METABOLIC-ACTIVATION OF N-HYDROXYARYLAMINES AND N-HYDROXYARYLAMIDES BY 16 RECOMBINANT HUMAN NAT2 ALLOZYMES - EFFECTS OF 7 SPECIFIC NAT2 NUCLEIC-ACID SUBSTITUTIONS

Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylati on of arylamine carcinogens and the metabolic activation of N-hydroxya rylamine and N-hydroxyarylamide carcinogens by O- and N,O-acetylation, respectively, Rapid and slow acetylator phenotype is regulated at the NAT2 locus, and each has been associated with differential risk to ce rtain cancers relating to carcinogenic arylamine exposures, We examine d arylamine N-acetylation, N-hydroxyarylamine O-acetylation, and N-hyd roxyarylamide N,O-acetylation catalytic activities of 16 different rec ombinant human NAT2 alleles expressed in an Escherichia coli JM105 exp ression system. NAT2 alleles contained nucleic acid substitutions at G (191)A (Arg(64)-->Gln), (CT)-T-282 (silent), (TC)-C-341 (Ile(114)-->Th r), (CT)-T-481 (silent), G(590)A (Arg(197)-->Gln), A(803)G (Lys(268)-- >Arg), Gs(857)A (Gly(286)-->Glu), and various combinations of substitu tions in the 870-bp NAT2-coding region. Expression of each NAT2 allele produced equivalent amounts of immunoreactive recombinant NAT2 protei n with differential levels of N-, O-, and N,O-acetylation activity. Ca talytic activities of each of the recombinant human NAT2 allozymes fol lowed the relative order N-acetylation > O-acetylation > N,O-acetylati on. Catalytic activation rates for the metabolic activation of N-hydro xy-2-aminofluorene and N-hydroxy-4-aminobiphenyl by O-acetylation and N-hydroxy-2-acetylaminofluorene by N,O-acetylation showed very strong correlations to the N-acetylation of 2-aminofluorene. NAT2 alleles wit h nucleic acid substitution (TC)-C-341 (NAT25A,*5B,*5C) expressed rec ombinant NAT2 allozymes, with the greatest reductions in metabolic act ivation of N-hydroxyarylamines and N-hydroxyarylamides by O- and N,O-a cetylation, respectively, NAT2 alleles with nucleic acid substitutions G(191)A (NAT214A,*14B) and G(590)A (NAT2*6A,*6B) expressed recombina nt NAT2 allozymes with more moderate reductions. NAT2 alleles with nuc leic acid substitution G(590)A (NAT27A,*7B) expressed recombinant NAT 2 allozymes with the smallest but yet significant reductions, NAT2 all eles with nucleic acid substitutions (CT)-T-282 (silent), (CT)-T-481 ( silent), and A(803)G (Lys(268)-->Arg) expressed recombinant NAT2 alloz ymes that did not have significant reductions in the metabolic activat ions of N-hydroxyarylamines and N-hydroxyarylamides. The differential capacity for the metabolic activation of N-hydroxyarylamines and N-hyd roxyarylamides by recombinant human NAT2 allozymes encoded by polymorp hic NAT2 alleles supports the hypothesis that acetylator phenotype may predispose to cancers related to activation of N-hydroxyarylamine and N-hydroxyarylamide carcinogens.