Flavonoids protect human retinal pigment epithelial cells from oxidative-stress-induced death.
OBJECTIVE
To determine whether specific dietary and synthetic flavonoids can protect human retinal pigment epithelial (RPE) cells from oxidative-stress-induced death.


METHODS
The efficacy and potency were determined of a variety of dietary and synthetic flavonoids on the survival of human ARPE-19 cells and primary human RPE cells treated with either hydrogen peroxide (H2O2) or t-butyl hydroperoxide (t-BOOH). We determined the effective concentrations (EC50s) and the toxicities (LD50s) of the flavonoids after 24 hours, by using the MTT assay. The efficacy of vitamins E and C on RPE cell survival were compared under identical conditions. The ability of specific flavonoids to protect RPE cells from cell death was determined at various time intervals after the cells were exposed to oxidative stress. The ability of flavonoids to block the accumulation of intracellular reactive oxygen species was examined with dichlorofluorescein (DCF) fluorescence. Finally, the ability of flavonoids to induce phase-2 detoxifying enzymes was tested by immunoblot analysis for the transcription factor Nrf2 and the phase-2 gene product heme-oxygenase 1.


RESULTS
Specific flavonoids protected human RPE cells from oxidative-stress-induced death with efficacies between 80% and 100% and potencies in the high-nanomolar and low-micromolar range. The toxicities of most of the effective flavonoids were low. The effective flavonoids included the dietary flavonoids fisetin, luteolin, quercetin, eriodictyol, baicalein, galangin and EGCG, and the synthetic flavonoids, 3,6-dihydroxy flavonol and 3,7 dihydroxy flavonol. Several flavonoids can protect RPE cells even when they are added after the cells have been exposed to oxidative stress. The flavonoids acted through an intracellular route to block the accumulation of reactive oxygen species. Many of these flavonoids induced the expression of Nrf2 and the phase-2 gene product heme-oxygenase 1 in human RPE cells.


CONCLUSIONS
The results identify a select group of flavonoids that protect RPE cells from oxidative-stress-induced death with a high degree of potency and low toxicity. Many of these flavonoids also induce the expression of phase-2 detoxification proteins which could function to provide additional protection against oxidative stress. This select group of flavonoids and the foods that contain high levels of these compounds may have some clinical benefit for patients with retinal diseases associated with oxidative stress.
Hanneken
Anne
A
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. ahanneke@scripps.edu
Lin
Fen-Fen
FF
Johnson
Jennifer
J
Maher
Pamela
P
D015153:Blotting, Western; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection / Q000187:drug effects*; D004032:Diet; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000494:pharmacology*; D051547:Heme Oxygenase-1 / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D005419:Flavonoids; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D013844:Thiazoles; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D051547:Heme Oxygenase-1; C022616:thiazolyl blue