Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition

The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis, The aim of this study was to determine in freshly isolated rat hepatocytes the roles of oxi dant stress and the mitochondrial permeability transition (MPT) in bile aci d-induced apoptosis, Hepatocytes isolated from adult male Sprague-Dawley ra ts were incubated for 4 hours in buffer containing the hydrophobic bile aci d, glycochenodeoxycholic acid (GCDC, 0-500 mu mol/L) or the hydrophilic bil e acid, glycocholic acid (GCA), and either the antioxidants, alpha tocopher ol, ebselen, or idebenone (a coenzyme Q analogue); or the MPT blockers, cyc losporin A, or bongkrekic acid, or a caspase-8 inhibitor. Apoptosis was ass essed hourly by nuclear morphologic changes of fixed cells by DAPI fluoresc ence microscopy and reactive oxygen species (ROS) generation by dichloroflu orescein fluorescence of hepatocytes. The percent of cells undergoing apopt osis increased in a time- and concentration-dependent manner in cells expos ed to GCDC, and to a much lesser extent to GCA, ROS generation preceded the onset of apoptosis. MPT blockers, caspase-8 inhibition, and antioxidants p revented apoptosis and reduced ROS generation by hepatocytes: Flow cytometr y analysis showed that MPT occurred within 1 hour of exposure of cells to 1 00 mu mol/L GCDC, prior to onset of significant apoptosis, In conclusion, R OS generation, MPT induction, and cytochrome c release are critical steps i n the induction of apoptosis by bile acids. Antioxidants may reduce liver i njury caused by low levels of bile acids by preventing the generation of ox idant stress and subsequent stimulation of the MPT and release of cytochrom e c from mitochondria.