Document 2JKRKbKwo5DOdYjbxewB5oML

Covance Studies The attachedreportsummarizes the resultsfrom the assessment of mitochondrialbioenergeticosf hepaticmitochondriaisolatedfrom ratstreatedfor3 weeks with one of4 doses(pluscontrolo)f dietaryFC10. What isobvious from the data is the abilityto detectdramaticdifferenceasmong the 13 animals tested. However, attemptsto group animals accordingto degreeofeffectfailedto correlate with theexposuredose. Furtherinquiryrevealedthatallanimals were euthanized with ca.,50 mg/kg pentobarbitawlh,ich we subsequentlydemonstrated to have profound effectosn inhibitinpgyruvateoxidationatcomplex I ofthe mitochondrial electrontransportchain. We attributoeur inabilittyo establisah dose-response correlatiotno thiseffectof pentobarbitalS.ubsequentattempts to eliminatethis confounding effectfrom the analyseswere unsuccessful.However, we remain convinced of the importanceof collectindgatadescribinmgitochondrialdeficitisn tissuesfrom exposed animalsand inour abilittyo detectsuch effectwsith sufficient sensitivitRya.ther than relyingon the isolatioonf high integrityi,ntact mitochondriaformeasuring coupled bioenergetipcropertiesw,e suggestmeasuring glycolytiacnd tricarboxylmiectabolitesin frozentissuesin orderto derivea profile of intermediarymetabolism for each exposed animal. From the relative concentrationosf the individualmetabolites(e.g.t,he oxoglutarate-to-pyruvaotre succinate-to-fumarartaetios)w,e can estimaterelativedegrees of metabolic dysfunctioninexposed animals'.Ratherthan requiringone ofus to be presentonsiteat thetime of sacrificwee, can perform such analyseson tissuesthatare collected in liquidnitrogenand kept frozen for shippingto our laboratory.This is considerablymore convenientforallpartiesinvolvedand we are anxious to discuss potentialopportunitietso continueto contributeto the invivomonitoring studies. 'Colson, C. and Klapper,M.H. (1979).Sequentialmeasurements of glycolytiacnd tricarboxyliaccidcycle metabolitesin singlesample aliquots.Anal.Biochem. 97, 394399. PROBE DESCREPTIONS Theintroduction. Ile rationaolfethefollowinegxperimenwtasstodeterminaesmany "independencth"aracterisotfics energeticosf rnitochondriaas itispossiblefrom a singleprobe and havingtime limitationIs>.uetothis,the specific experimentalprocedureand conditionswere designeclThe p.%Tuvate-supportedrespiratioannd the membrane potentiawlem measured ina simpleslightlhyypotonicincubationmedia.Other compounds, suchas ADP, Ce, and succmate,were added to mitochondna atsaturatmgconcentrationasnd atchfferenttimesbut atleast2 rnm afterthe rmtochondna were introducedto thepyruvate--contauungmedium. No udu-bitorosf oxidativephosphorylauon,the respiratorcyhain,Ca@'transporlor uncouplerswere useclAlso,no attemptswere tal= toreducetheconcentration of freefhtL3a,cidsco-isolatewdithmitochondriaor generatedduringincubation. The logicoftheexperimentaldesignwas to measure in a singleprobethemost vulnerablecharacteristiocfs nutochondna,such as pynwate-supportedrespiratioannd oxidativephosphorylationa,nd the most "ftLsoWtive", easilyreproduciblpearameterssuch as succinate-supportreedspiratioannd Ce u-ansporlUnder such hmtauons, thereisno possibilityo obtainthe precisecharacteristifcosr any of the parameters.However, the combinatory analysiosf allthemeasured parametersallowstoestimte.atfnv approximationt,heenergeticosfmitochondna,and make some conclusionasbouttheinvivoconditionsi,na tissuew,here themitochondda were isolatefdrom. The interpretatioofnany in vitrodata,and especialltyhe backward deductionof in vivo conditionsin a tissuefrom