Advertisement

Archives of Toxicology

, Volume 92, Issue 6, pp 1939–1952 | Cite as

Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures

  • Robim M. Rodrigues
  • Laxmikanth Kollipara
  • Umesh Chaudhari
  • Agapios Sachinidis
  • René P. Zahedi
  • Albert Sickmann
  • Annette Kopp-Schneider
  • Xiaoqi Jiang
  • Hector Keun
  • Jan Hengstler
  • Marlies Oorts
  • Pieter Annaert
  • Eef Hoeben
  • Eva Gijbels
  • Joery De Kock
  • Tamara Vanhaecke
  • Vera Rogiers
  • Mathieu Vinken
Toxicokinetics and Metabolism

Abstract

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

Keywords

Bosentan BSEP HepaRG Cholestasis Transcriptomics Proteomics Metabolomics Adverse outcome pathway. 

Abbreviations

ABCB11

ATP-binding cassette subfamily B member 11

ABCC2

ATP-binding cassette subfamily C member 2

ADH

Alcohol dehydrogenase

AOP

Adverse outcome pathway

BCA

Bicinchoninic acid assay

BCAA

Branched chain amino acid metabolites

BSEP

Bile salt export pump

CA

Cholic acid

CAR

Constitutive androstane receptor

CPMG

Carr–Purcell–Meiboom–Gill

CYP2B6

Cytochrome P450 2B6

CYP3A4

Cytochrome P450 3A4

DAPI

4′,6-diamidino-2-phenylindole

DMSO

Dimethyl sulfoxide

FASP

Filter aided sample preparation

FDR

False discovery rate

FXR

Farnesoid X receptor

GCA

Glycocholic acid

HBSS

Hank’s balanced salt solution

IPA

Ingenuity pathway analysis

IL6

Interleukin 6

IC10

Inhibitory concentration of 10%

IC50

Inhibitory concentration of 50%

IL8

Interleukin 8

iTRAQ

Isobaric tags for relative and absolute quantification

KE(s)

Key event(s)

LC–MS/MS

Liquid chromatography–mass spectrometry/mass spectrometry

LFC

Log2-fold change

MIE

Molecular initiating event

MRP2

Multidrug resistance-associated protein 2

MTT

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

NMR

Nuclear magnetic resonance

NTCP

Sodium–taurocholate cotransporting polypeptide

OATP1B1/3

Organic anion transporter 1B1/3

OSTα/β

Organic solute transporter α andβ

PCA

Principal component analysis

phosSTOP

Phosphatase inhibitor

PSMs

Peptide-spectrum matches

PSW-ANOVA

Probe sliding window-analysis of variance

PTX

Proteomics

PXR

Pregnane X receptor

RT

Room temperature

ROS

Reactive oxygen species

RXR

Retinoid X receptor

SDS

Sodium dodecyl sulphate

SLCO1B1

Solute carrier organic anion transporter family member 1B1

TAC

Affymetrix transcriptome analysis console

TCX

Transcriptomics

TFA

Trifluoroacetic acid

TNF

Tumor necrosis factor

Notes

Acknowledgements

This work was financially supported by the grants of European Union (FP7)/Cosmetics Europe (SEURAT-1 projects DETECTIVE (HEALTH-F5-2010-266838) and HeMiBio (HEALTH-F5-2010-266777)), the European Research Council (Starting Grant 335476), the Fund for Scientific Research-Flanders (FWO grants G009514N, G010214N, G012318N, G020018N and 12H2216N), the University Hospital of the Vrije Universiteit Brussel-Belgium (“Willy Gepts Fonds” UZ-VUB) and the Center for Alternatives to Animal Testing (CAAT) at Johns Hopkins University Baltimore-USA. The authors also gratefully acknowledge the financial support from the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin, and the Bundesministerium für Bildung und Forschung. The authors like to thank Dr. Christophe Chesné (Biopredic) for making HepaRG cells available, Dr. Stefan Vinckier for assistance with confocal microscopy and Miss Tineke Vanhalewyn for technical assistance.

