From the 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
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ABSTRACT |
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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
(16 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, 16 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, 16 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.
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]16 [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]6 Miscellaneous Methods--
Other procedures are described in our
previous papers (11-13).
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]20 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]16 Classification of Various Steroids Based on Their Respective
Effects on the Oxidative and 5
For additional interesting information,
3
By the way, one may envisage a possibility that such a stimulatory
effect of group A steroids on the 5 Inhibition of P450-dependent [3H]PROG
Metabolism by Anti-rat NADPH P450 Reductase Antiserum--
We examined
the direct effects of representative steroids on the 5
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 5 [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 6
Furthermore, we examined the types of inhibition and the inhibitor
constant (Ki) values of unlabeled PROG and
3
In conclusion, the present study clearly shows that the male-specific
products, 16 It is well known that various 3-keto-4-ene steroids such as PROG,
TEST, and 4-AN are primarily metabolized into 16 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, 6-hydroxyl and 6
-hydroxyl) products mainly by the respective,
male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they
are primarily metabolized into the 5
-reduced products by
female-predominant 5
-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 16
-hydroxyprogesterone and
6
-hydroxyprogesterone inhibited the 5
-reductive
[3H]progesterone metabolism by adult male rat liver
microsomes without significantly inhibiting the CYP2C11 and
CYP3A2 activities producing themselves, whereas
3
-hydroxy-5
-pregnan-20-one and 5
-pregnane-3,20-dione not only
stimulated the 5
-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
-OH (in some cases, 2
-OH also) and
6
-OH products, mainly by the respective, male-specific cytochrome
P450 subforms (P450s), CYP2C11 and CYP3A2, whereas the female
metabolizes them primarily into the 5
-reduced products by
female-predominant 5
-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 5
-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).
-OH-P and 6
-OH-P, inhibited
female-predominant [3H]PROG 5
-reductase activity
without significantly inhibiting the CYP2C11 and CYP3A2
activities producing themselves, while 3
-OH-5
-P and 5
-P,
female-predominant products by the 5
-reductase, not only
stimulated this enzyme activity but also inhibited the male-specific
oxidative [3H]PROG metabolism.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-OH-P (88.22 ± 4.14%), [3H]6
-OH-P (75.39 ± 3.95%),
[3H]2
-OH-P (69.15 ± 5.11%),
[3H]17
-OH-P (53.74 ± 9.72%),
[3H]20
-OH-P (92.86 ± 2.92%),
[3H]5
-P (92.97 ± 6.83%) and
[3H]3
-OH-5
-P (97.45 ± 3.81%).
-OH-P,
exclusively formed by the supersomes, was 95.31 ± 3.81%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-OH-P and
5
-reductase-dependent metabolites,
[3H]5
-P and [3H]3
-OH-5
-P, were
formed in small amounts. However, the addition of 3.16 µM
NADPH (the third column), a common cofactor of
P450-dependent and 5
-reductase-dependent
metabolisms induced larger formations of the respective male-specific
CYP2C11- and CYP3A2-dependent oxidized metabolites,
[3H]16
-OH-P (rather than [3H]2
-OH-P)
and [3H]6
-OH-P, as compared with small increases of
the 5
-reduced metabolites. The mean ± S.D. values of these
products obtained from the 37 experimental batches were as follows:
[3H]16
-OH-P (7.37 ± 1.30 pmol/mg protein/5 min),
[3H]6
-OH-P (2.87 ± 0.62),
[3H]5
-P (4.85 ± 1.75), and
[3H]3
-OH-5
-P (1.35 ± 0.63). The ratio of
[3H]16
-OH-P to [3H]6
-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
5
-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]5
-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 5
-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).
[3H]Progesterone metabolism by adult male rat liver
microsomes
-OH-P and
[3H]6
-OH-P) and 5
-reductive (sum of formed
[3H]5
-P and [3H]3
-OH-5
-P)
metabolisms of [3H]PROG were examined (Fig.
