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Oxidative stress, microsomal and peroxisomal fatty acid oxidation in the liver of rats treated with acetone
Comparative Biochemistry and Physiology Part C 128 Ž2001. 503᎐509
Oxidative stress, microsomal and peroxisomal fatty acid oxidation in the liver of rats treated with acetone Myriam Orellana B.U , Viviana Guajardo, Julia Araya, Lilian Thieleman, Ramon ´ Rodrigo ICBM Programa de Farmacologıa Facultad de Medicina, Uni¨ ersidad de Chile, Santiago 7 Casilla 70086, ´ Molecular y Clınica, ´ Chile Received 6 October 2000; received in revised form 26 January 2001; accepted 29 January 2001
Abstract Parameters of oxidative stress, microsomal cytochrome P450 activity and peroxisomal fatty acid oxidation were studied in liver of rats following acetone Ž1% vrv. consumption for 7 days. Acetone treatment increased the activity of catalase and decreased the activities of superoxide dismutase ŽSOD. and glutathione peroxidase ŽGTPx., but did not significantly modify the liver content of malondialdehyde ŽMDA. and reduced glutathione. Also, acetone increased the total content of cytochrome P450, the microsomal lauric acid hydroxylation, aminopyrine N-demethylation and the peroxisomal -oxidation of palmitoyl CoA. These effects were similar to those found previously in starved and ethanol-treated rats, supporting the hypothesis that ketone bodies would be the common inducer of microsomal and peroxisomal fatty acid oxidation in these metabolic states. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Acetone; Aminopyrine; Cytochrome P450; Lauric acid; Liver; Oxidative stress
1. Introduction P450 is a family of isoenzymes that metabolize both xenobiotics and endogenous compounds such as fatty acids ŽOrellana et al., 1989., sex steroids, cholesterol and bile acids ŽValdes ´ et al., 1994.. Numerous studies indicate that ethanol and ace-
Abbre¨ iations: , 12-hydroxy lauric acid; 1, 11-hydroxy lauric acid U Corresponding author. Tel.: q56-2-6786066; fax: q56-2755580. E-mail address: [email protected] ŽM. Orellana B...
tone increase the activity of diverse species of cytochrome P450, particularly the P450 2E1 isoenzyme ŽRonis et al., 1993.. Apart from the metabolism of endogenous compounds, P450 2E1 is thought to play an important role in the metabolism of exogenous compounds such as acetone, ethanol, ether, CCl 4 and nitrosamines ŽKoop, 1992.. Induction of P450 2E1 by a variety of structurally unrelated chemicals, including its own substrates, as well as by ketotic conditions such as a high fat diet, fasting and diabetes mellitus has been reported, ŽHong et al., 1987; Koop, 1992; Yun et al., 1992.. In addition to these well-known properties, P450 2E1 can act as a
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M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509
gluconeogenic enzyme in the conversion of fat to carbohydrates ŽCasazza et al., 1984; Koop and Casazza, 1985; Lieber, 1997.. The peroxisome is a subcellular organelle particularly abundant in the tissues actively contributing to lipid metabolism, such as the liver, and carrying out different processes including fatty acid -oxidation and glyoxylate metabolism ŽOsmundsen et al., 1991; Van den Bosh et al., 1992.. The peroxisomal fatty acid -oxidative pathway differs from the mitochondrial pathway fundamentally in the first reaction in which FADH 2 is re-oxidized directly by oxygen generating H 2 O 2 , a harmful intermediate that is effectively destroyed by the action of a catalase located in this cellular compartment ŽOsmundsen et al., 1991.. This extra-mitochondrial route of oxidation becomes quantitatively important under conditions in which liver fatty acid flux rates exceed the normal capacity for esterification and mitochondrial -oxidation, namely, high-fat diets ŽNilsson et al., 1987; Yoo et al., 1991., uncontrolled diabetes mellitus, and starvation ŽYang et al., 1992.. Lauric acid is hydroxylated by isoenzymes P450 from the family 4A ŽOrtiz de Montellano et al., 1992. and by P450 2E1 in the and 1 positions, respectively ŽAmet et al., 1994., both induced in starvation. Furthermore, the hypolipidemic drugs clofibrate and bezafibrate were reported to induce both P450 4A1 and the enzymes of peroxisomal fatty acid oxidation ŽSharma et al., 1989.. From these data it was hypothesized that microsomal and peroxisomal oxidations may be interrelated ŽOrtiz de Montellano et al., 1992.. Thus, microsomal hydroxylation of the terminal carbon of a fatty acid, followed by successive oxidations catalyzed by alcohol and aldehyde dehydrogenases, would generate the corresponding dicarboxylic acid derivative that may be preferentially oxidized by peroxisomes ŽHawkins et al., 1987; Ockner et al., 1993.. Previously, we studied the effect of starvation ŽOrellana et al., 1992, 1993. and ethanol consumption on the microsomal cytochrome P450 and peroxisomal oxidations ŽOrellana et al., 1998a,b.. In both models studied, increases in the microsomal lauric acid hydroxylation catalyzed by cytochrome P450 and in the peroxisomal fatty acid -oxidation were found. A possible clue to the mechanism of this induction is the fact that the metabolic states occurring during fasting and
ethanol treatment result in elevated production of ketone bodies and increased activity of diverse species of cytochrome P450, particularly P450 2E1 ŽYang et al., 1992; Ronis et al., 1993., an isoform reported as a major generator of superoxide anion ŽMiller and Yang, 1984; Kosugi et al., 1986; Roberts et al., 1994.. The aim of this study was to assess whether acetone plays a role as a common inducer of both the microsomal and the peroxisomal fatty acid oxidation and in the generation of oxidative stress in the liver.
