title: Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A-mediated ortho-benzoquinone metabolite formation and glutathione depletion.
abstract: Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive ortho-benzoquinone metabolites formation and glutathione (GSH) depletion in LUT-induced cytotoxicity in primary rat hepatocytes. A reactive ortho-benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT-induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT-induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L-buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'-dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A-mediated reactive metabolite formation plays a critical role in LUT-induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT-mediated cytotoxicity in primary rat hepatocytes.
authors.affiliation: Department of Pharmaceutical Analysis, Key Laboratory of Drug Quality Control and Pharmacovigilance affiliated to Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China.
authors.forename: Fuguo
authors.initials: F
authors.lastname: Shi
authors.affiliation: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Department of Pharmaceutical Analysis, Key Laboratory of Drug Quality Control and Pharmacovigilance affiliated to Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China.
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Pharmaceutical Analysis, Key Laboratory of Drug Quality Control and Pharmacovigilance affiliated to Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Pan
authors.affiliation: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China.
authors.forename: Shiping
authors.initials: S
authors.lastname: Ma
authors.affiliation: Department of Pharmaceutical Analysis, Key Laboratory of Drug Quality Control and Pharmacovigilance affiliated to Ministry of Education, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ding
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000378:metabolism*; D019328:Buthionine Sulfoximine / Q000633:toxicity; D002460:Cell Line; D002853:Chromatography, Liquid; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007654:Ketoconazole / Q000494:pharmacology; D047311:Luteolin / Q000633:toxicity*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry
chemical_list: D016227:Benzoquinones; D065692:Cytochrome P-450 CYP3A Inhibitors; C004532:quinone; D019328:Buthionine Sulfoximine; D051544:Cytochrome P-450 CYP3A; D005978:Glutathione; D047311:Luteolin; D007654:Ketoconazole
keywords: CYP3A; GSH depletion; LC-MS/MS; cytotoxicity; luteolin; reactive metabolites