Alterations by perfluorooctanoic acid of glycerolipid metabolism in rat liver

Chemico-Biological Interactions 118 (1999) 69 – 83

Alterations by perfluorooctanoic acid of glycerolipid metabolism in rat liver Naomi Kudo a, Hiroki Mizuguchi b, Aya Yamamoto a,1, Yoichi Kawashima a,* a

Faculty of Pharmaceutical Sciences, Josai Uni6ersity, Keyakidai 1 -1, Sakado, Saitama 350 -0295, Japan b Research Laboratories, Torii Pharmaceutical, 1 -2 -1 Ohnodai, Midori-ku, Chiba-shi, Chiba 267 -0056, Japan Received 24 August 1998; accepted 2 January 1999

Abstract The effects of perfluorooctanoic acid (PFOA) feeding on hepatic levels of glycerolipids and the underlying mechanism were investigated. Feeding of rats with 0.01% of PFOA in the diet for 1 week caused an increase in the contents of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and triglyceride (TG), which were 2.2, 2.4, 2.4, 1.6 and 5.2 times over control, respectively, on the basis of whole liver. The activities of glycerol-3-phosphate acyltransferase, diacylglycerol kinase and PtdSer decarboxylase were significantly increased upon PFOA feeding, whereas the activities of CTP:phosphoethanolamine cytidylyltransferase and PtdEtn Nmethyltransferase were decreased. On the other hand, the activity of CTP:phosphocholine cytidylyltransferase was not increased by PFOA. Upon PFOA feeding, hepatic level of 16:0–18:1 PtdCho was markedly increased and, by contrast, the levels of molecular species of PtdCho which contain 18:2 were decreased, resulting in the reduced concentration of molecular species of serum PtdCho containing 18:2. The increase in the level of hepatic 16:0–18:1 PtdCho seemed to be due to 3-fold increase in the activities of both D9 desaturase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase. The mechanism by which

* Corresponding author. Tel.: +81-492-71-7676; fax: +81-492-71-7984; e-mail: [email protected]. 1 Present address: Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Ichigaya, Funakawa-machi, Shinjuku-ku, Tokyo 162-0286, Japan. 0009-2797/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 0 0 2 - 2

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PFOA causes the accumulation of glycerolipids in liver was discussed. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Perfluorooctanoic acid; Glycerolipid metabolism; Molecular species composition

1. Introduction Perfluorooctanoic acid (PFOA) is widely used as a lubricant, detergent and wetting agent [1]. PFOA causes a hepatomegaly and peroxisome proliferation in the liver of rodent as well as other peroxisome proliferators [2–4]. In addition to peroxisomal proteins, several proteins and enzymes that are localized in mitochondria and endoplasmic reticulum are efficiently induced by PFOA [5–8]. An increase in the supply of phospholipids is required for rapid proliferation of organelles. In fact, PFOA also causes a considerable increase in the levels of phospholipids and triglyceride (TG) in livers of rats [9] and mice [10]. Many enzymes participate in synthesis of phospholipids and TG. PFOA, therefore, may alter the activities of such enzymes, resulting in an increase in the levels of phospholipids and TG in the liver. The synthetic rate of TG from acetate was shown to be accelerated in the liver of rats treated with PFOA [11]. To date, however, few studies have focused on the effects of PFOA on the activities of enzymes that are involved in the synthesis of TG and phospholipids, except for the effect of PFOA on CDPcholine:diacylglycerol cholinephosphotransferase [12] and CTP:phosphocholine cytidylyltransferase [13]. In the present study, the effects of PFOA feeding on the levels of individual glycerolipids and their molecular species composition were analyzed in rat liver and the metabolic changes in biosynthesis of glycerolipids were also determined to clarify the mechanism by which PFOA caused an increase in glycerolipids in liver.

