A Multi‐Parametric Fluorescent Assay for the Screening and Mechanistic Study of Drug‐Induced Steatosis in Liver Cells in Culture

Laia Tolosa1, M. José Gómez‐Lechón2, M. Teresa Donato3

1 Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS‐La Fe), Valencia, 2 CIBERehd, FIS, Barcelona, 3 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.15
DOI:  10.1002/cptx.20
Online Posting Date:  May, 2017
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Abstract

Human hepatic cells have been used for drug safety risk evaluations throughout early development phases. They provide rapid, cost‐effective early feedback to identify drug candidates with potential hepatotoxicity. This unit presents a cell‐based assay to evaluate the risk of liver damage associated with steatogenic drugs. Detailed protocols for cell exposure to test compounds and for the assessment of steatosis‐related cell parameters (intracellular lipid content, reactive oxygen species production, mitochondrial impairment, and cell death) are provided. A few representative results that illustrate the utility of this procedure for the screening of drug‐induced steatosis are shown. © 2017 by John Wiley & Sons, Inc.

Keywords: cultured cells; drug‐induced steatosis; fluorescent probes; high‐content screening; multi‐parametric assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Treatment of Cultured Cells with Test Compounds
  • Basic Protocol 2: Assessing Drug‐Induced Steatosis Using a High‐Content Screening Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Treatment of Cultured Cells with Test Compounds

  Materials
  • Phosphate‐buffered saline, pH 7.4 (PBS; see appendix 2A)
  • 50 mM sodium oleate stock solution in PBS
  • 50 mM sodium palmitate stock solution in PBS
  • Complete cell culture medium (e.g., Williams' E medium supplemented with 10% [v/v] fetal bovine serum, 2mM glutamine, and antibiotics)
  • Bovine serum albumin
  • Human hepatic cells cultured in 96‐well plates
  • Test compounds (positive [e.g., tetracycline and valproic acid] and negative steatosis inducers)
  • Dimethylsulfoxide (DMSO)
  • 37°C water bath
  • Cell culture incubator (37°C, 90% humidity, 5% CO 2)
  • Bath sonicator

Basic Protocol 2: Assessing Drug‐Induced Steatosis Using a High‐Content Screening Assay

  Materials
  • Fluorescent dyes:
    • 1 mg/ml Hoechst 33342 (e.g., Sigma‐Aldrich) dissolved in DMSO
    • 1 mg/ml propidium iodide (PI; e.g., Sigma‐Aldrich) dissolved in water
    • 10 µg/ml 4,4‐difluoro‐1,3,5,7,8‐pentamethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) 493/503 (e.g., Molecular Probes) dissolved in DMSO
    • 100 µM tetramethylrhodamine methyl ester (TMRM; e.g., Molecular Probes) dissolved in DMSO
    • 10 mg/ml 2′,7′‐dihydrodichlorofluorescein diacetate (DHCF‐DA; e.g., Molecular Probes) dissolved in DMSO
  • Complete cell culture medium (e.g., Williams' E medium supplemented with 10% [v/v] fetal bovine serum, 2mM glutamine, and antibiotics)
  • Previously treated cells and controls (see protocol 1)
  • 37°C water bath
  • Cell culture incubator (37°C, 90% humidity, 5% CO 2)
  • Aluminum foil, to cover plate
  • High‐content screening system and analysis software (e.g., Scan^R acquisition and analysis software)
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Figures

