Study On Embryo/neurodevelopmental Toxicity Of Bisphenol A Based On Human Embryonic Stem Cell Model And Its Exposure Limit

Bisphenol A（BPA）is a widely used organic compound with estrogen activity and can interfere with endogenous estrogen effect.However,the toxicity mechanism of BPA has not been clarified.At the same time,risk assessment methods have also been constantly developed,from the previous"external dose and response"analysis to the relevant data inclined to be included in the mode of action（MOA）as much as possible,so as to correlate the toxic effects based on the target tissue with the concentration of the active form of substances,and provide a dose measurement method related to biological reactions.The Physiologically based pharmacokinetics（PBPK）model is a powerful tool for such risk assessment.Embryonic stem cells can still self-renew and proliferate in vitro and are induced to differentiate into almost all types of cells of the body.They have been used in organ and tissue transplantation,cell replacement therapy,chemical toxicity assessment and other fields.The European Centre for the Validation of Alternative Methods（ECVAM）officially approved the embryonic stem cell test（EST）based on mouse embryonic stem cells in 2002.EST is an in vitro alternative evaluation model to predict the developmental toxicity of chemicals.However,EST uses mouse embryonic stem cell as the test system,when extrapolating its evaluation results to humans,there is still uncertainty caused by species differences.In addition,the traditional EST takes myocardial differentiation as the observation end point,and myocardial differentiation is mainly related to mesoderm,so it is impossible to detect the impact on other embryonic development.If the impact of compounds on non-myocardial differentiation is detected at the same time,it is expected to improve the accuracy and specificity of prediction.Our research team conducted biological information analysis on the toxic effects of BPA previously,and the results showed that BPA mainly acts on gonad,embryo and nervous system.Therefore,this study focuses on the test and evaluation of the embryotoxicity and neurodevelopmental toxicity of BPA.The traditional EST method was improved and optimized by using human embryonic stem cell H9（h ESC-H9）to replace mouse embryonic stem cell.And the developmental toxicity of BPA was evaluated with two observation endpoints of myocardial differentiation and neural differentiation,then the established h ESC-H9 model was used to explore the mechanism of embryotoxicity and neurodevelopmental of BPA.BPA concentrations and toxicity effect data were substituted into the model to carry out the MOA-based health risk assessment,and the exposure limits of BPA embryotoxicity and neurodevelopmental toxicity were obtained,which provides the basis for the setting of BPA health guidance value and risk management based on toxicity pathway.Part 1:Evaluation of embryotoxicity of bisphenol A based on human embryonic stem cell modelObjective:To establish the h ESC-H9 cell evaluation model and apply it to evaluate the embryonic developmental toxicity of BPA,in order to accumulate data for the application of h ESC in the toxicity evaluation alternative experiment,provide data for the optimization and development of EST method,and further improve the accuracy of embryonic developmental toxicity prediction.Methods:（1）Embryo differentiation:h ESC-H9 was cultured in trophoblast free culture.Embryonic body（EB）was formed using Aggre Well^TM culture plate.EB was further grown by suspension culture and then was transferred to adherent culture to induce natural differentiation of EB;（2）Nerve differentiation:h ESC-H9was cultured in trophoblast culture method.EB was formed by Aggre Well^TMculture plate.Collecting EBs and inducing them to form neural rosettes,replanting the neural rosette to form neural progenitor cells,further differentiating to neuronal precursors,collecting neuronal precursor cells,and then adherent culture after passage to enter the neuronal maturity stage.（3）Establishment and validation of h ESC model:the experiment was designed according to the traditional EST standard operating procedures,and the known developmental toxicity standard negative reference Penicillin G（PN-G）and standard positive reference 5-fluorouracil（5-FU）recommended by ECVAM were used to verify the model.