| Objective:By further exploring the toxic mechanisms of hexavalent chromium [Cr(Ⅵ)] and looking for the antidote for Cr(Ⅵ)-induced cytotoxicity, we aimed to provide experimental evidence for the prevention and cure of the occupational hazard and the health promotion for the people exposed to Cr(VI).Methods:The survival rates of L-02hepatocytes treated with different concentrations of Cr(Ⅵ) and CoQ10were detected by methyl thiazolyl tetrazolium (MTT) method. The apoptosis rates were measured by flow cytometry. Reactive oxygen species (ROS), O2’-and mitochondrial membrane potential (MMP) were detected using2’,7’-dichlorodihy-drofl-uorescin diacetate (DCFH-DA), dihydroethidium (DEH) and JC-1fluorescent probes, respectively. Methane dicarboxylic aldehyde (MDA), Superoxide dismutase (SOD) and ATP were measured by Microplate Reader corresponding kits.The concentration of Ca2+and the activity of membrane mitochondril transition pore (MPTP) were measured by fluorescence spectrophotometer using Flo-3M probe and calcein-AM-cobalt probe, respectively. Caspase-3and caspase-9activities were measured by corresponding kits. The protein expression levels of cytochrome C(CytC), B-cell lymphoma-2(Bcl-2) and Bcl-2Assaciated X protein (Bax) were measured by western blot. The mRNA expression levels of COQ2and COQ4were measured by real-time fluorescent quantitative PCR.Results:1. To assay the changes in cell viability after exposed to Cr(Ⅵ), we evaluated its dose effects on cultured L-02hepatocytes with or without CoQ10pretreatment. With the increasing concentrations of Cr(Ⅵ) ranging from0to8μM, the cell viability was significantly decreased (p<0.05). Although treatment with CoQ10increased cell viability at the concentrations of0-5μM, there was no statistical significance (p>0.05). However, the concentrations of CoQ10at2.5μM significantly decreased the cell viability (p<0.05).2. To measure the effects of CoQ10on programmed cellular death after Cr(Ⅵ)exposure, we analyzed cell apoptosis using annexin V-FITC and propidium iodide (PI) double staining methods after incubation for30min. With the increasing concentrations of Cr(Ⅵ) ranging from1to4μM, the cell viability was significantly increased (p<0.05). Pretreatment with CoQ10attenuated the Cr(Ⅵ)-induced increase of apoptosis, indicating the involvement of CoQ10in Cr(Ⅵ)-induced apoptosis.3.The results concerning CoQ10levels showed that Cr(Ⅵ) decreased CoQ10concentration in mitochondria of L-02hepatocytes compared to the control group (p<0.05). Pretreatment with CoQ10restored the level of CoQ10in comparison with the2uM Cr(Ⅵ) treatment group(p<0.05). Cr(Ⅵ) inhibited the mRNA expression of COQ2and COQ4which encoded the enzymes in the pathway of CoQ10synthesis, with a statistical significance (p<0.05). Pretreatment with CoQ10up-regulated genes of COQ2and COQ4, and there were statistical significance when compared to the2uM Cr(Ⅵ) treatment group(p<0.05).4. We measured ROS production using dihydroethidine (DHE) and DCFH-DA probes which detects superoxide and reaction oxygen species (ROS), respectively. Cr(Ⅵ) significantly enhanced the production of O2’ and ROS (p<0.05) compared to the control group, and pretreatment with2.5μM CoQ10prevented Cr(Ⅵ)-induced ROS accumulation. The levels of ROS were the same in CoQ10-treated cells and in the normal control cells (p>0.05).5. Treatment with Cr(Ⅵ) caused significant decrease of SOD compared with untreated control. Combination treatment of Cr(Ⅵ) with CoQ10restored SOD values to levels statistically comparable to control (p<0.05). MDA was served as an indicator of hepatocytes lipid peroxidation. Cr(Ⅵ) significantly increased MDA levels compared to control (p<0.05). Pretreatment with CoQ10reduced Cr(Ⅵ)-induced MDA increase significantly (p<0.05). 6. MPP can be detected using JC-1. With the concentration of Cr(Ⅵ) increasing, MPP was significantly decreased compared to the normal control (p>0.05), indicating the occurrence of mitochondrial membrane depolarization. Pretreatment with CoQ10relieved Cr(Ⅵ)-induced mitochondrial membrane depolarization. Calcein-AM-cobalt assay was performed to directly assess MPTP opening. The inner MPP was significantly increased in response to Cr(Ⅵ) stimuli in a concentration-dependent manner. The MPTP of the cells in the combination group of2μM Cr(Ⅵ) plus2.5μM CoQ10was significantly decreased compared to that of the cells incubated with2μM Cr(Ⅵ) alone (p<0.05).7. The cells were incubated with Flo-3M probe before detecting the intercellular Ca2+levels. Cr(Ⅵ) increased Ca2+levels in a concentration-dependent manner, with a statistical significance (p<0.05). Pretreatment with CoQ10significantly attenuated Ca2+overload compared to the group treated with2μM Cr(Ⅵ) alone (p<0.05). These results suggest that CoQ10could reduce mitochondrial Ca2+levels and block MPTP opening.8. To examine the effect of Cr(VI) on mitochondrial ATP production, L-02hepatocytes were treated with1,2,4μM Cr(Ⅵ) for24h or first pretreated with2.5μM CoQ10for2h, then added with2μM Cr(Ⅵ), and ATP levels were measured colorimetrically. Cr(Ⅵ) induced concentration-dependent decrease of ATP levels in L-02hepatocytes with a significant difference (p<0.05). Pretreatment with CoQ10reserved the Cr(Ⅵ)-induced ATP decrease with a statistical significance (p<0.05).9. Caspase-3and capase-9activities were analyzed as indexes of apoptosis execution in the intrinsic (mitochondrion-dependent) pathway. Treatment with Cr(Ⅵ) caused significant increase of caspase-3and caspase-9activities compared to control (p<0.05). Pretreatment with CoQ10attenuated both caspase-3and caspase-9activitation compared to the group treated with2μM Cr(Ⅵ) alone (p<0.05).10. Different concentrations of Cr(Ⅵ) significantly decreased of CytC protein expression in mitochondria but increased CytC protein expression in cytoplasm.Total intracellar CytC protein expression was inhibited by Cr(Ⅵ). Pretreatetment with CoQ10reserved both mitochondrial CytC and intracellular CytC protein expression, but decreased the CytC protein expression in cytoplasm.11.24h exposure of different concentrations of Cr(Ⅵ) induced significant concentration-dependent inhibition of Bcl-2and induction of Bax. Pretreatment with2.5μM CoQ10reserved Bcl-2expression and decreased Bax expression compared to the group treated with2μM Cr(Ⅵ) alone.Conclusion:1. CoQ10protects L-02hepatocytes against Cr(Ⅵ)-induced cytotoxicity through alleviating oxidative damage.2. CoQ10reduces the adverse effects after Cr(Ⅵ) exposure through maintaining the mitochondrial function.3. CoQ10protects against Cr(Ⅵ)-induced apoptosis via blocking mitochondrial-dependent cell apoptotic pathway. |
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