title: The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver.
abstract: To determine whether benzidine is acetylated in dog, like rat, the metabolism of benzidine was assessed with dog and rat liver slices. Slices were incubated with 0.05 mM [3M]benzidine for 4 h. Media and cellular DNA were analyzed for acetylated benzidine metabolites and adducts. In rat, benzidine was rapidly converted to acetylated metabolites. At 1 h, benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine represented 5%, 23%, and 54%, respectively, of the total radioactivity in media. Within 2 h, 75% of the radioactivity was N,N'-diacetylbenzidine. In dog, 45% of the radioactivity was present in metabolites more polar than benzidine by 4 h. No N-acetylated metabolites were observed in dog liver slice media. To identify acetylated benzidine DNA adducts, N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was prepared and identified by FAB MS. This nucleoside adduct was used to synthesize N-(deoxyguanosin-8-yl)-N-acetylbenzidine and N'-(deoxyguanosin-8-yl)-N-acetylbenzidine. Nucleoside adducts from slices incubated with [3H]benzidine were analyzed by HPLC. With this method of analysis, the 3H-material did not correlate with the synthetic adduct standards. To improve sensitivity and identify liver adducts, a 32P-postlabeling method was developed. 2'-Deoxyguanosine 3'-monophosphate adduct standards of acetylated benzidine were prepared. 32P-Postlabeling analysis demonstrated that rat liver contained only N'-(3'-monophosphodeoxyguanosine-8-yl)-N-acetylbenzidine after a 1- or 4-h exposure to benzidine. In contrast, no acetylated adducts were detected in dog. Results indicate that dog is a nonacetylator with respect to benzidine. The availability of acetylated benzidine nucleotide standards allowed unambiguous identification of N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine as the adduct present in rat liver slices. These nucleotide adduct standards will be useful in subsequent studies in animals and human.
authors.affiliation: VA Medical Center, St. Louis, MO 63125-4199, USA.
authors.forename: V M
authors.initials: VM
authors.lastname: Lakshmi
authors.forename: T V
authors.initials: TV
authors.lastname: Zenser
authors.forename: H D
authors.initials: HD
authors.lastname: Goldman
authors.forename: G G
authors.initials: GG
authors.lastname: Spencer
authors.forename: R C
authors.initials: RC
authors.lastname: Gupta
authors.forename: F F
authors.initials: FF
authors.lastname: Hsu
authors.forename: B B
authors.initials: BB
authors.lastname: Davis
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001560:Benzidines / Q000378:metabolism*; D002273:Carcinogens / Q000378:metabolism*; D018736:DNA Adducts / Q000096:biosynthesis*; D004285:Dogs; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D051381:Rats; D013057:Spectrum Analysis
chemical_list: D001560:Benzidines; D002273:Carcinogens; D018736:DNA Adducts; D000123:Acetyltransferases