A Highly Toxic Coplanar Polychlorinated Biphenyl Compound

Graduate School of Pharmaceutical Sciences, Kyushu University 62, ... of liver homogenates was significantly reduced on day 5 of PenCB administration,...
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Chem. Res. Toxicol. 1999, 12, 1158-1165

A Highly Toxic Coplanar Polychlorinated Biphenyl Compound Suppresses ∆5 and ∆6 Desaturase Activities Which Play Key Roles in Arachidonic Acid Synthesis in Rat Liver† Kimihiko Matsusue,‡ Yuji Ishii, Noritaka Ariyoshi,§ and Kazuta Oguri* Graduate School of Pharmaceutical Sciences, Kyushu University 62, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Received June 15, 1999

The effect of 3,3′,4,4′,5-pentachlorobiphenyl (PenCB) on the synthesis of unsaturated fatty acids was studied in male Wistar rats. The arachidonic acid (20:4) content in the total lipids of liver homogenates was significantly reduced on day 5 of PenCB administration, while those of linoleic acid (18:2) and bishomo-γ-linolenic acid (20:3) were increased. These changes in the total lipids of liver homogenates were observed following doses of PenCB ranging from 0.5 to 25 mg/kg of body weight. The same changes in these fatty acids were seen with four subtypes of microsomal glycerophospholipids in the liver. The marked reduction in the molar ratio of 20:4 to 18:2 in the lipids suggests alteration of the activity of the enzymes responsible for the synthesis of unsaturated fatty acids. The activity of ∆5 and ∆6 desaturases (arachidonic acid synthetase) in the liver microsomes was 17 and 13% of that of pair-fed control animals, whereas the activity of 1-acylglycerophosphorylcholine or 1-acylglycerophosphate acyltransferase, which transfers 20:4 or 18:2 to phospholipids, was not affected by the treatment. Thus, the reduction in the level of 20:4 that was observed can be explained by a reduction in desaturase activity. These results are evidence that the coplanar PenCB has a significant effect on the reduced synthesis of physiologically essential long-chain unsaturated fatty acids.

Introduction Polychlorinated biphenyls (PCBs)1 are ubiquitous environmental contaminants and were widely used as industrial chemicals (1, 2). Coplanar PCBs have been shown to produce toxic effects similar to that of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), and 3,3′,4,4′,5-pentachlorobiphenyl (PenCB, IUPAC PCB 126) is the most toxic congener of all PCBs (3). Coplanar PCBs are currently causing considerable anxiety as are TCDD contaminants (4, 5). We know a great deal about a trigger binding protein, the aryl hydrocarbon receptor (Ah receptor) (6, 7), but much less about the mechanism for the detailed expression of toxicity. Previous researchers have demonstrated that the exposure of animals, including † Presented in part at the 22nd Symposium on Environmental Toxicology, Niigata, Japan, October 1996 [Matsusue, K., et al. (1997) Jpn. Toxicol. Environ. Health 43, s-19 (abstract)]. * To whom correspondence should be addressed: Graduate School of Pharmaceutical Sciences, Kyushu University 62, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: +81-92-642-6585. Fax: +81-92-642-6588. E-mail: [email protected]. ‡ Present address: Division of Chemotherapy, National Kyushu Cancer Center, 3-1-1 Notame, Minami-ku, Fukuoka 815-1375, Japan. § Present address: Graduate School of Pharmaceutical Sciences, Hokkaido University, N12W6, Sapporo, Hokkaido 060-0812, Japan. 1 Abbreviations: PCB, polychlorinated biphenyl; PenCB, 3,3′,4,4′,5pentachlorobiphenyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; Ah receptor, aryl hydrocarbon receptor; 20:5, eicosapentaenoic acid; 18:3, R-linolenic acid; 14:0, myristic acid; 22:6, docosahexaenoic acid; 20:4, arachidonic acid; 18:2, linoleic acid; 20:3, bishomo-γ-linolenic acid; 16: 0, palmitic acid; 18:1, oleic acid; 17:0, margaric acid; 18:0, stearic acid; fp1, NADH-cytochrome b5 reductase; GPC, 1-acylglycerophosphorylcholine acyltransferase; GP, 1-acylglycerophosphate acyltransferase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.

