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Effect of clofibrate treatment on glutathione content and the activity of the enzymes related to peroxide metabolism in rat liver and heart
Int. J:Biochem. Vol. 19, No. 2, pp. 187-192, 198'7
0020-711X/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
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EFFECT OF C L O F I B R A T E T R E A T M E N T O N G L U T A T H I O N E C O N T E N T A N D THE ACTIVITY OF THE E N Z Y M E S R E L A T E D TO P E R O X I D E M E T A B O L I S M IN RAT LIVER A N D H E A R T VASILY D. ANTONENKOV, VLADIMIR A. GUSEV and LEONID F. PANCHENKO All-Union Research Center for Medico-Biological Problems of Prevention of Alcohol Abuse and Alcoholism, Kropotkinsky per. 23, Moscow, 119034, U.S.S.R. (Received 23 June 1986)
Abstract--l. Clofibrate treatment was shown to increase the content of reduced glutathione in rat liver and kidney, but did not alter the glutathione level in heart, brain, spleen and small intestine. 2. Clofibrate did not affect the activity of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase in rat liver and heart. 3. The drug decreased the activity of glutathione-S-transferase in the cytosolic fraction of liver homogenate. 4. Glutathione-S-transferase activity in small intestine was also reduced. 5. The administration of clofibrate decreased the content of polypeptides with mol. wt of 22,000 and 24,000 (possible monomers of glutathione-S-transferase) in the cytosolic fraction of liver cells.
INTRODUCTION It is well known that administration of the hypolipidaemic drug clofibrate to rodents causes proliferation of hepatic and heart peroxisomes (Fahimi et al., 1980; Reddy et al., 1982) and increases the activity of catalase and peroxisomal cyanideinsensitive palmitoyl-CoA oxidizing system (Lazarow and de Duve, 1976; Norseth and Thomassen, 1983). This system contains an H202-producing acyI-CoA oxidase which catalyses the first and rate-limiting step in fl-oxidation reactions sequence. Moreover, clofibrate treatment causes a marked increase of microsomal cytochrome P-450 (Tamburini et al., 1984), which is known to be a major source of 02and H202 in the liver cells (Chance et aL, 1979). It has been reported that clofibrate as well as other hypolipidaemic agents induced hepatocellular carcinomas in rats and mice (Reddy et al., 1980). According to the view of Reddy et al. (1982) the carcinogenic effect of hypolipidaemics is associated with the activation of peroxisomal hydrogen peroxide-producing oxidases and accumulation of toxic peroxides and oxygen radicals. However, there is little data concerning the effect of clofibrate on the activity of enzymes related to peroxide metabolism, which may represent a protective mechanism against the subversive action of the toxic peroxides and oxygen radicals on the membranous and other structures of the cell. In the present report we study the effects of clofibrate administration to rats on the activity of liver and heart catalase, acyl-CoA oxidase and some other peroxisomal oxidases. Glutathione content and the activity of enzymes acting as a defence against an "oxidative stress" (Cadenas and Sies, 1985) were determined at the same time. We also investigated the
effect of clofibrate treatment on the subcellular distribution of reduced glutathione and glutathione-Stransferase activity in rat liver homogenates. MATERIALS AND M E T H O D S
Chemicals
Glutathione (reduced and oxidised forms); glutathione reductase, type III; t-butyl hydroperoxide; epinephrine and adrenochrome were obtained from Sigma Chemical Co. l-Chloro-2,4-dinitrobenzene was from Aldrich Chemical Co. The sources of all other reagents were given previously (Antonenkov et al., 1985). Animals and tissue fractionation
Wistar male rats (200-250 g) were injected intraperitoneally with saline (control) or clofibrate in a dose of 400 mg/kg once daily in the course of 16 days. Animals were anesthetized with light ether and killed by decapitation after 16-18 hr starvation. Heart and liver were perfused with saline to wash out erythrocytes. The organs were immediately removed, weighed and homogenized in 9 vol of icecold 20 mM Tris-acetate, pH 7.2 containing 0.25 M sucrose (Panchenko et al., 1976). To prepare a tissue homogenate of the small intestine, its initial segment (from the pyloric portion of the stomach) 10 cm long was removed. Heart homogenates were centrifuged for I0 min at 1000 g (max) to remove unbroken cells, nuclei and cell debris ("postnuclear" homogenate). The subcellular fractionation of liver homogenate was performed by differential centrifugation according to de Duve et al. (1955) with slight modifications (Panchenko et al., 1985). Purified peroxisomes were obtained by centrifugation of the "light" mitochondrial fraction in a discontinuous sucrose-density gradient (Antonenkov et al., 1985). Enzyme assays
Acyl-CoA oxidase (EC 1.3.99.3) activity was measured at 37°C with palmitoyl-CoA as substrate (Horie et al., 1981). Catalase (EC 1.11.1.6) L
B.C 19/2.--F
188
VASILY D. ANTONENKOV et al. Table I. Effect of clofibrate treatment on the activities of peroxisomal enzymes in rat liver and heart bomogenates Activity (/zmol/min per g tissue) Control Clofibrate
Enzyme Liver
Catalse, units AeyI-CoA oxidase Urate oxidase o-Amino acid oxidase Hydroxy acid oxidase Protein (mg/g tissue)
(10) 39.8-+ 1.2 (15) 0.46_+0.04 (7) 3.0 _+0.2 (7) 0.12_+0.02 (7) 0.'36_+0.03 (17) 147.6_+4.3
52.8 + 1.9'** 2.26 _+0.11*** 2.7 + 0.2 0.04 _+0.01 *** 0.24 _+0.01 ** 160.2_+4.5"
Heart
Catalase, units AcyI-CoA oxidase Protein (mg/g tissue)
(18) 0.76_+0.04 1.28 _+0.09*** (10) 0.031 _+0.004 0.054_+0.006** (15) 70.0_+3.1 79.5 _+4.0
The number of animals in each group is given in parentheses. Each value is the mean -+ SD. up < 0.05; ,up < 0.01; **up < 0.001.
1.1.3.1), D-amino acid oxidase (EC 1.4.3.3; Hayashi et al., 1971), urate oxidase (EC 1.7.3.3; Leighton et al., 1968) and marker enzymes of subcellular particles (Leighton et al., 1968; Antonenkov et al., 1985) were assayed according to the published procedures. Superoxide dismutase (EC 1.15.1.1) was assayed in terms of its ability to inhibit the oxygen-dependent oxidation of epinephrine to adrenochrome (Misra and Fridovich, 1972). Glutathione peroxidase (EC 1.11.1.9) activity was determined using 0.8 mM H202 or 1.0mM t-butyl hydroperoxide as substrates (Lawrence and Burk, 1976). Glutathione reductase (EC 1.6.2.4; Pinto and Bartley, 1969), glucose-6-phosphate dehydrogenase (EC 1.1.1.49; Zakim et aL, 1970) and glutathione-S-transferase (EC 2.5.1.18) with l-chloro-2,4dinitrobenzene as substrate (Habig et al., 1974) were measured by standard procedures. The activities o f heart enzymes were measured in "postnuclear" homogenates. The activity of the enzymes (except for catalase and superoxide dismutase) was expressed in micromoles/min per g of tissue. The catalase (Panchenko et al., 1976) and superoxide dismutase (Misra and Fridovich, 1972) activities were calculated as described earlier. Other methods
Electrophoresis in the presence of sodium dodecyl sulphate was performed according to Weber and Osborn (1969), using 10% polyacrylamide gels. For calibration of the gels the molecular weight standard proteins from Sigma Chemical Co. were used. Reduced glutathione was determined in whole homogenates using Ellman's reagent (Sedlak and Lindsay, 1968). Protein content was measured following the method of Lowry et aL (1951) with bovine serum albumin as standard. Statistical significance for difference between means from control and clofibratetreated groups was estimated by Student's t-test.
RESULTS M a r k e d h e p a t o m e g a l y was p r o d u c e d following the a d m i n i s t r a t i o n o f clofibrate to rats. T h e relative liver weight increased 1.5 times c o m p a r e d to t h a t o f c o n t r o l g r o u p ( P < 0.001, n = 11). T h e p r o t e i n c o n c e n t r a t i o n was also i n c r e a s e d in liver h o m o g e n a t e s (Table 1). A t the s a m e time clofibrate d i d n o t alter the relative h e a r t w e i g h t a n d p r o t e i n c o n t e n t in the w h o l e h o m o g e n a t e s o f this organ. T a b l e 1 s h o w s t h e effects o f h y p o l i p i d a e m i c d r u g o n the activities o f pero x i s o m a l e n z y m e s . T h e activity o f catalase, as well as a c y I - C o A o x i d a s e w a s i n d u c e d in the liver a n d h e a r t by t r e a t m e n t with clofibrate. T h e d r u g d i d n o t affect the activity o f h e p a t i c u r a t e oxidase, w h e r e a s the activities o f D - a m i n o acid o x i d a s e a n d L-~t-hydroxy acid o x i d a s e were d e c r e a s e d to 33 a n d 6 7 % o f the c o n t r o l level, respectively. T h e s e results are in g o o d a g r e e m e n t with earlier r e p o r t s ( L a z a r o w a n d de D u v e , 1976; N o r s e t h a n d T h o m a s s e n , 1983). T h e activity o f all the oxidases s t u d i e d was m a r k e d l y lower in the h e a r t m u s c l e t h a n in the liver. T h e m y o c a r d i a l D - a m i n o acid o x i d a s e a n d L-ct-hydroxy acid o x i d a s e activities were l o w e r t h a n 2-3 n m o l / m i n p e r g o f tissue a n d the u r a t e o x i d a s e activity was n o t d e t e c t e d b o t h in the c o n t r o l o r clofibrate t r e a t e d rats. I n c o n t r a s t to t h e m a r k e d increase in the activity o f catalase, the a d m i n i s t r a t i o n o f clofibrate did n o t affect the activity o f s o m e o t h e r e n z y m e s acting as a d e f e n c e a g a i n s t a n " o x i d a t i v e s t r e s s " in rat liver a n d h e a r t (Table 2). T h e total a n d the specific activities o f s u p e r o x i d e d i s m u t a s e , g l u t a t h i o n e p e r o x i d a s e with H 2 0 2 a n d t - b u t y l h y d r o p e r o x i d e as substrates, glu-
