| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104-6048
Address all correspondence and requests for reprints to: Dr. Bernard H. Shapiro, Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, Pennsylvania 19104-6048. E-mail: shapirob{at}vet.upenn.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase (type 1). The gender-dependent
expression of the P450s can be reversed by exposing male rats to the
continuous feminine plasma GH profile and females to the episodic
masculine GH profile. Under these conditions, females will now express
the male-specific isoforms and suppress the female-dependent forms,
whereas the opposite will occur in the males. Nevertheless, it is not
clear whether the levels of expression or suppression are comparable in
male and female rats exposed to the same sex-dependent GH profiles. In
the present study, we have renaturalized the circulating feminine GH
profile in euthyroid-maintained, hypophysectomized female and male rats
at six concentrations ranging from 3–100% of normal. Continuous
monitoring of GH levels revealed indistinguishable plasma profiles in
females and males at each dosage administered. In the case of females,
restoration of the feminine-like plasma GH profile at a concentration
that was 3% of the normal level restored expression levels
(i.e. mRNA, protein, and/or catalytic activity) of
female-dependent CYP2C12, 2A1, and 5-reductase to 50% or greater of
normal and fully suppressed expression of male-specific CYP2A2, 2C11,
and 3A2. Twice the dosage of the hormone (6% of normal) was required
to restore female-predominant CYP2C7 to 50% of normal in
hypophysectomized female rats. In contrast, we found that all of the
measured isoforms were significantly less responsive to the inductive
and suppressive effects of the feminine-like GH profile when
administered to male rats. While suppression of the male-specific
isoforms (i.e. CYP2A2, 2C11, and 3A2) in male rats
required concentrations of GH in the feminine profile 2–3 times
greater than were effective in female rats, no dosage of the hormone
was as effective in inducing female-dependent P450s
(i.e. CYP2A1, 2C7, and 2C12) in males as in females.
Clearly, the continuous feminine GH profile was more effective at
inducing and suppressing gender-dependent isoforms of hepatic P450
when restored to female rats, where it is normally secreted, than in
males. As GH profiles appear to be the sole factor responsible for
regulating the sexually dimorphic expression of hepatic P450 isoforms
in adult rats, the differential responsiveness of male and female rats
to the feminine GH profile are likely to be inherently induced by
irreversible imprinting during a critical developmental period. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
200–300 ng/ml plasma) every 3.5–4 h.
Between the peaks, GH levels are undetectable. In female rats, the
hormone pulses are more frequent and irregular and are of lower
magnitude than those in males, whereas the interpulse concentrations of
GH are always measurable (2, 3).
In the rat, P450 responses to GH regulation are almost as variable as
the number of GH-dependent isoforms. That is, expression of the major
female-specific3 CYP2C12 (as
well as the non-P450 5-reductase) is dependent on the feminine
profile of continuous GH secretion. Exposure to the masculine profile
of episodic GH release as well as the absence of the hormone from the
circulation (e.g. hypophysectomy) results in the complete
suppression of CYP2C12 (6, 7, 8, 9). In a somewhat similar vein,
female-predominant CYP2C7 expression is also dependent on the feminine
GH profile and is completely suppressed in the hypophysectomized rat.
