ERS Mediated by GRP-78/PERK/CHOP Signaling Is Involved in Fluoride-Induced Ameloblast Apoptosis.

Fluoride can be widely ingested from the environment, and its excessive intake could result in adverse effects. Dental fluorosis is an early sign of fluoride toxicity which can cause esthetic and functional problems. Though apoptosis in ameloblasts is one of the potential mechanisms, the specific signal cascade is in-conclusive. High-throughput sequencing and molecular biological techniques were used in this study to explore the underlying pathogenesis of dental fluorosis, for its prevention and treatment. A fluorosis cell model was established. Viability and apoptosis rate of mouse ameloblast-derived cell line (LS8 cells) was measured using cell counting kit-8 (CCK-8) assay and flow cytometry analysis. Cells were harvested with or without 2-mM sodium fluoride (NaF) stimulation for high-throughput sequencing. Based on the sequencing data, subcellular structures, endoplasmic reticulum stress (ERS), and apoptosis related biomarkers were verified using transmission electron microscopy, quantitative real-time polymerase chain reaction, and Western blotting techniques. Expression of ERS markers, apoptosis related proteins, and enamel formation enzymes were detected using Western blotting after addition of 4-phenylbutyrate (4-PBA). NaF-inhibited LS8 cells displayed time- and dose- dependent viability. Additionally, apoptosis and morphological changes were observed. RNA-sequencing data showed that protein processing in endoplasmic reticulum was obviously affected. ERS and apoptosis were induced by excessive NaF. Downregulation of kallikrein-related peptidase 4 (KLK4) was also observed. Inhibition of ERS by 4-PBA rescued the apoptotic and functional protein changes in cells. Excessive fluoride induces apoptosis by activating ERS, which is mediated by GRP-78/PERK/CHOP signaling. Key proteinase is present in maturation-stage enamel; KLK4 was also affected by fluoride, but rescued by 4-PBA. This study presents a possibility for therapeutic strategies for dental fluorosis, while further exploration is required.

Fluoride Exposure Provokes Mitochondria-Mediated Apoptosis and Increases Mitophagy in Osteocytes via Increasing ROS Production.

Fluoride is a persistent environmental pollutant, and its excessive intake causes skeletal and dental fluorosis. However, few studies focused on the effects of fluoride on osteocytes, making up over 95% of all bone cells. This study aimed to investigate the effect of fluoride on osteocytes in vitro, as well as explore the underlying mechanisms. CCK-8, LDH assay, fluorescent probes, flow cytometry, and western blotting were performed to examine cell viability, apoptosis, mitochondria changes, reactive oxygen species (ROS) and mitochondrial ROS (mtROS), and protein expressions. Results showed that sodium fluoride (NaF) exposure (4, 8 mmol/L) for 24 h inhibited the cell viability of osteocytes MLO-Y4 and promoted G0/G1 phase arrest and increased cell apoptosis. NaF treatment remarkably caused mitochondria damage, loss of MMP, ATP decrease, Cyto c release, and Bax/Bcl-2 ratio increase and elevated the activity of caspase-9 and caspase-3. Furthermore, NaF significantly upregulated the expressions of LC-3II, PINK1, and Parkin and increased autophagy flux and the accumulation of acidic vacuoles, while the p62 level was downregulated. In addition, NaF exposure triggered the production of intracellular ROS and mtROS and increased malondialdehyde (MDA); but superoxide dismutase (SOD) activity and glutathione (GSH) content were decreased. The scavenger N-acetyl-L-cysteine (NAC) significantly reversed NaF-induced apoptosis and mitophagy, suggesting that ROS is responsible for the mitochondrial-mediated apoptosis and mitophagy induced by NaF exposure. These findings provide in vitro evidence that apoptosis and mitophagy are cellular mechanisms for the toxic effect of fluoride on osteocytes, thereby suggesting the potential role of osteocytes in skeletal and dental fluorosis.

