Co-occurrence, MODIFIED MYCOTOXINS, EMERGING MYCOTOXINS and interactions
Mariano Gorrachategui Animal nutrition consultant and president of CESFAC
1
Co-occurrence of mycotoxins We know that a certain mycotoxin can be produced by several types of fungi and that the same toxigenic fungus can produce several mycotoxins. Moreover, nowadays there are different sources of raw materials. Cereals, plant protein crops, and oilseeds grow and are harvested in a diverse range of climatological conditions, determining the growth of the fungi and the toxins produced. The storage and transport conditions also affect the fungus and toxins that can be produced during that time.
Under these conditions, it’s easy to understand that the odds of finding only one mycotoxin contaminating raw materials or feed are slim. Thus, the term “cooccurrence” is increasingly being used when referring to the simultaneous presence of two or more mycotoxins in the same sample.
There are many publications in literature that highlight the co-occurrence of mycotoxins: In Germany, Goertz et al. (2010) found corn to be simultaneously contaminated with at least 14
Fusarium mycotoxins: Deoxynivalenol (DON) and its acetylated forms Zearalenone (ZEA) Moniliformin (MON) Beauvericin (BEA) Nivalenol (NIV)
Eniantins (ENNs)
Other studies have revealed the
Fumonisins (FBs)
simultaneous presence of several
HT2 Toxin
mycotoxins in samples from
Streit and col. (2013a) indicated in their global monitoring report that 72% of the samples of feed and raw materials were contaminated with more than one mycotoxin. The same authors (Streit et al.,
2013b) observed 83 samples of feed and raw materials to be contaminated with 7 to 69 mycotoxins per sample, having detected up to 169 different compounds.
European countries (Almeida et al.,
2011; Blajet-Kosicka et al., 2014; Driehuid et al., 2008; Labuda et al., 2005a, 2005b; Monbaliu et al., 2010), finding a high percentage of samples to be contaminated with trichothecenes (DON, AcDon, T2, HT2) and FBs at the same time, as well as with ZEA in many cases.
2
In Spain, the study published by IbĂĄĂąez-Bea et al. (2012)
DON 95% of the samples
regarding the simultaneous presence of AFB1, ZEA, and OTA in samples of barley harvested in 2007 and 2008, revealed:
AFB1 + ZEA + OTA 27% of the samples
The presence of the three mycotoxins in 27% of the samples The presence of AFB1, together
AFB1 + ZEA y/o OTA 43% of the samples
Mycotoxin contamination analysis in barley 2007-2008
AFB1 + OTA + DON 29% of the samples AFB1 + OTA + DON + ZEA 26% of the samples
with any one of the other two in 43% of the samples The same authors detected DON in 95% of the samples and two mycotoxins from this group in 43% of the samples.
More recent data (Biomin, 2017) from the analysis of 1.378 samples indicated that 94% of the samples contained more than 10 mycotoxins
Taken together, the results of these studies reveal that 96% of the samples contained three or more mycotoxins. The most frequent combinations are: AFB1, OTA, and DON in
and metabolites and that the average was of 28 mycotoxins. In another study, Raj et al. (2017) after screening 113 samples of maize from Serbia and Bosnia and Herzegovina for mycotoxin contamination, found 28% of the samples to be contaminated with more than one mycotoxin. In another study the authors reported 76% samples contaminated with one or more mycotoxins (Raj et al., 2019).
29% of the samples AFB1, OTA, DON, and ZEA
Regarding fodder, recently there has also been
en 26% of the samples
data published (Panasiuk et al., 2019).
3
Modified mycotoxins In addition to co-occurrence, there is another phenomenon, the presence of the so-called “masked mycotoxins”. This term was first coined by Gareis et al. (1990) in reference to some cases of mycotoxicosis in which, the clinical signs observed in the animals couldn’t be explained by the low content of the mycotoxins detected in the feed.
