Biomonitoring mycotoxins in production animals - Perspectives & Challenges

BIOMONITORING MYCOTOXINS IN PRODUCTION ANIMALS Pe r s p e c t i ve s & C h alle n g e s

Professor Carlos Augusto Fernandes de Oliveira Department of Food Engineering, Faculty of Zootechnics and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil.

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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.

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.

Mycotoxins & Mycotoxicosis Mycotoxicosis is a disease

Importantly, while food &

associated with exposure to dietary

feedstuffs may contain individual

mycotoxins, causing immune

mycotoxins, contamination by

suppression and target organ

multiple mycotoxins in these

toxicity with lesions mainly in

products is quite common and

liver, kidneys, epithelial tissue

has become an important human

(skin and mucous membranes),

and animal health concern due

and central nervous system,

to the possible combined effects

depending on the type of toxin.

of different mycotoxins.

The main groups of toxigenic

Toxicity of some individual

fungi and their respective

mycotoxins may be

mycotoxins belong to the genus:

increased in a synergistic,

Aspergillus: A. flavus, A. parasiticus and A. nomius: aflatoxins

additive, or antagonistic way, when they occur as cocontaminants and are ingested by different animal species.

Fusarium: fumonisins, trichothecenes, moniliformins and zearalenone Aspergillus ochraceus: ochratoxins Penicillium: ochratoxins

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AFLATOXINS

OCHRATOXIN A

FUMONISINS

Among the mycotoxins affecting

Ochratoxin A (OTA) is an

28 structurally related fumonisins

farm animals, the aflatoxins

important nephrotoxic mycotoxin

have been isolated and identified,

are hepatotoxic, teratogenic

with immunotoxic, teratogenic,

although fumonisin B1 (FB1) is

and genotoxic compounds, also

carcinogenic and perhaps neurotoxic

the most predominant and toxic

classified as carcinogenic to humans

effects causing liver and kidney

form produced by the fungi.

(Group 1) by the International

cancer in numerous animal species.

Agency for Research on Cancer.

FB1 has been shown to be hepatotoxic, nephrotoxic and

The aflatoxins were identified

carcinogenic in several animal

in 1961, aflatoxin B1 (AFB1)

studies, also causing species-

being the main type of toxin

specific diseases including

produced by Aspergillus

porcine pulmonary edema and

under natural conditions.

equine leukoencephalomalacia.

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

ZEARALENONE

trichothecenes and often co-exist with ZEN in feed materials such

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

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).

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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.

EXPOSURE BIOMARKERS A biomarker of exposure refers to

The first mycotoxin biomarker used in

the quantification of the specific

production animals was aflatoxin M1

compound, its metabolites or

(AFM1) in milk of lactating animals

interaction products in a body

fed rations containing aflatoxin B1

compartment or fluid, which indicates (AFB1), as illustrated in Figure 1. the presence and magnitude of

In spite of several existing

exposure to the agent.

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.

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

Liver metabolism

AFM1 excreted in milk = 0,3-6,2% AFB1 ingested (Veldman et al., 1992)

Ingestion

Aflatoxin B1 in the ration Aflatoxin M1 in milk Excretion in milk

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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).

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.

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).

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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

PHASES OF BIOTRANSFORMATION

time.

Thus, depending on the toxicokinetics of a given

In all animal species, biotransformation occurs in two phases:

mycotoxin after ingestion, some

Phase I, is mainly based on hydrolysis, reduction, and

biomarkers could be approached

oxidation reactions.

to indicate the magnitude and level of its dietary exposure

Phase II, involves conjugation of the products formed

by means of quantification of

in Phase I.

its metabolites or interaction products in body fluids.

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TOXICOKINETICS OF AFLATOXIN B1 For AFB1, the resulting

Cytochrome P450 enzymes in the

metabolites in Phase I include:

liver also transform AFB1 in

Hydroxylated products: AFM1 and aflatoxin Q1 Demethylated products:

AFB1-8,9-epoxide, its procarcinogen form, which is covalently bound to nucleic acids, mainly DNA (which yields the adduct AFB1-N7-guanine),

aflatoxin P1

and to serum albumin (which yields

A product from the reduction by

et al., 2017).

the adduct AFB1-lysine) (Di Gregorio

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.

In this context, non-metabolized AFB1, its adducts, AFB1-lysine in blood serum and AFB1-N7guanine in urine, as well as its metabolite AFM1 in urine and milk, may be considered as validated biomarkers of AFB1 exposure.

