CHLORZOXAZONE 6-HYDROXYLASE AND P-NITROPHENOL HYDROXYLASE AS THE MOSTSUITABLE ACTIVITIES FOR ASSAYING CYTOCHROME-P450 2E1 IN CYNOMOLGUS MONKEY LIVER

Western blot analyses of liver microsomes from 13 male and 12 female m onkeys demonstrated that in each sample a variable amount of a cytochr ome P450 (P450) protein, likely monkey P450 2E1, cross-reacted with an ti-rat P450 2E1 antibodies. Therefore, the involvement of monkey 2E1 i n the oxidation of typical substrates for 2E1 from other species, such as dimethylnitrosamine (DMN), p-nitrophenol (pNP), chlorzoxazone (CLZ ), and aniline, was investigated. Kinetic studies using microsomes fro m five male and five female monkeys showed that CLZ and pNP hydroxylat ions were monophasic, with apparent K-M values of 77 and 14 mu M, resp ectively, whereas aniline hydroxylation and DMN demethylation were mul tiphasic, suggesting that P450s other than 2E1 were involved in cataly zing the latter two reactions. When correlation analyses were performe d using several monooxygenase activities determined in male and female monkey liver specimens, it was found that immunodetectable 2E1 conten ts were highly correlated (r greater than or equal to 0.75) with CLZ a nd pNP hydroxylations, weakly correlated (r = 0.6) with aniline hydrox ylation, and not correlated with DMN demethylation or other monooxygen ase activities; CU hydroxylation was strongly correlated with pNP hydr oxylation, weakly correlated with aniline hydroxylation, and not corre lated with DMN demethylation. Inhibition experiments showed that CU an d pNP hydroxylations were immunoinhibited by 60-80% by anti-rat P450 2 E1 and were inhibited by the prototypical 2E1 inhibitor 4-methylpyrazo le with IC50 values of 1.5 and 13 mu M, respectively. In conclusion, t he findings provide evidence that P450 2E1 is constitutively and equal ly expressed in male and female monkey liver and it exerts a major rol e only in hydroxylation of CU and pNP.