What does the commonly used DCF test for oxidative stress really show?

H₂DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate ‘cellular oxidative stress’. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H₂DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2′,7′-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H₂DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H₂DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H₂O₂, directly oxidizes H₂DCF. Consequently, oxidation of H₂DCF requires the presence of either cytochrome c or of both redox-active transition metals and H₂O₂. Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H₂DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H₂O₂, showing that by itself it is unable to oxidize H₂DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.