ALDH3A1 Prevents Apoptosis of Corneal Cells Induced by DNA Damaging Agents and Oxidative Stress

Abstract

Abstract: : Purpose: We have previously shown that ALDH3A1 protects human corneal epithelial cells against UV– and 4–hydroxynonenal–induced oxidative damage. The aim of this study was to determine whether ALDH3A1 can protect against cellular oxidative damage induced by DNA damaging agents (mitomycin C and VP–16) and oxidative stress (H₂O₂). Methods: Human corneal epithelial and rabbit corneal keratocyte cell lines lacking endogenous enzyme were stably transfected with the human ALDH3A1. Transfected cells were characterized for ALDH3A1 expression by Western blot analysis and enzymatic activity. The cytotoxic effects of mitomycin C, VP–16, and H₂O₂ were evaluated by MTT, 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 as an indicator of oxidative stress. Results: ALDH3A1–trasfected cells were found to be 3–fold more resistant to the cytotoxic effects of all three agents compared to the vector–transfected cells tested, as determined by MTT assays. In addition, DNA fragmentation assays revealed that treatment with either mitomycin C (50 µg/ml), VP–16 (100 µM), or H₂O₂ (500 µM) induced apoptosis in vector–transfected cells, but not in ALDH3A1–expressing cells. It was also found that ALDH3A1 expression was not affected and prevented 4–hydroxynonenal–protein adduct formation following 16 hrs treatment with mitomycin C, VP–16, and H₂O₂. However, total GSH levels were slightly depleted in both mock–transfected and ALDH3A1–expressing cells. Conclusions: These data support our long standing hypothesis about the protective role of ALDH3A1 against oxidative damage.

Keywords: cornea: epithelium • cornea: stroma and keratocytes • apoptosis/cell death