Sulforaphane induces thioredoxin through the antioxidant-responsive element and attenuates retinal light damage in mice.
OBJECTIVE
Thioredoxin (Trx) is a multifunctional endogenous redox regulator that protects cells against various types of cellular or tissue stresses. This study was conducted to test whether sulforaphane (SF), a naturally occurring isothiocyanate that is highly concentrated in broccoli sprouts, induces Trx in retinal tissues and whether pretreatment with SF protects against light-induced retinal damage in mice.


METHODS
Expression of Trx in mouse retina was analyzed by Western blot and immunohistochemistry. Retinal damage was induced by exposure to white light at 6000 lux for 2 hours. To estimate retinal cell damage, the number of cell nuclei and the percentage of TUNEL-positive cells were counted in the outer nuclear layer and the retinal pigment epithelial (RPE) layer and the electroretinograms recorded. To analyze further the mechanism of Trx induction by SF, cultured human K-1034 RPE cells were used.


RESULTS
Both intraperitoneal and oral SF induced Trx protein in the neural retina and RPE. The maximum induction of Trx was observed with intraperitoneal SF 0.5 mg/d for 3 days. After exposure to light, mice pretreated with SF had a significantly lower percentage of TUNEL-positive RPE and photoreceptor cells, a significantly higher number of RPE and photoreceptor nuclei, and greater amplitude of ERG a- and b-waves than in the saline-treated mice. In K-1034 cells, 1 microM SF induced Trx protein, whereas 10 microM SF did not damage cells or augment cellular peroxide production, tested by a lactate dehydrogenase (LDH) release assay and 2',7'-dichlorofluorescein diacetate (DCFH-DA)/flow cytometry, respectively. In the luciferase reporter assay, the antioxidant-responsive element (ARE) played a role in SF-induced Trx expression. In the electrophoretic mobility shift assay, SF induced binding of Nrf2, small Maf, and c-Jun to the ARE of the Trx gene.


CONCLUSIONS
SF induced Trx in murine retina and effectively reduced retinal light damage. Evidence suggests that the ARE is involved in the mechanism of Trx induction by SF in RPE cells.
Tanito
Masaki
M
Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo, Kyoto 606-8507, Japan.
Masutani
Hiroshi
H
Kim
Yong-Chul
YC
Nishikawa
Mai
M
Ohira
Akihiro
A
Yodoi
Junji
J
D000284:Administration, Oral; D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D000975:Antioxidants / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007274:Injections, Intraperitoneal; D017879:Isothiocyanates; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008027:Light; D050985:MafK Transcription Factor; D051379:Mice; D008807:Mice, Inbred BALB C; D051267:NF-E2-Related Factor 2; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D011833:Radiation Injuries, Experimental / Q000378:metabolism / Q000517:prevention & control*; D020218:Response Elements / Q000187:drug effects*; D012160:Retina / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D012164:Retinal Diseases / Q000378:metabolism / Q000517:prevention & control*; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D013879:Thioredoxins / Q000096:biosynthesis*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
D016588:Anticarcinogenic Agents; D000975:Antioxidants; D004268:DNA-Binding Proteins; D017879:Isothiocyanates; D050985:MafK Transcription Factor; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D009687:Nuclear Proteins; D013454:Sulfoxides; D013861:Thiocyanates; D015534:Trans-Activators; D013879:Thioredoxins; D007770:L-Lactate Dehydrogenase; C016766:sulforaphane