Masked mycotoxins are defined as “mycotoxins that are not detectable through standard routine analytical techniques”. Years later, Berthiller et al., (2013) and, particularly, Rychlik et al. (2016) introduced the terms following terms: “Matrix-associated mycotoxins” refer to mycotoxins that are associated with oligosaccharides and starch, and that are physically trapped or bound by covalent bonds, as in the case of Fumonisins. “Modified mycotoxins” including: “Biologically” modified mycotoxins, for example, by conjugation, with polar compounds, mainly β-glycoside, sulfate or even glutathione, and that would fall under the denomination of “masked”. “Chemically” modified mycotoxins, produced as a consequence of thermal processes or other kinds of processes that take place during the production of feed.
EFSA (2014a) refers to “modified mycotoxins” as all the forms that have been structurally modified in relation to their “parental compound” o the free mycotoxin.
4
Emerging mycotoxins The development of methods based on chromatographic techniques and mass spectrophotometry enables us to reliably detect smaller quantities of more and more compounds. Therefore, we are discovering new molecules that may have toxic effects and interactions that we know little about, making it important to study them in order to produce safe food. We are referring to the so-called “emerging mycotoxins”. Although this term hasn’t been clearly
Aspergillus metabolites such as sterigmatocystin (STE) and emodin (EMO). The Penicillium metabolite, mycophenolic acid (MPA).
Alternaria metabolites, that include more than 70 toxins. The best-known ones are alternariol (AOH), monomethyl alternariol ether (AME), altenuene (ALT), altertoxin (ATX) and tenuazonic acid (TeA) (GruberDorninger et al., 2017).
defined, in general, we refer to:
Fusarium metabolites such as eniantins (ENNs), beauvericin (BEA), moniliformin (MON), fusaproliferin (FP), fusidic acid (FA), culmorin (CUL), and butenolide (BUT).
This list is a non-exclusive list and, although these mycotoxins are not routinely screened for at the moment and they are not contemplated in animal feed legislation, there can be issues of toxicity or interactions between them.
5
Information relative to animal
What do we know about the toxicity of emerging mycotoxins?
they have not been linked to any transcendent pathologies in
ENNs & BEA
animals (Marín García, 2010). According to EFSA (2014b), there is a low probability that acute exposure to BEA and ENNs has adverse effects on the health of livestock and companion animals. It is also improbable to see adverse effects due to chronic exposure
ALTERNARIA METABOLITES
ENNs colonize cereals and can accumulate in grains. However,
sensitivity to Alternaria toxins (EFSA,
2011) is scarce and doesn’t allow to estimate the levels of tolerance for individual toxins and their combinations. There is only some information about toxicity in birds for evaluating the risk of these mycotoxins. EFSA concludes that it is improbable for AOH to pose a risk in broilers, but it is not entirely possible to exclude it as a risk for the species. Lack of toxicological data prevents us from drawing conclusions regarding other species.
in birds, although there isn’t enough information to assess them in other species. Regarding MON toxicity (EFSA, 2018), the available data on birds, pigs,
Interactions between mycotoxins
and minks indicates that exposure to MON via consumption of feed
MON
poses a low or insignificant risk for these species under current feeding practices. For the rest of the species, EFSA concludes that the risk is low
Sometimes, we observe symptoms that are hard to explain by the presence of only one or various mycotoxins, or by the amount of mycotoxins that is in the feed (Trenholm et al., 1983). These issues have been linked to the interactions between several mycotoxins, many of them that are not analytically determined.
or insignificant, but there isn’t enough information available on
This basic theory also explains the well-known fact that naturally
its toxicity to assess the risk.
contaminated food/feed is more toxic than the ones equally contaminated with purified mycotoxins (Trenholm et al., 1994).
6
Klaasen and Eaton (1991) classify the effects of the interactions between mycotoxins as: Less than additive Additive Synergic Enhanced Antagonistic
Synergic effects of mycotoxins In general, we know or sense that
Furthermore, after reviewing 112
Stoev et al. (2010) came to a
in most cases there are additive
publications on the toxicological
similar conclusion when studying
or synergic effects (Speijers and
interactions between mycotoxins,
nephropathies in poultry and
Speijers, 2004; Pedrosa, 2010). Many authors have highlighted this additivity, synergy or enhancement:
Grenier and Oswald (2011) found synergies and additivities associated with performance in most of the studies published. However, in relation to other parameters, especially biochemical ones, the results are more variable, ranging from synergic to antagonistic effects for the same combination of toxins.
pigs that could not be explained
Grenier et al. (2011) demonstrated in corn that the liver tumors initiated by AFB1 are exacerbated by the presence of FB1.
only by the content of OTA, under the limit recommended by the EU, finding the explanation in the simultaneous presence of OTA, FB1, and penicillic acid (PCA).
