iSelenium in Nutrition and HealthUntitled-1 1/8/2007, 6:38 PM 1iiUntitled-1 1/8/2007, 6:38 PM 2iiiSELENIUM IN NUTRITION ANDHEALTHPeter F. SuraiUntitled-1 1/8/2007, 6:38 PM 3ivNottingham University PressManor Farm, Main Street, ThrumptonNottingham NG11 0AX, United Kingdomwww.nup.comNOTTINGHAMFirst published 2006 PF SuraiAll rights reserved.No part of this publicationmay be reproduced in any material form(including photocopying or storing in anymedium by electronic means and whether or nottransiently or incidentally to some other use ofthis publication) without the written permissionof the copyright holder except in accordance withthe provisions of the Copyright, Designs andPatents Act 1988.Applications for the copyrightholders written permission to reproduce any partof this publication should be addressed to the publishers.British Library Cataloguing in Publication DataSelenium in Nutrition and HealthPF SuraiISBN 1-904761-16-XDisclaimerEvery reasonable effort has been made to ensure that the material in this book is true, correct, completeandappropriateatthetimeofwriting.Nevertheless,thepublishersandauthorsdonotacceptresponsibility for any omission or error, or for any injury, damage, loss or financial consequences arisingfrom the use of the book.Typeset by Nottingham University Press, NottinghamPrinted and bound by Cromwell Press, Trowbridge, WiltshireUntitled-1 1/8/2007, 6:38 PM 4vCONTENTSPreface xix1. Antioxidant systems in animal bodyIntroduction 1Free radicals and reactive oxygen and nitrogen species 1Three levels of antioxidant defence 7Antioxidant-prooxidant balance in the body and oxidative stress 25Conclusions 32References 342. Molecular mechanisms of Se action: SelenoproteinsIntroduction 45Selenoprotein family 45Glutathione peroxidase 51Cytosolic glutathione peroxidase (cGSH-Px or GSH-Px1) 51Negative effectors of GSH-Px activity 56Upregulation of GSH-Px 57Gender-specific expression of GSH-Px 58Tissue-specific expression of GSH-Px 58Response to dietary selenium 59Alterations of GSH-Px expression 60GSH-Px regulation and proteasomes 66Phospholipid glutathione peroxidase (GSH-Px4, PH-GSH-Px) 67PH-GSH-Px overexpression 69Plasma glutathione peroxidase (pGSH-Px, GSH-Px3) 73Gastrointestinal glutathione peroxidase(GI-GSH-Px, GSH-Px2) 74Specific sperm nuclei glutathione peroxidase (sn-GSH-Px) 74GSH-Px6 76Glutathione peroxidases and their biological roles 76Non-Se glutathione peroxidases 79Thioredoxin reductase 82Iodothyronine deiodinases (ID) 89Other selenoproteins 92Selenoprotein W 92Selenoprotein P 10 95Selenoprotein P12 96Selenophosphate synthetase-2 9715-kDa selenoprotein 9718-kDa selenoprotein 97Untitled-1 1/8/2007, 6:38 PM 5viContentsMitochondrial capsular selenoprotein 98Selenoprotein N 99Selenoprotein R 100Selenoprotein S 100Selenoprotein U 101Selenoprotein T and selenoprotein X 101Selenium-vitamin E interactions: antioxidant properties and beyond 103Prooxidant properties of selenite and possible antioxidant protection 109by selenomethionineReferences 1163. Selenium in food and feed: selenomethionine and beyondIntroduction 151Selenium in soils and plants 151Plants as major sources of selenium for animals and human 156Selenium absorption and metabolism 161Selenium status and bioavailability 171Effectors of selenium absorption, metabolism and bioavailability 181Selenium and feeding behaviour 184Selenium sources for animal and human consumption 184Supplemental sources of selenium 191References 1984. Selenium and immunityIntroduction 213Immune system and its evaluation 214Phagocyte functions 224Antibody production 228Lymphocyte functions 233In vitro effects of Se on immune cells 240Disease resistance 243Seleniumdeficiency and viruses 251Mechanisms of immunomodulating properties of selenium 252Immunocommunication, free radicals and selenium 257Conclusions 260References 2625. Selenium and semen qualityIntroduction 279Fatty acid composition of avian and mammalian semen 279Lipid peroxidation in semen 281Untitled-1 1/8/2007, 6:38 PM 6viiSelenoproteins and their role in antioxidant protection in semen 286Conclusions 304References 3056. Selenium and mycotoxinsIntroduction 317Mycotoxins of concern 318Increased lipid peroxidation as a consequence of mycotoxicosis 324Mycotoxins and apoptosis 326Mycotoxins and immune system 330Protective effect of selenium against mycotoxicosis 342Conclusions 347References 3477. Selenium in poultry nutritionIntroduction 363Selenium deficiency 364Selenium and chicken embryo development 374Effect of organic selenium in the maternal diet on antioxidant 385defences of the developing chicksSelenium and digestive and immune system development in birds 397Selenium requirement for breeders 400Selenium requirement for broilers 405Se and meat quality 411Se and egg quality 415Practical approaches to improve selenium status of poultry 418Conclusions 422References 4248. Selenium in pig nutritionIntroduction 445Se-deficiency 446Se requirement 454Selenium applications in pig production 459Maternal effect on the progeny: organic selenium vs selenite 459Selenium nutrition of pigs at weaning period 469Selenium in grower-finisher swine and meat quality 473Selenium-vitamin E combinations 474Selenium and iron injection 475Conclusions 476References 479ContentsUntitled-1 1/8/2007, 6:38 PM 7viii9. Selenium in ruminant nutritionIntroduction 487Selenium deficiency in ruminants 488Selenium deficiency in dairy and beef industries 518Important features of Se metabolism in ruminants 521Meeting selenium requirements 528Methods for Se status assessment 531Routes of selenium supplementation 537Practical applications of Se nutrition of ruminants 538Selenized yeast vs selenite 546Conclusions 559References 56110. Selenium in nutrition of other animals: horses, dogs, cats and fishSelenium in horse nutrition 589Introduction 589Se deficiency 589Se excess 590Se requirement 592Selenium and GSH-Px 592Se metabolism and effective sources 593Antioxidant defences and exercise 595Se and maternal effect 597Se and immunity 599Conclusions 599Se for companion animals 601Introduction 601Antioxidants and animal health 601Food quality 602Anorexia in companion animals and its connection to antioxidants 603Antioxidant deficiencies in companion animals 604Organic selenium vs sodium selenite 605Selenium requirement 607Taurine for cats: antioxidant properties and beyond 608Antioxidants and immune system 612Conclusions 615Selenium in fish nutritionIntroduction 616ContentsUntitled-1 1/8/2007, 6:38 PM 8ixSe deficiency 616Se toxicity 618Se requirement 620Health and immunity 621Selenium and fish quality 622Effective forms of Se in fish diets 624Se-fish as human food 626Conclusions 627References 62811. Selenium and human healthIntroduction 643Nutrition and health 643Are there ideal diets? 644Omega-3 PUFAs 651Selenium: global perspective 654Se distribution and reserves in human body 657Selenium deficiency 658Selenium requirement 662Se status assessment 664Selenium and health 665Cancer 666Selenium and cancer 671Epidemiological observations and prospective studies 672Case-control studies of Se levels in tissues of cancer patients 682Laboratory animal studies of anticancer effect of Se 687Human intervention trials with selenium 694Selenium and cancer treatment 705Molecular mechanisms of anticancer effects of selenium 708Selenium and cardiovascular diseases 720Selenium and other diseases 729Reproductive disorders 729Selenium and asthma 731Selenium and rheumatoid arthritis 732Selenium and diabetes 733Selenium and HIV 735Selenium and pancreatitis 736Selenium, brain function, mood and neurodegenerative diseases 738Selenium and radiation 741Selenium and ageing 746Adverse health effects of excess selenium in humans 751ContentsUntitled-1 1/8/2007, 6:38 PM 9xConclusions 754References 75512.Se-enriched eggs, milk and meat as functional foods and a solution toselenium deficiencyIntroduction 809Different strategies to address Se deficiency in human 810Selenium-enriched products 814Improving the image of the egg 817Selenium-enriched eggs as a route toward improving human 821selenium statusSe-enriched meat and milk 827Se-enriched eggs, meat and milk as functional food 837Conclusions: back to nature? 842References 84413.Antioxidant-prooxidant balance in the intestine: from healthy gut togeneral healthIntroduction 857The gastrointestinal tract (GIT) as a major site of antioxidant action 857Prooxidants in the GIT 859Peroxidized PUFAs 859Oxysterols 861Iron ions 862Nitrites and nitrates 864Heavy metals 865Persistent organic pollutants 867Mycotoxins 868Alcohol 870Immune system 871Combinations of prooxidants in the GIT and their detrimental effects 873Antioxidant defences in the GIT 874Vitamin E 874Coenzyme Q 876Carotenoids 880Vitamin A 882Ascorbic acid (AA) 882Glutathione 883Flavonoids 883Other poly(phenolics) 885Spices and essential oils 885ContentsUntitled-1 1/8/2007, 6:38 PM 10xiSelenomethionine 886Synthetic antioxidants 886Specific place for Se-dependent enzymes in antioxidant defence of GIT 886Other antioxidant mechanisms 887What should be changed to have a healthy diet? 892Antioxidant-prooxidant balance in the intestine and animal health 895Conclusions 896References 89714. Conclusions: Looking ahead 923References 949Index 955ContentsUntitled-1 1/8/2007, 6:38 PM 11xiiTo my wife Helen, my daughter Katie and my son Antonwho gave me an inspiration for writing this bookUntitled-1 1/8/2007, 6:38 PM 12xiiiABBREVIATIONSAA Ascorbic acidAb AntibodyACS Acute coronary syndromesAD Alzheimers diseaseAFB1 Aflatoxin B1ALA Alpha-linolenic acidApo-AI Apoprotein AIApoB Apoprotein BApoII Apoprotein IIAR Androgen receptorBDI scores Beck Depression InventoryscoresBHT Butylated hydroxytolueneBHA Butylated hydroxyanisoleCAT CatalaseCE Cholesteryl estersCH CholesterolCHD Coronary heart diseaseCIND Cognitive impairment non-dementiaCoQ Coenzyme QCon A Concanavalin ACPK Creatine phosphokinaseCRP C-reactive proteinCSF Cerebrospinal fluidCVD Cardiovascular diseasesDAA Dehydroascorbic acidDAT Dementia of the Alzheimer typeDHA Docosahexaenoic acidDM Dry matterDMBA 7,12-Dimethylbenz[a]anthraceneDON DeoxynivalenolDPA Docosapentaenoic acidDTA Docosatetraenoic acidDTH Delayed-type hypersensitivityECs Endothelial cellsED Exudative diathesisEPA Eicosapentaenoic acidFB1 Fumonisin B1FCR Feed conversion ratioUntitled-1 1/8/2007, 6:38 PM 13xivFFA Free fatty acidsGC-MS Gas Chromatography with Mass SpectrometryGI-GSH-Px Gastrointestinal glutathione peroxidaseGIT Gastrointestinal tractGOT Glutamic oxalacetic transaminaseGR Glutathione reductaseGSH Reduced glutathioneGSSG Oxidised glutathioneGSH-Px Glutathione peroxidaseGST Glutathione S-transferaseHA HemagglutinationHb HemoglobinHCC Hepatocellular carcinoma5-HETE 5-hydroxyeicosatetraenoic acid15-HPET 15-hydroperoxy analogueHI Hemagglutination inhibitionHDL High density lipoproteinHNE 4-hydroxynonenalHO-1 Heme oxygenaseHPLC High Performance LiquidChromatographyHSP Heat shock proteinsID Iodothyronine deiodinaseIFN InterferonIL-1 Interleukin 1IL-6 Interleukin 6IL-2R Interleukin 2 receptorIMI Intramammary infectionKD Keshans diseaseLA Linoleic acidLE LymphedemaLNA Alpha-linolenic acidLDL Low density lipoproteinLDH Lactate dehydrogenase5- LO 5-LipoxigenaseLOOH Lipid hydroperoxidesLP