Lauric acid hydroxylase activity and cytochrome P450 IV family proteins in human liver microsomes

Roles of human cytochrome P450 (P450 or CYP) 2C9, 2C19, and 3A4 in the oxidation of progesterone and testosterone were studied in recombinant P450 enzymes and in human liver microsomes.In vitroinhibition experiments showed that progesterone and its 17α- and 21-hydroxylated metabolites and 11-deoxycortisol suppressed the CYP2C19-dependentR-warfarin 7-hydroxylation activities, with progesterone being the most active. These steroid chemicals also inhibited CYP2C9-dependentS-warfarin 7-hydroxylation activities though lesser extents seen with those in CYP2C19 enzyme. Progesterone was found to be a competitive inhibitor of CYP2C19 and CYP2C9 in human liver microsomes. Recombinant CYP2C19 catalyzed progesterone to form 21-hydroxyprogesterone as a major product and 16α- and 17α-hydroxyprogesterone as minor products. CYP2C9 also had progesterone 21-hydroxylation activities, although the activities were lower than those catalyzed by CYP2C19.V_max/K_mratios for the progesterone 21-hydroxylation activity of CYP2C19 were determined to be 13- and 32-fold higher than those of CYP2C9 and 3A4, respectively. CYP3A4 oxidized progesterone to form 16α-, 6β-, and 2β-hydroxyprogesterone as major products and 21-hydroxyprogesterone as a minor product, but did not produce detectable levels of 17α-hydroxyprogesterone. Immunoinhibition experiments suggested that anti-CYP2C9 (which inhibits both CYP2C9 and CYP2C19 catalytic activities) suppressed the progesterone 21-hydroxylation activities catalyzed by liver microsomes of humans and monkeys and that anti-CYP2C11 inhibited the progesterone 21-hydroxylation activities catalyzed by liver microsomes of male rats. CYP2C19 was also found to oxidize testosterone at 17-position to form androstenedione. Androstenedione formation catalyzed by liver microsomes of humans and monkeys and of male rats was suppressed by anti-CYP2C9 and anti-CYP2C11, respectively. These results suggest that CYP2C19 plays important roles in the oxidation of progesterone and testosterone in human liver microsomes, although the physiological significance of these metabolic pathways remains unclear. CYP2C9 may have some, but lesser extent than those by CYP2C19, of the catalytic roles for the metabolism of progesterone and testosterone by human liver microsomes.

The goal of the current study was to evaluate the effects of arachidonic acid, as a representative polyunsaturated fatty acid, on the viability of a Hep G2 cell line, which has been transduced to express human cytochrome P4502E1 (CYP2E1). Arachidonic acid produced a concentration- and time-dependent toxicity to Hep G2-MV2E1-9 cells, which express CYP2E1, but little or no toxicity was found with control Hep G2-MV-5 cells, which were infected with retrovirus lacking human CYP2E1 cDNA. In contrast to arachidonic acid, oleic acid was not toxic to the Hep G2-MV2E1-9 cells. The cytotoxicity of arachidonic acid appeared to involve a lipid peroxidation type of mechanism since toxicity was enhanced after depletion of cellular glutathione; formation of malondialdehyde and 4-hydroxy-2-nonenal was markedly elevated in the cells expressing CYP2E1, and toxicity was prevented by antioxidants such as α-tocopherol phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), propylgallate, ascorbate, and diphenylphenylenediamine, and the iron chelator desferrioxamine. Transfection of the Hep G2-MV2E1-9 cells with plasmid containing CYP2E1 in the sense orientation enhanced the arachidonic acid toxicity, whereas transfection with plasmid containing CYP2E1 in the antisense orientation decreased toxicity. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis, as demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA laddering experiments. Trolox, which prevented toxicity of arachidonic acid, also prevented the apoptosis. Transfection with a plasmid containing bcl-2 resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently promotes apoptosis and cell toxicity when the cells are enriched with polyunsaturated fatty acids such as arachidonic acid. The Hep G2-MV2E1-9 cells appear to be a valuable model to study interaction between CYP2E1, polyunsaturated fatty acids, reactive radicals, and the consequence of these interactions on cell viability and to reproduce several of the key features associated with ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment.

