Effects of chlorohydroxyfuranones on 3-methylcholanthrene-induced neoplastic transformation in the two-stage transformation assay in C3H 10T1/2 cells

3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) are chlorination byproducts in disinfected drinking water. These compounds are positive in genotoxicity tests in vitro. We have previously shown that 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) can induce malignant transformed foci in the two-stage cell transformation assay in C3H 10T1/2 cells in vitro in both the initiation and promotion phases. In the present study we compared the effects of CMCF, MCF and MCA in the same assay. C3H 10T1/2 mouse embryonic fibroblasts were exposed to these chlorohydroxyfuranones (CHFs) at three different concentrations in the initiation phase or the promotion phase of the assay. In the latter experiments 3-methylcholanthrene (MC, 5 µg/ml) was used as the initiating chemical. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.3 µg/ml) was used as a positive control promoter. At the end of the assay (6 weeks from the start), the transformation foci were counted and scored after fixation and staining of the cells. When added at the initiation phase of the assay on their own, CMCF and MCF, but not MCA, increased the transformation foci formation. TPA added in the promotion phase did not modify the responses of CMCF and MCF but TPA increased the number of foci in MCA-treated cells. When CHFs were added during the promotion phase to the MC-initiated cells, MCF and MCA enhanced the development of the transformation foci. The effect of CMCF was equivocal since at higher concentrations CMCF actually decreased the number of the MC-induced foci. Including the previous data for MX in this assay and considering the lowest active concentrations, the initiation activity of the foci formation decreased in the order MX >CMCF >MCF, i.e. with the decreasing number of chlorine atoms of the methyl group in the 4-position of the CHF molecule (two, one, and zero, respectively). In contrast, the activity in the promotion phase did not follow the same pattern. MX, MCF and MCA were all active over the same concentration range. Hence, in addition to MX, MCF and MCA may also possess some potential to promote tumor development.