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Abstract #5881 Published in IGR 2-1
S-nitrosoglutathione photolysis as a novel therapy for antifibrosis in filtration surgery
Tannous M; Hutnik CML; Tingey DP; Mutus BInvestigative Ophthalmology and Visual Science 2000; 41: 749-755
PURPOSE: To determine whether a novel peroxynitrite-based photosensitizer S-nitrosoglutathione (GSNO) can produce specific in vitro light-induced cell death of both standard animal lung and human Tenon's capsule (TC) fibroblasts and to compare this effect with that produced by the established photodynamic porphyrin precursor 5-aminolevulinic acid (ALA). METHODS: V79-4 Chinese hamster lung and human TC fibroblasts were established in tissue culture. GSNO, together with its radioactive tritiated and fluorescent dansylated derivatives, were synthesized. The labelled molecules were prepared to determine the time course of uptake into the fibroblasts. Uptake was monitored by scintillation counting for the tritiated GSNO and confocal fluorescence microscopy for the dansylated GSNO. The uptake of ALA and biosynthesis of its photosensitive product were determined by fluorescence emission spectroscopy of a separate set of fibroblasts. Once uptake was established, both cell lines were incubated with varying concentrations of GSNO or ALA as a function of time (0, 4, or 24 hours) before light exposure (200 msec pulsed visible light, 0.068 W per pulse, for ten minutes at a distance of 10 cm). After ten minutes of irradiation, the cells were washed and exposed to fresh tissue culture medium. The effect of the treatment was determined 24 hours later by measuring cell viability. RESULTS: A two-minute drug treatment time (0 hours' incubation) with GSNO, followed by ten minutes of irradiation, resulted in approximately 78% of fibroblast cell death at the lowest concentration of GSNO used compared with the control, which was exposed to light, but no GSNO. The higher concentrations of GSNO, or longer drug treatment times before irradiation, did not statistically increase cell death. Maximal cell death was thus obtained using the lowest GSNO concentration (50 mM) and drug treatment time (two minutes). In contrast, the well-established photosensitizer ALA killed only approximately 4% of cells at the lowest concentration and drug treatment time tested. At drug treatment times of four hours and less, increased concentrations of ALA did not produce cell death of more statistical significance. It was not until 24 hours of drug treatment that comparable amounts of cell death were produced by ALA and GSNO. In all experiments, similar results were obtained with the animal lung and human TC fibroblasts, suggesting that the source of the fibroblast had no effect on the outcome. The differences in treatment effects between GSNO and ALA were statistically significant under all conditions tested. CONCLUSIONS: GSNO is able to cause light-specific cell death of human TC fibroblasts at drug treatment times (two minutes) and irradiation times (ten minutes) that would be compatible with its use in glaucoma filtering surgery. This in vitro performance was superior to that of the well-established photosensitizer ALA, which required treatment times longer than four hours to approach the light-specific cell death produced by only two minutes of GSNO treatment.
Dr. B. Mutus, Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset, Windsor, Ontario N9B 3P4, Canada
Classification:
12.8.10 Woundhealing antifibrosis (Part of: 12 Surgical treatment > 12.8 Filtering surgery)
