title: Functional analysis of arylamine N-acetyltransferase 1 (NAT1) NAT1*10 haplotypes in a complete NATb mRNA construct.
abstract: 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.
authors: Department of Pharmacology and Toxicology, James Graham Brown Cancer Center and Center for Environmental Genomics and Integrative Biology, University of Louisville, Louisville, KY 40202, USA.
authors: Lori M
authors: LM
authors: Millner
authors: Mark A
authors: MA
authors: Doll
authors: Marcus W
authors: MW
authors: Stepp
authors: J Christopher
authors: JC
authors: States
authors: David W
authors: DW
authors: Hein
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000107:Acetylation; D000611:Aminobiphenyl Compounds / Q000494:pharmacology; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000235:genetics*; D016466:CHO Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006224:Cricetinae; D018736:DNA Adducts; D004260:DNA Repair; D006239:Haplotypes; D056945:Hep G2 Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics*; D009154:Mutation; D016366:Open Reading Frames; D011110:Polymorphism, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D012307:Risk Factors; D014162:Transfection / Q000379:methods
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000611:Aminobiphenyl Compounds; D018736:DNA Adducts; D007527:Isoenzymes; D012333:RNA, Messenger; C006757:4-biphenylamine; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; D007041:Hypoxanthine Phosphoribosyltransferase