Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses.

OBJECTIVES: Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. METHODOLOGY: In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. RESULTS: In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. CONCLUSION: Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Solutions Containing a Statherin-Derived Peptide Reduce Enamel Erosion in vitro.

The effect of solutions containing a statherin-derived peptide (Stn15pSpS) on the protection against enamel erosion in vitro was evaluated. Bovine enamel specimens were divided into 4 groups (n = 15/group): (1) deionized water (negative control), (2) Elmex Erosion Protection™ (positive control), (3) 1.88 × 10-5 M Stn15pSpS, and (4) 3.76 × 10-5 M Stn15pSpS. The solutions were applied on the specimens for 1 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH-cycling protocol 4 times/day, for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The solutions were applied again during pH-cycling, 2 times/day for 1 min after the first and last erosive challenges. Enamel loss (µm) was assessed by contact profilometry. Data were analyzed by Kruskal-Wallis and Dunn's test (p < 0.05). The best protection against erosion was conferred by Elmex Erosion Protection that significantly differed from all the other treatments, followed by the solutions containing Stn15pSpS, regardless of the concentration. However, 3.76 × 10-5 M Stn15pSpS did not differ from the negative control. The solution containing the lower concentration of Stn15pSpS protected against erosion in vitro, which should be confirmed using protocols that more closely resemble the clinical condition.

Gels containing statherin-derived peptide protect against enamel and dentin erosive tooth wear in vitro.

The effect of gels containing a statherin-derived peptide (Stn) on the protection against enamel and dentin erosive tooth wear (ETW) in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 15 and 18/group for enamel and dentin, respectively) that were treated with Chitosan or Carboxymethylcellulose (CMC) gels containing Stn15pSpS at 1.88 × 10-5 M or 3.76 × 10-5 M. Chitosan or CMC gels without active ingredients served as negative controls, while chitosan gel containing 1.23% F (as NaF) and acidulated phosphate fluoride gel (1.23% F) served as positive controls. The gels were applied on the specimens for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The gels were applied again during pH cycling, 2 times/day for 4 min after the first and last erosive challenges. Enamel and dentin loss (µm) were assessed by contact profilometry. Scanning electron microscopy (SEM) was analyzed using a cold field emission. Data were analyzed by two-way ANOVA (for chitosan and CMC gels, separately) and Tukey's multiple comparison test. SEM images showed changes to enamel topography after application oft the gels containing Stn or F. Regarding CMC-based gels, for enamel, none of the treatments significantly reduced ETW in comparison with placebo; for dentin, however, gels containing Stn, regardless the concentration, significantly reduced the ETW. Moreover, Chitosan-based gels, regardless the Stn concentration, were able to protect enamel and dentin against ETW. Gels containing Stn might be a new approach to protect against ETW.

Metabolic effect of low fluoride levels in the islets of NOD mice: integrative morphological, immunohistochemical, and proteomic analyses

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Protective effect of calcium silicate toothpaste on enamel erosion and abrasion in vitro.

