Execution of superoxide-induced cell death by the proapoptotic Bcl-2-related proteins Bid and Bak.

Alcoholic liver disease leads to hepatocyte death, liver cirrhosis, and organ dysfunction and is a major cause of death in the United States (22, 23, 37). Hepatocyte injury is most prominent in the centrilobular region of the liver acinus and stems from both necrotic and apoptotic cell death (17, 18, 32). However, the mechanism(s) by which alcohol leads to cell death is unclear. A prominent feature of alcohol exposure is the production of reactive oxygen species (ROS). Phagocytic NAD(P)H oxidase, xanthine (X) oxidase (XO), and the mitochondrial respiratory chain are the major sources of ROS under both physiological and pathological conditions. In hepatocytes, XO and mitochondria are likely sources of ROS during oxidative stress (10). Specifically, ROS contribute to alterations in mitochondrial morphology and bioenergetics. However, little is known about how alcohol-induced ROS production directly affects mitochondrial function and hepatocyte injury. ROS are important mediators of apoptosis in liver diseases and are generated by all mammalian cells as by-products of abnormal metabolism and in response to paracrine factors such as ethanol (EtOH). Pathologically, both apoptosis and necrosis are initiated by ROS (2, 8, 20, 45). This damage is especially important in vascular and epithelial cells, which subsequently initiate a series of local chemical reactions and genetic alterations that ultimately result in amplification of the initial ROS-mediated tissue damage and/or cytotoxicity (9, 14, 30, 31). It is estimated that normal levels of ROS, generated in the basal region of cellular tissues from approximately 5% of the metabolized oxygen, are efficiently detoxified by endogenous enzymatic free-radical scavengers such as superoxide (O2.−) dismutase (SOD), glutathione peroxidase, and catalase (Cat) (9, 45). However, under conditions associated with excess production of ROS, such as acute and chronic EtOH exposure, the rate of ROS generated by tissues can exceed the capacity of endogenous oxidant defense mechanisms to detoxify ROS and prevent deleterious radical-mediated reactions. An important target of cellular ROS is the mitochondria, resulting in mitochondrial dysfunction and permeabilization of the outer mitochondrial membrane and leads to the release of apoptotic proteins, including cytochrome c (1, 11, 30, 48). Specifically, we have demonstrated that the oxidant O2.− can lead to mitochondrial depolarization by facilitating cytochrome c release (27, 30). Moreover, the exogenous addition of cytochrome c rescued the mitochondria from this O2.−-induced depolarization, indicating that the primary mechanism of mitochondrial dysfunction via O2.− is cytochrome c release. Yet, the mechanism whereby O2.− facilitates outer mitochondrial membrane permeabilization and cytochrome c release is not clear. A potential pathway in which O2.− may target the mitochondria is via the multidomain proapoptotic Bcl-2 family of proteins. Recent studies to characterize animals deficient in the Bcl-2 family members Bax and Bak suggest that these two proteins play essential but redundant roles in initiating mitochondrial apoptotic events (6, 25, 44, 49, 51, 55). However, despite their high homology, Bax and Bak have distinct subcellular localization and functional regulation. Bax is largely a cytosolic protein that must undergo conformational change to initiate its translocation to mitochondria prior to the initiation of apoptosis (33). In contrast, Bak is a resident mitochondrial protein whose proapoptotic activity can be activated by proapoptotic BH3 peptides (50). Although controversy exists about the induction of apoptosis in the liver of mice following chronic consumption of EtOH via a Lieber-DeCarli liquid diet, recent investigations have demonstrated that chronic EtOH consumption increases the expression of antiapoptotic Bcl-2 and Bcl-xL proteins by an interleukin-6-dependent mechanism (19). Upregulation of proapoptotic Bax proteins has also been observed in patients with alcoholic liver disease, but the modification of BH3-only proteins has not been determined (19). Here, we used a novel approach to identify the cytosolic components required for mitochondrial dysfunction during oxidative stress. The O2.−-mediated mitochondrial phase of apoptosis is mainly dependent on Bid and Bak but not Bax. Further, we demonstrate that mitochondrial membrane potential (ΔΨm) loss and the release of cytochrome c via this pathway can be antagonized by Bcl-xL. We also show that O2.−-evoked caspase-8 activation and Bid cleavage require caspase-2. Alcohol-induced O2.− generation sensitizes cells to proinflammatory cytokine-mediated apoptosis. Consistent with these findings, Bid processing has been manifested in aged, chronically alcohol-fed animals.