Biomonitoring mycotoxins
in production animals
Perspectives & Challenges

Biomarkers of exposure have been extensively used for improving the assessment of exposure to dietary mycotoxins in humans. Recently, biomarkers have also been proposed for biomonitoring mycotoxins in production animals.

Professor Carlos Augusto Fernandes de Oliveira

Department of Food Engineering, Faculty of Zootechnics and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil.

In this article, the main biomarkers used for animal biomonitoring of single or multiple mycotoxin exposure are presented, as well as the potential application of these biomarkers for diagnostic purposes and for evaluating the efficacy of chemo-protective interventions, such as mineral adsorbents.

 

 

Micotoxins & Mycotoxicosis

Mycotoxicosis is a disease associated with exposure to dietary mycotoxins, causing immune suppression and target organ toxicity with lesions mainly in liver, kidneys, epithelial tissue (skin and mucous membranes), and central nervous system, depending on the type of toxin.

The main groups of toxigenic fungi and their respective mycotoxins belong to the genus:

  • Aspergillus:
    • A. flavus, A. parasiticus y A. nomius: aflatoxins
    • Fusarium: fumonisins, trichothecenes, moniliformins and zearalenone
    • Aspergillus ochraceus
  • Penicillium: ochratoxins

Importantly, while food & feedstuffs may contain individual mycotoxins, contamination by multiple mycotoxins in these products is quite common and has become an important human and animal health concern due to the possible combined effects of different mycotoxins.

Toxicity of some individual mycotoxins may be increased in a synergistic, additive, or antagonistic way, when they occur as cocontaminants and are ingested by different animal species.

AFLATOXINS       

Among the mycotoxins affecting farm animals, the aflatoxins are hepatotoxic, teratogenic and genotoxic compounds, also classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer.

The aflatoxins were identified in 1961, aflatoxin B1 (AFB1) being the main type of toxin produced by Aspergillus under natural conditions.

OCHRATOXIN A   

Ochratoxin A (OTA) is an important nephrotoxic mycotoxin with immunotoxic, teratogenic, carcinogenic and perhaps neurotoxic effects causing liver and kidney cancer in numerous animal species.

  FUMONISINS  

28 structurally related fumonisins have been isolated and identified, although fumonisin B1 (FB1) is the most predominant and toxic form produced by the fungi.

FB1 has been shown to be hepatotoxic, nephrotoxic and carcinogenic in several animal studies, also causing species-specific diseases including porcine pulmonary edema and equine leukoencephalomalacia.

ZEARALENONE   

Zearalenone (ZEN) is a mycotoxin which binds competitively to estrogen receptors, leading to estrogenic abnormalities and generative syndromes, especially in pigs.

DEOXYNIVALENOL   

Deoxynivalenol (DON), correspondingly named vomitoxin because of its emetic effects after ingestion, is another mycotoxin produced by Fusarium species, which belongs to the class B trichothecenes and often co-exist with ZEN in feed materials such as corn, oats, barley, and wheat.

The acute exposure to DON also causes abdominal pain, salivation, diarrhea, leukocytosis, and gastrointestinal hemorrhage (Oliveira et al., 2014).

 

The challenge of mycotoxicosis diagnosis

Since the aflatoxins’ discovery in the early 1960s’, the assessment of negative effects of mycotoxins on production animals has been based on the observation of signs and symptoms of intoxication, including decreased performance parameters, combined with the mycotoxin contamination data in feed and/or ingredients.

In spite of several existing mycotoxicosis diagnosis criteria, these classical approaches are associated with important limitations such as the variability of individual susceptibility to mycotoxins and their heterogeneous distribution in feed.

EXPOSURE BIOMARKERS

A biomarker of exposure refers to the quantification of the specific compound, its metabolites or interaction products in a body compartment or fluid, which indicates the presence and magnitude of exposure to the agent.
The first mycotoxin biomarker used in production animals was aflatoxin M1 (AFM1) in milk of lactating animals fed rations containing aflatoxin B1 (AFB1), as illustrated in Figure 1.

Figure 1. . Metabolic pathway of aflatoxin B1 conversion into aflatoxin M1 in dairy cows

The available data on toxicokinetics of several mycotoxins in animal models indicate that exposure to mycotoxins can be accurately measured by biomarkers in several bio-specimens, especially in serum.

