Biomarkers for Antioxidant Defense and Oxidative Damage

Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications Biomarkers for Antioxi...

Author: Giancarlo Aldini | Kyung-Jin Yeum | Etsuo Niki | Robert M. Russell

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Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications

Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications Edited By

Giancarlo Aldini Kyung-Jin Yeum Etsuo Niki Robert M. Russell

A John Wiley & Sons, Inc., Publication

Edition f rst published 2010 © 2010 Blackwell Publishing Blackwell Publishing was acquired by John Wiley & Sons in F ebruary 2007. Blackwell’s publishing program has been mer ged with Wiley’s global Scientif c, Technical, and Medical business to for m Wiley-Blackwell. Editorial Off ce 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial off ces, for customer ser vices, and for infor mation about how to apply for per mission to reuse the cop yright material in this book, please see our Website at www.wiley.com/wiley-blackwell. Authorization to photocopy items for inter nal or personal use, or the inter nal or personal use of specif c clients, is g ranted by Blackwell Publishing, provided that the base fee is paid directl y to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been g ranted a photocopy license by CCC, a separate system of pa yments has been ar ranged. The fee code for users of the Transactional Reporting Service is ISBN-13: 978-0-8138-1535-0/2010. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, ser vice marks, trademarks, or registered trademarks of their respecti ve owners. The publisher is not associated with an y product or vendor mentioned in this book. This publication is designed to pro vide accurate and authoritative information in regard to the subject matter co vered. It is sold on the understanding that the publisher is not eng aged in rendering professional ser vices. If professional advice or other expert assistance is required, the ser vices of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Biomarkers for antioxidant defense and o xidative damage : principles and practical applications / edited by Giancarlo Aldini . . . [et al.]. p. ; cm. Includes bibliographical references and inde x. ISBN 978-0-8138-1535-0 (hardback : alk. paper) 1. Antioxidants. 2. Oxidative stress. 3. Active oxygen. 4. Biochemical markers. I. Aldini, Giancarlo. [DNLM: 1. Biological Markers. 2. Antioxidants–physiology. 3. Oxidative Stress–physiology. QW 541 B6159 2010] RB170.B566 2010 613.2 ′86–dc22 2010011386 ISBN 9780813815350 A catalog record for this book is a vailable from the U.S. Library of Cong ress. Set in 9.5 on 11 pt Times New Roman by Toppan Best-set Premedia Limited Printed in Singapore Disclaimer The publisher and the author mak e no representations or w arranties with respect to the accurac y or completeness of the contents of this w ork and specif cally disclaim all w arranties, including without limitation warranties of f tness for a par ticular purpose. No warranty may be created or extended by sales or promotional materials. The advice and strate gies contained herein ma y not be suitable for every situation. This work is sold with the understanding that the pub lisher is not engaged in rendering le gal, accounting, or other professional ser vices. If professional assistance is required, the ser vices of a competent professional person should be sought. Neither the pub lisher nor the author shall be liab le for damages arising herefrom. The fact that an or ganization or Website is refer red to in this w ork as a citation and/or a potential source of fur ther information does not mean that the author or the pub lisher endorses the infor mation the organization or Website may provide or recommendations it ma y make. Further, readers should be a ware that Internet Websites listed in this w ork may have changed or disappeared betw een when this work was written and w hen it is read. 1 2010

Contents

Preface vii Contributors ix 1.

Antioxidant Activity and Oxidative Stress: An Overview Kyung-Jin Yeum, Robert M. Russell, and Giancar lo Aldini

2.

Enzymatic Antioxidant Defenses 21 Sayuri Miyamoto, Hirofumi Arai, and Junji Terao

3.

Antioxidants as Biomarkers of Oxidative Stress 35 Ikuyo Ichi and Shosuke K ojo

4.

LDL Oxidation as a Biomar ker of Antioxidant Status 51 Mohsen Meydani, EunHee Kong, and Ashley Knight

5.

The Isoprostanes: Accurate Markers and Potent Mediators of Oxidant Injury in Vivo 65 Joshua D. Brooks, Brian E. Co x, Klarissa D. Hardy, Stephanie C. Sanc hez, Sonia Tourino, Tyler H. Koestner, Jocelyn R. Hyman-Howard, and Ginger L. Milne

6.

Hydroxyoctadecadienoic Acid (HODE) as a Mar ker of Linoleic Acid Oxidation 85 Yasukazu Yoshida and Etsuo Niki

7.

Oxysterols: Potential Biomarkers of Oxidative Stress 99 Luigi Iuliano and Ulf Diczfalusy

8.

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomarkers 117 Françoise Guéraud

3

v

vi Contents 9.

Oxidative Modif cation of Proteins: An Overview 137 Paul J. Thornalley and Naila Ra bbani

10. Immunochemical Detection of Lipid P eroxidation-specif c Epitopes 157 Koji Uchida 11. Mass Spectrometric Strategies for Identif cation and Characterization of Carbonylated Peptides and Proteins 173 Marina Carini and Marica Orioli 12.

Nitrotyrosine: Quantitative Analysis, Mapping in Pr oteins, and Biological Signif cance 199 José M. Souza, Silvina Bartesa ghi, Gonzalo Peluffo, and Rafael Radi

13. Ubiquitin Conjugates: A Sensitive Marker of Oxidative Stress 219 Fu Shang and Allen Taylor 14. Covalent Modif cations of Albumin Cys34 as a Biomarker of Mild Oxidative Stress 229 Giancarlo Aldini, Kyung-Jin Yeum, and Giulio Vistoli 15. Protein S-glutathionylation and S-cysteinylation 243 Graziano Colombo, Aldo Milzani, Roberto Colombo , and Isabella Dalle-Donne 16. DNA Oxidation, Antioxidant Effects, and DNA Repair Measured with the Comet Assay 261 Mária Dušinská and Andrew R. Collins 17. Hydroxylated Nucleotides: Measurement and Utility as Biomar kers for DNA Damage, Oxidative Stress, and Antioxidant Eff cacy 283 Phyllis E. Bowen 18. Exocyclic DNA Adducts as Biomarkers of Antioxidant Defense and Oxidative Stress 319 Roger W.L. Godschalk Index 333

Preface

Oxidative damage is kno wn to be associated with the aging process and v arious chronic diseases such as atherosclerosis, diabetes, cataract, macular degeneration, and Alzheimer’s disease. Oxidative damage may be either a cause or an effect; therefore, its accurate measurement using sensitive and specif c biomarker is greatly needed by the scientif c community. When oxidative damage acts as a causati ve f actor of disease, its biomark ers can be a useful tool to better understand the pathogenic mechanisms involved, f nd novel drug targets, and evaluate effective defense strate gies such as phar maceuticals, nutraceuticals, or food and deri vatives. When oxidative stress is the disease ’s effect, its measurement could pro vide early prediction of the onset and pro gression for the disease. Hence, o xidative stress biomark ers can be used for diagnosis, prognosis, and treatment eff cacy. Although signif cant methodolo gical adv ances ha ve been made since the de velopment of thiobarbituric acid reactive substances (TBARS) and total carbonyl assays, which were widely used in 1980s and 1990s for measuring o xidative damage, the achievement of a gold standard method to determine antioxidant defense/oxidative damage status is not yet at hand. The development and application of v arious biomark ers for measuring antio xidant defense/o xidative damage is an evolving research area. Biomarkers of oxidative damage can be classif ed as either direct or indirect methods. Direct methods measure the oxidation products involving substrates such as lipids, proteins, and nucleic acids. Indirect methods measure antio xidant status b y analyzing endogenous levels of enzymatic and nonenzymatic antioxidants, as well as the resistance of a biolo gical matrix to an induced o xidative stress. This book describes the methodolo gical principles of cur rent state -of-the-art methods for measuring antio xidant acti vity/oxidative stress and their practical applications. In par ticular, the f rst three chapters describe conventional biomarkers as well as recent advances for measuring antioxidants and antio xidant capacity. Basic concepts and methodolo gies of widel y used assays and their applications are criticall y evaluated. In addition, f actors affecting antioxidant capacity in a biological system as well as the biological relevance of hydrophilic and lipophilic capacity assays are discussed. Determination of o xidative damage using lipid pero xidation products and metabolites is co vered in chapters 4 through 7. In par ticular the follo wing specif c biomark ers for lipid pero xidation are considered: isoprostanes (arachidonic acid C20:4 o xidation products),

vii

viii Pref

ace

hydroxyloctadecaenoic acid (linoleic acid C18:2 o xidation products), o xysterols (cholesterol oxidation products), and reactive carbonyl species from lipid peroxidation. Chapters 9 through 11 cover an outstanding area in biomark er discovery, that of protein o xidation. After an introduction on oxidative modif cations of proteins, recent advances in the measurement of carbonyls adducted proteins b y antibodies and mass spectrometr y are discussed. These chapters are followed by discussions of the recent de velopment on protein tyrosine nitration (Chapter 12) and ubiquitin -conjugates (Chapter 13) to deter mine mild and se vere o xidative damage. Biomarkers that have recently been developed thanks to cutting -edge technology, which determines oxidative modif cations of protein thiols, are presented in chapters 14 and 15. Well reco gnized biomark ers as w ell as recentl y de veloped biomark ers for DN A o xidative damage and their application in humans are discussed in chapters 16 through 18. In particular, methodological aspects for the measurement of DN A o xidation (comet assa y, h ydroxylated nucleotides, and exocyclic DNA adducts) are described, together with their implication in relation to chronic diseases. We believe that the cur rent book will be of a g reat interest to scientists w ho are in volved in basic research on o xidation, applied scientists e valuating the ef fects of nutraceuticals or pharmaceutical compounds on antio xidant activity/oxidative status, and ph ysicians who want to understand the de gree of oxidative damage in patients with specif c chronic diseases.

Contributors

Giancar lo Aldini Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via L. Mangiagalli 25 20133 Milan, Italy [email protected] Hir ofumi Arai Department of Applied and Environmental Chemistry Kitami Institute of Technology Koen - cho165 Kitami 090 - 8507,Japan [email protected] - it.ac.jp SilvinaBartesaghi Departamento de Histología, Departamento de Bioqu ímica, and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] Ph yllis E. Bowen Department of Kinesiology and Nutrition University of Illinois at Chicago, m/c 994 Chicago, IL 60612 USA pbo [email protected] oshua J D. Brooks Department of Phar macology University of Califor nia San Diego San Diego, CA 92093 USA [email protected]

ix

x Contributors MarinaCarini Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via L. Mangiagalli 25 20133 Milan, Italy [email protected] Andr ew R. Collins Department of Nutrition University of Oslo PB 1046 Blinder n 0316 Oslo, Norway a.r [email protected] Gr aziano Colombo Department of Biology University of Milan Via Celoria 26 20133 Milan, Italy [email protected] Rober to Colombo Department of Biology University of Milan Via Celoria 26 20133 Milan, Italy [email protected] BrianE. Cox Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA brien.e.co [email protected] Isa bella Dalle - Donne Department of Biology University of Milan via Celoria 26 20133 Milan, Italy [email protected] UlfDiczfalusy Karolinska Institutet Department of Laborator y Medicine Division of Clinical Chemistr y Karolinska University Hospital, Huddinge C1.74 SE -141 86 Stockholm, Sweden [email protected]

Contributors

M á ria Du š insk á CEE Health Effects Group Norwegian Institute for Air Research PB 100 2027 Kjeller , Norway [email protected] and Department of Experimental and Applied Genetics Slovak Medical University Limbova 12 833 03 Bratisla va, Slovakia Ro ger W.L. Godschalk Department of Health Risk Analysis and Toxicology Maastricht University Universiteissingel 50 6200MD Maastricht, The Netherlands R.Godschalk@GRA T.unimaas.nl ran F ç oiseGu é arud INRA Institut National de la Recherche Argonomigue, UMR 1089 X énobiotiques BP 93173 31027 Toulouse Cedex 3, France [email protected] KlarissaD. Hardy Department of Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA klarissa.d.hardy@v anderbilt.edu ocelyn J R. Hyman - Ho ward Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA [email protected] Ikuy o Ichi Department of Health Chemistr y Graduate School of Phar maceutical Sciences The University of Tokyo Bunkyo - ku,Tokyo 113 - 0033Japan [email protected] - tok yo.ac.jp LuigiIuliano Sapienza University of Rome Department of Experimental Medicine, Unit of Vascular Medicine Vascular Biology and Mass Spectrometry Lab Corso della Republica 79 04100 Latina, Italy [email protected]

xi

xii Contributors Ashle y Knight Vascular Biology Laboratory Jean Mayer, USDA Human Nutrition Research Center on Aging at Tufts University 711Washington Street Boston, MA 02111 USA knight.ashle [email protected] yler T H. Koestner Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA tyler [email protected] Shosuk e Kojo Department of Food Science and Nutrition NaraWomen ’s University Nara 630 - 8506Japan [email protected] - wu.ac.jp EunHeeKong Department of Family Medicine College of Medicine Kosin University Seo - Gu,Busan, South Korea [email protected] MohsenMeydani Vascular Biology Laboratory Jean Mayer USDA—Human Nutrition Research Center on Aging Tufts University 711Washington St. Boston, MA 02111 USA mohsen.me [email protected] Ging er L. Milne Division of Clinical Phar macology Departments of Medicine and Phar macology Vanderbilt University School of Medicine Nashville, TN 37232 USA Ginger [email protected] AldoMilzani Department of Biology University of Milan Via Celoria 26 20133 Milan, Italy [email protected]

Contributors

Sa yuri Miyamoto Departamento de Bioqu í mica Institute de Qu í mica Universidade de S ã oPaulo CP 26077, CEP 05513 -970 S ão Paulo SP, Brazil [email protected] EtsuoNiki Health Research Institute National Institute of Advanced Industrial Science and Technology (AIST) Ikeda, Osaka 563 - 8577Japan etsuo - [email protected] MaricaOrioli Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via Mangiagalli 25 20133 Milan, Italy [email protected] GonzaloPeluffo Departamento de Bioqu ímica and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] NailaRabbani Systems Biology, Protein Damage, and Systems Biolo gy Research Group Clinical Sciences Research Institute University of Warwick University Hospital Coventry CV2 2DX, UK N [email protected] RafaelRadi Departamento de Bioqu ímica and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] r Rober t M. Russell Offce of Dietar y Supplements, National Institutes of Health 6100 Executive Blvd. Bethesda, Maryland 20892 USA [email protected] r

xiii

xiv Contributors StephanieC. Sanchez Division of Clinical Phar macology Vanderbilt University School of Medicine Nashville,TN 37232 - 6602USA Stephanie.sanchez@v anderbilt.edu FuShang Jean Mayer USDA—Human Nutrtion Research Center on Aging Tufts University 711Washington St. Boston, MA 02111 USA [email protected] os J é M. Souza Departamento de Bioqu ímica and Center for F ree Radical and Biomedical Research Facultad de Medicina Universidad de la Rep ública Avda. General Flores 2125 11800 Montevideo, Uruguay [email protected] Allen Taylor Jean Mayer USDA—Human Nutrition Research Center on Aging Tufts University 711Washington Street Boston, MA 02111 USA allen.ta [email protected] unji J Terao Department of Food Science Graduate School of Nutrition and Bioscience Institute of Health Biosciences University of Tokushima Kuramoto - cho3 Tokushima 770 - 8503Japan terao@nutr .med.tokushima - u.ac.jp aul P J. Thornalley Systems Biology, Protein Damage and Systems Biolo gy Research Group Clinical Sciences Research Institute University of Warwick University Hospital Coventry CV2 2DX, UK [email protected] Sonia Tourino Institute for Advanced Chemistry of Catalonia CSIC (ICAQ - CSIC) Barcelona, Spain [email protected]

Contributors

K oji Uchida Laboratory of Food and Biodynamics Graduate School of Bioag ricultural Sciences Nagoya University Nagoya 464 - 8601Japan [email protected] - u.ac.jp Giulio Vistoli GiulioVistoli, Ph.D. Dipartimento di Scienze Farmaceutiche “Pietro Pratesi ” Università degli Studi di Milano Via L. Mangiagalli 25 20133 Milan, Italy [email protected] yKung - JinYeum Jean Mayer USDA—Human Nutrition Research Center on Aging Tufts University 711Washington St. Boston, MA 02111 USA K [email protected] asukazu Y Yoshida Health Research Institute National Institute of Advanced Industrial Science and Technology 2217 - 14Hayashi - cho,Takamatsu 761 - 0395,Japan oyshida - [email protected]

xv

Biomarkers for Antioxidant Defense and Oxidative Damage: Principles and Practical Applications

Chapter1 Antioxidant Activity and Oxidative Stress: An Overview K yung - Jin eYum , RobertM. Russell and ,

Giancar lo Aldini

INTR ODUCTION Oxidative stress is in volved in the process of aging (Kre gel and Zhang 2007) and v arious chronic diseases such as atherosclerosis (F earon and Faux 2009), diabetes (Ceriello and Motz 2004), and eye disease (Li et al. 2009a), whereas fruit and vegetable diets rich in antio xidants such as pol yphenols, vitamin C, and carotenoids are cor related with a reduced risk of such chronic diseases (Christen et al. 2008, Dauchet et al. 2006, Dherani et al. 2008). An excessive amount of reacti ve o xygen/nitrogen species (R OS/RNS) leading to an imbalance betw een antioxidants and oxidants can cause oxidative damage in vulnerable targets such as unsaturated fatty acyl chains in membranes, thiol groups in proteins, and nucleic acid bases in DNA (Ceconi et al. 2003). Such a state of “oxidative stress ” is thought to contribute to the patho genesis of a number of human diseases (Thannickal and F anburg 2000). Sensitive and specif c biomarkers for antioxidant status/oxidative stress are essential to better understand the role of antio xidants and oxidative stress in human health and diseases, thereb y maintaining health and establishing effective defense strategies against oxidative stress. Several assays to measure “total” antioxidant capacity of biolo gical systems ha ve been de veloped to investigate the in volvement of o xidative stress in patholo gical conditions or to e valuate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antio xidant capacity in the aqueous compar tment of plasma. Consequently, w ater-soluble antio xidants such as ascorbic acid , uric acid , and protein thiols mainly inf uence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids show little inf uence over the many results. However, there are new approaches to def ne the total antioxidant capacity of plasma, w hich ref ect the antioxidant network between waterand fat-soluble antioxidants. Revelation of the mechanism of action of antio xidants and their true antioxidant potential can lead to identifying proper strate gies to optimize the antio xidant defense systems in the body . Methodological aspects of v arious antio xidant capacity assa ys ha ve been e xtensively discussed recentl y (Magalhaes et al. 2008). This chapter focuses on impor tant antio xidants in biological systems, f actors affecting bioavailability of antio xidants and, therefore, antio xidant capacity, and basic principles of v arious biomark ers for antio xidant capacity and their applications. Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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4 Chapter

1

OXIDATIVE STRESS AND ANTIOXIDANTS IN A BIOLOGICAL SYSTEM ROS are continuousl y generated b y nor mal metabolism in the body (Gate et al. 1999) and these ROS are necessary to maintain biological homeostasis through various functions such as vasoregulation and v arious cellular signal transduction (Hensle y and Flo yd 2002). However, overproduction of these ROS can also cause damage to the macromolecules necessar y for cell structure and function. Cellular production of ROS such as superoxide anion (O2•−), hydroxyl radical (HO• ),peroxyl radical (R OO•), and alk oxyl radical (R O•) occurs from both enzymatic and non -enzymatic reactions. Mitochondria appear to be the most impor tant subcellular site of R OS production, in particular of O 2•− and H 2 O2 in mammalian organs. The electron transfer system of the mitochondrial inner membrane is a major source of supero xide production when molecular oxygen is reduced b y a single electron. Supero xide can then dismutate to for m h ydrogen pero xide (H2 O2), and then can fur ther react to for m the hydroxyl radical (HO •) and ultimately water. In addition to intracellular membrane-associated oxidases, soluble enzymes such as xanthine oxidase, aldeh yde o xidase, dih ydroorotate deh ydrogenase, f avoprotein deh ydrogenase, and tryptophan dioxygenase can generate R OS during catal ytic c ycling. Auto-oxidation of small molecules such as dopamine, adrenaline (epinephrine), f avins, and quinols can be an important source of intracellular ROS production as well. In most cases, the direct product of such auto oxidation reactions is the supero xide anion (Thannickal and F anburg 2000). Any compound that can inhibit oxidation of external oxidants is considered to be an antioxidant. This is a relati vely simple def nition but, at times, it becomes v ery diff cult to e valuate whether a compound actuall y has an antio xidant action, par ticularly in vivo . It is still not clear w hat kinds of R OS play a role in the patho genesis of human disease and where the major sites of R OS action occur. There is, ho wever, convincing evidence that lipid peroxidation is related to human patholo gy, such as in atherosclerosis (V alkonen and K uusi 1997). The actions of antio xidants in biolo gical systems depend on the nature of o xidants or ROS imposed on the systems, and the acti vities and amounts of antio xidants present and their cooperative/synergistic interactions in these systems. Numerous epidemiological studies have indicated that diets rich in fr uits and vegetables are correlated with a reduced risk of chronic diseases (Czer nichow et al. 2009, Hung et al. 2004, Liu et al. 2001, Liu et al. 2000). It is probab le that antioxidants, present in the fr uits and vegetables such as polyphenols, carotenoids, and vitamin C, prevent damage from harmful reactive oxygen species, w hich either are continuousl y produced in the body during nor mal cellular functioning or are deri ved from exogenous sources (Gate et al. 1999). The possible protective effect of antioxidants in fruits and vegetables against ROS has led people to consume antio xidant supplements such as β - carotene,α-tocopherol, and/or multi vitamins. It is not sur prising to note that more than 11% of US adults age 20 y ears or older consume at least 400 IU of vitamin E per da y from supplements (F ord et al. 2005). However, inter vention studies ha ve failed to show a consistent benef cial effect of antioxidant supplements such as vitamin E (Lee et al. 2005) or β-carotene (Baron et al. 2003, Omenn et al. 1996) against chronic diseases. How can we explain these apparent contradictory results between observational studies and intervention trials? It is interesting to note that although se ven and a half years of supplementation with a combination of antio xidants (vitamin C, β-carotene, zinc, and selenium) did not af fect the risk of metabolic syndrome, baseline concentrations of ser um vitamin C and β - carotenewere negatively associated with metabolic syndrome in a generall y well-nourished population (Czernichow et al. 2009). It is probab le that the generall y w ell-nourished population maintains optimal ranges of antio xidants through a balanced dietar y fr uit and v egetable intak e. However, high doses of a single or limited mixture of antioxidant supplements may not affect the already saturated in vivo antioxidant network, but rather could result in an imbalance in the antio xidant

Antioxidant Activity and Oxidative Stress: An Overview

5

network and could possib ly even act as pro -oxidants. A recent prospecti ve study sho wing an inverse association of baseline plasma antioxidant concentrations with the risk of heart disease and cancer also suppor ts the benef cial effect of a balanced antio xidant status, w hich can be attained by eating diets high in fr uits and vegetables (Buijsse et al. 2005).

MARKERS OF ANTIOXIDANT CAPACITY IN A BIOLOGICAL SYSTEM Several human studies ha ve f ailed to sho w a direct cor relation between the ph ysiologic consumption of dietar y f at-soluble antio xidants and subsequent changes in antio xidant capacity (Castenmiller et al. 1999, Pellegrini et al. 2000). For example, it has even been suggested that carotenoids may not act as antio xidants in vivo (Rice - Ev ans et al. 1997 ).These suggestions derive from the lack of proper analytical methods for measuring antioxidant capacity. Inasmuch as conventional methods, such as total radical trapping antio xidant parameter (TRAP), oxygen radical absorbance capacity (ORA C), etc., use primaril y h ydrophilic radical generators and measure primarily antioxidant capacity in the aqueous compartment of plasma, they are unable to deter mine the antio xidant capacity of the lipid compar tment (Cao et al. 1993, Lussignoli et al. 1999). Therefore, it is not surprising that most of the methods used to measure pur ported “total antio xidant capacity ” of plasma are not af fected b y lipophilic antio xidants, such as carotenoids (Cao et al. 1998b, Castenmiller et al. 1999, Pellegrini et al. 2000). This can be e xplained by the f act that plasma carotenoids, w hich are deepl y embedded in the core of lipoproteins, are not a vailable for reaction with aqueous radical species or fer ric complexes used in these assa ys. In addition, an assa y to measure total antio xidant capacity in a biolo gical sample such as plasma must consider the hetero geneity of the sample, w hich consists of both h ydrophilic and lipophilic compar tments that contain w ater-soluble and f atsoluble antioxidants, respectively. Possible cooperative/synergistic interactions among antioxidants in biological samples should not be o verlooked. Azo initiators are a class of radical inducers (w hich contain the –N= N –group) widely used in experiments in vitro to generate radical species. The azo initiators decompose at a temper ature-controlled rate to give carbon -centered radicals, which react rapidly with O 2 to yield the peroxyl radical (ROO• ). R − N = N − R → N 2 + 2R i R i + O 2 → ROOi Peroxyl radicals deri ved from azo initiators can induce the lipid pero xidation cascade and can also damage proteins. Depending on the lipophilicity of the azo initiators [2,2 ′ - azobis - (2 amidinopropane) dihydrochloride (AAPH) is w ater soluble whereas 2,2 ′ - azobis(2,4 - dimeth ylvaleronitrile (AMVN) and ,2 ′ - azobis(4 - metho xy - 2,4 - dimeth ylvaleronitrile) (MeO - AMVN)are lipophilic], the pero xyl radicals are generated in the aqueous or lipid phase of the sample, respectively. The choice of the site of radical generation is of g reat impor tance, because the activities of antio xidants present in both the lipid and aqueous compar tments depend on the localization of the attacking radical species (Y eum et al. 2003). Table 1.1 shows the cur rently available assays to deter mine antioxidant capacity in h ydrophilic and lipophilic en vironments in biolo gical samples such as plasma. When used alone, those assays (Cao et al. 1993, Valkonen and Kuusi 1997) that use hydrophilic radical initiators and probes are insuff cient for deter mining the antio xidant activity of carotenoids, w hich are deeply embedded in the lipoprotein core of biolo gical samples. There have been attempts to determine the activity of fat-soluble antioxidants by measuring the antioxidant activity of lipid extracts dissolv ed in an or ganic solv ent (Prior et al. 2003). This approach, ho wever, cannot appreciate the possible interactions between the fat-soluble and water-soluble antioxidants. The alternative approach of producing radicals in the lipid compar tment of w hole plasma and monitoring lipid pero xidation by a lipophilic probe (Aldini et al. 2001) allows measurement

Table 1.1. Assays to determine antioxidant capacity in biological systems.

6 Assa y

Radical inducer

Oxidizab le substrate (probe)

W avelength

Plasma susceptibility against exogenous pro-oxidant induced oxidation (hydrophilic assay) TRAP AAPH DCFH λ ex = 480, λ em = 526 R - Ph ycoerythrin λ ex = 495, λ em = 595 ORA C Crocinbleaching

AAPH AB AP

R - Ph ycoerythrin

λ ex = 495, λ em = 595 445nm

Plasma quenching ability of stable/pre-formed radicals (hydrophilic assay) TEA C ABTS+ • 734nm FRAP

F e3+

593nm

Plasma susceptibility against exogenous pro-oxidant induced oxidation (lipophilic assay) LipophilicORAC AAPH Fluorescein λ ex = 485, λ em = 520 Lipophilicantioxidant AAPH DPHPC λ ex = 354, λ em = 430 activity T AP MeO - AMVN BODIPY581/591 λ ex = 500, λ em = 520 TRAP:Total radical - trappingantioxidant parameter. ORA C: Oxygen radical - absorbingcapacity. TEAC: Trolox equivalent antioxidant capacity. FRAP:Ferric - reducingability of plasma. T AP: Total antioxidant performance. AAPH,ABAP: 2,2′ - Azobis - (2 - amidinopropane)dih ydrochloride. ABTS:2,2′ - Azinobis(3 - ylbenzothiazoline eth 6 - sulphonate). AUC: Area under the cur ve. MeO - AMVN: 2,2′ - Azobis(4 - metho xy - 2,4 - dimeth ylvaleronitrile). DCFH:2′ ,7′ - Dichlorodih ydrof uorescein. DPHPC:1 - P almitoyl - 2 - ((2 - (4 - (6 -ylphen - trans - 1,3,5 -xatrienyl)phenyl)ethyl) he - carbon yl - sn -ycero gl - 3 - phosphocholine. BODIPY581/591: 4,4 - Difuoro - 5 - (4 - phen yl - 1,3 - butadien yl) - 4 - bora - 3a,4a - diaza - s - indacene - 3 - undecanoic acid. Modifed from Yeum et al. 2009b.

Calculation Lagtime A UC Absorbance

Absorbance

Reference V alkonen and Kuusi 1997 Ghiselli et al. 1995 Caoet al. 1995 T ubaro et al. 1998 Kampa et al. 2002

Absorbance

Milleret al. 1993 Re et al. 1999 Benzieand Strain 1996

A UC Lagtime

Prioret al. 2003 Ma yer et al. 2001

A UC

Aldiniet al. 2001

Antioxidant Activity and Oxidative Stress: An Overview

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of the actual “total” antio xidant acti vity including possib le interactions among antio xidants located in the h ydrophilic and lipophilic compar tments, because the interference of lar ge amounts of protein (e.g. albumin) in the h ydrophilic compar tment can be o vercome b y this approach. HYDR OPHILIC ANTIOXIDANT CAPACITY ASSAYS There are mainly two hydrophilic approaches to deter mine the antioxidant capacity in plasma. The f rst approach measures the antioxidant capacity in plasma using hydrophilic assays in the presence of o xidants that act as pro -oxidants. These assa ys deter mine the susceptibility of plasma against o xidation induced b y added pro -oxidants (radical inducers) and monitored b y an exogenous oxidizable substrate (probe). The oxidation of the probe is theoretically inhibited by the antioxidants present in plasma during the induction period. The TRAP and ORAC assays are presently the most widel y used methods for measuring antio xidant capacity in biolo gical systems such as ser um and tissues. Dichlorof uorescein - diacetate,phycoerythrin (R - P e), and crocin-based assays also are included in this category of assays. Specif cally, plasma or serum, when challenged with a h ydrophilic radical inducer such as 2,2 ′ - azobis(2,4 - amidinopropane) dihydrochloride (AAPH), can be monitored b y a h ydrophilic o xidizable substrate such as 2′ ,7′ - dichlorodih ydrof uorescein (DCFH) (V alkonen and K uusi 1997), crocin (Kampa et al. 2002, Tubaro et al. 1998), or R-Pe (Cao and Prior 1999). Antioxidant capacity can be expressed in various ways such as lag phase, area under the cur ve, or competition kinetics. AAPH is a hydrophilic azo-compound that spontaneously decomposes at 37°C with a known rate constant ( Ri = 1.36 × 10−6 [AAPH] mol/liter/sec), gi ving rise to carbon -centered radicals that then react with oxygen, yielding the corresponding peroxyl radicals. DCFH, which can be oxidized to highl y f uorescent (Exc 480 nm, Em 526 nm) dichlorof uorescein by peroxyl radicals, is used as an o xidizable substrate in the TRAP assay (Valkonen and K uusi 1997). R -Pe is a protein isolated from Corallina off cinalis, and is used as the o xidizable substrate in the TRAP (Ghiselli et al. 1995) and ORA C (Cao and Prior 1999) assays. R -Pe is a f uorescent protein that emits in the visib le region (Exc 495 nm, Em 595 nm) and is characterized b y f uorescence quenching upon reaction with pero xyl radicals. Crocin, isolated from saf fron and characterized by a polyene chain with a high extinction coeff cient, has been used as an oxidizable substrate in the assa y developed by Tubaro (Tubaro et al. 1998) and then automated b y Kampa (Kampa et al. 2002) in the crocin b leaching assay. The reaction of crocin with pero xyl radical leads to a loss of the double bond conjugation and hence to bleaching that can be readily monitored at 445 nm. The second approach to measure antio xidant capacity in plasma using a h ydrophilic assay is to quench a stab le and pre -formed radical that does not act as a pro -oxidant. The trolo x equivalent antio xidant capacity (TEA C) assa y, w hich w as repor ted b y Miller et al. (1993), determines the antio xidant capacity of plasma b y measuring the ability of plasma to quench the radical cation of 2,2′ - azinobis(3 - ylbenzothiazoline eth - 6 - sulfonate) (ABTS). The quenching reaction is monitored by measuring the decay of the radical cation at 734 nm. The ferric reducing ability of plasma (FRAP) assa y has received a g reat deal of attention because of its quick and simple methodolo gy (Benzie and Strain 1996). The FRAP assa y measures the reduction of the fer ric ion to fer rous ion at lo w pH, w hich causes a colored fer rous-tripyridyltriazine complex to form. FRAP values can be obtained by comparing the absorbance change at 593nm in test reaction mixtures with those containing the fer rous ion in a kno wn concentration. LIPOPHILICANTIOXIDANT CAPACITY ASSAYS Two decades ago, Niki (1990) introduced AAPH and AMVN as the sources of w ater- and lipid-soluble pero xyl radicals respecti vely. As sho wn in the w ork of Massaeli et al. (1999), where preincubation of LDL with f at-soluble antio xidants increased the protecti ve ef fect

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against free radicals w hile preincubation with w ater-soluble antio xidants did not sho w an y effect, the importance of lipophilicity vs. hydrophilicity in antioxidants and free radical generating systems for deter mining antio xidant capacity has been reco gnized. It has also been demonstrated (Yeum et al. 2003) that the acti vities of antio xidants present in both the lipid and aqueous compar tments depend on the localization of the attacking radical species. In an effort to understand the biological signif cance of lipophilic antioxidants, several recent studies ha ve paid attention to the antio xidant capacity in the lipid compar tment of plasma. Mayer et al. (2001) proposed a continuous spectroscopic method using selecti ve f uorescence markers to monitor the aqueous and lipid phases in human ser um. In par ticular, diphen ylhexatriene-labeled proprionic acid w as used as an appropriate probe for the aqueous phase because it preferentially binds to albumin, w hile diphenylhexatriene-labeled phosphatidylcholine, which incorporates into lipoproteins, monitors the lipid compartment oxidizability. AAPH was selected as the radical inducer for both compar tments. By using this method, the authors reported that supplementation of human serum with quercetin, rutin, vitamins E and C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the o xidation mark er and the h ydrophobicity of the antio xidant. That is, f atsoluble antioxidants such as quercetin and vitamin E sho wed higher protective effects against lipoprotein oxidation, whereas water-soluble lutin and vitamin C more eff ciently protected the aqueous phase. An improved TEAC assay has been repor ted b y Re et al. (1999). By using a pre -formed radical mono -cation of ABTS and an appropriate solv ent system, the assa y is applicab le to both hydrophilic and lipophilic systems. The ORAC assay has also been e xpanded to ref ect lipophilic antioxidants by using randoml y methylated β-cyclodextrin (RMCD) as a solubility enhancer, AAPH as a radical initiator , and f uorescein as an oxidizable substrate (Huang et al. 2002). Recently, this updated ORA C assay was applied to human plasma (Prior et al. 2003) and the authors repor ted that lipophilic antio xidants represent less than 30% of the total antioxidant capacity of the protein -free plasma. For the lipophilic ORAC assay, lipophilic antioxidants w ere e xtracted b y he xane, dried , and resuspended in 7% RMCD solution (50% acetone/50% w ater, v/v). Ho wever, this assa y, w hich par titioned h ydrophilic and lipophilic antioxidants, may not be rele vant to a tr ue biological system in w hich active communication occurs among hydrophilic and lipophilic antio xidants. Aldini et al. (2001) repor ted a method that measures antio xidant capacity in both the hydrophilic and lipophilic compar tments of plasma and allo ws for interaction betw een the antioxidants in the two compartments. A lipophilic radical generator coupled with a selective f uorescent probe capab le of detecting lipid pero xidation w as used to measure the lipid compar tment. 2,2 ′ - azobis(4 - metho xy - 2,4 - dimeth ylvaleronitrile) (MeO - AMVN),which decomposes at 37 °C, w as selected as a lipid -soluble radical inducer , and 4,4 -dif uoro - 5 - (4 phenyl - 1,3 - butadien yl) - 4 - bora - 3a,4a - diaza s - indacene - 3 - undecanoic acid (BODIPY581/591) was used as a selective lipophilic oxidizable substrate (Drummen et al. 2002, Pap et al. 1999). The signif cantly higher rate constant of MeO -AMVN as compared to that of AMVN allows it to easily achieve lipid peroxidation in a biological system (Aldini et al. 2001). An oxidationsensitive f uorescent probe, BODIPY 581/591, w hich has a high quantum yield and readil y enters membranes (Dr ummen et al. 2002), provided the sensiti ve and selecti ve measurement of o xidation in the lipid compar tment of plasma. The selecti ve incor poration of BODIPY 581/591 into the indi vidual lipoprotein fractions, VLDL, LDL, and HDL, of human plasma has been further conf rmed (Yeum et al. 2003). A signif cant correlation (p < 0.0001) between plasma carotenoid concentration and antio xidant capacity deter mined by this assay was found in subjects w ho participated in a dietar y intervention trial with high fr uit and v egetable diets (Yeum et al. 2005). It is interesting to note that a high amount of single antio xidant ( > 15 mg of α - tocopherol) has been reported to be required to show a difference in antioxidant capacity, whereas less than a half serving of fruits and vegetables resulted in signif cant difference in antioxidant capacity

9

Antioxidant Activity and Oxidative Stress: An Overview

in a recently reported cross-sectional study (Talegawkar et al. 2009). This observation supports the impor tance of syner gistic action among the numerous antio xidants found in foods vs. a single antio xidant supplement. Another notab le impro vement of this assa y is that it requires a much lo wer dilution of plasma (5 to 10 × dilution) as compared to those of pre viously reported assays, which require 100 ×, 150 ×, and 250 × dilutions for the FRAP (Benzie and Strain 1996), ORAC (Cao et al. 1995), and TRAP (Ghiselli et al. 1995) assays, respectively. One of the dra wbacks of con ventional assa ys to measure antio xidant capacity has been the high dilution of plasma resulting in v ery low concentrations of antio xidants in the reaction mixtures. APPLICATION OF HYDROPHILIC AND LIPOPHILIC ANTIOXIDANT CAPACITY ASSAYS When hydrophilic assays are applied , the majority of the antio xidant capacity of plasma can be accounted for b y protein (10% to 28%), uric acid (7% to 60%), and ascorbic acid (2% to 27%), whereas the ef fect of vitamin E ( <10%) is minimal (Benzie and Strain 1996, Cao and Prior 1998, Tubaro et al. 1998, Valkonen and K uusi 1997, Wayner et al. 1987) as sho wn in Table 1.2 . As discussed previously, these assays mainly measure the antioxidant capacity of the aqueous compartment, because the radicals produced in the h ydrophilic compar tment and probes are also located in the hydrophilic compartment oxidized by aqueous peroxyl radicals. α - ocopherol T (vitamin E), w hich has its chroman head g roup oriented to ward the lipoprotein membrane, may par ticipate some what in the antio xidant action through interaction with w ater-soluble antioxidants such as ascorbic acid. Ho wever, it is clear that carotenoids, w hich are deepl y embedded in the lipid core, cannot participate in the antioxidant effect under these experimental Table 1.2. Estimated percent contribution of plasma antioxidants in various antioxidant capacity assays.

Estimatedpercent contribution Plasma antioxidant W ater - solub le antioxidant Protein Uricacid Ascorbicacid aFt - solub le antioxidant T ocopherols Carotenoids

Plasma normal range (µ mol)

ORA C3 1,2 TRAP

otal T

C3 PCA * TEA

5 Crocin

800 – 1,000 150 – 450 30 – 150

21 – 24 58 9 – 14

28 7 <2

0 39 7

28 19 3

10 60 15

21 51 27

20 – 50 0.5 – 3

7–9 —

<1 —

— —

<2 —

5 —

— —

* Ser um non - proteinfractions extracted with perchloric acid (PCA). Valkonen and Kuusi 1997 . 2 Wayner et al. 1987 . 3 Cao et al. 1995 . 4 Benzie and Strain 1996 . 5 Tubaro et al. 1998 . Modifed from Yeum et al. 2004. 1

4 FRAP

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conditions. The lack of contribution of f at-soluble antio xidants can also be ascribed to the relatively lower amount of f at-soluble antioxidants than w ater-soluble antioxidants in plasma, although it should be reco gnized that the antio xidant activity of f at-soluble antioxidants can be g reatly enhanced b y syner gistic interactions with w ater-soluble and other f at-soluble antioxidants. Thus, foods such as g reen tea (Benzie and Szeto 1999, Seraf ni et al. 1996), cocoa (Rein et al. 2000), red wine (Seraf ni et al. 1998, Tubaro et al. 1998), coffee (Natella et al. 2002), and stra wberries (Cao et al. 1998a) that contain considerab le amounts of w ater-soluble polyphenols signif cantly increase plasma antio xidant capacity as deter mined by hydrophilic antioxidant capacity assa ys. It is interesting to note that se veral studies ha ve pointed out that the increase in plasma antio xidant capacity obser ved after the consumption of f avonoid - rich foods such as wine w as due to a plasma uric acid increase not caused b y the f avonoids (Caccetta et al. 2000, Day and Stansbie 1995, Lotito and F rei 2006). On the other hand, diets rich in carotenoids (e.g. l ycopene or β - carotene)do not affect antioxidant capacity as measured b y the h ydrophilic TRAP, FRAP, or ORA C assays (Bohm and Bitsch 1999, Bub et al. 2000, Pellegrini et al. 2000). In spite of the consistent f ailure to show the modif cation of antio xidant capacity b y consumption of a high carotenoid diet (P ellegrini et al. 2000) or supplementation with carotenoid in humans (Li et al. 2009b), it is note worthy that there is considerab le and consistent e vidence from e xperiments in vitro for antio xidant actions of carotenoids (Miller et al. 1996, Palozza and Krinsk y 1992a), including their geometrical isomers (Bohm et al. 2002), tested in solv ent systems in vitro .

BIOLOGICALSIGNIFICANCE OF ANTIOXIDANT INTERACTIONS The actions of antio xidants in biolo gical systems such as plasma depend on (1) the nature of o xidants or R OS imposed on the biolo gical systems, (2) the acti vities and amounts of antioxidants, and (3) their cooperati ve/synergistic interactions. It is still not clear w hat kinds of ROS play a role in human patholo gies and w here the major sites of R OS action occurs. In an attempt to gain a better understanding of the biolo gical actions of antioxidants, the activity of single antioxidants or various combinations of antio xidants have been studied o ver the last decade (Burke et al. 2001, Mortensen and Skibsted 1997, Palozza and Krinsk y 1992b). Most of these studies were carried out in homogeneous solvent systems (i.e. either aqueous or lipid) (Niki et al. 1984) or artif cial membranes (liposomes, micelles) in buf fer solutions (Fukuzawa et al. 1997, Woodall et al. 1995), or by using isolated LDLs (Carroll et al. 2000), cells (Palozza et al. 2004), and tissue preparations (P alozza and Krinsk y 1992b). However, these types of model systems are far different from an actual biological system such as human serum/plasma, in that plasma is a heterogeneous entity consisting of hydrophilic and lipophilic compartments and contains high concentrations of other components such as protein ( ∼ 600µ mol/L). Interactions of dif ferent antioxidants in plasma ha ve also been studied e xtensively over the past decade. In par ticular, work has focused on both the interactions betw een hydrophilic and lipophilic antioxidants, such as ascorbic acid andα-tocopherol (Niki et al. 1995), or carotenoids and ascorbic acid (Burk e et al. 2001), and betw een lipophilic antio xidants (carotenoids and α-tocopherol) (Mortensen and Skibsted 1997, Palozza and Krinsk y 1992b). The combination of α - tocopheroland β-carotene has been repor ted to act cooperati vely as w ell to slo w down MDA formation initiated by the aqueous pero xyl radical, AAPH, in a li ver microsomal membrane preparation (Palozza and Krinsky 1992b). β-Carotene added to prefor med lipid bilayers produced much less of an antio xidant ef fect than β-carotene incor porated in the liposomes during bila yer for mation (Lieb ler et al. 1997). It is possib le that α - tocopherolreduces β•) as well as β - caroteneradical cations (β - •C+), as has carotene peroxyl radicals (LOO-β - C - OO been shown in a homogeneous solution (Mortensen and Skibsted 1997). In addition, β - carotene may recycle α - tocopherolfrom the α - tocophero xyl radical (α - O T•) through electron transfer

Antioxidant Activity and Oxidative Stress: An Overview

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(Bohm et al. 1997), although this possib le mechanism of action should be fur ther studied , because the reduction potential of β-carotene is reported to be lower than that of α - tocopherol (Buettner 1993, Edge et al. 2000). In addition, a synergistic antioxidant activity of lycopene in combination with vitamin E in a liposome system has been repor ted (Shi et al. 2004). It has been reported that β-carotene, which is located in the lipophilic core of the membrane bilayer, can directl y interact with w ater-soluble antioxidants. Because β - carotenecan be converted into β-carotene peroxyl radical cations by scavenging radical species in a heterogeneous micellar environment (Hill et al. 1995), the more polar β - caroteneradical cation (β - •C+ ) can be reoriented to ward the h ydrophilic compar tment, allo wing ascorbic acid to repair the βcarotene radical (El -Agamey et al. 2004). Other w ork (Burke et al. 2001) has also sho wn an interaction between β-carotene radical cations and ascorbic acid. Ascorbic acid can also spare uric acid, and uric acid not onl y scavenges radicals but can also stabilize ascorbic acid b y iron chelation (Sevanian et al. 1991). It has been repor ted that the major g reen tea pol yphenols, (-)-epigallocatechin-(3)-gallate (EGCG) located in the aqueous phase, also can rec ycle αtocopherol through an H -transfer mechanism (Aldini et al. 2003). Daily supplementation with moderate doses of combined antio xidants (100 mg vitamin C, 100 mgvitamin E, 6 mgβ - carotene,and 50 µg selenium) has been repor ted to increase plasma antioxidant capacity and decrease chromosome aber rations signif cantly in l ymphocytes (Volkovova et al. 2005). On the other hand , a meta -analysis of randomized trials with antio xidant supplements suggested that high doses of β-carotene (Vivekananthan et al. 2003) or αtocopherol (Miller et al. 2005) led to signif cant increases in mortality due to all causes and no effect against coronary heart disease risk (Eidelman et al. 2004, Knekt et al. 2004). It is likely that physiological doses of a combination of w ater-soluble and fat-soluble antioxidants, which can be successfull y obtained b y adequate fr uit and v egetable intake, are required to estab lish an effective antioxidant network in vivo . It should be also reco gnized that e ven though v arious combinations of “two” antioxidants in physiologic concentrations showed additive/synergistic interactions within and betw een the hydrophilic and lipophilic compartments in vitro (Niki 1987, Yeum et al. 2009), the much more complex in vivo system, in w hich many different antioxidants such as uric acid and protein already exist, is generall y maintained in homeostasis. Thus, the potenc y of the entire antio xidant network is not subject to swift modif cations through supplementation of a single antioxidant or their combinations w hen given in physiologic doses to health y people.

BIOLOGICALRELEVANCE OF ANTIOXIDANT SUPPLEMENTATION It has been believed that dietary supplementation with antioxidants can be a part of a protective strategy to minimize the o xidative damage in vulnerab le populations, such as the elderl y. It should be pointed out that the metabolism and functions of antio xidants in vivo and in vitro may not be the same. F or example, antioxidant nutrients can interact with each other during gastrointestinal absorption and metabolism (Kostic et al. 1995, Paetau et al. 1997, van den Berg and van Vliet 1998, White et al. 1994). Although epidemiological evidence continues to accumulate showing that diets high in fr uits and v egetables are associated with a reduced risk of chronic diseases such as cardio vascular disease (Gaziano et al. 1995, Hu 2003, Hung et al. 2004, Mayne 2003, Osganian et al. 2003), several attempts to alter overall antioxidant activity by supplementing antio xidant nutrients or implementing dietar y modif cation in health y subjects (Castenmiller et al. 1999, Jacob et al. 2003, Pellegrini et al. 2000) have not been successful. Considering that the biolo gical antioxidant network in health y subjects already contains adequate amounts of w ater- and f at-soluble antio xidants w orking in an interacti ve manner , further increases of single or small combinations of antio xidants within a ph ysiologic range might not af fect the o verall in vivo antio xidant netw ork (Czer nichow et al. 2009, Li et al. 2009b). It should also be appreciated that syner gistic interactions with respect to antio xidant

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activity as well as biological functions can occur not onl y among well recognized antioxidants (e.g. vitamin C, vitamin E) but also betw een f avonoids such as soy and green tea (Bertipaglia de Santana et al. 2008), and between micromineral and ph ytochemicals such as selenium and sulforaphane (Campbell et al. 2007). As reported by Valtuena et al. (2008), food selection based on “total antioxidant capacity ” values for foods can modify antio xidant intake without altering mark ers of oxidative stress or total antioxidant activity in plasma. Thus, antioxidant supplementation may alter other markers of biological function such as systemic inf ammation and li ver function without an y changes in various markers of antioxidant capacity or lipid pero xidation (Valtuena et al. 2008).

ANTIO XIDANTS AND GENE INTERACTIONS Considering the f act that the total antio xidant capacity of plasma is the susceptibility of the biological system against e xogenous free radicals, endo genous antioxidants and lipid prof les may be the primar y factors in deter mining the reduction potential of the plasma. In addition, enzymatic antioxidant defense systems as w ell as genetic v ariance should not be o verlooked (Figure 1.1 ). Although vitamin E supplementation has been recommended to pre vent cardio vascular diseases (Jialal and Devaraj 2000, Lonn et al. 2005, Vivekananthan et al. 2003), a meta-analysis conducted by Miller et al. (2005) indicated that a high dose of vitamin E (>400 IU) was associated with a higher incidence of all causes of mor tality. The study b y Milman et al. (2008) provides a clue for this discrepanc y: diabetic patients with hapto globin 2 –2 genotype, w ho were supplemented with 400 IU of vitamin E, showed a protective effect against cardiovascular events. It is probab le that high -dose antio xidant administration ma y benef t indi viduals w ho are under high o xidative stress because hapto globin 2 allele protein products are inferior antioxidants as compared to that of Haptoglobin 1 (Asleh et al. 2005, Bamm et al. 2004, MelamedFrank et al. 2001). Furthermore, a recent study by Cahill et al. (2009) indicated that glutathione S-transferease genotypes can inf uence the association betw een dietar y vitamin C and ser um ascorbic acid, which in tur n could affect the antioxidant capacity. Finally, it has been repor ted that dietar y antio xidant supplementation (b lueberry and apple juice mixture) impro ves the

Lipid profiles Water soluble antioxidant

ROS/RNS

Fat-soluble antioxidant

Total antioxidant capacity

Enzymes

Genetic variance

Figure 1.1. Postulated factors affecting total antioxidant capacity. ROS/RNS, reactive oxygen species/reactive nitrogen species.

Antioxidant Activity and Oxidative Stress: An Overview

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nucleotide excision repair capacity in individuals carrying multiple low-activity alleles, indicating that polymorphisms in a certain gene ( XPA G23A) can predict the value of dietary antioxidants for the nucleotide e xcision repair capacity (Langie et al. 2009). Therefore, the bioavailability of antio xidants, the b lood response to supplementation with antio xidants, and the oxidative stress associated genomic stability can all be af fected by the genetic v ariance of individuals.

SUMMAR Y Evidence has accumulated that high fr uit and vegetable intakes are associated with lo wer risk of chronic diseases such as cardio vascular diseases, and e ye diseases such as cataract. It is possible that antio xidants such as pol yphenols, vitamin C, and carotenoids in fr uits and v egetables can pre vent or reduce the damage from e xcessive amounts of free radicals that are produced in the body. However, intervention studies have failed to show a consistent benef cial effect of high doses of antio xidant supplementation against chronic diseases. One possib le explanation for these apparently contradictory results between observational studies and intervention trials is that the antio xidant system in vivo, which is f nely balanced, requires the right amount, possib ly an optimal range, of both h ydrophilic and lipophilic antio xidants to w ork properly. The optimal ranges of antioxidants might be achieved best by a balanced dietary fruit and vegetable intake, but not b y a high dose of onl y one or a limited mixture of antio xidant supplements, which could cause an imbalance of the antio xidant machiner y leading in some cases to a pro-oxidant effect. In addition, other phytochemicals abundant in fruits and vegetable may not only exert unique biological functions, but may also interact synergistically with well recognized antioxidants to promote antioxidant effects. Furthermore, genetic variances, which have been repor ted to affect the bioavailibility of antioxidants such as vitamin C (Cahill et al. 2009) and vitamin E (Milman et al. 2008) and the response to the dietar y antioxidant supplementation (Langie et al. 2009) may affect overall antioxidant capacity in humans. Various biomarkers to deter mine the antio xidant capacity in a biolo gical system ha ve been developed and advanced. However it seems that there is not yet one system that predicts health outcomes, due to the v arious factors affecting the antio xidant capacity in a biolo gical system such as interactions of antioxidants, genetic variance, and the origin of reactive oxygen species. Therefore, an impor tant future direction of research w ould be to elucidate ho w best to improve our body defense systems against o xidative damage, w hich in tur n might reduce the risk of chronic diseases, by means of dietar y modif cation rather than by taking large amounts of antioxidant supplements. The advances in developing proper markers to evaluate the overall antioxidant network including both w ater- and f at-soluble antioxidants and their interactions in a biolo gical system w ould suppor t such ef forts. F inally, continuous ef fort to understand gene-nutrient interactions ma y provide a clue to inconsistent results of v arious inter vention studies with antioxidants to prevent or delay the process of aging or cer tain chronic diseases.

A CKNOWLEDGEMENT This work has been supported in part by the BioGreen 21 Program (Code #20070301034009), Rural De velopment Administration, K orea and the U .S. Depar tment of Agriculture, under Agreement 1950-51000-065-08S. The contents of this publication do not necessarily ref ect the views or policies of the U .S. Depar tment of Agriculture, nor does mention of trade names, commercial products, or or ganizations imply endorsement by the U.S. Government.

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CaoG , Russell RM , Lischner N , Prior RL .1998a . Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderl y women. J Nutr 128 : 2383 – 2390 . CaoG , Booth SL , Sado wski JA , Prior RL .1998b. Increases in human plasma antioxidant capacity after consumption of controlled diets high in fr uit and vegetables. Am J Clin Nutr 68 : 1081 – 1087 . CaoG ,V erdon CP , W u AH ,W ang H , Prior RL .1995 .Automated assay of oxygen radical absorbance capacity with the COB AS FARA II . Clin Chem 41 : 1738 – 1744 . Car roll YL , Corridan BM , Morrissey PA . 2000 . Lipoprotein carotenoid prof les and the susceptibility of low density lipoprotein to o xidative modif cation in healthy elderly volunteers. Eur J Clin Nutr 54 : 500 – 507 . CastenmillerJJ , Lauridsen ST , Dragsted LO , avn het Hof KH , Linssen JP ,et al. 1999 .β - carotene does not change mark ers of enzymatic and nonenzymatic antio xidant activity in human b lood. J Nutr 129 : 2162 – 2169 . CeconiC , BorasoA , Car gnoni A , F errari R .2003 . Oxidative stress in cardiovascular disease: myth or f act? Arch Biochem Biophys 420 : 217 – 221 . Ceriello A , Motz E .2004 . Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardio vascular disease? The common soil h ypothesis revisited. Arterioscler Thromb Vasc Biol 24 : 816 – 823 . ChristenWG , Liu S , Gl ynn RJ , Gaziano JM , Buring JE .2008 . Dietary carotenoids, vitamins C and E, and risk of cataract in w omen: a prospective study. Arch Ophthalmol 126 : 102 – 109 . Czer nichow S ,V ergnaud AC , Galan P , Arnaud J , aFvier A et , al. 2009 . Effects of long - term antioxidant supplementation and association of ser um antioxidant concentrations with risk of metabolic syndrome in adults . Am J Clin Nutr 90 : 329 – 335 . DauchetL ,Amouy el P , Hercber g S , Dallonge ville J . 2006 . Fruit and vegetable consumption and risk of coronar y heart disease: a meta -analysis of cohor t studies . J Nutr 136 : 2588 – 2593 . Da y A , Stansbie D . 1995 . Cardioprotective effect of red wine may be mediated by urate . Clin Chem 41 : 1319 – 1320 . DheraniM , Murthy GV , Gupta SK ,Y oung IS , Maraini G et , al. 2008 . Blood levels of vitamin C, carotenoids and retinol are in versely associated with cataract in a Nor th Indian population . Invest Ophthalmol Vis Sci 49 : 3328 – 3335 . Dr ummen GP , avn Liebergen LC , Opden Kamp JA , P ost JA . 2002 . C11 - BODIPY(581/591),an oxidation - sensiti ve f uorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology. Free Radic Biol Med 33 : 473 – 490 . EdgeR , Land EJ , McGarvey DJ , Burk e M ,T ruscott TG .2000 .The reduction potential of the •+ /β-carotene couple in an aqueous micro -heterogeneous environment. FEBS Lett β - Carotene 471 : 125 – 127 . EidelmanRS , Hollar D , Hebert PR , Lamas GA , Hennek ens CH .2004 . Randomized trials of vitamin E in the treatment and pre vention of cardiovascular disease . Arch Intern Med 164 : 1552 – 1556 . El - Agame y A , CantrellA , Land EJ , McGarvey DJ , T ruscott TG .2004 .Are dietary carotenoids benef cial? Reactions of carotenoids with o xy-radicals and singlet o xygen. Photochem Photobiol Sci 3 : 802 – 811 . earon F IM , aFux SP . 2009 . Oxidative stress and cardiovascular disease: novel tools give (free) radical insight . J Mol Cell Car diol 47 : 372 – 381 . ord F ES ,Ajani UA , MokdadAH .2005 . Brief communication: The prevalence of high intake of vitamin E from the use of supplements among U .S. adults . Ann Intern Med 143 : 116 – 120 . Fukuza wa K , Matsuura K ,T okumura A , SuzukiA ,T erao J . 1997 . Kinetics and dynamics of singlet oxygen scavenging by α-tocopherol in phospholipid model membranes . Free Radic Biol Med 22 : 923 – 930 . GateL , P aul J , Ba GN , T ew KD , T apiero H .1999 . Oxidative stress induced in pathologies: the role of antioxidants. Biomed Pharmacother 53 : 169 – 180 .

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GazianoJM , Manson JE , Branch LG , Colditz GA ,W illett WC et , al. 1995 .A prospective study of consumption of carotenoids in fr uits and vegetables and decreased cardio vascular mortality in the elderly. Ann Epidemiol 5 : 255 – 260 . Ghiselli A , Serafni M , Maiani G ,Azzini E , F erro - LuzziA .1995 .A f uorescence - basedmethod for measuring total plasma antio xidant capability. Free Radic Biol Med 18 : 29 – 36 . Hensle y K , Flo yd RA .2002 . Reactive oxygen species and protein oxidation in aging: a look back, a look ahead . Arch Biochem Biophys 397 : 377 – 383 . HillTJ , Land EJ , McGarvey DJ , SchalchW , T inkler JH et , al. 1995 . Interactions between carotenoids and CCl 3 O2• radical . J Am Chem Soc 117 : 8322 – 8326 . HuFB . 2003 . Plant - basedfoods and prevention of cardiovascular disease: an overview . Am J Clin Nutr 78 : 544S - 551S . HuangD , Ou B , Hampsch - oodill W M , Flanagan JA , Deemer EK .2002 . Development and validation of oxygen radical absorbance capacity assa y for lipophilic antio xidants using randomly methylated beta -cyclodextrin as the solubility enhancer . J Agric Food Chem 50 : 1815 – 1821 . HungHC , Joshipura KJ , Jiang R , Hu FB , Hunter D ,et al. 2004 . Fruit and vegetable intake and risk of major chronic disease . J Natl Cancer Inst 96 : 1577 – 1584 . JacobRA ,Aiello GM , Stephensen CB , Blumber g JB , Milbury PE et , al. 2003 . Moderate antioxidant supplementation has no ef fect on biomarkers of oxidant damage in health y men with low fruit and vegetable intakes. J Nutr 133 : 740 – 743 . JialalI , De varaj S .2000 .Vitamin E supplementation and cardiovascular events in high - risk patients . N Engl J Med 342 : 1917 – 1918 . KampaM , NistikakiA ,TsaousisV , Maliaraki N , Notas G et , al. 2002 .A new automated method for the deter mination of the Total Antioxidant Capacity (TAC) of human plasma, based on the crocin bleaching assay. BMC Clin Pathol 2 : 3 . KnektP , Ritz J , P ereira MA , O ’ Reill y EJ , Augustsson K et , al. 2004 .Antioxidant vitamins and coronary heart disease risk: a pooled anal ysis of 9 cohor ts. Am J Clin Nutr 80 : 1508 – 1520 . ostic K D , WhiteWS , Olson JA . 1995 . Intestinal absorption, serum clearance, and interactions between lutein and β-carotene when administered to human adults in separate or combined oral doses . Am J Clin Nutr 62 : 604 – 610 . Kre gel KC , Zhang HJ . 2007 .An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations . Am J Physiol Regul Integr Comp Physiol 292 : R18 – 36 . LangieSA ,W ilms LC , Hamalainen S , Kleinjans JC , Godschalk RW ,et al. 2009 . Modulation of nucleotide excision repair in human l ymphocytes by genetic and dietar y factors. Br J Nutr 1 – 12 . LeeIM , Cook NR , Gaziano JM , Gordon D , Ridk er PM et , al. 2005 .Vitamin E in the primary prevention of cardiovascular disease and cancer: the Women’s Health Study: a randomized controlled trial . JAMA 294 : 56 – 65 . LiL , Duk er JS ,Y oshida Y , Niki E , Rasmussen H et , al. 2009a . Oxidative stress and antioxidant status in older adults with earl y cataract . Eye (Lond) 23 : 1464 – 1468 . LiL , Chen CY , Aldini G , Johnson EJ , Rasmussen H et , al. 2009b. Supplementation with lutein or lutein plus g reen tea extracts does not change o xidative stress in adequatel y nourished older adults . J Nutr Biochem . Lieb ler DC , Stratton SP , Ka ysen KL .1997 .Antioxidant actions of β-carotene in liposomal and microsomal membranes: role of carotenoid -membrane incorporation and α - tocopherol Arch . Biochem Biophys 338 : 244 – 250 . LiuS , Lee IM ,Ajani U , Cole SR , Buring JE et , al. 2001 . Intake of vegetables rich in carotenoids and risk of coronar y heart disease in men: The Physicians’ Health Study. Int J Epidemiol 30 : 130 – 135 . LiuS , Manson JE , Lee IM , Cole SR , Hennek ens CH et , al. 2000 . Fruit and vegetable intake and risk of cardiovascular disease: the Women’s Health Study. Am J Clin Nutr 72 : 922 – 928 .

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LonnE , Bosch J , Y usuf S , Sheridan P , P ogue J ,et al. 2005 . Effects of long - term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial . Jama 293 : 1338 – 1347 . LotitoSB , F rei B . 2006 . Consumption of f avonoid-rich foods and increased plasma antio xidant capacity in humans: cause, consequence, or epiphenomenon? Free Radic Biol Med 41 : 1727 – 1746 . LussignoliS , F raccaroli M ,Andrioli G , Brocco G , Bella vite P . 1999 .A microplate - based colorimetric assay of the total pero xyl radical trapping capability of human plasma . Anal Biochem 269 : 38 – 44 . MagalhaesLM , Se gundo MA , Reis S , Lima JL .2008 . Methodological aspects about in vitro evaluation of antioxidant properties. Anal Chim Acta 613 : 1 – 19 . MassaeliH , Sobrattee S , Pierce GN . 1999 .The importance of lipid solubility in antioxidants and free radical generating systems for deter mining lipoprotein proxidation. Free Radic Biol Med 26 : 1524 – 1530 . Ma yer B , Schumacher M , Brandstatter H ,W agner FS , Hermetter A .2001 . High - throughput f uorescence screening of antio xidative capacity in human ser um. Anal Biochem 297 : 144 – 153 . Mayne ST. 2003. Antioxidant nutrients and chronic disease: use of biomark ers of exposure and oxidative stress status in epidemiolo gic research . J Nutr 133 : 933S – 940S . Melamedrank - F M , Lache O , Ena v BI , SzafranekT , Le vy NS et , al. 2001 . Structure - function analysis of the antio xidant properties of haptoglobin. Blood 98 : 3693 – 3698 . MillerER , 3rd , P astor - Barriuso R , Dalal D , Riemersma RA ,Appel LJ ,et al. 2005 . Meta analysis: high-dosage vitamin E supplementation ma y increase all -cause mortality. Ann Intern Med 142 : 37 – 46 . MillerNJ , Rice - Ev ans C , Da vies MJ , GopinathanV , MilnerA .1993 .A novel method for measuring antioxidant capacity and its application to monitoring the antio xidant status in premature neonates . Clin Sci (Colch) 84 : 407 – 412 . MillerNJ , Sampson J , Candeias LP , Bramle y PM , Rice - Ev ans CA .1996 .Antioxidant activities of carotenes and xanthophylls. FEBS Lett 384 : 240 – 242 . MilmanU , Blum S , Shapira C ,Aronson D , Miller- Lotan R et , al. 2008 .Vitamin E supplementation reduces cardiovascular events in a subg roup of middle -aged individuals with both type 2 diabetes mellitus and the hapto globin 2 –2 genotype: a prospecti ve double-blinded clinical trial . Arterioscler Thromb Vasc Biol 28 : 341 – 347 . Mor tensen A , Skibsted LH .1997 . Relative stability of carotenoid radical cations and homologue tocopheroxyl radicals. A real time kinetic study of antio xidant hierarchy. FEBS Lett 417 : 261 – 266 . NatellaF , Nardini M , Giannetti I , Dattilo C , Scaccini C .2002 . Coffee drinking inf uences plasma antioxidant capacity in humans . J Agric Food Chem 50 : 6211 – 6216 . NikiE .1987 . Interaction of ascorbate and α - tocopherol Ann . N Y Acad Sci 498 : 186 – 199 . NikiE .1990 . Free radical initiators as source of water - or lipid - solub le peroxyl radicals . Methods Enzymol 186 : 100 – 108 . NikiE , SaitoT , Ka wakami A , KamiyaY . 1984 . Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C . J Biol Chem 259 : 4177 – 4182 . NikiE , No guchi N , Tsuchihashi H , Gotoh N . 1995 . Interaction among vitamin C, vitamin E, and beta - carotene Am . J Clin Nutr 62 : 1322S - 1326S . OmennGS , Goodman GE ,Thornquist MD , Balmes J , Cullen MR et , al. 1996 . Effects of a combination of beta carotene and vitamin A on lung cancer and cardio vascular disease . N Engl J Med 334 : 1150 – 1155 . OsganianSK , Stampfer MJ , Rimm E , Spie gelman D , Manson JE et , al. 2003 . Dietary carotenoids and risk of coronar y artery disease in w omen. Am J Clin Nutr 77 : 1390 – 1399 . aetau P I , Chen H , Goh NM ,WhiteWS .1997 . Interactions in the postprandial appearance of β-carotene and canthaxanthin in plasma triac ylglycerol-rich lipoproteins in humans . Am J Clin Nutr 66 : 1133 – 1143 .

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alozza P P , Krinsk y NI .1992a .Antioxidant effects of carotenoids in vivo and in vitro : an overview . Methods Enzymol 213 : 403 – 420 . alozza P P , Krinsk y NI .1992b. β - Caroteneand α-tocopherol are synergistic antioxidants. Arch Biochem Biophys 297 : 184 – 187 . alozza P P , Serini S , DiNicuolo F , Boninse gna A ,T orsello A et , al. 2004 .β - Carotene exacerbates DNA oxidative damage and modif es p53 -related pathways of cell proliferation and apoptosis in cultured cells e xposed to tobacco smok e condensate . Carcinogenesis 25 : 1315 – 1325 . apP EH , Drummen GP , W inter VJ , K ooij TW , Rijk en P ,et al. 1999 . Ratio -uorescence f microscopy of lipid o xidation in living cells using C11 -BODIPY(581/591). FEBS Lett 453 : 278 – 282 . ellegrini P N , Riso P , P orrini M .2000 .Tomato consumption does not affect the total antioxidant capacity of plasma . Nutrition 16 : 268 – 271 . PriorRL , Hoang H , Gu L ,W u X , Bacchiocca M et , al. 2003 .Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORA CFL)) of plasma and other biological and food samples . J Agric Food Chem 51 : 3273 – 3279 . ReR , P ellegrini N , Prote ggente A , P annala A ,Y ang M et , al. 1999 .Antioxidant activity applying an improved ABTS radical cation decolorization assa y. Free Radic Biol Med 26 : 1231 – 1237 . ReinD , P aglieroni TG , P earson DA , W un T , Schmitz HH et , al. 2000 . Cocoa and wine polyphenols modulate platelet acti vation and function . J Nutr 130 : 2120S – 2126S . Rice -ans Ev CA , Sampson J , Bramle y PM , Hollo way DE .1997 .Why do we expect carotenoids to be antioxidants in vivo ? Free Radic Res 26 : 381 – 398 . Seraf ni M , GhiselliA , F erro - LuzziA .1996 .In vivo antioxidant effect of g reen and black tea in man . Eur J Clin Nutr 50 : 28 – 32 . Seraf ni M , Maiani G , F erro - LuzziA .1998 .Alcohol - freered wine enhances plasma antioxidant capacity in humans . J Nutr 128 : 1003 – 1007 . vanian Se A , Da vies KJ , Hochstein P . 1991 . Serum urate as an antioxidant for ascorbic acid . Am J Clin Nutr 54 : 1129S – 1134S . ShiJ , KakudaY , Y eung D . 2004 .Antioxidative properties of lycopene and other carotenoids from tomatoes: synergistic effects. Biofactors 21 : 203 – 210 . alegawkar T SA , Beretta G ,Y eum K - J, Johnson EJ , CarithersTC et , al. 2009 .Total antioxidant performance is associated with diet and ser um antioxidants in par ticipants of the diet and physical activity substudy of the Jackson Hear t Study. J Nutr 139 : 1964 – 1971 . ThannickalVJ , aFnburg BL .2000 . Reactive oxygen species in cell signaling . Am J Physiol Lung Cell Mol Physiol 279 : L1005 – 1028 . ubaro T F , GhiselliA , Rapuzzi P , Maiorino M , Ursini F . 1998 .Analysis of plasma antioxidant capacity by competition kinetics . Free Radic Biol Med 24 : 1228 – 1234 . alkonen V M ,K uusi T . 1997 . Spectrophotometric assay for total peroxyl radical - trapping antioxidant potential in human ser um. J Lipid Res 38 : 823 – 833 . altuena V S ,P ellegrini N , F ranzini L , Bianchi MA ,Ardigo D ,et al. 2008 . Food selection based on total antioxidant capacity can modify antio xidant intake, systemic inf ammation, and liver function without altering mark ers of oxidative stress . Am J Clin Nutr 87 : 1290 – 1297 . anv den Berg H ,avn Vliet T . 1998 . Effect of simultaneous, single oral doses of β - carotenewith lutein or lycopene on the β-carotene and retinyl ester responses in the triac ylglycerol-rich lipoprotein fraction of men . Am J Clin Nutr 68 : 82 – 89 . ivekananthan V DP , P enn MS , Sapp SK , HsuA ,T opol EJ . 2003 . Use of antioxidant vitamins for the prevention of cardiovascular disease: meta -analysis of randomised trials . Lancet 361 : 2017 – 2023 . olkovova V K , Barancok ova M , Kazimiro va A , CollinsA , Raslo va K et , al. 2005 .Antioxidant supplementation reduces inter -individual variation in markers of oxidative damage . Free Radic Res 39 : 659 – 666 .

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ayner W DD , Burton GW , Ingold KU , Barcla y LR , Lock e SJ . 1987 .The relative contributions of vitamin E, urate, ascorbate and proteins to the total pero xyl radical -trapping antioxidant activity of human blood plasma . Biochim Biophys Acta 924 : 408 – 419 . WhiteWS , Stace wicz - Sapuntzakis M , Erdman JW, Jr. , Bo wen PE .1994 . Pharmacokinetics of beta-carotene and canthaxanthin after ingestion of indi vidual and combined doses b y human subjects. J Am Coll Nutr 13 : 665 – 671 . oodall W AA , Britton G , Jackson MJ . 1995 .Antioxidant activity of carotenoids in phosphatidylcholine vesicles: chemical and str uctural considerations . Biochem Soc Trans 23 : 133S . eum Y K - J, Beretta G , Krinsk y NI , Russell RM ,Aldini G .2009a . Synergistic interactions of antioxidant nutrients in a biolo gical model system . Nutrition 25 : 839 – 846 . eum Y K - J, Aldini G , Russell RM , Krinsk y NI .2009b.Antioxidant/prooxidant actions of carotenois. In Carotenoids Vol 5: Nutrition and Health. Britton G, Liaaen -Jensen, Pfander H, eds. Basel, Switzerland: Birkh ä userVerlag. eum Y K - J, Aldini G , Johnson EJ , Russell RM , Krinsk y NI .2005 .Effect of feeding and then depleting a high fr uit and vegetable diet on o xidizability in human ser um. Champaign, IL: AOCS Press. eum Y K - J, Russell RM , Krinsk y NI ,Aldini G .2004 . Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compar tments of human plasma . Arch Biochem Biophys 430 : 97 – 103 . eum Y K - J, Aldini G , Chung H - Y, Krinsk y NI , Russell RM .2003 .The activities of antioxidant nutrients in human plasma depend on the localization of attacking radical species . J Nutr 133 : 2688 – 2691 .

Chapter2 Enzymatic Antioxidant Defenses Sayuri Miyamoto , Hir ofumi Ar ai and , unji J eTrao

INTR ODUCTION Oxidative stress causes the cellular and e xtracellular redox state to be shifted to the o xidative side by the disruption of balance between the generation of reactive oxygen species (ROS) and their elimination. Antioxidative enzymes play a crucial role in maintaining a homeostatic redox state and their def ciency or overexpression may cause irreversible damage to the tissues of the body. ROS include the superoxide anion radical (O 2•−), hydroxyl radical(•OH), hydrogen peroxide (H2 O2), and singlet molecular o xygen ( 1 O2). The superoxide anion radical can be ef fectively reduced through a reaction with superoxide dismutase (SOD). The resulting hydrogen peroxide is then con verted to nonto xic water by catalase or glutathione pero xidase (GPx). In humans, the combination of SOD and GPx has long been kno wn to be a po werful tool for the deto xif cation of superoxide anions generated within the cell. Any hydrogen peroxide which escapes this detoxif cation system may yield hydroxyl radicals by a Fenton-type reaction. It should be noted that the h ydroxyl radical is the most highl y reactive ROS, and cannot be eliminated b y antioxidants or antioxidant enzymes. Also, a singlet molecular oxygen-scavenging enzyme has not been detected in an y biological system. Although ROS can oxidize biological components such as nucleic acids, proteins, and lipids, the human body also possesses repair enzymes to eff ciently eliminate o xidized molecules. These antio xidative enzymes ma y be e xpressed at higher levels under conditions of o xidative stress. Attack of ROS on the lipids in biomembranes and plasma proteins is an important issue both from the viewpoint of biomarkers for oxidative stress as well as for antioxidant defense. Lipid peroxidation products ha ve long been used as sensiti ve and simple biomark ers for measuring oxidative stress in biolo gical systems. Thiobarbituric acid reacti ve substance (TB ARS) is a well known classical biomark er that is widel y used for in vitro and in vivo studies. In recent years, isoprostanes as w ell as shor t chain carbonyl compounds are frequentl y used for in vivo assays of o xidative stress in human b lood and urine. These products are deri ved from the primary products of lipid pero xidation, that is, lipid h ydroperoxides or lipid h ydroperoxyendoperoxides which are for med by the reaction of R OS with the pol yunsaturated f atty acid moiety of esterif ed lipids. Thus, lipid hydroperoxide-eliminating enzymes, including GPx and phospholipase A2, should be tak en into account w hen quantifying lipid pero xidation products as biomarkers of oxidative stress. This chapter focuses on the sequential processes of elimination of ROS and lipid h ydroperoxide by antioxidative enzymes. Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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SUPER OXIDE DISMUTASE ( SOD )AS AN ROS SCAVENGING SYSTEM SOD is an enzyme (EC 1.15.1.1) discovered by McCord and Fridovich, which plays an important role in the defense mechanism of biolo gical cells e xposed to o xygen (McCord and Fridovich 1969). SOD catal yzes the dismutation of supero xide anion radical (O 2•−) into an oxygen molecule and a hydrogen peroxide. This reaction is recognized as an antioxidant system that protects cells from supero xide toxicity. There are several types of SOD, depending on the type of metal ion. Three major isoforms of mammalian SOD have been identif ed with different tissue distributions (Zelko 2002 ). Cu/Zn-SOD (SOD1) exists in the cytoplasm, lysosomes, and nuclear compartments of mammalian cells (Crapo et al. 1992, Keller et al. 1991, Liou 1993). In humans, the liver has a relatively high amount and acti vity of SOD1 (Halliw ell and Gutteridge 2007). Human SOD1 is a homodimer containing one copper ion and one zinc ion in each 16 -kDa subunit which consists of 153 amino acids. The copper ion is held b y interaction with imidazolate ligands of the histidine residues in SOD1 in the enzymatic acti ve site. The zinc ion (Zn 2+) contributes to the stabilization of the enzyme. The following reaction is catal yzed by SOD1: Cu 2 + Zn-SOD + O i2− → Cu + Zn-SOD + O 2 Cu + Zn-SOD + Oi2− + 2H + → Cu 2 + Zn-SOD + H 2 O 2 + 2Oi− 2 + 2H → O 2 + H 2 O 2

The activity of SOD1 is nearly independent of pH in the range of 5.0 to 9.5, and the reaction rates are almost diffusion-limited (∼ 109 M−1 s−1) at physiological pH. It has been suggested that a variety of single amino acid mutations in SOD1 are link ed to f amilial amyotrophic lateral sclerosis (Liochev and Fridovich 2007, Valentine et al. 2005). Mn-SOD (SOD2) from humans is a homotetramer with an indi vidual 22 -kDa subunit that has one ion of Mn at the active center (Weisiger and Fridovich 1973). SOD2 is generally located in the mitochondrial matrix and catal yzes the same reaction as SOD1. In humans, the acti vity of SOD2 in the renal cor tex is higher than in other tissues (Halliw ell and Gutteridge 2007). Extracellular (EC) -SOD (SOD3) is found in e xtracellular f uids such as human plasma and lymph, and e xists as a tetramer of identical 30 -kDa subunits in most species (Nozik -Grayck et al. 2005). SOD3 is also a copper and zinc containing enzyme that acts as a supero xide scavenger to protect tissues from e xtracellular o xidative damage, although SOD3 is distinct from SOD1 described abo ve. SOD3 contains a high aff nity domain for heparin, w hich contributes to its localization in the e xtracellular matrix (Fattman et al. 2003). The polymorphism at the heparin-binding domain results in an increase in the plasma concentration of SOD3, and this has been sho wn to increase the risk of ischemic hear t disease (Juul et al. 2004). Many studies suggest that o xidative stress is in volved in the patho genesis of cardiovascular diseases, including heart attacks, cerebral ischemia, and atherosclerosis, which is characterized by a local thick ening of the v essel wall of the ar teries. Oxidation of lo w density lipoprotein (LDL) and uptak e of o xidized LDL b y macrophages is also belie ved to be associated with atherosclerosis (Hansson et al. 2006). It has been demonstrated that SOD3 acti vity and its protein expression are increased in macrophage -rich atherosclerotic lesions of apolipoprotein E – defcient mice (Fukai et al. 1998). On the other hand , the enzymatic acti vity of SOD3 w as decreased in atherosclerotic lesions of human aor ta as compared with nor mal aorta segments of the same indi vidual (Luoma et al. 1998). In patients with coronar y artery disease, vascular SOD3 activity was reduced w hile activities of SOD1 and SOD2 w ere similar in the coronar y arteries of the patients and in the control subjects (Landmesser et al. 2000). Infammation is a complex biological response to harmful stimuli such as infection of pathogens in the vascular system, and can induce various diseases including atherosclerosis.Activated leukocytes release mediators including c ytokines, eicosanoids, and nitric o xide (NO). Excess

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NO can react with superoxide to form peroxynitrite anion, a powerful ROS. It has been reported that o verproduction of tumor necrosis f actor (TNF) -α, w hich is mainl y released b y macrophages, is associated with a decrease in SOD1 e xpression, whereas TNF-α ,interleukin (IL) - 1, IL-4, and IL -6 activate SOD2 (Afonso et al. 2006, Kiningham et al. 2001). Exposure to combinations of interferon -γ and TNF -α has been sho wn to increase SOD3 secretion in der mal f broblasts and SOD3 e xpression along with inducib le NO synthase in rat type II pneumoc yte (Brady et al. 1997 ,Marklund 1992 ). Diabetes mellitus (DM) is a metabolic disorder mark ed b y h yperglycemia due to insuff ciency of secretion or action of insulin. Uncontrolled DM can lead to man y complications in various tissues in the vascular system (accelerated atherosclerosis). It has been determined that DM increases o xidative stress, w hich results in lipid pero xidation and o xidative degradation of glycated protein (Stephens et al. 2009). It has also been demonstrated that human SOD1 can be inactivated by glycation and fragmentation in vitro (Ookawara et al. 1992). However, the effect of DM on SOD acti vity in vivo is controversial, and depends on tissues, gender , and the species of the biolo gical objects (Maritim et al. 2003).

GLUT ATHIONE PEROXIDASE ( GP xAS ) AN ROS SCAVENGING SYSTEM GPx (EC 1.11.1.9) is a gl ycoprotein containing a single selenoc ysteine residue at the acti ve center of each subunit. To protect biological organisms from oxidative damage, GPx catalyses the reduction of hydrogen peroxide and lipid hydroperoxides to water and their cor responding alcohols, respectively, as follows (Mat és et al. 1999): ROOH + 2GSH → ROH + GSSG + H2 O2 where reduced monomeric glutathione (GSH) is essential as a h ydrogen donor, and GSH is oxidized to glutathione disulf de (GSSG). There are f ve main mammalian isozymes, w hich vary in the str ucture (amino acid sequence and subunit), tissue distribution (li ver, kidne y, erythrocyte, blood plasma, among others), location (c ytoplasm, intestine, e xtracellular f uid), and substrate specif city (hydrogen peroxide and lipid hydroperoxides) (Halliwell and Gutteridge 2007 ). It has been suggested that GPX has anti -inf ammatory activity in the cardiovascular system (Chu et al. 1993). An increase in c ytosolic GPx is link ed to a lo wer risk of cardio vascular disease (Blankenberg et al. 2003). Under conditions of DM, GPx acti vity can be increased in blood, erythrocyte, aorta, liver, kidney, and pancreas, w hile it is decreased in retina and hear t (Maritim et al. 2003). The mechanisms behind the ef fects of DM on GPx acti vities are still unclear.

GLUT ATHIONE PEROXIDASES ( GP xAND ) PHOSPHOLIPASES AS A LIPID HYDROPEROXIDE-ELIMINATION SYSTEM Lipid hydroperoxides (LOOH) can be for med in food and in biolo gical systems by enzymatic and non-enzymatic mechanisms. Several classes of hydroperoxides including fatty acid hydroperoxides, phospholipid h ydroperoxides, cholesterol h ydroperoxides, and cholester yl ester hydroperoxides have been detected and characterized both in vitro and in vivo . These hydroperoxides ma y accumulate under se veral patholo gical conditions and cells ha ve de veloped mechanisms to eliminate and repair these o xidized lipids. In order to counteract the accumulation of LOOH, cells are normally endowed with enzymes that catal yze the reduction of h ydroperoxides to the cor responding alcohols. The reducti ve detoxif cation of free fatty acid hydroperoxides is mediated by at least three classes of enzymes: glutathione pero xidases (GPx) (Brigelius -Flohé 1999, Ursini et al. 1995), glutathione

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S-transferases (GST) (Yang et al. 2001), and peroxiredoxins (Prx) (Rhee et al. 2005). In addition to these enzymes the elimination/repair of h ydroperoxides formed in membranes can also be mediated b y the action of phospholipase A2. In this section w e describe the impor tance of glutathione peroxidases and phospholipases in the elimination of LOOH. Glutathione peroxidases are antioxidant enzymes that contain a selenocysteine residue in the active site (Floh é et al. 1973). This amino acid residue par ticipates in the catal ytic c ycle whereby the hydroperoxide is reduced to alcohol and GSH is o xidized to GSSG. The activity of GPx depends on the constant suppl y of GSH w hich is regenerated from GSSG in the presence of glutathione reductase and NADPH. To date, f ve isoforms of GPx have been identif ed: the classical or c ytosolic GPx (cGPx or GPx1) w as the f rst and most abundant selenoprotein to be identif ed; the gastrointestinal GPx (GIGPx or GPx2); the plasma GPx (pGPx or GPx3); the phospholipid GPx (PHGPx or GPx4); and the sperm nuclei GPx (snGPX) (Brigelius-Flohé 1999, K ühn and Borcher t 2002). These enzymes dif fer in their substrate specif city, size, amino acid sequence, localization, and e xpression level in dif ferent tissues and/or cell types (Table 2.1 ). Elimination and repair of o xidatively modif ed phospholipids in membranes is criticall y important for the nor mal function of the cells. The sn-2 position of the gl ycerol backbone of phospholipids is mostl y comprised of unsaturated f atty acids, w hich are readil y oxidized by reactive oxygen species yielding f atty acid hydroperoxides. Unlike free fatty acid hydroperoxides that can be eliminated b y all isoforms of the GPx f amily (Brigelius -Flohé 1999, Ursini et al. 1995), as w ell as GST (Zhao et al. 1999) and Prx (Rhee et al. 2005), the elimination of esterif ed fatty acid hydroperoxides in situ is restricted to some specif c enzymes. Reductive detoxif cation of phospholipid hydroperoxides (PLOOH) can be accomplished by direct and/or indirect mechanisms (F igure 2.1). In the direct mechanism, the f atty acid hydroperoxide in the phospholipid is reduced in situ by the action of phospholipid glutathione per oxidase (PHGPx or GPx4) (Thomas et al. 1990, Ursini et al. 1985) or by the action of an α - type GST (Hurst et al. 1998, Yang et al. 2001). Alternatively, in the indirect mechanism, the PLOOH is f rst h ydrolyzed b y phospholipase A2 and the released f atty acid h ydroperoxide is then reduced by GPx (Vankuijk et al. 1987) Recently, it has been demonstrated that PLOOH can also be reduced in situ by 1 - CysPrx, an enzyme w hich contains both phospholipase A2 and GPx acti vity (Fisher et al. 1999). The relative impor tance of these enzymes in the elimination of PLOOH v aries according to the tissue type and expression level and activities of the enzymes involved in its elimination/repair mechanism. Studies conducted by Bao and Williamson (1996) revealed that the main elimination of PLOOH in HepG2 cells occurs by direct reduction to hydroxy-phospholipids by PHGPx or by GST α, and that PLA 2/selenium-dependent GPx does not pla y a signif cant role in the reduction. A similar result w as also obtained in a computer simulation study w hich estimated that the rate of reduction of lipid h ydroperoxide by PHGPx is 10 4-fold g reater than those of PLA2 and GPx -1 in rat li ver mitochondria (Antunes et al. 1995). PHGPx was f rst described b y Ursini et al. in 1982 (1995) and it is the onl y GPx f amily isoform capable of reducing PLOOH in situ. This enzyme is also capable of reducing thymine hydroperoxides (Bao et al. 1997, Tan et al. 1986), cholesterol, and cholesterol ester h ydroperoxides (Thomas et al. 1990). PHGPx e xists in tw o for ms: the long for m (L -PHGPx) that is located in mitochondria and the shor t form (S -PHGPx) that is located in the c ytosol (Pushpa Rekha et al. 1995). The mitochondrial form is located in the inter membrane space and studies have shown that its o verexpression prevents oxidant induced apoptosis (Nomura et al. 1999). This antioxidant property has been associated with the ability of PHGPx to reduce cardiolipin hydroperoxides, which prevents the release of c ytochrome c from the mitochondria. In contrast to the obser vations described b y Bao and Williamson (1996), the in vestigation of PLOOH elimination using TLC-blotting technique to specif cally monitor lipid hydroperoxides (Terao et al. 2001) revealed that gastric mucosa PLOOH elimination is mainl y mediated 2003). The TLC-blotting by the consecuti ve action of PLA 2 and GPx (Miyamoto et al.

Table 2.1. Characteristics of glutathione peroxidases.

GPxisoforms

Commonname

GPx1

Cytosolicor classical GPx (cGPx) GastrointestinalGPx (GIGPx) PlasmaGPx (pGPx)

GPx4

GPx5

GPx2 GPx3

Proteinsize

Cellularlocation

issue T or organ

23 – 25 kDa subunit Homotetramer 22kDa subunit Homotetramer 23 – 25 kDa subunit Homotetramer

Cytosol

Erythrocytes liver, kidney, lung Li ver, large intestine

Phospholipid hydroperoxide GPx (PHGPx)

19kDa Monomer

Cytosol Mitochondria Nuclei

Epididymalnon - selenium GPx (eGPx)

24 – 25 kDa

Secretedprotein membrane associated

Cytosol Extracellular

Kidne y HepG2, Caco - 2 cells T estis

Caputepididymidis

Substrates H2 O2 Fatty acid hydroperoxides H2 O2 Fatty acid hydroperoxides H2 O2 Fatty acid hydroperoxides H 2 O2 Fatty acid hydroperoxides Phospholipid hydroperoxides Cholesterol hydroperoxides Cholesterol ester hydroperoxides V ery low activity toward hydroperoxides

25

26 Chapter

2 HO

N+

O O P

N+

O

O

O

O

O O O P O O

+

HO

PLA2

O

GPx

O

O

HO

O

OOH

O

FAOOH

LP

OH

FAOH

N+

OOH

PHGPx

O

O

O O O P O O

O

PLOOH

OH PLOH

Figure 2.1. Possible routes for the elimination of phospholipid hydroperoxide by antioxidant enzyme system. PLOOH = phospholipid hydroperoxides, PLOH = phospholipid-hydroxides, LP = lysophopholipid, FAOOH = fatty acid hydroperoxides, FAOH = fatty acid hydroxides.

technique allowed direct visualization of hydroperoxides on the blotted membranes. Using this technique, it w as possible to obser ve the appearance of a f atty acid h ydroperoxide spot after incubation of PLOOH with the gastric mucosa indicating the h ydrolysis of the h ydroperoxide by PLA2. In addition we also observed the complete reduction of the released fatty acid hydroperoxide in the presence of an e xcess amount of GSH w hich strongly suggested the par ticipation of GPx enzymes (Miyamoto et al. 2003). The par ticipation of PLA 2 in the elimination/repair mechanism of o xidatively damaged membranes was f rst proposed by the group of Sevanian (Sevanian et al. 1983). Phospholipase A2 comprises a f amily with more than 20 isofor ms (Six and Dennis 2000) that can be broadl y classif ed into three families based on the Ca 2+ requirement and localization, the calcium independent (iPLA 2), and the calcium dependent c ytosolic (cPLA 2) and secretor y PLA 2 (sPLA2 ). The PLA 2 enzyme hydrolyzes the sn-2 ester bond of gl ycerophospholipids yielding free f atty acids and l ysophospholipids, which is a critical step for eicosanoid production and for membrane phospholipid repair and remodeling processes. Studies using model membranes sho wed that PLA 2 preferentially hydrolyzed oxidized phospholipids in membranes (Kamba yashi et al. 1998, Vankuijk et al. 1987). It has been proposed that an altered conformation of PLOOH with

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the exposure of the hydroperoxide group to the membrane interface would facilitate the access of PLA 2 to the sn-2 bond (Vandenberg et al. 1993). The importance of both GPx enzymes and PLA 2 in the elimination of lipid h ydroperoxides is demonstrated by recent molecular studies in w hich the enzyme w as either overexpressed or inhibited. Overexpression of PHGPx has been sho wn to reduce the R OS-induced toxicity in mice models of atherosclerotic (Guo et al. 2008) and neurode generative diseases (Ran et al. 2006). Transgenic mice o verexpressing PHGPx sho wed smaller atherosclerotic lesions (Guo et al. 2008) and were more resistant to oxidative stress induced by hydroperoxides and amyloidbeta peptides (Ran et al. 2006). The importance of PLA2 against hydroperoxide mediated toxicity is also e videnced by recent studies w hich point to an impor tant role of iPLA 2 (iPLA2β and iPLA2γ) in repairing pero xidized phospholipids in mitochondria. Selezne v et al. sho wed that overexpression of iPLA 2β in insulinoma cells and Chinese hamster o vary cells was protective against staurosporine-induced apoptosis, a process that is kno wn to involve oxidation of lipids by mitochondrially generated ROS (Seleznev et al. 2006).

PER OXIREDOXINS ( P rx ) Prx represent a f amily of ubiquitous and abundant non -selenium thiol specif c antioxidant enzymes that have gained attention since their discovery approximately 10 years ago (Hofmann et al. 2002, Rhee et al. 2005, Wood et al. 2003) . Most of their functional properties are ascribed to their ability to reduce a wide range of hydroperoxides, including hydrogen peroxide and free or phospholipid associated fatty acid hydroperoxides (Hofmann et al. 2002, Kang et al. 1998). A peroxynitrite reductase activity has also been described for Prx (Br yk et al. 2000). Recently in vitro studies ha ve suggested that Prx can also act as redo x sensors (Veal et al. 2007) and have chaperone acti vity w hich protects cells against heat -induced protein agg regation (Jang et al. 2004). Mammalian cells express six Prx isoforms (Prx1 to 6), which are classif ed as typical 2 -Cys (Prx1 to 4), a typical 2 -Cys (Prx5), and 1 -Cys (Prx6) enzymes according to the number of conserved cysteines required for their catalytic activity (Rhee et al. 2005). The catalytic mechanism of Prx involves a redox sensitive cysteine residue (Cys-SPH, the peroxidatic cysteine) that is oxidized to a sulfenic acid (Cys -SOH) during h ydroperoxide reduction. The way by which Cys-SOH is reduced back to a thiol is w hat differentiates the three classes (Wood et al. 2003). Brief y, this step in volves the for mation of an intramolecular or inter molecular disulf de bond between the Cys -SOH and another c ysteine residue (Cys -SRH, the resolving c ysteine). The recycling of the disulf de requires an electron donor, which in the case of 2-Cys Prx is provided by thioredoxin. The oxidized thioredoxin is then re generated by a thioredoxin reductase using NADPH. The most widely recognized and abundant types of Prx found in prokaryotes and mammalian cells are the 2 -Cys Prx (Rhee et al. 2005). Studies using genetic manipulations re vealed that these enzymes ha ve important roles in protecting against DN A damage, o xidative stress, and cancer (Lee et al. 2003, Neumann et al. 2003). Moreover, a recent study demonstrated that 2-Cys Prx has impor tant roles in stress resistance, stress signaling, and aging (Olaho va et al. 2008). Whether these ef fects are a consequence of its pero xide scavenging activity or due to its redox sensing or chaperone acti vity, or a combination of all these ef fects, is still unclear . Earlier studies on Prx enzymolo gy suggested that Prx ha ve a broad specif city and were relatively poor pero xidases with moderate catal ytic eff ciency (∼ 105 M−1 s−1), compared with catalase ( ∼ 106 M−1 s−1) and GPx ( ∼ 108 M−1 s−1) (Rhee et al. 2005). However, recent studies on the kinetic properties of yeast, bacterial, and human Prx revealed a much higher catalytic eff ciency with rate constants in the order of 10 7 M−1 s−1 for tur nover of inorganic and primar y hydroperoxide (Ogusucu et al. 2007, Parsonage et al. 2008, Peskin et al. 2007). Besides 2 -Cys Prx, cells also e xpress 1 -Cys peroxiredoxins. Peroxiredoxin 6 (Prx6) is the only mammalian 1 -Cys member (Manevich and Fisher 2005). It is a bifunctional protein with

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both GPx and PLA 2 activities in the same enzyme (Chen et al. 2000). Different from the other Prx isoforms, Prx 6 does not use thioredo xin as an electron donor . To date, the ph ysiological electron donor for Prx 6 pero xidase activity has not been clearl y established. GSH seems to act as a reductant for human recombinant protein but its par ticipation has been contro versial (Fisher et al. 1999). Recently, ascorbate has been identif ed as a electron donor for yeast 1-Cys Prx (Monteiro et al. 2007). Prx6 is e xpressed in all major or gans, and at a par ticularly high le vel in the lung, w here it is important for phospholipid tur nover and lung surf actant homeostasis (Manevich and Fisher 2005). The role of Prx6 as an antio xidant enzyme in the deto xif cation of h ydroperoxides is supported by cellular and animal studies. Prx6 is capable of reducing H2 O2 as well as fatty acid and phospholipid hydroperoxides (Chen et al. 2000). The estimated rate constant for reduction of all hydroperoxides is in the range of 2 to 5 × 106 M−1 s−1. Overexpression of Prx6 in animals models has been sho wn to increase their sur vival under h yperoxic conditions (W ang et al. 2006). In another study , k eratinocytes from Prx6 -overexpressing mice w ere more resistant against UVA- and UVB -induced cell death. This protecti ve function w as ascribed to Prx6 mediated detoxif cation of hydroperoxides, which are generated in response to UV ir radiation (Kumin et al. 2006). Taken together the accumulated in vitro and in vivo studies indicates that Prx constitutes a class of antio xidant enzymes that are criticall y impor tant not onl y because of their ability to reduce hydroperoxides but also because of their potential role to ser ve as redox sensors.

A P RAOXONASE - 1( PON -AND 1) PAF ACETYLHYDROLASE IN HIGH - DENSITYLIPOPROTEIN ( HDL ) Atherosclerosis is a degenerative disease involving formation of fatty streak and f brosis in the artery tissue. Its initial event is believed to be oxidative modif cation of low density lipoprotein (LDL) in the subendothelial space (Witztum and Steinberg 1991). Oxidation of LDL occurs at the unsaturated fatty acid moiety in phospholipids and cholesteryl esters resulting in the formation of bioacti ve peroxidized lipids. Uptak e of o xidized LDL b y macrophages via sca venger receptors leads to foam cell formation (Glass and Witztum 2001). Thus, the prevention of LDL oxidation and elimination of o xidized LDL is an ef fective strategy to decrease the occur rence of atherosclerotic diseases. Epidemiolo gical studies have demonstrated that high density lipoprotein (HDL) is an anti -atherosclerotic factor. It functions as a re verse cholesterol transpor t which reduces the tissue cholesterol le vel. In addition, recent studies clarif ed that HDL has a protective effect against LDL o xidation (Negre-Salvayre et al. 2006). Paraoxonase-1 (PON-1) is an HDL -associated enzyme able to hydrolyze organophosphates, aromatic carboxylic esters, and carbamates. Watson et al. (1995) indicated that HDL associated PON-1 can reduce and/or h ydrolyze peroxidized phospholipids. PON -1 is synthesized in the liver and mostl y translocated into the HDL fraction b y the binding to apo -A1 in the b loodstream. A sulfhydryl group present in the active center of this enzyme seems to be responsib le for its antioxidant activity (Jaouad et al. 2006). PON-1 also protects LDL and HDL from lipid peroxidation by eliminating peroxidized phospholipids from each lipoprotein par ticle by converting them to h ydrophilic l ysophospholipids and f atty acid h ydroperoxides or shor t chain carbonyls (Mackness et al. 2000). Epidemiological studies also suggest that the reduction in PON-1 activity is related to oxidative stress induced b y aging, smoking, and obesity (Sarkar et al. 2006, Garin et al. 2005, Durrington et al. 2001). It should be emphasized that the mechanism of action of PON -1 in the h ydrolysis of pero xidized phospholipids is the same as that of platelet acti vating f actor (PAF) acetylhydrolase. PAF-acetylhydrolase present in human plasma is also kno wn as lipoprotein associated phospholipase A2 (Lp -PLA2). This enzyme is distributed in both LDL and HDL and produces l ysophosphatidylcholine and o xidatively modif ed shor t aldehyde or nonesterif ed fatty acids with potent pro -inf ammatory and proathero genic bioactivities (Gorelick

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2008). Ho wever, the HDL -associated enzyme is belie ved to par ticipate in the protection of LDL from o xidative modif cation by promoting the clearance of monoc yte derived dendritic cells of which the migration is impaired by PAF and PAF-like oxidized phospholipids (Karasawa 2006). Recently, Kriska et al (2007) claimed that PON -1 is not ef fective in hydrolyzing phospholipid hydroperoxides to l ysophospholipids and f atty acid h ydroperoxides. They suggested that the hydrolytic action of HDL is derived from concomitant PAF-acetylhydrolase. No direct reduction of phospholipid h ydroperoxides in HDL or HDL fractions has been demonstrated. Further studies are required to full y understand the ph ysiological function of PON -1 and PAF acetylhydrolase in HDL for the protection of LDL o xidation.

A CKNOWLEDGEMENT SM is a member of the INCT de Processos Redox em Biomedicina-Redoxoma (CNPq/FAPESP/ CAPES).

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arsonage P D , Karplus PA , P oole LB . 2008 . Substrate specif city and redox potential of AhpC, a bacterial peroxiredoxin. Proc Natl Acad Sci USA 105 ( 24 ):8209 – 14 . eskin P AV , F elicia ML , P aton LN , Maghzal GJ , Hampton MB , W interbourn CC .2007 .The high reactivity of peroxiredoxin 2 with H 2 O2 is not ref ected in its reaction with other o xidants and thiol reagents . J Biol Chem 282 ( 16 ):11885 – 92 . Pushpa - Rekha TR , BurdsallAL , Oleksa LM , Chisolm GM , Driscoll DM .1995 . Rat Phospholipid hydroperoxide glutathione peroxidase. J Biol Chem 270 ( 45 ):26993 – 9 . RanQ , Gu M , Remmen HV , Strong R , Roberts JL , RichardsonA .2006 . Glutathione peroxidase 4 protects cor tical neurons from o xidative injury and amyloid toxicity. J Neurosc Res 84 ( 1 ): 202 – 8 . RheeSG , Chae HZ , Kim K .2005 . Peroxiredoxins: A historical overview and speculative preview of novel mechanisms and emer ging concepts in cell signaling . Free Radic Biol Med 38 ( 12 ): 1543 – 52 . SarkarPD , Shi vaprakash TM , Madhusudhan B . 2006 .Association between paraoxonase activity and lipid levels in patients with premature coronar y artery disease . Clin Chim Acta 373 ( 1 – 2 ): 77 – 81 . Selezne v S , Zhao C , Zhang XH , Song K , Ma ZA .2006 . Calcium - independentphospholipase A2 localizes in and protects mitochondria during apoptotic induction b y staurosporine . J Biol Chem 281 ( 31 ):22275 – 88 . vanian Se A , Muakkassah -elly K SF , Montestruque S .1983 .The inf uence of phospholipase A2 and glutathione peroxidase on the elimination of membrane lipid pero xides. Arch Biochem Biophys 223 ( 2 ):441 – 52 . SixDA , Dennis EA .2000 .The expanding superfamily of phospholipase A(2) enzymes: classif cation and characterization . Biochim Biophys Acta 1488 ( 1 – 21): – 19 . StephensJW , Khanolkar MP , Bain SC .2009 .The biological relevance and measurement of plasma markers of oxidative stress in diabetes and cardio vascular disease . Atherosclerosis 202 ( 2 ):321 – 9 . anT KH , Me yer DJ , Coles B , K etterer B . 1986 .Thymine hydroperoxide, a substrate for rat Se - dependentglutathione - pero xidase and glutathione transferase isoenzymes . FEBS Lett 207 ( 2 ): 231 – 3 . erao T J , Miy oshi M , Miyamoto S .2001 .Thin - yer la chromatography blotting for the f uorescence detection of phospholipid h ydroperoxides and cholester yl ester hydroperoxides. J Chromat B 765 ( 2 ):199 – 203 . ThomasJP , Maiorino M , Ursini F , GirottiAW . 1990 . Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane -damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol h ydroperoxides. J Biol Chem 265 ( 1 ):454 – 61 . UrsiniF , Maiorino M , Brigelius - FlohRé ,Aumann KD , Ro veri A , Schombur g D , Floh éL , P acker L .1995 . Diversity of glutathione peroxidases . In Methods Enzymol . Academic Press . pp. 38 : UrsiniF , Maiorino M , Gre golin C .1985 .The selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochim Biophys Acta 839 ( 1 ):62 – 70 . alentine V JS , Doucette PA , ZittinPotter S .2005 . Copper- zincsuperoxide dismutase and amyotrophic lateral sclerosis . Ann Rev Biochem 74 ( 1 ):563 – 93 . andenberg V JJM , Denkamp J , Lubin BH , K uypers FA . 1993 . Conformational - changesin oxidized phospholipids and their preferential h ydrolysis by phospholipase -A(2)—a monolayer study. Biochem 32 ( 18 ):4962 – 7 . ankuijk V FJGM ,Se vanian A ,HandelmanGJ , DratzEA .1987 .Anew role for phospholipase - A2 — protection of membranes from lipid-peroxidation damage. Trends Biochem Sci 12 ( 1 ):31 – 4 . eal V EA , Da y AM , Mor gan BA . 2007 . Hydrogen peroxide sensing and signaling . Molecular Cell 26 ( 1 ):1 – 14 . ang W Y , Phelan SA , Mane vich Y , F einstein SI , F isher AB . 2006 .Transgenic mice overexpressing peroxiredoxin 6 show increased resistance to lung injur y in hyperoxia. Am J Resp Cell Molec Biol 34 ( 4 ):481 – 6 .

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atson W AD , Na vab M , Hama SY , Se vanian A , Prescott SM , Staf forini DM , McIntyreTM ,Ladu BN , F ogelman AM , Berliner JA . 1995 . Effect of platelet activating factor - acetylh ydrolase on the formation and action of minimall y oxidized low density lipoprotein . J Clin Invest 95 ( 2 ): 774 – 82 . eisiger W RA , F ridovich I .1973 . Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization . J Biol Chem 248 ( 13 ):4793 – 6 . iW tztum JL , Steinber g D . 1991 . Role of oxidized low density lipoprotein in atherogenesis . J Clin Invest 88 ( 6 ):1785 – 92 . ood W ZA , Schroder E , Harris JR , P oole LB . 2003 . Structure, mechanism and regulation of peroxiredoxins . Trends Biochem Sci 28 ( 1 ):32 – 40 . ang Y Y , Cheng JZ , Singhal SS , Saini M , P andya U , A wasthi S and , A wasthi YC .2001 .Role of glutathione S -transferases in protection against lipid pero xidation. Overexpression of hGSTA2– 2 in K562 cells protects against h ydrogen peroxide-induced apoptosis and inhibits JNK and Caspase 3 activation. J Biol Chem 276 ( 22 ):19220 – 30 . Zelk o , I. N. , Mariani ,T. J. and F olz , R. J. 2002 . Superoxide dismutase multigene family: a comparison of the CuZn -SOD (SOD1), Mn -SOD (SOD2), and EC -SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med. 33 : 337 – 49 . ZhaoT , Singhal SS , Piper JT , Cheng J , P andya U , Clark - WronskiJ , A wasthi S ,A wasthi YC . 1999. The role of human glutathione S -transferases hGSTA1–1 and hGSTA2–2 in protection against oxidative stress . Arch Biochem Biophys 367 ( 2 ):216 – 24 .

Chapter3 Antioxidants as Biomarkers of Oxidative Stress Ikuyo Ic hi and

Shosuke K ojo

RADICALREACTIONS IN THE CELL To evaluate the role of antioxidants, we analyzed radical reactions in the cell, as in F igure 3.1. Radical species designated as X • initiate radical reactions in the membrane b y abstracting a hydrogen atom of the lipid (LH). The resulting radical (L •) reacts with o xygen at a dif fusioncontrolled rate, for ming alkyl peroxy radical, LOO •, which is scavenged by α - tocopherol(αToc) to for m lipid h ydroperoxide (LOOH) and the α - tocopher yl radical (Toc• ). Toc• is recovered to α-Toc at the interf ace of the membrane and aqueous phase via reduction b y vitamin C (AsA) (T anaka et al. 1997), w hich is con verted to monodeh ydroascorbic acid (MDAsA) and dehydroascorbic acid (DAsA). MDAsA and DAsA are reduced back to AsA by various reactions utilizing NADPH or glutathione (GSH) (Linster and v an Schaftingen 2007). LOO• also abstracts a h ydrogen atom from lipids to generate LOOH and LOO • , which propagates lipid peroxidation. LOOH has a suff cient lifetime to mig rate in the cell and modif es cellular components independently or with metal ions. In this w ay, LOOH extends radical reactions to cellular constituents such as DN A and proteins apar t from the membrane. In summary, superoxide, hydrogen peroxide (H 2 O2), hydroxyl radical, alk oxy radical, alk yl peroxy radical, and LOOH are all R OS (reacti ve o xygen species). Supero xide is generated during reduction of o xygen in the respirator y chain in mitochondria and is con verted by SOD (superoxide dismutase) or spontaneous dismutation into H 2 O2, w hich generates reacti ve hydroxyl radicals via reaction with redo x active metal ions. To pre vent o xidative stress, the cell has defense mechanisms such as antio xidants and enzymes. GSH pero xidase (GPX) decomposes LOOH and h ydrogen peroxide using GSH to form o xidized glutathione (GSSG), w hich is reduced to GSH b y GSSG reductase using NADPH. The oxidation product, NADP+, is recycled to NADPH by oxidizing glucose. Figure 3.1 shows that the oxidative stress is f nally compensated for by the reductive power of glucose. Oxidative stress is induced w hen the o xidative po wer becomes superior to the antio xidant system in the cell. Based on Figure 3.1, oxidative stress def nitely affects the level of physiological antioxidants and/or the ratio of oxidized/reduced antioxidants, although it is well established that the reduced state is predominant for AsA and GSH in the cell. In this chapter w e focus on antio xidants as biomarkers in relation to human diseases and review studies of animal experiments and human subjects. Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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NADP+ NADPH GSSG Reductase GSSG

GSH

AsA

GPX LOH

LOOH

α-Toc•

Metal ion Migration DNA

MDAsA or DAsA Membrane

α-Toc LOOH

L• + O2

LOO•

Proteins Aldehydes H2O + O2

Initiation Lipid Peroxidation Catalase, GSH PX •

LH + X•

H2O2

SOD

O2–

Metal ion OH

Figure 3.1. Radical reactions in the cell.

DR UG - INDUCEDHEPATITIS AS A MODEL TO EVALUATE OXIDATIVE STRESS CARBONTETRACHLORIDE ( CC4 l) - INDUCEDHEPATITIS In the study of radical reactions in biolo gy, one of the most studied models is dr ug-induced hepatitis. CCl 4 is a long -known typical hepatotoxin causing centrizonal necrosis (Zimmer man 1978 ).CCl4-induced hepatic injur y is assumed to in volve tw o phases. The initial phase is generation of radicals and the second phase is activation of Kupffer cells, which release various pro - infammatory mediators (Edwards et al. 1993). The activation of Kupffer cells is a common feature of dr ug-induced hepatitis caused b y thioacetamide, D -galactosamine, alcohol, acetaminophen, etc. (Ramadori et al. 2008). In the initial phase, CCl 4 undergoes reductive activation to trichlorometh yl radical ( • CCl3 ), which initiates lipid pero xidation, as sho wn below (Plaa 2000). In this equation F e(II) represents hemoprotein, especially cytochrome P450, which exists in the endoplasmic reticulum of the liver. Among the P450 isozymes CYP2E1 ma y be responsib le for the acti vation based on resistance of CYP2E1 knock out mice toward CCl 4 (Wong et al. 1998). CCl4 + Fe (II ) → • CCl3 + Cl − + Fe (III ) 1998). In this Although CCl4 induces typical necrosis, it also causes apoptosis (Shi et al. connection, CCl4 increases LOOH in mitochondria and decomposes heme a, a prosthetic group of cytochrome oxidase (Ikeda et al. 1998), suggesting that other Fe(II) species in the respiratory chain are also responsible for the reductive activation of CCl 4 .CCl4 induces disordering of the phospholipid bilayer in mitochondria (Megli and Sabatini 2004) and mitochondrial permeability transition (Hernandez - Munozet al. 2003 ),which leads to caspase - 3 - dependent apoptosis of hepatocytes (Sun et al. 2001).

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Based on these studies, CCl 4 is a typical to xin causing se vere oxidative stress in the li ver, and CCl 4-induced hepatitis has been used as the standard e xperimental model to e xamine the eff ciency of new biomarkers of oxidative stress (Song et al. 2008). BIOMARKERSOF OXIDATIVE STRESS IN CC4 lINTOXICATION Among the biomark ers of o xidative stress, thiobabituric acid -reactive substances (TB ARS) have been used most frequentl y. TBARS w ere sho wn to increase in the rat li ver after CCl 4 intoxication in man y studies, w hile the y did not increase in others (Recknagel and Ghoshal 1966, Terao et al. 1984, Yoshida et al. 2005). Because the nature and the eff ciency of TBARS and malondialdehyde (MDA) as mark ers of radical reactions w ere questioned e ven in simple oxidation of soybean oil (Kishida et al. 1993), many efforts have been made to establish a more reliable indicator of radical reactions, including specif c determination of LOOH applicable to CCl4 intoxication (Miyazawa et al. 1990, Ikeda et al. 1998, Yoshida et al. 2005). Among antioxidants, hepatic AsA, determined by a specif c and sensitive method involving chemical derivatization (Kishida et al. 1992), decreases at an earl y stage of CCl 4 intoxication (Sun et al. 2001), as explained more fully below. Concerning liver GSH during CCl 4 intoxication, all possibilities, i.e., a decrease (Cor rales et al. 1992, Irita et al. 1994, Wang et al. 1997, Ohta et al. 1999), an increase (Di Simplicio and Mannervik 1983, Gharisch and Me yer 1985), and no change (P arola et al. 1992) have been reported. Shimuzu et al. (1989) repor ted that the potentiation of hepatic injur y by CCl 4 was not related to hepatic GSH content, although GSH pretreatment decreased the acute liver injury by CCl 4 (Arosio et al. 1997). CHANGEIN A s AND A OTHER EVENTS CAUSED BY CC4 l CCl4 causes many events along with oxidative stress such as loss of calcium sequestration and catabolic enzymes, loss of ener gy supply, production of c ytokines, and inhibition of protein synthesis (Weber et al. 2003). Some of these e vents cor relate with mitochondrial damage as discussed above, but it is still an enigma ho w o xidative stress leads to cell death. The ne xt paragraphs describe a typical time course of e vents closely related to o xidative stress, w here changes in AsA and MAPKs (mito gen-activated protein kinases), w hich w ere acti vated b y oxidative stress both in cells (Sumba yev and Yasinska 2005) and in the li ver (Mendelson et al. 1996), are compared. In a typical in vivo experiment, CCl 4 (2 ml/kg body weight) was orally administered to rats. The necrotic process in the li ver w as acti ve 12 hours after CCl 4 into xication and continued thereafter as evidenced by high plasma AST and ALT (Iida et al. 2007a). After tw o to 24 hours of CCl 4 administration, the hepatic AsA concentration decreased signif cantly, indicating that o xidative stress w as signif cantly enhanced as earl y as 2 hours minal kinase) and after CCl 4 intoxication and thereafter . Phosphor ylated JNK (c -Jun NH 2 - ter phospho-ERK1/2 (extracellular signal -regulated kinase 1/2) w ere signif cantly increased transiently one to three hours after treatment with CCl4, while phosphorylated p38 MAPK decreased signif cantly one to 24 hours after CCl 4 treatment. These results indicate that the change in MAPKs preceded the changes in AsA by about one hour (Iida et al. 2007a). Since ERK is acti vated by 100 µM hydrogen peroxide, namely as low a level as 100 µ M of reactive oxygen species, in cultured rat hepatoc ytes (Rosseland et al. 2005), it is diff cult to detect a change of 100 µ M in 1,800 µM of AsA in the li ver, e ven if all h ydrogen pero xide caused an equivalent decrease in vitamin C. Therefore, MAPKs are more sensitive to oxidative stress than AsA. This conclusion indicates that the cell is equipped with a physiological protein network to respond to a lo w concentration of ROS as described in the ne xt section concerning the role of H 2 O2 in insulin function.

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H2 O2 AS AN INTRACELLULAR MEDIATOR ROS ha ve been considered to be deleterious to biolo gical systems. Ho wever H 2 O2 at a lo w concentration functions as a k ey intracellular mediator of hor mones such as insulin, EGF , PDGF, NGF, angiotensin II, VEGF, IL -1β ,TNFα, and IL -6 (Droege 2002, Valko et al. 2007). Concerning the pivotal role of H2 O2 in insulin signal transduction, a symbolic study (McClung et al. 2004) demonstrated that mice o verexpressed GPX1, a selenoprotein that reduced H 2 O2 , developing h yperglycemia and insulin resistance, w hich w as associated with a signif cant reduction in the insulin -stimulated phosphorylations of insulin receptors in the li ver. R OLE OF H2 O2 IN INSULIN FUNCTION As described above, H 2 O2 enhances protein phosphor ylation of insulin receptors. One mechanism by which H2 O2 regulates cellular processes is the reversible inhibition of protein tyrosine phosphatases via re versible oxidation of their catal ytic cysteine residue (Cys -215), which has a low pKa, and therefore e xists as –S−, and is highl y susceptible to o xidation by H 2 O2 (Denu and Tanner 1998 ). By incubation of protein tyrosine phosphatase 1B (PTP1B) with a stoichiometric amount of H2 O2, Cys-215 was oxidized to highly reactive sulfenic acid ( -SOH, formally –S+ − OH),which reacted with the N -H g roup of neighboring Ser -216 for ming a sulfen yl-amide species, the structure of which was analyzed by X-ray crystallography (Salmeen et al. 2003). The sulfenylamide protein w as quantitatively reduced back to the acti ve enzyme b y GSH (Salmeen et al. + in the protein. 2003), showing the presence of a redo x couple between –S− and – S Based on these studies, the h ypersensitive guard or antioxidant in the cell against R OS may be proteins with highl y reactive Cys residue (W interbourn and Hampton 2008), oxidation of which triggers cellular signals of hor mones and MAPKs. PHYSIOLOGICALCONSEQUENCE OF ROS GENERATION Even if the most sensiti ve antenna is highl y reactive Cys residue(s) in the protein, o xidation of the thiol group does not always cause an actual physiological effect on the cell. For example, oral administration of α-Toc to rats 12 hours before CCl 4 administration did not af fect the activations of JNK, ERK1/2, and p38 MAPK (w hich took place 1.5 hours after CCl 4 administration) at all, but the α - oc T treatment signif cantly ameliorated li ver necrosis 24 hours after T also signif cantly restored the CCl4 intoxication based on plasma AST and ALT levels. α - oc T hepatic AsA concentration 12 and 24 hours after CCl 4 intoxication, demonstrating that α - oc functioned as an antio xidant (Iida et al. 2009). These results demonstrate that e ven though MAPKs are full y activated at an earl y stage by a small amount of ROS, these activations do not directly lead to cellular necrosis. The necrotic process may be initiated w hen oxidative stress is g reat enough to consume AsA in the cell. Therefore, AsA can be an ef fective marker of oxidative stress to e valuate actual physiological consequences by ROS (Kojo 2004). HEP ATITIS CAUSED BY THIOACETAMIDE ( TA ) TA is a typical hepatotoxin causing centrilobular necrosis (Zimmer man 1978, Diez-Fernandez et al. 1996). Ledda-Columbano et al. (1991) reported that TA induced apoptosis in the rat liver based on histochemical observations. Apoptosis caused by TA involves activation of caspase -3 along with e xtensive necrosis (Sun et al. 2000). In the necrotic process radical reactions are involved, as e videnced by signif cant decreases in AsA and α-Toc and an increase in LOOH (Sun et al. 2000). TA administration also reduced the GSH content (T unez et al. 2005) and GSH/GSSG ratio (Andres et al. 2003) in the rat li ver.

