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Micromolar Fluoride Alters Ameloblast Lineage Cells in vitro
Q. Yan,
Y. Zhang,
W. Li and
P.K. DenBesten*
Department of Orofacial Sciences, University of California at San Francisco, 513 Parnassus Ave. S-704, San Francisco, CA 94143-0422, USA
Correspondence: * corresponding author, Pamela.denbesten{at}ucsf.edu
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ABSTRACT
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Fluorosed enamel is caused by exposure to fluoride during tooth formation. The objective of this study was to determine whether epithelial ameloblast-lineage cells, derived from the human enamel organ, are directly affected by micromolar concentrations of fluoride. Cells were cultured in the presence of fluoride, and proliferation was measured by BrdU incorporation. The effect of 0, 10, or 20 µM fluoride on apoptosis was determined by the flow cytometry apoptotic index. The effects of fluoride on gene expression were investigated by SuperArray microarray analysis and real-time PCR. Fluoride had a biphasic effect on cell proliferation, with enhanced proliferation at 16 µM, and reduced proliferation at greater than 1 mM F. Flow cytometry showed that both 10 µM and 20 µM NaF significantly increased the apoptotic index of ameloblast-lineage cells. There was no general effect of fluoride on gene expression. These results indicate multiple effects of micromolar fluoride on ameloblast-lineage cells.
Key Words: ameloblasts • proliferation • apoptosis • fluoride • in vitro
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INTRODUCTION
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Teeth exposed to higher-than-optimal fluoride concentrations have various degrees of enamel hypomineralization, known as enamel fluorosis. However, the mechanism(s) responsible for the formation of enamel fluorosis remain unclear (Aoba and Fejerskov, 2002; Robinson et al., 2004). Fluoride is likely to have multiple effects, including altered protein/mineral interactions. However, it is also possible that fluoride directly affects the cells of the enamel organ, including the ameloblasts (DenBesten et al., 1985; DenBesten and Thariani, 1992; Smith et al., 1993; Zhang et al., 2006).
Several studies have shown effects of fluoride on enamel organ cells. Gibson and co-workers (Li et al., 2005) used organ culture to show that 2 mM fluoride affected the Rho/ROCK signal transduction pathway, resulting in elevated F-actin in ameloblasts. Millimolar levels of fluoride were also found to induce endoplasmic reticulum stress, apoptosis, and caspase-mediated DNA fragmentation in enamel organ epithelial-derived cells (Kubota et al., 2005). In zebrafish exposed to fluoride, there was evidence of perturbations in growth factor signaling and apoptosis (Bartlett et al., 2005).
However, it is well-known that the effects of fluoride are highly dose-dependent. Therefore, for the biologically relevant effects of F in cells of the enamel organ to be determined, it is important that the fluoride concentrations used in these studies be similar to those found in vivo. The relevant F concentrations can only be estimated, but, based on both serum fluoride concentrations and analysis of enamel fluid (Aoba and Moreno, 1987), we propose that fluoride exposure to cells of the enamel organ is limited to micromolar levels. Serum fluoride levels in rodents with relatively constant plasma fluoride concentrations of approximately 5 µM resulted in hypomineralized fluorotic enamel (Angmar-Månsson and Whitford, 1982, 1984), and fluorosis was induced in pig enamel at serum fluoride levels ranging from 5 µM to 12 µM at the time of the animals’ death (Richards et al., 1985). We hypothesized that similar micromolar amounts of fluoride would result in fluorosis in humans. In this study, we determined whether micromolar levels of fluoride can affect cell proliferation, apoptosis, and gene expression in ameloblast-lineage cells isolated from the human enamel organ.
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MATERIALS & METHODS
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Tooth organs were freshly collected from 21- to 22-week-old human fetal tissue obtained under approved guidelines set by the University of California at San Francisco. The tooth organs, including teeth ranging from the bell stage (incisors) to the cap stage (second molars), were dissected within 3 hrs of collection, and the cells were isolated and cultured as previously described (Yan et al., 2006).
