Document p4XbDgbdrDYqDD7xENqY9boa

AR225-0171 Kendall8. WallaceJ,anPuha.rDy,2,7D,A20B0T0 Effect of acute FC administration on catalase and acylCoA oxidase expression `The following report summarizes the effects of in vivo administration of a single acute dose of N-E(FOSE or PFOS on catalase and acylCoA oxidase gene expression and enzyme activity in liver tissue from exposed rats and guinea pigs. Tissues analyzed in this study were derived from the 3M in-house study "NTBK 116471, pg 100 (a copy of the 3M "Study/Record" is attached) and included approximately 1 g of frozen liver from each of two male and two female rats and guinea pigs that received a single injection of 40 mg/kg PFOS, 160 mg/kg N-Et-FOSE, or an equivalent volumeof 2% tween 80 (vehicle control) on March 1, 1999. All animals were killed on March 4, three days following dosing. Portions of the respective livers were quick frozen in liquid nitrogen and shipped on dry ice to Duluth for analysis. Enzyme Sample Preparation ~ The enzyme fraction consisted of the 6,000 g supernatant of a 10% (wt/vol) homogenateof 0.5-1.0g frozen liver tissue in 300 mM mannitol-10 mM HEPES-1 mM EGTA (pH 7.2). Protein concentration was estimated according to the methodofBradford using commercial bovine serum albumin as standard. L-CoA Oxidase Assay ~ The equivalent of ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH,PO,-0.02 % Triton X100 (pH 7.4) containing 1 mM p-hydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 uM FAD, and 60 iM lauryl-CoA (LCoA). The reactions were allowed to incubate at 37C for 30 min in a shaking water bath and terminated by adding 3 volumes of 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration of HO, generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescence was calibrated with commercial H,0, and the results are expressed as nmol peroxide generated/min/mg `mitochondrial protein (Table 1). Protein was quantitated by the Bradford method. Catalase Assay - The activityof catalase was estimated by a modification of the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 4245-52, 1979), which is based on the direct measurement of H,0, disappearance as quantified spectrophotometrically at 240 nm. In this procedure, the tissue sample was diluted in 50 mM potassium phosphate (pH 7.0). The medium was warmed to 27C and the reaction initiated by adding 10.3 mM H,O,. The progress of the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E**'=43.6 mM cm) and expressed as units/mg protein (Table 1). One unit of activity is defined as that amount of enzyme which catalyzes the decomposition of 1 mole of H,0, per min. 04198 1 Kendall 8. WallaceJ,anPuha.ryD,27D,2A0B0T0 Northern Blot Analyses - Quantitation of mRNA for both acylCoA oxidase (ACoA) and catalase were performed by Northern blot analysis of quick frozen liver samples from treated rats and guinea pigs. Approximately 1 g of frozen liver was powderized in liquid nitrogen using a cmoorntcaern/tpreasttiloen.quTaonttailfieRdNsApecwtarsoprhoetcoomveetrreidcaulsliyngatt2h6e0nPmE.RFTEhCeTRNRANAwTMas iesloelcattrioonphkoirtesaenddonthea r1a%ndaogmalryos[e*Pg]el,fabbleoltedtroalnisgfoenrruecdletootiadecseltlhualtosweermeemPbCrRanaemplainfdiehdybfrriodmizperditmoertshetococar.r,es3p5o0ndbiansge asuetqoureandicoegraopfhictahlelyruessipnegcpthivoesphroa-timlaigvierng gseonfet.ware.mRNA band density was quantified RESULTS: Rats -- Both male and substantial difference female rats express in enzyme activities LCoAO and catalase between the sexes. activities, The same and there is is true for no the dcoonsntoittuetxipvreesesxptrheesmsieosnsaogfemfRoNr PACOfoArOb.oth enzymes, with the exception that unexposed male rats bAoctuhtemeaxlpeosaunrde tfoeNm-alEet-rFatOsS,Ebcuatusceastaaladsoeubalcitinvgoitfythise usnpcechiafnigceadc.tiviAtsysoofciLaCtoedAOwiitnhlitvheirs firsoma