2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal steroid metabolism and serum estradiol of pregnant rats

Experiments were conducted to evaluate the effects of administration of low, but fetotoxic quantities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy on steroid metabolism in liver microsomes. Oral administration of 1 microgram X kg-1 X day-1 of TCDD to pregnant rats on days 7-19 of gestation reduced maternal weight gain during pregnancy. Analysis of litters on day 20 showed that fetuses from TCDD-treated dams had a 66% incidence of visceral lesions characterized by intestinal hemorrhage. Liver microsomes prepared from TCDD-treated dams on day 20 of gestation exhibited a 2- to 3-fold increase in cytochrome P-450 content which was accompanied by a shift in the absorbance optimum of the dithionite reduced-CO spectrum to 448 nm. Catechol estrogen formation activity was decreased by 50-75% in hepatic microsomes from TCDD-treated dams. In contrast 7 alpha-hydroxylation of testosterone increased nearly 4-fold, while 16 alpha- and 6 beta-hydroxylase activities were unchanged in microsomes following exposure to TCDD. Thus, the inhibition of catechol estrogen formation associated with TCDD treatment did not reflect a general decrease in microsomal steroid hydroxylase activities. Insofar as catechol estrogen formation is physiologically a major pathway for estrogen metabolism, serum concentrations of 17 beta-estradiol were measured in a second group of pregnant rats treated with TCDD on days 4-15 of gestation. Serum estradiol levels were not different between control and treated dams at this stage of pregnancy. Thus, the present study does not support a link between TCDD-mediated inhibition of catechol estrogen formation measured in vitro in liver microsomes and altered circulating estradiol levels in vivo during pregnancy.     

The binding of isocyanides to cytochrome P-450 from mouse hepatic microsomes

The spectral interactions of a number of isocyanides with cytochrome P-450 were investigated. An interaction between the hydrophobic nature of the side chain and the spectral interaction was apparent. The concentration of the isocyanide affected the stability of the complex. Phenyl isocyanide dichloride was determined to be an acceptable replacement for ethyl isocyanide for the characterization of cytochrome P-450.     

Effects of cytochrome P-450 monooxygenase inducers on mouse hepatic microsomal metabolism of testosterone and alkoxyresorufins

The effects of treatment with phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), pregnenolone-16 alpha-carbonitrile (PCN), 3-methylcholanthrene (3-MC) and isosafrole on the hepatic microsomal formation of nine monohydroxy metabolites of testosterone and the O-dealkylation of the ethyl and pentyl ethers of resourfin were evaluated in adult male C57BL/6J and DBA/2NCR mice. In both strains, phenobarbital, TCPOBOP and PCN induced testosterone 2 beta-, 6 beta-, 15 beta- and 16 beta-hydroxylases up to 5-fold, while phenobarbital and TCPOBOP increased the rate of dealkylation of pentoxyresorufin by approximately 30-fold. However, phenobarbital and TCPOBOP did not exhibit identical patterns of induction for the testosterone oxidation reactions. Hepatic microsomes from C57BL/6J mice treated with TCPOBOP displayed a depression in 6 alpha-testosterone hydroxylase activity, which was also observed in PCN-treated animals, whereas phenobarbital-treated mice exhibited an elevation in this monooxygenase activity. A dose of TCPOBOP (0.5 mumol/kg) previously demonstrated to represent an ED50 for mouse aminopyrine N-demethylase activity was also found to approximate the ED50 for pentoxyresorufin O-dealkylase activity in the C57BL/6J mouse. Isosafrole or 3-MC treatment had little effect on testosterone metabolism or pentoxyresorufin O-dealkylase activity in either strain, while 3-MC induced ethoxyresorufin O-deethylase activity in C57BL/6J but not DBA/2NCR mice. This study confirms that TCPOBOP is a potent cytochrome P-450 inducer which most closely resembles phenobarbital in its mode of action. However, TCPOBOP and phenobarbital do not evoke identical modulations of cytochrome P-450-dependent monooxygenases in mice.     

Effects of bromocriptine on hepatic cytochrome P-450 monooxygenase system

We have evaluated the in vitro effects of bromocriptine (Br), on the hepatic cytochrome P-450 monooxygenase system of rats pretreated with saline phenobarbitone (PB) and beta-naphthoflavone (BNF). Br inhibited ethoxyresorufin O-dealkylase (EROD) activity in liver microsomes of rats pretreated with saline and PB but not in BNF pretreated animals. Maximum inhibition of EROD activity by Br in the microsomes of saline and PB pretreated rats were 50%-60% of the control. In contrast, a dual effect was observed on aminopyrine N-demethylase activity (APD) by Br in microsomes of saline, PB and BNF pretreated rats. At a low concentration (25 microM), Br inhibited the activity of APD to a similar extent in all pretreatment groups; however, with higher concentrations of Br (50 microM to 300 microM), enhancement of APD activity was observed. Br (300 microM) increased the APD activity to 2-3 times the control level in microsomes of rats pretreated with saline, PB or BNF. Spectral studies revealed a Type II binding of Br to cytochrome P-450 from microsomes of saline and PB pretreated rats. A reverse type I binding was observed for BNF induced microsomes. In addition, Br also enhanced NADPH cytochrome c (P-450) reductase activity to a similar extent in all pretreatment groups. These results suggest that the inhibition of EROD activity may be due to direct binding by Br to certain isozymes of cytochrome P-450 and that the enhancing effect of Br on APD activity may be in part due to the activation of the NADPH cytochrome c reductase component of the cytochrome P-450 monooxygenase system.     

In vivo effects of erythromycin, oleandomycin and erythralosamine derivatives on hepatic cytochrome P-450

Rats have been treated with several derivatives of the erythromycin, erythralosamine or oleandomycin series, in order to compare their ability to induce cytochrome P-450 and to form stable 456 nm-absorbing cytochrome P-450 metabolite complexes. The data obtained confirm that the cytochromes P-450 induced in rats by various macrolides are similar to that induced by pregnenolone 16 alpha-carbonitrile: the cytochrome P-450 IIIA1 isozyme. It showed that: (i) formation of a stable inhibitory 456 nm-absorbing cytochrome P-450 complex is not a prerequisite for cytochrome P-450 induction but enhances induction by stabilization of the IIIA isozyme. Therefore, the best inducers lead also to the maximal in vivo amounts of cytochrome P-450 metabolite complex (except for 2'MBEM); (ii) affinity for cytochrome P-450 IIIA1 is not directly involved for induction; and (iii) hydrophobicity favors induction and formation of complexes. Structural factors are also involved.     

Effects of intravenous emulsified perfluorochemicals on hepatic cytochrome P-450

Intravenous infusion of emulsified perfluorodecalin in rats caused a large increase in hepatic cytochrome P-450 concentration which persisted for many weeks. In contrast, hepatic cytochrome P-450 concentration was not changed significantly after infusion of perfluorotributylamine, but subsequent administration of phenobarbital caused the usual increase of cytochrome P-450. The cytochrome P-450 activity for demethylation of benzphetamine was decreased slightly after perfluorodecalin but was unchanged after perfluorotributylamine. The difference in the effects of these perfluorochemicals on hepatic cytochrome P-450 may be related to the difference in the time these compounds are retained in the liver.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal cytochrome P-448-mediated enzyme activities.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-³H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.     

2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated depression of rat testicular heme synthesis and microsomal cytochrome P-450

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chlorance, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 μg/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular δ-aminolevulinic acid (ALA) synthase were detected. The incorporation of [¹⁴C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.     

Metabolism of dichlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of ten individual dichlorobiphenyls (DCBs) has been investigated as part of a major study of the role of metabolism in the toxicity of polychlorinated biphenyl (PCB) pollutant mixtures. The DCBs were metabolized to monohydroxy and dihydrodiol metabolites and unstable metabolites of intermediate polarity. DCBs with both chloro substituents on the same ring, one or both of which were ortho substituents, were susceptible to the same regioselectivities for hydroxylation by control, phénobarbital (PB)- or β-naphthoflavone (BNF)-induced cytochromes P-450 (principally in the 4-position), with the greatest rates of hydroxylation arising with PB-induced cytochrome P-450. In contrast, DCBs with no ortho chlorosubstituents had regioselectivities for hydroxylation by control and PB-induced cytochrome P-450 which differed from that of BNF-induced cytochromes P-450; the greatest rates of hydroxylation were with BNF-induced systems. DCBs with one chloro substituent on each ring were metabolized, with the site of hydroxylation being under the electronic influence of the chloro substituent. With 4,4'-DCB, 60 per cent of the hydroxylated DCB metabolite underwent an NIH shift [G. Guroff, J. W. Daly, D. M. Jerina, J. Renson, B. Witkop and S. Udenfriend, Science157, 1524 (1967)]. The BNF-induced system produced the highest rates of dihydrodiol fomation that were eliminated by an epoxide hydratase inhibitor. The results indirectly prove that arene oxides are intermediates in DCB metabolism and are possibly the source of DCB mutagenicity. The PCBs 2,4,2'4'- and 3,4,3',4'-tetrachlorobiphenyl induced the same effects as PB and BNF respectively. Thus, PCBs differentially affect the metabolism of their individual components and are, possibly, responsible for enhancing their own toxicity by inducing enhanced rates of formation of arene oxide intermediates.     

