Attenuation of retinal photooxidative damage in thioredoxin transgenic mice Masaki Tanito a,b , Hiroshi Masutani a , Hajime Nakamura a , Shin-ichi Oka a , Akihiro Ohira b , Junji Yodoi a, * a Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogoin Sakyo-ku, Kyoto 606-8507, Japan b Department of Ophthalmology, Shimane Medical University of Medicine, Shimane 693-8501, Japan Received 28 January 2002; received in revised form 3 March 2002; accepted 18 March 2002 Abstract Thioredoxin (TRX) is an endogenous redox (reduction/oxidation) regulator that has cytoprotective effects against various types of oxidative stresses. Exposure to excessive levels of white light induces retinal photoreceptor damage. To test the cytoprotective effect of overexpressed TRX against retinal photooxidative damage, both TRX transgenic (trx- tg) mice and C57BL/6 (wild type) mice were exposed to intense white fluorescent light. The amounts of oxidized and tyrosine-phosphorylated proteins decreased in the neural retinas of the trx-tg mice compared to the wild type mice after light exposure. The electroretinographic amplitudes were higher and the formation of oxidized DNA was lower in trx-tg mice compared to wild type mice after light exposure. These results suggest that overexpression of TRX suppresses retinal photooxidative damage. TRX intensification may be a useful therapeutic strategy to prevent retinal photic injury. q 2002 Published by Elsevier Science Ireland Ltd. Keywords: Thioredoxin; Photoreceptor; Photic injury; Tyrosine phosphorylation; Electroretinogram; 8-Hydroxy-2-deoxyguanosine Excessive light may enhance the progression and severity of human age-related macular degeneration and some forms of retinitis pigmentosa [2], and the hazards of exposure to full-spectrum light from the operating microscope used in ophthalmic practice can cause photic maculopathy [7]. Exposure to excessive levels of white light induces photo- receptor damage, and free radicals including reactive oxygen species play a crucial role in such damage [13–15]. Thioredoxin (TRX) is a small ubiquitous protein (mole- cular weight, 13 kDa) with two redox-active half-cystine residues, -Cys-Gly-Pro-Cys-, in its active center [6]. TRX is upregulated in response to a wide variety of oxidative stresses [8]. We found that TRX is significantly upregulated in retinal tissue in response to retinal ischemia-reperfusion injury [10]. TRX scavenges singlet oxygen and hydroxyl radicals by itself [3] and hydrogen peroxide in association with peroxiredoxin [1]. TRX transgenic (trx-tg) mice are more resistant to focal cerebral ischemia [17] compared with wild type mice. Trx-tg mice are C57BL/6 mice (wild type) that carry the human TRX transgene under the control of b-actin promoter and express the human TRX throughout the body including the brain [17]. We found that human TRX also is expressed in the entire retina of trx-tg, but not in that of wild type mice by immunohistochemistry and Western blotting with anti-human TRX antibody (Figs. 1A,B). The methods of retinal sample preparation, immunohistochemistry, and Western blotting were previously described [10,17]. Therefore, we evaluated whether or not trx-tg mice were more resistant to intense light induced retinal damage. All procedures in this study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. First, we analyzed endogenous mouse TRX before and after light exposure in retinas from trx-tg and wild type mice by Western blotting with anti-mouse TRX antibody [10,17]. Before light exposure, 3-–4-week-old male mice were dark adapted for 48 h, and their pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops (Santen, Osaka, Japan). The unanesthetized mice were exposed to 8000 lux of diffuse, white fluorescent light (National, Osaka, Japan) for 2 h in cages with a reflec- Neuroscience Letters 326 (2002) 142–146 0304-3940/02/$ - see front matter q 2002 Published by Elsevier Science Ireland Ltd. PII: S0304-3940(02)00314-2 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 181-75-751-4024; fax: 181-75- 761-5766. E-mail address: yodoi@virus.kyoto-u.ac.jp (J. Yodoi).