Document NeBDV58Ey39oKqEVNoK010Enp

PerfluorosulfonamiMdeetabolisminRatvs..Hum-an--Heya R C H CH S 0 -2N -%. C H 2 CH2- FC]OR,H FC8,07R,H FX]2 ND R Rgll O-Glu H C H2C H3 C@F,,- S 0 @N 0,H, CH2CH 3 0 2 , H 2,,@@@ I(FC-807) R.,H xii(Fc-95)dt,, stocksolution R-S 02N'oe "fi-fi-O-H 22 ][I(FC_10) FCIOR,H _WCH2%CC H3 FC807ND (method ?) FX]2 ND Xl][i LY andxlfound in stock solution 0- s FCIOR C H 2C H 3 R - S 0 @N % cH2 c= 0 H IV P-CIOND FC807RND FX]2 ND IX in Oh R-S 021@@H -fi-fi-O-H 4 2 2 FC]ORH VI FC807R,H R I,1'-iFX12R,H c R S 0 i-l< 'H DC(FX-12) FC]OR?,H? FU8107RH,? foundxlinstock CH2 c H 3 R S 0 2 -N c -C H2 0 0- V FC]OR,H FC807 R,H FX]2 ND IH R-SO:N."' CHO 2 VII FC]ORH(trace) FC807RH(trace) FX]2 ND H R - S 0,N-' c-coo_ RH ' FC]OR,H VIII FC807RH R-S 0 r-N H2 R XI FCIO R?,H?(Ohincu R, 1-1. FC807PH FX]2 R?,H? r@.@,111 @ R-S03- FC]OR?(,OHhincubate) FC807P,1-1(9) XII(FC- FX]2 R7,H? -12 RH i. FC-807 11. FC-10 IX. FX- 12 xii FC-95 R H Blue: Cri-en: Rat metabolites Htunan metabolites Detectedamong metaboliteosf FC-807 (1) Detectediiiiiqsiinie,t-ibii[i4tkefs"FeC'-(I1t1f) ? compound found in stockor 0 hourincubate. Updated2/SM basedon 1/27/9r8eiportfroon ABS using a more sensitive metkod (SRM than previous dmft report& T-6295.23;ST-46 Protocol. 05/04100 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study Nos. T-6295.23; ST-46 E-xRloratotkIn-VitroPercutaneous Absort)tionStudy of TheoRhylUne, Salicvh Acid, orooctylsulfonatea,nd Annnonium Perfluorooctanoatein SkinEdiic ReconstitutedUidernds Mode Study Objective: This study isdesignedto gainfamiliaritwyith the SkinEthicReconstitutedEpidermis Model by conductinga simpleabsorptionstudyon two previouslycharacterized compounds (theophyllinaend salicyliacidina paperby Doucet,et.al.)a,s wellas one fluorochemicaclompound (perfluorooetylsulfonKa@t)e.,Supportinganalyticawlork for thisstudywillbe done inthenew CorporateToxicologyLaboratoryinBuilding236 as partof method and instrumentqualificatioAn.MTT assaywillbe conductedattheend oftheabsorptiosntudytodetermineifSkinEthicviabilitwyas affectedby applicatioonf the testcompounds. Research CUent: 3M SpecialtyChemicals Division 3M Center,Building236 SaintPauL MN 55144 Sponsor. Study Location: 3M SpecialtyChemicalsDivision 3M Center,Building236 SaintPauL MN 55144 3M StrategiTcoxicologyLaboratory 3M Center,Building270-3S-06room SB314 SaintPaul,MN 55144 Study Director.- John Butenhoff,Ph.D. SeniorLaboratoryManager 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPauL MN 55144 Ph.:651-733-1962FAX: 651-733-1773 Study Toxicologist.-Kathy Thompson, M.P.H. SeniorToxicologist 3M MedicalDept./CorporateToxicology 3M Center,Building220-2E-02 SaintPauL MN 55144 PIL:651-733-9578FAX: 651-733-1773 Proposed Study Timetine StartDate: Wednesd@ay,April12th, 2000 End Date: Friday,April14th,2000 AnalyticalCompletion Date: TBD FinalReport Completion Date: TBD T-6295.23;ST-46 Protocol. 05/04/00 Regulatory Compliance. This study will be performed in the 3M Strategic Toxicology Laboratory under a defmed protocoland classifieads a "ClassC Study" as explained inTOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. Test Materkd: The fluorochemicaltestmaterialwillbe provided by Dan Hakes, Product Responsibility Liaison,3M Chemicals Division. The drug testmaterialwillbe suppliedby Sigma. Identification: Names: PerfluorooctylsulfonatWe, (PFOS, K@ salt) TbeophyRine (THE) SahcylicAcid (SA) Molecular Formulas: C8Fl7sO3-K@ (PFOS) C7-H8-N4-02 (THE) C7-H6-03 (SA) Lot Number: Documentatiownillbekepton filbeytheSponsorfor PFOS (Lot 217) and by the CorporateToxicology AnalyticalLaboratory forTHE and SAPurity: Documentation willbe kept on fileby the Sponsor forPFOS, and by the Corporate Toxicology AnalyticalLaboratory forTBE and SA. Stability: Documentation willbe kept on filewith theSponsor forPFOS, and by the Corporate Toxicology AnalyticalLaboratory forTHE and SA. Storage Conditions: Upon receipt,testmaterialwillbe storedtightlysealedatroom temperature. Characteristics: Information on synthesismethods, composition or othercharacteristics thatdefme thetestmaterialwillbe kept on filewith the Sponsor for PFOS, and by theCorporate Toxicology AnalyticalLaboratory forTHE and SA. SkinEthic Preparation Procedure: The SkinEthic ReconstitutedEpidermis wW be prepared immediately upon arrival as per'ihemanufacturersinstruction(sseeAttachment One). Absorption testing willbegin on April 13,2000. All handlingof SkinEthicmaterialwillbe done using aseptictechniques. 2 T-6295.2S3T;-46PfOtOCOI. 05/04/00 Materials: o Twelve SkinEthicHuman ReconstituteEdpidermis,Size0.63 cm@, and twelveblank, filters o SkinEthicMaintenanceMedium (suppliebdy manufacturer) o Additionaelmpty 12 wellcultureplates(BectonDickinson) o HeatingBlock(37'C) Li Incubator (37'C, 5%CO2, saturated humidity) Reagents: ReceptorMedium -PhosphateBufferedSaline(GibcoBRL Cat #14200-075,Lot 1027781),1.5% Bovine Serum Albumin (Sigma,A- 8022 Lot#39HO69 1)added for FC compound Compound applicatiovnehicle- Milli-QWater 1% Soap Solution(Tween 20,Mallinckrodtl,ot#H285 N13H32) Trypsin(Sigma,lot#39HO952, cat#T-7418) MI-F(Sigmal,ot#39H5076c,at#M-2128) Procedure. c3 Unpack and maintainSkinethicReconstituteEdpidermis(REp) skincellculturesand blankfiltewrellsaccordingtomanufacturer'isnstructioinnscontrolletdemp and humidityincubator. Beforeproductapplicatioenx,pose REp and blankfiltewrellsto standardroom environmentalconditionsfor2 hourstoequilibratweithambientatmosphere. Place0.6ml receptormedium ineach wellof 12-wellcultureplates. TransferREp and blankfiltergsentlywithforcepstoeachwelland placeon heating block. :I Evenlyapply200 gl ofTHE and SA compound solutiontsoREp and blankfilter surfaceintriplicat2e0,0 @Llof PFOS compound solutioninquadruplicatea,nd 200 gl of blankapplicatiovnehicle(water)totheremainderand startimer: 1. THE appliedas saturatewdatersolutionof6 mg/ml 2. SA appliedassaturatweadtersolutiofn2 mg/ml 3. PFOS appliedasa solutioonf0.025mg/ml (50gM) TransferREp and blankfiltegrentlywithforcepstonew wen containinrgeceptor medium after0.25hr,0.5hr,I hr,2 hr.4 hr,and 6 hr. At end of experiment,placethereceptormedium ofeach platewellintoa polypropylenevialforlateranalysis. 3 T-6295.23;ST-46 Protocol. 05/04/00 WashtheREpandblanfkiltseurrfa3cteimewsith200gLof1% soapsolutiaonnd, rinse3 timeswithsame volume of water.Gentlywipe cleanedsurfacewith a cotton tip.Save washingfluidsand tipsina polypropylenevialforlateranalysis. Conduct MTr Reductionassayon one sample foreach testcompound and two samplesfornegativecontrol(waterapplicatioonnly)to determineSE viability. Cut remainingskinmembranes and blank filterisntosmallpieceswithscissorasnd digestfor48 hourswithTrypsinat40'C. Afterhomogenizationand centrifugation (1000g) for10 min,filtesrupernatantthrougha 0.45gm filtearnd saveina polypropylenevialforlateranalysis. Number of analyticaslamples generated:total=182: Surface Washing Fluids: A- PFOS: 8att=6hrs B. THE: 6att=6hrs C. SA-- 6 att=6hrs D. Water: 4 att=6 hrs ReceptorMedium: E. PFOS: 8 each att = 0.25 hrs,0.5hrs,1.0hrs,2.0hrs,4.0hrs,6.0hrs F. THE: 6 each att = 0.25 hrs,0.5hrs,1.0hrs,2.0hrs,4.0brs,6.0hrs G. SA: 6 each att = 0.25 hrs,0.5hrs,1.0hrs,2.0hrs,4.0hrs,6.0hrs H. Water: 4 each att = 0.25 hrs,0.5hrs,1.0hrs,2.0hrs,4.0hrs,6.0hrs DigestFilteredSupernatant: I. PFOS: 6att=6hrs J. THE: 4 att= 6 hrs IC SA: 4 att= 6 hrs Ship sampleson dryiceto: Dave Ehresman, BS ToxicologySpecialist 3M MedicalDept./CorporateToxicology 3M Center,Building236-Cl46 SainPtAULMN 55144 Ph: 651-736-8410 foranalysiosf thetestcompound and thepresumed metabolite(s3-methylxanthine1,,3,7trimethyluriacid,and 1,3-dimethyluriacidforTHE, gentisiaccidforSA@,none for PFOS). 4 T-6295.23;ST-46 PrOtOcOl. 05/04100 Data Analysis: Analytical results will be used to derive maximal flux rate in Rg/hour/cm2. The flux rate obtainedforTHE and SA willbe compared to thatreportedin thepaper by Doucet, et.aL Mass balance recovery shouldbe 90 10%. MTT resultswillbe used to determine if skinviabilitwyas affected(ascompared to negativecontrol)by applicationof thetest compounds. ResponsibiUties: Kathy Thompson willbe responsiblfeorperformingtheexperimentand sending specimens for analysis. Dave Ehresman will be responsible for analyticalevaluation of the samples. Kathy Thompson will draft a fmal report and ensure the report receives appropriate 3M review before a finalreport is issued, to be signed by Kathy Thompson and Dave Ehresman. References: Doucet, 0., N. Garcia and L. Zastrow. Skin Culture Model: a Possible Alternative to the Use of Excised Human Skin for Assessing In Vitro Percutaneous Absorption. Toxicology in Vitro 12 (1998) 423-430. 5 T-6295.23;ST-46 ProtocOl. Signatures: John Butenhoff, Ph.D. SeniorLaboratory Manager Study Director Kathy Thompson, M.P.H. SeniorToxicologist Study Toxicologist 05/04/00 Date Date Sponsor Representative Date 6 DT15 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY - ST-43S:tandarPdrocedurfeorLiveSrubcelulFarractionat-ion StudyObjeFfive: Perfluorochemciocmaploundssuchasperfluoroocstualnfeona(tPeFOS)have beenlinkedwithreductioninserum cholesterollevelasnd livehras beenidentified asthe primary target site. The exact celluhtr mechanism of how perfluorochemical compounds interactwith livertissuesisunknown and itisnecessaryto look into the sub-cellulafrractions. Thepurposoefthisstudyisthereforeto (1)isolateliversub-cellulafrractionsand (2)to quantitatethe PFOS contentsineach fractionT.he correlationbetween subcellularPFOS contentsand serum biochemicalmarkers willbe furtherinvestigated. This protocolispartof an ongoing study inthe 3M StrategicToxicology Laboratory designed to understand the effectsof perfluorooctanesulfonamidesand theirmetabolites,includingPFOS, on intracellulfaarttyacidtransportand metabolism,mitochondrialfunction,and cholesterolsynthesis.This protocol provides a means of combining our resources and focus analyticaclapabffitieosn the same setof tissues. Study Location: 3M StrategicToxicology Laboratory 3M Center,Building270-SB-314 SaintPauL MN 55144-1000 Sponsor: 3M SpecialtyChemicals 3M Center,Building236 SaintPaul MN 55144-1000 Study Director. Andrew M. Seacat Ph.D. 3M Medical Dept. Corporate Toxicology 3M Center Building220-2E-02 SaintPauL NIN 55144-1000 Ph.:651-575-3161,FAX: 651-733-1773 Study Toxicoloot: Sue Chang, M.S. Advanced Research Toxicologist M Medical Dept. / Corporate Toxicology 3M Center Building220-2E-02 SaintPaul,MN 55144-1000 Ph.:651-736-2212, FAX: 651-733-1773 Methods & Materials: DT15 Regulatory Compliance: This is an exploratory in nature and thus classifiedas non-GLP as explained in TOX SOP 0950, StrategicToxicology Lab GLP Program Procedure. Test Mitterial: The sp6nsor has provided samples of PFOS to theinvestigatorsA.nalytical documentation of the startingmaterialwillbe theresponsibilitoyf thesponsor. A chemicalcomposition specificatiosnheet willbe kept on file. Anii-hals: Species: Rat Strain: Sprague Dawley Source: Harlan Laboratories,Inc. Age at initiationof treatment: 10-12 weeks old Weight at initiationof treatment: approximately250-300g Animal Handling Cares and Specimen Collection: Pleasesee Study No. DT- 15 Specimen Handling /Processing: For perfluorosulfonamidemetaboliteanalysisl,iversamples willbe fractionized according to the procedures describedbelow. The isolatedsub-ceuularfractions willbe packed in dry iceand shipped to Dr. Kris Hansen at3M Environmental Lab. Kris Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue St.PauL MN 55133-3331 Ph.:651-778-6081, Fax: 651-778-6176. Procedures: Buffer needed: 0.25M sucrose in0.05M Tris-HCL pH 7.4 Equipment needed: Tissue grinder Centrifuge Polypropylenecentrifugetubes (1.5ml and 50 ml) Animal: Rat liversfrom both controland PFOS-treated groups. DT15 (NOTE - All procedures should be carried out at 0 - 40C 1 Suspend and homogenize theliverin0.25 M Sucrose in 0.05 M Tris-HCI buffer (pH 7.4)using20% weight/volume ratio,or approximately5 volumes of the liver weights; The homogenization should be carriedout on ice ataU time to prevent the enzymes and tissueproteinsfrom degradation. Homogenize on ice atlow speed using a 50 ml Tenebrock tissuegrinderwith the hollow handle of thepestlefillewdith crushed ice.Homogenize at least10 strokes,or untilthe suspensionisvisiblyhomogeneous. Record the totalvolume and the corresponding weights of the homogenates. Obtain two 1.5ml microcentrifugetubes and labelthem "I". Aliquot 1 ml of the homogenate intoeach tube. Store the tubes frozen forfurtheranalysis. Transferthe remaining of the homogenates intotwo 50-ml centrifugetubes. Note: For thisstepand stepsbelow following the cenuifugation, make sure to .measure and record the volumes and weights for each supernatant fraction. 2. Spin the whole homogenate at 700 x g for 10 minutes at4C. Supernatants: Decant supernatantfluidintonew centrifugetubes on ice.Weigh and recordthetotalvolume. Make two I ml aliquotof the supernatantsintotwo 1.5ml microcentrifugetubes labeled"2". Store frozen. PeRet: The resultingpelletcontainsdebrisand unbroken ceM. Add 1 ml of bufferper 1 gram of originalsample, re-suspend the pelletin 0.25 M Sucrose in 0.05 M Tris-HC1 buffer(pH 7.4)and labelitas "Plasma membrane". Make two I ml aliquotof the suspension intotwo 1.5ml microcentrifugetubes labeled"3". Store frozen. 3. Spin the supernatantsat 2500 x g for 10 minutes at 4C. Supernatants: Decant supernatantfluidintonew centrifugetubes on ice.Weigh and recordthe totalvolume. Make two 1 ml aliquotof the supernatantsintotwo 1.5ml microcentrifugetubeslabeled"4". Store frozen. PeUet: The resultingpelletcontainsnucleiand unbroken cells.Add I ml of bufferper I gram of originalsample,re-suspendthe pelletin 0.25 M Sucrose in0.05 M Tris-HCI buffer(pH 7.4).Label itas DT15 44nuclei".Make two 1 ml aliquotof the suspension intotwo 1.5ml microcentrifugetubes labeled"5". Store frozen. 4. Centrifugethe supernatantfluidobtainedfrom step3 at 10,000 x g for 15 minutes at4C. Supernatant: Decant supernatantfluidintoa new centirfugetube.Weigh and record thetotalvolume. Make two 1 ml aliquotof the supernatants intotwo 1.5ml microcentrifugetubes labeled"6". Store frozen. Pellet: The resultingpelletcontainsmitochondria.Add 1 ml of bufferper 1 gram of originalsample, re-suspend the pelletin0.25 M Sucrose in0.05 M Tris-HC1 buffer(pH 7.4). Label itas "nlitochondria". Make two 1 ml aliquotof the mitochondrialsuspension intotwo 1.5rnlmicrocentrifugetubes labeled"r'. Store frozen. 