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STUDY OFTHEEFFECT OF PERFLUOROBUTANESULFONATE,
5
PEPRERFFLLUUOORROOHOEXCATNAESNUELSFOUNLAFTOEONNASNAODTME |
ASPECTS OFCHOLESTEROLMETABOLISH INVITRO,
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NO_epor_of sFtudyiSWEOn T alverson TNCreport
16042008
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PERFSLTUUODRYOBOUFTTAHNEEESFUFLEFCOTNOAFTE,
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PERFLUOROHEXANESULFONATE AND
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PERFLUOROOCTANESULFONATE ON SOME
ASPECTS OF CHOLESTEROL METABOLISM IN VITRO.
Study/ project number: 3M#01/ 011.2517
TNO Quality of Life TNO Pharma 2Th00e1N8eCotEhxeL2aa1nic5sn, PFraoxne"1133117711551188 11940540
ACHHoocCrgoeveando Nobel
a3s,siMgednimceanltdeofpariment `JSoyhmoSnuittoern.nof.Ph.0.CI, DAST `HCaodrcpoarlatoepSacrieinmteisntt S3U0PCaeurliMeirnSneusiontag506212404--0150-0W0.02 UPhsoane: +1.651733 1982 FEomcail jTouotenrhoifi@cmnm com SOrtLyoduseciorC:oren SFitnaals,and te1:8 April 2005 PrBBoaaonrsuzs versioTZneocmaazcmnoszos Numbeorfpages: 34
Ne HAuetahdoorfitzhaetDibeoypnaDrrt.mHenMt.GvPrrincen, and Metabolic Diseases _
Signatureand date:
Abie oo seg
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TNO porofsudy SWAOT
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Finaiveren
Contents
Contents.
Testing facity
Contributors 1. Agreement and protocol 2. Objective ofthe study 3. M3e1t.hodsCompounds
32 Assays 32.1. Cholesterol synthesis in primary rat hepatocytes 322. Bile acid synthesis in primary rat hepatocytes 32.3. Ble acid uptake with hamster ileum pieces
33 Approval Animal Experiment Committee TNO
4 R4.e1s.utsCholesterol synthesis inprimary at hepatocytes
4423.. Blilee aacicd usynttheesiistihnapritmacry reatmhepiatcoecystes
5. Conclusions
5.1. Cholesterol synthesis in primary rat hepatocytes 52. Bile acid synthesis in primary rat hepatocytes. 53. Bile acid uptake with hamster leum pieces.
6. References.
Appendix 1, Cholesterol synthesis in primary at hepatocytes 1.1 Appendix: primary data experimen1t
11.32., AApoppeonndtiix:: pprriimmaarryy ddaattaa ecxppeerriimneentn3t2
1.4 Appendix: compost data
THe
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[NO-steudypWooTriof Fmalvewson
Appendi2x, Bile acid synthesis in primary at hepatocytes 21 Appendix pimary data experimen1t 22. Appendix: primary data experimen2t 23. Appendix: primary data experiment 3 24 Appendixcomposite data
Appendix 3, Bile acd uptake with hamster leu pieces
33.21. AAppppeennddiixx:: pprriimmaarryyddaattaa eexxppeerriimmeenntt 21
33. Appendix: primary data experiment 3
34. Appendix composite data
Testing facilty TNO Qualty of Lie Business Unit Biomedical Research DepartofmVaescnultar and Metabolic Diseases
Gaubius Laboratory
P.0.Box 2215 2301 CE Leiden The Netherlands phone: +31715181479
fax +3171 5181304
Contributors Studydirector
Or. LH. Cohen Phone: +3171 5181469 E-mail: LH Cohen@pg no nl
Experimentalwork:
