Biological effects of sodium fluoride varnishes used in remineralisation of enamel: An in vitro study.

AIM: During the last three decades, fluoride varnishes have been recognised as effective strategies for caries prevention in the young-child population and have contributed to a decrease in its prevalence worldwide. The present study aimed to assess in vitro the level of cytotoxicity and genotoxicity in human primary pulp fibroblasts (DPFs) of two NaF varnishes. MATERIALS AND METHODS: Four experimental assays were carried out (MTS, Mitotracker® system [mitochondrial function and morphology], Live/Dead®, and Comet) to assess the morphology, viability, and genotoxicity of two NaF varnishes (Duraphat® and Clinpro White®, both at two different concentrations). The essays were conducted on cultured pulp fibroblasts, grouped in four experimental and two control groups. Collected data were analysed by one-way ANOVA followed by the post hoc Bonferroni test. RESULTS: Some morphological changes of DPFs could be detected after the NaFVs stimulation. Most DPFs incubated in Duraphat (22.6 mg/L) maintained their morphological characteristics, except for a small decrease in cell size and shorter cytoplasmic projections (filopodia); DPFs treated with Clinpro White Varnish (22.6 mg/L) presented a morphology and size similar to the control group. DPFs exposed to Duraphat (113 mg/L) exhibited significant morphological alterations with considerable cell size increases and DPFs treated with Clinpro White Varnish (113 mg/L) showed a slight cell size increase without noticeable morphological anomalies. The Duraphat (22.6 mg/L) and Clinpro White Varnish (22.6 mg/L) groups promoted 31% and 35% cell proliferation, respectively, whereas DPFs proliferation with Duraphat (113 mg/L) decreased up to 59%, and cell proliferation with Clinpro White Varnish (113 mg/L) was similar to that of control. CONCLUSION: All tested varnishes induced changes in the fibroblastic mitochondria. In general, Duraphat was less biocompatible and caused a change in the number of mitochondria compared to Clinpro White Varnish.

The effect of reduced glutathione on the toxicity of silver diamine fluoride in rat pulpal cells.

INTRODUCTION: Due to its ability to arrest untreated dental caries, silver diamine fluoride (SDF) has been advocated for indirect pulp capping procedures. However, the high concentrations of silver and fluoride in SDF raise concerns about its biocompatibility to pulpal tissues. OBJECTIVES: This study aimed to investigate the effect of SDF on the viability, alkaline phosphatase (ALP) activity, and morphology of pulpal-like cells (RPC-C2A) and to evaluate the influence of reduced glutathione (GSH) on SDF-induced cytotoxicity and deposit formation on dentin. METHODOLOGY: The cytotoxicity of diluted 38% SDF solutions (10-4 and 10-5), with or without the addition of 5 mM or 50 mM GSH, was evaluated at 6 and 24 hours. Cell viability was detected using WST-8 and the effect on ALP activity was performed using an ALP assay kit. Cell morphology was observed using a phase-contrast microscope. Scanning electron microscopy analysis was conducted to evaluate the effect of GSH incorporation or conditioning on SDF-induced deposit formation on dentin discs. Cytotoxicity data were analyzed by two-way analysis of variance (ANOVA) and Tukey post hoc tests (p<0.05). RESULTS: There were significant differences between the groups. The results demonstrated that all tested SDF dilutions caused a remarkable cytotoxic effect, while the addition of GSH prevented SDF-induced damage at 6-hour exposure time in the higher dilution of SDF. Dentin treated with plain SDF or GSH-incorporated SDF solution showed deposit formation with occluded dentinal tubules, unlike the other groups. CONCLUSION: SDF severely disturbed the viability, mineralization-ability, and morphology of pulpal-like cells, while controlled concentrations of GSH had a short-term protective effect against SDF-induced damage. GSH showed an inhibitory effect on SDF-induced dentinal deposit formation. Further research is warranted to evaluate the effect of GSH on caries-arresting, anti-hypersensitivity, and antibacterial functions of SDF.

Temporal Exposure of Dermal Fibroblasts to Silver Diamine Fluoride and Fluorine NMR Quantitation.

