H2DCF Oxidation Seems to be Dependent on Free Iron and Cytochrome C as Studied in ARPE-19 Cells

Abstract

Purpose: : The dihydrodichlorofluorescein diacetate (H₂DCF-DA) technique is often used to assay cellular "oxidative stress". H₂DCF-DA is a non-fluorescent, lipophilic ester that is split by unspecific esterases intracellularly. One of the reaction products is the hydrophilic alcohol H₂DCF that is trapped within the cell where it may be oxidized into fluorescent DCF by a process which is assumed to involve unspecified "reactive oxygen species (ROS)". We have previously demonstrated a weak mitochondrial DCF-fluorescence in normal human ARPE-19 cells, while strong cytosolic fluorescence was found only in cells with apoptotic morphology. Here we point out possible underlying mechanisms for these findings.

Methods: : Human immortalized ARPE-19 cells, grown on coverslips, were exposed to 10 µM H₂DCF-DA for 30 min and then studied by confocal laser scanning microscopy following mounting in Hank’s buffered salt solution (HBSS) or in 100 µM of the lysosomotropic detergent MSDH, which rapidly induces lysosomal membrane permeabilization (LMP). In separate experiments, cells were exposed to the lysosomotropic, metachromatic fluorochrome acridine orange (AO) for 15 min and then mounted in MSDH as above. The dependence of H₂DCF oxidation on free ionic iron and cytochrome c was investigated in test tube experiments.

Results: : Cells that were exposed to H₂DCF-DA showed a weak mitochondrial-like fluorescence pattern, while those exposed to H₂DCF-DA + MSDH displayed a time-dependent strong increase in cytosolic fluorescence that paralleled a decrease in the number of intact AO-containing lysosomes. Test tube experiments proved both ionic iron and cytochrome c to be potent catalysts of H₂DCF-oxidation.

Conclusions: : It appears that LMP is either an upstream or a potentiating event in apoptosis that results in release of redox-active iron and lytic enzymes, which attack mitochondria with ensuing release of cytochrome c. H₂DCF-oxidation seems to rely both on iron-mediated Fenton-type reactions and enzymatic oxidation by cytochrome c in the presence of hydrogen peroxide and is dependent on initial LMP. The weak mitochondrial fluorescence pattern of normal ARPE-19 cells seems to originate in the intermembraneous space of the mitochondria, to which H₂DCF has easy access through the fenestrations of the outer membrane, where it encounters cytochrome c and hydrogen peroxide.

Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium • microscopy: confocal/tunneling