51. Production of chlorzoxazone glucuronides via cytochrome P4502E1 dependent and independent pathways in human hepatocytes
- Author
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Marie-Anne Robin, Elise Vene, Christina Gade, Pascal Loyer, Manuel Vlach, Simon Bucher, Helle Holst, Nicolas Quesnot, Thomas Gicquel, Samuel Santos Valença, Nutrition, Métabolismes et Cancer (NuMeCan), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Bispebjerg Hospital, Institut National de la Sante et de la Recherche Medicale (Inserm, France), Region Bretagne, Ligue contre le Cancer, Comite Departemental des Cotes d'Armor [22], Institut National de la Recherche Agronomique (INRA)-Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Universidade do Porto = University of Porto
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Male ,0301 basic medicine ,[SDV]Life Sciences [q-bio] ,Health, Toxicology and Mutagenesis ,Metabolite ,Glucuronidation ,Hydroxylation ,Toxicology ,030226 pharmacology & pharmacy ,03 medical and health sciences ,chemistry.chemical_compound ,Glucuronides ,0302 clinical medicine ,Cytochrome P-450 Enzyme System ,In vivo ,Enzymatic hydrolysis ,medicine ,Animals ,Humans ,CYP2E1 ,Glucuronosyltransferase ,Chromatography, High Pressure Liquid ,Drug metabolism ,Chemistry ,Hep G2 Cells ,General Medicine ,Recombinant Proteins ,Culture Media ,Mice, Inbred C57BL ,Chlorzoxazone ,030104 developmental biology ,Biochemistry ,[SDV.TOX]Life Sciences [q-bio]/Toxicology ,Hepatocytes ,Glucuronide ,UGT ,medicine.drug - Abstract
International audience; CYP2E1 activity is measured in vitro and in vivo via hydroxylation of the Chlorzoxazone (CHZ) producing the 6-hydroxychlorzoxazone (OH-CHZ) further metabolized as a glucuronide excreted in urine. Thus, the quantification of the OH-CHZ following enzymatic hydrolysis of CHZ-derived glucuronide appears to be a reliable assay to measure the CYP2E1 activity without direct detection of this glucuronide. However, OH-CHZ hydrolyzed from urinary glucuronide accounts for less than 80% of the CHZ administrated dose in humans leading to postulate the production of other unidentified metabolites. Moreover, the Uridine 5'-diphospho-glucuronosyltransferase (UGT) involved in the hepatic glucuronidation of OH-CHZ has not yet been identified. In this study, we used recombinant HepG2 cells expressing CYP2E1, metabolically competent HepaRG cells, primary hepatocytes and precision-cut human liver slices to identify metabolites of CHZ (300 μM) by high pressure liquid chromatography-UV and liquid-chromatography-mass spectrometry analyses. Herein, we report the detection of the CHZ-O-glucuronide (CHZ-O-Glc) derived from OH-CHZ in culture media but also in mouse and human urine and we identified a novel CHZ metabolite, the CHZ-N-glucuronide (CHZ-N-Glc), which is resistant to enzymatic hydrolysis and produced independently of CHZ hydroxylation by CYP2E1. Moreover, we demonstrate that UGT1A1, 1A6 and 1A9 proteins catalyze the synthesis of CHZ-O-Glc while CHZ-N-Glc is produced by UGT1A9 specifically. Together, we demonstrated that hydrolysis of CHZ-O-Glc is required to reliably quantify CYP2E1 activity because of the rapid transformation of OH-CHZ into CHZ-O-Glc and identified the CHZ-N-Glc produced independently of the CYP2E1 activity. Our results also raise the questions of the contribution of CHZ-N-Glc in the overall CHZ metabolism and of the quantification of CHZ glucuronides in vitro and in vivo for measuring UGT1A activities.
- Published
- 2018
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