OBJECTIVE: Silver diamine fluoride has been advocated as a caries arresting material for Early Childhood Caries (ECC) and has received considerable public attention as the "silver bullet". However, cytotoxicity tests on the current concentrations of Silver Diamine Fluoride (SDF) to soft tissue have not been thoroughly assessed and analyzed at selected time intervals. The level of fluoride that is present within human cells has yet to be quantified. Preliminary SDF toxicity studies in our lab determined exposures of Dermal Fibroblasts to 0.03% SDF for 18 hours resulted in 100% cytotoxicity and complete monolayer loss. Endpoint titration of SDF determined that morphologic cytotoxic effects were ameliorated at input SDF levels lower than 0.002%. Because of the small culture sample volumes, we were unable to effectively assay fluoride concentrations using commercially available assays. In this study we attempted to assess fluoride levels in culture supernatants in a temporal fashion using quantitative Nuclear Magnetic Resonance (NMR). STUDY DESIGN: Dermal Fibroblast (DF) cells were grown in 24 well cluster plates fitted with 0.4 micron TranswellTM inserts to confluency in 0.9mL of DF culture media. Then the DF cells were challenged with 0.1 mL of SDF in sterile water in the Transwell chamber to achieve a final concentration of 0.03% SDF. Cultures were reincubated for 30 minutes, 1, 2, 4 and 8 hours. At the selected time points Transwell inserts were removed. SDF culture media was removed and replaced with fresh media and allowed to re-incubate up to 8 hours. Harvested SDF culture media was centrifuged at 15,000 x g to remove any resulting SDF precipitates and supernatants were harvested and stored at -70°C for fluoride assay. After 8 hours, media was aspirated from all wells and DF cells were fixed and then stained with methylene blue and assessed for cytotoxicity. Harvested supernatants were assessed for fluoride content. While SDF is soluble in pure water, it precipitates instantly in the presence of other media constituents and 0.85% saline. Transwells inserts capture the precipitate but allow soluble SDF and constituents pass through to the cell monolayer. NMR was used to assess SDF (fluoride) prepared in water, in DF media or in normal saline at the same concentrations used in the DF cell studies. The 19F NMR spectra were acquired at 25 °C on an Agilent DD2 500 MHz spectrometer equipped with a 5mm HFX z gradient probe operating at 470.3 MHz for fluorine. For quantitative measurements, all spectra were collected with 64 scans and a delay of 5 seconds. The spectrum width is 220 ppm with offset at resonance of -110 ppm. The processing and analyzing were done by MNOVA. The dataset consists 45371 complex points and is zero-filled to the size of 128k points after applying 5Hz exponential line broadening. The 19F chemical shift was referenced indirectly based on proton chemical shift, which was referenced with respect to the water proton signal of 4.75 ppm at 25°C. RESULTS: Visible DF cell morphology changes begin to appear as early as 1 hour exposure to 0.03% SDF in Transwells and continue with degradation of cell morphology through 8 hours exposure at which point 100% of the cell monolayer is lost. The 8 hour image shows complete cell loss which is consistent with earlier studies using 24 hour exposures at 0.03% concentration. Note that the actual concentration of SDF affecting cell viability is shown in this study to be far lower than the 0.03% input because of the aggregate precipitation captured with the Transwell inserts. In this study, our NMR fluoride assessments showed that only 6-12 % of the input SDF fluoride reaches the lower cell chamber. CONCLUSIONS: Considering that the SDF reagent is applied orally at ~40%, these results warrant more refined testing to identify true lower limit of toxicity end points of SDF. SDF should be utilized only by trained professionals and never contact soft tissue. NMR may be utilized to determine relative amounts of fluoride both in cell culture media and within fluoride exposed cells.