2. Materials and methods

2.1. Materials [Methyl-14C]phosphocholine was purchased from Amersham; [methylC]choline, L-[14C(U)]glycerol-3-phosphate (153.2 Ci/mol) and cytidine diphospho[methyl-14C]choline (55.5 Ci/mol) were from Du Pont-New England Nuclear. S-Adenosyl-L-[methyl-14C]methionine (47 Ci/mol) was from ICN Biomedicals. Phosphocholine, CDP-choline, glycerol-3-phosphate, palmitoyl-CoA, phospholipase C (Cl. welchii ) and bovine serum albumin (BSA) (essentially fatty acid free) were purchased from Sigma, St. Louis, MO. N-Methyl-phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) (from egg) were purchased from NuCheck Prep.; CTP was purchased from Yamasa Biochemicals (Osaka, Japan); S-adenosyl-L-methionine was from Boeringher Mannheim (Germany); Tween 20 14

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was from Wako Chemicals (Osaka, Japan); Triton X-100 (reduced) was from Nakarai Tesque (Kyoto, Japan); PFOA was from Aldrich. Diacylglycerol was prepared enzymatically from egg PtdCho according to Wood and Snyder [14] and purified as described by Ishidate et al. [15]. NADH, NADPH, ATP and CoA were obtained from Oriental Yeast (Tokyo, Japan). All other chemicals were of analytical grade.

2.2. Animals Male Wistar rats of 6 weeks of age were obtained from SLC (Hamamatsu, Japan). After acclimatization for 1 week, the rats were fed a standard diet (CE-2, Clea, Tokyo, Japan) or a diet containing PFOA for 1 week. The diet was admixed with PFOA at the concentrations of 0.0025, 0.005, 0.01, 0.02 and 0.04% (w/w).

2.3. Preparation of subcellular fractions Rats were killed by decapitation under light ether anesthesia, and the blood samples were collected in centrifuge tubes for serum isolation at 09:00–11:00 h in the morning. The liver was excised and perfused with cold 0.9% NaCl. The liver was cut up into three pieces. One of them was frozen in liquid nitrogen and stored at −80°C until use for lipid analyses. The second part of the liver was homogenized in 4 vol. of 0.25 M sucrose/1 mM EDTA/10 mM Tris–HCl, pH 7.4. The homogenates were centrifuged at 18 000× g for 20 min and the supernatant was recentrifuged under the same conditions. The resulting supernatant was used as 18 000× g-supernatant. Microsomes and cytosol were prepared as described previously [16]. For the preparation of a mitochondrial fraction, the third part of the liver was homogenized in nine volumes of 0.25 M sucrose, 0.1 mM EDTA, 10 mM Tris – HCl buffer (pH 7.4). The homogenates were centrifuged at 600× g for 10 min. The supernatant was centrifuged at 5000× g for 10 min. The pellet was suspended in the original volume of the homogenizing buffer and recentrifuged under the same conditions. The resulting pellet was washed again in the same manner. The pellet obtained was resuspended in one tenth of the original volume of 0.25 M sucrose containing 10 mM Tris–HCl (pH 7.4) and was used as a mitochondrial fraction. Microsomes and cytosol for the assay of CTP:phosphocholine cytidylyltransferase were according to Pelech et al. [17]. All operations were carried out at 0 –4°C. Concentrations of protein were measured by the method of Lowry et al. [18] with bovine serum albumin as a standard.

2.4. Enzyme assays Glycerol-3-phosphate acyltransferase activity in microsomes was determined according to Yamada and Okuyama [19] using palmitoyl-CoA and [14C]glycerol-3phosphate. Diacylglycerol acyltransferase was assayed by the method of Bell and Miller [20] using [14C]palmitoyl-CoA and dioleoylglycerol added in ethanol. In brief, the reaction mixture of 0.36 ml contains 6 nmol [1-14C]palmitoyl-CoA, 3.2

