Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

Effects of actinomycin D on developing hamster molar tooth germs in vitro.

The aim of this study was to evaluate the toxic effects of actinomycin D on the developing hamster tooth germ in organ culture. Hamster tooth germs during early secretory amelogenesis were exposed in vitro for 24 h to 10(-9) M-5 x 10(-5) M actinomycin D. Actinomycin D dose-dependently (> or = 10(-7) M) decreased the tooth germ dry weight but mineralization was affected only by doses > or = 10(-5) M. However, the uptakes of TCA-insoluble 32P and [3H]thymidine were significantly reduced dose-dependently from > or = 10(-8) M actinomycin D, indicating that the drug inhibits the synthesis of phosphate-containing macromolecules as well as DNA synthesis. Histologically, 10(-8) M actinomycin D was the lowest dose which was not toxic to any cell type in the developing tooth germ. At 10(-7) M actinomycin D, the most sensitive cells were the proliferating pre-odontoblasts followed by pre-ameloblasts; the mature secretory ameloblasts and odontoblasts appeared unaffected. Higher doses resulted in increased cytotoxicity to the secretory cells and, eventually, total degeneration of most cells. The data suggest that children treated for cancer during tooth development using anti-chemotherapy cocktails containing actinomycin D (serum levels > 10(-7) M) may develop defects later on in the mature dentition as a direct consequence of the toxicity of the drug to the tooth organ.

Effects of vincristine on the developing hamster tooth germ in vitro.

Vincristine is one of the cytostatic drugs present in cocktails commonly used for the treatment of cancer in children. The aim of this study was to evaluate biochemically and histologically the toxic effects of this drug on the developing tooth in vitro using the organ culture model in order to be able to predict what damage the drug can induce in the developing teeth from children undergoing anti-neoplastic chemotherapy. The most profound effect of the drug (10(-8)M-10(-4)M vincristine) on the developing tooth germ was the induction of mitotic arrests at the cervical loop and in the inter-cuspal regions. The 10(-4)M-10(-6)M vincristine doses were cytotoxic to most cells in the developing tooth germ. The 10(-7)M vincristine dose apart from induction of mitotic arrests, did not appear to be cytotoxic to the mature differentiated secretory cells. However, this dose induced incomplete nuclear polarization of the differentiating ameloblasts and odontoblasts. At 10(-8)M vincristine, the only effect observed were mitotic arrests; the secretory cells did not appear to have been affected at all. On the other hand, mineralization (TCA-soluble 45Ca and 32P uptake) was dose-dependently decreased from 10(-7)M vincristine upwards. 10(-9)M vincristine, the lowest dose tested, did not induce any changes in the developing tooth germ. The organ culture data indicate that 10(-9)M vincristine is the highest (safe) dose which does not induce any toxic effects in the developing hamster tooth germ.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of a single dose of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) on presecretory ameloblast differentiation in rat incisors.

A single, high dose of HEBP to rats results in a triad of lesions along the mineralizing front of the incisor enamel. One type of lesion is a shallow groove crossing the apical enamel surface. The purpose of this study was to explore the pathogenesis of this "demarcation groove", and to characterize changes in the involved regions of amelogenesis. Rats were given a subcutaneous dose of 10 mg/kg body weight of HEBP and sacrificed by vascular perfusion at intervals ranging from 1 to 36 hours. Mandibular incisors were processed for light and electron microscopy. The region of ameloblasts facing dentin was divided into two subregions: A region of ameloblasts facing unmineralized dentin, comprising a posterior (Aud/p) and an anterior portion (Aud/a), and a region of ameloblasts facing mineralized dentin (Amd). The progressive apical mineralization of the predentin was arrested up to 12 hours after injection of HEBP, while ameloblasts related to already mineralizing dentin continued to differentiate and secrete enamel matrix. At 8 hours the dentin and enamel layers had assumed a common apical border at the start of Amd, marking the position of the future demarcation groove. The length of Aud/p remained constant, Aud/a doubled in length, and Amd was drastically reduced up to 24 hours after injection of HEBP. The normal migration rate of the ameloblasts was unaffected by HEBP. Accumulations of ameloblast secretory products occurred at certain time intervals between the cell apices, but no morphological changes were recorded in the organelles. Most of the changes observed may be indirect in nature resulting from the physico-chemical effect of HEBP on normal mineralization of dentin and enamel. However, further studies are needed to elucidate possible direct cellular effects on ameloblasts.