Lipid peroxidationLPL Lipoprotein lipaseLPO Lipid peroxidationLPS LipopolysaccharideLTA Lymphocyte transformation assayLTB Leukotriene BAbbreviationsUntitled-1 1/8/2007, 6:38 PM 14xvMAP MicroangiopathyMCS Mitochondrial capsular selenoproteinMDA MalondialdehydeMD Mareks diseaseMet MethionineMODA Milan overall dementia assessmentNDV Newcastle disease virusMIF Migration inhibitory factorMIP-2 Macrophage inflammatory protein 2MCP Mitochondrial capsule proteinMSA Methylseleninic acidMsrA and MsrB Methionine sulfoxide reductasesNE Nutritional encephalomalaciaNK cells Natural killer cellsNMD Nutritional muscular distrophyNPO 2'-(4-nitrophenoxy)oxiraneNRC National Research CouncilNRP Nutrient Requirements of PoultryOA Ochratoxin AOPN OsteopontinOVA OvalbuminPBMC Peripheral blood mononuclear cellsPC PhosphatidylcholinePCA Prostate cancerPFC test Plaque-forming cell testPGE2Prostaglandin E2pGSH-Px Plasma glutathione peroxidasePAM Prematurely aging micePL PhospholipidPLC Primary liver cancerPMN Polymorphonuclea cells, neutrophilsPG ProstaglandinPHA PhytohemagglutinPH-GSH-Px Phospholipid glutathione peroxidasePIH Pregnancy induced hypertensionPLA2 Phospholipase A2POPs Persistent organic pollutantsPRECISE Prevention of Cancer by Intervention with SePSA Prostate-specific antigenPUFA Polyunsaturated fatty acidPWM Pokeweed mitogenAbbreviationsUntitled-1 1/8/2007, 6:38 PM 15xviRA Rheumatoid arthritisRBC Red blood cellsRDA Recommended Dietary AllowancesROS Reactive oxygen speciesRNS Reactive nitrogen speciesRP Retained placentaRR Relative riskRSV Respiratory syncytial virusSCC Somatic cell countSCCs Ssquamous carcinoma cellsSDG SelenodiglutathioneSeCys SelenocysteineSel-Plex Selenium enriched yeastSECIS Selenocysteine insertion sequenceSELECT The Selenium and Vitamin E Cancer Prevention TrialSeN Selenoprotein NSeMet SelenomethionineSeP Selenoprotein PSeR Selenoprotein RSeS Selenoprotein SSeW Selenoprotein WSeRU Se-responsive unthriftinessSFA Saturated fatty acidssn-GSH-Px Sperm nuclei glutathione peroxidaseSOD Superoxide dismutaseSPS Selenophosphate synthetase-2SRBC Sheep red blood cellsSST Sperm storage tubulesSptrx-1 Sperm-specific thioredoxinSTZ StreptozotocinT3 TriiodthyronineT4 ThyroxineTBA Thiobarbituric acidTBARS Thiobarbituric acid reactive substancesTh T helperTG TriacylglycerolTNF-a Tumour necrosis factor aTPA 12-O-tetradecanoylphorbol-13-acetateTrx ThioredoxinTPx Thioredoxin peroxidaseTR Thioredoxin reductaseAbbreviationsUntitled-1 1/8/2007, 6:38 PM 16xviiTRAIL Tumor necrosis factor-related apoptosis-inducing ligandTxl-2 Thioredoxin-like protein 2VEGF Vascular endothelial growth factorVLDLVery low density lipoproteinvMDV Virulent Mareks disease virusWMD White muscle diseasep-XSC Xylene selenocyanate(1,4-phenylenebis(methylene)selenocyanate)YSM Yolk Sac MembraneZea ZearalenoneAbbreviationsUntitled-1 1/8/2007, 6:38 PM 17xviiiUntitled-1 1/8/2007, 6:38 PM 18xixPREFACEAmong many minerals selenium has a special place being the most controversial traceelement. Indeed a narrow gap between essentiality and toxicity and environmental issuesfrom the one hand and global selenium deficiency from the other hand, fuel research inthis field. There were several breakthroughs in selenium research. The first one was adiscovery of Se essentiality in early 1960th. The second one was a discovery in 1973that glutathione peroxidase is a selenoprotein. The third one came almost 30 years laterwith characterisation of main selenoproteins in human and animal body and furtherunderstanding the role of selenium in nutrition and health. Indeed, this third breakthroughisreallyaseleniumrevolutioncreatingmanyhypotheses,stimulatingnewresearchand providing practical applications in medicine and agriculture. New insight in therole of free radicals as signalling molecules, understanding the role of nutrients in geneexpressionandmaternalprogramming,tremendousprogressinhumanandanimalgenome work created new demands for further research related to biological roles ofselenium. For the last few years several comprehensive monographs and reviews havebeen published addressing various Se-related issues. However, most of them were dealingwith Se roles in human health. However, animal food-producing industry is developingvery quickly and a great body of information was accumulated indicating importanceofSeinmaintenanceofanimalhealth,productiveandreproductiveperformance.Unfortunately up to now there was no comprehensive review combining all major aspectsof selenium in relating to animal and human health. Indeed a chain: Se in soil - seleniumin plants-selenium in animals- selenium in human was not well characterised.Therefore the goal of this volume is to provide up to date information about theroles of Se in nutrition and health of human and farm animals, including poultry, pigs,ruminants, horses, cats, dogs and fish.In the first chapter a special emphasis is given totheroleofseleniumasanessentialpartoftheintegratedantioxidantsystemofthebodywithregulatoryfunctionsprovidingnecessaryconnectionsbetweendifferentantioxidants. In fact selenium is called the chief executive of the antioxidant defence.The second chapter is addressing molecular mechanisms of Se action describing majorfunctionsoftheselenoproteins.Indeed,thefamilyofselenoproteinsincludes25members and functions of many of them are still not well understood.Selenium infoodandfeedisdescribedinthethirdchapter.Themainideaofthischapteristocharacterise main Se sources in relation to new knowledge about Se speciation in variousfood and feed ingredients. It seems likely, that in grains and some other important foodUntitled-1 1/8/2007, 6:38 PM 19xxingredients selenomethionine is the main Se form. The idea was put forward that duringevolution the digestive system of human and animals was adapted to natural form ofselenium consisting of SeMet and other organic selenocompounds. Therefore this formof Se is more efficiently assimilated in the body than inorganic forms of selenium. Infact SeMet is considered to be the storage form of selenium in the body. Accumulationof the Se reserves in the body as a result of organic selenium consumption is consideredas an adaptive mechanism providing additional antioxidant defences in stress conditions.Thefourthchapterisdevotedtotheroleofseleniuminimmunity.Itisdifficulttooverestimated immunomodulating properties of selenium and increased resistance tovarious diseases of human and animals is a result of optimal Se status. The possibilityof virus mutation in the body of animals or human deficient in selenium is of greatimportance for understanding mechanisms of spreading such diseases as AIDS, chickeninfluenza, etc. New findings in the field of male reproduction in relation to Se status aredescribed in the chapter 5. From the data presented it is clear that Se has special importantrolesinmaintaininganimalreproduction.Itiswellknownthatantioxidantsplayimportantrolesinpreventingvariousstresses.Mycotoxinsareconsideredtobethebiggest feed-related stress in farm animals. Therefore, possible protective role of Se inmycotoxicosis is described in the chapter 6. Next four chapters are devoted to practicalapplications of Se in animal nutrition. The data presented indicated importance of Se ingrowth, development and reproduction of poultry, pigs, ruminants, horses, cats, dogsandfish.Themainideaofthechaptersistoshowpossibilitiesofimprovementofproductive and reproductive performance of animals and animal produce quality byoptimal Se supplementation. Indeed, organic selenium in the form of selenized yeast isproven to be the most effective form of Se supplementation for poultry, farm animals,companion animals and fish.Role of selenium in human health is described in thechapter11.SpecificemphasisisgiventoanticancerpropertiesofSewithdetailedanalysis of possible molecular mechanisms of such Se action. Beneficial role of Se inotherdiseases,includingcardio-vasculardiseases,diabetes,arthritis,pancreatitis,reproductive disorders as well as protective role of selenium against radiation are alsodescribed. Thelinkbetweenanimalindustryandhumanhealthisdescribedinthechapter 12 devoted to production of Se-enriched eggs, meat and milk. Indeed, productionof a range of Se-enriched products is considered as an important solution for global Sedeficiency.Se-enrichedeggsarealreadyonsupermarketshelvesinmorethan25countries worldwide with millions such eggs sold daily. The technologies of producingSe-meat and milk are also developed and await practical applications. An importantissue of antioxidant-prooxidant balance in the digestive tract is considered in the chapter13. It seems likely that this balance has been overlooked by scientists. However, fromthe one hand this balance could explain a beneficial effect of fruit and vegetables inhuman diet. On the other hand, this balance is a key for general health. In the conclusivechapter of the book the general trends for future research in the field of the Se biologyare shown. Information provided in this book will be of practical importance to medicalPrefaceUntitled-1 1/8/2007, 6:38 PM 20xxiand pharmaceutical industry professionals, dieticians, egg, meat and milk producers,animalscientists,feedformulatorsaswellasforstudentsofbiological,medical,veterinary and other related sciences at universities and colleges.Muchofthematerialinthisbookisbasedonideasandconceptsthatinmanyinstances have been developed from work carried out in the Department of BiochemistryandNutritionandfurtherinthe AvianScienceResearchCentreattheScottishAgricultural College. I am, therefore, grateful to the SAC for providing an environmentto develop these ideas, and to the Scottish Executive Environment and Rural AffairsDepartment for its generous support of this research.I understand that my views onthe role of selenium in nutrition and health are sometime different from those of otherscientists and therefore I would appreciate very much receiving any comments fromreaders which will help me in my future research.I would like to thank my colleagues with whom I have had a pleasure to collaborateandsharemyideasrelatedtonaturalantioxidantsandseleniuminparticular,whohelpedmeatvariousstagesofthisresearchbyprovidingreprintsoftheirrecentpublications. I am also indebted to the Alltech Inc. for providing organic selenium forexperimentsandsomeimportantinformationandideasandtotheWorldsPoultryScience Association for the Research Award.P.F. SuraiPrefaceUntitled-1 1/8/2007, 6:38 PM 21xxiiUntitled-1 1/8/2007, 6:38 PM 22AntioxidantSystemsinAnimalBody111ANTIOXIDANTSYSTEMSINANIMALBODYSelf-preservationisthefirstlawofnatureIntroductionFor the majority of organisms on Earth, life without oxygen is impossible. Animals,plantsandmanymicroorganismsrelyonoxygenfortheefficientproductionofenergy.However,thehighoxygenconcentrationintheatmosphereispotentiallytoxicforlivingorganisms.Itisinterestingthatoxygentoxicitywasfirstdescribedinlaboratoryanimalsin1878(seeKnight,1998).Forthelastfewyearsfreeradicalresearchhasgeneratedvaluableinformationforfurtherunderstandingnotonlydetrimental,butalsobeneficialroleoffreeradicalsincellsignalingandotherphysiologicalprocesses.Thebenefitorharmoffreeradicalsultimatelydependontheleveloftheirproductionandefficiencyofantioxidantdefence.Free