Cereals are infected by a range of biological contaminants, including fungi and mycotoxins. Humans ingesting cereals are therefore exposed to a mixture of natural toxins. Deoxynivalenol (DON) is present in almost all samples of cereal-based food. Previous estimations of the dietary exposure in Norway indicate that certain population groups may have an intake close to or exceeding the Tolerable Daily Intake of DON; and mycotoxin levels in grains seem to increase with climate change. An improved estimate of the human exposure is therefore needed to ensure that the current and expected levels in food pose no risk for consumers.
In the MycoMix project, samples of human blood and urine from individuals with a known diet were analysed for mycotoxins, and a database showing the occurrence of trichothecenes (DON, T-2 toxin, HT-2 toxin) in food was assembled. A total of 179 samples of urine from 47 individuals were analysed and only 2 individuals did not have any quantifiable DON metabolites in the urine. The intake of trichothecenes from grain-based food products was estimated from both biomonitoring and food data, and major sources of trichothecenes in the diet were identified. The estimated intake was then compared to the currently established TDIs. Typical toxic effects in animals caused by feed-borne DON and combined T-2 and HT-2 are feed refusal, gastrointestinal disturbances, impairment of the immune system and the reduced formation of blood cells, observed at exposure levels comparable to those expected in humans. Naturally infected grains were found to be more toxic than feed with the corresponding levels of the individual toxins, suggesting synergistic effects.
The project has focussed especially on the consequences of exposure to cooccurring mycotoxins and their potential interaction with DON. Selected fungal metabolites, from Fusarium (enniatin B; ENNB, 2-amino-14,16-dimethyloctadecan-3-ol; AOD, zearalenone; ZEA), Alternaria sp. (alternariol; AOH) and Aspergillus (sterigmatocystin; ST) were tested alone and in realistic mixtures/concentrations in in vitro cell models for the human immune system (e.g. macrophages).
MycoMix was funded by the Norwegian Research Council.
Partners: Norwegian Veterinary Institute, National Institute of Public Health, Norway and Michigan State University, USA
Project period: 2012–2016
Publications:
- Eriksen, G. S.; Knutsen, H. K.; Sandvik, M.; Brantsæter, A.-L., Urinary deoxynivalenol as a biomarker of exposure in different age, life stage and dietary practice population groups. Environment International 2021, 157, 106804.
Norwegians are exposed to DON in the same range as reported for other countries and children have a higher exposure than adults. Furthermore, intake estimates based on urinary DON concentration are useful for evaluation of the exposure at population level, but due to outliers, the estimates for individuals are uncertain. There are also uncertainties regarding the intake estimates both, from food consumption and from urinary DON concentration, and it was not possible to decide which approach provides the most accurate exposure estimate. - Solhaug, A., Karlsøen, L. M., Holme, J. A., Kristoffersen, A. B., & Eriksen, G. S. Immunomodulatory effects of individual and combined mycotoxins in the THP-1 cell line. Toxicology in Vitro 2016, 36, 120–132.
Differentiation of monocytes into macrophages is important for modulating immunological responses. Effects on the PMA-induced differentiation of THP-1 monocytes into macrophages were therefore investigated for the frequently co-occurring mycotoxins; 2-amino-14,16-dimethyloctadecan-3-ol (AOD), alternariol (AOH), enniatin B (ENNB), deoxynivalenol (DON), sterigmatocystin (ST) and zearaleone (ZEA). In THP-1 monocytes, single mycotoxins were found to affect cell proliferation and led to increased cytotoxicity at higher concentrations. ZEA and AOH induced cell cycle arrest in the G2/M-phase, while ST led to S-phase arrest. AOH, ZEA and DON inhibited the PMA-induced differentiation process from monocytes into macrophages as measured by expression of cell surface receptors (CD14, CD11b and CD71) at non-toxic concentrations. By applying the concentration addition (CA) and independent action (IA) prediction models the combination effects of mycotoxins were found to be mainly additive, but the combination of AOH and ZEA had somewhat synergistic effects at low concentrations. Furthermore, AOH and ZEA were found to inhibit lipopolysaccharide (LPS)-induced TNFα secretion, while DON increased the response. This is suggested to be caused by different regulation of the TNFα gene expression by the mycotoxins. - Solhaug, A., Eriksen, G.S, Holme, J.A. Mechanisms of action and toxicity of the mycotoxin alternariol: A review. Basic & Clinical Pharmacology & Toxicology 2016, 119, 533–539.