（4）The application of h ESC model:Based on model validation,Balb/c-3T3 and h ESC-H9 cells in the logarithmic growth stage were treated with different concentrations of BPA for 7 days.The cytotoxicity test was used to detect the 50%inhibitory proliferation concentration(IC₅₀)of BPA of the two cells.According to the pre-experiment results,cells with h ESC-H9 embryonic differentiation and neural differentiation were treated with BPA at different concentrations for 10 days.Quantitative Real-time polymerase chain reaction（q PCR）was used to detect the expression of alpha myosin heavy chain（α-MHC）of h ESC-H9 embryonic differentiation and the expression of nestin of h ESC-H9 neural cells.The effect curve was fitted to obtain the 50%inhibition of differentiation(ID₅₀)of the two differentiated cells.IC₅₀ of Balb/c-3T3 and h ESC-H9 and ID₅₀ of two kinds of differentiated cells were respectively substituted into the linear discriminant function formula for calculation,and the embryotoxicity of BPA was evaluated.Results:h ESC-H9 cells grew well by culturing with trophoblast-free culture method.Embryonic differentiation and neural differentiation cells showed good differentiation status.The validation results of the h ESC evaluation model showed that PN-G was non embryotoxic and 5-FU was strongly embryotoxic,which was consistent with the ECVAM evaluation results.The h ESC evaluation model was used to evaluate the embryotoxicity of BPA.The results showed that the IC₅₀ of BPA to h ESC-H9 was 135.20μM.IC₅₀was 63.2μM for Balb/c-3T3.The ID₅₀ of BPA for h ESC-H9 embryo differentiation was 54.63μM;The ID₅₀ of BPA for h ESC-H9 nerve differentiation was 100.80μM.The data of the two types of differentiation end points were substituted into the linear function formula to obtain the same results:II>I>III.According to the criteria,the h ESC model evaluated BPA as a weak embryotoxicity substance.Conclusions:An EST evaluation model based on h ESC-H9 was established and validated.The established model is used to evaluate BPA as a weak embryotoxicity substance,which is the same as the classical EST evaluation results.It provides a scientific basis for the application of h ESC in the evaluation of embryotoxicity and lays a foundation for the further exploration of the embryotoxicity and neurotoxicity mechanism of BPA.Part 2:Study on the mechanism of embryonic development of bisphenol AObjective:To explore the embryonic toxicity and mechanism of BPA based on the embryonic development model of human embryonic stem cells constructed in Part 1.Methods:The steps of cell culture and embryo differentiation were the same as those in Part 1.The height and weight of Chinese adult women were taken as physiological parameters in the pregnancy PBPK model of BPA,the dietary exposure of Chinese people to BPA,and the temporary tolerable daily intake（t-TDI）of BPA established by the European Food Safety Authority（EFSA）in 2015as the external exposure to calculate the concentration of BPA in the amniotic fluid after oral exposure to BPA.BPA exposure groups were set and 0.1%dimethyl sulfoxide（DMSO）as the solvent control group,10 n Mβ-Estradiol（17β-Estradiol,E2）as a positive control group.Several inhibitors G15,ICI182780,SP600125 and SB203580 were used for key pathway validation.h ESC-H9 during embryonic differentiation was exposed to BPA for 14 days.PI single staining flow cytometry was used to detect the effect of BPA on cell cycle.Sequencing of eukaryotic participating transcriptome was used to analyze the possible key pathways of BPA affecting h ESC-H9 embryonic differentiation.q PCR and Western blot were used to detect the effect of genes and protein expression.Results:With the BPA dietary exposure of Chinese people and the t-TDI established by EFSA in 2015 as the external exposure,the area under the concentration versus time curve（AUC）of BPA in amniotic fluid for 24 hours was calculated.0.05 n M and 1 n M were obtained through dividing AUC by 24.Based on this dose and the pre-experimental results,five concentration groups of BPA（0.05 n M、1 n M、100 n M、10μΜ、30μΜ）were designed in this study.The results of cell cycle showed that BPA could induce the cell cycle of differentiated h ESC-H9 cells to be disordered.The proportion of G0/G1 phase cells in the combined exposure group of G protein-coupled receptors（GPR30）inhibitor G15and 10?M BPA was significantly decreased,and the proportion of G2/M phase cells was significantly increased.The difference was statistically significant.The sequencing