Enamel proteases in secretory and maturation enamel of rats ingesting 0 and 100 PPM fluoride in drinking water

Dental enamel formed during ingestion of high levels of fluoride in drinking water has an increased organic content in the maturation stage, which may be due to a delay in the breakdown of amelogenins during the early-maturation stage of enamel formation. This delay in the breakdown of amelogenins in fluorosed enamel suggests an effect of fluoride on enamel proteases which hydrolyze the early secreted enamel proteins. In this study, we compared the proteases present in fluorosed and control secretory-stage and maturation-stage enamel. Enamel was demineralized and separated in SDS gels containing 0.1% gelatin. After incubation in 100 mmol/L Tris-HCl, pH 8, with 10 mmol/L CaCl2, the gels were stained with Coomassie Blue, and proteases were seen as clear zones of degraded gelatin. Similar bands of proteolytic activity were seen in fluorosed and in control enamel. In the maturation stage, more proteases were present than in the secretory stage of enamel formation. Less digestion of gelatin substrate occurred in several proteases found in the fluorosed maturation-stage enamel as compared with the control maturation-stage enamel. This suggests that the amount of protease secreted or the activity of the proteases may be altered in fluorosed maturation-stage enamel.     

Biological mechanisms of fluorosis and level and timing of systemic exposure to fluoride with respect to fluorosis.

Enamel fluorosis can occur following either an acute or chronic exposure to fluoride during tooth formation. Fluorosed enamel is characterized by a retention of amelogenins in the early-maturation stage, and by the formation of a more porous enamel with a subsurface hypomineralization. The mechanisms by which fluoride affects enamel development include specific effects on both the ameloblasts and on the developing enamel matrix. Maturation-stage ameloblast modulation is more rapid in fluorosed enamel as compared with control enamel, and proteolytic activity in fluorosed early-maturation enamel is reduced as compared with controls. Secretory enamel appears to be more susceptible to the effects of fluoride following acute fluoride exposure, such as may occur with the use of fluoride supplements. However, both human and animal studies show that the transition/early-maturation stage of enamel formation is most susceptible to the effects of chronic fluoride ingestion at above-optimal levels of fluoride in drinking water.     

Changes in the fluoride-induced modulation of maturation stage ameloblasts of rats

The maturation stage of enamel development is characterized by a cyclic modulation of the ameloblasts between bands of smooth-ended cells and longer bands of ruffle-ended cells. There are cyclic patterns of calcein staining of and 45Ca uptake in the enamel associated with this cellular modulation. Rats were given 0, 75, 100, or 150 ppm fluoride in their drinking water. Fluoride disrupted the cyclic patterns of the maturation stage, resulting in fewer bands of smooth-ended ameloblasts, fewer calcein-stained stripes, and fewer cycles of 45Ca uptake. When animals were given water containing 0 ppm fluoride following ingestion of water containing 100 ppm fluoride, the pattern of calcein staining returned to that of the control enamel. The disruption of the cyclic patterns in the maturation stage and the increased protein content of maturation enamel seem to be among the early events in the development of fluorosis.     

Mechanism and timing of fluoride effects on developing enamel

Fluoride appears to specifically interact with mineralizing tissues, causing an alteration of the mineralization process. In enamel, fluorosis results in a subsurface hypomineralization. This hypomineralized enamel appears to be directly related to a delay in the removal of amelogenins at the early-maturation stage of enamel formation. The specific cause for this delay is not known, although existing evidence points to reduced proteolytic activity of proteinases that hydrolyze amelogenin. This delay in hydrolysis of amelogenins could be due to a direct effect of fluoride on proteinase secretion or proteolytic activity, or to a reduced effectiveness of the proteinase due to other changes in the protein or mineral of the fluorosed enamel matrix. The formation of dental fluorosis is highly dependent on the dose, duration, and timing of fluoride exposure. The early-maturation stage of enamel formation appears to be particularly sensitive to the effects of fluoride on enamel formation. Although the risk of enamel fluorosis is minimal with exposure only during the secretory stage, this risk is greatest when exposure occurs in both secretory and maturation stages of enamel formation. The risk of fluorosis appears to be best related to the total cumulative fluoride exposure to the developing dentition.     

