Induction of cytochrome P-450c and P-450d by metyrapone in the primary culture of rat hepatocytes

The effects of metyrapone on qualitative changes in cytochrome P-450-dependent drug metabolizing activities in primary cultures of rat hepatocytes were investigated. Metyrapone apparently increased benzo(a)pyrene hydroxylation and maintained both ethoxycoumarin-O-deethylation and propoxycoumarin-O-depropylation, whereas it had little effect on methoxycoumarin-O-demethylation. Furthermore, P-450d (high spin type of P-448) as well as P-450c (low spin type of P-448) were induced by metyrapone, while P-450b and P-450e were not. In conclusion, metyrapone act as a 3-methylcholanthrene-like inducer in the primary cultures of rat hepatocytes.     

Postnatal development of constitutive forms of cytochrome P-450 in liver microsomes of male and female rats

In a previous paper (1), we reported on the purification of constitutive forms of cytochrome P-450, namely P-450-male and P-450-female, from liver microsomes of male and female rats, respectively. Immunochemical examinations of these hemoproteins showed that P-450-male and P-450-female were detectable specifically in respective liver microsomes of adult male and female rats. The synthesis of P-450-male was apparently dependent on testosterone, and that of P-450-female was dependent on estradiol.Thus, in this study we examined the postnatal development of P-450-male and P-450-female. We show herein that P-450-female is primarily synthesized before the occurrence of P-450-male in male rats. This and other results support the view that the synthesis of unknown pre-existing forms of cytochrome P-450 is depressed in association with the appearance of P-450-male and P-450-female during postnatal periods before sexual maturation.     

Effects of a hormone-supplemented medium on cytochrome P-450 content and mono-oxygenase activities of rat hepatocytes in primary culture

Cytochrome P-450 content and associated mono-oxygenation activities (7-ethoxycoumarin-O-deethylase, biphenyl-4-hydroxylase and 7-ethoxyresorufin-O-deethylase) of rat hepatocytes were found to decrease during the first 48 hr in primary culture in control (WOBA-M₂) medium. However, by culturing the hepatocytes in a hormone-supplemented medium (AB medium), all of these enzymes were maintained at higher levels after 12. 24 and 48 hr in culture. In particular. 7-ethoxyresorufin-O-deethylase activity was markedly enhanced after 12 and 24 hr in culture in AB medium to levels greater than that in isolated hepatocytes. Metabolic capacities of the cytochromes P-450 present in hepatocytes cultured in WOBA-M₂ medium vs AB medium were also quantitatively different at 12, 24 and 48 hr when specific activities/pmole of hemoprotein were compared. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiments further suggested that a cytochrome P-450-related protein was maintained to a greater extent in AB medium than in WOBA-M₂ medium. It is proposed that AB medium may maintain a higher cytochrome P-450 concentration in cultured primary rat hepatocytes by increasing both the rate of hcme synthesis and the synthesis of a cytochrome P-450-related protein.     

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.     

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 cytochrome P-450 inducers on the perazine metabolism in a primary culture of human hepatocytes

The metabolism of perazine in a primary culture of human hepatocytes after treatment of cells with TCDD (a CYP1A1/2 inducer) or rifampicin (mainly a CYP3A4 inducer) were studied in vitro. The concentrations of perazine and its main metabolites (perazine 5-sulfoxide, N-desmethylperazine) formed in hepatocytes were assayed in the extracellular medium using the HPLC method. TCDD and rifampicin induced the formation of perazine 5-sulfoxide, however, such an effect was not observed in the case of N-desmethylperazine. The accumulation of perazine 5-sulfoxide in the extracellular medium was enhanced until up to 4 h by rifampicin, and until up to 8 h byTCDD. After 24 h, perazine and perazine 5-sulfoxide were not detected in the extracellular medium of the inducer-treated cultures, except for perazine 5-sulfoxide in the TCDD-treated cultures The obtained results indicate that CYP1A2 and CYP3A4 are involved in the perazine metabolism via 5-sulfoxidation pathway.     