5. Centrifugethe supernatantfluidobtainedfrom step4 at30,000 x g for10 minutes at 4C. Supernatants:Decant supernatantfluidintonew centrifugetubes on ice.Weigh and record thetotalvolume. Make two 1 ml aliquotof the supernatantsintotwo 1.5ml microcentrifugetubes labeled"8". Store frozen. Pellet: The resultingpelletcontainslysosomes. Add I ml of bufferper 1 gram of originalsample, re-suspend the pelletin 0.25 M Sucrose in 0.05 M Tris-HCI buffer(pH 7.4) and labelitas "lysosome". Make two 1 ml aliquotof thelysosome suspensionintotwo 1.5ml mierocentrifugetubes labeled"9". Store frozen. 6. Centrifugethesupernatantfluidobtainedfrom step5 above at 105,000 x g for60 minutes at 40C. Supematants: Decant supernatantfluidintonew centrifugetubes on ice.Weigh and record the totalvolume. Label the supernatantas "cytosol". Make two 1 ml aliquotof the supernatainnttstowo1.5ml microcentrifugteubeslabeled"10". Storefrozen. Pellet: The resultinpgelletcontainsmicrosomes.Add 1 ml ofbufferper 1 gram of originaslample,re-suspendthepelleitn0.25M Sucrosein 0.05M Tris-HCIbuffer(pH 7.4)and labelitas"microsome". Make two 1 ml aliquotof themicrosomalsuspensionintotwo 1.5 ml microcentrifugteubeslabeled"11". Storefrozen. Data: 3M Study Number Date Animal #, sex,and strain Dose group Weightoflivesrample(g) Volume ofsucrose-TriHsCI added (mL) Fraction Whole homogenate Plasma membrane sup. Fraction weight before aliquot (Minus) amount savedfor LC-MS (ML) (Minus) amout saved for gelelectr. (ML) 700 x g sup Nucleisup. 2500 xg sup i SUP Mitochondrial sup. 10K x 9 S22 sup. 30K x g SUR Cytosol 105K x g sup DT15 Note ResponsibiUti,es: Andrew Seacat and Sue Chang willbe responsibleforperformingthe experiment and sendingspecimensforanalysis. DT15 Signatures: Andrew M. Seacat, Ph.D. Toxicology Specialist Study Director Sue Chang, M.S. Advanced Research Toxicologist Study Toxicologist Date Date Sponsor Representative Date 01/21/2000 Perfluorooctane Sulfonic Acid Induced HMG-COA Reductase Inhibition in PreanantRats and Rat Pp2s. Deanna J.Luebker,M.S.,AdvancedResearchToxicologist CorporateToxicology3,M MedicalDepartment 3M Center,Building220-2E-02,SaintPauL MN 55144 Phone (651)737-1373 FAX (651)736-2285 Dilu-,bk-er@mmm.com PREFACE Thisprojecitsdesignetdoservebothasa 3M researcphrojecatndasa projecttofulfill therequirementosfa Ph.D.thesiisnToxicologythroughtheUniversitoyfMinnesota Twin CitiesT.hisproposalisbeingsubmittedwiththeunderstandintghatpublicatioonf theresultwsillbe subjectoa frst-right-of-revbiye3wM. PROPOSED PROJECT AND ITS PURPOSE The objectivoefthisstudyistogainknowledgeintothemechanism ofactionby which perfluorooctasnuelfoniaccid(PFOS) inducesadverseeffectosn peri-/postnatal developmentinratsT.he hypothesitshatPFOS actsviahihibitionf3-hydroxy-3methylglutaryl-CorAeductas(eHMG-COA reductaset)h,erate-hmitinegnzyme of cholesteroslynthesisw,illbe examined. An attemptwillbe made topreventPFOS toxicitbyy co-administratimnegvalonatet,heimmediateproductofHMG-COA reductase.HMG-COA reductaseactivitwyillbecompared betweendams andtheirpups and toagematchedpositivaendnegativecontrols.Assayswillbe performedto determineifPFOS actsdirectloyn HMG-COA reductasei,ndirectvliyaactivationf AMP-activatedproteikninase(AMP-PK) orthroughinducingan increasienAMP levels. Otherparametersw,hich willbe followedwhere possiblei,ncludebody weight,liver weight,liveraf.idserumPFOS, liverand serum lipidasnd miocardiafliberinjuryand/or inflammation. INTRODUCT40N Perfluorooctasnuelfoniaccid(PFOS) istheultimatmeetabolitoefperfluoroalkayclids andtheirderivativeas,classofchemicalsproducedby 3M and broadlyutilizeadcross multiplemarkets.The largeproductionvolume and broad-basedutilizatioofnthisclass of chemicalsjustifitehseneed fora comprehensiveunderstandinogf thepotential adversehealtheffecttsheymay pose.BecausePFOS isthebreakdown productofthese chemicalsr,esearchfocusingon itseffectasnd mechanism(s)ofactionisofprimary D. J.Luebker Page 1 of 16 01/21/2000 concern.Thisproposalisaimed atgainingknowledge intothemechanism(s) by which PFOS induceseffectson peri-/postnatdaelvelopment inrats. Figure I : Chemical Structure of PFOS CF3-(CF@,@@FT o0 General effectsseenin3M PFOS reproductionstudiesinratsarereduced matemal body weight gain,increasedpercentstillborannd decreasedpup survival(Case,presentation, 1999). The primarytargetorgan identifiefdrom adultratstudiesisthe liverE.ffectsof PFOS treatmentinadultratsincludedecreasedbody weight gain,increasedliverweight, decreasedserum cholesteroltriglycerideasnd bflirubiannd mild peroxisome proliferationT.he firsdtetectablebiologicaleffectseen inadultratPFOS studiesis reduced