Mechanisms and in vitro models of drug-induced cholestasis

  • Eva Gijbels
  • Vânia Vilas-Boas
  • Neel Deferm
  • Lindsey Devisscher
  • Hartmut Jaeschke
  • Pieter Annaert
  • Mathieu VinkenEmail author
Review Article


Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided.


Drug-induced cholestasis Liver Mechanisms In vitro models 



α-Naphthyl isothiocyanate


Activating transcription factor


Bile salt export pump


Cholestatic index


Cytochrome P450


Drug-induced cholestasis index


Drug-induced liver injury


Deoxyribonucleic acid


Extracellular matrix


Early growth response factor-1


Endoplasmic reticulum


Farnesoid X receptor


Induced pluripotent stem cells


Inositol-requiring protein 1α


Kelch-like ECH-associated protein 1


Microtubule-associated protein 1 light chain 3


Multidrug resistance protein


Mixed lineage kinase domain-like


Multidrug resistance-associated protein


Nucleotide-binding and oligomerization leucine-rich repeat protein 3


Nuclear-related factor 2


Organic anion transporting polypeptides


Protein kinase RNA-like endoplasmic reticulum kinase


Precision-cut liver slice(s)


Pregnane X receptor


Receptor interacting protein


Rho-associated protein kinase


Reactive oxygen species


Toll-like receptor 9


Uridine diphosphate glucuronosyltransferase



This work was supported by the Grants of the European Research Council, the Center for Alternatives to Animal Testing at Johns Hopkins University Baltimore, USA, the Fund for Scientific Research, Flanders and the University Hospital of the Willy Gepts Fonds UZ, Brussels.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.


