Imprinting of hepatic microsomal cytochrome P-450 enzyme activities and cytochrome P-450IIC11 by peripubertal administration of testosterone in female rats.

The influence of peripubertal exposure to physiological doses of testosterone on the adult androgen responsiveness of hepatic microsomal cytochrome P-450 was investigated. Male and female Sprague-Dawley rats were sham-operated or gonadectomized before puberty, at 25 days of age. They were injected subcutaneously with testosterone enanthate (5 mumol/kg/day) during the pubertal time period, on days 35-49. Responsiveness to this same dose of testosterone was tested by administering the compound during adulthood, on days 81-89. The females provided a model that had not been exposed to neonatal androgen imprinting, in contrast to the males. Testosterone 2 alpha-hydroxylase activity and cytochrome P-450IIC11, which are normally expressed only in adult males, were expressed in the gonadectomized females administered testosterone during puberty with no further exposure to the hormone for the next 40 days. The levels found were similar to those in the gonadectomized male group. When the combined pubertal and adult testosterone regimen was used, a synergistic effect was produced; the 2 alpha-hydroxylase activity reached control male levels in both gonadectomized and sham-operated females and, in addition, cytochrome P-450IIC11 attained control male levels in the gonadectomized females. Testosterone 6 beta-hydroxylase and erythromycin N-demethylase activities were used as indicators of the cytochrome P-450IIIA subfamily. These activities were significantly increased only in the females treated with testosterone during both the pubertal and adult periods, reaching control male levels of 6 beta-hydroxylation. A similar effect, but in the opposite direction, was found with testosterone 7 alpha-hydroxylase, an enzyme activity indicative of cytochrome P-450IIA1. A decrease in this enzyme was produced in the females administered testosterone during both time periods, resulting in levels equivalent to those found in control males. In general, a highly significant interaction was found between the pubertal and adult treatment periods for the females, indicating a chronic effect of the pubertal exposure. The experiments with castrated males did not result in synergistic interactions, although there was some evidence of an additive effect. The results of this study support the hypothesis that the peripubertal period is a time during which testosterone imprinting of both increased basal levels and adult androgen responsiveness of some hepatic cytochrome P-450 enzymes can occur in the female rat.     

Profile of territrem metabolism and cytochrome P-450 3A expression in liver microsomes from Wistar rats of both genders as a function of age

This study determined territrem metabolites after incubation of territrem A, B, or C with NADPH and liver microsomes from Wistar rat of both genders aged 2 to 76 wk. The liver microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity, and CYP3A1 and CYP3A2 protein and mRNA levels were also analyzed. Male rats had significantly higher liver microsomal cytochrome P-450 content and NADPH-cytochrome P-450 reductase activities than females at 14 to 26 wk. Microsomal cytochrome P-450 content was decreased in senescence in both genders compared with postpubertal and adulthood stages. The activity of 6beta-testosterone hydroxylase in male rats, which was significantly higher than those in females at all ages, decreased after 52 wk. After 26 wk, the levels of CYP3A1 protein markely declined in both genders, which resulted in a large gender difference (male greater than female). The protein levels and mRNA of CYP3A2 were constitutively expressed in 2- to 52-wk-old male rats, but they decreased after 76 wk, and decreased in females after 6 wk. The expression of CYP3A1 or CYP3A2 in males are generally higher than in females. The metabolites of territrems MA1, MAX, MA2, MB2, MB4, and MC were measured by high-performance chromatography (HPLC). Formation of MA1, MAX, and MA2 decreased after 52 wk in males, and MAX and MA2 were not formed after 6 wk in females. The amount of MB2 formed in females was less than in males, but the amount of MC (TRC metabolites) formed in females was higher than in males. The gender differences in metabolism of TRA were related to the protein and mRNA expression of CYP3A2. The protein levels and mRNA expression of CYP3A2 and efficiency of territrems metabolism were decreased after 76 wk. The results suggested that the effects of age and gender on territrem metabolism are due to differences in CYP3A1 and CYP3A2 expression in the liver microsomes.     

Sex-related differences in rat hepatic cytochromes P450 expression following treatment with phenobarbital or 3-methylcholanthrene

The induction of hepatic cytochromes P450 and metabolic effects have been examined in male and female Sprague-Dawley rats following treatment with either phenobarbital or 3-methylcholanthrene. Hepatic cytochrome P450 levels were higher in males than in females by ≈40%. Treatment of male and female rats with phenobarbital or 3-methylcholanthrene resulted in an ≈1.6- and 2-fold increase, respectively, in heptic microsomal cytochrome P450 levels in both sexes, relative to untreated animals. Immunoblot analyses were performed to compare sex-related changes in P450 levels. Hepatic P450IIB1 levels in males were greater than those in females following phenobarbital treatment. 3-Methylcholanthrene-induced male hepatic microsomes exhibited greater levels of P450 IA1 and IA2 than female microsomes, whereas uninduced microsomes from males or females failed to exhibit a band. Mab PCN 2-13-1 against P450IIIA recognized an intense band in uninduced hepatic microsomes from males whereas no band was recognized in uninduced microsomes from female rats. The levels of P450IIIA in males were increased 2 to 3-fold following treatment with phenobarbital. while the increase of IIIA levels in females by phenobarbital was minimal, as monitored by immunoblot analysis. Solid phase radioimmunoassay using monoclonal antibodies supported the results of immunoblot analysis. Phenobarbital treatment caused a 6.5-fold increase in the monoclonal antibody binding to IIB1 in males, whereas treatment of females with phenobarbital resulted in a 12-fold increase of IIB1 binding, relative to respective controls. The relative increase of IA levels by 3-methylcholanthrene was also greater in females than in males (10-vs. 8-fold) although the levels of induced IA were comparable in both sexes, as assessed by radioimmunoassay. Radioimmunoassay also showed that hepatic IIE1 level was 1.5-fold higher in males than in females and that either phenobarbital or 3-methylcholanthrene treatment caused 80% to 40% decrease in IIE1 levels, relative to control, in both sexes. Sex-related metabolic activities were examined in hepatic microsomes. Hexobarbital hydroxylase activity was 2- to 3-fold higher in uninduced microsomes from males than that from females. This hydroxylase activity was increased 2- and 3-fold in males and females, respectively, following phenobarbital treatment, as compared to controls. Addition of anti-P450IIB1 antibody to phenobarbital-induced hepatic microsomes from males and females produced 64% and 84% inhibition of hexobarbital oxidation, respectively. Aryl hydrocarbon hydroxylase activity was increased ≈12- and 26-fold in males and females. respectively, following 3-methylcholanthrene treatment relative to controls. The anti-P450IA antibody inhibitable rate of aryl hydrocarbon hydroxylase activity was comparable in both sexes following 3-methylcholanthrene treatment (≈70%). These results demonstrate that levels of hepatic P450IIB1 or P450IA are greater in male than in female for untreated, phenobarbital- or 3-methylcholanthrene treated rats. In addition, the relative increase of P450IIB1 or IA by phenobarbital or 3-methylcholanthrene is more significant in females.     

Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16alpha-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.     

Three patterns of cytochrome P450 gene expression during liver maturation in mice

The neonatal period of liver development is an often overlooked phase of development. For instance, ontogeny of xenobiotic-metabolizing enzymes can markedly affect biotransformation as the liver matures. To systematically examine the ontogenic gene expression patterns of cytochrome P450 genes (P450) in mice, the gene expression profiles of 19 xenobiotic-metabolizing P450 in Cyp1 to 4 families were determined. The mRNA levels in C57BL/6 mouse livers were quantified using branched DNA technology at the following ages: gestational day 17 (2 days before birth) and postnatal days 0, 1, 3, 5, 10, 15, 20, 30, and 45. Among the 13 P450 genes expressed in mouse livers, three distinct ontogenic expression patterns were identified by cluster analysis. Genes in group 1 (Cyp3a16 as well as 3a41b in male) were expressed in the perinatal period, but they were essentially nondetectable by 30 days of age. Genes in group 2 (Cyp2e1, 3a11, and 4a10 as well as 3a41b in female) quickly increased after birth and reached maximal expression levels by day 5. Genes in group 3 (Cyp1a2, 2a4, 2b10, 2c29, 2d22, 2f2, 3a13, and 3a25) were expressed at low levels until days 10 to 15, but they markedly increased at day 20 to a high and stable level. In conclusion, the developmental expression of P450 in mouse liver can be divided into three patterns, suggesting that different mechanisms are responsible for the expression of P450 during liver maturation.     

