SeminarsinCancerBiology14(2004)473 - PDF document

SeminarsinCancerBiology14(2004)473–486 EnvironmentalandchemicalcarcinogenesisGeraldN.Wogan,StephenS.Hecht,JamesS.FeltonAllanH.Conney,LawrenceA.LoebBiologicalEngineeringDivision,MassachusettsInstituteofTechnology,Room26-009,Cambridge,MA02139,USA Peoplearecontinuouslyexposedexogenouslytovaryingamountsofchemicalsthathavebeenshowntohavecarcinogenicormuta-genicpropertiesinexperimentalsystems.Exposurecanoccurexogenouslywhentheseagentsarepresentinfood,airorwater,andalsoendogenouslywhentheyareproductsofmetabolismorpathophysiologicstatessuchasinammation.Ithasbeenestimatedthatexposuretoenvironmentalchemicalcarcinogensmaycontributesignicantlytothecausationofasizablefraction,perhapsamajority,ofhumancancers,whenexposuresarerelatedto“life-style”factorssuchasdiet,tobaccouse,etc.Thischaptersummarizesseveralaspectsof Correspondingauthor.Tel.:16172533188;fax:16172580499.E-mailaddresses:wogan@mit.edu(G.N.Wogan),hecht002@umn.edu(S.S.Hecht),felton1@llnl.gov(J.S.Felton),aconney@rci.rutgers.edu(A.H.Conney),laloeb@u.washington.edu(L.A.Loeb).1044-579X/$–seefrontmatter©2004ElsevierLtd.Allrightsreserved.doi:10.1016/j.semcancer.2004.06.010 G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486syntheticprocesses.Baseduponthedisparitybetweentheinfrequencyofspontaneousmutationsandthelargenumbersofmutationsreportedinhumantumors,ithasbeenpostulatedthatcancersmustexhibitamutatorphenotype,whichwouldrepresentanearlyeventincancerprogression.Amutatorphenotypecouldbegeneratedbymutationsingenesthatnormallyfunctiontoguaranteegeneticstability.ThesemutationspresumablyariseviaDNAdamagebyenvironmentalorendogenousagents,butitremainstobedeterminedwhethertheacquisitionofamutatorphenotypeisanecessaryeventduringtumorprogression.©2004ElsevierLtd.Allrightsreserved.Keywords:Aatoxin;Livercancer;Hepatitisviruses;Tobaccocarcinogens;Lungcancer;Heterocyclicamines;Coloncancer;Carcinogenmetabolism;Geneticpolymorphism;Mutatorphenotype 1.Validationofacausalrelationshipbetweenaßatoxinexposureandhepatocellularcarcinomariskinhumans:amolecularepidemiologyparadigmdemonstratingthepowerofbiomarkersTheMonographsProgramontheEvaluationofCarcino-genicRiskstoHumansoftheInternationalAgencyforResearchonCancer(IARC)publishesauthoritativecarcino-genicriskassessmentsbasedonexaminationbyexpertsofallrelevantinformationtoassessthestrengthofavailableevidencethatexposurestothechemicalscouldaltertheincidenceofcancerinhumans.Todate,theseevaluationshaveidentiedatotalof88agents,mixturesandexposuresthatareclassiedinGroup1,“carcinogenictohumans”.Includedare:64agentsandgroupsofagents(22drugs;14environmentalchemicals;14radiation;10viruses,bacteriaandparasites;and4inorganicbers);12mixtures;and13exposurecircumstances[].Nearlyalloftheseriskswererstidentiedthroughobservationalepidemiology,thenveriedbysupplementarystudiesinan-imalsandotherexperimentalsystems.Themold-producedaatoxinsareamongthefewenvironmentalchemicalsinthislistthatwererstidentiedascarcinogensinanimals,andsubsequentlyshowntoposecarcinogenicriskstohu-mansthroughepidemiologicstudies.Extensiveresearchhasproducedacomprehensivedatabaseaddressingrisksresultingfromthehighprevalenceoftheircontaminationofmajorfoodstaplesinmanypartsoftheworld,togetherwiththeircarcinogenicpotencyinanimals.Indeed,theaatoxin-livercancerriskrelationshipisamongthemostextensivelydocumentedexamplesdemonstratingthesig-nicanceofawidelydisseminatedenvironmentalchemicalcarcinogenasadeterminantofincreasedriskforamajorformofcancer.Continuingresearcheffortsstimulatedbytheirdiscoveryintheearly1960sproducedanextensivebodyofevidenceregardinghumanhealthrisksresultingfromaatoxinexposure.Collectively,epidemiologicdatatogetherwithevidencefrommanytypesofexperimentalmodelsdenestheroleofaatoxinexposureinHCCcau-sation.Itisinformativetoreviewthisinformationforper-spectiveregardingthemultifactorialetiologyofthediseaseand,inparticular,thecriticalroleofwell-validatedmolec-ularbiomarkersinestablishingthecausalexposure-riskChronicinfectionsbythehepatitisB(HBV)orhepatitisC(HCV)virusesaremajorriskfactorsforthegreatma-jorityofHCCcasesworldwideorldwide.TheyalsoarelargelyresponsibleforthegeographicalpatternofHCCincidence,HBVbeingthedominantcauseindevelopingcountriesofsubSaharanAfricaandAsia,whileHCVisthemajorriskfactorindevelopedcountrieswithahighorintermediatein-cidence.Evidencesupportingtheseconclusionshasrecentlybeensummarizedsummarized.CarrierratesofHBVinAfricanandAsianpopulationsmaybeashighas20%,andtheinfectionisacquiredearlyinlifeasaresulteitherofperinatalinfec-tionbyacarriermotherorbyhorizontalpassagefromin-fectioussiblings.MalesacquiringcarrierstatusearlyinlifeareatveryhighriskofdevelopingHCC,withalifetimerel-ativerisk(RR)of100calculatedforTaiwanesemen.Thus,chronicinfectionwithHBVhasbeenstatedtobethesin-glemostcommoncauseofglobalHCCHCC.TheimportanceofHCVinfectioninthecausationofHCChasbeenrecog-nizedmorerecently,andinteractiveeffectsbetweenthecar-cinogenicityofHBVandHCVhavebeendemonstratedinmost,butnotall,populationsinwhichithasbeenstudied.MechanismsthroughwhichtheseinfectionscauseHCCarestillunknown,althoughbothdirectandindirectactionsarethoughttobeinvolved.ThepotencyofHBVinfectionovershadowedrecogni-tionofthesignicanceofaatoxinexposureasacauseofHCC,evidenceofwhichhasmountedoveraperiodofseveraldecades.Aatoxinsbelongtoalargegroupofmy-cotoxins,toxicmetabolitesthatcontaminatefoodandfeedcommoditiesduringgrowthofcertainspoilagemolds.Inadditiontocausingacutetoxicity,aatoxinsarealsolivercarcinogensinexperimentalanimalsandextensivequalitycontrolmeasuresarenecessarytominimizelevelsinhumanfoods.Aatoxin-contaminatedfeedwasdiscoveredtobealivercarcinogeninratsevenbeforetheactiveagentwasisolatedandcharacterized.SubsequentexperimentswithchemicallypuretoxinshowedthatHCCwasinducedinsensitivespecieswhenaatoxinB),themajorcom-ponentofmixturestypicallyfoundinfoodrawmaterials,wasfedatlevelsaslowas15ppb(g/kg)inthediet.Bioas-saysinvariousspeciesofsh,birds,rodentsandsub-humanprimateseventuallyrevealedthatAFBisalivercarcino-geninallanimalstested.Althoughthereiswidevariationinsensitivity,nocompletelyrefractoryspecieshasbeen G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486identied.Thesedataclearlyimplicateaatoxinasapo-tentiallivercarcinogeninhumans,andtheplausibilityofthisimplicationissupportedbymuchadditionalexperi-mentalevidencevidence.Aatoxinisstronglymutagenicintestsystemsrangingfrombacteriatohumancellsinculture,requiringmetabolicactivationbycytochromeP450;path-waysofaatoxinmetabolismaresimilarincellsandtissuesofsusceptibleanimalsandhumans,includingtheepoxi-dationpathwayresultingincovalentbindingtoDNA;theDNAadductprole,withtheaatoxin--guanineadduct-gua)representingthemajoradduct,isidenticalinanimalandhumancellsmutagenizedbyaatoxin;adductlevelinliverDNAisquantitativelyrelatedtoaatoxindoseandtotumoryield;andchemopreventionofDNAadductformationinhibitstumorigenesisinexperimentalanimals.Aparadigmforvalidatingcausalrelationshipsutiliz-ingmolecularepidemiologyisoutlinedinScheme1[7]Thecarcinogenicpotencyofaatoxininanimalstogetherwiththeirfrequentcontaminationofhumanfoodsstimu-latedcross-sectionalepidemiologicinvestigationstoassessrelationshipsbetweenexposureandincidenceofHCC.Collectively,studiesconductedinsubSaharanAfricaandAsiabetween1965and1985revealedahighlysignicantassociationbetweenAFBintake,calculatedfromanalysisoffoodsasconsumed,andHCCincidenceestimatedfromcancerregistrydatadata.Basedontheseepidemiologicdata,togetherwiththebodyofexperimentalevidenceoutlinedabove,AFBwasdesignatedasaknownhumancarcinogen(group1)byIARCin19931993.Parenthetically,itisnote-worthythatthisdesignationwasassignedintheabsenceofinformationabouttheprevalenceofHBVinfectioninthepopulationsstudied. Chemical