Self-augmentation Effect of Male-specific Products on Sexually Differentiated Progesterone Metabolism in Adult Male Rat Liver Microsomes
2001; Elsevier BV; Volume: 276; Issue: 7 Linguagem: Inglês
10.1074/jbc.m003355200
ISSN1083-351X
AutoresAkihiko YAMADA, Morio Yamada, Yukihisa Fujita, Takashi Nishigami, Keiji Nakasho, Kunio Uematsu,
Tópico(s)Hormonal and reproductive studies
ResumoIt is well known that several 3-keto-4-ene steroids such as progesterone and testosterone are metabolized in a gender-specific or -predominant manner by adult rat liver microsomes. In the male, these steroids are primarily metabolized into two oxidized (16α-hydroxyl and 6β-hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5α-reduced products by female-predominant 5α-reductase in the female. These sexually differentiated enzyme activities are largely regulated at the transcription level under endocrine control. In the present study, we show that unlabeled 16α-hydroxyprogesterone and 6β-hydroxyprogesterone inhibited the 5α-reductive [3H]progesterone metabolism by adult male rat liver microsomes without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, whereas 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione not only stimulated the 5α-reductive metabolism producing themselves but also inhibited the male-specific oxidative metabolism. This finding compels us to propose a novel hypothesis that adult male rat liver microsomes may possess a self-augmentation system regulated by the male-specific products on sexually differentiated steroid metabolism, besides regulation by gene expressions of the related enzymes. It is well known that several 3-keto-4-ene steroids such as progesterone and testosterone are metabolized in a gender-specific or -predominant manner by adult rat liver microsomes. In the male, these steroids are primarily metabolized into two oxidized (16α-hydroxyl and 6β-hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5α-reduced products by female-predominant 5α-reductase in the female. These sexually differentiated enzyme activities are largely regulated at the transcription level under endocrine control. In the present study, we show that unlabeled 16α-hydroxyprogesterone and 6β-hydroxyprogesterone inhibited the 5α-reductive [3H]progesterone metabolism by adult male rat liver microsomes without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, whereas 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione not only stimulated the 5α-reductive metabolism producing themselves but also inhibited the male-specific oxidative metabolism. This finding compels us to propose a novel hypothesis that adult male rat liver microsomes may possess a self-augmentation system regulated by the male-specific products on sexually differentiated steroid metabolism, besides regulation by gene expressions of the related enzymes. progesterone 17β, 5α-androstane-3α,17β-diol 5β-androstan-17β-ol 4-androstene-3,17-dione 4-androsten-3-one-17β-carboxylic acid corticosterone cytochrome P450 11β-(OH)2-5α-P, 3α,11β-dihydroxy-5α-pregnan-20-one growth hormone 5α-pregnane-3,20-dione 3α-hydroxy-5α-pregnan-20-one testosterone X-hydroxyprogesterone adrenocorticotropic hormone It is well established that activities of many steroid-metabolizing enzymes in adult rat liver microsomes are sexually differentiated. The male primarily metabolizes various 3-keto-4-ene steroids such as progesterone (PROG),1 TEST, and 4-AN into the two oxidized products, 16α-OH (in some cases, 2α-OH also) and 6β-OH products, mainly by the respective, male-specific cytochrome P450 subforms (P450s), CYP2C11 and CYP3A2, whereas the female metabolizes them primarily into the 5α-reduced products by female-predominant 5α-reductase (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 2Blanck A. Åström A. Hansson T. Cancer Res. 1986; 46: 5072-5076PubMed Google Scholar, 3Waxman D.J. Morrissey J.J. LeBlanc G.A. Endocrinology. 1989; 124: 2954-2966Crossref PubMed Scopus (92) Google Scholar, 4Swinney D.C. Drug Metab. Dispos. 1990; 18: 859-865PubMed Google Scholar, 5Murray M. Cantrill E. Martini R. Farrell G.C. Arch. Biochem. Biophys. 