radicals and reactive oxygen and nitrogen speciesFreeradicalsareatomsormoleculescontainingoneormoreunpairedelectrons.FreeradicalsarehighlyunstableandreactiveandarecapableofdamagingbiologicallyrelevantmoleculessuchasDNA,proteins,lipidsorcarbohydrates.Theanimalbodyisunderconstantattackfromfreeradicals,formedasanaturalconsequenceofthebodysnormalmetabolicactivityandaspartoftheimmunesystemsstrategyfordestroyinginvadingmicroorganisms.TheinternalandexternalsourcesoffreeradicalsareshowninTable1.1.Recentlycollectivetermsreactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS)havebeenintroduced(HalliwellandGutteridge,1999)includingnotonlytheoxygenornitrogenradicals,butalsosomenon-radicalreactivederivativesofoxygenandnitrogen(Table1.2).Didyouknowthatfreeradicalsareproducedasaresultofphysiologicalmetabolisminthebody?Untitled-2 1/8/2007, 6:44 PM 12SeleniuminNutritionandHealthTable 1.1 Internal and external sources of free radicals (Adapted from Furst, 1996 and Halliwell, 1996).Internallygenerated External sourcesMitochondria (ETC) Cigarette smokePhagocytes RadiationXanthineoxidase UV lightReactions with Fe2+ or Cu+PollutionArachidonatepathways Certain drugsPeroxisomes Chemical reagentsInflammation IndustrialsolventsBiomoleculeoxidation(adrenaline,dopamine, tetrahydrofolates, ect.)Table 1.2Reactive oxygen and nitrogen species (Adapted from Halliwell and Gutteridge, 1999).Radicals Non-radicalsAlkoxyl, RO* Hydrogen peroxide, H2O2Hydroperoxyl, HOO* Hypochlorous acid, HOClHydroxyl, *OH Ozone, O3Peroxyl, ROO* Singlet oxygen,1O2Superoxide, O2* Peroxynitrite, ONOO-Nitric oxide, NO* Nitroxyl anion, NO-Nitrogen dioxide, NO2* Nitrous acid, HNO2Superoxide(O2*-)isthemainfreeradicalproducedinbiologicalsystemsduringnormalrespirationinmitochondriaandbyautoxidationreactionswithhalf-lifeat37Cintherangeof1x10-6 second.Superoxidecaninactivatesomeenzymesdue to formation of unstable complexes with transition metals of enzyme prostheticgroups,followedbyoxidativeself-destructionoftheactivesite(ChaudiereandFerrari-Iliou,1999).Dependingoncondition,superoxidecanactasoxidizingorareducingagent.Itisnecessarytomentionthatsuperoxide,byitself,isnotextremely dangerous and does not rapidly cross lipid membrane bilayer (Kruidenierand Verspaget,2002).However,superoxideisaprecursorofother,morepowerfulROS.Forexampleitreactswithnitricoxidewithaformationofperoxynitrite(ONOO-),astrongoxidant,whichleadtoformationofreactiveintermediatesduetospontaneousdecomposition(Kontos,2001;Mruketal.,2002).InfactONOO-wasshowntodamageawidevarietyofbiomolecules,includingproteins(viaUntitled-2 1/8/2007, 6:44 PM 2AntioxidantSystemsinAnimalBody3nitrationoftyrosineortriptophanresiduesoroxidationofmethionineorselenocysteineresidues),DNAandlipids(Groives,1999).Superoxidecanalsoparticipateintheproductionofmorepowerfulradicalsbydonatinganelectron,andtherebyreducingFe3+andCu2+toFe2+andCu+,asfollows:O2-+Fe3+/Cu2+Fe2+/Cu+ +O2FurtherreactionsofFe2+andCu+withH2O2areasourceofthehydroxylradical(*OH)intheFentonreaction:H2O2+Fe2+/Cu+*OH+OH-+Fe3+/Cu2+ThesumofreactionofsuperoxideradicalwithtransitionmetalsandtransitionmetalswithhydrogenperoxideisknownastheHaber-Weissreaction.Itisnecessarytounderlinethatsuperoxideradicalisadouble-edgedsword.Itisbeneficialwhenproducedbyactivatedpolymorphonuclealeukocytesandotherphagocytesasanessentialcomponentoftheirbactericidalactivitiesbutinexcessitmayresultintissuedamageassociatedwithinflammation.Didyouknowthatsuperoxideradicalisthemajorradicalproducedinbiologicalsystems?Hydroxylradicalisthemostreactivespecieswithanestimatedhalf-lifeofonlyabout10-9second.Itcandamageanybiologicalmoleculeittouches,however,itsdiffusioncapabilityisrestrictedtoonlyabouttwomoleculardiametersbeforereacting(Yu,1994).Therefore,inmostcasesdamagingeffectofhydroxylradicalis restricted to the site of its formation. In general, hydroxyl radical can be generatedin human/animal body as a result of radiation exposure from natural sources (radongas,cosmicradiation)andfromman-madesources(electromagneticradiationand radionuclide contamination). In fact in many cases hydroxyl radical is a triggerofchainreactioninlipidperoxidation.Didyouknowthathydroxylradicalisshort-livedbutmostpowerfulradicalinbiologicalsystems?Therefore,ROS/RNS(Table1.3)areconstantlyproducedinvivointhecourseofthephysiologicalmetabolismintissues.Itisgenerallyacceptedthattheelectron-Untitled-2 1/8/2007, 6:44 PM 34SeleniuminNutritionandHealthTable 1.3 Effect of free radicals on DNA damages (Adapted from Diplock, 1998).Radical EffectROO* GuanineoxidizedOH* All four bases are affectedO2*-, H2O2No base changesONOO-Xanthine, hypoxanthine,8-nitroguanine are affectedtransportchaininthemitochondriaisresponsibleformajorpartofsuperoxideproductioninthebody(HalliwellandGutteridge,1999).Mitochondrialelectrontransportsystemconsumesmorethan85%ofalloxygenusedbythecelland,becausetheefficiencyofelectrontransportisnot100%,about1-3%ofelectronsescapefromthechainandtheunivalentreductionofmolecularoxygenresultsinsuperoxideanionformation(Halliwell,1994;Singaletal.,1998;Chowetal.,1999). About1012O2moleculesprocessedbyeachratcelldailyandiftheleakageofpartiallyreducedoxygenmoleculesisabout2%,thiswillyieldabout2x1010molecules of ROS per cell per day (Chance et al., 1979). An interesting calculationhasbeenmadebyHalliwell(1994),showingthatinthehumanbodyabout1.72kg/yearofsuperoxideradicalisproduced.Instressconditionitwouldbesubstantiallyincreased.Clearly,thesecalculationsshowedthatfreeradicalproductioninthebodyissubstantialandmanythousandbiologicalmoleculescanbeeasilydamagedifarenotprotected.RecentlytheroleofmitochondriaasapermanentsourceofROShasbeenquestioned(StaniekandNohl,2000).Theactivationofmacrophagesinstressconditionsisanotherimportantsourceoffreeradicalgeneration.ImmunecellsproduceROS/RNSandusethemasanimportantweapontodestroypathogens(Schwarz,1996;KettleandWinterbourn,1997;SeeChapter6).Didyouknowthatabout2x1010moleculesofROSareproducedpercellperday?Themostimportanteffectoffreeradicalsonthecellularmetabolismisduetotheirparticipationinlipidperoxidationreactions.Thefirststepofthisprocessiscalled the initiation phase, during which carbon-centered free radicals are producedfromaprecursormolecule,forexampleapolyunsaturatedfattyacid(PUFA):

InitiatorLHL*Untitled-2 1/8/2007, 6:44 PM 4AntioxidantSystemsinAnimalBody5Theinitiatorinthisreactioncouldbythehydroxylradical,radiationorsomeothereventsorcompounds.Inpresenceofoxygentheseradicals(L*)reactwithoxygenproducingperoxylradicalsstartingthenextstageoflipidperoxidationcalledthepropagationphase:L* +O2LOO*Atthisstagearelativelyunreactivecarbon-centeredradical(L*)isconvertedtoahighlyreactiveperoxylradical. Aresultedperoxylradicalcanattackanyavailableperoxidazablematerialproducinghydroperoxide(LOOH)andnewcarbon-centeredradical(L*):LOO* + LHLOOH + L*Thereforelipidperoxidationisachainreactionandpotentiallylargenumberofcyclesofperoxidationcouldcausesubstantialdamagetocells.Inmembranestheperoxidazable material is represented by PUFAs. It is generally accepted that PUFAsusceptibilitytoperoxidationisproportionaltoamountdoubleboundsinthemolecules.Infact,docosahexaenoicacid(DHA,22:6n-3)andarachidonicacid(AA,20:4n-6)areamongmajorsubstratesoftheperoxidationinthemembrane.ItisnecessarytounderlinethatthesamePUFAsareresponsibleformaintenanceofphysiologicallyimportantmembranepropertiesincludingfluidityandpermeability.Thereforeasaresultoflipidperoxidationwithinthebiologicalmembranestheirstructureandfunctionsarecompromised.ProteinsandDNAarealsoimportanttargetsforROS.Didyouknowthatinthehumanbodyabout1.72kg/yearofsuperoxideradicalisproduced?IthasbeenshownthattheDNAineachcellofaratishitbyabout100,000freeradicalsadayandeachcellsustainsasmanyas10,000potentiallymutagenic(ifnotrepaired)lesionsperdayarisingfromendogenoussourcesofDNAdamage(AmesandGold,1997;Helbocketal.,1998;Ames,2003;Diplock,1994).Therefore,someoxidativelesionsescaperepairandthesteadystatelevelofoxidativelesionsincreasedwithage,andanoldrathasaccumulatedabout66,000oxidativeDNAlesionspercell(Ames,2003).Oxidation,methylation,deaminationanddepurinationarefourendogenousprocessesleadingtosignificantDNAdamagewithoxidationtobemostsignificantoneandapproximately20typesofUntitled-2 1/8/2007, 6:44 PM 56SeleniuminNutritionandHealthoxidativelyalteredDNAmoleculeshavebeenidentified.ThechemistryofattackbyROSonDNAisverycomplexandlesionsinchromatinincludedamagetobases,sugarlesions,singlestrand-breaks,basiclesionsandDNA-nucleoproteincross-links(Diplock,1994).Didyouknowthatanoldrathasaccumulatedabout66,000oxidativeDNAlesionspercell?Thecomplexstructureofproteinsandavarietyofoxidizablefunctionalgroupsoftheaminoacidsmakethemsusceptibletooxidativedamage.Infact,theaccumulationofoxidizedproteinshasbeenimplicatedintheagingprocessandinotherage-relatedpathologies.Arangeofoxidizedproteinsandaminoacidshasbeencharacterisedinbiologicalsystems(Table1.4;Kehrer,2000;Deanetal.,1997).Table 1.4Oxidized protein and amino acids found in biologic systems (Adapted from Kehrer, 2000;Dean et al., 1997).2-Oxohistidine Hydro(pero)xyleucine3-chlorotyrosine Hydro(pero)xyvaline3-Nitrotyrosine N-Formylkynureinine5-Hydroxy-2-aminovalericacid KynurenineAminomalonicacid o- and m-tyrosineDimers of hydroxylated aromaticamino acids p-HydroxyphenylacetaldehydeDopa ProteincarbonylsIngeneraltheaccumulationofoxidizedproteinsdependsonthebalancebetweenantioxidants,prooxidantsandremoval/repairmechanisms.Oxidationofproteinsleads to the formation of reversible disulfide bridges. More severe protein oxidationcausesaformationofchemicallymodifiedderivativese.g.shiffsbase(TiroshandReznick,2000).Nitricoxide,hydroxylradical,alkoxylandperoxylradicalsaswellascarbon-centeredradicals,hydrogenperoxide,aldehydesorotherproductsoflipidperoxidationcanattackproteinmolecules.Usuallyoxidativemodificationofproteinsoccursbytwodifferentmechanisms:asite-specificformationofROSviaredox-activetransitionmetalsandnon-metal-dependentROS-inducedoxidationofaminoacids(TiroshandReznick,2000).Themodificationofaproteinoccursbyeitheradirectoxidationofaspecificaminoacidintheproteinmoleculeorcleavageoftheproteinbackbone.InbothcasesUntitled-2 1/8/2007, 6:44 PM 6AntioxidantSystemsinAnimalBody7biologicalactivityofthemodifiedproteinswouldbecompromised.Thedegreeofproteindamagedependsonmanydifferentfactors(Gruneetal.,1997): thenatureandrelativelocationoftheoxidantorfreeradicalsource; natureandstructureofprotein; theproximityofROStoproteintarget; thenatureandconcentrationsofavailableantioxidants.Free radicals are implicated in the initiation or progression phase of various diseases,includingcardiovasculardisease,someformsofcancer,cataracts,age-relatedmaculardegeneration,rheumatoidarthritisandavarietyofneuro-degenerativediseases(Hogg,1998;McCord,2000;Table1.5).Ingeneral,itiswidelybelievedthatmosthumandiseasesatdifferentstagesoftheirdevelopmentareassociatedwithfreeradicalproductionandmetabolism.Normally,thereisadelicatebalancebetweentheamountoffreeradicalsgeneratedinthebodyandtheantioxidantstoprotectagainstthem. For the majority of organisms on Earth, life without oxygenisimpossible,animals,plantsandmanymicro-organismsrelyingonoxygenfortheefficientproductionofenergy.However,theypayahighpriceforpleasureoflivinginanoxygenatedatmospheresincehighoxygenconcentrationintheatmosphereispotentiallytoxicforlivingorganisms.DidyouknowthatfreeradicalsdamagenotonlylipidsbutalsoDNAandproteins?Three