Metabolic reduction of novel 3,4-dichloro-5-nitrofurans in Salmonella typhimurium

To gain insight on biochemical mechanisms of mutagenesis and carcinogenesis by the experimental carcinogens, 5-nitrofurans, a new series of 3,4-dichloro-5-nitrofurans, comprised of 3,4-dichloro-5-nitro-2-acetylfuran (I), 3,4-dichloro-5-nitro-2-bromoacetylfuran (II), methyl 3,4-dichloro-5-nitro-2-furoate (III), were synthesized and tested for their activation to mutagenic forms in the standard plate assay using Salmonella typhimurium TA98, TA100, and TA100NR, a derivative of TA100 deficient in nitroreductase activity. The mutagenic responses in TA98 were 2- to 6-fold lower compared to TA100. Furthermore, I and II were less active in TA100NR, while compound III was about four times more mutagenic in TA100NR compared to the parent strain TA100. Incubation of III with NADPH and bacterial lysates showed that the extent of reduction was greater in TA100 compared to TA100NR. High-pressure liquid chromatography analysis of the ethyl acetate extract obtained from incubation of III with lysates of TA100 revealed the formation of four metabolites with retention times of about 4.0, 5.7, 10.0, and 14.3 minutes. The spectroscopic and chromatographic properties of the components with retention times of 10.0 and 14.3 minutes were identical to two derivatives obtained by chemical reduction of III, and thus represent nitroreduction products. These derivatives have been identified as cis- and trans-oxime isomers of methyl 3,4-dichloro-2-furoate, based on spectroscopic analyses. These oximes were not mutagenic for TA100. Furthermore, III was more mutagenic under anaerobic conditions, suggesting that secondary superoxide or nitroanion free radicals generated from nitroreduction are not responsible for the mutagenicity of III. In addition, the higher mutagenic response in TA100NR, and the lack of mutagenic activities of the amino and the oxime analogs of III suggest that the mutagenic activation of III might be due to the nitroso intermediate or involve mechanisms other than nitroreduction.