1991; 286: 618-624Crossref PubMed Scopus (41) Google Scholar, 6Legraverend C. Mode A. Wells T. Robinson I. Gustafsson J-Å. FASEB J. 1992; 6: 711-718Crossref PubMed Scopus (136) Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar). Expressions of these sexually differentiated enzyme activities are largely regulated at the transcription level under endocrine control, with the secretory pattern of GH playing a major role. Intermittent and pulsatile (i.e. male pattern) GH secretion induces CYP2C11 gene expression, whereas a more continuous female pattern repressesCYP2C11 and CYP3A2 gene expressions and conversely induces 5α-reductase gene expression (6Legraverend C. Mode A. Wells T. Robinson I. Gustafsson J-Å. FASEB J. 1992; 6: 711-718Crossref PubMed Scopus (136) Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar). Furthermore, sex hormones are thought to affect indirectly these gene expressions by acting on the hypothalamo-pituitary axis that controls the sexually dimorphic pattern of GH secretion (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 2Blanck A. Åström A. Hansson T. Cancer Res. 1986; 46: 5072-5076PubMed Google Scholar, 3Waxman D.J. Morrissey J.J. LeBlanc G.A. Endocrinology. 1989; 124: 2954-2966Crossref PubMed Scopus (92) Google Scholar, 6Legraverend C. Mode A. Wells T. Robinson I. Gustafsson J-Å. FASEB J. 1992; 6: 711-718Crossref PubMed Scopus (136) Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar). In the course of our investigation on structural requirements of substrates and/or inhibitors for active sites of CYP2C11 and CYP3A2 in male rat liver microsomes (to be published elsewhere), we unexpectedly found that male-specific products, 16α-OH-P and 6β-OH-P, inhibited female-predominant [3H]PROG 5α-reductase activity without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, while 3α-OH-5α-P and 5α-P, female-predominant products by the 5α-reductase, not only stimulated this enzyme activity but also inhibited the male-specific oxidative [3H]PROG metabolism. In the present paper, we extend these findings and suggest a novel self-augmentation effect of the male-specific products on sexually differentiated steroid metabolism in adult male rat liver microsomes, not involving gene expressions of the related enzymes. [1,2-3H]PROG (specific activity, 49.2 Ci/mmol) was obtained from PerkinElmer Life Sciences and purified by a paper chromatographic system of hexane, saturated with formamide. Unlabeled steroids were purchased from Sigma and Steraloids Inc. (Wilton, NH). Goat anti-rat NADPH P450 reductase antiserum and rat CYP3A2 supersomes were purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan), and Whatman No. 1 filter papers used for paper chromatographies were from Whatman Ltd. Other reagents were of analytical grade. Male Wistar rats, originally provided by Japan Charles River K. K., were bred in our colony. They were castrated on the 70th day after birth and used 3–4 weeks later. The liver microsomes were prepared as described previously (11Yamada M. Nishigami T. Nakasho K. Nishimoto Y. Miyaji H. Hepatology. 1994; 20: 1271-1280Crossref PubMed Scopus (39) Google Scholar). The experiments were performed according to institutional guidelines for the care and use of laboratory animals. Effects of various unlabeled steroids on [3H]PROG metabolism by liver microsomes were examined, according to our previously described procedure (12Yamada M. Indo K. Nishigami T. Nakasho K. Miyaji H. J. Biol. Chem. 1990; 265: 11035-11043Abstract Full Text PDF PubMed Google Scholar). Briefly, the microsomal suspension (400–600 μg of protein/2.2 ml, total volume of the reaction mixture) was preincubated with [3H]PROG (20 nm) in the absence or presence of an unlabeled steroid (0.0316–10 μm) at 36 °C for 30 min. Then NADPH (3.16 μm) was added, and the reaction mixture was incubated for a further 5 min. After the incubation, two identical samples were mixed and extracted with toluene. In some cases, before the above described incubation procedure, microsomal suspension (250 μg of protein/1.1 ml, total volume of the reaction mixture) was preincubated with goat anti-rat NADPH P450 reductase antiserum (50 μl) at 25 °C for 30 min in order to inhibit P450-dependent oxidative [3H]PROG metabolism. The toluene-extractable [3H]PROG metabolites (more than 98%) were isolated by various paper chromatographic systems and then identified by the recrystallization method (13Yamada M. Matsumoto K. Endocrinology. 