levels of antioxidant defenceDuringevolutionlivingorganismshavedevelopedspecificantioxidantprotectivemechanisms to deal with ROS and RNS (Halliwell and Gutteridge, 1999). Thereforeitisonlythepresenceofnaturalantioxidantsinlivingorganismswhichenablethemtosurviveinanoxygen-richenvironment(Halliwell,1994).Thesemechanismsaredescribedbythegeneraltermantioxidantsystem.Itisdiverseandresponsiblefortheprotectionofcellsfromtheactionsoffreeradicals.Thissystemincludes: naturalfat-solubleantioxidants(vitaminsA,E,carotenoids,ubiquinones,etc.); water-solubleantioxidants(ascorbicacid,uricacid,taurine,etc.) antioxidantenzymes:glutathioneperoxidase(GSH-Px),catalase(CAT)andsuperoxidedismutase(SOD).Untitled-2 1/8/2007, 6:44 PM 78SeleniuminNutritionandHealthTable 1.5 Free radical involvement in the development of human diseases (adapted from Surai andSparks, 2001; McCord, 2000; Hogg, 1998 and references there).Liver EyeReperfusion RetionopathyofprematurityToxic effects of chemicals: halogenated Photicretinopathy hydrocarbons, quinones,iron, Maculardegeneration acetaminophen, ethanol Ocular hemorrhageEndotoxin CataractsKidney MuscleAutoimmunenephrosis:inflammation MusculardystrophyToxic effects of chemicals:amino- Over-exercisingglycosides, heavy metalsLung SkinNormobaric hyperoxic injury Radiation (UV or ionising)Bronchopulmonarydisplasia Thermal injuryToxic effects of chemicals: paraquat, Toxic effects of chemicals:bleomicintetracyclinesstimulatingEmphysema photosensitizationAsbestosis ContactdermatitisIdiopathicpulmonaryfibrosis PorphyriaHeart and cardiovascular system Brain and nervous systemAtherosclerosis Parkinsons diseaseHemochromatosis Alzheimers diseaseReperfusion: after infraction or Tardivedyskinesiatransplant NeuronalceroidlipofuscinosisSelenium deficiency (Keishan disease) NeurotoxinsToxic effects of chemicals: ethanol, Hypertensive cerebrovascular injurydoxorubicin AllergicencephalomyelitisMyocardialinfarction MultiplesclerosisGastrointestinal tract Inflammatory-immune systemReperfusion GlomerulonephritisToxic effects of chemicals: nonsteroidal Vasculitisand anti-inflammatory agents, alloxan,iron Autoimmune diseasePancreatitis, Colitis, Intestinal ischemia, Lupus erythermatosusGastric ulcers Reumatroid arthritisBlood Miscellaneous/generalMalaria AgingVarious anemias AIDS, Cancer, DiabetesProtoporphyrinphotooxidation InflammationToxic effects of chemicals:phenyl- Traumahydrazine, primaquine and related drugs,Ischemia/reperfusionsulfonamides, lead etc. RadiationinjuryFavism Rheumatoid arthritis and lupusFanconis anemia Toxic effects of chemicals: alloxan(diabetes), iron overloadAcute pancreatitis, AmyloidosisUntitled-2 1/8/2007, 6:44 PM 8AntioxidantSystemsinAnimalBody9 thiolredoxsystemconsistingoftheglutathionesystem(glutathione/glutathionereductase/glutaredoxin/glutathioneperoxidaseandathioredoxinsystem(thioredoxin/thioredoxinperoxidase/thioredoxinreductase)(fordetailsseeChapter2).Theprotectiveantioxidantcompoundsarelocatedinorganelles,subcellularcompartmentsortheextracellularspaceenablingmaximumcellularprotectiontooccur.Thusantioxidantsystemofthelivingcellincludesthreemajorlevelsofdefence(Niki,1996;Surai,1999;Surai,2002):ThefirstlevelofdefenceisresponsibleforpreventionoffreeradicalformationbyremovingprecursorsoffreeradicalsorbyinactivatingcatalystsandconsistsofthreeantioxidantenzymesnamelySOD,GSH-PxandCATplusmetal-bindingproteins (Figure 1.1).Since the superoxide radical is the main free radical producedinphysiologicalconditionsinthecell(Halliwell,1994)superoxidedismutase(EC1.15.1.1)isconsideredtobethemainelementofthefirstlevelofantioxidantdefenseinthecell(Surai,1999).Thisenzymedismutatesthesuperoxideradicalinthefollowingreaction: SOD2O2* +2H+

H2O2+O2First level of defence:Prevention of radical formationSuperoxide dismutase, glutathione peroxidase, catalse,glutathione and thioredoxin systems andMetal-binding proteinsSecond level of defence:Prevention and restrictition of chain formationand propagationVitamins A, E, C, Carotenoids, Ubiquinols, Glutathione, Uric acidThird level of defence:Excision and repair of damaged parts of moleculesLipases, Peptidases, Proteases, Transferases,DNA-repair enzymes etcFigure1.1Threelinesofantioxidantdefenceinanimalcells(adaptedfromSurai,1999).Untitled-2 1/8/2007, 6:46 PM 910SeleniuminNutritionandHealthSuperoxidedismutasewasdiscoveredbyMcCordandFridovichin1969asanenzymaticactivityinpreparationsofcarbonicanhydraseormyoglobinthatinhibitedtheaerobicreductionofcytochromecbyxanthineoxidase.Therefore,haemocuprein,whichwasdiscoveredmuchearlier,becamecopper-zincsuperoxidedismutase (Bannister, 1988). This discovery opened new era in free radical research.Atpresent,threedistinctisoformsofSODhavebeenidentifiedinmammals,andtheirgenomicstructure,cDNA,andproteinshavebeendescribed(Zelkoetal.,2002).SOD1,orCu,Zn-SOD,wasthefirstenzymeofthisfamilytobecharacterisedandisacopperandzinc-containinghomodimerthatisfoundalmostexclusivelyinintracellularcytoplasmicspaces.Itexistsasa32kDahomodimerandispresentinthecytoplasmandnucleusofeverycelltypeexamined(Zelkoetal.,2002).Didyouknowthattherearethreedifferentformsofsuperoxidedismutaseinmammaliancells?Thesecondmemberofthefamily(SOD2)hasmanganese(Mn)asacofactorandthereforecalledMn-SOD.Itwasshowntobea96kDahomotetramerandlocatedexclusivelyinthemitochondrialmatrix,aprimesiteofsuperoxideradicalproduction(HalliwellandGutteridge,1999).ThereforetheexpressionofMn-SODisconsideredtobeessentialforthesurvivalofaerobiclifeandthedevelopmentofcellularresistancetooxygenradical-mediatedtoxicity(Fridovich,1995).Mn-SODisinducableenzymeanditsactivityisaffectedbycytokinesandoxidative stress. In fact, Mn-SOD has been shown to play a major role in promotingcellulardifferentiationandinprotectingagainsthyperoxia-inducedpulmonarytoxicity(Fridovich,1995).ThebiologicalimportanceofMn-SODisillustratedinTable1.6.In1982,athirdSODisozymewasdiscoveredbyMarklundandcoworkersandcalledextracellularsuperoxidedismutase(EC-SOD),duetoitsexclusive extracellular location. EC-SOD is a glycoprotein with a molecular weightof135,000kDawithhighaffinityforheparin.However,therearesomespecies-specificvariationsinmolecularweight.EC-SODispresentinvariousorganismsasatetrameror,lesscommonly,asadimerandcontainsonecopperandonezincatompersubunit,whicharerequiredforenzymaticactivity(Fattmanetal.,2003).TheexpressionpatternofEC-SODishighlyrestrictedtothespecificcelltypeandtissueswhereitsactivitycanexceedthatofCu,Zn-SODorMn-SOD.ThefourthformoftheenzymeFe-SODwasisolatedfromvariousbacteriabutnotfoundinanimaltissues(Michalski,1992).Furthermore,anoveltypeofnickel-containingSODwaspurifiedtoapparenthomogeneityfromthecytosolicfractionsofStreptomycessp(Younetal.,1996). ThebiosynthesisofSODs,inmostbiologicalUntitled-2 1/8/2007, 6:46 PM 10AntioxidantSystemsinAnimalBody11systems,iswellcontrolled.Infact,exposuretoincreasedpO2,increasedintracellularfluxesofO2-,metalionsperturbation,andexposurestoseveralenvironmentaloxidantshavebeenshowntoinfluencetherateofSODsynthesisinbothprokaryoticandeukaryoticorganisms(Hassan,1988).Table 1.6 The biological importance of Mn-SOD (adapted from Mates et al., 1999).N Mn-SOD manipulationm Effects1 Inactivation of Mn-SOD genes in Increases mutation frequency in aerobicE. coli conditions2 Elimination of Mn-SOD gene in Increases its sencitivity to oxygenSaccharomycetes cervisiae3 Lack of expression in Mn-SOD Dilatedcardiomyopathyandneonatallethalityknock-outmice4 Effect of TNF Selectively induces Mn-SOD in various mousetissues and cultural cells5 Transfection of Mn-SOD cDNA into Rendered the cells resistant to paraquat, TNF andculturalcells adriamycin-inducedcytotoxicity6 Expression of human Mn-SOD Protectsagainstoxygen-inducedpulmonarygenes in transgenic mice injuryandadriamycin-inducedcardiactoxicityThehydrogenperoxideformedbySODactioncanbedetoxifiedbyGSH-PxorCATwhichreduceittowaterasfollows: GSH-PxH2O2+2GSHGSSG+2H2O Catalase2H2O22H2O+O2Catalase(EC1.11.1.6)isatetramericenzymeconsistingoffouridenticalsubunitsof60kDacontainingasingleferriprotoporphyringrouppersubunit.Itplaysanimportantroleintheacquisitionoftolerancetooxidativestressintheadaptiveresponseofcells(Matesetal.,1999).Inmammaliancells,NADPHisboundtocatalaseprotectingitfrominactivationbyH2O2 (ChaudiereandFerrari-Illiou,1999).SinceGSH-PxhasamuchhigheraffinityforH2O2thanCAT(Jonesetal.,1981) and wider distribution in the cell (catalase is located mainly in peroxisomes),theH2O2removalfromthecellisverymuchdependentonGSH-Px.DetailsofGSH-Px action are shown in Chapter 2. Recently it has been shown that thioredoxinperoxidasesarealsocapableofdirectlyreducinghydrogenperoxide(Nordbergand Arner, 2001). It is interesting that the levels of antioxidant enzymes are regulatedbygeneexpressionaswellasbypost-translationalmodifications(FugiandTaniguchi,1999).Untitled-2 1/8/2007, 6:46 PM 1112SeleniuminNutritionandHealthDidyouknowthataspartofGSH-PxandThioredoxinreductaseSeregulatesthefirstandsecondlevelsofantioxidantdefence?Transitionmetalionsalsoacceleratethedecompositionoflipidhydroperoxidesintocytotoxicproductssuchasaldehydes,alkoxylradicalsandperoxylradicals:LOOH + Fe2+ LO*+ Fe3+ + OH-LOOH + Fe3+ LOO* + Fe2+ + H+Therefore,metal-bindingproteins(transferrin,lactoferrin,haptoglobin,hemopexin,metallothionenin,ceruloplasmin,ferritin,albumin,myoglobin,etc.)alsobelongtothefirstlevelofdefence.Itisnecessarytotakeintoaccountthatironandcopperarepowerfulpromotersoffreeradicalreactionsandthereforetheiravailabilityincatalyticformsiscarefullyregulatedinvivo(Halliwell,1999).Thereforeorganismshaveevolvedtokeeptransitionmetalionssafelysequesteredinstorageortransportproteins.Inthiswaythemetal-bindingproteinspreventformationofhydroxylradicalbypreventingthemfromparticipationinradicalreactions.Forexample,transferrinbindstheiron(about0.1%ofthetotalbodyreserves), transports it in the plasma pool and attaches it to the transferrin receptor.Theimportantpointisthatironassociatedwithtransferrinwillnotcatalysefreeradicalreaction.Ferritinisconsideredtobeinvolvedinironstorage(about30%oftotalbodyreserves)withinthecytosolinvarioustissuesincludingliverandspleen.Majorpartofironinthebody(55-60%)isassociatedwithhemoglobinwithinredcellsandabout10%withmyoglobininmuscles(Galey,1997). Arangeofotheriron-containingproteins(mainlyenzymes)canbefoundinthebodyincludingNADHdehydrogenase,cytochromeP450,ribonucleotidereductase,prolinehydroxylase,tyrosinehydroxylase,peroxidases,catalase,cyclooxygenase,aconitase,succinatedehydrogenase,etc.