Mechanism of Oxidative DNA Damage Induced by a Heterocyclic Amine, 2‐Amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline

Abstract

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.

Effect of chrysin, a flavonoid compound, on the mutagenic activity of 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) and benzo(a)pyrene (B(a)P) in bacterial and human hepatoma (HepG2) cells

“…The CYP-mediated metabolism of heterocyclic aromatic amines and polycyclic aromatic hydrocarbons including B(a)P is known to yield reactive oxygen species [ROS (Murata et al 1999;Yo et al 2002)]. Therefore, another possible mechanism for the reduction in the PhIP and B(a)P-induced MN frequency in HepG2 cells may be scavenging of ROS induced by the mutagens.…”

Oxidative DNA Damage Induced by an N‐Hydroxy Metabolite of Carcinogenic 4‐Dimethylaminoazobenzene

“…24) Several studies indicate that NADH may react nonenzymatically with some xenobiotics and mediate their reduction. [25][26][27] The biological importance of NADH and NADPH as nuclear reductants has been pointed out. 28) N-OH-AAB plus Cu(II) induced piperidine-labile sites preferentially at thymine and cytosine residues.…”

“…However, we previously detected site-specific DNA damage, including hotspots, by the N-hydroxy metabolite of a heterocyclic amine, via a mechanism simi- lar to that of N-OH-AAB. 27) Further researches may reveal the correlation between the site specificity and the carcinogenic process.…”

“…9,32) It is reasonable to consider that N-hydroxy derivatives of carcinogenic amino-aromatic compounds such as amino azo dyes, naphthylamine and heterocyclic amines, play important roles in the carcinogenic process through H 2 O 2 formation. 27,32) …”

The Effect of Flavonoids in Soybean Products in Lymphocytes from IBD and Colon Cancer Patients After Treatment with Food Mutagens and Hydrogen Peroxide

“…The genotoxicity of HCA originates from their activation by a series of reactions involving cytochrome P450 when the parent compound is converted to an electrophilic derivative such as a nitrenium ion that covalently binds to DNA resulting in DNA adducts and subsequently in nucleotide alterations and chromosomal aberrations (Goldman and Shields, 2003;Hatch et al, 2001). IQ induces unscheduled DNA synthesis in liver cells and shows strong mutagenic properties in the Salmonella typhimurium test system, which contribute to its classification as a potent carcinogen (Maeda et al, 1999;Murata et al, 1999;Weisburger et al, 1986) Also in addition to the formation of DNA adducts, oxidative damage to the DNA itself plays a crucial role in the carcinogenic process of food mutagens (Maeda et al, 1999;Murata et al, 1999). Heterocyclic amines like IQ are able to generate free radicals in the presence of NADPH and cytochrome b5 reductase (Maeda et al, 1999).…”