Carcinogen in AnimalsSuspect HumanCarcinogen/Disease LinkageDetermine RelationofBiomarker toExposureand Disease inExperimental AnimalsLongitudinal Study ofBiomarkers in HumansValidated ExposureMarkerValidated RiskMarkerCase-control StudiesCohort StudiesClinical TrialsCross-sectional Studyof Biomarker Levels inExposed HumansIdentify and Develop Methodologies forMeasuring Chemical Specific BiomarkersModulation ofBiomarkerand Disease inAnimalChemoprevention Studies Scheme1.Aparadigmforvalidatingcausalrelationshipsbetweencarcinogenexposureandcancerriskutilizingmolecularepidemiology.Statisticalassociationofcarcinogenexposurewithcancerincidencedoesnotprovidedirectevidenceofacause-effectrelationship.Consequently,despitethemountingdatabasesupportingitsbiologicalplausibility,thesignicanceofaa-toxinasariskfactorforhepatocellularcarcinomainhumansremaineduncertain.Lackofcapabilitytoassessaatoxinexposureofindividualswithinstudypopulationswasrec-ognizedasaseriouslimitationoftheaboveepidemiologicstudies.Inresponsetothisneed,aatoxin-DNAandserumalbuminadductsweredevelopedandvalidatedasbiomark-erscapableofdetectingexposureonanindividualbasisinlargenumbersofsubjectssubjects.Inratsandotherexperi-mentalanimals,aatoxinismetabolizedinlivertooxidizedderivatives,including,thatareexcretedinurineandbile,withasmallproportion(about1%oftheingesteddose)beingactivatedthroughtheepoxidetoformcovalentadductsinDNA.AFB-gua,themajoradduct,isefcientlyre-movedfromDNAandexcretedinurinewhereit,aswellasothermetabolites,canbedetectedbyasensitiveana-lyticalprocedureinvolvingimmunoafnity/HPLCpurica-tionanduorescencedetection.Aatoxinadductsarealsoformedinserumalbuminandcanbedetectedbyimmunoas-say.Measurementsofurinaryandserumaatoxinadductlevelsreectrecent(72h)orchronic(11days)exposures,respectively.Formationofaatoxin-DNAadductsinliver,excretionoftheurinaryadductandformationoftheserumalbuminadductarehighlycorrelated,andmeasurementsofthetwobiomarkersprovidecomplementaryexposureinfor-Earlystudiesinpopulationsconsumingcontaminateddietsshowedthathumanshadthemetaboliccapacitytoproduceaatoxinmetabolitespreviouslydetectedin G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486experimentalanimals.Subsequentdose-responsestudiesinsmallgroupsofsubjectsinthePRCandTheGambia,WestAfrica,areaswhereaatoxincontaminationoffoodsisprevalent,measuredbothdietaryaatoxinintakeandlevelsofurinaryaatoxinbiomarkersers.UrinaryAFBandAFM1excretionshowedadose-dependentrelationshiptoaatoxinintake,andadditionalstudiesshowedasimilarrelationshipwithadductsinserumalbumin.Importantly,thekineticsofformationandexcretionofAFB-guainurinewerefoundtobesimilarinhumansandrats.Combineddatafromstudiesinratsandexposedhumansthereforeindicatedthaturinaryandserumadductlevelswerevalidbiomarkersofexposureandbiologicallyeffectivedose,strengtheningthevalidityofcross-speciesextrapolationinassessmentofHCCriskposedbyaatoxiningestion.ThevalidatedbiomarkerswereemployedinanevaluationofHBVandaatoxinasindependentandinteractiveriskfactorsforHCCin�18,000maleresidentsofShanghaiShanghai.Datafromthisnestedcase–controlstudyrevealedastatisticallysignicantincreaseintheRRof3.4forHCCcasesinwhomaa-toxinbiomarkers,butnoevidenceofHBVinfection,weredetected.ForHBsAg-positiveindividualswithoutaatoxinbiomarkers,theRRwas7,whereasforthosepositiveforbothaatoxinandHBVbiomarkerstheRRwas59.Thesendingshavebeenconrmedinsubsequentstudiesofsim-ilardesigninTaiwanan,andinasubsequentprospectivestudyinthePRCPRC,strengtheningtheconclusionthataatoxinisacausativeagentforHCCandsubstantiallyampliestheriskcreatedbyHBVinfection.Thesedataindicateindependentcausalrelationshipsbetweenthepres-enceofaatoxin-andHBV-specicbiomarkersandtheriskofHCC,andalsodemonstratethepowerofvalidatedaatoxinbiomarkerstodeneapreviouslyunrecognizedcarcinogen–viralinteractionintheinductionofthedisease.Mechanismsthroughwhichaatoxinexertsitscarcino-genicityandmultiplicativeinteractionwithHBVinfectioninHCCinductionhavenotyetbeenelucidated,andaresub-jectsofactivecurrentresearch.OnepathwaythroughwhichaatoxinmaycontributetoHCCriskrelatestoitscapacitytoinduceG:CtoT:Atransversionsasapredominantmutation.Thistypeofmutationhasbeenidentiedathighfrequencyinthep53tumorsuppressorgene,withspecicclusteringatthethirdbaseofcodon249,inHCCoccurringinpopu-lationsexposedtohighlevelsofdietaryaatoxinaatoxin.FewsuchmutationswerepresentinHCCofpatientsresidinginregionsoflowaatoxinexposure.Signicantly,AFBbeenshownpreferentiallytoinducethep53codon249G:CtoT:Amutationinculturedhumanhepatocytes.Thesere-sultsprovidefurthersupportfortheplausibilityofaatoxinasanetiologicalfactorinHCC.EvidencethataatoxinincreasesriskofHCCinpatientswithHBVhepatitiswouldsuggestthatmeasurestoreduceexposuretoaatoxinmightbebenecialtomenwithHBVhepatitisandcouldbeevaluatedinsuitablydesignedpro-tocols.Reductionofexposurepersethroughdietarymod-icationisdifcult,butprotectionthroughmodulationofaatoxinmetabolismmaybemorereadilyachieved.IthasrecentlybeenreportedreportedinaphaseIIchemopreventiontrialbeingconductedinQidong,PRC,thatintermittentorsustainedadministrationofoltipraz,aninducerofphase2metabolizingenzymes,signicantlyincreasedbiomarkersofaatoxindetoxication.Theseresultshighlightthefea-sibilityofthisorrelatedapproachesasachemopreventivestrategyinpopulationsathighriskforHCC.2.Evaluationoftobaccocarcinogens:amodelforenvironmentalcarcinogenesisTobaccoproductsprovideaclearexampleofcancercau-sationbyalife-stylefactorinvolvingcarcinogenexposure.Thereareoveronebillionsmokersandhundredsofmillionsofsmokelesstobaccousersworldwide.Tobaccouseisbyfarthemostwidespreadlinkbetweenexposuretoknowncar-cinogensanddeathfromcancer,andliketheaatoxin-HCCrelationship,canbeconsideredamodelforunderstandingmechanismsofcancerinductionbyexogenouschemicalcar-2.1.TobaccoproductsandcancerTheIARCMonographentitled‘TobaccoSmokeandIn-voluntarySmoking’,tobepublishedin2004,concludedthefollowingbasedonanevaluationoftheworld’sliteratureliterature.Cigarettesmokingincreasestheriskofallhistologi-caltypesoflungcancer.Itcausescanceroftheoralcav-ity,andthisriskisgreatlyincreasedbytheuseofsmoke-lesstobaccoorbyalcoholconsumptionincombinationwithsmoking.Cigarettesmokingisalsocausallyassociatedwithlaryngeal,oropharyngealandhypopharyngealcancer,andincreasestherisksforsinonasalandnasopharyngealcancer.Cigarettesmokingiscausallyassociatedwithcanceroftheesophagus,bothsquamouscellcarcinomaandadenocarci-noma.Furthermore,cigarettesmokingcausescancerofthestomach,liver,andpancreas,aswellastransitionalcellcar-cinomasofthebladder,ureterandrenalpelvis,andrenalcellcarcinoma.Finally,cigarettesmokingisalsoacauseofsquamouscellcervicalcarcinomaandmyeloidleukaemia,andtheriskofcolorectalcancercanalsobeincreasedbysmoking.Environmentaltobaccosmoke(ETS)causeslungcancer.Smokelesstobaccoproductsareestablishedcausesoforalcavitycancercancer.2.2.TobaccocarcinogensandcancerThecentralroleoftobaccocarcinogensandtheirDNAadductsintobacco-inducedcancerisillustratedinScheme22.Carcinogensarethekeyconnectionbetweennicotineaddictionandcancer.Nicotineaddictionisthemajorreasonwhypeoplecontinuetousetobaccoproducts.Whilenicotineitselfisnotcarcinogenic,eachcigaretteordipofsmokelesstobaccocontainsamixtureofcarcinogens,tumorpromoters, G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486 