Archives of Toxicology

, Volume 88, Issue 5, pp 1097–1107 | Cite as

Toxicity of the mycotoxin citrinin and its metabolite dihydrocitrinone and of mixtures of citrinin and ochratoxin A in vitro

  • Wolfram Föllmann
  • Claudia Behm
  • Gisela H. Degen
In vitro systems


Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by several species of the genera Aspergillus, Penicillium and Monascus. Both can be present as contaminants in various food commodities and in animal feed. The occurrence and toxicity of OTA and human exposure have been intensively studied, but for CIT such data are scarce by comparison. Recently, dihydrocitrinone (DH-CIT) was detected as main metabolite of CIT in human urine, and co-occurrence of CIT and OTA was shown in human blood plasma (Blaszkewicz et al. in Arch Toxicol 87:1087–1094, 2013). In light of these new findings, we have now investigated the toxicity of the metabolite DH-CIT in comparison with CIT and analysed the effects of mixtures of CIT and OTA in vitro. The cytotoxic potency of DH-CIT (IC50 of 320/200 μM) was distinctly lower compared with CIT (IC50 of 70/62 μM) after treatment of V79 cells for 24 and 48 h. Whereas CIT induced a concentration-dependent increase in micronucleus frequencies at concentrations ≥30 μM, DH-CIT showed no genotoxic effect up to 300 μM. Thus, conversion of CIT to DH-CIT in humans can be regarded as a detoxification step. Mixtures of CIT and OTA exerted additive effects in cytotoxicity assays. The effect of CIT and OTA mixtures on induction of micronuclei varied dependent on the used concentrations between additive for low μM concentrations and more-than-additive for high μM concentrations. Effects on cell cycle were mostly triggered by OTA when both mycotoxins were used in combination. The implications of our and related in vitro studies are discussed with respect to in vivo concentrations of CIT and OTA, which are found in animals and in humans.


