CYP2C11 Research Articles

Reduction of thyroid hormone may participate in the modulation of cytochromes P450 2C11 and 3A2 by retinol

Both hypothyroidism and retinol supplementation in rats induce CYP 3A2 and suppress CYP 2C11. Therefore studies were performed to evaluate the role of thyroid hormones in the modulation of P450 expression by retinol. Adult male Sprague-Dawley rats were given retinol as a single oral dose of 75 mg kg . Rats were killed and hepatic microsomes prepared at 24, 48, 72, and 96 hr following treatment. The catalytic activity of 2C11 was reduced maximally by retinol at 48 hr (by 30%) whereas 3A2 activity was elevated maximally at 24 hr (by 30%). The serum concentration of testosterone was not altered at any time point. However, retinol produced a decline in the concentration of thyroxine by 35% and 43% at 24 and 48 hr, respectively. These data suggest that administration of large doses of retinol may alter hepatic microsomal enzyme expression by perturbation of plasma thyroid hormone levels.

Cytochrome P450 mediated bioactivation of methyleugenol to 1'- hydroxymethyleugenol in Fischer 344 rat and human liver microsomes

Cytochrome P450 mediated metabolism of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol has been investigated in vitro. Kinetic studies undertaken in liver microsomes from control male Fischer 344 rats revealed that this reaction is catalyzed by high affinity (Km of 74.9 +/- 9.0 microM, Vmax of 1.42 +/- 0.17 nmol/min/nmol P450) and low affinity (apparent Km several mM) enzymic components. Studies undertaken at low substrate concentration (20 microM) with microsomes from livers of rats treated with the enzyme inducers phenobarbital, dexamethasone, isosafrole and isoniazid indicated that a number of cytochrome P450 isozymes can catalyze the high affinity component. In control rat liver microsomes, 1'-hydroxylation of methyleugenol (assayed at 20 microM substrate) was inhibited significantly (P < 0.05) by diallylsulfide (40%), p-nitrophenol (55%), tolbutamide (30%) and alpha-naphthoflavone (25%) but not by troleandomycin, furafylline, quinine or cimetidine. These results suggested that the reaction is catalyzed by CYP 2E1 and by another as yet unidentified isozyme(s) (most probably CYP 2C6), but not by CYP 3A, CYP 1A2, CYP 2D1 or CYP 2C11. Administration of methyleugenol (0-300 mg/kg/day for 5 days) to rats in vivo caused dose-dependent auto-induction of 1'-hydroxylation of methyleugenol in vitro which could be attributed to induction of various cytochrome P450 isozymes, including CYP 2B and CYP 1A2. Consequently, high dose rodent carcinogenicity studies are likely to over-estimate the risk to human health posed by methyleugenol. The rate of 1'-hydroxylation of methyleugenol in vitro in 13 human liver samples varied markedly (by 37-fold), with the highest activities being similar to the activity evident in control rat liver microsomes. This suggests that the risk posed by dietary ingestion of methyleugenol could vary markedly in the human population.

Cytochrome P450 2C11:Escherichia coliExpression, Purification, Functional Characterization, and Mechanism-Based Inactivation of the Enzyme

The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16α-hydroxylase of the untreated rat liver. The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL). Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme. Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1%. For these reasons, we chose to heterologously express the enzyme inEscherichia coli.The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression inE. coli.The enzyme was isolated and purified fromE. colimembranes in relatively high yields (≈60%) and relatively high specific content (19 nmol/mg protein). The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL. Studies with14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation. On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both [22-14C]SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein. The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid. Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates.

Effects of metyrapone on expression of CYPs 2C11, 3A2, and other 3A genes in rat hepatocytes cultured on matrigel