Role of Glycolysis/Gluconeogenesis and HIF-1 Signaling Pathways in Rats with Dental Fluorosis Integrated Proteomics and Metabolomics Analysis.

Fluoride is widely distributed, and excessive intake will lead to dental fluorosis. In this study, six offspring rats administrated 100 mg/L sodium fluoride were defined as the dental fluorosis group, and eight offspring rats who received pure water were defined as the control group. Differentially expressed proteins and metabolites extracted from peripheral blood were identified using the liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry, with the judgment criteria of fold change >1.2 or <0.83 and p < 0.05. A coexpression enrichment analysis using OmicsBean was conducted on the identified proteins and metabolites, and a false discovery rate (FDR) < 0.05 was considered significant. Human Protein Atlas was used to determine the subcellular distribution of hub proteins. The Gene Cards was used to verify results. A total of 123 up-regulated and 46 down-regulated proteins, and 12 up-regulated and 2 down-regulated metabolites were identified. The significant coexpression pathways were the HIF-1 (FDR = 1.86 × 10−3) and glycolysis/gluconeogenesis (FDR = 1.14 × 10−10). The results of validation analysis showed the proteins related to fluorine were mainly enriched in the cytoplasm and extrinsic component of the cytoplasmic side of the plasma membrane. The HIF-1 pathway (FDR = 1.01 × 10−7) was also identified. Therefore, the HIF-1 and glycolysis/gluconeogenesis pathways were significantly correlated with dental fluorosis.

Progress of Signaling Pathways, Stress Pathways and Epigenetics in the Pathogenesis of Skeletal Fluorosis.

Fluorine is widely dispersed in nature and has multiple physiological functions. Although it is usually regarded as an essential trace element for humans, this view is not held universally. Moreover, chronic fluorosis, mainly characterized by skeletal fluorosis, can be induced by long-term excessive fluoride consumption. High concentrations of fluoride in the environment and drinking water are major causes, and patients with skeletal fluorosis mainly present with symptoms of osteosclerosis, osteochondrosis, osteoporosis, and degenerative changes in joint cartilage. Etiologies for skeletal fluorosis have been established, but the specific pathogenesis is inconclusive. Currently, active osteogenesis and accelerated bone turnover are considered critical processes in the progression of skeletal fluorosis. In recent years, researchers have conducted extensive studies in fields of signaling pathways (Wnt/ß-catenin, Notch, PI3K/Akt/mTOR, Hedgehog, parathyroid hormone, and insulin signaling pathways), stress pathways (oxidative stress and endoplasmic reticulum stress pathways), epigenetics (DNA methylation and non-coding RNAs), and their inter-regulation involved in the pathogenesis of skeletal fluorosis. In this review, we summarised and analyzed relevant findings to provide a basis for comprehensive understandings of the pathogenesis of skeletal fluorosis and hopefully propose more effective prevention and therapeutic strategies.

Elemental Status and Lipid Peroxidation in the Blood of Children with Endemic Fluorosis.

The study aimed to assess the levels of trace elements, minerals, and toxic elements as well as lipid peroxidation biomarkers (lipid acyl hydroperoxides, 2-thiobarbituric acid reactive substances (TBARS)) in the blood of children with chronic fluorosis from endemic fluorosis areas (Sosnivka village, Lviv region, western Ukraine). The results were compared with healthy children from Staryi Sambir (Lviv region, western Ukraine), whose drinking water contained permissible levels (< 1 ppm) of fluoride. Thirty-one children from the Sosnivka village in the Lviv region, including 16 females and 15 males aged 7-10 years, with clinically diagnosed fluorosis, were recruited for the study. The children had been exposed to fluoride (> 1.5 ppm) through drinking water for more than 5 years. In the blood, eight macro- and microelements (calcium, zinc, potassium, iron, copper, selenium, manganese, chromium), five additional elements (sulfur, bromine, chlorine, nickel, strontium), and four toxic elements (lead, mercury, cadmium, mercury) were assessed with the X-ray fluorescence method. The results of our study demonstrated a 14-fold decrease in the copper level, a 2.5-fold decrease in the calcium and zinc levels, and a 2-fold decrease in the selenium level in the blood of children with chronic fluorosis compared with the healthy children from the non-fluorosis area. In turn, a 1.7- and 1.4-fold increase in the strontium and lead content, respectively, was noted. The sulfur, chlorine, potassium, calcium, copper, zinc, and selenium levels in the blood samples of children with chronic fluorosis were lower than the reference value. The children had higher blood TBARS levels, while the acyl hydroperoxide levels were non-significantly increased in comparison with healthy children living in the non-fluorosis area. Additionally, the bromine level was correlated positively with the selenium level and acyl hydroperoxides. However, more studies are needed to clarify the relationship between blood mineral status, oxidative stress biomarkers, and chronic fluorosis.

Protections against toxicity in the brains of rat with chronic fluorosis and primary neurons exposed to fluoride by resveratrol involves nicotinic acetylcholine receptors.

Protection of Resveratrol (RSV) against the neurotoxicity induced by high level of fluoride was investigated. Sprague-Dawley (SD) rats and their offspring, as well as cultures of primary neurons were divided randomly into four groups: untreated (control); treated with 50 mg RSV/kg/ (once daily by gavage) or (20 M in the cultured medium); exposed to 50 ppm F- in drinking water or 4 mmol/l in the cultured medium; and exposed to fluoride then RSV as above. The adult rats were treated for 7 months and the offspring sacrificed at 28 days of age; the cultured neurons for 48 h. For general characterization, dental fluorosis was assessed and the fluoride content of the urine measured (by fluoride-electrode) in the rates and the survival of cultured neurons monitored with the CCK-8 test. The spatial learning and memory of rats were assessed with the Morris water maze test. The levels of α7 and α4 nicotinic acetylcholine receptors (nAChRs) were quantified by Western blotting; and the activities of superoxide dismutase (SOD) and catalase (CAT), and the levels of malondialdehyde (MDA) and H2O2 assayed biochemically. The results showed that chronic fluorosis resulted in the impaired learning and memory in rats and their offspring, and more oxidative stress in both rat brains and cultured neurons, which may be associated the lower levels of α7 and α4 nAChR subunits. Interestingly, RSV attenuated all of these toxic effects by fluorosis, indicating that protection against the neurotoxicity of fluoride by RSV might be in mechanism involved enhancing the expressions of these nAChRs.

Fluoride exposure alters Ca2+ signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca2+ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca2+ stores and store-operated Ca2+ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca2+ homeostasis or increase the expression of ER stress-associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca2+ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump during Ca2+ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Fluoride resistance in fibroblasts is conferred via reduced susceptibility to oxidative stress and apoptosis.

Chronic fluoride exposure from drinking water may result in endemic fluorosis. To better understand the mechanisms by which some people are resistant to fluorosis, here we investigated the effect of treatment with NaF (sodium fluoride) on production of reactive oxygen species (ROS), morphological changes in mitochondria, the mRNA expression of Fas ligand (Fas-L), and the protein expression of cleaved caspase-3 in regular L-929 cells and fluoride-resistant (FR) L-929 cells. While morphological changes indicative of apoptosis and a network of fragmented mitochondria were observed in regular L-929 cells after NaF treatment, there were no morphological changes in FR L-929 cells after NaF treatment. Treatment with 10 mm NaF induced a significant difference in the production of ROS, triggered the expression of cleaved caspase-3, and upregulated the mRNA expression of Fas-L in regular L-929 cells. However, there was no significant production of ROS in FR L-929 cells. Additionally, cleaved caspase-3 and upregulated Fas-L were not detected in FR L-929 cells. These results suggest that FR fibroblasts are resistant to oxidative stress and apoptosis induced by fluoride.

