Document n2mbzqVb7DBBKBqm89DYJKqw

BiochemicaalndinlolecuMleacrhanistiSctudieosfN-Alkyl Perfluorosulfonamides KendaR B.Wallace,Ph.D.D,.A.B.T. Department of Biochemistry& Molecular Biology Universityof Minnesota School of Medicine Duluth,MN 55812 218n26-8899 FAX 726-8014 kwallaceC&d.unm.edu LONG-RANGE MONITOR CONSUMER GOAL: ESTABLISH A SCIENTIFICALLY-BASED METHOD TO FOR POTENTIAL HEALTH RISKS ASSOCIATED WITH WORKER OR EXPOSURE TO PERFLUOROALKYL ACIDS AND THEIR DERIVATRVES. SHORT-TERM GOAL (3-5years): 1. DEFINE RELIABLE BIOMARKERS FOR ASSESSING BOTH EXPOSURE AND BIOLOGICAL REACT=Y. 2. DEFINE THE MOST APPROPRIATE EXPERIMENTAL SYSTEM (IN TERMS OF BEST REPRESENTING THE HUMAN SITUATION) FOR ESTABLISHING RELATIONSHIPS BETWEEN EXPOSURE DOSE AND BIOLOGICAL EFFECTS. There existsconsiderableexperimentalevidence demonstratingthatperfluoroalkylacids and theirderivativeshave thepotentialof bein- toxicto humans, which raisesseriousconcern forthe safe production,use and disposalof thesechemicals in thework place,by the consumer, and in the environment. Many of the currentand pending regulatorydecisionsfor theseproducts are based on experimentalevidence aatheredfrom rats.forwhich thereis stron-evidence for the inductionof peroxisome proliferatiomne,tabolicwastin9 and carcinoa-enesis[1,2,31.However, the evidence for comparable effectsin othermammalian species,includi0naprimates,is less convincin0c,which sua=0aests profound and important differencesamonc0, species. Therefore, although ratsmay be themost convenient and extensivelystudiedspecies,theevidence may not April8, 1997 yieldaccuratiendicatioonfspotentialdversehuman healtehffectsA. moreprudentmeans of manaainc, potentialhuman healthrisksassociatedwith theproductionand use of these important products requirescarefulselectionof a representativey,et convenient surrogatespecies test organism. The factthatcherrlicarlesiduesof perfluoroalkyalcidshave been detectedin seraof production workers [4]adds uraency to theneed to understandpotentialhealthrisks,to establish guidelinesto safeguardhuman health,and toestimatemargins of safetyforexposed populations. In order to address the surro-atespeciesissue,itisfirstnecessaryto establishone or more reliableindicatorsof toxictissuedamage upon which the speciescomparisons can be made. The proposed investigatioins designed to providean understandingof the underlying mechanisms of toxictissueinjuryand to identifyreliablbeiomarkersof tissuedamage thatcan be used forrisk-management purposes. EM[NODIATE OBJECTIVES (24 months): 1. Define the "dominant" mechanism by which perfluoroalkyalcidsand theirderivatives manifest the well documented "metabolicwastina" effectobserved in experimental animalsin vivo. 2. Based on an understandi0naof the biologicalmechanism of action,identifypotential biomarkers thatcan be used to providereliableand quantitativeestimatesof exposures and the biologicalconsequences associatedwith such exposures. 3. Usin0- the biomarkersdescribedforAim 2, determinewhether differenceesxistamong speciesin theirsensitivittyo perfluoroalkyalcidsand theirderivativeasnd in the cellular mechanism by which thetoxicityismanifested. NOTE: The researchplan isbeina submitted with the understandinct,hatpublicationof the results willbe subjectto a 30 day first-right-of-revbiyewthesponsor. The sponsor willprovide allrequiredquantitieosf testchemicals. Selected tissue samples from the in vivo exposures will be archived for subsequent histologicalexarr@nationand traceresidueand metaboliteanalyses,both tobe performed by the sponsor. The sponsor willbe briefedon theproaressof the fundedprojecton a quarterlbyasis. In additionto the 18 month contract,an unrestricte-diftof $100.000 isrequestedto support more empiricalexploratoryresearchon relatedtopics. INTRODUCTION April8, 1997 Perfluoroalkyalcidsand theirderivativersepresenta lar,,aend importantclassof synthetichemicals,with productionamountin- toseveralthousandsofpounds each year. These compounds constitutae broad market includinasurfactanatnd foan-iina-pplicationass well as potentpesticidaalctivityP.rudentregulationof theproduction,distributiosnt,oragea,pplication and disposalof theseproductsnecessitateas thorou-hunderstandingof theprobabilitaynd natureof any potentiaaldversehealtheffects.This proposalisdirectedat improving the confidenceand judiciousnesswith which such riskmanacement decisionsaremade. Although each member of thisclasspossessesdistincsttructuraalnd physicalchemical propertiest,hey sharevery similarbiologicalactivitiewshen administeredathiah doses to experimentalsubjects.From a structure-activbiatsyis,thissuggeststhattheN-alkylsubstituent, aloncrwith the lengthand branchinaof thecarbon chain,determinetheratesofabsorptionand thebiologicadlistributio(npharmacokinetics)of thecompound but thatthebiolocicarleactivity (toxicityr)esidesinthesulfonicacidor sulfonamidethatisconunon toallmembers of theclass [5].A fullycomprehensivecharacterizatiofn therisksassociatedwith thisclassof chemicals would requirerigorousinvestiaatioonf the biologicarleactivitoyf each member compound. Such an analysiswould be extremelycostly,both intermsofresourcesand time.However, Dr. S. C. Gordon has proposed a tentativemetabolicpathway formany of the N-alkylated perfluorooctanesulfonamideswhich consistsof successiveN-dealkylationlseading to