Effects Of Polybrominated Diphenyl Ethers On Steroidogenesis And The Toxicity Study Using Mouse Embryonic Stem Cells

Polybrominated Diphenyl Ethers (PBDEs) are a group of brominated flame retardant (BFR) chemicals and have been widely used in textile and furniture, building materials and electronics and other potential flammable product because of their thermal stability and high flame retardant efficiency. Since1970, production and use of PBDEs has resulted in environmental pollution globally. Environmental epidemiology data have shown that PBDEs exist widely in various environmental media, and may through the digestive tract, respiratory and skin et al enter the body. Now we can detect PBDEs in the human body blood, milk and fat and other organizations. PBDEs have the property of low degradation, environmental stability, high fat-solubility and biological amplification functions, which can be transferred through food chain to allow the organisms of higher-level grade to be poisoned and finally bring dangers to human health. The current study suggests that, PBDEs might be carcinogenicity, reproductive toxicity, neurotoxicity and endocrine toxicity in animals test, and the main target organs of the effects including the nervous system, adipose tissue, thyroid and reproductive system, etc. Therefore, in the United States and Europe and other places, low-brominated PBDEs such as BDE-47,99,153and so on have been produced and used restrictly. However, the high bromide PBDEs, such as BDE-209, is still widely used because of its good flame-retardant, biotoxicity of uncertainty. However, BDE209in environment easily happened to take off the bromine reaction, generate low bromine compounds and the adverse effect of BDE-209still not allows ignoring. In present study, we choose BDE-47and BDE-209as the representative PBDEs, and mouse tumor ley dig cells (MLTC-1) and mouse embryonic stem cell (D3) were used to determine the effects of PBDEs on progesterone production and examine the cytotoxicity and the pluripotency maintaining respectively.Part I Effects of BDE-47/BDE-209on Steroidogenesis and related pathwayObjectiveTo investigate the effect of BDE-47/BDE-209on steridogenesis and the role of cAMP-dependent cascade in the inhibited progesterone production of MLTC-1cells by BDE-47/BDE-209.Methods1. The mouse Ley dig tumor cells (MLTC-1) were used as the experimental model.2. The cell viability of MLTC-1cells affected by BDE-47/BDE-209was analyzed using the method of MTT.3. MLTC-1cells were treated with different concentrations of BDE-47/BDE-209for24h. Then hCG (0.1U/L) was added into the medium. The level of progesterone in the medium was measured by RIA.4. MLTC-1cells were treated with different concentrations of BDE-47/BDE-209for 24h. Medium was then replaced by BDE-47/BDE-209-free media for24h following the stimulation with hCG.5. MLTC-1cells were treated with different concentrations of BDE-47/BDE-209for24h. Then0.1U/L hCG,30ng/ml CT or lOμmol/L forskolin were added into the medium for additional4h. The level of progesterone was measured by RIA and cAMP level was measured by ELISA kits.6. MLTC-1cells were treated with different concentrations of BDE-47/BDE-209for24h. Then500μmol/L8-Br-cAMP,25μmol/L22R-HC or10μmol/L pregnenolone were added into the medium for additional4h. The level of progesterone was measured by RIA.7. The mRNA expression of StAR/30-HSD/P450scc in MLTC-1cells was determined by Real-time PCR.8. Protein expression of StAR/3β-HSD/P450scc in MLTC-1cells was assessed by western blot.Results1. The doses of BDE-47/BDE-209were selected in virtue of the MTT result. They were control (0.1%solvent),0.04,0.2,1,5, and25μmol/L.2. BDE-47/BDE-209inhibited progesterone production in dose-dependent manner with the stimulation of hCG.3. After BDE-47/BDE-209was removed; the progesterone production was similar in the cells previously treated by various concentrations of BDE-47/BDE-209.4. The progesterone production declined in dose-dependent manner as the concentrations of BDE-47increased when stimulated by forskolin. But there is no significant difference when stimulated by CT. Meanwhile, BDE-209has the same results.5. Dose-dependent attenuation of cAMP formation was observed after24h exposure to BDE-47when MLTC-1cells were stimulated with hCG and forskolin. However, in the presence of CT, BDE-47treatment resulted in almost equivalent amounts of cAMP production at different doses. BDE-209treatment can not see any significant difference when stimulated by hCG/forskolin/CT.6. When stimulated with8-Br-cAMP, the progesterone production