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mmol MgCl2, 0.4 mg BSA and 8 mg microsomal proteins in 0.5 M Tris–HCl (pH 8.0). The mixture was preincubated at 23°C for 5min. Then the reaction was started by adding 50 nmol dioleoylglycerol which was dissolved into 40 ml ethanol. The activity of CDP-choline:diacylglycerol cholinephosphotransferase in microsomes was determined by the method of Ishidate et al. [15] using CDP-[methyl-14C]choline and diacylglycerol (from egg) added in Tween dispersion. CTP:phosphocholine cytidylyltransferase was assayed in 18 000× g supernatant, cytosol and microsomes according to Ishidate et al. [15] using [14C]phosphocholine; the activities in 18 000× g supernatant and cytosol were measured in the absence as well as in the presence of lipid vesicles comprising oleic acid and egg PtdCho [21]. CTP:phosphoethanolamine cytidylyltransferase in cytosol was assayed according to Sundler [22] using [14C]ethanolamine phosphate. PtdEtn N-methyltransferase in microsomes was assayed according to Audubert and Vance [23] using [14C]S-adenosyl methionine in the absence as well as in the presence of 1.2 mM N-methylPtdEtn. The activities of choline kinase and ethanolamine kinase were determined in cytosol by the method of Ishidate et al. [15] using [14C]choline and [14C]ethanolamine, respectively. The activity of phosphatidylserine (PtdSer) decarboxylase in mitochondria was measured using phosphatidyl-[3-14C]serine according to Houweling et al. [24]. Stearoyl-CoA desaturase was assayed essentially according to Oshino and Sato [25] as described previously [5]. The rate constant for the re-oxidation of NADH-reduced cytochrome b5 was measured in the presence (k) and in the absence (k − ) of stearoyl-CoA; the rate constant for D9 desaturation was given by k + = k−k − . Microsomal 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase was assayed spectrophotometrically using oleoyl-CoA as a substrate as described previously [6].

2.5. Lipid analyses Lipid was extracted from hepatic homogenates and serum by the method of Bligh and Dyer [26]. Tripentadecanoin or methylpentadecanoate were used as internal standards for quantification of TG and phospholipids, respectively. Neutral lipids were separated by TLC on silica gel G plates (Merck, Darmstadt, Germany) developed with n-hexane/diethyl ether/acetic acid (80:30:1, v/v/v). Phospholipids were separated by TLC on silica gel G developed with chloroform/methanol/acetic acid/water (50:37.5:5:3.5). After extraction of each lipid from silica gel, fatty acids were converted to their methyl esters and analyzed by GLC as described previously [27]. Molecular species was determined in PtdCho and PtdEtn according to Blank et al. [28] with some modifications as described previously [29].

2.6. Statistical analysis ANOVA was used to test the significance of the difference between means. Where the difference was significant, the statistical significance of the difference between any two means was determined using She´ffe’s multiple range test. Statistical significance between control and 0.02% PFOA-fed rats was analyzed by Student’s t-test or Welch’s test after F-test for two means.

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3. Results

3.1. Animals Feeding of the diet containing PFOA did not affect food consumption during the periods under the present experimental conditions. The difference of the body weight between experimental groups was not statistically significant (body weights of control, 0.0025% PFOA, 0.005% PFOA, 0.01% PFOA, 0.02% PFOA and 0.04% PFOA-feeding groups were 17998, 173 9 5, 178 9 3, 179 9 6, 174 9 8 and 164 9 12 g, respectively). Hepatomegaly was observed upon PFOA feeding, and liver weights of control, 0.0025% PFOA, 0.005% PFOA, 0.01% PFOA, 0.02% PFOA and 0.04% PFOA-feeding groups were 11.059 0.78, 12.159 0.54, 14.2690.20, 15.72 91.18, 15.859 1.29 and 15.0491.18 g, respectively.

3.2. Changes in glycerolipid contents Effects of PFOA-feeding on hepatic contents of glycerolipids were studied (Fig. 1). When compared on the basis of per g liver, contents of PtdCho, PtdEtn, phosphatidylinositol (PtdIns) and TG were increased, whereas PtdSer content was not changed (Fig. 1A). The increase in PtdEtn, PtdCho, PtdIns and TG was observed in a dose-dependent manner from 0.0025 to 0.01% PFOA and reached

Fig. 1. Effects of perfluorooctanoic acid (PFOA)-feeding on the contents of glycerolipids in rat liver. Rats were fed a diet containing PFOA (0.025 – 0.04%, w/w) for 1 week. Hepatic glycerolipid contents were expressed per gram liver (A) and per whole liver (B). Closed circles, phosphatidylcholine (PtdCho); open circles, phosphatidylethanolamine (PtdEtn); closed triangles, phosphatidylinositol (PtdIns); open triangles, phosphatidylserine (PtdSer); and open diamonds, triglyceride (TG), respectively. Values represent mean 9 S.D. for four to six rats. Some data points contain error bars within the size of the symbols. ANOVA was used to test the significance of the difference between means. Where the difference was significant, the statistical significance between any two means was determined using She´ffe’s multiple range test. *, Significantly different from respective controls (P B 0.05).

