Human N-acetylation of benzidine: role of NAT1 and NAT2.
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 microM, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 microM, 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.
Zenser
T V
TV
Veterans Affairs Medical Center, St. Louis, Missouri 63125-4199, USA.
Lakshmi
V M
VM
Rustan
T D
TD
Doll
M A
MA
Deitz
A C
AC
Davis
B B
BB
Hein
D W
DW
D000107:Acetylation; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism*; D001483:Base Sequence; D001560:Benzidines / Q000378:metabolism* / Q000493:pharmacokinetics; D005260:Female; D005838:Genotype; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000033:anatomy & histology / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010641:Phenotype; D011994:Recombinant Proteins / Q000378:metabolism
D001560:Benzidines; D007527:Isoenzymes; D011994:Recombinant Proteins; C029876:benzidine; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; C478900:NAT2 protein, human; C034938:N-acetylbenzidine