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Phosphorylated JNK signif cantly increased 12 hours after treatment with TA w hen li ver AsA decreased signif cantly, indicating that o xidative stress and the acti vation of JNK took place almost simultaneously. Phosphorylated ERK 2 was signif cantly increased six to 12 hours after TA injection before a signif cant decrease in AsA was obser ved, similar to the case of CCl4. Phosphorylated p38 MAPK was signif cantly decreased 24 hours after TA administration (Kishioka et al. 2007). DMSO (dimethyl sulfoxide) treatment totall y inhibited the change of these MAPKs b y TA and at the same time totally restored the liver AsA level, demonstrating that DMSO effectively ameliorated the o xidative stress caused b y TA, resulting in the pre vention of li ver necrosis (Kishioka et al. 2007 ). HEP ATITIS CAUSED BY D - GALA CTOSAMINE ( D - GALN) A high dose of D -Galn causes necrosis of the li ver by UTP depletion and inhibition of protein synthesis (Plaa 1991). Administration of D -Galn to rats caused caspase -3-dependent apoptosis in the liver when LOOH concentration increased signif cantly (Sun et al. 2003). Phosphorylated JNK and phosphorylated ERK started increasing three hours after D-Galn treatment when liver AsA star ted decreasing, indicating that o xidative stress and the acti vation of JNK and ERK occurred simultaneously (Nishioka et al. 2006). A decrease in li ver GSH content b y D -Galn has also been repor ted (Seckin et al. 1995). DMSO treatment inhibited liver necrosis and signif cantly restored the liver AsA level after D-Galn injection, demonstrating that DMSO effectively ameliorated the oxidative stress caused by D -Galn, resulting in the inhibition of necrosis of the li ver. However, DMSO treatment did not affect the change of MAPKs b y D -Galn (Iida et al. 2007b). Intoxication with CCl 4, TA, or D -Galn has been used as a model of hepatitis. They cause liver necrosis along with apoptosis b y elevating oxidative stress as e videnced by a signif cant decrease in AsA and an increase in LOOH. They also acti vate MAPKs. Ho wever, the acti ve radicals may be dif ferent because the eff ciency of antio xidants ( α - oc T and DMSO) against liver damage was considerably different. HEPATIC DAMAGE CAUSED BY ETHANOL Ethanol-induced oxidative stress in the li ver involving CYP2E1 induction and abnor mal cytokine metabolism are w ell documented (McClain et al. 2004, Lu and Cederbaum 2008). The effects of ethanol on mitochondrial GSH, as with total GSH, remain contro versial (Dey and Cederbaum 2006). In contrast to animal studies, reducing li ver damage mark ers in the b lood is the main tar get in human studies. The plasma concentrations of α - oc, T AsA, selenium, and GSH are repor ted to be lo wer in alcoholics than in controls (Lecomte et al. 1994; Loguercio and Federico 2003 ).

VIR US - INDUCEDHEPATITIS IN HUMAN SUBJECTS The plasma AsA concentration in chronic hepatitis, cir rhosis, and hepatoma patients is signif cantly lo wer than that in health y control subjects (Y amamoto et al. 1998, Murakami et al. 2006). The serum AsA level is also signif cantly lower in children with acute hepatitis A than in healthy subjects when compared to age and se x matched controls (Cemek et al. 2006).

DIABETESIN ANIMALS Diabetes mellitus is characterized by a series of complications that affect many organs. Oxygen free radicals have been implicated in the patho genesis of diabetes as w ell as its complications (Niedowicz and Daleke 2005).

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AsA concentrations in the brain, hear t, lung, li ver, kidne y, and plasma of streptozotocin induced diabetic rats decreased signif cantly compared to those of a control g roup (Sun et al. 1999). In addition, the tissue concentration of LOOH, w hich w as deter mined b y a specif c method involving chemical deri vatization with triar ylphosphine and HPLC (T okumaru et al. 1995 ),increased signif cantly in the hear t, li ver, kidne y, and muscle of these diabetic rats compared to controls (Sun et al. 1999). These results demonstrate that o xidative stress is enhanced in these tissues b y diabetes. In the liver of diabetic rats, DAsA represented 15% of the total AsA, whereas no DAsA was detected in normal rats. The total amount of AsA was also decreased in the diabetic condition. In the diabetic li ver, the acti vity of glucose -6-phosphate deh ydrogenase (G6PDH), w hich provides NADPH for reduction of DAsA, decreased signif cantly compared to the control rats, while the activity of glutathione reductase w as unchanged (Bode et al. 1993). Hepatic AsA regeneration signif cantly decreased in the diabetic rats in spite of increased expressions of AsA re generating enzymes, and AsA generation w as fur ther reduced as the diabetic state continued (Kashiba et al. 2002). Among the enzyme activities that reduce DAsA to AsA, the NADPH-dependent regeneration of AsA in the li ver was signif cantly suppressed in the Goto -Kakizaki diabetic rat (Kashiba et al. 2000). Urinary e xcretion of AsA w as repor ted to be signif cantly augmented , suggesting that increased urinar y excretion of AsA along with impaired hepatic and renal AsA regeneration contributed to the decreased le vels of AsA in plasma and tissues of diabetic rats (Kashiba et al. 2002 ).

DIABETES IN HUMAN SUBJECTS Many studies ha ve shown that the b lood AsA level is signif cantly lower in diabetic patients compared to control subjects and no signif cant differences are obser ved in the ser um DAsA level between patients and controls (Nagano et al. 1996). Patients with microangiopath y (MA) ha ve signif cantly lo wer plasma concentrations of AsA (42.1 ± 19.3µM) compared with those without MA (55.6 ± 20 µM), who in tur n have signif cantly lo wer concentrations of AsA than control subjects (82.9 ± 30.9µ M). Patients with MA have signif cantly elevated DAsA/AsA ratios compared with those without MA and control subjects, but no signif cant differences are obser ved in D AsA concentrations among these g roups (Sinclair et al. 1991). In spite of diabetic patients ha ving lo wer plasma AsA concentrations, the eff cacy of AsA supplements in treatment of diabetes or its complications is still controversial (Kuroki et al. 2003, Chertow 2004). Diabetic patients with angiopath y or endothelial dysfunction also ha ve signif cantly lo wer α-Toc/cholesterol ratio (Skrha et al. 2003 ). The GSH level in erythrocytes of diabetic subjects is repor ted to be signif cantly lower than that of control subjects. Cys-93 of the β chain of hemoglobin (Hb) forms a disulf de bond with GSH generating glutathion yl Hb (HbSSG), w hich signif cantly increases in diabetic patients, hemodialysis patients, and h yperlipidemic patients. HbSSG le vels are signif cantly associated with the duration of diabetes and Hb1c and are ne gatively cor related with GSH le vel in erythrocytes (Sampathkumar et al. 2005; Niwa 2007).

HEMODIAL YSIS PATIENTS AsA is removed from the plasma of patients undergoing hemodialysis. The occurrence of AsA def ciency has complicated the management of dial ysis patients since the be ginning of renal replacement therapy (Sullivan and Eisenstein 1970). The plasma AsA level decreases to less than 11 µM after dialysis (Jackson et al. 1995, Galli et al. 2004). AsA plasma concentrations between 11 and 28 µM represent marginal AsA status and at this le vel there is a risk of clinical AsA def ciency (Johnston et al. 2001). However,

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cautions regarding AsA treatment for dialysis patients have been discussed (Berns and Mosenkis 2005 ). In patients with chronic renal insuff ciency, the er ythrocyte contents of SOD , GSH, GPX, and α - oc T are reduced signif cantly compared to control subjects (Locatelli et al. 2003). The pro gressive deterioration of kidne y function gi ves rise to an increase of both α - CEHC (α - carbo xyethyl - yhdroxychroman) and γ-CEHC, vitamin E metabolites in plasma (Galli et al. 2004 ).

ISCHEMIA/REPERFUSION( I / RINJURY ) In I/R injury of the liver (Ito et al. 1999, Porttakal and Inal-Erden 1999), heart (Leichtweis and Ji 2001), kidney (Chen et al. 2008), and brain (Ri vera et al. 2008), the tissue GSH content is signif cantly depressed. The decrease in GSH or increase in the ratio of GSSG/GSH af fects functions of many proteins via glutathion ylation (Shelton and Mie yal 2008). For example, the 70 -kDa FAD-binding subunit of succinate ubiquinone reductase (comple x II) in mitochondria is intrinsicall y S -glutathionylated. This GSH moiety is eliminated in the post-ischemic m yocardium. The de -glutathionylated protein e xhibits lo wer electron transfer activity and increased formation of thiyl radical by superoxide (Chen et al. 2007). It is a mystery why ischemia, an o xidative condition, clea ves the disulf de bond reducti vely. On the other hand, reversible glutathionylation of complex I occurs within mitochondria following oxidation of the GSH pool (Taylor et al. 2003). This elevates superoxide production, which increases the amount of H 2 O2 that is released from mitochondria to c ytoplasm resulting in enhanced o xidative stress. In reperfused and nonreperfused cerebral ischemia, D AsA (given intravenously before ischemia) exerted dose -dependent neuroprotection, w hile AsA had no ef fect (Huang et al. 2001). This effect was ascribed to the transpor table nature of D AsA through the b lood brain bar rier. In ischemia of the rat li ver (De Tata et al. 2005) and heart (Guaiquil et al. 2004), pretreatment with D AsA confer red a signif cant protection. D AsA ma y be ef fective because man y cells possess the ability to tak e up and reduce D AsA in the b lood (Wilson 2002). In addition, a signif cant benef cial effect of AsA on I/R injury of the liver (Ozaki et al. 1995, Lee et al. 2007) and heart (Molyneux et al. 2002) has been repor ted.

MYOCARDIAL INFARCTION IN HUMAN SUBJECTS Plasma le vels of AsA, α - oc, T GSH, and β - caroteneare signif cantly lo wer in patients with acute myocardial inf arction than those in controls (Singh et al. 1994, Muzakova et al. 2001, Senthil et al. 2004). The administration of AsA has been repor ted to improve myocardial eff ciency in patients with hear t failure after myocardial infarction (Shinke et al. 2007). However, many intervention studies show no clear benef cial effect of antioxidant vitamins as food supplements in the primar y pre vention of m yocardial inf arction or strok e (Asplund 2002, Padayatty et al. 2003), although plasma AsA may be useful in identifying those at high risk of strok e (Myint et al. 2008). It is possib le that vitamins C and E are ef fective only for subjects with oxidative stress that is o ver a cer tain threshold (Block et al. 2008).

LEUKEMIA IN HUMAN SUBJECTS Oxidative stress in cancerous cells is ele vated (T oyokuni et al. 1995). Higher le vels of DNA base lesions in l ymphocytes are obser ved in children with acute l ymphoblastic leukemia (ALL) than in the l ymphocytes of control children. The le vels of GPX, catalase, and SOD in l ymphocytes of ALL patients are lo wer than those in l ymphocytes of controls, supporting the view that oxidative stress is ele vated in these malignant cells (Sentuerk er et al. 1997 ).

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Chronic leukemic patients (CML [chronic m yelogenous leukemia] and CLL [chronic l ymphocytic leukemia]) show a signif cant decrease in plasma AsA concentration and a signif cant increase in blood GSH concentration as compared with controls. Plasma D AsA concentration is signif cantly increased and the D AsA/AsA ratio is also ele vated compared to controls (Al Gayyar et al. 2007). A decrease in ser um GSH in CLL patients has also been repor ted (Bakan et al. 2003), and the GSH content in CLL l ymphocytes increases signif cantly compared with lymphocytes from nor mal subjects (Ferraris et al. 2006). Whereas AsA intake in patients with ALL was more than twice that in controls, plasma and urinary AsA concentrations w ere more than 10 times and 2.5 times higher , respecti vely, in controls than those in patients with ALL, indicating that AsA utilization is augmented in children with ALL (Ne yestani et al. 2007). Plasma AsA concentrations are decreased , w hereas DAsA concentrations are increased in patients of ALL, Hodgkin ’s disease, and non -Hodgkin’s lymphoma (Abou-Seif et al. 2000). These results indicate enhanced oxidative stress in leukemia patients.

α - OCOPHERYL T QUINONE α-Toc yields many products by radical reactions. The main catabolic pathw ay of α - oc T in the liver is de gradation by side chain o xidation, while two-electron oxidation of α - oc T yields αtocopheryl quinone (TQ) (T erentis et al. 2002). In homo genates of human aor tic tissues and carotid plaque specimens, the absolute amounts of protein -standardized o xidized α - oc T and lipids increased with increasing disease se verity. At all stages TQ w as the major o xidation product of α-Toc. In plasma samples from patients subjected to aor tic crossclamping, TQ increased during ischemia (Mur phy et al. 1992). TQ increased in rat tissues under h yperoxic conditions (Kanaza wa et al. 2000). Moreover, ethanol feeding reduced li ver α-Toc content and caused a mark ed increase in li ver TQ, especially in the microsomal fraction (Kawase et al. 1989). In diabetic rats, the ratio of TQ/α - oc T in the erythrocyte membrane was increased (Shinozaki et al. 2002). The ratio of TQ/α - oc T and/or TQ level may be a good marker of oxidative stress, but only limited studies are available.

COENZYME Q ( C o Q ) Coenzyme Q10 (CoQ10), a highl y h ydrophobic quinone deri vative, is present in reduced (CoQH2: ubiquinol) and o xidized (CoQ: ubiquinone) for ms. The redox cycling between CoQ and CoQH 2 is essential for o xidative phosphor ylation in the mitochondrial respirator y chain (Crane et al. 1957). The other impor tant function of CoQ10 is to ser ve as a potent radical scavenger. CoQH 2, a hydroquinone derivative and a w ell-established radical scavenger, is f rst oxidized during o xidation of LDL (lo w-density lipoprotein) in vitro (Stocker et al. 1991) and rescues Toc• to re generate α-Toc (Sohal 2004). In this conte xt CoQ10 supplementation w as anti - athero genic in apolipoprotein E - defcient mice via inhibiting lipid pero xidation (Thomas et al. 2001). CoQ10 depletion is associated with the patho genesis of other diseases. F or e xample, the depletion of CoQ10 intercepts m yocardial ener gy generation in mitochondria, resulting in chronic heart failure (Mortensen 2003). The percentage of o xidized CoQ10 (ubiquinone -10) vs. total CoQ10 in plasma of patients with hepatitis, cir rhosis, and hepatoma w as signif cantly higher than that in nor mal subjects, while the total CoQ10 le vel was similar among these four g roups (Yamamoto et al. 1998). The CoQ10 level in the cor tex region of the brain in patients with P arkinson’s disease (PD) is reported to be lower than that in age -matched controls (Hargreaves et al. 2008) and CoQ10 administration has retarded the functional deterioration in PD patients in one study (Shultz et al. 2002). Further studies are required to determine whether treatment with CoQ10 produces an improvement of PD.

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CONCLUDINGREMARKS The role of o xidative stress is being clarif ed in man y diseases. In animal e xperiments, determination of antioxidants in tissues is possib le and useful as o xidative stress biomarkers, while a reliable marker in the blood is required for clinical use. However, oxidative stress in an organ is not al ways ref ected in the b lood. For example, severe oxidative stress in the li ver may not be ref ected in the antioxidant level in plasma (Sun et al. 2001). At present, practical biomarkers of oxidative stress in the blood are extremely limited. It is also important to distinguish whether the decrease of antio xidative nutrients in the b lood is caused b y oxidative stress or simpl y by malnutrition in patients. In the case of antio xidant def ciency, supplementary antioxidative nutrients ma y be benef cial, although many intervention studies show no clear effect of antioxidants on the prevention or treatment of diseases where oxidative stress is proposed in the pathogenesis. Further studies are needed to disco ver a reliab le biomarker of o xidative stress and also to clarify the benef ts and the limits of antio xidant therapies.

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okumaru T S ,Tsukamoto I , Iguchi H , K ojo S .1995 . Specifc and sensitive determination of lipid hydroperoxides with chemical deri vatization into 1 -naphthyldiphenylphosphine oxide and high - performance liquid chromatography . Anal Chim Acta 307 : 97 – 102 . oyokuni T S , Okamoto K ,Y odoi J , Hiai H .1995 . Persistent oxidative stress in cancer . FEBS Lett 358 : 1 – 3 . unez T I , Munoz MC ,V illavicencio MA , Medina FJ , dePrado EP , Espejo I , Barcos M , Salcedo M ,F eijoo M , Montilla P . 2005 . Hepato -and neurotoxicity by thioacetamide: Protective effect of melatonin and dimeth ylsulfoxide. Pharmacol Res 52 : 223 – 228 . alko V M , Leibfritz D , Moncol J , Cronin MTD , Mazur M ,T elser J . 2007 . Free radicals and antioxidants in nor mal physiological functions and human disease . Int J Biochem Cell Biol 39 : 44 – 84 . ang W PY , Kanek o T , Tsukada H , Nakano M , NakajimaT , SatoA .1997 .Time course of hepatic injuries induced by chloroform and by carbon tetrachloride: comparison of biochemical and histopathological changes . Arch Toxicol 71 : 638 – 645 . eber W LWD , Boll M , StampfA. 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a to xicological model . Crit Rev Toxicol 33 : 105 – 136 . iW lson JW . 2002 .The physiological role of dehydroascorbic acid . FEBS Lett 527 : 5 – 9 . iW nterbourn CC , Hampton MB . 2008 .Thiol chemistry and specif city in redox signaling . Free Radic Biol Med 45 : 549 – 561 . ong W FWY , ChanWY , Lee SST . 1998 . Resistance to carbon tetrachloride - inducedhepatotoxicity in mice which lack CYP2E1 e xpression. Toxicol Appl Pharmacol 153 : 109 – 118 . amamoto Y Y, Y amashita S , Fujisa wa A , K okura S ,Y oshikawa T . 1998 . Oxidative stress in patients with hepatitis, cir rhosis, and hepatoma e valuated by plasma antioxidant. Biochem Biophys Res Comm 247 : 166 – 170 . oshida Y Y , Itoh N , Ha yakawa M , Piga R , Cynshi O , Jishage K , Niki E .2005 . Lipid peroxidation induced by carbon tetrachloride and its inhibition b y antioxidant as evaluated by an oxidative stress marker, HODE . Toxicol Appl Pharmacol 208 : 87 – 97 . Zimmerman HJ. 1978. Hepatotoxicity, The Adverse Effects of Dr ugs and Other Chemicals on the Liver. New York: Appleton - Century - Crofts.

Chapter4 LDL Oxidation as a Biomar ker of Antioxidant Status Mohsen Me ydani , EunHee K ong and , Ashle y Knight

INTR ODUCTION During the past four decades, se veral hypotheses have evolved about the cause of atherosclerosis, including vascular response to injur y (Ross and Glomset 1973), vascular wall retention of low density lipoprotein (LDL) (W illiams and Tabas 1998), and o xidative modif cation of LDL (Steinberg, Parthasarathy et al. 1989). While each hypothesis points to its critical initiating event, all share common complex cellular events, which include several components of inf ammation, such as monoc ytes/macrophages, T cells, and inf ammatory cytokines. Therefore, atherosclerosis is now believed to be an inf ammatory disease of ar teries (Libby 2002). According to the o xidative modif cation hypothesis of atherosclerosis, w hich was proposed by Steinberg et al. in 1989 (Steinberg, Parthasarathy et al. 1989), native LDL is not atherogenic; rather, oxidatively modif ed LDL is the main cause and source of lipids that accumulate during atherogenesis. Native LDL is entrapped in the sub -endothelial space of ar terial walls where it is o xidatively modif ed b y cellular and e xtracellular o xidants. Oxidati vely modif ed LDL in turn attracts circulating monoc ytes into the intimal space, w here they become resident monocytes-macrophages (Quinn, P arthasarathy et al. 1987) in sub -endothelial space e xpressing scavenger receptors through w hich they inter nalize oxidized LDL, accumulate lar ge amount of lipids, and become lipid -laden foam cells. While monocyte/macrophages are considered to be a major source of reacti ve o xygen and nitro gen, specious, v ascular smooth muscle cells (VSMC), endothelial cells, and adv entitial f broblasts also contribute to the production of oxidants (Griendling, Sorescu et al. 2000, Rey and Pagano 2002). Several sources of o xidants in the v ascular w all have been identif ed to contribute to the oxidation of LDL, including NADPH oxidases (Babior, Lambeth et al. 2002), myeloperoxidase (Savenkova, Mueller et al. 1994), lipoxygenases (Mur ray and Brash 1988), nitric o xide synthase (Cirino, F iorucci et al. 2003), and mitochondrial respirator y chain (Ballinger , Patterson et al. 2002). Further, a variety of stimuli induces the production of se veral inf ammatory cytokines and g rowth f actors in v ascular cells, including interleukin (IL)1, tumor necrosis f actor (TNF)α, platelet derived growth factor (PDGF), thrombin, angiotensin II, vascular endothelial growth factor (VEGF) and mechanical sheer stress (Ushio -Fukai, Tang et al. 2002). Because plasma contains robust antio xidant defenses and LDL itself contains lipid solub le antioxidant defenses such as α-tocopherol and carotenoids, o xidation of LDL is belie ved less likely to occur in circulation; rather , LDL o xidation mainly occurs in the v ascular wall, and Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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probably onl y a small fraction of o xidized LDL ma y transit from sub -endothelial space to circulation. In vitro, when oxidation only occurs in the lipid components of LDL, it is ter med as “minimally modif ed LDL” (mmLDL), which is capable of inducing monocyte chemotactic protein (MCP) -1 by monocytes and smooth muscle cells (SMC) and attracting inf ammatory cells to the site of acti vation (Cushing, Berliner et al. 1990). However, if the LDL is se verely oxidized, which in addition to oxidation of lipid component includes changes in LDL apolipoprotein B -100, it is then ter med “o xidized-LDL” (o x-LDL) and has higher electrophoretic mobility, higher density, and higher free cholesterol content than native LDL. Ox-LDL attracts monocytes (Quinn, P arthasarathy et al. 1987) and T lymphocytes (McMur ray, P arthasarathy et al. 1993), and it inhibits macrophages’ motility, so that the y become resident macrophages in the sub -endothelial space accumulating lipids (Morel, DiCorleto et al. 1984, Quinn, Parthasarathy et al. 1987). Ox -LDL stimulates proliferation of SMC (Stik o-Rahm, Hultgardh Nilsson et al. 1992) and induces auto-antibodies and immune complexes, which are recognized and taken up by macrophages. (Griff th, Virella et al. 1988, Yla-Herttuala, Palinski et al. 1989, Parums, Brown et al. 1990) Thus, oxidized LDL is more athero genic than nati ve LDL and is readily recognized by the scavenger receptors on monoc yte-derived macrophages. However, elevated oxLDL in circulation is strongl y associated with ele vated LDL and triglyceride levels, for example, in prediabetic patients (Kopprasch, Pietzsch et al. 2002), indicating that the more LDL par ticles in circulation, the more chance for these par ticles to become oxidized. An enhanced susceptibility to o xidation in diabetes might be an underl ying mechanism responsible for se veral vascular diseases that are obser ved in diabetics. Fur ther, several studies have found an inverse relationship between the susceptibility of LDL in in vitro oxidation with the se verity and pro gression of coronar y atherosclerosis in dyslipidemic patients (Regnstrom, Nilsson et al. 1992) Because LDL contains large amounts of cholesterol esters of linoleic and arachidonic acids, its continuous o xidative modif cation in the intimal space results in the accumulation of o xidized products, w hich e ventually lead to a full -blown spectr um of atherosclerosis. Se veral hydroxy products of cholesterol esters of linoleic and arachidonic acids as w ell as o xidized products of cholesterol have been isolated from human atherosclerotic lesions. When the concentration of cholesterol increases, the levels of specif c oxysterols increase as does the severity of atherosclerosis (Car penter, Taylor et al. 1993, Brown and Jessup 1999). While LDL is well recognized as atherogenic, the severity of atherogenicity of LDL depends on the size of LDL particles, which vary in size and density. The presence of small, dense LDL particles (sdLDL) in circulation is considered to be a high risk factor for coronary heart disease (CHD). These particles are highly susceptible to oxidative modif cation, and their atherogenicity is probab ly related to their small size so that the y can penetrate through endothelium and sub-endothelial space in the arteries. Thus, oxidation of sdLDL may signif cantly contribute to the deleterious effects of oxLDL in atherogenesis. sdLDL par ticles may be present in the cir culation of patients with metabolic syndrome (Garin, Kalix et al. 2005), and increased circulating le vels of sdLDL par ticles are associated with f aster pro gression of stenosis and g reater accumulation of o xLDL in the aor tic valve (Mohty, Pibarot et al. 2008). Diabetics also sho w high levels of sdLDL, w hich makes their o verall total LDL susceptibility to o xidation higher than in nor mal control subjects. (T an, Ai et al. 1999) The presence of sdLDL in population studies is regarded as a good predictor of CHD risk (Arsenault, Lemieux et al. 2007). The presence of o x-LDL in vivo has been identif ed by antibodies that reco gnize epitopes of ox-LDL, which are mainly found in macrophages within atherosclerotic lesions, but not in normal ar teries (Glavind, Har tmann et al. 1952, Brooks, Steel et al. 1971, Car penter, Taylor et al. 1993). Even though oxidation of LDL mainly occurs in the vascular wall, human plasma from patients with coronar y syndrome has sho wn immunoreacti vity w hen incubated with epitopes of o xidized LDL, indicating the presence of o xidized LDL in circulation (P alinski, Horkko et al. 1996). High levels of autoantibodies against ox-LDL have been demonstrated in human plasma, which is associated with the se verity of coronary artery disease and peripheral

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atherosclerosis (Parums, Brown et al. 1990, Ehara, Ueda et al. 2001). The level of ox-LDL as measured b y ELISA in patients with stab le CHD w as high and w as signif cantly correlated with plasma LDL and TG le vels and in versely cor related with HDL le vels (W einbrenner, Cladellas et al. 2003). In a follo w-up study of CVD patients, in vivo levels of o xLDL were well correlated with the numbers of LDL particles. The higher the levels of LDL in circulation, the more oxLDL was detected and inversely correlated with f ow-mediated dilation (FMD) of the brachial ar tery (v an der Zw an, Teerlink et al. 2009). Therefore, deter mination of LDL resistance to in vitro oxidation has been re garded as an indication of the in vivo susceptibility of LDL par ticles to oxidation in the v ascular walls.

LDLOXIDATION AS AN INDEX OF ANTIOXIDANT STATUS AND OXIDATIVE STRESS The oxidation of LDL is a complex process, which includes formation of lipid hydroperoxides, modif cation of apolipoprotein B , and loss of antio xidants such as α - tocopherol.Several methods of LDL oxidation have been developed to simulate in vivo conditions. The susceptibility of LDL to in vitro oxidation provides some clues to the role of nutrients and antio xidants on LDL athero genicity. To test the in vitro susceptibility of LDL to o xidation, it is necessar y to isolate LDL par ticles without introducing an ar tifact, and then to initiate o xidation and monitor the course of o xidation in a reproducib le manner so that the results can be compared with those repor ted in the literature. ISOLA TION OF LDL Lipoprotein particles can be isolated from fresh or frozen plasma by different methods. For the purpose of in vitro LDL o xidation, it is impor tant to a void auto -oxidation or modif cation of any component of LDL par ticles during the isolation. Therefore, the initial step, w hich is the collection of blood from human subjects, is an impor tant step. Because a small amount of red blood cells (RBC) is hemol yzed during the blood draw, which releases iron from hemo globin, it is necessar y to sequester and chelate the iron b y drawing blood into a tube containing ethylenediamine tetraacentic acid (EDTA). EDTA, in addition to being an anticoagulant, traps the iron and prevents it from initiating a F enton type reaction, producing o xygen free radicals and oxidizing LDL. Following separation of plasma by centrifuging of blood at 4 °C at 2,000 × g for 10 minutes, LDL can be isolated from the v arious lipoprotein sub -classes in several steps using shor t-time or long -time centrifugation procedures (Himber , Buhler et al. 1995, McDowell, McEneny et al. 1995). 0.9 mL of isolated EDTA plasma is pipetted into a centrifugation tube and its density is adjusted to 1.300 mg/mL with KBr (0.4451 g). This is then overlaid with 2.1 mL of 150 mM NaCl and centrifuged at 541,000 × g for tw o hours at 4 °C. The LDL la yer is aspirated and mixed (1:1 v/v) with a KBr solution containing 1% EDT A, w hich pro vides a density of 1.063 mg/mL. The mixture is centrifuged again at 541,000 × g for two hours at 4 °C. The presence of EDTA in LDL preparation, e ven in a small amount, interferes with in vitro copper-induced LDL o xidation, a common pro -oxidant used in an LDL o xidation assa y. Therefore, all of the EDT A should be remo ved from the LDL preparation. To do this, the collected LDL is either extensively dialyzed (pore size of 50,000 Daltons) for at least 24 hours in PBS buf fer containing 0.2 mM EDTA at 4 °C in dark or cleaned up b y using a Sephade x PD-10 column equilibrated with PBS using a buf fer (F-LDL) (Carru, Zinellu et al. 2004). The LDL fraction can be also aspirated and r un through an Econ -PacI0DG column (Bio -Rad Laboratories). In addition to EDT A, compounds such as aporotinin, chloramphenicol, and phen yl methyl sulfonyl f uoride have been recommended to prevent auto-oxidation of LDL (Galle and Wanner 1998). Using a v ertical rotor (Beckman VTi-65), it is possib le to reduce isolation time to 1.5

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hours. The purity of LDL can be conf rmed by gel electrophoresis. It is important to remember that the slightest change in density of KBr or separation g radient may change the resolution of the isolated layers. With a long con ventional centrifugation procedure, LDL is subject to possible auto -oxidation, which may give a f alse reading in the LDL o xidation assay such as an increase in baseline conjugated dienes le vels and shor tened lag -time of o xidation. Therefore, the use of a v ertical rotor and shor t term centrifugation is recommended. MONIT ORING IN VITRO LDL OXIDATION Lipids and protein moiety of lipoprotein in LDL particles in circulation are well protected from oxidation by the presence of lipoph ylic antioxidants within the LDL par ticle as well as by the presence of robust antioxidants in plasma. Despite the presence of lipoph ylic antioxidants, the lipid component of LDL particles can undergo oxidative modif cation in vivo within the arterial wall and contribute to the de velopment of f atty streaks and atherosclerosis. Ho wever, it is believed that the presence of high le vels of lipoph ylic antio xidants such as tocopherols and carotenoids in LDL par ticles as w ell as antio xidants present in plasma protect LDL par ticles from oxidation. Therefore, testing the susceptibility of LDL par ticles to o xidative insult in a test tube was suggested as one w ay to evaluate in vivo susceptibility of LDL of indi viduals to oxidative insult. This is the most frequent method used to deter mine resistance of LDL to oxidation. Due to the presence of lipoph ylic antioxidants in LDL par ticles, it takes more than a da y to oxidize isolated LDL par ticles at room temperature. Ho wever, by adding an o xidizing agent such as transition metals, iron or copper sulfate, the oxidative chain reaction is initiated in LDL particles and the progression of LDL oxidation is monitored spectrophotometrically (Esterbauer, Striegl et al. 1989). Although free copper ion (Cu2+) is not present in blood circulation, copperinduced oxidation has been commonl y used to in vestigate the o xidative potential of LDL in many studies. The copper ion may act in part by reacting with lipid hydroperoxides to produce chain radicals (Gutter 2007) i.e., LOOH + Cu 2 + → LOOi + Cu + + H +

(4.1)

It has been reported that copper-mediated peroxidation occurs independently of O2 ,aqueous HO−, and H2 O2, and may involve direct reduction of Cu2+ to Cu+1 by LDL particles. Experiments have shown that incubation of LDL with cupric ion resulted in Cu+1 formation. The mechanism of copper -induced LDL o xidation re viewed b y Burkitt (2001) has y et to be elucidated. Experimentally, oxidation by Cu 3+ sulfate is achieved by incubating 200 mg/mL isolated LDL with phosphate - buf fered saline containing 5 mMCu3+ sulfate at 37°C for 20 hours (Steinbrecher, Witztum et al. 1987). The length of incubation at 37°C depends on the LDL protein levels used in the reaction. The kinetics of copper -induced pero xidation and copper reduction are signif cantly inf uenced by the presence of lipid -soluble antioxidants and peroxidizable substances. Visioli et al. have suggested that polyunsaturated fatty acids (PUFA) in phospholipids of LDL par ticles are not the only substrates that under go peroxide formation, but PUFA in triglycerides and in the cholesterol ester in the core of the par ticles may undergo oxidation, leading to e xtensive peroxidation of all the LDL lipids (V isioli, Bordone et al. 2000). The presence of ser um albumin in copper -induced LDL o xidation has been in vestigated for its interaction with copper ion. It was suggested that the presence of minor amounts of serum albumin in samples may introduce variability in LDL o xidation. This can be overcome by increasing the concentration of copper in the assa y. Ho wever, b y using a w ater-soluble free radical generator such as 2,2 ′ - azobis (2-amidinoprpane) dih ydrochloride (AAPH) in in vitr o LDL o xidation, the ser um albumin interference will be minimal (Schnitzer , Pinchuk et al. 1997). Free iron is not present in blood circulation. Iron in circulation is sequestered in fer retin and hemoglobin; therefore, it is not freel y available to oxidize LDL in circulation. Ho wever, LDL •−

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Conjugated Diene (nmol/mL)

oxidation in vivo may occur through hemo globin (Hb) catalyzed heme -initiated globin radical formation. Hemoglobin (Hb), which is released b y intravascular hemolysis of red b lood cells, is a strong lipid o xidant through oxidation of its bound F e2+ to Fe3+ (Miller, Altamentova et al. 1997). Ho wever, there is a plasma protein called hapto globin (Hp), w hich binds with high aff nity to Hb, for ming a comple x that pre vents o xidative damage b y Hb (Zuw ala-Jagiello 2006). The Hb-Hp complex is then recognized and degraded by the reticuloendothelial system via endoc ytosis through the hemo globin sca venger receptor CD163, as w ell as b y other receptors. A higher plasma o x-LDL/LDL ratio has been obser ved in indi viduals with genetic defects in heptaglubulin. In humans, the Hp gene e xists mainly as two alleles, Hp1 and Hp2, leading to haptoglobin 1 -1, 1 -2, and 2 -2 genotypes. The magnitude of Hp binding capacity to Hb and the prevention of o xidative stress is in the order of Hp1 -1 > Hp1 - 2> Hp2 - 2.A Hp2 - 2polymorphism (Brouwers, Langlois et al. 2004), a genotype with lower antioxidant activity, occurs in the Western population with a frequenc y of about 36% (Bo wman and K urosky 1982). Therefore, LDL par ticles of indi viduals with Hp2 -2 pol ymorphism are more prone to iron initiated oxidation. Accordingly, supplemental intak e of antio xidants such as vitamin E ma y reduce the risk of CVD in these indi viduals (Singh, Devaraj et al. 2005, Rainwater, Mahaney et al. 2007). Oxidative reactions initiated in LDL par ticles in vitro initiate a chain reaction in the PUF A of phospholipid, cholesterol ester , and trigl ycerides of par ticles for ming conjugated dienes, which can be detected b y the changes in the optical density of light at 234 nm. Continuous monitoring and recording the conjugated diene formation generate a kinetic prof le of the LDL oxidation diag ram as sho wn in F igure 4.1. The o xidation kinetic prof le can be e xamined comparatively for the effect of dietary saturated and unsaturated f atty acids as well as antioxidants and dr ugs on LDL susceptibility to o xidation. The in vitro o xidation kinetic prof le consists of se veral parameters, w hich provide some information on the in vivo oxidative status of indi viduals and the potential of LDL resistance to oxidation, which is affected by dietary antioxidants and lipids. As depicted in F igure 4.1, the initial light absorbance of LDL preparation at 234 nm (point A star t of the assa y) represents the amount of conjugated dienes, o xidized lipids, present in LDL par ticles in vivo in circulation, assuming that during the isolation of LDL no o xidation has occurred. Therefore, the protection of LDL from o xidative modif cation and the introduction of ar tifact during the preparation of LDL par ticles are impor tant for evaluating the status of LDL in vivo .In vivo oxidation of LDL might be related to sustained o xidative stress, which is associated with lo w levels of endo genous and dietar y antioxidants, inf ammation, intoxication, and dr ugs. 2.5 D

2.0 1.5 C

1.0 0.5 0.0

–0.5

A 0

E

B 50

100

150

200

250

300

Time (min)

Figure 4.1. In vitro kinetic prof le of LDL oxidation. (A) The initial in vivo level of conjugated dienes in LDL particles. (A to B). Duration of lag time to oxidation. (C) Propagation of oxidation (rate of oxidation). (D) Maximum oxidation.

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The second parameter is the lag time, which is def ned as the interval (minutes) between the interception of the linear least-square slope of the curve with the initial-absorbance axis, (points A to B). The duration of the lag time is deter mined by the amount of antio xidants present in LDL such as vitamin E, carotenoids, retin ylstearate, and phytof uene. The higher the le vels of lipophylic antioxidants, the longer the lag time would be. This means that as superoxide radicals are produced by Fenton-type reaction in the test tube, the y are quenched by LDL antioxidants until all antioxidant reserves in the LDL par ticles are depleted. To simulate in vivo conditions, w here LDL par ticles are e xposed to w ater soluble antioxidants in circulation, the addition of ascorbic acid or urate in micromolar concentrations prolongs the lag time in a concentration -dependent manner . Ho wever, these increasing ef fects of vitamin C or urate are subject -specif c, with a strong interindi vidual v ariation. Once the antioxidants are depleted from the par ticles, the oxidation of LDL will proceed and accelerate to reach maximum o xidation (points B to D). The third parameter is maximal rate of oxidation (C), which can be calculated from the slope of the absorbance cur ve during the propagation phase (e xpressed as mol dienes • min −1 •g−1 of LDL-protein), again using the molar absorptivity for conjugated dienes. The maximal and total (maximal minus initial) amounts of dienes produced during o xidation of a par ticular LDL sample depend on the content of pero xidation substrates, i.e., the pol yunsaturated fatty acids such as linoleic acid (18:2) and arachidonic acid (20:4) and the n -3 fatty acids such as eicosapetaenoic acid (EPA) and docosahe xaenoic acid (DHA). F atty acids are inte gral components of triac ylglycerides, phospholipids, and cholester yl esters. Variations in the total amount of dienes produced during LDL o xidation are thus a ref ection of an indi vidual’s LDL f atty acid content and consequently of dietar y fatty acid intake. The four th parameter is maximal o xidation (max), the highest point of increase in optical density (D), which ref ects complete depletion of antioxidants and total oxidation of lipids after which oxidized fatty acids start to decompose (the decline of the curve after maximal oxidation point (D)). The time that it tak es to reach maximal o xidation (T-max) depends on the rate of oxidation (C); the slo wer rate of o xidation, due for e xample, to the presence of saturated and monounsaturated fatty acids, takes a longer time to reach T-max. In addition to monitoring conjugated diene for mation during LDL oxidation, other sensitive methods ha ve been de veloped to detect specif c lipid h ydroperoxides resulting from LDL oxidation. Diphen yl-1-pyrenylphosphine (DPPP) is a probing compound , w hich reacts with hydroperoxide to gi ve rise to a f uorescent emitting molecule DPPP =O (Akasaka, Ijichi et al. 1992). DPPP can be incor porated into the LDL par ticle using the dispersion reagent Pluronic F-127, and its reaction with dif ferent forms of hydroperoxides in LDL following the initiation of o xidation using azo radical generator (see belo w) can be monitored b y the increase in f uorescence intensity of the LDL (Okimoto, Warabi et al. 2003). While this method is sensitive and specif c for measuring in vivo le vels of h ydroperoxides present in LDL particle or h ydroperoxide for mation during LDL o xidation, b y f ar the copper -induced LDL oxidation and monitoring of the conjugated dienes for mation is the most frequentl y used method. ORGANICRADICAL GENERATORS AND LDL OXIDATION In addition to transition metals-induced LDL oxidation, organic water-soluble and lipid-soluble free radical generators have been used to initiate and produce oxidative stress in a constant rate and to measure the resistance of LDL par ticles to o xidative insult. AAPH (2,2 ′ - azobis(2 amidinopropane) hydrochloride) is a w ater soluble azo compound , which by ther mal decomposition produces molecular nitrogen and two carbon radicals in the aqueous phase at a constant rate (Niki 1990) surrounding the LDL particle, and thus produces a more or less random attack on the surface of the LDL par ticle. This is somewhat similar to what may occur in the circulation and tissues when LDL is exposed to oxidative insults produced by activated macrophages.

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To examine the resistance of LDL to o xidative insult generated b y AAPH, 100 mg protein of LDL/mL is incubated with 50 mM AAPH and human ser um albumin (hSA, 0.25 mg/mL) in 0.1 M phosphate buf fer (pH 7.4) at 37 °C. The oxidation of LDL is monitored with a spectrophotometer at 420 nm every f ve minutes for 60 minutes or longer , after w hich o xidation is stopped by cooling the tubes with cr ushed ice. AMVN (2,2 -azobis(2,4-dimethylvaleronitrile) is a lipid -soluble azo compound and thus is partitioned in the lipid domain of the LDL par ticle, and it dissociates spontaneousl y to for m carbon-centered radicals (Niki 1990). This radical generator is used to determine the resistance of LDL to lipid-soluble oxidants. The assay consists of about 100 mg/mL LDL protein in 0.1 M phosphate buffer at 37 °C. Oxidation is initiated b y the addition of 200 µ L of 10 mMAMVN, and change in absorbance is monitored at 420nm for 60 minutes or longer. In this assay system, deferoxamine mesylate (100 mM) can be added to chelate transition metal ions to prevent their interference in the lipid pero xidation process. CELL - MEDIA TED OXIDATION OF LDL IN VITRO LDL is susceptible to oxidation by several cells of the vascular and immune systems including endothelial cells, smooth muscle cells (SMC), macrophages, T cells, and activated monocytes. Cell-mediated oxidation of LDL has been used to more closel y simulate in vivo oxidation of LDL in an in vitro system. The principle behind using immune cells in the assa y is based on the oxidation of LDL b y reactive oxygen species, w hich is produced b y an o xidative burst of the activated cells. Purif ed LDL (200 µg protein/mL) is incubated with acti vated cells (5 to 10 × 106 cells/mL) in a GBSS buf fer at 37 °C for six hours. Activation of cells can be initiated by the addition of acti vators such as lipopol ysaccharide (LPS), or phorbol -12-myristate-13acetate (PMA), or chemotactic peptide from bacterial proteins, or y east cell w all preparation. Cell-mediated oxidation of LDL is ar rested by refrigeration and the addition of EDT A and a powerful organic antioxidant such as buthylated hydroxytoluene (BHT). The oxidation of LDL is measured by the formation of conjugated dienes (Scaccini and Jialal 1994). Other investigators have used serum free Ham ’s F -10 cell culture medium supplemented with fer rous sulfate, phenol red, and glutamine (Car penter, van der Veen et al. 1997).

SIGNIFICANCEOF WATER - SOLUBLEAND LIPID - SOLUBLE ANTIOXIDANTS TO LDL OXIDATION As stated abo ve, LDL is the major car rier of cholesterol, esterif ed cholesterol, trigl ycerides, phospholipids, and fat-soluble vitamins such as vitamins E, K, and carotenoids. There has been compelling evidence suggesting that the o xidation of LDL is one of the potential mechanisms by which high levels of LDL contribute to the de velopment of atherosclerosis. Here, in brief, we discuss the contribution and signif cance of the lipid -soluble compounds car ried in LDL particles and those of water-soluble antioxidants present in plasma and tissue on LDL oxidation and the association with risk of diseases. VIT AMIN EAND LDL OXIDATION Vitamin E is lipid solub le and w ell known for its antio xidant activity. Because of its h ydrophobic nature, vitamin E is mainly located in the core of the lipoprotein par ticles. On average, each LDL par ticle may car ry six to 12 molecules of tocopherol (Stock er, Bowry et al. 1991, Esterbauer, Gebicki et al. 1992). Supplemental intak e of vitamin E increases the vitamin E content of LDL par ticles and increases their resistance to o xidation (lag time to o xidation). Enrichment of LDL par ticles with vitamin E, according to Stock er et al. (Stock er, Bo wry et al. 1991, Thomas, Leichtweis et al. 2001), not onl y does not protect LDL from o xidation, but rather increases the susceptibility of LDL par ticles to lipid pero xidation in isolated LDL.

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This has raised great concern in those who believed that a high intake of vitamin E supplement reduces the risk of CVD . However, this may not occur in vivo due to the presence of CoQ10 H2 in LDL par ticles. Stocker et al. demonstrated that co -enrichment of LDL with CoQ10 H2 and tocopherol not onl y prevents the prooxidant effect of vitamin E supplementation but also increases LDL resistance to o xidation (Thomas, Leichtweis et al. 2001). Several repor ts have shown no cor relation between the LDL content of vitamin E and the lag time of LDL oxidation (Salonen, Salonen et al. 1985, Kok, de Bruijn et al. 1987, Esterbauer, Puhl et al. 1991). In coronary artery disease (CAD), despite the presence of the high le vels of vitamin E in the LDL par ticles in a patient under going coronar y angio graphy, the in vitr o susceptibility of LDL to o xidation was higher than those of control subjects. This has been attributed to the presence of an o xidation resistance component other than vitamin E in LDL and other factors such as density of LDL (small, dense LDL) or f atty acid content, which may contribute to the v ariability of LDL o xidation (Halevy, Thiery et al. 1997). CAR OTENOIDS AND LDL OXIDATION Because carotenoids are lipid soluble and are carried in LDL, it was thought that their benef cial effect was to protect LDL from o xidation and thus to contribute to the pre vention of CVD. It was assumed that with carotenoid supplementation, o xidation of LDL is pre vented; thus, it is possible to prevent the development of atherosclerosis (Diaz, F rei et al. 1997). Direct addition of β-carotene, canthaxanthin, and zeaxanthin to LDL suspension suppressed LDL o xidation either b y using copper or b y acti vating macrophages in vitr o (Jialal, Norkus et al. 1991, Carpenter, van der Veen et al. 1997). This effect of carotenoids on increasing the resistance of LDL to o xidation has also been demonstrated in health y subjects supplemented with β-carotene or l ycopene and in c ystic f brosis patients who received a β-carotene supplement (Allard, Royall et al. 1994, WinklhofferRoob, Puhl et al. 1995, Agarwal and Rao 1998, Fuhrman, Volkova et al. 2000). Early studies have also sho wn that supplementation of diabetic patients with a combination of β - carotene, vitamin C, and vitamin E for eight w eeks decreased their susceptibility of LDL to copper – induced oxidation in vitro (Anderson, Gowri et al. 1999), and the evidence suggested that this antioxidant vitamin supplementation w as benef cial in reducing the risk of CVD . Ho wever, other studies have shown no ef fect of β-carotene supplementation on increasing resistance of LDL oxidation in vitro (Princen, van Poppel et al. 1992). POL YPHENOLICS AND LDL OXIDATION Consumption of pol yphenolics present in fr uits, vegetables, and drinks ha ve also been tested on the susceptibility of LDL to o xidation. Short–term supplementation with garlic in humans increased resistance of LDL to copper-induce oxidation, presumably due to the presence of the polyphenolic antioxidants in garlic (Lau 2001). Consumption of 400 mL of red wine for tw o weeks substantially increased the lag time of copper -induced LDL o xidation. However, consumption of w hite wine increased susceptibility of LDL to o xidation (Fuhr man, Lavy et al. 1995). The effect of red wine on reducing the LDL susceptibility to o xidation was reported to be due to an increase in LDL -associated polyphenolic compounds with antioxidant properties, which are present in red wine (F rankel, Kanner et al. 1993, Fuhr man, La vy et al. 1995, Nigdikar, Williams et al. 1998, Ivanov, Carr et al. 2001). The antio xidant acti vity of se veral natural, pol yphenolic antio xidants has been tested b y adding compounds directly onto the LDL preparation and by monitoring the conjugated dienes formation after initiating oxidation. The addition of several phenolic antioxidant fractions from different berry juices to LDL particles in vitro has also shown decreased copper-induced oxidation of LDL, in par ticular Chilean ber ry repor ted to be more protecti ve than stra wberry and blackberry juice (Miranda -Rottmann, Aspillaga et al. 2002). Phenolic compounds e xtracted

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from r ye and oats ha ve been sho wn to retard copper -induced LDL o xidation (Andreasen, Landbo et al. 2001, Chen, Milbur y et al. 2004). PUF A AND LDL OXIDATION Urcini group (Bonanome, Pagnan et al. 1992) reported that healthy volunteers who consumed a diet rich in monounsaturated f atty acids increased their resistance to LDL in vitro oxidation initiated with AAPH. In contrast, the y repor ted that consumption of a diet rich in PUF A increased the rate of LDL oxidation. They demonstrated that the peroxidation rate of LDL was inversely cor related with the oleic acid to linoleic acid ratio in LDL par ticles. It appears that as the level of saturation of f atty acid increases in LDL par ticles, the rate of in vitro oxidation of LDL ma y decrease, and the presence of PUF A may accelerate o xidation of LDL in vitro and probably in vivo . Nestel and colleagues (Nestel, Pomeroy et al. 1997) reported that daily intake of α - linolenic acid by obese volunteers improved the systemic ar terial circulation, but increased the susceptibility of LDL to o xidation. The authors have suggested that a decrease in LDL resistance to oxidation with n-3 PUFA consumption might have been due to higher utilization of antioxidant in vivo . However, the in vitro o xidizability of n -3 PUFA from plant origins or from f sh oil does not necessarily translate into the adverse effect of n -3 PUFAs on arterial function or their atherogenicity (Whitman, F ish et al. 1994, Nestel, P omeroy et al. 1997). However, reduction in total f at intake appears to be ef fective in reducing susceptibility of LDL in vitro oxidation. Patients under going an atherosclerosis treatment pro gram for three months, w hich included exercise, stress management, and f at intak e reduction b y 10%, had increased vitamin E and β-carotene levels in LDL particles, and thus had reduced susceptibility to copper-induce oxidation (Nestel, Pomeroy et al. 1997; Parks, German et al. 1998).

THEASSOCIATION OF ox LDLAND IN VITRO LDL OXIDATION WITH HEALTH STATUS It has been estab lished that the higher the LDL cholesterol in the b loodstream, the higher the levels of o xLDL in circulation (K opprasch, Pietzsch et al. 2002). The in vivo level of o xLDL has been e xamined in relation to brachial ar tery response to f ow - mediateddilation (FMD). Van der Zw an et al. (2009) have recently repor ted that the ratio of o xLDL per LDL par ticle correlated with FMD response in that the higher ratio of o xLDL/LDL, the lo wer change in FMD levels. The enhanced susceptibility to o xidation may underlie the excess vascular disease observed in patients with diabetes. Diabetic patients have higher concentrations of sdLDL, which is more susceptible to oxidation, thus the lag phase of LDL o xidation is shorter in these patients compared to non -diabetic subjects (Anderson, Go wri et al. 1999, Tan, Ai et al. 1999). Recently, it was repor ted that an increase in the propor tion of sdLDL in circulation is associated with a greater accumulation of o xLDL in the aor tic valve, and thus the pro gression rate of stenosis was faster (Mohty, Pibarot et al. 2008). While cigarette smoking increases o xidative stress and ma y reduce vitamin C le vel in plasma, it w as reported that vitamin E le vels in LDL w ere not af fected by cigarette smoking (Princen, van Poppel et al. 1992). Thus, smoking appears not to alter in vitro LDL o xidation (Princen, van Poppel et al. 1992). However, smoking ma y increase the in vivo level of autoantibody titer of MD A-modif ed LDL as measured (Kw on, Kwon et al. 2002). Despite the cor relation of antio xidant levels in LDL with increased resistance of LDL to oxidation, the resistance of LDL to o xidation does not always translate into a benef cial effect of LDL enrichment with antioxidants. Hodis et al. (2002) reported that supplementing normolipidemic subjects with 400 IU of vitamin E for three y ears increased the plasma vitamin E level and lag time of in vitro copper -induced LDL o xidation, but vitamin E supplementation

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had no ef fect on the pro gression of carotid ar tery intima -media thickness (IMT). While early studies have shown that combined supplementation of β-carotene, vitamin C, and vitamin E decreased susceptibility of LDL to copper –induced oxidation in vitro (Anderson, Gowri et al. 1999), recent clinical trials using the combination of these antioxidants did not show any benef t on CVD (Cook, Albert et al. 2007).

CONCLUSION The concept of in vitr o LDL o xidation has emer ged based on the o xidative h ypothesis of atherosclerosis, where LDL o xidation and o xidative stress ha ve been identif ed as the major contributing f actors in the de velopment of the disease. Accordingly, o xidizability of LDL particles in vivo and in vitro was suggested to be a good biomark er to assess the risk of CVD . The o xidizability of LDL par ticles not onl y depends on the presence of multiple lipoph ylic antioxidants in the par ticle, but also on the antio xidant network within the par ticle and on the network of antioxidants present in the hydrophilic environment interfacing with LDL particles. The presence of oxLDL or antibodies against oxLDL in circulation establishes some value for the status of oxidative stress in vivo. The measurement of in vitro LDL susceptibility to oxidation only represents the antio xidant capacity of lipid -soluble antioxidants present in the LDL particle; however, it does not tak e into account the interaction that ma y occur between hydrophilic antioxidants present in plasma and in tissues. Ho wever, the association of dietar y antioxidants with LDL susceptibility to oxidation does not strongly correlate with disease or health status, and the total e vidence does not suppor t in vitro LDL o xidation as a reliab le marker of oxidative stress or a sur rogate marker of CVD.

A CKNOWLEDGEMENT This chapter is based on the w ork suppor ted b y the U .S. Depar tment of Agriculture, under agreement No. 58 - 1950 - 7 - 707. Any opinions, f ndings, conclusions, or recommendations expressed in this chapter are those of the author(s) and do not necessaril y ref ect the vie w of the U.S. Depar tment of Agriculture. We w ould also lik e to thank Stephanie Marco for her assistance in the preparation of this chapter .

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NestelPJ , P omeroy SE et , al. 1997 .Arterial compliance in obese subjects is improved with dietary plant n -3 fatty acid from f axseed oil despite increased LDL o xidizability. Arterioscler Thromb Vasc Biol 17 ( 6 ):1163 – 70 . NigdikarSV , W illiams NR et , al. 1998 . Consumption of red wine polyphenols reduces the susceptibility of low-density lipoproteins to o xidation in vivo .Am J Clin Nutr 68 ( 2 ):258 – 65 . NikiE. 1990 . Free radical initiators as source of water - or lipid - solub le peroxyl radicals . Methods Enzymol 186 : 100 – 8 . Okimoto Y, W arabi E et , al. 2003 .A novel method of following oxidation of low - density lipoprotein using a sensiti ve f uorescent probe, diphenyl - 1 -yrenylphosphine p . Free Radic Biol Med 35 ( 6 ):576 – 85 . alinski P W , Horkk o S et , al. 1996 . Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E -def cient mice. Demonstration of epitopes of o xidized low density lipoprotein in human plasma . J Clin Invest 98 ( 3 ):800 – 14 . arks P EJ , German JB ,et al. 1998 . Reduced oxidative susceptibility of LDL from patients participating in an intensi ve atherosclerosis treatment pro gram. Am J Clin Nutr 68 ( 4 ):778 – 85 . arums P DV , Bro wn DL et , al. 1990 . Serum antibodies to oxidized low - densitylipoprotein and ceroid in chronic periaor titis. Arch Pathol Lab Med 114 ( 4 ):383 – 7 . PrincenHM ,avn Poppel G et , al. 1992 . Supplementation with vitamin E but not beta - carotene in vivo protects low density lipoprotein from lipid pero xidation in vitro. Effect of cigarette smoking . Arteriosclerosis and Thrombosis 12 : 554 – 62 . QuinnMT , P arthasarathy S et , al. 1987 . Oxidatively modif ed low density lipoproteins: A potential role in recr uitment and retention of monoc yte/macrophages during atherogenesis. Proc Natl Acad Sci USA 84 : 2995 – 2998 . Rainw ater DL , Mahane y MC et , al. 2007 .Vitamin E dietary supplementation signif cantly affects multiple risk f actors for cardiovascular disease in baboons . Am J Clin Nutr 86 ( 3 ): 597 – 603 . Re gnstrom J , Nilsson J ,et al. 1992 . Susceptibility to low - densitylipoprotein oxidation and coronary atherosclerosis in man . Lancet 339 : 1183 – 6 . Re y FE , P agano PJ . 2002 .The reactive adventitia: f broblast oxidase in vascular function . Arterioscler Thromb Vasc Biol 22 ( 12 ):1962 – 71 . RossR , Glomset JA . 1973 .Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a k ey event in the genesis of the lesions of atherosclerosis . Science 180 ( 93 ): 1332 – 9 . SalonenJT , Salonen R et , al. 1985 . Serum fatty acids, apoliproteins, selenium and vitamin antioxidants and risk of death from coronar y artery disease . Am J Cardiol 56 : 226 – 31 . venkova Sa ML , Mueller DM et , al. 1994 .Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid pero xidation in low density lipoprotein . J Biol Chem 269 ( 32 ):20394 – 400 . ScacciniC , Jialal I .1994 . LDL modif cation by activated polymorphonuclear leukocytes: a cellular model of mild o xidative stress . Free Radic Biol Med 16 : 49 – 55 . SchnitzerE , Pinchuk I et , al. 1997 .The effect of albumin on copper - inducedLDL oxidation . Biochim Biophys Acta 1344 ( 3 ):300 – 11 . SinghU , De varaj S et , al. 2005 .Vitamin E, oxidative stress, and inf ammation. Annu Rev Nutr 25 : 151 – 74 . Steinber g D, P arthasarathy S et , al. 1989 . Beyond cholesterol: modif cations of low density lipoprotein that increase its athero genicity. New Engl J Med 320 : 915 – 24 . SteinbrecherUP , W itztum JL et , al. 1987 . Decrease in reactive amino groups during oxidation or endothelial cell modif cation of LDL. Cor relation with changes in receptor -mediated catabolism. Arteriosclerosis 7 ( 2 ):135 – 43 . Stik o - RahmA , Hultgardh - Nilsson A et , al. 1992 . Native and oxidized LDL enhances production of PDGF AA and the surf ace expression of PDGF receptors in cultured human smooth muscle cells. Arterioscler Thromb 12 ( 9 ):1099 – 109 .

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Stock er R , Bo wry VW ,et al. 1991 . Ubiquinol - 10protects human low density lipoprotein more eff ciently against lipid pero xidation than does alpha -tocopherol. Proc Natl Acad Sci USA 88 ( 5 ):1646 – 50 . anT KC ,AiVH et , al. 1999 . Infuence of low density lipoprotein (LDL) subfraction prof le and LDL oxidation on endothelium -dependent and independent v asodilation in patients with type 2 diabetes . J Clin Endocrinol Meta b 84 ( 9 ):3212 – 6 . ThomasSR , Leichtw eis SB ,et al. 2001 . Dietary cosupplementation with vitamin E and coenzyme Q(10) inhibits atherosclerosis in apolipoprotein E gene knock out mice . Arterioscler Thromb Vasc Biol 21 ( 4 ):585 – 93 . Ushio - Fukai M ,T ang Y ,et al. 2002 . Novel role of gp91(phox) - containingNAD(P)H oxidase in vascular endothelial g rowth factor-induced signaling and angio genesis. Circ Res 91 ( 12 ): 1160 – 7 . anv der Zwan LP , T eerlink T ,et al. 2009 . Circulating oxidized LDL: determinants and association with brachial f ow - mediateddilation . J Lipid Res 50 ( 2 ):342 – 9 . isioli V F , Bordone R et , al. 2000 .The kinetics of copper - inducedLDL oxidation depend upon its lipid composition and antio xidant content . Biochem Biophys Res Commun 268 ( 3 ):818 – 22 . einbrenner W T , Cladellas M et , al. 2003 . High oxidative stress in patients with stable coronary heart disease . Atherosclerosis 168 ( 1 ):99 – 106 . WhitmanSC , F ish JR et , al. 1994 . n - 3Fatty acid incorporation into LDL particles renders them more susceptible to oxidation in vitro but not necessaril y more atherogenic in vivo . Artherioscler Thromb 14 : 1170 – 6 . iW lliams KJ , T abas I .1998 .The response - to - retention hypothesis of atherogenesis reinforced . Curr Opin Lipidol 9 ( 5 ):471 – 4 . iW nklhoffer - Roob BM , Puhl H et , al. 1995 . Enhanced resistance to oxidation of low density lipoproteins and decreased lipid pero xide formation during b -carotene supplementation in c ystic f brosis. Free Radic Biol Med 18 : 849 – 59 . Yla - Her ttuala S , P alinski W ,et al. 1989 . Evidence for the presence of oxidatively modif ed LDL in human atherosclerotic lesions . Ciruculation 80 : II - 160 . Zuw ala - Jagiello J. 2006 . Haemoglobin scavenger receptor: function in relation to disease . Acta Biochim Pol 53 ( 2 ):257 – 68 .

Chapter5 The Isoprostanes: Accurate Markers and Potent Mediators of Oxidant Injury in Vivo oJshua D. Br ooks , BrianE. Co x , KlarissaD. Har dy , Stephanie C. Sanc hez , SoniaoTurino ,yler T H. K oestner ,ocelyn J R. Hyman - Ho ward and , Ging er L. Milne

INTR ODUCTION Free radicals derived from molecular oxygen have been implicated in a variety of human conditions and diseases including atherosclerosis, aging, cancer , and neurode generation, and these highly reactive free radicals are postulated to be major contributors to human pathophysiologies given their ability to damage impor tant cellular biomolecules such as DN A, RNA, proteins, and lipids (Halliw ell and Gutteridge 1990, Kadiiska et al. 2005). Measuring o xidative stress and the associated damage in humans requires an accurate quantif cation of either the free radicals or the oxidant-injured molecules, and a number of methodologies exist for this purpose. Many of these techniques, however, lack sensitivity and specif city when used to assess oxidant stress in vivo . In recent y ears, the Biomark ers of Oxidati ve Stress Study (BOSS), a multi investigator study sponsored b y the National Institutes of En vironmental Health Sciences (NIEHS), found that the most accurate method to assess o xidative stress status in vivo is the quantif cation of urinar y or plasma isoprostanes (Kadiiska et al. 2005). Isoprostanes, or IsoPs, are a series of prostaglandin-like isomers formed from the free-radical catalyzed o xidation of the pol yunsaturated f atty acid arachidonic acid. Ov er the last tw enty years, studies have def ned the basic chemistry and biochemistry involved in the formation and metabolism of the IsoPs. Further, a number of IsoPs have been found to possess potent biological activity and, thus, are also lik ely mediators of o xidant injury (Fam and Mor row 2003). In more recent y ears, additional related compounds deri ved from v arious polyunsaturated f atty acids, including the ome ga-3 f atty acid eicosapentaenoic acid , have been disco vered to for m as products of the IsoP pathw ay. It is the pur pose herein to summarize cur rent kno wledge regarding these impor tant products of lipid pero xidation.

THE FORMATION OF ISOPROSTANES The f rst class of IsoPs, called the F 2-IsoPs, w as disco vered in 1990 b y Drs. Jack Rober ts and Jason Mor row and were so named because the y, analogous to prostaglandin F 2α (PGF2α ), Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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Figure 5.1. Mechanism of Formation of F2-Isoprostanes. IsoPs are formed following arachidonic acid oxidation via a six -step mechanism. The four potential regioisomers are depicted, each of which has sixteen stereoisomers (not shown).

contained an F-type prostane ring and two carbon-carbon double bonds (Morrow et al. 1990b). A mechanism to e xplain the for mation of F 2-IsoPs from arachidonic acid is presented in Figure 5.1 . This mechanism was based on the work proposed by Pryor et al. for the generation of bic ycloendoperoxide intermediates (1976). As seen in the mechanism, free-radical induced abstraction of a h ydrogen atom from one of three bisall ylic carbons yields the for mation of a lipid free radical that then reacts with molecular o xygen to for m a pero xyl radical. This radical undergoes a series of 5 -exo c yclizations follo wed b y the addition of a second molecule of e molecules. Highly unstable, oxygen to the carbon backbone of the compound to form PGG2 - lik these bicycloendoperoxides are then reduced to F 2-IsoPs. Based on this mechanism of for mation, four F 2-IsoP regioisomers are generated , and these re gioisomers are named the 5 -, 12 -, 8 - ,or 15 - seriesF2-IsoPs, so denoted for the number of the carbon atom to w hich the hydroxyl side chain is added (Morrow et al. 1990b, Taber et al. 1997). Alternative nomenclature systems have been proposed as well in which the abbreviation iP is used for IsoP and the re gioisomers are denoted as III -VI based on the number of carbons betw een the omega carbon and the f rst double bond (Rokach et al. 1997b). Although the initial abstraction of any bisallylic hydrogen atom is equally likely, the different IsoP regioisomers are not for med in equal amounts. When arachidonic acid is o xidized either in vitro or in vivo , the 5 - and 15 -series regioisomers are for med in signif cantly greater abun-

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dance than the 8 - and 12 -series re gioisomers. One e xplanation for this dif ference has been recently elucidated in studies by the authors’ laboratory that demonstrated that the arachidonyl hydroperoxides that gi ve rise to the 8 - and 12 -series re gioisomers readil y under go fur ther oxidation to yield a no vel class of compounds that contains both bic ycloendoperoxide and cyclic pero xide moieties; these compounds w ere ter med dio xolane-IsoPs and ha ve been reported to for m in vivo (Yin et al. 2004). 5 - and 15 -series regioisomers cannot under go this further oxidation and can, therefore, accumulate at higher concentrations in tissues and f uids as they represent ter minal oxidation products of arachidonic acid. One important structural distinction between IsoPs and CO X-derived prostaglandins is that the former contain side chains that are predominantly oriented cis to the prostane ring whereas the latter possess e xclusively trans side chains (Mor row et al. 1990b). In this re gard, however, the authors’ lab has repor ted that prostaglandins can be for med via the IsoP pathw ay because smaller amounts of endopero xides containing trans side chains are generated in vitro and in vivo by free radical mechanisms (Gao et al. 2003, Yin et al. 2007). In this case, prostaglandins derived via the IsoP pathw ay can be distinguished from those for med by the c yclooxygenase enzymes because the free -radical pathw ay generates a racemic mixture of prostaglandins whereas the enzymatic pathway yields enantiomerically pure compounds. A second impor tant difference between IsoPs and prostaglandins is that w hile prostaglandins are generated onl y from free arachidonic acid , IsoPs are for med primarily in situ esterif ed to phospholipids and are subsequently released b y the enzyme platelet acti vating factor-acetylhydrolase (PAF-AH) after oxidation has occur red (Morrow et al. 1992a, Stafforini et al. 2006). Thus, while prostaglandins can only be quantif ed in the free for m, IsoPs can be quantif ed either as free acids or bound to phospholipid membranes.

FORMA TION OF ISOPROSTANES WITH ALTERNATIVE RING STRUCTURES Further analogous to prostaglandin for mation, different classes of IsoPs can for m depending on the cellular conditions in which they are generated; these classes usually differ in the structure of their prostane rings, and str uctures of characterized IsoPs can be seen in F igure 5.2. WhileF2-IsoPs are generated through reduction of the bicyclic endoperoxide ring, PGE2 -and e compounds (E2 /D2-IsoPs) are generated when the bicyclic endoperoxide intermediPGD2 - lik ate undergoes a rear rangement. Like their prostaglandin analo gues, E 2 /D2 - IsoPscan undergo subsequent spontaneous deh ydration to yield A2 /J2-IsoPs or, collectively, the c yclopentenone IsoPs (Chen et al. 1999a, Chen et al. 1999b, Mor row et al. 1998). IsoPs resemb ling thromoboxanes A 2 and B 2 have also been described in the literature (Mor row et al. 1996). The ring structure of a class of IsoPs is independent of the regio- and stereochemistry of the sidechains, and, as a result, the full complement of re gio- and stereoisomers e xist for each class of IsoP.

Q UANTIFICATION OF F2 - ISOPR OSTANES Over the past decade, quantif cation of F 2-IsoPs has been sho wn to be the most reliab le biomarker of o xidative injury in vitro and in vivo . F2-IsoPs, because of their stability , afford the most accurate measure of o xidant stress (Kadiiska et al. 2005, Milne et al. 2005b). Levels in tissue and f uids can be accurately measured using a gas chromatographic/negative ion chemical ionization mass spectrometric (GC/NICI -MS) methodology by employing stable isotope dilution. F2-IsoPs are stable, robust molecules that are detectable in all human tissues and biological f uids, including plasma, urine, bronchoalv eolar lavage f uid, and cerebrospinal f uid (Morrow et al. 1999). The quantif cation of F 2-IsoPs in urine and plasma, ho wever, provides the most convenient and least intr usive way to measure system o xidative stress. For quantif cation pur, and other poses, we measure an abundantl y forming regioisomer of the F 2 - IsoPs,15 - F 2t - IsoP F2-IsoPs that co -elute with this compound. This method is e xtremely sensiti ve with a lo wer

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Figure 5.2. Isoprostanes of different ring types formed by non -enzymatic oxidation of arachidonic acid. Multiple regioisomers form for each class of IsoP shown above, but only one regioisomer is depicted for the sake of simplicity.

limit of detection in the range of appro ximately 1 to 5 pico grams using a deuterated inter nal standard. The MS methodolo gy is also e xtremely specif c. Its dra wbacks are that it is labor intensive and requires considerab le expenditures on equipment. It should be noted that se veral alternative GC/MS assa ys have been de veloped by different investigators (Pratico et al. 1998, Rokach et al. 1997a, 1997b ). In addition to these GC/MS assays, a number of liquid chromato graphic-MS (LC/MS) methods for F 2 - IsoPshave been developed. LC/MS methodologies allows for sample preparation that is simpler than with the GC/MS because no derivatizations are required, and for monitoring of all F2 - IsoPregioisomers at once. Despite major adv ancements in LC/MS technolo gy, however, these assa ys are often much less sensiti ve than comparab le GC/MS methods. Fur thermore, of the man y LC/MS methodologies pub lished, to date onl y three ha ve been v alidated for quantif cation of these molecules in biolo gical f uids (Haschk e et al. 2007, Sircar and Subbaiah 2007, Taylor et al. 2006 ). Immunological approaches ha ve also been de veloped to quantify IsoPs. Antibodies ha ve been generated against 15-F2t-IsoP, and at least three immunoassay kits are commercially available. While mass spectrometric methods are considered the best methods for anal ysis, immunoassays have expanded research in this area because of their low cost and relative ease of use. Only limited information, however, is currently available regarding the precision and accuracy of these assays. In addition, little data exist comparing IsoP levels determined by immunoassays to MS. While Wang et al. of fered one e xample of an MS -validated immunoassay to measure

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urinary F 2-IsoPs (Wang et al. 1995), the authors’ own experience has not v alidated the commercially available kits. These kits, as w ell as their prostaglandin immunoassa y analogs, also suffer from a lack of specif city, and the sensiti vity of these kits v aries substantially among manufacturers and among users. STORAGE AND HANDLING OF BIOLOGICAL FLUIDS AND TISSUES FOR OSTANE QUANTIFICATION F2 - ISOPR When monitoring oxidative stress from in vivo samples, another drawback for any methodology measuring F2-IsoPs as an index of endogenous lipid peroxidation is that F2 - IsoPscan be generated ex vivo in biolo gical f uids (e.g., plasma) in w hich arachidono yl-containing lipids, the precursors to F2-IsoPs, are present. This occurs not only when biological f uids are left standing at room temperature but also during storage of lipid -containing biological f uids and tissues at -20°C. Studies have found, however, that the for mation of IsoPs does not occur during storage of biological f uids at −70°C for up to six months. Further, formation of IsoPs ex vivo does not occur if biolo gical f uids are processed immediatel y after procurement and if the free radical scavenger, butylated hydroxytoluene (BHT), is added to the or ganic solvents during extraction and hydrolysis of tissue phospholipids (Mor row et al. 1992a, Morrow et al. 1990a). Therefore, samples obtained for anal ysis should either be processed immediatel y or stored at − 70 ° C. Ideally, samples should be rapidly frozen in liquid nitrogen prior to placing them in the freezer. This is especially important when storing tissue samples for analysis of esterif ed IsoPs, because tissue samples simply placed at −70°C that were not snap frozen beforehand may contain inner areas that remain in a liquid state for a period of time. OSTANE - CONT AINING EXTRACTION AND HYDROLYSIS OF F 2 - ISOPR PHOSPHOLIPIDS IN TISSUES A summary of our methodology used for the quantif cation of 15-F2t-IsoP is provided in Figure 5.3. To 0.25 to 0.5 g of tissue is added 10 mL of ice -cold Folch solution (chlorfor m:methanol, 2:1, v/v) containing 0.005% BHT . The tissue is then homo genized with a b lade homogenizer (PTA 10S generator , Brinkman Instr uments, Westbury, NY) for one minute and the mixture sealed under nitro gen for 30 minutes with vigorous v ortexing every 10 minutes. 2 mL of salt solution (0.9% NaCl in H 2O) is added after the 30 minutes, and the solution is v ortexed and centrifuged at 800 g for 10 minutes at room temperature, after w hich the upper aqueous la yer is discarded and the lower organic layer is carefully separated from the intermediate semi-solid layer. Acidify biological fluid or hydrolyzed tissue extract to pH 3 ↓ Add deuterated internal standard {[2H4]-15-F2t-IsoP} ↓ C-18 and silica Sep-Pak extraction ↓ Derivatization to PFB esters ↓ TLC of F2-IsoPs as PFB esters ↓ Derivatization to trimethylsilyl ether derivatives ↓ Analysis by GC/NICI-MS

Figure 5.3. Outline of the procedure used for the extraction, purif cation, derivatization, and mass spectrometric analysis of F 2-Isoprostanes from biological sources.

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The or ganic phase contains the e xtracted lipids and is e vaporated under nitro gen gas to dryness. 0.5 mL of methanol containing BHT (0.005%) and 0.5 mL of aqueous KOH (15%) is added to the residue and incubated at 37 °C for 30 minutes to allo w for hydrolysis and release of F 2-IsoPs from the phospholipid membranes. After h ydrolysis, the mixture is acidif ed to pH ≤ 3 using 1 M HCl and diluted to a f nal volume of 12 mL using pH 3 H 2 O. The methanol in the solution must be diluted to ≤5% to ensure proper column e xtraction of F 2 - IsoPsin the subsequent purif cation phase. This acidif cation is necessary as well to ensure that the F2 - IsoPs are found in anionic for m to allo w for better adherence of the compounds to the solid phase extraction columns used for purif cation. Measuring F2 - IsoPsin biological f uids such as plasma and urine, as opposed to in tissues, utilizes a dif ferent methodology to this point. Biolo gical f uids are simpl y acidif ed to pH 3 using 1 M HCl and are ready for solid phase e xtraction after adding inter nal standard. Gi ven the extreme sensitivity of this assa y, it is not often necessar y to assay more than 1 to 3 mL of biological f uid for specif c and accurate results. PURIFICATION, DERIVATIZATION, AND QUANTIFICATION OF F2 - ISOPR OSTANES Following acidif cation of the sample, 1 ng of the deuterated internal standard [2 H4 ] - 15 2t- F- IsoP (8 - iso - PGF 2α, Cayman Chemical, Ann Arbor, MI) is added. After the addition of the inter nal ak column (Waters Associates, standard, the mixture is v ortexed and applied to a C 18 Sep - P Milford, MA) that has been preconditioned moments beforehand with 5 mL methanol followed by 7 mL pH 3 water. The sample and subsequent solvents are eluted through the Sep-Pak using a vacuum manifold system. The column is then washed sequentially with 10 mL of pH 3 water followed by 10 mL of heptane. The F 2-IsoPs are then eluted from the column using 10 mL of ethyl acetate:heptane (50:50, v/v). The ethyl actate/heptane eluate from the C 18 Sep -Pak is then dried brief y over anhydrous Na2 SO4 and added to a silica Sep -Pak column (W aters Associates) that has been pre washed with 5 mL of eth yl acetate. After the sample is loaded , the car tridge is w ashed with 5 mL of ethyl acetate follo wed by the elution of F 2-IsoPs with 5 mL of eth yl acetate:methanol (50:50, v/v). The ethyl acetate/methonal eluate is e vaporated under a stream of nitro gen. The F2-IsoPs are then con verted to the pentaf uorobenzyl (PFB) ester b y treatment with a mixture of 40 µL of 10% pentaf uorobenzyl bromide in acetonitrile and 20 µL of 10% N ,Ndiisopropylethylamine in acetonitrile at 37 °C for 20 minutes. These reagents are then dried under a stream of nitrogen. The remaining residue is re-eluted into 50µL of chloroform:methanol (3:2, v/v) and then subjected to thin la yer chromato graphy (TLC) using chlorofor m:ethanol (93:7, v/v) as the solvent. As a TLC standard, approximately 5 µg of the methyl ester of PGF 2α are chromatographed on a separate plate simultaneousl y. (The meth yl ester of PGF 2α , rather than the PFB ester, is used as the TLC standard to prevent sample contamination, because the methyl ester of PGF2α will not interfere with quantif cation since F2-IsoPs are analyzed by PFB esters.) After chromato graphy, the standard is visualized b y spra ying the plate with a 10% solution of phosphomol ybdic acid in ethanol follo wed b y heating. Compounds mig rating in the region of the methyl ester of PGF2α (Rf ∼ 0.15) and the adjacent areas 1cm above and below are scraped and e xtracted from the silica gel with eth yl acetate. The ethyl acetate is dried under a stream of nitro gen, and the F 2-IsoPs are then converted to the trimeth ylsilyl ether deri vative b y adding 20 µ L of N,O - bis - (trimeth ylsiyl) trif uoroacetamide (BSTFA) and 10 µL of dimeth ylformamide follo wed b y a f ve - minuteincubation at 37°C. The reagents are then dried under a stream of nitro gen, and the derivatized F 2 - IsoPsare dissolved in 10 µL of undecane (w hich has been dried o ver calcium h ydride) for anal ysis by GC/MS. The F2-IsoPs are chromatographed on an Agilent 5973 mass spectrometer with a computer interface using a 15 -m DB1701 fused silica capillar y column. This column is used because it

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Abundance Endogenous F2-IsoPs 2400 m/z 569 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Time → 5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55 Abundance 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Time →

* 5.26

[2H4]-15-F2t-IsoP m/z 573

+

5.00 5.05 5.10 5.15 5.20 5.25 5.30 5.35 5.40 5.45 5.50 5.55

Figure 5.4. Analysis of F2-Isoprostanes in plasma obtained from a rat 4 hours after treatment with CCl4 (2 mL/kg orogastrically) to induce endogenous lipid peroxidation. The m/z 573 ion current chromatogram represents the [ 2H4]-8-iso-PGF2α internal standard. The m/z 569 ion current chromatogram represents endogenous F 2-IsoPs. The peak in the upper chromatogram represented by the asterisk ( *) is the one routinely used for quantif cation of the F 2-IsoPs. The peak represented by the plus ( +) can be comprised of both F 2-IsoPs and cyclooxygenase -derived PGF2α. The concentration of F2-IsoPs in the plasma in this sample was calculated to be 83pg/mL.

gives e xcellent separation of indi vidual re gioisomers in comparison to others that are commercially available. The column temperature is pro grammed to increase from 190 °C to 300 °C at 20 °C per minute. Methane is used as the car rier gas for NICI. The ion source temperature is 200 °C. The ion monitored for endo genous F 2-IsoPs is the carbo xylate anion m/z 569 (loss of CH 2 C6 F5). The cor responding carbo xylate anion for the deuteriated inter nal standard is m/z 573. The sensiti vity of the mass spectrometer is check ed dail y b y injecting a standard consisting of 40 pg each of PGF 2α and [ 2 H4 ] - 15 2t--FIsoP. A sample chromato gram is pro vided in Figure 5.4 . METHODOLOGY TO QUANTIFY OTHER CLASSES OF ISOPR OSTANES While F2-IsoPs are cur rently the gold standard for measuring o xidative stress in vivo, assays also exist for the measurement of the other classes of IsoPs mentioned earlier , and commercially

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available internal standards are available for each. While the protocols are originall y based on the methodology for F 2-IsoPs, the dif ferent ring str uctures have required assa y modif cations for accurate measurements. Quantif cation of the D/E -ring and A/J-ring IsoPs utilizes only the C18 Sep -Pak e xtraction method but requires an e xtra deri vatization immediatel y after solid phase extraction. This derivatization requires the resuspension of dried eluant in 300 µL of 3% methoxyamine HCl solution (in p yridine) w hich is to be left at 37 °C for 30 minutes. The product is then dried under a stream of nitro gen and dissolv ed in pH 3 w ater and acidif ed to pH 3 before an ethyl acetate extraction. Subsequent TLC also uses a solvent of hexanes:acetone (70:30, v/v). The area scraped from the TLC also varies depending on the methyl ester standard used. These methodologies also monitor the presence of multiple re gioisomers rather than one single stereoisomer as in the F 2-IsoP method and monitor dif ferent m/z depending on the derivatized str ucture of the molecule and its deuterated inter nal standard (Chen et al. 1999a, Chen et al. 1999b, Morrow et al. 1998). A/J-ring IsoPs are also highl y reactive and are onl y measurable from tissue samples. They are h ydrolyzed from the phospholipid backbone using bee v enom phospholipase A2 (PLA2 ) instead of base h ydrolysis given the reacti ve nature of their α ,β - unsaturatedcarbonyl (Chen et al. 1999a, Chen et al. 1999b).

ISOPROSTANES AS AN INDEX OF OXIDATIVE STRESS IN VIVO A tr ue utility of the F 2-IsoPs is in the quantif cation of lipid pero xidation and, thus, o xidant stress in vivo (Fam and Mor row 2003, Milne et al. 2007). Measurements of the F 2 - IsoPshave revolutionized our ability to quantify o xidative injur y in vivo , and, in f act, nor mal levels of F2-IsoPs in health y humans ha ve been def ned (Fam and Mor row 2003, Milne et al. 2007). Def ning these levels allows for both the assessment of the ef fects of diseases on endo genous oxidant tone and the deter mination of the e xtent to w hich v arious therapeutic inter ventions affect levels of oxidative stress. Increased F2-IsoP levels have been detected in a v ariety of diseases in humans, including atherosclerosis, cancer , pulmonar y disease, rheumatoid ar thritis, Alzheimer’s disease, and diabetes, and these measurements ha ve helped implicate o xidative stress in these conditions, demonstrating a close link betw een oxidative stress and inf ammation (Morrow 2003, Musiek et al. 2005b). A THEROSCLEROSIS AND ASSOCIATED RISK FACTORS Atherosclerosis remains the most w ell-characterized disease state associated with o xidative stress, and studies have correlated this disease and its risk factors with an increase in the levels of IsoP for mation. Cigarette smoking, for instance, ma y lead to multiple patholo gies including acceleration of atherosclerosis and respirator y disease. Increased le vels of F 2-IsoPs are found in smok ers (Helmersson et al. 2002, Montuschi et al. 2000, Morrow et al. 1995), and, while former smokers ers, studies have shown are found to have higher endogenous levels of F 2 - IsoPsthan non - smok that cessation of smoking can reduce F 2-IsoP belo w the le vels of those of cur rent smok ers (Wohlin et al. 2007). Diabetic patients also exhibit higher levels of systemic oxidative stress. Compared to healthy populations, patients suf fering from Type II diabetes ha ve been found to ha ve elevated levels of 15 - F 2t-IsoP in both ser um and plasma (Da vi et al. 2003, Gopaul et al. 2000, Murai et al. 2000). In f act, in a lar ge cross -sectional study in elderl y men, it w as shown that the 24 -hour urinary level of F 2 - IsoPswas signif cantly higher in men with Type II diabetes than in age matched controls (Helmersson et al. 2004).

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Hypertension also puts strain on the o xidant tone of the human body. A serious clinical risk factor for the de velopment of atherosclerosis, h ypertensive patients ha ve been found to ha ve higher F 2-IsoP levels than those of nor mal patients (Cracowski et al. 2001, Lopes et al. 2003, Minuz et al. 2002). Treatment of hypertension appeared to modulate o xidative stress; a recent study repor ted that both plasma and urinar y F 2 - IsoPswere signif cantly lo wered in treated versus untreated hypertensive men, but not in w omen. Urinary F 2-IsoPs were also found to be more than two times higher in h ypertensive patients than in controls (Craco wski et al. 2002). Obesity has been associated with o xidative stress as w ell. At least three recent repor ts have provided evidence that ele vated systemic o xidant stress ma y be an impor tant mechanism b y which obesity increases the incidence of atherosclerotic cardio vascular disease (Block et al. 2002, Davi et al. 2002, Keaney et al. 2003). In a study by Keaney et al. (2003), it was reported that an association e xists between increasing body mass inde x and increasing systemic o xidative stress. Using the quantif cation of urinary F2-IsoPs, the authors demonstrated that in nearly 3,000 patients, enhanced IsoP for mation in men and w omen is strongl y associated with an increased body mass inde x. A more recent repor t with adolescents demonstrated that urinar y are signif cantly related to body mass index and found a signif cant interaction between F2 - IsoPs BMI and insulin resistance. In insulin-resistant teens, F2-IsoPs were found to be at much higher levels (Sinaiko et al. 2005). Unpublished studies perfor med by Rober ts et al., ho wever, have demonstrated that obese patients can see a signif cant decrease in endogenous F2 - IsoPproduction through a simple decrease in calorie intak e. These and other studies, tak en together, have collectively indicated that obesity is closel y related to o xidative stress. Many ha ve also sho wn that F 2 - IsoPlevels are signif cantly increased in atherosclerotic plaques compared to nor mal vascular tissue, suggesting that these compounds ma y play a role in the patho genesis of this disease (Gniw otta et al. 1997, Pratico et al. 1997). Systemic IsoP levels increase signif cantly after angioplasty , suggesting that local for mation of these compounds contributes to total body production in this population (Reill y et al. 1997). In addition, it has been suggested that if atherosclerotic lesions are a source of circulating IsoPs, levels may be increased in humans with an e xtensive burden of the disease compared with non -diseased individuals and could pro ve to be a useful atherosclerotic biomark er, although lar ger studies will be needed to conf rm this possible utility.

ISOPR OSTANES AS A MEDIATOR OF OXIDATIVE - STRESS RELATED DISEASES While the use of IsoPs as biomark ers of o xidative stress has been lar gely discussed in the literature, more recent studies ha ve begun to focus on IsoPs as a possib le active mediator of the biological consequences associated with oxidative stress. Studies def ning the exact mechanisms of IsoP bioacti vity, however, are still in their inf ancy. OSTANES PR O - INFLAMMA TORY ACTIVITIES OF F2 -AND E2 - ISOPR Two IsoPs that ha ve been e xtensively tested for biolo gical activities are an abundant F 2 - IsoP (i.e. 8 -isoregioisomer, 15 - F2t-IsoP, and an abundant re gioisomer of E 2 - IsoPs,the 15 - E 2t - IsoP PGE2) (Mor row et al. 1994a, Mor row et al. 1994b, Mor row et al. 1998). These compounds possess potent biolo gic ef fects and are belie ved to be mediators of o xidative-stress related pathophysiologies. Both of these IsoPs ha ve been sho wn to be potent v asoconstrictors in a variety of vascular beds, including the kidne y, lung, hear t, retina, por tal vein, brain, and l ymphatic system (Banerjee et al. 1992, Fukunaga et al. 1993, Kang et al. 1993, Lahaie et al. 1998, Sinzinger et al. 1997, Takahashi et al. 1992). The vasoconstrictive effects of 15-F2t-IsoP led to the f nding that this compound is an agonist of the TxA2 receptor (Banerjee et al. 1992, Mor row et al. 1992b, Takahashi et al. 1992). The

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vasocontraction response to 15-F2t-IsoP has also been reported to be dependent on extracellular Ca2+ from both L -and T - typeCa2+ channels, and, perhaps, also on protein kinase C (Fukunaga et al. 1993, Raal et al. 1999). It is also still debated if the ef fects of 15 -F2t - IsoPare mediated only b y the thrombo xane receptor or b y a no vel “IsoP” receptor (Fukunaga et al. 1993, Fukunaga et al. 1997, Longmire et al. 1994). Re views b y Mor row et al., ho wever, ha ve demonstrated that after for mation, IsoPs still esterif ed to phospholipids are not as acti ve as the free for m. With the e xistence of h ydrolyzing enzymes in di verse tissue, the esterif ed IsoPs are rapidly metabolized to their bioactive free form. When such compounds are available as free acids at high concentrations, which usually occurs after the pathophysiologic conditions mentioned earlier, they are f rst thought to af fect the inte grity and f uidity of the membrane and, consequentl y, of adjacent tissues, causing a state of in vivo o xidative strain before subsequently modulating receptor -mediate signaling cascades (Mor row 2006). It was recently reported, however, that administration of 15 -F2t - IsoPin rabbits induced COX - mediatedPGF2α formation (Basu 2006), and thus, induce production of TxA2. The ability of 15 -F2t - IsoPto induce production of these molecules has been h ypothesized to also be responsib le for some of the vasoconstrictive effects associated with the F 2 - IsoPsand oxidative stress. Another , the 15 - F F2 - IsoP 2c - IsoP(12 -iso - PGF 2α), has been sho wn to acti vate the PGF 2α receptor and induce hypertrophy in cardiac smooth muscle cells (K unapuli et al. 1997, Kunapuli et al. 1998 ). Recent evidence suggests that the constrictor effect of 15 -E2t - IsoPin pulmonary vasculature is mediated via the PGE 2 (EP3) receptor (Janssen and Tazzeo 2002). There is also e vidence that 15 - E 2t-IsoP possesses additional biolo gical activities, including induction of osteoclastic differentiation leading to bone resor ption, inhibition of platelet agg regation in human w hole blood, and enhancement of neurotransmission in the choliner gic nerves through acti vation of the PGF 2α (FP) receptor (Cranshaw et al. 2001, Csiszar et al. 2002, Paredes et al. 2007, Tintut et al. 2002 ). BIOLOGICALACTIVITIES OF THE CYCLOPENTENONE (A2 /J2 ) - ISOPR OSTANES Recent studies have also suggested that another class of IsoPs, the cyclopentenone IsoPs, exert potent biolo gical acti vities as w ell. The f rst total synthesis of an arachidonic acid -derived cyclopentenone IsoP w as completed in 2002 and led to the study of the bioacti vity of these molecules. Gi ven their α ,β-unsaturated c yclopentenone ring str ucture, these molecules are highly reactive electrophiles that readil y form Michael adducts with cellular thiols, including glutathione and c ysteine residues, much lik e their prostaglandin analo gs (Chen et al. 1999a, Milne et al. 2004, Musiek et al. 2005a). Studies have shown that in macrophages, the arachidonic acid - deri ved 15 - A2 - IsoPspotently suppress LPS - inducedinf ammatory signaling via inhibition of the NF κB pathw ay (Musiek et al. 2005a). As a result of this inhibition, these compounds have also been sho wn to reduce e xpression of both inducib le nitrogen oxide synthase (iNOS) and c yclooxygenase-2 (COX-2) in response to LPS as w ell as inhibit release of several pro - infammatory cytokines (Milne et al. 2005a, Musiek et al. 2005a). 15-J2 - IsoPsalso induce similar anti -inf ammatory ef fects, but, unlik e 15 -A2-IsoPs, are also ab le to acti vate PPARγ with an EC 50 in the lo w nanomolar range. This receptor modulates a wide v ariety of processes, and 15 - 2J-IsoPs are postulated to acti vate these processes including inf ammatory signaling and f atty acid metabolism (Musiek et al. 2005a). Theanti - infammatory effects of the A2 /J2-IsoPs were also shown to be balanced by cytotoxic effects. The cyclopentenone IsoPs were shown to increase the le vels of oxidative stress generated in macrophages, and J 2 - IsoPs,while not A2-IsoPs, w ere sho wn to induce apoptosis in macrophages (Musiek et al. 2006). Further studies also demonstrated that the apoptotic effects of A2-IsoPs in neurons, indicating that the ef fects of these compounds, like their prostaglandin analogs, are cell type and concentration dependent (Musiek et al. 2006).

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FACTORS REGULATING OXIDATIVE STRESS AND ISOPROSTANE FORMATION Given that the IsoPs are not onl y biomark ers but also mediators of o xidative-stress related diseases, multiple in vestigators ha ve focused on possib le therapeutic inter ventions that can decrease endogenous production of IsoPs. In fact, medical treatments of the diseases associated with oxidative stress have had some success in inhibiting IsoP for mation. Antioxidant supplementation, anti -diabetic treatments, cessation of smoking, w eight loss, and e ven a decrease in daily caloric intake have been shown to decrease endogenous IsoP formation (Davi et al. 1999, Roberts et al. 2007). Antioxidant therapy in par ticular has been studied in detail in humans, with vitamin E ( α-tocopherol) as the most widespread antio xidant studied. Studies b y Levine et al. and Rober ts et al. demonstrated , however, that that dail y doses of 1,600 IU or g reater were required to statisticall y affect plasma le vels of F 2-IsoPs (Levine et al. 2001, Rober ts et al. 2007). Recent studies have also demonstrated that daily intake of 200 to 2,000 IU a day for eight w eeks did not af fect concentration of F 2-IsoPs (Meagher et al. 2001). Vitamin E w as shown to not af fect F 2-IsoP levels in moderate smok ers either (Smedman et al. 2004). Other vitamin antio xidants such as vitamin C ha ve also been sho wn to ha ve no ef fect in humans (Levine et al. 2001). OMEGA-3 FATTY ACIDS AND FISH OIL AS A THERAPEUTIC AGENT IN OXIDATIVE STRESS -RELATED CONDITIONS AND DISEASES Evidence from both epidemiological studies and clinical trials has shown that increased intake of f sh oil, specif cally the ome ga-3 fatty acids, ha ve benef cial effects on diseases associated with oxidative stress as w ell, especially in patients suf fering from cardiovascular diseases. As early as 1980, studies suggested that the low mortality rates from coronary heart diseases among Greenland Eskimos compared with Danes ma y be due to the Eskimos’ high consumption of seafood (Bang et al. 1980). Since that time a number of cohor t studies ha ve been pub lished that demonstrate a cardioprotective effect from f sh consumption (Alber t et al. 1998, Daviglus et al. 1997a, Daviglus et al. 1997b, Gillum et al. 2000, Hu et al. 2002, Kromhout et al. 1985, Kromhout et al. 1995, Lemaitre et al. 2003). These epidemiolo gical studies ha ve pro vided evidence that f sh consumption f avorably affects coronary heart disease mor tality. In suppor t of these studies, the h ypothesis that consumption of ome ga-3 pol yunsaturated fatty acids found in marine f sh oils act to protect indi viduals from cardiovascular disease has been tested in a number of randomized control inter ventional trials. One of the larger prospective studies to date w as the Gr uppo Italian per lo Studio della Sopra vvivenzia nell -Infarto (GISSI)-Prevention study. In this study , 11,324 patients with pre -existing hear t disease w ere randomized to 300 mg/d vitamin E, 850 mg/d EPA + DHA, both, or neither. After 3.5 years of follow-up, the g roup given the ome ga-3 PUFAs alone demonstrated a 15% reduction in the primary endpoints of death, non -fatal myocardial infarction, and non -fatal stroke. This study also found that there was a 20% reduction in all causes of mortality as well as a 45% reduction in sudden cardiac death compared to the control diet g roup. Interestingl y, in these studies vitamin E supplementation w as found to pro vide no additional benef t (GISSI 1999). Studies have also demonstrated that a major ome ga-3 fatty acid found in f sh oil, eicosapentaenoic acid (EP A, C20:5), alone is helpful in treating cardio vascular disease patients. A large-scale study to determine the effects of the EPA, entitled the Japan EPA Lipid Intervention Study (JELIS), sought to deter mine the effect(s) dietary EPA supplementation would have on hypercholesterolemic patients suf fering from coronar y ar tery disease. 18,645 patients with a total cholesterol of 6.5 mmol/L or g reater were recruited throughout Japan, and patients w ere randomly assigned to recei ve either 1,800 mg of EPA daily with a statin treatment or statins alone. After a f ve-year follow-up, major coronar y events were decreased 19% in the EPA-fed group vs. the control statin g roup, indicating the promise of f sh oil supplementation in the

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F2-IsoPs (pg g/mL)

75

50

* 25

0 - EPA

+ EPA

Figure 5.5. Endogenous levels of F2-Isoprostane formation from hearts of mice after eight weeks of 0.56% EPA diet supplementation. Data are expressed as means ± S.D. (n = 5). *, p < 0.05 as compared with control.

prevention of cardiac e vents in patients with high cholesterol (Y okoyama and Origasa 2003, Yokoyama et al. 2007, Yokoyama et al. 2000). Multiple other smaller trials ha ve also sho wn additional cardioprotecti ve effects associated with EPA (Eritsland et al. 1996, v on Schack y et al. 1999). Work conducted b y Gao et al. quantif ed the effect of EPA supplementation on arachidonate content and F 2 - IsoPlevels in the heart tissue. As seen in F igure 5.5, levels of F 2-IsoPs decreased dramaticall y by up to 64%, suggesting that EP A ef fectively decreases le vels of pro -inf ammatory F2 - IsoPsformed from arachidonate (Gao et al. 2006 ). Despite what we know about the varied biological activities exerted by the omeag-3 PUFAs, the underl ying molecular mechanisms responsib le for these acti vities are unkno wn. Se veral hypotheses by which these molecules or their metabolites could af fect physiological processes in nor mal health as w ell as in o xidative-stress-associated diseases ha ve been prof fered, and, while the decrease of pro -inf ammatory lipid mediators of o xidative stress has been sho wn to be helpful, recent studies ha ve demonstrated that non -enzymatic peroxidation products of the omega-3 fatty acid EPA are themselves bioactive and are responsible for some of the biological benef t of f sh oil in oxidative - stress - related diseases.

EICOSAPENT AENOIC ACID - DERIVEDISOPS Compared with the well-characterized pathway of arachidonic acid oxidation, the free radical initiated oxidation of the omega-3 fatty acid EPA has only recently been systematically examined. Anggard and colleagues pro vided some of the f rst e vidence that similar IsoP -like compounds could be for med from EPA, although conclusi ve structural identif cation was not provided (Nourooz -Zadeh et al. 1997). Based on arachidonic acid o xidation, the peroxidation of EPA was hypothesized to yield a signif cantly more complex array of products than arachidonic acid owing to the f act that EPA contains an additional unsaturated carbon -carbon bond (Porter et al. 1995). Thef rst systematic characterization of EPA-derived IsoPs was carried out recently with the These compounds differ structurally description of PGF2α-like compounds termed the F3 - IsoPs. from the F2-IsoPs due only to an additional carbon-carbon double bond. The pathway of formation of F 3-IsoPs, based on the for mation of F 2 - IsoPs,is shown in Figure 5.6 . Even with the f rst systematic description of an EP A-derived IsoP, vir tually no studies existed examining the biolo gical activities of these compounds. One limited repor t providing no data stated that the EP A-derived IsoP, 15 -F3t-IsoP, possessed acti vity that is signif cantly different from the 15 -F2t-IsoP acid in that the EP A-derived compound does not af fect human platelet shape change (Pratico, Sm yth et al. 1996). The lack of acti vity of 15 -F3t - IsoPis

Figure 5.6. Mechanism of formation of EPA -derived F 3-Isoprostanes. The mechanism of F 3IsoPs is analogous to that of arachidonic -acid-derived F 2-IsoPs. The six potential regioisomers formed are shown, but, for the sake of simplicity, only one stereoisomer is shown for each regioisomer.

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% Contraction

75

50

25

0

–10

–9

–8 –7 Concentration (M)

–6

–5

Figure 5.7. The vasoconstrictive effects of EPA-derived 15-F3t-Isoprostane are less potent than those of arachidonic acid -derived 15 -F2t-Isoprostane. The EC 50 for the vasoconstrictive effects of 15 -F3t-IsoP was found to be 2 µm, while 15 -F2t-IsoP had an EC 50 0.2 µM.

consistent with observations regarding EPA-derived 3 -series prostaglandins in that these latter compounds e xert w eaker agonist or no ef fects in comparison to arachidonic acid -derived 2-series prostaglandins (Kulkarni and Srinivasan 1985, Balapure, Rexroad et al. 1989). Recent , while still vasoconstudies by the authors’ lab also demonstrated that synthesized 15 -F3t - IsoP strictor acting through the TP receptor lik e 15 -F2t-IsoP, exerts bioactivity only at much higher concentrations as seen in F igure 5.7; this data suppor ts the claim that EP A-derived products exert weaker effects. It is also of interest to note that w hile F 3-IsoPs are not found in animals fed a con ventional diet, they are found for ming in a 10 -fold greater abundance than the F 2 - IsoPswhen f sh oil is added to the diet, indicating that the y are generated at higher le vels and preferentiall y to the F2-IsoPs, perhaps highlighting another possib le benef t of f sh oil diet supplementation (Gao et al. 2006). Analogous to arachidonic acid o xidation, multiple classes of IsoPs, w hich can be seen in Figure 5.8, can also for m from EPA oxidation. A3 /J3-IsoPs, also kno wn as the EP A-derived c yclopentenone IsoPs, ha ve been def nitely described to for m in abundance in vivo in settings of o xidative stress as w ell. They have been found to exert potent anti -inf ammatory activity much like their arachidonic acid counter parts through inhibition of NF κB (Brooks et al. 2008). The authors ha ve also recentl y found that EPA-derived c yclopentenone IsoPs e xert potent immunomodulator y ef fects. When administered to LPS -stimulated macrophages, 15 -A3t - IsoPinhibited NFκB activation via impair ment of I κα degradation and I κK activity. Further, 15 -A3t - IsoPattenuated LPS - inducedexpression of the NF κB-responsive proteins iNOS and CO X-2 w hile b locking nitric o xide and PGD 2 production in a dose -dependent manner at biolo gically rele vant micromolar concentrations. The cyclopentenone ring on this compound was shown to be necessary for bioactivity, and the ability to under go Michael adduction to a specif c cysteine residue on the I κK complex was found to be impor tant for inhibition of LPS -induced inf ammation. These f ndings demonstrate that the mode of action is similar to other α β , - unsaturatedcarbonyl-containing compounds such as PGA 2 and A2 /J2-IsoPs (Musiek et al. 2005a, Rossi et al. 2000). These effects were shown to be PPARγ-independent, and, unlike their arachidonic acid

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Figure 5.8. Isoprostanes of different ring structures formed by the non -enzymatic oxidation of EPA.

analogs, 15 - A 3t-IsoP did not promote lipid pero xidation in RAW cells. These f ndings demonstrated a novel mechanism by which a specif c product of EPA peroxidation exerts is benef cial biological acti vities. Another recent repor t b y Gao et al. also deter mined that a J -ring-IsoP formed from EPA peroxidation induced the NF -E2 related f actor 2 (Nrf2) -based antioxidant response through inhibition of K eap1, a negative regulator of Nrf2 (Gao et al. 2007).

SUMMAR Y The discovery of the IsoPs as products of non -enzymatic lipid pero xidation has been a major contribution to the f eld of o xidative stress and free radical chemistr y. Our understanding of the IsoP pathway continues to expand, providing new insights into the nature of lipid peroxidation in vivo , and re vealing new molecules that e xert potent biolo gical actions. Basic research into the biochemistry and pharmacology of the IsoPs, coupled with clinical studies emplo ying these molecules as biomark ers, should continue to pro vide important insights into the role of oxidant stress in human ph ysiology and pathophysiology.

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Mor row JD , MintonTA , Mukundan CR , Campbell MD , Zack ert WE et , al. 1994b. Free radical induced generation of isoprostanes in vivo . Evidence for the for mation of D -ring and E -ring isoprostanes. J Biol Chem 269 : 4317 – 26 . Mor row JD , MintonTA , Roberts LJ 2nd .1992b. The F2 - isoprostane,8 - epi - prostaglandin F2 alpha, a potent agonist of the v ascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist . Prostaglandins 44 : 155 – 63 . Mor row JD , Scruggs J , ChenY , Zack ert WE , Roberts LJ 2nd .1998 . Evidence that the E2 isoprostane, 15 - E2t - isoprostane (8 - iso - prostaglandin E2), is formed in vivo .J Lipid Res 39 : 1589 – 93 . Murai Y , HishinumaT , Suzuki N , Satoh J , T oyota T , Mizugaki M .2000 . Determination of urinary 8 -epi-prostaglandin F(2alpha) using liquid chromato graphy-tandem mass spectrometr y: increased excretion in diabetics . Prostaglandins Other Lipid Mediat 62 : 173 – 81 . MusiekES , Breeding RS , Milne GL , Zanoni G , Morrow JD , McLaughlin B. 2006 . Cyclopentenone isoprostanes are no vel bioactive products of lipid o xidation which enhance neurodegeneration. J Neurochem 97 : 1301 – 13 . MusiekES , Gao L , Milne GL , HanW , Ev erhart MB ,et al. 2005a . Cyclopentenone isoprostanes inhibit the inf ammatory response in macrophages . J Biol Chem 280 : 35562 – 70 . MusiekES ,Y in H , Milne GL , Morrow JD. 2005b. Recent advances in the biochemistry and clinical relevance of the isoprostane pathw ay. Lipids 40 : 987 – 94 . Nourooz - Zadeh J , Halliw ell B , Anggard EE. 1997 . Evidence for the formation of F3 - isoprostanes during peroxidation of eicosapentaenoic acid . Biochem Biophys Res Commun 236 : 467 – 72 . aredes P C ,T azzeo T , Janssen LJ . 2007 . E - ringisoprostane augments cholinergic neurotransmission in bovine trachealis via FP prostanoid receptors . Am J Respir Cell Mol Biol 37 : 739 – 47 . orter P NA , Caldw ell SE , Mills KA .1995 . Mechanisms of free radical oxidation of unsaturated lipids . Lipids 30 : 277 – 90 . PraticoD , Barry OP , La wson JA , Adiyaman M , Hw ang SW ,et al. 1998 . IPF2alpha - I:an index of lipid peroxidation in humans . Proc Natl Acad Sci USA 95 : 3449 – 54 . PraticoD , Iuliano L , MaurielloA , Spagnoli L , La wson JA ,et al. 1997 . Localization of distinct F2-isoprostanes in human atherosclerotic lesions . J Clin Invest 100 : 2028 – 34 . Pratic D ò , Sm yth EM ,V ioli F , F itzGerald GA. 1996 . Local amplif cation of platelet function b y 8-Epi prostaglandin F2alpha is not mediated b y thromboxane receptor isofor ms. J Biol Chem 271 : 14916 – 24 . yor Pr WA , Stanle y JP , Blair E .1976 .Autoxidation of polyunsaturated fatty acids: II. A suggested mechanism for the for mation of TBA-reactive materials from prostaglandin -like endoperoxides. Lipids 11 : 370 – 9 . RaalFJ , Pilcher GJ , V eller MG , K otze MJ , Jof fe BI .1999 . Effcacy of vitamin E compared with either simvastatin or ator vastatin in preventing the progression of atherosclerosis in homozygous familial hypercholesterolemia. Am J Cardiol 84 : 1344 –A7 6 ,. Reill y MP , Delanty N , Ro y L , Rokach J , Callaghan PO ,et al. 1997 . Increased formation of the isoprostanes IPF2alpha -I and 8 -epi-prostaglandin F2alpha in acute coronar y angioplasty: evidence for oxidant stress during coronar y reperfusion in humans . Circulation 96 : 3314 – 20 . Rober ts LJ 2nd , Oates JA , Linton MF , aFzio S , Meador BP ,et al. 2007 .The relationship between dose of vitamin E and suppression of o xidative stress in humans . Free Radic Biol Med 43 : 1388 – 93 . RokachJ , Khanapure SP , Hw ang SW , Adiyaman M , La wson JA , F itzGerald GA. 1997a .The isoprostanes: a perspective. Prostaglandins 54 : 823 – 51 . RokachJ , Khanapure SP , Hw ang SW , Adiyaman M , La wson JA , F itzGerald GA. 1997b. Nomenclature of isoprostanes: a proposal . Prostaglandins 54 : 853 – 73 . Rossi A , Kapahi P , Natoli G ,T akahashi T , ChenY ,et al. 2000 .Anti - inf ammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase . Nature 403 : 103 – 8 .

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Sinaik o AR , Steinber ger J , MoranA , Prineas RJ , V essby B ,et al. 2005 . Relation of body mass index and insulin resistance to cardio vascular risk f actors, inf ammatory factors, and oxidative stress during adolescence . Circulation 111 : 1985 – 91 . SinzingerH , Oguo gho A , Kaliman J . 1997 . Isoprostane 8 - epi - prostaglandin F2 alpha is a potent contractor of human peripheral l ymphatics. Lymphology 30 : 155 – 9 . SircarD , Subbaiah PV . 2007 . Isoprostane measurement in plasma and urine by liquid chromatography-mass spectrometry with one -step sample preparation . Clin Chem 53 : 251 – 8 . Smedman A ,V essby B , Basu S .2004 . Isomer- specifc effects of conjugated linoleic acid on lipid peroxidation in humans: re gulation by alpha -tocopherol and cyclo-oxygenase-2 inhibitor. Clin Sci (Lond) 106 : 67 – 73 . Staf forini DM , Sheller JR , Blackw ell TS , SapirsteinA ,Y ull FE et , al. 2006 . Release of free F2 - isoprostanesfrom esterif ed phospholipids is catal yzed by intracellular and plasma platelet activating factor acetylhydrolases. J Biol Chem 281 : 4616 – 23 . aber T DF , Morrow JD , Roberts LJ 2nd .1997 .A nomenclature system for the isoprostanes . Prostaglandins 53 : 63 – 7 . akahashi T K , NammourTM , Fukunaga M , Ebert J , Morrow JD ,et al. 1992 . Glomerular actions of a free radical -generated novel prostaglandin, 8 -epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thrombo xane A2 receptors . J Clin Invest 90 : 136 – 41 . aylor T AW , Bruno RS , F rei B , T raber MG .2006 . Benefts of prolonged g radient separation for high-performance liquid chromatography-tandem mass spectrometr y quantitation of plasma total 15 - seriesF - isoprostanes Anal . Biochem 350 : 41 – 51 . intut T Y, P arhami F , TsingotjidouA ,T etradis S ,T errito M , Demer LL .2002 . 8 - Isopro staglandin E2 enhances receptor -activated NFkappa B ligand (RANKL) -dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathw ay. J Biol Chem 277 : 14221 – 6 . onv Schacky C ,Angerer P , K othny W , Theisen K , Mudra H. 1999 .The effect of dietary omega- 3 fatty acids on coronar y atherosclerosis. A randomized, double-blind, placebo -controlled trial . Ann Intern Med 130 : 554 – 62 . ang W Z , Ciabattoni G , Creminon C , La wson J , F itzgerald GA et , al. 1995 . Immunological characterization of urinar y 8 -epi-prostaglandin F2 alpha e xcretion in man . J Pharmacol Exp Ther 275 : 94 – 100 . ohlin W M , Helmersson J , Sundstrom J , Arnlov J , V essby B ,et al. 2007 . Both cyclooxygenase and cytokine - mediatedinf ammation are associated with carotid intima -media thickness . Cytokine 38 : 130 – 6 . inY H , Gao L ,T ai HH , Murphey LJ , P orter NA , Morrow JD . 2007 . Urinary prostaglandin F2alpha is generated from the isoprostane pathw ay and not the c yclooxygenase in humans . J Biol Chem 282 : 329 – 36 . inY H , Morrow JD , P orter NA . 2004 . Identifcation of a no vel class of endopero xides from arachidonate autoxidation. J Biol Chem 279 : 3766 – 76 . okoyama Y M , Origasa H .2003 . Effects of eicosapentaenoic acid on cardiovascular events in Japanese patients with h ypercholesterolemia: rationale, design, and baseline characteristics of the Japan EPA Lipid Inter vention Study (JELIS) . Am Heart J 146 : 613 – 20 . okoyama Y M , Origasa H , Matsuzaki M , Matsuza wa Y , SaitoY ,et al. 2007 . Effects of eicosapentaenoic acid on major coronar y events in hypercholesterolaemic patients (JELIS): a randomised open -label, blinded endpoint analysis. Lancet 369 : 1090 – 8 . okoyama Y Y , Saito M , SaitoT , Y uguchi T , Sa wataishi M et , al. 2000 . Synergistic antiproliferative effect of delta 12 -prostaglandin J2 (delta 12 -PGJ2) and hyperthermia on human esophageal cancer cell lines . Hum Cell 13 : 23 – 33 .

Chapter6 Hydroxyoctadecadienoic Acid (HODE) as a Mar ker of Linoleic Acid Oxidation aYsukazu oYshida and

Etsuo Niki

INTR ODUCTION Lipid peroxidation was f rst studied extensively in the f eld of food science and technology and later chemistry, biology, and medicine (Niki et al. 2005). There is increasing experimental and clinical evidence showing that lipid peroxidation induced by active oxygen and nitrogen species is involved in the pathogenesis of various disorders, diseases, cancer, and even aging (Halliwell and Gutteridge 2007). Moreo ver, the biolo gical role and ef fects of lipid o xidation products have received much attention in this decade. It is no w accepted that lipid peroxidation induces the disturbance of f ne structure and the functional loss of biological membranes and that many lipid peroxidation products e xert cytotoxic effects. At the same time, lipid pero xidation products are thought to act as cellular regulators and signaling messengers (Leonarduzzi et al. 2000, Tang et al. 2002, Gutierrez et al. 2006, Poli et al. 2008). Polyunsaturated fatty acids are known to be very susceptible to oxidation. Cholesterol is also an impor tant substrate of lipid pero xidation in vivo (Diczfalusy 2004). Linoleates are one of the abundant and reacti ve lipids in vivo . Like arachidonates and cholesterol, linoleic acid and its phospholipids and cholester yl esters are o xidized by three distinct mechanisms: enzymatic oxidation, free radical -mediated o xidation, and non -enzymatic, non -radical o xidation (Niki et al. 2005). Each type of o xidation yields specif c products and requires specif c antioxidants to be inhibited. One of the greatest needs in this area is the development of biomarkers for reliable measurement of oxidative stress in humans (Blumber g 2004). Such biomarkers should be effective for monitoring a healthy state and also for assessing the benef cial effects of antioxidants contained in foods, fruits, beverages, drugs, and supplements. They may be used also to examine whether oxidative stress is in f act involved in diseases and w hether antioxidants can prevent, lessen, or ameliorate the disease state. Numerous biomark ers for o xidative stress ha ve been proposed (Dalle-Donne et al. 2006), including the o xidation products of linoleates. It w as found that isoprostanes, isomers of prostaglandins, are produced by the free-radical-mediated peroxidation of arachidonic acid independent of cyclooxygenase (Morrow et al. 1992) and they are accepted as a reliable biomarker for lipid o xidation (Chapter 5). Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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MECHANISM AND DYNAMICS OF LINOLEATE OXIDATION As mentioned above, free linoleic acid and its esters are oxidized by three distinct mechanisms, which are reviewed brief y below. ENZYMA TIC OXIDATION Lipoxygenase (LO X), c yclooxygenase (CO X), and c ytochrome P -450 (CYP) are impor tant enzymes involved in lipid oxidation (Elbekai and El -Kadi 2006). LOX may be most important for the o xidation of linoleates. A characteristic of the o xidation b y LO X is the specif city (Schneider et al. 2007, Yamamoto 1992); that is, the o xidation of linoleic acid and esters b y LOX gives regio - stereo , - and , enantio - specifc products. For example, the oxidation of linoleic acid by 15 - LO X gives 13(S) - 9 - cis,11 - trans ydroperoxyoctadecadienoic -h acid (HPODE) specif cally. On the other hand , the free radical -mediated oxidation of linoleic acid gi ves both 9 and 13 -cis,trans- and trans, trans -HPODE and the y are racemic; that is, the same amounts of R and S forms. Therefore, the ratio of S/R forms in HPODE or its reduced product hydroxyoctadecadienoic acid (HODE) higher than 1 suggests the contribution of enzymatic o xidation by LOX. However, it should be noted that the specif city depends on the type of enzymes, substrates, and reaction milieu and that it decreases in the oxidation of linoleate esters in lipoprotein particles in plasma (Yamashita et al. 1999). FREERADICAL - MEDIA TED OXIDATION OF LINOLEATES The mechanism of oxidation of linoleic acid and esters mediated b y free radicals, or autoxidation, is no w w ell understood and documented (P orter et al. 1995). It proceeds b y chain mechanism—one initiating free radical can o xidize man y molecules of linoleates and other lipids. The chain-carrying active species is the lipid peroxyl radical, which is much less reactive than h ydroxyl and alk oxyl radicals and therefore attacks bisall ylic h ydrogen selecti vely. Bisallylic hydrogen of pol yunsaturated lipids is roughl y 100 times more reacti ve than all ylic hydrogen and 10 4 times more reactive than saturated alkyl hydrogen (Noguchi and Niki 1999). The susceptibility of lipids to free radical chain o xidation increases with increasing numbers of double bonds. The mechanism of oxidation of linoleic acid is shown in Figure 6.1. The oxidation of linoleates in phospholipids and cholester yl esters proceeds b y the same mechanism. As described above, the peroxyl radicals attack bisall ylic hydrogen at the 11 position of linoleates to gi ve a carbon-centered pentadienyl radical, to which an oxygen molecule adds rapidly to yield a lipid peroxyl radical. Oxygen reacts with pentadien yl radical at either the 9 or 13 position, not at the 11 position, to gi ve a ther mochemically more stab le conjugate diene. The reaction of oxygen and pentadien yl radical is re versible, and the pero xyl radical ma y decompose to gi ve oxygen and a pentadienyl radical, which has a thermochemically more stable trans, trans form. These hydrogen abstraction, oxygen addition, and adverse reactions continue in sequence until the lipid peroxyl radicals are stabilized b y reacting with another pero xyl radical to gi ve stable products or with chain-breaking antioxidant such as vitamin E. Thus, the oxidation of linoleates proceeds by a straightforward mechanism as shown in Figure 6.1 to give four conjugated diene hydroperoxides: 13 - yhdroperoxy - 9(Z), 11(E) - octadecadienoic acid (13 - (Z,E) - HPODE), 13 - yhdroperoxy - 9(E),11(E) - octadecadienoicacid (13 - (E,E) - HPODE), 9 - yhdroperoxy - 10(E), 12(Z) - octadecadienoicacid (9 - (E,Z) - HPODE), and 9 - yhdroperoxy - 10(E),12(E) - octadecadienoic acid (9 - (E,E) - HPODE). The amounts of 9 - HPODEand 13 - HPODEare equal. In contrast to linoleates, the o xidation of polyunsaturated fatty acids having more than tw o double bonds such as arachidonic acid and docosahe xaenoic acid (DHA) proceeds b y much more complicated mechanisms because there are more reactive bisallylic hydrogens, and intramolecular addition reactions tak e place to gi ve cyclic peroxides.

Hydroxyoctadecadienoic Acid (HODE) as a Mark er of Linoleic Acid Oxidation

13 R2 •OO

HOO

(108)

70

9-(E,E)-HPODE

R1 OO•

(108) •

OOH

70 (108)

1 9 x 106 1.9

625

625

9 (Z E) HPODE 9-(Z,E)-HPODE

•

•

OOH

H

70 (108)

13 (Z E) HPODE 13-(Z,E)-HPODE

9

11 H

87

OO•

(108)

70

(108)

•OO

OO•

OOH

HOO

13-(E,E)-HPODE

11-(Z,Z)-HPODE

Figure 6.1. The oxidation mechanism of linoleates.

NON - ENZYMA TIC, NON - RADICALOXIDATION OF LINOLEATES Another type of oxidation of linoleates is oxidation by non-enzymatic non-radical active species such as singlet o xygen, ozone, and m yeloperoxidase (MPO). Singlet o xygen oxidizes unsaturated lipids primarily by “ene -reaction” to give hydroperoxides with concomitant double bond migration, with minor side reactions such as 1,4 -addition and 1,2 -addition to gi ve cyclic peroxide and dioxetane, respectively. The oxidation of linoleates b y singlet oxygen gives 9 -, 10 -, 12 - ,and 13 - HPODE;10 - and 12 - HPODEare the specif c products b y singlet o xygen. The oxidation by singlet o xygen may be impor tant for skin and e ye. Ozone o xidizes unsaturated lipids to gi ve ozonide and clea vage products. The oxidation of lipids b y singlet o xygen and ozone proceeds stoichiometricall y; that is, in contrast to the free -radical-mediated oxidation, each molecule of singlet o xygen and ozone o xidizes one molecule of lipid. MPO is another important oxidant (Panasenko et al. 2007). It reacts with hydrogen peroxide in the presence of chloride to gi ve hypochlorous acid. Hypochlorite reacts with amino acids rapidly and it also reacts with h ydrogen pero xide to gi ve singlet o xygen w hile with lipid hydroperoxides to gi ve alk oxyl and/or pero xyl radicals. Thus, MPO ma y induce both non radical and radical o xidation. Lipid hydroperoxides are not stable but undergo various secondary reactions to give chemically reactive products such as aldeh ydes, and the y are substrates of se veral enzymes such as glutathione peroxidases and lecithin -cholesterol acyltransferase (LCAT). Therefore, although HPODE is for med as the major primar y product in the o xidation of linoleates, the le vel of HPODE does not show the level of lipid oxidation, but it is determined by the balance between formation, metabolism, clearance, and e xcretion.