Mr. W. van Duyvenvoorde Mrs. E. Hoegee-de Nobel Mrs. E. Piterman Mrs. C. van Thiel
TY
tv
16022008
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fTNO-repor of study #01
Finalverson
16042005
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1. AGREEMENT AND PROTOCOL
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Thhase breeesenaernchteargerdeoenme1n6t-0f1-r20t0h3e.prTehseenptropjreocjtechtabsebteweeennpeTrNfOormaendd a3cMc,orMdeidnigcatlo tDheepaprrottmoecnotl of 208-2004, which has been attached to the research agreement as Annex |
2. OBJECTIVE OF THE STUDY
3anMimhaalssotubdsieesr.vedIt tihsatthceertpauirnpopseeruoofrotahlekyplrseisfeonntatsetsudhyavteo gcahionlesitnesrioglhtloiwnteoritnhgeefpfoescstisblien mheepcahlaicricshmoleosftearcotlions.ynCthheosliess,terionlcrelaosweeriinnghecpaanticbebicleauasceidd isnynstehveesriasl owrayisn:hibiitnihoinbioifonthoef intestinal uptake of cholesterol or bile acids wil all lead to reduction of circulating cholesterol
Ipnritmhaeryprceusleunrtesotfudryatthheepaetffoeccyttsesofatnhde ocnombpioeuancdidsuopntackheoliensthearmosltaenrdebuilme apciiedcessynitnhevsiirs iins
investigated
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I
3. METHODS
31 Compounds
pT7e0hr3ef8l)tu,horrpeooeotcattseassntieuscmuofmpfpeonorafutuneodrs(o,PhFepOxoaStn;aessMsuTilDufImoDn-pae2tr0ef8l(;uPoFFrHoCSb-;uSt0aM0nT0eD-sI0uD2lf-5o62n-0a67t);e Lh(-aP9vF0eB5S1b;)eeManTnDdsIuDpp-opt2la0ise6sd;ibuLym-
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3hMe,pMroejdeicctalwaDsepoanrt1m-e1n2t-,20S0i4n.cDeutrhiengdetlhievepreyroffotrhmaencceomopfotuhendpsrojweacst adnedlacyoerdr,estphoendsitnargtlyo,f
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icnomtphoeunapdp2e0n8d,ic2e0s7 aonfdh2i0s8, rreepsopretcttivheelsye compounds have been designated as test
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`Tehxeperteismtenctofmrpesohulnydmsadweerseolduitisosnoslvoefdthien tDesMtScOomaptouandcosncheanvterabteieonn uofse1d00 mM. In each
32 Assays 3He2p4a.tocytesChwoleeestiesroollatseydnftfhoemsifsreisnhlpyriombatraiynreadtrhaetpfaitveorcaytnedsbrought into culture, After about 20 the cells were refreshed and incubaled with 3 diferent concentrations (up to `c1ao0cn0ettruaoMtl)e awonfadsthreperf3eesrteeennscttefcoaormH3pMhoGuf-nordCsolaAibnerldieundpgulcioctfaatsneeewfiolnryh3is.by5onrth.h(eAssfiitmzeveradsatca0ht.oil5neh)s-tpwerraeosiln,icunAbcsaotripaoonrp,aots[ei'td"iCv}ie-n tlTihhpeiedsefexrpaecextripioemner,nitmm.aeinnt[ls*y`wCcelorlnaetbaerileneipdnegaltiseptidedrstohlwr,eeeraentaielmxyetszreawdcittfehodr,sienpscaaoprroapntoierflaiyetidesdoalrnaadtdeidotahhceteipvnaiottnoy-cs[ya1t,p2eo]snifiable
3He2p2atocytesBiwleeraeciidsoslyanttedhefsriosminfrpershilmyaroyblraaitnsherpaattloicveyrteasnd brought ino culture, Afer `teasbtoutco28mhpotuhnedcsellisnwdeurpelirceaftreesfhoerd 2anh.d iCnoclulbeactteeddwictehlls3 dainffderemnetdcioancweentrreatainoanslyozfetdheon3 STyhnetsheeseixzpeedribmieenatcsiwdserbey rge.Lpce.at[3e].d three times with separately isolated hepatocytes.
