Document DD75n1kXDv2xBp1vjkgkmEYRd

At-,a titI;n;:'_-, 65 14q,Llscvi4s:cricncePuhii%hcrBs.%'.All i BBALIP -;.OIl The mechanism underlyingthe hypolipemic effect of perfluorooctanoiaccid(PFOA), perfluorooctanesulphonic acid(PFO.SA) and clofibriaccid Bente Haughom and OysteinSpydevold Iristiruorfei4edicalBiochemistrv,Universi@o%f- Oslo,Oslo (Nor.*v@i-) (Received 12 June 1992) Key words: Lipidmetabolism;TriacylglyceroCih;olesterolH;epatocyte;(Rat liver) 'ne influenceof the peroxisomal prolif.-ratopresrfluorooctanoicacid (PFOA), perfluorooctanesulphonic acid (PFOSA) and cloribriaccidon lipidmetabolism in ratswas studied.Dietary treatment of male Wistar ratswith these three comt)ounds resulted in ra id and pronotinced reduction in both cholesteroland triacylitlyceroilnsicru@m. @e concentrationOf livertriacylglycerols was increased by about 300% by PFOSA. Free cholesterolwas increased by both periluoro-compounds. Cholesterylesterwas reduced to 50% by PFOSA as well by clorib;at-Icn.-hepatocytes from fed rats.allthe compounds resultedin reduced cholesterol svnthesisfrom acetate.pyruvate and hydroxymethyrgl-uiarateb,ut there was no reduction of synthesisfrom mevalonic acid.The qxidationof paimitate was alsoincreased in allgroup .The perfluorocompounds, but not clofibrateq,aused some reduction in fatrvacid svnthesis.The activitonf, liverHMG-COA rcductasewas reduced to 50% or lessin alltreatment groups and allthree compounds led 7o lower activityof acyl-CoA: ch lesterola-cyltransfe:ras(eA--C-A-MC.hanges in other enzymes related to lipid metabolism were in@c-onsistenTtn.e present data suggest that the hypoliiie-mieffectof these compounds may, at leastpartly,be mediated viaa common mechanism; impaired production of lipoproteinparticlesdue to reduced synthesisand esterificatioonf cholesteroltogether with enhanced oxidationof fattyacidsin the liver. Introduction Many hvpolipemic drugs cause proliferationof peroxisomes and i!Lcreasethe actinityof the peroxisomal ,B-oxidationin rats (1-3].Chemir-ally,these drugs constitutea heterogeneous group including clofibrate,tibric acid,niadenate and long-chain acvlthioaceticacids, tiadenol,long-chain thia acids and MEDICA 16 (1-61. It has been suggested that the increase in the fatty acyl-CoA oxidizing system contributes to the hypolipemic effect of these drugs (7,81.The dominating mechanism underlying reduction of serum triacylglycerolsand cholesterol by these drugs is,however, uncertain. Other mechanisms \;,-hicmhay be important for the hypolipemic effect include; reduced hepatic synthesisof fattyacids and cholesterol(9-121; reduced Correspondence to:0. Spydevold.Institutoef.MedicalBiochemistry. P.O. Box II12.Blindern,N-031 " Oslo. ',orway. Abbreviations:PFOA. perfluoroociani,iac@d:PFOSA. perfluoromlane sulphonicacid-.ACAT. ac-vl-CoA:chu;ctterolac-yltransfcrase; HMG. hydrox'ymethylgluturicacic!. tc*.;i-decylthioucaectidc; VLDL very-low-dcnsitlyipoprotcins. triacylglycerolrelease by the liver [13,14];increased rate of VLDL degradation [6]; increased uptake or reduced release of fattyacids by adipose tissue [15,16] and increased excretion of cholesterol into bile and feces [17]. Ikeda et al.[18,19]observed that perfluorooctanoic acid (PFOA) and perfluorooctanc sulphonic acid (PFOSA) efficientlyinduced the peroxisomal B-oxidation in ratsthat were fed these compounds (0.02% in the diet).Just et al.