Briefly, tooth organs were first digested in collagenase/dispase (2 mg/mL in PBS) for 1 hr at a continuous agitation of 35 rpm at 37°C, then washed with PBS, followed by further digestion with STV (0.05 mM trypsin, 0.025% versene, in phosphate-buffered saline) for 5 min. The cell suspensions were filtered through a 70-µm strainer (Falcon, San Jose, CA, USA), and plated at 5x104 cells/mL on 10-cm Primaria (Becton Dickinson, Franklin Lakes, NJ, USA) tissue culture plates supplied with KGM-2 medium with 0.05 mM Ca2+ recorded as passage 0 (p0). Cells with an epithelial phenotpe were further selected by trypsin-impregnated cloning rings. The cells were subcultured, and either P1 or P2 cells were used for subsequent experiments.
Cell Characterization by Immunohistochemistry
Cells grown on glass chamber slides were fixed in 95% methanol and 5% acetic acid for 30 min at –20°C. Non-specific staining was blocked with 10% fetal bovine serum plus 0.1% bovine serum albumin (BSA) and 0.1% TritonX-100 for 30–60 min at room temperature. Primary antibodies were diluted in the same blocking solution, and immunostaining was performed with mouse anti-human DSP (1:200 dilution; gift from L. Fisher, NIDCR), rabbit anti-recombinant amelogenin (1:2000 dilution), rabbit anti-human TNF (1:200 dilution, Sigma-Aldrich Inc., St. Louis, MO, USA), and rabbit anti-human kallikrein 4 (1:500 dilution, QED Bioscience Inc., San Diego, CA, USA). Pre-immune immunostaining of control samples was performed with mouse IgG and rabbit IgG (Sigma). The primary antibody was labeled with a cocktail including the secondary antibody, anti-rabbit IgG-Alexfluor 594 conjugated (Molecular Probe, Eugene, OR, USA) plus anti-mouse IgG-FITC conjugated (Sigma, St. Louis, MO, USA) for 40 min in the dark. Nuclei were counterstained with 0.5 µg/mL Hoechst 33342 (Molecular Probe, Eugene, OR, USA) in the dark for 8 min. After being mounted, the sections were immediately photographed with a QIMAGING digital camera attached to a Nikon Eclipse 300 microscope equipped with SimplePCI software (Compix Inc, Sewickley, PA, USA).
The DeadEndTM Fluorometric TUNEL System (Promega Co., Madison, WI, USA) was used to detect the apoptotic cell population in our in vitro-cultured ameloblast-lineage cells, following the manufacturer’s instructions. Green fluorescence identified apoptotic cells (fluorescein-12-dUTP) in a red background counterstained by propidium iodide (PI, Molecular Probes, Eugene, OR, USA) under fluorescence microscopy.
Cell Proliferation
Cells were seeded in 96-well flat-bottomed plates at a density of 2x104 cells/well. After 24 hrs, growth factors were withdrawn for 20 hrs to synchronize the cells, then the medium was refreshed by KGM-2 supplemented with NaF at final concentrations of 0, 4, 16, 64, 256, or 1024 µM, respectively, with 6 repeat assays for each group. The cells were incubated for 48 hrs at 5% CO2, at 37°C. BrdU incorporation into the cells, as a measure of relative cell proliferation, was measured with the use of a BrdU chemiluminescence kit (Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions.
Comparative Expression of Selected mRNAs
Osteogenesis Microarray
We used the commercially available osteogenesis microarray system, SuperArray (SuperArray, Inc., Bethesda, MD, USA; www.superarray.com), to screen for a general effect of fluoride on gene expression, since many of the genes contained in this array were also present in our ameloblast-lineage cells. A GEArray Q series human osteogenesis gene array kit was obtained from SuperArray. RNA (3 µg) from P1 or P2 ameloblast-lineage cells derived from 21-week samples was used as a template to generate Biotin-16-dUTP-labeled cDNA probes, according to the manufacturer’s instructions. The cDNA probes were denatured and hybridized at 60°C with the osteogenesis SuperArray membrane. We then washed the membrane, exposed it with a chemiluminescent substrate, and analyzed it by scanning the x-ray film and importing the image into Adobe Photoshop as a TIFF file. The image file was inverted, and spots were digitized with ScanAlyze software (Shareware, http://rana.lbl.gov/EisenSoftware.htm), and normalized when the background was subtracted as the average intensity value of 3 spots containing plasmid DNA (PUC18). The average of 2 GAPDH spots was used as a positive control, set as the baseline value with which the signal intensities of other spots were compared. With these normalized data, the signal intensities from the membranes were compared via the GEArray Analyzer program (SuperArray Corp.). Each array was repeated three times.