  1. Afonso MB, Rodrigues PM, Simao AL et al (2016) Activation of necroptosis in human and experimental cholestasis. Cell Death Dis 7:e2390CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aleo MD, Shah F, He K et al (2017) Evaluating the role of multidrug resistance protein 3 (MDR3) inhibition in predicting drug-induced liver injury using 125 pharmaceuticals. Chem Res Toxicol 30:1219–1229CrossRefPubMedGoogle Scholar
  3. Ali I, Welch MA, Lu Y et al (2017) Identification of novel MRP3 inhibitors based on computational models and validation using an in vitro membrane vesicle assay. Eur J Pharm Sci 103:52–59CrossRefPubMedPubMedCentralGoogle Scholar
  4. Allen K, Kim ND, Moon JO et al (2010) Upregulation of early growth response factor-1 by bile acids requires mitogen-activated protein kinase signaling. Toxicol Appl Pharmacol 243:63–67CrossRefPubMedGoogle Scholar
  5. 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 178:175–186CrossRefPubMedPubMedCentralGoogle Scholar
  6. Annaert PP, Brouwer KL (2005) Assessment of drug interactions in hepatobiliary transport using rhodamine 123 in sandwich-cultured rat hepatocytes. Drug Metab Dispos 33:388–394CrossRefPubMedGoogle Scholar
  7. Anthérieu S, Chesné C, Li R et al (2010) Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab Dispos 38:516–525CrossRefPubMedGoogle Scholar
  8. Anthérieu S, Bachour-El Azzi P, Dumont J et al (2013) Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human HepaRG cells. Hepatology 57:1518–1529CrossRefPubMedGoogle Scholar
  9. Arduini A, Serviddio G, Escobar J et al (2011) Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions. Am J Physiol Gastrointest Liver Physiol 301:G119–G127CrossRefPubMedGoogle Scholar
  10. Bachour-El Azzi P, Sharanek A, Burban A et al (2015) Comparative localization and functional activity of the main hepatobiliary transporters in HepaRG Cells and primary human hepatocytes. Toxicol Sci 145:157–168CrossRefPubMedPubMedCentralGoogle Scholar
  11. Bale SS, Vernetti L, Senutovitch N et al (2014) In vitro platforms for evaluating liver toxicity. Exp Biol Med 239:1180–1191CrossRefGoogle Scholar
  12. Baze A, Parmentier C, Hendriks DFG et al (2018) Three-dimensional spheroid primary human hepatocytes in monoculture and coculture with nonparenchymal cells. Tissue Eng Part C Methods 24:534–545CrossRefPubMedGoogle Scholar
  13. Begriche K, Massart J, Robin MA, Borgne-Sanchez A et al (2011) Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. J Hepatol 54:773–794CrossRefPubMedGoogle Scholar
  14. Bell CC, Hendriks DF, Moro SM et al (2016) Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease. Sci Rep 6:25187CrossRefPubMedPubMedCentralGoogle Scholar
  15. Bell CC, Lauschke VM, Vorrink SU et al (2017) Transcriptional, functional, and mechanistic comparisons of stem cell-derived hepatocytes, HepaRG cells, and three-dimensional human hepatocyte spheroids as predictive in vitro systems for drug-induced liver injury. Drug Metab Dispos 45:419–429CrossRefPubMedPubMedCentralGoogle Scholar
  16. Bell CC, Dankers ACA, Lauschke VM et al (2018) Comparison of hepatic 2D sandwich cultures and 3D spheroids for long-term toxicity applications: a multicenter study. Toxicol Sci 162:655–666CrossRefPubMedPubMedCentralGoogle Scholar
  17. Benien P, Swami A (2014) 3D tumor models: history, advances and future perspectives. Future Oncol 10:1311–1327CrossRefPubMedGoogle Scholar
  18. Birben E, Sahiner UM, Sackesen C et al (2012) Oxidative stress and antioxidant defense. World Allergy Organ J 5:9–19CrossRefPubMedPubMedCentralGoogle Scholar
  19. Bhamidimarri KR, Schiff E (2013) Drug-induced cholestasis. Clin Liver Dis 17:519–531CrossRefPubMedGoogle Scholar
  20. Bhat TA, Chaudhary AK, Kumar S et al (2017) Endoplasmic reticulum-mediated unfolded protein response and mitochondrial apoptosis in cancer. Biochim Biophys Acta 1867:58–66Google Scholar
  21. Brophy CM, Luebke-Wheeler JL, Amiot BP et al (2009) Rat hepatocyte spheroids formed by rocket technique maintain differentiated hepatocyte gene expression and function. Hepatology 49:578–586CrossRefPubMedPubMedCentralGoogle Scholar
  22. Burban A, Sharanek A, Hue R et al (2017) Penicillinase-resistant antibiotics induce non-immune-mediated cholestasis through HSP27 activation associated with PKC/P38 and PI3K/AKT signaling pathways. Sci Rep 7:1815CrossRefPubMedPubMedCentralGoogle Scholar
  23. Burban A, Sharanek A, Guguen-Guillouzo C et al (2018) Endoplasmic reticulum stress precedes oxidative stress in antibiotic-induced cholestasis and cytotoxicity in human hepatocytes. Free Radic Biol Med 115:166–178CrossRefPubMedGoogle Scholar
  24. Burbank MG, Burban A, Sharanek A et al (2016) Early alterations of bile canaliculi dynamics and the rho kinase/myosin light chain kinase pathway are characteristics of drug-induced intrahepatic cholestasis. Drug Metab Dispos 44:1780–1793CrossRefPubMedGoogle Scholar