[20]reported that the perfluorocarboxylicacids alterhepatic lipidmetabolism and reduce serum lipidlevelsshowing that these compounds also belong to the group of peroxisomal proliferatorswith hypolipemic effect.The perfluorinated compounds are particularlyinterestingsince they obviously are not subject to ordinary metabolic modifications. The effects of these compounds must, therefore, be due to effectsof the compounds per se. Peroxisomal inducers may bring about the hypolipemic effect by affectingdifferent steps in lipid metabolism. However. itseems likelythat,some important steps in lipid metabolism are common targetsfor these compounds. The unphysiological character of the perfluoro compounds makes itpossible that theireffect on lipidmetabolism followsa patternthatwould make it easierto understand possiblemechanism for the lipidreductioneffectof peroxisomalprolifcrators. In the search for a possiblecommon mechanism underiving the hypolipemic effectof pcroxisomal inducers, we have compared hepatic fattyacid metabolism,cholesterolsynthesisand the activitieosf enzymes relatedto these metabolic processdsin the liverof rats fed perfluorooctanesulphonic acid(PFOSA), perfluorooctanoicacid (PFOA) and clofibrate. Methods and Materials Ani?nals Male Wistar ratswere used. They were divided into fivegroups. One group was allowed normal food ad libitum(controlTats).Three other groups were given food which contained 0.3% clofibriaccid,0.02% perfluorooctanoicacid or 0.02% perfluorooctane sulphonic acid,respectivelyn.e dietswere prepared by soaking standard food (in pelletform) in diethylether in which the compounds had been dissolved.The fifth group was restrictedin food intaketo that consumed by the PFOSA group. The stock diet was used for these paired feeding experiments.The average body weight when the experiments startedwas 269 g and the average dailyfood consumption per ratin the different groups were; control:23.7 g, clofibrate2:3.1 g, PFOA: 22.7g,PFOSA: 20.0g. Materials (2-1"C]Mevalonic acid, [i-IAC]pyruvic acid and (2- 14 C)pyruvicacid were from New England Nuclear. [1_14 C]aceticacid was obtained from Amersham (UK). Clofibratewas from Fluka (Buchs, Switzerland).Perfluorooctanoicacid was purchased from AldrichChemic (Steinheim, Germany) and Perfluoro-octane sulphonic acid was from Fluorochem (Old Glossop, UK). Other chemicals were from Sigma (St. Louis, MO, USA). Preparatioorfi hepatocytes The effectsof dietarytreatmentwere studied in hepatocytesisolatedfrom ratsfed the dictsfor I week and for the study of directeffectsof the compounds. hepatocyteswere isola,,efdrom rats fed the standard diet.Isolationof hepatocyteswas performed by perfusion with collagenase,according to Berry and Friend [21],with the modificationsdescribed by Seglen [221. Oxidationof palmitateand conl-ersionof labelledsub. strateintolipids Fatty acid oxidationwas measured according to Christiansenet al.[23)with 0.5 mM palmitateas substrate.FaM acids and cholesterols@,nthesizedfrom radioactiveprecursorswere extracted from the cell suspension after90 min incubation.The reactionwas stopped by the additionof 5% saturatedKOH in ethanol and the mixture was heated at 90*C for I h. The nonsaponifiablelipids were extracted with petroleum ether.7le suspension was then acidified with HCI. Tle extractswere evaporated to dryness and the radioactivelipidresiduewas dissolvedin 100 ul hexane. 'rbelipidextractswere chromatographed with hcxane/diethylether/aceticacid (80:20:1) on silica gel thin-layerplates.The spots corresponding to cholesteroland fattyacidswere identifiedby standards and were then isolatedfor measurement of radioactivity. Enzyme assays and measurements of DNA, protein and lipids Pyruvate dehydrogenase was estimated by measuring the "C02 liberatedwhen hepatocyttswere incubated with 5 MM [1_14C]pyruvate for 30 min at 37*C. Acetate thiokinasewas measured as described by Jones and Lipman [24).Liver microsomes were prepared according