Abstract
Purpose: :
Considerable evidence supports the role of oxidative stress-induced cell damage and lipid peroxidation in the pathogenesis of significant retinal degenerative disease processes, such as age-related macular degeneration. We previously demonstrated that N-Acetylcysteine amide (NACA), a novel antioxidant, protects human retinal pigment epithelium (hRPE) against oxidative cell death. We investigated whether NACA could inhibit oxidative stress-induced injury to the RPE in vitro and prevent retinal degeneration in vivo.
Methods: :
ARPE-19 cells were incubated in tert-butylhydroperoxide (tBHP) to induce oxidative stress. Cells were pre-incubated in NACA (or media control) for 24 hours, washed, then incubated in tBHP for 4 hours. We measured levels of reactive oxygen species (ROS), glutathione peroxidase, and catalase enzyme activity using direct assays. We measured levels of lipid peroxidation using reversed-phase HPLC to determine malondialdehyde (MDA) levels. Transepithelial electrical resistance (TEER) reflects cellular integrity and overall cell health and was measured as a functional end point. NACA is currently being evaluated in rd10/rd10 mice using P14 and P23 electroretinograms as well as histology. All p-values were calculated using Student’s t-test.
Results: :
BHP-induced RPE lipid peroxidation, as demonstrated by MDA production, was completely inhibited by NACA pre-incubation (p<0.0001). NACA pre-treatment significantly enhanced activity of the critical cell detoxification enzyme glutathione peroxidase (p<0.0001) while protecting hRPE cells from tBHP-induced ROS production thus restoring catalase concentrations to unstressed control levels (p<0.001). Functional deterioration of cell monolayer health due to tBHP exposure and oxidative stress, as measured by TEER, was also inhibited by NACA pre-treatment (p<0.0001). Early mouse testing has demonstrated excellent tolerance with the highest concentrations tested (550mg/kg/day) with promising potential in prevention of outer nuclear layer cell death and maintenance of ERG response.
Conclusions: :
We have demonstrated that NACA can protect human RPE against oxidative cell damage by inhibiting lipid peroxidation, scavenging ROS, and increasing intracellular antioxidative potential in vitro. These results combined with NACA’s excellent systemic tolerance in rodents, suggest that this compound may be a critical agent to delay or prevent retinal degenerative disease states.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • antioxidants