Document MwRbY0b09GOnqqR5zaKL70aj

Covance Studies AR226-0/68 The attached report summarizes the results from the assessment of `mitochondrial bioenergetics of hepatic mitochondria isolated from rats treated for 3 weeks with one of 4 doses (plus control) of dietary FC10. What is obvious from the data is the ability to detect dramatic differences among the 13 animals tested. However, attempts to group animals according to degree of effect failed to correlate with the exposure dose. Further inquiry revealed that all animals were euthanized with ca., 50 mg/kg pentobarbital, which we subsequently demonstrated to have profound effects on inhibiting pyruvate oxidation at complex I of the mitochondrial electron transport chain. We attribute our inability to establish a dose-response correlation to this effect of pentobarbital. Subsequent attempts to eliminate this confounding effect from the analyses were unsuccessful. However, we remain convinced of the importance of collecting data describing mitochondrial deficits in tissues from exposed animals and in our ability to detect such effects with sufficient sensitivity. Rather than relying on the isolation of high integrity, intact `mitochondria for measuring coupled bioenergetic properties, we suggest measuring glycolytic and tricarboxylic metabolites in frozen tissues in order to derive a profile of intermediary metabolism for each exposed animal. From the relative concentrations of the individual metabolites (eg. the oxoglutarate-to-pyruvate or succinate-to-fumarate ratios), we can estimate relative degrees of metabolic dysfunction in exposed animals`. Rather than requiring one of us to be present on- site at the time of sacrifice, we can perform such analyses on tissues that are collected in liquid nitrogen and kept frozen for shipping to our laboratory. This is considerably more convenient for all parties involved and we are anxious to discuss potential opportunities to continue to contribute to the in vivo monitoring studies. mCeotlasboonl,iCc.ainndsiKnlgalpepsearm,pMleH.ali(q1u9ot7.9.ASle.queBniionclhemme.su97r,n3e9n43s9o9f.glyco nd wiasbonylic ac yee 04184 PROBE DESCRIPTIONS `Theintroduction. `The rationaleofthe following experiments was to determine as many "independent" characteristics of `energeticsofmitochondriaasit i possiblefrom asingleprobeandhavingtime limitations.Duetothis, thespecific `experimental procedure andconditionswere designed.Thepyruvate supportedrespiration andthe membrane `potential weremeasuredin asimpleslightlyhypotonic incubation media.OthercompsuochausADnP,dCasTM,a,nd `succinate,wereadded tomitochondriaatsaturatingconcentrationsandatdifferenttimesbut a least 2minafterthe `mitwoerecinthrodoucend10dtherpyiruvaate~containing medium. No inhibitorsof oxidative phosphorylatitohne,: respiratorychain,CaTM transport,or uncouplers wereused.Also, no attempts weretaken oreduce theconcentration offreefattyacidsco-isolatedwithmitochondriaorgeneratedduringincubation. `Thelogico theexperimentaldesignwas to measurein a singleprobethemost vulnerable characteristicsof `mitochondria, suchaspyruvater-espsiruatiponpanodorxitdaetidve phosphorylation,andthe most "insensitive", easilyreproducibleparameterssuchas succinate-supported respiration and CaTMtransport.Undersuchlimitations, thereismopossibility toobtaintheprecise characteristicsfor anyoftheparameters. Howtehevcomebinrato,ry `analysisofalthemeasuredparametersallowstoestimate,at firstapproximation,theenergetics ofmitochondria, and `makesomeconclusionsaboutthe invivoconditions, n a tissue,where the mitochondriawere isolatedfrom. `Theinterpretation ofanyinvitro data, andespecialythebackward deductionof invivoconditionsin a tissuefrom thedataobtainedwithisolatedmitochondria,isalways akindof "mightbe" guessing.Itisrather temptingtothinginterms like"badmitochondria", say,iftheycan notoxidize asubstratefastenough,or "good mitoicfthheyexoertned adnRrCIimoraet"hen,20. Wewouldpreferanothermodeofthinking, namely that `mitochondriaaregood whenthe energeticscharacteristicstheyexertfitthecellular demands.Andtheparticular valuesformitochondrial energeticsusually depend on theparticularexperimentalconditions.Duetothis,itsemtso be necessarytoremind shortlythepeculiaritiesofmitochondrial biochemistry underlying the experimental `condweihtaviecohonsesn. `Themost vulnerable functionsofmitochondriaare pyruvate ~ supportedrespirationand oxidative `phosphorylation. The oxidationofpyruvatebylivermitochondriadepends on theavailabiltyofCoASH inthe `mitochondrialmatrix,ontheoverallactivityofthe TCAcycle, andon theactivityofrespiratorychain (especially, on theCompIalctievitxy). Inaddition 10these, maximalrates ofpyruvateoxidationusually require thepresenceof catalytic amountsof malateinthe incubation medium (tostimulatethe completetumoverofTCA cycle). Also,itis `underallostericcontrolbyof naturally occurringendogenouscompounds,suchasfreefatyacids,acetyl-CoA,and ATP inhibitingthe pyruvate dehydrogeacntaivsitey. Therespirationrate of pyrvate-sumpiptoocrhotnedrdia `phosphorylating ADPdepenondalslthe mentioned parametersplusthe activitiesofoxidative phosphorylation system (Pi transporter, ADP/ATP-translocase,and ATP-synthase). Theselatterare also can be negatively regulated byfreelongchainfaryacidsandlong-chainacyl-CoA. `The oxidationofsuccinate snotsovulnerable.ThesensitivityofsuccinatedehydrogenaseandComplexIII 10freefattyacidsismuchlessthen thatofpyruvatedehydrogenaseandComplex1.Practically, underany"regular" `experimental conditionstherateofsuccinate-supportedrespiration i limitedonlybytransmembraneelectrochemical Pprowh gradient.Theonlyknow strong endogenousinhibitorofsuccinate dehydrogenaseis oxaloacetic acid. 0418s `However,intheabsenceofmalateandthepresenceofsufficientamountsofpyruvatein theincubation medium,the. accumulation ofoxaloacetatecouldnotbe Pexpecied. `Therespirationratethatcanbeobservedduring Ca2+uptakeispracticallyinsensitive toanyendogenous. `compounds,whichmaybeco-isolatedwithmitochondria.Providingthatphosphateconcentration issufficient,the respiration rateduringCa2+transportcanbeconsideredasthemaximal possiblerateof oxidationof arespiratory substratebytherespiratorychain.Itislimited mainlybytheactivity of aCa2+transportingsystem,whichis kinetically faster, thenanyothertransportsysteminmitochondria, including thetransportofthe majorityof respiratory substrates, andmuchfasterthenADPtransport.However,when a NAD ~ dependentsubstrateisused,the `respirationmaybecomeinhibiteddue tofastopeningof aCa2+-inducedporeintheinnermitochondrialmembrane, andsubsequentreleaseanddilutionofmitochondrNiAaDl. Forsuccinatrees-pirsatiuonp,tpheosarmeteveendt `wouldprovidethe fastestrespirationratebecausethesuccinate dehydrogenaseis a FAD-dependentenzyme,and `poreopening completelydischargethetransmembrane