Study On Effect Of High Fluoride Exposure On Human Serums OPG Levels

ObjectiveEndemic fluorosis is a whole-body chronic poisoning disease caused by long term excessive consumption of fluoride.It is mainly divided into bone damage and non-bone damage.Bone damage is more harmful to the body and it can lead to accelerated bone turnover,bone sclerosis,osteomalacia,osteoporosis,bone soft tissue and bone and joint cartilage degeneration.Dental fluorosis and skeletal fluorosis are the main symptoms of chronic fluorosis,and skeletal fluorosis is the most serious clinical manifestations of chronic fluorosis.Endemic fluorosis 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 2008,there were about 118 million people living in the disease areas.Osteoprotegerin(OPG) is a member of the tumor necrosis factor receptor (TNFR) superfamily,and it represents a secretory basic glycoprotein that exists in a 60-kd monomeric form and a disulfide-linked homodimeric form of 120 kd.It has also been detected in a cell surface-associated form with some cell types,although sequence analysis failed to detect a classical hydrophobic transmembrane domain,which is typical for all other members of the TNFR superfamily.It is produced and released by activated osteoblast cells,OPG functions as a soluble decoy receptor for RANKL and competes with RANK for RANKL binding.Consequently,OPG is an effective inhibitor of osteoclast maturation and osteoclast activation in vitro and in vivo.The balance between RANKL-RANK signaling and the levels of biologically active OPG regulates development and activationof osteoclasts and bone metabolism.All factors that inhibit or increase bone resorption via osteoclasts act via regulation of RANKL-RANK and/or OPG-RANKL interactions RANKL/OPG system as a key cytokine network involved in the regulation of bone cell biology,osteoblast-osteoclast and bone-immune cross-talks,and maintenance of bone mass.In addition to providing substantial and detailed insights into the pathogenesis of various metabolic bone diseases,the administration of OPG may become a promising therapeutic option in the prevention and treatment of benign and malignant bone disease.Although the potential roles of OPG in bone turnover have been tested in various systems,it is still unclear that influence of subchronic exposure to excessive fluoride on level of OPG expression in bone tissue.To explore the potential effects of OPG in pathologic bone turnover in fluorosis,we designed the experiment.Materials and Methods1.Experiment subjectsWe randomly selected Fengtian country of Zhangwu,Jianye country of Linghai, Wang shanbao country of Kang ping,Huang huangdi country of Faku in Liaoning Province as our survey field spots.We investigated farmers aged over 16 years and collected their urine and fasting blood samples.Based on the value of urinary fluoride and dental fluorosis,the subjects were divided into two groups,fluorosis group and control group.2.Fluoride examinationCollecting the urine samples and record the name,number and volume,stored in -20℃refrigerator,measuring with a CSB-F-I fluoride ion electrode.3.Serum OPG testingBlood samples of 5ml was collected,standed two hours,separated the serum with 3000 rpm centrifugation for 20 minutes,stored in -70℃refrigerator.We used double-antibody sandwich ABC-ELISA method to test the levels of human serum OPG. With anti-human OPG monoclonal antibody coated on the ELISA plate,the standard materials and samples of OPG and monoclonal antibody binded,then added streptavidinhorseradish peroxidase conjugate.After halfhour incubation and the last washing step,the remaining conjugate was allowed to react with the substrate H₂O₂-tetramethylbenzidine.The reaction was stopped by addition of acidic solution and absorbance of the resulting yellow product was measured at 450 nm.The absorbance was proportional to the concentration of osteoprotegerin.A standard curve was constructed by plotting absorbance values versus osteoprotegerin concentrations of standards,and concentrations of unknown samples were determined using this standard curve.4.Statistical analysisWe use non-parametric test,x² test,t test or correlation analysis,do the statistical analysis with the software of SPSS16.0.Results1.Genaral status of peopleThere was no significant difference in the constitution ratio of gender,income and culture,and no significant difference in age.2.Dental fluorosis comparedThere was no significant difference in dental fluorosis divided by gender.3.Urinary fluoride levels comparedThere was no significant difference in urinary fluoride by group and gender.4.Correlation between dental fluorosis degree and urinary fluoride levelThe urinary fluoride level positively correlated with the dental fluorosis degree.5.Serum OPG levels comparedThere were no significant difference in different fluoride exposure and gender with the level of OPG,but higher in old age groups than the young,and there were no significant correlation between urinary fluoride and the level of OPG..ConclutionsThere was no significant difference in different fluoride exposure and gender,but higher in old age groups than the young,suggesting that the level of OPG and the time of fluoride exposure might have the relationship,pending further study.