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Microsomal and peroxisomal fatty acid oxidation in bile duct ligated rats: A comparative study between liver and kidney
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Gen. Pharmac. Vol. 28, No. 4, pp. 525-529, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Microsomal and Peroxisomal Fatty Acid Oxidation in Bile Duct Ligated Rats: A Comparative Study between Liver and Kidney Myriam Orellana,* Nicolas Avalos, Montserrat Abarca and Elena Vald& DEPARTMENT OF BIOCHEMISTRY, FACULTY OF MEDICINE, UNIVERSITY OF CHILE, SANTIAGO 7, CHILE, [FAx: 56-2-735-55-80]
ABSTRACT. 1. Microsomal cytochrome P-450 and peroxisomal fatty acid oxidation was studied in the kidney of rats 7 days after bile duct ligation (BDL) and a comparative study between kidney and liver was done. 2. Only in the liver did cholestasis decrease the cytocrome P-450 content and the peroxisomal fatty acid I~-oxidation, the catalase activity, and the microsomal metabolism of lauric acid and aminopyrine. 3. In contrast, cholestasis did not influence these activities in the kidney. The microsomal and peroxisomal activities studied responded in a coordinate way to cholestasis. 4. These results could suggest the possibility of a cause-and-effect relationship between microsomal cytochrome P-450 and peroxisomal activity. CEN PHAF~MAC28;4:525--529, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Aminopyrine, catalase, cytochrome P-450, bile duct ligation, lauric acid, peroxisomal 13-oxidation, rat kidney INTRODUCTION Cytochrome P-450, found predominantly in the liver but also in many other tissues, such as the kidney, functions as the terminal oxidase of the microsomal monooxygenase enzymatic system and as the binding site for substrate. Cytochrome P-450 is a family of isoenzymes that metabolizes both, xenobiotics and endogenous compounds such as fatty acids (Orellana et al., 1989), sex steroids (Valdds et al., 1994), cholesterol, and bile acids (Pandak et al., 1994). Fatty acid oxidation is catalyzed by the mitochondrial and peroxisomal [3-oxidation system. The hydroxylation in a terminal carbon (o position), and its last oxidation catalyzed by an alcohol dehydrogenase generates the corresponding dicarboxylic acids, preferentially chain-shortened by the peroxisomes (Osmundsen et al., 1991; Zaar, 1992). Under normal conditions, the peroxisomal [3-oxidation is only a minor pathway for fatty acid oxidation, but it is enhanced during starvation (Orellana et al., 1993), diabetes (Horie et al., 1981), and treatment with hypolipidemic drugs (Hawkins et al., 1987). Studies about the effect of several peroxisome proliferators demonstrated that compounds that maximally induce microsomal fatty acid hydroxylation also are the best inducers of peroxisomal p a l m i t o y l - C o A oxidation (Sharma et al., 1989). The microsomal function has been extensively studied in the liver, but our knowledge of both the constitutive and inducible content and activity of kidney cytochrome P-450s remains very limited. The levels of cytochrome P-450 in the kidney have been previously shown to be much lower than those seen in liver, the renal cortex being the region that presents the highest levels of cytochrome P-450 (Ellin et al., 1972; Jakobsson et al., 1970). Certain forms of cytochrome P-450 seem to be induced by starvation in kidney and liver microsomes (Imakoa et al., •990). Isozymes P-450 involved in the to-hydroxylation of fatty acids (Gonzalez, 1990) are particularly induced by starvation. In a previous study, we * To whom correspondence should be addressed. Received 12 March 1996; revised 27 May 1996; accepted 11 June 1996.
showed an increase in the microsomal lauric acid o3-hydroxylation and the peroxisomal fatty acid [3-oxidation in the liver (Orellana et al., 1992) and kidney of starved rats, with a high correlation between both processes (Orellana et al., 1993). Indeed, these results suggest the possibility of a cause-and-effect relationship between the two phenomena (Ortiz de Montellano et al., 1992). Experimental cholestasis produced by bile duct ligation (BDL) for 7 days has been associated with decreased concentrations of hepatic microsomal cytochrome P-450 and decreased hepatic microsomal oxidative drug metabolism. (Schacter et al., 1983). The liver of cholestasic rats is well characterized in terms of structure and function (Babany et al., 1985; Schacter et al., 1983), but only limited information about kidney microsomal and peroxisomal fatty acid oxidation is available. We studied the effect of cholestasis by BDL on the kidney microsomal metabolism of lauric acid and aminopyrine, peroxisomal fatty acid [3-oxidation, and catalase activity. We carried out a comparative study between kidney and a previous work in liver (Orellana et al., in press) about the response to experimental cholestasis. The aim of this study was to investigate the possible relation between the microsomal cytochrome P-450 and peroxisomal activity. MATERIALS A N D METHODS Animals
Mature male wistar rats weighing 200-250 g were used. Under ether anesthesia, the bile duct was ligated in 2 places 1 cm apart with nonresorbable sutures and then resected in between (BDL group), as reported by Franco et al. (1979). Sham-operated rats were used as controls. Both groups were killed 7 days after surgery. All animals had free access to standard pellets and water. MICROSOMAL CYTOCHROME P-450 CONTENT. Hepatic microsomes were prepared as described elsewhere (Orellana et al., 1989), and at least 3 rats were used for each liver or kidney microsomal sample. Microsomal protein levels were measured according to the
526 method of Lowry with bovine serum albumin as standard, and the total cytochrome P-450 content was measured as described by Omura and Sato (1964). LAURIe ACID HYDROXYLATION. To determine the microsomal activity to hydroxylate laurie acid, microsomal fractions were incubated in a buffer mixture of 50 mM Tris, pH 7.5, 150 mM KCI, 10 mM MgC12; 8 mM sodium isocitrate, 0.25 IU/ml isocitrate dehydrogenase, and lmM NADP. The final protein concentration was 1 mg/ml. After temperature equilibration at 30°C for 3 min, [1-14C] laurie acid (0,6 mCi/mmol) was added to a final concentration of 0.1 mM. After a 5-rain incubation (a range where the reaction was linear with time) 1-ml aliquots of the reaction mixture were removed and the metabolites extracted 3 times with 2 ml of diethyl ether containing 0.05 ml IN He1. Then, the organic phases were combined and evaporated under nitrogen for their analysis by highperformance liquid chromatography (HPLC). Laurie acid and its c01- and t0-hydroxy derivatives were resolved by a reverse-phase HPLC technique, as previously described (Orellana et al., 1992), using a Novapack Cls from Waters (0.39×30 cm, 4-p.m particles). AMINOPYRINE N-DEMETHYLATION.The determination of the microsomal Aminopyrine N-demethylation activity was performed according to the method described by Nash (1953). The reaction mixture contained: 10 mM aminopyrine, 50 mM buffer Tris pH 7.5, 150 mM KCI, 10 mM MgCI2, 8 mM sodium isocitrate, 0.25 IU/ml isocitrate dehydrogenase, 1 mM NADP, and 1.5 mg of liver or 3.0 mg of kidney microsomal protein in a final volume of 1 ml. Incubations were performed at 37°C for 20 min. PEROXlSOMALFATTYACID[~-OXIDATION. The peroxisomal fatty acid [~-oxidation was measured in a 20% homogenate as cyanideinsensitive reduction of NAD+palmitoyl-CoA as substrate, as described by Bronfman et al. (1979).
