Fluoride and children's IQ: evidence of causation lacking.

DATA SOURCES: Human, animal, and in vitro studies. Extensive literature search of multiple bibliographic databases, trial registries, major grey literature sources and bibliographies of identified studies. STUDY SELECTION: The authors aimed to identify studies which could be used to determine the maximum safe level for fluoride in drinking water. To identify new studies published since a 2016 Australian review, the search period was 2016 to July 2021. Studies which evaluated the association between either naturally or artificially fluoridated water (any concentration) and any health outcomes were included. No restrictions on study design or publication status. Articles published in a 'non-Latin language' were excluded. Screening of abstracts and full texts was in duplicate. For IQ and dental fluorosis, a top-up search was conducted between 2021 and Feb 2023. DATA EXTRACTION AND SYNTHESIS: Extensive data extraction. Risk of bias assessment using the OHAT tool. A narrative synthesis of the results was carried out. RESULTS: The review included 89 studies in humans, 199 in animals and 10 reviews of in vitro studies. Where there was consistent evidence of a positive association, in relation to a water fluoride concentration of <20 ppm (mg F/L), and where studies were judged to be acceptable or high quality, health effects were taken forwards for further examination of causality using Bradford Hill's 9 criteria. Of the 39 health outcomes reviewed, 4 were further assessed for causality. The authors reported 'strong' evidence of causality for dental fluorosis and reductions in children's IQ scores, 'moderate' strength evidence for thyroid dysfunction, 'weak' for kidney dysfunction, and 'limited' evidence for sex hormone disruption. CONCLUSIONS: The authors conclude that moderate dental fluorosis and reductions in children's IQ scores are the most appropriate health outcomes to use when setting an upper safe level of fluoride in drinking water. For reductions in children's IQ, the authors acknowledge a biological mechanism of action has not been elucidated, and the dose response curve is not clear at lower concentrations, limiting the ability to set an upper safe threshold.

Association between fluoride intake from drinking water and severity of dental fluorosis in Northern and Western Mexico: systematic review and meta-analysis.

BACKGROUND: Dental fluorosis (DF) is caused by excessive exposure to fluoride during odontogenesis and leads to various changes in the development of tooth enamel. Some regions in Mexico are considered endemic fluorosis zones due to the high fluoride content in drinking water. The objective of this study was to perform a systematic review and meta-analysis to identify the association between the concentration of fluoride in drinking water and the severity of dental fluorosis in northern and western Mexico. METHODS: This protocol was registered in the PROSPERO database (ID: CRD42023401519). The search for information was carried out in the PubMed/Medline, Scopus, SpringerLink, and Google Scholar databases between January 2015 and October 2023. The overall relative risk was calculated using the inverse of variance approach with the random effects method. The RoB 2.0 tool was used to construct risk plots. RESULTS: Eleven articles were analyzed qualitatively, and most of the included studies presented at least one level of DF severity; six articles were analyzed quantitatively, dividing them into two regions. In North region it was observed a higher prevalence of severe TF cases, corresponding to ≥ TF 5 category (4.78) [3.55, 6.42]. In the West region, most of the included studies presented a higher prevalence of less severe cases, corresponding to ≤ TF 4, in comparison with the North region (0.01) [0.00, 0.52], interpreted as a protective effect. CONCLUSION: The concentrations of fluorides in drinking water are reportedly high in these regions and are directly related to the severity of dental fluorosis experienced by the inhabitants. In the Northern region exists a major concentration of fluoride in drinking water compared with the Western region as well as a prevalence of higher severity cases of dental fluorosis.

Topical fluoride as a cause of dental fluorosis in children.

