Document rBMmNbqR3YagEXmn6D8DVM9kv

AN INVESTIGATION OF THE EFFECTS OF FLUOROCARBONS FATTY ACID-BINDING PROTEIN. DEANNA J.NABBEFELD Masters Thesis Kev ON LIVER FCI: PerfluorooctanSeulfonicAcid (PFOS) FC2: Ammonium Perfluorooctonat(eAPFO) FC3: N-ethylperfluorooctanSeulfonamide Ethanol (N-EtFOSE) FC4: N-ethvlperfluorooctanSeulfonamide (N-Et FOSE Amide: FX- 12) AN INVESTIGATION OF THE EFFECTS OF FLUOROCARBONS ON LIVER FATTY ACID-BINDING PROTEIN. A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF THIE UNRVERSM OF ME4NESOTA BY DEANNA J.NABBEFELD IN FULFELLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCEENCE/ENVIRONMENTAL HEALTH APPJI, 1998 AN INVESTIGATION OF THE EFFECTS OF FLUOROCARBONS FATTY ACED-BINDING PROTEIN. ON LIVER ABSTRACT The objectiveof thisstudywas to investigattehe hypothesisthatcertainFluorocarbons (FCs)bindliverfattyacid-bindinpgrotein(L-FABP) and displacendogenous fattyacids (FAs) as an initiaelventleadingto peroxisomeproliferatioTnh.e goalsof the studywere to assesstheeffectof FCs on L-FABP fiinctioansevaluatedby theabflitoyf the fluorescenFtA analogue 11 - (5-dimethylaminonapthalenesulphon-ylu)ndecanoicacid (DALTDA) to b'mdto L-FABP isolatefdrom ratsand guineapigstreatedand not treated withFC I invivo;and to assessthepotency ofFC 1,FC2, FC3 and FC4 forbindingto LFABP. Resultsshow a decreasedmaximum bindingcapacityofL-FABP from FC I treatedrats withoutan increaseinKd. The most potentL-FABP binderwas FC 1,followed by FC4 and (withequalIC5os)FC3 and FC2. ResultsforguineapigL-FABP samples were inconclusiveT.his may be because guineapig samples were onlypartiallpyurifiedt;hus resultinigna highdegree ofinterferencferom remainingcelluladrebrisand a lower concentrationof L-FABP, asa proportionoftotalproteina,s compared to ratsamples. INTRODUCTION STUDY OBJEC'RIVES This studywas designed toinvestigattehehypothesisthatcertainfluorocarbons(FCs) bindto liverfattyacid-bindinpgrotein(L-FABP) and displaceendogenous fattyacids (FAs) as an initiaelvent leadingto peroxisome proliferatioTno.examine thishypothesis, thekineticsofFA and FC bindingto L-FABP were investigatewdithan in vitrobinding assayusingthefluorescenFtA analogueI I - (5-dimethylaminonapthalenesulphony-l) undecanoicacid(DAUDA). FC I and FC2, known peroxisomeproliferatorasn,d FC3 and FC4, suspectperoxisome proliferatorwse,re examined. Wyeth- 14,643(WY), a well known peroxisome proliferatowra,s used as thepositivecontroland methanol asthe negativecontrol.Oleicacid,a FA known to bindto L-FABP with a veryhigh affinitwya,s used to measure the maximum L-FABP binding.(FigureI - structuresL)-.FABP from male rats(consideredtobe strongrespondersto peroxisome proliferatorasn)d male guineapigs(consideredto be weak or non-respondersto peroxisome proliferators) (Svoboda,Grady and Azamoff, 1967;Orton elal.,1984;Lake and Gray, 1985;Elcombe and NEtchell,1986),treatedor not treatedwith FC I in vivo,were examined. The goalsofthestudywere as follows: 1)to assesstheeffectofFCs on L-FABP function,asevaluatedby theabilitoyf DAUDA to bindto L-FABP isolatedfrom ratsand guineapigs;and 2) toassessthepotency of thevariousFCs forbindingto L-FABP. The firsgtoal was accompifishedas follows: a.L-FABP from ratsand guineapigs,treatedand not treatedwith FC 1,was isolated; b.themaximum bindingcapacityor receptornumber (Bmax) of each L-FABP sample and thedissociatiocnonstantoraffinit(yKd) of DAUDA foreach L-FABP sample were calculateda;nd c.theconcentrationofoleicacidwhich inhibite5d0% of specific DALTDA bindingto isolateLd-FABP samples,theoleicacidIC5oforeach sample,was measured. The second goal was achievedby calculatintghe IC50of each FC forthe bindingof DALTDA tothe isolatedcontrolratL-FABP sample. 2 FIGURE I - STRUCTURFS. FCI FC2 FC3 FC4 %eth- 14,643 H3 PC NH -HH3, --o cI SCH2COOH Methanol CH3--OH DALJDA N(CH3)_, OleicAcid CHr-(CH2)7-@H=CH--(CH2)7--COOH I 02NH(CH2),OCOOH 3 BACKGROUND LivER FAT7TAciD-BiNDiNG PRoTEiN The exact role of L-FABP, a member of the intracellularlipid-bindingprotein (ILBP) family,isunclear (Bass, Kaikaus and Ockner, 1993). Itisfound predominately in the liver,although itis also presentin the small intestinaalnd colonic enterocytes,gastric brush border,and enteroendocrine ceus (Bass, 1985; Bass, 1988; Sweetser, Heuckeroth and Gordon, 1987; Vincent and MuDer-Eberhard, 1985; Chan el al.,1985; Gordon el al., 1982; Sorof and Custer, 1987). Accepted functionsof L-FABP includebinding and transportingFAs within the cell,regulatinghpid metabolism, and protectingthe cellby maintainingthe concentration of free fattyacids(FFAS) below toxiclevels(Bass et al., 1993). L-FABP isunique to the ILBP family in that ithas a largerbinding cavity (Thompson et al.,1997); broader figandspecificit(ybindsmultiplehydrophobic compounds such as heme, certaineicosanoids,bilirubint,hyroxine,steroids,specific carcinogens and peroxisome profiferatorass well as FAs) (Kaikaus, Bass and Ockner, 1990; Ockner et al., 1972; Rolf el al.,1995; Thumser, Voysey and Wdtor@ 1994; Khan and Sorof, 1990; Lev@ Gatmaitan and Area, 1969); and the abilityto bind two molecules per proteinwhile other iLBPs bind only one (Thompson el al.,1997). The crystalstructureof rat L-FABP (Thompson et al, 1997) revealstwo short antiparallel a-heficespositionedover one end of an I I-strandedantiparalie0l-barrel.This differsfi7om other iLBPs, which are 10-stranded,but does not signfficantlaylterthe conformation of the protein. A cavityisformed withinthe 0-b=el thatservesas an internalizedligand 4 bindingsitewithpolarand nonpolarresiduesand bound water.Inadditiontoa normal gap between thetwo P-strands,L-FABP has a second gap formed by missing hydrogen bonds. The functionofthisgap isunknown, but themissinghydrogen bonds increasethe rangeof motion inL-FABP compared to otheriLBPs.Thislocalizecdonformational flexibilimtay contributetothebroad figandspecificietxyhibitebdy L-FABP. Two L-FABP bindingsitesexista,nd interacatllostericaUCyr.ystalstructureosfthe proteinpreparedwith oleicacidhave characterizetdheprimarybindingsiteby an internalizecdarboxylateand a U-shaped hydrocarbon chain.Fattyacidsbound inthe primarybindingsiteinteracwtith Arg,22,a conservedresidueinalliLBPs; and are surroundedby proteinatoms,structurawlater and nearbyatoms ofthesecond bound FA. The oleicacidintheprimarybindingsiteisinvolvedinhydrogen bond interactionastthe 124 . carboxylgroup with Ser9,Arg'22and Ser The secondarybindingsiteischaracterized by having thecarboxylateof thesecond oleicacidnear thesurface,and the hydrocarbon tailinsertedtoward the centerofthemolecule and between theU-shaped hydrocarbon chainintheprimarybindingsite.The carboxylateatthissiteissolvent-accessiblbeu,t still involvedina network of hydrogen bonds withresiduesformingtheentranceto the primarybindingcavity.The two ligandsareinphysicalcontactand itisbelievedthatthey influenceeach othersrelativaeffinitieSst.ructuradlatasuggestthe second sitemay not existuntdthe primary siteisfffledo,rthatthe priorpresenceof a FA inthe primarysite may be requiredforanythinglargerthata C 14FA to bindthe secondarysite(Thompson el al.,1997). 