M. Orellana et al. TABLE 1. Effect of BDL on kidney microsomal protein and total cytochrome P-450 content Group
Microsomal protein (mg/g kidney)
Cytochrome P-450 (nmol/mg protein)
Sham BDL
9.27 _+ 2.03 9.92 _+ 1.20
0.12 _+ 0.04 0.11 _+ 0.05
Values are the means -+ SD of at least 8 different experiments. At least 3 rats per group were used in each experiment.
was about 24% of liver value (the liver control value was 0.50_+0.09 nmol/mg microsomal protein). The effect of BDL for 7 days on the kidney microsomal cytochrome P-450 activity is shown in Table 2. The metabolism of a fatty acid such as laurie acid and a drug such as aminopyrine was determined. Kidney laurie acid to- and tol-hydroxylation and aminopyrine N-demethylation were not affected by BDL. In all groups, the major laurie acid metabolite obtained was the to-hydroxy derivative whose production was about twice that of tol-hydroxy laurie acid. The values of total laurie acid hydroxylation catalized by liver and kidney microsomes were similar in control rats (Fig. 2). However, in the liver a significant decrease to about 61% of control value was seen in the BDL group. As the kidney's total cytochrome P-450 content is lower than the liver's, the kidney has a higher turnover rate than the liver in laurie acid hydroxylation (2,34 and 10.66 nmoles product/min/nmol cytochrome P-450 in the control liver and kidney, respectively). The aminopyrine N-demethylation was not affected by cholestasis in rat kidney (Table 2). In contrast, this activity in liver decreased to about 54% of control value in the BDL group (Fig. 3). The very low aminopyrine N-demethylation of the kidney contrasted with its high laurie acid hydroxylation activity. Moreover,
CATALASEACTIVITY. Catalase activity was measured in a 20% homogenate according the method of Chance et al. (1979).
0,6
STATISTICALANALYSES. Results are expressed as means_+SD. All data were analyzed by the Student's t-test according to Snedecor and Cochran (1967).
Materials Laurie acid, NADPH, isocitrate dehydrogenase, sodium isocitrate, NAD, FAD, DTT, Palmitoyl-CoA, BSA fatty acid free, CoA, and Nicotinamide, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). [1-14C] Laurie acid (56 mCi/mmol) was from Amersham (Arlington Heights, IL). All other chemicals were obtained from commercial sources and were of the highest purity available.
0,5
i'i°. 4o 2._u
RESULTS Seven days after surgery, the body, liver, and kidney weights (g) were similar to the control values (220_+15; 11.80_+1.71 and 2.08_+0.28 respectively). The effectiveness of BDL for 7 days to produce cholestasis was evidenced by the significant increase in serum bile acids obtained in this group (627--67 ~M). As shown in Table 1, the kidney microsomal protein content (mg/g) was not altered significantly by BDL. Similar results were seen in liver (9.72-+ 1.57 and 8.38-+ 1.36 for control and BDL values, respectively). Cytochrome P-450 content was not affected by BDL in kidney, whereas in the liver it decreased to 34% of control value (Fig. 1). The cytochrome P-450 content in kidney of control rats
0,1
Liver
Kidney
FIGURE 1. Effect of BDL on total cytochrome P-450 content of rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means±SD of at least 8 different experiments. At least 3 rats per group were used in each experiment. *Significantly different from control at P<0.05.
Liver and Kidney Microsomal and Peroxisomal Fatty Acid Oxidation
527
TABLE 2. Effect of BDL on lauric acid hydroxylation and aminopyrine N-demethylation catalyzed by rat kidney microsomes Lauric acid hydroxylation (nmol/min/mg protein) Group
to1
to
Total
Aminopyrine N-demethylation (nmol/min/mg protein)
Sham BDL
0.44 ± 0.10 0.45 ± 0.07
0.84 ± 0.12 1.06 +_ 0.28
1.28 ± 0.33 1.51 ± 0.31
0.07 -+ 0.01 0.08 ± 0.02
Values are the means-+SD of at least 8 different experiments. At least 3 rats per group were used in each experiment.
