Spectrofluorometric determination of intracellular levels of reactive oxygen species in drug-sensitive and drug-resistant cancer cells using the 2′,7′-dichlorofluorescein diacetate assay

This article examines a non-invasive spectrofluorometric method using the 2′,7′-dichlorofluorescein diacetate (DCHF-DA) assay for quantifying the intracellular reactive oxygen species (ROSi) produced in four cultured cancer cell lines: drug-sensitive (K562) and drug-resistant (K562/adr) human erythromyelogenous leukemia cell lines, and drug-sensitive (GLC4) and drug-resistant (GLC4/adr) human small cell lung carcinoma cell lines. The oxidation of the probe to the fluorescent dichlorofluorescein (DCF) was continuously monitored by following the DCF fluorescence intensity as a function of time using a standard spectrofluorometer in the presence of an extracellular DCF fluorescence quencher (Co2+). By fitting the spectrofluorometric data to a kinetic model based on the following two reactions: (i) deacetylation of DCHF-DA to the oxidant-sensitive compound 2′,7′-dichlorofluorescein (DCHF) by cellular esterase enzymes (pseudo-first-order rate constant: ke) and (ii) oxidation of DCHF by ROSi (second-order rate constant: k2), the parameters intervening in DCF formation, ke and the product of k2 by the ROSi concentration, were quantitatively determined for the different cell lines studied. The results revealed that the intracellular esterase content or activity is similar in K562, K562/adr, and GLC4 cells, but 5-fold higher in GLC4/adr cells. The product k2[ROSi] was found to be similar in the four cell lines considered, with a mean value of (5.3±0.9)×10−7 cell−1 s−1. Assuming that H2O2 (in combination with peroxidases) is the primary responsible species for DCHF oxidation in intact cells, and using the rate constant value k2=790±62M-1s-1 established in our laboratory for the reaction of DCHF with H2O2 in the presence of horseradish peroxidase, the mean value of the intracellular levels of ROSi in those cells was estimated to be 0.67±0.16 nM per cell. Such a value compares favorably to H2O2 intracellular steady-state concentrations that have been estimated in the literature for a few other cell types.