Bisphenol A Represses Dopaminergic Neuron Differen

Authors: Boxian Huang Song Ning Qinjing Zhang Aiqin Chen Chunyan Jiang Yugui Cui Jian Hu Hong Li Guoping Fan Lianju Qin Jiayin Liu

Publish Date: 2016/06/07

Volume: 54, Issue: 5, Pages: 3798-3812

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Abstract

Bisphenol A BPA is a ubiquitous compound emerging as a possible toxicant during embryonic development Human embryonic stem cell hESC promises a valuable model for evaluating the effects of environmental chemicals on human prenatal development In our study 1 μM BPA were applied to hESCderived embryoid bodies hEBs and effects of BPA on neural cell differentiation were investigated The expression level of insulinlike growth factor 1 IGF1 and marker genes for ectoderm neuron progenitor cells and dopaminergic DA neurons were all repressed upon BPA exposure The population of hESCderived neural precursor cells NPCs and DA neurons were decreased Furthermore yield of DA neuronsecreted tyrosine hydroxylase TH and dopamine were also reduced When recombinant IGF1 supplied BPAcaused repressions were partially or completely relieved Our further methylation microarray analysis indicated that there was a higher methylation level on the promoter of SRYrelated HMGbox 5 SOX5 a possible enhancer of IGF1 Consistently next quantitative polymerase chain reaction qPCR results confirmed that SOX5 expression was downregulated Our investigation suggests that BPA represses DA neuron differentiation mainly through downregulating IGF1 expression which may attribute to the altered methylation level on the promoter of IGF1 upstream genes Our findings first elaborate the mechanism of IGF1mediated BPA effects on neuronal differentiation which is helpful to illuminate the unique mechanism of BPA toxicity on prenatal neurodevelopmentThis work was supported by the grants from the State Major Research Program of China 2012CB944902 National Natural Science Foundation of China 81370764 Jiangsu projects BL2012009 FXK201221 and the PAPD and China Scholarship Council 201307060009 Wu Jieping Research Funds BE2012653 We thank Wisconsin International Stem Cell Research Institute USA for providing us H9 hESCsLQ and JL carried out theoretical analysis LQ BH and AC have been involved in experimental design laboratory analysis data collection and statistical analysis LQ and BH wrote the manuscript BH SN QZ AC JH and CJ conducted overall experiments JL YC HL and GF reviewed the manuscriptProtocol for differentiation DA neurons from hESCs hESCs were induced differentiation into DA neurons through 2 stages stage I induction of NPCs stage II differentiation into DA neurons according to previous report with slight modification There were 3 steps in the stage I hEBs in all the three steps were cultured in BDM except that in Step 2 and 3 05  N2 and 1  N2 plus 10 ng/ml bFGF were added respectively Stage II included 2 steps Step 1 NPCs were differentiated into neural cells by cultured for 2 weeks in the neuron induced medium 1 composed of B27 1  N2 200 ng/ml SHH and 100 ng/ml bFGF8 Step 2 neural cells were differentiated into DA neurons by cultured for 4 days in the neuron induced medium 2 composed of B27 1  N2 200 ng/ml SHH 100 ng/ml bFGF8 200uM AA and 50 mg/ml heparin Cells were exposed to 01  DMSO 1 μM E2 1 μM BPA or 1 μM BPA plus 01 μM IGF1 respectively during all differentiation stages and steps NPCs Neuronal precursor cells SNM Spherical neural mass DA dopaminergic hEBs hESCsderived embryoid bodies BDM basic differentiation media hESC culture medium without bFGF bFGF basic fibroblast growth factor SHH Sonic hedgehog AA ascorbic acid DMSO dimethylsulfoxide BPA Bisphenol A IGF1 Insulinlike growth factor 1 E2 estrogen GIF 30 kb1 μM BPA did not affect hEBs growth a Apoptosis and proliferation rates of hEB were evaluated by FACS assay First panel annexin V staining assay 01 μM and 1 μM BPA had no significant effects on the early apoptosis of hEBs but 10 μM and 100 μM BPA significantly increased the early apoptosis rate of hEBs Second panel TUNEL assay 01 μM and 1 μM BPA had no significant effects on the late apoptosis of hEBs but 10 μM and 100 μM BPA significantly increased the late apoptosis rate of hEBs Third panel Ki67 staining assay 01 μM and 1 μM BPA had no significant effects on hEBs proliferation but 10 μM and 100 μM BPA significantly decreased the proliferation rate of hEBs b The morphology of hEBs exposed to 1 μM BPA was similar to that of DMSO group while hEBs treated by 10 μM and 100 μM BPA presented abnormal appearance Scar bar 100 μm n = 3 error bars indicate SD p  005 p  001 p  0001 GIF 109 kb

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