5 FLuoRocARBoNs Fluorocarbo(nFsCs)arecompoundsstructuraalnlaylogoutsohydrocarbownisththe hydrogens replacedwith fluon'nesT.he FCs under investigatiornesemblelong chainFAs, having a hydrophobic tailand a polarhead group. The tailsof FCs are more rigidin structurethan the tailsof FAs, however, and thus the conformational flexibilitoyf FCs is more restrictetdhanthatofFAs (Zisman,1964). FCs have uniquechemicaland physical propertiesuch as beingvery heat stablei,nertand chemicallyand electricalnloynreactive (Bryce, 1964; Bankes, 1970; George and Anderson, 1986; Gfflfland, 1992; Clark et al., 1973). Such characteristics make them ideal for use in many consumer products and industrialprocedures (Bryce, 1964).FCs are components of products includinghousehold cleaners,leathertreatments,insecticidesand fire-extinguishinfgoams; used as surfactants in the aqueous polymerization of fluorinatedmonomers; and used in industrialprocesses such as insulatingc,ooling,wetting,and corrosion inhibition(Bryce, 1964; Bankes, 1970-1 George and Anderson, 1986; CTUWand, 1992; Clark et al.,1973). Despite the usefulnessof these chemicals, some are known to cause mitochondrial inmbition,cholestasis, peroxisome proliferationand tumor formation in rodents (GilWand and Mandel, 1996; Langely, 1990; Ikeda et al.,1985 -P1astoor et al.,1987; Harrisionelal.,1988; AbdeHatif et al.,199 1). Permadi etal.(1993) suggest chain lengthof FC s influencesthe severityof effect,finding the greatestsignificanceexhibitedby Cg compounds followed closelyby Clo compounds, and increasinglylesssevere consequences exhibited with shorterchained molecules. The work of Fellerand Intrasuksri(1993) agreed with thatof Permadi elal.(1993),and added 6 thata carboxylicfunctionwas importantforthe stimulationof peroxisome prioliferation. Similarresultswere reportedby Kennedy etal.(1998),who analyzedFCs ranging in lengthfrom 4-9 carbons forthe effectof structureon toxicity.They found Cg FCs to produce effectast a 10-foldlower dose than C6 FCs, and shortchainedFCs to be theleast toxic. Although the effectsseen inrodents have not been seen in humans, the potentialfor cumulativeand long-term human toxicityresultingfrom continuousexposure to low concentrationsof FCs isof concern (Gillilanadnd MandeL 1996; Gilliland1,992). PEROXTSOMES & PEROYaSOMEPROLIFER,4 TION According to Small (1993),peroxisomes (alsocalledmicrobodies or,in plants, glyoxysomes) are singlemembrane-limited cytoplasmicorganeflespresentin most eukaryoticcefls.The major functionof pero)dsomes isthe 0-oxidationof FAs and FA derivative(sMannaerts and Van Veldhoveri,1993).Pero)dsomes containno DNA, rather, theirproteinsare synthesizedon freepolyribosomes inthe cellcytosoland imported into pre-exist'mpgerox3somes post-transiationallIyt.isbelievedthatnew peroxisomes form by fissionfrom existingperoxisomes. Pero)dsomes have been shown to proliferatfeollowingexposure to a diverseclassof chemicalsreferredto as peroxisome proliferator(sGreen, Issemann and Tugwood, 1993). The mechanism by which thisoccurs isunclear.Peroxisome profiferatioinsthought to be mediated by peroxisome proliferatoarctivatedreceptors(PPARs), nuclearhormone receptorswhich,upon bindingligand,recognizespecifiDcNA sequence motifslocated upstream of theperoxisome profiferatotrargetgenes (peroxisome proliferatorresponse 7 elements(PPREs)), and activatespecifigcene transcriptio(nIsseman and Green, 1990; Dryer etal.,1993). Due to thediversitoyf peroxisome proliferatorsshown to activate PPAR (Green elaL, 1993),speculationexistsover a directmodulation of PPAR by peroxisome proliferatorasn,d an indirectmechanism issuggested. In additionto chemical and xenobloticperoxisome proliferatornsa,turalfactorssuch as a highfatdiet,starvation and diabetes(Flatmark etal.,1988;Ishiiet al.,1980; Ishi@Horie and Suga, 1980; Horie, Fukumori and Suga, 1991; GottlicherW,idmark and Gustafsson,1992) have been shown to cause peroxisome profiferatioTnh.is correlationbetween peroxisome proliferatioannd FA metabolism suggests thatPPAR servesan importantrolein Epid homeostasis (Vanden Heuval, 1996). Itisprobable,thus,thatPPAR activatiornepresentsa physiological responseto a biologicalstimulus,likelya factorinvolvedinFA metabolism (Green etal., 1993). Possiblestimuli/PPAR figandsincludesteroidsF,As and derivativeosf FA metabolism,and cholesterolmetabolites(Green elal.,1993). Targetgenes includethose foracyl-CoA oxidase (Tugwood etal,1992; Fellerand Intrasuksri1,993),L-FABP (Isseman etal.,1992) and P450 FVAI genes (Green etal.,1993). Significanitnterestsurrounds the issueof peroxisome proliferatiobnecause some peroxisome proliferatorhsave been shown to cause hepatocellulacrarcinomas in laboratoryrodents (Moody elal, 199 1-Vanden Heuval, 1996).The mechanism by which peroxisome proliferatorcsause cancer inrodents isunknown. They are classifieads a novel classof epigenicchemicalcarcinogen(Vanden Heuvel, 1996),are nomnutagenic in the Ames assay and do not appear to bind DNA (Conway etal.,1989; Cohen and Grasso, 198 1;Reddy and Lalwani, 1983, Stott,1988; Reddy and Rao, 1989; Lake et al.,1990). 8 Multiplemechanisms have been proposed to explainperoxisome proliferator-inducleidver tumor forinatiomincludingoxidativestress(Reddy and Rao, 1989),enhanced cell replicatio(nMarsman elal.,1988) and promotion of spontaneouslyformed lesions (Schulte-HermannetaL, 1989).Green elaL (1993) propose pero)dsomeproliferatorasre @lcompletecarcinogens"which exhibita combinationofinitiatio(noxidativeradical production)and promotion (livemritogenesis)p,ossiblyleadingto sustainedDNA repbcationdepending on the compound and dose.Doubt about a causalrelationship between peroxisome proliferatioand carcinogenesiisn rodentsexistsh,owever, and the relevanceto human healthisunclear(Tucker and Orton, 1993). Mammalian speciesdifferintheirresponseto peroxisome proliferato(rLsake and Gray, 1985;Rodricks and TurnbulL 1987).Ratsare consideredstrongresponders,and guinea pigsand nonhuman primateslow tonon-responders(Svoboda etal.,1967; Orton etal., 1984-1Lake and Gray, 1985;Elcombe and NEtchefl,1986). Sfightto no increasein peroxisomeswere found inhuman patientstreatedwithcolfibrat(eHanefeld,Kemmer and Kadner, 1983) and fenofibrat(eBlumcke elal.,1983),drugs used inthetreatmentof hypercholesterolemiaand known pero)dsome proliferatoirnsrodents.Many hypothesize thatifa causalrelationshidpoes existbetween peroxisome proliferatioand hepatocarcinogenesiist,isspecifitco rodentsand not a riskto man (Tucker and Orton, 1993). Other wefldocumented effectosfperoxisome profiferatoirnsrodentsincludeinhibitioonf mitochondria0-oxidation(Elcombe and Mitchell,1986-1Eacho and Foxworthy, 1988; 9 Foxworthy and Eacho, 1988-1Lock, Mitchelland Elcombe, 1989-1Wallace,1998), inductionofperoxisomal0-oxidationand co-o)ddatioinntheER (Reddy and Lalwani, 1983,Hawkins elal.,1987),inductionof L-FABP expression(Bass,Manning and Ockner, 1985;Das, Gourisankarand Mukhedea, 1989-1FleischneretaL, 1975), cholestasi(sElcombe and Mitchell,1986;Foxworthy and Eacho, 1988; Lock elal.,1989-1 Van Rafelghem etal.,1988) and hepatomegaly(Moody etal.,199 1). FA rry A ciD CA TABoLism iN THE HEPA TocyTE Free fattyacids(FFAs),formed by thebreakdown oftriacylglycerosltsoredinadipocytes, arecarriedinthe blood by serum albuminand transportedintohepatocytesby what is thoughttobe a plasma membrane bound fattyacid-bindinpgrotein(FABPpm) (Stremmel, Strohmeyerand Berk, 1986;Stremmel etal.,1985). Once