Archives of Toxicology

, Volume 89, Issue 8, pp 1337–1346 | Cite as

Toxicological interactions between the mycotoxins deoxynivalenol, nivalenol and their acetylated derivatives in intestinal epithelial cells

  • Imourana Alassane-Kpembi
  • Olivier Puel
  • Isabelle P. OswaldEmail author
In vitro systems


In case of mycotoxin contaminations, food and feedstuff are usually contaminated by more than one toxin. However toxicological data concerning the effects of mycotoxin combinations are sparse. The intestinal epithelium is the first barrier against food contaminants and this constantly renewing organ is particularly sensitive to mycotoxins. The aim of this study was to investigate the effects of deoxynivalenol (DON) and four other type B trichothecenes (TCTB), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX) alone or in combination on intestinal epithelial cells. Proliferating, non-transformed IPEC-1 cells were exposed to increasing doses of TCTB, alone or in binary mixtures and mycotoxin-induced cytotoxicity was measured with MTT test. The toxicological interactions were assessed using the isobologram-Combination index method. The five tested mycotoxins and their mixtures had a dose-dependent effect on the proliferating enterocytes. DON–NIV, DON–15-ADON and 15-ADON–3-ADON combinations were synergistic, with magnitude of synergy for 10 % cytotoxicity ranging from 2 to 7. The association between DON and 3-ADON also demonstrated a synergy but only at high doses, at lower doses antagonism was noted. Additivity was observed between NIV and FX, and antagonism between DON and FX. These results indicate that the simultaneous presence of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of TCTB in the diet.


Combination index Cytotoxicity Non-transformed intestinal cell Mycotoxin Synergy Trichothecenes 







Combination index




Median effect dose


Dose reduction index


Fraction affected




Inhibitory concentration 50 %


3,(4,5-Dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide





The authors are grateful to Pr. T.C. Chou, Memorial Sloan-Kettering Cancer Center, New York City for kind help in data analysis, and Pr F. A. Abiola, Institut des Sciences Biomédicales Appliquées, Cotonou (Republic of Benin) for fruitful discussion. The authors thank Dr. Woodley for language editing. This work was supported by the ANR-CESA project DON & Co. I.A.K. was supported by a doctoral fellowship from the Government of the Republic of Benin.

Conflict of interest

Authors declare no conflict of interest.

Supplementary material

204_2014_1309_MOESM1_ESM.pptx (289 kb)
Supplementary material 1 (PPTX 289 kb)


  1. Alassane-Kpembi I, Kolf-Clauw M, Gauthier T, Abrami R, Abiola FA, Oswald IP, Puel O (2013) New insights into mycotoxin mixtures: the toxicity of low doses of Type B trichothecenes on intestinal epithelial cells is synergistic. Toxicol Appl Pharmacol 272:191–198PubMedCrossRefGoogle Scholar
  2. Almond GW (1996) Research applications using pigs. Vet Clin North Am Food Anim Pract 12:707–716PubMedGoogle Scholar
  3. Arunachalam C, Doohan FM (2013) Trichothecene toxicity in eukaryotes: cellular and molecular mechanisms in plants and animals. Toxicol Lett 217:149–158PubMedCrossRefGoogle Scholar
  4. Bianco G, Fontanella B, Severino L, Quaroni A, Autore G, Marzocco S (2012) Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study. PLoS One 7:e52051PubMedCentralPubMedCrossRefGoogle Scholar
  5. Boedeker W, Backhaus T (2010) The scientific assessment of combined effects of risk factors: different approaches in experimental biosciences and epidemiology. Eur J Epidemiol 25:539–546PubMedCrossRefGoogle Scholar
  6. Bony S, Olivier-Loiseau L, Carcelen M, Devaux A (2007) Genotoxic potential associated with low levels of the Fusarium mycotoxins nivalenol and fusarenon X in a human intestinal cell line. Toxicol In Vitro 21:457–465PubMedCrossRefGoogle Scholar
  7. Brosnahan AJ, Brown DR (2012) Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations. Vet Microbiol 156:229–237PubMedCentralPubMedCrossRefGoogle Scholar
  8. Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58:621–681PubMedCrossRefGoogle Scholar
  9. Chou TC (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70:440–446PubMedCrossRefGoogle Scholar
  10. Danicke S, Hegewald AK, Kahlert S, Kluess J, Rothkötter HJ, Breves G, Doll S (2010) Studies on the toxicity of deoxynivalenol (DON), sodium metabisulfite, DON-sulfonate (DONS) and de-epoxy-DON for porcine peripheral blood mononuclear cells and the Intestinal Porcine Epithelial Cell lines IPEC-1 and IPEC-J2, and on effects of DON and DONS on piglets. Food Chem Toxicol 48:2154–2162PubMedCrossRefGoogle Scholar
  11. De Boevre M, Jacxsens L, Lachat C, Eeckhout M, Di Mavungu JD, Audenaert K, Maene P, Haesaert G, Kolsteren P, De Meulenaer B, De Saeger S (2013) Human exposure to mycotoxins and their masked forms through cereal-based foods in Belgium. Toxicol Lett 218:281–292PubMedCrossRefGoogle Scholar
  12. De Vos M, Huygelen V, Casteleyn C, Van Cruchten S, Van Ginneken C (2012) Alternative models to study the intestinal barrier function of piglets. Altern Lab Anim 40:P26–P27PubMedGoogle Scholar
  13. Desjardins AE (2009) From yellow rain to green wheat: 25 years of trichothecene biosynthesis research. J Agric Food Chem 57:4478–4484PubMedCrossRefGoogle Scholar
  14. Desjardins AE, McCormick SP, Appell M (2007) Structure-activity relationships of trichothecene toxins in an Arabidopsis thaliana leaf assay. J Agric Food Chem 55:6487–6492PubMedCrossRefGoogle Scholar
  15. Diesing AK, Nossol C, Panther P, Walk N, Post A, Kluess J, Kreutzmann P, Dänicke S, Rothkötter HJ, Kahlert S (2011) Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2. Toxicol Lett 200:8–18PubMedCrossRefGoogle Scholar
  16. Eckard S, Wettstein FE, Forrer HR, Vogelgsang S (2011) Incidence of Fusarium species and mycotoxins in silage maize. Toxins 3:949–967PubMedCentralPubMedCrossRefGoogle Scholar
  17. Ficheux AS, Sibiril Y, Parent-Massin D (2012) Co-exposure of Fusarium mycotoxins: in vitro myelotoxicity assessment on human hematopoietic progenitors. Toxicon 60:1171–1179PubMedCrossRefGoogle Scholar
  18. Gauthier T, Wang X, Dos Santos JS, Fysikopoulos A, Tadrist S, Canlet C, Artigot MP, Loiseau N, Oswald IP, Puel O (2012) Trypacidin, a spore borne toxin from Aspergillus fumigatus, induces cytotoxicity in lung cells. PLoS One 7:e29906PubMedCentralPubMedCrossRefGoogle Scholar
  19. Gonzalez-Vallina R, Wang H, Zhan R, Berschneider HM, Lee RM, Davidson NO, Black DD (1996) Lipoprotein and apolipoprotein secretion by a newborn piglet intestinal cell line (IPEC-1). Am J Physiol 271:249–259Google Scholar
  20. Goossens J, Pasmans F, Verbrugghe E, Vandenbroucke V, De Baere S, Meyer E, Haesebrouck F, De Backer P, Croubels S (2012) Porcine intestinal epithelial barrier disruption by the Fusarium mycotoxins deoxynivalenol and T-2 toxin promotes transepithelial passage of doxycycline and paromomycin. BMC Vet Res 8:245PubMedCentralPubMedCrossRefGoogle Scholar