Table 2. Effect of clofibrate treatment on the activity of enzymes related to peroxide metabolism in rat liver and heart Activity (,umol/min per g tissue) Liver Heart Enzyme and substrate Control Clofibrate Control Clofibrate Superoxide dismutase, units (9) 2550 + 220 2950 + 256 (6) 634 + 79 712 _+ 110 Glutathione peroxidase with H202 (6) 45.0 _+9.0 49.3 _+6.1 (6) 25.6 + 3.1 24.8 _+2.0 with t-butyl hydroperoxide (12) 99.6 _+7.1 97.5 _+5.7 (6) 31.3 + 2.9 29.3 _+ 1.3 Glutathione reductase (8) 7.96_+0.48 7.95_+0.21 (8) 0.90 + 0.06 0.92_+0.06 Glutathione-S-transferase (12) 64.1 _+3.9 43.9_+ 2.7* (13) 4.05_ 0.24 4.39_+0.31 Glucose-6-phosphat¢ dehydrogenase (9) 4.78 +_0.38 4.93 + 0.47 (7) 0.34 _+0.02 0.34_+0.03 Glutathione (#mol/g tissue) (15) 1.98_+0.12 3.60+0.21" (14) 1.13+0.06 1.21-+0.04 Each value represents means + SD for the numbers of animals in parentheses; *P < 0.001 for difference between enzyme activities in clofibrate-treated and control groups.
189
Action of clofibrate tathione reductase and glucose-6-phosphate dehydrogenase were unchanged. Treatment with clofibrate caused a significant decrease in the activity of glutathione-S-transferase in the liver homogenate, whereas the drug did not change the enzyme activity in the rat heart. The administration of clofibrate increased the concentration of reduced glutathione in the liver (Table 2). At the same time the drug failed to induce glutathione content in the heart. A single dose of clofibrate (400 mg/kg) led to an elevation of hepatic glutathione concentration after 20hr (by 17%), however glutathione-S-transferase activity at these conditions did not change. To clarify the possible effects of clofibrate on the subcellular distribution of glutathione, glutathioneS-transferase and glutathione peroxidase, the subcellular fractions were isolated by differential centrifugation of rat liver homogenates. The cytosolic
fractions from the livers of control and clofibratetreated animals contained 25.4 and 44.3 % of the total reduced glutathione, respectively. The extent of glutathione elevation was greater in the soluble than in the mitochondrial fractions. Differential centrifugation showed that more than 85% of the glutathione-S-transferase activity in both the experimental and the control groups was located in the soluble fraction of liver homogenates. No redistribution of the enzyme activity between cytosol and subcellular particles was observed. Similarly, the drug did not affect the subcellular distribution of glutathione peroxidase with t-butyl hydroperoxide as substrate (Fig. 1). To achieve more complete separation of peroxisomes from mitochondria and other subeellular particles isopycnic centrifugation of the "light" mitochondrial fraction in a multi-stage sucrose density gradient was used. In both, the
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Fig. 1. Differential centrifugation of the normal and clofibrate-treated rat liver homogenate. The livers were homogenized and fractionated into nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and cytosolic (S) fractions as described in the text. The abscissa represents the cumulative percentage of protein (percentage of normal homogenate). Solid line: results for the control group; dotted line: results for the clofibrate-treated group. Each value represents the mean of four separate experiments. (A) Catalase; (B) lactate dehydrogenase; (C) reduced glutathione; (D) glutathione-S-transferase; (E) glutathione peroxidase; (F) glutamate dehydrogenase; (G) acid phosphatase; (H) arylesterase. The enzyme recoveries in all fractions varied between 85 and 110% compared to activity in the whole homogenate.
190
VASlLY D. ANTONENKOV el a].
rl I i (1)
one of the glutathione-S-transferase isoenzymes (Ketley e t al., 1975). The effect of clofibrate on the glutathione content and glutathione-S-transferase activity in different tissues is shown in Table 3. In addition to increase of glutathione concentration in rat liver, there is also a slight but significant elevation in the kidney. The glutathione-S-transferase activity was decreased not only in the liver, but also in the small intestine. The enzyme activity reduced 1.9-fold on administration of clofibrate. No decrease was detected in brain, heart, spleen and kidney.
