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

  • Toxicokinetics and Metabolism
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Abstract

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.

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Abbreviations

CHZ:

Chlorzoxazone

OH-CHZ:

6-hydroxychlorzoxazone

CHZ-O-Glc:

Chlorzoxazone-O-glucuronide

CHZ-N-Glc:

Chlorzoxazone-N-glucuronide

CYP450:

Cytochrome P450

DMSO:

Dimethyl sulfoxide

XMEs:

Xenobiotic metabolizing enzymes

UGT:

Uridine 5′-diphospho-glucuronosyltransferase

HPLC-UV:

High pressure liquid chromatography-UV

LC-MS:

Liquid-chromatography-mass spectrometry

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Acknowledgements

We thank Drs. Anne Corlu and Caroline Aninat (Institut NuMeCan, Inserm U1241, Rennes) for fruitful suggestions and Pr. Valérie Paradis (Département de Pathologie, Hôpital Bichat-Beaujon, Paris) and Pr. Véronique Catros (Service de Biologie cellulaire et cytogénétique, Hôpital Pontchaillou, Rennes) for helping us with the preparation of human liver slices. The authors would like to dedicate this article to Dr. Marie-Anne Robin who passed away during the course of this study. Marie-Anne Robin was an exceptional scientist and friend whose enthusiasm for life and belief in science will continue to inspire all of us who were privileged to know her.

Funding

This work was funded by the Institut National de la Santé et de la Recherche Médicale (Inserm, France). Nicolas Quesnot received a fellowship from the Région Bretagne and Ligue contre le Cancer, Comité Départemental des Côtes d’Armor (22).

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Author notes

  1. Nicolas Quesnot and Simon Bucher have contributed to this work equally.

Authors and Affiliations

  1. Inserm, INRA, Univ Rennes, Institut NUMECAN (Nutrition Metabolisms and Cancer) UMR-A 1341, UMR-S 1241, 35000, Rennes, France

    Nicolas Quesnot, Simon Bucher, Manuel Vlach, Elise Vene, Thomas Gicquel, Marie-Anne Robin & Pascal Loyer

  2. Department of Clinical Pharmacology, Bispebjerg Hospital, 23 Bispebjerg Bakke, 2400, Copenhagen, NV, Denmark

    Christina Gade & Helle Holst

  3. Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Nicolas Quesnot & Samuel Valença

  4. Institut NuMeCan, Inserm U1241, Hôpital Pontchaillou, 35033, Rennes, France

    Pascal Loyer

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  1. Nicolas Quesnot
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Correspondence to Pascal Loyer.

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Quesnot, N., Bucher, S., Gade, C. et al. Production of chlorzoxazone glucuronides via cytochrome P4502E1 dependent and independent pathways in human hepatocytes. Arch Toxicol 92, 3077–3091 (2018). https://doi.org/10.1007/s00204-018-2300-2

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