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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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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DOI: https://doi.org/10.1007/s00204-018-2214-z