Document 2NR2yO32Kp4opoQ9owBz3k3R5

'he -` Oeeiei'ie t H i e /jienM iu A < lu. 1 12 b v 1 ; A ' - i-,; bNcvicr Seif net Publishers B V All nnhis reserved <XI()5-27(i<l/9;/S(jyiXl B13ALIP 54011 N 'C ;-- a " ': --p--s A.-,..-*-: -'-I The mechanism underlying the hypolipmie effect of perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid Bente Haughom and 0ystein Spydevold Institute o f Medical Biochemistry, Universi!}- o f Oslo. Oslo (Norway) (Received 12 June 1992) Key words: Lipid metabolism; Triacylglycerol; Cholesterol; H epatocyte; (R at liver) The influence of the peroxisomal proliferators perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid on lipid metabolism in rats was studied. Dietary treatment of male Wistar rats with these three compounds resulted in rapid and pronounced reduction in both cholesterol and triacvlelvcerols in serum. The concentration of liver triacvlglycerols was increased by about 300% by PFOSA. Free cholesterol was increased by both perfluoro compounds. Cholesteryl ester was reduced to 50% by PFOSA as well by clofibrate. In hepatocytes from fed rats, all the compounds resulted in reduced cholesterol synthesis from acetate, pyruvate and hydroxymethyl glutarate, but there was no reduction of synthesis from mevalonic acid. The oxidation of palmitate was also increased in all groups. The perfluoro compounds, but not clofibrate, caused some reduction in fattv acid synthesis. The activity of liver HMG-CoA reductase was reduced to 50% or less in all treatment groups and all three compounds led to lower activity of acyl-CoA: cholesterol acyltransferase (ACATj. Changes in other enzymes related to lipid metabolism were inconsistent. The present data suggest that the hypolipemic effect of these compounds may, at least partly, be mediated via a common mechanism; impaired production of lipoprotein particles due to reduced synthesis and esterification of cholesterol together with enhanced oxidation of fatty acids in the liver. Introduction Many hypolipemic drugs cause proliferation of per oxisomes and increase the activity of the peroxisomal /3-oxidation in rats [1-3]. Chemically, these drugs con stitute a heterogeneous group including clofibrate, tibric acid, niadenate and long-chain acylthioacetic acids, tiadenol, long-chain thia acids and M EDICA 16 [1-6]. It has been suggested that the increase in the fatty acyl-CoA oxidizing system contributes to the hy polipemic effect of these drugs [7,3]. The dominating mechanism underlying reduction of serum triacyiglycerols and cholesterol by these drugs is, however, uncertain. O ther mechanisms which may be important for the hypolipemic effect include; reduced hepatic synthesis of fatty acids and cholesterol [9-12]; reduced C orrespondence lo: 0 . Spydevold. lnssitute of Medical Biochemistry. P.O, Box 1112. Blindern. N-0317 Oslo. Norway. Abbreviations: PFO A . perfluorooctanoic ac:C: PFOSA. perfluorooclane sulphonic acid; ACA T, acyl-CoA: cholesterol acyltransferase; HM C. hydroxymethyl glularic acid: T TA . leira-decyllhioacttic acid; VLDL. vcry-low-density lipoproteins. triacylglycerol release by the liver [13,14]; increased rate of V L D L degradation [6]; increased uptake or reduced release of fatty acids by adipose tissue [15,16] and increased excretion of cholesterol into bile and feces [17], Ikeda et al. [18,19] observed that perfluorooctanoic acid (PFO A ) and perfluorooctane sulphonic acid (PFOSA) efficiently induced the peroxisomal /3-oxida tion in rats th at were fed these compounds (0.02% in the diet). Just et al. [20] reported that the perfluorocarboxylic acids alter hepatic lipid metabolism and reduce serum lipid levels showing that these compounds also belong to the group of peroxisomal proliferators with hypolipemic effect. The perfluorinated compounds are particularly interesting since they obviously are not subject to ordinary metabolic modifications. The ef fects of these