Condition of mineralized tooth tissue in a population of 15-year-old adolescents living in a region of Ukraine with slightly exceeded fluorine concentration in the water.

INTRODUCTION: Fluorine is a common element in nature; however, the difference between a beneficial dose and a toxic dose for the organisms is small. The main source of fluoride for humans is water in addition to food. OBJECTIVE: The aim of this study was to estimate the degree of severity of pathological changes, namely, caries or fluorosis, in the mineralized tooth tissue of 15-year-old adolescents with respect to their hygienic and nutritional habits, and the content of fluorine in drinking water, soil and plant products. MATERIAL AND METHODS: 100 adolescents aged 15 from schools in Sosnilka, L'viv region, were examined. The condition of mineralized tooth tissue was evaluated by the caries severity, expressed by the mean number D3MFT, caries frequency and value of the SIC index. Fluorine in plant material and soil were determined according to the PN-G-04543:1982 standard, and water according to the PN-EN ISO 10304 - 1: 2009+ AC: 2012 standard. RESULTS: Severity of caries disease expressed by the D3MFT number in the examined group of 15-year-olds was 3.39; in the group of girls - 3.08, and in the group of boys - 3.76. In the examined group, the average number of teeth with fluorosis was 7.59. Value of the SIC index among the examined population of students (n-31) was 6.26: 5.89 (n-18) for girls and 7.31 (n-13) for boys. Fluorine concentration in the water was 0.78 - 1.25(mg·dm-3). In the soil, it also did not vary across the sampled areas and amounted, on average, to 176 mg·dm-3. The biggest fluorine content noted in the dry mass of beetroots was 3.50 (mg F· kg), and the lowest - 3.34 (mg F· kg). CONCLUSIONS: Close to optimal fluorine content lowers caries severity and frequency of fluorosis. Optimal fluorine content in drinking water and food does not require additional diet supplementation.

Atorvastatin attenuates spinal cord injury by chronic fluorosis in rats.

The aim of the study was to explore the effect of atorvastatin on improvement of the function of the spinal cord in rats with chronic fluorosis. Sixty 3-month-old Wistar rats were separated randomly into three groups: normal group (N group), control group (C group) and atorvastatin group (A group). The Basso Beattie and Bresnahan scale and oblique board test showed that the rats in A group got higher score and better hind-limb motor function than C group. Immunohistochemistry and western blotting revealed that compared with N group, matrix metalloproteinase 9 (MMP-9) and p53 were highly expressed and myelin basic protein (MBP) was low expressed in spinal cord of C group. Meanwhile, MMP-9 and p53 expression were decreased and MBP was upregulated by atorvastatin compared with C group. In conclusion, the improvement of the function of the spinal cord in rats can be found when they were treated with atorvastatin.

Disrupted Iron Storage in Dental Fluorosis.

Enamel formation and quality are dependent on environmental conditions, including exposure to fluoride, which is a widespread natural element. Fluoride is routinely used to prevent caries. However, when absorbed in excess, fluoride may also lead to altered enamel structural properties associated with enamel gene expression modulations. As iron plays a determinant role in enamel quality, the aim of our study was to evaluate the iron metabolism in dental epithelial cells and forming enamel of mice exposed to fluoride, as well as its putative relation with enamel mechanical properties. Iron storage was investigated in dental epithelial cells with Perl's blue staining and secondary ion mass spectrometry imaging. Iron was mainly stored by maturation-stage ameloblasts involved in terminal enamel mineralization. Iron storage was drastically reduced by fluoride. Among the proteins involved in iron metabolism, ferritin heavy chain (Fth), in charge of iron storage, appeared as the preferential target of fluoride according to quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry analyses. Fluorotic enamel presented a decreased quantity of iron oxides attested by electron spin resonance technique, altered mechanical properties measured by nanoindentation, and ultrastructural defects analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. The in vivo functional role of Fth was illustrated with Fth+/- mice, which incorporated less iron into their dental epithelium and exhibited poor enamel quality. These data demonstrate that exposure to excessive fluoride decreases ameloblast iron storage, which contributes to the defective structural and mechanical properties in rodent fluorotic enamel. They raise the question of fluoride's effects on iron storage in other cells and organs that may contribute to its effects on population health.

