Oxidative detoxication of carcinogenic 2-nitroanisole by human, rat and rabbit cytochrome P450.

OBJECTIVES: The detoxifying metabolism of a potent rodent carcinogen, 2-nitroanisole (2-NA) by human, rabbit and rat cytochromes P450 (P450) was investigated. Comparison between P450s of experimental animals and humans is essential for the extrapolation of animal carcinogenicity data to the human situation and to assess health risk. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 man.

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition.

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.

Identification of a genotoxic mechanism for the carcinogenicity of the environmental pollutant and suspected human carcinogen o-anisidine.

2-methoxyaniline (o-anisidine) is an industrial and environmental pollutant and a bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated with 2 independent methods, 32P-postlabeling and 14C-labeled o-anisidine, to show that o-anisidine binds covalently to DNA in vitro after its activation by human hepatic microsomes. We also investigated the capacity of o-anisidine to form DNA adducts in vivo. Rats were treated i.p. with o-anisidine (0.15 mg/kg daily for 5 days) and DNA from several organs was analyzed by 32P-postlabeling. Two o-anisidine-DNA adducts, identical to those found in DNA incubated with o-anisidine and human microsomes in vitro, were detected in urinary bladder (4.1 adducts per 10(7) nucleotides), the target organ, and, to a lesser extent, in liver, kidney and spleen. These DNA adducts were identified as deoxyguanosine adducts derived from a metabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine. This metabolite was identified in incubations with human microsomes. With 9 human hepatic microsomal preparations, we identified the specific CYP catalyzing the formation of the o-anisidine metabolites by correlation studies and by examining the effects of CYP inhibitors. On the basis of these analyses, oxidation of o-anisidine was attributed mainly to CYP2E1. Using recombinant human CYP (in Supersomes) and purified CYPs, the participation of CYP2E1 in o-anisidine oxidation was confirmed. In Supersomes, CYP1A2 was even more efficient in oxidizing o-anisidine than CYP2E1, followed by CYP2B6, 1A1, 2A6, 2D6 and 3A4. The results, the first report on the potential of the human microsomal CYP enzymes to activate o-anisidine, strongly suggest a carcinogenic potential of this rodent carcinogen for humans.

Carcinogenic pollutants o-nitroanisole and o-anisidine are substrates and inducers of cytochromes P450.

2-Methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole) are important pollutants and potent carcinogens for rodents. o-Anisidine is oxidized by microsomes of rats and rabbits to N-(2-methoxyphenyl)hydroxylamine that is also formed as the reduction metabolite of o-nitroanisole. o-Anisidine is a promiscuity substrate of rat and rabbit cytochrome P450 (CYP) enzymes, because CYPs of 1A, 2B, 2E and 3A subfamilies oxidize o-anisidine. Using purified CYP enzymes, reconstituted with NADPH: CYP reductase, rabbit CYP2E1 was the most efficient enzyme oxidizing o-anisidine, but the ability of CYP1A1, 1A2, 2B2, 2B4 and 3A6 to participate in o-anisidine oxidation was also proved. Utilizing Western blotting and consecutive immunoquantification employing chicken polyclonal anti bodies raised against various CYPs, the effect of o-anisidine and o-nitroanisole on the expression of the CYP enzymes was investigated. The expression of CYP1A1/2 was found to be strongly induced in rats treated with either compounds. In addition, 7-ethoxyresorufin O-deethylation, a marker activity for both CYP1A1 and 1A2, was significantly increased in rats treated with either carcinogen. The data demonstrate the participation of different rat and rabbit CYP enzymes in o-anisidine oxidation and indicate that both experimental animal species might serve as suitable models to mimic the o-anisidine oxidation in human. Furthermore, by induction of rat hepatic and renal CYP1A1/2, both o-nitroanisole and o-anisidine influence their carcinogenic effects, modifying their detoxification and/or activation pathways.

Enzymes involved in the metabolism of the carcinogen 2-nitroanisole: evidence for its oxidative detoxication by human cytochromes P450.

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent carcinogen for rodents. Determining the capability of humans to metabolize 2-NA and understanding which human cytochrome P450 (P450) enzymes are involved in its activation and/or detoxification are important to assess an individual's susceptibility to this environmental carcinogen. We compared the ability of hepatic microsomal samples from different species including human to metabolize 2-NA. Comparison between experimental animals and human P450 enzymes is essential for the extrapolation of animal carcinogenicity data to assess human health risk. 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, we 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, we attribute most of the 2-NA oxidation metabolism in human microsomes to P450 2E1. These results, the first report on the metabolism of 2-NA by human P450 enzymes, clearly demonstrate that P450 2E1 is the major human enzyme oxidizing this carcinogen in human liver.

Identification of a genotoxic mechanism for 2-nitroanisole carcinogenicity and of its carcinogenic potential for humans.

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated in this study. Here we have used two independent methods, (32)P-post-labeling and (3)H-labeled 2-NA, to show that 2-NA binds covalently to DNA in vitro after reductive activation by human hepatic cytosol and xanthine oxidase (XO). We also investigated the capacity of 2-NA to form DNA adducts in vivo. Male Wistar rats were treated i.p. with 2-NA (0.15 mg/kg body wt daily for 5 days) and DNA from several organs was analyzed by (32)P-post-labeling. Two 2-NA-specific DNA adducts, identical to those found in DNA incubated with 2-NA and human hepatic cytosol or XO in vitro, were detected in the urinary bladder (3.4 adducts/10(7) nt), the target organ, and, to a lesser extent, in liver, kidney and spleen. The two DNA adducts found in rat tissues in vivo were identified as deoxyguanosine adducts derived from a 2-NA reductive metabolite, N-(2-methoxyphenyl)hydroxylamine. This reactive metabolite of 2-NA was identified in incubations with human hepatic cytosol, besides 2-methoxyaniline (o-anisidine). The results of our study, the first report on the potential of human cytosolic enzymes to contribute to the activation of 2-NA by nitroreduction, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.

Mechanism of peroxidase-mediated oxidation of carcinogenic o-anisidine and its binding to DNA.

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [14C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The 32P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to 32P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the 32P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3'-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the 32P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.