The nervous system and liver are two major targets of environmental pollutants.The nervous system requires years to fully development and performs sophisticated biological processes T hus subtle interference from exogenous chemicals may induce severe developmental neural toxicity. The l iver is the organ responsible for many essential functions including the metabolisms of drugs and xenobiotics, and therefore is the targets of many environmental pollutants. Accumulating evidence has shown that a number of pollutants can permeate the placenta barrier and potentially affect embryonic development To comprehensivly assess the health risks of environmental pollutants, it is cruci al to consider the effects on the development of the nervous system and liver cells, such as hepatocytes. However , up to now, limited information is available on this topic . Therefore, we performed the following major aims
(1) Study t he effects of two wid ely used phathalates (DEP and DBP) on the early stages of the nervous system development using mouse embryonc stem cells (mESCs). We showed that both DEP and DBP could decrease mESC viability, with IC 50 of 1.0 ±0.3 mM and 0.26 ± 0.05 mM, respectively. Mec hnistically, DBP induce d intracellular ROS accumulation, cause d cell membrane damage and activate d the caspase 3/7 enzymes, while DEP only induced ROS accumulation. These data suggest that DBP has a more robust acute toxicity than DEP . Moreover, l ow non cytotoxic
concentrations of DEP and DBP (10 μM) might disrupt the early stages of embryo development by dysregulating the expression of markers of three primary germ layers.In fact, during mESC spontaneous differentiation , DEP and DBP disrupt ed the expression of genes responsible for the development of endoderm ( Gata6 , Foxa2 ),mesoderm ( T Hand1 ) and ectoderm Fgf5 , Otx2 ), and significantly altered neural ectoderm markers ( Pax6 , Nestin , Sox1 , Sox3 ). Neural progenitor cells induction assay further confi rmed the potential toxic effects of DEP and DBP on the development of the nervous system. The results can help better understand the associations between phathalates exposure and health problems, and most significantly remind us of the importance of addit ional health risk tests for these two largely used chemicals.
(2)Assess the effects of five halogenated flame retardants (BDE 47, BDE 209,TBBPA, TBBPS, TCBPA) on the nervous system w ith human embryonic stem cell differentiation assay We utilized RNA seq and qRT PCR analys e s and found out that these chemicals disturb ed the neural development process, and may affect the axon growth/ guidance , neuron transmission processes . In addition, these flame retardants may dysregulate the WNT and AHR signaling pathway s. Interestingly, BDE 209 showed similar tocicity with BDE 47 , whereas TBBPS and TCBPA may not be safe alternatives to TBBPA.
(3)Evaluate the effects of thyroid hormones (THs, T3 and T4) during the specification of h epatocytes from human embryonic stem cells. We demonstrated that TH exposure yielded to dsyfuntional hepatocytes, with impaired glycogen storage ability, lipid droplets accumulation and abnormal expression levels of AFP , ALB ,HNF4A , APOA1 , PPARA , CYP3A4 , C YP2C9 , etc. Transcript omics analys e s identified over a thousand of differentially expressed genes (DEGs) in samples treated with 30nM THs , s uggesting the hepatocyte differentiation process is very sensitive to THs.TH treatments mainly affected signaling pathways at late differentiation stage s , and dysregulated metabolism and chemical respnding biological processes, including “cholesterol metabolic process”, “lipoprotein metabolic process”, “cholesterol homeostasis”, “glycine, serine and threonine metaboli sm”, “drug metabolism cytochrome P450”, “carbon metabolism”, “fat digestion and absorption”, “PPAR signaling pathway”, “ABC transporters”, etc. Furthermore, the activation of the PPAR signaling cascades with an agonist of PPAR (GFT505) attenuate d T3 dep endent dysregulation of lipid relative genes PPARA , APOA1 , APOA2 , PPARR , APOA4 ,FABP7 , APOA3 , during hepatocyte differentiation
(4) Establish a set of biomarkers to screen the developmental hepato toxicity of TH analogues Among the all DEGs related to TH actions during hepatocytes differentiation and function s, we selected 69 of them as potential biomarkers. To validate these biomarkers, we used two famous TH analogues (BDE 47 and BDE 209)and treated ifferentiating cells with various concentrations (10 nM 100 nM 1 μM)of these two chemicals. After 19 days of hepatic differentiation, 25 out of the 69 potential biomarkers were responsive to at least one BDE. These 25 genes were
enriched in biological processes including “ chemical carcinogenesis”, “drug metabolism cytochrome P450”, “steroid hormone biosynthesis”. 17 genes were responsive to both BDE 47 and BDE 209, and includ ed APOA1 , AHR , PROX1 , RXRA ,CYP2C9 , CYP2C8 , HNF4A , CYP1A1 , CYP3A7 , THRA , THRB , PPARA , PROZ ,CYP19A 1 , GNMT , HSD3B1 and HPD T hus, these genes may serve as biomarkers for the prediction of unknown developmental hepatotoxicants
In conclusion, we employed embryonic stem cell based differentiation assays to evaluate the effects of environmental pollutants on the development of the nervous system and the most aboundant type of cells of the liver, hepatocytes. Both phathalates (DEP and DBP) and halogenated fl ame retardants (BDE 47, BDE 209,TBBPA, TBBPS, TCBPA) may disrupt the development of the nervous system. These results provide direct evidence for the compre he nsive understanding of the potential health risks of these chemicals. Furthermore, by employing t hyroid hormones ’treatment during human embryonic stem cell differentiation assay s , we identified a set of biomarkers to predict unknown hepatotoxicants. These biomarkers were validated with two toxicants, BDE 47 and BDE 209, providing a reference for the screening of