Supplementary material

204_2018_2214_MOESM1_ESM.pptx (3.1 mb)
Supplementary material 1 (PPTX 3216 KB)
204_2018_2214_MOESM2_ESM.docx (23 kb)
Supplementary material 2 (DOCX 23 KB)
204_2018_2214_MOESM3_ESM.docx (24 kb)
Supplementary material 3 (DOCX 24 KB)
204_2018_2214_MOESM4_ESM.docx (27 kb)
Supplementary material 4 (DOCX 26 KB)

References

  1. Alvarez L, Jara P, Sánchez-Sabaté E, Hierro L, Larrauri J, Díaz MC, Camarena C, De la Vega A, Frauca E, López-Collazo E, Lapunzina P (2004) Reduced hepatic expression of farnesoid X receptor in hereditary cholestasis associated to mutation in ATP8B1. Hum Mol Genet 13:2451–2460CrossRefPubMedGoogle Scholar
  2. Azzi B-E, Sharanek P, Burban A, Li A, Guével R, Abdel-Razzak RL, Stieger Z, Guguen-Guillouzo B, Guillouzo C, A (2015) Comparative localization and functional activity of the main hepatobiliary transporters in HepaRG cells and primary human hepatocytes. Toxicol Sci 145:157–168CrossRefGoogle Scholar
  3. Begriche K, Massart J, Robin MA, Borgne-Sanchez A, Fromenty B (2011) Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. J Hepatol 54:773–794CrossRefPubMedGoogle Scholar
  4. Beuers U, Trauner M, Jansen P, Poupon R (2015) New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond. J Hepatol 62:S25-S37CrossRefGoogle Scholar
  5. Bremer J (1983) Carnitine-metabolism and functions. Physiol Rev 63:1420–1480CrossRefPubMedGoogle Scholar
  6. Burbank MG, Sharanek A, Burban A, Mialanne H, Aerts H, Guguen-Guillouzo C, Weaver RJ, Guillouzo A (2017) From the cover: mechanistic insights in cytotoxic and cholestatic potential of the endothelial receptor antagonists using HepaRG Cells. Toxicol Sci 157:451–464CrossRefPubMedGoogle Scholar
  7. Burkhart JM, Schumbrutzki C, Wortelkamp S, Sickmann A, Zahedi RP (2012) Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. J Proteomics 75:1454–1462CrossRefPubMedGoogle Scholar
  8. Chatterjee S, Richert L, Augustijns P, Annaert P (2014) Hepatocyte-based in vitro model for assessment of drug-induced cholestasis. Toxicol Appl Pharmacol 274:124–136CrossRefPubMedGoogle Scholar
  9. Chen F, Ananthanarayanan M, Emre S, Neimark E, Bull LN, Knisely AS, Strautnieks SS, Thompson RJ, Magid MS, Gordon R, Balasubramanian N, Suchy FJ, Shneider BL (2004) Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity. Gastroenterology 126:756–764CrossRefPubMedGoogle Scholar
  10. Dawson S, Stahl S, Paul N, Barber J, Kenna JG (2011) In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans. Drug Metab Dispos 40:130–138CrossRefPubMedGoogle Scholar
  11. Demeilliers C, Jacquemin E, Barbu V, Mergey M, Paye F, Fouassier L, Chignard N, Housset C, Lomri NE (2006) Altered hepatobiliary gene expressions in PFIC1: ATP8B1 gene defect is associated with CFTR downregulation. Hepatology 43:1125–1134CrossRefPubMedGoogle Scholar
  12. Dickhut C, Radau S, Zahedi R (2014) Fast, efficient, and quality-controlled phosphopeptide enrichment from minute sample amounts using ditanium Dioxide. In: Martins-de-Souza D (ed) Shotgun proteomics, Springer, New York, pp 417–430CrossRefGoogle Scholar
  13. Dingemanse J, van Giersbergen PL (2004) Clinical pharmacology of bosentan, a dual endothelin receptor antagonist. Clin Pharmacokinet 43:1089–1115CrossRefPubMedGoogle Scholar
  14. Fattinger K, Funk C, Pantze M, Weber C, Reichen J, Stieger B, Meier PJ (2001) The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin Pharmacol Ther 69:223–231CrossRefPubMedGoogle Scholar
  15. Forman BM, Goode E, Chen J, Oro AE, Bradley DJ, Perlmann T, Noonan DJ, Burka LT, McMorris T, Lamph WW, Evans RM (1995) Identification of a nuclear receptor that is activated by farnesol metabolites. Cell 81:687–693CrossRefPubMedGoogle Scholar
