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Further studies on 20β-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in liver microsomes of male rats
Life Sciences 67 (2000) 2075Ð2082
Further studies on 20b-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in liver microsomes of male rats Hidenori Takada, Amiko Uchida, Rika Kamizono, Masaki Otagiri, Yorishige Imamura* Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1, Oe-honmachi, Kumamoto 862-0973, Japan
Abstract Further characterizations of 20b-hydroxysteroid dehydrogenase (20b-HSD) present in liver microsomes of male rats were examined. A signiÞcant relationship was observed between 20b-HSD and acetohexamide reductase (AHR) activities in liver microsomes of male rats. The hepatic microsomal 20b-HSD and AHR preferentially required NADPH as a cofactor. When NADPH was replaced by NADH, NADP or NAD at the same concentration, these reductase activities were little detected. The hepatic microsomal 20b-HSD and AHR activities in streptozotocin-induced diabetic rats were much lower than those in the corresponding controls. The hepatic microsomal 20b-HSD and AHR activities appeared as one main peak, respectively, on DEAE-Sephacel column chromatography, and the peak of 20b-HSD activity was in good agreement with that of AHR activity. Based on these results, we conclude that 20b-HSD present in liver microsomes of male rats functions as AHR, and exhibits a carbonyl reductase-like activity. © 2000 Elsevier Science Inc. All rights reserved. Keywords: 20b-hydroxysteroid dehydrogenase; Acetohexamide reductase activity; Carbonyl reductase-like activity; Hepatic microsomal enzyme; Male rat
Introduction Hydroxysteroid dehydrogenases are a series of enzymes responsible for the biosynthesis and degradation of endogenous steroids. Recently, several hydroxysteroid dehydrogenases have been reported to mediate the metabolism of xenobiotic compounds [1Ð5]. For example, 11b-hydroxysteroid dehydrogenase type-1 (11b-HSD-1) catalyzes the ketone-reduction of metyrapone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is the most potent carcinogenic compound contained in cigarette smoke [6], in liver microsomes of * Corresponding author. Fax: 181-96-362-7690. E-mail address: [email protected] (Y. Imamura) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 9 3 -1
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mice, indicating that the enzyme functions as carbonyl reductase, one of drug-metabolizing enzymes [7,8]. Carbonyl reductase has been considered to belong to the aldo-keto reductase superfamily, similar to aldehyde reductase and aldose reductase, based on its functional properties [9]. However, recent structural investigations have revealed that carbonyl reductase is classiÞed into the short-chain dehydrogenase/reductase (SDR) superfamily [10Ð13]. In fact, 11b-HSD-1 present in liver microsomes of mice is a member of the SDR superfamily [7]. 20b-Hydroxysteroid dehydrogenase (20b-HSD) [EC 1.1.1.53] is an NADP(H)-dependent oxidoreductase which catalyzes the conversion of progesterone and 17a-hydroxyprogesterone [14]. Since progesterone and 17a-hydroxyprogesterone are the precursors of biologically active steroids such as androgens, estrogens and corticoids, 20b-HSD is probably involved in the regulation of biosynthesis of these hormones. Tanaka et al. [15] have demonstrated that 20b-HSD puriÞed from neonatal pig testes has carbonyl reductase-like structure and activity. However, information on the structure and functions of 20b-HSD present in the liver has been very limited. Our previous paper [5] has shown that 20b-HSD catalyzes the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats, suggesting that the hepatic microsomal 20b-HSD exhibits acetohexamide reductase (AHR) activity (carbonyl reductase-like activity). In the present study, some attempts were made to elucidate further the characterizations of 20b-HSD present in liver microsomes of male rats.
Materials and methods Chemicals Progesterone (4-pregnene-3,20-dione), 4-pregnene-20a-ol-3-one and 4-pregnene-20b-ol3-one were purchased from Sigma Chemical Co. (St. Louis, MO). Acetohexamide was a gift from Shionogi Co. (Osaka, Japan). Hydroxyhexamide was synthesized from acetohexamide according to the method of Girgis-Takla and Chroneos [16]. NADPH, NADP, NADH, NAD, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Oriental Yeast Co. (Tokyo, Japan). Streptozotocin was purchased from Sigma Chemical Co. PiperazineN,N9-bis(2-ethanesulfonic acid (PIPES) and 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid (CHAPS) were the products of Dojindo (Kumamoto, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. All other chemicals were of reagent grade. Animals and treatments Male Fischer 344 (Fischer) and Sprague-Dawley (SD) rats at 9 weeks of age were purchased from Japan SLC (Shizuoka, Japan). In order to induce experimental diabetes, streptozotocin at a dose of 60 mg/kg was intravenously administered to male SD rats at 9 weeks of age. Streptozotocin injection was prepared by freshly dissolving in 0.1 M citrate buffer (pH 4.5). An equivalent volume of 0.1 M citrate buffer (pH 4.5) was given to control animals. Experiments were performed 10 days after streptozotocin administration. Only rats showing more than 400 mg/dl in serum glucose level were used [17]. Serum glucose was determined using a glucose test kit (Wako Pure Chemical, Osaka, Japan).
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Preparation of liver microsomes Male Fischer rats at 9 weeks of age, male SD rats and diabetic rats induced from male SD rats were killed by decapitation. After perfusion with ice-cold 1.15% KCl solution, the livers were immediately removed and homogenized in a Potter-Elvehjem homogenizer with 3 volumes of 10 mM sodium-potassium phosphate buffer containing 1.15% KCl (pH 7.4). The homogenates were centrifuged at 10,000g for 20 min and the resulting supernatants were centrifuged at 105,000g for 60 min to obtain the pellets. The microsomal pellets were suspended in 10 mM sodium-potassium phosphate buffer containing 1.15% KCl (pH 7.4) and were recentrifuged at 105,000g for 60 min. The microsomal pellets obtained were used for the enzyme assay. Assay of 20a- and 20b-HSD activities The assay of 20b-HSD activity was conducted in an NADPH-generating system consisting of progesterone (0.1 mM), NADP (0.25 mM), glucose-6-phosphate (6.25 mM), glucose6-phosphate dehydrogenase (0.25 U), MgCl2 (6.25 mM), enzyme (microsomal suspension) and 100 mM sodium-potassium phosphate buffer (pH 7.4) in a Þnal volume of 2.0 ml. In studying the cofactor requirement, 20a- and 20b-HSD activities were assayed using progesterone at a concentration of 0.1 mM as the substrate, and NADPH, NADP, NADH or NAD at a concentration of 0.25 mM was added to the mixture except glucose-6-phosphate (6.25 mM), glucose-6-phosphate dehydrogenase (0.25 U) and MgCl2 (6.25 mM). The mixture was incubated at 378C for 10 min and the reaction was stopped by adding 0.5 ml of 1.0 N HCl to the mixture. The reduction products (4-pregnene-20a-ol-3-one and 4-pregnene-20b-ol-3one) were determined by HPLC according to a slightly modiÞed method of Swinney et al [18]. Protein concentration was measured by the method of Bradford [19] with bovine serum albumin as the standard. Assay of AHR activity The assay of AHR activity (carbonyl reductase-like activity) was conducted in an NADPHgenerating system as described above. However, acetohexamide at a concentration of 1.0 mM was used as the substrate. The reduction product (hydroxyhexamide) was determined by HPLC according to the method of Takagishi et al. [20]. DEAE-Sephacel column chromatography After perfusion with ice-cold 1.15% KCl solution, the livers of male Fischer rats at 9 weeks of age were immediately removed and homogenized in a Potter-Elvehjem homogenizer with 3 volumes of 20 mM PIPES (pH 6.8) containing 0.5 mM EDTA, 0.1 mM dithiothreitol (DTT) and 1.15% KCl. All subsequent procedures were performed at 3 to 58C. The homogenates were centrifuged at 10,000g for 20 min and the resulting supernatants were centrifuged at 105,000g for 60 min to obtain the microsomal pellets. The microsomal pellets were suspended in 20 mM PIPES (pH 6.8) containing 0.5 mM EDTA and 0.1 mM DTT. To the microsomal suspension, 10% CHAPS solution was added slowly with stirring to a Þnal concentration of 0.6% for 60 min. The supernatant fraction was collected by centrifugation at 105,000g for 60 min, and dialyzed against 20 mM PIPES (pH 6.8) containing 0.5 mM EDTA,
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0.1 mM DTT, 0.1% CHAPS and 0.1 M NaCl (buffer A). The dialyzed enzyme solution was applied to a DEAE-Sephacel column (2.5 3 12 cm) equilibrated with buffer A. After the column was washed with buffer A, the enzyme was eluted with a linear gradient of NaCl (0.1Ð 0.5 M) in buffer A. 20b-HSD and AHR activities were assayed as described above. Statistical analysis StudentÕs t-test was used to analyze differences between two groups. A p value of 0.05 or less was considered to be signiÞcant. Results and discussion Our previous paper [21] has shown that there is an individual variation of AHR activity (carbonyl reductase-like activity) in liver microsomes of male Fischer rats. In the present study, we found such an individual variation in the hepatic microsomal 20b-HSD activity. Furthermore, a signiÞcant relationship was observed between these two enzyme activities, as shown in Fig. 1 (r 5 0.623, p , 0.01). These results suggest that 20b-HSD and AHR present in liver microsomes of male rats are the same enzyme. Since Fischer rat strain is inbred, the individual variation may result from the quantitative difference in the expression of 20bHSD gene. We have recently revealed that 20b-HSD present in liver microsomes of rats is a male-speciÞc and androgen-dependent enzyme [5]. Thus, it is possible that the amount of androgens secreted from the testes is involved in the individual variation. From this point of view, additional studies are in progress to elucidate the mechanism for the individual variation in the hepatic microsomal 20b-HSD activity. We have recently demonstrated that the activity of 20a-HSD in liver microsomes of male rats is much lower than that of 20b-HSD in liver microsomes of male rats [5]. Fig. 2 shows the cofactor requirement for the reductase activities of 20a-HSD, 20b-HSD and AHR
Fig. 1. Relationship between 20b-HSD and AHR activities in liver microsomes of male Fischer rats.