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Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the liver cytosolic fractions. Electrophoregram of the polypeptide composition of cytosol from clofibratetreated (A) and control (B) rat livers. The ordinate represents the absorbance at 550 nm. The vertical arrows indicate the position of the polypeptides with mol. wt of 22,000-24,000 (1) and 12,000 (2). normal and clofibrate-treated rats the presence of reduced glutathione, as well as glutathione peroxidase activity with t-butyl hydroperoxide as substrate, were detected in the mitochondrial fraction, but not in peroxisomes (data not shown). Figure 2 shows the electrophoretic profiles of protein from the liver cytosolic fractions. In accordance with the fndings reported earlier (Antonenkov et al., 1982), the concentration of polypeptide with mol. wt of approx. 12,000 was increased by administration of clofibrate. This polypeptide corresponds to the fatty acid binding protein (Z protein). At the same time the content of the polypeptides with mol. wt of approx. 24,000 and 22,000 was significantly reduced. There is evidence indicating that these polypeptides correspond to the monomers of the cytosolic Y protein or ligandin (Antonenkov et al., 1982), which represents
According to the results of our investigation clofibrate does not exert any significant effect on the enzymes which may lend protection against an "oxidative stress", in rat liver and heart tissue with the exception of peroxisomal catalase. Under the action of the drug no changes in the total and specific activities of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6phosphate dehydrogenase were observed. Earlier Ciriolo et al. (1982) reported about a 20% reduction of the superoxide dismutase and glutathione peroxidase specific activity in liver homogenates of the clofibrate-treated (300 mg/kg, 30 days) rats. The discrepancy between these results may be due to different courses of the drug administration. Clofibrate markedly increased concentration of the reduced glutathione in rat liver. This agrees well with the data of Foliot et al. (1984). Apart from liver tissue, glutathione content was slightly elevated in kidney but in the other organs changes were not detected. The increase of the reduced glutathione in the liver cells may facilitate the elimination of peroxides via glutathione peroxidase pathway (Chance et al., 1979). On the other hand, the elevation of the glutathione concentration could be related to a clofibrate-induced hepatomegaly. Thus, it was shown that glutathione is increased in the regenerating liver after partial hepatectomy in rats (Cockerile et al., 1983). Data about the influence of clofibrate on glutathione-S-transferase activity and ligandin content in the liver represent a particular interest. Clofibrate was shown to decrease the ligandin concentration in rat liver (Fleischner et al., 1975) and to reduce the glutathione-S-transferase activity (Foliot et al., 1984). The fall in the enzyme activity depends on the dose and duration of the drug treatment period (Foliot et al., 1984) and corresponds to the
Table 3. Effect of clofibrate treatment on glutathione content and glutathione-S-transferase activity in rat tissues Glutathione content (/~mol/g tissue) Tissue Liver Kidney Heart Brain Spleen Small intestine
Control 3.28 + 0.24 2.07+0.10 1.13_+0.07 1.09 + 0.25 1.06-+0.22 2.17 _+0.25
Clofibrate 4.88 + 0.26** 2.50+0.06 1.02_+0.0.5 0.90 + 0.06 0.93-+0.13 2.23 + 0.19
Enzyme activity (vmol/min per g tissue) Control
Clofibrate
107.5 _+ 5.4 13.3+0.7 3.4_+0.3 10.2 + 0.8 3.9_+0.4 10.3 _+0.7
53.2 -+ 2.4*** 12.5+ 1.3 3.6_+0.3 9.4 -+ 0.8 3.7_+0.5 5.4 _+0.8**
Means _+ SD are given for four animals in each group; *P < 0.05; **P < 0.01: ***P < 0.001.
Action of clofibrate diminution of the two cytosolic polypeptide fractions with mol. wt of 22,000 and 24,000 (see Fig. 2). It is reasonable to assume that these polypeptides represent the subunits o f various isozymes of glutathioneS-transferases (Antonenkov et al., 1982; Boyer et al., 1983) including those named Y-protein or ligandin (Bhargava et al., 1980). It is known that some of glutathione-S-transferases which consist of YaYc or YcYc dimers possess a pronounced glutathione peroxidase activity with organic hydroperoxides as substrates. At the same time the other isozymes (e.g. YbYb) are very p o o r peroxidases (Boyer et aL, 1983). It is remarkable that despite significant reduction of glutathione-S-transferase activity in the liver, clofibrate does not affect the activity of glutathione peroxidase with t-butyl hydroperoxide as substrate. Based on these data it is conceivable that the drug predominantly influences the content of isozymes with a low peroxidase but high glutathione-Stransferase activity. Several peroxisome proliferators including clofibrate have been shown to produce tumours when administered to rats for prolonged periods (Reddy et al., 1980, 1982). Relatively low doses of the drug promotes hepatocarcinogenesis induced by dimethylnitrozamine (Mochizuki et al., 1982). Our data about the lack of clofibrate effect on enzymes which may represent a protective mechanism against the subversive action of peroxides and oxygen radicals are consistent with the views of Reddy et al. (1982) who supposed a possible role of these toxic agents in carcinogenic action of peroxisome proliferators. At the same time considering the putative mechanisms o f carcinogenesis attention showed be paid to a significant reduction glutathione-S-transferase activity in the liver of the clofibrate-treated rats. There is ample evidence concerning implication of this enzyme in the detoxication of highly reactive carcinogens (Smith et al., 1977; Kaplowitz, 1980) as well as about its possible involvement in the anticarcinogenic effect of antioxidants such as butylated hydroxytoluene or butylated hydroxyanisole (Cha and Heine, 1982). Interestingly, under the experimental conditions employed we have not observed any elevation of the rate of lipid peroxidation in the mitochondrial or microsomal fraction of rat liver homogenates. At the same time the activation of the non-enzymic lipid peroxidation was unexpectedly registered in the nuclei-free homogenate and total particulate fraction of the heart (Antonenkov et al., manuscript in preparation). Further investigations are needed to make reasonable explanations for these findings.