However, exposure to the masculine profile allows expression of CYP2C7
at 25–40% normal female levels (3, 8, 10, 11). Expression of the
major male-specific CYP2C11 requires the episodic on/off masculine
profile of GH secretion. Although the feminine pattern of continuous GH
secretion blocks CYP2C11 expression, total GH depletion from the
circulation allows CYP2C11 expression at 15–25% of intact male levels
(3, 9, 12, 13). After hypophysectomy, female-predominant CYP2A1
(female/male, 3:1) concentrations decline, but remain above male
levels and are restored to intact female-like levels with continuously
administered GH (14, 15). Although the expression levels of CYP2C7,
CYP2C11, CYP2C12, and CYP2A1 are greatest when exposed to their
gender-dependent GH profiles, other isoforms are optimally expressed in
the absence of GH. Male-specific CYP2A2 and CYP3A2 are maximally
expressed in the hypophysectomized rat and disappear when GH is
secreted constantly, but are only partially suppressed, relative to the
high levels observed in hypophy-sectomized rats, under the
influence of episodic GH (14, 16, 17). Although there are additional
examples demonstrating that the expression or suppression of each
isoform of P450 is likely to be regulated by a different signal in the
sexually dimorphic GH profile, it is clear that GH per se is
the activational factor responsible for gender differences in P450
expression. Whether these gender differences in P450 levels are also
dependent upon imprinting is not as clear. Several studies have
examined sexually dimorphic imprinting of P450 isoforms, but their
results are not easily interpreted. Although these studies all
concluded that neonatal testosterone imprinting of the differentiating
liver was responsible for the sexually dimorphic expression of several
P450 isoforms, the permanence or irreversibility of the imprinting was
either not established (18, 19, 20) or was tested by administering
testosterone and not GH to neonatally castrated adult rats (11, 21, 22). The presence of inherent, possibly imprinted, sexual dimorphisms
in rat hepatic P450s is examined here by comparing the effects of the
renaturalized feminine-like plasma GH profile, restored at six
concentrations ranging from 3–100% of normal, in regulating the
expression of several male- and female-dependent isoforms of P450 in
hypophysectomized, euthyroid-maintained male and female rats.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Repetitive blood samples (10 µl) were obtained at 15-min intervals from unrestrained, unstressed, and completely conscious rats outfitted with our mobile catheterization apparatus (23, 26). Six-hour plasma GH profiles were determined using a RIA with a sensitivity of 2–3 ng/ml. Procedural details and statistical validation of the assay have been reported previously (27).
RNA analysis
Total hepatic RNA was isolated by using a single step guanidium
thiocyanate method (28). Ten micrograms of RNA was electrophoresed
under formaldehyde-denaturing conditions on 1% agarose and transferred
to GeneScreen nylon membranes (DuPont-New England Nuclear,
Boston, MA). The Northern blots were probed and reprobed with either
32P-labeled oligonucleotide probes or CYP2C11/complementary
DNA (cDNA) (29) probes, using hybridization and high stringency washing
conditions as described previously (30). The nucleotide sequence of
oligonucleotide probes for CYP2A1, CYP2A2, CYP2C7, CYP2C12 (30), CYP3A2
(31), and type 1 steroid 5-reductase (8) have been reported. We used
antisense oligonucleotide sequence
5'-CTC-AGC-ATC-TGG-AGC-GGT-ATC-TGC-3' to identify GH receptor (GHR)
messenger RNA. This probe is complementary to the cDNA nucleotides
1934–1957 bp of GHR (GenBank accession no. J04811) (32) and does not
recognize GH-binding protein (GHBP) messenger RNA (mRNA). To identify
GHBP, we used antisense sequence
5'-GTT-GTC-AAT-CTC-TTG-ATG-TGG-GTG-CTG-3' complementary to the splice
variant cDNA nucleotides 995-1021 bp (GenBank accession no. S49003)
encoding the GHBP hydrophilic tail (32), which does not recognize GHR
mRNA (33). Insulin-like growth factor (IGF-I) mRNA was detected using a
rat antisense oligonucleotide probe
(5'-ATA-GCC-TGT-GGG-CTT-GTT-GAA-GTA-AAA-GCC-3') complementary to the
22–31 amino acid residues from B and C domains, respectively (34). The
consistency of RNA loadings between samples was confirmed by ethidium
bromide staining of 18S and 28S ribosomal RNAs and was verified using
an 18S oligonucleotide probe (35). The hybridized mRNA signals were
quantified by scanning the autoradiographs and were normalized to the
18S ribosomal RNA signals in each lane.
Western blots
Hepatic microsomes were prepared from individual rat livers (36)
and then assayed for individual P450s by Western blotting and/or by
measurement of their selective catalytic activities (30, 37). Briefly,
10 µg microsomal protein were electrophoresed on 0.75-mm-thick
SDS-polyacrylamide (7.5%) gels and electroblotted onto nitrocellulose
filters. The blots were probed with monoclonal antirat CYP2C11 (Oxford
Biomedical Research, Oxford, MI) and antirat CYP2C12/13 (provided by
Dr. Marika Rönnholm, Huddinge University Hospital, Huddinge,
Sweden) mouse IgG, polyclonal antirat CYP2C7 (provided by Dr. Stelvio
M. Bandiera, University of British Columbia, Vancouver, Canada),
and antirat CYP3A1/2 (Human Biologics, Phoenix, AZ) rabbit IgG and were
detected with an enhanced chemiluminescence kit (Amersham,
Arlington Heights, IL) (38).
