will die (an infarct). It is convenient to think of the retina as having three blood supplies: the retinal blood supply, the choroidal blood supply and the optic nerve head blood supply. Ischemia cause by one or the other of these blood supplies being affected has been implicated in a variety of ocular diseases (Figure 2).

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OXIDATIVE STRESS Oxidative stress is imposed on cells as a result of one of three factors: (1) an increase in oxidant generation, (2) a decrease in antioxidant protection, or (3) a failure to repair oxidative damage (Figure 1). Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Examples are hydroxyl radical, superoxide, hydrogen peroxide, and peroxynitrite. The main source of ROS in vivo is aerobic respiration, although ROS are also produced by peroxisomal b-oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism, and tissue specific enzymes. Under normal conditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD), catalase, or glutathione (GSH) peroxidase. The main damage to cells results from the ROSinduced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA. Additionally, oxidative stress and ROS have been implicated in retinal ischemic disease states, such as occurs in diabetic retinopathy, ophthalmic artery occlusion, central retinal artery occlusion and glaucoma.

RELATIVE RESISTANCE OF THE RETINA TO ISCHEMIA COMPARED WITH THE BRAIN One striking difference between the retina and brain is the relative resistance of the retina to an ischemic insult. There is universal agreement that the retina survives considerably longer than the brain (Table 1). A few minutes of cerebral ischemia in the human results in widespread injury and death, but the primate retina can suffer up to 100 minutes of central retinal artery occlusion without permanent injury.1 Furthermore, the retina exhibits a regionalised sensitivity to ischemia, with the outer layers less sensitive than the inner layers. Significantly neuroglobin, a neurone-specific respiratory protein distantly related to haemoglobin and neuroglobin, is present in high amounts in the retina. The estimated concentration is 100-fold greater than in the brain and particularly located to the retinal photoreceptors.2 In ischemic models on holangiotic retinae that obstruct both retinal and choroidal, such as the rat elevated pressure-induced model,3 the photoreceptors incur less functional and structural injury than the inner retina. In contrast, in the merangiotic rabbit retina the reverse situation occurs, indicating fundamental inter-species variation in pathophysiology.4,5 The explanation for this phenomenon in vascularised retinae remains obscure, but may relate to the exceptional ability of the photoreceptors to extract energy from available sources anaerobically.6,7 Similarly, this explanation may extend to the other retinal layers

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Table 1 Retinal Tolerance Time (Minimum Period of Ischemia to Cause Irreversible Damage) in Experimental Models of Retinal Ischemia(a)

Tolerance time(b)

Model of retinal ischemia

Species

Animal centrifugation or asphyxiation Section of central retinal artery Occlusion of central retinal artery

Rat Rat Monkey Monkey Rat Rat Rat

<3 min <4–5 h 2–3.5 h 100 min <30 min <60 min 15 min

Mouse Rabbit Cat Monkey Rabbit

30–60 min <30 min 60–90 min <90 min 60–90 min 40 min

Ligature of ophthalmic vessels Occlusion of optic nerve bundle Elevated intraocular pressure (
systolic blood pressure)

In vitro ischemia

(a) Adapted in part from.1 (b) Retinal tolerance time is the minimum period of ischemia required to cause irreversible damage.

when survival times are compared with the brain. This possible explanation is based on three facts: (1) local energy: substrates are present (the vitreous contains considerable amounts of glucose, and there is a species-dependent store of glycogen in the retina)8; (2) these energy substrates are depleted during periods of retinal ischemia,9,10 (3) the isolated retina can efficiently extract adenosine triphosphate (ATP) from glycolysis as long as glucose is plentiful, even in the complete absence of oxygen.6,7 Alternatively, it is possible that retinal neurones are intrinsically more resistant to ischemia than cerebral neurones; however this ‘‘explanation’’ tells us nothing of any underlying mechanisms. Finally, the so called no-reflow phenomenon may explain the discrepancy.11,12 This term describes the situation in the oedematous brain after a period of ischemia: the rigid cranium cannot accommodate the swollen brain, which compresses the microvasculature producing ongoing ischemia despite restoration of macroscopic blood flow. In contrast, upon cessation of retinal ischemia, oedematous retina does not obstruct the microvasculature, as the thin retinal tissue has space (the vitreous cavity) to expand into. Any contribution of retinal metabolism to its relative insensitivity to ischemia is an area that could conceivably be clinically exploited, but to date has received little attention. Buchi et al. (1991)13 using a pressure ischemia model in rats showed that if 5% dextrose was used as the infusate instead of 0.9% saline, then the histological injury to the retina for a given duration of ischemia was

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markedly reduced. Romano et al. (1993)14 using a photothrombosis retinal ischemia model in rats, showed that a single intravitreal injection of glucose immediately prior to the ischemic insult attenuated the early histological changes compared to the saline-injected eyes. Similarly, using an isolated retina model, Romano et al. (1998)15 showed that glucose markedly reduced N-methyl-Daspartate (NMDA)-induced excitotoxic injury. Results from our own laboratory support the findings of Buchi et al. (1991)13 and demonstrate remarkable functional (by electroretinography) and structural protection when glucose is used as the infusate.16 Furthermore, when hypoglycaemia is induced or a nonmetabolisable analogue of glucose is used (2-deoxyglucose), the retinal injury is greater than that observed with saline17 supporting the notion that the protective mechanism is metabolic. This area of research awaits further study. RETINAL ISCHEMIA AND NEUROLOGICAL MECHANISMS Mammalian retinal ischemia results in irreversible morphological and functional changes. These are the consequence of depleted ATP stores, due to deprivation of both glucose and oxygen, though transient loss of these substrates is not immediately lethal. The cell death is the result of an extremely complex (not completely understood) cascade of biochemical responses initiated by energy failure. The tissue damage and functional deficits that follow periods of transient ischemia reflect the combined effects of several, often interrelated pathophysiological pathways. These result in drastic changes in ion movements, neurotransmitter levels and metabolites. Reperfusion may also damage cells that, until that moment, had sustained only reversible injury and were potentially salvable. This concept of oxygen restoration to ischemic tissue amplifying injury sustained during oxygen deprivation has its origins in experimental myocardial studies from the 1960s.18 Leakage of LDH, a marker of cell death caused by retinal ischemia, increases after reintroduction of oxygen in the continued glucose absence.19 It is useful to consider that stroke lesions consist of a densely ischemic focus, the ischemic core, surrounded by a better perfused area, the ischemic penumbra.20 Cells in the focus are usually doomed unless reperfusion is quickly instituted. In contrast, penumbral cells may remain viable for several hours and can be saved by reperfusion or by drugs that prevent the infarction extending into the penumbral zone.20 During the past decade a considerable amount of experimental work has been devoted to the elucidation of the mechanisms of ischemic neuronal injury, but there is still much debate over the underlying processes causing the injury. One area of interest is the role of glutamate and aspartate, whose extracellular concentrations increase markedly during ischemia.21 It is generally accepted that glutamate release during the early phase of brain ischemia triggers events leading to irreversible injury, not only in those areas in which oxygen supply is critically reduced, but also in regions of seemingly less disturbed energy

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metabolism i.e. the penumbra of focal ischemia. However there is still much controversy over the extent of their role in the pathophysiology of neural ischemia. Moreover, neuronal bodies (grey matter) and axons (white matter) are not affected in precisely the same way by ischemia.22 It is important to remember that much of the work studying ischemic neuronal and axonal damage has been carried out on tissues derived from defined brain regions. So care is important when drawing comparisons with what happens in the retina. In particular, the way photoreceptors respond to ischemia in light and dark conditions may be unique. Moreover, the thinness of the retina, its well defined blood systems and the large glycogen supply associated with the Mu¨ller cells provides the tissue with a unique energy supply when compared with the brain, so making the response of tissues to ischemia not the same. THE ROLE OF FREE RADICALS IN RETINAL ISCHEMIA Many cascades generated by glutamate and glucose/oxygen deprivation result in the formation of free radicals23 and it has been proposed that free radicals are important mediators in damage caused by retinal ischemia.24,25 Reperfusion injury after ischemia appears paradoxical, but oxygen-derived and other free radicals are principally formed when reduced compounds, which accumulate during ischemia, are reoxidized (Figures 3, 4). There is evidence that this free radical burst, produced during the early stage of reperfusion, overwhelms normal cellular antioxidant defense mechanisms, causing oxidative stress and a variety of types of tissue injury.26 There are many ways in which free radicals can be formed during ischemia-reperfusion, but the burst of superoxide radicals (·O2) which occurs during the early stage of reperfusion is thought to occur by the following pathway. During ischemia, degradation of ATP leads to the formation of hypoxanthine, and increases in intracellular calcium in neurones activate the Ca2þ-dependent protease calpain. Calpain converts xanthine dehydrogenase into xanthine oxidase and upon reperfusion the latter enzyme oxidizes the accumulated hypoxanthine to uric acid resulting in the release of O2. These two molecules react by the Haber-Weiss mechanism to yield the highly toxic hydroxyl radical (·OH). This reaction is catalyzed by iron, which is released from its protein-bound stores at the low pH generated during ischemia. In addition, O2 interacts with nitric oxide (NO·), which is produced in considerable amounts following ischemia, leading to the formation of peroxynitrite, nitrosyl radical and eventually OH.26 It is not just from the mitochondrial systems of neuronal cells that free radicals are generated, activation of glial cells and infiltrating leukocytes release inflammatory mediators, such as arachidonic acid, nitric oxide and cytokines, which all play major roles in the formation of free radicals following ischemia. An early indication that excessive free radical formation may be detrimental to the retina was the finding that iron-ascorbate perfusion rapidly attenuates the isolated rat retina’s b-wave and causes lipid peroxidation.27 However, the first studies to provide evidence for the involvement of free

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Figure 3 Formation of reactive oxygen species and antioxidant mechanisms. Oxygen is converted to superoxide (O2·) by oxidative enzymes in the endoplasmic reticulum (ER), mitochondria, plasma membrane, peroxisomes and cytosol. Superoxide is converted to H2O2 by dismutation and then to OH· by the copper and iron catalysed Fenton reaction. H2O2 is also derived directly from oxidases and peroxisomes. Free radicals cause lipid peroxidation, protein damage and DNA damage. Superoxide catalyses the reduction of iron so enhancing OH generation by the Fenton reaction. The major antioxidant enzymes are superoxide desmutase, catalase and glutathione peroxidase. GSH – reduced glutathione; GSSG – oxidised glutathione; NADPH – reduced nicotinamide adenine dinucleotide phosphate.

radicals in retinal damage after ischemia were performed by Szabo and colleagues who showed that administration of superoxide dismutase (SOD) affords protection against ischemia-induced histological damage28 and the ionic imbalance that occurs in the reperfusion period.29 The work of Szabo and colleagues showed, albeit indirectly, that the superoxide free radical is generated in such quantities during retinal ischemia-reperfusion that the normal endogenous levels of superoxide dismutase expressed by the retina are overwhelmed and are unable to protect the tissue from oxidative damage caused by this radical. Subsequent studies supported the involvement not only of superoxide in ischemic injury to the retina but also of hydrogen peroxide and the hydroxyl radical.30–32 Furthermore, the inability of endogenous free radical quenching mechanisms to cope with the demand posed following ischemia is illustrated by the capacity of a variety of free radical scavengers, such as extract of Ginkgo biloba,28 a-lipoic acid,33 vitamin E,34,35 thioredoxin,36 an ascorbic acid

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Figure 4 Production and reactions of nitrogen-derived radicals. (A) Nitric oxide synthase (NOS) produces nitric oxide radicals in the conversion of L-arginine to L-citrulline. This compound acts as an important homeostatic modulating agent under physiological conditions via its vasodilator, antioxidant, antiplatelet and antineutrophil actions. In the event that superoxide radicals are present (·O2), usually as a result of rapid tissue reoxygenation subsequent to an ischemic event, peroxynitrite is formed which rapidly decomposes to highly reactive oxidant species that can cause tissue injury. Under physiological conditions, there is a critical balance between cellular concentrations of NO, ·O2and superoxide dismutase activity which favour NO production. In pathological conditions such as reperfusion following an ischemic event, the formation of ONOO is favoured. The latter compound can be rapidly detoxified if it is combined with reduced glutathione (GSH) to form S-nitrosoglutathione (GSNO), but this depends upon the cellular antioxidant defence system being functional and this is generally overwhelmed during tissue reperfusion. (B) Reactions and formation of nitric oxide radicals as a result of ischemia-reperfusion. The combined production of nitric oxide derived radicals and failure of cellular antioxidant defence will lead to widespread macromolecular damage and cell death.

derivative,37 mannitol38 and the iron chelator desferrioxamine39 to protect the retina from ischemia-reperfusion injury. Although it can be inferred from all of these studies that there is an elevated level of free radicals in the retina after ischemia, direct measurement of free radical formation has only been performed by Muller and co-workers,30 who showed increased free radical formation both during and after ischemia, and by Szabo et al (1997)40 who subjected diabetic retinas to ischemia and documented increased levels of free radicals during the reperfusion phase.

