Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis

Abstract

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.

The role of lysosomes in iron metabolism and recycling

“…Under normal conditions, there are no indications of any significant amount of low mass redox-active iron that is in juxtaposition to DNA (Doulias et al, 2003, Kurz et al, 2004, Tenopoulou et al, 2005. However, as was recently demonstrated, under conditions of oxidative stress lysosomal rupture will occur, iron will be relocated, and DNA damage initiated (Doulias et al, 2003, Kurz et al, 2004, Persson et al, 2005, Tenopoulou et al, 2005.…”

“…When stored iron is needed for synthesis of iron-containing structures, it has to be released from ferritin. Recent research has shown that the major mechanism for this iron release involves lysosomal digestion (Bridges and Hoffman, 1986, Garner et al, 1998, Kidane et al, 2006, Konijn et al, 1999, Kurz et al, 2011, Kwok and Richardson, 2004, Persson et al, 2003, Radisky and Kaplan, 1998, Roberts and Bomford, 1988, Sakaida et al, 1990, Tenopoulou et al, 2005, Vaisman et al, 1997, Yu et al, 2003b, Zhang et al, 2010. Iron is then relocated to the cytoplasm, probably by the participation of DMT1.…”

Vitamin C modulation of H2O2-induced damage and iron homeostasis in human cells

“…Under oxidative stress conditions this labile ferrous iron may cause lipid peroxidation [62], destabilising the lysosomal membrane and diffusing into the nucleus, where it can take part in Fenton reactions. Whilst DFO could protect cells against H2O2-induced cell damage by keeping lysosomal iron in a non-reactive form [58,63], it is possible that AA, at low pH, would promote endosomal/lysosomal membrane destabilization and the concomitant release of low molecular weight iron chelates into the cytosol.…”

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

“…One hypothesis postulates it to be liberated directly in the cytosol under the influence of a feedback mechanism [10,29,30]. Another hypothesis that recently has gained strong support suggests a lysosomal pathway involving ferritin autophagy followed by degradation inside the lysosomal compartment and transfer of the liberated iron to the cytosol [8,[31][32][33][34][35][36][37][38][39][40][41][42]. Finally, ferritin degradation by proteasomes has been suggested, as well as a combination of lysosomal and proteasomal degradation [10,42,43].…”

“…[8,10,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]72]). To further analyze the possible change in autophagy mediated by iron-starvation, we exposed cells to the lipophilic ester calcein-AM, which, after passage through the plasma membrane, is cleaved by cytosolic esterases to form the fluorochromic, hydrophilic alcohol calcein, which is an iron-chelator that is widely used to assay cytosolic labile iron [70].…”