Hepatocytes cultured on matrigel express many liver-specific functions, but the levels and activities of the predominant male-specific rat hepatic CYPs, 3A2 and 2C11, decline rapidly in culture. Metyrapone maintains the level of total cytochrome P450 of rat hepatocytes in primary culture, but the mechanism underlying this effect has not been completely elucidated. The present study sought to determine whether metyrapone acts solely to stabilise CYP proteins in rat hepatocytes cultured on matrigel, or whether it also influences mRNA levels of the encoding genes. Metyrapone maintained the level of total cytochrome P450 in cultured hepatocytes so that values were > 200% of those found in untreated control cells 24 hr after isolation. At this time, CYP3A2-mediated testosterone 6β-hydroxylation was approximately 7-fold higher in hepatocytes cultured in the presence of metyrapone than in control cells, and CYP2C11-dependent testosterone 2α- and 16α-hydroxylation activities were between 2 and 3-fold greater. The results inferred from catalytic activities were supported by immunoquantitation of CYP3A and 2C11 proteins. The trend of increased CYP protein levels in metyrapone-treated cells continued throughout the 48-hr culture period. In control cells, CYP3A2 and 2C11 mRNA levels fell abruptly in culture to reach values at 24 hr that were < 30% of those in freshly isolated cells; addition of metyrapone failed to arrest this fall. However, treatment of cells with metyrapone considerably elevated levels of one or more CYP3A subfamily mRNA species, as detected by a riboprobe based on the cDNA for CYP3A1 (‘CYP3A1-like mRNA’) that were demonstrated, by another riboprobe, not to be CYP3A2 or RNCYP3AM. RT-PCR of mRNA prepared from cultured hepatocytes, followed by restriction mapping of the cloned cDNAs was used to characterise the CYP3A induced by metyrapone. This revealed that elevated levels of the CYP3A1-like mRNA were attributable to induction of RL33/cDEX mRNA; there were no CYP3A1 cDNAs isolated from these cells. These data are interpreted as indicating that metyrapone stabilises the expression of cytochrome P450 in culture by both pre- and posttranslational mechanisms. The particular mechanism employed is gene-specific, whereby even the highly homologous genes CYP3A2, RL33/cDEX and, possibly, RNCYP3AM are subject to different types of regulation in the presence of metyrapone.

Reproductive Toxicity and Growth Effects in Rats Exposed to Lead at Different Periods during Development

The reproductive toxicity and growth effects of developmental lead exposure were assessed using a rat model in which 0.6% (w/v) lead acetate was administered in the drinking waterad libitum.Three series of experiments were conducted in which lead exposure was initiated beginningin utero,prepubertally, or postpubertally. Lead effects were measured on reproductive physiology and endocrinology, sexually dimorphic hepatic testosterone hydroxylation, and growth rates in both male and female animals. In male animals secondary sex organ weights were significantly decreased only in animals exposed prepubertally. In addition, serum testosterone levels were significantly suppressed, most severely in animals exposed fromin utero(in thein uterogroup). Little effect was observed in adult female rats. However, in female animals exposed prepubertally, delayed vaginal opening and disrupted estrus cycling was observed. More severe reproductive disruption was accompanied by suppression of circulating estradiol in thein uterogroup. Effects on circulating sex steroids were accompanied by variable effects on circulating luteinizing hormone (LH) levels, pituitary LH, and pituitary LHβ mRNA, suggesting a dual site of lead action: (a) at the level of the hypothalamic pituitary unit, and (b) directly at the level of gonadal steroid biosynthesis. Prepubertal growth in both sexes was suppressed 25% in thein uterogroup. However, pubertal growth rates were significantly suppressed only in male animals and postpubertal growth was not significantly different from controls in any of the experiments, despite continued exposure to high lead levels in the drinking water. In addition, at age 85 days, male-specific hepatic hydroxylation of testosterone at positions 2α and 16α, which is catalyzed by a cytochrome P450 isozyme CYP 2C11, itself regulated by sexually dimorphic growth hormone secretion, was unaffected. This suggests that the growth effects of lead are possibly due to a delay in the development of sex-specific pituitary growth hormone secretion patterns rather than a persistent developmental defect. Thus, the reproductive and growth effects of lead are complex and sex-dependent, and appear to involve multiple sites on the hypothalamic–pituitary–gonadal axis.

