Phenotype of the most common "slow acetylator" arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent

Abstract

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II cytosolic enzyme responsible for the activation or deactivation of many arylamine compounds including pharmaceuticals and environmental carcinogens. NAT1 is highly polymorphic and has been associated with altered risk toward many cancers. NAT1*14B is characterized by a single nucleotide polymorphism in the coding region (rs4986782; 560G>A; R187Q). NAT1*14B is associated with higher frequency of smoking-induced lung cancer and is the most common "slow acetylator" arylamine NAT1 genetic variant. Previous studies have reported decreased N- and O-acetylation capacity and increased proteasomal degradation of NAT1 14B compared with the referent, NAT1 4. The current study is the first to investigate NAT1*14B expression using constructs that completely mimic NAT1 mRNA by including the 5'- and 3'-untranslated regions, together with the open reading frame of the referent, NAT1*4, or variant, NAT1*14B. Our results show that NAT1 14B is not simply associated with "slow acetylation." NAT1 14B-catalyzed acetylation phenotype is substrate-dependent, and NAT1 14B exhibits higher N- and O-acetylation catalytic efficiency as well as DNA adducts after exposure to the human carcinogen 4-aminobiphenyl.

Fig. 1.

NATb/NAT1*4 and NATb/NAT1*14B constructs. a, schematic of NAT1 genomic structure and most common RNA transcribed by the NATb promoter. b, constructs including 5′-UTR, ORF (exon 9), and 3′-UTR.

Fig. 2.

In situ ABP N-acetylation in yeast cultures recombinantly expressing NAT1*4 and NAT1*14B. Each bar illustrates mean ± S.E.M. for nanomole of acetylated ABP per million cells after three separate collections. Significant differences between NAT1*4- and NAT1*14B-expressing yeast cultures were determined by Student's t test. *, p < 0.05; **, p < 0.001.

Fig. 3.

Western blot to determine relative protein expression of NAT1 4 and NAT1 14B. a, representative Western blot. b, densitometric analysis of Western blot of 28 μg of total protein loaded. Loading either 28 or 14 μg of total protein lysate, NAT1 14B resulted in a ∼4-fold less NAT1 protein than NAT1 4 (**, p < 0.001). Bars represent mean ± S.E.M. for three Western blots, and significance was determined by Student's t test.

Fig. 4.

ABP-induced cytotoxicity (a) and dG-C8-ABP adducts (b) in cells stably transfected with NAT1*4 and NAT1*14B. Significantly more cytotoxicity was observed for NAT1 14B than NAT1 4 after all ABP exposures between 1.56 and 12.5 μM. Significantly more adducts were observed after all exposures examined in cells expressing NAT1 14B than in cells expressing NAT1 4. Values were adjusted for baseline values of UV5/1A1 cells. Bars represent mean ± S.E.M. for three determinations, and significance was determined by Student's t test. *, p < 0.05; **, p < 0.001; ***, p < 0.0001.