... The detoxifying metabolism of a potent rodent carcinogen, 2-nitroanisole (2-NA) by human, rabbit and rat cytochromes P450 (P450) was investigated ... HPLC with UV detection was employed for the separation and characterization of 2-NA metabolites formed by hepatic microsomes, human recombinant P450s and purified rat and rabbit P450s. An O-demethylated metabolite of 2-NA, 2-nitrophenol (2-NP), and two oxidation products of this metabolite [2,5-dihydroxynitrobenzene (2,5-DNB) and 2,6-dihydroxynitrobenzene (2,6-DNB)] were generated by microsomes and P450s from the species investigated, but at different levels. All the metabolites are detoxication products. 2-NP is the major metabolite generated by rabbit and rat microsomes, but 2,5-DNB is the predominant product in human microsomes. Using human recombinant P450s and purified rodent P450s, we found that human P450 2E1, 1A1 and 2B6 as well as orthologous animal P450s were the most efficient enzymes oxidizing 2-NA to 2-NP, while P450 2E1 and 1A1 were the most effective in the formation of 2,5-DNB and 2,6-DNB. In human hepatic microsomes, 2-NA was oxidized mainly by P4502E1. 2-NA and its reductive metabolite o-anisidine induced rat hepatic and renal P450 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1), thus modifying their own detoxication and/or activation pathways. The data demonstrated the participation of orthologous P450s in 2-NA oxidation by all species and indicated that the rat and rabbit might serve as suitable models to mimic 2-NA oxidation in /humans/.
2-Nitrophenol (2-NP) is the major detoxification metabolite of ... 2-nitroanisole (2-NA). Characterization of the products of 2-NP metabolism by rat hepatic microsomes containing cytochromes P450 (CYPs) and identification of the major CYP enzymes participating in this process are aims of this study ... Rat hepatic microsomes oxidize 2-NP to its hydroxylated metabolite, 2,5-dihydroxynitrobenzene (2,5-DNB). No nitroreductive metabolism leading to the formation of o-aminophenol was evident when using rat hepatic microsomes. Selective CYP inhibitors and hepatic microsomes of rats pre-treated with specific CYP inducers were used to characterize CYPs oxidizing 2-NP in rat livers. Based on these studies, we attribute most of 2-NP oxidation in rat liver to CYP2E1 and 3A, followed by CYP2D and 2C. Among recombinant rat CYP enzymes tested in this study, CYP2E1 and 2C11 were the most effective enzymes oxidizing 2-NP. Oxidation of 2-NP by rat CYP2E1 exhibits the Michaelis-Menten kinetics, having the Km value of 0.35 mM. The results found in this study ... demonstrate that CYP2E1 is the major enzyme oxidizing this compound in rat liver.
... The ability of hepatic microsomal samples from different species including human to metabolize 2-nitroanisole (2-NA) /was compared/ ... Human hepatic microsomes generated a pattern of 2-NA metabolites, reproducing that formed by hepatic microsomes of rats and rabbits. An O-demethylated metabolite of 2-NA (2-nitrophenol) and two ring-oxidized derivatives of this metabolite (2,6-dihydroxynitrobenzene and 2,X-dihydroxynitrobenzene) were produced. No nitroreductive metabolism leading to the formation of o-anisidine was evident with hepatic microsomes of any species. Likewise, no DNA binding of 2-NA metabolite(s) measured with either tritium-labeled 2-NA or the (32)P-postlabeling technique was detectable in microsomes. Therefore, hepatic microsomal P450 enzymes participate in the detoxication reactions of this environmental carcinogen. Using hepatic microsomes of rabbits pretreated with specific P450 inducers, microsomes from Baculovirus transfected insect cells expressing recombinant human P450 enzymes, purified P450 enzymes, and selective P450 inhibitors, /the authors/ found that human recombinant P450 2E1, 1A1, and 2B6, as well as orthologous rodent P450 enzymes, are the most efficient enzymes metabolizing 2-NA. The role of specific P450 enzymes in the metabolism of 2-NA in human hepatic microsomes was investigated by correlating specific P450-dependent reactions with the levels of 2-NA metabolites formed by the same microsomes and by examining the effects of specific inhibitors of P450 enzymes on 2-NA metabolism. On the basis of these studies, ... most of the 2-NA oxidation metabolism in human microsomes /was attributed/ to P450 2E1. These results ... clearly demonstrate that P450 2E1 is the major human enzyme oxidizing this carcinogen in human liver.
After receiving either 5 mg/kg bw or 50 mg/kg bw (14)C-labeled o-nitroanisole p.o. (male F344 rats), metabolites in the urine after 7 days included 63% o-nitrophenylsulfate, 11% o-nitrophenylglucuronide, 1.5% o-nitrophenol, 0.6% o-anisidine. /Translated from German/
Evaluation: There is inadequate evidence in humans for the carcinogenicity of 2-nitroanisole. There is sufficient evidence in experimental animals for the carcinogenicity of 2-nitroanisole. Overall evaluation: 2-Nitroanisole is possibly carcinogenic to humans (Group 2B).
Male F344 rats were injected iv with 25 mg/kg bw (14)C-labeled o-nitroanisole. 15 min post-administration maximum tissue concentration was reached. Elimination bi-phasic. The first elimination phase had a half-life of 1-2 hr, the second elimination phase was longer and was tissue-dependent. /Translated from German/
Male F344 rats received 4 or 50 mg/kg bw (14)C-labeled o-nitroanisole p.o. Within 24 hr 73% of the 5 mg/kg bw dose and 69% of the 50 mg/kg bw dose was eliminated in the urine. Within 7 days 71-78% in the urine and 7% in the feces was eliminated. /Translated from German/
Skin absorption in humans is possible. An increase in the substance can be detected in the urine in skin studies. Metabolites are formed by hydrolytic cleavage and are derivatives of o-nitrophenol. /Translated from German/
... An intravenous dose of 25 mg/kg bw was used for pharmacokinetic studies. Following a 25 mg/kg bw intravenous injection of (14C)2-nitroanisole, blood, tissues and excreta were collected at times ranging from 15 min to seven days. The distribution of 2-nitroanisole derived 14C to tissues (muscle, 20%; skin, 10%; fat, 6.8%; blood, 6.5%; liver, 4.8%; plasma, 3.1 %; kidney, 2.8%; and small intestine, 1.9%) occurred rapidly following administration. Peak tissue concentrations were reached in all tissues within 15 min. Urinary and fecal elimination were similar to that found after oral administration (urine, 86% by seven days; feces, 9% by seven days). ... Biliary excretion was similar to fecal elimination, indicating a lack of enterohepatic recirculation. Urine collected for 24 hr after intraperitoneal administration of 25 mg/kg bw 2-nitroanisole had a profile similar to that observed after oral administration (63% 2-nitrophenyl sulfate, 11 % 2-nitrophenyl glucuronide, 1.5% 2-nitrophenol and 0.6% ortho-anisidine).
