Effect of UVA Light on Antioxidant Enzyme mRNA Levels in Human Lens Epithelial Cells Cultured at 3% and 20% O2

Abstract

Abstract: : 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.

Keywords: antioxidants • stress response • radiation damage: light/UV