the dataobtainedwithisolatedmitochondriai,salways a kind of "might be" guessing,Itisrather. temptingto thinginterms like"bad mitochondria"s,ay,ifthey can not oxidizea subsb-atbea enough,or "good raitochondriai"f,theyexertedan RCI more then20. We would preferanothermode of thinking,namely that niitochondriaare good when the energeticcsharacteristitchsey exertfitthe celluladremands. And the particular valuesformitochondriaelnergeticussuallydepend on theparticulaerxperimentalconditionsD.ue tothis,itseems to be necessaryto remmd shortlythe peadonties of mitochondnalbiochenustryunderlyingthe experimental conditionswe have chosen. The most vulnerablefunctionsof mitochondriaare Vvruvate- supportedrespiratioannd oxidative phosphorylatiorTlhe oxidationof pyruvateby livermitochonddadepends on theavaiLVaihtoyf COASH in the mitochondnalniauix,on theoverallactivitoyftheTCA cycle,and an theactivitoyfrespiratorcyhain(especialloyn, theComplex I activityI)n. additionto these,maximal ratesof pymvate oxidationusuallyrequirethe presenceof catalytiacmounts of malatein theincubationiedium (tostimulattehe completeturnoverof TCA cycle)A.lso,itis under allostericcontrolby of naturalloyccurringendogenous compounds, such as fi-efeattyacids,acetyl-CoA,and ATP inhibitintghe pynivatedehydrogenaseactivityT.he respiratiornateof pyrmte-supported nutochondna phosphorylatmgADP depends on allthe mentioned parametersplus the activitieosf oxidativephosphorylation system(PitransporteArD.P/ATP-Uanslocase,and ATP-synthase).These lattearrealsocan be negativelyregulated by freelongchainfutyacidsand long-chainacyl-CoAThe oxidationofsuocinateisnot sovulnerable'.Me sensitiviotfysuccinatedehydrogenaseand Complex III to freefattvacidsismuch lessthenthatof pmwate dehydrogenaseand Complex 1.Practicalluyn,der any "regular" experimentalconditiontsherateof succmate-supporterdespiratioinsimted onlyby uansmembrane elwuwh=cal proift gradient The only know strongendogenous inhibitorof succmate dehydrogenaseis oxaloaceticacid However, intheabsenceofmalateand thep=nce ofsufficieanmtountsofpyiwate inthemcubaton nl,,,, the accumulationof oxaloacrtatceouldnotbe (2)eVecw& '[berespmon ratethatcan be observedduringCa2+ uptakeispracticalliynsensitivteo any endogenous cc)mpounds,which may be co-isolatewdith mitochondriaP.rovidingthatphosphateconcenMUon ismfftcientt,he respiratiornateduringCa2+ transporctan be consideredas themaxmw possiblerateof o)adatlonof a respiratory substratbey the respiratorcyhain-h islimitedrnawy by the acuvityof a Ca2+ transportinsgystem which is kineticallfyastert,hen any otherMisport system in mitochondriai,ncludingthe transportof the majorityof respiratorsyubmwm, and much fastetrhenADP wansporl However, when a NAD - dependentsubstratiesused,the respiratiomnay become inhibiteddue tof@g opemng of a Ca2+ -inducedporeintheinnernutochonddt membrane, and subsequentreleaseand cbluuonof nutochondnalNAD. For succinate-suppottreedspiratiotnh,e same event would providethe fastesmtgnrationratebecausethe suc=te dehydrogenaseisa FAD-dq=dcnt mzyme, and poreopeningcompletel)dischargtehe==embrane protongradient (2)duetoldnetilcimitatitoonTCA cyclearisinfgrom overreductioonfNAD and bg removalofoxaloacetyaccid stimulatetdi)p,vnrrate-cl-rivaecdetyl-CoA- MATERL4LS AND METHODS Theisolatioofnmitocbondria. Nfitochondriwaere isolatefdrom liverpieces(-1.59 wet weight)Of adult male and e femal ratsby a conventionadlifferenticaelntrifugatipornocedure.Liverpieceswere cooledin 20 mi Of isolatiomnedium (210 mM mannitol,10 mM sucrose,5mM HEPES-KOH (pH 7.4),1 mM EGTA). Cooled