Effect of N-acetyltransferase (NAT1 ) polymorphism on mutagenesis and DNA adduct formation.

摘要

Polymorphic variation in xenobiotic metabolizing enzymes is hypothesized to moderate individual susceptibility to environmental carcinogens. These carcinogens include the aromatic amines such as 2-aminofluorene and 4-aminobiphenyl, and heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine found in cigarette smoke and/or the western diet. N-acetyltransferase 1 (NAT1) catalyzes the biotransformation of these carcinogens via N-acetylation and O-acetylation. N-acetylation is typically an inactivation pathway, followed by urinary excretion. However, these compounds are activated through cytochrome p450 (CYP) hydroxylation of the amine group, followed by NAT1-catalyzed O-acetylation. These activation steps result in the formation of electrophilic intermediates that can react with DNA to form adducts. Differing rates of activation by NAT1 enzymes encoded by genetically variant alleles can modulate cancer risk. This study compares the NAT1*4 reference allele to important NAT1 alleles associated with increased risk of many cancers including urinary bladder, colorectal, breast, lung, and prostate cancers and non-Hodgkin lymphoma. However, the functional effect of these NAT1 alleles is poorly understood at the molecular level. In the present work, transient transfections of NAT1*4, NAT1*10, and NAT1*10B pcDNA5/FRT constructs were performed into COS-1 cells and nucleotide repair deficient (UV5) Chinese hamster ovary (CHO) cells expressing human CYP1A1 (UV5/CYP1A1/CHO) cells. Stable transfections were also carried out in UV5/CYP1A1/CHO cells. Measurements of arylamine N-acetylation were used to assess the effects of each allele on enzyme activity. All NAT1 transfected cells showed increased acetylation activity compared to vector-only transfected cells. However, no difference in acetylation capacity was observed between NAT1*4, NAT1*10, or NAT1*10B constructs that were transiently or stably transfected. Ongoing studies involve the use of novel constructs that closely mimic in vivo RNAs by cloning human NAT1 5' and 3' untranslated regions as well as the coding region. Complete characterization of stably transfected UV5/CYP1A1/CHO cells expressing human NAT1 alleles will include measuring N- and O-acetylation activity, quantitation of mRNA levels, mutagenicity, and DNA adduct formation following exposure to aromatic and heterocyclic amine carcinogens. This study will elucidate the role of important genetically variant NAT1 alleles on cancer risk after exposure to these environmental and dietary carcinogens.