The Salmonella typhimurium (TA100) mutagenic compound, mucochloric acid [3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA)], was inactivated by in vitro N-acetylcysteine (NAC). The reaction of MCA with NAC at pH7 was second order and gave products 4, 5, and 6a that resulted from the displacement of chlorine from C-3 or C-4 of MCA. The sodium borohydride treatment of product 4 gave the same product (7) as was obtained by treating 3,4-dichloro-2(5H)-furanone with NAC. The treatment of MCA with (R)-(+)-cysteine gave the bicyclic product 9a, in which the two chlorine atoms of MCA were still present. This product was slightly more mutagenic than MCA, whereas product 5 was less mutagenic than MCA and product 4 was nonmutagenic in the Salmonella typhimurium (TA100) assay.

The mutagenic 2(5H)-furanones resulting from the chlorination of lignohumic substances in water disinfection and paper pulp bleaching are known to be inactivated by thiols. The objectives of the present study were to characterize the kinetics of an inactivating reaction, isolate and characterize products, and determine their mutagenicity in relation to the starting, mutagenic 2(5H)-furanones. The Salmonella typhimurium (TA100) mutagenicity of mucochloric acid (MCA) had a mean value of 2800 revertants/mumol from four assays and was twice as potent as the C-5 isopropyl ether of MCA (MCA-IPE), whose mutagenicity was determined in the same four assays. A second-order reaction of MCA with GSH at pH 7 was observed. The major product, making up 70% of the total product mixture, was identified as a 1.5:1 mixture of two diastereomers formed by sulfur displacement of the C-4 Cl atom from MCA. The major diastereomer was isolated from the 1.5:1 mixture. Connectivity of GSH to the MCA moiety in the product was established by 2D long-range coupling NMR and fully coupled 13C NMR. On the basis of circular dichroism, the major diastereomer had the S configuration at the hydroxyl-bearing, C-5 ring carbon. MCA-IPE reacted with GSH and N-acetylcysteine (NAC), giving 1:1 mixtures of two diastereomers, again by displacement of the C-4 Cl atom from MCA. A single diastereomer was isolated from the 1:1 MCA-IPE plus NAC reaction. Its structure, determined by X-ray crystallography, had the 5R,8R configuration and was in agreement with the ross structure deduced from the NMR analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The inactivation of the drinking water mutagen mucochloric acid (MCA) by reduced glutathione (GSH) was linked to the formation of an MCA-GSH conjugate, a nonmutagen in the Salmonella typhimurium (TA100) plate incorporation assay. Anaerobic formation of MCA-GSH is found now to be associated with oxidized glutathione (GSSG) and unconverted MCA. The anaerobic reaction of GSH with MCA in the presence of the radical trap 2-methyl-2-nitrosopropane (tNB; "tert-nitrosobutane") gives rise to an electron paramagnetic resonance (EPR) resulting from the overlapping spectra of two radical adducts. The first species exhibited hyperfine coupling constants of aN = 13.65 G and aH beta = 0.73 G. The second radical adduct exhibited a three-line signal of aN = 12.8 G. The first species is assigned to an adduct of the MCA radical because deuteration of MCA (5-deuterio-MCA) caused the beta-hydrogen hyperfine coupling to collapse. The second radical adduct is unaffected by the deuteration of MCA. Thus, the involvement of both GSSG and a carbon-centered MCA radical in the action of MCA on GSH is indicated.

A difference in biological response to enantiomers is not an uncommon observation and is, therefore, to be expected in various manifestations of genotoxicity. The bacterial mutagen mucochloric acid (2,3-dichloro-5-hydroxy-2(5H)-furanone) has one chiral center, at C-5, but this mutagen exists in racemic form because of the facile stereoisomerization occurring by the mechanism of ring-chain tautomerism. Two readily synthesized enantiomeric analogs of mucochloric acid, as well as the racemic form of the two, were prepared from mucochloric acid and (R)-(+)-, (S)-(-)-, and (R,S)-(+/-)-cysteine. Using Salmonella typhimurium (TA100), the enantiomeric compounds were assayed together in four dose/response assays along with mucochloric acid, the reference mutagen. In three of the same four assays, the racemic form was also assayed. Neither statistically significant differences in mutagenicity, as determined in slope responses, nor distinctions from the plotted curves were observed among the two enantiomers and their racemic form. Therefore, no enantiospecific interaction between enantiomers and chiral DNA or enzymes involved in repair or replication could be concluded.