Constitutive Cytochrome P450 Isozymes Research Articles

Lack of effect of methotrexate on the expression of constitutive hepatic cytochromes P450 in the male rat.

1. The effects of methotrexate (MTX) on the expression of selected constitutive cytochrome P450 (CYP) isozymes in the liver of male rats at the catalytic activity and mRNA levels were examined. 2. Male rats received a single intraperitoneal injection of MTX (4 mg/kg) or vehicle and were killed, 1, 2, 7 or 14 days following drug administration. 3. Hepatic microsomes were used for determination of total CYP content, NADPH-CYP reductase activity, aminopyrine N-demethylase activity, and androstenedione (AD) hydroxylation activity; total RNA was also isolated from liver and was used for hybridization analysis of CYP isozyme expression at the mRNA level. 4. MTX did not affect any of the following parameters at any time-point in comparison with the corresponding vehicle control: body weight, liver weight, hepatic microsomal protein content, total CYP content, NADPH-CYP reductase activity, aminopyrine N-demethylase activity, AD 6 beta- and 7 alpha-hydroxylase activity, and CYP3A2 mRNA content. 5. The major male-specific CYP isozyme, 2C11, was down-regulated by MTX treatment as revealed by a marginal (25%), but statistically significant decrease in AD 16 alpha-hydroxylase activity at day 14 and a marginal (18%), but statistically significant decrease in CYP2C11 mRNA content at day 14. 6. In comparison with other antineoplastic drugs that have been examined, MTX appears to possess a lesser capacity for modulation of hepatic CYP enzymes.

The rat cytochrome P450 C‐M/F (CYP2D) subfamily: Constitutive P450 isozymes in male and female

Cytochrome P450 (CYP) isozymes have been cloned from various tissues and species and form the cytochrome P450 superfamily. We have isolated cytochrome P450 C-M/F, which we regard as a major form of the constitutive cytochrome P450 isozyme. Cytochrome P450 C-M/F was highly active in the 2- and 16α-hydroxylation of estrogens, as well as in the N-demethylation of ethyl-morphine and benzphetamine. It was essentially uninducible by phenobarbital, 3-methylcholan-threne or β-naphthoflavone. Immunochemical studies demonstrated the presence of cytochrome P450 C-M/F in the liver of male and female rats and in the kidney of male rats. This is consistent with P450 C-M/F activity towards estrogens, since they are metabolized in the liver and kidney and excreted into the urine. Highly similar genes were identified by immunoscreening, and comprised the CYP2D sub-family. Five genes—CYP2D1, CYP2D2, CYP2D3, CYP2D4 and CYP2D5—have been isolated from rats. In spite of the high similarity, there is an obvious difference in substrate specificity between even the most similar isozymes. Among allelic variants reported for CYP2D1 and CYP2D2, P450IID1v is a mutant with decreased activity specifically towards bufuralol, due to a single amino acid substitution. Biochemical studies have demonstrated enzyme activities of CYP2D1 and CYP2D2 isozymes, while those of CYP2D3, CYP2D4 and CYP2D5 remain unknown. It is suggested that CYP2D isozymes play a role in maintaining homeostasis of the organism since they are constitutively expressed in the livers of both sexes and in the kidneys of males. Understanding their endogenous substrates should help to clarify their roles.

Effects of thiamine deficiency on hepatic cytochromes P450 and drug-metabolizing enzyme activities

To elucidate the mechanisms by which thiamine deficiency affects hepatic microsomal monooxygenase activities, the effect of thiamine deficiency on two constitutive cytochrome P450 isozymes, P450IIE1 and P450IIC11, was investigated, using weanling male Sprague-Dawley rats. The clinical signs of thiamine deficiency were apparent after feeding a thiamine-deficient diet for 3 weeks. Thiamine deficiency caused an increase in P450IIE1, which was determined by N-nitrosodimethylamine demethylase assay and immunoquantitation of P450IIE1. This increase in the P450IIE1 level was mainly attributed to thiamine deficiency per se but not to dietary restriction. Ketone bodies were not elevated in thiamine-deficient rats, whereas ketone bodies were elevated and may have served as inducing factors in calorically restricted pair-fed animals. Injections of pyruvate or pyrithiamine in addition to thiamine deficiency did not potentiate the induction effect. On the other hand, thiamine deficiency did not affect the level of P450IIC11 during the 3 weeks of feeding the thiamine-deficient diet. In addition, thiamine deficiency increased cytosolic glutathione S-transferase activity but not steroid isomerase activity. The present study demonstrates the specificity of thiamine deficiency per se in the induction of P450IIE1 which does not involve an increase in the ketone body level.

