Mechanism Study Of Mitochondrial Respiratory Chain Dysfunction In Apoptosis And Premature Senescence In Cr(Ⅵ)-treated Hepatocytes

BackgroundChromium and its compounds are non-negligible pollutant that have became a serious threat to public health in the world with increasing use in diverse industrial processes, including metallurgy, electroplating, leather tanning and chroming. Occupational exposure to chromium is associated with several adverse effects of health, such as contact dermatitis, nasal perforation, and bronchiogenic cancer. Cr(Ⅵ) can induce toxic and chronic hepatitis and severe liver damage. The main cytotoxicity of Cr(Ⅵ) is apoptosis induction. Although it is believed that ROS plays an important role in the toxicity of Cr(Ⅵ), the related mechanisms still remain unclear, for example, how and where ROS are produced, how does Cr(Ⅵ) induce the activation of p53and NF-kB? In addition to explore the mechanisms for Cr(Ⅵ)-induced apoptosis, we also placed high emphasis on Cr(Ⅵ)-induce premature senescence. Premature senescence, which has been studied a lot recently, is totally different from apoptosis. The occupational exposure to Cr(Ⅵ) is low dose and long term, that is why the research on Cr(Ⅵ)-induced premature senescence has great significance. Premature senescence can be also viewed as an intrinsic cellular barrier against tumorigenesis, while apoptosis is programmed cell death. The present research which focused on both the apoptisis and premature senescence, has depicted an original cellular toxic phenomenon in response to Cr(Ⅵ) that may constitute a protective mechanism to the tumorigenic effect of chromium in humans.Methods1. Cr(Ⅵ) induces apoptosis in L-02hepatocytesUsing MTT method to detect the effect of different doses of Cr(Ⅵ)(0-512μM) exposure on L-02hepatocytes survival rate, and choose proper Cr(Ⅵ) treated concentrations (survival rate>70%) for the following experiments. Cr(Ⅵ)-induced DNA damage was examined by DNA-Ladder method. Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used to detect the occurrence of apoptosis in the cells. Annexin V-FITC/PI double stain was used to detect the apoptotic cells in both the early stage and late stage. Western blotting was processed in the study to detect the effect of Cr(Ⅵ) on the expression levels of PARP, Caspase-8, Caspase-9and Caspase-3to further analyze the apoptic pathway. The activity of Caspase-3was also measured using colorimetry. By analyzing the expression levels of heat shock proteins HSP70and HSP90after Cr(Ⅵ) exposure to explore the correlation between HSP and caspase-3.By detecting the levels of GSH, MDA and SOD to study the effect of Cr(Ⅵ) on cellular antioxidant system and the capability of the hepatocytes to produce oxygen radicals.2Cr(Ⅵ) induces the dysfunction of mitochondrial respiratory chain in L-02hepatocytesL-02hepatocytes were processed for mitochondria extraction after Cr(Ⅵ) exposure. The indices of mitochondrial respiration such as state3and state4respiration rates, respiratory control rate (RCR) were measured using Clark oxygen electrode by recording oxygen consummation (expressed as nmol/min/mg protein). L-02hepatocytes were treated with Cr(Ⅵ)(0,4,8,16,32μM) for24h. The mitochondria were used to detect the activities of mitochondrial respiratory chain complex (MRCC) Ⅰ-Ⅴ using the related kits. RNA was extracted and reversely transcripted into DNA. The mRNA expression levels of MRCC Ⅰ-Ⅴ were measured using real-time PCR. The protein expression levels of MRCC Ⅰ-Ⅴ were also detected using Western bloting. The mitochondrial membrane potential (Δψm), PTP open rate and membrane permeability, and the activities of LDH, ALT and AST were also measured in L-02hepatocytes after Cr(Ⅵ) exposure. The cellular content of adenine nucleotides (ATP, ADP, AMP) in Cr(Ⅵ)-treated hepatocytes were measured using high performance liquid chromatography (HPLC), and ATP/ADP ratio and energy charge (EC) were also calculated.3. The effect of the dysfunction of mitochondrial respiratory chain on Cr(Ⅵ)-induced apoptosis in L-02hepatocytesL-02hepatocytes were treated with PBS or16,32μM Cr(Ⅵ) for24 h, incubated with fluoresent probe CM-H2DCFDA and then were processed for the determination of ROS production. MRCC Ⅰ inhibitor Rotenone (ROT) which can induce ROS accumulation was used as positive control. The reduction