Biomarkers of mycotoxins exposure in swine

Mycotoxins as a worldwide menace to swine health and performance

Various mycotoxins can be detected in grains used as pig feedstuffs worldwide and their concurrent presence has been widely reported.

They are secondary metabolites of certain fungi (genera Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps)¹ and are produced before harvest of grains (fungi as plant pathogens), or during storage (fungi growing saprophytically).

Some are considered extremely significant for pig health and performance. Namely, aflatoxins (Afs: B1, B2, G1 and G2, deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FBs: FB1, FB2, FB3) and ochratoxin A (OTA) are considered significant for their devastating effects on pig production worldwide.

Other mycotoxins such as T-2, nivalenol, or ergot alkaloids have been suggested as somewhat significant for swine, especially in particular geographical regions^2,3.

Data at a global scale showed that up to 80% of feed and food crops are contaminated with mycotoxins, and that co-contamination of grains with more than one mycotoxin is common^4-6.

Biomarkers OR feed analysis

The term “biomarker”, which equals “biological marker”, refers to a broad subcategory of objective indications of a medical state that can be measured accurately and reproducibly^9,10.

Therefore, as regards mycotoxins exposure, the predominant characteristic of a biomarker is its accurate and reproducible measurement in biological matrices which could provide information on the total level of exposure to mycotoxins.

Assessment of particular biomarkers provides information on individual exposure, whereas it could assist in the explanation of biological consequences of mycotoxins in animals, observed as toxic effects¹¹.

Such an approach is increasingly used in monitoring human exposure to mycotoxins^12,13, which is reported as human biomonitoring (HBM), whereas a quite similar animal biomonitoring (ABM) process could be also successful in assessing animal exposure¹⁴.

Nevertheless, it is true that feed analysis for the presence of mycotoxins has a number of disadvantages when compared to biomonitoring as a diagnostic tool for mycotoxicosis cases. These include facts that may affect the validity of feed analysis results in comparison to the true mycotoxin contamination levels.

Such facts are the uneven distribution of mycotoxins in feed, the risk of improper sampling procedures, the absence of testing of modified or conjugated forms in feed which can convert back to the parent forms and hence contribute to their adverse effects, as well as the lack of information on individual animal exposure from the feed analysis results.

However, a critical point as regards diagnosis of mycotoxin cases in pigs under field conditions, is that clinical signs of mycotoxin exposure can appear when the contaminated feed has already been consumed.

Thus, diagnosis in such cases becomes complicated or even impossible¹⁵. That is one of the significant advantages of biomonitoring when compared to the classical feed analysis approach¹⁶.

On the other hand, analytical methodology is largely lacking multi-mycotoxin methods for pig excreta and only a limited availability is found for pig urine or other matrices, while as already reported the exposure to a mixture of mycotoxins through feed in pigs is widely observed^6,15.

Toxicokinetic characteristics of major mycotoxins in swine

Differences in metabolic properties and toxicokinetics of each mycotoxin play a significant role in selecting specific biomarkers, whereas their interactions when present together in contaminated feed, have not been fully investigated.

⇰ In order to discuss possible utilisation of biomarkers of exposure of the most important mycotoxins for pigs, major aspects of their metabolic and toxicokinetic properties are presented.

AFs

AFs are produced mainly by Aspergillus flavus, A. parasiticus and A. nomius and are detected usually in maize, peanuts and cottonseed.

The most common AFs are B1, B2, G1 and G2.

AFB1 is the most significant in terms of toxicity in swine¹⁷. AFs apart from hepatotoxicity, they have mutagenic, and possibly teratogenic properties in animals.

As regards pigs, 20-50 % of the administered dose of AFB1 is excreted as AFB1 and AFM1 via urine. The amount of AFM1 is 20% of the total excreted dose¹⁸. Previously Luthy et al.¹⁹ had also detected 20% of the administered dose in pig urine and 50-62% in pig faeces.

AFs and, in particular, AFB1 are considered the most potent naturally occurring carcinogens. AFB1 is metabolised predominantly in the liver by a number of cytochrome P450 enzymes, generating several hydroxy-metabolites, such as AFM1, AFQ1 and AFP1 and two significantly reactive epoxides, AFB1 exo- 8,9-epoxide and AFB1 endo-8,9-epoxide^20-22.