1974; 94: 777-784Crossref PubMed Scopus (44) Google Scholar). Because of the limited expense, the amounts of various [3H]PROG metabolites were estimated, based on the mean values of purified efficiencies obtained from the recrystallization method in the first 10 and several important experimental batches. The mean ± S.D. values of purified efficiencies were as follows: unchanged [3H]PROG (99.26 ± 2.59%), [3H]16α-OH-P (88.22 ± 4.14%), [3H]6β-OH-P (75.39 ± 3.95%), [3H]2α-OH-P (69.15 ± 5.11%), [3H]17α-OH-P (53.74 ± 9.72%), [3H]20α-OH-P (92.86 ± 2.92%), [3H]5α-P (92.97 ± 6.83%) and [3H]3α-OH-5α-P (97.45 ± 3.81%). In order to examine the direct effects of some unlabeled steroids on the oxidative [3H]PROG metabolism, we used rat CYP3A2 supersomes, microsomes (82.5 μg of protein/1.1 ml, total volume of the reaction mixture) of insect cells (BTI-TN-5B1-4) containing the cDNA-expressed rat CYP3A2, rat NADPH P450 reductase, and human cytochrome b 5. Other experimental conditions were the same as those using the rat liver microsomes. The purified efficiency of [3H]6β-OH-P, exclusively formed by the supersomes, was 95.31 ± 3.81%. Other procedures are described in our previous papers (11Yamada M. Nishigami T. Nakasho K. Nishimoto Y. Miyaji H. Hepatology. 1994; 20: 1271-1280Crossref PubMed Scopus (39) Google Scholar, 12Yamada M. Indo K. Nishigami T. Nakasho K. Miyaji H. J. Biol. Chem. 1990; 265: 11035-11043Abstract Full Text PDF PubMed Google Scholar, 13Yamada M. Matsumoto K. Endocrinology. 1974; 94: 777-784Crossref PubMed Scopus (44) Google Scholar). In the present study, the respective final concentrations of [3H]PROG and NADPH were adjusted to be 20 nmand 3.16 μm, although these were approximately 2–4 orders of magnitude lower than those of customary enzyme assay systems (4Swinney D.C. Drug Metab. Dispos. 1990; 18: 859-865PubMed Google Scholar, 5Murray M. Cantrill E. Martini R. Farrell G.C. Arch. Biochem. Biophys. 1991; 286: 618-624Crossref PubMed Scopus (41) Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar). The reasons are as follows. 1) When the final concentration of ethanol (used for solubilizing [3H]PROG and an unlabeled steroid) exceeded 2% (v/v), this induced aggregation of the microsomes, 2A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, and K. Uematsu, unpublished results. and Wiebel et al. (14Wiebel F.J. Leuts J.C. Diamond L. Gelboin H.V. Arch. Biochem. Biophys. 1971; 144: 78-86Crossref PubMed Scopus (337) Google Scholar) have shown that some P450-dependent enzyme activities could be affected by more than 1% (v/v) of ethanol. Therefore, ethanol concentration was fixed to be 0.68% (v/v) in the present study, by which some unlabeled steroids became insoluble in the reaction mixture at their final concentrations over 1.0 μm. 2) The [3H]PROG concentration of 20 nm used seems physiological rather than those of the customary systems, since the plasma PROG concentration is estimated to be about 10 nm in adult male rats (15Corpéchot C. Young J. Calvel M. Wehrey C. Veltz J.N. Touyer G. Mouren M. Prasad V.V.K. Banner C. Sjövall J. Baulieu E.E. Robel P. Endocrinology. 1993; 133: 1003-1009Crossref PubMed Scopus (0) Google Scholar, 16Lancel M. Faulhaber J. Holsboer F. Rupprecht R. Am. J. Physiol. 1996; 271: E763-E772Crossref PubMed Google Scholar). 3) The yields of unidentifiable [3H]PROG metabolites, included in both the water-soluble and toluene-extractable fractions, increased in a dose-dependent manner when either lower concentrations of [3H]PROG or higher concentrations of NADPH were used.2 The [3H]PROG metabolism of the representative result for the 37 experimental batches performed in the present study is shown in Table I. In the microsomes alone, without additions of NADPH and an unlabeled steroid (the second column), only [3H]20α-OH-P and 5α-reductase-dependent metabolites, [3H]5α-P and [3H]3α-OH-5α-P, were formed in small amounts. However, the addition of 3.16 μmNADPH (the third column), a common cofactor of P450-dependent and 5α-reductase-dependent metabolisms induced larger formations of the respective male-specific CYP2C11- and CYP3A2-dependent oxidized metabolites, [3H]16α-OH-P (rather than [3H]2α-OH-P) and [3H]6β-OH-P, as compared with small increases of the 5α-reduced metabolites. The mean ± S.D. values of these products obtained from the 37 experimental batches were as follows: [3H]16α-OH-P (7.37 ± 1.30 pmol/mg protein/5 min), [3H]6β-OH-P (2.87 ± 0.62), [3H]5α-P (4.85 ± 1.75), and [3H]3α-OH-5α-P (1.35 ± 0.63). The ratio of [3H]16α-OH-P to [3H]6β-OH-P agreed well with that of CYP2C11 to CYP3A2 content in adult male rat liver microsomes (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar). However, the ratio of the sum of oxidized to 5α-reduced products seemed to be severalfold to 10-fold lower than those of other investigators' data (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar). This discrepancy may be partly related to the fact that we used adult male rats castrated for 3–4 weeks (in order to decrease endogenous steroids and increase [3H]5α-reduced metabolites), because such a postpubertal castration is known to induce a partial feminization of liver microsomal steroid metabolisms by repressing theCYP2C11 and CYP3A2 gene expressions and conversely stimulating the 5α-reductase gene expression (2Blanck A. Åström A. Hansson T. Cancer Res. 1986; 46: 5072-5076PubMed Google Scholar, 17Ribeiro V. Lechner M.C. Arch. Biochem. Biophys. 