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TBBPA及其替代物TBBPS和TCBPA胚胎发育毒性的评估
Alternative TitleEvaluation of the Developmental Toxicity of TBBPA and Its Substitutes TBBPS and TCBPA
梁少君
Subtype博士
Thesis AdvisorFrancesco Faiola(费凡)
2019-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
KeywordTbbpa,tbbps,tcbpa,胚胎发育毒性,胚胎干细胞 Tbbpa, Tbbps, Tcbpa, Embryonic Developmental Toxicity, Embryonic Stem Cells
Abstract

      四溴双酚A(TBBPA)是目前使用最广泛的溴代阻燃剂,在环境和人体体液样本中高频检出。TBBPA在人脐带血样本中的高检出率和相对较高的浓度引起了人们对TBBPA的胚胎发育毒性的关注。目前的研究结果表明,TBBPA可能影响动物的胚胎发育,但其胚胎发育毒性尚无定论。四溴双酚S(TBBPS)和四氯双酚A(TCBPA)是TBBPA的替代物,与TBBPA有相似的化学结构。目前,TBBPS和TCBPA的发育毒性评估较少。三种阻燃剂对人胚胎发育的影响尚无直接相关的评估数据。
      胚胎干细胞可以在体外形成拟胚体或者定向分化成任何组织细胞,例如肝细胞、神经细胞和心肌细胞等。在特定的诱导条件下,胚胎干细胞分化系统可以在体外模拟胚胎的早期发育,或者某一组织细胞的发育过程。基于此系统,本研究从多角度评估了人体暴露和环境相关浓度的TBBPA、TBBPS和TCBPA对胚胎发育的影响。
      首先,本研究建立28天小鼠拟胚体体外分化模型,模拟小鼠胚胎早期发育的过程。基于此模型和转录组测序,本研究分析了TBBPA对拟胚体分化过程中不同阶段的影响,推测TBBPA潜在的发育毒性靶器官。1-100 nM的TBBPA在拟胚体发育早期阶段(第4天和第9天)影响发育相关的转录调控。在第12天和第18天,TBBPA上调神经相关的基因转录表达。在第18天和第28天,TBBPA下调心脏和骨骼肌发育相关的基因表达。
      然后,本研究基于小鼠胚胎干细胞的神经定向分化模型,评估了TBBPA和其替代物TBBPS和TCBPA对神经发育的影响。和拟胚体毒性评估结果一致,TBBPA异常地促进神经前体阶段的发育。TBBPS和TBBPA的毒性效应相似,显著地促进神经相关基因的表达。TCBPA明显抑制神经发育相关基因的表达。TBBPA轻微地干扰了影响神经早期发育的重要信号通路NOTCH(正调节)和WNT(负调节)信号通路相关基因的表达;TBBPS则明显抑制了WNT通路;TCBPA下调了NOTCH效应分子的表达,并增强WNT信号通路。因此,TBBPA、TBBPS和TCBPA通过干扰WNT和NOTCH信号通路影响小鼠神经的早期发育。
      随后,本研究在人胚胎干细胞神经发育系统上对上述研究结果和其毒性机制深入验证。人体暴露和环境相关浓度(1-100 nM)的三种化合物对神经干细胞没有明显的细胞毒性。本研究发现三种污染物可能通过干扰GSK3β细胞信号影响神经多能性基因SOX3、SOX2和NES的表达。此外,这三种污染物通过干扰甲状腺激素的细胞信号,影响和神经元生成相关基因SOX3和TUBB3的表达。因此,1-100 nM的TBBPA、TBBPS和TCBPA可能通过干扰GSK3β和甲状腺激素信号通路影响人神经干细胞的多能和分化基因的表达。
      基于人胚胎干细胞的体外心肌细胞分化模型,本研究评估了TBBPA对人心肌细胞和骨骼肌细胞生成的影响。1-100 nM的TBBPA显著的降低分化细胞的跳动,并且下调心肌细胞成熟相关基因的表达。此结果表明TBBPA影响心肌细胞的分化。此研究结果和上述小鼠拟胚体模型的毒性评估结果一致。
      小鼠拟胚体模型的毒性评估结果显示,TBBPA有潜在的肝脏发育毒性。本研究基于体外人胚胎干细胞的肝实质细胞样细胞发育模型,评估了TBBPA及其替代物TBBPS和TCBPA对人肝脏实质细胞分化的影响。TBBPA、TBBPS和TCBPA影响肝实质细胞样细胞的发育,并且该影响开始于肝脏发育过程中的限制性内胚层和肝脏前体细胞阶段。基于转录组数据分析,本研究发现,TBBPA、TBBPS和TCBPA可能干扰限制性内胚层阶段的TGF-beta信号通路和肝脏发育过程中的SERPINs家族基因的表达。此外,该研究发现,TCBPA的肝脏发育毒性强于TBBPA。
      综上所述,环境和人体暴露剂量下的TBBPA可能会主要影响胚胎的神经、肌肉系统和肝脏的早期发育。TBBPS和TCBPA对神经和肝脏等发育的影响明显强于TBBPA,这说明TBBPS和TCBPA并不是TBBPA安全的替代物。

