Robustness testing and optimization of an adverse outcome pathway on cholestatic liver injury


Adverse outcome pathways (AOPs) have been recently introduced as tools to map the mechanisms underlying toxic events relevant for chemical risk assessment. AOPs particularly depict the linkage between a molecular initiating event and an adverse outcome through a number of intermediate key events. An AOP has been previously introduced for cholestatic liver injury. The objective of this study was to test the robustness of this AOP for different types of cholestatic insult and the in vitro to in vivo extrapolation. For this purpose, in vitro samples from human hepatoma HepaRG cell cultures were exposed to cholestatic drugs (i.e. intrahepatic cholestasis), while in vivo samples were obtained from livers of cholestatic mice (i.e. extrahepatic cholestasis). The occurrence of cholestasis in vitro was confirmed through analysis of bile transporter functionality and bile acid analysis. Transcriptomic analysis revealed inflammation and oxidative stress as key events in both types of cholestatic liver injury. Major transcriptional differences between intrahepatic and extrahepatic cholestatic liver insults were observed at the level of cell death and metabolism. Novel key events identified by pathway analysis included endoplasmic reticulum stress in intrahepatic cholestasis, and autophagy and necroptosis in both intrahepatic as extrahepatic cholestasis. This study demonstrates that AOPs constitute dynamic tools that should be frequently updated with new input information.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Adverse outcome pathway


ATP-binding cassette family




Activation transcription factor


Bile acid(s)


Bile duct ligation


Bile salt export pump


Cholic acid


Constitutive androstane receptor


C–C chemokine receptor type


Chenodeoxycholic acid


CCAAT-enhancer-binding protein homologous protein


Cholestatic index




Cyclosporin A


Colony stimulating factor




Cytochrome P450


Drug-induced liver injury


Dimethyl sulfoxide


Deoxycholic acid


Fos proto-oncogene


Farnesoid X receptor


Glycocholic acid


Glycochenodeoxycholic acid


Glycodeoxycholic acid


Glutathione S-transferase


Interleukin (1 receptor-like 1)


Ingenuity Pathway Analysis


Interleukin 1 receptor-associated kinase


Jun proto-oncogene


Microtubule-associated protein 1 light chain 3β


Mitogen-activated protein kinase-activated protein kinase


Multidrug resistance protein


Mixed lineage kinase domain-like pseudokinase


Multidrug resistance-associated protein


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide




Nuclear receptor(s)


Sodium-taurocholate co-transporting polypeptide


NAD(P)H quinone dehydrogenase


Organic anion transporting peptide(s)


Organic solute transporter


Pregnane X receptor


Receptor interacting serine/threonine kinase


Standard deviation


Serpin E1


Small heterodimer partner


SH3 domain containing GRB2 like, endophilin B1


Solute carrier (organic anion transporter) family






  1. Afonso MB, Rodrigues PM, Simão AL et al (2016) Activation of necroptosis in human and experimental cholestasis. Cell Death Dis 7:e2390

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ankley GT, Bennett RS, Erickson RJ et al (2010) Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment. Environ Toxicol Chem 29:730–741

    CAS  PubMed  Google Scholar 

  3. 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 57:1518–1529

    PubMed  Google Scholar 

  4. Arduini A, Serviddio G, Tormos AM et al (2012) Mitochondrial dysfunction in cholestatic liver diseases. Front Biosci 4:2233–2252

    Google Scholar 

  5. 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 145:157–168

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Bale SS, Vernetti L, Senutovitch N et al (2014) In vitro platforms for evaluating liver toxicity. Exp Biol Med 239:1180–1191

    Google Scholar 

  7. Begriche K, Massart J, Robin MA et al (2011) Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. J Hepatol 54:773–794

    CAS  PubMed  Google Scholar 

  8. Bhat TA, Chaudhary AK, Kumar S et al (2017) Endoplasmic reticulum-mediated unfolded protein response and mitochondrial apoptosis in cancer. Biochim Biophis Acta 1867:58–66

