Functional effects of CYP1A2, NAT1, and NAT2 genetic variants in nucleotide excision repair-deficient human fibroblasts : implications for toxicological risk from environmental arylamines.

Abstract

Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the detoxification and/or activation of aromatic and heterocyclic amine carcinogens by two pathways. This metabolism reaction can lead to the detoxification by Nacetylation, or bioactivation by a-acetylation often preceded by CYP450 hydroxylation. Human NAT2 polymorphisms are characterized by rapid, intermediate, and slow acetylator phenotypes, thus resulting in differences in the rate of arylamine metabolism and consequently cancer risk. We have constructed nucleotide excision repair-deficient human cell model expressing human CYP1A2 and human NAT1and NAT2 in order to investigate carcinogen metabolism and cancer susceptibility in human cells. In this study we introduce the utilization of SV40-transformed human fibroblasts (GM4429) to examine the functional effects of human NAT2 haplotypes, in the presence of human NAT1. Many carcinogens, such as 4- aminobiphenyl, are metabolized by both NAT1 and NAT2, consequently both isozymes have toxicologically significant functions in the metabolism of arylamines. Furthermore, both NAT1 and NAT2 isozymes are expressed in human liver, where they are significant to carcinogen detoxification and/or activation consequences. With this model, we can examine the role of NAT1 and NAT2 carcinogen metabolism and genotoxicity in a NER-deficient human cell. In this study, we investigate novel inhibitors of NAT1 and NAT2. Previous studies have identified approximately 150 NAT inhibitor candidates following computer-based in silico screening of approximately 20 million compounds. The inhibitory properties of these compounds were tested using a High Performance Liquid Chromatography assay specific for human NAT1 and NAT2. A novel compound was identified that is an effective inhibitor of NAT1 (100%) and NAT2 (90%). This inhibitor was selective for NAT1 (IC50 about 1 µM), compared to an IC50 of 82.2µM (SMZ) for NAT2. Further studies will determine potency and efficacy of NAT inhibitors to reduce DNA adduct formation and mutagenesis. We have also successfully constructed and characterized NER-deficient human fibroblast cells with stable expression of human NAT2 alleles (NAT2*4, NAT2*S8, or NAT 2*78) and CYP1 A2. These models are used to better understand the effects of NAT2 haplotypes on carcinogen metabolism and DNA adduct formation in human cells