The mechanisms of AOH induced toxicity have extensively investigated during an earlier project (Toxicological characterization of selected secondary fungal metabolites in Norwegian grain) and in the current studies. These results, in addition to published work, have been summarised in a review. - Solhaug A, Wisbech C, Christoffersen TE, Hult LO, Lea T, Eriksen GS, Holme J. The mycotoxin alternariol induces DNA damage and modify macrophage phenotype and inflammatory responses. Toxicology Letters 2015, 239, 9–21. (NSFT publication prize)
In this paper we show that AOH induced marked phenotypic changes in macrophages, both in RAW 264.7 mouse macrophage cell line, primary mouse peritoneal macrophages and primary human macrophages derived from peripheral blood monocytes. Although AOH induced somewhat similar changes in cell morphology in the different macrophages, the cytokine profile, cell surface receptor expression and endocytic activity were somewhat different. The AOH induced changes could not be directly linked to an initial AOH-induced ROS production, cell cycle arrest or autophagy as seen as a consequence of AOH-induced double stranded DNA breaks (DSBs). However, AOH-induced DSBs will result in a complex DNA damage response and a link towards macrophage differentiation is still a likely explanation. - Solhaug, A.; Torgersen, M.L.; Holme, J.A.; Lagadic-Gossmann, D.; Eriksen, G.S. Autophagy and senescence, stress responses induced by the DNA-damaging mycotoxin alternariol. Toxicology 2014, 326, 119–129.
We found that the mycotoxin AOH induces autophagy in RAW 264.7 macrophages, possibly through the Sestrin2-AMPK-mTOR-S6K pathway. The induction of autophagy is most probably associated to AOH-induced DNA damage, due to association with topoisomerase activity rather than the production of ROS. After prolonged AOH exposure the cells entered senescence. These in vitro experiments suggest possible modulator effects of AOH on immune cells.
Presentations:
- 25. mai 2016. Invited oral presentation. Effects of mycotoxins on human health. NSFT vårmøte. Folkehelseinstituttet.
- 28–31. jan 2016. Invited oral presentation. A Solhaug. The mycotoxin alternariol induces DNA damage and modifies macrophage phenotype and inflammatory responses. NSFT Vintermøte, Beitostølen.
- 12–17. sep 2015. Poster presentation. A Solhaug. The mycotoxin alternariol induces DNA damage and differentiation of primary human macrophages. Eurotox. Porto, Portugal.
- 26–28. mai 2015. No presentation. Cell-based Assays for screening. Workshop. Hamburg, Germany.
- 17. mar 2015. Oral presentation of project. NFR, Forskerseminar, miljøpåvirkning og helse. Holmen Fjordhotell.
- 22–26. mar 2015. Poster presentation. JA Holme. Autophagy and senescence, stress responses induced by the DNA-damaging mycotoxin alternariol. Society of toxicology, San Diego, CA, USA.
- 29. jan – 1. feb 2015.
- Oral presentation. LM Karlsøen. Immunomodulary effects of single and combined mycotoxins using THP-1 cells in vitro as a model system. NSFT Vintermøte, Beitostølen.
- Invitert presentasjon. A Solhaug. Mekanismer for toksiske effekter av mykotoksiner. NSFT Vintermøte, Beitostølen.
- Invitert foredrag. GS Eriksen. Utgjør naturlig forekommende toksiner en større risiko enn kjemikalier? NSFT Vintermøte, Beitostølen.
- 18–19. nov 2014. 2 oral presentations:
- A Solhaug. Mycotoxins effects toxicity and differentiation of macrophages.
- GS Eriksen. Human exposure to mycotoxins from grain. Nordic-Baltic Fusarium seminar, Helsinki, Finland.
- 18–19. mar 2014. Oral presentation of project. NFR, Forskerseminar, miljøpåvirkning og helse. Triaden, Lørenskog.
- 24–27. jan 2014. Invited presentation. A Solhaug. Flowcytometri – Nyttig verktøy i toksikologisk forskning. NSFT Vintermøte, Beitostølen.
- 4. okt 2013. No presentation. EuroSciCon, O2 arena, London. Cell Cycle Analysis: the research and the technology.
- 3. okt 2013. No presentation. EuroSciCon, O2 arena, London. Analysis of autophagy regulation: Discussion of recent research and new technologies.
- 27–31. mai 2013. Invited presentation. A Solhaug. Toxicity of alternaria toxins, with a special focus on the effects of alternariol on macrophages. MycoRed international Conference, Martina Franca, Italy.
- 18–19. mar 2013. Oral presentation of project. A Solhaug. NFR, Forskerseminar, miljøpåvirkning og helse. Triaden, Lørenskog.
- 10–14. mar 2013. Poster presentation. A Solhaug. Alternariol induces differentiation of macrophages. Society of toxicology, San Antonio, Texas, USA.
- 6–7. feb 2013. Oral presentation. GS Eriksen. Mykotoksiner hvor går grensa. Bioforskkonferansen. Hamar.
- 13–15. nov 2012. Keynote lecture. Gunnar S Eriksen. Effects of combinations of Fusarium toxins on different animal species. Nordic-Baltic Fusarium seminar, Uppsala, Sweden.
Master theses
- Line Merete Karlsøen (2015). “Immunomodulatory effects of single and combined mycotoxins using THP-1 cells in vitro as a model system.” Master at UiO.
- Cathrine Wisbech (2013). “Toxicity of various mycotoxins to immune cells in vitro, with focus on morphological and phenotypic changes.” Master at UiO.