Phenomena such as the ones previously described justify the need to carry out multi-mycotoxins analysis in order to understand the effects that are seen in the field.
7
Antagonistic effects of mycotoxins Although most results reveal the
Thus, antagonism has been
Some publications reveal antagonistic
additive or synergic effects of
observed between:
effects associated with the
mycotoxins, it should be noted that antagonistic effects can also be seen (Koshinsky
and Khachatourians, 1992; Bernhoft et al., 2004).
DON and FB1 (Ficheux et al.,
2012; Wam et al., 2013a) DON and ZEA (Bensassi et al.,
simultaneous presence of three or more mycotoxins. For example, DON, NIV y FB1; NIV, ZEA, FB1 and DON, NIV, ZEA, and FB1 (Wam et al., 2013).
2014; Wam et al.,2013) DON and T2 (Thuvander et
Yang et al., (2017) demonstrated the
al., 1999; RuĂz et al., 2011)
antagonistic effect between some
DON and DAS (Thuvander et al., 1999)
modified DON toxins (acetylated
NIV and DON or FB1 (Wam et al., 2013) NIV and ZEA (Wam et al., 2013)
derivatives), such as 15-ADON + NIV and 15-ADON + FX.
BEA with DON or T2 (Ruiz et al., 2011)
Interactions between mycotoxins – In vitro vs In vivo Most of these studies have been
A clear example that highlights the
conducted in in vitro scenarios,
importance of the experimental design
with cell viability as the main
is the study carried out by Klaric et
parameter, although there
al. (2012) to asses the interactions
are other criteria, such as:
between OTA and citrinin (CIT)
Apoptosis and cell necrosis
in a model with PK15 kidney epithelial cells from pigs, determining
DNA damage
the final effect via cell viability,
Oxidative damage
apoptosis, necrosis, and genotoxicity,
Immunotoxicity Obviously, the combined toxic effects that are
obtaining the following results: Synergic effect on cell viability, apoptosis, and necrosis. Antagonistic effect for genotoxicity.
observed will depend on the experimental design:
Another factor that has complicated
1. Type of cells that are exposed
the interpretations up to now is that the response that is observed
2. Exposure time
in vivo does not always correlate
3. Dosage and relation
with what is seen in vitro.
between mycotoxins 4. Final points and tests used 5. Statistical aspects of the models
To illustrate this phenomenon, we can refer to the example of the interaction between DON and T-2.
8
In vitro studies reveal antagonism (EFSA, 2002), probably due to the competence for binding sites.
There are other published examples,
It is clear that the effects of the
such as the interaction between DON
combination of mycotoxins cannot
and ZEA, in which Swamy et al. (2002)
be predicted based solely on their
highlight their in vivo synergic effects in
individual effects and that, in
However, results from in vivo
piglets, while Ji et al. (2017) demonstrate
addition to additivities and synergies,
studies with mice (Schiefer et
their antagonistic effects in mice.
there can also be antagonisms.
al., 1986) demonstrate that the negative effects of DON in the animals are exacerbated in the presence of T-2, while in vivo studies with pigs (Friend et al., 1992) indicate that DON combined with T-2 present antagonism (with T-2 at half of the dose of DON).
Regarding the loss of growth that is
It is very difficult to predict these
sometimes observed in vivo in response
responses as they are dose,
to the presence of mycotoxins, Andretta
species, and toxin-dependent,
et al. (2015) point out that it is due to an increase in the energy required for the animals’ maintenance, which is supported by Pastorelli et al. (2012).
in addition to the variability due to methodology-related factors.