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Table 1 The effects of perfluorooctanoic acid (PFOA)-feeding on the activities of enzymes involved in the biosynthesis of glycerolipidsa Enzymes

(nmol/min/mg protein) Control

PFOA-fed

Glycerol-3-phosphate acyltransferase Diaclyglycerol acyltransferase Choline kinase

10.2 90.84 5.77 90.95 2.46 90.41

16.9 9 1.17*** 10.55 9 0.51*** 2.90 9 0.31

CTP:phosphocholine cytidylyltransferase 18 000×g sup (+vesicle) Cystsol (−vesicle) Cytosol (+vesicle) Microsomes

3.68 9 0.21 0.29 90.10 4.40 90.49 0.98 90.30

3.71 9 0.31 0.05 9 0.00* 4.51 90.35 0.28 90.05*

CDP-choline:diacylglycerol cholinephosphotransferase Ethanolamine kinase CTP:phosphoethanolamine cytidylyltransferase Phosphatidylethanolamine (PtdEtn) N-methyltransferase Phosphatidylserine (PtdSer) decarboxylase

31.6 9 3.30 1.46 90.02 4.88 90.52 9.80 90.20 0.57 9 0.07

36.7 92.14 1.62 90.25 4.01 9 0.28** 8.58 9 0.32** 0.73 9 0.03**

* PB0.05; ** PB0.01; *** PB0.001 are the statistically significant differences between control and PFOA-treated rats, respectively. a Male Wistar rats were fed a standard diet or a diet containing 0.02% (w/w) PFOA for 1 week. Results are means 9S.D. for three to five animals.

maximum at 0.01% PFOA in the diet. No further increase in the levels of PtdEtn, PtdCho and PtdIns was observed over 0.02% PFOA in the diet (analyzed by She´ffe’s multiple range test). The absolute mass of PtdCho, PtdEtn, PtdIns, PtdSer and TG in whole liver of rats fed the diet that contained 0.01% PFOA were 2.2, 2.4, 2.4, 1.6 and 5.2 times, respectively, over control (Fig. 1B), since PFOA induced hepatomegaly in rats.

3.3. Acti6ities of the enzymes in6ol6ed in the formation of glycerolipids To determine whether the synthesis of glycerolipids is stimulated by PFOA, the activities of enzymes which are involved in the biosynthesis of glycerolipids were compared between control and rats fed a diet containing 0.02% PFOA (Table 1). The treatment with PFOA significantly reduced CTP:phophocholine cytidylyltransferase in microsomes. Cytosolic CTP:phosphocholine cytidylyltransferase activity was significantly lower in PFOA-treated rats than in control rats when assayed in the absence of vesicles, whereas the treatment of rats with PFOA did not change the activity when assayed in the presence of vesicles. We also determined the activity of this enzyme using 18 000×g supernatant to estimate total activity of the tissue, since CTP:phosphocholine cytidylyltransferase is known to be translocated

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from cytosol to membrane. When assayed in the presence of vesicles, the activities were not significantly different to each other. The activities of choline kinase and choline phosphotransferase were not altered by the treatment with PFOA. Significantly lower activity was observed in PtdEtn N-methyltransferase in the rats treated with PFOA compared to control rats. As for the enzymes which are involved in the biosynthesis of PtdEtn, the activity of ethanolamine phosphate cytidylyltransferase was reduced by the feeding of rats with PFOA whereas ethanolamine kinase was not affected. The activity of PtdSer decarboxylase was significantly higher in PFOA-fed rats than in control rats. The activities of glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase, which are involved in the synthesis of TG, were significantly higher than those seen in control rats.

