Identification of Intracellular Sources Responsible for Endogenous Reactive Oxygen Species Formation

The endogenous reactive oxygen species ("ROS") formation is associated with many pathologic states such as inflammatory diseases, neurodegenerative diseases, brain and heart ischemic injuries, cancer, and aging. The purpose of this study was to investigate the endogenous sources for "ROS" formation in intact isolated rat hepatocytes, in particular, peroxisomal oxidases, monoamine oxidase, xanthine oxidase, cytochrome P450, and mitochondria electron transport. The rat hepatocyte catalyzed oxidation of 2',7'-dichlorofluorescin to form the fluorescent 2,7'-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen species ("ROS") formation by intact isolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous "ROS" formation was markedly increased in catalase inhibited or GSH depleted hepatocytes, and was inhibited by "ROS" scavengers or desferoxamine. Endogenous "ROS" formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased "ROS" before cytotoxicity ensued. This suggests endogenous "ROS" formation can largely be attributed to oxygen reduction by reduced mitochondrial electron transport components and reduced cytochrome P450 isozymes. Addition of monoamine oxidase substrates increased antimycin A-resistant respiration and "ROS" formation before cytotoxicity ensued. On the other hand peroxisomal substrates readily induced "ROS" formation and were cytotoxic towards catalase inhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H₂O₂ formed in the peroxisomes. The consequences of upregulation of peroxisomal oxidases are discussed.