humans, to PenCB or TCDD causes a marked disorder in lipid metabolism (8-10). These chemicals have received a great deal of attention as they increase the level of triglycerides or cholesterol in liver or blood. The fatty acid composition is altered in many diseases as a consequence of the illness itself, although the detailed mechanism remains unknown (11, 12). A balance of polyunsaturated fatty acids in cellular phospholipids is required to maintain the physiological functions of cells. Fatty acids liberated by lipase, in addition, are modulators of enzyme activity (13, 14) or gene transcription (15, 16). Most fatty acids retaining these functions are long-chain unsaturated fatty acids. Guitart et al. (17) have demonstrated that PCB concentrations in the liver of striped dolphins correlate with the alteration in fatty acid composition. Thus, the unusual lipid metabolism following administration of TCDD or PenCB needs to be monitored for accurate evaluation of the change in lipid composition as well as lipid levels. We have shown that PenCB-treated rats exhibit unusual changes in the fatty acid composition of whole liver or in four subtypes of glycerophospholipid in liver homogenates (18). The most important change was the reduction in the level of arachidonic acid (20:4) and the increase in the level of linoleic acid (18:2). However, no attempt has yet been made to investigate the mechanism behind the change in linoleic acid (18:2) and arachidonic acid (20:4) synthesis. Arachidonic acid (20:4) is synthesized from linoleic acid (18:2) by ∆6 desaturase, an elongation enzyme, and ∆5 desaturase in the liver endoplasmic reticulum. The step with ∆6 desaturase is

10.1021/tx990104r CCC: $18.00 © 1999 American Chemical Society Published on Web 11/04/1999

Suppression of Desaturases by Coplanar PCB

the rate-limiting step in 20:4 synthesis (19). Therefore, the suppression of 20:4 synthesis may result from a reduction in ∆6 desaturase activity in PenCB-treated rats. Further unsaturated fatty acids synthesized by desaturases are transferred by acyltransferase to the lipids. Acyltransferase is another important determinant regulating the fatty acid composition of lipids. In fact, a change in the activity of acyltransferase was shown to modulate the fatty acid composition of phospholipids (20). In light of the fact that there are acyltransferase isoforms (21, 22), PenCB may also affect the activity of the isoforms that utilize 20:4 or 18:2 most efficiently as a substrate. The report presented here focuses on the effect of PenCB on the desaturase activities responsible for arachidonic acid synthesis and acyltransferase activities responsible for the incorporation of arachidonic acid into glycerophospholipids. The experiments described in this paper show that PenCB causes a significant reduction in the proportion of 20:4 and the molar ratio of 20:4 to 18:2 in the glycerophospholipids of rat liver microsomes, and that the effect is due to a marked reduction in the activity of ∆5 and ∆6 desaturases.

Experimental Procedures Materials. PenCB was synthesized by the method of Saeki et al. (23). PenCB is a hazardous compound, and procedures were carried out in a chemical hazard chamber. The purity of PenCB was >98%, confirmed by GC using an electron-capture detector, MS, and NMR. [1-14C]Linoleic acid (59 mCi/mmol) and [1-14C]bishomo-γ-linolenic acid (47 mCi/mmol) were purchased from Amersham International and New England Nuclear (Boston, MA), respectively. Animal Treatment. Male Wistar rats (7 weeks old) were given a single intraperitoneal injection of PenCB dissolved in corn oil at a dose of 0.5, 1, 5, 10, and 25 mg/kg, and the same volume of corn oil alone was injected into PenCB-treated rats. To compare the effects of reduced food consumption produced by PenCB treatment, both free- and pair-fed controls were examined. Free-fed controls received vehicle and had free access to food and water. Pair-fed animals received vehicle and the same amount of chow as that consumed by PenCB-treated rats. The livers were removed from the PenCB-treated and control animals 5 days after injection, and none of the animals was fasted prior to being sacrificed. Liver homogenates and microsomes were prepared by standard procedures. Fatty Acid Composition. Whole liver homogenates or microsomes were extracted by the method of Bligh and Dyer (24). Isolation and purification of the phospholipid classes were performed according to the method of Fine and Sprecher (25) with slight modifications (18). The purified phospholipids were saponified according to the method of Korte et al. (26) with slight modifications (18). HPLC analysis of fatty acids using 3-(bromomethyl)-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone was carried out by the method of Yamaguchi et al. (27). The HPLC equipment used in this study was as follows. A Hitachi L-6200 intelligent pump was equipped with a fluorescence detector (Hitachi model L-7480 FL detector, Hitachi, Tokyo, Japan), a column (YMC Pak C8, YMC, Kyoto, Japan), a column heater (Sugai model U-620, Sugai, Tokyo, Japan), an autosampler (Hitachi model L-7200), and a data integrator (Hitachi model D-2500). The flow rate and column temperature were 2 mL/ min and 33 °C, respectively. The fluorescent derivatives of fatty acids were separated by HPLC using an acetonitrile density gradient as follows: 70 to 72% from 0 to 10 min, 72% from 10 to 17 min, 80% from 17 to 27 min, 80 to 95% from 27 to 32 min, 95% from 32 to 37 min, and 70% from 37 to 42 min. Enzyme Analyses. 1-Acylglycerophosphorylcholine or 1acylglycerophosphate acyltransferase activity was assayed by the method of Yamashita et al. (21).

Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1159 ∆5 and ∆6 desaturase activities were assayed by the method of Garda et al. (28) with slight modification. The reaction mixture contained 0.3-0.8 mg of microsomal protein, 0.25 M sucrose, 0.25 M KCl, 0.05 M phosphate buffer (pH 7.4), 0.7 mM N-acetylcystein, 0.04 M NaF, 0.33 mM nicotinamide, 1.3 mM ATP, 0.24 mM coenzyme A, 1 mM NADH, 5 mM MgCl2, and 30 µM fatty acid (0.2 µCi of [1-14C]linoleic acid or [1-14C]bishomoγ-linolenic acid) in a final volume of 0.2 mL. The reaction was stopped by adding 1 mL of 10% KOH/methanol after incubation for 15 (∆5 desaturase) or 20 min (∆6 desaturase) at 37 °C. After the addition of 50 µL of dibutylhydroxytoluene (15 mg/mL) and 75 µg of margaric acid (17:0) to each tube of unlabeled substrate and product of each desaturation reaction, the samples were saponified at 70 °C for 1 h and acidified with 0.2 mL of concentrated HCl. The free fatty acids were extracted twice with 2 mL of n-hexane; the organic solvent was evaporated, and the residues were methylated with 14% BF3 at 80 °C for 30 min. The methyl esters of the fatty acids were extracted twice with 2 mL of n-hexane and evaporated. The methyl esters of the fatty acids dissolved in chloroform were applied to TLC plates (Merck, Kieselgel 60, Art. 5582) impregnated with 10% AgNO3. The 10% AgNO3 TLC plates were activated at 80 °C for 1 h before use. The developing solvents were 90:10 (v/v) benzene/ethyl acetate (∆6 desaturase assay) and 90:10:1 (v/v) benzene/ethyl acetate/ acetic acid (∆5 desaturase assay), and detection of the labeled fatty acids separated on TLC was performed with a Bio-Imaging Analyzer (BAS 1000, Fuji Film, Tokyo, Japan). Content or Activity of Microsomal Electron-Transferring Protein. The content of cytochrome P450 or b5 was determined by the method of Omura and Sato (29). NADPHcytochrome c reductase and NADH-cytochrome b5 reductase activities were measured by the methods of Yasukochi and Masters (30) and Strittmatter (31), respectively.