aminopyrine N-demethylation was higher in the liver than in the kidney (the liver value is about 20-fold the kidney value obtained in the control rats). The effect of BDL on peroxisomal [3-oxidation of palmitoyl-CoA in the kidney and liver is shown in Table 3 and Fig. 4, respectively. This activity in the liver was about 4-fold the kidney value, and it decreased to 50% of control value in the BDL group. In contrast, in the kidney, BDL did not influence the peroxisomal 13-oxidation, a response that also occurred with microsomal lauric acid hydroxylation and aminopyrine N-demethylation. The kidney catalase activity, measured as a control parameter of another peroxisomal activity, was not significantly affected by cholestasis (Table 3). Only the liver catalase activity decreased in the BDL group to 61% of control value (Fig. 5). The liver catalase activity was about 2.4-fold higher than the kidney values in the control group. DISCUSSION The results reported indicate that cholestasis, 7 days after BDL, decreases peroxisomal fatty acid [3-oxidation, lauric acid hydroxylation, aminopyrine N-demethylation, and catalase activities in the rat liver, whereas these activities in the kidney are not affected by BDL. These findings agree with previous data pointing out a decreased hepatic microsomal cytochrome P-450 content after 7 days of BDL (Babany et al., 1985; Schacter et al., 1983). It is remarkable that the
response of microsomal and peroxisomal activities to cholestasis was in a coordinate way: both activities were decreased by BDL in the liver and did not affect the kidney. Our results in the BDL group are in agreement with those previously reported by Nishimura et al. (1988) and Babany et al. (1985). These authors found a decrease in the microsomal metabolism of exogenous substrates, such as aniline, aminopyrine and 7-etoxy coumarin in rat liver, 7 days after BDL. Dueland et al. (1991) reported that cirrhosis produced after 4 weeks of BDL (Reichen, 1993) resulted in the selective impairment of microsomal cytochrome P-450 isozymes. They found a decrease in the metabolism of aminopyrine and an increase in the cholesterol 7-a hydroxylase activity. About the effect of cholestasis on the microsomal metabolism of lauric acid and peroxisomal fatty acid [3-oxidation, there are no major reports. Krahenbul and Brass (1991), studying the fuel homeostasis in rats with cirrhosis induced by BDL and after a 24-hr fast, showed mitochondrial defects in fatty acid oxidation, suggesting changes in hepatic lipid metabolism induced by BDL. Our results showing a decreased peroxisomal fatty acid I~-oxidation in the liver of BDL rats
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1,4
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-k
0,8
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&o,4-
0,2 -
1
0,4 0,2
&\< ,>:×
0
I
Liver
Liver
Kidney
FIGURE 2. Effect of BDL on microsomal lauric acid hydroxylation by rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means±SD of at least 8 different experiments. At least 3 rats per group were used in each experiment. *Significantly different from control at P<0.05.
Kidney
F I G U R E 3 . Effect of (BDL) on microsomal aminopyrine N-demethylation by rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means+SD of at least 8 different experiments. At least 3 rats per group were used in each experiment. *Significantly different from control at P
528
M. Orellana et al.
TABLE 3. Effect of BDL on kidney peroxisomal fatty acid 13-oxidation and catalase activity Group
[3-Oxidation (nmol/min/mg protein)
Catalase activity (k/mg protein)"
Sham BDL
1.56 _+ 0.28 1.13 _+ 0.40
0.32 _+ 0.06 0.28 _+ 0.10
0,9 0,8 0,7w
~o,6-
Values are the means -+ SD of at least 20 rats. "k, Catalase first-order kinetic constant.
~ 0,5-
give further support to this last hypothesis. The liver catalase activity also is decreased by BDL. Sukhpal et al. (1992), showed a reduction of the enzymes glutathione peroxidase, glutathione transferase, and catalase activity in the liver of rats 21 days after BDL. The total liver and plasma glutathione and liver vitamin E contents also are decreased by BDL. These results suggest a shift in the prooxidantantioxidant balance in favor of lipid peroxidation, a hypothesis that may explain in part the decrease in the liver microsomal and peroxisomal activities studied here. Therefore, the decreased hepatic microsomal metabolism in cholestasis would appear to be caused not only by the competition of accumulated bile acid with xenobiotics for common binding sites for cytochrome P-450, but also by decreased levels of microsomal electron transport system component, such as NADPH and NADH reductase (Nishiura et al., 1988). Schacter et al. (1983) proposed that a decreased hepatic microsomal cytochrome P-450 concentration in cholestasis is partially the result of decreased cytochrome P-450 synthesis. Increased serum cholesterol and bile acid are characteristically found in rats rendered cirrhotic or cholestasic by BDL, but these values are higher in the cholestasic rats (Babany et al., 1985). Likely, the high level of serum bile acids in BDL rats could cause the lower liver microsomal fatty acid oxidation. Abnormal lipid composition has been described in the liver plasma and microsomal membranes
8-
0 0,30,2 0,1
m m
0
4
Liver
Kidney
FIGURE 5. Effect of BDL on catalase activity of rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means+SD of at least 20 rats, k, Catalase first-order kinetic constant. *Significantly different from control at P < O . 0 5 .
of rats with increased serum bile acids. It has been shown that bile acids change the phospholipids:cholesterol ratio affecting the fluidity of liver microsomal membrane (Bengochea et al., 1987; Reichen et al, 1992). In turn, a decrease in membrane fluidity is associated with a diminished microsomal function (Buters et al., 1993). Our resuits in the liver agree with this last theory. In the kidney, there are no major reports about the effect of BDL on the microsomal and peroxisomal functions. A decreased susceptibility toward induction of kidney microsomal activities by several typical liver inducers, such as phenobarbital and clofibrate, has been shown (Hawkins et al., 1987; Sharma et al., 1989). Therefore, kidney response to exogenous inducers is known to be different from what it is in the liver (Okey, 1990), but its response to endogenous factors is poorly known.
6
ti SUMMARY
i!3. 2 ~-
2-
Liver
Kidney
FIGURE 4. Effect of (BDL) on peroxisomal fatty acid 13-oxidation by rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means--SD of at least 20 rats. *Significantly different from control at P<0.05.