Cytotoxicity Genotoxicity Metabolite Mixtures 


  1. Babich H, Borenfreund E (1991) Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay. Appl Environ Microbiol 57:2101–2103PubMedPubMedCentralGoogle Scholar
  2. 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 53:423–430PubMedCrossRefGoogle Scholar
  3. Behm C, Föllmann W, Degen GH (2012) Cytotoxic potency of mycotoxins in cultures of V79 lung fibroblast cells. J Toxicol Environ Health A 75:1226–1231PubMedCrossRefGoogle Scholar
  4. Bernhoft A, Keblys M, Morrison E, Larsen HJ, Flåøyen A (2004) Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes. Mycopathologia 158:441–450PubMedCrossRefGoogle Scholar
  5. Blaszkewicz M, Muñoz K, Degen GH (2013) Methods for analysis of citrinin in human blood and urine. Arch Toxicol 87:1087–1094PubMedCrossRefGoogle Scholar
  6. Bouslimi A, Bouaziz C, Ayed-Boussema I, Hassen W, Bacha H (2008a) Individual and combined effects of ochratoxin A and citrinin on viability and DNA fragmentation in cultured Vero cells and on chromosome aberrations in mice bone marrow cells. Toxicology 251:1–7PubMedCrossRefGoogle Scholar
  7. Bouslimi A, Ouannes Z, El Golli E, Bouaziz C, Hassen W, Bacha H (2008b) Cytotoxicity and oxidative damage in kidney cells exposed to the mycotoxins ochratoxin A and citrinin: individual and combined effects. Toxicol Mech Methods 18:341–349PubMedCrossRefGoogle Scholar
  8. Chang CH, Yu FY, Wu TS, Wang LT, Liu BH (2011) Mycotoxin citrinin induced cell cycle G2/M arrest and numerical chromosomal aberration associated with disruption of microtubule formation in human cells. Toxicol Sci 119:84–92PubMedCrossRefGoogle Scholar
  9. Chu FS (1971) Interaction of ochratoxin A with bovine serum albumin. Arch Biochem Biophys 147:359–366PubMedCrossRefGoogle Scholar
  10. Countryman PI, Heddle JA (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutat Res 41:321–332PubMedCrossRefGoogle Scholar
  11. Creppy EE, Lorkowski G, Beck G, Röschenthaler R, Dirheimer G (1980) Combined action of citrinin and ochratoxin A on hepatoma tissue culture cells. Toxicol Lett 5:375–380PubMedCrossRefGoogle Scholar
  12. Cui J, Xing L, Li Z, Wu S, Wang J, Liu J, Wang J, Yan X, Zhang X (2010) Ochratoxin A induces G2 phase arrest in human gastric epithelium GES-1 cells in vitro. Toxicol Lett 193:152–158PubMedCrossRefGoogle Scholar
  13. Degen GH (2011) Tools for investigating workplace-related risks from mycotoxin exposure. World Mycotoxin J 4:315–327CrossRefGoogle Scholar
  14. Degen GH, Gerber MM, Obrecht-Pflumio S, Dirheimer G (1997) Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures. Arch Toxicol 71:365–371PubMedCrossRefGoogle Scholar
  15. Degen GH, Muñoz K, Hengstler JG (2013) Occurrence of mycotoxins in breast milk. In: Zibadi S, Watson RR, Preedy V (eds) Handbook of Dietary and Nutritional Aspects of Human Breast Milk. Wageningen Academic, Wageningen, The Netherlands, pp 813–831 Google Scholar
  16. Dönmez-Altuntas H, Dumlupinar G, Imamoglu N, Hamurcu Z, Liman BC (2007) Effects of the mycotoxin citrinin on micronucleus formation in a cytokinesis-block genotoxicity assay in cultured human lymphocytes. J Appl Toxicol 27:337–341PubMedCrossRefGoogle Scholar
  17. Dorn S, Bolt HMB, Thevis M, Diel P, Degen GH (2008) Induction of micronuclei in V79 cells by the anabolic steroids tetrahydrogestrinone and trenbolone. Arch Toxicol 82:257–263PubMedCrossRefGoogle Scholar
  18. Duarte SC, Pena A, Lino CM (2011) Human ochratoxin A biomarkers—from exposure to effect. Crit Rev Toxicol 41:187–212PubMedCrossRefGoogle Scholar
  19. Dunn BB, Stack ME, Park DL, Joshi A, Friedman L, King RL (1983) Isolation and identification of dihydrocitrinone, a urinary metabolite of citrinin in rats. J Toxicol Environ Health 12:283–289PubMedCrossRefGoogle Scholar
  20. EC/SCOOP—European Commission (2002) Assessment of dietary intake of ochratoxin A by the population of EU Member States. Report of the Scientific Cooperation, Task 3.2.7. Directorate-General Health and Consumer Protection, European Commission.
  21. EU Commission Regulation No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J L 364, 20.12.2006, p 5Google Scholar
  22. European Food Safety Authority (EFSA) (2006) Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. EFSA J 365:1–56Google Scholar
  23. European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM) (2012) Scientific Opinion on the risks for public and animal health related to the presence of citrinin in food and feed. EFSA J 10:2605Google Scholar
  24. Fenech M (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res 285:35–44PubMedCrossRefGoogle Scholar
  25. Flajs D, Peraica M (2009) Toxicological properties of citrinin. Arh Hig Rada Toksikol 60:457–464PubMedCrossRefGoogle Scholar
  26. Föllmann W, Lebrun S, Kullik B, Koch M, Römer HC, Golka K (2000) Cytotoxicity of ochratoxin A and citrinin in different cell types in vitro. Mycotoxin Res 16:123–126PubMedCrossRefGoogle Scholar
  27. Föllmann W, Behm C, Degen GH (2007) Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells. Mycotoxin Res 23:101–109PubMedCrossRefGoogle Scholar