perfluorooctanseulfonicacidas thefinalcommon metabolite(Fi0-.1).In view oftheconver0-in0a metabolicpathway,itmay be possibleto gainan accurateindicatioonf potentiahlealthrisksby examinincronly selectedmetabolitesthatare believedto be primarilyresponsiblefor the biologicalactivitoyf theentireclass.Thus, intheinteresotf efficiencyt,hisinitiailnvestigation is limitedto testingN-ethyl perfluorooctanseulfamidoethanol,N-ethylperfluorooctane sulfamidoacetate,thesulfonamideof perfluorooctanea,nd perfluorooctanseulforliacid..The structureosfall4 proposedtestcompounds arebracketedinficure1. Perfluorooctanoiacidwill serve as a positivecontrolfor allexposures. A riaorousunderstandincoyf the bioloaical reactivitioefs thesemetaboliteswillprovidethe most efficienmteans of aaininca, better understandin-the potentiahlealthrisksassociatedwith the entireclassof N-substituted perfluorooctanesulfonan-iides. 3 Acute and chronic toricirovfN-alkylperfluoroocranesulfonamides- April8, 1997 Both the sulfonicacid and the N-ethyl sulfonamide of perfluorooctaneare essentially non-toxicon an acute basis. Only at very high oraldoses (2-5 glka) are vague signs of gastrointestindailscomfortand weight lossobserved. The primaryconcern with acuteexposure to these compounds istheirpersistencein thebody, the half-livebseing between I and 11 days depending on the dosin- scheduleand the tissueexamined [6,7].fvfanyof thesechemicals bioconcentraecin the liverP.erfluorooctaneN-ethylsulfonamideisrapidlyde-ethylatedin vivo and itisthe des-ethylsulfonamidethataccumulatesin liver.This biopersistencreaisesserious concerns about the potentialcumulative and long-term toxicityassociatedwith continuous exposures to low concentrationsof thesechemical agents. Both perfluorooctaneN-ethyl sulfonamide and sulfonicacid elicitprofound subchroaic toxicitiesregardlessof the route of administration.Ninety-day feeding studiesin ratscaused deaths at doses of 100-150 ppm. Although high sub-lethaldoses cause assortedneurological, musculoskeletal,derrnatolo-icand hemopoetic abnormalities,the most consistentsymptoms observed at the lowest doses are anorexia,wei-ht loss,emaciation,and elevationof liver enzymes in theplasma (8-12].This apparenthepatotoxicitiysobserved in rats,rabbitsd,ogs and monkeys and ismost prevalentin males, possiblydue to themore rapidrenalelirrlinatioofnthe metabolite in females. Regardless, there are no substantivehistopathologyor gross morphological changes thataccompany thehepatotoxicityobserved atlow doses of eitherthe Nethylsulfonamide or the sulfonicacid of perfluorooctan(e9,12].This,by itselfi,sstrong evidence sua-estinathattheobserved toxicityismore a manifestationof metabolicor functional deteriorationr,atherthan structuradlamage. Prloposedmechanisms of toxicit-y Many of thesiansand symptoms associatedwith intoxicatiobny perfluoroalkyalcidsand theirderivativess,uch as the anorexiaand wei-ht loss,resemblethatof a metabolicdisorder.In deed perfluorooctanesulfonamide,like many other weak acids,has been demonstrated to uncouple mitochondrialrespiratioinn vitro[1,2].Itispresumed,but has yet to be confirmed, thatthisleadsto mitochondrialdepolarizationand thedepletionof ATP incellculturesas well as in vivo. Whether perfluorooctane sulfonic acid also uncouples mitochondrial oxidative phosphorylationhas yet to be determined. Itis,however, wellestablishedthatthe sulfonicacid is a potent peroxisome proliferatorin vivo in rats,which is evident as an hypertrophic hepatomecralyaccompanied by proliferatioonf both mitochondrialand microsomal membranes 4 April8, 1997 (3]. Associated with thisisthe stimulationof a number mitochondrialenzyme activities, includinaMn-superoxide dismutase and fattyacyl CoA-oxidase. The reportedinterferencewith spermato-enesisand inhibitioonf sperm motilityby N-ethyl perfluorosulfonan-@diesconsistent with an effecton inhibitinrardtochondriablioenergetics[10]. The accumulationof triacylglyceroalnsd freecholesterolin liverimpliesinterference with fattyacid or lipidmetabolism as a primary mode of expressionof the toxicity[13,141. In deed, the effectsof perfluorooctanesuffonicacid on fattyacid and cholesterolsynthesisrnin:Lics those of the HMG-COA reductaseinhibitorsw,hich are some of the most effectiveagents in treatinghypercholesterolemiacslinicallyI.tmay be more than coincidencethatmany of these lipid-loweringagents,such as the fibricacidsand the phthalicacid plastisizerasr,e classical peroxisome proliferatorsA.lthou-h circumstantialt,hislends stron-supportf.or an important effectof the perfluorocompoundson lipidmetabolism. Haughom and Spydevold (13] suggest that the hypoliperriiecffectof perfluorooctanesulfonicacid resultsfrom itsinhibitionof mitochondrialcamitineacyl-CoA Eransferasaectivityw,hich isrequiredforthetransportof long chain fattyacidsforbeta-oxidatiownithinthemitochondrialmatrix. Althou-h not much isknown reaardingthebiolo-icalactionof perfluoroalkyalcidsand theirderivativest,hereisconsiderableinformationavailableforotherperoxisome proliferators. As a class,these agents are known to interferweith mitochondrialrespiratio(nalthoughthe precisemechanism isnot known), to inhibitfattyacyl-CoA synthesis,acylcamitinetranslocase and n-iitochondrifaalttyacidbeta-oxidationa,nd cholesterolsynthesis.Peroxisome proliferators alsostimulatetheexpressionof severalimmediate earlyresponsegenes (such as c-fosand c-jun), CYP4, cyclin-dependentkinases,proliferatinncuyclearantiaen,and theperoxisome proliferatoractivatedreceptor,which isa licand-activatenducleartranscriptiofnactorthatup-regulatesthe expression of genes thattranscribevarious lipidmetabolizingenzymes (15-17]. Itis the expression of many of theseearlyresponsegenes thathas been implicatedin thenon-genotoxic carcinocreniacctivityof peroxisome proliferatorsW.hether thesesame cenes areactivatedby the perfluoroalkylacidsand theirderivativehsas yet to be determined. Regardless,the similarityin biolocricarlesponsewith classicperoxisome proliferatorpsrovidesample opportunitietso unravel many of the unresolvedquestionsconcemina theirmechanism of action. Itiscurious,and perhaps unfortunatefrom a reculatorystand-pointt,hatthe vast majority of knowledge gained for perfluoroalkyalcidsand theirderivativesas well as for the classic peroxisome proliferatorissderivedfrom the laboratoryrat.The concern liesin the factthat, although stronglypredictedby ratdata,thereislittleevidence forthe proliferationf liver peroxisomes in humans exposed to thesecompounds. In fact,therearea number of non-primaee species,such as guinea pigs and dogs. that don't respond to conventional peroxisome proliferator[s18-2t1. This thenraisesdoubts re%-zlrdint-hevaliditoyf usin- ratdata to establish 5 Apdl 8, 1997 rccrulatorpyolicy for these a-ents. Of particularconcern is the debate over whether the pcroxisome proliferatorisn,cludinathe N-alkyl perfluorosulfonamidesa,re non-genotoxic carcinoc,ens(22].The outcome willhave profound effectson the regulationof theseproducts, particularliyn thecontextof theadditivesor residuesinfoodstuffs(e.g.N,-ethylperfluorooctane sulfamido ethanol). Resolutionof thisapparent speciesdifferencewarrants immediate and aggressiveattention. RESEARCH PLAN SPECIFIC ADIS 1. Determine whether perfluoroalkylacidsand theirderivativeisnterferewith mitochondrial electrontransportand/orfattyacid metabolism by isolatedrathepaticmitochondria. 2. Identifymolecularand peroxisomalbiomarkers of N-ethylperfluorooctanesulfan-iido ethanolexposure in vivoinrats. 3. Compare and contrasttheresponse of hepatocytecellculturesisolatedfrom rats,guinea pigs and primatesexposed ex vivo to perfluorooctanesulfonicacidand itsN-alkyl derivativesand to compare the molecularand peroxisomalbiomarkers and the histopathologyin ratsand aulneapi-s in response toinvivo exposure toN-ethyl perfluorooctanesulfamido ethanol. 6 EXPERliN]IENTAL APPROACH April8, 1997 I DETERMINE WHETHER N-ETHYL PERFLUOROOCTAINE SULFAMIDO ACETATE, PERFLUOROOCTANE SULFONAMIDE, AND/OR PERFLUORO- OCTANE SULFONIC ACID INTERFERES WITH CELLULAR ENERGY 'i4.viETABOLISM liV VITRO. Mitochondrial bioenergetics - N-Ethyl perfluorooctane sulfonamide uncouples oxidative phosphorylation in kidney rriitochondria in vitro [1,2]. The authors attribute this activity to the des-ethyl metabolite. We propose to expand on thisobservationto determine whether the perfluorooctanesulfonicacid and itssulfonarriiddeerivativesuncouple mitochondrialrespiratioinnthe liverand to conduct a fullscalecharacterizatioonf theeffectsof thesea-entson rrutochondriablioenergeticsin vitro. The uncoupling protonophoricactivitywillbe assessedby the cherriical-inducdeedpolarization of n-iitochondriamlembrane potentials,timulationof cyanide-insensitivsetate4 respirationand acidificatioonf the mitochondrialmatrix. All experimentswillbe performed with freshly isolatedratlivermitochondria.Mitochondrialmembrane potentialwill be measured using a TPP+-selectiveelectrode.Oxyaen consumption willbe analyzedpolarographicalliyn a closed- chamber system employina an oxygen-selectiveClarke-type electrode. Protonophoric acidificatioonf the mitochondrialmatrixwillbe assessedwith an high-sensitivpeH electrode. Fattyacid metabolism - We have preliminaryevidence demonstrating that weak acids (both substitutedand unsubstitutedacrylatesp,hthalicacids,valproicacid,salicyliaccid,etc.)inhibit he mitochondrial camitine acyltransferasIerequiredfor fattyacid oxidation.Depending on chain length,this inhibitionof translocaseactivityresultsfrom the depletionof eitherthe cytosolicor mitochondrialcoenzyme A due to the formationof thioesterosf CoA with the corresponding acid. We expect thatmany perfluoroalkyalcidsand theirderivativeasrealsocapableof forming thioestersof coenzyme A, therebyinhibitincf,attyacidoxidation.Itis quitepossiblethatthis interferenceWith fattyacid metabolism may be responsible for the well-characterized peroxisome proliferatinegffectreportedforman y of theperfluoroalkanes. 7 April8, 1997 Accordin.-lyw,e propose to study the effectof perfluorooctanesulfonicacid and its sulfonamide derivativeson palrnitiaccid oxidationby intactratliverinitochondria.We will attempt to distinauiswhhether any deficitisdue to inhibitioonf acyl-CoA synthaseor camitine acyl-CoA transtocaseby varyin- the substratefor beta-oxidationbetween palmiticacid, palmitoyl CoA, and palmitoyl carnitine. We will also determine whether the pcrfluorocompounds depletemitochondrialcoenzyme A. suCa,9estingthe formationof a thioester. Furthermore, we can identifya specificeffecton fattyacid oxidationby demonstrating an inhibitionof mitochondrialrespiratiounsin- palmitoylcamitineas substrateb,ut not when either glutamate or succinateare substrates.This isolatesthe deficito the acyl-CoA dehydrogenase flavoprotein,independentof effectson any otherportionof the rrlitochondrieallectrontransport chain. 