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. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Cells Tissues Organs. 2015 Sep 19;200(6):413–423. doi: 10.1159/000438699

Fluoride Modulates Parathyroid Hormone Secretion In Vivo and In Vitro

Chaitanya P Puranik a, Kathleen A Ryan b, Zhaoyu Yin c, E Angeles Martinez-Mier d, John S Preisser c, Eric T Everett e
PMCID: PMC4679577  NIHMSID: NIHMS708704  PMID: 26381618

Abstract

The study objective was to investigate fluoride’s effects on iPTH secretion. Thryo-parathyroid complexes (TPCs) from C3H (n=18) and B6 (n=18) mice were cultured in Ca2+ optimized medium. TPCs were treated with 0, 250 or 500µM NaF for 24hrs and secreted iPTH assayed by ELISA. C3H (n=78) and B6 (n=78) mice were gavaged once with distilled or with fluoride (0.001mg [F−]/g body weight) water. At serial time points (0.5–96hrs) serum iPTH, fluoride, total calcium, phosphorus and magnesium levels were determined. Expression of genes involved in mineral regulation via bone-parathyroid-kidney (BPK) axis such as: Pth, Casr, Vdr, Pthlh, Fgf23, αKlotho, Fgf1rc, Tnfs11, Pth1r, Slc34a1, Slc9a3r1, Clcn5 and Pdzk1 were determined in TPCs, humerii and kidneys at 24hrs. An in vitro decrease in iPTH was seen in C3H and B6 TPC at 500µM (p<0.001). In vivo levels of serum fluoride peaked at 0.5hr in both C3H (p=0.002) and B6 (p=0.01). In C3H, iPTH decreased at 24hrs (p<0.0001) returning to baseline at 48hrs. In B6, iPTH increased at 12hrs (p<0.001) returning to baseline at 24hrs. Serum total calcium, phosphorus and magnesium did not change significantly. Pth, Casr, αKlotho, Fgf1rc, Vdr and Pthlh were significantly up-regulated in C3H TPC as compared to B6. Conclusions, fluoride’s effects on TPC in vitro were equivalent between the two mouse strains. However, fluoride demonstrated an early strain dependent effect on iPTH secretion in vivo. Both strains demonstrated a differences in the expression of genes involved in BPK axis suggesting a possible role in physiologic handling of fluoride.

Keywords: fluoride, skeletal fluorosis, parathyroid hormone, bone, inbred mouse strains

Introduction

Fluoride has recognized actions on bone homeostasis [Everett, 2011]. Yan et al investigated in two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), fluoride actions on bone homeostasis [Yan et al., 2007]. Short term systemic exposure to fluoride for C3H resulted in increased osteoclastogenesis as evidenced by increases in serum osteoclast biomarkers: intact parathyroid hormone (iPTH), soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) and tartrate-resistant acid phosphatase 5b (TRAP5b) along with decrease in serum osteoprotegerin (OPG) levels [Yan et al., 2007]. Osteoclast numbers along bone surfaces and osteoclast potential of bone marrow cells were increased in C3H as well. However, similar fluoride exposure in B6 mice favored anabolic responses with increases in serum alkaline phosphatase (ALP) activity, proximal tibia trabecular and vertebral bone mineral density.

PTH is responsible for calcium homeostasis and is released in response to hypocalcemia to normalize serum calcium levels [Potts, 2005]. PTH is an important component of the bone-parathyroid-kidney (BPK) feedback loop responsible for mineral homeostasis [Bergwitz and Juppner, 2010; Torres and De Brauwere, 2011]. Skeletal fluorosis results from systemic exposures to fluoride leading to calcification of ligaments, bone deformities, fractures, functional limitations, disturbed mineral balance and occasional pseudo-hyperparathyroidism [Teotia and Teotia, 1973; Teotia et al., 1998]. The mechanisms underlying altered PTH levels due to fluoride exposure are not understood. We hypothesize that PTH secretion is influenced by direct effects of fluoride on the parathyroid gland. Our in vitro and in vivo experiments were directed to investigate early events following fluoride exposure on PTH secretion.

Material and Methods

Animals

Ninety-six, 5–6 week old male C3H/HeJ (C3H) and C57BL/6J (B6) mice (The Jackson Laboratory, Bar Harbor, ME, USA) were placed on a constant nutrition no fluoride diet #5861 [Test Diet®, Richmond, IN, USA; fluoride: 0ppm, calcium: 0.60%, phosphorus: 0.57%, Vit D3: 2.2 IU/g, and Energy: 4.09 (kcal/g)2] and provided distilled drinking water, ad libitum, for one week to acclimatize. Mice were housed in The Division of Lab Animal Medicine facility at The University of North Carolina at Chapel Hill, an AAALAC accredited unit. Mice were placed in 12:12 light and dark cycles at 21°C ambient temperature. All procedures involving vertebrate animals were approved by the Institutional Animal Care and Use Committee at The University of North Carolina at Chapel Hill.

Parathyroid gland dispersed cell culture

Mice were euthanized by CO2 and ventral neck dissection was performed to isolate and harvest thyro-parathyroid complex (TPC) after micro-dissection. TPCs were finely minced and transferred to 2ml centrifuge tube with digestion buffer containing 1mg/mL of collagenase (Stem Cell Technologies, Vancouver, BC, Canada) in dispase solution (Stem Cell Technologies). TPC were incubated at 37°C for 3hrs with intermittent pipetting for homogeneous dispersion of cells. Dispersed cells were plated on to 6-well plates and incubated at 37°C at 5% CO2 in 2ml pre-warmed alpha MEM (Sigma Aldrich, St. Louis, MO, USA) media with 10% FBS (Clontech Laboratories, Mountain View, CA, USA) and 1% antibiotic-antimycotic (Sigma-Aldrich, St. Louis, MO, USA). Total calcium, magnesium, and phosphorus levels in media were adjusted to that of serum in each strain of mice: C3H (calcium: 10.6mg/dL, phosphorus: 8.7mg/dL, and magnesium: 2.1mg/dL) and B6 (calcium: 10.4mg/dL, phosphorus: 10.3mg/dL, and magnesium: 2.9mg/dL). TPC from C3H and B6 were prepared on two separate occasions using a total of 12 mice per strain. An initial study using two F concentrations (0 and 500µM). This was repeated using additional mice and three F concentrations examined (0, 250 or 500µM). Media supernatants were collected at 24hrs, centrifuged and stored at −80°C until analyzed.

Gavage and sample collections

Following acclimatization, n=72 mice from both strains were randomly divided in twelve groups of six mice (0.5, 1, 3, 6, 12 or 24hrs) based on time of sacrifice after gavage dose with control and test groups based on fluoride levels (0 or 100ppm) in gavage. Additionally, six mice from both strains were assigned to no-gavage at baseline group. At the time of gavage, mice were weighed and the gavage dose was calculated for each mouse (0.001mg [F−]/g body weight). Mice were anesthetized with ketamine-HCl (90mg/kg) and xylazine (14mg/kg) i.p. Sera were collected from anesthetized mice and stored at −80°C until use. TPCs, kidneys, and humerii were harvested, snap-frozen in liquid nitrogen, and stored at −80°C.

ELISA and biochemistry assays

Media supernatants and sera were analyzed for iPTH using 1–84 PTH ELISA kits (Immutopics, San Clemente, CA, USA). The intra- and inter-assay precision (coefficient of variation) for iPTH ELISA was 3.2 and 8.4, respectively. Each ELISA plate had 6 standard and 2 control solutions for determination of R2 values. The threshold value R2 value was set at 0.95. All ELISA plates demonstrated R2 values >0.98. Each ELISA plate was read with 6 samples each from control and test groups in triplicates.

Serum levels of fluoride were determined using direct fluoride microanalysis method [Vogel et al., 1990]. In this method micropipettes pulled from glass capillary tubes are combined with a custom-made micro-pipette holder and used to dispense nanoliter-sized standards and serum samples (diluted 9:1 with TISAB III). Serum samples are extracted over a light table using the micropipette/micropipette holder and dispensed onto the surface of an inverted fluoride electrode (F-ISE) under a layer of mineral oil to prevent loss of fluoride. Using a micromanipulator and microscope for viewing, the reference electrode is touched to the standard or sample drop, resulting in an electrometer mV reading. Both the F-ISE and hand-pulled reference electrode are connected to an electrometer and computer with plot program monitoring/recording software. Samples are run in triplicate, with periodic electrode conditioning and standard checks.

Calcium, phosphorus, and magnesium were determined by the Animal Clinical Laboratory, University of North Carolina at Chapel Hill and the Clinical Pathology Laboratory, College of Veterinary Medicine at North Carolina State University.

RNA extractions and cDNA preparation

RNA was extracted from TPCs, kidneys, and humerii samples using RNeasy Midi kit (Qiagen Inc., Valencia, CA, USA). Quantitative analysis of RNA sample was performed using Agilent Bioanalyzer using Nano Labchip at Genomic Core Facility, University of North Carolina at Chapel Hill. All RNA sample had RIN values 7.5–10 range. High Capacity Reverse transcription kit (Applied Biosciences, Carlsbad, CA, USA) was used to prepare cDNA. After preparation, cDNA was stored at −80°C.

qPCR

cDNA samples free of genomic DNA contamination were subjected to qPCR for suite of genes including parathyroid hormone (Pth), calcium sensing receptor (Casr), alpha-Klotho (αKlotho), fibroblast growth factor receptor 1c (Fgf1rc), vitamin D receptor (Vdr), parathyroid hormone like hormone (Pthlh), tumor necrosis factor 11 (Tnfs11), fibroblast growth factor 23 (Fgf23), parathyroid hormone receptor 1 (Pth1r), chloride channel 5 (Clcn5), Solute carrier 9 member 3 regulator 1 (Slc9a3r1), PDZ domain containing 1 (Pdzk1) and Solute carrier family 34 member 1 (Slc34a1). Expression of these genes was normalized using two house-keeping genes comprising of beta actin (Actb) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Thyroperoxidase (Tpo) was used as surrogate marker for the quality of TPC dispersed cell culture. All the primers were from SA Biosciences (Qiagen Inc., Valencia, CA, USA). Data were analyzed using 2−ΔΔCt method [Livak and Schmittgen, 2001; Yuan et al., 2006].

Statistical analysis

Data were reported as mean ± SE. One-way ANOVA was used to compare the effect of calcium concentration on iPTH secretion in the TPC dispersed cell culture model. For the in vitro experiments, six groups each consisting of six mice were compared using two-way ANOVA based on strain (C3H or B6) and levels of fluoride exposure (0, 250 or 500µM). For the in vivo experiments, 24 groups were compared using three-way ANOVA based on strain (C3H or B6) levels of fluoride exposure (0 or 100ppm) and time points (0.5, 1, 3, 6, 12 and 24hrs). A preliminary two-way ANOVA was first used to confirm the absence of a time effect in water gavage when baseline gavage (0hrs) was included for both strains of mice (total n=84) which would justify the omission of baseline gavage data at 0hrs in order to maintain balance in three-way ANOVA. Three-way ANOVAs were also evaluated for serum total calcium, phosphorus, and magnesium levels for each strain followed by treatment with 0 or 100µM fluoride concentrations at 0.5, 1, 3, 6, 12, and 24hrs. A natural log transformation was used for iPTH and serum fluoride (but not for calcium, magnesium or phosphorous) to address skewness. ANOVA main effect and interaction p-values ≤0.05 were considered statistically significant, followed by Bonferroni adjustments for post-hoc comparisons of levels of factors SAS version 9.3 (SAS Inc., Cary, NC, USA) was used for statistical analysis.

Results

Responsiveness of TPC dispersed cell culture model

The TPC (Fig. 1A) dispersed cell culture model was utilized in order to determine the direct effects of fluoride on the parathyroid gland. All TPC dispersed cell cultures demonstrated Tpo gene expression (a surrogate marker for TPC dispersed cell culture quality) with Ct valuse 24.8±2.1. Responsiveness of the dispersed cell culture model to changes in the extracellular calcium, a known PTH mediator was used to validate the culture model. TPCs cultured in varying calcium concentrations (hypocalcemic: 1.1mg/dL, normocalcemic: 10.5mg/dL or hypercalcemic: 25mg/dL) demonstrated changes in iPTH secretion into the media (p<0.001). Based upon a Bonferroni-corrected significance level of 0.05/3=0.017 and after 24hrs of incubation at hypocalcemic condition, iPTH secretion (129.4±27.1pg/mL) was significantly increased (p=0.007) as compared to the normocalcemic condition (49.2±6.2pg/mL) (Fig. 1B). The hypercalcemic condition lead to a further statistically significant decline in iPTH relative to the normocalcemic condition (p=0.012). Although not shown, TPCs in culture remain responsive to changes in calcium for 3 days.