(Galey,1997).Despiteanimportanceofironinvariousbiochemicalreactions,ironcanbeextremelydangerouswhennotcarefullyhandledbyproteins.Infact,inmanystressconditionsareleaseoffreeironfromitsnormalsitesanditsparticipationinFentonchemistrymediatedamagestocells.ForexamplesuperoxideradicalcanreleaseironfromferritinandH2O2degradesthehemeofhemoglobintoliberateironions(Halliwell,1987).Didyouknowthatmetal-bindingproteinsbindFeandCuandpreventtheirparticipationinfreeradicalproduction?Ceruloplasminisanothermajorproteinmediatingfreeradicalmetabolismbeingacopper-bindingprotein.UnderphysiologicalconditionsitbindssixorsevenUntitled-2 1/8/2007, 6:46 PM 12AntioxidantSystemsinAnimalBody13copperionspermoleculepreventingtheirparticipationinfreeradicalgeneration.About5%ofhumanplasmacopperisboundtoalbuminortoaminoacidsandtherestisboundtoceruloplasmin.Furthermoreceruloplasminpossessesantioxidantpropertiesitselfbeingabletoscavengesuperoxideradical(Yu,1994).Therefore,itisnowquiteclearthatmetalsequestrationisanimportantpartofextracellularantioxidantdefence.UnfortunatelythisfirstlevelofantioxidantdefenceinthecellisnotsufficienttocompletelypreventfreeradicalformationandsomeradicalsdoescapethroughthepreventivefirstlevelofantioxidantsafetyscreeninitiatinglipidperoxidationandcausingdamagetoDNAandproteins.Thereforethesecondlevelofdefenceconsists of chain-breaking antioxidants - vitamin E, ubiquinol, carotenoids, vitaminA,ascorbicacid,uricacidandsomeotherantioxidants.Glutathioneandthioredoxinsystemsalsohaveasubstantialroleinthesecondlevelofantioxidantdefence(fordetailsseeChapter2).Chain-breakingantioxidantsinhibitperoxidationbykeepingthechainlengthofthepropagationreactionassmallaspossible.Therefore,theypreventthepropagationstepoflipidperoxidationbyscavengingperoxylradicalintermediatesinthechainreaction:LOO*+ TocToc*+LOOH(LOO*islipidperoxylradical;Toc-tocopherol,Toc*-tocopheroxylradical,LOOHlipidhydroperoxide)VitaminE,themosteffectivenaturalfreeradicalscavengeridentifiedtodate,isthe main chain breaking antioxidant in the cell. However, hydroperoxides, producedinthereactionofvitaminEwiththeperoxylradical,aretoxicandifnotremoved,impairmembranestructureandfunctions(GutteridgeandHalliwell,1990).Infacts,lipidhydroperoxidesarenotstableandinthepresenceoftransitionmetalions can decompose producing new free radicals and cytotoxic aldehydes (Diplock,1994).ThereforehydroperoxideshavetoberemovedfromthecellinthesamewayasH2O2,butcatalaseisnotabletodetoxifythesecompoundsandonlySe-dependentGSH-Pxcandealwiththemconvertinghydroperoxidesintonon-reactiveproducts(Brigelius-Flohe,1999)asfollows:

GSH-PxROOH+2GSHROH(non-toxic)+H2O+GSSGThus,vitaminEperformsonlyhalfthejobinpreventinglipidperoxidationbyscavengingfreeradicalsandforminghydroperoxides.ThesecondpartofthisimportantprocessofantioxidantdefenceisduetoSe-GSH-Px.Itisnecessarytounderline,thatvitaminEandseleniumworkinatandem;andevenveryhighdosesofdietaryvitaminEcannotreplaceSewhichisneeded(intheformofUntitled-2 1/8/2007, 6:46 PM 1314SeleniuminNutritionandHealthGSH-Pxandthioredoxinreductase)tocompletethesecondpartofantioxidantdefenceasmentionedabove.Thus,SeasanintegralpartoftheGSH-Pxandthioredoxinreductasebelongstothefirstandsecondlevelsofantioxidantdefence.DidyouknowthatvitaminEperformsonlyhalfthejobinpreventinglipidperoxidationbyscavengingfreeradicalsandforminghydroperoxidesandSe-GSH-Pxisabsolutelyessentialtocompleteantioxidantdefence?CoenzymeQisconsideredtobeanimportantantioxidant,whichissynthesisedin vivo (see chapter 10) and is an important integral part of the antioxidant defencesysteminthecell.Carotenoidsrecentlywereincludedintofamilyofnaturalantioxidants.Theyexhibittheirmaximumantioxidantactivityatlowoxygenpressures,whichprevailinhealthytissues.Ithasbeenrecentlyhypothesisedthatcarotenoidsarenotthemajorantioxidantplayersthemselvesbutratherareanimportantpartoftheantioxidantsystem(Surai,2002).Thereforeantioxidantinteractionsincludingtheirrecyclingprovideaneffectiveandreliablesystemofdefencefromfreeradicalsandtoxicproductsoftheirmetabolism.VitaminCisahydrophilicantioxidantfunctioninginanaqueousenvironmentandpossessinghighfree-radical-scavengingactivity(Yu,1994).ItdirectlyreactswithO2-andOH*andvariouslipidhydroperoxidesandistakingpartinthevitamin E recycling (Yu, 1994; Halliwell, 1996). Ascorbic acid is protective againstanumberofROS(CarrandFrei,1999;Halliwell,1999a,1996;Table1.7).Themajoradvantagesofascorbateasanantioxidanthavebeendescribedasfollows(CarrandFrei,1999): Bothascorbateandascorbylradicalhavelowreductionpotentialsandcanreactwithmostotherbiologicallyrelevantradicalsandoxidants; Ascorbylradicalhasalowreactivityasaresultofresonancestabilisationofunpairedelectronandreadilydismutatestoascorbateanddehydroascorbicacid(DAA); AscorbylradicalandDAAcanbeconvertedintoactiveascorbateformbyenzyme-dependentorindependentpathways.Inparticular,ascorbylradicalcanbereducedbyNADH-dependentsemidehydroascorbatereductaseorbythioredoxinreductase. AtthesametimeDAAcanbereducedto AAbyGSH,lipoicacidorglutaredoxin.Untitled-2 1/8/2007, 6:46 PM 14AntioxidantSystemsinAnimalBody15Table 1.7 Antioxidant properties of ascorbate (Adapted from Halliwell, 1996; 1999a).Scavenges O2* and HO2*Scavenges water-soluble peroxyl (ROO*) radicals. Lypophylic ascorbate esters can scavengelipid-soluble ROO* radicalsScavengesthiylandsulphenylradicalsPrevents damage by radicals arising by attack of OH* or ROO* upon uric acidPowerful scavenger of hypochlorous acidInhibitsdamagebyperoxinitritePowerful scavenger and quencher of singlet oxygenScavenges OH* radicalsScavengesnitroxideradicalsMay regenerate -tocopherol from -tocopheryl radicals in membranes and lipoproteinsProtectsplasmalipidsagainstperoxidationinducedbyactivatedneutrophilsProtects membranes and lipoproteins against lipid peroxidation induced by cigarette smokePowerful scavenger of O3 and NOO* in human body fluidsInhibits oxidative damage by drug-derived radicalsScavenges free radicals on proteinsGlutathione (GSH) is the most abundant non-protein thiol in avian and mammaliancells,andconsideredtobeanactiveantioxidantinbiologicalsystemsprovidingcellswiththeirreducingmilieu(Meister,1992).CellularGSHplaysakeyroleinmanybiologicalprocesses(SenandPacker,2000): thesynthesisofDNAandproteins; cellgrowthandproliferation; regulationofprogrammedcelldeath; immuneregulation; thetransportofaminoacids; xenobioticmetabolism; redox-sensitivesignaltransduction.Furthermore, GSH thiolic group can react directly with (Lenzi et al., 2000; MeisterandAnderson,1983): H2O2; superoxideanion; hydroxylradicals; alkoxylradicals; hydroperoxidesUntitled-2 1/8/2007, 6:46 PM 1516SeleniuminNutritionandHealthTherefore,acrucialroleforGSHisasfreeradicalscavenger,particularlyeffectiveagainstthehydroxylradical(BainsandShaw,1997),sincetherearenoenzymaticdefencesagainstthisspeciesofradical.UsuallydecreasedGSHconcentrationintissuesisassociatedwithincreasedlipidperoxidation(Thompsonetal.,1992).Furthermore in stress conditions GSH prevents the loss of protein thiols and vitaminE(PalamandaandKehrer,1993)andplaysanimportantroleasakeymodulatorofcellsignaling(ElliottandKoliwad,1997).Animalsandhumanareabletosynthesiseglutathione.Taurine,asulphurcontainingaminoacid(2-aminoethanesulfonicacid)issynthesisedfrommethionineandcysteineinthepresenceofvitaminB6andfound in almost all tissues in mammals. It is the most abundant intracellular aminoacidinhumanswhichisnotincorporatedintoproteins. Antioxidantpropertiesoftaurinewereshowninvariousmodelsystemsinvivoandinvitro(Cozzietal.,1995;Haberetal.,2003;Sethupathyetal.,2002).However,antioxidanteffectoftaurineisnotrestrictedtoPUFAs.Forexample,Ogasawaraetal.(1993;1994)showedaninhibitingeffectoftaurineagainstthemodificationofprotein,aswellasanantioxidativeeffectthroughthereactionsoftaurinewithaldehydesinvivo.Itseemslikelythatinmanycasesinbiologicalsystemstaurinecouldinteractwithotherantioxidantsbeinganimportantpartofanintegratedantioxidantsystem.Forexample,taurinesupplementationofratsrestoredkidneyGSHcontentandGSH-Px activity and reduced MDA production levels in the kidney tissue followingcisplatintreatment(SaadandAl-Rikabi,2002).Instreptozotocin-diabeticrats,dietarytaurinesupplementationamelioratesMDAlevels,GSSG/GSH,andNAD+/NADH(ObrosovaandStevens,1999).TaurinereducedsignificantlyadecreaseofglutathioneantioxidantsystemactivityprotectingtissuesagainstGSHpooldepletionandpreventingadecreaseofglutathionereductaseandglutathioneperoxidaseactivitiesinacuteseverehypoxia(Mankovskaetal.,1998).Uricacidistraditionallyconsideredtobeametabolicallyinertend-productofpurinemetabolisminman,withoutanyphysiologicalvalue.However,thisubiquitouscompoundhasproventobeaselectiveantioxidant(Becker,1993;MaplesandMason,1988)whichcan: reactwithhydroxylradicalsandhypochlorousacid,itselfbeingconvertedtoinnocuousproducts serveasanoxidisablecosubstratefortheenzymecyclooxygenase protectagainstreperfusiondamageinducedbyactivatedgranulocytes preventsoxidativeinactivationofendothelialenzymesinstressconditions chelatetransitionmetalionsandscavengingROSSomespecificenzymeswhichhydrolyseoxidisedbasespreventingtheirincorporationintoDNAcanalsobeconsideredasapartofthesecondlevelofUntitled-2 1/8/2007, 6:46 PM 16AntioxidantSystemsinAnimalBody17antioxidantdefence(Slupphaugetal.,2003).Theroleofubiquinonesanduricacidinthefarmanimalandpoultrydevelopmenthasnotyetbeenstudied.However,eventhesecondlevelofantioxidantdefenceinthecellisnotabletopreventdamagingeffectsofROSandRNSonlipids,proteinsandDNA.Inthiscase,thethirdlevelofdefenceisbasedonsystemsthateliminatedamagedmoleculesorrepairthem.Thislevelofantioxidantdefenceincludeslipolytic(lipases),proteolytic(peptidasesorproteases)andotherenzymes(DNArepairenzymes,ligases,nucleases,polymerases,proteinases,phospholipasesandvarioustransferases).Sincemaintainingtheintegrityofthegenomeisofthevitalimportance,livingorganismshaveevolvedarangeofsystemsthatcanrecognise,signalthepresenceof,andrepairthevariousformsofDNAdamage.Infact,DNArepairisoneofthefundamentalprocessesoflife(130humanDNArepairgeneshavebeenidentified;Woodetal.,2001)andifthesystemsarecompromiseddevastatingconsequenceswouldbeexpected.Inordertodealwiththedeleteriouseffectsofsuchlesions,leadingtogenomicinstability,cellshaveevolvedanumberofDNArepair mechanisms. They include the direct reversal of the lesion,mismatch repair,thebaseexcisionrepair,nucleotideexcisionrepair,nucleotideincisionrepair,transcription-coupledrepair,globalgenomerepair,translesionsynthesis,homologousrecombinationandnon-homologousend-joining(Grosetal.,2002;Slupphaugetal.,2003).Theserepairpathwaysareuniversallypresentinlivingcellsandextremelywellconserved.Didyouknowthat130humanDNArepairgeneshavebeenidentified?Therefore,DNArepairsystemsincludeanumberofenzymaticprocessesrangingfrombaserecognitionandexcisiontoligationofDNAstrands.Inparticular,theDNAglycosylasesrecognisedamagedpurinesandpyrimidinesandhydrolysethebondlinkingtheabnormalbasetothesugar-phosphatebackbone(HalliwellandGutteridge,1999);the5I-apurinicendonucleasesprocessstrandbreaks,sitesofbaseloss,andtheproductsofDNAglycosylase/apuriniclyaseaction.DNApolymerasefillsintheone-nucleotidegapwiththecorrectbase.DNAligasescompletetherepairprocessbysealingthe3IendofthenewlysynthesisedstretchofDNAtotheoriginalportionoftheDNAchain(Cardozo-Pelaezetal.,2000;Wallaceetal.,1997;CroteauandBohr,1997).ItisbelievedthatmostdamagedorinappropriatebasesinDNAareremovedby excision repair, while a minority are repaired by direct damage reversal (Krokanetal.,2000).TheimportanceoftheseDNArepairsystemsisconfirmedbytheUntitled-2 1/8/2007, 6:46 PM 1718SeleniuminNutritionandHealthfact that defects in these can result in cell death and hypersensitivity to endogenousorenvironmentalmutagens(Jackson,1998).Thereforeremovingmutageniclesions in DNA is a vital task for repair systems. In general, the repair DNA damagemechanismsinbacteriaarewelldefined,whereasinhighereukaryotesthegenesandproteinsresponsibleforrepairawaitfurtherinvestigation(CroteauandBohr,1997).ItseemslikelythatDNArepairisintegratedwithcellcycleregulation,transcriptionandreplicationandusesomecommonfactors(Slupphaugetal.,2003).However,theenzymesofthethirdlevelofantioxidantdefencedonotachievecompleterepairorremovalofdamagedDNAmoleculesandthiscouldlead to arrest of cell cycle and cell death. In fact, programmed cell death (apoptosis)isinvolvedinmaintenanceofthegeneticintegritybyremovinggeneticallyalteredcells.SeleniuminanorganicformcandirectlyorindirectlyeffectDNAintegrityandrepair.Forexample,elderlymaledogswerefedonthedietsupplementedwithseleniumintheformofSeMetorhigh-seleniumyeastat3g/kgor6g/kgbodyweightperdayfor7monthsandacomparisonwasmadewithunsupplementedcontroldogs.TheextentofDNAdamageinprostatecellsandinperipheralbloodlymphocytes,asdeterminedbythealkalinecometassay,wasloweramongtheselenium-supplementeddogsthanamongthecontroldogs(Watersetal.,2003).Furthermore,Seoetal.