Role of aromatic amine acetyltransferases, NAT1 and NAT2, in carcinogen-DNA adduct formation in the human urinary bladder

Abstract

The metabolic activation and detoxification pathways associated with the carcinogenic aromatic amines provide an extraordinary model of polymorphisms that can modulate human urinary bladder carcinogenesis. In this study, the metabolic N-acetylation of p-aminobenzoic acid (PABA) to N-acetyl-PABA (NAT1 activity) and of sulfamethazine (SMZ) to N-acetyl-SMZ (NAT2 activity), as well as the O-acetylation of N-hydroxy-4-aminobiphenyl (OAT activity; catalyzed by NAT1 and NAT2), were measured in tissue cytosols prepared from 26 different human bladder samples; then DNA was isolated for determination of NAT1 and NAT2 genotype and for analyses of carcinogen-DNA adducts. Both PABA and OAT activities were detected, with mean activities +/- SD of 2.9 +/- 2.3 nmol/min/mg protein and 1.4 +/- 0.7 pmol bound/mg DNA/min/mg protein, respectively. However, SMZ activities were below the assay limits of detection (< 10 pmol/min/mg protein). The levels of putative carcinogen-DNA adducts were quantified by 32P-postlabeling and averaged 2.34 +/- 2.09 adducts/10(8) deoxyribonucleotide phosphate (dNp). Moreover, the DNA adduct levels in these tissues correlated with their NAT1-dependent PABA activities (r = 0.52; P < 0.01) but not with their OAT activities. Statistical and probit analyses indicated that this NAT1 activity was not normally distributed and appeared bimodal. Applying the NAT1:OAT activity ratios (N:O ratio) allowed arbitrary designation of rapid and slow NAT1 phenotypes, with a cutpoint near the median value. Within each of these subgroups, NAT1 correlated with OAT (P < 0.05); DNA adduct levels were elevated 2-fold in individuals with the rapid NAT1 or NAT1/OAT phenotype. Examination of DNA sequence polymorphisms in the NAT1 gene by PCR have demonstrated that an NAT1 polyadenylation polymorphism is associated with differences in tissue NAT1 enzyme activity; accordingly, NAT1 activity in the bladder of individuals with the heterozygous NAT1*10 allele was 2-fold higher than in subjects homozygous for the putative wild-type NAT1*4 allele. Likewise, DNA adduct levels in the mucosa of the urinary bladder were found to be 2-fold (P < 0.05) higher in individuals with the heterozygous NAT1*10 allele (3.5 +/- 2.1 adducts/10(8) dNp) as compared to NAT1*4 homozygous (1.8 +/- 1.9 adducts/10(8) dNp). Thus, these data provide strong support for the hypothesis that NAT1 activity in the urinary bladder mucosa represents a major bioactivation step that converts urinary N-hydroxy arylamines to reactive N-acetoxy esters that form covalent DNA adducts.(ABSTRACT TRUNCATED AT 400 WORDS)