compounds must, therefore, be due to effects of the compounds per se. Peroxisomal inducers may bring about the hy polipemic effect by affecting different steps in lipid metabolism. However, it seems likely that.som e im por tant steps in lipid metabolism are common targets for these compounds. The unphysiological character of the perfluoro com pounds makes it possible that their effect m OGCOa- on lipid metabolism follows a pattern that would make it easier to understand possible mechanism for the lipid reduction effect of peroxisomal proliferators. In the search for a possible common mechanism underlying the hvpolipemic effect of peroxisomal in ducers, we have compared hepatic fatty acid metabo lism, cholesterol synthesis and the activities of enzymes related to these metabolic processes in the liver of rats fed perfluorooctane sulphonic acid (PFOSA), perfluorooctanoic acid (PFOA) and clofibrate. Methods and Materials Animals Male W istar rats were used. They were divided into five groups. O ne group was allowed norma! food ad libitum (control rats). T h re e other groups were given food which contained 0.3% clofibric acid, 0.02% perfluorooctanoic acid or 0.02% perfluorooctane sul phonic acid, respectively. T he diets were prepared by soaking standard food (in pellet form) in diethylether in which the com pounds had been dissolved. The fifth group was restricted in food intake to that consumed by the PFO SA group. T he stock diet was used for these paired feeding experiments. The average body weight when the experim ents started was 269 g and the average daily food consumption per rat in the different groups were; control: 23.7 g, clofibrate: 23.1 g, PFOA: 22.7 g, PFOSA: 20.0 g. Materials [2-14C]M evalonic acid, [1-u C]pyruvic acid and [2- MC]pyruvic acid were from New England Nuclear. [ l- 14C]acetic acid was obtained from Am ersham (UK). Clofibrate was from Fluka (Buchs, Switzerland). Perfluorooctanoic acid was purchased from AldrichChemie (Steinheim, Germany) and Perfluoro-octane sulphonic acid was from Fluorochem (Old Glossop, UK). O th er chemicals w ere from Sigma (St. Louis, MO, USA). Preparation o f hcpatocytes The effects of dietary treatm ent were studied in hepatocytes isolated from rats fed the diets for 1 week and for the study of direct effects of the compounds, hepatocytes were isolated from rats fed the standard diet. Isolation of hepatocytes was performed by perfu sion with collagenase, according to Berry and Friend [21], with the modifications described by Seglen [22]. Oxidation o f palmitate and conversion o f labelled sub strate into lipids Fatty acid oxidation was m easured according to Christiansen et al. [23] with 0.5 mM palmitate as sub strate. Fatty acids and cholesterol synthesized from radioactive precursors were extracted from the cell suspension after 90 min incubation. The reaction was stopped by the addition of 5% saturated K O H in ethanol and the mixture was heated at 90C for 1 h. The nonsaponifiable lipids were extracted with petroleum ether. The suspension was then acidified with HCI. The extracts were evaporated to dryness and the radioactive lipid residue was dissolved in 100 1 hexane. The lipid extracts were chrom atographed with hexane/diethyl e th e r/a c e tic acid (8 0 :2 0 :1 ) on silica gel thin-layer plates. The spots corresponding to cholesterol and fatty acids were identified by standards and were then isolated for measurement of radioactiv ity. Enzyme assays and measurements o f DNA, protein and lipids Pyruvate dehydrogenase was estim ated by m easur ing the ,4C 0 2 liberated when hepatocytes were incu bated with 5 mM [ l - I4C]pyruvate for 30 min at 37C. Acetate thiokinase was m easured as described by Jones and Lipman [24], Liver microsomes were prepared according to Easom and Zammit [25], and HM G-CoA reductase was m easured according to Drevon et al. [26]. CDPcholine : 1,2-diacyIglycerol