BACKGROUND: This is an update of a review first published in 2010. Use of topical fluoride has become more common over time. Excessive fluoride consumption from topical fluorides in young children could potentially lead to dental fluorosis in permanent teeth. OBJECTIVES: To describe the relationship between the use of topical fluorides in young children and the risk of developing dental fluorosis in permanent teeth. SEARCH METHODS: We carried out electronic searches of the Cochrane Oral Health Trials Register, CENTRAL, MEDLINE, Embase, three other databases, and two trials registers. We searched the reference lists of relevant articles. The latest search date was 28 July 2022. SELECTION CRITERIA: We included randomized controlled trials (RCTs), quasi-RCTs, cohort studies, case-control studies, and cross-sectional surveys comparing fluoride toothpaste, mouth rinses, gels, foams, paint-on solutions, and varnishes to a different fluoride therapy, placebo, or no intervention. Upon the introduction of topical fluorides, the target population was children under six years of age. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane and used GRADE to assess the certainty of the evidence. The primary outcome measure was the percentage prevalence of fluorosis in the permanent teeth. Two authors extracted data from all included studies. In cases where both adjusted and unadjusted risk ratios or odds ratios were reported, we used the adjusted value in the meta-analysis. MAIN RESULTS: We included 43 studies: three RCTs, four cohort studies, 10 case-control studies, and 26 cross-sectional surveys. We judged all three RCTs, one cohort study, one case-control study, and six cross-sectional studies to have some concerns for risk of bias. We judged all other observational studies to be at high risk of bias. We grouped the studies into five comparisons. Comparison 1. Age at which children started toothbrushing with fluoride toothpaste Two cohort studies (260 children) provided very uncertain evidence regarding the association between children starting to use fluoride toothpaste for brushing at or before 12 months versus after 12 months and the development of fluorosis (risk ratio (RR) 0.98, 95% confidence interval (CI) 0.81 to 1.18; very low-certainty evidence). Similarly, evidence from one cohort study (3939 children) and two cross-sectional studies (1484 children) provided very uncertain evidence regarding the association between children starting to use fluoride toothpaste for brushing before or after the age of 24 months (RR 0.83, 95% CI 0.61 to 1.13; very low-certainty evidence) or before or after four years (odds ratio (OR) 1.60, 95% CI 0.77 to 3.35; very low-certainty evidence), respectively. Comparison 2. Frequency of toothbrushing with fluoride toothpaste Two case-control studies (258 children) provided very uncertain evidence regarding the association between children brushing less than twice per day versus twice or more per day and the development of fluorosis (OR 1.63, 95% CI 0.81 to 3.28; very low-certainty evidence). Two cross-sectional surveys (1693 children) demonstrated that brushing less than once per day versus once or more per day may be associated with a decrease in the development of fluorosis in children (OR 0.62, 95% CI 0.53 to 0.74; low-certainty evidence). Comparison 3. Amount of fluoride toothpaste used for toothbrushing Two case-control studies (258 children) provided very uncertain evidence regarding the association between children using less than half a brush of toothpaste, versus half or more of the brush, and the development of fluorosis (OR 0.77, 95% CI 0.41 to 1.46; very low-certainty evidence). The evidence from cross-sectional surveys was also very uncertain (OR 0.92, 95% CI 0.66 to 1.28; 3 studies, 2037 children; very low-certainty evidence). Comparison 4. Fluoride concentration in toothpaste There was evidence from two RCTs (1968 children) that lower fluoride concentration in the toothpaste used by children under six years of age likely reduces the risk of developing fluorosis: 550 parts per million (ppm) fluoride versus 1000 ppm (RR 0.75, 95% CI 0.57 to 0.99; moderate-certainty evidence); 440 ppm fluoride versus 1450 ppm (RR 0.72, 95% CI 0.58 to 0.89; moderate-certainty evidence). The age at which the toothbrushing commenced was 24 months and 12 months, respectively. Two case-control studies (258 children) provided very uncertain evidence regarding the association between fluoride concentrations under 1000 ppm, versus concentrations of 1000 ppm or above, and the development of fluorosis (OR 0.89, 95% CI 0.52 to 1.52; very low-certainty evidence). Comparison 5. Age at which topical fluoride varnish was applied There was evidence from one RCT (123 children) that there may be little to no difference between a fluoride varnish application before four years, versus no application, and the development of fluorosis (RR 0.77, 95% CI 0.45 to 1.31; low-certainty evidence). There was low-certainty evidence from two cross-sectional surveys (982 children) that the application of topical fluoride varnish before four years of age may be associated with the development of fluorosis in children (OR 2.18, 95% CI 1.46 to 3.25). AUTHORS' CONCLUSIONS: Most evidence identified mild fluorosis as a potential adverse outcome of using topical fluoride at an early age. There is low- to very low-certainty and inconclusive evidence on the risk of having fluorosis in permanent teeth for: when a child starts receiving topical fluoride varnish application; toothbrushing with fluoride toothpaste; the amount of toothpaste used by the child; and the frequency of toothbrushing. Moderate-certainty evidence from RCTs showed that children who brushed with 1000 ppm or more fluoride toothpaste from one to two years of age until five to six years of age probably had an increased chance of developing dental fluorosis in permanent teeth. It is unethical to propose new RCTs to assess the development of dental fluorosis. However, future RCTs focusing on dental caries prevention could record children's exposure to topical fluoride sources in early life and evaluate the dental fluorosis in their permanent teeth as a long-term outcome. In the absence of these studies and methods, further research in this area will come from observational studies. Attention needs to be given to the choice of study design, bearing in mind that prospective controlled studies will be less susceptible to bias than retrospective and uncontrolled studies.

Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models.

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (µCT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by µCT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

Progress in research on the role of fluoride in immune damage.

Excessive fluoride intake from residential environments may affect multiple tissues and organs; however, the specific pathogenic mechanisms are unclear. Researchers have recently focused on the damaging effects of fluoride on the immune system. Damage to immune function seriously affects the quality of life of fluoride-exposed populations and increases the incidence of infections and malignant tumors. Probing the mechanism of damage to immune function caused by fluoride helps identify effective drugs and methods to prevent and treat fluorosis and improve people's living standards in fluorosis-affected areas. Here, the recent literature on the effects of fluoride on the immune system is reviewed, and research on fluoride damage to the immune system is summarized in terms of three perspectives: immune organs, immune cells, and immune-active substances. We reviewed that excessive fluoride can damage immune organs, lead to immune cells dysfunction and interfere with the expression of immune-active substances. This review aimed to provide a potential direction for future fluorosis research from the perspective of fluoride-induced immune function impairment. In order to seek the key regulatory indicators of fluoride on immune homeostasis in the future.

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.

The influence of the acid water of the Banyupait River on the community health in Bantal village, Asembagus, Indonesia.

The pH of Mount Ijen crater water is 0-2, resulting in water that is acidic and sulfurous. A fault near the Mount Ijen Crater causes seepage so that acidic water flows into the Banyupait River. Chemical elements and heavy metals originating from the river pollute groundwater and plants. As a result, people around the river consume heavy metals. This research aims to determine the quality of river water and groundwater, as well as determine community factors that are susceptible to dental fluorosis. The methodology used is field mapping and laboratory analysis. For water samples, the Atomic Absorption Spectrophotometer (AAS) method is used. The pH of river water at the research location exceeds the quality standard, namely pH 4-5.5. Meanwhile, groundwater fluorine and sulfate elements exceed quality standards, namely fluorine of 0.6171 mg/L and 0.6870 mg/L, and sulfate ranging from 325-683 mg/L. These two elements cause symptoms of dental fluorosis. Meanwhile, the community factors most susceptible to dental fluorosis are people in the adult age category, and the last level of education is elementary school. This is because the Banyupait River water and groundwater are exposed to fluorine and sulfate water originating from seepage from the Mount Ijen Crater.

Protective effects of sodium butyrate on fluorosis in rats by regulating bone homeostasis and serum metabolism.

Fluorosis due to high fluoride levels in drinking water profoundly affects the development of human skeletal and dental structures. Sodium butyrate (NaB) has been found to regulate overall bone mass and prevent pathological bone loss. However, the mechanism of NaB action on fluorosis remains unclear. In this study, a rat model of fluorosis induced by 100 mg/L sodium fluoride was used to investigate the impact of NaB on bone homeostasis and serum metabolomics. It was found that NaB significantly reduced the levels of bone resorption markers CTX-Ⅰ and TRACP-5B in fluorosis rats. Moreover, NaB increased calcium and magnesium levels in bone, while decreasing phosphorus levels. In addition, NaB improved various bone microstructure parameters, including bone mineral density (BMD), trabecular thickness (Tb. Th), trabecular bone separation (Tb. SP), and structural model index (SMI) in the femur. Notably, NaB intervention also enhanced the antioxidant capacity of plasma in fluorosis rats. Furthermore, a comprehensive analysis of serum metabolomics by LC-MS revealed a significant reversal trend of seven biomarkers after the intervention of NaB. Finally, pathway enrichment analysis based on differential metabolites indicated that NaB exerted protective effects on fluorosis by modulating arginine and proline metabolic pathways. These findings suggest that NaB has a beneficial effect on fluorosis and can regulate bone homeostasis by ameliorating metabolic disorders.