was significantly inhibited by both BDE-47and BDE-209.7. BDE-47significantly suppressed22R-HC/pregnenolone-driven progesterone production; and the inhibitory effect of BDE-209on progesterone formation disappeared in the presence of22R-HC/pregnenolone.8. The expressions of StAR mRNA and protein had no change in MLTC-1cells as the doses of BDE-47/BDE-209increased.9. BDE-47treatment inhibited P450scc mRNA and protein levels in a dose-dependent manner, while only observed3β-HSD mRNA level decreasing but not protein level. As to BDE-209, we saw P450scc/3β-HSD mRNA level declining but not protein level.Conclusion1. BDE-47significantly inhibited steroidogenesis in MLTC-1cells, and the cells could recover most of their steroidogenic activity after the removal of BDE-47. And this action involved the impairment of the cAMP-dependent cascade by attenuating cAMP formation. Also, BDE-47suppressed P450scc and3β-HSD activity, expression and/or protein levels which might partly be explained by the involvement of the cAMP-dependent cascade.2. BDE-209significantly inhibited steroidogenesis in MLTC-1cells, and the cells could recover most of their steroidogenic activity after the removal of BDE-209. However this action did not involve the impairment of the cAMP-dependent. Part II Effects of BDE-47on mouse embryonic stem cells and related mechanism in vitroObjectiveTo investigate the cytotoxicity of BDE-47on mouse embryonic stem cells and determine the effect of BDE-47on the cell pluripotency maintaining and the related mechanism behind them.Methods1. The D3mouse embryonic stem cell was used as the experimental model.2. D3mESCs cells were treated with different concentrations of BDE-47for24h or48h. Then the cell morphology was observed.3. D3mESCs cells were treated with different concentrations of BDE-47for24h or48h. Then the cell viability of D3mESCs cells affected by BDE-47was analyzed using the method of MTT.4. D3mESCs cells were treated with different concentrations of BDE-47for24h. Then the cell cycle was analysed by flow cytometry.5. D3mESCs cells were treated with different concentrations of BDE-47for24h. The cell apoptosis was examined by Flow cytometry and DeadEndTM fluoremetry TUNEL system. Real time PCR was used to determine the mRNA level of P53after the treatment by BDE-47.6. D3mESCs cells were treated with different concentrations of BDE-47for24h. The effect of BDE047on alkaline phosphatase was analysed by AP staining kits and AP activity assay. 7. D3mESCs cells were treated with different concentrations of BDE-47for24h. The expression of the transcription factors Oct-4/Sox-2/Nanog were examined by cyto-immunofluorescence staining assay.8. The mRNA level of Oct-4/Sox-2/Nanog and the expression of microRNA-145/34a were determined by Real-time PCR after different concentrations of BDE-47treatment.9. The protein level of Oct-4/Sox-2/Nanog was determined by western blot after different concentrations of BDE-47treatment.Results1. After exposed to BDE-4724h, the D3mESCs cell morphology was changed at25μM and100μM. Comparing with controls, the cell density were obviously reduced, cell atrophy were show up, and part of the cloning edge collapse.2. MTT results showed that with the24h-treatment of BDE-47, D3mESCs cell viability were not affect significantly, but with48h treatment, cell activity was inhibited at100μM. Thus, we selected0.04μM,1μM,25μM, and100μM as the exposed dose and24h as the exposed time.3. Cell cycle analysis revealed that BDE-47did not affect the distribution of t he cell cycle stage in D3mESCs with the treatment of BDE-47.4. The cell apoptosis analysis by flowcyto metry showed that the apoptosis eel-1count increased at25μM and above. DeadEndTM fluoremetry TUNEL syste-m results showed the same results. Besides, P53Expression levels rised after BDE-47treatment.5. After exposed to BDE-4724h, the AP staining and AP activity were both i-nhibited.6. Cyto-immunofluorescence staining results showed the expression of the trans cription factors Oct-4/Sox-2/Nanog was declined. 7. After treatment with BDE-4724h, the protein/mRNA level of the transcripti on factors Oct-4/Sox-2/Nanog were both decreased. The expression of mmu-m iR-145/mmu-miR-34a was both declined after BDE-47treatment.Conclusion1、BDE-47could cause the cell morphology change, cell viability decreas and cell apoptosis.2、BDE-47could disturb the D3mESCs cell pluripotency maintaining a nd Significantly inhibit the expression of transcription factor Oct-4/Sox-2/Nanog, and the mechanism may relate to boost the related microRNA (mmu-miR-145and mmu-miR-34a) high expression.