Inhibition of polyaromatic hydrocarbon-DNA adduct formation by naturally occurring plant phenols in epidermis and lungs of SENCAR mice

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Inhibition of polyaromatic hydrocarbon-DNA adduct formation by naturally occurring plant phenols in epidermis and lungs of SENCAR mice
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  [CANCERRESEARCH47,767-773,February1,1987] InhibitionofPolycyclicAromaticHydrocarbon-DNAAdductFormationinEpidermisandLungsofSENCARMicebyNaturallyOccurringPlantPhenols1 MukulDas,2WasiuddinA.Khan,ParthasarathyAsokan,DavidR.Bickers,andHasanMukhtar3 DepartmentsofDermatology,UniversityHospitalsofCleveland,CaseWesternReserveUniversity,andVeteransAdministrationMedicalCenter,Cleveland,Ohio44106 ABSTRACT Naturallyoccurringplantphenolssuchastannicacid,quercetin,myricetin,andanthraflavicacidareknowntoinhibitthemutagenicityofseveralbay-regiondiol-epoxidesofpolycyclicaromatichydrocarbons(PAHs).Thebindingofbay-regiondiol-epoxidesofPAHstotargettissueDNAisthoughttobeessentialfortheinitiationofcancerbythesecompounds.InthisstudyweinvestigatedtheeffectoftheseplantphenolsonPAH-DNAadductformationintheepidermisandlungofSENCARmice.Invitroadditionoftannicacid,quercetin,myricetin,andanthraflavicacid(25¿IM)oanincubationsystemcontainingepidermalmicrosomespreparedfromeithercontrolor3-methylcholanthrene-pretreatedmiceinhibitedbenzo(a)pyrenebindingtocalfthymusDNAby63-64,38-43,36-37,and27-33 ,respectively.Asingletopicalapplicationoftannicacid,quercetin,myricetin,andanthraflavicacidatadoseof400nmol/kgbodyweightresultedintheinhibitionof| II|hcn/o(u)p\renebindingtoepidermalDNA(48-73 )andprotein(51-63 ).Thesamedoseoftheseplantphenols(400/tmol/kg)causedevengreaterinhibitionof(±>[3H]-7/3,8a-dihydroxy-7,8-dihydrobenzo(a)pyreneand|3H]-7,12-dimeth-ylbenz(<z)anthracenebindingtoepidermalDNAandprotein.Theformationof(+)-7j8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene-deoxyguanosineadductswassubstantiallydiminishedinbothepidermis(62-86 )andlungs(38-84 ).Theseresultsindicatethattannicacid,quercetin,myricetin,andanthraflavicacidarepotentinhibitorsofcarcinogenbindingtoepidermalandlungDNAandsuggestthattheseplantphenolscouldproveusefulinmodifyingtheriskoftumorinductionbyPAHssuchasbenzo(<i)pyreneand7,12-dimethyl-benz(a)anthrar.eneinthesetwotissues. INTRODUCTION PAHs4arewidelydistributedenvironmentalpollutants 1)andareknowntocausecanceroftheskinandlunginexperimentalanimalsandpossiblyinhumans 2-4).ThePAHsrequiremetabolicactivationtochemicallyreactivespecieswhicharethoughttoexertmutagenicandcarcinogeniceffectsinbiologicalsystems 5).OneofthePAHs,BP,ismetabolizedbythecytochromeP-450-dependentmixedfunctionoxidasesystemandepoxidehydrolasetoBPDE 5),ahighlyreactiveelectrophilicmetabolitewhichcanbindtocellularmacromole-cules 6-8).ThemetabolismofBPhasbeenshowntobestereoselective,producingamixtureoffourenantiomersofBPDE,withformationofthe +)-an/f -enantiomerpredominatingovertheothers 5).Only +)-anti-BPDEismutagenicinmammaliancellsandcarcinogenicinnewbornmice 9,10). Received3/31/86;revised9/11/86;accepted10/22/86.Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpagecharges.Thisarticlemustthereforebeherebymarkedadvertisementinaccordancewith18U.S.C.Section1734solelytoindicatethisfact.1SupportedinpartbyNIHGrantsES-1900,CA-38028,andAM-34368,byAmericanInstituteforCancerResearchGrant86A61,andbyresearchfundsfromtheVeteransAdministration. 