Effects And Mechanisms Of Representative Poly-and Perfluorinated Compounds On The Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

Per-and polyfluoroalkyl substances(PFASs)have been widely used in water-resistant coatings,food packaging,fire fighting foam and other industrial and civilian fields with their good stability and oil-and water-repelling properties.PFASs are detected in various environmental media and human serum from all over the world due to the mass production and use,as well as the characteristics of persistent organic pollutants.Many countries have strictly controlled and phased out the production and emissions of perfluorooctane sulfonate(PFOS),perfluorooctanoic acid(PFOA)and related chemicals,but they are still produced and used in some industries.In addition,PFOS substitutes have been developed rapidly,and the production and use increased dramatically.But the environmental pollution levels and toxicology research on PFOS subsitutes are limited,and their environmental health risk is unknown.Dalian has been reported of higher PFASs concentrations in various environmental media,and even human serum,than most of other regions in China.Therefore,the environmental risk of PFASs in this area has attracted extensive concern.Animal experiments and human anatomical results suggested that bone tissue may be the target tissue of PFASs.Epidemiological survey showed that human serum PFASs concentrations were associated with skeletal health.Human bone marrow mesenchymal stem cells(hBMSCs)are common progenitor cells of osteoblasts and adipocytes,which play an important role in maintaining bone health and can provide an effective detection system for studying the effects of environmental pollutants on bone health and potential molecular mechanisms.In this study,the temporal trend of environmental pollution of typical perfluorinated acids(PFAAs)and their substitutes was studied by monitoring PFASs in the atmospheric particles of Dalian.Based on the present study and other research results,five representative PFASs at high pollution level were selected to study their effects and potential mechanism on the hBMSCs based on cytotoxicity,differentiation,gene and protein expression.The main contents include,(1)The level and temporal trend of PFASs in the atmosphere of Dalian were studied.The results showed that the level of PFAAs increased in 2006-2008 and then decreased,ranging from 35.7 pg/m3 to 860 pg/m3.Perfluorocarboxylic acid(PFCAs)were the main substances,among which the level of PFOA was the highest with the average concentration of 71.7 pg/m3.The concentration of PFOS was the highest in perfluoroalkyl sulfonates(PFSAs),followed by PFHxS and PFBS.In 2007-2014,the concentration of PFOS alternatives-chlorofluorophyl ether sulfonate(Cl-PFESA,with trade name F-53B)increased substantially,ranging from 85.9-722 pg/m3,accounting for 37.9% to 95% of the total PFASs.(2)Alkaline phosphatase(ALP)activity,calcium nodules and key gene expression during osteogenesis differentiation were analyzed to assess the effects of PFASs exposure on osteogenic differentiation of hBMSCs.PFOS had no significant effects on ALP activity at day 7,while F-53 B,PFOA,PFHxS and PFBS inhibited the ALP activity in a certain concentration range.PFOS inhibited the formation of calcium nodules in a dose-dependent manner at 0.2-100 nM during osteogenic differentiation of h BMSCs.when 100 nM of PFOS caused a decrease of 65%,compared with the control group.F-53 B exhibited an enhancement on the formation of calcium nodules.The results of Real-time Quantitative PCR(RT-qPCR)showed that the expressions of Runx2,ON and OPN were down-regulated by PFOS exposure.At 100 nM,ON and OPN were down-regulated by 47% and 26%,respectively.The high concentration of PFHxS down-regulated BMP2 and OPN,and PFBS inhibited the expresion of Runx2,ON and OPG,while F-53 B and PFOA induced upregulation of osteogenic differentiation-related genes.The results indicated that perlfuorosulphonates and F-53 B,PFOA could have opposite effects on osteogenesis differentiation.In addition,PFOS significantly down-regulated bone turnover marker OPG in a dose-dependent manner in 0.2-100 nM.Expose to 100 nM of PFOS,OPG was down-regulated by 41%.F-53 B and PFHxS up-regulated bone turnover marker RANKL.PFASs exposure induced RANKL/OPG ratio above 1,suggesting that PFASs may affect the differentiation and activity of osteoclasts via OPG/RANKL/RANK pathway.(3)Effects of PFASs exposure on adipogenic differentiation of hBMSCs was assessed by the formation of lipid droplets and the expression of key adipogenic genes.Oil Red O staining showed that PFOS,PFHx S and low-concentration of PFBS exposure increased the formation of lipid droplets during adipogenic differentiation of hBMSCs.PFOS and PFHx S increased lipid droplets with increasing concentration in 0.2-100 nM.F-53 B and PFOA exposure inhibited the formation of lipid droplets,and the inhibitory effect of F-53 B was dose-dependent.The results of RT-qPCR showed that PFOS and PFHxS up-regulated the expression of PPAR?,C/EBP? and LPL.PFOA down-regulated PPAR ? and LPL expression,and F-53 B up-regulated CEBP? and LPL expression.The results indicated that PFOS and PFHxS exposure promoted adipogenic differentiation of hBMSCs,while PFOA inhibited the adipogenic differentiation.And F-53 B may affect the formation of lipid droplets mainly by influencing the translation of adipogenic key genes during adipogenic differentiation of hBMSCs.(4)The potential molecular mechanism of the effects of PFOS exposure on hBMSCs differentiation was studied by assessing the expression of Runx2,PPAR? and ?-catenin in different stages of hBMSCs differentiation.Moreover,the key gene and protein levels involved in the classical Wnt/?-catenin pathway were measured.And LiCl,an agonist for the classical Wnt/?-catenin pathway was used.In the early stage of osteogenic differentiation,PFOS exposure down-regulated the expression of Runx2 and ?-catenin with consistent trend.And LiCl weakened or even reverse the downregulation of ?-catenin,OPN and ON induced by PFOS exposure.In the late stage of osteogenesis differentiation,Runx2 was significantly down-regulated,while PPAR? was significantly up-regulated in a dose-dependent manner and up-regulated to 3.12 folds at 200 nM PFOS.?-catenin exhibited no significant changes at the gene and protein levels.It is suggested that the classical Wnt/?-catenin pathway plays a key role in the early stage of PFOS-induced effects on osteogenesis differentiation of hBMSCs,and the regulation of PPAR? plays an important role in the late stage.In summary,PFASs at the human exposure relevant dose caused effects on the osteogenic and adipogenic differentiation of hBMSCs at the cell phenotype,key gene expression and protein levels,where the mechnisms may be related with influence on differentiation and activity of osteoclasts through OPG/RANKL/RANK pathway.The results indicated that PFASs may affect bone health by interfering with the balance between osteogenesis and adipogenic differentiation of hBMSCs and the differentiation and activity of the osteoclasts.