he
40134
TGeportof study SWAGT ~F~ alvemen1602008
T3h2e3uptake Bmielaesaucriedmuepnttaskewewriethpehrafmosrtmeerdialcecuomrdpiinegcetso. a modification of the procedure
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atasurdoecshcorliabteed(TbCyA)Kurwaitha fertesahll.y (o4)btaIitneisd bhaasmesdleorn itlheeum sppeiceicfeisc. aTsshoeciaatsisooncalofioInH]is
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cloarbreelcetdeTdCAfo.r nTohnrsepeecciofniccenutprtaatkieonsbyofasosnoecitaetsitoncoimnptohuenpdreinsetnrcipelicoaftean(theexcinetsesstionfalnboinl-e
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aEcxipderiumpteanktes-wgreardeiernetpehataesd ttohesbetimteask.en into account) per one fleum were ested
3Th3e AnimalAEpxppreorviamlenAtniCmoamlmEixtpeeeri(mDeEnCt)CoofmTmiNtOtehsasTNapOproved the use of animals for thisstudyon 19-11-2004 under number DEC77.
4 Resurs
4T1h.e threeChtoelstesctoemrpoolusnydnsthheasvies bienepnritmeastreydriantthherpeeationcdyetpeesndent performed experiments,
using three diferent isolationsofrat ver microsomes. The primary data oblained in these
i
experiments are added to this report in Appendix 1.
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Trehsepecrievseulltys. oTfhethceomtphroeseitiencduerpveensdoefnttheexrpeereimeenxtpseriamreenstshoawren giinvefnignurfeig4u.r1e.14.1t.o4.4.1.3,
f{
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test compounds 3M on cholesterol synthesis
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Fig. 41.1. mEeafnvfoafleuPeFcsBSt,raPnFgHeSaraengdivPeFnOS on cholesterol synthesis in experimen1t;
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15.4.2 EffofeePcFBetS, PFHS an--d--PFOS on cholesterol synthesisnsxperiment 2 J--
test compounds 3M on cholesterol synthesis
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Fig. 413. mEeafnfvoafelPuFceEst,anPgEeHSreangdePnEOS on cholesterol synthesis in experiment3
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test compounds 3M on cholesterol synthesis --
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Fig. 41.4. SCyonmtphoessiitsefrcoumrvtehsoefetihnedeepfefnecdtenotflPyFpSe,rfPorFmHeSd adnetderPmFiOnaStioonnsc.holesterol
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The values aethemeanofth indidual experimental valu&SeEMs.
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The reference inhibitor of cholesterol synthesis, simvastatin, inhibited in al three
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lexepaerriimnheinbtistioansoefxcpheoclteesdterwoilthsaynnthIeCs-ivsaaluethe< c1o0ncneMn.irTathieontsesutpco(0mp1o0u0ndusM sahnodweudndneor
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rteesstulctoenddiintivoanlsueussefdo.r tIhnecuibnactoiropnorawtiihontohfe[d"iCflf-earecnettactoencientnotrsatteiroolnsswohficchomwpeoruensdimiPlFarOtSo
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othbelaiconnetdrowlitvhalcuoemspo(suenldassP1F0B0S%ainndtPhFeHfSi.gurTehs)e. eRxepmlaanraktaibolneiswenorteetahseyhtioghgeirvevasliunecse
tbheeaenffienccrseeaesmesofnocthotloebsetedroelpesnydntehnetsiosn, tbhuetccoanncenbteraatnioenfoffectthoenctohmeposuunbdstsr.atIet mpaooylnaost
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wheell,cosimnpcoeu[nCd-saicnetthaeteincisubuasteiodnfomretdhiiusmmeianscurreeamseedntt.heItincfoluulxdofbeabtehla,t otfh,eapermeasetnvcleyo,f
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`raenddulcoreddetghreadgaelnieornatoifonenodfoegnednooguesnoorusexaocegteynlo-uCsoA;fattthyeacliadtst.er Acdadnitboenatlheexapveariilambeinlttsy
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Would have to be performed to give more insight in this matter.