2RecipientofSchering-PloughFoundationfellowshipawardfromtheDer matologyFoundation.3Towhomrequestsforreprintsshouldbeaddressed,atVeteransAdministrationMedicalCenter,10701EastBoulevard,Cleveland,OH44106.4Theabbreviationsusedare:PAH,polycyclicaromatichydrocarbon;BP,benzo a)pyrene;BPDE,oneoftwopossiblediastereomers untior\rn)ofbenzo a)pyrene-7,8-diol-9,10-epoxide; +)-a/rti-BPDE, +)-7/3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo a)pyrene,orBPDE-I;dGuo,deoxygua-nosine;DMBA,7,12-dimethylbenz a)anthracene;MCA,3-methylcholanthrene;BP-7,8-diol, ±)-70,8a-dihydroxy-7,8-dihydrobenzo a)pyrene;HPLC,highpressureliquidchromatography. Thisstereospecificitywasalsoobservedinatwostageskintumorigenicityassay,inwhichthe +)-awf/ -enantiomerwasfoundtobe60-foldmorepotentthantheother3enantiomersasatumorinitiatorinCD-IandSENCARmice 11).Similarly,the +)-antiisomerwasatleast59timesmorepotentthanthe  - -antiisomerinitsstereospecificbindingtoDNAandRNA inmouseskin 6,8),humancolonexpiants 12),hamsterembryocells 13,14),andhamsterepidermalcells 7)exposedtoBP.ApositivecorrelationexistsbetweentheDNAbindingofbay-regiondiol-epoxidesofPAHssuchasBPinmouseskinandlungandtheircarcinogenicpotency 6,8,15).FollowingtopicalapplicationofBPtomouseskininvivoortreatmentofcellsororganexpiantsintissueculture,themajorDNAadductformedisBPDE-Iboundthroughatrans-additiontotheexo-cyclicaminogroupofdeoxyguanosine 16).Priorstudiesfromourlaboratoryhaveshownthatellagicacid,anaturallyoccurringplantphenol,isapotentinhibitorofepidermalmicrosomalarylhydrocarbonhydroxylaseactivityandoftheenzyme-mediatedbindingofBPtobothcalfthymusDNAinvitroandepidermalDNAinvivo 17).EllagicacidwasalsoshowntoinhibitthecovalentbindingofBPtomouseepidermalDNAinorganculture 18).Dixitetal. 19)haveshownthatellagicacidinhibitsthemetablicactivationandthebindingofBPandBP-7,8-dioltoDNAinculturedmouselung.Furthermore,wehaveshownthatellagicacidisapotentinhibitorofBPmetabolismanditssubsequentglucuronidation,sulfation,andcovalentbindingtoDNAinculturedBALB/cmousekeratinocytes 20).ItisalsoknownthattopicalapplicationofellagicacidtoskinofBALB/cmiceaffordsprotectionagainstMCA-inducedskincarcinogenesis 21).Lesea 22)hasshownthatparenteraladministrationofferrulic,chlorogenic,andellagicacidinhibitsBP-inducedlungtumorformationinA/Jmice.Usingastandardtwostage initiationandpromotion)skincarcinogenesisexperimentalprotocolinwhichDMBAwasusedasacarcinogen,Lesea 22)hasalsoshownthattopicallyappliedellagicacidinhibitsskintumorformationinCD-Imice.Recently,ellagicacidhasalsobeenfoundtoprotectmiceagainstskincarcinogenesisinducedbytopicallyappliedBP-diol-epoxide 23).Inarecentpublicationwehaveshownthattannicacid,quercetin,myricetin,andanthraflavicacidarepotentinhibitorsofepidermalcytochromeP-450-dependentmonooxygenasesincludingarylhydrocarbonhydroxylaseinSENCARmice 24).InthisstudywehaveanalyzedthecapacityoftheseplantphenolstoinhibitPAH-DNAadductformationintheskinandlungsofSENCARmice. MATERIALSANDMETHODS Chemicals.[<7-3H]BP specificactivity,25Ci/mmol)and[G-3H]-DMBA specificactivity,26Ci/mmol)werepurchasedfromAmershamSearle,Chicago,IL.