FLOW CYTOMETRIC DETECTION OF HYDROGEN-PEROXIDE PRODUCTION INDUCED BY DOXORUBICIN IN CANCER-CELLS

2',7'-Dichlorofluorescin diacetate (DCFH-DA) has been previously used to study the oxidative burst of neutrophils induced by different stimu li. The method is based on the fact that DCFH-DA diffuses through the cell membrane and it is hydrolyzed by intracellular esterases to DCFH, which remains trapped within the cells. DCFH, a nonfluorescent compou nd, is able to react with free radical products, particularly with hyd rogen peroxide, and to generate the fluorescent 2',7'-dichlorofluoresc ein (DCF). By flow cytometric detection of DCF fluorescence, an indire ct measure of reactive oxygen species production in single cells may b e obtained. Using a modified procedure to load cells of the human colo n adenocarcinoma cell line LoVo with DCFH-DA, a significant fluorescen ce increase above the basal fluorescence level has been detected after treatment with doxorubicin doses as low as 0.4 mu M. This increase is not detectable when the cells are preloaded with catalase, using a sc raping method, and it is not due to doxorubicin own fluorescence. Thes e experiments prove that the increase of DCF fluorescence intensity ob served during doxorubicin treatment is not due to technical artifacts but it is attributable to free radicals produced in the cells by the d rug.