Relationship Of Gene Expression Changes Involved In Energy Metabolism And Apoptosis In Hepatocytes Induced By Hexavalent Chromium

BackgroundHexavalent chromium (Cr(Ⅵ)) is one common heavy metal pollutant, existing widely in people's living environment as the result of industrial process. Recently, Cr(Ⅵ) pollution was also found existing in crops, hydrobiont, and drinking water in some regions of China, and the oral Cr(Ⅵ) hepatotoxicity was proposed due to liver is the main organ of biological metabolism. After entering blood circulation through digestive tract, Cr(Ⅵ) could damage both the structure and the function of hepatocytes. The toxicity of Cr(Ⅵ) has been confirmed by the in vivo study showing that Cr(Ⅵ) could induce hepatocyte ultra-structure disruption and apoptosis, and also by the in vitro study that Cr(Ⅵ) could induce mitochondria damage and cell apoptosis in L-02hepatocytes. Mitochondria are the main site for ATP synthesis. ATP, which can be produced by the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) in the inner mitochondrial membrane, is essential for cells survival. Meanwhile, the alteration of mitochondria structure or function leads to the release of apoptosis induced factor (AIF) and cytochrome c, which further activate caspases family and then initiate caspases-dependent apoptosis. Therefore, the damage of mitochondria not only leads to disorder of cell energy metabolism, but also induces apoptotic cell death. Further investigation of Cr(Ⅵ)-induced energy metabolism dysfunction and apoptosis on the molecular level could be of great help to clarify the toxicological mechanism of Cr(Ⅵ), and also to provide a reference for biological monitoring and prevention of chromium poisoning.ObjectTo preliminarily explore the interference effect of Cr(Ⅵ) on hepatocytes energy metabolism related genes expression levels and their association with apoptosis after different concentrations and different time periods of Cr(Ⅵ) exposure, and further to provide some novel clues of molecular mechanisms for Cr(Ⅵ)-induced apoptosis. Methods1. Measurement of cell survival rate and ATP levelsL-02hepatocytes were exposed to0,2,4,8,16,32μM Cr (VI) for12,24, or36h, and then were processed to evaluate cell survival rate or cellular ATP content by spectropcthotometry or bioluminescence assay, respectively. And the results were analyzed by statistical approach.2. Analysis of mRNA levels of energy metabolism-related genesL-02hepatocytes were treated with Cr (VI) for12,24, or36h. The mRNA levels of energy metabolism related genes were measured by reverse transcription qualitative PCR, and analyzed by the method of2-ΔΔCT. The genes analyzed in the present study include mitochondria genome-encoded genes such as mitochondria respiratory chain complex Ⅰ(NADH1),Ⅳ(COX1),Ⅴ(ATP-6S), and the nuclear-encoded genes such as hexokinase2(HK2), M2pyruvate kinase (PKM2), mitochondria voltage-dependent anion channel (VDAC1), and adenine nucleotide translocator1(ANT1).3. Detection of cellular ROS levels and apoptosis ratesROS levels in the hepatocytes after Cr (VI) exposure were determined by the method of loading fluorescent probe2',7'-dichlorofluorescin diacetate (DCFH-DA) in situ at37℃for20minutes (min), the fluorescence intensity of DCF was then measured by Enzyme-labelled Meter with an excitation wavelength of488nm and an emission wavelength of535nm. After trypsinization, cells were inclubated with FITC labeled Ca-dependent phospholipids binding protein (Annexin V), and then were treated with propidium iodide (PI) for double staining. The cell apoptosis rate was analyzed by recording the fluorescence intensity of Annexin V and PI using flow cytometry with the excitation/emission wavelengths of488/530nm.4. Construction of siVDAC1hepatocytesNucleotides159-177of the hVDACl coding sequence were chosen as target for siRNA. The hVDAC1-siRNA sequence was created by using the two complimentary oligonucleotides, each containing19nucleotides target sequence of hVDAC1(159-177). Then hVDAC1-siRNA sequence was cloned into the pSilencerTM4.1plasmid, named as siVDAC1plasmid. Then L-02hepatocytes were transfected with plasmid pSilencerTM4.1-siVDAC1by lipofectaminTM2000. The knock-down effiency of VDAC1in hepatocytes was confirmed by RT-qPCR for VDAC1mRNA level or by immunoblot using monoclonal anti-VDAC1antibodies for VDAC1protein level.5. Toxicological effect of Cr(Ⅵ) on siVDACl hepatocytesThe experiment was designed into two groups as following:①The L-02hepatocytes were transfected with plasmid pSilencerTM4.1-siVDAC1and cultured for48h, exposed to Cr(Ⅵ) for24h;②The L-02hepatocytes were transfected with empty plasmid pSilencerTM4.1and cultured for48h, exposed to Cr(Ⅵ) for24h. The Cr(Ⅵ) treatment concentrations were0,2,8,32μM. Levels of VDAC1mRNA, ROS, ATP and the apoptosis rate were measured by RT-qPCR, fluorometry, bioluminescence assay, and flow cytometry, respectively.6. Toxicological effect of Cr(Ⅵ) on NAC-pretreated hepatocytesThe experiment was designed into two groups as following:①Cr(Ⅵ) alone treatment group;②20mM N-acetyl-cysteine (NAC) pretreatment+Cr(Ⅵ) treatment group. The time period of NAC pretreatment was6h. The Cr(Ⅵ) treated concentrations as well as the detection methods were the same as "Method-5".Results1. Quantity-effect and time-effect of Cr(Ⅵ) on the ATP levels in L-02hepatocytes①The ATP levels increased at12h, a decreased at24h, and a slightly increased again at36h following4,8,16,32μM Cr(Ⅵ) treatment, corresponding to a "V-shaped" curve.