[G-3H]BP-7,8-diol specificactivity,395mCi/mmol)wasobtainedfromtheMidwestResearchInstitute,KansasCity,MO,throughtheChemicalRepositoryoftheNationalCancerInstitute.RadiolabeledBP,BP-7,8-diol,andDMBAwerepurifiedbyasilicagel ParlisiI10/mi:WatersAssociates)columnwithhexaneastheelutingsolventandsubsequentlybyreversephaseHPLCusingaDuPontZorbaxODScolumn 76.2mmx25cm)elutedwithmethanohwater767  INHIBITIONOFPAH-DNAADDUCTFORMATIONBYPLANTPHENOLS (19:1,v/v).Thepurityofthesecompoundswasgreaterthan99 asjudgedbyHPLC.Phenol(>99 pure),tannicacid,myricetin,andanthraflavicacidwereobtainedfromAldrichChemicalCo.,Milwaukee,WI.Quercetin,MCA,NADPH,protease(typeXI),m-cresol,8-hy-droxyquinoline,andRNaseA(typeIII-A)werepurchasedfromSigmaChemicalCo.,St.Louis,MO.SephadexLH-20wasaproductofPharmaciaFineChemicals,Piscataway,NJ.AllsolventsusedwereofHPLCgradeandwerepurchasedfromFisherScientificCo.,Fairlawn,NJ.Allotherchemicalswereobtainedinthepurestformcommerciallyavailable.TreatmentofAnimals.Six-week-oldfemaleSENCARmice,obtainedfromtheNCI-FrederickCancerResearchFacility,Bethesda,MD,wereused.ThemicewereshavedwithelectricclippersandNairdepilatorywasapplied1daypriortothebeginningoftheexperiment.Forthein vitroDNAbindingstudies onegroupofanimalsreceivedasingle topicalapplicationofMCA(50mg/kg)in0.1mlofacetonewhiletheothergroupreceivedanequalvolumeofacetoneandservedascontrol.ForstudiesontheinvivoeffectoftheplantphenolsonPAHbindingtoDNA,animalsweretreatedbytopicalapplicationofdifferentdosesofthecompounds(50-400/imol/kgbodyweight)in0.1mlofacetoneand/ordimethylsulfoxide.Animalstreatedwithanidenticalvolumeofvehicleservedascontrols.Onehaftertreatmentwiththeplantphenolstheanimalsweretreatedwithasingletopicalapplicationof5nmolofthe[3H]PAHs.PreparationofEpidermalandLungHomogenates.Animalstreatedwithorwithoutplantphenolswerekilledbycervicaldislocation24haftertreatmentwiththe|'IIjI'.MI.Skinandlungswereremovedandwashedinice-cold0.1Mphosphatebuffer,pH7.4.Theepidermiswasseparatedfrmthedermisbyheattreatmentoftheskinat52°Cor30sasdescribedearlier(25).Allsubsequentoperationswerecarriedoutat0-4°C.Theepidermisandlungsweremincedwithscissorsin0.1Mphosphatebuffer,pH7.4,containing10HIMEDTAasdescribedearlier(26).ThemincedtissuewashomogenizedwithaPolytrontissueho-mogenizer(BrinkmannInstruments,Westbury,NY)andusedfortheisolationofDNA.Forinvitrostudies,epidermalmicrosomeswerepreparedfromcontrolandMCA-pretreatedanimalsasdescribedpreviously(25).DNAIsolationfromEpidermisandLungs.DNAwereextractedfromepidermalandlunghomogenatesessentiallyasdescribedbyKatesandBeeson(27),withanadditionalincubationstepusingproteaseK(0.5mg/ml).AsecondextractionwasperformedusingKirby'sphenol(28)beforeprecipitationwithice-cold100 ethanol.TheDNAwasthendigestedwithRNaseA(1000units/ml),washed3timeswithacetone,anddriedunderastreamofnitrogen.TheextractedDNAwasdissolvedin5mlof0.lMsodiumchloride,pH7.0,andestimatedbymeasuringitsabsorptionat260nm.ThepurityoftheDNAwasassessedbytheabsorbanceratiosA26o/^28o»1-98and^200/^230*2.21(29).AliquotswerecountedonaPackardTri-Carb460CDliquidscintillationspectrometertodeterminetheamountof[3H]PAHsboundtotissueDNA.HPLCAnalysisofBP-DNAAdducts.IsolatedsamplesofDNAfromepidermisandlungsofanimalstreatedwithplantphenolsand[3H]BPwerehydrolyzedsequentiallyusingDNaseI,snakevenomphosphodi-esterase,andalkalinephosphatase(30)andtheDNAhydrolysatesweresubsequentlyanalyzedbyHPLC.PriortoinjectionontoHPLCallDNAsampleswereprocessedthroughasmallcolumnofSephadexLH-20.SephadexLH-20(poresize,25-100urn)waswashedwithdistilledwaterinabeakerandrefrigeratedfor1daypriortouse.Asmallcolumn(8.0x0.6cm)ofSephadexLH-20waspreparedinaPasteurpipet.TheDNAhydrolysateswereappliedtothecolumnandwashedwith20mlofwater.ThelesspolarBP-DNAadductswerethenelutedwith30mlofmethanol.Themcthanol-solublematerialfromthispreparatorysteprepresented80-85 ofthetotalDNA-boundradioactivityandwasdriedundernitrogenanddissolvedin0.1mlofmethanol.AWatersAssociatesmodel204liquidChromatograph,fittedwithaWatersAssociatesCig-fiBondapakcolumn(3.9mmx15cm)wasusedfortheanalysisoftheradiolabeledBP-DNAadducts.IdentificationoftheBPDE-I-dGuoadductwasbasedonastandardkindlysuppliedbyDr.R.M.Santi-Ila,CollegeofPhysiciansandSurgeons,ColumbiaUniversity,NewYork,NY.Thecolumnwaselutedatambienttemperaturewitha40-minlineargradientof50-75 methanolinwaterataflowrateof0.5ml/min.Theeluatesweremonitoredat254nm,fractionsofapproximately0.2mlwerecollecteddropwiseandtheradioactivityofeachfractionwasdeterminedonaPackardTri-Carb460CDliquidscintillationspectrometer. InVitroBP DNABinding.Theincubationsystemusedwassimilar tothatdescribedbyHesseetal.(31).Theincubationwascarriedoutinthepresenceandabsenceof25¿iMoncentrationsoftheplantphenols.Afterincubation,thereactionmixtureswerecentrifugedat105,000xgtoisolatethemicrosomesanddigestedwithsodiumdodecylsulfatetoextractanytracesofprotein.TheextractionprocedurewassimilartothatdescribedbyLeseaetal.(32).DNAestimationandbindingtoBPwerethencarriedoutasdescribedabove.CovalentBindingof|'II|PAIIstoEpidermalProtein.Theproteinfromanaliquotofepidermalhomogenatefrom|'111PAM-treatedmicewasprecipitatedbytheadditionof10 trichloroaceticacid.Theprecipitatedproteinwascentrifugedandwashedthreetimeswithethylacetateandsubsequentlythreetimeswithacetone.Thelastacetonewashcontainedvirtuallynomeasurableradioactivity.Theproteinthusobtainedwasdissolvedin1mloflNNaOH.TheproteinsampleswereassayedaccordingtothemethodofLowryetal.(33)andthealiquotswerecountedtodeterminetheamountof|'II¡I'MIboundtocellularprotein. RESULTSEffectofPlantPhenolsonEpidermalMicrosomalEnzyme-mediatedBindingof[3H]BPtoCalfThymusDNAinVitro.EpidermalmicrosomespreparedfromanimalspretreatedwithtopicallyappliedMCAincreasedthebindingofBPtocalfthymusDNAby75-91 ascomparedtountreatedcontrols.Theinvitroadditionoftannicacid,quercetin,myricetin,andanthraflavicacid,eachataconcentrationof25/¿M,otheepidermalmicrosomalincubationsystemfromeithercontrolorMCA-pretreatedanimalsresultedin63-64,38-43,36-37,and27-33 inhibitionofBPbindingtocalfthymusDNA,respectively(Table1).EffectofinVivoApplicationofPlantPhenolsontheCovalentBindingof|3HJPAHstoEpidermalDNAandProtein.Theeffectofasingletopicalapplicationofvaryingdosesoftannicacid,quercetin,myricetin,andanthraflavicacidonthebindingofBPmetabolitestoepidermalDNAandproteinisshowninFig. \A.Eachofthefourplantphenolssubstantiallyinhibitedthe bindingof[3H]BPtoepidermalDNAandprotein.Theobservedinhibitoryeffectswereconcentrationdependent.Topicalapplicationoftannicacid,quercetin,myricetin,andanthraflavicacid(400¿imol/kgbodyweight)tomiceresultedin73,64,63,and48 