The effect of fluoride on enamel and dentin formation in the uremic rat incisor

Renal impairment in children is associated with tooth defects that include enamel pitting and hypoplasia. However, the specific effects of uremia on tooth formation are not known. In this study, we used rat mandibular incisors, which continuously erupt and contain all stages of tooth formation, to characterize the effects of uremia on tooth formation. We also tested the hypothesis that uremia aggravates the fluoride (F)-induced changes in developing teeth. Rats were subjected to a two-stage 5/6 nephrectomy or sham operation and then exposed to 0 (control) or 50 ppm NaF in drinking water for 14 days. The effects of these treatments on food intake, body growth rate, and biochemical serum parameters for renal function and calcium metabolism were monitored. Nephrectomy reduced food intake and weight gain. Intake of F by nephrectomized rats increased plasma F levels twofold and further decreased food intake and body weight gain. Uremia affected formation of dentin and enamel and was more extensive than the effect of F alone. Uremia also significantly increased predentin width and induced deposition of large amounts of osteodentin-like matrix-containing cells in the pulp chamber. In enamel formation, the cells most sensitive to uremia were the transitional-stage ameloblasts. These data demonstrate that intake of F by rats with reduced renal function impairs F clearance from the plasma and aggravates the already negative effects of uremia on incisor tooth development

The Acid Test of Fluoride: How pH Modulates Toxicity

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, eIF2α, 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 eIF2α in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH∼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

Reduced Protein Expression of the Na+/Ca2++K+-Exchanger (SLC24A4) in Apical Plasma Membranes of Maturation Ameloblasts of Fluorotic Mice

Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca2+-transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca2+ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization

Caries inhibitory effect of fluoridated sugar in a trial in Indonesia

The document attached has been archived with permission from the Australian Dental Association. An external link to the publisher’s copy is included.Background: In some regional areas of Indonesia, caries prevalence is increasing rapidly. As water, salt or milk fluoridation were not considered suitable for use throughout Indonesia, and fluoridated tooth paste is mostly too expensive, a fluoride cocrystallised sugar containing 10ppm fluoride was prepared. Its efficacy in inhibiting caries development was tested in a field trial. Methods: The field trial was established in Medan, Sumatera. All dietary background data necessary to ensure the safety of a trial were collected. Subjects chosen were 176 children who were residents of two orphanages and a boarding school for children of poor rural families. The trial used a double-blind format. Close monitoring of fluoride consumption was maintained, and fluoride excretion rates were assessed six monthly by urinary fluoride analysis. Results: Records of total tooth surface caries present initially and after 18 months of sugar supply showed that the children using fluoridated sugar had significantly fewer carious lesions than those who used normal sugar. Conclusion: This result indicates that sugar might be considered as a further vehicle for supplementary dietary fluoride in communities where there is a high caries prevalence or high caries risk and little exposure to fluoride.Mulyani, J McIntyr

Alternative splicing of amelogenin mRNA from rat incisor ameloblasts.

Amelogenin proteins are a major component of the developing enamel matrix, and are likely to have a key role in the control of enamel biomineralization. The heterogeneity of amelogenin is in part due to alternative splicing of the amelogenin RNA transcripts. Several patterns of alternative splicing have been described in mouse, bovine, porcine, and human enamel, with all alternatively spliced products having homologous 5' and 3' sequences within the coding regions. In these studies, we have used anchored PCR to identify alternatively spliced amelogenin cDNA sequences in the rat. We found amelogenin cDNAs that could be divided into two groups based on their 3' sequence. Group 1 cDNAs had a novel terminal sequence that has not been previously identified, while group 2 cDNAs were similar to those previously identified in other animal species. We identified a sequence, identical to the novel 3' amelogenin cDNA sequence, in rat genomic DNA downstream from the previously identified exon 7. The putative amelogenin proteins predicted for the two groups differed in their predicted isoelectric points, with the novel group 1 proteins having a more basic isoelectric point

Identification of two additional exons at the 3' end of the amelogenin gene.

Alternative splicing of the amelogenin gene generates a family of proteins secreted by ameloblasts that are primarily responsible for tooth enamel formation. Here the presence of two additional exons, downstream of exon 7, which are followed by an alternate polyadenylation site, is reported. Long polymerase chain reaction was used with a rat genomic amelogenin template to amplify sequences downstream from the primary polyadenylation site. Within the amplified sequences, two exons of 45 bp (exon 8) and 110 bp (exon 9) were identified. The presence of these additional exons in the human and mouse genome was indicated by Southern blot analysis. Antibodies raised against a synthetic peptide corresponding to a sequence encoded by exon 9 positively stained ameloblasts in sections of developing murine and porcine unerupted teeth. These results suggest that an amelogenin protein(s) that includes sequences derived from exons 8 and 9 is synthesized by the ameloblasts

Identification of two additional exons at the 3' end of the amelogenin gene.

Alternative splicing of the amelogenin gene generates a family of proteins secreted by ameloblasts that are primarily responsible for tooth enamel formation. Here the presence of two additional exons, downstream of exon 7, which are followed by an alternate polyadenylation site, is reported. Long polymerase chain reaction was used with a rat genomic amelogenin template to amplify sequences downstream from the primary polyadenylation site. Within the amplified sequences, two exons of 45 bp (exon 8) and 110 bp (exon 9) were identified. The presence of these additional exons in the human and mouse genome was indicated by Southern blot analysis. Antibodies raised against a synthetic peptide corresponding to a sequence encoded by exon 9 positively stained ameloblasts in sections of developing murine and porcine unerupted teeth. These results suggest that an amelogenin protein(s) that includes sequences derived from exons 8 and 9 is synthesized by the ameloblasts

Activation of recombinant bovine matrix metalloproteinase-20 and its hydrolysis of two amelogenin oligopeptides.

Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin. The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP). MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli. The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography. The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments. The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme. Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20. Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP. These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin