CoQ10 Deficiency May Indicate Mitochondrial Dysfunction in Cr(VI) Toxicity

Abstract

To investigate the toxic mechanism of hexavalent chromium Cr(VI) and search for an antidote for Cr(VI)-induced cytotoxicity, a study of mitochondrial dysfunction induced by Cr(VI) and cell survival by recovering mitochondrial function was performed. In the present study, we found that the gene expression of electron transfer flavoprotein dehydrogenase (ETFDH) was strongly downregulated by Cr(VI) exposure. The levels of coenzyme 10 (CoQ10) and mitochondrial biogenesis presented by mitochondrial mass and mitochondrial DNA copy number were also significantly reduced after Cr(VI) exposure. The subsequent, Cr(VI)-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species (ROS) accumulation, caspase-3 and caspase-9 activation, decreased superoxide dismutase (SOD) and ATP production, increased methane dicarboxylic aldehyde (MDA) content, mitochondrial membrane depolarization and mitochondrial permeability transition pore (MPTP) opening, increased Ca²⁺ levels, Cyt c release, decreased Bcl-2 expression, and significantly elevated Bax expression. The Cr(VI)-induced deleterious changes were attenuated by pretreatment with CoQ10 in L-02 hepatocytes. These data suggest that Cr(VI) induces CoQ10 deficiency in L-02 hepatocytes, indicating that this deficiency may be a biomarker of mitochondrial dysfunction in Cr(VI) poisoning and that exogenous administration of CoQ10 may restore mitochondrial function and protect the liver from Cr(VI) exposure.

Keywords: L-02 hepatocytes; apoptosis; coenzyme Q10; hexavalent chromium Cr(VI); mitochondrial membrane potential (MMP); reactive oxygen species (ROS).

Figure 1

(A) Effect of different doses of Cr(VI) exposure on L-02 hepatocyte viability. Cells were cultured with different concentrations of Cr(VI), and the cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously; (B) Effect of different doses of CoQ10 exposure on L-02 hepatocyte viability; (C) The CoQ10 content in the mitochondria of L-02 hepatocytes treated with CoQ10 and Cr(VI). The data were presented as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 2

CoQ10 attenuates oxidative damage induced by Cr(VI). (A) Quantification of ROS levels. Effect of CoQ10 on Cr(VI)-induced ROS accumulation in L-02 hepatocytes and quantitation by fluorescence spectrophotometry; (B) After L-02 hepatocytes were treated with Cr(VI) (0~4 μM) for 24 h, with or without CoQ10 pretreatment for 2 h, O₂⁻ generation was detected with dihydroethidium; (C) CoQ10 reduced the oxidative damage induced by Cr(VI). The cells were treated with Cr(VI) (0~4 μM) for 24 h, with or without CoQ10 pretreatment, and MDA was detected by the MDA detection kit as the end product of lipid oxidation. SOD was measured using the total superoxide dismutase activity assay, which involves the inhibition of superoxide-induced chromogen chemiluminescence by SOD; (D) Effect of CoQ10 on Cr(VI)-induced ROS accumulation. The cells were incubated with 5 μM of CellROX^® Green Reagent for 30 min and observed under a confocal microscope using a 40× objective. Brighter green fluorescence indicated greater ROS accumulation. The data are presented as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 2

CoQ10 attenuates oxidative damage induced by Cr(VI). (A) Quantification of ROS levels. Effect of CoQ10 on Cr(VI)-induced ROS accumulation in L-02 hepatocytes and quantitation by fluorescence spectrophotometry; (B) After L-02 hepatocytes were treated with Cr(VI) (0~4 μM) for 24 h, with or without CoQ10 pretreatment for 2 h, O₂⁻ generation was detected with dihydroethidium; (C) CoQ10 reduced the oxidative damage induced by Cr(VI). The cells were treated with Cr(VI) (0~4 μM) for 24 h, with or without CoQ10 pretreatment, and MDA was detected by the MDA detection kit as the end product of lipid oxidation. SOD was measured using the total superoxide dismutase activity assay, which involves the inhibition of superoxide-induced chromogen chemiluminescence by SOD; (D) Effect of CoQ10 on Cr(VI)-induced ROS accumulation. The cells were incubated with 5 μM of CellROX^® Green Reagent for 30 min and observed under a confocal microscope using a 40× objective. Brighter green fluorescence indicated greater ROS accumulation. The data are presented as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 3

Cr(VI) triggers significant mitochondrial biogenesis loss. (A) NAO staining was used to analyze the mitochondrial mass using a microplate reader; (B) quantitative real-time PCR analysis was applied to detect the mtDNA copy number. The data are presented as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 4

Cr(VI) induces mitochondrial depolarization, MPTP opening, Ca²⁺ overload, and ATP level decrease, and these outcomes are attenuated by CoQ10. (A) Effect of CoQ10 on Cr(VI)-increased mitochondrial membrane potential in L-02 hepatocytes. The mitochondrial membrane potential was examined by JC-1 staining; (B) The activity of MPTP was detected using the calcein-AM-cobalt assay; (C) the Ca²⁺ concentration was measured with Flo-3M by fluorescence spectrophotometry; (D) cells were treated with Cr(VI) (0–4 μM) for 24 h, with or without CoQ10 pretreatment for 2 h, and the ATP levels in L-02 hepatocytes were assessed. The data are presented as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 5

Cr(VI) induces cytochrome c release from the mitochondria to the cytoplasm. (A) Merged images of the mitochondria (red), Cyt c (green) and nucleus (blue) after exposure to Cr(VI) for 24 h in L-02 hepatocytes. Cyt c (green) and mitochondria (red) localization (yellow) indicates that Cyt c is still inside mitochondria. The separation of Cyt c and mitochondria suggests that Cyt c is no longer within the mitochondria and has been released into the cytoplasm, scale bar: 10 μm; (B) CoQ10 prevents Cyt c release to the cytoplasm; Cyt c protein expression was measured by Western blotting. COXIV and β-actin were used as loading controls; (C) The relative protein levels were calculated by Image J software. Experiments were repeated three times and showed similar results.

Figure 6

Cr(VI) induces caspase-3 and caspase-9 activation and unbalanced Bcl-2/Bax expression in response to apoptotic stimuli, and CoQ10 counteracts these outcomes. Cells were treated with Cr(VI) (0~4 μM) for 24 h, with or without CoQ10 pretreatment for 2 h. Caspase-3 (A) and -9 (B) activities were detected using a microplate reader; (C) The expression of Bcl-2 and Bax was measured by Western blotting and the relative protein levels were calculated by Image J software (D). The data are expressed as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 7

Cr(VI) induces apoptosis in L-02 hepatocytes, and CoQ10 antagonizes apoptosis. (A) Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Both early apoptotic and late apoptotic cells were assessed in the cell death determinations. The experiments were repeated three times; (B) Quantification of apoptotic cells. Data were obtained from flow cytometry assays and were expressed as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.

Figure 7

Cr(VI) induces apoptosis in L-02 hepatocytes, and CoQ10 antagonizes apoptosis. (A) Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Both early apoptotic and late apoptotic cells were assessed in the cell death determinations. The experiments were repeated three times; (B) Quantification of apoptotic cells. Data were obtained from flow cytometry assays and were expressed as mean ± SD (n = 6). * p < 0.05 compared with the control group; # p < 0.05 compared with the 2 μM Cr(VI) treatment group.