Effect of neonatal exposure of 17beta-estradiol and tamoxifen on hepatic CYP3A activity at developmental periods in rats

We evaluated hepatic CYP3A activity during development in male and female rats and the effect of neonatal exposure of 17beta-estradiol and tamoxifen. In untreated and olive oil-treated (control) rats, hepatic CYP3A activities evaluated by erythromycin metabolism in vitro increased several-fold from age 2 to 9 weeks in males. In contrast, activity in females remained at a low and constant level from 2 to 15 weeks. Exposure of 17beta-estradiol to neonates at a dose of 10 micromol/kg daily for 3 days on day 1-3 (approximately) or 4-6 (approximately) after birth significantly increased hepatic CYP3A activity during the developmental period in both males and females, and a greater influence was observed in females exposed during days 4-6 (approximately). Pubertal exposure of 17beta-estradiol (7-weeks old, 10 micromol/kg daily for 3 days) also increased hepatic CYP3A activity, but only in females. Neonatal exposure to tamoxifen (10 micromol/kg daily for 3 days) showed no appreciable effect in either males or females. In conclusion, a marked sex-difference was observed in hepatic CYP3A activity, and exposure of 17beta-estradiol to neonates increased hepatic CYP3A activity during the developmental period, especially in female rats.     

Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.     

Tamoxifen Alters Hepatic Cytochrome P450 Enzyme Expression and Circulating Growth Hormone Levels in Intact Male Rats

PURPOSE: To investigate the effects of acute tamoxifen treatment on hepatic cytochrome P450 (CYP) expression and circulating thyroid and growth hormone (GH) levels in intact adult male rats. METHODS: Rats were injected subcutaneously with peanut oil (vehicle) or tamoxifen at a dosage of 20 or 200 mg/kg for 2 consecutive days. Blood for GH measurements was collected on day 34. Rats were sacrificed at 37 days after treatment, trunk blood was collected, and hepatic microsomes were prepared. RESULTS: Mean body weight of rats treated with tamoxifen at 200 mg/kg was decreased compared to vehicle-treated rats throughout the 5-week period after treatment. Hepatic CYP2A1-dependent testosterone 7alpha-hydroxylase activity and CYP2A1 protein content were increased, whereas CYP2C11-mediated testosterone 2alpha- and 16alpha-hydroxylase activities and CYP2C11 protein content were decreased significantly following tamoxifen administration. Peak plasma GH levels were 60% lower and nadir plasma GH levels were 30% higher in tamoxifen-treated relative to vehicle-treated rats. In contrast, serum triiodothyronine and thyroxine levels were not affected by tamoxifen treatment. CONCLUSIONS: Hepatic CYP enzyme expression was altered and body weight was decreased in adult male rats 5 weeks after treatment with tamoxifen. This alteration corresponded to changes in plasma GH levels.     

Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression.

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERalpha expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5alpha-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5alpha-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5alpha-reductase activity and a decrease in the formation of 16alpha-OH-, 2alpha-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERalpha expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERalpha expression, although these activities cannot be directly related to estrogenic activity.     

Toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic cytochrome P450 in F344 rats

The subacute toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic microsomal cytochrome P450 (P450) were investigated in male and female F344 rats. Rats were given 0.25, 0.5, and 1.0% terbutol for 28 days. Liver weights of male and female rats increased at all dose levels. The compound did not affect activity or amount of serum biochemical markers related to hepatic damage. The concentrations of terbutol in rat serum were less than 0.1 microM, and its major metabolites in serum were 2,6-di-tert-butyl-4-carboxyphenyl N-methyl-carbamate and 2,6-di-tert-butyl-4-carboxyphenol. In male rats, P450 and cytochrome b5 (b5) contents, and NADPH cytochrome c reductase (fp2) activity in liver microsomes were increased about 2-fold by 1% terbutol administration for 7 to 28 days. Among the P450-dependent monooxygenase activities in liver microsomes, 7-benzyloxyresorufin-O-debenzylase (BROD) activity was greatly increased by 100-fold, and 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), and aminopyrine-N-demethylase (APND) activities were elevated 2- to 3-fold. 7-Methoxyresorufin-O-deethylase (MROD), erythromycin-N-demethylase (EMND), estradiol 2-hydroxylase (ED2H), chlorzoxazone 6-hydroxylase (CZ6H), and lauric acid omega-hydroxylase (LAOH) activities were unchanged. For the activities of testosterone hydroxylation, testosterone 16beta-hydroxylase (T16BH) activity was markedly increased by 30-fold, and testosterone 6beta-hydroxylase (T6BH) and testosterone 7alpha-hydroxylase (T7AH) activities were slightly elevated. Testosterone 2alpha-hydroxylase (T2AH) activity was not affected. Terbutol 4-methylhydroxylase (T4MH) activity was increased 9-fold by 1% terbutol. In an immunoinhibition study, T4MH activity in liver microsomes from 1% terbutol-treated rats was decreased about 50% by polyclonal anti-rat CYP2B1, whereas polyclonal anti-rat CYP2A1 and CYP2C11 did not affected the activity. These results indicate that terbutol increased CYP2B subfamily in rat liver microsomes, and that the compound did not cause serious hepatic damage.     