cholinephosphotransferase, EC 2.7.8.2 and lysolecithin acyltransferase table i Body and liver weights and lit er lipid content in rats fed different diets fo r 7 days Clofibrate was given as 0.39c (w /w ) and PFOA and PFOSA as 0.02% in the diet. Values are given as means S .E . T here were four observations in each group. Fisher's F-valucs are given; * P < 0.05; * * P < 0.01 vs. control group. Body weight (g) Liver weight (g) Liver triacylglycerols (^ im o l/g liver) Liver non-estcrified cholesterol l^ m o l/g liver) Liver cholesterol ester (^ .m o l/g liver) Control 305 2 11.3 0.4 4.1 0.5 4.4 0 .4 0.58 0.08 Clofibrate 298 3 16.1 0.8 ** 4.0 0.7 4.5 0.3 0.24 0.02 * * PFOA 2SS 6 * 18.8 0.7 ** 3.9 0 .3 ft.5 0.4 * PFOSA 275 6 ** 15.9 = 0.7 * 13.8 18 " ' 7.6 0.3 * ' 0.27 = 0.03 ` Slock diet limited fed 2S2 ; 7 ** 4.1 r 0.6 15 OOOOffli (EC 2.3.1.23) was m easured as described by Parthasaruthv ei al. [27], Acyl-C oA : cholesterol acvltransferase (ACAT) was measured according lo Rustan et al. [28] and the synihesis of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine was measured as described by Vance [29]. O ther enzymes were m easured as described earlier [30]. DNA was measured by the m ethod of Labarca and Paigen [31] and protein was estim ated by the biuret method or by the method of Lowry et al. [32], Liver lipids were extracted with chloroform / methanol (2:1. v/v). Triacylglycerols was determined directly on the dried extract with a kit m ethod (Nyco, Oslo, Norway). Free and esterifled cholesterol was determined by Nycotest kit method for cholesterol (Nyco) after separation of the extract on thin-layer chromatography. Serum cholesterol and triacylglycerols was m easured directly by the kit methods. Results Table I shows that 0.02% PFO A or PFO SA in the diet resulted in a lower body weight after 7 days of feeding as com pared with the control group. The clofibrate diet (0.3%) did not affect the rat weight. The group with restricted food intake to that of the PFOSA group had about the same weight as the PFOSA group. All the compounds resulted in a 40-60% increased liver weight. Similar effect on the liver weight has been observed earlier in rats fed clofibrate [9,10,33]. Changes in serum and liver lipids by clofibrate, PFOA and PFOSA Fig. 1A shows that all three diets significantly re duced serum cholesterol. In all treatm ent groups, cholesterol was significantly reduced (to 50-70% of control) after 24 h. Dietary treatm ent for 2 weeks resulted in further cholesterol reductions by 70% or more. In agreement with other observations, fasting for 2 days did not bring about significant cholesterol changes. 67 Fig. 1. Effect of clofibrate. PFOA and PFOSA on the serum lipids. (A) Cholesterol: (B) triacylglycerols; ts. fasted; , 0.3% clofibrate; , 0.02% PFO A : . 0.02% PFOSA. The data represent mean S.E. of four observations. Fig. IB shows the effects of clofibrate, PFOA and PFO SA on triacylglycerols in the rat serum. None of the compounds resulted in significant changes after 1 day of treatm ent. A fter 7 days, clofibrate and PFO A resulted in reduction to about 60% of control value and no further reduction was obtained with further treatm ent. PFO SA reduced triacylglycerols to about 50% and 30% of control value after 1 and 2 weeks of treatm ent. T he significant reduction obtained by fast ing for 2 days was as expected. In the rats fed stock diet but restricted to that consumed by the PFOSA group, the serum triacylglycerols was 2.43 0.13 mM after 1 week of treatm ent (not shown) which is not significantly lower than observed in the control group (fed stock diet ad libitum). Table I shows that PFOSA increased the liver tri acylglycerols content to more than 3-times the control value. This effect of PFOSA on triacylglycerols was clearly in contrast to the effect of clofibrate and PFO A which did not affect the content of triacylglycerols and clearly indicates that PFOSA