Date on Master's Thesis/Doctoral Dissertation

8-2011

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Committee Chair

Hein, David W.

Author's Keywords

Arylamine; Carcinogen metabolism; 4-aminobiphenyl; N-acetyltransferase

Subject

Aromatic amines; Carcinogenesis

Abstract

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.

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