Objective  To examine the chloroacetic acid induced oxidative stress and the related effect on mitochondrial pathway related apoptosis in human normal bronchial epithelial 16HBE cells.

Method  Cell viability, apoptosis, and superoxide dismutase (SOD) activity were determined in 16HBE cells exposed to 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mmol/L chloroacetic acid for 24 h in vitro. Reactive oxygen species (ROS) were assayed after 16HBE cells were treated with the above-mentioned concentrations for 0.25, 0.5, 1, 8, 24 h, respectively. Mitochondrial membrane potential and expression levels of Bcl-2 and Bax mRNA were measured after 8 and 24 h of treatment. Expression levels of apoptosis-related proteins including Bcl-2, Bax, cytochrome C, Caspase-9, Caspase-3, and PARP-1 were measured after 16HBE cells treated with 1.5 and 2.5 mmol/L chloroacetic acid for 24 h.

Result  The cell viability rate, SOD activity, and mitochondrial membrane potential decreased in a dose-dependent manner with increasing concentrations of chloroacetic acid (r=-0.902, -0.732, and -0.863, respectively, P<0.05); but the cell apoptosis rate increased in a dose-dependent manner (r=0.914, P<0.05). In comparison with the control group, remarkable reductions of 12%, 20%, and 30% for cell viability, 9%, 21%, and 30% for SOD activity, and 11%, 18%, and 24% for mitochondrial membrane potential were found in the 1.5, 2.0, and 2.5 mmol/L chloroacetic acid treated groups (P<0.05) respectively; moreover, the cell apoptosis rates were 3, 4, and 7 times of the control group respectively. The intracellular ROS levels increased first and then decreased; peak values shown after 0.5 h of exposure in the 1.0, 1.5, 2.0, 2.5 mmol/L treatment groups, and significantly increased by 15%, 35%, 48%, and 55% respectively in comparison with the controls (P<0.05); the ROS levels in various concentration groups gradually dropped after 1h. There were significant dose-response relationships between ROS levels and concentrations of chloroacetic acid at each designed time point of exposure (r=0.756, 0.893, 0.735, 0.667, and 0.653, P<0.05). There was a distinct positive correlation between cell apoptosis rate and ROS level (r=0.826, P<0.05). After 8 and 24 h of exposure, the Bcl-2 and Bax mRNA expression levels both showed dose-response relationships with exposure concentrations (r=-0.634 and 0.754 at 8 h, r=-0.773 and 0.823 at 24 h, P<0.05). After 24 h of exposure, there was a significant decrease of the Bcl-2 mRNA and protein expression levels but a remarkable increase of the Bax mRNA and protein expression levels in 1.5 and 2.5 mmol/L treatment groups in contrast to the controls (P<0.05). There were significant positive correlations between the ROS level and the mRNA expression level of Bax (r=0.886 and 0.824, P<0.05), and negative correlations between the ROS level and the mRNA expression level of Bcl-2 (r=-0.862 and -0.815, P<0.05). After 24 h of exposure, compared with the control group, the protein expression levels of cytochrome C, activated Caspase-3, and activated Caspase-9 were distinctly increased and the PARP-1 protein level was significantly decreased in the 2.5 mmol/L chloroacetic acid treated group (P<0.05).

Conclusion  Chloroacetic acid could trigger oxidative stress in 16HBE cells and result in cell apoptosis via activating mitochondrial pathway with elevated expression level of Bax and decreased expression level of Bcl-2.