1992; 293: 147-152Crossref PubMed Scopus (69) Google Scholar). It should, however, be noted that there were several reports showing similar results to ours, using intact male rats (2Blanck A. Åström A. Hansson T. Cancer Res. 1986; 46: 5072-5076PubMed Google Scholar, 18Mode A. Norstedt G. Simic B. Eneroth P. Gustafsson J.Å. Endocrinology. 1981; 108: 2103-2108Crossref PubMed Scopus (138) Google Scholar).Table I[3H]Progesterone metabolism by adult male rat liver microsomesMicrosomes (μg protein)414414414414414414414NADPH (μm)3.163.163.163.163.163.16Unlabeled steroid (1 μm)PROG16α-OH-P11β-OH-P3α-OH-5α-P3α, 11β-(OH)2-5α-P5Metabolite (formation)%%%%%%%%Water-soluble0.210.771.610.870.801.980.491.02Toluene-extractable Unchanged PROG97.0392.1075.2285.2979.5970.5081.4476.08 16α-OH-P<0.08<0.217.14 (ox)1-aThe yields of these oxidized (ox) and 5α-reduced (red) metabolites, formed under the experimental condition of which the microsomal suspension was incubated with [3H[progesterone in the presence of NADPH and absence of an unlabeled compound (the third column), were regarded as control throughout the present study.4.257.367.342.061.97 6β-OH-P<0.08<0.262.56 (ox)1-aThe yields of these oxidized (ox) and 5α-reduced (red) metabolites, formed under the experimental condition of which the microsomal suspension was incubated with [3H[progesterone in the presence of NADPH and absence of an unlabeled compound (the third column), were regarded as control throughout the present study.1.882.543.291.401.65 17α-OH-P<0.07<0.11<0.38<0.18<0.30<0.42<0.15<0.24 2α-OH-P<0.24<0.241.10<0.891.111.24<0.42<0.54 20α-OH-P<0.061.10<0.71<0.37<0.811.03<0.731.24 3α-OH-5α-P<0.371.052.06 (red)1-aThe yields of these oxidized (ox) and 5α-reduced (red) metabolites, formed under the experimental condition of which the microsomal suspension was incubated with [3H[progesterone in the presence of NADPH and absence of an unlabeled compound (the third column), were regarded as control throughout the present study.0.691.232.513.274.17 5α-P<0.422.214.26 (red)1-aThe yields of these oxidized (ox) and 5α-reduced (red) metabolites, formed under the experimental condition of which the microsomal suspension was incubated with [3H[progesterone in the presence of NADPH and absence of an unlabeled compound (the third column), were regarded as control throughout the present study.1.511.586.916.088.53The microsomal suspension was preincubated with [3H]progesterone (20 nm) in the absence or presence of a 1 μm concentration of an unlabeled steroid at 36 °C for 30 min. Then NADPH (3.16 μm) was added or not, and the mixture was incubated for a further 5 min.1-a The yields of these oxidized (ox) and 5α-reduced (red) metabolites, formed under the experimental condition of which the microsomal suspension was incubated with [3H[progesterone in the presence of NADPH and absence of an unlabeled compound (the third column), were regarded as control throughout the present study. Open table in a new tab The microsomal suspension was preincubated with [3H]progesterone (20 nm) in the absence or presence of a 1 μm concentration of an unlabeled steroid at 36 °C for 30 min. Then NADPH (3.16 μm) was added or not, and the mixture was incubated for a further 5 min. The dose-dependent effects of representative, unlabeled steroids on the P450-dependent oxidative (sum of formed [3H]16α-OH-P and [3H]6β-OH-P) and 5α-reductive (sum of formed [3H]5α-P and [3H]3α-OH-5α-P) metabolisms of [3H]PROG were examined (Fig.1). Both PROG and 16α-OH-P effectively inhibited the formation of [3H]5α-reduced metabolites in a very similar, dose-dependent manner. The former steroid inhibited also the oxidative metabolism, but the latter did not inhibit it. Most interestingly, 3α-OH-5α-P and 3α,11β-(OH)2-5α-P, compared with PROG, not only showed stronger inhibitory effects on the oxidative metabolism but also conversely stimulated the 5α-reductive metabolism. We found