2. Materials and methods 2.1. Animals Mature male Wistar rats Ž Rattus nor¨ egicus. weighing 200 g at day of initiation of treatment received an aqueous acetone solution Ž1% vrv. ad libitum as sole drinking fluid for 7 days. A daily supplementation of 1 ml of this solution was also administered by gastric tube feeding and an equivalent volume of tap water was given to control rats. 2.2. Biochemical parameters 2.2.1. Microsomal cytochrome P450 content Hepatic microsomes were prepared by ultracentrifugation as described elsewhere ŽOrellana et al., 1989. and the livers of at least three rats were used for each microsomal sample. Microsomal protein was measured by the method of Lowry et al. Ž1951. using bovine serum albumin ŽBSA. as a standard. The total cytochrome P450 content was measured according to the method of Omura and Sato Ž1964.. 2.2.2. Lauric acid hydroxylation For the determination of this activity, microsomal fractions were incubated in a buffer mixture of 50 mM Tris, pH 7.5; 150 mM KCl; 10 mM MgCl 2 ; 8 mM sodium isocitrate; 0.25 IUrml isocitrate dehydrogenase and 1 mM NADP. The final protein concentration was 1 mgrml. After temperature equilibration at 30⬚C for 3 min, w1- 14 Cx lauric acid Ž0.6 mCirmmol. was added to a final concentration of 0.1 mM. After a 5-min incubation Ža range where the reaction was linear with time. 1-ml aliquots of the reaction mixture were removed and the metabolites extracted three
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509
times with 2 ml of diethyl-ether containing 0.05 ml 1 N HCl. Subsequently the organic phases were combined and evaporated under nitrogen for analysis by HPLC. Lauric acid and its 1- and -hydroxy derivatives were resolved by a reverse phase HPLC technique as previously described ŽOrellana et al., 1992. using a Novapack C 18 from Waters Ž0.39= 30cm, 4 m particle size.. 2.2.3. Aminopyrine N-demethylation The determination of this microsomal activity was performed according to the method described by Nash Ž1953. by using 1.5 mgrml of liver microsomal protein. 2.2.4. Peroxisomal fatty acid -oxidation This was measured in a 20% homogenate as a cyanide-insensitive reduction of NADq, using palmitoyl CoA as substrate ŽBronfman et al., 1979.. 2.2.5. Catalase To assess this activity, the breakdown of H 2 O 2 was determined directly by following spectrophotometrically the decrease of absorbance at 240 nm according to the method of Chance et al. Ž1979.. 2.2.6. Superoxide dismutase This activity was determined using a commercial kit ŽRandox Laboratories Ltd., UK.. This method employs xanthine and xanthine oxidase to generate superoxide radicals which react with 2Ž4-iodophenyl.-3-Ž4-nitrophenol.-5-phenyltetrazolium chloride ŽINT. to form a red formazan dye detected at 505 nm. 2.2.7. Glutathione peroxidase This activity was measured by a method based on the reduction of GSSG with NADPH by following the decrease of absorbance at 340 nm spectrophotometrically ŽFlohe 1984.. ´ and Gunzler, ¨ 2.2.8. Lipid peroxidation The lipid peroxides were determined by the thiobarbituric acid reaction measuring the production of malondialdehyde ŽMDA. ŽOhkawa et al., 1979.. 2.2.9. Reduced glutathione The content of reduced glutathione ŽGSH. was determined spectrophotometrically at 410 nm by
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using 5.5-dithionitrobenzoic acid ŽDTNB. as described by Tietze Ž1969. and Griffith Ž1980.. 2.3. Statistical analysis Values were expressed as mean " S.D. Statistical significance was analyzed by the Student’s t-test for unpaired data. The differences were considered significant when P- 0.05. 2.4. Materials Lauric acid, NADPH, isocitrate dehydrogenase, sodium isocitrate, NAD, FAD, dithiothreitol, palmitoyl CoA, BSA fatty acid free, CoA, and nicotinamide were purchased from Sigma Chemical Co. ŽSt Louis, Missouri, USA.. w1- 14 Cx lauric acid Ž56 mCirmmol. was purchased from Amersham ŽArlington, Heights, IL, USA.. All other chemicals were obtained from commercial sources and were of the highest purity available.
3. Results After 7 days of acetone consumption the body weight of the rats had increased to 12.5% of control values ŽTable 1.. Neither the weights of the liver nor the kidney, nor the liver microsomal protein content were altered significantly by the acetone treatment. However, the total cytochrome P450 content was increased by 94% above control values ŽTable 2.. The effect of acetone consumption on the activity of liver microsomal cytochrome P450 is shown in Table 3. The oxidation of fatty acid was assessed by lauric acid hydroxylation. The drug metabolism was assessed by aminopyrine N-demethylation. After acetone treatment the total Table 1 Effect of acetone treatment on body, liver and kidney weights Žg. a Group
Body
Liver
Kidney
Control
200 " 10 Ž20. 225 " 11b Ž22.
10.2" 0.27 Ž20. 10.8" 1.20 Ž22.
0.99" 0.05 Ž20. 1.03" 0.09 Ž22.
Acetone
a Data are mean " S.D. Number of rats is shown in parentheses. b Significantly different from control at P- 0.05.
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509
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Table 2 Effect of acetone treatment on liver microsomal protein and total cytochrome P450 content a Group
Microsomal protein Žmgrg liver.
Cytochrome P450 Žnmolrmg protein.
Control
10.20" 1.10 Ž15. 9.90" 1.20 Ž16.
0.50" 0.12 Ž6. 0.97" 0.10b Ž8.
Acetone a
Data are mean " S.D. Number of preparations is shown in parentheses. b Significantly different from control at P- 0.05.
lauric acid hydroxylation and aminopyrine N-demethylation were increased by 320 and 368% over control values, respectively. Although the major lauric acid metabolite obtained in every group was the -hydroxy derivative, the acetone treatment increased the 1-hydroxy lauric acid proportion to approximately 1.27 times that of the -hydroxy proportion. Peroxisomal -oxidation of palmitoyl CoA was studied to investigate the possible correlation between the microsomal and peroxisomal fatty acid oxidations. Peroxisomal -oxidation of palmitoyl CoA was increased by 41% over control values in the acetone-treated group ŽTable 4.. Also, the activity of catalase, the antioxidant enzyme present in peroxisomes, was increased by 136% over control values by acetone treatment ŽTable 4.. As shown in Table 5, the acetone treatment decreased the activities of the antioxidant enzymes superoxide dismutase ŽSOD. and glutathione peroxidase ŽGTPx. to 63 and 64% of control values, respectively. Regarding the effect of acetone on the index of lipoperoxidation assessed by the MDA level and the liver content of reduced glutathione ŽGSH., these parameters were not modulated significantly by acetone.
4. Discussion Data presented in this work indicate that acetone consumption increases the content of cytochrome P450, the microsomal biotransformation activity, the peroxisomal fatty acid oxidation, and the catalase activity in the liver. On the other hand, the acetone treatment decreased the hepatic activity of SOD and GTPx without altering the content of GSH and MDA of the liver. These results suggest that the effect of acetone in increasing the P450 activity and peroxisomal fatty acid oxidation is not related to significant changes in the oxidative stress status of the liver. The effect of acetone on microsomal, peroxisomal and catalase activities were similar to those found previously in starved and ethanol-treated rats, suggesting a common action mechanism. There are no reports about the effect of acetone on the parameters of oxidative stress and peroxisomal fatty acid oxidation, except for those concerning the effect on microsomal cytochrome P450 activity. In fact, cytochrome P450 induction by acetone treatment was paralleled by a significant increase in cytochrome P450 activity, in agreement with previous reports ŽMiller and Yang, 1984; Yang et al., 1992.. The acetone-induced lauric acid hydroxylation was characterized by a 4-fold enhancement. These changes seem to be due to the adaptive increase in specifically related P450 isoenzymes, namely P450 4A1 catalyzing the -hydroxylation of lauric and arachidonic acids ŽMa et al., 1993. and P450 2E1 involved in the 1-hydroxylation ŽLaethem et al., 1993; Amet et al., 1994.. Acetone induction of several P450 isoenzymes Ž2B1, 2B2, 2C11, 2C6, 3A, 1A1 and 1A2. could explain the increase in the microsomal activity for aminopyrine N-demethylation.