    CAS  Google Scholar 

  9. Bissio E, Lopardo GD (2013) Incidence of hyperbilirubinemia and jaundice due to atazanavir in a cohort of hispanic patients. AIDS Res Hum Retroviruses 29(3):415–417

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Botla R, Spivey JR, Aguilar H et al (1995) Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. J Pharmacol Exp Ther 272:930–938

    CAS  PubMed  Google Scholar 

  11. 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 115:166–178

    CAS  PubMed  Google Scholar 

  12. Burbank MG, Sharanek A, Burban A et al (2017) Mechanistic insights in cytotoxic and cholestatic potential of the endothelial receptor antagonists using HepaRG cells. Toxicol Sci 157:451–464

    CAS  PubMed  Google Scholar 

  13. Chatterjee S, Richert L, Augustijns P et al (2014) Hepatocyte-based in vitro model for assessment of drug-induced cholestasis. Toxicol Appl Pharmacol 274:124–136

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  15. Dawson S, Stahl S, Paul N et al (2012) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab Dispos 40:130–138

    CAS  PubMed  Google Scholar 

  16. Dewaele D, Annaert P, Hoeben E (2019) LC-MS/MS analysis of bile acids in in vitro samples. Methods Mol Biol 1981:15–23

    CAS  PubMed  Google Scholar 

  17. Dragovic S, Vermeulen NPE, Gerets HH et al (2016) Evidence-based selection of training compounds for use in the mechanism-based integrated prediction of drug-induced liver injury in man. Arch Toxicol 90:2979–3003

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Gao L, Lv G, Guo X et al (2014) Activation of autophagy protects against cholestasis-induced hepatic injury. Cell Biosci 4:47

    PubMed  PubMed Central  Google Scholar 

  19. Gijbels E, Vilas-Boas V, Deferm N et al (2019) Mechanisms and in vitro models of drug-induced cholestasis. Arch Toxicol 93:1169–1186

    CAS  PubMed  Google Scholar 

  20. Goldberg DS, Forde KA, Carbonari DM, Lewis JD, Leidl KBF, Reddy KR, Haynes K, Roy J, Sha D, Marks AR, Schneider JL, Strom BL, Corley DA, Lo Re V (2015) Population-representative incidence of drug-induced acute liver failure based on an analysis of an integrated health care system. Gastroenterology 148(7):1353–1361.e3

    PubMed  PubMed Central  Google Scholar 

  21. Gores GJ, Miyoshi H, Botla R et al (1998) Induction of the mitochondrial permeability transition as a mechanism of liver injury during cholestasis: a potential role for mitochondrial proteases. Biochim Biophys Acta 1366:167–175

    CAS  PubMed  Google Scholar 

  22. Halilbasic E, Baghdasaryan A, Trauner M (2013) Nuclear receptors as drug targets in cholestatic liver diseases. Clin Liver Dis 17:161–189

    PubMed  PubMed Central  Google Scholar 

  23. 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 6:35434

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Henkel AS, LeCuper B, Olivares S et al (2017) Endoplasmic reticulum stress regulated hepatic bile acid metabolism in mice. Cell Mol Gastroenterol Hepatol 3:261–271

    PubMed  Google Scholar 

  25. 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 899:135–145

    CAS  Google Scholar 

  26. Jones SC, Kortepeter C, Brinker AD (2018) Postmarketing surveillance of drug-induced liver injury. Drug-induced liver toxicity. Methods Pharmacol Toxicol.

