Archives of Toxicology

, Volume 91, Issue 4, pp 1859–1870 | Cite as

In vivo cardiomyocyte response to YTX- and AZA-1-induced damage: autophagy versus apoptosis

  • Sara F. Ferreiro
  • Natalia Vilariño
  • Cristina Carrera
  • M. Carmen Louzao
  • Germán Santamarina
  • Antonio G. Cantalapiedra
  • J. Manuel Cifuentes
  • Andrés Crespo
  • Luis M. Botana
Organ Toxicity and Mechanisms


Yessotoxins (YTX) and azaspiracids (AZAs) are marine toxins produced by phytoplanktonic dinoflagellates that get accumulated in filter feeding shellfish and finally reach human consumers through the food web. Both toxin classes are worldwide distributed, and food safety authorities have regulated their content in shellfish in many countries. Recently, YTXs and AZAs have been described as compounds with subacute cardiotoxic potential in rats owed to alterations of the cardiovascular function and ultrastructural heart damage. These molecules are also well known in vitro inducers of cell death. The aim of this study was to explore the presence of cardiomyocyte death after repeated subacute exposure of rats to AZA-1 and YTX for 15 days. Because autophagy and apoptosis are often found in dying cardiomyocytes, several autophagic and apoptotic markers were determined by western blot in heart tissues of these rats. The results showed that hearts from YTX-treated rats presented increased levels of the autophagic markers microtubule-associated protein light chain 3-II (LC3-II) and beclin-1, nevertheless AZA-1-treated hearts evidenced increased levels of the apoptosis markers cleaved caspase-3 and -8, cleaved PARP and Fas ligand. Therefore, while YTX-induced damage to the heart triggers autophagic processes, apoptosis activation occurs in the case of AZA-1. For the first time, activation of cell death signals in cardiomyocytes is demonstrated for these toxins with in vivo experiments, which may be related to alterations of the cardiovascular function.


Apoptosis Autophagy Azaspiracid Cardiotoxicity Subacute Yessotoxin 



The research leading to these results has received funding from the following FEDER cofunded grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA.

Supplementary material

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Supplementary material 1 (DOCX 5244 kb)