inthe cellF,FAs arepickedup by L-FABP and,under routineconditionst,he majorityaretransportedto the nutochondriaforP-oxidationa, processby which FAs aredegraded to acetyl-CoA by the sequentiarlemoval of two carbonsegments(Moran and Scrimgeour,1994).NEtochondfial 0-oxidationiscoupled to thegenerationof highenergyphosphatebonds viaoxidative phosphorylationa,nd resultsinthesynthesisof ATP and ketone bodies.In orderto gain entryintothe mitochondfia,FA must firsbte convertedto acyl-CoA estersby acyl-CoA synthetasesF,A specificenzymes locatedinthenu'tochondfiaolutermembrane (Singh, Derwas and Poulos,1987).The rateofFA entryintothemitochondriaisregulatedby camitineacyl-transferasIe,a second enzyme locatedintheoutermembrane of the rnitochondfiaw,hich convertsacyl-CoA esterstoacylcamitine(sMurthy and Pande, 1987).Once insidethernitochondriaa,cylcamitineasreconvertedback to by acyl-CoA 10 estersby camitineacyltransferas1e1,and degraded by mitochondrial0-oxidationto acetylCoA (McGarry and Foster,1980;Bieber,1988). Acetyl-CoA isshuttledintothe cytosol by the citratteransportsystemforcholesteroalnd lipidsynthesi(sStryer,1994).The key factorregulatintghe rateofmitochondrial0-o3ddationistheamount ofFA enteringthe rrutochondriwahich, as statedabove,iscontrouedby camitineacyl-transfera1s.eThe activitoyf camitineacyl-transferaIsiescontrouedby theabundance ofmalonyl-CoA, the firsctonunittedintermediateinFA synthesi(sMcGarry and Foster,1980).According to Bass elal.(1993),under conditionsofincreasedFA biosynthesism,alonyl-CoA productionisincreasedM.alonyl-CoA isproduced from acetyl-CoAina reaction catalyzedby acetyl-CoA carboxylasea,n enzyme controfiebdy reversiblpehosphorylation respondingtohormone signalsand thepresenceof fattyacyl-CoA (Moran and Scrimgeour,1994).When fattyacyl-CoA levelsarelow,acetyl-CoAcarboxylaseactivity ishigh. Thisenhancesthe conversionofacetyl-CoAto malonyl-CoA. When malonylCoA isplentifutlh,e activitoyfcamitine-acyltransferIasieslimited.This causesthe rate ofrnitochondria0l-oxidationtodecrease;FFAs to accumulate;acyl-CoA productionand hence cholesteroslynthesisto slow;and the ratesof alternatreoutesof FA catabolism, peroxisomal0-oxidationand a)-oxidatioinntheendoplasrr@rceticulum(ER), to increase (Lock etal.,1989). Pero)dsomal0-oxidationisnorma4 responsiblfeorcatabolizinmgost,ifnot all,ofthe verylongchainfattyacidsbrought intothe hepatocyte(Singhelal.,1981;Singh etal, 1984;Lazo etal.,1990;Jakobsand Wanders, 199 1).Thissystemisalsocapableof oxidizingmedium and longchainFAs and previouslyactivatedCoA estersof medium and II longchaindicarboxyliaccids.Under normal conditionsh,owever, mitochondrial0oxidationisthe dominant routeof catabolismforsuch substrate(sSinghetal.,1987). Peroxisomal0-o)ddationproceedsthrough similarstepsas does rr@tochondria0loxidationh,owever, importantdifferenceesxist.Firstt,he enzymes used ineach process aredifferenptroteins(Hashimoto,1987). Secondly,peroxisomal0-oxidationdoes not degrade FAs to theirtwo carbon fragmentsas does rnitochondn'a0l-oxidation;rather, pero)dsomal0-o)ddationstopsaftera few cycles,onlyshorteningthecarbon chain (Lazarow, 1978;Thomas etal.,1980). Thirdly,peroxisomal0-oxidationisnot coupled toan electrontransportchainand oxidativephosphorylationasismitochondrial oxidation(Lq7a ow and de Duve, 1976;N4annaertsetal.,1979). Thus, while rrutochondriaPl-oxidationproduces ATP and ketone bodies,peroxisomal0-o)ddation produces hydrogen peroxideand heat.The rateof pero)dsomal0-oxidationisthoughtto be controlledby substratesupply,specificaltlhye activitoyf acyl-CoA oxidase,which reducesmolecularoxygen to hydrogen peroxideinthe firssttepofperoxisonial oxidation(Mannaerts etal.,1979;Myazawa etal.,1983). Like substratefsor mitochondrial0-oxidations,ubstratefsorpero)dsomal0-oxidationmust be esterifietdo theiracyl-CoA derivativesh;owever, peroxisomalP-oxidationisnot dependent on camitineacyltransferas1e,as isn-@tochondriaPl-oxidation(Mannaertsand Van Veldhoven, 1993). co-OxidafioinntheER, a P450 rvalmediatedprocess,isresponsibleforconverting monocarboxylicacidsto dicarboxyliaccids.DicarboxylicacidsareactivatedintheER by 12 dicarboxylyi-CoAsynthetasea,n enzyme absentinrm'tochondriaand peroxisomes. CoA estersof dicarboxyliaccidsarealmostentireldyependent on mitochondrial0-o)ddationfor catabolism(Suzukietal.,1989). The ER alsooxidizesbfleacidintermediateasnd isable to esterifvyerylong chainfattyacids(Singhand Poulos,1988;Lazo etal.,1990). A prerequisitoef esterificatiiosnactivatioonfFAs to theirCoA derivative(sMannaertsand Van Veldhoven, 1993). co-Oxidatioinnthe ER isenhanced incasesofFA overload(eg uncontroflediabetes)or inhibitioonfmitochondria0l-o)ddafion(Mortensenand Gregersen,198 1;Golden and Kean, 1984;Mortensen,1986;Vianey - Liaud etal.,1987); and likeperoxisonial0-oxidationa,)-oxidatioinsnot dependenton camitineacyl transferasIe(Mannaertsand Van Veldhoven, 1993). HypoTHFsis As statedabove,exposure toFCs leadsto mitochondrialinhibitiocnh,olestasis, pero)Usome proliferatioarn@d tumor formation*inrodents.Three of theseendpointsmitochondrialinhibitionc,holestasiasnd pero)dsome proliferati-onare directllyinkedto FA metabolism.This studywas designedto testthehypothesisthatan initiasltepinFCinducedpero)dsome proliferatiiosndisplacementof FAs from L-FABP by FCs. This hypothesisissupportedby thefactthatL-FABP has been shown to bindnongenotoxic peroxisome profiferatorisn,cludingcertainFCs, in vitro(Vanden Heuvel, 1996; Issemann etal.,1992),with relativsetrengthsofbindingthatparalletlheirabilittyo elicit peroxisome proliferatio(nBrandesetal.,1990;Kanda elal.,1990;Cannon and Eacho, 1991).According to thetheoryunder question,upon displacementofFAs from L-FABP, theintraceuulalrevelsof fattyacyl-CoA would decrease.Thiswould increasetheactivity 13 of acetyl-CoA carboxylaseand enhance the conversion of acetyl-CoA to malonyl-CoA. An increaseinthe levelof malonyl-CoA would repressthe activityof camitine acyltransferas1e,and inhibimtitochondrial0-oxidation.(0-oxidationinthe ER would be enhanced, increasingthe production of dicarboxylicacids.PPARs would be activated,by the bindingof FAs or metabolic intermediatessuch as dicarboxyhc acids,and specificgene transcriptioonf acyl-CoA oxidase,L-FABP and P450 IVAI would be induced.A positive relationshibpetween the amount of L-FABP and the rateof peroxisomal 0-o)ddafionhas been found (Appelkvistand DaUner, 1980),and the levelof acyl-CoA o)ddase isthought to determinetherateof peromisomal P-o)ddafion(Mannaerts etal.,1979; Nbyazawa etaL, 1983).Thus, increasedtranscriptioonf acyl-CoA o)ddaseand L-FABP would increase ratesof peroxisomal 0-o)ddationand elicipteroxisome prohferation.Inducfion of P45c@vA, genes would furtherincreasethe rateof e)-o)ddationinthe ER. Cholesterolsynthesis would eventuallycease in responseto rnitochondriailnhibitioannd lackof acetyl-CoA production,and decreased esterificatiboyn the ER due to decreasedacyl-CoA. This would lead to cholestasisT.he mechanisms by which carcinogenesiscould be induced or promoted willnot be discussed. 14 MATERIALS AND METHODS MATERIALS Wyeth-14,643 (WY) was obtainedfrom ChemSyn ScienceLaboratories,Lexena, KS; FCs were provided by 3M SpecialitCyhenu'calsDivision,St.Paul,MN; OptifluorLSCcocktailwas obtainedftom the Packard InstrumentCompany, Meriden, CT, ANUCON YM-5 membrane was purchasedfrom Amicon Corporation,Lexington,MA; BCA Protein Assay was obtainedfrom PierceCherificaClompany, Rockford, IL;and II-(SDimethylaminonapthalenesulphonyl)-undecanoiaccid(DAUDA) was purchased from Molecular Probes, Eugene, OR. AJI otherchemicalswere obtainedfrom VWR Scientific, West Chester,PA. ANP@uLs A" TREATMENT Male ratsand guinea pigs,6-8 weeks of age, weighing between 150 and 250 grams were purchased fi7omCharles River Labs, Wilmingtor@ NiA. Following an adaptationperiod of one week afterarrivalat3K animalswere weighed, ear-taggedand exposed. The treatmentgroups consistedof the following: 1.Guinea Pig VehicleControl- Tween 80, 2% (n = 4); 2. Rat VehicleControl - Tween 80,2% (n = 4); 3. Guinea Pig FC I - FC I in Tween 80, 2% (n = 4);and, 4. Rat FC I - FC I inTween 80, 2% (n = 4). Alltreatmentswere adniinisterebdy intraperitone(ailp)injection.The vehiclecontrol groups were dosed at 5ml / kg body weight 2% Tween 80. The FC I groups were dosed at 15 5 n-d/kg body weight with a suspensionof 32 mM FC I in Tween-80, 2% (86 mg FC I kg body weight) - Allanimalswere housed individualliyn controlledenvironments and observed formortalityand clinicaslignsof toxicityduringthe firsftour hours afterdosing, at 24 hours,and dailythereafterforthe durationof the study.Animals were sacrificed with C02 12 days afterdosing.Body weights and selectedorgan weights (liverk,idneys, testes)were recorded atnecropsy.Organ tissuesand body fluidswere storedfrozen at70'C forbiochemical analysisor in I0*/obuff@redformaldehyde for subsequent histologicaalnalysis.Selectedfiverswere perfused with and storedingluteraldehydefor fiiturheistologicaalnalysisby fightmicroscopy. PURMCATION OF L-FABP Proteinpurificatiownas performed atthe Universityof San Francisco,CA (UCSF) Liver Research Center. Three frozenlivers(ratFC 1,guinea pig vehiclecontrol,and guinea pig FC 1)were shipped indry icefrom 3M to UCSF. These liversand one freshliver,from a non-treatedrat(control)sacrificeadt UCSF, were purified. Frozen livers(approximately IOg each)were thawed and weighed. The freshliver (approximatelyIOg) was isolatedand perfusedwith isotonicsaline.Each liverwas homogenized 300/o(w/v) inice-coldIOmM potassium phosphate buffer,pH 7.4,using a Teflon-glassPotter-Elvehjemtissuehomogenizer. The homogenates were centrifugedfor 20 minutes at 10,000g ina Sorvallsuperspeed RC2-B centrifugemaintained at 40C. The supernatantswere removed and subsequentlycentrifugedfor one hour at 3840,000 rpm (4'C) ina Beckman L7 UltracentrifugeT.he resultingsupernatant(cytosol)was labeled with 0.5gCi of I_14C oleateto tracethe L-FABP duringpurification, 16 P,urificatisotnepswere performed ina cold room at40C. The cytosolwas loaded on a Sephadex G50 M column (5 x 60 cm) equdibratedwith IOmM potassium phosphate buffer,pH 7.4. Proteinwas elutedfrom thecolumn ata flow rateof approximately 1.4 ml/minute.One hundred fractionswere collected(approximately14.5mi/fraction). OptifluorLSC-cockt" (5 n-d)was added to a 20gl aliquotof each fractionand a Packard Tn*-carb4530 scintillaticoonunter was used to assessL-FABP activity.Fractionswith LFABP activitywere pooled and concentratedto approximately5ml usinga vacuum filter apparatusfittedwith an ANUCON YM-5 membrane. Guinea pigsamples (controland FC I treated)were concentratedand frozenatthispoint.Rat samples (controland FC I treated)were furtherpurifiedas follows. The concentratedsolutionwas loaded on a Sephadex G50 (fine)gelfiltratiocnolumn (2.5 x 45 cm) equilibratewdith IOmM potassium phosphate buffer,pH 7.4. The flow ratewas approximately0.9 mi/n@nute. Sixtyfractionswere collectedat a volume of 3.48mi/fractionT.he fractionscontainingL-FABP activitwyere pooled and concentrated to approximately 5n-@.The concentratedcytosolwas dialyzedovernightat 4'C against 30mM Tris-HCL pH 9,using a SpectraporeMembrane MWCO 3,500. The sample was then appliedto a DEAE-ceflulose column (Whatman DE-52, 1.25x 15cm or 2.5 x 15cm) previouslyequilibratewdith 30mM Tris-HCI,pH 9, (degassed).The column was eluted with 30mM Tris-HCL pH 9, (degassed)followedby a lineargradientof NaCl (0-0.2W in 30mM Tris-HCL pH 9. The flow ratewas approximatlyIml/minute.Twenty fractions, 7.8 mi each,were couected. 17 Homogeneity of the finalratcontroland ratFC I fractionwsas assessedusing sodium dodecyl sulfat/e polyacrylamide-geellectrophoresi(sSDS PAGE) analysisa,nd confirmed by the appearanceof a dominant proteinband atmolecularweight (MW) 14,000 Daltons (Da.)(Figure4). Proteinconcentratioonf allsampleswas determinedusingthe bicinchoniaccid(BCA) proteinassayby Piercewith BSA as thestandard(Table 1). None of thesamples were defipidated. FiGuRE 3 - SDS PAGE ANALYSIS OF FRACNONS FROM RAT LRVER CYTOSOL. Marker M, valuesare shown. FOLLOWING PURIFICANON OF L-FAB RAT CONTROL L-FABP AK -as -57 -36 -18 RAT FC IL-FABP 44 -57 -34 -14 18 TABLE I - P'ROTF-TNCONCENTRATIONProteinconcentrationwas determinedusing the BCA proteinassaywith BSA as the standard. Values are a mean standarddeviationof 2 trials. FRACTIONS -1-LI)'AL PKO itiN CONCENTRATION (gg/ml) PURI_FIED Rat Control Rat FC 1 PAR 77ALL Y PURIFIED Guinea PigVehicleControl Guinea Pig FC 1 123.1 13.7 308.8 1.4 1755.7 10.1 1021.0 129.7 FLUORESCENCE MEASURENENTS Fluorescencemeasurements were based on the work of Wilkinson and Wilton (1986). AU assayswere carriedout at room temperatureusing a slitwith of 5mn ina SPEX 1681 0.22m spectrometer,SPEX IndustriesI,ncorporated.A stocksolutionof DALTDA, 0.1mM, was prepared by slowlyadding 50mM potassium phosphate (KH2PO4) buffer,pH 7.2,to ImM DAUDA inmethanol. AU furtherdilutionosf DAUDA were in 50mm KH2PO4, pH 7.4. AU dilutionsof L-FABP samples were in50mM KH2PO4, pH 7.4. AR FCs, WY and oleicacid were dissolvedinmethanol. AU measurements were made after binding had reached equilibrium. FLUORESCENCE CHARA CTERIZA TION Emission and Excit Ltion laxima and AverageMaximum Fluorescence Intensiiy The maximum emission and excitatiownavelengths (nm) and average maximum fluorescenceintensit(yFI)(cpm) were determinedfor I;LM DAUDA bindingto each L- 19 FABP sample.L-FABP, from originaulndilutedstock,was added to 2n@ IgM DALJDA in aliquotsof 1.6-114@il(dependingon theconcentratonof protein)untilno furtherchange inemissionor excitatiownavelengthor Fl was detected.The rangeof protein concentrationasnalyzedforeach L-FABP sample was 0.1gM-3 gM. Excitationscans, from 250-400nm usingan emissionwavelength of 500nm, and emissionscans,from 350600mn upon excitatioant350nm, were performed witheach additionF.l (Em. 500nm, Ex.,350run)was measured followingeachadditionofL-FABP. Curves of FI versus concentrationof L-FABP, representinagn average standarddeviationof 3 trialsw,ere constructedforeach L-FABP sample. The threehighestFl valuesforeach curve were averagedto determinetheaverage maximum FI forDALTDA bindingto each sample. SpecificDALTDA Binding TotalbindingofDAUDA (0-8pM) toeach L-FABP samplewas determinedby adding220pl aliquotosf 0.1mM DALTDA to2ml IgM L-FABP. IncreasedFl (Em. 500nm, Ex., 35Onm) due to the bindingof DALTDA to proteinwas measured. Nonspecificbindingwas assessedby saturatingL-FABP bindingsiteswith oleicacidand performingthe same titratioAnf.iquots,2-20pl,of 0.1mM DAUDA were added to a 2mi