protongradient. s2tidmuuelattoekdibnyetpiycrluivmaittea-tdieorntivoeTdaCcAectyycll-eCaorAi.singfrom overreductionofNAD andfast removalofoxaloacetycacid 04186 MATERANDIMEATHLODSS The staoftmitiochoondrnia. conventMiiontaolchdiofnfedrrenitaiwalecreenrsiluagtaetdiofnrpormoceIdvuerre.eLsivsrp(i-c1e.5s gvweeretwceoilgehtino2f0admulltofmsoaoanndlfmeemdeailuemra(t21s0bmyba manic,10mMsucrose, SMHEPES-KOH(H7.9), |mM EGTA,Cooled isewasmincedwithsorsand waamsohetdoroinveewniPtohs2e0s7hmoomfogenliatzieorn(mTeedfilounmp,etlhe,ndgiasedbewaikherthensdmleamceeddioumicaen.dThheomotgiesneiztemd oer d45riasteicuowwimaths approimately 1:20 (gm).Thisprocurewasappliedsequential to 8 verspecimens,Thetim intervalbetwen `homogenizingofthefirstand thelastliverpiecesdidnotexceed(') 9min.Thehomogenateswere simultaneously 1`c0enmtirnif.ugTehdfeopre1l0msiwnerx7e0re0sgu.spen=de4dCi,nan2dmmiltoocfhwoansdhriianlgpmeleldeitsuwmer(e21r0ecmovMermedabmytcean,tri1f0ugmatMiosnuactr1os0e,,00S0 gMx ceHnEviPuEgeSd-a 1Kp0HO007H0.4,g)x.T1h0emsiuns.pTehnesiionnaslomfitmisthoocnhdoinadlrpiealwtersewdeirleueteidstpoen3d5emdlinwwiatshhtihnegsmeadmieumme(d0iaupmr,otaenidn `concentrTathiiosnoifso8la0t-i1on20 mocgcxumri"waansdrsetpoeraetdeodnoircet.heThseecisoonldatsioonfprocievdeurepteoiomkeenxasc.tTlhye1phrooucre.ssingofthe fsst (1s2a:m3pl5eAs M1a-n8)dwcaosmpcloemtpeldewteidia ~| h1o0w:s35TAhMemeaansutrheempernotcsewseinrosfatrhoedsetc2on0d0sPtM(.samples 9 16)wasstaredat Measurements Miochondsalmembranepoentl (8%)was estimste fromTPP" ionsdisoationmeasuredwitha FP" selectivTelheirtatoe.oMiftaoxcyhgoenndcroinalsummepmtbieoannebypmeitnoachlonvdarsicawlasemeiasaurdeescwriitbehadchsacnwdb-emraed.e Clk ypeoxygen corde Boththmitochondriamembrane poinialantherspirtion aewere cordedsimulncouslyusing a `multichannelincubationchamberequippedwith amagneticstirrer.Thevolume ofthechamberwas 1.8ml. All se`qeuxepnetriimaelntasddwietiroenpseorffkonromwednaatoruootmotfeTmPpPer'aCtIu-rbeel(o2r2ethCe).adTdihteioTn oPfPmifsoecnbsiotnidvteiael(escetrFoidge 1w)a.Tshcealriebsrpaitreadtiobny aeswerecalculated assumingteil oxygenconcen obecad10240KM. KPO,Tahnedinc1u0bmatMiopnyrmoevdaituemwcaosnwtsainiinnga2l1le0xmpeMrimmeanntsi.cTh,es10ammMe svuoclruomsees,ofSimibtoHhEoPndEsSiaKlOsHus,pepnisio7n6,(40m0b) wereadded,regardlessofthe proteinconcentrations. -- ProteinconcentrationsweredeterminedaftertheexperimentbytheBradfordassay. Bovine serumalbumin aa, Addons,Whermeron.teadditionswere afollowing:mMsuccinic, 200kMADP, and264i CE 0 rs mgt 39S i pan thismodificationoftheconversions isoltionprocedurecanbeexpected todecrease he qualityofmitochondria. Accordtioonugr 04187 Theresults "Table 1showstherelationofsamplelabelingusdinthe followingextoth abel ofliverspecimens. Table 1. The sample label:s pFTF T Tr i m Vmll m Somral fbtevl [mon lE f mTus n Tz |G |1o3wn[o 4 m[|1oy 5n, | f Lf 1 Cm wf jf "Thesampisweremeasuredinthe followingimesequence:3 4 ~5 6 ~8~12-13 ~ 11 - 14-2 ~1-7-1s Anaveragetimespentforcachprobewas bout12min,withapproximately 5minintervalbetweenprobes,All `measurements werecompletedwithin Bours. Table 2showsthvaluesof he respirationrateabtainedinthe preofvsarieousnaddcitieons. `Tabl2e. The respiration parameters measured. seeProbeFDoerstchreiipnicounbsacthiaopntmeer.dDiautmaaanrdoetxphrrecsosneddiitninonmso,lscOeM/amteirni/amlgso&fMpreotthcoind.sV.Fpor,tthheseraqtueeonfcpeyrofuvaaddtiet--ions, suurpipnogrtCeadreuspptiaraktei;onV;oVcey,s,uctchienaatteeofsurpesppoirrtaetdionesprraotdiuocnedibnythAepDrPesaednlcieioofnp(y5r.a3v)a;teVnendithtehreeaobfsernecsepiofraottiohner