help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pampori, N. A.
Right arrow Articles by Shapiro, B. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pampori, N. A.
Right arrow Articles by Shapiro, B. H.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Endocrinology Vol. 140, No. 3 1245-1254
Copyright © 1999 by The Endocrine Society

ARTICLES

Gender Differences in the Responsiveness of the Sex-Dependent Isoforms of Hepatic P450 to the Feminine Plasma Growth Hormone Profile1

Nisar A. Pampori2 and Bernard H. Shapiro

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
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most of the constitutive hepatic P450 isoforms expressed in the rat exhibit dramatic gender differences. Whereas only male hepatocytes contain CYP2A2, 2C11, and 3A2, only female hepatocytes express CYP2C12 and 3- to 4-fold greater levels of CYP2C7. This sexually dimorphic expression of hepatic P450 isoforms is regulated by the gender-dependent secretory GH profiles, i.e. episodic in males and continuous in females. In the case of the feminine GH profile, the continuous presence of the hormone in the circulation completely suppresses male-specific CYP2A2, 2C11, and 3A2, while stimulating full expression of female-dependent CYP2A1, 2C7, 2C12, and non-P450 testosterone 5{alpha}-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{alpha}-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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEXUAL DIMORPHISMS are encountered at every level of biological investigation, i.e. at the behavioral, anatomic, physiological, biochemical, and molecular. In fact, many of the dimorphisms have become definitions for gender, e.g. sexual differences in genitalia, reproductive behaviors, and hormone levels. Sexual dimorphisms are produced by either imprinting or activational factors, or very often by a combination of the two. Imprinting, which can only occur at a species-specific critical developmental period, results in an irreversible dimorphism. In contrast, sexual dimorphisms established by activational events are independent of critical times and are reversible; they are capable of being induced multiple times during the animal’s life (1). Although many of the imprinting and activational events determining anatomic and behavioral sexual dimorphisms have been defined, considerably less is known about factors regulating gender differences expressed at the molecular level. In this regard, gender differences in hepatic drug metabolism occur in numerous species, including fish, birds, and mammals. From the few species in which studies have been extended to the molecular level, it seems that sexual dimorphisms in drug metabolism are due to the existence of multiple forms of hepatic cytochrome P450 (P450; CYP) whose gender-dependent expression is regulated by GH (2). Rat liver, which has received the preponderance of investigational attention, is known to contain at least a dozen sex-dependent isoforms of P450 that are regulated by the gender-dependent profiles of circulating GH (3, 4, 5). Male rats secrete GH in episodic bursts (~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{alpha}-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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animals were housed in the University of Pennsylvania Laboratory Animal Resources facility under the supervision of certified animal medicine veterinarians and were treated according to a research protocol approved by the university’s institutional animal care and use committee. Rats [Crl:CD(SD)BR] were hypophysectomized by the vendor (Charles River Laboratories, Inc., Wilmington, MA) at 8 weeks of age and were observed in our facilities for 5 weeks. The effectiveness of the surgery was verified by the lack of weight gain over this period and the absence of pituitaries or fragments at necropsy at the end of the study (i.e. 102–107 days of age).4 Hormone replacement experiments with rat GH (rGH; 1.8 IU/mg) via ip implanted osmotic pumps (Alza Corp., Palo Alto, CA) were started when the rats were 13–14 weeks old and continued for 6 days (23). Concurrently, all hypophysectomized animals received T4 continuously via separate sc implanted osmotic pumps at a dose (0.8 µg/h·kg BW) that produced the euthyroidism (24) required for maintaining normal concentrations of NADPH-cytochrome P450 reductase, a microsomal enzyme requisite for the expression of P450 catalytic activity (25). At the time of necropsy, the pumps were removed and found to contain the expected residual amounts of GH and T4.

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{alpha}-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{alpha}- and 6ß-hydroxylases, reflective of the activity levels of CYP2C11 and CYP3A2 proteins, respectively, and female-specific testosterone 5{alpha}-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH replacement
As expected (2, 3), plasma GH profiles in intact female rats were characterized by frequent and irregularly occurring pulses (~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. 1Go).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 1. Plasma levels of circulating rGH obtained from individual, undisturbed, catheterized, intact and hypophysectomized (HYPOX) rGH-replaced female and male rats at 15-min intervals for 6 consecutive h. Hypophysectomized rats were implanted peritoneally with osmotic minipumps set to continuously deliver rGH at the rates (micrograms of rGH per h/kg BW) indicated (center). Depicted next to each infusion rate are the resulting circulating profiles and calculated plasma rGH mean concentrations (single point ± SD error bar) normalized by subtracting plasma values obtained from untreated hypophysectomized rats. •, Values determined by RIA; {circ}, 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.)

