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M. Tanito, H. Masutani, Y.C. Kim, M. Nishikawa, A. Ohira, J. Yodoi; Induction of Thioredoxin via Antioxidant Responsive Element, and Attenuation of Retinal Light Damage in Mice by Sulforaphane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1672.
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Purpose: Thioredoxin (Trx) is a multifunctional endogenous redox regulator that protects cells against various types of cellular stresses. We tested 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 white light exposure at 6000 lux for 2 hours. To estimate retinal cell damage, we counted the number of cell nuclei and the percentage of TUNEL–positive cells in the outer nuclear layer and the retinal pigment epithelial (RPE) layer and recorded electroretinograms (ERGs). To further analyze 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/day for 3 days. After light exposure, mice pretreated with SF had a significantly lower percentage of TUNEL–positive RPE and photoreceptor cells, significantly higher numbers of RPE and photoreceptor nuclei, and greater amplitudes 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 LDH releasing assay and DCFH–DA/flowcytometry, 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 mouse retina and effectively reduced retinal light damage. ARE is involved in the mechanism of Trx induction by SF in RPE cells.
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