Results A significant change in the 20:4 and 18:2 composition in whole liver homogenates and in the phospholipids of liver homogenates has been demonstrated at a dose of 25 mg of PenCB/kg (18). In this study, a dose-dependent alteration in the proportion of saturated (16:0 and 18:0) or n - 6 unsaturated fatty acids (18:2, 20:3, and 20:4) in whole liver was observed (Figure 1). In comparison with the pair-fed control groups, a significant reduction in the level of 16:0 or 18:0 was observed at doses of 0.5 mg/kg, as well as at doses of 5 and 25 mg/kg. The levels of 18:2 and 20:3 increased in a dose-dependent manner following PenCB treatment, while the level of 20:4 decreased. Significant alteration in the levels of three fatty acids was observed even at a dose of 0.5 mg/kg. Most fatty acids taken up or synthesized in the liver are known to be incorporated by acyltransferase into phospholipids. Figure 2 shows typical chromatograms of fatty acids in microsomal phosphatidylcholine of rat liver. The profile of the peaks (Figure 2C) of the fatty acids of microsomal phosphatidylcholine in PenCB-treated rats exhibits a significant difference compared with those of the free-fed (Figure 2A) or pair-fed (Figure 2B) control groups. There was a significant reduction in the magnitude of the peak of 20:4 (Figure 2, peak 5) and a significant increase in that of 18:2 (Figure 2, peak 6) in the PenCB-treated groups. The peak area of bishomo-γlinolenic acid (20:3, peak 7) or stearic acid (18:0, peak 11) also increased. Figure 3 shows the fatty acid composition in four subtypes of glycerophospholipids from liver microsomes of rats. In all subtypes of glycerophospholipids, the proportion of 20:4 was reduced significantly compared to those of pair-fed control groups, while the proportion of 18:2 and 20:3 increased. Figure 4 shows

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Figure 1. Effect of 3,3′,4,4′,5-pentachlorobiphenyl on the mole percentage of palmitic acid (16:0), stearic acid (18:0), linoleic acid (18:2), bishomo-γ-linolenic acid (20:3), and arachidonic acid (20:4) in whole rat liver. Male Wistar rats were given a single intraperitoneal injection of 0, 0.5, 1, 5, 10, and 25 mg of PenCB/kg, and liver homogenates were prepared from the PenCB-treated animals 5 days later. Each symbol represents the mean ( SE of the percentages for the free-fed control groups (0 mg of PenCB/kg). Values (micromoles per gram of liver) obtained by HPLC are given as the mole percentage of the main long-chain fatty acid (myristic, palmitic, stearic, oleic, R-linolenic, eicosapentaenoic, docosahexaenoic, linoleic, bishomo-γ-linolenic, and arachidonic acids) in liver homogenates from rats treated with each PenCB dose. White or black circles represent data for pair-fed controls (n ) 3) or PenCBtreated (n ) 4) groups, respectively. Details of the HPLC conditions are given in the text. Values significantly different from those of free-fed controls: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001. Values significantly different from those of pair-fed controls: (†) p < 0.05, (††) p < 0.01, and (†††) p < 0.001.

that the correlation between body weight gain and the proportion of 18:2 or 20:4 in whole liver. The correlation coefficients (r ) 0.647) were not very high; however, a significant correlation was observed (p < 0.0006, Figure 4). To examine the mechanism behind these results, the activity of the enzymes which control fatty acid composition was measured. 1-Acylglycerophosphorylcholine (GPC) or 1-acylglycerophosphate acyltransferase (GA) acylates fatty acids at the C-2 position of the 1-acylglycerophospholipid or 1-acylglycerophosphate. GPC or GA utilizes 20:4 or 18:2 efficiently as a substrate (21). As far as the enzyme activities were concerned, no notable difference was observed between the control and PenCB-treated groups (Figure 5). Figure 6 shows the molar ratio of arachidonic acid to linoleic acid (20:4 to 18:2) as an indicator of arachidonic acid synthesis (32, 33). The ratio of 20:4 to 18:2 decreased significantly even at a dose of 0.5 mg/kg. Furthermore, in all phospholipid fractions, the 20:4 to 18:2 ratio in the PenCB-treated groups was reduced by about 50%, although the reason for the high value of phosphatidylinositol in the pair-fed control groups is unknown (Table 1). Next, we examined the activity of the ∆5 and ∆6 desaturases which are 20:4 synthetic enzymes (Figure 7). The activity in liver microsomes from PenCB-treated rats was markedly reduced. ∆6 and ∆5 desaturase

activities were reduced to 13% of those of the free-fed and pair-fed control group, 10% of those of the free-fed control group, and 17% of those of the pair-fed control group. Since both desaturases need an electron flux supplied by microsomal electron-transfer proteins, the effect of PenCB on the content or activity of microsomal electrontransfer proteins was determined (Table 2). It is wellknown that PenCB is a potent inducer of cytochrome P450 (3, 34). This cytochrome P450 content, as a positive control, increased about 4-fold in comparison with those of the two control groups. In comparison with pair-fed controls, although the cytochrome b5 content in the PenCB-treated rats increased about 1.3-fold, no significant change in NADH-cytochrome b5 reductase (fp1) activity was observed.