Fluoride binding by matrix proteins in rat mineralizing tissue.

Chronic fluoride exposure in vivo results in alterations in the formation of mineralizing tissues. One possible mechanism for the formation of fluorosed tooth enamel and bone is a binding of fluoride to matrix proteins, resulting in an alteration in their structure and function. Studies were designed to investigate fluoride binding to matrix proteins in vivo and their possible role in fluorosis. Rats were given either 0 or 100 parts/10(6) fluoride in drinking water for 6 weeks to allow the formation of fluorotic mineralizing tissues. The animals were killed by CO2 inhalation, and the enamel and bone were analysed for fluoride and calcium. Matrix binding by fluoride in enamel was determined after extraction of proteins from undemineralized matrix. In bone, the matrix was demineralized and F, Ca and P were determined in both ashed and unashed samples. The studies showed ionic binding of fluoride to the matrix in both enamel and bone, possibly associated with calcium binding by the matrix. There was no difference in the amount of matrix-bound fluoride in control as compared to fluorosed bone or maturation-stage enamel. This indicates that although matrix proteins can bind fluoride, it is not likely that this mechanism is important in the formation of fluorosed mineralizing tissues.     

The effects of chronic high fluoride levels on forming enamel in the rat

Sixty-gramme rats were given either $\text{0, 75, 100}\text{or}\text{150}\text{parts}\text{10}{}^6$ fluoride in their drinking water. After five weeks, the fluoride, the phosphorus and the protein contents of the enamel were compared in control and experimental animals at three stages of enamel development. The mineral content was reduced in pigmented enamel from animals given $\text{75}\text{parts}\text{10}{}^6$ or more fluoride in their drinking water. The fluoride content was elevated in all stages of fluorosed enamel development. At the lowest fluoride level ($\text{75}\text{parts}\text{10}{}^6$), a larger proline content was found in the proteins of the maturing, fluorosed enamel but there was no increase in the protein content. In animals given $\text{100}\text{parts}\text{10}{}^6$ fluoride in their drinking water, the proline content of the protein was greater in maturing, fluorosed enamel, and the total protein content of the post-secretory enamel (maturing and pigmented) was greater than in the controls. These observations indicate that, with increasing levels of fluoride in drinking water, there was an initial delay in the loss of the amelogenin proteins followed by a decreased removal of total protein from the enamel. These results indicate that fluoride interfered with the normal post-secretory, pre-eruptive development of enamel.     

Effect of prenatal and postnatal fluoride on the human deciduous dentition. A literature review

Fluoride passes from the mother to fetal teeth. Much of the fluoride is taken up in secretory enamel, probably by the forming mineral apatite crystals. Some is retained with residual proteins. The low concentration of fluoride in the inner enamel is incorporated mainly during the secretory stage, while the enhanced concentration in the surface enamel is produced during the much longer maturation stage. Mature, hard enamel is generally absent during fetal life. The clinical question is whether prenatal fluoride imparts an additional benefit to the universally accepted effect of postnatal fluoride. In general, surface enamel fluoride levels of deciduous teeth increase with increasing pre- and postnatal fluoride administration. A consistent level of caries protection has been reported with pre- and postnatal administration of fluoride unrelated to the acquisition of fluoride in the surface enamel. Many children develop enamel opacities in their deciduous dentition related by various factors to enamel mineralization disturbances in drinking water areas even low in fluoride. Accumulation of fluoride due to an increased fluoride intake is a feature of fluorosed enamel in the deciduous as well as permanent dentition. The resulting mature fluorosed enamel retains a relatively high proportion of immature matrix proteins onto the crystal surface. The degree of fluorosis of the deciduous dentition is less compared with that of the permanent dentition, due probably to a partial protection afforded by the maternal loss of fluoride, formerly known as the "placental barrier".     

