Intracellular retention of toxic bile salts contributes to hepatocellular injury during cholestasis. We have recently demonstrated that toxic bile salts directly induce apoptosis in hepatocytes. As oxidative stress has been implicated in many models of apoptosis, our aim was to determine if oxidative injury is a critical event during bile-salt-induced hepatocyte apoptosis. Cultured rat hepatocytes incubated with 50 microM glycochenodeoxycholate (GCDC) exhibited the characteristic morphological features of apoptosis such as nuclear fragmentation and cellular fragmentation into organelle-containing membrane-bound apoptotic bodies. After a 3-hr incubation, apoptosis was observed in 60 +/- 8% of cells compared to <1% in controls. GCDC-induced apoptosis was associated with lipid peroxidation as demonstrated by an increase in 8-isoprostane release. The antioxidant lazaroid U83836E inhibited 8-isoprostane generation during GCDC-induced hepatocye apoptosis. In addition, U83836E also reduced GCDC-mediated apoptosis by 70% as assessed using both stringent morpholgic (nuclear fragmentation) and biochemical (determination of DNA strand breaks) criteria. In summary, during treatment of hepatocytes with GCDC, (1) apoptosis is associated with lipid peroxidation, and (2) the antioxidant lazaroid U83836E inhibits both lipid peroxidation and apoptosis. In conclusion, these data suggest that oxidative stress contributes to bile-salt-induced apoptosis. We speculate that antioxidants may be useful in ameliorating liver injury during chronic cholestasis.