AFM1 AFM1 & AFLATOXICOL

TOXICOKINETICS OF FUMONISINS 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

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).

usually lower than 6%. Moreover, FB1 has a short half-life (< 24 h) and less than 2% recovered in urine.

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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

TOXICOKINETICS OF OCHRATOXIN

TOXICOKINETICS OF DON

in 24 hours (Gambacorta et al. 2013).

Trichothecenes including DON may

The ochratoxin group comprises 7

be easily and quickly absorbed

related toxins, although only OTA has

in the gastrointestinal tract upon

been found as a natural contaminant

exposure. Studies in several animal

of grains.

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

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).

deoxynivalenol-glucoronide (DON-

It is also biotransformed by

GlcA), and excreted in urine.

cytochrome P450 enzymes to their less toxic hydroxyochratoxin

Both urinary DON and DONGlcA are considered validated

A metabolites, mainly ochratoxin alpha (OTα).

biomarkers for assessing the dietary exposure to DON (Nagl

et al, 2015).

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.

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Application of Mycotoxin 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

DIFFERENTIAL DIAGNOSIS OF MYCOTOXICOSIS

most common specimen used for biomonitoring of mycotoxins in humans, as sample collection is

In production animals, the first

However, up to the present,

easily obtained in a non-invasive

important application of biomarkers

few studies have been carried

manner.

of exposure to mycotoxins would

out to determine dose-response

be the differential diagnosis

curves between symptoms of

of mycotoxicosis, as signs and

mycotoxicosis in production animals

symptoms of different mycotoxins

and biomarker concentrations.

are not evident and characteristic.

Probably the cost of analysis is

Thus, in an ideal condition,

an important obstacle for routine

preliminary diagnosis of a given

application of biomarkers in the

mycotoxicosis may be confirmed by

confirmation of mycotoxicosis

the levels of metabolites detected in

diagnosis, as it requires

plasma or urine of affected animals.

specialized laboratory equipment and highly qualified personnel to yield reliable results.

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MYCOTOXIN ADSORBENT 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.

However, experimental in vivo protocols are generally costly and

In this context, the use of

labor-intensive, as they involve

biomarkers to estimate the

the administration of toxins at

mycotoxin bioavailability of

different levels, with and without

adsorbent efficient in in vivo

the adsorbent, in order to assess

assays may reduce costs, besides

the effect on animal productivity.

being more practical, also helping the standardization of the

Besides, these assays

experimental trials, and making

The most important criterion for

generally require collection

it possible to assess the effect of

adsorbent assessment is the stability

of histopathological and

adsorbents in field conditions.

of the adsorbent-toxin bond in a

clinical data, among other

wide range of pH, as it is expected

issues that add to the cost

that it continues to function

and labor (European Food

throughout the gastrointestinal tract.

Safety Authority, 2010).

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).

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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.

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

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.

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.

reduction was compatible with

The possibility of having

the protective effect of the

results in shorter time

adsorbent against the negative

(Di Gregorio et al., 2017).

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.

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

These results indicate that

are not commercially available

AFB1-lysine has potential as

(Jager et al., 2016) but may be synthesized in specialized laboratories (Sass et al., 2014).

an AFB1 specific biomarker

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

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

for evaluating the efficacy of chemo-protective interventions in pigs and broilers.

adduct in farm animals.

Analysis

AFB1-lysine

AFB1 Adsorbent

Liver P450 enzymes

AFB1-albumin in the blood

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Biomarkers of Exposure to Multiple Mycotoxins in the Diet Recently, liquid chromatography-

Nowadays, liquid chromatography-

In this context, analytical

tandem mass spectrometry

tandem mass spectrometry

methods for determination

(LC-MS / MS) aiming at multiple

(HPLC-MS), operating with an

of biomarkers of several

analytes was successfully

electrospray ionizing source (ESI) is

mycotoxins in pig plasma have

introduced in mycotoxin analysis,

unquestionably the most successful

been developed (Devreese et al.

including in the assessment of

analytical tool used in quantitative

adequate biomarkers for the

and qualitative determination of

evaluation of human exposure.

mycotoxins in natural samples.

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.

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.

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

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.

it possible that a wider range of

The use of serum biomarkers

molecules are analyzed in a single

to estimate the mycotoxin

device.

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.

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