(2002a)showedthatseleniumintheformofSeMetinducesaDNArepairresponseinnormalhumanfibroblastsinvitro, and protects cells from DNA damage. A possible mechanism for the inducibleDNArepairresponsewasshowntobeenhancedrepaircomplexformationinselenomethionine-treatedcells.Similarly,treatmentwithSeMeteitheroninitiationoronselection/promotion,orduringtheentireexperimentshowedthatSeMetwasmosteffectiveinregulatingthecellularantioxidantdefencesystems,DNAchainbreakcontrolandreducingaberrantcryptfociinthecolorectaltissuesofrats(Mukherjeeetal.,2001).DidyouthatSeMetcanaffectDNA-repairenzymesystems?ProtectiveeffectofSeagainstDNAdamagedependsonthedoseused.Forexample,measurementsinacinarcellsinSyriangoldenhamsterssuggestedamorerapidrepairofsingle-strandbreaksDNAinhamstersprefed2.5ppmSethaninthoseprefed0.1ppmSe(Birtetal.,1988).IthasalsobeenshownthatseleniumintheformofSeMetcanactivatethep53tumorsuppressorproteinbyaredox mechanism that requires the redox factor Ref1 (Seo et al., 2002). Specifically,SeMetinducedsequence-specificDNAbindingandtransactivationbyp53andthereforetheDNArepairbranchofthep53pathwaywasactivated.RecognitionUntitled-2 1/8/2007, 6:46 PM 18AntioxidantSystemsinAnimalBody19and signalling of DNA damage is an important event for the induction of subsequentcellularresponsessuchasincreasedrepair,cellcyclearrestandapoptosis.RecognitionofDNAbreaksisaccomplishedbyagroupofphosphatidylinositol-3-kinases.ThesekinasesareATM(ataxiatelangiectasiamutated),ATR(ataxiatelangiectasiarelated)andthecatalyticsubunitofDNAPK(Christmannetal.,2003). ATRand ATMcanbindtoDNAendsofdamagedDNA,whichresultsinactivationofthekinaseactivity.ItisinterestingthattreatmentwithSeMetwasshowntoenhanceATRandCHK2geneexpressioninculturedhumanthyroidepithelialcells(Kennedyetal.,2004).TheauthorssuggestedthatSeMetmaypreventradiation-inducedadversebiologicaleffectsbyenhancingtheDNArepairmachineryinirradiatedcells.ThereforeitisclearthatSeMethasauniquespecificeffectontheDNArepairsystemaswellasprovidesaprotectionagainstDNAdamageandthiseffectisnotthecaseorevencanbeoppositewhenseleniteisused.Inspiteofimportantrolesofproteinoxidationinpathogenesisofthedevelopmentofvariousdiseases,mechanismsforthecontrolofproteinoxidationandtheirrepairhavenotbeenwellstudiedandthishasbeenatopicofgreatinterestforthelastfewyears.Theoxidativedamagetoproteinsisassociatedwithalterationoftransportproteinsandiondis-balance,disruptiontothereceptorsandimpairsignaltransduction,enzymeinactivationetc.ItisbelievedthatconversionofSHgroupsintodisulphidesandotheroxidizedspecies(e.g.oxyradicals)isoneoftheearliesteventsduringtheradical-mediatedoxidationofproteins.Therefore,thioredoxinplusthioredoxinreductasedealwiththesechangesbyreducingproteindisulphidestothiolsandregulatingredox-sensitivetranscriptionfactors(Deanetal.,1997).Itisinterestingthatreversibleoxidationofcysteinecouldbeanimportantcellularredoxsensorinsomeproteins(Finkel,2000).Methionineresiduesinproteins are also very susceptible to oxidation with methionine sulfoxide formation,whichwasdetectedinnativeproteins(Gaoetal.,1998).Thiscouldaffectactivityof various proteins (Table 1.8). In fact, almost all forms of ROS oxidize methionineresiduesofproteinstoamixtureoftheR-andS-isomersofmethioninesulfoxide(Stadtmanetal.,2002).Methioninesulfoxidereductase(Msr)canreduceeitherthefreeortheprotein-boundmethioninesulfoxidebacktomethionine.ThereforeMsrisconsideredarepairmechanismfordealingwiththeproductofreactionofoxidantswithmethionineresidues(Levineetal.,1996).Theauthorshypothesizedthatmethionineresiduesfunctionasalastchanceantioxidantdefencesystemforproteins.Itwasshownthatinbacterialglutaminesynthetasesurface-exposedmethionineresiduessurroundingtheentrancetotheactivesitearepreferentiallyoxidisedandotherresidues(e.g.cystein)withinthecriticalregionsoftheproteinareprotectedwithoutlossofcatalyticactivityoftheprotein(Levineetal.,1996).Indeed,duetoMsractivitythemethionine-methioninesulfoxidepaircanfunctionUntitled-2 1/8/2007, 6:46 PM 1920SeleniuminNutritionandHealthTable 1.8 Proteins and peptides with activity altered by methionine oxidation (adapted from Levine et al.,2000).Adrenocorticotropichormone Interferon-2b-1-antitrypsin Interferon -2-antitrypsin Interleukin6-2-macroglobulin Keratinocyte growth hormoneAmyloidbetapeptide LypoxygenaseAntiflammin LutropinAntitrombin LysozymeApolipoprotein MucusproteinaseinhibitorBombesin Neuropeptide YBugarotoxin OvoinhibitorCalcitonin Parathyroid hormoneCalmodulin PepsinChemotactic peptide f-Met-Leu-Phe PhosphoglucomutaseCholecystokinin PlasminogenactivatorinhibitorChorionicsomatomammotropin PotassiumchannelChymotrypsin ProlactinComplementC5 RibonucleaseCytochrom c peroxidase RibosomalproteinL12Cytochrome c SecretoryleukocyteproteinaseinhibitorEchistatin Small heat shork proteinEnkephalin SnakevenoncardiotoxinFactor VII Stem cell factorFibronectin SubtilisinGlucagon ThrombomodulinGlutaminesynthetase TissueplasminogenactivatorGrowth hormone TriptophanaseHemoglobin VasoactiveintestinalpeptideHIV-2 proteasecatalytically.MsrAispresentinmostlivingorganisms,isencodedbyasinglegeneandthemammalianenzymehasbeendetectedinalltissuesstudied.Inparticularitisfoundinthecytosolandmitochondriaofratlivercells(Vougieretal.,2003).Msrisconsideredtohaveatleastthreeimportantfunctionincellularmetabolism including antioxidant defence, repair enzyme and a regulator of certainenzymefunctionandpossiblyparticipationinsignaltransduction(Bar-NoyandMiskovitz,2002;Stadtmanetal.,2002).Inparticular,mousethatlackstheMsrAgene(Moskovitzetal.,2001): exhibitsenhancedsensitivitytooxidativestress hasashorterlifespanUntitled-2 1/8/2007, 6:46 PM 20AntioxidantSystemsinAnimalBody21 developsanatypicalwalkingpattern accumulateshighertissuelevelsofoxidisedproteinunderoxidativestress islessabletoup-regulateexpressionofthioredoxinreductaseunderoxidativestressOn the other hand, overexpretion of the MsrA gene in the nervous system markedlyextendsthelifespanofthefruitflyDrosophila(Ruanetal.,2002),humanT-cells(Moscovitzetal.,1998)andmouse(Moscovitzetal.,2001).Furthermore,theauthorsshowedthatMsrAtransgenicfliesaremoreresistanttoparaquat-inducedoxidativestress.InfactMsrisalsoconsideredtobeapotentialmissinglinkinthepost-translationalmodificationcycleinvolvedinthespecificoxidationandreductionofmethionineresiduesincellularsignallingproteinswhichcanchangecellular excitability (Hoshi and Heinemann, 2001). This could well be the regulatorymechanismsimilartoproteinphosphorylation.ThegeneralschemeofantioxidantfunctionofMsrisshowninFigure1.2.Protein-MetSOProtein-MetMsr -redMsr -oxThioredoxin-oxThioredoxin-redNADPHNADP+CO2+PentoseG-6-PRSredRSoxFigure1.2Functionofmethioninesulfoxidereductase(adaptedfromLevineetal.,2000).RSoxrepresentsareactivespecieswhichisreducedwithconcomitantoxidationofmethionineresidueintheprotein.Thioredoxin-redandox-arereducedandoxidisedthioredoxin.Msr-oxandred-aremethioninesulfoxidereductase.MsrAhasbeenknownforalongtime,anditsrepairingfunctioniswellcharacterised,however,recently,anewmethioninesulfoxidereductasewascharacterised(Grimaudetal.,2001).ItwasreferredtoasMsrBanditwasshownthatthegeneofMsrBispresentingenomesofeubacteria,archaebacteria,andeucaryotes.Therefore,inmammalstwomethioninesulfoxidereductases,MsrAand MsrB, are expressed with different substrate specificity (Grimaud et al., 2001).Theycatalyzethethioredoxin-dependentreductionoftheS-isomerandR-isomerofmethioninesulfoxidetomethionine,respectively.Untitled-2 1/8/2007, 6:46 PM 2122SeleniuminNutritionandHealthDidyouknowthatmammalianmethioninesulfoxidereductaseBhasbeenidentifiedasaselenoprotein,previouslyknownasselenoproteinR?RecentlythemajormammalianMsrBhasbeenidentifiedasaselenoprotein(Maskovitzetal.,2002;Kryukovetal.,2002).InfactithasbeenfoundthatselenoproteinRisazinc-containingsterteo-specificMsr(Kryukovetal.,2002).Furthermore, it has been shown that there was a loss of MsrB activity in the MsrA/mouseinparallelwithlossesinthelevelsofMsrBmRNAandMsrBprotein(MoskovitzandStadtman,2003).Therefore,theauthorsuggestedthatMsrAmighthave a role in MsrB transcription. Moreover, Se deficiency in mouse was associatedwithasubstantialdecreaseinthelevelsofMsrB-catalyticactivity,MsrBprotein,andMsrBmRNAinliverandkidneytissues(MoskovitzandStadtman,2003).IthasbeenreportedthathumanandmousegenomespossessthreeMsrBgenesresponsibleforsynthesisofthefollowingproteinproducts:MsrB1,MsrB2andMsrB3(KimandGladyshev,2003).Inparticular,MsrB1(SelenoproteinR)waspresentinthecytosolandnucleusandexhibitedthehighestmethionine-R-sulfoxidereductaseactivityduetopresenceofselenocysteine(Sec)initsactivesite.OthermammalianMsrBsarenotselenoproteinsandcontaincysteineinplaceofSecandwerelesscatalyticallyefficient(KimandGladyshev,2003).DidyouknowthatSeintheformSeMetisinvolvedinregulationofDNA-repairenzymesandasapartofMsrBregulatesproteinrepairingsystemandthereforeregulatesthethirdlevelofantioxidantdefence?ThereducedglutathioneitselfcanalsoparticipateinmaintenanceofproteinSHgroups. At the same time the thioredoxin system has alkyl hydroperoxide reductaseactivity.Proteindisulphideisomeraseisalsoinvolvedinre-pairingofSHgroupsinproteins(Deanetal.,1997).Furthermore,thecellscangenerallyremoveoxidizedproteinsbyproteolysis.Infact,damagedproteinsaredegradedbytheproteasome,multicatalyticproteinase(anintracellular,nonlysosomalthreoninetypeprotease,EC3.4.99.46),whichisresponsiblefordegradationofthemajorityofcytosolicproteins(Rocketal.,1994).Itiswellrecognisednowthattheproteasomeisthemajorenzymaticsysteminchargeofcellularcleansingandplaysakeyroleinthedegradationofdamagedproteinscontrollingthelevelofalteredproteinsineukaryoticcells(Friguetetal.,2000).Itissuggestedthatenhancedsusceptibilitytodegradationbyproteinasesisemployedasacriterionofunfolding(Deanetal.,1997),however,heavilyUntitled-2 1/8/2007, 6:46 PM 22AntioxidantSystemsinAnimalBody23oxidizedproteinsarecharacterisedbyanincreasedresistancetoproteolyticattackbymostproteinases.Theproteasomecomplexrecogniseshydrophobicaminoacidresidues,aromaticresidues,andbulkyaliphaticresiduesthataremodifiedduringtheoxidativestressandcatalysetheselectiveremovalofoxidativelymodifiedcellproteins(Gruneetal.,1997).Byminimisingproteinaggregationandcross-linkingandbyremovingpotentiallytoxicproteinfragmentsproteasomeis an active part of the cellular defence system against oxidative stress. The