Use of monoclonal antibodies to cytochrome P450s to indicate the critical dealkylation and the P450s involved in methyl- n-amylnitrosamine mutagenicity in the presence of induced rat liver microsomes

The mutagenicity for Salmonella typhimurium TA 1535 of the carcinogen methyl- n-amylnitrosamine (MNAN) was examined in the presence of rat liver microsomes from uninduced and induced rats. The number of mutations followed the order phenobarbital- and Aroclor-induced > 3-methylcholanthrene- and isoniazid-induced > uninduced microsomes. The MNAN metabolite 4-hydroxy-MNAN was not mutagenic. Using each type of induced liver microsomes, we examined the effect on MNAN mutagenicity of four monoclonal antibodies (MAbs) that inhibit cytochrome P450s. The MAbs inhibited MNAN mutagenicity in seven MAb-microsome combinations by up to 49%. Taken together, these results indicated that CYP (P450) 1B1 2B2 was responsible for one half and CYP 2C11 for one quarter of MNAN mutagenicity with phenobarbital-induced microsomes, CYP 1A1 1A2 accounted for about 40% of the mutagenicity with 3-methylcholanthrene-induced microsomes, CYP 2B1 2B2 accounted for half and CYP 1A1 1A2 and 2C11 for smaller proportions of the mutagenicity with Aroclor-induced microsomes, and CYP 1A1 1A2 accounted for about 30% of the mutagenicity with isoniazid-induced microsomes. With isoniazid-induced microsomes, MAb 2-66-3 to CYP 2B1 2B1 caused an unexpected 219% increase and MAb 1-68-11 caused a moderate increase in MNAN mutagenicity. The test MAbs also inhibited the microsome-catalyzed demethylation and depentylation of MNAN by up to 83%, confirming previous results. Four comparisons between individual mutagenic and metabolic results supported the view that depentylation of MNAN was more critical for its mutagenicity than was demethylation, e.g., with 3-methylcholanthrene- and Aroclor-induced microsomes, MAb 1-7-1 to CYP 1A1 1A2 inhibited mutagenesis and depentylation, but did not affect demethylation.

Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail ( Colinus virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs)

Ergosterol biosynthesis inhibiting fungicides (EBIFs) have complex effects on the hepatic microsomal monooxygenase systems of vertebrate species, having been described as mixed inducers and inhibitors of cytochrome P450. In the current study, we examined the effects of two EBIFs in clinical use, clotrimazole and ketoconazole, and two agricultural EBIFs, propiconazole and vinclozolin, on hepatic monooxygenase activities and P450 apoprotein expression in the male Sprague-Dawley rat and the male bobwhite quail. EBIFs produced Type II binding spectra with hepatic microsomes from both species and were effective inhibitors of methoxyresorufin O-demethylase, an activity selective for P450 isozymes in gene family 1. However, the EBIFs varied widely in their effectiveness as inducers of P450 isozymes in gene families 1, 2, 3 and 4, both within the same species and between species. In the rat, clotrimazole was the most effective inducer, increasing expression of CYP 3A isozymes over 450-fold, CYP 2B1/2 30-fold and CYP 1A1/2 12-fold and suppressing expression of CYP 2C11 nearly 70%. By contrast, in the quail, clotrimazole was the least effective inducer. In quail, vinclozolin and propiconazole elevated total P450 content 10- and 7-hold, respectively. The induction response also appeared to be mixed, but in this case consisted of a 5-fold induction of P450s in gene family 1A, a 3-fold induction of P450s in gene family 3A and 4A, and induction of protein(s) from gene family 2, cross-reactive with antisera against rat CYP 2C11 and CYP 2A1. A protein that was cross-reactive with antibodies raised against rat CYP 2B1 was decreased with EBIF treatment. In conclusion, EBIFs have complex patterns of induction, suppression and inhibition of cytochrome P450 isozymes in both mammals and birds, which vary according to both the fungicide and the species.

Induction of Cytochrome P450 1A1 and 1A2 by Hyperoxia

The effect of hyperoxia (O2>95%) for 48 hours on the induction of pulmonary and hepatic cytochrome P450 has been investigated in adult male rats. Northern blot analysis using six "specific" oligonucleotide probes indicated that CYP 1A1 and CYP 1A2 mRNAs in liver and CYP 1A1 mRNA in lung were significantly increased by hyperoxic exposure, whereas the major constitutive P450 mRNAs, CYP 2C11 in liver and CYP 2B1 in lung, were decreased. Since induction of CYP 1A1 has only been reported with the use of exogenously administered xenobiotics, further studies were carried out to confirm the results obtained with Northern blot analysis. cDNAs were synthesized for CYP 1A1 and 1A2 in the liver and CYP 1A1 in the lungs and amplified by reverse PCR. These results indicate that these cDNAs were amplified significantly more in the hyperoxia group than in the control animals. Furthermore, CYP 1A1 and 1A2 proteins in liver and CYP 1A1 protein in lungs as well as the corresponding monooxygenase activities were increased by hyperoxia. Hyperoxic induction of CYP 1A1 and 1A2 is the first demonstration of nonexogenous CYP 1A induction in animals and indicates the needs to pursue the changes of Ah receptor-ligand-DNA interaction in hyperoxia.

