Sodium Fluoride Causes Oxidative Stress and Apoptosis in Cementoblasts

Objectives: Toothpastes of up to 5000ppm fluoride is available in several countries for patients with high risk of root caries; however, the effects of fluoride on cementoblasts have received less attention. The objective of the study was to determine whether NaF would induce apoptosis in OCCM-30 cells (immortalized murine cementoblasts) and if so, its underlying mechanisms.
Methods: The OCCM-30 cells were stimulated with different concentration of NaF. The MTT assay was conducted to detect the cell viability after NaF stimulation. Hoechst staining was used to determine changes of nuclear morphology. Real-time quantitative RT-PCR and Western blotting were performed to investigate the mRNA and protein expression of caspase-3,-8,-9, cleaved Poly (ADP-ribose) polymerase (PARP) and Fas-ligand (Fas-L), a ligand of death receptor. CA-DCF-DA [5(6)-Carboxy-2’,7’-dichlorofluorescein diacetate] combined with fluorescent microscope imaging and fluorescence-activated cell sorting analysis were used to measure the generation of reactive oxygen species (ROS) in OCCM-30 cells after the NaF stimulation.
Results: The result showed that NaF decreased the cell viability of OCCM-30 cells in a time- and dose-dependent manner. Apoptotic morphological changes of OCCM-30 cells exposed to NaF, such as cell shrinkage, nuclear condensation, and fragmentation, were observed. In addition, 10 mM NaF induced the expression of cleaved caspase-3,-8,-9 and cleaved Poly (ADP-ribose) polymerase (PARP). The mRNA expression of the Fas-L was also upregulated in cells exposed to 5 mM NaF. Furthermore, 10 mM NaF stimulation resulted in a significant generation of ROS in the OCCM-30 cells.
Conclusions:
The results suggest that NaF induces apoptosis in OCCM-30 cells through both of the extrinsic death receptor-dependent and oxidative stress-related intrinsic apoptotic pathway.