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As previously mentioned, xanthine oxidase has been established as an important source of oxygen free radicals in certain ischemia-reperfusion injuries and there is evidence for a similar action in the retina. Xanthine oxidase activity has been reported to increase 5-fold within 10 minutes of reperfusing the ischemic rat retina, while concentrations of hypoxanthine and xanthine, respectively the substrate and product of xanthine oxidase, increase in a time-related fashion following ischemia-reperfusion32,41 and enhance free radical formation.42 Furthermore, administration of allopurinol or oxypurinol, blockers of xanthine oxidase, both result in significant improvement of the ERG after ischemia.41,43 These combined data suggest that xanthine oxidase-mediated processes contribute to the functional anomalies of retinal ischemia. However, there is limited evidence to suggest that xanthine oxidase may not be an important source of free radicals after retinal ischemia. Faberowski et al (1989)44 reported that allopurinol provided no significant protection against ischemia produced by transient ligation of the optic nerve, while Szabo et al (1993)45 found that allopurinol was only neuroprotective when administered in combination with extract of Ginkgo biloba. The reported variations may be due to either the allopurinol doses used, or the manner of its administration, or to the methods of evaluating injury. One potential source of free radicals in the retina following ischemia are polymorphonuclear leukocytes. In the brain, neutrophils oxidize NADPH to generate superoxide and are important free radical donors during and after focal ischemia.46 Agents that prevent the accumulation or activation of neutrophils are protective.47 In the retina, infiltration of neutrophils occurs during the early phase of reperfusion, probably in response to increased levels of cytokines and free radical formation. Although there is no direct evidence for increased free radical formation from neutrophils, blocking leukocyte accumulation has been shown to afford protection to the ischemic retina.48 Oxygen-derived free radicals cause extensive cellular damage in the brain. One of the main mechanisms by which this occurs is by attacking unsaturated fatty acids, which leads to lipid peroxidation of membranes. This will result in loss of membrane fluidity, cell swelling, oedema and feed forward production of more free radicals. Many additional mechanisms of damage have been ascribed to free radicals generated during ischemia-reperfusion, including attacking sulphhydryl protein bonds, which leads to the destruction of amino acids and polypeptide chains, fragmentation of DNA molecules, which leads to activation of poly(ADP-ribose) polymerase, activation of cytokines and NF-kB, which are likely to be instrumental in upregulations of iNOS and COX-2 and subsequent release of glutamate, and effects on Ca2þ homeostasis.47 Interestingly, a clear association between neuronal cell death and formation of free radicals and lipid peroxides in the retina subjected to ischemia-reperfusion has only recently been demonstrated (Celebi et al., 2002).35 In all of these studies, ischemia-reperfusion induced free radical formation, lipid peroxidation and neuronal injury and were largely preventable by administration of free radical scavengers35,36 and a novel metal chelate.32

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THE FREE RADICAL NITRIC OXIDE IN RETINAL ISCHEMIA Nitric oxide is an important neuromediator throughout the CNS, and is implicated in many physiological processes in the retina.49 It is synthesised from L-arginine via the action of nitric oxide synthetase (NOS) and three distinct isoforms of NOS have been characterised. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are Ca2þ-dependent and are constitutively expressed by a variety of nervous tissues and by endothelial cells of blood vessels, respectively. Inducible or immunologic NOS (iNOS) is Ca2þ-independent and is not generally found under normal physiological conditions but, as the name suggests, is induced by certain stimuli. In the normal retina, nNOS is synthesised by a variety of neurones,50 eNOS is only found in retinal vessels,51 while iNOS has been detected at low levels in Mu¨ller cells and the RPE.52 In the last decade, a significant body of evidence has indicated an involvement of NO in the pathogenesis of ischemic damage in the brain and retina. In models of focal and global ischemia in the brain, all three NOS isoforms are induced in the post-ischemic period, leading to a sustained production of NO. Protein levels of nNOS and eNOS are elevated shortly after the onset of ischemia, presumably due to the rise in intracellular Ca2þ, while the induction of iNOS is delayed by several hours. Many studies have attempted to determine the impact of NOS modulation on brain ischemia and, in general, the results indicate that nNOS and iNOS are detrimental but eNOS is beneficial to neurones.53 Increases in expression of all of the NOS isoforms have also been reported in the retina post-ischemia, although inevitably there is some variation between studies. This is likely the consequence of the different models used and the different durations of the ischemic insults. Increases in nNOS, generally in cells of the inner retina, have been shown following high IOP-induced ischemia54 and in the 2-vessel occlusion model of ischemia,55 although surprisingly, not after optic nerve bundle occlusion.56 In fact, levels of nNOS were seen to decline appreciably in this model of ischemia.57 Increases in eNOS have been detected after high IOP ischemia51 and optic nerve ligation.57 Substantial elevations of iNOS in the retina have been shown using the high IOP model of ischemia,58 in the 2-vessel occlusion model of ischemia,55 after optic nerve bundle occlusion,56 in a murine model of ischemic proliferative retinopathy,59 and in patients with diabetic retinopathy and ocular ischemic disease.60 The source of NO produced by iNOS are activated macrophages and other invading inflammatory cells, as well as retinal astrocytes and Mu¨ller cells.58 The question as to whether the post-ischemia increase in NO production is beneficial or detrimental to the retina has proved difficult to answer. Some studies suggest that activation of NOS causes cell death, whereas others have reported the opposite. This is partly due to the lack of specificity of the majority of pharmacological tools employed to date, partly due to the variety of different

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experimental protocols, and partly to the complexity of the NO system in the retina: all three isoforms of NOS are present and inducible in different cell types of the retina and the different NOS isoforms will be preferentially involved in the different stages of ischemia-reperfusion. The case for a positive effect of NO on the post-ischemic retina was first advanced by Veriac et al. (1993)61 who showed that recovery of the b-wave of the ERG in rabbits was more rapid after administration of a nitric oxide donor, yet delayed after administration of the NOS inhibitor L-NNA. These results were duplicated in rats by Hangai et al. (1999)57 who showed additionally that histological as well as functional damage to the retina was aggravated by L-NNA. The authors suggested that the detrimental effect of L-NNA results from delaying the onset of retinal reperfusion. It would seem logical to suppose, therefore, that activation of eNOS shortly after ischemia increases retinal perfusion and contributes to neuronal survival, as has been shown in the brain.62 However, L-NNA is also detrimental to the retina in an isolated retinal preparation of ischemia,63 and moreover, unlike in the brain, eNOS-deficient mice are not more susceptible to retinal ischemic damage.64 The group of Lipton have conducted a number of detailed studies over recent years which demonstrate, also, that charged cellular NO-derived equivalents (NOþ or NO) have the ability to S-nitrosylate (transfer the NO group to) the sulfhydryl group of a cysteine residue, specifically in the active site of the ‘‘apoptotic executioner’’ enzyme, caspase-3 and on the external face of the NMDA receptor. In both cases, activity of the target proteins are decreased and neuroprotection is afforded. NO itself, however, shows no such activity, preferentially acting on superoxide species to produce peroxynitrite which can cause neurodegeneration.47 Notwithstanding these reports, the majority of studies, as in the brain, have shown that inhibition of NOS leads to histological and functional protection of the ischemic retina. The nonspecific inhibitors aminoguanidine,58 L-NAME,65 L-NNA66 and L-NMMA66 have all been shown to reduce neuronal injury caused by ischemia/reperfusion. The involvement of the iNOS isoform in the injury process can be inferred from the study of Hangai et al. (1996),56 who showed that administration of the relatively specific inhibitor of iNOS, L-NIO, partly ameliorated damage caused by CRAO. Clearer proof was provided by the study of Neufeld et al. (2002),58 who found that chronic administration of the potent and selective iNOS inhibitor, SC-51, afforded substantial protection against retinal ischemia, and a greater degree of protection than aminoguanidine. It is also of significance that in elevated IOP models of glaucoma there is an activation of iNOS in rat retinal non-neuronal cells and the formed NO causes degeneration of ganglion cells.67 It should also be borne in mind that activation of the NMDA receptor during an ischemic insult will lead to an increase in the levels of intracellular calcium and this itself is known to induce expression of and activate each NOS isoform, either directly (nNOS) or via the activation of calcium-dependent protein kinase C (PKC) isoforms.47

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PROBABLE EVENTS ASSOCIATED WITH RETINAL ISCHEMIA The complex pathophysiology of retinal ischemia underscores the dynamic relationship of the retina and its vascular supply. Incipient reduction of choroidal, retinal or optic nerve head blood supply alone will cause different retinal pathologies to that occurring when all supplies are affected at the same time. When blood flow is disturbed, the normal homeostatic mechanisms linking metabolic demands and haemodynamics are altered, affecting the different retinal cell-types in some way. Depletion of cellular energy stores beyond a critical threshold (that may vary for every retinal cell-type) triggers waves of depolarisation and a series of molecular events known as the ‘‘ischemic cascade’’ (that may vary for every retinal cell-type), irrespective of the initial mechanism of vascular compromise. Thus different types of vascular changes in the optic nerve head blood supply are suggested to lead to similar retinal pathologies, as proposed to be the case in glaucoma.68 The complex pathophysiology of retinal ischemia underscores the dynamic relationship of the retina and its vascular supply. Incipient reduction of choroidal, retinal or optic nerve head blood supply alone will cause different retinal pathologies to that occurring when all supplies are affected at the same time. When blood flow is disturbed, the normal homeostatic mechanisms linking metabolic demands and haemodynamics are altered, affecting the different retinal cell-types in some way. Depletion of cellular energy stores beyond a critical threshold (that may vary for every retinal cell-type) triggers waves of depolarisation and a series of molecular events known as the ‘‘ischemic cascade’’ (that may vary for every retinal cell-type), irrespective of the initial mechanism of vascular compromise. Thus different types of vascular changes in the optic nerve head blood supply are suggested to lead to similar retinal pathologies, as proposed to be the case in glaucoma.68 Although early perfusion may prevent permanent retinal injury, the limited tolerance of neurones to hypoxic stress imposes a restricted time window on thrombolytic therapy, although this is the most effective way to treat stroke.69 A time-defined therapeutic window could be wider for retinal than for brain neurones. The possible causes of retinal ischemia suggest a number ofobvious targets for therapy (Figure 6), one being simply to restore the nutrient supply. However, such an approach is always associated with a temporal delay, during which time the retina undergoes ischemic damage. Therefore, specific pharmacological strategies need to be developed to arrest any of the many putative cascades generated during ischemia. The ‘‘ischemic cascade’’ is a complex succession of interrelated pathological changes at the cellular and molecular level whose knowledge derives mainly from studies on brain tissues.47,70,71 This cascade may be summarised as follows (Figure 5). Reduction of energy caused by loss of normal blood flow results in cell membrane depolarisation and excessive release of neurotransmitters. These include excitatory amino acids such as glutamate that activate specific receptors, leading to deranged ion fluxes and the detrimental effects of intracellular Ca2þ

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Figure 5 Cascade of events thought to occur in retinal ischemia. An interruption in the supply of blood to the retina leads to tissue ischemia which causes rapid failure of energy production and oxidative stress. This causes a number of biochemical events as outlined in the figure. Key steps include the failure of the Naþ/Kþ-ATPase pump, membrane depolarisation, cytoplasmic accumulation of sodium and calcium ions and the formation of destructive free radical species. The summed cellular response of these processes, if left unchecked, is cell death. This can occur by the classical and rapid necrotic process or by longer-duration apoptosis.

accumulation. Ca2þ interacts with intracellular proteins causing, amongst other events, degradation of structural cellular elements. Impairment of normal homeostasis then leads to cytotoxic oedema and anaerobic glycolysis will lead to progressive acidosis especially during prolonged hypoxia, compounding oedema formation and causing mitochondrial dysfunction. An accumulation of NO and the degradation of membrane phospholipids will contribute to the generation of neurotoxic free radical species. Inflammatory mediators will be activated to cause secondary neuronal injury through release of cytokines, phospholipases and chemokines. Superimposed waves of gene expression and growth factors will eventually induce cell death, mediated by a family of cysteine proteases (caspases). It should be emphasised, however, that precise knowledge of the temporal relationship and pathological relevance of these myriad processes remains unclear.