to Easom and Zammit [25],and HMG-COA reductase was measured according to Drevon et al. (26]. CDPcholine: 1,2-diacylglyceroclholinephospho- transferaseE,C 2.7.8.2and lysolecithiancyltransferase TABLE I Body and lit-erweights and lit-erLipidcontent in rats fed differentdietfsor 7 days Cloribratc was given as 0.315,('wc/w) and PFOA and PFOSA as 0.02% in the diet.Values are given as means S.E. There in each group. Fisher's P-values are given; * P < 0.05. * *P < 0.01 vs. control group. were four observations Control Cloribrate - Bod)-weight (g) Liverweight (g) 305 2 11.3 0.4 298 3 16.1 0.8 Uvcr triacvlglyccrti(lsAmol/g li%-cr) Livernon-csicririedcholesterol(,umal,lgliver) Livercholesiert)clster(Amui,,g li%;cr) 4.1 0-1 4.4 0.4 0.58 0.08. 4.0 0.7 4-5 0.3 0.24 0.02 PFOA :,q 18.3 6 0.7 3.9 O..l o.5 (1.4 PFOSA 275 15.9--0."# 13.8 7.6 0.27 18 0.3 0.0--1 Stock diet limitedfcd 2S2 +7" 4.1 = 0.6 67 (EC 2.3.1.23w)as mcasurcd asdcscribcdbv Parthasar- athy et al. [271.Acyl-CoA: cholesterol acyltransferase (ACAT) was measurcd according to Rustan et al.[281 and the svnthesis of phosphatidyiserine, phosphatidvlcthanola mine and phosphatidylcholine was measured as described by Vance [29).Other enzymes were measured as described earlier [30]. DNA was measured by the mithod of Ubarca and Paigen [311 and protein was estimated by the biuretmethod or by the method of I-owry et al.['321. Liver lipids were extracted with chloroform/ methanol (2:1. v/v).Triacylglycerolwsas determined directlyon the dried extractwith a kitmethod (Nyco, Oslo, Norway). Free and esterifiedcholesterol was determined by Nycotest kit method for cholesterol (Nyco) after separation of the extract on thin-layer chromatography. Serum cholesteroland triacylglycerols was measured directlyby the kit methods. Results Table I shows that 0.02% PFOA or PFOSA in the diet resulted in a lower body weight after 7 days of feeding as compared with the control group. ne cloribrate diet (0.3%) did not affectthe rat weight. The group with restrictedfood intake to thatof the PFOSA group had about the same weight as the PFOSA group. All the compounds resulted in a 40-60% increased liverweight. Similareffecton the liverweight has been observed earlierin rats fed clofibrate[9,10,331. Changes in serum and lit:elripidsby clofibrate,PFOA and PFOSA Fig. 1A shows that all three dietssignificantlryeduced serum cholesterol.In all treatment groups, cholesterolwas significantlryeduced (to 50-70% of control) after 24 h. Dietar-ytreatment for 2 weeks resulted in further cholesterolreductions by 70% or more. In agreement with other observations,fastingfor 2 days did not bring about significantcholesterol changes. 20 4 A I E B T c- 1 5 3 0 L 10 0 L u2 'o 05 .c 0 00 0 7 14 Days 0 1L 00 -7 14 Days Fig. 1.Effectof cloribrateP.FOA and PFOSA on the serum lipids. (A) Cholesterol(:B) triacylglycero&l.s;fasted-0., 0.3% cloribrate; 0.02% PFOA. a. 0.02% PFOSA. ne datarepresentmean t S.Eof four observations. Fig. 1B shows the effectsof clofibrate,PFOA and PFOSA on triacylglycerolisn the rat serum. None of the compounds resulted in significantchanges after I day of treatment. After 7 days, clofibrate and PFOA resulted in reduction to about 60% of control value and no further reduction was obtained with further treatment. PFOSA reduced triacylglyceroltso about 50% and 30% of controlvalue after I and 2 weeks of treatment. The significantreduction obtained by fast- ing for 2 days was as expected. In the rats fed stock diet but restrictedto that consumed by the PFOSA group, the serum triacylglycerolwsas 2.43 0.13 mM after 1 week of treatment (not shown) which is not significantllyower than observed in