site of Cr(Ⅵ) in mitochondria was determined by measuring the Cr(Ⅵ) reduction rate after the mitochondria were exposed to different substrates of MRCC. The antioxidant NAC was used to inhibit ROS since we inferred that ROS plays an important role in Cr(Ⅵ)-induced hepatocytes toxicity. The effect of NAC pre-treatment on cellular ATP content, p53mRNA and protein levels, the apoptotic pathway and HSP expressions in L-02hepatocytes after different doses of Cr(Ⅵ) exposure were measured. Cell cycle distribution of hepatocytes was also examined by flow cytometry. In order to explore the reasons for Cr(Ⅵ)-induced cell cycle distribution changes, by using real-time PCR and western blotting, we checked the expression levels of S phase checkpoint proteins (Tof1, Mrc1) and S phase related genes (CDK2, Cyclin E), as well as G2/M phase checkpoint proteins (BubR1, Mad2) and G2/M phase related proteins (Cyclin B, CDC25). We also used p53specific inhibitor PFT-a to block the function of p53in order to confirm if Cr(Ⅵ)-induced cell cycle arrest is depend on p53. Thus we measured the effect of PFT-a on the expression levels of p53, Mrc1, BubR1and cell cycle distribution in L-02hepatocytes exposed to different doses of Cr(Ⅵ). In addition to p53, we also examined the effect of Cr(Ⅵ) on NF-kB, PI3K/AKT pathway with or without ROS treatment.4. The effect of the dysfunction of mitochondrial respiratory chain on Cr(Ⅵ)-induced premature senescenceL-02hepatocytes were treated with PBS or10nM Cr(Ⅵ) twice a week for24h for a total period of4weeks. SA-β-Gal activity was measured to indicate the occurrence of premature senescence. Cell cycle distribution was determined by flow cytometry. The identification of apoptosis and premature senescence was achieved by checking the percentages of senescent, apoptotic, necrotic and growing cells in L-02hepatocytes after Cr(Ⅵ) exposure. L-02hepatocytes after4weeks of treatment were analyzed for ROS production utilizing oxidant-sensitive fluorogenic probe CM-H2DCFDA. We also used the MitoSOX probe to measure specifically the mitochondrial superoxide level since MRCC blockade leads generally to superoxide production. To investigate the mechanism for the elevated ROS level, we determined the activities of MRCC Ⅰ-Ⅳ. We also measured the effect of Cr(Ⅵ) on p53mRNA levels and phosphorylated protein levels, as well as senescence pathway (p53-p21WAF1/CIP1, Rb-p16INK4a) and pro-survival genes, S phase related genes in hepatocytes with or without NAC pre-treatment. We further checked if Cr(Ⅵ)-induced premature senescence was completely depend on p53function. The transfection system containing shRNA to p53was used to knockout p53in hepatocytes. The non-target scramble (Scr)-transfected and p53shRNA-transfected hepatocytes were treated with PBS or10nM Cr(Ⅵ) twice a week for24h for4consecutive weeks. We then measured the effect of p53knocking-out on the occurrence of premature senescence, the percentage of senescent cells, the expression levels of pro-survival genes and S phase related genes in L-02hepatocytes after Cr(Ⅵ) exposure.Results1. Cr(VI) induces apoptosis in hepatocytesCr(VI) induced a concentration-dependent loss of cell viability in L-02hepatocytes. Considering the survival rate should be in a proper range (>70%) and the demand of statistic analysis, we chose four concentrations of Cr(Ⅵ)(4,8,16,32μM) for the following experiments. The apoptotic hepatocytes showed the characteristic DNA fragement after Cr(Ⅵ) exposure. TUNEL assay confirmed that DNA damage was increased in a dose-dependent manner. Both the early stage and late stage of apoptitic cells were increased with the increasing Cr(Ⅵ) exposure. The caspase8related Fas apoptitc pathway as well as caspase-9related non-Fas apoptotic pathway were all participated in Cr(Ⅵ)-induced apoptosis. Cr(Ⅵ) targeted HSP70and HSP90to regulated caspase-3activity. The measurement of GSH, MDA and SOD levels revealed that Cr(Ⅵ) significantly affected cellular antioxidant system and the ability of hepatocytes to generate ROS.2. Cr(Ⅵ) induces the dysfunction of mitochondrial respiratory chain in L-02hepatocytesCr(Ⅵ) inhibited state3respiration and RCR, but had no effect on state4respiration. Different doses of Cr(Ⅵ) exposure significantly inhibited mRNA and protein levels of MRCC Ⅰ and Ⅱ, especially the former. In the Cr(Ⅵ)-treated hepatocytes, Δψm