  21. Heath JP (1996) Epithelial cell migration in the intestine. Cell Biol Int 20:139–146PubMedCrossRefGoogle Scholar
  22. Hedman R, Pettersson H, Lindberg JE (1997) Absorption and metabolism of nivalenol in pigs. Arch Tierernahr 50:13–24PubMedCrossRefGoogle Scholar
  23. Ireland JJ, Roberts RM, Palmer GH, Bauman DE, Bazer FW (2008) A commentary on domestic animals as dual-purpose models that benefit agricultural and biomedical research. J Anim Sci 86:2797–2805PubMedCrossRefGoogle Scholar
  24. Koh SY, George S, Brozel V, Moxley R, Francis D, Kaushik RS (2008) Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli. Vet Microbiol 130:191–197PubMedCrossRefGoogle Scholar
  25. Kouadio JH, Dano SD, Moukha S, Mobio TA, Creppy EE (2007) Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells. Toxicon 49:306–317PubMedCrossRefGoogle Scholar
  26. Larsen JC, Hunt J, Perrin I, Ruckenbauer P (2004) Workshop on trichothecenes with a focus on DON: summary report. Toxicol Lett 153:1–22PubMedCrossRefGoogle Scholar
  27. Loiseau N, Debrauwer L, Sambou T, Bouhet S, Miller JD, Martin PG, Viadere JL, Pinton P, Puel O, Pineau T, Tulliez J, Galtier P, Oswald IP (2007) Fumonisin B1 exposure and its selective effect on porcine jejunal segment: sphingolipids, glycolipids and trans-epithelial passage disturbance. Biochem Pharmacol 74:144–152PubMedCrossRefGoogle Scholar
  28. Lucioli J, Pinton P, Callu P, Laffitte J, Grosjean F, Kolf-Clauw M, Oswald IP, Bracarense AP (2013) The food contaminant deoxynivalenol activates the mitogen activated protein kinases in the intestine: interest of ex vivo models as an alternative to in vivo experiments. Toxicon 66:31–36PubMedCrossRefGoogle Scholar
  29. Madhyastha MS, Marquardt RR, Abramson D (1994) Structure-activity relationships and interactions among trichothecene mycotoxins as assessed by yeast bioassay. Toxicon 32:1147–1152PubMedCrossRefGoogle Scholar
  30. Maresca M (2013) From the gut to the brain: journey and pathophysiological effects of the food-associated trichothecene mycotoxin deoxynivalenol. Toxins 5:784–820PubMedCentralPubMedCrossRefGoogle Scholar
  31. Maresca M, Yahi N, Younes-Sakr L, Boyron M, Caporiccio B, Fantini J (2008) Both direct and indirect effects account for the pro-inflammatory activity of enteropathogenic mycotoxins on the human intestinal epithelium: stimulation of interleukin-8 secretion, potentiation of interleukin-1beta effect and increase in the transepithelial passage of commensal bacteria. Toxicol Appl Pharmacol 228:84–92PubMedCrossRefGoogle Scholar
  32. Marzocco S, Russo R, Bianco G, Autore G, Severino L (2009) Pro-apoptotic effects of nivalenol and deoxynivalenol trichothecenes in J774A.1 murine macrophages. Toxicol Lett 189:21–26PubMedCrossRefGoogle Scholar
  33. McCormick SP, Alexander NJ, Proctor RH (2013) Trichothecene triangle: toxins, genes, and plant disease. In: Gang DR (ed) Phytochemicals, plant growth, and the environment. Springer, New York, pp 1–17CrossRefGoogle Scholar
  34. Meissonnier GM, Laffitte J, Loiseau N, Benoit E, Raymond I, Pinton P, Cossalter AM, Bertin G, Oswald IP, Galtier P (2007) Selective impairment of drug-metabolizing enzymes in pig liver during subchronic dietary exposure to Aflatoxin B1. Food Chem Toxicol 45:2145–2154PubMedCrossRefGoogle Scholar
  35. Parent-Massin D (2004) Haematotoxicity of trichothecenes. Toxicol Lett 153:75–81PubMedCrossRefGoogle Scholar
  36. Pestka JJ, Zhou HR, Moon Y, Chung YJ (2004) Cellular and molecular mechanisms for immune modulation by deoxynivalenol and other trichothecenes: unraveling a paradox. Toxicol Lett 153:61–73PubMedCrossRefGoogle Scholar
  37. Pestka JJ, Yike I, Dearborn DG, Ward MD, Harkema JR (2008) Stachybotrys chartarum, trichothecene mycotoxins, and damp building-related illness: new insights into a public health enigma. Toxicol Sci 104:4–26PubMedCrossRefGoogle Scholar
  38. Pinton P, Braicu C, Nougayrede JP, Laffitte J, Taranu I, Oswald IP (2010) Deoxynivalenol impairs porcine intestinal barrier function and decreases the protein expression of claudin-4 through a mitogen-activated protein kinase-dependent mechanism. J Nutr 140:1956–1962PubMedCrossRefGoogle Scholar
  39. Pinton P, Tsybulskyy D, Lucioli J, Laffitte J, Callu P, Lyazhri F, Grosjean F, Bracarense AP, Kolf-Clauw M, Oswald IP (2012) Toxicity of deoxynivalenol and its acetylated derivatives on the intestine: differential effects on morphology, barrier function, tight junction proteins, and mitogen-activated protein kinases. Toxicol Sci 130:180–190PubMedCrossRefGoogle Scholar
  40. Poapolathep A, Sugita-Konishi Y, Doi K, Kumagai S (2003) The fates of trichothecene mycotoxins, nivalenol and fusarenon-X, in mice. Toxicon 41:1047–1054PubMedCrossRefGoogle Scholar
  41. Poapolathep A, Sugita-Konishi Y, Phitsanu T, Doi K, Kumagai S (2004) Placental and milk transmission of trichothecene mycotoxins, nivalenol and fusarenon-X, in mice. Toxicon 44:111–113PubMedCrossRefGoogle Scholar
  42. Ribeiro DH, Ferreira FL, da Silva VN, Aquino S, Correa B (2010) Effects of aflatoxin B1 and fumonisin B1 on the viability and induction of apoptosis in rat primary hepatocytes. Int J Mol Sci 11:1944–1955PubMedCentralPubMedCrossRefGoogle Scholar
  43. Schinkel AH, Jonker JW (2003) Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 55:3–29PubMedCrossRefGoogle Scholar
  44. Schollenberger M, Muller HM, Ernst K, Sondermann S, Liebscher M, Schlecker C, Wischer G, Drochner W, Hartung K, Piepho HP (2012) Occurrence and distribution of 13 trichothecene toxins in naturally contaminated maize plants in Germany. Toxins 4:778–787PubMedCentralPubMedCrossRefGoogle Scholar
  45. SCOOP (2003) Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states Reports on tasks for scientific cooperation SCOOP Task 3210. p 606.
  46. Speijers GJ, Speijers MH (2004) Combined toxic effects of mycotoxins. Toxicol Lett 153:91–98PubMedCrossRefGoogle Scholar
  47. Streit E, Schatzmayr G, Tassis P, Tzika E, Marin D, Taranu I, Tabuc C, Nicolau A, Aprodu I, Puel O, Oswald IP (2012) Current situation of mycotoxin contamination and co-occurrence in animal feed-focus on Europe. Toxins 4:788–809PubMedCentralPubMedCrossRefGoogle Scholar
  48. Streit E, Schwab C, Sulyok M, Naehrer K, Krska R, Schatzmayr G (2013) Multi-mycotoxin screening reveals the occurrence of 139 different secondary metabolites in feed and feed ingredients. Toxins 5:504–523PubMedCentralPubMedCrossRefGoogle Scholar
  49. Sundstol Eriksen G, Pettersson H, Lundh T (2004) Comparative cytotoxicity of deoxynivalenol, nivalenol, their acetylated derivatives and de-epoxy metabolites. Food Chem Toxicol 42:619–624PubMedCrossRefGoogle Scholar
  50. Tep J, Videmann B, Mazallon M, Balleydier S, Cavret S, Lecoeur S (2007) Transepithelial transport of fusariotoxin nivalenol: mediation of secretion by ABC transporters. Toxicol Lett 170:248–258PubMedCrossRefGoogle Scholar
  51. Thompson WL, Wannemacher RW Jr (1986) Structure-function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon 24:985–994PubMedCrossRefGoogle Scholar
  52. Van Der Fels-Klerx HJ, Klemsdal S, Hietaniemi V, Lindblad M, Ioannou-Kakouri E, Van Asselt ED (2012) Mycotoxin contamination of cereal grain commodities in relation to climate in North West Europe. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 29:1581–1592CrossRefGoogle Scholar
  53. Vandenbroucke V, Croubels S, Martel A, Verbrugghe E, Goossens J, Van Deun K, Boyen F, Thompson A, Shearer N, De Backer P, Haesebrouck F, Pasmans F (2011) The mycotoxin deoxynivalenol potentiates intestinal inflammation by Salmonella typhimurium in porcine ileal loops. PLoS One 6:e23871PubMedCentralPubMedCrossRefGoogle Scholar
  54. Videmann B, Tep J, Cavret S, Lecoeur S (2007) Epithelial transport of deoxynivalenol: involvement of human P-glycoprotein (ABCB1) and multidrug resistance-associated protein 2 (ABCC2). Food Chem Toxicol 45:1938–1947PubMedCrossRefGoogle Scholar
  55. Wan LY, Turner PC, El-Nezami H (2013) Individual and combined cytotoxic effects of Fusarium toxins (deoxynivalenol, nivalenol, zearalenone and fumonisins B1) on swine jejunal epithelial cells. Food Chem Toxicol 57:276–283PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Imourana Alassane-Kpembi
    • 1
    • 2
    • 3
  • Olivier Puel
    • 1
    • 2
  • Isabelle P. Oswald
    • 1
    • 2
    Email author
  1. 1.UMR 1331 Toxalim, Research Center in Food ToxicologyINRAToulouse cedex 03France
  2. 2.INP, UMR 1331, ToxalimUniversité de ToulouseToulouseFrance
  3. 3.Hôpital d’Instruction des ArméesCotonouBénin

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