The Effect Of Bromodichloromethane On Micronucleus And Its Underlying Mechanisms Related To Cell Proliferation

Bromodichloromethane (BDCM) is one of the trihalomethanes (THMs) that are considered the most prevalent classes of disinfection by-products in finished drinking water. In addition to BDCM, THMs includes chloroform, chlorodibromomethane (DBCM) and bromoform. According to previous reports, THMs could induce tumors of large intestine, kidney and liver in chronic rodent bioassays. When administered by gavage in a two-year study, BDCM produced renal and liver tumors in B6C3F1mouse, and renal and intestinal tumors in F344rat. Moreover, some epidemiological studies have shown that lifetime exposure to chlorinated water containing THMs is associated with increased risks of the bladder and colorectal cancers. However, the mechanism of carcinogenesis for THMs remains largely unknown. Carcinogens are generally considered to increase the risk of cancer by two different mechanisms: genotoxic and nongenotoxic mechanisms. Previous studies demonstrated that BDCM was mutagenic after activation by glutathione S-transfersase theta (GSTT1-1). Multiple experimental studies also have revealed that abnormal organ toxicity, cell proliferation and DNA methylation was observed in cancer target tissues after exposure to BDCM at a carcinogenic dose. These imply that nongenotoxic mechanisms may play an important role in the carcinogenic activity of BDCM.Although many of the initial genotoxicity tests for THMs resulted in negative responses, later studies showed that THMs were mutagenic after activation. The metabolically potential human hepatoma cell line HepG2is one of the most promising cell lines in genotoxic studies, and it can be used to assess the genotoxicity of direct and indirect mutagens. The cytokinesis-block micronucleus test (CBMNT) is a method for measuring chromosome breakage and/or whole chromosome loss. Thus, the present study used CBMNT to investigate the clastogenic effect of the four THMs on HepG2cells.Based on the above genotoxic study, BDCM, a classical form of the brominated THMs, was selected to explore the nongenotoxic mechanisms of this chemical. We aimed to investigate the time-dependent effect of BDCM on cell proliferation in the different tissues of male F344rats, and to explore whether E-cadherin mediated cell-cell adhesion and the Wnt signaling pathway are involved in cell proliferation related to BDCM.This study is composed of the following two parts:Part I The clastogenisity of the four THMs on HepG2cellsObjective:To investigate the clastogenic effect of the four THMs (chloroform, BDCM, DBCM and bromoform) on HepG2cells.Method:CBMNT was carried out to evaluate the effect of the THMs above on the frequency of micronuclei and nuclear division index (NDI) at four concentrations in HepG2cells. Dimethyl sulfoxide (DMSO) and benzo(a)pyrene were selected as solvent control and positive control, respectively. Results: Comparing with the solvent control, chloroform, BDCM and DBCM were found to induce statistically significant increases in the frequency of micronuclei in HepG2cells at the concentrations of10000,10000and3000μmol/L, respectively. No positive result was observed in HepG2cells treated with bromoform.Conclusion:Chloroform, BDCM and DBCM induced clastogenic activity in HepG2cells. These THMs are identified as genotoxic compounds.Part II The mechanisms of BDCM on cell proliferation in the different tissues of male F344ratsObjective:To investigate the mechanisms of BDCM on cell proliferation via E-cadherin mediated cell-cell adhesion and the Wnt signaling pathway in the different tissues (colons, kidneys and livers) of male F344rats.Method:A total of64male F344rats were randomly distributed into two groups, and were administered BDCM at doses of0,100mg/kg in corn oil for5days per week. Then groups of8rats were euthanized after1,4,8and12weeks of exposures, and the colon, kidney and liver were collected. Formalin fixed tissues were processed routinely, and stained with hematoxylin and eosin for pathological examination. DNA methylation status in the promoter of APC and E-cadherin was evaluated by bisulfite sequencing PCR (BSP). The mRNA content of β-catenin, APC, E-cadherin, c-jun, c-myc and cyclin D1was monitored by real-time RT-PCR. The protein expression of APC and E-cadherin was determined by immunohistochemistry. The level of cell proliferation was assessed by proliferating cell nuclear antigen (PCNA) immunolabelling.Results:No clinical signs of toxicity were observed except for progressively decreased body weight gain in rats treated with BDCM.Hypermethylation in the promoter of E-cadherin was observed in the colorectal epithelium of rats after exposure to BDCM for12weeks, associated with a decrease in E-cadherin mRNA and protein expression. Consistent with these, elevation of cell proliferation was seen in the colon at the same time point. However, no pathological alteration related to BDCM was observed in colons during the study periods.In kidneys, BDCM caused not only a decrease of E-cadherin mRNA, but also an increase of transcripts of the proliferative target genes c-myc and cyclin D1. But suppression of E-cadherin mRNA and protein expression occurred in the absence of obvious changes in DNA methylation status. Furthermore, BDCM induced mild nephrotoxicity characterized by cellular swelling, microvesicular vacuolation, individual karymegaly and occasionally sloughed necrotic epithelial cells at8and12weeks. Additionally, BDCM induced statistically significant increases of cell proliferation in the epithelial cells of the proximal tubule at4,8and12weeks.In livers, margin hepatotoxicity, including occasional swelling and steatosis, was observed after exposure to BDCM for12weeks. Although there were a transient decrease of P-catenin and c-myc mRNA in the liver after exposure to BDCM for8weeks, these abnormality returned to the baseline at12weeks. In addition, no change in the methylation status of APC and E-cadherin was obtained related to BDCM treatment, neither the mRNA and protein expression did.Conclusion:BDCM administered by gavage increased cell proliferation by suppression of E-cadherin mRNA and protein expression in colons. In addition, BDCM induced an elevation of cell proliferation in the epithelial cells of the proximal tubule via suppression of E-cadherin mRNA and protein expression and transcriptional activation of c-myc and cyclin D1, downstream genes of the Wnt pathway. However, no obvious alteration of nongenotoxic activity related to BDCM treatment was observed in livers during the study periods. Therefore, E-cadherin mediated cell-cell adhesion and the Wnt signaling pathway are involved in BDCM-induced cell proliferation in the different tissues of male F344rats, supporting nongenotoxic mechanisms for its carcinogenic activity in rodents.