Discussion In the studies presented here, we have demonstrated that PenCB treatment causes a marked reduction in ∆6 or ∆5 desaturase activity (Figure 7) in terms of a marked decrease in the fatty acid 20:4 to 18:2 ratio (Figure 6). This reduction appears to contribute largely to the reduction in the proportion of 20:4 in the whole liver (Figure 1) or microsomal glycerophospholipids (Figure 3 or 5). Although changes in hepatic fatty acid composition may be associated with the activity of many enzymes,

Suppression of Desaturases by Coplanar PCB

Figure 2. HPLC chromatograms of fatty acids in microsomal phosphatidylcholine of rat liver in the free-fed control (A), pairfed control (B), and PenCB-treated groups (C). Male Wistar rats were given a single intraperitoneal injection of 25 mg of PenCB/ kg, and the liver microsomes were prepared from the PenCBtreated (n ) 5) and control (n ) 4) animals 5 days later. Details of the HPLC conditions are given in the text: peak 1, eicosapentaenoic acid (20:5, n - 3); peak 2, R-linolenic acid (18:3, n - 3); peak 3, myristic acid (14:0); peak 4, docosahexaenoic acid (22: 6, n - 3); peak 5, arachidonic acid (20:4, n - 6); peak 6, linoleic acid (18:2, n - 6); peak 7, bishomo-γ-linolenic acid (20:3, n - 6); peak 8, palmitic acid (16:0); peak 9, oleic acid (18:1, n - 9); peak 10, margaric acid (17:0), as internal standard; and peak 11, stearic acid (18:0).

attention should be paid to the enzyme activity specifically affecting the hepatic distribution of 18:2 or 20:4.

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∆6 desaturase catalyzes a rate-limiting step in the biosynthesis of arachidonic acid. The reduction in the level of ∆6 desaturase which converts 18:2 (n - 6) to γ-18:3 (n - 6) can explain the increase in the level of 18:2 or the reduction in the level of 20:4. In fact, the change in ∆6 desaturase activity relates directly to the proportion of 18:2 and 20:4 or to de novo 20:4 synthesis in rat liver (35). It appears that the pattern of 20:3 change differs from that of 18:2 or 20:4 in the terms of the PenCB dose dependence (Figure 1). Although the reason is not clear, not only a reduction in ∆5 desaturase activity but also a change in the activity of the elongation enzymes, which convert from γ-18:3 to 20:3, may also contribute to this result. The activity of the elongation enzymes was not measured in this study because it did not appear to be a rate-limiting step in 20:4 synthesis (19). We demonstrated that PenCB affects the composition of unsaturated fatty acids more than that of saturated counterparts (Figure 1). This result appears to indicate that PenCB affects the activity of desaturase more than that of fatty acid synthase (saturated fatty acid synthase) found in cytosol. Our in vivo and in vitro results suggest that PenCB suppresses 20:4 synthesis, through a marked reduction in the activity of ∆5 and ∆6 desaturases. As an alternative mechanism, we also considered a reduction in GPC and GP acyltransferase, an enzyme which uses 20:4 and 18:2 most efficiently as a substrate (21), but no marked difference between the two control and PenCB-treated groups was observed (Figure 4). The substrate for desaturase is fatty acyl-CoA, not a free fatty acid. The quantity of fatty acyl-CoA in cells could be controlled by a balance between the activities of fatty acyl-CoA synthetase and hydrolase (36). However, the change in fatty acid composition in the study presented here does not appear to involve modulation of these

Figure 3. Effect of 3,3′,4,4′,5-pentachlorobiphenyl on the fatty acid composition of glycerophospholipid subtypes in rat liver microsomes. Values are given as the mole percentage (means ( SE) of the main fatty acids in rat liver. The dose of PenCB and preparation of microsomes are described in the legend of Figure 2. N.D. means not detectable (detection limit of 70 nmol/g of liver). Values significantly different from those of free-fed controls: (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001. Values significantly different from those of pair-fed controls: (†) p < 0.05, (††) p < 0.01, and (†††) p < 0.001.

