Document 2NRYnJMDENyY16vEqMKNnJ2v6

KendalBl.WallaceP,h.D.,DABT Januar2y7,2000 Effectof acuteFC administratioonn catalaseand acylCoA oxidaseexpression The following report summarizes the effects of in vivo administration of a single acute dose of N-ETFOSE or PFOS on catalaseand acylCoA oxidase gene expression and enzyme activityin livertissuefrom exposed ratsand guinea pigs. Tissues analyzed in thisstudy were derived from the 3M in-housestudy "NTBK 116471, pg 100" (acopy of the 3M "Study/Record" isattached) and included approximately I a of frozen liverfrom each of two male and two female ratsand guinea pigs thatreceived a singlein.iectioonf 40 mg/kg PFOS, 160 mg/kg N-Et-FOSE, or an equivalentvolume of 2% tween 80 (vehiclecontrol)on March 1,1999. All animals were killed on March 4, threedays followingdosing. Portionsof the respectiveliverswere quick frozen in liquidnitrogenand shipped on dry iceto Duluth foranalysis. Enzyme Sample Preparation - The enzyme fractionconsistedof the 6,000 g supernatant of a 10% (wt/vol)homogenate of 0.5-1.0g frozen livertissuein 300 mM mannitol-10 MM HEPES-1 mN4 EGTA (pH 7.2). Proteinconcentrationwas estimatedaccording,to the method of Bradford usinCg)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,P04-0.02 % TritonX 100 (pH 7.4)containing1 mM p-hydroxyphenylacetate(PHPA), 4 units/n-pderoxidase,20 pM FAD, and 60 pM lauryl-CoA (LCoA). The reactionswere allowed to incubateat 37'C for30 min in a shaking water bath and terminatedby adding 3 volumes of 2 mM KCN in 100 mM sodium carbonate(pH 10.5).The concentrationof H,O, Cenerateddurina the reactionwas estimatedfrom the fluorescenceof PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescencewas calibratedwith commercial H,O, and the resultsare expressed as nmol peroxide generated/min/mg mitochondrialprotein(Table 1).Proteinwas quantitatedby the Bradford method. CatalaseAssay - The activitoyf catalasewas estimatedby a modificationof the orioinalmethod published by Claiborne and Fridovich (J.Biol.Chem. 254, 4245-52, 1979),which isbased on the directmeasurement of H,O, disappearance as quantifiedspectrophotometricallyat 240 nm. In thisprocedure,the tissuesample was dilutedin 50 mM potassium phosphate (pH 7.0). The medium was warmed to 27'C and thereactioninitiatebdy adding 10.3 MM H20,. The progress of the reactionwas monitored at 240 nm for 5 min. Catalaseactivitywas estimated from the initiallinearrate( E240=43.6mM-'cm") and expressedas units/mgprotein(Table 1). One unitof activityis defined as thatamount of enzyme which catalyzesthe decomposition of I'Pmole of H,O, per min. KendallB.Wallace,Ph.D.,DABT January27,2000 NorthernBlotAnalyses- QuantitatiofnMRNA forbothacylCoAoxidase(ACOAO) and catalasweereperformedby Northerbnlotanalysiosfquickfrozenlivesramplesfromtreaterdats and guinea piIgs. Approximately 1 g of frozenliverwas powderized in liquidnitrogenusing a mortar/pestleT.otalRNA was recoveredusing the PERFECT RNA" isolationkitand the concentrationquantifiedspectrophotometricallayt260nm. The RNA was electrophoresedon a 1% agarose gel,blottransferretdo a cellulosemembrane and hybridizedto the corresponding randomly [12pl labeledoli-onucleotidetshatwere PCR amplifiedfrom primers to ca.,350 base sequence of the respectiverat livergene. MRNA band density was quantified autoradiographicalluysingphospho-imaging software. RESULTS: Rats - Both male and female ratsexpress LCOAO and catalaseactivitiesa,nd there is no substantialdifferencein enzyme activitiebsetween the sexes. The same is true for the constitutiveexpressionof MRNA forboth enzymes, with the exceptionthatunexposed male rats do not expressthe message forPCOAO. Acute exposure toN-ET-FOSE causesa doublingof the specificactivitoyf LCOAO in liverfrom both male and female rats,but catalaseactivityis unchanged. Associatedwith thisis a proportionat2e-foldincreaseinthe concentratioonf MRNA encoding forPCOAO, but no change in catalaseMRNA, in liversfrom both sexes. Again, thereisno remarkable sex differencein responsetoN-ET-FOSE exposure,at-leaswtith thislimitednumber ofanimalstested(n=2). Exposure of ratsto PFOS elicitas similardoublingof LCOAO activitfyor both sexes,and possiblya slightincreaseincatalaseactivitiyn liverfrom male rats.Hepatic catalaseactivitiyn female