Self-augmentation Effect of Male-specific Products on Sexually Differentiated Progesterone Metabolism in Adult Male Rat Liver Microsomes*

Akihiko YamadaDagger §, Morio Yamada, Yukihisa Fujita, Takashi NishigamiDagger , Keiji NakashoDagger , and Kunio UematsuDagger

From the Dagger  Second Department of Pathology, and the  Department of Chemistry, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan

Received for publication, April 19, 2000, and in revised form, August 28, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (16alpha -hydroxyl and 6beta -hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5alpha -reduced products by female-predominant 5alpha -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 16alpha -hydroxyprogesterone and 6beta -hydroxyprogesterone inhibited the 5alpha -reductive [3H]progesterone metabolism by adult male rat liver microsomes without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, whereas 3alpha -hydroxy-5alpha -pregnan-20-one and 5alpha -pregnane-3,20-dione not only stimulated the 5alpha -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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, 16alpha -OH (in some cases, 2alpha -OH also) and 6beta -OH products, mainly by the respective, male-specific cytochrome P450 subforms (P450s), CYP2C11 and CYP3A2, whereas the female metabolizes them primarily into the 5alpha -reduced products by female-predominant 5alpha -reductase (1-10). 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 represses CYP2C11 and CYP3A2 gene expressions and conversely induces 5alpha -reductase gene expression (6, 8-10). 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 (1-3, 6, 7).

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, 16alpha -OH-P and 6beta -OH-P, inhibited female-predominant [3H]PROG 5alpha -reductase activity without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, while 3alpha -OH-5alpha -P and 5alpha -P, female-predominant products by the 5alpha -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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [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.

Preparation of Adult Male Rat Liver Microsomes-- 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 (11). The experiments were performed according to institutional guidelines for the care and use of laboratory animals.

[3H]PROG Metabolism by Rat Liver Microsomes-- Effects of various unlabeled steroids on [3H]PROG metabolism by liver microsomes were examined, according to our previously described procedure (12). 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 (13). 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]16alpha -OH-P (88.22 ± 4.14%), [3H]6beta -OH-P (75.39 ± 3.95%), [3H]2alpha -OH-P (69.15 ± 5.11%), [3H]17alpha -OH-P (53.74 ± 9.72%), [3H]20alpha -OH-P (92.86 ± 2.92%), [3H]5alpha -P (92.97 ± 6.83%) and [3H]3alpha -OH-5alpha -P (97.45 ± 3.81%).

[3H]PROG Metabolism by Rat CYP3A2 Supersomes-- 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 b5. Other experimental conditions were the same as those using the rat liver microsomes. The purified efficiency of [3H]6beta -OH-P, exclusively formed by the supersomes, was 95.31 ± 3.81%.

Miscellaneous Methods-- Other procedures are described in our previous papers (11-13).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Evaluation of the Present Assay System for [3H]PROG Metabolism by Rat Liver Microsomes-- In the present study, the respective final concentrations of [3H]PROG and NADPH were adjusted to be 20 nM and 3.16 µM, although these were approximately 2-4 orders of magnitude lower than those of customary enzyme assay systems (4, 5, 7, 9). 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,2 and Wiebel et al. (14) 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 (15, 16). 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]20alpha -OH-P and 5alpha -reductase-dependent metabolites, [3H]5alpha -P and [3H]3alpha -OH-5alpha -P, were formed in small amounts. However, the addition of 3.16 µM NADPH (the third column), a common cofactor of P450-dependent and 5alpha -reductase-dependent metabolisms induced larger formations of the respective male-specific CYP2C11- and CYP3A2-dependent oxidized metabolites, [3H]16alpha -OH-P (rather than [3H]2alpha -OH-P) and [3H]6beta -OH-P, as compared with small increases of the 5alpha -reduced metabolites. The mean ± S.D. values of these products obtained from the 37 experimental batches were as follows: [3H]16alpha -OH-P (7.37 ± 1.30 pmol/mg protein/5 min), [3H]6beta -OH-P (2.87 ± 0.62), [3H]5alpha -P (4.85 ± 1.75), and [3H]3alpha -OH-5alpha -P (1.35 ± 0.63). The ratio of [3H]16alpha -OH-P to [3H]6beta -OH-P agreed well with that of CYP2C11 to CYP3A2 content in adult male rat liver microsomes (1, 9). However, the ratio of the sum of oxidized to 5alpha -reduced products seemed to be severalfold to 10-fold lower than those of other investigators' data (1, 7, 8, 10). 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]5alpha -reduced metabolites), because such a postpubertal castration is known to induce a partial feminization of liver microsomal steroid metabolisms by repressing the CYP2C11 and CYP3A2 gene expressions and conversely stimulating the 5alpha -reductase gene expression (2, 17). It should, however, be noted that there were several reports showing similar results to ours, using intact male rats (2, 18).