ROLE OF ANTIOXIDANTS AGAINST LIPID OXIDATION With increasing evidence that shows the involvement of lipid o xidation in the pathogenesis of various diseases, the role and capacity of antio xidants against lipid o xidation have recei ved much attention. The three different mechanisms of oxidation described above require different

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types of specif c antioxidants. For example, vitamin E is a more potent free -radical-scavenging antioxidant than carotenoids, but the re verse is tr ue against singlet o xygen. Enzymatic oxidation can be inhibited b y deactivating the enzyme and remo ving the compounds that activate the enzyme. For example, lipid hydroperoxides activate LOX by oxidizing ferrous ion to fer ric ion, Fe(II) to Fe(III). Therefore, the reduction of h ydroperoxides by GPx, for example, results in the inhibition of LO X-mediated oxidation. Free-radical-mediated oxidation of lipids can be inhibited by inhibiting the formation of free radicals and scavenging the free radicals. The former class of antioxidants is called preventive antioxidants and includes enzymes such as GPx and catalase, w hich reduce h ydroperoxides and hydrogen peroxide, respectively, and supero xide dismutase (SOD), w hich removes superoxide. The latter class of antio xidants refer red to as radical -scavenging antio xidants has attracted much attention. Another type of antio xidant is the photo -stabilizer and singlet o xygen quencher . Singlet oxygen is for med in general b y photochemical reaction, especiall y on the skin, and so either physical or chemical stabilization of light is effective for inhibition of singlet oxygen formation. Carotenoids are well known quenchers of singlet o xygen. The assessment of antio xidant capacity has been the subject of e xtensive studies (Yeum et al. 2004, Prior et al. 2005, Niki et al. 2008). The capacity of antio xidants for scavenging free radicals in vitro has been measured by versatile methods, often by following the decay of reference probe (Omata et al. 2008). It should be noted that the antioxidant capacity for scavenging free radicals does not al ways correlate with the capacity for inhibition of lipid o xidation, and that the antioxidant capacity in vivo is determined by many factors. As described later, HODE may be used as a reliab le biomarker for assessment of antio xidant capacity in vivo . Several compounds induce the phase II antio xidant compounds and/or enzymes, and these compounds may also play an impor tant role in the total antio xidant network in vivo .

MEASUREMENTOF HODE With increasing evidence indicating the involvement of lipid oxidation in various disorders and diseases, the de velopment of biomark ers for lipid o xidation has gained increasing attention. The detection and identif cation of lipid o xidation products are easier and more reliab le than the detection of reacti ve oxygen species, reacti ve nitrogen species, and other acti ve oxidants by using various probes and techniques such as f uorescence, chemiluminescence, and electron spin resonance. The o xidation products of linoleates ha ve been measured with high perfor mance liquid chromatography (HPLC) using UV absor ption at 230 to 235 nm due to conjugated diene. However, recent achie vements on mass spectrometr y have shown that coordination ion -spray mass spectrometry (CIS-MS) and electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time - of -ight f (MALDI -TOF) mass spectrometr y can be used as po werful tools to detect and identify complex mixtures of lipid oxidation products (Schiller et al. 2004). The o xidation products of linoleates are no w measured b y gas chromato graphy (mass spectrometry [GC -MS]) or high perfor mance liquid chromato graphy (tandem mass spectrometr y [LC-MS/MS]) with high accuracy and sensitivity. They are expensive and time-consuming and the development of simpler and more con venient detection and quantif cation systems such as enzyme-linked immunosorbent assay (ELISA) are needed. The oxidation of linoleates gi ves various products with dif ferent for ms. The authors ha ve tried to measure them inclusi vely by reduction with sodium boroh ydride followed by saponif cation with potassium h ydroxide (Yoshida et al. 2008a). This treatment con verts both free and ester for ms of h ydroperoxides, h ydroxides, and k etones to HODE, refer red to total as HODE (tHODE). The protocol of this method is summarized in F igure 6.2. Free and ester forms of 8-isoprostane PGF2α and hydroperoxyeicosatetraenoic acid (HPETE) are measured simultaneously as t8 - iso - PGF 2α and tHETE, respecti vely. Fur thermore, the free

Hydroxyoctadecadienoic Acid (HODE) as a Mark er of Linoleic Acid Oxidation

9-(E,Z)-HODE COOH

HO

Biomarkers tHODE and t8-iso-PGF2a Analysis of biological samples

COOH

COOH

HO

OH

13-(Z,E)-HODE

HO COOH

COOH OH HO

9-(E,E)-HODE

89

13-(E,E)-HODE

OH

8-iso-PGF2a

in plasma: Ester forms [PL, CE, TG]-18:2OOH [PL, CE, TG]-18:2=O [PL, CE, TG]-18:2OH Free forms 18:2OOH, 18:2=O, 18:2OH

Reduced (NaBH4) and saponified (KOH) sample ↓ pH=3 (HCl) ↓ C18 Sep-Pak (precondition; methanol, H2O) Sample load, wash; H2O, CH3CN/ H2O, reduction elution; Hex/AcOEt/iPA ↓ Hydroxides NH2 Sep-Pak (precondition; Hex) [PL, CE, TG]-18:2OH Sample load, wash; Hex/AcOEt, CH3CN, 18:2OH elution; AcOEt/MeOH/AcOH ↓ saponification evaporation ↓ tHODE (18:2OH) TMS (60 °C, 1hr) ↓ PCT/JP2004/018645 GC-MS Yoshida and Niki, Free Rad. Res. 38, 787 (2004)

Figure 6.2. The analytical method of tHODE and t8-isoprostagrandin F2α. Physiological samples were reduced with sodium borohydride and then saponif ed by potassium hydroxide, followed by solid phase extraction. tHODE assessed by this method accounts for a considerable amount of the oxidized linoleic acid.

and different ester forms of parent lipids are quantif ed by GC -MS and/or LC -MS/MS as total linoleates (t18:2), arachidonates (t20:4), and cholesterol (tCh) to measure the ratio of oxidation products to parent lipids. This ratio is useful for assessing the e xtent of oxidation in vivo. GC - MS METHOD FOR t HODEMEASUREMENT Plasma and erythrocytes separated from whole blood are used for the analyses of tHODE, other lipid o xidation products, and antio xidants as described belo w. The er ythrocytes are w ashed twice with a four -fold v olume of saline to remo ve plasma and w hite b lood cells and then adjusted to a hematocrit v alue (HV) of 40% with saline. The er ythrocytes (HV ca. 40%) are extracted with a four -fold volume of methanol containing 100 µ M 2,6 - ditert - - butyl - 4 - meth ylphenol (BHT) by vortexing and centrifugation (20,400 × g at 4 °C for 10 minutes) and immediately subjected to analyses of tHODE, total 7-hydroxycholesterol (t7-OHCh), and antioxidants. Tissues such as li vers and brains are also collected after perfusion with saline and stored at − 80 ° Cuntil analysis. Internal standards, 8 - iso - PGF 2α -4dand 9 - HODE -4,dand methanol are added to the plasma and the e xtracts from er ythrocytes, followed by reduction with an e xcessive amount of sodium boroh ydride at room temperature for 5 minutes under nitro gen. The liver and brain are homo genized (Polytron PT-3100, Kinematica AG, Lucer ne, Switzerland) in saline (liver or brain:saline = 1:3, w/w), and aliquots are diluted with saline. The inter nal standards and methanol are added to this solution follo wed b y reduction as described above. The reduced sample is then mixed with 1 M KOH in methanol under nitrogen and incubated for 30 minutes in the dark at 40 °C in a shak er. The sample is centrifuged (3,000 × g at 4 °C for 10 minutes) and the super natant is diluted with a four -fold volume of water and acidif ed (pH 3) using 2 N HCl. The acidif ed sample is centrifuged (3,000 × g at 4°C for 10 minutes) and the super natant is subjected to solid -phase extraction. The solution is

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evaporated under nitro gen and the sil ylating agent N,O - bis(trimethylsilyl)trif uoroacetamide (BSTFA) is added to the dried residue. The solution is vigorousl y mixed with a v ortex mixer for 1 minute and incubated for 60 minutes at 60°C to obtain the trimethylsilyl esters and ethers. The eluent obtained is diluted with acetone and then an aliquot of this sample is injected into a gas chromatograph (GC 6890 N, Agilent Technologies Co., Ltd., Califor nia, USA) equipped with a quadr upole mass spectrometer (5973 Netw ork, Agilent Technologies Co., Ltd.) and a fused-silica capillary column (HP -5MS, 5% phenyl methyl siloxane, 30 m × 0.25 mm,Agilent Technologies Co., Ltd.). Helium is used as the car rier gas at a f ow rate of 1.2 ml/minute. The temperature of the column is pro grammed from 60 °C to 280 °C at a rate of 10 °C/minute. The injector temperature is set at 250 °C and the temperatures of transfer lines to the mass detector and ion source are 250 °C and 230 °C, respectively. Electron ener gy of EI mass spectrometr y is set at 70 eV. HODE and 8 -iso-PGF2α are identif ed on the basis of their retention times and mass patterns (m/z = 440, 369, and 225 for HODE, and 571 and 481 for 8 -iso-PGF2α );precursor ions at 440 and 481 are selected for the quantif cation of HODE and 8 -iso-PGF2α , respectively, using the inter nal standards 9 -HODE-d4 (m/z = 444) and 8 - iso - PGF 2α -4d(m/z = 485). These precursor ions are the most sensiti ve among the detected ions. With this method, 9 - (E,Z) -and 13 - (Z,E) - HODE,9 - (E,E) - HODE,13 - (E,E) - HODE,and 8 - iso - PGF 2α are measured simultaneously and isolated on the GC-MS chromatograph. By using these methods, ar tif cial oxidation of lipids during the sample w ork-up is minimized and the experimental errors are within ± 10%. LC - MS /METHOD MS FOR t HODEMEASUREMENT Physiological samples such as li ver and brain are homo genized with four × weight of saline with pestles and tubes (Bel -Art Products, NJ, USA) for 2 minutes. The homogenized sample and plasma are mixed with saline. Subsequently, methanol containing internal standards 8-isoPGF2α -4d,13 - HODE -4 d,7α - OHCh 7- ,d7β - OHCh 7-, d16-hydroxyhexadecanoic acid, and butylated h ydroxytoluene (BHT) are added to the samples. This is follo wed b y the reduction of hydroperoxides and ketones with an excess amount of sodium borohydride at room temperature for 5 minutes. Next, the reduced sample is mixed with 1 M KOH in methanol under nitrogen and incubated in a shaker for 30 minutes in the dark at 40 °C. The mixture is cooled on ice and acidif ed with 10% acetic acid in w ater and e xtracted with chlorofor m and eth yl acetate (chlorofor m/ethyl acetate = 4/1, v/v). The sample is mix ed with a v ortex mixer for 1 minute and centrifuged at 1,500 × g for 5 minutes at 4°C. The chloroform and ethyl acetate layer is concentrated to around 1 ml after the remo val of the w ater layer and di vided equally into tw o por tions. The divided chloroform and ethyl acetate solution is evaporated to dryness under nitrogen. The derivatized sample is reconstituted with methanol and w ater (methanol/water = 70/30, v/v, 250 µl), and a portion of the sample (10 µl) is subjected to the LC -MS/MS analysis. LC is carried out on an ODS column (Hypersil Gold , 1.9 µ m,100 × 2.1 mm,Thermo Fisher Scientif c, California, USA) in a column oven (LC column casket CS-300B, Chromato Science Co., Ltd., Osaka, Japan) set at 30°C. The LC apparatus consists of an automatic sampler (automatic sampling system AS-100, Bio -Rad Laboratories Co. Ltd., Tokyo, Japan), three pumps (Gilson models 306 and 307, Gilson Co. Inc., Ohio, USA), and a dynamic mixer (811D, Gilson Co. Inc.). The eluent condition is a g radient comprising solv ent A, 2 mM ammonium acetate in water, and solvent B, methanol and acetonitrile (methanol/acetonitrile = 5/95), at a f ow rate of 0.2 ml/minute. The initial composition of the g radient is 80% A and 20% B and held for 2 minutes and then changed to 50% A and 50% B after 45 minutes. MS analysis is carried out using a Thermo Finnigan TSQ Quantum Discovery Max, a triplequadrupole mass spectrometer (Thermo Fisher Scientif c) f tted with an electrospray ionization (ESI) source. ESI is car ried out at a needle v oltage of 4.2 kV. Nitrogen is used as the sheath gas (17 psi) and auxiliar y gas (12 units). The capillary is heated to 280 °C and the mass spec-

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trometers are optimized to obtain the maximum sensitivity. A specif c precursor - to - product - ion transition is carried out by selected reaction monitoring (SRM) after collision-induced dissociation in the ne gative mode. Argon is used as the collision gas and the collision cell pressure is set 1.5 mTorr. The precursor, product ions, and collision ener gy are deter mined after the optimization of MS/MS as follo ws: m/z = 353.5 and 192.6 to 193.6 at 29 eV for 8 -iso-PGF2α , m/z = 357.0 and 196.5 to 197.5 at 29 eV for 8 -iso-PGF2α -4d, m/z = 319.0 and 114.5 to 115.5 at 10 eVfor 5 - HETE,m/z = 319.3 and 162.8 to 163.8 at 13 eV for 12 -HETE, m/z = 319.3and 202.5 to 203.5 at 10 eV for 15 -HETE, m/z = 295.0 and 194.6 to 195.6 at 21 eV for both 13 - (Z,E) - HODE and 13 - (E,E) - HODE, m/z = 295.0 and 170.5 to 171.5 at 24 eV for both 9 - (E,Z) - HODE and 9 - (E,E) - HODE, m/z = 295.0 and 182.6 to 183.6 at 22eV for both 10-(Z,E)HODE and 12 - (Z,E) - HODE, and m/z = 299.0 and 197.6 to 198.6 at 26 eV for 13 -HODE-d4 . The standard samples of HODE isomers (9- and 13-(Z, E) and (E, E)-HODE), HETE isomers (5 - ,12 - ,and 15 - HETE),8 - iso - PGF 2α , 7α - OHCh,7β - OHCh,cholesterol, and linoleic acid (18:2) are added to the human plasma to deter mine the factors for quantif cation. The concentrations of HODE, HETE isomers, and 8 -iso-PGF2α are determined by using 13 -HODE-d4 and 8 - iso - PGF 2α -4das inter nal standards. The f actors are 0.14 (9 -(Z,E)-HODE), 0.21 (9 -(E,E)HODE), 0.052 (12 - (Z,E) - HODE),0.066 (10 - (Z,E) - HODE),0.14 (13 - (Z,E) - HODE),0.11 (13 - (E,E) - HODE), 0.70 (5 - HETE),3.71 (12 - HETE),1.17 (15 - HETE),and 0.85 (8 - iso - PGF 2α ) by the SRM method of LC -MS/MS. The inter nal standards 7 α - OHCh 7- ,d7β - OHCh 7- ,d and HHDE are used for quantifying 7 α - OHCh,7β-OHCh and Ch, and 18:2. The f actors were found to be 1.0 (7 α - OHCh),1.0 (7β-OHCh), 15 (Ch), and 1.6 (18:2) b y the single ion monitoring (SIM) method of GC-MS. Apparently, the factors depend on the recovery eff ciency of the extraction with solvents as well as the sensitivities of targeted fragment ions. The quantif cation er rors in the anal ysis of biolo gical samples such as plasma and li ver are generall y within 5%. AN ALYSIS OF t 7 - OHC t hC, AND h , t 18:2BY GC - MS The other por tion of the chlorofor m and ethyl acetate solution described abo ve is also e vaporated to dr yness under nitro gen. A silylating agent N ,O-bis(trimethylsilyl)-trif uoroacetamide (BSTFA, 30 µl) is added to the dried residue. The solution is vigorousl y mixed by vortexing for 0.5 minute and incubated for 60 minutes at 60 °C to obtain trimethylsilyl esters and ethers. An aliquot of this sample is injected into a gas chromato graph (GC 6890 N , Agilent Technologies, P alo Alto, CA) that is equipped with a quadr upole mass spectrometer (5973 Network, Agilent Technologies). A fused-silica capillary column (HP -5MS, 5% phenyl methyl siloxane, 30 m × 0.25 mm, Agilent Technologies) is used. Helium is used as a car rier gas at a f ow rate of 1.2 ml/minute. Temperature programming was carried out from 60 °C to 280 °C at 10°C/minute. The injector temperature is set at 250°C, and the temperatures of the transfer line to the mass detector and ion source are 250 °C and 230 °C, respectively. Electron energy is set at 70 eV. 7α - OHCh,7β-OHCh, 24S -OHCh, Ch, and 18:2 are identif ed on the basis of their retention times and mass patter ns; ions ha ving m/z = 456 for 7α - OHChand 7β - OHCh,413 for 24S -OHCh, 458 for Ch, and 337 for linoleic acid are selected for the quantif cation. 7αOHCh, 7β - OHCh,24S - OHCh,and Ch are identif ed quantitatively by using 7 α - OHCh 7- and d 7β - OHCh 7- as d internal standards, and 18:2 w as quantif ed by using 16 -hydroxyhexadecanoic acid (HHDE) as an inter nal standard.

ASSESSMENT OF ANTIOXIDANT CAPACITY IN VIVO BY t HODE As described abo ve, the assessment of free -radical-scavenging antioxidant capacity in vivo is an impor tant subject and has been studied e xtensively (Yeum et al. 2004, Prior et al. 2005, Niki et al. 2008). The antio xidant capacity in vi vo is deter mined b y se veral f actors such as

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bioavailability, metabolism, localization, distribution, f ate of antio xidant-derived radical, and interaction with other antioxidants as well as the reactivity toward free radicals. It is impor tant to elucidate each factor by basic studies for sound inter pretation of experimental results, but it is quite diff cult to estimate the antio xidant capacity in vivo from these indi vidual f actors. Practically, it is essential to examine the effects of the antioxidant in appropriate animal model studies and human intervention studies. Biomarkers are necessary for these studies and HODE is a potential biomark er for the assessment of antio xidant capacity to inhibit lipid o xidation and attenuate oxidative stress in vi vo. Theoretically, the capacity of sca venging peroxyl radicals b y the antio xidant can be e valuated from the effect of the antioxidant on the level of HODE and other lipid oxidation products in biological f uids and/or tissues. The eff cacy for scavenging peroxyl radicals by the antioxidant in vivo can be estimated from the ratio of cis,trans -HODE to trans,trans -HODE, which is expressed by the following equation:

( cis,trans-HODE trans,trans-HODE ) = κinh c1 IH [ ] + κ p c1 [ LH ] + c2

(6.1)

where κinh and κp are the rate constants for sca venging peroxyl radicals by the antioxidant and chain propagation, IH and LH are antio xidant and substrate, and c1 and c2 are constants, respectively. Thus, an antioxidant capacity in vivo can be estimated by a stereo isomer ratio of HODE (cis,trans -HODE/trans,trans-HODE). Fur ther, this ratio ma y be useful for e valuating benef cial ef fects of foods, species, be verages, supplements, and dr ugs as w ell as antioxidants.

t HODE LEVELS IN HEALTHY SUBJECTS AND DISEASED PATIENTS A number of studies ha ve been perfor med to measure the le vel of lipid o xidation products in humans. Above all, thiobarbituric reacti ve substances (TB ARS), malonaldeh yde (MD A), HODE, isoprostanes, and oxysterols have been measured most frequently. Ethane and pentane in exhaled gas are also used as biomark ers of lipid oxidation in vivo. Each method has its own merits and demerits. Ho wever, usuall y these le vels are not measured simultaneousl y from a single subject, making it diff cult to compare the levels of different lipid oxidation products as biomarkers. Only a fe w recent studies measured se veral parameters simultaneousl y from the same subjects (Yoshida et al. 2007a, Lee et al. 2008). The levels of tHODE, t8 - iso - PGF 2α, and t7 -OHCh in plasma and er ythrocytes from 44 healthy human subjects were measured to assess the level of lipid oxidation in humans (Yoshida et al. 2007a). Plasma and er ythrocytes were treated with sodium boroh ydride and potassium hydroxide to convert both free and ester forms of linoleic acid, arachidonic acid, and cholesterol to free for ms. The average concentrations of tHODE, t8 -iso-PGF2α, and t7 -OHCh in plasma were 203, 0.727, and 243 nmol/L and in er ythrocytes 1,917, 12.8 and 5,226 nmol/packed L, respectively. The ratios of tHODE and t7 -OHCh to the parent substrates w ere 194 and 3,519 µmol tHODE/mol linoleates and 40.9 and 686 µmol t7-OHCh/mol cholesterol in plasma and erythrocytes, respectively. Thus, the le vel of lipid o xidation products in er ythrocytes was higher than that in plasma. In another e xperiment, the le vels of tHODE, t8 -iso-PGF2α, and t7 -OHCh in human LDL were measured. LDL isolated from nor mal human plasma was subfractionated into three fractions, LDL -1, LDL -2, and LDL -3, according to the surf ace electronegativity of LDL par ticle with anion-exchange HPLC (AE-HPLC) (Kitano et al. 2007). Each fraction consisted of 75.1%, 19.3%, and 5.6% of total LDL. The concentrations of tHODE, t7 -OHCh, and t8 -iso-PGF2α in each LDL subfraction were assessed after extraction, followed by reduction and saponif cation. It w as found that the le vels of tHODE, t8 -iso-PGF2α, and t7 -OHCh cor related w ell with negative charge of LDL par ticles (Table 6.1). These results clearl y indicate that the e xtent of oxidation increases in the order of LDL -1 < LDL - 2<< LDL - 3.

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Hydroxyoctadecadienoic Acid (HODE) as a Mark er of Linoleic Acid Oxidation Table 6.1. Lipid peroxidation products in human LDL fractions.

LDLfraction

tHODE

t8 - iso - PGF 2α

t7 - OHCh

LDL - 1 LDL - 2 LDL - 3

1,935(227) 4,657(782) 3,6434(3670)

5.8(8.7) 33.1(18.3) 162.1(66.6)

2100(116) 5703(181) 18314(344)

The concentrations in pmol/mg apo B . The numbers in parentheses are the ratio of products to the corresponding parent lipid, in µmol product/mol parent lipid.

It has been repor ted from numerous studies that the e xtent of lipid o xidation is ele vated in the diseased patients compared to normal subjects (Bachi et al. 1996, Dalle-Donne et al. 2006). Similar association has been obser ved for the o xidation of proteins and DN A. Several results showing the association betw een tHODE and diseases are sho wn below. The o xidative modif cation h ypothesis proposed b y Steinber g and his colleagues ( 1989) indicates that the o xidative modif cation of LDL is an impor tant initial event for the de velopment of atherosclerosis. Huge numbers of in vitro and animal studies suppor t this hypothesis. Lipid oxidation products ha ve been measured in human atherosclerotic lesions. It w as found in many studies that human atherosclerotic lesions contained increased amounts of lipid oxidation products when compared with non -atherosclerotic vessel wall (Jira et al. 1998, Platico et al. 1997). For example, it was reported that the contents in human atherosclerotic lesion w ere 4.7 mmol HODE/mol linoleic acid and 13.6 µmol isoprostane/mol aracidonic acid (after hydrolysis), which were much higher than the levels in normal umbilical vein (Gniwotta et al. 1997). The relationship of o xidative stress to cataracts w as studied (Li et al. 2009). Forty healthy men and postmenopausal women aged 50 to 70 years (F25, M15) underwent eye examinations. Blood samples w ere collected for anal yzing major w ell-known antioxidants, tHODE, and t8 iso - PGF 2α using GC -MS. Twenty-seven (F17, M10) of the 40 subjects w ere diagnosed to have early cataracts at the onset of the study, which were regarded as age appropriate lens opacities. There was no signif cant difference in plasma major antio xidants and lipid peroxidation determined by malondialdehyde as well as t8 -iso-PGF2α between the groups with and without early cataract. However, isomers of 9 - and 13 -(Z,E)-HODE levels were signif cantly higher in subjects with early cataract as compared with those of non -cataract subjects (P < 0.05). In another study, the le vels of lipid o xidation products w ere measured in plasma and li ver of hepatitis C - and B -infected patients and compared with those of control subjects (Y oshida et al. 2008a). It was found that tHODE, tHETE, and t7 -OHCh were the major products in both plasma and li ver and that the le vel of t8 - iso -PGF2α w as much smaller . The le vels of lipid oxidation products in plasma and li ver of patients w ere more ele vated than those of health y subjects. The role of o xidative stress in neurode generative diseases has recei ved much attention recently and ele vated levels of lipid o xidation products ha ve been obser ved in patients with neurological diseases (Montine et al. 2002, Roberts et al. 2005, Su et al. 2008). We also found that tHODE level and oxidatively modif ed peroxiredoxin - 2and - 6in Alzheimer ’sdisease were signif cantly higher than those in health y controls, and that the y increased with increasing clinical dementia ratings (Yoshida et al. 2009). It should be noted that, as is often pointed out, accurate deter mination of lipid o xidation products in human samples is diff cult and g reat care should be tak en. A potential generation or loss of lipid o xidation products during sampling, storage, isolation, and anal ysis may take place. The use of appropriate internal standard is recommended. Furthermore, the diet contains various lipid o xidation products and the measurement of their le vels in biolo gical f uids and tissues may be confounded b y diet unless the subjects are f asted, and therefore b lood should be withdrawn after suff cient fasting.

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t HODE LEVELS IN ANIMAL EXPERIMENTS There are no w abundant repor ts showing that the le vels of o xidation products of lipids and modif ed proteins and DN A are ele vated under o xidative stress conditions. The benef cial effects of antioxidants in vivo have been assessed in various animal experiments under normal conditions and under o xidative stress. This is impor tant because it is diff cult to estimate the antioxidant capacity from the results of in vitro studies. It is essential to e xamine the ef fects of antio xidants in animal model e xperiments and to elucidate the capacity and underl ying mechanisms of antioxidant action in vivo. Appropriate biomarkers are indispensable and HODE may serve this pur pose. It was previously found that the intraperitoneal administration of 2,2 -azobis (2 -amidinopropane) dihydrochloride (AAPH) to mice induced oxidative stress in vivo (Terao and Niki 1986). AAPH generates free radicals b y spontaneous ther mal decomposition without biotransfor mation. More recent studies sho wed that the administration of azo compounds to mice increased the le vels of tHODE and t8 -iso-PGF2α w hile decreasing the (cis,trans -HODE/trans,transHODE) ratio in mouse li ver (Table 6.2, Yoshida et al. 2005b). It was conf rmed that the remo val of vitamin E from the diet also increased the le vels of tHODE and decreased the cis,trans-HODE/trans,trans-HODE ratio, whereas the administration of natural and synthetic antioxidants such as vitamin E isoforms, chlorogenic acid, caffeic acid, coffee, and 2,3 - dih yro - 5 -ydroxy h - 4,6 - di -t ter - butyl2,2 - dipentylbenzo ylfuran (BO - 653)reduced the tHODE le vel and at the same time increased the cis,trans -HODE/trans,trans-HODE ratio (Yoshida et al. 2007b, Yoshida et al. 2008b, 2008c ). In another study , it w as found that coenzyme Q9 decreased the tHODE and t8-iso-PGF2α levels in plasma, erythrocytes, liver, and brain of mice fed a vitamin-E-free diet in a dose dependent manner (Yoshida et al. 2006a). Coenzyme Q9 increased the stereo isomer ratio mentioned abo ve. It is known that a choline def cient diet induces liver damage. Signif cant increases in tHODE and t8 - iso - PGF 2α in li ver and plasma w ere observed when mice w ere fed a choline -def cient diet (Yoshida et al. 2006b). This increase in tHODE and t8 - iso -PGF2α w as decreased to a normal level when α-tocopherol or BO-653 was mixed with the diet. The decrease of the HODE stereo isomer ratio b y a choline -def cient diet w as also reco vered b y these antio xidants. However, the increase in plasma glutamic-pyruvic transaminase and fatty acids in liver induced by a choline -def cient diet was not recovered by the antioxidants. Similar protective effects of antioxidants were observed against the liver damage induced by carbon tetrachloride (Yoshida et al. 2005a), a well established liver toxin. These results clearly show that the o xidative stress induced b y free radicals, o xidative toxic compounds, and decreases in antio xidants all enhance lipid o xidation, and that free radical scavenging antio xidants ameliorate lipid o xidation. Fur thermore, the antio xidant capacity in vivo may be assessed from these e xperiments. Obviously, it is impor tant to e xamine whether the level of lipid oxidation measured by the biomarkers is associated with the disease state and if the antio xidants prevent or ameliorate the diseases in appropriate animal e xperiments and well programmed human studies. Table 6.2. Effects of free radicals on lever levels of tHODE and t8 -iso-PGF2α in nmol/g tissue

tHODE ((Z,E) - HODE/ (E,E) - HODE) t8 - iso - PGF 2α

Control

AAPH

AIPH

15.5 2.3 0.11

57.0 * 1.5 0.25 *

33.3 * 1.2 * 0.19 *

AAPH was give to mice b y per oral administration w hile AIPH was dissolved in drinking w ater to mice once a day for two weeks *means a statistical signif cance, p <0.05

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CONCLUSION Lipid oxidation gives complex products including h ydroperoxides, cleavage products such as aldehydes, and polymeric materials. Further, these products modify proteins and DNA to yield diverse products. Their application as biomark ers for diagnosis of disease pro gression, evaluation of therapies, and health e xamination has been the focus of intensi ve studies. The usefulness and accuracy of lipid oxidation products such as HODE as biomarkers should be validated in the future studies.

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NikiE , OmataY , FukuharaA , SaitoY , Y oshida Y . 2008 .Assessment of radical scavenging capacity and lipid pero xidation inhibiting capacity. J Agric Food Chem 56 : 8255 – 8260 . No guchi N , Niki E .1999 . Chemistry of active oxygen species and antioxidants . In Antioxidant Status, Diet, Nutrition, and Health . P apas AM ed. , Boca Raton : CRC Press , pp. 3 – 20 . Omata Y , SaitoY , Y oshida Y , Niki E .2008 . Simple assessment of radical scavenging capacity of beverages . J Agric Food Chem 56 : 3386 – 3390 . anasenko P OM ,V akhrusheva TV , Vlaso va II , Chekanov AV , Barano v YV , Ser gienko VI .2007 . Role of myeloperoxidase - mediatedmodif cation of human b lood lipoproteins in atherosclerosis environment . Bull Exp Boil Med 144 : 428 – 431 . PlaticoD , Iuliano L , MaurielloA , Spagnoli L , La wson JA , Rokach J , Maclouf J , V ioli F , F itzgerald GA .1997 . Localization of distinct F2 - isoprostanesin human atherosclerotic lesions . J Clin Invest 100 : 2028 – 2034 . oli P G , Schaur RJ , SiemsWG , Leonarduzzi G .2008 . 4 - Hydro xynonenal: a membrane lipid oxidation products of medicinal interest . Medic Res Rev 28 : 569 – 631 . orter P NA , Caldw ell SE , Mills KA .1995 . Mechanisms of free radical oxidation of unsaturated lipids . Lipids 30 : 277 – 290 . PriorRL ,W u X , Scaich K .2005 . Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietar y supplements . J Agric Food Chem 53 : 4290 – 302 . Rober ts II LJ , F essel JP , Da vies SS .2005 .The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid pero xidation. Brain Pathol 15 : 143 – 148 . SchillerJ , S üR ,Arnhold J , Fuchs B , Lessig J , M ü llerM , P etkovic M , Spalteholz H , Zsch önig r O , Arnold K .2004 . Matrix - assistedlaser desorption and ionization time - of -ight f (MALDITOF) mass spectrometr y in lipid and phospholipid research . Prog Lipid Res 43 : 449 – 488 . SchneiderC , Pratt DA , P orter NA , BrashAR .2007 . Control of oxygenation in lipoxygenase catalysis . Chem Biol 14 : 473 – 488 . Steinber g D, P arthasarathy S , Care w TE , Khoo JW , W itztum JL .1989 . Beyond cholesterol: modif cation of low density lipoprotein that increase its athero genicity. New Engl J Med 320 : 915 – 924 . SuB , W ang X , NunomuraA , Moreira PI , Lee HG , P erry G , Smith MA , Zhu X .2008. Oxidative stress signaling in Alzheimer ’s disease . Curr Alzheimer Res 6 : 525 – 532 . ang T DG , La E , K ern J , K ehrer LP . 2002 . Fatty acid oxidation and signaling in apoptosis . Biol Chem 383 : 425 – 442 . erao T K , Niki E .1986 . Damage to biological tissues induced by radical initiator 2,2 - azobis(2 amidinopropane) dihydrochloride and its inhibition b y chain breaking antio xidants. J Free Radic Biol Med 2 : 193 – 201 . amamoto Y S. 1992 . Mammalian lipoxygenases: molecular structures and functions . Biochim Biophys Acta 1128 : 117 – 131 . amashita Y H , NakamuraA , No guchi N , Niki E , K uhn H .1999 . Oxidation of low density liporoprotein and plasma b y 15 -lipoxygenase and free radicals . FEBS Lett 445 : 287 – 290 . eum Y K - J, RussellRM ,Krinsk y NI ,AldiniG. 2004 .Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma . Arch Biochem Biophys 430 :97 – 103 . oshida Y Y , Itoh N , Ha yakawa M , Piga R , Cynshi O , Jishage K , Niki E. 2005a . Lipidperoxidation induced by carbon tetrachloride and its inhibition b y antioxidant as evaluated by an oxidative stress marker, HODE . Toxic Appl Pharmacol 208 : 87 – 97 . oshida Y Y , Ha yakawa M , Niki E. 2005b. Total hydroxyoctadecadienoic acid as a marker for lipid peroxidation in vivo .BioFactors 24 : 7 – 15 . oshida Y Y , Ha yakawa M , HabuchiY , Niki E. 2006a . Evaluation of the dietary effects of coenzyme Q in vi vo by the oxidative stress marker, hydroxyoctadecadienoic acid and its stereo isomer ratio . Biochim Biophys Acta 1760 : 1558 – 1568 . oshida Y Y , Itoh N , Ha yakawa M , HabuchiY , Inoue R , Chen Z - H Cao , J , Cynshi O, Niki E. 2006b. Lipid peroxidation in mice fed a choline -def cient diet as e valuated by total hydroxyoctadecadienoic acid . Nutr 22 : 303 – 311 .

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oshida Y Y , SaitoY , Ha yakawa M , HabuchiY , ImaiY , Sa wai Y , Niki E. 2007a . Levels of lipid peroxidation in human plasma and er ythrocytes: Comparison between fatty acids and cholesterol. Lipids 42 : 439 – 449 . oshida Y Y , Ha yakawa M , HabuchiY , Itoh N , Niki E. 2007b. Evaluation of lipophilic antioxidant eff cacy in vivo by the biomarkers hydroxyoctadecadienoic acid and isoprostane . Lipids 42 : 463 – 472 . oshida Y Y, K odai S ,T akemura S , MinamiyamaY , Niki E. 2008a . Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples . Anal Biochem 379 : 105 – 115 . oshida Y Y , Ha yakawa M , Cynshi O , Jishage K , Niki E. 2008b. Acceleration of lipid peroxidation in α-tocopherol transfer protein -knock out mice follo wing the consumption of drinking w ater containing a radical initiator . J Oleo Sci 57 : 577 – 583 . oshida Y Y , Ha yakawa M , Niki E. 2008c . Evaluation of the antioxidant effects of coffee and its components using the biomark ers hydroxyoctadecadienoic acid and isoprostane . J Oleo Sci 57 : 691 – 697 . oshida Y Y, Y oshikawa A , KinumiT , Oga wa Y , SaitoY , Ohara K ,Y amamoto H , ImaiY , Niki E . 2009. Hydroxyoctadecadienoic acid and o xidatively modif ed peroxiredoxins in the b lood of Alzheimer’s disease patients and their potential as biomark ers. Neurobiol Aging 30 : 174 – 185 .

Chapter7 Oxysterols: Potential Biomarkers of Oxidative Stress Luigi Iulianoand

Ulf Diczfalusy

INTR ODUCTION Cholesterol is an impor tant constituent of biolo gical membranes but also a precursor for the biosynthesis of bile acids and steroid hor mones. Cholesterol is susceptib le to o xidation, and easily forms oxygenated derivatives known as o xysterols. A great interest in o xysterols arose when it was shown that they exerted inhibitory actions on cholesterol biosynthesis (Kandutsch et al. 1978), a f nding that subsequently became known as the “oxysterol hypothesis.” Additional roles for o xysterols in human ph ysiology include their par ticipation in bile acid biosynthesis, their function as transpor t forms of cholesterol, and their roles as re gulators of gene transcription (Russell 2000). For a comprehensive review on oxysterols see Schroepfer ( 2000). Oxysterols can be formed by three different mechanisms: autoxidation, oxidation secondary to lipid pero xidation, and b y cholesterol metabolizing enzymes. The f act that cholesterol is oxidized by oxygen and other reactive oxygen species has led to the hypothesis that oxysterols may be used as biomark ers for o xidative stress (Yoshida & Niki 2004), in analo gy with the use of o xidation products of pol yunsaturated f atty acids (isoprostanes) as o xidative stress markers (Morrow 2005 ).

FORMA TION OF OXYSTEROLS Exposure of cholesterol to o xygen, in vitro and in vivo , leads to the for mation of numerous cholesterol autoxidation products (Smith 1981). Some of the more common autoxidation products are sho wn in F igure 7.1. These include 7 α -and 7β -ydroxycholesterol, h formed via their 7-hydroperoxy inter mediates, 7 -ketocholesterol, for med b y deh ydration of the inter mediary cholesterol 7 - yhdroperoxides, cholesterol 5,6α -and 5,6β-epoxides, and their common h ydrolysis product cholestane 3 β ,5α ,6β-triol. The ease of for mation of these o xysterols is also a problem in that their ar tifactual formation during storage, handling, and anal ysis of biological samples can easily take place (Smith 1981). Oxysterols are for med secondar y to lipid pero xidation, and the all ylic hydrogens at C7 of the cholesterol molecule are especially easily abstracted, resulting in a carbon centered radical. This radical reacts rapidly with molecular oxygen, leading to the for mation of 7 -hydroperoxyand 7 -hydroxycholesterols (Lund et al. 1992). Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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Figure 7.1. Common cholesterol autoxidation products. I: 7 α-hydroxycholesterol, II: 7 -ketocholesterol, III: 7β-hydroxycholesterol, IV: cholesterol-5α,6α-epoxide, V: cholestane-3,5α,6β-triol, VI: cholesterol-5β,6β-epoxide, VII: 5,6 -secosterol.

Figure 7.2. Enzymatic transformations of cholesterol into oxysterols. CYP7A1: cholesterol 7 αhydroxylase, CYP3A4: cytochrome P450 3A4, CYP46: cholesterol 24 -hydroxylase, CYP27A1: sterol 27 -hydroxylase, Ch -25-OHlase: cholesterol 25 -hydroxylase.

The third mode of for mation of oxysterols is through enzymatic metabolism of cholesterol. There are numerous cholesterol metabolizing enzymes, man y of w hich belong to the c ytochrome P450 (CYP) f amily (Pikuleva 2006). Figure 7.2 shows several enzymatic transfor mations of cholesterol into dif ferent o xysterols. Cholesterol 7 α -ydroxylase h (CYP7A1) is a

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hepatic enzyme that con verts cholesterol into 7 α -ydroxycholesterol, h an important intermediate in bile acid biosynthesis (Bj ö rkhem&Eggertsen 2001 Part ). of the 7α -ydroxycholesterol h formed in the li ver leaks out into the circulation, and its concentration in plasma cor relates to the activity of CYP7A1 (Bj örkhem et al. 1987). The drug-metabolizing enzyme CYP3A4 has been shown to con vert cholesterol into 4 β -ydroxycholesterol h (Bodin et al. 2001 ).We have suggested that this o xysterol can be used as an endo genous marker of CYP3A4 and CYP3A5 activity (Diczf alusy et al. 2008). The brain -specif c cholesterol 24 -hydroxylase (CYP46A1) converts cholesterol into 24S -hydroxycholesterol, a transpor t for m of cholesterol, w hich is important for maintenance of cholesterol homeostasis in the brain (L ütjohann et al. 1996). The mitochondrial enzyme sterol 27-hydroxylase (CYP27A1) is important for the side-chain oxidation during bile acid biosynthesis (Ja vitt 2002). In contrast to all the cholesterol metabolizing enzymes described abo ve, cholesterol 25-hydroxylase is not a c ytochrome P450 enzyme. This enzyme, w hich converts cholesterol into 25 -hydroxycholesterol, belongs to a g roup of enzymes that utilize o xygen and diiron cofactors to catalyze hydroxylation reactions (Lund et al. 1998). Several oxysterols have been used to monitor oxidative processes in vitro, such as oxidation of low density lipoprotein (Breuer et al. 1996, Dzeletovic et al. 1995a, Patel et al. 1996). The situation in vivo is more comple x because some of the o xysterols can be for med either b y cholesterol auto xidation or b y enzymatic con version of cholesterol. Measurement of these oxysterols in tissues will thus not re veal their mode of for mation. This is the case, e.g., 7 α -ydroxycholesterol, h 4β -ydroxycholesterol, h and 25 - yhdroxycholesterol. However, the major part of 4β -and 7α-hydroxycholesterol in the circulation is formed by the enzymes cholesterol 7 α-hydroxylase and cytochrome P450 3A4, respectively. The sidechain oxidized oxysterol 25 -hydroxycholesterol is a classical cholesterol auto xidation product (Smith 1981) but it can also be for med by the enzyme cholesterol 25 -hydroxylase (Lund et al. 1998). In pig and mouse liver it is formed as a by-product during enzymatic cholesterol oxidation by CYP27 (Li et al. 2007, Lund et al. 1993). It has not been sho wn, however, whether this mechanism operates in humans. The two oxysterols 7 β-hydroxycholesterol and 7 -ketocholesterol are assumed to be specif c cholesterol auto xidation products because no enzymes ha ve been found that catal yze their formation. It has been sho wn, ho wever, that in humans there is an intercon version of 7 βhydroxycholesterol and 7 -ketocholesterol as shown in Figure 7.3 (Larsson et al. 2007). The oxysterol 3 β -ydroxy h - 5 -xoo - 5,6 - secocholestan - 6(5,6 - al - secosterol)has attracted great interest recentl y. This o xysterol has been proposed as an ozone e xposure biomark er and is formed in the lung w hen cholesterol is e xposed to ozone (Pr yor et al. 1992, Pulfer & Murphy 2004). In addition, it has been identif ed in human atherosclerotic ar teries (Wentworth et al. 2003). Some uncertainty regarding the mode of formation of 5,6-secosterol has arisen, however, since it was shown that it is not e xclusively formed by cholesterol ozonolysis but can also be formed by Hock cleavage of cholesterol 5 α -ydroperoxide h (Brinkhorst et al. 2008 ).

Figure 7.3. In humans there is an interconversion of 7 β-hydroxycholesterol and 7 -ketocholesterol. This interconversion is probably due to the enzyme 11 β-hydroxysteroid dehydrogenase (11β-HSD).

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TRANSPORT OF OXYSTEROLS IN THE CIRCULATION The oxysterols are transpor ted in the human circulation in dif ferent ways, depending on the polarity of the oxysterol. Non-polar oxysterols such as 4α -4,β -24 , - and , 27 - yhdroxycholesterol are mainl y transpor ted in high density (HDL) and lo w density (LDL) lipoproteins. The 4-hydroxylated oxysterols are distributed in the lipoprotein fractions in the same w ay as cholesterol, with approximately two-thirds in the LDL fraction (Bodin et al. 2001), while 24 - and 27-hydroxycholesterols are equally distributed between the LDL - and HDL -fractions (Babiker & Diczfalusy 1998). More polar oxysterols are transported to a higher degree in the lipoproteinfree fraction in plasma. While onl y 41% of the cholestane -3β ,5α ,6β-triol is found in the lipoprotein-free fraction of plasma, 96% of the more polar oxysterol 3β -ydroxy h - 5 - cholestenoic acid is transpor ted in this fraction (Babik er 1998). The plasma enzyme lecithin -cholesterol acyltransferase (LCA T) catal yzes the esterif cation of o xysterols to long -chain f atty acids at the 3 β-hydroxyl g roup (Lin & Morel 1996, Szedlacsek et al. 1995). Most o xysterols in plasma for m monoesters, but 27 -hydroxycholesterol ma y for m a 3 β ,27 - diester(Szedlacsek et al. 1995). Esterifcation of o xysterols decreases their polarity and increases their association to lipoproteins. 3β - Hydro xy - 5 - cholestenoic acid, which is not esterif ed in plasma, is associated with lipoproteins to a v ery small extent (Babiker 1998).

TOXICITY OF OXYSTEROLS TO CELLS Several oxysterols have been reported to be cytotoxic to cultured cells, e.g. rabbit aortic smooth muscle cells (P eng et al. 1979), U937 cells (a human monoc ytic cell line) (Lemaire -Ewing et al. 2005, O’Callaghan et al. 2001), human monocyte/macrophages (Clare et al. 1995), smooth muscle cells from human umbilical ar teries (Zhou et al. 1993), and HL -60 cells (a human monocytic cell line) (Aupeix et al. 1995). The toxicity may be due to inhibition of 3 -hydroxy3-methylglutaryl (HMG) CoA reductase, the rate -limiting enzyme in cholesterol synthesis (Peng et al. 1979) or induction of apoptosis (A yala-Torres et al. 1997, Nishio & Watanabe 1996). Micromolar concentrations of oxysterols were used in many investigations of oxysterol toxicity. The concentrations of the quantitati vely dominating o xysterols in human plasma are in the nanomolar range (Dzeleto vic et al. 1995b). Experiments in human monoc yte-macrophages re vealed that w hen cholesterol w as added simultaneously with the c ytotoxic 26 -hydroxycholesterol, the to xicity was inhibited (Clare et al. 1995). Likewise, it w as found that w hen murine J774A.1 macrophages w ere treated with pure 7 -ketocholesterol, it triggered an o xidative burst leading to initiation of apoptosis, but cotreatment with 7β-hydroxycholesterol or an oxysterol mixture counteracted the proapoptotic effect of 7 -ketocholesterol (Biasi et al. 2004). It has recentl y been repor ted that ATP-binding cassette transpor ters (ABCs) promote eff ux of o xysterols from cells and in this w ay protect cells from oxysterols-induced cell death. ABCA1 has thus been shown to mediate a rapid f ux of 25 -hydroxycholesterol from intact cells (T am et al. 2006) and ABCG1 promotes f ux of 7β-hydroxycholesterol (Engel et al. 2007) and 7 -ketocholesterol (Terasaka et al. 2007) from different cell types. Another transporter, organic anion transporting polypeptide 2 (oatp2), has been implicated in the transport of 24S-hydroxycholesterol over the blood-brain barrier (Ohtsuki et al. 2007). As pointed out abo ve, a lar ge fraction of the plasma o xysterols is esterif ed. Esterif cation immobilizes the oxysterols in the lipoproteins, limiting their access to cells. It is thus diff cult to extrapolate the in vitro data on o xysterol toxicity to the in vivo situation.

MEASURMENTOF OXYSTEROLS Determination of oxysterols in human plasma is diff cult due to the simultaneous presence of cholesterol in a concentration that ma y be 10,000 to 100,000 times higher than the concentration of the o xysterol. Ev en the slightest o xidation of cholesterol during sampling, sample

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preparation, or analysis may lead to artifactual oxysterol formation in concentrations exceeding those originally present in the sample. It is therefore important to separate cholesterol from the oxysterols as early as possible during sample preparation. It is also impor tant to minimize the risk for cholesterol auto xidation b y adding antio xidants and/or chelating agents to pre vent oxidation. Oxysterols are present in plasma in nanomolar concentrations (Dzeletovic et al. 1995b). Most oxysterols do not contain an y chromophore, thus making it impossib le to detect them b y UVabsorption at the low concentrations found in living tissues. Therefore, high performance liquid chromatography (HPLC) with UV detection has had limited use for o xysterol anal ysis. An exception is 7 α -ydroxy h - 4 - cholesten - 3 - one, which can be easily detected at 254 nm (Axelson et al. 1988). Hydro xycholesterols can be con verted into o xysterols containing a 3 -keto-∆4 structure absorbing UV-light at 235 to 240 nm by incubation with microbial cholesterol oxidase (Teng & Smith 1995, Zhang et al. 2001). The labile cholesterol h ydroperoxides can be deter mined b y HPLC with electrochemical detection. The detection method is specif c for the hydroperoxides, and the common h ydroxycholesterols or 7 -ketocholesterol do not gi ve an y signal with this detection system (K orytowski et al. 1991). In addition, cholesterol h ydroperoxides are unstab le and 7 β-hydroperoxycholesterol is therefore unsuitab le for quantitation in clinical settings. ANALYSIS OF OXYSTEROLS BY GC -MS The most widel y used method for o xysterol analysis is gas liquid chromato graphy (GC). The oxysterols are usually derivatized to trimethylsilyl ether derivatives before analysis to improve their stability and chromato graphic proper ties. Gas chromato graphy gives good resolution of the different sterols, and can be used in combination with a mass spectrometric detector isotope dilution methodology, which increases the specif city and accurac y. The authors ha ve developed a method based on isotope -dilution GC -MS using deuterium -labeled inter nal standards (Dzeletovic et al. 1995b) suitable for deter mination of plasma o xysterols. The principal steps of the method , which deter mines nine dif ferent oxysterols and utilizes individual deuteriumlabeled internal standards, are outlined in Figure 7.4. A mix of deuterium-

Figure 7.4. Principal steps of a method based on isotope -dilution gas chromatography -mass spectrometry (GC -MS) to determine oxysterols in human plasma. Deuterium -labelled internal standards are added to 1 ml of plasma. The sample is exposed to mild alkaline hydrolysis to cleave oxysterol esters. After solvent extraction oxysterols are separated from cholesterol by solid phase extraction. The oxysterol fraction is f nally derivatized to trimethylsilyl ethers and analyzed by gas chromatography -mass spectrometry.

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labeled internal standards is added to 1 ml of EDTA-plasma, which is then subjected to mild alkaline h ydrolysis. The h ydrolysis conditions are critical because too harsh conditions will result in the decomposition of 7 -ketocholesterol (Park et al. 1996), while too mild conditions will result in incomplete h ydrolysis of 27 -hydroxycholesterol. The h ydrolysis is perfor med under an iner t atmosphere to pre vent cholesterol auto xidation. After solv ent e xtraction the sample is subjected to solid phase e xtraction on a silica car tridge to separate o xysterols from cholesterol. The o xysterol fraction is deri vatized to trimeth ylsilyl ethers and anal yzed b y GC-MS using the selected ion monitoring mode. Use of individual deuterium-labelled standards is a great advantage when analyzing multiple oxysterols simultaneously. The oxysterols differ in polarity and therefore ha ve different chromatographic proper ties in the solid phase e xtraction step. Fur thermore, the stability of the oxysterols to the h ydrolysis conditions dif fers signif cantly. Therefore, use of indi vidual deuterium-labelled inter nal standards is highl y prefer red o ver other inter nal standards, e.g., 19-hydroxycholesterol, which has been used frequentl y. The deuterium -labelled internal standards can be synthesized as described in Dzeleto vic et al. (1995b) but some of them are commercially available and others are a vailable through custom synthesis. ANALYSIS OF OXYSTEROLS BY LC -MS/MS In recent years, liquid chromatography—tandem mass spectrometry (LC-MS/MS)—has developed rapidly and a number of methods ha ve been de veloped for the anal ysis of o xysterols in tissues. LC -MS/MS with electrospray ionization (ESI) has been used for the deter mination of oxysterols (McDonald et al. 2007), but generall y oxysterols show poor ionization eff ciency with this ionization technique. Therefore, o xysterols ha ve been deri vatized to impro ve the ionization properties and thereby increase the sensiti vity of the method considerab ly. Derivatization of oxysterols into picolinyl esters increased the sensiti vity 1,000 times compared to the underivatized analytes (Honda et al. 2008). Another strategy to improve ionization properties of oxysterols for LC -ESI-MS analysis includes an enzymatic step in w hich oxysterols containing a 3 β -ydroxy h -∆5 str ucture are con verted into 3 -oxo-∆4 steroids b y cholesterol oxidase follo wed b y deri vatization with the Girard P reagent to gi ve Girard P h ydrazones (Griff ths et al. 2006). This method has been applied to determination of oxysterols in rat brain (Karu et al. 2007). Still another w ay to increase the sensiti vity 1,000 -fold is the deri vatization of oxysterols with N ,N-dimethylglycine to yield mono - or diesters, w hich are then anal yzed by LC - ESI - MS (Jiang et al. 2007 ). Oxysterol anal ysis using atmospheric pressure chemical ionization (APCI) has also been reported (Burkard et al. 2004, DeBarber et al. 2008), but the sensiti vity is not comparab le to the ESI methods using derivatization. The oxysterol 5,6 -secosterol, containing a reactive aldehyde function, can be deri vatized with 2,4 -dinitrophenylhydrazine to obtain an increase in sensitivity down to picogram levels when analyzed by APCI (Wentworth et al. 2003).

OXYSTEROLS AS MARKERS OF OXIDATIVE STRESS IN VITRO OXIDATION OF LDL Oxidative stress can be def ned as a per turbation of a steady state condition in w hich the free radical f ux is balanced b y the antio xidant defenses. The steady state condition occur ring in the healthy organism can be disturbed w hen the f ux of free radicals increases and/or antio xidants become reduced. F ree radicals escaping the antio xidant system attack an y biomolecule, but the most sensiti ve are lipids. A well-studied model of lipid pero xidation is the o xidation of low density lipoprotein (LDL), w hich is very relevant for the initiation and de velopment of atherosclerosis. As a consequence of lipid pero xidation, a series of o xidation products are formed, including malondialdehyde (MDA), which modif es apoB, resulting in the recognition

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of the modif ed LDL by the scavenger receptor (Esterbauer et al. 1992). Other aldehydes produced during LDL oxidation, such as 4-hydroxynonenal, have even higher reactivity compared to MDA. Free-radical-mediated LDL o xidation produces se veral dif ferent types of o xidized lipids other than aldehydes, including oxidized phospholipids with PAF-like structure (Subbanagounder et al. 2000), isoprostanes (Morrow et al. 1990), and oxysterols (Björkhem & Diczfalusy 2002). Oxidized lipids, found in atherosclerotic plaques, LDL, or plasma, ma y be deter mined b y specif c and sensitive methods based on mass spectrometr y (Iuliano et al. 2003). Isoprostanes are generated b y free radical attack of arachidonic acid , and are isomeric to enzymaticall y formed eicosanoids. There is considerable interest in the biological activity of isoprostanes and other congeners (Craco wski et al. 2001, Pratic ò 1999). In addition to their functional role in oxidative-stress-induced cellular events, isoprostanes have particular relevance as noninvasive markers of oxidative stress status in vivo. Oxysterols are oxygenated derivatives of cholesterol, which have been shown to possess many potent biological activities of relevance in atherogenesis (Brown & Jessup 1999), and are produced during LDL oxidation (Dzeletovic et al. 1995a). Many studies ha ve detected o xysterols in human atherosclerotic plaques. In f act, much of the interest in the role of o xysterols in atherosclerosis can be traced to the suggestion that atherogenic ef fects of cholesterol in animal models ma y ha ve resulted from contaminating oxysterols in the cholesterol supplied with the diet (Imai et al. 1976). In an analogy to isoprostanes, there is considerab le interest in o xysterols as non -invasive markers of o xidative stress in vivo . It is generall y considered that 7 β -ydroxycholesterol h and 7 - ektocholesterol are produced by free -radical attack on cholesterol in vivo in mammals. Thus, 7 β -ydroxycholesterol h and 7 -ketocholesterol are cur rently used as mark ers of o xidative stress in vivo and are deter mined by sensitive and specif c mass spectrometr y methods (Iuliano et al. 2001, Iuliano et al. 2003 ,Micheletta et al. 2004a ,Porkkala - Saratahoet al. 2000 ). O XYSTEROLS IN ATHEROSCLEROTIC PLAQUES Atherosclerosis is a comple x, ir reversible disease of the ar terial tree causing an e xcess of morbidity and most of the deaths due to myocardial infarction and stroke. Two elements associated with all the stages of the disease are lipids and macrophages, w hich have a crucial role in the initiation and progression of the atherosclerotic process. Striking evidence suggests that the atherosclerotic process initiates in the intima with the accumulation into macrophages of lipids (Stary 2000) deri ved from LDL w hich has penetrated the endothelial bar rier. In the intima, LDL is retained and tak en up by macrophages, which progressively transform into foam cells, which eventually die and liberate their lipid content. The presence of o xidized derivatives of cholesterol-linoleate in human atheroma was reported by Harland et al. (1973). Oxysterols have very short half -lives relative to cholesterol and are present in v ery low concentrations in biological systems. They are almost invariably accompanied by an approximately 10 6 - foldexcess of cholesterol with the e xception of atheroma, w here the ratio of 7 -ketocholesterol to cholesterol is approximately 1 : 1,000 (Micheletta et al. 2004b). A mixture of oxysterols is present in atherosclerotic plaques, includingα7 -ydroxycholesterol, h cholesterol- 5β ,6β - epo xide, cholesterol - 5α ,6α - epo xide, 7β -ydroxycholesterol, h 7 - ektocholesterol, and 27 -hydroxycholesterol (Car penter et al. 1995, Garcia -Cruset et al. 1999, Garcia Cruset et al. 2001). Oxysterols, especiall y 7 β -ydroxycholesterol h and 7 - ektocholesterol, are major toxic components found in o xidized LDL and atheromas (Hughes et al. 1994). Other oxysterols, such as 25 -hydroxycholesterol, appear at lo w levels in the lesions w hile high concentrations of 27 -hydroxycholesterol ha ve been found in human atherosclerotic lesions (Mattsson Hult én et al. 1996). The high concentration of 27 -hydroxycholesterol in atherosclerotic lesions is a consequence of enzymatic transformation of cholesterol by macrophages. This can be thought of as a defence mechanism to eliminate e xcess cholesterol from the lesions (Bj ö rkhemet al. 1994 ).

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OXYSTEROLS VS. OTHER MARKERS: ISOPROSTANES, TBARS Both isoprostane and oxysterol research have produced unequivocal evidence of the occurrence of free - radical - mediated lipid oxidation in vivo from measurements in the b loodstream and in atherosclerotic plaques. The ar tifactual generation of lipid o xidation products during tissue processing has long been a matter of concer n. Most of the data on lipid pero xidation in plaques is based on the thiobarbituric acid assa y (TBARS), which is a relatively sensitive assay for many aldehydes. TBARS is the most widely used assay in oxidative stress studies because it is cheap and simple to perfor m in any laboratory. It requires onl y a boiling w ater bath for the color reaction and a spectrophotometer . However, it lacks specif city and is intrinsicall y prone to ar tifactual lipid pero xidation at the high processing temperature (100 °C). Therefore, its use should be limited to in vitro studies, such as LDL oxidation, to monitor lipid pero xidation. In vivo studies require specif c methods to measure compounds of interest, not only to detect the occurrence of oxidative stress but also to unravel biological activities of specif c oxidative stress indicators. In this conte xt, the unsur passed methodology is mass spectrometr y. Application of isotope dilution methods to the f eld of oxidative stress studies has allowed the quantif cation of several isoprostanes and o xysterols in vivo . To unequi vocally e xclude the occur rence of processing artifacts in these studies, a split -sample technique with deuterium -labelled arachidonic acid or cholesterol has been used to ascer tain that no ar tifactual pero xidation occurs during tissue processing (Chisolm et al. 1994, Iuliano et al. 2003, Pratic ò et al. 1997). Actually, mass spectrometr y assay of isoprostanes are repor tedly the gold standard method to study o xidative stress in vivo (Milne et al. 2007). This concept is based on the follo wing issues: (1) isoprostanes are generated via free radical oxidation of arachidonic acid, and (2) are chemically stable; (3) their formation can be inhibited by antioxidants both in vitro and in vivo ; and (4) the isotope dilution method used to measure isoprostanes is specif c. All of these issues apply to free radical -mediated oxidation products of cholesterol, o xysterols. Therefore, measurement of 7 β -ydroxycholesterol h and 7 - ektocheolestrol by isotope - dilutionmass spectrometry should be analo gous to the isoprostane methodolo gy to study o xidative stress in vivo . In addition, the concentrations of oxysterols in the circulation are 100 to 1,000 times higher than the concentrations of isoprostanes. OXYSTEROLS IN EXPERIMENTAL ANIMAL MODELS One of the issues raised in connection with the o xidized LDL hypothesis of atherosclerosis is the potential contribution of o xidized lipids in the diet. F eeding New Zealand White (NZW) rabbits an oxysterol-rich (5%) diet resulted in a 100% increase in f atty streaks, the initial lipid lesion of atherosclerosis, in the aor ta (Staprans et al. 1998). In another study, a diet containing 6% oysterols given to NZW rabbits pro voked a 64% increase in total aor tic cholesterol compared to the pure cholesterol control animals (V ine et al. 1998). Accelerated atherosclerosis was also observed in LDL-receptor-def cient mice and in ApoE-def cient mice fed an oxysterolcontaining diet (Staprans et al. 2000). Macrophages from atherosclerotic ApoE-/- mice, w hich are under o xidative stress, w ere reported to contain more o xysterols than macrophages from control animals, and to release two-fold more superoxide anions and oxidize LDL 2.5-fold more than control (C57BL6) mice. Dietary supplementation of vitamin E to ApoE-/- mice resulted in a reduction in macrophage oxysterol content that w as associated with a reduction in supero xide anion release and LDL oxidation, compared with unsupplemented E - / mice (Rosenblat &Aviram 2002 ). 7-Oxygenated oxysterols are increased in diabetic rats. After four w eeks of streptozotocin injection in rats there w as a signif cant increase in 7 α -ydroperoxy h - ,7β -ydroperoxy,7 h αhydroxy - and 7β-hydroxycholesterol and 7 -ketocholesterol in the kidne ys, hear t, and li ver compared to control rats (Y oshioka et al. 2005). An increase in o xysterols was found in the

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myocardium of diabetic rats, suggesting that o xysterols may play a role in the de velopment of diabetic cardiomyopathy (Matsui et al. 1997). Experiments were perfor med in rats and mice in w hich acetylated LDL or a ch ylomicron k was administered intraveremnant-like emulsion (both enriched with [ 14 C]7 -etocholesterol) nously. In both species there was a rapid metabolism of 7 -ketocholesterol, which was excreted in feces, and there w ere no signs of accumulation of 7 -ketocholesterol in the aor ta. These studies suggested that dietar y 7 -ketocholesterol mak es little contribution to atherosclerosis (Lyons & Brown 2001 Lyons , et al. 1999 ).

O XYSTEROLS AND CLINICAL CONDITIONS O XYSTEROLS AND NEURODEGENERATIVE DISORDERS Aberrant cholesterol metabolism has been implicated in Alzheimer’s disease and other neurological disorders (Vaya & Schipper 2007). The human brain contains about 20 mg cholesterol/g tissue, exceeding that of any other organ in the body. The central nervous system accounts for 2% of the w hole body mass, y et contains 25% of the total unesterif ed body cholesterol. Oxysterols seem to be k ey re gulators of brain cholesterol metabolism. The b lood brain barrier prevents exchange of brain cholesterol with lipoprotein cholesterol in the circulation. There is a slow continuous synthesis of cholesterol in the brain. In order to maintain cholesterol homeostasis, the brain must eliminate cholesterol, and one impor tant mechanism to achie ve this is the enzymatic con version of cholesterol into 24S -hydroxycholesterol, which can transverse the b lood-brain bar rier and be transpor ted in the circulation to the li ver for fur ther metabolism (Lütjohann et al. 1996). In humans, 24S -hydroxycholesterol is only synthesized in the brain and the concentration of this o xysterol in the circulation ma y therefore be used to monitor changes in cholesterol homeostasis in the brain. The enzyme responsible for the synthesis of 24S -hydroxycholesterol, CYP46, is located to the neurons (Lund et al. 1999). Neurodegenerative diseases therefore result in lo wer production of 24S -hydroxycholesterol, as has been shown for Alzheimer’s disease and multiple sclerosis (Bretillon et al. 2000; Leoni et al. 2002). Although most 24S -hydroxycholesterol for med in the brain transv erses the b lood-brain barrier, a small fraction ( <1%) is eliminated via the cerebrospinal f uid. For reasons not completely understood, the level of 24S -hydroxycholesterol in the cerebrospinal f uid is increased in patients with neurolo gical diseases and the concentration is more sensiti ve to changes in brain cholesterol homeostasis than the concentration of circulating 24S -hydroxycholesterol (Leoni et al. 2004). A recent study suggests that the concentration of 24S -hydroxycholesterol in cerebrospinal f uid may have a potential as a biomarker for mild cognitive impairment (Leoni et al. 2006). In neuronal cell cultures, o xidative stress accr uing from Aβ administration promotes the oxidation of cholesterol to 7 β-hydroxycholesterol. This oxysterol is neuroto xic at nanomolar concentrations and ma y therefore contribute to Aβ-related neurodegeneration in the brains of Alzheimer ’s disease subjects (Nelson & Alkon 2005 ).Recently, 5,6 - secosterol has been detected in human brains with Alzheimer disease, and has been shown to dramatically accelerate amioidogenesis in vitro (Zhang et al. 2004). O XYSTEROLS AND ATHEROSCLEROSIS There are relatively few studies on the use of plasma o xysterols as biomarkers for atherosclerosis. One study deter mined unesterif ed plasma o xysterol concentrations in patients with severe coronar y atherosclerosis and in controls and found no dif ference (v an P oppel et al. 1997). The authors ha ve found a signif cant dif ference in plasma 7 β -ydroxycholesterol h in patients with carotid atherosclerosis compared to controls (Micheletta et al. 2004b). In a study

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in Finnish men it w as found that ser um 7 β-hydroxycholesterol was the best predictor of progression of carotid atherosclerosis w hen more than 30 v ariables were tested (Salonen et al. 1997). Fur thermore, supplementation with vitamin E decreased the circulating le vel of 7 βhydroxycholesterol (Micheletta et al. 2004b, P orkkala-Sarataho et al. 2000). It should be emphasized, however, that the absolute concentration dif ferences in these studies w ere small, and although it w as possib le to sho w statisticall y signif cant dif ferences betw een g roups of patients and controls, 7 β-hydroxycholesterol could not be used as a biomark er for clinical purposes in a single patient. There is a differential distribution of oxysterols in the atherosclerotic lesions at the coronary or carotid levels. 27 -hydroxycholesterol and 7 -ketocholesterol levels in the carotid lesions are similar to those of coronary lesions. In contrast, the amount of 7β -ydroxycholesterol h is higher in the coronaries compared to carotids (0.14 vs . 0.02 µg/mg tissue) (V aya et al. 2001). This differential distribution may explain the different manifestation of the atherosclerotic disease, which exhibits predominant carotid presentation in some individuals but predominant coronary disease in others. Moreo ver, a dif ferential distribution of plasma 7 β -ydroxycholesterol h and 7-ketocholesterol in patients with carotid and coronar y atherosclerosis has been found (Micheletta et al. 2004b). There are no data in the literature concerning the levels of oxysterols in peripheral v ascular disease, compared to the coronar y and carotid manifestations of atherosclerosis. O XYSTEROLS AND LIVER DISEASE A g rowing body of e vidence suppor ts a role for o xidative stress in li ver damage attributab le to a number of patholo gical agents. Reacti ve oxygen species act as proapoptotic, proinf ammatory, and prof brogenic mediators in dif ferent models of li ver injur y, and thus pla y an important role in all of the pathoph ysiological steps that lead to li ver cir rhosis (Poli 2000). Patients with chronic li ver disease have increased levels of plasma 7 β -ydroxycholesterol h and 7-ketocholesterol, and reduced le vels of α - tocopherol(Corradini et al. 2005 ).Furthermore, in comparison with nor mal li ver, cir rhotic li ver contains higher concentrations of 7 ketocholesterol and 7 β-hydroxycholesterol and lo wer levels of α - tocopherol(Corradini et al. 2005; Iuliano et al. 2003). Oxidative stress is implicated in the patho genesis of hepatic ischemia -reperfusion injur y, a major determinant of poor graft function after liver transplantation. The authors prospectively investigated the association betw een the plasma preoperati ve oxidative stress and the occur rence of poor g raft function after li ver transplantation (Cor radini et al. 2005). The plasma concentration of 7β -ydroxycholesterol h was signif cantly higher in transplants with subsequent poor graft function compared with those with initial good g raft function. In a lo gistic re gression model, the 7 β-hydroxycholesterol concentration in plasma w as an independent predictor of poor g raft function with an odds ratio of 1.17 (95% CI, 1.02 to 1.33, P = 0.028). It is note worthy that in this study on patients with li ver cir rhosis the o xysterol levels w ere increased w hile the cholesterol le vels w ere decreased. In patients with f amilial combined h yperlipidemia (see belo w) w ho ha ve increased cholesterol le vels w e ha ve observed increased o xysterol levels. When the o xysterol levels in the h yperlipidemia patients were related to cholesterol, the o xysterol/cholesterol ratio w as still ele vated, suggesting that the elevated oxysterol levels in the tw o patient g roups were not dependent on the cholesterol level. O XYSTEROLS AND DIABETES Increased production of reacti ve oxygen species has been implicated in the initiation and progression of diabetes mellitus, including diabetic neuropath y (F igueroa-Romero et al. 2008), and it is likely that oxidative stress accounts for the unexplained increase in cardiovascular risk

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observed in diabetes (Stephens et al. 2008). Several studies ha ve focused on measuring sur rogate markers of o xidative stress in diabetes, but the majority of these studies adopted non specifc mark ers. Plasma 7 -ketocholesterol, 7 α -ydroxycholesterol, h 7β -ydroxycholesterol, h 27 - yhdroxycholesterol, and 5α ,6α-epoxy-cholestanol w ere studied in patients with diabetes mellitus and h ypercholesterolemia. Plasma 7 -ketocholesterol w as signif cantly higher in patients with diabetes or hypercholesterolemia than in the control group (Murakami et al. 2000). The increased le vel of 7 -ketocholesterol in patients with type 2 diabetes mellitus w as conf rmed in a recent study (Endo et al. 2008). It is likely that specif c markers of oxidative stress such as isoprostanes and o xysterols could be usefull y incorporated in the future into risk prediction in diabetes and cardio vascular disease. O XYSTEROLS AND HYPERLIPIDEMIA Only a fe w published studies are found in the literature concer ning oxysterol levels in hyperlipidemias. In a recent ar ticle (Arca et al. 2007) the authors ha ve repor ted on plasma 7 βhydroxycholesterol and 7 -ketocholesterol in patients with f amilial combined h yperlipidemia (FCHL). FCHL is the most common inherited lipid disorder associated with an increased risk of atherosclerosis that is not full y explained by the metabolic disturbances of these patients. FCHL patients showed markedly increased levels of oxysterols and reduced alpha -tocopherol/ total lipids compared to controls. These differences were independent of the presence of clinical atherosclerosis and persisted after cor rection for hyperlipidemia. Two drugs, atorvastatin and fenof brate, were used in this study to elucidate the mechanism of increased o xidative stress status in FCHL patients. Both dr ugs signif cantly improved the lipid prof le and caused a comparable decrease in plasma oxysterols, with the normalization of 7-ketocholesterol and a signif cant reduction of 7 β -ydroxycholesterol. h Atorvastatin and fenof brate ha ve dif ferent str uctures, and it is unlik ely that the ef fects on o xidative stress w ere dependent on putative antioxidant action, which is usually implied when markers of oxidative stress are reduced b y treatments. The authors suggested that the increased production of o xysterols seen in FCHL indi viduals is the consequence of an altered lipid status rather than the result of upregulated cholesterol biosynthesis.

CONCLUSIONS Oxidative stress plays a central role in diverse pathophysiological settings. Free radicals attack almost all biomolecules, but lipids, being the most abundant components of cell architecture, are the preferential targets. Oxidized lipids are of g reat interest because they serve as markers to assess oxidative stress status in vivo and due to their potent biolo gical activities that confer to these molecules the status of o xidative stress effectors. Free radical attack on cholesterol gi ves rise to an ar ray of cholesterol o xidation products, oxysterols, which are structurally similar to the parent compound. Mass spectrometric measurements of oxysterols, especially 7 β -ydroxycholesterol h and 7 - ektocholestrol, have shown interesting results in a number of e xperimental and clinical conditions and these o xysterols are promising biomarkers for future studies on o xidative stress in vivo .

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Rosenb lat M ,A viram M .2002 . Oxysterol - inducedactivation of macrophage NADPH - xoidase enhances cell -mediated oxidation of LDL in the atherosclerotic apolipoprotein E def cient mouse: inhibitory role for vitamin E . Atherosclerosis 160 : 69 – 80 . RussellDW. 2000 . Oxysterol biosynthetic enzymes . Biochim Biophys Acta 1529 : 126 – 35 . SalonenJT , Nyyss ö nenK , Salonen R , P orkkala - Sarataho E ,T uomainen T- P, Diczfalusy U , Bj ö rkhemI. 1997 . Lipoprotein oxidation and progression of carotid atherosclerosis . Circulation 95 : 840 – 5 . SchroepferJr GJ. 2000 . Oxysterols: Modulators of cholesterol metabolism and other processes . Physiol Rev 80 : 361 – 554 . SmithLL. 1981 . Cholesterol autoxidation . New York : Plenum Press . StapransI , P an X - M Rapp , JH , F eingold KR. 1998 . Oxidized cholesterol in the diet accelerates the development of aor tic atherosclerosis in cholesterol -fed rabbits . Arterioscler Thromb Vasc Biol 18 : 977 – 83 . StapransI , P an X - M Rapp , JH , Grunfeld C , F eingold KR. 2000 . Oxidized cholesterol in the diet accelerates the development of atherosclerosis in LDL receptor - and apolipoprotein E -def cient mice. Arterioscler Thromb Vasc Biol 20 : 708 – 14 . Stary HC. 2000. Lipid and macrophage accumulations in ar teries of children and the de velopment of atherosclerosis . Am J Clin Nutr 72 : 1297S – 306S . StephensJW , Khanolkar MP , Bain SC .2008 .The biological relevance and measurement of plasma markers of oxidative stress in diabetes and cardio vascular disease . Atherosclerosis 202 : 321 – 9 . SubbanagounderG , Leitinger N , Schw enke DC ,W ong JW , Lee H , Rizza C ,W atson AD , F aull KF , F ogelman AM , Berliner JA . 2000 . Determinants of bioactivity of oxidized phospholipids. Specif c oxidized fatty acyl groups at the sn -2 position . Arterioscler Thromb Vasc Biol 20 : 2248 – 54 . SzedlacsekSE ,W asowicz E , Hulea SA , Nishida HI , K ummerow FA , NishidaT . 1995 . Esterifcation of oxysterols by human plasma lecithin -cholesterol acyltransferase. J Biol Chem 270 : 11812 – 9 . am T SP , Mok L , Chimini G ,V asa M , Deele y RG .2006 .ABCA1 mediates high - affnity uptake of 25-hydroxycholesterol by membrane vesicles and rapid eff ux of oxysterol by intact cells . Am J Physiol Cell Physiol 291 : C490 – C502 . eng T JI , Smith LL .1995 . High - perfor mance liquid chromatographic analysis of human erythrocyte oxysterols as ∆4 - 3 etone - k derivatives . J Chromatogr 691 : 247 – 54 . erasaka T N,W ang N , Yv an - Charvet L ,T all AR. 2007 . High - densitylipoprotein protects macrophages from oxidized low-density lipoprotein -induced apoptosis by promoting eff ux of 7 - ektocholesterol via ABCG1 . Proc Natl Acad Sci USA 104 : 15093 – 8 . anv Poppel G ,avn de Vijver LPL , K osmeyer - SchuilT , Johanns ESD , KardinaalAFM ,avn de Bovenkamp P , Kruyssen DACM , K ok FJ. 1997 . Plasma oxysterol and angiographically determined coronary atherosclerosis: a case -control study. Biomarkers 2 : 373 – 8 . aya V J,A viram M , Mahmood S , Ha yek T , Grenadir E , Hof fman A , Milo S .2001 . Selective distribution of oxysterols in atherosclerotc lesions and human plasma lipoproteins . Free Rad Res 34 : 485 – 97 . aya V J , Schipper HM .2007 . Oxysterols, cholesterol homeostasis, and Alzheimer disease . J Neurochem 102 : 1727 – 37 . entworth W Jr. P , Nie va J , T akeuchi C , Galv e R ,W entworth AD , Dille y RB , DeLaria GA, Sa ven A , Babior BM , Janda KD , EschenmoserA , Lerner RA. 2003 . Evidence for ozone formation in human atherosclerotic ar teries. Science 302 : 1053 – 6 . ine V DF , Mamo JCL , Beilin LJ , MoriTA , Croft KD . 1998 . Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to o xidation and increase aortic cholesterol concentration of rabbits . J Lipid Res 39 : 1995 – 2004 . oshida Y Y , Niki E .2004 . Detection of lipid peroxidation in vivo :Total hydroxyoctadecadienoic acid and 7 -hydroxycholesterol as oxidative stress marker. Free Rad Res 38 : 787 – 94 .

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oshioka Y N , Adachi J , UenoY , Y oshida K .2005 . Oxysterols increase in diabetic rats . Free Radic Res 39 : 299 – 304 . ZhangQ , P owers ET , Nie va J , Huf f ME , Dendle MA , Bieschk e J , Glabe CG , Eschenmoser A , W entworth P, Jr , Lerner RA , K elly JW. 2004 . Metabolite - initiatedprotein misfolding may trigger Alzheimer’ s disease . Proc Natl Acad Sci USA 101 : 4752 – 7 . ZhangZ , Li D , Blanchard DE , Lear SR , Erickson SK , SpencerTA . 2001 . Key regulatory oxysterols in liver: analysis as ∆4-3-ketone derivatives by HPLC and response to ph ysiological perturbations. J Lipid Res 42 : 649 – 58 . ZhouQ , SmithTL , K ummerow FA . 1993 . Cytotoxicity of oxysterols on cultured smooth muscle cells from human umbilical ar teries. Proc Soc Exp Biol Med 202 : 75 – 80 .

Chapter8 Lipid Peroxidation Originating α ,β - unsatur ated Aldehydes and Their Metabolites as Biomarkers F ran ç oise Gu éaud r

INTR ODUCTION One of the consequences of o xidative stress is the stimulation of F enton chemistry that gi ves rise to the oxidation of cellular lipids and the formation of lipid radicals that in turn can initiate the lipid peroxidation process. This chain reaction begins with the for mation of a single lipid – hydroperoxide molecule that gives rise to a huge production of lipid -hydroperoxide molecules if supero xide and free iron are present. This reaction can be stopped b y reducing the lipid hydroperoxides (achieved by class α Glutathione S-transferases [GSTs]) to lipid alcohols, but when the antioxidant defenses are overwhelmed, lipid-hydroperoxides can undergo degradation to form aldehydes, saturated ketones, and unsaturated aldeh ydes. There are numerous aldeh ydic compounds for med after o xidative stress/lipid pero xidation. Among α ,β-unsaturated aldehydes, which are more reacti ve compounds than alkanals, there are 2 -alkenals, alkadienals, alkatrienals, h ydroxy- or o xo-alkenals, and malondialdeh yde (MDA) (Esterbauer et al. 1990). They are of particular interest due to their quantitative importance and their reacti vity. Some of these α ,β-unsaturated alkenals are used as biomark ers of lipid o xidation/oxidative stress. However, due to their chemical reacti vity toward other molecules and their chemical instability, they are not easy to measure and their measurement ma y not ref ect the “cellular burden” of the aldeh yde (V olkel et al. 2005). Some of these aldeh ydes such as acrolein (2-propenal), 4 -hydroxyalkenals such as 4 -hydroxynonenal (HNE), or o xoalkenals such as 4-oxononenal (ONE) are electrophilic compounds and are readil y conjugated with the endo genous nucleophilic compound glutathione ( γ - Glu - Cys y; - Gl GSH), rendering them less chemically acti ve and more easil y e xcretable. This reaction can occur spontaneousl y but is also catalyzed by GSTs. Ho wever, GSH conjugates are not nor mally excreted unchanged in urine or in feces. The glutathione par t of these conjugates is fur ther metabolized into mercapturic acid derivatives (mercapturic acid pathway, [MAP]) and the conjugates are excreted and eliminated into urine. In this chapter , w e focus on the main reacti ve compounds and their urinar y metabolites, particularly mercapturic acids (MAs), which are stable compounds that can be measured using noninvasive sampling techniques and that represent promising biomarkers in the f eld of oxidative stress/lipid peroxidation research. Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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ALDEHYDES AS BIOMARKERS OF LIPID PER OXIDATION MDA has been one of the f rst compounds to be used as an inde x of lipid pero xidation. It has been widely and is still used for this pur pose. However, this compound can also arise from nonlipid biomolecules. It ma y also come from enzymatic lipid o xidation as a side product of eicosanoid formation (Janero 1990), which is why its use as a lipid peroxidation biomarker has been criticized. HNE is considered to be the most abundant 4 -hydroxyalkenal formed during lipid peroxidation. The literature concer ning this compound is no w e xtensive. Ho wever, Blair ’s g roup showed that ONE was also a major lipid -hydroperoxide–derived bifunctional electrophile. The 6-carbon analogue of HNE, 4 -hydroxyhexenal (HHE), may serve also as a biomark er of lipid peroxidation, par ticularly in n -3 polyunsaturated f atty acid - (PUFA) rich tissues, such as the CNS, as this compound w as shown to be increased in the spinal cords of patients suf fering from sporadic amyotrophic lateral sclerosis (Shibata et al. 2004). Although lipid pero xidation does not seem to be a major source of acrolein (Ste vens and Maier 2008), and its measurement is especiall y used as a biomark er of e xposure to en vironmental sources, par ticularly cigarette smok e, Uchida ’s g roup sho wed that its protein -bound form could be used as a biomark er of endo genous lipid pero xidation (Uchida et al. 1998). Figure 8.1 shows the main α ,β-unsaturated alkenals used as lipid pero xidation biomarkers.

SOURCES MDA comes from the o xidation of PUFAs bearing more than tw o double bonds such as linolenic acid, arachidonic acid , eicosapentaenoic acid (EP A), and docosahe xaenoïc acid (DHA) (Draper et al. 2000, Pryor et al. 1976). Hexanal, HNE, and ONE come from the o xidation of PUFAs of the ome ga-6 f amily such as linoleic, γ-linolenic, and arachidonic acids. Propanal and HHE come from the o xidation of PUFAS of the ome ga-3 family such as α - linolenicacid O

O

O

O Malondialdehyde

Crotonaldehyde

Acrolein

OH O

4-hydroxy-nonenal

OH R

O

OH O

4-hydroxy-alkenal 4-hydroxy-hexenal

O O

4-oxo-nonenal

Figure 8.1. Different α,β-unsaturated aldehydes originating from the lipid peroxidation process.

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomark ers 119

and EPA and DHA (Guichardant et al. 2004, Van Kuijk et al. 1990). All of these compounds have an endo genous and e xogenous origin because f atty acids are present in the body and in foodstuffs. Acrolein is belie ved to arise from pol yamine metabolism and also from lipid pero xidation but the mechanisms involved are not fully understood. However, the major sources of acrolein seem to be exogenous, like the heating/baking of carbohydrates; the incomplete combustion of petrol, wood, plastic or tobacco products; and the frying of foods in oil (Esterbauer et al. 1991, Stevens and Maier 2008). FORMA TION HNE is a de gradation product of h ydroperoxides of ome ga-6 fatty acids with 4 -hydroperoxy2E-nonenal as the inter mediary compound. Two major pathways are involved that should also apply to other f atty acid h ydroperoxides (Schneider et al. 2001). ONE is also a de gradation product of hydroperoxides of omega-6 fatty acids (Lee and Blair 2000). The formation of MDA involves the e xistence of a h ydroperoxy epidio xide or a h ydroperoxy bic ycloperoxide (Esterbauer et al. 1990). The formation of acrolein depends on the source of the compound because precursors can be carbohydrates, amino acids, and lipids. Enolization of hydroxyacetone formed after 3,4-retro aldol clea vage of glucose seems to be an impor tant pathw ay of for mation of acrolein from carbohydrates (Stevens and Maier 2008). From lipids, acrolein can arise from the glycerol part of triacyl and diac yl glycerides and its for mation from f atty acids is still a matter of debate (Stevens and Maier 2008). Esterbauer et al. proposed that acrolein would come from the center of the aliphatic chain of arachidonic acid after tw o carbon -carbon bond cleavages (Esterbauer et al. 1991 ).

ASSA Y SINGLEDETERMINATION Malondialdeh yde ( MDA ) Among aldehydic products of lipid peroxidation, MDA is one of the better known and has been the most used as a mark er of o xidative stress. It has been measured since the 1960s in man y different biolo gical tissues and f uids, and in foods. Detection and quantif cation ha ve been achieved with various methods described in the literature (Del Rio et al. 2005). Its colorimetric determination by the thiobarbituric acid reactive substances (TBARS) assay (Yagi 1976), using colorimetric (532 to 535 nm) or f uorimetric (e xcitation at 532 nm and emission at 553 nm) detection, has been widel y used because this method is easy and ine xpensive (Figure 8.2). However, this method has been criticized because of the lack of specif city of the TBARS reaction because thiobarbituric acid can react with all carbon yls that include aldehydes as well as sugars, amino acids, and bilir ubin, to produce a pink to red color that can interfere with measurements of MD A (Knight et al. 1988). To o vercome this prob lem, chromato graphic techniques coupled with sensiti ve detection ha ve been de veloped, allo wing separation and identif cation of the MDA-TBA complexes (Bird et al. 1983, Chirico 1994, Grotto et al. 2007, Lepage et al. 1991, Nielsen 1997, Templar et al. 1999). Most techniques used HPLC coupled with UV-visible detection of the TBA-complexes at 532 nm, but f uorimetric detection has also been used (Agarwal and Chase 2002, Del Rio et al. 2003, Fukunaga et al. 1993). As the treatment of samples to obtain the TBA complexes is car ried out at a high temperature that ma y generate artif cial oxidation of the samples, a protein precipitation step is usuall y added prior to the TBA reaction. Other deri vatization methods than the TBARS method have been de veloped to overcome the prob lem of ar tif cial oxidation of the matrix encountered in the sample preparation step (1 h at 100 ° C).

120 Chapter

8

O

+2

Malondialdehyde

S

Acid, heat

H N

S

O

HN O Thiobarbituric acid

OH HO

N

O

SH

N

N

+ 2 H2O

N OH

OH

Malondialdehyde/thiobarbituric acid pigment

Figure 8.2. The TBARS assay: two molecules of thiobarbituric acid react with one molecule of MDA and give a pigmented and f uorescent compound. HN

NH2

OH

NO2 O

+

4-hydroxynonenal NO2 Dinitrophenylhydrazine

OH

O2N N

N H

NO2 + H2O

Dinitrophenylhydrazone derivative

Figure 8.3. Principle of the DNPH derivatization of aldehydes: example of 4 derivatization.

-hydroxynonenal

MDA, like other aldeh ydes, can also be measured in f uids and tissues after deri vatization with 2,4 -dinitrophenylhydrazine (DNPH) (Pilz et al. 2000). DNPH reacts with aldeh ydes to form h ydrazone deri vatives that are stab le, non volatile, and ha ve an intense y ellow color (Figure 8.3). Sim and collaborators used HPLC with UV detection for the measurement of MD A in plasma, after DNPH deri vatization, with meth yl-MDA as an inter nal standard (Sim et al. 2003). Other deri vatization techniques ha ve been used , including diaminonaphtalene (Steghens et al. 2001 ), 1 - meth yl - 2 - phen ylindole (Gerard - Monnier et al. 1998 ), or 9 - fuorenylmethoxycarbonyl hydrazine (Mao et al. 2006). A LC -MS technique was developed for the identif cation of deri vatized MDA (Peiro et al. 2005) and se veral laboratories de veloped GC -MS techniques to measure MD A, using pentaf uorophenylhydrazine derivatization (Yeo et al. 1994) or phen ylhydrazine deri vatization and the use of a deuterated inter nal standard (Cighetti et al. 1999 ). Karatas et al. described a method to measure free MD A directly, without deri vatization in deproteinized plasma with HPLC separation and UV detection set at 254 nm (Karatas et al. 2002). High performance capillary electrophoresis (HPCE) has been also used to quantify free MDA directly (Wilson et al. 1997). Very recently, Lord et al. developed an on -line solid phase analytical deri vatization method coupled with HPLC and f uorescence detection to measure MDA (Lord et al. 2008).

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomark ers 121

The problem with the determination of MDA, and it is also tr ue for other lipid peroxidation products, is that the basal values obtained in a given tissue, for example in human plasma, may vary more than 10 times, according to the methodolo gy used, the laborator y, the preparation and derivatization steps, or e ven the nature of the b lood anticoagulant used, leading to doubts about the biolo gical signif cance of the data (Del Rio et al. 2005). The TBARS assay is still widely used although the most recent methods are much more accurate and reliab le, probably because the latter require sophisticated de vices and specialized personnel. 4 - Hydr oxyalkenals and HNE In many studies, HNE and other 4-hydroxyalkenal concentrations in different tissues and f uids were determined after derivatization with DNPH in the presence of butylated h ydroxytoluene (BHT) to prevent artifactual peroxidation (Esterbauer et al. 1989). Some authors used CH 2 Cl2 extraction and a thin la yer chromatography (TLC) pre -purif cation of 4 -hydroxyalkenals followed by HPLC with UV detection at 360 to 380 nm. Others replaced the solv ent extraction step by an Extrelut column extraction and the TLC purif cation step by column chromatography on silica gel (Strohmaier et al. 1995). Peak identif cation was based on the retention times of DNPH standards. Using this methodolo gy, HNE has been deter mined in many rat tissues and biof uids. Because 4 -hydroxyalkenals ha ve an intensi ve absor ption maximum at 220 nm, direct determination of HNE b y HPLC is possib le, after CH 2 CL2 e xtraction of the aldeh yde and subsequent cleaning of the samples by solid phase extraction on octadecyl-bonded silica (ODS) columns. P eak identif cation can be achie ved b y retention times of standards and b y mass spectrometry. GC-MS techniques are also widely used because they are very sensitive; HNE is derivatized with pentaf uorobenzyl-hydroxylamine to for m the pentaf uorobenzyl - xoime (PFB - xoime) of HNE, followed by silylation of the h ydroxyl group (Kinter et al. 1992, Selley et al. 1989, van Kuijk et al. 1986). Some authors impro ved the technique with the use of deuterated HNE standard (Selle y 1997, v an K uijk et al. 1995). Recentl y, Stopfor th et al. measured HNE in human urine using stir bar sorptive extraction-thermal desorption-GC-MS technique (Stopforth et al. 2006). A method using c yclohexanedione derivatization has also been emplo yed (Figure 8.4), followed by HPLC separation and f uorescence detection (Yoshino et al. 1986). Liu et al. employed HPLC with laser -induced f uorescence detection to measure HNE after deri vatization with a tagging reagent, 4 - (2 - carbazo ylpyrrolidin - 1 - yl) - 7 - nitro - 2,1,3 xadiazole, - benzo in rat hepatocytes (Liu et al. 1999). All of these methods for the deter mination of aldehydic lipid peroxidation products using GC -MS have been extensively reported and detailed in the v ery complete review of Spiteller et al, pub lished in 1999 (Spiteller et al. 1999). Gioacchini et al. de veloped an electrospra y (ESI) -MS method for the direct anal ysis of HNE in biolo gical f uids at the nanomolar range, with the use of a deuterated standard (Gioacchini et al. 1999). Very recentl y, Warnke et al. measured , b y LC -ESI-MS HNE, the reduced metabolite of ONE (w hich has the same mass as HNE) and the glutathione conjugate of HNE in mice lacking or that w ere not a specif c transpor ter for glutathione conjugates, namely RLIP76 (Warnke et al. 2008). Levels were determined for irradiated and non-irradiated tissue samples. Des Rosiers et al. proposed a reduction of the aldeh yde function to stab le alcohol using sodium boroh ydride, and fur ther detection b y GC -MS. They used this method for se veral aldehydes including HNE and MD A (Des Rosiers et al. 1993). This reduction w as also used by this group to measure HNE bound to thiol proteins with Raney Nickel to cleave the thioether linkages (Veronneau et al. 2002). Guichardant et al. measured the oxidized metabolites of HNE and HHE in human urine, namel y 4 -hydroxy-nonenoic acid (HN A) and 4 -hydroxyhexenoic acid (HHA), respectively, using NICI - GC - MS (Guichardant et al. 2004 ).

122 Chapter

8 Aldehyde O

R

O

+ O

O

H

Cyclohexanedione H

N

O

H

Ammonia

O

R

O + 3 H2O

N H

Dimedone derivative

Figure 8.4. Principle of the cyclohexanedione derivatization of aldehydes.

MUL TIPLE DETERMINATION Several groups have achieved the determination and measurement of multiple aldehydes originating from lipid pero xidation. This multiple deter mination seems par ticularly attracti ve because lipid peroxidation originating aldehydes are often specif c from their fatty acid (or fatty acid family precursor) and because these aldeh ydes play an impor tant role in the to xic effects of lipid peroxidation. Cordis et al. measured dif ferent aldeh ydes including HNE using DNPH deri vatization, pentane extraction, and HPLC combined with multichannel UV detection. P eak identif cation was achie ved using GC -MS (Cordis et al. 1994). With this method , Draper et al. measured several aldehydes including MDA in rat urine under conditions of pero xidative stress (Draper et al. 2000). Moreover, the different hydrazone derivatives share a common fragment ion that can be used for single ion monitoring (SIM) detection of aldeh ydes. Some authors used c yclohexanedione derivatization followed by SPE extraction and HPLC separation for multiple deter mination of aldeh ydes in b lood plasma and tissue homo genates (Holley et al. 1993, Matsuoka et al. 1996). Some authors improved the sensitivity of the method with miniaturization of the deri vatization procedure (Baile y et al. 1997) or with the use of MS-MS (O’Brien-Coker et al. 2001, Williams et al. 2005). Williams et al. used this methodology to measure acrolein and HNE concentration in brain in early Alzheimer’s disease and mild cognitive impair ment (Williams et al. 2006.). Thomas et al. de veloped a GC -MS analysis of aldehyde dinitrophen ylhydrazones, including acrolein and HNE (Thomas et al. 1995). Luo et al. (Luo et al. 1995) determined 22 saturated and unsaturated aldehydes including HNE and MDA using PFB -oxime derivatives and trimeth ylsilylation, and GC -MS detection. Br uenner et al. (Br uenner et al. 1996) compared the deri vatization method (PFB -oxime or o ximeTBDMS) followed by GC/MS quantif cation for the simultaneous detection of hexanal, nonanal, and HNE in plasma and li ver samples to gether with the use of deuterated inter nal standards. De Zwart et al. measured the urinar y excretion of se veral aldehydes including MD A in rats treated with diquat or N-nitrosodimethylamine using GC with electron capture detection after derivatization with PFB (de Zw art et al. 1999). Rauli et al. validated the measurement of MDA and HNE in plasma by NICI-GC-MS (Rauli et al. 1998). Andreoli et al. simultaneousl y measured se veral classes of aldeh ydes in e xhaled

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomark ers 123

breath condensate in smokers using LC/APCI-MS/MS after derivatization with DNPH (Andreoli et al. 2003), w hile others used solid phase microe xtraction with on -f ber deri vatization to determine volatile aldehydes by GC-MS in blood plasma (Deng and Zhang 2004) and in human breath (Svensson et al. 2007). Recently, Kawai et al. perfor med a lipidomic GC -MS analysis for lipid peroxidation-derived aldehydes in model lipid hydroperoxide and ferrous iron preparations and in mice treated with bromobenzene, an experimental model of in vivo lipid peroxidation (Kawai et al. 2007). These authors monitored all aldehydes using PFB derivatization. PFB derivatives exhibited a characteristic fragment ion at m/z 181. Using fur ther derivatization of the polar functional g roups into their trimeth ylsilyl (TMS) deri vatives, the authors w ere able to classify the aldeh ydes into f ve g roups by the selected ion monitoring of aldeh yde class specifc fragment ion. In their in vivo study, these authors w ere ab le to identify at least 33 aldehydes. This new targeted oxidized lipidomic approach represents a po werful tool to better understand the implication of lipid pero xidation in various diseases.

GLUTATHIONE CONJUGATES AS BIOMARKERS OF LIPID PEROXIDATION FORMA TION The tripeptide glutathione (Glu -Cys-Gly) (GSH) is the most abundant lo w molecular w eight thiol in mammalian cells (Blair 2006). This compound is involved in the reduction of reacti ve oxygen species and lipid h ydroperoxide by glutathione related enzymes. The sulfhydryl group of cysteine has nucleophilic proper ties that make this compound impor tant for the deto xif cation of electrophilic compounds. Because lipid peroxidation originating α ,β-unsaturated alkenals are electrophilic compounds, due to the aldeh ydic function conjugated with the α ,β double bound, they readily conjugate with the nucleophilic compound glutathione through a Michael addition on the doub le bond, probably on the C -3 position for 4 -hydroxyalkenals (Figure 8.5). Such Michael additions can also occur with amino acid residues of proteins, such as cysteine, histidine, arginine, and lysine. HNE adducted proteins are used as lipid pero xidation biomarkers (Uchida et al. 1993). They can be measured either b y immunohistochemistry, mass spectrometr y, Western blotting or b y ELISA; this point is de veloped elsewhere in this book. Urinar y metabolites of protein -HNE adducts have been measured in obese Zuck er rats using LC -MS/MS working in the multiple reaction monitoring (MRM) mode (Orioli et al. 2007). In the same w ay, MDA can covalently bind proteins and phospholipids as phosphatidylethanolamine and phosphatidylserine and derivatives can be measured in urine (Draper et al. 2000). Glutathione conjugation is often seen as the f rst and major step in the biotransfor mation of these aldeh ydes (Alar y et al. 2003, Alary, Gueraud et al. 2003). Conjugation with glutathione is a w ay for the cell to eliminate those c ytotoxic aldehydes. In vitro, this conjugation can occur spontaneousl y, but this reaction can be se veral hundred times increased in the presence of GSTs. The isofor m GST A4-4 w as sho wn to be specif c for HNE conjugation (Hubatsch et al. 1998). Knock -out animals for GST specif c for HNE had a reduced ability to conjugate HNE and sho wed a lo wer sur vival time than wild -type controls w hen treated with an oxidative stress inducer (Engle et al. 2004). Enzymes responsible for GSH conjugates export from the cell also seem to ha ve an impor tant role vis a vis o xidative stress. Reichard et al. e videnced the multi -drug resistance protein 2 (MRP2) as a transpor ter for HNE -GSH adducts in the li ver (Reichard et al. 2003) and Awatshi’s g roup showed that the Ral -binding GTPase acti vating protein RLIP76 w as a specif c transpor ter for GSH conjugates (A wasthi et al. 2002). Overexpression of this protein, and probab ly on the other hand K O animals for this protein, can dramaticall y modify HNE biotransfor mation and metabolism (Cheng et al. 2001) but also the cell resistance to ward o xidative injuries such as from radiation (Y ang et al. 2003).

124 Chapter

8 OH

Glu Cys

SH

+

C5H11

C5H11

GSTs

O

Glu

HNE

Glutathione (GSH)

C5H11

Cys

S

OH

S

Gly

Gamma-glutamyl transpeptidase

O

O

Cys

Gly

GS-HNE (hemiacetal form)

OH

Peptidase

C5H11

Cysteinylglycinase

O

OH

S

Gly

Cys

Cysteine-HNE C5H11

O

OH

C5H11

O

OH

N-acetyl-transferase

HOOC

S

S

CH2

CH2

CH

NH2

Cysteine-HNE

HOOC

CH

NHCOCH3

N-acetyl-L-cysteine-HNE or HNE-mercapturic acid

Figure 8.5. The mercapturic acid pathway: example of 4 -hydroxynonenal (HNE).

Thus, measuring GS -HNE conjugates can pro vide a ref ection of the production, to gether with detoxif cation capacities. It can be interesting to simultaneousl y have a measurement of HNE/protein and HNE/DN A adducts that w ould represent the par t of HNE that w ould have escaped the metabolism/defence capacities. Falletti et al. Simultaneously measured HNE-DNA adducts and GS -HNE conjugate in human monocytes using HPLC -MS/MS, and observed that GSH w as much more reacti ve to ward HNE than DN A and that HNE -DNA adducts w ere increased if cell GSH content has been artif cially depressed by buthionine sulfoximine (BSO). The same obser vation has been made in the li ver of rats treated with BSO in drinking w ater (Chung et al. 2005). Such a depressed GSH intracellular concentration could occur under condition of o xidative stress and these authors suggested that o xidative stress could g reatly increase HNE -DNA adducts (Falletti et al. 2007, Falletti and Douki 2008). ASSA Y HNEand ONE Glutathione conjugates of lipid pero xidation products ha ve been measured b y HPLC -MS in the liver of rats treated with iron nitriloacetate, a compound that induces oxidative stress (Volkel et al. 2005). These authors showed a f ve-fold increase in the concentration of the HNE -GSH adducts. Boon et al. de veloped an HPLC method combined with electrochemical detection to measure HNE -GSH adducts (Boon et al. 1999). Such adducts w ere also sho wn to be

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomark ers 125

increased in the post -mortem brain of patients suffering from severe Alzheimer’s disease using ESI-MS/MS (Volkel et al. 2006). Orioli et al. measured HNE -GSH and HNE protein adducts using LC-ESI-MS-MS in muscle homogenates (Orioli et al. 2005). Hontzacko et al. simultaneously measured in rat brain mitochondria HNE enantiomers, HNE - and 1,4 dihydroxynonaneGSH (the reduced metabolite of HNE-GSH) adducts, and 4-hydroxynonenoic acid, the oxidized metabolite of HNE, in a single r un using LC -ESI-MS-MS (Honzatko et al. 2007). A novel GSH conjugate originating from the lipid oxidation product ONE has been recently evidenced. This conjugate is quite dif ferent from the HNE -GSH adducts previously described, in which the addition reaction occurs at the C -3 position. In this case, the conjugation occurs at the C1 position and is then followed by intramolecular cyclization and dehydration reactions, giving a thiadiazabic yclo-ONE-GSH adduct. This no vel adduct has been measured using LC-MRM/MS in endothelial cells treated with ONE but also in the same cells under o xidative stress conditions (Jian et al. 2007). This recently discovered adduct could ser ve as a ne w biomarker of intracellular o xidative stress. Acr olein The main primar y metabolite of acrolein is also its glutathione conjugate (Ste vens and Maier 2008 ).

MERCAPTURIC ACIDS DERIVATIVES AS BIOMARKERS OF LIPID PEROXIDATION Mercapturic acids (MAs) are S - substitutedn - acetyl - Lysteine -c derivatives. Considered as end products of the metabolism of endo genous or x enobiotic electrophilic compounds, the y were evidenced more than a centur y ago as urinar y metabolites of en vironmental electrophilic pollutants. Glutathione-, cysteine-, and mercapturic acid -conjugates are thioethers. A measurement of total thioether e xcretion in urine w ould give an idea of the global production or e xposure to electrophilic compounds (endo genous or x enobiotic) and deto xif cation of these compounds through GSH conjugation. However, this method lacks sensiti vity and selectivity and does not provide any information about the nature of the compounds. As a result, the deter mination of indi vidual MAs w ere used as tools in to xicity studies (1,2-dibromoethane, ethylene oxide, vinyl chloride) for more than thirty years (Perbellini et al. 2002, Seutter -Berlage et al. 1977, Vermeulen 1989). The literature concer ning MAs as biomarkers for professional e xposure monitoring is rather impor tant and dif ferent methods ha ve been developed for their deter mination. Determination of MAs of endogenous electrophilic compounds formed after oxidative stress has been much less in vestigated in the literature than deter mination of MAs of industrial pollutants. However, MAs of HNE, and probably of all 4-hydroxy- or 4-oxo-alkenals, and acrolein are the main metabolites found in urine (Alar y et al. 1995, Alary et al. 1998, Draminski et al. 1983, Linhart et al. 1996) and their deter mination could pro vide a nonin vasive index of lipid peroxidation (Kuiper et al. 2008, Peiro et al. 2005). FORMA TION Mercapturic acid pathway (MAP) is considered to be the major route of excretion of glutathione conjugates. Other routes are for mation of meth ylsulf des, meth ylsulfoxides, and meth ylsulfones. This pathw ay is generall y considered to be an inter -organ biotransfor mation process (Inoue et al. 1984) with the conjugation being essentiall y hepatic and the remo val of the glutamyl moiety by γ-glutamyl transpeptidases almost exclusively occurring in the kidney. Glycine is then remo ved by cysteinylglycinase and aminopeptidases, w hich are present mostl y in the

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kidney but also in the li ver. N -acetylation of c ysteine is catal ysed by N - acetyltransferases,in the kidney and in the li ver, giving MAs derivatives which are excreted into urine (Figure 8.5). However, some authors sho wed that the w hole process can be intrahepatic (Hinchman and Ballatori 1994). But MAs can be deacetylated and the cysteine part can be removed by β -yases, l giving back the parent compound that can e xert its to xicity in the kidne y, where the deacetylation takes place, catalyzed by acylases (Monks et al. 1990). 4 - Hydr oxyalkenals 1,4-dihydrononene mercapturic acid (DHN -MA), which is the reduced mercapturic acid conjugate of HNE, is the major metabolite of HNE in rat urine (F igure 8.6). Other MAs, such as HNE - MAand 4 - yhdroxynonenoic lactone mercapturic acid (HNA - lactone - MA), were also found in urine after HNE administration (Alary et al. 1995, de Zwart et al. 1996), together with more polar metabolites that result from the ome ga oxidation of the side chain of HNE (Alar y et al. 1998) by cytochrome P450 4A enzymes (Gueraud et al. 1999). Winter et al. reported the existence of the MA of 4 -hydroxyhexenal in rat urine after HHE administration in the por tal vein (Winter et al. 1987 ). Acr olein The main metabolites of acrolein found in urine are 3-hydroxypropylmercapturic acid (HPMA) and carboxyethylmercapturic acid (CEMA), w hich are respecti vely the reduced and o xidized metabolites of S-(3-oxopropyl)mercapturic acid (OPMA), w hich is the mercapturic acid of acrolein (Linhart et al. 1996, Parent et al. 1998, Stevens and Maier 2008). ASSA Y The f rst methods to deter mine mercapturic acids and thioethers in urine as biomark ers of exposure to alk ylating agents used deproteinization of the samples, alkaline h ydrolysis of the thioethers to the cor responding thiophenols, and assay of the liberated and the free SH -groups following the method of Ellman (Ellman 1959 )using dithio - bis(2 - nitrobenzoic acid)(DTNB) with detection at 412nm (Seutter-Berlage et al. 1977). More recent detection techniques require extraction and/or purif cation steps followed by separation/detection techniques. Extraction of mercapturic acids from biolo gical f uids (urine, bile) requires acidif cation of samples at pH lower than 2 combined with e xtraction with organic solvents. Separation/detection techniques

HNE

Oxidation

OH

or

O

Reduction OH

Omega é oxidation

OH

GSH conjugation S

O HO

Mercapturic acid pathway

NH O

DHN-MA

Figure 8.6. Main metabolic pathways of 4 -hydroxynonenal (HNE) and formation of 1,4 dihydroxynonene mercapturic acid (DHN -MA).

-

Lipid Peroxidation Originating α,β-unsaturated Aldehydes and Their Metabolites as Biomark ers 127

include HPLC, GC -MS after deri vatization, GC with sulphur selecti ve f ame - photometric detection, or other detection methods (v an Welie et al. 1992, van Welie et al. 1989) Concerning MAs originating from lipid pero xidation products, Jones et al. deter mined HNE-MA using GC -electron capture negative ion chemical ionization -MS (Jones et al. 1993). The author ’s g roup developed an LC -MS/MS/MS method for the measurement of DHN -MA in urine using a deuterated inter nal standard (Rathahao et al. 2005), and w e compared the urinary excretion of MDA, DHN-MA, and 8 -isoPGF2α in rats treated with a lipid pero xidation inducer (Peiro et al. 2005). Very recently, Kuiper et al. measured MAs of ONE and HNE and their oxidized and reduced derivatives, namely 4-oxo-nonenoic acid, HNA, 4-oxo-nonenol, and 1,4-dihydroxynonene, in rat urine b y LC -MS/MS after treatment of the animals with CCl 4 (Kuiper et al. 2008). HPMA, a major metabolite of acrolein, has been measured in human urine by LC -MS-MS and its e xcretion was much higher in smok ers than in non -smokers (Mascher et al. 2001). Schettgen et al. performed a simultaneous determination of several MAs including HPMA using LC -MS/MS (Schettgen et al. 2008) Most of these techniques require comple x and time -consuming preparation steps to gether with complicated and e xpensive detection methodolo gies. Therefore, more con venient measurement methods, such as immunoassa ys, have been de veloped. Marco et al. de veloped an ELISA for the detection of MA metabolites of naphthalene (Marco et al. 1993). The author ’s group developed a competiti ve enzyme immunoassa y for the deter mination of DHN -MA in urine, using acetylcholineesterase bound DHN-MA as tracer (Gueraud et al. 2006) (Figure 8.7). This assay has been v alidated against LC -MS/MS/MS methodology previously developed in the laboratory of the author ’s group (Rathahao et al. 2005). Different g roups used the metabonomics approach on MAs using high resolution NMR spectroscopy or MS. Nicholson ’s group detected only the metabolite of acrolein HPMA in the urine of rats treated with the all ylic compound allyl formate, using 1 H - NMR(Athersuch et al. 2006). Mall y et al. Simultaneousl y measured both DHN -MA and HNE -MA b y LC -MS/MS Competitive DHN-MA immunoassay

18 hours 4°C

1: Microtiter plates coated with mouse monoclonal anti-rabbit IgG

2: Incubation with immunized rabbit polyclonal serum, AChE-DHN-MA tracer, urine sample, or standard

Absorbance at 414 nm

3: Washing of the plate to remove unbound compounds

: Mouse anti-rabbit IgG

4: Addition of Ellman’s reagent to quantify the enzyme activity of the tracer : Acetylcholineesterase -DHN-MA tracer

: DHN-MA

: Specific antibody against DHN-MA

Figure 8.7. Principle of the competitive immunoassay for the determination of 1,4 dihydroxynonene mercapturic acid (DHN -MA) in urine.

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and the 1 H - NMRmetabonomic prof ling of the same urine samples of tw o rodent models of oxidative kidney damage. Although DHN-MA excretion was shown to be increased in the acute model of nephrotoxicity, 1 H - NMRmetabonomic prof ling did not re veal any alteration in MA excretion (Mally et al. 2007). Other authors improved the NMR metabolite screening sensitivity using LC -MS metabonomic prof ling. Wagner et al. applied this technique to de velop an integrated approach for LC -MS metabolic prof ling of MAs (W agner et al. 2006, Wagner et al. 2007). Undoubtedly, this “mercapturomics” approach will ha ve a promising future.

APPLICA TIONS Detection and measurement of α ,β-unsaturated aldehydes has been done in a variety of tissues and biof uids under numerous patholo gical or o xidative stress conditions. F or most of these aldehydes, determination is also possib le in foods. Determination of the MAs of lipid pero xidation secondar y products is limited to urine, but measuring biomark ers in urine has se veral adv antages including nonin vasiveness of the sampling and stability of the biomark ers because the y are usuall y end -products. Moreo ver, measurements in urine o ver a 24 -hour period can pro vide an inte grated inde x of o xidative stress.

LIMIT ATIONS One of the major dra wbacks to the use of α ,β-unsaturated aldehydes as biomarkers is the f act that they are currently present in foodstuffs, in very variable amounts. Moreover, they can also be formed in the digesti ve tract during digestion. These exogenous compounds may cross the intestinal bar rier and be absorbed and ma y interfere with the measurement of the aldeh ydes that are endo genously for med. Environmental exposure to some pollutants can also interfere with the measurement of endo genous compounds, as is the case for acrolein in human studies (Stevens and Maier 2008). Another f act that can be seen as a disadv antage to the use of those compounds as lipid oxidation biomarkers is that their use is limited to recent e xposure, contrary to their protein or DNA adducts, w hich can ref ect semi -chronic or chronic e xposure (v an Welie et al. 1992). Contrary to their mercapturic acid end -products, most of the α ,β - unsaturatedaldehydes are reactive and unstab le compounds. This can be also an impor tant dra wback to their use as biomarkers. Concerning compounds that deri ve from one or more enzymatic biotransfor mation step(s), such as GSH conjugates or MAs, their formation and subsequent measurement may depend on the presence of functional enzymes in volved in those transfor mation steps. For instance, some authors showed that GST pol ymorphism may inf uence MAs excretion in the case of occupational exposure to to xic compounds in human studies (Haufroid and Lison 2005). However, the perfect oxidative stress biomarker combining specif city, sensitivity, stability, noninvasiveness, and reliability, and in w hich measurement is easy , fast, and ine xpensive, does not e xist. Compromises are necessar y and the use of dif ferent biomark ers originating from dif ferent sources as lipid o xidation, protein oxidation, or nucleic acid o xidation is recommended.

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Liu YM , Jinno H , K urihara M , Miyata N , T oyo ’ okaT. 1999 . Determination of 4 - yhdroxy - -2 nonenal in primar y rat hepatocyte cultures by liquid chromatography with laser induced f uorescence detection . Biomedical Chromatography 13 : 75 – 80 . LordHL , Rosenfeld J , Raha S , Hamadeh MJ . 2008 .Automated derivatization and analysis of malondialdehyde using column s witching sample preparation HPLC with f uorescence detection . Journal of Separation Science 31 : 387 – 401 . LuoXP , Y azdanpanah M , Bhooi N , Lehota y DC .1995 . Determination of aldehydes and other lipid peroxidation products in biolo gical samples by gas chromatography-mass spectrometry. Analytical Biochemistry 228 : 294 – 8 . Mall y A ,Amber g A , Hard GC , DekantW . 2007 .Are 4 - yhdroxy - 2(E) - nonenal derived mercapturic acids and (1)H NMR metabonomics potential biomark ers of chemically induced oxidative stress in the kidne y? Toxicology 230 : 244 – 55 . MaoJ , Zhang H , Luo J , Li L , Zhao R et , al. 2006 . New method for HPLC separation and f uorescence detection of malonaldeh yde in nor mal human plasma . Journal of Chromatography B, Analytical Technologies in the Biomedical and Lif e Sciences 832 : 103 – 8 . MarcoMP , Nasiri M , K urth MJ , Hammock BD . 1993 . Enzyme - link ed immunosorbent assay for the specif c detection of the mercapturic acid metabolites of naphthalene . Chemical Research in Toxicology 6 : 284 – 93 . MascherDG , Mascher HJ , Scherer G , Schmid ER .2001 . High - perfor mance liquid chromatographic-tandem mass spectrometric deter mination of 3 -hydroxypropylmercapturic acid in human urine . Journal of Chromatography. B, Biomedical Applications 750 : 163 – 9 . MatsuokaM , Imado N , MakiT , Banno K , SatoT . 1996 . Determination of free aliphatic aldehydes in plasma b y high -performance liquid chromatography of the 1,3 -cyclohexanedione derivatives . Chromatographia 43 : 501 – 6 . MonksTJ , Anders MW , DekantW , Ste vens JL , Lau SS ,avn Bladeren PJ. 1990 . Glutathione conjugate mediated toxicities. Toxicology and Applied Pharmacology 106 : 1 – 19 . NielsenF , Mikk elsen BB , Nielsen JB , Andersen HR , Grandjean P . 1997 . Plasma malondialdehyde as biomarker for oxidative stress: reference inter val and effects of life -style factors . Clinical Chemistry 43 : 1209 – 14 . O ’ Brien -erCok IC , P erkins G , MalletAI. 2001 .Aldehyde analysis by high performance liquid chromatography/tandem mass spectrometr y. Rapid Communications in Mass Spectr ometry 15 : 920 – 8 . OrioliM ,Aldini G , Benfatto MC , aFcino RM , Carini M .2007 . HNE Michael adducts to histidine and histidine -containing peptides as biomark ers of lipid -derived carbonyl stress in urines: LC - MS/MSprof ling in Zucker obese rats . Analytical Chemistry 79 : 9174 – 84 . OrioliM ,Aldini G , Beretta G , aFcino RM , Carini M .2005 . LC - ESI - MS/MS determination of 4 - yhdroxy - trans - 2 - nonenal Michael adducts with cysteine and histidine - containingpeptides as early markers of oxidative stress in e xcitable tissues . Journal of Chromatography B, Analytical Technologies in the Biomedical and Lif e Sciences 827 : 109 – 18 . arent P RA , P aust DE , Schrimpf MK ,T alaat RE , Doane RA et , al. 1998 . Metabolism and distribution of [2,3 -14C]acrolein in Sprague -Dawley rats. II. Identif cation of urinar y and fecal metabolites. Toxicological Sciences 43 : 110 – 20 . eiro P G ,Alary J , Cra vedi JP , Rathahao E , Ste ghens JP , Gueraud F . 2005 . Dihydroxynonene mercapturic acid, a urinar y metabolite of 4 -hydroxynonenal, as a biomark er of lipid peroxidation . Biofactors 24 : 89 – 96 . erbellini P L ,V eronese N , Princi valle A .2002 . Mercapturic acids in the biological monitoring of occupational exposure to chemicals . Journal of Chromatography B, Analytical Technologies in the Biomedical and Lif e Sciences 781 : 269 – 90 . PilzJ , Meinek e I , Gleiter CH .2000 . Measurement of free and bound malondialdehyde in plasma by high -performance liquid chromatography as the 2,4 -dinitrophenylhydrazine derivative. Journal of Chromatography. B, Biomedical Applications 742 : 315 – 25 .

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yor Pr WA , Stanle y JP , Blair E .1976 .Autoxidation of polyunsaturated fatty acids: II. A suggested mechanism for the for mation of TBA-reactive materials from prostaglandin -like endoperoxides. Lipids 11 : 370 – 9 . RathahaoE , P eiro G , Martins N , Alary J , Gueraud F , Debrauw er L .2005 . Liquid chromatography-multistage tandem mass spectrometr y for the quantif cation of dihydroxynonene mercapturic acid (DHN - MA), a urinary end - metaboliteof 4 - yhdroxynonenal . Analytical and Bioanalytical Chemistry 381 : 1532 – 9 . RauliS , Puppo MD , Magni F , Kienle MG .1998 .Validation of malondialdehyde and 4 - yhdroxy 2 - trans - nonenal measurement in plasma by NICI - GC - MSJournal . of Biochemistry 123 : 918 – 23 . ReichardJF , Doorn JA , Simon F , T aylor MS , P etersen DR .2003 . Characterization of multidrug resistance-associated protein 2 in the hepatocellular disposition of 4 -hydroxynonenal. Archives of Biochemistry and Biophysics 411 : 243 – 50 . SchettgenT , MusiolA , KrausT . 2008 . Simultaneous determination of mercapturic acids derived from ethylene oxide (HEMA), propylene oxide (2 -HPMA), acrolein (3 -HPMA), acrylamide (AAMA) and N ,N-dimethylformamide (AMCC) in human urine using liquid chromatography/tandem mass spectrometr y. Rapid Communications in Mass Spectr ometry 22 : 2629 – 38 . SchneiderC ,T allman KA , P orter NA , BrashAR .2001 .Two distinct pathways of formation of 4-hydroxynonenal. Mechanisms of nonenzymatic transfor mation of the 9 - and 13-hydroperoxides of linoleic acid to 4 -hydroxyalkenals. Journal of Biological Chemistry 276 : 20831 – 8 . Selle y ML. 1997 . Determination of the lipid peroxidation product (E) - 4 -ydroxy h - 2 - nonenal in clinical samples by gas chromatography—negative-ion chemical ionisation mass spectrometr y of the O -pentaf uorobenzyl oxime. Journal of Chromatography. B, Biomedical Applications 691 : 263 – 8 . Selle y ML , Bartlett MR , McGuiness JA , HapelAJ , Ardlie NG. 1989 . Determination of the lipid peroxidation product trans - 4 -ydroxy h - 2 - nonenal in biological samples by high - performance liquid chromatography and combined capillar y column gas chromato graphy-negative-ion chemical ionisation mass spectrometr y. Journal of Chromatography 488 : 329 – 40 . Seutter - Berlage F , avn Dorp HL , K osse HG , Henderson PT. 1977 . Urinary mercapturic acid excretion as a biolo gical parameter of e xposure to alkylating agents . International Archives of Occupational and Environmental Health 39 : 45 – 51 . ShibataN , Y amada S , Uchida K , HiranoA , Sak oda S et , al. 2004 .Accumulation of protein bound 4 -hydroxy-2-hexenal in spinal cords from patients with sporadic am yotrophic lateral sclerosis. Brain Research 1019 : 170 – 7 . Sim AS , Salonikas C , Naidoo D , W ilcken DE .2003 . Improved method for plasma malondialdehyde measurement by high -performance liquid chromatography using methyl malondialdehyde as an inter nal standard . Journal of Chromatography B, Analytical Technologies in the Biomedical and Lif e Sciences 785 : 337 – 44 . SpitellerG , K ern W , Spiteller P . 1999 . Investigation of aldehydic lipid peroxidation products by gas chromatography – massspectrometry . Journal of Chromatography. A 843 : 29 – 98 . Ste ghens JP , avn Kappel AL , Denis I , Collombel C. 2001 . Diaminonaphtalene, a new highly specif c reagent for HPLC -UV measurement of total and free malondialdeh yde in human plasma or ser um. Free Radical Biology and Medicine 31 : 242 – 9 . Ste vens JF , Maier CS .2008 .Acrolein: sources, metabolism, and biomolecular interactions relevant to human health and disease . Molecular Nutrition and Food Research 52 : 7 – 25 . Stopfor th A , Bur ger BV , CrouchAM , Sandra P . 2006 . Urinalysis of 4 - yhdroxynonenal, a marker of oxidative stress, using stir bar sor ptive extraction-thermal desorption-gas chromatography/ mass spectrometry. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences 834 : 134 – 40 .

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StrohmaierH , Hinghofer- Szalka y H , Schaur RJ. 1995 . Detection of 4 - yhdroxynonenal (HNE) as a physiological component in human plasma . Journal of Lipid Mediator s and Cell Signalling 11 : 51 – 61 . Sv ensson S , Larstad M , Broo K , OlinAC . 2007 . Determination of aldehydes in human breath by on - fbre derivatization, solid -phase microextraction and GC -MS. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Lif e Sciences 860 : 86 – 91 . emplar T J, K on SP , MilliganTP , Ne wman DJ , Raftery MJ . 1999 . Increased plasma malondialdehyde levels in glomer ular disease as deter mined by a fully validated HPLC method . Nephrology, Dialysis, Transplantation 14 : 946 – 51 . ThomasMJ , RobisonTW , Samuel M , F orman HJ . 1995 . Detecting and identifying volatile aldehydes as dinitrophenylhydrazones using gas chromato graphy mass spectrometr y. Free Radical Biology and Medicine 18 : 553 – 7 . UchidaK , Kanematsu M , Sakai K , MatsudaT , Hattori N ,et al. 1998 . Protein - boundacrolein: potential markers for oxidative stress . Proceedings of the National Academy of Sciences of the United States of America 95 : 4882 – 7 . UchidaK , Szw eda LI , Chae HZ , Stadtman ER .1993 . Immunochemical detection of 4-hydroxynonenal protein adducts in o xidized hepatocytes. Proceedings of the National Academy of Sciences of the United States of America 90 : 8742 – 6 . an V Kuijk FJ , Holte LL , Dratz EA. 1990 . 4 - Hydro xyhexenal: a lipid peroxidation product derived from oxidized docosahexaenoic acid . Biochimica et Biophysica Acta 1043 : 116 – 8 . anv Kuijk FJ , Siak otos AN , F ong LG , Stephens RJ , Thomas DW. 1995 . Quantitative measurement of 4 -hydroxyalkenals in oxidized low-density lipoprotein by gas chromatographymass spectrometry . Analytical Biochemistry 224 : 420 – 4 . anv Kuijk FJ , Thomas DW , Stephens RJ , Dratz EA. 1986 . Occurrence of 4 - yhdroxyalkenals in rat tissues determined as pentaf uorobenzyl oxime derivatives by gas chromatography-mass spectrometry . Biochemical and Biophysical Research Communications 139 : 144 – 9 . anv Welie RT , avn Dijck RG ,V ermeulen NP , avn Sittert NJ. 1992 . Mercapturic acids, protein adducts, and DNA adducts as biomark ers of electrophilic chemicals . Critical Reviews in Toxicology 22 : 271 – 306 . anv Welie RT , avn Duyn P , V ermeulen NP. 1989 . Determination of two mercapturic acid metabolites of 1,3 -dichloropropene in human urine with gas chromato graphy and sulphur selective detection . Journal of Chromatography 496 : 463 – 71 . ermeulen V NP. 1989 .Analysis of mercapturic acids as a tool in biotransformation, biomonitoring and toxicological studies . Trends in Pharmacological Sciences 10 : 177 – 81 . eronneau V M , Comte B , Des Rosiers C. 2002 . Quantitative gas chromatographic - mass spectrometric assay of 4 -hydroxynonenal bound to thiol proteins in ischemic/reperfused rat hearts . Free Radical Biology and Medicine 33 : 1380 – 8 . olkel V W , Alv arez - Sanchez R ,W eick I , Mall y A , DekantW , P ahler A. 2005 . Glutathione conjugates of 4 -hydroxy-2(E)-nonenal as biomarkers of hepatic o xidative stress -induced lipid peroxidation in rats . Free Radical Biology and Medicine 38 : 1526 – 36 . olkel V W , SiciliaT , P ahler A , GsellW , T atschner T ,et al. 2006 . Increased brain levels of 4 - yhdroxy - 2 - nonenal glutathione conjugates in severe Alzheimer ’s disease . Neurochemistry International 48 : 679 – 86 . agner W S , Scholz K , Done gan M , Burton L ,W ingate J , V olkel W . 2006 . Metabonomics and biomarker discovery: LC -MS metabolic prof ling and constant neutral loss scanning combined with multivariate data analysis for mercapturic acid anal ysis. Analytical Chemistry 78 : 1296 – 305 . agner W S , Scholz K , Sieber M , K ellert M ,V oelkel W . 2007 .Tools in metabonomics: an integrated validation approach for LC -MS metabolic prof ling of mercapturic acids in human urine . Analytical Chemistry 79 : 2918 – 26 . arnke W MM ,W anigasekara E , Singhal SS , Singhal J , A wasthi S ,Armstrong DW . 2008 .The determination of glutathione - 4 -ydroxynonenal h (GSHNE), E - 4 -ydroxynonenal h (HNE), and

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E - 1 -ydroxynon h - 2 - en - 4 -(HNO) one in mouse liver tissue by LC - ESI - MSAnalytical . and Bioanalytical Chemistry 392 : 1325 – 33 . iW lliams TI , Lo vell MA , L ynn BC .2005 .Analysis of derivatized biogenic aldehydes by LC tandem mass spectrometr y. Analytical Chemistry 77 : 3383 – 9 . iW lliams TI , L ynn BC , Mark esbery WR , Lo vell MA .2006 . Increased levels of 4 - yhdroxynonenal and acrolein, neurotoxic markers of lipid pero xidation, in the brain in Mild Co gnitive Impairment and early Alzheimer ’s disease . Neurobiology of Aging 27 : 1094 – 9 . iW lson DW , Metz HN , Gra ver LM , Rao PS .1997 . Direct method for quantif cation of free malondialdehyde with high -performance capillary electrophoresis in biolo gical samples . Clinical Chemistry 43 : 1982 – 4 . iW nter CK , Se gall HJ , JonesAD . 1987 . Distribution of trans - 4 -ydroxy h - 2 - xenal he and tandem mass spectrometric detection of its urinar y mercapturic acid in the rat . Drug Metabolism and Disposition 15 : 608 – 12 . agi Y K. 1976 .A simple f uorometric assay for lipoperoxide in blood plasma . Biochemical Medicine 15 : 212 – 6 . ang Y Y , Sharma R , Sharma A ,A wasthi S ,A wasthi YC .2003 . Lipid peroxidation and cell cycle signaling: 4 -hydroxynonenal, a key molecule in stress mediated signaling . Acta Biochimica Polonica 50 : 319 – 36 . eo Y HC , Helbock HJ , Ch yu DW , Ames BN . 1994 .Assay of malondialdehyde in biological f uids by gas chromatography-mass spectrometry. Analytical Biochemistry 220 : 391 – 6 . oshino Y K , MatsuuraT , Sano M , Saito S ,T omita I .1986 . Fluorometric liquid chromatographic determination of aliphatic aldeh ydes arising from lipid pero xides. Chemical and Pharmaceutical Bulletin (Tokyo) 34 : 1694 – 700 .

Chapter9 Oxidative Modif cation of Proteins: An Overview P aul J. Thornalle y and

Naila Ra bbani

INTR ODUCTION PROTEIN OXIDATION IN PHYSIOLOGICAL SYSTEMS-DAMAGING, DELIBERATE, AND DEFENSIVE The o xidation of proteins in ph ysiological systems occurs b y spontaneous auto xidation of cysteinyl thiols, interaction of proteins with reacti ve o xidizing inter mediates, and deliberate and controlled reactions catalyzed by oxidases (Thornalley 2006a). The spontaneous autoxidation of c ysteine and interaction of proteins with o xidizing inter mediates are thought to be deleterious as they lead to reversible and irreversible inactivation of proteins which are repaired and replaced. It is wasteful of metabolic energy consumed in repair, replacement, and degradation of the damaged protein. It ma y also be har mful if there is functional loss of proteins of signif cant extent and duration. When oxidative damage to proteins is directed to in vading micro -organisms or tumors as part of the host defense, then oxidative damage may be considered to be benef cial. Deliberate, benef cial, and regulated protein oxidation occurs in enzymatic oxidative crosslinking of extracellular matrix proteins. F or e xample, l ysyl o xidase catal yzed for mation of p yridinolines in collagen and desmosines in elastin (Lucero and Kagan2006) and dual oxidase catalyzed formation dityrosine residues in cuticle collagen and other proteins (Edens et al. 2001). There is also a vie w that lo w le vels of o xidative damage to proteins induce a hor metic response: a low level of o xidative damage acti vating a stronger anti -stress gene response that is eventually protective. Hormesis is the benef cial effects of a treatment that at a higher intensity is har mful (Calabrese et al. 1999). Agents inducing hor mesis are called “hormetins” and many bioactive compounds considered to be benefcial for maintenance of good health although not essential for ph ysiological function ma y be of this type. Lo w levels of protein o xidation may be involved in signalling for hormetic responses. This might explain observations that are currently judged contro versial of bioacti ve compounds at high doses that ha ve adverse toxic effects, whereas at lo wer doses the y are benef cial (Lamber t et al. 2007). For hor mesis, this dose-dependent behavior is expected, and will be accepted providing there are plausible mechanistic explanations for benef cial and adv erse effects. Hor mesis has been proposed to pla y a role in healthy aging (Gems and P artridge 2008). Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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PHYSIOLOGICALOXIDIZING AGENTS The o xidation of proteins in ph ysiological systems occurs b y spontaneous auto xidation of cysteinyl thiols (Saez et al. 1982) and b y interaction of proteins with o xidizing agents such as h ydrogen pero xide H 2 O2, h ydroperoxides R OOH, h ypochlorite ClO − , peroxynitrite, and other o xidizing reacti ve inter mediates (h ydroxyl radical HO •, carbonate radical anion CO3 •−, and others) (F inkel and Holbrook 2003). These processes ma y be catal yzed b y trace redox acti ve metal ions such as iron (III) or fer ric ion F e3+, and Cu (II) or cupric Cu 2+ ion (Castellani et al. 2004). Hydrogen peroxide in ph ysiological systems originates mainl y from the dismutation of superoxide O2 •− formed by ineff ciency of mitochondrial respiration (Chance et al. 1979), superoxide-forming enzymes, such as phagoc yte and v ascular NADPH oxidases (Lassegue and Clempus 2003), uncoupling of enzymatic reactions (for e xample, nitric o xide synthases) (Du et al. 1999), and auto xidation of reducing metabolites (F inkel and Holbrook 2003 ). Hypochlorite is for med b y the enzymatic reaction of m yeloperoxidase in neutrophils (Naskalski et al. 2002). Extracellular proteins are more susceptib le to o xidation than cellular proteins because the non -enzymatic and enzymatic acti vities of antio xidants mostl y have an intracellular location. Ho wever, one of the sources of highest f ux of h ydrogen peroxide generation, mitochondria (Mur phy 2009), is also intracellular . Although the c ytoplasm in ph ysiological systems is maintained under reducing conditions b y the presence of high concentrations of lo w molecular mass peptide thiols (usuall y glutathione, GSH), there is nevertheless a residual lo w le vel of o xidative damage to both cellular and e xtracellular proteins.

OXIDATION OF CYSTEINE RESIDUES The thiol g roup of cysteine residues in proteins is usuall y the most easil y oxidized site within proteins (Figure 9.1). It is also an oxidation of proteins that is most easily reversed, particularly when oxidized to disulf de. This reversal occurs in cells for glutathione -mixed disulf des catalyzed by glutaredoxin (Gallogly et al. 2009) and in plasma where most disulf des are albumin cysteinyl - mix ed disulf des b y c ysteine/disulf de e xchange. Fur ther o xidation occurs to for m cysteine sulfenic acid cys–S-OH (Luo et al. 2005) or sulphenylamide derivatives (van Montfort et al. 2003). Cysteinyl sulfenic acid and sulfenyl-amide derivatives may play a role in the redox regulation of cysteine-dependent enzymes (Poole et al. 2004). Effective oxidants are hydrogen peroxide, h ypochlorite, pero xynitrite, and other reacti ve o xygen species (R OS) and reacti ve nitrogen species (RNS). Exposure of rat hear t tissue to h ydrogen pero xide ex vivo , for e xample, for med c ysteine sulfenic acid residues in the follo wing proteins: m yosin hea vy and light chains, aconitase, acyl-CoA dehydrogenase, ATP synthase, aspar tate aminotransferase, actin, tropom yosin, troponin T, ADP, ATP car rier protein T1, malate deh ydrogenase, L -lactate dehydrogenase, cytochrome c oxidase, and myoglobin (Kiley and Storz 2004). Cysteine sulfenic acid residues are readily reversed by reduction with GSH, thioredo xin, and glutaredoxin. Protein sulfenic acid for mation is in volved in the re gulation of tyrosine phosphatases, per oxiredoxins, methionine sulfo xide reductases, and other proteins (P oole et al. 2004). GSH reacts with c ysteine sulfenic acid residues to for m a glutathion yl mixed disulf de, a process called protein thiolation. Protein thiolation is also considered to mediate redo x regulation of enzymes and other proteins (Giustarini et al. 2004). Disulf de and sulfenic acid residue for mation are readil y reversed by reduction with GSH. Cysteine residues ma y also be o xidized to cysteine sulf nic acid residues, cys–SO2H. This is mainly an irreversible oxidation (Poole et al. 2004 ).Sulf redoxin repairs sulf nic acid formation in peroxiredoxins (Woo et al. 2005). Further oxidation to a sulfonic acid or c ysteic acid c ys–SO3H residues is possib le but requires strong oxidants and is an ir reversible oxidation (Poole et al. 2004).

Oxidati

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ve Modif cation of Proteins: An Overview

Oxidative damage

Repair

CO SH

NH

RNS

R'SH

R'S-SR'

Cysteine

CO

ROS/ RNS

NH

CO S NO

H2O HNO S-Nitrosocysteine

GSH, Thioredoxin, Glutaredoxin

R' NH

S

Mixed disulfide (protein thiolation)

CO NH

S

S OH

Cysteine sulfenic acid ROS

R'SH

OH2

Repair of sulfinic acid (for Peroxiredoxins, catalyzed by sulfiredoxin)

CO NH

S OH O

Cysteine sulfinic acid

CO O NH

S OH

Catabolism

O Cysteine sulfonic acid (cysteic acid)

Figure 9.1. Oxidation and repair of cysteine residues in proteins. For the corresponding free adducts at physiological pH, the N-terminal amino group is protonated −NH3+ and the C-terminal carbonyl is a carboxylate −CO2− moiety.

OXIDATION OF METHIONINE RESIDUES Methionine residues in proteins are susceptible to oxidation, leading to the formation of R -and S-epimers of methionine sulfo xide (MetSO) and methionine sulfone MetSO 2 residues (Figure 9.2). MetSO residues are for med by exposure to hydrogen peroxide (Finch et al. 1993), hypochlorite (Panzenbock and Stock er 2005), and pero xynitrite, but in the latter case the yield of MetSO residues w as decreased mark edly b y ph ysiological concentrations of carbon dio xide (Tien et al. 1999). Severe oxidizing conditions are thought to be required to for m methionine sulfone (Chowdhury et al. 1995). MetSO is a major quantitati ve marker of protein o xidation in cellular and extracellular proteins. Some examples of MetSO residue detection are gi ven in Table 9.1. MetSO residue for mation in ph ysiological systems can be repaired , ho wever, b y enzymatic reduction catal yzed b y methionine sulfo xide reductase (Msr) (W eissbach et al. 2005). The physiological co-factor providing reducing equivalents to sustain the reducing reaction is thioredoxin, which is itself reduced b y NADPH catalyzed by thioredoxin reductase.

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Repair

Oxidative damage CO S CH3 NH Methionine

Oxidized thioredoxin MsrA

MsrB

ROS/ RNS Thioredoxin

CO

O S CH3

NH Methionine (S)-sulfoxide

+

CO

CH3 S O

NH Methionine (R)-sulfoxide

ROS

CO

O S CH3

NH

O

Methionine sulfone

Figure 9.2. Oxidation and repair of methionine residues in proteins.

There are tw o forms of Msr: MsrA reduces the S-epimer of MetSO and MsrB reduces the R-epimer (Boschi-Muller et al. 2005, Hansel et al. 2005). Msr isozymes reduce MetSO residues in proteins and MetSO free adduct (Mosk ovitz et al. 1996, Boschi-Muller et al. 2005). Human Msr is expressed in all tissues, with the highest e xpression in the hear t, skeletal muscle, liver, and cerebellum (Hu et al. 2005, K uschel et al. 1999, Jung et al. 2002, Hansel et al. 2003). mRNA splice variants of MsrA targets the enzyme to mitochondria and to nuclear and cytosolic subcellular locations (Hansel et al. 2002, Vougier et al. 2003, Hansel et al. 2005). The expression of MSR in mammalian kidne y is usually high and therefore MetSO f ltered by the glomer uli and reclaimed in the pro ximal tubules is reduced eff ciently to methionine. The urinary excretion of MetSO in human subjects is therefore v ery low: 0.02 nmol/mg creatinine (Ahmed et al. 2005). MetSO escapes reduction in renal f ailure with renal replacement therapy b y dial ysis, leading to mark ed increases in e xcretion of MetSO in dial ysate; it is increased 16 -fold in peritoneal dial ysis patients (Agalou et al. 2005).

O XIDATION OF TYROSINE RESIDUES Tyrosine residues adjacent in proteins ma y be o xidized to for m a crosslink ed dimer , 2,2′ - dih ydroxy - 5,5′ - bis(β - carbo xy -β-aminoethyl) diphen yl or dityrosine (McCor mick et al. 1998) (Figure 9.3A). When for med from tyrosine residues in proteins, this represents a non -

Table 9.1. Detection of protein oxidation adduct residues and free adducts in biomedical research: methionine sulfoxide.

Adduct

Sample

Species

Comment

Reference

MetSO residue

Plasmaprotein

Human

Hemo globin

Human

(Ahmedet al. 2005 ) (Ahmedet al. 2005 )

Skincollagen

Human

Lensprotein

Bo vine

Cardiac mitochondrial proteins

Human

Glomeruli, retina, nerve, skeletal muscle, plasma

Rat

Normal healthy controls Normal healthy controls and patients with diabetes Increasewith aging. mmol MetSO/mol Met = 1.6 × age (yrs) + 20.7 Hydro gen peroxide oxidation. α A crystallin: met - 1 oxidized. α B crystallin: met - 1and met - 68oxidized Multiplemitochondrial proteins modif ed, identif ed by proteomics Normal control and streptozotocin induced diabetic rats

Plasma

Human

Urine

Human

MetSOfree adduct

Normal healthy controls Normal healthy controls

B.

A.

(F inley et al. 1998 )

(T aylor et al. 2003 ) (Thornalley et al. 2003 ) (Ahmedet al. 2005 ) (Ahmedet al. 2005 )

CO

CO CH2

(W ells - Knecht et al. 1997 )

CH2

OH NH

NH Tyrosine

Phenylalanine ROS, RNS or Duox

ROS

ROS

NH CO

CH2

HO

CO CO

CO CH2

NH CH2

OH

NH

CH2 NH

HO o-Tyrosine

OH m-Tyrosine

Dityrosine

Figure 9.3. (A) Oxidative dimerization of tyrosine residues in proteins. (B) Hydroxylation of phenylalanine residues in proteins.

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Table 9.2. Detection of protein oxidation adduct residues and free adducts in biomedical research: dityrosine and o -tyrosine.

ype T of adduct

Sample

Species

Comment

Reference

Dityrosine residues

Carotidintimal protein

Human

(Fuet al. 1998 )

Lensprotein

Human

Increasedlevels in atherosclerotic plaques

Heart and muscle

Rat

Heart, skeletal muscle, brain and liver Cuticle

Mouse C.elegans

Urine

Human

Skincollagen

Human

Dityrosinefree adduct o -yrosine T residues

Cytosolicand mitochondrial protein in control Decreasedformation in dietary restriction Duo x shown as precursor of cuticle protein dityrosine Normal healthy controls Increasewith aging. o -yr T (µ mol/mol Phe) = 0.88 × age (years) + 1.2

(Souzaet al. 2000 ) (Leeuw enburgh et al, 1999 ) (Leeuw enburgh et al. 1997 ) (Edenset al. 2001 ) (Ahmedet al. 2005 ) (W ells - Knecht et al. 1997 )

sulfhydryl crosslink. Dityrosine is for med b y e xposure of proteins to h ydrogen pero xide, including peroxidase catalyzed reactions, hypochlorite and peroxynitrite, the eff cacy of formation depending on the pro ximity of tw o tyrosine residue side chain moieties for crosslink formation (Aeschbach et al. 1976, Krishnan et al. 2003, Ushijima et al. 1984). Dityrosine formation stimulated b y pero xynitrite w as f avored at lo w, steady state concentrations of peroxynitrite (Pfeiffer et al. 2000). Dityrosine residue for mation in cellular proteins w as suggested as a mark er of protein o xidation (Giuli vi and Da vies 2001). Dityrosine residues are formed enzymatically by the dual oxidase/peroxidase activity of the phagocyte oxidase subunit gp91phox. Dityrosyl residues are for med in cuticle collagen and other proteins to stabilize the cuticular extracellular matrix (Edens et al. 2001). Some examples of dityrosine residue detection are given in Table 9.2 .

OXIDATION OF PHENYLALANINE RESIDUES Phenylalanine residues in proteins ma y be o xidized in iron - (III) and copper -catalyzed (II) oxidations with hydrogen peroxide, leading to the formation of o and - m - tyrosine (Leeuwenburgh et al. 1997a, Leeuwenburgh et al. 1997b) (Figure 9.3B). o -and m - tyrosineresidue concentrations were increased in mitochondrial proteins of hear t muscle after e xercise (Leeuwenburgh et al. 1999a). Antioxidants have little ef fect on the le vels of o-tyrosine in urine of aging and exercised rats (Leeuw enburgh et al. 1999b). Some e xamples of o -and m - tyrosineresidue detection are given in Table 9.2.

O XIDATION OF TRYPTOPHAN RESIDUES Tryptophan (tr p) and tr yptophan residues are susceptib le to o xidation by hydrogen peroxide, hypochlorite, and pero xynitrite under ph ysiological conditions (Simat and Steinhar t, 1998,

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Kato et al. 1997). The major products of tr p o xidation w ere o xindolylalanine, 3a -hydroxy1,2,3,3a,8,8a - he xahydropyrrolo - [2,3 - b]indole - 2 - carbo xylic acid, N - formyl - yknurenine (NFK), dioxindolylalanine, k ynurenine (K yn), and 5 -hydroxytryptophan. The rate of o xidation w as slower for trp residues in proteins than for free tr p, where oxindolylalanine and NFK were the main degradation products (Simat and Steinhar t 1998) (Figure 9.4). NFK may be formed by oxidation of trp with peroxidase compound III (Ximenes et al. 2001) and model iron (III) comple xes (Itakura et al. 1994). NFK is f uorescent with e xcitation and emission wavelength maximum of 325 nm and 434 nm, respectively. Kynurenine is also f uorescent, but it is a w eaker f uorophore than NFK, with e xcitation and emission w avelength maximum of 365 nm and 480 nm, respectively (Fukunaga et al. 1982). Some examples of NFK OH

N H

N H

CO2-

3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid

CO

OH

CH2 NH

ROS RNS

N H

ROS RNS

5-Hydroxytryptophan

CO CH2 NH

ROS RNS

N H

CO CH2

Tryptophan NH ROS, RNS or Tryptophan 2,3-dioxygenase (free amino acid only)

O

N H

Oxindolylalanine

ROS

CO2CH2 NH3+

OH

CO

O N

CH2

O H

H

NH

O

N H Dioxindolylalanine

N-Formylkynurenine ROS or Formamidase (free amino acid only)

CO2H

CO2CH2 NH3+

NH2 O

Kynurenine

N

CO2H

Quinolinic acid

Figure 9.4. Oxidation of tryptophan residues in proteins.

NAD+

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Table 9.3. Detection of protein oxidation adduct residues and free adducts in biomedical research: N -formylkynurenine.

Adduct

Sample

Species

Comment

Reference

NFK residues

Plasmaprotein

Human

Normal controls

Lensprotein

Bo vine

(Ahmedet al. 2005 ) (F inley et al. 1998 )

Collagentype IV Cardiac mitochondria

Human Human

Cerebro - spinal f uid protein

Human

Urine

Human

Cerebro - spinal f uid

Human

NFKfree adduct

α A - cr ystallins oxidized by hydrogen peroxide: W9 and W60 modif ed. P eroxynitrite oxidation Multiplemitochondrial proteins modif ed, identif ed by proteomics, especially complex I subunit B17.2 Health y controls: 3.17 ± 0.50 mmol/mol trp; increased in Alzheimer ’s disease. Normal controls Health y controls: 391 ± 196 nM

(Katoet al. 1997 ) (T aylor et al. 2003 ,Castegna et al. 2003 )

(Ahmedet al. 2004 ) (Ahmedet al. 2005 ) (Ahmedet al. 2004 )

residue detection are given in Table 9.3. Some of the other oxidized amino acids adducts studied as mark ers of protein o xidation are 3,4 -dihydroxyphenylalanine (dopa), h ydroxyleucine, and hydroxyvaline (Fu et al. 1998a, Fu et al. 1998b).

OTHER RELATED MODIFICATIONS OF OXIDIZED PROTEINS Histidine residues in proteins suf fering severe oxidation may be o xidized to a 2 -imidazolone derivative (Cheng et al. 1992). This adduct has been detected in copper - (II) ion -catalyzed oxidation of apolipoprotein B100 of human lo w density lipoprotein (LDL) b y mass spectrometric peptide mapping (Obama et al. 2007). Saccharide residues of glycated proteins such as glycated haemoglobin and plasma proteins are susceptib le to o xidation. Such proteins contain N -1,-deoxyfructosamine residues: N εfr - amino fructosyl-lysine residues when lysine residues are glycated by glucose and N α - uctosyl acid residues when N -terminal amino acids are gl ycated. The major product for med by oxidation is N ε - carbo xymethyl -ysine l (CML) residues, and Nε-carboxymethyl-valine (CMV) residues in gl ycated hemo globin (Ahmed and Thornalley, 2007). CML residues are typicall y about 1% of the le vel of gl ycated proteins (Ahmed et al. 2005) and therefore indicate that the o xidative de gradation of gl ycated proteins is relati vely slow under physiological conditions, par ticularly when redox active ferric and cupric ions are chelated (Smith and Thornalley 1992). CML free adduct is found in high concentrations in urine, where absor ption of CML from digestion of protein in ther mally-processed foods ma y be an impor tant source (Ahmed et al. 2005, Liardon et al. 1987). CML and CMV are adv anced glycation endproducts (A GEs) with for mation involving an oxidative step and hence ha ve been called “glycoxidation adducts. ” Another gl ycoxidation

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adduct is pentosidine, which is typically less than 0.1% of the level of glycated protein (Ahmed et al. 2005). These relatively minor protein adduct residues in plasma and tissues are link ed to glycemic and o xidative status and hence are most useful biomark ers in diabetes and related disorders (Ahmed and Thornalley 2007). α ,β-Dicarbonyl compounds are formed as intermediates in the degradation of fructosamine residues and may be trapped and detected by derivatization with radiolabeled amino guanidine, for ming 5,6 -disubstituted 1,2,4 -triazine (Thor nalley and Minhas 1999). Reactive carbon yl compounds for med from lipid pero xidation such as malondialdeh yde (MDA) and 4 -hydroxynonenal (4HNE) react with proteins to for m characteristic adducts. Adducts for med b y the reaction of MD A with proteins ha ve been characterized recentl y b y mass spectrometry and are N -propenal- and dih ydropyridine-adducts of l ysine residues (Ishii et al. 2006). An important adduct of 4HNE with proteins is a c yclic Michael addition adduct with histidine residues (Uchida and Stadtman 1992, Nadkar ni and Sa yre 1995). This adduct has been detected in apolipoprotein B100 of LDL b y mass spectrometric peptide mapping (Obama et al. 2007). There is extensive literature on the biological effects of MDA and 4HNE, many of which are thought to be mediated via for mation of protein adducts. Fur ther research is required to assess whether the levels of MDA and 4HNE adducts found in situ in physiological systems are suff cient to activate pharmacological responses.

PROTEIN CARBONYLS, ADVANCED PROTEIN OXIDATION PRODUCTS, AND NON - TR YPTOPHAN FLUORESCENCE AS MARKERS OF PROTEIN OXIDATION “Protein carbon yls” ha ve been emplo yed as a measure of protein o xidation (Berlett and Stadtman 1997). Total protein carbon yls ha ve been deter mined b y deri vatization with 2,4 dinitrophenylhydrazine (2,4 -DNP). The related h ydrazones for med ha ve been detected b y characteristic absorbance at 360 and 390 nm (Levine et al. 1994); immunodetection (Nakamura and Goto 1996), including related ELISA (Buss et al. 1997); or reduction with tritiated borohydride (Yan and Sohal, 2000). Protein carbon yls are considered to be mainl y 2 -aminoadipic semialdehyde (AASA) for med from o xidative deamination of l ysine and glutamic semialdehyde (GSA) formed by oxidation of proline and arginine residues (Requena et al. 2001). AASA is also for med enzymatically by lysyl oxidase (Lucero and Kagan 2006). AASA may oxidize further to 2 -aminoadipic acid, and this has been detected in human skin collagen (Sell et al. 2007). These anal ytes ha ve been detected discretel y after reduction to 6 - yhdroxy - 2 - aminocaproicacid (HACA) and 5 - yhdroxy - 2 - amino valeric acid (HAVA) (Requena et al. 2001). The GSA residue content of rat hear t mitochondria was 20 -fold higher than AASA (Pamplona et al. 2002). Immunochemical detection of protein carbon yls suggested that the peak cellular concentration was in the cytosol close to the plasma membrane (Jung et al. 2006). There is doubt whether the measure of protein carbon yls is reliab le b y either the GS -MS or ELISA methods. Pre analytical reduction in the GC-MS method involved incubating samples at high pH, which may increase oxidation. The ELISA method has high variation and sometimes gives higher estimates for reduced proteins than for putati ve test o xidized proteins (Marangon et al. 1999). There is also a risk of oxidative degradation of the derivatizing agent 2,4-DNP leading to overestimates. In a recent attempted validation, protein carbonyl measurement of protein oxidation was unresponsive in models of o xidative stress (Kadiiska et al. 2005b). “Advanced oxidation protein products ” (AOPPs) are a fur ther indirect measure of protein oxidation in w hich molecular species contributing to the assessment ha ve been incompletel y def ned. It is a measure of protein o xidation products to o xidize iodide to iodine (W itkoSarsat et al. 1996), thought to be related to N -chloramines derivatives formed by oxidation of protein with hypochlorite generated by myeloperoxidase (Capeillere-Blandin et al. 2004). It is not clear

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if this has signif cance for oxidative damage of proteins. Recently interference of triglycerides in the assay has been reco gnized (Valli et al. 2007). Non - yptophan tr protein f uorescence has been associated with protein damage by its association with advanced glycation endproducts (AGEs) (Monnier et al. 1984) and for mation of the aging pigment lipofuscin (Sohal 1981). Formation of AGEs often involves production of trace amounts of the intense f uorophores, pentosidine (Sell and Monnier 1989), and v esperlysine (f uorophore LM-1) (Tessier et al. 1999), and the weaker f uorophore argpyrimidine (Shipanova et al. 1997). Protein oxidation may lead to the oxidation of tryptophan to form the f uorophores NFK and k ynurenine (Fukunaga et al. 1982). Interaction of proteins with lipid pero xidation products such as malondialdeh yde also leads to the for mation of f uorophores such as 3,5 diformyl - 1,4 - dih ydropyridin - 4 - ylyridinium -p derivatives (Yamada et al. 2001 ) and related crosslinks (Itakura et al. 1996). These and other compounds (retinoid deri vatives in retinal tissue, for e xample) (Eldred and Lask y 1993, Spar row et al. 1999) are thought to contribute to the f uorescence of lipofuscin, although the mixture of f uorophores is comple x (Li et al. 2006 ). A qualitative assessment of chemicall y undef ned markers of protein o xidation is pro vided in the above assays. Detection of chemicall y def ned protein carbonyls, AOPPs, and oxidative f urophores directl y with minimal sample processing b y stab le isotopic dilution anal ysis by liquid chromatography with tandem mass spectrometric detection (LC -MS/MS) is required to provide secure estimates of these mark ers of protein o xidation in tissues and body f uids. Care must be tak en in all procedures to a void protein o xidation during pre -analytic processing.

ADVANCES IN QUANTITATION OF PROTEIN DAMAGE The assessment of o xidative damage to proteins requires measurement of multiple trace amounts (fmol to pmol) of o xidized amino acids in the presence of 10 3 -to 106 - foldexcess of related unmodif ed amino acid , a challenge of the anal ytical methodolo gy emplo yed. Physiological samples are also labile; o xidation adduct anal ytes ma y be for med readil y b y inappropriate sample processing. Immunoassa ys have been employed for the measurement of protein oxidation adduct analytes but have often given unreliable and overestimated values of analyte content. This is due to (1) poor antigen specif city, (2) introduction of anal yte into the assay matrix in blocking proteins (to decrease non-specif c binding of the antibody), (3) formation of analyte during sample processing, and (4) lack of validation of matrix effects (particular interferences). Fluorescence-based assays are appropriate but are restricted to the minority of protein oxidation adduct analytes that are f uorescent. Fluorophores should also be resolv ed by a chromatographic step if quantitative measurement is desired. High perfor mance liquid chromatography (HPLC) with electrochemical detection has been used for estimation of dityrosine residues but has led to mark ed overestimation (Hensley et al. 1998). These issues, in relation to detection of oxidation adducts, have been discussed else where (Thornalley 2006a). The gold standard reference method for analysis of protein oxidation adducts is LC -MS/MS with isotopic dilution anal ysis, as described (Thor nalley et al. 2003, Thornalley 2006b). This or immunoassays cor roborated to it are preferab le. When such procedures are implemented widely, it ma y lead to a profound adv ance in understanding of o xidation damage to proteins in physiological systems; compare, for example, use of LC -MS/MS assay of isoprostanes relative to the use of the thiobarbituric acid reacti ve substances (TBARS) assays for estimation of lipid peroxidation in o xidative stress. Protein o xidation adduct residues are deter mined after exhaustive enzymatic h ydrolysis. Protein o xidation-free adducts (o xidized amino acids) are determined in ultraf ltrates of physiological f uids, typically using a 10 – 12 - kDa cut - of f microspin f lter to prepare the ultraf ltrate. Many research teams are now employing this technology but commercial a vailability to nor mal and stab le isotopic -substituted standards is required to

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improve access to this technique. Other techniques, par ticularly immunoassay, are more practicable for high sample throughput but the protocol and use in a particular sample matrix should be v alidated to the reference LC -MS/MS method before use. Recent de velopments include application of ultra high perfor mance liquid chromatography with tandem mass spectrometric detection (UPLC - MS/MS),nanof ow liquid chromatography, and robotic automation of enzymatic hydrolysis procedures.

PROTEASOMAL AND LYSOSOMAL PROTEOLYSIS OF OXIDIZED PROTEINS Oxidative damage of proteins w as originall y vie wed as post -translational modif cations that accumulated mostly on e xtracellular proteins. Oxidation adduct residues w ere thought to be formed slowly in proteins throughout life and the protein residue content of o xidation adduct residues found w as considered to represent a life -long accumulation of these adducts. While this applies for chemicall y stable oxidation adducts for med on long -lived proteins, it is no w appreciated that some o xidation adducts such as disulf des and MetSO are readil y repaired in cellular proteins and all o xidation adducts ma y also be for med on cellular and shor t-lived extracellular proteins. Protein damage b y o xidation is implicated in protein misfolding. Misfolded proteins are degraded by the proteasome to ensure the high quality of intracellular proteins (Goldberg 2003). Formation of MetSO residues in calmodulin, for example, led to degradation by the proteasome where the rate of de gradation was related to changes in secondar y structure (Ferrington et al. 2001). The median half -life of cellular proteins w as 32 hours (Ger ner et al. 2002). The degradation of oxidized proteins releases oxidation free adducts: MetSO, dityrosine, NFK, and others for repair, further metabolism, and e xcretion. Proteins damaged b y oxidation are misfolded and under go proteasomal proteol ysis by the 20S proteasome in a ubiquitin-independent process (Grune et al. 1996, Hernebring et al. 2006). Recent f ndings suggest ubiquitination and the 26S proteasome ma y also be in volved (Dudek et al. 2005). Lysosomal proteol ysis is also impor tant for de gradation of long -lived cellular proteins, endoc ytosed e xtracellular proteins (Goldber g et al. 1997), and chaperone -mediated autophagy (CMA) of cellular proteins (F ranch et al. 2001). Damaged mitochondrial proteins are degraded by Lon proteases and peptides released into the cytosol (Tatsuta and Langer 2008). Decrease of oxidative damage content of cellular proteins with increased cellular 20S proteasome acti vity (Her nebring et al. 2006) is consistent with tar geting of o xidized proteins for proteasomal degradation. Incorporation of oxidized amino acids into proteins targets the modif ed protein for cellular proteol ysis (Dunlop et al. 2008). The mechanism of o xidized protein presentation to the proteasome and l ysosomes is not full y understood , but heat shock protein -90 (hsp90) and others are lik ely in volved in presentation to the proteasome and constituti vely e xpressed for m of heat shock protein 70 (hsc70) involved in presentation of o xidized proteins to the l ysosomes in the CMA pathw ay. Hsp90 enhanced the de gradation of o xidized calmodulin b y the 20S proteasome (Whittier et al. 2004) and protected the 20S proteasome from o xidative inactivation (Conconi et al. 1998). Most CMA -targeted proteins contain a peptide sequence related to KFERQ, w hich is present in approximately 30% of c ytosolic proteins (Dice 2007). Hsc70 stimulates protein translocation across membranes and interacts with other chaperone proteins: heat shock protein 40, hsp90, and hsc70 –hsp90 organizer protein. The main function of the chaperones is to unfold the tar get protein prior to its transpor t across the l ysosomal membrane. CMA is acti vated during o xidative stress and the processing of tar get proteins is enhanced when oxidized (Kiff n et al. 2004). CMA activity declines in aging (Cuervo and Dice 2000); preservation of the CMA acti vity was associated with lower intracellular accumulation of damaged proteins, resistance to protein damage, and impro ved organ function (Zhang and Cuervo 2008 ).

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PROTEOLYTIC DEBRIS OF OXIDIZED PROTEINS: OXIDATION - FREEADDUCTS The tur nover of proteins damaged b y o xidation de gradation b y cellular proteol ysis releases oxidation free adducts (Thor nalley et al. 2003). They are also for med by digestion of oxidized proteins in food and absorbed from the gastrointestinal tract (Ahmed and Thornalley 2007). Protein oxidation free adducts (oxidized amino acids) are exported from cells, leak into plasma, and are e xcreted in the urine. Indeed , o xidized amino acids are present in all body f uids, including cerebrospinal f uid and syno vial f uid (Ahmed et al. 2004, Ahmed et al. 2006). Oxidation free adducts are the major for m in w hich irreversible oxidative damage to proteins is excreted from the body in urine (Thor nalley et al. 2003, Ahmed et al. 2005, Kirschbaum, 2001 ). MetSO had low renal clearances (0.22 ml/min) (Ahmed et al. 2005), ref ecting the eff cient renal reduction of MetSO by MetSO reductase (Weissbach et al. 2002). Loss of renal function leads to profound increases in protein oxidation free adducts in plasma. This was found experimentally after bilateral nephrectom y (Rabbani et al. 2007) and clinicall y in patients with end stage renal disease (Agalou et al. 2005). The plasma concentration of dityrosine increased less markedly in experimental bilateral ureteral ligation than in bilateral nephrectom y, demonstrating the ability of kidne y to remove damaged amino acids from the circulation (Rabbani et al. 2007 ). NFK free adduct is found in plasma, urine, and other ph ysiological f uids in vivo (Buxton and Guilbault 1974). NFK free adduct is also for med enzymatically by tryptophan 2,3 -dioxygenase (TDO) (Knox and Mehler 1950). It is usually found together with kynurenine formamidase (KFA), w hich converts NFK to k ynurenine (P abarcus and Casida 2002, Seifer t 1993). This is par t of the k ynurenine pathway that for ms k ynurenic acid as w ell as 3 -hydroxykynurenine and quinolinic acid, which are intermediates involved in NAD+ synthesis (Heyes et al. 1997, Schw arcz and P ellicciari 2002) (F igure 9.4). TDO e xpression w as induced via tumor necrosis factor -α and interferon -γ dependent pathw ays in response to endoto xin (Fujigaki et al. 2001). Kynurenine pathway metabolites were also increased in uremia (Pawlak et al. 2001). TDO and KFA act on tr yptophan and NFK free adduct, respecti vely, and not tr yptophan and NFK residues.

PROTEOMICS STUDIES OF OXIDIZED PROTEINS Proteomics of protein o xidation has emplo yed se veral techniques to tag o xidized proteins and mass spectrometr y proteomics to then identify the tagged protein. Techniques used include: (1) thiol o xidation by alkylation of residual thiols and reducti ve tagging of disulf des (Lind et al. 2002 ), (2) two - dimensional, sequential non - reducing/reducing SDS - A P GE (diagonal electrophoresis) (Brennan et al. 2004), (3) 2,4 -DNP modif cation of carbon yls and immunodetection/pull-down of 2,4 -DNP-constaining proteins (Caste gna et al. 2002), and others (Barelli et al. 2008). There are prob lems with the con ventional proteomics approach that require attention and scr utiny of the comple x data outputs from mass spectrometr y analysis: 1. Oxidationof peptides during pre - anal ytic processing and matrix - assistedlaser desorption (MALDI) and electrospray ionisation (ESI) 2. Useof high temperature and pH in pre - anal ytic processing 3. Useof oxidized blocking proteins in immunodetection. Oxidation of methionine, tyrosine, and tr yptophan residues in peptides and proteins is commonly observed during electrophoresis and MALDI and ESI ionisation. Oxidation of alkylated cysteine residues produced during reducti ve alk ylation of limited proteol ysis digests also occurs, producing no vel fragmentation pathw ays, and compromises peptide detection (Steen

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and Mann 2001). For future analysis, specif c detection of oxidized amino acid residues within peptides of limited proteol ysis digests by nanof ow label - freeor isotopically - labelledpeptides is required, and care must be tak en to control for protein o xidation during all steps of pre analytic processing.

BIOMARKERS The use of biomarkers as surrogate assessment of clinical endpoints offers several advantages: (1) indication of earl y, pre -clinical stages or risk of chronic disease, (2) the y are present in a large population, w hich decreases subject recr uitment problems, (3) the y are measured with noninvasive or minimall y invasive procedures (sampling b lood and urine), (4) the y are often highly responsive to therapeutic inter ventions, and (5) the y may be relati vely inexpensive to measure. Because w e predict that protein o xidation plays an impor tant role in man y chronic and de generative diseases, it is e xpected that measures of protein o xidation ma y be robust biomarkers. This requires clinical v alidation. The Biomark ers of Oxidati ve Stress Study (BOSS) v alidated mark ers of o xidative stress in specif c animal models of o xidative stress: (1) carbon tetrachloride poisoning, (2) environmental exposure to ozone, (3) systemic exposure to endotoxin, and (4) continuous skin e xposure to cumene h ydroperoxide. It remains unclear how these models translate to oxidative stress in the clinical setting. Unfortunately no measures of protein oxidation emerged as robust biomark ers of oxidative stress (Kadiiska et al. 2005a). This ma y be due to lack of anal ytical robustness in the techniques used in biomark er measurement. Analysis of oxidized amino acid residue excretion in urine is a procedure that is analytically robust, involves minimal sample processing, and is noninvasive for protein oxidation biomarker assessment. This w as perfor med b y stab le isotopic dilution anal ysis LC -MS/MS after urine ultraf ltration and indicated increased oxidative damage in clinical diabetes and end stage renal disease on peritoneal dialysis but not in patients pre-dialysis with chronic renal failure (Ahmed et al. 2005, Agalou et al. 2005). The future challenge is to build on this and similar biomarkers of protein oxidation with careful and thorough clinical v alidation.

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Chapter10 Immunochemical Detection of Lipid Peroxidation - specifc Epitopes K oji Uc hida

INTR ODUCTION Oxidative stress is increasingl y seen as a major upstream component in the signaling cascade involved in many cellular functions, such as cell proliferation, inf ammatory responses, adhesion molecule stimulation, and chemoattractant production. A growing body of e vidence suggests that man y of the ef fects of cellular dysfunction under o xidative stress are mediated b y products of non -enzymatic reactions, such as the pero xidative degradation of polyunsaturated fatty acids. Lipid pero xidation in tissue and tissue fractions represents a de gradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases including atherosclerosis, diabetes, cancer , and rheumatoid arthritis, as well as drug-associated toxicity, postischemic reoxygenation injury, and aging. The peroxidative breakdown of pol yunsaturated fatty acids has also been implicated in the pathogenesis of many types of li ver injury and especially in the hepatic damage induced b y several toxic substances. There is increasing evidence that reactive aldehydes, such as alkanals, 2-alkenals, 4-hydroxy2 - alk enals, 4 - xoo - 2 - alk enals, and ketoaldehydes (Figure 10.1 ),endogenously generated during the process of lipid peroxidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues (Esterbauer et al. 1991, Uchida 2003). More importantly, these aldeh ydes have been implicated as causati ve agents in c ytotoxic processes initiated by the exposure of biological systems to oxidizing agents. Compared to free radicals, the aldeh ydes are relati vely stab le and can dif fuse within or e ven escape from the cell and attack targets far from the site of the original e vent. These aldehydes can covalently react with biomacromolecules, such as proteins, to generate adducts that could serve as useful biomarkers for lipid peroxidation (Uchida 2003). The lipid peroxidation - specifc adducts generated on protein molecules ha ve been used as the biomarkers for o xidative stress. The most popular approach for the detection of lipid per oxidation - specifc adducts generated on protein molecules must be the use of antibodies. Antibodies are raised b y immunizing animals with car rier proteins, such as k eyhole limpet Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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10 O

R

O O

H 2-Alkenals

R H α-Ketoaldehydes

OH O R

OH

H O

4-Hydroxy Hydroxy--2-alkenals

R H

O

α-Hydroxyaldehydes

O R H 4-Oxo Oxo--2-alkenals

Figure 10.1. Sources of lipid peroxidation -specif c epitopes. O NH2

HN

R

H2O2 R-CHO

N H

O

Lysine

N H

O

N-Acylated lysine

Figure 10.2. H2O2-mediated protein N-acylation by alkanals.

hemocyanoin (KLH), that w ere treated with lipid pero xidation products and tar get specif c antigenic str uctures (lipid pero xidation-specif c epitopes) generated on the amino acid side chains of proteins. Taking advantage of the f act that the lipid pero xidation-specif c epitopes are excellent immunogens that are capable of stimulating adaptive immune response, a number of monoclonal antibodies against these epitopes ha ve been developed. This chapter is an overview of studies on lipid pero xidation-specif c epitopes, focusing on their chemical str uctures and development of monoclonal antibodies.

LIPIDPEROXIDATION - SPECIFICEPITOPES ALKAN ALS Alkanals, such as he xanal, are most abundantl y for med in lipid pero xidation. Upon reaction with proteins, these aldehydes react with lysine residues to form an imine or Schiff base adduct. Due to the reversible nature of such unconjugated Schif f bases, these aldehydes have received relatively little attention as the causati ve agent for modif cation of nucleophilic biomolecules. However, Ishino et al. (2008) ha ve recentl y estab lished a no vel mechanism of irreversible covalent protein modif cation by aldehydes, in w hich H 2 O2 and alk yl hydroperoxides mediate the binding of saturated aldeh ydes to the l ysine residues of protein to generate str ucturally unusual N - ac ylation products (Figure 10.2 ).

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CHO

NH2

+

N

N

CHO

N H

O

Lysine

N H

O

FDP--lysine FDP

N H

O

MP MP--lysine

Figure 10.3. Formation of FDP -lysine and MP -lysine upon reaction of lysine residue with acrolein.

2 - ALKEN ALS 2-Alkenals represent a group of highly reactive aldehydes containing two electrophilic reaction centers. 2-Alkenals can react by a Michael addition mechanism with the nitrogens of the imidazole ring of histidine and the sulfh ydryl group of cysteine to generate β - substitutedpropanal adducts (Figure 10.3). Upon reaction of acrolein with the ε-amino group of l ysine, the major product is not the β-substituted propanal or Schif f ’s base crosslinks but Nε - (3 - myl for - 3,4 dehydropiperidino)lysine (FDP -lysine), which requires the attachment of tw o acrolein molecules to one l ysine side chain (F igure 10.3) (Uchida et al. 1998a, Uchida et al. 1998b). In addition, Furuhata et al. (Furuhata et al. 2002) revealed the electrophilic potential of FDPlysine and established a novel mechanism of protein thiolation in w hich the FDP -lysine generated in the acrolein -modif ed protein reacts with sulfh ydryl groups to for m thioether adducts. In a later study , Nε - (3 - meth ylpyridinium)lysine (MP -ysine), l resulting from the Schiff base formation of acrolein with the ε-amino g roup of l ysine, w as also identif ed (Fur uhata et al. 2003). The formation of these lysine-pyridinium species in proteins may result in the placement of a f xed, positi ve char ge on the ε-amino g roup. Moreover, in contrast to the f act that the FDP-type adducts are unstab le inter mediates against nucleophilic addition, the p yridinium adducts are highl y stab le end products. The for mation of the p yridinium adducts is also a dominant pathway for the modif cation of the primar y amine with 2 -alkenals, such as crotonaldehyde, 2 -hexenal, and 2 -octenal (Ichihashi et al. 2001). 4 - HYDR OXY - 2 - ALKEN ALS 4 - Hydro xy - 2 - alk enals represent the most prominent lipid peroxidation - specifc aldehydes. In a similar manner to 2 -alkenals, 4-hyroxy-2-alkenals are also potent electrophiles and preferentially react with cysteine, histidine, and lysine residues of proteins (Uchida 2003). The mechanism of modif cation of these amino acids with 4 -hydroxy-2-alkenals has been e xtensively studied on 4 -hydroxy-2-nonenal (HNE), and it has been estab lished that HNE primarily forms adducts ha ving a hemiacetal str ucture via the Michael addition mechanism (F igure 10.4). Because 4-hydroxy-2-alkenals generated in lipid peroxidation are a racemic mixture of 4R -and 4S-isomers, the c yclic hemiacetal adducts contain chiral centers at C -2, C -3, and C -4 of the 4-hydroxy-2-alkenal moiety. Accordingly, the c yclic hemiacetal adducts could be composed of at least eight conf gurational isomers. HNE - CYSTEINE ADDUCTS HNE, among the reacti ve aldehydes, is a major product of lipid pero xidation and is belie ved to be lar gely responsib le for the c ytopathological ef fects obser ved during o xidative stress (Esterbauer et al. 1991). HNE exerts these effects because of its facile reactivity with biological

160 Chapter

10 OH

OH

O

O

H

HNE

X

H

XH

Protein OH O

X

HNE adducts Figure 10.4. Formation of hemiacetal -type Michael adducts upon reaction of protein with HNE. XH represents nucleophilic amino acid side -chains (Cys, His, Lys).

materials, particularly the sulfhydryl groups of proteins. The reaction of HNE with sulfh ydryl groups leads to the for mation of thioether adducts that fur ther under go c yclization to for m cyclic hemiacetals. Although HNE also for ms Michael adducts with the imidazole moiety of histidine residues and the ε-amino g roup of l ysine residues (Uchida 2003), the for mation of thiol-derived Michael adducts, stabilized as the c yclic hemiacetal, is considered to constitute the main reactivity of HNE, due to the nucleophilic potential of the sulfhydryl group compared with those of the imidazole and amine g roups. Balogh et al. (Balo gh et al. 2008) recently characterized the stereochemical conf gurations of the HNE-glutathione adduct by NMR experiments in combination with simulated annealing structure determinations. To differentiate between various modes of carbonyl group formation, a method for the detection and quantif cation of protein carbon yl g roups associated with the conjugation of protein sulfh ydryl g roups with lipid pero xidation products w as pre viously developed (Uchida and Stadtman 1992a). This method is based on the reduction of the adducts with NaB[3 H]H4 to stable radioactive derivatives followed by cleavage of the thioether linkage upon treatment with Raney nickel. Although this procedure is not specif c for the HNE-cysteine adducts, it can provide a means of determining the fraction of total free carbonyl groups introduced into proteins via reaction of α ,β-unsaturated aldehydes with protein sulfh ydryl groups. HNE - HISTIDINE ADDUCTS Structural information on the nature of the HNE modif cation of histidine side chain w as f rst reported by Uchida and Stadtman (Uchida and Stadtman 1992b). The HNE -histidine Michael adduct is readily isolable and is stabilized toward retro-Michael reaction, apparently due to the poorer leaving group ability of imidazole o ver amine at neutral conditions. It w as f rst speculated that the HNE -histidine adduct w as a mixture of the isomeric for m of the Nπ -and Nτsubstituted adducts of the imidazole ring; ho wever, on the basis of the NMR spectral anal ysis ylation). of the adducts, it appeared that the reaction e xclusively occurs at one position ( Nτ - alk The observation that reduction of the aldehyde group of the primary Michael addition product with sodium boroh ydride converts them to the h ydroxy deri vatives that are stab le to strong acid h ydrolysis for med the basis of methods for the identif cation and quantif cation of the

Immunochemical Detection of Lipid P eroxidation-specif c Epitopes

161

HNE-histidine Michael adduct of proteins b y conventional amino acid anal ytical techniques (Uchida and Stadtman 1992b). It w as shown by means of these techniques that at least 80% of the histidine residues that were lost when human plasma low density lipoprotein (LDL) was treated with HNE w ere accounted for as the Michael addition product (Uchida et al 1993). HNE YSINE -L ADDUCTS The reaction of HNE with protein-based lysine ε-amino groups has been documented in several instances (Esterbauer et al. 1991). In particular, it has been suggested that covalent modif cation of LDL b y HNE ma y be of pathoph ysiologic impor tance. Upon reaction with l ysine, HNE forms multiple products, including the HNE -lysine Michael adduct, p yrrole-type adduct, and f uorescent cross -linking-type adducts. Isolation and a proposed str ucture of the HNE -lysine Michael adduct were f rst reported by Uchida and Stadtman (Uchida and Stadtman 1993) and Szweda et al. (Szw eda et al. 1993). Later , str uctural def nition of the HNE -lysine Michael adduct was done b y Nadkarni and Sa yre, demonstrating that the HNE -lysine Michael adduct is predominantly produced in the presence of a lar ge excess of l ysine over HNE and that the use of equimolar quantities affords mainly adducts containing two (or more) molecules of HNE per amine molecule (Nadkar ni et al. 1995). The pyrrole adduct is the f rst Schiff base -derived HNE -amine adduct (Sa yre et al. 1993), which is apparentl y v ery stab le and represents the most stab le end product of HNE protein modif cation. The amine -based HNE adducts that are for med rapidly are later shown to represent simple amine Michael adducts and the Schif f base Michael adduct 1:2 cross-links (Nadkarni et al. 1995). On the other hand , Esterbauer and his colleagues demonstrated that treatment of LDL with HNE generates the same lipofuscin -like f uorescence properties as seen in o xidized LDL (Esterbauer et al. 1986). This f nding suggested that HNE could be a major contributor to the f uorescence generated in o xidized LDL. The chemical nature of the f uorophore arising from HNE protein modif cation had remained elusive; however, Itakura et al. (Itakura et al. 1998) have identif ed for the f rst time the major lipofuscin - lik e f uorophore deri ved from HNE and l ysine to be the 3 -hydroxy-3-imino-1,2dihydropyrrole derivative and found that the f uorescent properties of this pigment are similar to those of the o xidized LDL. Later, the same adduct w as also repor ted by Xu and Sa yre (Xu and Sayre 1998) and Tsai et al (Tsai et al. 1998). A pathway for the formation of the f uorophore f rst proposed w as that ε-amino g roup of l ysine reacts readil y with C -1 and C -3 of HNE via Schiff base for mation and Michael addition, respecti vely, to for m the initial 1:2 HNE -amine intermediate, which is subsequently converted into the f uorophore via two oxidation steps and intermolecular c yclization. Mechanistic studies on the HNE -derived f uorophore formation have proposed an alter native mechanism, involving two 2e o xidations following initial Schif f base formation (Xu and Sa yre 1998). 4 -Oxo-2-nonenal (ONE), w hich forms this f uorophore far more ef fectively than HNE, has been recentl y identif ed as the lipid pero xidation product (Lee and Blair 2000), suggesting that ONE rather than HNE ma y be a potential source of this f uorophore in biological systems. 4 -XO O - 2 - ALKEN ALS It has been estab lished that the free -radical-initiated degradation of n6 -polyunsaturated f atty acids generates ONE, the 4 -keto cousin of HNE (Rindgen et al. 1999, Lee and Blair 2000). Upon reaction with proteins, ONE selecti vely modif es the nucleophilic side chains of l ysine, histidine, cysteine, and ar ginine (Doorn and P etersen 2002). The predominant initial reaction appears to in volve the Michael addition to the central ONE doub le bond, more at C3 than at C2, to gi ve substituted 4 -oxononanals. These adducts are relati vely unstab le and could be further converted to stab le long -lived products, w hich include dih ydrofuran, dihydropyrrole, and isomeric 4 -ketoamide derivatives originating from the reaction of ONE with l ysine (Zhu

162 Chapter

10

and Sayre 2007) and a substituted imidazole deri vative with ar ginine (Oe et al. 2003). ONE also for ms furan deri vatives upon its reaction with c ysteine and histidine deri vatives (Zhang et al. 2003, Yocum et al. 2005). Shimozu et al. (Shimozu et al. 2009) have recently characterized the products, including the 2 -cyclopentenone and thiomor pholine derivatives, that originated from the initial ONE -cysteine Michael adducts. O THER SHORT - CHAINALDEHYDES Other impor tant reactive aldehydes originating from the lipid pero xidation include k etoaldehydes, such as malondialdehyde (MDA). MDA occurs in biological materials in various covalently bound for ms (Esterbauer et al. 1991). It has been suggested that MD A primarily forms adducts with lysine residues of proteins or with amine head g roups of phospholipids, such as phosphatidylserine or phosphatidylethanolamine. The major reaction of MDA comprises addition to primar y amines, generating Nε - (2 - propenal)l ysine (Figure 10.3 )(Chio and Tappel 1969). This adduct has been detected as the major form in which endogenous MDA is excreted in rat and human urine (McGir r et al. 1985, Draper et al. 1988). MDA also for ms some f uorescent products, such as the dih ydropyridine-type adduct, a model of f uorescent components in lipofuscin (F igure 10.5) (Kikuga wa and Ido 1984, Slatter et al. 1998). Other f uorescent products include N N , ’ - disubstituted1 - amino - 3 - iminopropene - type and pyridyldihydropyridine - typelysine -ysine l cross - links(Chio and Tappel 1969 ,Itakura et al. 1996 ). COREALDEHYDES The primary products of lipid pero xidation, lipid hydroperoxides, can undergo carbon -carbon bond cleavage via alkoxyl radicals in the presence of transition metals, giving rise to the formation of short-chain, unesterif ed aldehydes, or a second class of aldeh ydes still esterif ed to the parent lipid. These esterif ed aldeh ydes are commonl y ter med core aldeh ydes (Itabe 1998). Although these compounds ha ve received less attention, the for mation of phospholipid - and cholesteryl ester -core aldehydes during the o xidation of LDL has been demonstrated (W atson et al. 1997, Kamido et al. 1992). Itabe et al. (Itabe et al. 1994) provided evidence that oxidized phospholipids form complexes with lysine residues on proteins due to the presence of 9 -oxononanoylphosphatidylcholine. In addition, the Schiff-base adducts of phospholipid core -aldehydes with lysine residues of myoglobin (Ra vandi et al. 1997) and th yroglobin (W ang and Tai 1990) ha ve been identif ed. Furthermore, the binding of phospholipids to apo B during LDL o xidation was directly shown by measuring the phosphorous incor porated into the protein (Gillotte et al. 2000). In ter ms of cholester yl ester -core aldehydes, Kamido and colleagues (Kamido et al. 1992) isolated and identif ed 9 - xoononanoylcholesterol (9 - ONC)and 5 - xoovaleroylcholesterol (5 CH3 OHC

NH2

N OHC

N H

O

Lysine

CHO

CHO

N H

O

DHP--lysine DHP

Figure 10.5. Formation of DHP -lysine upon reaction of lysine residue with MDA.

Immunochemical Detection of Lipid P eroxidation-specif c Epitopes

163

OVC) as the o xidation products of cholester yl linoleate and cholester yl arachidonate, respectively, in o xidized LDL. The oxidation of HDL has been sho wn to result in the for mation of primarily 5 -OVC and 9 -ONC and , to a lesser e xtent, the cor responding 7 -ketocholesterol derivatives (Kamido et al. 1995). In line with the potential pathoph ysiological role of these compounds, the cholesteryl ester- and 7 -ketocholesteryl ester-core aldehydes of varying chain lengths were identif ed in human atheromas (Hoppe et al. 1997, Karten et al. 1998). Covalent binding of cholesteryl ester-core aldehyde to proteins has been ascertained by LC-MS analysis (Hoppe et al. 1997). These f ndings strongly suggest that the for mation of lipid -protein complexes could occur via interactions betw een the carbon yl g roups of the phospholipid - and cholesteryl ester-core aldehydes and lysine residues on proteins.

IMMUNOCHEMICAL DETECTION OF LIPID PEROXIDATION - SPECIFICEPITOPES Table 10.1 lists the lipid - pero xidation - specifc monoclonal antibodies that ha ve been established by the author ’s group at Nagoya University. As space is limited, in the following studies on the de velopment of monoclonal antibodies against HNE -histidine conf gurational isomers are discussed. The follo wing sections discuss the de velopment of monoclonal antibodies against HNE - histidineconf gurational isomers. STR UCTURAL BASIS OF HNE - HISTIDINE CONFIGURATIONAL ISOMERS Due to the presence of three chiral centers at C -2, C -4, and C -5 in the tetrahydrofuran moiety, the HNE -histidine Michael adduct w as suggested to be composed of at least eight conf gurational isomers (Figure 10.6) (Uchida and Stadtman 1992b, Nadkarni and Sayre 1995). In addition, the previous observations that (1) the HNE-Nα-acetylhistidine Michael adduct was detected as two peaks upon reverse-phase HPLC analysis and (2) four peaks w ere detected in the acid hydrolysis followed by the amino acid analysis of their OPA derivatives (Uchida and Stadtman reac1992b) also suggested the multiplicity of primary products in the HNE/Nα - acetylhistidine tion. However, with complicating diastereoscopic splittings b y three chiral centers, the proton NMR spectr um of R,S-HNE-histidine adduct w as too comple x to anal yze directly (Nadkar ni and Sayre 1995 ). To f acilitate the str uctural characterization of HNE -histidine isomers, Hashimoto et al. (Hashimoto et al. 2003) prepared R - HNEand - S - HNE- Nα - acetylhistidineadducts separately, both of w hich provided two peaks with relati ve amounts of 1:2 in the re verse-phase HPLC analysis. With regard to the reacti vity of HNE enantiomers to ward the histidine deri vative, no signif cant differences in the rate of formation of the Michael adducts between two HNE enantiomers were observed, suggesting that the chilarity of the C -4 hydroxy group may not af fect the reactivity at the C -3 double bond with the imidazole g roup of the histidine deri vative. The 600 MHz NMR anal ysis of the four peaks re vealed that each peak contained a pair of diastereomers. In addition, the absolute conf gurations of eight isomers w ere deter mined by NOE analysis and the signals of the isomers in the one -dimensional proton NMR spectr um of the R,S - HNENα- -acetylhistidine Michael adduct w ere fully assigned. The assignment of the eight isomers of the HNE -Nα - acetylhistidineMichael adducts conf rmed the pre vious proposal of Nadkarni and Sa yre made on the basis of an indirect comparati ve experiment (Nadkar ni and Sayre 1995 ). In molecular orbital calculation of the model compound of the HNE -histidine adduct -h - 5 - meth yltetrahydrofuran - 4 - yl)imidazole],there was a correlation [5′ - meth yl -Nt - (2 ydroxy between the trend of the relati ve stability and the O —C-2 bond length in the tetrah ydrofuran moiety (Hashimoto et al. 2004). In addition, the relati ve ener gies calculated b y molecular orbital methods cor related well with the relati ve amounts of the eight isomers in the NMR analysis. These obser vations suggest that the electron delocalization features on the o xygen

Table 10.1. Line-up of lipid peroxidation -specif c monoclonal antibodies.

Lipidperoxidation products

mAb

Epitope

References

13H1

9 - HODE

Unpub lished

9H2

13 - HODE

Unpub lished

12H8

12 - HETE

Unpub lished

Oxidized lipids 9 - Hydro xy - 10E,12Z octadecadienoic acid (9 - HODE) 13 - Hydro xy - 9Z,11E octadecadienoic acid (13 - HODE) 12 - Hydro xyeicosatetraenoic aci (12 - HETE) Leuk otoxin (epoxylinoleic acid) 7 -etocholesterol K (7 - KC)

21D1

Leuk otoxin

Unpub lished

35A - 8

7 - KC

Unpub lished

Protein - boundaldehydes Acrolein(ACR)

5F6

A CR -ysine l

Crotonaldeh yde (CRA

82D3

Malondialdeh yde (MDA)

1F83

4 - Hydro xy - 2 - xenal he (HHE) 2 - Hydro xyheptanal (2HH)

53

4 - Hydro xy - 2 - nonenal (HNE) 4 - Hydro xy - 2 - nonenal (HNE) 4R - 4 - Hydro xy - 2 - nonenal ((R ) - HNE) 4S - 4 - Hydro xy - 2 - nonenal ((S ) - HNE) Protein - boundcore aldehyde 9 - Oxononano ylcholesterol (9 - ONC) DNA - boundaldehydes Acrolein(ACR) 4 - Oxo - 2 - nonenal (ONE)

164

3C8 HNEJ2 2C12 R310 S412

Uchidaet al. (1998) PNAS 95, 4882. CRA ysine -l Ichihashiet al. (2001) JBC 276, 23903. Fluorescent Yamada et al. (2001) MDA -ysine l J Lipid Res 42, 1187. HHE - histidine Y amada et al. (2004) J Lipid Res 45, 626. 2 - HHysine -l Itakuraet al. (2003) BBRC 308, 452. HNE - histidine T oyokuni et al. (1995) FEBS Lett. 359, 189. Fluorescent Itakuraet al. (2000) HNE -ysine l FEBS Lett . 473, 249. R( ) - HNE - histidine Hashimotoet al. (2003) JBC 278, 5044. S( ) - HNE - histidine Hashimotoet al. (2003) JBC 278, 5044.

2A81

9 - ONCysine -l

21

A CR deoxyadenosine ONE deoxyguanosine

6A3

Ka wai et al. (2003) JBC 278, 21040. Kawai et al. (2003) JBC 278, 50346. Kawai et al. (2002) Carcinogenesis 23, 48.

Immunochemical Detection of Lipid P eroxidation-specif c Epitopes H N

NHNH-COCH3

NH--COCH3 NH

N

COOH

165

COOH

N

N

H

H O O

OH

OH

NH--COCH3 NH

N

COOH N

O

OH

Figure 10.6. Formation of HNE -histidine Michael adduct upon reaction of histidine residue with HNE.

atom of tetrahydrofuran moiety may ref ect the relative amounts of isomers. The delocalization of the electron pair on the o xygen atom of the tetrah ydrofuran ring increases the strength of the covalent bonding nature and in tur n decreases the polarization of the bond and stabilizes the whole molecule. Thus, the conf guration of the tetrahydrofuran ring may affect the electron delocalization features, which contribute to the stability of the HNE -histidine adduct. ANTIBODIESAGAINST HNE - HISTIDINE CONFIGURATIONAL ISOMERS Because of the increasing interest in HNE and HNE modif cation of proteins under o xidative stress, it seemed useful to prepare an antibody interacting specif cally with the HNE moiety or with the HNE-amino acid conjugates in proteins; such antibodies have been prepared by immunizing rabbits with HNE-treated KLH (Uchida et al. 1994), in which HNE adducts of histidine, lysine, and c ysteine serve as the antigenic sites. Later , Toyokuni et al. (T oyokuni et al. 1995) raised the monoclonal antibody (HNEJ2) against HNE -modif ed KLH and found that the antibody cross - reactedspecif cally with HNE-modif ed proteins and had a higher aff nity for HNEhistidine adduct than for HNE -lysine and HNE -cysteine adducts. This monoclonal antibody was attested to be specif c for the HNE -histidine Michael adduct. In a later study , Esterbauer ’s g roup, independentl y follo wing the same line of research, reported on an antibody , which also seems to be highl y specif c for HNE bound to histidine residues (Waeg et al. 1996). On the other hand, antibodies against the HNE -lysine adduct have also been raised. Sa yre et al. (Sa yre et al. 1996) raised and epitopicall y characterized the specif c pol yclonal antibodies reco gnizing the l ysine-based 2 -pentylpyrrole adducts. The pol yclonal and monoclonal antibodies against HNE -lysine f uorophore (3 - yhdroxy - 3 - imino - 1,2 dihydropyrrole) have also been prepared against the f uorophore-protein conjugate (Tsai et al. 1998, Itakura et al. 2000). On the other hand, most of the antibodies, including HNEJ2, directed to the HNE -modif ed protein have been raised against a protein treated with racemic HNE. Therefore, nothing w as known about which of the two enantiomers bound to proteins is generated in vitro and in vivo . However, Hashimoto et al. (Hashimoto et al. 2004) successfully raised monoclonal antibodies, R310 and S412, against R - HNE - treated and S - HNE - modif ed KLH, respectively (Figure 10.7). It was obser ved that R310 and S412 sho wed the highest aff nity for the R - HNE - treated and S-HNE-treated proteins, respectively, and scarcely reacted with the proteins treated with other

166 Chapter

10

A

B

OH

OH O

O R

S

H R-HNE M

0

1

2

3

H

S-HNE 4

5

0

R310

1

2

3

R-HNE 4

5 (mM)

M

0

1

2

3

S-HNE 4

5

0

1

2

3

4

5 (mM)

S412

Figure 10.7. Specif city of monoclonal antibodies R310 and S412 to the R-HNE-modif ed and S-HNE-modif ed proteins. (A) Immunoreactivity of R310 toward R-HNE-modif ed and S-HNEmodif ed BSA. Upper, SDS -PAGE. Lower, immunoblot. (B) Immunoreactivity of S412 toward R-HNE-modif ed and S -HNE-modif ed BSA. Upper, SDS -PAGE. Lower, immunoblot. In panels A and B, BSA (1 mg/ml) was incubated with R - or S -HNE (0 to 5 mM) in 1 ml of 50 mM sodium phosphate buffer, pH 7.4, for 24 hours at 37 °C.

aldehydes. The lack of cross -reactivity of the antibodies for the 2 -nonenal-treated protein can be ascribed to the absence of the 4 -hydroxy group, which leads the primary Michael adduct to the tetrahydrofuran derivative through an intra -molecular cyclization. This and the observation that both antibodies did not cross react with the proteins that had been treated with the HNE analogs, such as 4 - yhdroxy - 2 - pentenal, 4 - yhdroxy - 2 - xenal, he 4 - yhdroxy - 2 - heptenal, 4 - yhdroxy 2-octenal, and 4 -hydroxy-2-decenal, suggest that both tetrah ydrofuran and butyl moieties of the HNE -histidine adduct may be critical for the antibody binding. In addition, the binding of these antibodies to the HNE -treated proteins w as selecti vely inhibited b y HNE -histidine adducts, suggesting that the imidazole ring is also in volved in the antibody binding. -h -5Thus, the author ’s g roup proposes that R310 and S412 reco gnize the Nτ - (2 ydroxy butyltetrahydrofuran-4-yl)imidazole as the common epitopes. Fur thermore, the antibodies’ ability to reco gnize the conf gurations of the tetrah ydrofuran moiety of the HNE -histidine adduct was characterized, showing that R310 and S412 preferentiall y reacted with the mixture of 2 R, 4S, 5R and 2 S, 4S, 5R isomers and the mixture of 2 R, 4S, 5S and 2 S, 4S, 5S isomers, respectively (Figure 10.8). Based on the common conf gurations in each mixture, the author ’s group suggested that both antibodies might reco gnize the conf gurations at C -4 and C -5 of the tetrahydrofuran ring in the adduct, i.e., R310 and S412 reco gnize the 4 S ,5R and 4 S ,5S conf gurations, respectively. Cr ystallization of R310 F ab in the presence of the HNE -histidine adduct shows that the adduct binds to a h ydrophobic pocket in the g roove, the antigen -binding site, consisting of 6 CDRs (F igure 10.9) (Akagawa et al. 2006). The crystal structure of R310 F ab showed that the antibody primaril y binds the HNE -histidine adduct b y sandwiching betw een

167

Immunochemical Detection of Lipid P eroxidation-specif c Epitopes 1.2 His

His 2RS,4R,5R

1.0 OH

O

R

2RS,4R,5R

O

OH

0.8

2RS,4R,5S

B/Bo

R

His

His

2RS,4R,5S

0.6

2RS,4S,5S

0.4 02 0.2

R

OH

O 2RS,4S,5R

R

O

2RS,4S,5R

OH 0 10-4

2RS,4S,5S

10-3

10-2

10-1

100

Competitor (mM)

Figure 10.8. Binding of a monoclonal antibody R310 to the conf histidine adducts.

A

gurational isomers of HNE -

B

Figure 10.9. X-ray crystallographic analysis of R310 Fab fragment. (A) The crystal structure of a recombinant R310 Fab with the R -HNE-histidine adduct. The R -HNE-histidine is shown in wireframe model. (B) The hydrophobic domains at the antigen -binding site of the antibody. Courtesy of Dr. Sohei Ito (University of Shizuoka).

hydrophobic domains comprised of several critical stacking Tyr and Phe residues. Thus, hydrophobic desolvation may underlie the obser ved negative enthalpy of binding. The presence of immunoreacti ve materials with R310 and S412 in vivo was demonstrated in the kidne y of rats e xposed to F e3+-NTA. It has been suggested that o xidative stress is one of the basic mechanisms of F e3+-NTA-induced acute renal injur y and is closel y associated with renal carcinogenesis. The present study using R310 and S412 demonstrated that, in agreement with the previous observation on racemic HNE (Toyokuni et al. 1995), both R - HNEand A S-HNE epitopes were mainly detected in the pro ximal tubules, the tar get organs of Fe3+ - NT (Figure 10.10). Ho wever, the intracellular localizations of these epitopes w ere mark edly different. The S-HNE epitopes w ere detected in c ytosols and in some of the nuclei, w hereas the R-HNE epitopes were mainly detected in the nuclei. Although the mechanism of the distinct

168 Chapter

10

R310

S412

Figure 10.10. Immunohistochemistry of renal cortex with R310 (Left), and S412 (Right) (serial sections, ×400). The immunoreactivities appeared in some of the renal proximal tubular cells 3 hours after the administration of 15 mg Fe/kg body weight of Fe 3+-NTA. The immunoreactivities with S412 were mainly detected in the cytoplasm and in some of the nuclei, whereas the immunoreactivities with R310 were mainly detected in the nuclei.

localization of R -and S-HNE epitopes is cur rently unknown, these results in vite speculation that there ma y be a dif ferential mechanism for production of R -and S-HNE or there ma y be specif c targets of each enantiomer in the cells. Clearl y, it is impor tant to establish the mechanism for the differential cellular distributions ofR and - S-HNE epitopes in the cells. Furthermore, the characterization of the biolo gical consequences of their distributions also merit immediate attention.

CONCLUSION On the basis of a large number of reports concerning the detection of lipid-peroxidation-specif c adducts as biomarkers in human diseases, there is no doubt that the steady -state levels of lipid peroxidation products increase under pathophysiological states associated with oxidative stress. Considerable progress has also recentl y been made to ward understanding the mechanisms of action of lipid pero xidation products. The quantitative and anal ytical importance of lipid -peroxidation - specifc adducts has prompted the de velopment of methods to specif cally analyze these adducts because of understanding of their chemical nature, for mation pathway, and distribution level in vivo . Immunological detection is a po werful tool that can be used to e valuate the presence of a desired target and its subcellular localization. The major advantages of this technique over the chemical approaches are the e valuation of small numbers of cells or archi val tissues that ma y otherwise not be subject to analysis. The antibodies developed through this procedure bind not only to the modif ed protein used as the immunogen, but to a variety of other proteins on which the same epitope is found. Thus, antibodies generated against a modif ed protein with lipid peroxidation - specifc products recognize a variety of similarly modif ed proteins. Such properties mak e the antibodies directed to the epitopes useful and reliab le for the immunolo gical evaluation of lipid pero xidation in vitro and in vivo .

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UchidaK , Stadtman ER. 1992a . Modifcation of histidine residues in proteins b y reaction with 4 - yhdroxynonenal . Proc Natl Acad Sci USA 89 : 4544 – 4548 . UchidaK , Stadtman ER. 1992b. Selective cleavage of thioether linkage in protein modif ed with 4 - yhdroxynonenal . Proc Natl Acad Sci USA 89 : 5611 – 5615 . UchidaK , Stadtman ER .1993 . Covalent attachment of 4 - yhdroxynonenal to glyceraldehyde - 3 phosphate dehydrogenase: a possible involvement of intramolecular and inter molecular cross- linking reactions . J Biol Chem 268 : 6388 – 6393 . UchidaK ,T oyokuni S , Nishika wa K , Ka wakishi S , Oda H , Hiai H , Stadtman ER .1994 .Michael addition - type4 - yhdroxy - 2 - nonenal adducts in modif ed low density lipoproteins: mark ers for atherosclerosis. Biochemistry 33 : 12487 – 12494 . UchidaK , Kanematsu M , Sakai K , Matsuda M , Hattori N , MizunoY , Suzuki D , MiyataT , No guchi N , Niki E , Osa wa T. 1998a . Protein - boundacrolein: potential markers for oxidative stress. Proc Natl Acad Sci USA 95 : 4882 – 4887 . UchidaK , Kanematsu M , MorimitsuY , Osa wa T , No guchi N , Niki E. 1998b. Acrolein is a product of lipid pero xidation reaction: for mation of acrolein and its conjugate with l ysine residues in oxidized low-density lipoprotein . J Biol Chem 273 : 16058 – 16066 . UchidaK , Szw eda LI , Chae HZ , Stadtman ER .1993 . Immunochemical detection of 4 - yhdroxy - 2 nonenal - modif ed proteins in o xidized hepatocytes. Proc Natl Acad Sci USA 90 : 8742 – 8746 . aeg W G , Dimsity G , Esterbauer H .1996 . Monoclonal antibodies for detection of 4 - yhdroxynonenal modif ed proteins . Free Radic Res 25 : 149 – 159 . ang W CJ , T ai HH .1990 .A facile synthesis of an aldehydic analog of platelet activating factor and its use in the production of specif c antibodies . Chem Phys Lipids 55 : 265 – 273 . atson W AD , Leitinger N , Na vab M , aFull KF , Horkk o S ,W itztum JL , P alinski W , Schw enke D , Salomon RG , ShaW , Subbanagounder G , F ogelman AM , Berliner JA . 1997 . Structural identif cation by mass spectrometr y of oxidized phospholipids in minimall y oxidized low density lipoprotein that induce monoc yte/endothelial interactions and e vidence for their presence in vivo .J Biol Chem 272 : 13597 – 13607 . XuG , Sa yre LM .1998 . Structural characterization of a 4 - yhdroxy - 2 - alk enal - deri ved f uorophore that contributes to lipopero xidation-dependent protein cross -linking in aging and de generative disease. Chem Res Toxicol 11 : 247 – 251 . ocum Y AK , OeT , Y ergey AL , Blair IA .2005 . Novel lipid hydroperoxide - deri ved hemoglobin histidine adducts as biomark ers of oxidative stress . J Mass Spectrom 40 : 754 – 764 . ZhuX , Sa yre LM .2007 . Long - ved li 4 - xoo - 2 - enal - deri ved apparent lysine Michael adducts are actually the isomeric 4 -ketoamides. Chem Res Toxicol 20 : 165 – 170 . ZhangWH , Liu J , Xu G ,Y uan Q , Sa yre LM .2003 . Model studies on protein side chain modif cation by 4 - xoo - 2 - nonenalChem . Res Toxicol 16 : 512 – 523 .

Chapter11 Mass Spectrometric Strategies for Identif cation and Characterization of Carbonylated Peptides and Proteins Marina Cariniand

Marica Orioli

INTR ODUCTION On the basis of the cur rent knowledge and proven experience, carbonylation is an ir reversible, non - enzymaticmodif cation of proteins leading to a change in their acti vity or function. Although the overall biology of o xidative protein modif cations remains comple x and incompletely def ned, protein carbon ylation and the chemistr y of the reactions that gi ve rise to car bonyl groups are quite w ell characterized (Stadman and Le vine 2006). In par ticular, protein carbon ylation induced b y reactive carbonyl species (RCS) generated by peroxidation of polyunsaturated fatty acids has gained an even greater importance in recent years, in view of the emerging deleterious role of the RCS/protein adducts in the etiolo gy and/ or progression of several human diseases. These include cardiovascular (atherosclerosis, long term complications of diabetes) and neurode generative (Alzheimer ’s disease, P arkinson’s disease, and cerebral ischemia) diseases. Oxidative decomposition of pol yunsaturated f atty acids (PUF As) initiates chain reactions that lead to the for mation of a variety of RCS (three to nine carbons in length), the most reactive and c ytotoxic being α ,β - unsaturatedaldehydes [4 - yhdroxy -trans - 2 - nonenal (HNE) and acrolein (A CR)], di -aldehydes [malondialdeh yde (MD A) and gl yoxal (GO)], and k etoaldehydes [4 - xoo -trans-2-nonenal (ONE)]. Most of the biolo gical ef fects of inter mediate RCS are due to their capacity to react with the nucleophilic sites of proteins, binding to the sulfhydryl group of cysteine (Cys), the ε-amino group of lysine (Lys), or the imidazole g roup of histidine (His) residues to for m Michael or Schiff base protein adducts, known as advanced lipoxidation end - products(ALEs). HNE is by far the best recognized and most studied cytotoxic product arising from peroxidation of ome ga-6 PUFAs, and proteins and peptides represent the most impor tant g roup of HNE-targeted biomolecules. It w as estimated that 1% to 8% of the HNE for med in cells will modify proteins (Siems and Gr une 2003). Protein modif cation most lik ely represents one of the main mechanisms b y which HNE can inf uence physiological as w ell as patholo gical Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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processes. For this reason, this aldeh yde has been implicated as causati ve agents in c ytotoxic processes initiated b y the e xposure of biolo gical systems to o xidizing agents (Aldini et al. 2007a ). The origin, reacti vity, and the biolo gical effects of HNE and other RCS, as w ell as the relevance of RCS -modif ed proteins in human diseases, ha ve been e xtensively reviewed (Carini et al. 2004, 2006 ; Dalle -Donne et al. 2006; Aldini et al. 2006a, 2007a ; Poli et al. 2008). Mass spectrometry (MS), owing to the advances in instrumental solutions, has played a key role in detection and characterization of peptides and proteins co valently modif ed by RCS in vitro in the last ten y ears, especiall y for def nition of the stoichiometr y of the protein/RCS adduct for mation, and for characterization of the sites of modif cation. In this conte xt, f ve years ago the authors pub lished a review that demonstrated the usefulness of MS in the study of non -enzymatic peptide and protein carbon ylation b y HNE (Carini et al. 2004). In recent years MS has gained much more impor tance, because coupled with computational and functional studies, it has pro vided key information to biologists in e xplaining the effects of modif cations by RCS on protein function and in understanding the relative consequences at the cell level. Moreover, the role of MS is no w particularly important because the majority of e xperiments are cur rently driving broad initiati ves in biomark er discovery. These efforts are be ginning to identify ne w carbon ylated anal ytes that are sensiti ve and selecti ve indicators of diseases. In light of the adv ancement in mass spectrometric resolution and sensiti vity, this chapter outlines a general approach for using MS to detect and characterize RCS -modif ed proteins, particularly focusing on two main objectives: (1) to extend the applicability of MS technologies to other lipid pero xidation-derived RCS b y considering not onl y α ,β - unsaturatedaldehydes such as HNE and ACR, but also k eto-aldehydes (ONE), α β , - unsaturatedketones such as 15 - deo xy -∆12,14 - prostaglandinJ2 (15d - PGJ2), and dialdeh ydes (MDA), and (2) to outline the progress in MS applications, w hich have shifted from in vitro to ex - vivostudies.

GENERALAND NEW APPROACHES FOR MS CHARACTERIZATION OF COVALENTLY MODIFIED PROTEINS Mass spectrometry strategies for the identif cation of protein oxidative modif cations may vary depending on the type of modif cation and the a vailable MS instr umentation. In an y e vent, most approaches involve the analysis of either intact proteins (top -down approach) or peptides generated from enzymatic digestion (bottom -up or peptide mapping approach) (Reid and McLuckey 2002). Both these approaches (Figure 11.1) and their relative advantages and limitations have been recently reviewed (Srebalus Barnes and Lim 2007), as have the capabilities of the different available MS instr umental solutions. The main advantages afforded by MS in the top -down approach include complete sequence coverage and identif cation of labile post -translational modif cations (PTMs), but anal ysis of large proteins require appropriate ionization techniques and mass anal yzers with suitab le resolving power and mass accuracy. Matrix -assisted laser desor ption/ionization (MALDI -MS) or ESI (Electrospray Ionization) -MS are the most used ionization techniques in the proteomic f eld for determination of intact proteins. A key step in enabling top-down approaches has been the ability to assign tandem mass spectrometer product ion identities, w hich can be done via high resolving power or through product ion charge state manipulation. Only Fourier transform ion cyclotron resonance (FTICR) (Bogdanov and Smith 2005) and the more recently introduced Orbitrap mass analyzers fulf l these requirements, providing the highest resolution, mass accuracy, and peak capacity, and providing isotopic resolution of fragment ions generated b y MS/ MS analysis, thus to obtain reliab le MS/MS sequencing of intact proteins (McLaf ferty et al. 2001, Bogdanov and Smith 2005, Meng et al. 2005, Zhai et al. 2005). Some other specialized

175

Mass Spectrometric Strategies for Identif cation Protein Digested peptides (Bottom-up approach)

Intact mass (Top-down approach) advantages

1) Cys residues denaturation, reduction, and alkylation

• Sequence coverage • Indentification of labile post-translational modifications (PTMs)

2) Enzymatic digestion

requirements

Peptide mixture

• Appropriate ionization techniques • Resolving power and mass accuracy

MALDI-MS

LC-ESI-MS

MALDI-MS/MS LC-ESI-MS/MS

• MALDI-MS • ESI-MS

High resolution • FTICR-MS • ORBITRAP Low resolution • QTrap • QQQ • hybrid qTOF

Figure 11.1. Characterization of covalently modif ed proteins: top -down and bottom -up mass spectrometric approaches.

ion trap instr uments reach the same perfor mance (Stephenson and McLuck ey 1998, VerBerkmoes et al. 2002, Amunugama et al. 2004, Pitteri and McLuck ey 2005), and some examples of top - do wn approaches using quadrupole/time - of -ight f (TOF) (Nemeth - Ca wley and Rouse 2002 ),and time - of -ight/time f - of -ight f (TOF/TOF) instr uments (Lin et al. 2003) have been also described. Despite its potential, FTICR instruments have found a limited application in the characterization of RCS -covalently modif ed proteins. In the w ork by Rauniyar et al. (2007), the authors were able to accurately measure HNE adduct formation on apomyoglobin, using a hybrid linear ion trap -7-Tesla FTICR mass spectrometer. The pattern of adduction and the deter mination of component molecular weights were possible without deconvolution and additional data manipulation, as is con versely required to anal yze the comple xity of the ESI mass spectra obtained using a QQQ mass anal yzer (Bolgar and Gask ell 1996). Another e xample is represented b y the work of Eliuk et al. (2007) who used FTICR-MS to investigate the structural consequences of reacting the c ytosolic brain isofor m of creatine kinase with HNE (see Characterization of Carbonylated Proteins In Vitro , below). Recent advances in top -down characterization of intact proteins in volve the use of electron capture dissociation (ECD) for complete sequence co verage and retention of labile post translationalmodif cations (PTMs). ECD, an additional mode of dissociation for large, multiply charged intact protein ions, in volves dissociation processes distinct from collision -induced dissocation (CID) and other related dissociati ve ‘heating’ techniques (Reid and McLuck ey 2002). ECD provides complementary (N–Cα amine-bond cleavage, which results in the formation of c- and z-type fragment ions), and perhaps more valuable sequence information, because unstable modif cations are typically retained on the amino acid residue during the fragmentation process (Bolgar and Gask ell 1996). The value of ECD dissociation o ver CID for the MS/MS deter mination of the modif cation site by HNE on o xidized insulin B chain has been recentl y repor ted (Rauniyar et al. 2007). The same authors (Rauniyar et al. 2009) introduced neutral loss -triggered ECD tandem mass spectrometry (NL -ECD-MS/MS) for the characterization of HNE -modif cation sites (neutral loss of 156 Da cor responding to an HNE molecule) in peptides using a h ybrid linear ion

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trap-FTICR mass spectrometer. ECD has been shown to retain the HNE g roup during MS/MS of the precursor ion, f acilitating the cor rect localization of the modif cation site. The use of ECD for MS/MS analysis is not, however, widely implemented for this pur pose, because only FTICR mass spectrometers w ould allow for the reliab le and eff cient application of this technique to induce dissociation of protonated peptides and proteins. Technical improvements in MS instrumentation provided new mass spectrometers with high sensitivity, resolution, and mass accurac y, such as the ne w mass analyzer Orbitrap, developed in 2000 by Makarov. Interf acing this mass anal yzer to ESI has been sho wn to pro vide good sensitivity, resolution (150,000 fwhm), and mass accuracy (1 to 2 ppm) (Hardman and Makarov 2003). MS/MS capability w as achieved by interf acing the Orbitrap with a linear quadr upole ion trap (L TQ): the resulting h ybrid Orbitrap has been successfull y demonstrated for proteomics applications (Yates et al. 2006), but v ery few examples have been repor ted to study protein carbon ylation. Dose -response inacti vation b y HNE of hB AT (human bile acid CoA:amino acid N -acyltransferase) and CKBB (c ytosolic brain isofor m of creatine kinase), associated with site - specifc modif cations, has been demonstrated b y using this approach, leading to identif cation of 14 HNE adducts on hB AT and 17 on CKBB (Bar nes et al. 2008). Other less e xpensive instr umental solutions than FTICR are generall y a vailable in most laboratories, including orthogonal acceleration time - of -ight f (oaT OF), quadr upole ion trap, triple quadr upole (QQQ), and quadr upole/oaTOF (h ybrid qT OF), that are suitab le for the analysis of small or moderatel y sized intact proteins. This is because their resolving po wer is not suff cient for dif ferentiation of modif cations that result in small mass changes on e ven moderate sized proteins. Therefore, the bottom -up or peptide mapping approach is the most commonly used to study PTMs. As shown in F igure 11.1, this generall y involves denaturation, reduction, and alk ylation of Cys residues prior to enzymatic digestion of the protein. The resulting peptide mixture is then analyzed using MALDI -MS or ESI -MS coupled to liquid chromato graphy. Tandem MS sequence analysis of digest peptides, via CID, post-source decay or other alternative approaches, is generally used to sequence digest peptides for site -specif c identif cation of RCS -modif ed amino acid residues in the bottom -up approach. This is mainly carried out by MALDI MS/MS or LC -ESI-MS/MS with data dependent acquisition or of f-line nanospray MS/MS. CID fragmentation, involving generation of characteristic b and y ions follo wing the preferential cleavage of CO -NH bonds, can be eff ciently perfor med using QQQ, ion trap, and h ybrid qTOF instruments on digested peptides no lar ger than 5kDa, thus guaranteeing complete sequence coverage (Kelleher 2004). Several examples of their application are repor ted below ( in vitro experiments).

CHARACTERIZATION OF CARBONYLATED PROTEINS IN VITRO: REACTION MECHANISMS, FUNCTIONAL STUDIES, AND BIOMARKERS IDENTIFICATION MS continues to be e xtensively applied in in vitro studies in volving exposure of an isolated protein to RCS, but with additional, no vel targets, i.e. to disco ver new reliable and earl y biomarkers of oxidative damage, study the reacti vity of a tar get protein to RCS other than HNE, or elucidate disease mechanism(s) at the protein le vel. Identif cation of proteins susceptible to carbonylation, assessment of the precise nature of the modif cation(s), and identif cation of modif cation sites might pro vide insight on the f actors affecting protein function. Generall y, these studies, which involve exposure of a purif ed protein to RCS, also comprise the e valuation of the impact of such modif cation(s) on protein function (i.e., the paper b y Siegel et al. 2007), demonstrating the ability of HNE to promote Alzheimer protof bril formation). This point is cr ucial to estab lish w hether carbon ylation of specif c proteins is causati ve, correlative, or consequential of o xidative stress -associated conditions, because carbon ylation

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does not necessaril y result in protein function alteration. When the str uctural modif cations affect proteasome activity, such as altering the subunit composition or o xidative modif cation, the knowledge of such modif cations is critical to understanding the proteasome function under both normal and pathological conditions. The papers considered in the following section cover these important aspects. HNE The paper b y Isom et al. (2004) was developed with the aim to gain deeper insight into the effects of HNE adduction under in vitro conditions on c ytochrome c function. MALDI -TOF MS enabled the establishment of the addition of four HNE residues; peptide mass fngerprinting (MALDI-TOF-MS and LC -ESI-MS/MS) pro vided sequence infor mation that w as used to determine the specif c adduction sites: His33, Lys87, and R38. Mapping these results onto the X-ray crystal structure of nati ve cytochrome c indicated that HNE adduction could ha ve signif cant effects on tertiary structure, electron transport, and ultimately, mitochondrial dysfunction. The same MALDI-TOF-MS approach was applied to conf rm covalent adduction of HNE to the thioredoxin system, one of the core re gulation enzymes of cells’ function, playing a key role in redox regulation and being in volved in many signaling pathways (Fang and Holmg ren 2006 ). To understand the mechanism of action of HNE as a promoter of Alzheimer protof bril formation, its in vitro interaction with the am yloid β peptide (A β) was fully investigated by MALDI-TOF-MS analysis (Siegel et al. 2007). HNE adduction hastens the for mation of Aβ 1 – 40protof brils, producing more agg regates, as demonstrated b y a v ariety of techniques (thiof avin T f uorescence, size exclusion chromatography, static light scattering, atomic force microscopy). It also inhibits the con version of these protof brils into less to xic long, straight f brils, sustaining toxicity, exacerbating neuronal death, and ultimatel y leading to AD. LC-ESI-MS/MS was employed to determine the specif c location(s) of HNE modif cation(s) of Erk-2 (extracellular signal-regulated kinase), with the f nal aim to investigate the mechanism of HNE -mediated inhibition of kinase signalling (Sampe y et al. 2007). By incubating mouse recombinant Erk -2 with increasing HNE concentrations (up to 100 µM), the major adduct species was identif ed at the His178 within the kinase phosphor ylation lip, a crucial residue in the shift from the inacti ve to acti ve confor mation of both Erk -1 and Erk -2. This infor mation provides new insight into the molecular mechanisms b y which HNE modulates Erk -1/2 signal transduction and potentially gene expression in hepatocytes. FTICR-MS (direct infusion experiments) and MS/MS technologies (both NanoMate ESI and LC-ESI r uns) ha ve been applied to in vestigate the str uctural consequences of reacting the cytosolic brain isofor m of creatine kinase with HNE at pathoph ysiologically relevant concentrations (10 – 300µM) (Eliuk et al. 2007). Functional studies indicated that the dose -dependent reduction of enzyme activity was correlated with HNE adduct formation on specif c amino acid residues, including the acti ve site residues His66, His191, Cys283, and His296. Semi quantitation of the peptide containing HNE -modif ed Cys283 w as perfor med relati ve to the peptide containing unmodif ed Cys283 at dif ferent HNE concentrations. The peak intensities of both the unmodif ed and HNE -modif ed Cys283 quadr uply char ged tr yptic peptide 266 SKDYEFMWNPHLGYILTCPSNLGTGLR292 were nor malized to the peak intensity of the peptide 33 VLTPELYAELR43, which does not contain HNE -modif able residues. A similar semi-quantitative approach was used by the authors to detect the modif cation sites on human ser um albumin (HSA) (Aldini et al. 2006b). By using a LC -ESI-MS/MS approach (quenching-peptide method using a triple quadr upole system), the authors found that HSA treated with HNE gi ves rise to the for mation of 11 dif ferent adducts, in volving nine nucleophilic sites. The most reactive are Cys34 and His146 (gi ving HNE -Michael adducts) adducts, and Lys199, which mainly reacts by forming the Schiff base adduct. These HSA modif cations are suitable tags of HNE-adducted albumin and could be useful biomarkers of circulating HNE

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and oxidative and carbonylation damage in humans. The high reactivity of HSA to ward HNE also has been demonstrated b y Szapacs et al. (2006) using an L TQ linear ion trap instr ument for LC - ESI - MS/MS analysis. Two - dimensionalSDS - A P GE followed by immunoblot analysis (using polyclonal antibodies against HNE -modif ed proteins) and MALDI -TOF mass f ngerprint analysis of the isolated and digested protein allowed the authors to conclude that HSA is the major nucleophilic plasma target of HNE (Aldini et al. 2008a). The same conclusion was drawn by Tallman et al. (2007), working with a LTQ linear ion trap instr ument equipped with a microelectrospra y source and using a headg roup biotinylated phosphatidylcholine: HSA is the major plasma protein tar get of electrophilic aldeh ydes generated b y the o xidative decomposition of the biotin ylated lipid. Combined molecular modelling and kinetics studies (carried out by LC-ESI-MS/MS) indicated that HSA, through nucleophilic residues, and in par ticular Cys34, can act as an endo genous detoxifying agent of circulating RCS (Aldini et al. 2008a). The rate constant for reaction of Cys34 with HNE was found to be almost one order of magnitude higher than that of glutathione (GSH), and the dif ferences in reacti vity are w ell-explained b y molecular modeling results, which conf rmed the signif cant acidity of Cys34 thiol g roup, and unveiled a remarkable similarity betw een the residues sur rounding Cys34 and the catal ytic site of cer tain glutathione transferases. C AR AND OTHER RCS Several studies have been also perfor med on acrolein (ACR), a highly toxic lipid peroxidation aldehyde and a strong cross -linking agent of cellular proteins. Among α ,β - unsaturatedaldehydes, ACR is b y f ar the strongest electrophile and therefore sho ws the highest reacti vity toward nucleophiles (Carini et al. 2006, Aldini et al. 2007a). Sources, metabolism, and biomolecular interactions of ACR relevant to human health and disease ha ve been recently reviewed (Stevens and Maier 2008). ACR is kno wn to for m four dif ferent types of adducts, namel y aldimine - , propanal - , methylpyridinium (MP) - , and formyl - deh ydropiperidino (FDP) - type adducts, resulting in increments of 38, 56, 76 and 94 Da, respecti vely, in the mass value of the peptide (Figure 11.2 ). To understand the mechanisms of o xidative stress -induced protein agg regation, Ishii et al. (2007) characterized the ACR modif cation of chain B from bo vine insulin by mass spectrometry (MALDI -TOF-MS and LC -ESI-MS/MS ion trap system), using as a model peptide the insulin B chain from sequence FVNQHLC * GSHL VEALYLVC * GERGFFYTPKA(C * :Cys SO3H). This contains four main possible modif cation sites of ACR: the N-terminal amino acid residue (Phe1), two His residues (His5 and His10), and one L ys residue (Lys29). MS analysis

Figure 11.2. Acrolein covalent adducts to Cys, His, and Lys residues. FDP -lysine, N -(epsilon)3(formyl-3,4-dehydropiperidino)lysine, MP -lysine, N(epsilon) -(3-methylpyridium) lysine.

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of ACR-treated peptide indicates for mation of all types of adducts, the aldimine being the most prominent. To identify the cross -linking sites, the ACR-treated peptide was digested with a protease and the resulting peptides w ere anal ysed b y LC -ESI-MS/MS. Inter - and intra molecular cross -linking adducts were identif ed between amino g roups (N -terminus [Phe1] or Lys29) and the His residues (His5 or His10). The data allo wed proposing a mechanism of protein cross -linking b y ACR, w hich in volves tw o reactions, namel y Michael addition and Schiff base for mation. MALDI - OF T - MSand nanospray LC - ESI - MS/MS demonstrated that ACR inhibits cytokine gene expression in human T lymphocytes by alkylating two amino acids (Cys61 and Arg307) in the DNA binding domain of NF -kappaB1 (p50 subunit), resulting in inhibition of p50 DNA binding. Crotonaldehyde reacted with Cys61, but not Arg307, whereas the saturated aldehydes in cigarette smok e did not react with p50. These experiments demonstrate that aldeh ydes in cigarette smoke can re gulate gene e xpression by direct modif cation of a transcription f actor (Lambert et al. 2007). MALDI-TOF/TOF of digested peptides demonstrated that ACR (and HNE) selecti vely modif ed the nonactive site Cys73 of thioredoxin-1 (Trx1), a protein recently found to regulate antioxidant function in endothelial cells (Go et al. 2007). The modif cation resulted in loss of catalytic activity, inhibition of thioredo xin reductase -1 (TrxR1), and stimulation of monoc yte adhesion to endothelial cells, an earl y event of atherosclerosis. Moreover, MS/MS anal ysis (infusion e xperiments) w as used to assess the distribution of ACR adducts, and also explore the adduct-trapping reactivity of cytoprotective hydrazino drugs (Kaminskas et al. 2007). To clarify the chemistr y underlying the ability of h ydralazine to trap ACR-protein adducts, the reactivity of hydralazine with ACR-modif ed PPE (preproenkephalin fragment 128 to 140) w as assessed, using ESI -MS to monitor the loss of adducted peptide in the presence of h ydralazine and detect reaction products for med by the dr ug. The papers by Shao et al. (2005a, 2005b) are a clear example of how mass spectrometry can contribute to elucidating the pathogenetic role of protein carbonylation. MALDI-TOF and LCESI-MS/MS demonstrated that Lys226, located near the center of helix 10 in apoA -I, was the major site modif ed by ACR, with for mation of FDP -lysine. This region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid, and the conversion of Lys226 by ACR is associated quantitati vely with decreased cholesterol eff ux from cells via the ATPbinding cassette transpor ter A1 pathw ay. Immunohistochemical studies with a monoclonal antibody re vealed co -localization of apoA -I with ACR adducts in human atherosclerotic lesions. Hence, ACR might interfere with nor mal re verse cholesterol transpor t b y HDL b y modifying specif c sites in apoA -I, and might contribute to atherogenesis by impairing cholesterol removal from the ar tery wall. The authors used a combined approach (LC -MS/MS, functional and confor mational analyses) to study the effect of ACR (and HNE) on actin polymerization and to clarify the molecular constraints induced by aldehyde adduction, ref ecting on actin functioning (Aldini et al. 2005, Dalle-Donne et al. 2007). Actin, a relati vely abundant protein in most mammalian cells, has been chosen because is one of the main carbon ylated proteins in vivo. Carbonylation has been shown to cause the disr uption of the actin c ytoskeleton and the loss of the monola yer barrier function. Cys374 (o wing to its signif cant accessib le surf ace and substantial thiol acidity) represents the primar y target site of both α β , - unsaturatedaldehydes, but actin ’s capability to polymerize is not compromised by Cys374 adduction (no alteration in protein folding). Unlike HNE, also ACR reacts with actin His87 and His173 (and minimally with His40), and this kind of modif cation inhibits pol ymerization in a dose -dependent manner . Molecular modeling analyses indicated that structural distortions of the ATP-binding site, induced by ACR-Michael adducts on His residues, could e xplain the changes in the pol ymerization process. Asignif cant unfolding of actin str ucture was also induced by 15-deoxy-∆12,14 - prostaglandin J2 (15d - PGJ2), a prostanglandin that, unlik e other classes of eicosanoids, is characterized b y the presence of an electrophilic α ,β-unsaturated carbon yl g roup in the c yclopentenone ring

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(Aldini et al. 2007b). In par ticular, 15d -PGJ2, by selectively binding to Cys 374 (mass spectrometric evidences), distorts the actin subdomains 2 and 4, which def ne the nucleotide binding sites impeding the nucleotide exchange (computational studies). This can explain the disruption of the actin cytoskeleton, F -actin depolymerization and impairment of G -actin polymerization (as demonstrated b y Western blot and functional anal yses). All of these obser vations provide an insight into the role of 15d -PGJ2 as a c ytotoxic compound in neuronal cell dysfunction (neuroblastoma cells). The results obtained b y a combined LC -ESI-MS/MS and computational approach demonstrated the high reacti vity of both albumin and actin to ward α ,β aldeh ydes, and estab lished the stoichiometr y of reaction with HNE and ACR, as w ell as the amino acid residues more susceptible to carbonyl attack (Aldini et al. 2005, 2006b ; Dalle -Donne et al. 2007). Based on these premises, a new mass spectrometric approach, based on LC-MS/MS analysis of tag HNE/ ACR - modifed peptides of carbonylated albumin and actin, has been proposed , and the advantages over the con ventional methods for RCS and RCS -adducted protein anal yses discussed (Aldini et al. 2007c). The steps reported in Figure 11.3 allow detection and quantitation of the

Figure 11.3. Procedure for the quali/quantitative analysis of ACR - or HNE -adducted peptides as tag of carbonylated albumin (A) and actin (B). *Indicates the site of adduction. NaBH4 reduction before enzymatic digestion guarantees peptide adducts stabilization. The adducted Cys374– Phe375 dipeptide (Michael adduct to Cys 374) is the tag peptide for actin modif ed by α,β-unsaturated aldehydes, which is detected by LC -MS in selected ion monitoring (SIM) mode, using the [M +H]+ at m/z 327 as f lter ion (calculated monoisotopic ion: m/z 327.13) for ACR or -adducted peptides LQQC *PF (obtained by the [M +H]+ at m/z 427 for HNE. The HNE trypsin+chymotrypsin digestion) and LK *CASLQK (trypsin digestion) are the two tag peptides of HNE-treated albumin. They are detected by tandem mass spectrometry using the suitable diagnostic product ions and the protonated molecular ions as parent ions. Reproduced with permission from Aldini et al. (2007c). Copyright 2007 © Maney Publishing, http://maney.co.uk/index. php/journals/rer/ and http://www.ingentaconnect.com/content/maney/rer.

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tag peptides of carbon ylated HSA in human ser um (F igure 11.3A) and actin in cell/tissues (Figure 11.3B). Quantitative analysis can be carried out by using isotopically labelled carbonylated peptides prepared by reacting deuterated HNE with the synthesized peptides. The LC-ESIMS/MS method for the measurement of carbonylated tag peptide can be applied to the digested proteins isolated from the biolo gical samples or directl y to the digested sample (one -shot procedure). HSA also has been found to be modif ed by MDA, and LC-ESI-MS/MS of digested peptides was used to identify the modif cation sites (Ishii et al. 2008). Six peptides, which contained an N-propenal adduct at L ys136, Lys174, Lys240, Lys281, Lys525, and Lys541, were characterized. Lys525, which is located at the center of positi vely charged grooves (as demonstrated by analysis of electrostatic surface potential of a 3-D model structure of HSA), is the most reactive residue for MDA modif cation. This indicates that the modif cation of proteins by lipid-derived aldehydes may be signif cantly inf uenced by the electrostatic potential of the protein surf ace. More recently, ONE has been demonstrated to be another direct product of lipid o xidation, arising independently and not from oxidation of HNE. The ONE reactivity toward nucleophilic sites of proteins has been recentl y re viewed (Sayre et al. 2006). The adducts betw een ONE and hemo globin (Hb) chains w ere characterized b y MALDI -TOF-MS anal ysis of the intact proteins and b y a combination of LC -ESI-MS/MS and MALDI -TOF-MS/MS analysis of the tryptic peptides (Yocum et al. 2005). Covalent modif cations were found on His of both Hb chains, His20 from α-Hb, and His63 from β-Hb, the tw o most solv ent accessib le residues (molecular modelling data). MS/MS data allo wed elucidating the reaction mechanism, based on that described for the reaction of HNE with proteins, in volving: (a) nucleophilic Michael addition, (b) hydration of the resulting aldeh yde, (c) cyclization, and (d) tw o sequential dehydration reactions to gi ve stab le furan deri vatives (F igure 11.4). These Hb modif cations can serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage, and ma y ref ect an indirect measurement of the potential for damage in vivo . MALDI-TOF-MS and postsource deca y (PSD) -TOF-MS analyses have been used to demonstrate that ONE mediates not onl y adduct for mation, but also o xidative decarboxylation of II [Ang N-terminal aspartic acid on angiotensin (Ang) II and des-Ile5 ,His6 ,Pro7, and Phe8 - Ang II (1 –4)], a reaction w hich is not mediated b y other lipid pero xidation-derived aldehydes such as HNE and trans - 4,5 - epo xy - 2(E) - decenal. The initial reaction of ONE with an N - terminal

Figure 11.4. 4-Oxo-trans-2-nonenal (ONE) adduction to hemoglobin: reaction mechanism.

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α-amino group of Ang II or Ang II (1 to 4) results in the for mation of a Schiff base intermediate. The resulting inter mediate under goes tautomerization and decarbo xylation follo wed b y hydrolysis to pro vide an α-keto amide (p yruvamide) moiety at the N ter minus of Ang II and Ang II (1 to 4) (Lee et al. 2008). An analysis of the decomposition of 13 -hydroperoxy-octadecadienoic acid (13 -HPODE, the precursor of α ,β-unsaturated aldehydes) in the presence of a single protein has been sho wn to provide some new insights concer ning potential biomark ers for lipid h ydroperoxide-mediated macromolecule damage. Cytochrome c was used as protein model to determine the most abundant adduct formed following decomposition of 13 -HPODE (Williams et al. 2007). The use of ESI-TOF-MS (anal ysis of the intact proteins) and a combination of ESI -iontrap/MSn with quadrupole-TOF-MS/MS analysis of the tr yptic and ch ymotryptic peptides allo wed recognition, in addition to HNE and ONE, of another major and highl y electrophilic lipid h ydroperoxide - deri ved species, 9,12 - dio xo - 10(E) - dodecenoic acid (DODE).

IDENTIFICA TION AND CHARACTERIZATION OF CARBONYLATED PROTEINS IN BIOLOGICAL MATRICES: FROM IN VITRO TO EX VIVO STUDIES In the last fe w years, many efforts have been made to identify in a biolo gical milieu the more susceptible protein(s) to carbon ylation, with the f nal aim to understand the impact of such modif cation(s) on cell/tissue function. The general approach for characterization of covalently modif ed proteins in a biolo gical matrix, summarized in F igure 11.5, in volves detection of carbonylated proteins b y Western blot immunoassay, using aspecif c antibodies such as anti dinitrophenylhydrazone (DNP) follo wing reaction of carbon yls with 2,4 -dinitro-phenylhydrazine (DPNH) (Dalle-Donne et al. 2005), or more specif c mono/polyclonal antibodies, followed by in -gel digestion and mass spectrometr y. Antibodies specif c for HNE or ACR-Michael adducts ha ve been the most widel y used for detecting protein covalently modif ed by α β , -unsaturated aldehydes in cells and tissues (Uchida et al. 1998, Neely et al. 1999, Ji et al. 2001, Levonen et al. 2004, Tamamizu-Kato et al. 2007). Anti-HNE antibodies sho w cross -reactivity with other lipid electrophiles, such as 4 -hydroxy2-decenal and 4 -hydroxy-2-octenal (Vila et al. 2008). In this conte xt, mass spectrometr y is of Biological matrices 2D gel map In vitro (cells/tissues + to RCS)

Western blot

Ex vitro • Tissues • Fluids

RCS antibodies*

Protein identification Database search De novo sequencing

Relative Abundance

Spot excision 100 80 60 40 20 0

In-gel trypsin digestion Mass spectrometry

200 300 400 500 600 700 m/z

Figure 11.5. General approach for characterization of RCS matrices.

-modif ed proteins in biological

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paramount impor tance, because it can assess the critical sites of proteins that are modif ed, while antibodies do not distinguish betw een different modif ed sites. Although several oxidized proteins ha ve been successfull y identif ed, identif cation/conf rmation of the sites of carbonylation often has not been achieved. This task is made diff cult by the numerous carbon ylated amino acid residues that could be for med and b y the low stoichiometry of the reactions in volved. Therefore, to o vercome the challenges of mapping sites of HNE (and other RCS) modif cation within complex mixtures, several specif c strategies involving HNE deri vatization, aff nity enrichment methodolo gies (F enaille et al. 2002, Ho and Gaskell 2003, Carini et al. 2004) followed by MS/MS characterization, derivatization (Fenaille et al. 2004a), precursor ion scanning (Sangv anich et al. 2003), or 18O labeling (Sun and Anderson 2005) have been developed. DERIV ATIZATION/ENRICHMENT A series of papers published by the group of Yang before 2004 suggested that DNPH derivatization of protein carbon yls is successful in localizing the site of carbon yl appearance in proteolytic peptides using mass spectrometr y (Yang et al. 1997a, 1997b, 1999a, 1999b, 2001 ). Fenaille’s group reported in 2002 a method for immunoaff nity capture of DNPH-derivatized HNE and MD A-adducted tr yptic peptides, and the y were successful in identifying a DNPH HNE-His Michael adduct on m yelin basic protein (F enaille et al. 2002). Yuan et al. (2007), using a model carbon yl-containing peptide, re vealed that once the DNPH h ydrazone is separated from e xcess reagent the deri vatives dissociate in solution to an e xtent that depends on time and pH. Thus, it appears that simple derivatization with DNPH is unsuitable for determining peptide -based carbonyls using a protocol in w hich the oxidized protein is derivatized with DNPH prior to proteol ysis. The solution proteol ysis steps and dilute acid conditions typicall y used for re versed-phase LC - ESI - MS analysis are apparently suff cient to cause signif cant reversibility, as w as found for h ydrazide-carbonyl deri vatives. In the absence of de veloping a protocol that stabilizes DNPH deri vatives b y reduction (DNPH -derived h ydrazones are reducib le with NaBH 3 CN), one alternative to detect peptide carbon yls is to anal yze the unfractionated proteol ytic digests directly b y MALDI -TOF-MS, w here the e xcess DNPH reagent ser ves as the matrix. This strategy was developed to identify HNE -modif ed peptides (Michael adducts) within unfractionated tr yptic digests (F enaille et al. 2004a). This method con verts HNE -modif ed peptides into the cor responding h ydrazones, and the resulting products can be detected with a high sensitivity compared to underivatized peptides because of the high desor ption/ionization yield of the hydrazone derivatives. This approach has been also applied to monitor milk protein o xidative modif cations during industrial treatments (F enaille et al. 2005), in vie w of both product quality and aller genicity issues (carbonylation tends to increase the natural aller genicity of milk proteins). Protein residues modif ed by RCS contribute to the for mation of new immunologically reactive structures as a consequence of their reacti vity (cross -linked protein species, resistance to proteol ytic digestion). Using MALDI/DNPH and nanoESI-MS/MS, it was found that β - lacto globulin (one of the major milk aller gens in nati ve for m) is the preferential carbon ylation tar get during industrial treatment of milk and that His146 is the HNE -modif ed residue (Figure 11.6 ). Similar to the proteomic study of other PTMs to proteins, RCS-modif ed components generally mak e up a relati vely small propor tion of the total proteins within a comple x biological sample. Hence, methods for enriching RCS -adducted proteins over nonadducted proteins prior to LC -MS/MS anal ysis ha ve been de veloped to enhance the o verall identif cation of lo wer abundance protein adducts. Because traditional 2 -D gel electrophoresis/mass spectrometric technologies, although widel y used in the f eld of proteomics, can reliab ly detect onl y high abundance proteins (Gygi et al. 2000), several alternative shotgun proteomic approaches ha ve been developed to solve detection and enrichment prob lems.

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Figure 11.6. NanoESI-MS/MS mass spectrum of m/z 653.3 ([M + 2H] 2+) corresponding to the peptide fragment (139 to 148) from β-lactoglobulin containing an HNE-modif ed His residue (H*). Reproduced with permission from Fenaille et al. (2005). Copyright 2005 © WILEY -VCH.

Biotin hydrazide, highly specif c for free aldehyde groups on proteins (Yan et al. 1998), has been used as a repor ter tag for proteins adducted b y lipid o xidation products (Sore ghan et al. 2003, Yoo and Re gnier 2004, Mirzaei and Re gnier 2005) and HNE (Grimsr ud et al. 2007) in vitro and in vivo . The main adv antage of this method o ver immunochemical approaches is that it recognizes RCS -protein adducts ir respective of the type of adduct for med. In addition, the biotin tag provides enrichment of the labelled sample via biotin–avidin binding prior to MS analysis. For example, an on -line microcapillar y reverse-phase LC -MS/MS analysis coupled with a hydrazide biotin -streptavidin methodology was developed to identify protein carbon ylation in rat brain of aged mice (Sore ghan et al. 2003). Aff nity enrichment using monomeric a vidin aff nity chromatography followed by LC -MS/MS analysis of tr yptic peptides w as developed to study protein carbon ylation in li ver homo genates from rats dosed with 2 -nitropropane (Mirzaei and Re gnier 2005), study the ef fects of o xidative stress on y east proteome (Mirzaei and Regnier 2007) (in this case avidin selected oxidized protein mixtures were further fractionated by LC before proteol ysis to reduce the comple xity of peptide mixtures), and in vestigate protein modif cation by HNE in adipose tissue from obese insulin -resistant mice (Grimsr ud et al. 2007). Using transfer rin as a model protein, the g roup of Mirzaei (Mirzaei and Re gnier 2006a) developed an alter native enrichment procedure w hich involves (a) derivatization of aldehydes and ketones in oxidized proteins with the Girard P reagent, (GPR, 1 - [2-hydrazino-2-oxoethyl] pyridinium chloride), which introduces an additional positive charge at the site of carbonylation to enhance ionization eff ciency in MS anal ysis, (b) proteol ysis enrichment of the deri vatized peptide using strong cation exchange chromatography at near-neutral pH, and (c) identif cation of modif cation sites b y MALDI -TOF/TOF anal ysis. This approach, although adv antageous over other labelling strategies, does not allow overcoming problems associated with identif cation of modif ed peptides in comple x mixtures of unmodif ed peptides. Hence, a ne w strategy for labelling protein carbon yls w as de veloped b y the same authors (Mirzaei and Re gnier 2006b). This strategy was based on the use of light and hea vy isotope coded GPR that allo ws not only conf dent identif cation of carbon ylated proteins via multi -step f ltering process, but also their relati ve quantif cation. Although promising, to date this approach has been applied only to protein models exposed to oxidizing agents, in which the bulk of protein carbonyls are generated by direct oxidation of Lys, Arg, Pro, and Thr residues into the cor responding ketone or aldeh yde deri vatives. Its usefulness in detection and quantitation of co valently modif ed proteins by RCS remains to be demonstrated.

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Figure 11.7. Labeling of HNE -adducted proteins with biotin using click chemistry (azido and alkinyl derivatives) for subsequent enrichment and identif cation by proteomic analysis.

Mayer et al. made se veral efforts to f nd alter native strategies to h ydrazine-based reagents for aff nity enrichment and MS detection of proteins modif ed by α ,β - unsaturatedaldehydes through Michael addition reaction. In 2007 Cha vez et al. repor ted the use of N′ - amino oxymethylcarbonyl-hydrazino D -biotin, a biotin ylated h ydroxylamine deri vative that for ms with the aldeh yde/keto group an o xime derivative. The increased stability of the o xime compared to the hydrazone derivative allows the reduction step, necessary to convert the hydrazone to the chemicall y more stab le hydrazine, to be omitted (Y oo and Re gnier 2004, Mirzaei and Regnier 2005, Sore ghan et al. 2003). The usefulness of this approach, in combination with Western blot analysis followed by in -gel tr ypsinization and MALDI -MS/MS, has been demonstrated in the identif cation of o xidatively modif ed proteins from interf brillar hear t mitochondria from young and old rats (Chung et al. 2008). The use of azido and alk ynyl deri vatives of HNE, in conjunction with click chemistr y (copper-catalyzed Huisgen 1,3 dipolar c ycloaddition reaction), has been recentl y proposed as a ne w strate gy for conjugating biotin to HNE -adducted proteins in the enrichment process before aff nity purif cation (Vila et al. 2008). This has been sho wn to be a v aluable approach to probe the reacti vity of HNE with proteins in vivo . The click reaction employs a Cu(I) catalyzed cyclization of an azide with an alk yne to for m a stab le triazole. The overall scheme of reaction is shown in Figure 11.7. A label -free strategy based on a solid -phase hydrazide (SPH) reagent for the enrichment of HNE - modifed peptides within comple x mixtures has been also de veloped (Roe et al. 2007). HNE - modifed peptides are captured on h ydrazide-coated glass beads via the for mation of a covalent but re versible hydrazone bond enab ling their specif c enrichment and release under acidic conditions. This front -end enrichment strate gy, combined with neutral loss -dependent acquisition of (MS 3) spectra and pulsed Q dissociation (PQD) operation on a linear ion trap mass spectrometer, provided a comprehensive catalogue of amino acid sites susceptible to HNE modif cation in treated y east protein mixtures. The overall procedure is summarized in F igure 11.8. The main adv antage of this approach o ver con ventional labelling techniques, w hich does not involve the addition of e xtra molecular tags, is the simplif cation of MS/MS spectra

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Figure 11.8. Enrichment of HNE -modif ed peptides within complex mixtures by the solid -phase hydrazide (SPH) approach. Capture of HNE -modif ed peptides is carried out under mildly acidic conditions to capitalize on the reactivity of hydrazide with aldehydes at this pH, while limiting the reactivity of other nucleophiles, and also to maximize the formation of hydrazone bonds relative to their hydrolysis. The reacted beads are then washed with reaction buffer, high salt, and organic solvents in succession to limit the occurrence of nonspecifcally bound peptides in the fnal peptide analysis. These wash steps are conducted under mild pH conditions in which the hydrazone formed between the SPH and HNE -modif ed peptide remains stable. The bound peptides are f nally released by heating the beads in strong acid (10% formic acid): the hydrazone bonds are reversed, and the released HNE -modif ed peptides are readily analyzed by mass spectrometer. Adapted with permission from Roe et al. (2007). Copyright 2007 © American Chemical Society.

interpretation. In addition, unlike gel-based methodologies, it allows scaling up the enrichment step starting from a high amount of proteins. Codreanu et al. (2008) identif ed protein tar gets of HNE in RK O cells treated with 50 or 100 µM HNE. HNE Michael adducts w ere biotinylated by reaction with biotinamidohe xanoic acid h ydrazide and captured with strepta vidin, and the captured proteins w ere resolv ed b y 1D-SDS PAGE, digested with trypsin, and identif ed by LC-ESI-MS/MS. Of the 1,500 proteins identif ed, 417 displa yed a statisticall y signif cant increase in adduction with increasing HNE exposure concentration. An additional 18 biotin h ydrazide-modif ed, HNE adducted peptides were identif ed by specif c capture using anti -biotin antibody and anal ysis by high resolution LC - ESI - MS/MS. Protein interaction network analysis indicated several subsystems impacted by HNE, including the 26S proteasomal and CCT chaperonin systems in volved in protein folding and degradation, as well as the COP9 signalosome, translation initiation comple x, and a large network of ribonucleoproteins. PRECURSORION SCANNING Development of specif c mass spectrometric detection methods is another impor tant strategy to study protein modif cations. For example, the precursor ion-scanning mode technique, using

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a triple-stage quadrupole instrument, is a powerful tool in this area, because it permits selective detection of tar get modif ed peptides bearing a common chemical moiety in the presence of multiple unmodif ed structures and str ucturally unrelated compounds. Compared to a con ventional ESI-MS approach, this methodology signif cantly increases the specif city and sensitivity of the procedure by f ltering out chemical noise and other unnecessary ions from the spectrum. Scanning of precursors of m/z 268, a product ion characteristic of the presence of HNE modifed His residues, has been used b y Sangvanich et al. (2003) to detect this kind of modif cation in tr yptic fragments of lo w density lipoproteins e xposed to o xidative stress. F enaille et al. (2004b) used this approach for the specif c and sensiti ve identif cation of peptides containing hexanal - modifed L ys residues within an unfractionated tr yptic digest of he xanalmodifed apomyoglobin. We recently developed a precursor ion scanning technique using a triple -stage quadr upole to detect free and protein -bound histidine (His) residues modif ed by RCS in urine from obese Zucker rats, a nondiabetic animal model characterized by obesity and hyperlipidemia, in which RCS for mation plays a k ey role in the de velopment of renal and cardiac dysfunction (Orioli et al. 2007). The immonium ion of His at m/z 110 w as used as a specif c product ion of His containing peptides to generate precursor ion spectra, follo wed by MS 2 acquisitions of each precursor ion of interest for str uctural characterization. Using this approach, three no vel adducts, w hich are e xcreted in free for m only, have been identif ed. Two originate from the conjugation of HNE to His, follo wed by reduction/oxidation of the aldehyde: His - 1,4 - dih ydroxynonane (His - DHN),His - 4 -ydroxynonanoic h acid (His HNA), and car nosine-HNE. The last w as reco gnized in pre vious in vitr o studies as a ne w potential biomarker of carbonyl stress (Orioli et al. 2005). No free His -HNE was found in the urine, which was detected only in protein hydrolysates. The same LC-MS/MS method, working in multiple reaction monitoring (MRM) mode, has been de veloped, validated, and applied to quantitatively prof le all of the identif ed peptide adducts in Zuck er urine. In view of the importance of Cys34 in the RCS vascular detoxif cation (see Characterization of Carbon ylated Proteins In Vitro, above), an LC -ESI-MS/MS approach based on precursor ion scanning w as developed to characterize the co valent modif cations of Cys34 caused b y a variety of structurally different RCS such as HNE, ACR, crotonaldehyde, glyoxal (GO), methylglyoxal (MGO), and nonenal (Aldini et al. 2008b). HSA is isolated and digested enzymatically to generate the peptide LQQC *PF containing the modif ed tag residue ( *Cys34). The resulting LQQCPF modif ed peptides are identif ed by LC -ESI-MS/MS in precursor ion scan mode and then characterized in product ion scan mode. The method w as f rst e valuated to identify and characterize the Cys34 co valent adducts of HSA incubated with HNE, ACR, and 4-hydroxy-hexenal. It w as then emplo yed to study the Cys34 modif cation of human plasma incubated with mildl y o xidized LDL, and the LQQCPF adducts with HNE and ACR w ere easily identif ed. Studies are in pro gress to appl y this methodolo gy to reco gnize changes of HSA in human diseases in volving systemic o xidation/carbonylation, focusing on end -stage renal disease, in w hich RCS -induced tissue damage is a kno wn feature. SELECTED EXAMPLES OF IN VITRO STUDIES: CELLS, TISSUES, FLUIDS, AND PROTEIN FRACTIONS EXPOSED TO RCS Ferrington and Kapphahn (2004) demonstrated that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purif ed from liver, the proteolytic core of the proteasome, and elucidated the molecular details responsib le for the loss in acti vity. Subunits containing HNE adducts were identif ed by a conventional approach following 2-D gel electrophoresis, Western immunoblotting, and anal ysis b y MALDI -TOF-MS. The a6/C2 subunit w as found to be uniquely modif ed at a time w hen only the chymotrypsin-like activity is inhibited. This inhibition would have a direct ne gative impact on man y key cell functions re gulated by the proteasome because it is primaril y the chymotrypsin-like activity that deter mines the rate of protein

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breakdown. These results w ere conf rmed only in par t (Farout et al. 2006), because w orking with a combined LC -ESI-MS/MS and MALDI -TOF/TOF approach, it w as found that more than one specif c subunit of the 20S proteasome (from liver and heart) is targeted for modif cation b y HNE. In an y case, the restricted number of subunits found to be modif ed, and the differences between liver and hear t, conf rm that HNE modif cation is not a random process but strictl y depends on protein accessibility (i.e. dif ferences in protein str ucture confer differential susceptibility to HNE modif cation). LC-MS/MS (using a Q -TOF micro ™ mass spectrometer) in combination with a ne wly developed sample preparation procedure (PVDF membrane as an alter native to the commonly used in -gel digestion protocol) has been sho wn to be a useful method to in vestigate oxidative modif cations of highly hydrophobic proteins such as apolipoprotein B-100 (apoB-100) isolated from LDL of health y subjects (Obama et al. 2007). This approach, resulting in impro ved sequence co verage, led to the identif cation of a His residue modif ed b y HNE in copper induced oxidation of LDL. Figure 11.9 schematically reports the on-membrane sample preparation procedure, w hich in volves LDL spotting on the PVDF membrane, remo val of lipid components b y solv ent w ashing, reduction and alk ylation of immobilized proteins on the membrane, trypsin digestion, and LC -MS/MS analysis of collected peptides. In-gel trypsin digestion and LC -MS/MS analysis were applied to demonstrate in Neuro2a, SH-SY5Y, COS-7, and HeLa cells that RCS modif cation by 4 - yhdroxy - 2 - alk enals (but not by ketoaldehydes) of ubiquitin C -terminal h ydrolase L1 (UCH -L1), and subsequent abnor mal interaction of RCS -modif ed UCH -L1 with multiple proteins (including tubulin), constitutes one of the causes of sporadic P arkinson’s disease (Kabuta et al. 2008). A shotgun -based approach that in volves both conventional data -dependent and neutral loss (NL) - dri ven MS3 data acquisition on a h ybrid linear ion trap -Fourier transform ion c yclotron resonance mass spectrometer (nanoelectrospra y ionization) w as applied for the rapid characterization of covalent modif cations of rat brain mitochondrial proteins b y HNE (Stevens et al. 2007). Several sites of HNE modif cation on 15 unique proteins w ere identif ed, which corresponded e xclusively to Michael adduct for mation on His residues. A number of HNE -

Figure 11.9. Schematic diagram of the on-membrane sample preparation procedure for analyzing trypsin-digested apolipoproteins from native LDL and/or modif ed LDL. Adapted with permission from Obama et al. (2007). Copyright 2007 © WILEY -VCH Verlag GmbH and Co.KGaA.

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modifed peptides produced a predominant HNE NL fragment -ion signal upon CID; therefore, NL - dri ven MS3 data -dependent acquisition w as found to be a v aluable method to enhance fragmentation information for these par ticular modif ed peptides. EX VIVO STUDIES Using proteomic techniques, the HNE (and peroxynitrite)-modif ed serum proteins from young and old F ischer 344 rats w ere investigated by Kim et al. (2006). Two-dimensional gel electrophoresis/Western blot analysis of nitrotyrosine and HNE-His revealed that serum proteins were differentially modif ed by ONOO − and HNE. Sixteen of the modif ed proteins, identif ed by MALDI-TOF-MS are involved in blood coagulation, lipid transport, blood pressure regulation, and protease inhibition. Furthermore, nitration and HNE adduction were found to increase with age, lending suppor t to the o xidative stress h ypothesis of aging. Proteins containing HNE adducts w ere identif ed in both the macular and peripheral re gions during four pro gressive stages of age -related macular degeneration (Ethen et al. 2007). The proteins were resolved by 2-D gel electrophoresis before detection of HNE -adducted proteins. Modif ed proteins w ere identif ed by MALDI -TOF-MS (and the total content of HNE adducts w as compared using a slot blot immunoassay). The for mation of ethanolamine phospholipids (PE) Michael adducts has been repor ted to occur in human blood platelets in response to o xidative stress and in retinas of streptozotocin induced diabetic rats (Bacot et al. 2007). PEs coupled with dif ferent h ydroxyalkenals, 4 - yhdroxy - 2(E) - xenal he (HHE), HNE, and 4 - yhdroxy - dodecadienal(HDDE), characterized by an alternative mass spectrometric approach (gas chromato graphy-mass spectrometry measurement of their ethanolamine moieties), could be used as specif c markers of membrane disorders occurring in pathoph ysiological states with associated o xidative stress, and might af fect cell function. Carbone et al. (2005a) identif ed HNE - and ONE - modifed proteins in a model of ethanol induced oxidative stress through a proteomic approach applied to liver fractions prepared from rats fed a combination high -fat/ethanol diet involving 2 -D gel electrophoresis, immunodetection of HNE adducts, in -gel digestion, and peptide mass f ngerprinting (LC - ESI - MS/MS). Several cr ucial proteins w ere found to be modif ed b y ONE (and HNE), among them the essential heat shock protein 90 (Hsp90, modif cation of Cys 572). In vitro chaperoning experiments performed to assess the functional ef fect of ONE modif cation on purif ed recombinant human Hsp90 indicated a signif cant decrease of the chaperoning eff ciency of the modif ed protein. By applying the same proteomic approach to mitochondrial protein isolated in the liver fractions from the same animals (rat model of earl y stage alcoholic liver disease), the research group found that another endoplasmic reticulum chaperone, protein disulf de isomerase, w as consistently modif ed b y HNE, especiall y under conditions of reduced GSH concentrations (Carbone et al. 2005b), providing unequivocal evidence that modif cation of intracellular k ey proteins contribute to disease pro gression. A close association of HNE -protein modif cations with the initiation of light -induced retinal degeneration has been also repor ted (Tanito et al. 2006). A conventional proteomic approach was used to understand the molecular mechanism(s) underlying the retinal degeneration induced by photooxidative stress in rats: 2-D gel electrophoresis, Western blot followed by in-gel digestion of 2 -DE gel spots, and peptide mass f ngerprinting by MALDI - T OF - MS.Nine retinal proteins, specif c in se veral functional cate gories (energy metabolism, gl ycolysis, chaperone, phototransduction, and RNA processing), were found to be modif ed by HNE. By immunohistochemistry, localization of three identif ed proteins o verlapped with immunoreacti vity of HNE - modifed proteins in light -exposed retinas. As outlined by the authors, the results cannot be considered conclusi ve because onl y antibodies specif c to His -Michael adducts w ere used, while other protein modif cations such as Cys - and L ys-Michael adducts can also occur in light - damagedretinas.

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MALDI-TOF-MS (and MS/MS) following in-gel enzymatic digestion were used to identify HNE-immunopositive proteins in retinal preparations from young and old rats and in rat retinal pigment epithelium (Kappahan et al. 2006). The HNE -adducted proteins fell into three major categories: chaperone/cell protection (heat shock co gnate 70, α A, αB, and β B2 crystallin), energy metabolism (triose phosphate isomerase, α enolase, aldolase C), and fatty acid transport FABP5. Identif cation of HNE -modif ed proteins in tw o alter native human model systems, human retinal pigment epithelial cells in culture and human donor e yes, indicated that triose phosphate isomerase and α enolase are generall y modif ed. HNE - deri ved modif cations of selective targeting of enzymes involved in glycolysis, energy production, and CO 2 hydration have been demonstrated to inhibit muscle contractile perfor mance in the v entilatory muscles of rats. Two-dimensional electrophoresis, immunob lotting, and mass spectrometry (LC-Q-Tof) of digested proteins were used to identify selective proteins targeted by HNE inside the diaphragm of rats under tw o conditions: severe sepsis and during strenuous muscle contractions elicited b y severe inspirator y resistive loading (Hussain et al. 2006 ). LC-ESI-MS/MS follo wing a con ventional proteomic approach w as used to in vestigate protein modif cation by HNE as a consequence of obesity (in adipose tissue from obese insulinresistant mice) and its potential relationship to the development of insulin resistance (Grimsrud et al. 2007). Biotin hydrazide was used as a chemical tag to couple proteins with free carbon yl groups and horseradish pero xidase-conjugated streptavidin for their b lotting detection. Biotin hydrazide - modifed adipose proteins from obese mice w ere captured using a vidin-Sepharose aff nity chromato graphy, proteol ytically digested , and subjected to mass spectrometr y for identif cation. Several adipose-regulatory proteins involved in cellular stress response, lipotoxicity, and insulin signaling (f atty acid-binding protein, glutathione S -transferase M1, peroxiredoxin 1, glutathione pero xidase 1, eukar yotic elongation f actor 1 –1, f lamin A) were found to be carbonylated. Tryptic digestion follo wed b y MALDI -TOF-MS anal ysis w as used to identify catalase as the main protein modif ed by HNE in red blood cells from systemic lupus erythematosus (SLE) patients. RCS- modifed catalase could be one of the reasons for lower enzymatic activity among SLE subjects, w hich in tur n could f avor the accumulation of deleterious h ydrogen peroxide. Furthermore, HNE -products are potential neoantigens and could be in volved in the patho genesis of SLE (D ’zousa et al. 2008). A conventional approach, based on 2D -gel electrophoresis, Western blot, and subsequent immunochemical detection of HNE -modif ed proteins, followed by a MALDI -TOF analysis of peptides resulting from in -gel digestion, allo wed identif cation (in autoptic brain specimens from mild co gnitive impair ment subjects) of 11 HNE -modif ed specif c tar get proteins w hich w ere identical to those found in not onl y AD, but also other neurodegenerative diseases (Reed et al. 2008).

CONCLUSIONS As seen by the numerous e xamples reported in the Characterization of Carbon ylated Proteins In Vitro section, abo ve, conventional mass spectrometric approaches continue to ha ve a fundamental role in detection and characterization of peptides and proteins co valently modif ed by RCS, especially for def nition of the stoichiometry of the protein/RCS adduct formation and for characterization of the sites of modif cation. However, in the last few years MS has gained much more impor tance because, in conjunction with biolo gical data, it has pro vided a deeper insight into the effects of modif cation by RCS on protein function, allowing the understanding of consequences of such modif cations at the cell level. The role of MS is particularly important at present because the majority of e xperiments are cur rently driving broad initiati ves in biomarker discovery and identif cation in a biological milieu of the more susceptible protein(s) to carbonylation, with the f nal aim to understand the impact of such modif cation(s) on cell/tissue function. This point is cr ucial to estab lish w hether carbon ylation of specif c proteins is

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causative, cor relative, or consequential to o xidative stress -associated conditions. Therefore, it is mandator y to impro ve the methodolo gies in ter ms of both deri vatization/enrichment procedures and mass spectrometric approaches. This will be signif cantly improved with the introduction of newly developed high resolution mass spectrometers. SELDI - OF T - MS(surface - enhancedlaser desorption/ionization time - of -ight) f mass spectrometry that combines chip -based solid -phase (chemically or biochemicall y modif ed) chromatographic enrichment of proteins from complex samples with TOF-MS is another promising high-throughput approach for identifying ne w biomark ers in v arious body f uids (Kiehntopf et al. 2007, Gagnon et al 2008). This approach has pro ved to be par ticularly useful, as MB MALDI - OF T - MS,for peptidome prof ling of human urine (F iedler et al. 2007). Hence, ProteinChip technology, in combination with moder n mass spectrometr y, allows the comple x search for biomark ers, molecular interactions, post -translational modif cations, and signaling pathways through a proteomic platform alternative to the more traditional 2 -D polyacrylamide gel electrophoresis. With the binding of specif c antibodies to the chip surf ace, SELDI also will be promising in detection of carbon ylated proteins, but no applications in the f eld of protein carbonylation have been developed. From the e xamples repor ted in this sur vey, it is e vident that until no w, MS methods ha ve been mainl y applied for detection of carbon ylated protein in in vitr o and ex vivo systems. Conversely, only a few reports dealing with a quantitative approach are available today, despite the growing importance of protein carbon ylation in disease pro gression, where detection and quantif cation of the target protein of this type of modif cation may serve as a useful biomarker for the early diagnosis of RCS -mediated diseases. In this context, HPLC-ESI-MS/MS of tryptic digests can play a key role in quantif cation of carbonyl - modifed proteins, and it will gain signif cant impor tance in routine protein/peptide quantif cation within a fe w years if the methods, still requiring tedious and time -consuming sample preparation procedures, will be implemented b y automated sample processing, and b y the use of ne w systems that will increase the speed of anal ysis. Traditional tryptic digests can require up to 18 hours, but the newly commercially available ultra-micro spin columns containing highly purif ed tr ypsin immobilized on a chemicall y modif ed silica suppor t to minimize non - specifc adsor ption can complete the same digest in onl y fe w minutes. The tw o main problems that in the authors’ opinion remain unresolv ed are (a) the digest step, w hich is assumed but not demonstrated to be quantitati ve and reproducib le, and (b) the limited a vailability of appropriately labelled inter nal standards. In conclusion, w e belie ve that MS will pla y a cr ucial role in the future in enhancing the actual knowledge of RCS reacti vity, metabolism, signalling, and the modulator y effect in the various human or gans, as w ell as in quantitati ve prof le changes in these modif cations with disease states or aging. This should pro vide a solid backg round to the elucidation of the RCS contribution to the patho genesis of se veral human chronic diseases, and will lik ely promote advanced and oriented applications not onl y in diagnosis and pre vention, but also in molecular treatment of human diseases. Undoubtedl y, new MS instr umentation developments will provide indispensable tools with impro ved resolution, sensiti vity, and mass accurac y for the str uctural characterization of lar ge, hetero geneous proteins adducted b y highl y reacti ve RCS.

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Chapter12 Nitrotyrosine: Quantitative Analysis, Mapping in Pr oteins, and Biological Signif cance oJs é M. Souza ,Silvina Bartesa ghi , Gonzalo P eluffo and , Rafael Radi

INTR ODUCTION Protein tyrosine nitration has been associated with se veral pathologies (Beckman et al. 1994, Greenacre and Ischiropoulos 2001, Ischiropoulos 1998, Pacher et al. 2007, Peluffo and Radi 2007, Radi et al. 2001, Souza et al. 2008b, Turko and Murad 2002) such as cardio vascular disease, neurodegeneration, inf ammation, and diabetic complications, and has been re vealed as a strong biomark er of o xidative stress in vivo and a predictor of disease pro gression and severity (Peluffo and Radi 2007, Zhang et al. 2001a, Zhang et al. 2001b, Zheng et al. 2005) in several pathological conditions. The product of this reaction, 3 -nitrotyrosine (3-NT), is a post translational modif cation found in proteins, w hich has dif ferent effects in the modulation of protein activity. Protein tyrosine nitration could result in dramatic changes in protein str ucture and function w hich can lead to a loss or a gain of function (Radi 2004), promote protein aggregation and affect degradation, and may alter phosphorylation cascades (Kong et al. 1996, Verhaar et al. 1999) and immunological responses (Herzog et al. 2005, Radi 2004). Elucidating the pathw ays and ef fects of 3 -NT for mation in pathoph ysiological conditions, as a means to understand the mechanism of human patholo gy, is the ultimate goal of this f eld. Interdisciplinary studies using dif ferent techniques and approaches can mo ve us closer to answer whether protein 3 -NT is responsible for the development of a pathological outcome or if it is only a side effect due to the increase in the for mation of nitrating agents in vivo. In this chapter the authors pro vide a biochemical foundation of 3 -NT for mation; anal yze dif ferent methodologies for measuring, quantitating, and mapping 3 -NT in proteins; and discuss the possible consequences of 3 -NT formation in biological processes.

FOUND ATION OF 3 - NITR OTYROSINE FORMATION The pathw ays of 3 -NT for mation in cells under biolo gically-relevant conditions ha ve been reviewed recentl y (Bar tesaghi et al. 2007, P eluffo and Radi 2007, Radi 2004, Souza et al. Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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2008b). The nitration of protein tyrosine residues arises from the reactions of nitric o xide (•NO)-derived oxidants such as pero xynitrite (ONOO −) and nitro gen dioxide ( •NO2) with the amino acid tyrosine, a nor mal constituent residue in proteins(appro ximately 3.2% in average). Tyrosine nitration can occur biologically by a variety of routes, mainly based on free radical pathways, and in volves at least tw o consecutive reactions: (1) the inter mediate for mation of the tyrosyl phenoxyl radical by one -electron oxidation of tyrosine and (2) the combination of the phenoxyl radical with • NO2 derived from (a) peroxynitrite decomposition (Radi et al. 2001), (b) hydrogen peroxide (H 2 O2)-dependent nitrite o xidation catalyzed by hemeperoxidases (e.g. myeloperoxidase) (Hazen et al. 1999), eosinophil pero xidase (W u et al. 1999), (c) aerobic oxidation of •NO (Wink et al. 1993), and (d) nitrite under acidic conditions (Olah et al. 1989) to yield 3 -NT (Bartesaghi et al. 2007). The oxidation of tyrosine to yield tyrosyl radical ma y be achieved by a number of o xidants such as hydroxyl radical ( •OH), carbonate radical (CO3•−), or high oxidation states of transition metal complexes (Me (n+ 1)+ =O; compound I of hemeperoxidases) (Radi 2004). In addition, the participation of the lipid-derived peroxyl radicals (LOO• ;E °LOOH/LOO• = 1.02 V)as one - electron oxidants which participate in the nitration pathw ay in hydrophobic biocompartments has been recently demonstrated (Bartesaghi et al. 2007) (Bartesaghi et al. 2010). Indeed, these alternative one-electron oxidants could play important roles in membranes and lipoproteins w here, due to the high concentration of unsaturated f atty acids, lipid pero xidation reactions tak e place and yield large amounts of LO • and LOO • radicals. Importantly, • NO2 can diffuse and concentrate in lipidic en vironments, potentiall y f avoring o xidation and nitration reactions (Moller et al. 2005 ). An alter native mechanism for the for mation of 3 -NT without the par ticipation of • NO2 involves the reaction of tyrosyl radical with •NO to form 3 -nitrosotyrosine followed by a twoelectron o xidation to yield 3 -NT with the inter mediate for mation of imino xyl radical (Radi 2004). This mechanism may be relevant in transition metal-containing proteins that can oxidize 3-nitrosotyrosine such as prostaglandin H synthase -2 (Gunther et al. 1997). An additional tyrosine nitration pathway, the impor tance of which in biology is ill -def ned, is the reaction of ONOO - and transition metal centers that ma y promote a radical -independent electrophilic aromatic nitration through the for mation of a comple x that operates as a car rier of nitronium cation (NO 2+) (de Queiroz et al. 2006, Radi 2004). The biochemistry and mechanisms of per oxynitrite- and hemepero xidase-dependent tyrosine nitration ha ve been re viewed recentl y (Souza et al. 2008b). Concomitant to the formation of 3 -NT, other side products can be for med. The combination of two tyrosyl radicals yields the dimerization product 3,3 ′ - dityrosine,a naturally occurring cross-linked amino acid w hich has also been used as a footprint of o xidative stress. Indeed , the formation of 3,3′-dityrosine has been shown in many proteins such as MnSOD (MacMillanCrow et al. 1998), f brinogen (Nowak et al. 2007), α synuclein (Souza et al. 2000, Zhou et al. 2008), and amyloid β peptide (Smith et al. 2007), and its for mation is associated with se veral neurodegenerative diseases. In addition, both pero xynitrite- and hemepero xidase-dependent pathways can lead to tyrosine h ydroxylation to 3 -hydroxytyrosine (DOPA) (Bar tesaghi et al. 2006, Santos et al. 2000). Overall protein tyrosine o xidation (i.e., nitration, dimerization, and hydroxylation) represents a footprint of o xidative stress and has been implicated in a v ariety of degenerative diseases as w ell as in the aging process (Xu et al. 2006).

TYROSINE ANALOGS AS PROBES FOR FOLLOWING NITRATION AND OXIDATION REACTIONS Most of the mechanistic studies on tyrosine nitration have been performed in aqueous solution. Due to the lo w solubility of tyrosine in w ater, when higher concentrations are required , more soluble tyrosine analogs such as p -hydroxyphenylacetic acid (pHPA), N-acetyltyrosine, or short

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tyrosine-containing peptides ha ve been e xtensively used (Beckman et al. 1992, Mani et al. 2003, Ye et al. 2007). Ho wever, fe w studies ha ve been perfor med in h ydrophobic en vironments, despite the f act that many protein tyrosine residues sho wn to be nitrated either in vivo or in vitro are associated to non -polar compartments such as biomembranes and lipoproteins: red blood cell membranes (Velsor et al. 2003), erythrocyte membrane band 3 (Mallozzi et al. 1997), microsomal gluthathione s -transferase (Ji et al. 2006), apo -B (Hazen et al. 1999, Hsiai et al. 2007), and apo -A (Zheng et al. 2004, Zheng et al. 2005). Factors controlling tyrosine nitration and dimerization in h ydrophobic environments have been revealed, and the y differ from those in aqueous solution (Bar tesaghi et al. 2007, Zhang et al. 2001a) (Bartesaghi et al. 2010). In this regard, several hydrophobic tyrosine probes have been recentl y de veloped to study tyrosine nitration in membranes. Dif ferent tyrosine esters have been e valuated (Zhang et al. 2001a), all of them with a tert butyl moiety that can be anchored in the bilayer. From different hydrophobic tyrosine analogs, N - t BOC L - tyrosinetert butyl ester (BTBE) was successfully incorporated with large yields to model phosphatidylcholine membranes (Bar tesaghi et al. 2006, Zhang et al. 2001a) and even to red b lood cell membranes (Romero et al. 2007), resulting a useful probe to study tyrosine nitration and other oxidation processes in membranes. Lipid composition and en vironmental factors such as CO 2 , transition metals, and pH, among others, in tyrosine nitration yields ha ve been e valuated (Bartesaghi et al. 2006, Romero et al. 2007, Zhang et al. 2001a). Indeed, BTBE incor porated to hydrophobic structures can be exposed to different oxidizing and nitrating species, and reaction products (i.e. 3 -nitroBTBE, 3,3 ′ - diBTBE,and 3 - yhdroxy - BTBE)can be analyzed after organic e xtraction b y RP -HPLC separation with UV -Vis detection for the nitro -derivative, f uorimetric detection for the dimerization product and mass spectrometr y detection for the hydroxylated derivative (Bartesaghi et al. 2006). In addition to BTBE, Kal yanaraman and co -workers have developed 23 amino acid transmembrane peptides containing tyrosine residues in dif ferent depths (i.e. Y-4, Y-8, and Y-12) (Zhang et al. 2003). These peptides ha ve hydrophilic and char ged amino acids in the N - and C -terminus (e.g. Lys) and a h ydrophobic sequence in the central re gion (repeats of Ala-Leu) with the tyrosine residue located in dif ferent positions. Str uctures of the h ydrophobic tyrosyl probes have been shown elsewhere (Bartesaghi et al. 2007, Zhang et al. 2003). These peptides more closel y resemble the str ucture of transmembrane domains in proteins and have been used to assess how the location of the tyrosine residue inf uences the oxidation/ nitration yields (e.g., solvent exposed or superf cial residues vs. residues away from the aqueous phase). For e xample, pero xynitrite-dependent tyrosine nitration increased w hen the tyrosine was deeply located in the bilayer; it was higher for Y-12, whereas MPO/nitrite/H2 O2 - dependent nitration w as g reater w hen the residue w as closer to the aqueous inter phase (Y-4) (Zhang et al. 2003). Because the physicochemical factors controlling tyrosine nitration, dimerization, and hydroxylations in h ydrophobic biocompar tments have unique characteristics (e.g. e xclusion of k ey antioxidants nor mally present in aqueous phases such as glutathione, high concentration of unsaturated fatty acids, and f avored partitioning of •NO and •NO2), the use and v alidation of these hydrophobic tyrosine probes help to understand the nitration mechanisms within membrane bilayers and lipoproteins (Bartesaghi et al. 2007). In this regard, BTBE is a valuable tool because it can be used for a g reat number of biochemical studies and it pro vides quantitative data with a relatively simple experimental protocol. As the determination of 3-NT in membrane proteins is complex (see below), the use of probes such as BTBE in the model and even natural membranes allows the study of tyrosine nitration mechanisms in a more controlled situation. On the other hand , transmembrane peptides closel y resemble a transmembrane domain, and thus are very useful for studying the rele vance for nitration yields of the intramembrane location of tyrosine residues, the role of neighboring amino acids (i.e., c ysteine and methionine), as well as the inf uence of the lipidic composition.

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METHODS FOR QUANTIFICATION OF 3 -NITROTYROSINE IN BIOLOGICAL SAMPLES A number of methods w ere developed for the separation, detection, and quantitation of 3 -NT in biological samples (Table 12.1). These methods can be grouped as mass spectrometry-based methods and non -mass spectrometr y methods. The latter hetero geneous g roup includes the immunochemical techniques using specif c antibodies and high-performance liquid chromatography (HPLC) coupled with dif ferent detection devices. Protein 3-NT is a good immunogenic antigen and it allows the generation of polyclonal and monoclonal antibodies (Y e et al. 1996). Immunohistochemical detection of 3 -NT in f xed tissues or cells slices is widel y used and seems to be more sensiti ve than the Western b lot methods of the protein homogenates, probably due to antigen dilution effect (Viera et al. 1999). Antibody-based methods pro vide less quantitati ve data, although enzyme -linked immunoabsorbent assays (ELISA) w ere developed using nitrated albumin as a standard to obtain more quantitative results (Khan et al. 1998, ter Steege et al. 1998) (Table 12.2). Total protein h ydrolysis is a k ey step in the quantitation process of 3 -NT. The most commonly applied hydrolysis method is acid hydrolysis, in which the proteins are incubated in the presence of 6 N HCl at 110 °C for 16 to 24 hours, usuall y in vacuo or under a nitro gen atmosphere, and in the presence of 1% phenol to sca venge oxidants that ma y destroy Tyr residues (Fountoulakis and Lahm 1998). This is a v ery eff cient method to obtain the complete protein hydrolysis but it also has cer tain side ef fects that should be tak en into account (see belo w). The alternatives to acid hydrolysis are the alkaline hydrolysis and protease hydrolysis methods. Alkaline hydrolysis is perfor med with 4 M NaOH heated at 120 °C for 16 hours (F rost et al. 2000) and protease h ydrolysis is done in sodium acetate pH 6.5 to 7.2 with freshl y prepared pronase incubated at 50 °C for 18 hours (Hensle y et al. 1998, Shigenaga et al. 1997). Reverse-phase (RP) -HPLC columns allow the separation of free tyrosine from 3 -NT due to the higher h ydrophobic character of 3 -NT over tyrosine. Se veral detection systems, such as ultraviolet absorption and electrochemical (ECD) and f uorescence detections can be applied. ECD and f uorescence detection methods are based in deri vatization of 3 -NT (Table 12.1). The ECD detection implies the 3 -NT transfor mation to an N - acetylderivative followed by the reduction of the nitro tyrosine g roup to an amino g roup ( -NH3) b y ditionite reduction. These tw o conditions are needed to obtain an increase in the retention time in the column and lo wer the o xidation potential in the ECD detector ( + 37 mV) (Shigenaga et al. 1997 ) (Table 12.1 ). 3-NT can be measured b y ECD without deri vatization using a dual channel detector or a coulumetric multiple ar ray detector with se veral (8 to 12) channels. In the dual ECD the upstream electrode is set at a reduction potential of −900 mV and the downstream electrode at +600 mV (Sodum et al. 2000). For the multiple channels ECD the channels are set with increasing potentials betw een 100 and 950 mV; 3 -NT is detected betw een 700 to 950 mV due to its high oxidation potential (Crow 1999, Hensley et al. 1998). With a single cell ECD at an 800mV potential, it is nearly impossible to detect 3 -NT because of the high backg round noise, and the coulometric ECD ar ray detector allows “cleaning” the sample at the lo wer potentials and then decreasing the interference at the higher v oltages. Fluorescence deri vatization of 3 -NT can be done using typical amino acid reagents as 4 - fuoro - 7 - nitrobenzo - xa 2 --ol,3 - diazole (NBD - F)and following the 470 nmand 530 nmexcitation and emission w avelengths, respecti vely (Table 12.1). Similar NBD -F deri vatization can be done and the samples separated b y capillar y electrophoresis coupled to laser -induced f uorescence (Massip et al. 2002). P eptide containing 3 -NT residues w ere deri vatized with 4-aminomethylbenzene sulfonic acid prior to reduction of the nitro g roup to an amino g roup to for m a f uorescence adduct with an e xcitation and emission of 360 nm and 490 nm, respectively (Sharov et al. 2008). These derivatization methods are diff cult due to the multiple steps and they do not allo w intra -experimental monitoring of ar tifactual tyrosine nitration.

Table 12.1. Methods for 3 -nitrotyrosine detection.

Method

Detectedcompound

UVabsorbance

Signature Aromaticabsorption at 274, 350, or 420 nm

OH NO2

H +

H3N C COOH

3-nitrotyrosine

Electrochemical detection (ECD)

OH NH3

Half -xidation o potential + 37 mV

H CH3COHN C COOH

N-acetyl-3aminotyrosine

Fluorescence

NO2 OH

H C

Excitationat 470 nm and emission at 530 nm

COOH

NH N O N NO2

7-nitrobenzo-2-oxa-1,3diazole-3-nitrotyrosine

Gaschromatography mass spectrometry (GC - MS)

DMBTS O

Detected as a fragment of m/z 518

NO2

H F7C3COHN

C

C O -------------------O DMBTS

N-heptafluorobutyryl-O, O-di(t-butyldimethylsilyl)3-nitrotyrosine

Liquidchromatography mass spectrometry (LC - MS)

OH NO2

Detectedas a transition 227/181

H +

H3N C --------------COOH

3-nitrotyrosine

203

204 Chapter

12

Table 12.2. Limits of quantitation and basal plasma levels of protein 3 -nitrotyrosine.

Method

Limitof quantitation

ELISA (sandwich) ELISA (competitive) HPLC - UV

0.2nM 3.8nM

HPLC - ECD

0.25pmol

HPLC f uorescence

6nM

200nM

Mass spectrometry - based GC - MS 0.07fmol

References terSteege et al. 1998 Khanet al. 1998 Kaurand Halliwell 1994 Shigenaga et al. 1997 Kamisaki et al. 1996

Le vels of protein 3 - NTin plasma as µ mol 3 - NT/molTyr Undetectab le 5.6+ (120 nM) — 0.37(8 nMa ) 1.4+ (31 nM)

Gautet al. 2002

8.9(192 nMa) 8.0 (172 nMa )

GC - tandem MS

0.125nM

Schw edhelm et al. 1999

1.2(26 nMa) 1.6 (33.4 nMa )

LC - MS

100fmol

0.6(13 nMa )

LC - MS

3.2fmol

Nicholls et al. 2005 Gautet al. 2002

0.8 to 3.2 (17.3 to 69 nMa )

References terSteege et al. 1998 Khanet al. 1998 — Shigenagaet al. 1997 Kamisakiet al. 1996 Delatouret al. 2002b Pennathur et al. 2004 K eimer et al. 2003 Tsikas et al. 2003 Ahmedet al. 2005 Lorchet al. 2003

The original data are repor ted in nM units and w ere transformed to µ mol 3 - NT/molTyr using 0.64 mM of serum albumin plasma concentration and 3.1% of Tyr frequency. a Calculated from the original data repor ted in µmol 3 -NT/mol Tyr units assuming the same estimate as for + but in an opposite w ay. +

Mass-spectrometry-based methods are applied to measure 3 -NT by two main approaches: with or without 3 -NT derivatization (Table 12.1). For gas chromatography-mass spectrometry (GC-MS), 3-NT is derivatized to obtain more volatile and highly electron-capturing molecules which are easier to detect. By using appropriate f uorine-containing deri vatization agents, a specif c 3 -NT adduct can be for med and detected in the mass spectrometer . As an e xample, Table 12.1 shows the adduct for med with the derivatization reagents ethyl heptaf uorobutyrate and N -t (- butyldimeth ylsilyl) -N - meth yltrif uoroacetamide (TBDMS) that generate the Nheptafuorobutyryl -O ,O - di( t-butyldimethylsilyl) deri vatives of 3 -NT, w hich are measured in the selected ion monitoring mode (SIM) as a fragment of m/z 518 (F rost et al. 2000, Gaut et al. 2002). Other derivatization reactions and adducts can be used in a similar w ay (reviewed in Ryberg and Caidahl 2007). The GC-MS methods can be improved by GC-tandem MS using the same principle as GC -MS, but adding the fragmentation patter n of the adducted residue in a selected reaction monitoring or multiple reaction monitoring scan mode (Ryberg and Caidahl 2007, Schwedhelm et al. 1999, Tsikas and Caidahl 2005).

Nitrotyrosine: Quantitative Analysis, Mapping in Proteins, and Biolo gical Signif cance

205

Combination of HPLC and mass spectrometr y (LC -MS) allo ws the quantitation of 3 -NT without any derivatization reaction after the complete protein h ydrolysis. The amino acids are separated by reverse-phase chromatography, and electrospray ionization (ESI) is coupled with the HPLC to monitor the 3 -NT ions. As in the case of GC -tandem MS, the pattern of fragmentation of 3 -NT can be specif cally followed using the selected reaction monitoring or multiple reaction monitoring scan mode. F or example, tyrosine and 3 -NT have [M + H]+ ion masses of 182 and 227 and generated fragments of 136 and 181, respecti vely (Delatour et al. 2002a, Nicholls et al. 2005) (Figure 12.1, Table 12.1). Monitoring the transition 182/136 and 227/181 by multiple reaction monitoring mode, tyrosine and 3 -NT, respectively, can be easil y detected in the same HPLC r un (Nicholls et al. 2005). Both mass spectrometric methods, GS -MS and LC -MS, allow the introduction of kno wn T and [13 C6 ] - NO T before the amounts of isotope -labeled internal standards such as [ 13 C6 ] -yr 2 -yr protein h ydrolysis, and in the case of GC before the deri vatization reactions. Isotopicall ylabeled inter nal standards are identical to the anal ytes, apar t from their hea vier masses, and behave identicall y during the w hole sample processing. The stab le-isotope-dilution methods allow an increase in the accurac y of the quantitati ve measurements due to a compensation for analyte loss during the sample h ydrolysis. In addition, the inter nal standards allow the e valuation of ar tifactual tyrosine nitration during the sample processing, mainl y during the acid hydrolysis, where contaminant nitrite ma y induce nitration. Experiments with sample suppleT may show the mentation with [ 13 C6]-Tyr or with the uni versally-labeled isotope [ 13 C915 N1 ] -yr T or [13 C915 N1 ] - NO presence of [ 13 C6 ] - NO 2 -yr 2-Tyr due to ar tifactual generation of 3 -NT. This last issue is critical and ma y induce an o verestimation of the real le vels of tyrosine nitration in biological samples, where the •NO end product nitrite accumulates. To avoid artifactual acid tyrosine nitration, alkaline hydrolysis (Frost et al. 2000) or protease hydrolysis (with Pronase) methods (Hensley et al. 1998, Shigenaga et al. 1997) may be used.

MAPPING3 - NITR OTYROSINE IN PROTEINS The global anal ysis of protein 3 -NT generation in human samples or e xperimental models of disease is a useful parameter to correlate clinical output, pharmacology, or genetic manipulation and nitro xidative stress (Szabo et al. 2007). A deeper anal ysis involves the identif cation of specif c proteins modif ed by tyrosine nitration and the nitration site(s) within each protein. In this approach, the protein of interest is digested b y proteol ytic enzymes, typicall y tr ypsin (Figure 12.2 ). If the identity of the protein is unkno wn, the resulting peptide mixture can be directl y analyzed b y MALDI -TOF mass spectrometr y and the generated f amily of mass scans can be compared to existing protein databases. Under this condition it ma y be diff cult to obser ve the nitrated peptide due to se veral issues: (1) the same unmodif ed native peptide ma y suppress the signal from the nitrated peptide, (2) the nitrated peptide ma y be less prone to desor ption, and (3) the nitro moiety group can be lost during the ionization in the MALDI-TOF (see below). In early work, proteomic anal ysis of nitrated proteins relied on the identif cation of nitrated proteins by 2D Western blotting and mass spectrometr y was used to identify the protein, not the site of nitration (Aulak et al. 2001). The combination of HPLC separation of the tr ypsin-digested protein with electrospra y ionization increases the chances of identifying the site of tyrosine nitration. 3 -NT–containing peptides have greater retention times compared to the nati ve, unmodif ed peptides. Free 3 -NT and nitrated peptide sho w an absorbance peak at 430 nm at alkaline pH, w hich changes to 350 nm at acidic pH. F or in vitr o e xperiments the nitrated peptide can be follo wed b y the absorbance at 350 nm during the HPLC separation, although 3 -nitrotryptophan or prosthetic group-adducted to peptide can also absorb at 350 nm (Cassina et al. 2000, Souza et al. 1999). Nitrated peptides have an increase in mass of 45 Da in relation to the native peptide; the MALDI mass spectra also show the characteristic loss of-16 Da two times—[M+H−16]+ and [M+H−32]+

A

OH

H +H N 3

C COOH 182

OH

OH

H +

H +

H3N C

C COOH

I Immonium i iion 136

165

+

+

HCOOH

NH3

B

OH NO2

H +H N 3

C COOH

227

OH

OH NO2

+H N 3

NO2

H

H

C

+C

Immonium ion 181 + HCOOH

COOH 210 + NH3

Figure 12.1. Fragmentation patterns of tyrosine and 3-nitrotyrosine by electrospray mass spectrometry. Tyrosine and 3 -NT are injected in a quadrupole ion -trap MS (Applied Biosystems) in 1% acetic acid, 50% methanol. Enhanced product ion mode was used to analyze the fragment pattern of tyrosine (A) and 3 -NT (B).

206

Nitrotyrosine: Quantitative Analysis, Mapping in Proteins, and Biolo gical Signif cance

207

Protein Mixture (Nitrated and non-nitrated proteins) Complete protein hydrolysis: • Acid hydrolysis • Alkaline hydrolysis • Protease Enrichment of nitrated protein: • Immune precipitation or affinity columns • 2D gel electrophoresis and Western blots

HPLC separation: • UV absorption • EDC detection • Fluorescence detection

Trypsin digestion

Trypsin digestion Enrichment of nitrated peptide: • Immune affinity column • Reduction and biotin tagging • Derivatization with SATA and thiopropyl beads

Mass Spectrometry detection: • GC-MS • GC-tandem MS • LC-MS

Quantitative Data 3-NT/tyrosine 3-NT/mg of protein

Protein identification and 3-NT mapping Mass fingerprint with >15% sequence coverage → Protein ID Native Peptide + 45 Da → Nitrated peptide MS/MS spectrum → peptide sequence with 3-NT mapping

Figure 12.2. Quantitative analysis and mapping of protein 3 -nitrotyrosine; general protocol to quantitate, identify, and map the site of tyrosine nitration in proteins. The protein mixture can be hydrolyzed to quantitate the total amount of 3-NT; it can also be subjected to enrichment methods for nitrated proteins or be analyzed directly after trypsin hydrolysis.

–16

40000

–16

Relative Intensity

993.2353 30000

1009.2204 994.2418

20000

1010.2217 978.0002 979.2639

10000

995.2437 996.2437

1011.2215

0 970

980

990 Mass (m/z)

1000

1010

92 Figure 12.3. MALDI spectra of the cytochrome c nitrated peptide EDLIA97NO2YLK99. The nitrated peptide was purif ed by RP-HPLC from a mononitred cytochrome c species and analyzed by a Applied Biosystems MALDI -TOF. Reproduced with permission from Batthyany et al. 2005.

(Batthyany et al. 2005) (F igure 12.3)—probably due to photochemical reactions induced b y the presence of the nitro g roup (Petersson et al. 2001). This lability of the nitro g roup in the MALDI ma y decrease the sensiti vity in a peptide mixture. In the case of ESI spectra, no fragmentation is obser ved in the nitrated peptide. MS/ MS of the selected nitrated peptide allows the mapping of the site of tyrosine nitration through

208 Chapter

12

the analysis of ions (b and y) generated after peptide bond r upture that allo ws sequencing of the peptide (Petersson et al. 2001). In addition, the product ion spectrum of the nitrated peptide shows the distinctive immonium ion of 3 -NT with an m/z of 181 (P etersson et al. 2001) (Figure 12.1B). This immonium ion of 3-NT allo ws the identif cation of nitrated peptides using the precursor ion scanning mode, which permits a fast scan of peptides that have a mass loss 181 m/z. The precursor ion scanning may generate some false positive results but it is a rapid method to detect the nitrated peptides which need fur ther conf rmation by MS/MS (Petersson et al. 2001). Due to the lo w abundance of nitrated protein and nitrated peptides generated after protein trypsin hydrolysis, methods have been developed to enrich and selecti vely capture the nitrated species. These methods are based on the dif ferent reacti vity of the 3 -aminotyrosine residue generated after sodium dithionite reduction of the 3 -NT residue. The amino g roup of the 3-aminotyrosine has a much lo wer pKa (pKa 4.8) as compared with other amino g roups such as the lysine (pKa 10.4 to 11.1) or the guanidinium g roup of arginine (pKa 12.0), or the amino terminal of the pol ypeptide chain (pKa 6.8 to 8.0) present in proteins (Creighton 1993). The 3 -aminotyrosine can be labeled specif cally at pH 5.0 with sulfosuccinimidyl -2(biotinamido) ethyl-1,3-dithiopropionate, a bifunctional reagent with an N -ydroxysuccinimide h ester capable of reacting with the amino g roup and a biotin tag, link ed by a disulf de bridge. Most of the other protein amino groups are protonated at this pH and therefore it will not react (Nikov et al. 2003). The protein is then tr ypsinized and the biotin ylated peptides aff nitypurifed b y a strepta vidin-column, with elution of the tagged peptides b y reduction of the disulf de linker (Nikov et al. 2003). To avoid the reaction of other protein amino g roups, a similar strate gy was used in w hich all of the primar y amino g roups were acetylated with acetic anh ydride before the reduction, then 3-NT residues were reduced to the cor responding 3-aminotyrosine residue. The 3-aminotyrosine residues were reacted with N -succinimidyl-S-acetylthioacetate (SATA) at pH 5.0, and the tagged protein or peptide generated a free thiol using h ydroxylamine, which could then be aff nity - purifed by thiopropyl sepharose beads (Zhang et al. 2007). Using a similar approach, Sharo v et al. reacted 3 -aminotyrosine residue with benzylamine (and benzylamine derivatives) to form a f uorescence benzoxazole as mentioned above (Sharov et al. 2008), and Amoresano et al. reacted with dansyl chloride to also for m a f uorescent derivative (Amoresano et al. 2007). All of these adducted 3 -aminotyrosine peptides ha ve a specif c mass dif ference that can be follo wed by MS and ma y generate a ne w family of ions, depending on the modif cation. F or e xample, the dansyl deri vatives of the aminotyrosine 3 peptides create a fragment of m/z 234 w hich fur ther generate an m/z of 170 in the MS mode. By using the precursor ion scanning mode of 170 or the combination of 234 → 170 wed (Amoresano et al. 2007) in MS 3, the identif cation of the nitrated peptides is allo (Figure 12.2 ). Proteomic methods to identify nitrated proteins w ere f rst applied in LPS -treated rats as an inf ammatory model using tw o-dimensional gel electrophoresis and Western blots with anti nitrotyrosine antibody to select the nitrated proteins (F igure 12.2) (Aulak et al. 2001). These authors were able to identify 40 proteins based on MALDI -TOF analysis of the obtained peptides from the trypsin-digested spots, but without the mapping of the nitrated site in each protein (Aulak et al. 2001). A shotgun strate gy with po werful mass spectrometers has allo wed the analysis of endo genously nitrated proteins of mouse brain b y tw o-dimensional LC -MS/MS with the combination of a c ysteinyl-enrichment fraction plus a global fraction depleted of cysteinyl-peptides, and with the map of the site of tyrosine nitration (Sacksteder et al. 2006). Of the 7,792 identif ed proteins, 29 proteins that were nitrated in tyrosine residues were mapped (corresponding to 0.4%), preferentiall y corresponding to mitochondrial and c ytoskeletal locations (Sacksteder et al. 2006). Later, the same g roup using the 3 -NT enrichment method (the SATA described above) identif ed the sites of nitration of 102 proteins in mouse brain (Zhang et al. 2007 ).

Nitrotyrosine: Quantitative Analysis, Mapping in Proteins, and Biolo gical Signif cance

209

BIOLOGICAL EFFECTS OF 3 -NITROTYROSINE IN PROTEINS The reported levels of proteins 3 -NT found in nor mal plasma and measured as µ molof 3 - NT/ mol of tyrosine are in the range of 0.4 to 6 3 -NT residues per 106 tyrosines, which corresponds to the nM range (betw een 8 and 190 nM) (Table 12.2). The basal le vels of tyrosine nitration observed in plasma, cells, and tissues (Ischiropoulos 2003, Knyushko et al. 2005, van der Loo et al. 2000, Xu et al. 2006), and in proteomic anal ysis of endo genous nitrated proteins (Sacksteder et al. 2006, Zhang et al. 2007) represents the ph ysiological f ux of • NO - deri ved oxidants. Although speculative, the “basal nitration” may be part of a non-yet described signaling pathway analogous to w hat is obser ved for HIF 1 α and NF -kappa B acti vation by physiological low levels of oxidants such as O 2•− , H2 O2 and presumably ONOO − . An increase in protein tyrosine nitration has been associated with patho genesis in se veral human and animal models of disease. Fur thermore, 3 -NT levels have been used as a mark er to monitor the progress of disease and the effcacy of pharmacological treatment in some human trials (Brennan et al. 2002, Shishehbor et al. 2003, Zheng et al. 2004). In this regard, an important issue that deser ves some consideration w hen dealing with protein 3 -NT levels is that in pathological conditions 3 -NT tends to be “concentrated” mainl y in one or a fe w proteins. For example, the le vels of nitrated apo -B100 found in plasma LDL increase up to 90 -fold when the sample is anal yzed from the atherosclerotic lesion (840 vs. 9 µ mol 3 - NT/moltyrosine, lesions vs. matched plasma, respecti vely) (Leeuwenburgh et al. 1997). It remains a challenge to estab lish a direct link betw een patholo gical e vents and tyrosine nitration in specif c proteins. Three possible scenarios can be envisioned in this regard: (1) loss of function, (2) gain of function, and (3) in se veral cases tyrosine nitration might just re veal an increase in nitro xidative stress without altering protein function. A classic e xample of loss of function after tyrosine nitration is the site-specif c nitration of tyrosine 34 in MnSOD (Table 12.3). Nitrated MnSOD has been detected , for e xample, in samples from human chronic allograft nephropathy and it cor relates with the decrease in enzyme acti vity (MacMillan-Crow et al. 1996). More recently, nitrated MnSOD has been detected in aged v ascular tissue by the T - MnSOD(Xu et al. 2006 ). use of an antibody specif cally directed to NO 2 -yr34 In order to be of biolo gical importance, the loss of function requires that a high propor tion of the tar get protein be nitrated. As mentioned above, the measured le vels of 3 -NT over total tyrosine residues is in general lo w. However, it does not e xclude the possibility that a high nitration yield for a specif c protein could be reached and af fect the biolo gical function, as is the case for MnSOD. The gain of function after tyrosine nitration is an alter native option to e xplain a signif cant biological effect for a small propor tion of nitrated proteins. These involve the increase or the manifestation of a ne w function after the protein is nitrated and w ould allow a small por tion of nitrated protein to amplify their ef fects over the unmodif ed proteins. For instance, nitration of α-synuclein has been found in Le wy bodies and other protein agg regates in P arkinson’s disease and related synucleinopathies (Giasson et al. 2000). Monomeric nitrated α - synuclein is able to accelerate the rate of f bril for mation of the unmodif ed α - synuclein(Hodara et al. 2004), which may hypothetically occur when a small amount of α-synuclein is nitrated at the beginning of the disease. Other important examples substantiating the gain-of-function concept include cytochrome c, f brinogen, and nerve growth factor (NGF), among others (Table 12.3). Protein tyrosine nitration may have additional biological consequences: (1) increase degradation by the proteasome system, (2) af fect phosphor ylation cascade signaling, and (3) induce immunological responses, as re viewed recently (Souza et al. 2008b).

CONCLUSIONSAND PERSPECTIVES Protein 3 -NT has emer ged as a biomark er of nitro xidative stress in disease processes. Tyrosine nitration depends on free radical reactions dri ven by •NO-oxidants. The specif city

210 Table 12.3. Effect of tyrsosine nitration on selected proteins.

Protein

Nor mal Function

Cellularlocation

NitrationEffect

References

Cytochromec

Electrontransfer and apoptosis

Mitochondrial intermembrane space

Batth yany et al. 2005 , Cassina et al. 2000, Souza et al. 2008a

Clotformation Serine/threonine kinase GSHperoxidase activity Presynaptic protein Dephosphorylation

Plasma Cytosoland membrane Endoplasmic reticulum Presynaptic terminals CNS Cytosol

Higherperoxidatic activity, inhibition of apoptosome activation Higheraggregation T ranslocation and activation Higheractivity

V adseth et al. 2004 Balafanova et al. 2002 Jiet al. 2006

Higheraggregation

Hodaraet al. 2004

Inhibition of NF -κ B Axonalstructure

Cytosol

F ibrinogen Proteinkinase Cε Glutathione - S transferase 1 α - Synuclein Protein Phosphatase A2 Iκ Bα Neuroflament L

Cytosol

Nerve growth factor MnSOD

Neurotrophic factor Supero xide dismutation

Extracellular space Mitochondrial matrix

Prostac yclin synthase

Prostac yclin synthesis

Endoplasmic reticulum

Higheractivity Acti vation of NF -κ B Inhibitassembly of neurof lament Neuronalapoptosis Decreasedactivity Decreasedactivity

Cro w et al. 1997 P ehar et al. 2006 MacMillan - Cro w et al. 1996, Xu et al. 2006 Zouet al. 1997

Protein

Nor mal Function

Cellularlocation

NitrationEffect

References

yTrosine hydroxylase Prostaglandin endoperoxide H synthase Glutamine synthetase

Synthesisof L - DOP A Synthesisof PGH2

Cytosol

Decreasedactivity

Ribonucleotide reductase Glutathione reductase CytochromeP450 2BI Ornithine decarboxylase Proteinkinase C Succin yl - CoA:3 oxoacid CoA transferase

Condensationof glutamate and ammonia Reductionof ribonucleotides Reductionof GSSG Metabolismof xenobiotics Putrescine synthesis Serine/threonine kinase Acetoacetyl - CoA formation

Cytosol

Decreasedactivity

Blanchard -illion F et al. 2001 Goodwinet al. 1998 , Trostchansky et al. 2007 Berlettet al. 1998

Cytosol

Decreasedactivity

Guittetet al. 1998

Cytosol

Decreasedactivity

Sa vvides et al. 2002

Endoplasmic reticulum Cytosol

Decreasedactivity

Roberts et al. 1998

Decreasedactivity

Seidelet al. 2001

Cytosol

Decreaseactivity

Knappet al. 2001

Mitochondrial matrix

Decreaseactivity

Marcondeset al. 2001

Endoplasmic reticulum

Decreasedactivity

211

212 Chapter

12

of the nitration process o ver a gi ven protein and tyrosine residues is a result of a comple x variety of f actors, among w hich are the nature of the nitrating species, protein str ucture, and environment where the process tak es place (e.g., h ydrophilic vs. hydrophobic). The quantitati ve anal ysis of 3 -NT is still tedious and prone to ar tifacts. Nonetheless, the development of methods such as MS -based bioanalytical techniques allows the deter mination of actual 3 -NT values in biolo gical samples both under basal and patholo gical conditions. In addition, depending on the nature of the required infor mation, semi -quantitative methods for 3-NT detection (e.g., immunochemicall y-based) can be useful. F inally, 3 -NT can constitute a mediator of disease as w ell as a risk f actor because nitrated proteins ma y par ticipate in cell/ tissue dysfunction. Ho wever, signif cant work is still required to fur ther identify specif c proteins, sites of nitration, and biochemical mechanisms to link protein tyrosine nitration to pathogenesis in a causati ve way.

A CKNOWLEDGEMENTS This w ork w as suppor ted b y g rants from Ho ward Hughes Medical Institute (HHMI), International Centre of Genetic Engineering and Biotechnolo gy (ICGEB) to Raf ael Radi, and Agencia Nacional de In vestigación e Inno vación Fondo Clemente Estab le to Jos é M. Souza (No. 491) and Silvina Bar tesaghi (No. 362). Silvina Bar tesaghi was par tially suppor ted by a fellowship from PEDECIB A and Agencia Nacional de In vestigación e Inno vación, Ur uguay. Rafael Radi is a Ho ward Hughes Inter national Research Scholar.

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Chapter13 Ubiquitin Conjugates: A Sensitive Marker of Oxidative Stress Fu Shangand AllenaTylor

INTR ODUCTION Ubiquitin is a highly conserved 76 amino acid polypeptide that is ubiquitously expressed in all eukaryotes. The best understood function of ubiquitin is to tag proteins for de gradation by the ubiquitin-proteasome pathway (UPP) (Glickman and Ciechanover 2002). The UPP is the major non-lysosomal proteolytic pathway within cells (Ciechanover 2003, Glickman and Ciechanover 2002, Shang and Taylor 2004). In this pathw ay, proteins destined for de gradation must be conjugated with a chain of multiple ubiquitins in order to be reco gnized and de graded by a large protease complex called the 26S proteasome. The proteasome complex consists of a 20S proteolytic core and typically two regulatory 19S caps. The 19S cap recr uits ubiquitin conjugates to the proteasome, and clea ves ubiquitin moieties from the substrate. It then unfolds the polypeptide and feeds it through the narrow channel of the proteolytic chamber of the 20S core (DeMartino and Slaughter 1999). Ubiquitin conjugation, or ubiquitination, is a highly ordered process in which a ubiquitin-activating enzyme (E1) f rst activates and transfers ubiquitin to a ubiquitin -conjugating enzyme (E2), w hich then acts in concert with one of a lar ge family of ubiquitin protein ligases (E3) to transfer ubiquitin to a l ysine residue on the tar get substrate (Glickman and Ciechano ver 2002, Pickar t 2001). In most cases, multiple ubiquitins are conjugated to the initial ubiquitin moiety to form polyubiquitin chains. A chain of at least four ubiquitin moieties is often required for substrate reco gnition by the 26S proteasome comple x (Beal et al. 1996, Lam et al. 2002, Pickart 1997). The UPP is an impor tant protein quality control system (Goldber g 2003, Grune et al. 2005, Marques et al. 2006), w hich selecti vely de grades mutant, misfolded , or damaged proteins (Marques et al. 2004, Shang et al. 2001, Ward et al. 1995). Timely removal of abnor mal or damaged proteins b y the UPP is essential for cells to withstand and reco ver from v arious environmental stresses (Dudek et al. 2005, Shang et al. 2005). However, the UPP itself is also a target of such stresses. All of the classes of ubiquitination enzymes (E1, E2, and some E3) and deubiquitinating enzymes (enzymes w hich release ubiquitin moieties from ubiquitin conjugates) have a c ysteine in their acti ve sites, and therefore the acti vities of these enzymes are subject to redo x regulation (Jahngen -Hodge et al. 1997, Obin et al. 1998). In addition, other types of modif cations, such as S -nitrosylation, can inactivate these enzymes (Yao et al. 2004). Reactive oxygen species and reacti ve lipid pero xidation products, such as 4 -hydroxynonenal Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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P R O T E I N S

Figure 13.1. The dynamics of ubiquitin conjugates in cells and tissues. In the process of ubiquitin-mediated protein degradation, ubiquitin is f rst conjugated to protein substrates via sequential reactions catalyzed by E1, E2, and E3. ATP is required for activation of ubiquitin in this process. The ubiquitin conjugates are either degraded by the 26S proteasome with the release of free ubiquitin and degraded products or deubiquitinated by a large family of deubiquitinating enzymes (also called isopeptidases). The steady state levels of endogenous ubiquitin conjugates are the net balance between the rate of ubiquitin conjugation and the rate of degradation and/or deubiquitination. The rate of ubiquitination depends on the availability of substrates; activity of ubiquitin -conjugating enzymes such as E1, E2s, and E3s; and ATP levels. The rate of turnover of ubiquitin conjugates in cells or tissues is determined by the activity of the 26S proteasome and deubiquitinating enzymes. Mild oxidative stress may increase levels of ubiquitin conjungates in cells by increasing substrate availability, enhancing activities of E1 and E2s, or by inhibiting proteasome activity. Therefore, the increase of ubiquitin conjugates is a sensitive marker of mild oxidative stress. However, severe oxidative stress may decrease the levels of ubiquitin conjugates by depleting cellular ATP or by inhibiting activities of E1, E2s; and E3s. Thus, a decline in levels of ubiquitin conjugates may also indicate severe oxidative stress.

(HNE), also impair the proteasome (Caballero et al. 2003, Conconi et al. 1998, Gr une et al. 2005, Ishii et al. 2005, Okada et al. 1999, Shringarpure et al. 2000). Formation of ubiquitin -protein conjugates is a prerequisite for de gradation by the UPP. The steady state levels of endogenous ubiquitin conjugates are the net balance betw een the rate of ubiquitin conjugation and the rate of de gradation and/or deubiquitination (F igure 13.1). The rate of ubiquitination depends on availability of substrates, the activity of ubiquitin conjugating enzymes, such as E1, E2s, and E3s, andATP levels. The rate of turnover of ubiquitin conjugates in cells or tissues is determined by the activity of the proteasome and deubiquitinating enzymes (also called isopeptidases). Whereas the proteasome degrades the ubiquitinated substrates with the release of free ubiquitin, deubiquitinating enzymes remo ve ubiquitin from the substrates and spare the substrates from de gradation. Although all of these enzymes could be inacti vated by oxidative stress, the susceptibilities of these enzymes to o xidative stress differ. For example, mild o xidative stress up -regulates the ubiquitin conjugating acti vity and promotes formation of ubiquitin conjugates (Shang et al. 1997b, Shang et al. 2001, Zhang et al. 2008). Because o xidized proteins are the prefer red substrates for ubiquitination (Dudek et al. 2005), mild o xidative stress w ould likely increase the substrate a vailability for ubiquitination (Shang et al. 1997b). In comparison, the proteasome is more susceptib le to o xidative stress than ubiquitin -conjugating enzymes. Physiologically relevant oxidative stress could inacti vate the proteasome in man y cell types (Zhang et al. 2008). In general, le vels of endo genous ubiquitin conjugates in the cells or tissues increase in response to mild o xidative stress or other cellular insults. Therefore, an increased le vel of endogenous ubiquitin conjugates is a useful mark er of ph ysiologically rele vant o xidative stress (Adamo et al. 1999, Dudek et al. 2005, F igueiredo-Pereira et al. 1997, Ramanathan et al. 1999, Scrofano et al. 1998a, Scrofano et al. 1998b, Shang et al. 2006, Shang et al. 1997a, Shang et al. 1997b, Shang et al. 2001, Shang and Taylor 1995). This chapter demonstrates that levels of ubiquitin conjugates increase in response to various oxidative stressors in dif ferent types of cells and tissues. The authors used H 2 O2 and photo oxidative stress to induce mild o xidative stress in cultured cells. To induce o xidative stress in vivo, the authors used paraquat, a potent supero xide radical generator, to generate supero xide.

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Neocarzinostatin, a dr ug that induces DN A doub le strand breaks and protein damage (Edo et al. 1991), was also used to stress l ymphoblastoid cells.

MA TERIALS AND METHODS N-ethyl-maleimide (NEM) w as obtained from Aldrich Chemical Co. (Mil waukee, WI, USA). 4 - (2 - Aminoeth yl) - benzenesulfon ylf uoride h ydrochloride (AEBSF) w as obtained from Calbiochem-Novabiochem Cor p. (La Jolla, CA, USA). o -Phosphoric acid w as obtained from Fisher Scientif c (Fair Lawn, NJ, USA), perchloric acid from JT Bak er Inc. (Phillipsbur g, NJ, USA), bathophenanthroline -disulfonic acid (BPDS) from Sigma Chemical Compan y (St. Louis, MO, USA), neocarzinostatin (NCS) from Ka yaku Co. (Tokyo, Japan), anti -rabbit-HRP antibody from Jackson ImmunoResearch (W est Gro ve, PA, USA), anti -DNP antibody from Dako Corp. (Carpenteria, CA, USA), Super Signal from Pierce (Rockford, IL, USA), 125 I(NaI) from PerkinElmer (Boston, MA, USA), and SDS -PAGE reagents from BIORAD (Hercules, CA, USA). All other chemicals w ere obtained from Sigma. All buffers and chemical reagents were of the highest purity a vailable. EXPOSURETO H2 O2 Retinal pigment epithelial cells, lens epithelial cells, and rat lenses w ere treated with either constant generated lo w le vels of H 2 O2 (10 to 50 µM) (the glucose/glucose o xidase system) (Shang et al. 2001) or a single bolus of the indicated le vel (see f gures 12.1 to 12.4) of H 2 O2 in serum-, pyruvate-, and phenol -red-free DMEM for the times indicated. EXPOSURETO PARAQUAT Twenty-three-month-old Emory mice were injected i.p. with a single dose of 20mg/kg paraquat dissolved in 0.9% saline 24 hours prior to sacrif ce. Non -stressed animals w ere injected with 0.9% saline. F ood was removed from the cages 12 hours before the animals w ere sacrif ced by CO 2 inhalation. Li vers were immediately excised and frozen in liquid nitro gen and stored at − 80 ° Cuntil processing. EXPOSURETO LIPOFUSCIN - MEDIA TED PHOTOOXIDATION Confuent ARPE19 cultures w ere fed isolated lipofuscin g ranules ( ∼300/cell) and maintained in basal medium for se ven days. Control cultures lacking lipofuscin w ere maintained under similar conditions. The medium was replaced with photosensitizer free medium and the cultures were either e xposed to b lue light (409 to 490 nm, 2.8 mW/cm2) from a sunlight source or maintained in the dark. Cells w ere lysed at zero, tw o, and four hours post e xposure. EXPOSURETO NCS Lymphoblastoid cells (L40) were grown in suspension cultures in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells at a concentration of 0.6 × 106 cells/mlwere treated with 80 ng/ml NCS for 0, 15, and 30 minutes and one, tw o, four, and eight hours. The cells were then collected and ubiquitin conjugates w ere detected by Western blotting. DETECTION OF ENDOGENOUS UBIQ UITIN CONJUGATES Levels of endogenous ubiquitin conjugates w ere determined by Western blotting as described previously (Shang and Taylor 1995). Brief y, cells w ere l ysed in situ with SDS - loading buffer and boiled immediately to inactivate any proteases or deubiquitinating enzymes. Tissues

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were homo genized in 50 mM Tris-HCl buf fer, pH 7.6, containing 2 mM AEBSF, 10 mM N-ethylmaleimide, 5 mM EDTA, 1% NP-40, and 0.1% SDS.After centrifugation at 13,000RPM for 10 minutes, the supernatant was mixed with an equal volume of 2 × SDS - gel loading buffer and boiled for three minutes. Proteins w ere resolved by SDS -PAGE on 10% to 12% gels and transferred to nitrocellulose. The blots were probed with antibody to ubiquitin. The specif cally bound antibody was detected by chemiluminescent detection using the Super Signal kit. DE NOVO UBIQUITIN CONJUGATION To determine the ability to for m ubiquitin conjugates, cells were lysed in 50 mM Tris-HCl, pH 7.6, containing 1 mM DTT . The conjugation acti vity w as deter mined using endo genous enzymes and substrates with e xogenous 125 I - ubiquitin.Brief y, the assa y was conducted in a f nal volume of 25 µl, containing (f nal concentrations) ∼ 10 mg/mlcell supernatant, 50 mMTris buffer, pH 7.6, 2 mM ATP, 1 mM DTT, 5 mM MgCl2 , and 4 µ M 125 I - labeledubiquitin. The mixture was incubated at 37 °C for 20 minutes, and then the reaction w as stopped by addition of 25 µ l 2 × SDS-PAGE loading buf fer. After boiling at 100 °C for three minutes, aliquots of the mixture w ere resolv ed b y SDS -PAGE. The de no vo - for med ubiquitin conjugates were visualized by autoradiography.

RESUL TS UBIQUITIN CONJUGATES IN RESPONSE TO OXIDATIVE STRESS A variety of oxidative stressors increase the le vels of endogenous ubiquitin conjugates in difxidants in biolo gy. ferent types of cells and tissues. H 2 O2 is one of the most common o Therefore, the authors f rst investigated the ef fects of H 2 O2 on le vels of ubiquitin conjugates in cultured cells and tissues. Cultured retinal pigment epithelial cells were exposed to different concentrations of constantly generated H 2 O2 for four hours and le vels of ubiquitin conjugates in the cells w ere determined. As shown in Figure 13.2A, the majority of ubiquitin conjugates in the cells w ere of high mass and the y mig rated on top of SDS -gels. Levels of endo genous ubiquitin conjugates increased ∼2.5-fold when the cells w ere exposed to an a verage of 10 µ M H2 O2 for four hours (F igure 13.2A). Exposure to 50 µ M H2 O2 increased the le vels of endogenous ubiquitin conjugates by ∼ three-fold. Consistent with results found in cultured RPE cells, exposure of intact lenses to H2 O2 also increased the levels of ubiquitin conjugates by ∼ two - fold (Figure 13.2 B). Other types of o xidative stressors also increase le vels of ubiquitin conjugates in cells or tissues. For example, treatment of mice with paraquat (a superoxide generator) induces marked enhancements of ubiquitin conjugates in the li ver (F igure 13.2C). Lipofuscin is a class of granular y ellow-brown lipid -containing w aste materials that are accumulated in aged cells and tissues. Accumulation of lipofusion triggers photo -oxidation by generating singlet oxygen upon light e xposure. In human RPE cells, e xposure of lipofuscin -loaded cells to shor t wavelength light (400 to 500 nm) markedly increased the le vels of ubiquitin conjugates in the cells (Figure 13.2D). In contrast, exposure to light alone had little ef fect on levels of ubiquitin conjugates (Figure 13.2D). Together, these data suggest that elevated levels of ubiquitin conjugates are a general response to a v ariety of oxidative stressors. The radiomimetic neocarzinostatin, an anticancer dr ug that induces DN A doub le strand breaks and protein damage (Edo et al. 1991), also increases le vels of ubiquitin conjugates in l ymphoblastoid cells. Le vels of ubiquitin conjugates in the cells increased as soon as 30 minutes after treatment with neocarzinostatin (Figure 13.2E). Levels of ubiquitin conjugates peaked two hours after e xposure to neocarzinostatin (F igure 13.2E). Subsequentl y, the cells recovered and le vels of ubiquitin conjugates g radually retur ned to the nor mal le vel (F igure 13.2 E).

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Figure 13.2. Ubiquitin conjugates accumulate in cells in response to various types of oxidative stressors. (A) Human retinal pigment epithelial cells (RPE) were treated with constantly generated 10 or 50 µM H2O2 for four hours. The cells were lysed and endogenous ubiquitin conjugates were determined by Western blotting using an anti -ubiquitin antiserum. (B) Lenses from three month-old rats were treated with a single bolus of 500 µM H 2O2 for 30 minutes. Proteins were extracted from the cortex of these lenses and levels of endogenous ubiquitin conjugates were determined as described in (A). (C) Twenty -three-month-old Emory mice were injected i.p. with 20 mg/kg paraquat dissolved in 0.9% saline 24 hours prior to sacrif ce. Endogenous ubiquitin conjugates in the soluble fraction were detected in 50 µg protein from each exposure group as described in (A). (D) Human RPE cells were preloaded with lipofuscin and then exposed to blue light for the time indicated. Levels of endogenous ubiquitin conjugates were detected as described in (A). (E) Lymphoblastoid cells (L-40) from a normal human at a concentration of 0.5 × 106 cells/ ml were treated with 80 ng/ml of NCS for zero to four hours. Equal amounts of soluble protein (50 µg) from each exposure group were separated by SDS -PAGE and transferred to nitrocellulose. Endogenous ubiquitin conjugates were detected ad described in (A).

OXIDATIVE STRESS INCREASES SUBSTRATE AVAILABILITY, ALTERS UBIQUITIN CONJUGATION CAPABILITY There are se veral possibilities to e xplain why ubiquitin conjugate le vels increase in response to oxidative stresses. These include (1) increased substrate a vailability, (2) increased ubiquitin conjugating acti vity, and (3) decreased proteasome acti vity and/or deubiquitinating acti vity. As shown in Figure 13.3, exposure of lens epithelial cells to mild (100 µ M) H2 O2 for only 30 minutes, follo wed b y a four -hour recovery, resulted in an ∼ eight -fold increase in ubiquitin conjugates (compare lane 3 vs. 1). But by 24 hours of recovery, levels of ubiquitinated proteins were indistinguishable from levels in pre -stressed samples (Shang et al. 1997b). Thus, the data indicate that oxidative stress is associated with a transient accumulation of additional substrates for conjugation. Oxidati ve stress w as also associated with for mation of protein carbon yls (Shang et al. 2001, #1254, Dudek et al. 2005, #2473). The formation and clearance of o xidatively modif ed proteins occurs in a time frame coincident with clearance of ubiquitin conjugates, indicating that increased le vels of ubiquitin conjugates in response to o xidative stress is partially due to increased substrate a vailability.

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Figure 13.3. Increased substrate availability for ubiquitination upon exposure to oxidative stress. Lens epithelial cells were exposed to a single bolus of 100 µM H 2O2 for 30 minutes and then were cultured in H 2O2-free medium to allow the cells to recover from oxidative damage. The activities of E1 and E2s were determined by a thiol ester assay. Equal amounts of protein (300 µg) were used in each assay. Lanes 1 to 3 without addition of purif ed E1, and lanes 4 to 6 with addition of 10 micro -units of purif ed E1. Lanes 1 and 4, control cells; lanes 2 and 5, cells treated with 100 µM H 2O2 for 30 minutes; lanes 3 and 6, cells treated with 100 µM H 2O2 for 30 minutes and then recovered for four hours.

In addition to increased le vels of substrates, mild o xidative stress also increases the acti vity of E1, the f rst and rate -limiting enzyme of the ubiquitin conjugating system. As sho wn in Figure 13.3 (compare lanes 3 vs. 1), reco very from o xidative stress is associated with an increase in levels of E1 ∼ Ub thiol esters, indicating that there is a h yperactivation of E1 upon recovery from mild o xidative stress. As with the o xidation-induced increase in ubiquitin conjugates, the h yperactivation of E1 is abated b y 24 hours reco very (not sho wn). Fur thermore, addition of purif ed E1 to the conjugation assay signif cantly increases the conjugating activity. These data indicate that increased ubiquitin conjugating acti vity ma y also contribute to the stress-induced increase in le vels of ubiquitin conjugates. THE PROTEASOME IS A TARGET OF MILD OXIDATIVE STRESS The proteasome is more susceptib le than ubiquitin -conjugating enzymes to sustained ph ysiologically relevant levels of oxidative stress. As shown in Figure 13.2A, physiologically relevant levels of sustained oxidative stress resulted in accumulation of ubiquitin conjugates in the cells. In order to search for the mechanism w hereby sustained o xidative stress increases the le vels of endo genous ubiquitin conjugates, the authors e valuated the ef fect of sustained o xidative stress on ubiquitin conjugating acti vity. As shown in Figure 13.4A (left panel), exposure to 10 µ MH2 O2 for four hours increased the ubiquitin conjugating activity (as indicated by de novo-formed ubiquitin conjugates) by 150%. Exposure to higher levels of H2 O2 (50 µM) increased the conjugating activity by 80%. Thiolester assays showed that the level of E1 ∼ ubiquitin thiolester did not change follo wing exposure to the sustained o xidative insult (F igure 13.4A, right panel), indicating that E1 acti vity was not affected b y these le vels of o xidative stress. Thiolester assays detected at least four dif ferent active E2s in RPE cells and the levels of all of these E2 ∼ ubiquitin thiolesters increased ∼ 60% upon exposure to 10 µ MH2 O2. When exposed to 50 µ MH2 O2 for four hours, the levels of these E2s∼ubiquitin thiolesters retur ned to the le vels found in control cells. These results indicate that low levels (non-toxic levels), but not high levels (toxic levels), of oxidative stress stimulate some E2s of the ubiquitin conjugating machiner y. To fur ther explore the causes of the ele vated levels of endo genous ubiquitin conjugates in response to o xidative stress, the authors e valuated the ef fect of o xidative stress on three peptidase activities of the proteasome. Exposure of the cells to an a verage of 10 µ MH2 O2 for four hours had no detectab le ef fect on an y peptidase acti vity of the proteasome, although these

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Figure 13.4. Sustained physiologically relevant oxidative stress inactivates the proteasome. Human RPE cells were exposed to constantly generated 10 or 50 µM H 2O2 for four hours. The cells were then lysed and the supernatant was used to determine ubiquitin conjugating activity, and E1 and E2 activities by thiolester assays (A).Proteasome activity was determined using fuorogenic peptides as substrates (B). The proteasome activity of the cells that were not treated with H 2O2 was arbitrarily def ned as 100% and the rest were expressed as relative activities normalized with controls.

levels of H 2 O2 resulted in a signif cant increase in le vels of endo genous ubiquitin conjugates and conjugating acti vity. Ho wever, treatment of the cells with 50 µ M H2 O2 for four hours resulted in ∼50% inhibition of ch ymotrypsin-like and tr ypsin-like activities. The peptidylglutamyl peptide hydrolase activity of the proteasome w as inhibited by ∼30% under these conditions (F igure 13.4B). Thus, the data indicate that w hereas the upre gulation of ubiquitin conjugating activity in response to lo w levels of o xidative stress ma y explain the o xidationinduced increase in le vels of endogenous ubiquitin conjugates, inacti vation of the proteasome by sustained oxidative stress is another cause of the ele vated levels of ubiquitin conjugates in response to higher le vels of oxidative stress.

CONCLUSION The UPP pla ys impor tant roles in a v ast number of cellular functions, including protein quality control and signal transduction (Shang and Taylor 2004). A functional UPP is required for the cells to cope with v arious stresses, including hea vy metals (Jungmann et al. 1993, Tsirigotis et al. 2001), amino acid analogs, and oxidation (Dudek et al. 2005, Shang et al. 2005). However, an extensive oxidative insult is also likely to damage or impair the function of critical components of the UPP (Caballero et al. 2003, Conconi et al. 1998, Ding and K eller 2001, Ishii et al. 2005, Jahngen -Hodge et al. 1997, Obin et al. 1998, Okada et al. 1999, Yao et al. 2004). The UPP is an impor tant protein quality control mechanism that selecti vely degrades various damaged proteins, including o xidized proteins. Formation of ubiquitin -protein conjugates is a prerequisite for ubiquitin -mediated degradation. Fluctuation in le vels of ubiquitin protein conjugates in cells ref ects the balance betw een the rate of for mation and the rates of degradation and deubiquitination. Mild oxidative stress increases the rate of ubiquitin conjugating b y increasing substrate a vailability and enhancing acti vities of ubiquitin -conjugating enzymes (Dudek et al. 2005, Marques et al. 2004, Shang et al. 1997b). Sustained physiologically relevant levels of oxidative stress could inactivate the proteasome and reduce the degradation of ubiquitinated substrates, contributing to the accumulation of ubiquitin conjugates in cells or tissues (Caballero et al. 2003, Cecarini et al. 2007, Conconi et al. 1998, Dasuri et al. 2008,; Ding and K eller 2001, Ishii et al. 2005, Okada et al. 1999, Yao et al. 2004, Zhang

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et al. 2008). Therefore, increased le vel of endo genous ubiquitin conjugates is a sensiti ve and general marker of mild o xidative stress. However, e xtensive o xidative stress ma y reduce the le vels of ubiquitin conjugates in the cells by inactivating ubiquitin conjugating enzymes (Jahngen -Hodge et al. 1997, Obin et al. 1998). Because the for mation of ubiquitin conjugates is ATP-dependent, depleting of ATP by severe oxidative stress also contributes to the decline in le vels of ubiquitin conjugates (Shang and Taylor 1995). Thus, whereas elevated levels of ubiquitin conjugates generally indicate mild oxidative stress, a dramatic decline in le vels of ubiquitin conjugates ma y also be a result of severe oxidative stress. When a decline is observed in cells or tissue, other markers of oxidative stress should be measured to ascer tain that the decline in ubiquitin conjugates is related to severe oxidative stress.

TECHNICALDETAILS AND CAUTIONS The tur nover of ubiquitin conjugates in cells and tissues is a rapid process due to an acti ve proteasome and deubiquitinating enzymes. To accuratel y deter mine the le vels of ubiquitin conjugates in cells or tissues, the samples should be processed quickl y. For cultured cells, an ideal method is to collect the cells with SDS -gel loading buffer and boil the samples immediately. Tissues should be f ash frozen immediately after harvest and stored at − 80 ° C until further processing. To preserve the ubiquitin conjugates in the tissue during processing, tissues should be homo genized in a buf fer that contains protease inhibitors. The authors found that 2 mM AEBSF together with 10 mM N -ethylmaleimide and 5 mM EDTA in the buf fer is suff cient to preserve ubiquitin conjugates in dif ferent tissues. Commerciall y a vailable protease inhibitor cocktails that contain both serine/threonine - and c ysteine-protease inhibitors are also suitab le for blocking the proteasome and deubiquitinating enzymes. Detergents are required to ef fectively extract ubiquitin conjugates from cells or tissues. A buffer that contains 1% NP -40 and 0.1% SDS can e xtract more than 90% of the conjugates in most tissues. Unlik e most other proteins, ubiquitin conjugates in SDS -loading buffer are not very stable. They disappear g radually at temperatures abo ve 4 °C. For long -term storage, the samples prepared in SDS -gel loading buffer should be k ept at − 80 ° C.

A CKNOWLEDGMENTS This work is supported partially by NIH grants EY11717 (to Fu Shang) and EY13250 (to Allen Taylor), and USDA CRIS 1950 - 51000 - 060 - 01A.

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Chapter14 Covalent Modif cations of Albumin Cys34 as a Biomar ker of Mild Oxidative Stress Giancar lo Aldini yung ,K - Jin eYum and ,

GiulioV istoli

INTR ODUCTION Human ser um albumin (HSA) is the most abundant protein in plasma, amounting to almost 60% of total circulating proteins. It is a globular protein, containing 35 Cys residues, with 34 of them in disulf de bonds. HSA therefore has only one free cysteine residue, Cys34, that makes up the largest pool of thiols in the circulation (80%). In health y adults, about 70% to 80% of the Cys34 in albumin contains a free sulfh ydryl group; the rest for ms a disulf de with compounds lik e cysteine, homocysteine, or glutathione, or is oxidized to irreversible forms such as sulf nic (− SO2H) and sulfonic acid (− SO3 H)derivatives. HSA plays various roles including the maintenance of osmotic pressure and transpor t of exogenous and endogenous substances such as fatty acids, bilirubin, and steroids (Peters 1995). Epidemiological studies ha ve estab lished an in verse relationship betw een ser um albumin concentration and mor tality risk. The various biological mechanisms proposed to e xplain the benef cial effects of higher albumin concentrations include an antioxidant effect (Gillum 2000). HSA is the main antio xidant of plasma and e xtracellular f uids, and this is mainl y due to the Cys34 residue, w hich sca venges o xidants such as h ydroxyl and pero xyl radicals, h ydrogen peroxide, and pero xynitrite (Carballal et al. 2003, Roche et al. 2008), and for ms co valent adducts with lipid - xoidation electrophilic by - products such as 4 - yhdroxy -trans - 2 - nonenal (HNE) (Aldini et al. 2006). Cys34’s strong radical scavenging and carbonyl quenching activity can be explained by: (1) its high acidity , resulting from the sur rounding amino acids ’ ability to stabilize the thiolate anion, and (2) the signif cant solv ent accessibility of the thiolate anion, as w e pre viously reported (Aldini et al. 2008b) (see below). The antioxidant role of HSA in vivo is conf rmed by repor ts of a signif cant reduction of mercaptoalbumin in dif ferent o xidative-based physiopathological conditions such as chronic renal failure (Matsuyama et al. 2009), coronary heart disease (Kadota et al. 1991), liver diseases (Oettl et al. 2008), ph ysical e xercise (Lamprecht et al. 2008), and aging (Era et al. 1995). Mercaptoalbumin depletion is associated with the formation of some oxidation products which have been identif ed and characterized in vitr o and in vivo . These include dimeric for m (Ogasawara et al. 2006), mixed disulf de with small -molecular-weight thiols such as c ysteine Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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and homoc ysteine (Sengupta et al. 2001), ir reversible sulf nic and sulfonic acid deri vatives (Carballal et al. 2003), and the co valent Michael adduct with HNE (Aldini et al. 2006, Aldini et al. 2008b). Thus, mercaptoalbumin depletion and detection of the cor responding reaction products serve as valuable markers of systemic o xidative stress. This chapter describes albumin Cys34 and its o xidized for ms as potential biomark ers of oxidative damage. Molecular mechanisms of mercaptoalbumin as an e xtracellular antioxidant are discussed, as are the main o xidation products. Recent analytical approaches for measuring mercaptoalbumin and its reaction products are reported, along with their applications in several physiopathological conditions.

CYS34 REACTIVITY: A MOLECULAR INSIGHT From a structural point of view, Cys34 is located betw een the helices Ia -h2 and Ia -h3, as conf rmed by X -ray studies (Sugio et al. 1999). Although Cys34 is in a f airly superf cial region, the resolved str ucture clearly shows that its sulfh ydryl function is inser ted in a deep cre vice and is completel y buried b y the close side -chains of Pro35, His39, Val77, and Tyr84. This arrangement ma y pre vent the sulfh ydryl g roup from reacting with e xternal counter parts. In light of these str uctural data, Cys34 ’s known reactivity implies that the domain Ia under goes a conformational shift, probably due to isomerization of Pro35, w hich renders the Cys34 sulfhydryl function accessible, enabling it to react with other molecules. Apart from the accessibility , potentiometric studies and kinetic anal yses (Christodoulou 1994, Le wis 1980) all ag ree that the Cys34 sulfh ydryl g roup is much more acid than free cysteine, with a pK 3 between 6.5 and 7.0. This signif cant acidity can be e xplained in terms of the sur rounding residues w hich increase the polarity of the thiol g roup g rabbing the proton (e.g., Asp38, His39, and Tyr84), w hile also stabilizing the subsequent thiolate anion (e.g., His39, Lys41, and Tyr84). Recent modeling studies (Aldini et al. 2007b) described a possib le mechanism for Cys34 ’s acidity: in a f rst step the thiol proton is g rabbed by His39 or, more probably, by Tyr84, whose phenate anion is stabilized by the nearby Lys41. In the second step, Cys34 thiolate is stabilized by ion pairing with L ys41 and π - sulphurinteraction with His39. There is an intriguing similarity betw een the architecture of k ey residues around Cys34 in HSA and the active site of glutathione S -transferase M1a -1a (Patskovsky 2006). In both cases the reactive thiol group’s acidity is enhanced by the closeness of one tyrosine and one histidine residue, being ab le to accept the thiol proton and stabilize the thiolate anion. Again, both structures ha ve a positi vely char ged residue near the tyrosine (a l ysine in albumin and an arginine in glutathione S-transferase) that boosts the polarity of the phenol group and stabilizes the thiolate anion. This analogy suggests that the alb umin can act through an auto -enzymatic activity to maximize the reacti vity of the Cys34 thiol g roup toward reactive carbonyl species (RCS), and implies that at ph ysiological pH there is an equilibrium betw een neutral thiol and anionic thiolate in w hich both for ms are markedly populated. Mutagenesis studies (Stewart 2005 have ) conf rmed the k ey roles of both Tyr84 and His39 in Cys34 ’s abnor mally low pK 3. Tyr84 plays a triple role: it enhances the nucleophilicity of the sulphur of Cys34, contributes to its high redo x potential, and simultaneously inf uences its accessibility. Molecular dynamics simulations ha ve shown that the accessibility is also inf uenced by Cys34 ’s ionization state because the thiolate promotes its e xposure, as evidenced by comparing the average surface of the sulphur atom in the molecular dynamics (MD) simulation of thiolate with that of the neutral thiol function.

CYS34 ACTS AS AN ANTIOXIDANT HSA has signif cant antioxidant activity, as recently reviewed by Roche et al. (2008). Albumin is the principle circulating antio xidant in plasma, w hich is e xposed to continuous o xidative

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stress. Albumin’s antio xidant acti vity is mainl y related to its ability to bind metal ions and scavenge free radicals. Direct e vidence of this acti vity is pro vided by HSA’s signif cant contribution in assa ys developed to measure the ser um/plasma antioxidant activity. For instance, the antioxidant contribution of HSA accounts for almost 30% in assa ys measuring the antioxidant activity in aqueous compar tments (TRAP, ORAC) as reviewed by Yeum et al. (2004). Strong, though indirect, e vidence of albumin ’s antioxidant activity in vivo is found in the increased o xidative status and ser um o xidizability in patients with lo w le vels of albumin (Delimaris et al. 2008) and the benef ts of albumin administered to septic patients. This is, in part, attributed to an increase in antio xidant activity, related to thiol repletion (Quinlan et al. 2004). Besides its plasma concentration, str uctural modif cations of HSA induced b y glucose or free radicals impair the antio xidant defenses mediated b y albumin, leading to w orse oxidative damage (Faure et al. 2008a). Albumin’s antioxidant activity is primaril y explained by the sca venging ability of reduced Cys34. Using an in vitro model based on copper -mediated o xidation of human lo w-density lipoproteins (LDL), Bourdon (Bourdon et al. 2005) proposed that Cys34 mainl y works as a free radical sca venger w hile Met residues mainl y act as metal chelators. Cys34 sca venges several reactive oxygen, carbon, and nitro gen species. For instance, it is o xidized by the twoelectron oxidants hydrogen peroxide and pero xynitrite and is also the tar get for one -electron oxidants such as the hydroxyl radical, the carbonate anion radical, and nitrogen dioxide (Turell et al. 2009a ).

CYS34 AS A DETOXIFYING AGENT FOR REACTIVE CARBONYL SPECIES Besides acting as a direct antio xidant, albumin Cys34 is also in volved in the deto xif cation of lipid pero xidation b y products such as α β , - unsaturatedaldehydes, forming carbonylated adducts. Carbonylated albumin has been identif ed in oxidative stress conditions such as kidney disease and diabetes, and is an estab lished biomarker of oxidative stress (Aldini et al. 2007a). Several studies ha ve look ed into the carbon ylation mechanism, w hich mainl y in volves the covalent adduction of electrophilic RCS to dif ferent accessible and nucleophilic sites of HSA. The authors reported the adduction of Cys34 with RCS (Aldini et al. 2006, Aldini et al. 2008b). When HNE w as spiked in human plasma, it rapidl y disappeared (within 40 seconds) and no phase I metabolites were detected, suggesting that its main fate was due to an adduction mechanism. Proteomics identif ed albumin (HSA) as the main protein tar get, fur ther conf rmed by the signif cantly reduced HNE quenching of de -albuminated plasma (Aldini et al. 2008b). LC - ESI - MS/MS analysis identif ed Cys34 and L ys199 as the most reacti ve adduction sites of HSA, through the for mation of Michael and Schif f base adducts, respecti vely. The rate constant of HNE trapping by albumin was 50.61 ± 1.89 M−1 s−1, and that of Cys34 (29.37M−1 s−1 ) was one order of magnitude higher than glutathione (GSH)(3.81 ± 0.17 M−1 s−1), as e xplained by the molecular modeling studies reported above. Cys34 carbonylation was further conf rmed in biological samples by studying the covalent modif cations of Cys34 after human plasma was incubated with mildl y o xidized LDL, and using a precursor -ion scanning approach (Aldini et al. 2008a). Two main peaks were identif ed as Cys34 -containing peptides adducted by HNE and acrolein (ACR), conf rming that these are the main aldehydes released from oxidized LDL, and that Cys34 is the tar get site of electrophilic aldeh ydes.

CYS34OXIDIZED FORMS Judging from its reactivity and accessibility, Cys34 is a reactive antioxidant as well as a detoxifying residue of electrophilic species. Several oxidation products of Cys34 have been characterized in vitro and in vivo by different analytical techniques. The main oxidation product of HSA is a sulfenic acid derivative, formed by the reaction of Cys34 with radical and oxidizing species

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Figure 14.1. Main oxidative covalent modif cations of HSA Cys34.

such as hydroxyl radicals, peroxynitrite, and hydrogen peroxide (Turell et al. 2009a). Detection of sulfenic acid is complicated b y its reacti vity because it can be fur ther oxidized to sulf nic and sulfonic acids or can react with aminothiols, for ming mix ed disulf des (F igure 14.1). Carballal et al. were the f rst to demonstrate the formation of a sulfenic acid derivative of HSA treated with hydrogen peroxide, by using mass spectrometry (MS) and dimedone as a derivatizing agent (Carballal et al. 2003). The HSA sulfenic acid deri vative w as stab le in phosphate buf fer at 4 °C, with more than 95% remaining after one hour , but g radually disappeared at 25 °C and 37 °C. Further analysis showed that at 37 °C the f rst-order rate constant of spontaneous decay was 1.7 ± 0.3× 10−3 s−1 . Albumin sulfenic acid undergoes several reactions: it can be further oxidized to sulf nic (SO2 H) and sulfonic acid (SO 3H) or it can rapidl y react with plasma aminothiols such as c ysteine (21.6 ± 0.2 M−1 s−1), glutathione (2.9 ± 0.5 M−1 s−1), homocysteine (9.3 ± 0.9 M−1 s−1), and cysteinglycine (55 ± 3 M−1 s−1) to for m the cor responding mixed disulf des (Turell et al. 2008). Another class of modif ed Cys34 for ms that could potentiall y arise in vivo are the Michael covalent adducts of Cys34 with electrophilic RCS. Ho wever, although it is w ell known that albumin undergoes carbonylation damage in se veral oxidative-based pathological conditions, so f ar there is no direct e vidence of Cys34 carbon ylation in ex vivo samples. Onl y in vitro experiments have been repor ted, and these ha ve shown that Cys34 is a reacti ve target of car bonylation and rapidl y reacts with se veral RCS, including α ,β - unsaturatedaldehydes (Aldini et al. 2008b). As described belo w, more sensiti ve MS strate gies are needed to f nally demonstrate Cys34 carbonylation in different physio-pathological conditions.

AN ALYTICAL STRATEGIES TO MEASURE MERCAPTOALBUMIN AND CYS34 OXIDATIVE MODIFICATIONS Spectrophotometric, chromatographic, electrophoretic and MS methods ha ve been described for measuring mercaptoalbumin and the Cys34 o xidized forms, as summarily described below (Table 14.1 ). ALB UMIN SHTITRATION Mercaptoalbumin is usuall y anal yzed b y spectrophotometr y, using Ellman ’s reagent (5,5′-dithiobis(2-nitrobenzoic acid) and albumin isolated from human plasma b y aff nity chromatography. In man y cases, HSA is not isolated and the assa y is done directl y on plasma.

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Table 14.1. Analytical strategies to measure mercaptoalbumin and Cys34 oxidative modif cations

Assa y HSASH titration

HSAdisulf de Re versibly oxidized non - mercaptoalbumin Strongl y oxidized non - mercaptoalbumin Co valent adducts of Cys34

Method Spectrophotometry (Ellman ’s reagent) One - dimensional polyacrylamide gel (maleimide - PEO2 - biotin as derivatizing agent) HPLC(anion exchange column) SDS gel electrophoresis HPLC(anion - xechange hydrophobic column) Direct infusion MS HPLC(anion - xechange hydrophobic column) LC - ESI - MS/MS LC - ESI - MS/MS

arget T Mercaptoalbumin

MonomericHSA and disulf de relative contents Disulfdes with small molecular weight such as Cys and homoCys Sulfnic and sulfonic acid derivatives RCSadducts to Cys34

Because Cys34 for ms the largest pool of reduced thiols in plasma, this ma y provide a reliable indicator of mercaptoalbumin content. F ree sulfhydryl (SH) g roup titration of HSA can also be done using maleimide -PEO2-biotin as a deri vatizing agent. After reaction with this agent, the sample is separated in one -dimensional pol yacrylamide gel according to Laemmli, and without mercaptoethanol. Biotin is then revealed with streptavidin conjugated with horseradish peroxidase. A calibration cur ve is plotted using kno wn concentrations of albumin and a mean error of less than 5% has been repor ted (Musante et al. 2006). ALB UMIN DISULFIDE Monomeric albumin and albumin disulf de relative contents were determined by HPLC using an anion e xchange column, an elution g radient, and a f uorescent detector (Greilber ger et al. 2008). Albumin dimer can be deter mined b y SDS gel electrophoresis on 10% (w/v) gel in non-reducing conditions and using Coomassie brilliant b lue as the staining agent (Ogasa wara et al. 2006 ). CHR OMATOGRAPHIC ANALYSIS OF ALBUMIN REDOX VARIANTS Albumin is hetero geneous, par ticularly as re gards the redo x status of the Cys34 thiol. The predominant for m is mercaptoalbumin (HMA, about 70% of total albumin), follo wed by the reversibly oxidized non -mercaptoalbumin (disulf des with small - molecular- w eight thiols such as Cys and homoc ysteine, HNA1), accounting for about 20%, and b y a small amount (about 1%) of strongly oxidized non-mercaptoalbumin (sulf nic and sulfonic acid deri vatives, HNA2) (Turell et al. 2009b). This heterogeneity can be determined by HPLC using an anion -exchange hydrophobic column to separate albumin into the three fractions (HMA, HN A1, HNA2) (Era et al. 1988, Sogami et al. 1984). Fluorescence and UV detectors are normally used to follow the elution of the HSA fractions, and chromatographic analysis usually takes at least 30 minutes. Separation probab ly depends

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on conformational changes related to the thiol redox status. Results for each fraction are usually reported as a percentage of total albumin. This assay gi ves onl y a general indication of the oxidative state of Cys34, because it separates tw o mixtures of o xidized species, the re versible and irreversible oxidized forms. It gives no str uctural information about the o xidized forms. DIRECT INFUSION MASS SPECTR OMETRY OF INTACT ALBUMIN Direct infusion MS in volves directl y infusing a solution of isolated albumin dissolv ed in a denaturing solvent, such as a mixture of water, acetonitrile, and formic acid, into the ESI source coupled to the MS anal yzer. The resulting MS spectr um shows several multicharged ions, and the molecular weights (MW) of albumin and derivatives are then determined by deconvolution analysis. The deconvoluted MS spectrum of albumin from healthy subjects usually gives three distinct peaks at 66,446, 66,566 and 66,610 Da, attributed to mercaptoalbumin and to the cor responding adducts with Cys and glucose in a ratio of appro ximately 50 : 25 : 25 (Beck et al. 2004 )(Figure. 14.2 ). This MS approach was used to check the purity of albumin for clinical use. Bar -Or (Bar-Or et al. 2005a) identif ed se veral post -translational modif cations of six commercial albumin preparations for clinical use, using positi ve electrospra y ionization and time -of-f ight mass spectrometry (ESI-TOF). All human serum samples obtained from uremic patients had a higher content of bound Cys in position Cys34 than those of health y controls (57.2% vs. 22.9%, p < 0.02). In addition, there w as a high content of nitrosylated albumin. Similar results w ere obtained using an ESI source coupled to an ion trap mass spectrometer (Kleino va et al. 2005).

Figure 14.2. Direct infusion mass spectrometry of intact albumin. Direct infusion MS involves directly infusing a solution of isolated albumin dissolved in a denaturing solvent into the ESI source coupled to the MS analyzer. The resulting MS spectrum shows several multicharged ions, and the molecular weights (MW) of albumin and derivatives are then determined by deconvolution analysis.

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Direct infusion MS of isolated HSA was also used to demonstrate markedly higher maternal plasma le vels of c ysteinylated albumin in pre gnancies with intrauterine g rowth restriction compared to nor mal pregnancies (44.7% vs. 20.9%) (Bar -Or et al. 2005b). The authors recently used ESI -MS direct infusion to follow the covalent changes of HSA in non-diabetic end-stage renal disease patients before and after hemodialysis (HD). Cysteinylated HSA was signif cantly higher than in age -matched subjects and HD induced almost complete recovery of mercaptoalbumin through massive reduction of the cysteinylated form (manuscript in preparation). Direct infusion MS is a useful technique for studying the co valent adduction of Cys34 in vitr o. The authors used this approach to demonstrate that Cys34 is a reacti ve nucleophilic center that can for m covalent adducts with several electrophilic species including HNE (Aldini et al. 2007b). Direct infusion MS to detect co valent changes of HSA of fers several advantages but also suffers some dra wbacks. Among the for mer, sample preparation is rapid and onl y requires a simple purif cation step w hich usually consists of HSA isolation b y blue gel aff nity chromatography followed by desalting. The MS anal ysis is also speedy because it does not require any chromatography and usuall y the spectr um is recorded within one minute of sample infusion. Among the limits, HSA and its derivatives cannot be quantif ed in absolute terms and the results for the HSA adducts are gi ven as percentages. Another limit is that onl y HSA adducts reaching at least 5% to 10% (depending on the resolution of the MS analyzer) can be detected. This means direct infusion MS can be used for detecting the most abundant covalent modif cations of HSA, such as the c ysteinylated and glycated forms, but not for minor modif cations. LC - ESI - MS/MS OF ENZYME - DIGESTEDALBUMIN The LC -ESI-MS/MS approach in volves separating the mixture of albumin peptides obtained by enzymatic digestion (usuall y using tr ypsin as the proteol ytic enzyme) by HPLC connected through an ESI interf ace to the MS detector operating in the data -dependent scan mode. MS/ MS analysis of the eluted peptides means one can retrie ve the y and b fragments (arising from the amide bond cleavage), and on the basis of these v alues the site and type of modif cation is unequivocally re vealed. This approach detected se veral Cys34 modif cations in vitr o and ex vivo . Using dimedone as the deri vatizing agent, it w as unequi vocally demonstrated that Cys34 sulfenic acid for med when HSA was treated with an o xidizing agent such as h ydrogen peroxide or peroxynitrite (Carballal et al. 2003). LC-ESI-MS/MS was also used to demonstrate that Cys34 is the preferential nucleophilic tar get site of electrophilic carbon yl species such as HNE (Aldini et al. 2006). In ex vivo studies, LC -ESI-TOFMS anal ysis showed that the disulf de - bondedcysteine at the thiol Cys34 of reduced HSA w as the major str uctural change in o xidized HSA in health y human plasma (Kawakami et al. 2006). Musante et al. used LC -ESI-MS/MS to map the covalent modif cations of HSA in patients with nephritic syndrome (Musante et al. 2006, Musante et al. 2007). They showed that plasma albumin in patients with acti ve focal se gmental glomerulosclerosis undergoes massive and stab le oxidation with chemical o xidation of the sole free SH of Cys34 to a sulfonic g roup. This involves some impor tant changes to the protein such as the net char ge as a result of additional ne gative residues, and loss of free SH titration. The authors are no w working on dif ferent sensitive and specif c MS approaches to detect unknown covalent modif cations of Cys34. The authors ha ve described an MS strate gy based on the precursor -ion scanning technique and using a triple quadr upole as the MS anal yzer (Aldini et al. 2008a). The method specif cally detects unkno wn modif ed peptides containing the Cys34 residue and was originally evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4 -hydroxy-hexenal, HNE, and ACR. Then it was employed to study the Cys34 modif cation of human plasma incubated with mildl y oxidized LDL, and the Cys34 adducts with HNE and ACR were easily identif ed.

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In other experiments, plasma was oxidized by 2,2 ′ - azobis(2 - amidinopropane) HCl (AAPH) or by Fe2+ /H2 O2. In both conditions, the sulf nic derivative of Cys34 w as identif ed and char acterized, indicating that the method is suitab le not only for studying RCS -modif ed albumin, but also to check the o xidative state of Cys34 as a mark er of o xidative damage. The main advantage of this procedure is that it can detect unkno wn Cys34 adducts. A second MS approach that the authors are no w working on is based on nanoLC coupled to the LTQ Orbitrap XL ™ hybrid FTMS (F ourier transfor m mass spectrometer) as the mass analyzer (F igure 14.3). This h ybrid MS system is capab le of high mass resolution (up to 100,000 at m/z 400) and mass measurement accuracies of less than 2 ppm for complex peptide mixtures (P erry et al. 2008). Cys34 co valent adducts and o xidized for ms are searched in a database of predicted v ariable modif cations on Cys residues such as sulfenic, sulf nic, and sulfonic derivatives, α ,β - unsaturatedMichael adducts, S - nitrosylation,and S - (carbo xymethyl) cysteine through the reaction of alpha -dicarbonyl compounds with the thiol g roup. The limit of this approach is that it can only identify predicted changes. However, it offers certain advantages. In par ticular, it is f ast (a database search b y a 6 multi cpu system tak e less than 10 minutes), accurate, and v ery sensiti ve thanks to the high perfor mance of the Orbitrap mass analyzer. The authors recentl y employed this approach to rapidl y identify the co valent modif cations of Cys34 in albumin e xposed to a w hole-phase cigarette smoke extract. The covalent Michael adducts of Cys34 with ACR and crotonaldeh yde were identif ed, as w ell as the sulf nic and

Figure 14.3. LC-ESI-MS approach for the identif cation of unknown (precursor -ion scanning analysis) and predicted (data -dependent scan mode analysis) modif cations of Cys34.

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sulfonic acid deri vatives (Colombo et al. 2010). The authors are no w appl ying these two MS approaches to identify the Cys34 modif cations in dif ferent ph ysio-pathological conditions in volving o xidative stress such as aging, diabetes, metabolic syndrome, and nephropathy.

MERCAPT OALBUMIN AND THE REVERSIBLE AND IRREVERSIBLE OXIDIZED FORMS AS MARKERS OF OXIDATIVE DAMAGE Mercaptoalbumin depletion, accompanied b y signif cant increases in the re versible and ir reversible oxidized forms, has been observed in several oxidative-based diseases. Mercaptoalbumin depletion is an estab lished f nding in hemodial ysis patients and is enhanced in relation with the level of renal dysfunction among patients with chronic renal f ailure (Terawaki et al. 2004). A close cor relation w as found betw een HN A2, ser um protein carbon ylation, and de gree of renal dysfunction in predial ysis chronic kidne y disease patients, suggesting that ir reversible Cys34 o xidation of albumin is in volved in ser um protein carbon ylation (Matsuyama et al. 2009 ). Some of the o xidized Cys34 for ms are re versed after dial ysis. Soejima et al. repor ted that the HMA fraction of HSA w as signif cantly lower in maintenance HD patients than in age matched healthy subjects and that it increased three to f ve hours after star ting HD (Soejima et al. 2004). Using a direct infusion MS approach, the authors e xtended these results, f nding that the reduction of mercaptoalbumin in HD patients is due to for mation of the c ysteinylated derivative, a reversible oxidized form which is signif cantly reduced with concomitant for mation of mercaptoalbumin after HD (manuscript in preparation). Mercaptoalbumin depletion has been obser ved in other o xidative-based diseases including diabetes (Suzuki et al. 1992), diabetic retinopathy (Kawai et al. 2001), and liver diseases (Oettl et al. 2008). Faure et al. repor ted that obstr uctive sleep apnea (OSA) syndrome patients had signif cantly fewer albumin thiol groups than those of healthy volunteers and this was correlated to the se verity of OSA e xpressed by the noctur nal oxygen desaturation (F aure et al. 2008a). Greilberger reported an increase in albumin disulf de in neurode generative patients with mild cognitive impair ment and Alzheimer’s disease compared to an age -matched control g roup (Greilberger et al. 2008). Mercaptoalbumin depletion, accompanied by increases in reversible and/or irreversible HSA modif cations, is signif cantly reduced not only in established oxidative stress associated pathological conditions, such as uremia and diabetes, but also in ph ysiological states in which there is mild o xidative stress, such as aging (Era et al. 1995) and ph ysical e xercise (Lamprecht et al. 2008). In healthy elderly people, the fraction of mercaptoalbumin was signif cantly lower than in health y young males, indicating that in the elderl y HSA becomes more o xidized than in younger subjects (Era et al. 1995). Conf rming this, a signif cant age - dependentreduction in the plasma protein sulfh ydryl g roups, and age -dependent increases in c ysteinylated and homocysteinylated proteins were reported in healthy humans (Giustarini et al.2006). Lamprecht et al. described the ef fect of single bouts of e xercise at three dif ferent intensities (70%, 75%, and 80% of maximal o xygen consumption, VO2) on the redo x state of HSA and on protein carbonyls (Lamprecht et al. 2008). Mercaptoalbumin decreased at all three intensities and w as primarily oxidized to mixed disulf de derivatives. It should be noted that in many cases the Cys34 oxidative state was not correlated with other accepted oxidative stress parameters, such as isoprostanes and carbon yls, which remained the same as controls, indicating that mercaptoalbumin and the o xidized for ms are biomark ers of mild oxidative stress. For example, OSA patients had signif cantly fewer albumin thiol g roups than healthy v olunteers, w hereas the onl y difference for urinar y prostaglandins w as a slight positive tendency.

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In Lamprecht ’s study , o xidative protein damage, measured b y protein carbon ylation, increased only at the highest exercise intensity (80% VO2) and the antioxidant activity of superoxide dismutase and glutathione peroxidase in erythrocytes hardly changed. The study’s authors concluded that the HSA redox system in plasma acts at the beginning of the radical scavenging chain, and protein carbonyls indicate the extent of overcharge of the plasma antioxidant systems (Lamprecht et al. 2008 ).

MERCAPT OALBUMIN AS A MARKER OF ANTIOXIDANT STATUS Besides being a promising biomarker of oxidative stress, mercaptoalbumin has also been used to e valuate the in vivo acti vity of antio xidants. Anraku et al. recentl y repor ted the ef fect of water-soluble chitosan, a water polymer derived from chitin, on plasma redo x status. Chitosan lowered the ratio of o xidized to reduced albumin and boosted plasma antio xidant acti vity (TAP). A signif cant cor relation w as found betw een TAP and the o xidized albumin ratio (Anraku et al. 2009). Faure et al. repor ted that albumin thiols of health y subjects w ere much higher than in patients with type 2 diabetes mellitus and that metfor min but not sulfon ylureas signif cantly boosted the albumin thiol g roups (Faure et al. 2008b).

CONCLUSIONS Evidence is still accumulating that mercaptoalbumin and the re versible and ir reversible oxidized for ms are reliab le and sensiti ve mark ers of mild o xidative stress. Ho wever, se veral questions still remain, particularly in regard to the in vivo stability of the Cys34 oxidized forms of albumin. The half-life of unmodif ed albumin is about 19 days and it is broken down mostly in muscle, skin, li ver, and kidne y. Modif ed albumin under goes a most eff cient catabolism. Chemically modif ed albumin (such as male ylated and for mylated), denatured albumin gener ated under o xidative stress conditions, or albumin altered in specif c amino acid residues is preferentially recognized over native protein and tak en up from circulating b lood by several receptors which are widely distributed among the body organs, resulting in rapid internalization and degradation (Turell et al. 2009a). Information is still lacking on the in vivo metabolic stability of Cys34 oxidized albumin and on the kinetics of mercaptoalbumin re generation. In addition, it is not clear w hether these processes depend on genetic prof les. This needs to be clarif ed to use Cys34 oxidized albumin as a biomark er of o xidative stress or to search for proteol ytic peptides containing the Cys34 residue in biological f uids. Most studies to date repor ting an increase in the o xidized form of albumin ha ve used chromatography to measure albumin redox variants. Only a few studies have used MS. The authors believe the two approaches can be complementar y: HPLC anal ysis gives a general indication of the albumin redox state and MS can identify and characterize the o xidized forms, providing valuable information not only for measuring oxidative stress but also to help clarify the mechanism of the o xidative damage. More MS studies are needed to map the main o xidized form(s) of Cys34 in dif ferent physiopathological conditions. This can be done using state -of-the-art high -resolution MS equipment such as the Orbitrap. The results will ser ve to identify specif c co valently adducted albumin derivatives in order to set up quantitati ve assays using deuterated standards.

A CKNOWLEDGMENTS This work was supported by funds from the Uni versity of Milan (PUR 2007, 2008) and from MIUR (PRIN 2007) and b y the BioGreen 21 Pro gram (Code #20070301034009), Rural Development Administration, Korea.

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GiustariniD , Dalle - DonneI , Lorenzini S , MilzaniA , Rossi R. 2006 .Age - relatedinf uence on thiol, disulf de, and protein -mixed disulf de levels in human plasma . J Gerontol A Biol Sci Med Sci 61 : 1030 – 8 . Greilber ger J , K oidl C , Greilber ger M , Lamprecht M , Schroecksnadel K et , al. 2008 . Malondialdehyde, carbonyl proteins and albumin -disulphide as useful o xidative markers in mild cognitive impairment and Alzheimer’s disease . Free Radic Res 42 : 633 – 8 . KadotaK ,Y ui Y , Hattori R , MuroharaY , Ka wai C .1991 . Decreased sulfhydryl groups of serum albumin in coronar y artery disease . Jpn Circ J 55 : 937 – 41 . Ka wai K ,Y oh M , Ha yashi T , Imai H , Ne gawa T ,et al. 2001 . Effect of diabetic retinopathy on redox state of aqueous humor and ser um albumin in patients with senile cataract . Tokai J Exp Clin Med 26 : 93 – 9 . Ka wakami A , K ubota K ,Y amada N , T agami U , T akehana K et , al. 2006 . Identifcation and characterization of oxidized human ser um albumin. A slight str uctural change impairs its ligand-binding and antioxidant functions . Febs J 273 : 3346 – 57 . Kleino va M , Belgacem O , P ock K , RizziA , BuchacherA ,Allmaier G .2005 . Characterization of cysteinylation of phar maceutical-grade human ser um albumin by electrospray ionization mass spectrometry and low-energy collision -induced dissociation tandem mass spectrometr y. Rapid Commun Mass Spectrom 19 : 2965 – 73 . LamprechtM , Greilber ger JF , Schw aberger G , Hofmann P , Oettl K .2008 . Single bouts of exercise affect albumin redox state and carbon yl groups on plasma protein of trained men in a workload - dependentmanner . J Appl Physiol 104 : 1611 – 7 . Le wis SD , Misra DC , Shafer JA . 1980 . Determination of interactive thiol ionisations in bovine serum albumin, glutathione and other thiols b y potentiometric difference titration , Biochemistry 19 , 6129 – 6137 . Matsuyama Y, T erawaki H ,T erada T , Era S .2009 .Albumin thiol oxidation and serum protein carbonyl formation are progressively enhanced with adv ancing stages of chronic kidne y disease. Clin Exp Nephrol . MusanteL , Bruschi M , Candiano G , P etretto A , Dimasi N ,et al. 2006 . Characterization of oxidation end product of plasma albumin ‘in vivo .’ Biochem Biophys Res Commun 349 : 668 – 73 . MusanteL , Candiano G , P etretto A , Bruschi M , Dimasi N ,et al. 2007 .Active focal segmental glomerulosclerosis is associated with massi ve oxidation of plasma albumin . J Am Soc Nephrol 18 : 799 – 810 . OettlK , StadlbauerV , P etter F , Greilber ger J , Putz - BankutiC et , al. 2008 . Oxidative damage of albumin in advanced liver disease . Biochim Biophys Acta 1782 : 469 – 73 . Ogasa wara Y , NamaiT , T ogawa T , Ishii K .2006 . Formation of albumin dimers induced by exposure to peroxides in human plasma: a possib le biomarker for oxidative stress . Biochem Biophys Res Commun 340 : 353 – 8 . atskovsky P Y, P atskovska L ,Almo SC , Listo wsky I .2006 .Transition state model and mechanism of nucleophilic aromatic substitution reactions catal yzed by human glutathione S -transferase M1a - 1a Biochemistry. . 45 : 3852 – 62 . erry P RH , Cooks RG , Noll RJ . 2008 . Orbitrap mass spectrometry: instrumentation, ion motion and applications . Mass Spectrom Rev 27 : 661 – 99 . eters P T. 1995 All . about Albumin. Biochemistry, Genetics, and Medical Applications. San Diego: Academic Press, ISBN 0 - 12 - 552110 - 3. QuinlanGJ , Mumb y S , Martin GS , Bernard GR , Gutteridge JM , Ev ans TW . 2004 .Albumin inf uences total plasma antio xidant capacity f avorably in patients with acute lung injur y. Crit Care Med 32 : 755 – 9 . RocheM , Rondeau P , Singh NR ,T arnus E , Bourdon E .2008 .The antioxidant properties of serum albumin . FEBS Lett 582 : 1783 – 7 . SenguptaS , Chen H ,T ogawa T , DiBello PM , MajorsAK et , al. 2001 .Albumin thiolate anion is an inter mediate in the for mation of albumin -S-S-homocysteine. J Biol Chem 276 : 30111 – 7 .

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Soejima A , Matsuza wa N , Ha yashi T , Kimura R , OotsukaT ,et al. 2004 .Alteration of redox state of human ser um albumin before and after hemodial ysis. Blood Purif 22 : 525 – 9 . So gami M , Nagoka S , Era S , Honda M , No guchi K .1984 . Resolution of human mercapt - and non - mercaptalbuminby high - performance liquid chromatography . Int J Pept Protein Res 24 : 96 – 103 . Ste wart AJ , Blindauer CA , Berezenk o S , Sleep D , T ooth D , Sadler PJ . 2005 . Role of Tyr84 in controlling the reactivity of Cys34 of human albumin , FEBS J. 272 : 353 – 62 . SugioS , KashimaA , Mochizuki S , Noda M , K obayashi K .1999 . Crystal structure of human serum albumin at 2.5 A resolution . Protein Eng 12 : 439 – 46 . SuzukiE ,Y asuda K ,T akeda N , Sakata S , Era S et , al. 1992 . Increased oxidized form of human serum albumin in patients with diabetes mellitus . Diabetes Res Clin Pr act 18 : 153 – 8 . erawaki T H ,Y oshimura K , Hase gawa T , MatsuyamaY , Ne gawa T ,et al. 2004 . Oxidative stress is enhanced in cor relation with renal dysfunction: e xamination with the redo x state of albumin . Kidney Int 66 : 1988 – 93 . urell T L , Botti H , Carballal S , F errer - Sueta G , Souza JM et , al. 2008 . Reactivity of sulfenic acid in human ser um albumin . Biochemistry 47 : 358 – 67 . urell T L , Botti H , Carballal S , Radi R ,Alv arez B. 2009a . Sulfenic acid — Akey intermediate in albumin thiol oxidation. J Chromatogr B Analyt Technol Biomed Life Sci 877 : 3384 – 3392 . urell T L , Carballal S , Botti H , Radi R ,Alv arez B. 2009b. Oxidation of the albumin thiol to sulfenic acid and its implications in the intra vascular compartment. Braz J Med Biol Res 42 : 305 – 11 . eum Y KJ , Russell RM , Krinsk y NI ,Aldini G .2004 . Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compar tments of human plasma . Arch Biochem Biophys 430 : 97 – 103 .

Chapter15 Protein S - glutathion ylation and S - cystein ylation Gr aziano Colombo , Aldo Milzani ,Roberto Colomboand , Isa bella Dalle - Donne

INTR ODUCTION The double - ace f signif cance of reacti ve oxygen species (R OS) and reacti ve nitrogen species (RNS) embraces not onl y their fundamental roles in modulating ph ysiological processes, but also their unw anted y et inevitable adv erse effect on molecule functions due to the potential destructive proper ties. In f act cellular macromolecules such as nucleic acids, carboh ydrates, and proteins, following reactions with ROS and/or RNS, can undergo reversible and/or irreversible modif cations that are ab le to harshly modify molecular str uctures and cellular functions. In particular, proteins represent the most abundant tar get of these R OS/RNS-mediated modif cations, which can often be associated with a permanent loss of protein function, as is usually observed in protein carbon ylation (Dalle -Donne et al. 2006), or can lead to accumulation of damaged proteins into cytoplasmic inclusions that are resistant to proteasomal de gradation, as observed in age -related neurodegenerative disorders (Giasson et al. 2002). The –SH groups of cysteinyl residues are the most reacti ve moieties that under take re versible and ir reversible oxidative modif cations within a protein (F igure 15.1). Under moderate oxidative stress conditions, protein thiol g roups (PSHs) form a physiologically reversible cysteine sulfenic derivative (PSOH) (Figure 15.1A), which can further increase its o xidation state in a more o xidative environment, leading to the for mation of ir reversible modif cations such as sulf nic (PSO2H) and sulfonic (PSO 3H) acids (F igure 15.1A). Alternatively, oxidation can result in a disulf de bridge, either intramolecular or inter molecular (Figure 15.1B). The importance of thiols in def ning tertiary protein structure and catalytic function is w ell conf rmed and accepted b y a lar ge number of literature w orks; alterations in the o xidation state of –SH g roups can deepl y modify protein str ucture and biolo gical activity. In order to pre vent the ir reversible effects of reacti ve species, cell e volution seems to ha ve contrived some molecular mechanisms in volving c ysteine thiol g roups, w hich are ab le to reversibly protect cysteinyl residues when oxidative stress overcomes the potentially dangerous threshold responsible for the loss of functional –SH redox state. Cysteine oxidation can indeed lead to the re versible for mation of mix ed disulf des betw een protein thiol g roups and lo wBiomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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Figure 15.1. Oxidative modif cations of protein thiols. (A) The oxidation of a cysteine residue within a protein can result in the formation of a cysteinyl radical (Cys –S•, not shown) or a sulfenic, sulf nic, or sulfonic acid derivative (the latter of which is always irreversible). (B) Alternatively, oxidation can result in a disulf de bridge (cystine). Protein disulf des can form under oxidative conditions between two adjacent proteins (intermolecular cystine) or between two adjacent sulfhydryl groups within a protein (intramolecular cystine), causing changes in protein aggregation and conformation. (C) Reaction between protein cysteinyl residues and low-molecular-mass thiols such as glutathione and free cysteine can yield protein –glutathione or protein –cysteine mixed disulf des, respectively, i.e., S-glutathionylated or S-cysteinylated proteins. Each of these protein thiol modif cations has the possibility of eliciting different cellular responses. Reprinted from Free Radical Biology and Medicine, 43(6), Dalle -Donne I, Rossi R, Giustarini D, Colombo R, Milzani A, S-glutathionylation in protein redox regulation, 883 -898, Copyright 2007, with permission from Elsevier.

molecular- mass(LMM) thiols (S-thiolation), particularly with glutathione (S - glutathion ylation) and free c ysteine ( S -ysteinylation) c (Figure 15.1 C).These two on - demandmodif cations, besides playing a protective role against irreversible oxidation of cysteinyl residues, can simultaneously represent a par ticularly f ne-tuned regulation mechanism dri ven by and depending on cellular redox state (Figure 15.2).

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Figure 15.2. Role of protein cysteine S-glutathionylation in redox signaling and oxidative stress responses. At low ROS/RNS levels (basal, or physiological, conditions), reversible Sglutathionylation of protein cysteine thiols (PSHs) regulates protein activity/function and can subserve cell signaling responses. Oxidative stress results from excessive generation of ROS/ RNS and/or impaired antioxidant defenses. After moderate elevation of ROS/RNS levels and/or moderate impairment of antioxidant defenses (moderate oxidative stress), reversible Sglutathionylation of PSHs maintains GSH within cells (often at the expense of temporary loss of protein function) and protects PSHs from irreversible oxidation. At high ROS/RNS levels and/or strong impairment of antioxidant defenses (severe oxidative stress), irreversible oxidation of PSHs can change protein function permanently, thus impairing the physiological redox regulation of proteins with redox -sensitive cysteines. Irreversible modif cations are usually associated with permanent loss of function and may lead to the elimination of the damaged proteins by the proteasome system or to their accumulation as insoluble aggregates. Reprinted from Free Radical Biology and Medicine, 43(6), Dalle -Donne I, Rossi R, Giustarini D, Colombo R, Milzani A, S-glutathionylation in protein redox regulation, 883-898, Copyright 2007, with permission from Elsevier.

At present, a g rowing body of literature suppor ts S-glutathionylation as both a positi ve and negative redox regulation trick in volved in protein acti vity and str ucture modulation. In f act, many reports show how metabolic enzymes (e.g., carbonic anhydrase III; Cabiscol and Levine, 1996), signaling proteins (e.g., protein kinase A and mitogen-activated protein kinase [MAPK]/ extracellular-signal-regulated kinase [ERK]; Humphries et al. 2002), transcription factors (e.g., c-Jun; Klatt et al. 1999), heat shock proteins (e.g., HSP60; Hamnell -Pamment et al. 2005), ion channels (e.g., r yanodine receptor type 2 RyR2; Sanchez et al. 2005), c ytoskeletal proteins (e.g., actin; Dalle -Donne et al. 2003a,b), and mitochondrial proteins (e.g., comple x 1 NADHubiquinone o xidoreductase; Taylor et al. 2003) can be selecti vely and re versibly Sglutathionylated owing to a specif c stimulus. In contrast, the picture concerning S-cysteinylation is actually less def ned, but some authors seem to suggest a similar re gulative and reversible role of this protein post -translational modif cation (Banks et al. 2008) and a possib le role as a ne w node in the circuitr y for biolo gical redox signaling (Jones et al. 2004). This chapter discusses the basic chemistr y of mix ed disulf de for mation and the most adv anced anal ytical techniques for detection of protein S - glutathion ylation and S-cysteinylation, as well as their biolo gical signif cance.

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REDO X HOMEOSTASIS: GLUTATHIONE/GLUTATHIONE DISULFIDE AND CYSTEINE/CYSTINE, TWO THIOL/ DISULFIDE REDOX COUPLES WITH DIFFERENT EQUILIBRIUM STATES Glutathione (GSH) and c ysteine (Cys) represent the most abundant LMM thiols in mammals. They show a different distribution between intra- and extracellular environments and they exert diverse roles in cellular physiology and biochemistry. Glutathione concentration is higher than the concentrations of cysteine, glutathione disulf de (GSSG), and cystine (CySS) (Figure 15.3) within cells, where GSH plays an essential role in maintaining the highly reduced environment. By contrast, free c ysteine is more plentiful than GSH in the e xtracellular environment, where it embodies an impor tant component in antio xidant defense. As a whole, the Cys/CySS redo x

Figure 15.3. Structure of the thiol and disulf de form of glutathione (GSH and GSSG, respectively) and the sulfur amino acid cysteine (Cys and cystine, or CySS, respectively) as well as the resulting S-glutathionylated and S-cysteinylated proteins following S-glutathionylation and S-cysteinylation reactions, respectively. In both glutathione forms, the peptide bond linking the amino-terminal glutamate and the cysteinyl residue is through the γ-carboxyl group and protects glutathione from degradation by serum aminopeptidases or intracellular proteases.

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couple predominates in e xtracellular f uids such as the plasma, w here it mainl y exists in the disulf de for m, w hereas the GSH/GSSG redo x couple pro vides control mechanisms within cells, where it is principall y present in the reduced for m (Figure 15.4). Extracellular thiols also constitute an impor tant component in antio xidant defense. These thiols are also found as LMM disulf des and protein mix ed disulf des. A recent study has demonstrated an age -dependent reduction in the plasma le vel of PSHs and an age -dependent increase in S-cysteinylated plasma proteins, which is accompanied by a decrease in the plasma Cys/CySS redox couple in health y humans, with the phenomenon becoming clearl y manifest as from 60 y ears (Rossi et al. 2008) (Figures 15.5 and 15.6). This suggests that there is some increase in oxidative events targeting protein thiols throughout adult life and/or that protection from oxidative damage by plasma thiol redo x buffer becomes less eff cient with age. Glutathione is a w ater-soluble tripeptide composed b y glycine, cysteine, and glutamic acid (L -γ - glutam yl - L -ysteinyl c - gl ycine, GSH) (Figure 15.3 ).It is generally present in all mammalian cells at a concentration ranging from 1 to 10 mM. In contrast, the e xtracellular GSH concentration, with the exception of bile, is relatively low: for example human plasma contains GSH at a concentration of 1 to 5 µM (Giustarini et al. 2003b, 2004 ) (Figure 15.4).

Figure 15.4. Intracellular and extracellular distribution of the glutathione and cysteine pools. In mammalian cells, such as red blood cells, GSH concentrations are millimolar and GSSG, cysteine, and cystine (CySS) concentrations are micromolar. While GSH and its disulf de form, GSSG, constitute the most abundant cellular redox couple, the Cys/CySS couple predominates in the plasma.

Figure 15.5. The effect of age on the plasma cysteine/cystine (CysSH/[CysS] 2) redox couple in healthy human beings. Data are means ± SD. Reprinted from Journal of Cellular and Molecular Medicine, “Postprint”; DOI: doi:10.1111/j.1582-4934.2008.00417.x, Rossi R, Giustarini D, Milzani A, Dalle -Donne I, Cysteinylation and homocysteinylation of plasma protein thiols during ageing of healthy humans, Copyright 2008, with permission from Blackwell Publishing.

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Figure 15.6. Plasma levels of S-glutathionylated (PSSG) (A) and S-cysteinylated (CysSSP) (B) proteins in the plasma of healthy individuals for each age group considered in the study. All values are expressed as the percentage to total plasma protein thiols. Data are means ± SD. Reprinted from Journal of Cellular and Molecular Medicine, “Postprint”; DOI: doi:10.1111/j.1582-4934.2008.00417.x, Rossi R, Giustarini D, Milzani A, Dalle -Donne I, Cysteinylation and homocysteinylation of plasma protein thiols during ageing of healthy humans, Copyright 2008, with permission from Blackwell Publishing.

GSH is characterized by a strong electron -donating behavior, so its nature, accompanied b y direct interaction with ROS/RNS, leads the GSH cysteinyl thiol-losing electrons with the direct consequence that a glutathione molecule becomes o xidized. Generall y this e vent leads tw o oxidized GSH molecules to dimerize b y a disulf de bridge to generate GSSG (F igure 15.3). This linkage is characterized b y reversibility upon reduction; cells usuall y reconvert GSSG to GSH through an enzymatic reaction in volving glutathione reductase that requires N ADPH as cofactor (Hayes and McLellan 1999). Despite the f act that GSH demonstrates a ubiquitous distribution in the dif ferent cellular compartments, it also sho ws an inhomo geneous abundance among cellular districts; in f act, almost 90% of GSH is located in the c ytosol (1 to 10 mM), 10% in the mitochondria (5 to 10 mM), and a small amount is in the endoplasmic reticulum and the nucleus (Lu 2000). This wide-range cellular distribution of GSH is accompanied by an exclusive cytoplasmic synthesis that requires multiple transpor t mechanisms, w hich are not y et well def ned (Ballatori et al. 2005 ).These diverse and independent cell district - specifc GSH pools seem to ha ve different physiological relevances. For instance, mitochondrial and c ytoplasmic GSH pools pla y a different role in def ning cell sur vival, and e xperimental data suppor t a functional independence between the two pools (Shan et al. 1993). Mammalian cells can g row in culture medium if supplemented with CySS, e ven though no enzymes are actuall y kno wn to specif cally reduce CySS to Cys. Two e xplanations could explain this issue. F irst, a w eaker thiol -disulf de exchange reaction in w hich CySS is used is possible, because the GSH released b y cells into the medium can react with CySS to generate Cys. Although this reaction clearl y occurs in vivo , it remains a potential e xplanation because the estimated rate of the reaction seems to be too slow to counteract the rate of Cys metabolism in cells (Reed and Betty 1978). A second possibility in volves the enhancement of the CySS reduction due to nonspecif c enzymes such as glutaredo xin and thioredoxin, two enzymes that together can accomplish about 20% of CySS reduction (Manner vik et al. 1983). Using the Nernst equation, it is possible to def ne the redox potential of the two redox couples GSH/GSSG and Cys/CySS within cells (Jones et al. 2004, Schafer and Buettner 2001). The steady-state redox potential of the Cys/CySS couple is E h = −145 mV in HT29 cells, and it is suff ciently oxidized ( >90 mV) compared with that of the GSH/GSSG couple (E h = − 250 mV)

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to function as an o xidant in redox switching (Jones et al. 2004). The normally reductive intracellular conditions cause the concentrations of GSH and GSSG to be in a ratio of about 100 : 1, but this ratio varies in mammalian cells in vitro in association with differentiation, proliferation, and apoptosis (Moriar ty-Craige and Jones 2004). Also, the Cys/CySS couple can e xperience modulation of its redox state when cellular physiological and metabolic states change. During cell differentiation, for instance, both GSH/GSSG and Cys/CySS redox couples undergo oxidation but the intensity of change sho ws a dissimilar e xtent, suggesting that the coincidence of two redox couples evolved and act parallel in cells to control physiologically relevant processes depending on redox mechanisms (Jones et al. 2004). Pharmacological treatments of human cultured HT29 cells with GSH inhibitors or inducers support this h ypothesis. In these cells buthionine sulfo ximide, a w ell known inhibitor of the f rst enzyme involved in GSH synthesis (the glutamate:c ysteine ligase), is able to decrease the GSH/GSSG redox state to about 40 mV, but it has no ef fects on the Cys/CySS redo x state in proliferating HT29 cells (Jones et al. 2004). In contrast, benzyl isothiocyanate, a molecule that enhances GSH synthesis, causes an augmentation in GSH that is accompanied b y a small oxidation of the GSH/GSSG redo x couple ( ∼10 mV), whereas it supports an evident oxidation of the Cys/CySS redo x state ( ∼30 mV). This uncor related beha vior betw een the tw o redo x couples suggests an independent control of the tw o redox pathways supporting a complementary role of GSH/GSSG and Cys/CySS in cellular ph ysiology.

S - GLUT ATHIONYLATION AND S - CYSTEINYLA TION ARE THIOL - SELECTIVEPOST - TRANSLA TIONAL MODIFICATIONS Although a single protein can contain a large number of cysteinyl residues, not all of them play a relevant role in the formation of S - glutathion ylated or S -ysteinylated c proteins (Figure 15.3 ). This specif city is principall y due to the steric ar rangement of the c ysteine thiol inside the protein (accessibility) and the microenvironment surrounding the cysteinyl residue (reactivity). Accessibility is def ned by the secondar y and ter tiary structure of the protein and depends on the relative ease of the c ysteine to be e xposed to the solv ent, whereas reactivity is a more diff cult feature to predict. In f act, the cysteinyl residue needs to have a pK a lower than 7 to react with glutathione or cysteine. Most of the c ysteine residues within cellular proteins are characterized b y a pK a around 8; this means that the y are maintained in the protonated state in the reducing c ytoplasmic environment, making them not prone to o xidation. Conversely the great majority of c ysteines involved in S-thiolation are localized in a basic microen vironment (determined by surrounding amino acids) that shifts their pK a to a v alue near or belo w 7; this lower pK a keeps the cysteines in the thiolated for m at neutral pH and promotes their o xidative modif cations (Dalle Donne et al. 2009). Under continuous and high evolutionary pressure, the possibility to have strategic and functional cysteines modif able by oxidative stress selected many regulative mechanisms dependent on the oxidative state of the cysteinyl residues (Rhee et al. 2000). For instance, protein tyrosine phosphatase 1B (PTP1B) has a conser ved cysteine in its acti ve site that is par ticularly prone to oxidation and can shift betw een reduced and S - glutathion ylated. These two oxidative states of the acti ve site Cys are responsib le for the acti vation or deacti vation of the phosphatase because the enzyme is catal ytically inacti ve w hen such Cys e xists in the S - glutathion ylated state (Barrett et al. 1999). Cysteinylation, the co valent bond betw een a protein c ysteinyl residue and a free c ysteine (Figure 15.3), is a regulatory mechanism known to be involved in the presentation of antigenic peptide epitopes on MHC molecules that can re gulate the immune response to tumor antigens and contribute to autoimmune diseases, including type I diabetes (Kittlesen et al. 1998, Mannering et al. 2005). F or e xample, S-cysteinylation of the potentiall y onco genic protein kinase C (PKC) isozymes ε and γ has been sho wn to re gulate inactivation of their enzymatic activity and simultaneousl y stimulate the pro -apoptotic function of PKC -δ, making isozyme -

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specifc PKC re gulators an attracti ve target for the treatment of proliferati ve diseases such as cancer (Chu et al. 2003). The impor tance of S-cysteinylation as a means for thiol protection against o xidative stress is demonstrated in Bacillus subtilis , a Gram -positive bacterium that lacks GSH. In Bacillus subtilis, Cys represents the most abundant LMM thiol and the e xposure to the thiol -specif c oxidant diamide is accompanied b y a massi ve induction of Cys biosynthesis genes as w ell as others related to homeostasis of the intracellular thiol -disulf de state (Leichert et al. 2003) and consequent S-cysteinylation of man y cellular proteins (Hochg räfe et al. 2007). These results suggest that S-cysteinylation is a general mechanism to protect protein thiols from fur ther oxidation and ir reversible damage in B. subtilis and ma y pla y a role in redo x re gulation of several thiol -containing proteins. Among these B. subtilis S -ysteinylation c targets there are cobalamin - independentmethionine synthase, inorganic pyrophosphatase, and inosine - monophosphate deh ydrogenase, three proteins also kno wn to under go S - glutathion ylation in Escherichia coli (Cotg reave et al. 2002), human peripheral b lood mononuclear cells (F ratelli et al. 2004), and human endothelial -like cells (Lind et al. 2002), respectively.

POTENTIAL MECHANISMS OF PR OTEIN S - GLUT ATHIONYLATION AND S - CYSTEINYLA TION S - glutathion ylation and S -ysteinylation c are reversible post - translationalmodif cations that are not completely clarif ed. Whereas protein de glutathionylation is chief y catalyzed by glutaredoxins, no e vident enzymes or mechanisms ha ve been f rmly estab lished for the Sglutathionylation reaction. At present four potential mechanisms of S - glutathion ylation are proposed, occurring spontaneously or being catal yzed by specif c enzymes (Figure 15.7): • Thiol - disulf de exchange • Sulfenicacid intermediate formation • Thiylradical mechanisms • S - nitrosyl intermediate formation The f rst mechanism (F igure 15.7, mechanism A) seems to be too f ar of f from cellular physiology to occur: in fact the typical GSH/GSSG ratio in non -stressed cells is approximately 100 : 1 and it was estimated that a 50% protein S-glutathionylation occurs at a GSH/GSSG ratio as low as 1 : 1 (re viewed in Dalle -Donne et al. 2008). These studies suppor t the idea that a thiol - disulfde e xchange mechanism is an unlik ely method of P -SSG for mation in a nor mal cellular en vironment. The DN A binding protein c -Jun represents an e xception: c -Jun DN A binding ability is inhibited b y S-glutathionylation when GSH/GSSG ratios are relati vely high (∼13); this peculiarity mak es c -Jun a plausib le S-glutathionylated protein in cells b y a thiol disulfde exchange mechanism (Klatt et al. 1999). Protein- and GSH - sulfenic acid inter mediates are a direct consequence of the reaction between ROS or RNS and c ysteinyl residues of proteins and GSH, respecti vely. These intermediates are par ticularly unstable and tend to fur ther oxidize to sulf nic and sulfonic acids, or tend to combine with GSH or a protein thiol, respectively, to rapidly yield P-SSG (Figure 15.7, mechanisms B and C). The diff culty to detect sulfenic acids in vivo (Saurin et al. 2004), together with e vidence coming from in vitro experiments perfor med on mammalian PTP1B , seem to suggest the quick conversion of P-SOH to P-SSG by GSH in the cytoplasmic environment (Bar rett et al. 1999). S-glutathionylation of the c ysteine sulfenic deri vative prevents the enzyme from easy further oxidation to its irreversible sulf nic and sulfonic forms and constitutes an eff cient regulatory mechanism. The third mechanism that could generate S-glutathionylated proteins involves the formation of thiyl radical inter mediates (Figure 15.7, mechanisms D , E, and F), w hich are usuall y produced both in vivo and in vitro following the reactions betw een ROS and/or RNS and thiols.

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Figure 15.7. Proposed mechanisms of S-glutathionylation reaction. S -glutathionylated proteins (P-SSG) can be formed in response to changes in the GSH/GSSG ratio, through thiol/disulf de exchange reactions between a protein sulfhydryl group (P-SH) and GSSG (mechanism A). Thiol/ disulf de exchange reactions can also occur between a P-SH and a P-SSG (not shown). Indeed, minor changes in the intracellular GSH/GSSG ratio are unlikely to lead to substantial Sglutathionylation of proteins according to thiol –disulf de exchange involving GSSG and P -SHs (mechanism A), because the redox potentials of most cysteine residues are such that they would only be 50% S -glutathionylated at a very low GSH/GSSG ratio of 1 (i.e., an usually and very unlikely change in cellular GSSG concentration). In addition, the GSSG export by most cells as a protective mechanism against oxidative stress drastically limits large shifts in the intracellular redox potential under normal physiological conditions. S-glutathionylated proteins can arise from two electron oxidation of the protein thiol to sulfenic acid (P -SOH), followed by reaction with GSH (mechanism B) or from reactions between P -SH and glutathione sulfenic acid (GSOH) (mechanism C). S-Glutathionylated proteins can also be formed following activation (i.e., partial oxidation) of the P -SH or the sulfhydryl group on the cysteinyl portion of GSH. This can occur by a thiyl radical reaction, both through a single -step radical recombination (mechanism D) and through a two -step reaction, by one electron oxidation of the thiol on GSH or the P -SH to give the respective thiyl radical (mechanisms E and F), which further reacts with a P -SH or GSH, respectively, to yield P -SSG. The reaction of GS • with proteins to generate P -SSG is catalyzed by glutaredoxin, an enzyme normally acting as a reductant. Furthermore, P -SSG can arise from reactions between GSH and S-nitrosated proteins (P -SNO) (mechanism G) or from reactions between P -SH and S-nitrosothiols such as GSNO (mechanism H).

Thiyl radicals (RS •) are v ery shor t-lived sulfhydryl derivatives that can alter natively lead to P-SSG for mation both through a single -step radical recombination (i.e., GSH • and P -S•→ P SSG) and a tw o-step reaction, f rst producing a thiolated protein that consequentl y combines with molecular oxygen to yield P -SSG. These mechanisms are suppor ted by in vitro experimental data demonstrating ho w several redox-sensitive proteins under go S-glutathionylation in the presence of GS • in a manner that is glutaredoxin-dependent (Starke et al. 2003). Finally, a four th proposed mechanism h ypothesizes an S - nitrosylationstep, following a stimulus, that mak es the protein -SNO rapidl y reacti ve with GSH to generate P -SSG. Because S-nitrosylation also can occur to GSH, generating GSNO , an alter native pathw ay passes through the combination of P -SH and GSNO prior to generating P -SSG (Figure 15.7,

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mechanisms G and H). This second w ay is suppor ted by the rapid reacti vity of GSNO with isolated proteins such as GAPDH and papain in vitro (West et al. 2006) and b y detection of GSNO at micromolar concentrations in non -stressed tissue (reviewed in Gaston 1999). In contrast to the more investigated mechanisms concerning S - glutathion ylation, the cellular pathways necessary for S-cysteinylation are less e vident, and it is onl y a similarity in the biochemistry of GSH and Cys that leads us to suppose the four mechanisms e xplained above are possibly responsib le for protein S-cysteinylation. As for glutathione biochemistr y, enzymes that can generate c ysteinyl protein adducts are still unkno wn but, in contrast to GSH, the mechanism involving thiol -disulf de exchange seems to be the most plausib le event underling protein S-cysteinylation (Summa et al. 2007). In a c ystathionine β - synthase(CBS) def ciency mouse model, experimental observations suggest a possible metabolic mechanism accountable for the generation of the high plasmatic c ystine concentration that leads to straight Scysteinylation of albumin Cys34. In this mouse model a drastic decrease in S -ysteinylated c albumin concentration is accompanied b y a drastic increase in the for mation of Shomocysteinylated albumin, suggesting that CBS is an impor tant k ey in def ning the redo x equilibrium in plasma alb umin thiols (Bar -Or et al. 2004).

HOW IS IT POSSIBLE TO STUDY PROTEINS UNDERGOING S - GLUT ATHIONYLATION AND S - CYSTEINYLA TION REACTIONS? The study of S-thiolated proteins is a classic scientif c problem that can be investigated with a variety of research instr uments including high pressure liquid chromato graphy (HPLC), spectrophotometric assays, mass spectrometr y analysis, 1D or 2D electrophoretic separations, and radiolabeling approaches. This chapter discusses protein modif cations occur ring at c ysteinyl residues that in volve an e vident change in the o xidative state of the amino acid. Considering that S - glutathion ylation and S-cysteinylation are reactions leading to o xidation of c ysteine, in general a useful and rapid method to e valuate and compare global level of S - modif ed proteins after a specif c treatment, ph ysiological stimulus, or ph ysiopathological insult consists of a selective reduction of the cysteinyl residues using reducing agents such as dithiothreitol (DTT), dithioerythritol (DTE), β - mercaptoethanol (β-ME), sodium borohydride, or trialkylphosphines. These molecules can clea ve the covalent bond between protein residues and GSH or Cys. In order to be robust and repeatab le, this approach requires a preliminar y acidic sample purif cation step with the intent to remo ve and anal yze free solub le GSH and Cys usuall y present in the cellular e xtract. Once the possible endogenous LMM thiol contamination of the protein fraction has been a voided, the treatment with a reducing agent allo ws the release of covalently bound GSH and/or Cys from proteins, which is followed by a subsequent quantif cation step by chromatographic separation combined with f uorescent or colorimetric detection. The selection of the most appropriate reducing agent depends on the succeeding passages required to anal yze the cellular e xtract. In f act, although DTT, DTE, and β - MEare very eff cient disulf de bond clea ving agents, the y can also react with molecules used for GSH/Cys titration (Meister and Anderson 1983). In contrast, trialk ylphosphines are non -reactive toward many other functional groups (Burns et al. 1991) but their reducing eff ciency is inf uenced by temperature and reductant concentration (Krijt et al. 2001). Sodium borohydride also has some drawbacks, because it leads to an o verestimate of GSH and Cys contents as a result of its collateral property to also cleave proteins in acidic conditions; these acid-soluble proteins represent a consistent fraction of the supposed GSH signal (Brigelius et al. 1983). Furthermore, because the use of sodium borohydride can also interfere with GSH revealing agents, its extraction from the sample is frequentl y needed (Meredith 1983). In order to ha ve a robust GSH and/or Cys anal ysis and to minimize ar tifacts in a comple x protein mixture, such as cellular e xtracts, it is essential to consider the emplo yment of an

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immediate step of free sulfh ydryl group alkylation or ar ylation. N - eth yl maleimide (NEM) is available worldwide and is the most used agent in this b locking step. NEM can ir reversibly bind all free sulfhydryls within seconds, thus avoiding thiol-disulf de exchange reactions, which can generate variability among experiments due to handling of samples. Thiol alkylation with NEM before sample acidif cation also a voids GSH o xidation during acidif cation in the presence of hemoglobin and hemoproteins in general. A second impor tant point to be considered is indeed the presence of hemoproteins. During sample acidif cation, o xyhemoglobin, if not adequatel y handled , produces ar tif cially high amounts of GSSG and P-SSG, likely through ROS generation, which lead to an overestimation of S-glutathionylated proteins (Rossi et al. 2002, Giustarini et al. 2003a). These two fundamental factors concerning determination of P-SSG, and likely S-thiolated proteins in general, could be the reason w hy many studies repor t high physiological levels (around 50 to 200 µM) of Sglutathionylated proteins in b lood from health y humans (Muscat et al. 2004, Mandal et al. 2007) compared to those measured b y others, ranging around 2 to 3 µM (Giustarini et al. 2003a ). Once soluble GSH or Cys are eliminated, the protein pellet is acidif ed, and GSH or Cys are released from S-thiolated cysteinyl residues, and it is possib le to quantify the amount of GSH or Cys in solution both spectrophotometricall y and chromato graphically. Spectrophotometric methods employ two colorimetric reagents, i.e., 5,5 ′ - dithio - bis(2 - nitrobenzoic acid) (DTNB) (Dalle- Donne et al. 2003 )and 1 - chloro - 2,4 - dinitrobenzene (CDNB) (Rokutan et al. 1991 ), showing different thiol specif city and diverse aims. The f rst binds all thiols in a non -specif c manner, whereas the second one requires catal ysis of glutathione S - transferasesand is GSH specifc. Therefore, DTNB is recommended for a general assa y addressed to e valuate global levels of o xidized sulfhydryls in proteins, w hereas CDNB, because of its strict specif city for GSH, is suggested for the in vestigation of S - glutathion ylated proteins. Once GSH or Cys are released from S-thiolated proteins as a consequence of the reduction of the disulf de bond(s), the free reduced LMM thiols can be conjugated with a f uorescent molecule and a conceptuall y similar approach can be perfor med using HPLC to increase sensitivity, separation ability, and detection limit. This technique also allows for the measurement of GSH and Cys in the presence of reducing agents, because HPLC separation discriminates between the dif ferent molecules labeled with f uorescent reagents. The three f uorophores principally employed for this pur pose are monobromobimane (mBrB), dansyl chloride, and o - phthalaldhe yde (OPA). The f rst, used for quantif cation of S - glutathion ylated and Scysteinylated proteins in plasma, b lood, and primar y cultures of human neurons (Sebasti à et al. 2003, Sakhi et al. 2007, Rossi et al. 2008) (Figure 15.6), is the most largely employed thiol alkylating f uorophore due to its rapidity, reproducibility, and possibility to discriminate among the different thiols physiologically bound to proteins (Giustarini et al. 2003a,b). Dansyl chloride, despite a more complicated operati ve procedure, which consists of disulf de bond reduction, protein remo val b y sample acidif cation, alk ylation of the free sulfh ydryl g roup of the released LMM thiol by iodoacetic acid, and dansylation of the amino g roup of the LMM thiol with dansyl chloride, is equall y eff cient, and it was successfully used to deter minate glutathione and c ysteine concentrations in human plasma (Jones et al. 2000). OPA was also used in blood, tissues, and plasma (P aroni et al. 1995), but, in contrast to mBrB and dansyl chloride, some misgi vings emer ged about its specif city due to the possibility that some endo genous molecules (i.e., f uorogenic thiol and non -thiol compounds reacting with OP A and/or f uorescence-quenching substances) in specif c tissues such as li ver could generate interference (Floreani et al. 1997). Therefore, OPA should be used under specif c and strictl y controlled assay conditions (Senft et al. 2000). The addition of a GSH or Cys molecule to a pol ypeptide chain leads to an increase of the protein mass of + 306 Da and +119 Da, respectively, so that the mass dif ference can be ascer tained b y standard electrospra y ionization (ESI), matrix -assisted laser -desorption/ionization time - of -ight f (MALDI -TOF) mass spectrometr y (MS), or a fr uitful combination betw een

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HPLC and MS that merges a highly reproducible separation with elevated precision in peptide identif cation (Niwa et al. 2000). These analytical techniques do not need an y diff cult sample handling and can be emplo yed without laborious pretreatment of the protein mixture (see Dalle-Donne et al. 2008 for a recent re view on techniques cur rently used to detect P -SSG). A clear advantage of MS techniques is that the y allow the identif cation of the specif c cysteinyl residue to which GSH or Cys is bound in the S - thiolatedprotein. The above mentioned techniques are also reliable for the quantif cation of the global content of GSH/Cys - modifed proteins in comple x biolo gical samples. The possibility to def nitely detect w hich specif c proteins under go S - glutathion ylation or S -ysteinylation c is also very interesting in an ef fort to def ne the f ne-tuning adaptations that a cell assumes to contrast oxidative stress, defend its homeostatic equilibrium, and regulate cellular functions. One of the most commonl y used techniques for this specif c pur pose consists of the emplo yment of a radiolabeled substrate that can tag intracellular GSH or Cys pool. Considering that the tripeptide GSH is not permeable to cells, 35S-cysteine is the best alternative because it passes biological membranes and is e xtensively used for the biosynthesis of glutathione. To avoid labeling of new synthesized proteins, cells should be pretreated with cycloheximide to b lock protein synthesis, labeled with 35S-cysteine, and then subjected to o xidative stress (Grimm et al. 1985). Cellular protein e xtracts can be separated b y 1D or 2D electrophoresis under non-reducing conditions and S-thiolated proteins can be highlighted through autoradiography follo wed b y protein sequencing (F ratelli et al. 2003). Two lik ely dra wbacks for this approach are (1) the contemporar y labeling of S - glutathion ylated, S -ysteinylated, c and Scysteinylglycinylated proteins (although, within cells, P -SSG lar gely pre vails o ver other Sthiolated proteins) and (2) the impossibility to detect constituti ve S - thiolatedproteins because radiolabeled cysteine is added just prior the o xidative insult. Whereas the f rst limit does not allow specif c on-gel discrimination and requires the employment of a subsequent MS step for consistent identif cation, the second limit is conquerab le by the employment of specif c monoclonal antibodies developed against P-SSG (Dalle-Donne et al. 2005) (Figure 15.8). Antibodies against S-cysteinylated proteins ha ve recentl y been commercialized , but their eff ciency and specif city require testing. A

B

spectrin α ankyrin β band 3 protein 4.1 protein 4.2

250 kD 150 100 75 50

actin

37

GAPDH

25 20 MW 0

5 20 60 100

Time of incubation with diamide (min)

MW 0

5 20 60 100

Time of incubation with diamide (min)

Figure 15.8. Western blot analysis of membrane skeletal proteins of human red blood cells exposed to the thiol -specif c oxidant diamide. Protein samples were separated by 10% SDS PAGE under non -reducing conditions. Samples were then blotted onto PVDF and probed with anti-GSH monoclonal antibody (B). (A) The same blot shown in (B) stained for proteins with amido black. MW = molecular weight markers. Each panel shows a single representative experiment of three separate experiments. Reprinted from Blood Cells, Molecules, and Diseases, 37(3), Rossi R, Giustarini D, Milzani A, Dalle -Donne I, Membrane skeletal protein -Sglutathionylation and hemolysis in human red blood cells, 180-187, Copyright 2006, with permission from Elsevier.

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A brief note about 2D electrophoresis, w hich, when combined with specif c antibodies, is warranted. This technique is a v ery powerful approach due to both its recent easy -to-use protocols and its high resolution. This is particularly true for S - glutathion ylated proteins, because addition of GSH to a protein inser ts a ne w charge that mak es the protein ’s isoelectric point shift in the 2D map and allo ws a f ast qualitative identif cation of possib le protein tar gets of S - glutathion ylation. In addition to the use of direct detection methods, it is also possible to determine S -xidized o proteins by indirect molecular tools that are ab le to label modif ed cysteinyl residues. These approaches require an initial step of free sulfh ydryl g roup alk ylation with NEM, follo wed alternatively by reduction with DTT of S - modif ed cysteines, in the case of a global v aluation of S-thiolated proteins as a w hole, or selecti ve reduction of S - glutathion ylated residues with glutaredoxin - 3from E. coli (Lind et al. 2002). Once reduced, -SH groups can be labeled with f uorescent probes, such as f uorescein iodoacetamide (Chen et al. 2008), or easy -to-highlight tags, such as HPDP -biotin and NEM -biotin. These molecular f ags can be e videnced using streptavidin conjugated to horseradish pero xidase (HRP) and de veloping the signal with a standard colorimetric or chemioluminescent method. Another interesting approach emplo ys biotinylated glutathione eth yl ester (BGEE), a molecule that can go right through cellular membranes (Sullivan et al. 2000). In response to oxidative stress, BGEE, and consequentl y the biotin yl moiety, is incor porated within proteins. The separation of S - modif ed proteins can be perfor med through aff nity chromato graphy with streptavidin bound to agarose beads, and isolated proteins are then anal yzed through 1D or 2D SDS-PAGE (Sullivan et al. 2000). Only one doubt rises about the applicability of BGEE: its relati vely higher steric hindrance compared to that of GSH could diminish its accessibility to c ysteinyl residues, par ticularly to those that are not solv ent-exposed. A similar approach uses biotin ylated GSH to probe for protein S-glutathionylation during o xidative stress conditions, follo wed b y non -reducing Western b lots and strepta vidin–HRP to detect cellular P -SSG and strepta vidin–agarose to isolate P -SSG (Eaton et al. 2002). Finally, treatment of cells with the recentl y synthesized N,N - biotin yl GSSG mimics an increase in cellular GSSG, like that associated with some disease conditions and aging as w ell as redox signaling models. This increase in GSSG leads to thiol -disulf de exchange reactions, promoting protein S-glutathionylation, which is detected on non-reducing Western blots probed with streptavidin-HRP, whereas specif c P -SSG can be purif ed using streptavidin-agarose and identif ed using proteomic methods (Brennan et al. 2006).

CONCLUSION S - glutathion ylation and S-cysteinylation are two protein modif cations that occur during oxidative stress, but also in a number of physiological situations (i.e. basal conditions) in which they can produce discrete modulatory effects on protein function. Although the detailed mechanisms producing S - glutathion ylated and, especially, S-cysteinylated proteins, are not y et clear, these modif cations show a site-specif c localization and can modify function and structure of proteins in a re versible w ay. The most recent data on protein S - glutathion ylation and, likely, Scysteinylation, suggest not onl y a role in e vents can protect protein reacti ve c ysteines from irreversible o xidative modif cations b y R OS/RNS, but also an in volvement in a number of signaling responses whose cellular dynamics still need to be elucidated. Inact, f S - glutathion ylated and S-cysteinylated proteins are no w not onl y re garded as mere biomark ers of o xidative damage, but these modif cations can also be considered a common feature of redo x signal transduction and regulation of the acti vities of redox-sensitive thiol -proteins. It is sur mised that the actual kno wledge of S - glutathion ylation and S -ysteinylation c mechanisms represents onl y the tip of an iceber g of w hat the f ne-tuned cellular e volution and the astonishing cellular adaptation to life reall y are.

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REFERENCES Ballatori N , HammondCL ,CunninghamJB , KranceSM ,MarchanR 2005 . .Molecularmechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins. Toxicology and Applied Pharmacology 204 ( 3 ): 238 – 255 . BanksDD , Gadgil HS , Pipes GD , Bondarenk o PV , HobbsV , Sca vezze JL , Kim J , Jiang X , MukkuV , DillonT . 2008 . Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody impro ves homogeneity, stability and biological activity. Journal of Pharmaceutical Sciences 97 ( 2 ): 764 – 779 . Bar - Or D , Curtis CG , Sulli van A , Rael LT , Thomas GW , Craun M , Bar- Or R , Maclean KN , Kraus JP. 2004. Plasma albumin cysteinylation is regulated by cystathionine β - synthase . Biochemical and Biophysical Research Communications 325 ( 4 ): 1449 – 1453 . Bar rett WC , DeGnore JP , K onig S , aFles HM , K eng YF , Zhang ZY , Y im MB , Chock PB. 1999 . Regulation of PTP1B via glutathion ylation of the acti ve site cysteine 215 . Biochemistry 38 ( 20 ): 6699 – 6705 . BrennanJP , Miller JI , FullerW , W ait R , Be gum S , Dunn MJ , Eaton P . 2006 .The utility of N,N - biotin yl glutathione disulf de in the study of protein S -glutathiolation. Molecular and Cellular Proteomics 5 ( 2 ): 215 – 225 . BrigeliusR , Muck el C ,Ak erboom TP , Sies H .1983 . Identifcation and quantitation of glutathione in hepatic protein mix ed disulf des and its relationship to glutathione disulf de. Biochemical Pharmacology 32 ( 17 ): 2529 – 2534 . Bur ns JA , Butler JC , Moran J , Whitesides GM .1991 . Selective reduction of disulf des by tris(2 - carbo xyethyl)phosphine . Journal of Organic Chemistry 56 : 2648 – 2650 . CabiscolE , Le vine RL .1996 .The phosphatase activity of carbonic anhydrase III is reversibly regulated by glutathionylation. Proceedings of the National Academy of Sciences of the USA 93 ( 9 ): 4170 – 4174 . ChenSH , Hsu JL , Lin FS .2008 . Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometr y. Analytical Chemistry 80 ( 13 ): 5251 – 5259 . ChuF , W ard NE , O ’ BrianCA. 2003 . PKC isozyme S - ycsteinylation by cysteine stimulates the pro - apoptoticisozyme PKCδ and inactivates the oncogenic isozyme PKC ε .Carcinogenesis 24 ( 2 ): 317 – 325 . Cotg reave IA , Gerdes R , Schuppe -oistinen K I , Lind C. 2002 . S - glutathion ylation of glyceraldehyde-3-phosphate dehydrogenase: role of thiol o xidation and catalysis by glutaredoxin . Methods in Enzymology 348 : 175 – 182 . Dalle - Donne I ,Aldini G , Carini M , Colombo R , Rossi R , MilzaniA. 2006 . Protein carbonylation, cellular dysfunction, and disease pro gression. Journal of Cellular and Molecular Medicine 10 ( 2 ): 389 – 406 . Dalle - Donne I , Giustarini D , Colombo R , MilzaniA , Rossi R. 2005 . S - glutathion ylation in human platelets by a thiol -disulf de exchange - independentmechanism . Free Radical Biology and Medicine 38 ( 11 ): 1501 – 1510 . Dalle - Donne I , Giustarini D , Rossi R , Colombo R , MilzaniA. 2003a . Reversible S-glutathionylation of Cys374 re gulates actin f lament formation by inducing str uctural changes in the actin molecule . Free Radical Biology and Medicine 34 ( 1 ): 23 – 32 . Dalle - Donne I , MilzaniA , Gagliano N , Colombo R , Giustarini D , Rossi R. 2008 . Molecular mechanisms and potential clinical signif cance of S - glutathion ylation . Antioxidants and Redox Signaling 10 ( 3 ): 445 – 473 . Dalle - Donne I , Rossi R , Colombo G , Giustarini D , MilzaniA. 2009 . Protein S - glutathion ylation: a regulatory device from bacteria to humans . Trends in Biochemical Sciences 34 ( 2 ): 85 – 96 . Dalle - Donne I , Rossi R , Giustarini D , Colombo R , MilzaniA. 2003b. Actin S - glutathion ylation: evidence against a thiol -disulphide exchange mechanism . Free Radical Biology and Medicine 35 ( 10 ): 1185 – 1193 .

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Chapter16 DNA Oxidation, Antioxidant Effects, and DNA Repair Measured with the Comet Assay M á riaDu š inskand á Andr ew R. Collins

INTR ODUCTION The choice of biomark ers in a human biomonitoring study is — or should be —dependent on the nature of the study and the aims of the in vestigation. It is impor tant to be clear about w hat exactly a biomarker can tell us. DNA damage is generally regarded as a biomarker of exposure to environmental mutagens, w hich induce lesions in the DN A. These lesions, if not repaired by the cell, can result in mutations. If specif c genes are affected, they can also result in tumorigenic transformation. Though it is tempting to re gard DNA damage as a biomark er of cancer risk, and there is an ob vious underl ying mechanism, there is no e vidence from prospecti ve studies that a high le vel of DN A damage is associated with a high risk of cancer (with the exception of isolated cases such as af atoxin, where a high le vel of adducts measured in DNA is indicati ve of a high risk of hepatic cancer , especiall y in association with hepatitis infection). DNA oxidation can result from e xposure to v arious environmental mutagens, but probab ly the main cause of damage is attack b y endogenous free radicals, in par ticular superoxide radicals released from the respirator y chain in mitochondria. White blood cells (usuall y fractionated, as l ymphocytes) are the usual source of DN A to measure DN A o xidation; w hat is measured is the steady state level of damage, a balance between input (free radical attack) and cellular repair. An increase in repair rate should lead to a decrease in the steady state o xidation level, and vice v ersa. A low steady state le vel of damage ma y also ref ect a high le vel of antioxidant defenses, whether intrinsic (antioxidant enzymes, glutathione etc.) or derived from the diet (vitamin C, carotenoids, etc.) Thus, the relation betw een DNA damage and cancer risk is not a simple one. Furthermore, to obtain a full picture it is impor tant to consider not just DNA oxidation but also antio xidant status and the capacity of cells for DN A repair . The comet assay—essentially a method for detecting DN A strand breaks —has been adapted to measure all three of these. This chapter describes the comet assa y in its v arious modif ed forms and its application to the study of en vironmental and occupational e xposure to mutagens. It also deals with the Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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application of the technique to the question of nutritional f actors that can affect DNA stability and, potentially, cancer risk. Genetic f actors are seen as being v ery important, though there is a need for lar ge-scale investigations to gi ve enough v ariants for signif cant conclusions to be made. Finally, the chapter looks at the e vidence for changes in the le vels of DNA damage and repair with age and changes that are associated with chronic diseases.

PRINCIPLES OF THE COMET ASSAY AND APPLICATIONS TO BIOMARKER MEASUREMENT The comet assay (single cell gel electrophoresis, Figure 16.1) is known as a sensitive and simple method for measuring DN A damage (Collins 2004). Cells are embedded in a thin la yer of agarose on a microscope slide, and l ysed in a solution containing a non -ionic detergent and 2.5M NaCl. This removes membranes and most solub le cell contents, including histones, but leaves the DN A, which retains the supercoiled for m that it had in the nucleosomes. Because it is attached at inter vals to the nuclear matrix, the DN A can best be visualised as a series of constrained loops, rather than as a linear molecule; one loop is the stretch of DN A between two attachment points. The presence of breaks in the DNA loops relaxes their supercoiling and they can then e xtend under electrophoresis to for m the characteristic image of a comet tail (Collins 2004). The relative amount of DN A in the tail ref ects the number of relax ed loops and therefore the number of DN A strand breaks (SBs). A simple w ay of scoring comets is based on their appearance to the e ye (visual scoring). One hundred comets are scored in each gel, and assigned a v alue according to their classif cation, from zero (undamaged) to 4 (maximal e xtension of tail). Thus, the score of the whole gel has a value of between zero and 400 arbitrar y units. Computer-based image analysis is an alternative method. Fluorescence is measured over the head and tail of each comet, and preferab ly expressed as the percentage of DN A in the tail. There are other measures, notab ly tail length and tail moment. Tail length increases onl y over Cells embedded in agarose on microscope slide

Lysis: Triton X-100, 2.5 M NaCl ± Digestion with lesion-specific endonuclease, e.g. FPG for 8-oxoGua

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Figure 16.1. The comet assay. A schematic outline of the method, including digestion with lesion-specif c endonuclease to reveal base damage. (Inset graph from Collins et al. 2008 reprinted with permission from Oxford University Press).

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a narrow range of lo w damage levels. Tail moment, an amalgam of tail length and percentage of tail DNA, is not recommended as a quantitative measure because it has no generally accepted units and it is impossible to visualize from a tail moment value what the comet being described looks like. For most pur poses, relative damage levels are all that matters, and results can be left in the form of “percent of DN A in tail ” or “visual score; arbitrar y units.” However, sometimes it is important to kno w the real amount of damage that is present, in units such as breaks per 10 9 daltons, or per cell, or per million undamaged bases. In that case, it is necessar y to calibrate the assay. The assay can be calibrated using cells treated with dif ferent doses of ionizing radiation. A dose of about 10 Gy causes maximal relaxation of loops, with a majority of the DN A being in the tail, although there is some v ariation between different laboratories in the actual ratio of percentage of tail DN A to X -ray dose (Collins et al. 2008).

MEASURING DNA OXIDATION An early modif cation of the assay involved the application of specif c repair endonucleases to convert lesions to breaks. F or example, endonuclease III (EndoIII) w as used to measure o xidized p yrimidines (Collins et al. 1993), and for mamidopyrimidine DN A gl ycosylase (FPG) was used to measure o xidized purines, specif cally 8 -oxoGua and purine breakdown products, the formamidopyrimidines FaPyGua and FaPyAde (Du šinská and Collins 1996). This modif cation has been par ticularly useful for assessing the dynamic steady state backg round level of DNA oxidation in human l ymphocytes. Results from the analysis of lymphocyte DNA damage in a sample of 25 people (a subgroup of the Boyd Orr Cohort, described below) are shown in Figure 16.2 to illustrate the estimation of oxidized bases. In F igure 16.2a, the breaks detected on incubation with FPG are indicated for each subject b y the total height of the bar . This includes both SBs (including alkali -labile sites) and FPG -sensitive sites. SBs are estimated from a parallel incubation with buf fer alone, and are shown by the dark er lower part of each bar . The remaining breaks (the lighter par t of the bar , i.e., the dif ference betw een breaks with and without enzyme) are the net -enzymesensitive sites, and this is the parameter used as an inde x of base o xidation. Oxidized pyrimidines and purines have a common cause, i.e. o xidative damage, and so it is to be e xpected that the indi vidual values for FPG sites and EndoIII sites will cor relate, as is clear from F igure 16.2b. That the cor relation is not g reater is not sur prising, since the e xact mechanisms of free radical reaction dif fer from base to base —quite apart from the ine vitable variability in experimental determinations. Advantages of the comet assa y include its simplicity , speed, and lo w cost. Lik e most techniques, its value depends on how well it is learned and performed, and in the case of the comet assay an understanding of the principles is especiall y useful. There are, unfor tunately, various misunderstandings, notably the belief that simpl y r unning the comet assa y electrophoresis at neutral pH will detect only double strand breaks, while an alkaline pH also detects single strand breaks. There are other techniques, such as alkaline/neutral elution, w here this rule is true, but in the case of the comet assa y, based on the relaxation of supercoiling, denaturation of DN A is not necessar y to reveal a single strand break. This is clearly demonstrated by comparing the original version of single cell gel electrophoresis (Ostling and Johanson 1984), w hich w as car ried out at pH 10.0 (w ell belo w the pH needed to denature DNA), with the f rst alkaline comet assay, described by Singh et al. (1988). In both cases, ionizing radiation w as used to test the sensiti vity of the assa y, and in both cases the ef fect of a fraction of a Gy w as detected. Doub le strand breaks are for med with a yield about 1/20 of that of single strand breaks, so it is most unlik ely that such similar results would have been obtained if onl y double strand breaks w ere registered in the neutral assay.

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Figure 16.2. (A) Results from an experiment to measure base oxidation in lymphocyte DNA. Each bar represents one individual. The whole bar represents the total DNA breaks detected after incubation with FPG, which include the breaks present after buffer incubation (the lower, darker-shaded part of the bar). Net enzyme-sensitive sites are estimated by subtracting the mean buffer score from the overall damage score. (B) Net EndoIII-sensitive sites and net FPG-sensitive sites in a sample of healthy volunteers.

EUROPEAN STANDARDS COMMITTEE ON OXIDATIVE DNA DAMAGE ( ESCODD ):A VALIDATION EXERCISE The comet assay may be simple and fast, but is it accurate enough to be used in biomonitoring? The original methods used to measure DN A oxidation as a biomark er were chromatographic: GC-MS and HPLC with electrochemical detection. Results w ere expressed in dif ferent units, and methods w ere rarely compared. In the 1990s it became clear that GC -MS was routinely giving values for 8 -oxoGua in nor mal human w hite blood cells that w ere about 10 times (or more) greater than the v alues given by HPLC. When FPG -based methods came to be applied to this purpose, a further discrepancy appeared; the enzymic approach gave levels of 8-oxoGua that were an order of magnitude below the estimates by HPLC. This difference was seen when the same human l ymphocyte samples w ere anal yzed b y the tw o methods in one laborator y (Collins et al. 1997). Because of the percei ved signif cance of 8 -oxoGua as an indicator of oxidative stress, and as a possib le precursor of cancer , there w as clearl y an ur gent need to discover the cause of these discrepancies and estab lish the tr ue background level of damage.

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The European Standards Committee on Oxidati ve DNA Damage (ESCODD) w as set up in 1997. It w as a concer ted action suppor ted by the European Commission from 2000 to 2003, with about 25 members. ESCODD operated by sending standard samples to partners for analysis. These samples included solutions of 8 -oxodGuo, calf thymus DNA, oligonucleotides with known content of 8 -oxoGua, li ver, and HeLa cells with and without additional 8 -oxoGua induced by photosensitizer Ro 19 -8022 plus light. Only at this last stage could methods based on FPG be included, because they require intact cellular DNA. Figure 16.3 shows the comparative results of measurement of DN A base o xidation b y chromato graphic and FPG -based methods (mostly the comet assa y, but also including alkaline elution and alkaline unwinding, which are alter native methods for measuring DN A breaks) (ESCODD 2002). The range of estimates is wide (about 100-fold) with both chromatographic and FPG-based methods, though it seems that the latter are more closel y clustered around the median. Median v alues for the two approaches differ by a f actor of nearly seven. Varying amounts of damage w ere induced in HeLa cell DN A with Ro 19 -8022 plus light, and measured by either HPLC or FPG and the comet assay. When dose responses were plotted (8-oxoGua against dose of Ro 19 -8022 plus light), slopes were very similar, indicating similar eff ciency at detecting induced damage. Ho wever, y -axis intercepts w ere several times higher with HPLC than with the comet assa y, impl ying the occur rence of spurious o xidation in chromatographic sample preparation (Gedik et al. 2002). The f nal ESCODD trial in volved recr uiting subjects in par tners’ own countries to donate lymphocytes for analysis. Mean values from the population samples in different countries/laboratories v aried widel y, especiall y w hen HPLC w as used. Once again, there w as an order of magnitude dif ference betw een median v alues of the population sample means for the tw o approaches. It seems that the adv entitious o xidation that ar tif cially ele vates backg round damage measured by chromatography cannot be reliably eliminated. ESCODD concluded that the most lik ely true background level of 8 -oxoGua is betw een 0.3 and 4.2 per million uno xidized guanines, i.e., betw een the median v alues for the comet assa y and HPLC, calculated from the sample means in the v arious countries. This is much lo wer than suggested in man y published studies.

LIMIT ATIONS OF THE COMET ASSAY Although the comet assa y in combination with FPG probab ly gives a measure of backg round DNA oxidation that is close to the true value, i.e., it is relatively accurate, it is not very precise. Individual v alues for 8 -oxoGua are bound to be appro ximate. When comparisons are being made at the level of groups (such as exposed/nonexposed, patients/controls, or subjects of different ages), the lack of precision is not so impor tant. Since the ESCODD trial, w hen fewer than half of the participating laboratories were able to detect a dose response in DNA of HeLa cells treated with Ro 19 -8022 plus light, there has been a def nite improvement. In a recent similar inter -laboratory trial, seven of eight laboratories w ere able to detect the dose response in FPG-sensitive sites in A549 cells treated with Ro 19-8022 plus light (Johansson et al. 2010). Efforts are being made to reduce the v ariability seen in the comet assa y by measures such as calibration car ried out in each laborator y (w hich helps in inter -laboratory comparisons), standardization of protocols, and inclusion of reference standards. The best kind of reference standard is an inter nal standard. In the case of the comet assa y, these are cells with a kno wn level of damage that are embedded in the same gel with sample cells and are distinguishab le from the latter w hen visualized as comets after electrophoresis; for e xample, on account of a substantiall y higher or lo wer genome size and consequent dif ference in total f uorescence (an idea that has been de veloped b y G. Br unborg within the EC -funded project COMICS; http://comics.vitamib.com/ ). There is a concer n about the specif city of FPG. It is probab le that 8 -oxoGua is the main lesion detected in nor mal cells, but FPG is kno wn to reco gnize lesions other than 8 -oxoGua,

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Laboratory/method Figure 16.3. Analysis of oxidized base damage in HeLa cell DNA, using chromatographic techniques (A) or a method employing FPG to convert 8 -oxoGua to SBs. (B). In (A), HPLC with coulometric electrochemical detection was used except where indicated. In (B), all but two laboratories used the comet assay. Redrawn from ESCODD 2002 with permission from Oxford University Press .

notably the formamidopyrimidines (FaPy) that are formed by ring-opening of damaged purines (hence the enzyme ’s name). These FaPy products ma y arise from o xidative damage, but also by spontaneous ring -opening of Gua resulting from treatment with alk ylating agents (Speit et al. 2004). The lack of specif city should al ways be bor ne in mind. The mammalian analo gue of FPG, OGG1 (8 -oxoGua DNA glycosylase), has a nar rower specif city. In mark ed contrast to both FPG and EndoIII, it did not give any increase in breaks when incubated with nucleoids

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from cells treated with the alk ylating agent meth yl methane sulphonate (MMS) (Smith et al. 2006). Another worry is that FPG might underestimate damage because some lesions ma y be inaccessible, or ma y occur in clusters, re gistering in the comet assa y as a single break site. However, the ag reement with HPLC o ver the Ro 19 -8022 dose response slopes (described above) suggests that this is not the case, or that er rors of under - and o ver-estimation cancel each other out. Lymphocytes are obtained in a f airly nonin vasive w ay and are b y f ar the most common sample taken for measurement of DN A damage in humans. An important question is w hether they can be re garded as a sur rogate for cancer tar get tissues; i.e. do the y provide information that is rele vant to the process of carcino genesis? This question has not been satisf actorily answered. A systematic study is needed to compare damage levels in lymphocytes and in some other tissue, but the latter is diff cult to obtain. At the moment all that can be said is that oxidative DNA damage in lymphocytes probably ref ects the body ’s overall redox state, because the lymphocytes circulate throughout the body and are e xposed in all par ts of the body. The comet assay is labor -intensive and time -consuming in its standard for m. There are high throughput v ersions, for e xample 12 mini -gels on one slide, or 48 or 96 gels on a sheet of GelBond plastic f lm (de veloped and in use in the EC -funded projects COMICS and NewGeneris). The steps as f ar as electrophoresis are speeded up, but then scoring depends on a fast and eff cient automated image anal ysis system.

ASSESSINGANTIOXIDANT STATUS In addition to measuring the backg round level of o xidative DNA damage in human l ymphocytes, the comet assay can be used to assess antio xidant status in an ex vivo test. Lymphocytes are treated with H 2 O2 (typically 100 µM) for f ve minutes on ice (to prevent repair from occurring) and then SBs are assessed with the comet assa y. The best approach is to treat the cells after embedding them in agarose b y placing the slide in ice -cold H 2 O2 solution. This avoids the possibility of repair occur ring w hile the cells are in w arm agarose. There is an in verse relationship between DNA break frequency and antioxidant status of the cells.

MEASURING DNA REPAIR The v ersion of the comet assa y in w hich lesion -specif c endonucleases are used to measure particular kinds of damage is no w widel y used in human biomonitoring studies. It can be applied to the measurement of cellular DN A repair by treating the cells with DN A-damaging agent, incubating them, and measuring the damage remaining at inter vals to plot the kinetics of repair. This approach has been attempted with freshl y isolated lymphocytes, but they show very slow repair of DN A SBs or o xidized bases w hen compared with cell lines in culture. If lymphocytes are incubated for 24 or 48 hours before inducing damage, SB rejoining is accelerated (Collins et al. 2008), and others ha ve reported rapid rejoining in l ymphocytes stimulated with ph ytohemagglutinin (Schmezer et al. 2001). In vie w of this complicated situation, the authors sought a method for measuring l ymphocytes’ potential for repair b y de veloping an in vitro assay (Collins et al. 2001). The assay is related to the comet assa y with lesion -specif c enzymes, but instead of using def ned enzymes to detect and measure an unkno wn amount of damage, the authors tak e cells with a kno wn amount of a specif c kind of damage as a substrate to measure the unkno wn activity of repair enzymes in a cell-free extract. The extract may be prepared from lymphocytes collected during a human biomonitoring or inter vention study or from cells in culture. The substrate cells, after treatment with a specif c DNA-damaging agent, are embedded in agarose on a microscope slide and l ysed to produce nucleoids, as in the standard comet assa y. The cell-free extract is then incubated with this substrate, and the breaks introduced in the nucleoid DNA represent the repair acti vity of the e xtract. If the substrate cells w ere treated with Ro

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Table 16.1. Range of BER and NER activities in human lymphocytes.

Repairrate (arbitrary units) N Baseexcision repair Nucleotideexcision repair

36 36

Minimum

Maximum

Range ( - fold)

27 9.5

86 70

3.2 7.4

Blood samples w ere tak en from health y subjects o ver a period of se veral months during a crosso ver nutritional intervention trial (Gaivão et al. 2009). Samples analyzed here are from pre- and post-intervention periods and are not inf uenced by the intervention. Repair activities were measured with the in vitro comet assay based method, as described. Mean values from four lymphocyte samples from each individual were calculated.

19-8022 plus visible light, inducing 8-oxoGua in the DNA, then the assay measures the activity of OGG1, w hich is involved in base e xcision repair (BER) (Collins et al. 2001). (The e xtract lacked incision acti vity in a control e xperiment using cells from OGG1 − mice.) Langie et al. (2006) modif ed the assa y b y treating substrate cells with benzo(a)p yrene diolepo xide; the bulky DN A adducts so for med are repaired b y nucleotide e xcision repair (NER). UV(C) irradiation of substrate cells dimerises adjacent p yrimidines, which are also repaired b y NER. Thus, the nature of the damage in the substrate comet DN A def nes which variety of repair is detected. In a small study with 36 health y volunteers, the authors applied the in vitro assay for BER, using as substrate nucleoids from cells treated with Ro -19-8022 plus light, and nucleoids containing pyrimidine dimers induced by UV for NER. Repair rates for each subject measured in blood samples tak en on dif ferent occasions cor related signif cantly, indicating that each individual has a characteristic le vel of repair enzyme acti vity (Gaiv ão et al. 2009). No cor relation was found betw een BER and NER acti vities. There w as a considerab le v ariation betw een individuals, estimated at three -fold for BER and se ven-fold for NER (T able 16.1). The range was estimated b y comparing the mean v alues for each subject o ver the four sampling times. This range is wide enough to gi ve plenty of oppor tunity to e xplore the f actors af fecting it, whether they are genetic, or en vironmental. On these g rounds, DNA repair is highl y suitable as a biomarker in human biomonitoring.

SOMECONSIDERATIONS WHEN DESIGNING AND CARRYING OUT A BIOMONITORING TRIAL There are advantages to an approach that depends on the use of molecular biomarkers (molecular epidemiolo gy): it is generall y f ar quick er and requires f ar fe wer subjects than a trial in which disease or death is the measured outcome, and of course is much less costl y. Furthermore, if biomarkers are chosen with care, the y can pro vide much v aluable mechanistic infor mation about ef fects of e xposure to a par ticular agent, or the ph ysiological response of the body . However, w hile such studies are simpler , the y still require the obser vance of some general principles that under pin biomonitoring studies. In addition, there are some specif c conditions that apply to the use of the comet assa y. The number of subjects in a trial should be decided on the basis of a po wer calculation, which depends on the expected effect of a treatment or expected difference between differently exposed groups, the variability observed between individuals in the biomark er of interest, the probability that an obser ved ef fect has not arisen b y chance (nor mally set at 0.05), and the power required, e.g., 80% or 90% probability that a tr ue effect (or difference) will actually be observed. There are v arious models for calculating po wer. Often it is necessar y to mak e

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assumptions, or informed guesses, about the extent of the expected effect or the inter-individual variability if the biomarker has not been widely used before. Subject numbers vary from around 20 in a typical crosso ver nutritional inter vention, to hundreds w hen comparing occupationally exposed subjects with a control g roup, or thousands when genetic variation is to be tak en into account, especially when infrequent genetic pol ymorphisms or interactions betw een different genes are investigated. Background levels of DN A damage ma y vary with time of y ear (M øller et al. 1998, 2002 ; Dušinská et al. 2002); these are so -called seasonal ef fects. It is therefore impor tant, w hen organizing a trial, to take samples from cases (or exposed) and controls, or treated and placebo groups, in parallel, rather than in consecuti ve phases. Sample preparation and storage are cr ucially impor tant. Standard methods for isolating viable l ymphocytes, using a density step g radient (with L ymphoprep or similar), should be followed. Sometimes it is possib le to r un the comet assa y on these samples immediatel y, but more often they must be stored (and in an y case replicates should be stored e ven if immediate analysis is possib le). Aliquots of l ymphocytes in freezing medium (MEM or RPMI culture medium with fetal calf ser um and 10% DMSO) should be frozen slo wly to -80 °C and stored at that temperature or in liquid nitro gen. Duplicate gels should be prepared from each sample and for each end -point (SBs, H 2 O2sensitivity, FPG - and EndoIII -sensitive sites, DNA repair, etc.) Slides should be coded so that the scoring is performed blind. Negative and positive controls should be included in each comet assay experiment. These can conveniently be lymphocytes, prepared in bulk, and treated with X - ra ys or H2 O2 (positive control) or untreated (negative control) before being frozen in suitable aliquots and in suff cient number to last for the w hole period of the trial. Slides can be archi ved. After scoring, simpl y wash in w ater to remo ve stain, allo w to dr y on the bench, pack in bo xes, and store at room temperature. Image analysis provides data for each indi vidual comet. The distribution of comets within the sample ma y be of interest w hen inter preting the nature of the damage or the cellular response. But for statistical e valuation, comparing population g roups, or different phases in a supplementation trial, w hat matters is the o verall comet score for each sample, such as the median percentage tail DN A.

OCCUP ATIONAL, ENVIRONMENTAL, AND EXPERIMENTAL EXPOSURE TO GENOTOXIC AGENTS The comet assa y has been used as a biomark er assa y in studies of e xposure of humans to various en vironmental mutagens/carcino gens (estab lished or potential). In most but not all cases, exposure occurred as a consequence of a par ticular occupation. In addition to measurements of endogenous DNA damage, studies of antio xidant status and BER acti vity have been reported. Exposure of male workers (for four years on average) to organic solvent-based adhesives in a footwear factory was associated with an increase in l ymphocyte DNA damage (SBs) w hen compared with either unexposed controls or workers exposed to water-based adhesives (Heuser et al. 2005). Micronuclei (MN) (in binucleated l ymphocytes and exfoliated buccal cells) w ere not signif cantly different. Vodicka et al. (2004b) e xamined styrene -exposed w orkers and controls, cate gorizing the workers according to the le vel of styrene in their w orkplace; zero (control g roup), 1 to 50 mg/ m3, or higher than 50 mg/m3. There were 20 or more in each g roup. DNA SBs (though not EndoIII-sites) cor related ne gatively with e xposure le vel. The rate of SB rejoining after γirradiation as well as OGG1 activity (measured with the in vitro assay) increased with styrene level. Effects of e xposure in tw o f actories in volved in r ubber and plastic manuf acture w ere examined. Workers in a r ubber tire f actory (N =29), e xposed to a broad spectr um of

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polycyclic aromatic h ydrocarbons, w ere compared with tw o control g roups (administrati ve staff and laboratory personnel at the same factory; 22 subjects each) (Somorovská et al. 1999b). In a lamination plant, w here styrene w as the onl y contaminant, 17 hand laminators directl y exposed to styrene, 10 medium -exposed sprayers, and 18 controls w ere studied (Somoro vská et al. 1999a). In both factories, exposed workers had signif cantly elevated levels of DNA SBs compared with controls (p < 0.001). Signif cant correlations were found between SBs in l ymphocytes and years of exposure (r = 0.57, P < 0.001, r = 0.55, P < 0.001, respectively) in both studies. Occupational exposure to w ood dust is a reco gnized hazard. P alus et al. (1999) measured DNA damage in w hite b lood cells (WBC) from 35 w orkers in volved in w ooden fur niture manufacture and 41 control subjects. SBs were assessed in cells before and after incubation in medium at 37 °C. In the w oodworkers, the fraction of cells with comet tails w as double that seen in the controls. A slight but signif cant decrease in the fraction of damaged cells was seen when the cells w ere incubated , presumab ly ref ecting DN A SB repair in the WBC of woodworkers. Wood-dust exposure in the wooden furniture industry was also the subject of an investigation by Rekhadevi et al. (2009). MN and chromosome aber rations were measured in addition to DNA SBs by the comet assa y. Sixty car penters were compared with 60 non -exposed subjects matched by age, smoking, and alcohol consumption; all three indices of genetic damage w ere elevated in the carpenters’ blood. Age, smoking, alcohol consumption, and duration of exposure were signif cant factors in deter mining the le vel of DN A damage. Antioxidant enzyme le vels were signif cantly decreased in the e xposed subjects. Workers in a cigarette f actory, exposed to tobacco dust, w ere studied b y Zhu et al. (1999). They were compared with unexposed managerial workers. Exposed workers had slightly higher levels of DNA damage. Occupational exposure and smoking both had signif cant and independent effects on the le vel of damage. Tunnel constr uction w orkers are e xposed to dust (alpha -quartz and other par ticles from blasting), gases such as NO 2, diesel e xhaust, and oil mist. DN A damage (SBs and o xidized bases), sister-chromatid exchanges (SCE), and MN were measured in 39 underground workers and 34 unexposed subjects (Villarini et al. 2008). There were no signif cant differences in the levels of DNA damage or SCE frequenc y between the tunnel workers and controls, while MN were elevated in the e xposed subjects. The ef fect of e xposure to As and Pb in children li ving in a contaminated mining area in Mexico was investigated by Yáñez et al. (2003). Levels of As in urine and Pb in b lood were high compared to those found in children li ving in a less e xposed town, and DN A damage (SBs) was also elevated. Another study from Me xico showed that although DN A damage was higher in As- and Pb exposed children, there w as no association betw een DNA damage and the le vel of e xposure (M é ndez - G ó mez et al. 2008 ).DNA repair (rejoining of H2 O2 - inducedbreaks) was decreased in exposed children and the repair rate w as negatively associated with the level of As in urine. Workers exposed to cobalt dust, or to hard metal dust, w ere compared with matched une xposed controls from the same plants (De Boeck et al. 2000). No signif cant increase in DN A SBs in l ymphocytes (or in MN or urinar y 8 -oxodGuo) w as detected in w orkers e xposed to Co-containing dust compared with controls. DNA base o xidation, antio xidant status, and BER acti vity on a substrate of DN A with 8-oxoGua w ere measured b y Du šinská et al. (2004b) in l ymphocytes from for mer asbestos factory workers, compared with controls. DN A damage (o xidized pyrimidines) was higher in exposed men compared with non-exposed men (P = 0.04). There was also a positive association of SBs and alk ylation damage to DN A with age (P = 0.04 in both cases). Moreo ver, oxidized pyrimidines (P = 0.01) and alkylated bases (P = 0.001) strongly correlated with years of occupational exposure. When BER capacity w as measured, no difference was seen overall, though when the se xes w ere e xamined separatel y, asbestos -exposed w omen sho wed a signif cantly

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lower repair rate than female controls. Asbestos production ceased almost one y ear before the blood samples w ere taken, and it is sur prising to see such an ef fect on repair persisting o ver such a long period. Zhao et al. (2006) compared 104 asbestos -exposed w orkers and 101 control w orkers in Qingdao City (China) for asbestos -induced DN A damage and DN A repair capacity . DN A damage (SBs) w as signif cantly higher in asbestos -exposed workers than in control w orkers, and rejoining of H 2 O2-induced SBs was retarded in asbestos -exposed subjects. Workers cur rently e xposed to man -made mineral f bers (mineral w ool) w ere studied (Dušinská et al. 2004a, 2006b; Star uchova et al. 2006, 2007, 2008). SBs in l ymphocyte DNA were higher in all e xposed workers compared to controls, as w ell as in e xposed non -smokers compared with control non -smokers. Exposure had no ef fect on specif c base damage (o xidation or alk ylation). A strong association w as seen betw een base DN A damage (FPG - and EndoIII-sensitive sites) and antioxidant enzymes GST (negative) and GPX (positive) measured in lymphocytes; this cor relation was found in the w hole population as w ell as in man y subgroups (exposed, controls, men, women, smokers, nonsmokers) (Staruchova et al., 2008). There was no signif cant difference in DNA repair capacity between exposed and controls. However, an interesting f nding was the negative correlation between repair rate and MN frequenc y (the latter representing chromosome damage, and recentl y estab lished as a predicti ve mark er of cancer risk). It seems that unrepaired 8 -oxoGua contributes to the for mation of MN. In another study of mineral f ber exposure, 116 workers in a glass f ber factory were examined (36 controls, 80 e xposed) (Du šinská et al. 2006b, Kazimiro va et al. 2007, and Du šinská et al., submitted). DN A SBs w ere elevated in e xposed workers. EndoIII -sensitive sites w ere signif cantly higher in exposed subjects taken as a whole, and in exposed male workers (Figure 16.4). There was no difference in repair rates betw een exposed subjects and controls. There are few examples of experimental exposure of humans to environmental contaminants. However, brief e xposure to agents that are commonl y found in e veryday life is deemed to be acceptable, and so it w as possib le to test the ef fect of ultra -f ne par ticles (UFPs) in traff cpolluted city air. On separate occasions, volunteers were exposed to air from a busy city street, with and without f ltration. The air w as pumped into a small room in w hich the v olunteers stayed for 24 hours, with tw o periods of strenuous bic ycle exercise. UFP e xposure increased the levels of DN A SBs and FPG -sensitive sites, but OGG repair rate w as unaltered (Br äuner et al. 2007). An ele vated le vel of DN A damage (SBs) w as found in b lood samples from operators of diesel-powered tr ucks in a mine in Estonia, compared with surf ace w orkers at the mine Exposed

EndoIII sites/arbitrary units

100 75

Controls

p=0.08

p=0.03

50 25 0 All

Men

W omen

S

N-S

Figure 16.4. DNA damage (EndoIII -sensitive sites) in lymphocytes of workers in a glass f ber factory, and matched controls from the same town. Comparisons are shown for the whole group and for sub-groups: men, women, smokers (S), and non-smokers (N-S). Mean values are shown, plus SEM. (M. Dušsinská and colleagues, unpublished)

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(Knudsen et al. 2005). The underground workers were shown to have a 7.5-fold higher exposure to particle-associated 1 -nitropyrene (1 -NP). However, levels of 1 -NP DNA adducts, measured by 32P-postlabelling, did not dif fer between the two groups. Smoke from domestic w ood-burning stoves is a relati vely common, potentiall y hazardous human exposure. Experimental e xposure to wood smoke for four hours produced no increase in OGG acti vity or FPG -sensitive sites, but an increased concentration of OGG1 mRNA (Danielsen et al. 2008). DNA damage in b lood cells w as compared in outdoor and indoor w orkers in Me xico City and Puebla, Mexico (Tovalin et al. 2006). Personal exposure to v olatile organic compounds, PM (2.5; par ticulate matter of diameter 2.5 µm or less), and ozone w as monitored. Workers showing especially high levels of DNA damage also had signif cantly higher exposures to PM (2.5), ozone, and some v olatile organic compounds. DNA SBs and FPG -sensitive sites were measured in residents of Benin e xposed to different levels of urban air pollution: r ural subjects, suburban subjects, residents li ving near very busy roads, and “taxi-moto” drivers (Avogbe et al. 2005). Exposure was characterized by excretion of S-phenylmercapturic acid (S-PMA), a biomarker of benzene exposure, and by ambient UFP. DNA damage in blood cells (SBs and FPG-sensitive sites) increased in line with these pollution indicators. Tobacco smok e is, of course, the most pre valent environmental e xposure worldwide, and many studies have looked at smoking effects using the comet assay—either addressing smoking directly as a genoto xic exposure or in vestigating smoking as a potential confounder in occupational exposure studies. Results are conf icting. Hoffmann et al. (2005) carried out a meta analysis of 38 studies in which DNA SBs in lymphocytes were measured. Overall, signif cantly more DNA damage was seen in smokers than in non -smokers. Surprisingly, the difference was more convincing in the studies in w hich the comparison of smok ers and non -smokers was the main objecti ve than w hen smoking w as a confounding f actor. Also, studies in w hich visual scoring was used to assess comets w ere more likely to show a positive effect of smoking than were studies with damage assessed b y image anal ysis. This does not necessaril y indicate subjective bias in visual scoring; it is generall y found that visual scoring is more sensiti ve to low levels of damage.

EFFECTS OF NUTRITION ON DNA STABILITY The link betw een fr uit and v egetable consumption and reduced cancer incidence, as demonstrated by epidemiological trials, has long been attributed to the high content of antio xidants in these foods. After all, it is kno wn that o xidative damage can lead to mutations, w hich are the ra w material for carcino genesis. Oxidati ve DN A damage is measurab le in human cells (leuk ocytes or l ymphocytes) b y HPLC or FPG -based assa ys. It therefore mak es sense to in vestigate w hether DN A o xidation damage (and DN A repair capacity) is related to nutritional parameters deri ved from dietar y questionnaires or from plasma measurement of micronutrients. Nutritional information was obtained from the subjects in the mineral f ber exposure studies, via food frequenc y questionnaires. In addition, plasma le vels of selected micronutrients w ere measured. Altogether, 387 subjects (239 e xposed, 148 controls) w ere investigated. Intakes of fruits, vegetables, milk, and cereals were signif cantly, inversely correlated with FPG -sensitive sites in lymphocyte DNA in the w hole group, and similar cor relations were found in the subgroup of e xposed subjects. DN A repair rate (with the comet assa y-based in vitro BER assa y) positively correlated with consumption of white wine in all subjects, as w ell as in the e xposed group, and in all three monitoring locations. The DNA repair rate cor related negatively with consumption of v egetables in the control g roup o verall, and in all subjects in the asbestos monitoring study as well as in the e xposed subgroup in this location (Star uchova et al. 2006).

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The indi vidual cor relations w ere not strong (r = 0.2 or less), but the o verall picture clearl y indicates that nutritional f actors are signif cant determinants of DNA damage and repair. Measurement of concentrations of micronutrients in plasma gives more objective and reliable data than are obtainable from questionnaires, and accordingly stronger correlations were found with DNA damage and repair (Staruchova et al. 2006). Table 16.2 gives data for α - tocopherol, β-carotene, and retinol, cor related with FPG -sensitive sites. Retinol and α - tocopherolare positively associated with base o xidation, w hile β-carotene sho ws a ne gative cor relation. Correlations with EndoIII sites sho w a v ery similar patter n. In an earlier study (Collins et al . 1998 )a signif cant negative correlation was seen between plasma concentrations of lutein, βcarotene, or total carotenoids, and EndoIII -sensitive sites in l ymphocyte DN A. The authors pointed out that an association does not establish a cause. It could be that carotenoids are simply a good mark er of fr uit and v egetable consumption, and that something else in those foods is responsible for the protection against DNA damage. In that connection, the positive correlations of α-tocopherol and retinol with DN A oxidation might be related to their dietar y origins in animal and dair y products, and seeds and v egetable oils, respectively. The standard w ay to sho w whether an ing redient of the diet is causall y associated with a physiologial effect is to car ry out an inter vention trial. Thus, plant foods or pure antio xidant compounds, together or singl y, can be administered to human v olunteers to see w hether they can decrease the level of base oxidation in DNA or enhance the antioxidant status of the body. If so, this would apparently conf rm the so-called antioxidant hypothesis, and provide an explanation for the cancer -protective effects of fr uits and vegetables. Thef rst use of the comet assay to test the effects of an antioxidant supplement was reported by Green et al. (1994). They measured DNA SBs in white blood cells before and after breakfast, Table 16.2. Micronutrients and DNA damage.

Micronutrient α - ocopherol T

β - Carotene Retinol

Group Allsubjects Men Smok ers Non - smok ers Malecontrols Controlnon - smok ers Allsubjects Smok ers Exposednon - smok ers Allsubjects Exposed Control W omen Non - smok ers Exposedwomen Controlnon - smok ers Exposedsmokers

Numberof subjects 140 95 53 88 20 27 141 52 61 140 98 43 45 88 23 27 37

Cor relation with FPG - sites (r)

Probability

0.26 0.30 0.27 0.36 0.59 0.69 − 0.18 − 0.38 − 0.28 0.21 0.20 0.45 0.37 0.22 0.62 0.46 0.33

0.002 0.004 0.05 0.001 0.006 0.001 0.04 0.006 0.03 0.01 0.04 0.003 0.05 0.04 0.001 0.015 0.04

α - tocopherol,β-carotene, and retinol in plasma were assayed by HPLC, and DNA damage (FPG-sensitive sites) in l ymphocytes by the comet assa y. Signif cant cor relations only are sho wn for all subjects in the mineral wool study and for subg roups. Data from Star uchova et al. (2008).

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which included a dose of 500 mg of vitamin C, and found a signif cant decrease both in background SBs and in SBs induced b y γ-irradiation of the b lood sample. In a placebo -controlled parallel study design, both SBs and o xidized pyrimidines were measured (Duthie et al. 1996). Groups of about 50 male smok ers and non -smokers in their 50s w ere randomized, with half of each g roup receiving a dail y supplement of vitamin C, vitamin E, and β - carotene,and the other half taking a placebo. After 20 weeks, EndoIII-sensitive sites were signif cantly decreased in those taking the supplement compared with the placebo. In addition to measuring endo genous DN A damage, the authors treated the l ymphocytes in vitr o with H 2 O2 and found less damage in those from the supplemented g roup. Since then the authors have carried out various intervention trials. Comparing soy milk with rice milk and co w’s milk to look for possib le effects of f avonoids (phytoestrogens) in the so y milk, there w as a substantial and signif cant decrease in the g roup taking so y milk after four weeks (Mitchell and Collins 1999). A different kind of design w as adopted w hen looking for effects of kiwifr uit (Collins et al. 2003): this w as a crosso ver trial with onl y 14 v olunteers, who were divided into three g roups taking dif ferent “doses” of kiwifr uit (one, tw o, or three per day for three w eeks) in dif ferent orders, with w ashout periods betw een the supplementations. It was then possible to compare before and after biomark er values for each subject. The possibility of seasonal change, i.e., a change in a biomark er occurring with time but unrelated to the supplementation, w as a concer n, but the crosso ver design means that subjects took different supplements (doses) at the same time, and therefore seasonal effects were neutralized. Kiwifruit supplementation resulted in decreases in o xidized p yrimidines (measured with EndoIII), 8 -oxoGua (FPG -sensitive sites), and H 2 O2-induced DN A breaks, all of w hich are highly suggestive of an enhanced antio xidant status. Numerous other studies ha ve been car ried out using these biomark ers measured with the comet assa y. Roughl y half ha ve sho wn signif cant protection against DN A o xidation, w hile few if an y have convincingly demonstrated a potentiation of damage. It is still puzzling w hy some studies gi ve null results. In a careful trial b y M øller et al. (2003), parallel randomized groups of subjects recei ved a diet depleted of fr uits, vegetables, etc.; this diet plus 600 g/day of fruits and vegetables; or this diet plus vitamin tablets with antioxidants and minerals. During the 24 da ys of the study , plasma antio xidant levels fell in the depleted g roup and sta yed at a high level in the supplemented g roup, and y et there w ere no dif ferences in mark ers of DN A damage. Møller et al. suggest that in this group the inherent antioxidant defenses were suff cient to protect l ymphocytes against o xidative attack. Ho wever, in an o verview of man y studies, Møller and Loft (2006) have concluded that protective effects occur far more often than would be expected by chance. Thein vitro DNA repair assay has now been added to the battery of biomarker assays applied in nutritional studies. One w eek of supplementation with coenzyme Q10 resulted in a signif cant increase in OGG acti vity (Tomasetti et al. 2001). In the kiwifr uit study described abo ve (Collins et al. 2003), there was an increase in BER acti vity of extracts prepared from lymphocytes after one, two, or three kiwifruits (with no sign of a dose response). mRN A was isolated from lymphocytes of subjects who had shown a good response in the in vitro repair assay, and levels of expression of OGG1 and APE1 (AP -endonuclease 1, the enzyme that car ries out the next step after the gl ycosylase) were measured, but showed no difference between before and after samples. Guar nieri et al. (2008) found that slo w-release vitamin C capsules increased BER activity, while normal vitamin C supplements did not. Thus it seems that DN A o xidation is subject to modulation b y antio xidants or b y other phytochemicals present in fruits and vegetables. In addition to decreasing the incident damage, they can also af fect the rate at w hich damage is remo ved from the DN A. The v ariant of the in vitr o repair assa y w hich measures NER w as recentl y emplo yed to investigate possible effects of supplementation with b lueberry and apple juice (Langie et al. 2010). Although overall no ef fect was seen, in subjects car rying multiple pol ymorphisms of NER genes associated with reduced acti vity, there was an increase after supplementation.

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DN A DAMAGE, DNA REPAIR, AND AGE It has long been h ypothesised that aging ma y be caused b y the accumulation of damage to proteins, lipids, and nucleic acids, resulting in cellular and tissue dysfunction. One of the major causes of such damage is surel y endo genous o xidation. At least as f ar as DN A damage is concerned, the e vidence is not compelling. Applying an alkaline unwinding assa y (similar in some ways to the comet assa y) to human l ymphocytes, Barnett and colleagues repor ted more DNA breaks in 65- to 69-year-olds compared with 35- to 39-year-olds (Barnett and King 1995), but then found similar le vels in 75 - to 80 -year-olds to those in the y oungest age g roup (King et al. 1997). With the comet assa y, nonagenarians w ere found to ha ve similar le vels of SBs and oxidized bases as 40 - to 60 -year-olds (Hyland et al. 2002), while Mutlu -Türkoglu et al. (2003) reported a doubling in SBs betw een ages 21 and 40 and 61 and 85. In a comparison of l ymphocytes from three age -groups, 20 to 35, 63 to 70, and 75 to 82, a positive correlation was found between age and FPG-sensitive sites (Humphreys et al. 2007). The two older age groups were subgroups taken from the Boyd Orr Cohort, set up in the 1930s to study health ef fects related to household nutrition. Sur vivors of the cohor t were recalled in 2002 to investigate their current health status. More recently, the authors analyzed all available samples from that cohort (i.e., not just the two restricted age groups) and found no cor relation between base oxidation and age (within the age range 63 to 82). Another recent study, measuring 8 -oxodGuo in leuk ocytes with HPLC, found a 60% increase betw een g roups of health y subjects of mean age 31 and 67 (Siomek et al. 2007). In the authors ’ asbestos and mineral f ber exposure trials, 387 subjects (age 21 to 88) w ere studied; 239 were exposed to asbestos, mineral wool, or glass f ber and the rest were unexposed subjects. DNA base damage (FPG - and EndoIII -sensitive sites) increased with age, sho wing highly signif cant correlations when the whole population or subgroups of the population were analyzed (Du š insk et á al. 2006a Staruchova ; et al. 2008 ). Lymphocytes are not the most suitab le cell type to e xamine for long -term accumulation of DNA damage, because the y have a limited lifespan (a fe w months). Fur thermore, damage to DNA is perhaps not the best indicator , because the damage is subject to cellular sur veillance and continual repair (in contrast to proteins and lipids). A higher le vel of damage might be expected, however, if antioxidant defenses or DNA repair enzymes showed a deterioration with age. Comparing the three age g roups (Humphreys et al. 2007), there was a marginally signif cant positive correlation between age and DNA repair capacity (BER) and a g reater resistance to H 2 O2 damage in the v ery oldest sub -group of the Bo yd Or r Cohor t. Ho wever, w hen all Boyd Orr samples were recently analyzed, these patterns were not seen (Collins and Du šinská, unpublished). If there is a cor relation between DNA stability (a ter m that includes DNA damage, antioxidant resistance, and DN A repair) and age, it is lik ely to be w eak, at least w hen lymphocytes are the object of in vestigation. Animal studies, in w hich other tissues can be e xamined, have so far not re vealed any striking increases in damage with age (re viewed in Humphre ys et al. 2007 ).

INFLUENCE OF GENETIC POLYMORPHISMS ON DNA DAMAGE AND REPAIR Polymorphisms in genes for DN A repair ha ve been tested for ef fects on the enzyme acti vity measured with the comet assay; subjects homozygous for Cys at position 326 ofhOGG1 showed a lower repair activity compared with carriers of the Ser variant (Vodicka et al. 2007), but there was no detectable effect of polymorphisms in XRCC1 or XRCC3 (Vodicka et al. 2004a). Mateuca et al. (2005) examined whether variations in hOGG1, XRCC1 (genes for proteins involved in BER), and XRCC3 (coding for DNA double SB repair) contribute to inter -individual differences in genotoxic effects (MN, urinary 8-oxodG levels, and DNA SBs). Multivariate

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analysis with genotype, age, exposure status, type of plant, smoking, and their interaction terms as independent variables indicated that MN frequency and comet tail DNA were inf uenced by genetic polymorphisms. In the exposed and total populations, workers variant for both XRCC3 and hOGG1 had elevated MN frequencies. A caveat applies to these studies: onl y small numbers of subjects w ere used, which severely reduces the likelihood of detecting a real ef fect. TheXRCC1 gene, coding for a protein involved in BER, has a fairly common polymorphism at codon 399: substitution of T for G at this position changes Arg to Gln. Zhao et al. (2006), in their comparison of asbestos -exposed and non -exposed workers, found that comet scores were higher in w orkers with asbestosis with Gln/Gln than in those with Arg/Arg. This polymorphism w as also anal yzed in the authors ’ biomonitoring study in three Slo vak f actories producing mineral f bers (asbestos, glass f bers, and mineral wool) with a total of 387 e xposed and control subjects (Dušinská et al. unpublished). Subjects homozygous in the wild type allele GG (Arg/Arg) had signif cantly lower levels of oxidized purines compared with both GT heterozygotes and TT homozygotes (Arg/Gln or Gln/Gln). This association was seen in asbestos exposed subjects as well as in the whole asbestos group and in all women. However, SBs were higher in wild -type (Ar g/Arg) compared with v ariant. An association of SBs with XRCC1 variants was also seen in the glass f ber f actory. Sur prisingly, the authors found that XRCC1 variant is associated with higher DN A repair capacity for 8 -oxoGua; this was seen in exposed men from all three f actories. Slyskova et al. (2007) investigated, among other biomarkers, capacity to repair DNA oxidation damage b y using cell e xtract from l ymphocytes and the comet assa y in 24 lamination workers occupationall y e xposed to styrene and 15 une xposed controls. Signif cantly higher levels of DNA repair were observed in carriers of GSTM1+, compared to those with a deletion in GSTM1. In contrast to the results from the mineral f bers study, just described, the capacity for DNA repair w as signif cantly lower in indi viduals with the Gln/Gln v ariant genotype in XRCC1 than in those with Arg/Gln or wild -type Arg/Arg. A signif cantly lower repair capacity was also found in indi viduals with the wild -type Lys/Lys XPC genotype at L ys939Gln, as compared with those homozygous for the Gln/Gln v ariant genotype. However, the size of this study is reall y too small to dra w f rm conclusions concer ning the signif cance of genetic polymorphisms. Although XPA is kno wn as a protein in volved in NER of UV -induced damage and bulk y DNA adducts, it ma y also ha ve a role in the repair of o xidized bases (Du šinská et al., 2006). Participants in the authors’ study of occupational exposure to mineral f bers were also analyzed for the polymorphism 23A→G in the DNA repair gene XPA. Strong links were found between the variant XPA allele and elevated DNA oxidation damage in all subjects (P = 0.016,n = 354), as well as in sub -groups: women (P = 0.02, n = 150, both oxidized purines and p yrimidines), men (P = 0.004,n = 222), all controls (P = 0.05,n = 131), exposed women (P = 0.004,n = 75), exposed men (P = 0.009,n = 157),and non - smok ers (P = 0.015,n=238). OGG1 repair activity also increased with age, but w hen anal yzed according to XPA genotype, the increase w as observed only in those indi viduals with an A allele. In a study of 151 healthy, middle-aged men, smokers had higher levels of FPG- and EndoIIIsensitive sites in lymphocyte DNA, compared with non-smokers. However, when subjects were classif ed according to GST polymorphisms, the difference in FPG-sensitive sites was seen only in GSTP1 Val105Val homozygotes, w hile the dif ference in EndoIII -sensitive sites appeared only in those w ho were GSTT1 null (Du š insk et á al. 2001 )(Figure 16.5 ).

CONCLUSION The comet assa y represents a simple and accurate approach to measuring DN A damage and DNA repair, and it has been successfully used to estimate background levels of base oxidation,

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(a) 25

FPG sites (% DNA in tail)

Smokers

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15

10

5

0

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GSTP1 a/b

GSTP1 b/b

(b) EndoIII sites (% DNA in tail)

25 20

Smokers Non-smokers

15 10 5 0

GSTT1 +

GSTT1 null

Figure 16.5. Inf uences of genotype on levels of DNA damage in human lymphocytes. (A) FPGsensitive sites, mean values with SEM; subjects were classif ed according to GSTP1 genotype. Only the GSTP1 b/b homozygotes show a difference between smokers and non -smokers. (B) EndoIII-sensitive sites, mean values with SEM; subjects were classif ed according to GSTT1 genotype. Only subjects who are GSTT1 null show a difference between smokers and non smokers. Redrawn from Du šinská et al. (2001) with permission from Elsevier .

antioxidant resistance, and excision repair of oxidized bases. This approach has given valuable information about the de gree of o xidative stress in population g roups exposed to to xic environmental agents (often not v ery g reat), and about the ability of dietar y antio xidants, or an increase in fruit and vegetable consumption, to moderate the damage. The in vitro DNA repair assay based on the comet assa y is be ginning to elucidate some of the re gulatory mechanisms involved in maintaining genetic stability . However, there are man y gaps in our kno wledge, and inter pretation of the a vailable data can be hazardous. F or instance, there is no e vidence linking indi vidual DNA damage le vels with cancer risk, and it is b y no means clear that decreasing the already lo w level of DN A oxidation (e.g., by antioxidant supplementation) has an y effect at all on health or , specif cally,

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BER rate (arbitrary units)

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200 160 120 80 40 0 -40

0

50

100

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FPG sites (arbitrary units)

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Figure 16.6. Relationship between DNA damage (oxidized purines) and the activity of the repair enzyme that removes 8-oxoGua, OGG1. Lymphocytes were collected from subjects in the Boyd Orr Cohort. Collins AR, Du šinská M, unpublished results .

on avoidance of cancer. In fact, reactive oxygen species play an essential role in cell signalling pathways, and suppressing them too much might be deleterious. DNA repair capacity has been regarded as a marker of individual susceptibility. The assumption is that an individual with a low intrinsic rate of repair will be less able to deal with damage and therefore more prone to cancer . An inverse cor relation between repair rate and damage would be expected. Results show that individuals do seem to ha ve a characteristic repair rate, and also that repair rates v ary considerably in the population. But the lo gic might be different. Perhaps (and this surel y would not be a g reat surprise) a regulatory mechanism exists, so that repair genes are e xpressed, or enzymes acti vated, only if there is suff cient damage to justify the cellular effort. In that case, a positive correlation between damage and repair might be seen. So it is not at all clear w hether a high measured repair capacity is a “good” or a “bad” thing. Figure 16.6 displays the data on DN A damage and repair for the Bo yd Or r Cohor t. There is no correlation between the level of oxidized purines and the cellular capacity for repair of that damage. These are preliminar y results, and more data are needed before f rm conclusions can be drawn. There is plenty left for the comet assa y to achie ve, apar t from merel y monitoring DNA damage, in the f eld of human biomonitoring.

A CKNOWLEDGMENTS The skilled technical assistance of Turid Veggan is ackno wledged. Professor Richard Mar tin, PI on the Bo yd Orr Cohort at the Depar tment of Social Medicine, Uni versity of Bristol, provided the b lood samples for anal ysis as par t of the 2002 follo w-up funded b y the Wellcome Trust. Dr. Elise Whitley is thanked for providing the statistical analysis of the Boyd Orr Cohort data. We are g rateful to all those in volved in the F ibretox project, including subjects, medical personnel and researchers, for their dedicated par ticipation.

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Br ä uner EV , F orchhammer L , M ø llerP , Simonsen J , Glasius M et , al. 2007 . Exposure to ultraf ne particles from ambient air and o xidative stress -induced DNA damage . Env Health Persp 115 : 1177 – 82 . Collins AR. 2004 .The comet assay for DNA damage and repair . Molecular Biotechnology 26 : 249 – 61 . Collins AR ,AzquetaOscoz A , Brunborg G , Gai v ã o I , Gio vannelli L et , al. 2008 .The comet assay: topical issues . Mutagenesis 23 : 143 – 51 . Collins AR , Du š inskM á , Horvathova E , Munro E , Sa vio M , Stetina R .2001 . Inter- ndividual i differences in DNA base excision repair activity measured in vitro with the comet assa y. Mutagenesis 16 : 297 – 301 . Collins AR , Duthie SJ , DobsonVL .1993 . Direct enzymic detection of endogenous oxidative base damage in human l ymphocyte DNA. Carcinogenesis 14 : 1733 – 5 . Collins AR ,DuthieSJ , F illion L ,GedikCM ,V aughan N , W ood SG .1997 .Oxidative DNA damage in human cells: the inf uence of antioxidants and DNA repair. Biochem Soc Trans 25 : 326 – 31 . Collins AR , Harrington V , Dre w J , Melvin R .2003 . Nutritional modulation of DNA repair in a human intervention study. Carcinogenesis 24 : 511 – 5 . Collins AR , Olmedilla B , Southon S , Granado F , Duthie SJ . 1998 . Serum carotenoids and oxidative DNA damage in human l ymphocytes. Carcinogenesis 19 : 2159 – 62 . DanielsenPH , Br ä unerEV , Barregard L , S ä llstenG ,W allin M et , al. 2008 . Oxidatively damaged DNA and its repair after e xperimental exposure to wood smoke in healthy humans . Mutat Res 642 : 37 – 42 . DeBoeck M , Lardau S , Buchet JP , Kirsch -olders V M , Lison D. 2000 .Absence of signif cant genotoxicity in lymphocytes and urine from w orkers exposed to moderate le vels of cobalt containing dust: a cross -sectional study. Env Mol Mutagen . 36 : 151 – 60 . Du š insk Má , Barancok ova M , Kazimiro va A , Harrington V , V olkovova K et , al. 2004a . Does occupational exposure to mineral f bres cause DNA or chromosome damage? Mutat Res 553 : 103 – 10 . Du š insk Má , CollinsA .1996 . Detection of oxidized purines and UV - inducedphotoproducts in DNA of single cells, b y inclusion of lesion -specif c enzymes in the comet assa y. Altern Lab Animals 24 : 405 – 11 . Du š insk Má , CollinsA , Kazimiro va A , Barancok ova M , Harrington V ,et al. 2004b. Genotoxic effects of asbestos in humans . Mutation Research. 553 : 91 – 102 . Du š insk Má , Dzupink ov á Z ,Ws óvloá L , Harrington V , CollinsAR .2006a . Possible involvement of XPA in repair of o xidative DNA damage deduced from anal ysis of damage, repair and genotype in a human population study . Mutagenesis 21 : 205 – 211 . Du š insk Má , F icek A , Horsk áA , Ra švloá K , P etrovsk á H et , al. 2001 . Glutathione S - transferase polymorphisms inf uence the level of oxidative DNA damage and antio xidant protection in humans. Mutat Res 482 : 47 – 55 . Du š insk Má ,V allova B , Ursin yova M , Hladik ova V , Smolk ova B ,et al. 2002 . DNA damage and antioxidants; f uctuations through the y ear in a central European population g roup. Food and Chem Toxicol 40 : 1119 – 23 . DuthieSJ , MaA , Ross MA , CollinsAR .1996 .Antioxidant supplementation decreases oxidative DNA damage in human l ymphocytes. Cancer Res 56 : 1291 – 5 . ESCODD . 2002 . Comparative analysis of baseline 8 - xoo - 7,8 - dih ydroguanine in mammalian cell DNA, by different methods in dif ferent laboratories: an approach to consensus . Carcinogenesis 23 : 2129 – 33 . Gai v ã o I , PiasekA , Bre vik A , Shaposhnik ov S , CollinsAR .2009 . Comet assay - basedmethods for measuring DNA repair in vitro; estimates of inter - and intra -individual variation. Cell Biol Toxicol 25 : 45 – 52 . GedikCM , Bo yle SP , W ood SG ,V aughan NJ , CollinsAR .2002 . Oxidative stress in humans: validation of biomarkers of DNA damage . Carcinogenesis 23 : 1441 – 6 .

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GreenMHL , Lo we JE ,W augh APW , Aldridge KE .1994 . Effect of diet and vitamin C on DNA strand breakage in freshl y-isolated human white blood cells . Mutat Res 316 : 91 – 102 . Guar nieri S , Loft S , Riso P , P orrini M , Risom L et , al. 2008 . DNA repair phenotype and dietary antioxidant supplementation . Brit J Nutr 99 : 1018 – 24 . HeuserVD , deAndrade VM , da Silva J , Erdtmann B. 2005 . Comparison of genetic damage in Brazilian footwear-workers exposed to solvent-based or water-based adhesive. Mutat Res 583 : 85 – 94 . Hof fmann H , H ö gelJ , Speit G .2005 .The effect of smoking on DNA effects in the comet assay: a meta - anal ysis . Mutagenesis 20 : 455 – 466 . Humphre ys V , Martin RM , Ratclif fe B , Duthie S ,W ood S et , al. 2007 .Age - relatedincreases in DNA repair and antio xidant protection: A comparison of the Bo yd Orr Cohort of elderly subjects with a y ounger population sample . Age and Ageing 36 : 521 – 526 . HylandP , Duggan O , T urbitt J , Coulter J , W ikby A et , al. 2002 . Nonagenarians from the Swedish NONA Immune Study ha ve increased plasma antio xidant capacity and similar le vels of DNA damage in peripheral b lood mononuclear cells compared to y ounger control subjects . Exp Geront 37 : 465 – 73 . JohanssonC , M ø llerP , F orchhammer L , Loft S , Godschalk RWL ,et al. 2010 .An ECVAG trial on assessment of o xidative damage to DN A measured by the comet assa y. Mutagenesis 25 : 125 – 32 . Kazim ví ro á A , Barancok ov á M ,Ws óvloá L , Dusinsk áM. 2007 . Cytogenetic analysis of lymphocytes of workers occupationally exposed to rockwool and glass f bres. Molecularepidemiologic study . Chemicke Listy 101 : 192 – 193 . KingCM , Bristo w - Craig HE , Gillespie ES , Barnett YA. 1997 .In vivo antioxidant status, DNA damage, mutation and DN A repair capacity in cultured l ymphocytes from healthy 75 -80-yearold humans . Mutat Res 377 : 137 – 47 . KnudsenLE , Gask ell M , Martin EA , P oole J , Scheepers PT ,et al. 2005 . Genotoxic damage in mine workers exposed to diesel e xhaust, and the ef fects of glutathione transferase genotypes . Mutat Res 583 : 120 – 32 . LangieSAS , KnaapenAM , Brauers KJJ , avn Berlo D , avn Schooten F - J, GodschalkRWL. 2006 . Development and validation of a modif ed comet assay to phenotypically assess nucleotide excision repair. Mutagenesis 21 : 153 – 8 . LangieSA ,W ilms LC , H ä m ä l ä inen S , Kleinjans JC , Gosdchalk RW , avn Schooten FJ. 2010 . Modulation of nucleotide e xcision repair in human l ymphocytes by genetic and dietar y factors. Brit J Nutr 103 : 490 – 501 . MateucaR ,Aka PV , DeBoeck M , Hauspie R , Kirsch -olders V M , Lison D. 2005 . Infuence of hOGG1, XRCC1 and XRCC3 genotypes on biomark ers of genotoxicity in workers exposed to cobalt or hard metal dusts . Toxicol Lett 156 : 277 – 88 . M é ndez - G óJmez , Garc í aargas - V GG , L ó pez - rillo Car L , Calder ó n - Aranda ES , G ó mez A et , al. 2008. Genotoxic effects of environmental exposure to arsenic and lead on children in re gion Lagunera, Mexico. Ann NY Aca. Sci 1140 : 358 – 67 . MitchellJH , CollinsAR .1999 . Effects of a soy milk supplement on plasma cholesterol levels and oxidative DNA damage in men —a pilot study. Eur J Nutr 38 : 143 – 8 . Mutlu - Toglu ü rk U , Ilhan E , Oztezcan S , K uru A ,A ykac - T oker G , Uysal M. 2003 .Age- related increases in plasma malondialdeh yde and protein carbon yl levels and lymphocyte DNA damage in elderly subjects . Clinical Biochem 36 : 397 – 400 . M ø ller P , Loft S .2006 . Dietary antioxidants and benef cial effect on oxidatively damaged DNA. Free Rad Biol Med 41 : 388 – 415 . M ø ller P , Knudsen LE , F rentz G , Dybdahl M ,W allin H , Ne x ø BA . 1998 . Seasonal variation of DNA damage and repair in patients with non -melanoma skin cancer and referents with and without psoriasis . Mutat Res 407 : 25 – 34 . M ø ller P, W allin H , Holst E , Knudsen LE .2002 . Sunlight - inducedDNA damage in human mononuclear cells . FASEB J. 16 : 45 – 53 .

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M ø ller P, V ogel U , P edersen A , Dragsted LO , Sandstr ö mB , Loft S .2003 . No effect of 600 grams fruit and vegetables per day on oxidative DNA damage and repair in health y nonsmokers . Cancer Epidem Biomark Prev 12 : 1016 – 22 . OstlingO , Johanson KJ . 1984 . Microelectrophoretic study of radiation - inducedDNA damages in individual mammalian cells . Biochem Biophys Res Comm 123 : 291 – 8 . alus P J , Dziuba łwska to E , Rydzy´ ski n K. 1999. DNA damage detected b y the comet assa y in the white blood cells of w orkers in a w ooden furniture plant . Mutat Res 444 : 61 – 74 . Rekhade vi PV , Mahboob M , Rahman MF , Gro ver P . 2009 . Genetic damage in wood dust exposed workers . Mutagenesis 24 : 59 – 65 . SchmezerP , Rajaee - BehbahaniN , RischA ,Thiel S , RittgenW ,et al. 2001 . Rapid screening assay for mutagen sensiti vity and DNA repair capacity in human peripheral b lood lymphocytes. Mutagenesis 16 : 25 – 30 . SinghNP , McCo y MT , T ice RR , Schneider EL. 1988 .A simple technique for quantitation of low levels of DNA damage in indi vidual cells . Exp Cell Res 175 : 184 – 91 . Siomek A , Gack owski D , Rozalski R , DziamanT , SzpilaA et , al. 2007 . Higher leukocyte 8 - xoo - 7,8 - dih ydro - ′2-deoxyguanosine and lower plasma ascorbate in aging humans? Antiox Redox Signal 9 : 143 – 50 . yskova Sl J , Du š inskM á ,K uricova M , Soucek P , V odickova L et al . 2007 . Relationship between the capacity to repair 8 -oxoguanine, biomarkers of genotoxicity and individual susceptibility in styrene- exposed workers . Mutat Res 634 : 101 – 111 . SmithCC , O ’ Dono van MR , Martin EA .2006 . hOGG1 recognizes oxidative damage using the comet assay with g reater specif city than FPG or ENDOIII . Mutagenesis 21 : 185 – 90 . Somoro vsk á M ,Jahno v á E ,T ulinsk á J , Z á me ˇ ncíov k á M , Š manov ar á J ,et al. 1999a. Biomonitoring of occupational exposure to styrene in a plastics lamination plant . Mutat Res 428 : 255 – 269 . Somoro vsk á M , Szabo v á E ,V odi ˇc ka P , T ulinsk á J , Baranˇ ok c ov á M et , al . 1999b. Biomonitoring of genotoxic risk in w orkers in a r ubber factory: comparison of the comet assa y with cytogenetic methods and immunolo gy. Mutat Res 445 : 181 – 192 . SpeitG , Sch ü tzP , Bonzheim I ,T renz K , Hof fmann H .2004 . Sensitivity of the FPG protein towards alkylation damage in the comet assa y. Toxicol Lett 146 : 151 – 8 . Star uchov á M , CollinsAR ,V olkovova K , Mislano v á C ,K ovacikova Z et , al. 2008 . Occupational exposure to mineral f bres. Biomarkers of oxidative damage and antio xidant defence and associations with DNA damage and repair . Mutagenesis . 23 : 249 – 260 . Star uchov á M ,V olkovova K , Lajdo va A , Mislano va C , CollinsA et , al. 2006 . Importance of diet in protection against o xidative damage . Neuro Endocrinol Lett 27 Suppl 2 : 112 – 115 . Star uchov á M ,V olkovov á K , Misl’ano v á C ,K ov á cik ov á Z ,Ws óvloá L et al. 2007 .Oxidative damage and antioxidant defence in the plasma of people e xposed to mineral f bres. Chemicke Listy 101 : 273 – 274 . omasetti T M ,Alle va R , Bor ghi B , CollinsAR .2001 .In vivo supplementation with coenzyme Q10 enhances the reco very of human l ymphocytes from oxidative DNA damage . FASEB J 10.:1096/fj.00 - 0694fje. ovalin T H ,V alverde M , Morandi MT , Blanco S ,Whitehead L , Rojas E .2006 . DNA damage in outdoor workers occupationally exposed to environmental air pollutants. 5 : Occup Environ Med 63 : 230 – 6 . illarini V M , Moretti M , aFtigoni C ,Agea E , Dominici L et , al. 2008 . Evaluation of primary DNA damage, cytogenetic biomarkers and genetic pol ymorphisms for CYP1A1 and GSTM1 in road tunnel construction workers. J Toxicol Envir Health A . 71 : 1430 – 1439 . odicka V P, K umar R , Stetina R , San yal S , Soucek P ,et al. 2004a . Genetic polymorphisms in DNA repair genes and possib le links with DN A repair rates, chromosomal aber rations and single-strand breaks in DN A. Carcinogenesis 25 : 757 – 63 . odicka V P , Stetina R , P olakova V , T ulupova E , NaccaratiA et , al. 2007 .Association of DNA repair polymorphisms with DNA repair functional outcomes in health y human subjects . Carcinogenesis 28 : 657 – 64 .

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odicka V P, T uimala J , Stetina R , K umar R , Manini P ,et al. 2004b. Cytogenetic markers, DNA single-strand breaks, urinar y metabolites, and DN A repair rates in styrene -exposed lamination workers . Envir Health Persp 112 : 867 – 71 . Y á ñLez, Garc í a - Nieto E , Rojas E , Carrizales L , Mej í Ja ,et al. 2003 . DNA damage in blood cells from children exposed to arsenic and lead in a mining area . Environ Res 3 : 231 – 240 . ZhaoXH , Jia G , LiuYQ , Liu SW , Y an L et , al. 2006 .Association between polymorphisms of DNA repair gene XRCC1 and DN A damage in asbestos -exposed workers. Biomed Envir Sciences 19 : 232 – 238 . ZhuCQ , LamTH , Jiang CQ ,W ei BX , Lou X , LiuWW , Lao XQ , ChenYH .1999 . Lymphocyte DNA damage in cigarette f actory workers measured by the Comet assa y. Mutat Res 444 : 1 – 6 .

Chapter17 Hydroxylated Nucleotides: Measurement and Utility as Biomarkers for DNA Damage, Oxidative Stress, and Antioxidant Eff cacy Ph yllis E. Bo wen

INTR ODUCTION Hydroxylated nucleotides are damaged bases that occur as a result of endo genous or environmental insult from free radical attack, oxidation reactions, or ionizing radiation. The literature, using older analytical methods, estimates that cellular DN A receives a few hundred o xidative hits per day for each of the estimated 5 × 1013 cells in the human body (Halliw ell 2000). Most of this is actively repaired, leaving small residual amounts in DNA under normal circumstances, which can increase tremendousl y in tumors or with inf ammation, or en vironmental insult. Because several of these oxidized nucleotides can lead to mutations, there has been great interest over the last 25 y ears to f nd accurate w ays to measure the e xtent of DN A damage as an early biomarker of carcino genesis or to identify cancer risk f actors or substances that reduce carcinogenesis. Measurement of oxidized nucleosides or bases has been applied to other disease states, aging, toxicology, and the eff cacy of dietary supplements. Valavanidis et al., in a search using Google Scholar, were able to identify 56,300 papers on o xidative DNA damage, 6,180 for 8 -hydroxydeoxyquanosine (8OHdG), and 2,430 for 8 -oxodeoxyquanosine (8 -oxodG) b y the end of 2008 (Valavanidis et al. 2009). (Both of these names are used in the research literature to identify the same chemical compound.)Therefore, this review cannot be comprehensive, but rather focuses on issues of measurement and the utility of 8OHdG as a biomark er for various disease states, en vironmental exposure, and treatment modalities.

WHY FOCUS ON THE MEASUREMENT OF 8 - HYDR OXYDEOXYGUANOSINE (8 OH d G )? Of the hydroxylated bases, 8OHdG has been the prominent choice as a biomark er of oxidative DNA damage and as a general mark er of o xidative stress. Guanine is more easil y o xidized Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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compared to adenine, is a prevalent hydroxylated base in most tissues, leads to point mutations in replicating DNA, and is easier to assess in the pico gram quantities found in tissues because it is electrochemically active. However, Dizdaroglu et al. (Dizdaroglu et al. 2008) have pointed out that tw o additional purine o xidation products should not be o verlooked because the y are formed in relati vely large quantities and are the tar gets for the same enzyme repair systems. These are 4,6 - diamino - 5 -mfor- amidop yrimidine (FapyAde) and 2,6 - diamino - 4ydroxy -h -5formamidopyrimidine (FapyGua), the latter of w hich is formed from the same 8OHdG radical as 8OHdG. The ratio of 8OHdG to FaypGua depends upon the redox state of the cell (reduced at low oxygen partial pressure) and the presence of transition metals such as iron and copper , which ma y af fect the inter pretation of an y measurement of 8OHdG (Halliw ell 2000). Nevertheless, 8OHdG is the focus of this re view because much more is known about issues of measurement and inter pretation than an y of the other h ydroxylated bases. What is kno wn of other hydroxylated bases is addressed w here appropriate.

HYDR OXYLATED GUANINE BASE( HGB )NOMENCLATURE The nomenclature for oxidized guanine bases has not been standardized in the literature, which leads to some confusion. F or example, 8 -hydroxy-2′-deoxyguanosine (8OHdG), w hich is the primary focus of this re view, is also refer red to as 8 -oxy-2-deoxyguanosine (o xo8dG), xyguanosine (8 - xoo - dG),and 8 - xoo - 7,8 - dih ydro - ′2- deo xyguanosine 7,8 - dih ydro - 8 -xoo - 2′ - deo (8-oxodG). The reason for the “oxo” nomenclature is because 8OHdG e xists in its k eto form at neutral pH. To add to the confusion, guanine from RN A can also be o xidized to for m 8 - yhdroxy - 2′-guanosine (8 -OHGua or 8OHG), or the free base (without the sugar) can be oxidized to form 7,8 - dih ydro - 8 -xoo - guanine,also referred to as 8 - xoo - 7,8 - dih ydro - guanine, 8 - Oxo - guanine, 8 - xooG, and 8 - yhdroxyguanine (Wu et al. 2004 ).Figure 17.1 lists the acronyms that are be used in this re view and their str uctures.

