Bone Response to Fluoride Exposure Is Influenced by Genetics

Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue

Bone response to fluoride exposure is influenced by genetics.

Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue

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Quantification of ALP activity in plasma from A/J and 129P3/J mice treated with 0, 10 or 50 ppm F in the drinking water for 8 weeks.

<p>Results are shown as mean ± SD of enzymatic activity (nmol of p-NP per min per mg of total protein). ^*Represents significant differences between strains for each group (<i>p</i><0.05). No statistical differences were found for each strain after receiving F.</p

Western blot analysis of collagen type I.

<p>(A) Representative immunoblot showing collagen type I levels (upper) in A/J and 129P3/J mice after treatment with 0, 10 or 50 ppmF in drinking water for 8 weeks. The level of β-tubulin was used as control of sample loading (lower). (B) Values are mean ± SD of arbitrary values of four independent experiments quantified by densitometry analysis using the Image J Software (NIH Image). Arbitrary values from control A/J were standardized as 1. ^**Represents significant differences between strains for each treatment (<i>p</i><0.01). No statistical differences were found for each strain after receiving F.</p

Mineral apposition rate (MAR) of femur from A/J and 129P3/J mice after F exposure.

<p>(A) Upper squares: Representative images showing calcein (green) and alizarin complexone (red) labeled mid-diaphyseal cross-sections of 129P3/J mice. Lower squares: Representative images showing calcein (green) and alizarin complexone (red) labeled mid-diaphyseal cross-sections of A/J mice. Concentrations of F are indicated. (B) Values are mean ± SD of the measurement distances between the labels, n = 8/group. ^* Represents significant differences between strains for each group (<i>p</i><0.05); ^#<i>p</i><0.01 and ^##<i>p</i><0.001 represent significant differences of control and 10 ppmF-treated 129P3/J versus 50 ppmF-treated 129P3/J mice, respectively.</p

Total number of identified bone proteins with differences in abundance in each comparison, by label-free semi-quantitative differential expression analysis.

<p>*Ratio of the relative protein abundance between each comparison. Significant differences in protein abundance were considered when ratio ≤0.5 or ≥1.5. Ratios of ≤0.5 or ≥1.5 mean increase or decrease in group B in relation to group A, respectively.</p><p>Total number of identified bone proteins with differences in abundance in each comparison, by label-free semi-quantitative differential expression analysis.</p

Schemes of the protein-based mechanisms triggered by F in osteoblasts and osteoclasts.

<p>Identified proteins with greater abundance in 129P3/J in comparison to A/J related to bone metabolism are shown in red. (A) Exportin enhances transport of cfab/Runx-2 to the nucleus activating transcription of collagen type I genes. Cadherins promote intercellular adhesion enhancing osteoblast differentiation. Nox-mediated ROS production enhances RANK ligand (RANKL) that interacts to its receptor (RANK) to induce osteoclast differentiation. (B) The treatment of 129P3/J with high F promotes enhancement of Nox, CDH, cadherin, catetin and SHIP while (C) in A/J, F at high dose promotes enhancement of SHIP and reduction of exportin.</p