2. IDENTIFY POTENTIAL BIOMARKERS OF TOXIC TISSUE DAMAGE ASSOCIATED WITH IN V7'VO EXPOSURE TO N-ETHYL PERFLUOROOCTANE SULFA.IMMO ETHANOL Identificatioonf potentialbiomarkers of toxictissuedamac0e is based on a thorough understandingof themechanisms of actionof theseagentsas exploredin Aim I of thisproposal. Therefore,itisn'tpossibleatthistime todescribetheprecisedetailsre9ardin0-which biomarkers to use to monitor in vivoexposure and toxictissuedama-e. The ultimateexperimentalplan for identifyincs,pecificbiomarkers iscontingenton theoutcome of theexperimentslistedin Aim 1. The resultswill provide a sound basis for discussin-the most probablemarkers and for designing futureexperiments to addressdose-responserelationshipisn an attempt to define mar'a0ins of safetyinexposed populations. Biomarkers of mitochondrialuncouplingand liberatioonf oxygenfreeradicals- Althou-h theexpressionof oxidativestressin vitroisverywellcharacterizeda,ttemptsto identifya quantitativbeiomarker of tissuedamaae invivohave met with limitedsuccess.One of the most promising markers thatmost consistentlyieldscorrelationtso theextentof oxidative stressisthe accumulationof 8-hydroxydeoxy-uanosine(8OHdG) adductsto n-@tochondriaDlNA (mtDNA), which can be measured in circulatinp-eripherallymphocytes. There isa larsraend growing body of evidence thatdemonstratesa progressiveaccumulationof 8OHdG adducts to 8 April8, 1997 mtDNA with age (20-24],givingriseto thewidely held theoryof "mitochondriaalging" [23]. Further-more,the rateof "mitochondrialaaing" can be influencedby environmentalexposures. We've recentlydemonstrated an or-an-specificand preferentiaolxidationof cardiacmtDNA followinCg) acuteexposures to adriamycin in vivo. SimilarorCg7 anotropicoxidationsof mtDNA have been reportedformany othergenotoxic carcinogens.In fact,Takagi etal[24]reporta 2fold increase80HdG adductsof nuclearDNA (NDNA) in liversb,ut not kidneys,from rats treatedacutelywith perfluorooctanoicacid. Based on our experience,we expectthatthe 80HdG adducts of mtDNA would have been 2-10 times hi-herhad the n-iitochondrbieaen examined. For this aim, we will investiaate whether exposure of rats in vivo to N-ethyl perfluorooctanesulfan-ddoethanolcauses thepreferentiaolxidationof mtDNA in liver,more so than in otheroraans which exhibitminimal orcranopathy.Rats willbe injectedintraperitoneauy with varying doses of theperfluorocompound and killedatdifferentimesthereafterT.he tissues willbe harvested and the NDNA and mtDNA extractedfrom the correspondingcellfractions. 80HdG adducts will be quantifiedby HPLC/EC and the resultsexpressed as the ratioof mtDNA/nDNA adducts. We expect to observe a dose-dependentaccumulationof 80HdG adducts to MEDNA. Once we establishan effectivesingleacutedose, we plan to repeat the dosina experimentson a sub-chronicbasis,where the ratswillreceivemultiplelow dailydoses. Subsets of animals willbe killedat variousdosing intervalasnd the totalamount of DNA adducts expressed as a functionof the "cumulative"dose. To complete the dosimetry experiments,we willcharacterizethepersistenceof theDNA adducts.Rats willbe exposed to a cumulative dose of perfluorocompound thatis known to cause a modest degree of DNA oxidation.They willthenbe allowed varyinaperiodsof exposure-freerecoverypriorto sacrifice and measuring DNA adducts. Being thatn-iitochondrilackthe proficienDtNA repairenzymes found in thenucleus,we expect thatthe mtDNA adductswillpersistforsome time afterdosing. Such a phenomenon will be of crreatadvantaae in providing a lastingmeasure of past and repeatedexposure histories. We alsowillscreenforthe inductionof severalearlyresponse-enes thatareknown to be induced in response to mito0aenic and/or oxidativestress,includi-n-gthe classicperoxisome proliferator[s15-17].These includeGST Ya, metallothioneiInIA,c-fos,NFXB, GADD153 and GADD45, p53, HSP70, and GRP78, allof which are availablecommercially(Xenometrix). Differentiailnductionof these-enes willrevealimportantinsi-htintodiscretemodes of action, specificallydistinauishingbetween oxidativestress,-enotoxicity,a,nd inductionof cell proliferation. 9 April8, 1997 types willbe compared in terms of theirsensitivittyo chemical-induceddepolarizationof mitochondrialmembrane potentiald,epletionof ATP, increasedfattyacyl oxidase activity, inductionof the earlyresponsecrenesand the peroxisome proliferator-activatreedceptor,and cell death. The comparisons between specieswillbe both qualitativaend quantitative. The finalobjectivewillbe to compare (againboth qualitativelaynd quantitativelyt)he responseof guinea pigs and ratsexposed in vivo to N-ethylperfluorooctanseulfarnidoethanol exposure. The objectivewillbe to gauae the appropriatenessof the respectivespeciesas a representativseurrocatefor estimatinapotentialhuman healthrisks. Using the biomarkers identifieidnAim 2,we willcompare and contrastheeffectosf in vivoexposureto varyingdoses on the accumulation of 80HdG adducts to mtDNA, stimulationof earlyresponse genes, stimulationof fattyacyl CoA oxidaseactivitya,nd inductionof theperoxisome proliferatoractivatedreceptorgene. Dosing regimens willresemble those describedforAim 2 and the calculateedffectivdeosescompared