tissuewas minced with scissorasnd washed once with20 n-dofisolatiomnedium, thendilutedwiththe same medium and homogenized for-45 secwith a motor-&ivenPotterhomogenizer(Teflonpestleg,lassbeaker)and placedon ice.The tissueto medium ratiowas appro),dniate1l:y20(g:ml)T.his procedurewas appliedsequeraiauyto8 liverspecimens.The time intervabletween homogenizingof thefirstand thelastliverpiecesdid not exceed() 9 niin.The homogenates were simultaneously centrifugefdor 10 min x 700 g,I'= 40 C, and mitochondriaplelletwsere recoveredby cenu&ption at 10,000g x 10 min. The pelletswere resuspendedin 2 ml of washing medium (210 mM rnannitol1,0 mM sucrose,5mM HEPES-KOK pH 7.4).The suspensionsof mitochondriawere dilutedto 35 n-dwith the same medimn, and centrifugeadt 10,000g x 10 mim The finalmitochondrialpelletwsere resuspendedin washing medium toa protein concentratioonf80-120 mg x ml" and stomd on ice.The isolatiopnroceduretookexactlyI hour. Ms isolatiopnrocedurewas repeatedforthesecondsetof 8 liverspecimens.ne processingof thefim set (samplesI - 8)was completedat- 10:35AM and theprocessingofthe second set(samples9 - 16) was startedat -12:35 AM and completedwithinI hour.The measurementswere starteadt-2:00 PK Measumments. Mitochond?lalmembrane potentia(lAT) was estimatedfrom i'l@Fionsdisuftum measuredwith a TPP"- selectivelectrodeN.fitochondriamlembrane potentiawlas calculateads describedelsewhere. The rate of oxygen consumphon by mitochondriawas measured with a hand-made Clark -qM oxygen electrode. Both the mitochondnalmembrane potentiaalnd the respiratiornatewere recordedsimultaneousluysing a multichannelincubatiocnhamber equippedwitha magneticstu=. 'Me volume of the chamber was 1.9ml. AU experimentswere performedat room temperature(220 C). The T?F" -sensitivceiwuode was calibratebdy sequentiaaldditionosf known amounts of T?P'-Clbeforethe additionof nutochondna (seeFig,1).The respu-abon rateswere calculateadssuming theuutwl oxygen concentratiotnobe equalto240 JIM. The incubatiomnedium containin2g10 mM mannitol,10 mM sucrose,5mM BEPES-KOK pH 7.4,4 mM KH2PO4, and 10 mM pyravatewas used inallexperunents.The same volumes of mitochondrialsuspension(30 were addeclregardlessoftheproteinconcenuations. Proteinconcentrationwsere determinedaftertheexperimentby the Bradfordassay.Bovine serum albumin was used asa standarcl Additions.Where mentionecltheadditionswere as following:5 mM succinate2,00 @LM ADP, and 264 CaC12- (1)- thismodification ofthe conventional isoisfion procedure ciln be expecled to decrease the quality ofmkoc honeiria. Accordingto our exp-icnce, 10 min of storing ofhomogarlized tissue on ioe can produce approximxwy 20 - 300/o decrease in RCI, depending an the perfadness of ODOIIO& libemsults Table I shows therelatioonfsample labelingused inthefollowingtexttothelabelsOf liverspecimens- Table 1.The sample labels: 3 4 C90753 90@719@ C912-30 C909@35 91@024@--C--9--1-3--5-0@190@890@ m m f m m f m 8fcgl -FC91116655 f 11 C91205 f 12 C91430 f 13 14 15 C90814 C91135 Thesamplewseremeasureidnthefollowitnigmesequenc3e-:4- 5 - 6- 8- 12- 13- 11- 14- 2 -1-7-15. An averaguem spenftoreachprobewasabout12nuriw,ithapproximat5elmym intervbaeltweepnrobesA.R measurements were completedvntlun4 hours.Table 2 shows thevaluesoftherespiratiornateobtainedinthe presenceofvariousadditions. Table 2.The respiratiopnarametersmeasured. For theincubationmedium and otherconditionss,eeMaterials& Methods. For thesequenceof additions, seeProbe Descriptionschapter.Data areexpressedinmaol O@ ImWmg ofprotein.Vpyr,therateofpyruvate- supportedrespiratioVnd;p, therateofrespiratiopnroducedby