Neonatal exposure to Aroclor 1254: effects on adult hepatic testosterone hydroxylase activities

1. The effects of neonatal exposure to Aroclor 1254 (100 mumol/kg) on the metabolism of testosterone by adult male and female rats were determined by comparing their hepatic microsomal testosterone hydroxylase activities at 60, 90 and 120 days after the initial exposure. 2. The most pronounced effects in male rats were observed 90 days after treatment with Aroclor 1254, whereas in female rats the major changes in testosterone hydroxylase activities were observed after 60 days. 3. Ninety-day-old male rats neonatally treated with Aroclor 1254 exhibited decreased basal testosterone 7 alpha-hydroxylase and increased basal testosterone 16 alpha-, 2 alpha- and 15 beta-hydroxylase activities and androstenedione formation. In addition, the Aroclor 1254-mediated induction of testosterone 7 alpha- and 6 alpha-hydroxylase activities and androstenedione formation was decreased, and that of testosterone 2 beta- and 15 beta-hydroxylase activities was increased. 4. Sixty-day-old female rats exposed neonatally to Aroclor 1254 exhibited increased basal testosterone 16 alpha-, 2 beta-, 6 alpha- and 15 beta-hydroxylase activities and androstenedione formation, and increased Aroclor 1254-induced metabolism of testosterone at all positions except 16 alpha and 2 alpha. 5. Changes in testosterone hydroxylase activities indicative of permanent damage (or imprinting) in androgen metabolism, i.e. altered activities in 120-day-old animals, were observed only in male rats. These activities included basal testosterone 6 beta-, 16 alpha- and 2 alpha-hydroxylase activities and androstenedione formation.     

Inhibition and induction of human cytochrome P450 (CYP) enzymes

1. The present authors have previously developed a transgenic rat carrying a chimeric gene of the mouse whey acidic protein promoter and the structural portion of human growth hormone (GH) gene. Among this (hGH-TG) rat, a line (low GH rat) missing a male-specific pulsatile GH secretary pattern due to suppression of endogenous GH secretion and having a continuous low GH (hGH and rat GH) level in the peripheral circulation was identified. The latter rat was also characterized as having severe obesity with age. This strain (low Gh rat) was used to correlate the sex-specific secretory pattern of GH with the sex-specific expression of cytochrome P450 (CYP) in rat. 2. Comparisons were made between the low GH rat and the non-transgenic rat as to the expression of liver microsomal CYP isozymes. The following enzyme activities were assessed: testosterone (T) hydroxylation and oxidation; ethoxyresorufin O-dealkylation (EROD); bunitrolol (BTL) 4-hydroxylation and T5 alpha-reduction. Protein expression of CYP1A, CYP2C11, CYP2D, CYP2E1, CYP3A2 and CYP4A1 were also assessed by Western blot analysis. 3. Enzyme activities and protein expression of CYP2C11 (T16 alpha and 2alpha-hydroxylase and 17-oxidase activities) and CYP3A2 (T6beta and 2beta-hydroxylase activities) levels, which are known to be higher in the male than in the female rat, were significantly lower in the adult male low GH rat than in the control male rat. In contrast, CYP2A1 (T7 alpha hydroxylase) and T5-alpha-reductase activities, which are known to be specifically elevated in the female, were significantly higher in the adult male low GH rat than in the control male rat. Thus, the loss of male-specific secretory pattern of GH results in feminization of the pattern of expression of CYP and T5 alpha-reductase activity in the liver. 4. In contrast to other GH-deficient models so far studied, an increase in CYP4A1 and a decrease in CYP2E1 protein expression were observed in the low GH rat. These trends are consistent with the characteristic phenotype of obesity in the transgenic rat because CYP4A1 and CYP2E1 enhance fatty acid excretion and glyconeogenesis from fatty acids respectively.     

