dieizen-cart

777FAIIJUREOFCOPPER,TOINHIBITCARCINOG"EN,ESISBY2-A-MINOFLUORENEC.
M.
GOODALL*Ft-o,tntheHiiflhIda.
aDepartmewoCa,)icet-Research.
Unirersity(.
fOtago.
Diiedb,NerZeala)tdReceivedfoi-publicationAugust13,1964EXPERIMENTALprocedureswhichiiiterfereNN-iththeiiiductioiioftunioLtrsbvclicinicalcompo-tiiids,providethemeaiistoiiivestigatetheroleofho,8tfactollse,sisentialfor,orliniitiiig,carcinogenesis.
Inthecaseofthelivertttmoursiiiducedini-atsbychemicalcarcinogenssuchas2-amiiiofluoreiieaiiditsderivatives.
orthecarcinogeiiicazo-des,theprocedureswhichmostdramaticallyinhibithepatocarcinogenesisarethvroidablatioii(BielschowskaiidHall.
19-153),adrell-alectomy(Eversole,1957)orhypophysectomy(Cxriffiii.
Riiifretaiid("orsiglia,1953:O'NealaiidGrriffin,1957).
Theimportanceofezternalfactorsintheiiidtictioiiofhepatomasby4-dimethvlaminoazobenzeiie(DAB)ando-amiiio-azotoluenewasrecognisedloiigago-,dietswithahighcontentofproteinaiidof'i-iboflaviii,orenrichedwithliverextract,retardthedevelopmeiitandlowertheii-icidenceoflivertumours(Ando.
1938:Nakahara,FujiwaraaiidMori.
193(l):Nakahara,MoriandFujiwara,1939).
Thecompositioiiofthefoodhaslessinfltienceontumouryield,however.
inratstreatedwithAForAAF(Harris,1947).
SeveralauthorshavereportedthatratsfeddietshighincopperconteiitatthesametimeasthevwerefedDABor3'-methyl-DABshowedlessliverdamage,ainuchlongeriiitervalforlivertumourinductioii.
andastrikinglylowerincideiieeoflivertumours,wheiicomparedwithcontrolsreceivingdietsofiiormalorlowcopperconteiit(Sharpless,1946PedreroandKozelka,1951Clavtoii,KiiigandSpain,1953King,SpaiiiandClayton,1957;Howell,1958Fare,1963).
Astheseobservationshavebeeiicoiifinedtoazo-dyefeedingexperimentsit,.
;eemeddesirabletocheckwhethertheinhibitoryeffectofcoppercouldbeobtainedwithadifferenttypeofcarcinogeiiwhichcouldbeadministeredbvadifferentroute.
AfurthercoiisiderationpromptingthepresentexperimentswastheobservatioiiofHermannaiidKun(1961)thatthecoppercontentofratliverwasincreasedaboutthreetimesabovethenormalamountfollowinghypophvsectomv,aiidthatthiseffectwasabolishedbytheinjectionofgrowthhormone.
Inviewofthepreviouslyquotedresultswithcarcinogenicazo-dyesitseemedcoil-ceivablethatdisturbancesofcoppermetabolismmighthaveaccountedfortheextremeresistancetobothazo-dyeandaminofluorenehepatocarciiiogenesis-%N-hichisshowiibyhypophvsectomisedrats.
Forthesereasons-thefollowingexperimeiitwasperformed.
METHODSStartingwheiithevwere6weeksofage,24maleWistarratsweregiveiicupi-icacetate(hexahvdrate,B.
-D.
H.
0-tg.
/100ml.
)intheirdriiikiiigwater.
coii-Presentaci(ii-ess,Divisionof011cology.
Chieago11edicalSchool.
Chicago.
U.
S.
A.
778C.
'.
NI.
GOODALLtinuouslyiiiitilthelastanimalwaskilled38weekslater.
Sixaiiimalswerekeptwithoutadditionaltreatmeiitashistologicalcoiitrols.
Theothersweretreatedwith2-aminofluorene(AF),begiiiiiiiigafter2weeksofthecoppertreatmelittopermitaccumulatioiiofcopperintheliverbeforeexposuretothecarcinogell.
