Studies on metabolic activation of nitrofurans: Formation of active metabolites and their binding to cysteine and glutathione.

Abstract

When 14C-3-(5-nitro-2-furyl)-2-(2-furyl) acrylamide (AF-2) or 3H-methyl 3-(5-nitro-2-furyl)-2-phenylacrylate (MNFPA) was incubated with SH compounds such as cysteine and glutathione in purified milk xanthine oxidase-hypoxanthine system, the ethyl acetateextractable radioactivity from the reaction mixture was markedly decreased compared with the radioactivity obtained without the SH compound. When the radioactive water-soluble product from AF-2 was treated with 2, 4-dinitrofluorobenzene, considerable amounts of the radioactive product could be extracted with ethyl acetate from acidic but not alkaline solution. Furthermore, 3H-MNFPA and cysteine, and 14C-cysteine and MNFPA were incubated in the same enzyme system as above. After incubation, the water-soluble products were chromatographed on Amberlite XAD-2 columns and on silica gel thin-layer plates (n-butanol-acetic acid-water, 3 : 1 : 1) successively. As a result, the radioactive peaks of 3H and 14C were observed at the same Rf value of 0.53. These observations indicate that the active metabolites of AF-2 and MNFPA, which were produced during their enzymatic reduction, may react with the SH compounds such as cysteine and glutathione to form the water-soluble conjugates. The stoichiometry of reduction of these nitrofurans by the purified enzyme-xanthine system in the presence of cysteine showed that the active metabolites might be the hydroxylaminofurans. Two reduction products of MNFPA by partially purified milk xanthine oxidasehypoxanthine system were isolated and tentatively identified as methyl 6-cyano-4-oxo-2-phenyl-2-hexenoate and 3-(azacyclopenta-5-oxo-1, 3-dien-2-yl)-2-phenylacrylic acid.