HUMAN N-ACETYLATION OF BENZIDINE - ROLE OF NAT1 AND NAT2

These studies were designed to assess metabolism of benzidine and N-ac etylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism w as assessed using human recombinant NAT1 and NAT2 and human liver slic es. For benzidine and N-acetylbenzidine, K-m and V-max values were hig her for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzi dine and N-acetylbenzidine were 54 and 535, respectively, suggesting t hat N-acetylbenzidine is a preferred substrate for NAT1. The much high er NAT1 and NAT2 K-m values for N-acetylbenzidine (1380 +/- 90 and 471 +/- 23 mu M, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 mu M, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic p arameters over a 20-fold range of acetyl-CoA concentrations demonstrat ed that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intac t liver tissue metabolism using human liver slices. Samples incubated with either [H-3]benzidine or [H-3]N-acetylbenzidine had a similar rat io of N-acetylated benzidines (N-acetylbenzidine + N',N'-diacetylbenzi dine/benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N'-diacetylbenzidine. With [H-3]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreas ed the amount of N-acetylbenzidine produced, resulting in a decreased ratio of acetylated products. This is consistent with benzidine being a NAT1 substrate. N-Acetylation of benzidine or N-acetylbenzidine by h uman liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices p ossessing the NAT110 compared to the NAT1*4 allele. Thus, a combinati on of human recombinant NAT and liver slice experiments has demonstrat ed that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorph ic expression in human liver.