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Fig. 2. Cofactor requirement of 20a-HSD (A), and 20b-HSD (B) and AHR (C) activities in liver microsomes of male Fischer rats. 20a- and 20b-HSD activities were assayed using progesterone at a concentration of 0.1 mM. AHR activity was assayed using acetohexamide at a concentration of 1.0 mM. ND, not detected.
present in liver microsomes of male Fischer rats. The hepatic microsomal 20a-HSD activity was found to be higher in the presence of NADH rather than NADPH (Fig. 2A). On the other hand, the hepatic microsomal 20b-HSD required NADPH as a cofactor (Fig. 2B). When NADPH was replaced by NADH, NADP, or NAD at the same concentration, little or no enzyme activity was detected. Similarly, the hepatic microsomal AHR preferentially required NADPH as a cofactor (Fig. 2C). These results lead us to conclude that the hepatic microsomal 20a-HSD, unlike the hepatic microsomal 20b-HSD, cannot play a signiÞcant role in the ketone-reduction of acetohexamide. Streptozotocin-induced diabetes has been demonstrated to cause a decrease of serum testosterone levels, and to depress the expression of androgen-dependent cytochrome P450 (CYP) isoforms such as CYP2C11 in male rats [22]. 20b-HSD present in liver microsomes of male rats, as described above, is an androgen-dependent enzyme. Thus, we examined whether streptozotocin-induced diabetes can decrease the hepatic microsomal 20b-HSD activity in male SD rats. As expected, the hepatic microsomal 20b-HSD activity in the diabetic rats was much lower than that in the controls (Fig. 3A). A similar result was observed for the hepatic microsomal AHR activity (Fig. 3B), suggesting that 20b-HSD present in liver microsomes of male rats exhibits AHR activity (carbonyl reductase-like activity). In this study, the experimental diabetes was induced by intravenous administration of streptozotocin at a dose of 60 mg/kg. The induction of experimental diabetes was also attempted in male Fischer rats. However, intravenous administration of streptozotocin at the same dose (60 mg/kg) frequently caused their death in male Fischer rats, unlike in male SD rats. To obtain direct evidence that 20b-HSD present in liver microsomes of male rats has AHR activity (carbonyl reductase-like activity), the puriÞcation of the enzyme was performed. We at Þrst prepared microsomal pellets from the liver of male Fischer rats. The resulting mi-
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Fig. 3. Effect of streptozotocin-induced diabetes on 20b-HSD (A) and AHR (B) activities in liver microsomes of male SD rats. * p , 0.05, ** p , 0.01, signiÞcantly different from the corresponding control.
crosomal pellets were suspended and solubilized by adding CHAPS. Fig. 4 shows the elution proÞles of 20b-HSD and AHR activities from DEAE-Sephacel column of the solubilized liver microsomes. The 20b-HSD and AHR activities appeared as one main peak, respectively, and the peak of 20b-HSD activity was in good agreement with that of AHR activity. Further puriÞcation failed because of a marked loss of both 20b-HSD and AHR activities in the subsequent column chromatographic step. However, the results obtained in DEAESephacel column chromatography strongly support the idea that 20b-HSD present in liver microsomes of male rats exhibits AHR activity (carbonyl reductase-like activity). The present study has provided further evidence that 20b-HSD present in liver microsomes of male rats functions as AHR. So far, 20b-HSD has been puriÞed from only neo-
Fig. 4. DEAE-Sephacel column chromatography of solubilized liver microsomes of male Fischer rats. The column was eluted with a linear 0.1Ð0.5 M NaCl gradient. s, 20b-HSD activity; d, AHR activity.
