Co-occurrence, modified mycotoxins, emerging mycotoxins and interactions

Co-occurrence, MODIFIED MYCOTOXINS, EMERGING MYCOTOXINS and interactions

Mariano Gorrachategui Animal nutrition consultant and president of CESFAC

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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.

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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).

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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.

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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.

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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).

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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.

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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.

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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.

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