  28. Föllmann W, Behm C, Degen GH (2009) The emerging Fusarium toxin enniatin B: in vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins. Mycotoxin Res 25:11–19PubMedCrossRefGoogle Scholar
  29. Frisvad JC, Thrane U, Samson RA, Pitt JI (2006) Important mycotoxins and the fungi which produce them. Adv Exp Med Biol 571:3–31PubMedCrossRefGoogle Scholar
  30. Galtier P, Charpenteau JL, Alvinerie M, Labouche C (1979) The pharmacokinetic profile of ochratoxin A in the rat after oral and intravenous administration. Drug Metab Dispos 7:429–434PubMedGoogle Scholar
  31. Grosse Y, Baudrimont I, Castegnaro M, Betbeder AM, Creppy EE, Dirheimer G, Pfohl-Leszkowicz A (1995) Formation of ochratoxin A metabolites and DNA-adducts in monkey kidney cells. Chem Biol Interact 95:175–187PubMedCrossRefGoogle Scholar
  32. Heussner AH, Dietrich DR, O′Brien E (2006) In vitro investigation of individual and combined cytotoxic effects of ochratoxin A and other selected mycotoxins on renal cells. Toxicol In Vitro 20:332–341PubMedCrossRefGoogle Scholar
  33. Hult K, Plestina R, Habazin-Novak V, Radic B, Ceovic S (1982) Ochratoxin A in human blood and balkan endemic nephropathy. Arch Toxicol 51:313–351CrossRefGoogle Scholar
  34. IARC (International Agency for Research on Cancer) (1986) Citrinin: some naturally occurring and synthetic food components, furocoumarins and ultraviolet radiation. IARC Monogr Eval Carcinog Risk Chem Hum 40:67Google Scholar
  35. IARC (International Agency for Research on Cancer) (1993) Some naturally occurring substances: heterocyclic aromatic amines and mycotoxins. IARC Monogr Eval Carcinog Risk Chem Hum 56:397–444Google Scholar
  36. Kamp HG, Eisenbrand G, Schlatter J, Würth K, Janzowski C (2005) Ochratoxin A: induction of (oxidative) DNA damage, cytotoxicity and apoptosis in mammalian cell lines and primary cells. Toxicology 206:413–425PubMedCrossRefGoogle Scholar
  37. Klarić MS, Zelježić D, Rumora L, Peraica M, Pepeljnjak S, Domijan AM (2012) A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin. Arch Toxicol 86:97–107PubMedCrossRefGoogle Scholar
  38. Knasmüller S, Cavin C, Chakraborty A, Darroudi F, Majer BJ, Huber WW, Ehrlich VA (2004) Structurally related mycotoxins ochratoxin A, ochratoxin B, and citrinin differ in their genotoxic activities and in their mode of action in human-derived liver (HepG2) cells: implications for risk assessment. Nutr Cancer 50:190–197PubMedCrossRefGoogle Scholar
  39. Knecht A, Schwerdt G, Gekle M, Humpf H-U (2005) Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells. Mycotoxin Res 21:176–181PubMedCrossRefGoogle Scholar
  40. Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193PubMedCrossRefGoogle Scholar
  41. Mally A (2012) Ochratoxin A and mitotic disruption: mode of action analysis of renal tumor formation by ochratoxin A. Toxicol Sci 127:315–330PubMedCrossRefGoogle Scholar
  42. Palma N, Cinelli S, Sapora O, Wilson SH, Dogliotti E (2007) Ochratoxin A-induced mutagenesis in mammalian cells is consistent with the production of oxidative stress. Chem Res Toxicol 20:1031–1037PubMedCrossRefPubMedCentralGoogle Scholar
  43. Petkova-Bocharova T, Castegnaro M, Michelon J, Maru V (1991) Ochratoxin A and other mycotoxins in cereals from an area of Balkan endemic nephropathy and urinary tract tumours in Bulgaria in mycotoxin, endemic nephropathy and urinary tract tumours. In: Castegnaro M, Pleština R, Driheimer G, Chernozemsky IN, Bartsch H (eds.) IARC Scientific Publications 115:83–87Google Scholar
  44. Petrik J, Zanić-Grubisić T, Barisić K, Pepeljnjak S, Radić B, Ferencić Z, Cepelak I (2003) Apoptosis and oxidative stress induced by ochratoxin A in rat kidney. Arch Toxicol 77:685–693PubMedCrossRefGoogle Scholar
  45. Pfeiffer E, Gross K, Metzler M (1998) Aneuploidogenic and clastogenic potential of the mycotoxins citrinin and patulin. Carcinogenesis 19:1313–1318PubMedCrossRefGoogle Scholar
  46. Rached E, Hard GC, Blumbach K, Weber K, Draheim R, Lutz WK, Ozden S, Steger U, Dekant W, Mally A (2007) Ochratoxin A: 13-week oral toxicity and cell proliferation in male F344/N rats. Toxicol Sci 97:288–298PubMedCrossRefGoogle Scholar
  47. Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1:1458–1461PubMedCrossRefGoogle Scholar
  48. Sándor G, Busch A, Watzke H, Reek J, Ványi A (1991) Subacute toxicity testing of ochratoxin A and citrinin in swine. Acta Vet Hung 39:149–160PubMedGoogle Scholar
  49. Speijers GJA, Speijers MHM (2004) Combined toxic effects of mycotoxins. Toxicol Lett 153:91–98PubMedCrossRefGoogle Scholar
  50. WHO/IPCS (International Programme on Chemical Safety) (2001) Safety evaluation of certain mycotoxins in food. WHO Food Addit Ser 47:103–415Google Scholar
  51. Xu B, Jia X, Gu L, Sung C (2006) Review on the qualitative and quantitative analysis of the mycotoxin citrinin. Food Control 17:271–285CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Wolfram Föllmann
    • 1
  • Claudia Behm
    • 1
  • Gisela H. Degen
    • 1
  1. 1.Leibniz Research Centre for Working Environment and Human Factors (IfADo)DortmundGermany

Personalised recommendations