Effects of drinking water fluorosis on L-type calcium channel of hippocampal neurons in mice.

The study aimed to investigate the effects of drinking water fluorosis on L-type calcium channels (LTCCs) in mouse hippocampal neurons. A total of 60 newly weaned ICR male mice were randomly divided into control, low fluoride and high fluoride groups. After 3 and 6 months of exposure to fluoride, the patch clamp technique was used to detect the peak and relative values (I/Imax), steady-state activation curve ratio (G/Gmax), decay time constant, and tail current time constant of LTCCs currents in hippocampal CA1 region of mouse brain slices. Fluoride greatly reduced the serum and urinary calcium concentrations in mice, and the chronic fluorosis has a greater impact than subchronic fluorosis. The peak value of LTCCs current in pyramidal neurons of hippocampal CA1 area was significant and increased with the prolonged exposure time. The relative values of current and steady-state coefficients were changed greatly. The decay and tail current time increased significantly. High fluorine concentration indicates great peak value and open time of LTCCs opening. LTCCs are sensitive to fluoride exposure. The activation voltage of calcium channels induced by fluoride exposure is decreased, the opening time of calcium channels is prolonged, and the calcium influx per unit time increased, thereby overloading calcium concentration in neurons and this may be an explanation for intracellular calcium overload caused by fluoride. The imbalance of calcium metabolism caused by fluorosis may be a pathogenesis of brain injury induced by fluoride. Furthermore, the risk of brain damage from low-fluorine exposure cannot be ignored.

The Effect of Chronic Fluorosis on Calcium Ions and CaMKIIα, and c-fos Expression in the Rat Hippocampus.

This study investigated neurotoxicity of chronic fluorosis in the rat hippocampus. Newly weaning, male, Sprague-Dawley (SD) rats were administered 15, 30, and 60 mg/L sodium fluoride (NaF) solution (fluorine ion concentration 8.25, 16.50, and 33.00 mg/L, respectively), and tap water, for 18 months. The neurotoxicological mechanism was examined with a focus on intracellular calcium overload. Results showed that as the fluoride concentration increased, calcium ion concentration [Ca2+], the expression of calcium/calmodulin-dependent protein kinase II α (CaMKIIα), and the expression of catus proto-oncogene protein c-fos (c-fos) all tend to increase. Compared to the control group, Ca2+, CaMKIIα, and c-fos significantly increased (P < 0.05) in the moderate-fluoride and the high-fluoride groups. These results indicate that Ca2+/CaMKIIα/c-fos channel signal may be the molecular mechanism of central nervous system damage caused by chronic fluoride intoxication. Moreover, elevated Ca2+ concentration in the hippocampus may be the initiating factor of neuronal apoptosis induced by fluoride.

Fluoride, Thyroid Hormone Derangements and its Correlation with Tooth Eruption Pattern Among the Pediatric Population from Endemic and Non-endemic Fluorosis Areas.

