Collect. Czech. Chem. Commun. 2010, 75, 703-719
Published online 2010-06-30 19:27:02

Identification of human enzymes oxidizing a human metabolite of carcinogenic 2-nitroanisole, 2-nitrophenol. Evidence for its oxidative detoxification by human cytochromes P450

Martina Svobodováa, Markéta Martínkováa, Eva Freib and Marie Stiborováa,*

a Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague, Albertov 2030, 128 43 Prague 2, Czech Republic
b German Cancer Research Center, Im Neuenheimar Feld 280, 69120 Heidelberg, Germany


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.

Keywords: 2-Nitrophenol; 2-Nitroanisole; Cytochrome P450; Oxidation; Detoxification.

References: 34 live references.