Chlorzoxazone 6-hydroxylase and p-nitrophenol hydroxylase as the most suitable activities for assaying cytochrome P450 2E1 in cynomolgus monkey liver.
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 apparent KM values of 77 and 14 microgramsM, 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 microgramsM, 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.
Amato
G
G
Laboratory of Genetic and Biochemical Toxicology, Istituto di Mutagenesi e Differenziamento, Consiglio Nazionale delle Ricerche, Pisa Italy.
Longo
V
V
Mazzaccaro
A
A
Gervasi
P G
PG
D000818:Animals; D015153:Blotting, Western; D002753:Chlorzoxazone / Q000493:pharmacokinetics*; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D005260:Female; D006900:Hydroxylation; D008252:Macaca fascicularis; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009125:Muscle Relaxants, Central / Q000493:pharmacokinetics*; D009596:Nitrophenols / Q000493:pharmacokinetics*
D009125:Muscle Relaxants, Central; D009596:Nitrophenols; D019392:Cytochrome P-450 CYP2E1; D002753:Chlorzoxazone; C024836:4-nitrophenol