Scheme2.Schemelinkingnicotineaddictionandcancerviatobaccocarcinogens.andco-carcinogens.Mosttobaccocarcinogensrequiremetabolicactivationtoexerttheircarcinogeniceffects;therearecompetingdetoxicationpathwaysandthebal-ancebetweenmetabolicactivationanddetoxicationdiffersamongindividualsandaffectscancerrisk.ThisisshowninthecentraltrackofScheme2[16]MetabolicactivationleadstotheformationofDNAadducts,whicharecarcinogenmetabolitesboundcovalentlytoDNA.DNAadductsareabsolutelycentraltothecarcino-genicprocess.Iftheirformationisinhibitedorblocked,soiscarcinogenesiscarcinogenesis.IfDNAadductsescapecellularrepairmechanismsandpersist,theymayleadtomiscoding,resultinginpermanentmutations.CellswithdamagedDNAmayberemovedbyapoptosis,orprogrammedcelldeath.Ifapermanentmutationoccursinacriticalregionofanonco-geneortumorsuppressorgene,itcanleadtoactivationoftheoncogeneordeactivationofthetumorsuppressorgene.Multipleeventsofthistypeleadtoaberrantcellswithlossofnormalgrowthcontrolandultimatelytocancer.TheseeventsarealsoshowninthecentraltrackofScheme2ThechronicbarrageofDNAdamagebytobaccocarcino-gensinpeoplewhousetobaccoproductsiscompletelyconsistentwiththemultiplegeneticchangesobservedintobacco-inducedcancersandwithourcurrentunderstandingoftheroleofgeneticaberrationsincancerinduction.TheuppertrackofScheme2showsthatnicotineandtobacco-specicnitrosaminescanbinddirectlytocertainre-ceptorsleadingtoactivationofcellularregulatoryfactorssuchasAKT.Thiscanresultultimatelyindecreasedapop-tosis,increasedangiogenesis,andincreasedcelltransforma-tion.ThesechangesmayenhancetheeffectsofcarcinogensandtheirDNAadducts.ThelowertrackofScheme2lustratesthecontributionsofcofactorssuchastumorpro-motersandco-carcinogensintobaccoproducts,orirritationandviruses(forexample,intheoralcavity)whichmayen-hancetheactivityoftobaccocarcinogensthroughavarietyofmechanisms.Tobaccoproductscontainadiversearrayofchemicalcar-Table1presentsanoverviewofcarcinogensintobaccoproducts.Morethan60knowncarcinogenshavebeendetectedincigarettesmoke.SeveralcarcinogenslistedTable1havebeendetectedonlysporadically,butmostareroutinelyfound.AllofthecarcinogensinTable1havebeenformallyevaluatedbytheIARC,andineachcase,studiesineitherlaboratoryanimalsorinhumanshavepro-videdsufcientevidenceofcarcinogenicity.Thereisalargerangeofpotenciesandconcentrationsamongthesecarcino-gens.Ingeneral,thestrongercarcinogenssuchaspolycyclicaromatichydrocarbons(PAHs),nitrosamines,andaromaticaminesoccurinloweramountsincigarettesmoke(1–200ngpercigarette)thantheweakercarcinogenssuchasacetalde-hyde(nearly1mgpercigarette).Thetotalamountofcar-cinogensincigarettesmokeaddupto1–3mgpercigarette(similartotheamountofnicotine,0.5–1.5mgpercigarette),althoughmostofthistotaliscomprisedofweakercarcino-genssuchasacetaldehyde,catechol,andisoprene.Unburnedtobacco,includingcigarettetobacco,oralsnuff,chewingtobacco,andothersmokelesstobaccoproducts,Table1Carcinogensinsmokeandunburnedtobaco ChemicalclassNo.ofRepresentative TobaccosmokePAH14BaP,dibenz[a,h]anthraceneNitrosamines8NNK,NNNAromaticamines124-Aminobiphenyl,Aldehydes2Formaldehyde,acetaldehydePhenols2CatecholVolatilehydrocarbons3Benzene,1,3-butadieneNitrocompounds3NitromethaneOtherorganics8Ethyleneoxide,acrylonitrileInorganiccompounds9CadmiumTotal61UnburnedtobaccoChemicalclassNo.ofRepresentativePAH1BaPNitrosamines6NNK,NNNAldehydes2Formaldehyde,acetaldehydeInorganiccompounds7CadmiumTotal16 G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486Table2Evaluationofspeciccarcinogensascausesoflungcancerinsmokers Compound(s)PresenceincigarettesmokecarcinogenicityinrodentsuptakeHumanmetabolismandadductformationMolecularchangesinhumangenesOverall SpecicPAHs4443318Aza-arenes33112104443318Metals4411111Miscellaneousorganic4311110Freeradicals/oxidative3133111 Scores:1=inadequatedata;2=weakorequivocalevidence;3=someevidence,limitedstudies;4=clearevidence,strongreproduciblestudies.containsfewercarcinogensthancigarettesmokebecausemostinsmokeareformedduringcombustion(Table1LevelsofPAHinunburnedtobaccoaretypicallylow.Ni-trosamines,particularlythetobacco-specicnitrosamines4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)nitrosonornicotine(NNN),arebyfarthemostpreva-lentstrongcarcinogensinunburnedtobaccotobacco.ThelevelsofNNKandNNNinsmokelesstobaccoproductsarehundredstothousandsoftimeshigherthanthoseofcarcinogenicnitrosaminesinanyotherconsumerproductdesignedforingestioningestion.2.3.EvaluatingtheroleofspeciÞccarcinogensintobacco-relatedcancerWhenconsideringtherelationshipbetweentobaccocar-cinogenexposureandspecictypesofcancer,wehavethe“advantage”ofaknownexposureandaknowncancerend-point.Wearealsoaidedbythecomprehensivecharacteri-zationoftobaccoproductchemistrythatisavailableintheliterature.Tobaccouseisunfortunatelythelargestvoluntarycarcinogenexposureexperimentinhistory,andisstillon-going.Themajordisadvantage,fromthepointofviewofrelatingspeciccarcinogenstotobacco-inducedcancer,isthattheexposuresarealwaystomixturesoftobaccocar-cinogens,alongwithcofactorssuchastumorpromotersandcocarcinogens.Thisclearlycomplicatesthetaskofrelatingparticulartobaccocarcinogenstospeciccancertypes.Nevertheless,aweightoftheevidenceapproachcanbetakenen.Thisisillustratedforcigarettesmokecarcino-gensandlungcancerinTable2.Thecriteriausedforevalu-ationarethepresenceofthecompoundsincigarettesmoke,theirpulmonarycarcinogenicityinlaboratoryanimals,theirhumanuptake,metabolismandadductformation,theirpos-sibleroleincausingmolecularchangesinoncogenesorsup-pressorgenes,andotherrelevantdata.Usingthisapproachandassigningascoretoeachgroupofcompoundsasillus-tratedinTable2,theconclusionisthatconsiderableevi-dencefavorsPAHsandNNKasmajoretiologicalfactorsintobacco-inducedlungcancer(Table3PAHsarestronglocally-actingcarcinogens,andtobaccosmokefractionsenrichedinthesecompoundsarecarcino-carcino-.PAH-DNAadductshavebeendetectedinhu-manlungsamples,andmutationsintheTp53geneisolatedfromlungtumorsaresimilartothoseproducedinvitrobyPAHdiolepoxidemetabolites.NNKisastrongsystemiclungcarcinogeninrodents,inducinglungtumorsindepen-dentlyofitsrouteofadministrationadministration.ThestrengthofNNKisparticularlygreatintherat,inwhichtotaldosesaslowas6mg/kg(and1.8mg/kgwhenconsideredaspartofadose-responsetrend)haveinducedasignicantincidenceoflungtumors.Thiscomparestoanestimated1.1mg/kgdoseofNNKin40yearsofsmoking.DNAadductsde-rivedfromNNKortherelatedtobacco-specicnitrosamineNNNarepresentatahigherlevelinlungtissuefromlungcancerpatientsthancontrols,andmetabolitesofNNKarefoundintheurineofpeoplewhousetobaccoproductsorareexposedtoETSETS.Epidemiologicdataindicatethatasystemiccarcinogencauseslungcancerincigarsmokerswhodonotinhale;thisisconsistentwiththetumorigenicpropertiesofNNK.Thechanginghistologyoflungcan-cer,inwhichadenocarcinomahasnowovertakensquamouscellcarcinomaasthemostcommonlungcancertype,isalsoconsistentwiththeroleofNNK,whichproducespri-marilyadenocarcinomainrodents.NNKconcentrationsinTable3Rolesofspecictobaccocarcinogensintobacco-inducedcancersinhu- CancertypeLikelycarcinogeninvolvement LungPAH,NNK(major)1,3-butadiene,isoprene,ethyleneoxide,ethylcarbamate,aldehydes,benzene,metalsLarynxPAHNasalNNK,NNN,othernitrosamines,aldehydesOralcavitySmokersPAH,NNK,NNNSmokelesstobaccoNNK,NNNEsophagusNNN,othernitrosaminesLiverNNK,othernitrosamines,furanPancreasNNK,NNALCervixPAH,NNKBladder4-Aminobiphenyl,otheraromaticaminesLeukemiaBenzene G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486mainstreamsmokeincreased,whilethoseofBaPdecreased,asnitrateconcentrationsintobaccoincreasedoverthepe-riodof1959–1997duetotheuseoftobaccoblendscontain-inghigherlevelsofair-curedtobacco,useofreconstitutedtobacco,andotherfactors.Usingthisweightoftheevidenceapproach,theroleofvariouscarcinogensascausesofspecictobacco-inducedcancersotherthanlungcanalsobeestimated(Table3).Theparticulatephaseofcigarettesmokecausestumorsofthelarynxinhamsters—thismaybeattributedtoPAH.Tp53genemutationsidentiedintumorsofthehumanlarynxsupportaroleforPAHinthedevelopmentofthiscancer.Nitrosamines,aswellasacetaldehydeandformaldehyde,inducenasaltumorsinrodentsandarelikelycausesofsmoking-associatednasaltumors.Basedonanimalstudies,PAH,NNKandNNNarethemostlikelycausesoforalcan-cerinsmokers.NNKandNNN,perhapstogetherwithen-hancingagents,maycauseoralcancerinsmokelesstobaccousers,becausetheyarethemostprevalentstrongcarcino-gensintheseproductsandtheyinduceoraltumorsinrats,whenco-administered.Nitrosaminesarethemosteffectiveesophagealcarcinogensknown,andNNN,whichproducestumorsoftheesophagusinrats,isthemostprevalentni-trosaminecarcinogenincigarettesmoke.NNK,severalothercigarettesmokenitrosamines,andfu-ranareeffectivehepatocarcinogensinrats.NNKanditsma-jormetabolite4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL)aretheonlyknownpancreaticcarcinogenstowhichpeoplewhousetobaccoproductsareexposed,andbiochem-icaldatafromstudieswithhumantissuessupporttheirroleinsmokingrelatedpancreaticcancer.BiochemicalstudiesdemonstratethatNNKandPAHcanreachthecervixinhu-mans,andaremetabolicallyactivatedthere.DNAadductsderivedfromBaPandotherhydrophobiccompoundshavebeendetectedincervicaltissueofsmokers.Therefore,thesecompoundsmaycontributetotheetiologyofcervicalcan-cerinsmokers,incombinationwithhumanpapillomavirus.4-Aminobiphenyland2-naphthylamineareknownhumanbladdercarcinogens,andconsiderabledatafromhumanstudiessupporttheroleoftheseandotheraromaticaminesasthemajorcauseofbladdercancerinsmokers.Themostprobablecauseofleukemiainsmokersisbenzene,whichoccursinlargequantitiesincigarettesmoke,andisaknowncauseofacutemyelogenousleukemiainhumans.Thecriteriausedhereforevaluationoftherolesofspecictobaccocarcinogensintobacco-inducedcancer,namelythepresenceofthecompoundintobaccoproducts;itscarcino-genicityinlaboratoryanimals;itshumanuptake,metabolismandadductformation;itspossibleroleincausingmolecularchangesinoncogenesorsuppressorgenes;andotherrele-vantdatacanallbeappliedtoenvironmentalcarcinogens.Itisonlynecessarytosubstitute“presenceofthecompoundintobaccoproducts”for“presenceofthecompoundintheenvironment”.Inthisway,evaluationoftobaccocarcino-gensservesasamodelforevaluationofenvironmentalcar-Themajorgapinthisevaluationschemeistherelativepaucityofprospectiveepidemiologicstudiesthathaveusedmolecularbiomarkersofspecictobaccocarcinogens,suchascarcinogen-DNAadducts,carcinogen-proteinadducts,andcarcinogenmetabolitesinbloodorurine.Whilesev-eralstudieshaveexaminedlevelsofDNAadductsinsmokerswithrespecttodevelopmentofcertaincancers,theyusednonspecicmethodssuchasimmunoassayandP-postlabelling,resultsofwhichcannotbetracedtoindi-vidualcarcinogens.Prospectiveepidemiologicstudiesthatincorporatespecictobaccocarcinogenbiomarkerswouldprovidethemostconvincingevidencethatagivencarcino-genwasrelatedtocancerdevelopment.Furthermore,suchstudiesultimatelycouldprovideinformationthatmightbeincorporatedintoapredictivemodelofindividualcancersusceptibility.Suchmodelswouldbeextremelyusefulintobacco-relatedcancercontrol.2.4.SummaryTobaccocarcinogensandtheirDNAadductsareab-solutelycentraltocancerinductionbytobaccoprod-ucts.Thecontributionofspecictobaccocarcinogenstotobacco-inducedcancercanbeevaluatedbyaweightoftheevidenceapproach.Examplesweregivenforvari-ouscarcinogensandtobaccorelatedcancers,e.g.theroleofPAHandNNKinlungcancer.Factorsconsideredinthisapproachincludethepresenceofthecompoundintobaccoproducts,itscarcinogenicityinlaboratoryani-mals,itshumanuptake,metabolismandadductformation,itspossibleroleincausingmolecularchangesinonco-genesorsuppressorgenes,andotherrelevantdata.Allofthesefactorscanbeappliedtoevaluationofenviron-mentalcarcinogens.Itisonlynecessarytochange“pres-enceofthecompoundintobaccoproducts”to“presenceofthecompoundintheenvironment”.Theseevaluationswouldbemarkedlyfacilitatedbyprospectiveepidemiologicstudiesthatincorporatephenotypiccarcinogen-specicbiomarkers,butfewsuchstudieshavebeencarriedoutto3.Heterocyclicaminecarcinogensinourdiet:etiologicalagentsforhumancancer?Humanriskestimatesandcanceretiologyattributedtotheconsumptionofmutagensandcarcinogensinourfoodaredifculttoevaluate,asthesetoxicantscomefromnu-meroussourcesinourdiet.Asdiscussedabove,mycotox-ins,suchasaatoxinareformedbyfungigrowingonpoorlystoredgrainproductsandcanbestronglivercar-cinogens,especiallyinindividualsinfectedbythehepatitisBvirus.PAHsuchasbenzo[a]pyrene,ascombustionprod-ucts,arepresentinwoodresoramegrillingandcanbedepositedonfoodfromfatdrippingontothecoalsduringthistypeofcooking.Thesecompoundsaregenerallypotent