selectivedegradationofoxidativelydamagedproteinsenablescellstorestorevitalproteinsincludingenzymesduringphysiologicalmetabolismandduringmoderatestressconditions(Gruneetal.,1997).Oxidizedproteinsmayalsoberecognisedasforeignbytheimmunesystemwithcorrespondingantibodyformation(HalliwellandGutteridge,1999).Clearly,furtherworkisneededtoclarifymolecularmechanismsofthethirdlevelofantioxidantdefence. Alltheseantioxidantsareoperatinginthebodyinassociationwitheachotherforminganintegratedantioxidantsystem.Theco-operativeinteractionsbetweenantioxidantsinthecellarevitalformaximumprotectionfromthedeleteriouseffectsoffreeradicalsandtoxicproductsoftheirmetabolism.For-example,itiswellestablishedthatvitaminEisthemajorantioxidantinbiologicalmembranes,aheadquarterofantioxidantnetwork.Howeveritisusuallypresentthereinlowmolarratios(onemoleculeper2000-3000phospholipids)butvitaminEdeficiencyisdifficulttoinduceinadultanimals.ItisprobablyduetothefactthatoxidisedvitaminEcanbeconvertedbackintotheactivereducedformbyreactingwithotherantioxidants:ascorbicacid,glutathione,ubiquinolsorcarotenoids(Figure1.3).Thisfiguredemonstratesaconnectionofantioxidantdefencetothegeneralbodymetabolism(thepentosephosphatecycleisthemajorproducerofreducingequivalentsintheformofNADPH)andshowsinvolvementofothernutrientsinthisprocess.Forexample,dietaryproteinisasourceofessentialaminoacidsforglutathionesynthesis,riboflavinisanessentialpartofglutathionereductase,niacinisapartofNADPHandSeisanintegralpartofthioredoxinreductase.Atthesametimethiamineisrequiredfortransketolaseinthepentosephosphatepathway.Thus,amajorfindinginrecentyearsisthepossibilityofdirectorindirectvitaminErecyclingfromitsoxidisedradicalformbymeansofascorbate(Chanetal.,1991;Chan,1993),glutathione(Nikietal.,1982;Chan,1993),cysteine(Motoyamaetal.,1989),ubiquinols(FreislebenandPacker,1993;Chan,1993),lipoicacid(Packer,1998),estrogens(Mukaietal.,1990),carotenoids(PalozzaandKrinsky,1992;Bohmetal.,1997)andpotentiallyquercetinandcatechins(Pietta,2000),anthocyanins(Franketal.,2002)androsemaryextracts(Madsenetal.,1997).Enzymaticregenerationof-tocopherolhasbeenalsodescribed(Maguireetal.,1989;Kaganetal.,1998).TherateofreductionofphenoxylradicalinthemembranedecreasedintheorderofascorbicUntitled-2 1/8/2007, 6:46 PM 2324SeleniuminNutritionandHealthacid>cysteine>glutathione(Niki,1996).Therateofregeneration,orrecycling,ofthevitaminEradicalsthatformduringtheirantioxidantactionmayaffectbothitsefficiencyinantioxidantactionanditslifetimeinbiologicalsystemsandthegreaterrecyclingactivityisassociatedwithincreasedefficiencyofinhibitionoflipidperoxidation(Packer,1995).Whetheralltheseregenerationreactionstakeplaceinvivoawaitinvestigation.Duetoincompleteregeneration(theefficiencyofrecyclingisusuallylessthan100%)inbiologicalsystems,theantioxidantshavetobeobtainedfromthediet(e.g.vitaminEandcarotenoids)orsynthesisedinthetissues(glutathione).Vit .E-radicalVitamin EAADAAGSSH2GSHNADPHNADP+CO2+PentoseG-6-PGlucoseDiketo-L- gulonicacidVit .E quinoneMembraneTransport123ROO*ROOHLossLossLossSeRiboflavinNiacinThiaminFigure1.2RedoxcycleofvitaminE(AdaptedfromWinkleretal.,1994;Surai,1999)AsaresultofantioxidantactionofvitaminE,tocopheroxylradicalisformed.Thisradicalcanbereducedbacktoanactiveformof-tocopherolbycouplingwithascorbicacidoxidation.AscorbicacidcanberegeneratedbackfromtheoxidisedformbyrecyclingwithglutathionewhichcanreceiveareducingpotentialfromNADPH,synthesisedinthepentosephosphatecycleofcarbohydratemetabolism.EnzymesinvolvedinvitaminErecyclingareasfollows:1.Thioredoxinreductase;2.Glutathionereductase;3.Glucose-6-phosphatedehydrogenase.Duetoincompleteregeneration(theefficiencyofrecyclingisusuallylessthan100%)inbiologicalsystems,theantioxidantshavetobeobtainedfromthediet(vitaminEandcarotenoids)orsynthesisedinthetissues(ascorbicacidandglutathione).ThereforetheantioxidantprotectioninthecelldependsnotonlyonvitaminEconcentrationandlocation,butalsoreliesontheeffectiverecycling.Indeed,iftherecyclingiseffectivethenevenlowvitaminEconcentrationsareabletomaintainhighantioxidantprotectioninphysiologicalconditions.Forexample,thiscouldbedemonstratedusingchickenbrainasamodelsystem.Indeed,ourUntitled-2 1/8/2007, 6:47 PM 24AntioxidantSystemsinAnimalBody25data(Surai,2002)indicatethatthebrainischaracterisedbyextremelyhighconcentrationsoflongchainpolyunsaturatedfattyacidspredisposingthistissuetolipidperoxidation.Furthermore,braincontainsmuchlowerlevelsofvitaminEthanotherbodytissues.However,infreshchickenbrain,levelsofproductsoflipidperoxidationareverylow,whichcouldbeareflectionofaneffectivevitaminErecyclingbyascorbicacidwhichispresentinthistissueincomparativelyhighconcentrations.Antioxidantrecyclingisthemostimportantelementinunderstandingmechanismsinvolvedinantioxidantprotectionagainstoxidativestress.Therateofregeneration,orrecycling,ofthevitaminEradicalsmayaffectbothitsantioxidantefficiencyanditslifetimeinbiologicalsystems.Ascanbeseenfromdatapresentedabovetheantioxidantdefenceincludesseveraloptions(Surai,2002): Decreaselocalisedoxygenconcentration; Prevention of first-chain initiation by scavenging initial radicals (SOD, GSH-Pxandcatalase); Bindingmetalions(metal-bindingproteins); Decompositionofperoxidesbyconvertingthemtonon-radical,non-toxicproducts(Se-GSH-Px); Chain-breakingbyscavengingintermediateradicalssuchasperoxylandalkoxylradicals(vitaminsE,C,glutathione,uricacid,ubiquinol,bilirubinetc.); Repairandremovalofdamagedmolecules.Additionaldefensivemechanismsresponsibleformaintenanceofphysiologicalmetabolisminstressconditionsinclude(Surai,2002): Antioxidantrecyclingmechanisms; Redox-signallingandgeneexpressionwithanadditionalsynthesisofimportantantioxidantmolecules; Stress-proteinsynthesis(e.g.heatshockproteins); Apoptosis(canremovedamagedcellsandrestrictmutagenesis).Antioxidant-prooxidant balance in the body and oxidative stressInthebodyadelicatecriticalbalanceexistsbetweenantioxidantdefenceandrepairsystemsandfreeradicalgeneration(Figure1.4).Inphysiologicalconditionstherightandleftpartsoftheso-calledbalancesareinequilibriumi.e.freeradicalgenerationisneutralisedbytheantioxidantsystem.Exogenousfactorsareamongthemostimportantelements,whichincreaseanefficiencyoftheUntitled-2 1/8/2007, 6:47 PM 2526SeleniuminNutritionandHealthantioxidantsystemoftheorganism.NaturalandsyntheticantioxidantsinthefeedaswellasoptimallevelsofMn,Cu,ZnandSehelptomaintaintheefficientlevelsofendogenousantioxidantsinthetissues.Optimaldietcompositionallowstheantioxidantsofthefoodtobeefficientlyabsorbedandmetabolised.Optimaltemperature,humidityandotherenvironmentalconditionsarealsorequiredfortheeffectiveprotectionagainstfreeradicalproduction.Thepreventionofdifferentdiseases by antibiotics and other drugs is an integral part of the efficient antioxidantdefenceaswell.Increase of antioxidant defence Stress conditionsNatural antioxidants in the feed(Vitamins A, E, C, carotenoids, flavonoids,essential oils); Synthetic antioxidants in the feed (BHT,ethoxiquineetc.)Se,Mn Zn, CuDietoptimizationEnvironmental condition optimizationDisease prevention andtreatment by antibiotics andother drugsAntioxidant systems of the organismVitamins A, E, C, carotenoids, glutathione,ubiquinol , uric acid, antioxidant enzymes (SOD, GSH-Px, Catalase)Toxins, high PUFA,deficiencies of vitamin E, Se,Mn, Zn, Fe-overloadTemperature, humidity, hyperoxia,radiation, UV,microwave etc.Diseases: bacterial, viral,allergy etc.Free radical generationElectron-transport chain,Phagocytes,Xanthine oxidase etc.Membrane damageNutrient absorption decreaseNutrientimbalanceDecrease of productiveand reproductiveperformancesLipidperoxidat ion,damagest olipids, prot eins,DNAInjuries to heart -vascular, brain and nervous and muscle systemsMeat quality andshelf life reductionMilk taste deteriorationImmune incompetenceNutritionalEnvironmentalInternalFigure1.4Antioxidant-prooxidantbalanceintheorganism(adaptedfromSurai,1999).Differentstressconditionsareassociatedwithoverproductionoffreeradicalsandcauseoxidativestressi.e.adisturbanceintheprooxidant-antioxidantbalanceleadingtopotentialtissuedamage(Jaeschke,1995).Stressconditionscanbegenerallydividedintothreemaincategories.ThemostimportantpartisnutritionalstressconditionsincludinghighlevelsofPUFAs,deficienciesofvitaminE,Se,ZnorMn,Fe-overload,hypervitaminosisAandpresenceofdifferenttoxinsandtoxiccompounds(seechapter10).Asecondgroupofstressfactorsincludesenvironmentalconditions:increasedtemperatureorhumidity,hyperoxia,radiationetc.Internalstressfactorsincludevariousbacterialorviraldiseasesaswellasallergy.Alltheabove-mentionedconditionsstimulatefreeradicalgenerationbyadecreaseofcouplingofoxidationandphosphorylationinthemitochondriathatresultsinanincreasedelectronleakageandoverproductionofsuperoxideradical.Untitled-2 1/8/2007, 6:47 PM 26AntioxidantSystemsinAnimalBody27LivingcellspermanentlybalancetheprocessofformationandinactivationofROSandasaresultROSlevelremainslowbutstillabovezero.Adverseenvironmentalconditionsinitiateattemptsoforganismstoresisttheenvironmentthatbecamemoreaggressive(Skulachev,1998).Cellscanusuallytoleratemildoxidativestressbyadditionalsynthesisofvariousantioxidants(glutathione,antioxidantenzymes,etc.)inanattempttorestoreantioxidant/oxidantbalance.Atthesametime,energyexpendituresincreasedandrespirationactivatedleadingtotheincreasedyieldofROS(Skulachev,1998).Howevertheseadaptivemechanismshavelimitedability.Oncethefreeradicalproductionexceedstheabilityofantioxidantsystemtoneutralisethem,lipidperoxidationdevelopsandcausesdamagetounsaturatedlipidsincellmembranes,aminoacidsinproteinsandnucleotidesinDNAandasaresult,membraneandcellintegrityisdisrupted.Membranedamageisassociatedwithadecreasedefficiencyofabsorptionofdifferentnutrientsandleadstoanimbalanceofvitamins,aminoacids,inorganicelementsandothernutrientsintheorganism.Alltheseeventsresultindecreasedproductiveandreproductiveperformancesofanimals.Immunityincompetenceandunfavourablechangesinthecardio-vascularsystem,brainandneuronesandmusclesystemduetoincreasedlipidperoxidationmakethesituationevenworse.Asitwasshownaboveallantioxidantsinthebodyareworkingasateamresponsibleforantioxidantdefenceandwecallthisteamtheantioxidantsystem.Inthisteamonememberhelpsanotheroneworkingefficiently.Thereforeifrelationships in this team are effective, which happens only in the case of balanceddietandsufficientprovisionofdietaryantioxidantnutrients,thenevenlowdosesofsuchantioxidantsasvitaminEcouldbeeffective.Ontheotherhandwhenthisteamissubjectedtohighstressconditions,freeradicalproductionisincreaseddramatically.Duringthesetimes,withoutexternalhelpitisdifficulttopreventdamagetomajororgansandsystems.Thisexternalhelpisdietarysupplementationwithincreasedconcentrationsofnaturalantioxidants.Fornutritionist or feed formulator it is a great challenge to understand when the internalantioxidantteaminthebodyrequireshelp,howmuchofthishelptoprovideandwhattheeconomicreturnwillbe.Again,itisnecessarytorememberaboutessentialityofkeepingrightbalancebetweenfreeradicalproductionandantioxidantdefence.Indeed,ROSandRNShaveanothermoreattractivefaceparticipatinginaregulationofvarietiesofphysiologicalfunctions(Table1.9).Didyouknowthatallantioxidantsinthebodyareworkingasateamresponsibleforantioxidantdefenceandwecallthisteamtheantioxidantsystem?Untitled-2 1/8/2007, 6:47 PM 2728SeleniuminNutritionandHealthTable 1.9Some regulatory functions of free radicals (adapted from McCord, 2000; Droge, 2002; Yoonet al., 2002; Thundathil et al., 2003; de Lamirande and Gagnon, 2003).Type of radical Sourceofradical