Recombinant expression and catalytic analysis of rapid and slow acetylator Syrian hamster chimeric NAT2 alleles.

Abstract

Polymorphic aromatic amine N-acetyltransferase (NAT2) catalyzes the N-acetylation of aromatic amines and the metabolic activation of N-hydroxyarylamines (via O-acetylation) and N-hydroxy-N-acetylarylamines (via N,O-acetylation) to electrophilic intermediates that mutate DNA. Acetylation capacity in humans and other mammalian species such as Syrian hamsters is subject to a genetic polymorphism. NAT2 is regulated by a single gene (NAT2) containing a single coding exon of 870 bp. Syrian hamster slow acetylator differs from the rapid acetylator NAT2 coding region by three nucleotide substitutions at T36C, A633G, and C727T. We measured expression of immunoreactive NAT2 protein and aromatic amine N-acetylation. N-hydroxyarylamine O-acetylation and N-hydroxy-N-acetylarylamine N,O-acetylation by recombinant NAT2 proteins expressed from alleles containing all combinations of the T36C, A633G, and C727T substitutions. The C727T substitution, which creates an opal stop codon in slow acetylator NAT2, was the sole mutation responsible for substantial reduction in expression of a truncated NAT2 protein with reduced capacity for the deactivation of aromatic amines (N-acetylation) and the metabolic activation of N-hydroxyarylamines (O-acetylation) and N-hydroxy-N-acetylarylamines (N,O-acetylation). The reductions in aromatic amine N-acetylation correlated very highly with the reductions in metabolic activation of the corresponding N-hydroxyarylamines and N-hydroxy-N-acetylarylamines.