| Embryonic stem cells (ES cells) have pluripotency character and can differentiate into different cell lines. The differentiation procession of mES cells in vitro is similar to the development of embryo in vivo."Hanging drop culture-suspension culture-adherent culture" is the classic method of mouse ES cells directional differentiation into cardiomyocytes. ES cells are applied in cell therapy, the drug discovery and test of embryonic toxicological compounds. The embryonic stem cell test (EST) is one of three in vitro embryotoxic tests validated by the European Centre for the Validation of Alternative Methods (ECVAM). It is less expensive, need less time-consuming and don’t require large numbers of animals compared in vivo animal tests. Currently, the EST is the most promising in vitro alternative to the in vivo animal tests. The most commonly used is the cardiac mouse embryonic stem cell test (ESTc), which assesses the effect of developmental toxicants on cardiomyocyte differentiation. The cell lines used in the EST were mES cell line D3,3T3from balb/c mouse. Three end-points in the EST included IC50D3, IC503T3, ID50. They means respectively:the concentration of the tested compounds that inhibits50%viability of ES cells or3T3cells and the concentration of the tested compounds causes50%inhibition of differentiation of mES cells into cardiomyocytes. Based on the three endpoints, compounds were classified into three classes:non-embryotoxic, weakly or strongly.PFAS (Polyfluorinated Alkyl Substances) has been widely used in a variety of industrial and comsumer products. Due to their high-energy fluorine-carbon bonds, they are physicochemical stability and resistant to degradation. PFOS and PFOA have been detected in wildlife and human serum worldwide via environmental persistence, and distribution through food webs. Recent reports have suggested that PFAS might have developmental toxicity, reproductive toxicity, cardiovascular toxicity and neurotoxicity in experiment animal studies. But the developmental cardiotoxicity of PFAS using EST and molecular mechanisms is unkown.Stable isotope labeling of amino acids in cell culture (SILAC), deploying the in vivo incorporation of amino acids with substituted stable isotop elabeled amino acids into cell culture, was employed for mass spectrometry-based quantitative proteomics. SILAC is a new method of comparative proteomics recently which can directly capture low abundance protein and rapid qualitative and quantitative identification analysis to quickly identify important functional protein.In our study, we selected three compounds (PFOS, PFOA, PFBS) included in PFAS to test whether they have developmental cardiotoxicity. These results can reveal the cardiotoxicity targets of PFAS, and may provide a valuable attempt to expand the application of EST to the toxicology field.Aim:To assess developmental cardiotoxicity of three compounds (PFOS, PFOA, PFBS) included in PFAS. To establish the protein expression profiles of mouse embryonic stem cells (mES cells) differentiating into cardiomyocytes exposed PFOS and explore the mechanisms of developmental cardiotoxicity of PFOS. A comprehensive quantitative analysis of changes in proteins following exposure of the cells to DMSO or PFOS40μM for10d was performed using the stable isotope labeling of amino acids in cell culture (SILAC) approach.Methods:Using the hanging drop culture method, mES cells were differentiated into cardiomyocytes. The tested compounds were added into each sample from the day0. The EST system was established with the positive control5-FU, the negative control penicillinG and another control DPH. The tested compounds were PFOS, PFOA and PFBS. The embryotoxic potential was determined by three endpoints calculated from the IC503T3, IC50D3, and ID50D3.A MTT assay was performed to determine cell viability of3T3and mES cells and the cytotoxicity of the tested compounds was determined from the inhibiting concentration of50%(IC50) obtained through concentration-response curve. Statistical differentiation rate of myocardial cells to detect the effects of tested compounds on the differentiation of mES cells into cardiomyoctyes. The inhibition of differentiation (ID50) was calculated from the concentration-response curve. The three endpoints obtained from cytotoxic assay and differentiation assay were involved in three linear discriminant functions to assess classification embryotoxicity of the tested compounds.To elucidate how PFOS affects the development of embryo, we performed SILAC analysis and mass spectrometry-based quantitative proteomics on cardiomyocyte differentiation from mouse embryonic stem cells. With one-dimensional gel electrophoresis separation of proteins, stained using silver staining, scanned and Mass Spectrometry analysis to identify the different expression. We searched the proteins having at least1.5-fold changes with P<0.05, and made analysis involved in these pathways using GeneGO as well as KEGG pathway.In the identified proteins, we selected the ROS signaling cascades and DNA methylation system as a mechanism for the study. The related genes and proteins were detected by western blot and RT-PCR. Cardiac-specific proteins were analyzed by flow cytometry intracellular ROS levels were measured using the fluorescent dye DCF-DA.Results:IC50D3of penicillinG, DPH and5-FU was6981,246and0.36μM, IC503T3was6370、240and0.45μM, ID50D3was4626,99and0.31μM. The embryo toxic ity was classified as strongly, weakly, or non-embryotoxic by the prediction model. These were accord with the reported, which suggested that the EST system was abolished successfully. IC50D3of PFBS, PFOA and PFOS was4139,470,291μM, IC503T3was5980,484,435μM, ID50D3was808,223,40μM. PFOS and PFOA were classified as weakly embryotoxic compounds, while PFBS was classified as non-embryotoxicity compound, and PFOS was more toxic than PFOA. The different toxicity is related to the structure of the tested chemicals. The functional groups of PFAS when they had the same chain length indicate that PFAS with a sulfonate group had a larger toxic potential. And the PFAS with the same functional groups had a larger toxic potential when they had longer chain length.Our results showed PFOS affected EBs’morphologicariinl changes, which made the radius of EBs decrease. After PFOS treatment, the expression of Brachury, a-MHC, GATA4, MEF2C increased assessed by RT-PCR and western blot. The results of flow cytometry demonstrated PFOS decreased the expression of of a-actinin. Aftet the exposure of PFOS, we observed67up-regulated and109down-regulated with PFOS in protein expression profiles. The analysis about67up-regulated and109down-regulated proteins involved in GeneGO, KEGG pathway and network. It revealed that PFOS plays roles in31pathway and1296interactions with other proteins.PFOS could decrease expression of Dnmt3a and Dnmt3b increase expression of OCT4. Expression of Dnmt3b decreased from d3to d6and expression of Dnmt3a decreased at d6compared with control. The OCT4methylation and siliencing were inhibited because of decrease expression of Dnmt3a and Dnmt3b.On the other hand, PFOS could increase expression of SOD2, p-Hsp27and decrease ROS generation in EBs. Expression of SOD2decreased from d3to d5and expression of Dnmt3a decreased from d3to d10compared with control. ROS signaling cascades was essential for cardiomyocyte differentiation of mouse ES cells in vitro. ROS activated ERK1,2and p38MAPK leaded to the stimulation of cardiac differentiation from mouse mES cells. While with PFOS treated, phosphorylation of ERK and p38MAPK was lower and the expression of GATA4, MEF2C, α-MHC, a-actinin and MYH10decreased.Conclusion1. PFOS and PFOA indicated with weakly embryotoxicity, while PFBS had non-embryotoxicity, and the following order of toxicity:PFOS> PFOA> PFBS. The different toxicity was related to the structure of the tested chemicals. The functional groups of PFAS when they had the same chain length indicated that PFAS with a sulfonate group had a larger toxic potential. And the PFAS with the same functional groups had a larger toxic potential when they had longer chain length.2. The analysis about67up-regulated and109down-regulated proteins under PFOS exposured involved in GeneGO, KEGG pathway and network. It revealed that PFOS plays roles in31pathway and1296interactions with other proteins.3. PFOS made ROS in EBs decrease via the expression of SOD2and p-Hsp27increasing, hindered p38MAPK activity to affect cardiomyocyte differentiation from mouse embryonic stem cells. PFOS inhibited the silence of OCT4and differentiate of ES through decreasing the expression of Dnmt3b, Dnmt3a. |
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