Ethylbenzene-mediated induction of cytochrome P450 isozymes in male and female rats.

Male and female Holtzman rats were exposed to ethylbenzene, and the effect on liver microsomal activities was studied. Hydrocarbon- and sex-dependent effects on P450-dependent metabolism of drugs and aromatic hydrocarbons were investigated. Hydrocarbon treatment produced two patterns of induction in cytochrome P450-dependent activities: (1) induction common to both sexes; and (2) induction exclusively in females. Benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, p-nitroanisole O-demethylation and aromatic hydroxylation of toluene were induced in both sexes after rats were exposed to ethylbenzene. The rate of benzphetamine N-demethylation increased 4-fold in females and nearly doubled in males. The increase in O-deethylation of 7-ethoxycoumarin was 3-fold in females and doubled in males, while p-nitroanisole O-demethylation increased 4-fold in both sexes after exposure to ethylbenzene. Ethylbenzene had its greatest effect upon the formation of aromatic hydroxylated metabolites of toluene. Ethylbenzene exposure increased the rate of o-cresol formation by 4- and 9-fold in female and male rats, respectively. The formation rate of p-cresol was undetectable in either sex prior to hydrocarbon exposure; however, after the rats were given ethylbenzene, rates increased to 0.4 nmol/min/mg protein in females and to 0.9 nmol/min/mg protein in the males. Ethylbenzene exposure selectively induced aminopyrine demethylation, aniline hydroxylation, N,N-dimethylnitrosamine N-demethylation (DMNA) and aliphatic hydroxylation of toluene in females. Rates for aminopyrine, aniline, and DMNA were increased 50% over controls, while formation of benzyl alcohol from toluene was enhanced to 260% of control. Western immunoblotting indicated that ethylbenzene treatment induced cytochrome P450 2B1/2B2 to a greater extent in male rats and cytochrome P450 2E1 only in females. Ethylbenzene exposure did not affect significantly the level of cytochrome P450 1A1.     

Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat

The profile of hepatic microsomal cytochrome P450 expressed in the male and female rat was dramatically altered by streptozotocin-induced diabetes. In the diabetic male, P450 forms IIC11, IIC13, IIA2, and IIIA2 were suppressed and forms IIA1 and IIC12 were induced to the levels observed in the immature male rat. A 6- to 8-fold induction of P450 IIE1 was detected in both male and female diabetic rats. A member of the P450 IIIA family was also induced in the diabetic female rat. Accompanying the change in P450 profile in the diabetic male rat was reduction in circulating testosterone and tetraiodothyronine concentrations and a sharp diminution of the normally pulsatile pattern of growth hormone secretion. In contrast to the male rat, the growth hormone secretion pattern in the diabetic female rat was unchanged from control. The hormone and P450 profiles detected in the diabetic male rat suggest a reversion to an immature physiological state. Testosterone replacement treatments carried out for 2 weeks slightly but significantly affected the suppression of P450 IIC11 and reversed the changes in P450 IIA2, IIIA2, and IIC12 in the diabetic male, without altering the suppressed state of growth hormone secretion. However, 1 week of human growth hormone, administered intravenously every 4 hr to diabetic male rats, failed to significantly reverse the diabetes-induced changes in hepatic cytochromes P450, in particular forms IIC11 and IIE1, despite the presence of an episodic plasma hGH profile. An induction of P450 IIE1 in diabetic female rats, without a reduction in growth hormone secretion, suggests that its induction in diabetes in both sexes is not related to changes in growth hormone. In addition, the results of testosterone treatment on the expression of IIC12, IIA2, and IIIA2 in the diabetic male rat suggest a regulatory role for this hormone that does not involve the pituitary secretion of growth hormone. However, the lack of effect of human growth hormone treatment in the diabetic male on levels of individual P450 forms indicates that in diabetes there may be a change in the ability of the male rat hepatocyte to respond to a somatic signal, possibly as a result of the changes in other hormone factors.     

Sex-specific modulation of hepatic covalent DNA modifications (I-compounds) by the cytochrome P450 inducer, pregnenolone-16 alpha-carbonitrile.