3.4. Effect of PFOA on the content of indi6idual fatty acids in glycerolipids Effects of PFOA-feeding on the content of individual fatty acids were examined (Table 2). In PtdCho, PFOA increased the content of 16:0 and 18:1, which accounted for the net increase in PtdCho as observed in Table 2A. By contrast, the level of 22:6 in PtdCho was reduced by the feeding of 0.01% PFOA by 75%. In PtdEtn, increase in the contents of 18:0 and 20:4 was observed by the feeding of rats with PFOA as well as 16:0 and 18:1. The content of 22:6 in PtdEtn was reduced by the treatment with PFOA as was seen in PtdCho. In TG, the major components were 18:1, 16:0 and 18:2, in which levels were changed by the feeding of rats with PFOA in a similar manner. Among those fatty acids, the increase in the level of 18:1 was predominant and the content of 18:1 in TG in whole liver of rats fed 0.01% PFOA was five times higher than that of control rats. Analysis of molecular species revealed that an increase in the amount of PtdCho is mainly due to increase in 16:0 –18:1 species (Fig. 2A). Such an increase resulted in an increase in the proportion of the species that contained 18:1 and a decrease in the proportion of the species that contained 18:2 (Fig. 2B). By contrast, an increase in the amount of species that contained 20:4 was mainly responsible for an increase in the amounts of PtdEtn (Fig. 2C). Other molecular species that contained 18:2 and 18:1 were also increased upon PFOA-feeding. Therefore, the composition of molecular species of PtdEtn was not significantly altered except that the proportion of 16:0 – 22:6 species decreased (Fig. 2D).

3.5. Effect of PFOA on the enzymes in6ol6ed in modification of acyl composition of phospholipids Hepatic stearoyl-CoA desaturase activity, which catalyzes the conversion of 18:0 to 18:1, was 2.9 times higher in the rat fed 0.02% PFOA in the diet than that in control rats (Table 3). The activity of 1-acyl-GPC acyltransferase was markedly increased by the feeding of PFOA.

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Table 2 Effects of perfluorooctanoic acid (PFOA)-feeding on the contents of glycerolipids in rat livera Treatment

Control

Fatty acid

(mmol/g liver)

0.0025%

0.005%

0.01%

0.02%

0.04%

(A) Phosphatidylcholine (PtdCho) 16:0 10.619 1.26 11.2890.74 12.20 91.27 13.40 91.36 13.61 90.89 12.54 9 0.68 18:0 7.599 0.51 8.32 9 0.25 8.20 9 0.66 7.56 9 1.04 8.50 90.43 7.78 90.52 18:1 2.949 0.47 3.299 0.26 4.37 9 0.28* 5.25 90.65* 6.53 90.48* 6.32 90.37* 18:2 7.159 0.86 6.139 1.00 6.56 90.47 7.01 90.54 6.67 90.72 6.50 9 0.11 20:4 17.709 1.35 7.809 0.43* 7.53 90.74* 6.69 91.12* 7.26 90.45* 6.16 90.28* 22:6 4.369 0.78 1.239 0.34* 1.16 9 0.89* 1.07 90.19* 1.04 90.13* 0.89 90.43* (B) Phosphatidylethanlolamine (PtdEtn) 16:0 4.599 0.27 5.549 0.92 18:0 3.969 0.33 6.249 0.11* 18:1 0.869 0.16 1.16 9 0.46 18:2 1.409 0.19 1.62 9 0.17 20:4 3.849 0.38 6.75 9 0.49* 20:6 2.019 0.14 1.9690.11

5.26 9 0.38 5.61 90.70* 1.18 90.91 1.47 9 0.10 6.26 90.63* 1.57 90.21

5.58 9 0.24* 5.67 90.50* 1.31 90.59* 1.65 90.13 6.47 90.57* 1.49 90.19*

5.75 9 0.64* 6.05 90.55* 1.67 9 0.19* 1.70 90.41 6.97 90.70* 13.5 9 0.16*

5.15 90.27 5.84 90.31* 1.79 9 0.33* 1.57 90.41 6.63 90.27* 1.22 9 0.80*

(C) Phosphatidylserine (PtdSer) 16:0 0.339 0.03 0.339 0.02 18:0 1.359 0.16 1.459 0.07 18:1 0.199 0.02 0.25 90.00 18:2 0.429 0.03 0.519 0.03 20:4 0.949 0.11 0.839 0.06 22:6 0.389 0.03 0.379 0.02

0.39 90.06 1.14 9 0.11 0.20 9 0.06 0.32 9 0.15 0.81 9 0.16 0.36 9 0.03

0.46 9 0.02 1.18 90.10 0.22 90.03 0.03 9 0.05 0.73 90.08 0.41 90.04

0.43 9 0.07 1.15 90.18 0.22 90.05 0.26 9 0.04 0.6490.08 0.36 90.06

0.4290.14 1.94 90.42 0.26 90.10 0.29 90.12 0.92 90.28 0.64 9 0.17*

(D) Phosphatidylinositol (PtdIns) 16:0 0.679 0.06 1.049 0.07 18:0 2.6590.18 3.7090.20 18:1 0.129 0.02 0.19 9 0.02 18:2 0.319 0.03 0.299 0.04 20:4 2.219 0.02 0.169 0.01