Characterization of hepatic cytochrome P450 isozyme composition in the transgenic rat expressing low level human growth hormone

1. The present authors have previously developed a transgenic rat carrying a chimeric gene of the mouse whey acidic protein promoter and the structural portion of human growth hormone (GH) gene. Among this (hGH-TG) rat, a line (low GH rat) missing a male-specific pulsatile GH secretary pattern due to suppression of endogenous GH secretion and having a continuous low GH (hGH and rat GH) level in the peripheral circulation was identified. The latter rat was also characterized as having severe obesity with age. This strain (low Gh rat) was used to correlate the sex-specific secretory pattern of GH with the sex-specific expression of cytochrome P450 (CYP) in rat. 2. Comparisons were made between the low GH rat and the non-transgenic rat as to the expression of liver microsomal CYP isozymes. The following enzyme activities were assessed: testosterone (T) hydroxylation and oxidation; ethoxyresorufin O-dealkylation (EROD); bunitrolol (BTL) 4-hydroxylation and T5 alpha-reduction. Protein expression of CYP1A, CYP2C11, CYP2D, CYP2E1, CYP3A2 and CYP4A1 were also assessed by Western blot analysis. 3. Enzyme activities and protein expression of CYP2C11 (T16 alpha and 2alpha-hydroxylase and 17-oxidase activities) and CYP3A2 (T6beta and 2beta-hydroxylase activities) levels, which are known to be higher in the male than in the female rat, were significantly lower in the adult male low GH rat than in the control male rat. In contrast, CYP2A1 (T7 alpha-hydroxylase) and T5-alpha-reductase activities, which are known to be specifically elevated in the female, were significantly higher in the adult male low GH rat than in the control male rat. Thus, the loss of male-specific secretory pattern of GH results in feminization of the pattern of expression of CYP and T5 alpha-reductase activity in the liver. 4. In contrast to other GH-deficient models so far studied, an increase in CYP4A1 and a decrease in CYP2E1 protein expression were observed in the low GH rat. These trends are consistent with the characteristic phenotype of obesity in the transgenic rat because CYP4A1 and CYP2E1 enhance fatty acid excretion and glyconeogenesis from fatty acids respectively.     

Male‐specific induction of CYP3A2 in rats by zolmitriptan

We report here a novel observation that zolmitriptan induced CYP3A2 in male but not female rats. As part of our research programme to evaluate sex differences in the response to zolmitriptan, we studied the effects of zolmitriptan on CYP3A activity, protein and gene expression in male and female rats. Zolmitriptan was found to induce CYP3A activity, measured as testosterone and diazepam metabolism in‐vitro, as well as midazolam pharmacokinetics in‐vivo, in male but not female rats. The sex difference in response to zolmitriptan was further evaluated by analysis of CYP3A1/2 mRNA levels using real‐time PCR, and CYP3A1/2 protein levels using immunoblotting. Zolmitriptan preferentially induced CYP3A2 in male but not female rats. No obvious effects on CYP3A1 were observed at any dose in either sex. Thus, we concluded that the observed sex‐dependent induction of CYP3A by zolmitriptan was largely due to induction of CYP3A2 in male rats.     

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.     

Induction of various cytochromes CYP2B, CYP2C and CYP3A by phenobarbitone in non-human primates.

