Testosterone hydroxylase as multibiomarker of effect in evaluating vinclozolin cocarcinogenesis

In this work the modulation of the regio- and stereo-selective hydroxylation of testosterone by vinclozolin was studied in evaluating cocarcinogenic properties. Changes of cytochrome P450-(CYP)-catalysed drug metabolism was investigated in liver, kidney and lung microsomes of Swiss Albino CD1 mice of both sexes after single (625 or 1250 mg kg-1 b.w.) or repeated (daily 750 mg kg-1 b.w. for 3 days) i.p. administrations. Treatment of mice with a single dose of vinclozolin caused in a dose-dependent fashion from 2 1 to 14 1-fold increase in the 7-, 6- and 2-hydroxylations of testosterone in liver. Lower increase in extrahepatic tissues ranging from 2 3 to 8 1-fold for testosterone 6-, 16 -, 2- and 2- hydroxylase activity in the kidney or from 2 2 to 5 1-fold for 6-, 16 -, 16 - and 2- hydroxylase activity in the lung were observed. Repeated treatment with this fungicide did not substantially modify the extent and pattern of induction, the liver being the only tissue responsive (up to 7 6-fold increase, 7-hydroxylation) in both male and female. In the kidney (7-, 6-, 16 -, 2-, 7-hydroxylations) and lung (6-, 7-, 6-, 16 -, 16 - and 2- hydroxylations), a typical sex-dependent induction (up to 9 0-fold, 16 -hydroxylation in the lung, female) was achieved. In general, however, vinclozolin has a complex pattern of induction and suppression of CYP-dependent enzymes, as exemplified from the reduced expression of some hydroxylations depending upon dose, sex and organ considered. For example, after a single administration, 16 -hydroxylation was suppressed in liver (up to 78% loss in male, higher dose), whereas 16 -hydroxylation was reduced in kidney up to 50% in both sexes (at the higher dose). Glutathione S-transferase activity, measured as index of post-oxidative reactions, was markedly increased by vinclozolin in the liver (up to 5 2-fold, female) and kidney (up to 3 9-fold, female) but not in the lung. Because both phase I and phase II reactions were enhanced by vinclozolin treatment in liver and kidney, the ratio between activation/detoxification mechanisms was slightly affected. Conversely, this ratio was shifted toward activating mechanisms in the lung, sustaining, in part, the expression of certain type of tumours tissue-dependent. Taken together, these findings seem to indicate the cotoxic, cocarcinogenic and promoting potential of this fungicide.     

25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

The metabolism of estradiol 17-sulfate by pheochromocytoma tissue

The metabolism of estradiol 17-sulfate by subcellular localization enzymes of pheochromocytoma tissue obtained from a 41-year old female was investigated. In any incubations under the presence of NADH and NADPH, metabolites hydroxylated at the C-2, C-4, C-6 beta, C-7 alpha and C-7 beta positions were produced. These hydroxylations are considered to occur without cleavage of the sulfate group. The 2-hydroxylation at the substrate concentration of 100 microM by mitochondria, microsomes and cytosol fractions occurred at rates of 141, 222 and 167 pmol/mg protein/30 min, respectively; the corresponding rates for the 4-hydroxylation were 24, 40 and 38 pmol/mg protein/30 min. Mitochondrial 2- and 4-hydroxylations were enhanced by addition of calcium ion (Ca2+) into the incubation medium.     

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver,kidney and lung of CD1 mice

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.     

Evidence for the involvement of a distinct form of cytochrome p450 3a in the oxidation of digitoxin by rat liver microsomes

The preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1. J. Biochem. Toxicol, 7 (43–52).) described the regulation of two rat liver microsomal proteins (50- and 51-kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51-kDa 3A protein were usually paralleled by changes in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation. The present study demonstrates that age- and sex-dependent changes in the 50-kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50-kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2β-, 6β- and 15β-hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this coclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone-16α-carbonitrile (PCN), which induces both the 50-kDa and the 51-kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16- to 18-fold) in the 6β-hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN-induced rats with chloramphenicol caused a ～70% decrease in liver microsomal testosterone 6β-hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51-kDa protein) was also inhibited by chloramphenicol in a time- and reduced nicotinamite adenine dinucleotide phosphate (NADPH)-dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized trole-andomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes. We propose that the 2SbT-, 6β-, and 15β-hydroxylation of testosterone by rat liver microsomes is primarily catalyzed by the 51-kDa 3A proteins (either 3A1 or 3A2 depending on the source of microsomes), whereas digitoxin oxidation is primarily catalyzed by the 50-kDa protein.     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