Biological mechanisms of dental fluorosis relevant to the use of fluoride supplements

Fluorosis occurs when fluoride interacts with mineralizing tissues, causing alterations in the mineralization process. In dental enamel, fluorosis causes subsurface hypomineralizations or porosity, which extend toward the dentinal-enamel junction as severity increases. This subsurface porosity is most likely caused by a delay in the hydrolysis and removal of enamel proteins, particularly amelogenins, as the enamel matures. This delay could be due to the direct effect of fluoride on the ameloblasts or to an interaction of fluoride with the proteins or proteinases in the mineralizing matrix. The specific mechanisms by which fluoride causes the changes leading to enamel fluorosis are not well defined; though the early-maturation stage of enamel formation appears to be particularly sensitive to fluoride exposure. The development of fluorosis is highly dependent on the dose, duration, and timing of fluoride exposure. The risk of enamel fluorosis is lowest when exposure takes place only during the secretory stage, but highest when exposure occurs in both secretory and maturation stages. The incidence of dental fluorosis is best correlated with the total cumulative fluoride exposure to the developing dentition. Fluoride supplements can contribute to the total fluoride exposure of children, and if the total fluoride exposure to the developing teeth is excessive, fluorosis will result.     

ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL STUDY OF THE INFLUENCE OF FLUORIDE EXCESS ON THE DEVELOPMENT OF RAT INCISOR TOOTH BUDS

Little information is available on the pathogenesis of fluorosis during the fetal and initial postnatal period. In the present study, female rats received 0 (control), 7 or 100 ppm of sodium fluoride in drinking water, one week before breeding and throughout gestation and nursing periods. The hemimandibles of the offspring were collected at 0, 7 and 14 days of postnatal life (n = 5) and processed for morphological analyses by light and electron microscopy, immunohistochemical analysis for amelogenin and morphometric study of enamel matrix and ameloblasts of incisors. The results showed a decrease in matrix production at the secretory phase at all study periods for the 100 ppm group. In this same group, the secretory ameloblasts showed reduction of enamel matrix secretion, disorganization of mitochondrial crests, large vacuoles at the apical portion of the cytoplasm, retention of intracisternal material and dilatation of some cisterns in the rough endoplasmic reticulum. In the groups of animals aged 7 and 14 days, analysis of variance showed significant reduction (p<0.05) in cytoplasmic volume of 23.80% and 24.75%, respectively, in relation to the control group. The smooth-ended maturation ameloblasts exhibited a large number of vacuoles with electron-dense endocytic matrix, suggesting a delay in the resorption process. Immunohistochemical analysis showed no difference in the intensity and labeling pattern of the enamel matrix in any study group. Interestingly, in offspring at the age of 14 days for the 7 ppm group, there was an increase in the matrix length at the secretory phase. Therefore, part of the excessive dose of sodium fluoride given to the mother in drinking water can reach the offspring through the placenta and mother’s milk, causing morphological changes in ameloblasts and suggesting a reduction in secretion and a delay in matrix resorption.     

Diffusion of fluoride through the rat enamel organ in vitro

This study investigated the diffusion of fluoride through the enamel organ in vitro. The rat molar explants used were entirely in the secretory stage or predominantly in the maturation stage of enamel formation. The removal of the enamel organ or metabolic inhibition with iodoacetate caused significant increases in enamel fluoride uptake at both stages of enamel formation. Inhibition with dinitrophenol caused a significant increase only in the maturation phase. Uptake of fluoride in enamel was related to the fluoride concentration in the medium, except in the maturation stage explants, where increasing the medium fluoride concentration from 0.05 ppm to 0.08 ppm did not significantly increase fluoride uptake at any of the three observation times. The findings indicate that the enamel organ exists as a diffusion-limiting membrane to the movement of fluoride from the extracellular fluid compartment to the developing enamel.     

