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S-Nitrosoglutathione Photolysis as a Novel Therapy for Antifibrosis in Filtration Surgery

¹ From the Department of Chemistry and Biochemistry, University of Windsor, Ontario; and the ² London Health Sciences Centre, Department of Ophthalmology, Victoria Campus, London, Ontario, Canada.

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 labeled 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 10 minutes at a distance of 10 cm). After 10 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 2-minute drug treatment time (0 hours incubation) with GSNO, followed by 10 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 (2 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 4 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 (2 minutes) and irradiation times (10 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 4 hours to approach the light-specific cell death produced by only 2 minutes of GSNO treatment.