ROLE OF AROMATIC AMINE ACETYLTRANSFERASES, NAT1 AND NAT2, IN CARCINOGEN-DNA ADDUCT FORMATION IN THE HUMAN URINARY-BLADDER

The metabolic activation and detoxification pathways associated with t he carcinogenic aromatic amines provide an extraordinary model of poly morphisms that can modulate human urinary bladder carcinogenesis, In t his study, the metabolic N-acetylation of p-aminobenzoic acid (PABA) t o N-acetyl-PABA (NAT1 activity) and of sulfamethazine (SMZ) to N-acety l-SMZ (NAT2 activity), as well as the O-acetylation of N-hydroxy-4-ami nobiphenyl (OAT activity; catalyzed by NAT1 and NAT2), were measured i n tissue cytosols prepared from 26 different human bladder samples; th en DNA was isolated for determination of NAT1 and NAT2 genotype and fo r analyses of carcinogen-DNA adducts. Both PABA and OAT activities wer e detected, with mean activities +/- SD of 2.9 +/- 2.3 mmol/min/mg pro tein and 1.4 +/- 0.7 pmol bound/mg DNA/min/mg protein, respectively, H owever, SMZ activities were below the assay limits of detection (< 10 pmol/min/mg protein), The levels of putative carcinogen-DNA adducts we re quantified by P-32-postlabeling and averaged 2.34 +/- 2.09 adducts/ 10(8) deoxyribonucleotide phosphate (dNp). Moreover, the DNA adduct le vels in these tissues correlated with their NAT1-dependent PABA activi ties (r = 0.52; P < 0.01) but not with their OAT activities, Statistic al and probit analyses indicated that this NAT1 activity was not norma lly distributed and appeared bimodal, Applying the NAT1:OAT activity r atios (N:O ratio) allowed arbitrary designation of rapid and slow NAT1 phenotypes, with a cutpoint near the median value, Within each of the se subgroups, NAT1 correlated with OAT (P < 0.05); DNA adduct levels w ere elevated 2-fold in individuals with the rapid NAT1 or NAT1/OAT phe notype. Examination of DNA sequence polymorphisms in the NAT1 gene by PCR have demonstrated that an NAT1 polyadenylation polymorphism is ass ociated 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 putat ive wild-type NAT14 allele, Likewise, DIVA adduct levels in the mucos a of the urinary bladder were found to be 2-fold (P < 0.05) higher in individuals with the heterozygous NAT110 allele (3.5 +/- 2.1 adducts/ 10(8) dNp) as compared to NAT14 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 bioact ivation step that converts urinary N-hydroxy arylamines to reactive N- acetoxy esters that form covalent DNA adducts. Since previous studies have indicated that hepatic NAT2 activity is an important detoxificati on step for bladder carcinogenesis, one would predict that individuals who inherit slow NAT2 and rapid NAT1 (NAT110) genotypes would be at highest risk, Although our sample size was limited, this combined geno type indeed exhibited the highest adduct level (4.2 +/- 1.6 adducts/10 (8) dNp) and the highest NAT1 activity (5.8 +/- 2.5 nnol/min/mg protei n) among all other combined NAT1-NAT2 genotypes, Together, these data provide the first evidence that phenotypic and genotypic polymorphisms in both NAT1 and NAT2 are predictive of DNA adduct levels in human ur inary bladder.