Methods: HGEC (Ca9-22, HSRRB, Osaka) were incubated in 100, 150, 200 or 250ppm of NaF medium at 37oC. Concentrations of NaF were much lower than concentrations in clinical dose. After 24h incubation, HGEC were double stained with Annexin V and 7-AAD to detect apoptosis, and analyzed using a flow cytometer. Key apoptotic enzymes, caspase-8 and 9, and reactive oxygen species (ROS) also assessed by a flow cytometer using fluorescent markers. Fragmentation of DNA, Caspase-3, and cytotoxicity (LDH activity) were determined using ELISA.
Results: The levels of LDH and ROS increased with concentration dependently. Early apoptosis and caspase-3 activity were significantly increased in 150 and 200ppm NaF that in comparison with 100, 250ppm NaF and control. DNA fragmentation were also showed 5-7 times higher than the control. At 150 and 200ppm NaF, caspase-3, 8 and 9 activities were significantly increased by 40-50%.
Conclusion: The present results demonstrated that the range of NaF concentration causing apoptosis in HGEC is 150-200ppm. Results of caspase activities and ROS assay indicated that one of the apoptotic pathway caused by fluoride is mitochondrial-mediated.