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Production of chlorzoxazone glucuronides via cytochrome P4502E1 dependent and independent pathways in human hepatocytes

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

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  • Nicolas Quesnot
  • Simon Bucher
  • Christina Gade
  • Manuel Vlach
  • Elise Vene
  • Samuel Valença
  • Thomas Gicquel
  • Helle Holst
  • Marie-Anne Robin
  • Pascal Loyer
Vis graf over relationer

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.

OriginalsprogEngelsk
TidsskriftArchives of Toxicology
Vol/bind92
Udgave nummer10
Sider (fra-til)3077-3091
Antal sider15
ISSN0340-5761
DOI
StatusUdgivet - okt. 2018

ID: 55622315