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the ω- and ω-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate ω-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing ω-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate ω-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M_r= 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH₂-terminal amino acid sequence identical to that predicted from the humanCYP4A11cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b₅, CYP4A11 proved to be a potent laurate ω-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the ω-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate ω-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a loneK_mof 48.9 μMwith aV_MAXof 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate ω-hydroxylase activity while ω-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r= 0.89;P< 0.001) was found between immunochemically determined CYP4A11 content and laurate ω-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate ω-hydroxylating enzyme expressed in human liver.

The stability of cytochrome P450 enzymes, cytochrome b₅, and NADPH–cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at −80°C, (B) human liver microsomes suspended in 250 mMsucrose and stored at −80°C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at −80°C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b₅and the activity of NADPH–cytochrome c reductase decreased 20–40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4′- hydroxylation), CYP2D6 (dextromethorphan O-de- methylation), CYP2E1 (chlorzoxazone 6-hydroxyla- tion), CYP3A4/5 (testosterone 6β-hydroxylation), and CYP4A9/11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cyto- chromes P450 and b₅and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b₅, the activity of NADPH–cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at −80°C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b₅, and NADPH–cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at −80°C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b₅concentration, or NADPH–cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at −80°C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.

The effects of zileuton, a 5-lipoxygenase inhibitor, on hepatic peroxisomal enzyme activity as well as hepatic drug metabolizing activity in male and female CD-1 mice were assessed after oral administration of the drug (50, 150, or 450 mg/kg/day) for 14 days. The effects were compared to those in mice receiving clofibrate (CLOF; 462 mg/kg/day, po) or sodium phenobarbital (PB; 50 mg/kg/day, po). Zileuton pretreatment caused hepatomegaly and elevated liver peroxisomal KCN-insensitive palmitoyl CoA oxidase activity in a dose-dependent manner. However, these changes were marginal (≤121% increase), when compared to those elicited by CLOF (∼370% increase). In both sexes, zileuton pretreatment also caused a dose-dependent increase in the levels of liver microsomal cytochrome P450 2B and cytochrome P450 4A (CYP4A) proteins, and their associated monooxygenase activity. In the case of CYP4A, the induction of lauric acid 12-hydroxylase activity by zileuton was more pronounced in female (maximal 851% increase) than in male mice (maximal 111% increase). Based on the dose normalized response observed in CD-1 mice, zileuton can be considered a relatively weak inducer of peroxisome enzyme activities (cf. CLOF) and a moderate inducer of cytochromes P450. Moreover, zileuton exhibits characteristics of both a PB- and a CLOF-type hepatic enzyme inducer, especially in the female mice.

Guinea pig was previously classified as a species nonresponsive to peroxisome proliferators. However, none of the previous reports was based on pharmacokinetic data. Here, after a comparative pharmacokinetic study between guinea pig and rat, we evaluate the guinea pig liver peroxisomal response to ciprofibrate, a hypolipemic agent and a potent peroxisome proliferator in rat. (1) Pharmacokinetic results show that plasmatic concentrations of ciprofibrate are equivalent in guinea pig and rat when guinea pigs are treated with ciprofibrate at 30 mg/kg twice a day and rats are treated at 3 mg/kg once a day. (2) The treatment of guinea pigs at 30 mg/kg twice a day for 2 weeks leads to a significant increase in the liver peroxisomal palmitoyl-CoA oxidase activity (×1.6) and also in the microsomal ω-laurate hydroxylase activity (×1.8). These increases are in accordance with the changes in polypeptide patterns of isolated liver peroxisomes as well as in the immunoblotting of acyl-CoA oxidase. It is deduced that a weak, but significant, peroxisome proliferation can occur in guinea pig liver after a ciprofibrate treatment at dosages corresponding to equivalent plasmatic concentrations of the drug between guinea pig and rat. (3) The hybridization of guinea pig liver RNA with the rat liver-inducible acyl-CoA oxidase cDNA probe shows a decrease in the corresponding heterologuous mRNA content after treatment with ciprofibrate at 30 mg/kg twice a day. This result contrasts with the slight increase observed in immunodetection and in enzymatic assays, suggesting the existence of at least two different acyl-CoA oxidases in guinea pig liver peroxisomes.