was decreased, PTP open rate and the membrane permeability was increased. Cr(Ⅵ) also significantly reduced cellular ATP content, ATP/ADP ratio and EC.3. Effect of mitochondrial respiratory chain dysfunction on apoptosis in Cr(Ⅵ)-treated hepatocytesCr(Ⅵ) induced ROS accumulation in L-02hepatocytes in a dose-dependent manner. Cr(Ⅵ) reduction rate significantly increased in the mitochondria treated with MRCC Ⅰ substrates glutamate/malate (Glu/Mal), indicating that Cr(Ⅵ) reduction occurs at MRCC I. NAC treatment significantly blocked the induction of apoptosis by inhibiting ROS accumulation, rescuing ATP reduction and inhibiting the activation of apoptic pathway. p53expression levels was increased in Cr(Ⅵ)-treated groups in a dose-dependent manner, while NAC treatment significantly blocked p53. Cr(Ⅵ) activated caspase-3by targeting ROS to regulate heat shock proteins. Low dose of Cr(Ⅵ)(4μM) exposure induced S phase cell cycle arrest while higher dose of Cr(Ⅵ)(16,32μM) exposure induced G2/M phase cell cycle arrest in L-02hepatocytes. Further investigation revealed that Cr(Ⅵ) could target S phase checkpoint proteins (Tof1,Mrc1) and S phase related proteins (CDK2, Cyclin E) to induce S phase arrest, and target G2/M phase checkpoint proteins (BubR1,Mad2) and G2/M phase related proteins (Cyclin B,CDC25) to induce G2/M phase arrest. NAC and PFT-a pre-treatment significantly alleviated Cr(Ⅵ)-induced S phase or G2/M phase cell cycle arrest, indicating that Cr(Ⅵ) could target ROS-p53to induce cell cycle arrest. In addition to p53, ROS also regulated NF-kB and PI3K/AKT pathway to induce apoptosis in L-02hepatocytes after Cr(Ⅵ) exposure.4. Effect of mitochondrial respiratory chain dysfunction on premature senescence in Cr(Ⅵ)-treated hepatocytesLow dose and long-term exposure of Cr(Ⅵ) induces premature senescence in L-02hepatocytes. Senescent cells showed significant S phase cell cycle arrest and the increase of SA-β-Gal activity. Similar to ROS production mechanisms in apoptotic cells, Cr(Ⅵ) could selectively target the sensitive sites MRCC Ⅰ and Ⅱ in respiratory chain to induce ROS accumulation. After Cr(Ⅵ) treatment, p53expression significantly increased in the senescent cells and phosphorylation of p53at Ser15also exhibited a clear increase. NAC pre-treatment resulted in almost undetectable p53expression in both the control and Cr(Ⅵ) treatment group, indicating the role of ROS played in regulating p53expression. Western blotting for senescence pathway analysis revealed that p53-p21WAF1/CIP1pathway, but not Rb-p16INK4a pathway was involved in Cr(Ⅵ)-induced premature senescence. In senescent cells, ROS could target p53to regulate the prosurvival genes Bcl-2, Mcl-2to inhibit cell proliferation, and also to regulate S phase related proteins CDK2, Cyclin E to induce S phase cell cycle arrest. The pre-treatment of NAC significantly inhibit the occurrence of premature senescence in hepatocytes after Cr(Ⅵ) exposure. Knocking-out of p53in L-02hepatocytes blocked the occurrence of premature senescence. Although premature senescence was not detected, ROS was found accumulation in L-02-p53shRNA cells, which indicating that Cr(Ⅵ)-induced premature senescence is depend on ROS function, while ROS function is completely depend on p53.Conclusion1. Cr(Ⅵ) induces apoptosis in L-02hepatocytes. The caspase-8related Fas apoptitc pathway as well as caspase-9related non-Fas apoptotic pathway are all participated in Cr(Ⅵ)-induced apoptosis.2. Cr(Ⅵ) induces the inhibition of mitochondrial respiratory chain and the disturbance of energy metabolism in L-02hepatocytes.3. The dysfunction of mitochondrial respiratory chain lead to ROS accumulation. Cr(Ⅵ) targets and inhibits MRCC Ⅰ and Ⅱ to increase ROS cellular level, and the reduction of Cr(Ⅵ) occurs at MRCC I. Low dose of Cr(Ⅵ) exposure induces S phase cell cycle arrest while high dose of Cr(Ⅵ) exposure induces G2/M phase eel cycle arrest. Cr(Ⅵ)-induced apoptosis in L-02hepatocytes is depent on ROS-p53, NF-kB and PI3K/AKT pathway.4. Low dose and long-term Cr(Ⅵ) exposure induces premature senescence in L-02hepatocytes. p53-p21WAF1/CIP1pathway was involved in Cr(Ⅵ)-induced premature senescence. ROS targets and regulates pro-survival genes and S phase related proteins to induced premature senescence. Cr(Ⅵ)-induced premature senescence is depent on ROS-p53function.