Fluorides are present in the environment. Excessive systemic exposure to fluorides can lead to disturbances of bone homeostasis (skeletal fluorosis) and enamel development (dental/enamel fluorosis). The severity of dental fluorosis is also dependent upon fluoride dose and the timing and duration of fluoride exposure. Fluoride's actions on bone cells predominate as anabolic effects both in vitro and in vivo. More recently, fluoride has been shown to induce osteoclastogenesis in mice. Fluorides appear to mediate their actions through the MAPK signaling pathway and can lead to changes in gene expression, cell stress, and cell death. Different strains of inbred mice demonstrate differential physiological responses to ingested fluoride. Genetic studies in mice are capable of identifying and characterizing fluoride-responsive genetic variations. Ultimately, this can lead to the identification of at-risk human populations who are susceptible to the unwanted or potentially adverse effects of fluoride action and to the elucidation of fundamental mechanisms by which fluoride affects biomineralization.

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Fluoride's Effects on the Formation of Teeth and Bones, and the Influence of Genetics

Dental fluorosis occurs as a result of excess fluoride ingestion during tooth formation. Enamel fluorosis and primary dentin fluorosis can only occur when teeth are forming, and therefore fluoride exposure (as it relates to dental fluorosis) occurs during childhood. In the permanent dentition, this would begin with the lower incisors, which complete mineralization at approximately 2–3 years of age, and end after mineralization of the third molars. The white opaque appearance of fluorosed enamel is caused by a hypomineralized enamel subsurface. With more severe dental fluorosis, pitting and a loss of the enamel surface occurs, leading to secondary staining (appearing as a brown color). Many of the changes caused by fluoride are related to cell/matrix interactions as the teeth are forming. At the early maturation stage, the relative quantity of amelogenin protein is increased in fluorosed enamel in a dose-related manner. This appears to result from a delay in the removal of amelogenins as the enamel matures. In vitro, when fluoride is incorporated into the mineral, more protein binds to the forming mineral, and protein removal by proteinases is delayed. This suggests that altered protein/mineral interactions are in part responsible for retention of amelogenins and the resultant hypomineralization that occurs in fluorosed enamel. Fluoride also appears to enhance mineral precipitation in forming teeth, resulting in hypermineralized bands of enamel, which are then followed by hypomineralized bands. Enhanced mineral precipitation with local increases in matrix acidity may affect maturation stage ameloblast modulation, potentially explaining the dose-related decrease in cycles of ameloblast modulation from ruffle-ended to smooth-ended cells that occur with fluoride exposure in rodents. Specific cellular effects of fluoride have been implicated, but more research is needed to determine which of these changes are relevant to the formation of fluorosed teeth. As further studies are done, we will better understand the mechanisms responsible for dental fluorosis.

Chronic fluoride toxicity: dental fluorosis.

Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. These heavily polarized cells form a monolayer around the developing enamel tissue and move as a single forming front in specified directions as they lay down a proteinaceous matrix that serves as a template for crystal growth. Ameloblasts maintain intercellular connections creating a semi-permeable barrier that at one end (basal/proximal) receives nutrients and ions from blood vessels, and at the opposite end (secretory/apical/distal) forms extracellular crystals within specified pH conditions. In this unique environment, ameloblasts orchestrate crystal growth via multiple cellular activities including modulating the transport of minerals and ions, pH regulation, proteolysis, and endocytosis. In many vertebrates, the bulk of the enamel tissue volume is first formed and subsequently mineralized by these same cells as they retransform their morphology and function. Cell death by apoptosis and regression are the fates of many ameloblasts following enamel maturation, and what cells remain of the enamel organ are shed during tooth eruption, or are incorporated into the tooth’s epithelial attachment to the oral gingiva. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

Dental enamel formation and implications for oral health and disease.

Intake of excess amounts of fluoride during tooth development cause enamel fluorosis, a developmental disturbance that makes enamel more porous. In mild fluorosis, there are white opaque striations across the enamel surface, whereas in more severe cases, the porous regions increase in size, with enamel pitting, and secondary discoloration of the enamel surface. The effects of fluoride on enamel formation suggest that fluoride affects the enamel-forming cells, the ameloblasts. Studies investigating the effects of fluoride on ameloblasts and the mechanisms of fluorosis are based on in vitro cultures as well as animal models. The use of these model systems requires a biologically relevant fluoride dose, and must be carefully interpreted in relation to human tooth formation. Based on these studies, we propose that fluoride can directly affect the ameloblasts, particularly at high fluoride levels, while at lower fluoride levels, the ameloblasts may respond to local effects of fluoride on the mineralizing matrix. A new working model is presented, focused on the assumption that fluoride increases the rate of mineral formation, resulting in a greater release of protons into the forming enamel matrix.

