Document pJZ1BxDxz45MaJyq87nmww37

EFFECT OF SELECTED PERFLUORO-COMPOUNDS P-OXIDATION KendalBl.WallaceP,h.D.,DABT'December20,1999 ON MITOCHONDRIAL SUMMARY itwas hypothesizedthattheperfluorocheniicmaalys be structuramlimics oflongchain fattyacids and therebybe competitiveinhibitorosf mitochondrialfattyacid oxidation. Such inhibitionmay occur at the stageof formation of the CoA or carnitineesters(depletionof cytosolicCOASH, inhibitionof fattyacyl CoA synthetase[thiokinase]o,r inhibitionof camitine acyltransferase1),transportof the camitine esteracross the inner mitochondrial membrane, interferencewith the formationof the CoA esterwithinthe mitochondrialmatrix (depletionof matrix COASH or inhibitioonf camitineacyltransferasIeI),or inhibitioonf the mitochondrialPoxidationenzymes themselves (alternatelectronacceptorcomplex 11of the electrontransport chain). To testthis,we firstasked the generalquestionof whether the fluorochemicalsinhibit the oxidationof palmitoylcamitine,which isa measure of allstepsbeginning with the transport of the camitine esteracross the innermitochondrialmembrane. Thus, thisis a measure of the effectof the fluorochemicalson allsteps thatoccur within the mitochondrion,but does not reflectany potentialeffectof thecompounds on theextramitochondrialformation of the COASH or carnitineesters.Eight fluorochemicalcompounds were selectedfor thisstudy (N-EtFOSE, FC-129 (PFOSAA), FX-12, FC-95 (PFOS), PFOSA, FC-143 (PFOA), FC-228, and CMPD-8 (M556)). MATERIAL AND METHODS Rat livermitochondria were isolatedby differentiaclentrifugationand incubated in a medium containing200 mM mannitol,10 mM sucrose,5 niM HEPES (pH=7.4),I mM EGTA, 2 pM oligomycin,and 10 mM KH2PO4' Mitochondria were added at0.8 - 1 mg protein/ml.All of the testcompounds except FX-228 were added ata concentrationof 50 nmol/mg mitochondrial protein.FX-228 was added at 50 pg/ mg mitochondrialprotein.Palmitoylcarnitinweas added to a finalconcentrationof 40 VM. Glutamate + malate (finalconcentrationof 5 MM, each) and succinate+ rotenone (finalconcentration,5 mM and 2 VM, respectivelyw)ere pre-mixed and added as indicated.2,4-Dinitrophenolwas added at a finalconcentrationof 40 PM. Mitochondrial respiratiownas measured with a Clark-typeelectrode. RESULTS KendalBl.WallaceP,h.D.,DABT December20,1999 miio PC, MRO PC C-228 51 23 Glu DNP MRO PC DNP GiU mito Pf @C-9 N ' 9 @@D@NP 4 Glu 4B. 0-1 OH .$ I DNP Glu Suc Suc Suo' Sut go. C-i 0 0 1.12' 1.3 E .1triin Figure1.The effecotfrepresentatipveerfluorochemiccaolmpounds on respiratioofnratlivemritochondria oxidizingpalmitoylcarnitiMneed.ium compositionand additionseeMethods.Mitochondrialproteincontentwas I mg/ml. Numbers nearthecurvesindicatteherateofrespirationnm,ol 02 x min" x mg-1protein.Abbreviations: Mito,mitochondriaP;C, paimitoylcamitinDeN;P, 2,4-dinitrophenCo-l2;28,FX-228; Glu,glutamate+ malate;Suc, succinate+ rotenone;C-95N, PFOSA; C-IOH, FC-129 (PFOSAA). For explanationsp,leaseseethetext. DISCUSSION The efficiencyof 0-oxidationin ratliverrnitochondriacan be estimatedby measuring the rate of oxygen consumption in the presence of palmitoylcamitine(PC). This substrateis imported into the mitochondrialmatrix over the acylcarnitinetransporterand subsequently oxidizedthrough the P-oxidationpathway in thematrix of mitochondria.Insidethe matrix space, palmitoylcamitine is converted in palmitoyl-CoA by carnitineacyltransferase11 and then oxidizedby molecular oxygen with the participatioonf two complexes of respiratorychain, Complex I and Complex HI. Under conditionsof uncoupled respiratio(nfollowingthe addition of DNP), themaximal rateof mitochondrialrespirationin the presenceof palmitoylcamitineisa 2 KendalBl.WallaceP,h.D.,DABT December20,1999 