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J. E. Roberts, A. Wielgus, U. Andley, C. F. Chignell; Phototoxicity and Cytotoxicity of Fullerol in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2423.
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© ARVO (1962-2015); The Authors (2016-present)
To determine potential ocular toxicity and phototoxicity of hydroxylated fullerene [nano- C60(OH)24, fullerol] by measuring its cytotoxicity and its UVA and visible light induced phototoxicity of fullerol in vitro with human lens epithelial cells.
Human epithelial lens cells (cell line HLE B-3) were incubated in the dark with 1-50 µM fullerol for 16 hours. Uptake of fullerol was confirmed by the yellow color that was apparent in intact rat lenses after incubation with the drug. Accumulation of nano- C60(OH)24 in the cells was determined spectrophotometrically. After incubation with fullerol, cells were washed, overlaid with HBSS, and irradiated with 3.7 J cm-2 UVA (UV Houvalite F20T12/BL/HO PUVA) with cut-off filter to remove all radiation below 300 nm or 8.5 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 estimated using MTS and LDH assays. Damage to all irradiated and dark control cells was assessed by flow cytometry in combination with Annexin V-FITC / propidium iodide double staining, to identify apoptosis and necrosis induced by fullerol. In selected experiments, cells were pretreated with lutein (20 µM) or N-acetyl cysteine (1 mM) or L-ascorbic acid (1 mM) at 37oC for 18 h prior to irradiation.
Fullerol was taken up by the rats’ intact lenses and human lens epithelial cells. Fullerol was cytotoxic toward HLE cells maintained in the dark at concentrations higher than 20 µM. With exposure to either UVA or visible light, fullerol toxicity as determined by MTS and LDH assays was detected at much lower concentraions (>5 µM). Fullerol induced both apoptosis and necrosis in the cells, both in the dark and when irradiated. Although N-acetyl cysteine or L-ascorbic acid, were not effective, lutein offered some protection against photoinduced fullerol damage.
Fullerol is both cytotoxic and phototoxic to human lens epithelial cells. The ocular phototoxicity is in part due to singlet oxygen phototoxidation. Although acute toxicity of water soluble nano-C60 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used in the future to deliver drugs to the eye, they should be further tested for photo- and cytotoxicity.
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