The exo-epoxide interacts with the guanine part of DNA, resulting in the formation of AFB0-guanine adducts such as AF-N7-Gua which can be used (in urine) as a biomarker of exposure^20,21,23.

Both the endo- and exo-epoxide of AFB1 are toxic and lead to the formation of aflatoxin–albumin (AF-alb) in hepatocytes, which are observable in the sera of exposed animals and humans²⁰. These albumin adducts of AFs have been discussed as useful biomarkers of AFs-induced hepatotoxicity²⁰.

DON

On the other hand, DON as a member of the trichothecene B group, is produced by Fusarium fungi which are one of the most important phytopathogenic fungal genera groups. That group is also responsible for the production of ZEN and FBs¹⁵.

DON’s main mode of action includes the inhibition of protein synthesis by interference with the termination step of the polypeptide chain, which is a part of the elongation step of the protein synthesis that is carried out in the 60S ribosomal unit.

⇰ Acute DON intoxications in pigs are clinically observed as signs of abdominal pain, increased salivation, diarrhoea and emesis (3.6-40 mg DON/kg feed)²⁴, whereas lower DON contamination levels result in reduced feed intake and decreased weight gain, growth retardation, immuno-toxicity, impaired reproduction and development²⁵.

DON is rapidly (maximal plasma concentration after 15-30 min. of exposure) absorbed in pigs (approximately 90% of ingested dose). DON is distributed to all tissues and can undergo different detoxification pathways, including mainly microbial degradation in the gastro-intestinal tract and secondly a biotransformation in tissues such as the liver^15,26,27.

DON can be metabolised by intestinal microbes to de-epoxy-DON (DOM-1) that can be found in plasma or excreta[28]. In pigs, microbial formation of DOM-1 mainly occurs in the distal part of the digestive system, whereas DON is predominantly absorbed in the upper digestive tract, thus partially “avoiding” microbial de-epoxidation[28,29]. DON and DOM1 can be conjugated with glucuronic acid and sulphate while in pigs glucuronidation is more common³⁰.

The development of an exposure biomarker for DON by quantifying urinary free-DON and a DON-glucuronide combined as total urinary DON was suggested by observations in a rodent model³¹.

As regards pigs and the possible use of total urinary DON as a potential biomarker of exposure, it should be highlighted that the major excretion pathway for DON is indeed through urine, according to Prelusky et al.³² who reported that 54–85% of radioactive labelled DON administered intra-gastrically to pigs was excreted in urine within 24 hours.

It has been reported that about 40–60% of DON in urine is glucuronide conjugated[18,33], whereas the amount of DOM-1 in urine is approximately 2—5% of the DON in urine, and 1–5% of the DON intake^34-37. A linear increase in urine concentration of DON with increasing dietary DON concentration has been demonstrated³⁶. On the other hand, the amount of DON that reaches the faeces is negligible and includes 0.1 – 5% of the intake¹⁵.

FBs

The fumonisins are a family of toxins produced mainly by Fusarium verticillioides and F. proliferatum, with FB1 and FB2 as the most frequently observed, whereas FB1 represents approximately 70% of the natural contamination of maize³⁸.

In contrast to other Fusarium mycotoxins, FB1 is poorly absorbed from the gastro-intestinal tract of pigs with an oral bioavailability of approximately 4% of the administered dose^39,40.

Distribution to tissues has been found predominantly in the liver and kidney, besides the large intestine and brain and to a smaller extent lung, heart and adrenal gland.

FB1 also undergoes enterohepatic circulation, which extends its biological elimination half-life.

Its metabolism takes place by microorganisms in the small intestine and results in the formation of fully hydrolysed FB or aminophenol (AP) and partially hydrolysed FB1 (PHFB1).

⇰ FB1 is mainly excreted via faeces of pigs (>90%) with maximum concentration at 48h after administration and to a lesser extent via urine (± 5%) as FB1¹⁵.

FB1 has a sphingoid backbone and can inhibit ceramide synthase, resulting in the modulation of two physiologically important precursors in sphingolipid production: sphinganine (Sa) and sphingosine (So), thus inducing accumulation of sphinganine (Sa) and sphingosine (So) in tissues, serum and urine⁴¹ and an increase in the Sa/So ratio.