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COUNTERACTING OXIDATIVE STRESS AS A NEUROPROTECTIVE STRATEGY IN RETINAL ISCHEMIA Oxygen free radicals and other reactive oxygen species (ROS) can react detrimentally with most macromolecular constituents of the cell and lead to protein modification, lipid peroxidation and nucleic acid breakdown47 It is for this reason that cells have developed natural antioxidant defence mechanisms, which include the use of enzymes (catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase) and other compounds (ascorbate, uric acid, a-tocopherol, glutathione). In ischemia/reperfusion, oxygen free radicals are generated in excess of the natural cellular antioxidant defence systems and cellular damage or destruction can therefore occur. As a consequence, any form of treatment that canenhance the natural cellular antioxidant defence system will have a neuroprotective action in retinal ischemia. This has indeed been demonstrated to be the case in experimental studies (Figs. 6 and 7). Elevation of IOP to cause retinal ischemia in the rabbit generates oxygen-derived free radicals as well as retinal injury that can be attenuated by administration of the antioxidant

Figure 6 Therapeutic intervention strategies for ischemic retinal neurodegeneration. The pathway of damaging events outlined is based on that shown in Fig. 5. In this case, steps which can be modified to have an effect on the overall death process have been highlighted. Numbers refer to the strategies listed in the boxes at the sides of the figure and the points at which these strategies can be implemented are shown by the numbers highlighted on the outlined pathway.

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Figure 7 Reports supporting the view that reduction of oxidative stress attenuates retinal ischemia. HIOP – high intraocular pressure, ONBL – optic nerve bundle, 2VO – two vessel occlusion, 4VO – four vessel occlusion.

compound dimethylthiourea or other antioxidant treatments. Protection of the retina after ischemia/reperfusion has also been demonstrated with the following antioxidant strategies: vitamin E, a-lipoic acid, superoxide dismutase, catalase and EGB-761, CV-3611, desferrioxamine, mannitol and allopurinol. Antioxidant properties could also account for the ameliorative effects of calcium dobesilate, flupirtine, and trimetazidine in retinal ischemia. Antioxidants which can readily reach the intracellular compartment and are well tolerated in long-term therapy, such as EGB-761, a Ginkgo biloba extract, may theoretically be prescribed prophylactically as an oral therapy to patients at a high risk of developing retinal ischemia. Furthermore, several of the above-mentioned antioxidants, such as superoxide dismutase, catalase, manitol and desferrioxamine, are protective when administered just before reperfusion following retinal ischemia, which is of major interest for potential clinical use. However, the rapid clearance of superoxide dismutase and catalase by the kidneys could make the therapeutic use of both enzymes difficult. It should be noted that, apart from directly counteracting the molecular damage caused by free radicals, antioxidant compounds may also attenuate ischemic insults by decreasing the release of glutamate evoked by ischemia. CONCLUSION Retinal ischemia is a common cause of visual impairment and blindness. At the cellular level, ischemic retinal injury consists of a number of self-reinforcing destructive cascades, initiated by energy failure, to cause neuronal depolarisation,

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calcium influx and inevitably oxidative stress. The resulting cell death is by apoptosis and necrosis, pending on the intensity of the ischemic insult. Combating mild ischemic insults with appropriate antioxidants is one therapeutic strategy worthy of consideration. REFERENCES 1. Hayreh SS, Weingeist TA. Experimental occlusion of the central artery of the retina. IV: retinal tolerance time to acute ischaemia. Br J Ophthalmol 1980; 64:818–825. 2. Schmidt M, Giessl A, Laufs T, et al. How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina. J Biol Chem 2003; 278:1932–1935. 3. Osborne NN, Ugarte M, Chao M, et al. Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv Ophthalmol 1999; 43(suppl 1):S102–S128. 4. Osborne NN, Larsen AK. Antigens associated with specific retinal cells are affected by ischaemia caused by raised intraocular pressure: effect of glutamate antagonists. Neurochem Int 1996; 29:263–270. 5. Osborne NN, Schwarz M, Pergande G. Protection of rabbit retina from ischemic injury by flupirtine. Invest Ophthalmol Vis Sci 1996; 37:274–280. 6. Winkler BS. The electroretinogram of the isolated rat retina. Vision Res 1972; 12:1183–1198. 7. Stone J, Maslim J, Valter-Kocsi K, et al. Mechanisms of photoreceptor death and survival in mammalian retina. Prog Retin Eye Res 1999; 18:689–735. 8. Kuwabara T, Cogan D. Retinal glycogen. Arch Ophthalmol 1961; 66:680–688. 9. Weiss H. The carbohydrate reserve in the vitreous body and retina of the rabbit’s eye during and after pressure ischaemia and insulin hypoglycaemia. Ophthal Res 1972; 3:360–371. 10. Johnson NF. Retinal glycogen content during ischaemia. Albrecht von Graefes Arch Klin Exp Ophthalmol 1977; 203:271–282. 11. Ames A III, Wright RL, Kowada M, et al. Cerebral ischemia. II. The no-reflow phenomenon. Am J Pathol 1968; 52:437–453. 12. Fischer EG, Ames A III, Hedley-Whyte ET, et al. Reassessment of cerebral capillary changes in acute global ischemia and their relationship to the ‘‘no-reflow phenomenon’’. Stroke 1977; 8:36–39. 13. Buchi ER, Suivaizdis I, Fu J. Pressure-induced retinal ischemia in rats: an experimental model for quantitative study. Ophthalmologica 1991; 203:138–147. 14. Romano C, Price M, Bai HY, et al. Neuroprotectants in Honghua: glucose attenuates retinal ischemic damage. Invest Ophthalmol Vis Sci 1993; 34:72–80. 15. Romano C, Price MT, Almli T, et al. Excitotoxic neurodegeneration induced by deprivation of oxygen and glucose in isolated retina. Invest Ophthalmol Vis Sci 1998; 39:416–423. 16. Casson RJ, Wood JPM, Melena J, et al. The effect of ischemic preconditioning on light-induced photoreceptor injury. Invest Ophthalmol Vis Sci 2003; 44:1348–1354. 17. Casson RJ, Chidlow G, Wood JPM, et al. The effect of hyperglycemia on experimental retinal ischemia. Arch Ophthalmol 2004; 122:361–366. 18. Jennings RB, Sommers HM, Smyth GA, et al. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. Arch Pathol 1960; 70:68–78.

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Retinal Ischemia and Oxidative Stress

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19. Sims NR. Energy metabolism and selective neuronal vulnerability following global cerebral ischemia. Neurochem Res 1992; 17:923–931. 20. Leker RR, Shohami E. Cerebral ischemia and trauma. Different etiologies yet similar mechanisms: neuroprotective opportunities. Brain Res Rev 2002; 39:55–73. 21. Davalos A, Castillo J, Serena J, et al. Duration of glutamate release after acute ischemic stroke. Stroke 1997; 28:708–710. 22. Petty MA, Wettstein JG. White matter ischaemia. Brain Res Rev 1999; 31:58–64. 23. Pellegrini-Giampietro DE, Cherici G, Alesiani M, et al. Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage. J Neurosci 1990; 10:1035–1041. 24. Bonne C, Muller A, Villain M. Free radicals in retinal ischemia. Gen Pharmacol 1998; 30:275–280. 25. Osborne NN, Casson RJ, Wood JP, et al. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog Retin Eye Res 2004; 23:91–147. 26. Gilgun-Sherki Y, Rosenbaum Z, Melamed E, et al. Antioxidant therapy in acute central nervous system injury: current state. Pharmacol Rev 2002; 54:271–284. 27. Doly M, Braquet P, Bonhomme B, et al. Effects of lipid peroxidation on the isolated rat retina. Ophthalmic Res 1984; 16:292–296. 28. Szabo ME, Droy-Lefaix MT, Doly M, et al. Ischemia and reperfusion-induced histologic changes in the rat retina. Demonstration of a free radical-mediated mechanism. Invest Ophthalmol Vis Sci 1991; 32:1471–1478. 29. Szabo ME, Droy-Lefaix MT, Doly M, et al. Ischaemia- and reperfusion-induced Naþ, Kþ, Ca2þ and Mg2þ shifts in rat retina: effects of two free radical scavengers, SOD and EGB 761. Exp Eye Res 1992; 55:39–45. 30. Muller A, Pietri S, Villain M, et al. Free radicals in rabbit retina under ocular hyperpressure and functional consequences. Exp Eye Res 1997; 64:637–643. 31. Rios L, Cluzel J, Vennat JC, et al. Comparison of intraocular treatment of DMTU and SOD following retinal ischemia in rats. J Ocul Pharmacol Ther 1999; 15: 547–556. 32. Banin E, Berenshtein E, Kitrossky N, et al. Gallium-desferrioxamine protects the cat retina against injury after ischemia and reperfusion. Free Radic Biol Med 2000; 28:315–323. 33. Chidlow G, Schmidt KG, Wood JP et al. Alpha-lipoic acid protects the retina against ischemia-reperfusion. Neuropharmacology 2002; 43:1015–1025. 34. Block F, Schwarz M. Effects of antioxidants on ischemic retinal dysfunction. Exp Eye Res 1997; 64:559–564. 35. Celebi S, Dilsiz N, Yilmaz T, et al. Effects of melatonin, vitamin E and octreotide on lipid peroxidation during ischemia-reperfusion in the guinea pig retina. Eur J Ophthalmol 2002; 12:77–83. 36. Shibuki H, Katai N, Yodoi J, et al. Lipid peroxidation and peroxynitrite in retinal ischemia-reperfusion injury. Invest Ophthalmol Vis Sci 2000; 41:3607–3614. 37. Kuriyama H, Waki M, Nakagawa M, et al. Involvement of oxygen free radicals in experimental retinal ischemia and the selective vulnerability of retinal damage. Ophthalmic Res 2001; 33:196–202. 38. Gupta LY, Marmor MF. Mannitol, dextromethorphan, and catalase minimize ischemic damage to retinal pigment epithelium and retina. Arch Ophthalmol 1993; 111:384–388. 39. Ugarte M, Osborne NN. Zinc in the retina. Prog Neurobiol 2001; 64:219–249.