The effect of reduced glutathione on the toxicity of silver diamine fluoride in rat pulpal cells

Abstract Introduction Due to its ability to arrest untreated dental caries, silver diamine fluoride (SDF) has been advocated for indirect pulp capping procedures. However, the high concentrations of silver and fluoride in SDF raise concerns about its biocompatibility to pulpal tissues. Objectives This study aimed to investigate the effect of SDF on the viability, alkaline phosphatase (ALP) activity, and morphology of pulpal-like cells (RPC-C2A) and to evaluate the influence of reduced glutathione (GSH) on SDF-induced cytotoxicity and deposit formation on dentin. Methodology The cytotoxicity of diluted 38% SDF solutions (10-4 and 10-5), with or without the addition of 5 mM or 50 mM GSH, was evaluated at 6 and 24 hours. Cell viability was detected using WST-8 and the effect on ALP activity was performed using an ALP assay kit. Cell morphology was observed using a phase-contrast microscope. Scanning electron microscopy analysis was conducted to evaluate the effect of GSH incorporation or conditioning on SDF-induced deposit formation on dentin discs. Cytotoxicity data were analyzed by two-way analysis of variance (ANOVA) and Tukey post hoc tests (p<0.05). Results There were significant differences between the groups. The results demonstrated that all tested SDF dilutions caused a remarkable cytotoxic effect, while the addition of GSH prevented SDF-induced damage at 6-hour exposure time in the higher dilution of SDF. Dentin treated with plain SDF or GSH-incorporated SDF solution showed deposit formation with occluded dentinal tubules, unlike the other groups. Conclusion SDF severely disturbed the viability, mineralization-ability, and morphology of pulpal-like cells, while controlled concentrations of GSH had a short-term protective effect against SDF-induced damage. GSH showed an inhibitory effect on SDF-induced dentinal deposit formation. Further research is warranted to evaluate the effect of GSH on caries-arresting, anti-hypersensitivity, and antibacterial functions of SDF.

The enamel remineralization potential of fluoride varnishes containing arginine.

OBJECTIVES: To examine the effect of incorporating arginine in a 5% sodium fluoride (NaF) varnish on its remineralization potential and HGF-1 cytotoxicity. METHODS: Experimental varnishes were prepared using two arginine variants: l-arginine (Arg) and l-arginine monohydrochloride (Arg.HCl) at 2%, 4%, & 8% w/v. with control 5% NaF varnish. Sound enamel specimens with varnish treatments were subjected to a 4-day demineralization assay and surface roughness analysis using AFM (n = 3). Enamel specimens with artificial incipient caries-like lesions were subjected to a remineralization assay (n = 6) and an 8-day pH-cycling (n = 3). For remineralization assay, enamel F/Arg-uptake and Ca/PO43-i-content were estimated. During pH-cycling, EDX mapping for Ca/F-content and mineral density using micro-CT were assessed. The HGF-1 cytotoxicity of the varnishes was examined using MTT/CCK-8 assay. RESULTS: The Ca-content/F-uptake of the 2% Arg-NaF varnish treated specimens were significantly higher than other varnishes (p < 0.05). The 2% Arg-NaF had significantly higher remineralization potential than NaF (p < 0.001), suggesting Arg-Ca-F complex formation post-varnish application. The 8% Arg-NaF exhibited significantly more pronounced cytotoxicity to HGF-1 cells than 2% Arg-NaF (p < 0.001) and NaF varnish. CONCLUSIONS: Incorporation of 2% Arg in NaF varnish enhanced the enamel remineralization potential of NaF varnish; while 8% Arg in NaF varnish was cytotoxic to HGF-1. CLINICAL SIGNIFICANCE: A professionally deliverable l-arginine-enriched NaF varnish for caries prevention addresses a global public health priority. The synergism between l-arginine and NaF varnish counters the limited antimicrobial effects of fluorides and supports acquisition of a balanced oral microbiome. The combined arginine-fluoride varnish will provide the much-needed ecological-based approach for caries prevention.

Fluoride in Drinking Water and Skeletal Fluorosis: a Review of the Global Impact.

When safe and adequate exposure of an essential trace element is exceeded it becomes potentially toxic. Fluoride is one classic example of such a double edged sword which both plays a fundamental role in the normal growth and development of the body for example the consumption of levels between 0.5-1.0 ppm via drinking water is beneficial for prevention of dental caries but its excessive consumption leads to development of fluorosis. PURPOSE OF REVIEW: The abundance of fluorine in the environment as well as in drinking water sources are the major contributors to fluorosis. It is a serious public health concern as it is a noteworthy medical problem in 24 nations including India yet the threat of fluorosis has not been rooted out. The review focuses on recent findings related to skeletal fluorosis and role of oxidative stress in its development. The fluoride mitigation strategies adopted in recent years are also discussed. RECENT FINDINGS BASED ON CASE STUDIES: Recent findings revealed that consumption of fluoride at concentrations of 1.5 ppm is majorly responsible for skeletal fluorosis. The sampling from rural areas showed that 80% villages are having fluoride concentrations more than the WHO permissible limits and people residing in such areas are affected by the skeletal fluorosis and also in the regions of Africa and Asia endemic fluorosis have been accounted in the majority of the region affecting approximately 100 million people. Various mitigation programmes and strategies have been conducted all over the world using defluoridation. Fluorosis is a slow and progressive malady affecting our body and a serious concern to be taken into consideration and to be dealt with effectively. The fluoride toxicity although reversible, is a slow process and the side effects lack treatment options. The treatment options available are either not approachable or affordable in the rural areas commonly suffering from the fluoride toxicity. No specific treatments are available to date to treat skeletal fluorosis affectively; therefore, prevention is one of most safest and best approach to fight fluorosis. The current review lays emphasis on the skeletal fluorosis and its prevalence in recent years. It also includes the recent findings as well as the current strategies related to combat skeletal fluorosis and provides findings that might be helpful to promote the research in the field of effective treatment for fluorosis as well as development of easy and affordable methods of fluoride removal from water.

Sodium fluoride disrupts testosterone biosynthesis by affecting the steroidogenic pathway in TM3 Leydig cells.

Fluorine is an essential trace element to which humans and animals are exposed through water, food, air and products used for dental health. Numerous studies have reported the detrimental effects of fluoride on testicular function and fertility; however, the underlying mechanisms of testosterone biosynthesis remain unclear. In this study, Leydig cells, the primary cells responsible for the production and regulation of steroid hormones in the testis, were used to elicit effects of sodium fluoride on the steroidogenic pathway. Leydig cells were treated with 0, 0.1, 1, 10 and 100 mg/L sodium fluoride for 24 h, respectively. The result of the study showed that sodium fluoride significantly decreased cell viability and cell proliferation, increased cell cytotoxicity and decreased the amounts of testosterone and 3',5'-cyclic adenosine monophosphate levels in a concentration-dependent manner. Also, these results indicated that NaF suppressed the expression of steroidogenic genes (steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3ß-hydroxy dehydrogenase type I and 17ß-hydroxy dehydrogenase type III) and proteins (luteinizing hormone receptor, cholesterol side-chain cleavage enzyme, 3ß-hydroxy dehydrogenase), by changing the mRNA expression levels of the transcription factors (steroidogenic factor-1, GATA binding protein-4, nerve growth factor IB and nuclear receptor subfamily 0 group B member 1).

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.

Enteric innervation combined with proteomics for the evaluation of the effects of chronic fluoride exposure on the duodenum of rats.

Ingested fluoride (F) is absorbed mainly in the small intestine, which is controlled by the Enteric Nervous System (ENS). Although important intestinal symptomatology has been described after excessive F exposure, there have been no studies reporting the effects of F on the ENS. In this study, the effects of chronic F exposure were evaluated on the duodenums of rats through proteomic and morphological analyses. Concentrations of 0, 10, or 50 ppm of F were applied to the drinking water for 30 days. Immunofluorescence techniques were performed in the myenteric plexus of the duodenum to detect HuC/D, neuronal nitric oxide (nNOS), vasoactive intestinal peptide (VIP), calcitonin gene related peptide (CGRP), and substance P (SP). The 50 ppm F group presented a significant decrease in the density of nNOS-IR neurons. Significant morphological alterations were also observed in HUC/D-IR and nNOS-IR neurons; VIP-IR, CGRP-IR, and SP-IR varicosities for both groups (10 and 50 ppm F). Proteomic analysis of the duodenum demonstrated alterations in the expression of several proteins, especially those related to important biological processes, such as protein polymerization, which helps to explain the downregulation of many proteins upon exposure to 50 ppm of F.

Fluoride and arsenic exposure affects spatial memory and activates the ERK/CREB signaling pathway in offspring rats.