Serum aflatoxin B1-lysine (AFB1-lys), a digest product of AFB1-albumin used for human biomonitoring of aflatoxin exposure has been confirmed as a specific biomarker of aflatoxicosis in broilers and piglets (Di Gregorio et al., 2017).

For fumonisin B1 (FB1), experimental studies indicate that plasma and urinary FB1 are good biomarkers of early exposure of pigs to low dietary FB1 levels, although plasma is recommended to assess prolonged exposure (>14 days) (Souto et al., 2017).

LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS)

In recent years, the liquid chromatography tandem mass spectrometry (LC-MS/MS) based on the multi-analyte approach has been successfully introduced in the field of mycotoxins analysis, opening new perspectives for the evaluation of suitable biomarkers for mycotoxins mixtures.

 

Biomarkers for assessing mycotoxin exposure in production animals

An ideal biomarker should be:

  • Specific
  • Quantifiable
  • Detectable at low levels
  • Obtained by non-invasive and inexpensive techniques

These attributes should be determined for each potential toxicant based on its toxicokinetics, which refers to the study of absorption, distribution, metabolism/ biotransformation, and excretion (ADME) of toxicants in relation to time.

Thus, depending on the toxicokinetics of a given mycotoxin after ingestion, some biomarkers could be approached to indicate the magnitude and level of its dietary exposure by means of quantification of its metabolites or interaction products in body fluids.

PHASES OF BIOTRANSFORMATION

In all animal species, biotransformation occurs in two phases:

  • Phase I, is mainly based on hydrolysis, reduction, and oxidation reactions.
  • Phase II, involves conjugation of the products formed in Phase I.

TOXICOKINETICS OF AFLATOXIN B1

For AFB1, the resulting metabolites in Phase I include:

  • Hydroxylated products: AFM1 and aflatoxin Q1
  • Demethylated products: aflatoxin P1
  • A product from the reduction by cytoplasmic enzymes: aflatoxicol

All these compounds may be shed in urine, bile and feces (Oliveira et al., 2014). The excretion rate of AFM1 in the milk of dairy cows ranges from 0,3 to 6,2% of the AFB1 ingested, depending on the lactation stage and volume of milk produced. In domestic poultry, the main products of AFB1 biotransformation are AFM1 and aflatoxicol, which may be found in eggs.

Cytochrome P450 enzymes in the liver also transform AFB1 in AFB1-8,9-epoxide, its procarcinogen form, which is covalently bound to nucleic acids, mainly DNA (which yields the adduct AFB1-N7-guanine), and to serum albumin (which yields the adduct AFB1-lysine) (Di Gregorio et al., 2017).

In this context, non-metabolized AFB1, its adducts, AFB1-lysine in blood serum and AFB1-N7- guanine in urine, as well as its metabolite AFM1 in urine and milk, may be considered as validated biomarkers of AFB1 exposure.

 

TOXICOKINETICS OF FUMONOSINS

Fumonisins are the most recently discovered group of mycotoxins. Since they were isolated in 1988, they have been associated with diseases such as equine encephalomalacia and pulmonary edema in pigs.

The bioavailability of FB1 after ingestion in several animal species is usually lower than 6%. Moreover, FB1 has a short half-life (< 24 h) and less than 2% recovered in urine.

However, FB1 residues can be found in plasma and urine from pigs orally dosed with 3,1-9.,0 mg FB1/kg feed, with good correlations between the ingested FB1 and the residual levels in plasma or urine (Souto et al., 2017).

TOXICOKINETICS OF ZEARALENONE

Zearalenone is an estrogenic substance derived from resorcylic acid and produced by Fusarium species, such as F. roseum (F. graminearum), F. culmorum, and F. equisetum, among others.

In mammals, zearalenone can be reduced to its hydroxy stereoisomer analogues, α-zearalenol and β-zearalenol (α- and β-ZOL). A glucuronic acid conjugate, preferentially in the 14-hidroxyl phenol group, may also be formed.

In pigs, the plasma half-life of ZEA is 87 hours after the intravenous or oral routes. Piglets have shown an excretion rate of 37% of dietary ZEN in 24 hours (Gambacorta et al. 2013).

TOXICOKINETICS OF DON

Trichothecenes including DON may be easily and quickly absorbed in the gastrointestinal tract upon exposure. Studies in several animal species showed DON availability ranging from 50-60%, suggesting efficient absorption.