Effects of fluoride on matrix proteins and their properties in rat secretory enamel

This publication concerns the selective adsorption of rat enamel proteins onto hydroxyapatite, their solubility in aqueous solutions, and the effect that systemic fluoride has on these properties. The enamel proteins used as adsorbates were extracted in 0.5 mol/L acetic acid from the secretory enamel of the upper and lower incisors of SD rats (females, 200-220 g body weight). Equilibration of the proteins with hydroxyapatite was performed in two solutions: (i) 50 mmol/L acetate buffer at pH 6.0 and 0 degrees C, and (ii) 50 mmol/L Tris buffer containing 4 mol/L guanidine at pH 7.4 and room temperature. Enamel was dissected from animals, which were given either de-ionized water (control group) or water containing 25, 50, 75, or 100 ppm fluoride as NaF for four weeks. From these enamel samples, the proteins were extracted in sequence with 160 mmol/L NaCl and 3 mmol/L phosphate (pH 7.3), 50 mmol/L carbonate buffer (pH 10.8), and finally, with 0.5 mol/L acetic acid for dissolution of the enamel mineral. The F, Ca, and P contents of the various enamel samples were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoride incorporation into apatite crystals delays amelogenin hydrolysis

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.     

The dissolution rate of enamel in acid in developing rat incisors

This study was undertaken to determine the changes in this dissolution rate at different developmental stages after different fluoride dietary regimes. Four groups of Wistar rats received water with 0, 25, 50 and 100 parts/10(6) fluoride respectively for 10 weeks. Six exposed windows of 2 mm2 were prepared on the enamel surface of the upper incisors, corresponding to six different developmental stages. The acid-dissolution rates were determined at each window by using 1.4 M sodium acetate-hydrochloric acid buffer (pH 2.3). The rate of enamel dissolution was highest in the matrix-formation stage and dropped sharply in a step-wise fashion towards the stages of secondary mineralization and iron deposition. The dissolution rate in the maturation stage decreased significantly with increasing intake of fluoride. However, in the pigmented enamel, the opposite occurred. The iron pigmentation or the porosity in this region of fluorosed enamel might be responsible for the change in the dissolution rate of the pigmented enamel.     

Fluoride down-regulates the expression of matrix metalloproteinase-20 in human fetal tooth ameloblast-lineage cells in vitro

Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10  µ M fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10  µ M NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10  µ M NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.     

Fluoride-induced developmental changes in enamel and dentine of European roe deer (Capreolus capreolus L.) as a result of environmental pollution

Using macroscopic, microradiographic and scanning electron-microscopic methods, the effects of increased fluoride exposure on enamel and dentine formation were studied in fluorosed mandibular premolars and molars of roe deer from the heavily industrialized Ruhr area, Germany. Macroscopically, fluorosed teeth were characterized by opaque and stained enamel and in more severe cases also by enamel surface lesions, reduction or loss of enamel ridges on their occlusal surfaces and increased wear. Microradiographically, fluorosed enamel exhibited different degrees of subsurface hypomineralization, in part apparently indicating a fluoride effect during enamel maturation. In some specimens, a pronounced but varying enhancement of the pattern of Retzius lines was observed throughout the enamel, denoting strongly intermittent fluoride exposure during enamel matrix secretion. This variation in exposure was also reflected histologically in dentine, by bands of interglobular dentine and marked accentuation of incremental lines. Microradiography of sections through enamel surface hypoplastic lesions showed the enamel forming the bottom and partly also the walls of the lesions to be highly mineralized. Scanning electron microscopy showed that the outer enamel along the more pronounced hypoplastic lesions consisted of stacked, thin layers of ‘aprismatic’ enamel, indicating that the ameloblasts in these areas had lost the distal (rod-forming) regions of their Tomes' processes. These observations demonstrate that the origin of enamel hypoplasias in deer clearly differs from that in rodents, where fluoride induces the formation of subameloblastic cysts. The differences in the degree of fluorotic alteration between the teeth of a single tooth row could be related to the developmental sequence of the dentition in roe deer. The roe deer is thus considered to be a very sensitive and useful bioindicator of environmental pollution by fluorides.     

