Study On Neurodevelopmental Toxicity And Its Mechanism Of Organophosphates Based On Human Embryonic Stem Cell Derived Motor Neuron Model

TBEP and TCEP are two main types of organophosphorus flame retardants(OPFRs)that are frequently detected in environmental media or organisms.Mounting evidence in animal experiments proves that early life stage exposure to organophosphate flame retardants(OPFRs)affects the locomotor behavior and changes the transcriptions of central nervous system genes.However,their effect on human motor neuron(MN)development,which is necessary for body locomotion and survival,has not yet characterized before due to the lack of suitable in vitro human MN model system.And little is known about the effects of TBEP and TCEP on human motor neuron(MN)neurite outgrowth,which is particularly essential for MN development and function throughout life.Here,we established an in vitro MN differentiation model from human embryonic stem cells(hESCs),which were successively differentiated into neuroepithelial(NE)cells,motor neuron progenitor(pMN)cells,and terminally towards MNs.Then we adopted this model to test the effects of two OPFRs with different substitutions,including non-halogenated tris(2-butoxyethyl)phosphate TBEP and halogenated tris(2-chloroethyl)phosphate TCEP,on human MN differentiation and the possible mechanisms behind,and evaluated the toxicity of TBEP and TCEP at human body exposure level on human MN neurite growth.We first measured the changes in gene expression levels at different stages of differentiation after exposure to TBEP and TCEP.At the same time,the expression changes of cytoskeletal protein and presynaptic structural protein after exposure were further analyzed.Finally,the molecular level changes of Notch pathway,typical WNT/β-catenin pathway and TGFβ signal regulating neurite formation were investigated after exposure to TBEP and TCEP.The results are as follows:1.Our results showed that TBEP showed stronger activation on NE and subsequent pMN cell formation than TCEP.Intriguingly,both TBEP and TCEP significantly disrupted the generation of MNs from pMN stage even at concentrations in human body.Ultimately,we demonstrated that TBEP and TCEP interfered with MN differentiation by up-regulating NOTCH signaling and down-regulation of WNT signaling.2.We demonstrated for the first time that both TBEP and TCEP could disrupt MN neurite growth,whereas TBEP showing significantly more potent.Further analysis found the two OPFRs impaired the expression of cytoskeletal protein responsible for neurite outgrowth and presynaptic structural proteins in axon terminal.Mechanistically,our findings reveal that TBEP and TCEP exposure significantly affected the expression of key factors in the TGFβ signaling pathway,which plays crucial roles in neurite outgrowth.In conclusion,this is the first study to develop an in vitro human MN differentiation model for evaluation of OPFRs potentially detrimental to fetal MN development.This study provides new insights into the cellular and molecular mechanisms behind the toxicity of TBEP and TCEP on human MN differentiation and axonal growth,and raise concern about their potential harm in causing MN disorders.