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FULLEROL CYTOTOXICITY AND PHOTOTOXICITY IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS.
Citation:
ROBERTS, J. E., D. HU, C. F. CHIGNELL, D. S. MILLER, P. BILSKI, N. WALKER, W. K. BOYES, AND A. R. WIELGUS. FULLEROL CYTOTOXICITY AND PHOTOTOXICITY IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS. Presented at NIEHS Summer of Discovery Program, RTP, NC, July 26, 2006.
Description:
Purpose:
Water-soluble fullerenes [nano-C60 ,fullerol] have recently been shown to exhibit antibacterial and antiviral activity including inhibition of HIV protease. In addition to their potential to be used as drugs, they are also being tested as carriers to deliver drugs to the brain. To assess ocular toxicity of these nanoparticles which might limit their use, we determined the cytotoxicity and visible light induced phototoxicity of fullerol in vitro with human retinal pigment epithelial (hRPE) cells.
Methods:
Human RPE cells from donor eyes were incubated in the dark for 22 hours with 1-50 µM fullerol in F-12 medium supplemented with 10% FBS. Cells were washed and placed in Hanks Balanced Salt Solution. Then they were exposed to visible light emitted by 2 Philips, F40 AX50, 5000 K Advantage lamps equipped with a filter which transmitted only wavelengths above 400 nm. The visible light exposures used in our experiments (10.6-12 W/m2) . The light exposures used in the experiments were similar to the daily dose that reaches the human retina in intense sunlight. Cell viability and cytotoxicity were estimated using MTS and LDH assays, respectively. Dose and light dependent changes of TBARS concentration were monitored. Protein content was determined by the BCA method. The potential of fullerol to photoproduce 1O2 was measured by direct detection of its steady state phosphorescence at 1270 nm, achieved by using an Oriel 500 W Hg lamp that was equipped with an interference filter with maximal transmittance at 366nm.
Results:
The LDH assays have shown that fullerol is toxic toward RPE cells in the dark at concentrations of 20-50 µM. When cells preincubated with fullerol are exposed to light (> 400nm) cytotoxicity is increased. Increased TBARS production induced by light correlates to fullerol concentration in hRPE cells. We have also determined that fullerol produces singlet oxygen (¿ = 0.07) at a rate similar to lipfuscin (¿ = 0.09) the endogenous phototoxidizing agent in the human retina.
Conclusions:
Fullerol is both cytotoxic in the dark and phototoxic to hRPE cells at low concentrations and weak visible light. The mechanism of this damage is, at least in part, through singlet oxygen production resulting in lipid peroxidation. Lipid peroxidation is an important risk factor in the induction of retinal and choroidal neovascularization and diabetic retinopathy.