Heterogeneity of hepatic mixed function oxidases

The effects of NADH and increasing concentrations of potassium phosphate buffer, potassium chloride and potassium thiocyanate on several hydroxylations catalyzed by rat liver microsomes were studied. All the hydroxylations were stimulated by NADH in the presence of suboptimal concentrations of NADPH. The 7α-hydroxylation of cholesterol, the 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and the 16-hydroxylation of palmitic acid were inhibited by increasing concentrations of potassium phosphate buffer, potassium chloride and potassium thiocyanate to a greater extent than any of the other hydroxylations studied. This finding and the previous finding that these three hydroxylations are not stimulated by phenobarbital treatment suggest differences between these hydroxylations and most other microsomal hydroxylations in liver. The possibility is discussed that different types of cytochrome P-450 may be involved.     

Side chain hydroxylations in bile acid biosynthesis catalyzed by CYP3A are markedly up-regulated in Cyp27-/- mice but not in cerebrotendinous xanthomatosis.

The accumulation of various 25-hydroxylated C(27)-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27(-/-) mice do not show CTX-related abnormalities. Microsomal activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6beta-hydroxylase, a marker enzyme for CYP3A, in Cyp27(-/-) mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases and 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6beta-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27(-/-) mice, CYP3A activity was not up-regulated despite marked accumulation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in CTX.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

Summary The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).     

Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16α-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male > female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2β, 6β-, and 15β-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2β-, 6β-, and 15β-hydroxylation of testosterone.     

Effect of biliary obstruction on formation and metabolism of bile acids in rat

The effect of biliary obstruction in the rat on several hydroxylations involved in the formation and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The rate of 7α-hydroxylation of cholesterol increased two- to threefold between 24 and 48 hours after ligation of the bile duct and remained at this level the next 48 hours. During the first 24 hours of obstruction the rates of 1 2α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and 7α-hydroxylation of taurodeoxycholic acid decreased but returned to control levels between 24 and 48 hours after operation. The rate of 6β-hydroxylation of lithocholic acid and taurochenodeoxycholic acid increased gradually and reached a plateau between 24 and 48 hours at which time the rate was two to three times faster than in the controls. The increase in 6β-hydroxylase activity was reflected in the pattern of the bile acids excreted in urine. After 48 hours of obstruction β-muricholic acid accounted for 50% or more of the bile acids in urine.     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Captan impairs CYP-catalyzed drug metabolism in the mouse

To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6beta-(CYP3A), 6alpha-(CYP2A1 and CYP2B1) and 16alpha-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2alpha-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O*2-) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The adverse outcomes associated to CYP changes (e.g. cotoxicity, comutagenicity and promotion) may also have harmful consequences.     

Sex differences in the hydroxylation of cholecalciferol and of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in rat liver.

The effect of sex hormones on hydroxylation of cholecalciferol ('vitamin D3') and of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol has been investigated in female- and male-rat livers. The mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities were respectively 4.6- and 2.7-fold higher in female- than in male-rat livers. The microsomal 1 alpha-hydroxycholecalciferol 25-hydroxylase was 2.8-fold higher in male- than in female-rat liver. No significant difference was found in the microsomal 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. Liver microsomes (microsomal fractions) from male, but not from female, rats also catalysed 1-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. Injection of testosterone into female rats decreased the mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities, but not to a statistically significant extent. Testosterone treatment had no effect on the microsomal hydroxylases in female-rat liver. Injection of oestradiol valerate to male rats resulted in increased activities of both mitochondrial hydroxylases to the same levels as those of control females, while the microsomal enzyme activities decreased. The present results indicate that sex hormones exert a regulatory control on the mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes

We examined and compared enantioselectivity in the oxidation of propranolol (PL) by liver microsomes from humans and Japanese monkeys (Macaca fuscata). PL was oxidized at the naphthalene ring to 4-hydroxypropranolol, 5-hydroxypropranolol and side chain N-desisopropylpropranolol by human liver microsomes with enantioselectivity of [R(+)>S(-)] in PL oxidation rates at substrate concentrations of 10 microM and 1 mM. In contrast, reversed enantioselectivity [R(+)相似文献