the control group (fed stock diet ad libitum). Table I shows that PFOSA increased the livertri- acylglycerolscontent to more than 3-times the control value. This effect of PFOSA on triacylglycerolwsas clearlyin contrastto the effectof clofibrateand PFOA which did not affectthe content of triacylglycerolasnd clearlyindicatesthat PFOSA inhibitsthe excretion of triacylglycerolfsrom the liver.Both PFOA and in particularPFOSA increased the livercontent of non- esterifiedcholesterol.In contrast to this,a significant TABLE 11 Cont-orsionof labelledsubstratesinto"C-labelledcholesterobly hepatocytesof rafsfeddifferendtictsfor7 days Cloribratewas givenas 0.3c,'(cw./w)and PFOA and PFOSA as 0.02% in the diet.ne concentrationof (2-"'Clmevalonateand [3-1"C)hydroxymethylglutaratweas 0.5mm. Incubationswith [I-"'Clacetateand 12-"'Clpyruvatweere conducted with 5 mM of the labelledsubstratesand includedalsounlabelledglucose(10 m.M). Values are means:tS.E. There were fourobservationsin each group.Fisher'sP valuesare given * p < 0.05. p < 0.01. Substrate [I-"C]Acclate 12_ 14C]Pvruvate 14 Ciliyrjri)xmye-thylglutraate [2-"'C]Mcvulonatc Formation of "'C-labellecdholesterol(nmol substratecarbon-mg DNA- control cloribrate PFOA 147,6 21.4 h.2 14.6 1-,.9 3.3 2280 tlio 59 13 46 9.5 3.6:i 0.9 2160 280 3'.6 16.5' 35.2 9.7* 3.6-- 1.3' 1730 240 1-h-1) PFOSA 21.2 7.4* * 26.5 5.4* * 5.0 1.1** 2030 310 68 TABLE III C(.vii-er.vi(yi7(iflabelledsi4bsl-reis;,,(",C"-tahellfeadttyacidc and aridati(irfIrU- I"Clpal-traiIrn.hepatiw.%-tticfrsatsfed differendtiets .f(y"rda.rv The conccnlrationsof compounds inthedictand the concentrations of [I-"'Cldcetataend 12."'Clpyruvatweere as describedin Table 11. The concentrationof (U-14C]palmitatweas 0.5 mM. Values are means=S.E. There Were 4 obsc-%-aiionisn each group.Fisher'sP valuesare given:* P < 0.05, P < 0.01vs.controlgroup. TABLE IV Ctyrit-enic(;mf/I."'laccratiento"'('-labelflaetdiraritiavnd terokl%-hepa:oo-tcisntheprevence()fk%7w/ipc"tic'crprpiix)ttndv The concentratioonf [1-"'Clicetatweas i mM and theincuhaticins alsoincluded10 mM glucose.Each incuhationnask contained4 mr cellproteinand 1.5% ulbunlinin ; mi Krebs-Ht:nselcibticarbonate buffer.The concentration@f hypolipcmiccomp-L)undswits I mM. Values are means t:S.E.There were fourobservationsin each group. Fisher'sP valuesare given P < 0.01vs.controlgroup. Substrate "C-Labelled fattyacidformation (nmol substratecarbon-mgDNA -'-h - 1) control clofibrate PFOA PFOSA [I-"ClAcetate 252t28 180@-29 12-"C]Pyruvate 22121 322=23* 90@-54* 104:t28** 182@-55 128 21 [U-"'C)Paimitatoexidation (nmol substratoexidized-M&DNA control cloribratePFOA 50250 726@-36 * 56463 1-h - 1) PFOSA 961 102 * Addition None TTA Cloribriaccid PFOA PFOSA Formation of "'C-labelled cholesterol Formation of '4C-labelled fattyacid (nmol substratecarbonms DNA- 1-h-1) 114 -+ 8 1.4 0.07 57 0.7 63 11 65 7 313 14 7.5: 1.3 243 8 611 t2O 1908 29 reduction (to approx. 50%) in esterifiecdholesterolin the treatedrats(PFOA-treated ratsnot measured) was found. Conversionof labellesdubstrateisntocholesterboylhepatocyleosf ratsfed differendtiets Table II shows the incorporatioonf labelledcarbon atoms from differentllyabelledacetate,pyruvate, mevalonate and hydroxymethylglutariacid (HMG). The major pointemerging from thistableisthat the rate of cholesterolsynthesiswas significantrleyduced from allsubstrateswhich are proximalto the HMGCoA dehydrogenase*step in alltreatment groups. In contrast,cholesterolsynthesisfrom mevalonate was not reduced in any of the groups. Table IIIshows the *-nthesisof fattyacidsfrom pyruvateand acetate.Both PFOA and PFOSA treat- ment reduced lipidsynthesis(notsignificanftrom