Effect of nucleotide substitutions in N-acetyltransferase-1 on N-acetylation (deactivation) and O-acetylation (activation) of arylamine carcinogens: implications for cancer predisposition

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Abstract

Genetic polymorphism in N-acetyltransferase-1 (NAT1) is associated with increased risk of various cancers, but epidemiological investigations are compromised by poor understanding of the relationship between NAT1 genotype and phenotype. Human reference NAT14 and 12 known human NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region were cloned and expressed in yeast (Schizosaccharomyces pombe). Large reductions in the N-acetylation of 4-aminobiphenyl and the O-acetylation of N-hydroxy-2-aminofluorene were observed for recombinant NAT1 allozymes encoded by NAT114B, NAT115, NAT117, NAT119, and NAT122. Each of these alleles exhibited substantially lower expression of NAT1 protein than the reference NAT14 and the other NAT1 alleles. These results show an important effect of the NAT1 genetic polymorphism on the N- and O-acetylation of arylamine carcinogens, suggesting modification of cancer susceptibility following exposures to arylamine carcinogens.

Introduction

The carcinogenic action of aromatic amines requires metabolic activation resulting in electrophiles that bind to DNA [1]. The N-acetyltransferase enzymes (E.C.2.3.1.5) play an important role in the metabolic cascade, catalyzing both the N-acetylation of arylamines (usually deactivation) and, following N-oxidation, the O-acetylation (usually activation) of their N-hydroxyarylamine metabolites [2]. N-acetyltransferase-1 (NAT1) and N-acetyltransferase-2 (NAT2) both catalyze the N-acetylation and O-acetylation of model aromatic amine carcinogens such as 2-aminofluorene and 4-aminobiphenyl [3]. NAT1 and NAT2 both are encoded by single open reading frames of 870 base pairs that exhibit genetic polymorphism in human populations [4]. Over 20 different NAT1 and NAT2 alleles have been identified in human populations, many of which possess single nucleotide polymorphisms in their coding region [4]. Previous studies have investigated the role of NAT2 single nucleotide polymorphisms, alone [5] or in combination [6], [7], [8], [9], [10], [11], [12], [13] on the N- and O-acetylation of aromatic amine carcinogens. In comparison, less is known regarding the effect of NAT1 single nucleotide polymorphisms on aromatic amine carcinogen N- and O-acetylation. Since NAT1 catalyzes both the activation and/or deactivation of aromatic amine carcinogens, genetic variation in NAT1 catalytic activity may have implications for genetic predisposition to cancer following exposure to aromatic amine carcinogens. Understanding the functional significance of the nucleotide polymorphisms on deactivation and activation of aromatic amine carcinogens is important for interpretation of molecular epidemiological investigations in cancer susceptibility. Thus, we undertook this study to clone and recombinantly express reference NAT14 and 12 NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region. The variant human NAT1 alleles were compared to reference NAT14 with respect to protein expression and N- and O-acetyltransferase activities.

Section snippets

Materials and methods

Human reference (NAT14) and NAT1 allelic variants possessing nucleotide polymorphisms in the NAT1 coding region (Table 1) were amplified by polymerase chain reaction (PCR) from genomic DNA samples or previously constructed plasmids using primers engineered with restriction endonuclease sites to facilitate cloning as described previously [14]. Primers were designed to amplify the NAT1 coding region only. The yeast vector pESP-3 (Stratagene, La Jolla, CA) was digested with NdeI and AscI at 37 °C

Results

The N-acetylation of the aromatic amine carcinogen 4-aminobiphenyl was catalyzed by each of the NAT1 allozymes (Fig. 1). Substantial reductions in 4-aminobiphenyl N-acetyltransferase activities were observed for recombinant NAT1 14B (150-fold), NAT1 15 (42,000-fold), NAT1 17 (12,000-fold), NAT1 19 (1300-fold), and NAT1 22 (37,000-fold) compared with the reference NAT1 4. Very slight reductions ranging from 1.2 to 2.3-fold were observed with NAT1 11, NAT1 20, NAT1 21, NAT1 23, NAT1 24, NAT1 25

Discussion

NAT14 and each of the variant NAT1 alleles were cloned and expressed in the yeast expression system. In contrast to NAT14 and the other seven variant NAT1 alleles, NAT114B, NAT115, NAT117, NAT119, and NAT122 encoded NAT1 allozymes that were undetectable by Western blot analysis and did not catalyze the metabolic activation of N-hydroxy-2-aminofluorene above background. These five NAT1 alleles have been shown to express NAT1 mRNA at levels equivalent to NAT14 [14]. Despite the inability

Acknowledgements

Portions of this work constitute partial fulfilment of the Ph.D. in Pharmacology and Toxicology for Adrian J. Fretland at the University of Louisville. This study was partially supported by a United States Public Health Service Grant from the National Cancer Institute (R01-CA34627)

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