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194

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40. Szabo ME, Droy-Lefaix MT, Doly M. Direct measurement of free radicals in ischemic/reperfused diabetic rat retina. Clin Neurosci 1997; 4:240–245. 41. Roth S, Park SS, Sikorski CW, et al. Concentrations of adenosine and its metabolites in the rat retina/choroid during reperfusion after ischemia. Curr Eye Res 1997; 16:875–885. 42. Zhang H, Agardh CD, Agardh E. Increased catalase levels and hypoxanthineenhanced nitro-blue tetrazolium staining in rat retina after ischemia followed by recirculation. Curr Eye Res 1995; 14:47–54. 43. Peachey NS, Green DJ, Ripps H. Ocular ischemia and the effects of allopurinol on functional recovery in the retina of the arterially perfused cat eye. Invest Ophthalmol Vis Sci 1993; 34:58–65. 44. Faberowski N, Stefansson E, Davidson RC. Local hypothermia protects the retina from ischemia. A quantitative study in the rat. Invest Ophthalmol Vis Sci 1989; 30:2309–2313. 45. Szabo ME, Droy-Lefaix MT, Doly M, et al. Modification of ischemia/reperfusioninduced ion shifts (Naþ, Kþ, Ca2þ and Mg2þ) by free radical scavengers in the rat retina. Ophthalmic Res 1993; 25:1–9. 46. Matsuo Y, Kihara T, Ikeda M, et al. Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: effect of neutrophil depletion on extracellular ascorbyl radical formation. J Cereb Blood Flow Metab 1995; 15:941–947. 47. Lipton P. Ischemic cell death in brain neurons. Physiol Rev 1999; 79:1431–1568. 48. Tsujikawa A, Ogura Y, Hiroshiba N, et al. Retinal ischemia-reperfusion injury attenuated by blocking of adhesion molecules of vascular endothelium. Invest Ophthalmol Vis Sci 1999; 40:1183–1190. 49. Goldstein IM, Ostwald P, Roth S. Nitric oxide: a review of its role in retinal function and disease. Vision Res 1996; 36:2979–2994. 50. Shin DH, Lee HY, Kim HJ, et al. In situ localization of neuronal nitric oxide synthase (nNOS) mRNA in the rat retina. Neurosci Lett 1999; 270:53–55. 51. Cheon EW, Park CH, Kang SS, et al. Change in endothelial nitric oxide synthase in the rat retina following transient ischemia. Neuroreport 2003; 14:329–333. 52. Lopez-Costa JJ, Goldstein J, Saavedra JP. Neuronal and macrophagic nitric oxide synthase isoforms distribution in normal rat retina. Neurosci Lett 1997; 232:155–158. 53. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci 1997; 20:132–139. 54. Cheon EW, Park CH, Kang SS, et al. Nitric oxide synthase expression in the transient ischemic rat retina: neuroprotection of betaxolol. Neurosci Lett 2002; 330:265–269. 55. Kobayashi M, Kuroiwa T, Shimokawa R, et al. Nitric oxide synthase expression in ischemic rat retinas. Jpn J Ophthalmol 2000; 44:235–244. 56. Hangai M, Yoshimura N, Hiroi K, et al. Inducible nitric oxide synthase in retinal ischemia-reperfusion injury. Exp Eye Res 1996; 63:501–509. 57. Hangai M, Miyamoto K, Hiroi K, et al. Roles of constitutive nitric oxide synthase in postischemic rat retina. Invest Ophthalmol Vis Sci 1999; 40:450–458. 58. Neufeld AH, Kawai S, Das S, et al. Loss of retinal ganglion cells following retinal ischemia: the role of inducible nitric oxide synthase. Exp Eye Res 2002; 75:521–528. 59. Sennlaub F, Courtois Y, Goureau O. Inducible nitric oxide synthase mediates the change from retinal to vitreal neovascularization in ischemic retinopathy. J Clin Invest 2001; 107:717–725.

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60. Abu El-Asrar AM, Desmet S, Meersschaert A, et al. Expression of the inducible isoform of nitric oxide synthase in the retinas of human subjects with diabetes mellitus. Am J Ophthalmol 2001; 132:551–556. 61. Veriac S, Tissie G, Bonne C. Oxygen free radicals adversely affect the regulation of vascular tone by nitric oxide in the rabbit retina under high intraocular pressure. Exp Eye Res 1993; 56:85–88. 62. Lo EH, Hara H, Rogowska J, et al. Temporal correlation mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial nitric oxide synthase gene expression. Stroke 1996; 27:1381–1385. 63. Maynard KI, Chen D, Arango PM, et al. Nitric oxide produced during ischemia improves functional recovery in the rabbit retina. Neuroreport 1996; 8:81–85. 64. Vorwerk CK, Hyman BT, Miller JW, et al. The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity. Invest Ophthalmol Vis Sci 1997; 38:2038–2044. 65. Adachi K, Fujita Y, Morizane C, et al. Inhibition of NMDA receptors and nitric oxide synthase reduces ischemic injury of the retina. Eur J Pharmacol 1998; 350:53–57. 66. Lam TT, Tso MO. Nitric oxide synthase (NOS) inhibitors ameliorate retinal damage induced by ischemia in rats. Res Commun Mol Pathol Pharmacol 1996; 92:329–340. 67. Neufeld AH, Sawada A, Becker B. Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma. Proc Natl Acad Sci U S A 1999; 96:9944–9948. 68. Osborne NN, Melena J, Chidlow G, et al. A hypothesis to explain ganglion cell death caused by vascular insults at the optic nerve head: possible implication for the treatment of glaucoma. Br J Ophthalmol 2001; 85:1252–1259. 69. Stapf C, Mohr JP. Ischemic stroke therapy. Annu Rev Med 2002; 53:453–475. 70. Kristian T, Siesjo BK. Calcium in ischemic cell death. Stroke 1998; 29:705–718. 71. Nishizawa Y. Glutamate release and neuronal damage in ischemia. Life Sci 2001; 69:369–381.

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15 Reduction of Oxidative Stress in Retinal Disease Ulrich Schraermeyer, Petra Blitgen-Heinecke, Despina Kokkinou, and Tobias Schwarz Sektion fu¨r Experimentelle Vitreoretinale Chirurgie, Universita¨ts-Augenklinik Tu¨bingen, Tu¨bingen, Germany

Ju¨rgen Kopitz Zentrum fu¨r Pathologie, Abt. Angewandte Tumorbiologie, Klinikum der Ruprecht-Karls-Universita¨t, Im Neuenheimer Heidelberg, Germany

INTRODUCTION Melanin pigment is produced in the neuroectodermic retinal pigment epithelium (RPE) and neural crest-derived melanocytes in mammals.1 Melanin-synthesizing cells contain specific organelles, the premelanosomes, in which glycoproteinic transmembrane tyrosinase catalyzes melanin biosynthesis.2 The RPE is a functionally relevant single-layer of pigmented cells in the mammalian eye. Basic adult RPE functions are the formation of the outer bloodretina barrier, transepithelial transport, protection against reactive oxygen intermediates and light, storage of retinoids and turnover of the scuffed rod outer segment discs.3 Once fully differentiated, RPE cells do not divide and remain functional throughout the life of an individual. The RPE-choroid complex contains the highest melanin concentration of all human tissues. Disturbances in melanin biosynthesis have been implicated in genetic disorders, such as oculocutaneous

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albinism (OCA). Tyrosinase is the rate limiting enzyme of melanin biosynthesis and catalyses the first two steps of melanin synthesis: the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), and the oxidation of L-DOPA to DOPAquinone.4 Furthermore, a third enzymatic reaction was assigned to tyrosinase: the oxidation of 5,6-dihydroxyindole to 5,6-dihydroxyquinone.5 It has been postulated that melanogenesis in the RPE is restricted to prenatal periods, since tyrosinase, the key enzyme in melanin biosynthesis, was detected in early stage human embryos only and was absent long before gestation ends.6–9 This is the actual state of knowledge up to the present day. However, premelanosomes and early-stage-melanosomes have been found in adult RPE10–13 which led to this hypothesis being questioned. Tyrosinase activity has also been demonstrated in adult cultured bovine,14,15 porcine,11 mouse,16 rabbit,17 rat18 and human RPE cells.19 Additionally, tyrosinase activity was found in adult bovine RPE.20 Tyrosinase promoter activity was significantly up-regulated in in cultured human RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, in that study tyrosinase enzymic activity was not found.21 Recent studies show that phagocytosis of ROS induces gene expression in RPE cells.22 Our study was performed to examine the presence of tyrosinase protein as well as its enzymatic activity in adult mammalian RPE. Phagocytosis of shed photoreceptor outer segment distal discs is one of the most important functions of the RPE. Cellular functions of RPE cells such as disc shedding and subsequent phagocytosis are controlled by circadian rhythm.23 Therefore we investigated whether phagocytosis of ROS can increase tyrosinase expression in vitro. MATERIALS AND METHODS Organ Culture of Bovine RPE-Choroid Complexes Pigmented RPE-choroid complexes were isolated according to Schraermeyer and Stieve.15 In brief, cattle eyes from two year-old animals were transported on ice from a slaughter house to the laboratory. The eyes were washed once with Hanks’ balanced salt solution (HBSS) containing penicillin (100 U/ml) and streptomycin (0.1 mg/ml). All tissue culture media or solutions were purchased from Sigma (Deisenhofen, Germany). The anterior half of the eye was removed and discarded. The vitreous was removed, and the retina was gently floated off the RPE by pipetting HBSS into the subretinal space. The retinae were removed after cutting the optic nerve and used for isolation of the rod outer segments as described below. Specimens (1 mm3) of the RPE-choroid complex were kept in 24-well tissue culture plates (Cluster,24 Costar, Cambridge, UK). The complete tissue culture medium contained Dulbecco’s Modified Eagle Medium with 4500 mg/l glucose supplemented with 15% fetal calf serum, 25 mM HEPES buffer, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 3.7 g/l

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sodium bicarbonate and 4 mM L-glutamine. The cultures were gassed with 5% CO2 and the medium was changed completely once a week. In a separate set of experiments for immunodetection of tyrosinase RPE cells were cultured as monolayers after trypsinisation in the same manner. Isolation of Rod Outer Segments The method was performed as described by Schraermeyer and Stieve.15 In detail, isolated retinae were agitated for 2 min in KCl buffer (0.3 M KCl, 10 mM HEPES, 0.5 mM CaCl2, 1 mM MgCl2 and 48% sucrose) at pH 7.0 and then centrifuged at 2000 rpm in a tabletop centrifuge (type UJ 1, Christ, Germany) for 5 min. The supernatant was filtered through a tube gauze fingerling and then diluted with KCl-buffer (1:1) and centrifuged at 2500 rpm for 10 min. The pellet, containing the rod outer segments, was washed and centrifuged in complete culture medium before feeding to the RPE. Assay for Phagocytosis RPE cell monolayers or RPE-choroid complexes, adapted to the culture medium conditions for 2 weeks, were exposed to rod outer segments (rods from one eye/ 4 wells) that had been isolated and marked as described above. After 4 hrs the non-phagocytosed rod fragments were washed out and replaced by fresh medium. The medium was changed daily during the experiments. Electron-Microscopical Localization of Tyrosinase The enzyme tyrosinase was localized by electron-microscopical histochemistry.24,25 In brief, for localisation of tyrosinase, RPE cells that had been exposed to fragments of rod outer segments as described above were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 6.8) for 1h. RPE cells without feeding were treated in the same manner. Specimens were washed twice in sodium cacodylate buffer and kept at 48C overnight in this buffer, containing 5 mM L-dihydroxphenylalanine (L-DOPA) or, as a control, 5 mM D-DOPA (Sigma, Deisenhofen, Germany). Thereafter, these solutions were renewed, and the tissue pieces were incubated for a further 5hrs at 378C. The reacted RPE cells were washed in sodium cacodylate buffer and immersed for 1 h at room temperature in the same buffer containing a mixture of osmium tetroxide (1%) and potassium ferrocyanide (1.5%). Finally, the tissue pieces were dehydrated and embedded in Spurr’s resin for routine electron microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and observed examined with a Zeiss EM 902A electron microscope. Immunodetection of Tyrosinase The mouse anti-tyrosinase monoclonal antibody 2G10 was purchased from Chemicon Int. Inc. (Temecula, CA, USA) and is described by the manufacturer

