Mitochondria Represent a Weak Link in the Antioxidant Defenses of the RPE

Abstract

Purpose:: We have previously shown that RPE cells are more resistant to oxidative stress than other cell types (Jarrett et al, Free Radical Res, 2006). Mitochondria, the power house of the cell, account for the bulk of endogenously formed reactive oxygen species (ROS) and represent a major source for cellular damage. We investigated the degree of susceptibility of mitochondria to oxidative damage in RPE cells.

Methods:: Primary RPE cell cultures and the ARPE 19 cell line were exposed to the oxidative stressors tert-butylhydroperoxide (t-BOOH) and hydrogen peroxide (H₂0₂) (0.5 - 5 mM) for periods up to 48 hours. Mitochondrial respiration was determined by the MTT assay and mitochondrial fusion and fission was assessed using tetra-methyl rhodamine ethyl ester redistribution following photobleaching. MnZnSOD activity was determined in isolated mitochondria. Mitochondrial (mtDNA) and the nuclear DNA (nDNA) damage and repair were measured by QPCR. Mitochondrial targeted antioxidants were assessed for their ability to protect against ROS induced damage.

Results:: Mitochondrial respiration in RPE cells was significantly reduced by both t-BOOH and H₂0₂ exposure and this could be reduced by pre-incubation with mitochondrial-targeted antioxidants. Mitochondria showed a dose-dependent defect in fusion/fission following exposure to oxidants, which could be reversed using mitochondrial-directed antioxidants. MnZnSOD activity was significantly reduced in mitochondria isolated from cells stressed by oxidation compared to control treated cells. Mitochondrial DNA sustained a significantly greater extent of oxidant-induced DNA damage compared to the respective nDNA and this could be reduced by mitochondrial-targeted antioxidants. nDNA damage was repaired within 12 hours while mtDNA damage was never completely repaired during the 48 hour period examined.

Conclusions:: The mitochondria of RPE cells are susceptible to oxidative stress and represent a weak link in an otherwise efficient antioxidant and repair system. This vulnerability will contribute to RPE aging and dysfunction and may be prevented by pharmacological intervention with mitochondrial-targeted antioxidants.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • mitochondria