FIGURE 4.

Activation of JNK by mitochondrial ROS. A, APAP treatment induces increased H₂O₂ generation from mitochondria. C57BL/6 mice were treated with either APAP (400 mg/kg) or AMAP (600 mg/kg) dissolved in warm PBS. 1 h following APAP treatment, livers were taken and mitochondria was isolated using discontinuous Percoll gradient centrifugation, and H₂O₂ release from mitochondria was measured by monitoring fluorescence of p-hydroxyphenyl acetate oxidation in the presence of horseradish peroxidase. H₂O₂ measurements were made with mitochondria treated with complex I substrates (glutamate/malate 7.5 mm). B, H₂O₂ and inhibitors that increase mitochondrial H₂O₂ generation induce JNK activation in isolated primary cultured hepatocytes. Primary cultured hepatocytes were treated with various doses of H₂O₂ (300 and 400 μm for 30 min) or rotenone (2.5 and 5 μm for 60 min) or antimycin (5 and 10 μm for 60 min). Western blot analysis was performed using antisera against phospho-JNK, JNK, and actin as loading control.