 
GH administration to hypophysectomized female and male rats at the rate of 20 µg rGH/h·kg BW produced mean circulating concentrations (mean ± SD, 37.1 ± 2.8 and 38.9 ± 3.9 ng/ml, respectively) that were statistically no different from each other and no different from those in intact rats of either sex [38.8 ± 26.8 ng/ml (females) and 36.8 ± 67.0 ng/ml (males); Fig. 1Go]. 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. 1Go).

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. 1Go).

Hepatic CYP2C12
The female specificity of CYP2C12 was illustrated by its expression in intact female liver and its absence in male liver (Fig. 2Go). 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).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 2. Relative hepatic CYP2C12 mRNA and protein levels in intact and hypophysectomized rGH-replaced female and male rats. The levels of rGH replacement by continuous infusion are presented as a percentage of the normal feminine plasma GH profile illustrated in Fig. 1Go and determined in Results. Relative CYP2C12 mRNA and protein levels were determined by laser densitometry of actual Northern radiographs and Western enhanced chemiluminescence radiographs of at least five different livers for each treatment group (mean ± SD). ND, Not detected. *, P < 0.01 compared with identically treated females.

 
Hepatic 5{alpha}-reductase
Although not a P450 enzyme, hepatic gender- and GH-dependent expressions of 5{alpha}-reductase (type 1) are comparable to those of CYP2C12 (30, 40). This similarity was illustrated by a near lack of expression of 5{alpha}-reductase mRNA and dependent testosterone 5{alpha}-reductase activity in liver from intact male and hypophysectomized female rats (Fig. 3Go). Exhibiting an even greater hormone sensitivity than CYP2C12, 5{alpha}-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{alpha}-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. 2Go), but exhibiting less dramatic gender differences, administration of feminine GH profiles at 3–50% of normal was significantly less effective in inducing 5{alpha}-reductase expression in hypophy-sectomized males than females (Fig. 3Go). Only at the 100% replacement dose were 5{alpha}-reductase and associated testosterone 5{alpha}-reductase activity levels indistinguishable in both sexes and nearly equal to intact female-like levels.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 3. Relative hepatic testosterone 5{alpha}-reductase mRNA and catalytic activity levels were determined in intact and hypophysectomized (HYPOX) rGH-replaced female and male rats. Levels of rGH replacement by continuous infusion are presented as a percentage of the normal feminine plasma GH profile illustrated in Fig. 1Go and determined in Results. Relative 5{alpha}-reductase mRNA levels were determined by laser densitometry of the actual Northern radiographs, and the microsomal testosterone 5{alpha}-reductase activity of at least five different livers was measured for each treatment group (mean ± SD). ND, Not detected. *, P < 0.01 compared with identically treated females.

 
Hepatic CYP2C7
In contrast to female-specific CYP2C12, CYP2C7 is more appropriately designated as female predominant because the isoform is also expressed in male liver, albeit at significantly lower concentrations than in female liver (3, 8, 10, 11). In this regard, we found that males expressed CYP2C7 at about 25% of female levels (Fig. 4Go). Hypophysectomy reduced CYP2C7 expression in female and male livers to undetectable levels. In comparison to female-specific CYP2C12 and 5{alpha}-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. 4Go).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. Relative hepatic CYP2C7 mRNA and protein levels in intact and hypophysectomized (HYPOX) rGH-replaced female and male rats. The levels of rGH replacement by continuous infusion are presented as a percentage of the normal feminine plasma GH profile illustrated in Fig. 1Go and determined in Results. Relative CYP2C7 mRNA and protein levels determined by laser densitometry of actual Northern radiographs and Western enhanced chemiluminescense radiographs of at least five different livers for each treatment group (mean ± SD). *, P < 0.01 compared with identically treated females.

