The acid test of fluoride: how pH modulates toxicity

Abstract

Background: It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F(-)). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F(-). Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of F(-) into cells. Here, we asked if F(-) was more toxic at low pH, as measured by increased cell stress and decreased cell function.

Methodology/principal findings: Treatment of ameloblast-derived LS8 cells with F(-) at low pH reduced the threshold dose of F(-) required to phosphorylate stress-related proteins, PERK, eIF2alpha, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F(-) dose and pH. Luciferase secretion significantly decreased within 2 hr of F(-) treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F(-) in their drinking water exhibited increased stress-mediated phosphorylation of eIF2alpha in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH approximately 7.2). Intriguingly, F(-)-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected.

Conclusions: The low pH environment of maturation stage ameloblasts facilitates the uptake of F(-), causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis.

Figure 1. Low pH enhances F⁻-mediated stress.

(A) Immunoblots of LS8 cells treated with indicated doses of NaF for 2 hr at pH 7.4 or pH 6.6 were probed for phosphorylated JNK and c-jun. Actin bands are controls for protein loading. (B) Immunoblots of LS8 cells treated with 0.25 mM NaF for 2 hr or 4 hr were probed for phosphorylated forms of JNK, c-jun, PERK and eIF2α. Total eIF2α bands are controls for protein loading. In all cases, low pH enhanced stress protein activation.

Figure 2. Low pH further decreases the F⁻-mediated reduction in protein secretion.

(A) Recombinant Gluc was harvested from medium supernatant and directly treated with the indicated doses of NaF at 37°C for 6 hr. No significant decrease in Gluc activity was observed, demonstrating that F⁻,by itself does not inhibit Gluc activity (B) LS8-Gluc-CFP cells were treated with NaCl, NaF or the ER stress-inducing agent, tunicamycin for 6 hr; medium supernatant was then analyzed for Gluc activity (secretion). NaF and tunicamycin, but not NaCl, decreased Gluc secretion. (C) LS8-Gluc-YFP cells were treated with 0.25 mM NaF for 6 hr and imaged for YFP. NaF treatment localized the fusion protein within the peri-nuclear region. (D) LS8-Gluc-CFP cells were treated with the indicated doses of NaF for 24 hr and medium supernatants and cell lysates were immunoblotted and probed for Gluc. Actin served as the loading control. Note that F⁻ treatment resulted in intracellular accumulation of Gluc. (E) LS8-Gluc-CFP cells were treated with NaF at pH 6.6 or 7.4 for 2 hr. Gluc activity (secretion) in medium supernatant significantly decreased at pH 6.6 (p<0.05) (F) Cell proliferation, as measured by WST1 assay after 6 hr treatment, did not change significantly, indicating that the observed differences were not due to a proliferative advantage of one treatment group over another, in the short time period examined. All experiments were performed in triplicate and repeated three times. Scale bar for (C) represents 10 µm.

Figure 3. Maturation stage, but not secretory stage, ameloblasts from F⁻-treated rats exhibit stress.

Rats were supplied ad libitum with 0 or 100 ppm F⁻ in their drinking water. Immunohistochemistry was performed on incisor sections with antiserum specific for phosphorylated eIF2α. Note significant staining in maturation stage ameloblasts and in the papillary layer but not in secretory stage ameloblasts of F⁻-treated rats. No staining was observed in the untreated rats. Curly brackets indicate ameloblasts and square brackets indicate papillary layer. Scale bar represents 50 µm.

Figure 4. Decreased expression of maturation but not secretory stage-specific genes.

Rats were treated with 0, 50, 100 or 150 ppm F⁻ in their drinking water for 6 weeks. qPCR was performed on secretory and maturation stage enamel organs. Data shown is an average of three separate experiments, performed in triplicate. Data was normalized to the eEF1α1 expression control gene. Note the decreased expression of maturation stage genes, Klk4 and Amtn (p<0.05). Secretory stage genes (Amel, Ambn, Enam and Mmp20) did not exhibit any changes in expression.

Figure 5. Schematic showing our postulated mechanism for maturation stage ameloblast sensitivity to fluoride.

During the maturation stage, massive precipitation of hydroxyapatite occurs, releasing H⁺ ions. F⁻ can reversibly associate with H⁺ ions to form HF. Approximately 25-fold more HF is formed at pH 6.0 as compared to pH 7.4. HF diffuses into the cell more easily than F⁻ and flows down a steep concentration gradient from the acidic maturation stage enamel matrix into the neutral cytosol of the ameloblast. The neutral pH inside the cell causes reversion of HF to F⁻. Excess F⁻ within the cell interferes with ER homoestasis that may result in the dimerization and phosphorylation of PERK and its substrate, eIF2α. Consequently, protein synthesis is attenuated. ER stress can also lead to increased degradation of transcripts encoding secreted proteins such as Klk4. Collectively, decreased secretion of matrix-degrading enzymes such as KLK4 can lead to delayed resorption of enamel matrix proteins, resulting in the higher protein content observed in fluorosed enamel. ER, endoplasmic reticulum.