One important DON metabolite is deepoxy-deoxynivalenol (DOM-1), produced by intestinal microorganisms in several animal species, especially in ruminants. Moreover, DON can be sulfonated or conjugated with glucuronic acid resulting in deoxynivalenol-glucoronide (DONGlcA), and excreted in urine.

Both urinary DON and DONGlcA are considered validated biomarkers for assessing the dietary exposure to DON (Nagl et al, 2015).

TOXICOKINETICS OF OCHRATOXIN

The ochratoxin group comprises 7 related toxins, although only OTA has been found as a natural contaminant of grains.

  • After ingestion, OTA can remain in the serum linked to proteins and reach the kidneys, muscles, and liver.
  • It has a high plasma-protein binding potential (up to 99%), with an estimated plasma halflife of 35 days (Dietrich et al., 2005).
  • It is also biotransformed by cytochrome P450 enzymes to their less toxic hydroxyochratoxin A metabolites, mainly ochratoxin alpha (OTα).
Both OTα and OTA may be excreted in urine, although so far the urinary OTA has not been validated as a biomarker of dietary OTA.

Application of Mycotoxins Biomarkers in Production Animals

For practical reasons, biomarkers of exposure to mycotoxins of interest in production animals are those that may be analyzed in plasma, whereas urine is the most common specimen used for biomonitoring of mycotoxins in humans, as sample collection is easily obtained in a non-invasive manner.

DIFFERENTIAL DIAGNOSIS OF MYCOTOXICOSIS   

In production animals, the first important application of biomarkers of exposure to mycotoxins would be the differential diagnosis of mycotoxicosis, as signs and symptoms of different mycotoxins are not evident and characteristic.

Thus, in an ideal condition, preliminary diagnosis of a given mycotoxicosis may be confirmed by the levels of metabolites detected in plasma or urine of affected animals.

However, up to the present, few studies have been carried out to determine dose-response curves between symptoms of mycotoxicosis in production animals and biomarker concentrations.

Probably the cost of analysis is an important obstacle for routine application of biomarkers in the confirmation of mycotoxicosis diagnosis, as it requires specialized laboratory equipment and highly qualified personnel to yield reliable results.

MYCOTOXIN ADSORBET EFFICIENCY ASSESSMENT   

A possible, more attractive application of biomarkers of exposure to mycotoxins in production animals, which may show good cost-benefit relationship, is the evaluation of efficiency of adsorbents used in large scale to prevent the toxic effects of mycotoxins.

The most important criterion for adsorbent assessment is the stability of the adsorbent-toxin bond in a wide range of pH, as it is expected that it continues to function throughout the gastrointestinal tract.

As adsorbents may show wide variation in composition and physical-chemical properties, it is necessary to assess their efficacy using in vitro and in vivo assays (Di Gregorio et al., 2014).

However, experimental in vivo protocols are generally costly and labor-intensive, as they involve the administration of toxins at different levels, with and without the adsorbent, in order to assess the effect on animal productivity.

Besides, these assays generally require collection of histopathological and clinical data, among other issues that add to the cost and labor (European Food Safety Authority, 2010).

In this context, the use of biomarkers to estimate the mycotoxin bioavailability of adsorbent efficient in in vivo assays may reduce costs, besides being more practical, also helping the standardization of the experimental trials, and making it possible to assess the effect of adsorbents in field conditions.

APPLICATION OF AFM1 FOR ASSESSING ADSORBENT EFFICIENCY

The first biomarker used to evaluate adsorbent efficient probably was AFM1 in the milk of dairy cows.

A good example of this kind of approach is the study by Diaz et al. (2004) in which AFM1 concentration was reduced by 31-65% in the milk of cows fed diets containing four type of commercial adsorbents and 55 μg/kg AFB1.

Edrington et al. (1996) used three types of adsorbents (Hydrated Sodium Calcium Aluminum Silicate (HSCAS), activated charcoal, and acidic HSCAS) in the feed of colostomized turkeys intoxicated with 0,75 mg/kg AFB1, and observed reduction of 52-72% in urinary excretion rates of AFM1 48 hours after the ingestion of the contaminated feed.

APPLICATION OF AFB1-LYSINE FOR ASSESSING ADSORBENT EFFICIENCY

Carão (2016) and Di Gregorio et al. (2017) evaluated the efficiency of a commercial adsorbent based on HSCAS using the adduct AFB1-lysine in the serum of broilers and swine fed diets containing 500 and 1,100 μg/kg AFB1.