solutionof I4M LFABP and IOOGI oleicacid.SpecificbindingofDAUDA toeach L-FABP sample was determinedby subtractinngonspecificbindingfrom totalbinding. ANAL Ysis OF THE EFFECT OF FCs OF L-FABP CalculationofD ALTD A Binding ConstanIs Kd and Bmax valueswere detemiinedforeach protein.Specificbindingwas transformed to unitsofbound DAUDA (gM) by dividintghespecifiFcI (cpm) by themaximum Fl per 20 IPM DAUDA (cpm) foreach L-FABP sample. Computer assistednonlinearregression (GraFitVersion 3,ErithacusSoftware Limited)was used to constructcurvesof specific bound DAUDA versusfreeDAUDA representingan averageof 3-6 trialsT.he following equationwas used,Bound = ([L]x Bmax)/(Kd + [L]). Calculationof Oleic Acid ICio@s The concentrationof oleicacidwhich inhibits50% of specificDAUDA binding,the IC5o, was calculatedforeach combination of oleicacidand L-FABP sample. Cuvettes contained2ml lpM L-FABP and IpM DAUDA. Oleicacid,lmM in 10% methanol,was added in0.4-20 @Llaliquots.FI (cpm) (Em. 500run,Ex. 350nm) due tothe bindingof DAUDA to proteinfollowingeach additionwas measured. Curves of percentinhibition of specificDAUDA binding versusoleicacidconcentration,representingan average standarddeviationof 3-6 wWs correctedforthe effectof methanol, were constructed. ANALYSIS OF THE P07ENCYOF VApiousFCSFORBiNDiNGToL-FABP Compr,titiveBinding ExMriments -CalculationofIC5o:-s Cuvettescontained 2 ml IpM ratcontrolL-FABP and IpM DAUDA. Ligands,ImM (FCs and )&rYin 100% methanol,and methanol in 50mM KH2PO4. pH 7.2),were added in 0.4-200 aliquots.Fl (cpm) (Em. 500nm, Ex. 350nrn)due thebindingof DAUDA to proteinfollowingeach additionwas measured. Curves of percentinhibitioonf specific DAUDA binding versuscompetitorconcentrationr,epmsenting an average standard deviationof 3-6 trialcsorrectedfortheeffectof methanol were constructed.The concentrationof each competitorwhich inhibited50% of specificDAUDA binding,the IC5o,was calculated. 21 RESULTS & DISCUSSION FI,UOR&SCENCE MEASUREMENTS FLUORESCENCE CHARACTERIZATION Emissionand Excitation-Ma&iM.UM The emissionand excitatiosnpectraofeach proteinbindingDAUDA was analyzed to determinetheoptimalconditionsforthestudy. The maximum emissionand excitation wavelengthsforaU proteinswere approximately500 and 350runrespectivel(yTable2). Values forguinea pig L-FABP samples were slightlhyigherthanthoseforratsamples. This differencieslikelybecauseguineapigsamples were onlypartiallpyurified;thus,more celluladrebrisremained topotentiauyinterferweithDAUDA binding,and lessL-FABP as a proportionoftotalproteinwas present.A "blueshift"inbothemissionand excitation wavelength occurredupon theadditionofeach L-FABP sample toDAUDA. Excitation wavelengthshiftedfrom approximately330 to340nm (Figure4),and emissionwavelength shiftedfrom approximately550 to500nm (Figure5).Thisshiftinogfwavelengthis characteristoifcDAUDA bindingtoa nonpolarsiteon L-FABP (Wilidnsonand Wilton, 1986). According toThumser etal.(1994a),DAUDA only bindsone of thetwo oleatebindingsitesof L-FABP. Due tothestringenctonformationalrequirementsof primary sitebinding(internalizecdarboxylate/polagroup and a U-shaped hydrocarbon/hydrophobichain),DAUDA most likelybindsthesecondarysite.Similar shiftinogf fluorescencewavelengthand emissionand excitatiomnaxima were found by Wilkinsonand Wilton (1986)andThumser, Vosey and Wilton (1996). 22 TABLE 2 - ENflssioAND ExcITATION MAXIMA. Fluorescenceemissionmaxima (run)were measured upon excitatioant350nm. Excitation maxima (run)were measured usingan emissionwavelength of500nm. L-FABP was added to IW DALTDA untilno finiherchange inemissionor excitatiownavelengthwas detected.Each value isan average standarddeviationof 3 trials. SAWLE ENUSSION MAXRvfUM (nin) EXCITATION MAXWUMina) DALTDA only 551.33 1.53 331.00 1.00 Rat ControlL-FABP Rat FC I L-FABP 502.67 4.62 502.33 4.04 344.33 2.08 339.67 6.66 Guinea Pig VehicleControlL-FABP 511.00 7.81 340.67 5.13 Guinea Pig FC I L-FABP 511.00 7.81 333.67 1.15 FIGURE 4 - ExcrrATION MA)M,,4A. Fluorescenceexcitatiomnaxima (run)were measured usingan emissionwavelength of 500tun.L-FABP was added to2nilIgM DALTDA untd no furtherchange inexcitation wavelengthwas detected.Each curveisrepresentativoef 3 trial(sIgM DAUDA, 3gM LFABP). KEY: a = Guinea Pig FCI L-FABP; b = Guinea Pig Vehicle ContirolL-FABP; c Rat FCI L- FABP-, d Rat ControlL-FABP. d c b a Wavokwigth (mn) 360 3iO 4m 23 FIGURF, 5 - ENUSSION MAXIMA. Fluorescenceemissionmaxima (m) weremeasuredupon excitatiaotn350run.L-FABP was added to2tW IgM DAUDA untilno furtherchange inemissionwavelength was detected.Each curve is representative of 3 trials. KEY: a - 0 DAUDA. 0 L-FABP, 5omM KH2PO,; b = IgM DAUDA, 0 L-FABP-,c = IpM DAUDA. 0.IgM L-FABP; d = lpM DAUDA, 0.5pM L-FABP,, c IgM DALIDA. IgM L-FABP: f IgM DAUDA, 3pM L-FABP. RAT CONTROL L-FABP RAT FC I L-FABP f f e d d 4n GungEA PIG V c b a ONTROL L-FABP f c b a GUNEA PiG C IL-FABP f d c b e d c b a 24 Average Maximum Fluorescence Intensity The average maximum Fl (cpm) of Igm DALTDA bindingto L-FABP from FC I treated and non-treatedanimals did not substantlafldyiffer.The average maximum Fl was approximately850,000 cpm for ratL-FABP samples,and 660,000 cpm for guinea pig LFABP samples (Table3).Maximum Fl was reached foflowingthe additionof IPM rat controlL-FABP, 2.5gM ratFC I L-FABP, 2.5gM guinea pigvehiclecontrolL-FABP and 2gM guineapigFC I L-FABP (Figure6).The lower the affinitoyf receptorforprobe,the highertheconcentrationof receptorneeded forbinding,and viceversa(Matthews, 1993). Thus, thesedata suggest thatthe ratFC I L-FABP sample had a decreased affuiitfyor DAUDA as compared to the ratcontrolL-FABP sample,and thatthe guinea pig FC I sample had an increasedaffinitfyorDALTDA as compared to the guinea pig vehicle controlL-FABP sample. The average maximum FI forguinea pig samples was 77.6% lower thanthatfor ratsamples. This may be due to the impurityof the guinea pig samples resultinign a high degree of interferencienDAUDA bindingand a lower concentrationof L-FABP as a proportionof totalprotein. TABLE 3 -MAXMUM Fl OF IMM DAUDA. Cuvettescontaining2 rniIVM DAUDA were titratewdith ratand guineapig L-FABP samples (0.1mM) to determinethe maximum FI of Igm DALTDA bindingto each sample. Three trialpser proteinwere performed. Values are the average standarddeviationof the 3 highestaverage Fl valuesper protein. L-FABP SANTLE Rat Control Rat FC 1 Guinea Pig Vehicle Control Guinea Pig FC 1 MANIMUM Fl (cpm) 848,093 13,639 852,438 24,776 655,348 63,812 669,632 33,619 25 Fic,uRE6- AvEgAGEMAXD4umFloF ILI DAUDA.Flvs[L-FAB-EL Cuvettescontaining2 ml IpM DAUDA were Litratewdithratand guineapigL-FABP samples(0.1mM) todeterminethemaximum Fl of Ipm DALTDA bindingtoeach sample. Fl,due tothebindingofDAUDA toproteinw,as measuredaftereachaddition.Each curveisan averageof 3 trials. 