Nrf2 Transcription Factor Mediated Bisphenols-induced SK-N-SH Cell Damage And Its Mechanism

Objective: To observe the effects of bisphenol A(BPA)and bisphenol S(BPS)on the growth of human neuroblastoma SK-N-SH cell line.The aim of this study was to explore the role of Nrf2 signaling pathway on oxidative stress and apoptosis induced by BPA and BPS,and to further elucidate the mechanism of its cytotoxicity.Methods:1.The SK-N-SH cells were treated with different concentrations of BPS(0,50,100,150,200,250,300,350,400 ?mol/L)or BPA(0,50,100,150,200 ?mol/L)for 48 h and the cell viability was determined by MTT assay.The quantity of intracellular superoxide dismutase(SOD),glutathione reductase(GSH),reactive oxygen species(ROS)and malondialdehyde(MDA)were determined by spectrophotometry.The expression of apoptosis-related proteins(Bax,Bcl-2)and Nrf2 were detected by Western Blot.Cell cycle profile and apoptosis were assessed by flow cytometry.2.The SK-N-SH cells were pretreated with 20 ?mol/L t-BHQ,a Nrf2 activator,for 30 min and then incubated with BPS(100 ?mol/L)or BPA(100 ?mol/L)for another 48 h.The quantity of ROS,SOD,MDA and GSH were determined spectrophotometrically.The expression of apoptosis-related proteins(Bax,Bcl-2)and Nrf2 were detected by Western Blot.Cell cycle profile and apoptosis were assessed by flow cytometry.Results:1.The BPA IC50 of SK-N-SH cells was 164.54 ?mol/L after 48 h of BPA treatment.While the BPS IC50 of SK-N-SH cells was 345.43 ?mol/L after 48 h of BPA treatment.Significantly decreased levels of SOD and GSH and increased levels of ROS and MDA compared with control group were observed following treatment with 100 ?mol/L BPA.When SK-N-SH cells were treated with different concentrations of BPS,the ROS and MDA levels increased first and then decreased with the increase of the exposure dose,and reached the highest level at 200 ?mol/L.At the same time,the levels of SOD and GSH decreased.Meanwhile,the ratio of Bcl-2 to Bax was decreased,the expression of Nrf2 protein and mitochondrial membrane potential were decreased with a dose–response relationship.2.The combination treatments with t-BHQ and BPS/BPA increased SOD and GSH levels,Bcl-2 to Bax ratio,Nrf2 protein expression levels and mitochondrial membrane potential,decreased MDA and ROS levels than BPS/BPA treatment alone.3.The 100 ?mol/L BPS and the 100 ?mol/L BPA treatment alone group could induced cell cycle arrest and apoptosis.And the cell cycle arrest and apoptosis was alleviated in the combination treatments with t-BHQ and BPS/BPA group compared with BPS/BPA treatment alone.Conclusions:1.Bisphenol A and bisphenol S can reduced cell viability in SK-N-SH cells,induces the cellular oxidative stress,cell cycle profile and apoptosis.2.The Nrf2 transcription factor mediated the cell damage of BPS/BPA on SK-N-SH cells.