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

Abstract

The fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2′,7′-dichlorodihydrofluorescein (H₂DCF), product of intracellular hydrolysis of H₂DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H₂O₂) 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 H₂DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H₂O₂ 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 H₂DCF 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 H₂DCF oxidation.

• 2′,7′-dichlorodihydrofluorescein diacetate
• iron chelation
• salicylaldehyde isonicotinoyl hydrazone (SIH)
• labile iron pool

Abbreviations
DMSO	=	dimethyl sulfoxide
HBSS	=	Hank's buffered salt solution
H2DCF-DA	=	2′,7′-dichlorodihydrofluorescein diacetate
HPR	=	horseradish peroxidase
LIP	=	labile iron pool
ROS	=	reactive oxygen species
SIH	=	salicylaldehyde isonicotinoyl hydrazone
SOD	=	superoxide dismutase

Abbreviations
DMSO	=	dimethyl sulfoxide
HBSS	=	Hank's buffered salt solution
H2DCF-DA	=	2′,7′-dichlorodihydrofluorescein diacetate
HPR	=	horseradish peroxidase
LIP	=	labile iron pool
ROS	=	reactive oxygen species
SIH	=	salicylaldehyde isonicotinoyl hydrazone
SOD	=	superoxide dismutase