title: 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome P4501A1 and rapid or slow acetylator N-acetyltransferase 2.
abstract: 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) is one of the most potent and abundant mutagens in the western diet. Bioactivation includes N-hydroxylation catalyzed by cytochrome P450s followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). In humans, NAT2*4 allele is associated with rapid acetylator phenotype, whereas NAT2*5B allele is associated with slow acetylator phenotype. We hypothesized that rapid acetylator phenotype predisposes humans to DNA damage and mutagenesis from MeIQx. Nucleotide excision repair-deficient Chinese hamster ovary cells were constructed by stable transfection of human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A1 and NAT2 catalytic activities were undetectable in untransfected Chinese hamster ovary cell lines. CYP1A1 activity did not differ significantly (P > 0.05) among the CYP1A1-transfected cell lines. Cells transfected with NAT2*4 had 20-fold significantly higher levels of sulfamethazine N-acetyltransferase (P = 0.0001) and 6-fold higher levels of N-hydroxy-MeIQx O-acetyltransferase (P = 0.0093) catalytic activity than cells transfected with NAT2*5B. Only cells transfected with both CYP1A1 and NAT2*4 showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase mutagenesis following MeIQx treatment. Deoxyguanosine-C8-MeIQx was the primary DNA adduct formed and levels were dose dependent in each cell line and in the following order: untransfected < transfected with CYP1A1 < transfected with CYP1A1 and NAT2*5B < transfected with CYP1A1 and NAT2*4. MeIQx DNA adduct levels were significantly higher (P < 0.001) in CYP1A1/NAT2*4 than CYP1A1/NAT2*5B cells at all concentrations of MeIQx tested. MeIQx-induced DNA adduct levels correlated very highly (r2 = 0.88) with MeIQx-induced mutants. These results strongly support extrahepatic activation of MeIQx by CYP1A1 and a robust effect of human NAT2 genetic polymorphism on MeIQx-induced DNA adducts and mutagenesis. The results provide laboratory-based support for epidemiologic studies reporting higher frequency of heterocyclic amine-related cancers in rapid NAT2 acetylators.
authors.affiliation: Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Bendaly
authors.forename: Shuang
authors.initials: S
authors.lastname: Zhao
authors.forename: Jason R
authors.initials: JR
authors.lastname: Neale
authors.forename: Kristin J
authors.initials: KJ
authors.lastname: Metry
authors.forename: Mark A
authors.initials: MA
authors.lastname: Doll
authors.forename: J Christopher
authors.initials: JC
authors.lastname: States
authors.forename: William M
authors.initials: WM
authors.lastname: Pierce
authors.forename: David W
authors.initials: DW
authors.lastname: Hein
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D016466:CHO Cells / Q000187:drug effects / Q000201:enzymology; D002273:Carcinogens / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics* / Q000378:metabolism; D018736:DNA Adducts; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011810:Quinoxalines / Q000633:toxicity*; D053719:Tandem Mass Spectrometry; D014162:Transfection
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D011810:Quinoxalines; C036990:2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline; D019363:Cytochrome P-450 CYP1A1; D000123:Acetyltransferases; D001191:Arylamine N-Acetyltransferase; C478900:NAT2 protein, human