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

Abstract

3-Chloro-4-(chloromethyl)-5-hydroxy-2(5 H)-furanone (CMCF), 3-chloro-4-methyl-5-hydroxy-2(5 H)-furanone (MCF) and 3,4-dichloro-5-hydroxy-2(5 H)-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(5 H)-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 micro g/ml) was used as the initiating chemical. The phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA, 0.3 micro g/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.

Electrolyzed Reduced Water Supplemented with Platinum Nanoparticles Suppresses Promotion of Two-stage Cell Transformation

“…Balb/c 3T3 and C3H10T1/2 cells are sensitive to carcinogens compared with NIH/ 3T3 cells, which have been used for oncogene research. In vitro cell transformation assayed with Balb/c 3T3 and C3H10T1/2 cells, has long been recognized as being directly relevant to carcinogenesis (Kakunaga 1973;Heidelberger 1983;Kennedy 1983) and is regarded as a useful method to screen potential carcinogens (Dunkel et al 1981;Atchison et al 1982;Meyer 1983;Fang et al 2002;Laaksonen et al 2004). According to the two-stage cell transformation theory, normal cells first receive initiation, which is mainly caused by DNA damage, and then promotion, which enhances transformation (Tsuchiya and Makoto 1995).…”

A color discriminating broad range cell staining technology for early detection of cell transformation

“…When using this cell line for transformation assay, we counted type 2 and type 3 foci. [29] The results of foci formation in the experiment with C3H/10T1/2 cells are summarized in Table 2.…”