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natal pig testes [14]. Since the testicular 20b-HSD is known to exhibit carbonyl reductaselike structure and activity [4,15], the testicular enzyme, as well as the hepatic microsomal 20b-HSD in this study, may catalyze the ketone-reduction of acetohexamide. In addition, it has been reported that the product of 20b-HSD gene cloned from cDNA library of rainbow trout ovarian follicles has carbonyl reductase-like activity [23]. However, hepatic 20b-HSD rather than such extrahepatic 20b-HSDs would largely contribute to the reductive metabolism of drugs with a ketone group. Therefore, it is of interest to elucidate the structure and functions of 20b-HSD present in liver microsomes of male rats. We are currently isolating cDNA which encodes 20b-HSD with carbonyl reductase-like activity from liver microsomes of male rats. References 1. Maser E, Bannenberg G. 11b-Hydroxysteroid dehydrogenase mediates reductive metabo-lism of xenobiotic carbonyl compounds. Biochemical Pharmacology 1994: 47 (10): 1805Ð12. 2. Maser E. Xenobiotic carbonyl reduction and physiological steroid oxidoreduction: The pluripotency of several hydroxysteroid dehydrogenases. Biochemical Pharmacology 1995: 49 (4): 421Ð40. 3. Muramatsu S, Komokata Y, Tanaka Y, Takahagi H. Metabolism of pravastatin sodium by 3a-hydroxysteroid dehydrogenase. Biological and Pharmaceutical Bulletin 1997: 20 (11): 1199Ð203. 4. Nakajin S, Tamura F, Takase N, Toyoshima S. Carbonyl reductase activity exhibited by pig testicular 20bhydroxysteroid dehydrogenase. Biological and Pharmaceutical Bulletin 1997: 20 (11): 1215Ð8. 5. Takada H, Otagiri M, Imamura Y. 20b-Hydroxysteroid dehydrogenase catalyzes ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of adult male rats. Journal of Pharmacology and Experimental Therapeutics 1998: 287 (2): 504Ð7. 6. Hecht SS. Biochemistry, biology, and carcinogenicity of tabacco-speciÞc nitrosamines. Chemical Research in Toxicology 1998: 11 (6): 559Ð603. 7. Maser E, Oppermann UCT. The 11b-hydroxysteroid dehydrogenase system, a determinant of glucocorticoid and mineralocorticoid action: Role of type-1 11b-hydroxysteroid dehydro-genase in detoxiÞcation processes. European Journal of Biochemistry 1997: 249 (2): 365Ð9. 8. Jakoby WB, Ziegler DM. The enzymes of detoxication. Journal of Biological Chemistry 1990: 265 (34): 20715Ð8. 9. Flynn TG, Green NC. The aldo-keto reductases: An overview. In: Weiner H, Crabb DW, Flynn TG, editors. Advances in Experimental Medicine and Biology (volume 328): Enzymology and Molecular Biology of Carbonyl Metabolism 4. New York: Plenum Press, 1993. pp 251Ð257. 10. Wermuth B, Bohren KM, Heinemann G, von Wartburg J-P, Gabbay KH. Human carbonyl reductase: Nucleotide sequence analysis of a cDNA and amino acid sequence of the encoded protein. Journal of Biological Chemistry 1988: 263 (31): 16185Ð8. 11. Gonzalez B, Sapra A, Rivera H, Kaplan WD, Yam B, Forrest GL. Cloning and expression of the cDNA encoding rabbit liver carbonyl reductase. Gene 1995: 154 (2): 297Ð8. 12. Jšrnvall H, Persson B, Krook M, Atrian S, Gonzˆlez-Duarte R, Jeffery J, Ghosh D. Short-chain dehydrogenase/ reductase (SDR). Biochemistry 1995: 34 (18): 6003Ð13. 13. Jez JM, Flynn TG, Penning TM. A new nomenclature for the aldo-keto reductase superfamily. Biochemical Pharmacology 1997: 54 (6): 639Ð47. 14. Nakajin S, Ohno S, Shinoda M. 20b-Hydroxysteroid dehydrogenase of neonatal pig testis: PuriÞcation and some properties. Journal of Biochemistry 1988: 104 (4): 565Ð9. 15. Tanaka M, Ohno S, Adachi S, Nakajin S, Shinoda M, Nagahama Y. Pig testicular 20b-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity: cDNA cloning of pig testicular 20bhydroxysteroid dehydrogenase. Journal of Biological Chemistry 1992: 276 (19): 13451Ð5. 16. Girgis-Takla P, Chroneos I. Fluorometric determination of acetohexamide in plasma and tablet formulation using 1-methylnicotinamide. Analyst 1979: 104 (1235): 117Ð23.
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17. Watkins III JB, Dykstra TP. Alteration in biliary excretory function by streptozotocin-induced diabetes. Drug Metabolism and Disposition 1987: 15 (2): 177Ð83. 18. Swinney DC, Ryan DE, Thomas PE, Levin W. Regioselective progesterone hydroxylation catalyzed by Elever rat hepatic cytochrome p-450 isozymes. Biochemistry 1987: 26 (22): 7073Ð83. 19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Chemistry 1976: 72: 247Ð54. 20. Takagishi Y, Sato K, Tomita K, Sakamoto T. High-performance liquid chromatographic determination of acetohexamide and its metabolite hydroxyhexamide. Yakugaku Zasshi 1979: 99 (9): 961Ð3. 21. Imamura Y, Kozono Y, Mori Y, Takada H, Otagiri M. Individual variation of acetohexamide reductase activities in liver microsomes and cytosol of rats. Biological and Pharmaceutical Bulletin 1997: 20 (8): 924Ð6. 22. Thummel KE, Schenkman JB. Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat. Molecular Pharmacology 1990: 37 (1): 119Ð29. 23. Guan G, Tanaka M, Todo T, Young G, Yoshikuni M, Nagahama Y. Cloning and expression of two carbonyl reductase-like 20b-hydroxysteroid dehydrogenase cDNA in ovarian follicles of rainbow trout (Oncorhynchus mykiss). Biochemical and Biophysical Research Communications 1999: 255 (1): 123Ð8.
