Imaging of hydrogen peroxide generation in cultured cells using dichlorofluorescein derivatives

There is growing evidence that generation of reactive oxygen molecules (e.g., hydrogen peroxide, superoxide, hydroxyl and nitric oxide radicals) plays an important role in cell death. In this report we evaluated the effectiveness of the membrane permeable probe carboxy- dichlorodihydrofluorescein diacetate acetoxymethyl ester (C-DCDHF-DA-AM) for imaging the production of H ₂O ₂ in cultured cells. We examined the properties of three derivatives of the ester in saline droplets and compared the results with responses recorded from cells loaded with the ester. Results indicated that fluorescence was generated in cells and droplets by a photo-oxidative process involving H ₂O ₂. Videomicroscopy demonstrated that the cellular responses originated in small vesicles (presumably peroxisomes), with large responses filling the cytosol and enveloping the nucleus. We interpreted these responses as due to light-induced activation of flavin-containing oxidases, which generate H ₂O ₂ in peroxisomes, followed by diffusion of H ₂O ₂ throughout the cell. Escape of H ₂O ₂ from peroxisomes into Fe ²⁺-containing compartments could have dire consequences on cell viability due to the production of hydroxyl free radicals. Such a mechanism could underlie the phototoxic effects of visible light on cultured cells.