Abstract
Purpose: :
To determine potential ocular toxicity of [(γ-CyD)2/C60] in vitro in human lens epithelial cells.
Methods: :
γ-Cyclodextrin bicapped C60 [(γ-CyD)2/C60] (γ-CyD)2/fullerene) was prepared by the method of Yoshida, et al. Human epithelial lens cells (cell line HLE B-3) were incubated in the dark with 0.125-2 µM (γ-CyD)2/fullerene for 2 hours. Accumulation of (γ-CyD)2/fullerene in the cells was determined spectrophotometrically from its absorption spectra at 349 nm. After incubation, cells were washed, overlaid with HBSS, and irradiated with 15 J cm-2 UVA (UV Houvalite F20T12/BL/HO PUVA) with cut-off filter to remove all radiation below 300 nm or 5.4 J cm-2 visible light emitted by 2 lamps (Philips, F40AX50 5000 K Advantage, 40 W) equipped with a filter which transmitted only wavelengths above 400 nm. Cell viability was determined using the MTS assay. Apoptosis was assessed by flow cytometry following double staining with Annexin V-FITC / propidium iodide, to quantify apoptosis and necrosis induced by (γ-CyD)2/fullerene. Intracellular singlet oxygen induced protein peroxides were assayed using a modified FOX assay as described by Wright et al. In selected experiments, cells were pretreated the singlet oxygen quencher NaN3 (10 mM)) prior to irradiation. Singlet Oxygen production was measured by its phosphorescence emission spectra using a steady-state singlet oxygen spectrophotometer.
Results: :
No obvious effect on viability, apoptosis or the formation singlet oxygen peroxides was observed when lens cells were exposed to up to 2 µM (γ-CyD)2/fullerene in the dark or in the presence of visible light. However, UVA irradiation in the presence of up to 2 µM (γ-CyD)2/fullerene caused a concentration-dependent loss of mitochondrial activity, increase in apoptosis and formation of singlet oxygen-mediated protein peroxides, which were dramatically decreased when the singlet oxygen quencher NaN3 (10 mM) was added during irradiation, The production of singlet oxygen by (γ-CyD)2/fullerene was found to be as efficient (quantum yield in D2O was 0.76) as most PDT photosensitizers.
Conclusions: :
(γ-CyD)2/fullerene is highly phototoxic to HLE B-3 cells upon UVA irradiation. Although (γ-CyD)2/fullerene could potentially be used in photodynamic therapy, without further testing it may not be appropriate for clinical applications to treat normal cells.
Keywords: radiation damage: light/UV • oxidation/oxidative or free radical damage • photodynamic therapy