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as an antibody to human tyrosinase.26 The monoclonal anti-tyrosine hydroxylase antibody, clone TH-2 and IgG1 isotype, was from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany and was used as a control. This antibody was raised against an epitope of rat tyrosine hydroxylase, also present in human tyrosine hydroxylase. The secondary purified goat Cy3-conjugated anti-mouse IgG antiserum was obtained from Rockland Immunochemicals Inc., Gilbertsville, PA, USA. This antiserum had been raised against mouse IgG. Antibodies (3-8 mg/ml) were used on paraformaldehyde fixed RPE monolayers as described elsewhere.27 First antibodies were diluted at 1:200 and incubated in buffer containing 0.01 M sodium phosphate and 0.25 M NaCl for 30 minutes at room temperature. Second antibodies were used diluted 1:1000 and incubated in the same buffer for 1 hour. Cells were photographed under an Axioplan 2 mikroskop (Zeiss, Oberkochem, Germany). Image processing was performed with a Orca camera (Hamamatsu Photonics, Germany) and Openlab Software (Improvision, Tu¨bingen, Germany. Tyrosine Hydroxylase Activity of Tyrosinase Samples were homogenized (using a Potter-Elvehjem homogenizer at 10 strokes up-and-down at 1,200 rpm) in 100 mM potassium phosphate buffer, pH 7.4. Then 25 ml of the homogenate were mixed with 25 ml assay buffer containing 100 mM potassium phosphate, pH 7.4, 18.5 MBq L-[3,5-3H]tyrosine (specific radioactivity 1.83 GBq/mmol; Moravek Biochemicals, Brea, CA) and 1 mM L-DOPA (Sigma-Aldrich Chemie, Taufkirchen, Germany) and incubated for 24 hours at 378C. The reaction was stopped by adding 100 mg Celite 545 (Merck KGaA, Darmstadt, Germany) and 100 mg activated charcoal suspended in 1 ml 0.1 N HCl solution. After 1 hour shaking at room temperature, the samples were centrifuged (15,000
g, 5 min), 500 ml of the resulting supernatant were mixed with 10 ml of Ultima GoldTM scintillation cocktail and radioactivity was determined by a Packard TRI-CARB 2900TR liquid scintillation counter. Protein concentration of the samples was measured using the method of Lowry et al.28 applying bovine serum albumin as standard. Statistical Analysis Data are given as mean  standard deviation. Statistical evaluation was based on Student’s t-test for two populations. A double-sided p-value of less than 0.05 was considered statistically significant. RESULTS Electron Microscopic Localization of Tyrosinase by DOPA Histochemistry To optimize the in vitro conditions, we used organ cultures consisting of melanotic RPE-choroid complexes from bovine eyes. The RPE layer remains on Bruch’s membrane and under these physiological conditions the apical microvilli

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are preserved in vitro. ROS isolated from bovine eyes were phagocytized and immediately after exposure to ROS many phagosomes containing ROS were present within the RPE cells (Figure 1A). Tyrosinase activity was investigated by electron microscopic (EM) histochemistry. DOPA oxidase activity of tyrosinase was determined. Five hours after feeding with ROS, tyrosinase activity was demonstrated in RPE by DOPA-oxidase assay. DOPA-positive electron-dense vesicles, indicating the presence of tyrosinase, were present throughout the cytoplasm as shown in electron micrographs (Figure 1B, C). Without feeding DOPA positive vesicles were not found in RPE cells (Figure 1D). DOPA positive Golgi bodies were regularly seen 5 hours (Figure 1E) after feeding. Occasionally DOPA-positive vesicles were observed in close vicinity to melanosomes (Figure 1F). Twenty-four hours after feeding with ROS, DOPA-positive vesicles and Golgi bodies disappeared from the cytoplasm (Figure 1G). Lipid like droplets, 1-3 mm in diameter, were present in many RPE cells (Figure 1G, H). The lipid droplets were surrounded by membrane stacks that contained DOPA-positive material (Figure 1G, H). These organelles corresponded exactly to those found after immunocytochemistry with anti-tyrosinase antibodies as shown in Fig. 2D. DOPA oxidase activity was not detected in controls incubated with 5 mM D DOPA (data not shown). Antibody Staining of Tyrosinase in Bovine RPE Cells In a monolayer of cultured bovine RPE cells, control immunocytochemical experiments performed using antibodies against L-tyrosine 3-hydroxylase (EC 1.14.16.2) were negative (Figure 2A). Without feeding with ROS, using antityrosinase antibodies, no specific staining different from background levels was detected (Figure 2B). However, five hours after feeding slight specific staining was found (Figure 2C) corresponding to the electron microscopic DOPA staining shown in Fig. 1B, C. Twenty-four hours after feeding with ROS by immunocytochemistry with a monoclonal antibody, tyrosinase protein was predominately detected in ellipsoid organelles (Figure 3D). Tyrosine Hydroxylase Activity of Tyrosinase Using a highly specific radioactive assay, L-tyrosine 3-hydroxylase activity was determined in the same adult bovine melanotic RPE-choroid complexes after feeding with ROS (Figure 3A). By measuring the release of tritium from tritiated L-tyrosine in homogenized samples, we demonstrated that feeding with ROS increases tyrosinase enzyme activity in comparison to non-challenged RPE (15.8  2.5 to 725.0  70.9 fmol/mg/h, Figure 3) by a factor of 40. The difference between the ROS-fed and non-fed RPE was statistically significant ( p ¼ 0.003; n ¼ 3). In control samples, containing choroid without RPE layer, only low levels of tyrosinase activity were detected (4.9 fmol/mg/h, n ¼ 1, mean of

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Figure 1 A. Organ culture consisting of melanotic RPE-choroid complex from bovine eyes. Electron micrograph of a cross section immediately after feeding with ROS for 4 hrs. The RPE cell with many phagosomes (P) containing ROS. Under these culture conditions the apical microvilli (arrow) of an RPE cell are preserved in vitro since the RPE layer is cultivated on Bruch’s membrane (B). This natural environment may improve the functions of RPE in vitro. B, C. Phagocytosis of ROS induces tyrosinase activity in RPE. DOPAoxidase activity of tyrosinase demonstrated by EM histochemistry in RPE cells 5 hours after feeding with ROS. Many DOPA positive vesicles (arrows) are scattered throughout the cytoplasm (M = mitochondrion, N = nucleus). D. Electron micrograph taken from control cultures that were not exposed to ROS. DOPA-oxidase activity was not present in these cultures. E, F. DOPA-positive Golgi (G) bodies are seen 5 hrs after exposure to ROS. Golgi derived vesicles are close to melanosomes and suggest classical biosynthesis of this enzyme 5 hrs after feeding with ROS (arrow in 1F). G. Twenty-four hours after feeding tyrosinase activity surrounds large lysosomes (arrows) filled with lipid-like material (L) but is no longer seen in cytoplasmic vesicles. 1H. Tyrosinase is localized within membrane stacks (arrow) surrounding these lipid droplets (L). These droplets correspond to organelles staining positive for anti-tyrosinase antibodies in Fig. 2D.

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Figure 2 (See color insert.) For tyrosinase immunocytochemistry, the RPE monolayer was prepared and exposed to ROS for 4 hrs. The expression of tyrosinase was investigated before feeding with ROS, as well as 5 and 24 hours afterwards. 2A Five hours after feeding with ROS, no staining was visible with anti-tyrosine hydroxylase antibodies. 2B Without feeding with ROS no staining was found with anti-tyrosinase antibodies. 2C Five hours after feeding with ROS faint staining was observed with anti-tyrosinase antibodies corresponding to DOPA positive vesicles in Fig. 1B, 2D. Twenty-four hours after feeding with ROS intense staining of lysosome-like organelles (arrows) was found with antityrosinase antibodies. These organelles correspond to those shown in Fig. 1G, 1B.

3 measurements), which were not significantly increased after exposure to ROS (19.9 fmol/mg/h, n ¼ 1, mean of 3 measurements. DISCUSSION These results clearly demonstrate that phagocytosis of ROS can induce tyrosinase expression in RPE. Here, we provide EM histological and biochemical evidence for the presence and induction of tyrosinase activity after phagocytosis of ROS in adult mammalian RPE.

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Figure 3 Phagocytosis of ROS increased the activity of tyrosinase approximately 40-fold. Organ cultures consisting of melanotic bovine RPE-choroid complexes were exposed to ROS for 4 hrs. Twenty-four hours after feeding with ROS the cultures were homogenized and used for determination of tyrosinase activity. Without feeding with ROS tyrosinase activity was almost not detectable (*, p – 0.003). These results clearly demonstrate that tyrosinase activity was induced only after feeding with ROS.

The role of phagocytosis-induced tyrosinase expression is not yet clear. Several studies suggest that melanin biosynthesis can take place within lysosomes15,16,25,29,30 because melanosomes belong to the same lineage of organelles. Moreover, melanosomes contain most lysosomal enzymes31 and it was stated that the melanosome is a specialized lysosome.32 It is not surprising that synthesis of tyrosinase was induced by phagocytosis, because ROS uptake is associated with regulation of many genes in the RPE.22 Furthermore, lipids, particularly sphingolipids, induce melanogenesis by increasing the expression of tyrosinase and its related proteins in vitro.33 Therefore, we intend to investigate in future studies whether ROS lipids induce expression of tyrosinase and melanogenesis. Whereas the electron microscopic findings of tyrosinase activation early after feeding with ROS correspond to the classical scheme of melanogenesis with Golgi derived DOPA positive vesicles scattered throughout the cytoplasm of a pigment cell, there are unexpected findings after 24 hours: At this time point DOPA positive vesicles were not present, but tyrosinase was seen in membrane stacks surrounding lipid droplets (Figure 1G, F). As this was seen in all RPE cultures of this study with DOPA histochemistry and immunocytochemistry using anti-tyrosinase antibodies it is not an artifact but a real finding. The meaning of this observation remains unknown but may be in part explained by the following hypothesis: culture conditions decrease the ability of RPE cells to degrade the high amounts of ROS (Figure 1A) within 24 hours and lipid-like material accumulates in large lysosomes. This may be partially because in vivo entire rods are taken up, instead of

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shed tips. In vivo tyrosinase containing vesicles may fuse with phagosomes. Inside phagosomes it may react with superoxide as an radical trapping enzyme,34 may simply be targeted for degradation as an unfunctional enzyme or may have other yet unknown functions. In this study the content of lysosomes is so densely packed that the uptake of protein seems to be blocked, and the vesicles may fuse forming membrane stacks around the lysosomes. This would explain why the margins of the lipid vacuoles stain positive for tyrosinase whereas the inner layers do not (Figure 1G, F). This hypothesis is in agreement with findings of this and earlier studies, in which tyrosinase was detected in incompletely degraded phagosomes (not shown).15 Moreover, there is a connection between phagosomal degradation pathways and melanosomes in RPE cells, as it was found that material from phagosomes is transported to the melanosomes or melanogenesis taking place inside phagosomes.35 As phagosomes are related to endosomes, the hypothesis, earlier findings as well as the observations of this study fit into the general scheme of tyrosinase trafficking in melanocytes. In melanocytes tyrosinase is transported from transGolgi network to early endosomes and reaches via late endosomes lysosomes and premelanosomes.36 Tyrosinase has many variable functions, the mechanisms of which are still unknown. It is directly involved in light adaptation in zebra fish.37 Moreover, tyrosinase plays a role in glaucoma of zebra fish38 and mouse models.39 Tyrosinase is also present in most neurons of the murine brain40,41 without being melanogenic active and it promotes survival of catecholaminergic neurons.42 Tyrosinase or its metabolites are responsible for development of a fully functional macula39,43 and the normal crossing of optic nerve fibers.44 In addition immature children are more protected from retinopathy of prematurity (ROP) if they have a darker fundus resulting from melanin pigmentation.45,46 It is not known whether tyrosinase or its metabolites protect against oxidative stress in this disease. Moreover, black people are more protected from ARMD and a lower amount of Drusen, crosslinked proteins in Bruch’s membrane and lipofuscin in the RPE than white Caucasians.47–49 In ARMD it is not known whether this is caused by tyrosinase itself or its metabolites. The functional role of tyrosinase expression induced by phagocytosis is not known. The phagocytosis of ROS is associated with generation of reactive oxygen species such as superoxide anion radicals (O2 ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH ).50 Hydrogen peroxide was shown to increase tyrosinase activity in cultured human melanoma cells,51 to be a competitive inhibitor of Tyrosinase,52 and to induce tyrosinase.51,53 Furthermore, superoxide anions increase human tyrosinase activity.52 Tyrosinase may protect against oxidative stress by several mechanisms: firstly, by oxidation of tetrahydroisoquinones, when tyrosinase may promote their sinking into insoluble inert polymers; secondly, by utilizing superoxide anion as substrate, when it could act as a free radical trapping enzyme.34 



