Resveratrol prevents protein nitration and release of endonucleases from mitochondria during acetaminophen hepatotoxicity

Abstract

Overdose of acetaminophen (APAP) is a common cause of acute liver injury and liver failure. The mechanism involves formation of a reactive metabolite, protein binding, oxidative stress and activation of c-Jun N-terminal kinase (JNK), mitochondrial dysfunction, and nuclear DNA fragmentation caused by endonucleases released from damaged mitochondria. Previous work has shown that the natural product resveratrol (RSV) can protect against APAP hepatotoxicity in mice through prevention of lipid peroxidation and anti-inflammatory effects. However, these earlier studies did not take into consideration several fundamental aspects of the pathophysiology. To address this, we treated C57Bl/6 mice with 300 mg/kg APAP followed by 50 mg/kg RSV 1.5 h later. Our results confirmed that RSV reduced liver injury after APAP overdose in mice. Importantly, RSV did not inhibit reactive metabolite formation and protein bindings, nor did it reduce activation of JNK. However, RSV decreased protein nitration after APAP treatment, possibly through direct scavenging of peroxynitrite. Interestingly, RSV also inhibited release of apoptosis-inducing factor and endonuclease G from mitochondria independent of Bax pore formation and prevented the downstream nuclear DNA fragmentation. Our data show that RSV protects against APAP hepatotoxicity both through antioxidant effects and by preventing mitochondrial release of endonucleases and nuclear DNA damage.

Keywords: Acetaminophen; DNA fragmentation; Drug hepatotoxicity; Mitochondrial dysfunction; Oxidant stress; Resveratrol.

Figure 1. Resveratrol protects against acetaminophen (APAP) hepatotoxicity

Mice were treated with 300 mg/kg APAP followed by dimethyl sulfoxide (DMSO) vehicle or 50 mg/kg resveratrol (RSV) 1.5h later. Plasma and liver tissue were harvested at the indicated time points post-APAP. (A) Plasma alanine aminotransferase (ALT) activity. (B) H&E stained liver sections. Bars represent means ± SEM for n = 3–6 mice. *P < 0.05 vs. 0h. ^#P < 0.05 vs. APAP + DMSO treatment.

Figure 2. Resveratrol post-treatment does not affect acetaminophen (APAP)-protein binding

Mice were treated with 300 mg/kg APAP followed by dimethyl sulfoxide (DMSO) vehicle or 50 mg/kg resveratrol (RSV) 1.5h later. Liver tissue was harvested at 6h post-APAP. APAP-protein adducts were measured in both whole liver homogenates and isolated mitochondrial fractions. Bar graphs show mean ± SEM for n = 3–6 mice.

Figure 3. Resveratrol post-treatment does not affect glutathione depletion but does reduce oxidative stress and nitrotyrosine levels in the liver

Mice were treated with 300 mg/kg acetaminophen (APAP) followed by dimethyl sulfoxide (DMSO) vehicle or 50 mg/kg resveratrol (RSV) 1.5h later. Liver tissue was harvested at the indicated time points post-APAP. (A) Total hepatic glutathione (GSH) levels. (B) The percentage of total GSH in the oxidized form (GSSG). (C) Nitrotyrosine staining of liver tissue sections. (D) Western blot of whole liver homogenate using an anti-nitrotyrosine antibody. Bar graphs show mean ± SEM for n = 3–6 mice. *P < 0.05 vs. CTRL. ^#P < 0.05 vs. APAP + DMSO treatment.

Figure 4. Resveratrol does not prevent c-Jun N-terminal kinase (JNK) activation or mitochondrial translocation

Mice were treated with 300 mg/kg acetaminophen (APAP) followed by dimethyl sulfoxide (DMSO) vehicle or 50 mg/kg resveratrol (RSV) 1.5h later. (A) Liver tissue was harvested at 6 h post-APAP and mitochondrial and cytosol fractions were isolated and probed for the total and phosphorylated forms of the c-Jun N-terminal kinases 1/2. (B) Densitometry. Bar graphs show mean ± SEM for n = 3–4 mice. *P < 0.05 vs. CTRL. ^#P< 0.05 vs. APAP + DMSO treatment.

Figure 5. Resveratrol protects against acetaminophen (APAP) toxicity in vitro without preventing loss of mitochondrial membrane potential

Primary mouse hepatocytes were treated with 5 mM APAP and co-treated with either dimethyl sulfoxide (DMSO) vehicle or 100 µM resveratrol (RSV). (A) Lactate dehydrogenase (LDH) release into the cell culture medium. (B) Mitochondrial membrane potential, based on JC-1 red/green fluorescence ratio. Bar graphs show mean ± SEM for n = 3 hepatocyte preparations. *P < 0.05 vs. CTRL.

Figure 6. Resveratrol prevents release of endonucleases factor from mitochondria and nuclear DNA fragmentation during acetaminophen (APAP) hepatotoxicity

Mice were treated with 300 mg/kg APAP followed by dimethyl sulfoxide (DMSO) vehicle or 50 mg/kg resveratrol (RSV) 1.5h later. Liver tissue was harvested at 6 h post-APAP and mitochondrial and cytosol fractions were isolated. (A) Western blots for apoptosis-induced factor (AIF), endonuclease G (EndoG), and Bax in subcellular fractions. (B) Densitometry. Bar graphs show mean ± SEM for n = 3–4 mice. *P < 0.05 vs. CTRL. ^#P< 0.05 vs. APAP + DMSO treatment. (C) Nuclear DNA fragmentation in liver tissue sections as visualized by the TUNEL assay.

Figure 6. Resveratrol prevents release of endonucleases factor from mitochondria and nuclear DNA fragmentation during acetaminophen (APAP) hepatotoxicity

Mice were treated with 300 mg/kg APAP followed by dimethyl sulfoxide (DMSO) vehicle or 50 mg/kg resveratrol (RSV) 1.5h later. Liver tissue was harvested at 6 h post-APAP and mitochondrial and cytosol fractions were isolated. (A) Western blots for apoptosis-induced factor (AIF), endonuclease G (EndoG), and Bax in subcellular fractions. (B) Densitometry. Bar graphs show mean ± SEM for n = 3–4 mice. *P < 0.05 vs. CTRL. ^#P< 0.05 vs. APAP + DMSO treatment. (C) Nuclear DNA fragmentation in liver tissue sections as visualized by the TUNEL assay.