Male and female patas (Erythrocebus patas) and cynomolgus (Macaca fascicularis) monkeys were treated with phenobarbitone (PB) and examined for the induction of various cytochrome P450 (P450)-mediated drug metabolizing enzymes. Hydroxylation of testosterone at the 6 beta, 2 beta, and 15 beta positions, metabolites normally associated with CYP3A P450s increased 2- to 5-fold in PB-treated animals. Induction of this P450 family was confirmed by the use of polyclonal antisera directed against the human CYP3A enzymes which inhibited both induced and constitutive 6 beta- and 15 beta-hydroxylase activities in both species of monkeys. The enzymatic activities testosterone 16 beta-hydroxylation, pentoxyresorufin O-dealkylation [PROD], and benzyloxyresorufin O-dealkylation [BZROD] typically associated with the rodent CYP2B subfamily in rodents were also examined. Testosterone 16 beta-hydroxylation activity was highly induced up to 15-fold in both species of monkeys to maximal levels similar to those induced in PB-treated male rats. BZROD was similarly induced up to 10-fold in both species of monkeys, but the maximal levels of BZROD achieved were substantially lower than those obtained in PB-treated rats. Finally, PROD yielded an idiosyncratic response showing substantial induction (> 30-fold) in certain patas monkeys (4 out of 8) but minimal (< 5-fold) induction in the other patas monkeys (4 out of 8) or any of the cynomolgus monkeys (0 out of 8). Immunodetection of various cytochromes using polyclonal antisera directed against rat cytochromes confirmed the induction of CYP3A proteins as well as the induction of protein(s) immunologically cross-reactive with rat CYP2B P450s. BZROD, PROD, or testosterone 16 beta-hydroxylase activities, were not inhibited by antibody to CYP3A P450s. However, high concentrations of polyclonal antiserum directed against rat CYP2B inhibited all three activities in PB-induced patas monkeys. In contrast, this antiserum failed to inhibit the hydroxylation of testosterone at the 6 beta or 2 beta positions. Induction of the CYP2C subfamily was observed by immunochemical detection. Interestingly, induction of this subfamily appeared to be more pronounced in cynomolgus than in patas monkeys. Finally, we failed to observe significant sex-dependent differences in P450-mediated enzymatic activities in either control or induced monkeys. These results confirm the induction of a similar spectrum of P450 proteins by PB in non-human primates to that which has previously been observed in rodents.     

Further studies on the persistence of neonatal androgen imprinting on sex-specific cytochrome P-450, testosterone and drug oxidations

Neonatal castration completely suppressed the expression of P-450-male and expressed P-450-female; and testosterone treatment in a neonatal period partially reversed the effect of castration, i.e., neonatal imprinting (Kamataki et al., 1984; Waxman et al., 1985). In the present communication, we investigate the reversibility and persistency of neonatal imprinting on the expression of P-450-male and P-450-female. To our surprise, testosterone treatment at adulthood (8 weeks old) caused full expression of P-450-male and restored the activities of 2 alpha- and 16 alpha-testosterone hydroxylases in neonatally castrated rats. The levels of ethylmorphine N-demethylation, propoxycoumarin O-depropylation and benzo(a)pyrene hydroxylation were increased to the levels of adult male rats by adult testosterone-treatment. Moreover, treatment with testosterone of neonatally castrated rats at the age of 19 weeks did not cause a complete recovery of P-450-male content and drug-metabolizing activities. Testosterone administration into neonatal female rats did not significantly alter the contents of sex-dependent cytochrome P-450 and drug and steroid metabolizing activities in adulthood. Additional testosterone treatment in adulthood only slightly affected these parameters. All these results indicate that neonatal androgen imprinting on sex-dependent cytochrome P-450 and drug and steroid metabolizing activities in rat liver microsomes is not a permanent programming process and is modified by the presence and absence of sex-steroid hormones.     

Induction of hepatic CYP2B is a more sensitive indicator of exposure to aroclor 1260 than CYP1A in male rats

To evaluate the effect of exposure to an environmentally relevant polychlorinated biphenyl mixture, adult male rats were treated with Aroclor 1260 for 7 days and levels of several cytochrome P450 (CYP) enzymes were measured in liver microsomes prepared 3 days after the last dose. Treatment with Aroclor 1260 at dosages ranging from 0.5 to 50 mg/kg/day had no effect on body weight, but liver weight was increased significantly in rats treated with the two highest dosages. Of the monooxygenase activities examined, benzyloxyresorufin O-dealkylase and testosterone 16beta-hydroxylase activities were increased to the greatest extent with maximal induction of both activities reached at 5 mg/kg/day. Densitometric quantitation of blots probed with antibody against CYP2B revealed that CYP2B1 and CYP2B2 protein levels were increased approximately 55-fold and 16-fold, respectively, after treatment with Aroclor 1260 at 5 mg/kg/day. Ethoxyresorufin O-deethylase activity and CYP1A1 protein levels displayed linear dose-dependent increases, but the hepatic CYP1A1 content did not exceed 10% that of CYP2B1 at all dosages of Aroclor 1260. Microsomal CYP3A- and CYP2A1-mediated enzyme activities and protein levels were also increased by treatment with Aroclor 1260 but to a lesser extent, whereas CYP2C11-mediated enzyme activities and protein levels were reduced. A separate time-course study showed that induction of CYP2B, but not of CYP1A, enzymes persisted for at least 48 days after treatment with Aroclor 1260 at 10 mg/kg/day. In summary, the results indicate that induction of CYP2B enzymes is a more sensitive biomarker of exposure to Aroclor 1260 than CYP1A.     