Testosterone metabolism
Testosterone 2- and 6ß-hydroxylases, reflective of the
activity levels of CYP2C11 and CYP3A2 proteins, respectively, and
female-specific testosterone 5-reductase were assayed according to
our methods, as described previously (39).
Statistics
All data were subjected to ANOVA, and differences were
determined with t statistics and the Bonferroni procedure
for multiple comparison.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
50–100
ng/ml plasma) and brief interpulse periods of about 10–20 ng/ml. In
contrast, male rats secreted GH in episodic bursts (200–250 ng/ml)
every 3–4 h separated by prolonged interpulses containing no
detectable hormone levels (Fig. 1
).
|
]. There was a proportional relationship between the
dose of GH replacement by ip implanted osmotic minipumps and the
resulting mean plasma concentration of rGH. That is, when the dose of
administered rGH was reduced from 20 to 10 µg rGH/h·kg BW, the mean
plasma concentration of the hormone declined to about 50% of normal
[18.6 ± 2.8 ng/ml (females) and 19.1 ± 3.8 ng/ml
(males)]. Another reduction in GH replacement to 5 µg rGH/h·kg BW
produced circulating hormone levels that were approximately 25% of
normal [8.7 ± 2.2 ng/ml (females) and 9.1 ± 2.2 ng/ml
(males)]. A further 50% reduction in the rate of GH administration to
2.5 µg rGH/h·kg BW resulted in a decline of plasma rGH to about
12% of normal [3.9 ± 0.7 ng/ml (females) and 4.0 ± 0.7
ng/ml (males)]. GH replacement at rates of 1.25 and 0.625 µg
rGH/h·kg BW produced circulating GH concentrations below the
statistical sensitivity of the assay. However, because the higher rates
of GH replacement resulted in proportional and predictable plasma
hormone levels, we thought it reasonable to extrapolate (by linear
regression) circulating rGH concentrations for the hypophysectomized
rats with osmotic pumps secreting 1.25 and 0.625 µg rGH/h·kg BW to
approximately 6% [2.0 ± 0.4 ng/ml (females and males)] and
about 3% [1.0 ± 0.2 ng/ml (females and males)] of normal
circulating concentrations, respectively (Fig. 1).
The gender of the animal had no effect on the resulting plasma levels
of the hormone. That is, each dose of administered rGH produced
indistinguishable circulating concentrations and profiles of the
hormone in female and male hypophysectomized rats (Fig. 1).
Hepatic CYP2C12
The female specificity of CYP2C12 was illustrated by its
expression in intact female liver and its absence in male liver (Fig. 2). Furthermore, with the disappearance
of the feminine pattern of continuous GH secretion in the
hypophysectomized female rat, expression of the isoform was no longer
detectable. (CYP2C12 remained undetectable in the
hypophy-sectomized male rats.) Restoration to females of only 3%
of the levels characteristic of the feminine profile of GH secretion
was capable of restoring CYP2C12 expression (i.e. mRNA and
protein) to approximately 45% of normal. In contrast, 3% of the
normal GH profile was considerably less effective in hypophysectomized
males, inducing only half the amount of CYP2C12 mRNA and less than 10%
of the protein observed in similarly treated females. When plasma GH
concentrations in hypophysectomized females were increased to about 6%
of normal, expression levels of CYP2C12 were elevated by an additional
30% to about 75% of normal. Restoration of the continuous GH
secretory profile to 12–25% of female-like levels was sufficient to
fully restore expression levels of CYP2C12 mRNA and protein in
hypophysectomized female rats. In contrast, reproducing the feminine
hormonal profile from 6–50% of normal in hypophysectomized males
induced no more than 50% of the CYP2C12 mRNA and protein levels found
in intact females. It was only when the physiological (100%) feminine
GH profile was replicated in the males that CYP2C12 mRNA and protein
approached (although still less than) normal female levels (Fig.
