Archives of Toxicology

, Volume 89, Issue 12, pp 2305–2323 | Cite as

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

  • Juliana Garcia
  • Vera Marisa Costa
  • Alexandra T. P. Carvalho
  • Ricardo Silvestre
  • José Alberto Duarte
  • Daniel F. A. R. Dourado
  • Marcelo D. Arbo
  • Teresa Baltazar
  • Ricardo Jorge Dinis-Oliveira
  • Paula Baptista
  • Maria de Lourdes Bastos
  • Félix Carvalho
Molecular Toxicology


Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. α-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to α-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of α-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by α-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal α-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to α-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-α-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with α-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in α-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.


α-Amanitin RNA polymerase II Polymyxin B Liver Kidney 



Juliana Garcia, Vera Marisa Costa, Ricardo Dinis-Oliveira and Ricardo Silvestre thank FCT—Foundation for Science and Technology—for their PhD grant (SFRH/BD/74979/2010), Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/110001/2015) and Investigator grants (IF/01147/2013) and (IF/00021/2014), respectively. This work was supported by the Fundação para a Ciência e Tecnologia (FCT) – project PTDC/DTPFTO/4973/2014 – and the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Barbosa DJ, Capela JP, Oliveira JM et al (2012) Pro-oxidant effects of Ecstasy and its metabolites in mouse brain synaptosomes. Br J Pharmacol 165(4b):1017–1033PubMedCentralCrossRefPubMedGoogle Scholar
  2. Beck BD, Seeley M, Calabrese EJ (2014) The use of toxicology in the regulatory process. In: Kruger CL (ed) Wallace H, A. Haye’s principles and methods of toxicology. CRC Press, US, pp 35–87Google Scholar
  3. Beelman CA, Parker R (1995) Degradation of mRNA in eukaryotes. Cell 81(2):179–183CrossRefPubMedGoogle Scholar
  4. Broussard CN, Aggarwal A, Lacey SR et al (2001) Mushroom poisoning–from diarrhea to liver transplantation. Am J Gastroenterol 96(11):3195–3198PubMedGoogle Scholar
  5. Bushnell DA, Cramer P, Kornberg RD (2002) Structural basis of transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution. Proc Natl Acad Sci USA 99(3):1218–1222PubMedCentralCrossRefPubMedGoogle Scholar
  6. Case DA, Cheatham TE 3rd, Darden T et al (2005) The amber biomolecular simulation programs. J Comput Chem 26(16):1668–1688PubMedCentralCrossRefPubMedGoogle Scholar
  7. Chang I-M, Yamaura Y (1993) Aucubin: a new antidote for poisonous amanita mushrooms. Phytother Res 7(1):53–56CrossRefGoogle Scholar
  8. Cheung PCK (2010) The nutritional and health benefits of mushrooms. Nutr Bull 35(4):292–299CrossRefGoogle Scholar
  9. Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R (2003) Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta 329(1–2):23–38CrossRefPubMedGoogle Scholar
  10. Dores-Sousa JL, Duarte JA, Seabra V, Bastos Mde L, Carvalho F, Costa VM (2015) The age factor for mitoxantrone’s cardiotoxicity: multiple doses render the adult mouse heart more susceptible to injury. Toxicology 329:106–119CrossRefPubMedGoogle Scholar
  11. Essmann U et al (1995) A smooth particle mesh Ewald method. J Chem Phys 103Google Scholar
  12. Fineschi V, Di Paolo M, Centini F (1996) Histological criteria for diagnosis of amanita phalloides poisoning. J Forensic Sci 41(3):429–432CrossRefPubMedGoogle Scholar
  13. Gao C, Guo H, Downey L, Marroquin C, Wei J, Kuo PC (2003) Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells. Carcinogenesis 24(12):1871–1878CrossRefPubMedGoogle Scholar
  14. Garcia J, Carvalho AT, Dourado DF, Baptista P, de Lourdes Bastos M, Carvalho F (2014) New in silico insights into the inhibition of RNAP II by alpha-amanitin and the protective effect mediated by effective antidotes. J Mol Graph Model 51:120–127CrossRefPubMedGoogle Scholar
  15. Garcia J, Costa V, Carvalho A et al (2015a) Amanita phalloides poisoning: mechanisms of toxicity and treatment (accepted)Google Scholar
  16. Garcia J, Costa VM, Baptista P, Bastos MdL, Carvalho F (2015b) Quantification of alpha-amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection. J Chromatogr B 997:85–95CrossRefGoogle Scholar
  17. Guhaniyogi J, Brewer G (2001) Regulation of mRNA stability in mammalian cells. Gene 265(1–2):11–23CrossRefPubMedGoogle Scholar
  18. He J, Gao S, Hu M, Chow DS, Tam VH (2013) A validated ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of polymyxin B in mouse serum and epithelial lining fluid: application to pharmacokinetic studies. J Antimicrob Chemother 68(5):1104–1110PubMedCentralCrossRefPubMedGoogle Scholar
  19. Kaya E, Surmen MG, Yaykasli KO et al (2014) Dermal absorption and toxicity of alpha amanitin in mice. Cutan Ocul Toxicol 33(2):154–160CrossRefPubMedGoogle Scholar
  20. Koda-Kimble MA, Alldredge BK, Corelli RL, Ernst ME (2012) Koda-Kimble and young’s applied therapeutics: the clinical use of drugs. Wolters Kluwer Health/Lippincott Williams & Wilkins, BaltimoreGoogle Scholar
  21. Kollman PA, Massova I, Reyes C et al (2000) Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc Chem Res 33(12):889–897CrossRefPubMedGoogle Scholar
  22. Larson DR (2011) What do expression dynamics tell us about the mechanism of transcription? Curr Opin Genet Dev 21(5):591–599PubMedCentralCrossRefPubMedGoogle Scholar
  23. Lawrence T (2009) The nuclear factor NF-κB pathway in inflammation. Cold Spring Harb Perspect Biol 1(6):a001651PubMedCentralCrossRefPubMedGoogle Scholar
  24. Leclerc G, Leclerc G, Barredo J (2002) Real-time RT-PCR analysis of mRNA decay: half-life of beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines. Cancer Cell Int 2(1):1PubMedCentralCrossRefPubMedGoogle Scholar
  25. Leist M, Gantner F, Naumann H et al (1997) Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins. Gastroenterology 112(3):923–934CrossRefPubMedGoogle Scholar
  26. Morris GM, Huey R, Lindstrom W et al (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791PubMedCentralCrossRefPubMedGoogle Scholar
  27. Mowry JB, Spyker DA, Cantilena LR Jr, Bailey JE, Ford M (2013) 2012 annual report of the american association of poison control centers’ national poison data system (NPDS): 30th annual report. Clin Toxicol 51(10):949–1229CrossRefGoogle Scholar
  28. Murr MM, Yang J, Fier A, Kaylor P, Mastorides S, Norman JG (2002) Pancreatic elastase induces liver injury by activating cytokine production within kupffer cells via nuclear factor-kappa B. J Gastrointest Surg 6(3):474–480CrossRefPubMedGoogle Scholar
  29. Mydlik M, Derzsiova K (2006) Liver and kidney damage in acute poisonings. Bantao J 4(1):30–32Google Scholar
  30. Onufriev A, Bashford D, Case DA (2000) Modification of the generalized born model suitable for macromolecules. J Phys Chem B 104(15):3712–3720CrossRefGoogle Scholar
  31. Pinson CW, Daya MR, Benner KG et al (1990) Liver transplantation for severe amanita phalloides mushroom poisoning. Am J Surg 159(5):493–499CrossRefPubMedGoogle Scholar
  32. Poucheret P, Fons F, Dore JC, Michelot D, Rapior S (2010) Amatoxin poisoning treatment decision-making: pharmaco-therapeutic clinical strategy assessment using multidimensional multivariate statistic analysis. Toxicon 55(7):1338–1345CrossRefPubMedGoogle Scholar
  33. Reuner KH, Wiederhold M, Dunker P et al (1995) Autoregulation of actin synthesis in hepatocytes by transcriptional and posttranscriptional mechanisms. Eur J Biochem 230(1):32–37CrossRefPubMedGoogle Scholar
  34. Ross J (1995) mRNA stability in mammalian cells. Microbiol Rev 59(3):423–450PubMedCentralPubMedGoogle Scholar
  35. Schneider SM, Borochovitz D, Krenzelok EP (1987) Cimetidine protection against alpha-amanitin hepatotoxicity in mice: a potential model for the treatment of amanita phalloides poisoning. Ann Emerg Med 16(10):1136–1140CrossRefPubMedGoogle Scholar
  36. Schneider SM, Michelson EA, Vanscoy G (1992) Failure of N-acetylcysteine to reduce alpha amanitin toxicity. J Appl Toxicol 12(2):141–142CrossRefPubMedGoogle Scholar