Further studies on 20b-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in liver microsomes of male rats Hidenori Takada, Amiko Uchida, Rika Kamizono, Masaki Otagiri, Yorishige Imamura* Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1, Oe-honmachi, Kumamoto 862-0973, Japan
Abstract Further characterizations of 20b-hydroxysteroid dehydrogenase (20b-HSD) present in liver microsomes of male rats were examined. A signiÞcant relationship was observed between 20b-HSD and acetohexamide reductase (AHR) activities in liver microsomes of male rats. The hepatic microsomal 20b-HSD and AHR preferentially required NADPH as a cofactor. When NADPH was replaced by NADH, NADP or NAD at the same concentration, these reductase activities were little detected. The hepatic microsomal 20b-HSD and AHR activities in streptozotocin-induced diabetic rats were much lower than those in the corresponding controls. The hepatic microsomal 20b-HSD and AHR activities appeared as one main peak, respectively, on DEAE-Sephacel column chromatography, and the peak of 20b-HSD activity was in good agreement with that of AHR activity. Based on these results, we conclude that 20b-HSD present in liver microsomes of male rats functions as AHR, and exhibits a carbonyl reductase-like activity. © 2000 Elsevier Science Inc. All rights reserved. Keywords: 20b-hydroxysteroid dehydrogenase; Acetohexamide reductase activity; Carbonyl reductase-like activity; Hepatic microsomal enzyme; Male rat
Introduction Hydroxysteroid dehydrogenases are a series of enzymes responsible for the biosynthesis and degradation of endogenous steroids. Recently, several hydroxysteroid dehydrogenases have been reported to mediate the metabolism of xenobiotic compounds [1Ð5]. For example, 11b-hydroxysteroid dehydrogenase type-1 (11b-HSD-1) catalyzes the ketone-reduction of metyrapone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is the most potent carcinogenic compound contained in cigarette smoke [6], in liver microsomes of * Corresponding author. Fax: 181-96-362-7690. E-mail address: [email protected] (Y. Imamura) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 7 9 3 -1
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mice, indicating that the enzyme functions as carbonyl reductase, one of drug-metabolizing enzymes [7,8]. Carbonyl reductase has been considered to belong to the aldo-keto reductase superfamily, similar to aldehyde reductase and aldose reductase, based on its functional properties [9]. However, recent structural investigations have revealed that carbonyl reductase is classiÞed into the short-chain dehydrogenase/reductase (SDR) superfamily [10Ð13]. In fact, 11b-HSD-1 present in liver microsomes of mice is a member of the SDR superfamily [7]. 20b-Hydroxysteroid dehydrogenase (20b-HSD) [EC 1.1.1.53] is an NADP(H)-dependent oxidoreductase which catalyzes the conversion of progesterone and 17a-hydroxyprogesterone [14]. Since progesterone and 17a-hydroxyprogesterone are the precursors of biologically active steroids such as androgens, estrogens and corticoids, 20b-HSD is probably involved in the regulation of biosynthesis of these hormones. Tanaka et al. [15] have demonstrated that 20b-HSD puriÞed from neonatal pig testes has carbonyl reductase-like structure and activity. However, information on the structure and functions of 20b-HSD present in the liver has been very limited. Our previous paper [5] has shown that 20b-HSD catalyzes the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats, suggesting that the hepatic microsomal 20b-HSD exhibits acetohexamide reductase (AHR) activity (carbonyl reductase-like activity). In the present study, some attempts were made to elucidate further the characterizations of 20b-HSD present in liver microsomes of male rats.
