Abstract
Purpose: :
Visible light induces damage in ocular tissue through photo–oxidative stress. In these experiments, we assessed the photo–oxidative stress induced by broadband light as a function of wavelength. Further, we compared the protective actions of anti–oxidants and melanosomes against photo–oxidative stress.
Methods: :
Human lens epithelial cells were grown under standard conditions in 96–well plates. Broad– or narrow–band 5’ exposures to the cells were made with a Xenon arc lamp (0.3 W/cm2), with or without a band pass filter with transmission peak at 400nm, a longpass filter with a cutoff at 420nm, and various neutral density filters. Oxidative stress was measured with the fluorescent indicator, DCFH. The protective actions of 20 mM N–acetylcysteine (NAC), 500 µM ascorbic acid (Asc), and exogenous bovine RPE melanosomes, incorporated into the cells, were tested. A SPEX plate reader measured the DCF fluorescence in the cells following light exposure.
Results: :
For exposures matched in time and irradiance, wavelengths <400nm produced significantly more intracellular oxidative stress than did wavelengths >420nm (p<.001). The combination of these wavelength bands produced slightly more oxidation than did each band alone (difference not significant, p>.05). Melanosomes conferred a protective effect against broadband light (p =0.003). With wavelengths <400 nm, NAC and Asc each significantly reduced oxidative stress compared to melanosomes alone (NAC: p<.001; Asc: p<.001); combining the antioxidants with melanosomes enhanced their protective activity. With wavelengths >420 nm, melanosomes were significantly more protective than either NAC or Asc (both: p<0.05), while in combination, melanosomes enhanced the effect of NAC (p<0.05) but not Asc (p>0.05).
Conclusions: :
Broadband light induces photo–oxidative stress in lens epithelial cells; this stress is ameliorated by anti–oxidants. The results support (1) the putative involvement of chronic, broadband light exposure in accelerated aging and degeneration, and (2) the utility of developing effective antioxidants for preventing these processes in ocular tissues.
Keywords: radiation damage: light/UV • oxidation/oxidative or free radical damage • antioxidants