pyruvate in the PFOA group).No reductionin fattyacid synthesiswas found in hepatocytesfrom clofibratetreatedanimals.The tablefurthershows thatthe peroxisomal inducers,as expected,resultedin an increasedrate of palmitateoxidation(althoughPFOA did not resultin a significanitncrease)T.he experiments do not exclude the possibilithyatintracellular contentof the testedcompounds might have a direct (reversible)ffecton fattyacidand cholesteroslynthesisin vivo.Such effectscould have escaped our detectionsince the compounds might have been washed out duringthe cellisolationprocedure.To testpossible directinhibitoreyffectsof thesedrugs,the synthesiosf fattyacidsand cholesterolin hepatocyteswith additionsof the compounds to the incubationmedium were performed (TableIV).The tableshows thatI mM of clofibriaccid,PFOA and FPOSA inhibitedthe choles- TABLE V Activitoyf cn--ymesrelatedtothes)-nihesoifscholesteraonld fattyacidsin liveorf ratsfeddifferendtietfsor 7 days The concentrationosf compounds inthedietwere as describedin Table 1.Values aregivenas means S.E.There were four observationisn each group.* P < 0.05; P < 0.0:5; P < 0.01vs.controlgroup. Enzyme Pyruvat dehydrogenase Citratesynthase ATP-citraiclyase Acetatethiokinast Malate dehydrogenase(NADF) (decarharylating) Malate dehydrogenase G Iucose-&phosphate dehydtorenaw lsocitratdeehydrogenuse(\ADP) HMG-COA reductuse Activit(yAmol -(mg DNA -'-min control clofibrate 0.590.05 0.97 0.12 1.540.34 8.7 0.64 0.360.05 27.1 3.2 0.50 0.10 2.44 0.35 0.58t 0.04 0.94 0.06 0.78 0.38 5.7 0.85 0.83 0.05 ' 18.4 2.4* 0.170.03 * 2.71 @-0.56 (nmol-;Lgprotein-'-min -') 0.31 0.0.1 0.16 0.04 ' PFOA 0.42 0.08 0.80 0.05 0.75 17 0 3.5 0.5 1.-34 0.05 0 20.7 t 2.6 0.2d4 O.D4 * 2.7@ 0.19 0.Is--0.05 PFOSA 0.36 0.04' 1.1029 0.24 0.06 ' 5.7 0.28 * 0.21 0.01 19.6 3.0 0.11 0.01 3.2.,= 0.13 0.1t1O.01 69 tcrolsynthesisto the same extent (approx.iOl"/eS,u)r.prisinglyi.ctradccylthioaccatciicd (TTA). another induccr of pcroxisomal6-oxidationwith hypolipemiceffcct in rats [.,.41a.lmost completely inhibitedcholcsterol synthesis.The pronounced inhibitoryeffectof T7A on the fattyacid-synthesiosbserved by Skrede et al.[34]was alsoconfirmed.Clofibricacid reduced the fattyacid production by about 20%. However, a direct inhibitoryeffecton the fattyacid synthesisis not a property shared by all the tested compounds. Both perfluorinatedcompounds unexpectedly stimulatedthe rate of fattyacid synthesisstrongly.It is unlikelythat thiswas due to an inhibitionof Krebs cyclewith a concomitant increase in lipogenic precursors,since these compounds inhibitedthe cholesterolsynthesis.It seems more likelythat the compounds stimulatea rate-limitinegnzyme, e.g.,acetyl-CoA carboxylase.At lower concentrations(0.5and 0.1 mM), there were very small inhibitoryeffectsof clofibricacid, PFOA and PFOSA (data not shown). En--ymesrelatedto the synthesisof cholesteroalnd fatty acidsfrom pyrut-ateand acetate Table V shows that pyruvate dehydrogenase, citrate synthase and acetate thiokinase were only slightlyaffectedby treatment with the three compounds. ATPcitratelyase activitywas reduced to about 15% of controlvalues by PFOSA. PFOA reduced the activity of thisenzyme significantltyo 50%. The effectof the compounds on three NADPH-generating enzymes shows a remarkable pattern.All three compounds significantlyreduced the activityof glucose-6-phosphate dehydrogenase. PFOSA reduced the activityto 20% of c6ntrol values. In contrast,isocitratedehydrogenase was unaffectedby allthree compounds. The activitoyf malic enzyme was increased 2- and 3.5-foldby clofibrate and PFOA, respectively.Malate dehydrogenase, which isnot specificalliynvolved in