Fig. 1. TPC histology and iPTH levels in media supernatant.

Fig. 1

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(A) Histological section of the thyro-parathyroid complex (TPC) with arrows indicating parathyroid tissue. (B) iPTH ± SE (pg/mL) in C3H TPCs at three calcium concentrations; 1.1, 10.5 or 25mg/dL (n=3 in each group).

Effect of fluoride on TPCs

The F exposure experiments using TPCs from B6 and C3H mice were performed at two different times. An initial experiment using TPCs from B6 (n=6) and C3H (n=6) mice were cultured in media normalized to calcium, phosphorus, and magnesium levels present in the sera for each strain. Calcium, phosphorus and magnesium are known modulators of iPTH and can serve as confounding variables and hence were kept at the levels commonly seen in the sera of each strain. Hence, constant levels of calcium, phosphorus and magnesium was present in the test and control groups. This was followed by treatment with two fluoride concentrations (0 or 500µM). Media supernatants were collected at 24hrs and iPTH determined. TPCs from C3H demonstrated a significant (p<0.001) decrease in iPTH with increasing fluoride concentration from 0µM (145.7±13.1pg/mL) to 500µM (72.2±5.9pg/mL). TPCs from B6 demonstrated similar decrease (p<0.001) in iPTH with increasing fluoride concentrations from 0µM (88.3±6.1pg/mL) to 500µM (47.1.1±5.3pg/mL). A replicate experiment was performed using TPCs from B6 (n=6) and C3H (n=6) mice cultured in media normalized with calcium, phosphorus, and magnesium as described above then followed by treatment with varying fluoride concentrations (0, 250 or 500µM). Media supernatants were collected at 24hrs and iPTH determined. In the two-way ANOVA, main effects (both p<0.001) and interaction (p=0.042) were statistically significant indicating that iPTH differs significantly between the two strains, and the three fluoride levels, with the effect of fluoride exposure on iPTH being different for C3H than for B6. Based upon a Bonferroni-corrected significance level of 0.05/6=0.008, For C3H, all three levels of fluoride exposure are significantly different from one another with higher fluoride level leading to lower value of iPTH as follows: 0µM (128.5±10.6pg/mL) to 250µM (86.2±12.4pg/mL, p<0.001) or 500µM (52.3±8.9pg/mL, p<0.001). TPCs from B6 demonstrated less decrease in iPTH with increasing fluoride concentrations from 0µM (64.3±14.7pg/mL) to 250µM (50.9±12.2pg/mL) or 500µM (38.1±5.1pg/mL) with the only significant pairwise comparison relating to fluoride concentrations at 0 versus 500µM (p<0.001) (Fig. 2). For the initial and replicate studies C3H had higher initial iPTH levels in the media compared to B6 TPCs (p<0.01). TPCs from both strains showed F dose dependent decreases in iPTH secretion at 24hrs.

Fig. 2. iPTH levels in media supernatant (In vitro study).

Fig. 2

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Mean iPTH ± SE (pg/mL) in C3H and B6 TPCs after 24hrs of incubation at three fluoride concentrations; 0, 250 or 500µM (grey bar=C3H and black bar=B6, n=6 in each group; **p<0.005).

Serum iPTH after single fluoride dose via oro-gastric gavage

As in the in vitro experiments in vivo studies were done at two different instances. The initial experiment involved 42 mice per strain. Serum iPTH in C3H and B6 mice was measured at four time points (baseline, 6, 12 or 24hrs) after fluoride gavage (0.001mg [F−]/g body weight) or gavage with an equal volume of 0ppm fluoride water. At baseline with 0ppm fluoride water gavage, the hour effect was not significant for the main effect and interaction and therefore, the baseline observations were dropped from the analysis. Three way interaction between strain, hour and gavage was not significant, but all two way interactions were significant (strain*gavage: p=0.001, strain*hour: p<0.001, gavage*hour: p=0.009). Although significance not reached, at 6hr after 100ppm fluoride gavage, serum iPTH levels increased modestly for C3H (243.9±13.4pg/mL) and B6 (102.1±7.3pg/mL) as compared to baseline untreated levels (C3H: 210.5±16.2pg/mL and B6: 86.6±8.9pg/mL) or control levels (0ppm fluoride gavage) (C3H: 189.1±19.8pg/mL and B6: 77.1±4.8pg/mL). In the C3H strain, iPTH dropped below baseline at 24hrs (126.7±10.2pg/mL, p<0.001) (Fig. 3A); whereas, in B6, iPTH increased at 12hr (234.8±34.1pg/mL, p<0.001) after 100ppm fluoride gavage (Fig. 3B).