that various unlabeled steroids used could be divided into six groups, A, B, C, D, E, and F, based on their respective effects on the oxidative and 5α-reductive [3H]PROG metabolisms (Fig.2 and Table I). The group A steroids such as 3α-OH-5α-P and 5α-P showed inhibitory effects on the oxidative metabolism, while having stimulatory effects on the 5α-reductive metabolism producing themselves. The group B steroids, PROG and TEST, inhibited both metabolisms as probably alternative substrates. The group C steroids, 5β-A-17β-ol and 3β-OH-P, showed inhibitory effects on the oxidative metabolism with no effect on the 5α-reductive metabolism, and conversely, the group D steroids, COR and 11β-OH-P, showed stimulatory effects on the 5α-reductive metabolism with no effect on the oxidative metabolism, despite possessing a 3-keto-4-ene structure that might be catalyzed by the 5α-reductase. Other 3-keto-4-ene steroids (group E), 16α-OH-P and 6β-OH-P, inhibited only the 5α-reductive metabolism without the product inhibition effects on the oxidative metabolism producing themselves. It is noteworthy that 16α-OH-P, as well as 20α-OH-P and 4-AN-CA already reported by other investigators (19Kinoshita Y. Endocr. J. 1981; 28: 499-513Crossref Scopus (6) Google Scholar, 20Monsalve A. Blaquier J.A. Steroids. 1977; 30: 41-51Crossref PubMed Scopus (40) Google Scholar), were of the 3-keto-4-ene steroids showing the strongest inhibitory effect on the 5α-reductase activity. Finally, the group F steroids, 11α-OH-P and cholesterol, showed a slight effect or no effect on both of the metabolisms. For additional interesting information, 3α,11β-(OH)2-5α-P, a group A steroid, containing both a 3α-OH-5α-reduced structure and a C-11β-OH structure, showed an additively stimulatory effect on the 5α-reductive metabolism, as compared with its parental steroids, 3α-OH-5α-P and 11β-OH-P, and thus this steroid, although not actually produced in the liver, was the highest stimulator of the 5α-reductase activity. By the way, one may envisage a possibility that such a stimulatory effect of group A steroids on the 5α-reductive metabolism may result from the increasing utilizations of free [3H]PROG and NADPH, left over by their inhibitory effects on the oxidative [3H]PROG metabolism and vice versa. However, this possibility may be largely refuted by the results of the following two experiments using the anti-rat NADPH P450 reductase antiserum and rat CYP3A2 supersomes. We examined the direct effects of representative steroids on the 5α-reductive [3H]PROG metabolism, using the rat liver microsomes pretreated with goat anti-rat NADPH P450 reductase antiserum (Fig.3). By this means, more than 85% of the P450-dependent, oxidative [3H]PROG metabolism was inhibited, irrespective of the absence or presence of an unlabeled steroid. Under such an experimental condition, PROG and 16α-OH-P inhibited the 5α-reductive metabolism, while 11β-OH-P, 3α-OH-5α-P, and 3α,11β-(OH)2-5α-P stimulated it, as the intact microsomes did (see Fig. 2). This result clearly shows that the effects of these steroids on the 5α-reductive metabolism could be brought about by their intrinsic properties, not affected by the co-existence of P450-dependent metabolism in intact rat liver microsomes. For additional information, an addition of normal goat serum, as compared with the 130 mm KCl-based buffer (12Yamada M. Indo K. Nishigami T. Nakasho K. Miyaji H. J. Biol. Chem. 1990; 265: 11035-11043Abstract Full Text PDF PubMed Google Scholar), induced a tendency to decrease the oxidative metabolism and increase the 5α-reductive metabolism. Although the mechanism inducing such a tendency is wholly unclear at present, this may have been associated with lower stimulatory effects of 11β-OH-P, 3α-OH-5α-P, and 3α,11β-(OH)2-5α-P on the 5α-reductive metabolism by the antiserum-treated microsomes, compared with the intact microsomes. In order to examine also the direct effects of representative steroids on the male-specific P450-dependent [3H]PROG metabolism, we used rat CYP3A2, but not CYP2C11, supersomes, which were composed of the microsomes of insect cells containing the cDNA-expressed rat CYP3A2, rat NADPH P450 reductase, and human cytochrome b 5, since a recombinant CYP2C11 expression system has not come into the market, and we found that various unlabeled steroids showed a similar inhibitory pattern on rat liver microsomal [3H]PROG 6β-oxidation and 16α-oxidation, mainly catalyzed by CYP3A2 and CYP2C11, respectively (Fig. 