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System Microscopy of Stress Response Pathways in Cholestasis Research

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Experimental Cholestasis Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1981))

Abstract

Exposure to oxidative radical species and cytokine-mediated inflammatory stress are established contributors to hepatocyte cell death during cholestasis. Cellular counter measures against those stressors are called adaptive stress response pathways. While in early stages of the disease adaptive stress pathways protect the hepatocytes, in later stages during prolonged stressed conditions they fail. The quantitative imaging-based assessment of cellular stress response pathways using the HepG2 BAC-GFP response reporter platform is a powerful strategy to evaluate the impact of chemical substances and gene knockdown on activation of adaptive stress response pathways, hence allowing systematic screening for positive or negative influences on cholestasis progression. This protocol allows the application of a highly versatile screening tool for a systematic evaluation of the effect of compounds having cholestasis liability and affected genes during cholestatic injury on cellular adaptive stress pathway activation. The approach involves high-throughput live-cell visualization of GFP-tagged key proteins of the oxidative stress response/Nrf2 pathway and inflammatory cytokine signaling. Quantitative image analysis of temporal responses of individual cells is followed by informatics analysis. The overall practical approaches are discussed in this chapter.

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References

  1. Wink S, Hiemstra S, Huppelschoten S et al (2014) Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment. Chem Res Toxicol 27:338–355

    Article  CAS  Google Scholar 

  2. Copple BL, Jaeschke H, Klaassen CD (2010) Oxidative stress and the pathogenesis of cholestasis. Semin Liver Dis 30:195–204

    Article  CAS  Google Scholar 

  3. Yamamoto M, Kensler TW, Motohashi H (2018) The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis. Phys Rev 98:1169–1203

    CAS  Google Scholar 

  4. Okada K, Shoda J, Taguchi K et al (2009) Nrf2 counteracts cholestatic liver injury via stimulation of hepatic defense systems. Biochem Biophys Res Commun 389:431–436

    Article  CAS  Google Scholar 

  5. Tanaka Y, Aleksunes LM, Cui YJ et al (2009) ANIT-induced intrahepatic cholestasis alters hepatobiliary transporter expression via Nrf2-dependent and independent signaling. Toxicol Sci 108:247–257

    Article  CAS  PubMed Central  Google Scholar 

  6. Weerachayaphorn J, Luo Y, Mennone A et al (2014) Deleterious effect of oltipraz on extrahepatic cholestasis in bile duct-ligated mice. J Hepatol 60:160–166

    Article  CAS  PubMed Central  Google Scholar 

  7. Gujral JS, Farhood A, Bajt ML et al (2003) Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct–ligated mice. Hepatology 38:355–363

    Article  Google Scholar 

  8. Woolbright BL, Jaeschke H (2018) Mechanisms of inflammatory liver injury and drug-induced hepatotoxicity. Curr Pharm Rep 5:346–357

    Article  Google Scholar 

  9. Gehrke N, Nagel M, Straub BK et al (2018) Loss of cellular FLICE-inhibitory protein promotes acute cholestatic liver injury and inflammation from bile duct ligation. Am J Physiol Gastrointest Liver Physiol 314:G319–GG33

    Article  Google Scholar 

  10. Allen K, Jaeschke H, Copple BL (2011) Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am J Pathol 178:175–186

    Article  CAS  PubMed Central  Google Scholar 

  11. Qin P, Borges-Marcucci LA, Evans MJ et al (2005) Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes. Am J Physiol Gastrointest Liver Physiol 289:G267–GG73

    Article  CAS  Google Scholar 

  12. Cai S-Y, Boyer JL (2017) Studies on the mechanisms of bile acid initiated hepatic inflammation in cholestatic liver injury. Inflamm Cell Signal 4:e1561

    PubMed Central  Google Scholar 

  13. Wink S, Hiemstra SW, Huppelschoten S et al (2018) Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury. Arch Toxicol 92:1797–1814

    Article  CAS  PubMed Central  Google Scholar 

  14. Van den Hof WFPM, Coonen MLJ, van Herwijnen M et al (2014) Classification of hepatotoxicants using HepG2 cells: a proof of principle study. Chem Chem Res Toxicol 27:433–442

    Article  Google Scholar 

  15. Niemeijer M, Hiemstra S, Wink S et al (2018) Systems microscopy approaches in unraveling and predicting drug-induced liver injury (DILI). In: Chen M, Will Y (eds) Drug-induced liver toxicity, 1st edn. Springer Nature, New York

    Google Scholar 

  16. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  PubMed Central  Google Scholar 

  17. Du G (2012) FociPicker3D 3D and 2D particle counter. Institute of Modern Physics, CAS, China

    Google Scholar 

  18. Di Z, Herpers B, Fredriksson L et al (2012) Automated analysis of NF-kappaB nuclear translocation kinetics in high-throughput screening. PLoS One 7:e52337

    Article  CAS  PubMed Central  Google Scholar 

  19. Oppelt A, Kaschek D, Huppelschoten S et al (2018) Model-based identification of TNFα-induced IKKβ-mediated and IκBα-mediated regulation of NFκB signal transduction as a tool to quantify the impact of drug-induced liver injury compounds. npjsba 4:23

    Google Scholar 

  20. Ritz C, Baty F, Streibig JC et al (2016) Dose-response analysis using R. PLoS One 10:e0146021

    Article  Google Scholar 

  21. Davis JA, Gift JS, Zhao QJ (2011) Introduction to benchmark dose methods and U.S. EPA's benchmark dose software (BMDS) version 2.1.1. Toxicol Appl Pharmacol 254:181–191

    Article  CAS  Google Scholar 

  22. Akaike H (1973) Information theory and an extension of the maximum likelihood principle. In: Petrov BN, Csáki F (eds) Second international symposium on information theory. Akadémiai Kiadó, Tsahkadsor, Armenia, U.S.S.R.: Budapest, pp 267–281

    Google Scholar 

  23. Wickham H (2016) ggplot2—Elegant graphics for data analysis, vol XVI. Springer, New York, 260 pp

    Google Scholar 

  24. Poser I, Sarov M, Hutchins JR et al (2008) BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 5:409–415

    Article  CAS  PubMed Central  Google Scholar 

  25. Muyrers JP, Zhang Y, Testa G et al (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555–1557

    Article  CAS  PubMed Central  Google Scholar 

  26. Puigvert JC, de Bont H, van de Water B et al (2010) High-throughput live cell imaging of apoptosis. Curr Protoc Cell Biol Chapter 18:Unit 18.0.1-3

    Google Scholar 

  27. Yan K, Verbeek FJ (2012) Segmentation for high-throughput image analysis: watershed masked clustering, Berlin, Heidelberg, 2012. Springer, Berlin, Heidelberg, pp 25–41

    Google Scholar 

  28. R Development Core Team. (2013) R: a language and environment for statistical computing. R foundation for Statistical Computing

    Google Scholar 

  29. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184:39–51

    Article  CAS  Google Scholar 

  30. Hanshaw RG, Smith BD (2005) New reagents for phosphatidylserine recognition and detection of apoptosis. Bioorg Med Chem 13:5035–5042

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the FP7 DETECTIVE project (grant agreement 266838), IMI IP-DILI project (grant agreement 115336), and H2020 EU-ToxRisk project (grant agreement 681002).

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Authors and Affiliations

  1. Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands

    Johannes P. Schimming, Bas ter Braak, Marije Niemeijer, Steven Wink & Bob van de Water

Authors
  1. Johannes P. Schimming
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  2. Bas ter Braak
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  3. Marije Niemeijer
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  4. Steven Wink
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  5. Bob van de Water
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Corresponding author

Correspondence to Bob van de Water .

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Editors and Affiliations

  1. Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium

    Mathieu Vinken

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Schimming, J.P., ter Braak, B., Niemeijer, M., Wink, S., van de Water, B. (2019). System Microscopy of Stress Response Pathways in Cholestasis Research. In: Vinken, M. (eds) Experimental Cholestasis Research. Methods in Molecular Biology, vol 1981. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9420-5_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9420-5_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9419-9

  • Online ISBN: 978-1-4939-9420-5

  • eBook Packages: Springer Protocols

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