Our studies show that cholestasis by BDL decreases the microsomal and peroxisomal fatty acid oxidation in the liver, but does not affect these activities in the kidney. These results indicate that the kidney's response to experimental cholestasis is different from that of the liver. In addition, the renal microsomal and peroxisomal activities were lower than the hepatic one. Only the microsomal lauric acid metabolism was similar in both organs. The microsomal and peroxisomal activities studied in the liver responded in a coordinate way to cholestasis, whereas no change in any activity was observed in the kidney. These results could suggest the possibility of a causeand-effect relationship between the microsomal cytochrome P-450 and peroxisomal activity (Ortiz de Montellano et al., 1992). Further studies are needed to confirm this hypothesis. We thank Dr. Eugenia Del Villar for her assistance in the preparation of the manuscript and Dr. Antezana for her technical assistance. This work was supported by Grant B-3178 from the Department of Technical Investigation from the University of Chile and Grant 1950-699 from Fondecyt.
Liver and Kidney Microsomal and Peroxisomal Fatty Acid Oxidation
References Babany G., Descatoire V., Corvic M., Gendre S., Gegott C., Larrey D., Letteron P., Wandscheer J. C., Funk-Brentano C. and Pessayre D. (1985) Regulation of renal cytochrome P-450. Biochem. Pharmacol. 34, 311320. Bengochea L., Ouvifia G., Sozzani P. and Lemberg A. (1987) Protein and lipid disturbances in rat liver microsomal membranes after bile duct ligation. Biochem. Biophys. Res. Comm. 144, 980-985. Bronfman M., Inestroza N. and Leighton F. (1979) Fatty acid oxidation by human liver peroxisomes. Biochem. and Biophys. Res. Comm. 88, 10301036. Buters J. T. M., Zysset T. and Reichen J. (1993) Metabolism of antipyrine in vivo in two rat models of liver cirrhosis. Biochem. Pharmacol. 46, 983991. Chance B., Sies H. and Boveris A. (1979) Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-601. Dueland S., Reichen J., Everson T. and Davis R. A. (1991) Regulation of cholesterol and bile acid homeostasis in bile-obstructed rats. Biochem. J. 280, 373-377. Ellin A., Jakobsson S. V., Schenkman J. B. and Orrenius S. (1972) Cytochrome P-450 of rat kidney cortex microsomes: its involvement in fatty acid ~o and (~0-1)-hydroxylation. Arch. Biochem. Biophys. 150, 64-71. Franco D., Gigou M., Szekely M. and Bismuth H. (1979) Portal hypertension after bile duct obstruction. Arch. Surg. 114, 1064-1067. Gonzalez F. J. (1990) Molecular genetics of the P-450 superfamily. Pharmacol. Ther. 45, 1-38. Hawkins J. M., Jones W. E., Bonner F. W. and Gibson G. G. (1987) The effect of peroxisome proliferators on microsomal, peroxisomal, and mitochondrial enzyme activities in the liver and kidney. DrugMetab. Rev. 18, 441-551. Horie S., Ishii H. and Suga T. (1981) Changes in peroxisomal fatty acid oxidation in the diabetic rat liver. J. Biochem. 90, 1691-1696. Imaoka S., Yamaguchi Y. and Funae Y. (1990) Induction and regulation of cytochrome P-450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation. Biochem. Biophys. Acta 1036, 18-23. Jakobsson S., Thor H. and Orrenius S. (1970) Fatty acid inducible cytochrome P-450 of rat kidney cortex microsomes. Biophys. Biochem. Res. Comm. 39, 1073-1080. Krahenbuhl S. and Brass E. P. (1991) Fuel homeostasis and carnitine metabolism in rats with secondary biliary cirrhosis. Hepatology 14, 927-934. Nash T. (1953) The colorimetric estimation of formaldehyde by means the Hatzsh reaction. Biochem. J. 55,416-421. Nishiura S., Koga A. and Yanagisawa J. (1988) Effects of bile obstruction and decompression on hepatic microsomal function oxidase system in rats. Exp. Mol. Pathol. 49, 62-74. Okey B. A. (1990) Enzyme induction in the cytochrome P-450 system. Pharmacol. Ther. 45,241-298.
529 Omura T. and Sato R. (1964) The carbon-monoxide binding pigment by liver microsomes. J. Biol. Chem. 239, 2379-2385. Orellana M., Valdes E., Capdevila J. and Gil L. (1989) Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P-450. Arch. Biochem. Biophys. 274, 251-258. Orellana M., Fuentes O., Rosenbluth H., Lara M. and Vald6s, E. (1992) Modulation of rat liver peroxisomal and microsomal fatty acid oxidation by starvation. FEBS Letters 310, 193-196. Orellana M., Fuentes O. and Valdes E. (1993) Starvation effect on rat kidney peroxisomal and microsomal fatty acid oxidation. FEBS Lett. 322, 61-64. Orellana M., Avalos N., Abarca M. and Vald6s E. (in press). Microsomal and peroxisomal fatty acid oxidation in liver of rats with bile duct ligation and two thirds hepatectomy. Comp. Biochem. Physiol. Ortiz de Montellano P. R., Chan W. K., Tuck S. K., Kaikaus R. M., Bass N. M. and Peterson J. A. (1992) Mechanism-based probes of the topoiogy and function of fatty acid hydroxylases. FASEB J. 6, 695-699. Osmundsen H., Bremer J. and Pedersen J. I. ( 1991 ) Metabolic aspects of peroxisomal [3-oxidation. Biochem. Biophys. Acta. 1085, 141-158. Pandak W. M., Vlahcevic R., Heuman D. M., Redford K. S., Chiang Y. L. and Hylemon P. B. (1994) Effects of different bile acids salts on steadystate mRNA levels and transcriptional activity of cholesterol 7a-hydroxylase. Hepatology 19, 941-947. Reichen J. (1993) Mechanisms of cholestasis. In: Hepathic and Bile Secretion: Physiology and Pathophysiology, (Edited by Tavaloni N. and Berk P. D.) pp. 665-672. Raven Press, New York. Reichen J, Buters J. T. M. and Rooss F.J. (1992) Abnormal lipid composition of microsomes from cirrhotic rat l i v e r ~ o e s it contribute to decreased microsomal function? Experent/a 48, 482-486. Schacter B. A., Joseph E. and Fimeisz G. (1983) Effect of cholestasis produced by bile duct ligation on hepatic heme and hemoprotein metabolism in rats. Gastroenterology 84, 227-235. Sharma K., Lake B. G., Makowsky R., Bradshaw T., Earnshaw D., Dale J. W. and Gibson G. (1989) Differential induction of peroxisomal and microsomal fatty-acid-oxidizing enzymes by peroxisome proliferation in rat liver and kidney. Eur. J. Biochem. 184, 69-78. Snedecor G. W. and Cochran W. G. (1967) Statistical Methods. Iowa State University Press, Ames. Sukhpal S., Shackleton G., Ah-Sing E., Chakraborty J. and Bailey M. E. (1992) Antioxidant defenses in the bile duct-ligated rat. Gastroenterol, ogy, 103, 1625-1629. Vald~s E., Orellana M., Del Villar E. and Vargas M. (1994) Androstenedione metabolism in streptozotocin diabetes and fasting male rats: similarities and differences. Comp. Biochem. Physiol. 107A, 261-267. Zaar K. (1992) Structure and function of peroxisomes in the mammalian kidney. Eur. J. Cell Biol. 59, 233-254.
