Antioxidant Potential and Inhibition of Mitochondrial Permeability Transition Pore by Myricetin Reduces Aluminium Phosphide-Induced Cytotoxicity and Mitochondrial Impairments

Abstract

Oxidative stress and mitochondrial dysfunction are involved in the mechanisms of cardiac toxicity induced by aluminum phosphide (AlP). AlP-induced cardiotoxicity leads to cardiomyocyte death, cardiomyopathy, cardiac dysfunction, and eventually severe heart failure and death. Importantly, protecting cardiomyocytes from death resulting from AlP is vital for improving survival. It has been reported that flavonoids such as myricetin (Myr) act as modifiers of mitochondrial function and prevent mitochondrial damage resulting from many insults and subsequent cell dysfunction. In this study, the ameliorative effect of Myr, as an important antioxidant and mitochondrial protective agent, was investigated in cardiomyocytes and mitochondria isolated from rat heart against AlP-induced toxicity, oxidative stress, and mitochondrial dysfunction. Treatment of AlP (20 μg/ml) significantly increased cytotoxicity; reduced glutathione (GSH) depletion, cellular reactive oxygen species (ROS) formation, malondialdehyde (MDA) level, ATP depletion, caspase-3 activation, mitochondrial membrane potential (ΔΨm) collapse, and lysosomal dysfunction; and decreased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in intact cardiomyocytes. Also, treatment of AlP (20 μg/ml) significantly increased mitochondrial dysfunction and swelling in isolated mitochondria. Myr (80 µM) appeared to ameliorate AlP-induced cytotoxicity in isolated cardiomyocytes; significantly lessened the AlP-stimulated intracellular ROS and MDA production and depletion of GSH; and increased the activities of SOD, CAT, and GSH-Px. Furthermore, Myr (40 and 80 µM) lowered AlP-induced lysosomal/mitochondrial dysfunction, ATP depletion, and caspase-3 activation. In the light of these findings, we concluded that Myr through antioxidant potential and inhibition of mitochondrial permeability transition (MPT) pore exerted an ameliorative role in AlP-induced toxicity in isolated cardiomyocytes and mitochondria, and it would be valuable to examine its in vivo effects.

Keywords: antioxidant; cardiomyopathy; flavonoids; mitochondrial dysfunction; poisoning.

FIGURE 1

Ameliorative effect of Myr on AlP-induced cytotoxicity in isolated cardiomyocytes. (A) Isolated cardiomyocytes were treated or cotreated with the indicated concentrations (20 μg/ml) of AlP and Myr (20, 40, and 80 µM) for 3 h. Cell viability was examined after incubation using MTT assay. Myr inhibits AlP-induced cytotoxicity in isolated cardiomyocytes. (B) Isolated cardiomyocytes were treated or cotreated with Myr (20, 40, and 80 μM) and AlP (20 μg/ml) for 3 h. MDA production was measured by production of thiobarbituric acid (TBA) reactive substances (TBARS). (C) Isolated cardiomyocytes (10⁶ cells/ml) were incubated in CCT medium in conventional condition (37°C and 5% CO₂-air) for 3 h. Caspase-3 activity was determined by Sigma-Aldrich kit. Columns represent caspase-3 activity (μM pNA/min/ml) in isolated cardiomyocytes. (D) ATP/ADP ratios were determined by Luciferin/Luciferase assay as described in the Materials and Methods. Values represent mean ± SD (n = 3) of three independent experiments. ***p < 0.001 versus control; ###p < 0.001 versus AlP-treated cardiomyocytes, one-way ANOVA, Tukey’s test. Myr, myricetin; AlP, aluminum phosphide; MDA, malondialdehyde; STS, staurosporine; CCT, creatine–carnitine–taurine.

FIGURE 2

Myr inhibits AlP-induced ROS production in isolated cardiomyocytes. As shown, the fluorescence intensity of DCF is increased after exposure to AlP. The peak is moved to the right as compared with control, while cotreatment of Myr (40 and 80 µM) with AlP showed that the fluorescence intensity of DCF is decreased, and the peaks are moved to the left. Values represent mean ± SD (n = 3) of three independent experiments, ***p < 0.001 compared with control; ##p < 0.01; ###p < 0.001 compared with AlP-treated cardiomyocytes, one-way ANOVA, Tukey’s test. Myr, myricetin; AlP, aluminum phosphide; ROS, reactive oxygen species; DCF-DA, 2ʹ,7ʹ-dichlorofluorescin diacetate; BHT, butylated hydroxytoluene; H₂O₂, hydrogen peroxide.

