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.