Detection of Oxidative Stress in Lymphocytes Using Dichlorodihydrofluorescein Diacetate

Normal oxidative metabolism leads to the generation of reactive oxygen species (ROS), in particular, superoxide anion (O₂ −), and its dismutation product hydrogen peroxide (H₂ O₂ ), which may escape from the electron-transport chain (1 . In addition, oxidative stress may be generated through the action of specialized enzymes, e.g., NADPH oxidase, nitric oxide synthase, cycloxygenase, and lipoxygenase, which produce H₂ O₂ , O₂ −, NO, and lipid hydroperoxides [ROOH] (2 ,3 ). Toxicants can also induce oxidative stress by a variety of mechanisms. Compounds that uncouple or block the electron-transport chain lead to increased leakage of ROS from mitochondria to the cytosol. Redox cycling of metals can result in H₂ O₂ , hydroxyl radical (�OH), and thionyl radical (RS�) production, and can deplete cellular thiol pools (4 ). In addition, toxicants may directly inhibit antioxidant enzymes or compromise cellular reducing capacity by depletion of NAD(P)H, and glutathione (GSH) through the cytochrome P450 oxidoreductase and glutathione-S-transferase detoxification pathways, and through the action of the GSH peroxidase/reductase cycle (5 ). In cells with diminished natural antioxidant capacity, normal metabolic sources of ROS may overwhelm the redox balance and push cells into a condition of oxidative stress.