AIM: To comparatively evaluate the status of fluoride in the body with thyroid activity in the pediatric population of endemic fluorosis areas. The present study also attempted to elucidate whether any correlation exists between fluoride and thyroid hormone derangement with delayed tooth eruption. MATERIALS AND METHODS: A total of 400 pediatric subjects were included in the present study. All the patients were divided into two broad groups; groups A and B. Group A included 200 subjects who belonged to the endemic fluorosis area while Group B included remaining 200 subjects, who belonged to the fluorosis non-endemic area. Group B subjects were taken as control. Group A subjects were further divided into two study groups as follows: Group A1: 100 Pediatric subjects with dental fluorosis, and Group A 2: A total of one hundred pediatric subjects without dental fluorosis. Dean's index of fluorosis was calculated in all the patients. Blood samples were collected and were sent to a laboratory for assessment of thyroid hormone levels. All the results were subjected to statistical analysis by Statistical Package for the Social Sciences (SPSS) software. RESULTS: Mean thyroid stimulating hormone (TSH), water fluoride levels, urine fluoride levels and serum fluoride levels of subjects in group 1 were found to be significantly higher than that of subjects of group 2. Delayed tooth eruption was absent in subjects of group B while it was present in 100 subjects of group A. Thyroid hormone level derangement was seen in 54 percent subjects of group B, while it was seen in 67.5% subjects of group A. CONCLUSION: Positive correlation exists between fluorosis and thyroid functional activity. However; the tooth eruption pattern is independent up on the thyroid hormone derangement. CLINICAL SIGNIFICANCE: Delayed tooth eruption and alteration in thyroid hormone levels can occur in subjects of the endemic fluoride areas. Therefore, adequate measures should be taken for controlling such adverse effects.

Skeletal fluorosis in relation to drinking water in rural areas of West Azerbaijan, Iran.

Skeletal fluorosis resulting from high fluoride level in drinking water is a major public health problem. The present study evaluated the association between exposures to drinking water fluoride and skeletal fluorosis in 5 villages of Poldasht County, Iran. All the data and information on the prevalence of bone diseases were obtained from the Health Record Department, Poldasht Health Centre. To obtain the odds ratio of bone disease problem in different risk factors, when considering the cluster effect of rural area, logistic regression in a multilevel model was used. Results showed that skeletal fluorosis of people who live in areas with high fluoride concentration is 18.1% higher than that of individuals who live in areas with low fluoride concentration. Skeletal fluorosis (54.5%) was observed in the age group of 71 years and above, and was more commonly found in females than males. According to Unadjusted, individuals who consume ≤3 unit milk and dairy products per week have almost the same level of bone diseases as compared to those that consume more than 3 units. This study indicated that, skeletal fluorosis is a general health problem in these rural areas because the results revealed that high percentage of the studied population had symptoms of skeletal fluorosis.

Dental fluorosis, nutritional status, kidney damage, and thyroid function along with bone metabolic indicators in school-going children living in fluoride-affected hilly areas of Doda district, Jammu and Kashmir, India.

A case-control study was undertaken among the school children aged 8-15 years to know the presence and severity of dental fluorosis, nutrition and kidney status, and thyroid function along with bone metabolic indicators in Doda district situated at high altitude where drinking water was contaminated and heat stress. This study included 824 participants with an age of 8-15 years. The results of the study reviled that dental fluorosis was significantly higher in affected than control area children. Urinary fluoride was significantly higher (p < 0.05) in affected children as compared to the control area school children. Nutritional status of affected children was lower than control area children. The chronic kidney damage (CKD) was higher in affected than control school children. Thyroid function was affected more in affected than control area schools. Serum creatinine, total alkaline phosphatase, parathyroid hormone, 1, 25(OH)2 vitamin D, and osteocalcin were significantly higher in affected school children (p < 0.05) as compared to control school children, whereas there was no significant difference in triiodothyronine (T3), thyroxine (T4), and 25-OH vitamin D among the two groups. There was a significant decrease in thyroid-stimulating hormone (TSH) in the affected area school children compared to control. In conclusion, fluorotic area school children were more affected with dental fluorosis, kidney damage, along and some bone indicators as compared to control school children.

ClC-7/Ostm1 contribute to the ability of tea polyphenols to maintain bone homeostasis in C57BL/6 mice, protecting against fluorosis.

