The Mechanism Of Cr(Ⅵ)-induced Apoptosis In P53 Negative Cells Under Inhibiting Caspase Activation

Objective:To explore the mechanism of Cr(Ⅵ)-induced apoptosis in Hep3B cells under the condition of the absence of p53 and the inhibition of caspase in vitro,then further clarify ROS plays a role in pathway of mitochondria-dependent apoptosis induced by Cr(Ⅵ).Methods:MTT test was adopted to test the viability of Hep3B cells treated with Z-VAD-fmk,NAC,Cr(Ⅵ) respectively at different times and concentrations.Cell apoptosis was detected by using the single cell gel electrophoresiss(SCGE) to find the effective concentrations and times of Cr(Ⅵ) in Hep3B cells.The cultured Hep3B cells were randomly divided into five groups,namely control group,Cr(Ⅵ) alone,Z-VAD-fmk+Cr(Ⅵ),NAC+Cr(Ⅵ) and Z-VAD-fmk+NAC+Cr(Ⅵ).According to the results,Hep3B cells were incubated with Z-VAD-fmk at 10μmol/L,NAC at 10 mmol/L and Z-VAD-fmk+NAC for 1 hour respectively,and then treated with Cr(Ⅵ) at 20μmol/L for 6 hrs.Giemsa staining and electric microscope was performed to detect the morphological features of cells in each group;DNA damage was detected by using the SCGE;AnnexinV-FITC/PI double staining with FCM detection showed the cellular apoptosis and necrosis ratios;The mitochondrial permeability transition pore(PTP) and the mitochondrial transmembrane potential(△ψm) were determined by the fluorospectrophotometric method;The activity of SOD,GR and CAT and the levels of MDA and NO were detected by the colorimetric method.Results:1.The concentration-dependent decrease in cell survival rate of Cr(Ⅵ)-treated Hep3B cells was observed.The relationship between concentration and survival rate was significantly negative correlation (r=-0.893).2.Compared with control group,treatment of Cr(Ⅵ),Z-VAD-fmk+Cr(Ⅵ), NAC+Cr(Ⅵ) and Z-VAD-fmk+NAC+Cr(Ⅵ) resulted in significant DNA damage and increasing comet cells in Hep3B cells(p＜0.05),while NAC treatment could reduce the DNA damage caused by Cr(Ⅵ).3.Shrinking or swelling,and chromatin condensation were observed in treated groups,and the gaps between cells widened.Cell density decreased significantly,cell membrane and karyotheca were not intact or disappeared,and basophilic of chromatin declined.Loss of membrane microvilli,cytoplasmic hypervacuolization,apoptotic badies,condensation and margination of nuclear chromatin were observed under Electron Microscope.While NAC could protect cells from morphological changes induced by Cr(Ⅵ).4.Compared with control group,treatment of Cr(Ⅵ),Z-VAD -fmk+Cr(Ⅵ),NAC+Cr(Ⅵ) and Z-VAD-fmk+NAC+Cr(Ⅵ) resulted in significant apoptosis and necrosis in Hep3B cells(p＜0.05)while these changes could be significantly protected by NAC treatment.5.The mitochondrial transmembrane potential(△ψm) of Hep3B cells in the groups treated with Cr(Ⅵ),Z-VAD-fmk+Cr(Ⅵ),NAC+ Cr(Ⅵ) and Z-VAD-fmk+NAC+Cr(Ⅵ) were significantly decreased,and the open rate of PTP significantly increased(P＜0.05),compared with control group. The mitochondria damage was protected by NAC(p＜0.05).6.Compared with control group,the acticity of SOD,GR and CAT in groups treated with Cr(Ⅵ) alone,NAC+Cr(Ⅵ) and Z-VAD-fmk+NAC+Cr(Ⅵ) were significantly decreased,and the level of MDA and NO was significantly increased(P＜0.05).While the above changes could be protected by NAC(P＜0.05).Conclusions:The oxidation stress and apoptosis,as well as damage to mitochondiral membrane and genomic DNA in Hep3B cells caused by Cr(Ⅵ) at the concentration of 20μmol/L were observed in this study.The results show that apoptosis in p53 negative cells with caspase inhibited can be induced by Cr(Ⅵ).ROS plays an important role in pathway of mitochondria-dependent apoptosis induced by Cr(Ⅵ).