Purification of Multiple Forms of Cytochrome P450 from a Human Brain and Reconstitution of Catalytic Activities

The present study demonstrates the presence of multiple forms of cytochrome P450 (P450) in human brain obtained at autopsy, the purification of various isoforms to apparent homogeneity, and the monooxygenase activities in reconstituted systems. Sequential chromatography on octylamino-Sepharose 4B, DEAE-Sephacel, and DEAE-cellulose yielded four isoforms of P450 (A, B, C, and D) with specific contents of 11.0, 9.4, 12.5, and 8.3 nmol of P450/mg protein, respectively. While the forms A, B, and C were apparently homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the P450D was not homogeneous. The apparent molecular masses of the four forms of P450 were 60,200 Da (P450A), 60,900 Da (P450B), 60,200 Da (P450C), and 61,000 Da (P450D), respectively. NADPH cytochrome P450 reductase (reductase) was also partially purified from the brain microsomes. Immunoblot analysis of the four forms of human purified P450, using antisera to purified rat liver P450 (IIBL + 11B2), rat liver P450 (1A1 + 1A2), phenobarbital-inducible rat brain P450, human liver P450 IIE 1, P450 1A2, P450 IIC, and P450 IIIA4, indicated differential immunological cross-reactivity. The monooxygenase activities of the purified human brain P450s were demonstrated with various substrates (aminopyrine, morphine, aniline, 7-ethoxycoumaria, and nifedipine) as examined in reconstituted systems consisting of purified human brain P450, purified rat brain NADPH cytochrome P450 reductase, deoxycholate, phospholipid, and NADPH.

Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion.

The onset of the sexually dimorphic pattern of GH secretion and increased hepatic GH-binding capacity in rats at puberty is temporally correlated with the developmental induction of three hepatic cytochrome P-450s with steroid hydroxylase activity, P-450 IIC11, P-450 IIC12, and P-450 IIC13, and one cytochrome P-450 with vitamin A hydroxylase activity, P-450 IIC7. In this study we demonstrate that expression of the 2C11, 2C12, and 2C13 genes is modulated by GH at the level of transcriptional initiation both in vivo and in primary cultures of adult hepatocytes. In an effort to define the minimum sequence responsible for the inductive effects of GH, we have analyzed the ability of a 0.7-kilobase fragment isolated from the 5'-flank of the 2C12 gene, including the natural promoter, to drive transcription of a 320-basepair G-less cassette in vitro. We were unable to detect any substantial difference in RNA polymerase-II-dependent transcriptional efficiency toward the 2C12 promoter between liver nuclear extracts from normal and hypophysectomized rats of both sexes. This observation supports the assumption that the sequence information contained between bases -700 and 1 is sufficient to support basal transcription of the 2C12 gene. Sequence information residing 5' or 3' of the 0.7-kilobase 5'-flank or a higher ordered chromatin structure may be necessary for the sex-specific transcriptional activation of the 2C12 gene.

The microsomal monooxygenase system of regenerating liver: An examination of the role of estradiol in the demasculinization of drug metabolism produced by [formula omitted] partial hepatectomy

Declines in total cytochrome P450 content and in monooxygenase activities associated with some male specific isozymes of cytochrome P450 have been reported in the rat following 2 3 partial hepatectomy ( 2 3 PH). In the present study, we examined the effects of 2 3 PH on hepatic microsomal monooxygenase activities towards testosterone, the alkoxyresorufins, p-nitrophenol and carbon tetrachloride in male rats. Levels of P450 apoproteins were determined by Western blot analysis. The effects of hepatectomy and sham operations on plasma growth hormone (GH) pulse profiles and the effects of a single acute dose of estradiol (E2) were studied to determine the role of these factors in 2 3 PH mediated changes in oxidative metabolism. 2 3 PH produced substantial decreases in testosterone hydroxylation at positions 16α, 2α and 7α, but only a small decrease in hydroxylation at position 6β. Reductions in CYP 2C11 (P450h) and CYP 2A1 (P450a) expression were observed with Western blot analysis down to 19 and 41% of control values, respectively, but insignificant effects were observed on expression of CYP 3A (P450p family) proteins recognized by a polyclonal antibody raised against rat CYP 3A2 (P450pcn2). In contrast, acute E2 treatment caused a 2-fold increase in expression of CYP 2A1 apoprotein and significantly decreased expression of CYP 2E1 (P450j) apoprotein and dependent monooxygenase activities, but had no significant effect on expression of CYP 2C11. Both sham operations and 2 3 PH caused a temporary decrease in plasma GH concentrations, but secretion returned towards normal 24–48 hr after both operations. These data suggest that some factor other than GH or E2 must be involved in the selective suppression of some P450 isozymes observed after 2 3 PH.

In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

Administration of a diet supplemented with 0.06% vitamin E acetate to male rats over a 6-week period doubled hepatic microsomal stores of α-tocopherol over those in control (vitamin E adequate) rat liver. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity were significantly elevated in hepatic microsomes from vitamin E-supplemented rats to 111% and 123% of respective control values. Androstenedione 16α-hydroxylase activity was increased in these fractions (2.57 ± 0.31 nmol product/min/mg protein vs 1.81 ± 0.38 in controls) whereas activities of the 6β-, 7α- and 16β-hydroxylase pathways were unchanged. Immunoquantitation of the microsomal 16α-hydroxylase, P450 IIC11, indicated a corresponding increase in the hepatic content of the enzyme. In view of the established antioxidant role of tocopherols, the effects of dietary vitamin E manipulation on the concentration of protein sulphydryl groups and the susceptibility of microsomes to ferric sulphate-ADP-NADPH-mediated lipid peroxidation were also assessed. Dietary supplementation did not influence microsomal protein sulphydryl content (68 ± 10 nmol glutathione equivalents/mg protein) but decreased the extent of lipid peroxidation produced by the ferric sulphate-ADP-NADPH system in vitro. Further in vitro experiments demonstrated that vitamin E acetate (2 μM) protected protein sulphydryl groups and lipids against peroxidation in control microsomes and partially reduced the associated losses of P450-mediated steroid hydroxylase activities. Western immunoquantitation of P450 IIC11 revealed that exogenous vitamin E acetate protected completely against peroxidation-induced apoprotein loss. These studies establish that the in vitro protective effects of vitamin E acetate against sulphydryl and lipid peroxidation extend to protection of the P450 apoprotein but that enzyme activity is only partially protected. This finding suggests that peroxidation-dependent loss of P450 in vitro is mediated by haem degradation from the P450 holoenzyme and is not directly related to lipid/sulphydryl oxidation. In contrast, the in vivo effects of dietary vitamin E on drug metabolizing enzymes are regulatory in nature and are unrelated to effects on lipid peroxidation.

DNA binding, adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal, Kupffer and endothelial cells

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B1, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP+ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B1 to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B1 had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B1 in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B1. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2 alpha-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2 alpha-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B1 to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B1, a dose-dependent aflatoxin B1 binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1.

Catalytic activity of cytochrome P450 isozymes purified from rat liver in converting 11-oxo-Δ 8-tetrahydrocannabinol to Δ 8-tetrahydrocannabinol-11-oic acid

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-Δ 8-tetrahydrocannabinol (11-oxo-Δ 8-THC) to Δ 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-Δ 8-THC to Δ 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O 2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-Δ 8-THC to Δ 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-Δ 8-THC to Δ 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.

Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age

We have used immunohistochemical, immunoblotting and messenger RNA blotting approaches to study the distribution and quantitation of three cytochrome P-450 enzymes, namely P-450 IA2, P-450 IIC and P-450 IIIA and, for comparison, epoxide hydrolase and NADPH-cytochrome P-450 reductase in human liver. Age-related changes in both the amounts and the intralobular distributions of these enzymes were demonstrated, and the enzymes differ in these regards: In fetal liver, P-450 IA2 and P-450 IIC were very low, when present at all, whereas P-450 IIIA, epoxide hydrolase and the reductase were already abundant and found in all the hepatocytes. During the postnatal period, P-450 IIC dramatically increased and was observed in all hepatocytes, the centriobular ones being more intensely stained. P-450 IIIA was restricted to centrilobular and midzonal hepatocytes in normal adult liver. P-450 IA2 showed this same intralobular distribution; however, its presence was detected only several weeks or months after birth as judged both by immunohistochemical and immunoblotting techniques. Epoxide hydrolase and NADPH-cytochrome P-450 reductase were easily visualized in all hepatocytes regardless of the age of the donor; in child and adult livers, centrilobular hepatocytes were more intensely stained. Immunoreactive protein contents and corresponding messenger RNA levels correlated well with immunohistochemical observations. No major modification was seen in fibrotic liver, whereas both positive and negative cells were observed in cirrhotic liver nodules for all enzymes studied. (HEPATOLOGY 1991;13:1142–1151.)

Intralobular distribution and quantitation of cytochrome P-450 enzymes in human liver as a function of age

We have used immunohistochemical, immunoblotting and messenger RNA blotting approaches to study the distribution and quantitation of three cytochrome P-450 enzymes, namely P-450 IA2, P-450 IIC and P-450 IIIA and, for comparison, epoxide hydrolase and NADPH-cytochrome P-450 reductase in human liver. Age-related changes in both the amounts and the intralobular distributions of these enzymes were demonstrated, and the enzymes differ in these regards: In fetal liver, P-450 IA2 and P-450 IIC were very low, when present at all, whereas P-450 IIIA, epoxide hydrolase and the reductase were already abundant and found in all the hepatocytes. During the postnatal period, P-450 IIC dramatically increased and was observed in all hepatocytes, the centrilobular ones being more intensely stained. P-450 IIIA was restricted to centrilobular and midzonal hepatocytes in normal adult liver. P-450 IA2 showed this same intralobular distribution; however, its presence was detected only several weeks or months after birth as judged both by immunohistochemical and immunoblotting techniques. Epoxide hydrolase and NADPH-cytochrome P-450 reductase were easily visualized in all hepatocytes regardless of the age of the donor; in child and adult livers, centrilobular hepatocytes were more intensely stained. Immunoreactive protein contents and corresponding messenger RNA levels correlated well with immunohistochemical observations. No major modification was seen in fibrotic liver, whereas both positive and negative cells were observed in cirrhotic liver nodules for all enzymes studied.

Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6β-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver. 6β-Hydroxylation was the most important pathway of biotransfromation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2β-, 15β- and 15α-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6β-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6β-alcohol was the only metabolite formed from AD at a rate > 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti- P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2β-, 6β-, 15α- and 15β-positions (by 31–56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6β-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6β-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6β-hydroxylation.

Hydroxylation and formation of electrophilic metabolites of tienilic acid and its isomer by human liver microsomes: Catalysis by a cytochrome P450 IIC different from that responsible for mephenytoin hydroxylation

Tienilic acid (TA) is metabolized by human liver microsomes in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the hydroxylation of the thiophene ring of TA. Besides this hydroxylation, TA is oxidized into reactive metabolites which covalently bind to microsomal proteins. Oxidation of an isomer of tienilic acid (TAI), bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring, by human liver microsomes, gives a much higher level of covalent binding to proteins. Both covalent binding of TA and TAI metabolites are almost completely suppressed in the presence of glutathione. These three activities of human liver microsomes (TA 5-hydroxylation, covalent binding of TA and TAI metabolites) seem dependent on the same cytochrome P450 of the IIC subfamily, since (i) antibodies against human liver cytochromes P450 IIC strongly inhibit these three activities, (ii) there is a clear correlation between these activities in various human liver microsomes, and (iii) TA acts as a competitive inhibitor for TAI activation into electrophilic metabolites ( K i ≅ 25 μM) and TAI inhibits TA 5-hydroxylation. However cross inhibition experiments indicate that tienilic acid hydroxylation and mephenytoin hydroxylation, a typical reaction of some human liver P450 IIC isoenzymes, are not catalysed by the same member of the P450 IIC subfamily.

