Antioxidant Effect of Coenzyme Q10 in the Prevention of Oxidative Stress in Arsenic-Treated CHO-K1 Cells and Possible Participation of Zinc as a Pro-Oxidant Agent

Abstract

Oxidative stress is an imbalance between levels of reactive oxygen species (ROS) and antioxidant enzymes. Compounds with antioxidant properties, such as coenzyme Q10 (CoQ10), can reduce cellular imbalance caused by an increase in ROS. CoQ10 participates in modulating redox homeostasis due to its antioxidant activity and its preserving mitochondrial functions. Thus, the present study demonstrated the protective effects of CoQ10 against oxidative stress and cytotoxicity induced by arsenic (As). Antioxidant capacity, formation of hydroperoxides, generation of ROS, and the effect on cellular viability of CoQ10, were investigated to determine the protective effect of CoQ10 against As and pro-oxidant compounds, such as zinc. Cell viability assays showed that CoQ10 is cytoprotective under cellular stress conditions, with potent antioxidant activity, regardless of the concentration tested. Zn, when used at higher concentrations, can increase ROS and show a pro-oxidant effect causing cell damage. The cytotoxic effect observed for As, Zn, or the combination of both could be prevented by CoQ10, without any decrease in its activity at cellular levels when combined with Zn.

Keywords: Zinc; antioxidants; coenzyme Q10; free radical; oxidative stress.

Figure 1

Effect of As as Negative control (a) with positive controls of As 5 ppm (b), As 10 ppm (c), and 20 ppm (d) concentration on CHO-K1 cell morphology. Image taken with a camera under a 200× optical microscope.

Figure 2

Antioxidant activity of CoQ10, and CoQ10 + Zn determined using the ferric reduction power assay (FRAP). The extracts were analyzed, and the values were compared with the standard curve constructed with ascorbic acid (0–60 µg/mL).

Figure 3

DPPH radical reduction kinetics in the presence of CoQ10 (0.05 mg/mL) and CoQ10 + Zn (0.05 + 0.005 mg/mL, respectively), expressed from a standard ascorbic acid starting point (1 mg/mL).

Figure 4

Percentage of inhibition of DPPH radical in 5 s by the antioxidant activity of CoQ10 (0.5 mg/mL) and CoQ10 + Zn (0.5 + 0.05 mg/mL, respectively) compared to the ascorbic acid standard (1 mg/mL).

Figure 5

Violet crystal staining to determine viability in cells treated for 2 h with 0.5 mg/mL CoQ10, 0.05 mg/mL Zn, and 0.5 mg/mL CoQ10 + 0.05 mg/mL Zn compared with that seen with the same treatments in the presence of As at 5 ppm. (a) control, (b) As, (c) CoQ10, (d) CoQ10 + As, (e) Zn, (f) Zn + As, (g) CoQ10 + Zn, (h) CoQ10 + Zn + As. Image taken with a camera under a 200× optical microscope.

Figure 6

Determination of concentration of aqueous hydroperoxide in the presence of As at 5 ppm at concentrations different. (A) of CoQ10 1 at 0.5 mg/mL, CoQ10 2 at 1.0 mg/mL and CoQ10 3 at 5.0 mg/mL; (B) of Zn 1 at 0.05 mg/mL, Zn 2 at 0.1 mg/mL and Zn 3 at 0.5 mg/mL; and (C) at CoQ10 + Zn 1 at 0.5 + 0.05 mg/mL, CoQ10 + Zn 2 at 1.0 + 0.1 mg/mL and CoQ10 + Zn 3 at 5.0 + 0.5 mg/mL, respectively, and each antioxidant treatment combined with As at 5 ppm. Statistical analysis was performed by one-way ANOVA test, followed by Tukey’s post-hoc test. * (p < 0.05).

Figure 7

Protective effect of CoQ10 (A), Zn (B) and CoQ10 + Zn (C) pretreatment against As-induced oxidative stress in CHO-K1 cells. The CHO-K1 cells were exposed to 5 ppm of As for 24 h with or without test compounds pretreatment. ROS generation was measured by the DCFH-DA method using a multidetection reader and fluorescence microscope at 100× magnification. Statistical analysis was performed by one-way ANOVA test, followed by Tukey’s post-hoc test. The results are expressed as the means ± SEM of three independent experiments. * (p < 0.05) versus the same compound group without As, # (p < 0.05) versus between groups with As.

Figure 8

Cell viability by the MTT and Alamar Blue^® methods after 24 h of exposure in CHOK-1 cells treated with CoQ10 (a), Zn (b) and CoQ10 + Zn (c) in different concentrations. Statistical analysis was performed by one-way ANOVA test, followed by Tukey’s post-hoc test. The p-values are represented by * (p = 0.02–0.009) and ** (p ≤ 0.000).