lipidsynthesisw,as virtuallyunchanged by any of the compounds. ne activitoyf HMG-COA rcductasc.the ratc-limitinagnd regulatedstep in choicstcrolsynthcsisw,as rcduccd to 50% or lessin allthreetreatment groups. En--ynierselatedto thesvnihesisof ci?olcstere.svtlerand phospholipidsiiiratsfed'differendtiets Table VI shows thatacyl-CoA: cholesterolacyltransferase(ACAT), was significantldyownregulated by all three compounds. PFOSA, which had the strongest effect,reduced the activittyo one thirdof control.The downregulation of thisenzyme is in keeping with the reductionof livercholesterolester(Table 1).Tne table furthershows thatthe activitieosf two enzymes important in the phopholipid turnover,acyl-CoA: I-acylglycero-3-phosphocholincacyltransferascand CDPcholine:1.2-diacylglyceroclholinephosphotransferase, were not significantlaylteredin the ratsfed any of the hypolipemic drugs.'ne activityof phosphatidylserine synthase, phosphatidylethanomaline synthase and phosphatidylcholinesynthase.were also unaffected by any of the dietaryregimes utilized(notshown). Activitieosf en.-ymesrelatedto phospholipidsynthesisin the presenceof h)-polipemicdrugs Table VII shows that at 0.5 mM clofibricacid had littleffecton activitieosf the enzymes listedin the table.The five enzymes were all inhibitedby both perfluorinated.compounds. PFOA had a particular inhibitoryeffecton phosphatidylserinesynthase activity which was reduced to 18% of normal with 0.5 mM PFOA. PFOSA had strongestinhibitoryeffecton the activitoyf CDP-choline: 1,2-diacylglycercohlolinephosphotransferase,phosphatidyiserinesynthase and phosphatidylethanolamine synthase,which were reduced to 14%, 13% and 28% of control,respectivelywith 0.5 mM PFOSA. At lower concentration(0.1 mM) the inhibitoryeffectsof the perfluorinatedcompounds were very moderate (datanot shown). Clofibricacid had no significanetffectat 1 mM concentration. TABLE VI Aciii-ionf.en--)-r"nleastedtosynthesoi.sfcholestereyslterasnd phospholipiidnslit-eorfsratsfed hypolipemidcrugsfor 7 days The concentrationisnthe dietwere as describedinTable 1.Valuesarc givenas means @-S.E.There were fourobsmations in each group. *' P < 0.01vs.controlgroup. Enzyme Acyl-CoA Cholesterol acyltransfera(sAeCAT) (nmol/mg proteinpermin) Acyl-'CoA: I-acylglyccrc,@-' phosphocholineacyltransferase (nnit.)'lnipgrotcinpermin) CDP-cht)lineI:.:-diacvli;lvccrtil cht)iinc,nlitl%pht)trunsf;:r:i-.t (nmiilimg prt)tcipner min) Control 660 51 14.0- 1.3 10.4-- 1.3 Cloribrate 427 18 12.4 1.0 9.1 1.2 PFOA 3:3 39*1 1:.7= 1.3 .6= 0.7 PFOSA 2.17 41 11.8t 1.8 S.tl 1.0 70 TABLE VII Aciii-iti(eisfcti--.vmrcevlatedto ph(j3plitilinp*ildfthcsiisn titepreiriicr of ltipoliprmidrruj:s Concentratedstilutic)(n1s11mM) of cltifibric;sPcFiOdA. and PFOSA inDMSO were dilutedto 0.5mM finalconcentrationin a,-,Say.t%erm. (The controlswere added the same amount of pure DMSO.) The effectof the drugs on each of the enzymes were testedfour tim:s and in each of the experiment!.t.he aciivitiweesre calculatedas p r cent of the controlvalue.Values are givenas means --S@.-E. Control CloribricPFOA acid Acyl-CoA lysophosphatidyl transferase 100 CDP-choline: 1.2-diacylglycerol cholinephosphotransferast loo Phosphatidyiserinesynthase ioo Phosphatidylethanolmaine synthase 100 Phosphatidylcholinesynthase 100 90@-6 56t7 95 t 5 71 t 2 101=9 184 101 3 475 89 -@3 475 PFOSA 61.t5 14:t0.6 13 t 1.4 28:t2 483 The effect of the compounds on the activityof ACAT was tested only at 100 uM and with thisconcentration the ACAT activitywas unaffected by the compounds (not shown). Discussion Reduction of the steady-statelevelsof serum lipids may be visualized as the resultof downregulation of lipidsynthesisor increased clearance from plasma. The aim of this study was to evaluate the effectsof three differentperoxisomal proliferatorson a series of en- zymes involved in hepatic lipidsynthesis.The most important observations with allthree compounds was downregulation of HMG-COA reductase, the rate- limitingenzyme of cholesterol synthesisand of choles- terol esterificationenzvme (ACAT). The agreement between the observed alterationsin the enzyme activi- tiesand production of cholesteroland lipidsin intact livercells provides some support for the assumption that such enzyme measurements do reflectrealchanges in metabolic activityin the intactorgan and shed light on the mechanism of the hypolipemic effectsof the agents investigated in thisstudy. These compounds do not show a correlated effect on the lipogenic and cholesterogcnic pathways in the in vivo experiments. The in vitroexperiments indicate that there are direct, presumably reversible,effectson these pathways. These effectsare differentthan those observed after dietary manipulation. The latterprobably represent changes in enzyme concentrations since, at least,the changes in the cholestcrogenic pathway are correlated with the changes in HMG-COA rcductase. The perfluorinatcd compounds utilizedin thisstudy are strong local irritantsT.lic observed actions should not be rcgardcd as mcrc unspecifictoxic effectssince. inadditionto reductionof the activitoyf some enzyme systems.thesecompounds incrcasethe activitoyfothcr enzymes, e.g., cnzvmes of the peroxisomal fattv acid 0-oxidation system [18.19].Howcvcr. local irritation may explain the reduction of food intake and slower weight increase obser-@ed after feeding perfluorinatcd compounds. However, this probably contributes little to the reduction in serum triacvlglycerols,ince no significantreduction was observed in rats with restrictedfood intake.Increased liverweight correlates well with earlierstudies on peroxisomal proliferators [35,361. Cholesterol and fatty acid synthesis The reduction of cholesterolsvnthesis from differ- ent labelledsubstratesfitswell with the reduced activ- ity of HMG-COA reductase observed in this study. Three substrates,proximal to the reductase step,were incorporated into cholesterolat a reduced ratewhereas no reduction from mevalonate was observed in any of the treatment groups. Lowering of HMG-COA reduc- tase levelsby clofibrateisin accord with other observa- tions[9,37,381.Even though the three compounds tested in thisstudy reduce the HMG-COA reductase activity, thiseffect may not be a characteristicof allperoxiso- mal inducers.MEDICA 16, another compound in this group, does not act via an effecton this reductase (12,39),but rather inhibitsthe synthesisof cholesterol at a step distalto HMG-COA reductase. Our observa- tions with clofibrateare at variance with the data obtained by Azarnoff et al.[10]who found that choles- terol synthesis from mevalonic acid was reduced in liversof ratsfed clofibrate. In contrast to the inhibitoryeffectof MEDICA 16 on ATP-citrate Ivasc [39],clofibricacid, PFOA and PFOSA had essentiallyno directeffecton ATP-citrate lyaseat I mM (data not shown), but the drugs down- regulated the enzyme afterdietaryadministration.This effectmay contribute to reduced synthesis of choles- terolin vivo,since the enzyme isimportant in the main pathyway for cholesterolprecursor synthesis.Itisinter- esting that all three compounds reduced one of the NADPH generating enzymes (glucose-6-phosphate de- hydrogenase), while isocitratedehydrogenase (NADP) was unchanged. Reduced capacity for NADPH genera- tion can therefore hardly contribute to reduction in serum lipid levels. The