  16. Garzel B, Yang H, Zhang L, Huang SM, Polli JE, Wang H (2014) The role of bile salt export pump gene repression in drug-induced cholestatic liver toxicity. Drug Metab Dispos 42:318–322CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hartman JC, Brouwer K, Mandagere A, Melvin L, Gorczynski R (2010) Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich-cultured human hepatocytes. Can J Physiol Pharmacol 88:682–691CrossRefPubMedGoogle Scholar
  18. Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ (2007) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Meth 4:923–925CrossRefGoogle Scholar
  19. Kemp DC, Zamek-Gliszczynski MJ, Brouwer KL (2005) Xenobiotics inhibit hepatic uptake and biliary excretion of taurocholate in rat hepatocytes. Toxicol Sci 83:207–214CrossRefPubMedGoogle Scholar
  20. Kis E, Ioja E, Rajnai Z, Jani M, Méhn D, Herédi-Szabó K, Krajcsi P (2012) BSEP inhibition: in vitro screens to assess cholestatic potential of drugs. Toxicol In Vitro 26:1294–1299CrossRefPubMedGoogle Scholar
  21. Köck K, Ferslew BC, Netterberg I, Yang K, Urban TJ, Swaan PW, Stewart PW, Brouwer KL (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
  22. Labbe G, Pessayre D, Fromenty B (2008) Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies. Fundam Clin Pharmacol 22:335–353CrossRefPubMedGoogle Scholar
  23. Langhi C, Pedraz-Cuesta E, Haro D, Marrero PF, Rodríguez JC (2013) Regulation of human class I alcohol dehydrogenases by bile acids. J Lipid Res 54:2475–2484CrossRefPubMedPubMedCentralGoogle Scholar
  24. Le Vee M, Jigorel E, Glaise D, Gripon P, Guguen-Guillouzo C, Fardel O (2006) Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur J Pharm Sci 28:109–117CrossRefPubMedGoogle Scholar
  25. Lepist EI, Gillies H, Smith W, Hao J, Hubert C, Claire St, Brouwer RL 3rd, Ray KR (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:e87548CrossRefPubMedPubMedCentralGoogle Scholar
  26. Lynch CJ, Adams SH (2014) Branched-chain amino acids in metabolic signalling and insulin resistance. Nat Rev Endocrinol 10:723–736CrossRefPubMedPubMedCentralGoogle Scholar
  27. Manza LL, Stamer SL, Ham AJ, Codreanu SG, Liebler DC (2005) Sample preparation and digestion for proteomic analyses using spin filters. Proteomics 5:1742–1745CrossRefPubMedGoogle Scholar
  28. Mita S, Suzuki H, Akita H, Hayashi H, Onuki R, Hofmann AF, Sugiyama Y (2006) Inhibition of bile acid transport across Na+/taurocholate cotransporting polypeptide (SLC10A1) and bile salt export pump (ABCB 11)-coexpressing LLC-PK1 cells by cholestasis-inducing drugs. Drug Metab Dispos 34:1575–1581CrossRefPubMedGoogle Scholar
  29. Morel Y, Barouki R (1999) Repression of gene expression by oxidative stress. Biochem J 342:481–496CrossRefPubMedPubMedCentralGoogle Scholar
  30. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65:55–63CrossRefGoogle Scholar
  31. Muller PY, Milton MN (2012) The determination and interpretation of the therapeutic index in drug development. Nat Rev Drug Discov 11:751–761CrossRefPubMedGoogle Scholar
  32. Padda MS, Sanchez M, Akhtar AJ, Boyer JL (2011) Drug-induced cholestasis. Hepatology 53:1377–1387CrossRefPubMedPubMedCentralGoogle Scholar
  33. Palmisano G, Parker BL, Engholm-Keller K, Lendal SE, Kulej K, Schulz M, Schwämmle V, Graham ME, Saxtorph H, Cordwell SJ, Larsen MR (2012) A novel method for the simultaneous enrichment, identification, and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development. Mol Cell Proteomics 11:1191–1202CrossRefPubMedPubMedCentralGoogle Scholar
  34. Qiu X, Zhang Y, Liu T, Shen H, Xiao Y, Bourner MJ, Pratt JR, Thompson DC, Marathe P, Humphreys WG, Lai Y (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
  35. Reif R, Karlsson J, Günther G, Beattie L, Wrangborg D, Hammad S, Begher-Tibbe B, Vartak A, Melega S, Kaye PM, Hengstler JG, Jirstrand M (2015) Bile canalicular dynamics in hepatocyte sandwich cultures. Arch Toxicol 89:1861–1870CrossRefPubMedGoogle Scholar
