title: Knock down of gamma-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity.
abstract: 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. Gamma-glutamylcysteine synthetase (gamma-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 gamma-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 10(11) 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.
authors.affiliation: Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Akai
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hosomi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Minami
authors.forename: Koichi
authors.initials: K
authors.lastname: Tsuneyama
authors.forename: Miki
authors.initials: M
authors.lastname: Katoh
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D000082:Acetaminophen / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D056486:Chemical and Drug Induced Liver Injury / Q000175:diagnosis* / Q000209:etiology*; D004195:Disease Models, Animal; D004357:Drug Synergism; D020868:Gene Silencing; D005721:Glutamate-Cysteine Ligase / Q000172:deficiency / Q000235:genetics*; D005978:Glutathione / Q000032:analysis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats
chemical_list: D034741:RNA, Small Interfering; D000082:Acetaminophen; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione