Gallic acid protects rat liver mitochondria ex vivo from bisphenol A induced oxidative stress mediated damages

Abstract

Humans are often exposed to bisphenol A (BPA), the monomer of polycarbonate plastics and epoxy resins, through BPA contaminated drinking water, beverages and foods, packaged in polycarbonate plastic bottles and cans coated with epoxy resins due to leaching. Several research groups have reported that BPA may cause damage of mitochondria in liver, kidney, heart and brain cells by inducing oxidative stress. The antioxidant efficacy of gallic acid (GA), a polyphenol compound obtained from plants, against different toxicants induced oxidative stress has been well established. The aim of the present study was to examine the protective efficacy of GA against BPA induced oxidative damages of the rat liver mitochondria ex vivo. In our study, we have found a significant decrease in the intactness of mitochondria; a significant increase (P ≤ 0.001) in the levels of lipid peroxidation end product (i.e. malondialdehyde) and protein carbonylation product; and also a significant decrease (P ≤ 0.001) in the reduced glutathione content; when mitochondria were incubated with BPA (160 μM/ml) only. These results indicate that BPA probably causes damage to the cellular macromolecules through oxidative stress. We have observed significant counteractions (P ≤ 0.001) against BPA induced alterations in mitochondrial intactness, lipid peroxidation and protein carbonylation products formation and reduced glutathione content when mitochondria were incubated with BPA and GA (20 μg/ml/ 40 μg/ml/ 80 μg/ml) in combination in a dose-dependent manner. Gallic acid also showed significant restorations (P ≤ 0.001) of the activities of antioxidant enzymes, Krebs cycle enzymes, respiratory chain enzymes and thiolase when mitochondria were incubated with BPA and dosage of GA (20 μg/ml/ 40 μg/ml/ 80 μg/ml) in combination compared to BPA incubated mitochondria. Furthermore, GA significantly (P ≤ 0.001) counteracted the BPA induced decrease in tryptophan and NADH auto-fluroscence levels in mitochondria. This result suggests that GA protects the mitochondria probably by reducing the oxidative stress. Besides, GA protects the mitochondrial surface from BPA induced oxidative damages as viewed under the scanning electron microscope. Considering all the results, it can be concluded that GA shows potent efficacy in protecting the rat liver mitochondria ex vivo from BPA induced oxidative stress mediated damages.

Keywords: Antioxidant; Bisphenol A; Gallic acid; Krebs cycle enzymes; Mitochondria; Oxidative stress.

Fig. 1

Chemical structure of gallic acid (GA).

Fig. 2

Protective effect of gallic acid against bisphenol A-induced changes of intactness of rat liver mitochondria. Janus green B stained: (A–C) by phase contrast microscopy (400X magnification) and (D–F) by confocal microscopy (40X magnification); BPA = bisphenol A- incubated mitochondrial group; GA20-80= mitochondrial groups incubated with gallic acid at the dose of 20–80 μg/ml respectively; BPA-GA20-80= mitochondrial groups co-incubated with bisphenol A and gallic acid at the dose of 20–80 μg/ml respectively.

Fig. 3

Protective effect of gallic acid against bisphenol A-induced increase in the levels of (A) reactive nitrogen species and (C) conjugated diene and decrease in (B) swelling of rat liver mitochondria. BPA = bisphenol A- incubated mitochondrial group; GA20-80= mitochondrial groups incubated with gallic acid at the dose of 20–80 μg/ml respectively; BPA-GA20-80= mitochondrial groups co-incubated with bisphenol A and gallic acid at the dose of 20–80 μg/ml respectively; The values are expressed as Mean ± SE for six replicates in the experiment; ^* P ≤ 0.001 compared to control values using ANOVA; ^P ≤ 0.001 compared to bisphenol A- incubated values using ANOVA.

Fig. 4

Protective effect of gallic acid against bisphenol A-induced decrease in the activity of (A) acetoacetyl CoA thiolase, levels of (C) tryptophan and (D) NADH auto fluorescence and decrease in (B) di-tyrosine level and also (E) damages in mitochondrial DNA of rat liver mitochondria. BPA = bisphenol A- incubated mitochondrial group; GA20-80= mitochondrial groups incubated with gallic acid at the dose of 20–80 μg/ml respectively; BPA-GA20-80= mitochondrial groups co-incubated with bisphenol A and gallic acid at the dose of 20–80 μg/ml respectively; The values are expressed as Mean ± SE for six replicates in the experiment; ^* P ≤ 0.001 compared to control values using ANOVA; ^P ≤ 0.001 compared to bisphenol A- incubated values using ANOVA; ^@P ≤ 0.0001 compared to control values using ANOVA; ^#P ≤ 0.0001 compared to bisphenol A- incubated values using ANOVA.

Fig. 5

Protective effect of gallic acid against bisphenol A-induced alterations in (A–H) mitochondrial quantity (100X) and (I–M) mitochondrial quality (400X); Scanning electron microscopic study also depicts the changes in cyto-architecture of mitochondria (N–U); Arrow heads indicate the furrow formation on the surface of mitochondria by the treatment of bisphenol A; BPA = bisphenol A- incubated mitochondrial group; GA20-80= mitochondrial groups incubated with gallic acid at the dose of 20–80 μg/ml respectively; BPA-GA20-80= mitochondrial groups co-incubated with bisphenol A and gallic acid at the dose of 20–80 μg/ml respectively.

Fig. 6

Different forms of gallic acids which are present in the plant materials.

Fig. 7

Schematic representation showing the probable mechanisms in the protection of liver mitochondria from bisphenol A induced oxidative stress mediated damages by gallic acid. ETC = Electron transport chain; NO = Nitric oxide; O₂^˙− = Superoxide anion free radical; MnSOD = Manganese-superoxide dismutase; H₂O₂= Hydrogen peroxide; GPx = Glutathione peroxidase; GR = Glutathione reductase; H₂O = Water; GSH = Reduced glutathione; GSSG = Oxidised glutathione; MDA = Manoldialdehyde; OH˙= Hydroxyl radical; ONOO˙= Nitroperoxyl radical; mtDNA = Mitochondrial DNA.