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0090-9556/97/2508-0994-1000$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 8

Effect of Ovariectomy and Androgen on Phenobarbital Induction of Hepatic CYP2B1 and CYP2B2 in Sprague-Dawley Rats

Thomas K. H. Chang, Mellissa D. Anderson, Stelvio M. Bandiera, and Gail D. Bellward

Faculty of Pharmaceutical Sciences, The University of British Columbia

    Abstract
Abstract
Introduction
Results
Discussion
References

The purpose of this study was to investigate the impact of prepubertal ovariectomy and postpubertal administration of testosterone on inducibility of rat hepatic CYP2B1 and CYP2B2 by phenobarbital. Intact adult male and female Sprague-Dawley rats were injected ip with sodium phenobarbital (10 mg/kg) or saline (control) once daily on days 129-135 of age and sacrificed one day after the last dose. Hepatic microsomal androstenedione 16beta -hydroxylase activity, benzyloxyresorufin O-dealkylase activity, pentoxyresorufin O-dealkylase activity, and CYP2B1 protein levels were lower in phenobarbital-treated female rats than in phenobarbital-treated male rats. In contrast, there was no sex difference in inducibility of CYP2B2. The lesser inducibility of CYP2B1 in adult female rats was attributed to the presence of an intact ovary because prepubertal ovariectomy (day 25 of age) resulted in increased induction of CYP2B1 and its associated activities (androstenedione 16beta -hydroxylase, benzyloxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase) by phenobarbital. By comparison, postpubertal administration of testosterone enanthate (5 µmol/kg sc once daily on days 80-94 of age) did not enhance the inducibility of CYP2B1 or its associated activities in prepubertally ovariectomized adult (136-day-old) rats administered phenobarbital (10 mg/kg/day on days 129-135 of age). However, the androgen treatment did increase CYP2C11-dependent testosterone 2alpha -hydroxylase activity in the same microsomal samples. Overall, the results show a sex difference in phenobarbital induction of hepatic CYP2B1 but not CYP2B2 in adult Sprague-Dawley rats. They also indicate that prepubertal ovariectomy enhances the effect of phenobarbital on CYP2B1, whereas administration of testosterone enanthate postpubertally does not influence the inducibility of either CYP2B1 or CYP2B2 in prepubertally ovariectomized adult rats.

    Introduction
Abstract
Introduction
Results
Discussion
References

Hormones play a major role in regulating expression of many rat cytochrome P450 enzymes. In the case of the male-specific CYP2C111,2 and the female-specific CYP2C12, it is not the presence of GH per se but the sexually dimorphic pattern of GH secretion that regulates the hepatic expression of these enzymes (1). GH secretion is intermittent and pulsatile in the male rat, but it is more continuous in the female rat (2, 3). The male pattern of GH secretion induces CYP2C11 (4-6), whereas the female pattern stimulates CYP2C12 (5, 7). Studies of hormonal regulation of rat hepatic cytochrome P450 have focused mainly on the expression of the various constitutive enzymes, and much less is known about the endocrine control of inducible forms.

Hepatic CYP2B1 and CYP2B2 are expressed constitutively at very low levels in male and female rats (8, 9). These enzymes are subject to induction by a large number of structurally diverse compounds, including phenobarbital (8, 10, 11), clotrimazole (12, 13), diallyl sulfide (14), and certain halogenated biphenyl congeners (15-17). Hypophysectomy and GH replacement studies have suggested that this pituitary hormone suppresses phenobarbital induction of CYP2B3, and it appears that the female pattern of GH secretion is more inhibitory than the male pattern (9, 18). Interestingly, the effect of hypophysectomy on the responsiveness of CYP2B to phenobarbital is observed in Sprague-Dawley (9, 18) and Wistar Furth (19) rats, but not in Fischer 344 rats (19), suggesting a strain-dependent effect. Indeed, a strain difference in CYP2B expression has been reported (19-22). The suppressive influence of GH on hepatic CYP2B induction has also been shown in rats rendered GH deficient by neonatal administration of monosodium glutamate (23, 24). Furthermore, experiments with cultured rat hepatocytes have established that GH can directly inhibit the effect of phenobarbital on CYP2B mRNA (25) and protein expression (18, 26). Although GH negatively regulates barbiturate-mediated induction of CYP2B, it does not affect the constitutive hepatic expression of these enzymes, as shown in rats rendered GH deficient by neonatal monosodium glutamate administration (24) and in dwarf rats (27), which have very low circulating GH levels owing to a genetic defect in GH synthesis (28).