Materials and methods Chemicals Progesterone (4-pregnene-3,20-dione), 4-pregnene-20a-ol-3-one and 4-pregnene-20b-ol3-one were purchased from Sigma Chemical Co. (St. Louis, MO). Acetohexamide was a gift from Shionogi Co. (Osaka, Japan). Hydroxyhexamide was synthesized from acetohexamide according to the method of Girgis-Takla and Chroneos [16]. NADPH, NADP, NADH, NAD, glucose-6-phosphate and glucose-6-phosphate dehydrogenase were obtained from Oriental Yeast Co. (Tokyo, Japan). Streptozotocin was purchased from Sigma Chemical Co. PiperazineN,N9-bis(2-ethanesulfonic acid (PIPES) and 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid (CHAPS) were the products of Dojindo (Kumamoto, Japan) and Nacalai Tesque (Kyoto, Japan), respectively. All other chemicals were of reagent grade. Animals and treatments Male Fischer 344 (Fischer) and Sprague-Dawley (SD) rats at 9 weeks of age were purchased from Japan SLC (Shizuoka, Japan). In order to induce experimental diabetes, streptozotocin at a dose of 60 mg/kg was intravenously administered to male SD rats at 9 weeks of age. Streptozotocin injection was prepared by freshly dissolving in 0.1 M citrate buffer (pH 4.5). An equivalent volume of 0.1 M citrate buffer (pH 4.5) was given to control animals. Experiments were performed 10 days after streptozotocin administration. Only rats showing more than 400 mg/dl in serum glucose level were used [17]. Serum glucose was determined using a glucose test kit (Wako Pure Chemical, Osaka, Japan).
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Preparation of liver microsomes Male Fischer rats at 9 weeks of age, male SD rats and diabetic rats induced from male SD rats were killed by decapitation. After perfusion with ice-cold 1.15% KCl solution, the livers were immediately removed and homogenized in a Potter-Elvehjem homogenizer with 3 volumes of 10 mM sodium-potassium phosphate buffer containing 1.15% KCl (pH 7.4). The homogenates were centrifuged at 10,000g for 20 min and the resulting supernatants were centrifuged at 105,000g for 60 min to obtain the pellets. The microsomal pellets were suspended in 10 mM sodium-potassium phosphate buffer containing 1.15% KCl (pH 7.4) and were recentrifuged at 105,000g for 60 min. The microsomal pellets obtained were used for the enzyme assay. Assay of 20a- and 20b-HSD activities The assay of 20b-HSD activity was conducted in an NADPH-generating system consisting of progesterone (0.1 mM), NADP (0.25 mM), glucose-6-phosphate (6.25 mM), glucose6-phosphate dehydrogenase (0.25 U), MgCl2 (6.25 mM), enzyme (microsomal suspension) and 100 mM sodium-potassium phosphate buffer (pH 7.4) in a Þnal volume of 2.0 ml. In studying the cofactor requirement, 20a- and 20b-HSD activities were assayed using progesterone at a concentration of 0.1 mM as the substrate, and NADPH, NADP, NADH or NAD at a concentration of 0.25 mM was added to the mixture except glucose-6-phosphate (6.25 mM), glucose-6-phosphate dehydrogenase (0.25 U) and MgCl2 (6.25 mM). The mixture was incubated at 378C for 10 min and the reaction was stopped by adding 0.5 ml of 1.0 N HCl to the mixture. The reduction products (4-pregnene-20a-ol-3-one and 4-pregnene-20b-ol-3one) were determined by HPLC according to a slightly modiÞed method of Swinney et al [18]. Protein concentration was measured by the method of Bradford [19] with bovine serum albumin as the standard. Assay of AHR activity The assay of AHR activity (carbonyl reductase-like activity) was conducted in an NADPHgenerating system as described above. However, acetohexamide at a concentration of 1.0 mM was used as the substrate. The reduction product (hydroxyhexamide) was determined by HPLC according to the method of Takagishi et al. [20]. DEAE-Sephacel column chromatography After perfusion with ice-cold 1.15% KCl solution, the livers of male Fischer rats at 9 weeks of age were immediately removed and homogenized in a Potter-Elvehjem homogenizer with 3 volumes of 20 mM PIPES (pH 6.8) containing 0.5 mM EDTA, 0.1 mM dithiothreitol (DTT) and 1.15% KCl. All subsequent procedures were performed at 3 to 58C. The homogenates were centrifuged at 10,000g for 20 min and the resulting supernatants were centrifuged at 105,000g for 60 min to obtain the microsomal pellets. The microsomal pellets were suspended in 20 mM PIPES (pH 6.8) containing 0.5 mM EDTA and 0.1 mM DTT. To the microsomal suspension, 10% CHAPS solution was added slowly with stirring to a Þnal concentration of 0.6% for 60 min. The supernatant fraction was collected by centrifugation at 105,000g for 60 min, and dialyzed against 20 mM PIPES (pH 6.8) containing 0.5 mM EDTA,
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0.1 mM DTT, 0.1% CHAPS and 0.1 M NaCl (buffer A). The dialyzed enzyme solution was applied to a DEAE-Sephacel column (2.5 3 12 cm) equilibrated with buffer A. After the column was washed with buffer A, the enzyme was eluted with a linear gradient of NaCl (0.1Ð 0.5 M) in buffer A. 20b-HSD and AHR activities were assayed as described above. Statistical analysis StudentÕs t-test was used to analyze differences between two groups. A p value of 0.05 or less was considered to be signiÞcant. Results and discussion Our previous paper [21] has shown that there is an individual variation of AHR activity (carbonyl reductase-like activity) in liver microsomes of male Fischer rats. In the present study, we found such an individual variation in the hepatic microsomal 20b-HSD activity. Furthermore, a signiÞcant relationship was observed between these two enzyme activities, as shown in Fig. 1 (r 5 0.623, p , 0.01). These results suggest that 20b-HSD and AHR present in liver microsomes of male rats are the same enzyme. Since Fischer rat strain is inbred, the individual variation may result from the quantitative difference in the expression of 20bHSD gene. We have recently revealed that 20b-HSD present in liver microsomes of rats is a male-speciÞc and androgen-dependent enzyme [5]. Thus, it is possible that the amount of androgens secreted from the testes is involved in the individual variation. From this point of view, additional studies are in progress to elucidate the mechanism for the individual variation in the hepatic microsomal 20b-HSD activity. We have recently demonstrated that the activity of 20a-HSD in liver microsomes of male rats is much lower than that of 20b-HSD in liver microsomes of male rats [5]. Fig. 2 shows the cofactor requirement for the reductase activities of 20a-HSD, 20b-HSD and AHR
Fig. 1. Relationship between 20b-HSD and AHR activities in liver microsomes of male Fischer rats.
