A distinction between 3-methylcholanthrene and estrogen binding in the uterus.

D.O. Toft2andT.C. Spelsberg2'3DepartmentofObstetricsandGynecology,VanderbiltMedicalSchool,Nashville,Tennessee37232SUMMARYThebindingofmethylcholanthrene-3Htomacromolecularcomponentsoftheuterinecytosolfractionwasanalyzedandcomparedwiththespecificbindingofestradioltoitsreceptor.Methylcholanthrene-3Hbindstoacomponent whichsedimentsasa5Scomplexonsucrosegradientsandcanbeeasilydistinguishedfromthe8Sestradiol-receptorcomplex.Unlabeled estradiol does not interfere withmethylcholanthrene-3H binding, nor does unlabeledmethylcholanthrene haveanyeffectontheformationorsedimentationoftheestradiol-3H-receptorcomplex.Theseresultsnegatethepossibilitythatthecarcinogenactsinestrogenictargettissuesbydirectlyinterferingwiththenormalinteractionofestrogenswiththeirreceptorsites. INTRODUCTIONOverthepastdecade,manyinvestigatorshaveanalyzedthefateoftritium-labeledsteroidhormonesintheirtargettissues(14). Thisapproachhasbeenusedsuccessfullytoidentifyspecificbindingproteinsorreceptorswhichareprobablyinvolvedinthemechanismofhormoneaction.Recently,similarmethodshavebeenutilizedtostudytheinteractionofchemicalcarcinogenswithvariouscellularcomponents(9,16). Thebasisofthisapproachisthehypothesisthatcarcinogensinteract with and alter the activity ofspecificgrowth-regulatingsubstances,substanceswhich,possibly,arealsoinvolvedintheactionofsteroidhormones(4â€”6,8,13,20). Ithasbeensuggestedthatsomecarcinogensfunctionbyinteractingwithhormonereceptorsinawaythatalterstheirnormalactivityinthecell.Insupportofthisconcept,Hierowski and MadoA±(3) have reported thatmethylcholanthreneaffectsthebindingofestradiol-170intheratuterusbycompetingforhormonereceptorsitesinvitro.Thisevidencewasbasedontheobservationbytheseauthorsthatmethylcholanthrene causedareductioninbindingoflabeledestradiol-17/3tosolubleproteininvitrowhenitwasaddedtothecytosolpreparationatthetimethehormonewasadded.Inaddition,bothdimethylaminoazobenzene (11)and3-methylcholanthrene(16)havebeenshowntobindinvivoto'ThisworkwassupportedbyGrantCA13065-01fromtheNationalCancerInstitute,andGrantsNIHNICHD70-2165andAID/csd2491,aswellasAmericanCancerSocietyGrantP-576andFordFoundationGrant630-0141A.3MemberoftheCenterforPopulationResearchattheVanderbiltUniversitySchoolofMedicine. 3FellowoftheNationalGeneticsFoundation.ReceivedJune26,1972;acceptedSeptember5,1972.aproteininlivercytosolwhichisindistinguishablefromthecortisol metabolite Binder Iby fractionation onDEAE-SephadexA-50,Cellex-P,andSephadexG-75columns,andbyisoelectricfocusing.Incontrasttothesereports,studiesoftheinteractionsofestrogenswiththeirreceptorproteinsinrabbit(10)andmouse(17)uterihavesuggestedamarkedstereospecificityintheirinteractionsandalsosuggestthattheyseverelylimitthestructuralchangesthatpermitthestrongbindingdisplayedbyestrogeniccompounds.Inaddition,BrecherandWotiz(1)havedemonstratedthat3-methylcholanthreneandahostofothercompoundsfailedtocompetewithestradiol-17j3invitrofor theestrogenreceptorin low-speedsedimentsofratuterinehomogenates.Inthisstudy,attemptsweremadetocharacterizefurthertheinteractionof3-methylcholanthrene withtheuterineestrogenreceptor.Suchaninteractioncouldnotbeobserved,anditappearsthatthe2ligandsassociatewithcompletelyseparatebindingproteins.MATERIALSANDMETHODSImmaturefemalerats(TheHoltzmanCo.,Madison,Wis.)wereobtainedwhen21daysofageand usedwithin3days.Sexuallymaturefemalerats(morethan3monthsold)werealsousedinsomeexperiments.Theratswerekilledbycranialfracture,andtheuteriwereremoved,rinsedin0.9%NaClsolution,andhomogenizedinBufferA(0.05MTris-HCl,1mMEDTA,and0.01Mthioglycerol,pH7.5)withtheuseofaPolytronPt-10(BrinkmanInstruments,Inc.,Westbury,N.Y.). Thetissueconcentrationswere5immatureuterior0.25gofmatureuteripermlofbuffer. Allproceduresweredoneat4Â°. Thecytosolfractionswereobtainedbycentrifugation ofthehomogenates,firstfor10minat39,000X^,andthenfor1hrat149,000Xg.Wepreparedserumfrommaturefemaleratsbycollectingthebloodbycardiacpunctureandallowingittoremainonicefor30minforcoagulation,afterwhichitwascentrifugedfor10minat1,000Xg.Theserumwasthendilutedabout7-foldwithBufferAtogiveafinalproteinconcentrationthatwascomparabletothecytosolpreparations(8to10mg/ml). Estradiol-17(3-6,7-3H,40Ci/mmole,wasobtainedfromNewEnglandNuclear,Boston,Mass. ,andgenerallylabeled3-methylcholanthrene-3H,3.1Ci/mmole,wasobtainedfromAmersham/SearleCorp. ,ArlingtonHeights,111.Thecarcinogenwasshowntobegreaterthan95%purebythin-layerchromatographyinvarioussolventsystems.ThebindingoftheseisotopestomacromoleculeswasDECEMBER1972 2743