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

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.

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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

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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.

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Correspondence to Mathieu Vinken.

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Rodrigues, R.M., Kollipara, L., Chaudhari, U. et al. Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures. Arch Toxicol 92, 1939–1952 (2018). https://doi.org/10.1007/s00204-018-2214-z

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Keywords

  • Bosentan
  • BSEP
  • HepaRG
  • Cholestasis
  • Transcriptomics
  • Proteomics
  • Metabolomics
  • Adverse outcome pathway.