In previous studies we have demonstrated that transforming growth factor (TGF)-alpha/c-myc double transgenic mice exhibit an enhanced rate of cell proliferation, accumulate extensive DNA damage, and develop multiple liver tumors between 4 and 8 months of age. To clarify the biochemical events that may be responsible for the genotoxic and carcinogenic effects observed in this transgenic model, several parameters of redox homeostasis in the liver were examined prior to development of hepatic tumors. By 2 months of age, production of reactive oxygen species, determined by the peroxidation-sensitive fluorescent dye, 2',7'-dichlorofluorescin diacetate, was significantly elevated in TGF-alpha/c-myc transgenic hepatocytes versus either wild type or c-myc single transgenic cells, and occurred in parallel with an increase in lipid peroxidation. Concomitantly with a rise in oxidant levels, antioxidant defenses were decreased, including total glutathione content and the activity of glutathione peroxidase, whereas thioredoxin reductase activity was not changed. However, hepatic tumors which developed in TGF-alpha/c-myc mice exhibited an increase in thioredoxin reductase activity and a very low activity of glutathione peroxidase. Furthermore, specific deletions were detected in mtDNA as early as 5 weeks of age in the transgenic mice. These data provide experimental evidence that co-expression of TGF-alpha and c-myc transgenes in mouse liver promotes overproduction of reactive oxygen species and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-alpha/c-myc mouse model.