2).
|
-reductase-reductase (type 1) are comparable to those of
CYP2C12 (30, 40). This similarity was illustrated by a near lack of
expression of 5-reductase mRNA and dependent testosterone
5-reductase activity in liver from intact male and hypophysectomized
female rats (Fig. 3). Exhibiting an even
greater hormone sensitivity than CYP2C12, 5-reductase mRNA and its
catalytic activity were restored in hypophysectomized females to 75%
and 50% of normal, respectively, by only 3% of the mean concentration
of the female-like circulating GH profile. An increase in plasma GH
levels to approximately 6% of normal further elevated 5-reductase
expression, and 12% and above of normal GH concentrations seemed to
nearly fully restore hepatic expression of the reductase in
hypophysectomized female rats. Similar to the findings with CYP2C12
(Fig. 2), but exhibiting less dramatic gender differences,
administration of feminine GH profiles at 3–50% of normal was
significantly less effective in inducing 5-reductase expression in
hypophy-sectomized males than females (Fig. 3). Only at the 100%
replacement dose were 5-reductase and associated testosterone
5-reductase activity levels indistinguishable in both sexes and
nearly equal to intact female-like levels.
|
). Hypophysectomy reduced CYP2C7
expression in female and male livers to undetectable levels. In
comparison to female-specific CYP2C12 and 5-reductase, CYP2C7 seemed
to be less sensitive to GH regulation. Restoration of 3% of the GH
levels characteristic of the feminine plasma profile increased hepatic
CYP2C7 mRNA and protein in hypophysectomized females to 20% of normal,
which was below the expression levels found in intact males. Although
increases in the circulating concentrations of rGH produced
commensurate increases in CYP2C7 mRNA and protein, it was not until
hormone levels in hypophysectomized females were 50–100% of normal
that CYP2C7 expression was similar to intact levels. In dramatic
contrast to that observed in the treated females, CYP2C7 expression in
the hypophysectomized males was only minimally responsive to the
feminine pattern of GH replacement. Regardless of the dose of rGH
administered, including 100% of normal, induction concentrations of
CYP2C7 mRNA and protein never surpassed those found in intact males and
remained only 20% of normal female levels (Fig. 4).
|
-hydroxylase5) and found
virtually no expression in intact female rats (Fig. 5). Expression levels in
hypophysectomized rats of both genders were approximately 25% of the
intact male level. All dosages of osmotic pump-delivered GH were
equally effective as the lowest dosage (0.625 µg rGH/h·kg BW) in
completely blocking CYP2C11 expression in hypophysectomized female
rats. Compared with the females, however, the continuous feminine GH
secretory pattern was not as effective in blocking CYP2C11 expression
in hypophysectomized males (Fig. 5). At the lowest 3% rGH replacement
dose, measurable concentrations of CYP2C11 mRNA, protein, and catalytic
activity remained evident in the hypophysectomized males. Although
transcript levels of the isoform were undetectable when the males were
treated with rGH at doses of 6% and above, there remained a
persistent, albeit minimal, expression of the protein and its activity
at all rGH doses, suggesting some enhanced stability of the
isoform.
|
). The complete elimination of
circulating GH by hypophy-sectomy resulted in an overexpression of
CYP3A2 mRNA and protein in both sexes. Restorations of the feminine
secretory GH profile at 3%, 6%, 12%, 25%, 50%, and 100% of
normal were equally and completely effective in suppressing CYP3A2
expression in hypophysectomized females. CYP3A2 expression in
hypophysectomized males, on the other hand, was less responsive to the
inhibitory effects of the continuous GH profile (Fig. 6). Whereas 3%
of the normal feminine GH profile completely blocked CYP3A2 expression
in hypophysectomized females, similarly treated males continued to
express 40–50% normal CYP3A2 mRNA, protein, and activity levels.
Moreover, at the 6% replacement dose, expression levels of the isoform
in hypophysectomized males remained at 15–20% of normal. Only when
the dose of rGH was administered at 12% and above was CYP3A2
expression fully blocked in hypophysectomized males.
|
). Although as little as 3% of the normal
feminine GH profile completely blocked CYP2A2 expression in
hypophysectomized females, even twice that dose of rGH (i.e.
6%) allowed for the expression of about 40% of normal transcript
levels in the hypophysectomized males.
CYP2A1 is a female-predominant isoform whose mRNA levels were
severalfold higher in liver from intact females than in liver from
intact males (Fig. 7). Hypophysectomy
reduced CYP2A1 mRNA in female rat liver to concentrations intermediate
between those in intact males and intact females, but had no effect on
the characteristically low levels expressed in males. Restoration from
3–6% of the normal feminine plasma GH concentrations seemed to fully
restore female-like expression levels of CYP2A1 mRNA in
hypophy-sectomized females. In contrast, none of the administered
rGH doses, from 3–100%, elevated CYP2A1 transcript levels in
hypophysectomized males to female-like levels (Fig. 7).