Relatively little is known about the influence of gonadal hormones on phenobarbital induction of CYP2B. In a recent study (29), ovariectomy attenuated the effect of phenobarbital on hepatic CYP2B protein content, and this effect was more pronounced in the Wistar Furth strain than in the Fischer 344 strain. Castration inhibited the inducibility of CYP2B, but the effect was fully reversed by exogenous androgen administration. Studies from our laboratory have shown that responsiveness of CYP2C11 to androgen in prepubertally ovariectomized adult rats can be enhanced by prior exposure (1 month earlier) to testosterone enanthate (30, 31). To gain further insight into the influence of gonadal hormones on hepatic CYP2B inducibility, we investigated the effect of postpubertal androgen administration on phenobarbital induction of hepatic CYP2B1 and CYP2B2 in prepubertally ovariectomized adult Sprague-Dawley rats. The results obtained indicate a sex difference in phenobarbital induction of hepatic CYP2B1 but not CYP2B2. Whereas prepubertal ovariectomy enhanced the effect of phenobarbital on CYP2B1 and its associated enzyme activities, administration of testosterone enanthate postpubertally did not influence the inducibility of either CYP2B1 or CYP2B2 in prepubertally ovariectomized adult Sprague-Dawley rats.

Materials and Methods

Chemicals. Testosterone enanthate, testosterone, and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO). Authentic 2alpha - and 11beta -hydroxytestosterone metabolite standards were bought from Steraloids, Inc. (Wilton, NH). Benzyloxyresorufin, pentoxyresorufin, and resorufin were supplied by Molecular Probes, Inc. (Eugene, OR). Sodium phenobarbital and [4-14C]androstenedione (54 mCi/mmol) were obtained from BDH Chemicals (Toronto, Ontario, Canada) and Amersham Canada Ltd. (Oakville, Ontario, Canada), respectively. 17beta -N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha -androstan-3-one (4-MA) was a gift from Merck Sharp and Dohme Research Laboratories (Rahway, NJ).

Animals. Adult male and female Sprague-Dawley rats were purchased from Charles River Co. (Montreal, Quebec, Canada) and were allowed to acclimatize in our animal care facility for at least 7 days prior to initiation of treatment. Female rats were either ovariectomized or sham-operated at 25 days of age by the breeder. The rats were housed on corn cob bedding, provided with food and tap water ad libitum up to the time of sacrifice, and cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care.

Treatment of Animals. Intact male and female rats were injected ip with sodium phenobarbital (10 mg/kg) or saline (control) once daily for 6 consecutive days (days 129-135 of age). This injection protocol is known to produce sex differences in phenobarbital induction of CYP2B-mediated enzyme activity in rat liver (24, 32). In other experiments, prepubertally ovariectomized (day 25 of age) rats were injected sc with testosterone enanthate (5 µmol/kg) (30) or corn oil (vehicle) once daily on days 80-94 followed by phenobarbital administration on days 129-135 as described for the intact animals.

Preparation of Microsomes and Serum Samples. Rats were killed on day 136 of age. Livers were quickly excised, washed in ice-cold 1.15% KCl/50 mM Tris (pH 7.5), and used immediately to prepare microsomes by differential ultracentrifugation (33). The final microsomal pellet was suspended in 0.25 M sucrose and aliquots of the suspension were stored at -80°C until use. Blood was collected and allowed to clot at 4°C. Serum was prepared by centrifugation and then stored at -20°C until use.

Preparation of Anti-Rat CYP2B Antibodies. Polyclonal rabbit anti-rat CYP2B IgG was prepared as described previously (34). As reported elsewhere (35), this antibody preparation specifically detects CYP2B enzymes in rat liver microsomes.

Immunoquantitation of CYP2B1 and CYP2B2 Proteins. SDS-PAGE was performed according to the method of Laemmli (36) in a separating gel containing 7.5% acrylamide and a stacking gel containing 3% acrylamide. Proteins resolved by SDS-PAGE were transferred electrophoretically onto nitrocellulose membrane (37), using a Hoefer Transphor Apparatus (Model TE52) at a setting of 0.4 A for 2 hr at 4°C. Each blot was probed with polyclonal rabbit anti-rat CYP2B IgG at a concentration of 2 µg IgG/ml at 37°C for 2 hr with shaking. Alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:3000 dilution) was used to locate bound anti-CYP2B IgG. Immunoreactive CYP2B1 and CYP2B2 proteins were detected by immersing the nitrocellulose membrane in a solution containing 0.01% nitro blue tetrazolium, 0.005% 5-bromo-4-chloro-3-indolyl phosphate, 0.5 mM MgCl2, and 0.1 M Tris-HCl, pH 9.5. Assay conditions were optimized to ensure that color development did not proceed beyond the linear response range of the phosphatase reaction. Staining intensities of the bands were quantitated by densitometry (VISAGE 110 Bio Image Analyzer, Bio Image, Ann Arbor, MI) with purified rat CYP2B1 as standard as described previously (35).

Androstenedione 16beta -Hydroxylase Assay. Microsomal androstenedione 16beta -hydroxylase activity was determined by TLC (38). Reactions were carried out at 37°C in 200 µl incubation mixtures containing 100 mM HEPES (pH 7.4), 0.1 mM EDTA, 50 µM 14C-labeled androstenedione, 2.5 µM 4-MA [to inhibit steroid 5alpha -reductase (39)] and microsomes (15 pmol total cytochrome P450 for samples from phenobarbital-induced rats and 30 pmol total cytochrome P450 for samples from uninduced rats). Microsomal androstenedione metabolism was initiated by addition of NADPH (1 mM final concentration) and stopped 10 min later with 1 ml ethyl acetate. Reaction products were extracted with ethyl acetate and then chromatographed on silica gel TLC plates developed with dichloromethane/absolute ethanol (97:3, v/v) followed by ethyl acetate/chloroform (1:1, v/v). Metabolites were localized by autoradiography and quantitated by liquid scintillation counting.

Alkoxyresorufin O-Dealkylase Assays. Microsomal benzyloxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase activities were determined by a continuous spectrofluorometric assay (40). Each 2-ml incubation mixture contained 100 mM HEPES (pH 7.8), 5 mM MgCl2, 5 µM benzyloxyresorufin or pentoxyresorufin, microsomes (100 µg protein for samples from phenobarbital-induced rats and 300 µg protein for samples from uninduced rats) and 0.25 mM NADPH. Reactions were carried out at 37°C. Product formation was monitored for 3 min for induced samples and 5 min for uninduced samples. The amount of resorufin formed was determined spectrofluorometrically (530 nm excitation wavelength and 582 nm emission wavelength) in comparison with authentic resorufin standard.

Testosterone 2alpha -Hydroxylase Assay. Microsomal testosterone 2alpha -hydroxylase activity was measured by HPLC (41). Reactions were carried out at 37°C in 1 ml incubation mixtures containing 100 mM potassium phosphate (pH 7.4), 3 mM MgCl2, 0.1 mM EDTA, 0.25 mM testosterone, 2.5 µM 4-MA, and 0.5 mg microsomal protein. Microsomal testosterone metabolism was initiated by addition of NADPH (1 mM final concentration) and stopped 5 min later with 6 ml dichloromethane. The internal standard, 11beta -hydroxytestosterone (3 nmol), was then added to each incubation tube, and the mixture was extracted with dichloromethane. Separation and quantitation of monohydroxytestosterone metabolites were carried out by reversed phase HPLC as described previously (42).

