GENERATION OF HYDROPEROXIDES IN ISOLATED RAT HEPATOCYTES AND HEPATIC MITOCHONDRIA EXPOSED TO HYDROPHOBIC BILE-ACIDS

Background & Aims: The mechanisms causing liver injury in cholestatic diseases are unclear. The hypothesis that accumulation of hydrophobic bile acids in hepatocytes during cholestasis leads to generation of oxygen free radicals and oxidative injury was tested. The aim of this study was to determine if hydrophobic bile acid toxicity is associated with increased hydroperoxide generation in isolated rat hepatocytes and mitochondria. Methods: Hepatocytes were exposed to taurochenodeoxycholic acid (TCDC; 0-2000 mu mol/L) or taurocholic acid (TC; 1000 mu mol/L), and cellular injury, intracellular hydroperoxide generation, and thiobarbituric acid-reacting substances (TEARS) were measured. Isolated mitochondria were incubated with 400 mu mol/L chenodeoxycholic acid or 400 mu mol/L cholic acid, and hydroperoxide generation was measured fluorometrically. Results: Hepatocyte injury, hydroperoxide generation, and TEARS increased over 4 hours on exposure to TCDC but not TC. Hydroperoxide generation preceded hepatocyte injury and accumulation of TEARS. Preincubation of hepatocytes with the antioxidant, d-alpha-tocopheryl succinate, completely abrogated cellular injury, hydroperoxide, and TEARS generation. Hydroperoxide generation was increased in mitochondria exposed to chenodeoxycholic acid. Conclusions: Intracellular generation of hydroperoxides by mitochondria appears to be an early event in hydrophobic bile acid-induced hepatocyte toxicity. Antioxidants may be of benefit in cholestasis.