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...

Author: Zierhut Manfred | Enrique Cadenas | Narsing Rao

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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.

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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

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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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Preface

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


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

v

81


vi

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

197

Index

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

209


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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viii

Contributors

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.


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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Yap et al.

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.


Free Radical Biology, Mitochondrial Functions, and Nitric Oxide

3

(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. Inãrrea P. Purification and determination of activity of mitochondrial cyanide-sensitive superoxide dismutase in rat tissue extract. Methods Enzymol 2002; 349:106–114. 18. Inãrrea 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, Arnaíz 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.


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.

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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.



3 Nitric Oxide—Related Oxidants in Health and Disease Cecilia Gonza´lez de Ordunã 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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Gonza´lez de Ordunã and Lamas

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,


Nitric Oxide—Related Oxidants in Health and Disease

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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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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 mM1cm1 and 13.8 mM1cm1 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.


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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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.


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.



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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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.


[rakesh][D:/Informa_Publishing/Zierhut_H4433_112023/z_production/ z_3B2_3D_files/978-1-4200-4433-1_CH0008.3d] [28/1/08/12:17:47] [107–120]

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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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 naıë 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.



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.



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 0.41.0 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).


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).


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 ).


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

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