                              
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Table I
[3H]Progesterone metabolism by adult male rat liver microsomes
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.

Dose-dependent Effects of Representative Steroids on [3H]PROG Metabolism by Rat Liver Microsomes-- The dose-dependent effects of representative, unlabeled steroids on the P450-dependent oxidative (sum of formed [3H]16alpha -OH-P and [3H]6beta -OH-P) and 5alpha -reductive (sum of formed [3H]5alpha -P and [3H]3alpha -OH-5alpha -P) metabolisms of [3H]PROG were examined (Fig. 1). Both PROG and 16alpha -OH-P effectively inhibited the formation of [3H]5alpha -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, 3alpha -OH-5alpha -P and 3alpha ,11beta -(OH)2-5alpha -P, compared with PROG, not only showed stronger inhibitory effects on the oxidative metabolism but also conversely stimulated the 5alpha -reductive metabolism.



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Fig. 1.   Dose-dependent effects of representative unlabeled steroids on the oxidative and 5alpha -reductive [3H]progesterone metabolisms by adult male rat liver microsomes. 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 (control; 0% inhibition or 0% stimulation) or presence of an unlabeled steroid (3.16 × 10-8 to 10-5 M) at 36 °C for 30 min. Then NADPH (3.16 µM) was added, and the reaction mixture was incubated for a further 5 min. The data are mean values of at least three experiments for separate rats. a, the oxidized products (filled symbols, solid lines) are the sum of formed [3H]16alpha -OH-P and [3H]6beta -OH-P, and the 5alpha -reduced products (open symbols, broken lines) are the sum of formed [3H]5alpha -P and [3H]3alpha -OH-5alpha -P. Unlabeled steroids used are as follows: progesterone (, open circle ), 16alpha -hydroxyprogesterone (black-triangle, triangle ), 3alpha -hydroxy-5alpha -pregnan-20-one (black-diamond , diamond ), 3alpha ,11beta -dihydroxy-5alpha -pregnan-20-one (black-square, ).

Classification of Various Steroids Based on Their Respective Effects on the Oxidative and 5alpha -Reductive [3H]PROG Metabolisms by Rat Liver Microsomes-- 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 5alpha -reductive [3H]PROG metabolisms (Fig. 2 and Table I). The group A steroids such as 3alpha -OH-5alpha -P and 5alpha -P showed inhibitory effects on the oxidative metabolism, while having stimulatory effects on the 5alpha -reductive metabolism producing themselves. The group B steroids, PROG and TEST, inhibited both metabolisms as probably alternative substrates. The group C steroids, 5beta -A-17beta -ol and 3beta -OH-P, showed inhibitory effects on the oxidative metabolism with no effect on the 5alpha -reductive metabolism, and conversely, the group D steroids, COR and 11beta -OH-P, showed stimulatory effects on the 5alpha -reductive metabolism with no effect on the oxidative metabolism, despite possessing a 3-keto-4-ene structure that might be catalyzed by the 5alpha -reductase. Other 3-keto-4-ene steroids (group E), 16alpha -OH-P and 6beta -OH-P, inhibited only the 5alpha -reductive metabolism without the product inhibition effects on the oxidative metabolism producing themselves. It is noteworthy that 16alpha -OH-P, as well as 20alpha -OH-P and 4-AN-CA already reported by other investigators (19, 20), were of the 3-keto-4-ene steroids showing the strongest inhibitory effect on the 5alpha -reductase activity. Finally, the group F steroids, 11alpha -OH-P and cholesterol, showed a slight effect or no effect on both of the metabolisms.



