Fluoride reduces the expression of enamel proteins and cytokines in an ameloblast-derived cell line

Abstract

Objective:

To investigate the effects of two different fluoride concentrations on the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins by an ameloblast-derived cell line.

Methods:

Murine ameloblast-derived cells (LS-8), mouse odontogenic epithelia, were exposed to 1 or 5 ppm sodium fluoride (NaF) (0.46 and 2.25 ppm F, respectively) for 1, 3 and 7 days. The effect of NaF on the mRNA expression of enamel proteins was quantified; the secretion of cytokines, and interleukins, and the alkaline phosphatase (ALP) activity, into the cell culture medium was measured and compared to untreated controls. The effect on cell growth after 1- and 3-days in culture was measured using BrdU incorporation.

Results:

Fluoride at 2.25 ppm reduced mRNA expression of the structural enamel matrix proteins amelogenin (amel), ameloblastin (ambn), enamelin (enam), and the enamel protease matrix metallopeptidase-20 (MMP-20). Similarly several vascularisation factors (vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10), was also reduced by 2.25 ppm fluoride. ALP activity and proliferation were stimulated by 0.46 ppm fluoride but inhibited by 2.25 ppm fluoride.

Conclusions:

These results indicate that fluoride may impact on the expression of structural enamel proteins and the protease responsible for processing these proteins during the secretory stage of amelogenesis and go some way to explaining the mineralization defect that characterises fluorotic enamel.

1. Introduction

Numerous deleterious factors, including hereditary disorders and environmental factors have been implicated in underpinning the mechanism(s) responsible for enamel defects.¹ The physical properties and physiological function of enamel are directly related to the composition, orientation, disposition, and morphology of the mineral components within the tissue.² Fluoride is well known as a specific and effective caries prophylactic agent, and its systematic application has therefore been recommended widely.³ However, fluorosis, a disturbance of enamel development and mineralization, may occur when acute or chronic exposure to excessive amounts of fluoride takes place.⁴ The severity of the resultant hypoplastic or hypomineralized enamel defects depends on the age of the patient during fluoride exposure since the early stages of amelogenesis are more sensitive to perturbation by fluoride than the later stages.⁵ The development of fluorosis is believed to be caused by a disturbance of ameloblast function and a reduction in the amount of secreted enamel matrix⁶ or to the ability to degrade enamel matrix proteins during mineral phase maturation.⁷ The thickness and mineral content of enamel are a reflection of the unique molecular and cellular activities that take place during amelogenesis. Amelogenesis involves a secretory stage, during which ameloblasts elaborate a partially mineralized extracellular protein matrix that defines the crown morphology. Matrix metallopeptidase-20 (MMP-20) is co-secreted and serves to proteolytically modify the matrix. In the subsequent maturation stage, ameloblasts secrete a second proteolytic enzyme kallikrein-4 (KLK4), which completely degrades the matrix. The resultant fluid filled space is occluded by mineral as the enamel crystals grow significantly in response to an increased influx of mineral ions into the tissue by the maturation stage ameloblasts. Excess fluoride is thought to decrease the proteolytic activity of the enamel enzymes and reduce the processing of amelogenins causing the retention of organic material in the maturation stage enamel which inhibits the final mineralization of the enamel.⁸ Excessive fluoride may even initiate an endoplasmic reticulum stress response in ameloblasts that interferes with protein synthesis and secretion.⁹ In vivo effects of fluoride on tooth development have been extensively investigated¹⁰; however the nature of any direct effects on ameloblasts and the molecular mechanisms involved remains unclear. The aim of this study was to use an ameloblast-derived cell line (LS8) from odontogenic epithelia as an in vitro model to investigate how fluoride treatment affects cell proliferation, and the expression of enamel proteins, alkaline phosphatase (ALP), cytokines and interleukins.