900000- 800000 ----------------------- -------- 700000 -@ ------------- ---- ------ .. t 600000 ------------- .. ... ... ....... E 5ooc)oo- - - - - - - - - - - - - -- - - - - - - - - - - - - - - C0L - - - - - - - - - - - -- - - - - - = 400000 - - - - - - - - - - LL 300000 ------ - --- - ---------------- 200000- ----------------------- 100000 --------------------------- 0 0 0.1 0.3 0.5 1 1.5 2 2.5 3 [L-FAEF] (W) Ratcontrol 0 RatPFOS GuineaRg V. Control Gurea PigPFOS SpgcificDAUD Binding Because of itswell documented high affinityfor L-FABP (Thumser er al.,1994 a,b; Thumser and Wilton, 1994-.Thumser et al.,1996; Thumser and Wilton, 1995), oleicacid was chosenasthedisplacinlgigandtodeterminenonspecifibcindingofDAUDA toLFABP. Excessoleicacidwas added tooccupy allL-FABP bindingsitesand preventthe specifibcindingof DAUDA toL-FABP. DAUDA was titrateidntotheassayand FI,due tononspecifibcinding,was observed.Totalbindingwas determinedby performingthe assayintheabsenceof oleicacid;specifibcindingwas calculatebdy subtracting nonspecifibcindingfrom toW bindingA.bsoluteFl valuesaregiveninTable 4. The breakdown oftotalbindingintopercentspecifiacnd nonspecifibcindingisshown in Figure7. 26 TAgLE4 - lLiMDAUDA RNPNG To IUM L-FABP. TotalbindingofDAUDA toeach L-FABP sample was determinedby addingaliquotsof 0.1mM DAUDA to2ml IgM L-FABP. IncreasedFI due tothebindingof DAUDA to proteinwas measured. Nonspecificbindingwas measured in thepresenceof lOOpM oleic acid.SpecificbindingofDAUDA toeachL-FABP sample was determinedby subtracting nonspecificbindingfrom totalbinding.Values arean average standarddeviationof 3-6 trial(scpm). L-FABP SAMPLE Rat Control Rat FC 1 Guinea Pig V. Control Guinea Pig FC 1 TOTAL 818,000 124,000 302,000 81,000 219,000 138,000 162,000 15,000 NONSPECIFIC 78,000 26,000 96,000 21,000 92,000 23,000 94,000 65,000 SPECIFIC 740,000 124,(M 206,000 81,000 127,000 138,000 68,000 15,000 27 Fic;ug7E-DAU A BINDING. Total binding of DALTDA to each L-FABP sample was determined by adding aliquotsof 0.ImM DAUDA to 2mi IpM L-FAB P. IncreasedFl due to the binding of DAUDA to proteinwas measured. Nonspecific bindinc,was measured in the presence of IOOPM oleic acid.Specificbinding of DAUDA toeach L-FABP sample was determined by subtracting nonspecificbinding from totalbinding.Values are an average of 3-6trials. RAT CONTROL L-FABP Nonspecdic 10% Spocdic 90% RAT FC-i- L-FABP Nonspecific 320/a Speclic 68% GUINEA PIG VEHICLE CONTROL L-FABP Specdic 58% Nonspecific 42*/. GUINEA PIG FCa L-FABP specific 42% c;;@ Non spec ific 58% 28 Specificbindingof DALJDA toratcontrolL-FABP representedapproximately900/oof totalbindingw,hilespecifibcindingofDAUDA to ratFC I L-FABP accountedforonly 68% oftotalbinding.When a figandhas lower affinitfyora receptora, largerproportion of totalbindingisnonspecifi(cMatthews, 1993);thus,thesedatasuggestthe ratFC I LFABP samplehad a threefoldlower affinitfyorDAUDA thantheratcontrolL-FABP sample. Thisisconsistentwiththe resultsofthe averagemaximum Fl analysisof therat L-FABP samples,which alsosuggeststheaffinitoyf L-FABP forDALJDA was decreased intheFC I sample.Less ofa dfferencewas seenbetween thetwo guinea pig L-FABP samples.Specificbindingof IgM DALJDA to IgM L-FABP represented58% oftotal bindingintheguinea pigvehiclecontrolL-FABP sample,and 42% oftotalbindinginthe guineapig FC I sample.The data suggesttheguinea pigFC I L-FABP sample had lower affir@tfyorDALJDA thantheguineapigvehiclecontrolL-FABP sample. This isnot consistenwtiththe resultsofthe averagemaximum Fl analysisw,hich suggestthe relative affinitoyf theguinea pigFC I L-FABP sample was higherthanthatforthe guinea pig vehiclecontrolL-FABP sample. Thisinconsistencmyay be due tothe crudenessof the guineapig samplesgivingriseto a highdegree of interferencienthebindingof DALFDA to L-FABP. The overah crudenessof theguineapigL-FABP samples,as compared to the ratL-FABP samples,isreflectedina lower absolutetotalDALYDA binding(cpm), a higherpercent nonspecificbindingand a lower percentspecifibcinding. 29 A,VALYSIS OF THE EFFECTOF FCs ONL-FABP CalculatoifoDnAUDA BindiniC!onstants DAUDA bindingconstantswere calculatedforeach L-FABP sample. The purpose of this analysiswas to assesstheeffectof FC I on thefunctionalitoyf L-FABP: and toconfirm theresultshown thus far,which suggesttheaffinityof L-FABP forDAUDA was decreased inFCl treatedratL-FABP samples, and thatguinea pig L-FABP samples were too impure toaccuratelyanalyze. The maximum bindingcapacityor receptornumber (Bmax) of each L-FABP sample and thedissociatiocnonstantor affinit(yKd) of each L-FABP sample forDAUDA were determined using computer assistednonlinearregression(Table 5 and Figure 9). Specific bindingof DAUDA to each L-FABP sample was convertedtogM by dividingthe FI (cpm) due to specificDAUDA binding(Table4) by theaveragemaximum Fl (cpm) per IWM DAUDA foreach L-FABP sample (Table3). The Bmax of ratcontrolL-FABP was nearly14M. This agreeswith thework of Thumser etal.(1994 a,b),Thumser and Wilton (1994) and Tbumser et al.(1996)who allfound DAUDA to bind to one of theoleicacidbindingsiteson L-FABP. The Bmax fortherat FC I L-FABP sample was approximately0.35@LM, suggestingthecapacityof ratFC I LFABP to bind DAUDA was nearlyone thirdthatof ratcontrolL-FABP. One possible explanationforthisdecreased capacityisthatFC I isbound to L-FABP in the FCl sample, renderingfewer availablebindingsitesand allowinglessDAUDA tobind.The rigidtailof 30 FC I (Zisman, 1964) and stricctonformationalrequirementsforprimary sitebinding toLFABP (Thompson etal.,1997) suggestFC I bindingwould occur in thesecondary binding site.Another possibleexplanationisthatthevehicle(Tween 80,2%) somehow affected thebindingof DAUDA toL-FABP in theFC I treatedsample. This isunlikely,however, sinceTween 80 isa mild,non-ionicdetergent,designed toallow solubilizedproteinsto retaintheirnativestructure(Sigma, 1998);and because a low concentrationof Tween 80 used (seemethods). This does pointout,however, the importance of includinga vehicle controlin theexperiment. The Kds fortheratcontrolL-FABP and ratFC I L-FABP samples were not notably differentand were comparable to theKd of 0.38 t 0.02@LM found by Thumser etal. (1996). Thus, although thedataforaveragemaximum FI and percentspecificbinding suggesta decreasedaffinitoyf ratFCl L-FABP forDAUDA as compared to ratcontrol L-FABP, theKd and Bmax valuessuggestthe maximum binding capacity/number of binding sitesratherthan the binding affinitwyas affected. The Bmax of the guinea pig vehiclecontrolL-FABP sample was much lower than expected,0.28l@iM as compared toapproximately l@iM forthe ratcontrolL-FABP sample. The Kd forthe guinea pig vehiclecontrolL-FABP sample was higher than expected,0.48@iM as compared to approximately0.34M for the ratcontrolL-FABP. One possibleexplanationforthesedifferencesinresultsisthatratL-FABP and guinea pig L-FABP aredifferenl Although thecrystalstructureof guinea pig L-FABP has not been deduced, sequence identityforFABPs of the same tissuetype from differentspeciesis 31 approximately82-92% (R-ichierOig,ata and Kleinfeld,1994).One can,thereforee,xpect thebindingconstantsofcontrolguineapigand ratL-FABP tobe comparable.Other possibleexplanationsforthisdifferencaerethatthevehicle(Tween 80,2%) had some effecton theL-FABP, or thatthecrudenessof theguinea pigsample caused a greatdeal of interferencweith thebindingofDAUDA toL-FABP. As mentioned above,theaffectof Tween 80 ispresumably minimal; thus,thecrudenessof thesample islikelyto be to blame fortheunexpectedbinding constants. As discussedpreviouslyt,heresultfsorspecificbindingofDAUDA toguineapig L-FABP suggesttheguineapig FC I L-FABP sample had lower affinitfyorDAUDA thanthe guinea pigvehiclecontrolL-FABP sample. This suggestionwas countertotheresultsfor averagemaximum Fl,which indicatetheguineapigFC I sample had an increasedaff-inity forDAUDA ascompared totheguineapig vehiclecontrolL-FABP sample.These contradictordyata,when combined tocalculatea Kd and Bmax foreach guinea pig LFABP sample,resultedina Kd fortheguinea pigFC I L-FAB P sample thatwas about4.5 times thatof thevehiclecontrolsample, and a capacityof theguinea pigFC 1 L-FAB P sample thatwas about twicethatof theguinea pigvehiclecontrolL-FABP sample.These resultsareinconsistenatnd inconclusivel,ikelydue totheimpurityoftheguinea pig samples,as discussedabove. 