|
male), and hypophysectomy, with or
without GH replacement, had minimal effects on the expression levels of
the transcripts in either sex (data not presented). In contrast,
hepatic concentrations of IGF-I mRNA were clearly greater in intact
males than in intact females, although GH-dependent expression by the
feminine profile was similar in both sexes (data not presented). That
is, whereas IGF-I mRNA was nearly undetectable after hypophysectomy,
restoration of the feminine circulating GH profile at 3% and 6% of
normal was only slightly effective in restoring IGF-I expression in
hypophysectomized females and males. In fact, plasma GH had to be
restored to 25% of normal to induce female-like concentrations (which
were still considerably below those observed in males) of IGF-I mRNA in
both sexes. [The similar concentrations of 18S ribosomal RNA observed
for all treatment groups (Fig. 7) verifies the high consistency and
integrity of RNA loading on the Northern blots.] |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-reductase required about 12–25% physiological
hormone levels,6 and CYP2C7
mRNA required 50% of normal GH levels to reach female-like
concentrations (while CYP2C7 and 5-reductase proteins remained
somewhat below normal at all GH concentrations).
In previous studies using hypophysectomized female rats, administration
of human GH (without monitoring resulting plasma profiles) at
concentrations reflecting physiological or higher levels (as based upon
our findings presented in Fig. 1) increased CYP2C12 protein to 40–60%
of normal (6, 7) and mRNA to approximately 100% of normal (9),
5-reductase mRNA and its catalytic activity to about 70% of normal
(7, 8), CYP2C7 mRNA to 60–100% of normal (8, 10), and CYP2A1 mRNA to
near normal (7, 14, 15). Although in many of these earlier studies
hepatic enzyme concentrations were not restored to prehypophysectomy
levels,7 they did establish
the importance of the continuous feminine GH profile in directing
CYP2C12, CYP2C7, CYP2A1, and 5-reductase expression. Our
observations extend these studies and indicate that the signaling
concentrations in the feminine plasma GH profile regulating expression
of the female-dependent P450 isoforms and 5-reductase are remarkably
below physiological concentrations.
Although subnormal concentrations of circulating GH in females restored
normal levels of female-dependent CYP2C12, CYP2C7, CYP2A1, and
5-reductase, it is clear that the male-specific isoforms were more
sensitive to the suppressive effects of the hormone. Our observation
that as little as 3% of the circulating feminine GH profile could
completely suppress CYP2C11, CYP2A2, and CYP3A2 expression in female
rats illustrates the profound sensitivity of these male-specific P450
gene products to the inhibitory effects of the continuous feminine GH
profile. Previous studies in which human GH was infused (via sc placed
osmotic pumps) at a rate about 35-fold greater than our rate of 0.625
µg rGH/h·kg BW demonstrated equally effective suppression of
CYP2C11, CYP2A2, and CYP3A2 expression in hypophysectomized female rats
(3, 9, 12, 14). Accordingly, our findings indicate that the suppression
of P450s is more sensitive to GH regulation than is P450 expression.
Understandably, in suppression, one needs to interrupt only one step in
the expression mechanism, whereas induction of expression requires the
harmonious activation of all steps in the sequence.
The effectiveness of low circulating GH concentrations in feminizing
P450 expression may be explained by the high affinity of the GH
receptor for the hormone (Kd = 10-10
M) (42), which corresponds to half-maximal saturation of
the membrane receptor at a plasma GH concentration of 2 ng/ml or only
6% of the normal feminine level. Restoration of circulating GH levels
to hypophysectomized female rats at 6% of the physiological
concentration very effectively initiates expression of female-dependent
CYP2C12, CYP2A1, and 5-reductase to levels approaching, if not
reaching, normal. However, the suppressive effects of GH on
male-specific CYP2C11, CYP2A2, and CYP3A2 occur at what might be
considered nominal plasma concentrations. Because 3% of the
physiological GH concentration was so completely effective in blocking
CYP2C11, CYP2A2, and CYP3A2 in hypophysectomized females, it is not
unreasonable to speculate that even half of this concentration could be
effective. Thus, plasma concentrations of GH continuously binding 10%
or less of the GH receptor may be sufficient to signal the suppression
of the male-specific isoforms in females.