That mechanism has been recognized as the causal pathway for FBs toxicity. This leads to reduced nutrient absorption, anorexia, decreased daily weight gain and hepatotoxicity in pigs¹⁵.

Signs of encephalopathy and cardiac dysfunction with pulmonary oedema have been observed after administration of FB1⁴².

OTA

Aspergillus species mainly, and Penicillium species can produce ochratoxins with OTA having major nephrotoxic properties.

OTA is passively and rapidly absorbed from the stomach and the proximal duodenum after oral ingestion. Approximately 66% of oral administered OTA is absorbed in pigs^15,43,44.

OTA in the systemic circulation has a high affinity for plasma proteins, especially to albumin, therefore 99% of the absorbed fraction will be bound to plasma proteins in pigs (<0.2% OTA free fraction in serum).

Thus, the absorption of OTA from the gastrointestinal tract is stimulated resulting in prolongation of its presence in the body, slowing both its biotransformation and excretion half-life^15,45.

OTA can be metabolised in the kidney, liver and intestines whereas major metabolic pathways include hydrolysis, hydroxylation, lactone-opening and conjugation.

OTA can undergo phase II metabolism with the formation of glucuronic acid and sulphate conjugates, whereas only the glucuronic acid conjugates have been detected in the bile of pigs¹⁵.

The metabolite OTalpha (OTα) is formed by the cleavage of the peptide bond in OTA and is considered as a nontoxic product⁴⁶.

OTA largely remains unchanged after ingestion in pigs and can accumulate in the meat and organs due to high bioavailability, limited conversion rate into OTalpha and long half-life.

OTA is excreted via urine (via tubular secretion) and faeces (via biliary excretion). However, the occurrence of OTA reabsorption from the urine by all nephron segments has been reported, resulting in retarded excretion and accumulation of OTA in the kidney, where it exerts its main toxicity^44,47.

ZEN

ZEN is a macrocyclic β-resorcylic acid lactone with structural similarity to naturally occurring oestrogens, thus reproductive disorders in swine have been predominantly linked to ZEN toxicity, following the binding of ZEN to oestrogen receptors.

Hyperestrogenism in pigs observed at doses of 0.06 and 0.15 mg/kg feed includes signs of reddening, hyperaemia and oedematous swelling of the vulva, enlargement of the uterus with cyst formation on the ovarian and enlargement of the mammary glands, whereas vaginal or rectal prolapse in such cases has been also reported.

Gilts are significantly sensitive to the toxic effects of ZEN since their concentrations of 17- β-oestradiol are lower compared to sows.

On the other hand, signs in boars include atrophy of the testes, reduction of the concentration of spermatozoa and oedematous swelling of the preputium and mammarian complex. Moreover, embryotoxic and teratogenic properties of ZEN have been described^15,48.

ZEN is rapidly absorbed after oral exposure with an oral bioavailability of approximately 80% of the administered dose in pigs⁴⁸.

ZEN is metabolised via phase I and II reactions. ZEN metabolism in pigs includes a phase I reduction of ZEN, by the microbial organisms in the gastrointestinal tract of pigs and by 3α or 3β-hydroxysteroid dehydrogenases in the liver, oocytes and intestinal mucosa, to predominantly α-zearalenol (α-ZEL) and at a lower level to β-zearalenol (β-ZEL)^15,49, while also α-zearalanol (α- ZAL), β-zearalanol (b-ZAL) and zearalanone (ZAN) can be formed.

The α-ZEL is considered a bioactivation product as a more potent metabolite^2,49. ZEN and its phase I metabolites can undergo phase II conjugation with glucuronic acid and sulphate (through catalysis by by uridine 5′-diphospho-glucuronosyltransferases and sulfotransferases)⁴⁸. Both glucuronidation and sulfation are considered as detoxification pathways¹⁵.

As regards ZEN possible biomarkers of exposure, Olsen et al.⁵⁰ reported that 15.6% of ingested ZEN from 5 mg/kg feed was excreted in urine as ZEN (mostly as glucuronide conjugates) and α-ZEL within 8 h.

Similarly, Zöllner et al.⁵¹, estimated that approximately 14% of ZEN intake was excreted in urine either as parent toxin or as metabolites within 8 h.