4).2 When the CYP3A2 supersomes were incubated with [3H]PROG, an exclusively formed product was [3H]6β-OH-P (data not shown), and the inhibitory pattern of unlabeled steroids on the [3H]6β-OH-P formation resembled that obtained from the intact rat liver microsomes (Fig. 5).Figure 5Effects of representative unlabeled steroids on the [3H]progesterone 6β-oxidizing activity by rat CYP3A2 supersomes. Rat CYP3A2 supersomes (82.5 μg of protein/1.1 ml, total volume of the reaction mixture) were used instead of rat liver microsomes, and the concentration of an unlabeled steroid was fixed to be 1.0 μm in this experiment. Other experimental conditions were the same as shown in Fig. 1. The data are means ± S.D. of at least three experiments. The percentage of formation of exclusively formed [3H]6β-OH-P was estimated to be 13.98 ± 1.80% (37.28 pmol/mg of protein/5 min) in the control experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Furthermore, we examined the types of inhibition and the inhibitor constant (K i) values of unlabeled PROG and 3α-OH-5α-P against [3H]6β-OH-P formation by rat CYP3A2 supersomes, according to a simple graphic method using two [3H]PROG concentrations (21Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3298) Google Scholar). From this graphic presentation (so-called Dixon's plot) shown in Fig.6, it turned out that both of the unlabeled steroids behaved like a competitive inhibitor, theK i value of 3α-OH-5α-P was about 10-fold lower than that of PROG, and this K i value ratio agreed well with the IC27.5 (against [3H]6β-OH-P formation) and IC40 (against 16α-OH-P formation) value ratios obtained using rat liver microsomes (TableII). Since not only unlabeled PROG but also 3α-OH-5α-P (22Heinrichs W.L. Feder H.H. Colás A. Steroids. 1966; 7: 91-98Crossref PubMed Scopus (36) Google Scholar) must be metabolized into 6β-OH- and/or 16α-OH-products, it is most likely that these unlabeled steroids compete with [3H]PROG as alternative substrates, but not as true competitive inhibitors, for rat CYP3A2 and/or CYP2C11 with 3α-OH-5α-P possessing about 10-fold higher affinity for the substrate-binding pockets of these enzymes and that the effects of these unlabeled steroids on the CYP3A2 (and probably CYP2C11) activity also can be brought about by their intrinsic properties, independent of a difference of the microsomal structures between the rat liver and insect cells.Table IIThe quantitative parameters of inhibitory effects of unlabeled progesterone and 3α-OH-5α-pregnan-20-one against oxidative [3H]progesterone metabolismUnlabeled steroidRat liver microsomesRat CYP3A2 supersomes, [3H]6β-OH (K i)2-bThe K i values are estimated from three experiments.[3H]6β-OH (IC27.5)2-aThe IC27.5 and IC40 values, estimated from at least four experiments for separate rats, were defined as the molar concentrations (×10−6m) of unlabeled PROG or 3α-OH-5α-P causing a 27.5 and a 40% inhibition against the respective [3H]PROG 6β-oxidizing ([3H]6β-OH) and 16α-oxidizing ([3H]16α-OH) activities, and these inhibitory percentages were conveniently created as showing half-maximal inhibitions by the concentrations up to 1.0 μm, a maximally solubilizable concentration, of unlabeled 3α-OH-5α-P.[3H]16α-OH (IC40)2-aThe IC27.5 and IC40 values, estimated from at least four experiments for separate rats, were defined as the molar concentrations (×10−6m) of unlabeled PROG or 3α-OH-5α-P causing a 27.5 and a 40% inhibition against the respective [3H]PROG 6β-oxidizing ([3H]6β-OH) and 16α-oxidizing ([3H]16α-OH) activities, and these inhibitory percentages were conveniently created as showing half-maximal inhibitions by the concentrations up to 1.0 μm, a maximally solubilizable concentration, of unlabeled 3α-OH-5α-P.×10−6mPROG0.710.760.983α-OH-5α-P0.080.060.12The experimental conditions were the same as shown in Fig. 1 for rat liver microsomes and Fig. 6 for rat CYP3A2 supersomes.2-a The IC27.5 and IC40 values, estimated from at least four experiments for separate rats, were defined as the molar concentrations (×10−6m) of unlabeled PROG or 3α-OH-5α-P causing a 27.5 and a 40% inhibition against the respective [3H]PROG 6β-oxidizing ([3H]6β-OH) and 16α-oxidizing ([3H]16α-OH) activities, and these inhibitory percentages were conveniently created as showing half-maximal inhibitions by the concentrations up to 1.0 μm, a maximally solubilizable concentration, of unlabeled 3α-OH-5α-P.2-b The K i values are estimated from three experiments. Open table in a new tab