Molecular mechanisms of bromate-induced renal toxicity
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We tested the hypothesis that bromate (BrO3-), a water disinfection by-product (DBP), induces renal cell death via genotoxic and non-genotoxic mechanisms. Cytotoxicty was assessed using acute (100-400 ppm for 48 hr) and chronic doses (0-100 ppm for 28 days) of BrO3-. Molecular markers of cytotoxicity included mitogen activated protein kinases (MAPK), cell cycle regulating proteins (p53, p21 and cyclin B1) and histone 2AX (H2AX). Genotoxicity was assessed using 8-hydroxyl-deoxyguanosine (8-OHdG) staining. The ability of other DBPs to alter BrO3--induced cell death and the effect of BrO3- on epigenetics were also determined. Acute doses of BrO3- (100- 400 ppm) induced renal cell necrosis and G2/M arrest. G2/M arrest correlated to activation of p53, p21, cyclin B1, cdc2, p38 and ERK1/2 MAPK. Inhibition of p38 partially reversed BrO3--induced G2/M arrest and decreased p-p53, p21 and cyclin B1 expression. Activation of these proteins was inhibited by the ROS scavenger ascorbic acid, as was G2/M arrest; however, ascorbic acid had no effect on 8-OHdG staining. Exposure of cells to BrO3- in the presence of the DBPs sodium chlorite (NaClO2) and bromochloroacetic acid (BCAA) increased cell death, in correlation with 8-OHdG staining, but not to ROS formation. Increased cell death also correlated to decreased G2/M arrest and increased apoptosis. These data suggest that mixtures of DBPs enhance toxicity by switching cell death pathways. Epigenetic mechanisms of BrO3- cytotoxicity including DNA methylation and histone modification were studied. Methylation specific PCR and immunoblot showed that BrO3- (0-100 ppm) increased p21 promoter unmethylation and H2AX phosphorylation after 3 days. These effects were seen with concentrations as low as 100 ppb after 9 days or longer time of exposure. These data demonstrate the novel findings that BrO3- induced G2/M cell cycle arrest and expression of cell cycle and cell signaling proteins. Mixtures of BrO3- and other DBPs increased cell death and DNA damage, but not ROS formation. Relatively low doses of BrO3- induced epigenetic changes in p21, in the absence of 8-OHdG staining. These data identify novel mechanisms of action for BrO3- in kidney cells and will aid in the risk assessment of BrO3- to humans.