PhysiologicalprocessNitric oxide (NO*) Nitric oxide synthase Smooth muscle relaxation (control ofvascular tone) and various other cGMP-dependent functions; specific role in signaltransduction events leading to spermcapacitationSuperoxide(O2-*) NAD(P)H oxidase Control of ventilation; Control ofand related ROS erythropoiteninproductionandotherhypoxia-inductiblefunctions;Smoothmuscle relaxation; Signal transduction fromvarious membrane receptors/enhancement ofimmunologicalfunctionsSuperoxideand Any source Oxidative stress responses and therelated ROS maintenance of redox homeostasis; Anincreased intracellular ROS level stimulatescell proliferation, apoptosis anddifferentiationdependingontherelativeconcentration of oxidants in the cell;Promotesphysiologicalcapacitationinsperm allowing the acquisition of fertilizingcapacityROS Any source Activation of a variety of kinases includingSrc kinase family, preotein kinase C,mitogen-activated protein kinase (MAPK),receptor tyrosine kinases and transriptionalfactors such as AP-1 and NF-kB;Modificationofredox-sensitiveproteins(e.g. thioredoxin)affecting stress kinasesIncommercialanimalandpoultryproductionthereisarangeofstressorsandstress-conditions.Forexample,stressconditionsrelatedtooverproductionoffreeradicalsinpoultryproductioncanbesummarisedasfollows: Increasedintervalbetweenaneggbeinglaidanditscoolingdownforstorage.Eggsshouldbecollectedmorefrequentlyonwarmdays.Insuchconditionsfreeradicaldamagestolipidsandproteinscouldoccurandantioxidantprotectionwouldbebeneficial. EggstoragebeforeincubationcouldbeassociatedwithlipidperoxidationwithintheeggmembranescontaininghighlevelsofPUFAs.IncreasedSeUntitled-2 1/8/2007, 6:47 PM 28AntioxidantSystemsinAnimalBody29concentrationincombinationwithotherantioxidants(vitaminEandcarotenoids)couldbeaneffectivemeanstopreventdamagingeffectsoffreeradicalsproducedwithintheegg. Temperature,humidityandcarbondioxideconcentrationfluctuationsduringincubation.Theyalsocanaffectembryonicdevelopment,oxidationandphosphorylationintheembryonictissuesleadingtofreeradicalproduction.Forexample,highcarbondioxideconcentrationsduringtheincubationperiodcanjeopardisetheliveabilityoftheembryo(Decuypereetal.,2001). Day19ofembryonicdevelopmentisanimportantpointwhenriskoflipidperoxidationisveryhigh.AtthisstagechicktissuesarecharacterisedbycomparativelyhighlevelsofPUFAs(Speakeetal.,1998).Atthesametime,concentrationsofnaturalantioxidants(vitaminEandcarotenoids)havenotreachedmaximum(Surai,1999).Atthisstageofdevelopmentpipingoccurs;andoxygenavailabilityfortissuesincreases.Lowantioxidantstatusincombinationwithhightemperature,humidity,andPUFAscouldincreasesusceptibilitytolipidperoxidation.Didyouknowthatincommercialanimalproductionthereisarangeofstress-conditionsassociatedwithincreasedfreeradicalproduction? Hatchingtimeisconsideredasanenvironmentalstressforthechick. Atthispointnaturalantioxidantconcentrationshavereachedmaximum(Surai,1999),buthighlevelsoflipidunsaturationintissues,decreasingconcentrationofascorbicacid(canlimitvitaminErecycling)andhightemperatureandhumidityincreaseriskoflipidperoxidation. Delayincollectingchicksfromincubator.Sincenotallchicksarehatchedatthesametimebecauseofheterogeneityofthestartingmaterial(eggsfromolderbreedershatchearlierthanthosefromyoungflocksandchicksfromsmallereggshatchearlierthanthosefromlargeeggs;Decuypereetal., 2001), some would be in the incubator for 2-12 hours longer than others.Thisputspressureonantioxidantdefencecapacity.Furthermore,anydelayin food and/or water intake after hatching usually negatively affect a numberofperformanceparametersandadelayoccursinthematurationoftheenzymaticsystemsthatcontrolmetabolism(Decuypereetal.,2001)andfreeradicalproductionandprotectionagainstthem.Untitled-2 1/8/2007, 6:47 PM 2930SeleniuminNutritionandHealth Transportation from hatchery to farm is another source of stress. For breedingcompanieswherechickentransportationcouldinvolveseveralthousandmiles,averyhighdegreeofstressisassociatedwithtemperaturefluctuationanddehydration. Suboptimaltemperaturesinthepoultryhouse.Coldtoleranceaswellasfeathercoverisinfluencedbythyroidhormoneactivity,whichisSe-dependent. HighlevelsofammoniaandCO2inpoultryhouseasaresultofinadequateventilation.Thiscouldsubstantiallydecreaseantioxidantsystemefficiency. Diseasechallenge.Phagocyticimmunecellsthemselvesproducefreeradicalsintheprocessofkillinginternalisedpathogens.Withoutadequateantioxidantnutrientreserves,cellularmachinerycanbedamagedbythefreeradicalstherebyreducingtheeffectivenessoftheimmunecell.Inaddition,Seisconsideredtohaveaspecificroleinimmunesystemregulation,whichcouldbeindependentonitsantioxidantfunctions. Vaccinationisalsoasubstantialstress;andinsomecasesusingvitaminE,forexample,asavaccineadjuvantcanhelpimprovevaccinationefficiency. Inducedmoltingwithfeedwithdrawalisanimportantstressconditionwhendecreasedefficiencyofheterophilfunctionincreasesbirdsusceptibilitytovariousinfections(Kogutetal.,1999).Forexample,colonicinflammation,consistingofheterophilsinfiltratinglaminapropriaandepithelium,occurredmoreofteninmoltedinfectedchickensthaninunmoltedinfectedchickens(PorterandHolt,1993). Mycotoxinsinthefeedcansubstantiallydecreaseantioxidantassimilationfromthefeedandincreasetheirrequirementtopreventdamagingeffectsoffreeradicalsandtoxicproductsoftheirmetabolism,producedasaresultofmycotoxinexposure.Itisnowincreasinglyrecognisedthatatleast25%ofworldsgrainsarecontaminatedwithmycotoxins. Heavymetalsandothertoxicants(dioxine,pesticides,fungicides,herbicides,etc.)inthefeedcanalsocauseanoxidativestress,decreasingimmunocompetence,productiveandreproductiveperformancesandincreasingarequirementforantioxidants.Forexample,thiramawidelyuseddithiocarbamatefungicide,inculturedhumanskinfibroblastsinducedGSHdepletion,leadingtooxidativestress,lipidperoxidationandfinallyUntitled-2 1/8/2007, 6:47 PM 30AntioxidantSystemsinAnimalBody31celldeath(Cereseretal.,2001).Similarly,inarecentstudyforty-onemale,healthyagriculturalsprayers,exposedtopesticidesfor5years,werecomparedwithtwentyonecontrolsmatchedforageandeconomicstatuswithrespecttofreeradicalgeneration,lipidperoxidation,antioxidantstatusandconcentrationofcellularenzymesweredetermined(Prakasametal.,2001).SignificantlyincreasedTBARSlevelsanddecreasedconcentrationsofantioxidantssuchasGSH,alpha-tocopherol,ascorbicacidandceruloplasminwereobservedinsprayerpopulations,whencomparedtocontrols. When Mallard eggs were exposed to diquat dibromide, a commonlyusedaquaticherbicide,significantmanifestationsofoxidativestresswereapparentinhatchlingsandincludedincreasedhepaticlipidperoxidationand decreased brain reduced glutathione concentration (Sewalket al., 2001).Anotherherbicide ANITENIcausedanoxidativestressinpheasantkidneyandliverbydecreasingactivitiesofantioxidantenzymes(Holovskaetal.,1998). Environmentalpollutants cancauseanoxidativestressinbirds.Forexamplepolychlorinatedbiphenyls(PCBs)increasedaGSSG/GSHratiointheliverofAmericankestrels(Hoffmanetal.,1996).Whenchickeneggswereinjectedintotheaircellwith0.4-1.6mgPCB/kgeggincornoilpriortoincubationlipidperoxidationwassignificantlyincreasedintheembryonicliverandadiposetissuesimultaneouslywithsignificantdecreasesinGSH-Pxactivityinthesetissues(Jinetal.,2001). Anotherpollutantdioxinalsocauseddose-dependentincreasesintheproductionofsuperoxideanion,lipid peroxidation and DNA single-strand breaks rat liver and brain (Hassounetal.,2001). Oxidizedfatinthedietcancauseoxidativestressintheintestineincreasingantioxidantrequirementtopreventdamages.Forexampletheintakeofoxidized oil caused a growth depression and increased TBARS concentrationsinplasmaanddecreasedconcentrationsoftocopherols,lutein,beta-caroteneandretinolinplasmaandtissuesofbroilers(Enbergetal.,1996).Furthermore,toxicproductsoflipidperoxidationmaydamagethebrushborder membrane in the intestine (Kimura et al., 1984) decreasing absorptionofantioxidants.Whenachickendietincludesspentfatafteritshightemperature treatment, the fat usually contains peroxides and hydroperoxideswhichcancontributesubstantiallytooxidativestress.Thereforeitisnecessarytoevaluateabenefitanddisadvantagesofusingsuchfatsources. Extensivepreventivemedication(coccidiostatsorotherveterinarydrugsinthediet)candecreaseantioxidantassimilationfromthedietorincreaseUntitled-2 1/8/2007, 6:47 PM 3132SeleniuminNutritionandHealththeirrequirementtodealwithgeneratedstressconditions.Forexample,monensincanstimulatelipidperoxidationinthechickenliver(Salyietal.,1990).Similarly,oralfurazolidonetreatmentofchickenswasassociatedwithadecreasedvitaminEconcentrationandincreasedlipidperoxidationintheirliver(Sas,1993).Otherdrugs,forexampleallopurinol,canalsocause an oxidative stress in broilers and decrease their body weight (Klandorfetal.,2001). VitaminAexcessinthedietisshown(Suraietal.,1998;2000)tocauseanoxidativestressdecreasingvitaminEandcarotenoidconcentrationsintissuesandincreasingtissuesusceptibilitytolipidperoxidation.Thelistofpotentialstressescanvaryfromonepoultryfarmtoanother,butoverproductionoffreeradicalsandthecriticalneedforantioxidantprotectionarecommonfactors.Itisinteresting,thatinwild,birdsareoftenexposedtoelectromagneticfields,whichcauseoxidativestressandsuppressedplasmatotalprotein,hematocritandcarotenoidsinkestrels(FernieandBird,2001).Similarstressescanbeattributedtofarmanimalproduction.Somepotentialstress-conditions, related to the production of free radicals in the digestive tract of humanandanimalsarepresentedinChapter10.Didyouknowthatantioxidant-prooxidantbalanceinthecellisanimportantdeterminantofvariousphysiologicalfunctions?ConclusionsAntioxidant-prooxidantbalanceinthecellisanimportantdeterminantofvariousphysiologicalfunctions.Indeed,oxidativestressoccurswhenthisbalanceisdisturbedduetooverproductionoffreeradicalsorcompromisedantioxidantdefences.Freeradicaloverproductionandoxidativestressareconsideredasapathobiochemicalmechanisminvolvedintheinitiationorprogressionphaseofvarious diseases.. In animal production free radial generation and lipid peroxidationareresponsibleforthedevelopmentofvariousdiseasesaswellasforthedecreaseofanimalproductivityandproductquality(HurleyandDoane,1989;McDowell,2000).Dietaryantioxidantsmaybeespeciallyimportantinprotectingagainstthedevelopmentoftheoxidativestress.Ingeneral,ingestionofexcessiveamountsofantioxidantsispresumedtoshifttheoxidant-antioxidantbalancetowardtheantioxidantside.Thisissupposedtobebeneficial;however,thismayalsoadverselyaffectkeyphysiologicalUntitled-2 1/8/2007, 6:47 PM 32AntioxidantSystemsinAnimalBody33processesthataredependentonfreeradicals,includingprostaglandinproduction,cell division and differentiation (Azzi and Stocker, 2000). Recent evidence suggeststhatcellularoxidationcaninducechangesingeneexpressionduringnormaldevelopment.Conversely,antioxidantssuchasascorbate,glutathione,-tocopherolorcarotenoidsareinhibitorytodifferentiationinmanytypesofcells(AllenandVenkatraj,1992).Didyouknowthatfreeradicalshaveapleasantfacebeingessentialsforaregulationofvariousphysiologicalprocesses?Freeradicalsarenowconsideredtotakepartinsignaltransductioninthecellandatleasttwowell-definedtranscriptionfactors,nuclearfactorkBandactivatorprotein-1havebeenidentifiedtoberegulatedbytheintracellularredoxstate(SenandPacker,1996).Theregulationofgeneexpressionbyoxidants,antioxidants,andredoxstatehasemergedasano