Table 3 Effect of acetone treatment on liver microsomal activities catalyzed by cytochrome P450 Žnmolrmin per mg protein. a Microsomal activity Lauric acid hydroxylation 1 Total Aminopyrine N-demethylation a b
Control
Acetone
Acetonercontrol
0.45" 0.10 0.81" 0.12 1.26" 0.33 Ž21. 1.54" 0.50 Ž10.
2.19" 0.30b 3.10" 0.50b 5.29" 1.05b Ž21. 7.21" 1.20b Ž12.
4.87 3.83 4.20
Data are mean " S.D. Number of preparations is shown in parentheses. Significantly different from control at P- 0.05.
4.68
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509 Table 4 Effect of acetone treatment on liver peroxisomal activity a Group
Palmitoyl CoA -oxidation Žnmolrmin per mg protein.
Catalase activity Žkrmg protein. b
Control
6.80" 0.81 Ž11. 9.60" 1.20 c Ž12.
0.83" 0.04 Ž9. 1.96" 0.08 c Ž19.
Acetone
a Values are mean " S.D. Number preparations is shown in parentheses. k, catalase first-order kinetic constant. b k, catalase first-order kinetic constant. c Significantly different from control at P- 0.05.
Studies about the effect of several peroxisome proliferators demonstrate that those compounds that maximally induce microsomal fatty acid hydroxylation are also the best inducers of peroxisomal palmitoyl-CoA oxidation ŽHawkins et al., 1987; Ortiz de Montellano et al., 1992; Ockner et al., 1993; Amet et al., 1994.. In addition to the possible role of acetone-induced specific P450 isoenzymes in the determination of higher rates of microsomal fatty acid oxidation, such as in ethanol consumption, their importance in the depression of mitochondrial fatty acid oxidation and the deposition of sterified fatty acids in the liver should also be considered ŽAmet et al., 1994.. These factors are likely to favor higher rates of peroxisomal fatty acid oxidation, a process known to be stimulated by products of the -hydroxylation pathway ŽOckner et al., 1993.. Thus, microsomal -hydroxylation and peroxisomal fatty acid oxidation seem to be inter-related in the liver cell providing that the -hydroxy fatty acids produced by the former process are further oxidized to dicarboxylic acids by the enzymes of the alcohol dehydrogenase pathway ŽMa et al., 1993.. Table 5 Effect of acetone treatment on some parameters of liver oxidative stress a Group
Control
Acetone
SOD ŽUrmg protein. GTPx ŽUrmg protein. MDA Žnmolesrmg protein. GSH Žmolrg tissue.
36.88" 9.06 Ž8. 0.42" 0.07 Ž8. 6.80" 0.61 Ž8. 4.25" 1.09 Ž8.
23.27" 5.15b Ž10. 0.27" 0.04b Ž10. 5.91" 0.88 Ž8. 4.27 " 1.06 Ž9.
a Data are mean " S.D. Number of experiments are in parentheses. b Significantly different from control at P- 0.05.
507
In chronic ethanol consumption as well as in starvation, the induction of peroxisomal -oxidation and microsomal activities towards lauric acid support the view that the inductive response could be triggered by endogenous factors common to the metabolic state of starvation and of ethanol treatment, namely high hepatic levels of free fatty acids and a relative excess of endogenous acetone or ketone bodies. These may give rise to an induction of the cytochrome P450 4A family and the P450 2E1 isoenzyme, thereby increasing the rate of fatty acid hydroxylation and formation of dicarboxylic acids. In addition, the acetone effect of increasing the lipid catabolism was similar to that obtained from rats treated with peroxisome proliferators ŽHawkins et al., 1987; Sharma et al., 1989.. Moreover, our study indicates a correlation between the microsomal and peroxisomal oxidation of fatty acids by acetone treatment, suggesting that the regulation of microsomal and peroxisomal fatty acid oxidation are closely related. It has been reported that ketotic rats and humans excrete considerable amounts of dicarboxylic acids in the urine. These seem to be derived from endogenous fatty acid by -oxidation ŽOsmundsen et al., 1991; Ortiz de Montellano et al., 1992.. It has been proposed that dicarboxylic acids can be completely chain-shortened to succinate by peroxisomal -oxidation. Succinate synthesized in this manner can make some net synthesis of glucose from fatty acids possible, however, no such conversion has yet been directly demonstrated ŽOsmundsen et al., 1991; Wada and Usami, 1992.. Recent reports signaling the presence of 1,2-propanediol and 2,3-butanediol in blood drawn from both alcoholics and starved patients and in blood drawn from acetone-treated rats suggest that similar metabolic pathways may be responsible for the production of these compounds in these circumstances ŽCasazza et al., 1984; Koop and Casazza, 1985.. The chronic administration of ethanol or acetone to rats results in the induction of two microsomal activities ᎏ acetone mono-oxygenase which converts acetone to acetol and acetol mono-oxygenase which converts acetol to methylglyoxal. These reactions require oxygen and NADPH, suggesting that both may be P450-catalyzed. Two pathways have been postulated by which acetol from acetone is converted to glucose, one via methylglyoxal and the other via propanediol with lactate as an intermediate
508
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ŽKosugi et al., 1986.. The glyoxylate cycle provides a mechanism for the net conversion of acetyl units to carbohydrate in micro-organisms and fat-bearing seeds. In mammals glyoxylate can be metabolized by conversion to glycine in a reaction catalyzed by alanine. Glyoxylate aminotransferase is an enzyme present in both peroxisomes and mitochondria detected only in liver, and to a lesser extent, in kidney ŽVan den Bosh et al., 1992. and in this way the glyoxylate could enter to gluconeogenic pathway. It has been postulated that acetone may be a significant gluconeogenic precursor in fasting humans, accounting for 10% of the gluconeogenic demands of humans fasted by a 21-day process where the P450 2E1 induced by ethanol, acetone and starvation would be involved ŽLieber, 1997, 1999.. In agreement with data here reported, the acetone effect of increasing the microsomal and peroxisomal fatty acid oxidation was similar to that obtained previously in rats starved and rats treated with ethanol ŽOrellana et al., 1992, 1998b.. These results support the hypothesis that ketone bodies could be the common inducer of the microsomal and peroxisomal fatty acid oxidation in these metabolic states. We proposed that the final purpose of these linked fatty acid oxidations could be the generation of a precursor for the synthesis of glucose in these metabolic states. Further research is required to develop this hypothesis.
Acknowledgements We thank Maria Eugenia Rybak O, Diego Soto and Miladio Ruiz for their technical assistance. This work was supported by Grant EDID-98-002 from DID from the Universidad de Chile and Grants 1950-699 and 1990᎐784 from Fondecyt. References Amet, Y., Berthou, F., Goasduff, T., Salaun, J.P., Le Breton, L., Menez, J.F., 1994. Evidence that cytochrome P450 2E1 is involved in the Ž 1.-hydroxylation of lauric acid in rat liver microsomes. Biochem. Biophys. Res. Commun. 203, 1168᎐1174. Bronfman, M., Inestroza, N., Leighton, F., 1979. Fatty acid oxidation by human liver peroxisomes. Biochem. Biophys. Res. Commun. 88, 1030᎐1036.