    Article  Google Scholar 

  27. Kmiéc Z (2001) Cooperation of liver cells in health and disease. Adv Anat Embryol Cell Biol 161:1–151

    Google Scholar 

  28. Kolarić TO, Ninčević V, Smolić R et al (2019) Mechanisms of hepatic cholestatic drug injury. J Clin Transl Hepatol 7:86–92

    PubMed  PubMed Central  Google Scholar 

  29. Kostrubsky SE, Strom SC, Kalgutkar AS et al (2006) Inhibition of hepatobiliary transport as a predictive method for clinical hepatotoxicity of nefazodone. Toxicol Sci 90:451–459

    CAS  PubMed  Google Scholar 

  30. 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 66:961–976

    CAS  PubMed  Google Scholar 

  31. Lee WM (2013) Drug-induced acute liver failure. Clin Liver Dis 17:575–586

    PubMed  Google Scholar 

  32. Lepist EI, Gillies H, Smith W et al (2014) Evaluation of the endothelin receptor antagonists ambrisentan, bosentan, macitentan, and sitaxsentan as hepatobiliary transporter inhibitors and substrates in sandwich- cultured human hepatocytes. PLoS ONE 9:e87548

    PubMed  PubMed Central  Google Scholar 

  33. Liu R, Li X, Huang Z et al (2018) C/EBP homologous protein-induced loss of intestinal epithelial stemness contributes to bile duct ligation-induced cholestatic liver injury in mice. Hepatology 67:1441–1457

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Malhi H, Kaufman RJ (2011) Endoplasmic reticulum stress in liver disease. J Hepatol 54:795–809

    CAS  PubMed  Google Scholar 

  35. McGill MR, Yan HM, Ramachandran A et al (2011) HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity. Hepatology 53:974–982

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Manley S, Ni HM, Kong B et al (2014) Suppression of autophagic flux by bile acids in hepatocytes. Toxicol Sci 137:478–490

    CAS  PubMed  Google Scholar 

  37. Mariotti V, Strazzabosco M, Fabris L et al (2017) Animal models of biliary injury and altered bile acid metabolism. Biochim Biophys Acta 1864:1254–1261

    PubMed Central  Google Scholar 

  38. 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 118:485–500

    CAS  PubMed  Google Scholar 

  39. Nguyen KD, Sundaram V, Ayoub WS (2014) Atypical causes of cholestasis. World J Gastroenterol 20:9418–9426

    PubMed  PubMed Central  Google Scholar 

  40. Noor F (2015) A shift in paradigm towards human biology-based systems for cholestatic-liver diseases. J Physiol 593:5043–5055

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Oorts M, Baze A, Bachellier P et al (2016) Drug-induced cholestasis risk assessment in sandwich-cultured human hepatocytes. Toxicol In Vitro 34:179–196

    CAS  PubMed  Google Scholar 

  42. Parent R, Marion MJ, Furio L et al (2004) Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126:1147–1156

    PubMed  Google Scholar 

  43. Qu X, Zhang Y, Zhang S, Zhai J, Gao H, Tao L, Song Y (2018) Dysregulation of BSEP and MRP2 may play an important role in isoniazid-induced liver injury via the SIRT1/FXR pathway in rats and HepG2 cells. Biol Pharm Bull 41(8):1211–1218

    CAS  PubMed  Google Scholar 

  44. Rakotondravelo S, Poinsignon Y, Borsa-Lebas F et al (2012) Complicated atazanavir-associated cholelithiasis: a report of 14 cases. Clin Infect Dis 55:1270–1272

    CAS  PubMed  Google Scholar 

  45. Riede J, Poller B, Huwyler J et al (2017) Assessing the risk of drug-induced cholestasis using unbound intrahepatic concentrations. Drug Metab Dispos 45:523–531

    CAS  PubMed  Google Scholar 

  46. Rodrigues RM, Kollipara L, Chaudhari U et al (2018) Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures. Arch Toxicol 92:1939–1952

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Roman ID, Fernandez-Moreno MD, Fueyo JA et al (2003) Cyclosporin A induced internalization of the bile salt export pump in isolated rat hepatocyte couplets. Toxicol Sci 71:276–281