CONCLUSIONS
It goes without saying that the
Analysing a single mycotoxin may
The application of atoxic fungi
in vitro methodology must be standardized at an international level in order to expand our knowledge about the interactions between mycotoxins and to have access to comparable data.
not be enough to explain many
that grow on the crops, as well as
cases. However, excessive analytical
varieties that are resistant to the
information, if not correctly interpreted,
colonization of toxigenic fungi
isn’t a solution either, making it
or climate models that predict
important to carry out further studies.
the presence of mycotoxins are some of the methods that
Under these circumstances, without
are available to counteract the
These standards would only be
renouncing to other tools, the best
negative effects of mycotoxins
valid if they could predict the
way to reduce the risk is prevention
and significantly reduce the risk.
in vivo response in animals.
through good agricultural practices and risk analysis in the primary links of the food chain.
9
REFERENCES
Almeida I, Martins HM, Santos S, Costa JM y Bernardo F. (2011). Co-occurrence of mycotoxins in swine feed produced in Portugal. Mycotoxin Research, 27, 177–181. Andretta I, Kipper M, Hauschild L, Lehnen CR, Remus A y Melchior R. (2015). Meta-analysis of individual and combined effects of mycotoxins on growing pigs. Scientia Agricola, 73(4), 328-331. Bensassi F, Gallerne C, el Dein OS, Hajlaoui MR, Lemaire C. y Bacha H. (2014). In vitro investigation of toxicological interactions between the fusariotoxins deoxynivalenol and zearalenone. Toxicon, 84, 1-6. Bernhoft A, Keblys M, Morrison E, Larsen HJS y Flaoyen A. (2004). Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes. Mycopathologia, 158, 441-450. Berthiller F, Crews C, Dall’Asta C, Saeger SD, Haesaert G, Karlovsky P, Oswald IP, Seefelder W, Speijers G y Stroka J. (2013). Masked mycotoxins: A review. Molecular Nutrition & Food Research, 57, 165–186. Biomin. The Global Mycotoxin Threat 2017. https://info.biomin.net/acton/fs/blocks/showLandingPage/a/14109/p/p-004e/t/page/fm/17. Accedido 13/06/2019 Blajet-Kosicka A, Twaruzek M, Kosicki R, Sibiorowska E y Grajewski J. (2014). Co-occurrence and evaluation of mycotoxins in organic and conventional rye grain and products. Food Control, 38, 61-66 Driehuis F, Spanjer MC, Scholten JM, y Giffel, MC. (2008) Occurrence of Mycotoxins in Feedstuffs of Dairy Cows and Estimation of Total Dietary Intakes. Journal of Dairy Science, 91, 4261–4271. EFSA (2002). Opinion of the Scientific Committee on Food on Fusarium toxins. Part 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol. SCF/CS/CNTM/MYC/27 Final. EFSA (2011), Scientific Opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food. EFSA Panel on Contaminants in the Food Chain (CONTAM). EFSA Journal, 9 (10),2407. EFSA (2014a). Scientific Opinion on the risks for human and animal health related to the presence of modified forms of certain mycotoxins in food and feed1 EFSA Panel on Contaminants in the Food Chain (CONTAM). EFSA Journal, 12 (12), 3916. EFSA (2014b). Scientific Opinion on the risks to human and animal health related to the presence of beauvericin and enniatins in food and feed. EFSA Panel on Contaminants in the Food Chain (CONTAM). EFSA Journal, 12 (8),3802. EFSA (2018). Risks to human and animal health related to the presence of moniliformin in food and feed. EFSA Panel on Contaminants in the Food Chain (CONTAM), EFSA Journal, 16 (3), 5082. Ficheux AS, Sibiril Y y Parent-Massin D. (2012). Co-exposure of Fusarium