Other Microsomal Assays. Total cytochrome P450 content was determined from the sodium dithionite-reduced carbon monoxide difference spectrum, using a molar extinction coefficient of 91 cm-1 mM-1 (43). Microsomal protein concentration was determined using the Bio-Rad Protein Assay Kit.

Serum Testosterone Assay. Serum testosterone concentration was measured by solid-phase 125I radioimmunoassay with the ImmuChem Direct Testosterone kit (ICN Biomedicals, Inc., Costa Mesa, CA).

Statistics. The significance of the difference between the means of treatment groups was assessed by two-way ANOVA and, where applicable, was followed by the Newman-Keuls multiple range test. The level of significance was set a priori at p < 0.05.

    Results
Abstract
Introduction
Results
Discussion
References

Basal Levels and Inducibility of Hepatic CYP2B Enzyme Activities by Phenobarbital in Adult Male and Female Rats. The basal level of pentoxyresorufin O-dealkylase activity was 13-fold higher in liver microsomes from adult male rats than in those from adult female rats (table 1). In contrast, no sex difference in constitutive levels of androstendione 16beta -hydroxylase or benzyloxyresorufin O-dealkylase activities was found in hepatic microsomes isolated from adult male and female rats. Treatment of adult female rats with phenobarbital (10 mg/kg/day ip for 6 consecutive days) increased hepatic microsomal androstenedione 16beta -hydroxylase activity, benzyloxyresorufin O-dealkylase activity, and pentoxyresorufin O-dealkylase activity by 4-fold, 18-fold, and 83-fold, respectively. By comparison, phenobarbital treatment increased androstenedione 16beta -hydroxylase activity, benzyloxyresorufin O-dealkylase activity, and pentoxyresorufin O-dealkylase activity by 9-fold, 28-fold, and 12-fold, respectively, in hepatic microsomes from adult male rats.

                              
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TABLE 1
Effect of phenobarbital on hepatic microsomal CYP2B activity and protein levels in adult Sprague-Dawley ratsa

Differential Effect of Phenobarbital on Hepatic CYP2B1 and CYP2B2 Protein Levels. Androstenedione 16beta -hydroxylation (44), benzyloxyresorufin O-dealkylation (45,46), and pentoxyresorufin O-dealkylation (46-48) are catalyzed predominantly by one or more of the CYP2B enzymes in hepatic microsomes from phenobarbital-treated rats. Therefore, levels of CYP2B1 and CYP2B2 proteins were measured by immunoblot analysis (fig. 1) in the same microsomes used for enzyme activity determinations. As shown in table 1, administration of phenobarbital resulted in a higher level of CYP2B1 in adult male than in adult female rats, whereas CYP2B2 content after phenobarbital treatment was increased to the same extent for these two groups.


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  		Fig. 1.   Immunoblot of CYP2B1 and CYP2B2 proteins in hepatic microsomes from adult male and female rats treated with phenobarbital.

Adult male and female rats were treated as described in the footnote to table 1. Hepatic microsomes (each lane contains 10 pmol of total microsomal CYP isolated from one individual rat liver) and purified rat CYP2B1 (1 pmol applied per lane) were subjected to SDS-PAGE. Proteins were transferred electrophoretically onto nitrocellulose membrane. The immunoblot was probed with polyclonal rabbit anti-rat CYP2B IgG at a final concentration of 2 µg/ml followed by alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:3000 dilution). Immunoreactive CYP2B proteins were detected as described under Materials and Methods. Lanes A and R: purified rat CYP2B1 standard. Lanes B-E: hepatic microsomes from saline-treated adult female rats. Lanes F-I: hepatic microsomes from phenobarbital-treated adult female rats. Lanes J-M: hepatic microsomes from saline-treated adult male rats. Lanes N-Q: hepatic microsomes from phenobarbital-treated adult male rats. In lanes where two dark bands are visible, the upper and lower bands correspond to CYP2B2 and CYP2B1, respectively (35).