The mechanism of aphidicolin bioinactivation by rat liver in vitro systems

1. Aphidicolin is shown to undergo rapid metabolism by rat-liver microsomes resulting in its inactivation and loss of its DNA polymerase alpha/delta inhibition. Metabolism of aphidicolin was not observed with cytosolic enzymes of rat liver and was inconsistent with the involvement of microsomal 3 alpha-hydroxysteroid oxidoreductases. 2. Rates of aphidicolin inactivation as a function of microsomal enzyme induction (per nmol cytochrome P-450) followed the order: untreated microsomes greater than dexamethasone-induced greater than phenobarbital-induced greater than beta-naphthoflavone-induced greater than clofibrate-induced. 3. The principal metabolic process, constituting greater than 90% of the metabolic profile, produces 3-ketoaphidicolin 2, which exhibits approximately 10% of the activity of aphidicolin in inhibition of DNA polymerase alpha. This metabolic transformation, the oxidation of an alcohol to a ketone, is an unusual, but not unique conversion, for cytochrome P-450. 4. 3-Ketoaphidicolin 2 is an intermediate and ultimately undergoes 18-dehydroxymethylation to produce 18-noraphidicolinones 3, which are inactive in the inhibition of DNA polymerase alpha. 5. A specific constitutive cytochrome P-450 isozyme, involved in endogenous steroid regulation, was implicated as the species responsible for aphidicolin metabolism in vitro.

Cytochrome P450 isozyme distribution in normal and tumor-bearing hepatic tissue from rainbow trout ( Salmo gairdneri)

An immunohistochemical technique was used to localize the major constitutive cytochrome P450 isozyme, P450 LM 2, and the major β-naphthoflavone-inducible isozyme, P450 LM 4b, in the livers of untreated and aflatoxin B 1 (AFB 1)-initiated, tumor-bearing rainbow trout. In hepatic tissue sections from untreated trout, no regular anatomical pattern within the hepatic parenchymal cells could be discerned for either isozyme. Immunostaining was observed for P450 LM 2 along the sinusoidal border of some of the parenchymal cells, there was moderate staining within the cytoplasm of most cells, and there were focal areas of increased staining. There was intense, uniform immunostaining for P450 LM 2 within the cytoplasm of the bile duct cells, in the endothelial lining of arterioles, and along the epithelial surface of the gall bladder. Staining for P450 LM 4b in livers from untreated trout was barely detectable. In liver tissue sections from AFB 1-treated tumor-bearing fish, P450 LM 2 appeared to be reduced and P450 LM 4b was absent in the hepatocellular carcinoma nodules. An apparent increase in immunostaining for P450 LM 4b was observed in nonneoplastic cells juxtaposed next to neoplastic cells as well as in areas distant to the tumors. These results may indicate that the pattern of P450 isozymes is altered in nonneoplastic cells of tumor-bearing trout livers.

Localization of cytochrome P-450 in the head and trunk kidney of rainbow trout ( Salmo gairdneri)

Mature, untreated rainbow trout display a marked sex difference in the level and activity of the P-450-dependent mixed-function oxidase system with males demonstrating higher levels than females. This difference is accentuated in the trunk kidney, is specific for endogenous substrates such as lauric acid and sex steroids, and is correlated with greater levels of the constitutive cytochrome P-450 isozyme, LM 2. Using an immunohistochemical technique, the cellular localization of P-450 LM 2 and P-450 LM 4b, the major β-naphthoflavone inducible isozyme, in the head and trunk kidney of male, female, and juvenile trout has been demonstrated. Immunostaining for P-450 LM 2 was observed in the cytoplasm of cells in the second portion (P 2) of the proximal tubules of both male and female trout. In male fish, staining appeared to be more intense and involved most of the P 2 segments in a tissue section. In female fish, P 2 staining was moderate and involved fewer segments. Low staining intensity for P-450 LM 4b was observed in the cytoplasm of cells both in the first portion of the proximal tubules and in P 2 of male and female fish. Sexual dimorphism was not apparent. The finding of greater amounts of P-450 LM 2 in males and low levels of P-450 LM 4b in untreated male and female trout confirms previous biochemical studies. Localization of P-450 isozymes in the proximal tubules is consistent with findings in mammalian species. Moderate immunostaining by anti- P-450 LM 4b IgG also was observed in the interrenal cells of the head kidney of male, female, and juvenile trout.

Stereospecificity in the oxidation of phorate and phorate sulphoxide by purified FAD-containing mono-oxygenase and cytochrome P-450 isozymes.

1. Both the cytochrome P-450-dependent mono-oxygenase system and the FAD-containing mono-oxygenase catalyse the sulphoxidation of thioether-containing organophosphate insecticides. Using purified FAD-containing mono-oxygenase and purified cytochrome P-450 isozymes isolated from mouse liver microsomes, the stereospecificity of the oxidation of phorate to (+)-and (-)-phorate sulphoxide and the further oxidations of the (+)-and (-)-phorate sulphoxides to the sulphone, the oxon sulphoxide and the oxon sulphone were examined. 2. The FAD-containing mono-oxygenase catalysed the formation of (-)-phorate sulphoxide, while two cytochrome P-450 isozymes (cytochrome P-450-B2, a constitutive form, and cytochrome P-450-PB, the principal form induced by phenobarbital) produced (+)-phorate sulphoxide. The other three constitutive cytochrome P-450 isozymes examined yielded racemic mixtures. 3. The FAD-containing mono-oxygenase had the lowest Km for the sulphoxidation reaction, 32 microM, while the Km values for the cytochrome P-450 isozymes ranged from 67 microM to 250 microM. No additional oxidation of phorate sulphoxide by the FAD-containing monooxygenase was detected using either (+)-phorate sulphoxide or (-)-phorate sulphoxide as substrates. 4. In contrast, all five cytochrome P-450 isozymes tested formed additional oxidation products; the (+)-phorate sulphoxide was the preferred substrate for all cytochrome P-450 forms. 5. The final oxidation product, phorate oxon sulphone, was derived by desulphuration of phorate sulphone, with the formation of the oxon sulphoxide being a terminal pathway.

Evidence for distinct binding sites in the cumene hydroperoxide-dependent metabolism of benzo[a]pyrene catalyzed by cytochrome P-450

A few constitutive cytochrome P-450 isozymes in male rat liver microsomes catalyzed the metabolism of benzo[ a]pyrene (BP) in cumene hydroperoxide (CHP)-dependent reactions, which produced predominantly 3-hydroxyBP and BP quinones. This process varied with the concentration of CHP. At 0.05 mM CHP, 3-hydroxyBP was the major metabolite. An increase in CHP concentration reduced 3-hydroxyBP formation but increased the level of BP quinones. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene selectively inhibited quinone formation and enhanced 3-hydroxyBP formation. Naphthalene, phenanthrene and benz[ a] anthracene nonspecifically inhibited total metabolism. BP binding to microsomal protein correlated with quinone formation, suggesting a common precursor reactive intermediate. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less effective than that in the male. With females, quinones were the major metabolites at all CHP concentrations, and their formation was again modulated by pyrene. These data indicate that two distinct binding sites are responsible for the formation of 3-hydroxyBP and BP quinones.

Dependence of benzo[ a]pyrene metabolic profile on the concentration of cumene hydroperoxide with uninduced and induced rat liver microsomes

The effect of cumene hydroperoxide (CHP) in microsomal metabolism of benzo[ a]pyrene (BP) was studied using liver microsomes from mature male Wistar rats induced with phenobarbital (PB), 3-methylcholanthrene (MC), Aroclor 1254 or olive oil (uninduced). In contrast to NADPH-supported metabolism, these inducers did not increase the CHP-dependent metabolism. Total BP metabolism was dependent on CHP concentration and was maximal at 0.15 mM, except for PB-induced microsomes, which had a maximum at 0.5 mM CHP. At 0.05 mM CHP, the major metabolites were phenols. However, increasing CHP concentration enhanced the formation of dihydrodiols, quinones and protein-bound BP but reduced phenol production. At and above 0.15 mM CHP, the profile of BP metabolites was essentially constant, with at least 66% quinones but no more than 10% phenols. The effect of CHP on inhibition of phenol formation and enhancement of quinone formation was reversed by preincubation of microsomes with BP or by increasing BP concentration. These results suggest that CHP-dependent metabolism of BP is selectively mediated by constitutive cytochrome P-450 isozyme(s) and that two forms of BP binding sites exist in cytochrome P-450 isozymes and are responsible for the hydroxylation of BP at C-3 and C-6.

Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation.

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.