  1. Alfonso A, de la Rosa L, Vieytes MR, Yasumoto T, Botana LM (2003) Yessotoxin, a novel phycotoxin, activates phosphodiesterase activity. Effect of yessotoxin on cAMP levels in human lymphocytes. Biochem Pharmacol 65(2):193–208CrossRefPubMedGoogle Scholar
  2. Alfonso A, Roman Y, Vieytes MR et al (2005) Azaspiracid-4 inhibits Ca2+ entry by stored operated channels in human T lymphocytes. Biochem Pharmacol 69(11):1627–1636CrossRefPubMedGoogle Scholar
  3. Bishopric NH, Andreka P, Slepak T, Webster KA (2001) Molecular mechanisms of apoptosis in the cardiac myocyte. Curr Opin Pharmacol 1(2):141–150CrossRefPubMedGoogle Scholar
  4. Buja LM, Vela D (2008) Cardiomyocyte death and renewal in the normal and diseased heart. Cardiovasc Pathol 17(6):349–374CrossRefPubMedGoogle Scholar
  5. de la Rosa LA, Alfonso A, Vilarino N, Vieytes MR, Botana LM (2001) Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin. Biochem Pharmacol 61(7):827–833CrossRefPubMedGoogle Scholar
  6. Dhesi P, Tehrani F, Fuess J, Schwarz ER (2010) How does the heart (not) die? The role of autophagy in cardiomyocyte homeostasis and cell death. Heart Fail Rev 15(1):15–21CrossRefPubMedGoogle Scholar
  7. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516CrossRefPubMedPubMedCentralGoogle Scholar
  8. European Comission Regulation (2004) (EC) No. 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food animal origin. Off J Eur Commun Annex III, Sect VIII, Chap V, 2. L 226:60–61Google Scholar
  9. Fernandez-Araujo A, Tobio A, Alfonso A, Botana LM (2014) Role of AKAP 149-PKA-PDE4A complex in cell survival and cell differentiation processes. Int J Biochem Cell Biol 53C:89–101CrossRefGoogle Scholar
  10. Fernandez-Araujo A, Alfonso A, Vieytes M, Botana L (2015) Key role of phosphodiesterase 4A (PDE4A) in autophagy triggered by yessotoxin. Toxicology 329:60–72CrossRefPubMedGoogle Scholar
  11. Ferreiro SF, Vilariño N, Carrera C et al (2014a) In vivo arrhythmogenicity of the marine biotoxin azaspiracid-2 in rats. Arch Toxicol 88(2):425–434CrossRefPubMedGoogle Scholar
  12. Ferreiro SF, Vilariño N, Louzao MC, Nicolaou KC, Frederick MO, Botana LM (2014b) In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2. Toxicon 91:69–75CrossRefPubMedGoogle Scholar
  13. Ferreiro SF, Vilarino N, Carrera C et al (2016a) Subacute cardiotoxicity of yessotoxin. In vitro and in vivo studies. Chem Res Toxicol 29(6):981–990CrossRefPubMedGoogle Scholar
  14. Ferreiro SF, Vilarino N, Carrera C et al (2016b) Subacute cardiovascular toxicity of the marine phycotoxin azaspiracid-1 in rats. Toxicol Sci 151(1):104–114CrossRefPubMedGoogle Scholar
  15. Furey A, O’Doherty S, O’Callaghan K, Lehane M, James KJ (2010) Azaspiracid poisoning (AZP) toxins in shellfish: toxicological and health considerations. Toxicon 56(2):173–190CrossRefPubMedGoogle Scholar
  16. Gurusamy N, Das DK (2009) Autophagy, redox signaling, and ventricular remodeling. Antioxid Redox Signal 11(8):1975–1988CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gustafsson AB, Gottlieb RA (2008) Recycle or die: the role of autophagy in cardioprotection. J Mol Cell Cardiol 44(4):654–661CrossRefPubMedPubMedCentralGoogle Scholar
  18. James KJ, Fidalgo Saez MJ, Furey A, Lehane M (2004) Azaspiracid poisoning, the food-borne illness associated with shellfish consumption. Food Addit Contam 21(9):879–892CrossRefPubMedGoogle Scholar
  19. Jung CH, Ro SH, Cao J, Otto NM, Kim DH (2010) mTOR regulation of autophagy. FEBS Lett 584(7):1287–1295CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kemp CD, Conte JV (2012) The pathophysiology of heart failure. Cardiovasc Pathol 21(5):365–371CrossRefPubMedGoogle Scholar
  21. Kolattukudy PE, Niu J (2012) Inflammation, endoplasmic reticulum stress, autophagy, and the monocyte chemoattractant protein-1/CCR2 pathway. Circ Res 110(1):174–189CrossRefPubMedPubMedCentralGoogle Scholar
  22. Korsnes MS (2012) Yessotoxin as a tool to study induction of multiple cell death pathways. Toxins (Basel) 4(7):568–579CrossRefGoogle Scholar
  23. Korsnes MS, Hetland DL, Espenes A, Aune T (2006) Induction of apoptosis by YTX in myoblast cell lines via mitochondrial signalling transduction pathway. Toxicol In Vitro 20(8):1419–1426CrossRefPubMedGoogle Scholar
  24. Korsnes MS, Hetland DL, Espenes A, Aune T (2007) Cleavage of tensin during cytoskeleton disruption in YTX-induced apoptosis. Toxicol In Vitro 21(1):9–15CrossRefPubMedGoogle Scholar
  25. Lee Y, Gustafsson AB (2009) Role of apoptosis in cardiovascular disease. Apoptosis 14(4):536–548CrossRefPubMedGoogle Scholar
  26. Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129(7):1261–1274CrossRefPubMedPubMedCentralGoogle Scholar
  27. MCMahon T, Silke J (1996) Winter toxicity of unknown aetiology in mussels. Harmful Algae 14:2Google Scholar
  28. Miyamoto S, Rubio M, Sussman MA (2009) Nuclear and mitochondrial signalling Akts in cardiomyocytes. Cardiovasc Res 82(2):272–285CrossRefPubMedPubMedCentralGoogle Scholar
  29. Moulin M, Piquereau J, Mateo P et al (2015) Sexual dimorphism of doxorubicin-mediated cardiotoxicity: potential role of energy metabolism remodeling. Circ Heart Fail 8(1):98–108CrossRefPubMedGoogle Scholar
  30. Munday R, Aune T, Rossini GP (2008) Toxicology of the yessotoxins. In: Botana LM (ed) Seafood and freshwater toxins pharmacology. CRC Press Boca Ratón, Physiol Detect, pp 329–339Google Scholar
  31. Murata M, Shimatani M, Sugitani H, Oshima Y, Yasumoto T (1987) Isolation and structural elucidation of the causative toxin of the diarrhetic shellfish poisoning. Bull Jpn Soc Sci Fish 48:549–552CrossRefGoogle Scholar
  32. Nishida K, Otsu K (2008) Cell death in heart failure. Circ J 72(Suppl A):A17-21PubMedGoogle Scholar
  33. Petrovski G, Das S, Juhasz B, Kertesz A, Tosaki A, Das DK (2011) Cardioprotection by endoplasmic reticulum stress-induced autophagy. Antioxid Redox Signal 14(11):2191–2200CrossRefPubMedGoogle Scholar
  34. Reinartz M, Raupach A, Kaisers W, Godecke A (2014) AKT1 and AKT2 induce distinct phosphorylation patterns in HL-1 cardiac myocytes. J Proteome Res 13(10):4232–4245CrossRefPubMedGoogle Scholar
  35. Ronzitti G, Callegari F, Malaguti C, Rossini GP (2004) Selective disruption of the E-cadherin-catenin system by an algal toxin. Br J Cancer 90(5):1100–1107CrossRefPubMedPubMedCentralGoogle Scholar
  36. Rubiolo JA, Lopez-Alonso H, Martinez P et al (2014) Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3. Cell Signal 26(2):419–432CrossRefPubMedGoogle Scholar
  37. Satake M, Ofuji K, Naoki H et al (1998) Azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from Irish mussels, Mytilus edulis. J Am Chem Soc 120(38):9967–9968CrossRefGoogle Scholar
  38. Takemura G, Kanoh M, Minatoguchi S, Fujiwara H (2013) Cardiomyocyte apoptosis in the failing heart—a critical review from definition and classification of cell death. Int J Cardiol 167(6):2373–2386CrossRefPubMedGoogle Scholar
  39. Terao K, Ito E, Oarada M, Murata M, Yasumoto T (1990) Histopathological studies on experimental marine toxin poisoning—5. The effects in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon 28(9):1095–1104CrossRefPubMedGoogle Scholar
  40. Tillmann U, Elbrächter M, Krock B, John U, Cembella A (2009) Azadinium spinosum gen. et sp. nov. (Dinophyceae) identified as a primary producer of azaspiracid toxins. Eur J Phycol 44(1):63–79CrossRefGoogle Scholar
  41. Toker A, Marmiroli S (2014) Signaling specificity in the Akt pathway in biology and disease. Adv Biol Regul 55:28–38CrossRefPubMedGoogle Scholar
  42. Tubaro A, Dell’ovo V, Sosa S, Florio C (2010) Yessotoxins: a toxicological overview. Toxicon 56(2):163–172CrossRefPubMedGoogle Scholar
  43. Twiner MJ, Doucette GJ, Rasky A, Huang XP, Roth BL, Sanguinetti MC (2012a) Marine algal toxin azaspiracid is an open-state blocker of HERG potassium channels. Chem Res Toxicol 25(9):1975–1984CrossRefPubMedPubMedCentralGoogle Scholar
  44. Twiner MJ, Hanagriff JC, Butler S, Madhkoor AK, Doucette GJ (2012b) Induction of apoptosis pathways in several cell lines following exposure to the marine algal toxin azaspiracid. Chem Res Toxicol 25(7):1493–1501CrossRefPubMedGoogle Scholar
  45. Vilariño N, Nicolaou KC, Frederick MO et al (2006) Cell growth inhibition and actin cytoskeleton disorganization induced by azaspiracid-1 structure-activity studies. Chem Res Toxicol 19(11):1459–1466CrossRefPubMedGoogle Scholar
  46. Vilariño N, Nicolaou KC, Frederick MO et al (2008) Azaspiracid substituent at C1 is relevant to in vitro toxicity. Chem Res Toxicol 21(9):1823–1831CrossRefPubMedPubMedCentralGoogle Scholar
  47. Wesselborg S, Stork B (2015) Autophagy signal transduction by ATG proteins: from hierarchies to networks. Cell Mol Life Sci 72(24):4721–4757CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Sara F. Ferreiro
    • 1
  • Natalia Vilariño
    • 1
  • Cristina Carrera
    • 1
    • 3
  • M. Carmen Louzao
    • 1
  • Germán Santamarina
    • 2
    • 3
  • Antonio G. Cantalapiedra
    • 2
    • 3
  • J. Manuel Cifuentes
    • 4
  • Andrés Crespo
    • 1
  • Luis M. Botana
    • 1
  1. 1.Departamento de Farmacología, Facultad de VeterinariaUniversidade de Santiago de CompostelaLugoSpain
  2. 2.Departamento de Ciencias Clínicas Veterinarias, Facultad de VeterinariaUniversidade de Santiago de CompostelaLugoSpain
  3. 3.Hospital Veterinario Universitario Rof Codina, Facultad de VeterinariaUniversidade de Santiago de CompostelaLugoSpain
  4. 4.Departamento de Anatomía y Producción Animal, Facultad de VeterinariaUniversidade de Santiago de CompostelaLugoSpain

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