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Metabolic Activation of N-Hydroxyarylamines and N-Hydroxyarylamides by 16 Recombinant Human NAT2 Allozymes: Effects of 7 Specific NAT2 Nucleic Acid Substitutions¹

Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202-9037

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 (Arg⁶⁴Gln), C²⁸²T (silent), T³⁴¹C (IIe¹¹⁴Thr), C⁴⁸¹T (silent), G⁵⁹⁰A (Arg¹⁹⁷Gln), A⁸⁰³G (Lys²⁶⁸Arg), G⁸⁵⁷A (Gly²⁸⁶Glu), 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.

¹ This study was partially supported by United States Public Health Service Grant CA-34627 and United States Environmental Protection Agency Grant R821836. Preliminary reports of this work were presented at the Annual Meeting of the American Association For Cancer Research (1), March 1994, and the Tenth International Symposium on Drug Microsomes and Drug Oxidations (2), July 1994.

² To whom requests for reprints should be addressed, at Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, 501 North Columbia Road, Grand Forks, ND 58202-9037.

³ Present Address: National Institute for Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.

Received 2/24/95. Accepted 6/ 9/95.