  37. Tong TC, Hernandez M, Richardson WH 3rd et al (2007) Comparative treatment of alpha-amanitin poisoning with N-acetylcysteine, benzylpenicillin, cimetidine, thioctic acid, and silybin in a murine model. Ann Emerg Med 50(3):282–288CrossRefPubMedGoogle Scholar
  38. Vetter J (1998) Toxins of amanita phalloides. Toxicon 36(1):13–24CrossRefPubMedGoogle Scholar
  39. Vogel G, Tuchweber B, Trost W, Mengs U (1984) Protection by silibinin against amanita phalloides intoxication in beagles. Toxicol Appl Pharmacol 73(3):355–362CrossRefPubMedGoogle Scholar
  40. Weiser J, Shenkin PS, Still WC (1999) Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO). J Comput Chem 20(2):217–230CrossRefGoogle Scholar
  41. Wieland T (1983) The toxic peptides from amanita mushrooms. Int J Pept Prot Res 22(3):257–276CrossRefGoogle Scholar
  42. Wieland T, Faulstich H (1978) Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous amanita mushrooms. CRC Crit Rev Biochem 5(3):185–260CrossRefPubMedGoogle Scholar
  43. Wills BK, Haller NA, Peter D, White LJ (2005) Use of amifostine, a novel cytoprotective, in alpha-amanitin poisoning. Clin Toxicol (Phila) 43(4):261–267CrossRefGoogle Scholar
  44. Yamaura Y, Fukuhara M, Takabatake E, Ito N, Hashimoto T (1986) Hepatotoxic action of a poisonous mushroom, amanita abrupta in mice and its toxic component. Toxicology 38(2):161–173CrossRefPubMedGoogle Scholar
  45. Zavascki AP, Goldani LZ, Li J, Nation RL (2007) Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 60(6):1206–1215CrossRefPubMedGoogle Scholar
  46. Zhang XP, Zhang L, Chen LJ et al (2007) Influence of dexamethasone on inflammatory mediators and NF-kappaB expression in multiple organs of rats with severe acute pancreatitis. World J Gastroenterol 13(4):548–556PubMedCentralCrossRefPubMedGoogle Scholar
  47. Zhao YF, Zhai WL, Zhang SJ, Chen XP (2005) Protection effect of triptolide to liver injury in rats with severe acute pancreatitis. Hepatobiliary Pancreat Dis Int 4(4):604–608PubMedGoogle Scholar
  48. Zhao J, Cao M, Zhang J, Sun Q, Chen Q, Yang ZR (2006) Pathological effects of the mushroom toxin alpha-amanitin on BALB/c mice. Peptides 27(12):3047–3052CrossRefPubMedGoogle Scholar
  49. Zheleva A (2013) Phenoxyl radicals formation might contribute to severe toxicity of mushrooms toxin alpha-amanitin-an electron paramagnetic resonance study. TJS 11(1):33–38Google Scholar
  50. Zheleva A, Tolekova A, Zhelev M, Uzunova V, Platikanova M, Gadzheva V (2007) Free radical reactions might contribute to severe alpha amanitin hepatotoxicity-a hypothesis. Med Hypotheses 69(2):361–367CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Juliana Garcia
    • 1
  • Vera Marisa Costa
    • 1
  • Alexandra T. P. Carvalho
    • 2
  • Ricardo Silvestre
    • 3
    • 4
  • José Alberto Duarte
    • 5
  • Daniel F. A. R. Dourado
    • 2
  • Marcelo D. Arbo
    • 1
  • Teresa Baltazar
    • 1
  • Ricardo Jorge Dinis-Oliveira
    • 1
    • 6
    • 7
  • Paula Baptista
    • 8
  • Maria de Lourdes Bastos
    • 1
  • Félix Carvalho
    • 1
  1. 1.UCIBIO/REQUIMTE-Laboratory of Toxicology, Department of Biological Sciences, Faculty of PharmacyUniversity of PortoPortoPortugal
  2. 2.Department of Cell and Molecular Biology, Computational and Systems Biology, Biomedical CenterUppsala UniversityUppsalaSweden
  3. 3.School of Health Sciences, Life and Health Sciences Research Institute (ICVS)University of MinhoBragaPortugal
  4. 4.ICVS/3B’s-PT Government Associate LaboratoryBraga, GuimarãesPortugal
  5. 5.Faculty of Sport, CIAFELUniversity of PortoPortoPortugal
  6. 6.Department of Legal Medicine and Forensic Sciences, Faculty of MedicineUniversity of PortoPortoPortugal
  7. 7.Department of Sciences, IINFACTS-Institute of Research and Advanced Training in Health Sciences and TechnologiesAdvanced Institute of Health Sciences–North (ISCS-N), CESPU, CRLGandraPortugal
  8. 8.CIMO/School of Agriculture, Polytechnic Institute of BragançaBragançaPortugal

Personalised recommendations