I-compounds are recently discovered, age-dependent covalent DNA modifications, which are detectable by 32P-postlabeling assay for DNA adducts. The effects of the catatoxic antiglucocorticoid, pregnenolone-16 alpha-carbonitrile (PCN), on hepatic and renal I-compound levels have been studied in male and female Sprague-Dawley rats together with the levels of microsomal cytochrome P450 and rates of ethylmorphine N-demethylation. PCN (50 mg/kg ip) was dissolved in corn oil and administered to rats once daily for 4 days, and animals were killed at 1 day or 8 days after the last treatment. Hepatic and renal I-compounds were analyzed by 32P-postlabeling in control and PCN-treated animals at both time points. Microsomal cytochrome P450 and ethylmorphine N-demethylase activities were also determined. Total levels of liver nonpolar and polar I-compounds were reduced in female rats by 37 and 51%, respectively, compared to controls, at 1 day. Ten out of sixteen individual I-compounds were also markedly reduced in female rat liver DNA as a result of PCN administration. In contrast to females, total levels of liver I-compounds were not significantly altered in males by PCN at 1 day; however, two individual I-compounds were lowered. I-compound levels recovered 8 days after termination of PCN treatment in both males and females. Total levels of renal I-compounds were not affected by PCN treatment in either males or females. [3H]Methylthymidine incorporation studies showed an increase in mean DNA synthesis rate at 1 day in liver of both males and females, but this was significant in males only. Marked induction of hepatic microsomal cytochrome P450 (2.2-fold) and ethylmorphine N-demethylase (4.0-fold) activity was observed in female rats treated with PCN at 1 day as compared to controls. The extent of induction of these enzymes was much higher in females than males. At 8 days the levels of cytochrome P450 and ethylmorphine N-demethylase activity had returned to uninduced values. The results are consistent with a pivotal role for PCN-inducible cytochrome P450 in the metabolism of I-compounds.     

Induction of cytochrome P-450 isozymes upon repeated administration of nifedipine

In experiments on male Wistar rats it has been found that nifedipine administration at a dose of 10 mg/kg body weight i.p. daily for 20 days did not significantly increase the total amount of cytochrome P-450 but markedly increased the 7 alpha-, 16 beta- and 6 beta-hydroxylation of androstenedione in liver microsomes, suggesting the induction of cytochromes P-450IIA1, P-450IIB1, and P-450IIIA1, respectively. The induction of cytochrome P-450IIIB1 was also confirmed immunochemically with polyclonal antibodies against cytochrome P-450IIB1/B2.     

Immunochemical detection of human liver cytochrome P450 forms related to phenobarbital-inducible forms in the mouse

Polyclonal antibodies generated to four distinct mouse liver phenobarbital-inducible cytochrome P450 isoforms were used to analyse related forms in human liver. N-terminal sequence analysis and biochemical properties of the P450s used as antigens suggest that they belong to P450 subfamilies IIB (P450PBI), IA (P450PBII), IIC (P450PBIII) and IIA (P450Coh). In immunoblot analysis, anti-P450PBII detected a single protein presumed to be P450IA2 in all the human livers tested. No proteins corresponding with P450IA1 could be detected. Anti-PBIII and anti-P450Coh antibodies each detected one band (54 and 48 kDa, respectively) in the liver samples. No bands were revealed by anti-P450PBI antibody. Protein dot-immunobinding analysis showed that P450s immunodetectable by anti-P450PBII, anti-P450PBIII and anti-P450Coh antibodies are expressed in human liver (range 9 to 69 pmol P450/mg protein). In immunoinhibition experiments the activity of 7-ethoxyresorutin O-deethylase (EROD) was blocked up to 90% by the anti-P450PBII antibody. Aryl hydrocarbon hydroxylase (AHH) was inhibited only by anti-P450PBIII, and coumarin 7-hydroxylase (COH) only by anti-P450Coh antibody. Testosterone hydroxylations in positions 6 beta, 7 alpha, 15 alpha and 16 alpha were not affected significantly by any of the antibodies. These data suggest that the human liver P450IA2 is responsible for most of the elevated EROD activity, P450s in the IIC subfamily for constitutive AHH and P450s in the IIA subfamily for all of COH activity.     

