Both Cytochromes P450 2E1 and 3A Are Involved in the O-Hydroxylation of p-Nitrophenol, a Catalytic Activity Known To Be Specific for P450 2E1

Abstract

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.

THECYP2E1-HUMANIZED TRANSGENIC MOUSE: ROLE OF CYP2E1 IN ACETAMINOPHEN HEPATOTOXICITY

“…Measurement of chlorzoxazone and trimethadione metabolism by CYP2E1 has also been used as an indicator for liver diseases in vivo and in vitro (Dilger et al, 1997;Burckart et al, 1998;Mishin et al, 1998;Dupont et al, 2000). An alternative method for measuring CYP2E1 activity has been to analyze p-nitrophenol hydroxylation ( p-nitrocatechol formation) (Zerilli et al, 1997). In this study, complete inhibition of chlorzoxazone 6-hydroxylation was not achieved due to the contribution of other P450s such as CYP1A2 to the metabolism of chlorzoxazone (Carriere et al, 1993;Ono et al, 1995).…”

Isoform-Specific Regulation of Cytochromes P450 Expression by Estradiol and Progesterone

“…In estradiol-treated hepatocytes, similar increases in the P450 enzyme activity were observed for multiple probe substrates of CYP2C9 (diclofenac and tolbutamide) and CYP2E1 (p-nitrophenol and chlorzoxazone). Furthermore, cotreatment of hepatocytes with a phase II enzyme inhibitor (i.e., 2 mM salicylamide) to block the known glucuronidation of p-nitrophenol (Zerilli et al, 1997;Hanioka et al, 2001) did not abolish the increased p-nitrophenol hydroxylation upon estradiol treatment (data not shown). These results suggest that potential nonspecificity of the substrates unlikely contributes to the enhanced CYP2C9 and CYP2E1 activity by estradiol.…”

Minipig Cytochrome P450 2e1: Comparison With Human Enzyme

“…In line with this fact, there is in the minipig probably a contribution also from the CYP3A (Madden et al, 1998) as well as the CYP1A enzymes to the metabolism of chlorzoxazone (Bogaards et al, 2000) in the microsomal preparations. The second substrate, p-nitrophenol, is known to be metabolized by CYP2E1 as well as (with lower turnover number) by CYP3A enzymes in humans (Zerilli et al, 1997). Most recent results indicate an involvement of CYP2A6 and possibly also of CYP2C19 enzymes in this reaction (Monostory et al, 2004).…”