FIGURE 3

Myr inhibits AlP-induced mitochondrial membrane potential (ΔΨm) collapse in isolated cardiomyocytes. Isolated cardiomyocytes were treated or cotreated with the indicated concentrations (20 μg/ml) of AlP and Myr (20, 40, and 80 µM) for 3 h. Mitochondrial membrane potential (ΔΨm) collapse was examined after incubation using rhodamine 123 staining. Representative fluorescence intensity of rhodamine 123 staining. Data presented are the mean ± SD (n = 3 per group). ***p < 0.001 compared with control; ##p < 0.01; ###p < 0.001 compared with AlP-treated cardiomyocytes, one-way ANOVA, Tukey’s test. Myr, myricetin; AlP, aluminum phosphide; Cs.A, cyclosporine; CaCl₂, calcium chloride.

FIGURE 4

Myr inhibits AlP-induced lysosomal dysfunction in isolated cardiomyocytes. Isolated cardiomyocytes were treated or cotreated with the indicated concentrations (20 μg/ml) of AlP and Myr (20, 40, and 80 µM) for 3 h. Lysosomal membrane stability was examined after incubation using acridine orange fluorescence dye. Data are mean ± SD (n = 3) of three independent experiments. ***p < 0.001 significantly different from control, ##p < 0.01 significantly different from AlP-treated cardiomyocytes, one-way ANOVA, Tukey’s test. Myr, myricetin; AlP, aluminum phosphide; AO, acridine orange; t-BuOOH, tert-butyl hydroperoxide.

FIGURE 5

Myr inhibits AlP-induced GSH depletion in isolated cardiomyocytes. Isolated cardiomyocytes were treated or cotreated with the indicated concentrations (20 μg/ml) of AlP and Myr (20, 40, and 80 µM) for 3 h. GSH and GSSG levels were measured as previously described in the Materials and Methods section. (A) GSH content significantly decreased AlP-treated isolated cardiomyocytes, while cotreatment of Myr (40 and 80 µM) with AlP obviously increased the GSH content in isolated cardiomyocytes. (B) AlP significantly increased the GSSG content in isolated cardiomyocytes after 3 h of exposure, while Myr (40 and 80 µM) significantly decreased the GSSG content in the presence of AlP. Data are mean ± SD (n = 3) of three independent experiments. ***p < 0.001 significantly different from control, ###p < 0.001 significantly different from AlP-treated cardiomyocytes, one-way ANOVA, Tukey’s test. Myr, myricetin; AlP, aluminum phosphide; GSH, reduced glutathione; GSSG; oxidized glutathione.

FIGURE 6

Myr inhibits AlP-induced mitochondrial dysfunction (A) and swelling (B) in isolated mitochondria. (A) Isolated mitochondria were treated or cotreated with the indicated concentrations (20 μg/ml) of AlP and Myr (20, 40 and 80 µM) for 1 h. Succinate dehydrogenase activity was evaluated by MTT assay. Data showed AlP (20 μg/ml) to significantly decrease SDH activity as compared with control, while Myr (40 and 80 µM) significantly increased SDH activity as compared with AlP-treated cardiomyocytes. (B) Isolated mitochondria were treated or cotreated with the indicated concentrations (20 μg/ml) of AlP and Myr (20, 40, and 80 µM) for 1 h. Mitochondrial swelling was evaluated by monitoring absorbance at 540 nm. Data showed that AlP (20 μg/ml) significantly induced mitochondrial swelling as compared with control, while Myr (40 and 80 µM) significantly inhibits mitochondrial swelling as compared with AlP-treated cardiomyocytes. Data are mean ± SD (n = 3) of three independent experiments. ***p < 0.001 significantly different from control, ##p < 0.01, ###p < 0.001 significantly different from AlP-treated cardiomyocytes, one-way ANOVA and two-way ANOVA followed by post-hoc Tukey and Bonferroni test. Myr, myricetin; AlP, aluminum phosphide; SDH, succinate dehydrogenase activity.