Effect of Prepubertal Ovariectomy on Phenobarbital Induction of Hepatic CYP2B. A previous study (29) showed that ovariectomy performed during adult life attenuated the inductive effect of phenobarbital on hepatic CYP2B protein content in Wistar Furth rats, although the effect was less pronounced in Fischer 344 rats. In the present study, female Sprague-Dawley rats were ovariectomized or sham-operated at 25 days of age (prepuberty) and injected ip with phenobarbital (10 mg/kg) or saline once daily on days 129-136 (adult life). Prepubertal ovariectomy did not alter constitutive levels of hepatic microsomal androstenedione 16beta -hydroxylase activity (fig. 2A), benzyloxyresorufin O-dealkylase activity (fig. 2B), or pentoxyresorufin O-dealkylase activity (data not shown) in adult female rats. In contrast, prepubertal ovariectomy increased all three activities and CYP2B1 protein levels after phenobarbital treatment compared with the sham-operated group (fig. 2A-2C and data not shown). The same surgical operation did not alter the inducibility of CYP2B2 protein (fig. 2D).


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  		Fig. 2.   Effect of prepubertal ovariectomy on phenobarbital induction of hepatic microsomal CYP2B activities and proteins in adult female rats.

Female rats were ovariectomized or sham operated (control) on day 25 of age and injected ip with sodium phenobarbital (10 mg/kg) or saline (control) once daily on days 129-135. All the animals were sacrificed one day after the last injection. Results are expressed as mean ± SE for 8-10 individual rats per treatment group. aSignificantly different from the corresponding saline-treated group (p < 0.05). bSignificantly different from the phenobarbital-treated intact female rats (p < 0.05). A) Androstenedione (AD) 16beta -hydroxylase activity. B) Benzyloxyresorufin O-dealkylase (BROD) activity. C) CYP2B1 protein content. D): CYP2B2 protein content.

Effect of Prior Androgen Exposure on Phenobarbital Induction of Hepatic CYP2B in Prepubertally Ovariectomized Adult Female Rats. To investigate whether prior exposure to androgen influences phenobarbital induction of hepatic CYP2B, prepubertally ovariectomized rats were pretreated with testosterone enanthate (5 µmol/kg sc) or corn oil once daily on days 80-94 of age and then administered phenobarbital (10 mg/kg ip) or saline once daily on days 129-135. Prior androgen exposure did not increase hepatic microsomal androstenedione 16beta -hydroxylase activity (fig. 3A), benzyloxyresorufin O-dealkylase activity (fig. 3B), pentoxyresorufin O-dealkylase activity (data not shown), CYP2B1 protein content (fig. 3C), or CYP2B2 protein content (fig. 3D) in prepubertally ovariectomized adult female rats administered phenobarbital. However, androgen treatment increased CYP2C11-mediated testosterone 2alpha -hydroxylase activity in the same microsome samples, at a time when serum testosterone was undetectable (below the limit of detection of the assay, 2 ng/ml) (table 2).


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  		Fig. 3.   Effects of prior androgen exposure on phenobarbital induction of hepatic microsomal CYP2B activities and proteins in prepubertally ovariectomized adult female rats.

Prepubertally ovariectomized (day 25 of age) rats were administered testosterone enanthate (5 µmol/kg sc) or corn oil (control) on days 80-94 and subsequently injected with sodium phenobarbital (10 mg/kg/day ip) or saline (control) on days 129-135. All the animals were sacrificed one day after the last injection. Results are expressed as mean ± SE for 9 or 10 individual rats per treatment group. aSignificantly different from the corresponding saline-treated group (p < 0.05). A) Androstenedione (AD) 16beta -hydroxylase activity. B) Benzyloxyresorufin O-dealkylase (BROD) activity. C): CYP2B1 protein content. D): CYP2B2 protein content.

