METABOLIC-ACTIVATION OF N-HYDROXY-N,N'-DIACETYLBENZIDINE BY HEPATIC SULFOTRANSFERASE

Abstract

N-Hydroxy-N,N''-diacetylbenzidine (N-HO-DABZ) is an in vitro metabolite of benzidine in several rodent species, and may represent the proximate form of the carcinogen. Like other arylhydroxamic acids, N-HO-DABZ may be converted to an ultimate carcinogenic electrophile by metabolic O-sulfonation in hepatic cytosol. Liver cytosols from rats, mice and hamsters were assayed for their ability to catalyze the 3''-phosphoadenosine 5''-phosphosulfate-dependent metabolism of N-HO-DABZ and the formation of an adduct with methionine. For comparison, sulfotransferase activity for N-hydroxy-2-acetylaminofluorene (N-HO-AAF) was also measured. In the rat, N-HO-DABZ and N-HO-AAF were metabolized at rates of 2.5 and 4.3 nmol of arylhydroxamic acid lost/min per mg of protein, respectively. In the mouse, these rates were 0.5 nmol for N-HO-DABZ and < 0.05 nmol for N-HO-AAF. Sulfotransferase activity for these substrates in hamster liver cytosol could not be detected (< 0.05 nmol/min per mg). The inclusion of methionine in sulfotransferase incubation mixtures and subsequent heating resulted in the formation of methylmercapto arylamides from N-HO-DABZ and N-HO-AAF. From 20-40% of the N-HO-DABZ metabolized was trapped and recovered as an adduct, while 80-100% of the N-HO-AAF metabolized was similarly obtained. A methylmercapto-N,N''-diacetylbenzidine derivative was also obtained by reaction of N-acetoxy-N,N''-diacetylbenzidine with methionine. It was identified by high-pressure liquid chromatography, UV light, mass spectroscopic analyses and 13C NMR spectra as 3-methylmercapto-N,N''-diacetylbenzidine. Since the yield of the product from N-acetoxy-N,N''-diacetylbenzidine and methionine did not vary appreciably with pH (4-8), a reaction mechanism involving an electrophilic carbocation at position 3 is proposed. N-HO-DABZ can be esterified to an electrophilic reactant by hepatic sulfotransferases in the rat and the mouse and may be involved in the hepatocarcinogenicity of benzidine.