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A. M. Schimel, L. Abraham, N. Ercal, R. S. Apte; Protection of Retinal Pigment Epithelial Cells From Oxidative Injury by N-Acetylcysteine Amide, a Novel Thiol Antioxidant. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5939.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated oxygen tension, high polyunsaturated lipid content, focused light exposure, and other distinct biochemical visual events create a high oxidative stress environment for the human retinal pigment epithelium (hRPE). While the RPE has considerable antioxidant capability to neutralize this environment, the antioxidant capacity is overwhelmed in a number of retinal diseases including age-related macular degeneration (ARMD), subjecting the RPE to injury and cell death. We investigated whether a novel compound, N-acetylcysteine amide (NACA), protects against oxidative cell death in hRPE cells.
Preliminary experiments were performed to develop dose-response curves based on ARPE-19 cell line incubation in our oxidizing compound, tert-butylhydroperoxide (tBH), in various concentrations over 4 hours, as well as in various concentrations of NACA over 24 hours. Optimal concentrations were established and cells were preincubated in NACA for 24 hours followed by complete removal of NACA and incubation in tBH for 4 hours. Cells were subsequently stained with annexin V and propidium iodide and assessed using flow cytometry to evaluate cell death. Reduced glutathione (GSH) levels were measured on samples using ELISA. All experiments were run in triplicate.
There was no significant ARPE-19 toxicity due to NACA after incubation in all concentrations tested for 24 hours. Incubation in 0.2mM and 0.4mM tBH for 4 hours led to 85.83% and 21.06% cell viability respectively. Exposure to media for 28 hours yielded cell viability of 83.60%. Exposure to media for 24 hours followed by 0.4mM tBH yielded cell viability of 21.06%, while preincubation with 10mM NACA for 24 hours, followed by removal of NACA and subsequent exposure to 0.4mM tBH for 4 hours yielded cell viability of 88.84%. This demonstrated a 322% increase in cell survival with NACA preincubation. Greater than 100% increase in GSH levels were demonstrated in NACA pretreated cells exposed to tBH versus media pretreated cells exposed to tBH.
NACA provides remarkable protection of hRPE cells against oxidative cell death. Because of the considerable evidence supporting the role of oxidative stress in the pathogenesis of ARMD and NACA's superior chemical properties and bioavailability, this compound may be an important agent in the delay or prevention of this debilitating disease.
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