G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486carcinogensinexperimentalanimals,butarequiteubiqui-tousinourenvironment.Anotherimportantclassofcarcinogensinfoodistheheterocyclicamines.Thesecompoundsarepotentmutagensandmoderatelypotentcarcinogensatnumerousorgansitesinrodentsandintheliverofnon-humanprimates.Theyareproducedwhenmusclefoodsareheatedabove180Cforlongperiodsoftime.Thelevelsinmeatproductscanreachhundredsofpartsperbillion(ppb),butaregenerallylowerinchickenandbeefcookedtoawell-donestate.Byvirtueoftheirfrequentpresenceinmeat,theypresentaprob-leminwidespreadexposureinthedietofnon-vegetarians.Atleastsixteen,andpossiblymore,differentheterocyclicamineshavebeenisolatedfromcookedfoods.Fourofthesecompoundscanconsistentlybeidentiedinwell-donemeatproductsfromtheNorthAmericandiet.Themajor-ity(morethan75%)ofepidemiologystudiesdesignedtoevaluatethehealthsignicanceoftheseanimalcarcinogenshavelinkedconsumptionofwell-donemeatproductstocancerofthecolon,breastandstomach.However,acausallinkagehasnotbeenrmlyestablished,sincesomewell-designedstudiesfoundnostatisticallysignicantpositivecorrelationbetweenconsumptionofdietscontaininghet-erocyclicaminesandincidenceofcolonorothercancers.StudiesemployingDNAadductsandurinemetabolitesasmolecularbiomarkerssuggestthatindividualsmaydifferintheirsusceptibilitytothesecarcinogens.Polymorphismsinmetabolicactivationanddetoxication,aswellasDNArepairgenesmaycontributetothisvariability.Variableex-posuresandgeneticdifferencesinalargenumberofgenes,eachprobablyhavingsmallimpact(i.e.,lowpenetrance)complicateestimationofindividualrisks.Additionalvari- Scheme3.Thereareanumberofcomplexstepsbetweencarcinogenformationandcancer.Theyshouldmakemechanisticsenseandcontributetothepartsofanetiologicalpuzzlebeforeimplyingcausationofhumancancer.ablesareintroducedbymodulationofinternaldoseandexposurebyinteractionswithotherfoodsinthedietaswellasindividualdifferencesinadsorptionofthecarcinogens.Itisthereforeessentialtoputthesevariablesintoperspective,inassessmentofriskstothegeneralpopulationattendanttoconsumptionofdietarycarcinogens.Arelatedchemical,acrylamide,hasrecentlybeeniden-tiedinstarch-basedfoodssuchaspotatochipsandFrenchfriescookedusinghightemperaturedeep-fryingandbak-ingmethods.Thiscompoundisweakly-ornon-mutagenicininvitrotestsandweaklycarcinogenictoexperimen-talanimals,butincomparisontotheheterocyclicaminesarepresentinlarge(part-permillion)quantitiesinthesestarch-derivedproducts.Thesecompoundsdonothavethestronggenotoxicpropertiesoftheotherfood-bornecarcinogensdiscussedinthisChapter,suchasaatoxin,benzo[a]pyreneandtheheterocyclicamines,buttheexposurelevelsmaybequitehigh.Intermsofmodeofaction,currentknowledgeabouttheheterocyclicaminesindicatesthattheunderlyingmecha-nismsarecomparabletothoseinvolvedinthecarcinogenic-ityofmanyotherenvironmentalcarcinogens(seeScheme3Exposure,metabolism,DNAdamage,repairofthedamage,mutationsandtumorsallresultfromorareimpactedbytheetiologicalagent.Forheterocyclicaminesallthesestepstintotheetiologicalpathway.4.ExtrapolationofanimalcarcinogenesisstudiestohumansÑwhichhuman?MostchemicalcarcinogensarenotcarcinogenicpersebutmustbemetabolizedbyafamilyofcytochromeP450 G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486enzymestochemicallyreactiveelectrophilespriortoreact-ingwithDNAtoinitiateacarcinogenicresponse.ThesesamecytochromeP450enzymes–aswellasenzymesthatactonthemetabolicproductsofthecytochromesP450(e.g.glucuronyltransferase,glutathione-transferaseandothers)–alsometabolizechemicalsbyinactivationpathways,andtherelativeamountsofenzymesthatmetabolicallyactivateanddetoxifythechemicalwilldeterminewhetheritiscar-cinogenic.Becausebothgeneticandenvironmentalfactorsinuencethelevelsofenzymesthatmetabolicallyactivateanddetoxifychemicals,thesefactorscaninuencecarcino-genicrisk.4.1.SpeciesdifferencesinthemetabolismofcarcinogensanddrugsLargeinterspeciesdifferencesexistintheratesofmetabolismofforeignchemicalsandintheproleofmetabolitesformed.Sincespeciesdifferencesinthemetabolismofcarcinogenscaninuencethecarcinogenicresponse,thesedifferencesareimportanttoconsiderinextrapolationofanimalcarcinogenesisdatatohumans.Forinstance,the-hydroxylationof-2-uorenylacetamide(FAA;2-acetylaminouorene)toaproximatecarcinogenicmetaboliteoccursinrats,miceandhumansbutnotintheguineapigorSteppelemminglemming.Accordingly,FAAiscarcinogenicinratsandmicebutnotintheguineapigorSteppelemming.Sincehumans-hydroxylateFAA,itislikelythatFAAwouldbecarcinogenicinhumans.ThemetabolismofFAAtotheinactiveringhydroxylated7-OHFAAanditscarcinogenicN-OH-FAAmetaboliteinseveralanimalspeciesasmeasuredbyurinaryexcretionisshowninTable4.SinceFAAwasonceconsideredforhumanuseasapesticide,itisfortunatethattheinitialcarcinogenicitystudywiththiscompoundwasdoneintheratinsteadoftheguineapig.Theseandotherrelated A BCDEFGHJ Surgical Biopsy Samples 300020001000 4000300020001000 18012060 Revertants/mgRevertants/mg3-OHBP Formed(pmol/mg/min)min)a]pyrene7,8 -dihydrodiolBenzo[]pyreneAflatoxinBA BCDEFGHJIA BCDEFGHJ Fig.1.Interindividualdifferencesinthemetabolismofcarcinogensbysurgicalbiopsysamplesof10differenthumanlivers.Themetabolismofbenzo[a]pyrene(BP)touorescentphenols,expressedas3-hydroxy-BP(3-HOBP),andthemetabolismofaatoxinBandBP7,8-dihydrodioltomutagensweredeterminedbyincubationofthesubstratewithlivermicrosomesandNADPH.Eachliverbiopsywastakenformedicalreasons,andonlyhistologicallynormalsampleswereusedforthemetabolismstudies(takenfromrefs.refs.).Table4ComparisonofthemetabolismofFAAinseveralspecies SpeciesCarcinogenicityPercentofdose -OH–FAA7-OH–FAA Guineapig–072Steppelemming–Trace420.3–1519–271.8–2.316–2013–3015–2915–2035–395.20.91.511Monkey?1.8–2.79–18Man?4–1425–30 DatacompiledbyWeisburgeretal.al..observationsindicatethatitisimportanttoevaluatethepotentialcarcinogenicityofchemicalsinananimalspecieswiththesameproleofmetaboliccapabilitiesashumans.Comparativestudiesonthemetabolicproleofnewchem-icalsbyexperimentalanimalsandhumanscanbedonewithlivermicrosomes,puriedcytochromesP450and/orinvivo.Extrapolationofanimaldatatohumansshouldtakeintoconsiderationthelargeinterindividualdifferencesthatoccurinthemetabolismofchemicalsinthehuman4.2.InterindividualdifferencesinthemetabolismofdrugsandcarcinogensTherearelargeperson-to-persondifferencesintheratesofmetabolismofdrugsandcarcinogens.Largedif-ferencesinthemetabolicactivationofbenzo[a]pyrene7,8-dihydrodiolandaatoxintomutagenicmetabolitesandinthemetabolismofbenzo[a]pyrenetononcarcino-genicphenolicmetabolitesbytensurgicalliverbiopsysam-plesfromdifferentindividualsareshowninFig.1[25,26] G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486Person-to-persondifferencesinthemetabolismofdrugsandcarcinogensarecausedbybothgeneticandenvironmentalfactors.Manystudieshavedemonstratedpolymorphismsingenesthatcodeforspecicdrug-metabolizingenzymes,andmutationsinthesegenescanleadtoimpaireddrugmetabolismandaltereddrugactioninpatientspatients.Exam-plesofenvironmentalfactorsthatinuencethemetabolismofdrugsandcarcinogensinhumansincludediet,smoking,alcoholingestion,drugadministration,ingestionofherbalremedies,exposuretoenvironmentalpollutantsanddiseasedisease.Becauseoftheseconsiderations,determina-tionofanindividual’sabilitytometabolicallyactivateanddetoxifychemicalcarcinogensrequiresbothphenotypingandgenotypingmethods.Availabilityofsimplemethodsforlargescaletestingiscurrentlylimited,andaccordingly,itwillbeimportanttodevelopsimple,rapidandaccuratemethodsforbothgenotypingandphenotypingindividualsthatwillcontributetoassessmentofanindividual’sabil-itytometabolicallyactivateanddetoxifyenvironmentalchemicalcarcinogens.4.3.EffectofenvironmentalcontextoncarcinogenesisbychemicalsWhetherachemicalisacarcinogen,ananticarcino-genorneitherdependsontheenvironmentalcontext.Although3-methylcholanthreneisacarcinogeninmanyexperimentalanimalmodels,itinhibitsthehepatocarcino-geniceffectsof3andFAAbyenhancingthemetabolicdetoxicationofthesechemicalschemicals.AlthoughTCDD(dioxin)causeslivertumorsinratsandisatumorpromoterin7,12-dimethylbenz[a]anthracene-initiatedHRS/Jhairlesshairless,TCDDinhibitstheformationofspontaneousbreasttumorsinratsratsandinhibitsinitiationoftumorformationbyboth7,12-dimethylbenz[a]anthraceneandbenzo[a]pyreneinmouseskinbyenhancingthemetabolicdetoxicationofthesehydrocarbonshydrocarbons.Inadditionalstudies,topicalapplicationsof1,25-dihydroxyvitaminDorall-transretinoicacidinhibitTPA-inducedtumorpro-motionin7,12-dimethylbenz[a]anthracene-initiatedmouseskin,butthesecompoundsenhance7,12-dimethylbenz[a]-anthracene-inducedcompletecarcinogenesisinmouseskinTable55.Epidemiologystudiesindicatethatdailysupplementsof20–30mgof-caroteneareassociatedwithanincreasedriskoflungcancerinsmokerswhoalsodrinkalcoholicbever--.Incontrasttotheadverseeffectsof-caroteneinsmokerswhodrinkalcoholicbeverages,dailysupplementsof25mgof-caroteneinhibittherecurrenceofcolorectaladenomasinpersonswhodonotsmokeordrinkalcoholicbeverageserages.Theseresultsindicatethatachemicalmaybecarcinogenicinoneexperimentalsettingandananticar-cinogeninanother,demonstratingtheimportanceofenvi-ronmentalcontextasavariableinassessingcarcinogenicTable5Effectsof1andall-transretinoicacidontumorpromotionbyTPAandcompletecarcinogenesisbyDMBA ExperimentTreatment%TumorTumors/mouse 1TPA9220.0TPA(0.5nmol)633.9TPARA(2.0nmol)331.32DMBA631.20DMBA(0.5nmol)1005.67DMBARA(0.5nmol)802.57DMBARA(25nmol)938.40 Inexperiment1,CD-1micepreviouslyinitiatedwith200nmolof7,12-dimethylbenz[a]anthracene(DMBA)weretreatedtopicallywithtransretinoicacid(RA)or1,25-dihydroxyvitaminD)togetherwith5nmolTPAtwiceaweekfor15weeks.Inexperiment2,animalsweretreatedwithVD,RAorsolventvehicle1hpriortotreatmentwith50nmolDMBAtwiceaweekfor16weeks.MicethatweretreatedtwiceweeklywithVDorall-transretinoicacidintheabsenceofDMBAdidnotdevelopanytumors(takenfromrefs.refs.).5.Multiplemutationsincancers:sourcesand5.1.CancerisachronicdiseaseInthecaseofsolidtumorsthereisa20–40-yearintervalfromthetimeofexposureofanindividualtoachemicalorviralcarcinogenuntiltheclinicaldetectionofatumor.Bythetimeatumorisapparent,cancercellshaveacquiredtheabilitytodividewherenormalcellsoughtnot,toin-vadeadjacentcellulararchitectures,tometastasizeandtokillthehost.Manyofthesephenotypescanbetheresultofmutationsthataccumulateastumorsprogress.MutationscanbedenedasachangeinthenucleotidesequenceofDNA.ThesecanariseasaresultofDNAdamageorbytheincorporationofnon-complementarynucleotidesduringDNAsyntheticprocesses.SourcesofDNAdamagecanbebroadlydividedintotwocategories:thosethatresultfromexogenousagentssuchaschemicals,viruses,andirradiationandthosecausebyreactivemoleculesgeneratedbynormalcellularprocesses.NormalcellularprocessesthatdamageDNAincludethegenerationofreactiveoxygenandnitro-genspecies,alkylation,depurination,andcytidinedeamina-deamina-.ThemagnitudeofDNAdamagebynormalcel-lularprocessesisenormous;forexample,ithasbeenesti-matedthatapproximatelytenthousanddepurinatedsitesaregeneratedpercellperdaydayandanevenlargernumberofalterationsresultfromthegenerationofreactiveoxygenoxygen(Scheme4AgainstthisextensiveDNAdamageisanarmamentar-iumofDNArepairsystemswithoverlappingspecicities.ThesesystemscontinuouslymonitorthegenomeandrepairsitesofDNAdamage.Sofar,over130DNArepairgeneproductshavebeenidentiedidentied.PathwaysofDNAre-pairincludebase-excisionrepair,nucleotideexcisionrepair, G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486 Scheme4.EquilibriumbetweenDNAdamageandDNArepair.AbovethescreenarelistedexogenousandendogenoussourcesofDNAdamage.BelowthescreenarethesmallnumberofDNAalterationsthatescapeDNArepairandresultinmutagenesis.transcription-coupledrepair,mismatchrepair,andevendi-rectreversalofDNAdamage.Inaddition,cellcyclecontrolgenessuchasp53monitorcellreplication,haltingthecy-cleandasaresultallowingadditionaltimeforDNArepair,whenrequired.LossofcheckpointcontrolsresultinaberrantDNAsynthesisormitoticsegregation.ThehighefciencyofthesemechanismsforDNArepairguaranteesthatonlyafewofthetensofthousandsofDNAlesionsarestillpresentatthetimeofDNAreplicationthathavethepotentialtocausemutations.InnormalcellsDNAreplicationandchromosomalsegre-gationareexceptionallyaccurateprocesses.Measurementsofmutagenesisofcellsgrownincultureyieldvaluesofap-proximately2singlebase-substitutions/nucleotideinDNA/celldivisionor1mutationsgene/celldi-di-.Anevenlowerfrequencyhasrecentlybeendemonstratedusingstemcellsinculturecultureanditisgener-allybelievedthattumorsarisefromstemcells.Takingintoaccountthisverylowmutationfrequency,itseemsimproba-blethatthespontaneousmutationrateinnormalcellsissuf-cienttogeneratethelargenumbersofgeneticalterationsthatareobservedinhumancancercells.Ifoneassumesthatacancerarisesinasinglestemcell,thenthespontaneousmutationratewouldonlybeadequatetoaccountforlessthanonemutationpertumor.Baseduponthedisparitybetweentheinfrequencyofspon-taneousmutationsandthelargenumbersofmutationsre-portedinhumantumors,itwaspostulatedthatcancersmustexhibitamutatorphenotypephenotype.Theexpressionofamuta-torphenotypewouldbeanearlyeventincancerprogression.Itcouldarisebymutationsindifferentgenesthatnormallyfunctiontomaintaingeneticintegrity.AttractivecandidateswouldbemutationsinDNApolymerasesthatrenderthemerror-prone,mutationsinDNArepairgenesthatrendertheminefcientormutationsingenesinvolvedinchromosomalsegregation.MutationsinthesegenesorinthepathwaysinwhichtheyfunctioncouldexceedcellularcapacityforDNArepairandresultintheaccumulationofmultiplemutationsthroughoutthegenome.Amongstthethousandsofmuta-tionsthatensueinacancerwouldbemutationsinonco-genesandtumorsuppressorgenesthatconferamalignantphenotype.Theconceptofamutatorphenotypeisnotatvariancewiththetwo-hitmodelofKnudsenfortheoriginofretinoblastomaretinoblastomaorthelimitednumberofphenotypesthathavebeenpostulatedtoberequiredfortumorigenesistumorigenesis.Amutatorphenotypecouldaccountforthelargenum-bersofmutationspresentincancersandprovideamecha-nismforthegenerationofmutationsinalimitednumberofcriticalcancerrelatedgenes.Mutationsincancercellscoverawidespectrum,fromchromosomalalterationsthatencompassmillionsofnu-cleotidestopointmutationsthatinvolveonlyafewnu-cleotidesubstitutions.Multiplechromosomalalterationshavebeenidentiedinmosttypesoftumorsandinvolvetranslocations,deletions,amplicationsandaneuploidy.Whiletherearediagnosticchromosomalaberrationsthatoccurathighfrequenciesincertaintumors,thereisalsoastrikingheterogeneityofchromosomalalterationsincancercellswithinmosttumors.Insometumorsthereisevidenceforasequentialorderformutationsinkeygenesduringtu-morprogressionprogression.However,theorderofchromosomalalterationshasnotbeenreportedtobeinvariantortooccurwithinallcancercellsinatumor.Measurementsofthenumberofcopiesofsegmentsofthegenomeintumorcells(DNAcopynumber)andthelossofpiecesofDNA(lossofheterozygosity)haveestablishedthatmanytumorshar-borasmanyas40chromosomalalterations,eachinvolvingmillionsofgenesgenes.Carefulstudiesonisolatedsingletumorcellshavedocumentedthatlargenumbersofchangesthatoccurineachindividualcancercellcell.Therstdi-rectevidencetosupportamutatorphenotypeatthelevelofsmallchangesinnucleotidesequencewasprovidedbythedemonstrationthatpatientswithhereditarynon-polyposiscoloncancerexhibitedchangesinthelengthofrepetitive