results of the DMSO group,10?M BPA group and E2 group showed that the differential genes of 10?M BPA group versus DMSO group and E2 group versus DMSO group were enriched in the following KEGG pathways:protein digestion and absorption,ECM receptor interaction,P13K-Akt signal pathway,MAPK signal pathway and Ras signal pathway.Moreover,the differentially expressed genes in 10?M BPA group versus DMSO group were also enriched in the ovarian steroid production pathway.The results of q PCR showed that the expression of CDKN1C,HSD3B1,CYP19A1,CYP11A1,GPER1,CACNA1C and CGA changed non-monotonously with the increase of exposure dose,and the differences between groups were significant.Western blot results showed that compared with the blank control,the protein expression of CYP19A1 in 0.05 n M,1 n M,100 n M,10?M and E2 groups was significantly decreased（P<0.05）.Compared with the 10?M BPA group,the expression of CDKN1C,CYP11A1,CYP19A1 and HSD3B1 in 10?M+ICI182780 and 10?M+SP600125 inhibitor groups were all decreased.The difference in the expression of CDKN1C was statistically significant（P<0.05）,and the expression of P-ERK was significantly increased（P<0.05）.Conclusions:The effects of GPR30 inhibitor G15 on the cell cycle of BPA group cells and ER inhibitor ICI182780 on the protein content of BPA group cells suggest that both ER and GPR30,as estrogen receptors,may bind to BPA,inhibit the ERK/JNK pathway（indicated by the effect of JNK inhibitor SP600125 on the protein content of BPA group cells）,affect the expression of steroidogenic genes（HSD3B1,CYP11A1 and CYP19A1）,and thus interfere with hormone production and cause embryotoxic effects.Part 3:Study on the mechanism of neurodevelopmental toxicity of bisphenol AObjective:To explore the neurodevelopmental toxicity of BPA and its mechanism based on the neural differentiation model of human embryonic stem cells constructed in Part 1.Methods:The steps of cell culture and nerve differentiation were the same as those in Part 1.The input parameters of the BPA pregnancy PBPK model were the same as those in Part 2.The output parameters are the concentration of BPA in the fetal brain after human oral exposure to BPA.Five concentration groups of BPA were set,0.1%DMSO as the solvent control group,10 n M E2 as the positive control group.Several inhibitors U0126,ICI182780 and MK801 were used to carry out key pathway validation.h ESC-H9 during neural differentiation was exposed to BPA for 14 days.FITC/PI double staining flow cytometry was performed to detect the effect of BPA on cell apoptosis,and a PCR array was designed based on the biological information analysis results and relevant data of our research group on BPA in the early stage to detect the m RNA concentration of genes related to neurodevelopmental toxicity.4D-PRM targeted quantitative proteome was conducted to verify the protein content of related genes in the possible key pathway of neural differentiation.Results:Based on the internal exposure simulation of PBPK model and the pre-experimental results,five BPA concentration groups（0.014 n M、0.256 n M、50?M、75?M、100?M）were designed in this study.The results of cell apoptosis suggested that BPA could induce apoptosis in h ESC neural differentiated cells,but there was no concentration dependence.Compared with the 50?M BPA group,the proportion of early apoptosis cells in the 50?M BPA+MK801 group and the 50?M BPA+U0126 group increased significantly,and the proportion of late apoptosis cells in the 50?M BPA+U0126 group decreased significantly,with statistically significant differences（P<0.05）.The results of PCR array showed that the m RNA content of RPS6KA2,RCAN1,PTN,NTRK2,NFATC2,NFAT5,GR1A1,FABP5,EGR3,BCL2,ARAF,BDNF,GRIN2A,GRIN2D,GRIN3A and POMC showed differences among groups,but the expression amount of each gene changed non monotonically with the increase of exposure dose.The 4D-PRM targeted quantitative proteomics analysis of DMSO group,0.014 n M BPA group,50?M BPA group,100?M BPA group,50?M BPA+ICI182780 and 50?M BPA+MK801 group was conducted.The protein content of 35 selected proteins was determined and the difference between groups was verified.The results showed that with the increase of BPA concentration,the number of differential proteins in 0.014 n M,50μM and 100μM BPA groups increased gradually.Compared with DMSO control group,the expression of Survival of motor neuron-related-splicing factor 30（O75940）in 0.014 n M BPA group was significantly