inhibits the excretion of triacylglycerols from the liver. Both PFO A and in par ticular PFO SA increased the liver content of nonesterified cholesterol. In contrast to this, a significant TABLE II Conversion o f labelled substrates into ,JC-labelled cholesterol by hepatocytes o f rats fed different diets fo r 7 days Clofibrate was given as 0.3% (w /w ) and PFOA and PFOSA as 0.02% in the diet. The concentration of [O-'^Clmevalonate and [3- 14C]hydroxymethylglutaratc was 0.5 mM . Incubations with [ l - MC]aceiate and l2-H C]pyruvate were conducted with 5 mM of the labelled substrates and included also unlabelled glucose (10 m.M). Values are m ean s S .E . T h ere were four observations in each group. Fisher's P values are given ' P < 0.05; " P < 0.01. Substrate [1- IJC]Acetate 12-'J C]Pyruvate 13- l4C]Hydro;cy-mc!hylglu[urale [2- u C]M cvalonale Formation of u C-labelled cholesterol (nmol substrate carbon mg D N A -1 h ' 1) control clofibrate PFOA PFOSA 14~,6 21.4 7K.2 14.6 17.4- 3.3 2 280 150 59 13 * 46 9.5 * * 3.6 0 . 9 ' * 2160 280 37.6 16.5 * 35.2 9.7 * 3.6 1.3** 1 730 240 21.2 7.4 ** 26.5 5.4 * * 5.0 1.1** 2030 310 0000$^ TABLE III Conrcrsion o f labelled substrates into 1 C-labelled fatty acids and oxidation o f / U -14C/palrrutate by hcpattK~\ tes o f rats fe d different diets for 7 days The concentrations of compounds in the diet and the concentrations of ll* u C]acctaie and [2-u C]pyruvaie were as described in Table II. The concentration of [U -I4C!palmiiaie was 0.5 mM. Values are means r S .E . T here were 4 observations in each group. F isher's P values are given: * P < 0.05; * * P < 0.01 vs. control group. Substrate [ l - 14C lA cetaie (2- 14C]Pyruvate |4 C-Labelled fatty acid formation (nmol substrate carbon mgDNA " ' h " 1) control clofibrate PFOA PFOSA 252 28 180 --2S 90 = 54 * 104 2 8 * * 221 21 322 = 23 * 182 = 55 128 2 1 * [U -l4C]Palmitate oxidation (nm ol substrate oxidized-m gD N A "1h " ' ) control clofibrate PFOA PFOSA 5 0 2 50 726 = 36 * 564 6 3 861 102 * reduction (to approx. 50%) in esterified cholesterol in the treated rats (PFOA -treated rats not measured) was found. Conversion o f labelled substrates into cholesterol by hepatocytes o f rats fe d different diets Table II shows the incorporation of labelled carbon atoms from differently labelled acetate, pyruvate, m evalonate and hydroxymethylglutaric acid (H M G ). The m ajor point emerging from this table is that the rate of cholesterol synthesis was significantly reduced from all substrates which are proximal to the HM GCoA dehydrogenase step in all treatm ent groups. In contrast, cholesterol synthesis from mevalonate was not reduced in any of the groups. Table III shows the synthesis of fatty acids from pyruvate and acetate. Both PFOA and PFOSA treat- TA13LE IV Conrcrsion o f ! 1 n acetate into u C-labcilcd fa tty acids and etudes. ;crul by hepatocytes in the presence o f hypolipmie com pounds The conccnlration of [ I - l4C]acctute was 5 mM and the incubations also included 1 mM glucose. Each incubation flask contained 4 mg cell protein and 1.59? albumin in 5 ml Krebs-Hensclcit bicarbonate buffer. The concentration of hypolipcmic com pounds was 1 mM. Values are means S .E . T here were four observations in each group. Fisher's P values are given * * P <0.01 vs. ccnlrol group. Addition None TTA Clofibric acid PFOA PFOSA Formation of l4C - la b e lle d cholesterol Form ation of l4C - la b e ! le d fatty acid (nmol substrate carbon mg D N A 'l h _ I ) 114 8 1.4 = 0.07 * * 57 0.7 ' * 63 11 ** 65 7 ** 313 1 4 7.5 1.3 * * 243 8 * * 611 20 * * 1908 29 * * ment reduced lipid synthesis (not significant from pyru vate in the PFO A group). No reduction in fatty acid synthesis was found in hepatocytes from clofibratetreated animals. The table further shows that the per oxisomal inducers, as expected, resulted in an in creased rate of palmitate oxidation (although PFOA did not result in a