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Table 1. Effect of 3,3′,4,4′,5-Pentachlorobiphenyl on the Arachidonic Acid to Linoleic Acid Ratio in Total Lipids and Glycerophospholipids of Rat Liver Microsomesa microsomal lipid fractions

free-fed control (n ) 4)b

pair-fed control (n ) 4)b

PenCB-treated (n ) 5)b

total lipids phosphatidylcholine phosphatidylethanolamine phosphatidylserine phosphatidylinositol

1.40 ( 0.05 (100) 1.05 ( 0.03 (100) 1.56 ( 0.05 (100) 4.01 ( 0.50 (100) 7.02 ( 0.80 (100)

1.67 ( 0.19 (119) 1.63 ( 0.08 (155)c 1.96 ( 0.05 (126)d 4.52 ( 0.21 (113) 21.0 ( 7.5 (299)

0.81 ( 0.10 (58)d,e 0.61 ( 0.03 (58)c,f 0.75 ( 0.06 (48)c,f 1.96 ( 0.18 (49)d,f 2.44 ( 0.30 (35)c,g

a Values show the molar ratio (means ( SE) of arachidonic and linoleic acid in the total lipids and glycerophospholipids of rat liver microsomes. The dose of PenCB and preparation of microsomes are described in the legend of Figure 3. The values in parentheses are relative to those of the free-fed control groups ()100). b Number of rats used. c Values significantly different from those of free-fed controls (p < 0.001). d Values significantly different from those of free-fed controls (p < 0.01). e Values significantly different from those of pairfed controls (p < 0.01). f Values significantly different from those of pair-fed controls (p < 0.001). g Values significantly different from those of pair-fed controls (p < 0.05).

Figure 5. Effect of 3,3′,4,4′,5-pentachlorobiphenyl on the 1-acylglycerophosphorylcholine (A) and 1-acylglycerophosphate (B) acyltransferase activities of rat liver microsomes. Acyltransferase activity was measured with 20:4-CoA (A) and 18:2-CoA (B) as substrates. Each bar represents the mean ( SE of the percentages for the free-fed control groups ()100). The activities (units per milligram of microsomal protein) in the free-fed controls were 37.9 ( 0.4 (A) and 85.1 ( 0.4 (B). One unit of glycerolipid acyltransferase activity is defined as the formation of 1 nmol of the product per minute. The dose of PenCB and preparation of microsomes are described in the legend of Figure 2. Values significantly different from those of free-fed controls: (*) p < 0.05. Figure 4. Correlation between the suppression of body weight gain by PenCB and the mole percentage of unsaturated fatty acids in the whole liver of rats. Data are shown as scatter plots of the dose-dependent change in unsaturated fatty acid (mole percentage) vs body weight gain (g). In panel A, the correlation between body weight gain and the mole percentage of linoleic acid (18:2) is depicted, and in panel B, the correlation between body weight gain and the mole percentage of arachidonic acid (20:4) is depicted. Each symbol shows the data from a PenCBtreated animal. The dose of PenCB (ip) was as follows: (O) 0, (×) 0.5, (4) 1, (0) 5, (+) 10, and (]) 25 mg/kg. r is the correlation coefficient.

enzymes, because their activities are many times faster than the desaturation and have no influence on the acyltransferase (Figure 5), which utilizes fatty acyl-CoA as a substrate. The increase in phospholipase A2 activity may also reduce the proportion of 20:4 in phospholipids, although the increase in 18:2 cannot be explained. It is well-known that a cytosolic phospholipase A2 has a preference for 20:4 in the sn-2 position of phospholipids (37). However, we know nothing about the change in this enzyme activity in PenCB- or TCDD-treated animals. In addition, P450 1A1 and 1A2 are induced by PenCB treatment, and these have P450-dependent oxygenase, epoxygenase, or hydroxylation activity that is responsible for the metabolism of 20:4 (38). The proportion of 20:4 may have been reduced by a mechanism which depends on P450, although the increase in the level of 18:2 cannot be explained in this way (39). If PenCB elevates the phospholipase A2 activity or the P450-mediated metabolism of arachidonate, that probably acts in a cooperative