Epidemiological investigations indicate that certain ingredients in tea bricks can antagonize the adverse effects of fluoride. Tea polyphenols (TPs), the most bioactive ingredient in tea bricks, have been demonstrated to be potent bone-supporting agents. ClC­7 is known to be crucial for osteoclast (OC) bone resorption. Thus, in this study, we investigated the potential protective effects of TPs against fluorosis using a mouse model and explored the underlying mechanisms with particular focus on ClC­7. A total of 40, healthy, 3­week­old male C57BL/6 mice were randomly divided into 4 groups (n=10/group) by weight as follows: distilled water (control group), 100 mg/l fluoridated water (F group), water containing 10 g/l TPs (TP group) and water containing 100 mg/l fluoride and 10 g/l TPs (F + TP group). After 15 weeks, and after the mice were sacrificed, the long bones were removed and bone marrow-derived macrophages were cultured ex vivo in order to perform several experiments. OCs were identified and counted by tartrate­resistant acid phosphatase (TRAP) staining. The consumption of fluoride resulted in severe fluorosis and in an impaired OC function [impaired bone resorption, and a low mRNA expression of nuclear factor of activated T-cells 1 (NFATc1), ATPase H+ transporting V0 subunit D2 (ATP6v0d2) and osteopetrosis­associated transmembrane protein 1 (Ostm1)]. In the F + TP group, fluorosis was attenuated and OC function was restored, but not the high bone fluoride content. Compared with the F group, mature OCs in the F + TP group expressed higher mRNA levels of ClC­7 and Ostm1; the transportation and retaining of Cl­ was improved, as shown by the fluorescence intensity experiment. On the whole, our findings indicate that TPs mitigate fluorosis in C57BL/6 mice by regulating OC bone resorption. Fluoride inhibits OC resorption by inhibiting ClC­7 and Ostm1, whereas TPs attenuate this inhibitory effect of fluoride.

Proteomic analysis of liver in mice with different susceptibilities to fluorosis / Análise proteômica do fígado de camundongos com diferentes suscetibilidades à fluorose

Fluoride (F) is a potent anti-cariogenic element, but only an appropriate dose is effective to have therapeutic action, else systemic toxicity may be observed. Additionally, two factors, amount of F and time of exposure, drive its action. Surprisingly, the susceptibility to toxic effects of F is genetically determined. The present study identified the effects of F on the liver proteome of mice susceptible (A/J) or resistant (129P3/J) to the effects of F. Weanling male A/J (n=6) and 129P3/J mice (n=6) were housed in pairs and assigned to three groups given low-F diet and drinking water containing 0, 15 or 50 ppm F for 7 weeks. Liver proteome profiles were examined using nano-LC-ESI-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8). Difference in expression among the groups was determined using the PLGS software. In the control group (0 ppm F), most proteins with fold change were increased in A/J mice. Precisely the proteins related to energy flux and oxidative stress were quite significant in this context, suggesting the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress. Treatment with the lower F concentration provoked more pronounced alterations in fold change in liver proteins in comparison to the treatment with the higher F concentration. Strikingly, most of the proteins with fold change upon following 15 ppm F treatment, were increased in the A/J mice compared with their 129P3/J counterparts, thus suggesting attempt of the former to fight against the toxic effects of F. With respect to 50 ppm F, most proteins with fold change were decreased in the A/J mice compared with their 129P3/J counterparts, especially proteins related to oxidative stress and protein folding, which might be related to the higher susceptibility of the A/J animals to the deleterious effects of F. Our findings can provide new insights into the molecular mechanisms underlying genetic susceptibility to fluorosis by indicating key protein players which need to be better addressed in future experimental studies.(AU)

