Adduct formation has been considered to be a major causal factor of DNA damage by carcinogenic heterocyclic amines. By means of experiments with 32 P-labeled DNA fragments and an electrochemical detector coupled to a high-pressure liquid chromatograph, we investigated whether the N-hydroxy metabolite of 2-amino-3, Key words: Heterocyclic amine -MeIQx -Oxidative DNA damage -Copper -NADH Several heterocyclic amines isolated from cooked foods are among the most potent mutagens known.1, 2) 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is one such heterocyclic amine isolated from cooked beef, chicken and mutton.3, 4) Oral administration of MeIQx produces hepatocellular carcinomas and lung tumors in mice, [5][6][7] and hepatocellular carcinomas and squamous cell carcinomas of the Zymbal gland in rats. 5,8,9) An epidemiological study showed a significantly increased risk for cancers at all sites and for gastric cancer associated with the consumption of cooked fish. 4) Thus, most heterocyclic amines in food-pyrolysates were suggested to pose probable or possible carcinogenic risk to humans. 4)Regarding the mechanisms of DNA damage by carcinogenic heterocyclic amines, DNA adduct formation has been considered to be a major causal factor. That is, MeIQx is oxidized to the N-hydroxy derivative [MeIQx(NHOH)] in the liver by cytochrome P450 IA2 isozyme, and the latter is esterified by O-acetyltransferase to the N-acetoxy derivative, which reacts with DNA to form adducts. 10,11) DNA adducts generated by MeIQx were found in vitro and in vivo by means of the 32 P-postlabeling method.12) On the other hand, it has been reported that several antioxidants [13][14][15] significantly inhibited MeIQx-induced hepatocarcinogenesis in rats. Kato et al. 16) reported that the 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) level in rat liver increased dose-dependently with the concentration of MeIQx in the diet. These reports lead us to consider that reactive oxygen species may participate in heterocyclic amine-induced tumor development. Hayatsu and his colleagues 17,18) showed that the N-hydroxy derivative of 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2) produces intracellular reactive oxygen species that can damage DNA in mouse cells in culture. Therefore, DNA adduct formation is a prerequisite, but the DNA adducts themselves may not be sufficient for the carcinogenic action. There remains a possibility that oxidative DNA damage also plays a role in carcinogenesis induced by MeIQx.In this study, we investigated whether MeIQx(NHOH) can cause oxidative DNA damage or not, using 32 P-5′-endlabeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. We analyzed 8-oxodG formation in calf thymus DNA by MeIQx(NHOH) in the presence of Cu(II) and β-nicotinamide adenine dinucleotide (NADH). Furthermore, in order to clarify the mechanism of oxidative DNA damage, spectral changes during the autoxidation of MeIQx(NHOH) were measured, using UV-visible spectroscopy.
Nitro derivative (nitro-IQ) of a carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is known to be a potent mutagen as well as IQ, and nitro-IQ is believed to be activated enzymatically by nitroreductase. We investigated nonenzymatic reduction of nitro-IQ by an endogenous reductant NADH and the ability of inducing DNA damage by nitro-IQ. Nitro-IQ caused DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of NADH and Cu(II). Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). Nitro-IQ induced DNA cleavage frequently at thymine and cytosine residues in the presence of NADH and Cu(II). UV-vis spectroscopic study showed that no spectral change of Nitro-IQ and NADH was observed in the absence of Cu(II), while rapid spectral change was observed in the presence of Cu(II), suggesting that Cu(II) mediated redox reaction of nitro-IQ and NADH. These results suggest that nitro-IQ can be reduced nonenzymatically by NADH in the presence of Cu(II), and the redox reaction resulted in oxidative DNA damage due to the copper-oxygen complex, derived from the reaction of Cu(I) with H2O2. We conclude that nonenzymatic reduction of nitro-IQ and resulting in oxidative DNA damage can play a role in carcinogenesis of IQ.
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