Gen. Pharmac. Vol. 28, No. 4, pp. 525-529, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Microsomal and Peroxisomal Fatty Acid Oxidation in Bile Duct Ligated Rats: A Comparative Study between Liver and Kidney Myriam Orellana,* Nicolas Avalos, Montserrat Abarca and Elena Vald& DEPARTMENT OF BIOCHEMISTRY, FACULTY OF MEDICINE, UNIVERSITY OF CHILE, SANTIAGO 7, CHILE, [FAx: 56-2-735-55-80]
ABSTRACT. 1. Microsomal cytochrome P-450 and peroxisomal fatty acid oxidation was studied in the kidney of rats 7 days after bile duct ligation (BDL) and a comparative study between kidney and liver was done. 2. Only in the liver did cholestasis decrease the cytocrome P-450 content and the peroxisomal fatty acid I~-oxidation, the catalase activity, and the microsomal metabolism of lauric acid and aminopyrine. 3. In contrast, cholestasis did not influence these activities in the kidney. The microsomal and peroxisomal activities studied responded in a coordinate way to cholestasis. 4. These results could suggest the possibility of a cause-and-effect relationship between microsomal cytochrome P-450 and peroxisomal activity. CEN PHAF~MAC28;4:525--529, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Aminopyrine, catalase, cytochrome P-450, bile duct ligation, lauric acid, peroxisomal 13-oxidation, rat kidney INTRODUCTION Cytochrome P-450, found predominantly in the liver but also in many other tissues, such as the kidney, functions as the terminal oxidase of the microsomal monooxygenase enzymatic system and as the binding site for substrate. Cytochrome P-450 is a family of isoenzymes that metabolizes both, xenobiotics and endogenous compounds such as fatty acids (Orellana et al., 1989), sex steroids (Valdds et al., 1994), cholesterol, and bile acids (Pandak et al., 1994). Fatty acid oxidation is catalyzed by the mitochondrial and peroxisomal [3-oxidation system. The hydroxylation in a terminal carbon (o position), and its last oxidation catalyzed by an alcohol dehydrogenase generates the corresponding dicarboxylic acids, preferentially chain-shortened by the peroxisomes (Osmundsen et al., 1991; Zaar, 1992). Under normal conditions, the peroxisomal [3-oxidation is only a minor pathway for fatty acid oxidation, but it is enhanced during starvation (Orellana et al., 1993), diabetes (Horie et al., 1981), and treatment with hypolipidemic drugs (Hawkins et al., 1987). Studies about the effect of several peroxisome proliferators demonstrated that compounds that maximally induce microsomal fatty acid hydroxylation also are the best inducers of peroxisomal p a l m i t o y l - C o A oxidation (Sharma et al., 1989). The microsomal function has been extensively studied in the liver, but our knowledge of both the constitutive and inducible content and activity of kidney cytochrome P-450s remains very limited. The levels of cytochrome P-450 in the kidney have been previously shown to be much lower than those seen in liver, the renal cortex being the region that presents the highest levels of cytochrome P-450 (Ellin et al., 1972; Jakobsson et al., 1970). Certain forms of cytochrome P-450 seem to be induced by starvation in kidney and liver microsomes (Imakoa et al., •990). Isozymes P-450 involved in the to-hydroxylation of fatty acids (Gonzalez, 1990) are particularly induced by starvation. In a previous study, we * To whom correspondence should be addressed. Received 12 March 1996; revised 27 May 1996; accepted 11 June 1996.