Metabolism of monochlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of the monochlorobiphenyls (MCBs) 2-, 3- and 4-chlorobiphenyl, has been investigated as a model for the metabolism of polychlorinated biphenyl pollutants. MCB metabolism was catalyzed by cytochrome P-450, as indicated by a dependence on NADPH and O₂, inhibition by 2-diethylaminoethyl-2,2-diphenylpropylacetate (SKF 525-A), metyrapone and CO, and the formation of type I difference spectra, on the addition of MCBs to microsomes. All MCBs yielded a 4'-monohydroxy MCB as the major metabolite, as determined by mass and nuclear magnetic resonance spectroscopy, dechlorination to 4-hydroxybiphenyl, and high-pressure liquid chromatography retention times. Minor monohydroxy and dihydroxy metabolites were also produced from the MCBs. The regioselectivity of control cytochrome P-450 for metabolism of MCBs at the 4' position was not altered by preinduction of cytochrome P-450 with 2,4,2',4'-tetrachlorobiphenyl (TCB) or cytochrome P-448 with 3,4,3', 4'-TCB. 2-Chlorobiphenyl was metabolized only by control and induced cytochrome P-450; 3- and 4-chlorobiphenyl were metabolized by control and by induced cytochrome P-450 and P-448. Thus, the regioselectivity of metabolism of MCBs is independent of the chlorine position or the form of the induced cytochrome involved, but the extent of metabolism of polychlorinated biphenyls (PCBs) is determined by induction of the hepatic cytochromes P-450.     

The effects of cimetidine, ranitidine and famotidine on rat hepatic microsomal cytochrome P-450 activities

It has been questioned whether the interaction of H2-antagonists with cytochrome P-450 that is observed in vitro is also relevant for the in vivo situation. Until now the possibility that cytochrome P-450 may function with different modes of action has been neglected in this respect. We studied the effect of cimetidine, ranitidine and famotidine on the monoxygenase, the oxidase and the peroxidase action of cytochrome P-450. Biotransformation catalyzed by the monoxygenase and oxidase action of cytochrome P-450 was affected by cimetidine (probably via its ligand interaction with cytochrome P-450), whereas metabolism by the peroxidase mode of action of cytochrome P-450 was hardly influenced. Ranitidine and famotidine (both pharmacodynamically more potent than cimetidine) only slightly affected cytochrome P-450 activities.     

6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities

In addition to being one of the most toxic chemicals known, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent inducer of rat liver microsomal cytochrome P-4501A1 (P-450c). Previous studies have demonstrated that a high affinity, low capacity cytosolic receptor (the Ah receptor) mediates the activity of TCDD to induce cytochrome P-4501A1, which catalyzes benzo[a]pyrene hydroxylation [aryl hydrocarbon hydroxylase (AHH]) and 7-ethoxyresorufin O-dealkylation (EROD). The results of the present study indicate that 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) effectively competes with [3H]TCDD for binding to the Ah receptor in rat liver cytosol. The concentration of MCDF effecting 50% displacement of [3H]TCDD was 4.9 X 10(-8) M, which is approximately 50 times greater than the EC50 for unlabeled TCDD (approximately 1 X 10(-9) M). However, in contrast to TCDD, MCDF was only a weak inducer of AHH and EROD activity in rat hepatoma H-4-II cells in culture. When co-incubated, MCDF diminished in a concentration-dependent manner the ability of TCDD to induce AHH and EROD activity in vitro. Treatment of rats with 20-200 mumol/kg MCDF in vivo had little or no effect on liver microsomal AHH and EROD activity, whereas treatment of rats with 16 nmol/kg TCDD caused a 6- and a 70-fold induction of AHH and EROD activity, respectively. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce AHH and EROD activity in vivo. The partial antagonism produced by 50 mumol/kg MCDF could be partially overcome by doubling the dosage of TCDD from 16 to 32 nmol/kg. Immunochemical analysis of rat liver microsomes revealed that treatment of rats with 20-200 mumol/kg MCDF caused little or no induction of cytochromes P-4501A1 and P-4501A2 (P-450d), whereas these isozymes were induced 33- and 5-fold, respectively, in rats treated with 16 nmol/kg TCDD. When co-administered, MCDF diminished by approximately 50% the ability of TCDD to induce cytochrome P-4501A1 in vivo, which established that MCDF was not simply acting as an inhibitor of AHH and EROD activity. MCDF also antagonized the ability of TCDD to induce cytochrome P-4501A2, which suggests that the induction of both cytochromes P-4501A1 and P-4501A2 is regulated by the Ah receptor. These results indicate that MCDF binds with high affinity to the Ah receptor in rat liver cytosol and competitively blocks the binding of TCDD.(ABSTRACT TRUNCATED AT 400 WORDS)     

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。