New therapeutic approach: diphenyl diselenide reduces mitochondrial dysfunction in acetaminophen-induced acute liver failure

Abstract

The acute liver failure (ALF) induced by acetaminophen (APAP) is closely related to oxidative damage and depletion of hepatic glutathione, consequently changes in cell energy metabolism and mitochondrial dysfunction have been observed after APAP overdose. Diphenyl diselenide [(PhSe)2], a simple organoselenium compound with antioxidant properties, previously demonstrated to confer hepatoprotection. However, little is known about the protective mechanism on mitochondria. The main objective of this study was to investigate the effects (PhSe)2 to reduce mitochondrial dysfunction and, secondly, compare in the liver homogenate the hepatoprotective effects of the (PhSe)2 to the N-acetylcysteine (NAC) during APAP-induced ALF to validate our model. Mice were injected intraperitoneal with APAP (600 mg/kg), (PhSe)2 (15.6 mg/kg), NAC (1200 mg/kg), APAP+(PhSe)2 or APAP+NAC, where the (PhSe)2 or NAC treatment were given 1 h following APAP. The liver was collected 4 h after overdose. The plasma alanine and aspartate aminotransferase activities increased after APAP administration. APAP caused a remarkable increase of oxidative stress markers (lipid peroxidation, reactive species and protein carbonylation) and decrease of the antioxidant defense in the liver homogenate and mitochondria. APAP caused a marked loss in the mitochondrial membrane potential, the mitochondrial ATPase activity, and the rate of mitochondrial oxygen consumption and increased the mitochondrial swelling. All these effects were significantly prevented by (PhSe)2. The effectiveness of (PhSe)2 was similar at a lower dose than NAC. In summary, (PhSe)2 provided a significant improvement to the mitochondrial redox homeostasis and the mitochondrial bioenergetics dysfunction caused by membrane permeability transition in the hepatotoxicity APAP-induced.

Figure 1. Effects of treatment with (PhSe)₂ and NAC on the survival following lethal doses of acetaminophen.

Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.) and NAC (1200 mg/kg, i.p.). Survival was followed for 48 h, n = 10 per group.

Figure 2. Effects of treatment with APAP and (PhSe)₂ on oxidative damage markers in liver mitochondria of mice.

(A) TBARS. (B) Oxidized H₂DCF-DA. (C) Protein carbonyls. Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Data are expressed as means ± SEM, (n = 5). Significance was assessed by one–way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.

Figure 3. Effects of treatment with APAP and (PhSe)₂ on reduced glutathione (GSH) levels in liver mitochondria of mice.

Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Dates are expressed as means ± SEM, (n = 5). Significance was assessed by one-way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.

Figure 4. Effects of treatment with APAP and (PhSe)₂ on antioxidant enzyme activities in liver mitochondria of mice.

(A) Glutathione peroxidase (GPx) activity. (B) Glutathione reductase (GR) activity. (C) Mn Superoxide dismutase activity. Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Dates are expressed as means ± SEM, (n = 5). Significance was assessed by one-way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.

Figure 5. Effects of treatment with APAP and (PhSe)₂ on the mitochondrial membrane potential in liver mitochondria of mice.

(A) The traces are representative of five independent experiments. (B) Means of the five experiments mitochondrial transmembrane electrical potential (Δψ_m). Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Mitochondria (0.5 mg/ml) were incubated in the reaction medium containing 230 mM Mannitol, 70 mM Sucrose, 0.02 mM EDTA, 1 mM K₂HPO₄, 20 mM Tris-HCl, pH 7.4 and was energized by 5 mM Glutamate and 5 mM Succinate. The mitochondria and 2,4 DNP (100 µM) were added where indicated by arrows. Dates are expressed as means ± SEM, (n = 5). Significance was assessed by one-way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.

Figure 6. Effects of treatment with APAP and (PhSe)₂ on PTP opening in liver mitochondria based on swelling measurements.

(A) The traces are representative of five independent experiments. (B) Means of the five experiments swelling. Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Mitochondria (0.1 mg/ml) were incubated in the reaction medium containing 230 mM Mannitol, 70 mM Sucrose, 1 mM K₂HPO₄, 20 mM Tris-HCl, pH 7.4 and was energized by 5 mM Glutamate and 5 mM Succinate. The light scattering was monitored after adding CaCl₂ (100 µM). Dates are expressed as means ± SEM, (n = 5). Significance was assessed by one-way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.

Figure 7. Effects of treatment with APAP and (PhSe)₂ on mitochondria function markers in liver mitochondria of mice.

(A) Pyridine nucleotide autofluorescence (NAD(P)H redox). (B) Mitochondrial activity (MTT reduction). Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Dates are expressed as means ± SEM, (n = 5). Significance was assessed by one-way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.

Figure 8. Effects of treatment with APAP and (PhSe)₂ on the activity of respiratory chain enzymes in liver mitochondria of mice.

(A) Complex I (NADH dehydrogenase) activity. (B) Complex II (succinate dehydrogenase) activity. (C) Mitochondrial ATPase activity. Mice were given acetaminophen (600 mg/kg, i.p.) and 1 h after were treated with or without (PhSe)₂ (15.6 mg/kg, i.p.), and were killed at 4 h after the APAP treatment. Dates are expressed as means ± SEM, (n = 5). Significance was assessed by one-way analysis of variance (ANOVA), followed by Newman-Keuls's test for post hoc comparison. Significant differences are indicated by ^*p<0.05 when compared with control group. Significant difference is indicated by ^#p<0.05 when compared with APAP group.