Influence Of Subchronic Exposure To Excessive Fluoride On Level Of Bone Sialoprotein Expression In Rat Bone Tissue

ObjectiveEndemic fluorosis is a whole-body chronic poisoning disease caused by long-term excessive consumption of fluoride.Dental fluorosis and skeletal fluorosis are the main symptoms of chronic fluorosis.It is the most severe and widespread endemic disease in the world.This naturally occurring disease is global in scope,occurring on all continents and affecting many millions of people.In China,chronic fluorosis occurs in almost all provinces,autonomous regions and municipalities except Shanghai and Hainan.By the end of 2005,there were about 116 million people living in the disease areas,40 million people with dental fluorosis and 2.88 million with skeletal fluorosis.Bone sialoprotein(BSP)is one of the major non-collagenous glycosylated phosphoproteins found in bone and tooth.Structure-function studies have demonstrated that post-translational modifications and possible phosphorylation of BSP may be important for the regulation of in vitro cell attachment,mineralization,and bone resorption.Extensive studies on the phosphorylation sites of BSP and the protein kinases involved in the phosphorylation have suggested that one of the prime regulators of bone mineralization is likely to be the activity of microsomal casein kinaseâ…¡,which phosphorylates specific matrix phosphoproteins.Since BSP is highly expressed in mineralized tissues as a calcium-binding protein,a great deal of attention has been focused on the potential roles of this protein in the nucleation and growth of apatite crystals.Cell culture experiments using bone-derived osteoblast-like and bone marrow cells have revealed that BSP promotes both expression of osteoblastic phenotypes and calcification of newly synthesized organic matrix.Although the potential roles of BSP in bone turnover have been tested in various systems,it is still unclear that influence of subchronic exposure to excessive fluoride on level of BSP expression in bone tissue.To explore the potential effects of BSP in pathologic bone turnover in fluorosis,we designed the experiment.Materials and Methods1.Experimental animalsSixty male Wistar rats,weighed 80±10g at the beginning of the study,were randomly and equally divided into 4 groups.The animals in the control group were given drinking water containing 0.5 mg F/L.The other three groups received the water with 50,100 and 150 mg F/L,respectively.The animals were fed normal diet(26-30 mg/L fluoride).During the study,rats were housed in plastic cages suspend in stainless steel racks.The humidity ranged from 45%to 55%and the temperature remained about 20℃.Fluoride-treated water was administered to rats of 3 experimental groups for 3 months.At the end of each month five rats from four groups were sacrificed for urine, serum and bone samples.All the samples were kept at -20℃till further analysis.2.Fluoride examinationsFluoride contents in the urine,serum and bone samples were measured with a CSB-F-I fluoride ion electrode.3.Immunohistochemical analysisThe one third left lower of distal femur of rats were embedded in paraffin for HE staining and immunohistochemical staining.BSP was identified by the avidin-biotin peroxidase complex method with rabbit anti-rats BSP polyclonal antibodies. Immunohistochemistry was performed with a SABC kit.Tissue sections were deparaffinized in xylenes and rehydrated in a graded series of ethanol solution,then rinsed in PBS.Blocking of endogenous peroxidase was performed with 3%H₂O₂ in methanol,and non-specific serum binding sites were blocked with normal goat serum.Antibodies at a dilution of 1:100 were applied,and the tissue sections were incubated for 1 hour at room temperature.Sections were then incubated with biotinylated goat antirabbit antibody for 10 min followed by exposure to labelled horseradish peroxidase complex.Finally,the sections were treated with the substrate chromogen,3-amino-9-ethylcarbazole(AEC),counterstained with Haematoxylin and mounted.Control slides were processed in an identical manner except that either the primary or secondary antibodies were replaced by diluting buffer solution.After immunohistochemical staining,sections were analyzed in a random order with a computer-assisted image analysis system,analyzing 5 high power fields for each section.The mean optical integrated density were measured by computer-assisted image analysis system.4.Statistical analysisSPSS 12.0 statistical software package was used to analyze the experimental data. The values for all assays were analyzed by analysis of a one-way analysis of variance (ANOVA)followed by a protected least significant difference(LSD and SNK)test (two-tailed)(α=0.05).The linear correlation was analyzed by Pearson linear correlation (α=0.05).Results1.General status of ratsDuring the experiment,the rats' general status in the control group was better than the experimental groups.Most of the fluoride-treated rats had dental fluorosis in different degrees.It could been seen that the dose-response and time-response relationships of rat drinking water fluoride concentrations to the serious degrees and incidence rates of rat dental fluorosis.In addition,in this experiment,decreased body weight and lack of shiny fur were found in the experimental rats.Furthermore,along with time passing,the trend became more and more obvious.2.Urine,serum and bone fluoride levelsWhile fed with the same fluoride concentration,the rats' urine,serum and bone fluoride levels in fluoride-treated groups were significantly higher than those in the control group(P＜0.05).With the fluoride-treated time prolonged,the rats' urine,serum and bone fluoride levels became more and more higher.3.Morphology and immunohistochemistry(1)HE stainingThe bone tissue structures had changed in three experimental groups.With the increasing of fluoride concentrations and time in rats' drinking water,the bone cell quantities became more and more higher.The cortex bone cells were in disorder in the experimental groups.(2)ImmunohistochemistryBSP was mainly expressed in some osteoblasts and bone matrix cells in marrow cavity.The positive staining was the brown staining cytoplasm and the blue staining nuclei.The negative staining was only the blue staining nuclei.The expression of BSP in the fluoride-treated rats was enhanced in bone cortex.Furthermore,with the increasing fluoride concentrations and time,the expression of BSP was significantly enhanced.(3)Semi-quantitative analysis of BSP expressionThe statistical analysis showed that the mean optical integrated density(the mean integrated OD)in fluoride-treated groups was significantly higher than the control group.In the same concentration fluoride-treated group,the mean integrated OD was significantly higher.Along with time passing,the trend became more and more obvious.4.Correlation analysis of BSP expression level with bone fluoride and timeThere was significantly positive correlation relationship between the mean integrated OD of BSP in bone and bone fluoride level.ConclusionsIn this study,the drinking-water-type chronic fluorosis rat models in the different fluoride-treated dosages were successfully copied.Our results suggested that fluoride might promote the expression of BSP,and superfluous BSP might strengthen osteogenesis and matrix calcification.The positive correlation between fluoride levels and the expression of BSP indicated that fluoride and BSP might exist synergistic effect in fluorosis.With the increased expression of BSP,fluoride might cause the occurrence of fluorosis.For the first time,the relationship of the bone tissue expression of BSP to excessive fluoride was discussed and this information may help to understand the chronic fluorosis mechanism.