Effects Of TBBPA And Its Derivatives On Viability And Steroid Hormone Secretion Of BeWo Cells

Objective:The aim of this study was to compare the cytotoxic effects of TBBPA,TBBPABHEE,TBBPA-BGE,TBBPA-BAE and TBBPA-BDBPE on BeWo cells,the effects of TBBPA and TBBPA-BHEE on ATP content and mitochondrial membrane potential were explored to study the placentotoxicity mechanisms in BeWo cells.The purpose of this study was also to investigate the impact of TBBPA and TBBPA-BHEE on pregnenolone and progesterone secretion,and the genes and proteins expression of key enzyme in steroid hormones synthesis were detected to study related mechanisms in BeWo cells.Methods:1.MTS assay was used to detected cell viability of BeWo cells after treated with 0-100??M TBBPA,TBBPA-BHEE,TBBPA-BGE,TBBPA-BDBPE and 0-50??M TBBPA-BAE for 24 h and 48 h.2.The changes of mitochondrial membrane potential in BeWo cells were detected after treated with 0,0.05,1,10,100??M TBBPA and 0,0.05,1,10,25 ?M TBBPABHEE for 48 h.3.The intracellular ATP content was calculated in BeWo cells after treated with 0,0.05,1,10,100??M TBBPA and 0,0.05,1,10,25 ?M TBBPA-BHEE for 48 h.4.ELISA kits were used to quantify pregnenolone and progesterone levels in conditional culture medium after treated with 0,0.05,1,10,100??M TBBPA and 0,0.05,1,10,25 ?M TBBPA-BHEE for 48 h.5.The mRNA expression levels of cyp11a1 gene and 3?-hsd gene in BeWo cells were determinated by qPCR after treated with 0,0.05,1,10,100??M TBBPA and 0,0.05,1,10,25 ?M TBBPA-BHEE for 48 h.6.The protein expression levels of CYP11A1 and 3?-HSD in BeWo cells were determinated by western blot after treated with 0,0.05,1,10,100??M TBBPA and 0,0.05,1,10,25 ?M TBBPA-BHEE for 48 h.Results:1.Compared with control group,BeWo cell viability was significantly inhibited by TBBPA,TBBPA-BHEE,TBBPA-BGE and TBBPA-BAE for 24 h and 48 h exposure.Exposed to TBBPA-BDBPE for 24 h had nearly no effect on the viability of BeWo cells under the tested concentrations,but it induced cell viability inhibition at the concentration of 0.1-100 ?M for 48 h.2.Compared with control group,mitochondrial depolarization was induced by 100 ?M TBBPA,10 and 25 ?M TBBPA-BHEE for 48 h exposure.3.Compared with control group,the intracellular ATP content in BeWo cells was significantly decreased after exposed to 100 ?M TBBPA,10 and 25 ?M TBBPA-BHEE for 48 h.4.For TBBPA treatment,compared with control group,pregnenolone and progesterone levels increased only in the 100 ?M with 2.54 and 2.07 folds increasement,respectively.For TBBPA-BHEE treatment,the pregnenolone secretion increased in a dose-dependent manner after incubated with 0-25 ?M TBBPA-BHEE for 48 h,that increased by around 1.10,1.22,1.54 and 3.04 folds for 0.05 ?M,1 ?M,10 ?M and 25 ?M TBBPA-BHEE treatment groups,respectively.TBBPA-BHEE also significantly increased progesterone secretion in a dose-dependent manner,that increased by around 1.22,1.58 and 1.61 folds of control group for 1 ?M,10 ?M and 25 ?M TBBPA-BHEE treatment groups,respectively.5.The mRNA expression level of cyp11a1 gene in BeWo cells was slightly upregulated by 100 ?M TBBPA,10 and 25 ?M TBBPA-BHEE for 48 h exposure.0-100 ?M TBBPA exposure had non-significant effects on the mRNA expression of 3?-hsd gene,while that were significantly decreased in 10 and 25 ?M TBBPA-BHEE treatment groups.6.Exposed to 0-100 ?M TBBPA and 0-25 ?M TBBPA-BHEE for 48 h had nonsignificant effects on CYP11A1 and 3?-HSD protein expression.Conclusions:1.In the experimental range of concentration(0-100 ?M),TBBPA,TBBPABHEE,TBBPA-BGE and TBBPA-BAE(0-50 ?M)had an obvious inhibition on BeWo cell proliferation in a dose-and time-dependent manner.The sequence of cellular toxicity of tested chemicals on BeWo cells were TBBPA-BHEE > TBBPA-BAE > TBBPA-BGE > TBBPA > TBBPA-BDBPE,which was not entirely dependent on hydrophobicity.2.TBBPA and TBBPA-BHEE mediated BeWo cytotoxicity by inducing mitochondrial depolarization and reducing intracellular ATP content.TBBPA and TBBPA-BHEE may be apoptotic agonists of BeWo cells.3.TBBPA and TBBPA-BHEE promoted pregnenolone secretion as potential endocrine disrupting chemicals,and they can also stimulate progesterone secretion by increasing pregnenolone level.4.The mechanisms of TBBPA and TBBPA-BHEE on pregnenolone and progesterone promotion were independent of key enzymes expression.The mechanisms of TBBPA and TBBPA-BHEE on progesterone promotion were partly associated with the ability of tested chemicals on pregnenolone increase.