  36. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169CrossRefPubMedGoogle Scholar
  37. Sharanek A, Burban A, Humbert L, Bachour-El Azzi P, Felix-Gomes N, Rainteau D, Guillouzo A (2015) Cellular accumulation and toxic effects of bile acids in cyclosporine A-treated HepaRG hepatocytes. Toxicol Sci 147:573–587CrossRefPubMedGoogle Scholar
  38. Sharanek A, Burban A, Burbank M, Le Guevel R, Li R, Guillouzo A, Guguen-Guillouzo C (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
  39. Szalowska E, Stoopen G, Groot MJ, Hendriksen PJ, Peijnenburg AA (2013) Treatment of mouse liver slices with cholestatic hepatotoxicants results in down-regulation of Fxr and its target genes. BMC Med Genomics 10:6–39Google Scholar
  40. Taus T, Köcher T, Pichler P, Paschke C, Schmidt A, Henrich C, Mechtler K (2011) Universal and confident phosphorylation site localization using phosphoRS. J Proteome Res 10:5354–5362CrossRefPubMedGoogle Scholar
  41. Vatakuti S, Olinga P, Pennings JL, Groothuis GM (2017) Validation of precision-cut liver slices to study drug-induced cholestasis: a transcriptomics approach. Arch Toxicol 91:1401–1412CrossRefPubMedGoogle Scholar
  42. Vinken M, Landesmann B, Goumenou M, Vinken S, Shah I, Jaeschke H, Willett C, Whelan M, Rogiers V (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
  43. Vinken M, Knapen D, Vergauwen L, Hengstler JG, Angrish M, Whelan M (2017) Adverse outcome pathways: a concise introduction for toxicologists. Arch Toxicol 91:3697–3707CrossRefPubMedPubMedCentralGoogle Scholar
  44. Wagner M, Zollner G, Trauner M (2009) New molecular insights into the mechanisms of cholestasis. J Hepatol 51:565–580CrossRefPubMedGoogle Scholar
  45. Wang G, Shen H, Rajaraman G, Roberts MS, Gong Y, Jiang P, Burczynski F (2007) Expression and antioxidant function of liver fatty acid binding protein in normal and bile-duct ligated rats. Eur J Pharmacol 560:61–68CrossRefPubMedGoogle Scholar
  46. Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Meth 6:359–362CrossRefGoogle Scholar
  47. Zollner G, Trauner M (2006) Molecular mechanisms of cholestasis. Wien Med Wochenschr 156:380–385CrossRefPubMedGoogle Scholar
  48. Zollner G, Trauner M (2008) Mechanisms of cholestasis. Clin Liver Dis 12:1–26CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Robim M. Rodrigues
    • 1
  • Laxmikanth Kollipara
    • 2
  • Umesh Chaudhari
    • 3
  • Agapios Sachinidis
    • 3
  • René P. Zahedi
    • 2
  • Albert Sickmann
    • 2
    • 4
    • 5
  • Annette Kopp-Schneider
    • 6
  • Xiaoqi Jiang
    • 6
  • Hector Keun
    • 7
  • Jan Hengstler
    • 8
  • Marlies Oorts
    • 9
  • Pieter Annaert
    • 9
  • Eef Hoeben
    • 10
  • Eva Gijbels
    • 1
  • Joery De Kock
    • 1
  • Tamara Vanhaecke
    • 1
  • Vera Rogiers
    • 1
  • Mathieu Vinken
    • 1
  1. 1.Department of In Vitro Toxicology and Dermato-CosmetologyVrije Universiteit BrusselBrusselsBelgium
  2. 2.Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V.DortmundGermany
  3. 3.Institute of Neurophysiology and Center for Molecular Medicine CologneUniversity of CologneCologneGermany
  4. 4.Department of Chemistry, College of Physical SciencesUniversity of AberdeenAberdeenUK
  5. 5.Medizinische Fakultät, Medizinische Proteom-Center (MPC)Ruhr-Universität BochumBochumGermany
  6. 6.Division of BiostatisticsGerman Cancer Research CenterHeidelbergGermany
  7. 7.Computational and Systems Medicine, Department of Surgery and CancerImperial College LondonLondonUK
  8. 8.Leibniz Research Centre for Working Environment and Human Factors at the Technical University of DortmundDortmundGermany
  9. 9.Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological SciencesKatholieke Universiteit LeuvenLeuvenBelgium
  10. 10.BioNotus GCVNielBelgium

Personalised recommendations