TheaminofluorenewassvnthesisedaccordingtothemethodofDiels(1901)asmodifiedbvKuhn(1943)aiidadmiiiisteredasa4pei-ceiitsolutioiiinacetoiieC'AiialarpaintedontotheshaveddorsalskiiiiisiiigaNo.
6sableartist'sbr-Lishwhichwasfoundtodeliver3-0±0-1mg.
AFperapplicatioii.
Theratswerepaintedfourtimesweeklyfor22weeksuntiltheyhadreceivedatotaldoseofapproximatelv270mg.
AF.
Aiiothergroupof12ratsweretreatedinthesanie-%N-aywithAFbutdidnotreceivecoppersolutioii.
asadditioiialcoiitrols.
ThisamountofAFregularlviiiduceslivertumoursiniiitactmaleratsofourMolly,butnoiieatallinthyroidectomisedorhypophvsectomisedaiiimals.
Theconsumptioiiofcoppersolutioi-iwasmeasuredthroughouttheexperimeiit,aiidatiiecropsvsamplesoftheliversweretakeiiforassayofthetotalcoppercoiitentbythediethvldithiocarbamatemethodofEdeiiaiidG[reen(1940)aftei--%A,etashing.
Thedieicoi-isistedofwholewheatadlibitutn,aildamashcompose(Iofbran6,pollard7,skimmilkpo-%A-der4.
andmaizemeal3liartsbyweighti-espectively.
Thiswassupplemeiitedbvweeklyratioiisofmilk.
choppedcarrotandoccasioi-iallvlettuce.
Oiiceamoiitl.
0-5ml.
codliveroilper'ratwasaddedtothefoodbowl.
Theanimalswerehoused6toacageinaroomthermostaticallx-controlledat72±2'F.
Thevwereweighedandcarefttllvexamiiied"A-eekly,aii(INN-heiitumoursoftheliverwerefouiidbypalpatioii.
thevwerekilledwithcoalgas.
AtiiecropsysamplesoftheliverandotherorgansofabnormalappearaiicewerefixedinZenker-formalaiidembeddedin"Tissuematt"(FisherScieiitificCo.
).
Sectionswererotitinelystainedbyhaematoxyliiiai-ideosiii,aiidinselectedcasesalsobvthePAS-diastase.
I'aiiGrieson.
aiidLaidlaw'sreticultimtechiiiques.
RESUTLTSIntheaiiimalsreceiviiigbothAFaiidcupricacetatethei-e'%N-asiioapparelitiiihibitioiiorretardatioi-iofthecarcinogeiiicresponse.
Inoi-ieanimalkilledatthe18thweekforIiistoloicalcomparisonwiththecoiitrolgroups,therealrea(lx-weremicroscopicfociofneoplasticlivercells.
Inallof'theremainiiig17ratsofthisgroupkilled2-18weekslater,thereweremultiplehepatocellularcareiiiomas(Fig.
2)rangiiiginsizefrom0-2to3-5cm.
indiameter.
Mostofthehepatomaswereoftrabecularform,aiidinfivecasespulmoiiarvmetastaseswerealreadypresei-it.
Apartfromthetumours,theliverswerediffuseivhvpertrophiedilleachcaseaiidboremultiplebenigiicysticlesioiisofbiliarv'tiss'ue.
Themeaiiliverweightwas6-25±(S.
E.
)0-84g.
per100g.
bodv,comparedwith3-72±0-18a.
per100g.
innormaluntreatedratsofourcolonv.
Inthecoiitrolstreatedm-itliAFalonetheliversaveraged4-8g.
per100g.
thecoiitrolsreceiviiigcupricacetatealonehowever,alsoshoweddiffuseliverhypertrophyofeveiigreatei-degree(5-9g.
/100g.
).
Microscopicallv,therewasiioevideiiceofiieoplasiainratstreatedwithcupricacetateoiilyforperiodsofupto40weeksduratioil,bilt,theliversoftheseanimals(Fig.
3)oftenshowedamilddiffusecirrhosisaiidwere,richinglycogen.