ADP addition(St.3);Vm therateof respiration duringCa2+ Wtake; V. ,suminam- supportedrespiratioinnthepresenceofpymvate and in theabsenceof other Ca2+ transporte(dnmol)to theamount ofoxygen consum-eAa additions(unlesstatedotherwise)C;a/0,theratioof (nmol);ADP/0, theratiofADP phosphorylate(dmnol)totheamountofoxygenconmmed (iunol). Vpyr VADP VCR it 2 1 3 4 5 6 7 8t 11 12 3.7 3.1 2 6.8 5.5 4 5.7 0 4.6 2.1 0 P5.7 30.5 :505 @'102 r5 1 0 -15- --Fg- 1-9 44.5 94.3 -@3-2- -N5- -76- 4- 96 36 8 89 vsucc 10.4 14.7 13.6 33.4 *119 29.8 15.9 24 *69 10.6 -Ea/0- 7.6 7.3 4.3- N-D 'p72 ND AD] ib-r- I N. N.R- 3.6 N. PI ND 3.1 ND - notdetemiined N.R- - notresolved(seeProbeDescriptionchapter) p pvmmte -supportedrespiration * therespiratiornatewas measured in thepresenceof theexcessof Ca2+, which pore.so itshouldbe consideredas thenuxtmal rateof succinateoxidation. 13 141 1.8 2.7 . --r77- -727P14.2 15 1 2.7 34.1 46.9 *87 16.7 12.3 i45 4.7 0.7 1 inducedthe opening of Probe maintainthemembrane The sequenceof additionswas ADP, succinateC,a2+, Ca2+. Nfitochondriwaere ableto potentialof 144 mV by pvrtivaw oxidatioilHowever-,j@-DPdi@ddnnososltttiiu@ltma@mthuetelhateemrVe@sipriAriaoin,o@n;thoughinducedthe AT decreaseto 130 mV. The subsequentadditionof succinatestimulatetdherespiratioannd increasedtheAT to 170 mV. ne increaseinres*won and intheAT was biphasicm.cbcatingincompletephosphorylatioonfADP tothe moment oftheadditionofsuccinateO.nly steady-statvealuesforAT and respiratioanreshown. RCI for phosphorylatiownas about1.2.Subsequentadditionof Ca2+ induced-4 foldtransiensttimulatioonfrespiratioannd decreaseinAT to 149 mV. These mitochondriaconsumed 360 nmol Ca2+/mg of proteint,heporewas not observed. Interpretationt:hepymvate transporstystemor pyruvateoxidationisinhibitedt-h,eactivitoyfrespiratory chainwith succinatdeuringCa2+ uptakeis-2 foldlessthenusual(ortheCa2+ umisporting*stem isinhibitedt)h;e resistancoeftheseniitochondritaoporeopeningisintheuppertopofnormal range(oreven slightlhyigher)fora well- coupledratlivermitochondria. Probe 2. The sequence of additionswas as in Probe I(IT]rle@@iseponses@on AD@P, succi@nataen@d Ca2+@wereas@in Prob@e1,too@ 'Me AT value forpyrttvateoxidationwas 138 mV, the ADP -induced AT dwmw was to 127 mV, the succinatesupportedAT was 166 mV, and Ca2+ inducedthedecreaseinAT to 127 mV. InterpretationsT:he same as forProbe I forexceptthatapparentCa2+ capacity(theresistancteopore opening,determinedby the characteristic,d&eTcrease)was lessthenforProbe I (between200 and 360 mnol Ca2+/mg of protein). Probe 3. The sequenceof additionswas succaime,ADP, Ca2+. These nutochondna were unableto mamtm the &T with pyrlivateT.he AT with succinatweas alsolow, 144 mV. The additionof ADP inducedthe decreasein &T to 137 mV and irreversibslteimulatetdherespiratio(nrable2).The additionof Ca2+ fiutherstimulatetdherespiratiobnu,t inthiscasethestinudatiownas transien(tnormalresponse)M.r porewas opened by 164 mnol Ca2+/mg of protein. InterpretationT&h:ese mitochondria)uncoupledb,)thepymvate oxidationisinhibiteadtthe levelof wxisporl pyravatedehydrogenase,or Complex 1.Ile restof respiratorcyhain and/or succinatedehydrogenase activitayre inthenormalrange.The sensitivittoyCa2+ istoohigh.Itmay be due to accumulationofa poreinducingcompound likefreelong-cbaifnattyacids.The decreasein Ca/0 Crable1) isprobablydue to uncoupling and/orincompletephosphorylatioonf ADP tothemoment of Ca2+ addition. Probe 4. The seqwnceofaddifionwsasasinNk 3.ThesemitochondrwiearenotabletomaintaitnheAT by pyruvate oxidauon.The ADP in-eversibsllyigbtlsytimulatetdhe