2. Materials and methods

2.1. Cell Culture and treatment

Differentiated ameloblasts enter a secretory phase and are characterised by the presence of the Tomes’ process which displays the secretory surface responsible for enamel protein secretion. Once the maturation phase begins the Tomes’ process is lost as enamel protein secretion ceases. The mouse immortalized ameloblast-derived cell line, LS-8 exhibits a quasi-Tomes’ process and on this basis are characteristic of secretory phase ameloblasts.¹¹ However, whilst the LS8 cells express many of the gene associated with amelogenesis, they do not elaborate a typical rodent extracellular enamel matrix in culture.^9,12 LS-8 cells (passage 4) were maintained in Dulbecco’s Modified Eagle’s Medium (Sigma–Aldrich, St. Louis, USA) supplemented with foetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 mg/ml).¹² The cells were cultured at 37 °C and incubated with 1 ppm (24 μM) or 5 ppm (120 μM) sodium fluoride (Fluka BioUltra, Sigma–Aldrich, St. Louis, USA), corresponding to 0.46 and 2.25 ppm fluoride, respectively. Subsequently, both cell culture media and cells were harvested after a further incubation period of 1, 3 and 7 days at 37 °C. Experiments were performed in triplicate with controls consisting of identical experiments run concurrently but minus added sodium fluoride.

The effect of fluoride on cell proliferation, and the ameloblast differentiation markers ALP and enamel proteins (amel, enam, MMP-20, ambn)¹³ and cytokine secretion was measured. In addition, the effect of fluoride on calcium concentration in the cell culture medium was measured.

2.2. Cell proliferation

Cells were seeded in 96-well plates (1000 cells/well), and cultured for 24 h. Then the cells were washed once with serum-free medium prior to the addition of fresh medium with or without added NaF. After 4 h, 5-bromo-20-deoxyuridine (BrdU)-labelling solution (Roche Molecular Biochemicals, Mannheim, Germany) was added, and the cells cultured for an additional 18 h before incorporation of BrdU was measured as described by the manufacturer. Briefly, the labelling medium was removed and the cells were fixed and genomic DNA denaturated by adding 150 ml FixDenat per well for 30 min at room temperature. The FixDenat-solution was removed and 100 μl of peroxidase-conjugated anti-BrdU antibody solution was added per well and incubated at room temperature for 90 min. The cells were then washed three times with 200 μl washing solution before 100 μl of substrate; Luminol/4-idophenol was added. After 3 min, chemiluminescence was measured (RLU1/4 relative luminiscence units) using a microplate luminometer (Fluoroskan Ascent FL, Labsystems).

2.3. Lactate dehydrogenase (LDH) activity

Cells were cultured to 90% confluence prior to treatment. Cell viability was confirmed by monitoring the release of lactate dehydrogenase (LDH) into the medium at all harvest times. LDH was measured using the microplate based Cytotoxicity Detection Kit (LDH) (Boehringer, Mannheim, Germany). According to the manufacturer’s protocol, 50 μl aliquots of cell culture medium were used and quantified spectroscopically against a lactate standard curve using a microplate reader at 450 nm.

2.4. Alkaline phosphatase (ALP) activity

Cells were cultured to 90% confluence prior to treatment. ALP activity was quantified in the cell media harvested after either 1, 3 or 7 days culture by measuring the hydrolysis of p-nitrophenyl phosphate (pNPP) (Sigma) at 405 nm. Standard curves, constructed using calf intestinal alkaline phosphatase (CIAP) (Promega, Madison, WI, USA), were run in parallel for quantification purposes. The total protein content in the culture medium was determined using Sigma Microprotein PR assay kit with a Protein Standard Solution Calibrator (Sigma Diagnostics St. Louis, USA). Analyses were performed using a Cobas Mira chemistry analyzer (Roche Diagnostics, Germany). Intra-assay and inter-assay variability were less than 2.4% and 3.2%, respectively. The assay detection range was 10–2000 mg/l. ALP activity was calculated in terms of nmol of pNPP/min/mg of total protein in each individual sample. The activity was finally expressed as % of the controls (controls being 100%) at the different time points.

2.5. mRNA isolation

Cells were lysed in lysis/binding buffer (100 mM Tris–HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, pH 8.0, 0.5 mM dithiothreitol [DTT], and 1% sodium dodecyl sulphate [SDS]) and mRNA was isolated using magnetic beads [oligo (dT)₂₅] as described by the manufacturer (Dynal AS, Oslo, Norway). Beads containing mRNA were resuspended in 10 mM Tris–HCl, pH 8.0, and stored at −70 °C until use. 10 μl of the mRNA-containing solution was used to synthesize a first-strand complementary DNA (cDNA) using the iScript cDNA Synthesis Kit which contains both oligo(dT) and random hexamer primers (Bio-Rad, Hercules, CA, USA).