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Fig. 2.   Classification of various unlabeled steroids, based on their respective effects on the oxidative and 5alpha -reductive [3H]progesterone metabolisms by adult male rat liver microsomes. The experimental conditions were the same as shown in Fig. 1 except for the concentration of unlabeled steroids (1.0 µM in this experiment). The data are means ± S.D. of at least three experiments for separate rats. The sum of oxidized (black-square) or 5alpha -reduced () products is the same as shown in Fig. 1. CHOL, cholesterol.

For additional interesting information, 3alpha ,11beta -(OH)2-5alpha -P, a group A steroid, containing both a 3alpha -OH-5alpha -reduced structure and a C-11beta -OH structure, showed an additively stimulatory effect on the 5alpha -reductive metabolism, as compared with its parental steroids, 3alpha -OH-5alpha -P and 11beta -OH-P, and thus this steroid, although not actually produced in the liver, was the highest stimulator of the 5alpha -reductase activity.

By the way, one may envisage a possibility that such a stimulatory effect of group A steroids on the 5alpha -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.

Inhibition of P450-dependent [3H]PROG Metabolism by Anti-rat NADPH P450 Reductase Antiserum-- We examined the direct effects of representative steroids on the 5alpha -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 16alpha -OH-P inhibited the 5alpha -reductive metabolism, while 11beta -OH-P, 3alpha -OH-5alpha -P, and 3alpha ,11beta -(OH)2-5alpha -P stimulated it, as the intact microsomes did (see Fig. 2). This result clearly shows that the effects of these steroids on the 5alpha -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.



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Fig. 3.   Effects of representative unlabeled steroids on [3H]progesterone metabolism by adult male rat liver microsomes, pretreated with goat anti-rat NADPH P450 reductase antiserum. The microsomal suspension (250 µg of protein/1.1 ml, total volume of the reaction mixture) was preincubated with or without normal goat serum (50 µl) or goat anti-rat NADPH P450 reductase antiserum (50 µl) at 25 °C for 30 min, and then they were incubated with [3H]PROG (20 nM), an unlabeled steroid (1.0 µM), and NADPH (3.16 µM), according to the procedure shown in Fig. 1. a, 130 mM KCl-based buffer without goat serum (12). b, the antiserum-pretreated microsomal suspension, incubated without an unlabeled steroid, was used as control. The data are means ± S.D. of at least three experiments for separate rats. The sum of oxidized (black-square) or 5alpha -reduced () products is the same as shown in Fig. 1.

For additional information, an addition of normal goat serum, as compared with the 130 mM KCl-based buffer (12), induced a tendency to decrease the oxidative metabolism and increase the 5alpha -reductive metabolism. Although the mechanism inducing such a tendency is wholly unclear at present, this may have been associated with lower stimulatory effects of 11beta -OH-P, 3alpha -OH-5alpha -P, and 3alpha ,11beta -(OH)2-5alpha -P on the 5alpha -reductive metabolism by the antiserum-treated microsomes, compared with the intact microsomes.

[3H]PROG Metabolism by Rat CYP3A2 Supersomes-- 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 b5, 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 6beta -oxidation and 16alpha -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]6beta -OH-P (data not shown), and the inhibitory pattern of unlabeled steroids on the [3H]6beta -OH-P formation resembled that obtained from the intact rat liver microsomes (Fig. 5).



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Fig. 4.   Comparison of the effects of representative unlabeled steroids on the [3H]progesterone 6beta - or 16alpha -oxidizing activity by adult male rat liver microsomes. The formation of [3H]6beta -OH-P (black-square) or [3H]16alpha -OH-P () was determined according to the procedure shown in Fig. 1 except for the concentration of unlabeled steroids (1.0 µM in this experiment). The data are means ± S.D. of at least three experiments for separate rats.



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Fig. 5.   Effects of representative unlabeled steroids on the [3H]progesterone 6beta -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]6beta -OH-P was estimated to be 13.98 ± 1.80% (37.28 pmol/mg of protein/5 min) in the control experiments.

