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V.A. Padgaonkar, V.R. Leverenz, S.E. Pelliccia, F.J. Giblin; Effect of UVA Light on Antioxidant Enzyme mRNA Levels in Human Lens Epithelial Cells Cultured at 3% and 20% O2 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):300.
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Purpose: To investigate antioxidant gene expression in cultured immortalized human lens epithelial (HLE) cells following exposure to UVA radiation at two different O2 levels, 20% and a physiological level of 3%. Methods: Plates of 4-5 x 105 HLE cells (SRA01/04) were exposed to 4-7 mW/cm2 of UVA light (338- 400nm wavelength, peak at 365nm ) for one hour in PBS plus 5mM glucose at 3% or 20% O2 levels. Following UVA exposure, the cells were cultured normally in MEM containing 15% fetal bovine serum for different intervals of time. Control cells were also cultured normally. Total cellular RNA was isolated with TRIZOL (Gibco). Real Time RT-PCR with a QuantiTect SYBR Green RT-PCR kit (Qiagen) was used to evaluate changes in gene expression of various antioxidant enzymes. Cell viability was measured using a Coulter counter. Results: Exposure of the cells to UVA radiation at either 3% or 20% O2, followed by a post-radiation recovery period, increased the message for the antioxidant enzymes heme oxygenase-1 (HO-1), thioredoxin reductase (TR) and manganese superoxide dismutase (MnSOD) in the HLE cells. The response was time-dependent as well as dose-dependent. A maximum accumulation of transcript was observed at 4-6h of the post-radiation period, and this returned to control levels by 16h. Levels of increased expression at 3% O2 /4-7mW/cm2 were 7-10x (HO-1), 3-5x (TR) and 2-4x(MnSOD), compared to controls. There was no significant difference in antioxidant gene expression between the 20% and 3% O2 levels at a 4h recovery period. At 20% O2, in contrast to 3% O2 , significant cell death was observed. 20-25% cell death occurred at 20% O2 /4mw/cm2 UVA ,compared to less than 10% cell death observed at 3% O2 /4-7mW/cm2. A 6mW/cm2,or higher, UVA radiation at 20% O2 caused 90% cell death. Preliminary results indicate that a failure of TR to be induced by 6mW/cm2, or higher, UVA radiation at 20% O2 may be linked with the death of the cells. Conclusions: HLE cells avoid UVA-induced cell death at a physiological level of 3% O2 by up-regulating mRNA levels of vital antioxidant enzymes such as HO-1, TR and MnSOD. UVA light is significantly more damaging to HLE cells at 20% O2, compared to 3%. The exact cause of UVA-induced cell death at a level of 20% O2 is yet to be determined, but appears to be associated with insufficient TR induction.
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