2.6. Real-time PCR quantification

Reactions were performed and monitored using real time PCR (iCycler iQ, Bio-Rad, Hercules, CA, USA). The 2X iQ SYBR Green Supermix was based on iTaq DNA polymerase (Bio-Rad, Hercules, CA, USA). cDNA samples (1 μl in a total reaction volume of 25 μl) were analysed both for the genes of interest and the reference genes (β-actin and GAPDH). The cycling profile was as follows: samples were denatured at 94 °C for 5 min followed by 40 cycles of annealing at 57 °C for 30 s, primer extension at 72 °C for 30 s, and denaturing at 95 °C for 30 s. Finally, one 3 min extension cycle completed the reaction sequence. Reactions were performed in duplicate in a 96-well plate. Cycle threshold (Ct) values were obtained graphically. Gene expression was normalized to β-actin and GAPDH and presented as ΔCt values. Comparison of gene expression between untreated control samples and treated samples was derived from subtraction of control ΔCt values from treatment ΔCt values to give a ΔΔCt value, and relative gene expression was calculated as 2^−ΔΔCt and normalized to controls. The efficiency of each set of primers was always higher than 90%. The nucleotide sequences of the oligonucleotides are shown in Table 1. Ten microlitre of the reaction mixture was separated on 2% agarose gels and stained with ethidium bromide to verify the identity of the PCR products by comparison to their predicted size.

Table 1 –

Primers used in RT-PCR of LS8 cells.

Primer name	Refseq accession	Sequence
Amel	NM_009666.1	5′-TGAGGTGCTTACCCCTTTGAA 5′-GGAACTGGCATCATTGGTTGC
Ambn	NM_009664.1	5′-TTCTCCCACCGCATAACTCTTTC 5′-TTTGTTGTGTGCCATTGGTCCCCG
Enam	NM_017468.2	5′-TGGCAATGGACTTTACCCCTATC 5′-GCATCAGGCACAGTTGAGTTTGTAG
MMP-20	NM_013903.2	5′-TAAGAATGCTTGCTGCTCCA 5′-AGCCACCAGAGAGGATCAGA
GAPDH	NM_008084.2	5′-ACCCAGAAGACTGTGGATGG 5′-CACATTGGGGGTAGGAACAC
Actb	NM_007393.2	5′-GCTTCTTTGCAGCTCCTTCGT 5′-ATATCGTCATCCATGGCGAAC

Listed are primer name and direction, BLAST reference, and nucleic acid sequence. Amel, amelogenin; Ambn, ameloblastin; Enam, enamelin, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Actb, beta-actin.

2.7. Cytokine levels in the culture medium

Multianalyte profiling was performed using the Luminex-100 system and the XY Platform (Luminex Corporation, Austin, TX). Calibration microspheres for classification and reporter readings as well as sheath fluid were also purchased from Luminex Corporation. Acquired fluorescence data were analysed by the STarStation software (Version 2.0; Applied Cytometry Systems, Sheffield, UK). Prior to analysis, the samples were concentrated 10 times using MicrosepTM Centrifugal tubes with 3 KDa cut-off from Pall Life Science (Ann Armor, MI, USA). The concentrations of cytokines (FGF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-1α, TNF-α, and VEGF) in the cell culture medium were determined using the Murine Cytokine 20-plex from BioSource (Carmarillo, CA, USA). All analyses were performed according to the manufacturers’ protocols.

2.8. Statistical evaluation

Real-time PCR data (ΔCt values) and the protein analysis data passed normality and equal variance tests. Statistical comparison between groups and treatments was performed using the parametric one-way ANOVA test and post hoc Holm-Sidak tests (SigmaStat software; Systat Software Inc., San Jose, CA). A probability of less than or equal to 0.05 was considered significant. Data were presented as a percentage of untreated cells (=100%) at each time point of observation.

3. Results

3.1. LDH activity in the cell culture media was unaffected by F treatment

Lactate dehydrogenase activity (LDH) in the cell culture medium was unchanged after fluoride treatment (data not shown) at any of the time points used, indicating that fluoride at the concentrations used here had no major cytotoxic effects on the LS-8 cells.

3.2. Proliferation and ALP activity were enhanced by 0.46 ppm and reduced by 2.25 ppm fluoride

Cell growth, measured by BrdU incorporation, was enhanced by 0.46 and 2.25 ppm fluoride after 1 day exposure (to 154% ± 15.5 of control, p = 0.002) and 3 days (to 131% ± 6.9 of control, p < 0.001), and reduced by 2.25 ppm fluoride after 3 days (to 76% ± 5.3 of control, p = 0.003) compared to control (Fig. 1A).

Fig. 1 –

Effect of fluoride (0.46 and 2.25 ppm) on proliferation (A) and alkaline phosphatase activity (B) in the cell culture medium from LS8 cells (n = 6). The data is presented as mean ± SD and calculated as a percentage of untreated cells at each time point.

We observed small, but significant changes in ALP activity following fluoride treatment compared to controls. Fluoride at 0.46 ppm enhanced ALP activity after both 1 day (to 125.9% ± 7.3 of control, p = 0.026) and 3 days (to 120.9% ± 0.3 of control, p < 0.001), whereas fluoride at 2.25 ppm reduced the ALP activity (to 90.3% ± 1.8 of control, p < 0.001) after 3 days (Fig. 1B).

3.3. High dosages of fluoride reduced the expression of enamel proteins

After 7 days of exposure to the high dose of fluoride (2.25 ppm), relative gene expression of amel was significantly reduced (to 63% ± 1.8 of control, p < 0.001) (Fig. 2A), MMP-20 (to 43.8% ± 9.7 of control, p < 0.001) (Fig. 2B), enam (to 59.7% ± 3.5 of control, p = 0.002) (Fig. 2C), and ambn (to 76.8% ± 3.1 of control, p = 0.001) (Fig. 2D) compared to control. We observed a significant reduction in MMP-20 (to 70.7% ± 8.7 of control, p = 0.001; to 86.1% ± 2.7 of control, p = 0.04,) and of ambn mRNA expression (to 74.8% ± 4.4 of control, p = 0.012; to 78.5% ± 11.6 of control, p = 0.025) after 1-day exposure to both concentrations of fluoride (0.46 ppm and 2.25 ppm, respectively). However, the mRNA expression of enam was enhanced (to 168.6% ± 12.7 of control, p < 0.001; to 159.4% ± 11.3 of control, p < 0.001) after 1 day compared to control.

Fig. 2 –

Effect of fluoride (0.46 and 2.25 ppm) on mRNA expression of amelogenin (amel) (A), matrix metallopeptidase-20 (MMP-20) (B), enamelin (enam) (C) and ameloblastin (ambn) (D) in LS8 cells (n = 6). The data is presented as mean ± SD and calculated as a percentage of untreated cells at each time point.

3.4. Fluoride reduced the secretion of vascular signalling factors into the cell culture medium

The highest dosage of fluoride (2.25 ppm) reduced the concentration of monocyte chemoattractant proteins (MCP-1) in the cell culture medium to 7.9% ± 1.0 (p = 0.001) of control at 7 days (Fig. 3A). A similar down regulatory effect of fluoride was also found on the concentrations of vascular endothelial growth factor (VEGF) (to 9.2% ± 6.8 of control, p = 0.002) (Fig. 3B) and interferon inducible protein 10 (IP-10) (to 15.0% ± 9.12 of control, p = 0.003) (Fig. 3C) in the cell culture medium relative to control levels. The IP-10 concentration was also reduced after exposure to 0.46 ppm fluoride (to 50.0% ± 28.9 of control, p = 0.03) at day 7. Interestingly, 0.46 ppm fluoride significantly enhanced the VEGF concentration (to 155.6% ± 19.8 of control, p = 0.006) at day 1. Neither fluoride concentration had any significant effect on any of the other cytokines examined (FGF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, IL-13, IL-17, KC, MIG, MIP-1α, and TNF-α).