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EFFECT OF C L O F I B R A T E T R E A T M E N T O N G L U T A T H I O N E C O N T E N T A N D THE ACTIVITY OF THE E N Z Y M E S R E L A T E D TO P E R O X I D E M E T A B O L I S M IN RAT LIVER A N D H E A R T VASILY D. ANTONENKOV, VLADIMIR A. GUSEV and LEONID F. PANCHENKO All-Union Research Center for Medico-Biological Problems of Prevention of Alcohol Abuse and Alcoholism, Kropotkinsky per. 23, Moscow, 119034, U.S.S.R. (Received 23 June 1986)
Abstract--l. Clofibrate treatment was shown to increase the content of reduced glutathione in rat liver and kidney, but did not alter the glutathione level in heart, brain, spleen and small intestine. 2. Clofibrate did not affect the activity of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase in rat liver and heart. 3. The drug decreased the activity of glutathione-S-transferase in the cytosolic fraction of liver homogenate. 4. Glutathione-S-transferase activity in small intestine was also reduced. 5. The administration of clofibrate decreased the content of polypeptides with mol. wt of 22,000 and 24,000 (possible monomers of glutathione-S-transferase) in the cytosolic fraction of liver cells.
INTRODUCTION It is well known that administration of the hypolipidaemic drug clofibrate to rodents causes proliferation of hepatic and heart peroxisomes (Fahimi et al., 1980; Reddy et al., 1982) and increases the activity of catalase and peroxisomal cyanideinsensitive palmitoyl-CoA oxidizing system (Lazarow and de Duve, 1976; Norseth and Thomassen, 1983). This system contains an H202-producing acyI-CoA oxidase which catalyses the first and rate-limiting step in fl-oxidation reactions sequence. Moreover, clofibrate treatment causes a marked increase of microsomal cytochrome P-450 (Tamburini et al., 1984), which is known to be a major source of 02and H202 in the liver cells (Chance et aL, 1979). It has been reported that clofibrate as well as other hypolipidaemic agents induced hepatocellular carcinomas in rats and mice (Reddy et al., 1980). According to the view of Reddy et al. (1982) the carcinogenic effect of hypolipidaemics is associated with the activation of peroxisomal hydrogen peroxide-producing oxidases and accumulation of toxic peroxides and oxygen radicals. However, there is little data concerning the effect of clofibrate on the activity of enzymes related to peroxide metabolism, which may represent a protective mechanism against the subversive action of the toxic peroxides and oxygen radicals on the membranous and other structures of the cell. In the present report we study the effects of clofibrate administration to rats on the activity of liver and heart catalase, acyl-CoA oxidase and some other peroxisomal oxidases. Glutathione content and the activity of enzymes acting as a defence against an "oxidative stress" (Cadenas and Sies, 1985) were determined at the same time. We also investigated the
effect of clofibrate treatment on the subcellular distribution of reduced glutathione and glutathione-Stransferase activity in rat liver homogenates. MATERIALS AND M E T H O D S
Chemicals
Glutathione (reduced and oxidised forms); glutathione reductase, type III; t-butyl hydroperoxide; epinephrine and adrenochrome were obtained from Sigma Chemical Co. l-Chloro-2,4-dinitrobenzene was from Aldrich Chemical Co. The sources of all other reagents were given previously (Antonenkov et al., 1985). Animals and tissue fractionation
Wistar male rats (200-250 g) were injected intraperitoneally with saline (control) or clofibrate in a dose of 400 mg/kg once daily in the course of 16 days. Animals were anesthetized with light ether and killed by decapitation after 16-18 hr starvation. Heart and liver were perfused with saline to wash out erythrocytes. The organs were immediately removed, weighed and homogenized in 9 vol of icecold 20 mM Tris-acetate, pH 7.2 containing 0.25 M sucrose (Panchenko et al., 1976). To prepare a tissue homogenate of the small intestine, its initial segment (from the pyloric portion of the stomach) 10 cm long was removed. Heart homogenates were centrifuged for I0 min at 1000 g (max) to remove unbroken cells, nuclei and cell debris ("postnuclear" homogenate). The subcellular fractionation of liver homogenate was performed by differential centrifugation according to de Duve et al. (1955) with slight modifications (Panchenko et al., 1985). Purified peroxisomes were obtained by centrifugation of the "light" mitochondrial fraction in a discontinuous sucrose-density gradient (Antonenkov et al., 1985). Enzyme assays
Acyl-CoA oxidase (EC 1.3.99.3) activity was measured at 37°C with palmitoyl-CoA as substrate (Horie et al., 1981). Catalase (EC 1.11.1.6) L
B.C 19/2.--F
188
VASILY D. ANTONENKOV et al. Table I. Effect of clofibrate treatment on the activities of peroxisomal enzymes in rat liver and heart bomogenates Activity (/zmol/min per g tissue) Control Clofibrate
Enzyme Liver
Catalse, units AeyI-CoA oxidase Urate oxidase o-Amino acid oxidase Hydroxy acid oxidase Protein (mg/g tissue)
(10) 39.8-+ 1.2 (15) 0.46_+0.04 (7) 3.0 _+0.2 (7) 0.12_+0.02 (7) 0.'36_+0.03 (17) 147.6_+4.3
52.8 + 1.9'** 2.26 _+0.11*** 2.7 + 0.2 0.04 _+0.01 *** 0.24 _+0.01 ** 160.2_+4.5"
Heart
Catalase, units AcyI-CoA oxidase Protein (mg/g tissue)
(18) 0.76_+0.04 1.28 _+0.09*** (10) 0.031 _+0.004 0.054_+0.006** (15) 70.0_+3.1 79.5 _+4.0
The number of animals in each group is given in parentheses. Each value is the mean -+ SD. up < 0.05; ,up < 0.01; **up < 0.001.
