Date on Master's Thesis/Doctoral Dissertation

8-2020

Document Type

Master's Thesis

Degree Name

M.S.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, MS

Committee Chair

Hein, David W.

Committee Co-Chair (if applicable)

Kidd, LaCreis R.

Committee Member

Kidd, LaCreis R.

Committee Member

Siskind, Leah J.

Author's Keywords

N-acetyltransferase 2; hepatocytes; aromatic amines; carcinogens; cytotoxicity

Abstract

Arylamine N-acetyltransferases, NAT1 and NAT2, catalyze the detoxification and/or activation of drugs and aromatic amine carcinogens. Single nucleotide polymorphisms or SNPs result in different human NAT2 genotypes thus dividing the population into rapid, intermediate, and slow acetylators. We hypothesize allelic variants of NAT2 genotype will decrease levels of N-acetylation, cytotoxicity, oxidative stress, DNA adduct formation, and mutagenesis compared to the reference allele NAT2*4. Cryopreserved human hepatocytes expressing different NAT2 genotypes and NER-deficient Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and human NAT2*4, NAT2*5B or NAT2*7B have been used to investigate N-acetylation of different xenobiotics. In vitro and in situ N-acetylation products of hydralazine and isoniazid (INH) as drugs and 4-aminobiphenyl (ABP), β-naphthylamine (BNA), and 4, 4’-methylene bis (2-chloroaniline) (MOCA) as carcinogens were measured using high performance liquid chromatography (HPLC). Cell viability was measured using alamar blue assay. Our findings showed that N-acetylation rates of ABP, BNA and MOCA in rapid acetylators are higher than slow acetylators. Also, CHO cells expressing NAT2*4 (rapid acetylator) showed higher N-acetylation rates of hydralazine, INH, BNA and MOCA than cells expressing slow alleles NAT2*5B or NAT2*7B. N- acetylation rate of hydralazine in CHO cells expressing NAT2*5B was insignificantly higher compared to NAT2*7B N-acetylation rates of hydralazine. However, For BNA, N- acetylation rate in CHO cells expressing NAT2*5B was insignificantly lower compared to NAT2*7B suggesting genetic heterogeneity within the slow NAT2 phenotype. Treatment of cells with ABP, BNA and MOCA resulted in concentration dependent cytotoxicity in human hepatocytes and CHO cells. These data suggest that individual susceptibility to side effects or carcinogenicity can be modified by human NAT2 genotype. Moreover, NAT2 slow acetylator phenotype is not homogeneous, but rather multiple slow acetylator phenotypes exist resulting from different mechanisms inferred by various SNPs. Therefore, the investigations of NAT2 genotype/phenotype relationship could be more precise if heterogeneity within the “slow” NAT2 acetylator phenotype is incorporated into future lab-based, pre-clinical and clinical studies. These results are important for future studies to determine the role of NAT2 genotype on the toxic effects of aromatic amines such as DNA damage, DNA adducts and mutagenicity.

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