In swine, HSCAS reduced serum levels of AFB1-lysine in 53-72% between days 7 and 21 of continuous exposure to contaminated feed. This reduction was compatible with the protective effect of the adsorbent against the negative effects of AFB1 in animals (Di Gregorio et al., 2017).

However, the use of the same commercial adsorbent in broilers did not show satisfactory results in decreasing AFB1 toxic effects (Carão, 2016), as there was no reduction in AFB1-lysine concentration in the serum of intoxicated birds.

These results indicate that AFB1-lysine has potential as an AFB1 specific biomarker for evaluating the efficacy of chemo-protective interventions in pigs and broilers.

ADVANTAGES ASSOCIATED WITH THE USE OF SERUM AFB1-LYSINE FOR EVALUATING ADSORBENTS FOR AFLATOXINS:

  • Higher precision due to exposure assessments at the individual level.
  • Lower cost of in vivo studies because of lower required numbers of animals and laboratory analysis.
  • The possibility of having results in shorter time (Di Gregorio et al., 2017).

An overview of the procedures for assessing the adsorbent’s efficiency using AFB1-lysine adduct is presented in Figure 2.

However, an important limitation for the routine analysis of AFB1-lysine adduct is the requirement of reference standards, which are not commercially available (Jager et al., 2016) but may be synthesized in specialized laboratories (Sass et al., 2014).

Figure 2. Overview of procedures for assessing the adsorbent’s efficiency using serum AFB1-lysine adduct in farm animals.

Biomarkers of Exposure to Multiple Mycotoxins in the Diet 

Recently, liquid chromatographytandem mass spectrometry (LC-MS / MS) aiming at multiple analytes was successfully introduced in mycotoxin analysis, including in the assessment of adequate biomarkers for the evaluation of human exposure.

The development of new techniques has brought important contributions for biomarkers of multiple mycotoxins and it allows for:

  • The measurement of a more realistic data set on exposure, as in real conditions animals are exposed to a mixture of mycotoxins.
  • The potential application of risk assessments for combined mycotoxin and the possible effects of their interaction.

However, sample preparation continues to be a challenge for the development of methods of analysis for multi-mycotoxins due to matrix effects and the wide range of chemical properties of mycotoxins and their metabolites.

Nowadays, liquid chromatography tandem mass spectrometry (HPLC-MS), operating with an electrospray ionizing source (ESI) is unquestionably the most successful analytical tool used in quantitative and qualitative determination of mycotoxins in natural samples.

Particularly, the use of LC-MS/ MS leads to increased gains in sensitivity and analytical selectivity, once methods based on MS/MS use data on the molecular ion of a given analyte and of its product ions, providing a maximum confidence scale for the identification of a target analyte.

Modern mass spectrometers are increasingly versatile in terms of possible combinations in a single device, different ionization sources, and different analyzers. The greatest advantage of existing equipment is that it enables more refined analytical development, making it possible that a wider range of molecules are analyzed in a single device.

In this context, analytical methods for determination of biomarkers of several mycotoxins in pig plasma have been developed (Devreese et al. 2012). However, the influence of matrix effects is the major challenge in developing reliable quantitative multi-analyte methods. Therefore, considerable efforts to control matrix effects should be carried out to obtain accurate results, namely, the inclusion of a sample cleanup step (e.g. using QuEChERS) and the compensation of the signal suppression/enhancement through the usage of matrix matched standards.

Biomarkers are important tools in the evaluation of mycotoxin exposure as they make it possible to confirm the diagnostic of mycotoxicosis and identify individual animals that are at risk but do not show signs of intoxication.

The use of serum biomarkers to estimate the mycotoxin bioavailability in in vivo adsorbent efficient assays looks promising to reduce the costs of these assays, especially for AFB1 and FB1, and possibly for DON.

However, further validation studies are still required to provide physiologically based toxicokinetics of serum biomarkers of combined mycotoxins in production animals.

REFERENCES

Carão, A. C. P. Determinação de biomarcadores de aflatoxina B1e aplicabilidade na avaliação de adsorventes em frangos de corte. 2016.145 f. Tese (Doutorado) – Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, 2016.

Devreese, M., De Baere, S., De Backer, P., Croubels, S., 2012. Quantitative determination of several toxicological important mycotoxins in pig plasma using multi-mycotoxin and analyte-specific high performance liquid chromatography-tandem mass spectrometric methods. J. Chromatogr. 1257, 74-80.

Diaz D. E. et al. Aflatoxin binders II: reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed . Mycopathologia, n. 157, p. 233-241, 2004.

Dietrich, D.R., Heussner, A.H., and O’Brien, E. (2005). Ochratoxin A: comparative pharmacokinetics and toxicological implications (experimental and domestic animals and humans). Food Additives and Contaminants, 22, 45–52.

Di Gregorio, M.C.; Jager, A.V.; Souto, P.C.M.C.; Costa, A.A.; Rottinghaus, G.E.; Passarelli, D.; Budiño, F.H.L.; Corassin, C.H.; Oliveira, C.A.F. (2017) Determination of serum aflatoxin B1-lysine to evaluate the efficacy of an aflatoxin adsorbing feed additive in pigs fed an aflatoxin B1-contaminated diet. Mycotoxin Research, 33:93-102.

Di Gregorio, M.C.; Neeff, D.V.; Jager, A.V.; Corassin, C.H.; Carão, A.C.P.; Albuquerque, R.; Azevedo, A.C.; Oliveira, C.A.F. (2014) Mineral adsorbents for prevention of mycotoxins in animal feeds. Toxin Reviews, 33:1-11.

Edrington, T. S. et al. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M, in turkey poults. Lack of effect by activated charcoal on aflatoxicosis. Toxicology Letters, n. 89, p. 115-122, 1996.

European Food Safety Authority (2010) Statement on the establishment of guidelines for the assessment of additives from the functional group “substances for reduction of the contamination of feed by mycotoxins”. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDA). EFSA J 8:1–8.

Gambacorta, M.S, Visconti, M., Powers, S., Cossalter, A.M, Pinton, P., Oswald, I.P., 2013. Validation study on urinary biomarkers of exposure for aflatoxins B1, ochratoxin A, fumonisin B1, deoxynivalenol and zearalenone in piglets. World Mycotoxin J. 6, 299-308.

Gonçalves, B.L.; Gonçalves, J.L.; Rosim, R.E.; Cappato, L.P.; Cruz, A.G.; Oliveira, C.A.F.; Corassin C.H. (2017) Effects of different sources of Saccaromyces cerevisae biomass on milk production, composition, and aflatoxin M1 excretion in milk from dairy cows fed aflatoxin B1. Journal of Dairy Science, 11:5701-5708.

Jager, A. V., Tonin, F. G., Baptista, G. Z., Souto, P. C., & Oliveira, C. A. (2016). Assessment of aflatoxin exposure using serum and urinary biomarkers in São Paulo, Brazil: a pilot study. International Journal of Hygiene and Environmental Health, 219, 294-300.

Nagl, V.; Schatzmayr, G. (2015). Deoxynivalenol and its masked forms in food and feed. Curr. Opin. Food Sci., 5, 43–49.

Oliveira, C.A.F.; Corassin, C.H.; Corrêa, B.; Oswald, I.P. (2014) Animal Health: Mycotoxins. In: Encyclopedia of Agriculture and Food Systems, 2nd Edition. Oxford, UK: Elsevier Limited, pp. 358-377.

Queiroz, O. C. M. et al. Effect of adding a mycotoxin-sequestering agent on milk aflatoxin M1 concentration and the performance and immune response of dairy cattle fed an aflatoxin B1-contaminated diet. Journal of Dairy Science, n. 95 , p. 5901-8, 2012.

Sass, D.C.; Jager, A.V.; Tonin, F.G.; Rosim, R.E.; Constantino, M.G.; Oliveira, C.A.F. (2015) Synthesis and purification of the aflatoxin B1-lysine adduct. Toxin Reviews, 34:53-59.

Souto, P.C.M.C.; Jager, A.V.; Tonin, F.G.; Petta, T.; Di Gregorio, M.C.; Cossalter, A.M.; Pinton, P.; Oswald, I.O.; Rottinghaus, G.E.; Oliveira, C.A.F. (2017) Determination of fumonisin B1 levels in body fluids and hair from piglets fed fumonisin B1–contaminated diets. Food and Chemical Toxicology, 108:1-9.

Xiong, J. L. et al. Transfer of dietary aflatoxin B1 to milk aflatoxin M1 and effect of inclusion of adsorbent in the diet of dairy cows. J Dairy Sci, n. 98, p. 2545-2554, 2015. 13



Micotoxicosis prevention
Sign up