The experimental conditions were the same as shown in Fig. 1 for rat liver microsomes and Fig. 6 for rat CYP3A2 supersomes. In conclusion, the present study clearly shows that the male-specific products, 16α-OH-P and 6β-OH-P, inhibited the female-predominant 5α-reductase activity without significantly inhibiting the male-specific CYP2C11 and CYP3A2 activities producing themselves. On the other hand, the female-predominant products, 5α-P and 3α-OH-5α-P, not only inhibited the male-specific P450 activities but also stimulated the 5α-reductase activity producing themselves, and such effects can be brought about by the intrinsic properties of these steroids. Thus, we can propose a novel hypothesis, as described under “Discussion,” on the regulation system of sexually differentiated steroid metabolisms in adult male rat liver. It is well known that various 3-keto-4-ene steroids such as PROG, TEST, and 4-AN are primarily metabolized into 16α- (in some cases, 2α- also) and 6β-oxidized products mainly by the respective, male-specific P450 subforms, CYP2C11 and CYP3A2, in male rat liver microsomes, whereas they are primarily metabolized into the 5α-reduced products by female-predominant 5α-reductase in the female (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 2Blanck A. Åström A. Hansson T. Cancer Res. 1986; 46: 5072-5076PubMed Google Scholar, 3Waxman D.J. Morrissey J.J. LeBlanc G.A. Endocrinology. 1989; 124: 2954-2966Crossref PubMed Scopus (92) Google Scholar, 4Swinney D.C. Drug Metab. Dispos. 1990; 18: 859-865PubMed Google Scholar, 5Murray M. Cantrill E. Martini R. Farrell G.C. Arch. Biochem. Biophys. 1991; 286: 618-624Crossref PubMed Scopus (41) Google Scholar, 6Legraverend C. Mode A. Wells T. Robinson I. Gustafsson J-Å. FASEB J. 1992; 6: 711-718Crossref PubMed Scopus (136) Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar), and it is known that expressions of these sexually differentiated enzyme activities are largely regulated in transcription level under endocrine control of which GH plays a major role (6Legraverend C. Mode A. Wells T. Robinson I. Gustafsson J-Å. FASEB J. 1992; 6: 711-718Crossref PubMed Scopus (136) Google Scholar,8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar). In the present in vitro study using adult male rat liver microsomes (Tables I and II; Figs. Figure 1, Figure 2, Figure 3, Figure 4) and rat CYP3A2 supersomes (Figs. 5 and 6; Table II), we showed for the first time that two major male-specific oxidized PROG metabolites, 6β-OH-P and especially 16α-OH-P, strongly inhibited the female-predominant 5α-reductase activity without significantly showing the inhibitory effects on the CYP3A2 and CYP2C11 activities producing themselves, and these events may be further enhanced by high levels of CYP2C11 andCYP3A2 gene expressions in the male (6Legraverend C. Mode A. Wells T. Robinson I. Gustafsson J-Å. FASEB J. 1992; 6: 711-718Crossref PubMed Scopus (136) Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar, 17Ribeiro V. Lechner M.C. Arch. Biochem. Biophys. 1992; 293: 147-152Crossref PubMed Scopus (69) Google Scholar). On the other hand, 5α-P and especially 3α-OH-5α-P not only inhibited both the CYP2C11 and CYP3A2 activities but also stimulated the 5α-reductase activity producing themselves. However, such adverse effects of the 5α-reduced products on the male pattern metabolism may be attenuated by a scanty expression of the 5α-reductase gene in the male (8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar). Thus, our results compel us to propose a very interesting hypothesis, summarized in Fig. 7, that adult male rat liver microsomes may possess a self-augmentation system by the male-specific products on sexually differentiated steroid-metabolizing activities, coupled with the regulation system by gene expressions of the related enzymes under endocrine control. In other words, the results may also explain the reason why adult male rat liver should preserve not only much higher levels of CYP2C11 and CYP3A2 gene expressions but also lower 5α-reductase gene expression, as compared with the female. Furthermore, it is of great interest and importance to investigate whether the female rat liver also possesses such a self-augmentation system, although the present results strongly suggest that at least female-predominant 5α-reductase activity (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 3Waxman D.J. Morrissey J.J. LeBlanc G.A. Endocrinology. 