4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% (+/- 11) and 13% (+/- 16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.

The induction effects of pyrazole and dexamethasone (known to be specific to P450 2E1 and 3A enzymes, respectively), given alone or simultaneously, were studied in rat liver and kidney microsomes. Pyrazole treatment induced the catalytic activity and the amount of P450 2E1 enzyme in both organs. Immunoreactive P450 2E1 and 4-nitrophenol 2-hydroxylation increased 8- and 13-fold, respectively (versus control), in the kidney, but only 2.4- and 2.7-fold (versus control) in the liver after pyrazole treatment. As assessed by nifedipine oxidation activity, dexamethasone treatment increased the P450 3A catalytic activity approximately 4-fold (versus control) in the liver, but not in the kidney, suggesting that P450 3A was not inducible in the kidney. Pyrazole decreased P450 3A activity in the liver but did not modify it in the kidney. A combination of both chemicals induced both enzymes, but to a lesser extent than treatment with each single chemical compound. Furthermore, the 2-hydroxylation of p-nitrophenol, considered one of the most specific substrates for monitoring the level of P450 2E1, was mediated also by P450 3A, at least in dexamethasone-treated rats. Finally, this experimental work demonstrated that P450 3A induction is organ-specific, and it also demonstrated the lack of specificity of p-nitrophenol as a P450 2E1 substrate.

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