inhibitionof[3H]BP-DNAadductformationand63,61,58,and51 inhibitionof[3H]BPbindingtoepidermalprotein,respectively(Fig.IA).Thecumulativeeffectofthreerepeatedtopicalapplicationsoftheplantphenolsoninvivo[3H]BP Table1Effectofplantphenolsonepidermalmicrosomalenzyme-mediated covalentbindingofpHJBPtocalfthymusDNAinvitro Covalentbinding(pmol/mgDNA)°Control','dl'in-M('A-inducedTreatmentmicrosomeshibitionmicrosomes ofinhibitionAcetone62.6±.9*Dimethylsulfoxide67.0±4.3Tannicacid23.5±2.1Quercetin35.7±2.7Myricetin40.2±4.2Anthraflavicacid44.7±3.36343 3633119.6±10.611 4.0±9.943.5±4.174.5±5.874.9±6.982.6±6.864383727 Theincubationmixturecontained12mgcalfthymusDNA,10mgepidermalmicrosomalprotein,8.8mgNADPH,5nmol|'IIJBP.and25pMplantphenolsinafinalvolumeof10mlof50HIMTrisbuffercontaining25IHMKt'1-5HIMMgClj,pH7.5.AfterlhofincubationtheDNAwasisolatedandpurifiedasdescribedin MaterialsandMethods. hMean±SEoffourindividualvalues. 768  INHIBITIONOFP H DN DDUCTFORM TIONBYPL NTPHENOLS 12TonnicacidUuercetm Myricetin nthraflavicacid 200300400100TOPICALAPPLICATION pmol/kg 1 Fig.1.Effectoftopicalapplicationofplantphenolson[3H]BPbindingtoepidermalDNAandproteininSENCARmice.A,concentration-dependenteffectofasingleinvivoapplicationoftheplantphenols.Animalsreceivedasingletopicalapplicationofthedesiredconcentrationoffannieacid,quercetin,imrieetin,andanthraflavicacidinacetoneordimethylsulfoxide(DMSO)lhpriortotheapplicationof[3H]BP.Animalswerekilled24haftertreatmentwiththehydrocarbonasdescribedin MaterialsandMethods. II.effectofmultiplein vivotopicalapplicationsoftheplantphenols.Animalsreceived3topicalappli cationsof400«tmol/kgbodyweightoffannieacid,quercetin,myricetin,andanthraflavicacidat6-hintervals.Onehafterthelastapplicationofplantphenolstheanimalsreceivedasingletopicalapplicationof[3H]BPandwerekilled24hlaterasdescribedin MaterialsandMethods. Datarepresentmeanoffourindividualvalues;bars,SE. bindingtoepidermalDNAandproteinisshowninFig.IB.Animalsweretreatedwithtopicallyappliedplantphenols(400//mol/kg)threetimesat6-hintervalsbetweeneachapplication.Thisresultedin56,77,64,and45 inhibitionin[3H]BP-DNAadductformationand53,68,61,and48 inhibitionin[3H]BPbindingtoprotein(Fig.IB)inepidermis,respectively.Ingeneraltheeffectofrepeatedapplicationsoftheplantphenolswasnotsubstantiallydifferentfromthatobservedfollowingasingleapplication.Theeffectofasingletopicalapplicationoftheplantphenolson[3H]BP-7,8-dioland[3H]DMBAbindingtoepidermalDNAandproteinisdepictedinFig.2.Adoseof400/¿mol/kgoftannicacid,quercetin,myricetin,andanthraflavicacidresultedin98,52,95,and63 inhibitionof[3H]BP-7,8-diolbindingtoepidermalDNAand78,44,54,and36 inhibitionofthecovalentbindingof[3H]BP-7,8-dioltoepidermalprotein,respectively(Fig.2/1).Tannicacid,quercetin,myricetin,andanthraflavicacidatadoseof400/¿mol/kgesultedin87,79,64,and33 inhibitionof[3H]DMBA-DNAadductformationinmouseepidermisand43,55,50,and54 inhibitionof[3HJ-DMBAbindingtoepidermalprotein,respectively(Fig.2B). I I i » £-b° ?5So1 1 1 J« XA I zoS 12_v3i-i-ÃŽ üÜ O ili Fig.2.Effectofasingleinvivotopicalapplicationofplantphenolson[3H)BP-7,8-dioland(3H]DMBAbindingtoepidermalDNAandproteininSENCARmice.Animalsreceivedasingletopicalapplication(400fimol/kgbodyweight)oftannicacid,quercetin,myricetin,andanthraflavicacidandlhlaterweretreatedwith[3H]BP-7,8-diolor[3H)DMBA.Animalswerekilled24hafterthetreatmentofthehydrocarbon.Datarepresentmeansoffourindividualvalues; bars