serum cholesterolA.t high doses of PFOS inrats,toxicitryesponse curves become very steepand an apparentwastingsyndrome occurs which culminatesindeath (3M CorporateToxicology,unpublished,1999). Haughom and Spydevold (1992)investigatetdhe mechanism underlyingthehypohpemic effectof PFOS inrats.They found PFOS to down regulate3-hydroxy-3-methylglutarylCoA reductase(HMG-COA reductase)activittyo 35% ofcontrolHMG-COA reductaseis a microsomal enzyme which controlsthe rate-hmitingstepincholesteroslynthesist,he conversionofHMG-COA tomevalonate (HMG-COA + 2 NADPH + 2 W -*mevalonate + 2NADPH+ + C'oA). The amount of HMG-COA reductasepresentina celliscontrolled via negativefe@dbackregulationthroughrepressedgene transcriptio(nEdwards etal., 1983),inhibitioonf MRNA translatio(nNakanishietal.,1988)and stimulatioonf therate of degradation(Gilletal.,1985). The primarymechanism by which thisenzyme is controlledacuttlyisreversiblpehosphorylation(Gibson,1985;Beg etal.,1987;Hardie and Carling,1997)with phosphorylationinactivatintgheenzyme. Othercontrol mechanisms includereversibltehiol-disulfifdoermation(Roitelmanand Shechter, 1984a),allostefaictivatiobny NAD(P)H (Roitelmanand Shechter,1984b) and alterationisnmembrane fluidit(yMitropoulosand Venkatesan,1985). AMP-activatedproteinkinase(AMP-PK) isthemajor HMG-COA reductasekinaseinrat liver(CarlingetaL, 1989). Itiscontrolledby reversiblpehosphorylationw,ith phosphorylatiofatctivatintghekinase.AMP-activate proteinkinasekinase(AMP-PKK) istheupstream component inthishighlyconservedkinasecascade(Hardieand Carling, 1997;Hardie,Carlingand Carlson,1998). Elevationof AMP can activatAeMP-PK by arostericalalcytivatinAgW-PKK, by bindingtoAMP-PK and making ita poorer substratfeorproteinphosphatasesb,y bindingto AMP-PK and making ita better substratfeortheAMP-PKK and by allostericauayctivatinAgMP-PK (Cortonetal., 1995;Hawley etal.,1995;Davies etal.,1995).The AMP-PK cascadeistherefore activatedunder conditionswhere AMP iselevatedand ATP isdepressed.The D. J.Luebker Page2 of16 01121/2000 consequencesofAMP-PK activatioinncludeinactivationf ATP-consuming anabolic pathways (e.g.f,attyacidsynthesivsiaphosphorylatioonf acetylCoA carboxylasea)nd steroslynthesitv.iaphosphorylatioonf HMG-COA reductase(Cortonetal.,1995;Henin etaL, 1995).T6 cascadealsoactivatetsheATP-generatingcatabolipcathway offatty acidoxidationviadepressionof malonyl-CoA levels(Merrileltal.,1997;Hayashietal., 1998).AMP-PK couldthusacttobalancethesupplyof ATP tothedemand of the nucleotid(eHdrdieetal.,1999).Thismay be importantduringperiodsofcellulasrtress associatewdithATP depletions,uch as treatmentwithmetabolicpoisonsinthe hepatocyte(Corton,Gillespiaend Hardie,1994). Figure 2: Reversible Phosphorylation of HMG-COA Solidline= acfivadond,offedline= inacfivafion Reductass AMP-PK ] - - - - - - - - - - - HKG-CoAReducmw Prot@emPMhos atases InhibitioonfHMG-COA reductaseactivitiyncreasetshecelluladremand forcholesterol Thisresultisnan increaseinhepaticlow densitylipoprotei(nIDL) receptorasnd an increaseinclearanceofplasma LDL by theliver(Kovanen etal.,1981).Because LDL carriemsost of thecholesteroilnhumans, inhibitioonfHMG-COA reductaseresultisna decreaseinplasma cholesterolC.holesteroLhowever, isan importantprecursorto hormones neoded forthemaintenanceof pregnancy,parturitiolna,ctatioannd matemal behavior(Numan, 1988;Freeman, 1988;Hodgen & Itskovitz1,988)and isrequiredfor normal in utero and postnatal development m mammals (Belknap & Dietschy, 1988; Yount & McNamara, 1991). Inhibitionof HMG-COA reductasealsoleadsto a deficiencyof mevalonate and isoprenoidssuch as dohchol, isopentyladenine, ubiquinone,haem A and many proteinsbound to famesyl and geranylgeranylresidues (Soma etal.,1992; Brewer etal.,1993; Corsiniet al.,1995). These isoprenoidsare involved in membrane synthesis,DNA replicationc,ellulargrowth and metabolism and proteinglycosylation(Quesney-Huneeus etal.,1983; Suranietal.,1983; Farnsworth et al.,1987; Brewer etal.,1993). D. J.Luebker Page 3 of 16 01/21/2000 Figure 3: HMG-COA inhibitionof EMG-COA Reductage Inhibition asee Inhibitioofnnievalonatperoduction Decreased isoprenoidsynthesis increasedcellular demand forcholesterol Increased he_p@aticc LDL receptors Increa,s@e]dclcel@eraar@a@ncceeoff plasm LDL by the liver(Kovanen etal., 1981) L -------- Decreased cholesterol wistyhntlhbesis teIrnfterfee@rnecnecewith in-uteroand postnatal development (Bellmap& Dietschy,1988; Yount & McNamara, 1991) Decreased synthesisof hormones importantto maintenance of pregnancy,parturition, lactatio&n matemal behavior(Numan, 1988; Freeman, 1988; Hodgen & Itskovitz1,988) Decreased plasum concentrationof LDL cholesterol(Henck etal.,1998; Nawroeld etal.,1995) Decreased synthesisof isoprenoldsother than cholesterol(Soma