Figure 6. Effect of 3,3′,4,4′,5-pentachlorobiphenyl on molar ratio of linoleic and arachidonic acids in the whole liver of rats. The dose of PenCB and preparation of homogenates are described in the legend of Figure 1. Each symbol represents the mean ( SE of the percentages for the free-fed control groups (0 mg of PenCB/kg). Values are given as the molar ratio of arachidonic and linoleic acid. White and black circles represent data for pair-fed controls and PenCB-treated groups, respectively. Values significantly different from those of free-fed controls: (*) p < 0.05 and (***) p < 0.001. Values significantly different from those of pair-fed controls: (†) p < 0.05 and (†††) p < 0.001.

fashion with suppression of the ∆5 and ∆6 desaturases to reduce the proportion of arachidonate in phospholipids. However, it is noteworthy that the former two mechanisms cannot account for the increase in the levels of 18:2 and 20:3, as precursors of 20:4. Thus, we conclude that

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Table 2. Effect of 3,3′,4,4′,5-Pentachlorobiphenyl on the Activities of NADH-Cytochrome b5 Reductase and NADPH-Cytochrome c Reductase, and Contents of Cytochrome b5 and P450 in Rat Liver Microsomes treatment

NADH-cytochrome b5a reductase

NADPH-cytochrome ca reductase

cytochrome b5b

cytochrome P450b

free-fed control (4)c pair-fed control (4)c PenCB-treated (5)c

3.30 ( 0.18 (100) 2.62 ( 0.19 (79)d 1.85 ( 0.17 (56)e,f

54.0 ( 2.0 (100) 38.0 ( 2.0 (70)g 41.0 ( 1.0 (76)e

0.29 ( 0.02 (100) 0.28 ( 0.02 (97) 0.38 ( 0.02 (131)g,h

0.76 ( 0.05 (100) 1.00 ( 0.03 (132)g 4.31 ( 0.23 (567)e,h

a Values show means ( SE of micromoles per minute per milligram of microsomal protein. The dose of PenCB and preparation of microsomes are described in the legend of Figure 3. The values in parentheses are relative to those of the free-fed control groups ()100). b Values show means ( SE of nanomoles per milligram of microsomal protein. The dose of PenCB and preparation of microsomes are described in the legend of Figure 3. The values in parentheses are relative to those of the free-fed control groups ()100). c Number of rats used. d Values significantly different from those of free-fed controls (p < 0.05). e Values significantly different from those of free-fed controls (p < 0.001). f Values significantly different from those of pair-fed controls (p < 0.05). g Values significantly different from those of free-fed controls (p < 0.01). h Values significantly different from those of pair-fed controls (p < 0.001).

Figure 7. Effect of 3,3′,4,4′,5-pentachlorobiphenyl on the activity of ∆5 (A) and ∆6 (B) desaturases of rat liver microsomes. Each bar represents the mean ( SE of the percentages for the free-fed control groups ()100). The activities (picomoles per minute per milligram of microsomal protein) in the free-fed controls were 47.3 ( 3.0 (A) and 6.56 ( 0.92 (B). The dose of PenCB and preparation of microsomes are described in the legend of Figure 2. Values significantly different from those of free-fed controls: (***) p < 0.001. Values significantly different from those of pair-fed controls: (†††) p < 0.001.