SE.A [3H]BP 7 8 diolbinding;B [3H]DMBAbinding.DMSO dimethyl sulfoxide. InVivoEffectofPlantPhenolsonBPDE I dGuoAdduct FormationinEpidermisandLung.TheeffectofasingletopicalapplicationofplantphenolsonBPDE-I-dGuoadductformationintheepidermisofSENCARmiceisshowninFig.3.InthisstudyepidermalDNAwasisolatedfromanimalstreatedwiththeplantphenolsorfromanimalstreatedwiththevehiclealone.EqualamountsofDNAweredigestedandpassedthroughasmallcolumnofSephadexLH-20asdescribedin MaterialsandMethods. ThelesspolarBP-DNAadductswereelutedwithmethanolandanalyzedonHPLC.TheHPLCprofileoftheBP-DNAadductsshowedamajorpeakandaminorpeak.Basedonreferencestandards,themajorpeakwasidentifiedasBPDE-I-dGuo(Fig.3A).Asingletopicalapplicationof400¿tmol/kgoftannicacid(Fig.3Ä),quercetin(Fig.3C),myricetin(Fig.3D),andanthraflavicacid(Fig.3E)inhibitedBPDE-I-dGuoadductformationinepidermisby79,62,86,and80 ,respectively.Furthermore,inanimalstreatedwiththeplantphenolstheotherunidentifiedpeakwaseithersubstantiallydiminishedorundetectable(Fig.3).TheinhibitoryeffectofeachplantphenolonBPDE-I-dGuoadductformation 769  1800160014001200 û 1000o 800600400200 INHIBITIONOFPAH DNAADDUCTFORMATIONBYPLANTPHENOLS 800  onti BPOE dGgo   600 è400 200B ITITI 2 4 6 8 2 2O4O6O8OIOO 2 O_O 8 6 4 2 l 20406080100120FRACTIONNUMBER E 20406080100120FRACTIONNUMBER20406080100120FRACTIONNUMBER Fig.3.EffectofasingletopicalapplicationofplantphenolsoninvivoBPDE-I-dGuoadductformationinepidermisofSENCARmice.Followingasingletopicalapplication(400/¿mol/kgbodyweight)ofplantphenolsand[3H]BPasdescribedinFig.1,DNAwasisolatedfromtheepidermisandequalamountsfromeachsampleweredigestedandpassedthroughasmallSephadexLH-20columnasdescribedin MaterialsandMethods. TheHPLCprofili'oftheepidermalBPDE-I-dGuoadductsisshownincontrol(• }.annieacid(£)-,quercetin(C)-,myricetin(D)-,andanthraflavicacid(/:')treatedanimals. wasgreaterthanthatobservedfortotal[3H]BP-DNAadductformationshowninFig.1.SincelungisanotherorgansusceptibletocarcinogenesisbyPAHsincludingBP,wefurtherinvestigatedtheeffectofcutaneousapplicationoftheplantphenolsonthebindingof[3H]BPtolungDNA.TheeffectofasingletopicalapplicationofplantphenolsontotalBP-DNAbindingisshowninTable2.Tannicacid,quercetin,myricetin,andanthraflavicaciddiminishedtotalBP-DNAadductformationinlungsby65,51,58,and59%,respectively(Table2).InfurtherexperimentstheeffectofcutaneousapplicationoftheplantphenolsonlungBPDE-I-dGuoadductformationwasassessed.Animalstreatedwithonlyacetoneordimethylsulfoxidepriortotreatmentwith[3H]BPshowedtwounidentifiedpeaks,oneatthebeginningofthechromatogramandasecondwhichelutedafterBPDE-I-dGuo(Fig.4A).ThesepeakshavealsobeenobservedbyDixit etal. 19)inmouselungexplants.Asingletopicalapplication of400¿¿mol/kgftannicacid(Fig.45),quercetin(Fig.4C),myricetin(Fig.4D),andanthraflavicacid(Fig.4E)diminishedBPDE-I-dGuoadductformationinlungsby84,38,53,and69%,respectively.Noneoftheplantphenolsalteredthefirstunidentifiedpeak,whereasthesecondunidentifiedpeakwas Table2Effectafasingletopicalapplicationofplantphenolsontheinvivo bindingoff HJBPtolungDNAinSENCARmice Followingasingletopicalapplication(400fimol/kgbodyweight)ofplantphenolsand[3H]BPasdescribedinFig.1,DNAwasisolatedfromthelung.Covalentbindingwasestimatedasdescribedin