The gaseous signalling molecule, hydrogen sulfide (H₂S) has antioxidant, anti-inflammatory and anti-apoptotic properties. Since oxidative stress has been implicated in the pathogenesis of cataracts and lenticular hydrogen peroxide (H₂O₂) is elevated in some cataract patients, the present study investigated the ability of H₂S-releasing compounds to prevent H₂O₂-induced cataract formation in cultured bovine lenses. Lenses were cultured in either Dulbelcco’s Modified Eagle Medium (DMEM; control); H₂O₂ (50 mM); ascorbic acid (AA; 3 mM) (positive control); and the H₂S-releasing compounds (diallyl trisulfide [DATS] or GYY4137) in the presence of H₂O₂ (50 mM). Lens opacity was determined using a plate reader to measure transmittance. Lens glutathione content (GSH), superoxide dismutase (SOD) activity and lactate dehydrogenase (LDH) cytotoxicity were assessed before and after treatment with the H₂S-releasing compounds. Both DATS (10⁻⁷M - 10⁻⁴M) and GYY4137 (10⁻⁷M - 10⁻⁴M) significantly (p<0.001) attenuated H₂O₂ (50 mM)-induced loss in transmittance, with DATS (10⁻⁴M) and GYY4137 (10⁻⁷M) achieving a maximal reversal of opacity by 56.86±0.01% (n = 6) and 8.39 ± 0.11% (n = 6) after 120 hours, respectively. These observations were corroborated by photographic evaluation, where DATS (10⁻⁵M - 10⁻⁴M) and GYY4137 (10⁻⁷M - 10⁻⁵M)-treated lenses had relatively clear grids after 120 hours, compared to H₂O₂ (50 mM)-treated lenses. The H₂O₂ (50 mM)-induced decline in total GSH content and total SOD activity were significantly (p<0.001; n=5) reversed by DATS (10⁻⁴M) and GYY4137 (10⁻⁷M). After 24 hours, DATS (10⁻⁴M) and GYY4137 (10⁻⁷M) significantly (p<0.001; n=4) reduced cytotoxicity of primary bovine lens epithelial cells by 33.88 ± 4.59% and 36.19 ± 10.53%, respectively. Both H₂S-releasing compounds protected cultured bovine lenses against oxidative stress-induced cataract formation. The slow-releasing H₂S compound, GYY4137 was more potent than DATS in restoring lenticular total GSH content and total SOD activity along with reducing H₂O₂ (50mM)-induced cytotoxicity.

Background: We have evidence that hydrogen sulfide (H₂S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H₂S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H₂S-releasing compounds against hydrogen peroxide (H₂O₂)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H₂S biosynthesis called L-cysteine, with the fast H₂S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H₂S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H₂O₂ (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H₂S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H₂S-releasing compounds. Results: Exposure of the isolated retinas to H₂O₂ produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p < 0.001; n = 12) prevented H₂O₂-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H₂O₂ damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H₂O₂ alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H₂O₂-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H₂S-releasing compounds and the substrate, L-cysteine, can prevent H₂O₂-induced lipid peroxidation in an isolated bovine retina.

Both hydrogen sulfide and endocannabinoids can protect the neural retina from toxic insults under in vitro and in vivo conditions. Purpose: The aim of the present study was two-fold: (a) to examine the neuroprotective action of cannabinoids [methanandamide and 2-arachidonyl glycerol (2-AG)] against hydrogen peroxide (H₂O₂)-induced oxidative damage in the isolated bovine retina and (b) to evaluate the role of endogenously biosynthesized hydrogen sulfide (H₂S) in the inhibitory actions of cannabinoids on the oxidative stress in the bovine retina. Methods: Isolated neural retinas from cows were exposed to oxidative damage using H₂O₂ (100 µM) for 10 min. When used, tissues were pretreated with methanandamide (1 nM–100 nM) and 2-AG (1–10 µM) for 30 min before a 10 min treatment with H₂O₂ (100 µM). In some experiments, retinas were pretreated with inhibitors of the biosynthesis of H₂S [cystathionine β-synthase/cystathionine γ-lyase (CBS/CSE), aminooxyacetic acid, AOAA 30 µM, or 3-mercaptopyruvate sulfurtransferase (3MST), α-keto-butyric acid, KBA 1 mM] and the CB1-receptor antagonist, AM251 (100 nM) for 30 min before treatment with methanandamide (1 nM–100 µM). Enzyme immunoassay measurement of 8-epi PGF2α (8-isoprostane) levels was performed to assess lipid peroxidation in retinal tissues. Results: In the presence of H₂O₂ (100 µM), methanandamide (1 nM–100 µM) and 2-AG (1–10 µM) significantly (p < 0.001) blocked the H₂O₂-induced elevation in 8-isoprostane levels in the isolated bovine retina. In the presence of the CB1 antagonist AM251 (100 nM), the effect of methanandamide (1 nM) on the H₂O₂-induced 8-isoprostane production was significantly (p < 0.001) attenuated. While AOAA (30 µM) had no significant (p > 0.05) effect on the inhibition of H₂O₂-induced oxidative stress elicited by methanandamide, KBA (1 mM) reversed the neuroprotective action of methanandamide. Conclusions: The cannabinoids, methanandamide and 2-AG can prevent H₂O₂-induced oxidative stress in the isolated bovine retina. The neuroprotective actions of cannabinoids are partially dependent upon the activation of the CB1 receptors and endogenous production of H₂S via the 3-MST/CAT pathway.