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42 Bile acid synthesis in primaryrat hepatocytes
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T`ehxepertihmreentes,teusstingcothmrpeoeudnidffserehntaviesolabteieonns otfersatedverinmitchrroeseomiensd.epTehnedpernitmarpyedrfaotrameidn
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tThheesereesxupletsriomfentthse otbhtraeieneidndaerpeeandddenetd teoxpteirsirmeepnotrst ianrAeppsehnodwinxi2n. figure 4.2.1 10 423,
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respectively.Thecomposite curvesof the three experiments are given in igure 4.2.4
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aIncitdhesytnhirheeeseisxpweirhimaeinttseteherceomipsoaucnodnsc.entTrhaitsiiosnmdoesptenprdoennoturnecnedd wtiotwhatrhdes hhiigghheesrtbile
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mscouansjctoearnitbnreladetibaoycniadofisnhcimfotomuipnsoteuh)enidcsoPrmepFloaOstSiivte(il1yo0nmo0ofurtMeh).einTicnhrdeeiavcisdheudaanlcgobemilpinaatrchiededs:boilfe-cahmcouilrdaitcseyhnoatlnhtdeesoi(-sinies
cmuhraincgheoslaitnea(ctsieveitAyopfpreantdeixli2)m.inTgheenczayumseeso,fwthhiechinacrreeamseemibnebrisl oafcitdhesycnytthoecsihsrommaeyP-be
4ch5o0lefsatmeirloyl. easstewreilflicaasticonhainsgheisbiitneds.ubstrate availabiiy.E.0. the later is the case when
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4In3oneBilieleaucmidthurpeteakceonwcietnhtrhaatimosntserofilaenuem cpoiemcpeosund each with thee different ileum
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spiideece1s0wtehreecteecstuemd.siSdien,ctehtehceoumpbtiankaetoifonauofrotcrihpollicaaltee linucrmeapseesclsineaarrelycfhroosmetnheinjesjuucnhuam
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iwnatyhtesheaetxhpeearvimeernatgse acronetarodlduepdttaokethiiss mreoproerot inlAepspsetnhdeisxa3m,e. The primary data obtained
rTehsepecrteisvuelltys.Tofhethceomtphoreseitiendceuprevnedseonfttehexptehreimeeenxtpserairmeensthsowarne ignivfeingunrefi4g3u.r1e 40.3.44.3.3,
test compounds 3M on TCA uptake
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Fig. 434. TChhaoemmspvtoaeslruieteseuacrmuertvpheieesmcoeefsatfnhoreofemfthfteehcrtteheoefisPnddFieBfpSee,rnodPneEneHtxSlpyearpniadmrePfnoFtrsOm+SedSoEndeMTteCrAmiunpattaikoenswith
eUfnfdecetr otnhetehxepesrpiecmiefnitcalascsooncdiiattiioonns oufsetda,urtohcehotleastte cwoimthpohuanmdsstesrhofwleunmo poirecheas.rdlyPFaBnSy vsahroiawteidonainmtihniosrsdyesctreemaseat the highest concentration tested, but this may be due o the
5. Conclusions
T5h1e tCeshtolceostmoprooulnsdysntshheoswiesdinnporicmlaerary concentrations and conditions used.
rianthihbietpiaotnoocfyttehse
cholesterol
synthesis
at
the
wIansthoebsperrevseedn.cAeddoiftPioFnBaSl eaxnpderPiFmHenSt,sawohuilghderhaivnecotropobreatpieonrfoofrm["eCdl-foacgeitvaetemoirteo sitnesrioglhst in this matter.
tT5hh2reereec_ioBsimalpeocauocnnicddessn.tyrnTaththiiesosniisdsmeiopnsetnpdrpiermnoatnrotyruenrnacdtetdhoewwpiaahrtdotschyhetihegihsgehrebsitlecoancciednstyrnatthieosniosfwPiFthOSt,he(100 ti1hMn)de.icviyAdttuoatlchhibsrilocemoneacciPedn-st4.r5aC0thifaoanmnigtleyh,esraeiniawsceatldildviiattsyioocnfhalarlanytgeaelssimmiiantlislnugbceshtnarznaygtmeeeasivn,atiwhlheaibcicolhtmypaormseaimytiebomenbotefhrtesheof baciaclucesoaemcpiodafnstiyhenetdihnebscyirsieantscheriesianlsbeeidalfdeatscocailddebsicylrneetahacesisedidse.xcchIroneltceirsoetnae.rsoeSldilnbecivleeelcashcoildesstyenrtohlesiisthseosfutbesntrate for pInerofrodrermteod ipnrowvheicthhibsimeeacchiadnsiysnmtohefsaicstiaonnd.falnoiwmaclan(eb.eg.mmoonuistoer)edstvuidaiebsiecacnanbuelation and
fecal bile acids (mass and composition) can be analyzed.