Fig. 3. Serum iPTH levels at 0, 6, 12 or 24 hrs.

Fig. 3

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Mean serum iPTH ± SE (pg/mL) after 0 or 100ppm fluoride gavage in C3H and B6 mice sacrificed at serial time points; 0, 6, 12 or 24hrs compared to untreated mice (grey bar=untreated/baseline group, white bar=0ppm group and black bar=100ppm group) (n=6 in each group; Boneferroni-corrected significance level *p<0.008, **p<0.001).

The in vivo studies were replicated using an additional 78 mice per strain. As before serum iPTH was measured at various time points (0, 0.5, 1, 3, 6, 12 or 24hrs) in C3H and B6 mice after fluoride gavage (0.001mg [F−]/g body weight) or gavage with an equal volume of 0ppm fluoride water. A preliminary two-way ANOVA restricted to baseline gavage 0 µM fluoride at six time points and strain as the second factor gave a non-significant interaction and non-significant time effect consistent with the assumption of no differences between mean serum iPTH for baseline gavage (0hrs) and 0µM fluoride at any time point. In the three-way ANOVA, the three-way interaction was not statistically significant, but all two way interactions were significant (strain*gavage: p=0.017, strain*hour: p<0.001, gavage*hour: p<0.001). This means that each factor has effects on iPTH that vary across the levels of a second factor, when averaged over the third factor. Based upon a Bonferroni-corrected significance level of 0.05/6=0.008, fluoride levels 0ppm and 100ppm have statistically different mean iPTH only at 0.5 hours (p=0.004) and 24 hours (P<.0001) for either strain. Specifically, at 0.5 hour, gavage with fluoride level 100ppm gives C3H (358.4±19.4pg/mL) and B6 (149.9±21.2pg/mL as compared to control (0ppm fluoride gavage) levels (C3H: 244.4±35.5pg/mL and B6: 98.6±6.4pg/mL) groups. However, gavage with fluoride level 0ppm gives higher mean iPTH at hour 24 (p≤.001).. In the C3H strain iPTH dropped below baseline at 24hrs (82.0±10.7pg/mL, p=0.002) (Fig. 4A); whereas, in B6, iPTH increased at 12hr (176.9±20.8pg/mL, p<0.001) and then returned to baseline at 24hrs (95.6±19.9pg/mL) (Fig. 4B). The peak iPTH value in C3H (358.4±19.4pg/mL) was twice that of peak iPTH values in B6 (176.9±20.8pg/mL).

Fig. 4. Serum iPTH levels at 0.5, 1, 3, 6, 12 or 24 hrs.

Fig. 4

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Mean serum iPTH ± SE (pg/mL) after 0 or 100ppm fluoride gavage in C3H (A) and B6 (B) mice sacrificed at serial time points; 0.5, 1, 3, 6, 12 or 24hrs compared to untreated mice (grey bar=untreated/baseline group, white bar=0ppm group and black bar=100ppm group) (n=6 in each group; *p<0.05, **p<0.005).

Since the iPTH levels in C3H mice dropped below baseline at 24hrs additional time points up to 96 hours were collected. The serum iPTH in C3H mice after single 100ppm fluoride gavage returned to baseline at 48hrs (Fig. 5). These data demonstrated a difference in fluoride’s effects on iPTH secretion in two mice strains; C3H and B6.

Fig. 5. Serum iPTH levels 48–96hrs.

Fig. 5

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Mean serum iPTH ± SE (pg/mL) after 0 or 100ppm fluoride gavage in C3H harvested at extended time points; 48, 72 or 96hr compared to untreated mice (grey bar=untreated/baseline group, white bar=0ppm group and black bar=100ppm group) (n=6 in each group; **p<0.005).