A Bayesian benchmark concentration analysis for urinary fluoride and intelligence in adults in Guizhou, China.

Environmental fluoride exposure has been linked to numerous cases of fluorosis worldwide. Previous studies have indicated that long-term exposure to fluoride can result in intellectual damage among children. However, a comprehensive health risk assessment of fluorosis-induced intellectual damage is still pending. In this research, we utilized the Bayesian Benchmark Dose Analysis System (BBMD) to investigate the dose-response relationship between urinary fluoride (U-F) concentration and Raven scores in adults from Nayong, Guizhou, China. Our research findings indecate a dose-response relationship between the concentration of U-F and intelligence scores in adults. As the benchmark response (BMR) increased, both the benchmark concentration (BMCs) and the lower bound of the credible interval (BMCLs) increased. Specifically, BMCs for the association between U-F and IQ score were determined to be 0.18 mg/L (BMCL1 = 0.08 mg/L), 0.91 mg/L (BMCL5 = 0.40 mg/L), 1.83 mg/L (BMCL10 = 0.83 mg/L) when using BMRs of 1 %, 5 %, and 10 %. These results indicate that U-F can serve as an effective biomarker for monitoring the loss of IQ in population. We propose three interim targets for public policy in preventing interllectual harm from fluoride exposure.

Analysis of indicators of caries of permanent teeth in children according to the endemic features of the Poltava region, Ukraine.

OBJECTIVE: Aim: This research is focused at analyzing the indicators and substantiating the peculiarities of caries prevention in permanent teeth in schoolchildren of Poltava region, taking into account the endemic features of the Poltava region. PATIENTS AND METHODS: Materials and Methods: A comparative study was conducted among 608 pupils of secondary schools in Kremenchuk, who consumed drinking water with fluoride concentrations in the lower limits of the norm, and 1214 pupils of secondary schools in Poltava, who consumed drinking water with fluoride concentrations in the optimal upper limits of the norm. RESULTS: Results: The rates of caries in permanent teeth in children living in a region with fluoride concentrations in drinking water in the optimal-upper normal range are several times lower than in children of the same age living in a region with fluoride concentrations in drinking water in the lower normal range, and a significant increase in the prevalence and intensity of caries is observed from 7 to 9 to 12 years of age, as teeth after eruption are most vulnerable to caries. In a region where the fluoride concentration in drinking water is in the optimal-upper range of the norm, children with early forms of fluorosis have the lowest prevalence and intensity of caries. CONCLUSION: Conclusions: Endemic features of the region directly affect the prevalence and intensity of the caries process. In regions with a fluoride concentration in drinking water within the optimal upper limits of the norm, prevention of caries in permanent teeth in children should be carried out taking into account the presence of fluorosis.

The association between Dental Fluorosis and COL1A2 gene polymorphism among a Tunisian Population.

Dental fluorosis (DF) is a prevalent developmental defect of tooth enamel caused by exposure to excessive fluoride, with the severity dependent on various factors. This study aimed to investigate the association between DF and a specific genetic polymorphism (rs412777) in the COL1A2 gene among a Tunisian population. A case-control study was conducted from July to November 2022, involving a total of 95 participants including 51 cases and 44 controls. Dental examinations and genetic analysis were performed to assess the relationship between the COL1A2 gene polymorphism and DF.The results of allelic distribution revealed that A allele carriers were significantly protected against (DF) when compared to those with the C allele (C vs. A, p = 0.001; OR = 0.375 (0.207-0.672)). This suggests a strong correlation between the presence of the C allele and the risk of developing DF. Additionally, significant association between the CC genotype of rs412777 and an increased risk of DF was found under both codominant and dominant genetic models (P = 0.002 and P < 0.001 respectively).The findings suggest that genetic predisposition plays a relevant role in the development of DF. Further research is needed to explore the potential use of genetic markers for DF and their implications for public health. This study provides the first insights into the genetic factors associated with DF in the Tunisian population, contributing to our understanding of this prevalent dental condition.

