Chlorinated drinking water contains several chlorohydroxyfuranone (CHF) by-products whose contribution to cancer risk is not presently known. 3,4-Dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), and 3- chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) were studied for the induction of DNA damage, using the alkaline single-cell gel (SCG)/comet assay, and for chromosome damage, using sister-chromatid exchange (SCE) and chromosome aberration (CA) tests, in Chinese hamster ovary (CHO) cells. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the known genotoxic chlorination by-product and a rat carcinogen, was used as a reference chemical. The SCG analyses were done using concentrations that caused little or no cytotoxicity compared to that of the concurrent control cultures. In the cytogenetic tests, the CHFs were tested up to maximum cytotoxicity. MX and MCA were the most cytotoxic of the compounds in CHO cells followed by CMCF and MCF. All of the CHFs induced DNA damage, SCEs and CAs (mainly chromatid-type breaks and exchanges) in a concentration-related manner, with the exception that MCA was a weak inducer of SCEs. There were no significant differences between the lowest concentration of MX, MCA, and CMCF to cause DNA damage (SCG assay). Based on comparisons of the slopes of regression lines, MX was somewhat more potent than either MCA or CMCF, and MCF was clearly less potent than the other three compounds in the assay. The order of potency was MX > CMCF > MCA > MCF in inducing SCEs and MX > MCA > CMCF > MCF in inducing CAs. The data show that there are differences in the potency of genotoxicity among the CHFs tested. In many cases, however, the extent of maximum effect observed was comparable between the compounds. The results suggest that besides MX other CHFs should be considered in the evaluation of genotoxic risks associated with the consumption of chlorinated drinking water.

Syngeneic or allogeneic cells transformed by BK virus (BKV) were used to immunize C57BL/6J mice. After in vitro stimulation, lymphocytes prepared from the spleens of immunized mice were used in in vitro cytotoxicity tests. The results of these tests revealed the presence of a cell surface antigen, presumably corresponding to the viral transplantation antigen, common to all tested BKV- and SV40-transformed cell lines of C57BL/6J origin. An allogeneic cell line transformed by BKV also contained the same antigen. Immunization, i.e., in vivo priming, did not require syngeneic transformed cells, whereas cytolysis was only observed when the virus-specific antigen on target cells was associated with the same H2 haplotype as was expressed by effector cells. An additional unidentified antigen was shared by some of the BKV-transformed cell lines and cell lines transformed by simian adenovirus SA7.

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3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone (MX) is a mutagenic by-product found in chlorinated drinking water. It is a multi-site carcinogen in Wistar rats although the mechanisms of action of the carcinogenesis remain unresolved. We evaluated the ability of MX to promote development of transformation foci in a two-stage cell transformation assay in vitro. C3H 10T1/2 mouse embryonic fibroblasts were exposed to 3-methylcholanthrene (MC, 5 microg/ml) in the initiation phase and to MX (0.5, 1 or 2 microg/ml) or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, the positive control, 0.3 microg/ml) during the promotion phase of the assay on dishes. In other experiments, the cells were exposed to MX (0.5, 5 or 10 microg/ml) only in the initiation phase. At the end of the assay (6 weeks from the start of the assay), the transformation foci were counted and scored after fixation and staining of the cells. MC increased the total number of transformation foci per dish and the number of malignant type III foci, and TPA further promoted this phenomenon. When MX was added during the promotion phase in the MC-initiated cells, it promoted the development of the transformation foci in a dose-dependent manner. MX alone (added as an initiator) also slightly increased the development of the foci, including the malignant forms (type II and III), but the effect was not dose-dependent. In contrast to MC-induced foci, TPA did not promote the development of MX-initiated foci, it even decreased their number. The results suggest that MX may also have potential to promote tumor development.

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.