Real-time PCR
For analysis of gene expression by real-time PCR, teeth were grouped according to their developmental sequence. Group 1 included primary incisors, group 2 included primary canines and first molars, and group 3 included primary second molars. After the first subculture, we synchronized the cells by removing growth factors for 20 hrs. When the cells reached 50% confluence, fluoride was added to the media to a final concentration of 0 or 10 µM (triplicate samples). After 48 hrs, mRNA was extracted with the use of an RNeasy Protect Mini Kit (Qiagen, Valencia, CA, USA) and further purified by RNaseOUTTM (Invitrogen, Carlsbad, CA, USA) and RQ1 RNase-Free DNase (Promega, Madison, WI, USA). An Agilent 2100 bioanalyzer was used to test the quality of the isolated RNA. SuperScriptTM II Reverse Transcriptase was applied to reverse-transcribe 20 ng of RNA into cDNA, and random primers (Invitrogen) were used in nested real-time PCR to determine mRNA expression levels. (See APPENDIX for further details.)
Flow-cytometry-based Apoptosis Assay
P1 ameloblast-lineage cells, pooled from 21- and 22-week-old fetal enamel organs, were grown to 50% confluence. The cells were starved for 20 hrs with 1:1 KBM-2 and HEPES, and then plated with KGM-2, with either 0, 10, or 20 µM F, for 48 hrs. The apoptotic population was determined by a flow cytometric assay as previously described (Hamel et al., 1996) and measured in triplicate. For each assay, cells were trypsinized from a 100-mm culture dish and re-suspended in 2 mL ice-cold PBS containing 2% fetal bovine serum, with 1 µg/mL PI, to detect cells without intact membranes, and 2.5% PBS-based cell dissociation buffer (Invitrogen, Carlsbad, CA, USA). Cells were kept on ice until 10 to 15 min before the addition of Hoechst 33342 (Ho342). The solution was brought to room temperature, and Ho342 was added to the cell suspension to a final concentration of 7 µg/mL. After 6 min, a dual-laser FACStar+ cell-sorter (Becton Dickinson, San Jose, CA, USA) was used to detect viable cells (R2, PI–, and Ho342+), as well as cells without intact membranes (R3, PI+). The apoptotic index of both passages was calculated as follows: Apoptotic index = (R4+R5)/(R4+R5+R2), where R4 refers to an early-apoptotic population (PI– and increased Ho342 fluorescence), R5 is the late-apoptotic population (intermediate intensity of PI and decreased Ho342 fluorescence), and R2 is the viable cell population.
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RESULTS
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Immunohistochemistry
Immunohistochemistry showed DSP in all ameloblast-lineage cells. Amelogenin immunostaining was limited to larger, more differentiated, cells, similar to our previously published results (DenBesten et al., 2005; Yan et al., 2006). Some cells stained for KLK-4 and for TNF(Figs. 1A–1D ).
Real-time PCR
Triplicate nested real-time PCR results showed that both amelogenin and MMP-20 mRNA were present, though at relatively low levels compared with other mRNAs amplified in this assay. DSPP, an early marker of ameloblast differentiation, was consistently down-regulated by F exposure in cells harvested from second primary molars (group 1) (Figs. 1E, 1F ), and TNF was up-regulated in this same group of teeth. Expression of DSPP and TNF was not altered in cells harvested from the more developmentally advanced teeth (incisors, first molars, and canines).