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The Impact of Fluoride on Ameloblasts and the Mechanisms of Enamel Fluorosis

Fluoride is ubiquitously present throughout the world. It is released from minerals, magmatic gas, and industrial processing, and travels in the atmosphere and water. Exposure to low concentrations of fluoride increases overall oral health. Consequently, many countries add fluoride to their public water supply at 0.7–1.5 ppm. Exposure to high concentrations of fluoride, such as in a laboratory setting often exceeding 100 ppm, results in a wide array of toxicity phenotypes. This includes oxidative stress, organelle damage, and apoptosis in single cells, and skeletal and soft tissue damage in multicellular organisms. The mechanism of fluoride toxicity can be broadly attributed to four mechanisms: inhibition of proteins, organelle disruption, altered pH, and electrolyte imbalance. Recently, there has been renewed concern in the public sector as to whether fluoride is safe at the current exposure levels. In this review, we will focus on the impact of fluoride at the chemical, cellular, and multisystem level, as well as how organisms defend against fluoride. We also address public concerns about fluoride toxicity, including whether fluoride has a significant effect on neurodegeneration, diabetes, and the endocrine system.

Principles of fluoride toxicity and the cellular response: a review.

Genetic background influences fluoride's effects on osteoclastogenesis

Systemic exposure to greater than optimal fluoride (F) can lead to dental fluorosis (DF). Parental A/J (DF-susceptible) and 129P3/J (DF-resistant) inbred mice were used for histological studies and to generate F2 progeny. Mice were treated with 0 or 50 ppm F in their drinking water for 60 days. A clinical criterion (modified Thylstrup and Fejerskov categorical scale) was used to assess the severity of DF for each individual F2 animal. Parental strains were subjected to histological examination of maturing enamel. F treatment resulted in accumulation of amelogenins in the maturing enamel of A/J mice. Quantitative trait loci (QTL) detection was performed using phenotypic extreme F2 animals genotyped for 354 single nucleotide polymorphism-based markers distributed throughout the mouse genome followed by χ2 analysis. Significant evidence of association was observed on chromosomes 2 and 11 for a series of consecutive markers (p < 0.0001). Further analyses were performed to examine whether the phenotypic effects were found in both male and female F2 mice or whether there was evidence for gender-specific effects. Analyses performed using the markers on chromosomes 2 and 11 which were significant in the mixed-gender mice were also significant when analyses were limited to only the male or female mice. The QTL detected on chromosomes 2 and 11 which influence the variation in response to fluorosis have their effect in mice of both genders. Finally, the QTL in both chromosomes appear to have an additive effect.

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Detection of Dental Fluorosis-Associated Quantitative Trait Loci on Mouse Chromosomes 2 and 11

Fluoride (F) in various chemical forms and exposures has been repeatedly shown to have actions on the development and homeostasis of mineralized tissues (1, 2). Excessive systemic fluoride during critical periods of tooth enamel development (amelogenesis) can result in an undesirable developmental defect of tooth enamel, dental fluorosis (enamel fluorosis). Dental fluorosis (DF) is characterized by increased porosity (subsurface hypomineralization) with a loss of enamel translucency and increased opacity (3). It is generally accepted that increasing DF severity correlates with increasing F exposure. DF remains highly prevalent world-wide. As recent as 2005 23% of persons in the United States aged 6–39 years had very mild or greater enamel fluorosis (4). We have demonstrated that genetic background plays an integral role in responses of mineralized tissues to systemic fluoride (5–10). Genetically diverse inbred strains of mice when provided equivalent exposures to F in their environment show strain specific DF severities (5, 9). DF can be considered a complex condition influenced by environmental (F exposure) and host factors (genetics). The later is determined by multiple genes with additive effects (11). Quantitative trait locus (QTL) detection mapping has been extensively used in mapping loci associated with complex traits. Generally large panels of animals and large QTL effects improve the power to detect and map QTLs. Two traditional QTL mapping approaches that can yield large panels are F2 and backcross strategies. In mice F2 panels are created from a two generation cross between two parental inbred strains which have measurable differences for a particular trait. In the case of DF we used the 129P3/J (DF resistant strain) and the A/J (DF susceptible strain)(5, 11). Recently we identified QTLs on chromosomes (Chr) 2 and 11 in mice that are strongly associated with DF susceptibility (11). On Chr 2 a QTL designated as Dfs1 (MGI Accession ID: MGI: 4418088) was detected with an interval of approximately 77.62 Mb and flanked by markers rs13476589 and UT_2_156.443943. A second QTL on Chr 11 designated Dfs2 (MGI Accession ID: MGI: 4418089) was detected with an interval of approximately 74.29 Mb and flanked by markers rs3708339 and rs6161623. The QTLs Dfs1 and Dfs2 combined contain over 2600 genes. The present study had two goals. First, to refine the two DF QTL intervals through the use of increased marker densities on Chr 2 and Chr 11 and by two strain haplotype analysis between the parental A/J and 129P3/J strains. Second, to test a candidate gene Accn1 which encodes an amiloride-sensitive cation channel 1, neuronal (degenerin) and resides on Chr 11 near the QTL peak detected.