Fig. 3 –

Effect of fluoride (0.46 and 2.25 ppm) on the concentration of monocyte chemoattractant proteins (MCP-1) (A), vascular endothelial growth factor (VEGF) (B), and interferon inducible protein 10 (IP-10) (C) in the cell culture medium from LS8 cells (n = 6). The data is presented as mean ± SD and calculated as a percentage of untreated cells at each time point.

4. Discussion

Mean DMFT values fall from a value of 7 to 3.5 when fluoride is added to drinking water at 1.0 ppm in temperate regions. At drinking water fluoride concentrations of 1 ppm, around 20% of children exhibit dental fluorosis but at a level that is not cosmetically obvious to the children or their parents.¹⁴ Increasing the fluoride concentration to 2.6 ppm continues to decrease caries incidence but this decrease is slight and is associated with increasing severity of fluorosis. The margin between the beneficial effects of fluoride and the occurrence of dental fluorosis is small and increasing fluoride intake, either by increasing drinking water fluoride concentration per se, or by increasing consumption of water fluoridated at 1 ppm (e.g. in hot climates) can tip the balance in favour of fluorosis (WHO).

The mechanism by which excess fluoride leads to the eruption of fluorotic enamel is still not completely clear. Previous studies have centred on the potential effects of fluoride on the maturation stage of amelogenesis since this is the developmental stage at which amelogenesis appears to be most susceptible to excessive fluoride. Rodent models have been widely used to study fluorosis but the levels of dietary fluoride required to drive up serum fluoride concentrations to levels leading to fluorosis are typically an order of magnitude greater than in humans (e.g. 50 ppm fluoride added to drinking water) presumably due to rodents having a greater excretory capacity for fluoride. Cell and organ culture systems have also being used to study fluorosis but the appropriateness and physiological relevance of the fluoride dose used in a particular study is of prime importance if biologically meaningful data is to be obtained. Human plasma fluoride concentrations ranging from 0.5 to 1.5 μmol/l (~0.001–0.03 ppm) are typical for populations served with drinking water containing 1.0 ppm fluoride.¹⁵

Naturally, when excess fluoride is ingested, plasma levels are increased (e.g. up to 10 or 20 μmol/l (0.19–0.38 ppm) where drinking water contains 10 ppm fluoride). In the present cell culture based study we have elected to apply fluoride at 0.46 or 2.25 ppm which is considerably higher than plasma fluoride levels found even under extremely heavy dietary fluoride loads. However, ameloblasts are in intimate contact with the enamel matrix they secrete, and the enamel fluid present in this matrix may contain fluoride levels above those found in plasma. For example, secretory stage enamel fluid in normally fed pigs contains around 5 μmol/l fluoride (~0.1 ppm).¹⁶ Making the assumption that the enamel fluid fluoride levels increase in proportion to plasma levels a high fluoride plasma load 10 times normal levels would drive enamel fluid fluoride up to 50 μmol/l, or about 1 ppm. The fluoride levels we have used here (0.46 or 2.25 ppm) bracket this theoretical concentration of fluoride found in fluorotic enamel fluid.

We found that 2.25 ppm fluoride reduced both the gene expression and the secretion of vascularisation factors, whereas fluoride at 0.46 ppm enhanced the ALP activity and proliferation of ameloblasts.^17–20 We observed enhanced cell proliferation at 0.46 ppm fluoride, but no effects at 2.25 ppm compared to untreated cells. Our findings are in accordance with Yan and co-workers who found a bell-shaped dose effect for fluoride on the proliferation of primary human enamel organ epithelial cells. Proliferation rate was maximum at 0.3 ppm fluoride (16 μM) whereas fluoride concentrations >19.2 ppm (1024 μM) inhibited proliferation.²¹ In contrast, Sharma and co-workers reported that fluoride concentrations between 2.4 ppm and 18.75 mM (125 μM to 1 mM fluoride) had no effect on the proliferation of LS8 cells.⁹

Administration of calcium has previously been reported to induce cell differentiation and up-regulate of amelogenin.²² Fluoride might oppose this effect by binding calcium.