 
Hepatic CYP2C11
Like its female counterpart, CYP2C12, CYP2C11 is considered a male-specific isoform because its expression is limited to males (3, 9, 12, 13). We measured hepatic CYP2C11 expression at the mRNA, protein and specific catalytic levels (i.e. CYP2C11-dependent testosterone 2{alpha}-hydroxylase5) and found virtually no expression in intact female rats (Fig. 5Go). 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. 5Go). 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.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 5. Relative hepatic CYP2C11 mRNA, protein, and catalytic activity levels were determined in intact and hypohysectomized (HYPOX) rGH-replaced female and male rats. The levels of rGH replacement by continuous infusion are presented as a percentage of the normal feminine plasma GH profile illustrated in Fig. 1Go and determined in Results. Relative CYP2C11 mRNA and protein levels were determined by laser densitometry of actual Northern radiographs and Western enhanced chemiluminescence radiographs, and microsomal CYP2C11-dependent testosterone 2{alpha}-hydroxylase levels of at least five different livers for each treatment group were measured (mean ± SD). ND, Not detected. *, P < 0.01 compared with identically treated females.

 
Hepatic CYP3A2
CYP3A2 is a male-specific isoform whose expression (mRNA, protein, and specific testosterone 6ß-hydroxylase activity) was nearly undetectable in intact female liver (Fig. 6Go). 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. 6Go). 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.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 6. Relative hepatic CYP3A2 mRNA, protein, and catalytic activity levels were determined in intact and hypophysectomized (HYPOX) rGH-replaced female and male rats. The levels of rGH replacement by continuous infusion are presented as a percentage of the normal feminine plasma GH profile illustrated in Fig. 1Go and determined in Results. Relative CYP3A2 mRNA and protein levels were determined by laser densitometry of actual Northern radiographs and Western enhanced chemiluminescence radiographs, and microsomal CYP3A2-dependent testosterone 6ß-hydroxylase levels of at least five different livers for each treatment group were measured (mean ± SD). ND, Not detected. *, P < 0.01 compared with identically treated females.

 
Other gender-dependent hepatic P450 isoforms
Like CYP3A2, CYP2A2 is a male-specific isoform (16, 17), whose transcript was undetectable in intact female liver but was expressed at normal or above normal levels in the livers of hypophysectomized female and male rats (Fig. 2Go). 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. 7Go). 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. 7Go).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 7. Relative hepatic CYP2A2 and CYP2A1 mRNA levels were determined in intact and hypophysectomized (HYPOX) rGH-replaced female and male rats. The levels of rGH replacement by continuous infusion are presented as a percentage of the normal feminine plasma GH profile illustrated in Fig. 1Go and determined in Results. Northern blot analyses used 32P-labeled oligonucleotide probes specific for each mRNA. Bottom, The same Northern blot reanalyzed with a 32P-labeled oligonucleotide probe specific for 18S ribosomal RNA was used as a control to indicate equal loading of the RNA in all lanes. At least five different livers were analyzed for each treatment group.

 
Hepatic non-P450 mRNAs
We observed very little sexually dimorphic expression of hepatic GHR and GHBP mRNAs (female >= 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. 7Go) verifies the high consistency and integrity of RNA loading on the Northern blots.]


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In agreement with our previous report (40), we found that plasma GH concentrations in female rats too low to be assayable are capable of maintaining the feminine hepatic profile of P450 isoforms. In all cases, restoration of the feminine GH profile in hypophysectomized females at 3% of the physiological concentration induced significant, albeit usually below normal, increases in the female-dependent P450s. CYP2A1 was restored to normal female levels with only 3% of the physiological concentration of the hormone; normal expression levels of CYP2C12 and 5{alpha}-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{alpha}-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. 1Go) increased CYP2C12 protein to 40–60% of normal (6, 7) and mRNA to approximately 100% of normal (9), 5{alpha}-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{alpha}-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{alpha}-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{alpha}-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{alpha}-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{alpha}-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{alpha}-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{alpha}-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
 
We appreciate the generosity of Drs. Marika Rönnholm, Agneta Mode, and Jan-Åke Gustafsson in supplying the antibody to rat CYP2C12, and that of Dr. Stelvio M. Bandiera in supplying the antibody to rat CYP2C7. Materials used to assay rGH were obtained through the National Hormone and Pituitary Program and A. F. Parlow. We also thank Ms. Mubeen Pampori for excellent technical assistance.


