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J. Biol. Chem., Vol. 282, Issue 33, 23996-24003, August 17, 2007

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Knock Down of -Glutamylcysteine Synthetase in Rat Causes Acetaminophen-induced Hepatotoxicity^*

Sho Akai, Hiroko Hosomi, Keiichi Minami, Koichi Tsuneyama, Miki Katoh, Miki Nakajima, and Tsuyoshi Yokoi¹

From the Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan and Department of Diagnostic Pathology, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Sugitani 930-0194, Toyama, Japan

Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. -Glutamylcysteine synthetase (-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat -GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10¹¹ pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.

Received for publication, April 3, 2007 , and in revised form, June 7, 2007.

^* This work was supported in part by Research on Toxicogenomics, Health and Labor Science research grants from the Ministry of Health, Labor, and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. Tel./Fax: 81-76-234-4407; E-mail: tyokoi{at}kenroku.kanazawa-u.ac.jp.

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