In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat Fusako Usuki a, * , Akira Yasutake b , Fujio Umehara c , Hidehiro Tokunaga d , Miyuki Matsumoto a , Komyo Eto a , Shoichi Ishiura e , Itsuro Higuchi c a Department of Clinical Medicine, National Institute for Minamata Disease, 4058-18 Hama, Minamata 867±0008, Japan b Biochemistry Section, National Institute for Minamata Disease, 4058-18 Hama, Minamata 867±0008, Japan c Third Department of Internal Medicine, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890, Japan d Department of Surgical Pathology, Kumamoto University School of Medicine, 2-39-1 Kurokami, Kumamoto 860-8555, Japan e Department of Life Sciences, Graduate School of Arts and Sciences,University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153, Japan Received 20 January 2001; received in revised form 15 March 2001; accepted 21 March 2001 Abstract Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the ®rst in vivo effectiveness of antiox- idant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clari®ed that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Methylmercury-intoxication; Mitochondrial electron transport system; Apoptosis; Oxidative stress; Antioxidant; Trolox (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid); In vivo protection Methylmercury (MeHg) is a well-established neurotoxi- cant, known to be the cause of Minamata disease, a condi- tion characterized by ataxia, visual and hearing disturbances, sensory disturbances, convulsions, memory disturbances, muscle weakness and wasting, and muscle cramp [5,6]. MeHg pollution is a continuous environmental hazard to human health, especially in ®sh-eating and physi- cally stressed populations around the world. Recently, the possible effects of low level prenatal MeHg exposure on neurodevelopment have been reported [3]. We previously reported that MeHg exposure affects skeletal muscle directly, i.e. induces a decrease in the enzyme activities of mitochondrial energy metabolism such as cytochrome c oxidase (CCO) and succinate dehydrogenase (SDH) [16]. In an in vitro study we showed that low levels of MeHg induce apoptosis in cultured myogenic cells, the mediators of which are reactive oxygen species [17], and reported that MeHg activated one of the MAPK pathways, stress-acti- vated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) [18]. Here we assess a role of MeHg-mediated oxida- tive stress in the in vivo pathological process of MeHg intoxication using antioxidant Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), a water soluble vitamin E analog. Twenty-®ve male Wistar strain rats (age 10 weeks: weight 300±325 g) were randomly divided into ®ve groups and treated as follows: (1) MeHg (2) MeHg 1 low dose Trolox (1 mg/kg) (3) MeHg 1 high dose Trolox (2.5 mg/kg) (4) high dose Trolox (2.5 mg/kg) (5) control without MeHg and Trolox. MeHg was administered in water polluted by 20 ppm Hg in the form of MeHg-glutathione (1:1) complex every day for 28 days. The average intake was 600 mg Hg/rat per day. Trolox (SIGMA±Aldrich) was dissolved in 0.154 N NaOH, neutralized by 0.154 N HC1, and administered by intraperitoneal injection 5 days/week for 4 weeks of MeHg exposure. Animals not treated with Trolox were injected with saline. The experimental protocol was approved by the Animal Committee of the National Neuroscience Letters 304 (2001) 199±203 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)01764-5 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 181-966-63-3111; fax: 181-966- 611145. E-mail address: usuki@nimd.go.jp (F. Usuki).