OXIDATION PATHWAYS FOR THE INTRACELLULAR PROBE 2',7'-DICHLOROFLUORESCIN

The oxidation of 2',7'-dichlorofluorescin (DCFH) to a fluorescent prod uct is currently used to evaluate oxidant stress in cells. However, th ere is considerable uncertainty as to the enzymatic and nonenzymatic p athways that may result in DCFH oxidation. Iron/hydrogen peroxide-indu ced DCFH oxidation was inhibited by catalase or by the hydroxyl radica l scavenger dimethylsulfoxide; however, superoxide dismutase (SOD) had no effect on DCFH oxidation. The formation of hydroxyl radical (indic ated by the oxidation of salicylic acid to 2,3-dihydroxybenzoic acid) was proportional to DCFH oxidation, suggesting that the hydroxyl radic al is responsible for the iron/peroxide-mediated oxidation of DCFH. Ut ilizing a superoxide generating system consisting of hypoxanthine/xant hine oxidase, oxidation of DCFH was unaffected by SOD, catalase or des feroxamine, and stimulated by removing hypoxanthine from the reaction mixture. In contrast, SOD or elimination of hypoxanthine abolished sup eroxide formation. In addition, potassium superoxide did not support t he oxidation of DCFH. Thus. superoxide is not involved in DCFH oxidati on. Boiling xanthine oxidase eliminated its concentration-dependent ox idation of 1 mu M DCFH, indicating that xanthine oxidase can en2ymatic ally utilize DCFH as a high affinity substrate. Kinetic studies of the oxidation of DCFH by xanthine oxidase indicated a K-m(app) of 0.62 mu M. Hypoxanthine competed with DCFH with a K-i(app) of 1.03 mM. These studies suggest that DCFH oxidation may be a useful indicator of oxida tive stress. However, other types of cellular damage may produce DCFH oxidation. For example, conditions or chemicals that damage intracellu lar membranes may release to the cytoplasm oxidases or peroxidases tha t might use DCFH as a substrate, similar to xanthine oxidase