   Footnotes
 
1 This work was supported by NIH Grant GM-45758. Back

2 Present address: Scripps Research Institute, La Jolla, California 29087. Back

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. Back

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). Back

5 Less specific testosterone 16{alpha}-hydroxylase activity was in agreement with testosterone 2{alpha}-hydroxylase activity (data not presented). Back

6 Interestingly, as hypophysectomy completely blocks hepatic CYP2C12 and testosterone 5{alpha}-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{alpha}-reductase levels are considerably more sensitive markers than body weight gain (40 ) in evaluating GH ablation in female rats. Back

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 ). Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Adler NT 1981 Neuroendocrinology of Reproduction, Physiology and Behavior. Plenum Press, New York, pp 555
  2. Shapiro BH, Agrawal AK, Pampori NA 1995 Gender differences in drug metabolism regulated by growth hormone. Int J Biochem Cell Biol 27:9–20[CrossRef][Medline]
  3. Legraverend C, Mode A, Wells T, Robinson I, Gustafsson J-Å 1992 Hepatic steroid hydroxylating enzymes are controlled by the sexually dimorphic pattern of growth hormone secretion in normal and dwarf rats. FASEB J 6:711–718[Abstract]
  4. Waxman DJ 1992 Regulation of liver specific steroid metabolizing cytochromes P450: cholesterol 7{alpha}-hydroxylase, bile acid 6ß-hydroxylase, and growth hormone-responsive steroid hormone hydroxlase. J Steroid Biochem Mol Biol 43:1055–1072[CrossRef]
  5. Schenkman JB 1992 Steroid metabolism by constitutive cytochromes P450. J Steroid Biochem Mol Biol 43:1023–1030[CrossRef]
  6. MacGeoch C, Morgan ET, Gustafsson J-Å 1985 Hypothalamo-pituitary regulation of cytochrome P45015ß apoprotein levels in rat liver. Endocrinology 117:2085–2092[Abstract]
  7. Waxman DJ, Morrissey JJ, LeBlanc GA 1989 Female-predominant rat hepatic P-450 forms j (IIE1) and 3 (IIA1) are under hormonal regulatory controls distinct from those of the sex-specific P-450 forms. Endocrinology 124:2954–2966[Abstract]
  8. Ram PA, Waxman DJ 1990 Pretranslational control by thyroid hormone of rat liver steroid 5{alpha}-reductase and comparison to the thyroid dependence of two growth hormone-regulated CYP2C mRNAs. J Biol Chem 265:19223–19229[Abstract/Free Full Text]
  9. Legraverend C, Mode A, Westin S, Ström A, Eguchi H, Zaphiropoulos PG, Gustafsson J-Å 1992 Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion. Mol Endocrinol 6:259–266[Abstract]
  10. Westin S, Ström A, Gustafsson J-Å, Zaphiropoulos PG 1990 Growth hormone regulation of the cytochrome P-450 IIC subfamily in the rat: inductive, repressive and transcriptional effects on P450f (IIC7) and P-450PB1 (IIC6) gene expression. Mol Pharmacol 38:192–197[Abstract]
  11. Bandiera S, Dworschak C 1992 Effects of testosterone and estrogen on hepatic levels of cytochrome P450 2C7 and P450 2C11 in the rat. Arch Biochem Biophys 296:286–295[CrossRef][Medline]
  12. Morgan ET, MacGeoch C, Gustafsson J-Å 1985 Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16{alpha}-hydroxylase cytochrome P-450 apoprotein in the rat. J Biol Chem 260:11895–11898[Abstract/Free Full Text]
  13. Janeczko R, Waxman DJ, LeBlanc GA, Morville A, Adesnik M 1990 Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constituitive male-specific form of cytochrome P450. Mol Endocrinol 4:295–303[CrossRef][Medline]
  14. Waxman DJ, Ram PA, Notani G, LeBlanc GA, Alberta JA, Morrissey JJ, and Sundseth SS 1990 Pituitary regulation of male-specific steroid 6ß-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver: suppresive influence of growth hormone and thyroxine acting at a pretranslational level. Mol Endocrinol 4:447–454[CrossRef][Medline]