  25. Cai J, DeLaForest A, Fisher J et al (2012) Protocol for directed dedifferentiation of human pluripotent stem cells toward a hepatocyte facte. In: StemBook (Harvard Stem Cell Institute)Google Scholar
  26. Cai SY, Ouyang X, Chen Y et al (2017) Bile acids initiate cholestatic liver injury by triggering a hepatocyte-specific inflammatory response. JCI Insight 2:e90780CrossRefPubMedPubMedCentralGoogle Scholar
  27. Castell JV, Jover R, Martinez-Jimenez CP et al (2006) Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies. Expert Opin Drug Metab Toxicol 2:183–212CrossRefPubMedGoogle Scholar
  28. Chatterjee S, Annaert P (2018) Drug-induced cholestasis: mechanisms, models and markers. Curr Drug Metab 19:808–818CrossRefPubMedGoogle Scholar
  29. Chatterjee S, Richert L, Augustijns P et al (2014) Hepatocyte-based in vitro model for assessment of drug-induced cholestasis. Toxicol Appl Pharmacol 274:124–136CrossRefPubMedGoogle Scholar
  30. Chen X, Zhang C, Wang H et al (2009) Altered integrity and decreased expression of hepatocyte tight junctions in rifampicin-induced cholestasis in mice. Toxicol Appl Pharmacol 240:26–36CrossRefPubMedGoogle Scholar
  31. Copple BL, Jaeschke H, Klaassen CD (2010) Oxidative stress and the pathogenesis of cholestasis. Semin Liver Dis 30:195–204CrossRefPubMedGoogle Scholar
  32. Cuperus FJ, Claudel T, Gautherot J et al (2014) The role of canalicular ABC transporters in cholestasis. Drug Metab Dispos 42:546–560CrossRefPubMedGoogle Scholar
  33. Das S (2018) Chapter 7: extrapolation of in vitro results to predict human toxicity. In Vitro Toxicol 2018:127–142CrossRefGoogle Scholar
  34. Dixon PH, Weerasekera N, Linton KJ et al (2000) Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking. Hum Mol Genet 9:1209–1217CrossRefPubMedGoogle Scholar
  35. De Bruyn T, Chatterjee S, Fattah S et al (2013) Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 9:589–616CrossRefPubMedGoogle Scholar
  36. de Lima TVM, Tagliati CA (2014) Hepatobiliary transporters in drug-induced cholestasis: a perspective on the current identifying tools. Expert Opin Drug Metab Toxicol 10:581–597CrossRefGoogle Scholar
  37. Donato MT, Jover R, Gomez-Lechon MJ (2013) Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering. Curr Drug Metab 14:946–968CrossRefPubMedGoogle Scholar
  38. Du Y, Han R, Wen F et al (2008) Synthetic sandwich culture of 3D hepatocyte monolayer. Biomaterials 29:290–301CrossRefPubMedGoogle Scholar
  39. Erlinger S (2015) NTCP deficiency: a new inherited disease of bile acid transport. Clin Res Hepatol Gastroenterol 39:7–8CrossRefPubMedGoogle Scholar
  40. European Association for the Study of the Liver (2009) EASL Clinical practice guidelines: management of cholestatic liver diseases. J Hepatol 51:237–267CrossRefGoogle Scholar
  41. Fickert P, Trauner M, Fuchsbichler A et al (2002) Cytokeratins as targets for bile acid-induced toxicity. Am J Pathol 160:491–499CrossRefPubMedPubMedCentralGoogle Scholar
  42. Fraczek J, Bolleyn J, Vanhaecke T et al (2013) Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch Toxicol 87:577–610CrossRefPubMedGoogle Scholar
  43. Fukuda J, Sakai Y, Nakazawa K (2006) Novel hepatocyte culture system developed using microfabrication and collagen/polyethylene glycol microcontact printing. Biomaterials 27:1061–1070CrossRefPubMedGoogle Scholar
  44. Gao L, Lv G, Guo X et al (2014) Activation of autophagy protects against cholestasis-induced hepatic injury. Cell Biosci 4:47CrossRefPubMedPubMedCentralGoogle Scholar
  45. Gao Y, Zhang X, Zhang L et al (2017) Distinct gene expression and epigenetic signatures in hepatocyte-like cells produced by different strategies from the same donor. Stem Cell Reports 9:1813–1824CrossRefPubMedPubMedCentralGoogle Scholar
  46. Garzel B, Yang H, Zhang L et al (2014) The role of bile salt export pump gene repression in drug-induced cholestatic liver toxicity. Drug Metab Dispos 42:318–322CrossRefPubMedPubMedCentralGoogle Scholar
  47. Godoy P, Hewitt NJ, Albrecht U 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
  48. Gong Z, Zhou J, Zhao S et al (2016) Chenodeoxycholic acid activates NLRP3 inflammasome and contributes to cholestatic liver fibrosis. Oncotarget 7:83951–83963PubMedPubMedCentralGoogle Scholar
  49. Greupink R, Nabuurs SB, Zarzycka B et al (2012) In silico identification of potential cholestasis-inducing agents via modeling of Na(+)-dependent taurocholate cotransporting polypeptide substrate specificity. Toxicol Sci 129:35–48CrossRefPubMedGoogle Scholar
  50. Gripon P, Rumin S, Urban S et al (2002) Infection of a human hepatoma cell line by hepatitis B virus. Proc Nati Acad Sci USA 99:15655–15660CrossRefGoogle Scholar
  51. Guguen-Guillouzo C, Guillouzo A (2010) General review on in vitro hepatocyte models and their applications. Methods Mol Biol 640:1–40CrossRefPubMedGoogle Scholar
  52. Guguen-Guillouzo C, Corlu A, Guillouzo A (2010) Stem cell-derived hepatocytes and their use in toxicology. Toxicology 270:3–9CrossRefPubMedGoogle Scholar
  53. Gujral JS, Farhood A, Bajt ML et al (2003) Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 38:355–363CrossRefPubMedGoogle Scholar
  54. Gujral JS, Liu J, Farhood A et al (2004) Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation. Hepatology 40:998–1007CrossRefPubMedGoogle Scholar
  55. Gunness P, Mueller D, Shevchenko V et al (2013) 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci 133:67–78CrossRefPubMedGoogle Scholar
  56. Hassanein T, Frederick T (2004) Mitochondrial dysfunction in liver disease and organ transplantation. Mitochondrion 4:609–620CrossRefPubMedGoogle Scholar
  57. Halilbasic E, Baghdasaryan A, Trauner M (2013) Nuclear receptors as drug targets in cholestatic liver diseases. Clin Liver Dis 17:161–189CrossRefPubMedPubMedCentralGoogle Scholar
  58. Hannan NRF, Segeritz CP, Touboul T et al (2013) Production of hepatocyte like cells from human pluripotent stem cells. Nat Protoc 8:430–437CrossRefPubMedPubMedCentralGoogle Scholar
  59. Hasirci V, Berthiaume F, Bondre DP et al (2001) Expression of liver-specific functions by rat hepatocytes seeded in treated poly (lactic-co-glycocholic) acid biodegradable foams. Tissue Eng 7:379–386CrossRefGoogle Scholar
  60. Hendriks DFG, Puigvert LF, Messner S et al (2016) Hepatic 3D spheroid models for the detection and study of compounds with cholestatic liability. Sci Rep 6:35434CrossRefPubMedPubMedCentralGoogle Scholar
  61. Hengstler JG, Utesch D, Steinberg P et al (2000) Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab Rev 32:81–118CrossRefPubMedGoogle Scholar
  62. Hengstler JG, Hammad S, Ghallab A et al (2014) In vitro systems for hepatotoxicity testing. Vitro Toxicol Syst 2014:27–44CrossRefGoogle Scholar
  63. Henkel AS, LeCuyer B, Olivares S et al (2017) Endoplasmic reticulum stress regulates hepatic bile acid metabolism in mice. Cell Mol Gastroenterol Hepatol 3:261–271CrossRefPubMedGoogle Scholar
  64. Hoffmaster KA, Turncliff RZ, Lecluyse EL et al (2004) P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide. Pharm Res 21:1294–1302CrossRefPubMedGoogle Scholar
  65. Huang P, Zhang L, Gao Y et al (2014) Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 14:370–384CrossRefPubMedGoogle Scholar
  66. Humbert L, Maubert MA, Wolf C et al (2012) Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients. J Chromatogr B Analyt Technol Biomed Life Sci 899:135–145CrossRefPubMedGoogle Scholar
  67. Hyogo H, Tazuma S, Kajiyama G et al (1999) Transcytotic vesicle fusion is reduced in cholestatic rats: redistribution of phospholipids in the canalicular membrane. Dig Dis Sci 44:1662–1668CrossRefPubMedGoogle Scholar
  68. Imagawa K, Takayma K, Isoyama S et al (2017) Generation of a bile salt export pump deficiency model using patient-specific induced pluripotent stem cell-derived hepatocyte-like cells. Sci Rep 7:41806CrossRefPubMedPubMedCentralGoogle Scholar
  69. Jaeschke H (2011) Reactive oxygen and mechanisms of inflammatory liver injury: present concepts. J Gastroenterol Hepatol 1:173–179CrossRefGoogle Scholar
  70. Jaeschke H, Krell H, Pfaff E (1983) No increase of biliary permeability in ethinylestradiol-treated rats. Gastroenterology 85:808–814PubMedGoogle Scholar
  71. Jaeschke H, Trummer E, Krell H (1987) Increase in biliary permeability subsequent to intrahepatic cholestasis by estradiol valerate in rats. Gastroenterology 93:533–538CrossRefPubMedGoogle Scholar
  72. Jansen PL, Ghallab A, Vartak N et al (2017) The ascending pathophysiology of cholestatic liver disease. Hepatology 65:722–738CrossRefPubMedGoogle Scholar
  73. Kawamoto T, Ito Y, Morita O et al (2017) Mechanism-based risk assessment strategy for drug-induced cholestasis using the transcriptional benchmark dose derived by toxicogenomics. J Toxicol Sci 42:427–436CrossRefPubMedGoogle Scholar
  74. Keller GM (1995) In vitro differentiation of embyronic stem cells. Curr Opin Cell Biol 7:862–869CrossRefPubMedGoogle Scholar
  75. Kelm JM, Fussenegger M (2004) Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol 22:195–202CrossRefPubMedGoogle Scholar
  76. Kelm JM, Timmins NE, Brown CJ et al (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180CrossRefPubMedGoogle Scholar
  77. Kia R, Sison RLC, Heslop J et al (2013) Stem cell-derived hepatocytes as a predictive model for drug-induced liver injury: are we there yet? Br J Clin Pharmacol 75:885–896CrossRefPubMedGoogle Scholar
  78. Kim ND, Moon JO, Slitt AL et al (2006) Early growth response factor-1 is critical for cholestatic liver injury. Toxicol Sci 90:586–595CrossRefPubMedGoogle Scholar
  79. Köck K, Ferslew BC, Netterberg I et al (2014) Risk factors for development of cholestatic drug-induced liver injury: inhibition of hepatic basolateral bile acid transporters multidrug resistance-associated proteins 3 and 4. Drug Metab Dispos 42:665–674CrossRefPubMedPubMedCentralGoogle Scholar
  80. Kotsampasakou E, Ecker GF (2017) Predicting drug-induced cholestasis with the help of hepatic transporters—an in silico modeling approach. J Chem Inf Model 57:608–615CrossRefPubMedPubMedCentralGoogle Scholar
