Biomarkers of mycotoxin exposure in swine

Assessment of particular biomarkers provides information on individual exposure and could assist in explaining the biological consequences of mycotoxins in animals, observed as toxic effects[^11]. Such an approach is increasingly used in monitoring human exposure to mycotoxins[^12][^13], which is reported as human biomonitoring (HBM), whereas a similar animal biomonitoring (ABM) process could also be successful in assessing animal exposure[^14].

Nevertheless, feed analysis for the presence of mycotoxins has several disadvantages when compared to biomonitoring as a diagnostic tool for mycotoxicosis cases. These include factors that may affect the validity of feed analysis results in comparison to true mycotoxin contamination levels, such as the uneven distribution of mycotoxins in feed, the risk of improper sampling procedures, and the absence of testing for modified or conjugated forms in feed that can convert back to the parent forms and contribute to their adverse effects. Additionally, feed analysis does not provide information on individual animal exposure.

However, a critical point regarding the 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. This makes diagnosis in such cases complicated or even impossible[^15]. This is one of the significant advantages of biomonitoring compared to the classical feed analysis approach[^16].

On the other hand, analytical methodology is largely lacking multimycotoxin methods for pig excreta, and only 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]. Therefore, it seems that the use of biomarkers in mycotoxicosis cases could become a significant key for early understanding of exposure levels and for the diagnosis of mycotoxicosis in swine.

The present review highlights the major biomarkers that could be evaluated in swine mycotoxicosis cases and addresses possible future prospects.

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 the possible utilization of biomarkers of exposure for the most important mycotoxins in pigs, major aspects of their metabolic and toxicokinetic properties are presented.

Aflatoxins (AFs)

AFs are produced mainly by Aspergillus flavus, A. parasiticus, and A. nomius and are usually detected 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 swine[^17]. AFs, apart from hepatotoxicity, 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 dose[^18]. Previously, Luthy et al.[^19] also detected 20% of the administered dose in pig urine and 50-62% in pig feces.

AFs, and in particular AFB1, are considered the most potent naturally occurring carcinogens. AFB1 is metabolized predominantly in the liver by a number of cytochrome P450 enzymes, generating several hydroxymetabolites, such as AFM1, AFQ1, and AFP1, and two significantly reactive epoxides, AFB1 exo-8,9-epoxide and AFB1 endo-8,9-epoxide[^20][^21][^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 humans[^20]. These albumin adducts of AFs have been discussed as useful biomarkers of AFs-induced hepatotoxicity[^20].

Deoxynivalenol (DON)

DON, as a member of the trichothecene B group, is produced by Fusarium fungi, one of the most important phytopathogenic fungal genera groups, also responsible for the production of ZEN and FBs[^15]. DON’s main mode of action includes the inhibition of protein synthesis by interfering with the termination step of the polypeptide chain, which is a part of the elongation step of protein synthesis carried out in the 60S ribosomal unit.

Acute DON intoxications in pigs are clinically observed as signs of abdominal pain, increased salivation, diarrhea, and emesis (3.6-40 mg DON/kg feed)[^24], whereas lower DON contamination levels result in reduced feed intake, decreased weight gain, growth retardation, immunotoxicity, and impaired reproduction and development[^25].

DON is rapidly absorbed in pigs (maximal plasma concentration after 15-30 min. of exposure) with approximately 90% of the ingested dose being absorbed. DON is distributed to all tissues and can undergo different detoxification pathways, including microbial degradation in the gastrointestinal tract and biotransformation in tissues such as the liver[^15][^26][^27]. DON can be metabolized 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 DOM-1 can be conjugated with glucuronic acid and sulfate, while in pigs, glucuronidation is more common[^30]. The development of an exposure biomarker for DON by quantifying urinary free-DON and DON-glucuronide combined as total urinary DON was suggested by observations in a rodent model[^31].

