title: Role of cytochrome P450-mediated metabolism and identification of novel thiol-conjugated metabolites in mice with phenytoin-induced liver injury.
abstract: Phenytoin, 5,5-diphenylhydantoin (DPH), is widely used as an anticonvulsant agent. Severe hepatic injury rarely occurs in patients who received DPH. The development of liver injury is thought to be caused by reactive metabolites; however, the metabolites suggested to contribute to hepatotoxicity have not yet been detected in vivo and their effect on developing the liver injury is largely unknown. We recently demonstrated that DPH treatment decreased hepatic glutathione (GSH) contents, and GSH-depleted condition exacerbated DPH-induced liver injury in mice. The aim of the present study was to identify the reactive metabolite and to investigate the role of P450-mediated metabolisms in DPH-induced liver injury. We identified a novel GSH-conjugated (GS)-DPH, a conjugate of putative electrophilic arene oxide intermediate with GSH, in the bile of mice with DPH-induced liver injury. In plasma, cysteine- or N-acetylcysteine-conjugated DPH was detected, and these thiol conjugates levels were correlated with the plasma alanine aminotransferase (ALT) levels. These changes were significantly reduced by pretreatment with P450 inhibitor. Furthermore, the increases of hepatic P450 activities were in parallel with elevation of plasma thiol conjugates levels. These findings suggest that the arene oxide intermediate, which can be converted to thiol conjugates, is involved in DPH-induced liver injury.
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: esasaki@stu.kanazawa-u.ac.jp.
authors.forename: Eita
authors.initials: E
authors.lastname: Sasaki
authors.affiliation: DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda, Saitama 335-8505, Japan. Electronic address: iwamura.atsushi@mf.mt-pharma.co.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwamura
authors.affiliation: Department of Diagnostic Pathology, Graduate School of Medicine and Pharmaceutical Science for Research, University of Toyama, Sugitani, Toyama 930-0194, Japan. Electronic address: ktsune@med.u-toyama.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Tsuneyama
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: tatsuki@p.kanazawa-u.ac.jp.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan. Electronic address: nmiki@p.kanazawa-u.ac.jp.
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda, Saitama 335-8505, Japan. Electronic address: kume.toshiyuki@mu.mt-pharma.co.jp.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kume
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan; Department of Drug Safety Sciences, Nagoya University Graduate School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Electronic address: tyokoi@med.nagoya-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D065767:Activation, Metabolic; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D000927:Anticonvulsants / Q000378:metabolism*; D001646:Bile / Q000378:metabolism; D056486:Chemical and Drug Induced Liver Injury / Q000097:blood / Q000201:enzymology* / Q000473:pathology / Q000517:prevention & control; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D005978:Glutathione / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008810:Mice, Inbred C57BL; D010672:Phenytoin / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000097:blood / Q000378:metabolism*; D013997:Time Factors; D008565:Membrane Proteins
chemical_list: D000111:Acetylcysteine; D000410:Alanine Transaminase; D000927:Anticonvulsants; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; D010672:Phenytoin; D013438:Sulfhydryl Compounds; C400322:cytochrome P-450 CYP2C subfamily; C496508:Cyp3a11 protein, mouse; D008565:Membrane Proteins
keywords: Arene oxide; Cytochrome P450; Glutathione; Hepatotoxicity; Phenytoin; Reactive metabolite