Abstract
Purpose::
4-Hydroxynonenal (HNE), a metastable lipid peroxidation product, is highly toxic to various cell types if not detoxified. Because of its constant exposure to light, the ocular lens continuously generates reactive oxygen species which, under conditions of oxidative stress, may lead to formation of excessive lipid peroxidation products. We have shown that HNE is generated in abundance in the lens epithelium under conditions of oxidative stress and that oxidation of HNE by aldehydes dehydrogenase A1 (ALDH1A1) is a major detoxification route of this lipid aldehyde. We have therefore overexpressed ALDH1A1 in human lens epithelial cells (HLECs) to investigate the resistance of these cells against oxidation-induced toxicity.
Methods::
Immortalized HLECs were either obtained from ATCC or were a generous gift from Dr. Usha Andley, Washington University, St. Louis, MO. The cells were transfected with either the empty expression vector, pcDNA3.1 or ALDH1A1 vector (pDrive-hALDH1-γEF) using lipofectamine as the transfection reagent. Expression of ALDH1A1 was verified by Western blot analysis as well as by measuring the oxidation of 3HHNE. The cells were exposed to oxidative stress (250 µM H2O2 or 50 mM glucose) for 16 hrs to evaluate the resistance of ALDH1A1-transfected HLECs against oxidative stress. Cell viability was determined by trypan exclusion. Oxidative stress-induced formation of Protein-HNE , induction of apoptosis and expression of ALDH1A1 was evaluated by immunohistochemistry.
Results::
As judged by Western blots, HLECs transfected with ALDH1A1 showed approximately ten-fold increase in the expression of ALDH1A1. These cells also oxidized 3HHNE to a greater extent as compared to the control cells. Overexpression of ALDH1A1 was also associated with increased resistance of HLECs against oxidative damage, including apoptosis.
Conclusions::
The results suggest that under oxidative stress, HNE produced in the lens epithelium can cause toxicity and thus contribute to oxidation-induced cataractogenesis and that ALDH1A1 is a critical enzyme in maintaining the lens clarity. Specific activators of this enzyme may therefore be anticataractogenic.
Keywords: cataract • oxidation/oxidative or free radical damage • enzymes/enzyme inhibitors