Regarding 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. Prelusky et al.[^32] reported that 54–85% of radioactive labeled DON administered intragastrically to pigs was excreted in urine within 24 hours. 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
demonstrated[36]. On the other hand, the amount of DON that reaches the faeces is negligible and includes 0.1 – 5% of the intake[15].

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 maize. In contrast to other Fusarium mycotoxins, FB1 is poorly absorbed from the gastrointestinal tract of pigs, with an oral bioavailability of approximately 4% of the administered dose. Distribution to tissues has been found predominantly in the liver and kidney, besides the large intestine and brain, and to a smaller extent in the 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 hydrolyzed FB or aminophenol (AP) and partially hydrolyzed FB1 (PHFB1). FB1 is mainly excreted via feces in pigs (>90%) with maximum concentration at 48 hours after administration and to a lesser extent via urine (approximately 5%) as FB1.

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 the accumulation of sphinganine (Sa) and sphingosine (So) in tissues, serum, and urine, and an increase in the Sa/So ratio. That mechanism has been recognized as the causal pathway for FB toxicity. This leads to reduced nutrient absorption, anorexia, decreased daily weight gain, and hepatotoxicity in pigs. Signs of encephalopathy and cardiac dysfunction with pulmonary edema have been observed after administration of FB1.

According to EFSA recommendations, the sphinganine/sphingosine ratio in blood should be held as the gold standard of FB biomarkers in swine.

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 orally administered OTA is absorbed in pigs. OTA in the systemic circulation has a high affinity for plasma proteins, especially 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 the prolongation of its presence in the body, slowing both its biotransformation and excretion half-life. OTA can be metabolized in the kidney, liver, and intestines, with major metabolic pathways including hydrolysis, hydroxylation, lactone-opening, and conjugation. OTA can undergo phase II metabolism with the formation of glucuronic acid and sulfate conjugates, whereas only the glucuronic acid conjugates have been detected in the bile of pigs.

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

ZEN

ZEN is a macrocyclic β-resorcylic acid lactone with structural similarity to naturally occurring estrogens, thus reproductive disorders in swine have been predominantly linked to ZEN toxicity, following the binding of ZEN to estrogen receptors. Hyperestrogenism in pigs observed at doses of 0.06 and 0.15 mg/kg feed includes signs of reddening, hyperemia, and edematous swelling of the vulva, enlargement of the uterus with cyst formation on the ovaries and enlargement of the mammary glands, whereas vaginal or rectal prolapse in such cases has also been reported.

Gilts are significantly sensitive to the toxic effects of ZEN since their concentrations of 17-β-estradiol are lower compared to sows. On the other hand, signs in boars include atrophy of the testes, reduction of the concentration of spermatozoa, and edematous swelling of the preputium and mammary complex. Moreover, embryotoxic and teratogenic properties of ZEN have been described.

ZEN is rapidly absorbed after oral exposure with an oral bioavailability of approximately 80% of the administered dose in pigs. ZEN is metabolized via phase I and phase 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), while also α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), and zearalanone (ZAN) can be formed. The α-ZEL is considered a bioactivation product as a more potent metabolite. ZEN and its phase I metabolites can undergo phase II conjugation with glucuronic acid and sulfate (through catalysis by uridine 5’-diphospho-glucuronosyltransferases and sulfotransferases). Both glucuronidation and sulfation are considered detoxification pathways.

• Olsen et al. 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 hours.
• Zöllner et al. estimated that approximately 14% of ZEN intake was excreted in urine either as the parent toxin or as metabolites within 8 hours.
• Dänicke et al. found 30% urine excretion of ZEN within 6 hours after female pigs received a bolus of 1 mg ZEN/kg body weight, with up to 70.4% excreted within 72 hours.
• 40% of the administered dose of ZEN can be recovered in urine (26±10%) and feces (14±3%) in pigs as ZEN or its metabolites 48 hours after administration.
• Fecal samples have been shown to contain ZEN and α-ZEL, which are also present in plasma, while urine contains ZEN and ZEN-GlcA.