In agreement with earlier reports (3, 43, 44), we observed no gender- or GH-dependent effects on GHR and GHBP mRNAs, whose expression may be GH regulated by posttranscriptional events (3, 45). As expected (43, 46), hepatic IGF-I mRNA levels were sexually dimorphic (male > female), declined to very low concentrations after hypophysectomy, and were restored to normal female-like expression levels with the continuous administration of GH. Although we found that 5 µg rGH/h·kg BW (25% of normal) could restore female-like levels of IGF-I mRNA in both sexes, the continuous administration of 10 times this amount of an equivalently active bovine GH preparation was similarly effective in restoring hepatic IGF-I in female hypophysectomized rats (46).
In contrast to the gender-independent regulation of IGF-I mRNA by the
feminine GH profile, we observed a dramatic sexual dimorphism in
expression levels of hepatic CYP2A1, 2A2, 2C7, 2C11, 2C12, 3A2, and
testosterone 5-reductase when exposed to the same continuous
feminine GH profiles. Induction levels of those enzymes dependent upon
the feminine GH profile for expression (e.g. CYP2C7, 2C12,
and 5-reductase) or at least for full expression (CYP2A1) were
significantly greater in female livers exposed to the same feminine
hormone profiles as male livers. In agreement, a single,
physiologic-like dose of human GH administered by osmotic minipumps to
hypophysectomized rats induced twice the amount of CYP2C12 mRNA (9) and
3 times the amount of CYP2C7 mRNA (10) in female livers compared with
male livers. In the case of the male-specific isoforms of P450, whether
dependent upon the masculine episodic GH profile for expression
(e.g. CYP2C11) or effectively expressed in the absence of GH
(e.g. CYP2A2 and 3A2), all were completely suppressed by the
feminine continuous GH profile (see introduction). In this regard, the
male-specific isoforms in liver from female rats were significantly
more responsive to the suppressive effects of the same feminine GH
profiles administered to male rats. Actually, it was at the lowest
hormone replacement concentrations (i.e. 3% and 6%) that
we observed the greatest gender differences. By the time the feminine
GH profile was replaced at 25% of the normal level, suppression of the
male-specific isoforms was fairly complete in both sexes. In fact, it
is this latter observation that may partially explain the lack of
attention directed toward studies examining the sexually dimorphic
responsiveness of P450 isoforms to GH regulation. [This is in contrast
to the many reports describing gender differences in individual P450
expression (2, 3, 4, 5).] The preponderance of studies investigating the
effects of GH regulation on P450 expression administer a single,
maximally effective (usually physiological or supraphysiological) dose
of human or bovine GH to hypophysectomized animals. In light of the
dramatic changes produced by GH per se in either sex, the
smaller, sexually dimorphic responses are generally overlooked. Of
course, the additional fact that the vast majority of P450 studies are
limited to a single sex obviously precludes the possibility of
identifying sexual dimorphisms.
In agreement with our earlier reports demonstrating phenobarbital- and
GH-independent, pre- and posttranscriptional gender differences
regulating CYP2B1 and 2B2 expression (47, 48) it seems reasonable to
conclude that rat hepatic P450 isoforms can exhibit sexually dimorphic
responses to the same inducers. This sexual dimorphism could be
explained by the existence of some unidentified hormone or factor
limited to one sex that modifies that gender’s response to GH.
Although such open ended possibilities are difficult to completely
resolve, there is considerable contradictory evidence. With the
possible exception of thyroid hormone (25, 31), which was replaced at
euthyroid levels, GH, secreted in sexually dimorphic profiles, is the
sole endogenous factor regulating expression of the gender-dependent
isoforms examined in this study (2, 3, 4, 9, 10, 40). This conclusion is
supported in the present report by our finding that expressions of
female-dependent CYP2C7 and 2C12 and 5-reductase were completely
suppressed in the hypophysectomized rat, whereas replacement of the
feminine GH profile alone completely restored expression of the enzymes
in females. Moreover, we have shown that male-specific isoforms of P450
were completely restored to normal male-like levels in
hypophysectomized male rats by restoration of the masculine episodic
plasma GH profile without any other hormones or factors (49, 50).
What seems to be a more reasonable explanation is that GH response elements in male and female hepatocytes (e.g. GH receptor, signal transduction pathways, and nuclear binding sites) express different sensitivities to the hormone profiles. Clearly, a severalfold greater number of GH receptors must be occupied in male livers to produce the same effects on P450 expression as observed in female livers exposed to the same feminine GH profile. Accordingly, if we assume that male and female hepatocytes employ the same GH-dependent mechanisms to regulate, for example, CYP2C12 expression or CYP2C11 suppression, one or more of these mechanistic steps in male hepatocytes may be less responsive to the feminine hormone profile. As the gender differences in the expression levels of the P450s are GH independent, it seems reasonable to conclude, based upon earlier findings (11, 21, 22), that the sexually dimorphic responsiveness of the hepatic isoforms of P450 are inherently expressed due to irreversible imprinting at a critical developmental period.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Present address: Scripps Research Institute, La Jolla,
California 29087.