HYDROXYLATED NUCLEOTIDE FORMATION, REPAIR, AND METABOLISM In order to use DN A damage products such as the h ydroxylated nucleotides as biomark ers of DNA damage, o xidative stress, disease states, or the eff cacy of v arious inter ventions, it is important to understand , in some detail, ho w these products are for med, excised from DN A, and excreted. It is a v ery dynamic process. FORMA TION Evans et al. ha ve re viewed the chemistr y of for mation in some detail and focused on the hydroxyl radical as a prominent culprit (Ev ans et al. 2004), especially if the oxidative stress is endogenously generated. The highl y reacti ve h ydroxyl radical ( •OH), w hich can be deri ved from the supero xide radical (O 2−•) or h ydrogen peroxide (H 2 O2), targets organic compounds either by addition or subtraction. In the case of purines and p yrmidines, it adds to the doub le bonds at second order rate constants of 4.5 to 9 × 109 M−1 Sec−1. This means that roughl y 40% of a mole of guanosine could be o xidized per second gi ven suff cient quantities of •OH. The hydroxyl radical can also abstract a h ydrogen atom from each of the doub le bonds of 2′ deoxyribose. The chemistr y is quite comple x because adduct radicals are for med that gi ve w ay to a large ar ray of more stab le compounds, depending upon the a vailability of o xygen, other oxidants, reducing agents, and general redo x properties of the en vironment. For example, the 8-hydroxydeoxygaunosine adduct radical that gi ves rise to 8OHdG can be for med b y three different adduct radicals. More than 24 major o xidative DN A damage products ha ve been identif ed, many of which are hydroxylated. Figure 17.2 is an abbre viated version of the path-

Hydro

285

xylated Nucleotides O N

HN H2N

O

OH N

N H

H2N

H2N

H2N

dR 8-hydroxydeoxyguanosine(8OHdG)

NH2

OH

dR

NHCHO

N

N

8-hydroxydeoxyadenosine (8OHdA) O

N

NH

dR 4,6-diamino-5-formamidopyrimidine (FapyAde)

NHCHO

HN H2N

N

R 8-hydroxyguanosine (8OHG)

N N

OH N

NH2 HN

N

HN

OH N

N H

8-oxo-guanine O

N N

O N

8-hydroxyguanine (8OHgua) O HN

H N

HN

N

NH

R = ribose dR = deoxyribose

dR 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua)

Figure 17.1. Structure of oxidized purines and the acronyms used in this review.

ways displayed over a number of f gures by Evans et al. (Ev ans et al. 2004). Because of the extreme reactivity of the h ydroxyl radical, it must be presumed that its generation must be in close proximity to the base it attacks. In the case of nuclear DN A damage, it remains une xplained how a highl y reactive hydroxyl radical, most lik ely generated from mitochondriall ygenerated supero xide resulting from nor mal respiration, comes in reacti ve pro ximity to the nucleus. Whether other o xidants can directl y produce hydroxylated bases is still a source of debate. Older literature identif es the pronated for m (pK a = 6.8) of peroxynitrite (ONO 2−), the product formed from nitric oxide and superoxide, capable of direct attack independent of peroxynitrite’s ability to for m some h ydroxyl radical. Interestingl y, pero xynitrite is more reacti ve to ward 8OHdG than unoxidized dG (Marnett 2000, Barney et al. 1999). Hydrogen peroxide, superoxide, and singlet o xygen do not directl y react with DN A bases, e xcept for guanine, w hich is a unique target for singlet oxygen attack (Devasagayam et al. 1991) and may explain the higher background concentrations of 8OHdG found in man y tissues and f uids (Wiseman 1995 , Halliwell 1998). Peroxynitrite and hydrogen peroxide are less reacti ve and have a longer biological half -life and g reater dif fusibility, so it is presumed that such compounds and other reactive o xygen and nitro gen species ma y be the original generators of localized h ydroxyl

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17 C5-OH adduct or C4-OH adduct radical O

O HN H2N

+

N

+

OH

HN

OH

N

N

H2N

H2N

–e–, –H+ oxidation

O

N N

H

+e–, +H+ reduction

HN

OH N

N

C8-OH-adduct radical

Guanine (2dG)

HN

OH

dR

dR

O

N

N

H2N

N

O

H N

H

N

OH

+

N

N

dR +e–, +H+ reduction

ring opening

dR = 2′-deoxyribose These reactions can also occur with guanine, guanosine, or adenine

NHCHO

HN

H2N

dR

dR 8-hydroxydeoxyguanosine (8OHdG)

ring opening

O NHCHO

HN H2N

N

NH

dR 2,6-diamino-4-hydroxy-5-formamidopyridine (FapyGua)

Figure 17.2. Formation of 8 -hydroxydeoxyguanosine and 2,6 -diamino-4-hydroxy-5-formamidopyridine from guanine. Reactions occur in free purines and those that are contained in DNA. Modif ed from Evans and Cook et al. 2004.

radical. Recentl y, Goto et al. ha ve e xplored the ability of 2,2 ′ - azobis(2 - amidonopropane) (AAPH), a hydrogen peroxide generator, to produce 8OHdG in calf thymus DNA (commercial lots contain small levels of DNA oxidation products) and found that it w as formed in measurable quantities, but pure dG w as not susceptib le. The presence of o xidized deo xythymidine was necessary for the oxidation of dG to 8OHdG. They speculated that peroxidized lipids could serve the same role in vivo (Goto et al. 2008). INTERNAL SOURCES OF HYDR OXYL RADICAL FORMATION Generation of the h ydroxyl radical is a common biolo gical process. The major endo genous source is the result of the re gular formation of the supero xide anion, a uni valent reduction of the triplet state of molecular o xygen w hich is mediated b y ubiquitous enzymes such as the

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NAD(P)H oxidases and xanthine o xidase of mitochondria and spontaneousl y from the for mation of semi -ubiquinone as part of the mitochondrial electron transpor t system. The mitochondrial generation rate is estimated at 4 to 7 nmol/minute−1 /mg protein−1 (Droge 2002). Most of the generated supero xide is con verted to nonradical h ydrogen pero xide b y supero xide dismutases and then to w ater by glutathione pero xidase or catalase. Biolo gical tissues can also convert it to singlet o xygen ( 1 O2). However, in the presence of fer rous and cuprous ions (and accelerated by ascorbic acid), h ydrogen peroxide can be con verted to the h ydroxyl radical via Fenton chemistry. DN A REPAIR Kasai et al. (Kasai et al. 1986) were among the f rst to note that there must be a repair process for the 8OHdG lesion. X -ray radiated mice w ere killed e very 15 minutes after radiation and liver DNA was extracted and assessed for 8OHdG. What was notable is that the 8OHdG/dG ratio w as lo wer in animals killed just 15 minutes after radiation compared to those killed immediately after, and was almost maximally lower ( ∼ 43% lower) 30 minutes after radiation, indicating that repair w as rapid and substantial. The implication is that an y measurement of 8OHdG in intact cells or tissues must be inter preted as the balance between the level of oxidative attack and rate of repair at the moment of sample collection (assuming no fur ther artifactual generation). Therefore, understanding the dynamics of e xcision and repair is impor tant for the interpretation of 8OHG or 8OHdG as liberated products resulting from repair (urine and plasma) or as residual v alues in intact DN A as biomark ers of o xidative stress, damage, or mutation. The issue of ar tifactual production during measurement is discussed later . Extensive study of the v arious repair enzymes in E. coli , yeast, mammals, and humans has been conducted with the identif cation of specif c proteins and amino acid sequences for several of these. The f rst to be disco vered and characterized w as the enzyme for mamidopyrimidine DNA glycosylase (FPG or MutM), w hich e xcises 8 -OHG, FapyGua, and F apyAde from the DNA of E. coli (Boiteux et al. 1990). A completely different protein glycosylase, yOGG1, was identif ed in yeast and later the mammalian and human homolo gues of OGG1 w ere identif ed and cloned. The human for m of hOGG1 has been considered the predominant repair enzyme for mammalian repair of 8OHdG lesions and has at least four isofor ms. The two major for ms are α-OGG1 (OGG1 type 1a), w hich is localized in the nucleus, and β-OGG1 (type 2a), which is found in the mitochondria (Hirano 2008). All forms have a specif city for 8OHdG:C pairs contained in doub le stranded DN A. The mechanism is a multistep process that starts with the removal of the base from the sugar phosphate backbone producing an apurinal site (AP). The enzymes also ha ve an AP lyase activity, producing a break in the phosphate bond between nucleotides. A separate AP-endonuclease prepares the site for purine addition. The resulting one -nucleotide gap is repaired mostl y b y shor t-patch base e xcision repair (BER) (Ev ans et al. 2004). F apyGua is also eff ciently repaired b y this gl ycosylase (Dizdaroglu et al. 2008). Organization of DN A in its chromatin str ucture, extent of o xidative damage, and damage other than o xidative damage can af fect DN A repair mechanisms (Ev ans et al. 2004). Furthermore, one polymorphism (Cys326 variant) of hOGG1 exhibited substantially lower BER activity than the wild-type (Kohno et al. 1998) and is present in abut 15% of Caucasian populations but ma y be lo wer in Asian populations (P ark et al. 2004). The major product of gl ycosylase repair enzymes should theoreticall y be the o xidized base (8OHGua) rather than the derivative of the 2 ′-deoxynucleoside (8OHdG), y et 8OHdG can be readil y found in human urine using the highl y specif c LC -MS techniques (Ev ans et al. 2004). Its origin in urine is discussed later. Formamidopyrmidine DN A gl ycosylase (FPG), the E. coli gl ycosylase that reco gnizes 8OHdG and also ring -opened purines such as the for manidopyrimidines (Fapy), has been used to indirectly measure the number of 8OHdG sites in intact cells using the comet assay (Chapter

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16). After the l ysis of single cells embedded in agarose gel, one set is e xposed to the FPG, which removes the damaged base, making an apurinic site (AP site) vulnerable to single strand breaks in the comet assa y alkali digestion step. The percentage of DN A in the tails of cells exposed to electrophoresis is compared to cells not exposed to FPG and the value is the estimate of the number of 8OHdG lesions in the nuclear DN A of the cell. When FPG and the comet assay were used to determine the basal level of 8OHdG in human lymphocytes in the multicenter collaboration of the European Standards Committee on Oxidative DNA Damage (ESCODD), the FPG -based assay using comet for detection ga ve a mean value for background oxidation of 0.3 8OHdG per 106 for leukocytes, which was 10-fold lower than the median v alues obtain by chromatographic techniques such as HPLC with electrochemical detection (Collins et al. 2008). These measurement discrepancies are discussed in greater detail later. Although it is generally agreed that the OGG1 glycosylase is a dominant DNA repair enzyme directed toward 8OHdG lesions, it is not the onl y repair mechanism. OGG1 knock -out mice (OGG1− /−) excreted 26% less 8OHGua than their wild -type controls but there was no effect on 8OHdG excretion. Rozalski et al. (Rozalski et al. 2005) suggested that if OGG1 were the only glycosylase then the knock -out mice would have negligible 8OHGua in their urine. Therefore, there must be some other back -up glycosylase activity which cannot completel y compensate for the loss of OGG1, hence the measured decrease in urinar y e xcretion. The f act that the amount of 8OHdG in the urine was unchanged in the OGG1 − /− mice indicates that its presence comes from another repair mechanism entirely. A few have been proposed, but their importance is unclear. For e xample, nucleotide e xcision repair (NER)that is principall y directed to ward more bulky lesions can act upon DNA-bound 8OHdG, generating an oligomer that is 24 to 32 nucleotides in length, but these do not appear in the urine, indicating the possibility of fur ther metabolism which frees the 8OHdG (Cooke et al. 2008). Another possibility are endonucleases, one of w hich has been characterized because it lacks an y gl ycosylase acti vity but produces 3,5-8-oxoGDP, which would have to be fur ther broken down by nucleotidases (Bessho et al. 1993 ). HYDR OXYLATED GUANINE BASE ( HGB )METABOLISM Until recently, it was generally thought that once e xcised from DNA, the HGB w ere not reincorporated into newly forming or existing DNA. Therefore, they would appear f rst in plasma and then excreted in the urine (Loft et al. 1995). However, new evidence indicates considerable reincorporation of 8OHdG into DNA, thanks to new techniques in accelerator mass spectrometry that can measure sub -attomole ( < 10− 15) amounts of 14 C - labeledbases including 8OHdG. MCF-7 breast cancer cells w ere dosed with 14C-labeled 8OHdG with an incor poration of 3.4 fmol/µg DNA and 13 fmol of 8OHG/µg RNA. These levels are close to the current estimates of residual levels of 8OHdG in DN A in unstressed cells. The pathw ay for reincor poration be gins with dephosphor ylation of 8OHdGPs b y purine nucleoside phosphorylase (PNP). This results in 8OHGua, w hich is then rephosphor ylated to 8OHdGTP by hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and then by diphosphate reductase (RR) to 8OHdGTP, which is acted upon b y DNA polymerase for DNA incorporation. Comparisons with 2 ’dG incor poration indicated little discrimination betw een the oxidized and uno xidized base in this salv age pathway. The T1/2 for cellular repair w as about two da ys, presumab ly via hOGGT1 and the BER mechanism. Sur prisingly, the creation of oxidatively stressed MCF -7 cells through estradiol administration actuall y lowered 14 C8OHdG incorporation. This was a result of tremendous upregulation of the enzyme MTH1 that converts 8OHdGTP back to 8OHdGMP as par t of the Nudix -related pyrophosphate hydrolases which protect the cell from oxidized base incorporation into DNA (Mundt et al. 2008 Hah et al. 2007). This rec ycling of 8OHdG and lo wered rec ycling with o xidative stress cer tainly clouds the interpretation of directly measured 8OHdG in DNA of cells, but the recycling might be a rela-

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xylated Nucleotides

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tively small ef fect compared to o xidative insult via h ydroxyl radical attack on dG already located in DNA.

WHERE HAVE HYROXYLATED GUANINE BASES ( HGB) BEEN MEASURED? Two major approaches ha ve been tak en; one is to measure 8OHdG as it resides in DN A and the other is to measure free HGBs (especially 8OHdG) in body f uids. It is important to understand the origins of 8OHdG found in an y compartment in order to select the most appropriate one for answering the research question at hand. EXOGENOUS HGB FOUND IN URINE, PLASMA, AND SALIVA For investigators wishing to explore whole body attack on DNA as a measure of oxidative load and extent of repair , urinar y excretion of v arious hydroxylated nucleic acids holds par ticular interest. The bases that appear in the urine o ver a gi ven time period (usuall y 24 hours) are related to the e xtent of o xidative attack combined with the e xtent of repair , presumably over the entire tissue mass. It is assumed that because of their small size and w ater solubility that most of these will be f ltered from the plasma by the kidney glomerulus and not be reabsorbed in the renal tubules. In fact, urinary recovery of IV-injected 8OHdG in pigs is almost complete in four hours (Loft et al. 1995). Therefore, several investigators have focused on the measurement of various oxidized bases that appear in the urine in humans and other animals. Cooke et al. (Cook e et al. 2008) ha ve recentl y re viewed papers that quantif ed specif c urinary o xidized bases and found se ven guanine deri vatives, three adenine deri vatives, f ve thymine derivatives, and tw o cytosine derivatives. There are lik ely many more that could be found in the urine. Weimann et al. (W eimann et al. 2002) measured the three major guanine bases b y HPLC -MS/MS and found that total e xcretion w as 212 nmol/24 hours with RN Aderived 8OHG predominating at 64%, repair -derived 8OHGua at 23%, and 8OHdG at 13%. The source of urinar y 8OHdG is still somewhat of a mystery, especially because it is the most often measured and it is the major target of the N45.1 antibody which is the foundation of most ELISA kits, the most repor ted method for the measurement of 8OHdG in urine. The possible sources for e xtracellular 8OHdG that ultimatel y land in urine could be diet, cell death, or some repair mechanism other than a gl ycosylase. Cooke et al. discussed these possibilities in their review (Cooke et al. 2008) and presented evidence that dietary 8OHdG is not appreciably absorbed. While the relative importance of cell tur nover or apoptosis is anecdotal at best, these investigators believe it is unimportant. They argue that the most likely source for e xtracellular 8OHdG production are the human Nudix h ydrolases (nuclear diphosphate linked to another moiety , X: NDP -X) NUDT1 and NUDT 15 (and ma ybe NUDT 5), w hich prevent 8-OHdG triphosphate from being incorporated into DNA by converting it to the monophosphate form. Remember that despite this elimination pathw ay, there is still considerab le reincor poration of 8OHdG into DN A. Perhaps a nucleotidase can con vert it to 8OHdG, w hich can be easil y eliminated from the cell because it is char ge-neutral (Cooke et al. 2008). In f act, cell culture studies performed by Haghdoost et al. suggest that this nucleotide pool is a signif cant target for oxidative stress (Haghdoost et al. 2006). Therefore, the measurement of urinar y 8OHdG is unlikely to ha ve resulted from 8OHdG deri ved from damaged DN A, but it still might be a measure of whole body oxidative stress. The utility of understanding both the measurement and meaning of guanine -based urinar y excretion products is still promising and has stimulated the formation of the European Standards Committee on Urinary (DNA) Lesion Analysis (ESCULA) which is currently conducting stable isotope-labeled DNA feeding studies with knock-out mice and markers of cell turnover (Cooke et al. 2008).

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The measurement of ser um or saliva should ref ect the same sor ts and distributions of compounds found in urine but there ha ve been f ar fewer studies using these sources of e xogenous HBG. The levels are so low that they are often below the detection limit for direct methods of analysis, but antibody -based methods can be used. Whole sali va consists of the secretions of sali vary glands and e xfoliated mucosal cells, serum, and e ven blood cells if lesions e xist. Takane et al. w ere the f rst to deter mine 8OHdG levels in saliva by ELISA. They found 8OHdG to be 1.9-fold higher in subjects with periodontal disease (Takane et al. 2002). Su et al. also found 8OHdG in sali va by an ELISA assa y and found it to be much higher in their control subjects compared to Takane et al. (42.7 ng/mL vs. 1.65 ng/mL). They attributed the discrepancy to differences in how saliva was collected. Takane et al. collected paraff n-stimulated saliva which was immediately frozen. Su et al. used non stimulated saliva collected at least 30 minutes after meal or be verage ingestion in the mor ning and k ept refrigerated for less than one hour , and then frozen before anal ysis. Both g roups centrifuged samples and used the super natant and used the same commercial ELISA kit. Lik e Takane et al., Su et al. found that their non -smoking subjects with periodontal disease had 1.6-fold higher levels of 8OHdG which mirrored the 1.5 -fold higher levels of 8 -epi-PGF2α (a lipid peroxidation product) seen in these same subjects (Su et al. 2009). ENDOGENOUS8 OH d FOUND G IN CELLS AND TISSUES The measurement of 8OHdG that remains in cells more directl y assesses residual damage to cellular DNA that has not y et been repaired. Theoretically, such measures point more directl y to risk to the genome. The measurement of w hole tissue is prob lematic because most tissues are complex mixtures of a number of cell types, some of which are more prone to DNA damage and carcinogenic processes. Therefore, the amount of 8OHdG or the ratio of 8OHdG/dG that is obtained by the analysis of a whole tissue sample may become more a function of the number of cells in that small bit of tissue that are more subject to o xidative damage. Histochemical assessment has the capability of locating the cell types that are most damaged and counting those cells or damage sites. Histochemical techniques are discussed later . The most commonl y measured cells from humans ha ve been circulating leuk ocytes where the w hole b lood sample is used , red b lood cells are hemol yzed, and DN A is e xtracted. The rationale for using the w hole sample is to eliminate steps for specif c cell separation into l ymphocytes or monoc ytes to lo wer the probability of ar tifactual 8OHdG de velopment. A fe w laboratories have e valuated l ymphocytes and monoc ytes. One of the questions that arises is whether the anticoagulants EDT A or heparin best preser ve the DN A from fur ther o xidative damage. The author ’s research associates ha ve obser ved that a cleaner nuclear pellet can be obtained using EDTA-treated cells and h ypothesized that heparin -treated samples would have slightly higher levels of DNA damage because of traces of hemo globin that tend to remain in these samples. A comparison of 31 health y subjects with samples collected with both anticoagulants showed high cor relations and similar mean v alues with a slightl y higher variance for those samples collected in heparin (Bor thakur et al. 2008). Exfoliated buccal cells are another candidate for anal ysis in humans because the y are easy to collect and represent a tissue type that is more subject to carcino genesis. Cells are usuall y collected by soft -bristled toothbr ush by the subjects themselv es. The author ’s research g roup explored w hether buccal mucosal cells (BMC) har vested at dif ferent times from a baseline harvest would have different levels of 8OHdG because these cells could be more damaged as they became k eratinized and ready for e xfoliation. The author ’s research g roup found that BMCs collected three da ys after a baseline collection in health y subjects had lo wer 8OHdG/ dG ratios (measured by HPLC-EC) compared to those collected seven days later. Furthermore, BMC 8OHdG/dG w as three - to four -fold higher than found in leuk ocytes collected at the same time, and there w as no cor relation between BMC and leuk ocyte values (Borthakur et al. 2008 ).

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These higher le vels mak e sense because these epithelial cells are e xposed to all sor ts of oxidatively acti ve compounds during their life c ycle and are lik ely to ha ve less antio xidant protection. Few investigators have taken advantage of these readil y accessible cells. Romano et al. e valuated BMC in 109 health y volunteers using the monoclonal antibody 1F1 to detect 8OHdG histochemically. They were able to differentiate smokers from non -smokers and those living in urban areas from those living in rural or suburban locations by differences in staining density of the cells (Romano et al. 2000). MIT OCHONDRIAL VS. NUCLEAR DNA DAMAGE The amount of oxidized bases that remain in DNA theoretically represents the residual damage that has escaped repair at the moment the sample is tak en. Its ratio to uno xidized nucleotides represents the balance between oxidative attack and e xtent of repair. Damage to nuclear DN A (nDNA) has been the focus of most research because of the consequences of mutation, especially for cancer . Ho wever, until recentl y it w as thought that mitochondrial DN A (mtDN A) was subject to much greater oxidative attack and damage because of its proximity to superoxide generation in the electron transpor t chain and its lack of histone protection. Early reports of 8OHdG in mitochondria w ere 16-fold higher than nuclear 8OHdG (Richter et al. 1988) and later repor ts found higher concentrations, w hile others found no dif ferences or even lower values (Lim et al. 2005). The variance in repor ted values of 8OHdG (measured as 8OHgua by GC -MS) and other h ydroxylated bases in mitochondria are so lar ge that methodological issues revolving around artifact generation during extraction and isolation of mtDNA uncontaminated by nDNA must play an important part in the measurement variance. It is now known that mitochondria ha ve eff cient repair enzymes, especiall y the OGG (type 2a) w hich may be more bioactive because of the lack of histones protecting mtDNA. Given that the levels of oxidative lesions in mtDNA might be no higher that those in nDN A, and that mtDNA constitutes only about 1% of cellular DNA (Lim et al. 2005), the measurement of whole cell DNA damage predominantly ref ects damage to nDN A.

METHODS OF MEASUREMENT IN VARIOUS BIOLOGICAL COMPARTMENTS Limitations in the methods for the measurement of the extremely small quantities of hydroxylated nucleotides normally found in tissues, cells, and body f uids have been a source of concern for the last 20 y ears. Primaril y, the concer n has focused on the de velopment of ar tifactual hydroxylated nucleotides during the course of sample e xtraction and measurement because of the disproportionate amount of unoxidized base in samples. Various methods produced manyfold dif ferences in the le vel of backg round 8OHdG in leuk ocytes, commerciall y a vailable DNA, and cells in culture. The European Standards Committee on Oxidati ve DNA damage (ESCODD) was set up in 1997 to identify problems, devise standardized techniques, and reach a consensus on true background levels in normal cells. The committee has published a number of ar ticles in volving the concer ted action of 27 European laboratories and one from Japan (Collin et al. 1997 ;ESCODD 2002 ;Riis 2002 ;ESCODD 2002 , 2003 ).The methodology includes (1) direct measurement of o xidized bases (mostly 8OHdG) found in DN A which has been hydrolyzed and separated by gas chromatography (GC) or high perfor mance liquid chromatography (HPLC) and identif ed and quantif ed by mass spectroscopy (MS) or electrochemical detection (EC), (2) indirect measurement by the employment of a glycosylase such as FPG that removes the oxidized base and is measured by DNA strand breaks, or (3) the development and use of pol yclonal or monoclonal antibodies for par ticular oxidized bases. The presence of in situ h ydroxylated bases either in nuclear or mitochondrial DN A have been measured in tissues, circulating leuk ocytes, and cells in culture either b y the direct methods or histochemicall y by the employment of antibodies. F ree hydroxylated bases found

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in serum, urine, or sali va presumably come, predominantly, from oxidized bases liberated via active repair, cell tur nover, or the o xidation of GTP. Although each anal ytical method has its problems and caveats, each also has strengths for par ticular applications. These are discussed in the conte xt of the description of each anal ytical approach. Guetens et al. pro vide a much more extensive and specif c review of the methodological issues (supported by data on various hydrolysis procedures, incubation temperatures, pH, etc.) sur rounding measurement. This review’s careful study is recommended for all w ho use or wish to use 8OHdG as a biomark er for their research (Guetens et al. 2002). DIRECT MEASUREMENT OF HYDR OXYLATED BASES Gaschromatography - massspectroscopy ( GC - MS ) Pioneering work on the identif cation of DNA oxidation products using GC-MS techniques has been perfor med in the laborator y of Dizdaro glu (Dizdaroglu 1986). In these techniques, o xidized bases remaining in DN A are liberated b y acid h ydrolysis (historically by for mic acid). A mixture of proteinases, endo - and e xonucleases plus alkaline phosphatase can also be used to liberate the nucleosides. Bases, nucleosides, and nucleotides are not suff ciently volatile, so they must be con verted to v olatile derivatives before GC separation. Trimethylsilylation and tert-butyldimethylsilylation are most often used for this deri vatization. Fused silica capillar y columns are used to separate the deri vatized bases. Electron -ionization (EI) mass spectra of the Me3Si derivatives of the DNA base products are suff ciently specif c to provide unequivocal identif cation. For the low levels (e.g., femtomole levels) of complex mixture of DNA oxidation products that may result from ionizing radiation or carcinogen exposure, the GC-MS is carried out using selected ion monitoring (SIM). The MS is pro grammed to monitor a number of characteristic ions and the time of elution. Retention times can be measured with g reat accuracy. A larger number of DNA base products can be monitored in a single run using the same derivatized hydrolysate and only small amounts of DN A (0.1 to 0.4 µg) are required for anal ysis (Dizdaro glu 1991). Careful quantitation requires the simultaneous use of stab le isotopes of the compounds of interest or a closel yrelated compound w hich goes through the same h ydrolysis and deri vatization procedures and is calculated as inter nal standards. Quantif cation has often been limited to damage products for which isotopes were commercially available, such as 8OHdG. If acid h ydrolysis is used, 8 hydroxyguanine (8OHGua) is liberated , but if the typical enzyme h ydrolyses are used , the nucleoside (8OHdG) is produced. The competing method at the time of active use of the GC/MS techniques was the HPLC-EC methodology, w hich w as dependent upon the measurement of an electrochemicall y acti ve compound. 8OHdG was not only one of the more abundant DNA oxidation products but easily underwent redo x reactions, w hich made it a prime candidate for HPLC -EC assa ys. What became clear as measurements w ere made using the tw o procedures w as that v alues using GC-MS were 10 -fold higher than those using the HPLC -EC technique for 8OHdG anal ysis (Halliwell 1992 ). Cadet et al. (Cadet et al. 1998) carefully evaluated the source of higher levels, including the possibility that the HPLC -EC technique underestimated the le vel of 8OHdG present. They found that unmodif ed guanine present in the non-purif ed DNA hydrolysate was partly responsible for its ar tifactual o xidation to 8OHGua during the sialization step at the rate of one oxidized base per thousand normal bases. When much gentler hydrolysis procedures were used along with a pre -purif cation step to g reatly reduce the number of uno xidized bases before derivatization, the GC -MS technique produced v alues very similar to an impro ved HPLC -EC assay that also produced lo wer values for 8OHdG than those originall y repor ted using older techniques with more complicated h ydrolysis procedures. These obser vations also appl y to 8OHG, 8 - OH - dih ydroadenine, 5 - yhdroxycytosine, 5 - OH - meth yluracil, and 5 - formyluracil.

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Liquidchromatography – massspectroscopy ( LC - MS ) High performance liquid chromato graphy coupled with specif c identif cation of ion products by mass spectroscopy would appear to offer all the benef ts of the GC -MS procedures without the need for deri vatization. As is most often practiced , DNA samples are subjected to enzyme hydrolysis to liberate the nucleosides, just as in the HPLC -EC assays, and thus suffer from the possible ar tifactual de velopment of 8OHdG from the o verwhelming amount of nati ve dG present in an y sample. As with GC -MS methods, the lar ge amount of dG must be eliminated by separate column clean -up procedures before ion products are produced b y electrospra y technology to avoid further development of ar tifactual 8OHdG. The most recent adv ances have used automated column s witching technology in w hich the dG is eluted, the 8OHdG or other base damage products are retained , and the column is then automatically switched at a predeter mined time (e.g., eight minutes) to the anal ytical column. It is here that the much smaller amounts of damage nucleosides and their isotopicall y-labeled internal standards are separated and electro -sprayed for negative or positive ionization and the mass products separated and quantif ed (Singh et al. 2009). The dG must be deter mined sepao rately by UV detection and 8OHdG —rationalized to its inter nal standard (e.g., 15 N15 ]8 -xo dG)—is expressed as a ratio of the number of unoxidized dG molecules in the injected sample. The reliability of even these newer approaches to LC-MS-MS is insuff cient for many applications where small changes in in vivo oxidative stress are produced or modulated b y antioxidant inter vention in human subjects. Betw een-day coeff cients of v ariation for these latest methods using commercial calf th ymus DNA were around 15% to 20% for 8OHdG and 10% to 14% for 8OHdA, although both o xidized nucleosides increase linearl y with increasing exposure to meth ylene b lue plus light e xposure. 8OHdG w as about 29 -fold more pre valent than 8OHdA but was highly correlated with 8OHdG at every exposure level (r = 0.999)(Singh et al. 2009). ESCODD efforts conducted over the period from 1998 to 2002 to compare the perfor mance of various laboratories and assa y approaches on common samples found that the tw o laboratories repor ting values using LC -MS-MS approaches for pig li ver and HeLa cell DN A were widely divergent, with one laborator y producing the highest v alues of any measurement technique (Collins et al. 2004). However, there are applications in w hich both GC -MS and LC MS-MS are the most appropriate methodolo gy, especially in the v alidation of other methods and their ability to distinguish isotopically labeled molecules that can be used as stalking horses for determining the sources of ar tifactual oxidation in the enzyme h ydrolysis procedures. Highperformance liquid chromatography – electr ochemical detection ( HPLC – EC ) Of the direct measures of 8OHdG, high performance liquid chromatography with electrochemical detection is the most popular. Of the 28 laboratories participating in the ESCODD validation studies, HPLC with coulometric electrochemical detection predominated. Coulometric detection is distinguished from amporometric detection b y using a porous electrode w hich oxidizes the entire amount of the anal yte in question instead of just the anal yte in close pro ximity to the surf ace of a solid electrode. DN A samples are enzymaticall y h ydrolyzed to indi vidual nucleosides and then separated b y reverse phase HPLC, usuall y by dual channel detection, in which the unoxidized nucleosides are detected b y UV on one channel and 8OHdG under goes a redox reaction and the v oltage shift is measured coulometricall y on the other channel. The level of 8OHdG is e xpressed as a ratio to the uno xidized dG in the injected sample. The ESCODD laboratories using this assay worked together to develop a validated standardized h ydrolysis protocol w hich reduced inter -laboratory v ariance, but the de velopment of artifactual 8OHdG was still unacceptably high at an estimated 13 -fold. In addition, only six of 19 laboratories detected and quantif ed the dose response for calf th ymus DN A e xposed to three levels of a photosensitizer (producing DN A damage)sent to the laboratories for anal ysis.

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Only one of the laboratories using LC-MS-MS and none of the laboratories using GC-MS were able to detect the dose response (Collins et al. 2004). Even under the best of circumstances the HPLC-EC method ga ve about 2.5 -fold higher measurements of 8OHdG/dG compared to the use of FPG enzyme nicking to produce strand breaks in live cells (Collins et al. 2004). Despite the persistently higher values for the HPLC -EC method there w as a high cor relation between values obtained b y laboratories using both the HPLC -EC and the FPG -based methods on the same samples. The HPLC -EC approach is more prob lematic w hen it comes to measuring urine. Urine contains man y electrochemicall y acti ve compounds, and depending upon the urine sample, there are se veral interfering peaks that mak es deter mination of 8OHdG undependab le except through e xtensive purif cation procedures before separation and anal ysis. This is generall y achieved b y passing the centrifuged urine sample through a preliminar y column (generall y multifunction including gel f ltration, reverse phase, and ion -exchange) where the 8OHdG is retained. It is then eluted (e.g., acetic acid) and the 8OHdG peak is collected , a fraction of which is automatically injected onto the analytical column where it is detected coulometrically (Shimoi et al. 2002 ). ANTIBOD Y- B ASED ASSAYS Strictly speaking, the use of an antibody to identify and quantify an antigen such as 8OHdG is a direct method of measurement. Ho wever, the issues and approaches for their uses are unique compared to the separation and detection techniques discussed so f ar. Enzyme - link ed immunosorbent assays ( ELISA ) The diff culty in appl ying the earl y HPLC -EC methods to the urinar y e xcretion of 8OHdG, and the g rowing suspicion (at that time) of the ar tifactual production of 8OHdG during measurement, as well as the possibility of pinpointing the location of DN A damage in tissue sections, led the development of antibodies to 8OHdG. The f rst poly- and monoclonal antibodies showed considerab le cross -reactivity with 8OHG from RN A (De gan et al. 1991, Yin et al. 1995). Osawa et al. (Osawa T et al. 1995) developed the most successful monoclonal antibody, N45.1, w hich is the basis of most commerciall y a vailable ELISA kits toda y. They did the preliminary work to establish the specif city of N45.1, comparing its performance to the HPLCEC analysis of 8OHdG, and then de veloped the f rst histochemical assay that could be applied to paraff n sections of tissue (in their case, kidne y)(Toyakuni et al. 1997). Compounds such as the unoxidized bases, 8OHdA, guanine, and 8OHGua do not react with the antibody. However, there was some cross -reactivity with 8OHG but the binding is v ery weak because it requires two orders of magnitude higher le vels of 8OHG to detect its reaction with the antibody . Weimann et al. (Weimann et al. 2002) have shown that 8OHG is present at 48 nmol/24 hours and 8OHdG is present at 28 nmol/24 hours in urine of health y adults; therefore, 8OHG is unlikely to interfere in ELISA -based studies of human urine. The cur rent ELISA assa y (the one produced b y the Japanese Institute for the Control of Aging [JICA]) predominates in the pub lished literature and is a competiti ve binding assa y in which protein -bound 8OHdG is f xed to the bottom of 96 -well plates. The sample and N45.1 antibody is applied and after a wash-step color is developed through the application of a second antibody bound to a chromophore. The color intensity is in versely proportional to the amount of 8OHdG in the sample. Despite the apparent specif city for 8OHdG b y the N45.1 antibody, the JICA ELISA kit still gi ves values that are four - to 10 -fold higher than that of an y of the chemically-based methods, although the cor relations with the HPLC -EC method are modestly good (r = 0.46 and r = 0.73) in tw o out of three pub lications. Strict temperature control with low-temperature overnight incubation and the de velopment of both high and lo w sensiti vity kits by JICA has helped lo wer the values obtained (Cooke et al. 2008, Evans et al. 2008).

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The source of the higher ELISA -based values remains a m ystery, although fur ther analysis of N45.1 specif city has identif ed 8OHGMP as an equi valent competitor with 8OHdG (Evans et al. 2008). Although there is ob vious cross -reactivity with unkno wn compounds, another possibility is the presence of oligomers containing 8OHdG (products of repair mechanisms) in urine that are not evaluated as part of the chemical assays. The pros and cons of this possibility have been thoroughly discussed by Cook et al. (Cook e et al. 2008) in their re view but cannot be resolved with the data a vailable. These authors also note that the nature of the competiti ve binding assay may make it prone to interference with high molecular w eight compounds such as carbohydrates and proteins, which would result in inappropriately high values that have been noted for a subset of urine samples in population studies. Given that 8OHdG represents only a small portion of oxidized guanine bases in urine, a less specif c antibody ma y be appropriate as a measure of generalized o xidative stress. Trevigen (Gaithersburg, MD) distributes the monoclonal antibody 1F7 that reacts equall y with 8OHdG, 8OHG, and 8OHGua, but when used in a competitive binding ELISA correlates very well with the JICA ELISA kit (r = 0.9) (Wu LL et al. 2004). Histochemicaldetection With the existence of a f airly specif c antibody for 8OHdG, the possibility of a histochemical assay to be applied to both fresh and paraff n sections of tissue became practical. Such an approach overcomes two problems of most of the other methods: (1) the de velopment of ar tifactual 8OHdG during the course of DN A hydrolysis (although it does not a void its de velopment during tissue preparation) and (2) the inability to localize damage to specif c cells in tissues in w hich se veral cell types are present and onl y one cell type is more vulnerab le to oxidative stress or neoplasm. Toyokuni et al. (Toyakuni et al. 1997) developed the f rst quantitative immunohistochemical assay for 8OHdG deter mination in paraff n-embedded tissue sections using N45.1 as the antibody. The antibody has also been used in freshl y f xed tissue sections (Dekk er et al. 2005). Although different laboratories ha ve adapted the assa y, it in volves the deparaff nization with xylene; then ethanol follo wed by microwave treatment; then a vidin, biotin, and horse ser um incubation to b lock non -specif c binding. Slides are then treated with the N45.1 anti -8OHdG antibody, washed, then treated with a biogenated link antibody and alkaline phosphatase which is subsequently blocked by an alkaline phosphatase substrate w hich reacts with the alkaline phosphatase labeling antibody to yield a bro wn color at the antigen site. Slides are then counterstained (usually by hemoto xylin) to stain the rest of the cell str ucture in the tissue being examined. Although Toyokuni et al. (T oyakuni et al. 1997) used alkaline phosphatase in the original histochemical use of N45.1, others ha ve used a standard immunopero xidase (which includes hydrogen pero xide) method because endo genous alkaline phosphatase acti vity is diff cult to inhibit. However this approach may produced more artifactual background staining. In a direct comparison of both the alkaline phosphatase and immunoperoxidase methods on prostate tissue in the author ’s laboratory group, it was found that the immunoperoxidase produced signif cant artifactual staining for 8OHdG (Bo wen et al. 2002). The antibody has also been adapted for f ow c ytometry applications using leuk ocytes, and when compared to the HPLC -EC method gave a correlation of r = 0.395,p < 0.001(Peng et al. 2007 ). INDIRECTMETHODS These assay techniques can be classif ed as indirect because the y depend upon reactions that cause DNA fragments or isotopicall y labeled phosphate substances.

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Repair enzyme methods measur ed as DNA chain breaks As described above, the repair enzyme-based measurement of DNA strand breaks is the method that produces the lo west estimates of g round-level 8OHdG in commerciall y available DNA, HeLa cells, leuk ocytes, and tissues. A more detailed description of the procedures ma y be useful. Quantif cation is based upon the use of a radiation -based quantif cation curve worked out years ago in w hich 1 Gy of X - or γ-radiation produces 0.31 breaks for 10 6 Da of cellular DNA (Ahnstrom 1981). Cells are carefully lysed, producing intact cellular DNA, and then half are incubated with the E. coli glycosylase FPG, w hich liberates the 8OHGua from its sugar backbone leaving an AP site that is con verted to a strand break b y an associated AP lyase. E. coli Endonuclease III is also used instead of FPG, but it is not as specif c for 8OHdG, being mainly a pyrimidine glycosylase. Using the comet assay (alkaline gel electrophoresis) for the quantif cation of breaks, the cells are f rst embedded in agarose on a microscope slide and then l ysed, leaving supercoiled DNA which is then incubated with FPG or buf fer alone and then subjected to nuclear uncoiling via high pH (pH > 13is suff cient to convert AP sites to breaks). When subjected to electrophoresis these loops caused by breaks move faster, producing a leading tail (hence the term comet), and the length and the density of the tail can be vie wed by f uorescence microscopy using a DNAstaining dye such as ethidium bromide. The amount of 8OHdG is calculated b y the difference in tail length or percentage of DN A in the tail or the product of the tw o measurements (tail moment) between FPG -incubated cells vs. control cells incubated in buf fer alone. These are quantif ed using the radiation standard cur ve for the number of breaks (Collin et al. 2008). Another approach is to measure strand breaks b y alkaline elution or alkaline unwinding. With alkaline elution, cells are lysed to liberate DNA, then incubated with FPG above a microporous f lter and then eluted with an alkaline buf fer; the smaller DNA fragments pass through the f lter f rst so the rate of elution is a measure of the e xtent of strand breakage. These values are again compared to the standard DN A radiation cur ve. Alkaline unwinding e xposes FPG incubated and control cells to a def ned period of lysis at high pH, producing strand separation. The cellular DNA is then neutralized, causing intact DNA to recoil to double strands but leaves the strands with breaks as single strands. These are separated into single and doub le stranded fragments on a h ydroxyapetite column. Quantif cation is based on the radiation -produced standard curve for the number of strand breaks. The assumption is that each strand break represents an AP site w here 8OHdG w as excised by the FPG gl ycosylase (Collins et al. 2004). These three methods of measuring strand breaks gi ve comparable results when they are compared for the assessment of 8OHdG using a common radiation -based standard cur ve (Collins et al. 1997). More recently, Smith et al. compared FPG gl ycosylase, Endonuclease III, and hOGG1 in their ability to detect DN A damage specif cally ascribed to oxidative damage in a mouse l ymphocyte cell line. Using percent tail DNA intensity in the comet assay as the outcome measure, they found that all three enzymes produced linear increases in tail intensity with increasing gamma radiation (a known producer of the hydroxyl radical). Endonuclease III w as unresponsive to KBrO 3, another kno wn o xidizer with uncer tain mechanism. hOGG1 w as the onl y enzyme that w as unresponsive to tw o alkalating agents that tend to produced DN A damage through methylation. Smith et al. concluded that hOGG1 had g reater specif city for the detection of 8OHdG caused b y oxidative damage (Smith et al. 2006). The question remains as to w hy this indirect approach gi ves lower v alues than the direct assays using HPLC or GC or antibodies. Proponents of the DN A chain break assays point out that isolated li ving cells are minimall y manipulated and measurements can be made immediately upon har vesting, and so are less subject to ar tifactually generated 8OHdG. On the other hand, more than one 8OHdG could be contained within a single brok en strand, especially if the damaged bases occur in close pro ximity. Indeed, FPG has been sho wn to cover about f ve nucleotides surrounding the damaged nucleotide, thus e xcluding excision of another o xidized

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nucleotide within that area (Sattler et al. 2000). Under this circumstance, counting repair enzyme-mediated DNA strand breaks w ould be an underestimate of the amount of 8OHdG present. Most applications are focused on detecting lo w levels of DNA damage where several 8OHdG in close pro ximity are less lik ely. 32

P - postla beling methods

The labeling of nucleosides liberated from DNA with radioactive isotope-labeled phosphorous (32P) is a highly sensitive method for identifying and quantifying all sor ts of DNA adducts and has been applied to the measurement of 8OHdG (Zeisig et al. 1999, Guetens et al. 2002). The basic technique in volves the enzymatic h ydrolysis of DN A samples, and in the case of the measurement of 8OHdG, its separation from unoxidized dG before incubation with 32 P - labeled ATP plus kinase. The presence of dG in the mixture leads to the ar tifactual production of 8OHdG. The purif cation of 8OHdG can be accomplished b y HPLC column separation or the use of thin la yer chromato graphy (TLC) with electrophoresis. In the latter case, onl y the 8OHdG migrates and can be collected and labeled. After incubation with ( γ 32- P) ATP plus T4 pol yis treated with nuclease p1 to nucleotide kinase, the 32 P - labeled8OHdG 3,5 ’ - diphosphate produce its 5 ’ 32-P 8OHdG monophosphate, w hich is more easil y separated b y HPLC or tw odimensional polyethylene-diamine cellulose TLC, and γ - radiation - counted. The attributes of this approach is that v ery small DNA samples of 2 to 5 µg, as opposed to the 30 to 50 µg required for HPLC -EC or GC -MS, can be used. The practical sensiti vity is about one 8OHdG/dG × 106 in a 1 - to 2 -µg DNA sample. Unfor tunately, this method is as subject to the de velopment of ar tifactual 8OHdG as the other assa y approaches and is highl y dependant on the elimination of dG before labeling. The multi -step nature of these methods resulted in poor reliability and inability to detect dose response in the ESCODD comparisons, which has led to the abandonment of this approach for the measurement of 8OHdG in recent years (ESCODD 2000 ). O THER METHODS Due to the dissatisf action with existing methods, a number of other methods ha ve been developed, most of w hich tak e adv antage of e xisting modalities with an ef fort to o vercome their limitations. F or e xample, Sattler et al. (Sattler et al. 2000) measured the number of FPG damaged DNA complexes using an antibody to the comple x and e valuated the e xtent of chemiluminescence in plasmids with kno wn amounts of damage. P eoples and Kar nes (Peoples 2005) discussed the various attempts to use capillary electrophoresis as a separation technique, especially for urine samples. It has limitations because of the need for small samples w hich limits the technique ’s ability to detect lo w levels of damage products. The need for lo w cost, high throughput for lar ge epidemiological studies or e ven clinical applications has led to the e xploration of electrodes that specif cally sense the presence of 8OHdG. Li et al. developed a methylthiophene modif ed glassy carbon electrode which is much more sensitive than the standard amphoteric electrodes for electrochemical detection and can be used directly for the detection of 8OHdG in urine (Li et al. 2007). Ersoz et al. w ent a step further and imprinted 8OHdG onto a methacr yloylamidohistidine-platinum (II) monomer for the chelating of 8OHdG in samples, then dropped it onto a quar tz crystal microbalance whose frequency w as detected electronicall y. When samples such as plasma w ere e xposed to the chelating monomer the frequenc y of the microbalance changed depending upon the mass of the 8OHdG that w as chelated. The limit of detection w as e xcellent and the a verage percent recovery w as 80% but the ability of the monomer to select 8OHdG from either guanine or guanosine was 20 -fold, which may not be suff ciently selective (Ersoz et al. 2008). Although these approaches need fur ther de velopment, the need to deter mine w hether 8OHdG is a

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predictive biomark er of disease or therapeutic inter vention becomes more rele vant with the possibility of simple, lo w cost assay procedures than can be applied clinicall y.

THE PRACTICAL USE OF HYDR OXYLATED BASES AS BIOMARKERS Halliwell and Whiteman (Halliwell 2004) laid out criteria for the ideal biomark er of oxidative damage. The core criterion w as that it had to predict the later de velopment of disease. They listed additional “technical criteria ” as (a) must identify a major par t of a f xed percentage of ongoing damage in vivo ,(b) inter - assa y coeff cient of variation (CV) for the same sample must be smaller than the dif ferences between samples (subjects), (c) should not v ary widely in the same subject under stab le conditions over time, (d) measurements must be chemicall y robust, (e) must not be confounded b y dietar y contributions, (f) stab le on storage, either b y loss or artifact development. How does 8OHdG measure up to these criteria? The technical issues are addressed f rst since w hether 8OHdG or other DN A o xidation product predicts disease or effective pre vention or treatment rests upon w hether this biomark er is stab le and robustl y measurable. ASSA Y RELIABILITY From the extensive discussion concerning the problems with most of the e xisting methods for the measurement of 8OHdG, one w ould assume that the quantitation of 8OHdG w ould have less utility than its original promise. However, a large amount of literature exists in many areas where the quantity of 8OHdG or ratio of 8OHdG/dG in urine, ser um, tissues, and cells has been able to: (1) distinguish disease from non -disease, (2) mark the exposure to various oxidative environments or (3) trace the ameliorati ve effects of known antioxidants. What accounts for this paradox? One of the issues the ESCODD group was trying to resolve was whether the v arious methods could gi ve valid (accurate) v alues for the amount of background DNA damage that might e xist at the moment of collection in non -diseased or o xidatively unstressed samples. This is different than reliability, which refers to the repeatability of the measurement on the same sample w hich is usuall y repor ted as within assa y and betw een assay CVs in a gi ven laboratory. Although the elimination of all ar tifactual 8OHdG should be the goal of any robust assay, the biomarker might be useful if the amount of artifactual 8OHdG is roughly similar from sample to sample. The reporting of assay CVs in research reports is helpful and should be < 10%,but it doesn ’t solve the prob lem that each sample ma y gi ve rise to dif ferent en vironments for ar tifactual development, which will increase between-sample variance and/or produce measurement bias. Therefore, the careful approach would be to report individual assay CVs using sample material that represents all treatment or disease states measured. Disappointingl y fe w pub lications, regardless of the assa y used for the measurement of 8OHdG or its counter parts, report assay CVs. Even fewer repor t the tissue or f uid used to assess assa y CV, and none, including the author’s laborator y, have used sample material from dif ferent disease states or treatments for separate CVs. Investigators using the HPLC -EC assay for leuk ocytes report intra - and inter -assay CVs of 5% to 10%, but most do not specify w hether they used calf -thymus DNA, pooled leukocytes, or other DNA material to determine their assay reliability. One of the few to address this issue was Breton et al., w ho measured peripheral blood mononuclear cells (PBMCs) collected from esophageal cancer patients and controls. They e valuated a number of DN A-isolation and 8OHdG-liberating procedures including the ESCODD -recommended method. They were able to achieve low 8OHdG/dG ratios for their control subjects close to the median le vels reported as backg round for the ESCODD consensus. Whereas their calf th ymus DNA inter -assay CV was less than 10%, the best intra -day and inter -day CVs they could obtain using PBMCs w ere

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16% and 17%, respectively (Breton et al. 2005). For LC-MS with column switching using liver DNA from CCl 4–treated rats, intra -assay CVs w ere 15.1% for chaotropic procedures, but the expected increase in 8OHdG in treated animals compared to controls w as barel y detectab le (Singh et al. 2009). Others have reported intra -day CVs of 2.5% to 6.9% and interda y CVs of 4.1% to 8% for mouse li ver or calf th ymus DNA (Chao et al. 2008). The comet assa y, w hich depends upon human scoring of tail length, typicall y has CVs between 20% and 50% and tw o-thirds of this variance could be attributed to inter -assay rather than intra -assay er ror (Moller 2006). Inclusion of the FPG incubation to obtain 8OHdG sensitive sites produced assa y CVs of 7% to 100% for no radiation e xposure to 0.4% to 26% with 10 Gy X -ray e xposure for 10 in vestigators (f ve e xperienced and f ve inexperienced) given the same set of lung epithelial cell slides to e valuate. Experienced e valuators produced lower CVs, using their own calibration curves, but all investigators could detect dose response relationships (Moller 2004). Ev en though these CVs seem lar ge compared to the other approaches to measuring 8OHdG, the relati ve absence of ar tifact, the assay’s sensitivity to the existence of damage, and the small sample amounts needed ma y mitigate its lo wer assa y reliability. The measurement of 8OHdG in urine using the ELISA method is the ne xt most often reported assay, and presumab ly reliability measurements will ha ve been made on urine samples. It is often e xpressed using a timed or untimed sample as a function of urinar y creatinine since multiple 24 -hour urine collections are diff cult to obtain. Hakim et al. repor ted an intra -assay CV of 4.9% for 8OHdG and 3.6% for creatinine (Hakim et al.2008), while Parise et al. reported CVs of 6.4% and 8.2% without and with creatinine -adjustment, respecti vely (P arise et al. 2005). Whether other investigators are able to achieve the same CVs with urine using the same commercial ELISA kits is unkno wn. Wilbur et al. reported CVs of 1% to 17% (Wilbur et al. 2004) while Thompson et al. reported CVs of less than 7% (Thomson et al. 2007). A correlation of r = 0.334, p = 0.002 was found for urine from 84 adolescents between the ELISA and HPLC -EC assays by two different laboratories using both methods, but CVs for the assays were not reported (Peng et al. 2007). Using an HPLC-EC with SPE cartridge clean-up, Pilger et al. found a cor relation of r = 0.96(n = 18 subjects) for repeat measures on the same samples anal yzed tw o da ys or six months apar t (Pilger et al. 2002) and an intra -assay CV of 3% and an inter -assay CV of 14% (Pilger et al. 2000). Reliability of the GC -MS-NCI method after solid phase e xtraction of the 8OHdG to eliminate dG was reported as a CV of less than 9% for urine (Lee et al. 2008). For HPLC -EC using column s witching, the intra -day CV w as 8% and inter -day CV w as 10% (Loft et al. 1992). Cho et al. measured a number of urinar y nucleosides in triplicate in w omen with and without breast cancer using the column -switching LC -MS approach. Their intra -assay CVs ranged from 2.28% to 11.74% and the inter -assay CVs ranged from 4.36% to 11.5%, and four of the nucleosides measured (including 8OHdG and 5OHdU) were signif cantly higher in breast cancer patients (Cho et al. 2006). In careful hands, most of the measurement approaches are relatively reliab le within a gi ven laborator y for a v ariety of f uids and cells. Histochemical techniques are not as amenable to the same sort of reliability assessments because each section of the slide represents a dif ferent set of cells. Given that those laboratories that repor t assay CVs are ab le to k eep er rors under 10%, the question is which assay may be more sensitive to differences in 8OHdG predicted by investigator hypotheses? Comparisons have been made using the ESCODD -improved methodology for HPLC-EC and the FPG modif ed comet assa y. Hofer et al. assessed l ymphocytes collected from 94 healthy young people (19 to 31 y ears old) and found no cor relation between 8OHdG/ dG by HPLC-EC, net FPG sites/dG, or single strand breaks (SSB) plus alkali-labile sites (ALS). The variance was g reater for 8OHdG/dG and le vels were 3.9 -fold higher for 8OHdG/dG as measured by HPLC -EC. The standard comet assa y (SSB plus ALS) was more sensiti ve than the other approaches in picking up signif cant differences by gender and fr uit intake (Hoffer et al. 2006 ).

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ST ABILITY IN STORAGE In the course of the ESCODD methods v alidation studies, standardized calf thymus DNA was prepared to send out to v arious laboratories. Ov ernight freeze -drying of this material led to a two-fold increase in 8OHdG/dG assessed by HPLC-EC. Repeated analysis over a 53-day period of the freeze-dried DNA stored at room temperature in air determined that values were remarkably constant up to day 50 but later results were erratic. Storage under nitrogen did not protect the DNA from o xidation (Wood et al. 2000). Casual urine samples stored at -80 ° C, 4 ° C, and 25 °C for 24 hours w ere not dif ferent in 8OHdG v alue, variance, or cor relations between the different storage temperatures measured b y tw o-stage HPLC -EC. Fur thermore, urine stored 200 days vs. 1,000 days at -80 °C was not different (Maysumoto et al. 2008). However, storage at - 20°C for a six -year interval reduced the cor relation to r = 0.64 (Poulsen et al. 1998 ). RELATIVELY STABLE VALUES OVER TIME IN THE SAME INDIVIDUAL Oxidative attack to DN A and its repair are ongoing biolo gical processes; therefore, there is great concern about whether this biomarker is suff ciently stable to mark the chronic oxidative stress of disease or treatment response or the ef fect of environmental oxidative stress. Several investigators asked this question, mostl y for urine measurements. Urine samples obtained from 20 health y individuals were assayed, using an immunoaff nity column - modifed HPLC -EC assa y, and then w ere anal yzed on separate da ys with a same sample correlation of r 2 = 0.91. When urine was collected from the same indi viduals 120 days later, the r 2 w as 0.53, indicating some consistenc y for each indi vidual (P ark et al. 1992). However, Pilger et al. found that 24 -hour urine collected daily for 10 days in two subjects had intra-individual CVs of 37% and 57% (Pilger et al. 2002). This implies that a single urine sample likely does not ref ect a steady state of o xidative stress, e ven for health y individuals. This is not uncommon for biolo gical mark ers of disease. F or e xample, a single measure of plasma cholesterol concentration is a w eak predictor of a repeat sample tak en close in time and urinary creatinine values in daily 24-hour urine collections have a CV of 13%, even under the best collection conditions. Can urinary 8OHdG le vels be used to char t changes over time? In 68 health y subjects followed for six 24 -hour urine collections spaced four to eight w eeks apar t, the intra -individual CVs ranged from 18% to 106%, but the g roup means over time varied only 14%. The correlation from one collection to the ne xt ranged from -0.06 to 0.41. Only in collections f ve and six were the cor relations statisticall y signif cant (Pilger et al. 2000). Additionally, Pilger et al. found that although spot urine adjusted for creatinine ga ve similar g roup mean v alues as 24 hour creatinine adjusted urine collections, the spot urine samples could not distinguish smokers from non -smokers, whereas the 24 -hour urine samples did (Pilger et al. 2002). There seems to be considerab le diur nal v ariation in 8OHdG e xcretion. Kanabrocki et al. performed a careful study in light -/dark-cycle-adapted healthy men (n = 7) and men with Type 2 diabetes (n = 4). Meals and meal timing w ere strictly controlled and timed urine collections were every three hours. Amplitude (one -half the extent of change e xplainable by rhythmicity) of ELISA -measured 8OHdG w as 8.6 pmol/kgBW/three -hour urine v olume (p = 0.004) with maximal excretion occurring early in the evening and the nadir reached about 6 a.m. Although there w as no statistical dif ference betw een men w ho w ere clinicall y health y or those with diabetes, probably because of the small sample sizes, the visual dif ference in the g raphs were striking. They followed the same diur nal patter n but the four men with diabetes had higher urinary excretions and their diur nal pattern was more exaggerated (Kanabrocki et al. 2002). Another study by the same investigator found a similar circadian pattern of 11 subjects with multiple sclerosis and 10 health y women. Yet, there w as no dif ference in 8OHdG e xcretion between the two groups (Kanabrocki 2006). This observation is consistent with the accr ual of oxidative insult during the acti vities of daily living with quick repair follo wed by more quies-

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cent repair as o xidative insult is lo wer during the hours of sleep. Miw a et al. found similar 8OHdG v ariability in spot urine collected o ver two consecuti ve days in f ve indi viduals but there was no detectable diurnal pattern (Miwa et al. 2004). Exercise studies point to rather quick repair w hich is f rst ref ected in cells and tissues followed by increased urinary excretion (Okamura et al. 1997). It appears that the diurnal variation might be controlled with consistentl y-timed urine collections. Miw a et al. found an r 2 of 0.75 between 24-hour urine samples and time-controlled morning spot urine collected from 42 nonsmoking healthy subjects (Miwa et al. 2004). The author’s research group studied variations in leukocyte 8OHdG measured by HPLC-EC over a 24 -hour period in 32 men and w omen with and without Type 2 diabetes. The g roup’s intra-assay and between-assay CVs were 4% and 7.7% on a pool of heparinized human leuk ocytes (Borthakur et al. 2008) and all samples for an indi vidual subject w ere assayed within a single daily run. Subjects were fed timed standardized meals based upon their measured metabolic needs and the protocol was repeated twice, spaced at least one month apart with or without an acute bout of moderate e xercise. Figure 17.3 shows the variability over the 24 -hour period for three indi viduals for their e xercise and no e xercise protocols. The hourly variability was much greater than the assay CV but individuals tended to have characteristic basal 8OHdG/dG ratios at the earl y morning fasting blood draw for both protocols (unpub lished results). The author’s research group also observed occasional drastic spikes at single time points for an individual, as seen in the third panel. Given that the HPLC-EC assay are prone to artifactual development of 8OHdG, it is diff cult to deter mine when and w hen not to eliminate a v alue from the data set, especially when, as with this individual, the values are characteristically high at tw o separate dates of data collection. These data point to the advisability of collecting samples in the earl y morning while the subject is f asting. SEASONAL EFFECTS ON STABILITY AND ABSORPTION OF FOOD - B ASED 8 OH d G Early studies using the administration of radio -labeled 8OHdG in rats b y gastric intubation found that less than 2% of the administered dose could be detected in the urine o ver a sevenday collection period (Shigenaga et al. 1989), pointing to poor absor ption of pre -formed 8OHdG from foods or other sources. A more e xtensive in vestigation of pre -formed stab le isotopes of 8OHdG and 8OHG in health y men found no urinar y excretion of 15N for up to 14 days after the dose (Cook e et al. 2005). Therefore, 8OHdG contained in the diet is not a confounding factor. The effect of various food -based oxidants and antioxidants that might be par t of a diet are discussed later . Seasonal effects in residual 8OHdG in cells and urinary excretion have been noted by several investigators. Dusinska et al. measured lymphocyte strand breaks with and without glycosylase incubation monthl y o ver a one -year period in 11 health y middle -aged male residents of Slovakia. Crude strand breaks and lymphocytes treated with FPG or Endonuclease III followed the same monthly pattern of rises and falls, with the mean winter month values higher than the summer months for all three measurements. The higher winter values in this study could ha ve been linked to g reat industrial pollution (Duinska et al. 2002). Whether summer or winter seasons produce the greatest DNA damage has not been resolved and probably depends upon the assa y and other en vironmental factors and seasonal beha viors that vary with season. The strongest e vidence that sunlight e xposure might ha ve an ef fect on circulating cells because of e xposure to UV light as the y pass through the der mis, especially in light -skinned individuals, was presented b y Moller et al. They carefully sampled b lood in the morning over a one-year period in 21 healthy men and women from Copenhagen, separated monocytes, and calculated percentage of tail DN A using the comet assa y. Inter nal control for each assay using mouse l ymphoma cells deter mined that there w as no assay drift over the study time period. They found a positive association with daily sunlight inf ux, and individual

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Figure 17.3. Hourly variation in leukocyte 8OHdG/dG in three subjects (top panel = subject with type 2 diabetes, bottom two panels = control subjects). Subjects either moderately exercised or rested in the morning and followed with three timed meals. Beginning and ending data points are from fasting blood samples taken 24 hours apart. Exercise and sedentary protocols occurred at least one month apart. (Unpublished data.)

exposure being strongest for the three- to six-day periods before sampling but could be detected up to 50 da ys beforehand (r = 0.47 - 0.51,P < 0.0001). Intra -individual variance over time was large even when controlling for sunlight exposure and could be mainly attributed to assay reliability. The seasonal variation could not be attributed to changes in fr uit intake (Moller 2002). Although this study did not specifcally measure 8OHdG by FPG treatment, the high correlation between total strand breaks and FPG -chain breaks (Duinska et al. 2002) implies that 8OHdG generation might also be induced with sunlight e xposure, especiall y because the 8OHdG damage product uniquel y occurs with singlet o xygen exposure, a prominent reacti ve oxygen species generated with sunlight.

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The presence of inf ammatory disease might amplify the seasonal ef fect. Patients with systemic lupus erythematosus had higher urinary 8OHdG excretions (by ELISA) during late May through early September (a period of high-intensity sunlight in Japan), whereas healthy control subjects showed no seasonal effect. The lupus patients could not be distinguished from control subjects during the winter months (Maeshima et al. 2002). SMOKING, GENDER, AGE, AND PHYSICAL ACTIVITY AS CONFOUNDING VARIABLES Loft et al. were among the f rst to describe smoking, gender, and age as modulators of urinar y 8OHdG in health y subjects. Through re gression anal ysis of 83 randoml y selected people, smokers e xcreted 50% more and males e xcreted 29% more 8OHdG than non -smokers and females, respectively. BMI had an in verse effect (Loft et al. 1992). Presumably the larger fatfree mass (more cells) of males could explain the gender and BMI effect. Since that time other investigators have had to account for these variables as confounders in their own data. Whereas smoking is almost uni versally found to increase both urinar y excretion and residual 8OHdG in DNA (Pilger 2006), investigators have not found consistent ef fects for gender, age or BMI. Although urinary 8OHdG could distinguish a g roup of heavy vs. light cigarette smok ers there was no cor relation with urinar y nicotine equivalents (Lowe et al. 2009). There is a lar ge amount of literature that has e xplored the question of e xercise-induced oxidative stress and has repor ted 8OHdG concentrations/ratios/outputs as a biomark er. Acute bouts of intense e xercise in trained and untrained rats or humans can increase both leuk ocyte and urine 8OHdG between one hour to one week after the exercise (Orhan et al. 2004, Umegaki et al. 2000, Almar et al. 2002, Tsai et al. 2001, Okamura et al. 1997). However, physical training lowers 8OHdG levels (Parise et al. 2005), presumably because of the induction of enzymes that mitigate the generation of h ydroxyl radicals and DN A repair in muscle tissue (Radak et al. 2002, Nakatani et al. 2005, Sato et al. 2003). From these data, it might be useful to treat exercise training status as a confounding v ariable, but sor ting b y training le vel re vealed no difference in 8OHdG e xcretion in men and w omen (Bloomer et al. 2008). A number of other studies found that e xercise w as not mark ed b y dif ferences in 8OHdG le vels in leuk ocytes (Sacheck et al. 2003), serum (Bloomer et al. 2005, 2006 ), and urine (W ilbur et al. 2004), but many of these subjects had par ticipated in exercise training prior to the study . UTILITYOF 8 OH d AS G A BIOMARKER IN DISEASE There are two dynamics that deter mine the inherent ability of the presence of a DN A damage product, such as 8OHdG, to predict the propensity for the development of disease; for example, a specif c type of cancer. Is the biomarker sensitive? That is, can it identify all future or present cases of the disease in those with the disease? Is the biomark er specif c? Can it identify all those without the disease in the class of indi viduals without the disease? A biomarker with the most predictive value should be both sensiti ve and specif c. Oxidative DNA damage probab ly comes earl y in the initiation and pro gression of carcinogenesis, although DNA damage also may occur as a result of the presence of cancer cells. The presence of 8OHdG in the DN A strand induces guanosine, c ytosine to thymine, adenine (GC to-TA) transversion-type point mutation and is belie ved to have a k ey role in cancer de velopment (Cheng et al. 1992). The presence of 8OHdA has a similar mutagenicity with a mutation inducibility of ∼ 1% (Kamiya et al. 1995, Kamiya et al. 1995). Continuing with the e xample of DNA damage as cancer -causing, one might theoreticall y propose that DN A damage would be a sensiti ve biomarker because it identif es the f rst defects that lead to neoplasm. But it is not very specif c because other processes need to occur for a neoplasm to develop. Furthermore, residual DNA damage results from a lar ge number of other causes (o xidative stress, inf ammation, loss of DN A repair enzymes) that ma y not result in a par ticular cancer. The later a

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biomarker is e xpressed in the carcino genic process, the more lik ely it is to a void providing a false positive. That is, its low level should clearly identify those without cancer. It is important to realize w here o xidative DN A damage occurs in an y disease process for w hich it is an intended biomarker and that such damage could result from man y different causes. Another issue that confuses an y marker of o xidative DNA damage is that o xidative DNA damage is known to induce apoptosis (Kowaltowski et al. 2001), but greater apoptosis decreases the number of cells sporting measurable DNA damage. Therefore, an inducer of oxidative DNA damage in cells with competent apoptosis machiner y will result in lower levels of 8OHdG and even 8OHdG/dG. Fur thermore, comet tails are produced in earl y apoptotic cells, and antio xidants that suppress apoptosis could be misinter preted in directly protecting DNA from oxidative damage (Choucroun et al. 2001).

UTILITY OF 8OHDG AS A PREDICTOR OF DISEASE For an 8OHdG to be used clinicall y, hopefully it could identify indi viduals who had not y et been diagnosed with a disease or w ere at e xcessive risk for the disease so that pre ventive strategies could be implemented. There is a f airly large amount of literature that indicates that 8OHdG in tissue, leuk ocytes, urine, ser um, and cerebral spinal f uid tends to be ele vated in those with cancer (Breton et al. 2005, Hsu et al. 2009, Yang et al. 2009, Kuo et al. 2007, Yano et al. 2009), diabetes (Xu et al. 2004, Nakanishi et al. 2004, Nishikawa et al. 2003), and diseases of oxidative stress and inf ammation (Chang et al. 2004, Bashir et al. 1993, Fukahara et al. 2008, Teunissen et al. 2002). Several reviews describe man y of these studies (Hw ang 2007, Halliwell 2007, Valavanidis et al. 2009, Wu et al. 2004, Loft et al. 2006). Loft and Moller (Loft et al. 2006) ha ve pointed out that these higher le vels are lik ely to be associated with reverse causality. It is unfor tunate that most of these studies did not choose cut -points that might distinguish cases from controls and then use these cut -points to calculate the sensiti vity and specif city of their 8OHdG assa y to identify subjects with and without disease. Onl y Malins et al. (Malins et al. 1993, 2001) have reported sensitivity and specif city for detecting cancer in prostate tissue or breast tissue vs. nor mal tissues using the ratio of Lo g10 (8OHAde + 8OHGua)/ (FapyAde + FapyGua) measured b y GC -MS (for mic acid -based GC -MS measures the bases rather than the nucleosides). Their rationale for e xpressing the h ydroxypurines as a function of their cor responding ring -opened F apy-pyrimidines is that the presence of residual F apy compounds are b lockers of DN A synthesis (Grazie wicz et al. 2000, O ’Conner et al. 1988), which would decrease cancer cell propagation. They estimated sensitivity and specif city using a number of trial cut -points for their ratio and came up with a cut point of 0.65 for the prostate cancer series and a cut -point ratio of 0.50 for their breast cancer series vs. nor mal tissue samples. Whereas the amount of 8OHGua/mg DN A did not distinguish cases from control in either the breast or prostate cancer series, the ratios did , with sensiti vities of 91% and 82% and specif cities of 97% and 93% for breast and prostate cancer , respecti vely. These impressi ve predictive v alues are on tissues that w ere already diagnosed b y patholo gical obser vation so have little diagnostic utility, but these studies demonstrate an approach that should be used for all reports of 8OHdG or other biomarkers as a predictor of disease. Because 8OHdG in leuk ocytes and urine w as removed from the site of o xidative stress and DN A damage, the y should have lower predictive values, but repor ts will adv ance our understanding of the utility of one method of measurement, tissue, or application o ver another for the identif cation of v arious disease processes. In order to a void issues of re verse causality, 8OHdG should be measured before disease is manifest. Only one such cohor t study could be found in the literature for an y disease. Danish men and women provided a spot urine at study entry and then were followed for three to seven years for lung cancer incidence. The 260 cases w ere matched for gender , age, and smoking

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duration and the spot urines were assessed for 8OHdG by HPLC-EC as a function of creatinine excretion (inter - assa y CV = 2.7%). Overall, cases did not have higher 8OHdG excretion levels with or without adjustment for smoking, and OGG1 genotype had no effect. However, the eight never-smoker cases with equi valent secondar y smok e e xposure e xcreted 70% more 8OHdG than their matched control subjects, and their incidence rate ratio was 11.8/doubling of 8OHdG excretion (p = 0.03) (Loft et al. 2006). Anecdotal e vidence w as presented b y Kanabrocki et al. (Kanabrocki et al. 2006), w ho described two individuals whose diurnal excretion of 8OHdG had been studied f ve years prior to the time the y developed breast or prostate cancer . Their 8OHdG at that time e xceeded the 95% upper prediction limit for the health y subjects in the study . It appears that confounding factors such as smoking, in the case of lung cancer , may limit the predicti ve value of 8OHdG for cancer and perhaps other diseases. The inclusion of 8OHdG as a general biomark er of oxidative damage should be encouraged in future longitudinal studies pro vided its collection can be consistent and confounding v ariables controlled in the statistical anal ysis.

UTILITYOF 8 OH d AS G A MEASURE OF ENVIRONMENTAL INSULT Pilger and Rudiger (Pilger 2006) reviewed the use of 8OHdG as a mark er of o xidative DNA damage resulting from occupational or en vironmental e xposures. They pro vided tab les of studies that e valuated smoking, benzene, asbestos, silica, and a number of hea vy metals exposures. The predominant materials tested w ere urine and leuk ocytes from e xposed and unexposed individuals. There is also a lar ge body of literature that uses the comet assa y for the e valuation of toxic exposures (Gallo et al. 2008). For benzene, interactions with gender and the timing of the collection ma y confuse the ability of 8OHdG to detect benzene e xposure. F or e xample, urine collected in late e vening after a shift w as higher in 8OHdG than pre -shift values for benzene exposed workers, and pre-shift measurement could not distinguish those workers that had been exposed to benzene (Nilsson et al. 1996), which points to the need to consider w hen repair or catabolism of these o xidation products are taking place in relation to sample collection. Likewise, asbestos f bers cause the generation of 8OHdG in vitro, but some human studies have found that the y mark exposure and others ha ve not. Here leuk ocytes seem to be a better indicator than urine 8OHdG, and asbestos-associated iron exposure may play an important role in asbestos-related carcinogenesis (Jiang et al. 2008) and may be a confounding f actor for this biomarker. Early animal studies found two- to three -fold higher levels of 8OHdG after one to f ve days in lung tissue collected from rats with a single intratracheal installation of silica. Leuk ocyte 8OHdG w as slightl y higher but not signif cantly dif ferent from control rats (Y amono et al. 1995). These results remind us that tissues close to the insult ma y incur damage w hile other cells, such as leukocytes, may not. In human studies, lymphocyte and urinary 8OHdG measurements were higher in silica -exposed workers but the biomark er could not distinguish w orkers with pneumoconiosis or silicosis. In one study of silicosis patients w ho had e xcessively high levels of leukocyte 8OHdG, their urinar y excretion of 8OHdG w as very low, pointing to poor repair as an impor tant factor in the de velopment of silicosis (Pilger 2006). Of the hea vy metals, chromium and cobalt ha ve been most studied b y the measurement of 8OHdG or FPG -dependent strand breaks. F ine par ticulate matter and air pollution e xposure have also been studied. Both of these contain hea vy metals besides other to xic materials. Hexavalent chromium is carcino genic and can induce 8OHdG and DN A strand breaks in leukocytes in vitro, and se veral research g roups ha ve found higher 8OHdG e xcretion le vels in chromium-exposed individuals, but this has not been uni versally observed (Pilger 2006). Cobolt, which could par ticipate in Fenton-type production of R OS like iron, does not seem to increase urinar y 8OHdG e xcretion, but one in vestigator found that this could be e xplained

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by a decrease in DN A repair acti vity (Pilger 2006). In f act, hea vy-metal interference with OGG1 quantity or activity has been a common theme of research fndings for arsenic, cadmium, chromium, manganese, and lead (Hirano 2008). The implication here is that urinar y 8OHdG excretion ma y be decreased (although 8OHGua is the major b y-product of OGG1 acti vity) while residual 8OHdG in cellular DN A would be decreased. Exposure to f ne par ticulate matter less than 2.5 µ m (PM2.5) w hich contains a v ariety of substances, including heavy metals, appears to be mark ed by increased 8OHdG e xcretion but the increase tends to be f eeting and is more intense post-exposure on work shifts (Pilger 2006, Wei et al. 2009). Cigarette smoking is a great confounder in some of these studies. Photocopier toner emits f ne particulates in the form of carbon black, titanium oxide, amorphous silica, and volatile or ganic compounds. Se veral studies e xplored the implications of this e xposure in workers in toner manufacturing plants compared to controls. Urinary 8OHdG was not different than that of the control g roup in a preliminar y analysis of the data (Kitmura et al. 2009). Volatile organic compounds such as aromatic hydrocarbons are also associated with particulate matter, general air pollution, diesel e xhaust, and a v ariety of industrial and occupational exposures. Some of these e xposures would at f rst appear mild, yet result in increased 8OHdG in either leuk ocytes or urine. F or e xample, among 202 non -smoking Chinese kitchen staf f compared to service staff, urinary 8OHdG/g creatinine was almost 50% higher and cor related with their exposure to cooking oil fumes as measured by urinary 1-hydroxypyrene, and female workers appeared more vulnerab le (Pan et al. 2008), a repeating theme in some of the these studies. General exposure to air pollution in 960 adults dw elling in South K orean urban areas w as measured by their excretion of a number of markers of polycyclic aromatic hydrocarbon (PAH), toluene/xylene, phthalates, and bisphenol A. These were correlated by linear modeling controlling for a number of v ariables such as age, gender , smoking, body w eight, and exercise level. Urinary 8OHdG w as highly associated in the multiple re gression analysis controlling for the above characteristics for all e xposures but PAH (Hong et al. 2009). Cho et al. (Cho et al. 2008) measured a number of rat urinar y nucleosides to deter mine whether a metabolomic patter n would better predict oxidative stress and the time course of its development using bisphenol A, an endocrine disruptor and environmental contaminant known to cause DN A damage. Of the 14 nucleosides measured , se veral of w hich w ere not DN A damage products, 8OHdG and 5OH-methyl-uridine were the only metabolites that were responsive to the bisphenol A insult both in dose and time course. They did not measure either 8OHGua or 8OHG. From this brief discussion, it should be clear to the reader that aside from the methodological issues in the measurement of 8OHdG, the man y confounding f actors and the acti ve role of repair and factors that not only include genetic differences but also direct assaults on the repair mechanism that 8OHdG is a confounded screening indicator of prob lematic toxic exposures. It is best measured immediatel y after the e xposure.

UTILITYAS A BIOMARKER OF THERAPEUTIC IMPROVEMENT Many investigators measure 8OHdG in cells, tissues, and urine as a biomark er of therapeutic improvement from a presumably oxidatively stressed situation or as a result of disease associated with o xidative stress. Others ha ve used health y subjects or animals. Among the m yriad problems already identif ed in the use of 8OHdG as a biomark er, an additional prob lem is whether the inter vention would ameliorate a condition in w hich DNA damage is already at a low backg round le vel. For e xample, since e xercise is thought to produce o xidative stress, a number of investigators have explored antioxidant supplementation as an ameliorative strategy and used 8OHdG as a biomark er, among others. It is no sur prise that if exercise did not affect 8OHdG levels, the addition of an antio xidant would be no cor rection (Bloomer et al. 2006,

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Huang et al. 2000, Subudhi et al. 2004). However, urinary 8OHdG was higher after a hea vily active basketball game and was 60% decreased post-game when the players were supplemented with cysteine (a precursor of glutathione) for 30 da ys prior to the game (Tsakiris et al. 2006). Astaxanthin, a carotenoid, produced lower 8OHdG in gastrocnemius and hear t muscle in rats run to exhaustion and killed 24 hours later (Aoi et al. 2003). The intervention studies, besides using healthy or oxidatively stressed individuals or animals, generally fall into categories such as known or suspected antio xidants, whole diet modif cations, and caloric restriction. RESPONSEOF 8 OH d TO G ANTIOXIDANT INTERVENTIONS The earlier intervention studies focused on the nutrient antio xidants such as vitamins C and E and β-carotene and their mixtures, but later studies ha ve explored other dietar y components that may or may not act primaril y as antioxidants such as the pol yphenolic compounds prevalent in fr uits and v egetables. Se veral e xtensive and systematic re views have been pub lished that list studies and their outcomes for v arious antio xidant inter ventions, mostl y measuring chain breaks via comet assa y (with or without FPG incubation to obtain 8OHdG sites) or urinary 8OHdG excretion (Moller et al. 2002, 2004; Loft et al. 2008). The highlights of these reviews are summarized here with some additional studies. Most of the antio xidant studies ha ve used apparentl y healthy people as subjects. Because 8OHdG in DN A or 8OHdG e xpelled from DN A and found free in body f uids is a result of rapid oxidative insult and repair , antioxidant interventions can track immediate insult. Single dose antioxidant studies using the comet assa y with or without nicking enzymes indicate that their ameliorative effect can be measured within two to eight hours of the antio xidant dose for vitamin C and lycopene, but the effect is f eeting (Moller 2002). These studies are confounded by the initial le vel of damage and each subject ’s antioxidant status. Positive longer ter m single -antioxidant interventions are limited and seem to tak e as much as six months of supplementation. The multiple antioxidant and food-based antioxidant studies fare better, with about 50% of them repor ting a positi ve effect. These reviewers obser ve, as does the author ’s research g roup in their studies, that those with higher le vels of leuk ocyte 8OHdG or chain breaks tend to be more responsi ve to the antio xidants. Most positive studies see an effect by one month of supplementation (Moller 2004, Loft et al. 2008). Of the urinar y 8OHdG studies reviewed with acceptable design scores, 11 showed a decrease with antioxidant supplementation while 17 repor ted no ef fect, with a 50 -50 split for those using antio xidantcontaining foods (Loft et al. 2008). Most of these studies used the 8OHdG ELISA assa y. Of interest is w hether the predominant h ydroxylated guanine base in urine, 8OHGua (the result of hOGG1 excision), is responsive to antioxidant intervention given the shrouded origin of urinar y 8OHdG. Macho wetz et al. measured the ef fect of three oli ve oils with dif fering phenolic content in a randomized cross -over design (three -week interventions with tw o-week washouts) on the e xcretion of 8OHdG, 8OHG, and 8OHGua measured b y HPLC -MS. Only urinary 8OHdG decreased b y 13% (p = 0.008) with oli ve oil supplementation w hich w as independent of its phenolic content. Sur prisingly, the e xcretion pattern of these HGBs v aried greatly for subjects from different European regions with 8OHdG being excreted at almost the same levels as the other tw o HGBs (Machowetz et al. 2007). Studies using presumab ly o xidative-stressed indi viduals because of disease or a stressing event have problematic design issues because if those with high initial le vels are selected, the second (outcome) measurement is lik ely to regress toward the mean independent of the inter vention effect. Therefore, these studies are biased to ward the discovery of a protecti ve effect. Few studies ha ve used multiple measurements o ver time to o vercome re gression toward the mean. One study that o vercame that limitation e valuated smok ers w ho had higher le vels of strand breaks and FPG -sensitive sites than healthy subjects. They also had lower plasma levels of vitamin C. A 250-mg dose of slow-release ascorbic acid was tracked at four-, six- and eighthours post dosing at the be ginning of the study and four w eeks after regular supplementation.

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The slow release for mulation of vitamin C w as able to decrease FPG -sensitive sites b y four hours and remained low thereafter, but the regular formulation of vitamin C was ineffective in the chronic dosing situation. Both Endonuclease III and FPG incubation were more responsive than just measuring DNA chain breaks. Vitamin C supplementation did not affect mRNA levels of hOGG1 (Moller 2004). A number of animal studies co vering a v ariety of prob lems from radiation to aging/senescence to hepatoto xicity ha ve track ed the ameliorati ve ef fects of antio xidants b y 8OHdG in tissues (Hasegawa et al. 1995, Alvarado et al. 2006, Tomofuji et al. 2009, Lemon et al. 2008). The power of these studies is the y are able to link the lo wering of 8OHdG in the tissues with improved pathology. RESPONSEOF 8 OH d TO G DIET INTERVENTIONS, INCLUDING CALORIC RESTRICTION Whole diet interventions are problematic because they often depend upon the ability of enrolled subjects to adhere to the prescribed diet. Adherence levels are often quite modest. One study that got excellent adherence was the Women’s Healthy Eating and Living (WHEL) study which was composed of women who had completed therapy for breast cancer one to four y ears prior to study. A subg roup (n = 179) volunteered 24 -hour urine samples before and one y ear after an intensive increase in fruit and vegetable consumption. Although their 8OHdG/creatinine (by ELISA) decreased, the difference was not statistically signif cant but was inversely associated with their level of vitamin E intak e (Thomson et al. 2005). Another study found that twenty-four days of fruit and vegetable depletion, or increased fruit and vegetable consumption (600 g/day for 24 days), or equivalent levels of vitamins and minerals had no effect on leukocyte FGP-mediated strand breaks or urinar y 8OHdG in healthy nonsmokers (Moller et al. 2003). The author ’s research g roup measured leuk ocyte 8OHdG/dG in healthy young people over a 30 -day period when complete liquid formula diets were their sole source of nutrition. The g roup w as e valuating a ne w for mulation of Ensure that contained higher antioxidant levels and lower polyunsaturated fatty acid levels. There was no difference between the ne w and old for mulation, but 8OHdG/dG pro gressively decreased (as did the inter - indi vidual coeff cient of variation) over the 30-day period (Chen et al. 1999). The author’s research group speculated that the decrease was due to the regulation of lifestyle of the college student participants during the study and the nutritional completeness of the liquid for mulas. Changing whole diets is diff cult, and given the association of different dietary patterns with disease such as cancer and hear t disease, the use of 8OHdG as a mark er of optimal diet in cross-sectional population studies is tempting. Such an anal ysis was reported for a v ery small sample of adults (n = 71) from Florence, Ital y. Giovannelli et al. e valuated food frequenc y questionnaires and obtained leuk ocytes from a subg roup of v olunteers from the EPIC study (European Prospective Investigation into Cancer and Nutrition), so they had extensive lifestyle data. The outcome was leukocyte FPG-mediated percent tail DNA in the comet assay. Although they explored gender, height, weight, smoking, and seasonal and urban/r ural effects and found no association except for height and season, they did not control their dietary analysis for these variables. Surprisingly, they found increases in 8OHdG sites uncovered by FPG with increasing consumption of all v egetables, tomatoes, cof fee, sugar, and vitamin E, with onl y cr uciferous vegetables associated with lo wer FPG sites (Gio vannelli et al. 2002). Given that none of the intervention studies with foods or antio xidants found increased chain breaks or 8OHdG urine excretion, this study points to the diff culty of using 8OHdG as a biomark er in population studies. Thesediff culties are compounded in presumab ly oxidatively stressed individuals. Machado et al. evaluated a number of oxidative stress biomarkers, including plasma 8OHdG by ELISA, in 43 patients with non -alcoholic steatohepatitis (NASH) and 33 control subjects and explored associations with v arious dietar y parameters in a cross -sectional study . The mean plasma

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8OHdG concentration in the controls w as actually higher than the in the patients with N ASH, although several of the NASH patients had the highest levels of anyone. No dietary associations were detected (Muchado et al. 2008). Aside from the confounding v ariable previously identif ed, the additional concer n is whether or not o xidative DNA damage is related to this disease at all. One of the rationales for w hy caloric restriction promotes longe vity is that it slo ws the rate of superoxide production and subsequent hydroxyl radical production. Therefore, several investigators have evaluated 8OHdG in DNA with caloric restriction. These have mostly been animal studies. Generall y, the amount of 8OHdG increases with the age of the animal, and caloric restriction (mostly a 40% reduction in ener gy intake) lowers 8OHdG le vels in all tissues but mostly those that are not di viding as quickly such as brain (Randerath et al. 1993, Sohal et al. 1994, Kaneko et al. 1997, Barja 2002). These f ndings consistent with theor y point to tissue 8OHdG being a good biomark er of o xidative stress despite the lik ely artifactual development of 8OHdG that lik ely occurred in these older studies. Can 8OHdG in circulating leuk ocytes track this phenomenon just as w ell? Wolf et al. used both caloric restriction and alter nate-day f asting to e xplore how well peripheral l ymphocyte 8OHdG, measured histochemicall y, track ed brain, hear t, sk eletal muscle, intestine, and li ver 8OHdG in 12 - month - old and 24 - month - old Sprague - Da wley rats. Although all tissues showed increased 8OHdG with age and decreased 8OHdG with both 40% caloric restriction and alternate-day fasting, lymphocytes at the highest baseline 8OHdG and w ere more responsi ve to both aging and caloric restriction (W olf et al. 2005). In nor mal weight middle -aged men, a two-week 20% energy-restricted diet produced no signif cant change in leuk ocyte 8OHdG/dG or urinary 8OHdG, although e xcretion tended to increase (V elthuis-Te Wierik et al. 1995). A short-term f ast (three to 11 da ys) f ailed to change urinar y 8OHdG in health y w omen e ven though two markers of lipid pero xidation decreased (Bar tsch et al. 2006). In addition to the lower levels of caloric restriction in the human studies, issues of adherence and genetic v ariability and environmental control likely attenuate the ef fect.

CONCLUSIONSAND SUGGESTIONS The use of 8OHdG as a biomark er of o xidative DNA damage or generalized o xidative stress is clouded b y a lack of clarity to its origin in DN A (subject to o xidation and rec ycling from the GTP pool) and in body f uids. Yet, because it has been the focus of so much research, a great deal is known of its limitations and utility in a v ariety of circumstances. This allows the possibility for judicious study design, sample collection, storage, and measurement ef forts for the aler t investigator. Although all of the assa y techniques are prob lematic and prone to the artifactual development of 8OHdG, g reat strides have been made to overcome the major measurement problems. Assay reliability within a gi ven laboratory can be suff ciently managed to distinguish study effects. The following suggestions may improve the utility of this biomark er: 1. Thoroughly study the origin, repair, and metabolism of HGBs and select the most appropriate tissue or biolo gical f uid to answer the research question at hand. 2. Select the most appropriate anal ytical method for the research question and tak e steps to apply the best approaches to reduce ar tifacts. 3. Determine the reliability of the assa y selected on similar tissues or f uids used for data collection and repor t coeff cients of variation in all research repor ts. 4. Control for confounding f actors through robust study design, including: timing of sample collection; gender; en vironmental hazards; and lifestyle f actors such as smoking, alcohol use, and e xtreme e xercise close to sampling times. Those that cannot be controlled through elimination should be tak en into consideration during the statistical anal ysis of the data.

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5. Where possible, measure genetic differences in DNA repair enzyme activity to help explain differences in subsets of human subjects. 6. When using 8OHdG as a biomark er for therapeutic eff cacy, design studies to r ule out regression toward the mean as the reason for dif ferences in outcome.

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KitmuraH ,T erunuma N , K urosaki S , Hata K , Ide R , K uga H , Kakiuchi N , Masuda M, T otsuzaki T , OsatoA , Uchino B , Kitahara K , Iw asaki A ,Y oshizumi K , MorimotoY , Kaisi H , Murase T , HigashiT . 2009 . Cross - sectionalstudy on respiratory effect of toner - xeposed work in manufacturing plants, Japan: pulmonar y function, blood cells and biochemical mark ers. Hum Exp Toxicol 28 ( 6 – 7 ): 331 – 338 . ohno K T , Shinmura K ,T osaka M ,T ani M , Kim SR , Sugimura H , NohmiT , Kasai H ,Y okota J . 1998. Genetic polymorphisms and alter native splicing of the hOGG1 gene, that is in volved in the repair of 8 -hydroxyguanine in damaged DN A. Oncogene 16 ( 3219 – 3225 ): 3219 . owaltowski K AJ , Castilho RF , V ercesi AE .2001 . Mitochondrial permeability transition and oxidative stress . FEBS Letters 495 : 12 – 15 . uo K HW , Chou SY , HuTW , W u FY , Chen DJ . 2007 . Urinary 8 - yhdroxy - 2′ - deo xyguanosine (8-OHdG) and genetic pol ymorphism in breast cancer patients . Mutat Res 631 ( 1 ): 62 – 68 . LeeLS ,W ise SD , Chan C , P arsons TL , Fle xner C , Lietman PS .2008 . Possible differential induction of phase 2 enzyme and antio xidant pathways by American ginseng, Panax quinquefolius .J Clin Pharmacol 48 : 599 – 609 . LemonJA , Rollo CD , Boreham DR .2008 . Elevated DNA damage in a mouse model of oxidative stress: impacts of ionizing radiation and a protecti ve dietary supplement . Mutagenesis 23 ( 6 ): 473 – 482 . LiTH , JiaWL ,W ang HS , Liu RM .2007 . Electrochemical performance of 8 - yhdroxy - 2′deoxyguanosine and its detection at pol y(3-methylthiophene) modif ed glassy carbon electrode . Biosens Bioelectron 22 ( 7 ): 1245 – 1250 . LimKS ,Aseeland KJ , Whiteman M , JennerA , Halliw ell B . 2005 . Oxidative damage in mitochondrial DNA is not e xtensive. Ann NY Acad Sci 1042 . LoftS , Larsen PN , RasmussenA , F ischer - NielsenA , Bondesen S , Kirkagaard P , Rasmussen LS , Ejlersen E ,T ornoe K , Ber gholdt R et , al. 1995 . Oxidative DNA damage after transplantation of the liver and small intestine in pigs . Transplantation 59 ( 1 ): 16 – 20 . LoftS , Moller P , Cook e MS , Rozalski R , Olinski R .2008 .Antioxidant vitamins and cancer risk: is oxidative damage to DN A a relevant biomarker? Eur J Nutr 47 ( Suppl 2 ): 19 – 28 . LoftS , Sv oboda P , Kasei H ,TjonnelandA ,V ogel U , Moller, Overvad K , Raaschou - Nielse n O. 2006 . Prospective study of 8 - xoo - 7,8 - dih ydro - ′2-deoxyguanosine excretion and the risk of lung cancer . Carcinogenesis 27 ( 6 ): 1245 – 1250 . LoftS ,V istisen K , Ew ertz M ,TjonnelandA , Ov ervad K , P oulsen HE .1992 . Oxidative DNA damage estimated by 8 -hydroxydeoxyguanosine excretion in humans: inf uence of smoking, gender and body mass inde x. Carcinogenesis 13 ( 12 ): 2241 – 2247 . Lo we FJ , Gre gg EO , McEw an M. 2009 . Evaluation of biomarkers of exposure and potential harm in smokers, former smokers and never-smokers. Clin Chem Lab Med 47 ( 3 ): 311 – 320 . Macho wetz A , P oulsen HE , Gruendel S ,W eimann A , F ito M , Marrugat J , dela Torre R , Solonen JT , Nyyssonen K , Mursu J , Nascetti S , GaddiA , Kiese wetter H , Baumler H , Selmi H , Kaikk onen J , Zunft HJF , Co vas MI , K oebnick C .2007 . Effect of olive oils on biomarkers of oxidative DNA stress in Nor thern and Souther n Europeans . FASEB J 21 : 45 – 52 . MaeshimaE , Liang XM , Otani H , Mune M ,Y ukawa S .2002 . Effect of environmental changes on oxidative deoxyribonucleic acid (DNA) damage in Systemic Lupus Er ythematosus. Arch Environ Health 57 ( 5 ): 425 – 428 . MalinsDC , Holmes EH , P olissar NL , Gunselman SJ . 1993 .The etiology of breast cancer. Characteristic alterations in h ydroxyl radical -induced DNA base lesions during onco genesis with potential for e valuating incidence of risk . Cancer (Phila.) 71 : 3036 – 3043 . MalinsDC , Johnson PM ,WheelerTM , Bark er EA , P olissar NL ,V inson MA .2001 .Age - rela ted radical-induced DNA damage is link ed to prostate cancer . Cancer Res 61 : 6025 – 6028 . Mar nett LJ. 2000 . Oxyradicals and DNA damage . Carcinogenesis 21 ( 3 ): 361 – 370 . Ma ysumoto Y , Oga wa Y , Y oshida R , ShimamoriA , Kasai H .2008 . the stability of the oxidative stress marker, urinary 8 - yhdroxy - ′2 - deo xyguanosine (8 - OHdG),when stored at room temperature. JOccup Health 50 : 366 – 372 .

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Chapter18 Exocyclic DNA Adducts as Biomarkers of Antioxidant Defense and Oxidative Stress Ro ger W.L. Godsc halk

INTR ODUCTION EXOCYCLIC DNA ADDUCTS AS BIOMARKERS IN MOLECULAR EPIDEMIOLOGICAL STUDIES Epidemiological studies have shown that chronic inf ammation is often link ed to the de velopment of malignant diseases in the in volved tissues. F or instance, inf ammation of the li ver induced by hepatitis B and C vir uses is related to an increased risk of de veloping liver cancer (Farazi 2006). Another example is the persistent inf ammation in chronic obstructive pulmonary disease (COPD), w hich leads to an increased lung cancer risk, independent from smoking behavior (Barnes and Celli 2009). Additional examples have been listed in Table 18.1. Mechanistically, it is thought that chronic inf ammation leads to persistent o xidative stress and subsequent e xcess of lipid pero xidation (LPO) (F igure 18.1), w hich can cause massi ve DNA damage. DN A damage that is not repaired before cell di vision can lead to mutations due to mispairing during DN A replication opposite the damaged nucleotide. Accumulation of mutations is a hallmark for the initiation and pro gression of carcino genesis. DNA damage can be induced b y a direct interaction of reacti ve o xygen and nitro gen species (R OS/RNS), generated during inf ammatory conditions, or these R OS/RNS can f rst interact with lipids to for m reacti ve LPO products that can interact with DN A to for m pro -mutagenic lesions, including so-called exocyclic DNA adducts (Medeiros 2009). These DNA lesions can theoretically be used as earl y endpoints (i.e., biomark ers) in inter vention studies, because the y seem to pla y an essential role in the causal pathw ay of inf ammation-driven diseases leading to malignancies. The use of such biomark ers in epidemiolo gical settings is called molecular epidemiolo gy, which aims to inte grate techniques of anal ytical chemistr y, biochemistr y, molecular biolo gy, and epidemiolo gy (Schulte and P erera 1993). Molecular epidemiolo gy has the potential to contribute to cancer research in a number of areas, including assessment of exposure to endogenous genoto xic compounds and the earl y identif cation of indi viduals at potentiall y high cancer risk. The general scheme of molecular epidemiology is presented in Figure 18.2, which shows that stab le DNA adducts induced b y LPO can be used as biomark ers for e xposure to Biomarkers for Antioxidant Defense and Oxidative Dama ge: Principles and Pr actical Applications Edited by Giancarlo Aldini, Kyung-Jin Yeum, Estuo Niki, and Rober t M. Russell ©2010 Blackwell Publishing Ltd.

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Table 18.1. Some examples that show a relationship between inf ammation and subsequent tumorigenesis. Inf ammation can be induced by bacteria, viruses, parasites or chemicals.

Source of inf ammation V iral Bacterialand parasitic

Chemicals Unkno wn

Cancersite HepatitisB virus H. pylori S. typhi/paratyphi O.viverrini (liver f uke) Sch. Hematobium Cigarettesmoke (COPD) Asbestosis Excessive alcohol use (cir rhosis) Crohns’disease Ulcerative colitis Pancreatitis

Li ver cancer Gastriccancer Gall bladder cancer Cholangiocarcinoma Bladder cancer Lungcancer Lung cancer Liver cancer Bo wel cancer Bowel cancer Pancreas cancer

Figure 18.1. The process of lipid peroxidation (LPO) initiated by free radicals that are, for instance, produced during chronic inf ammatory diseases. LPO leads to the formation of reactive aldehydes (4 -hydroxy-2-nonenal [HNE] and malondialdehyde [MDA]) that can subsequently react with DNA to form exocyclic DNA adducts (Figure 18.3).

DNA-damaging compounds that ha ve been generated during inf ammation (exposure assessment) and that interventions that modulate DNA adduct levels could in theory also change the cancer risk accordingly (risk assessment/prevention). In theory, exposure biomarker analysis comprises the assessment of agents or their metabolites either in tissues, secreta, e xcreta, or an y combination of these to e valuate e xposure as compared with an appropriate reference (Schulte and P erera 1993). However, the direct measurement of ROS/RNS in humans is diff cult due to the shor t half -life of these radicals (such as the h ydroxyl radical OH •). LPO products are more stab le and ha ve therefore been used as biomarkers for oxidative stress, for instance by assessing thiobarbituric acid reactive substances

Exocyclic DNA Adducts as Biomarkers of Antioxidant Defense and Oxidati ve Stress Exposure to genotoxins Inflammation

Early endpoint (biomarkers) ROS Lipid peroxidation

DNA damage

321

Disease Cancer

Mutations

Prevention

Risk assessment

Exposure assessment

Intervention

Figure 18.2. The general scheme of molecular epidemiology representing the continuum between exposure to genotoxins and ultimately the development of disease. Stable DNA adducts induced by oxidative stress or lipid peroxidation can be used as early endpoints (biomarkers) to study the eff cacy of interventions to prevent inf ammation-driven malignant diseases.

(TBARS) and isoprostanes in body f uids or tissues. Ho wever, their measurement does not necessarily represent the biolo gically ef fective dose, w hich is the dose that actuall y af fects DNA in the tissue in w hich tumors ultimatel y may occur. Therefore, the most promising biomarker seems to be the measurement of DNA adducts, because it takes into account individual differences in exposure to ROS/RNS and LPO products, detoxif cation by dietary or enzymatic antioxidants, as well as cell turnover and DNA repair. However, several aspects of DNA adduct measurements need to be kno wn in detail for the ef fective and reliab le use of these DN A adducts as biomarkers for oxidative stress or to test the eff cacy of inter ventions.

CRITERIA FOR DNA ADDUCTS AS BIOMARKERS OF EXPOSURE TO OXIDATIVE STRESS AND LIPID PEROXIDATION PRODUCTS MECHANISMSOF EXOCYCLIC DNA ADDUCT FORMATION Chronic inf ammatory processes produce e xcess ROS that can directl y interact with DN A to form various types of DN A lesions. The most studied type of these o xidative DNA lesions is 7 - yhdro - 8 -xoo - ′2-deoxyguanosine (8 -oxo-dG), and se veral methodolo gies ha ve been de veloped to measure 8 -oxo-dG in human tissues and urine. Unfor tunately, the (background) levels of 8-oxo-dG were severely overestimated in earlier studies due to its artif cial formation during DNA isolation and/or anal ysis. Although there ha ve been some attempts to o vercome this problem and the actual backg round levels are more clear no w (ESCODD 2002, Collins et al. 2004), the assessment of LPO -derived DNA lesions seems to be more reliab le because the y are less sensitive to ar tif cial formation during the complete procedure of anal ysis. LPO-derived reactive aldehydes are formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, and these aldeh ydes can damage DN A either b y reacting directly with DNA bases or by generating even more reactive bifunctional intermediates, which form so -called exocyclic DNA adducts (Medeiros 2009). DNA lesions for med by the reactive aldehyde 4 -hydroxy-2-nonenal (HNE) are among the most intensel y studied DNA lesions and are thought to contribute to the mutagenic and carcino genic effects associated with inf ammation (Nair et al. 2007a). HNE generates exocyclic adducts with a f ve - memberedring (so - called etheno-DNA adducts) attached to the DN A bases; the str uctures of these DN A lesions which have been detected in human and animal tissues are sho wn in F igure 18.3 and include xyadenosine (ε dA), 3,N4 - etheno ′- -2 deo xycytidine (ε dC), 1,N2 - etheno ′- 2 1,N6 - etheno ′- -2 deo 2 2 xyguanosine (N2 ,3ε dG)(Medeiros 2009 Nair , deoxyguanosine (N εdG), and N ,3 - etheno ′- -2 deo et al. 2007a). One of these etheno -DNA adducts ( εdA) was found to be more mutagenic than 8-oxo-dG in Hela cells that w ere transfected with plasmids containing a single DN A adduct, leading to A→T mutations (Levine et al. 2000).

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PARENT NUCLEOTIDES NH2 N

N

N N dR dA

O

NH2 N

N

N dR

N O dR dC

HNE

NH

HNE

N

NH2

dG

HNE

MDA

ADDUCTED NUCLEOTIDES N

N N N N dR εdA

O N

N N dR εdC

N O

N dR

N N N H 1,N2εdG

N

NH

N dR

N 2

N 3εdG

N

O N N dR

N N

N

M1dG

Figure 18.3. Formation of exocyclic DNA adducts from the LPO products 4 -hydroxy-2-nonenal (HNE) and malondialdehyde (MDA).

Other reactive aldehydes, such as malondialdehyde (MDA), can also react with DNA to form exocyclic DNA adducts. For MDA, the major adduct formed is 3 - (2 - deo xy - b - D ythro - er - pentafuranosyl)pyrimido[1,2 - a]purin - 10(3H) - one deoxyguanosine (M1 dG) (Figure 18.3 )(Marnett 1999 ).Also, the M1dG adduct is considered to be a pro -mutagenic lesion because its presence in DNA can lead to mispairing of nucleotides during DN A synthesis and as a consequence mutations are formed. M1dG was found to be mutagenic in both bacteria and mammalian cells, predominantly leading to base pair substitutions (G →T and G →A) and frameshift mutations when using shuttle v ectors containing a site -specif c lesion in COS -7 cells (VanderVeen et al. 2003 ). A signif cant cor relation between LPO and subsequent for mation of etheno -DNA adducts was observed (Chung et al. 1996), and inhibition of LPO b y, for instance, dietar y antioxidant vitamins was also found to decrease exocyclic DNA adduct formation (Hagenlocher et al. 2001, Van Helden et al. 2009). High intak e of dietar y omega-6 polyunsaturated f atty acids, w hich are sensitive for LPO, has been associated with increased le vels of both etheno -DNA adducts and M 1dG (Fang et al. 1996, Nair et al. 1997). Etheno - DN A adducts and M1dG are both for med by LPO products; therefore, one w ould expect that their levels will correlate, but this was not conf rmed in all studies (Kadlubar et al. 1998, Arab et al. 2009). A lack of correlation might be attributed to differences in DNA repair; etheno-DNA adducts are repaired by base excision repair (BER) (Gros et al. 2003, Hang et al. 1996 ),whereas M1dG is thought to be repaired b y nucleotide e xcision repair (NER) in both bacterial and mammalian cells (Fink et al. 1997, Johnson et al. 1997, VanderVeen et al. 2003). However, it is also likely that differences in formation underlie the lack of cor relation between both types of e xocyclic DNA lesions. Ne xt to the for mation of M 1dG by MDA, Dedon and co-workers (1998) proposed a mechanism in which M1dG adducts can also be for med through the for mation of base propenals during o xidative stress. The study b y Kadlubar et al. (1998) is in f avor of this h ypothesis and M 1dG could be for med primarily by reaction of DN A with a base propenal, because the y observed a good cor relation between M 1 dG and 8 - xoo - dG,but not with the levels of εdA and εdC. Thus, the formation of M1dG is less clearly related to LPO. Alternative mechanisms have also been postulated for the formation of etheno-DNA adducts. The upregulation of enzymes that metabolize arachidonic acid (such as c yclooxygenases and

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lipoxygenases) was shown to increase the formation of εdA and εdC at distinct stages of a skin tumor model induced b y dimeth ylbenzanthracene (DMB A)(Nair et al. 2000). Ho wever, the exact reactive metabolites that are responsib le for DNA adduct formation in this experimental system still need to be identif ed. Circumstantial e vidence available from in vitro and in vivo studies points to ward the in volvement of h ydroxyalkenals (Bar tsch 1999) that are produced during lipoxygenase-catalyzed arachidonic acid metabolism. TECHNIQ UES TO MEASURE EXOCYCLIC - DN A ADDUCTS AND THEIR VALIDATION Sensitive and highl y specif c methods ha ve been de veloped to detect and quantify e xocyclic DNA adducts in human and animal tissues. These methods ha ve f acilitated the detection of in vivo background levels possibly arising from endogenous LPO products as w ell as study of their formation and role in experimental and human carcinogenesis. The scientif c literature on this subject has already been comprehensi vely reviewed (Nair 1999, Barbin 2000). The prinimmunohistochemicipal techniques for exocyclic-DNA adduct detection are: 32 P - postlabeling, cal staining, immuno -slotblot, gas chromato graphy-mass spectrometr y (GC -MS), and liquid chromatography - mass spectrometry (LC - MS or LC - ES - MS/MS)(reviewed in Nair 1999 , Bartsch 2000, Nair et al. 2007a). The technical validation of some of these methods in humans has resulted in potentially useful biomarkers for testing of the eff cacy of an intervention aimed at reducing oxidative stress and LPO . The main limitations of these methods are the lack of inter -laboratory comparisons with the same method and lack of conf rmation of adduct levels with two different methods on the same DNA sample. F or instance, recent data did not sho w a cor relation between M 1dG adducts as determined by immuno -slotblot and an immuno -enriched 32 P - postlabelingtechnique (unpublished data). Another problem is the dif ferent detection limits of the methods and the amount of DNA required for anal ysis, which is often limited for biopsy material and WBC samples methods that are obtainable in ethically approved human studies. For example, 32 P - postlabeling for exocyclic-DNA adducts require small amounts of DNA (1 to 25 µg), while mass spectrometry still requires up to 100 µg of DN A for anal ysis. Ho wever, it should be mentioned that techniques based on mass spectrometr y are continuousl y improving and smaller amounts of DNA could be applicab le in the near future. New developments also have made it possible to measure the presence of small amounts of adducted nucleosides/nucleotides in biolo gical f uids (Hanaoka et al. 2002, Sun et al. 2006). The possibility to assess DNA adducts in urine is especially valuable for large-scale molecular epidemiology, because such samples can be obtained in a non-invasive way. However, it should be noted that the source of DN A adducts in urine is still a matter of debate; the y can originate from DNA lesions that were excised from the DNA during DNA repair, dietary intake, as well as from damage to the cellular nucleotide pool. Therefore, the infor mation obtained from urinary analysis is less clear re garding potential health risks as compared to the anal ysis of exocyclic-DNA adducts in tar get tissues. SURR OGATE TISSUES Practical and ethical considerations limit the types of tissues a vailable for anal ysis of DN A adducts in humans. Most tar get organs for LPO -induced genotoxicity cannot be reached for routine sampling. Nonetheless, man y studies in w hich e xocyclic-DNA adducts ha ve been assessed in inf amed tissues actuall y used tar get tissues, such as colon, aor ta, pancreas, and liver (Nair et al. 2007a). Studies that used easil y accessible sur rogate tissues, such as w hite blood cells (WBC), also sho wed that w hite blood cells can accumulate DN A adducts in suff cient amounts to f nd differences between exposed and unexposed subjects. In fact, the highest

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Table 18.2. Levels of etheno -DNA and M 1dG adducts in various tissues and cells of humans. Data obtained from Nair 2007.

Or gan Li ver Esophagus Gastricmucosa P ancreas Colon Lung * Breast Leuk ocytes

Etheno - DN A adduct levels (adductsper 108 parent nucleotides) < 0.05 – 4 < 0.01 – 4 – 0.1 – 6 0.1 – 9 0.3 – 14 – 0.3 – 25

M 1 dG 5 – 120 – 3 – 65 1 – 50 nd * * – 120 10± 4 0.5 – 56 2 – 287

*Etheno-DNA adduct levels in lung of smok ers and nonsmokers. No effect of smoking w as found. Godschalk et al. 2002. * * nd: not detected.

levels of e xocyclic-DNA adducts w ere found in WBC as compared to other or gans/tissues (Table 18.2 ). Of course, w hen WBC were used as sur rogate tissue, one w ould expect that etheno -DNA adducts in WBC would ref ect the levels of etheno -DNA adducts in target tissues. In the study by Speina et al. (2003) both WBC and lung samples w ere analyzed, and leuk ocytes accumulated a signif cantly higher number of etheno-DNA adducts than lung tissue. Although ε dCand εdA levels were highly cor related, the authors unfor tunately did not repor t on the cor relation between surrogate (leukocytes) and lung tissue. A recent study on adduct formation in pregnant women and their newborn children showed a good correlation between the formation of ethenoDNA adducts in both mother and neonate (Arab et al. 2009), indicating that that LPO -related DNA damage is induced similarly in fetus and mother, and that adducts in blood of the mother can be used to predict etheno -DNA adduct formation in the neonate. Although direct evidence for a correlation between DNA adducts in target and surrogate tissue is lacking, it is likely that such a cor relation exists. DEFINEDKINETICS AND VARIATION IN TIME Knowledge of dose -response relationships and adduct persistence is essential for e xposure monitoring studies and inter ventions, which use DNA adduct levels as endpoints (Schulte and Perera, 1993). For example, researchers should kno w how, and ho w fast, adduct le vels might change upon differences in exposure or after an intervention to select the most appropriate time for sampling. Thus, understanding the kinetics of DN A adducts in sur rogate and target tissues guides the inter pretation of DNA adduct data (Schulte and P erera, 1993). The dose-response relationship between the level of LPO and subsequent for mation of exocyclic DNA adducts in vivo has not been studied in detail, but it is clear that DN A adduct measurements can distinguish betw een two or more dif ferent exposure levels as compared to control samples. Most of the studies simpl y compared samples obtained from diseased vs . control subjects and indeed found statisticall y signif cant dif ferences; in tissues in volved in several types of inf ammatory diseases, the le vels of e xocyclic DNA adducts w ere higher as compared to controls, with relati ve differences up to one or tw o orders of magnitude (Bar tsch 2006 ). However, it is currently unknown what the half-life of exocyclic-DNA adducts is after reducing the level of LPO b y an inter vention or complete remo val of the inf ammation by effective

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treatment of the disease. Nonetheless, this is important information for designing future (intervention) studies and choosing the frequenc y and timing of sampling. A recent inter vention study in subjects who suffered from chronic infection with Opisthorchis viverrini (OV) indeed revealed that the urinar y excretion of etheno -DNA adducts was signif cantly reduced within a period of tw o months b y treatment for the infection with praziquantel (Dechakhamphu et al. 2008). These data prove that the assessment of (urinar y) exocyclic DNA adducts can be used to monitor the eff cacy of inter ventions. Ho wever, more infor mation on adduct kinetics in tissues is still needed and deser ves further attention in future studies. The stability of etheno -DNA adducts depends on the life span of the cells in w hich adducts are measured , but also on dif ferences in DN A repair . White b lood cells consist of se veral subtypes with various life spans (days to years). Although it is known that, for instance, levels of bulk y DNA lesions can dif fer between the subtypes of cells (Godschalk et al. 1998), no information is cur rently available on exocyclic-DNA adduct formation. However, it would not be surprising if these cells actuall y vary in the accumulation of etheno -DNA adducts, because they may also differ in their DN A repair capacity. Etheno-DNA adducts are repaired b y base e xcision repair (BER), and for each adducted nucleotide, another gl ycosylase is in volved: εdA is repaired b y 3 -methyladenine DNA glycosylase and εdC is repaired b y a mismatch specif c th ymidine-DNA gl ycosylase (Gros et al. 2003, Hang et al. 1996). The action of these glycosylases results in apurinic/apyrimidinic sites that are subsequently repaired by the mammalian apurinic/ap yrimidinic endonuclease (APE1/ Ref1) (Demple 2005). APE1/Ref1 also functions as a redox factor, facilitating the DNA binding capability of several transcription factors such as NF-kB, which were also found to be involved in inf ammation-related diseases (Winther 2005). The author and colleagues pre viously found that decreased le vels of etheno -DNA adducts in aor ta of an atherosclerotic mouse model coincided with an increased e xpression of APE1/Ref1, suggesting that repair w as enhanced (Godschalk et al. 2007). Overall, APE1/Ref1 may represent an impor tant link between oxidative stress, DNA damage, and subsequent gene e xpression. Due to the in volvement of different DNA glycosylases for the repair of εdA and ε dC, their relative levels can vary between diseased and non-diseased subjects, as well as between tissues of a single indi vidual (regardless of w hether they have a disease). F or instance, the ε dA/ε dC ratio was different in patients with familial adenomatous polyposis (FAP) as compared to colon samples of patients suf fering from ulcerati ve colitis (Schmid et al. 2000, Nair et al. 2006). Moreover, the repair rates also differed between white blood cells, tumor tissue, and uninvolved tumor-adjacent tissues (Speina et al. 2003). It was additionally reported that the ratio betw een εdA and εdC can v ary due to a direct interaction of the 3 -methyladenine DNA gl ycosylase with εdC without actually repairing it, making it impossib le for the mismatch -specif c thymidine-DNA glycosylase to reach the εdC lesion to repair it eff ciently (Gros et al. 2004). This could be one e xplanation why εdC levels are usuall y 1.5 - to nine -fold higher than the le vels of ε dA. SOURCESOF INTER -AND INTRA - INDIVIDU AL VARIATIONS Sources of inter - and intra -individual variations should be characterized before DN A adducts can be ef fectively applied as biomark ers in lar ge-scale studies (Schulte and P erera 1993). It can be expected that etheno-DNA adduct formation may be subject to a greater variability than the assessment of LPO products in body f uids, because the body actively participates in ROSproduction and subsequent LPO and the elimination of LPO products and LPO -derived DNA adducts. In other words, two individuals with similar levels of oxidative stress and subsequent LPO might still ha ve dif ferent e xocyclic-DNA adduct le vels in their tissues or leuk ocytes. Variations ma y be due to dif ferences in metabolic phenotypes, possib ly related to genetic polymorphisms in a variety of enzymes involved in the formation or detoxif cation of ROS and LPO products or DN A repair.

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Many genetic pol ymorphisms have been identif ed in genes related to o xidative stress and the repair of DN A damage related to o xidative stress. It is, ho wever, beyond the scope of this manuscript to gi ve a detailed o verview of all genetic pol ymorphisms that could af fect the formation of e xocyclic-DNA adducts. Some interesting candidates will be mentioned. With regard to detoxif cation of LPO -products, especially glutathione S -transferases (GST), A4 and P1 seem to be most relevant. GSTA4 is recognized as one of the predominant enzymes responsible for the deto xif cation of the etheno -DNA adduct for ming aldeh yde HNE, and GSTP1 catalyzes the detoxif cation of base propenals, which are known to be involved in the formation of M 1dG (Berhane et al. 1994). A candidate gene pol ymorphism in the repair of etheno -DNA adducts is XRCC1, which interacts with DN A ligase III, pol ymerase β , and poly(ADP - ribose) polymerase (PARP) in the base e xcision repair pathway. A specif c genetic pol ymorphism in XRCC1 was related to the occur rence of specif c p53 mutations that were detected in vinyl-chloride-exposed workers (Li et al. 2003). The genotoxicity of vin yl chloride is predominantl y elicited via the for mation of etheno -DNA adducts. In fact, etheno -DNA adducts w ere f rst described to be induced b y vinyl chloride. Later it w as found that LPO could induce the same DN A lesions. In general, genetic pol ymorphisms are likely to be in volved in the v ariation in exocyclic-DNA adduct levels as obser ved in a cer tain population, but this needs fur ther study. Lifestyle is another important reason for inter-individual differences in adduct levels. Several dietary f actors modulated DN A adduct for mation in tissues/cells or the e xcretion of etheno DNA adducts in urine. F or example, the induction of CYP2E1 b y the consumption of alcohol was recently found to increase the le vels of εdC and εdA (Wang et al. 2009), and the intak e of antioxidants, which reduce the levels of ROS and LPO, was found to simultaneously reduce the levels of exocyclic DNA adducts (Hagenlocher et al. 2001, Van Helden et al. 2009). Dietary habits were found to affect the urinary excretion of etheno-DNA adducts (Hanaoka et al. 2002). Thus, diet (or dietary interventions) can inf uence DNA adduct levels, modulating the processes that lead to malignant transfor mation of cells. The studies b y Fang et al. (1996) and Nair et al. (1997) showed that a diet rich in pol yunsaturated f atty acids increased e xocyclic-DNA adduct for mation in WBC, predominantl y in females. These results were later conf rmed in studies on laboratory rodents (Fang et al. 2007). On the other hand , high intak e of f at (Western type of diet) decreased etheno -DNA adduct formation in the aorta of pro-atherosclerotic ApoE knock-out mice, which was probably linked to increased DNA repair, because the expression of APE1/Ref1 was also signif cantly increased (Godschalk et al. 2007). Although increased e xpression of BER -related enzymes seems to indicate a protective mechanism, it should be mentioned that an imbalanced increase in Ape1/ Ref1 acti vity w as found to result in microsatellite instability during chronic inf ammation (Hofseth et al. 2003). In other words, “overcompensation” by BER may not always be benef cial. Moreo ver, in these y oung ApoE knock -out animals, atherosclerotic lesions w ere still absent, which could ha ve affected the results because etheno -DNA adducts w ere found to be signif cantly increased in human aor tas car rying severe atherosclerotic plaques, especiall y in those of active smokers (Nair et al. 2007b). Smoking is an important source of oxidative stress and thus for inter -individual differences; one single puf f of cigarette smok e seems to contain up to 10 14 radicals that could initiate the process of LPO (Pr yor 1993). Ho wever, smoking did not af fect the le vels of etheno -DNA adducts (Godschalk et al. 2002) nor repair acti vities (Speina et al. 2003) of εdA and ε dC in human lung. On the other hand, a signif cant difference in repair capacity was observed between two histolo gical types of lung cancer —squamous cell carcinoma and adenocarcinoma. In individuals suffering from lung adenocarcinoma, εdA and εdC repair activities in normal lung and blood leuk ocytes were signif cantly lower than in patients suf fering from squamous cell carcinoma (Speina et al. 2003). Moreover, repair capacity for εdA was signif cantly lower in blood leukocytes of lung cancer patients than in leuk ocytes of health y volunteers, which was

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even more pronounced betw een health y v olunteers and patients de veloping inf ammationrelated adenocarcinoma (Speina et al. 2003). Sources for intra-individual variation in etheno-DNA adduct levels have not yet been studied, but it is evident that long -term changes, for instance, in dietar y habits and disease status, ma y also have consequences for the adduct le vels as found in a par ticular person.

COMP ARISON OF VARIOUS METHODOLOGIES REFLECTING DIFFERENT PROCESSES IN INFLAMMATION - DRIVENGENOTOXICITY Several kinds of biomarkers, or combinations of biomarkers, have been used to assess exposure to LPO products and subsequent for mation of exocyclic DNA adducts. Comparing one potential biomarker to another can pro vide useful infor mation on mark er characteristics, but does not constitute validation (Schulte and Perera 1993). With the methodologies that are cur rently available, the le vel of LPO and its end products can be assessed (for instance TBARS, iso prostanes, or MD A le vels) in biolo gical matrices and simultaneousl y adduct for mation and removal in the same biolo gical samples can be deter mined. Together these biomark ers ma y yield valuable information on the causal pathway between chronic inf ammation and the occurrence of cancer, and may also identify the most promising pre ventive measures. The w orking h ypothesis is indicated in F igure 18.4. Exoc yclic-DNA adduct for mation increases with increasing oxidative stress, but cells may initially try to compensate, for instance, by improving DNA repair. Only when the inf ammation persists or increases in the course of the disease, adduct le vels star t to accumulate because compensation becomes inadequate. Moreover, DNA repair (BER as w ell as NER) can be impaired b y continuous oxidative stress and LPO products (Bar tsch 2006). Together with dere gulation of cell homeostasis during inf ammation, the resulting genetic changes may act as a driving force in disease pathogenesis. Thus, DNA damage caused by ROS, RNS, and LPO end products provide promising molecular signatures for risk prediction and potential tar gets for pre ventive measures. Pre ventive measures that modulate the for mation of e xocyclic-DNA adducts ma y thus also modulate the processes that lead to malignanc y (i.e., changing the slopes of the lines as indicated in F igure 18.4 ). 3

lev vel

1

2

Time or progression of disease

Figure 18.4. Progression of an inf ammatory disease produces an excess of reactive oxygen/ nitrogen species (indicated as gray area). This increased oxidative stress also increases the formation of exocyclic-DNA adducts (1). Although DNA repair may initially try to compensate (2), the persistent oxidative stress is known to decrease DNA repair capacity. In combination with other relevant changes during inf ammation, such as cell turnover, the conversion of DNA lesions into mutations (3) steadily increases, leading to progression of the disease. Dietary factors/ interventions could affect the slopes of these lines, and thus the process of carcinogenesis.