Other Abstract

      Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant, and frequently detected in the environment and human body fluid samples. The high detection rate and relatively high concentrations of TBBPA in human umbilical cord blood samples have attracted people's attention to its likely embryonic developmental toxicity. Tetrabromobisphenol S (TBBPS) and tetrachlorobisphenol A (TCBPA) are substitutes of TBBPA, and have similar chemical structures compared to TBBPA. Current findings suggest that TBBPS and TCBPA may have similar toxicity as TBBPA, and prticularly disrupt endocrine systems. However, the effects of the three flame retardants on human embryonic development are still unclear.
      Embryonic stem cells (ESCs) can form embryoid bodies in vitro and differentiate into any tissue cells, such as hepatocytes, neurons, cardiomyocytes, etc. With defined induction conditions, the ESCs differentiation system can simulate the early stage of embryo development or the tissue development process in vitro. Based on this system, in this study we evaluated the effects of TBBPA, TBBPS and TCBPA at human exposure and environment relevant concentrations, on embryonic development.
      First, we employed a mouse embryoid body (mEB)- based 28-day differentiation process to mimic in vitro the process of embryo development. Based on this model and whole transcriptome sequencing (RNA-seq), we analyzed the effects of TBBPA on different stages of embryoid bodies formation. TBBPA, at 1-100 nM, affected development-related transcriptional regulators at early stages of the differentiation (day 4 and day 9). On day 12 and 18, TBBPA upregulated the expression of neuron-related genes. On day 18 and 28, TBBPA down-regulated genes associated with heart and skeletal muscle development.
      Then, we evaluated the effects of TBBPA and its substitutes TBBPS and TCBPA, on neural development based on the neural specification model from mouse ESCs. Consistent with the results of the embryoid body experiments, TBBPA abnormally upregualated the expression of neural development related genes at the neural precursors’ stage. The toxic effects of TBBPS were similar, significantly promoting the expression of neurogenesis related genes. However, TCBPA significantly inhibited the expression of neurodevelopment-related genes. Mechanismically, TBBPA slightly interfered with the important signaling pathways NOTCH (positive regulation) and WNT (negative regulation). TBBPS significantly inhibited the expression of WNT pathway-related genes. TCBPA down-regulated the expression of NOTCH effectors, but enhanced the expression of WNT signaling pathway-related genes.
      Subsequently, we further evaluated the effects of TBBPA, TBBPS and TCBPA on neural development based on human neural stem cells (hNSCs). The three compounds, at human and environmental concentrations (1-100 nM), did not exert cytotoxicity to hNSCs. However, the three pollutants may interfere with GSK3β signaling, thereby affecting the expression of the neural multipotent genes SOX3, SOX2 and NES. In addition, these three pollutants affected the expression of SOX3 and TUBB3, related to neurogensis, in hNSCs differentiation process, by interfering with the thyroid hormone cellular signaling pathway. Therefore, these results suggest that TBBPA, TBBPS and TCBPA may affect the neural development of human embryos.
      Based on an hESC cardiomyocyte differentiation model, we investigated the effects of TBBPA on human cardiomyocyte and skeletal muscle cell genesis. 1-100 nM TBBPA significantly reduced the number of beating cells. Moreover, it reduced the expression of genes related to the maturation of cardiomyocytes. These results indicate that TBBPA affected the maturation of cardiomyocytes, which was consistent with the toxicity assessment results obtained with the mEB system.
      The results of the toxicity assessment with the mEB model showed that TBBPA had potential hepatic developmental toxicity. Thus, we also evaluated the effects of TBBPA and its substitutes TBBPS and TCBPA, on human liver development, based on the in vitro hepatocyte specification from hESCs. TBBPA, TBBPS and TCBPA affected hepatocyte specification by altering definitive endoderm and hepatoblast differentiation. With RNA-seq analyses, we found that TBBPA, TBBPS and TCBPA interfered with the TGF-beta signaling pathway at the definitive endoderm stage, and SERPIN family genes expression in the hepatic developmental process. Additionally, the hepatic developmental toxicity of TCBPA was stronger than that of TBBPA.
      To sum up, TBBPA, at environment and human exposure relevant concentrations, may mainly affect the early stages of development of the nervous and muscle systems, and liver cells. The adverse effects of TBBPS and TCBPA on nerve and liver development were stronger than those of TBBPA. Therefore, TBBPS and TCBPA are not safe alternatives to TBBPA.

Pages177
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42244
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
梁少君. TBBPA及其替代物TBBPS和TCBPA胚胎发育毒性的评估[D]. 北京. 中国科学院生态环境研究中心,2019.
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