1.1.3.1), D-amino acid oxidase (EC 1.4.3.3; Hayashi et al., 1971), urate oxidase (EC 1.7.3.3; Leighton et al., 1968) and marker enzymes of subcellular particles (Leighton et al., 1968; Antonenkov et al., 1985) were assayed according to the published procedures. Superoxide dismutase (EC 1.15.1.1) was assayed in terms of its ability to inhibit the oxygen-dependent oxidation of epinephrine to adrenochrome (Misra and Fridovich, 1972). Glutathione peroxidase (EC 1.11.1.9) activity was determined using 0.8 mM H202 or 1.0mM t-butyl hydroperoxide as substrates (Lawrence and Burk, 1976). Glutathione reductase (EC 1.6.2.4; Pinto and Bartley, 1969), glucose-6-phosphate dehydrogenase (EC 1.1.1.49; Zakim et aL, 1970) and glutathione-S-transferase (EC 2.5.1.18) with l-chloro-2,4dinitrobenzene as substrate (Habig et al., 1974) were measured by standard procedures. The activities o f heart enzymes were measured in "postnuclear" homogenates. The activity of the enzymes (except for catalase and superoxide dismutase) was expressed in micromoles/min per g of tissue. The catalase (Panchenko et al., 1976) and superoxide dismutase (Misra and Fridovich, 1972) activities were calculated as described earlier. Other methods
Electrophoresis in the presence of sodium dodecyl sulphate was performed according to Weber and Osborn (1969), using 10% polyacrylamide gels. For calibration of the gels the molecular weight standard proteins from Sigma Chemical Co. were used. Reduced glutathione was determined in whole homogenates using Ellman's reagent (Sedlak and Lindsay, 1968). Protein content was measured following the method of Lowry et aL (1951) with bovine serum albumin as standard. Statistical significance for difference between means from control and clofibratetreated groups was estimated by Student's t-test.
RESULTS M a r k e d h e p a t o m e g a l y was p r o d u c e d following the a d m i n i s t r a t i o n o f clofibrate to rats. T h e relative liver weight increased 1.5 times c o m p a r e d to t h a t o f c o n t r o l g r o u p ( P < 0.001, n = 11). T h e p r o t e i n c o n c e n t r a t i o n was also i n c r e a s e d in liver h o m o g e n a t e s (Table 1). A t the s a m e time clofibrate d i d n o t alter the relative h e a r t w e i g h t a n d p r o t e i n c o n t e n t in the w h o l e h o m o g e n a t e s o f this organ. T a b l e 1 s h o w s t h e effects o f h y p o l i p i d a e m i c d r u g o n the activities o f pero x i s o m a l e n z y m e s . T h e activity o f catalase, as well as a c y I - C o A o x i d a s e w a s i n d u c e d in the liver a n d h e a r t by t r e a t m e n t with clofibrate. T h e d r u g d i d n o t affect the activity o f h e p a t i c u r a t e oxidase, w h e r e a s the activities o f D - a m i n o acid o x i d a s e a n d L-~t-hydroxy acid o x i d a s e were d e c r e a s e d to 33 a n d 6 7 % o f the c o n t r o l level, respectively. T h e s e results are in g o o d a g r e e m e n t with earlier r e p o r t s ( L a z a r o w a n d de D u v e , 1976; N o r s e t h a n d T h o m a s s e n , 1983). T h e activity o f all the oxidases s t u d i e d was m a r k e d l y lower in the h e a r t m u s c l e t h a n in the liver. T h e m y o c a r d i a l D - a m i n o acid o x i d a s e a n d L-ct-hydroxy acid o x i d a s e activities were l o w e r t h a n 2-3 n m o l / m i n p e r g o f tissue a n d the u r a t e o x i d a s e activity was n o t d e t e c t e d b o t h in the c o n t r o l o r clofibrate t r e a t e d rats. I n c o n t r a s t to t h e m a r k e d increase in the activity o f catalase, the a d m i n i s t r a t i o n o f clofibrate did n o t affect the activity o f s o m e o t h e r e n z y m e s acting as a d e f e n c e a g a i n s t a n " o x i d a t i v e s t r e s s " in rat liver a n d h e a r t (Table 2). T h e total a n d the specific activities o f s u p e r o x i d e d i s m u t a s e , g l u t a t h i o n e p e r o x i d a s e with H 2 0 2 a n d t - b u t y l h y d r o p e r o x i d e as substrates, glu-
Table 2. Effect of clofibrate treatment on the activity of enzymes related to peroxide metabolism in rat liver and heart Activity (,umol/min per g tissue) Liver Heart Enzyme and substrate Control Clofibrate Control Clofibrate Superoxide dismutase, units (9) 2550 + 220 2950 + 256 (6) 634 + 79 712 _+ 110 Glutathione peroxidase with H202 (6) 45.0 _+9.0 49.3 _+6.1 (6) 25.6 + 3.1 24.8 _+2.0 with t-butyl hydroperoxide (12) 99.6 _+7.1 97.5 _+5.7 (6) 31.3 + 2.9 29.3 _+ 1.3 Glutathione reductase (8) 7.96_+0.48 7.95_+0.21 (8) 0.90 + 0.06 0.92_+0.06 Glutathione-S-transferase (12) 64.1 _+3.9 43.9_+ 2.7* (13) 4.05_ 0.24 4.39_+0.31 Glucose-6-phosphat¢ dehydrogenase (9) 4.78 +_0.38 4.93 + 0.47 (7) 0.34 _+0.02 0.34_+0.03 Glutathione (#mol/g tissue) (15) 1.98_+0.12 3.60+0.21" (14) 1.13+0.06 1.21-+0.04 Each value represents means + SD for the numbers of animals in parentheses; *P < 0.001 for difference between enzyme activities in clofibrate-treated and control groups.