Purification and aminopyrine monooxygenase activity of liver microsomal cytochrome P-450 from alloxan-induced diabetic rats.

We purified two diabetes-inducible and insulin-sensitive forms of cytochrome P-450, named P-450AL-1 and AL-2, from the liver microsomes of alloxan-diabetic male rats, using sodium cholate solubilization, octylamino-Sepharose 4B chromatography, and HPLC with diethylaminoethyl-5PW and hydroxyapatite columns. The purified forms gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 50,000 for P-450AL-1 or 48,500 for P-450AL-2. The CO-reduced spectral maximum of these forms was at 452 nm for P-450AL-1 and 451 nm for P-450AL-2. The two purified forms had the low-spin state of heme in the oxidized form. Both P-450AL-1 and AL-2 were active in the metabolism of aniline, benzphetamine, and 7-ethoxycoumarin. However, the catalytic activity of P-450AL-2 for these substrates was obviously higher than that of AL-1. The NH2-terminal sequences of P-450AL-1 and AL-2 differed from each other, and did not agree with those of the other P-450 forms purified from diabetic rats previously. Furthermore, we examined the metabolism of aminopyrine in a reconstituted system with the purified cytochromes P-450. The diabetes-inducible forms of P-450 had high aminopyrine 3-hydroxylation and low N-demethylation activities. These findings provide clear evidence supporting our previous results, which have shown an increase in 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e and a decrease in 4-monomethylaminoantipyrine in intact diabetic rats.     

Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.

Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.     

Regulation of heme metabolism and cytochrome P-450 levels in primary culture of rat hepatocytes in a defined medium

Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of delta-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30-40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19-24 hr when its activity was about eight times the original activity. In 2-4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of delta-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 microM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme.     

Induction of cytochrome P-450, cytochrome b-5, NADPH-cytochrome c reductase and change of cytochrome P-450 isozymes with long-term trichloroethylene treatment

Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected with TCE, 1.0 g/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21%) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52,000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase in similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56,000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB.     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450

1. Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing monooxygenase and the stoichiometry between substrate, O₂ and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity.

2. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines.

3. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.     

Hypo activity of cytochrome P-450 after triacetyloleandomycin administration

In a preceding communication we reported that triacetyloleandomycin (TAO) induces its own transformation into a metabolite forming a stable complex with the iron (II) of reduced cytochrome P-450; the complex is unstable in the ferric state and disrupted by potassium ferricyanide. In this communication, we report the effects of TAO administration on various monooxygenase activities. Repeated administration of TAO, 1 mmole/kg i.p. daily for 4 days, markedly prolonged the hexobarbital sleeping time, decreased the in vivo disappearance rate of hexobarbital from the liver, and decreased the in vitro activity of hexobarbital hydroxylase, benzo(a)pyrene hydroxylase, ethylmorphine N-demethylase and the in vitro conversion of 17β-estradiol into water-soluble metabolites. Hypoactivity of musomal enzymes was not detected 1 hr after a single dose and was moderate 24 hr after a single dose of TAO, 1 mmole/kg i.p. In vitro, addition of 0.3 mM TAO to the incubation mixture decreased hexobarbital hydroxylase activity by only 6% in control musomes or musomes from rats killed 1 hr after a single dose of TAO, but decreased it by 30% in musomes from rats killed 24 hr after a single dose of TAO and by 60% in musomes from rats killed after repeated doses of TAO. Addition of 50 μM potassium ferricyanide to the musomes did not modify monooxygenase activities in control musomes but increased them in musomes from rats treated with repeated doses of TAO. It is concluded that the administration of TAO decreases several monooxygenase activities and that hypoactivity is at least partly related to the in vivo formation, in TAO-induced rats, of an inactive cytochrome P-450-TAO metabolite complex.