32 TABLE 5 - DAUDA 13INDTNG CONSTANTS@ Values are an averace standarddeviationof 3 trialass calculatedby computer assisted nonlinearregressio'n.Bound = ([L]x Bmax)/(Kd + [LI). L-FABP SAMPLE Bmax(jiM) Rat Control 0.987 0.016 Rat FC 1 0.345 0.012 Guinea Pig Vehicle Control 0.281 0.043 Guinea Pig FC 1 0.413 0.060 Kd (g 0.278 0.020 0.260 0.063 0.480 0.391 2.240 0.852 FiGuRE 8 - SPECERC BouND vs FREE DAUDAComputer assistednonlinearregressionwas used to constructcurves of specificbound DAUDA versusfreeDAUDA (Bound = ([L]x Bmax)/(Kd + [L]))E.ach curve isan average of 3-6 trials.The reduced chi square value foreach regressionwas as follows:rat controlL-FABP, 3.83e-16;ratFCI L-FABP, 7.83e-16;guinea pigvehiclecontrolLFABP, 6.12e-15;and guinea pig FC 1 L-FABP, 1.67e-15. RAT CONTROL L-FABP RAT F,@@ L-FABP 1.000.80 --------- -------- a 0.60------- ------------- c 0.40 --- -------- 02 0 Average CD 0.20 -- ----- Calculated - ---- ------------- 0.00 o@07 0.25 0.43 0.91 0.14 0.19 0.24 0.29 Free (IjM) 0.35 0.30 ------------ X. 0.25 ------- -------------- -B 0.20 -0 ----------------- o.is 20 0.10 . . . . . . . . . . 0 Average 0.05 - - - - - - - - - - CalcugWdj 0.00 1 0.17 0.29 0.76 0.12 0.17 0.22 0.27 0.32 Free(pM) GUINEA PIG CONTROL L-FABP 0.50- 0 0.40- - Average CalcuWW ---- 0.30 - - - - - - - - - - - - - - 0-0 . 0 0.20 - - - - - - - - - 0. 00 0.10 - - - - - -0 - - - - - - - - - - - 0.00- 1 1 1 iii 0.05 0.35 1.29 2.42 3.35 4.34 5.67 7.58 Free(pM) GUINEA PIG Fc-I- L-FABP 0.35 0.30---- - - - - - - 0.25 --- - - - 0.,>O --- - - - - - - --- 0 6- 0 --- a 2 0.15 --- - - - 0 0.10 0.05 0 - - - - - - - - - - - -- - - - - 0 Average - - - - - - CaJcuUded 0.00 0.09 0.47 1.35 2.26 3.22 4.25 5.68 7.68 Free(IjM) 33 C-alculaLioofnOteicAcidIC,,O@s An IC5owas calculatefdoreach combinationofoicicacidand L-FABP sample toassess thefunctionaliotfyL-FABP from FC I-treateadnd non-treaterdatsand guineapigs.Oleic acid,ImM in 10% methanol,was titraeiendto2 ml IgM L-FABP and IPM DAUDA. Fl (cpm),due thebindingofDAUDA toproteinw,as measuredfollowingeach addition. Curves ofpercentinhibitionfspecifiDcAUDA bindingversusoleicacidconcentration were constructe(dFigure9).Table6 shows thepercentinhibitioonfspecifiDcAUDA bindingby 24M oleicacidand theIC5ovaluesforeach L-FABP sample.As discussedin theintroductioonl,eicacidiscapableofbindingbothprimaryand secondaryL-FABP bindingsitesa;nd once a ligandhasbound theprimarysiteb,indingtothesecondarysiteis facilitat(eTdhompson etal.,1997).Thisrelativeluynconstrainedbindingof oleicacidto L-FABP isreflecteidna sharpdecreaseinFI (cpm) and an increaseinDALTDA-specific bindinginhibitiounpon theadditioonfmicro-molarquantitieosfoleicacidtosolutionosf L-FABP and DALJDA. Ninetyone percentofspecifiDcAUDA bindingtoratcontrolL-FABP was inhibitebdy 24M oleicacid;and theIC5oofoleicacidfortheratcontrolL-FABP sample was 0.01gM. Thispercentinhibitiownas higherthanthatfound by Tliumseretal.(1994b),'numser et al.(1996)and Thumser and Wilton(1995),of 80.5%,76% and 80% respectiveluyn,der similarconditionsT.he percentinhibitiownas much lower(52%) and IC5o much higher (0.5@LM)fortheratFC 1L-FABP sampleascompared totheratcontrolL-FABP sample. ThissuggeststheabihtyofL-FABP from theFCI-treatedrattobindoleicacidwas decreased.One explanationforthisdecreaseinfunctionalitiys,thatFC I was bound toL- 34 FABP intheFC I treatedsample,renderingfewer sitesavailabletobindoleicacidand requiringmore oleicacidtoachieve50% inhibitionT.his agreeswiththeresultspresented fortheanalysisofaveragemaximum Fl,specificDAUDA bindingand DAUDA binding constantsinratL-FABP samples. The percentinhibitioonfspecificDAUDA bindingtotheguineapigvehiclecontrolLFABP sample upon additionof 24M oleicacidwas 93%; thatfortheguineapig FCI sample was 63%. This issimilartotheresultfsortheratsamples;however,thecurvesof percentinhibitiovnersusoleicacidconcentratiofnorratand guinea pigsamples were very differen(tFigure9). The curvesforratsampleswere hyperbolicwhilethecurvesfor guinea pigsamples were more linearand scattered.The resultingIC5ovaluesforboth guinea pig L-FABP samples were thesame, 0.24M oleicacid.The dataareambiguous, and arelikelydue to thecrudenessof theguineapig samples. TABLE 6 - OLEic Acip Icio's& PERCENT INHrBrrION OF SpEcigc DAUDA BINDING, The ICsoofoleicacidforeach L-FABP sample was calculatebdy adding oleicacid,ImM in 10% methanol,toa solutionof lpM L-FABP and IpM DAUDA. Values areIC5o'sof oleicacidforeach L-FABP sample and percentinhibitioonf specifiDcAUDA bindingby 2gM oleicacid. L-FABP Sample OleicAcid IC5o (gM) % Inhibitionby 2pM oleicacid Rat Control 0.01 91 Rat FC 1 0.5 52 Guinea Pig Vehicle Control 0.2 93 Gu'mea Pig FC 1 0.2 63 35 FiGuRE 9- %.DAIJDA INHIBMON VS [OLEIC ACID] - Oleicacid,ImM in 10% methanol, was added toa solutionof lpm L-FABP and IPM DAUDA. Curves representan average of 3-6 trialscon-ectedfor the effecotfmethanol. RAT L-FABP 100% 8(r/o---- --------0 60*/o-- ----------- o RatControl 400/a------- ----- -45- Ratrl_lI 20% ... ... ..... 0% --1 -----1-------!-----i------ 0.000.020.10020 1 2 [oloicacid)(W) GUINEA PIG L-FABP 100*/.- 80*-/ -------------- 0 R 60-*-/--------- --- .E 40-/.-------- ------ 20% ---- ---- ---------------- 0.00 0.02 0.10 0.20 1 2 [olwc add] (IjM) GuinPeiag V.Control GuinPeiag F4-7- ANALYSIS OF THE POTENCY OF FCS FOR BINDING To L-FABP Comi&titive Binding Expgriments - Calculation of ICso's An IC5oof eachFC, WY and methanolwas calculatefdorDAUDA bindingtotherat controlL-FABP sample.Each ligand ImM, in 100,clmoethanol or 50mM KH2PO4, pH 7.2)was titrateidnto2ml IpM ratcontrolL-FABP and IpM DAUDA. The additionof 36 competitorto L-FABP and DALTDA induceda shiftoferru'ssiwoanvelength back towards thecurveofDAUDA with no L-FABP (Figure10).Thisisindicativoef DALJDA being displacedfrom a nonpolar bindingsiteon L-FABP, such asthe secondarybindingsite.To varyingdegrees,similarshiftinagnd decreasesinFl occurredupon additionof each competitor.Fl (cpm),due the bindingof DALJDA toproteinw,as measured following eachadditionofcompetitor;and curvesof percentinhibitioonfspecifiDcALJDA binding versusfigandconcentrationconstructed(Figure11).Table 8 givesthepercentinhibition of specifiDcALTDA bindingby IOpM competitorand the IC5ovaluesforeach competitor. FC I was the strongestinhibitoorf specifiDcALJDA binding,with690/oinhibitiounpon the additionof IOW and an IC5oof4.9pM. FC4 was thenextstrongestw,ith 51% inhibition upon theadditionof IOpM and an IC5,oDf 9.7pM. Wyeth followedFC4, with 500/o inhibitiounpon theadditionof 10@LK and an IC5oof IOW. FC3 and FC2 both inhibited 43% of specifiDcALJDA bindingupon theadditionof IOW and had IC5o'sgreaterthan Io@im. Thumser and Wdton (1996) suggestthatligandsforL-FABP need to have both a hydrophobicand a hydrophihcdomain. Each FC and VTY fitsthisdescriptiotno varying degrees.Due to the rigidtailoftheFCs and the bulkystructuroefWY, itislikelythat bindingof each of thesecompetitorsto L-FABP occursinthesecondarybindingsiteon L-FABP. ItisconceivablethattheFCs bindwith theirrigidhydrophobicCF tails, analogoustothe fle)dblCeH taflofoleicacid,inthe secondarybindingsiteand thatthe polarhead groups of FCs aresolventexposed as isthecarboxylategroup on oleicacid. 