| Objective:To explore the relation of apoptosis induced by hexavalent chromium[Cr(Ⅵ)]and calcium homeostasis disequilibrium in Hep3B hepatocytes in which p53 is absent,and to investigate its mechanisms of action in vitro test.Methods:Hep3B hepatocytes were cultured,and MTT test was adopted to test the viability of Hep3B hepatocytes treated with Cr(Ⅵ), DZ and BAPTA/AM under 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 hepatocytes.In the following test,Hep3B hepatocytes were incubated with DZ at 50μmol/L and BAPTA/AM at 20μmol/L for 1 hour respectively,and then treated with Cr(Ⅵ) at 20μmol/L for 6 hours.The cultured Hep3B hepatocytes were randomly divided into five groups, namely control group,Cr(Ⅵ) alone,DZ+Cr(Ⅵ),BAPTA/AM+Cr(Ⅵ), and DZ+BAPTA/AM+Cr(Ⅵ).Giemsa staining was performed to detect the morphological features in each group;DNA damage was detected by using the single cell gel electrophoresiss(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 intracellular free calcium ion concentration([Ca2+]i) were detected by fluorescent spectrophotometry, using Fura-2/AM as fluorescence probe respectively;The activities of Ca2+-ATPase in cell membrane and mitochondrial membrane were determined by colorimetry method.Results:1.The concentration-dependent decreases in cell survival rate of Cr(Ⅵ)-treated Hep3B hepatocytes was observed(p＜0.05),the relationship between concentration and survival rate was significantly negative correlation(r=-0.948).The survival rate of 20μmol/L Cr(Ⅵ) treated group that after DZ and BAPTA/AM pretreatment was significantly higher than 20μmol/L Cr(Ⅵ) treated group.2.Changes in morphology of Hep3B hepatocytes caused by each treatment:observation by light microscope,most cells treated by 20μmol/L Cr(Ⅵ) for 6hs were observed to shrink or swell,chromatin condensed,the vacuolus in cytoplasm and the size of gaps between cells increased.These changes became more obvious of cell density decreased significantly,cell membrane and karyotheca were not intact or disappeared,and basophilic of chromatin declined.cells treated by 20μmol/L Cr(Ⅵ) for 6hs after the pretreatment by DZ and BAPTA/AM, most of them had a well growth condition,some of them had vacuolus in cytoplasm,and some karyotheca were not intact or disappeared.3.DNA damage of Hep3B hepatocytes caused by each treatment: Compared with control group,treatment of Cr(Ⅵ),DZ+Cr(Ⅵ), BAPTA/AM+Cr(Ⅵ),DZ+BAPTA/AM+Cr(Ⅵ) resulted in significant DNA damage and increasing number of comet cells in Hep3B hepatocytes(p＜0.05),while DZ and BAPTA/AM treatment can reduce the DNA damage results from Cr(Ⅵ).Loss of membrane microvilli, swelling of mitochondria,deprivation of cristae,appearance of vacuolus in cytoplasm and appearance of lucent area in cell nucleus were observed under Electron Microscope with Cr(Ⅵ) treated.Pretreatment of DZ would protect mitochondria.4.Apoptosis of Hep3B hepatocytes caused by each treatment: Compared with control group,treatment of Cr(Ⅵ),DZ+Cr(Ⅵ), BAPTA/AM+Cr(Ⅵ),DZ+BAPTA/AM+Cr(Ⅵ) resulted in significant apoptosis and necrosis in Hep3B hepatocytes(p＜0.05),while these changes can be significantly protected by DZ and BAPTA/AM treatment.5.Effect of mitochondria membrane in Hep3B hepatocytes caused by each treatment:The mitochondrial transmembrane potential(△ψm) of Hep3B hepatocytes in the groups treated with Cr(Ⅵ),DZ+Cr(Ⅵ), BAPTA/AM+Cr(Ⅵ),DZ+BAPTA/AM+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 DZ and BAPTA/AM(P＜0.05). 6.Effect of intracellular[Ca2+]i in Hep3B hepatocytes caused by each treatment:Compared with control group,intracellular[Ca2+]i was significantly increased in the Hep3B hepatocytes treated with Cr(Ⅵ), DZ+Cr(Ⅵ),BAPTA/AM+Cr(Ⅵ),DZ+BAPTA/AM+Cr(Ⅵ)(P＜0.05). Compared with Cr(Ⅵ) treaed group,intracellular[Ca2+]i was decreased in the Hep3B hepatocytes treated with DZ and BAPTA/AM(P＜0.05).7.Effect of the activities of cell membraneous ATPase in Hep3B hepatocytes caused by each treatment:The activities of cell membraneous ATPase in Cr(Ⅵ) and BAPTA/AM+Cr(Ⅵ) treated groups were significantly lower than that of control group(P＜0.05).The activities of mitochondria membraneous ATPase in each DZ and BAPTA/AM pretreated groups were significantly higher than that of Cr(Ⅵ) treated group(P＜0.05).The activities of mitochondria membraneous ATPase in each treatment groups were significantly lower than that of control group (P＜0.05).And the activities of ATPase can be protected by DZ and BAPTA/AM.Conclusions:Treatment of Cr(Ⅵ) at the concentration of 20μmol/L could significantly cause apoptosis of Hep3B hepatocytes and damage to mitochondiral membrane and genomic DNA.It also led to the increase of the intracellular free calcium ion concentration([Ca2+]i) and the reduction of the activities of Ca2+-ATPase in cell membrane and mitochondrial membrane of Hep3B hepatocytes.DZ and BAPTA/AM had propotional antagonistic effect to the results above.The results show that the apoptosis pathway with Cr(Ⅵ) can be induced by mitochondira-dependent pathway without p53,and calcium homeostasis disequilibrium plays an important role in the apopotosis induced by Cr(Ⅵ).
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