showed an increase in the microsomal lauric acid o3-hydroxylation and the peroxisomal fatty acid [3-oxidation in the liver (Orellana et al., 1992) and kidney of starved rats, with a high correlation between both processes (Orellana et al., 1993). Indeed, these results suggest the possibility of a cause-and-effect relationship between the two phenomena (Ortiz de Montellano et al., 1992). Experimental cholestasis produced by bile duct ligation (BDL) for 7 days has been associated with decreased concentrations of hepatic microsomal cytochrome P-450 and decreased hepatic microsomal oxidative drug metabolism. (Schacter et al., 1983). The liver of cholestasic rats is well characterized in terms of structure and function (Babany et al., 1985; Schacter et al., 1983), but only limited information about kidney microsomal and peroxisomal fatty acid oxidation is available. We studied the effect of cholestasis by BDL on the kidney microsomal metabolism of lauric acid and aminopyrine, peroxisomal fatty acid [3-oxidation, and catalase activity. We carried out a comparative study between kidney and a previous work in liver (Orellana et al., in press) about the response to experimental cholestasis. The aim of this study was to investigate the possible relation between the microsomal cytochrome P-450 and peroxisomal activity. MATERIALS A N D METHODS Animals
Mature male wistar rats weighing 200-250 g were used. Under ether anesthesia, the bile duct was ligated in 2 places 1 cm apart with nonresorbable sutures and then resected in between (BDL group), as reported by Franco et al. (1979). Sham-operated rats were used as controls. Both groups were killed 7 days after surgery. All animals had free access to standard pellets and water. MICROSOMAL CYTOCHROME P-450 CONTENT. Hepatic microsomes were prepared as described elsewhere (Orellana et al., 1989), and at least 3 rats were used for each liver or kidney microsomal sample. Microsomal protein levels were measured according to the
526 method of Lowry with bovine serum albumin as standard, and the total cytochrome P-450 content was measured as described by Omura and Sato (1964). LAURIe ACID HYDROXYLATION. To determine the microsomal activity to hydroxylate laurie acid, microsomal fractions were incubated in a buffer mixture of 50 mM Tris, pH 7.5, 150 mM KCI, 10 mM MgC12; 8 mM sodium isocitrate, 0.25 IU/ml isocitrate dehydrogenase, and lmM NADP. The final protein concentration was 1 mg/ml. After temperature equilibration at 30°C for 3 min, [1-14C] laurie acid (0,6 mCi/mmol) was added to a final concentration of 0.1 mM. After a 5-rain incubation (a range where the reaction was linear with time) 1-ml aliquots of the reaction mixture were removed and the metabolites extracted 3 times with 2 ml of diethyl ether containing 0.05 ml IN He1. Then, the organic phases were combined and evaporated under nitrogen for their analysis by highperformance liquid chromatography (HPLC). Laurie acid and its c01- and t0-hydroxy derivatives were resolved by a reverse-phase HPLC technique, as previously described (Orellana et al., 1992), using a Novapack Cls from Waters (0.39×30 cm, 4-p.m particles). AMINOPYRINE N-DEMETHYLATION.The determination of the microsomal Aminopyrine N-demethylation activity was performed according to the method described by Nash (1953). The reaction mixture contained: 10 mM aminopyrine, 50 mM buffer Tris pH 7.5, 150 mM KCI, 10 mM MgCI2, 8 mM sodium isocitrate, 0.25 IU/ml isocitrate dehydrogenase, 1 mM NADP, and 1.5 mg of liver or 3.0 mg of kidney microsomal protein in a final volume of 1 ml. Incubations were performed at 37°C for 20 min. PEROXlSOMALFATTYACID[~-OXIDATION. The peroxisomal fatty acid [~-oxidation was measured in a 20% homogenate as cyanideinsensitive reduction of NAD+palmitoyl-CoA as substrate, as described by Bronfman et al. (1979).
M. Orellana et al. TABLE 1. Effect of BDL on kidney microsomal protein and total cytochrome P-450 content Group
Microsomal protein (mg/g kidney)
Cytochrome P-450 (nmol/mg protein)
Sham BDL
9.27 _+ 2.03 9.92 _+ 1.20
0.12 _+ 0.04 0.11 _+ 0.05
Values are the means -+ SD of at least 8 different experiments. At least 3 rats per group were used in each experiment.
was about 24% of liver value (the liver control value was 0.50_+0.09 nmol/mg microsomal protein). The effect of BDL for 7 days on the kidney microsomal cytochrome P-450 activity is shown in Table 2. The metabolism of a fatty acid such as laurie acid and a drug such as aminopyrine was determined. Kidney laurie acid to- and tol-hydroxylation and aminopyrine N-demethylation were not affected by BDL. In all groups, the major laurie acid metabolite obtained was the to-hydroxy derivative whose production was about twice that of tol-hydroxy laurie acid. The values of total laurie acid hydroxylation catalized by liver and kidney microsomes were similar in control rats (Fig. 2). However, in the liver a significant decrease to about 61% of control value was seen in the BDL group. As the kidney's total cytochrome P-450 content is lower than the liver's, the kidney has a higher turnover rate than the liver in laurie acid hydroxylation (2,34 and 10.66 nmoles product/min/nmol cytochrome P-450 in the control liver and kidney, respectively). The aminopyrine N-demethylation was not affected by cholestasis in rat kidney (Table 2). In contrast, this activity in liver decreased to about 54% of control value in the BDL group (Fig. 3). The very low aminopyrine N-demethylation of the kidney contrasted with its high laurie acid hydroxylation activity. Moreover,
CATALASEACTIVITY. Catalase activity was measured in a 20% homogenate according the method of Chance et al. (1979).
0,6
STATISTICALANALYSES. Results are expressed as means_+SD. All data were analyzed by the Student's t-test according to Snedecor and Cochran (1967).
Materials Laurie acid, NADPH, isocitrate dehydrogenase, sodium isocitrate, NAD, FAD, DTT, Palmitoyl-CoA, BSA fatty acid free, CoA, and Nicotinamide, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). [1-14C] Laurie acid (56 mCi/mmol) was from Amersham (Arlington Heights, IL). All other chemicals were obtained from commercial sources and were of the highest purity available.
0,5
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RESULTS Seven days after surgery, the body, liver, and kidney weights (g) were similar to the control values (220_+15; 11.80_+1.71 and 2.08_+0.28 respectively). The effectiveness of BDL for 7 days to produce cholestasis was evidenced by the significant increase in serum bile acids obtained in this group (627--67 ~M). As shown in Table 1, the kidney microsomal protein content (mg/g) was not altered significantly by BDL. Similar results were seen in liver (9.72-+ 1.57 and 8.38-+ 1.36 for control and BDL values, respectively). Cytochrome P-450 content was not affected by BDL in kidney, whereas in the liver it decreased to 34% of control value (Fig. 1). The cytochrome P-450 content in kidney of control rats
0,1
Liver
Kidney
FIGURE 1. Effect of BDL on total cytochrome P-450 content of rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means±SD of at least 8 different experiments. At least 3 rats per group were used in each experiment. *Significantly different from control at P<0.05.