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Fig. 2. Cofactor requirement of 20a-HSD (A), and 20b-HSD (B) and AHR (C) activities in liver microsomes of male Fischer rats. 20a- and 20b-HSD activities were assayed using progesterone at a concentration of 0.1 mM. AHR activity was assayed using acetohexamide at a concentration of 1.0 mM. ND, not detected.
present in liver microsomes of male Fischer rats. The hepatic microsomal 20a-HSD activity was found to be higher in the presence of NADH rather than NADPH (Fig. 2A). On the other hand, the hepatic microsomal 20b-HSD required NADPH as a cofactor (Fig. 2B). When NADPH was replaced by NADH, NADP, or NAD at the same concentration, little or no enzyme activity was detected. Similarly, the hepatic microsomal AHR preferentially required NADPH as a cofactor (Fig. 2C). These results lead us to conclude that the hepatic microsomal 20a-HSD, unlike the hepatic microsomal 20b-HSD, cannot play a signiÞcant role in the ketone-reduction of acetohexamide. Streptozotocin-induced diabetes has been demonstrated to cause a decrease of serum testosterone levels, and to depress the expression of androgen-dependent cytochrome P450 (CYP) isoforms such as CYP2C11 in male rats [22]. 20b-HSD present in liver microsomes of male rats, as described above, is an androgen-dependent enzyme. Thus, we examined whether streptozotocin-induced diabetes can decrease the hepatic microsomal 20b-HSD activity in male SD rats. As expected, the hepatic microsomal 20b-HSD activity in the diabetic rats was much lower than that in the controls (Fig. 3A). A similar result was observed for the hepatic microsomal AHR activity (Fig. 3B), suggesting that 20b-HSD present in liver microsomes of male rats exhibits AHR activity (carbonyl reductase-like activity). In this study, the experimental diabetes was induced by intravenous administration of streptozotocin at a dose of 60 mg/kg. The induction of experimental diabetes was also attempted in male Fischer rats. However, intravenous administration of streptozotocin at the same dose (60 mg/kg) frequently caused their death in male Fischer rats, unlike in male SD rats. To obtain direct evidence that 20b-HSD present in liver microsomes of male rats has AHR activity (carbonyl reductase-like activity), the puriÞcation of the enzyme was performed. We at Þrst prepared microsomal pellets from the liver of male Fischer rats. The resulting mi-
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Fig. 3. Effect of streptozotocin-induced diabetes on 20b-HSD (A) and AHR (B) activities in liver microsomes of male SD rats. * p , 0.05, ** p , 0.01, signiÞcantly different from the corresponding control.
crosomal pellets were suspended and solubilized by adding CHAPS. Fig. 4 shows the elution proÞles of 20b-HSD and AHR activities from DEAE-Sephacel column of the solubilized liver microsomes. The 20b-HSD and AHR activities appeared as one main peak, respectively, and the peak of 20b-HSD activity was in good agreement with that of AHR activity. Further puriÞcation failed because of a marked loss of both 20b-HSD and AHR activities in the subsequent column chromatographic step. However, the results obtained in DEAESephacel column chromatography strongly support the idea that 20b-HSD present in liver microsomes of male rats exhibits AHR activity (carbonyl reductase-like activity). The present study has provided further evidence that 20b-HSD present in liver microsomes of male rats functions as AHR. So far, 20b-HSD has been puriÞed from only neo-
Fig. 4. DEAE-Sephacel column chromatography of solubilized liver microsomes of male Fischer rats. The column was eluted with a linear 0.1Ð0.5 M NaCl gradient. s, 20b-HSD activity; d, AHR activity.