3 The terms sex dependent, sex predominant or
dominant, and sex specific are often used indiscriminately. We use sex
or gender dependent to imply that expression levels are dependent upon
the existence of gender; sex or gender predominant indicates that
expression levels, regardless of magnitude, are consistently great in
one gender; and sex or gender specific implies that expression is
basically restricted to only one gender.
4 Despite the absence of detectable pituitary
tissue examined by dissecting scope at necropsy, 30–40% of the
hypophysectomized females exhibited inappropriate body weight gain at
2–3 weeks after surgery, necessitating their exclusion from the study.
In contrast, we found that less than 15% of male rats show any body
weight gain after hypophysectomy (personal observations).
5 Less specific testosterone 16-hydroxylase
activity was in agreement with testosterone 2-hydroxylase activity
(data not presented).
6 Interestingly, as hypophysectomy completely
blocks hepatic CYP2C12 and testosterone 5-reductase expression, and
restoration of as little as 3% of the normal feminine GH profile
restores the transcript and protein concentrations to 50% or greater,
it appears that CYP2C12 and 5-reductase levels are considerably more
sensitive markers than body weight gain (40 ) in evaluating GH ablation
in female rats.
7 This was possibly a result of the use of bovine
and human GH instead of rat GH, which are not necessarily all equally
effective (6 14 41 ), and the probably inconsistent absorption
kinetics of GH when administered via sc rather than ip implanted
osmotic pumps (23 ).
Received August 20, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxylase,
bile acid 6ß-hydroxylase, and growth hormone-responsive steroid
hormone hydroxlase. J Steroid Biochem Mol Biol 43:1055–1072[CrossRef]
-reductase and comparison to
the thyroid dependence of two growth hormone-regulated CYP2C mRNAs.
J Biol Chem 265:19223–19229-hydroxylase cytochrome P-450 apoprotein in the
rat. J Biol Chem 260:11895–11898-hydroxylase, P-450a (P450IIA), by thyroid hormone and growth
hormone in rat liver. J Biochem 108:599–603
4-steroid
5-reductase, flavin-containing monooxygenase, and sex-specific
cytochromes P-450. J Biol Chem 261:10728–10735-hydroxylase
(P-45016) in male rat liver. Acta Endocrinol (Copenh) 118:314–320This article has been cited by other articles:
 |
|
 |
|
  D. J. Waxman and M. G. Holloway Sex Differences in the Expression of Hepatic Drug Metabolizing Enzymes Mol. Pharmacol., August 1, 2009; 76(2): 215 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Thangavel and B. H. Shapiro Inherent Sexually Dimorphic Expression of Hepatic CYP2C12 Correlated with Repressed Activation of Growth Hormone-Regulated Signal Transduction in Male Rats Drug Metab. Dispos., September 1, 2008; 36(9): 1884 - 1895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Wauthier and D. J. Waxman Sex-Specific Early Growth Hormone Response Genes in Rat Liver Mol. Endocrinol., August 1, 2008; 22(8): 1962 - 1974. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Dhir, C. Thangavel, and B. H. Shapiro Attenuated Expression of Episodic Growth Hormone-Induced CYP2C11 in Female Rats Associated with Suboptimal Activation of the Jak2/Stat5B and Other Modulating Signaling Pathways Drug Metab. Dispos., November 1, 2007; 35(11): 2102 - 2110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lin, J. Tang, X. Q. Liu, Y. Jiang, and J. Zheng Deacetylclivorine: A Gender-Selective Metabolite of Clivorine Formed in Female Sprague-Dawley Rat Liver Microsomes Drug Metab. Dispos., April 1, 2007; 35(4): 607 - 613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Thangavel, W. Dworakowski, and B. H. Shapiro INDUCIBILITY OF MALE-SPECIFIC ISOFORMS OF CYTOCHROME P450 BY SEX-DEPENDENT GROWTH HORMONE PROFILES IN HEPATOCYTE CULTURES FROM MALE BUT NOT FEMALE RATS Drug Metab. Dispos., March 1, 2006; 34(3): 410 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. N. Dhir, W. Dworakowski, C. Thangavel, and