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


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.


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





Alanine transaminase


Aspartate transaminase


Arbitrary unit


Area under the curve


Bile canaliculi


Body weight


Liver extract




Cytochrome P450 monooxygenase(s)


Green fluorescent protein


Central vein


Hematoxylin-eosin staining

KH buffer

Krebs–Henseleit buffer


Liver sinusoidal endothelial cells


Non-parenchymal cells


Propidium iodide


Pyrrolizidine alkaloid(s)


Primary mouse hepatocytes


NADPH-cytochrome P450 reductase




Time to maximum


Tetramethylrhodamine ethyl ester


Veno-occlusive disease



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.

Supplementary material

204_2019_2582_MOESM1_ESM.docx (1.8 mb)
Supplementary material 1 (DOCX 1883 kb)
204_2019_2582_MOESM2_ESM.mp4 (4.1 mb)
Supplementary material 2 (MP4 4230 kb)
204_2019_2582_MOESM3_ESM.mp4 (13.9 mb)
Supplementary material 3 (MP4 14279 kb)
204_2019_2582_MOESM4_ESM.mp4 (10.1 mb)
Supplementary material 4 (MP4 10369 kb)
204_2019_2582_MOESM5_ESM.mp4 (16.3 mb)
Supplementary material 5 (MP4 16714 kb)


  1. Baker DC, Pfister JA, Molyneux RJ, Kechele P (1991) Cynoglossum officinale toxicity in calves. J Comp Pathol 104(4):403–410CrossRefGoogle Scholar
  2. Bodi D, Ronczka S, Gottschalk C et al (2014) Determination of pyrrolizidine alkaloids in tea, herbal drugs and honey. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 31(11):1886–1895. CrossRefPubMedGoogle Scholar
  3. Chen Z, Huo JR (2010) Hepatic veno-occlusive disease associated with toxicity of pyrrolizidine alkaloids in herbal preparations. Neth J Med 68(6):252–260PubMedGoogle Scholar
  4. Constien R, Forde A, Liliensiek B et al (2001) Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line. Genesis 30(1):36–44CrossRefGoogle Scholar
  5. Copple BL, Rondelli CM, Maddox JF, Hoglen NC, Ganey PE, Roth RA (2004) Modes of cell death in rat liver after monocrotaline exposure. Toxicol Sci 77(1):172–182. CrossRefPubMedGoogle Scholar
  6. Datta DV, Khuroo MS, Mattocks AR, Aikat BK, Chhuttani PN (1978) Herbal medicines and veno-occlusive disease in India. Postgrad Med J 54(634):511–515. CrossRefPubMedPubMedCentralGoogle Scholar
  7. DeLeve LD, Shulman HM, McDonald GB (2002) Toxic injury to hepatic sinusoids: sinusoidal obstruction syndrome (veno-occlusive disease). Semin Liver Dis 22(1):27–42. CrossRefPubMedGoogle Scholar
  8. DeLeve LD, Ito Y, Bethea NW, McCuskey MK, Wang X, McCuskey RS (2003a) Embolization by sinusoidal lining cells obstructs the microcirculation in rat sinusoidal obstruction syndrome. Am J Physiol Gastrointest Liver Physiol 284(6):G1045–G1052. CrossRefPubMedGoogle Scholar
  9. Deleve LD, Wang X, Tsai J, Kanel G, Strasberg S, Tokes ZA (2003b) Sinusoidal obstruction syndrome (veno-occlusive disease) in the rat is prevented by matrix metalloproteinase inhibition. Gastroenterology 125(3):882–890CrossRefGoogle Scholar
  10. Denk W, Strickler J, Webb W (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dusemund B, Nowak N, Sommerfeld C, Lindtner O, Schafer B, Lampen A (2018) Risk assessment of pyrrolizidine alkaloids in food of plant and animal origin. Food Chem Toxicol 115:63–72. CrossRefPubMedGoogle Scholar
  12. Ebmeyer J, Behrend J, Lorenz M et al (2019a) Pyrrolizidine alkaloid-induced alterations of prostanoid synthesis in human endothelial cells. Chem Biol Interact 298:104–111. CrossRefPubMedGoogle Scholar
  13. Ebmeyer J, Braeuning A, Glatt H, These A, Hessel-Pras S, Lampen A (2019b) Human CYP3A4-mediated toxification of the pyrrolizidine alkaloid lasiocarpine. Food Chem Toxicol 130:79–88. CrossRefPubMedGoogle Scholar
  14. Ebmeyer J, Franz L, Lim R et al (2019c) Sensitization of human liver cells toward fas-mediated apoptosis by the metabolically activated pyrrolizidine alkaloid lasiocarpine. Mol Nutr Food Res. CrossRefPubMedGoogle Scholar
  15. EFSA (2016) Dietary exposure assessment to pyrrolizidine alkaloids in the European population. EFSA J 14(8):50Google Scholar
  16. Fox DW, Hart MC, Bergeson PS, Jarrett PB, Stillman AE, Huxtable RJ (1978) Pyrrolizidine (Senecio) intoxication mimicking Reye syndrome. J Pediatr 93(6):980–982. CrossRefPubMedGoogle Scholar
  17. Fu PP, Xia Q, Lin G, Chou MW (2004) Pyrrolizidine alkaloids—genotoxicity, metabolism enzymes, metabolic activation, and mechanisms. Drug Metab Rev 36(1):1–55. CrossRefPubMedGoogle Scholar
  18. Gao H, Li N, Wang JY, Zhang SC, Lin G (2012) Definitive diagnosis of hepatic sinusoidal obstruction syndrome induced by pyrrolizidine alkaloids. J Dig Dis 13(1):33–39. CrossRefPubMedGoogle Scholar
  19. Gordon GJ, Coleman WB, Grisham JW (2000) Bax-mediated apoptosis in the livers of rats after partial hepatectomy in the retrorsine model of hepatocellular injury. Hepatology 32(2):312–320. CrossRefPubMedGoogle Scholar
  20. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. CrossRefPubMedGoogle Scholar
  21. Helmy A (2006) Review article: updates in the pathogenesis and therapy of hepatic sinusoidal obstruction syndrome. Aliment Pharmacol Ther 23(1):11–25. CrossRefPubMedGoogle Scholar
  22. Henderson CJ, McLaughlin LA, Osuna-Cabello M et al (2015) Application of a novel regulatable Cre recombinase system to define the role of liver and gut metabolism in drug oral bioavailability. Biochem J 465(3):479–488. CrossRefPubMedGoogle Scholar
  23. Kakar F, Akbarian Z, Leslie T et al (2010) An outbreak of hepatic veno-occlusive disease in Western Afghanistan associated with exposure to wheat flour contaminated with pyrrolizidine alkaloids. J Toxicol 2010:313280. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Koni PA, Joshi SK, Temann UA, Olson D, Burkly L, Flavell RA (2001) Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow. J Exp Med 193(6):741–754CrossRefGoogle Scholar
