Abstract
Abstract: :
Purpose: RPE are located in a highly oxidative environment suggesting that mitochondrial (mt)DNA repair must be significant. When exposed to light, selective mtDNA damage is observed. Sustained mtDNA damage leads to mitochondrial dysfunction and cell death. Base excision repair is the main pathway used by mammalian cell mitochondria; DNA polymerase γ is involved in this process. The purpose of this study is to determine the effect of oxidative stress in the mitochondrial repair enzyme DNA polymerase γ. Methods: ARPE–19 cells incubated in 10% DMEM–F12 medium were treated with H2O2/TNFα at one hour intervals up to 6h. DNA polymerase γ was localized immunohistochemically. Briefly, cells were fixed with ice–cold methanol, washed in PBS, blocked and incubated with DNA polymerase γ primary antibody. A fluorescent secondary antibody was used and images were obtained by deconvolution microscopy. Linear pixel analysis of cell intensities was performed. Cells were also processed for Western blot analysis and probed for DNA polymerase γ. Results: Immunohistochemistry showed that DNA polymerase γ increased in RPE cells within 1h after treatment with H2O2/TNFα, peaking at 4h and declining to control levels after 6h. The measured fluorescent intensity measured for 4h is increased two fold above controls. This 4h DNA polymerase γ peak was confirmed by Western blot analysis. The rapid appearance and increase in DNA polymerase γ protein suggests activation of an early response gene, and the rapid protein decrease may imply a peroxisome–mediated degradation. Conclusions: RPE are endowed with inducible mtDNA repair. Under normal conditions this event may be important for the preservation of cell integrity and function. However, inadequate or inefficient mtDNA repair due to excessive demand may result in RPE cell death. Oxidative stress–induced mitochondrial DNA damage is a possible mechanism for RPE aging and age–related macular degeneration. Therefore, pharmacological or genetic manipulation of repair genes may contribute to ameliorate retinal degenerations.
Keywords: oxidation/oxidative or free radical damage • neuroprotection • retinal pigment epithelium