  81. Krell H, Höke H, Pfaff E (1982) Development of intrahepatic cholestasis by alpha-naphthylisothiocyanate in rats. Gastroenterology 82:507–514PubMedGoogle Scholar
  82. Lakshmipathy U, Verfaillie C (2005) Stem cell plasticity. Blood Rev 19:29–38CrossRefPubMedGoogle Scholar
  83. Landry J, Bernier D, Ouellet C et al (1985) Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J Cell Biol 101:914–923CrossRefPubMedGoogle Scholar
  84. Lang C, Meier Y, Stieger B et al (2007) Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury. Pharmacogenet Genomics 17:47–60CrossRefPubMedGoogle Scholar
  85. Laverty HG, Antoine DJ, Benson C et al (2010) The potential of cytokines as safety biomarkers for drug-induced liver injury. Eur J Clin Pharmacol 66:961–976CrossRefPubMedGoogle Scholar
  86. Lerche-Legrand C, Toutain HJ (2000) Precision-cut liver slices: characteristics and use for in vitro pharmaco-toxicology. Toxicology 153:221–253CrossRefGoogle Scholar
  87. Li T, Apte U (2015) Bile acid metabolism and signaling in cholestasis, inflammation, and cancer. Adv Pharmacol 74:263–302CrossRefPubMedPubMedCentralGoogle Scholar
  88. Li P, He K, Li J et al (2017) The role of Kupffer cells in hepatic diseases. Mol Immunol 85:222–229CrossRefPubMedGoogle Scholar
  89. Linkermann A, Green DR (2014) Necroptosis. N Engl J Med 370:455–465CrossRefPubMedPubMedCentralGoogle Scholar
  90. Malhi H, Kaufman RJ (2011) Endoplasmic reticulum stress in liver disease. J Hepatol 54:795–809CrossRefPubMedGoogle Scholar
  91. Manley S, Ni HM, Kong B et al (2014) Suppression of autophagic flux by bile acids in hepatocytes. Toxicol Sci 137:478–490CrossRefPubMedGoogle Scholar
  92. Mariotti V, Strazzabosco M, Fabris L et al (2017) Animal models of biliary injury and altered bile acid metabolism. Biochim Biophys Acta 1864:1254–1261CrossRefPubMedCentralGoogle Scholar
  93. Messner S, Agarkova I, Moritz W et al (2013) Multi-cell type human liver microtissues for hepatotoxicity testing. Arch Toxicol 87:209–213CrossRefPubMedGoogle Scholar
  94. Messner S, Fredriksson L, Lauschke VM et al (2018) Transcriptomic, proteomic, and functional long-term characterization of multicellular three-dimensional human liver microtissues. Applied In Vitro Toxicology 4:1–12CrossRefGoogle Scholar
  95. Mitchell C, Mahrouf-Yorgov M, Mayeuf A et al (2011) Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease. Lab Invest 91:273–282CrossRefPubMedGoogle Scholar
  96. Morgan RE, Trauner M, van Staden CJ et al (2010) Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol Sci 118:485–500CrossRefPubMedGoogle Scholar
  97. Moscona A (1961) Rotation-mediated histogenetic aggregation of dissociated cells. A quantifiable approach to cell interactions in vitro. Exp Cell Res 22:455–475CrossRefPubMedGoogle Scholar
  98. Mottino AD, Cao J, Veggi LM et al (2002) Altered localization and activity of canalicular Mrp2 in estradiol-17β-d-glucuronide-induced cholestasis. Hepatology 35:1409–1419CrossRefPubMedGoogle Scholar
  99. Mottino AD, Hoffman T, Crocenzi FA et al (2007) Disruption of function and localization of tight junctional structures and Mrp2 in sustained estradiol-17β-D-glucuronide-induced cholestasis. Am J Physiol Gastrointest Liver Physiol 293:G391–G402CrossRefPubMedGoogle Scholar
  100. Mueller SO, Guillouzo A, Hewitt PG et al (2015) Drug biokinetic and toxicity assessments in rat and human primary hepatocytes and HepaRG cells within the EU-funded Predict-IV project. Toxicol In Vitro 30:19–26CrossRefPubMedGoogle Scholar
  101. Natale A, Boeckmans J, Desmae T et al (2018) Hepatic cells derived from human skin progenitors show a typical phospholipidotic response upon exposure to amiodarone. Toxicol Lett 284:184–194CrossRefPubMedGoogle Scholar
  102. Nguyen KD, Sundaram V, Ayoub WS (2014) Atypical causes of cholestasis. World J Gastroenterol 20:9418–9426CrossRefPubMedPubMedCentralGoogle Scholar
  103. Ni X, Gao Y, Wu Z et al (2016) Functional human induced hepatocytes (hiHeps) with bile acid synthesis and transport capacities: a novel in vitro cholestatic model. Sci Rep 6:38694CrossRefPubMedPubMedCentralGoogle Scholar
  104. Noor F (2015) A shift in paradigm towards human biology-based systems for cholestatic-liver diseases. J Physiol 593:5043–5055CrossRefPubMedPubMedCentralGoogle Scholar
  105. Olinga P, Elferink MG, Draaisma AL et al (2008) Coordinated induction of drug transporters and phase I and II metabolism in human liver slices. Eur J Pharm Sci 33:380–389CrossRefPubMedGoogle Scholar
  106. Olson H, Betton G, Robinson D et al (2000) Concordance of the toxicity of pharmaceuticals in humans and in animals. Regul Toxicol Pharmacol 32:56–67CrossRefPubMedGoogle Scholar
  107. Oorts M, Richert L, Annaert P (2015) Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture. J Pharmacol Toxicol Methods 73:63–71CrossRefPubMedGoogle Scholar
  108. Oorts M, Baze A, Bachellier P et al (2016) Drug-induced cholestasis risk assessment in sandwich-cultured human hepatocytes. Toxicol In Vitro 2016:179–186CrossRefGoogle Scholar