A monoclonal antibody to rat liver cytochrome P450 IIC11 strongly and regiospecifically inhibits constitutive benzo[a]pyrene metabolism and DNA binding

The monoclonal antibody MAb 1-68-11, prepared to constitutive cytochrome P450 IIC11 (2c/RLM5) from male Sprague-Dawley rat liver, was used to study the contribution of the class of cytochrome P450s epitopically related to P450 IIC11 to the regiospecific metabolism of benzo[a]pyrene (BP) and its binding to DNA. The effect of MAb 1-68-11 was determined on the conversion of BP to BP-9,10-dihydrodiol, BP-7,8-dihydrodiol, BP-4,5-dihydrodiol, BP phenols, and BP quinones, and on the P450-dependent DNA binding catalyzed by P450 in microsomes from uninduced male and female Wistar and Sprague-Dawley rat livers, as well as 3-methylcholanthrene- and phenobarbital (PB)-induced male Wistar rat livers. In liver microsomes from untreated male rats, MAb 1-68-11 inhibited BP-9,10-dihydrodiol formation by 80%; in liver microsomes from untreated female rats, the inhibition was 100%. BP-7,8-dihydrodiol formation was inhibited from 38 to 77% in microsomes from males and 50% in those from females. In microsomes from PB-induced rats, inhibition of the 9,10-dihydrodiol and the 7,8-dihydrodiol was 90% and 73%, respectively, whereas BP-4,5-dihydrodiol formation was enhanced 80%. In microsomes from 3-methylcholanthrene-treated rats, no inhibition of MAb 1-68-11 was observed on either the metabolism of BP or its binding to DNA. In contrast, the binding of BP to DNA was completely inhibited by MAb 1-68-11 in microsomes from uninduced male Wistar rats and 70% in PB-induced microsomes. 32P-postlabeling analysis showed that formation of the major stable adduct, BP diol epoxide bound at C-10 to the 2-amino of deoxyguanosine, was strongly inhibited in uninduced and PB-induced microsomes. Formation of the major labile BP-DNA adduct 7-(benzo[a]pyren-6-yl) guanine (BP-N7Gua) was inhibited about 60% in microsomes from untreated male Wistar rats. These results show that MAb 1-68-11 regiospecifically inhibits cytochrome P450 IIC11 and epitopically related P450s that metabolize BP at the 7,8 and 9,10 positions. MAb 1-68-11 also inhibits enzyme-catalyzed binding of BP to DNA in the specific formation of BP-N7Gua and adducts detected by the 32P-postlabeling technique.

Structure-activity relationships in the in vitro modulation of rat hepatic microsomal androst-4-ene-3,17-dione hydroxylase activities by derivatives of 5α- and 5β-androstane

The relationships between structure and inhibitory potency toward microsomal cytochrome P-450 ( P-450)-mediated androst-4-ene-3,17-dione hydroxylase activities were investigated in rat liver with a series of 5α- and 5β-androstane derivatives. 5β -Reduced steroids (containing a cis-A/B ring junction) were more potent inhibitors than the 5α-reduced epimers (containing a trans-A/B ring junction) except in the case of the 17β-hydroxy-substituted derivatives. The most effective inhibitor was 5β-androstane-3β-ol which exhibited I 50 values of 7 and 27 μM against androstenedione 16α- and 6β -hydroxylase activities, which are catalysed by P-450 IIC11 and IIIA2, respectively. In general, these two pathways of steroid hydroxylation were more susceptible to inhibition than the 7α- and 16β -hydroxylase pathways. The 7α-hydroxylase enzyme ( P-450 IIA1) was only inhibited by 5β -reduced steroids that contained an oxygenated function at C17. All of the test compounds elicited type I spectral binding interactions with P-450 in oxidised microsomes. The most effective steroid inhibitors generally exhibited the greatest capacity to interact with P-450. Additional studies with one of the more potent compounds, 5β-androstane-3β-ol-17-one, revealed that the inhibition kinetics were competitive and that preincubation of the inhibitor with NADPH-supplemented microsomes prior to substrate (androstenedione) addition decreased the extent of inhibition observed. These findings are consistent with the assertion that the inhibition of hepatic steroid hydroxylases by 5β-androstanes involves an effective competitive interaction with the steroid substrate at the P-450 active site. Since the relative overproduction of 5β-reduced metabolites of certain androgens has been reported in clinical conditions, such as androgen insensitivity, it now appears important to investigate the hepatic drug oxidation capacity of patients with hormonal abnormalities.