Demonstration by redox fluorometry that sulforaphane protects retinal pigment epithelial cells against oxidative stress.
OBJECTIVE
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.
Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Marisol
M
del V Cano
Johann M
JM
Reyes
Choul Y
CY
Park
Xiangqun
X
Gao
Keisuke
K
Mori
Roy S
RS
Chuck
Peter L
PL
Gehlbach
D002470:Cell Survival; D002478:Cells, Cultured; D019610:Cytoprotection; D005420:Flavoproteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D017879:Isothiocyanates; D008856:Microscopy, Fluorescence; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D005420:Flavoproteins; D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; D009249:NADP; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; C016766:sulforaphane