Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis.
Jurkat cells in culture were exposed to oxidative stress in the form of continuously generated hydrogen peroxide, obtained by the addition of glucose oxidase to the medium. This treatment induced a rapid, dose-dependent increase in the ICIP (intracellular calcein-chelatable iron pool). Early destabilization of lysosomal membranes and subsequent nuclear DNA strand breaks were also observed, as evaluated by the Acridine Orange relocation test and the comet assay respectively. Somewhat later, these effects were followed by a lowered mitochondrial membrane potential, with release of cytochrome c and apoptosis-inducing factor. These events were all prevented if cells were pretreated with the potent iron chelator DFO (desferrioxamine) for a period of time (2-3 h) long enough to allow the drug to reach the lysosomal compartment following fluid-phase endocytosis. The hydrophilic calcein, a cleavage product of calcein acetoxymethyl ester following the action of cytosolic esterases, obviously does not penetrate intact lysosomal membranes, thus explaining why ICIP increased dramatically following lysosomal rupture. The rapid decrease in ICIP after addition of DFO to the medium suggests draining of cytosolic iron to the medium, rather than penetration of DFO through the plasma membrane. Most importantly, these observations directly connect oxidative stress and resultant DNA damage with lysosomal rupture and the release of redox-active iron into the cytosol and, apparently, the nucleus.
Tenopoulou
Margarita
M
Laboratory of Biological Chemistry, University of Ioannina Medical School, 451 10 Ioannina, Greece.
Doulias
Paschalis-Thomas
PT
Barbouti
Alexandra
A
Brunk
Ulf
U
Galaris
Dimitrios
D
D017209:Apoptosis / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D004249:DNA Damage / Q000502:physiology*; D003676:Deferoxamine / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000502:physiology*; D007502:Iron Chelating Agents / Q000494:pharmacology*; D019169:Jurkat Cells; D008247:Lysosomes / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000502:physiology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D013696:Temperature; D013995:Time
D005452:Fluoresceins; D007502:Iron Chelating Agents; D006861:Hydrogen Peroxide; D007501:Iron; D003676:Deferoxamine; C007740:fluorexon