1). Both PROG and 16
-OH-P effectively inhibited the formation of [3H]5
-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, 3
-OH-5
-P and
3
,11
-(OH)2-5
-P, compared with PROG, not only
showed stronger inhibitory effects on the oxidative metabolism but also
conversely stimulated the 5
-reductive metabolism.
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Fig. 1.
Dose-dependent effects of
representative unlabeled steroids on the oxidative and
5 -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]16
-OH-P and [3H]6
-OH-P,
and the 5
-reduced products (open symbols,
broken lines) are the sum of formed
[3H]5
-P and [3H]3
-OH-5
-P.
Unlabeled steroids used are as follows: progesterone (
,
),
16
-hydroxyprogesterone (
,
),
3
-hydroxy-5
-pregnan-20-one (
,
),
3
,11
-dihydroxy-5
-pregnan-20-one (
,
).
-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 5
-reductive
[3H]PROG metabolisms (Fig.
2 and Table I). The group A steroids such
as 3
-OH-5
-P and 5
-P showed inhibitory effects on the oxidative metabolism, while having stimulatory effects on the
5
-reductive metabolism producing themselves. The group B steroids,
PROG and TEST, inhibited both metabolisms as probably alternative
substrates. The group C steroids, 5
-A-17
-ol and 3
-OH-P, showed
inhibitory effects on the oxidative metabolism with no effect on the
5
-reductive metabolism, and conversely, the group D steroids,
COR and 11
-OH-P, showed stimulatory effects on the 5
-reductive
metabolism with no effect on the oxidative metabolism, despite
possessing a 3-keto-4-ene structure that might be catalyzed by the
5
-reductase. Other 3-keto-4-ene steroids (group E), 16
-OH-P and
6
-OH-P, inhibited only the 5
-reductive metabolism without the
product inhibition effects on the oxidative metabolism producing
themselves. It is noteworthy that 16
-OH-P, as well as 20
-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
5
-reductase activity. Finally, the group F steroids, 11
-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
5 -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 (
) or
5
-reduced (
) products is the same as shown in Fig. 1.
CHOL, cholesterol.
,11
-(OH)2-5
-P, a group A steroid, containing both
a 3
-OH-5
-reduced structure and a C-11
-OH structure, showed an
additively stimulatory effect on the 5
-reductive metabolism, as
compared with its parental steroids, 3
-OH-5
-P and 11
-OH-P, and
thus this steroid, although not actually produced in the liver, was the
highest stimulator of the 5
-reductase activity.
-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.
-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 16
-OH-P
inhibited the 5
-reductive metabolism, while 11
-OH-P,
3
-OH-5
-P, and 3
,11
-(OH)2-5
-P stimulated it,
as the intact microsomes did (see Fig. 2). This result clearly shows that the effects of these steroids on the 5
-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
( ) or 5
-reduced (
) products is the same as shown in Fig.
1.
-reductive metabolism. Although the mechanism inducing such a
tendency is wholly unclear at present, this may have been associated with lower stimulatory effects of 11
-OH-P, 3
-OH-5
-P, and
3
,11
-(OH)2-5
-P on the 5
-reductive metabolism by
the antiserum-treated microsomes, compared with the intact microsomes.
-oxidation and
16
-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]6
-OH-P (data not shown), and
the inhibitory pattern of unlabeled steroids on the
[3H]6
-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
6 - or 16
-oxidizing
activity by adult male rat liver microsomes. The formation of
[3H]6
-OH-P (
) or [3H]16
-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
6 -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]6
-OH-P was estimated to be
13.98 ± 1.80% (37.28 pmol/mg of protein/5 min) in the control
experiments.
-OH-5
-P against [3H]6
-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 3
-OH-5
-P was about 10-fold lower
than that of PROG, and this Ki value ratio agreed
well with the IC27.5 (against [3H]6
-OH-P
formation) and IC40 (against 16
-OH-P formation) value ratios obtained using rat liver microsomes (Table
II). Since not only unlabeled PROG but
also 3
-OH-5
-P (22) must be metabolized into 6
-OH- and/or
16
-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
3
-OH-5
-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
3 -OH-5
-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 3
-OH-5
-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]6
-OH-P formation,
pmol/mg of protein/5 min) in the presence of various concentrations of
unlabeled PROG or 3
-OH-5
-P. The Ki value is
given by the point of intersection of the respective
straight lines obtained using two
[3H]PROG concentrations.