                              
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TABLE 2
Hepatic microsomal CYP2C11-mediated testosterone 2alpha -hydroxylase activity and serum testosterone concentration in prepubertally ovariectomized 136-day-old rats administered testosterone enanthate on days 80-94 of agea

    Discussion
Abstract
Introduction
Results
Discussion
References

Phenobarbital is known to induce hepatic microsomal androstenedione 16beta -hydroxylase (11, 24) and pentoxyresorufin O-dealkylase (9) activities to a greater extent in adult male rats than in adult female rats. In this study, a treatment regimen of phenobarbital (10 mg/kg/day ip for 6 days) previously shown to produce a sex differentiated inducibility of rat hepatic CYP2B (24) increased benzyloxyresorufin O-dealkylase in addition to these two activities to a higher level in male than in female rats, although the fold-increase was greater in female rats for pentoxyresorufin O-dealkylase activity. Since CYP2B enzymes contribute extensively to each of these enzyme activities in hepatic microsomes from phenobarbital-treated rats (44-48), levels of CYP2B1 and CYP2B2 proteins were measured by immunoblot analysis with CYP2B subfamily-specific antibodies. Phenobarbital increased hepatic microsomal CYP2B1 to a greater extent in adult male rats than in adult female rats, whereas no sex difference was observed for the inducibility of CYP2B2. Our protein data are consistent with the Northern blot results reported by Agrawal and Shapiro (32). In that study, phenobarbital administration to Sprague-Dawley rats using the same dosing protocol as the one used in the present study resulted in higher hepatic CYP2B1 mRNA levels in adult male rats than in adult female rats, whereas no sex difference was found in CYP2B2 mRNA levels after inducer treatment. However, studies with the same strain of rats and using the same treatment regimen (10 mg/kg/day ip for 6 days) (32) or a regimen with a higher dose (80 mg/kg/day ip for 3 days) (9) of phenobarbital have shown a sexually differentiated response of both proteins to the barbiturate. The reason for the discrepancy in the effect of phenobarbital on CYP2B2 protein levels is not known.

GH negatively regulates phenobarbital induction of rat hepatic CYP2B (9, 18, 23, 24). It has been suggested that GH pulse amplitude, rather than pulse frequency or the interpulse period, is the signaling element that is responsible for the suppressive effect of GH on CYP2B inducibility (24). This proposal is based on the observation that the extent of induction of CYP2B by phenobarbital is less in control male rats than in male rats administered monosodium glutamate (2 mg/g body weight) neonatally, which reduces GH pulse amplitude by ~90% without altering other features of the male pattern of GH secretion (49, 50). In the present study, ovariectomy enhanced phenobarbital induction of hepatic CYP2B1 protein and its associated enzyme activities in adult rats, whereas Larsen and Jefcoate (29) showed that ovariectomy did not increase, but rather decreased, the effect of phenobarbital on CYP2B protein levels in peripubertal rats. However, in that study (29) levels of the individual CYP2B1 and CYP2B2 proteins were not reported. Collectively, these data are consistent with the notion that GH pulse amplitude signals the suppressive effect of GH on CYP2B induction because ovariectomy is known to decrease GH pulse amplitude in adult rats (51), but increases GH pulse amplitude in peripubertal rats (52). A novel finding from the present study is that ovariectomy did not influence CYP2B2 inducibility, suggesting that GH pulse amplitude is not a cellular signal that regulates hepatic expression of this protein. Consistent with this proposal, hepatic CYP2B2 protein is induced by phenobarbital to a similar extent in hypophysectomized male and female rats in which there is absence of circulating GH (9).

