Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells

Abstract

2',7'-Dichlorofluorescin diacetate (DCFH-DA) has been previously used to study the oxidative burst of neutrophils induced by different stimuli. 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 compound, is able to react with free radical products, particularly with hydrogen peroxide, and to generate the fluorescent 2',7'-dichlorofluorescein (DCF). By flow cytometric detection of DCF fluorescence, an indirect measure of reactive oxygen species production in single cells may be obtained. Using a modified procedure to load cells of the human colon adenocarcinoma cell line LoVo with DCFH-DA, a significant fluorescence increase above the basal fluorescence level has been detected after treatment with doxorubicin doses as low as 0.4 microM. This increase is not detectable when the cells are preloaded with catalase, using a scraping method, and it is not due to doxorubicin own fluorescence. These experiments prove that the increase of DCF fluorescence intensity observed during doxorubicin treatment is not due to technical artifacts but it is attributable to free radicals produced in the cells by the drug.