Cytochrome P450 mono-oxygenase gene expression and protein activity in cultures of adult cardiomyocytes of the rat

There are a substantial number of drugs acting either directly or indirectly on the heart, but surprisingly, little is known about the metabolic capacity of heart muscle cells. We therefore investigated the gene expression and protein activity of cytochrome P450 isozymes in cultures of adult cardiomyocytes of the rat. Semi-quantitative CYP gene expression pattern suggests CYP1A1 and CYP2B1/2 to be key players in cardiomyocytes and upon treatment with Aroclor 1254 approximate 4 fold inductions could be observed for both gene families, when compared with appropriate controls. The mRNA expression of most genes was sustained for prolonged periods of time, e.g. up to 120 h in culture and in the case of the CYP3A1 gene an approximate 10 fold induction was observed at the higher Aroclor 1254 dose level (10 microM) in 24 h old cultures. The constitutively expressed genes, e.g. CYP2C11 and CYP2E1 are expressed throughout the entire culture period (5 days) and did not respond to Aroclor 1254 treatment. CYP4A1 was mainly expressed in freshly isolated cardiomyocytes of control animals and its expression declined rapidly in culture. There was good agreement between gene expression and translated protein activity using 7-ethoxyresorufin and testosterone as substrates. The data reported herein should foster the routine use of freshly isolated and cultivated cardiomyocytes for drug profiling and toxicity studies.     

Rat liver cytochrome P-450 isozymes as catalysts of aldrin epoxidation in reconstituted monooxygenase systems and microsomes

To explore which rat liver cytochrome P-450 species are involved in aldrin epoxidation, we have studied the catalytic activities of a series of cytochrome P-450 isozymes purified from untreated and inducer-treated Sprague-Dawley rats. Of ten cytochrome P-450 forms analyzed, seven isozymes, listed in order of decreasing activity, catalyzed aldrin epoxidation: P-450UT-A, P-450PB-C, P-450UT-H, P-450PB-B, P-450PCN-E, P-450UT-F, and P-450PB-D. P-450UT-I, P-450BNF-B, and P-450ISF-G were not very active at all. A novel aldrin metabolite, endo-dieldrin, was formed by cytochrome P-450UT-F in a 6-fold excess over dieldrin, which is the exo-isomer. The activity of aldrin epoxidase furthermore was assayed in liver microsomes from Sprague-Dawley rats of diverse physiological status and after pretreatment with various inducers resulting in a peculiar pattern of cytochrome P-450 isozymes. Untreated animals, at an age of 3 weeks, showed similar enzyme activities in both genders. During maturation, the activity of males increased by 3-fold, while the activity in females did not significantly change during this period. Pretreatment with pregnenolone-16-alpha-carbonitrile or dexamethasone strongly increased the activity in females. Pretreatment with dexamethasone did not increase the activity of males. A 50% depression of epoxidase activity was noted for males pretreated with 5,6-benzoflavone. Phenobarbital pretreatment increased the activity of females by 12-fold and of males by 2-fold. Males responded to pretreatment with polychlorinated biphenyls in a strain dependent fashion: enzyme activity was increased 2-fold in Sprague-Dawley rats but was not altered in Wistar rats. "Theoretical" values of microsomal epoxidase activity were calculated for weanling and adult Sprague-Dawley rats from turnover numbers and published data on the relative abundance of aldrin epoxidizing P-450 isozymes (Waxmann et al., Biochemistry 24, 4409, 1985). These values agreed with the activities determined. A similar statement can be made for male rats of both strains pretreated with inducers, when the ratio of enzyme activity of pretreated to control animals was used as a basis of comparison. The activity ratio of females pretreated with pregnenolone-16-alpha-carbonitrile, dexamethasone and phenobarbital, however, was much higher than the ratio calculated. Our results reveal that aldrin epoxidation is a reaction indicative of male specific and of phenobarbital-inducible cytochrome P-450 isozymes in rat liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phenobarbital and dexamethasone induce expression of cytochrome P-450 genes from subfamilies IIB, IIC, and IIIA in mouse liver.