    CAS  PubMed  Google Scholar 

  48. Schoemaker MH, Conde de la Rosa L, Buist-Homan M et al (2004) Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways. Hepatology 39:1563–1573

    CAS  PubMed  Google Scholar 

  49. Seok J, Warren HS, Cuenca AG et al (2013) Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci USA 110:3507–3512

    CAS  PubMed  Google Scholar 

  50. Sharanek A, Bachour-El Azzi P, Al-Attrache H et al (2014) Different dose-dependent mechanisms are involved in early cyclosporine-A induced cholestatic effects in HepaRG cells. Toxicol Sci 141:244–253

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Sharanek A, Burban A, Humbert L et al (2015) Cellular accumulation and toxic effects of bile acids in cyclosporine A-treated HepaRG hepatocytes. Toxicol Sci 147:573–587

    CAS  PubMed  Google Scholar 

  52. 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 45:1292–1303

    CAS  PubMed  Google Scholar 

  53. Tag CG, Sauer-Lehnen S, Weiskirchen S et al (2015) Bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis. J Vis Exp 96:52438

    Google Scholar 

  54. 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 41:1633–1641

    CAS  PubMed  Google Scholar 

  55. Van Campenhout S, Van Vlierberghe H, Devisscher L (2019) Common bile duct ligation as model for secondary biliary cirrhosis. Methods Mol Biol 1981:237–247

    PubMed  Google Scholar 

  56. Van den Hof WF, Ruiz-Aracama A, Van Summeren A et al (2015) Integrating multiple omics to unravel mechanisms of cyclosporin A induced hepatotoxicity in vitro. Toxicol In Vitro 29:489–501

    PubMed  Google Scholar 

  57. Vatakuti S, Olinga P, Pennings JLA, Groothuis GMM (2017) Validation of precision-cut liver slices to study drug-induced cholestasis: a transcriptomics approach. Arch Toxicol 91(3):1401–1412

    CAS  PubMed  Google Scholar 

  58. Villeneuve DL, Crump D, Garcia-Reyero N et al (2014) Adverse outcome pathway (AOP) development I: strategies and principles. Toxicol Sci 142:312–320

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 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 136:97–106

    CAS  PubMed  Google Scholar 

  60. Woolbright BL, Jaeschke H (2012) Novel insight into mechanisms of cholestatic liver injury. World J Gastroenterol 18:4985–4993

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Yasumiba S, Tazuma S, Ochi H et al (2001) Cyclosporin A reduces canalicular membrane fluidity and regulates transporter function in rats. Biochem J 354:591–596

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Zhang D, Chango TJ, Everett DW et al (2005) In vitro inhibition of UDP glucuronosyltransferases by atazanavir and other HIV protease inhibitors and the relationship of this property to in vivo bilirubin glucuronidation. Drug Metab Dispos 33:1729–1739

    CAS  PubMed  Google Scholar 

  63. Zhang J, Kan H, Cai L et al (2016) Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat and mouse. Chem Biol Interact 255:45–54

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 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 3:231–251

    CAS  PubMed  Google Scholar 

  65. Zollner G, Trauner M (2006) Molecular mechanisms of cholestasis. Wien Med Wochenschr 156:380–385

    PubMed  Google Scholar 

Download references


This work was supported by grants of the Research Foundation Flanders, Belgium and the Scientific Fund Willy Gepts, Belgium and the Center for Alternatives to Animal Testing at Johns Hopkins University, USA.

Author information



Corresponding author

Correspondence to Mathieu Vinken.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Electronic supplementary material 1 (DOCX 340 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gijbels, E., Vilas‐Boas, V., Annaert, P. et al. Robustness testing and optimization of an adverse outcome pathway on cholestatic liver injury. Arch Toxicol 94, 1151–1172 (2020).

Download citation


  • Adverse outcome pathways
  • Drug-induced cholestasis
  • Mechanistic toxicology
  • Transcriptomics