Abstract
Purpose: :
Previously we obtained evidence that in avian corneal epithelial (CE) cells, ferritin is a nuclear component and that ferritin in this location protects DNA from UV-induced damage. As UV irradiation is known to produce reactive oxygen species (ROS), and as ferritin is known to ameliorate such damage by sequestering free iron and thus decreasing the formation of hydroxyl radicals (through the Fenton reaction), we concluded that the protection afforded by nuclear ferritin is, at least in part, through this mechanism. In the present study we have tested this hypothesis further by examining whether nuclear ferritin can prevent damage by the ROS hydrogen peroxide (H2O2).
Methods: :
Primary cultures of CE cells were exposed to H2O2 , and DNA strand breaks were measured by in situ 3’-end labeling (ISEL). For comparison, a closely related epithelial cell type which does not have nuclear ferritin - the skin epithelial (SE) cell - was also tested. To examine whether the damage to DNA involved the iron-mediated Fenton reaction, the concentration of iron in the medium was increased before exposure to H2O2 , and to determine whether the nuclear ferritin was directly responsible for preventing this damage, its production was inhibited before exposure to H2O2 . In addition, to determine whether cytoplasmic ROS scavenger enzymes also participated in this protection of the nuclear DNA from damage, we inhibited the two major scavenger enzymes for H2O2 (i.e., catalase and glutathione peroxidase) before treatment.
Results: :
CE cells, as compared to SE cells, were much more resistant to H2O2 -induced damage. The damage to SE cells, but not CE cells, was exacerbated by increased iron, and the protection of CE cells to damage was lost when the production of nuclear ferritin was inhibited. Also, in the presence of inhibitors of scavenger enzymes, the nuclear DNA of the CE cells showed no detectible increase in damage by H2O2 treatment.
Conclusions: :
These studies suggest that the nuclear ferritin of CE cells can prevent H2O2 damage to nuclear DNA, and one mechanism for this protection is through sequestration of iron, thus inhibiting the formation of Fenton reaction-produced hydroxyl radicals. This may represent a unique mechanism for protecting DNA from oxidative damage, as it appears to act independent of ROS scavenger enzymes.
Keywords: cornea: epithelium • differentiation • cornea: basic science