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SUMMAR Y AND PERSPECTIVES Infammation is ine vitably link ed to increased le vels of o xidative stress, w hich can lead to direct and indirect types of DN A damage. Direct types of DN A damage, such as 7 -hydro-8oxo - 2′-deoxyguanosine, have often been used as biomark ers of o xidative stress, but accurate measurements were often hampered b y ar tif cial for mation of these DN A lesions due to the manipulation of samples during DN A isolation and the actual anal ysis. On the other hand, the indirect types of DNA damage, such as that induced b y lipid peroxidation products, seem less sensitive for such ar tifacts and have therefore received attention as alter natives for the assessment of oxidative stress and to test the eff cacy of interventions. These types of DNA damages include so-called exocyclic-DNA adducts, which can be measured by highly sensitive methods. Their presence w as shown in tissues of health y humans and animals, and in tissues in volved in several types of inf ammatory diseases, these le vels were one or tw o orders of magnitude higher. Moreover, dietary antioxidants were found to lo wer exocyclic DNA adduct levels. Overall, it is clear that assessment of e xocyclic DNA adducts has at least three theoretical advantages as earl y endpoints in inter vention studies: f rst, DNA adducts are thought to pla y a signif cant role in the causation of cer tain types of cancer, and therefore represent a measure for exposure to ROS/LPO-products as well as risk. Second, they can smooth the e xtreme variability in e xposure to ROS and LPO products, w hich is typical for chronic inf ammatory diseases (for instance, during e xacerbations) and therefore they can reduce the monitoring ef fort. Third, DNA adducts may account for inter -individual differences in for mation and repair. On the other hand, sampling and analytical demands as well as the imprecision of assays may lead to signif cant measurement er rors, which will tend to attenuate the underl ying relationships. Therefore, the success of future studies in the application of DN A adducts depends on the development and systematic use of w ell validated techniques and biomark ers. Identifcation of individuals with high levels of exocyclic DNA adducts may result in a better selection of appropriate study populations in future molecular epidemiological studies. Progress is to be expected from studies in which promising intermediate risk markers such as exocyclic DNA adducts and susceptibility (genetic as w ell as phenotypic) are combined , providing reasonably short and cost -effective intervention studies. Thus, e xocyclic DN A adducts ha ve the potential to be used as promising inter mediate molecular mark ers of inf ammation-driven human carcino genesis and to test the eff cacy of chemopreventive agents therein. Some aspects still need to be addressed in more detail. F or instance, do adduct le vels in sur rogate tissues ref ect the le vels in tar get organs (also after an intervention), and w hat are the e xact sources of intra - and inter -individual v ariation? Nonetheless, it can be concluded that there is no w suff cient e vidence to eff ciently apply exocyclic-DNA adducts as biomark ers in inter vention studies.

REFERENCES ArabK , P edersen M , Nair J , Meerang M , Knudsen LE , Bartsch H .2009 .Typical signature of DNA damage in w hite blood cells: a pilot study on etheno adducts in Danish mother -newborn child pairs . Carcinogenesis 30 ( 2 ):282 – 285 . Barbin A. 2000 . Etheno - adduct - ming for chemicals: from mutagenicity testing to tumor mutation spectra. Mutat Res 462 : 55 – 69 . Bar nes PJ , Celli BR .2009 . Systemic manifestations and comorbidities of COPD . Eur Respir J 33 ( 5 ): 1165 – 1185 . Bartsch H. 1999. Exocyclic adducts as ne w risk markers for DNA damage in man . In Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis . Singer B , Bartsch H eds. , IARC Scientif c Publication 150, Lyon, France : International Agency for Research on Cancer . pp 1 – 16 . Bar tsch H , Nair J . 2000 . Ultrasensitive and specif c detection methods for e xocylic DNA adducts: markers for lipid pero xidation and oxidative stress . Toxicology 153 : 105 – 114 .

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omega 6 -polyunsaturated fatty acid intake in postmenopausal Japanese w omen. Int J Cancer 100 ( 1 ): 71 – 75 . HangB , Chenna B , Rao S , Hang B , ChennaA , Rao S , Singer B . 1996 . 1,N6 - ethenoadenineand 3,N4-ethenocytosine are excised by separate human DN A glycosylases. Carcinogenesis 17 : 155 – 157 . HofsethLJ , Khan MA ,Ambrose M , Nik olayeva O , Xu - elliver W M , Kartalou M , Hussain SP, Roth RB , Zhou X , Mechanic LE , Zurer I , RotterV , Samson LD , Harris CC. 2003 .The adaptive imbalance in base e xcision-repair enzymes generates microsatellite instability in chronic inf ammation, J Clin Invest 112 : 1887 – 1894 . JohnsonKA , F ink SP , Marnett LJ . 1997 . Repair of propanodeoxyguanosine by nucleotide excision repair in vivo and in vitro .J Biol Chem 272 : 11434 – 11438 . KadlubarFF , Anderson KE , Haussermann S , Lang NP , Barone GW , Thompson PA , MacLeod SL , Chou MW , Mikhailo va M , Plastaras J , Marnett LJ , Nair J , V elic I , Bartsch H. 1998 . Comparison of DNA adduct levels associated with o xidative stress in human pancreas . Mutat Res 405 : 125 – 133 . Le vine RL ,Y ang IY , Hossain M , P andya GA , GrollmanAP , Moriya M .2000 . Mutagenesis induced by a single 1,N 6-ethenodeoxyadenosine adduct in human cells . Cancer Res 60 : 4098 – 4104 . Li Y , Marion M - J, RundleA , Brandt - RaufPW. 2003 .A common polymorphism in XRCC1 as a biomarker of susceptibility for chemicall y induced genetic damage . Biomarkers 8 ( 5 ): 408 – 414 . Mar nett LJ. 1999 . Chemistry and biology of DNA damage by malondialdehyde . IARC Sci Publ 17 – 27 . MedeirosMH. 2009 . Exocyclic DNA Adducts as Biomarkers of Lipid Oxidation and Predictors of Disease. Challenges in De veloping Sensitive and Specif c Methods for Clinical Studies . Chem Res Toxicol 22 ( 3 ):419 – 425 . NairJ , V aca CE ,V elic I , Mutanen M ,V alsta LM , Bartsch H .1997 . High dietary omega -6 polyunsaturated fatty acids drastically increase the for mation of etheno -DNA base adducts in white blood cells of female subjects . Cancer Epidemiol Biomarkers Prev 6 ( 8 ): 597 – 601 . NairJ. 1999 . Lipid peroxidation - inducedetheno - DN A adducts in humans . IARC Sci Publ 55 – 61 . NairJ , F ü rstenber ger G , B üger r F , Marks F , Bartsch H .2000 . Pro - mutagenicetheno - DN A adducts in multistage mouse skin carcino genesis: correlation with lipoxygenase- catalyzed arachidonic acid metabolism . Chem Res Toxicol 13 ( 8 ): 703 – 709 . NairJ , Gansauge F , Be ger H , Dolara P , W inde G , Bartsch H .2006 . Increased etheno - DN A adducts in affected tissues of patients suf fering from Crohn ’s disease, ulcerative colitis, and chronic pancreatitis . Antioxid Redox Signal 8 ( 5 – 6 ): 1003 – 1010 . NairU , Bartsch H , Nair J. 2007a . Lipid peroxidation - inducedDNA damage in cancer - prone inf ammatory diseases: a re view of published adduct types and le vels in humans . Free Radic Biol Med 43 ( 8 ): 1109 – 1120 . NairJ , DeFlora S , IzzottiA , Bartsch H. 2007b. Lipid peroxidation - deri ved etheno - DN A adducts in human atherosclerotic lesions . Mutat Res 621 ( 1 – 2 ): 95 – 105 . yor Pr WA , Stone K .1993 . Oxidants in cigarette smoke. Radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann NY Acad Sci 686 : 12 – discussion 27 ; 27 – 8. SchmidK , Nair J , W inde G ,V elic I , Bartsch H .2000 . Increased levels of promutagenic ethenoDNA adducts in colonic pol yps of FAP patients . Int J Cancer 87 ( 1 ): 1 – 4 . SchultePA , P erera FP . 1993 . Molecular epidemiology. Principles and practices . San Diego, USA : Academic Press . SpeinaE , Zieli´ nska M , BarbinA , Gack owski D , K owalewski J , Grazie wicz MA , Siedlecki JA , Oli´ nski R ,T udek B . 2003 . Decreased repair activities of 1,N6 - ethenoadenineand 3,N4ethenocytosine in lung adenocarcinoma patients . Cancer Res 63 ( 15 ): 4351 – 4357 . SunX , KarlssonA , Bartsch H , Nair J . 2006 . New ultrasensitive 32 P - postlabellingmethod for the analysis of 3,N 4 - etheno ′- -2 deo xycytidine in human urine . Biomarkers 11 ( 4 ): 329 – 340 .

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Index Header note: Italicized page locators indicate a photo/f gure; tables are noted with a t.

A

AAPH, 8 free radicals generated b y, 94 production of 8OHdG in calf th ymus DNA and, 286 AAPH assay, 7 AASA, protein oxidation and, 145 ABCs. See ATP-binding cassette transpor ters ABTS, 8 ABTS assay, 7 Acid hydrolysis, for quantif cation of 3-NT in biological samples, 202, 204–205 ACR-adducted peptides as tag of carbon ylated albumin and actin, procedure for quali/ quantitative analysis of, 180 Acrolein, 164t adducts formed from, 178 formation of FDP-lysine and MP-lysine upon reaction of lysine residue with, 159 GC-MS analysis of, 122 main metabolites of primary, 125 in urine, 126 origination of, from lipid pero xidation process, 118 source of, 119 Actin, S-glutathionylation and, 245 Actin cytoskeleton, carbonylation and disr uption of, 179 Acute lymphoblastic leukemia, 41 Adhesion molecule stimulation, o xidative stress and, 157 Adrenaline, auto-oxidation of, 4 Advanced glycation endproducts, 144 non-tryptophan protein f uorescence and, 146 Advanced lipoxidation end-products, 173 Advanced protein oxidation products, as mark ers of protein oxidation, 145–146 Af atoxin, hepatic cancer risk and , 261 Age/aging DNA damage, DNA repair and, 275 free radicals and, 65

future research on antio xidants and, 13 healthy, hormesis and, 137 lipid peroxidation and, 85, 157 mercaptoalbumin depletion and, 237 as modulator of urinar y 8OHdG in health y subjects, 303 oxidative damage and, vii oxidative stress and process of, 3 reduction in PON-1 acti vity and oxidative stress induced by, 28 2-Cys Prx and, 27 AGEs. See Advanced glycation endproducts Ahmed, N., 141t, 142t, 144t, 204 Air pollution, exposure to, evaluation of, 306 Albumin antioxidant activity and, evidence of, 231 enzyme-digested, LC-ESI-MS/MS of, 235–237 Albumin disulf de, measuring mercaptoalbumin and Cys34 oxidative modif cations and, 233 Albumin SH titration, measuring mercaptoalbumin and Cys34 o xidative modif cations with, 232–233 Aldehydes applications, 128 assay: single deter mination 4-hydroxyalkenals and HNE, 121 malondialdehyde, 119–121 as biomarkers of lipid pero xidation, 117 core, 162–163 glutathione conjugation and biotransfor mation of, 123 limitations, 128 multiple determination, 122–123 principle of cyclohexanedione derivatization of, 121, 122 principle of DNPH deri vatization of: 4-hydroxynonenal derivatization, 120 reactive, lipid peroxidation and, 157 Aldehydic compounds, numerous for mation of, after oxidative stress/lipid peroxidation, 117

333

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x

Aldimine-formyl-dehydropiperidino-type adducts, from acrolein, 178 Aldini, G., 8 ALEs. See Advanced lipoxidation end-products Alkadienals, 117 Alkaline elution, repair enzyme methods measured as DNA chain breaks and, 296 Alkaline hydrolysis, for quantif cation of 3-NT in biological samples, 202, 204–205 Alkaline/neutral elution, 263 Alkaline unwinding assay, 275 Alkanals H2O2-mediated protein N-acylation by, 158 lipid peroxidation and, 157, 158 Alkatrienals, 117 Alkinyl derivatives, labeling of HNE-adducted proteins with biotin and use of, for subsequent enrichment and identif cation by proteomic analysis, 185 Alkoxy radical, 4 radical reactions in the cell and , 35 Alkyl peroxy radical, radical reactions in the cell and, 35 ALL. See Acute lymphoblastic leukemia Alpha,beta-unsaturated aldehydes detection and measurement of applications, 128 limitations of, as biomark ers, 128 diff culty in measurement of, 117 origination of, from lipid pero xidation process, 118 Alpha-hydroxyaldehydes, lipid peroxidationspecif c epitopes and, 158 Alpha-ketoaldehydes, lipid peroxidation-specif c epitopes and, 158 Alpha-linolenic acid, 118 LDL oxidation and, 59 Alpha-OGG1 (type 1a), 287 Alpha-tocopherol DNA damage and, 273, 273t interaction of, in plasma, 10, 11 Alpha-tocopheryl, myocardial infarction in human subjects and plasma le vels of, 41 Alpha-tocopheryl quinone, products b y radical reactions yielded by, 42 Alpha-tocopheryl radical, radical reactions in the cell and, 35 Alzheimer protof bril formation, HNE and promotion of, 176, 177–178 Alzheimer’s disease HNE-GSH adducts and, 125 increased albumin disulf de and, 237 increased F 2-IsoP levels in, 72 oxidative stress and, 93 oxysterols and, 107 RCS/protein adducts and, 173

Amino acid transmembrane peptides, with tyrosine residues in dif ferent depths, 201 AMVN, 57 Amyotrophic lateral sclerosis, 118 Andreoli, R., 122 Angiotensin II, 51 Animal experiments, tHODE levels in, 94 Animal models, experimental, oxysterols in, 106–107 Animals, diabetes mellitus in, 39–40 Anraku, M., 238 Antibodies, detection of lipid pero xidationspecif c adducts and, 157–158 Antibody-based assays, 294–295 enzyme-linked immunosorbent assays, 294–295 histochemical detection, 295 Antioxidant capacity, markers of, in biolo gical system, 5, 7 Antioxidant capacity assays, estimated percent contribution of plasma antio xidants in, 9 t Antioxidant capacity in biolo gical systems, assays in determination of, 5, 6 t Antioxidant enzyme system, possib le routes for elimination of phospholipid h ydroperoxide by, 26 Antioxidant hypothesis, 273 Antioxidant interactions, biological signif cance of, 10–11 Antioxidant interventions, response of 8OHdG to, 307–308 Antioxidants achieving optimal ranges of, 13 apparently contradictory results on, in observational studies and inter vention trials, 13 in biological system, oxidative stress and, 4–5 combined, biological signif cance of interactions in, 10–11 comet assay and assessing status of, 267 Cys34 as, 230–231 def ned, 4 in diet, reduced risk of chronic diseases and , 3, 4, 11, 13 in foods, impor tance of synergistic action among, 8–9 gene interactions and, 12–13 mercaptoalbumin and evaluation of in vivo activity of, 238 metabolism and functions of, in vivo and in vitro, 11 role of, against lipid o xidation, 87–88 Antioxidants as biomarkers of oxidative stress, 35 alpha-tocopheryl quinone, 42 coenzyme Q, 42 concluding remarks, 43 diabetes in animals, 39–40 diabetes in human subjects, 40

335

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drug-induced hepatitis as model to e valuate oxidative stress, 36–39 biomarkers of oxidative stress in CCl 4 intoxication, 37 carbon tetrachloride-induced hepatitis, 36–37 change in AsA and other e vents caused by CCl4, 37 hepatic damage caused b y ethanol, 39 hepatitis caused by D-galactosamine, 39 hepatitis caused by thioacetamide, 38–39 H2O2 in insulin function, 38 physiological consequence of R OS generation, 38 hemodialysis patients, 40–41 ischemia/reperfusion injury, 41 leukemia in human subjects, 41–42 myocardial infarction in human subjects, 41 radical reactions in the cell, 35 virus-induced hepatitis in human subjects, 39 Antioxidant status balanced, fruit and vegetable diet and, 4–5 LDL oxidation as index of, 53–57 Antioxidant supplementation biological relevance of, 11–12 decreased endogenous IsoP for mation and, 75 Antioxidant therapies, clarifying benef ts/limits of, 43 Antioxidative enzymes, key role of, in homeostatic redox, 21 AOPPs. See Advanced oxidation protein products APCI. See Atmospheric pressure chemical ionization Ape1/Ref1 activity, chronic inf ammation, and imbalanced increase in, 326 Apomyoglobin, measuring HNE adduct for mation on, 175 Apoptosis, caused by thioacetamide, 38 Arachidonic acid isoprostanes of different ring types for med by non-enzymatic oxidation of, 68 oxidation of polyunsaturated fatty acids and, 86 Arachidonic acid-derived 15-F 2t-isoprostane, vasoconstrictive effects of EPA-derived 15-F3t-isoprostane vs. those of, 78 Arachidonic acid oxidation, via six-step mechanism, mechanism of for mation of F 2 isoprostanes after, 66 Arg307, alkylating, demonstrating ACR inhibition of cytokine gene expression in human T lymphocytes and, 179 Arginine, 123 AsA diabetes in animals and concentrations of, 40 in diabetic patients, 39 hemodialysis patients levels of, 40–41 myocardial infarction in human subjects and plasma levels of, 41

Asbestos-exposed women, study of, 270–271 Asbestos-exposed workers inf uence of genetic pol ymorphisms on DNA damage and repair in, 276 study on, 271 Asbestos exposure trials, age and, 275 Ascorbic acid, hydrophilic assays, antioxidant capacity of plasma and, 9 As-exposed children, in Me xico, studies of, 270 Assay reliability, hydroxylated bases as biomarkers and, 298–299 Assays to determine antioxidant capacity in biolo gical systems, 5, 6t hydrophilic antioxidant capacity, 7 lipophilic antioxidant capacity, 7–9 Atherosclerosis def ned, 105 evolution of, hypotheses related to, 51 free radicals and, 65 increased F 2-IsoP levels in, 72 initial event in, 28 isoprostanes as index of oxidative stress in vivo and, 72–73 lipid peroxidation and, 4, 157 oxidative hypothesis of, 60 oxidative modif cation of LDL and, 93 oxidative stress and patho genesis of, 22 oxidative stress and process of, 3 oxysterols and, 107–108 oxysterols and severity of, 52 RCS/protein adducts and, 173 Atherosclerotic lesions at coronary or carotid le vels, differential distribution of oxysterols in, 108 overexpression of PHGPx in transgenic mice and, 27 Atherosclerotic plaques, oxysterols in, 105 Atmospheric pressure chemical ionization, oxysterol analysis with, 104 Atorvastatin, oxidative stress status in FCHL patients and, 109 ATP-binding cassette transpor ters, protection from oxysterol-induced cell death and, 102 Automated column switching technology, 293 Autoxidation, oxysterol formation and, 99 Autoxidation products, common, 99, 100 Azido derivatives, labeling of HNE-adducted proteins with biotin and use of, for subsequent enrichment and identif cation by proteomic analysis, 185 Azo initiators, 5

B

Bacillus subtilis, importance of S-cysteinylation as means of thiol protection against oxidative stress demonstrated in, 250

336 Inde

x

Balafanova, Z., 210 t Balogh, L. M., 160 Bao, Y., 24 Barnett, Y. A., 275 Basal value variations, lipid peroxidation determinations and, 121 Base excision repair etheno-DNA adducts repaired b y, 322, 325 OGG1 measurement and, 268 range of, in human l ymphocytes, 268t Batthyany, C., 210t Benef cial protein oxidation, 137 Benzene exposure, comet assay and evaluation of, 305 Benzyl isothiocyanate, 249 BER. See Base excision repair Berlett, B. S., 211t Berry juices, LDL o xidation and, 58 Beta-carotene, 4, 274 DNA damage and, 273, 273t interaction of, in plasma, 10, 11 myocardial infarction in human subjects and plasma levels of, 41 response of 80HdG to, 307 Beta-carotene radical cations, 11 Beta-mercaptoethanol, 252 Beta-OGG1 (type 2a), 287 BGEE. See Biotinylated glutathione ethyl ester BHT. See Butylated hydroxytoluene Bile acid biosynthesis, o xysterols and, 99 Biological matrices general approach to characterization of RCS-modif ed proteins in, 182 identif cation and characterization of carbonylated proteins in, 182–190 Biological systems assays to deter mine antioxidant capacity in, 5, 6t markers of antioxidant capacity in, 5, 7 Biomarker measurement, comet assa y and applications to, 262–263 Biomarkers choice of, in human biomonitoring studies, 261 of oxidative damage, classif cation of, vii of oxidative stress in carbon tetrachloride intoxication, 37 practical use of h ydroxylated bases as, 298–304 assay reliability, 298–299 relatively stable values over time in the same individual, 300–301 seasonal effects on stability and absor ption of food-based 80HdG, 301–303 smoking, gender, age, and ph ysical activity as confounding variables, 303 stability in storage, 300 utility of 80HdG as biomark er in disease, 303–304

for reliable measurement of o xidative stress in humans, need for, 85 as surrogate assessment of clinical endpoints, advantages with, 149 Biomarkers of Oxidative Stress Study, 65, 149 Biomembranes, attack of R OS on lipids in, 21 Biomonitoring, accuracy of comet assa y in, 264 Biomonitoring trials, considerations related to, 268–269 Biotin hydrazide as chemical tag, in in vestigation of protein modif cation by HNE as consequence of obesity, 190 as reporter tag for proteins adducted b y lipid oxidation products, 184 Biotinylated glutathione ethyl ester, 255 Bisphenol A, exposure to, evaluation, 306 Blair, I. A., 118 Blanchard-Fillion, B., 211t Blueberry and apple juice mixture, gene interactions and, 12–13 BODIPY 581/591, 8 Boon, P. J., 124 BOSS. See Biomarkers of Oxidative Stress Study Bottom-up approach, mass-spectrometr y and, in characterization of covalently modif ed proteins, 174, 175 Bourdon, E., 231 Boyd Orr cohort lymphocytes from, relationship betw een DNA damage and activity of repair enzyme that removes 8-oxoGua, OGG1, 278, 278 samples, DNA damage, DNA repair, and age in, 275 Brain cholesterol metabolism, o xysterols and regulation of, 107 Breast cancer utility of 80HdG as predictor of, 304, 305 Women’s Healthy Eating and Li ving study and, 308 Bruenner, B. A., 122 Brunborg, G., 264 BSO. See Buthionine sulfoximine Burkitt, M. J., 54 Buthionine sulfoximine, 124, 249 Butylated hydroxytoluene, 57, 69, 121

C

Cadet, J., 292 Cahill, L.E., 12 Calcium dependent cytosolic phospholipase A2 (cPLA2), 26 Calcium independent phospholipase A2 (iPLA 2), 26 Caloric intake, lowering, decreased endogenous IsoP formation and, 75

337

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Caloric restriction, response of 80HdG to, 308–309 Cancer DNA adducts and cer tain types of, 328 DNA repair capacity and, 278 free radicals and, 65 increased F 2-IsoP levels in, 72 lipid peroxidation and, 85, 157 2-Cys Prx and, 27 Capillary electrophoresis, 297 Carballal, S., 232 Carbon-centered radicals, for mation of, 57 Carbone, D. L., 189 Carbonic anhydrase III, S-glutathionylation and, 245 Carbon tetrachloride, change in AsA and other events caused by, 37 Carbon tetrachloride (CCl 4)-induced hepatitis, phases of, 36–37 Carbon tetrachloride intoxication, biomarkers of oxidative stress in, 37 Carbon tetrachloride poisoning, Biomark ers of Oxidative Stress Study and, 149 Carbonylated proteins characterization of in vitro: reaction mechanism, functional studies, and biomarkers identif cation, 176–182 ACR and other RCs, 178–182 HNE, 177–178 Carbonylated proteins in biolo gical matrices ex vivo studies, 189–190 identif cation and characterization of: from in vitro to ex vivo studies, 182–190 derivatization/enrichment, 183–186 precursor ion scanning, 186–187 in vitro studies: cells, tissues, f uids, and protein fractions exposed to RCS, 187–189 Carbonylation, protein modif cation and, 173 Carboxyethylmercapturic acid, as a main metabolite of acrolein in urine, 126 Carcinogenesis, accumulation of mutations and initiation of, 319 Carcinogens, environmental, comet assay used as biomarker assay in studies of, 269 Cardiovascular disease oxidative stress and patho genesis of, 22 protein tyrosine nitration and, 199 RCS/protein adducts and, 173 vitamin E studies and, 12 Carotenoids, 3, 4, 88 duration of lag time and , 56 LDL oxidation and, 58 as quenchers of singlet o xygen, 88 “total antioxidant capacity” and, 5 in vitro experiments and antioxidant actions of, 10

Cassina, A. M., 210t Castegna, A., 144t Catalase, as main modif ed protein by HNE in red blood cells of systemic lupus erythematosus patients, 190 Catalytic function, def ning, thiols and, 243 Cataracts, oxidative stress and, 93 CDNB, spectrophotometric methods, study of S-glutathionylation and S-cysteinylation and, 253 Cell-mediated oxidation, of LDL in vitro, 57 Cell proliferation, oxidative stress and, 157 Cells dynamics of ubiquitin conjug ates in, 220 endogenous 80Hdg found in, 290–291 exposed to RCS: e xamples of in vitro studies, 187–189 human, levels of M 1dG adducts and ethenoDNA adducts in, 324 t oxysterols and toxicity to, 102 radical reactions in, 35, 36 ubiguitin conjugates in, accurately determining levels of, 226 CEMA. See Carboxyethylmercapturic acid Central nervous system, n-3 pol yunsaturated fatty acid-rich tissues and, 118 Cerebral ischemia oxidative stress and patho genesis of, 22 RCS/protein adducts and, 173 Chain mechanism, free radical-mediated o xidation of linoleates and, 86 Chaperone-mediated autophagy, of cellular proteins, 147 Chavez, J., 185 CHD. See Coronary heart disease Chemoattractant production, oxidative stress and, 157 Chilean berry juice, LDL o xidation and, 58 Chitosan, antioxidant status and, 238 Cho, S. H., 299, 306 Cholestane 3beta, 5alpha,6beta-triol, 99, 100 Cholesterol, 100 lipid peroxidation and, 85 PHGPx and reduction of, 24 susceptibility of, to o xidation, 99 Cholesterol autoxidation products, common, 99, 100 Cholesterol ester hydroperoxides, PHGPx and reduction of, 24 Cholesterol hydroperoxides, 23 Cholesterol metabolizing enzymes, o xidation formation and, 99 Cholesterol 25-hydroxylase, 101 Cholesteryl ester-core aldehydes, 162 Cholesteryl ester hydroperoxides, 23 Choline def cient diets, liver damage and, 94

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Chromatographic analysis of albumin redo x variants, measuring mercaptoalbumin and Cys34 oxidative modif cations and, 233–234 Chromium-exposure, comet assay and evaluation of, 305 Chronic diseases. See also Diseases future research on antio xidants and, 13 lack of consistent benef cial effects of antioxidant supplementation and, 13 oxidative damage and, vii oxidative stress and process of, 3, 4 Chronic myelogenous leukemia, 42 Ch-25-oHlase, 100 Cigarette factory workers, study of, 270 Cigarette smoke extract, whole-phase, identifying covalent modif cations of Cys34 in albumin exposed to, 236–237 Circulation, oxysterols transported in, 102 CIS-MS. See Coordination ion-spray mass spectrometry c-Jun, S-glutathionylation and, 245 CKBB, identif cation of 14 HNE adducts on, 176 CMA. See Chaperone-mediated autophagy CML. See Chronic myelogenous leukemia CML residues, oxidized proteins and, 144 CMV residues, oxidized proteins and, 144 CNS. See Central ner vous system Cobalt-exposure comet assay and evaluation of, 305 study of, 270 Cocoa, plasma antioxidant capacity and, 10 Codreanu, S. G., 186 Coenzyme Q10, disease and depletion of, 42 Coffee, plasma antioxidant capacity and, 10 Comet assay, 261 accuracy of, in biomonitoring, 264 advantages of, 263 antioxidant status assessed with, 267 biomonitoring trial considerations and, 269 coeff cients of variation and, 299 future uses for, in biomonitoring f eld, 278, 278 inf uence of genetic pol ymorphisms on DNA damage and repair measured with, 275–276 limitations of, 265–267 nutrition’s effects on DNA stability and, 274 occupational, environmental, and experimental exposure to genotoxic agents and, 269–272 principles of, and applications to biomark er measurement, 262–263 repair enzyme methods measured as DN A chain breaks and, 296–297 response of 80HdG to diet inter ventions and, 308

schematic outline of method, 262 simplicity and accuracy with, 276–277 toxic exposures evaluated with, 305 Comets, scoring, 262 Competitive immunoassay principle, for determination of DHN-MA in urine, 127 Complex 1 NADH-ubiquinone oxidoreductase, S-glutathionylation and, 245 Computer-based image analysis, comet assay and, 262 Cooke, M. S., 289, 295 Coomassie brilliant blue staining agent, albumin disulf de, measuring mercaptoalbumin and Cys34 oxidative modif cations and, 233 Coordination ion-spray mass spectrometr y, measurement of HODE and, 88 Copper-induced oxidation, polyphenolics and, 58, 59 Copper-mediated peroxidation, monitoring in vitro LDL oxidation and, 54 Corallina off cinalis, 7 Cordis, G. A., 122 Core aldehydes, 162–163 Coronary heart disease, small, dense LDL particles and, 52 Coronary vascular disease, oxidizability of LDL particles in vivo and in vitro and, 60 Covalently modif ed proteins mass spectrometry characterization of, general and new approaches with, 174–176 top-down and bottom-up mass spectrometric approaches, 174, 175 Cow’s milk, f avonoids and, 274 COX. See Cyclooxygenase COX-derived prostaglandins, ISoPs vs., 67 Crocin, 7 Crocin bleaching assays, 7 determining antioxidant capacity in biolo gical systems and, 6t Crotonaldehyde, 164t origination of, from lipid pero xidation process, 118 Crow, J. P., 210t Cumene hydroperoxide, continuous skin e xposure to, Biomarkers of Oxidative Stress Study and, 149 Cu/Zn-SOD (SOD1), reaction catal yzed by, 22 Cyclohexanedione derivatization of aldehydes, principle of, 121, 122 HNE and 4-hydroxyalkenal determination and, 121 Cycloheximide, cells pretreated with, 254 Cyclooxygenase, lipid oxidation and, 86 Cyclopentenone (A 2/J2)-isoprostanes, biological activities of, 74

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CYP46, 107 CYP450. See Cytochrome P-450 CYP3A4, 100, 101 CYP3A5, 101 CYP7A1, 100, 101 CYP27A1, 100 CYP46A1, 100, 101 CYP2E1 induction, ethanol-induced o xidative stress in liver and, 39 Cys residue, acrolein co valent adducts to, 178 CySS, mammalian cell g rowth in culture medium when supplemented with, 248 Cys61, alkylating, demonstrating ACR inhibition of cytokine gene expression in human T lymphocytes and, 179 Cys-SOH, reduction of, back to a thiol, 27 Cysteine, 123 abundance of, in mammals, 246 mercaptoalbumin depletion and, 229–230 N-acetylation of, 126 Cysteine acid-conjugates, 125 Cysteine/cystine redox couple age and effect on, 247 equilibrium states of, 246–249 structure of sulfur amino acid for m of, 246 Cysteine oxidation, effects of, 243–244 Cysteine pools, intracellular and e xtracellular distribution of, 247 Cysteine residues oxidation and repair of, in proteins, 139 oxidation of, 138 Cysteinylation, def ned, 249 Cys34 as antioxidant, 230–231 covalent modif cations of, as biomark er of mild oxidative stress, 229–238 as detoxifying agent for reacti ve carbonyl species, 231 identifying covalent modif cations of, in albumin exposed to whole-phase cigarette smoke extract, 236–237 importance of, in RCS v ascular detoxif cation of carbonylated proteins in vitro, 187 LC-ESI-MS/MS approach for identif cation of unknown and predicted modif cations of, 236 main oxidative covalent modif cations of, 232 oxidized forms of, 231–232 questions remaining in studies of, 238 rate constant for reaction of, with HNE, 178 Cys34 oxidative modif cations analytical strategies to measure mercaptoalbumin and, 232–237, 233 t albumin disulf de, 233 albumin SH titration, 232–233 chromatographic analysis of albumin redo x variants, 233–234

direct infusion mass spectrometr y of intact albumin, 234–235 LC-ESI-MS/MS of enzyme-digested albumin, 235–237 Cys34 reactivity, molecular insight into, 230 Cytochrome P-450, lipid o xidation and, 86 Cytochrome P450 f amily, cholesterol metabolizing enzymes in, 100, 100–101 Cytokine gene expression, in human T lymphocytes, experiments demonstrating, 179 Cytokines, inf ammation and release of, 22 Cytoskeletal proteins, S-glutathionylation and, 245 Cytosolic GPx, 24

D

Danes, low mortality rates due to coronar y heart diseases among Greenland Eskimos vs. among, 75 Dansyl chloride, study of S-glutathionylation and S-cysteinylation and, 253 Dehydroascorbic acid (DAsA), 35 Delatour, T., 204 Deliberate protein oxidation, 137 De novo ubiquitin conjugation, 222 Des Rosiers, C., 121 Detergents, for extracting ubiquitin conjugates from cells or tissues, 226 de Zwart, L. L., 122 D-galactosamine, hepatitis caused b y, 39 DHA. See Docosahexaenoic acid DHN-MA (1,4-dihydroxynonene mercapturic acid) principle of competitive immunoassay for determination of, in urine, 127 DHP-lysine, formation of, upon reaction of l ysine residue with MDA, 162 Diabetes in human subjects, 40 increased F 2-IsoP levels in, 72 lipid peroxidation and pathogenesis of, 157 mercaptoalbumin depletion and, 237 oxidative stress and process of, 3 oxysterols and, 108–109 protein tyrosine nitration and, 199 sdLDL levels and, 59 systemic oxidative stress and, 72 Diabetes mellitus albumin thiols and, 237, 238 in animals, 39–40 oxidative stress and, 23 Diabetic patients, sdLDL le vels in, 52 Diabetic retinopathy, mercaptoalbumin depletion and, 237 Dialysis, some oxidized Cys34 for ms reversed after, 237

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Diaminonaphtalene (88), 120 Dichlorof uorescein-diacetate assay, 7 Diene hydroperoxides, conjugated, oxidation of linoleates and, 86, 87 Dienes, LDL oxidation and, 56 Diet, DNA adduct levels and, 326 Diet interventions, response of 8OHdG to, 308–309 Dihydrofuran, 161 Dihydropyrrole, 161 Dimethyl sulfoxide treatment, hepatitis caused b y thioacetamide and, 39 Dioxindolyalanine, 143 Dioxolane-IsoPs, 67 Diphenyl-hexatriene-labeled propionic acid, 8 Diphenyl-1-pyrenylphosphine, in vitro monitoring of LDL oxidation and, 56 Direct infusion mass spectrometr y of intact albumin, 234, 234–235 measuring mercaptoalbumin and Cys34 oxidative modif cations with, 233t Direct methods of biomark ers of oxidative damage, vii Diseases. See also Chronic diseases detection of lipid-peroxidation-specif c adducts as biomarkers in, 168 exposure to genotoxins and development of, 321 increase in protein tyrosine nitration and , 209 lipid peroxidation and, 85 oxidative-based, mercaptoalbumin depletion and, 237 protein tyrosine nitration and, 199 utility of 80HdG as biomark er in, 303–304 utility of 80HdG as predictor of, 304–305 Disulf de bridge, oxidation and, 243 Disulf des, thiol oxidation by alkylation of residual thiols and reducti ve tagging of, 148 Dithioerythritol, 252 Dithiothreitol, 252 Dityrosine, formation of, 142 Dityrosine residues, detection of protein o xidation adduct residues and free adducts in, 142 t Dizdaroglu, M., 284 DM. See Diabetes mellitus DMSO. See Dimethyl sulfoxide treatment DNA adducts criteria for, as biomarkers of exposure to oxidative stress and lipid pero xidation products, 321–327 def ned kinetics and v ariation in time, 324–325 mechanisms of exocyclic DNA adduct formation, 321–323 sources of inter- and intra-individual variations, 325–327

surrogate tissues, 323–324 techniques to measure e xocyclic-DNA adducts and their v alidation, 323 DNA-bound aldehydes, 164t DNA chain breaks, repair enzyme methods measured as, 296–297 DNA damage age and, 275 comet assay and measurement of, 262 direction of reactive oxygen and nitrogen species and, 319 inf ammation, oxidative stress and, 328 inf uence of genetic pol ymorphisms on, 275–276 inf uences of genotype on le vels of, in human lymphocytes, 277 in blood samples from, 271–272 in lymphocytes of workers in glass f ber factory and matched controls from same to wn, 271 micronutrients and, 273, 273 t occupational, environmental, and experimental exposure to genotoxic agents and, 269–272 relationship between activity of repair enzyme that removes 8-oxoGua, OGG1 and, 278 2-Cys Prx and, 27 DNA oxidation, 261 European Standards Committee on o xidative DNA damage: validation exercise, 264–265 measurement of comet assay, accuracy of, 264 comet assay, advantages with, 263 comet assay, limitations of, 265–267 measuring, 263–265 net enzyme-sensitive sites, 263, 264 DNA repair age and, 275 hydroxylated nucleotides and, 287–288 inf uence of genetic pol ymorphisms on, 275–276 measuring, 267–268 DNA repair capacity, cancer and, 278 DNA stability, nutrition and, 272–274 DNA strand breaks (SBs), 262 DNPH, MDA measured in f uids and tissues after derivatization with, 120 DNPH derivatization of aldehydes, principle of: example of 4-hydroxynonenal derivatization, 120 Docosahexaenoic acid MDA and, 118 oxidation of polyunsaturated fatty acids and, 86 Dopamine, auto-oxidation of, 4

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Dose-response relationship, DNA adduct levels and, 324–325 Double strand breaks (DN A), 263 Drug-associated toxicity, lipid peroxidation and, 157 DTE. See Dithioerythritol DTNB, spectrophotometric methods, study of S-glutathionylation and S-cysteinylation and, 253 DTT. See Dithiothreitol Dusinská, M., 270, 301 Dzeletovic, S., 104

utility of, as predictor of disease, 304–305 utility of, improving, 309–310 8-hydroxyguanine (8OHgua), 285 8-hydroxyguanosine (8OHG), 285 8-oxo-dG, study of, 321 8-oxo-guanine, 285 Electron capture dissociation 3-NT measurement/detection with, 202, 203 top-down characterization of intact proteins and, 175, 176 Electron transfer system, of mitochondrial inner membrane, superoxide production and, 4 Electrospray ionization LC-MS/MS with, for deter mination of oxysterols, 104 measurement of HODE and, 88 Electrospray ionization-MS, deter mination of intact proteins and, 174 Electrospray mass spectrometr y, fragmentation patterns of tyrosine and 3-NT b y, 205, 206 11beta-hydroxysteroid dehydrogenase, interconversion of 7betahydroxycholesterol and 7-ketocholesterol, 101 11-(Z,Z)-HPODE, oxidation of linoleates and, 86, 87 ELISA. See Enzyme-linked immunosorbent assay Eliuk, S. M., 175 Ellman’s reagent, measuring mercaptoalbumin and Cys34 oxidative modif cations with, 232, 233t Endogenous 8OHdG, in cells and tissues, 290–291 Endogenous oxidation, age and, 275 Endogenous ubiquitin conjugates, detection of, 221–222 Endonuclease III (EndoIII), 263 Endotoxins, exposure to, Biomarkers of Oxidative Stress Study and, 149 Ensure, 308 Environmental exposure to genotoxic agents, 269–272 Environmental insult, utility of 80HdG as measure of, 305–306 Enzymatic antioxidant defenses, 21–29 glutathione peroxidase as an R OS scavenging system, 23 glutathione peroxidases and phospholipases as a lipid hydroperoxide-elimination system, 23–24, 26–27 introduction, 21 paraoxonase-1 and PAF acetylhydrolase in high-density lipoprotein, 28–29 peroxiredoxins, 27–28 superoxide dismutase as an R OS scavenging system, 22–23

E

ECD. See Electron capture dissociation Edens, W. A., 142t EDTA. See Ethylenediamine tetraacetic acid Ehtanol-induced oxidative stress model, HNEand ONE-modif ed proteins identif ed in, 189 Eicosanoids, inf ammation and release of, 22 Eicosapentaenoic acid cardiovascular disease patients and, 75 isoprostanes of different ring str uctures formed by non-enzymatic oxidation of, 79 MDA and, 118 Eicosapentaenoic acid-derived isoprostanes, 76, 78–79 8-hydroxydeoxyadenosine (80HdA), 285 8-hydroxydeoxyguanosine (80HdG), 285 assay reliability and, 298–299 DNA repair and, 287–288 endogenous, found in cells and tissues, 290–291 focusing on measurement of, 283–284 food-based, seasonal effects on stability and absorption of, 301–303 formation of, from guanine, 286 hourly variation in, in three subjects, e xercise vs. sedentary, 302 hydroxylated guanine base metabolism and , 288–289 mitochondrial vs. nuclear DNA damage and, 291 nomenclature related to, 284 relatively stable values over time in same individual, 300–301 smoking, gender, age, and ph ysical activity as confounding variables and, 303 utility of, as biomark er in disease, 303–304 utility of, as biomark er of therapeutic improvement, 306–309 response to antioxidant interventions, 307–308 response to diet inter ventions, including caloric restriction, 308–309 utility of, as measure of en vironmental insult, 305–306

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Enzymatic oxidation, oxidation of linoleic acid and, 85, 86 Enzyme-linked immunosorbent assay, 88 antibody-based assays, 8OHdG and, 294–295 measurement of 8OHdG in urine with use of, 299 measure of protein carbon yls and, 145 quantif cation of 3-NT in biolo gical samples and, 202 EPA. See Eicosapentaenoic acid EPA-derived F3-isoprostanes, mechanism behind formation of, 77 EPA-derived 15-F 3t-isoprostane, vasoconstrictive effects of vs. those of arachidonic acid-derived 15-F 2t-isoprostane, 78 EPA diet, F 2-isoprostane formation from hear ts of mice after eight w eeks of 0.56% supplementation with, 76 EPIC study, 308 Epididymal non-selenium GPx, characteristics of, 25t Ersoz, A., 297 Escherichia coli, proteins known to undergo S-glutathionylation in, 250 ESCODD. See European Standards Committee on Oxidative DNA Damage ESCULA. See European Standards Committee on Urinary Lesion Analysis ESI. See Electrospray ionization Esterbauer, H., 119, 161, 165 Esterif ed aldehydes (core aldehydes), 162–163 Esterif ed IsoPs, 74 Ethanol, hepatic damage caused b y, 39 Ethanolamine phospholipids Michael adducts, formation of, in human platelets in response to oxidative stress, 189 Etheno-DNA adducts, 321 alternative mechanisms related to for mation of, 322–323 base excision repair and repair of, 322, 325 correlation between LPO and subsequent formation of, 322 levels of, in v arious tissues and cells of humans, 324t smoking and, 326 Ethylenediamine tetraacetic acid, isolation of LDL and, 53 E2- isoprostanes, pro-inf ammatory activities of, 73–74 European Commission, 265 European Standards Committee on Oxidati ve DNA Damage, 288, 298 role of, 291 validation exercise, 264–265 European Standards Committee on Urinar y Lesion Analysis, 289 Evans, D. M., 284, 285

Exercise mercaptoalbumin depletion and, 237 sedentary vs., hourly variation in 8-hydroxydeoxyquanosine in three subjects, 302 Exocyclic DNA adduct for mation, mechanisms of, 321–323 Exocyclic DNA adducts, 328 as biomarkers in molecular epidemiolo gical studies, 319–321 measurement techniques for and v alidation of, 323 Experimental exposure to genotoxic agents, 269–272 Extracellular-SOD (SOD3), 22 Extracellular thiols, antioxidant defense and, 247 Ex vivo studies, of modif ed proteins, 189–190 Eye disease, oxidative stress and process of, 3

F

Familial combined hyperlipidemia, oxysterols and, 109 Fang, Q., 326 FAOH. See Fatty acid hydroxide FAOOH. See Fatty acid hydroperoxides FapyAde, 263, 284, 285, 287 FapyGua, 263, 284, 285 DNA repair and, 287 formation of, from guanine, 286 Fat-soluble antioxidants, lack of contribution of, in plasma antioxidants, 10 Fatty acid hydroperoxides, 23, 26 Fatty acid hydroxide, 26 Fatty acids, 56 Faure, P., 237, 238 FCHL. See Familial combined hyperlipidemia FDP-lysine, 178 formation of, upon reaction of l ysine residue with acrolein, 159 FDP-type adducts, 159 Fenaille, F., 183, 187 Fenof brate, oxidative stress status in FCHL patients and, 109 Fenton chemistry, stimulation of o xidative stress and, 117 Fenton-type reaction, 53 superoxide radical production and, 56 Ferrington, D. A., 187 FE3+-NTA-induced acute renal injur y, oxidative stress and, 167 15-E2t-IsoP, 73, 74 15d-PGJ2, role of, as c ytotoxic compound in neuronal cell dysfunction, 180 Fine particulate matter, exposure to, comet assa y and evaluation of, 306 Finley, E. L., 141 t, 144t

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Fischer rats, young and old, proteomic techniques for studying HNE-modif ed proteins from, 189 Fish consumption, f avorable effect from, relative to coronary heart disease mor tality, 75 Fish oil, as therapeutic agent in o xidative stress-related conditions and diseases, 75–76 5alpha-hydroperoxide, 3beta-hydroxy-5-oxo-5,6secocholestan-6-al formation and Hock cleavage of, 101 5-hydroxytryptophan, 143 5-oxovaleroylcholesterol, 162–163 5,6alpha-epoxides, 99, 100 5,6beta-epoxides, 99, 100 5,6-secosterol, neurodegenerative disorders and, 107 Flavins, auto-oxidation of, 4 Flavonoids biological functions occur ring among, 12 soy milk vs. rice milk vs. cow’s milk and, 274 Flow-mediated dilation, 59 Fluids, exposed to RCS: e xamples of in vitro studies, 187–189 Fluorescence-based assays, quantifying protein damage and, 146 Fluorescence benzoxazole, 208 Fluorescence derivatization, of 3-NT, 202, 203 FMD. See Flow-mediated dilation Food-based 80HdG, seasonal ef fects on stability and absorption of, 301–303 Formamidopyrimidine DNA glycosylase endonuclease III and measurement of, 263 indirect measurement of 80HdG sites in intact cells using comet assa y and, 287–288 specif city of, concer n about, 265–267 4alpha-OH-cholesterol, 100 4beta-hydroxycholesterol, 101 4-hydroxyalkenals determination of, 121 origination of, from lipid pero xidation process, 118 in urine, after HNE administration, 126 4-hydroxyhexanals as biomarker of lipid pero xidation, 118 origination of, from lipid pero xidation process, 118 4-hydroxynonenals, 105, 145 characterization of in vitro: reaction mechanism, functional studies, and biomarkers identif cation, 177–178 determination of, 121 formation of, 119 GC-MS analysis of, 122 lipid peroxidation and, 118 main metabolic pathways of, 126 mercapturic acid pathway and example of, 124

origination of, from lipid pero xidation process, 118 protein modif cation and, 173–174 source of, 118 4-hydroxy-2-alkenals lipid peroxidation and, 159 lipid peroxidation-specif c epitopes and, 157 4-hydroxy-2-hexenals, 164t 4-hydroxy-2-nonenals, 164t exocyclic DNA adducts for med from, 322 Fourier transform ion cyclotron resonance, characterization of RCS-covalently modif ed proteins and, 174, 175 4-oxononenals, 117 free-radical-initiated degradation of n6-polyunsaturated fatty acids and generation of, 161 lipid peroxidaiton and, 118 novel adduct originating from, 125 origination of, from lipid pero xidation process, 118 source of, 118 4-oxo-trans-2-nonenal (ONE) adduction, to hemoglobin: reaction mechanism, 181 4-oxo-2-alkenals lipid peroxidation and, 161–162 lipid peroxidation-specif c epitopes and, 157, 158 4R-4-hydroxy-2-nonenal, 164t 4S-4-hydroxy-2-nonenal, 164t FPG. See Formamidopyrimidine DNA glycosylase FPG-sensitive sites age and, 275 DNA oxidation measurement and, 264 FRAP, determining antioxidant capacity in biological systems and, 6t FRAP assay, 7, 9 estimated percent contribution of, in plasma antioxidants, 9t Free iron, 54 Free-radical-mediated LDL oxidation, oxidized lipids produced by, 105 Free radical-mediated oxidation, oxidation of linoleic acid and, 85, 86 Free radical-mediated oxidation of linoleates, linoleate oxidation and, 86 Free radical-mediated oxidation of lipids, inhibiting, 88 Free radical pathways, tyrosine nitration and, 200 Free radicals chronic inf ammatory disease and process of lipid peroxidation initiated by, 320 diseases and conditions attributed to, 65 effects of, on le ver levels of tHODE and t8-iso-PGF2alpha in nmol/g tissue, 94 t lipids attacked by, 109

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Fridovich, I., 22 Fruit and vegetable consumption cancer-protective effects of, 273 DNA stability and, 272–274 reduced risk of chronic diseases and , 3, 4, 5, 11, 13 response of 80HdG to, 308 response of 80HdG to pol yphenols in, 307 FTICR, 174 F2-isoprostane formation endogenous levels of, from hear ts of mice after eight weeks of 0.56% EPA diet supplementation, 76 F2 isoprostanes analysis of, in plasma obtained from rat 4 hours after treatment with CCl 4 to induce endogenous lipid peroxidation, 71 discovery of, 65 extraction and hydrolysis of phospholipids in tissues with, 69–70 mechanism of discovery of, 66 outline of procedure used for e xtraction, purif cation, derivatization, and mass spectrometric analysis of, from biolo gical sources, 69 pro-inf ammatory activities of, 73–74 purif cation, derivatization, and quantif cation of, 70–71 quantif cation of, 67–69 storage and handling of biolo gical f uids and tissues for quantif cation of, 69 Fu, S., 142 t Furuhata, A., 159

Gas chromatography-mass spectroscopy, direct measure of hydroxylated bases and, 292 Gastrointestinal GPx, 24 characteristics of, 25 t Gaut, J. P., 204 GC-electron capture negative ion chemical ionization-MS, HNE-MA deter mination with, 127 GC-MS. See Gas chromatography-mass spectrometry GC/MS assays, alternative, 68 GC/NICI-MS. See Gas chromatographic/negative ion chemical ionization mass spectrometric methodology Gel electrophoresis, purity of LDL conf rmed by, 54 Gel preparation, biomonitoring trials and , 269 Gender, as modulator of urinar y 80HdG in healthy subjects, 303 Gene interactions, antioxidants and, 12–13 Genetic polymorphisms, inf uence of, on DN A damage and DNA repair, 275–276 Gene transcription, oxysterols and regulation of, 99 Genotoxicity, inf ammation-driven, comparing methodologies ref ecting different processes in, 327 Genotoxins exposure to and disease de velopment, 321 occupational, environmental, and experimental exposure to, 269–272 Gioacchini, A. M., 121 Giovannelli, L., 308 Girard P hydrazones, 104 Glass f ber factory, DNA damage in l ymphocytes of workers in, and matched controls from same town, 271 Glass f ber trials, age and, 275 Glutathione abundance of, in mammals, 123, 246 characteristics of, 248 composition of, 247 equilibrium states of, 246–249 intracellular and extracellular distribution of, 247 myocardial infarction in human subjects and plasma levels of, 41 structure of thiol and disulf de form of, 246 Glutathione conjugates assay acrolein, 125 HNE and ONE, 124–125 as biomarkers of lipid pero xidation, 123–125 formation of, 123–124 Glutathione disulf de, 23 Glutathione/glutathione disulf de equilibrium states of, 246–249 structure of thiol and disulf de form of, 246

G

Gain of function, tyrosine nitration in specif c proteins and, 209 Gao, L., 76, 79 Garlic, LDL oxidation and, 58 Gas chromatographic/negative ion chemical ionization mass spectrometric methodology, 67 Gas chromatography-mass spectrometry aldehyde dinitrophenylhydrazones analyzed with, 122 exocyclic DNA adduct detection and, 323 HNE and 4-hydroxyalkenal determination and, 121 lipidomic analysis, for lipid pero xidationderived aldehydes, 123 MDA measurement and, 120 oxidation products of linoleates measured b y, 88 oxysterol analysis with, 103, 103–104 tHODE measurement with, 89–90 of 3-NT, 203 t7-OHCh, tCh, and t18:2 anal yzed with, 91

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Glutathione peroxidase, 21, 23 characteristics of, 25t def ned, 24 forms of, 24 phospholipases and, as lipid h ydroperoxideelimination system, 23–24, 26–27 Glutathione-S-transferase M1a-1a, architectural similarities between key residues around Cys34 and active site of, 230 Glutathione-S-transferases, 24, 117 Glycoxidation adducts, 144 Goodwin, D. C., 211 t Goto, M., 286 GPx. See Glutathione peroxidase Greenland Eskimos, low mortality rates due to coronary heart diseases among, vs. among Danes, 75 Green tea biological functions occur ring between soy and, 12 plasma antioxidant capacity and, 10 Greilberger, J., 237 Gruppo Italian per lo Studio della Sopravvvivenzia nell-Infarto (GISSI)Prevention study, omega-3 fatty acids and f sh oil in, 75 GSH. See Glutathione GS-HNE conjugates, measuring, 124 GSH peroxidase, radical reactions in the cell and , 35 GS-MS method, measure of protein carbon yls and, 145 GSSG. See Glutathione disulf de GSTA4, 326 GST polymorphisms, FPG- and EndoIII-sensiti ve sites in lymphocyte DNA in, smokers vs. non-smokers and, 276, 277 GSTs. See Glutathione-S-transferases Guanine, formation of 8-hydroxydeoxyguanosine and FapyGua from, 286 Guarnieri, S., 274 Guetens, G., 292 Guittet, O., 211t

Health status, oxLDL and in vitro LDL oxidation associated with, 59–60 Heart attacks, oxidative stress and patho genesis of, 22 Heat shock protein-90 (hsp90), 147 Heat shock proteins, S-glutathionylation and, 245 HeLa cell DNA, analysis of oxidized base damage in, 266 Hemiacetal-type Michael adducts, upon reaction of protein with HNE, 160 Hemodialysis patients AsA levels and, 40–41 mercaptoalbumin depletion and, 237 Hemoglobin, 4-oxo trans-2-nonenal (ONE) adduction: reaction mechanism, 181 Hemoproteins, study of S-glutathionylation and S-cysteinylation and, 253 Hepatic cancer risk, af atoxin and, 261 Hepatitis carbon tetrachloride-induced, 36–37 D-galactosamine and, 39 thioacetamide and, 38–39 virus-induced, in human subjects, 39 Hepatitis B lipid oxidation product levels and, 93 liver inf ammation and, 319 Hepatitis C lipid oxidation product levels and, 93 liver inf ammation and, 319 HepG2 cells, main elimination of PLOOH in, 24 Heptaglubulin, higher plasma o x-LDL/LDL ratio in individuals with genetic defects in, 55 Hexanals lipid peroxidation and, 158 source of, 118 HGB. See Hydroxylated guanine base HHE. See 4-hydroxyhexanal High-density lipoprotein paraoxonase-1 and PAF acetylhydrolase in, 28–29 protective effect of, against LDL o xidation, 28 High performance capillary electrophoresis, quantifying free MDA directly with, 120 High performance liquid chromatography estimation of dityrosine residues and , 146 oxidation products of linoleates measured with, 88 High performance liquid chromatographyelectrochemical detection, direct measure of hydroxylated bases and, 293–294 High pressure liquid chromato graphy, study of S-thiolated proteins and, 252 His residue, acrolein co valent adducts to, 178

H

HACA, protein oxidation and, 145 Hakim, I. A., 299 Halliwell, B., 204, 298 Haptoglobin, 55 Haptoglobin 1, 12 Hard metal dust, w orkers exposed to, study of, 270 Harland, W. A., 105 Hashimoto, M., 163, 164 t, 165 HAVA, protein oxidation and, 145 hBAT, 14 HNE adducts identif ed on, 176 HbSSG levels, diabetes in human subjects and , 40

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Histidine, 123 Histidine residues, in proteins, 144 Histochemical detection, antibody-based assa ys, 80HdG and, 295 HNE-adducted peptides as tag of carbon ylated albumin and actin, procedure for quali/ quantitative analysis of, 180 HNE adducted proteins, as lipid pero xidation biomarkers, measurement of, 123 HNE-adducted proteins with biotin, labeling of, by using click chemistr y for subsequent enrichment and identif cation by proteomic analysis, 185 HNE-cysteine adducts, lipid pero xidation and, 159–160 HNE-histidine adducts binding of R310 to conf gurational isomers of, 167 lipid peroxidation and, 160–161 HNE-histidine conf gurational isomers antibodies against, 165–168 structural basis of, 163, 165 HNE-histidine isomers structural basis of facilitating, 163 molecular orbital calculations, 163, 165 HNE-histidine Michael adduct, for mation of, upon reaction of histidine residue with HNE, 165 HNE-lysine adducts, lipid pero xidation and, 161 HNEs. See 4-hydroxynonenals Hodara, R., 210t HODE. See Hydroxyoctadecadienoic acid Hodis, H. N., 59 Hoffmann, H., 272 hOGG1 gene, inf uence of genetic pol ymorphisms on, 275, 276 Homocysteine, mercaptoalbumin depletion and, 230 Hontzacko, A., 125 Hormesis, def ned, 137 Hormetins, 137 HPCE. See High perfor mance capillary electrophoresis HPETE. See Hydroperoxyeicosatetraenoic acid HPLC. See High perfor mance liquid chromatography; High pressure liquid chromatography HPLC (anion exchange column), measuring mercaptoalbumin and Cys34 o xidative modif cations with, 233t HPLC (anion-exchange hydrophobic column), measuring mercaptoalbumin and Cys34 oxidative modif cations with, 233t HPLC-ESI-MS/MS, of tr yptic digests, key role with, in quantifying carbon yl-modif ed proteins, 191

HPLC-MS, glutathione conjugates measured b y, in liver of rats treated with iron nitriloacetate, 124 HPMA. See 3-hydroxypropylmercapturic acid Hp1 allele, 55 Hp2 allele, 55 HSA. See Human ser um albumin Hsc70, 147 HT29 cells, human cultured, pharmacological treatments of, with GSH inhibitors or inducers, 249 H2O2 as intracellular mediator, 38 role of, in insulin function, 38 ubiquitin conjugates and exposure to, 221 Human serum albumin abundance of, in plasma, 229 antioxidant role of, in vivo, 229–230 direct infusion MS used to detect co valent changes in, advantages and disadvantages with, 235 modif cation of, by MDA, 181 semi-quantitative approach used to detect modif cation sites of, HNE and , 177–178 Human subjects diabetes in, 40 leukemia in, 41–42 myocardial infarction in, 41 virus-induced hepatitis in, 39 Hybrid linear ion trap-7-Tesla FTICR mass spectrometer, 175 Hybrid qTOF. See Quadrupole/oaTOF Hydrogen peroxide, 21, 232, 285 in physiological systems, origination of, 138 radical reactions in the cell and , 35 Hydroperoxide-mediated toxicity, importance of PLA2 in, 27 Hydroperoxyeicosatetraenoic acid, 88 Hydrophilic antioxidant capacity assays, 7 application of, 9–10, 9 t Hydrophilic compartment of plasma, measuring antioxidant capacity in lipophilic compartment and, 8 Hydrophobic environments, factors controlling tyrosine nitration and dimerization in, 201 Hydroxy-alkenals, 117 Hydroxylated bases direct measure of, 292–294 gas chromatography-mass spectroscopy, 292 high performance liquid chromatographyelectrochemical detection, 293–294 liquid chromatography-mass spectroscopy, 293 practical use of, as biomark ers, 298–304 assay reliability, 298–299 relatively stable values over time in the same individual, 300–301

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seasonal effects on stability and absor ption of food-based 80HdG smoking, gender, age, and ph ysical activity as confounding variables, 303 stability in storage, 300 utility of 80HdG as biomark er in disease, 303–304 Hydroxylated guanine bases metabolism, 288–289 nomenclature for, 284 where they have been measured, 289–291 endogenous 80HdG found in cells and tissues, 290–291 exogenous HGB found in urine, plasma, and saliva, 289–290 mitochondrial vs. nuclear DNA damage, 291 Hydroxylated nucleotides, 283–310 antibody-based assays, 294–295 enzyme-linked immunosorbent assays, 294–295 histochemical detection, 295 def ned, 283 direct methods of h ydroxylated bases, 292–294 gas chromatography-mass spectroscopy, 292 high performance liquid chromatographyelectrochemical detection, 293–294 liquid chromatography-mass spectroscopy, 293 DNA repair and, 287–288 formation of, 284–286 hydroxylated guanine base metabolism, 288–289 indirect methods, 295–297 repair enzyme methods measured as DN A chain breaks, 296–297 32 P-postlabeling methods, 297 internal sources of h ydroxyl radical for mation, 286–287 methods of measurement in v arious biological compartments and, 291–298 other methods, 297–298 Hydroxyl radicals, 4, 21, 232 extreme reactivity of, 285 formation, internal sources of, 286–287 hydroxylated nucleotide for mation and, 284 radical reactions in the cell and , 35 Hydroxyoctadecadienoic acid assessment of antioxidant capacity in vivo by, 91–92 levels in animal e xperiments, 94 levels in healthy subjects and diseased patients, 92–93 as marker of linoleic acid o xidation, 85–95 mechanism and dynamics of linoleate oxidation, 86–87 role of antioxidants against lipid o xidation, 87–88

measurement of, 88–91 analysis of t7-OHCh, tCh, and t18:2 b y GC-MS, 91 GC-MS method for, 89–90 LC-MS/MS method for, 90–91 Hyperlipidemia, oxysterols and, 109 Hypertension, systemic oxidative stress and, 73 Hypochlorite, formation of, 138

I

Ikatura, K., 164 t Image analysis, biomonitoring trials and, 269 Immonium ion, of 3-NT, 206, 208 Immunochemical detection, of protein carbon yls, 145 Immunochemical detection of lipid-pero xidationspecif c epitopes antibodies against HNE-histidine conf gurational isomers, 165–168 structural basis of HNE-histidine conf gurational isomers, 163, 165 Immunohistochemical staining, exocyclic DNA adduct detection and, 323 Immunological approaches, IsoP quantif cation and, 68–69 Immunological detection, advantages with, 168 Immunological responses, inducing, protein tyrosine nitration and, 209 Immuno-slotblot, exocyclic DNA adduct detection and, 323 IMT. See Intima-media thickness Indirect methods of biomark ers of oxidative damage, vii Inf ammation chronic, malignant disease and, 319 diseases induced by, 22 protein tyrosine nitration and, 199 tumorigenesis and, 320t Inf ammation-driven genotoxicity, comparison of methodologies ref ecting different processes in, 327 Inf ammatory disease, seasonal ef fects on stability and absorption of food-based 80HdG and , 303 Inf ammatory process, excess of reactive oxygen/ reactive nitrogen species and pro gression of, 327, 327 Inf ammatory responses, oxidative stress and, 157 Insulin function, role of H 2O2 in, 38 Insulin signal transduction, H 2O2 and pivotal role in, 38 Inter-individual variations, DNA adducts as biomarkers in large-scale studies and, 325–327 Interleukin 1, 51 Intervention trials, nutrition studies and , 273, 274

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Intima-media thickness, carotid ar tery, antioxidants and effect on progression of, 60 Intra-individual variations, DNA adducts as biomarkers in large-scale studies and, 325–327 Ion channels, S-glutathionylation and, 245 Iron nitriloacetate, glutathione conjugates measured by HPLC-MS in li ver of rats treated with, 124 Ischemia/reperfusion injury, 41 Ishii, T., 178 Ishino, K., 158 Isom, A. L., 177 Isomeric 4-ketoamide derivatives, 161 Isopeptidases, 220 Isoprostanes, 21, 65–79, 85 COX-derived prostaglandins vs., 67 def ned, 65 of different ring str uctures formed by nonenzymatic oxidation of EPA, 79 of different ring types for med by nonenzymatic oxidation of arachidonic acid, 68 eicosapentaenoic acid-derived, 76, 78–79 extraction and hydrolysis of F 2-isoprostanecontaining phospholipids in tissues, 69–70 factors regulating oxidative stress and formation of, 75–75 omega-3 fatty acids and f sh oil as a therapeutic agent in o xidative stressrelated conditions, 75–76 formation of, 65–67 with alternative structures, 67 as index of oxidative stress in vivo, 72 atherosclerosis and associated risk f actors, 72–73 interest in biological activity of, 105 introduction to, 65 LPO products and assessment of, in body f uids or tissues, 321 mass spectrometry assay of, 106 as a mediator of o xidative-stress related diseases, 73–74 biological activities of the c yclopentenone (A2/J2)-isoprostanes, 74 pro-inf ammatory activies of F 2- and E2-isoprostanes, 73–74 methodology to quantify classes of, 71–72 purif cation, derivation, and quantif cation of F2-isoprostanes, 70–71 quantif cation of F 2-isoprostanes, 67–69 storage and handing of biolo gical f uids and tissues for F 2-isoprostane quantif cation, 69 summary remarks, 79

Isotope dilution methods, o xidative stress studies and, 106 Itakura, K., 161

J

Japanese Institute for the Control of Aging, 294 Ji, Y., 210t JICA. See Japanese Institute for the Control of Aging Jones, A. D., 127

K

Kamido, H., 162 Kamisaki, Y., 204 Kanabrocki, E. L., 300, 305 Kapphahn, R. J., 187 Karatas, F., 120 Karnes, H. T., 297 Kasai, H., 287 Kato, Y., 144t Kaur, H., 204 Kawai, Y., 123 Keaney, J. F., Jr., 73 Keap1, EPA peroxidation and, 79 Keimer, R., 204 Ketoaldehydes, lipid peroxidation and, 157 Keyhole limpet hemocyanoin, 157–158 Khan, J., 204 Kidney, Msr expression in, 140 Kim, C. H., 189 Kiwifruit, intervention trial on, 274 KLH. See Keyhole limpet hemocyanoin Knapp, L. T., 211t Kuiper, H. C., 127 Kupffer cells, carbon tetrachloride-induced hepatitis and activation of, 36 Kynurenine, 143, 148

L

Lag time, def ned, 56 Lamprecht, M., 237, 238 Langie, S. A. S., 268 LCAT. See Lecithin-cholesterol acyltransferase LC-ESI-MS/MS method based on precursor ion scanning, for characterizing covalent modif cations of Cys34, 187 employing, to deter mine specif c locations of HNE modif cations of Erk-2, 177 high reactivity of HSA to ward HNE demonstrated with use of L TQ linear ion trap instrument for, 178 for measurement of carbon ylated tag peptide applied to digested proteins, 181 measuring mercaptoalbumin and Cys34 oxidative modif cations with, 233 t

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LC-MS, quantif cation of 3-nitrotyrosine in biological samples and, 205 LC-MS metabonomic prof ling, NMR metabolite screening sensitivity improved with, 128 LC/MS methods. See Liquid chromatographic-MS methods LC-MS/MS. See Liquid chromatography tandem mass spectrometry LDL oxidation in vitro kinetic prof le of, 55, 55 in vitro monitoring copper-mediated peroxidation and, 54 DPPP and, 56 initial light abosrbance of LDL preparation, 55 kinetic prof le of, 55, 55 lag time and, 56 maximal oxidation (max) and, 56 maximal rate of o xidation and, 56 in vitro monitoring of, 54–56 LDL oxidation as biomarker of antioxidant status, 51–60 association of oxLDL and in vitro LDL oxidation with health status, 59–60 cell-mediated oxidation of LDL in vitro, 57 conclusion, 60 introduction, 51–53 isolation of LDL, 53–54 monitoring in vitro LDL oxidation, 54–56 organic radical generators and LDL o xidation, 56–57 signif cance of water-soluble and lipid-soluble antioxidants to LDL o xidation carotenoids and LDL o xidation, 58 polyphenolics and LDL o xidation, 58–59 PUFA and LDL o xidation, 59 vitamin E and LDL o xidation, 57–58 LDLs. See Low density lipoproteins Lecithin-cholesterol acyltransferase, 87, 102 Ledda-Columbano, G. M., 38 Leeuwenburgh, C., 142 t Leukemia, in human subjects, 41–42 Leukotoxin (epoxylinoleic acid), 164 t Levine, M., 75 LI. See Lysopholipid Li, T. H., 297 Lifestyle, inter-individual differences in adduct levels and, 326 Linear quadrupole ion trap high reactivity of HSA to ward HNE demonstrated with use of, 178 Orbitrap interfaced with, 176 Linoleate oxidation, 86–87 enzymatic oxidation and, 86 free radical-mediated oxidation of linoleates, 86–87

non-enzymatic, non-radical oxidation of linoleates, 87 Linoleates abundance of, in vivo, 85 oxidation mechanism of, 87 Linoleic acid oxidation hydroxyoctadecadienoic acid as mark er of, 85–95 mechanisms for, 85, 86–87 Linolenic acid, MDA and, 118 Lipid hydroperoxide, radical reactions in the cell and, 35 Lipid hydroperoxide-elimination system, glutathione peroxidases and phospholipases as, 23–24, 26–27 Lipid hydroperoxides, 23, 162 Lipid oxidation complex products from, 95 role of antioxidants against, 87–88 Lipid oxidation products in human samples, accurate determination of, g reat care taken in, 93 Lipid peroxidation aldehydes as biomarkers of, 118 assay acrolein, 125 HNE and ONE, 124–125 atherosclerosis and, 4 chronic inf ammatory diseases and process of, 320 Cys34 and detoxif cation of by-products of, 231 as degradative process in tissue and tissue fractions, 157 endogenous, analysis of F 2 isoprostanes in plasma obtained from rat 4 hours after treatment with CCl 4 for inducing of, 71 excess of, inf ammation and, 319 extensive study of, 85 formation of, 123–124 glutathione conjugates as biomarkers of, 123–125 measuring multiple aldehydes originating from, 122–123 mercapturic acids derivatives as biomarkers of, 125–128 oxidation secondary to, 99 Lipid peroxidation cascade, peroxyl radicals and, 5 Lipid peroxidation products criteria for DNA adducts as biomark ers of exposure to, 321–327 in human LDL fractions, 93 t use of, 21 Lipid-peroxidation-specif c adducts, quantitative and analytical importance of, 168

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Lipid-peroxidation-specif c epitopes, 158–163 alkanals, 158 4-hydroxy-2-alkenals, 159 4-oxo-2-alkenals, 161–162 HNE-cysteine adducts, 159–160 HNE-histidine adducts, 160–161 HNE-lysine adducts, 161 immunochemical detection of, 163, 165–168 antibodies against HNE-histidine conf gurational isomers, 165–168 structural basis of HNE-histidine conf gurational isomers, 163, 165 sources of, 158 2-alkenals, 159 Lipid-peroxidation-specif c monoclonal antibodies, line-up of, 164 t Lipids, free radical attack on, 109 Lipid-soluble antioxidants signif cance of, to LDL o xidation, 57–59 carotenoids and LDL o xidation, 58 polyphenolics and LDL o xidation, 58–59 PUFA and LDL o xidation, 59 vitamin E and LDL o xidation, 57–58 Lipofuscin accumulation, ubiquitin conjug ates in response to, 222 Lipofuscin-mediated photooxidation, ubiquitin conjugates and exposure to, 221 Lipophilic antioxidant activity, determining antioxidant capacity in biolo gical systems and, 6t Lipophilic antioxidant capacity assays, 7–9 application of, 9–10, 9 t Lipophilic compartment of plasma, measuring antioxidant capacity in h ydrophilic compartment and, 8 Lipophilic ORAC, determining antioxidant capacity in biological systems and, 6t Lipophylic antioxidants, duration of lag time and , 56 Lipoprotein associated phospholipase A2, 28 Lipoxygenases, 51, 86 Liquid chromatography-mass spectrometry exocyclic DNA adduct detection and, 323 of 3-NT, 203 Liquid chromatography-mass spectroscopy, direct measure of h ydroxylated bases and, 293 Liquid chromatography tandem mass spectrometry derivatized MDA identif ed with, 120 with isotopic dilution anal ysis, for analysis of protein oxidation adducts, 146 oxidation products of linoleates measured b y, 88 oxysterol analysis by, 104 Liquid chromatograpy-mass spectrometry, for IsoP quantif cation, 68

Liquid chromatrography tandem mass spectrometry, tHODE measurement and, 90–91 Liu, Y. M., 121 Liver ethanol-induced oxidative stress in, 39 ischemia/reperfusion injury of, 41 Liver damage, choline def cient diets and, 94 Liver diseases mercaptoalbumin depletion and, 237 oxysterols and, 108 Loft, S., 274, 303 Longevity, caloric restriction and, 309 LOO, radical reactions in the cell and , 35 LOOH. See Lipid hydroperoxides Lorch, S. A., 204 Lord, H. L., 120 Loss of function, tyrosine nitration in specif c proteins and, 209 Low density lipoproteins atherosclerosis and oxidative modif cation of, 93 cell-mediated oxidation of, in vitro, 57 complex process behind o xidation of, 53 elevated, oxLDL and, in prediabetic patients, 52 isolation of, 53–54 oxidation of, 28 oxidation of, atherosclerosis and, 22 oxidative modif cation hypothesis of atherosclerosis and, 51 in vitro oxidation of, 104–105 Low-molecular-mass (LMM) thiols, 243–244 LOX. See Lipoxygenase LPO-derived reactive aldehydes, formation of, 321 LTQ. See Linear quadropole ion trap Lung, Prx6 in, 28 Lung cancer etheno-DNA adducts, inter-individual differences and, 326–327 smoking and limit on predicti ve value of 80HdG as predictor of, 305 Luo, X. P., 122 Lymphocyte DNA, results from e xperiment to measure base oxidation in, 264 Lymphocyte DNA damage, analysis of, 263 Lymphocytes DNA damage and lifespan of, 275 human, inf uences of genotype on le vels of DNA damage in, 277 isolating, biomonitoring trials and, 269 obtaining, measuring DNA damage and, 267 range of BER and NER acti vities in, 268 t Lymphoprep, 269 Lysine, 123 Lysophospholipid, 26

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Lysosomal proteolysis, of oxidized proteins, 147 Lys residue, acrolein co valent adducts to, 178

knowledge about RCS reacti vity, metabolism, signalling and modulator y effects, 191 pathogenetic role of protein carbon ylation elucidated with, 179 top-down and bottom-up approaches, in characterization of covalently modif ed proteins, 174, 175 Mass spectrometry-based methods, for quantif cation of 3-NT in biolo gical samples, 202, 204–205 Mateuca, R., 275 Matrix-assisted laser desor ption, 148 Matrix-assisted laser desor ption and ionization time-of-f ight determination of intact proteins and , 174 mapping 3-nitrotyrosine in proteins and , 205 measurement of HODE and, 88 Mayer, B., 8 McCord, J. M., 22 MDA. See Malondialdehyde MDAsA. See Monodehydroascorbic acid Mechanical sheer stress, 51 MeO-AMVN, 8 Mercaptoalbumin analytical strategies to measure Cys34 oxidative modif cations and, 232–237, 233t albumin disulf de, 233 albumin SH titration, 232–233 chromatographic analysis of albumin redo x variants, 233–234 direct infusion mass spectrometr y of intact albumin, 234–235 LC-ESI-MS/MS of enzyme-digested albumin, 235–237 depletion of, oxidation products for med relative to, 229–230 as marker of antioxidant status, 238 questions remaining in studies of, 238 reversible and ir reversible oxidized forms, as markers of oxidative damage and, 237–238 Mercapturic acid pathway example of 4-hydroxynonenal, 124 as major route of e xcretion of glutathione conjugates, 125–126 Mercapturic acids, 117 def ned, 125 Mercapturic acids derivatives assay, 126–128 as biomarkers of lipid pero xidation, 125–128 acrolein, 126 formation, 125–126 4-hydroxlalkenals, 126 “Mercapturomics” approach, deter mining mercapturic acids and thioethers in urine and, 128

M

MA. See Microangiopathy MacMillan-Crow, L. A., 210t MALDI. See Matrix-assisted laser desor ption MALDI-MS. See Matrix-assisted laser desor ption/ ionization MALDI spectra, of c ytochrome c nitrated peptide 92 EDLIA97NO2YLK99, 207 MALDI-TOF. See Matrix-assisted laser desorption and ionization time-of-f ight MALDI-TOF-MS application of, to conf rm covalent adduction of HNE to thioredoxin system, 177 demonstrating ACR inhibition of c ytokine gene expression in human T lymphocytes with, 179 Malignant disease, chronic inf ammation and, 319 Malins, D. C., 304 Mally, A., 127 Malondialdehyde, 117, 145, 164 t derivatization techniques for, 120 DHP-lysine formation upon reaction of l ysine residue with, 162 exocyclic DNA adducts for med from, 322, 322 formation, 119 HSA modif ed by, 181 as index of lipid pero xidation, 118 lipid peroxidation and, 162 measuring, 119–121 origination of, from lipid pero xidation process, 118 sources of, 118–119 tHODE levels in healthy and diseased subjects and, 92 in vitro oxidation of LDL and, 104, 105 MAP. See Mercapturic acid pathw ay MAPK/ERK. See Mitogen-activated protein kinase/extracellular-signal-regulated kinase MAPKs. See Mitogen-activated protein kinases Marco, M. P., 127 Marcondes, S., 211t MAs. See Mercapturic acids Massaeli, H., 7 Mass spectrometers, technical impro vements in MS instrumentation, 176 Mass spectrometry detection/characterization of RCS-modif ed proteins with, 174 fundamental role of, in detection and characterization of peptides and co valently modif ed proteins by RCS, 190 identif cation and characterization of carbonylated proteins in biolo gical matrices and use of, 182–183

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Metabolic syndrome, vitamin supplementation and, 4 Metabonomics approach, deter mining mercapturic acids and thioethers in urine and , 127 Methacryloylamidohistidine-platinum (II) monomer, 80HdG imprinted onto, 297 Methionine residues oxidation and repair of, in proteins, 140 oxidation of, 139–140 sulfone, 139 Methionine sulfoxide, 139 detection of protein o xidation adduct residues and free adducts in biomedical research, 141t low renal clearance with, 148 Methionine sulfoxide reductase, 139 two forms of, 140 Methyl-MDA, 120 Methylpyridinium-formyl-dehydropiperidino-type adducts, 178 Methylthiophene modif ed glassy carbon electrode, 297 MetSO. See Methionine sulfoxide Mexican mining area, study of children and exposure to As and Pb in, 270 Michael addition adduct with histidine residues, 145 Micheal adducts, 74, 78 Microangiopathy, plasma concentrations of AsA and, 40 Micronutrients, DNA damage and, 273, 273t Milk protein oxidative modif cations, during industrial treatment, monitoring, 183 Miller, E.R., 12 Miller, N. J., 7 Milman, U., 12 Mineral f ber-exposed workers, inf uence of genetic polymorphisms on DNA damage and repair in, 276 Mineral f ber exposure, studies on, 271 Mineral f ber trials, age and, 275 Miniaturization of derivatization procedure, multiple determination for aldehydes and, 122 Minimally modif ed LDL, 52 Mirzaei, H., 184 Misfolded proteins, protein damage b y oxidation and, 147 Mitochondrial DNA damage, nuclear DN A damage vs., 291 Mitochondrial proteins, S-glutathionylation and, 245 Mitochondrial respiratory chain, 51 Mitogen-activated protein kinase/extracellularsignal-regulated kinase, S-glutathionylation and, 245 Mitogen-activated protein kinases, 37

Miwa, M., 301 mmLDL. See Minimally modif ed LDL Mn-SOD (SOD2), 22 Modif ed proteins. See also 3-nitrotyrosine covalently, general and ne w approaches for MS characterization of, 174–175 ex vivo studies of, 189–190 precursor ion scanning and study of, 186–187 Molecular epidemiological studies, exocyclic DNA adducts, 319–321 Molecular epidemiology, general scheme of, representing continuum between exposure to genotoxins and disease de velopment, 321 Moller, P., 274, 301 M1dG, omega-6-polyunsaturated fatty acids and increased levels of, 322 M1dG adducts, levels of, in v arious tissues and cells of humans, 324 t Monobromobimane, study of S-glutathionylation and S-cysteinylation and, 253 Monoclonal antibodies against HNE-histidine conf gurational isomers, 165–168 lipid-peroxidation-specif c, line-up of, 164 t Monodehydroascorbic acid, 35 Morrow, J. D., 74 Morrow, Jason, 65 Mortality, antioxidants, gene interactions and, 12 Mortality risk, inverse relationship between serum albumin concentration and, 229 MP-lysine, 178 formation of, upon reaction of l ysine residue with acrolein, 159 MPO. See Myeloperoxidase MRM. See Multiple reaction monitoring MS. See Mass spectrometr y MS-MS, multiple deter mination for aldehydes and, 122 Msr. See Methionine sulfoxide reductase Multiple reaction monitoring, 123 Multiple sclerosis, oxysterols and, 107 Musante, L., 235 Mutagens, environmental, comet assay used as biomarker assay in studies of, 269 Myeloperoxidase, 51 non-enzymatic, non-radical oxidation of linoleates and, 87 Myocardial infarction atherosclerosis and, 105 in human subjects, 41

N

N-acetyltransferases, N-acetylation of c ysteine catalyzed by, 126 N-acetyltyrosine, 200 Nadkarni, D. V., 163

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NADPH oxidases, 51 Nair, J., 326 Nanof ow liquid chromatography, quantifying protein damage and, 147 Nanospray LC-ESI-MS/MS, demonstrating ACR inhibition of cytokine gene expression in human T lymphocytes with, 179 Naphthalene, ELISA for detection of MA metabolites of, 127 NASH. See Non-alcoholic steatohepatitis National Institutes of En vironmental Health Sciences, 65 NCS, ubiquitin conjugates and e xposure to, 221 NEM. See N-ethyl maleimide N-(epsilon)-3(formyl-3,4-dehydropiperidino) lysine, 178 N-(epsilon)-(3-methylpyridium) lysine, 178 NER. See Nucleotide excision repair Nernst equation, 248 Nestel, P. J., 59 Net EndoIII-sensitive sites, 263, 264 Net enzyme-sensitive sites, estimating, 264 N-ethyl maleimide, 253 Neuroblastoma cells, role of 15d-PGJ 2 in, 180 Neurodegeneration free radicals and, 65 protein tyrosine nitration and, 199 Neurodegenerative diseases increased albumin disulf de and, 237 oxidative stress and, 93 oxysterols and, 107 New Zealand White (NZW) rabbits, o xysterols in, 106 NFK. See N-formyl-kynurenine NFK free adduct, 148 N-formyl-kynurenine, 143, 143 detection of protein o xidation adduct residues and free adducts in, 144 t Nicholls, S. J., 204 NICI-GC-MS, MDA and HNE measurement in plasma validated by, 122 NIEHS. See National Institutes of En vironmental Health Sciences Niki, E., 7 9-(E,E)-HPODE, oxidation of linoleates and, 86, 87 9-f uorenylmethoxycarbonyl hydrazine, 120 9-hydroxy-10E,12Z-octadecadienoic acid, 164t 9-oxononanoylcholesterol, 162, 163, 164 t 9-(Z,E)-HPODE, oxidation of linoleates and, 86, 87 Nitrated peptide, cytochrome c, MALDI spectra of, 207 Nitration reactions, tyrosine analo gs as probes for following, 200–201 Nitric oxide, inf ammation and release of, 22 Nitric oxide synthase, 51

Nitrogen dioxide, 200 Nitrotyrosine biological effects of 3-nitrotyrosine in proteins, 209 effects of tyrosine nitration on selected proteins, 210–211t foundation of 3-nitrotyrosine for mation, 199–200 mapping 3-nitrotyrosine in proteins, 205, 207–208 perspectives on, 209, 212 quantif cation methods for 3-nitrotyrosine in biological samples, 202, 203, 204–205 quantitative analysis, mapping in proteins, and biological signif cance, 199–212 tyrosine analogs as probes for follo wing nitration and oxidation reactions, 200–201 NL-driven MS 3 data-dependent acquisition, v alue of, in enhancing fragmentation infor mation for modif ed peptides, 189 N,N-biotinyl GSSG, increase in GSSG and , 255 NO. See Nitric oxide Non-alcoholic steatohepatitis, 308, 309 Non-enzymatic, non-radical oxidation of linoleates, 87 oxidation of linoleic acid and , 85, 87 Non-mass spectrometry-based methods, for quantif cation of 3-NT in biolo gical samples, 202, 204–205 Non-polar oxysterols, transportation in circulation, 102 Non-tryptophan f uorescence, as markers of protein oxidation, 145–146 n-3 polyunsaturated fatty acid-rich tissue, central nervous system and, 118 Nuclear DNA damage, mitochondrial DN A damage vs., 291 Nucleophilic sites of proteins, ONE reacti vity toward, 181 Nucleotide excision repair, range of acti vities, in human lymphocytes, 268t Nutrition, DNA stability and ef fects of, 272–274

O

oaTOF. See Orthogonal acceleration time-of-f ight Obese Zucker rats, precursor ion scanning technique to detect free, protein-bound histidine residues modif ed by RCS in urine from, 187 Obesity LC-ESI-MS/MS used to in vestigate protein modif cation by HNE as consequence of, 190 oxidative stress and, 73 reduction in PON-1 acti vity and oxidative stress induced by, 28

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Obstructive sleep apnea, decrease in albumin thiol groups and, 237 Occupational exposure to genotoxic agents, 269–272 Octadecyl-bonded silica columns, 121 Omega-6-polyunsaturated fatty acids, increased levels of etheno-DNA adducts and M 1dG, 322 Omega-3 fatty acids, as therapeutic agents in oxidative stress-related conditions and diseases, 75–76 ONE. See 4-oxononenals 1-Cys Prx, PLOOH reduced in situ by, 24 1,4-dihydroxynonene mercapturic acid, formation of, 126 One-dimensional polyacrylamide gel, measuring mercaptoalbumin and Cys34 o xidative modif cations with, 233 t 1-methyl-2-phenylindole, 120 ONE reactivity, toward nucleophilic sites of proteins, 181 On-membrane sample preparation procedure, for analyzing trypsin-digested apolipoproteins from native LDL and/or modif ed LDL, 188, 188 o-phthalaldehyde, study of S-glutathionylation and S-cysteinylation and, 253 Opisthorchis viverrini, exocyclic DNA adducts and, study of patients suf fering from, 325 ORAC. See Oxygen radical absorbance capacity ORAC assay, 7, 8, 9 estimated percent contribution of, in plasma antioxidants, 9t Orbitrap, interfacing with linear quadr upole ion trap, 176 Organic radical generators, LDL o xidation and, 56–57 Orioli, M., 125 Orthogonal acceleration time-of-f ight, 176 OSA. See Obstructive sleep apnea Osawa, T., 294 o-tyrosine residues, detection of protein o xidation adduct residues and free adducts in, 142 t OV. See Opisthorchis viverrini OVC. See 5-oxovaleroylcholesterol Oxidation of cysteine residues, 138 of methionine residues, 139–140 of phenylalanine residues, 142 polyunsaturated fatty acids and, 85 of tryptophan residues, 142–144 of tyrosine residues, 140, 142 Oxidation-free adducts, proteolytic debris of oxidized proteins and, 148 Oxidation modif cation of proteins, 137–149 advances in quantif cation of protein damage, 146–147

biomarkers, 149 other related modif cations, 144–145 oxidation of cysteine residues, 138 oxidation of methionine residues, 139–140 oxidation of phenylalanine residues, 142 oxidation of tr yptophan residues, 142–144 oxidation of tyrosine residues, 140, 142 physiological oxidizing agents, 138 proteasomal and lysosomal proteolysis of oxidized proteins, 147 protein carbonyls, advanced protein oxidation products, and non-tr yptophan f uorescence as markers of protein o xidation, 145–146 protein oxidation in physiological systems: damaging, deliberate, and defensi ve, 137 proteolytic debris of o xidized proteins: oxidation-free adducts, 148 proteomics studies of o xidized proteins, 148–149 Oxidation reactions, tyrosine analo gs as probes for following, 200–201 Oxidation stress, LDL o xidation as index of, 53–57 Oxidative damage criteria for ideal biomark er of, 298 future research needs on, 13 GPx and protecting biolo gical organisms from, 23 Oxidative modif cation hypothesis, 93 Oxidative stress. See also Antioxidants as biomarkers of oxidative stress antioxidants in biological system and, 4–5 biomarkers for, 85 cataracts and, 93 comet assay and infor mation about degree of, 277 criteria for DNA adducts as biomark ers of exposure to, 321–327 def ned, 104 diabetes mellitus and, 23 factors regulating isoprostane for mation and, 75–76 further studies needed on, 43 genetic polymorphisms identif ed in genes related to, 326 importance of S-cysteinylation as means for thiol protection against, 250 increased substrate availability for ubiquitination upon exposure to, 224 inf ammation and increased le vels of, 328 levels of endogenous ubiquitin conjugates in cells or tissues and, 220 mild, covalent modif cations of Cys34 as biomarker of, 229–238 mild, proteasome as tar get of, 224–225, 225 neurodegenerative diseases and, 93 overview, 3–13

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pathogenesis of disease and, 22 persistent, inf ammation and, 319 protein tyrosine nitration and, 199 results for ubiquitin conjug ates in response to, 222–225 increase in substrate a vailability and alteration in ubiquitin conjug ation capability, 223–224 proteasome as target of mild o xidative stress, 224–225 role of, in di verse pathophysiological settings, 109 S-glutathionylation and, 244, 245 signaling cascade in cellular functions and , 157 smoking as source of, 326 stimulation of Fenton chemistry and, 117 2-Cys Prx and, 27 ubiquitin conjugate accumulation in cells in response to, 223 ubiquitin conjugates as sensitive markers of, 219–226 in vivo, isoprostanes as inde x of, 72–73 atherosclerosis and associated risk f actors, 72–73 Oxidative stress-induced protein agg regation, understanding mechanisms of, 178–179 Oxidative stress/lipid peroxidation, aldehydic compounds formed after, 117 Oxidative stress-related conditions/diseases, omega-3 fatty acids and f sh oil as therapeutic agents in, 75–76 Oxidative-stress related diseases, isoprostanes as mediator of, 73–74 Oxidized amino acid residue e xcretion in urine, analysis of, 149 Oxidized lipids, 164t Oxidized nucleosides, measurement of, 283 Oxidized proteins proteasomal and lysosomal proteolysis of, 147 proteomics studies of, 148–149 tagging, 148 Oxidized purines, str ucture of, and acron yms used in text, 285 Oxidized pyrimidines endonuclease III and measurement of, 263 workers exposed to, study of, 270 Oxindolylalanine, 143 oxLDL, elevated LDL and tricl ycerides and, 52 Oxo-alkenals, 117 Oxygen radical absorbance capacity, 5 determining antioxidant capacity in biolo gical systems and, 6t Oxysterol hypothesis, 99 Oxysterols, 99–109 clinical conditions and, 107–109 atherosclerosis, 107–108 diabetes, 108–109

hyperlipidemia, 109 liver disease, 108 neurodegenerative disorders, 107 conclusion, 109 enzymatic transformations of cholesterol into, 100 formation of, 99–101 introduction, 99 as markers of oxidative stress, 104–105 in atherosclerotic plaques, 105 in experimental animal models, 106–107 other markers vs.: isoprostanes, TBARS, 106 in vitro oxidation of LDL, 104–105 measurement of, 102–104 analysis by GC-MS, 103–104 analysis by LC-MS/MS, 104 toxicity of, to cells, 102 transport of, in the circulation, 102 Ozone, environmental exposure to, Biomarkers of Oxidative Stress Study and, 149

P

PAF-acetylhydrolase, in high-density lipoprotein, 28–29 PAH. See Polycyclic aromatic hydrocarbons Palus, J., 270 Paraoxonase-1, in high-density lipoprotein, 28–29 Paraquat ubiquitin conjugates and exposure to, 221 ubiquitin conjugates in response to, 222 Parkinson’s disease CoQ10 level in cor tex region of patients with, 42 RCS/protein adducts and, 173 Pb-exposed children, in Me xico, studies of, 270 PCA assay, estimated percent contribution of, in plasma antioxidants, 9t PD. See Parkinson’s disease PDGF. See Platelet derived growth factor Pehar, M., 210 t Pennathur, S., 204 Pentaf uorobenzyl, 70 Pentaf uorophenylhydrazine derivatization, MDA measurement and, 120 Pentosidine, 145 Peoples, M. C., 297 Peroxiredoxin 6 (Prx6), 27–28 Peroxiredoxins, 24 Peroxyl radicals, 4, 5 Peroxynitrite, 200, 232, 285 Peroxynitrite anion, for mation of, 23 PFB. See Pentaf uorobenzyl PFB derivatization, aldehydes monitored with, 123

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Phenylalanine residues hydroxylation of, in proteins, 141 oxidation of, 142 Phenylhydrazine derivatization, MDA measurement and, 120 PHGPx forms of, 24 overexpression of, 27 Phospholipase A2, 24 classif cation of, 26 participation of, in elimination/repair of oxidatively damaged membranes, 26 Phospholipid GPx, 24 Phospholipid hydroperoxides, 23, 26 characteristics of, 25t possible routes for elimination of, b y antioxidant enzyme system, 26 reductive detoxif cation of, 24 Phospholipids, sn-2 position of gl ycerol backbone of, 24 Phospholipids hydroxide, 26 Phosphorylated JNK, 37 Phosphorylation cascade signaling, protein tyrosine nitration and, 209 Phthalates, exposure to, evaluation, 306 Phycoerythrin assay, 7 p-hydroxyphenylacetic acid, 200 Physical activity, as modulator of urinar y 80HdG in healthy subjects, 303 Physiological antioxidants, oxidative stress and level of, 35 Physiological oxidizing agents, 138 Phytoestrogens, soy milk vs. rice milk vs. cow’s milk and, 274 Phytof uene, duration of lag time and , 56 Pilger, A., 299, 300, 305 Plasma exogenous HGB found in, 289–290 hydrophilic approaches for deter mining antioxidant capacity in, 7 measuring antioxidant capacity in both hydrophilic and lipophilic compar tments of, 8 Plasma antioxidants, estimated percent contribution of antioxidant capacity assays in, 9t Plasma GPx, 24 characteristics of, 25 t Plasma oxysterols, analysis of, by GC-MS, 103, 103–104 Plastics manufacturing workers, study of, 269–270 Platelet derived growth factor, 51 PLOH. See Phospholipids hydroxide PLOOH. See Phospholipid hydroperoxides Polar oxysterols, transportation in circulation, 102

Polycyclic aromatic hydrocarbons, exposure to, evaluation, 306 Polyphenols, 3, 4, 13 LDL oxidation and, 58–59 plasma antioxidant capacity and, 10 response of 80HdG to, 307 Polyunsaturated fatty acids, 54 increased exocyclic-DNA adduct for mation in WBCs and, 326 LDL oxidation and, 59 lipid peroxidation and, 118, 157 oxidation of, 86 oxidative decomposition of, chain reactions in wake of, 173 susceptibility of, to o xidation, 85 PON-1. See Paraoxonase-1 Postischemic reoxygenation injury, lipid peroxidation and, 157 Post-translational modif cations, top-down characterization of intact proteins and , 176 P-postlabeling technique, exocyclic DNA adduct detection and, 323 Propanal, source of, 118 Propanal-formyl-dehydropiperidino-type adducts, 178 Prostate cancer, utility of 80HdG as predictor of, 304, 305 Protease hydrolysis, for quantif cation of 3-NT in biological samples, 202, 204–205 Proteasomal proteolysis, of oxidized proteins, 147 Protein aggregation, oxidative stress-induced, understanding mechanisms of, 178–179 Protein-bound aldehydes, 164t Protein-bound core aldehyde, 164t Protein carbonylation, 173 mass spectrometry and elucidating patho genetic role of, 179 Protein carbonyls, as markers of protein oxidation, 145–146 ProteinChip technology, protein carbonylation f eld and, 191 Protein damage, advances in quantif cation of, 146–147 Protein deglutathionylation, catalysis of, 250 Protein disulf des, formation of, 244 Protein fractions, exposed to RCS: e xamples of in vitro studies, 187–189 Protein ion scanning, protein modif cation study with, 186–187 Protein kinase C isozymes, S-cysteinylation of, 249 Protein misfolding, protein damage b y oxidation and, 147 Protein modif cation, top-down and bottom-up mass spectrometry approaches and, 174, 175

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Protein oxidation in physiological systems: damaging, deliberate, and defensive, 137 protein carbonyls, advanced protein oxidation products, and non-tr yptophan f uorescence as markers of, 145–146 Protein phosphorlylation of insulin receptors, H2O2 and enhancement of, 38 Proteins. See also Carbonylated proteins; Modif ed proteins biological effects of 3-nitrotyrosine in, 209 hydrophilic assays, antioxidant capacity of plasma and, 9 hydroxylation of phenylalanine residues in, 141 oxidation and repair of cysteine residues in, 139 oxidation and repair of methionine residues in, 140 oxidation of tr yptophan residues in, 143 oxidative dimerization of tyrosine residues in, 141 oxidized, proteasomal and l ysosomal proteolysis of, 147 oxidized, proteolytic debris of: o xidation-free adducts, 148 oxidized, proteomics studies of, 148–149 selected, effect of tyrosine nitration on, 210–211t Protein sulfenic acid for mation, role of, 138 Protein thiol g roups age-dependent reduction in plasma le vel of, 247 under moderate oxidative stress conditions, 243 Protein thiols, oxidative modif cations of, 244 Protein 3-NT, immunohistochemical detection of, 202 Proteomic methods, for identifying nitrated proteins, 208 Proteomics studies, of o xidized proteins, 148–149 Proteosomes, as target of mild o xidative stress, ubiquitin-conjugate enzymes and, 224–225, 225 Proteosome system, protein tyrosine nitration and increased degradation by, 209 Prx, abundant types of, in prokar yotes and mammalian cells, 27 Prx isoforms, mammalian cells and e xpression of, 27 Pryor, W. A., 66 PSHs. See Protein thiol g roups PSOH, formation of, 243 PTMs. See Post-translational modif cations PUFAs. See Polyunsaturated fatty acids Pulmonary disease, increased F 2-IsoP levels in, 72 Purine nucleoside phosphor ylase, reincorporation of 80HdG into DN A and, 288

Pyridinium adducts, for mation of, 159 Pyrole adduct, 161

357

Q

Quadrupole ion trap, 176 Quadrupole/time-of-f ight instruments, 175 Quercetin, 8 Quinolinic acid, 143 Quinols, auto-oxidation of, 4

R

Radical reactions in cells, 35, 36 Radiomimetic neocarzinostatin, ubiquitin conjugate response to, 222 Randomly methylated beta-cyclodextrin, 8 Raney Nickel, measuring HNE bound to thiol proteins with, 121 Rats, streptozotocin-induced diabetic, for mation of ethanolamine phospholipids Michael adducts in retinas of, 189 Rauli, S., 122 Rauniyar, N., 175 RCS mass spectrometry and knowledge about reactivity, metabolism, signalling and modulatory effects of, 191 mass spectrometry’s multifaceted role in detection and characterization of peptides and covalently modif ed proteins by, 190–191 selected examples of in vitro studies: cells, tissues, f uids, and protein fractions exposed to, 187–189 RCS-modif ed proteins, general approach to characterization of, in biolo gical matrices, 182 Re, R., 8 Reactive aldehydes lipid peroxidation and, 157 LPO-derived, formation of, 321 Reactive carbonyl species Cys34 as detoxifying agent for, 231 protein carbonylation induced by, disease and, 173 Reactive nitrogen species double-face signif cance of, 243 oxidation of cysteine residues and, 138 Reactive oxygen species, 35 biological signif cance of antioxidant interactions and, 10 continuous generation of, 4 double-face signif cance of, 243 generation of, physiological consequence of, 38 oxidation of cysteine residues and, 138 oxidative stress and types of, 21

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Reactive oxygen species/reactive nitrogen species DNA damage and, 319 oxidative damage and e xcessive amounts of, 3 postulated factors affecting total antioxidant capacity of, 12 progression of inf ammatory disease and e xcess of, 327, 327 short half-life of, 320 Redox signaling, S-glutathionylation and, 244, 245 Red wine LDL oxidation and, 58 plasma antioxidant capacity and, 10 Regulated protein oxidation, 137 Reichard, J. F., 123 Renal cortex, activity of SOD2 in, 22 Renal failure, chronic, mercaptoalbumin depletion and, 237 Renal function loss, increases in protein o xidation free adducts in plasma, 148 Renal insuff ciency, chronic, er ythrocyte contents of SOD, GSH, GPX, and alpha-T oc, 41 Repair enzyme methods, measurement of, as DNA chain breaks, 296–297 Retinal degeneration, light-induced, HNE-protein modif cations and initiation of, 189–190 Retinal pigment epithelial cells, ubiquitin conjugate response to o xidative stress and, 223 Retinol, DNA damage and, 273, 273t Retinylstearate, duration of lag time and , 56 Reverse-phase-HPLC columns, quantif cation of 3-NT in biological samples and, 202 Rheumatoid arthritis increased F 2-IsoP levels in, 72 lipid peroxidation and pathogenesis of, 157 R-HNE epitopes, detection of, in nuclei, 167 R-HNE histidine, 167 Rice milk, f avonoids and, 274 Ring structures, alternative, isoprostane for mation with, 67 Ring types, different, isoprostane for mation by non-enzymatic oxidation of arachidonic acid, 68 RMCD. See Randomly methylated beta-cyclodextrin RNS. See Reactive nitrogen species Roberts, E. S., 211 t Roberts, Jack, 65 Roberts, L. J., 2nd, 73, 75 Robotic automation, of enzymatic h ydrolysis procedures, nanof ow liquid chromatography, 147 Roche, M., 230 ROS. See Reactive oxygen species ROS/RNS. See Reactive oxygen species/reactive nitrogen species

R310, 166 binding of, to conf gurational isomers of HNE-histidine products, 166, 167 immunohistochemistry of renal cor tex with, 168 presence of immunoreactive materials with, 167 R310 Fab fragment, X-ray crystallographic analysis of, 166, 167 Rubber tire workers, study of, 269–270 Rudiger, H. W., 305 Rutin, 8 Ryanodine receptor type 2 RyR2, S-glutathionylation and, 245

S

Saccharide residues, of gl ycated proteins, 144 Saliva, exogenous HGB found in, 289–290 Sample preparation and storage, biomonitoring trials and, 269 Sangvanich, P., 187 Sattler, U., 297 Savvides, S. N., 211t Sayre, L. M., 161, 163, 165 SBs. See Strand breaks (DN A) Schiff base adducts, alkanals and , 158 Schwedhelm, E., 204 S-cysteinylated plasma proteins, age-dependent increase in, 247 S-cysteinylated proteins, plasma le vels of, in plasma of healthy individuals per age group, 248 S-cysteinylation, 244, 245 concluding remarks about, 255 importance of, as means of thiol protection against oxidative stress, 250 potential mechanisms of, 250–252 as thiol-selective post-translational modif cations, 249–250 S-cysteinylation reaction, studying proteins undergoing, 252–255 sdLDL levels, diabetes and, 52, 59 SDS gel electrophoresis, measuring mercaptoalbumin and Cys34 o xidative modif cations with, 233t Seasonal effects biomonitoring trials and, 269 on stability and absor ption of food-based 80HdG, 301–303 Secretory phospholipase A2 (cPLA 2), 26 Seidel, E. R., 211 t SELDI-TO-FMS mass spectrometr y, for identifying new biomarkers in various body f uids, 191 Selenium, biological functions occur ring between sulforaphane and, 12

359

Inde

x

Selenocysteine residue, in glutathione peroxidases, 24 Seleznev, S., 27 Sevanian, A., 26 7alpha-hydroxycholesterol, 99, 100, 101 7alpha-OH-cholesterol, 100 7 beta-hydroxycholesterols, 99, 100, 101 atherosclerotic plaques and, 105 11 beta-hydroxysteroid dehydrogenase and interconversion of, 101 liver disease and, 108 7-hydroperoxycholesterol, 99, 100 7-hydroxycholesterol, 99, 100 as promising biomarker for studies on o xidative stress in vivo, 109 7-ketocholesterol, 99, 100, 101, 163, 164 t atherosclerotic plaques and, 105 11 beta-hydroxysteroid dehydrogenase and interconversion of, 101 liver disease and, 108 as promising biomarker for studies on o xidative stress in vivo, 109 type 2 diabetes mellitus and , 109 S412 immunohistochemistry of renal cor tex with, 168 presence of immunoreactive materials with, 167 S-glutathionylated proteins, plasma le vels of, in plasma of healthy individuals per age group, 248 S-glutathionylation, 244 concluding remarks about, 255 potential mechanisms of, 250–252 role of, in redo x signaling and o xidative stress response, 244, 245 as thiol-selective post-translational modif cations, 249–250 S-glutathionylation reaction proposed mechanisms of, 251 studying proteins undergoing, 252–255 Shao, B., 179 Sharov, V. S., 208 Shigenaga, M. K., 204 S-HNE epitopes, detection of, in c ytosols and some nuclei, 167 Short-chain aldehydes, other, lipid peroxidation and, 162 Short chain carbonyl compounds, 21 Short form-PHGPx, 24 Signaling proteins, S-glutathionylation and, 245 Silica-exposure, comet assay and evaluation of, 305 SIM. See Single ion monitoring Sim, A. S., 120 Single ion monitoring, of aldeh ydes, 122 Singlet molecular oxygen, 21

Singlet oxygen, formation of, 88 Sleep, lower oxidative insult during hours of, 301 Sleep apnea, obstr uctive, decrease in albumin thiol groups and, 237 Slides, archiving, biomonitoring trials and, 269 Slyskova, J., 276 Small, dense LDL par ticles, coronary heart disease and, 52 SMC. See Smooth muscle cells Smith, C. C., 296 Smokers non-smokers vs. determining mercapturic acids and thioethers in urine and, 127 DNA damage in, 272 FPG- and EndoIII-sensitive sites in lymphocyte DNA in, 276, 277 micronutrients, DNA damage and, 273t Smoking etheno-DNA adducts, inter-individual differences and, 326 exposure to f ne particulate matter and, 306 increased F 2-IsoP levels in, 72 increase in oxidative stress and, 59 as modulator of urinar y 80HdG in health y subjects, 303 reduction in PON-1 acti vity and oxidative stress induced by, 28 Smoking cessation, decreased endo genous IsoP formation and, 75 Smooth muscle cells, 52 S-nitrosyl intermediate formation, S-glutathionylation and, 250, 251, 251 SOD. See Superoxide dismutase Sodium borohydride, 252 Soejima, A., 237 Solid-phase hydrazide reagent, label-free strate gy based on, for enrichment of HNE-modif ed peptides within complex mixtures, 185, 186 Souza, J. M., 142t, 210t S-oxidized proteins, deter mining by indirect molecular tools, 255 Soy, biological functions occur ring between green tea and, 12 Soy milk, f avonoids and, 274 Spectrophotometric methods, study of S-glutathionylation and S-cysteinylation and, 253 Spectrophotometry, measuring mercaptoalbumin and Cys34 oxidative modif cations with, 232, 233t Sperm nuclei GPx, 24 S-phenylmercapturic acid, exposure to urban air pollution and excretion of, 272 SPH reagent. See Solid-phase hydrazide reagent S-PMA. See S-phenylmercapturic acid

360 Inde

x

Stable-isotope-dilution methods, quantif cation of 3-nitrotyrosine in biological samples and, 205 Stadtman, E. R., 160, 161 Staurosporine-induced apoptosis, overexpression of iPLA 2beta in insulinoma cells and Chinese hamster ovary cells and, 27 Steinberg, D., 51, 93 Stir bar sor ptive extraction-thermal desorptionGC-MS technique, HNE in human urine measured with, 121 Stocker, R., 58 Stopforth, A., 121 Strand breaks (DNA) estimating, 263 seasonal effects on stability and absor ption of food-based 80HdG and, 301 Strawberries, plasma antioxidant capacity and, 10 Stress resistance and signaling, 2-Cys Prx and , 27 Stroke, atherosclerosis and, 105 Styrene-exposed workers inf uence of genetic pol ymorphisms on DNA damage and repair in, 276 studies of, 269, 270 Su, H., 290 Sulfenic acid, detection of, 232 Sulfenic acid inter mediate formation, as potential mechanism of S-glutathionylation, 250, 251 Sulf nic acids, for mation of, 243 Sulfonic acids, for mation of, 243 Sulforaphane, biological functions occur ring between selenium and, 12 Superoxide anion radicals, 21, 22 Superoxide anions, 4 detoxif cation of, SOD and GPx and , 21 Superoxide dismutase, 21, 88 discovery of, 22 mammalian, three major isofor ms of, 22 as ROS scavenging system, 22–23 Superoxide production, electron transfer system of mitochondrial inner membrane and , 4 Superoxides, radical reactions in the cell and , 35 Superoxide toxicity, protecting cells from, 22 Surrogate tissues, DNA adducts in humans and , 323–324 Systemic lupus er ythematosus, tryptic digestion followed by MALDI-TOFMS analysis for identifying catalase as main modif ed protein by HNE in red b lood cells and, 190 Sytosolic GPx, characteristics of, 25 t Szapacs, M. E., 178

Tail length, 299 comet assay and, 262–263 Tail moment, comet assa y and, 263 Takane, M., 290 Tallman, K. A., 178 TAP, determining antioxidant capacity in biological systems and, 6t Taylor, S. W., 141t, 144t TBARS assay. See Thiobarbituric acid-reactive substances assay TEAC, determining antioxidant capacity in biological systems and, 6t TEAC assay, 7, 8 estimated percent contribution of, in plasma antioxidants, 9t t8-iso-PGF2alpha, 88 analytical method of, 89 Tertiary protein str ucture, def ning, thiols and, 243 Therapeutic improvement utility of 80HdG as biomark er of, 306–309 response of, to antio xidant interventions, 307–308 response of, to diet inter ventions, including carloric restrictions, 308–309 tHETE, 88 Thiadiazabicyclo-ONE-GSH adduct, 125 Thin layer chromatography with electrophoresis, purif cation of 8OHdG with, 297 Thioacetamide, hepatitis caused b y, 38–39 Thiobarbituric acid reactive substances, LPO products and assessment of, in body f uids or tissues, 320–321 Thiobarbituric acid-reactive substances assay, vii, 37 lipid peroxidation in plaques data and , 106 MDA measurement and, 119 tHODE levels in healthy and diseased subjects and, 92 two molecules of thiobarbituric acid reacting with one molecule of MD A giving pigmented and f uorescent compound, 120 use in in vitro and in vivo studies, 21 Thioethers, 125 Thiol-derived Michael adducts, for mation of, 160 Thiol-disulf de exchange, as potential mechanism of S-glutathionylation, 250, 251 Thiol group of cysteine residues in proteins, 138 Thiols, oxidative modif cations of, 244 Thiol-selective post-translational modif cations, S-glutathionylation and S-cysteinylation as, 249–250 Thiol-specif c oxidant diamide, Western blot analysis of membrane sk eletal proteins of human red blood cells exposed to, 254 13-(E,E)-HPODE, oxidation of linoleates and, 86, 87

T

TA. See Thioacetamide Tagging oxidized proteins, 148

361

Inde

x

13-hydroxy-9Z,11E-octadecadienoic acid, 164t 32 P-postlabeling methods, measurement and purif cation of 80HdG and, 297 13-(Z,E) -HPODE, oxidation of linoleates and, 86, 87 Thiyl radical mechanisms, S-glutathionylation and, 250–251, 251 Thomas, M. J., 122 Thompson, C. A., 299 Thornalley, P. J., 141t 3-aminotyrosine, trypsinizing, 208 3 beta-hydroxy-5-oxo-5,6-secocholestan-6-al, formation of, 101 3-hydroxy-3-imino-1,2-dihydropyrrole derivative, f uorescent properties of, 161 3-hydroxypropylmercapturic acid, as a main metabolite of acrolein in urine, 126 3-hydroxytyrosine, 200 3-nitrotyrosine (3-NT) alternative mechanism for for mation of, without participation of NO 2, 200 biological effects of, in proteins, 209 formation, foundation of, 199–200 fragmentation patterns of, 205, 206 limits of quantitation and basal plasma le vels of, 204t mapping in proteins, 205, 207, 207–208 quantif cation methods of, in biolo gical samples, 202, 203, 204–205 quantitative analysis and mapping of, 207 side products for med concomitant to for mation of, 200 Thrombin, 51 Thymine hydroperoxides, PHGPx and reduction of, 24 Time-of-f ight/time-of-f ight instruments, 175 Tissues dynamics of ubiquitin conjug ates in, 220 endogenous 80Hdg found in, 290–291 exposed to RCS: e xamples of in vitro studies, 187–189 human, levels of M 1dG adducts and ethenoDNA adducts in, 324 t ubiguitin conjugates in, accurately determining levels of, 226 TLC-blotting technique, PLOOH elimination with, 24 TLC purif cation step by column chromatography on silica gel, 121 TMS derivatives. See Trimethylsilyl derivatives Tobacco dust exposure, investigation of, 270 Tobacco smoke, comet assay and studying ef fects of, 272 TOF/TOF. See Time-of-f ight/time-of-f ight instruments Toluene, exposure to, evaluation, 306

Top-down approach, mass-spectrometr y and, in characterization of covalently modif ed proteins, 174, 175 Total antioxidant activity, ROS/RNS and, 12 Total antioxidant capacity, antioxidant intake and food selection based on, 12 Total carbonyl assays, vii Total HODE (tHODE) measurement, 88 analysis of t7-OHCh, tCh, and t18:2 b y GC-MS, 91 analytical method of, 89 assessment of antioxidant capacity in vivo by, 91–92 effects of free radicals on le ver levels of, 94 t GC-MS method for, 89–90 LC-MS/MS method for, 90–91 levels of, in animal e xperiments, 94 levels of, in health y subjects and diseased patients, 92–93 Total protein hydrolysis, quantif cation process of 3-NT and, 202 Total radical trapping antio xidant parameter, 5 Toyokuni, S., 164 t, 295 Transcription factors, S-glutathionylation and, 245 Transgenic mice, overexpression of PHGPx in, 27 Transmembrane peptides, amino acid, with tyrosine residues in dif ferent depths, 201 TRAP. See Total radical trapping antio xidant parameter TRAP assay, 7 determining antioxidant capacity in biolo gical systems and, 6t estimated percent contribution of, in plasma antioxidants, 9t Trialkylphosphines, 252 Triglyceride levels oxLDL and, in prediabetic patients, 52 Trimethylsilyl derivatives, aldehyde monitoring and, 123 Triple quadropole, 176 Trostchansky, A., 211t trp oxidation, major products of, 143 Trypsin-digested apolipoproteins, from nati ve LDL and/or modif ed LDL, on-membrane sample preparation procedure for anal ysis of, 188, 188 Tryptic digests, HPLC-ESI-MS/MS of, in k ey role of quantifying carbon yl-modif ed proteins, 191 Tryptophan, 143 Tryptophan residues oxidation of, 142–144 in proteins, 143 Tsikas, D., 204 Tubaro, F., 7 Tumorigenesis, studies showing relationship between inf ammation and, 320t

362 Inde

x

Tumor necrosis f actor-alpha, 51 decrease in SOD1 e xpression and, 23 Tumor necrosis f actor-alpha, IL-1, IL-4 and IL-6, SOD2 activated by, 23 Tunnel construction workers, study of, 270 12-hydroxyeicosatetraenoic acid, 164t 25-hydroxycholesterol, 101 27-hydroxycholesterol, atherosclerotic plaques and, 105 24-OH-cholesterol, 100 25-OH-cholesterol, 100 27-OH-cholesterol, 100 24S-hydroxycholesterol, neurodegenerative disorders and, 107 2-alkenals, 117 lipid peroxidation and, 159 lipid peroxidation-specif c epitopes and, 158 2-Cys Prx, role of, in protecting against DN A damage, oxidative stress, and cancer , 27 2D electrophoresis, study of S-glutathionylation and S-cysteinylation and, 255 Two-dimensional, sequential non-reducing/ reducing SDS-PAGE (diagonal electrophoresis), tagging of o xidized proteins and, 148 Two-dimensional gel electrophoresis, identifying nitrated proteins with, 208 2,4-DNP modif cation of carbonyls and immunodetection/pull-down of 2,4-DNPcontaining proteins, 148 2-hydroxyheptanal, 164t Tyr84, Cys34’s low pK 3 and, 230 Tyrosine, 200 fragmentation patterns of, 205, 206 Tyrosine analogs, as probes for follo wing nitration and oxidation reactions, 200–201 Tyrosine nitration biological occurrence of, routes for , 200 effect of, on selected proteins, 210–211 t Tyrosine residues oxidation of, 140, 142 oxidative dimerization of, in proteins, 141 Tyrosyl radical, oxidation of tyrosine and, 200

exposure to NCS, 221 exposure to paraquat, 221 results, 222–225 oxidative stress increases substrate availability and alters capability of ubiquitin conjugates, 223–224 proteasome as target of mild o xidative stress, 224–225 in response to o xidative stress, 222 technical details and cautions, 226 Ubiquitin-conjugating enzyme (E2), 219, 220 Ubiquitin-proteasome pathway, 219 cellular functions and impor tant roles of, 225 Ubiquitin protein ligases (E3), 219, 220 Uchida, K., 118, 160, 161, 164 t Ultra-f ne particles, testing effect of, 271 Ultra high perfor mance liquid chromatography with tandem mass spectrometric detection, quantifying protein damage and, 147 UPLC-MS/MS. See Ultra high perfor mance liquid chromatography with tandem mass spectrometric detection UPP. See Ubiquitin-proteasome pathway Urban air pollution, study of residents e xposed to, 272 Uremia, mercaptoalbumin depletion and, 237 Uric acid, hydrophilic assays, antioxidant capacity of plasma and, 9 Urine, exogenous HGB found in, 289–290 UV absorbance, 3-NT measurement/detection with, 203

U

Ubiquitin, ubiquitous expression of, in all eukaryotes, 219 Ubiquitin-activating enzyme (E1), 219, 220 Ubiquitination, def ned, 219 Ubiquitin conjugates, 219–226 dynamics of, in cells and tissues, 220 materials and methods, 221–222 de novo, 222 endogenous, detection of, 231–231 exposure to H 2O2, 221 exposure to lipofuscin-mediated photooxidation, 221

V

Vadseth, C., 210t Valavanidis, A., 283 Valtuena, S., 12 Vascular endothelial g rowth factor, 51 Vascular smooth muscle cells, 51 Vegetable and fr uit consumption cancer-protective effects of, 273 DNA stability and, 272–274 response of 80HdG to, 308 VEGF. See Vascular endothelial g rowth factor Vinyl chloride, genotoxicity of, for mation of etheno-DNA adducts and, 326 Virus-induced hepatitis, in human subjects, 39 Visioli, F., 54 Vitamin C, 3, 4, 8, 13, 274 lack of effect on F 2-IsoP levels, 75 response of 80HdG to, 307, 308 Vitamin E, 4, 8, 13, 88, 274 duration of lag time and , 56 gene interactions and, 12 hydrophilic assays, antioxidant capacity of plasma and, 9 individuals with Hp2-2 pol ymorphism and, 55

363

Inde

x

interaction of, in plasma, 10, 11 lack of effect on F 2-IsoP levels in moderate smokers, 75 LDL oxidation and, 57–58 oxysterols in atherosclerosis studies and , 108 response of 80HdG to, 307, 308 smoking, LDL and, 59 tHODE levels in animal e xperiments and, 94 Vitamin supplementation, contradictor y results in studies and trials and, 4 Vodicka, P., 269 Volatile organic compounds exposure to, evaluation, 306 study on exposure to, 272 VSMC. See Vascular smooth muscle cells

Western blots, nitrated proteins identif ed with, 208 WHEL study. See Women’s Healthy Eating and Living study White blood cells, as sur rogate tissue, ethenoDNA adducts and, 323–324 Whiteman, M., 298 White wine, LDL o xidation and, 58 Wilbur, R. L., 299 Williams, T. I., 122 Williamson, G., 24 Wolf, F. K., 309 Women’s Healthy Eating and Li ving study, 308 Wood-burning stoves, domestic, study on smok e from, 272 Wood dust, occupational e xposure to, 270

W

Wagner, S., 128 Warnke, M. M., 121 Water-soluble antioxidants, 3 signif cance of, to LDL o xidation, 57–59 carotenoids and LDL o xidation, 58 polyphenolics and LDL o xidation, 58–59 PUFA and LDL o xidation, 59 vitamin E and LDL o xidation, 57–58 Watson, A. D., 28 WBCs. See White blood cells Weight loss, decreased endo genous IsoP formation and, 75 Weimann, A., 289 Wells-Knecht, M. C., 141 t, 142t Western blot analysis, of membrane sk eletal proteins of human red b lood cells exposed to thiol-specif c oxidant diamide, 254 Western blot immunoassay, identif cation and characterization of carbonylated proteins in biological matrices and use of, 182 Western blot methods of protein homo genates, quantif cation of 3-NT in biolo gical samples and, 202

X

XPA, repair of o xidized bases and, 276 XRCC1 gene, 326 inf uence of genetic pol ymorphisms on, 275, 276 XRCC3 gene, inf uence of genetic pol ymorphisms on, 275, 276 Xu, G., 161 Xu, S., 210t Xylene, exposure to, evaluation, 306

Y

Yamada, T., 164t Yáñez, L. Yang, C. Y., 183

Z

Zhao, X. H., 271 Zhu, C. Q., 270 Zou, M., 210 t Zucker rats, obese, precursor ion scanning technique to detect free, protein-bound histidine residues modif ed by RCS in urine from, 187

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