The Effects Of Perfluorooctane Sulfonate On Mouse Embryonic Stem Cells And Mouse Embryoid Bodies

Perfluorooctane sulfonate (PFOS) is one of the persistent organic pollutants (POPs), which caused widespread environmental concerns in recent years. Due to its hydrophobic and oleophobic, it is widely used in paints, textiles, toothpaste, pharmaceuticals, electronics, petrochemical, fire extinguishing agents, adhesives and insecticides. In light of its environmental persistence, bioaccumulation, and potential toxicity, PFOS exposure generates great concern about global pollution. It has been identified in various environmental sectors, including air, sewage sludge, snow, lake, and surface runoff water. PFOS is also commonly detected in maternal serum, amniotic fluid, umbilical cord blood, breast milk, nail, hair, urine and semen. Currently, there are many reports about the reproductive and developmental toxicity of PFOS, but they mainly focused on the study of birth weight, behavioral intelligence, etc., little is known about the underlying molecular mechanisms. In this study, we conducted a comprehensive assessment of PFOS by two in vitro models (mouse embryonic stem cells and mouse embryoid bodies), and further clarified the molecular mechanisms by epigenetic regulation (miRNA, PcG).Part I Effects of Perfluorooctane sulfonate on pluripotency factors of mouse embryonic stem cells AbstractMouse embryonic stem cells (mESCs) are ideal models for developmental toxicity testing of environmental contaminants in vitro. Perfluorooctane sulfonate (PFOS) poses potential risks to reproduction and development. However, the mechanism by which PFOS affects early embryonic development is still unclear. In this study, mESCs were exposed to PFOS for 24 h, and then general cytotoxicity and pluripotency factors were evaluated. MTT assay showed that neither PFOS (0.2μM, 2μM,20μM,200μM) nor control medium (0.1% DMSO) treatments affected cell viability. Furthermore, there were no significant differences in cell cycle and apoptosis between the PFOS treatment and control groups. There was no significant differences in alkaline phosphatase staining (AP). However, we found that the mRNA and protein levels of pluripotency markers (Sox2, Nanog) in mESCs were significantly decreased following exposure to PFOS for 24 h, while there were no significant changes in the mRNA and protein levels of Oct4. Accordingly, the expression levels of mmu-miR-145 and mmu-miR-490-3p, which can regulate Sox2 and Nanog, were significantly increased. Chrm2, the host gene of mmu-miR-490-3p, was positively associated with mmu-miR-490-3p expression after PFOS exposure. Dual luciferase reporter assay suggested that mmu-miR-490-3p directly target Nanog. In summary, this study firstly clarify the toxic effects of PFOS exposure to mESCs and the possible mechanisms. These results suggested that PFOS could disturb the expression of pluripotency factors in mESCs, while mmu-miR-145 and mmu-miR-490-3p played key roles in modulating this effect.Part Ⅱ Effects of Perfluorooctane sulfonate on differentiation factors of mouse embryoid bodies AbstractEmbryonic stem cells were cultured in differentiation medium (free of LIF) can undergo spontaneous differentiation, and they can development in spatial and temporal patterns of early embryonic development during the whole process, and then formed three-dimensional aggregates embryoid body (EB). The formation of EB is similar to the early stages of embryo implantation. Thus, EB is often used as an in vitro model to study the differentiation associated gene expressions and early embryonic development. In the present study, after PFOS exposure to EB, we collected the EBs at 2day,4day,6day. We tested the morphology, pluripotency factors, differentiation factors, miRNA and PcG of mEBs. The results showed that compared to the control, PFOS exposure did not affect the morphology of EB. The expression levels of pluripotent factors (Oct4, Sox2, Nanog) were significantly increased. Accordingly, the expression levels of mmu-miR-134, mmu-miR-145 and mmu-miR-490-3p, which can regulate pluripotency factors were significantly decreased. We found that the differentiation markers (Sox 17, FOXA2, SMA, Brachyury, Nestin, Fgf5) of mEBs were significantly decreased following exposure to PFOS, while the subunit of PcG (Cbx4, Cbx7, Ezh2) were significantly increased. In summary, this study firstly clarify the toxic effects of PFOS exposure to mEBs and the possible mechanisms. These results suggested that PFOS could disturb the expression of pluripotency factors and differentiation markers in mEBs, while miRNA and PcG played key roles in modulating this effect.