Search

Human arylamine N-acetyltransferase (NAT) is a phase II cytosolic enzyme that occurs as two isozymes, NAT1 and NAT2. This family of polymorphic enzymes catalyzes the detoxification and/or activation of many aromatic and heterocyclic amine drugs and carcinogens. These metabolism reactions can lead to detoxification of xenobiotics by N-acetylation, or bioactivation by O-acetylation which is preceded by cytochrome P450 (CYP) hydroxylation. This study evaluates the role of human arylamine N-acetyltransferase in carcinogen metabolism as it pertains to substrate selectivity, kinetic activity towards arylamine and alkylaniline substrates, and toxicological risk. Here we introduce the use of immortalized human fibroblasts (GM4429) to investigate the effects of combinations of human NAT1, human NAT2 haplotypes (NA T2*4, NA T2*S8, or NAT2*78), and varying CYP1 A2 enzymatic activity on carcinogen metabolism. We determined the apparent Michaelis-Menten constants of human NAT1 and human NAT2 for several well-characterized and putative environmental carcinogens. We utilized these data to investigate NAT substrate selectivity for these compounds of interest. NAT1 has been implicated in several cancers including urinary bladder, colorectal, lung, and breast cancer. Studies suggest that NAT1 plays an important role in cell growth and survival, as well as in cell proliferation and cell invasion, which are hallmarks of metastatic cancer. Here we describe the use of computational screening to identify effective, novel small molecule inhibitors of the molecular target NAT1. Our lead compound, Compound 10, suppressed carcinogen metabolism and 4-aminobiphenyl (ASP) -induced DNA adducts. The results in this study show that upon NAT1 inhibition there is a significant decrease in ASP activation, cell invasion, and cell proliferation in human breast adenocarcinoma cells. We also show that NAT1 inhibition in human breast cancer cells resulted in mitotic arrest, which supports findings suggesting that NAT1 plays an important role in human breast cancer progression. Our results indicate that human NAT1 is a molecular target for cancer therapy.

N-acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4-aminobiphenyl (ASP). NAT1 catalyzes N-acetylation of arylamines as well as the 0-acetylation of N-hydroxylated arylamines. 0-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5'-untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5'-UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ASP, pcDNA5/FRT plasmid constructs were prepared for transfection of full length human mRNAs including the 5'-UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3'-UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N- and 0-acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (p < 0.05). Consistent with NAT1 expression and activity, ASP-induced DNA adducts and hypoxanthine phosphoribosy/ transferase mutants were higher (p < 0.05) in NATb/NA T1 *4 than in NAT a/NAT1*4 transfected cells following exposure to ABP. These NATa and NATb mRNA constructs have also been used to study variant NAT1 alleles, including NAT1 *14B and NA T1 *10. NA T1 *14B is the most common allele associated with reduced N-acetylation activity and has been associated with increased risk for smoking induced lung cancer. NATb/NAT1 *14B transfected cells resulted in lower Vmax for PABA, ABP, and N-OH-ABP compared to cells transfected with NATb/NAT1*4. However, cells transfected with NATb/NAT1*14B resulted in increased Vmax/Km for ABP and N-OH-ABP. Cells transfected with NATb/NAT1*14B also resulted in increased ABP-induced DNAadducts compared to cells transfected with NATb/NAT1 *4 transfected cell. This indicates that NAT1 14B has lowered capacity for N- and 0- ABP acetylation at high substrate concentrations but higher capacity at low substrate concentration when compared to NAT14. NAT1 14B Vmax/Km compared to NAT1 4 was lower for PABA but higher for ABP and N-OH-ABP. This indicates that NAT1 14B is not simply associated with lowered acetylation, but is substrate dependent. Another variant allele, NA T1 *10 is the most common variant allele in many populations and has been characterized by increased acetylation activity in colon and bladder. NA T1 *10 has been associated with increased cancer risk for prostate, breast, urinary bladder cancer, gastric adenocarcinoma, colon cancers and non-Hodgkin's lymphoma. Following sequencing of NAT1*10 genomic sources, additional polymorphisms (A1642C, f1CT1647, C1716T, and A 1735T) were observed in one source. This allele is referred to as NA T1 *1 OB in this dissertation. Cells transfected with NATb/NA T1 *10 and NATb/NA T1 *1 OB resulted in higher NAT1 activity, protein, mRNA, ABP-induced mutants and DNA adducts than cells transfected with NATb/NA T1 *4. Differences between NAT1 4, NAT1 10, and NAT1 10B were also observed in NATa constructs. These studies illustrate the importance of determining NAT1 phenotypes and cancer risk based on mRNA type, substrate type and concentration.

2,5-Diphenyloxazole (PPO) was tested as a potential alternate inducer for the aryl hydrocarbon hydroxylase (AHH) system. Its apparent lack. of carcinogenicity and toxicity provide a possible system for investigation of enzyme systems related to chemical carcinogenesis without exposure of the researcher to potent carcinogenic compounds. These studies found PPO to be an inducer of AHH in cultured human lymphocytes. When PPO was utilized as a substrate for the AHH assay system, the major metabolites produced were strongly fluorescent. A simple fluorometric assay was developed which employed PPO as the substrate and which measured constitutive activity more efficiently than similar assays using benzo(a)pyrene as the substrate. Quantitation of both basal and induced lymphocyte AHH metabolism of PPO may be applicable to human population studies and may provide a tool to determine possible genetic variables with respect to carcinogen metabolism related to cancer risk.