succmate-supportedrespiratiotrhleadditionof Ca2+ was withoutfurthedrfect.'Mc resistancteoporeopeningwas higherthen 135 mnol Ca2+/mg ofprotein. InterpretatioNnfsi:tochondwreiraeuncoupleadnd inhibitweidthbothsubsntesH.owever,pvnivam oxidatiownas lessmlubitedthenintheprobesdescribeadbove(comparetheVpyr valuesinTable1).The amount of Ca2+ requirefdorporeopeningbearno infomiatioinnthiscase(theproteinconcenwationw.hich was measured aftetrheexperimentw,as toohigh,1.97 mg/ml,sotheaddedamountofCa2+ was ftmfficient). Probe5. The sequenceof additionswas ADP. Ca2+. Ca2+, and succinateT.hese mitocbondriawere ableto maintainAW of 143 mV by pyruvateoxidationT.he additionof ADP transientlsytimulatetdherespiratioannd decreasedthe AF to 115 mV, the RCI was 2.7.The measurementof the AW revealedan unusualpatterna,fterthe completionof ADP phosphorylatiotnheAT increasedto thelugherlevel,thanthatbeforetheADP addition(a hyperpolarisationne). responseofrespiratioonn theCa2+ additionwas unusuallyslowerthentheresponseon ADP (seeTable 2).The pore was opened by 243 nmol Ca2+/ mg ofprotein.Succinatc-mpportedrespiratiownas recordedafterthepore opening, so itreflecttshenmxirnw activitoyf succinatedehydrogenase+ therestof respiratorcyhain,and itisin thenormal range. InterpretationsT:hese mitochondriawere coupleclthe respiratiownith both substratewsas not significantilnyhibitecHlowever, theresponseon Ca2+ was unusua itmight indicatseome alteratioin the Ca2+ uwisportingsystem.Actuallyt,he"goo-S'pyruvateoxidationmay alsoreflecttheavailabiliotfyfreeCOASH in the nutochondnalmatrix,thatcan resultfsrom theinhibitioonf fattyamds P-o)adafion. Probe 6. The sequenceof additionswas ADP, succinateC,a2+. These mitochondnamaintainedthe AIY of 122 mV by pynivateoxidationA.DP slightlsytimulatedther-espiratiTohrel responseon thesuccinateC,a2+ additionswere like theseinProbe 3,forexceptthat155 nmol Ca2+/mg ofproteindidnot open thepore (normalresponse). InterpretationsT:hese mitochondriawere uncoupled,and the pyrtivatcoxidationinhibitecTlhe data provideno uiformationon the sensitivittoyporeby the same reasonsas inProbe4 (msufficienCta2+ loading). Probe 7. The sequence of additionswas ADP, succinateC,a2+, Ca2+, Ca2+. The responseswere essentiallays in Probes 1,2. The AIP forpyruvateoxidauon was 143 mV, and thatforsuccinate-supportreedspiratiownas 167 mV. Ca2+ opened theporeintherange306 -465 nmol/mg ofprotein(seeProbe 1). InterpretationsS:ee Probe 1,Probe2. Probe S. The ordy succmatewas added becausethesenutochondna appearedto be unable to nmultam the AT both with pynivateand with succinateS.uccmate-supporterdespuuuon was partiallmyinbited Itshouldbe notedalso,thatthe yieldof mitochondriafrom thisliverspecimen was the lowes@ the initiacloncentratioonf mitochondriain suspensionwas 75 mg/ml and thatinprobewas 1.25mgtml Interpretationsf:or a regularratlivermitochondria,1.25 mg/ml concentrationin probe is more then sufficiteonotbservaellthemitochondrfiuanlctiowc@@@@@@@@@@ Probe 11. The sequenceofadditionswas ADP. Ca2+, and n=cwate. These nutochondnawere ableto nwntam the &W of 136 mV by p)Tmte o@ddanorlThe additionof ADP slightlbyut reversiblsetimulatetdherespiratiobnu,t thatof Ca2+ m,as,%ithouetffectH.owever, theadditionof Ca2+ inducedcompleted=ergv-atloz4ad the mbsequent succ=tc additiondid notincreasetheA'P whereasstunulatetdherespiration. InterpretationsT:he absenceofthestimulatioonf pvmvate oxidatiobny theCa2+ additionisprobablydue to theopeningofpore (seeburoduction).ThseensitivitoyCa2+ isunusuallyhigh,lessthen 150 mnol Ca2+/ mg of proteirals inProbe3.