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(Investigative Ophthalmology and Visual Science. 2008;49:2606-2612.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0960
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Demonstration by Redox Fluorometry that Sulforaphane Protects Retinal Pigment Epithelial Cells against Oxidative Stress

Marisol del V. Cano,1 Johann M. Reyes,1 Choul Y. Park,1,2 Xiangqun Gao,3 Keisuke Mori,4 Roy S. Chuck,1 and Peter L. Gehlbach1

1From the Departments of Ophthalmology and 3Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland; the 2Department of Ophthalmology, Dongguk University School of Medicine, Ilsan, South Korea; and the 4Department of Ophthalmology, Saitama Medical University, Iruma, Saitama, Japan.

PURPOSE. To quantify the effects of oxidant challenge on the redox state of adult human retinal pigment epithelial cells using microscopic autofluorescence spectroscopy and to determine whether treatment with the isothiocyanate sulforaphane protects these cells against oxidative stress.

METHODS. Oxidative stress was evoked in ARPE-19 cells by H2O2 and tert-butyl hydroperoxide. Reduced nicotinamide nucleotides NAD(P)H were assessed by excitation at 366 nm with measurement of fluorescence at 450 nm. Oxidized flavoproteins were assessed by excitation at 460 nm with measurement of fluorescence at 540 nm. The ratio of these measurements served as the index of cellular redox status.

RESULTS. Redox ratio and cell viability decreased in a dose-dependent manner after oxidant exposure. ARPE-19 cells treated with sulforaphane maintained significantly higher redox ratio and cell viability. The ratio for sulforaphane-treated cells after exposure to 0.64 mM H2O2 was 2.64 ± 0.19 compared with 1.77 ± 0.16 in untreated cells (P = 0.001). At 1.2 mM H2O2, the redox ratio of sulforaphane-treated cells was 2.30 ± 0.18 compared with 1.76 ± 0.13 in untreated cells (P = 0.02). Similar results were observed after insult with tert-butyl hydroperoxide.

CONCLUSIONS. Redox fluorometry provides quantitative information on the redox status of living cells. Sulforaphane protects ARPE-19 cells from oxidative injury by induction of antioxidant phase 2 genes. The findings in this study describe a useful method for assessing antioxidant effects in live cells and support phase 2 gene induction as a potential treatment strategy for macular degeneration and diseases in which oxidative injury plays a causative role.






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Copyright © 2008 by the Association for Research in Vision and Ophthalmology