mycotoxins: in vitro myelotoxicity assessment on human hematopoietic progenitors. Toxicon, 60, 1171-1179. Friend DW, Thompson B K, Trenholm HL, Boermans HJ, Hartin, KE y Panich PL (1992). Toxicity of T-2 toxin and its interaction with deoxynivalenol when fed to young pigs. Canadian Journal of Animal Science, 72,703–711. Gareis M, Bauer J, Thiem J, Plank G, Grabley S y Gedek B. (1990) Cleavage of Zearalenone-Glycoside, a “Masked” Mycotoxin, during Digestion in Swine. Journal of Veterinary Medicine, 37,236–240. Goertz A, Zuehlke S, Spiteller M, Steiner U, Dehne HW, Waalwijk C, Vries I y Oerke EC. (2010). Fusarium species and mycotoxin profiles on commercial maize hybrids in Germany. European Journal of Plant Pathology, 128, 101–111. Grenier B, Loureiro-Bracarense A P, Schwartz H, Cossalter AM, Schatzmayr G, Moll WD y Oswald IP. (2010). Hydrolysis of Fumonisin B1 strongly reduced toxicity for piglets at the intestinal and systemic levels. 6th World Mycotoxin Forum. November 8-10. Noordwijkerhout, Netherlands. Grenier B y Oswald IP.(2011). Mycotoxin co-contamination of food and feed: Meta-analysis of publications describing toxicological interactions. World Mycotoxin Journal, 4, 285–313. Gruber-Dorninger C, Novak B, Nagl V y Berthiller F. (2017). Emerging Mycotoxins: Beyond Traditionally Determined Food Contaminants. Journal of Agricultural and Food Chemistry, 65, 7052−7070. Ibáñez-Vea M, González-Peñas E, Lizarraga E y López de Cerain A. (2012). Co-occurrence of mycotoxins in Spanish barley: A statistical overview. Food Control, 28, 295–298. Ji J, Zhu P, Cui P, Fuwei P, Zhang Y, Li Y, Wang J y Sun X. (2017). The Antagonistic Effect of Mycotoxins Deoxynivalenol and Zearalenone on Metabolic Profiling in Serum and Liver of Mice. Toxins, 9, 28. Raj J, Farkaš H, Čepela R, Pol I, Bošnjak-Neumüller J and Vasiljević M (2018) A survey on mycotoxins detected in corn samples received from Serbia and Bosnia & Herzegovina during August to November 2017. The World Mycotoxins Forum 10th Conference, March 11-14, 2018, Amsterdam, The Netherlands (Poster no 57). Page: 116). Raj J, Farkaš H, Cepela R, Pol I, Bošnjak-Neumüller J, and Vasiljević M (2019). High level of Fumonisin B1 detected in corn samples received from Serbia during August to November 2018, 22 nd European Symposium on Poultry Nutrition (ESPN 2019), June 10-13, 2019 at Gdansk, Poland Proceedings pg 287.
10
Klaric MS, Zeljezic D, Rumora L, Peraica M, Pepeljnjak S y 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. Archives Toxicology, 86, 97-107. Klaassen CD y Eaton CL. (1991). Principles of Toxicology in: Toxicology, The Basic Science of Poisons. M. O. Amdur, J. Doull, and C. D. Klaassen, ed. Pergamon Press, Inc., Maxwell House, Fairview Park, Elmsford, NY, 12–49 Koshinsky HA y Khachatourians GG. (1992). Bioassay for deoxynivalenol based on the interaction of T2-toxin with trichothecene mycotoxins. Bulletin of Environmental Contamination and Toxicology, 49 (2), 246-51. Labuda R, Parich A, Vekiru E, y Tancinová D. (2005a). Incidence of Fumonisins, Moniliformin and Fusarium species in Poultry Feed from Slovakia. Annals of Agricultural and Environmental Medicine, 12, 81–86. Labuda R, Parich A, Berthiller F y Tančinová D. (2005b). Incidence of trichothecenes and zearalenone in poultry feed mixtures from Slovakia. The International Journal of Food Microbiology, 105, 19–25. Marín Garcia, P. (2010). Tesis. Análisis de factores ecofisiológicos que influyen en la expresión de genes relacionados con la biosíntesis de toxinas en especies de Fusarium. Facultad de Farmacia. UNIVERSIDAD