1989; 124: 2954-2966Crossref PubMed Scopus (92) Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar, 18Mode A. Norstedt G. Simic B. Eneroth P. Gustafsson J.Å. Endocrinology. 1981; 108: 2103-2108Crossref PubMed Scopus (138) Google Scholar) may be further enhanced by its products, 5α-P and especially 3α-OH-5α-P. As regards these, an important question for future study is to elucidate the reason why adult male rat liver microsomes must metabolize PROG first into more hydrophilic products, 16α-OH-P and 6β-OH-P, while the female must metabolize it into more hydrophobic products, 5α-P, under the strictly regulated systems described above. By the way, a similar scenario may occur on the androgen metabolism, since 3-keto-4-ene androgens such as TEST and 4-AN are also known to be catalyzed sex-dependently by the same enzyme systems (1Dannan G.A. Guengerich F.P. Waxman D.J. J. Biol. Chem. 1986; 261: 10728-10735Abstract Full Text PDF PubMed Google Scholar, 2Blanck A. Åström A. Hansson T. Cancer Res. 1986; 46: 5072-5076PubMed Google Scholar, 3Waxman D.J. Morrissey J.J. LeBlanc G.A. Endocrinology. 1989; 124: 2954-2966Crossref PubMed Scopus (92) Google Scholar,5Murray M. Cantrill E. Martini R. Farrell G.C. Arch. Biochem. Biophys. 1991; 286: 618-624Crossref PubMed Scopus (41) Google Scholar, 7Chang T.K.H. Bellward G.D. J. Pharmacol. Exp. Ther. 1996; 278: 1383-1391PubMed Google Scholar, 8Pampori N.A. Shapiro B.H. Mol. Pharmacol. 1996; 50: 1148-1156PubMed Google Scholar, 9Shimada M. Murayama N. Nagata K. Hashimoto H. Ishikawa H. Yamazoe Y. Arch. Biochem. Biophys. 1997; 337: 34-42Crossref PubMed Scopus (26) Google Scholar, 10Pampori N.A. Shapiro B.H. Endocrinology. 1999; 140: 1245-1254Crossref PubMed Google Scholar, 18Mode A. Norstedt G. Simic B. Eneroth P. Gustafsson J.Å. Endocrinology. 1981; 108: 2103-2108Crossref PubMed Scopus (138) Google Scholar), and the effects of various 3-keto-4-ene and 5α-reduced androgens, especially TEST and 5α-A-3α,17β, on the [3H]PROG metabolism showed a similar pattern to those of various 4-pregnene and 5α-pregnane steroids described here (Fig. 2).2 As regards another interesting finding obtained from the present study, it has been reported that endogenous COR production in rat adrenal cortex is suppressed by exogenously administrated COR or cortisol inin vivo and in cell culture systems and that this inhibition probably results from the various effects of these steroids, namely inhibiting ACTH secretion from the pituitary, decreasing ACTH sensitivity of adrenal cortex (23Birmingham M.K. Kurlents E. Endocrinology. 1958; 62: 47-60Crossref PubMed Scopus (43) Google Scholar), and stimulating the adrenal 5α-reductase activity metabolizing COR into its 5α-reduced products (24Carsia R.V. Scanes C.G. Malamed S. Endocrinology. 1984; 115: 2464-2472Crossref PubMed Scopus (21) Google Scholar). However, several recent studies have clearly shown that the two 11β-OH corticosteroids, COR and cortisol, are of the poorest substrate group for 5α-reductases of various organs probably including the adrenal cortex itself (19Kinoshita Y. Endocr. J. 1981; 28: 499-513Crossref Scopus (6) Google Scholar, 20Monsalve A. Blaquier J.A. Steroids. 1977; 30: 41-51Crossref PubMed Scopus (40) Google Scholar, 25Normington K. Russell D.W. J. Biol. Chem. 1992; 267: 19548-19554Abstract Full Text PDF PubMed Google Scholar), and we showed in the present study that COR and 11β-OH-P, but not 11α-OH-P, rather stimulated [3H]PROG 5α-reductase activity of rat liver microsomes (Fig. 2). These results suggest that the C-11β-OH group of a steroid molecule may strongly disturb access of the steroid to the active site of the 5α-reductase, and we can propose another possibility that adrenal cortex may possess a short negative feedback system of which the excessively produced COR (and probably cortisol) inhibits its own production by stimulating the 5α-reduction of PROG (but not COR itself), the major precursor of COR. In conclusion, we can propose two novel hypotheses on 1) the self-augmentation system on sexually differentiated steroid metabolism in adult male rat liver and 2) a short negative feedback system of COR production in adrenal glands. Although the action mechanisms operating these regulatory systems are largely unclear at present, an attempt to clarify them is currently under investigation in our laboratory. We thank Dr. Nobuyuki Terada for helpful suggestions and also thank Ayako Kuhara and Fumiko Kozuki for technical assistance.
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