Disturbance In PPARs Regulation In Human Bone Marrow Mesenchymal Stem Cells Induced By Novel Substitutes To Per- And Polyfluoroalkyl Substances

The potential health risks of per-and polyfluoroalkyl substances(PFASs)alternatives are not well-known.Since peroxisome proliferators-activated receptors(PPARs)are the primary molecular targets of PFASs,it is of great significance to evaluate the disturbance in PPARs caused by novel PFASs substitutes for the prediction and screening of their toxicity.In the present study,the in vitro differentiation model of human bone marrow mesenchymal stem cells was used to investigate the effects of PFASs substitutes on various subtypes of PPARs in undifferentiated and differentiated cells,as well as to elucidate the relationship between abnormal regulation of PPARs and the imbalance of multidirectional cell differentiation.Tested compounds included alternatives to perflurooctane sulfonate(PFOS),chlorinated polyfluorinated ether sulfonate(Cl-PFESA)and perfluorohexane sulfonate(PFHxS),and alternatives to perfluorooctanoic acid(PFOA),hexafluoropropylene oxide trimer acid(HFPO-TA)and hexafluoropropylene oxide dimer acid(HFPO-DA).The main research contents and results include:(1)At non-cytotoxic concentrations,0.1 ?M,1 ?M and 10 ?M,exposure to PFOS,PFOA and their substitutes for 7 days stimulated the gene expression of three PPARs subtypes in undifferentiated cells,whereas PFOS and PFHxS posed stronger activation on PPAR?,and Cl-PFESA posed stronger activation on PPAR?.PFOA exhibited stronger effects on PPAR?,while HFPO-DA and HFPO-TA exhibited stronger effects on PPAR?.(2)Exposure to PFOS and PFHxS for 7 days at non-cytotoxic concentrations stimulated the gene expression of three PPARs subtypes in differentiated cells,with strong effects on PPAR?.Cl-PFESA activated PPAR? and PPAR?,without significant effects on PPAR?.PFOA and its substitutes HFPO-DA and HFPO-TA upregulated gene expression of PPARs in differentiated cells,consistently causing relatively stronger effects on PPAR?.Except Cl-PFESA,the test substances repressed gene expression of the osteogenic differentiation-specific transcription factor RUNX2,and upregulated the gene expression of adipogenic differentiation-specific transcription factor CEBP?.PFOS,PFHxS,HFPO-DA and HFPO-TA consistently increased RANKL/OPG,suggesting enhanced osteoclastogenesis,while Cl-PFESA decreased RANKL/OPG.(3)After exposure of differentiated cells for 14 days,the test compounds,except Cl-PFESA,inhibited the formation of calcium nodules(phenotype marker of osteogenic differentiation),and promoted lipid droplet formation(phenotype marker of adipogenic differentiation).Variations in phenotypic marker were consistent with PPAR? activating,down-regulation of RUNX2 gene,and up-regulation of CEBP? gene.On the contrary,Cl-PFESA promoted the formation of calcium nodules,without significant effects on lipid droplet formation,which was consistent with the absent changes in PPAR? gene expression and relatively stronger activating to PPAR? and OPG.The tested novel PFASs substitutes stimulated the gene expression of PPARs subtypes in human bone marrow mesenchymal stem cells,which in turn interfered the regulation of cell differentiation genes,resulting in imbalance of osteogenic/adipogenic differentiation.Due to the diversity in the effects on various PPARs subtypes,the tested compounds led to different cell differentiation direction,which may be associated with different harmful health outcomes.Our results provide scientific evidence and new method for identifying the toxicity mechanisms and health risks of novel PFASs alternatives.