  15. Yamzoe Y, Ling X, Murayama N, Gong D, Nagata K, Kato R 1990 Modulation of hepatic level of microsomal testosterone 7{alpha}-hydroxylase, P-450a (P450IIA), by thyroid hormone and growth hormone in rat liver. J Biochem 108:599–603[Abstract/Free Full Text]
  16. Waxman DJ, LeBlanc GA, Morrissey JJ, Staunton J, Lapenson DP 1988 Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression. J Biol Chem 263:11396–11406[Abstract/Free Full Text]
  17. Waxman DJ, Ram PA, Pampori NA, Shapiro BH 1995 Growth hormone regulation of male-specific rat liver P450s 2A2 and 3A2: induction by intermittent growth hormone pulses in male but not female rats rendered growth hormone deficient by neonatal monosodium glutamate. Mol Pharmacol 48:790–797[Abstract]
  18. Chung LWK, Chao H 1980 Neonatal imprinting and hepatic cytochrome P-450. I. Comparison of testosterone hydroxylation in a reconstituted system between neonatally imprinted and nonimprinted rats. Mol Pharmacol 18:543–549[Abstract/Free Full Text]
  19. Waxman DJ, Dannan GA, Guengerich FP 1985 Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific expression. Biochemistry 24:4409–4417[CrossRef][Medline]
  20. Dannan GA, Guengerich FP, Waxman DJ 1986 Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of {Delta}4-steroid 5{alpha}-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450. J Biol Chem 261:10728–10735[Abstract/Free Full Text]
  21. Gustafsson J-Å, Gustafsson SA, Ingelman-Sundberg M, Pousette Å, Stenberg Å, Wrange Ö 1974 Sexual differentiation of hepatic steroid metabolism in the rat. J Steroid Biochem 5:855–859[CrossRef]
  22. Gustafsson J-Å, Stenberg Å 1974 Neonatal programming of androgen responsiveness of liver of adult rats. J Biol Chem 249:719–723[Abstract/Free Full Text]
  23. Pampori NA, Agrawal AK, Shapiro BH 1991 Renaturalizing the sexually dimorphic profile of circulating growth hormone in hypophysectomized rats. Acta Endocrinol (Copenh) 124:283–289[Medline]
  24. Emerson CH, Lew R, Braverman E, DeVito WJ 1989 Serum thyrotropin concentrations are more highly correlated with serum triiodothyronine concentrations than with serum thyroxine concentrations in thyroid hormone-infused thyroidectomized rats. Endocrinology 124:2415–2418[Abstract]
  25. Ram PA, Waxman DJ 1992 Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. J Biol Chem 267:3294–3301[Abstract/Free Full Text]
  26. MacLeod JN, Shapiro BH 1988 Repetitive blood sampling in unrestrained and unstressed mice with a chronic indwelling right atrial catheterization apparatus. Lab Anim Sci 38:603–608[Medline]
  27. Shapiro BH, MacLeod JN, Pampori NA, Morrissey JJ, Lapenson DP, Waxman DJ 1989 Signalling elements in the ultradian rhythm of growth hormone regulating expression of sex-dependent forms of hepatic cytochrome P450. Endocrinology 125:2935–2944[Abstract]
  28. Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  29. Pampori NA, Shapiro BH 1994 Over-expression of CYP2C11, the major male-specific form of hepatic cytochrome P450, in the presence of nominal pulses of circulating growth hormone in adult male rats neonatally exposed to low levels of monosodium glutamate. J Pharmacol Exp Ther 271:1067–1073[Abstract/Free Full Text]
  30. Waxman DJ 1991 Rat hepatic P450IIC subfamily expression using catalytic, immunochemical and molecular probes. Methods Enzymol 206:249–267[Medline]
  31. Ram PA, Waxman DJ 1991 Hepatic P450 expression in hypothyroid rats: differential responsiveness of male-specific P450 forms 2a (IIIA2), 2c (IIC11), and RLM2 (IIA2) to thyroid hormone. Mol Endocrinol 5:13–20[CrossRef][Medline]
  32. Baumbach WR, Horner DL, Logan JS 1989 The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. Genes Dev 3:1199–1205[Abstract/Free Full Text]
  33. Gabrielsson BG, Carmignac DF, Flavell DM, Robinson ICAF 1995 Steroid regulation of growth hormone (GH) receptor and GH-binding protein messenger ribonucleic acids in the rat. Endocrinology 136:209–217[Abstract]