Liver and Kidney Microsomal and Peroxisomal Fatty Acid Oxidation
527
TABLE 2. Effect of BDL on lauric acid hydroxylation and aminopyrine N-demethylation catalyzed by rat kidney microsomes Lauric acid hydroxylation (nmol/min/mg protein) Group
to1
to
Total
Aminopyrine N-demethylation (nmol/min/mg protein)
Sham BDL
0.44 ± 0.10 0.45 ± 0.07
0.84 ± 0.12 1.06 +_ 0.28
1.28 ± 0.33 1.51 ± 0.31
0.07 -+ 0.01 0.08 ± 0.02
Values are the means-+SD of at least 8 different experiments. At least 3 rats per group were used in each experiment.
aminopyrine N-demethylation was higher in the liver than in the kidney (the liver value is about 20-fold the kidney value obtained in the control rats). The effect of BDL on peroxisomal [3-oxidation of palmitoyl-CoA in the kidney and liver is shown in Table 3 and Fig. 4, respectively. This activity in the liver was about 4-fold the kidney value, and it decreased to 50% of control value in the BDL group. In contrast, in the kidney, BDL did not influence the peroxisomal 13-oxidation, a response that also occurred with microsomal lauric acid hydroxylation and aminopyrine N-demethylation. The kidney catalase activity, measured as a control parameter of another peroxisomal activity, was not significantly affected by cholestasis (Table 3). Only the liver catalase activity decreased in the BDL group to 61% of control value (Fig. 5). The liver catalase activity was about 2.4-fold higher than the kidney values in the control group. DISCUSSION The results reported indicate that cholestasis, 7 days after BDL, decreases peroxisomal fatty acid [3-oxidation, lauric acid hydroxylation, aminopyrine N-demethylation, and catalase activities in the rat liver, whereas these activities in the kidney are not affected by BDL. These findings agree with previous data pointing out a decreased hepatic microsomal cytochrome P-450 content after 7 days of BDL (Babany et al., 1985; Schacter et al., 1983). It is remarkable that the
response of microsomal and peroxisomal activities to cholestasis was in a coordinate way: both activities were decreased by BDL in the liver and did not affect the kidney. Our results in the BDL group are in agreement with those previously reported by Nishimura et al. (1988) and Babany et al. (1985). These authors found a decrease in the microsomal metabolism of exogenous substrates, such as aniline, aminopyrine and 7-etoxy coumarin in rat liver, 7 days after BDL. Dueland et al. (1991) reported that cirrhosis produced after 4 weeks of BDL (Reichen, 1993) resulted in the selective impairment of microsomal cytochrome P-450 isozymes. They found a decrease in the metabolism of aminopyrine and an increase in the cholesterol 7-a hydroxylase activity. About the effect of cholestasis on the microsomal metabolism of lauric acid and peroxisomal fatty acid [3-oxidation, there are no major reports. Krahenbul and Brass (1991), studying the fuel homeostasis in rats with cirrhosis induced by BDL and after a 24-hr fast, showed mitochondrial defects in fatty acid oxidation, suggesting changes in hepatic lipid metabolism induced by BDL. Our results showing a decreased peroxisomal fatty acid I~-oxidation in the liver of BDL rats
1,8 1,6
2
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1,8 t
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i,
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1,4
0
1,
-k
0,8
¢" O g'~
&o,4-
0,2 -
1
0,4 0,2
&\< ,>:×
0
I
Liver
Liver
Kidney
FIGURE 2. Effect of BDL on microsomal lauric acid hydroxylation by rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means±SD of at least 8 different experiments. At least 3 rats per group were used in each experiment. *Significantly different from control at P<0.05.
Kidney
F I G U R E 3 . Effect of (BDL) on microsomal aminopyrine N-demethylation by rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means+SD of at least 8 different experiments. At least 3 rats per group were used in each experiment. *Significantly different from control at P
528
M. Orellana et al.
TABLE 3. Effect of BDL on kidney peroxisomal fatty acid 13-oxidation and catalase activity Group
[3-Oxidation (nmol/min/mg protein)
Catalase activity (k/mg protein)"
Sham BDL
1.56 _+ 0.28 1.13 _+ 0.40
0.32 _+ 0.06 0.28 _+ 0.10
0,9 0,8 0,7w
~o,6-
Values are the means -+ SD of at least 20 rats. "k, Catalase first-order kinetic constant.
~ 0,5-
give further support to this last hypothesis. The liver catalase activity also is decreased by BDL. Sukhpal et al. (1992), showed a reduction of the enzymes glutathione peroxidase, glutathione transferase, and catalase activity in the liver of rats 21 days after BDL. The total liver and plasma glutathione and liver vitamin E contents also are decreased by BDL. These results suggest a shift in the prooxidantantioxidant balance in favor of lipid peroxidation, a hypothesis that may explain in part the decrease in the liver microsomal and peroxisomal activities studied here. Therefore, the decreased hepatic microsomal metabolism in cholestasis would appear to be caused not only by the competition of accumulated bile acid with xenobiotics for common binding sites for cytochrome P-450, but also by decreased levels of microsomal electron transport system component, such as NADPH and NADH reductase (Nishiura et al., 1988). Schacter et al. (1983) proposed that a decreased hepatic microsomal cytochrome P-450 concentration in cholestasis is partially the result of decreased cytochrome P-450 synthesis. Increased serum cholesterol and bile acid are characteristically found in rats rendered cirrhotic or cholestasic by BDL, but these values are higher in the cholestasic rats (Babany et al., 1985). Likely, the high level of serum bile acids in BDL rats could cause the lower liver microsomal fatty acid oxidation. Abnormal lipid composition has been described in the liver plasma and microsomal membranes
8-
0 0,30,2 0,1
m m
0
4
Liver
Kidney
FIGURE 5. Effect of BDL on catalase activity of rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means+SD of at least 20 rats, k, Catalase first-order kinetic constant. *Significantly different from control at P < O . 0 5 .
