Direct oxidation of 2′,7′-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production

Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2′,7′-dichlorodihydrofluorescein (DCFH₂), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH₂-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH₂ to DCF without an ROS intermediate. Incubation of DCFH ₂ with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2′,7′-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH₂ with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.