betweenspecies.To complement this,we willalsoexamine thetissueshistologicalfloyrevidenceof peroxisome proliferatiionone or both species.Itis these experiments thatwilldetermine the suitabilitoyf the ratas a representativesurrogate speciesfor regulatorypurposes. SUMMARY - The ultimategoal of the proposed invest0ioatioins to provide a scientificalsloyund basisfor managing the potentialrisksassociatedwith human exposures to perfluoroalkylacids and their derivatives.The existingevidence suggeststhat regulationon the basis of peroxisome proliferatioannd non-cyenotoxicarcinogenicitymay be overlyrestrictivien thatthese responses areunique toratsand mice. There islittleevidenceto supportthisbiologicalreactivitiyn guinea pigs,docs, or primates,includinahumans. The proposed investigatioinsdesigned to provide a scientifibcasisto supportor refutesuch a claim. The individualaims willwalk the investigation through the successionofstepnseededtoidentifvyalibdiomarkertsomake theappropriate surrogatespeciescomparisons. As such,theinvestiaatiohnas the potentiaolf havina a profound impact on thereaulationof theseimportantproducts,not only in desioningeffectivemonitoring proarams 0 to insureworker safety,but alsoforsetti0n0-auidelinesto safeguardconsumer health. The ur-ency forsuch informationishiahlightedby the factthat,1)perfluoroalkyalcidsand their derivativesareknown to have thepotentiatlo be toxic,and 2)residuheasvebeendetecteidnthe seraof productionworkers. The pressingquestionis: What isthe margin of safetyfor these individuals?The proposed investiaatiownillprovide informationcr-itictaol answerinc,this question. April8, 1997 REFERENCES I 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.- 24. 25. Schnelimann, R.G. and,'vlanninaR..,O. (1990) Biochim. Biophys.Acta 1016: 344-348. Keller,B.J.,Marsman, D.S.,Popp, J.A.,and Thurman, R.A. (1992) Biochim. Biophys. Acta 1102:237-244. Pastoor,T.P.,Lee. K.P.,Perri.i'vf.Aa.n,d GilliesP,.J.(1987) Exptl.Mol. Pathol.47: 98109. Anon. (1979) Tech. Report No. 723Q, 3M CentralAnalyticalLaboratory,St.Paul,MN. Goecke-Flora,C.M. and Reo, N.V. (1996) Chem. Res.Toxicol.9:689-695. Johnson, J.D.and Ober, R.D. 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(1996) Toxicol.Appi.Pharrnacol.140:322-327. -a io,;ne-rn/FlIbo 10 ATTACHMENT A May 7, 1()()8 Bi,--)chemical-ind Moleculir Mechanistic Studies of N-alkyl Perfltiorosulfonamides Kendall B. Wallace, Pli.D. Univt-.rsitoyf imini-tesotSaciio(-)olf Mekiicine Dep,irtnieiiotf 13ioctieiiiii&trMyolectilarBiolt-)gy TI-ieinitialphase of this investigationrevealed that all of tl-te perfltiorl)chemicals(PFC's) examined wort! capable of interferingwith the biocnergcti(-propt-rtit!osf freshlyisolatediiitacrtiitliver mituchondria iti i.,itrcH)o.wever, [I-tperecisemechiiiis-mby which each PFC iiiterict(w@iLtiil the mitochondriiil membranes differed,the activitiersanging from a specific protonophoi-ic uncoupling uf oxiditive pi-iosphorylatic)tnL)i) full-blown detergeiit-likcd!isorganization of the membranes. Regirdless of the ,,pt-!Cific mc)leculiir mechiinism, the results indicate thiit -,ufficientlyhigh cr-)i,)ceiitratoifoiain.ys of the PFC's have the pot(-ntilo)f inhibitingoxiliative pi-it-)spliorylationnli ATI-I-;ynthe.,;iIst,i.sprestjiiiedtl-iaittthe cellor tissue 1,L@ve.t1h,e.r(.-,-@u(tciinegficiotf ATP will be m,.inife.-,tiLs!dan interference with the regulationan(i integritic)onf the varic)ttcstiergy-dependent intermediiry metabolic pathwiy-.@,cellsi&,,nilin-g,,iiciedllcycle et)ntrolpoints, including the balance between A'1'1'-,icpcnLicitiiptoptl),sainsd cellprolifercition. At this poii-ita,ll ttiitIiiib-t,:enestablished is that the Pf-C'shL)vL!thk- pt)tei)ti,titl-i)nterfert-witl-imitochondriil biociiergoticasnli preSLIn-liblcyell i-iiiiiibeiriid fLLnctiotip,rovicied the conc(,nti-atioit,i,s;ufficietitly Whctht,r such c(incei-itritioanrse ichieved iii expus-Liresceniric)sh,-is not yct beefi estiiblisht!LiA.ccorLiingly,two pressing tltiestionsthit reiiiaiii-ii-e: 1) Is the itiliibitionc-)fmitt)cht)ndriiiibil)energeticsin zjitroby tht- vilriou.,; PFC:'s niiiiiift,-,@tie.da,ibi(,)cnergetidceficitand interferencewith metiib(.)Iic I'M/ltD@j@Sll:bl ID:BlochemzMol8io FAX:218-726-8014 PAGE Nlay 7. 1999 regulation at 1-iigi-ileervels (.)bfiol(),i;icail)rginizition, SLich as in cell intact tisslies,or whole organisms? iinLI2) Arc tile nominally effective concentrations within (lierange to @virrlintconc(!rn for human expo.'i'Lires.1 inherent iii this latter qltestion is the silit-,Ibiloiftyisolated rat liver mitochoiitiri,t to predict adverse human health outcomes ittT@i7?0. We propose to extend tht@inve.,;tigi)tiotnt)address the,,4equestit-)n!b;@, iissessing the response of prii-neircyitittirecs)fisolated hepatocytes to selected PFC's i@it)itro(Gr@iv ot al.,1983). 'I'liscpecificcle.-,,iigsitit) thi!effects of expo.,ing cells to inliividual PFC:'.o'n; vliric)usindicators of cellbiL i ergeties, including changes in oxygen consumption, mituchundrial membrane PL)tential,ATI'3 pht-,sphc)rylationpotential, and cellviibility.In addition, we intend to incorporate into the-,-cell studi(-,m,e@islires designed tc)assess the effects of PFC's oi-ithe expression of geiies believed to be rosponsibit! for moliiatiiig the effect.,o;f rol@iteccihemi(-.iilosi-iperoxisome prt)liferitioi-lti,pili metaboli,,:;mand ce-11proliferiti(-)nT.he ei-itirsetiiteof genes to be anilyzcd is believeli to be under the control of the "peroxisume prutiferatorresponse elenients" (PPRE) ancl includ(! per(-)xisc)malfittyacyl CL)A uxidase, cytosolic fatty-,icibdii-idingproteiii(.rABr), niicrosonizilCYP,,,,,mitochondrial HMG- Ct-)A syntliiise,iictiviitii@iigLiip(-)cyptret.)tt!(i-n,iP2)a,iitia@)oproteiiisAl and CUI (Liitruffeand Viimocq, 1997; Gonzilez, 1997). All of these proteirishlive been implicated t(.bie responsible for tl-ieffectsof pc.-roxisomc,proliferatol-S(in -ifterelilipid metzibc)lism itivivo and miy prove to be valltiiblmearkers ,)fthe bi(-)Iogicarlesponse to PFC expost-iresin the corresponding species. Finally, siiicethere art! Lirg-uments qtiestioning the v,-kliditoyf datiifroni rzitsinLi rrii(-tto, pri-dictthe prciliferitioioif peroxis()iiiesIncl pr(-)m(-)ti(-()nftumorigene,,ii-i%iiliuniiii.-w;e, propose to iiiclLlLicllthL!StLidy Lit!signthe opportunity t(icompare in,.@coi)triistl-icmetabolic iiii,g-einetic ro.spt-)nsoef rlitiiiil[iILIm@in hepatt)cyte cell cxjltttrestO Pr'C eXPOSLire l@lzitro. TI-iere is substiintialevidence thit expOSLirei of iit)n-hL[miinprimat,,-,t.orel,itL-tC.Li)MPL)LII-ILILSiue,-;not induce the proliferiitionof perL)xi,.;L)Mes, fic@,atomeg,ily, ind tLLnitr)igenesis Lis is characteristicof rats iiid mice. 10:13tochem/MoiBio FAX:218-726-8014 PAGE 6 Nlay 7. 1998 EpiLiemiolc)gicaelvidence Concurs Witi-tIileSLIggCStionthitriitsiiiLmiicl!are 1-)cio-r,tirrt)gLiftoerstl-iheuiiiin rt!spon.,iBc,.isc(kiin publisi-ieedxperimental evilic!nce, c-)Ltrorigii-i.@).l,Liggcstiotl@vis thit gllinei pigs itre i-nore representative of the 1-iiin-ian rt!spt)nscto exposure t(.3 perc)xisome proliferators.We inten@ito tos-tthisbv ii-icluliipnrginiarycitItLtroefs guinei pig hcpatocytes ii-tihe celleXpOl-,Lterxcperiiiiei-itTsl.-ieintei-iits to coniplire ,lnlicni-itrastthe r(!spons(o@f rat,gllineapig aiid humaii hepatocytes to exp,Lisurt@tso the iiiLlividLiP.F1C1-'s.('()mpLiri.,;w(i)lnl,,be qliiiititatiavneli based iL)ndo-,e-relitecthiiiiges in I-)ioeiierge-tiincd g(@nt-tiicndicator,-o;f a metabolic resor,)n.,t;teiPFC.*empt)s,,tl@e. FXPERIMENTAL DESIGN isolated rat,guii-teipig, and human hepitc)cvteswill be purcha,@;cdfrk)nl a c(-)mmon veiidor in order to iiiinimizethe possibilitoyf artifactual differei-tcaemsong the differentcelltypes due tf)cellisolationprocedures. The cellswillbe suspended in I-lepeq-ba,,;meidnimum meliiiiat 37"C and clistributta-mdong , wells LitiiLiciisitoyf zipproxiiiilit1e0l6ycells/mi. Cells in suspension willbe exp(-)sericiti-iientiiviliiiP-F,Ci'is for up to 24 hr. The ct)nct:ntriitioif-)cniciiI"f-Cwillbe varied in order to estiblishan effective rcingeof conc-t!ntr-,itifoonrseach end-p()int(biot!n(!r).!o,regtki!cne t!xpressi(-)n) for eaciiPFC. '['hirsiingeof effectiveet)ticcritrLitwiiolnl,s,erve @istticbasisfor qu,intit.itivcel)rnp;irisons)mongst the izidividLIZIII)I-'C'@S(nd for tsscssing differencesiiisensitivitietsiftlit3-,;pecies-.speccieflilctyp(!.sT.he diiratic)onf CXPOSLir(-w,illbe -ilctitebtyi the kineticsoi'tiierespoiiseili,i-(isi-elitionsliiIpL) indicLiti(-f)onr.,g;r(-)scsliingesin cellviability. The biot!nerguticpiirlimetertso be assessed dtiritit)t.i,e,COLir-s(e)fthe cell exp-L)surc,isnclude L)xygt,.cnonstiiiiptic)ttiix,ltc)clit)sidirii.e@iilibr@ipnoetunti@it, ATI" pht)--,phi)rvl@itpio(t,e)ntial,and cell viibility. Whole cell(-)xygen COIISL[Mpti(iwnillbe mL)i-iiti.i)nrcaciminnei- similartt.t)hat(-mplc)yedfc)rthe '98 11:52 ID:Blochem/moieio FA',@:@-1@B-726-8 @n14 PAGE 7 ivfuv7. 1998 niitk)(:hondriaC.ell-w4ill be suspended in a stirrei,clc)-ikc@ldiinibcr intl)Which Isii-iserteadClark-typeoxygen clectrc)rimcetere(-tio a cL)ntinuL)tls strip-c-h,irrLt-ct)rderT.he cellsuspending niedium willbe stipplem(-.nteiLviith glucL)st-i,nd insulin to provide the requisite nictibolicfLICI-q.OnCt! n equilibritinis establishe,.sie,quential-idditioiiosf tticselectetIi'l-Cwill be I m,ilicind iny change in the riteof oxygen cc)nstimptic)nrt-vorded.liiorder to confirm ai-ilnlori-nalizeiny effectoil mitoclit-itidrrieaslpir@ltl()atlll, re,ictil)wnisll be terminated by fir.-ajdtding antimycin A fL)IloweLbiy I:CCI-1. Oxygen consltmption in the presence of iiitimyciiAi will-;crvct:c)reflectiiL)i)n-iitL)chc)nLirrica,l,pirL%titi)sn,one might expect if the il,@.,net in questioi-i tindergl)escytochrome P450-dependent (-ixygenatiozoir redox cycling,for extimpte. 'I'hLr,ateof (-)xygenci)n$LiMptionin the pr(!-,tncoLf,the Lincoupler, FCCI-1,will provide @tmoa.,;uroef any directeffectof th(@indivi(illPiFlCl c)n the efectrc)ntr-Linsporcthiiin. The effectof the iiidividitiPirC's oi-iinitoci-ioiitiiriii@einlibrz)rlc pc-)tentio,filthe cellsin Culturewillbe monitored cozitinLl(-)LoIiilltyi-IbLa-.!L;iosf the LiistribLItico)nfthe citionicflii(.irometdryiec, rh(idiniine123 (Rhl23) as we liavepublished previOLISl(yPalineireitial.,1990).Cullswillb(!prt4-loided with Rhl23, folloivedby washing, and then -tilocatetLti)inLiividLiaelxposure wells. Agiii-i,ii-icreasincgoncentrations of the re.,;pk!ctPiFvC(! will be added sequentiallyanc.1the flLior(!,scenrceecorded. The reictionswillbe terminated by adding FCCP to achieve a fulldepolarizitioto-fi iner-nbriinpeotontii], follow(,dby .Iigitonitifi)issurecOMplete LiiSSOILIti(O)lfClCIIiiicinbritne@sIRIL( releiisoef nt-)t-i-speclizfiitcenftlut.)rcscence. CLIIIvi@isilitwyill be recorded in piraliel with mitocii(.)ii,.Iriil i-neiiibr.ipnoetentialLl-';itnhge fILtort)nictriincliie@itporL)piLiiumit)Liid(ePI) -ic*c,.it')trLngto NtiiLnia rci p roc(!