increased,the expression of GTPase NRas（P01111）in 50?M BPA group was significantly increased,and the expression of Ribosomal protein S6 kinase alpha-3（P51812）was significantly decreased;The expressions of P01111,Calmodulin-1（P0DP23）and Caspase-3（P42574）were significantly up-regulated,and the expressions of P51812 and Guanine nucleotide-binding protein G（i）subunit alpha-1（P63096）were significantly down regulated in the 100?M BPA group.Compared with 50?M BPA group,the expression of P51812,P63096 and Calcium uniporter protein,mitochondrial（Q8NE86）in 50?M BPA+ICI182780 group was significantly reduced;The expressions of Guanine nucleotide-binding protein G（i）subunit alpha-2（P04899）,Neurofilament light polypeptide（P07196）,Protein kinase C alpha type（P17252）,Dual specificity mitogen-activated protein kinase kinase 2（P36507）,P51812,P63096,Apoptosis regulator BAX（Q07812）,Calcium/calmodulin-dependent protein kinase type IV（Q16566）and Q8NE86were significantly decreased in the 50?M BPA+MK801 group.Conclusions:The results of this study showed that the number of differential proteins between ER inhibitor group and BPA group was limited,suggesting that although ER mediates the neurodevelopmental toxicity of BPA,it may not be the only receptor of action.The content of GTPase NRas protein in 50?M and 100?M BPA groups was significantly higher than that in DMSO group.Combined with the literature data,it suggests that BPA may also act as a GTPase ligand,activate GTPase NRas,and trigger the downstream signal cascade reaction,including ERK1/2 signal pathway and NMDAR-mediated LTP pathway.Part 4:Evaluation of exposure limit of bisphenol A combined with in vitro test and PBPK modelObjective:To combine the experimental data of Part 2 and Part 3 with PBPK based in vitro to in vivo extrapolation（IVIVE）to derive human equivalent dose（HED）and further obtain reference doses（Rf Ds）.Methods:The PCR data of embryotoxicity and neural differentiation toxicity in Part 2 and Part 3 was tested for significance of difference,and then the qualified data and the corresponding BPA concentration were included in the Bayesian benchmark dose（BBMD）analysis to build a dose response relationship model.The benchmark dose lower confidence limit（BMDL）was taken,and the baseline response（BMR）was set at 5%.The BMDL₅ value of each gene was set as the starting point（POD）.In order to reconstruct exposure,100000 iterations of Markov Chain Monte Carlo（MCMC）simulation were performed using R statistical software.The maximum steady-state concentration（Cmax,ss）and its quartile value of each gene can be obtained by running four Markov chains（MCs）.POD and Cmax,ss were substituted into the formula to get HEDs.The quintile of HEDs is taken as threshold dose（TD）,and divided it by the safety factor 10 to get Rf Ds.Results:From the PCR data of embryotoxicity test and neurodevelopmental toxicity test,the minimum Rf Ds（μg/kg/d）were 4.940（HSD3B1）and 5.181（nuclear factor of activated T cells 2,NFATC2）respectively.It is close to the t-TDI（4?g/kg·bw）formulated by EFSA in 2015.Conclusions:This study built a dose-response relationship model based on the embryonic toxicity and neurodevelopmental toxicity data of h ESC,and the minimum Rf Ds calculated by PBPK model are 4.940?g/kg/d and 5.181?g/kg/d,respectively,which are not significantly different and close to the t-TDI（4?g/kg·bw）formulated by EFSA in 2015.This result shows that the combination of in vitro toxicological dose-response data and PBPK model can be a potential alternative method to support the decision-making of chemical risk assessment.SummaryIn this study,we first successfully constructed an embryonic developmental toxicity evaluation model using h ESC and applied it to the developmental toxicity evaluation of BPA.Then the mechanism of embryonic toxicity and neurodevelopmental toxicity of BPA was explored by referring to the embryonic differentiation and neurodifferentiation methods in the established model.The dose-response data of embryotoxicity and neurodevelopmental toxicity of BPA was integrated with PBPK model to obtain the Rf Ds of BPA embryonic developmental toxicity and neurodevelopmental toxicity.This study can provide a basis for comprehensively elucidating the embryotoxicity and neurodevelopmental toxicity of BPA and its mechanism of action and MOA,and provide a reference for the application of in vitro toxicological substitution experiments and dose-response data in risk assessment.