MaterialsandMethods. TreatmentAcetoneDimethylsulfoxideTannicacidQuercetinMyricetinAnthraflavicacidCovalentbinding(pmol/mgNA)1.38±0.15 1.29±0.110.49±0.030.68±0.050.58±0.040.56±0.06%ofinhibition65 515859  Mean±SEoffourindividualvalues. eitherundetectableorsubstantiallydecreasedfollowingtopicalapplicationoftheseplantphenols(Fig.4). DISCUSSION StructuralalterationsinDNAproducedbycertainchemicalsmaychangeparentstrandtemplatesresultinginmutationsormalignanttransformationofcells.TheabilityofcellstorepairthesetypesofchemicallyinducedDNAdamagebytheexcisionrepairpathwaypriortocellreplicationconstitutesacriticalmechanismforprotectionagainstmutagenesisandcarcinogenesis(34).InnormalhumanfibroblastsalinearrelationshiphasbeendemonstratedbetweentheextentofBPDE-I-DNAad-ductspresentatthetimeofDNAreplicationandBPDE-I-inducedmutationfrequency(35).MutationscanbeeliminatedincellspermittedtoremaininconfluenceuntilBPDE-Iadductsareremovedbyexcisionrepairenzymes(35).Furtherevidenceforthishascomefromstudiesshowingthatthelengthoftimeforwhichexcisionrepair-deficientxerodermapigmentosumcellsremaininconfluencehasnoeffectonthefrequencyofBPDE-I-inducedmutations(36).DeficientDNArepairprocesseshavebeenidentifiedinindividualswithxerodermapigmentosum(36)andataxiatelangiectasia(37)andthisdefectappearstopredisposethemtoneoplasia(6,15).Ithasbeensuggestedthatthelevelsandpersistenceofspecificcarcinogen-DNAadductsinatargettissuecorrelatewithsusceptibilitytoneoplasia.Forexample,thepersistenceofBPDE-I-dGuoadductandtheconfigurationalchangeintheDNAmoleculeassociatedtherewithappearstocorrelatewithsusceptibilitytoskinandlungtumorinductioninmice(6,15,38).EastmanandBresnick(39)havesuggestedthatMCAmetabolite-DNAadductsaremorepersistentinthelungsofmousestrainssusceptibletoPAH-inducedpulmonaryneoplasiathaninotherstrains.Furthermore,SlagaandBracken(40)haveshownthatcertainantioxidantsincludingbutylatedhydroxyanisoleinhibit 770  INHIBITIONOFPAH-DNAADDUCTFORMATIONBYPLANTPHENOLS Û. o 3UU8007006005004OO30020O100A- t-lanli-BPDEclGuo-•--_1  .1400300.a.0 200100- B.-1L .11 111- c_-1 I1i iii20 406080100120204060801002300^200o1001-Lu II- EIL 1 20406080100FRACTIONNUMBER 120 20406080100FRACTIONNUMBER12020406080100FRACTIONNUMBER 120 Fig.4.EffectofasingletopicalapplicationofplantphenolsoninvivoBPDE-I-dGuoadduciformationinlungofSENCARmice.DNAwasisolatedfromthelungfollowingtopicalapplicationoftheplantphenols.TheHPLCprofileofthelungBPDE-I-dGuoadductsisshownincontrolill.tannicacid Hi-,quercetinI i,myricetin />),andanthraflavicacid f)-treatedanimals.ForotherdetailsseeFig.3. tumorinitiationbyDMBAinatwostageskintumorigenesisetal. 44)whoshowedthatin[3H]BP-treatedmice,thecovalentsystemwhichcorrelateswiththeinhibitionof[3H]DMBA-DNAadduciformationintheskin.Knowledgethatinterferencewithmacromoleculeadductformationcouldinfluencecancerriskpromptedustoassesstheeffectofthetopicalapplicationoftannicacid,quercetin,myricetin,andanthraflavicacidonthecovalentbindingof[3H]BP,[3H]BP-7,8-diol,and[3H]DMBAtoDNAandproteinisolatedfromtheepidermisandlungofSENCARmice.Theseplantphenolsweremaximallyeffectiveindiminishingthecovalentbindingof[3H]BP-7,8-dioltoepidermalDNAandofthesetannicacidwasmosteffectiveinthisregard.Ourrecentstudieshaveshownthattannicacid,quercetin,myricetin,andanthraflavicacidarepotentinhibitorsofarylhydrocarbonhydroxylase,7-ethoxyresorufinO-deethylase,and7-ethoxycou-marinO-deethylaseactivitiesintheepidermisofSENCARmicebothinvitroandinvivo 