Experimental studies on cellular mechanisms of the carcinogenicity of 3-Chloro-4-(Dichloromethyl)-5-Hydroxy-2(5H)-Furanone (MX)

Several different disinfection by-products (DBPs) are formed during the chlorination of raw water. The DBPs which pose a cancer risk to man are not known. One candidate group is the chlorohydroxyfuranones (CHFs). Of the CHFs, 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4-(chloromethyl)-5hydroxy-2(5H)-furanone (CMCF), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) are all genotoxic in mammalian cells in vitro. MX is also carcinogenic in Wistar rats. This thesis focuses on the underlying cellular mechanisms involved in MX-induced carcinogenicity. Point mutations were analyzed in the p53 tumor suppressor gene (exons 4-7) and the Ki-, Ha-, and N-ras oncogenes (exons 1-2) of the liver tumors of the MX carcinogenicity study in rats. The expression of p53 protein was examined immunohistochemically in the liver and thyroid gland tumors. In addition, the expression of p21 Ki-ras protein was determined in several thyroid gland tumors. No mutations were detected in ras genes in 50 rat liver tumors. The mutation frequency of p53 gene was low (4 mutations). Moreover, the mutations had no consistent pattern, scattering to different codons and positions of the codon. Immunohistochemical analyses revealed that hepatocellular adenomas and carcinomas did not overexpress p53 protein. Instead, all the cholangiomas and cholangiocarcinomas, which originate from the bile duct epithelial cells, overexpressed p53 protein. The epithelial cells of the hyperplastic bile ducts of aged rats overexpressed p53 protein independently of the MX treatment. The MXinduced thyroid tumors did not have an abnormal expression of p53 or p21 Ki-ras proteins. In other experiments, the effects of MX, CMCF, MCA, and MCF were investigated on gap junctional intercellular communication (GJIC), first in Balb/c 3T3 mouse fibroblast cells and then in WB-F344 rat liver epithelial cells, the target cells of MX tumorigenicity. Inhibition of GJIC is a common mechanism among tumor promoters. Concentrationand time-responses for inhibition of GJIC by CHFs were studied in both cell lines. In addition, the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling pathways in the inhibition of GJIC by CHFs was defined in WB-F344 cells. The effects of CHFs on the gap junction structural protein, connexin43 (Cx43), and its mRNA levels were also determined. All the studied CHFs inhibited GJIC dose-dependently in both cell lines. The orders of potency to inhibit GJIC in Balb/c 3T3 and WB-F344 cells were MX>MCA>CMCF>MCF and MX>CMCF≈MCA>MCF, respectively. In Balb/c 3T3 cells MX inhibited GJIC already at nanomolar concentrations. In WB-F344 cells, the inhibition of GJIC by CHFs was reversible and was dependent upon the activation of the MAPK signaling pathway. CHFs decreased the expression of the Cx43 protein without altering its mRNA level. These results suggest that the main mechanism by which CHFs inhibit GJIC in the liver cells was to decrease the expression of Cx43. Altogether, the results suggest that point mutations in p53 and ras genes do not contribute to the MX-induced liver and thyroid tumorigenesis in rats. In contrast, inhibition of GJIC may be one mechanism by which MX can promote tumor development.