Furthermore, we examined the types of inhibition and the inhibitor constant (Ki) values of unlabeled PROG and 3alpha -OH-5alpha -P against [3H]6beta -OH-P formation by rat CYP3A2 supersomes, according to a simple graphic method using two [3H]PROG concentrations (21). 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, the Ki value of 3alpha -OH-5alpha -P was about 10-fold lower than that of PROG, and this Ki value ratio agreed well with the IC27.5 (against [3H]6beta -OH-P formation) and IC40 (against 16alpha -OH-P formation) value ratios obtained using rat liver microsomes (Table II). Since not only unlabeled PROG but also 3alpha -OH-5alpha -P (22) must be metabolized into 6beta -OH- and/or 16alpha -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 3alpha -OH-5alpha -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.



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Fig. 6.   Graphic determinations of the types of inhibition and the inhibitor constant (Ki) values of unlabeled progesterone and 3alpha -OH-5alpha -pregnan-20-one. Rat CYP3A2 supersomes (82.5 µg of protein/1.1 ml) were incubated with 4.46 nM (closed symbols) or 18.5 nM (open symbols) of [3H]PROG in the presence of unlabeled PROG (0.1, 0.316, 0.667, and 1.0 µM) or 3alpha -OH-5alpha -P (0.01, 0.02, 0.0316, 0.05, 0.1, 0.2, and/or 0.316 µM). Other experimental conditions were the same as shown in Fig. 5. The vi values show reaction velocities ([3H]6beta -OH-P formation, pmol/mg of protein/5 min) in the presence of various concentrations of unlabeled PROG or 3alpha -OH-5alpha -P. The Ki value is given by the point of intersection of the respective straight lines obtained using two [3H]PROG concentrations.


                              
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Table II
The quantitative parameters of inhibitory effects of unlabeled progesterone and 3alpha -OH-5alpha -pregnan-20-one against oxidative [3H]progesterone metabolism
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, 16alpha -OH-P and 6beta -OH-P, inhibited the female-predominant 5alpha -reductase activity without significantly inhibiting the male-specific CYP2C11 and CYP3A2 activities producing themselves. On the other hand, the female-predominant products, 5alpha -P and 3alpha -OH-5alpha -P, not only inhibited the male-specific P450 activities but also stimulated the 5alpha -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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is well known that various 3-keto-4-ene steroids such as PROG, TEST, and 4-AN are primarily metabolized into 16alpha - (in some cases, 2alpha - also) and 6beta -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 5alpha -reduced products by female-predominant 5alpha -reductase in the female (1-10), 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 (6, 8-10).

In the present in vitro study using adult male rat liver microsomes (Tables I and II; Figs. 1-4) and rat CYP3A2 supersomes (Figs. 5 and 6; Table II), we showed for the first time that two major male-specific oxidized PROG metabolites, 6beta -OH-P and especially 16alpha -OH-P, strongly inhibited the female-predominant 5alpha -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 and CYP3A2 gene expressions in the male (6, 8-10, 17). On the other hand, 5alpha -P and especially 3alpha -OH-5alpha -P not only inhibited both the CYP2C11 and CYP3A2 activities but also stimulated the 5alpha -reductase activity producing themselves. However, such adverse effects of the 5alpha -reduced products on the male pattern metabolism may be attenuated by a scanty expression of the 5alpha -reductase gene in the male (8, 10). 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 5alpha -reductase gene expression, as compared with the female.