A mode of action for induction of liver tumors by Pyrethrins in the rat

High doses of Pyrethrins produce liver tumors in female rats. To elucidate the mode of action for tumor formation, the hepatic effects of Pyrethrins have been investigated. Male Sprague-Dawley CD rats were fed diets containing 0 (control) and 8000 ppm Pyrethrins and female rats' diets containing 0, 100, 3000 and 8000 ppm Pyrethrins for periods of 7, 14 and 42 days and 42 days followed by 42 days of reversal. As a positive control, rats were also fed diets containing 1200-1558 ppm sodium Phenobarbital (NaPB) for 7 and 14 days. The treatment of male rats with 8000 ppm Pyrethrins, female rats with 3000 and 8000 ppm Pyrethrins and both sexes with NaPB resulted in increased liver weights, which were associated with hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis was also increased by treatment with Pyrethrins and NaPB. The treatment of male and female rats with Pyrethrins and NaPB produced significant increases in hepatic microsomal cytochrome P450 (CYP) content and a marked induction of CYP2B-dependent 7-pentoxyresorufin O-depentylase and testosterone 16beta-hydroxylase activities. Significant increases were also observed in CYP3A-dependent testosterone 6beta-hydroxylase activity. The hepatic effects of Pyrethrins were dose-dependent in female rats with 100 ppm being a no effect level and on cessation of treatment were reversible in both sexes. This study demonstrates that Pyrethrins are mitogenic CYP2B form inducers in rat liver. The mode of action for Pyrethrins-induced rat liver tumor formation appears to be similar to that of NaPB and some other non-genotoxic CYP2B inducers of hepatic xenobiotic metabolism.     

Intestinal expression and metabolic activity of the CYP3A subfamily in female rats

The intestinal expression of the CYP3A subfamily was investigated in female rats, and the intestinal metabolism of two CYP3A substrates, testosterone and rifabutin, was examined and compared between males and females. CYP3A1/23 and CYP3A2 intestinal expression was barely detected in male and female rats. Although CYP3A9 was predominantly expressed in the female rat liver, its expression in the intestine was not different between the two sexes. The rate of testosterone 6beta-hydroxylation in the female intestine was similar to that for males. Rifabutin was also metabolized at similar rates in both intestines, although the metabolic rate was greater in the female liver. These results indicate that the intestinal drug metabolizing activity of the CYP3A subfamily is similar between males and females, and that CYP3A9 is involved in the intestinal metabolism of CYP3A substrates in both sexes.     

Sex difference in induction of hepatic CYP2B and CYP3A subfamily enzymes by nicardipine and nifedipine in rats

Male and female of F344 rats were treated per os with nicardipine (Nic) and nifedipine (Nif), and changes in the levels of mRNA and protein of hepatic cytochrome P450 (P450) enzymes, CYP2B1, CYP2B2, CYP3A1, CYP3A2, CYP3A9, and CYP3A18 were examined. Furthermore, hepatic microsomal activities for pentoxyresorufin O-dealkylation (PROD) and nifedipine oxidation, which are mainly mediated by CYP2B and CYP3A subfamily enzymes, respectively, were measured. Analyses of RT-PCR and Western blotting revealed that Nic and Nif induced predominantly CYP3A and CYP2B enzymes, respectively. As for the gene activation of CYP2B enzymes, especially CYP2B1, Nif showed high capacity in both sexes of rats, whereas Nic did a definite capacity in the males but little in the females. Gene activations of CYP3A1, CYP3A2, and CYP3A18 by Nic occurred in both sexes of rats, although that of CYP3A9 did only in the male rats. Although gene activations of CYP3A1 and CYP3A2 by Nif were observed in both sexes of rats, a slight activation of the CYP3A9 gene occurred only in female rats, and the CYP3A18 gene activation, in neither male nor female rats. Thus, changes in levels of the mRNA or protein of CYP2B and CYP3A enzymes, especially CYP2B1 and CYP3A2, were closely correlated with those in hepatic PROD and nifedipine oxidation activities, respectively. The present findings demonstrate for the first time the sex difference in the Nic- and Nif-mediated induction of hepatic P450 enzymes in rats and further indicate that Nic and Nif show different specificities and sex dependencies in the induction of hepatic P450 enzymes.     