significant increase). The experi ments do not exclude the possibility that intracellular content of the tested compounds might have a direct (reversible) effect on fatty acid and cholesterol synthe sis in vivo. Such effects could have escaped our detec tion since the compounds might have been washed out during the cell isolation procedure. To test possible direct inhibitory effects of these drugs, the synthesis of fatty acids and cholesterol in hepatocytes with addi tions of the compounds to the incubation medium were performed (Table IV). The table shows that 1 mM of clofibric acid, PFO A and FPOSA inhibited the choles- TABLE V Activity o f enzymes related to the synthesis o f cholesterol and fa tty acids in liver o f rats fe d different diets fo r 7 days The concentrations o f compounds in the diet were as described in Table I. Values are given as m eans + S.E. There were four observations in each group. * P < 0.05: * * P < 0.05; P < 0.01 vs. control group. Enzyme Pyruvat dehydrogenase Citrate synthase A TP-cilraie lyase Acetate thiokinase Malate dehydrogenase (NADP) (decarhoxylating) Malate dehydrogenase Glucosc-6-phosphate dehydrogenase Isocitralc dehydrogenase (NADP) HMG-CoA reductase Activity (im ol-(m g D N A "'-m in " ') control clofibrate 0.59 0.05 0.97 0.12 1.54 0.34 8.7 0.64 0.36 0.05 27.1 3 .2 0.50 0.10 2.44 0.35 0.58 0.04 0.94 0.06 0.78 0-38 5.7 0.85 0.83 0.05 * * 18.4 2 .4 * 0.17 0 .0 3 * 2.71 = 0.56 (n m o l-jig protein " 1min " 1) 0.31 0.03 0 .!6 0.04 * PFOA 0.42 0.08 0.80 0.05 0.75 17 * 3.5 0.5 * * 1.34 0.05 ** 20.7 2.6 0.23 0.04 * 2.73 0.19 0.15 0.05 * PFOSA 0.36 0.04 * 1.10 29 0.24 0.06 ' * 5.7 0.28 ** 0.21 0.01 19.6 3.0 0.11 0.01 ** 3.23 0.13 0.11 0.01 * 0000$$ tcrol synthesis to the same extent (approx. 50%). Sur prisingly. tctradecvlthioacctic acid (TTA). another in ducer of peroxisomal /3-oxidation with hypolipemic ef fect in rats [3.4], almost completely inhibited choles terol synthesis. The pronounced inhibitory effect of TTA on the fatty acid synthesis observed by Skrede et al. [34] was also confirmed. Clofibric acid reduced the fatty acid production by about 20%. However, a direct inhibitory effect on the fatty acid synthesis is not a property shared by all the tested compounds. Both perfluorinated compounds unexpectedly stimulated the rate of fatty' acid synthesis strongly. It is unlikely that this was due to an inhibition of Krebs cycle with a concomitant increase in lipogenic precursors, since these compounds inhibited the cholesterol synthesis. It seems more likely that the compounds stimulate a rate-limiting enzyme, e.g., acetyl-CoA carboxylase. At lower concentrations (0.5 and 0.1 mM), there were very small inhibitory effects of clofibric acid, PFOA and PFOSA (data not shown). Enzymes related to the synthesis o f cholesterol and fatty acids from pyruvate and acetate Table V shows that pyruvate dehydrogenase, citrate synthase and acetate thiokinase were only slightly af fected by treatm ent with the three compounds. ATPcitrate lyase activity was reduced to about 15% of control values by PFOSA. PFO A reduced the activity of this enzyme significantly to 50%. The effect of the compounds on three NADPH-generating enzymes shows a rem arkable pattern. AJ1 three compounds sig nificantly reduced the activity of glucose-6-phosphate dehydrogenase. PFOSA reduced the activity to 20% of control values. In contrast, isocitrate dehydrogenase was unaffected by all three compounds. The activity of malic enzyme was increased 2- and 3.5-fold by clofibrate and PFOA, respectively. Malate dehydrogenase, which is not specifically involved in lipid synthesis, was virtually unchanged by any of the compounds. The activity of HM G-CoA reductase, the rate-lim iting and regulated step in cholesterol synthesis, was reduced to 50% or less in all three treatm ent groups. Enzymes related to the synthesis o f cholcsteryl ester and phospholipids in rats fed