Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species

Dihydrocalcein (H-2-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H-2-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H-2-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5 min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H-2-calcein but increased the oxidation of H-2-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H-2-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H-2-DCF. In contrast, DPI inhibited H-2-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H-2-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H-2-calcein, but not H-2-DCF inhibited complex I activity. These observations indicate that H-2-DCF is an indicator for intracellular ROS, whereas the oxidation of H-2-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.