Casazza, J.P., Felver, M.E., Veech, R.L., 1984. The metabolism of acetone in rat. J. Biol. Chem. 259, 231᎐236. Chance, B., Sies, H., Boveris, A., 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 538᎐539. Flohe, ´ L., Gunzler, W.A., 1984. Assays of glutathione peroxidase. In: Colowic, S.P., Kaplan, N.O. ŽEds.., Methods in Enzymology, 105. Academic Press, New York, pp. 114᎐121. Griffith, O.W., 1980. Determination of glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207᎐212. Hawkins, J.M., Jones, W.E., Bonner, F.W., Gibson, G.G., 1987. The effect of peroxisome proliferators on microsomal, peroxisomal, and mitochondrial enzyme activities in the liver and kidney. Drug Metab. Rev 18, 441᎐551. Hong, J.Y., Pan, J., Dong, Z., Ning, S.M., Yang, C.S., 1987. Regulation of N-nitrosodimethylamine demethylase in rat liver and kidney. Cancer Res. 47, 5948᎐5953. Koop, D.R., 1992. Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J. 6, 724᎐730. Koop, D.R., Casazza, J.P., 1985. Identification of ethanol-inducible P450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J. Biol. Chem. 260, 13607᎐13612. Kosugi, K., Scofield, R.F.:, Chandramouli, V., Kumaran, K., Schumann, W.C., Landau, B.R., 1986. Pathways of acetone’s metabolism. J. Biol. Chem. 25, 3952᎐3957. Laethem, R.M., Balazy, M., Falck, J.R., Laethem, C., Koop, D.R., 1993. Formation of 19Žs.-, 19 䊛-, and 18 䊛-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1. J. Biol. Chem. 268, 12912᎐12918. Lieber, C.S., 1997. Cytochrome P450 2E1: its physiological and pathological role. Physiol. Rev. 77, 517᎐544. Lieber, C.S., 1999. Microsomal ethanol-oxidizing system ŽMEOS.: The first 30 years Ž1968᎐1998., alcoholism:. Clin. Exp. Res. 23 Ž6., 991᎐1007. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265᎐275. Ma, X., Baraona, E., Lieber, C.S., 1993. Alcohol consumption enhances fatty acid Ž -oxidation, with a greater increase in male than in female rats. Hepatology 18, 1247᎐1253. Miller, K.W., Yang, C.S., 1984. Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting, acetone and ethanol. Arch. Biochem. Biophys. 229, 483᎐491. Nash, T., 1953. The colorimetric estimation of
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509
formaldehyde by means of the Hatzsh reaction. Biochem. J. 55, 416᎐421. Nilsson, A., Pridz, K., Rortveit, T., Christiansen, E.N., 1987. Studies on the interrelated stimulation of microsomal -oxidation and peroxisomal -oxidation in rat liver with partially hydrogenated fish oil diet. Biochim. Biophys. Acta 920, 114᎐119. Ockner, R.K., Kaikaus, R.M., Bass, N.M., 1993. Fattyacid metabolism and the pathogenesis of hepatocellular carcinoma: review and hypothesis. Hepatology 18, 669᎐676. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351᎐358. Omura, T., Sato, R., 1964. The carbon-monoxide binding pigment by liver microsomes. J Biol. Chem. 239, 2379᎐2385. Orellana, M., Valdes, ´ E., Capdevila, J., Gil, L., 1989. Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P450. Arch. Biochem. Biophys. 274, 251᎐258. Orellana, M., Fuentes, O., Rosenbluth, H., Lara, M., Valdes, ´ E., 1992. Modulation of rat liver peroxisomal and microsomal fatty acid oxidation by starvation. FEBS Lett. 310, 193᎐196. Orellana, M., Fuentes, O., Valdes, ´ E., 1993. Starvation effect on rat kidney peroxisomal and microsomal fatty acid oxidation. FEBS Lett. 322, 61᎐64. Orellana, M., Valdes, J., Rodrigo, R., ´ E., Fernandez, ´ 1998a. Effects of chronic ethanol consumption on extra-mitochondrial fatty acid oxidation and ethanol metabolism by rat kidney. Gen. Pharmacol. 30, 719᎐723. Orellana, M., Rodrigo, R., Valdes, ´ E., 1998b. Peroxisomal and microsomal fatty acid oxidation in liver of rats following chronic ethanol consumption. Gen. Pharmacol. 31, 817᎐820. Ortiz de Montellano, P.R., Chan, W.K., Tuck, S.K., Kaikaus, R.M., Bass, N.M., Peterson, J.A., 1992. Mechanism-based probes of the topology and function of fatty acid hydroxylases. FASEB J. 6, 695᎐699. Osmundsen, H., Bremer, J., Pedersen, J.I., 1991. Metabolic aspects of peroxisomal Ž-oxidation.. Biochim. Biophys. Acta 1085, 141᎐158.
509
Roberts, B.J., Shoaf, S.E., Jeong, K.-S., Song, B.J., 1994. Induction of CYP2E1 in liver, kidney, brain and intestine during chronic ethanol administration and withdrawal: evidence that CYP2E1 possesses a rapid phase half-life of 6 hours or less. Biochem. Biophys. Res. Comm. 205, 1064᎐1071. Ronis, M.J., Huang, J., Crouch, J. et al., 1993. Cytochrome P450 cyp 2E1 induction during alcohol exposure occurs by a two-step mechanism associated with blood alcohol concentrations in rats. J. Pharm. Exp. Ther. 264, 944᎐950. Sharma, K., Lake, B.G., Makowsky, R. et al., 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. Tietze, F., 1969. Enzymic method for quantitative determination of nanograms amounts of total and oxidized glutathione. Anal. Biochem. 27, 502᎐522. Valdes, ´ E., Orellana, M., Del Villar, E., Vargas, M., 1994. Androstenedione metabolism in streptozotocin diabetes and fasting male rats: similarities and differences. Comp. Biochem. Physiol. 107A, 261᎐267. Van den Bosh, H., Schutgens, R.B.H., Wanders, R.J.A., Tager, J.M., 1992. Biochemistry of peroxisomes. Annu. Rev. Biochem. 62, 157᎐197. Wada, F., Usami, M., 1992. Studies of fatty acid oxidation: antiketogenic effect and gluconeogenicity of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats. Biochem Biophys Acta 487, 261᎐268. Yang, C., Brady, J., Hong, J., 1992. Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity. FASEB J. 6, 737᎐744. Yoo, J.S.H., Ning, S.M., Pantuck, C.B., Pantuck, E.J., Yang, C.S., 1991. Regulation of hepatic microsomal cytochrome P450IIE1 level by dietary lipids and carbohydrates in rats. J. Nutr. 121, 959᎐965. Yun, Y.P., Casazza, J.P., Sohn, D.H., Veech, R.L., Song, B.J., 1992. Pretranslational activation of cytochrome P450IIE during ketosis induced by a high fat diet. Mol. Pharmacol. 41, 474᎐479.