We found that 0.46 ppm fluoride increased ALP activity slightly whereas ALP activity was reduced by 2.25 ppm fluoride. The stimulatory effect of 1 ppm fluoride has on ALP activity and proliferation has previously been described in pulp cells.²³ A reduction in ALP activity by high concentrations of fluoride has also previously been described.²¹

We observed that 0.46 ppm fluoride had no effect on enamel protein gene expression in the ameloblast-derived cell line whereas 2.25 ppm fluoride reduced the mRNA expression of amel, ambn and enam. These enamel proteins, as well as MMP-20, are critical for the structural organization of apatite crystals during enamel mineralization, and reduced enamel protein gene expression would lead to reduced enamel matrix production resulting in the production of defective enamel. The effects of fluoride on enamel development through reduced enamel protein expression, as described here, may be just a contributory factor involved in fluorosis since fluoride has also been found to inhibit downstream protein synthesis and secretion by inducing endoplasmic reticulum stress in ameloblasts⁹ and low dosages of fluoride have been found to down-regulate MMP-20 expression in vitro which could compromise correct extracellular enamel protein processing.²⁴

Our in vitro study only modelled some cellular effects of fluoride, and did not explore the post secretory aspects of amelogenesis such as enamel crystal nucleation and growth. Clinically, it might be difficult to differentially diagnose hypomaturation enamel defects caused by either fluorosis or amelogenesis imperfecta,²⁵ and some of the mechanisms involved in hereditary enamel malformations like amelogenesis imperfecta (AI) lead to a reduction in amel, enam and/or MMP-20 expression.²⁶

We measured the effects of fluoride on several cytokines and found that vascular signalling factors in LS8 cells were reduced in a dosage and time related manner after exposure to 2.25 ppm fluoride. The cytokines IP-10, VEGF and MCP-1 are fundamental for the processes of development, proliferation and tissue differentiation. Fluoride reduced the levels of all three cytokines by 60–90% relative to the controls. Vascular endothelial growth factor (VEGF) is an important signalling protein involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature.²⁷ Monocyte chemoattractant protein-1 (MCP-1) acts as a chemokine to recruit mononuclear cells to the dental follicle,²⁸ and IP-10 is an antiangiogenic protein that selectively affects endothelial cells with respect to apoptosis and proliferation.²⁹ The distribution of blood vessels in the initial stage of tooth development has been shown to be important for differentiation of the tooth germ. Quite apart from any role vascularisation may play in the differentiation of the tooth germ it is also a key event in the maturation stage of amelogenesis itself. The enamel organ becomes more vascularised with fenestrated capillaries during maturation presumably in response to the increased requirement for mineral ions as the ameloblasts pump calcium and phosphate into the enamel matrix to bring about complete mineralization of the enamel. Any compromise in the ability of the enamel organ to supply mineral ions to the maturation stage enamel could disrupt final maturation and lead to the eruption of porous fluorotic enamel.

We can only speculate on the implications of our findings. Tooth enamel is the hardest and most highly mineralized (95%) hard tissue and after the maturation stage, the ameloblasts undergo apoptosis and the erupted acellular enamel, unlike bone or other tissues of the body, is not able to undergo cell directed regeneration and repair. Enamel is unique since after eruption it is exposed to the oral environment, and is immediately susceptible to wear and demineralization due to acid attack (via the processes of caries or erosion). Paediatric patients with enamel hypomineralization or hypoplasia often suffer from sensitive or decayed teeth and fluoride prophylaxis is widely recommended. If tooth formation is already compromised due to illness or hereditary factors, administration of high fluoride dosages might induce an additional reduction of enamel protein expression in any unerrupted permanent teeth still undergoing amelogenesis. This work suggests that excessive amounts of fluoride may reduce the expression of enamel proteins which are essential for normal enamel development. Our results indicate that changes in the expression of cytokines, as well as enamel proteins might contribute to the detrimental effect of excess fluoride on enamel development.

The LS8 cells are an immortalized ameloblast-like cell line that expresses enamel-specific genes such as amelogenin and ameloblastin.^11,30 Molecular mechanisms found in cell lines do not always mimic the expression in primary cells, or in the complex milieu of a developing organ in vivo, however, some of the effects we have observed may explain some of the clinical characteristics of fluorosis. The use of an in vitro system and an ameloblast cell-line limits the interpolation of our results. However, we hypothesise that a reduction in VEGF, IP-10, MPC-1, amel, ambn, enam and MMP-20 expression are important factors underpinning the pathological mechanism responsible for fluorosis.