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natal pig testes [14]. Since the testicular 20b-HSD is known to exhibit carbonyl reductaselike structure and activity [4,15], the testicular enzyme, as well as the hepatic microsomal 20b-HSD in this study, may catalyze the ketone-reduction of acetohexamide. In addition, it has been reported that the product of 20b-HSD gene cloned from cDNA library of rainbow trout ovarian follicles has carbonyl reductase-like activity [23]. However, hepatic 20b-HSD rather than such extrahepatic 20b-HSDs would largely contribute to the reductive metabolism of drugs with a ketone group. Therefore, it is of interest to elucidate the structure and functions of 20b-HSD present in liver microsomes of male rats. We are currently isolating cDNA which encodes 20b-HSD with carbonyl reductase-like activity from liver microsomes of male rats. References 1. Maser E, Bannenberg G. 11b-Hydroxysteroid dehydrogenase mediates reductive metabo-lism of xenobiotic carbonyl compounds. Biochemical Pharmacology 1994: 47 (10): 1805Ð12. 2. Maser E. Xenobiotic carbonyl reduction and physiological steroid oxidoreduction: The pluripotency of several hydroxysteroid dehydrogenases. Biochemical Pharmacology 1995: 49 (4): 421Ð40. 3. Muramatsu S, Komokata Y, Tanaka Y, Takahagi H. Metabolism of pravastatin sodium by 3a-hydroxysteroid dehydrogenase. Biological and Pharmaceutical Bulletin 1997: 20 (11): 1199Ð203. 4. Nakajin S, Tamura F, Takase N, Toyoshima S. Carbonyl reductase activity exhibited by pig testicular 20bhydroxysteroid dehydrogenase. Biological and Pharmaceutical Bulletin 1997: 20 (11): 1215Ð8. 5. Takada H, Otagiri M, Imamura Y. 20b-Hydroxysteroid dehydrogenase catalyzes ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of adult male rats. Journal of Pharmacology and Experimental Therapeutics 1998: 287 (2): 504Ð7. 6. Hecht SS. Biochemistry, biology, and carcinogenicity of tabacco-speciÞc nitrosamines. Chemical Research in Toxicology 1998: 11 (6): 559Ð603. 7. Maser E, Oppermann UCT. The 11b-hydroxysteroid dehydrogenase system, a determinant of glucocorticoid and mineralocorticoid action: Role of type-1 11b-hydroxysteroid dehydro-genase in detoxiÞcation processes. European Journal of Biochemistry 1997: 249 (2): 365Ð9. 8. Jakoby WB, Ziegler DM. The enzymes of detoxication. Journal of Biological Chemistry 1990: 265 (34): 20715Ð8. 9. Flynn TG, Green NC. The aldo-keto reductases: An overview. In: Weiner H, Crabb DW, Flynn TG, editors. Advances in Experimental Medicine and Biology (volume 328): Enzymology and Molecular Biology of Carbonyl Metabolism 4. New York: Plenum Press, 1993. pp 251Ð257. 10. Wermuth B, Bohren KM, Heinemann G, von Wartburg J-P, Gabbay KH. Human carbonyl reductase: Nucleotide sequence analysis of a cDNA and amino acid sequence of the encoded protein. Journal of Biological Chemistry 1988: 263 (31): 16185Ð8. 11. Gonzalez B, Sapra A, Rivera H, Kaplan WD, Yam B, Forrest GL. Cloning and expression of the cDNA encoding rabbit liver carbonyl reductase. Gene 1995: 154 (2): 297Ð8. 12. Jšrnvall H, Persson B, Krook M, Atrian S, Gonzˆlez-Duarte R, Jeffery J, Ghosh D. Short-chain dehydrogenase/ reductase (SDR). Biochemistry 1995: 34 (18): 6003Ð13. 13. Jez JM, Flynn TG, Penning TM. A new nomenclature for the aldo-keto reductase superfamily. Biochemical Pharmacology 1997: 54 (6): 639Ð47. 14. Nakajin S, Ohno S, Shinoda M. 20b-Hydroxysteroid dehydrogenase of neonatal pig testis: PuriÞcation and some properties. Journal of Biochemistry 1988: 104 (4): 565Ð9. 15. Tanaka M, Ohno S, Adachi S, Nakajin S, Shinoda M, Nagahama Y. Pig testicular 20b-hydroxysteroid dehydrogenase exhibits carbonyl reductase-like structure and activity: cDNA cloning of pig testicular 20bhydroxysteroid dehydrogenase. Journal of Biological Chemistry 1992: 276 (19): 13451Ð5. 16. Girgis-Takla P, Chroneos I. Fluorometric determination of acetohexamide in plasma and tablet formulation using 1-methylnicotinamide. Analyst 1979: 104 (1235): 117Ð23.
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