the most probable reason for the reduced proportion of arachidonate in phospholipids caused by PenCB is a decrease in the level of arachidonate synthesis through the suppression of ∆5 and ∆6 desaturase activity. Further, in combination with the suppression of both types of desaturase activity, to some degree, the extent of utilization of arachidonate is possibly increased through the induction of P450s by PenCB which is able to catabolize 20:4. Borlakoglu et al. (40, 41) have shown that Arochlor 1254, a commercial PCB mixture, increases the activity of ∆5 and ∆6 desaturases. These results do not agree with the studies presented here. However, since Arochlor 1254 is a PCB mixture containing numerous congeners, it is not clear which of these induce the change in the activity of ∆5 and ∆6 desaturases. The effect of Arochlor 1254 given to rats may differ from that of PenCB. Although Arochlor 1254 did not induce ethoxyresorufin O-deethylase activity in one report (41), PenCB induced this activity markedly in another (34). Thus, the results may reflect the effect of non-coplanar type PCBs and not coplanar ones such as the PCB used in the present study. In fact, most of the congeners in Arochlor 1254 are noncoplanar, binding chlorine at the ortho position (42). The mechanism for the reduction in the activity of ∆6 or ∆5 desaturases in PenCB-treated rats is unclear. We have not yet determined if the reduction in ∆6 or ∆5 desaturase activity seen in this study is due to a decreased level of mRNA or to other factors. Only a few attempts have so far been made to identify the detailed characteristics or amino acid sequences of these purified enzymes, although there is one report of ∆6 desaturase purification (43). ∆6 and ∆5 desaturase are integral proteins of the microsomal membrane, and they need an

electron flux supplied mainly by NADH through the amphiphilic protein, fp1, and cytochrome b5. Therefore, we measured the content and activity of microsomal electron-transfer proteins in the PenCB-treated rats. No significant change in fp1 activity was observed between the PenCB-treated and pair-fed control groups, and only the content of cytochrome b5 increased (Table 2). These results indicate that changes in cytochrome b5 content or fp1 activity in the PenCB-treated rats do not contribute to a reduction in ∆6 or ∆5 desaturase activity. A possible explanation for the reduction in the activity of ∆5 and ∆6 desaturases may be hormonal alterations produced by PenCB treatment. The activity of both desaturases is reduced by an increase in the levels of glucocorticoids, glucagon, and thyroxin (44), and TCDD increases these hormonal levels (45-47). Furthermore, evidence has been obtained which shows that in vitro microsomal ∆6 desaturation of fatty acids requires a cytosolic protein factor to achieve full activity. This cytosolic factor binds to a small extent to the microsomal membrane (48) and has lipoprotein properties (49). It is not clear whether PenCB elicits a change in the amount of this factor. There is still a possibility that PenCB is able to inhibit ∆5 and ∆6 desaturases directly, and this effect cannot be excluded in this in vivo study. It is well-known that PenCB and TCDD are effective inducers of numerous enzymes and that the induction is mediated by aryl hydrocarbon receptor (Ah receptor) (6, 7). However, PenCB or TCDD treatment also reduces the activity or mRNA of some enzymes (46, 50-52). It is not clear whether the reduction in desaturase activity is mediated by the Ah receptor; there may be a mechanism, as yet unknown, mediated possibly by the Ah receptor responsible for the reduction in activity of these enzymes. The 20:4 is one of the essential fatty acids. PenCB may be able to cause essential fatty acid deficiency because 18:2 cannot be fully utilized to markedly reduce ∆6 desaturase activity. Essential fatty acid deficiency has been shown to cause body weight loss (53), a typical toxic effect of PenCB or TCDD. We have found that the PenCB dose-dependent reduction in the level of 20:4 and increase in the level of 18:2 are significantly correlated with the suppression of body weight gain (Figure 4). This hypothesis needs more detailed investigation. The 20:4 released from phospholipids is known to be transformed to a variety of eicosanoids through the cyclooxygenase and lipoxygenase pathways. Nothing is known about the activity of these enzymes in PenCB-treated rat liver. However, prostaglandin synthesis is affected not only by enzymes such as phospholipase, cyclooxygenase, and lipoxygenase but also by the 20:4 content of phospholipids (54). Furthermore, it should be noted that a reduction in

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the level of hepatic phospholipid or triglyceride 20:4 results in an increase in the level of triglyceride (55, 56). PenCB and TCDD treatment markedly increases the level of hepatic triglyceride in rat liver (4, 57). The results obtained in the study presented here may explain the marked changes in lipid metabolism following exposure to coplanar PCB. Further investigations are needed to correlate our data with PenCB toxicity.

Acknowledgment. We are grateful to Ms. Michie Inoue for her excellent assistance with this work. This work was supported in part by the Ministry of Health and Welfare, Japan.

Note Added in Proof During the revision of the manuscript, Cho et al.2 reported the cloning of ∆6 desaturase in mice and humans, although rat ∆6 and ∆5 desaturases remain to be cloned.

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