Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.     

Glucocorticoids inhibit interconversion of 7-hydroxy and 7-oxo metabolites of dehydroepiandrosterone: a role for 11beta-hydroxysteroid dehydrogenases?

The cytochrome p450-dependent formation and subsequent interconversion of dehydroepiandrosterone (DHEA) metabolites 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA was observed in human, pig, and rat liver microsomal fractions. Rat liver mitochondria and nuclei also converted DHEA to 7 alpha-OH-DHEA and 7-oxo-DHEA, but at a lower rate. With NADP(+), and less so with NAD(+), rat, pig, and human liver microsomes and rat liver mitochondria and nuclei converted 7 alpha-OH-DHEA to 7-oxo-DHEA. This reaction was inhibited by corticosterone and the 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) inhibitor carbenoxolone (CBX). The conversion of 7 alpha-OH-DHEA to 7-oxo-DHEA by rat kidney occurred at higher rates with NAD(+) than with NADP(+) and was inhibited by corticosterone. With NADPH, 7-oxo-DHEA was converted to unidentified hydroxylated metabolites and low levels of 7 alpha-OH-DHEA by rat liver microsomes. In contrast, pig liver microsomal fractions reduced 7-oxo-DHEA to nearly equal amounts of 7 alpha- and 7 beta-OH-DHEA, while human fractions produced mainly 7 beta-OH-DHEA. Dehydrocorticosterone inhibited the reduction to both isomers by pig liver microsomes, but only to 7 alpha-OH-DHEA by human microsomes; CBX inhibited both reactions. Rat kidney did not reduce 7-oxo-DHEA with either NADPH or NADH. These results demonstrate that DHEA is first converted in liver to 7 alpha-OH-DHEA, which is subsequently oxidized to 7-oxo-DHEA in both liver and kidney. In liver, interconversion of 7-oxo-DHEA and 7-OH-DHEA isomers is largely catalyzed by 11 betaHSD1, while in kidney 11 betaHSD2 (NAD(+)-dependent) and 11 betaHSD3 (NADP(+)-dependent) likely catalyze the unidirectional oxidation of 7 alpha-hydroxy-DHEA to 7-oxo-DHEA. Distinct species-specific routes of metabolism of DHEA and the interconversion of its metabolites obviate extrapolation of animal studies to humans.     

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.     

Ontogenic development of liver progesterone metabolism in female sheep. Contribution of cytochrome P4502B and P4503A subfamilies.

Age-related changes in progesterone hepatic metabolism were measured in Lacaune ewes in the foetal, neonatal (1 and 4 weeks), growing (7 months), pregnant (11 months) and adult (6 years) stages. 6 beta-Hydroxylation and 20 alpha-reduction were found to be the most efficient metabolic process in ovine microsomes. These activities were detected in 3-month-old foetuses and they increased rapidly during the first month of life, in a similar manner to the developmental expression of the cytochrome P4503A subfamily. 16 alpha- and 21-hydroxylation of progesterone were characterized by low, constant turn over in sheep liver microsomes during development. The hepatic ovine P4502B isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, hydroxylapatite and CM cellulose chromatographic separations. This hemoprotein had an apparent molecular weight of 51 kDa and was characterized by spectral data, NH2-terminal amino-acid sequence, immunological and catalytic properties. The relative contribution of this form and of the previously purified ovine P4503A subfamily was investigated in liver progesterone metabolism by immunoinhibition studies using polyclonal antibodies raised in rabbits and from the existence of induction and of significant correlations between microsomal activity and specific P450 content. In sheep liver microsomes, it would appear that cytochrome P4502B is involved in progesterone 21-hydroxylation whereas P4503A participates in the 6 beta- and 16 alpha-hydroxylation and possibly in the reductive conversion of progesterone in its 20 alpha-hydroxy derivative.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism

The metabolism of the dihydropyridine calcium antagonist and vasodilator nifedipine has been reported to exhibit polymorphism among individual humans (Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M., Vermeulen, N. P. E., and Breimer, D.D. (1984) Biochem. Pharmacol. 33, 3721-3724). Nifedipine oxidation has been shown to be catalyzed by cytochrome P-450 (P-450) enzymes. Reconstitution, immunoinhibition, and induction studies with rat liver indicated that the forms designated P-450UT-A and P-450PCN-E are the major contributors to microsomal nifedipine oxidation. The P-450 which oxidizes nifedipine (P-450NF) was purified to electrophoretic homogeneity from several human liver samples. Antibodies raised to P-450NF were highly specific as judged by immunoblotting analysis and inhibited greater than 90% of the nifedipine oxidase activity in human liver microsomes. A monoclonal antibody raised to the human P-450 preparation reacted with both human P-450NF and rat P-450PCN-E. Immunoblotting analysis of 39 human liver microsomal samples using anti-P-450NF antibodies revealed the same 52,000-dalton polypeptide, corresponding to P-450NF, with only one of the microsomal samples showing an additional immunoreactive protein. The level of nifedipine oxidase activity was highly correlated with the amount of P-450NF thus detected using either polyclonal (r = 0.78) or monoclonal (r = 0.65) antibodies, suggesting that the amount of the P-450NF polypeptide may be a major factor in influencing the level of catalytic activity in humans as well as rats. Cytochrome b5 enhanced the catalytic activity of reconstituted P-450NF, and anti-cytochrome b5 inhibited nifedipine oxidase activity in human liver microsomes. P-450NF also appears to be a major contributor to human liver microsomal aldrin epoxidation, d-benzphetamine N-demethylation, 17 beta-estradiol 2- and 4-hydroxylation, and testosterone 6 beta-hydroxylation, the major pathway for oxidation of this androgen in human liver microsomes.     

