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Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

ABSTRACT: This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.

SUBMITTER: Sinclair P

PROVIDER: S-EPMC1138346 | biostudies-other | 1989 Feb

REPOSITORIES: biostudies-other

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Project description:Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was assayed in cultures of chick-embryo hepatocytes by the changes in composition of porphyrins accumulated after addition of excess 5-aminolaevulinate. Control cells accumulated mainly protoporphyrin, whereas cells treated with 3,4,3',4'-tetrachlorobiphenyl or 2,4,5,3',4'-pentabromobiphenyl accumulated mainly uroporphyrin, indicating decreased activity of the decarboxylase. 3-Methylcholanthrene and other polycyclic-hydrocarbon inducers of the P-448 isoenzyme of cytochrome P-450, did not affect the decarboxylase in the absence of the biphenyls. Induction of P-448 was detected as an increase in ethoxyresorufin de-ethylase activity. Pretreatment of cells with methylcholanthrene decreased the time required for the halogenated biphenyls to inhibit the decarboxylase. The dose response of methylcholanthrene showed that less than 40% of the maximal induction of cytochrome P-448 was needed to produce the maximum biphenyl-mediated inhibition of the decarboxylase. In contrast, induction of the cytochrome P-450 isoenzyme by propylisopropylacetamide had no effect on the biphenyl-mediated decrease in decarboxylase activity. Use of inhibitors of the P-450 and P-448 isoenzymes (SKF-525A, piperonyl butoxide and ellipticine) supported the concept that only the P-448 isoenzyme is involved in the inhibition of the decarboxylase by the halogenated biphenyls. The effect of preinduction with methylcholanthrene to enhance inhibition of the decarboxylase was also shown by the increased rate at which porphyrin accumulated from endogenously synthesized 5-aminolaevulinate after treatment of cells with the combination of propylisopropylacetamide and the biphenyls. Antioxidants, chelators of iron, and chromate affected the decrease in decarboxylase activity only if they prevented the induced increase in cytochrome P-448. We conclude that the P-448 and not the P-450 isoenzyme of cytochrome P-450 plays an obligatory role in the inhibition of uroporphyrinogen decarboxylase caused by halogenated biphenyls.

Project description:Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein. While the apparent Km values for the 2C9/10 isoenzymes (71.6-131.7 microM) were comparable with the range of apparent Km values previously observed in human liver microsomes, the apparent Km for 2C8 (650.5 microM) was appreciably higher. The 2C8 enzyme also showed quite different sulphaphenazole inhibition characteristics. The 4-hydroxylation of phenytoin was also more efficiently catalysed by the 2C9/10 enzymes. These enzymes showed similarities in kinetics of phenytoin hydroxylation and sulphaphenazole inhibition compared with human liver phenytoin hydroxylase. Also of interest was the observation that, among the 2C9 variants, small differences in amino acid composition could appreciably affect both tolbutamide and phenytoin hydroxylations. The amino acid substitution Cys-144-->Arg increased both the rates of tolbutamide and phenytoin hydroxylations, while the Leu-359-->Ile change had a greater effect on phenytoin hydroxylation. We conclude that: (1) although 2C8 and 2C9/10 proteins metabolize tolbutamide. only 2C9/10 proteins play a major role in human liver; (2) 2C9/10 proteins also appear to be chiefly responsible for phenytoin hydroxylation; and (3) subtle differences in the amino acid composition of these 2C9/10 proteins can affect the functional specificities towards both tolbutamide and phenytoin.

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Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

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