However, Dänicke et al.⁵³ reported 30% urine excretion of ZEN within 6 h after female pigs had been given a single bolus of 1 mg ZEN/kg body weight, whereas up to 70.4 % of the dose was excreted in urine within 72 h.

Taken together, 40% of the administered dose of ZEN can be recovered in urine (26±10%) and faeces (14± 3%) in pigs, as ZEN or its metabolites at 48h after administration¹⁵.

Faecal samples have been shown to contain ZEN and α-ZEL, which are also present in plasma, whereas in urine ZEN and ZEN-GlcA are detected^51,52.

Biomarkers of major mycotoxins in swine

As previously suggested biomarkers are defined as substances measured in biological systems linked to effect, exposure or susceptibility⁵³, while the measurement of these markers in biological matrices is known as biomonitoring.

Biomonitoring has two major applications which include exposure assessment and in vivo efficacy testing of candidate mycotoxin detoxifiers¹⁵. Biomarkers can be categorised based on their characteristics and use as follows^15,54:

• ⇰ Biomarkers of exposure: They provide evidence of exposure of individuals to xenobiotics (such as mycotoxins) through an estimation of the concentrations of these compounds or their metabolites in biological matrices. They are often the mycotoxin itself or/and its phase I and II metabolites or interaction products.
• ⇰ Biomarkers of effect: They are connected with the measurement of biochemical, physiological, or behavioural alterations in the organism which can be attributed to a possible or established health effect.
• ⇰ Biomarkers of susceptibility: They are used as indicators of an ability (inherent or acquired) to respond to exposure to xenobiotics.

Based on the above-mentioned differences among species and metabolic/toxicokinetic properties of various mycotoxins, the European Food Safety Authority (EFSA)⁵⁵ has recommended endpoints/ biomarkers (Table 1) as proper validation endpoints of the efficacy of a feed additive which claims adsorbing or modifying capability against mycotoxins.

The selection of the appropriate endpoint should be based on the mycotoxin and target species, whereas the availability of sensitive analytical methods validated for the specific matrices should be taken into account.

However, it has been suggested that the abovementioned biomarkers may not always be the most optimal for each mycotoxin¹⁵.

DON concentrations in blood serum could be a relevant biomarker for exposure, but on the other hand, DON undergoes an extensive phase II biotransformation to DON-GlcA in pigs, therefore, that metabolite could be considered as more precise biomarker for exposure than DON itself³⁰.

Furthermore, even though biomarkers for exposure have been defined for particular mycotoxins in some animal species, the identification of the suitable biomarker for each mycotoxin in a certain species and specific biological matrix is often missing¹⁵.

The most widely used biological matrices are urine and blood, whereas biomarkers can be also detected, in hair, saliva and faeces.

• ⇰ Urine, plasma and faeces can be collected easily in living pigs, whereas bile or organs can be also useful matrices, but they are collected post-mortem. Thus, biomonitoring on easily accessible matrices, which can be collected through non-invasive and non-stressogenic processes, is gaining more interest in recent years.
• ⇰ Dried blood spots (DBS), hair and saliva are among such matrices, which have reduced storage and transport requirements. Until today methods are available for mycotoxins in DBS and hair of animals and humans, whereas for the detection in saliva they are not fully available¹⁵.

Assessment of DBS for multi-mycotoxin biomarker analysis in pigs has been previously reported⁵⁶. In that study, a method was developed and validated for the determination of 23 mycotoxins and their phase I and II metabolites in DBS of pigs, whereas under in vivo exposure conditions, a strong correlation was observed between plasma and DBS concentrations. Findings supported DBS as an interesting micro-sampling technique for use as a non-invasive biomonitoring system of mycotoxins exposure in swine.

Moreover, recent findings suggested that circulating microRNAs (MiRNAs: small non-coding RNAs) have remarkable potential to serve as indicators for pathological processes in tissues, such as the case of toxic effects induced by ZEN⁵⁷ and DON⁵⁸, OTA and AFB1⁵⁹.

MiRNAs can be viewed as characteristic biomarkers whose levels can be directly measured in serum, urine, tissues and saliva^57,59.