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Fig. 7.   Supposed self-augmentation system on sexually differentiated progesterone metabolism in adult male rat liver. 16alpha -OH-P and 6beta -OH-P are produced by male-specific microsomal P450s, CYP2C11 and CYP3A2, respectively. Both products inhibit PROG 5alpha -reduction without significant product-inhibition effects on the above P450 activities, and these events may be further enhanced by high levels of CYP2C11 and CYP3A2 gene expressions in the male. Actually, 16alpha -OH-P may make a higher contribution to the inhibitory effect on the 5alpha -reductase activity than 6beta -OH-P, judging from the result shown in Fig. 2 and a higher expression of CYP2C11 gene than CYP3A2 gene (1, 9). On the other hand, 5alpha -P and 3alpha -OH-5alpha -P are produced by the 5alpha -reductase and subsequently cytosolic (and, to a lesser degree, microsomal) 3alpha -hydroxysteroid dehydrogenase (26-28), respectively. Although these products, especially 3alpha -OH-5alpha -P, not only inhibit the CYP2C11 and CYP3A2 activities but also stimulate the 5alpha -reductase activity, such adverse effects of these 5alpha -reduced products on the male pattern metabolism may be actually attenuated by lower expressions of both the 5alpha -reductase (1, 3, 7, 8, 10, 18) and 3alpha -hydroxysteroid dehydrogenase (27, 28) genes in the male, compared with the female. 16alpha -OH-P, 6beta -OH-P, or 3alpha -OH-5alpha -P can be further metabolized into its glucuronide or sulfate and eventually excreted into urine and/or bile. It is most likely that the same self-augmentation system operates on the androgen metabolism. m, microsomal enzyme. c, cytosolic enzyme. §, the magnitudes of sexually differentiated enzyme activities in adult male rat liver, compared with the female, are as follows: CYP2C11 (>30-fold higher) (1-3, 9), CYP3A2 (>20-fold higher) (1, 9), 5alpha -reductase (~10-fold lower) (2, 3, 7), 3alpha -hydroxysteroid dehydrogenase (2-3-fold lower) (27, 28), UDP-glucuronosyl transferase (~1.5-fold higher) (29), and hydroxysteroid sulfotransferase (4-6-fold lower) (30, 31).

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 5alpha -reductase activity (1, 3, 7, 8, 10, 18) may be further enhanced by its products, 5alpha -P and especially 3alpha -OH-5alpha -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, 16alpha -OH-P and 6beta -OH-P, while the female must metabolize it into more hydrophobic products, 5alpha -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 (1-3, 5, 7-10, 18), and the effects of various 3-keto-4-ene and 5alpha -reduced androgens, especially TEST and 5alpha -A-3alpha ,17beta , on the [3H]PROG metabolism showed a similar pattern to those of various 4-pregnene and 5alpha -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 in in 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 (23), and stimulating the adrenal 5alpha -reductase activity metabolizing COR into its 5alpha -reduced products (24). However, several recent studies have clearly shown that the two 11beta -OH corticosteroids, COR and cortisol, are of the poorest substrate group for 5alpha -reductases of various organs probably including the adrenal cortex itself (19, 20, 25), and we showed in the present study that COR and 11beta -OH-P, but not 11alpha -OH-P, rather stimulated [3H]PROG 5alpha -reductase activity of rat liver microsomes (Fig. 2). These results suggest that the C-11beta -OH group of a steroid molecule may strongly disturb access of the steroid to the active site of the 5alpha -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 5alpha -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.


    ACKNOWLEDGEMENTS

We thank Dr. Nobuyuki Terada for helpful suggestions and also thank Ayako Kuhara and Fumiko Kozuki for technical assistance.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: The Second Dept. of Pathology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Tel.: 81-798-45-6427; Fax: 81-798-45-6426; E-mail: a-yamada@hyo-med.ac.jp.

Published, JBC Papers in Press, September 19, 2000, DOI 10.1074/jbc.M003355200

2 A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, and K. Uematsu, unpublished results.


    ABBREVIATIONS

The abbreviations used are: PROG, progesterone; 5alpha -A-3alpha , 17beta , 5alpha -androstane-3alpha ,17beta -diol; 5beta -A-17beta -ol, 5beta -androstan-17beta -ol; 4-AN, 4-androstene-3,17-dione; 4-AN-CA, 4-androsten-3-one-17beta -carboxylic acid; COR, corticosterone; P450, cytochrome P450; 3alpha , 11beta -(OH)2-5alpha -P, 3alpha ,11beta -dihydroxy-5alpha -pregnan-20-one; GH, growth hormone; 5alpha -P, 5alpha -pregnane-3,20-dione; 3alpha -OH-5alpha -P, 3alpha -hydroxy-5alpha -pregnan-20-one; TEST, testosterone; X-OH-P, X-hydroxyprogesterone; ACTH, adrenocorticotropic hormone.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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