COVALENT BINDING OF BENZIDINE AND N-ACETYLBENZIDINE TO DNA AT THE C-8 ATOM OF DEOXYGUANOSINE INVIVO AND INVITRO

Abstract

Benzidine, a human urinary bladder carcinogen, induces hepatic tumors in mice and rats. [3H]benzidine was administered in drinking water to mice for 1 wk, and the covalent binding of the carcinogen to hepatic DNA was then determined. A single carcinogen:DNA adduct was detected which decreased in concentration by .apprx. 50% at 1 day after treatment and then remained at a nearly constant level for at least 7 days. Injection of radiolabeled benzidine or N-acetylbenzidine into rats also resulted in a single carcinogen:DNA adduct that was chromatographically identical to that obtained in mouse liver. While administration of benzidine and N-acetylbenzidine resulted in high levels of the adduct in rat hepatic DNA, injection of N,N''-[ring-14C]diacetylbenzidine did not give detectable binding (< 0.3 residue/mg DNA). The same carcinogen:DNA adduct found in rat and mouse liver was prepared synthetically by: (a) hydrolysis of calf thymus DNA reacted with N-hydroxy-N''-acetylbenzidine at pH 5; and (b) reaction of N-acetoxy-N,N''-diacetylbenzidine with deoxyguanosine and subsequent selective deacetylation of the product with methanolic ammonia. The in vitro and in vivo products were found to have identical high-pressure liquid chromatography retention times and to exhibit similar pH-dependent solvent partitioning characteristics. Mass and nuclear magnetic resonance spectral data of the synthetic products established the structure of the hepatic adduct as N-(deoxyguanosin-8-yl)-N''-acetylbenzidine. The structural isomer, N-(deoxyguanosin-8-yl)-N-acetylbenzidine, was synthesized by treatment of N-(deoxyguanosin-8-yl)-N,N''-diacetylbenzidine (the intermediate in b) with carboxylesterase and was chromatographically distinct from the in vivo adduct. The nonacetylated derivative, N-(deoxyguanosin-8-yl)benzidine, was synthesized by carboxylesterase treatment of N-(deoxyguanosin-8-yl)-N''-acetylbenzidine and was shown not to occur in rat and mouse liver DNA. The metabolic activation of benzidine to an ultimate carcinogen in rats and mice does not involve N-hydroxybenzidine or sulfotransferase-catalyzed activation of N-hydroxy-N,N''-diacetylbenzidine. The remaining pathways for metabolic conversion of benzidine to an ultimate carcinogenic species are discussed in relation to liver and urinary bladder carcinogenesis.