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CONCLUSION Although the function of tyrosinase, or whether it is melanogenetically active or not in RPE cells remains unknown, here it is shown for the first time with three independent methods that tyrosinase can be induced by phagocytosis in RPE cells of postnatal vertebrates. REFERENCES 1. Marks MS, Seabra MC. The melanosome: membrane dynamics in black and white. Nat Rev Mol Cell Biol 2001; 2:738–748. 2. Seiji M, Fitzpatrick TB, Simpson RT, et al. Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes. Nature 1963; 197:1082–1084. 3. Boulton M, Dayhaw-Barker P. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye 2001; 15:384–389. 4. Lerch K. Monophenol monooxygenase from Neurospora crassa. Methods Enzymol 1987; 142:165–169. 5. Korner A, Pawelek J. Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin. Science 1982; 217:1163–1165. 6. Miyamoto M, Fitzpatrick TB. On the nature of pigment in retinal pigment epithelium. Science 1957; 126:449–450. 7. Carr RE, Siegel IM. The retinal pigment epithelium in ocular albinism. In: Zinn KM, Marmor MF, eds. The Retinal Pigment Epithelium. Cambridge, Massachusetts, London: Harvard University Press, 1979:413–423. 8. Sarna T. Properties and function of the ocular melanin—a photobiophysical view. J Photochem Photobiol B 1992; 12:215–258. 9. Smith-Thomas L, Richardson P, Thody AJ, et al. Human ocular melanocytes and retinal pigment epithelial cells differ in their melanogenic properties in vivo and in vitro. Curr Eye Res 1996; 15:1079–1091. 10. Young RW. The daily rhythm of shedding and degradation of rod and cone outer segment membranes in the chick retina. Invest Ophthalmol Vis Sci 1978; 17: 105–116. 11. Dorey CK, Torres X, Swart T. Evidence of melanogenesis in porcine retinal pigment epithelial cells in vitro. Exp Eye Res 1990; 50:1–10. 12. Schraermeyer U. Does melanin turnover occur in the eyes of adult vertebrates? Pigment Cell Res 1993; 6:193–204. 13. Schraermeyer U, Heimann K. Current understanding on the role of retinal pigment epithelium and its pigmentation. Pigment Cell Res 1999; 12:219–236. 14. Basu PK, Sarkar P, Menon I, et al. Bovine retinal pigment epithelial cells cultured in vitro: growth characteristics, morphology, chromosomes, phagocytosis ability, tyrosinase activity and effect of freezing. Exp Eye Res 1983; 36:671–683. 15. Schraermeyer U, Stieve H. A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle. Cell Tissue Res 1994; 276:273–279. 16. Novikoff AB, Leuenberger PM, Novikoff PM, et al. Retinal pigment epithelium. Interrelations of endoplasmic reticulum and melanolysosomes in the black mouse and its beige mutant. Lab Invest 1979; 40:155–165.

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17. Varela JM, Stempels NA, Vanden Berghe DA, et al. Isoenzymic patterns of tyrosinase in the rabbit choroid and retina/retinal pigment epithelium. Exp Eye Res 1995; 60:621–629. 18. Weisse I. Changes in the aging rat retina. Ophthalmic Res 1995; 27(suppl 1): 154–163. 19. Aronson JF. Human retinal pigment cell culture. In Vitro 1983; 19:642–650. 20. Dryja TP, O’Neil-Dryja M, Pawelek JM, et al. Demonstration of tyrosinase in the adult bovine uveal tract and retinal pigment epithelium. Invest Ophthalmol Vis Sci 1978; 17:511–514. 21. Abul-Hassan K, Walmsley R, Tombran-Tink J, et al. Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells. Pigment Cell Res 2000; 13:436–441. 22. Chowers I, Kim Y, Farkas RH, et al. Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake. Invest Ophthalmol Vis Sci 2004; 45:2098–2106. 23. LaVail MM. Circadian nature of rod outer segment disc shedding in the rat. Invest Ophthalmol Vis Sci 1980; 19:407–411. 24. Schraermeyer U. Localization of peroxidase activity in the retina and the retinal pigment epithelium of the Syrian golden hamster (Mesocricetus auratus). Comp Biochem Physiol B 1992; 103:139–145. 25. Schraermeyer U. Evidence for melanogenesis in the retinal pigment epithelium of adult cattle and golden hamster. Comp Biochem Physiol B 1992; 103:435–442. 26. Cuomo M, Nicotra MR, Apollonj C, et al. Production and characterization of the murine monoclonal antibody 2G10 to a human T4-tyrosinase epitope. J Invest Dermatol 1991; 96:446–451. 27. Schraermeyer U, Enzmann V, Kohen L, et al. Porcine iris pigment epithelial cells can take up retinal outer segments. Exp Eye Res 1997; 65:277–287. 28. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275. 29. Nakagawa H, Rhodes AR, Momtaz TK, et al. Morphologic alterations of epidermal melanocytes and melanosomes in PUVA lentigines: a comparative ultrastructural investigation of lentigines induced by PUVA and sunlight. J Invest Dermatol 1984; 82:101–107. 30. Schraermeyer U. Transport of endocytosed material into melanin granules in cultured choroidal melanocytes of cattle—new insights into the relationship of melanosomes with lysosomes. Pigment Cell Res 1995; 8:209–214. 31. Diment S, Eidelman M, Rodriguez GM, et al. Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells. J Biol Chem 1995; 270: 4213–4215. 32. Orlow SJ. Melanosomes are specialized members of the lysosomal lineage of organelles. J Invest Dermatol 1995; 105:3–7. 33. Mallick S, Singh SK, Sarkar C, et al. Human placental lipid induces melanogenesis by increasing the expression of tyrosinase and its related proteins in vitro. Pigment Cell Res 2005; 18:25–33. 34. Valverde P, Manning P, McNeil CJ, et al. Activation of tyrosinase reduces the cytotoxic effects of the superoxide anion in B16 mouse melanoma cells. Pigment Cell Res 1996; 9:77–84.

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35. Schraermeyer U, Peters S, Thumann G, et al. Melanin granules of retinal pigment epithelium are connected with the lysosomal degradation pathway. Exp Eye Res 1999; 68:237–245. 36. Hearing VJ. Biogenesis of pigment granules: a sensitive way to regulate melanocyte function. J Dermatol Sci 2005; 37:3–14. 37. Page-McCaw PS, Chung SC, Muto A, et al. Retinal network adaptation to bright light requires tyrosinase. Nat Neurosci 2004; 7:1329–1336. 38. Link BA, Gray MP, Smith RS, et al. Intraocular pressure in zebrafish: comparison of inbred strains and identification of a reduced melanin mutant with raised IOP. Invest Ophthalmol Vis Sci 2004; 45:4415–4422. 39. Libby RT, Smith RS, Savinova OV, et al. Modification of ocular defects in mouse developmental glaucoma models by tyrosinase. Science 2003; 299:1578–1581. 40. Tief K, Hahne M, Schmidt A, et al. Tyrosinase, the key enzyme in melanin synthesis, is expressed in murine brain. Eur J Biochem 1996; 241:12–16. 41. Tief K, Schmidt A, Beermann F. New evidence for presence of tyrosinase in substantia nigra, forebrain and midbrain. Mol Brain Res 1998; 53:307–310. 42. Higashi Y, Asanuma M, Miyazaki I, et al. Inhibition of tyrosinase reduces cell viability in catecholaminergic neuronal cells. J Neurochem 2000; 75:1771–1774. 43. Summers CG. Vision in albinism. Trans Am Ophthalmol Soc 1996; 94:1095–1155. 44. Rachel RA, Mason CA, Beermann F. Influence of tyrosinase levels on pigment accumulation in the retinal pigment epithelium and on the uncrossed retinal projection. Pigment Cell Res 2002; 15:273–281. 45. Tadesse M, Dhanireddy R, Mittal M, et al. Race, Candida sepsis, and retinopathy of prematurity. Biol Neonate 2002; 81:86–90. 46. Saunders RA, Donahue ML, Christmann LM, et al. Racial variation in retinopathy of prematurity. The Cryotherapy for Retinopathy of Prematurity Cooperative Group. Arch Ophthalmol 1997; 115:604–608. 47. Gregor Z, Joffe L. Senile macular changes in the black African. Br J Ophthalmol 1978; 62:547–550. 48. Friedman DS, Katz J, Bressler NM, et al. Racial differences in the prevalence of age-related macular degeneration: the Baltimore Eye Survey. Ophthalmology 1999; 106:1049–1055. 49. Hollyfield JG. IOVS 2004; 45:2289 (ARVO E-Abstract). 50. Dorey CK, Khouri GG, Syniuta LA, et al. Superoxide production by porcine retinal pigment epithelium in vitro. Invest Ophthalmol Vis Sci 1989; 30:1047–1054. 51. Karg E, Odh G, Wittbjer A, et al. Hydrogen peroxide as an inducer of elevated tyrosinase level in melanoma cells. J Invest Dermatol 1993; 100(2 suppl): 209S–213S. 52. Wood JM, Schallreuter KU. Studies on the reactions between human tyrosinase, superoxide anion, hydrogen peroxide and thiols. Biochim Biophys Acta 1991; 1074:378–385. 53. Gomez-Sarosi LA, Rieber MS, Rieber M. Hydrogen peroxide increases a 55-kDa tyrosinase concomitantly with induction of p53-dependent p21 waf1 expression and a greater Bax/Bcl-2 ratio in pigmented melanoma. Biochem Biophys Res Commun 2003; 312:355–359.

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Index

[Antioxidants] cooperative effects and cataract, 96–100 defence system, 13 definition, 13 diabetic retinopathy and, 159–164 dietary, 15, 20 modulation of cardiovascular disease, 11–23 oxidative events as cause of disease, 19–20 properties of melanin, 150–152 selection of, 22 strategies for future therapies, 18 supplementation and diet, 20–21 systems in mitochondria, 4 vitamins, 14–18. See also Antioxidant vitamins Antioxidant Supplementation in Atherosclerosis Prevention study (ASAP), 18 Antioxidant vitamins atherosclerosis and, 16–18 cardiovascular disease and, 11–23 epidemiological and clinical studies, 17–18 experimental and animal studies, 16–17

ABCR4, 168 Adriamycin, 86 Age-related macular degeneration (AMD), 97, 167–175 antioxidant and, 168, 171 dry stage, 167–168 incidence of, 167–168 and photoreactivity of lipofuscin, 169–170 melanosome, 171 RPE susceptibility and adaptation to ROS, 171–172 wet stage, 168 Alkoxyl radical, 72, 96–97 Allopurinol, 185, 191 Alpha-tocopherol acetate, 160 Alzheimer’s disease, 40, 97 Amadorins, 98 Amadori rearrangements, 87–88 AMD. See Age-related macular degeneration (AMD) Aminoguanidine, 98 AMPA-receptors, 99 Amyotrophic lateral sclerosis, 40 Anoxia, 177–179 Anthraquinones, 86 Antioxidants AMD and, 168, 171 basal oxidant tone requirement, 19

209

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210 Antioxidative strategies, for protection of cataract, 94–100 cooperative effects of antioxidative, 96–100 from oxygen stress, 94–96 Antioxidative therapies, strategies for, 22 AP-1, 12, 109 APOE, 168 Apoptosis signal-regulating kinase-1 (ASK-1), 7 ARE-Nrf2 signaling pathway, 14 Ascorbic acid, 13–15, 21, 57, 59–60, 65, 160, 183, 190 Atherogenesis, 15–16 Atherosclerosis, 11, 22–23, 40, 97, 100 and antioxidant vitamins, 16–18 and oxidative stress, 15–16 Autocrine signaling, and IFNg stimulation in EAU, 126–127

Beaver Dam Eye Study, 168 Benzoquinones, 86 Bestrophin, 168 Bilirubin, 13–14 Blue Mountain Eye Study, 168 Bone marrow-derived macrophages (BM-Mj), generation of, 122 Bordetella pertussis toxin (PTX), 122 Bovine RPE-choroid complexes antibody staining of tyrosinase in, 201 organ culture of, 198–199, 202 Bowman’s layer, 56 Brain, resistance to ischemia, 179–181 Bullous keratopathy, 61–63 Buthionine sulfoximine (BSO), 47–48

Caffeine, 99 Calcium-dependent protein kinase C (PKC), 187 Calcium dobesilate, 191 Cancer and oxidative stress, 11, 40 Cardiovascular disease antioxidants atherosclerosis and vitamins, 16–18 modulation of, 11–23 vitamins, 11–23

Index [Cardiovascular disease] epidemiological and clinical studies, 17–18 oxidative stress and, 15–16 ROS and, 12–13 Carnitin, 15 b-carotene, 99, 160 Carotenoids, 13–14, 21, 81–82, 98, 168–169 Carrageenan-gamma radiation, 100 Caspase-3, 187 Catalase, 13, 35, 71–72, 179, 190–191 Cataract diabetic events, 87–89 effects of antioxidants, 96–100 herbal extracts, 97–100 LDL oxidation, 96–98 effects of light, 83–86 experimental induction, 89 first biochemical signs of, 89–90 formation by light, 85 forms of, 83–84 intrinsic light reactions, 90 models for investigating topical penetration rates, 91–94 modes of induction, 83–84 naphthalin induced, 89 prevention in model reactions in vitro and ex vivo, 90–92 protection by antioxidative strategies, 94–100 cooperative effects of antioxidative, 96–100 from oxygen stress, 94–96 protection from oxygen stress, 94–96 by detoxifying enzymes, 94–95 by phenolic derivatives, 95–96 protein glycosylation, 87–89 reductive events and, 86–87 Cataractogenesis, mechanisms of, 89 Ccl2, 168 CD18, 15 CD11b, 15 CD4+ T cell, 122, 142 CD4+ Th1 mediated disease, 121. Cell redox environment, 4

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Index Cell signaling basal ROS tone for, 19 and gene expression, 8 Cellular glutathione analysis with CDNB, 50 DTNB, 48–50 mBBr, 48–50 concentrations modulation and determination, 45–52 determination methods, 48–50 diazenedicarboxylic acid reaction with, 47 experimental modulation, 46–48 menadione and concentration of, 50–52 reaction of diamide with, 47 reaction of diethyl maleate with, 47–48 Central retinal artery occlusion, 179 Ceruloplasmin, 13 Chemotaxis, 15 Chesapeake Bay Study, 168 Chlorodiazepoxides, 85 1-chloro-2,4-dinitrobenzoic acid (CDNB), 50 Chlorothiazines, 85 Choroidal neovascularization (CNV), 168 Collagen, 15 Cornea. See also Corneal diseases antioxidants, 58–60 ascorbic acid, 59–60 superoxide dismutases (SOD), 58–59 endothelium of, 57–58 stroma of, 57 Corneal diseases bullous keratopathy, 61–63 endothelial cell loss after phacoemulsification cataract surgery, 58, 64–65 endothelial cell loss in inflammatory eye diseases, 63–64 epithelium cell loss, 56–57 after phacoemulsification cataract surgery, 64–65 in inflammatory eye diseases, 63–64 Fuchs endothelial dystrophy, 61–63 keratoconus, 60–61 oxidants in, 55–65 ROS and, 55, 63–65

211 CR-6 (3,4-dihydro-6-hydroxy-7-methoxy2,2-dimethyl-1(2H)-benzopyran), 161–164 Cyclic photosystem I, 81 Cyclooxygenases-1 (Cox 1), 35 Cyclooxygenases-2 (Cox 2), 35 Cytochrome c, 169 displacement in EAU, 138–139, 142 location and function in mitochondria, 141–142 Cytokine analysis, 123 Cytosolic copper-zinc-containing SOD (SOD1), 58

Descemet’s membrane, 57 Desferrioxamine, 191 Detoxification, by enzymatic processes, 94–95 Diabetes mellitus, 159 Diabetic retinopathy, 179 and antioxidants, 159–164 electroretinogram and, 162–164 peroxynitrite scavengers, 160–162 Diclofenac, 99 Dietary antioxidants, 15, 20 Diethyl maleate (DEM) cellular glutathione reaction with, 47–48 glutathione levels in cells exposed to, 52 Dihydro-thioctic acid, 91 Dihydroxyfumaric acid (DHF), autoxidation of, 88 Dihydroxyphenylalanin (DOPA), 89 5,50 -Dithiobis-2-nitrobenzoic acid (DTNB), 48–49

Ebselen, 160, 164 Electroretinogram (ERG), and diabetic retinopathy, 162–164 Electroretinography and ischemia, 181 Ellman’s reagent, 48 ELOVLA4, 168 Endothelialleukocyte adhesion molecule (ELAM-1), 75 Endothelial nitric oxide synthase (eNOS), 12, 21–22, 108, 112, 186–187 Endotoxin induced uveitis (EIU), 111

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212 Estrogens, 85 Eumelanin, 148, 153–154 Experimental autoimmune uveoretinitis (EAU) autocrine signaling and IFN g stimulation in, 126–127 cytochrome c displacement from electron transport assembly in, 138–139, 142 IFNg stimulation in, 126–127 infiltrating macrophage function during, 123–125 localization of nitrated retinal protein in, 137–139 nitration and release of cytochrome c in, 139 nitric oxide in, 107–114 pathogenesis, 111–113 pathology, 108–111 therapeutic strategies to reduce tissue damage, 113–114 TNF activation and, 121–128 peroxynitrite role in, 139–142 retinal microenvironment during, 123–125 retinal morphology and protein nitration during, 135–136 superoxide and nitric oxide in, 132–139 TNFa neutralising activity, 125 Extracellular SOD (SOD3), 58 corneal endothelium of, 63 immunohistochemical staining for, 58–59 in KC, 61 Fasþ T cells, 113 Fenton reaction, 34 Ferritin, 13 Fibulin-3, 168 Flavonoids, 14, 21 Flavoprotein (FP)-oxidoreductases, 86 FLICE-inhibitory protein (FLIP), 108, 114 Flupirtine, 191 FPLC chromatography, 90–91

Index Free radicals, 34 biology, 1–8 melanin interaction with, 150–152 Fuchs endothelial dystrophy, 61–63 Furocumarines, 85

Ganglion apoptosis, oxidative stress in, 76–78 Ganglion cell apoptosis, 74, 76–78 axons and glaucoma, 74–75 death in glaucoma, 72–74 Gene expression, and cell signaling, 8 Ginkgo biloba extracts (GBE), 97–100, 183, 185 Ginkgolide A, 100 Ginkgolide B, 99–100 Ginkgolide J, 99 Glaucoma, 179, 188 ganglion cell death in, 72–74 initiation for loss of vision in patients of, 72–73 mitochondria and, 74–75 oxidative stress in ganglion apoptosis, 76–78 involvement in, 71–78 and raised IOP, 75–76 pathogenesis, 71–78 plasma analysis in POAG for, 75 retinal ganglion cell axons and, 74–75 risk factors in, 72–73 Glutamate-induced cytotoxicity, 99 Glutamate-induced neurotoxicity, 100 g-Glutamylcysteine synthetase (GCS), 47–48 g-Glutamylcysteinylglycine (GSH). See Glutathione Glutathione disulfide (GSSG), 4, 39, 46–52 Glutathione (GSH), 4, 13–14, 71–72, 75–76, 98, 100, 164, 184. See also Cellular glutathione oxidative stress and, 76 structure, 45–46 Glutathione peroxidase, 13, 35, 179, 190 Glutathione reductase, 13

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Index Glutathione S-transferase isoenzymes, 75 Glutathione S-transferases (GSTs), 47, 50, 95 Gluthathione redox cycles, 35 Glycaemia, 161 GPx activity, 161–162 Green tea, 160 Griseofulvin, 85 GSH peroxidase. See Glutathione peroxidase

Hard exudates, 168 Health and disease, nitric oxide related oxidants in, 33–41 Herbal extracts, effect on cataract, 97–100 HIF-1, 12 HTRAI, 168 Human RPE melanin, 154–155. See also Retinal pigment epithelium (RPE) Hydrogen peroxide, 12, 33–35, 71–72, 75, 111, 171–172, 179, 183, 205 in cell signaling and gene expression, 8 Hydroperoxide, 72 6-hydroxykynurenic acid (6-HKA), 99 Hydroxyl radical, 12, 33–34, 71–72, 179, 183, 205 Hyperglycaemia, 161 Hypericum perforatum extracts, 97 Hypochlorite, 12 Hypochlorous acid, 12, 34–35 Hypoglycaemia, 181 Hypoxia, 177–178

IL-1, 109 IL-1b, 15, 110–111 IL-2, 110–112, 122–123, 127 IL-4, 15, 107, 109, 112–113 IL-6, 110–111 IL-8, 111 IL-10, 107, 112, 114, 123 IL-12, 123 IL-17, 113 IL-23, 113

213 Inducible NO synthase (iNOS or NOS2), 108–114, 185, 187 immunohistochemical analysis of macrophages during EAU, 123–124 INF-a, 109 INF-g, 107, 109–114, 121–128 stimulation in EAU and autocrine signaling, 126–127 Inflammatory eye diseases, corneal endothelial cells loss in, 63–64 Interferon-gamma. See INF-g Interleukins 4. See IL-4 Interleukins 1b. See IL-1b Interphotoreceptor retinoid-binding protein (IRBP) peptide 1–20, 122 Intraocular pressure (IOP), 71–72, 75–76 Intravascular ultrasonography study (IVUS), 18 Intrinsic light reactions, and cataract, 90 IRBP. See Interphotoreceptor retinoidbinding protein (IRBP) peptide Iron II+ oxidation process, 82 Ischemia, 177–178 retina and brain resistance to, 179–181. See also Retinal ischemia Ischemic cascade, 188

Janus protein tyrosine kinases, 12 Juglone, 86

Kelch-like ECH-associated protein 1 (Keap 1), 14 Keratoconus (KC), 60–61 a-Keto-S-methyl-butyric acid (KMB), 90 Kynurenic acid, 99

L-arginine, 112, 140, 184, 186 L-citrulline, 184 L-3,4-dihydroxyphenylalanine (L-DOPA), 198–200 LDL oxidation, effects on cataract, 96–98

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214 LDL protection, cooperative effects of rutin and ascorbate in, 97–98 Lipid peroxidation, 99 radicals, 34–35 Lipofuscin, 169–172 photoreactivity of, 169–170 Lipoic acid, 13–14, 160, 183, 191 L-NAME, 112, 187 L-tyrosine, 198, 201 Lutein, 160, 164, 169, 171

Macrophage cultures, cytokine stimulation of, 122–123 Macular degeneration, ROS role, 167–172 Manganese superoxide dismutase, 40 Mannitol, 184, 191 Melanin, 169 biosynthesis, 147–152, 197–198 and oxidative reactions, 147–155 properties antioxidant properties, 150–152 for photoprotection, 147–152 pro-oxidant and phototoxic action, 153–155 rate constants of interaction with free radicals and singlet oxygen, 150–151 Melanogenesis, 148 Melanosomes, 148 photoreactivity of, 171 in RPE cells, 205 Menadione (2-methyl-1,4-naphthoquinone) glutathione levels in cells exposed to, 52 Michael addition of thiols/thiolates to, 51 redox cycling of, 50–52 Metalloproteins, 40 Metallothioneins, 71–72 Michael addition of thiols/thiolates, to menadione, 51 Mitochondria, 22, 140–142 antioxidant systems, 4 cellular sources of superoxide anion, 2, 12 features and cell function, 5–6 generation of signaling molecules, 6–8 and glaucoma, 74–75 location and function of cytochrome c in, 141–142

Index Mitochondrial Manganese-containing SOD (SOD2), 58 Mitochondrial respiratory chain, 6–7 Mitochondrial SOD, 35 Mitogen-activated protein kinases, 12 Mitosis, 58, 62 Monobromobimane (mBBr), 48–49 Muller cells, 112, 163, 183, 186 Myocardial contractile failure, 40 Myricetin, 99

N-acetyl cysteine, 160 NADPH oxidase, 2, 12, 36, 94 NAD(P)H oxidoreductases (diaphorases), redox substrates of, 87 NADPH-quinone reductase, 95 Naphthoquinones, 86 Neural cell apoptosis, 160–161 Neurodegenerative disorders, 11 Neuronal hypoxia, 98 Neuronal NO synthase (nNOS or NOS1), 108, 186 NFkB, 12, 109, 185 Nitrated retinal proteins identification of, 133–137 localization in EAU, 137–139 Nitrate-respiration, 82 Nitric oxide, 12, 34–35, 71–72 biological effects, 109 in cell signaling and gene expression, 8 in EAU, 107–114, 132–139 pathogenesis, 111–113 pathology, 108–111 TNF activation and, 121–128 effects on immune function, 108–109 generation of RNS and, 33 induced tissue damage, 113–114 induce protein S-glutathionylation, 39 induction in EAU, 111–113 quantification of synthesis of, 123 related oxidants in health and disease, 33–41 therapeutic strategies to reduce tissue damage by, 113–114 univalent oxidation of, 2–3 Nitric oxide synthases (NOS), 37, 108–114, 132, 140, 142, 163, 184, 186

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Index Nitrofuran, 86 Nitrogen, oxidation of, 2–3 Nitrosyl radical, 182 NMDA-receptors, 99 NOS2 KO mice model, 111 NOS uncoupling, 37 Nrf-2-Keap1-ARE activators, 20 NSAIDs (non-steroidal anti-inflammatory drugs), 94

Ocular diseases, and ROS, 94 Ophthalmic artery occlusion, 179 Oxidants, in corneal diseases, 55–65 Oxidative reactions, and melanin, 147–155 Oxidative stress and atherosclerosis, 15–16 concept of, 1–2, 71 in ganglion apoptosis, 76–78 glutathione and, 76 involvement in glaucoma pathogenesis, 71–78 as neuroprotective strategy in retinal ischemia, 190–191 in pathogenesis of retinal ischemia, 177–192 and raised IOP, 75–76 reduction in retinal disease, 197–206 ROS and RNS and, 33 Oxygen toxicity, 82 univalent reduction of, 2–3 Oxygen stress cataract protection from, 94–96 by detoxifying enzymes, 94–95 by phenolic derivatives, 95–96

Paraquat, 86, 171 Parkinson’s disease, 40, 97 Pathogen-associated molecular pattern (PAMP), 127 Pecking order principle, 96–97 Penetration rates, determination of, 91–94 Penicillin, 198 Peroxiredoxins, 13 Peroxyl radical, 71–72

215 Peroxynitrite, 12, 34–35, 72, 111–112, 179, 182 and ocular inflammation, 131–142 role in EAU, 139–142 scavengers and diabetic retinopathy, 160–162 Phacoemulsification cataract surgery, corneal endothelial cell loss after, 64–65 Phagocytosis assay for, 199 induced tyrosinase expression, 204 of ROS, 112, 198, 201, 203–204 Phenolic redox reactions, 96 Phenothiazines, 85 Pheomelanin, 148, 153 Phosphoglycerate mutase, 140 Phosphoinositide-3-kinase, 12 Phospholipase C-g1, 12 Photo-ageing, 149 Photodynamic drugs, 84–85 Photoprotection, melanin properties for, 147–152 Photoreactivity of lipofuscin, 169–170 of melanosomes, 171 of retina, 168–169 Photoreceptor cells, 140 Photosystem I and II, 81–82 Photothrombosis retinal ischemia model, 181 Phototoxic action, of melanin, 153–155 Polyphenols, 13–14, 81 Porphyrin, 169 Postranslational modifications in proteins by ROS and RNS, 38. See also Protein post-translational modifications Primary open-angle glaucoma (POAG), 71, 75 Progesterones, 85 Pro-oxidant, properties of melanin, 153–155 Propolis, 99 Protein glycosylation, and cataract, 87–89 Protein post-translational modifications by reactive oxygen and nitrogen species, 3–4

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216 [Protein post-translational modifications] by ROS and RNS, 37–41 S-Glutathionylation, 37–39 S-nitros(yl)ation, 38–40 tyrosine nitration, 38, 40–41 Pycnogenol, 99, 160 Pyridorin, 98 Pyridoxamine, 98 Pyrroloquinolin quinones (PQQ), 86 Quercetin, 99 Radiation-induced cataract, 100 Reactive nitrogen species (RNS). See also ROS and RNS generation of, 3, 33 postranslational protein modifications, 33–34 protein post-translational modifications by, 3–4 Reactive oxygen species (ROS). See also ROS and RNS AMD and, 167–172 cell damage by, 179 and corneal diseases, 55, 63–65 intracellular concentrations of, 71 mediated cell damage, 12 ocular diseases and, 94 phagocytosis of, 112, 198, 201, 203–204 and photoreactivity of lipofuscin, 169–170 melanosome, 171 retina, 168–169 protein post-translational modifications by, 3–4 RPE susceptibility and adaptation to, 171–172 signaling pathways, 12 sources, 12–13, 77 specific removal of, 5 Redox cell signaling, 3 Reductive events, and cataract, 86–87 Respiratory burst, 83

Index Retina photoreactivity of, 168–169 resistance to ischemia, 179–181. See also Retinal ischemia tyrosine-nitrated proteins in, 138, 142 Retinal ganglion cell axons, 71, 74–75 and glaucoma, 74–75 Retinal ischemia counteracting oxidative stress as neuroprotective strategy, 190–191 events associated with, 188–189 free radical role, 182–185 model of, 179–180 neurological mechanisms, 181–182 nitric oxide free radical in, 186–187 ocular diseases implicated in, 178 oxidative stress role in pathogenesis of, 177–192 therapeutic intervention strategies for, 190 Retinal myeloid cell isolation, 122 Retinal pigment epithelium (RPE), 110–112, 153–155, 185, 197–204 susceptibility and adaptation to ROS, 171–172 Retinal proteins in uveitis, nitration of, 132–135 Riboflavine, 83 ROS and RNS characteristics of, 33–35 formation of, 35–37 oxidative stress and, 33 postranslational modifications, 38 protein modification produced, 37–41 S-Glutathionylation, 37–39 S-nitros(yl)ation, 38–40 tyrosine nitration, 38, 40–41 sources, 12–13, 36 Rotterdam study, 168 RPE65, 168

Scavenger receptors (SRA), 15 Selenite-induced cataract, 99 Selenium, 160 Sensory retinal detachment, 168 S-Glutathiolation. See S-glutathionylation

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Index S-Glutathionylation, 3–4, 34, 37–39 Signaling molecules, mitochondrial generation of, 6–8 Singlet oxygen, 71–72, 83, 86 melanin interaction with, 150–152 S-nitrosation. See S-nitros(yl)ation S-nitros(yl)ation, 3, 34, 38–40 Sodium arsenite, 171 Staurosporine (ST)-induced neuronal apoptosis, 99 STNFr-Ig therapy, 125–126 Streptomycin, 198 Subarachnoid hemorrhage, 99 Sulfonamides, 85 Sulfonic urea, 85 Superoxide anion, 12, 34, 71–72, 75, 205 in cell signaling and gene expression, 8 in EAU, 132–139 sources of, 2, 33 Superoxide dismutases (SODs), 13, 34–35, 57–59, 71–72, 76, 95, 99, 140, 160, 168, 172, 179, 183, 190–191

Taurine, 160 T cell apoptosis, 113–114 Tertbutylhydroperoxide, 171 Tetracyclines, 85 TGF-b, 107, 109, 111–112, 122–125 Th1 cytokines, 107, 110, 112, 124 Th2 cytokines, 108–109, 112 Thiol-disulfide oxidoreductases, 13 Thioredoxin, 183 T lymphocytes, 110 TNF-a, 76, 109–110, 113–114, 121, 122–128, 142 TNFRp55-/- mice, 122, 126–127 role in autocrine signaling following IFN g stimulation in EAU, 126–127 a-Tocopherol, 13–15, 17–19, 21, 96, 161, 171, 190 b-Tocopherol, 15 d-Tocopherol, 15 g-Tocopherol, 15 Toxic lipoprotein degradation, 95 Transferrin, 13 Transition metal catalyzed oxidations, 88

217 Triacetylphenolisatin, 85 Trimetazidine, 191 Trolox, 57, 160 Tumour Necrosis Factor-alpha (TNF-a). See TNF-a Tyrosinase antibody staining in bovine RPE cells, 201 DOPA oxidase activity of, 200–201 electron-microscopical localization of, 199–201 functions, 205–206 immunocytochemistry, 203 immunodetection of, 199–200 phagocytosis-induced expression, 204–205 rod outer segments isolation, 199 ROS impact on activity of, 204 tyrosine hydroxylase activity of, 200–201, 203 Tyrosine nitration, 38, 40–41 Ubiquinol, 13–14 Ubisemiquinone, autoxidation of, 2 Univalent reduction of oxygen, mechanisms, 2–3 Uric acid, 13–14, 190 Uveitis, nitration of retinal proteins in, 132–135 VCAM-1, 15 Vitamin A, 71–72 Vitamin C, 13–15, 17, 21, 57, 59, 81, 98–99, 160, 168 Vitamin D, 71–72 Vitamin E, 13–15, 17, 19, 21, 57, 71–72, 81, 98, 168, 183, 191 Voltage dependent anion channels (VDAC), 7 Xanthine oxidase, 12, 36, 56, 185 Xanthurenic acid, 85–86 Zeaxanthin, 169, 171 Zeta-crystallin, 95 Zinc, 160

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about the book… Free radicals are molecules with an unpaired electron in the outer shell or an electron that was damaged from either attack or from a poor splitting bond. After a free radical is formed it will continue to attack other molecules, which usually results in the damage of tissue or destruction of a healthy cell. Free radicals arise normally through metabolism. However, sometimes the body’s immune system will create them on purpose to neutralize viruses and bacteria. Free radicals are implicated in many ophthalmic disorders including uveitis, optic nerve damage, retinal ischemia, and macular degeneration. Free Radicals in Ophthalmic Disorders presents the most current knowledge pertaining to the role of free radicals/oxidants in ocular disorders, and the use of antioxidants in the prevention of these disorders. Written by today’s leading ocular scientists and clinicians Free Radicals in Ophthalmic Disorders • gives comprehensive coverage of the role of free radicals/oxidants in ocular disorders • covers the use of antioxidants to prevent oxidative stress and ocular tissue damage • examines external factors that may result in the stimulation and heightened occurrence of free radicals/oxidants about the editors... MANFRED ZIERHUT is Associate Professor of Ophthalmology, University Eye Hospital, Tubingen, Germany. Dr. Zierhut received his M.D. from the University of Hannover, Germany, and has published 102 articles, co-authored 24 books, and completed over 3000 surgeries in ophthalmology. ENRIQUE CADENAS is Professor of Pharmacology and Pharmaceutical Sciences and Associate Dean of Research Affairs at the University of Southern California School of Pharmacy, Los Angeles. He is also Professor of Biochemistry at the Keck School of Medicine, University of Southern California. Dr. Cadenas received his M.D. in Medicine and his Ph.D. in Biochemistry/ Biophysics from the University of Buenos Aires, Argentina, and his main focus of research, besides free radicals, covers oxidative stress, mitochondrial dysfunction, aging, and neurodegenerative diseases. He is the author of over 200 peer-reviewed papers.

Printed in the United States of America

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Zierhut • Cadenas • Rao

NARSING A. RAO is Professor of Ophthalmology and Pathology at the Keck School of Medicine, and the first chair holder of the Stieger Vision Research Endowed Chair of Doheny Eye Institute, University of Southern California, Los Angeles and Director of the Intraocular Inflammation/ Uveitis Service and the Director of the Ophthalmic Pathology Laboratories at the Doheny Eye Institute. Dr. Rao was awarded his M.D. from Osmania University and completed his internship at Osmania General Hospital, Hyderabad, India. Following a year of rotating internships in upstate New York, he completed two residencies in pathology and ophthalmology at Georgetown University, Washington, D.C. and a fellowship in ophthalmic pathology at the Armed Forces Institute of Pathology, Washington, D.C. Dr. Rao is involved in both research aspects and the clinical treatment of inflammatory ocular diseases affecting the uveal tract, vitreous, retina and sclera and immune disorders affecting the eye. Dr. Rao has published over 375 peer-reviewed articles in U.S. and international journals and has authored or edited four books.

Free Radicals in Ophthalmic Disorders

Ophthalmology

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Edited by

Manfred Zierhut Enrique Cadenas Narsing A. Rao

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2/18/2008 8:36:22 AM