ipnrocpaotratliaosneatmeR2N-fAo,ld iinnclrievearsse finrotmhebcootnhcesnetxreast.ionAgoafinm,RtNheAreeniscondoinrgemfaorrkPaCbloeAOs,exbduitfnfeorcenhcaengien response to N-Et-FOSE exposure, at-least with this limited numberofanimals tested (n=2). pEoxspsoisbulryea oslfigrhattsintcorePaFseOSin eclaitcailtassea ascitmiivlitayr idnoulibvleirnfgroomf mLaClOeAraOts.actHievpiattyicfocratbaoltahsesaecxteisv,ityanidn female rats a doubling does not appear to be affected of mRNA band intensity for by PFOS pCOAO, exposure. Unlike N-Et-FOSE, which caused acute exposure to PFOS caused a 3-6 fold increase in pronounced iPnCfOeAmaOlemcRomNpAareexdprteosmsaiolne.ratTs,hiasndenihnabnoctehdseexxepsrersessiuoltnedofinmneossgargeaetewraLsCmOoArOe enzyme activity than was observed athalfthe mRNA level for rats exposed to N-Et-FOSE. Guinea Pigs dramatically - The different response to guinea from that observed pigs for to exposure to N-Et-FOSE and rats. The constitutive activity of PFOS was LCoAO in wunaesxp1.o5s-e3dtigmueisnehaigphiegrs.ofHcoiwtehveerr,setxhewaacstivveirtyyoflnoewi(tnheearr eLnOzyDmevawluaess)stwihmeurleataesdcfaotlallaosweinagctaicvuittey exposure to is the fact N-Et-FOSE that guinea or PFOS in pig mRNA vivo. Perhaps encoding for the most PCoAO dramatic difference was undetectable, between species even following `ewxeproesuusriengtohyNb-rEitdi-zFaOtiSoEn oprroPbFeOsSd.evePlaorpteodfatghaeinesxtptlhaenasteiqounenfocretfhoirsroabtsleirvveartAioCnOmAaOybbeecatuhsaet twhee sequence of the guinea pig enzyme is not known at this time. It may be that the guinea pig sequence is sufficiently different hybridization to the probe for the that the PCR rat sequence. product lacks Although this cmoamyplienmdeenetdarbietyanfoarrtiafdaectq,uatthee 04199 Kendal8l. WallaceJ,anPuha.rDy.,37D,2A0B00T a`bmsReNncAeoarfeccoonnsstiisttuetnitvewliythextphreelsoswedanmdesusnaigneduacnibdlteheeniznaybmielitaycttiovisttyiminulgautieneexappriegsss.ion of PCOAQ CONCLUSIONS: stimulate both the These data provide strong transcriptional and translational evidence that: 1) N-Et-FOSE expressionof acyCoA oxidase in and PFOS rats in vivo, tahnedse2)ttwhoerefliusoaromcahremkiecdaldsi.ffeTrehnecseeinretshueltrsesapreonvseeorfyrcaotnssiasntdengtuiwnietah ptihges stuogignesvitvioonextphoastutrheesteo dfelmuoornoscthreamtiecdalfocrotmhpeoculnasdssicaarle""ppeerrooxxiissoommeepprroolliiffeerraattoorr"s"chienmircataslsa,ndg,uimnuecahpliigkse awrhearteshiasstabnteetno ntheicsroefpfseyctfionfdifnlgusoraonchdemtihcuaslceaxnpnoosturceos.mmeWnet woenrewhneotthegirveenxptohseurdeatatofeoirthoerrgaangewnetigchatusseodr bhaespeadtoomnegoaulrydoartao,thweer isnudsipceacttiotnhsatthtahteareeviadsesnocceiawtieldlwrietvheaplertohxeicsloamsseicparlolsifiegrnastioofn.peHrooxwiesvoemre,. proliferation in rats, but not guinea pigs, caused by these acute exposures. 04200 | ew mma a oe C EE wCem -- Em EhEe r pe e eE aa a e e a f f paf ps e t ie ----!h parepa} a aFa E E mE fa E faa| hw me p a ee a e E H e f EEe r E m e a E ee e e t e bE oree ee h t eato bw a me mmfp h e --e af r e t ee e E {E Ef FaeEf m Eoof a o mhiE rem e| o mr a] e m een p EeaE b i He eee a a w fmdse -- a e --e e-- ee E er -- e RR RE | 82 S8 . 2 b ESEE 2=g 3 3| SEE ES=cS3 5#382 5| c 2 2 25 Methylene Blue Stained Blot ; . rross me gp veFCohJ arose veri } vena rrooss, "AE newose wewVoensiec"e; ence Fis Bs -- i3 1 Ee Radioggrraapplh of P-32 Lableled 4pCoAO Probe - : : 3 s3 aa3 aId 5 M31 g#ig1 . i 2 2 MethBylule Setanineed Blot ---- 2 HE RTE s [erosd; Es -=a2 g| HI F= 2sg. 2) | F Es081 5e8o 2 | gf & 3= 2 5 5 gf 5 werose 7fl IR 3 nevrerncoeis&eod 08 Boif vee SR, 3 3 eros ETT nevos Ehud : -- vance 8ine A= venco: 08 & neros;e Sule LS oennce"c' {ESER RadiogorfaPp-3h2 Labeled pCoAO Probe | ~ Be a TSETpiaYin ee ih : --: Lisfaaglia itoh sar INEogee; po BA Sa SR RT 2 = SygiileaBe ce HI J "a Ee > Tgo i th