VALIDATION OF 4-NITROPHENOL AS AN IN-VITRO SUBSTRATE PROBE FOR HUMAN LIVER CYP2E1 USING CDNA EXPRESSION AND MICROSOMAL KINETIC TECHNIQUES

The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylatio n of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigate d using cDNA expression and liver microsomal kinetic and inhibitor tec hniques. 4NP hydroxylation by human liver microsomes and cDNA-expresse d human CYP2E1 exhibited Michaelis-Menten kinetics; the respective app arent K-m values were 30 +/- 7 and 21 mu M. Mutual competitive inhibit ion was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent K-m and K-i values for each individual compou nd. 4NP and CZ hydroxylase activities in microsomes from 18 liver dono rs varied to a similar extent (3.3- and 3.0-fold, respectively) and 4N P hydroxylase activity correlated significantly (r(s) greater than or equal to 0.75, P < 0.005) with both CZ hydroxylation and immunoreactiv e CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbam ate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxy lation by cDNA-expressed CYP2E1 and human liver microsomes in parallel . Probes for other human CYP isoforms namely (alpha-naphthoflavone, co umarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) ca used <15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed la rgely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme.