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The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice

  • Stefanie Hessel-PrasEmail author
  • Albert Braeuning
  • Georgia Guenther
  • Alshaimaa Adawy
  • Anne-Margarethe Enge
  • Johanna Ebmeyer
  • Colin J. Henderson
  • Jan G. Hengstler
  • Alfonso Lampen
  • Raymond Reif
Organ Toxicity and Mechanisms

Abstract

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.

Keywords

Hepatotoxicity Veno-occlusive disease 2-Photon microscopy Xenobiotic metabolism Liver necrosis 

Abbreviations

3-MC

3-Methylcholanthrene

ALT

Alanine transaminase

AST

Aspartate transaminase

AU

Arbitrary unit

AUC

Area under the curve

BC

Bile canaliculi

BW

Body weight

LE

Liver extract

CLF

Cholyl-lysyl-fluorescein

CYP

Cytochrome P450 monooxygenase(s)

GFP

Green fluorescent protein

CV

Central vein

HE

Hematoxylin-eosin staining

KH buffer

Krebs–Henseleit buffer

LSEC

Liver sinusoidal endothelial cells

NPC

Non-parenchymal cells

PI

Propidium iodide

PA(s)

Pyrrolizidine alkaloid(s)

PMH

Primary mouse hepatocytes

POR

NADPH-cytochrome P450 reductase

t1/2

Half-life

tmax

Time to maximum

TMRE

Tetramethylrhodamine ethyl ester

VOD

Veno-occlusive disease

Notes

Acknowledgements

This work was supported by the German Research Foundation (Grant Number LA1177/12-1), by the German Federal Institute for Risk Assessment (Grant Numbers 1322-591 and 1322-624) and the BMBF funded project LiSyM. ERL mice were generated under Cancer Research UK Programme Grant C4639/A10822 awarded to Professor C.R. Wolf. Additionally, we thank Ms. Brigitte Begher-Tibbe (Leibniz Research Center for Working Environment and Human Factors, Technical University Dortmund, Germany) for Immunostaining and for 3 D reconstructions. Special thanks to Ms. Gisela H. Degen for scientific discussion and proof reading of the manuscript.

Funding

This work was supported by the German Research Foundation (Grant Number LA1177/12-1), by the German Federal Institute for Risk Assessment (Grant Numbers 1322-591 and 1322-624) and the BMBF Funded project LiSyM. ERL mice were generated under Cancer Research UK Programme Grant C4639/A10822 awarded to Professor C.R. Wolf.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Stefanie Hessel-Pras
    • 1
    Email author
  • Albert Braeuning
    • 1
  • Georgia Guenther
    • 2
  • Alshaimaa Adawy
    • 2
  • Anne-Margarethe Enge
    • 1
  • Johanna Ebmeyer
    • 1
  • Colin J. Henderson
    • 3
  • Jan G. Hengstler
    • 2
  • Alfonso Lampen
    • 1
  • Raymond Reif
    • 2
  1. 1.Department Food SafetyGerman Federal Institute for Risk AssessmentBerlinGermany
  2. 2.Leibniz Research Centre for Working Environment and Human FactorsTechnical University DortmundDortmundGermany
  3. 3.Systems Medicine, Jacqui Wood Cancer CentreUniversity of Dundee, School of MedicineDundeeUK

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