S-nitrosoglutathione photolysis as a novel therapy for antifibrosis in filtration surgery

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) fibrobla sts and to compare this effect with that produced by the established photod ynamic porphyrin precursor 5-aminolevulinic acid (ALA). Methods. V79-4 Chinese hamster lung and human TC fibroblasts were establish ed in tissue culture. GSNO, together with its radioactive tritiated and flu orescent dansylated derivatives, were synthesized. The labeled molecules we re prepared to determine the time course of uptake into the fibroblasts. Up take was monitored by scintillation counting for the tritiated GSNO and con focal fluorescence microscopy for the dansylated GSNO. The uptake of ALA an d biosynthesis of its photosensitive product were determined by fluorescenc e emission spectroscopy of a separate set of fibroblasts. Once uptake was e stablished, both cell lines were incubated with varying concentrations of G SNO 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 dist ance 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 det ermined 24 hours later by measuring cell viability. Results. A 2-minute drug treatment time (0 hours incubation) with GSNO, fol lowed by 10 minutes of irradiation, resulted in approximately 78% of fibrob last 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 statis tically 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 approximatel y 4% of cells at the lowest concentration and drug treatment time tested. A t drug treatment times of 4 hours and less, increased concentrations of ALA did not produce cell death of more statistical significance. It was not un til 24 hours of drug treatment that comparable amounts of cell death were p roduced by ALA and GSNO. In all experiments similar results were obtained w ith 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 condi tions tested. Conclusions. GSNO is able to cause light-specific cell death of human TC fi broblasts at drug treatment times (2 minutes) and irradiation times (10 min utes) that would be compatible with its use in glaucoma filtering surgery. This in vitro performance was superior to that of the well-established phot osensitizer ALA, which required treatment times longer than 4 hours to appr oach the light-specific cell death produced by only 2 minutes of GSNO treat ment.