Effect of N-Acetyltransferase 2 Polymorphism on Tumor Target Tissue DNA Adduct Levels in Rapid and Slow Acetylator Congenic Rats Administered 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine or 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline

Abstract

ABSTRACT:2-Amino-3,8-dimethylimidazo-[4,5-f ]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are suspected human carcinogens generated in well done meats. After N-hydroxylation, they are O-acetylated by N-acetyltransferase 2 (NAT2) to electrophiles that form DNA adducts. dG-C8-MeIQx and dG-C8-PhIP adducts have been identified in human tissues. In the female rat, administration of PhIP leads to mammary and colon tumors, whereas MeIQx induces liver tumors. Both humans and rats exhibit NAT2 genetic polymorphism yielding rapid and slow acetylator phenotypes. Because O-acetylation is an activation pathway, we hypothesized that MeIQx-and PhIP-induced DNA damage would be greater in tumor target tissues and higher in rapid than slow NAT2 acetylators. Adult female rapid and slow acetylator rats congenic at the Nat2 locus received a single dose of 25 mg/kg MeIQx or 50 mg/kg PhIP by gavage, and tissue DNA was isolated after 24 h. Deoxyribonucleoside adducts were identified and quantified by capillary liquid chromatography-tandem mass spectrometry using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. dG-C8-PhIP DNA adducts were highest in colon, lowest in liver and did not significantly differ between rapid and slow acetylator congenic rats in any tissue tested. In contrast, dG-C8-MeIQx adducts were highest in liver and significantly (p < 0.001) higher in rapid acetylator liver than in slow acetylator liver. Our results are consistent with the tumor target specificity of PhIP and MeIQx and with increased susceptibility to MeIQx-induced liver tumors in rapid NAT2 acetylators. -3,8-dimethylimidazo-[4,5-f ]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) are potent and abundant mutagens in the human diet, formed during high temperature cooking of meats (Keating and Bogen, 2004). They also have been detected in processed food flavorings, beer, wine, cigarette smoke, smoke condensate formed during frying of beef patties and bacon, and in aerosol from cooking of stir-fried fish (National Toxicology Program, 2005). PhIP has been detected in airborne particles, diesel-exhaust particles, and incineration ash from garbage-burning plants (Manabe et al., 1993). 2-AminoBoth MeIQx and PhIP induce tumors in the rat (Sugimura et al., 2004) and are designated as "reasonably anticipated to be a human carcinogen" (National Toxicology Program, 2005). MeIQx induces liver tumors in mice (Ohgaki et al., 1987) and up to 100% incidence of hepatic tumors in rats (Kato et al., 1988;Kushida et al., 1994). PhIP target organ specificity differs from MeIQx in the rat because it induces tumors in colon, prostate, and mammary tissue (Sugimura et al., 2004). MeIQx-and PhIP-induced DNA adduct formation and carcinogenesis require N-hydroxylation, which occurs at relatively high rates in humans (Stil1well et al., 1999;Turesky 2002). N-Hydroxy-MeIQx and -PhIP are further O-acetylated by N-acetyltransferase 2 (NAT2) to a...

Effect of Hepatic Cytochrome P450 (P450) Oxidoreductase Deficiency on 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA Adduct Formation in P450 Reductase Conditional Null Mice

“…These results are consistent with colon as a tumor target organ of PhIP in rodents (Sugimura et al, 2004). Previous studies have identified PhIP-DNA adducts in rat and mouse colon (Kaderlik et al, 1994;Snyderwine et al, 2002;Metry et al, 2009), and they have also been identified in human colon (Malfatti et al, 2006).…”

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

“…No PhIP-C8-dG adducts were detected in control animals dosed with corn oil alone. Previous studies have shown higher levels of PhIP-C8-dG adducts are detected in colon tissue when compared to the liver, which is consistent with the colon being the target tissue for tumour development [29,30].…”

“…LC-mass spectrometry has made sufficient gains in sensitivity for the detection of DNA adducts resulting in it being a viable alternative to other detection methods such as 32 P-postlabelling and immunoassays, to provide more accurate and precise results through the use of stable isotope internal standards [27,28]. Recently, LC-tandem mass spectrometry (MS/MS) methods using deuterated stable isotope internal standards and off-line pre-purification of the PhIP-C8-dG adduct using solid phase extraction or online column-switching LC have been described for the detection and quantitation of adducts in cells and DNA treated in vitro with PhIP, and also in the liver and colon of rats treated in vivo with PhIP [20,22,[29][30][31]. Mass spectrometry based methods offer a number of advantages with respect to the identification and quantitation of DNA adducts, as they rely on the detection of adduct-specific ions and characteristic transitions of the ions being monitored, respectively, and because quantification is based upon the use of isotopicallylabelled internal standards.…”

Curcumin inhibits PhIP induced cytotoxicity in breast epithelial cells through multiple molecular targets

“…Tail moment is defined as the product of the tail length and the fraction of total DNA in the tail. Tail moment incorporates a measure of the smallest detectable size of migrating DNA (reflected in the comet tail length) and the number of relaxed/broken pieces represented by the intensity of the DNA found in the tail [34]. OTM in control cell populations ranged from 0.0 to 0.92 with an average of 0.155 ± 0.022 (SEM).…”

“…Previous studies have shown that PhIP induces the production of reactive oxygen species (ROS) and DNA adduct formation [6,7,15,16]. Phytochemicals like curcumin are able to inhibit DNA adduct formation [34]. We hypothesized that curcumin may be a potential food additive that may be inhibitory to PhIP-induced carcinogenicity by inhibiting ROS production, DNA adduct formation and DNA strand breaks.…”