Findings through transcriptomics approaches indicated that ZEN can induce dose-dependent alterations of several uterine MiRNAs, whereas such effect was partly reflected in serum of exposed animals, thus providing evidence for a novel mycotoxin biomarker discovery⁵⁷.

Evaluation of DON-induced changes in the MiRNAs expression in porcine liver, jejunum and serum, proved a time dependent serum MiRNAs response to DON which declined after removal of contaminated diets.

Based on those findings four MiRNAs were identified as candidates for the detection of DON toxicity in porcine serum⁵⁸.

It seems that the use of biomarkers involved in critical signalling pathways and connected to the mode of action of mycotoxins may help predict their potential toxicity and the progression of associated mycotoxicosis, as well as to develop effective biomonitoring approaches⁶⁰.

Previous studies in pigs have provided evidence of multiple biomarkers of exposure to mycotoxins which correlate with the mycotoxins ingestion levels received:

• ⇰ The total DON concentration in serum and the sum of ZEN and metabolites in urine both have correlated well with the intake of the toxins from the feed⁶¹.
• ⇰ DON concentration in serum and DON and DOM1 concentrations in urine increase in a dose-response-related manner as dietary DON-concentration increases[52]. Therefore, they are suitable biomarkers of exposure in pigs, whereas DON-GlcA in serum could be considered as a better biomarker for exposure¹⁴.
• ⇰ DON-glucuronide (DON-GlcA) and ZENglucuronide (ZEN-GlcA) in plasma, DON and ZEN-GlcA in urine and, ZEN in faeces (late excretion due to enterohepatic circulation of ZEN) have been reported as optimal biomarkers for DON and ZEN⁵⁴. According to the review by Tkaczyk, and Jedziniak¹⁴, ZEN and its metabolites α- and β-ZEL in urine, as well as ZEN and its metabolites: α-, β-ZEL, and ZAN in faeces, or the plasma levels of GlcA metabolites (phase II metabolites) of ZEN, α-ZEL, and β-ZEL can also be suitable biomarkers of exposure in pigs.

• ⇰ Both plasma and urine FB1 levels are considered as adequate biomarkers of early exposure to low dietary levels and plasma is recommended under conditions of prolonged exposure (>14 days) in pigs. A correlation between FB1 in feed and hair has been also reported but was lower compared to urine and plasma⁶².
• ⇰ The disruption of the sphingolipid biosynthesis can also be used as a biomarker for FB1 exposure, though it can be considered also as a biomarker of effect. The ratio Sa/So will increase in serum and tissues in a dose-dependent manner^63,64.
• ⇰ Measurement of AFB1-lysine concentration in serum demonstrated that longer exposure to AFB1 results in higher levels of AFB1-lysine, thus that the lysine adduct might be a good biomarker for long term AFB1 exposure⁶⁵. In addition, AFB1-N7- guanine and AFM1 in urine have been also suggested as possible biomarkers for AFB1 exposure in pigs¹⁵. Tkaczyk et al.¹⁴ identified AFB1, AFM1, and AFB2 as biomarkers of AFB1 exposure in the urine of pigs.
• ⇰ The concentrations of OTA in blood and urine have been reported as good markers for OTA contamination in various organs¹⁵.

Future prospects

Several issues should be further clarified through research on the field of mycotoxicosis such as the occurrence and fate of modified or masked mycotoxins, as well as the interactions among mycotoxins when present as mycotoxin mixtures in contaminated pig feed.

The use of biomonitoring and the assessment of respective biomarkers seems a promising tool in understanding such complex procedures and their effects on health and performance of swine.

Early diagnosis of mycotoxicosis under field conditions based on biomarkers assessment in biological matrices should be well connected with respective feed contamination levels in order to lead to proper and timely performed counteracting measures regarding contaminated feed and the animals.

It should be stated that accurate – sensitive methods (at reasonable cost) of quick biomonitoring testing in biological matrices, received with minimal animal restraint should be the focus of the future research efforts on the field of mycotoxicosis diagnostics and especially in cases of combined mycotoxicosis exposure.

Nevertheless, taking into account the complexity of sampling preparation for such analysis and the concurrent presence of multiple mycotoxins with different toxicokinetic properties, it seems that the preparation of such technology would not be an easy task.

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