Sulforaphane Inhibits Redox Fluorometry Shifts in Retinal Pigment Epithelium Cells Following Oxidative Challenge

Abstract

Purpose: : Oxidative injury to retinal pigment epithelium (RPE) has been implicated in the development of age–related macular degeneration. Autofluorescence spectroscopy has been used to assess cellular redox state, change. Here redox spectroscopy is used to image living RPE and to quantify response to oxidant challenge. RPE cells pretreated with the isothiocynate, sulforaphane, are compared to untreated cells.

Methods: : ARPE–19 cells were pretreated with 4 micromolar sulforaphane in media for 24 hours. Sulforaphane treated and untreated cells were exposed to a range of H₂O₂ concentrations, for 2 hours. Individual cells from each plate were then imaged. Reduced pyridine nucleotides were detected by excitation in the region of 366 nm with fluorescence emission measurement in the region of 450 nm. Oxidized flavoproteins were detected by excitation at 460 nm and measuring fluorescence at 540 nm. The ratio of these measurements served as the index of redox state.

Results: : The ratio of reduced nicotinamide nucleotide to oxidized flavoprotein decreased in a dose dependent manner following H₂O₂ exposure. RPE pretreated with sulforaphane maintained a significantly higher ratio at the concentrations of H₂O₂ tested. The redox ratio for sulforaphane treated cells following 0.59 mM H₂O₂ exposure was 2.64 ± 0.19 as compared to 1.77 ± 0.16 in untreated cells, (p = 0.001). At 1.17 mM H₂O₂, the redox ratio of sulforaphane treated cells was 2.30 ± 0.18, compared to 1.76 ± 0.13 in untreated cells (p = 0.02). At an H₂O₂ concentration of 2.34 mM, treated cells maintain a higher viability that untreated cells. The correlation between the nicotinamide nucleotides in redox fluorometry and the measured chemical values of NADH/NADPH was r = 0.85.

Conclusions: : To our knowledge this is the first report utilizing redox fluorometry to quantitatively evaluate oxidative injury in RPE. We present evidence that the technique has sufficient sensitivity to detect a cytoprotective effect conferred by sulforaphane pretreatment. Sulforaphane protection of RPE from oxidative damage merits further study in the setting of age–related macular degeneration.

Keywords: retinal pigment epithelium • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • antioxidants