Caries prevalence and water fluoridation in Israel: a cross-sectional study.

OBJECTIVES: To assess the effect of Community Water Fluoridation (CWF) in the prevalence of dental caries and dental fluorosis in 12-year-old children living in Israel. Considering that CWF is important in the prevention of dental caries. Between 2002 and 2014, the water in communities of at least 5,000 individuals was fluoridated. In 2014, CWF in Israel stopped. METHOD AND MATERIALS: Data on 12-year-old children from all areas in Israel from the national cross-sectional epidemiological survey conducted in 2011 to 2012 were stratified by city water fluoridation and by city and school socioeconomic status. Two dependent variables were defined: (1) DMFT index of caries experience in the permanent dentition; (2) dental fluorosis in central incisors using the Thylstrup-Fejerskov classification of fluorosis. RESULTS: Data from 2,181 12-year-olds were analyzed. The average DMFT was 1.17 ± 1.72, and 49% were caries-free. Based on DMFT, the caries experience was significantly higher in nonfluoridated cities (1.38 vs 0.98 in fluoridated cities) and there were more caries-free children in fluoridated cities (56.4% vs 40.6% in nonfluoridated). DMFT was higher in cities with lower socioeconomic status than high socioeconomic status (1.29 vs 1.05, respectively, P < .001) and there were fewer caries-free children in low socioeconomic status cities (44.5% vs 53.0% in high socioeconomic status cities, P < .0001). Almost all the 10.3% of children with signs of fluorosis (scoring at least 1 in the Thylstrup-Fejerskov index), had questionable to mild fluorosis (9.3%). CONCLUSIONS: CWF is a cheap, simple method of dental health protection that reaches all socioeconomic levels, and cessation of water fluoridation reduced the health of Israel's children. CLINICAL SIGNIFICANCE: Water fluoridation provides substantial caries prevention, by reaching a substantial number of people. The relevance of this work is for policymakers to consider CWF as clinically proven method for reducing health inequalities.

Systematic review of epidemiological and toxicological evidence on health effects of fluoride in drinking water.

INTRODUCTION: Fluoride is a naturally occurring substance that is also added to drinking water, dental hygiene products, and food supplements for preventing dental caries. Concerns have been raised about several other potential health risks of fluoride. OBJECTIVE: To conduct a robust synthesis of evidence regarding human health risks due to exposure to fluoride in drinking water, and to develop a point of departure (POD) for setting a health-based value (HBV) for fluoride in drinking water. METHODS: A systematic review of evidence published since recent reviews of human, animal, and in vitro data was carried out. Bradford Hill considerations were used to weigh the evidence for causality. Several key studies were considered for deriving PODs. RESULTS: The current review identified 89 human studies, 199 animal studies, and 10 major in vitro reviews. The weight of evidence on 39 health endpoints was presented. In addition to dental fluorosis, evidence was considered strong for reduction in IQ scores in children, moderate for thyroid dysfunction, weak for kidney dysfunction, and limited for sex hormone disruptions. CONCLUSION: The current review identified moderate dental fluorosis and reduction in IQ scores in children as the most relevant endpoints for establishing an HBV for fluoride in drinking water. PODs were derived for these two endpoints, although there is still some uncertainty in the causal weight of evidence for causality for reducing IQ scores in children and considerable uncertainty in the derivation of its POD. Given our evaluation of the overall weight of evidence, moderate dental fluorosis is suggested as the key endpoint until more evidence is accumulated on possible reduction of IQ scores effects. A POD of 1.56 mg fluoride/L for moderate dental fluorosis may be preferred as a starting point for setting an HBV for fluoride in drinking water to protect against moderate and severe dental fluorosis. Although outside the scope of the current review, precautionary concerns for potential neurodevelopmental cognitive effects may warrant special consideration in the derivation of the HBV for fluoride in drinking water.

Potential risk of dental fluorosis associated with different baby formulas and water brands marketed in Spain.

