Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice

Histopathology of liver 24h after administration of acetaminophen. Mice were given acetaminophen (APAP, 350mg/kg, i.p.) together with or without cyclosporin A (CsA, 50mg/kg, i.p.), and were killed 24h after the treatment. Liver sections were subjected to hematoxylin–eosin staining.

Time course of hepatic GSH and GSSG contents after administration of acetaminophen. Mice were given acetaminophen (APAP, 350mg/kg, i.p.) together with or without cyclosporin A (CsA, 50mg/kg, i.p.), and were killed at various time points. Contents of GSH and GSSG in liver homogenates were determined. The results are means±SE of 3–6 mice. *P<0.05, **P<0.01, Significantly different from corresponding APAP(−) groups.

Mitochondrial GSH, GSSG and ATP contents after administration of acetaminophen. Mice were given acetaminophen (APAP, 350mg/kg, i.p.) together with or without cyclosporin A (CsA, 50mg/kg, i.p.), and were killed 8h after the treatment. Contents of GSH, GSSG and ATP in liver mitochondria were determined. The results are means±SE of 3–6 mice. *P<0.05, **P<0.01, Significantly different from corresponding APAP(−) groups. #P<0.05, ##P<0.01, Significantly different from corresponding CsA(−) groups.

Leakage of cytochrome c to cytosol after administration of acetaminophen. Mice were given acetaminophen (350mg/kg, i.p.) together with or without cyclosporin A (50mg/kg, i.p.), and were killed 24h after the treatment. Hepatic cytosol fractions from the mice were analyzed by Western blot analysis with antibody against cytochrome c. Each lane represents a sample from a single mouse. The results are representative blots from 3 to 5 mice.

Swelling of liver mitochondria from mice after administration of acetaminophen. Mice were given acetaminophen (350mg/kg, i.p.) and were killed 8h after the treatment along with control mice. Mitochondria (1mg/ml) of the mice were incubated in the reaction medium containing 125mM sucrose, 150mM KCl, 10mM HEPES-KOH, 2.5μM rotenone, 20μM CaCl₂, and was energized by 5mM succinate. Absorbance at 540nm was monitored after adding CaCl₂. The dotted line shows the incubation of mitochondria from acetaminophen-treated mice with 1μM cyclosporin A. The results are representatives from 3 to 5 experiments.

Swelling and depolarization of liver mitochondria from mice in vitro treated with NAPQI. Mitochondria (1mg/ml) of untreated mice were incubated in the reaction medium containing 125mM sucrose, 150mM KCl, 10mM HEPES-KOH, 2.5μM rotenone, 20μM CaCl₂, 5–50μM NAPQI and were energized by 5mM succinate. Absorbance at 540nm was monitored after adding NAPQI. Numbers in the figure are concentrations (μM) of NAPQI. The dotted line shows the incubation of mitochondria with 50μM NAPQI and 1μM cyclosporin A. The plots inserted are Δψ values obtained from the fluorescence intensity at the 505/535nm wavelength pair after incubation of the same mixture with 0.5mM rhodamine 123. The addition of chemicals (C, no addition; N, 50μM NAPQI; CsA, 50μM NAPQI+1μM cyclosporin A) are shown in the bottom. The results are representatives of 3 experiments.