In vitro metabolism of FK-506 in rat, rabbit, and human liver microsomes: identification of a major metabolite and of cytochrome P450 3A as the major enzymes responsible for its metabolism.

The metabolism of the immunosuppressant FK-506 was shown to be catalyzed primarily by cytochrome P450 isozymes of the P450 3A subfamily. Antibodies against rat P450 3A inhibited FK-506 metabolism by 82% in rat liver microsomes and by 35-56% in liver microsomes from humans, dexamethasone-induced rats, and erythromycin-induced rabbits. Poor species cross-reactivity of the antibodies, metabolic switching, and/or some metabolism by P450 isozymes other than P450 3A may be responsible for the incomplete inhibition observed. Besides anti-rat P450 3A, antibodies against rat P450 1A also appeared to have some inhibitory effect implicating these particular cytochrome P450 isozymes as having a minor role in FK-506 metabolism. The formation of 13-desmethyl FK-506, identified here as a major metabolite of FK-506 in all types of microsomes examined, was inhibited completely by anti-P450 3A in liver microsomes from dexamethasone-induced rats and erythromycin-induced rabbits but only partially in human and control rat liver microsomes.     

Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS(n)) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.     

Selective potent restriction of P450b- but not P450e-dependent 7,12-dimethylbenz[a]anthracene metabolism by the microsomal environment

The prototypic members of the rat liver cytochrome P450IIB subfamily, P450b and P450e, differ by only 13 amino acids and yet purified P450b is considerably more active than P450e for all known substrates. A unique regioselectivity difference between cytochromes P450b and P450e for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and a genetic deficiency in P450e expression in the Marshall (M520/N) rat strain have been exploited to determine the microsomal contributions of the respective forms toward the metabolism of DMBA. The total contribution to metabolism by each isozyme has been assessed based on the sensitivity to rabbit anti-P450b/e IgG and comparison with microsomal P450b and P450e content as measured by Western blots. Liver microsomes from untreated M520/N rats do not express detectable levels of P450e but express P450b at a level that is 2-fold higher than that of P450e in liver microsomes from untreated F344 rats (50 pmol/mg). However, only 4% of the constitutive DMBA metabolizing activity of liver microsomes from the M520/N rat strain could be inhibited by anti-P450b/e IgG. A 30-fold induction of hepatic P450b by phenobarbital (PB) was also completely ineffective in increasing P450b-dependent DMBA metabolism. PB treatment had no appreciable effect on either the levels of expression of P450b protein or P450b-dependent DMBA metabolism, in M520/N lung and adrenal microsomes. In contrast, PB treatment of F344 rats considerably increased P450b/e-dependent metabolism by liver, lung, and adrenal microsomes. The regioselectivity of the anti-P450b/e-sensitive metabolism (predominantly 12-methyl hydroxylation), however, indicated a much greater contribution from P450e than P450b in every tissue examined despite a several fold higher expression of P450b than of P450e. P450b was expressed constitutively in lung microsomes from both strains but again failed to exhibit appreciable DMBA metabolizing activity. Based on these activities and microsomal P450b contents, P450b consistently exhibited turnover numbers (0.02-0.15 nmol/nmol P450b/min) that were at least 10-fold lower than those of pure P450b. In contrast, the calculated turnover numbers for microsomal P450e were consistently comparable to those of pure P450e (approximately 1 nmol/nmol P450e/min).     

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*.     

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.     

Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).     

Hydroperoxidation by cytochrome P-450 oxygenase: metabolism of 9-methylfluorene

9-Methylfluorene was metabolized by rat liver microsomes to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene. The results were confirmed by using a reconstituted cytochrome P-450 oxygenase system purified from phenobarbital-induced rat liver which established its involvement. SKF-525A strongly inhibited the formation of both oxygenation products. Cytochrome P-450 alone brought about the conversion of the hydroperoxide to its alcohol. NADPH augmented the peroxidative reaction, but the presence of NADPH-cytochrome P-450 reductase was without effect. Certain microsomal preparations and reconstituted enzyme yielded little or no detectable amounts of hydroperoxide. This was due to a too rapid conversion of the hydroperoxide to its alcohol. The addition of metyrapone, a compound that inhibited such conversion, resulted in accumulation of 9-hydroperoxy-9-methylfluorene for positive identification. Incubation of 9-methylfluorene with microsomes and NADPH resulted in covalent binding of its metabolite to microsomal proteins. Incubation of 14C-labeled 9-hydroperoxy-9-methylfluorene caused covalent binding of label to proteins, RNA, and DNA.     