Serum fluoride levels after gavage

A calculated dose of fluoride was delivered by oro-gastric gavage. Serum fluoride concentrations were measured at serial time points (0.5, 1, 3, 6, 12 or 24hrs) after a single gavage dose. The fluoride levels in the serum peaked in both strains, C3H (61.0±5.6µM) and B6 (26.7±3.1µM), at 0.5hr (p<0.001) after gavage. Fluoride levels reached baseline (0ppm) in C3H (8.7±1.7µM) and B6 (3.5±0.4µM) after 6–9hrs (Fig. 6). The interaction between hour and strain are significant (p<0.0001) indicating that the pattern of fluoride kinetics are different across the strains. In particular, fluoride levels in B6 were significantly lower than C3H at 0.5, 1, 3 and 12hrs (p<0.001). The peak fluoride concentration in C3H (61.0±5.6µM) was nearly twice that compared to B6 (26.7±3.1µM).

Fig. 6. Serum fluoride levels.

Fig. 6

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Mean serum fluoride (F) ± SE (µM) after 100ppm fluoride gavage in C3H and B6 mice sacrificed at serial time points; 0.5, 1, 3, 6, 12 or 24hrs compared to baseline (no gavage), (grey bar=C3H and black bar=B6) (n=6 in each group; **p<0.005).

Serum total calcium, phosphorus, and magnesium levels

Serum total calcium levels were relatively consistent between 0 and 100ppm fluoride gavage mice in both C3H (Fig. 7A) and B6 mice (Fig. 7D). We observed a significant reduction (p<0.05) in serum total calcium only at 0.5hr in 100ppm fluoride gavage group (C3H: 8.7±0.2mg/dL and B6: 8.5±0.2mg/dL) as compared to 0ppm control (C3H: 8.9±0.1mg/dL and B6: 9.5±0.2mg/dL) or untreated baseline group (C3H: 9.3±0.1mg/dL and B6: 9.2±0.2mg/dL).

Fig. 7. Serum Ca, Pi, and Mg levels.

Fig. 7

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Mean serum calcium (Ca) ± SE, phosphorus (Pi) ± SE and magnesium (Mg) ± SE (mg/dL) after 0 or 100ppm fluoride gavage in C3H (A, B and C respectively) and B6 (D, E and F respectively) harvested at serial time points; 0.5, 1, 3, 6, 12 or 24hrs (grey bar=untreated/baseline group, white bar=0ppm group and black bar=100ppm group) (n=6 in each group; *p<0.05, **p<0.005).

The strain by gavage interaction in the two-way ANOVA for serum phosphorus levels was significant (p=0.034). Hour was removed from the model as it was not statistically significant. Therefore, when time was not considered, there was an overall difference between 0 and 100ppm fluoride gavage for C3H (p=0.009) (Fig. 7B) but not for B6 (Fig. 7E) mice. No statistically significant differences were observed in magnesium levels between 0 and 100ppm fluoride gavage mice at any time point for both C3H (Fig. 7C) and B6 (Fig. 7F) mice.

qPCR: Expression of genes involved in BPK feedback loop

We investigated the expression of various genes involved in the BPK feedback loop using TPC, humerii and kidneys. Expression of the genes in the tissues obtained from 100ppm fluoride gavage C3H and B6 mice group at 24hrs were normalized with an average of two house-keeping genes (Actb and Gapdh). Gene expression was further normalized to control 0ppm fluoride gavage group and fold difference of genes in TPC (Fig. 8A), humerii (Fig. 8B) and kidneys (Fig. 8C) were calculated for C3H and B6 mice using 2−ΔΔCt method. In TPC, Pth (p=0.001), Casr (p=0.012), αKlotho (p=0.003), Fgf1rc (p=0.035), Vdr (p=0.001) and Pthlh (p=0.03) were significantly up-regulated in C3H as compared to B6. In humerii, only Pthlh (p=0.001) was significantly up-regulated in C3H as compared to B6 mice. In kidney, Pth1r (p=0.005), Clcn5 (p=0.003), αKlotho (p=0.05), Fgf1rc (p=0.004) and Slc9a3r1 (p=0.04) were significantly down-regulated in C3H as compared to B6.

Fig. 8. Expression of genes involved in BPK feedback loop.

Fig. 8

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Fold-difference ± SE in C3H and B6 100ppm fluoride gavage group normalized to control, 0ppm fluoride group and house-keeping gene pool (Actb and Gapdh) in TPCs (A), humerii (B) and kidneys (C). (grey bar=C3H and black bar=B6) (n=6 in each group; *p<0.05, **p<0.001).