  109. Ozer J, Ratner M, Shaw M et al (2008) The current state of serum biomarkers of hepatotoxicity. Toxicology 245:194–205CrossRefPubMedGoogle Scholar
  110. Padda MS, Sanchez M, Akhtar AJ et al (2011) Drug-induced cholestasis. Hepatology 53:1377–1387CrossRefPubMedPubMedCentralGoogle Scholar
  111. Palmeira CM, Rolo AP (2004) Mitochondrially-mediated toxicity of bile acids. Toxicology 203:1–15CrossRefPubMedGoogle Scholar
  112. Parent R, Marion MJ, Furio L et al (2004) Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126:1147–1156CrossRefPubMedGoogle Scholar
  113. Parmentier C, Truisi GL, Moenks K et al (2013) Transcriptomic hepatotoxicity signature of chlorpromazine after short-and long-term exposure in primary human sandwich cultures. Drug Metab Dispos 41:1835–1842CrossRefPubMedGoogle Scholar
  114. Parmentier C, Hendriks DFG, Heyd B et al (2018) Inter-individual differences in the susceptibility of primary human hepatocytes towards drug-induced cholestasis are compound and time dependent. Toxicol Lett 295:187–194CrossRefPubMedGoogle Scholar
  115. Pauli-Magnus C, Meier PJ (2006) Hepatobiliary transporters and drug-induced cholestasis. Hepatology 44:778–787CrossRefPubMedGoogle Scholar
  116. Perez MJ, Briz O (2009) Bile-acid-induced cell injury and protection. World J Gastroenterol 15:1677–1689CrossRefPubMedPubMedCentralGoogle Scholar
  117. Przybylak KR, Cronin MT (2012) In silico models for drug-induced liver injury–current status. Expert Opin Drug Metab Toxicol 8:201–217CrossRefPubMedGoogle Scholar
  118. Qiu X, Zhang Y, Liu T et al (2016) Disruption of BSEP function in HepaRG cells alters bile acid disposition and is a susceptive factor to drug-induced cholestatic injury. Mol Pharm 13:1206–1216CrossRefPubMedGoogle Scholar
  119. Ramboer E, Vanhaecke T, Rogiers V et al (2013) Primary hepatocyte cultures as prominent in vitro tools to study hepatic drug transporters. Drug Metab Rev 45:196–217CrossRefPubMedGoogle Scholar
  120. Rathinam Vijay AK, Vanaja Sivapriya K, Waggoner L et al (2012) TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150:606–619CrossRefPubMedPubMedCentralGoogle Scholar
  121. Rodrigues RM, De Kock J, Branson S et al (2013) Human skin-derived stem cells as a novel cell source for in vitro hepatotoxicity screening of pharmaceuticals. Stem Cells Dev 23:44–55CrossRefPubMedPubMedCentralGoogle Scholar
  122. Rodrigues RM, Sachinidis A, De Boe V et al (2015) Identification of potential biomarkers of hepatitis B-induced acute liver failure using hepatic cells derived from human skin precursors. Toxicol In Vitro 29:1231–1239CrossRefPubMedGoogle Scholar
  123. Rodrigues RM, Branson S, De Boe V et al (2016) In vitro assessment of drug-induced liver steatosis based on human dermal stem cell-derived hepatic cells. Arch Toxicol 90:677–689CrossRefPubMedGoogle Scholar
  124. Russell WMS, Burch RL (1959) The principles of humane experimental techniqueGoogle Scholar
  125. Sasaki M, Yoshimura-Miyakoshi M, Sato Y et al (2015) A possible involvement of endoplasmic reticulum stress in biliary epithelial autophagy and senescence in primary biliary cirrhosis. J Gastroenterol 50:984–995CrossRefPubMedGoogle Scholar
  126. Schulz S, Schmitt S, Wimmer R et al (2013) Progressive stages of mitochondrial destruction caused by cell toxic bile salts. Biochim Biophys Acta 1828:2121–2133CrossRefPubMedGoogle Scholar
  127. Seglen PO, Reith A (1976) Ammonia inhibition of protein degradation in isolated rat hepatocytes. Quantitative ultrastructural alterations in the lysosomal system. Exp Cell Res 100:276–280CrossRefPubMedGoogle Scholar
  128. Sharanek A, Azzi PB, Al-Attrache H et al (2014) Different dose-dependent mechanisms are involved in early cyclosporine a-induced cholestatic effects in HepaRG cells. Toxicol Sci 141:244–253CrossRefPubMedPubMedCentralGoogle Scholar
  129. Sharanek A, Burban A, Burbank M et al (2016) Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs. Sci Rep 6:24709CrossRefPubMedPubMedCentralGoogle Scholar
  130. Sharanek A, Burban A, Humbert L et al (2017) Progressive and preferential cellular accumulation of hydrophobic bile acids induced by cholestatic drugs is associated with inhibition of their amidation and sulfatation. Drug Metab Dispos 45:1292–1303CrossRefPubMedGoogle Scholar
  131. Sison-Young RL, Mitsa D, Jenkins RE et al (2015) Comparative proteomic characterization of 4 human liver-derived single cell culture models reveals significant variation in the capacity of drug disposition, bioactivation and detoxication. Toxicol Sci 147:412–424CrossRefPubMedPubMedCentralGoogle Scholar
  132. Smith DJ, Gordon ER (1987) Membrane fluidity and cholestasis. J Hepatol 5:362–365CrossRefPubMedGoogle Scholar
  133. Snykers S, De Kock J, Rogiers V et al (2009) In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art. Stem cells 27:577–605CrossRefPubMedPubMedCentralGoogle Scholar
  134. Snykers S, De Kock J, Vanhaecke V et al (2011) Hepatic differentiation of mesenchymal stem cells: in vitro strategies. Methods Mol Biol 698:305–314CrossRefPubMedGoogle Scholar