ratsdoes not appeartobe affectedby PFOS exposure. Unlike N-Et-FOSE, which caused a doubling of MRNA band intensitfyorpCoAO, acuteexposure to PFOS caused a 3-6 fold increasein PCOAO MRNA expression. This enhanced expressionof message was more pronounced in female compared to male rats,and in both sexes resultedin no greaterLCOAO enzyme activittyhan was observedathalfthe MRNA levelforratsexposed toN-Et-FOSE. Guinea Pigs - The response to guinea pigs to exposure to N-ET-FOSE and PFOS was dramaticallydifferentfrom thatobserved for rats. The constitutivaectivityof LCOAO in unexposed guinea pigs of eithersex was very low (nearLOD values)whereas catalaseactivity was 1.5-3timeshigher.However, theactivitoyf neitherenzyme was stimulatedfollowingacute exposuretoN-ET-FOSE or PFOS invivo.Perhaps themost dramaticdifferencbeetween species is the factthatguinea pig MRNA encoding for PCOAO was undetectable,even following exposure to N-ET-FOSE or PFOS. Partof the explanationforthisobservationmay be thatwe were usinghybridizatiopnrobesdevelopedagainstthesequence forratliverACOAO because the sequence of the guinea pig enzyme isnot known at thistime. Itmay be thatthe guinea pig sequence is sufficientldyifferenthatthe PCR product lackscomplementarityfor adequate hybridizatiotno the probe forthe ratsequence. Although thismay indeed be an artifactt,he 2 KendalBl.WallaceP,h.D.,DABT Januar2y7,2000 absenceofconstitutiveelxypressedmessage and theinabilittyostimulateexpressionofPCOAO MRNA areconsistenwtiththelow and uninducibleenzyme activitiynguineapigs. CONCLUSIONS: These data provide strong evidence that: 1) N-ET-FOSE and PFOS stimulateboth thetranscriptionaanld translationeaxlpressionof acylCoA oxidaseinratsinvivo, and 2) thereisa marked differenceintheresponseof ratsand guineapigsto invivo exposure to these two fluorochemicals. These resultsare very consistentwith the suggestion that these fluorochemical compounds are "peroxisome proliferatorsi"n ratsand, much likewhat has been demonstrated for the classical"peroxisome proliferatorc"hemicals,guinea pigs are resistantto thiseffectof fluorochemicalexposures. We were not given the data for organ weights or necropsy findingsand thus cannot comment on whether exposure to eitheragent caused hepatomegaly or otherindicationsthatare associatedwith peroxisome proliferation.However, based on our data,we suspect thatthe evidence willrevealthe classicalsigns of peroxisome proliferationin rats,but not guinea pigs,caused by these acute exposures. 3 NTBK 116471 An@mal SPECES 9ROO463 rat 9R00464 ral 9ROO469 rat 9800470 rat 9ROO465 9R00466 9R00471 9ROO472 -rat Irat 1 I 'at rat 9R00467 rat 9R00468 rat 9R00473 rat 9ROO474 rat dosed 3/1/99 sac 314/99 5o ix m Veh m Veh 40 mg/kg PFOS 160 mg/kg N-ET-FOSE 2% tween 80 protebn 151.8 145.6 Activity LCOAO (nmol/min/mq protein) 4.7 5.3 calaim (Units/mQ protein) 219.9 158.5 F Vsh F Veh 188.3 4.7 153.3 6.8 141.1 204.7 m N-ET-POSE m N-El-FOSE- 157.5 134.9- 10.5 11.9 9.5-1 204.3 F N-ET-FOSE F N@ET-FOSE 165.8 215.8 10.3 9.3 186.1 171.9 m PFCS m PFUS 115.4 153.0- io.9 13.1 252.0 236.9 F PFOS F PFCS 143.1 236.6 9.6 11.2 204.1 155.6 MRNA gCoAQ MBNA (% Band Intensitvi- 0 0 caiaum (% Band Intensitv) 127 73 100 92 100 108 290 140 1 292 98 1 263 118 320 147 482 165 277 116 630 ISO - 404 147 - 9GO0045 Gpig m 9GO0046 cviq m 9GO0051 Gpi-Q F 9GO0052 Gp@ F 9GO0047 Gpic, m 9GO0048 Gpiq m 9GO0053 Goia F 9GO0054 Gpiq F 9GO0049 Gpi-a m 9000050 Gpiu m 9GO0055 Goig F 9GOOO 6 Gpla F Veh Veh Veh Veh N-ET-FOSE N-ET-FOSE N-Et-FC)SE N-ET-FOSE PFOS PFCS PFOS PFOS 175.2 251.7 180.3 222.1 212.7 269.1 176.4 218.3 153.2 222.1 127.8 171.6 2.3_ 1.9 2.6 1.2 3.0 1.4 2.6 1.4 ND 1.0 t 2 314.7 390.7 550.6 488.0 349.3 379.3 416.8 478.1 413.0 460.8 672.9 531 A 0 103 0 97 0 91 0 109 0 ? 0 92 0 105 0 104 - 0 ? 0 97 - 0 103 0 96 NorthernHybridizatioonf Rat LiverTissue Male Female zz mr m zz mm 6< -n -@n -0 -u 7m, 7m, -0 -0 00 mm 7 7 0 0 cn (n 0 0 c. c. cn cn m 0 m m w0 0 m M M Cl) CD CD (D OD Cl) m CL CL 0 rr (D 290 292 482 277 CO CO 253 320 630 404 CD BaridInterrA(y% ofAvailablCeortrols) 0 0 cr CD (D :3 CD cn a) > cu (D :3 a. c m N c a) MethyleneBlue StainedBlot PFOS PFOS, A N-ET-FOSE E (D U- N-Et-FOSE, Vehicle Vehicle.. PFOS N-ET-FOSE Vehicle Vehicle N-ET-FOSE Vehicle z RadiographofP-32 Labeled p