24).Thereforethedecreaseincovalentbindingof[3H]PAHstoepidermalDNAandproteinasshownherecouldbeduetoadecreaseintheproductionofreactivemetabolitesofthePAHsstudied.Tannicacid,quercetin,myricetin,andanthraflavicacidareeachhighlyactiveinhibitorsofthemutagenicityofBPDE 41,42).Anexaminationofthestructure-activityrelationshipfortheantimutagenicactivityofthehydroxylatedanthraquinones,cinnamicacidderivatives,andflavonoidsrevealedthatphenolichydroxylgroupsonthephenylsubstituentwereessentialforthiseffect 41,42).Otherstudieshaveshownthatellagicacid,anotherplantpol\phenol,isastrongantagonistofthemutagenicityofbay-regiondiol-epoxidesofPAHs 43).Ourdataindicatethat11-13pmol[3H]BP-DNAadducts/mgDNAareformedinepidermisofSENCARmice24hafterthetopicalapplicationoftheradiolabeledhydrocarbon.However,ourdataindicatethatthebindingof[3H]BPtocalfthymusDNAinvitroisabout5timeshigherthanthatofadductsformedinvivo.Thismaybeduetothepresenceofdetoxificationsystemsand/orDNArepairprocessesintheintactbiologicalsystem.The///vivoadductlevelsinepidermisobservedinourstudyareincloseagreementwiththoseobservedbyDigiovannibindingofthehydrocarbontoepidermalDNAreachedapeak24haftertreatment.Inadditionitwasshownthatthedetectableadductsdecreasedrapidlyto50 ofinitiallevelsat48handthatthereafterthelevelofboundBPdisappearedatamuchslowerrate 44).ThesestudiesindicatethatPAH-DNAadductsdisappearfromepidermalDNAwithabiphasicdecaycurve 44).KakefudaandYamamoto 45)providedevidencethatthebindingofBPDEtoadenineinDNAcausedlocaldenaturationofthenucleicacid,whereasbindingtoguanineproducednosuchdenaturation,eventhoughtherewasa10-foldgreaterbindingofBPDEtotheguanineresidues.SincetheBPDE-I-dGuoadductliesintheminorgrooveoftheDNAhelixandcauseslittle,ifany,distortionitmaybelessaccessibletoenzymesessentialforrepairprocesses 46).Otherstudieshaveshownboththerepairofadductsandthepersistenceofadductsforaslongas1yearinwholeskinpreparationsfollowingapplicationofPAHsathighdoses 29).Furthermore,itispossiblethatlossofDNAadductsfrommouseskincanbeaccountedforsolelybycellturnoverratherthanbyrepair.Sincepersistenceofthebay-regiondiol-epoxide-dGuoadductsandtherepairphenomenonareeachimportantdeterminantsofcancerinductiontheeffectsofplantphenolsontheseprocessesshouldbestudiedtomorepreciselydefinetheirmechanism s)ofaction.Phenolicantioxidantssuchasbutylatedhydroxyanisolandbutylatedhydroxytoluene,whenaddedtocommercialdiets,effectivelyinhibitPAH-inducedneoplasiaoflungs,forestom-ach,breast,andintestineinmice 47).Theprotectiveeffectsofthesecompoundsmaybeaccountedfor,atleastinpart,bytheirabilitytoenhancetheactivityofavarietyofdetoxificationenzymesand/ortoshiftthemetabolicprofileofcarcinogenssuchthattheintracellularconcentrationofthereactivemetabolitesisdiminished 47).ButylatedhydroxyanisolehasbeenshowntoinhibittumorinitiationbyDMBAinatwostageskintumorigenesisassaywhichcorrelateswithinhibitionof[3H]-DMBA-DNAadductformationinskin 40).RecentstudiesbyDixitetal. 19)haveshownthatellagicacidisastronginhibitor 771
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