Despite efforts to promote breastfeeding, many babies aged <6 months receive only baby formula reconstituted with bottled water. The intake of high levels of fluoride during amelogenesis has been associated with hypomineralization of the tooth enamel, with aesthetic and mechanical repercussions. The objective of this study was to determine the potential risk of dental fluorosis associated with baby formulas marketed in Spain. We measured 26 baby formulas frequently consumed in Spain; 17 brands for babies aged <6 months, 5 for those aged >6 months, and 4 ready-to-use brands. They were prepared with 4 types of water: distilled water and three brands of bottled water with different levels of fluoride. The fluoride concentration (mg/L or ppm F) was measured with an ion-specific electrode coupled to an ion analyzer. Each sample was prepared according to the manufacturer's instructions and analyzed in triplicate. A descriptive analysis was carried out. The minimum fluoride level found was 0.04 mg/L and the maximum was 1.02 mg/L. Considering the daily intake of these formulas, none exceeded the clinically-acceptable daily dose limit risk for fluorosis (0.07 mg F/day/kg body weight) when mixed with bottled water with a low level of fluoride (0.1 mg/L). However, when the same brands of formula were reconstituted with bottled water with a higher fluoride content (0.99 mg/L), they all exceeded the daily dose limit for the risk of fluorosis. As the potential risk of dental fluorosis associated with the formulas tested depends exclusively on the fluoride concentration of the waters used for reconstitution, formula packaging should contain a warning.

Poor oral health and the risk of esophageal squamous cell carcinoma in Malawi.

Esophageal squamous cell carcinoma (ESCC) is the second most common cancer in Malawi. Risk factors for this cancer in Malawi are poorly understood. Poor oral health has previously been linked to increased ESCC risk in other high-incidence regions, including parts of Eastern and Southern Africa. We assessed the relationship between oral health and ESCC risk in a sex, age and location frequency-matched case-control study based at two hospitals in Lilongwe, Malawi from 2017 to 2020. Trained interviewers used a structured questionnaire and direct observation to collect data on demographics; behaviors; oral hygiene habits; the sum of decayed, missing or filled teeth (DMFT score); oral mucosa status; lip depigmentation and dental fluorosis via a visual scale. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CI), adjusted for known and suspected ESCC risk factors. During the study period, 300 cases and 300 controls were enrolled. Subjects in the highest tertile of DMFT score (≥7) had an increased risk of ESCC with an adjusted OR of 1.96 (95% CI: 1.16-3.36) compared to those with a DMFT score of 0. Severe dental fluorosis was associated with a statistically nonsignificant increased risk of ESCC (adjusted OR = 2.24, 95% CI: 0.97-5.49) compared to individuals with no fluorosis. Associations with oral mucosa status, lip depigmentation and toothbrushing method and frequency were mostly null or uncertain. Poor oral health, indicated by a higher DMFT score, was associated with increased ESCC risk in Malawi. Dental fluorosis is another possible risk factor in this population, but further evaluation is necessary to clarify any effects of fluorosis on ESCC risk.

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.

Proteomics Sequencing Reveals the Role of TGF-ß Signaling Pathway in the Peripheral Blood of Offspring Rats Exposed to Fluoride.

The underlying mechanism of fluorosis has not been fully elucidated. The purpose of this study was to explore the mechanism of fluorosis induced by sodium fluoride (NaF) using proteomics. Six offspring rats exposed to fluoride without dental fluorosis were defined as group A, 8 offspring rats without fluoride exposure were defined as control group B, and 6 offspring rats exposed to fluoride with dental fluorosis were defined as group C. Total proteins from the peripheral blood were extracted and then separated using liquid chromatography-tandem mass spectrometry. The identified criteria for differentially expressed proteins were fold change > 1.2 or < 0.83 and P < 0.05. Gene Ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the oeCloud tool. The 177 upregulated and 22 downregulated proteins were identified in the A + C vs. B group. KEGG pathway enrichment analysis revealed that transforming growth factor-ß (TGF-ß) signaling pathway significantly enriched. PPI network constructed using Cytoscape confirmed RhoA may play a crucial role. The KEGG results of genes associated with fluoride and genes associated with both fluoride and inflammation in the GeneCards database also showed that TGF-ß signaling pathway was significantly enriched. The immunofluorescence in HPA database showed that the main expression sites of RhoA are plasma membrane and cytosol, while the main expression site of Fbn1 is the Golgi apparatus. In conclusion, long-term NaF intake may cause inflammatory response in the peripheral blood of rats by upregulating TGF-ß signaling pathway, in which RhoA may play a key role.

