Pitfalls in the use of common luminescent probes for oxidative and nitrosative stress

Lucigenin (LC2+, bis-N-methylacridinium) and 2',7' -dichlorofluorescin (DCFH,) are widely used as chemiluminescent or fluorescent probes for cellular oxidative stress, to reflect levels of superoxide (O-2(-)) and hydrogen peroxide, respectively. We report mechanistic studies that add to the growing evidence for the unsuitability of either probe except in very well-defined circumstances. The ability for lucigenin to generate superoxide via reduction of LC2+, to LC+ and redox cycling with oxygen depends on the reduction potential of the LC2+/LC+ couple. Redox equilibrium between LC+ and the redox indicator benzyl viologen is established in microseconds after generation of the radicals by pulse radiolysis and indicated E(LC2+/LC+) similar to -0.28 V vs. NHE. Reaction of LC+ with O-2 to generate O-2(-) was also observed directly similarly, occurring in milliseconds, with a rate constant k similar to 3 x 10(6) M-1 s(-1). Quinones act as redox mediators in LC+/O-2 redox cycling. Oxidation of DCFH2 to fluorescent DCF is not achieved by O-2(-) or H2O2, but NO2- reacts rapidly: k similar to 1 x 10(7) M-1 s(-1). Oxidation by H2O2 requires a catalyst: cytochrome c (released into the cytosol in apoptosis) is very effective (even 10 nM), Fluorescence reflects catalyst level as much as O-2(-) production.