G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486nucleotidesequencesinassociationwithmutationsingenesinvolvedinmismatchrepairrepair.ItispresumedthatthesechangesareduetoslippagebyDNApolymerases.Similarchanges,butatlowerfrequencies,havebeenreportedinavarietyoftumorsthatarenotknowntobeassociatedwithmutationsorsilencinginmismatchrepairgenesgenes.Amutatorphenotypecouldbegeneratedbymutationsingenesthatnormallyfunctiontoguaranteegeneticstability.ThesemutationspresumablyariseviarandomDNAdamagebyenvironmentalorendogenousagents.Onemechanismforthegenerationofamutatorphenotypewouldbeviamuta-tionsinDNArepairgenesthatresultindiminishedfunctionScheme4).Anumberofmousemodelshavebeencon-structedharboringdeletionsinDNArepairgenes.Manyoftheseresultinavarietyofcancers.Moreover,therearein-heritedhumandiseaseswithmutationsinDNArepairgenes.Themostinstructiveexampleisxerodermapigmentosum;mutationsingenesthatrepairUV-inducedDNAdamagere-sultinskincancerinindividualsexposedtoUV-irradiationUV-irradiation.Enhancedmutagenesiscanalsobetheresultofin-creasedmisincorporationbyDNApolymerases.AmousemodelhasbeenconstructedinvolvingthereplacementofthemajorreplicatingDNApolymerase-deltawithamutantallelethatlacksproofreadingactivity.Thesemiceexhibitahighincidenceoflymophomasaswellasavarietyofep-ithelialtumors.Thus,bothmousemodelsmodelsandhumandiseasedemonstratethattheexpressionofamutatorpheno-typecanbecausallyassociatedwithcancer.Thesestudiesestablishthatamutatorphenotypeissufcienttoresultinmalignancybutitremainstobedemonstratedthatitisanecessaryrequirementinhumans.Itisinstructivetoconsidertheargumentsthathavebeenraisedagainstamutatorphenotypehypothesisincancer.First,mostmutationsaredetrimentalandthusalargein-creaseinmutationfrequencywouldbelethal.Second,istheproposalthataneuploidyistheinitiatingeventintheconver-sionofnormalcellstocancercellscells.Third,mathemati-calmodelsoftumorprogressionincoloncancercanaccountforlargenumbersofmutationswithoutinvokingamuta-torphenotypephenotype.Finally,sequencingofcDNAintumorcelllineshassofarfailedtoreveallargenumbersofmuta-muta-.Itisappropriatetopointoutthatnormalcolonstemcellsundergolargenumbersofcelldivisionsandthisisnotageneralpropertyofmanytissuesthatgiverisetomalignancies.Thesequencingstudieswouldnotdetectran-domeventsafterthelastroundofclonalselection,anddonotencompassnon-synonymoussubstitutionsandsequencechangesinintronswheremutationaccumulationwouldbemostpronounced.Itremainstobedeterminedwhethertheacquisitionofamutatorphenotypeisanecessaryeventduringtumorpro-gression.Thepresenceofmultiplerandommutationsinhu-mantumorshasimportantimplications.First,itprovidesamonitorforthemalignantstateofatumorandmayallowforstraticationoftumors.Tumorsharboringfewermutationsmightbelesslikelytobecomeresistanttochemotherapeuticagents.Second,itmaybeutilizedtocalibratechronicex-posureofindividualstocarcinogensortomeasurethesus-ceptibilityofdifferentpopulationstodifferentcarcinogens.Third,thepresenceofthousandsofrandommutationsinindividualcancercells,suggeststhatwithinaclinicallyde-tectedtumor,comprising10cells,therearecancercellsthatharbormutationsrenderingthemresistanttoanychemother-apeuticagent.Lastly,iftheacquisitionorexpressionofamutatorphenotypeisrate-limitingfortumorprogression,theninhibitingmutationaccumulationmaydelaycarcino-genesis.Assuminga20-yearaverageprogressiontoclini-calcancerinadults,evenatwo-foldreductionintherateofmutationaccumulationwouldprovideasignicantclin-icaldelay,asmuchas20years.ThedesignandutilizationofdrugsthatreduceDNAdamagecoulddelaytheonsetofcancerandthussignicantlyreducecancerdeaths.AcknowledgementsStudiesintheauthors’laboratoriesweresupportedbythefollowinggrants:P01ES06052fromNIEHS(GNW);CA81301andCA92025fromNCIandRP-00-138fromtheAmericanCancerSociety(SSH);DOEcon-tractW-7405-ENG-48fromtheDepartmentofEnergy,P30CA93373,P01CA55861andCA94709fromNCI(JSF);CA49756,CA80759andP01CA88961fromNCI(AHC);CA80993andCA84087fromNCIandES11045fromNIEHS(LAL).References[1]Hepatitisviruses,vol.59.InternationalAgencyforResearchonCancerMonographsontheEvaluationofCarcinogenicRiskstoHumans,Lyon,France;1994.[2]KewMC.Hepatitisvirusesandhepatocellularcarcinoma.ResVirol[3]WoganGN.Aatoxinsasriskfactorsforhepatocellularcarcinomainhumans.CancerRes1992;52:2114s–8s.[4]BoschFX,MunozN.Prospectsforepidemiologicalstudiesonhep-atocellularcarcinomaasamodelforassessingviral-chemicalinter-actions.IARCScienticPub1988;89:427–33.[5]Somenaturallyoccurringsubstances:fooditemsandconstituents,heterocyclicaromaticaminesandmycotoxins,vol.53.InternationalAgencyforResearchonCancerMonographsontheEvaluationofCarcinogenicRiskstoHumans,Lyon,France;1993.[6]WoganGN.Molecularepidemiologyincancerriskassessmentandprevention:recentprogressandavenuesforfutureresearch.EnvironHealthPersp1992;98:167–78.[7]GroopmanJD,KenslerTW.Thelightattheendofthetunnelforchemical-specicbiomarkers:daylightorheadlight?Carcinogenesis[8]RossRK,YuanJ-M,YuMC,WoganGN,QianG-S,TuJ-T,etal.Urinaryaatoxinbiomarkersandriskofhepatocellularcarcinoma.Lancet1992;339:943–6.[9]QianG-S,RossRK,YuMC,YuanJ-M,GaoY-T,HendersonBE,etal.Afollow-upstudyofurinarymarkersofaatoxinexposureandlivercancerriskinShanghai,People’sRepublicofChina.CancerEpiBiomarkPrev1994;3:3–10. G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486[10]WangL-Y,HatchM,ChenCJ,LevinB,YouSL,LuSN,etal.AatoxinexposureandriskofhepatocellularcarcinomainTaiwan.IntJCancer1996;67:620–5.[11]SunZ,LuP,GailMH,PeeD,ZhangQ,MingL,etal.IncreasedriskofhepatocellularcarcinomainmaleHBsAgcarrierswithchronichepatitiswhohavedetectableaatoxinmetaboliteM1.Hepatology[12]GreenblattMS,BennettWP,HollsteinM,HarrisCC.Mutationsinthep53tumorsuppressorgene:cluestocanceretiologyandmolecularpathogenesis.CancerRes1994;54:4855–78.[13]WangJS,ShenX,HeX,ZhuYR,ZhangBC,WangWB,etal.Protectivealterationsinphase1andphase2metabolismofaatoxinbyoltiprazinresidentsofQidong,People’sRepublicofChina.JNatlCancerInst1999;91:347–54.[14]Tobaccosmokeandinvoluntarysmoking,vol.83.InternationalAgencyforResearchonCancerMonographsontheEvaluationofCarcinogenicRiskstoHumans,Lyon,France;2004.[15]Tobaccohabitsotherthansmoking:betelquidandareanutchewingandsomerelatednitrosamines,vol.37.InternationalAgencyforRe-searchonCancerMonographsontheEvaluationoftheCarcinogenicRiskofChemicalstoHumans,Lyon,France;1985.p.37–202.