COMPLUTENSE DE MADRID. Monbaliu S, van Poucke C, Detavernier CL, Dumoulin FdR, van De Velde M, Schoeters E, van Dyck S, Averkieva O, van Peteghem C y de Saeger S. (2010) Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. Journal of Agricultural and Food Chemistry, 58, 66–71. Panasiuk L, Jedziniak P, Pietruszka K, Piatkowska M y Bocian L. (2019). Frequency and levels of regulated and emerging mycotoxins in silage in Poland. Mycotoxin Research, 35,17–25. Pastorelli H, Milgen J, Lovatto P y Montagne L. (2012). Meta-analysis of feed intake and growth responses of growing pigs after a sanitary challenge. Animal, 6, 952-961. Pedrosa K. (2010). Synergistic effectc of mycotoxin contaminated feed. International Pig Topics, 25 (7), 7-9. Ruiz MJ, Franzova P, Garcia AJ y Font G. (2011). Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro. Toxicon, 58, 315-326. Rychlik M, Humpf HU, Marko D, Dänicke S, Mally A, Berthiller F, Klaffke H y Lorenz N. (2014). Proposal of a comprehensive definition of modified and other forms of mycotoxins including “masked” mycotoxins. Mycotoxin Research, 30, 197−205. Schiefer HB, Hancock DS y Bhatti AR. (1986) Systemic effects of topically applied trichothecenes. I. Comparative study of various trichothecenes in mice. Journal of Veterinary Medicine, 33a, 373- 383. Speijers GJA y Speijers MHM. (2004). Combined toxic effects of mycotoxins. Toxicology Letters, 153, 91-98. Stoev SD, Denev S, Dutton M, Njobeh P, Mosonik J, Steenkamp P y Petkov I. (2010). Complex etiology and pathology of mycotoxic nephropathy in South African pigs. Mycotoxin Research. 26,31-46. Streit E, Naehrer K, Rodrigues I y Schatzmayr G. (2013a). Mycotoxin occurrence in feed and feed raw materials worldwide: long-term analysis with special focus on Europe and Asia. Journal of the Science of Food and Agriculture, 93, 2892-2899. Streit E, Schwab C, Sulyok M, Naehrer K y Schatzmayr G. (2013b). Multi-Mycotoxin Screening Reveals the Occurrence of 139 Different Secondary Metabolites in Feed and Feed Ingredients. Toxins, 5, 504-523. Swamy HV, Smith TK, MacDonald EJ, Boermans HJ y Squires EJ, (2002b). Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on swine performance, brain regional neurochemistry, and serum chemistry and the efficacy of a polymeric glucomannan mycotoxin adsorbent. Journal of Animal Science, 80, 3257-3267. Thuvander A, Wikman C, y Gadhasson I. (1999). In vitro exposure of human lymphocytes to trichothecenes: Individual variation in sensitivity and effects of combined exposure on lymphocyte function. Food and Chemical Toxicology, 37, 639-648. Trenholm HL, Cochrane WP, Cohen H, Elliot JI, Farnworth ER, Friend DW, Hamilton RM, Standish JF y Thompson BK. (1983). Survey of vomitoxin contamination of 1980 Ontario white winter wheat crop: results of survey and feeding trials. Journal Association of Official Analytical Chemists, 66 (1), 92-97. Trenholm HL, Foster BC, Charmley LL, Thompson BK, Hartin KE, Coopock RW y Albassam MA. (1994). Effects of feeding diets containing Fusarium (naturally) contaminated wheat or pure deoxynivalenol (DON) in growing pigs. Canadian. Journal of Animal Science, 74, 361–369. Wan LY, Turner PC y El-Nezami H. (2013a). Individual and combined cytotoxic effects of Fusarium toxins (deoxynivalenol, nivalenol, zearalenone and fumonisins B1) on swine jejunal epithelial cells. Food and Chemical Toxicology, 57, 276-283. Yang Y, Yu S, Tan Y, Liu N y Wu A. (2017). Individual and Combined Cytotoxic Effects of Co-Occurring Deoxynivalenol Family Mycotoxins on Human Gastric Epithelial Cells. Toxins, 9, 96.
11