FIG. 6.

Effect of Antcin H on ROS generation. (A) Antcin H decreases ROS level of hepatocytes in vitro. Three hours after plating hepatocytes and pretreatment with Antcin H for 2 h, PMH were exposed to APAP (10 mM) for 1 h. Hepatocytes were then incubated for 30 min with DHE probe at 37°C and ROS detected by flow cytometry (left panel). “FL2-H” represents the intensity of fluorescence of DHE, and “Counts” is the cell numbers. Right panel shows summary of data from three experiments normalized to control. (*p < 0.05, n = 3 experiments). (B) Mitochondrial superoxide measured by MitoSOX increased after incubation of liver cytoplasm harvested 2 h after APAP (p-JNKc) containing p-JNK with normal mitochondria. Vehicle (DMSO) had no effect on ROS level, while Antcin H in DMSO significantly inhibited mitochondrial ROS production. “no MitoSOX” and “no p-JNK (normal cytoplasm)” are controls. (*p < 0.05 “no p-JNK” vs. “p-JNKc”; ^#p < 0.05 “p-JNK” vs. “p-JNKc+Antcin H,” n = 3 experiments). (C) Fifty micromolar DPPH with different Antcin H concentrations (0, 2.5, 5, 10, 24, 50, 75, 100, 150, 50, and 300 μM) in triplicate were wrapped in aluminum foil and kept at 30°C for 30 min in the dark. All measurements were done under dim light. Spectrophotometric measurements were done at 517 nm. BHA (50, 100, 50, and 300 μM) was used as a positive control. Radical scavenging activity (%) = {OD (Blank) − OD (Sample)}/OD (Control) × 100%. (D) Antimycin A inhibition of Complex III generated superoxide from mitochondria. FeTCCP (iron protoporphyrin) effectively inhibited superoxide production, whereas Antcin H did not affect superoxide level. ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars