Abstract
Western blot analyses of liver microsomes from 13 male and 12 female monkeys demonstrated that in each sample a variable amount of a cytochrome P450 (P450) protein, likely monkey P450 2E1, cross-reacted with anti-rat P450 2E1 antibodies. Therefore, the involvement of monkey 2E1 in 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 from five male and five female monkeys showed that CLZ and pNP hydroxylations were monophasic, with apparentKM values of 77 and 14 μM, respectively, whereas aniline hydroxylation and DMN demethylation were multiphasic, suggesting that P450s other than 2E1 were involved in catalyzing the latter two reactions. When correlation analyses were performed using several monooxygenase activities determined in male and female monkey liver specimens, it was found that immunodetectable 2E1 contents were highly correlated (r ≥ 0.75) with CLZ and pNP hydroxylations, weakly correlated (r = 0.6) with aniline hydroxylation, and not correlated with DMN demethylation or other monooxygenase activities; CLZ hydroxylation was strongly correlated with pNP hydroxylation, weakly correlated with aniline hydroxylation, and not correlated with DMN demethylation. Inhibition experiments showed that CLZ and pNP hydroxylations were immunoinhibited by 60–80% by anti-rat P450 2E1 and were inhibited by the prototypical 2E1 inhibitor 4-methylpyrazole with IC50 values of 1.5 and 13 μM, respectively. In conclusion, the findings provide evidence that P450 2E1 is constitutively and equally expressed in male and female monkey liver and it exerts a major role only in hydroxylation of CLZ and pNP.
Footnotes
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Send reprint requests to: Dr. Pier Giovanni Gervasi, Istituto di Mutagenesi e Differenziamento, C.N.R., via Svezia 10, 56124 Pisa, Italy.
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This work was partially supported by European Community Program BIOMED 2 PL95–0184 and partially supported by Menarini Ricerche S.p.A. (Pomezia, Italy).
- Abbreviations used are::
- P450
- cytochrome P450
- AnH
- aniline hydroxylase
- CLZ
- chlorzoxazone
- CLZ6H
- chlorzoxazone 6-hydroxylase
- DMN
- dimethylnitrosamine
- DMNd
- dimethylnitrosamine demethylase
- 8MP
- 8-methoxypsoralen
- 4MPy
- 4-methylpyrazole
- pNP
- p-nitrophenol
- pNPH
- p-nitrophenol hydroxylase
- Received June 19, 1997.
- Accepted January 9, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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