②The change of intracellular ATP level was not significant under2μM Cr(Ⅵ) treatment (P>0.05), while32μM Cr(Ⅵ) exposure led to the significant change of ATP level in L-02hepatocytes (P<0.05), the toxic threshold was4μM. 2. Interference of Cr(Ⅵ) on mRNA levels of mitochondria respiratory chain complex or glucolysis key enzyme genes①After Cr(Ⅵ) treatment for12h, changes of NADH1and ATP-6S genes mRNA levels were not significant, while COX1gene mRNA expression decreased obviously compared with control group. After Cr(VI) treatment for24h, mRNA levels of NADH1, ATP-6S genes increased distinctly, while COX1gene mRNA level was still low (P <0.05). After Cr(Ⅵ) treatment for36h, NADH1mRNA level decreased obviousl, while COX1and ATP-6S genes mRNA levels increased significantly.②Following Cr(Ⅵ) treatment for12h, mRNA levels of HK2and PKM2changed slightly, after Cr(Ⅵ) treatment for24and36h, both of HK2and PKM2mRNA were of lower levels compared with that of control, and the inhibition rate of mRNA level was more than90%in majority of Cr(Ⅵ)-treated groups.3. Effect of Cr(Ⅵ) on the mRNA levels of genes in mitochondrial membrane VDAC1and ANT1①Level of VDAC1mRNA in cells treated with Cr(Ⅵ) was lower than that of control at12hours, then showed a slight increase at24h, and increased significantly at36h (P<0.05)②Following Cr(VI) treatment for12and24h, adenine nucleotide translocator1(ANT1) mRNA level decreased significantly. After Cr(Ⅵ) treatment for36hours, ANT1mRNA level increased significantly (P <0.05)4. Effect of Cr(Ⅵ) on L-02hepatocytes transfected with VDACD1siRNACompared with the wild type L-02hepatocytes, the VDAC1siRNA transfected hepatocytes showed that nuclear VDAC1mRNA expression levels was inhibited in the32μM Cr(Ⅵ) exposure group. The cell apoptosis rate of VDAC1siRNA hepatocytes treated with Cr(Ⅵ) although decreased, was still higher than the rate in the hepatocytes transfected with VDAC1siRNA but without Cr(Ⅵ) treatment (P<0.05). The content of cellular ATP showed obvious increase, but still lower than that of the untreated wild type L-02hepatocytes (P<0.05), while the cellular ROS levels in the wild type hepatocytes and the VDAC1siRNA-transfected hepatocytes after Cr(Ⅵ) exposure had no significance (P>0.05). The correlation between Cr(Ⅵ) doses and ROS, ATP levels as well as apoptosis rate were found in the quantity-effect manners in both of the hepatocytes.5. Protective effect of antioxidant NAC pretreatment against the cytotoxicity of Cr(Ⅵ)Compared with the non-NAC pretreated hepatocytes, the NAC pretreated hepatocytes showed significantly decrease of cellular ROS levels, VDAC1mRNA levels and cell apoptosis rate after32μM Cr(Ⅵ) exposure, but the cellular ATP content obviously recovered (P<0.05). And a significant quantity-effect manner was found between the Cr(Ⅵ) doses and the cellular ROS levels, VDAC1mRNA levels, ATP cellular content as well as cell apoptosis rate after NAC pretreatment.Conclusion1. The results suggest that ATP levels in L-02hepatocytes could be affected by Cr(Ⅵ). The significant decrease of ATP levels at24h after Cr(Ⅵ) exposure indicates the disruption of mitochondrial energy metabolism, and the slight increase of ATP levels at36h shows the partial recovery of mitochondrial function.2. After Cr(Ⅵ) exposure, the mRNA levels of mitochondrial respiratory chain related gene NADH1, ATP-6S showed no change at the time point of12h, but showed significant increase at time point of24h, which shows that Cr(Ⅵ) could interrupt the electrons transfer on the respiratory chain, and further leads to the compensatory increase of NADH main respiratory chain function.3. The mRNA levels of the nuclear-encoded mitochondrial membrane protein VDAC1were decreased at12h after Cr(Ⅵ) exposure, and began to increase at the time point of24h. The levels were significantly increased in all treated groups at36h after treatment, which suggests that Cr(Ⅵ) could cause the abnormality of mitochondrial membrane permeability by altering the genes expression of VDAC1, and further results in the dysfunction of cellular energy metabolism.4. The regulation of VDAC1gene in the hepatocytes could rescue Cr(Ⅵ)-induced decrease of cellular ATP content and apoptosis to a certain extent, which further confirms the correlation between VDAC1gene and the energy metabolism dysfunction as well as apoptosis in the hepatocytes after Cr(Ⅵ) exposure.5. After pretreated with antioxidant NAC in the hepatocytes, the VDAC1mRNA levels and cell apoptosis rate were significantly decreased along with the decrease of ROS levels, and the ATP levels were recovered. The present study suggests that NAC could regulate VDAC1gene expression to protect the hepatocytes against the cytotoxicity of Cr(Ⅵ) by decreasing the ROS levels, and the best protective function of NAC occurs at the highest Cr(Ⅵ)-treated concentration.