  135. Soldatow VY, LeCluyse EL, Griffith LG et al (2013) In vitro models for liver toxicity testing. Toxicol Res (Camb) 2:23–39CrossRefGoogle Scholar
  136. Song JY, Van Marle J, Van Noorden CJ et al (1996) Redistribution of Ca2+, Mg2+-ATPase activity in relation to alterations of the cytoskeleton and tight junctions in hepatocytes of cholestatic rat liver. Eur J Cell Biol 71:277–285PubMedGoogle Scholar
  137. Song JY, Van Noorden CJF, Frederiks WM (1998) The involvement of altered vesicle transport in redistribution of Ca2+, Mg2+-ATPase in cholestatic rat liver. Histochem J 30:909–916CrossRefPubMedGoogle Scholar
  138. Song Z, Cai J, Liu Y et al (2009) Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 19:1233–1242CrossRefPubMedGoogle Scholar
  139. Spivey JR, Bronk SF, Gores GJ (1993) Glycochenodeoxycholate-induced lethal hepatocellular injury in rat hepatocytes. Role of ATP depletion and cytosolic free calcium. J Clin Invest 92:17–24CrossRefPubMedPubMedCentralGoogle Scholar
  140. Starokozhko V, Greupink R, van de Broek P et al (2017a) Rat precision-cut liver slices predict drug-induced cholestatic injury. Arch Toxicol 91:3403–3413CrossRefPubMedPubMedCentralGoogle Scholar
  141. Starokozhko V, Vatakuti S, Schievink B et al (2017b) Maintenance of drug metabolism and transport functions in human precision-cut liver slices during prolonged incubation for 5 days. Arch Toxicol 91:2079–2092CrossRefPubMedGoogle Scholar
  142. Stehlik C, Lee SH, Dorfleutner A et al (2003) Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation. J Immunol 171:6154–6163CrossRefPubMedGoogle Scholar
  143. Strnad P, Stumptner C, Zatloukal K et al (2008) Intermediate filament cytoskeleton of the liver in health and disease. Histochem Cell Biol 129:735–749CrossRefPubMedPubMedCentralGoogle Scholar
  144. Swift B, Pfeifer ND, Brouwer KLR (2010) Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev 42:446–471CrossRefPubMedPubMedCentralGoogle Scholar
  145. Szabo M, Veres Z, Baranyai Z et al (2013) Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict a risk of drug induced hepatotoxicity. PLoS One 8:e59432CrossRefPubMedPubMedCentralGoogle Scholar
  146. Szalowska E, Stoopen G, Groot MJ et al (2013) Treatment of mouse liver slices with cholestatic hepatotoxicants results in down-regulation of Fxr and its target genes. BMC Med Genom 6:39CrossRefGoogle Scholar
  147. de Vree JM, Jacquemin E, Sturm E et al (1998) Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci USA 95:282–287CrossRefPubMedGoogle Scholar
  148. Tagliacozzi D, Mozzi AF, Casetta B et al (2003) Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem mass spectrometry: a simple and rapid one-step method. Clin Chem Lab Med 41:1633–1641CrossRefPubMedGoogle Scholar
  149. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676CrossRefPubMedGoogle Scholar
  150. Takahashi Y, Hori Y, Yamamoto T (2015) 3D spheroid cultures improve the metabolic gene expression profiles of HepaRG cells. Biosci Rep 35:e00208PubMedPubMedCentralGoogle Scholar
  151. Tamaki N, Hatano E, Taura K et al (2008) CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury. Am J Physiol Gastrointest Liver Physiol 294:G498–G505CrossRefPubMedGoogle Scholar
  152. Tiao MM, Lin TK, Wang PW et al (2009) The role of mitochondria in cholestatic liver injury. Chang Gung Med J 32:346–353PubMedGoogle Scholar
  153. Tostões RM, Leite SB, Serra M et al (2012) Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology 55:1227–1236CrossRefPubMedGoogle Scholar
  154. Trauner M, Boyer JL (2003) Bile salt transporters: molecular characterization, function and regulation. Physiol Rev 83:663–671CrossRefGoogle Scholar
  155. Trauner M, Arrese M, Soroka CJ et al (1997) The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 113:255–264CrossRefPubMedGoogle Scholar
  156. Van den Hof WF, Coonen ML, van Herwijnen M et al (2014) Classification of hepatotoxicants using HepG2 cells: a proof of principle study. Chem Res Toxicol 27:433–442CrossRefPubMedGoogle Scholar
  157. van Zijl F, Mikulits W (2010) Hepatospheres: three dimensional cell cultures resemble physiological conditions of the liver. World J Hepatol 2:1–7CrossRefPubMedPubMedCentralGoogle Scholar
  158. Vartak N, Damle-Vartak A, Richter B et al (2016) Cholestasis-induced adaptive remodeling of interlobular bile ducts. Hepatology 63:951–964CrossRefPubMedPubMedCentralGoogle Scholar
  159. Vatakuti S, Pennings JL, Gore E et al (2016) Classification of cholestatic and necrotic hepatotoxicants using transcriptomics on human precision-cut liver slices. Chem Res Toxicol 29:342–351CrossRefPubMedGoogle Scholar
  160. Vatakuti S, Olinga P, Pennings JL et al (2017) Validation of precision-cut liver slices to study drug-induced cholestasis: a transcriptomics approach. Arch Toxicol 91:1401–1412CrossRefPubMedGoogle Scholar
  161. Vinken M, Elaut G, Henkens T et al (2006) Rat hepatocyte cultures: collagen gel sandwich and immobilization cultures. Methods Mol Biol 320:247–254PubMedGoogle Scholar