The Chronic Administration of Nicotine Induces Cytochrome P450 in Rat Brain

Abstract: The objective of these studies was to determine whether chronic administration of nicotine altered the cytochrome P450 (P450) monooxygenase system in rat brain. Male Sprague-Dawley rats received injections of nicotine bitartrate (1.76 mg/kg, s.c, twice daily for 10 days), and total cytochrome P450 content, the activity of N ADPH-cytochrome c reductase, and the activities and relative abundance of P4502B1 and P4502B2 (P4502B1/2) were determined in microsomal fractions from rat brain. The content of P450 increased significantly (p < 0.02) in all brain regions examined from nicotine-injected rats: the largest increase (208% of control) was in frontal cortex and the smallest increase (122% of control) in cerebellum. The activity of NADPH-cytochrome c reductase was unaltered by nicotine administration. Benzyloxyresorufin O-dealkylase (BROD) and pentoxyresorufin O-dealkylase (PROD) activities, mediated by P4502B1/2, increased significantly (p < 0.02) following nicotine administration; the largest increase (213-227% of control) was in frontal cortex. Western blots of microsomal proteins indicated that the increase in enzymatic activity was associated with an increase in content of P4502B1/2 immunoreactive proteins. In contrast to brain, total P450 content, activities of NADPH-cytochrome c reductase, BROD, and PROD, and levels of P4502B1 /2 immunoreactive proteins in liver were unaffected by chronic nicotine administration. Results indicate that chronic nicotine administration regulates the expression of P4502B1/2 in brain and that at the dose schedule used this effect occurs without a demonstrable effect on the hepatic P450 monooxygenase system.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Neurosteroids: metabolism in human intestine microsomes

Both dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA) are substrate for cytochrome P450 species and enzymes that produce 7alpha- and 7beta-hydroxylated metabolites in the brain and other organs. In contrast to DHEA and EpiA, the 7-hydroxylated derivatives were shown to mediate neuroprotection, and 7beta-hydroxy-EpiA was the most potent. The suggested use of any of these steroids as drugs administered per os for neuroprotection requires the assessment of their metabolism in the human intestine and liver. To achieve this, we produced radio-labeled 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA that were used as substrates in incubations with human intestine microsomes supplemented with reduced or oxidized cofactors. Identity of the radio-labeled metabolites obtained was determined by gas chromatography/mass spectrometry after comparison with authentic steroid references. The proportions of metabolites produced resulted from their radioactivity contents. The only metabolite obtained with DHEA, EpiA, 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA substrates was its 17beta-reduced derivative, thus inferring the presence of 17beta-hydroxysteroid oxidoreductases in the human intestine microsomes. In addition to the 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA substrates, their 17beta-reduced metabolites were obtained with 7beta-hydroxy-EpiA and 7alpha-hydroxy-EpiA, respectively. The identity of the enzyme responsible for the 7alpha-hydroxy-EpiA/7beta-hydroxy-EpiA inter-conversion is unknown. The incubation conditions used produced these metabolites in low but significant yields that suggest their presence in the portal blood before access to the liver.     

Obesity as a risk factor for drug-induced organ injury. VI. Increased hepatic P450 concentration and microsomal ethanol oxidizing activity in the obese overfed rat

The obese overfed rat effectively models many of the pharmacological changes in human obesity. Recent data show that the obese rat is unusually susceptible to liver damage by several metabolically activated drugs that may be more toxic in obese humans. Results of the present study suggest a specific molecular locus for this interaction. In obese rats, P450 content of liver and the microsomal concentration of P450 were elevated 88% and 31%, respectively, over nonobese controls. Increases in microsomal ethanol oxidation were of identical magnitude. The ethanol-inducible form of P450 that is responsible for microsomal ethanol oxidation, P450IIE1, bioactivates several drugs that are shown to cause increased injury in obese rats. Collectively, these findings indicate that specific forms of P450 may become up-regulated in obesity, increasing the risk of a biochemically defined spectrum of drug-induced organ injuries.     

MPTP, the neurotoxin inducing Parkinson's disease, is a potent competitive inhibitor of human and rat cytochrome P450 isozymes (P450bufI, P450db1) catalyzing debrisoquine 4-hydroxylation

In human liver microsomal preparations the neurotoxic chemical N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and several of its analogs competitively inhibited bufuralol 1'-hydroxylase activity of cytochrome P450bufI. This enzyme is the target of the common genetic polymorphism of drug oxidation known as debrisoquine polymorphism. Bufuralol 1'-hydroxylase activity was detectable in rat brain tissue. The activity was inhibited by antisera raised against a rat liver cytochrome P450 called P450db1. Immunoblotting experiments revealed the presence of a protein in rat and human brain microsomes with the same electrophoretic properties as the liver enzyme. These data suggest that P450bufI may be involved in the metabolism and neurotoxicity of MPTP.