et al.,1992;Brewer etal., 1993;Corsinietal., 1995) involvedin membrane synthesis, DNA replication, cellulargrowth and metabolism, protein glycosylatio(nQuesneyHuneeus etal.,1983; Suraniet a[.,1983; Farnsworthetal.,1987; Brewer etal.,1993) Statinsa, classofcompounds used to treathyperhpidemia,actas competitiveinhibitors ofHMG-COA reductase(Stryer,1995). Some statinsf,orexample lovastati(nMevinolin, MEVACOR) and simvastatin(synvinolinZ,OCOR), existas inactive"prodrugs"which must be enzymaticallyor chemicallyopened totheirrespectivecarboxylateforms to eliciitnhibitorayctivitaygainstHMG-COA reductase(Corsinietal.,1995). Other statinsf,orexample pravastati(neptastatiann)d fluvastati(nLESCOL), aredosed intheir activeo,pen ringform (Corsinietal.,1995).The activeopen-acidstructurersesemble thatofHMG-COA, thenormal substrateforthe reductase.Statinsbind tothe reductase, preventitfrom bindingto EB4G-CoA and thusblock the productionof mevalonate. D. J.Luebker Page 4 of 16 01/21/2000 Figure4: Chemical Structures- Statine& HMG-COA Exa=le Statins: Lovastatin(Mevinohn, MEVACOR) InactiveProdrug 0 0 C@@H @; H CH2 CH 3*"' C-@@H MevinolinicAcid ActiveInhibitor 0 HO 'COH i-@0- 0 0 H CH2 I,," C@ 3 Pravastatin ActiveInhibitor HO%,@CC02Na 0 0 CH 'H H CH3 CH2 HMG-COA: Normal HMG-COA reductasesubstrate CH3 HO I C-0- 0 SCOA HO The majority of statinsare known to be dev'elopmentary toxic to rats (Henck et al., 1998). Effects include increased malformations (Minsker et al.,1983); decreased postweaning survival and delayed development of reflexes and behavior (FDA, 1987); reduced fetal body weight (Wise et al.,1990a); reduced fetal and offspring body weights (Wise et al.,1990b); reduced offspring body weight and increased swim maze errors (Minsker et al.,1990); reduced offspring body weight and decrease neonatal survival (Hrab et al., 1994); and retarded skeletal development and reduced fetal body weights (FDA, 1993).Studieshave found thatco-admihistratioonf mevalonate,the immediate product of HMG-COA reductase,preventsor antagonizesvariousorgan toxicities resultingfrom HMG-COA reductaseinhibition(MacDonald etal.,1988; Kombrust etal., 1989; Minsker et al.,1983; Hrab et al.,1994). Minsker et al.(1983) found thatcoadministrationof cholesterolh,owever, had no effecton theteratogenicitoyf lovastatin (Mevinolin,MEVACOR). Some statinsarealsoknown to produce myocardialeffects. D. J.Luebker Page5 of16 01121/2000 Hrab etaL (1994) found significantcardiacmyopathy in pregnant ratsdosed with 24mg/kgldy of fluvastati(nLESCOL). AU animalsdying inthisdose group had cardiac lesionswhich were consideredtreatmentrelatedand believedtobe associatedtothe cause of death (Hrab etaL, 1994).The authorsconcluded thatinhibitioonf mevalonate and cholesteroslynthesiswas a significanftactorcontributingtothemyocardialdamage and/ordeathof thefluvastatitnreatedrats.Co-administrationofmevalonate complete blocked andforamelioratedthe mortalityc,ardiacmyopathy and otheradverseeffects seenwith fluvastati(nHrab etaL, 1994).In-vitrostudiesby GuijarroetaL (1998,1999) found thelipophilisctatinastorvastati(nLIPITOR), simvastatin(synvinolinZ,OCOR) and lovastati(nmevinolin,MEVACOR) toproduce apoptosisofratand human vascular smooth muscle cells(VSMC). Mevalonate administratiocnompletelypreventedthis apoptosis(GuijarroetaL, 1998,1999). Severalmevalonatemetaboliteswere evaluated todeterminewhich were involvedinthe statin-induceadpoptosis.Cholesterol, isopentenyladenine,ubiquinone(coenzyme QIO) and squalenefailedtopreventthe statiinnducedeffectswhilethe isoprenoidsfamesyl-pyrophosphate(FPP) and geranylgeranyl-pyrophosphat(eGGPP) fullyreversedtheeffects(GuijarToetaL, 1998, 1999).These studiessuggestthe adverseeffectsseen with HMG-COA reductase inhibitormsay be more a resultofdecreasedsynthesisof certainisoprenoidsthanof decreasecholesterolsynthesis. D. J.Luebker Page 6 of 16 01/2112000 Figure 5.-lsopronoid Biosynthetic Pathway Key. PP = pyrophosphate oe = shown topreventstatininducedVSMC apoptosisin-vitro' X = shown NOT topreventstatiinnducedVSMC apoptosisin-vitroj 2 * = shown topreventotherstatiinnducedadverseeffectsin-vivoand/orin-vitro 0 = shown NOT topreventotherstatininducedadverseeffectsin-vivoand/or in-vitro2 'Guijarroetal.,1998& 1999 2 MinSker elal.,1983; Stuanietal.,1983;Carson & Lennarz,1979:Hrab etal.,1994 MacDonald etal., 1988;Kornbrustetal.,1989,etc... + acetoacetyl-Co 3-hydroxy-3-methylgluteryl-(CHoMAG-CoAl ediictase Statins PFOS????? ------i-ve * Mevalonate I I Mevalon pp@ Diinethylall-yPlP Isopentyl- PP Isopentyaldenide(TRNA) F-x Proteins 4- Dolichols Isoprenoicacids 4- X LThiquinone (Coenzyme Q10) 6/ Geranylgeranyl pp Geranyt- PP V Farnesyl- PP x S ualene esterol Proteins Haem A lwenoic acids Steroidhormones VitaminD Bileacids Lipoproteins D. J.Luebker Page 7 of 16 01/21/2000 Smith,Learand Erickson(1995)foundno sex relatedifferenceisnHMG-COA reductasenzyme activitiynratslessthanor equalto28 daysold.However, significanthliygheractivitlyevelsarefound infemaleratsascompared to males followingpuberty(CarlsonM,itchelland Goldfarb,1978). Levin etaL (1989) investigatetdhedevelopmentalchangesinexpressionof genes involvedincholesterol biosynthesiasnd hpidtransporitnhuman and ratfetaland neonatalliversL.evelsof HMG-COA reductaseMRNA undergo largefluctuationdsuringratliverontogeny (McNamara, Quackenbush and Rodwell,1972;Leoni etal.,1984),beinghighpriorto birthd,eclinintgo low levelsduringsuckling(days1-13 postnatala)nd transiently increasinagtweaning (day 19-21 postnatal)I.n humans, however,thesemRNAs appear atan earlieprhaseoffetalife(byweek 8)and undergoonlyminimal(< 3 fold)changes inconcentratiodnuringtherestof theintrauteriansewellaspostnatallife(LevinetaL, 1989).Fetaland earlypostnatarlatsmay thusbe verysensitivteoinhibitorosfHMGCoA reductaseactivitwyhilefetaland earlypostnatahlumans may not. The studyoutlinedinthisproposalisdesignedto helpdeterminehow PFOS administratioinnpregnantratsresultisnreducedmatemal body weightgain,increased percentstillborpnups and decreasedpup survivalafterbirth.The hypothesistobe tested isthatPFOS actstoinhibiRtMG-COA reductaseactivitiynpregnantratsand/ortheir offsprinagnd thuscan behave as a developmentaltoxicantathigherdose levelsT.he resultosf thisstudy,alongwithwhat isalreadyknown abouthuman HMG-COA reductaseactivityw,illhelpdeterminethepotentiarliskPFOS holdsto be a developmentaltoxicantinhumans. Figure 6: Hypothesis IHMG- PF inhibitionofIHMG-COA Reductam in pups RESEARCH PLAN OBJECTIVES 1.DeterminewhetherhepaticHMG-COA pregnantrats. 2.DeterminewhetherhepaticHMG-COA PFOS treateddams. reductaseactivitiysinhibiteidnPFOS treated reductaseactivitiysinhibiteidnratpups from D. J.Luebker Page 8 of 16 01/21/2000 3. Investigattehemechanism by which PFOS actstoinhibiHtMG-COA reductase activity. a) DetermineifPFOS actsdirectloyn HMG-COA reductase. b) DetermineifPFOS actson AMP-PK. c) DetermineifPFOS inducesan increaseincellulaArMP levels. EXPERIMENTAL APPROACH Allresultwsillbe compared betweendams and theirpups toidentifayny relationship between dam effectand pup effectB.ody weight and totaliverweightwillbe recorded foreachdam and littears indicesof generalhealthand hepatomegany. Each group of dams and pups willbe compared toage matched positivecontrols(dosedwitha statin), negativecontrols(dosedwithvehicle)and mevalonatechallengedcontrolsO.ther parametersthatwillbe followedwhere possibleincludeliverand serum PFOS levels, liverand serum lipidlevelsand myocardialfiberinjuryand inflammationA.s deemed necessaryp,ortionsofthestudymay be sentto contractlabsforcompletion. Table 1: Dose Groups PFOS Groups Positive Control Groups Group 1 2 3 4 -5 6 Compound(s) PFOS PFOS + mevalonate PFOS vehiclecontrol statin statin+ mevalonate stativnehiclecontrol In-VitroPhas HMG-COA reductase The abilitoyfPFOS todirectliynhibiHtMG-COA reductasewillbe examined usingrat livermicrosomes. Microsomes willbe preparedfrom untreatedfemaleratswithand withoutsodium fluorid(eNaF) inallbuffersand assaymedia. NaF inhibits phosphoproteinphosphataseactivit(yKatoand Bishop,1972)and thus,inclusioonf NaF atallstagesof isolatioannd purificatiosneverelyinhibittsheconversionof inactive (phosphorylatedr)eductasteotheactive(unphosphorylatedf)orm (Nordstrom,Rodwell and Mitchelen,1977).Mcrosomes willbe dosed as listeidntableI and,followingan incubationperiod,assayedfortotaland activeHMG-COA reductaseby omissionand inclusionr,espectiveloyf,NaF. Activitywin be calculateads therate (picomoles/minutoef)formationof [3Hlmevalonatefrom [3HIHMG-COA permilligram of microsomalprotein.Lipidlevelsofthevariousdosed microsomes willbe measured usingcommerciallyavailablkeitsand/orHPLC. Cholesteroplroductionincultured hepatocytesa, biomarkerof HMG-COA reductaseactivitym,ay be used asa secondary measure of in-vitrHoMG-COA reductaseinhibition. D. J.Luebker Page 9 of 16 01/21/2000 AMP levels To determine whether PFOS induces an increaseincellularAMP in-vitroA,MP levelsof themicrosomes willbe quantifiedusingBPLC. AMP-PK To assesstheabilitoyfPFOS toactdirectltyo activatAeMP-PK in-vitroa,peptideassay developed by Davies,Carlingand Hardie (1989)which usesa syntheticpeptidesubstrate, SAMS peptide,willbe used. SAMS peptidewas developed based on the amino acid sequence (Ser79)which isphosphorylatedexclusivelbyy AMP-PK. AMP-PK specimens willbe preparedby partiallpyurifyingthecytosohc proteinfractioonf control ratliverasdescribedby CarlingetaL (1989).These specimenswillbe treatedasoutlined intable1. Followingan incubationperiod,AMP-PK activitwyillbe measured based