directmeasure of the activityof the P-oxidationmultienzyme complex and/orthe rateof palmitoylcarnitin(ePC) penetrationintomitochondrialmatrix.Hence, ifa compound inhibitsthe transportof PC and/or the P-oxidationenzymes, itwill decrease the rate of uncoupled respiration. None of the perfluorochemicalstested decreased the rate of PC-supported respirationin the presence of DNP, indicatingthatnone of the compounds inhibitedeitherthe transportor P-oxidationof fattyacidsby isolatedratlivermitochondriain vitro. A compound may alsoinhibitComplex I or Complex IIIdirectlyw,hich may complicate the interpretatioonf the data.However, thiscomplicationcan be revealby comparing theratesof palmitoylcarnitinoexidationby uncoupled mitochondria to thatof other substratesof Complex I and Complex III,such as glutamate/malateor succinate. The above mentioned considerationswere taken into account in designing our experimentalprocedure,as illustrateidn figure 1. First,we estimatedthe maximal rate of PC oxidationby uncoupled mitochondria by measuring themaximal rateof PC-supported uncoupled respiratio(ntheuncoupling was achieved by adding 2,4-bNP ata concentrationthatinduced the maximal rate of respirationin the presence of glutamate+malate).The additions of glutamate+malate (Complex I -dependent substrates)and succinate(Complex IIIsubstrate) yieldedthe values for themaximum uninhibitedratesof Complex I and Complex IIIdependent respirationr,espectively(Fig.1,curve a). The otherthreecurves of figure I illustrattehree possiblescenariosof effectson theuncoupling efficiencieosf the compounds of interest: curve b - If a compound (such as FX-228) does not inhibitPC oxidation,but itisa less efficienutncoupler of oxidativephosphorylationthan 2,4-DNP, then the additionof the latter would stimulatethecoupled respiratio(npriorto adding DNP), whereas therespirationratein the presence of 2,4-DNP plus FX-228 is nearly identicalto thatin the presence of DNP alone (compare with curve a); 0 curve c - If a compound such as the amide of FC-95 (PFOSA) is more efficientthan DNP at uncoupling mitochondrialoxidativephosphorylation,itproduces the maximal rate of respirationwhen added to mitochondria by itself.This ratecannot be furtherstimulatedby adding DNP (compare therespiratiornatesbefore and aftertheadding DNP); 0 curve d - The FC-129 (PFOSAA)-stimulated rate of respiration is also insensitive to DNP, but the rate of oxygen consumption is only about half of the maximal rate (30 nmol/min/mg afterDNP additioncompared to 47-51 nmol/min/mg for curves a, b, & c). Furthermore, the respirationrate was also suppressed following the additions of glutamate+malate (Glu) and succinate+rotenone(Suc),which indicatesthatFC-129 (PFOSAA) isan inhibitorof Complex I (and possiblyComplex HI) of the respiratorcyhain,ratherthan an inhibitorof PC oxidation. These experiments were allrepeatedin triplicatfeoralleightperfluorochemicalcompounds. For N-ETFOSE, PFOS, PFOA, and M556 the effectswere qualitativelsyimilarto those illustratefdor 3 KendalBl.WallaceP,h.D.,DABT December20,1999 FX-228 (curve b) for allthe compounds. FX-12 at 50 pM is a fairlystrong uncoupler of oxidativephosphorylationand affectedthe respirationsimilarto thatcaused by PFOSA (curve C). CONCLUSIONS In allof the experiments,the chosen concentrationsof the compounds were eitherequal to or several-foldhigher than what earlierwork demonstrated to be maximal uncoupling concentrations(referencereporton mitochondrialbioenergeties).Because allthe compounds were practicallywithouteffecton maximal ratesof PC oxidation,we conclude thatnone of the eightperfluorochemicalstestedalteredtherateof oxidationof palmitoylcarnitinbey isolatedrat livermitochondria *nvitro. 4