a(dudmiotli)o;nAsD(PunIlOes,ssthaetaediootohefrAwiDsPe)p;hCos4p0h,ortyhlatreadt(ooufmCl)a"tottrhaenaspmoorutendto(fmooxlyogetnhceoanmsouumnetdo(fnomxoylg.enconsumed [L TesTeA lTamaT ya as) NCI lI ll cl dl ad INCE INCI ll ld il ll lh dd dd id Il N ll ldhd [Cao [PE] e ees ee ca IE ND oideerined ld dlNl id NRpyr~unvoatteressoulpvpeodrtseederresopbireaDteisocnriptionchapter) pore,0it*s-htohulrdebspeircaotnisoindrearteedwaasthmeemaasxuirmeadlnrattheeporfessuecnccienoatfetohxeiedxatcieosnsof Ca2+,whichinducedtheopeningof 04188 e et. h08io ,Co Mst t in ee prinof 14 mYtypvtxeonHover DP otsvi erpRTE bougheidobe `mV. Theincreaseinrespirationandinthe A% wasbiphasic, indicating incomplete phosphoryoflAaDPttioothne `momentoftheadditionofsuccinate.Onlysteady-state valuesfor A'andrespirationareshown. RCIfor ens decir nAYe10a14s9me V.Thesemitochondriaconsumed 360nmol Ca2+/mgofprotein,theporewasnotobserved. ha T mr --oe eeeope --rsatsit--toe i"icotC e e eACP,Em Ci, Tos a ta 7chsi oee n m aSeAm i rtetyse a mmt tAeee e P?arT9 transport,pyruvatedehydrogoerCnoampsleex,1.Therestofrespiratorychainand/orsuccinatedehydrogenase e A e A TrA AP inducingcompoundlikefreelong-chainfattyacids.Thedecreasein Ca/O(Table 1)isprobablyduetouncoupling. Probe 4. heoomoe na tt ee To e entelTieSerei e bey prrs (Ca2+requiredfor poreopeningbearnoinformationinthis case (theproteinconcentration,whichwasmeasured C04189 Toescof ons visADP, 1, Cd sic Th chemivere iin of 143 mVbypyruvate oxidation.The addition ofADPtransientlystimulatedtherespirationanddecreasedthe AYto photon the 5 css te ig le,teto freAD adn perlarisio). The 115 mV,the RCIwas2.7. Themeasurementofthe AWrevealedanunusualpattern,afterthecompletionofADP responseofrespirationonthe Ca2+additionwasunusuallyslowerthentheresponse onADP(see Table 2). Thepore: `was openedby243 nmolCa2+/mgof protein. Succinate-supportedrespirationwasrecordedafterthepore opening, ne Inerprstons: The 50itreflectsthemaxiamctaivilty ofsuccinatedehydrogenase +therestofrespiratorychain,anditisinthenormal mii wer cole be rein vith bh sb as 1 puringtsntt.eA,Houverl, hedronrsotn ao2 aavns 4 ighcniitlssloifonCoheHCine chilma ct eeomoon cs ition. pThye cPerxnobeeo6o.f sAoDPnsvhtaAiDPm, ceitesr, Cpt..TThoeempcedikaind,Coae OdYioof n12v2emeVhbey einIPoreepr,ttoroncse:eTahts15mone/vegrepmrpiln,tso phephreGxminrnsi)bid Te dia provid mi fer by hesen es fnPo 4(s rinCo nig) TTeefPcrrorbeo7fv.sttontveADasP,14cmi,n,C02, Cts,inCtee. pToenetrmpiwornsvel1y67.iCParesse1n.2d poe [ heeg0r 6465 ly m of ein ore). Th nysci Probe8. as edcs hs chon sp ob ble sia OY bh wil 58ofmoons om pyranudwivtha succtinae te. i er pin Succinate-supported ate respiration me, he waspartially nin onion of inhibited. It shouldbenoted chsfn also,thatthe suspensionwas75mg/mlandthatinprobewas 1.25mg/ml iciPnoases1 e he iochondsi fncton To michond weeRBISry ome Interpretations:for aregularratlivermitochondria, 1.25mg/mlconcentrationinprobeis morethen Thebsyuprreeofvitaon.aTheAaDPt,iC2n4o,fADPcsihy.iTovsemircihiiedre:irepioni,iuhe 8tofoCfi16 tivointttfcsHeoveeor, trsnmofiCa2 drdepcinpintdoegston, de ivagati rotinpsiIinntnerroprefeptsatrioosnosictinTestC2 ely i ns:Theabsenceofthestimoufplyruavatteoxiidoatinon gbhy ksthe Ca2e+n 150additionisCaemgof probably due 04190