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V. Vasiliou, A. Pappa, W.J. Black, J.V. Jester, B.J. Day, E. Min, N. Lassen; The Corneal Crystallin ALDH3A1 Is a Multifunctional Protein That Protects Cells Against Oxidative Damage . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4926.
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© ARVO (1962-2015); The Authors (2016-present)
Aldehyde dehydrogenase 3A1 (ALDH3A1) is expressed at high concentrations in the mammalian cornea and is found both in epithelial cells and stromal keratocytes. We have recently shown that ALDH3A1 protects corneal epithelial cells against apoptosis induced by either UV light, 4–hydroxy–2–nonenal–(4–HNE) or DNA damaging agents. The aim of the present study was to elucidate the mechanisms by which ALDH3A1 protects against oxidative damage.
A rabbit corneal myofibroblastic cell line (TRK43), which lacks endogenous ALDH3A1 protein, was stably transfected with the human ALDH3A1 cDNA and subjected to oxidative stress induced by either H2O2 or the DNA damaging agents mitomycin C (MMC) and etoposide (VP–16). The apoptotic effects of MMC, VP–16, and H2O2 were evaluated by DNA fragmentation and Western blot analysis in both vector– and ALDH3A1–transfected cells. The levels of glutathione (GSH) were assessed by spectrophotometric and HPLC analysis. Protein oxidation was determined by the detection of carbonylated proteins using Western blot analysis.
DNA fragmentation assays revealed that H2O2, MMC, and VP–16 treatment induced apoptosis in vector–transfected cells but not in ALDH3A1–expressing cells. Apoptosis was associated with increased levels of 4–HNE–protein adducts, which were almost undetectable in ALDH3A1–expressing cells. Treatment with H2O2 resulted in a rise of reduced GSH levels, which was more pronounced in the ALDH3A1–expressing cells. Treatment with the DNA damaging agents caused GSH depletion in both vector– and ALDH3A1–transfected cells. However, the decrease was significantly less in ALDH3A1–expressing cells compared to vector–transfected cells. Increased oxidation of ALDH3A1 was found in the treated cells that were not associated with loss of the enzymatic activity.
These data suggest that ALDH3A1 is a multifunctional protein that protects corneal cells against cellular oxidative damage by metabolizing toxic products of lipid peroxidation such as 4–HNE, maintaining cellular GSH levels and redox balance, and by acting as antioxidant.
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