Health risk assessment in an area of dental fluorosis disease from high fluoride drinking water: a case study from southeastern Türkiye.

This study focuses on identifying fluoride (F‒) concentrations and its health risk assessment (HRA) in drinking water sources in south-eastern Türkiye. Groundwater quality was assessed using some graphical approaches such as Schoeller and Piper diagrams and GIS mapping. Average daily exposure dosages through oral and dermal contact exposure routes were considered to determine the potential health risk of F‒ in groundwater. Groundwater samples were taken from 53 points in spring, summer, autumn, and winter seasons. The results showed that the average annual F‒ concentrations in water resources in the study area were 0.26‒3.62 mg/L. According to the HRA results, the highest F‒ health risk in this region was observed in children, followed by teenagers and adults. This study indicated that there is a strong relationship between the high health risk (4.28 > 3.5) in children and dental fluorosis caused by high F‒ concentration in groundwater.

Association between polymorphism and haplotype of ATP2B1 gene and skeletal fluorosis in Han population.

To evaluate the association between ATP2B1 gene polymorphisms and skeletal fluorosis, a cross-sectional study was conducted. In China, 962 individuals were recruited, including 342 cases of skeletal fluorosis. Four TP2BA1 polymorphisms (rs2070759, rs12817819, rs17249754, and rs7136259) were analysed. The results suggested that rs17249754 and rs7136259 were associated with skeletal fluorosis. After controlling confounders, the protective effect of GG genotype in rs17249754 was apparent in individuals over 45 years old, female, with urine fluoride concentration below 1.6 mg/L, serum calcium above 2.25 mmol/L or serum phosphorus between 1.1 and 1.3. Heterozygote TC in rs7136259 increased the risk of skeletal fluorosis in subjects who are elderly, female, with urinary fluoride more than 1.6 mg/L, serum calcium more than 2.25 mmol/L and blood phosphorus between 1.1 and 1.3 mmol/L. Four loci were found to be tightly related by linkage disequilibrium analysis, and the frequency of distribution of haplotype GCGT was lower in the skeletal fluorosis group.

TGF-ß1/Smad3 Signaling Is Required to Alleviate Fluoride-Induced Enamel Hypomineralization.

Excessive fluoride intake during enamel development can affect enamel mineralization, leading to dental fluorosis. However, its potential mechanisms remain largely unexplored. In the present study, we aimed to investigate the impact of fluoride on the expressions of RUNX2 and ALPL during mineralization and the effect of TGF-ß1 administration on fluoride treatment. A dental fluorosis model of newborn mice and an ameloblast cell line ALC were both used in the present study. The mice of the NaF group, including the mothers and newborns, were fed with water containing 150 ppm NaF after delivery to induce dental fluorosis. The mandibular incisors and molars showed significant abrasion in the NaF group. Immunostaining, qRT-PCR, and Western blotting analysis indicated that exposure to fluoride markedly down-regulated RUNX2 and ALPL in mouse ameloblasts and ALCs. Besides, fluoride treatment significantly decreased the mineralization level detected by ALP staining. Furthermore, exogenous TGF-ß1 up-regulated RUNX2 and ALPL and promoted mineralization, while the addition of SIS3 could block such TGF-ß1-induced up-regulation. In TGF-ß1 conditional knockout mice, the immunostaining of RUNX2 and ALPL was weaker compared with wild-type mice. Exposure to fluoride inhibited the expressions of TGF-ß1 and Smad3. Co-treatment of TGF-ß1 and fluoride up-regulated RUNX2 and ALPL compared with the fluoride alone treatment, promoting mineralization. Collectively, our data indicated that TGF-ß1/Smad3 signaling pathway was necessary for the regulatory effects of fluoride on RUNX2 and ALPL, and the fluoride-induced suppression of ameloblast mineralization was mitigated by activating TGF-ß1/Smad3 signaling pathway.