  162. Vinken M, Landesmann B, Goumenou M et al (2013) Development of an adverse outcome pathway from drug-mediated bile salt export pump inhibition to cholestatic liver injury. Toxicol Sci 136:97–106CrossRefPubMedGoogle Scholar
  163. Vorrink SU, Ullah S, Schmidt S et al (2017) Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics. FASEB J 31:2696–2708CrossRefPubMedPubMedCentralGoogle Scholar
  164. Vorrink SU, Zhou Y, Ingelman-Sundberg M et al (2018) Prediction of drug-induced hepatotoxicity using long-term stable primary hepatic 3D spheroid cultures in chemically defined conditions. Toxicol Sci 163:655–665CrossRefPubMedPubMedCentralGoogle Scholar
  165. Wagner M, Zollner G, Trauner M (2009) New molecular insights into the mechanisms of cholestasis. J Hepatol 51:565–580CrossRefPubMedGoogle Scholar
  166. Wei Y, Rector RS, Thyfault JP et al (2008) Nonalcoholic fatty liver disease and mitochondrial dysfunction. World J Gastroenterol 14:193–199CrossRefPubMedPubMedCentralGoogle Scholar
  167. Woolbright BL, Jaeschke H (2012) Novel insight into mechanisms of cholestatic liver injury. World J Gastroenterol 18:4985–4993CrossRefPubMedPubMedCentralGoogle Scholar
  168. Woolbright BL, Jaeschke H (2017) The impact of sterile inflammation in acute liver injury. J Clin Transl Res 3(Suppl 1):170–188PubMedPubMedCentralGoogle Scholar
  169. Woolbright BL, Antoine DJ, Jenkins RE et al (2013) Plasma biomarkers of liver injury and inflammation demonstrate a lack of apoptosis during obstructive cholestasis in mice. Toxicol Appl Pharmacol 273:524–531CrossRefPubMedGoogle Scholar
  170. Woolbright BL, Li F, Xie Y et al (2014) Lithocholic acid feeding results in direct hepato-toxicity independent of neutrophil function in mice. Toxicol Lett 228:56–66CrossRefPubMedPubMedCentralGoogle Scholar
  171. Woolbright BL, Dorko K, Antoine DJ et al (2015) Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis. Toxicol Appl Pharmacol 283:168–177CrossRefPubMedPubMedCentralGoogle Scholar
  172. Woolbright BL, McGill MR, Yan H et al (2016) Bile acid-induced toxicity in HepaRG cells recapitulates the response in primary human hepatocytes. Basic Clin Pharmacol Toxicol 118:160–167CrossRefPubMedGoogle Scholar
  173. Yang K, Köck K, Sedykh A et al (2013) An updated review on drug-induced cholestasis: mechanisms and investigation of physicochemical properties and pharmacokinetic parameters. J Pharm Sci 102:3037–3057CrossRefPubMedPubMedCentralGoogle Scholar
  174. Yang K, Guo C, Woodhead JL et al (2016) Sandwich-cultured hepatocytes as a tool to study drug disposition and drug-induced liver injury. J Pharm Sci 105:443–459CrossRefPubMedPubMedCentralGoogle Scholar
  175. Yao X, Li Y, Cheng X et al (2016) ER stress contributes to alpha-naphthyl isothiocyanate-induced liver injury with cholestasis in mice. Pathol Res Pract 212:560–567CrossRefPubMedGoogle Scholar
  176. Yasumiba S, Tazuma S, Ochi H et al (2001) Cyclosporin A reduces canalicular membrane fluidity and regulates transporter function in rats. Biochem J 354:591–596CrossRefPubMedPubMedCentralGoogle Scholar
  177. Yu T, Wang L, Lee H et al (2014) Decreasing mitochondrial fission prevents cholestatic liver injury. J Biol Chem 289:34074–34088CrossRefPubMedPubMedCentralGoogle Scholar
  178. Zeilinger K, Freyer N, Damm G et al (2016) Cell sources for in vitro human liver cell culture models. Exp Biol Med (Maywood) 241:1684–1698CrossRefGoogle Scholar
  179. Zhang Y, Hong JY, Rockwell CE et al (2012) Effect of bile duct ligation on bile acid composition in mouse serum and liver. Liver Int 32:58–69CrossRefPubMedGoogle Scholar
  180. Zhu F, Li XX, Yang SY et al (2018) Clinical success of drug targets prospectively predicted by in silico study. Trends Pharmacol Sci 39:229–231CrossRefPubMedGoogle Scholar
  181. Zollner G, Trauner M (2006) Molecular mechanisms of cholestasis. Wien Med Wochenschr 156:380–385CrossRefPubMedGoogle Scholar
  182. Zollner G, Trauner M (2008) Mechanisms of cholestasis. Clin Liver Dis 12:1–26CrossRefPubMedGoogle Scholar
  183. Zollner G, Marschall HU, Wagner M et al (2006) Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations. Mol Pharm 3:231–251CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Eva Gijbels
    • 1
  • Vânia Vilas-Boas
    • 1
  • Neel Deferm
    • 2
  • Lindsey Devisscher
    • 3
  • Hartmut Jaeschke
    • 4
  • Pieter Annaert
    • 2
  • Mathieu Vinken
    • 1
    Email author
  1. 1.Department of In Vitro Toxicology and Dermato-CosmetologyVrije Universiteit BrusselBrusselsBelgium
  2. 2.Drug Delivery and DispositionDepartment of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N2LeuvenBelgium
  3. 3.Basic and Applied Medical Sciences, Gut-Liver Immunopharmacology Unit, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium
  4. 4.Department of Pharmacology, Toxicology and TherapeuticsUniversity of Kansas Medical CenterKansas CityUSA

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