Fluoride and arsenic are inorganic contaminants that occur in the natural environment. Chronic fluoride and/or arsenic exposure can induce developmental neurotoxicity and negatively influence intelligence in children, although the underlying molecular mechanisms are poorly understood. This study explored the effects of fluoride and arsenic exposure in drinking water on spatial learning, memory and key protein expression in the ERK/CREB signaling pathway in hippocampal and cerebral cortex tissue in rat offspring. Pregnant rats were divided into four groups. Control rats drank tap water, while rats in the three exposure groups drank water with sodium fluoride (100mg/L), sodium arsenite (75mg/L), and a sodium fluoride (100mg/L) and sodium arsenite (75mg/L) combination during gestation and lactation. After weaning, rat pups drank the same solution as their mothers. Spatial learning and memory ability of pups at postnatal day 21 (PND21) and postnatal day 42 (PND42) were measured using a Morris water maze. ERK, phospho-ERK (p-ERK), CREB and phospho-CREB (p-CREB) protein expression in the hippocampus and cerebral cortex was detected using Western blot. Compared with the control pups, escape latencies increased in PND42 pups exposed to arsenic and co-exposed to fluoride and arsenic, and the short-term and long-term spatial memory ability declined in pups exposed to fluoride and arsenic, both alone and in combination. Compared with controls, ERK and p-ERK levels decreased in the hippocampus and cerebral cortex in pups exposed to combined fluoride and arsenic. CREB protein expression in the cerebral cortex decreased in pups exposed to fluoride, arsenic, and the fluoride and arsenic combination. p-CREB protein expression in both the hippocampus and cerebral cortex was decreased in pups exposed to fluoride and arsenic in combination compared to the control group. There were negative correlation between the proteins expression and escape latency periods in pups. These data indicate that exposure to fluoride and arsenic in early life stage changes ERK, p-ERK, CREB and p-CREB protein expression in the hippocampus and cerebral cortex of rat offspring at PND21 and PND 42, which may contribute to impaired neurodevelopment following exposure.

Sodium fluoride induces hypertension and cardiac complications through generation of reactive oxygen species and activation of nuclear factor kappa beta.

Human exposure to sodium fluoride through its daily usage is almost inevitable. Cardiovascular and renal dysfunction has been associated with fluoride toxicity. Therefore, this study investigated the mechanism of action of sodium fluoride (NaF) induced hypertension and cardiovascular complications Forty male albino rats of an average of 10 rats per group were used. Group A received clean tap water. Toxicity was induced in Group B to D by administering graded doses of NaF through drinking water ad libitum for 10 days at 150 ppm, 300 ppm, and 600 ppm concentration respectively. Following administration of NaF, there was significant increase in systolic pressure, diastolic pressure and mean arterial pressure. Markers of oxidative stress; malondialdehyde, hydrogen peroxide, advance oxidation protein products, and protein carbonyl were significantly increased in dose-dependent pattern in the cardiac and renal tissues of rats together with significant decrease in the GST activity in NaF-treated rats compared to the control. Also serum markers of inflammation, cardiac, and renal damage including myeloperoxidase, xanthine oxidase, blood urea nitrogen, creatinine, Lactate dehydrogenase (LDH), and Creatinine kinase myocardial band (CK-MB) significantly increased indicating induction of oxidative stress, renal, and cardiac damage after exposure. Histopathology of the kidney and heart revealed aberrations in the histological architecture in NaF-treated rats. Also, immunohistochemistry showed higher expression of nuclear factor kappa beta (NF-kB) in the cardiac and renal tissues of rats administered NaF. Combining all, these results indicate NaF-induced hypertension through generation of reactive oxygen species and activation of renal and cardiac NF-kB expressions. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1089-1101, 2017.

The effect of fluoride on the structure, function, and proteome of a renal epithelial cell monolayer.

