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

Abstract

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.

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Abbreviations

AOP:

Adverse outcome pathway

ABC:

ATP-binding cassette family

ATA:

Atazanavir

ATF:

Activation transcription factor

BA(s):

Bile acid(s)

BDL:

Bile duct ligation

BSEP:

Bile salt export pump

CA:

Cholic acid

CAR:

Constitutive androstane receptor

CCR:

C–C chemokine receptor type

CDCA:

Chenodeoxycholic acid

CHOP:

CCAAT-enhancer-binding protein homologous protein

CIx:

Cholestatic index

CLF:

Cholyl-l-lysyl-fluorescein

CsA:

Cyclosporin A

CSF:

Colony stimulating factor

CYLD:

Cylindromatosis

CYP:

Cytochrome P450

DILI:

Drug-induced liver injury

DMSO:

Dimethyl sulfoxide

DCA:

Deoxycholic acid

Fos:

Fos proto-oncogene

FXR:

Farnesoid X receptor

GCA:

Glycocholic acid

GCDCA:

Glycochenodeoxycholic acid

GDCA:

Glycodeoxycholic acid

Gst:

Glutathione S-transferase

Il(1rl1):

Interleukin (1 receptor-like 1)

IPA:

Ingenuity Pathway Analysis

IRAK:

Interleukin 1 receptor-associated kinase

JUN:

Jun proto-oncogene

MAP1LC3β:

Microtubule-associated protein 1 light chain 3β

MAPKAPK:

Mitogen-activated protein kinase-activated protein kinase

MDR:

Multidrug resistance protein

MLKL:

Mixed lineage kinase domain-like pseudokinase

MRP:

Multidrug resistance-associated protein

MTT:

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

NEFA:

Nefazodone

NR(s):

Nuclear receptor(s)

NTCP:

Sodium-taurocholate co-transporting polypeptide

Nqo:

NAD(P)H quinone dehydrogenase

OATP(s):

Organic anion transporting peptide(s)

OST:

Organic solute transporter

PXR:

Pregnane X receptor

RIPK:

Receptor interacting serine/threonine kinase

SD:

Standard deviation

SERPINE1:

Serpin E1

SHP:

Small heterodimer partner

SH3GLB1:

SH3 domain containing GRB2 like, endophilin B1

SLC(O):

Solute carrier (organic anion transporter) family

SQSTM:

Sequestosome

UGT:

UDP-glucuronosyltransferase

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Acknowledgements

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.

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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). https://doi.org/10.1007/s00204-020-02691-9

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Keywords

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