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Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation

  • Molecular Toxicology
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Abstract

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug–drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

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Acknowledgements

The authors would like to acknowledge the Nikon Imaging Centre, University of Heidelberg, for providing the possibility to use their microscopically setup. Special thanks to Dr. Ulrike Engel for her support in optimizing the microscopically setup and her expert assistance and technical support. The authors would like to thank Saskia Schmitteckert for the substantial help in performing the experiments.

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  1. Jannick Theobald and Xinlai Cheng equal contributor.

Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Pharmaceutical Biology, Heidelberg University, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany

    Jannick Theobald, Xinlai Cheng, Ali Ghanem & Stefan Wölfl

  2. Department of Medicine II, Section Molecular Hepatology, Alcohol Associated Diseases, Medical Faculty Mannheim at Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167, Mannheim, Germany

    Haristi Gaitantzi, Katja Breitkopf-Heinlein & Steven Dooley

  3. Theoretical Biophysics, Department of Biology, Humboldt-Universität zu Berlin, Invalidenstraße 42, 10115, Berlin, Germany

    Edda Klipp & Judith Wodke

  4. microfluidic ChipShop GmbH, Stockholmer Str. 20, 07747, Jena, Germany

    Holger Becker

  5. Experimentelle Nephrologie, KIM III, Universitätsklinikum Jena, Am Nonnenplan 2, 07743, Jena, Germany

    Ralf Mrowka

  6. Department of Gastroenterology, Shanghai Institute of Liver Disease, Zhongshan Hospital, Fudan University, Building 19, Fenglin Road 179, Shanghai, China

    Guangqi Song

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  1. Jannick Theobald
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  2. Xinlai Cheng
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  3. Ali Ghanem
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Contributions

JT, Experimental design, performed experiments, performed data analysis, wrote the manuscript. XC, Performed experiments, experimental design, performed data analysis. AG, Experimental design, performed experiments. HG Isolated primary murine hepatocytes, performed experiments. GS Developed and provided iHep cells. EK Participated in experimental design. JW Participated in experimental design. HB, Developed and manufactured the Chip-design. RM, Participated in experimental design. KB-H Experimental design, corrected the manuscript. SD, Supervised experiments, corrected the manuscript. SW, Supervised experiments, experimental design, corrected the manuscript.

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Correspondence to Stefan Wölfl.

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BMBF SysTox: FKZ 031A303E iPS-Profiler: FKZ 01EK1612C, DFG Grant program (CH 1690/2-1).

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The authors declare that they have no competing financial interest.

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Theobald, J., Cheng, X., Ghanem, A. et al. Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation. Arch Toxicol 92, 1133–1149 (2018). https://doi.org/10.1007/s00204-017-2128-1

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