The N-acetylation of sulfamethazine and p-aminobenzoic acid by human liver slices in dynamic organ culture

Abstract

N-Acetyltransferase (NAT) polymorphism has been implicated in differences in the susceptibility of individuals to the toxicity of chemicals metabolized by this enzyme system. Investigation into the toxicological consequences of acetylator polymorphism and the mechanism of these effects in humans, however, has been greatly hindered due to the lack of a suitable human tissue culture system for determination of hepatic NAT activity and acetylator status of individuals. An in vitro system has been developed to study NAT activity using human liver slices in dynamic organ culture. Acetylation of para-aminobenzoic acid (PABA) and sulfamethazine (SMZ) by human liver slices was monitored by measuring the disappearance of the parent amine from the incubation medium using the colorimetric procedure of Bratton and Marshall. Presence of the acetyl conjugate was confirmed using HPLC. PABA acetylation rates varied from 0.72-2.52 nmol/hr/mg protein (N = 8). This small variation (less than 4-fold) is consistent with the classification of PABA as a monomorphic substrate. The variation in the rate of SMZ acetylation was greater than 20-fold (0.144-3.68 nmol/hr/mg protein; N = 9). This larger variation is characteristic of SMZ as a polymorphic substrate. A good correlation of N-acetylation activities for SMZ was also found between cytosol and slices prepared from the same human livers. The results obtained indicate that human liver slices in dynamic organ culture can be used for the determination of hepatic NAT activity in humans. These slices may be useful in toxicological studies that seek to relate N-acetylation of chemicals in the human liver with potential toxicity.