OBJECTIVES: To compare in vitro the effect of a toothpaste containing fluoride (F), calcium silicate (CaSi) and sodium phosphate salts to conventional toothpaste (NaF) on human enamel specimens submitted to erosive and abrasive challenges. METHODS: 48 sound and 48 enamel samples pre-treated with 1% citric acid were divided into 4 groups (n = 12): Group 1- Non-fluoride toothpaste; Group 2- NaF toothpaste (1450 ppmF); Group 3- CaSi toothpaste (1450 ppmF; MFP); Group 4- Erosion only. The samples were subjected to pH cycling (3 cycles/day; 90s; 1% citric acid, pH 3.6) and to abrasion for 7 days. After the 1st and the last cycle, they were submitted to abrasion (15s, 1.5N load), using a brushing machine, soft toothbrush and toothpaste slurry (1:3; 15ml/sample) and then immersed in the slurry for 45s. Samples were immersed in artificial saliva between the challenges. Enamel loss was evaluated using profilometry on days 3 and 7. Data were analysed by ANOVA and Tukey's test (p < 0.05). RESULTS: For sound enamel at baseline, mean (±SD) enamel loss (µm) for groups 1-4 on day 3 was 2.15 ± 0.35a, 1.20 ± 0.22b, 0.95 ± 0.19b and 1.98 ± 0.32a; on day 7 was 3.05 ± 0.40a, 2.07 ± 0.32b, 1.36 ± 0.33c and 3.69 ± 0.27d respectively. For acid-softened enamel at baseline, enamel loss on day 3 was 3.16 ± 0.19a, 2.17 ± 0.14b, 1.70 ± 0.11c and 3.04 ± 0.19a; on day 7 was 3.92 ± 0.25a, 3.07 ± 0.13b, 2.09 ± 0.15c and 3.87 ± 0.25a respectively. CONCLUSIONS: Both F toothpastes led to significantly higher enamel protection from short-term erosion and abrasion in comparison to the non-F toothpaste and erosion only. In the longer term, CaSi toothpaste conferred significantly higher protection than NaF toothpaste. CLINICAL SIGNIFICANCE: The results showed that for the longer term the CaSi toothpaste provided significantly higher protection than the NaF toothpaste, which indicates a good potential of the former to help prevent erosive tooth wear.

Increase of complex I and reduction of complex II mitochondrial activity are possible adaptive effects provoked by fluoride exposure.

Fluoride (F) can induce changes in the expression of several liver proteins, most of them localized in the mitochondria and its effect is dose- and time-dependent. This study analyzed the effect of distinct F concentrations and exposure periods on the mitochondrial activity of complex I-III and II-III in the liver. Thirty-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the duration of the treatment (20 or 60 days). They were subdivided into 3 subgroups (n = 6) according to the concentration of F (0 mg/L, 15 mg/L or 50 mg/L). After the experimental periods, the animals were anesthetized, liver mitochondria were isolated and stored for activity analyses. The determination of complexes II-III and I-III was based on the reduction of cytochrome c3+ to cytochrome c2+ performed spectrophotometrically. Bioinformatics analyses were performed using data from a previous study (Pereira et al., 2018). The mitochondrial complex I-III was significantly activated in the groups treated with 50 mgF/L for 20 days and 15 mgF/L for 60 days. The complex II-III was significantly reduced in the group treated with the higher F dose for 60 days. The networks indicated more changes in mitochondrial proteins in the group treated with the higher dose for 20 days; the reduction is probably linked to the activation of the complex I-III. The reduction in the complex II-III upon exposure to the higher F dose in the long term might be part of an adaptative mechanism of the body to counteract the deleterious effects of this ion on the energy metabolism.

Effects of low-level fluoride exposure on glucose homeostasis in female NOD mice.

Water fluoridation is an important public health measure for the control of dental caries. Recent animal studies have shown that low doses of fluoride (F) in the drinking water, similar to those found in public water supplies, increase insulin sensitivity and reduce blood glucose. In the present study we evaluated the effects of low-level F exposure through the drinking water on glucose homeostasis in female NOD mice. Seventy-two 6-week mice were randomly divided into 2 groups according to the concentration of F in the drinking water (0-control, or 10 mg/L) they received for 14 weeks. After the experimental period the blood was collected for analyses of plasma F, glucose and insulin. Liver and gastrocnemius muscle were collected for proteomic analysis. Plasma F concentrations were significantly higher in the F-treated than in the control group. Despite treatment with fluoridated water reduced plasma levels glucose by 20% compared to control, no significant differences were found between the groups for plasma glucose and insulin. In the muscle, treatment with fluoridated water increased the expression of proteins related to muscle contraction, while in the liver, there was an increase in expression of antioxidant proteins and in proteins related to carboxylic acid metabolic process. Remarkably, phosphoenolpyruvate carboxykinase (PEPCK) was found exclusively in the liver of control mice. The reduction in PEPCK, a positive regulator of gluconeogenesis, thus increasing glucose uptake, might be a probable mechanism to explain the anti-diabetic effects of low doses of F, which should be evaluated in further studies.