  34. Murphy LJ, Bell GI, Duckworth ML, Friesen HG 1987 Identification, characterization and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor I. Endocrinology 121:684–691[Abstract]
  35. Ramsden R, Sommer KM, Omiecinski CJ 1993 Phenobarbital induction and tissue-specific expression of the rat CYP2B2 gene in transgenic mice. J Biol Chem 268:21722–21726[Abstract/Free Full Text]
  36. Shapiro BH, Szcotka SM 1984 Androgenic repression of hexobarbitone metabolism and action in Crl:CD-1(ICR)BR mice. Br J Pharmacol 81:49–54[Medline]
  37. Agrawal AK, Pampori NA, Shapiro BH 1995 Thin-layer chromatographic separation of regioselective and stereospecific androgen metabolites. Anal Biochem 224:455–457[CrossRef][Medline]
  38. Pampori NA, Pampori MK, Shapiro BH 1995 Dilution of the chemiluminescence reagents reduces the background noise on Western blots. Biotechniques 18:588–590[Medline]
  39. Pampori NA, Agrawal AK, Waxman DJ, Shapiro BH 1991 Differential effects of neonatally administered glutamate on the ultradian pattern of circulating growth hormone regulating expression of sex-dependent forms of cytochrome P450. Biochem Pharmacol 41:1299–1309[CrossRef][Medline]
  40. Pampori NA, Shapiro BH 1996 Feminization of hepatic cytochrome P450s by nominal levels of growth hormone in the feminine plasma profile. Mol Pharmacol 50:1148–1156[Abstract]
  41. Ström A, Mode A, Zaphiropoulos PG, Nilsson AG, Morgan E, Gustafsson J-Å 1988 Cloning and pretranslational hormonal regulation of testosterone 16{alpha}-hydroxylase (P-45016{alpha}) in male rat liver. Acta Endocrinol (Copenh) 118:314–320[Abstract/Free Full Text]
  42. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330:537–543[CrossRef][Medline]
  43. Mathews LS, Enberg B, Norstedt G 1993 Regulation of rat growth hormone receptor gene expression. J Biol Chem 264:9905–9910[Abstract/Free Full Text]
  44. Carmignac DF, Gabrielsson BG, Robinson ICAF 1993 Growth hormone binding protein in the rat: effects of gonadal steroids. Endocrinology 133:2445–2452[Abstract]
  45. Bick T, Hochberg Z, Amit T, Isaksson OGP, Jansson J-O 1992 Roles of pulsatility and continuity of growth hormones (GH) administration in the regulation of hepatic GH-receptors, and circulating GH-binding protein and insulin-like growth factor-I. Endocrinology 131:423–429[Abstract]
  46. Isgaard J, Carlsson L, Isaksson OGP, Jansson J-O 1988 Pulsatile intravenous growth hormone (GH) infusion to hypophysectomized rats increases insulin-like growth factor-I messenger ribonucleic acid in skeletal tissue more effectively than continuous GH infusion. Endocrinology 123:2605–2610[Abstract]
  47. Shapiro BH, Pampori NA, Lapenson DP, Waxman DJ 1994 Growth hormone-dependent and -independent sexually dimorphic regulation of phenobarbital-induced hepatic cytochromes P450 2B1 and 2B2. Arch Biochem Biophys 312:234–239[CrossRef][Medline]
  48. Agrawal AK, Shapiro BH 1996 Phenobarbital induction of hepatic CYP2B1 and CYP2B2: pretranscriptional and post-transcriptional effects of gender, adult age and phenobarbital dose. Mol Pharmacol 49:523–531[Abstract]
  49. Waxman DJ, Pampori NA, Ram PA, Agrawal AK, Shapiro BH 1991 Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic P450. Proc Natl Acad Sci USA 88:6868–6872[Abstract/Free Full Text]
  50. Shapiro BH, Pampori NA, Ram PA, Waxman DJ 1993 Irreversible suppression of growth hormone-dependent cytochrome P450 2C11 in adult rats neonatally treated with monosodium glutamate. J Pharmacol Exp Ther 265:979–984[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pampori, N. A.
Right arrow Articles by Shapiro, B. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pampori, N. A.
Right arrow Articles by Shapiro, B. H.
Right arrowPubmed/NCBI databases
*Substance via MeSH

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