Afewratsfromeachtreatment-groupweresacrificedatequaldurationsupto22weeksforhistologicalcomparisons,aiidthedegreeofcirrhosisiiiducedbvcopperalonewasateachtimeofsimilarordertothatproducedby779FAILUREOFCOPPERINHim,rioNtreatmentwithAFaloiie;butafter2r)weeksof'coppertreatmeiitatolietheappearaiiceoftheliverrevertedalmosttoiiormal.
Inratsreceivingbothdrugs.
tiowever,theeirrliosiswasi-atliermoresevere.
ii-idicatiiigaiiadditiveeffectIIIthisrespect.
Thelateiitperiodsforthegrosslyrecogiiisablehepatomasintheratsi-eceiviiigbothcopperaiidAFraiigedfrom20to36weeks.
withameaiilatencyof32-6weeks;thediffereiieefromthemeaiilateiievof'34-0weeksillthecoiitrolgrouptreatedoiilvwithamiiiofluoreiieisstaiisticallyiiotsignificant.
I'heiiicideiiceaiiddistribtiiioiioftheextrahepaticiieoplasmsinduce(Ibv,7,AFalsoappearedtobeuiiehaiigedbvtheadditionaltreatmeiitwithcupricacetatetherewere8ratswitlicareiiiomasoftheexteriialeai-ducts(Zvmbalglaiidtumotirs).
3-with.
breastcareiiiomas.
aiidoiiewithatubulo-papillaryadellomaoftheItilig.
hitheanimalsreceiviiigbothdrugs.
Consumptionofthecupricacetatesolutioiivariedbetweeii1-5aiid6-5iiij.
per100g.
bodvweightperday,withanaveragedoseof2-5ml.
/100g.
ratperday.
,rherewasmoderategrowth-inhibitionbycoppertreatmeiitalone,butiiotsomuchasthatproducedbytheAFtreatmeiit.
andtheweightcurveofthegroul)treatedwithcupricacetateandAFincombinatioiiwascoincideiitwiththatofthegroupreceiviiigoiilvAF(Fig.
1).
ThusthedoseofAFperuliitbodyweightwassimilarinthetwogroups.
Thecopperanalysisatiiecropsyshowedincreases,offrom.
5upto17timestheiiormal(7-9p.
p.
m.
Cu;dryweight)amountsofliepaticcopperinthetwogroupswhichhadbeeiitreatedwithcupricacetate.
wheiicomparedwiththeuiitreatedorAF-treatedcontrols.
Copper-treatedratsofteiihadhyperemicaiidslightlyeiiiargedthvroids,sonietiiiieswithilliliol-degeiiei-ativelesionsinthefollictilarepithelitim(].
ig.
4).
-DISCUSSION'I'hepreseiitexperimentshaveshowiithattheinhihitionofazo-dvehepato-careinogenesisbyexcessdietarVcopperisnotageiieralphenomeiioiibutlikelytoberelatedrathertosomepeculiarityinthemetabolismoftheazo-dvecarciiio-YellsDABor3'-me-DAB.
NAith2-aminofluoreiieasthecarcinogen,liver-celltumotirswereindticedinallcopper-treatedaiiimalsatrisk.
andtheinductioiitimeteiided,ifaiivthiiig,tobeshortei-thaiiinthecoiitrols.
-Liver-celliiecrosisaiidcirrhosisincopper-treatedratshavepreviouslvbeeiireported(Mallory.
1925C-xubleretal.
.
1954'ATolff.
1960),results,whichthepresentobservatio;iscoiifirm,andasimilardiffusecirrhosisinliversofhumaiipatieiitswithWilsoii's(lisease(hepatolenticulardegeiieration)wasdescribedbvAildersoilaildPopper(I960).
WheiiratsweretreatedwithbothcupricacetaIeaiidaminofluoreiiethe(legreeofliverdamagewasapparentlyaccountableasduetosimplesummatioiioftheeffectsofthe(irugsgiveiiseparately.
Bvcoiitrast,thestudiesoftheeffectofcopperonazo-dvecarciiiogenesishaveallemphasisedtheprotectioiioftheliveragaiiistdamageaiidcirrhosis.
aswellasthemoreorlessstrikiiiginhibitiolloflivertumouriiid-tictioii.
aiidgreatlyprolongedlateiitperiodsforthefe-%A-tumoursocciirring.
AsHowell(1958)showedliowever.
the"protectiveeffectofthecopperwasiiotappareiitlviiicreasedbvraisiiio,thedoseofcopperafteradministratioilofthecarcinogen.
adbeguii.
Fromthepreviousm-orkciteditappearsthattheprotectiveeffectsofcopper-wereoiilydemoiistrablem-heiitheazo-dv,eaiidthecoppersalthadbeeiiiiiixedtogethereitherinthefoodmixture.
or.
lsewithiiitheiiitestiiialti-act.
aswotil(t780C.
M.
GOODALLstillbethecaseinHowell'salteriiate-feedingexperiment(op.
cit).
Undersuchconditionsdestructionofthecareinogeiioccurs(Kingetal.
,1957),althoughRowell(1958)consideredthiscoLildiiotaccountentirelyfortheobservedAA'IIIAiIIIIIi11IIIZ.
IIA9300.
280.
I4elIIIIirIia9260.
240FII0iAIIIIII,do220z200M0180m3::IIIIIALa,IfIIFlbO.
IIII&Iij140F120F10080bIIIIIIIIIII1-II1812lb202428323btStortAFWEEKSStortcupricocetoteFi(.
.
,.
J.
-Weightcurvesofuntreatedrats(1),ratstreatedwithcupricacetate(11),witliaminofluorene(IV),an(Iwithboth(irugs(111),showingthemoderategrowth-inhibitiolibythesedrugs.
inhibitionofliver-tumourinduction.
Thisexperimentaldeficiency,alreadyrecognisedbyKingetal.
(1957),hasbeenavoidedinthepresentexperiment'givingthecoppersaltpero8whiletheaminofluoreneenteredthebodypercutaiie-ously(GutmanandPeters,1957).
ThedoseofcupricacetateadministeredinEXPLANATIONOFPLATEFIG.
2.
-Invasivehepatocellularcarcinomainratstreatedwitliainiiioflitoi-eneandcupricacetate(28weeks).
H.
anciE.
x60.
Flc.
.
3.
-Liverofrattreatedfor40weekswithcupricacetatealone;theliveris1-ichinglycogeiiandthepoi-taltractsarestillhypercellular.
H.
andE.
x60.
FiG.
4.
-Slightlyhyperplasticthyroidofrattreatedfor35weekswithcupricacetatealone.
showingfocaldegenerationoffollicularepitheliumanddisruptionofafewindividualfollicles.
H.
andE.
x60.
Vol.
XVIIII,No.
4.
BRITISHJOURNALOFCANCER.
4Goodall.
FAILUREOFCOPPERINHIBITION781-thepreseiitexperimeiits(average2-0-Ing.
per100g.
bodvperday)equalsorexceedsthepresumeddosageinpreviousexperimei-itswithazo-dyes,andasaprelin'linaryperiodwasallowedforloadiiigtheliverwithcopperbeforeexposuretothecareinogeii,thetestforaiiyinhibitingeffectofcoppersaltsonamillo-fluorenecarcinogenesisappearstohavebeenadequate.
Finally,theincreaseinlivercopperconteiitresultingfromtreatmeiit'"-ithcupricacetateexceededconsiderablytheincreasesfoundbyHermannandKiiii(I961)tooccurafterhpophvsectomy,andthedisturbancesincoppermetabolismafterthisandothereiidocrineablationscanthereforenotaccountforthestrikiiio,Z--refractorinessofthelivertocarcinogensofboththeazo-dyeandaminofillorelietypes.
whichthoseoperatioiisindtice.
SUMMARYIncoiitrasttopreviousexperiinentswithoraltvadministeredazo-dvecarcinogens,ratsgivencupricacetatesolutionpero8werenotprotectedfromeitherliverinj'urvorhepatomainductionby2-aminofl-Lioreiieadministeredpercutaneously.
TheauthorisverygratefultoDr.
F.
Bielschowskyforhisadviceaiideii-couragement,toMissG.
Macdonaldforthecopperdeterminations,andtotheNewZealandBranchoftheBritishEmpireCaiicerCampaignforResearchaiidU.
S.
PublicHealthServiceG'raduateTrainiiigGrantNo.
CA-5070forfinai-icialstipport.
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