of rats with increased serum bile acids. It has been shown that bile acids change the phospholipids:cholesterol ratio affecting the fluidity of liver microsomal membrane (Bengochea et al., 1987; Reichen et al, 1992). In turn, a decrease in membrane fluidity is associated with a diminished microsomal function (Buters et al., 1993). Our resuits in the liver agree with this last theory. In the kidney, there are no major reports about the effect of BDL on the microsomal and peroxisomal functions. A decreased susceptibility toward induction of kidney microsomal activities by several typical liver inducers, such as phenobarbital and clofibrate, has been shown (Hawkins et al., 1987; Sharma et al., 1989). Therefore, kidney response to exogenous inducers is known to be different from what it is in the liver (Okey, 1990), but its response to endogenous factors is poorly known.
6
ti SUMMARY
i!3. 2 ~-
2-
Liver
Kidney
FIGURE 4. Effect of (BDL) on peroxisomal fatty acid 13-oxidation by rat liver and kidney. Open squares, controls; cross-hatched squares, BDL group. Values are the means--SD of at least 20 rats. *Significantly different from control at P<0.05.
Our studies show that cholestasis by BDL decreases the microsomal and peroxisomal fatty acid oxidation in the liver, but does not affect these activities in the kidney. These results indicate that the kidney's response to experimental cholestasis is different from that of the liver. In addition, the renal microsomal and peroxisomal activities were lower than the hepatic one. Only the microsomal lauric acid metabolism was similar in both organs. The microsomal and peroxisomal activities studied in the liver responded in a coordinate way to cholestasis, whereas no change in any activity was observed in the kidney. These results could suggest the possibility of a causeand-effect relationship between the microsomal cytochrome P-450 and peroxisomal activity (Ortiz de Montellano et al., 1992). Further studies are needed to confirm this hypothesis. We thank Dr. Eugenia Del Villar for her assistance in the preparation of the manuscript and Dr. Antezana for her technical assistance. This work was supported by Grant B-3178 from the Department of Technical Investigation from the University of Chile and Grant 1950-699 from Fondecyt.
Liver and Kidney Microsomal and Peroxisomal Fatty Acid Oxidation
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529 Omura T. and Sato R. (1964) The carbon-monoxide binding pigment by liver microsomes. J. Biol. Chem. 239, 2379-2385. Orellana M., Valdes E., Capdevila J. and Gil L. (1989) Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P-450. Arch. Biochem. Biophys. 274, 251-258. Orellana M., Fuentes O., Rosenbluth H., Lara M. and Vald6s, E. (1992) Modulation of rat liver peroxisomal and microsomal fatty acid oxidation by starvation. FEBS Letters 310, 193-196. Orellana M., Fuentes O. and Valdes E. (1993) Starvation effect on rat kidney peroxisomal and microsomal fatty acid oxidation. FEBS Lett. 322, 61-64. Orellana M., Avalos N., Abarca M. and Vald6s E. (in press). Microsomal and peroxisomal fatty acid oxidation in liver of rats with bile duct ligation and two thirds hepatectomy. Comp. Biochem. Physiol. Ortiz de Montellano P. R., Chan W. K., Tuck S. K., Kaikaus R. M., Bass N. M. and Peterson J. A. (1992) Mechanism-based probes of the topoiogy and function of fatty acid hydroxylases. FASEB J. 6, 695-699. Osmundsen H., Bremer J. and Pedersen J. I. ( 1991 ) Metabolic aspects of peroxisomal [3-oxidation. Biochem. Biophys. Acta. 1085, 141-158. Pandak W. M., Vlahcevic R., Heuman D. M., Redford K. S., Chiang Y. L. and Hylemon P. B. (1994) Effects of different bile acids salts on steadystate mRNA levels and transcriptional activity of cholesterol 7a-hydroxylase. Hepatology 19, 941-947. Reichen J. (1993) Mechanisms of cholestasis. In: Hepathic and Bile Secretion: Physiology and Pathophysiology, (Edited by Tavaloni N. and Berk P. D.) pp. 665-672. Raven Press, New York. Reichen J, Buters J. T. M. and Rooss F.J. (1992) Abnormal lipid composition of microsomes from cirrhotic rat l i v e r ~ o e s it contribute to decreased microsomal function? Experent/a 48, 482-486. Schacter B. A., Joseph E. and Fimeisz G. (1983) Effect of cholestasis produced by bile duct ligation on hepatic heme and hemoprotein metabolism in rats. Gastroenterology 84, 227-235. Sharma K., Lake B. G., Makowsky R., Bradshaw T., Earnshaw D., Dale J. W. and Gibson G. (1989) Differential induction of peroxisomal and microsomal fatty-acid-oxidizing enzymes by peroxisome proliferation in rat liver and kidney. Eur. J. Biochem. 184, 69-78. Snedecor G. W. and Cochran W. G. (1967) Statistical Methods. Iowa State University Press, Ames. Sukhpal S., Shackleton G., Ah-Sing E., Chakraborty J. and Bailey M. E. (1992) Antioxidant defenses in the bile duct-ligated rat. Gastroenterol, ogy, 103, 1625-1629. Vald~s E., Orellana M., Del Villar E. and Vargas M. (1994) Androstenedione metabolism in streptozotocin diabetes and fasting male rats: similarities and differences. Comp. Biochem. Physiol. 107A, 261-267. Zaar K. (1992) Structure and function of peroxisomes in the mammalian kidney. Eur. J. Cell Biol. 59, 233-254.