Lack of evidence for induction of CYP2B1, CYP3A23, and CYP1A2 gene expression by Panax ginseng and Panax quinquefolius extracts in adult rats and primary cultures of rat hepatocytes.

Treatment of rats with a single oral dose (10-30 mg/kg) of a crude Panax ginseng extract of unknown ginsenoside content has been reported to modestly increase hepatic microsomal cytochrome P450-mediated aminopyrine N-demethylation activity. In the present study, we compared the effect of P. ginseng and Panax quinquefolius extracts on rat hepatic CYP2B1, CYP3A23, and CYP1A2 gene expression. Adult male Sprague-Dawley rats (250-275 g) received, by oral gavage or i.p., P. ginseng extract [4% (w/w) total ginsenosides; 30 or 100 mg/kg/day for 1 or 4 days], P. quinquefolius extract [10% (w/w) total ginsenosides; 100 or 400 mg/kg/day for 21 consecutive days), or an equivalent volume (2 ml/kg) of the vehicle (0.9% NaCl or 0.3% carboxymethylcellulose) and were terminated 1 day after the last dose. P. ginseng and P. quinquefolius extracts did not affect body weight gain, absolute or relative liver weight, hepatic CYP2B1, CYP3A23, or CYP1A2 mRNA expression, or microsomal CYP2B-mediated 7-benzyloxyresorufin O-dealkylation (BROD) or CYP1A-mediated 7-ethoxyresorufin O-dealkylation (EROD) activity. In contrast, results from positive control experiments indicated that phenobarbital increased CYP2B1 mRNA and BROD activity, dexamethasone increased CYP3A23 mRNA, and beta-naphthoflavone increased CYP1A2 mRNA and EROD activity levels. Treatment of primary cultures of rat hepatocytes with either of the ginseng extracts (0.1-1000 microg/ml for 2 days) also did not affect CYP2B1 or CYP3A23 mRNA expression. Overall, our data indicate that P. ginseng and P. quinquefolius extracts do not increase rat hepatic CYP2B1, CYP3A23, or CYP1A2 gene expression.     

Selective and potent inhibition of human CYP2C19 activity by a conformationally targeted antipeptide antibody.

A conformationally targeted anti-peptide antibody was produced by immunizing a rabbit with a cyclized peptide corresponding to a loop region of human CYP2C19 (residues 250-261). In an enzyme-linked immunosorbent assay, the antibody bound strongly to recombinant CYP2C19 and poorly to recombinant CYP2C8, CYP2C9, and CYP2C18. In immunoblotting studies, the antibody bound strongly to recombinant CYP2C19 and weakly to recombinant CYP2C8. No binding to recombinant CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP2E1, and CYP3A4 was detected. In immunoinhibition experiments, the anti-peptide antibody targeted against CYP2C19 potently inhibited (S)-mephenytoin 4'-hydroxylase activity of human hepatic microsomal fraction (>90%). It had no appreciable effect on ethoxyresorufin O-deethylase (CYP1A2), tolbutamide methyl-hydroxylase (CYP2C9), dextromethorphan O-demethylase (CYP2D6), 4-nitrophenol hydroxylase (CYP2E1), or testosterone 6beta-hydroxylase (CYP3A4) activity of human hepatic microsomal fraction. However, large amounts of purified IgG fractions were able to inhibit up to 35% of paclitaxel 6alpha-hydroxylase (CYP2C8) activity. In conclusion, we have demonstrated that an anti-peptide antibody targeted against residues 250 to 261 of human CYP2C19 selectively and potently inhibited CYP2C19 activity of human hepatic microsomal fraction.