Abstract
Purpose: :
Oxidative stress is generally considered a major etiologic factor in AMD and, therefore, is often assayed following experiments on RPE cells in culture. The dihydro-dichloro-fluorescein diacetate (H2DCF-DA) technique is commonly used for this purpose. The ester H2DCF-DA is intracellularly cleaved, forming H2DCF that in its protonated, but not in its deprotonated, form permeates membranes to a limited degree. Its oxidation into DCF results in green fluorescence, which is usually measured by microplate readers without microscopy. It is assumed that oxidation of H2DCF results from the production of reactive oxygen species in general and that H2DCF is evenly distributed in the cell. Here we show that these assumptions may need to be modified.
Methods: :
Human immortalized ARPE-19 cells, grown on glass coverslips, were exposed for 30 min to 30 µM H2DCF-DA +/- 100 µM of the iron-chelator CP22 and then mounted and studied by confocal scanning and fluorescence microscopy, either directly or following exposure to 100 µM hydrogen peroxide for a few minutes. To visualize mitochondria, cells were separately exposed to tetramethylrhodamine ethyl ester. To prove the iron-dependent oxidation of H2DCF, it was exposed to FeCl3, cysteine and hydrogen peroxide in test tubes.
Results: :
The cells showed a DCF-mediated mitochondrial-type fluorescence that was slightly enhanced, but not changed, by a brief exposure to hydrogen peroxide. Exposure to CP22, slightly depressed the mitochondrial fluorescence. Occasional early apoptotic cells showed bright diffuse green fluorescence, while post-apoptotic necrotic cells presented weak fluorescence. The test tube experiments proved oxidation of H2DCF to be a function of iron catalyzed, Fenton-type reactions.
Conclusions: :
The weak acid H2DCF accumulates in the basic environment of the mitochondrial matrix, where its oxidation and appearing fluorescence indicates formation of hydroxyl radicals and, hence, the presence of redox-active iron (presumably heme, iron-sulphur clusters and some labile CP22-chelatable iron) and hydrogen peroxide. The pronounced, diffuse DCF-induced fluorescence from early apoptotic cells may be explained by opening of permeability transition pores in damaged mitochondria with resulting iron relocation to the cytoplasm. Interestingly, neither the mitochondrial affinity, nor the metal-dependent oxidation of H2DCF seems to be generally recognized. The results of this study emphasize that DCF-induced fluorescence cannot uncritically be interpreted as a general sign of cellular oxidative stress.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • microscopy: light/fluorescence/immunohistochemistry