37 To reasonby structuret,hevaryingpotenciesofeach FC to bindingL-FABP isdifficult. Thompson etal.(1997) suggestthatanythinglargerthana C14 moleculemay requireprior bindingintheprimary site.As was mentioned inthe introductionC,g and Clo FCs arethe most potentperoxisome proliferatorsI.tmay be thecasethatCs and Clo moleculesbind most readilytothe secondarybindingsiteon L-FABP, not requiringa molecule tobe bound intheprimary bindingsite.AU of the FCs examined inthepresentstudywere Cg molecules;thus,an analysisof chainlengthcannot be undertakenatthistime. The disparatepotenciesofbindingL-FABP between FCs do not appear tobe due to dfferencesinpolarityand hydrogenbonding abilityF.or example,FC2, having a very polarcarboxylatehead group,and capableof extensivehydrogen bonding,has a higher IC5othan FC4, which has a lesspolarhead group and lessabilittyo hydrogen bond (see Figure 1). The bindingofVrY to the secondarybindingsiteon L-FABP isdifficutlot reason. In placeofthehydrophobic tailp,resenton FCs and ofeicacid,VN has two aeromaticrings. The confonnafionalflexibiliotfyL-FABP may accommodate thisbulky structure internallayn,d positionthecarboxylateexternallyi,na solventexposed manner ableto participatienhydrogen bonding. Thompson etal.(1997)alsospeculatethata third bindingsitel,ocatedcompletelyon theexteriorof L-FABP, may exist.OrAy thefirsftew carbons of a molecule bindingto such a sitewould be bound inan organizedfashion,the remainingportionofthe ligandwould be disordered.This,althoughhighlyspeculative, may be thesiteof WY bindingto L-FABP. 38 Methanol was chosen as thenegativecontrolbecausea non-peroxisomeproliferator, similairnstructurteo theFCs beingresearchedh,as notbeen identifiedU-pon the additioonf IOpM methanol,2 1% ofspecifiDcAUDA bindingwas inhibitedT.he IC5(o)f methanolwas much greaterthan IOpM. Itisreasonableto suggestmethanoliscapableof weakly bindingthesecondaryL-FABP bindingsitew,ith theCH3 group internalllyocated and theOH group exposedto theexterioorftheprotein.The structuroefmethanol rendersitan unlikelcyandidateforloopingintoa U-shapeand participatiinngprimarysite binding. 39 FIGURE 10 - CHROMATO-GRAM- The additioonf competitortoL-FABP and DAUDA induceda shiftof en@ssion wavelengthback towardsthecurveof DALTDA withno L-FABP. Thisisindicativoef DALTDA beingdisplacedfrom a nonpolarbindingsiteon L-FABP. To varyingdegrees, simflasrhiftinagnd decreasesinFl occurredupon additionof eachcompetitor.The example shown isforFC2 bindingratcontrolL-FABP. KEY: a= lpMDAUDA, IpML-FABP;b= IPMDAUDA, I L-FABP, 10liMFC2;c= IPMDAUDA, IpM L-FABP, 1511M FC2; d = IpM DAUDA, 1pM L-FABP, 20ILM FC2; e IILM DAUDA; f 0 DAUDA, 0 L-FABP, 5()MM KH2P04. ISE+OfpIOE+05- o-- 450 a c d e f WaveWrqM (nm) 560 40 FiGuRE II - % HlDrriON OF S T-)A R rNnNC, "re-r -ID Each competitor, ImM, was added to l@iM L-FABP and IpM DAUDA. OR], easurements of FI were made upon each addition.Curves representan average of 3-6 trialscorrected for the effectof methanol. RAT CONTROL L-FABP 70.00*-/@ 60.0(-r-/------------------------50.0c-r-/------------------- -------------- 4o.o(-r-/ ----------------- ----- 30.0(-r-/------------- --- ......... 20.00*/--1---------- - . ......... 10.o(r/o------- ------ ------------ 0.0(r/. 0 0-02 0.1 0.2 1 2 3 4 5 6 7 8 9 10 [competitor] (pM) --O--WY i5 --IIF-F-C a TABLE 8 - I C29 INHEBMON OF SPE= DALTDA BZQING. The IC5o of each competitor for the ratL-FABP sample was calculatedby adding competitor (lmM) to IVM L-FABP and lp@A DAUDA. Values are the IC5o'sof each competitor and % inhibitionof specificDAUDA binding by 10@LM of each competitor. COMPETITOR FCI FC4 WY FC3 FC2 METHANOL IC50 4.9 9.7 10 >10 >10 >10 % INHEBMON 69 51 50 43 43 21 41 CONCLUSIONS & FUTURE DIRECTIONS Thisstudywas designedtoinvestigattheehypothesitshatcertaiFnluorocarbon(sFCs) bind to liverfattyacid-bindingprotein(L-FABP) and displaceendogenous fattyacids (FAs) as an initiaelvent in peroxisome proliferationT.o examine thishypothesis,the kineticsof FA and FC bindingto L-FABP were investigatedusing DAUDA, a fluorescentllyabeledoctanoicacidanalogue.L-FABP from male rats(consideredtobe strongrespondersto peroxisome proliferatorsa)nd male guinea pigs (consideredto be weak or non-responders to peroxisome prohferators)(Svoboda etal.,1967; Orton etal., 1984; Lake and Gray, 1985; Elcombe and Mitchell,1986) were examined. The firstgoal was to assesstheeffectof FCs on L-FABP functionas evaluated by the abilityof DAUDA to bind to L-FABP isolatedfrom ratsand guinea pigstreatedand not treatedwith FC I in vivo. Resultsshow a decreasedmaximum bindingcapacityof LFABP from FC1-treatedratswithout an increasein Kd. This was demonstrated by a lower Bmax, higheroleicacidIC5oand unchanged Kd in L-FABP from ratstreatedwith FCI as compared to samples from controlrats.These resultsare likelydue to FC I binding to the secondary bindingsiteon L-FABP; thuspreventingthebindingof DAUDA to L-FABP. Resultsforguinea pig L-FABP samples were inconclusive.This ispresumably because guinea pig samples were only partiallpyurifiedr,esultingin a highdegree of interference from remaining cellulardebrisand a lower concentrationof L-FABP, as a proportionof totalprotein,as compared to the more highlypurifiedratsamples.A second attempt will be made to analyze the differencein response to FCs seen in rodentand guinea pig LFABP. This analysiswillbe performedon fullypurifiedL-FABP samples. 42 The second goalof thestudywas toassessthepotency of thevariousFCs forbindingto L-FABP, assuggestedby IC50 values.Resultsindicatethemost potentL-FABP binderis FC 1,followedby FC4, WY and (withequalIC5os)FC3 and FC2. Binding of FCs toLFABP likelyoccursinthesecondarybindingsite;and thevarianceinpotency islikelydue to structuradlifferences. Futurework willfocuson correlatinngew datawith theresultosf thisstudy,and relating theeffectseen in rodentsand guineapigs to therelevancetheypose to human health.The possibilitoyfcompleting a new setofkineticasssaysusinga probe capableofbindingboth L-FABP bindingsiteswillbe lookedinto.This would helpcharacterizteheinteractioonf DALTDA with L-FABP and createa new setof resultsforanalysisH.uman L-FABP win be examined, and therelevanceto human healthof FC induced peroxisome proliferatioin rodentswillbe investigatedF.Cs ofdifferencthainlengthwillbe investigateda,nd the effectofchainlengthon abilittyobindL-FABP analyzed.The abilitoyf each FC toinduce peroxisome proliferatioinrodentL-FABP willbe assessedusingthemethod of Lazarow and de Duve (1976). An attemptwillbe made tocorrelattehiscapacitywith therelative potencyof each FC tobindL-FABP, as determinedinthecurrentstudy.Electro-Spray Mass Spectrometryanalysison thesame L-FABP samples as analyzedin thisstudyis currentlyunderway. 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