different diets Table VI shows that acyl-CoA: cholesterol acvltransferase (ACAT), was significantly downregulated by all three compounds. PFOSA, which had the strongest effect, reduced the activity to one third of control. The downregulation of this enzyme is in keeping with the reduction of liver cholesterol ester (Table I). T he table further shows that the activities of two enzymes impor tant in the phopholipid turnover, acyl-C oA : l-acylglycero-3-phosphocholine acyltransferase and CDPcholine : 1.2-diacylglycerol cholinephosphotransferase, were not significantly altered in the rats fed any of the hypolipemic drugs. The activity of phosphatidylserine synthase, phosphatidylethanom aline synthase and phosphatidylcholine synthase were also unaffected by any of the dietary regimes utilized (not shown). Activities o f enzymes related to phospholipid synthesis in the presence o f hypolipemic drugs Table VII shows that at 0.5 mM clofibric acid had little effect on activities of the enzymes listed in the table. The five enzymes w ere all inhibited by both perfluorinated. compounds. PFOA had a particular inhibitory effect on phosphatidylserine synthase activity which was reduced to 18% of normal with 0.5 mM PFOA. PFOSA had strongest inhibitory effect on the activity of CDP-choline : 1,2-diacylglycerol cholinephos photransferase, phosphatidylserine synthase and phosphatidylethanolamine synthase, which were reduced to 14%, 13% and 28% of control, respectively w'ith 0.5 mM PFOSA. At lower concentration (0.1 mM) the inhibitory effects of the perfluorinated compounds were very moderate (data not shown). Clofibric acid had no significant effect at 1 mM concentration. TABLE VI Activity o f enzymes related to synthesis o f cholcsteryl esters and phospholipids in livers o f rats fe d hypolipemic drugs fo r 7 days The concentrations in the diet were as described in Table I. Values are given as means S .E . T here were four observations in each group. * * P < 0.01 vs. control group. Enzyme Acyl-CoA Cholesterol acyltransferase lACAT) (nm ol/m g protein per min) Acyl-CoA: 1-acylglycero-3phosphocholine acyltransferase (nmol nig protein per min) C D P-eholine: 1.2-diucylglycerol cholincphosphoirensicrusc (nmol, mg protein per min) Control 660 51 i4 .o i.3 10.4 = 1.3 Clofibrate 427 1 8 ** ] 2.4 1.0 9.1 1.2 PFOA 323 3 9 " 12.7 = 1.3 7.6 0.7 PFOSA 237 4 1 * * 11.S 1.8 S.6 1.0 o o o o i # 1$ 70 t a b l e vii Actn Hies o f enzymes related to phospholipid synthesis in the presence o f hypolipcmic druys C oncentrated solutions (10 m.M) of clofibric acid. PFOA and PFOSA in DM SO were diluted to 0.5 mM final concentration in assay system (The controls were added the same amount of pure DMSO.) The effect of the drugs on each of the enzymes were tested four times and in each of the experiments, the activities were calculated as per cent of the control value. Values are given as means = S.. Control Clofibric PFOA PFOSA acid Acyl-CoA lysophosphatidyl transferase too C D P-choline: 1.2-diacylglyccrol cholinephosphotransferase too Phosphatidylserine synthase too Phosphatidyiethanolamine synthase too Phosphatidylcholine synthase too 90 = 6 56 7 61 5 95 5 71 2 14 + 0.6 101 = 9 18 = 4 13+1.4 101 3 47 5 28 2 89 = 3 47 5 48 3 The effect of the compounds on the activity of ACAT was tested only at 100 /iM and with this con centration the ACAT activity was unaffected by the compounds (not shown). Discussion Reduction of the steady-state levels of serum lipids may be visualized as the result of downregulation of lipid synthesis or increased clearance from plasma. The aim of this study was to evaluate the effects of three different peroxisomal proliferators on a series of en zymes involved in hepatic lipid synthesis. The most im portant observations with all three compounds was downregulation of HMG-CoA reductase, the ratelimiting enzyme of cholesterol synthesis and of choles terol esterification enzyme (ACAT). The agreement between the observed alterations in the enzyme activi ties and production of cholesterol and lipids in intact liver cells provides some support for the assumption that such enzyme measurements do reflect real changes in metabolic activity in the intact organ and shed light on the mechanism of the hypolipemic effects of the agents investigated in this study. These compounds do not show a correlated effect on the lipogenic and cholesterogenic pathways in the in vivo experiments. The in vitro experiments indicate that there are direct, presum ably reversible, effects on these pathways. These effects are different than those observed after dietary m anipulation. The latter probably represent changes in enzyme concentrations since, at least, the changes in the cholesterogenic pathway are correlated with the changes in HM G-CoA reductase. The pcrfluorinated compounds utilized in this study are strong local irritants. The obscivcd actions should not be regarded as mere unspecific toxic effects since. in addition to reduction of the activity of some enzyme systems, these compounds increase the activity of other enzymes, e.g., enzymes of the peroxisomal fatty acid /3-oxidation system [18.19], However, local irritation may explain the reduction of food intake and slower weight increase observed after feeding perfluorinatcd compounds. However, this probably contributes little to the reduction in serum triacvlglycerols, since no significant reduction was observed in rats with re stricted food intake. Increased liver weight correlates well with earlier studies on peroxisomal proliferators [35,36]. Cholesterol and fatty acid synthesis The reduction of cholesterol synthesis from differ ent labelled substrates fits well with the reduced activ ity of HM G-CoA reductase observed in this study. Three substrates, proximal to the reductase step, were incorporated into cholesterol at a reduced rate whereas no reduction from mevalonate was observed in any of the treatm ent groups. Lowering of HMG-CoA reduc tase levels by clofibrate is in accord with other observa tions [9,37,38]. Even though the three compounds tested in this study reduce the HMG-CoA reductase activity, this effect may not be a characteristic of all peroxiso mal inducers. M EDICA 16. another compound in this group, does not act via an effect on this reductase [12,39], but rather inhibits the synthesis of cholesterol at a step distal to HMG-CoA reductase. Our observa tions with clofibrate are at variance with the data obtained by Azarnoff et al. [10] who found that choles terol synthesis from mevalonic acid was reduced in livers of rats fed clofibrate. In contrast to the inhibitory effect of M EDICA 16 on ATP-citrate lyase [39], clofibric acid, PFOA and PFOSA had essentially no direct effect on ATP-citrate lyase at 1 mM (data not shown), but the drugs downregulated the enzyme after dietary administration. This effect may contribute to reduced synthesis of choles terol in vivo, since the enzyme is important in the main pathyway for cholesterol precursor synthesis. It is inter esting that all three compounds reduced one of the NADPH generating enzymes (glucose-6-phosphate de hydrogenase), while isocitrate dehydrogenase (NADP) was unchanged. Reduced capacity for NA DPH genera tion can therefore hardly contribute to reduction in serum lipid levels. The synthesis of fatty acids from acetate or pyruvate was not reduced in hepatocytes from rats fed clofibrate (Table III), but was significantly reduced in the hepatocytes from rats fed PFOSA. The reduced rate of fatty acid synthesis in the PFOA and PFOSA groups is probably not related to reduction in scrum iriacvlglyccrols. In rats fed PFOSA. there was accumulation of liver triacvlglycerols. The lack of reduction of falls acid synthesis by clofibrate suggests that other pro- OGOOIG H ccsscs in lipid metabolism must be more essential for the hvpolipemia. Tomarelli et al. [4(1] found increased synthesis of lipids from acetate in rats fed clofibric acid and also in rats fed the very potent hypolipemic com pound W Y-14.643. Synthesis o f cholcsieryl ester and phospholipids Liver exports lipids to other organs mainly as VLDL particles. In addition to apolipoproteins. triacylglycerols and free cholesterol, these panicles consist mainly of esterified cholesterol and phospholipids. Hence, reduced hepatic synthesis of these components might lead to reduced transport of lipids from the liver. The hepatic content of cholestervl ester was reduced both in clofibrate and PFOSA fed rats (PFO A fed rats were not tested) even where free cholesterol was not reduced (Table I). Similar effect was obtained by Avignan et al. [41] with the hypolipemic drug MER-29. Reduced cholestery! ester production is most likely a result of downregulation of the ACAT activity which was observed in all treatm ent groups. A direct in hibitory effect on the enzyme by the compounds is probably of less importance since 100 mM of the com pounds did not affect the enzyme activity (higher con centrations were not tested). Vance [29] argued that synthesis of phospholipids may be limiting for lipopro tein synthesis. In this study, we have not observed any downregulation of enzymes involved in the synthesis or metabolism of phospholipids. W e have observed only small direct effects of clofibric acid on these enzymes. It seems likely, however, that reduced phospholipid synthesis plays a role in the lipid-reducing effect of the perfluorinated compounds, since these had direct in hibitory effect on several im portant enzymes. Parthasarathy et al. [27] suggested from their studies that inhibition of phosphatidyl-choline synthesis, par ticularly by the lysolecithin acyltransferase pathway, may be related to a d ru g 's effectiveness in decreasing serum lipids. We found that the transferase was mod erately inhibited by the perfluorinated compounds. We also observed that this enzyme was virtually unaffected by 0.5 mM (Table VII) and also by 1 mM of clofibric acid. This agrees with the data reported by Parthasarathy et al. [27], O ur data do not support the hypothesis that reduced activity of lysolecothine acyl transferase plays a central role in the hypolipemic effect of the compounds we have tested. Inhibition of other enzymes of phospholipid synthesis may play a role. Reduced release o f lipids from the lirer In earlier studies, it has been reported that clofi- bratc reduces the release of lipids from the liver [13,14], A direct effect on the excretion process by clofibrate was not confirmed by accumulation of liver triacylglyccrols in elofibrate-fed. nor in PFOA-fed animals. A 71 reduced release of lipids from liver was probably an effect of PFOSA. however, since the compound in creased liver triacylglvccrols by about 200% in spite of reduced rate of fatty acid synthesis (Table IV). Since interference with t*he synthesis of cholestery! ester may cause reduced hepatic lipid output [42], it may be concluded that reduction in cholesterol synthe sis and esterification due to downregulation of HMGCoA reductase and ACAT together with enhanced fatty acid oxidation in the liver, are effects caused by clofibric acid as well as by the perfluorinated com pounds. This may reduce V LD L production by liver which plays a central role a fter the postprandial chylomicronemic period during which the liver is the dom inating organ for delivery of lipids to serum. In addition, the different hypolipemic drug may act by inhibiting the synthesis of other lipoprotein compo nents such as phosphatidylcholine [27]. Acknowledgements Financial support was received from Norwegian Council on Cardiovascular Diseases; Anders Jahres Foundation for promotion of Science, Norway; and The Insulin Fond, Copenhagen, Denmark. We thank D r. 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