Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation
- 424 Downloads
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.
KeywordsHepatocytes Fluorescence CYP450 enzymes In vitro systems Assay Liver models
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.
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.
Compliance with ethical standards
BMBF SysTox: FKZ 031A303E iPS-Profiler: FKZ 01EK1612C, DFG Grant program (CH 1690/2-1).
Conflict of interest
The authors declare that they have no competing financial interest.
Data availability statement
All data are is included in the manuscript.
- Gerets HHJ, Tilmant K, Gerin B, Chanteux H, Depelchin BO et al (2012) Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol Toxicol 28:69–87CrossRefPubMedPubMedCentralGoogle Scholar
- Godoy P, Hengstler JG, Ilkavets I, Meyer C, Bachmann A, Muller A, Tuschl G, Mueller SO, Dooley S (2009) Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology 49:2031–2043. https://doi.org/10.1002/hep.22880 CrossRefPubMedGoogle Scholar
- Godoy P, Hewitt N, Albrecht U, Andersen M, Ansari N et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87:1315–1530CrossRefPubMedPubMedCentralGoogle Scholar
- Guguen-Guillouzo C, Guillouzo A (2010) General review on in vitro hepatocyte models and their applications. In: Maurel P (ed) Hepatocytes. Methods in molecular biology (Methods and protocols), vol 640. Humana Press, pp 1–40Google Scholar
- Hewitt NJ, Gómez Lechón MJ, Houston JB, Hallifax D, Brown HS et al (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159–234CrossRefPubMedGoogle Scholar
- Hou YT, Ijima H, Matsumoto S, Kubo T, Takei T, Sakai S, Kawakami K (2010) Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes. J Biosci Bioeng 110:208–216. https://doi.org/10.1016/j.jbiosc.2010.01.016 CrossRefPubMedGoogle Scholar
- Kang YB, Sodunke TR, Lamontagne J, Cirillo J, Rajiv C et al. (2015) Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms. Biotechnol BioengGoogle Scholar
- Kim M, Lee JY, Jones CN, Revzin A, Tae G (2010) Heparin-based hydrogel as a matrix for encapsulation and cultivation of primary hepatocytes. Biomaterials 31:3596–3603. https://doi.org/10.1016/j.biomaterials.2010.01.068 CrossRefPubMedPubMedCentralGoogle Scholar
- Li W, Harper PA, Tang B-K, Okey AB (1998) Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene. Biochem Pharmacol 56:599–612CrossRefPubMedGoogle Scholar
- Zellmer S, Schmidt-Heck W, Godoy P, Weng H, Meyer C, Lehmann T, Sparna T, Schormann W, Hammad S, Kreutz C, Timmer J, von Weizsacker F, Thurmann PA, Merfort I, Guthke R, Dooley S, Hengstler JG, Gebhardt R (2010) Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes. Hepatology 52:2127–2236. https://doi.org/10.1002/hep.23930 CrossRefPubMedGoogle Scholar