Oxidative stress, microsomal and peroxisomal fatty acid oxidation in the liver of rats treated with acetone Myriam Orellana B.U , Viviana Guajardo, Julia Araya, Lilian Thieleman, Ramon ´ Rodrigo ICBM Programa de Farmacologıa Facultad de Medicina, Uni¨ ersidad de Chile, Santiago 7 Casilla 70086, ´ Molecular y Clınica, ´ Chile Received 6 October 2000; received in revised form 26 January 2001; accepted 29 January 2001
Abstract Parameters of oxidative stress, microsomal cytochrome P450 activity and peroxisomal fatty acid oxidation were studied in liver of rats following acetone Ž1% vrv. consumption for 7 days. Acetone treatment increased the activity of catalase and decreased the activities of superoxide dismutase ŽSOD. and glutathione peroxidase ŽGTPx., but did not significantly modify the liver content of malondialdehyde ŽMDA. and reduced glutathione. Also, acetone increased the total content of cytochrome P450, the microsomal lauric acid hydroxylation, aminopyrine N-demethylation and the peroxisomal -oxidation of palmitoyl CoA. These effects were similar to those found previously in starved and ethanol-treated rats, supporting the hypothesis that ketone bodies would be the common inducer of microsomal and peroxisomal fatty acid oxidation in these metabolic states. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Acetone; Aminopyrine; Cytochrome P450; Lauric acid; Liver; Oxidative stress
1. Introduction P450 is a family of isoenzymes that metabolize both xenobiotics and endogenous compounds such as fatty acids ŽOrellana et al., 1989., sex steroids, cholesterol and bile acids ŽValdes ´ et al., 1994.. Numerous studies indicate that ethanol and ace-
Abbre¨ iations: , 12-hydroxy lauric acid; 1, 11-hydroxy lauric acid U Corresponding author. Tel.: q56-2-6786066; fax: q56-2755580. E-mail address: [email protected] ŽM. Orellana B...
tone increase the activity of diverse species of cytochrome P450, particularly the P450 2E1 isoenzyme ŽRonis et al., 1993.. Apart from the metabolism of endogenous compounds, P450 2E1 is thought to play an important role in the metabolism of exogenous compounds such as acetone, ethanol, ether, CCl 4 and nitrosamines ŽKoop, 1992.. Induction of P450 2E1 by a variety of structurally unrelated chemicals, including its own substrates, as well as by ketotic conditions such as a high fat diet, fasting and diabetes mellitus has been reported, ŽHong et al., 1987; Koop, 1992; Yun et al., 1992.. In addition to these well-known properties, P450 2E1 can act as a
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gluconeogenic enzyme in the conversion of fat to carbohydrates ŽCasazza et al., 1984; Koop and Casazza, 1985; Lieber, 1997.. The peroxisome is a subcellular organelle particularly abundant in the tissues actively contributing to lipid metabolism, such as the liver, and carrying out different processes including fatty acid -oxidation and glyoxylate metabolism ŽOsmundsen et al., 1991; Van den Bosh et al., 1992.. The peroxisomal fatty acid -oxidative pathway differs from the mitochondrial pathway fundamentally in the first reaction in which FADH 2 is re-oxidized directly by oxygen generating H 2 O 2 , a harmful intermediate that is effectively destroyed by the action of a catalase located in this cellular compartment ŽOsmundsen et al., 1991.. This extra-mitochondrial route of oxidation becomes quantitatively important under conditions in which liver fatty acid flux rates exceed the normal capacity for esterification and mitochondrial -oxidation, namely, high-fat diets ŽNilsson et al., 1987; Yoo et al., 1991., uncontrolled diabetes mellitus, and starvation ŽYang et al., 1992.. Lauric acid is hydroxylated by isoenzymes P450 from the family 4A ŽOrtiz de Montellano et al., 1992. and by P450 2E1 in the and 1 positions, respectively ŽAmet et al., 1994., both induced in starvation. Furthermore, the hypolipidemic drugs clofibrate and bezafibrate were reported to induce both P450 4A1 and the enzymes of peroxisomal fatty acid oxidation ŽSharma et al., 1989.. From these data it was hypothesized that microsomal and peroxisomal oxidations may be interrelated ŽOrtiz de Montellano et al., 1992.. Thus, microsomal hydroxylation of the terminal carbon of a fatty acid, followed by successive oxidations catalyzed by alcohol and aldehyde dehydrogenases, would generate the corresponding dicarboxylic acid derivative that may be preferentially oxidized by peroxisomes ŽHawkins et al., 1987; Ockner et al., 1993.. Previously, we studied the effect of starvation ŽOrellana et al., 1992, 1993. and ethanol consumption on the microsomal cytochrome P450 and peroxisomal oxidations ŽOrellana et al., 1998a,b.. In both models studied, increases in the microsomal lauric acid hydroxylation catalyzed by cytochrome P450 and in the peroxisomal fatty acid -oxidation were found. A possible clue to the mechanism of this induction is the fact that the metabolic states occurring during fasting and
ethanol treatment result in elevated production of ketone bodies and increased activity of diverse species of cytochrome P450, particularly P450 2E1 ŽYang et al., 1992; Ronis et al., 1993., an isoform reported as a major generator of superoxide anion ŽMiller and Yang, 1984; Kosugi et al., 1986; Roberts et al., 1994.. The aim of this study was to assess whether acetone plays a role as a common inducer of both the microsomal and the peroxisomal fatty acid oxidation and in the generation of oxidative stress in the liver.
2. Materials and methods 2.1. Animals Mature male Wistar rats Ž Rattus nor¨ egicus. weighing 200 g at day of initiation of treatment received an aqueous acetone solution Ž1% vrv. ad libitum as sole drinking fluid for 7 days. A daily supplementation of 1 ml of this solution was also administered by gastric tube feeding and an equivalent volume of tap water was given to control rats. 2.2. Biochemical parameters 2.2.1. Microsomal cytochrome P450 content Hepatic microsomes were prepared by ultracentrifugation as described elsewhere ŽOrellana et al., 1989. and the livers of at least three rats were used for each microsomal sample. Microsomal protein was measured by the method of Lowry et al. Ž1951. using bovine serum albumin ŽBSA. as a standard. The total cytochrome P450 content was measured according to the method of Omura and Sato Ž1964.. 2.2.2. Lauric acid hydroxylation For the determination of this activity, microsomal fractions were incubated in a buffer mixture of 50 mM Tris, pH 7.5; 150 mM KCl; 10 mM MgCl 2 ; 8 mM sodium isocitrate; 0.25 IUrml isocitrate dehydrogenase and 1 mM NADP. The final protein concentration was 1 mgrml. After temperature equilibration at 30⬚C for 3 min, w1- 14 Cx lauric acid Ž0.6 mCirmmol. was added to a final concentration of 0.1 mM. After a 5-min incubation Ža range where the reaction was linear with time. 1-ml aliquots of the reaction mixture were removed and the metabolites extracted three
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509
times with 2 ml of diethyl-ether containing 0.05 ml 1 N HCl. Subsequently the organic phases were combined and evaporated under nitrogen for analysis by HPLC. Lauric acid and its 1- and -hydroxy derivatives were resolved by a reverse phase HPLC technique as previously described ŽOrellana et al., 1992. using a Novapack C 18 from Waters Ž0.39= 30cm, 4 m particle size.. 2.2.3. Aminopyrine N-demethylation The determination of this microsomal activity was performed according to the method described by Nash Ž1953. by using 1.5 mgrml of liver microsomal protein. 2.2.4. Peroxisomal fatty acid -oxidation This was measured in a 20% homogenate as a cyanide-insensitive reduction of NADq, using palmitoyl CoA as substrate ŽBronfman et al., 1979.. 2.2.5. Catalase To assess this activity, the breakdown of H 2 O 2 was determined directly by following spectrophotometrically the decrease of absorbance at 240 nm according to the method of Chance et al. Ž1979.. 2.2.6. Superoxide dismutase This activity was determined using a commercial kit ŽRandox Laboratories Ltd., UK.. This method employs xanthine and xanthine oxidase to generate superoxide radicals which react with 2Ž4-iodophenyl.-3-Ž4-nitrophenol.-5-phenyltetrazolium chloride ŽINT. to form a red formazan dye detected at 505 nm. 2.2.7. Glutathione peroxidase This activity was measured by a method based on the reduction of GSSG with NADPH by following the decrease of absorbance at 340 nm spectrophotometrically ŽFlohe 1984.. ´ and Gunzler, ¨ 2.2.8. Lipid peroxidation The lipid peroxides were determined by the thiobarbituric acid reaction measuring the production of malondialdehyde ŽMDA. ŽOhkawa et al., 1979.. 2.2.9. Reduced glutathione The content of reduced glutathione ŽGSH. was determined spectrophotometrically at 410 nm by
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using 5.5-dithionitrobenzoic acid ŽDTNB. as described by Tietze Ž1969. and Griffith Ž1980.. 2.3. Statistical analysis Values were expressed as mean " S.D. Statistical significance was analyzed by the Student’s t-test for unpaired data. The differences were considered significant when P- 0.05. 2.4. Materials Lauric acid, NADPH, isocitrate dehydrogenase, sodium isocitrate, NAD, FAD, dithiothreitol, palmitoyl CoA, BSA fatty acid free, CoA, and nicotinamide were purchased from Sigma Chemical Co. ŽSt Louis, Missouri, USA.. w1- 14 Cx lauric acid Ž56 mCirmmol. was purchased from Amersham ŽArlington, Heights, IL, USA.. All other chemicals were obtained from commercial sources and were of the highest purity available.
3. Results After 7 days of acetone consumption the body weight of the rats had increased to 12.5% of control values ŽTable 1.. Neither the weights of the liver nor the kidney, nor the liver microsomal protein content were altered significantly by the acetone treatment. However, the total cytochrome P450 content was increased by 94% above control values ŽTable 2.. The effect of acetone consumption on the activity of liver microsomal cytochrome P450 is shown in Table 3. The oxidation of fatty acid was assessed by lauric acid hydroxylation. The drug metabolism was assessed by aminopyrine N-demethylation. After acetone treatment the total Table 1 Effect of acetone treatment on body, liver and kidney weights Žg. a Group
Body
Liver
Kidney
Control
200 " 10 Ž20. 225 " 11b Ž22.
10.2" 0.27 Ž20. 10.8" 1.20 Ž22.
0.99" 0.05 Ž20. 1.03" 0.09 Ž22.
Acetone
a Data are mean " S.D. Number of rats is shown in parentheses. b Significantly different from control at P- 0.05.
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Table 2 Effect of acetone treatment on liver microsomal protein and total cytochrome P450 content a Group
Microsomal protein Žmgrg liver.
Cytochrome P450 Žnmolrmg protein.
Control
10.20" 1.10 Ž15. 9.90" 1.20 Ž16.
0.50" 0.12 Ž6. 0.97" 0.10b Ž8.
Acetone a
Data are mean " S.D. Number of preparations is shown in parentheses. b Significantly different from control at P- 0.05.
lauric acid hydroxylation and aminopyrine N-demethylation were increased by 320 and 368% over control values, respectively. Although the major lauric acid metabolite obtained in every group was the -hydroxy derivative, the acetone treatment increased the 1-hydroxy lauric acid proportion to approximately 1.27 times that of the -hydroxy proportion. Peroxisomal -oxidation of palmitoyl CoA was studied to investigate the possible correlation between the microsomal and peroxisomal fatty acid oxidations. Peroxisomal -oxidation of palmitoyl CoA was increased by 41% over control values in the acetone-treated group ŽTable 4.. Also, the activity of catalase, the antioxidant enzyme present in peroxisomes, was increased by 136% over control values by acetone treatment ŽTable 4.. As shown in Table 5, the acetone treatment decreased the activities of the antioxidant enzymes superoxide dismutase ŽSOD. and glutathione peroxidase ŽGTPx. to 63 and 64% of control values, respectively. Regarding the effect of acetone on the index of lipoperoxidation assessed by the MDA level and the liver content of reduced glutathione ŽGSH., these parameters were not modulated significantly by acetone.
4. Discussion Data presented in this work indicate that acetone consumption increases the content of cytochrome P450, the microsomal biotransformation activity, the peroxisomal fatty acid oxidation, and the catalase activity in the liver. On the other hand, the acetone treatment decreased the hepatic activity of SOD and GTPx without altering the content of GSH and MDA of the liver. These results suggest that the effect of acetone in increasing the P450 activity and peroxisomal fatty acid oxidation is not related to significant changes in the oxidative stress status of the liver. The effect of acetone on microsomal, peroxisomal and catalase activities were similar to those found previously in starved and ethanol-treated rats, suggesting a common action mechanism. There are no reports about the effect of acetone on the parameters of oxidative stress and peroxisomal fatty acid oxidation, except for those concerning the effect on microsomal cytochrome P450 activity. In fact, cytochrome P450 induction by acetone treatment was paralleled by a significant increase in cytochrome P450 activity, in agreement with previous reports ŽMiller and Yang, 1984; Yang et al., 1992.. The acetone-induced lauric acid hydroxylation was characterized by a 4-fold enhancement. These changes seem to be due to the adaptive increase in specifically related P450 isoenzymes, namely P450 4A1 catalyzing the -hydroxylation of lauric and arachidonic acids ŽMa et al., 1993. and P450 2E1 involved in the 1-hydroxylation ŽLaethem et al., 1993; Amet et al., 1994.. Acetone induction of several P450 isoenzymes Ž2B1, 2B2, 2C11, 2C6, 3A, 1A1 and 1A2. could explain the increase in the microsomal activity for aminopyrine N-demethylation.
Table 3 Effect of acetone treatment on liver microsomal activities catalyzed by cytochrome P450 Žnmolrmin per mg protein. a Microsomal activity Lauric acid hydroxylation 1 Total Aminopyrine N-demethylation a b
Control
Acetone
Acetonercontrol
0.45" 0.10 0.81" 0.12 1.26" 0.33 Ž21. 1.54" 0.50 Ž10.
2.19" 0.30b 3.10" 0.50b 5.29" 1.05b Ž21. 7.21" 1.20b Ž12.
4.87 3.83 4.20
Data are mean " S.D. Number of preparations is shown in parentheses. Significantly different from control at P- 0.05.
4.68
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509 Table 4 Effect of acetone treatment on liver peroxisomal activity a Group
Palmitoyl CoA -oxidation Žnmolrmin per mg protein.
Catalase activity Žkrmg protein. b
Control
6.80" 0.81 Ž11. 9.60" 1.20 c Ž12.
0.83" 0.04 Ž9. 1.96" 0.08 c Ž19.
Acetone
a Values are mean " S.D. Number preparations is shown in parentheses. k, catalase first-order kinetic constant. b k, catalase first-order kinetic constant. c Significantly different from control at P- 0.05.
Studies about the effect of several peroxisome proliferators demonstrate that those compounds that maximally induce microsomal fatty acid hydroxylation are also the best inducers of peroxisomal palmitoyl-CoA oxidation ŽHawkins et al., 1987; Ortiz de Montellano et al., 1992; Ockner et al., 1993; Amet et al., 1994.. In addition to the possible role of acetone-induced specific P450 isoenzymes in the determination of higher rates of microsomal fatty acid oxidation, such as in ethanol consumption, their importance in the depression of mitochondrial fatty acid oxidation and the deposition of sterified fatty acids in the liver should also be considered ŽAmet et al., 1994.. These factors are likely to favor higher rates of peroxisomal fatty acid oxidation, a process known to be stimulated by products of the -hydroxylation pathway ŽOckner et al., 1993.. Thus, microsomal -hydroxylation and peroxisomal fatty acid oxidation seem to be inter-related in the liver cell providing that the -hydroxy fatty acids produced by the former process are further oxidized to dicarboxylic acids by the enzymes of the alcohol dehydrogenase pathway ŽMa et al., 1993.. Table 5 Effect of acetone treatment on some parameters of liver oxidative stress a Group
Control
Acetone
SOD ŽUrmg protein. GTPx ŽUrmg protein. MDA Žnmolesrmg protein. GSH Žmolrg tissue.
36.88" 9.06 Ž8. 0.42" 0.07 Ž8. 6.80" 0.61 Ž8. 4.25" 1.09 Ž8.
23.27" 5.15b Ž10. 0.27" 0.04b Ž10. 5.91" 0.88 Ž8. 4.27 " 1.06 Ž9.
a Data are mean " S.D. Number of experiments are in parentheses. b Significantly different from control at P- 0.05.
507
In chronic ethanol consumption as well as in starvation, the induction of peroxisomal -oxidation and microsomal activities towards lauric acid support the view that the inductive response could be triggered by endogenous factors common to the metabolic state of starvation and of ethanol treatment, namely high hepatic levels of free fatty acids and a relative excess of endogenous acetone or ketone bodies. These may give rise to an induction of the cytochrome P450 4A family and the P450 2E1 isoenzyme, thereby increasing the rate of fatty acid hydroxylation and formation of dicarboxylic acids. In addition, the acetone effect of increasing the lipid catabolism was similar to that obtained from rats treated with peroxisome proliferators ŽHawkins et al., 1987; Sharma et al., 1989.. Moreover, our study indicates a correlation between the microsomal and peroxisomal oxidation of fatty acids by acetone treatment, suggesting that the regulation of microsomal and peroxisomal fatty acid oxidation are closely related. It has been reported that ketotic rats and humans excrete considerable amounts of dicarboxylic acids in the urine. These seem to be derived from endogenous fatty acid by -oxidation ŽOsmundsen et al., 1991; Ortiz de Montellano et al., 1992.. It has been proposed that dicarboxylic acids can be completely chain-shortened to succinate by peroxisomal -oxidation. Succinate synthesized in this manner can make some net synthesis of glucose from fatty acids possible, however, no such conversion has yet been directly demonstrated ŽOsmundsen et al., 1991; Wada and Usami, 1992.. Recent reports signaling the presence of 1,2-propanediol and 2,3-butanediol in blood drawn from both alcoholics and starved patients and in blood drawn from acetone-treated rats suggest that similar metabolic pathways may be responsible for the production of these compounds in these circumstances ŽCasazza et al., 1984; Koop and Casazza, 1985.. The chronic administration of ethanol or acetone to rats results in the induction of two microsomal activities ᎏ acetone mono-oxygenase which converts acetone to acetol and acetol mono-oxygenase which converts acetol to methylglyoxal. These reactions require oxygen and NADPH, suggesting that both may be P450-catalyzed. Two pathways have been postulated by which acetol from acetone is converted to glucose, one via methylglyoxal and the other via propanediol with lactate as an intermediate
508
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ŽKosugi et al., 1986.. The glyoxylate cycle provides a mechanism for the net conversion of acetyl units to carbohydrate in micro-organisms and fat-bearing seeds. In mammals glyoxylate can be metabolized by conversion to glycine in a reaction catalyzed by alanine. Glyoxylate aminotransferase is an enzyme present in both peroxisomes and mitochondria detected only in liver, and to a lesser extent, in kidney ŽVan den Bosh et al., 1992. and in this way the glyoxylate could enter to gluconeogenic pathway. It has been postulated that acetone may be a significant gluconeogenic precursor in fasting humans, accounting for 10% of the gluconeogenic demands of humans fasted by a 21-day process where the P450 2E1 induced by ethanol, acetone and starvation would be involved ŽLieber, 1997, 1999.. In agreement with data here reported, the acetone effect of increasing the microsomal and peroxisomal fatty acid oxidation was similar to that obtained previously in rats starved and rats treated with ethanol ŽOrellana et al., 1992, 1998b.. These results support the hypothesis that ketone bodies could be the common inducer of the microsomal and peroxisomal fatty acid oxidation in these metabolic states. We proposed that the final purpose of these linked fatty acid oxidations could be the generation of a precursor for the synthesis of glucose in these metabolic states. Further research is required to develop this hypothesis.
Acknowledgements We thank Maria Eugenia Rybak O, Diego Soto and Miladio Ruiz for their technical assistance. This work was supported by Grant EDID-98-002 from DID from the Universidad de Chile and Grants 1950-699 and 1990᎐784 from Fondecyt. References Amet, Y., Berthou, F., Goasduff, T., Salaun, J.P., Le Breton, L., Menez, J.F., 1994. Evidence that cytochrome P450 2E1 is involved in the Ž 1.-hydroxylation of lauric acid in rat liver microsomes. Biochem. Biophys. Res. Commun. 203, 1168᎐1174. Bronfman, M., Inestroza, N., Leighton, F., 1979. Fatty acid oxidation by human liver peroxisomes. Biochem. Biophys. Res. Commun. 88, 1030᎐1036.