1.37 9 0.24* 3.89 9 0.61 0.32 9 0.04 0.35 90.06 0.24 90.04

1.39 90.25* 3.41 9 0.64 0.44 90.11* 0.50 90.10* 0.24 9 0.01

1.44 9 0.12* 3.29 9 0.49 0.55 90.06* 0.46 9 0.08 0.27 90.01

1.50 9 0.198* 3.96 90.50* 0.79 90.13* 0.54 90.06* 0.28 90.05

(E) Triglyceride (TG) 16:0 5.889 0.83 18:0 0.579 0.17 18:1 4.429 0.46 18:2 2.069 0.44 20:4 0.169 0.05 22:6 0.119 0.05

14.1890.54* 14.67 91.62* 16.98 91.92* 11.33 91.13* 12.05 9 1.56* 0.57 90.3 0.57 90.10 0.83 9 0.10 0.47 9 0.08 0.52 90.08 12.88 90.88* 16.37 91.96* 22.61 91.57* 16.70 91.21* 19.35 9 2.63* 6.32 9 1.10* 5.77 9 1.21* 5.27 90.63 2.99 90.99 3.02 90.35 0.47 9 0.07* 0.39 90.11* 0.42 90.07* 0.22 90.07 0.24 9 0.02 0.389 0.09* 0.28 9 0.12 0.21 90.04 0.07 90.06 0.05 90.04

* ANOVA was used to test the significance of the difference between means. Where the difference was significant, the statistical significance between any two means was determined using She´ffe’s multiple range test. Significantly different from respective controls (PB0.05). a Rats were fed a diet containing PFOA (0.025–0.04%, w/w) for 1 week. Fatty acids which were esterified to PtdCho (A), PtdEtn (B), PtdSer (C), PtdIns (D) and TG (E) were quantified by GLC and expressed as mmol/g liver. Values represent mean 9 S.D. for four to six rats.

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3.6. Alteration of serum lipids by PFOA As shown in Table 4, PFOA significantly reduced the levels of serum PtdCho, TG and cholesterol. The reductions were 45% in PtdCho, 40% in cholesterol and 34% in TG. The composition of molecular species of PtdCho was compared between control and PFOA-fed rats (Fig. 3). In PtdCho of control serum, 16:0– 18:2 and 18:0 – 18:2 are predominant molecular species and accounted for approximately 50% of PtdCho (Fig. 3A). Compared to hepatic composition of PtdCho molecular species in control rats (Fig. 2B), the ratio of molecular species containing 18:2 to those containing 18:1 and 20:4 was higher in PtdCho of serum (1.07 in liver and 2.75 in serum). When rats were fed the diet containing PFOA, the mass of all PtdCho molecular species except for 16:0–18:1, 18:0– 18:1, 16:0 – 20:3, 18:0 – 20:4 and 16:0–16:0 was reduced (Fig. 3B). The reduction of 16:0 – 18:2 and 18:0 – 18:2 caused 58% of total reduction of PtdCho.

4. Discussion The aim of this study is to clarify the metabolic changes responsible for an increase in hepatic glycerolipids in the rat fed a diet containing PFOA. The present study demonstrated that PFOA caused elevation of the hepatic levels of phospholipids and TG. The elevation was the most prominent in TG (Fig. 1). Clofibrate did not cause TG accumulation in liver [16], despite that this drug is a peroxisome proliferator similar to PFOA. Two plausible mechanisms can be considered for the explanation of TG accumulation by PFOA in liver. First, TG synthesis in liver is up-regulated by PFOA-feeding. Pastoor et al. demonstrated that the treatment of rats with PFOA increased TG synthesis in vivo from [14C]acetate by increasing synthesis de novo of fatty acid [11]. Conflicting results, however, were provided by Davis et al. [30] who suggested the synthesis of fatty acid from [3H]2O was suppressed by perfluorodecanoic acid, a structurally related compound of PFOA. The present results demonstrated that PFOA-feeding caused an increase in the activities of TG-synthesizing enzymes which may be responsible for TG accumulation in liver. Moreover, the present study showed that PFOA increased the activity of D9 desaturase (Table 3) which supplied the increased amounts of 18:1 to TG formation. The second possibility is that PFOA causes an impairment of TG secretion in rat liver. The fact that PFOA-feeding caused the reduction of serum level of TG (Table 4) suggested this mechanism, although no direct evidence is available at the present time. PFOA causes hepatomegaly which is accompanied by a drastic increase in the number of peroxisomes [2 – 4]. Therefore, it seems likely that the increase in the synthesis of phospholipids should be required for supplying components of mem-