Osteogenesis SuperArray
Triplicate osteogenesis arrays showed relatively high levels of expression of cell plasma membrane proteins (see GEO, http://www.ncbi.nlm.nih.gov/geo/, accession #GSE5365, for details). We did not detect differences in expression levels of genes that were consistently expressed in all 3 assays, when comparing the 10-µM-NaF-treated group with control cells (Fig. 2).
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Figure 2. Osteogenesis-related genes expressed in relatively high levels by ameloblast-lineage cells derived from pooled teeth in the cap and bell stages. There were no differences detected between 10-µM-NaF-treated groups and the control cells. Average (SD) values of normalized signal strength of the gene tested: (Control) ITGA3, 0.42 (0.16); COL17A1, 0.29 (0.20); TNF, 0.32 (0.14); EGFR, 0.61 (0.25); FN1, 1.03 (0.39); GAPDH 1 (0); ITGB1, 0.39 (0.19); RUNX2, 0.71 (0.29); VEGF, 0.18 (0.05); (10-µM-NaF-treated groups) ITGA3, 0.22 (0.04); COL17A1, 0.18 (0.09); TNF, 0.22 (0.08); EGFR, 0.47 (0.06); FN1, 0.83 (0.28); GAPDH 1 (0); ITGB1, 0.40 (0.20); RUNX2, 0.74 (0.10); VEGF, 0.24 (0.22) (n = 3).
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Cell Proliferation
Exposure of ameloblast-lineage cells to different concentrations of fluoride, for 48 hrs, resulted in a biphasic effect on cell proliferation. Lower concentrations of fluoride promoted the ameloblast-lineage cells’ proliferation, with the peak around 16 µM, and the higher levels of fluoride (higher than 1 mM) drastically reduced cell proliferation (Fig. 3).
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Figure 3. Cell proliferation rates were measured by immunofluorescent staining of Brdu incorporation, quantitated as relative light units per sec (rlu/s). Fluoride concentrations less than 256 µM promoted ameloblast-lineage cell proliferation, with the peak effect at 16 µM, while the higher levels significantly inhibited cell proliferation relative to control cells. Average (SD) values of groups: 0 µm, 71.9 (19.5); 4 µm, 126.7 (62.5); 16 µm, 164.6 (44.7); 64 µm, 134.3 (48.2); 256 µm, 113.4 (38.9); 1024 µm, 20.5 (12.7); 4096 µm, 14.4 (4.8) (n = 6).
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Apoptosis Characterized by Flow Cytometry
Flow cytometry showed that both 10 µM fluoride and 20 µM fluoride significantly increased the apoptotic index of the in vitro-cultured cells derived from the 21-week-old fetal enamel organ for 2 consecutive passages (Fig. 4). However, for those derived from the 22-week-old fetal enamel organ, these fluoride concentrations increased the apoptotic index only in P1 cells. TUNEL staining indicated apoptosis in relatively few subpopulations of cells (Figs. 4E, 4F).
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Figure 4. Ameloblast-lineage cells exposed to fluoride. (A–C) Flow cytometry of ameloblast-lineage passage 2 cells isolated from 21-week-old fetal tissue exposed to: (A) 0 µM F, (B) 10 µM F, or (C) 20 µM F. PI, propidium iodide; Hoechst, Hoechst 33342. The cell group labeled R4 refers to an early-apoptotic population (PI- and increased Ho342 fluorescence), R5 is the late-apoptotic population (intermediate intensity of PI and decreased Ho342 fluorescence), and R2 is the viable cell population. (D) The apoptotic index shows that cells exposed to fluoride had significantly increased apoptosis. The average (SD) apoptotic index of each group: (21W) control, 6.87 (1.44); 10 µm NaF, 11.39 (2.70); 20 µm NaF, 13.84 (1.22); (22W) control, 4.98 (0.73); 10 µm NaF, 9.92 (0.99); 20 µm NaF, 11.43 (1.40) (n = 3). (E) TUNEL staining showed positive clusters of apoptotic cells in green. Scale bar = 20 µm.