(.i-ic,.t,i;n olir liiboritory,([",lli-eTtle1i1ri1996). ,;eqllenti@ziidlditi(-o)fntqhe indivilitiPiFiC]'saiittierniii-i@iotfitolt-iireeictioii with I-CCI"aiiddigitoi)iwnillI>,p-e-rformed ilistas Licscribtf!odr Rhl23 f]LiorL!.sccnc,,-U.nfortun@itely,PI apparently qlienches iume uf tile R1.1123 fiLlt)rc,sceipircoeh,ibiting the concurrent mciisui-ement of both membrii-ic 4 '@JV 11:52 IDSiochem/MoiBio FA'x.:218-726-8n,14 PAGE imuy 7, 1998 pt)tonti,ainl,-icell.,iibil'itntythe szime cells,fn.,,tettLhie,experI'me n ts %vilIbe ct,)nLiElCtiOnLiptrZI1101LtSingthe cellsfrom the CLiltLirseimple for both m(!a.sure.,;, The conceiitratintoifs the varit)LiisLlenineiiticiec)tid,evsilble bv HL-'LCOones, 1981). lst)liitheelpiatocytes froin citchof tiio3 specil-swill be eXPL)Sef,iillClIftLirteo viiryingcoticentratil)oifisthe iiiiLlividLPtFiCi'lS. The CXPOSLire.w,i;ll be stoppeliby @ilkiiiinizitnhge rt.-iictniiLe)(n.iiunw-iitliKol I to stiibiliztelio idcnine nLICICOtiLiCiSn the respcctivephospl-ILiryiitisoti@iiti,!. ,Samples frL)M #-iiicehxposure well will be centrifugedto remove dc!hri,-;, ne-Litrzilizaendd iiijecteodl-itio Waterq Ri)diilP-,iCk18 column. TndividLlili nLicleotiLieLsireelliteiziitI m)/min in 100 mM pil)tassiupmhc)spliit(epi-I6) fc)ll(iwebdy a gradieiitto 5% methai-iol. itiliividtiiailticlel)ticwielsl L-pe detected at 260 iim ajidqLlalltifibeyLiititegriititnhge peiikareiiilscl)mpared to known stindards. Activation of PPAR-responsive gtnes will be assessedby tiifferenti-,ii display 1-isingstandard moleciil-,ibriology teelinitltt(eW.a@n;g @ind Morais, 1997). The method cunsistsof exposing -,il3lspocio.-o;f I-t(@p,-itoctyotetshe correspoi-idij1i'g1,'CA.t pro-Lictermint-t.idmes,the reiclioiiwsillbe St(-)ppeiLni liqtji(nIitrogen an(ianiilyzedforthe enhtnceli exprt@-,.qioofni)selectgroup of gc.-noe.t-i,codingff,)preruxisomal (iiidmitochondrial proteins. This entiils i.qol,itioofntotiiRiNA ft-)Ilowebiyi gelseparatit)i@itidNortht-@rnhybridizition forselectelli-ni@NA's.Probes have been prepared by I-CR for the two "hi-)use- keeping" geties 0-actin iind glyceraldel-tvde3-@,tiosph@ttLoichyLirt-)gt!n@i.,;t! (GAPDH), wi-tich@ireSLippo-,-odluyn-indt.iciblaeillitilLIVS,111(n1lc-,isllrfoti)r gont- copy nlimbt-i-(cellntimber). Tile PF(:-inLiuceti@:tivLitiLu)nfiiiit)ther gelieswillbe normii(izetitL)the cupy number ft)rtlik!stcwt)genes iiioi-Lict.o,r qtiai-itilitivgealLyLgC th(!d(,greeo.f stimi-ilati(ci)figieiieexpressioi-oIn the bi!iis L)ftotiiglenomic DNA. Fxpti:iure-relatpero.liiferatioonf pt-rt)xisc)niwti!lslbe estimited oil tl-ie basis(-itfhe stimulated expression of the following geiies:perl)xisoniilf@itty 5 ;vo Ii;t)i ID:Biochem/Moleio Fg>@:218-726-8014 PAGE Mity 7. 1998 .icylCoA oxi(.i,i.c,y;teo,sc)lifcattyaci(ibinlilngprotein (FABP), microsonial CYI-IIVAl,llitOChL)ndriI,I-Mt(l-,-CoA synthisl,a,cti@,,,,iatliinigpocyteprotein (al-12), ,iiidcipoproteins Al and CIII (LatriLif@fien(.ViiimecLI,1997; Conzalez, 1997). ',vlitocl-ionkiprrioalliferationwill be isses@,,eoLiii the basisof the enhanced expression c)f citrite synthiise iii)d SLIccinate(-Ichvdrogen-ase(SDH). Amplificc-ttiiL(i)nfthe niitoclioi-idrigiti-i-nOITI(CLiitDNA per mitt)cht)rl(irita)sn oppc)!;cLtio an in(-reiisien iiiitoch(.)ncfrnilziimlber per cell)will bt!LissesseLbiy -inincrease iiigent,(-()pyIIL[nibefror mitocht)ndriiitiranscriptL)fcytL)chr()mcb (CY'Fb) ind NTD1 relativeio SDT-1 (a nltcleirencoded gene) Ll.,;iglelr_ic dosi experiments (Boultwood et al,,19961@. probes have been generated bv PCR for all3 genes. Increased expre-,,Ni(o-f)iciy'rb ai-idNDI reizitivteo SDH hitsbeei-iused as an indicatorof an incrt!a.,i;nc mtDNA copy nliniber in car(-fiiiteissue following Licutc izitoxicatit)n(-)frats with adriamycin (tinpLiblisheLolbservation). TI-icstimtilationof botliperoxisumiiiand mitocliondri-,iplroliferation i,s@)scribe4ito ,ilteredti-anscriptioiicaLlintrLil.To verify this, we will ,qlibc,tantiaotleir results of the gene dosing Lini differenti,-diil@;pl@iy expei-imcnts by nie,:istirintghe c(@)rrL!.-;potieciliznygjiieacti@,itiefsollowiiig eacl-(i.itfl-i%(,-LiriOLCleilltreatment piiriil]igmsT.he CXPOSLire incub.@itionw.i,l;l be stopped and the cellsrecovered by centrifugzition.Fatty acyl CoA cixi(.iaw activitywill be estimated from the cyanide insei-isitiovxeiliatiunof piilmitoyl CoA according to the spectrophutumetric teclinil]tiocf Lazarow ('198'1). Succinute LiehydrE)@,,oiizise (SDH), citrzitt!syiithise ind cytochrome c)xi(-izi.@,t2(aCrOeX) nicisiirt.-dby the correspot-iding -spectroptiotometi-ic iiiethoLf,(.P;ont-tingtoi-1i9,61;Trc)un(-(c!-tit).*,1996;Lash and jont:s,19@)3). 6 1):8iochern/Mo 18io -rs@)(:218-726-8C-14 PPGE IMAIARY May 7. 1998 '.rlipcroposed ijivestigationis ciesigned to take our CLirrent finliings regarding the effectsof PFC's oii the bioenergeticpropertiesof isc)lated mito(:honLiri,-mielmbranes to the next levelof biologicalintegration. The objective is two-fold:to determine whether the effect,o;f PFC's t)n isol,-ited mitl)chondritii.@i;iizinifestaesd bioenergeticdvsfiti-ictiaotitihe levelof whole cell ind to compare iinlicontrastthe response of fiepatocytefsrom different ..;pccictso Pf:cexposure in ctiltitreT.he metabolic and geneticstudieswill yieldvaluable understanliinginto the i-nechaiii.-,noif(bsi)ologicalacti,,,iatsy well as revealpotenti@illiymplirt-.ibiiiotiiizirkefrosrasse.,;siPiFiCg CXPO.-qLtirni-, I)ivo. The comparisons ami-)n.,@,specie-,w-ill flirthertest the quoition rcgzirliiitiiglcSLiitabiliotfy the ritas aiiexperiment,)]model for predicting Li,-iver-1,1-0c!lltii-l)LItCO11i1n0SIILLi-nateixsposed to peroxisoilleproliferatorsand whe.tht!rthe @.-,uinclipi@,,,may prove to be @iiiiore-;uitablSeLtrrogateT.hese data arL-essentialto extrapolatingdose-respl)nserelationshipsfrom experimental evidence to predictno effectlevelsantiiiiargiio-fisafetyforpotentialhilmall exposlires,especiallyin lightof our preliminary evidence which indicates that iq(-)Iat(h!eLpiatic mitochondriii from @,tiinclpiiS--i-.,,3;-6 timt!,@m;(-)r(@ i-esi.,;tta(n.tP)FC-induct!d interfvrcnriw,ith bi(ientrgetictshan are rat liveii-nitoch(-)ndr(iuanpublished observations). 7 iu -i.i@ochem/no ju io FAX:218-726-8014 PAGE Nlay 7, 1998 REFERENCES Birch-Machin, M., Jack,,ion,S. Kler, R.,;.,and TLirnbtill,L).,\4(.1993). '-;tudyof skeletalmuscle mitt-)chondrialcivsftinctit)n.lii:Nterlioi,-i,n Toxico]()A_ y vol. 2; kliti)cht)ndrial Dysf-Linc,tit@)ii (Las)i ind jont-.-,, cLi.,:i.) A(-ZILIL!MiC Press. f3OLl]twood,J.,Filder,C.,Mills,K.I.,Frod--,I),iIi"-.n',vlK.L,ibec,R., GiiL.,(,-r, A C;ale,l@.E.,Lit-ichD,.C.,Littlewood,T.J.,Nlosg,I-I.A.H.a,nli W,.iii-iscoat, J.S. (1996). Amplification t)fmit(ici)(.iiidrDiNiAl iii-ICLItnO)velt)id leukemia. Brit.J.H-,iemati-)9I5.:426-431. Goiizalez,F.I.(1997).Recent xtpdateot-ithe PPARrx-niillilioliseB,iochiii-iie 7();139-144. Gr.iy,T.J.8.L,tke, B-C,.B,eiimand,J.A.,Ft),steJr.,R.,tnd Gingt)lliS,.F).(1983). Peroxisome pr(-)Iiferitiinoipii-imarycLiftiLtrLe)sfrat 'rieputt,)cyteq. Toxicol.Appi. Pharmiicol.67:15-25. joi-iesD,.P. (1981).Determinationof pyriditiefiinticlec)tii,n@icce.l,le;xtrlctsby high-perfurmanee liqlticdhromatography. J.Cliromatogt-.225:446-449. l,atri.ifNf.ea.nd Vamecq, J. (1997).reroxisoiiiper()Iiferatta)nrdsperoxisc)nie pr(-)Iiferataocrtivatedreceptc)r(sPPARS) as reguliitorosf lipid motlibolism. 13iochimic79:81-@)4. Pitlmeira,C.M.,ML)i-(!noA,.J.M.,Mideiri, V-kf.(".z,indWallace,K.B. (1996). CL)titinuousmonitoring of niitt)chonciriiielmbraiipeotentialin hepatocyttC!ellSLISpellSioJn.sP.)iarmacolT.oxicL)MIe.tliods35:35-43. PernningtL)Gn., (1961).ALivantageosf -iphosphiitebtiffefrorOsC)4sollitions in fixation.J.Appi. Phys. 32:'1637-16-'19. Trounce, [.A.,Kim, Y.L.,Jtin,A.,i.a,nd WiliziceD,.C. (1996).Assessnientc)f mit(-)cht)iidtr)ixailiitivpeht)sphorviiitiiotiipiiitictniitusclebiopsies, lympht-ibl@istisn,d trcinsmitt)cl-it)nc@eilrliiliilties.Iii: in Enzymol. 264:484-509. Wzing, H. aiidMorais, R. (1997).Up-regLitatiLioifiniicle-,giernes in re-,-poi-ise to it-ihibititl))fnmitocliondrialDNA expres.,;iint)cnhicketicells. Riochini.Bic)phys.Acta 1352:325-334. kbw-2n/98 EffecotfN-AlkylPerfluorooctylsulfonoanm@iadteoschondrial BioenergeticIsn Vitro Kendall B. Wallace, Ph.D. Department of Biochemistry & Molecular Biology University of Minnesota School of Medicine ExecutiveSummary The following report summarizes research conducted the past 7 months concerning the effectsof 6 differentperfluorooctanonyl compounds (PFC's; supplied by The 3M Company) on the bioenergeticcharacteristicosf isolated rat liver mitochondria in vitro. For purposes of confidentiahty,the trade names and chemical structureswere not disclosed to those conducting the research and analyzing the results. Consequently, the identitiesof the individual compounds are codifiedthroughout the report. They are: PF10; PFLOH; PF12L; PF12M; PF95; PF143; FC10 the carboxylicacid of FC10 linearFX12 mixed FX12 FC95 FC143 [Resultsfor the suffonamide and the N-acetic acid of the sulfonamide are not summarized in thisreport.] AllsixPFC's examined todateexhibitesdome effecton mitochondriablioenergeticFsC.10 increasedthe passiveprotonleak,resultinign a stimulatioonf state4 (nonphosphorylatingr)espiratiownithoutuncouplingoxidativpehosphorylation.The acidof FC10 increasedmembrane permeabilittyo ion conductance,resultingin membrane depolarizatioand releaseof cytochromec to inhibirtespirationB.oth linearand mixed FX 12 were potentprotonophoreuncouplersof oxidativpehosphorylationB.oth FC95 and FC 143 exhibiteda generaldetergent-liekfefecton mitochondriamlembranes asreflectebdy a decreaseinrespiratorcyontrolwithoutuncouplingoxidativpehosphorylation. In conclusiona,lthoughallPFC's interferewdithmitochondriablioenergetictsh,eydiffered inbothpotencyand mechanism. Regardless,theultimateffecitsthesame; inhibitioonf AT? synthesis.At thecellulalrevelthismay wellmanifestitselafs failuroef assorted energy-dependenmtetabolicor cellsignalinpgathwaysleadingto disrupdonof metabohc regulatioannd cellcyclecontrol.Whether and by what mechanism thismay relatteo the allegedperoxisome proliferatiancgtivitaynd tumorigenicactivitoyf thesecompounds remainstobe defmed. ----Cl12CH3 f--" --TI-OT_T 2- FCI-r) increapsaescs-ipvrpotolieiaCla--'a6%04- CH2CH3 C8F17-So2-N < CH2COOH PF-'LO-H increasedpermeabilityto ion conductance @ 6 MM CH2CH3 Ll H FX12 protonophore uncoupler Cw6 VM (mixed and lineararecomparable) \\\*4 C8Fl7-SO2-NH2 H C8Fl7-SC)2-N< CH2COOH Ctiit7@d1111 C8Fl7-SO3- FC95 weak increasein membrane fluidity@ 10 tiM