Characterization of congenic mouse (NAT2*) and human (NAT1*) N-acetyltransferases.

Abstract

N-Acetyltransferase (NAT) N-acetylates various arylamine drugs (e.g., p-aminosalicylate, p-aminobenzoate, and isoniazid), and carcinogens (e.g., $\beta$-naphthylamine, 2-aminofluorene, and benzidine). It is well established that a polymorphism is associated with the human NAT2* locus. Phenotypic dissimilarities in acetylator status between individuals may have undesirable clinical consequences, such as impaired therapeutic efficacy in rapid acetylators and increased drug toxicity in slow acetylators. The aim of this thesis is twofold: (1) to provide molecular genetic and biochemical evidence for the genotypic and acetylator phenotypic identity of congenic mice with the parental strains of origin, and to further characterize the mouse NAT2*9 polymorphism; and (2) to rigorously investigate the biochemical events associated with structurally variant N-acetyltransferase alleles at the human NAT1* locus. Congenic mouse strains were developed in order to eliminate differences due to other polymorphic background genes, and these strains may give us an indication of the influence of the genetic background on the expression of the individual NAT* genes. The data demonstrate that congenic acetylator mouse strains were actually constructed, since the congenic strains were genotypically and phenotypically identical to the corresponding parental strains of origin. A novel mechanism of slow acetylation was disclosed, namely, that the Asp99Ile change in NAT2*9 from slow acetylator mice does not hinder the synthesis of hepatic NAT2 9 protein, but, rather, leads to the production of a conformationally modified NAT2 molecule that resists degradation by tissue proteases but is labile and catalytically impaired. Four human NAT1* variants have been identified in our laboratory, namely, NAT1*4, *10, *11, and *16. NAT1*4, *10, and *16 have identical coding regions, whereas NAT1*11 contains two amino acid substitutions; each NAT1* variant is associated with a unique 3$\sp\prime$ untranslated region. The coding region and 3$\sp\prime$ untranslated region events were studied in a eukaryotic expression system, whereas the coding region events were studied in a prokaryotic expression system. The results indicate that the 3$\sp\prime$ untranslated region may determine the NAT1 acetylator status of an individual by producing less NAT1 protein, this represents a novel mechanism of slow acetylation.