Abstract
These studies were designed to assess metabolism of benzidine and N-acetylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism was assessed using human recombinant NAT1 and NAT2 and human liver slices. For benzidine and N-acetylbenzidine, Km and Vmax values were higher for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzidine and N-acetylbenzidine were 54 and 535, respectively, suggesting that N-acetylbenzidine is a preferred substrate for NAT1. The much higher NAT1 and NAT2 Km values for N-acetylbenzidine (1380 ± 90 and 471 ± 23 µm, respectively) compared to benzidine (254 ± 38 and 33.3 ± 1.5 µm, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic parameters over a 20-fold range of acetyl-CoA concentrations demonstrated that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intact liver tissue metabolism using human liver slices. Samples incubated with either [3H]benzidine or [3H]N-acetylbenzidine had a similar ratio of N-acetylated benzidines (N-acetylbenzidine + N′,N′-diacetylbenzidine/benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N′-diacetylbenzidine. With [3H]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreased 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 human liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices possessing the NAT1*10 compared to the NAT1*4 allele. Thus, a combination of human recombinant NAT and liver slice experiments has demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorphic expression in human liver.
Footnotes
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↵1 This work was supported by the Department of Veterans Affairs (T. V. Z., B. B. D.), United States Public Health Service Grant CA-34627 (D. W. H), and United States Environmental Protection Agency Grant R821836 (D. W. H.).
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↵2 To whom requests for reprints should be addressed, at Veterans Affairs Medical Center (GRECC/11G-JB), St. Louis, MO 63125-4199. Phone: (314) 894-6510; Fax: (314) 894-6614.
- Received April 15, 1996.
- Accepted July 2, 1996.
- ©1996 American Association for Cancer Research.