Does the cellular labile iron pool participate in the oxidation of 2',7'-dichlorodihydrofluorescein?

The fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2',7'-dichlorodihydrofluorescein (H(2)DCF), product of intracellular hydrolysis of H(2)DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H(2)O(2)) in the presence of catalysts. The present study was aimed at answering the question whether the labile iron pool (LIP) may contribute to the oxidation of H(2)DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H(2)O(2) dependent on ferrous ions but not by peroxidase or superoxide dismutase in defined in vitro systems. When applied to cells, the probe inhibited considerably oxidation of H(2)DCF in V79 Chinese hamster fibroblasts and two murine lymphoma L5178Y(LY) sublines (LY-R, LY-S) differing in LIP level, the extent of inhibition being greater in the LY-R line of higher LIP level. These results demonstrate that LIP is a significant factor determining the rate of intracellular H(2)DCF oxidation.