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DISCUSSION
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The cells used for this in vitro study were derived from the enamel organ epithelium, and expressed mRNA for enamel matrix proteins, including DSPP and amelogenin. Expression of these matrix proteins suggested an ameloblast phenotype, though it is possible that they were precursor cells that may differentiate into various epithelial cells of the enamel organ. It is also possible that some of these cells were de-differentiated enamel organ epithelial cells, including ameloblasts. The studies reported here showed an effect of fluoride on both cell proliferation and apoptosis. These seemingly opposing effects of fluoride suggest that there may be different subpopulations of cells within these primary cultured cells.
The effects of fluoride on cells were highly concentration-dependent, and we found that fluoride had a biphasic effect on cell proliferation. The enhancement of cell proliferation at lower fluoride concentrations was similar to that reported in studies of osteoblasts, where micromolar levels of fluoride were shown to increase cell proliferation (Farley et al., 1983) through enhanced tyrosine kinase activity (Burgener et al., 1995). Thaweboon and co-workers reported a biphasic effect of fluoride at millimolar fluoride levels in dental pulp cells (Thaweboon et al., 2003). This biphasic effect of fluoride suggested that fluoride can affect cells by multiple pathways, some of which are more sensitive to lower concentrations of fluoride, and others that require higher fluoride concentrations.
We found no statistically significant effect of fluoride on mRNA expression in pooled ameloblast-lineage cells, as analyzed by cDNA array comparing 96 osteogenesis-related genes using a commercially available array, or by comparing tooth-specific genes in pooled teeth by real-time PCR. However, we did find DSPP, a marker of early ameloblast differentiation (MacDougall et al., 1998), down-regulated in the youngest cell group, isolated from the developing 2nd primary molars. The recent report by Maciejewska and coworkers (Maciejewska et al., 2006), showing variable fluoride effects on DSP protein in vivo, suggests that further studies of fluoride on DSPP expression at the early stages of ameloblast differentiation are warranted.
Several studies have shown that millimolar levels of fluoride can induce apoptosis in many cell types, including hepatic cells, epithelial lung cells, human leukemia HL-60 cells, etc. (Anuradha et al., 2000; Thrane et al., 2001; Refsnes et al., 2003).
In our study, we found that 10 µM NaF significantly increased the apoptotic index of in vitro ameloblast-lineage cells. The apoptotic index, used as a measure of apoptosis in this study, was determined by the relative numbers of R2, R3, and R4 cells. R2 cells were PI-negative and Ho342-positive, representing a group of cells with intact membrane and viability. R3 cells were PI-positive, necrotic cells without intact cytomembranes. R4 cells were an early apoptotic population with increased cell membrane permeability, indicated by an increased PI fluorescence as compared with R2. R5 cells were of intermediate PI intensity, with decreased Ho342 fluorescence, due to further dsDNA degradation and resultant lowered binding potency with Ho342. R6 were cell debris, as indicated by very low fluorescence in both PI and Ho342.
In these studies, we used micromolar levels of fluoride to determine whether fluoride can affect ameloblast-lineage cells at concentrations that may be found in serum. However, it is possible that the cell machinery found in these cells is also contained in the more differentiated ameloblasts found in the tooth organ. In that case, the effects of higher levels of fluoride found in the enamel fluid (such as in the transition stage of formation) (Weatherell et al., 1975) could also result in increased apoptosis of cells at critical stages of enamel formation. Further studies to determine the cellular mechanisms responsible for these effects of fluoride are needed to help us better understand the mechanisms of fluorosis.
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ACKNOWLEDGMENTS
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Supported by a Lee Hysan fellowship to Q. Yan and by NIH NIDCR R01 DE013508 to PKDB.
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication March 20, 2007.
Revision received November 22, 2006.
Accepted for publication November 29, 2006.
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Journal of Dental Research, Vol. 86, No. 4,
336-340 (2007)
DOI: 10.1177/154405910708600407
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ABSTRACT
INTRODUCTION