synthesisof fattyacids from acetate or pyruvate was not reduced in hepatocytes from ratsfed cloribrate (Table 111),but was significantlryeduced in the hepato- cytes from rats fed PFOSA. The reduced rate of fittv acid synthesis in the PFOA and PFOSA groups is probably not related to reduction in scrum triicyl- glyccrols.In rats fcd PFOSA. thcre'was 2iccumulation of livcr trizic%llglvccroTii.l%e. lack of rcduction of f;itIN' acid s@-nthcsisby clofibruicsuggcsts that othcr prt)- 71 ec,;.scisn lipidmetabolismmust be more csscniiaflor the hypolipemia.Tomarelliet al.[40]found increascd svnthesio,f,lipidsfrom acetatein ratsfedcloribriaccid and alsoin ratsfcdthe vcrvPotcnihypolipemiccompound \k*)'-14.643. S-vililioefscilsiolesteersyilerand phospholipids Liverexportslipidtso otherorgansmainlyas VLDL particlesI.n additionto apolipoproteintsr.iacylglycerolasnd freecholesterolt,hesepar-ticiceosnsist mainly of esterifiecdholesteroalnd phospholipids. Hence, reduced hepaticsynthesisof thesecomponents might leadtoreducedtransportof lipidfsrom the liver. The hepaticcontentof cholesterveisterwas reduced bothinclofibrataend PFOSA fed rats(PFOA fedrats were not tested)even where freecholesterowlas not reduced(Table1).Similareffectwas obtainedby Avignan et al.[411with the hypolipemicdrug MER-29. Reduced cholesteryelsterproductionismost likelya resultof downregulationof the ACAT activitwyhich was observed in alltreatmentgroups.A directinhibitor@e,ffecton the enz@-rneby the compounds is probablyof lessimportancesince100 gM of thecompounds did not affecthe enzyme activit(vhigherconcentrationwsere not tested).Vance (2@]argued that synthesiosf phospholipidsmay be limitinfgorlipoproteinsynthesisI.n thisstudy,we have not observedany downregulationof enzyrnesinvolvedinthe synthesiosr metabolismof phospholipidsW.e have observedonly smalldirecteffectosf clofibriaccidon theseenzymes. It seems likelyh,owever, thatreduced phospholipid synthesipslaysa rolein the lipid-reducinegffectof the perfluorinatecdompounds, sincethesehad directinhi@itoryeffecton several important enzymes. Parthasaratheyt al.(27)suggestedfrom theirstudies thatinhibitioonf phosphatidyi-cholisnyenthesisp,articularlbyy the lysolecithaicnyltransferapsaethway, may be relatedto a dr-ug'esffectivenesisn decreasing serum lipidsW.e found thatthe transferasweas moderatelyinhibitebdy the pcrfluorinatecdompounds. We alsoobserved thatthisenzyme was virtualluynaffected by 0.5mM (TableVII)and alsoby I mM of clofibric acid.This agrees with the data reportedby Parthasarathyet al.[271.Our data do not supportthe hypothesisthatreduced activitoyf lysolecothinaecyltransferaspelaysa centralrolein the hy.p olipemic effectof the compounds we have tested.Inhibitioonf other enzymes of phospholipidsn.nthesimsay play a role. Rediiced releaseof lipidvfrot?zihe lit-er In earlierstudiesi.thas been rc,-)orttchdatclori- bratcreducesthe releaseof lipidfsrom the liver[13,14]. A directeffecton the excretionprocessb%.clof-ibratc was not confirmedby accumulationof livertriacylglyccrolisnclofibrate-fcndo.rin PFOA-fed animals.A reduccd releaseof lipidsfrom liverwas probablyan effectof PFOSA. however,sincethe compound increasedlivertriacylglvccrboylsabout 200% in spiteof reducedrateof fattyacidsynthesi(sTableIV). Since interferencweith t@e synthesisof cholester-yl estermay cause reduced hepaticlipidoutput[42],it may be concludedthatreductionincholesteroslynthe- sisand esterificatidoune to downregulationofHMGCoA reductaseand ACAT togetherwith enhanced fattyacidoxidationinthe livera,re effectcsausedby clofibriaccid as wellas by the perfluorinatecdom- pounds. 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