title: Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates.
abstract: Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.
authors.affiliation: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Electronic address: sanoh@hiroshima-u.ac.jp.
authors.forename: Seigo
authors.initials: S
authors.lastname: Sanoh
authors.affiliation: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Santoh
authors.affiliation: Faculty of Pharmaceutical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Takagi
authors.affiliation: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kanayama
authors.affiliation: Faculty of Pharmaceutical Sciences, Hiroshima International University, 5-1-1 Hirokoshinkai, Kure, Hiroshima 737-0112, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Sugihara
authors.affiliation: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Yaichiro
authors.initials: Y
authors.lastname: Kotake
authors.affiliation: Tsukuba Research Center, Kuraray Co., Ltd., 41 Miyukigaoka, Tsukuba, Ibaraki 305-0841, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Ejiri
authors.affiliation: De Three Research Laboratories, 25-4 Saigo, Tsukuba, Ibaraki 305-0036, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Horie
authors.affiliation: Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kitamura
authors.affiliation: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ohta
mesh_terms: D000082:Acetaminophen / Q000633:toxicity*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015239:Arylsulfotransferase / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D005470:Fluorometry; D014453:Glucuronosyltransferase / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018874:Spheroids, Cellular / Q000187:drug effects* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D000082:Acetaminophen; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D014453:Glucuronosyltransferase; D015239:Arylsulfotransferase; C573795:Sult1a1 protein, rat; D005978:Glutathione
keywords: Acetaminophen; Hepatocytes; Imaging; Micro-space cell culture plate; Spheroids