Functional analysis of arylamine N-acetyltransferase 1 (NAT1) NAT110 haplotypes in a complete NATb mRNA construct

• Published in: Carcinogenesis (New York) 2012-02, Vol.33 (2), p.348-355
• Main Authors: Millner, Lori M., Doll, Mark A., Stepp, Marcus W., States, J.Christopher, Hein, David W.
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; 5' Untranslated Regions; Acetylation; Aminobiphenyl Compounds - pharmacology; Animals; Arylamine N-Acetyltransferase - genetics; Biological and medical sciences; Cancer Biomarkers and Molecular Epidemiology; Carcinogenesis, carcinogens and anticarcinogens; Cell Line, Tumor; Cells, Cultured; CHO Cells; Cricetinae; DNA Adducts; DNA Repair; Haplotypes; Hep G2 Cells; Humans; Hypoxanthine Phosphoribosyltransferase - genetics; Isoenzymes - genetics; Medical sciences; Mutation; Open Reading Frames; Polymorphism, Genetic - genetics; Promoter Regions, Genetic; Risk Factors; RNA, Messenger - genetics; Transfection - methods; Tumors
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/bgr273

N-acetyltransferase 1 (NAT1) catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1*10 is the most common variant haplotype and is associated with increased risk for numerous cancers. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in messenger RNAs (mRNAs) with distinct 5′-untranslated regions (UTRs). To best mimic in vivo metabolism and the effect of NAT1*10 polymorphisms on polyadenylation usage, pcDNA5/Flp recombination target plasmid constructs were prepared for transfection of full-length human mRNAs including the 5′-UTR derived from NATb, the open reading frame and 888 nucleotides of the 3′-UTR. Following stable transfection of NAT1*4, NAT1*10 and an additional NAT1*10 variant (termed NAT1*10B) into nucleotide excision repair-deficient Chinese hamster ovary cells, N- and O-acetyltransferase activity (in vitro and in situ), mRNA and protein expression were higher in cells transfected with NAT1*10 and NAT1*10B than in cells transfected with NAT1*4 (P < 0.05). Consistent with NAT1 expression and activity, cytotoxicity and hypoxanthine phosphoribosyl transferase mutants following 4-aminobiphenyl exposures were higher in NAT1*10 than in NAT1*4 transfected cells. Ribonuclease protection assays showed no difference between NAT1*4 and NAT1*10. However, protection of one probe by NAT1*10B was not observed with NAT1*4 or NAT1*10, suggesting additional mechanisms that regulate NAT1*10B. The higher mutants in cells transfected with NAT1*10 and NAT1*10B are consistent with an increased cancer risk for individuals possessing NAT1*10 haplotypes.