2-Nitrophenol (2-NP) is the major detoxification metabolite of an important industrial pollutant and a potent carcinogen, 2-nitroanisole (2-NA). Here, we characterized the product of 2-NP metabolism catalyzed by human, rat, rabbit and mouse hepatic microsomes containing cytochromes P450 (CYPs) and identified the major human CYP enzymes participating in this process. The 2-NP metabolite was characterized by mass spectrometry and co-chromatography on HPLC with a synthetic standard, 2,5-dihydroxynitrobenzene (2,5-DNB) to be 2,5-DNB. No nitroreductive metabolism leading to the formation of N-(2-hydroxyphenyl)hydroxylamine or o-aminophenol was evident by all tested hepatic microsomes. Likewise, no DNA binding of 2-NP metabolite(s) measured with the 32P-postlabeling technique was detectable in hepatic microsomes. Therefore, hepatic microsomal CYP enzymes participate in 2-NP metabolism that does not lead to its activation to species binding to DNA. Selective inhibitors of human CYPs were used to characterize CYPs oxidizing 2-NP in human livers. Based on these inhibitory studies, we attribute most of 2-NP oxidation in human liver to CYP2E1, 3A4, 2A6, 2C and 2D6. Among recombinant human CYP enzymes tested in this study, CYP2E1, 2A6 and 2B6 were the most effective enzymes oxidizing 2-NP. Oxidation of 2-NP by human CYP2E1 exhibits the Michaelis-Menten kinetics, having the Km value of 0.21 mM. The results found in this study, the first report on the metabolism of 2-NP by human hepatic microsomes and human CYP enzymes, demonstrate that CYP2E1 is the major enzyme oxidizing this compound in human.

Department of Biochemistry Faculty of Natural Sciences Charles University Prague Albertov 2030 128 43 Prague 2 Czech Republic

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