The synergistic effect of sodium chlorite and bromochloroacetic acid on BrO3−-induced renal cell death

Bromate (BrO3−) is a drinking water disinfection by-product (DBP) that induces renal cell death via DNA damage-dependent and -independent mechanisms. Drinking water contains other DBPs in addition to BrO3−. We tested the effect of two of these, sodium chlorite (NaClO2) and bromochloroacetic acid (BCAA), on BrO3− cytotoxicity in normal rat kidney (NRK) cells. NaClO2 and BCAA alone induced cytotoxicity at concentrations of over 20 ppm, while BrO3− was only moderately cytotoxic at concentrations of 200 ppm. Combining BrO3− with NaClO2 or BCAA alone enhanced cytotoxicity 1.5–4 fold. Exposing cells to all three compounds induced synergistic-like increases in cytotoxicity. This effect did not correlate to increases in reactive oxygen species (ROS), even though all three compounds induced ROS formation alone. NaClO2, but not BCAA, increased BrO3−-mediated DNA damage as measured by 8-hydroxydeoxyguanosine (8-OHdG) staining. In addition, NaClO2, but not BCAA, decreased BrO3−-induced G2/M cell cycle arrest. Both compounds increased apoptosis in the presence of BrO3− as assessed by annexin V, PI, and DAPI staining. This is in contrast to BrO3− treatment alone, which induced necrosis. Immunoblot analysis showed that both NaClO2 and BCAA increased p38 activation; however, consistent with 8-OHdG staining, only NaClO2 increased BrO3−-induced histone H2AX phosphorylation, a marker of DNA damage. In contrast, BCAA, but not NaClO2, increased BrO3−-induced phosphorylation of p53. These data support the novel finding that mixtures of DBPs increase BrO3−-induced renal cell death by DNA-dependent and -independent mechanisms, and could alter how the risk of these DBPs towards humans is assessed.

► Mixtures of water disinfection by-products (DPBs) enhanced renal cell death.
► Increased cell death correlated to increased DNA damage and changes in cell cycle.
► Mixtures of DBPs altered cell death mechanisms and cell signaling.
► These data suggest that assessment of the risk of DBPs should include mixtures.