O Fluoreto (F) é um potente elemento anti-cariogênico, mas é somente efetivo terapeuticamente em uma dose apropriada. Por outro lado, doses acima das recomendadas levam a toxicidade sistêmica. Em adição, dois fatores decidem sua efetividade de ação: quantidade de F e tempo de exposição. A suscetibilidade aos efeitos tóxicos do F é determinada geneticamente. O presente estudo avaliou os efeitos do F no proteoma do fígado de camundongos suscetíveis (A/J) ou resistentes (129P3/J) aos efeitos do F. Camundongos machos desmamados A/J (n=6) e 129P3/J (n=6) foram alojados em pares e divididos em três grupos tratados com ração com baixo teor de F e água contendo 0, 15, ou 50 ppm de F por 7 semanas. Perfis proteômicos do fígado foram examinados usando nano-LC-ESI-MS/MS. A função de proteínas foi classificada pelo processamento biológico GO (Cluego v2.0.7 + Clupedia v1.0.8). A diferença de expressão entre os grupos foi determinada usando o software PLGS. No grupo controle (0 ppm F), a expressão da maioria das proteínas foi aumentada nos camundongos A/J e precisamente as proteínas relacionadas ao fluxo de energia e estresse oxidativo foram significativas neste contexto, sugerindo portanto, a alta sucetibilidade destes camundongos aos efeitos do F, já que a exposição também induz o estresse oxidativo. O tratamento com baixa concentração de F provocou alterações mais pronunciadas em proteínas do fígado comparado ao tratamento com alta concentração de F. Notadamente, a maioria das proteínas encontradas no fígado dos animais tratados com 15 ppm de F foi aumentada em camundongos A/J comparados aos camundongos 129P3/J, demonstrando portanto, uma tentativa dos A/J de neutralizar os efeitos tóxicos do F. Já nos animais tratados com 50 ppm de F, a maioria das proteínas foi diminuída nos camundongos comparados aos seus pares 129P3/J, especialmente proteínas relacionadas ao estresse oxidativo e enovelamento de proteínas, o que pode estar relacionado à alta suscetibilidade dos animais A/J aos efeitos deletérios do F. Nossos achados podem fornecer novos insights que podem contribuir para a interpretação os mecanismos moleculares relacionados à suscetibilidade genética à fluorose, indicando proteínas chaves que precisam ser melhor estudadas em estudos futuros.(AU)

Fluorosis increases the risk of postmenopausal osteoporosis by stimulating interferon γ.

Estrogen deficiency in postmenopausal women frequently activates osteoclasts (OC), accelerates bone resorption, and leads to osteoporosis (OP). Previous studies have demonstrated that interferon γ (IFNγ) could increase bone resorption and may be involved in postmenopausal OP. Fluorosis also increased the risk of fractures and dental fluorosis, and fluoride may enhance osteoclast formation and induce osteoclastic bone destruction in postmenopausal women, but the underlying mechanisms are as yet unknown. Here, we show that serum fluoride and IFNγ levels are negatively correlated with bone mineral density (BMD) in postmenopausal women residing in a fluorotic area. Estrogen suppresses IFNγ, which is elevated by fluoride, playing a pivotal role in triggering bone loss in estrogen-deficient conditions. In vitro, IFNγ is inhibited by estrogen treatment and increased by fluoride in Raw264.7 cell, an osteoclast progenitor cell line. In ovariectomized (Ovx) mice, estrogen loss and IFNγ promote OC activation and subsequent bone loss in vivo. However, IFNγ deficiency prevents bone loss in Ovx mice even in fluoride conditions. Interestingly, fluoride fails to increase IFNγ expression in estrogen receptor α (ERα)-deficient conditions, but not in ERß-deficient conditions. These findings demonstrate that fluorosis increases the bone loss in postmenopausal OP through an IFNγ-dependent mechanism. IFNγ signaling activates OC and aggravates estrogen deficiency inducing OP. Thus, stimulation of IFNγ production is a pivotal ''upstream'' mechanism by which fluoride promotes bone loss. Suppression of IFNγ levels may constitute a therapeutic approach for preventing bone loss.

Fluoride induced endoplasmic reticulum stress and calcium overload in ameloblasts.

OBJECTIVE: The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis. METHODS: We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis. RESULT: High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride. CONCLUSIONS: These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis.