Our previous study demonstrated that polycationic liposomes are highly stable in the bloodstream and represent an effective agent for liver gene delivery. We report here that liposomemediated extracellular superoxide dismutase (EC-SOD) gene delivery successfully prevented acute liver injury in mice. The therapeutic efficacy of EC-SOD gene delivery by polycationic liposomes was determined against the toxicity of superoxide anions and hydroxyethyl radicals in HepG2 cells and in a mouse model of acute liver injury caused by D-galactosamine and lipopolysaccharide intoxication. Transfection of HepG2 cells with an EC-SOD plasmid led to a striking increase in superoxide dismutase activity in the medium. The transfected cells had much less cell death after reactive oxygen species exposure compared with untransfected or control plasmid-transfected cells. In a model of acute liver injury, serum alanine aminotransferase levels in mice receiving portal vein injections of EC-SOD lipoplexes were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes. Liver histology confirmed that there was less cell death in the EC-SOD lipoplex-treated group. Quantitative reverse transcriptase polymerase chain reaction showed a 55-fold increase in human EC-SOD gene expression in the liver of mice injected with EC-SOD lipoplexes. Serum superoxide dismutase activity in EC-SOD lipoplex-treated mice was higher than in the control groups; this was associated with higher liver glutathione levels and reduced lipid peroxidation. In conclusion, polycationic liposome-mediated EC-SOD gene delivery protects against reactive oxygen species toxicity in vitro and against lipopolysaccharide-induced acute liver injury in D-galactosamine-sensitized mice. (HEPATOLOGY 2004;40:195-204.) F ormation of reactive oxygen species (ROS) occurs in a variety of forms of liver injury and fibrogenesis. Common free radicals, such as superoxide anions (O 2 ⅐Ϫ ), hydroxyl radicals (HO ⅐Ϫ ), hydrogen peroxide (H 2 O 2 ) or hydroxyethyl radicals (HERs), are generated during drug toxicity, ischemia/reperfusion, and alcohol metabolism, in addition to reactive intermediate metabolites of hepatotoxins or drugs. 1 These ROS are responsible for necrosis and/or apoptosis of hepatocytes and sinusoidal endothelial cells, the activation of Kupffer cells (thus causing the second phase of liver inflammation), and the activation of hepatic stellate cells and subsequent hepatic fibrogenesis; therefore, antioxidative treatment appears to be an effective means of attenuating liver injury and fibrosis in liver diseases. 2 Our previous studies have shown that using antioxidants such as vitamins E and C or free radical scavengers such as catalase or superoxide dismutase (SOD) prevented carbon tetrachloride, D-galactosamine (GalN), or bromobenzene-induced acute hepatocellular damage. [3][4][5] Therefore, our speculation is that treatment with antioxidants or free radical scavengers