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The emerging Fusarium toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins

Mycotoxin Research volume 25, pages 11–19 (2009)Cite this article

Abstract

The Fusarium metabolite enniatin B is now recognized as a frequent contaminant of grains used for human foods and animal feeds. Yet, so far very limited data are available on its toxicity and that of other emerging Fusarium mycotoxins (Jestoi M, 2008, Crit Rev Food Sci Nutr 48:21-49). Thus, the mutagenic/genotoxic potential of enniatin B was investigated in a battery of short-term tests, and its cytotoxicity compared with that of several other mycotoxins. No mutagenicity was detected in the Ames assay with four Salmonella typhimurium strains, and in the HPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 cells, in either the presence or absence of an external metabolizing enzyme system (rat liver S9). For other types of genotoxicity, i.e., clastogenicity and chromosomal damage, studied in V79 cells by means of alkaline single-cell gel electrophoresis (Comet) assay and micronucleus assay, no significant genotoxic potential of enniatin B was revealed. However, the Fusarium metabolite exerts pronounced time- and concentration-dependent cytotoxic effects in V79 cells as determined by Alamar Blue reduction and by neutral red uptake assays. For instance, IC20 and IC50 values determined for enniatin B by neutral red assay for 48-h exposure are 1.5 μM and 4 μM. These values are higher than those of the more potent Fusarium toxin deoxynivalenol (IC20 0.7 μM, IC50 of 0.8 μM), but clearly lower than the IC values of several other mycotoxins tested in parallel. Their ranking of cytotoxicity in V79 cells was as follows: deoxynivalenol > enniatin B > patulin > ochratoxin A > zearalenone > citrinin. Moreover, enniatin B was found to induce nuclear fragmentation, a sign of apoptosis, already at low submicromolar concentrations. In summary, despite an apparent lack of mutagenic and genotoxic activity, enniatin B can cause pronounced cytotoxicity in mammalian cells, detectable at low micromolar concentrations.

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References

  • Babich H, Borenfreund E (1991) Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay. Appl Environm Microbiol 57:2101–2103

    CAS  Google Scholar 

  • Behm C, Degen GH, Föllmann W (2009) The Fusarium toxin enniatin B exerts no genotoxic activity, but pronounced cytotoxicity in vitro. Mol Nutr Food Res (in press)

  • Bonacker D, Stoiber T, Böhm KJ, Unger E, Degen GH, Thier R, Bolt HM (2004) Chromosomal genotoxicity of nitrobenzene and benzonitrile. Arch Toxicol 78:49–57

    PubMed  Article  CAS  Google Scholar 

  • Degen GH, Gerber MM, Hillebrand I, Föllmann W (1995) Untersuchungen zur Genotoxizität von Ochratoxin A in Zellkulturen von Schwein und Schaf. In: Landbauforschung, Sonderheft 157 (Proceedings of the 17th Mykotoxin-Workshop, Braunschweig, 15-17 May, 1995), pp 13-17

  • Dorn S, Bolt HM, Thevis M, Diel P, Degen GH (2008) Induction of micronuclei in V79 cells by the anabolic steroids tetrahydrogestrinone and trenbolone. Arch Toxicol 82:257–263

    PubMed  Article  CAS  Google Scholar 

  • Dornetshuber R, Heffeter P, Kamyar MR, Peterbauer T, Berger W, Lemmens-Gruber R (2007) Enniatin exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells. Chem Res Toxicol 20:465–73

    PubMed  Article  CAS  Google Scholar 

  • Firakova S, Proksa B, Sturdikova M (2007) Biosynthesis and biological activities of enniatins. Pharmazie 62:563–568

    PubMed  CAS  Google Scholar 

  • Föllmann W, Lucas S (2003) Effects of the mycotoxin ochratoxin A in a bacterial and a mammalian in vitro mutagenicity test system. Arch Toxicol 77:298–304

    PubMed  Google Scholar 

  • Föllmann W, Degen GH, Behm C (2007) Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultures cells. Mycotoxin Res 23:101–109

    Article  Google Scholar 

  • Fornelli F, Minervini F, Logrieco A (2004) Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9). J Invert Pathol 85:74–79

    Article  CAS  Google Scholar 

  • Gäumann E, Roth S, Ettlinger LE, Plattner PA, Nagel U (1947) Enniatin, ein neues, gegen Mykobakterien wirksames Antibiotikum. Experientia 3:202–203

    Article  Google Scholar 

  • Gutleb AC, Morrison E, Murk AJ (2002) Cytotoxicity assays for mycotoxins produced by Fusarium strains: a review. Environ Toxicol Pharm 11:309–320

    Article  CAS  Google Scholar 

  • Hiraga K, Yamamoto S, Fukuda H, Hamanaka N, Oda K (2005) Enniatin has a new function as an inhibitor of the Pdr5p, one of the ABC transporter in Saccharomyces cerevisiae. Biochim Biophys Res Commun 328:1119–1125

    Article  CAS  Google Scholar 

  • Ivanov VT, Evstratov AV, Sumskaya LV, Melnik EI, Chumburidze TS, Portnova SL, Balashova TA, Ovchinnikov YA (1973) Sandwich complexes as a functional form of the enniatin ionophores. FEBS Lett 36:65–71

    Article  CAS  Google Scholar 

  • Ivanova L, Uhlig S (2008) A bioassay for the simultaneous measurement of metabolic activity, membrane integrity, and lysosomal activity in cell cultures. Anal Biochem 379:16–19

    PubMed  Article  CAS  Google Scholar 

  • Ivanova L, Skjerve E, Eriksen GS, Uhlig S (2006) Cytotoxicity of enniatins A, A1, B, B1, B2 and B3 from Fusarium avenaceum. Toxicon 47:868–876

    PubMed  Article  CAS  Google Scholar 

  • Jestoi M (2008) Emerging Fusarium-mycotoxins fusaproliferin, beauvericin, enniatins, and moniliformin—a review. Crit Rev Food Sci Nutr 48:21–49

    PubMed  Article  CAS  Google Scholar 

  • Jestoi M, Rokka M, Yli-Mattila T, Parikka P, Rizzo A, Peltonen K (2004a) Presence and concentrations of the Fusarium-related mycotoxins beauvericin, enniatins and moniliformin in finnish grain samples. Food Addit Contam 21:794–802

    PubMed  Article  CAS  Google Scholar 

  • Jestoi M, Somma MC, Kouva M, Veijalainen P, Rizzo A, Ritieni A, Peltonen K (2004b) Levels of mycotoxins and sample cytotoxicity of selected organic and conventional grain-based products purchased from Finnish and Italian markets. Mol Nutr Food Res 48:299–307

    PubMed  Article  CAS  Google Scholar 

  • Kamyar M, Rawnduzi P, Studenik CR, Kouri K, Lemmens-Gruber R (2004) Investigation of the electrophysiological properties of enniatins. Arch Biochim Biophys 429:215–223

    Article  CAS  Google Scholar 

  • Klötzel M, Gutsche B, Lauber U, Humpf HU (2005) Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry. J Agric Food Chem 53:8904–8910

    PubMed  Article  Google Scholar 

  • Königs M, Lenczyk M, Schwerdt G, Holzinger H, Gekle M, Humpf HU (2007) Cytotoxicity, metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture. Toxicology 240:48–59

    PubMed  Article  Google Scholar 

  • Königs M, Schwerdt G, Gekle M, Humpf HU (2008) Effects of the mycotoxin deoxynivalenol on human primary hepatocytes. Molec Nutr Food Res 52:830–839

    Article  Google Scholar 

  • Kosiak B, Torp M, Skjerve E, Thrane U (2003) The prevalence and distribution of Fusarium species in Norwegian cereals: a survey. Acta Agric Scand Sect B: Soil and Plant Sci 53:168–176

    Google Scholar 

  • Logrieco A, Rizzo A, Ferracane R, Ritieni A (2002) Occurrence of beauvericin and enniatins in wheat affected by Fusarium avenaceum head blight. Appl Environ Microbiol 68:82–85

    PubMed  Article  CAS  Google Scholar 

  • Lohmann A (1988) Analytische, pharmakologische und mikrobiologische Untersuchungen des ionophoren Antibiotikums Fusafungin. PhD thesis, Westfälische Wilhelms Universität, Münster

  • Lorge E, Lambert C, Gervais V, Becourt-Lhote N, Delongeas JL, Claude N (2007) Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay. Toxicol Sci 96:214–217

    PubMed  Article  CAS  Google Scholar 

  • Michels G, Wätjen W, Weber N, Niering P, Chovolou Y, Kampkötter A, Proksch P, Kahl R (2006) Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells. Toxicology 225:173–182