Casazza, J.P., Felver, M.E., Veech, R.L., 1984. The metabolism of acetone in rat. J. Biol. Chem. 259, 231᎐236. Chance, B., Sies, H., Boveris, A., 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 538᎐539. Flohe, ´ L., Gunzler, W.A., 1984. Assays of glutathione peroxidase. In: Colowic, S.P., Kaplan, N.O. ŽEds.., Methods in Enzymology, 105. Academic Press, New York, pp. 114᎐121. Griffith, O.W., 1980. Determination of glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207᎐212. Hawkins, J.M., Jones, W.E., Bonner, F.W., Gibson, G.G., 1987. The effect of peroxisome proliferators on microsomal, peroxisomal, and mitochondrial enzyme activities in the liver and kidney. Drug Metab. Rev 18, 441᎐551. Hong, J.Y., Pan, J., Dong, Z., Ning, S.M., Yang, C.S., 1987. Regulation of N-nitrosodimethylamine demethylase in rat liver and kidney. Cancer Res. 47, 5948᎐5953. Koop, D.R., 1992. Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J. 6, 724᎐730. Koop, D.R., Casazza, J.P., 1985. Identification of ethanol-inducible P450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J. Biol. Chem. 260, 13607᎐13612. Kosugi, K., Scofield, R.F.:, Chandramouli, V., Kumaran, K., Schumann, W.C., Landau, B.R., 1986. Pathways of acetone’s metabolism. J. Biol. Chem. 25, 3952᎐3957. Laethem, R.M., Balazy, M., Falck, J.R., Laethem, C., Koop, D.R., 1993. Formation of 19Žs.-, 19 䊛-, and 18 䊛-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1. J. Biol. Chem. 268, 12912᎐12918. Lieber, C.S., 1997. Cytochrome P450 2E1: its physiological and pathological role. Physiol. Rev. 77, 517᎐544. Lieber, C.S., 1999. Microsomal ethanol-oxidizing system ŽMEOS.: The first 30 years Ž1968᎐1998., alcoholism:. Clin. Exp. Res. 23 Ž6., 991᎐1007. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265᎐275. Ma, X., Baraona, E., Lieber, C.S., 1993. Alcohol consumption enhances fatty acid Ž -oxidation, with a greater increase in male than in female rats. Hepatology 18, 1247᎐1253. Miller, K.W., Yang, C.S., 1984. Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting, acetone and ethanol. Arch. Biochem. Biophys. 229, 483᎐491. Nash, T., 1953. The colorimetric estimation of
M. Orellana B. et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 503᎐509
formaldehyde by means of the Hatzsh reaction. Biochem. J. 55, 416᎐421. Nilsson, A., Pridz, K., Rortveit, T., Christiansen, E.N., 1987. Studies on the interrelated stimulation of microsomal -oxidation and peroxisomal -oxidation in rat liver with partially hydrogenated fish oil diet. Biochim. Biophys. Acta 920, 114᎐119. Ockner, R.K., Kaikaus, R.M., Bass, N.M., 1993. Fattyacid metabolism and the pathogenesis of hepatocellular carcinoma: review and hypothesis. Hepatology 18, 669᎐676. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351᎐358. Omura, T., Sato, R., 1964. The carbon-monoxide binding pigment by liver microsomes. J Biol. Chem. 239, 2379᎐2385. Orellana, M., Valdes, ´ E., Capdevila, J., Gil, L., 1989. Nutritionally triggered alterations in the regiospecificity of arachidonic acid oxygenation by rat liver microsomal cytochrome P450. Arch. Biochem. Biophys. 274, 251᎐258. Orellana, M., Fuentes, O., Rosenbluth, H., Lara, M., Valdes, ´ E., 1992. Modulation of rat liver peroxisomal and microsomal fatty acid oxidation by starvation. FEBS Lett. 310, 193᎐196. Orellana, M., Fuentes, O., Valdes, ´ E., 1993. Starvation effect on rat kidney peroxisomal and microsomal fatty acid oxidation. FEBS Lett. 322, 61᎐64. Orellana, M., Valdes, J., Rodrigo, R., ´ E., Fernandez, ´ 1998a. Effects of chronic ethanol consumption on extra-mitochondrial fatty acid oxidation and ethanol metabolism by rat kidney. Gen. Pharmacol. 30, 719᎐723. Orellana, M., Rodrigo, R., Valdes, ´ E., 1998b. Peroxisomal and microsomal fatty acid oxidation in liver of rats following chronic ethanol consumption. Gen. Pharmacol. 31, 817᎐820. Ortiz de Montellano, P.R., Chan, W.K., Tuck, S.K., Kaikaus, R.M., Bass, N.M., Peterson, J.A., 1992. Mechanism-based probes of the topology and function of fatty acid hydroxylases. FASEB J. 6, 695᎐699. Osmundsen, H., Bremer, J., Pedersen, J.I., 1991. Metabolic aspects of peroxisomal Ž-oxidation.. Biochim. Biophys. Acta 1085, 141᎐158.
509
Roberts, B.J., Shoaf, S.E., Jeong, K.-S., Song, B.J., 1994. Induction of CYP2E1 in liver, kidney, brain and intestine during chronic ethanol administration and withdrawal: evidence that CYP2E1 possesses a rapid phase half-life of 6 hours or less. Biochem. Biophys. Res. Comm. 205, 1064᎐1071. Ronis, M.J., Huang, J., Crouch, J. et al., 1993. Cytochrome P450 cyp 2E1 induction during alcohol exposure occurs by a two-step mechanism associated with blood alcohol concentrations in rats. J. Pharm. Exp. Ther. 264, 944᎐950. Sharma, K., Lake, B.G., Makowsky, R. et al., 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. Tietze, F., 1969. Enzymic method for quantitative determination of nanograms amounts of total and oxidized glutathione. Anal. Biochem. 27, 502᎐522. Valdes, ´ E., Orellana, M., Del Villar, E., Vargas, M., 1994. Androstenedione metabolism in streptozotocin diabetes and fasting male rats: similarities and differences. Comp. Biochem. Physiol. 107A, 261᎐267. Van den Bosh, H., Schutgens, R.B.H., Wanders, R.J.A., Tager, J.M., 1992. Biochemistry of peroxisomes. Annu. Rev. Biochem. 62, 157᎐197. Wada, F., Usami, M., 1992. Studies of fatty acid oxidation: antiketogenic effect and gluconeogenicity of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats. Biochem Biophys Acta 487, 261᎐268. Yang, C., Brady, J., Hong, J., 1992. Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity. FASEB J. 6, 737᎐744. Yoo, J.S.H., Ning, S.M., Pantuck, C.B., Pantuck, E.J., Yang, C.S., 1991. Regulation of hepatic microsomal cytochrome P450IIE1 level by dietary lipids and carbohydrates in rats. J. Nutr. 121, 959᎐965. Yun, Y.P., Casazza, J.P., Sohn, D.H., Veech, R.L., Song, B.J., 1992. Pretranslational activation of cytochrome P450IIE during ketosis induced by a high fat diet. Mol. Pharmacol. 41, 474᎐479.