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Fig. 2. Effects of perfluorooctanoic acid (PFOA)-feeding on the molecular species composition of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) in rat liver. Rats were fed a standard diet (hatched bars) or a diet containing 0.02% PFOA (w/w, closed bars) for 1 week. Molecular species were analyzed on PtdCho (A and B) and PtdEtn (C and D). Values represent mmol/whole liver (A and C) and % composition of molecular species (B and D). Data was mean 9S.D. for four to six rats. *, Statistically significant difference between control and PFOA-fed rats (P B0.05).

brane. In fact, the contents of phospholipids, such as PtdCho, PtdEtn and PtdIns were increased by 2-fold in whole liver by PFOA-feeding (Fig. 1). The fold of the increase in PtdCho and PtdEtn was greater than that recently reported [31]. This

Table 3 The effects of perfluorooctanoic acid (PFOA)-feeding on the activities of enzymes responsible for 18:1 content in PtdChoa Enzymes

Control

PFOA-fed

Stearoyl-CoA desaturase k+ (min−1) Acyl-CoA:1-acyl-GPC acyltransferase (nmol/min/mg protein)

1.10 9 0.26

3.14 9 0.35*

69.4 9 14.1

208.9 916.1**

* PB0.01; ** PB0.001 are the statistically significant differences between control and PFOA-fed rats. a Microsomal fractions were prepared from livers of rats which were fed a standard diet or a diet containing 0.02% (w/w) PFOA for 1 week. Values are means 9S.D. for four animals.

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Table 4 Effects of perfluorooctanoic acid (PFOA)-feeding on the levels of serum lipidsa Serum lipid (mmol/ml)

Control

PFOA-fed

Total phospholipid Phosphatidylcholine (PtdCho) Triglyceride (TG) Cholesterol

2.45 9 0.37 1.58 9 0.27 2.00 90.19 1.78 90.19

1.63 90.08* 0.87 90.06* 1.32 9 0.19* 1.08 9 0.08**

* PB0.05; ** PB0.001 are the statistically significant differences between control and PFOA-fed rats. a Rats were fed a standard diet or the diet containing 0.02% PFOA (w/w) for 1 week. Values are means9 S.D. for four animals.

seems due to the different conditions of the treatment of PFOA. Accordingly, the metabolic changes in the synthesis of phospholipids were investigated, especially PtdCho and PtdEtn, major components of membrane. An increase in glycerol-3phosphate acyltransferase activity (Table 1) seems to be responsible for the formation of all glycerolipids including phospholipids and TG, since it governs the common route of glycerolipid synthesis. In addition, PFOA caused various alterations of enzyme activities within the routes of individual phospholipid synthesis in different manners (Table 2). As for the enzymes which are involved in the synthesis of PtdCho, the activities of choline kinase and CDP-choline:diacylglycerol cholinephosphotransferase were not altered by PFOA (Table 1). The activities of both microsomal and cytosolic CTP:phosphocholine cytidylyltransferase were reduced by PFOA-feeding when assayed in the absence of lipid vesicles (Table 1). The result is in accordance with the results observed by Reo et al. [13]. When cytosolic CTP:phosphocholine cytidylyltransferase was assayed in the presence of lipid vesicles, however, no significant change was observed between control and PFOAfed rats. Moreover, the activity of CTP:phosphocholine cytidylyltransferase in post-mitochondrial supernatant fraction was not different between control and PFOA-fed rats when assayed in the presence of lipid vesicles. Consequently, therefore, total amounts of the enzyme seem not to be altered by PFOA-feeding. CTP:phosphocholine cytidylyltransferase is thought to be translocated from cytosol to membrane where it is active [32] and CTP:phosphocholine cytydylyltransferase is a key determinant for the synthesis de novo of PtdCho [32]. Therefore, the results may indicate that PFOA does not elevate the synthesis de novo of PtdCho. An alternative pathway of PtdCho formation is catalyzed by PtdEtn N-methyltransferase, which is thought to be responsible for 20–40% of the PtdCho synthesized in hepatocytes in physiological conditions [33]. The present study demonstrated that PtdEtn N-methyltransferase activity was slightly reduced by PFOA (Table 1). These alterations suggest that PFOA causes the reduction of PtdCho synthesis, which is inconsistent with the results of mass determination of PtdCho. One possible

