The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice
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.
KeywordsHepatotoxicity Veno-occlusive disease 2-Photon microscopy Xenobiotic metabolism Liver necrosis
Area under the curve
Cytochrome P450 monooxygenase(s)
Green fluorescent protein
- KH buffer
Liver sinusoidal endothelial cells
Primary mouse hepatocytes
NADPH-cytochrome P450 reductase
Time to maximum
Tetramethylrhodamine ethyl ester
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.
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.
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