Metabolic Activation of N-Hydroxyarylamines and N-Hydroxyarylamides by 16 Recombinant Human NAT2 Allozymes: Effects of 7 Specific NAT2 Nucleic Acid Substitutions

Abstract

Human polymorphic N-acetyltransferase (NAT2) catalyzes the N-acetylation of arylamine carcinogens and the metabolic activation of N-hydroxyarylamine 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 certain cancers relating to carcinogenic arylamine exposures. We examined arylamine N-acetylation, N-hydroxyarylamine O-acetylation, and N-hydroxyarylamide N,O-acetylation catalytic activities of 16 different recombinant human NAT2 alleles expressed in an Escherichia coli JM105 expression system. NAT2 alleles contained nucleic acid substitutions at G¹⁹¹A (Arg⁶⁴→Gln), C²⁸²T (silent), T³⁴¹C (IIe¹¹⁴→Thr), C⁴⁸¹T (silent), G⁵⁹⁰A (Arg¹⁹⁷→Gln), A⁸⁰³G (Lys²⁶⁸→Arg), G⁸⁵⁷A (Gly²⁸⁶→Glu), and various combinations of substitutions in the 870-bp NAT2-coding region. Expression of each NAT2 allele produced equivalent amounts of immunoreactive recombinant NAT2 protein with differential levels of N-, O-, and N,O-acetylation activity. Catalytic activities of each of the recombinant human NAT2 allozymes followed the relative order N-acetylation > O-acetylation > N,O-acetylation. Catalytic activation rates for the metabolic activation of N-hydroxy-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 with nucleic acid substitution T³⁴¹C (NAT2*5A, *5B, *5C) expressed recombinant NAT2 allozymes, with the greatest reductions in metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by O- and N,O-acetylation, respectively. NAT2 alleles with nucleic acid substitutions G¹⁹¹A (NAT2*14A, *14B) and G⁵⁹⁰A (NAT2*6A, *6B) expressed recombinant NAT2 allozymes with more moderate reductions. NAT2 alleles with nucleic acid substitution G⁸⁵⁷A (NAT2*7A, *7B) expressed recombinant NAT2 allozymes with the smallest but yet significant reductions. NAT2 alleles with nucleic acid substitutions C²⁸²T (silent), C⁴⁸¹T (silent), and A⁸⁰³G (Lys²⁶⁸→ Arg) expressed recombinant NAT2 allozymes that did not have significant reductions in the metabolic activations of N-hydroxyarylamines and N-hydroxyarylamides. The differential capacity for the metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by recombinant human NAT2 allozymes encoded by polymorphic NAT2 alleles supports the hypothesis that acetylator phenotype may predispose to cancers related to activation of N-hydroxyarylamine and N-hydroxyarylamide carcinogens.