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explanation for this discrepancy is that PtdCho is used as a component of lipoprotein for secretion into circulation. This is supported by the fact that serum levels of PtdCho was markedly reduced by PFOA. The comparison of the molecular species of PtdCho between liver and serum of control rats indicated that a certain species such as 18:0 – 18:2 and 16:0–18:2 were selectively secreted to the circulation [34,35]. Molecular species analysis revealed that the species which contains 18:1 at the 2-position are specifically increased in hepatic PtdCho by PFOA-feeding (Figue 2). This event can be explained by an increase in the activities of both D9 desaturase and 1-acyl-GPC acyltransferase (Table 3), leading to an increase in 18:1 production and subsequent acyl exchange at 2-position in PtdCho. The altered proportion of PtdCho molecular species in the liver reflected directly the composition of molecular species of PtdCho in serum (Fig. 3). Namely, the

Fig. 3. Effects of perfluorooctanoic acid (PFOA)-feeding on molecular species composition of phosphatidylcholine (PtdCho) in rat serum. Serum was prepared from control rats (hatched bars) or the rats which were fed the diet containing 0.02% PFOA for 1 week (closed bars). Molecular species of PtdCho was compared between control and PFOA-treated rats. Values represents % composition of molecular species (A) and mmol/ml serum (B). Data was mean 9S.D. for four rats.

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concentrations of 18:2-containing species in serum were selectively reduced by PFOA-feeding whereas others were less changed. The conclusion is that hepatic PtdCho is increased due to a reduction in secretion to serum lipoproteins, possibly brought about by a decrease in the content of 18:2 components in PtdCho. As to the effects of PFOA on PtdEtn-synthesizing enzymes, the activities of CTP:phosphoethanolamine cytidylyltransferase was slightly decreased by PFOA. Since this enzyme is thought to catalyze the rate-limiting step of PtdEtn synthesis de novo [36], this decrease in the activity might lead to a decrease in hepatic content of PtdEtn. By contrast, PtdSer decarboxylase activity was significantly higher in PFOA-treated rats and the activity of PtdEtn N-methyltransferase was slightly reduced by PFOA. The species of 16:0–20:4 and 18:0–20:4 were specifically increased in hepatic PtdEtn by PFOA-feeding. The elevation of these species was not observed in PtdCho. This may be due to the reduction of the activity of PtdEtn N-methyltransferase. In fact, PtdEtn contained a higher proportion of 20:4-containing species than PtdCho, therefore, the conversion of PtdEtn to PtdCho is known to increase the proportion of this species in PtdCho [37]. Taken together, PFOAfeeding seems to stimulate the synthetic phathways of PtdCho–PtdSer to PtdEtn, but not the pathway of synthesis de novo, resulting in overall increase in hepatic level of PtdEtn. In conclusion, the results suggested the following effects of PFOA on the metabolism of glycerolipids: (i) hepatic PtdCho is increased due to a reduction in secretion of lipoprotein to circulation, possibly brought about by a decrease in the level of PtdCho species containing 18:2; (ii) hepatic PtdEtn is elevated due to an increase in the activity of PtdSer decarboxylase in concert with a decrease in PtdEtn N-methyltransferase activity; (iii) hepatic TG is elevated due to a reduction of secretion of TG into circulation and to an increase in the activity of DG acyltransferase. In the present study, it is demonstrated that PFOA extensively affected glycerolipid metabolism in rat liver. The alterations seem to be responsible for remarkable increase in the levels of glycerolipids in rat liver. In addition, PFOA caused a reduction of serum levels of PtdCho which may be due, at least in part, to alteration of molecular species composition of hepatic PtdCho.

Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research from the president of Josai University and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

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