Discussion

Skeletal fluorosis is characterized by a spectrum of radiographic bone changes ranging from osteoporosis to osteosclerosis [Wang et al., 1994]. Although, clinical cases of skeletal fluorosis are rare in the United States [Kurland et al., 2007; Whyte et al., 2008] in other parts of the world where skeletal fluorosis is more common, patients often demonstrate pseudo-hyperparathyroidism [Teotia and Teotia, 1973; Teotia et al., 1998; Gupta et al., 2001; Xu et al., 2010; Koroglu et al., 2011]. Epidemiologic studies show that individuals of the population exposed to similar fluoride levels demonstrate variable skeletal features ranging from excessive bone formation to bone resorption [Teotia et al., 1998; Harinarayan et al., 2006]. Therefore, the mechanisms that underlie fluoride’s actions on the bone and PTH are not clearly understood. Yan et al demonstrated a link between fluoride exposure and iPTH levels using the C3H and B6 inbred strains of mice [Yan et al., 2007]. Fluoride exposure in C3H led to enhanced osteoclastogenesis including elevated serum iPTH, whereas in B6 an anabolic action of fluoride was favored. Additionally these strains are widely studied for their differences in bone biology. C3H mice have high bone mass, peak bone density and alkaline phosphatase activity and low bone resorption capacity as compared to B6 mice [Chen and Kalu, 1999; Linkhart et al., 1999; Turner et al., 2000; Turner et al., 2001].

Direct or indirect effects of fluoride on the parathyroid gland have not been investigated. We developed a murine TPC dispersed cell culture model using inbred mouse strains with predictable iPTH secretion response to changes in calcium. Similar TPC dispersed cell culture models have been employed using bovine, human and rat parathyroid tissues to study parathyroid hormone regulation [Wongsurawat and Armbrecht, 1987; Nielsen et al., 1996; Nakajima et al., 2010]. Calcium, phosphorus, and magnesium are known to mediate changes in iPTH secretion and therefore, the levels of these in the media were kept equivalent with strain specific serum levels to prevent unwanted effects on iPTH secretion. In both C3H and B6 fluoride exposure resulted in dose dependent reductions in iPTH secretion into the media measured at 24hrs. This reduction was more evident for C3H which at baseline secretes almost twice the iPTH as compared to B6.

The fluoride kinetics observed for B6 and C3H strains were consistent with previous studies where serum fluoride reaches a peak within 20–30mins [Ekstrand et al., 1977; Whitford, 1994]. It is generally accepted that after absorption, fluoride is incorporated in newly mineralized bone or is excreted in the urine resulting in decline of serum levels reaching baseline by 6–8hrs [Ekstrand et al., 1980]. In order for fluoride to elicit a rapid iPTH response it is hypothesized that fluoride interacts with the parathyroid gland directly through modulating iPTH release from vesicles in the Chief cells. The exact molecular mechanism of this response is not understood. However, our in vitro findings suggest the presence of such a direct interaction of fluoride on the parathyroid glands.

In the current study, C3H mice have higher baseline serum iPTH compared to B6. This is consistent with our TPC baseline values. C3H mice are more responsive to the single fluoride exposure perhaps because reaching a higher peak serum fluoride level. This enhanced response is shown after the gavage when the levels of iPTH were higher in C3H as compared to B6. At 24hrs iPTH levels were lower than baseline for both strains. Both strains also showed bimodal responses to fluoride. For C3H, iPTH peaks at 0.5hr and declines below the baseline at 12hrs reaching a nadir at 24hrs then over the next 24hrs returns to baseline and remains at baseline at 96hrs. B6 mice show a similar but lower magnitude of response.

The modulation of iPTH secretion as a response to fluoride exposure may also be mediated by changes in calcium levels. Decreased serum calcium levels after acute hydrofluoric acid inhalation has been documented in humans [Zierold and Chauviere, 2012]. Acute exposure to fluoride through injection of hydrofluoric acid (1.6mg/Kg) in rats led to a decrease in serum ionized calcium and total calcium after 30 and 300mins respectively [Santoyo-Sanchez et al., 2013]. Similar decrease in plasma calcium was observed in rats due to acute fluoride exposure [Imanishi et al., 2009]. The reduced serum calcium is hypothesized to be due to the formation of CaF2 complexes resulting into a net reduction in calcium levels leading to a hypocalcemic response. In our study, we observed a transient decrease in calcium in both strains of mice only at 0.5hr with return to baseline within 1hr. There were no significant changes in phosphorus and magnesium at any time point.

