Abstract
Purpose: :
To determine whether ascorbate (vitamin C, AscH) can affect the susceptibility of retinal pigment epithelial cells in culture to light–induced toxicity mediated by all–trans–retinal (RAL) and, if so, to elucidate the mechanism responsible.
Methods: :
Photooxidative damage to ARPE–19 cells in culture was induced by irradiation with visible light in the presence of liposomes containing RAL and the addition of increasing concentrations of AscH (0–2 mM). To investigate effects of singlet oxygen quenchers and free radical scavengers, zeaxanthin (ZEA) and/or vitamin E (TOH) were added to liposomes. Cell viability was determined by assays of plasma membrane impermeability to propidium iodite and mitochondrial activity. Direct electron spin resonance spectroscopy was used to monitor light–induced kinetics of ascorbyl radical accumulation during irradiation of liposomal suspension in the presence of AscH. Loss of unsaturated lipids and iodometric assay of lipid hydroperoxides were used to monitor lipid peroxidation. Laser flash photolysis combined with transient absorption detection were used to investigate interactions of AscH with photoexcited RAL. Time resolved detection of singlet oxygen phosphorescence was used to determine the effect of ascorbate on quantum yields of singlet oxygen generation by photoexcited RAL.
Results: :
AscH at concentrations below 0.5 mM proved protective against RAL–mediated phototoxicity. Irradiation of cells in the presence of RAL and AscH at concentrations above 0.5 mM led to greater toxicity than in the absence of AscH. ZEA and TOH partly counteracted the toxic effects of ascorbate and led to inhibition of lipid peroxidation. Photoexcitation of RAL in the presence of AscH resulted in a shortening of excited RAL triplet state lifetime, decreased quantum yields of singlet oxygen generation and a formation of RAL anion radical and ascorbyl radical.
Conclusions: :
AscH can lead to an increase of phototoxic effects of RAL via shifting the mechanism of photooxidative damage from singlet oxygen–mediated to free radical–mediated. It can be speculated that there is a risk that high concentration of AscH in photoreceptor layer of the retina can exacerbate phototoxicity of RAL accumulated in photoreceptor outer segments after photobleaching of the visual pigment.
Keywords: oxidation/oxidative or free radical damage • radiation damage: light/UV • retinal pigment epithelium