High concentrations of fluoride in the body may cause toxic effects. Here, we investigated the effects of fluoride on the structure, function, and proteome of a cortical collecting duct epithelium in vitro. Kidney tubule cells (M-1) were chosen because the concentration of fluoride in the kidney is 4-5-fold higher than that in plasma. Mouse M-1 cell monolayers were incubated in fluoride-containing media, and the amiloride-sensitive short-circuit current and transepithelial resistance were measured. The Young's modulus of the epithelium was determined using atomic force microscopy, and the effect of fluoride on epithelial structure was assessed using scanning and transmission electron microscopy, and immunofluorescence. Differences in the expression of membrane proteins were evaluated using proteomics and bioinformatics. Fluoride exposure reduced both transepithelial Na+ transport and resistance. The IC50 for fluoride was ∼300 µM for both effects, and the half-times for the decays of ion transport and resistance were 8.4 h and 3.6 days, respectively. Fluoride treatment did not affect the sensitivity of Na+ transport to amiloride. The Young's modulus of the epithelium was also unaffected by fluoride; however, the functional effects of fluoride were accompanied by marked structural effects. Proteomic analysis revealed changes in expression of a number of proteins, and particularly mitochondrial proteins. Treatment with fluoride had profound effects on the structure, function and proteome of a model cortical collecting duct epithelium. Significantly, however, these effects were produced only at concentrations considerably higher than those likely to be encountered in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1455-1467, 2017.

Grading and quantification of dental fluorosis in zebrafish larva.

OBJECTIVE: The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. MATERIALS AND METHODS: After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. RESULTS: With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. CONCLUSIONS: Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis.

Impact of early developmental fluoride exposure on the peripheral pain sensitivity in mice.

Consumption of high concentration of fluoride in the drinking water would cause the fluorosis and chronic pain. Similar pain syndrome appeared in the patients in fluoride therapy of osteoporotic. The aim of the current study was to examine whether exposing immature mice to fluoride would modify the peripheral pain sensitivity or even cause a pain syndrome. We gave developmental fluoride exposure to mice in different concentration (0mg/L, 50mg/L and 100mg/L) and evaluated their basal pain threshold. Von Frey hair test, hot plate test and formalin test were conducted to examine the mechanical, thermal nociceptive threshold and inflammatory pain, respectively. In addition, the expression of hippocampal brain-derived neurotrophic factor (BDNF) was also evaluated by Western blotting. Hyperalgesia in fluoride exposure mice was exhibited in the Von Frey hair test, hot plate test and formalin test. Meanwhile, the expression of BDNF was significantly higher than that of control group. The results suggest that early developmental fluoride exposure may lower the basal pain threshold and be associated with the increasing of BDNF expression in hippocampus.

Vitamin A deficiency: An oxidative stress marker in sodium fluoride (NaF) induced oxidative damage in developing rat brain.

Fluoride induced oxidative stress through depletion in levels of various anti-oxidants such as glutathione, superoxide dismutase (SOD), fat soluble vitamins (D and E) with increased levels of lipid peroxidation (LPO) and fluoride aggravate the damage in rodents as well as in humans. Vitamins A, a fat soluble vitamin possess antioxidant property which plays a significant role in scavenging the free radicals species similar to vitamin D and E. Vitamin A is involved in neural tissue development and plasticity. The growing evidence about vitamin A being antioxidant in different biological reactions formed the basis to determine the effect of fluoride on its levels. The present study was conducted in Wistar rat pups. The pregnant wistar rats were dosed with 20 ppm sodium fluoride (NaF) from day one of pregnancy till the pups were aged day 30. The serum was collected from developing rat pups on regular intervals (14th, 21st, 30th day) and vitamin A levels were analyzed by High performance liquid chromatography (HPLC). Body weights, Behavioural studies and spectrophotometric estimation of SOD, LPO in brain lysates were also performed. The results showed significant decrease (p<0.001) in vitamin A in fluoride induced samples in comparison to the control samples suggesting that decreased levels of vitamin A can be used as another marker in fluoride induced toxicity studies.

The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Excessive fluoride reduces Foxo1 expression in dental epithelial cells of the rat incisor.