on the incorporatioonf radioactivitfyrom [y_32 P]AT? intotheSAMS peptide(DaviesetaL, 1989). In-ViioPha&e HMG-COA Reductase Activityin Pregnant Rats and Rat Pups IsolatioonfcellulasrubfractiontsodirectleyvaluateHMG-COA reductaseactivitryesults indissociatioonf thedrug from theenzyme, which returnscatalyticalalcytive(Corsiniet al.,1995). The most favorablemethod to detectin-vivoinhibitioonfHMG-COA reductaseisby determiningthe in-vivorateof incorporatioonf precursorsf,orexample acetatei,ntocholestero(lCorsinietaL, 1995).The potencyof PFOS toinhibithe of conversion 14C-acetateinto14C-cholesteroiln-vivo,thereforew,illbe used as a biomarker to determineIHMG-COA reductaseindams. These resultsshould paralletlhe inhibitioonf HMG-COA reductaseinhibitiofnound inthe in-vitraossays.Other biomarkers which have been used successfullyincludeurinaryand plasma mevalonate levels(CorsinietaL, 1995). Pregnant ratswillbe repeatdosed duringgestationaccordingtothe dose groups listedin table1.Priortoeuthanizationd,ams wiflbe dosed with 14C-acetate.Half ofalldams will be allowed tocome to term and halfwillbe euthanizedthemorning of day 21 of gestationand pups willbe removed. IfPFOS isa ITMG-COA reductaseinhibitort,he reduced matemal weight gain,increasedstillborannd decreasedpup survivalseen in previousstudiesshould be eliminatedoramelioratedinthe mevalonate supplemented dose group. Plasma and/orurinemay be collectedand analyzed formevalonate touse as additionalbiomarkersofHMG-COA reductaseactivitiynthedams. Plasma mevalonate levelswillserveasthe biomarker todeterminethedegree ofIB4G-CoA reductase inhibitioinneach litteLri.ver(from each dam and pooled foreach litterw)illbe used for AMP-PK activitdyeterminationl,iverlipidanalysisand AMP determinationS.era willbe collectedfrom each dam and pooled foreach littefrorPFOS determinationand serum D. J.Luebker Page 10 of 16 01/21/2000 lipidanalysis.Liverand serum high densityhpoprotein(HDL), low densitylipoprotein (LDL) and totalcholesterowlillbe determinedusingcommerciallyavailablkeitsand/or HPLC. Liverand serum PFOS determinationwillbe performed by the 3M EnvirorunentaLlaboratoryFluorineAnalyticalChemistryTeam (FACT) headed by Kris Hansen, Ph.D. Myocardialtissuewillbe examined by electronmicroscopy (EM) or other appropriatmeethod forfiberinjuryand/orinflammation. Liver AMP Levels in Pregnant Rats and Pups To determinewhether PFOS inducesan increaseincellulaArMP levelsin-vivol,iver AMP levelswillbe determinedusingHPLC. AMP-PK Activityin Pregnant Rats and Pups AMP-PK specimenswillbe preparedby partiallpyurifyingthecytosolicproteinfraction as describedby Carlingetal.(1989).AMP-PK activitwyillbe measured based on the [y_32 incorporatioonfradioactivitfyrom P]AT? intotheSAMS peptide(DaviesetaL, 1989). SUMMARY The studyoutlineidnd-dsproposalisaimedatgainingknowledgeintothemechanism(s) by which PFOS leadstoreduced matemal body weight gain,increasedpercentstillborn and decreasedpup survivalinrats.The hypothesisthatPFOS actsviainhibitioonfHMGCoA reductase,therate-limitinegnzyme ofcholesteroslynthesisw,illbe examined. PFOS has previouslybeen shown to down regulateHMG-COA reductaseactivitiynrats (Haughom and Spydevold,1992).SeveralHMG-COA reductaseinhibitor(sstatinsh)ave been shown toinducedevelopmentaleffectssimilartothoseseen withPFOS (Minsker et al.,1983;FDA, 1987;Wise etal.,1990a;Wise etal.,1990b;Minsker etal.,1990; Hrab etal.,1994;FDA, 1993).Studieshave found thatco-administratioonf mevalonate,the immediate product ofHMG-COA reductase,preventsor antagonizesvariousorgan toxicitiersesultinfgrom HMG-COA reductaseinhibitioinnratsand rabbits(MacDonald etal.,1988;Kombrust etal.,1989;Minsker etal.,1983;Hrab etal.,1994).An attempt willbe made topreventPFOS toxicitbyy co-administratinmgevalonate. HMG-COA reductaseactivitwyillbe compared between dams and theirpups and to age matched positiveand negativecontrols.Specimens willbe examined todetermine ifPFOS acts directloyn HMG-COA reductase,indirectlvyia activatioonf AMP-activatedprotein kinase(AMP-PK) or throughinducingan increaseinAMP levelsO.ther parameters, which willbe followedwhere possible,includebody weight,liverweight,liverand serum PFOS, liverand serum hpids and myocardialfiberinjuryand inflammation.Due tonormal developmentaldifferencesinHMG-COA reductaseactivityf,etaland early postnatalratsmay be much more sensitivteo HMG-COA reductaseinhibitiotnhanare fetaland earlypostnatalhumans (Levinetal.,1989).The resultosf thisstudy,takenwith what isalreadyknown about HMG-COA reductaseactivityw,illhelpdeterminethe mechanism by which high doses of PFOS cause adverseeffectsinpregnant ratsand their pups and the potentialriskPFOS holdsto be a developmental toxicantinhumans. D. 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