Cadherin-related neuronal receptors in incisor development

Cadherins are cell adhesion molecules that are critical for tissue development. In this report, we identified members of the cadherin family cadherin-related neuronal receptors (CNRs) 1 and 5 expressed in rat incisors by the differential display method. Quantitative RT-PCR revealed that CNR1 mRNA is expressed in the secretory stage but reduced in the early-maturation stage, while CNR5 mRNA is expressed in both these stages. In situ hybridization showed that strong expression of CNR1 is strong in the secretory stage, but reduced in the early phase and diminished in the late phase of the early-maturation stage. CNR5 mRNA is expressed almost at the same levels in the secretory and in the early phase of the early-maturation stages but is absent in the late phase of the early-maturation stage. Both CNR1 and 5 mRNA are continuously expressed in odontoblasts. Immunohistology showed that CNR proteins are expressed in the secretory and early-maturation stages of ameloblasts, but no protein expression at the late-maturation stage was observed. CNR proteins were continuously expressed in odontoblasts. We found that recombinant CNR1 binds dental epithelial and mesenchymal cells through N-terminal domain EC1 in vitro. These results suggest that CNR1 and CNR5 may play an important role in enamel and dentin formation, probably through cell-cell and/or cell-matrix interactions.     

Effects of fluoride on rat dental enamel matrix proteinases

Enamel fluorosis is characterised by increased porosity and a delay in the removal of enamel matrix proteins as the enamel matures. Amelogenin is the primary matrix protein in secretory-stage dental enamel. As enamel matures, amelogenins are hydrolysed by a number of enamel proteinases, including matrix metalloproteinase-20 (MMP-20 or enamelysin) and serine proteinase. Here, the effect of ingested fluoride on the relative activity of proteinases in the enamel matrix and the specific effect of fluoride on MMP-20 activity were examined. Proteinase activity relative to total enamel matrix protein was measured by fluorescence assay of enamel matrix dissected from rats given 0, 50, or 100 parts per 10(6) fluoride in their drinking water. To determine the specific effect of fluoride on the activity of MMP-20, the hydrolysis of a full-length recombinant human amelogenin by recombinant MMP-20 (rMMP-20) in the presence of 0, 2, 5, 10 or 100 microM fluoride was compared by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). In addition, a fluorescent peptide assay was developed to quantify enzyme activity against the tyrosine-rich amelogenin peptide cleavage site. In the late maturation stage, total proteinase activity per unit protein was lower in the fluoride-exposed rats than in the control rats. This in vivo finding indicates that fluoride ingestion can alter the relative amount of active proteinase in mature enamel. Hydrolysis of amelogenin at neutral pH by rMMP-20 was reduced in the presence of 100 microM F. In the peptide assay, rMMP-20 activity was significantly reduced by concentrations of fluoride as low as 2 microM at pH 6, with no significant effect at pH 7.2. These in vitro assays show that micromolar concentrations of fluoride can alter metalloproteinase activity, particularly when the pH is reduced to 6.0. These studies suggest that the effects of fluoride on enamel matrix proteinase secretion or activity could be involved in the aetiology of fluorosis in enamel and other mineralising tissues.     

Inhibition of experimental dental caries using an intraoral fluoride-releasing device

An intraoral fluoride releasing device designed to provide continual topical fluoride therapy for the prevention of dental caries was found to be effective in inhibiting caries in the rat model. Animals fitted with an intraoral device that released approximately 0.15 mg of fluoride per day developed 63% fewer carious enamel areas than animals receiving no treatment. Fluoridated drinking water (10 ppm) produced a 25% reduction in carious enamel areas. The fluoride-releasing device was more effective than ad libitum fluoridated drinking water in inhibiting caries on the approximal and sulcal surfaces. These results agree with the hypothesis that the continual presence of fluoride in oral fluids enhances the cariostatic effect of fluoride and, when combined with the results of earlier primate and human trials of the intraoral fluoride-releasing device, suggest that this fluoride delivery system can be developed into an effective anticaries agent.     

Effect of long-term administration of fluoride on the levels of EDTA-soluble protein and gamma-carboxyglutamic acid in rat incisor teeth

EDTA-soluble material, extracted from incisor teeth of rats given 50 ppm fluoride (NaF) in drinking water ad libitum from conception to age nine wk, contained significantly higher levels of protein (P less than 0.01) and gamma-carboxyglutamic acid (P less than 0.01) than did similar material from control rats. Tooth fluoride levels indicated that the incisors were mildly fluorosed. Analysis of these data indicates that mild fluorosis of rat incisor teeth involves not only the characteristic disturbances of pigmentation and mineral structure but also some alteration of the matrix protein.