[16]HechtSS.Tobaccocarcinogens,theirbiomarkers,andtobacco-inducedcancer.NatureRevCancer2003;3:733–44.[17]HechtSS.Tobaccosmokecarcinogensandlungcancer.JNatlCancerInst1999;91:1194–210.[18]HechtSS,HoffmannD.Tobacco-specicnitrosamines,animportantgroupofcarcinogensintobaccoandtobaccosmoke.Carcinogenesis[19]Polynucleararomaticcompounds.Part1,chemical,environmental,andexperimentaldata,vol.32.InternationalAgencyforResearchonCancerMonographsontheEvaluationoftheCarcinogenicRiskofChemicalstoHumans,Lyon,France;1983.p.33–91.[20]HoffmannD,SchmeltzI,HechtSS,WynderEL.Tobaccocarcino-genesis.In:GelboinH,Ts’oPOP,editors.Polycyclichydrocarbonsandcancer.NewYork:AcademicPress;1978.p.85–117.[21]Deutsch-WenzelR,BruneH,GrimmerG.Experimentalstudiesinratlungsonthecarcinogenicityanddose-responserelationshipsofeightfrequentlyoccurringenvironmentalpolycyclicaromatichydro-carbons.JNatlCancerInst1983;71:539–44.[22]HechtSS.Biochemistry,biology,andcarcinogenicityoftobacco-specicN-nitrosamines.ChemResToxicol1998;11:559–603.[23]HechtSS.Humanurinarycarcinogenmetabolites:biomarkersforinvestigatingtobaccoandcancer.Carcinogenesis2002;23:907–22.[24]WeisburgerJH,GranthamPH,VanhornEC,SteigbigelNH,RallDP,WeisburgerEK.ActivationanddetoxicationofN-2-uorenylacetamideinman.CancerRes1964;24:475–9.[25]ConneyAH,PantuckEJ,PantuckCB,BueningMK,JerinaDM,FortnerJG,etal.Roleofenvironmentanddietintheregulationofhumandrugmetabolism.In:EstabrookRW,LindenlaubE,editors.Theinductionofdrugmetabolism.NewYork:FKSchattauerVerlag;1979.p.583–605.[26]ConneyAH.Inductionofmicrosomalenzymesbyforeignchemi-calsandcarcinogenesisbypolycyclicaromatichydrocarbons:GHAClowesMemorialLecture.CancerRes1982;42:4875–917.[27]Ingelman-SundbergM.ImplicationsofpolymorphiccytochromeP450-dependentdrugmetabolismfordrugdevelopment.DrugMetabDispos2001;29:570–3.[28]ConneyAH.Inductionofdrug-metabolizingenzymes:apathtothediscoveryofmultiplecytochromesP450.AnnuRevPharmacolToxicol2003;43:1–30.[29]RichardsonHL,StierAR,Borsos-Nacht-NebelE.Livertumorinhi-bitionandadrenalhistologicresponsesinratstowhich3dimethylaminoazobenzeneand20-methylcholanthreneweresimulta-neouslyadministered.CancerRes1952;12:356–61.[30]ConneyAH,MillerEC,MillerJA.Themetabolismofmethylatedaminoazodyes.V.Evidenceforinductionofenzymesynthesisintheratby3-methylcholanthrene.CancerRes1956;16:450–9.[31]MillerEC,MillerJA,BrownRR,MacDonaldJC.Ontheprotectiveactionofcertainpolycyclicaromatichydrocarbonsagainstcarcino-genesisbyaminoazodyesand2-acetylaminouorene.CancerRes[32]KocibaRJ,KeyesDG,BeyerJE,CarreonRM,WadeCE,DittenberDA,etal.Resultsofatwo-yearchronictoxicityandoncogenicitystudyof2,3,7,8-tetrachlorodibenzo-p-dioxininrats.ToxicolApplPharmacol1978;46:279–303.[33]PolandA,PalenD,GloverE.TumourpromotionbyTCDDinskinofHRS/Jhairlessmice.Nature1982;300:271–3.[34]DiGiovanniJ,BerryDL,SlagaTJ,JuchauMR.Studiesontherela-tionshipsbetweeninductionofbiotransformationandtumor-initiatingactivityof7,12-dimethylbenz[a]anthraceneinmouseskin.In:JonesPW,LeberP,editors.Polynucleararomatichydrocarbons.AnnAr-bor,MI:AnnArborSciencePublishers;1979.p.553–68.[35]DiGiovanniJ,KishoreGS,SlagaTJ,BoutwellRK.2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)-inducedalterationsinoxida-tiveandnonoxidativebiotransformationofPAHinmouseskin:roleofanticarcinogenesisbyTCDD.In:BjorsethO,DennisAJ,editors.Polynucleararomatichydrocarbons:chemistryandbiologicaleffects.Columbus,OH:BattallePress;1980.p.935–54.[36]VermaAK,ShapasBG,RiceHM,BoutwellRK.Correlationoftheinhibitionbyretinoidsoftumorpromoter-inducedmouseepidermalornithinedecarboxylaseactivityandofskintumorpromotion.CancerRes1979;39:419–25.[37]WoodAW,ChangRL,HuangMT,UskokovicM,ConneyAH.,25-DihydroxyvitaminDinhibitsphorbolester-dependentchem-icalcarcinogenesisinmouseskin.BiochemBiophysResCommun[38]WoodAW,ChangRL,HuangM-T,BaggioliniE,PartridgeJJ,UskokovicM,etal.Stimulatoryeffectof1ontheformationofskintumorsinmicetreatedchronicallywith7,12-dimethylbenz[a]anthracene.BiochemBiophysResCommun[39]VermaAK,ConradEA,BoutwellRK.Differentialeffectsofretinoicacidand7,8-benzoavoneontheinductionofmouseskintumorsbythecompletecarcinogenesisprocessandbytheinitiation-promotionregimen.CancerRes1982;42:3519–25.[40]ConneyAH,LouY-R,XieJ-G,OsawaT,NewmarkHL,LiuY,etal.Someperspectivesondietaryinhibitionofcarcinogenesis:studieswithcurcuminandtea.ProcSocExpBiolMed1997;216:234–45.[41]OmennGS.Chemopreventionoflungcancer:theriseanddemiseofbeta-carotene.AnnuRevPublicHealth1998;19:73–99.[42]BaronJA,ColeBF,MottL,HaileR,GrauM,ChurchTR,etal.Neoplasticandantineoplasticeffectsofbeta-caroteneoncolorectaladenomarecurrence:resultsofarandomizedtrial.JNatlCancerInst[43]LoebLA.Endogenouscarcinogenesis:molecularoncologyintothetwenty-rstcentury—presidentialaddress.CancerRes[44]LindahlT.InstabilityanddecayoftheprimarystructureofDNA.Nature1993;362:709–15.[45]AmesBN,GoldLS,WilletWC.Thecausesandpreventionofcancer.ProcNatlAcadSciUSA1995;92:5258–65.[46]WoodRD,MitchellM,SgourosJ,LindahlT.HumanDNArepairgenes.Science2001;29:1284–9.[47]LoebLA.Amutatorphenotypeincancer.CancerRes2001;61:3230–[48]CervantesRB,StringerJR,ShaoC,TischeldJA,StambrookPJ.Embryonicstemcellsandsomaticcellsdifferinmutationfrequencyandtype.ProcNatlAcadSciUSA2002;99:3586–90.[49]LoebLA,SpringgateCF,BattulaN.ErrorsinDNAreplicationasabasisofmalignantchange.CancerRes1974;34:2311–21.[50]KnudsonAG.Hereditarycancer:twohitsrevisited.JCancerResClinOncol1996;122:135–40.[51]HanahanD,WeinbergRA.Thehallmarksofcancer.Cell G.N.Woganetal./SeminarsinCancerBiology14(2004)473Ð486[52]VogelsteinB,FearonER,KernSE,HamiltonSR,PreisingerAC,LeppertM,etal.Geneticalterationsduringcolorectal-tumordevel-opment.NEnglJMed1988;319:525–32.[53]KallioniemiA,KallioniemiO-P,PiperJ,TannerJM,StokkeT,ChenL,etal.DetectonandmappingofampliedDNAsequencesinbreastcancerbycomparativegnomichybridization.ProcNatlAcadSciUSA1994;91:2156–60.[54]KleinCA,Schmidt-KittlerO,SchardtJA,PantelK,SpeicherMR,RiethmullerG.Comparativegnomichybridization,lossofheterozy-gosity,andDNAsequenceanalysisofsinglecells.ProcNatlAcadSciUSA1999;96:4494–9.[55]PeruchoM.Cancerofthemicrosatellitemutatorphenotype.BiolChem1996;377:675–84.[56]JacksonAL,LoebLA.ThecontributionofendogenoussourcesofDNAdamagetothemultiplemutationsincancer.MutatRes[57]CleaverJE,KraemerKH.Xerodermapigmentosum.In:ScriverCR,BeudetAL,SlyWS,ValleD,editors.Themetabolicbasesofinher-iteddisease.NewYork:McGraw-Hill;1989.p.2949–71.[58]GoldsbyRE,HaysLE,ChenX,OlmstedEA,SlaytonWB,SpangrudeGJ,etal.HighincidenceofepithelialcancersinmicedecientforDNApolymerasedeltaproofreading.ProcNatlAcadSciUSA[59]DuesbergP,RauschC,RasnickD,HehlmannR.Geneticinstabilityofcancercellsisproportionaltotheirdegreeofaneuploidy.ProcNatlAcadSciUSA1998;95:13692–7.[60]TomlinsonI,BodmerW.Selection,themutationrateandcancer:ensuringthatthetaildoesnotwagthedog.NatureMed1999;5:11–[61]WangTL,RagoC,SillimanN,PtakJ,MarkowitzS,WilsonJK,etal.Prevalanceofsomaticalterationsinthecolorectalcancercellgenome.ProcNatlAcadSciUSA2002;99:3076–80.