BPK feedback loop has been extensively studied to understand the interaction of various genes involved in mineral homeostasis. The cross-talk between the bone, parathyroid and kidney is responsible for effective regulation of minerals. The effect of fluoride on these individual tissues after a single fluoride dose in these two strains of mice will help us to understand the effect of fluoride on regulatory aspects of mineral homeostasis. Change in gene expression patterns due to fluoride exposure have been studied in the past [Li and DenBesten, 1993; Nair et al., 2011; Pei et al., 2012] but our study has focused only on expression of the genes involved in calcium and phosphorus homeostasis and are part of BPK feedback loop. Fold differences in expression of Pth, Casr, αKlotho, Fgf1rc, Vdr and Pthlh in TPCs suggests that at the molecular level fluoride has a differential impact on parathyroid tissues in C3H and B6. The expression of these genes reflects a compensatory mechanism to counter changes in iPTH secretion. It is clear that fluoride effects are not limited to Pth but extends to Pth homologues such as Pthlh. Expression of Fgf1rc-αKlotho (receptor couple for Fgf23) and Pth1r are suggestive of an effort to intercept conservation of calcium and excretion of phosphorus in the kidneys to achieve homeostasis after initial iPTH secretion. Overall, there exists a difference in expression of BPK genes in the two strains of mice. This suggests a difference in physiologic handling of fluoride by the two strains. It will be interesting to study the effect of repeated fluoride dose on expression of the key genes in C3H and B6.

Our study investigated the early events involved in fluoride interaction on parathyroid gland in vitro and in vivo. Fluoride can act on the parathyroid gland directly to modulate iPTH secretion in vitro and in vivo. It is still not clear if the decrease in total serum calcium level is responsible for fluoride-mediated iPTH modulation. The effect of fluoride on iPTH secretion is rapid and observable even after single fluoride dose. The overall effects of fluoride are not limited to parathyroid gland but changes in expression of genes involved in BPK feedback loop suggest a broader impact of fluoride on multiple aspects of bone metabolism.

Acknowledgements

Ms. Staci Love is gratefully acknowledged for her help in vitro studies. Research reported in this publication was supported by the NIDCR of the National Institutes of Health under award number R01DE018104 to ETE

Abbreviations used in this paper

ALP

Alkaline phosphatase

B6

C57BL/6J strain

BPK

Bone-parathyroid-kidney axis

C3H

C3H/HeJ strain

Casr

Calcium sensing receptor

Clcn5

Chloride channel 5

Fgf1rc

Fibroblast growth factor receptor 1c

Fgf23

Fibroblast growth factor 23

iPTH

Intact parathyroid hormone

NaF

Sodium fluoride

OPG

Osteoprotegerin

Pdzk1

PDZ domain containing 1

Pth

parathyroid hormone

Pth1r

Parathyroid hormone receptor 1

Pthlh

Parathyroid hormone like hormone

Slc34a1

Solute carrier family 34 member 1

Slc9a3r1

Solute carrier 9 member 3 regulator 1

sRANKL

Soluble receptor activator of nuclear factor kappa-B ligand

Tnfs11

Tumor necrosis factor 11

TPC

Thyro-parathyroid complex

TRAP5b

Tartrate-resistant acid phosphatase 5b

Vdr

Vitamin D receptor

αKlotho

Alpha Klotho

Footnotes

Conflicts of interest

The authors do not have any conflicts of interest.

Authors’ roles

Study design: CPP and ETE

Study conduct: CPP and KAR

Data collection: CPP

Data collection and analysis: CPP, ZY, JSP and ETE

Serum fluoride analysis: EAM

Data interpretation: CPP and ETE

Drafting manuscript: CPP and ETE

Approval of final version of the manuscript: CPP and ETE

Contributor Information

Chaitanya P. Puranik, Email: chaitanya_puranik@unc.edu.

Kathleen A. Ryan, Email: kathleen_ryan@unc.edu.

Zhaoyu Yin, Email: zyin@live.unc.edu.

E. Angeles Martinez-Mier, Email: esmartin@iu.edu.

John S. Preisser, Email: jpreisse@bios.unc.edu.

Eric T. Everett, Email: eric_everett@unc.edu.

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