Background: Hydrogen sulfide (H₂S) is an endogenous transmitter with the potential to regulate aqueous humor dynamics and protect retinal neurons from degeneration. The aim of the present study was two-fold: (a) to evaluate the release of H₂S from two polysulfides, diallyl disulfide (DADS), and diallyl trisulfide (DATS); and (b) to investigate their ocular hypotensive actions in normotensive male and female rabbits in the presence and absence of GSH. Materials and Methods: H₂S was quantified hourly for up to 6 h using a H₂S-Biosensor (World Precision Instruments, Sarasota, Fl). Intraocular pressure (IOP) was assessed in normotensive New Zealand Albino rabbits using a pneumotonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY, USA). Results: In the presence of GSH, there was an increase in the in vitro release of H₂S produced by DADS and DATS. Both DADS and DATS also caused a dose-dependent reduction in IOP in male and female rabbits, in both treated and untreated eyes. For instance, in male animals, the presence of GSH (3% and 5%) significantly (p < 0.05, n = 5) enhanced the ocular hypotensive action of DADS (2%) and DATS (2%) from 14.02 ± 2.89% to 18.67 ± 5.6% and from 16.22 ± 3.48 to 23.62 ± 5.79%, respectively. Conclusions: GSH enhanced both H₂S release and ocular hypotensive action of the polysulfides in a manner that was dependent on the number of sulfur atoms present in each polysulfide. Furthermore, female animals were less sensitive to the IOP-lowering action of the polysulfides, when compared to their male counterparts.

Background: Hydrogen sulfide (H₂S)-releasing compounds can reduce intraocular pressure in normotensive rabbits by increasing aqueous humor (AH) outflow through the trabecular meshwork. In the present study, we investigated the contribution of endogenous H₂S and the role of intramurally generated prostaglandins in the observed increase in AH outflow facility in an ex vivo porcine ocular anterior segment model. Material and Methods: Porcine ocular anterior segment explants were perfused with Dulbecco’s Modified Eagle’s Medium maintained at 37 °C and gassed with 5% CO₂ and 95% air under an elevated pressure of 15 mmHg for four hours. Perfusates from the anterior segment explants were collected and immediately assayed for their H₂S and prostaglandin E₂ content. Results: Elevating perfusion pressure from 7.35 to 15 mm Hg significantly (p < 0.001) increased H₂S concentration in the perfusate from 0.4 ± 0.1 to 67.6 ± 3.6 nM/µg protein. In the presence of an inhibitor of cystathionine ß-synthase/cystathionine γ-lyase, aminooxyacetic acid (AOAA, 30 µM), or an inhibitor of 3-mercaptopyruvate sulfurtransferase, α-ketobutyric acid (KBA, 1 mM), the effects of elevated pressure on H₂S levels in the perfusate was significant (p < 0.001). Furthermore, flurbiprofen (30 µM) and indomethacin (10 µM) attenuated the elevated pressure-induced increase in H₂S levels in the perfusate. Interestingly, elevating perfusion pressure had no significant effect on PGE₂ concentrations in the perfusate. While the inhibition of H₂S biosynthesis by AOAA or KBA did not affect PGE₂ levels in perfusate, flurbiprofen (30 µM) caused a significant (p < 0.05) decrease in the concentration of PGE₂ under conditions of elevated perfusion pressure. Conclusions: We conclude that the elevated perfusion pressure-induced increase in H₂S concentrations depends upon the endogenous biosynthesis of H₂S and intramurally produced prostaglandins in the porcine anterior segment explants. While the concentration of PGE₂ in the perfusate under elevated perfusion pressure was unaffected by pretreatment with inhibitors of H₂S biosynthesis, it was reduced in the presence of an inhibitor of cyclooxygenase.