Enamel fluorosis is characterized by hypomineralization, and forkhead box O1 (Foxo1) is essential for mouse enamel biomineralization. This study investigated the effect of fluoride on Foxo1 expression and its implications for enamel fluorosis. Mandibular incisors were extracted from Sprague Dawley rats treated for 3 months with water containing 0, 50, or 100 p.p.m. F⁻. Immunohistochemistry was used to localize and quantify FOXO1 expression in dental epithelial layer cells of the incisors. The effect of fluoride on expression of Foxo1, kallikrein-4 (Klk4), and amelotin (Amtn) mRNAs was analyzed by real-time RT-PCR, and western blotting was used to measure total and nuclear FOXO1 protein levels in mature dental epithelial cells. The results revealed that nuclear FOXO1 was mainly localized in the transition and the mature ameloblasts and exhibited weaker expression in the rats exposed to fluoride. In addition to the reduced levels of Foxo1, Klk4, and AmtnmRNAs, the protein levels of total and nuclearFOXO1 were decreased in the mature dental epithelial cells exposed to fluoride. Thus, excessive fluoride may have an effect on the expression levels of Foxo1 in dental epithelial cells and thereby affect hypomineralization of the enamel during fluorosis.

Histopathological changes of renal tissue following sodium fluoride administration in two consecutive generations of mice. Correlation with the urinary elimination of fluoride.

The present study was designed to investigate the toxic effects (evaluated as histopathological changes) of sodium fluoride on the kidney in two consecutive generations of NMRI mice. An attempt to correlate the toxicity with the urinary elimination of fluoride has been made, as urinary fluoride excretion has been widely used as an indicator of fluoride intake and exposure. Six mixed (males and females) animal groups have been constituted by dividing the populations of mice derived from pregnant females (named "mothers" 0.5 mg sodium fluoride) treated with 0.5 mg sodium fluoride by daily gavage and pregnant females (named "mothers" 0.25 mg sodium fluoride) treated with 0.25 mg sodium fluoride by daily gavage; three types of sodium fluoride treatments were administrated: homeopathic, allopathic-homeopathic and allopathic. When the animals reached the adulthood, by randomization, they were selected in pairs for giving birth to the second generation of mice. No treatments were administrated to the second generation of mice; thus, the urinary elimination of fluoride in the second generation is attributed to exposure at sodium fluoride before birth. The administration of sodium fluoride to the first generation (F1) is realized until the mice reached the adulthood. For the first generation, the urine was collected at three times, every three weeks: at the age of four weeks, seven weeks and 11 weeks; single sampling urine, at the age of four weeks, has been conducted for the second generation. The urine samples have been analyzed using the ion selective electrode method for fluoride. For the histopathological examination, the animals were killed by cervical dislocation; the kidneys were collected in a 10% formalin solution. The preparation of samples for optical microscopy was realized with Hematoxylin-Eosin staining. The results indicate that the elimination of fluoride was similar (at the second evaluation, at 7-week-old of the first generation) for the both generations of mice. Histopathological observation of the kidney has revealed granular dystrophy of the renal tubules, necrosis of the endothelial cells and of the mesangial cells of renal glomerulus. The study indicates that different sodium fluoride treatments produce some pathological aspects of the kidneys and influence the urinary elimination of fluoride in two consecutive generations of mice. For the higher doses, the pathological changes of the kidney are more important, and the urinary elimination of fluoride is higher, especially for the allopathic doses.

Comparison of the toxicity of fluoridation compounds in the nematode Caenorhabditis elegans.

Fluorides are commonly added to drinking water in the United States to decrease the incidence of dental caries. Silicofluorides, such as sodium hexafluorosilicate (Na2 SiF6 ) and fluorosilicic acid (H2 SiF6 ), are mainly used for fluoridation, although fluoride salts such as sodium fluoride (NaF) are also used. Interestingly, only the toxicity of NaF has been examined and not that of the more often used silicofluorides. In the present study, the toxicities of NaF, Na2 SiF6 , and H2 SiF6 were compared. The toxicity of these fluorides on the growth, feeding, and reproduction in the alternative toxicological testing organism Caenorhabditis elegans was examined. Exposure to these compounds produced classic concentration-response toxicity profiles. Although the effects of the fluoride compounds varied among the 3 biological endpoints, no differences were found between the 3 compounds, relative to the fluoride ion concentration, in any of the assays. This suggests that silicofluorides have similar toxicity to NaF.