Purpose: Vigabatrin is an antiepileptic drug that acts by increasing the inhibitory neurotransmitter γ-aminobutyric acid (GABA) by suicidal inhibition of enzyme GABA transaminase (GABA-T). Vigabatrin dose in human produces peripheral visual field defects. Various preclinical studies have proven that chronic administration of vigabatrin leads to ocular toxicity by accumulation of vigabatrin in retina. Vigabatrin increases GABA concentrations much higher in retina compared to brain leading to retinal damage. The objective of the study was to measure reactive oxygen species generated by vigabatrin and to screen antioxidants with neuroprotective potential against vigabatrin induced in vitro retinal neuronal (R28) and pigmental epithelial (ARPE) cell toxicity.
Methods: In vitro cytotoxicity studies were performed using R28 and ARPE19 cells. R28 cells are immortal postnatal Day 6 rat retinal neuronal cells with characteristics of glial and neuronal cell markers. R28 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. ARPE19 is adult retinal pigment epithelial cell line derived from male normal human eyes. DMEM: F12 Medium supplemented with 10% fetal bovine serum is used for culture. The cytotoxicity studies were performed in 96 well plate seeded with 20000 cells (R28/ARPE19) per well in serum-free medium for 24 hours to attain a non-proliferative state. Each well was exposed to different concentrations of vigabatrin (50, 25, 12.5, 5, 2.5, 1 and 0.5 mM) in triplicate to measure ROS generation and to determine IC50 (concentration required to kill 50% of total cells incubated). The cells were treated with antioxidants (taxifolin, piperine, quercetin, curcumin, hesperidin, resveratrol and vitamin E) at 200, 100, 50, 25 and 12.5 mM, 1 h prior to addition of IC50 of vigabatrin. After incubation period of 24 h, cell viability in each well was quantified by using CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit and ROS was measured using OxiSelect™ Intracellular ROS Assay Kit.
Results: The ROS produced by vigabatrin in R28 and ARPE19 cells was prevented by pretreatment of cells with antioxidants. The R28 and ARPE19 cells exposed to 37.3 and 27.5 mM of vigabatrin for 24 h produced cytotoxicity of 50% and this concentration was used for assay. Pretreatment of R28 cells with taxifolin, piperine, quercetin, curcumin, hesperidin, resveratrol and vitamin E increased the cell viability to 92.9 ± 5.70, 75.9 ± 4.32, 62.5 ± 3.25, 65.2 ± 6.25, 88.2 ± 6.12, 69.2 ± 5.31 and 72.3 ± 3.26%, respectively in presence of 37.3 mM of vigabatrin. Pretreatment of ARPE cells with taxifolin, piperine, quercetin, curcumin, hesperidin, resveratrol and vitamin E increased the cell viability to 100 ± 8.50, 71.4 ± 3.12, 101 ± 2.13, 85.9 ± 5.57, 101 ± 9.98, 99.0 ± 5.01 and 98.7 ± 2.75 %, respectively in presence of 27.5 mM of vigabatrin.
Conclusion: Taxifolin, piperine and hesperidin showed potent cytoprotective effect on vigabatrin induced retinal neuronal cell toxicity. All antioxidants screened increased retinal pigmental epithelial cell viability in presence of vigabatrin. The results indicate that vigabatrin treatment supplemented with antioxidants would combat vigabatrin induced retinal cell toxicity.