The Role Of Mitochondria On Oxidative Damage Induced By Sodium Arsenite In Human Bronchial Epithelial Cells

Objective:To explore the role of mitochondrial DNA(mt DNA) in oxidative damage induced by sodium arsenite in human bronchial epithelial cells(HBE), to provide a new research approach in elucidate the molecular mechanism of arsenic toxicity.Method:(1) Establish the model of mitochondrial DNA-depleted HBE cells: according to the literatures, using Et Br to reduce mt DNA copy number of HBE cells. Identify mt DNA reduction by nutritional deficiencies, and real-time PCR, define the mt DNA-depleted HBE cells as ρ-HBE, and control group as ρ+HBE. According to the exposure doses of the method 2, using the way of western blot to detecte the level of proliferation protein NQO1.(2) The determination of sodium arsenite exposed dose sodium arsenite: take it the two groups of cells(ρ+HBE and ρ-HBE) as the research object, exposured by different doses of sodium arsenite(0~160μM), the cells’ viability was detected by MTT cell cytotoxicity assay kit. Using SPSS19.0 software to calculate the dose of sodium arsenite,which effect the cells at the viability over 70%, so as to determine the following sodium arsenite’s dose.(3) The detection of relevant indicators of oxidative stress: the ρ+HBE and ρ-HBE cells were exposed to different dose of sodium arsenite(0、1、2、4、8、16μM) for 24 h, then collecting the all cells. Using flow cytometry to determine the ROS levels. Thiobarbituric acid(TBA) assay were used to detect cells’ malondialdehyde(MDA) content. The activities of SOD in HBE cells were determined by using the WST-1. Using the DTNB to detect cell GSH content. Detecting the related proteins( including Nrf2, Keap1 and downstream hememono oxygenase protein expression) of Keap1/Nrf2-ARE antioxidant pathway by Western blot.(4) The detection of relevant proteins of apoptosis: All of cells were exposed to different concentrations of sodium arsenite(0, 1, 2, 4, 8, 16μM) for 24 h. The apoptosis related proteins(Bcl-2, Bax, Caspase9, Caspase3) were quantified by Western blot.Results:1. The establishment of mitochondrial DNA-depleted HBE cellsThe HBE cells were exposed to 50ng/ml Et Br for 7d, then culture at ρ0(mt DNA completely missing cells) induced medium for 3d, we could find that a large number of cells were dead. Real-time PCR results displayed that Et Br-induced cells’ mt DNA copy number were decreased about 97.30%. With different concentrations of sodium arsenite(0,1, 2, 4, 8, 16μM) treated for 24 h, the NQO1 protein levels in ρ+HBE cells were first increase and then decrease, compared with ρ+HBE cells, the ρ-HBE cells NQO1 protein levels showed a downward trend, and were lower than the control cells(P <0.05).2.The cells viability of HBE cells exposured by different dose sodium arseniteThe ρ+HBE and ρ-HBE cells were exposed to different dose of sodium arsenite(0、1、2、4、8、16μM) for 24 h, as the MTT cell toxicity results showing, along with the increase of the concentration of sodium arsenite, the cells viability of the two groups were both reduced. And at the same dose of sodium arsenite effect, the ρ-HBE cells viability was higher than the ρ+HBE cells. By using SPSS 19.0, we calculated the IC50 of arsenite doses,the ρ-HBE cells’ IC50 was 88.351μM, while ρ+HBE cells were 75.34μM, at the same time,the rate of cell proliferation sodium arsenite concentrations above 70% were≤26.83μM(ρ-HBE), ≤ 14.66μM(ρ+HBE). So we determined the sodium arsenite subsequent exposure doses were 0, 1, 2, 4, 8, 16μM.3. The oxidative stress levels of HBE cells exposured by different dose sodium arsenite.With increasing concentration of sodium arsenite, in ρ+HBE cells, the ROS and MDA levels were increased, while ρ-HBE cells were showing a down trend instead, but were higher than the untreated group, and were higher than the control group(ρ+HBE) cells(P<0.05). Along with the concentration of sodium arsenite increase in the two groups, SOD enzyme activity levels were gradually decreased, when less than 8μM sodium arsenite exposure, the SOD enzyme activity levels in ρ-HBE cells were higher than in ρ+HBE cells,while when it came to 16μM sodium arsenite, the SOD enzyme activity levels in ρ-HBE cells were lower than ρ+HBE cells(P<0.05). In the two groups, GSH, GSH/GSSG levels were increased first and then decreased along with the increased concentration of sodium arsenite, the GSH levels in ρ-HBE cells higher than in ρ+HBE cells at the dose of 0~4μM sodium arsenite, and then were lower than in ρ+HBE cells(P<0.05). The HO-1 protein levels were increased along with the exposed dose of sodium arsenite in both two groups,and in ρ-HBE cells, HO-1 levels were higher than in ρ+HBE cells(P<0.05). Along with the concentration of sodium arsenite increase in the two groups, the Nrf2 and Keap1 levels were increased first and then decreased, and in ρ-HBE cells, the two proteins’ levels showde a continued high level of state, and were higher than control(ρ+HBE) cells(P<0.05).4. The apoptosis related protein levels of HBE cells exposured by different dose sodium arsenite.With increasing concentration of sodium arsenite, the Bcl-2 and Bax protein levels were increased first and then decreased in ρ+HBE cells, but in ρ-HBE cells, the Bax, Bcl-2protein levels showed a decreasing trend, and in the exposure of 0~4μM sodium arsenite,the Bcl-2 protein levels in ρ-HBE cells were higher than in ρ+HBE cells, while in the exposure of 8~16μM, the Bcl-2 protein levels in ρ-HBE cells were lower than in ρ+HBE cells(P <0.05). The Bax protein levels in ρ-HBE cells were lower than in ρ+HBE cells when exposed by all dose of sodium arsenite. The Bcl-2/Bax ratios in the two groups were showing a downward trend after the first rise, and the ρ-HBE cells were higher than theρ+HBE cells when exposured at the same concentration of sodium arsenite(P <0.05). With increasing concentration of sodium arsenite, the protein levels of caspase9 and cleaved caspase9 were increased first and then decreased, and the two protein levels in ρ-HBE cells were both significantly lower than in the ρ+HBE cells(P <0.05). Along with the concentration of sodium arsenite increase in the two groups, the caspase3 and cleaved caspase3 protein levels showed a downward after the first rising trend, and the difference of the changes between the two groups were not significant.ConclusionMitochondrial DNA will alleviate oxidative damage of cells induced by sodium arsenite in some degree, and can induce apoptosis to remove damaged cells. Mitochondria play a protective role in cellular physiological activity.