The quantitative parameters of inhibitory effects of unlabeled
progesterone and 3-OH-5
-pregnan-20-one against oxidative
[3H]progesterone metabolism
-OH-P and 6
-OH-P, inhibited the female-predominant 5
-reductase activity without significantly inhibiting the
male-specific CYP2C11 and CYP3A2 activities producing themselves. On
the other hand, the female-predominant products, 5
-P and
3
-OH-5
-P, not only inhibited the male-specific P450 activities
but also stimulated the 5
-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
- (in some cases,
2
- also) and 6
-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
5
-reduced products by female-predominant 5
-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).
-OH-P and especially 16
-OH-P, strongly inhibited the female-predominant 5
-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, 5
-P and especially 3
-OH-5
-P not only inhibited both the CYP2C11 and CYP3A2 activities but also stimulated the 5
-reductase activity producing themselves. However, such adverse effects of the 5
-reduced products on the male pattern metabolism may
be attenuated by a scanty expression of the 5
-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 5
-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.
16 -OH-P and 6
-OH-P are produced by male-specific microsomal
P450s, CYP2C11 and CYP3A2, respectively. Both products inhibit PROG
5
-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, 16
-OH-P may make a higher contribution to the
inhibitory effect on the 5
-reductase activity than 6
-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, 5
-P and 3
-OH-5
-P are produced by the
5
-reductase and subsequently cytosolic (and, to a lesser degree,
microsomal) 3
-hydroxysteroid dehydrogenase (26-28), respectively.
Although these products, especially 3
-OH-5
-P, not only inhibit
the CYP2C11 and CYP3A2 activities but also stimulate the 5
-reductase
activity, such adverse effects of these 5
-reduced products on the
male pattern metabolism may be actually attenuated by lower expressions
of both the 5
-reductase (1, 3, 7, 8, 10, 18) and 3
-hydroxysteroid
dehydrogenase (27, 28) genes in the male, compared with the female.
16
-OH-P, 6
-OH-P, or 3
-OH-5
-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), 5
-reductase (~10-fold lower) (2,
3, 7), 3
-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 5-reductase activity (1, 3, 7, 8, 10, 18) may be
further enhanced by its products, 5
-P and especially 3
-OH-5
-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, 16
-OH-P and
6
-OH-P, while the female must metabolize it into more
hydrophobic products, 5
-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 5-reduced
androgens, especially TEST and 5
-A-3
,17
, on the
[3H]PROG metabolism showed a similar pattern to those of
various 4-pregnene and 5
-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
5-reductase activity metabolizing COR into its 5
-reduced products
(24). However, several recent studies have clearly shown that the two
11
-OH corticosteroids, COR and cortisol, are of the poorest
substrate group for 5
-reductases of various organs probably
including the adrenal cortex itself (19, 20, 25), and we showed in the
present study that COR and 11
-OH-P, but not 11
-OH-P, rather
stimulated [3H]PROG 5
-reductase activity of rat liver
microsomes (Fig. 2). These results suggest that the C-11
-OH group of
a steroid molecule may strongly disturb access of the steroid to the
active site of the 5
-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 5
-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.
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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;
5-A-3
, 17
, 5
-androstane-3
,17
-diol;
5
-A-17
-ol, 5
-androstan-17
-ol;
4-AN, 4-androstene-3,17-dione;
4-AN-CA, 4-androsten-3-one-17
-carboxylic
acid;
COR, corticosterone;
P450, cytochrome P450;
3
, 11
-(OH)2-5
-P,
3
,11
-dihydroxy-5
-pregnan-20-one;
GH, growth hormone;
5
-P, 5
-pregnane-3,20-dione;
3
-OH-5
-P, 3
-hydroxy-5
-pregnan-20-one;
TEST, testosterone;
X-OH-P, X-hydroxyprogesterone;
ACTH, adrenocorticotropic hormone.
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