Videos

Literature Cited

Literature Cited
  Amacher, D. E. (2011). Strategies for the early detection of drug‐induced hepatic steatosis in preclinical drug safety evaluation studies. Toxicology, 279, 10–18. doi: 10.1016/j.tox.2010.10.006.
  Amacher, D. E. & Chalasani, N. (2014). Drug‐induced hepatic steatosis. Seminars in Liver Disease, 34, 205–214. doi: 10.1055/s‐0034‐1375960.
  Amacher, D. E. & Martin, B. A. (1997). Tetracycline‐induced steatosis in primary canine hepatocyte cultures. Fundamental and Applied Toxicology, 40, 256–263. doi: 10.1006/faat.1997.2389.
  Antherieu, S., Rogue, A., Fromenty, B., Guillouzo, A., & Robin, M. A. (2011). Induction of vesicular steatosis by amiodarone and tetracycline is associated with up‐regulation of lipogenic genes in HepaRG cells. Hepatology, 53, 1895–1905. doi: 10.1002/hep.24290.
  Benet, M., Moya, M., Donato, M. T., Lahoz, A., Hervás, D., Guzmán, C., … Jover, R. (2014). A simple transcriptomic signature able to predict drug‐induced hepatic steatosis. Archives of Toxicology, 88, 967–982. doi: 10.1007/s00204‐014‐1197‐7.
  Donato, M. T. & Gomez‐Lechon, M. J. (2012). Drug‐induced liver steatosis and phospholipidosis: Cell‐based assays for early screening of drug candidates. Current Drug Metabolism, 13, 1160–1173. doi: 10.2174/138920012802850001.
  Donato, M. T., Martinez‐Romero, A., Jimenez, N., Negro, A., Herrera, G., Castell, J. V., … Gómez‐Lechón, M. J. (2009). Cytometric analysis for drug‐induced steatosis in HepG2 cells. Chemico‐Biological Interactions, 181, 417–423. doi: 10.1016/j.cbi.2009.07.019.
  Donato, M. T., Tolosa, L., Jimenez, N., Castell, J. V., & Gomez‐Lechon, M. J. (2012). High‐content imaging technology for the evaluation of drug‐induced steatosis using a multiparametric cell‐based assay. Journal of Biomolecular Screening, 17, 394–400. doi: 10.1177/1087057111427586.
  Fromenty, B. & Pessayre, D. (1997). Impaired mitochondrial function in microvesicular steatosis. Effects of drugs, ethanol, hormones and cytokines. Journal of Hepatology, 26(Suppl 2), 43–53. doi: 10.1016/S0168‐8278(97)80496‐5.
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  Gomez‐Lechon, M. J., Donato, M. T., Martinez‐Romero, A., Jimenez, N., Castell, J. V., and O'Connor, J. E. (2007). A human hepatocellular in vitro model to investigate steatosis. Chemico‐Biological Interactions, 165, 106–116. doi: 10.1016/j.cbi.2006.11.004.
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  Schoonen, W. G., Stevenson, J. C., Westerink, W. M., & Horbach, G. J. (2012). Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue. Toxicology In Vitro, 26, 511–525. doi: 10.1016/j.tiv.2012.01.004.
  Tolosa, L., Gomez‐Lechon, M. J., Jimenez, N., Hervas, D., Jover, R., & Donato, M. T. (2016). Advantageous use of HepaRG cells for the screening and mechanistic study of drug‐induced steatosis. Toxicology and Applied Pharmacology, 302, 1–9. doi: 10.1016/j.taap.2016.04.007.
  Tolosa, L., Pinto, S., Donato, M. T., Lahoz, A., Castell, J. V., O'Connor, J. E., & Gómez‐Lechón, M. J. (2012). Development of a multiparametric cell‐based protocol to screen and classify the hepatotoxicity potential of drugs. The Journal of Toxicological Sciences, 127, 187–198. doi: 10.1093/toxsci/kfs083.
  Tomida, T., Okamura, H., Satsukawa, M., Yokoi, T., & Konno, Y. (2015). Multiparametric assay using HepaRG cells for predicting drug‐induced liver injury. Toxicology Letters, 236, 16–24. doi: 10.1016/j.toxlet.2015.04.014.
  Xu, J. J., Henstock, P. V., Dunn, M. C., Smith, A. R., Chabot, J. R., & de Graaf, D. (2008). Cellular imaging predictions of clinical drug‐induced liver injury. The Journal of Toxicological Sciences, 105, 97–105. doi: 10.1093/toxsci/kfn109.s.
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