189
Action of clofibrate tathione reductase and glucose-6-phosphate dehydrogenase were unchanged. Treatment with clofibrate caused a significant decrease in the activity of glutathione-S-transferase in the liver homogenate, whereas the drug did not change the enzyme activity in the rat heart. The administration of clofibrate increased the concentration of reduced glutathione in the liver (Table 2). At the same time the drug failed to induce glutathione content in the heart. A single dose of clofibrate (400 mg/kg) led to an elevation of hepatic glutathione concentration after 20hr (by 17%), however glutathione-S-transferase activity at these conditions did not change. To clarify the possible effects of clofibrate on the subcellular distribution of glutathione, glutathioneS-transferase and glutathione peroxidase, the subcellular fractions were isolated by differential centrifugation of rat liver homogenates. The cytosolic
fractions from the livers of control and clofibratetreated animals contained 25.4 and 44.3 % of the total reduced glutathione, respectively. The extent of glutathione elevation was greater in the soluble than in the mitochondrial fractions. Differential centrifugation showed that more than 85% of the glutathione-S-transferase activity in both the experimental and the control groups was located in the soluble fraction of liver homogenates. No redistribution of the enzyme activity between cytosol and subcellular particles was observed. Similarly, the drug did not affect the subcellular distribution of glutathione peroxidase with t-butyl hydroperoxide as substrate (Fig. 1). To achieve more complete separation of peroxisomes from mitochondria and other subeellular particles isopycnic centrifugation of the "light" mitochondrial fraction in a multi-stage sucrose density gradient was used. In both, the
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Fig. 1. Differential centrifugation of the normal and clofibrate-treated rat liver homogenate. The livers were homogenized and fractionated into nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and cytosolic (S) fractions as described in the text. The abscissa represents the cumulative percentage of protein (percentage of normal homogenate). Solid line: results for the control group; dotted line: results for the clofibrate-treated group. Each value represents the mean of four separate experiments. (A) Catalase; (B) lactate dehydrogenase; (C) reduced glutathione; (D) glutathione-S-transferase; (E) glutathione peroxidase; (F) glutamate dehydrogenase; (G) acid phosphatase; (H) arylesterase. The enzyme recoveries in all fractions varied between 85 and 110% compared to activity in the whole homogenate.
190
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one of the glutathione-S-transferase isoenzymes (Ketley e t al., 1975). The effect of clofibrate on the glutathione content and glutathione-S-transferase activity in different tissues is shown in Table 3. In addition to increase of glutathione concentration in rat liver, there is also a slight but significant elevation in the kidney. The glutathione-S-transferase activity was decreased not only in the liver, but also in the small intestine. The enzyme activity reduced 1.9-fold on administration of clofibrate. No decrease was detected in brain, heart, spleen and kidney.
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Fig. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the liver cytosolic fractions. Electrophoregram of the polypeptide composition of cytosol from clofibratetreated (A) and control (B) rat livers. The ordinate represents the absorbance at 550 nm. The vertical arrows indicate the position of the polypeptides with mol. wt of 22,000-24,000 (1) and 12,000 (2). normal and clofibrate-treated rats the presence of reduced glutathione, as well as glutathione peroxidase activity with t-butyl hydroperoxide as substrate, were detected in the mitochondrial fraction, but not in peroxisomes (data not shown). Figure 2 shows the electrophoretic profiles of protein from the liver cytosolic fractions. In accordance with the fndings reported earlier (Antonenkov et al., 1982), the concentration of polypeptide with mol. wt of approx. 12,000 was increased by administration of clofibrate. This polypeptide corresponds to the fatty acid binding protein (Z protein). At the same time the content of the polypeptides with mol. wt of approx. 24,000 and 22,000 was significantly reduced. There is evidence indicating that these polypeptides correspond to the monomers of the cytosolic Y protein or ligandin (Antonenkov et al., 1982), which represents
According to the results of our investigation clofibrate does not exert any significant effect on the enzymes which may lend protection against an "oxidative stress", in rat liver and heart tissue with the exception of peroxisomal catalase. Under the action of the drug no changes in the total and specific activities of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6phosphate dehydrogenase were observed. Earlier Ciriolo et al. (1982) reported about a 20% reduction of the superoxide dismutase and glutathione peroxidase specific activity in liver homogenates of the clofibrate-treated (300 mg/kg, 30 days) rats. The discrepancy between these results may be due to different courses of the drug administration. Clofibrate markedly increased concentration of the reduced glutathione in rat liver. This agrees well with the data of Foliot et al. (1984). Apart from liver tissue, glutathione content was slightly elevated in kidney but in the other organs changes were not detected. The increase of the reduced glutathione in the liver cells may facilitate the elimination of peroxides via glutathione peroxidase pathway (Chance et al., 1979). On the other hand, the elevation of the glutathione concentration could be related to a clofibrate-induced hepatomegaly. Thus, it was shown that glutathione is increased in the regenerating liver after partial hepatectomy in rats (Cockerile et al., 1983). Data about the influence of clofibrate on glutathione-S-transferase activity and ligandin content in the liver represent a particular interest. Clofibrate was shown to decrease the ligandin concentration in rat liver (Fleischner et al., 1975) and to reduce the glutathione-S-transferase activity (Foliot et al., 1984). The fall in the enzyme activity depends on the dose and duration of the drug treatment period (Foliot et al., 1984) and corresponds to the
Table 3. Effect of clofibrate treatment on glutathione content and glutathione-S-transferase activity in rat tissues Glutathione content (/~mol/g tissue) Tissue Liver Kidney Heart Brain Spleen Small intestine
Control 3.28 + 0.24 2.07+0.10 1.13_+0.07 1.09 + 0.25 1.06-+0.22 2.17 _+0.25
Clofibrate 4.88 + 0.26** 2.50+0.06 1.02_+0.0.5 0.90 + 0.06 0.93-+0.13 2.23 + 0.19
Enzyme activity (vmol/min per g tissue) Control
Clofibrate
107.5 _+ 5.4 13.3+0.7 3.4_+0.3 10.2 + 0.8 3.9_+0.4 10.3 _+0.7
53.2 -+ 2.4*** 12.5+ 1.3 3.6_+0.3 9.4 -+ 0.8 3.7_+0.5 5.4 _+0.8**
Means _+ SD are given for four animals in each group; *P < 0.05; **P < 0.01: ***P < 0.001.
Action of clofibrate diminution of the two cytosolic polypeptide fractions with mol. wt of 22,000 and 24,000 (see Fig. 2). It is reasonable to assume that these polypeptides represent the subunits o f various isozymes of glutathioneS-transferases (Antonenkov et al., 1982; Boyer et al., 1983) including those named Y-protein or ligandin (Bhargava et al., 1980). It is known that some of glutathione-S-transferases which consist of YaYc or YcYc dimers possess a pronounced glutathione peroxidase activity with organic hydroperoxides as substrates. At the same time the other isozymes (e.g. YbYb) are very p o o r peroxidases (Boyer et aL, 1983). It is remarkable that despite significant reduction of glutathione-S-transferase activity in the liver, clofibrate does not affect the activity of glutathione peroxidase with t-butyl hydroperoxide as substrate. Based on these data it is conceivable that the drug predominantly influences the content of isozymes with a low peroxidase but high glutathione-Stransferase activity. Several peroxisome proliferators including clofibrate have been shown to produce tumours when administered to rats for prolonged periods (Reddy et al., 1980, 1982). Relatively low doses of the drug promotes hepatocarcinogenesis induced by dimethylnitrozamine (Mochizuki et al., 1982). Our data about the lack of clofibrate effect on enzymes which may represent a protective mechanism against the subversive action of peroxides and oxygen radicals are consistent with the views of Reddy et al. (1982) who supposed a possible role of these toxic agents in carcinogenic action of peroxisome proliferators. At the same time considering the putative mechanisms o f carcinogenesis attention showed be paid to a significant reduction glutathione-S-transferase activity in the liver of the clofibrate-treated rats. There is ample evidence concerning implication of this enzyme in the detoxication of highly reactive carcinogens (Smith et al., 1977; Kaplowitz, 1980) as well as about its possible involvement in the anticarcinogenic effect of antioxidants such as butylated hydroxytoluene or butylated hydroxyanisole (Cha and Heine, 1982). Interestingly, under the experimental conditions employed we have not observed any elevation of the rate of lipid peroxidation in the mitochondrial or microsomal fraction of rat liver homogenates. At the same time the activation of the non-enzymic lipid peroxidation was unexpectedly registered in the nuclei-free homogenate and total particulate fraction of the heart (Antonenkov et al., manuscript in preparation). Further investigations are needed to make reasonable explanations for these findings.
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