Circulating androgens influence CYP2B inducibility because a recent study reported that castration diminishes the effects of phenobarbital on hepatic microsomal CYP2B protein content and pentoxyresorufin O-dealkylase activity, and these effects can be fully reversed by exogenous androgen administration (29). We have reported that responsiveness of hepatic CYP2C11 to androgen in ovariectomized adult rats can be enhanced by prior exposure (approximately one month earlier) to testosterone enanthate (30, 31). In the present study, treatment of prepubertally ovariectomized rats with testosterone enanthate (days 80-94 of age) did not impact on the inductive effect of phenobarbital (administered on days 129-135) on hepatic CYP2B1 or CYP2B2. As indicated above, GH pulse amplitude seems to be an important cellular signal in hepatic CYP2B1 expression (24). In a previous study, treatment of neonatally ovariectomized rats with testosterone enanthate during the neonatal or peripubertal period resulted in an increase in peak concentration and a decrease in trough level of plasma GH in the adult animals (53). In the present study, testosterone enanthate pretreatment during adult life did not influence CYP2B1 inducibility, suggesting that postpubertal exposure to this androgen does not have a long-lasting effect on GH pulse amplitude. In contrast to the lack of an effect of testosterone enanthate pretreatment on CYP2B, it did increase CYP2C11-mediated testosterone 2alpha -hydroxylase activity in the same microsomal samples. In the case of CYP2C11, it is the absence of GH or presence of subdetectable levels of GH during the interpulse period (6), rather than the pulse amplitude (49, 54), that is a cellular signal for the hepatic expression of this cytochrome P450.

As mentioned above, CYP2B enzymes contribute extensively to androstenedione 16beta -hydroxylase, benzyloxyresorufin O-dealkylase, and pentoxyresorufin O-dealkylase activities in hepatic microsomes from phenobarbital-treated rats (44-48), although it is not known whether these activities reflect mostly CYP2B1 or CYP2B2. However, based on the overall pattern of response of these two proteins in hepatic microsomes from rats treated with phenobarbital (10 mg/kg/day for 6 days), the present study suggests that microsomal androstenedione 16beta -hydroxylase, benzyloxyresorufin O-dealkylase, and pentoxyresorufin O-dealkylase activities largely reflect CYP2B1 protein levels, with little or no contribution from CYP2B2. Consistent with this proposal, purified CYP2B1 is considerably more active than purified CYP2B2 in the oxidation of androstenedione (41, 55) and each of the two alkoxyresorufin compounds (46, 48). However, it should be noted that androstenedione 16beta -hydroxylase and pentoxyresorufin O-dealkylase activities in hepatic microsomes from uninduced adult male rats are catalyzed primarily by enzymes other than CYP2B (44, 56). For example, the male-specific CYP2C11 is largely responsible for pentoxyresorufin O-dealkylase activity in microsomes from untreated adult male rats (42, 56), which explains the observation that the constitutive level of hepatic microsomal pentoxyresorufin O-dealkylase activity is higher in adult male rats than in adult female rats (table 1). In contrast, benzyloxyresorufin O-dealkylase activity in hepatic microsomes from uninduced rats is still selective for CYP2B, as indicated by data from immunoinhibition experiments (46, 57).

In summary, phenobarbital increased hepatic CYP2B1 to a greater extent in adult male than in adult female Sprague-Dawley rats, whereas no sex difference was found in CYP2B2 inducibility. Prepubertal ovariectomy enhanced phenobarbital induction of CYP2B1 but not CYP2B2. Finally, testosterone enanthate pretreatment given approximately 1 month prior to phenobarbital did not influence the inducibility of either CYP2B1 or CYP2B2 in prepubertally ovariectomized adult rats.

    Acknowledgments

We thank Merck Sharp and Dohme Research Laboratories (Rahway, NJ) for the generous gift of 4-MA.

    Footnotes

Received December 20, 1996; accepted April 8, 1997.

   This work was supported by the Medical Research Council of Canada. M.D.A. was supported by a postgraduate scholarship (PGS-A) from the Natural Sciences and Engineering Research Council of Canada. Part of this study was presented at the XIth International Symposium on Microsomes and Drug Oxidations, Los Angeles, CA, July, 1996.

2   Individual cytochromes P450 are designated according to the systematic nomenclature (58).

3   The term CYP2B is used when the cited study did not distinguish between the individual CYP2B enzymes.

Send reprint requests to: Dr. G. D. Bellward, Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, B.C., V6T 1Z3, Canada.

    Abbreviations

Abbreviations used are: CYP, cytochrome P450; GH, growth hormone; 4-MA, 17beta -N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha -androstan-3-one; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

    References
Abstract
Introduction
Results
Discussion
References

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