B. H. Shapiro Sexually Dimorphic Regulation of Hepatic Isoforms of Human Cytochrome P450 by Growth Hormone J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 87 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Wiwi and D. J. Waxman Role of Hepatocyte Nuclear Factors in Transcriptional Regulation of Male-specific CYP2A2 J. Biol. Chem., February 4, 2005; 280(5): 3259 - 3268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nisar, C. S. Lane, A. F. Wilderspin, K. J. Welham, W. J. Griffiths, and L. H. Patterson A PROTEOMIC APPROACH TO THE IDENTIFICATION OF CYTOCHROME P450 ISOFORMS IN MALE AND FEMALE RAT LIVER BY NANOSCALE LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION-TANDEM MASS SPECTROMETRY Drug Metab. Dispos., April 1, 2004; 32(4): 382 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. N. Dhir and B. H. Shapiro Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats PNAS, December 9, 2003; 100(25): 15224 - 15228. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Agrawal and B. H. Shapiro Constitutive and Inducible Hepatic Cytochrome P450 Isoforms in Senescent Male and Female Rats and Response to Low-Dose Phenobarbital Drug Metab. Dispos., May 1, 2003; 31(5): 612 - 619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kalsotra, S. Anakk, C. L. Boehme, and H. W. Strobel Sexual Dimorphism and Tissue Specificity in the Expression of CYP4F Forms in Sprague Dawley Rats Drug Metab. Dispos., September 1, 2002; 30(9): 1022 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kaufhold, P. K. Nigam, R. N. Dhir, and B. H. Shapiro Prevention of Latently Expressed CYP2C11, CYP3A2, and Growth Hormone Defects in Neonatally Monosodium Glutamate-Treated Male Rats by the N-Methyl-D-Aspartate Receptor Antagonist Dizocilpine Maleate J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 490 - 496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Dhir, W. Dworakowski, and B. H. Shapiro Middle-Age Alterations in the Sexually Dimorphic Plasma Growth Hormone Profiles: Involvement of Growth Hormone-Releasing Factor and Effects on Cytochrome P450 Expression Drug Metab. Dispos., February 1, 2002; 30(2): 141 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Garcia, C. Thangavel, and B. H. Shapiro Epidermal Growth Factor Regulation of Female-Dependent CYP2A1 and CYP2C12 in Primary Rat Hepatocyte Culture Drug Metab. Dispos., February 1, 2001; 29(2): 111 - 120. [Abstract] [Full Text]
|
|
|
|
|
|
N. A. Pampori, A. K. Agrawal, and B. H. Shapiro Infusion of Gender-Dependent Plasma Growth Hormone Profiles Into Intact Rats: Effects of Subcutaneous, Intraperitoneal, and Intravenous Routes of Rat and Human Growth Hormone on Endogenous Circulating Growth Hormone Profiles and Expression of Sexually Dimorphic Hepatic Cyp Isoforms Drug Metab. Dispos., January 1, 2001; 29(1): 8 - 16. [Abstract] [Full Text]
|
|
|
|
 |
|
 N. A. Pampori and B. H. Shapiro Nominal Growth Hormone Pulses in Otherwise Normal Masculine Plasma Profiles Induce Intron Retention of Overexpressed Hepatic CYP2C11 with Associated Nuclear Splicing Deficiency Endocrinology, November 1, 2000; 141(11): 4100 - 4106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Agrawal and B. H. Shapiro Latent Overexpression of Hepatic CYP2C7 in Adult Male and Female Rats Neonatally Exposed to Phenobarbital: A Developmental Profile of Gender-Dependent P450s J. Pharmacol. Exp. Ther., June 1, 2000; 293(3): 1027 - 1033. [Abstract] [Full Text]
|
|
|
|
|
|
A. Yamada, M. Yamada, Y. Fujita, T. Nishigami, K. Nakasho, and K. Uematsu Self-augmentation Effect of Male-specific Products on Sexually Differentiated Progesterone Metabolism in Adult Male Rat Liver Microsomes J. Biol. Chem., February 9, 2001; 276(7): 4604 - 4610. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, below the sensitivity of the RIA.
Estimates were extrapolated from linear regression analysis of the
measurable values. Similar findings were obtained from two or three
additional animals in each treatment group. (Note the different scales
on the y-axes.)