Phenobarbital (PB) and dexamethasone (DEX) induce several liver-specific cytochrome P-450 mRNAs in rats. We examined the induction of P-450 mRNAs from families IIB, IIC, and IIIA by PB and DEX in six inbred mouse strains. P-450IIB1-related mRNA species were induced 3- to 13-fold by PB and 3- to 15-fold by DEX in all animals. P-450IIC6-related mRNA species were induced 3- to 7-fold by PB in males and females, and up to 5-fold by DEX in most males but not in female mice, in which DEX was inactive. P-450IIIA-related mRNA species were induced 2- to 7-fold by PB and 2- to 20-fold by DEX in all animals of either sex. In DBA/2J female mice, both inducers triggered a comparable early response (4 hr) at a low dose (10 mg/kg) for all three gene subfamilies, the maximum being reached between 8 and 18 hr of treatment with 100 mg/kg. Under the optimal induction conditions, coadministration of PB and DEX did not lead to any further increase in the responses. These results demonstrate the existence of analogies, as well as striking differences in the inductive effects of PB and DEX between rats and mice. They also indicate the possible involvement of these inducers in related inductive pathways for three cytochrome P-450 gene subfamilies in mouse liver.     

Metabolism of territrem B and C in liver microsomes from 14-wk-old Wistar rats is catalyzed by cytochrome P-450 3A

The metabolism of territrem B (TRB) and territrem C (TRC) in liver microsomes of 14-wk-old male and female Wistar rats was investigated. Metabolism of TRB to 4beta-hydroxylmethyl-4beta-demethylterritrem B (MB2), O-demethylation of the methoxy group of the aromatic moiety of TRB to form MB4 (same structure as TRC), and metabolism of TRC to 4beta-hydroxylmethyl-4beta-demethylterritrem C (MC) were observed in both genders. However, the amounts of MB2, MB4, and MC formed in females were much lower than in males. To investigate which cytochrome P-450 (CYP450) isoforms were involved in each step, four CYP450 isotype-specific inhibitors (furafylline, orphenadrine, cimetidine, and troleandomycin) and antibodies against CYP1A1, CYP2B1, CYP2C11, or CYP3A2 were used. Formation of MB2, MB4, and MC was markedly inhibited by cimetidine and troleandomycin, but less by furafylline and orphenadrine. Anti-CYP3A2 antibody completely inhibited MB, MB, and MC formation, while antibodies against CYP1A1, CYP2B1, or CYP2C11 produced no marked effect. Of the seven tested supersomes from baculovirus-transformed insect cells expressing rat CYP450 isoforms (1Al, 1A2, 2B1, 2C11, 2C12, 3A1, and 3A2), only those expressing CYP3A1 and CYP3A2 metabolized TRB and TRC. The amounts of MB2, MB4, and MC formed by male and female rat liver microsome preparations were related to the testosterone 6beta-hydroxylase activity and CYP3A1/2 protein content of the preparation. Immunoblotting showed that CYP3A1 was expressed in both genders, but at different levels, while CYP3A2 was only expressed in males. These results suggest that the formation of MB2, MB4, and MC in liver microsomes from 14-wk-old rats of either gender is mediated by CYP3A1 and CYP3A2.     

Developmental aspects of P450IIIA: prenatal activity and inducibility

We have used antiserum of defined specificity as well as a specific inducers and inhibitors of P450IIIA1(2) to determine the fetal occurrence and inducibility of this enzyme in rats. Apparently absent from uninduced fetal rat liver (or present in extremely low amounts) cytochrome P450IIIA1(2) becomes increasingly inducible as a function of gestational age. In adult rats, it is now apparent that there are at least two inducible members and one male-specific constitutive member of the IIIA subfamily. The ontogenesis of these enzymes from 2 weeks post partum to puberty has also been determined. The male-specific occurrence of P450IIIA2 subject to testosterone imprinting and maintenance has been proposed. Inconsistencies persist, however. Waxman et al. have proposed the perinatal occurrence in male and female rats with subsequent suppression in females, whereas others have not detected P450IIIA1(2) in uninduced perinatal rat liver. These differences remain unresolved and reflect the difficulties in defining the individual enzyme specificities for various substrates and of antiserum reactivity. Approaches recently applied to investigations of the IIB subfamily of cytochromes P-450 should contribute greatly to the elucidation of factors governing the ontogenesis of IIIA in rats and humans. Recently, cDNA probes capable of discriminating P450IIB1 and P450IIB2 (commonly referred to as P450s b and e, respectively) were utilized to discriminate the developmental regulation of these immune cross-reactive enzymes. cDNA probes specific for the constitutive and inducible P450IIIA enzymes should clarify the P450IIIA ontogeny in rats. However, in light of regulatory differences among the human and rat members of P450IIIA, it is apparent that the extrapolation of human biotransforming potential from results of animal models must be approached with great caution.