a
120f34
NOreporofstuoy WOT
Fmalvewon
Changes in liver cytochrome P450 enzymes can be analyzed as well
15062005
5Th3e teBsitlecaocmipdouupntdaskeswhiotwh hiaomsortehrairldelyumanpyieecfefsect on the uptake of taurocholate in hamster ileum pieces under the experimental conditions used
6. Rererences
[1] tChoehcehnolLeHs,tevraonlVsiyinetthAe,siRsoiondhenubmuarnghLe.pJaatnosmeancLeMllC,ineGrHifefpioGen2M.lesPsratvhaasntastiimnvaisnthaitbiitned acnodenlzovyamsteatAin,rewdhuictcahsise arenfdlescqtueadlienntehesyunptrheagsuel.atBiionoocfh3e-mhyPdhraorxmya-c3o-lm1e9th9y3l;gi45u:ta2ry2!032208,
[2] VefafnecVtsieotfA3K-,hyvdaronxTyh-i3e-lmeGtChFy,lgHluutiasrmyakn-cRoHe,nzMymoeshaargeeduHc,taYsaepiSnhHi,biCtoorhseonnLsHt.eroDlifferent skyinnthesis in various humancelltypes. Biochimica et Biophysica Acta 1995; 1254; 105-
[3] PmroinnocleanyeHrM,culMteuirjeesr oPf, rHaotfhsetpeaetoB.cyDteexsabmyetinhdauscotinoen roefgcuhloalteesstebriolle a7ciadlpshyan-thhyedsriosxyilnase.
|
Biochem J 1989; 262: 341-348,
[4] KKounroaKt.Ha,,aSnudzuKkoih.aSr.m,a,OTb.h,atAa.nYo.v,elIkceldaas.Tso.,faHpaisceaglaswoad,iTu.r,-Kdietpaeynadmean,tK.biIenaabcai.dT. transporter inhibitors: the amphiphilic 4-0x0-1-phenyi-1 4-dinydroquinoline derivatives,
|| |
Bioorg. Med Chem.Lett. 14 (2004) 1183-1186.
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17]
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E CO . - T a --
!
APPENDIX 1 GHOLESTEROL SYNTHESIS IN PRIMARY RAT HEPATOCYTES
Appendix1.1
experimen1t
ncaompsen
EcCpm] pom
mTte | ml aste . ri 7i ws
eainon isisToor asaee |--ssaer oT]
retonersdt [a iozeesToasse | saammos--||--sso-- | ---- ]
controlethanol
[2|201 314|acoa7|mm
er
tccensttcocaompnpoocusnnd2o0a0as06tnti1oo0oo0s4mM[||3sSa34eim88ziass|||| ooo0zs.ssm1o9
|||| 1rtr7eri0oo4sp0es1s||||
mm 148103
|
|
owames
[3 fer)
sa [wr]
|
ertcpommp0asiaotw[[rsa stea||woorme ||m rteesnuess||vmios || i msewt|| wmswsTwaeao]
| |
ceetocarrmp22po00a 77otaomu[a soames||ozozer |itusesone| |E woes | mee d | se {wr| |
pmonceeammpnn2eo0rascaoty P[ aeesa|o loses o || ttoopuz sr|| e is
Fa lws| sareeasss|
|
o emtceomme 2tp0taos[n zr| 0os8m asonrs|m r s] re
el i nT
eaSvraectro:nt [[P oosoto{T oameo T||aaeso ||w sar ||r 5|tre]] |
Ssmaantoeao:tnw [Ee77n0|r om | wwe0|Ai R wmwe |ress mss ar]
|
Bp,
|
140134
|
|
)
|
ATpGpernedpoirxt 1o.f2Saag SHOT experiment 2
Fave
im0sans
|
'
compo
C dom | protienmign|mgprotein | permgprotein | %ofcontro | %ofcontrol | range|
et 50 Eee
control DMSO
Lam am
320090 i
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Appendix 3.4 Composite data experiments 1-3
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18-04-2008
test compound
% of control.
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