/pdf/fine-mapping-of-dental-fluorosis-quantitative-trait-loci-in-4ltslbjdjl.pdf

Fine mapping of dental fluorosis quantitative trait loci in mice.

Excessive systemic exposure to fluoride (F) can lead to disturbances in bone homeostasis and dental enamel development. We have previously shown strain-specific responses to F in the development of dental fluorosis (DF) and in bone formation/mineralization. The current study was undertaken to further investigate F responsive variations in bone metabolism and to determine possible relationships with DF susceptibility. Seven-week-old male mice from FVB/NJ, C57BL/6J, C3H/HeJ, A/J, 129S1/SvImJ, AKR/J, DBA/2J, and BALB/cByJ inbred strains were exposed to NaF (0 or 50 ppm as F–) in drinking water for 60 days. Sera were collected for F, Ca, Mg, PO4, iPTH, sRANKL, and ALP levels. Bone marrow cells were subjected to ex vivo cell culture for osteoclast potential and CFU colony assays (CFU-fibroblast, CFU-osteoblast, CFU-erythrocyte/granulocyte/macrophage/megakaryocyte, CFU-granulocyte/macrophage, CFU-macrophage, and CFU-granulocyte). Femurs and vertebrae were subjected to micro-CT analyses, biomechanical testing, and F, Mg, and Ca content assays. DF was evaluated using quantitative fluorescence and clinical criteria. Strain-specific responses to F were observed for DF, serum studies, ex vivo cell culture studies, and bone quality. Among the strains, there were no patterns or significant correlations between DF severity and the actions of F on bone homeostasis (serum studies, ex vivo assays, or bone quality parameters). The genetic background continues to play a role in the actions of F on tooth enamel development and bone homeostasis. F exposure led to variable phenotypic responses between strains involving dental enamel development and bone metabolism.

/pdf/phenotypic-variation-of-fluoride-responses-between-inbred-25cfykie2g.pdf

Phenotypic variation of fluoride responses between inbred strains of mice.

Systemic exposure to greater than optimal fluoride (F) can lead to dental fluorosis. Parental A/J (dental fluorosis susceptible) and 129P3/J (dental fluorosis resistant) inbred mice were used for histological studies and to generate F2 progeny. Mice were treated with 0ppm or 50ppm F in their drinking water for 60 days. A clinical criterion (modified Thylstrup and Fejerskov categorical scale) was used to assess the severity of dental fluorosis for each individual F2 animal. Parental strains were subjected to histological examination of maturing enamel. Fluoride treatment resulted in accumulation of amelogenins in the maturing enamel of A/J mice. Quantitative trait loci (QTL) detection was performed using phenotypic extreme F2 animals genotyped for 354 SNP-based markers distributed throughout the mouse genome followed by Chi square analysis. Significant evidence of association was observed on chromosomes 2 and 11 for a series of consecutive markers (p<0.0001). Further analyses were performed to examine whether the phenotypic effects were found in both male and female F2 mice or whether there was evidence for gender-specific effects. Analyses performed using the markers on chromosomes 2 and 11 which were significant in the mixed-gender mice were also significant when analyses were limited to only the male or female mice. The QTL detected on chromosomes 2 and 11 which influence the variation in response to fluorosis have their effect in mice of both genders. Finally the QTL in both chromosomes appear to have an additive effect.

Dong Yan

Papers

Genetic background influences fluoride's effects on osteoclastogenesis

Detection of Dental Fluorosis-Associated Quantitative Trait Loci on Mouse Chromosomes 2 and 11

Fine mapping of dental fluorosis quantitative trait loci in mice.

Phenotypic variation of fluoride responses between inbred strains of mice.

Detection of dental fluorosis associated QTL on mouse chromosomes 2 and 11