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V.A. Padgaonkar, V.R. Leverenz, A.V. Bhat, S.E. Pelliccia, F.J. Giblin; A Normal Level of GSH Is Important for Defending Human Lens Epithelial Cells Against UVA Radiation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):842.
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Purpose:Substantial amounts of UVA light can reach the human lens epithelium where it can generate reactive oxygen species. Here we have investigated the role of GSH in protecting cultured human lens epithelial cells (LECs) against UVA–induced damage. Methods: Plates of 5 x 105 human LECs (SRA 01/04) were used in the study. The cells were exposed to a physiologically relevant level of UVA light, 7mW/cm2 (338–400nm wavelength, peak at 365nm), for 1h at 36oC, in either PBS plus 5mM glucose or MEM (without serum), at either 3% O2 (physiological) or 20% O2 (non–physiological, but typically used for LEC culture). In certain experiments, the cells were depleted of GSH by 60% by a 10 min pretreatment with 0.02mM CDNB (1–chloro–2, 4–dinitrobenzene). Activities of glutathione reductase (GR), catalase, glutathione peroxidase (GSHPx) and thioredoxin reductase (TrxR) were assayed by standard techniques. Real Time RT–PCR was used to analyze upregulation of mRNA message for various antioxidant enzymes. Changes in morphology were evaluated by light microscopy. Results:Normal HLE cells treated with 7mW/cm2 of UVA light for 1h, at either 3% or 20% O2, in either PBS or MEM, showed no subsequent change in morphology or cell death. Although catalase was 70% inactivated by the UVA light, there were no effects on the activities of GR, GSHPx or TrxR. Significant upregulation of message was seen at 8h for heme oxygenase–1(HO–1), TrxR and MnSOD at 3% O2, and a greater effect was observed at 20% O2. GSH–depleted cells treated with UVA light at 3% O2 in PBS showed significant effects on morphology, as well as 20% cell death and a 35% decrease in TrxR activity, compared to cells treated only with CDNB. No effects on the activities of GR or GSHPx were seen in these cells. Conducting the UVA exposure in 20% O2 increased all of the effects, and also caused a 25% loss of GSHPx activity. Upregulation of message for HO–1 was 6–8 fold higher in GSH–depleted cells exposed to UVA light, compared to normal cells treated identically, at 3% and 20% O2 levels. Use of MEM instead of PBS during the UVA exposure of GSH–depleted cells produced major cell death at either 3% or 20% O2. These damaging effects could be partially prevented with use of the iron–chelator deferoxamine. Conclusions: A normal level of GSH is necessary for human LECs to defend against a 7mW/cm2 level of UVA light in either 3% or 20% O2. When challenging LECs with UVA light, consideration must be given to both the level of O2, as well as the type of culture medium, that are used.
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