    PubMed  Article  CAS  Google Scholar 

  • O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426

    PubMed  Article  CAS  Google Scholar 

  • Placinta CM, D’Mello JPF, Macdonald AMC (1999) A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim Feed Sci Technol 78:21–37

    Article  CAS  Google Scholar 

  • Pfuhler S, Albertini S, Fautz R, Herbold B, Madle S, Utesch D, Poth A (2007) Genetic toxicity assessment: employing the best science for human safety evaluation part IV: Recommendation of a working group of the Gesellschaft fuer Umwelt-Mutationsforschung (GUM) for a simple and straightforward approach to genotoxicity testing. Toxicol Sci 97:237–240

    PubMed  Article  CAS  Google Scholar 

  • Scherkenbeck J, Jeschke P, Harder A (2002) PF1022A and related cyclodepsi–peptides. Curr Top Med Chem 2:759–777

    PubMed  Article  CAS  Google Scholar 

  • Schollenberger M, Müller HM, Rüfle M, Suchy S, Plank S, Drochner W (2006) Natural occurrence of 16 Fusarium toxins in grains and feedstuffs of plant origin from Germany. Mycopathologia 161:43–52

    PubMed  Article  Google Scholar 

  • Speit G, Schütz P, Bonzheim I, Trenz K, Hoffmann H (2004) Sensitivity of the FPG protein towards alkylation damage in the comet assay. Toxicol Lett 146:151–158

    PubMed  Article  CAS  Google Scholar 

  • Stock S (2004) Das Mykotoxin Ochratoxin A: Untersuchungen zu in vitro Genotoxizität und Metabolismus. Dissertation (Thesis), Justus-Liebig-Universität, Giessen. http://geb.uni-giessen.de/geb/volltexte/2005/2188/

  • Uhlig S, Torp M, Heier BT (2006) Beauvericin and enniatin A, A1, B and B1 in Norwegian grain: a survey. Food Chemistry 94:193–201

    CAS  Google Scholar 

  • Uhlig S, Jestoi M, Parikka P (2007) Fusarium avenaceum—the North European situation. Int J Food Microbiol 119:17–24

    PubMed  Article  CAS  Google Scholar 

  • Wätjen W, Debbab A, Hohlfeld A, Chovolou Y, Kampkötter A, Edrada RA, Ebel R, Hakiki A, Mosaddak M, Totzke F, Kubbutat M, Proksch P (2009) Enniatins A1, B and B1 from an endophytic strain of Fusarium tricintum induce apoptotic cell death in H4IIE hepatoma cells accompanied by inhibition of ERK phosphorylation. Mol Nutr Food Res (in press)

  • Wolf A, Kutz A, Plöttner S, Behm C, Bolt HM, Föllmann W, Kuhlmann J (2005) The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints. Toxicology 207:255–269

    PubMed  Article  CAS  Google Scholar 

  • Zühlke S, Dehne HW, Herebian D, Spiteller M (2008) Mykotoxinmultimethode für die Landwirtschaft. LaborPraxis, Januar 2008, 22-25

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Acknowledgements

The authors gratefully acknowledge the excellent technical assistance of Ingrid Ester, Anke Jung and Brigitte Begher-Tibbe. Many thanks go to R. Dietrich for helpful discussions on morphological alterations induced by ionophoric cyclic peptides. This study was supported by a grant from the Ministerium für Innovation, Wissenschaft, Forschung und Technologie des Landes Nordrhein-Westfalen, Germany, for a collaborative project entitled ”Multikomponentenanalyse für Mykotoxine in Getreide und Futtermitteln” within EU-Ziel 2-program, NRW 2000-2006, Technologie- und Innovationsprogramm.

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  1. Leibniz-Institut für Arbeitsforschung an der TU Dortmund (IfADo), Leibniz Research Centre for Working Environment and Human Factors, Ardeystr. 67, 44139, Dortmund, Germany

    Wolfram Föllmann, Claudia Behm & Gisela H. Degen

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  1. Wolfram Föllmann
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  2. Claudia Behm
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  3. Gisela H. Degen
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Correspondence to Wolfram Föllmann.

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Föllmann, W., Behm, C. & Degen, G.H. The emerging Fusarium toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins. Mycotox Res 25, 11–19 (2009). https://doi.org/10.1007/s12550-008-0002-y

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  • DOI: https://doi.org/10.1007/s12550-008-0002-y

Keywords

  • Cytotoxicity
  • Enniatin B
  • Fusarium toxins
  • Genotoxicity assays