  25. Luckert C, Hessel S, Lenze D, Lampen A (2015) Disturbance of gene expression in primary human hepatocytes by hepatotoxic pyrrolizidine alkaloids: a whole genome transcriptome analysis. Toxicol In Vitro 29(7):1669–1682. CrossRefPubMedGoogle Scholar
  26. Mendel VE, Witt MR, Gitchell BS et al (1988) Pyrrolizidine alkaloid-induced liver disease in horses: an early diagnosis. Am J Vet Res 49(4):572–578PubMedGoogle Scholar
  27. Merz KH, Schrenk D (2016) Interim relative potency factors for the toxicological risk assessment of pyrrolizidine alkaloids in food and herbal medicines. Toxicol Lett 263:44–57. CrossRefPubMedGoogle Scholar
  28. Mohabbat O, Younos MS, Merzad AA, Srivastava RN, Sediq GG, Aram GN (1976) An outbreak of hepatic veno-occlusive disease in north-western Afghanistan. Lancet (London, England) 2(7980):269–271CrossRefGoogle Scholar
  29. Mulder PPJ, Sánchez PL, These A, Preiss-Weigert A, Castellari M (2015) Occurrence of pyrrolizidine alkaloids in food. EFSA Support Publ. 12(8):859E. CrossRefGoogle Scholar
  30. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605. CrossRefPubMedGoogle Scholar
  31. Park BK, Kitteringham NR, Maggs JL, Pirmohamed M, Williams DP (2005) The role of metabolic activation in drug-induced hepatotoxicity. Annu Rev Pharmacol Toxicol 45:177–202. CrossRefPubMedGoogle Scholar
  32. Pei QL, Kobayashi Y, Tanaka Y et al (2002) Increased expression of multidrug resistance-associated protein 1 (mrp1) in hepatocyte basolateral membrane and renal tubular epithelia after bile duct ligation in rats. Hepatol Res 22(1):58–64CrossRefGoogle Scholar
  33. Reif R, Karlsson J, Gunther G et al (2015) Bile canalicular dynamics in hepatocyte sandwich cultures. Arch Toxicol 89(10):1861–1870. CrossRefPubMedGoogle Scholar
  34. Reif R, Ghallab A, Beattie L et al (2017) In vivo imaging of systemic transport and elimination of xenobiotics and endogenous molecules in mice. Arch Toxicol 91(3):1335–1352. CrossRefPubMedGoogle Scholar
  35. Ruan J, Yang M, Fu P, Ye Y, Lin G (2014) Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis. Chem Res Toxicol 27(6):1030–1039. CrossRefPubMedGoogle Scholar
  36. Steenkamp V, Stewart MJ, van der Merwe S, Zuckerman M, Crowther NJ (2001) The effect of Senecio latifolius a plant used as a South African traditional medicine, on a human hepatoma cell line. J Ethnopharmacol 78(1):51–58. CrossRefPubMedGoogle Scholar
  37. Stegelmeier BL, Gardner DR, James LF, Molyneux RJ (1996) Pyrrole detection and the pathologic progression of Cynoglossum officinale (houndstongue) poisoning in horses. J Vet Diagn Invest 8(1):81–90. CrossRefPubMedGoogle Scholar
  38. Stegelmeier BL, Edgar JA, Colegate SM et al (1999) Pyrrolizidine alkaloid plants, metabolism and toxicity. J NatToxins 8(1):95–116Google Scholar
  39. Stillman AS, Huxtable R, Consroe P, Kohnen P, Smith S (1977) Hepatic veno-occlusive disease due to pyrrolizidine (Senecio) poisoning in Arizona. Gastroenterology 73(2):349–352CrossRefGoogle Scholar
  40. Tandon HD, Tandon BN, Mattocks AR (1978) An epidemic of veno-occlusive disease of the liver in Afghanistan Pathologic features. Am J Gastroenterol 70(6):607–613PubMedGoogle Scholar
  41. Waizenegger J, Braeuning A, Templin M, Lampen A, Hessel-Pras S (2018) Structure-dependent induction of apoptosis by hepatotoxic pyrrolizidine alkaloids in the human hepatoma cell line HepaRG: single versus repeated exposure. Food Chem Toxicol 114:215–226. CrossRefPubMedGoogle Scholar
  42. Wang X, Kanel GC, DeLeve LD (2000) Support of sinusoidal endothelial cell glutathione prevents hepatic veno-occlusive disease in the rat. Hepatology 31(2):428–434. CrossRefPubMedGoogle Scholar
  43. WHO (1988) WHO IPCS International Programme on Chemical Safety: Pyrrolizidine alkaloids Environmental Health Criteria, vol 80. World Health Organization, GenevaGoogle Scholar
  44. Xiong A, Yang F, Fang L et al (2014) Metabolomic and genomic evidence for compromised bile acid homeostasis by senecionine, a hepatotoxic pyrrolizidine alkaloid. Chem Res Toxicol 27(5):775–786. CrossRefPubMedGoogle Scholar
  45. Yang M, Ruan J, Fu PP, Lin G (2016) Cytotoxicity of pyrrolizidine alkaloid in human hepatic parenchymal and sinusoidal endothelial cells: firm evidence for the reactive metabolites mediated pyrrolizidine alkaloid-induced hepatotoxicity. Chem Biol Interact 243:119–126. CrossRefPubMedGoogle Scholar

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

Personalised recommendations