Altered Calcium Homeostasis by Fluoride In Enamel Cells

Objectives: Dental fluorosis (DF) develops during the period of dental enamel formation if enamel forming cells (ameloblasts) are exposed to high levels of fluoride. Characteristic brown stains, mottled and opaque enamel are features associated with DF. It is estimated that over 15% of the US population present with DF of varying degrees of severity. The source of this fluoride is commonly drinking water, tooth paste and oral rinses. Previous reports indicated that in patients with DF, their enamel appeared hypominerilized, suggesting a possible alteration in calcium transport. Here we investigate the effects of fluoride in intracellular calcium homeostasis in enamel cells.
Methods: The cytosolic calcium dye Fura2-AM and the genetically encoded calcium indicator CEPIA (red), targeting the endoplasmic reticulum (ER), were used to monitor changes in calcium concentration in the well-known enamel cell line LS8 cells. Cells were treated with concentrations of 0.5 mM and 1 mM of sodium fluoride (NaF) for 0.5 hr, 1hr, 2hr and 24 hrs. Cells were then imaged in the Flexstation III plate reader or a Nikon fluorescent microscope to determine changes in calcium concentration following the application of the ER calcium pump (SERCA) inhibitor, thapsigargin. We also analyzed, by quantitative real time PCR, whether this treatment induced cell stress or altered the expression of enamel genes (e.g. AmelX, Enam, Ambn). Protein level differences were determined by Western Blot analysis.
Results: Thapsigargin-induced ER calcium release differed in NaF treated cells from controls when targeting both cytosolic and ER calcium. Cell stress markers were also upregulated in NaF treated cells. There was also a marked change in the expression of key enamel genes.
Conclusions: These preliminary data suggest that a potential factor affecting enamel mineralization in DF could be associated with altered calcium homeostasis.