The effect of acivicin, an inhibitor of gamma-glutamyl transpeptidase on mouse molar development in vitro.

Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme found on the surface of cells having secretory or resorptive functions. GGT has been found to be strongly localized in the stellate reticulum of the developing tooth. It has been proposed that the role for GGT in tooth development is related to the transport of amino acids into the cell via the gamma-glutamyl cycle. In order to ascribe a role for GGT and the stellate reticulum in the developing tooth, the activity of GGT was inhibited by a daily 1 hour application of the glutamine analog, acivicin (Upjohn), to mouse first molar tooth germs in serum free organ culture for periods of 5 to 8 days. Acivicin treatment effectively arrested tooth development. Additionally, tooth germs were allowed to recover for 4 to 7 days following a 4 day treatment with acivicin or they were incubated in media supplemented with additional glutamine or nucleosides during the acivicin treatment. Tooth germs were able to recover when returned to control medium. However, treated teeth were smaller in size than the controls. Glutamine partially compensated for the acivicin treatment. Nucleotide supplemented media appeared to almost completely override the inhibitory effect of acivicin. It appears that the inhibition by acivicin is primarily due to its effect on DNA and RNA synthesis. The inhibition of the gamma-glutamyl cycle by acivicin was unaffected by the addition of glutamine or nucleosides. Therefore, inhibition of the gamma-glutamyl cycle and GGT does not seem to significantly affect the development of teeth at the stages studied.

Effects of beta-D-xyloside on morphogenesis and cytodifferentiation in cultured embryonic mouse molars.

Embryonic mouse molars were grown on a semi-solid medium supplemented with 2 mM beta-D-xylopyranoside (beta-xyloside), a specific inhibitor of proteoglycan synthesis. The induced glycosaminoglycan depletion in the extracellular matrix was monitored by immunohistochemistry employing monoclonal antibodies to chondroitin 4- and chondroitin 6-sulfates. beta-Xyloside inhibited formation of the dental bell and delayed the appearance of the first odontoblasts. Odontoblast functional differentiation proceeded in the absence of chondroitin sulfate in the basement membrane. Predentin secreted in the presence of beta-xyloside triggered the polarization of ameloblasts, but did not allow the maintenance of polarized odontoblasts. These results support the hypothesis that, in the tooth germ, chondroitin sulfate proteoglycans participate in the regulation of cell kinetic-dependent morphogenesis (Mark et al., 1990. Differentiation 43, 37-50). On the other hand, the possibility that chondroitin sulfate might play a role in odontoblast terminal differentiation is definitively ruled out.

Effects of glycosaminoglycans on in vitro mouse dental cells.

Trypsin-dissociated dental papillae and enamel organs removed from the first lower molars of day-18 and day-19 mouse embryos were cultivated for 2 to 4 days on Millipore filters in DMEM supplemented with 15 per cent fetal calf serum and chondroitin sulphate and hyaluronic acid, individually or together. Three concentrations of glycosaminoglycans (GAGs) were also added to the media (01, 0.2, 0.4 mg/ml). Control cultures were made in the absence of GAGs, and additional experiments performed in which the presence of GAGs was associated with serum-free medium. Elongated and polarized odontoblasts showing synthetic activity were only observed in the presence of serum containing medium supplemented with 0.1 or 0.2 mg/ml of the GAGs. [3H]-thymidine autoradiography demonstrated that these cells were already post-mitotic at the onset of the culture. Polarized ameloblasts were never observed. These data provide evidence that GAGs are able to maintain the polarized state of cultured odontoblasts.