BIOMARKERS OF MAJOR MYCOTOXINS IN SWINE

As previously suggested, biomarkers are defined as substances measured in biological systems linked to effect, exposure, or susceptibility, 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 detoxifiers. Biomarkers can be categorized based on their characteristics and use as follows:

• 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 and/or its phase I and phase II metabolites or interaction products.
• Biomarkers of effect: They are connected with the measurement of biochemical, physiological, or behavioral 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.

The European Food Safety Authority (EFSA) has recommended endpoints/biomarkers as proper validation endpoints for assessing the efficacy of feed additives that claim to adsorb or modify mycotoxins.

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

1. It has been suggested that the aforementioned biomarkers may not always be the most optimal for each mycotoxin.
2. DON concentrations in blood serum could be a relevant biomarker for exposure; however, DON undergoes extensive phase II biotransformation to DON-GlcA in pigs. Therefore, DON-GlcA might be considered a more precise biomarker for exposure than DON itself.

Biomarkers for exposure have been defined for particular mycotoxins in some animal species, but the identification of suitable biomarkers for each mycotoxin in specific species and biological matrices is often missing.

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

Urine, plasma, and feces can be easily collected from living pigs, making them practical for biomonitoring. In contrast, bile and organs, which are also useful matrices, can only be collected post-mortem. This has led to increased interest in using non-invasive and non-stressogenic methods for biomonitoring, focusing on easily accessible matrices.

Dried blood spots (DBS), hair, and saliva are emerging as viable matrices due to their reduced storage and transport requirements. While methods for detecting mycotoxins in DBS and hair of animals and humans are available, detection methods for saliva are not yet fully developed.

Recent studies have reported the use of DBS for multi-mycotoxin biomarker analysis in pigs. A validated method was developed for determining 23 mycotoxins and their metabolites in DBS, with findings showing a strong correlation between plasma and DBS concentrations. This supports the use of DBS as a non-invasive biomonitoring technique.

Moreover, circulating microRNAs (MiRNAs) have shown potential as biomarkers for pathological processes induced by mycotoxins such as ZEN, DON, OTA, and AFB1. MiRNAs can be measured in various biological matrices, including serum, urine, tissues, and saliva.

Transcriptomics research has revealed that ZEN can cause dose-dependent changes in uterine MiRNAs, with some of these changes reflected in serum. For DON, changes in serum MiRNAs were observed to be time-dependent and declined after the removal of contaminated diets. Four MiRNAs were identified as potential biomarkers for DON toxicity in porcine serum.

The use of biomarkers linked to critical signalling pathways and the mode of action of mycotoxins can aid in predicting their potential toxicity and the progression of associated mycotoxicosis, as well as in developing effective biomonitoring approaches. Previous studies in pigs have highlighted several biomarkers of exposure that correlate with mycotoxin intake levels:

1. Total DON concentration in serum and the sum of ZEN and its metabolites in urine both correlate well with toxin intake from the feed. DON concentration in serum and DON and DOM1 concentrations in urine increase in a dose-response manner as dietary DON concentration rises. Therefore, they are suitable biomarkers of exposure in pigs, with DON-GlcA in serum being considered a better biomarker for exposure.
2. DON-glucuronide (DON-GlcA) and ZEN-glucuronide (ZEN-GlcA) in plasma, as well as DON and ZEN-GlcA in urine, and ZEN in feces (due to enterohepatic circulation) have been reported as optimal biomarkers for DON and ZEN.
3. ZEN and its metabolites α- and β-ZEL in urine, along with ZEN and its metabolites α-, β-ZEL, and ZAN in feces, or plasma levels of GlcA metabolites of ZEN, α-ZEL, and β-ZEL can also be suitable biomarkers of exposure in pigs.
4. Both plasma and urine FB1 levels are considered adequate biomarkers of early exposure to low dietary levels of FB1, with plasma being recommended for prolonged exposure (greater than 14 days). A correlation between FB1 in feed and hair has been reported, though it is lower compared to urine and plasma.
5. Disruption in sphingolipid biosynthesis due to FB1 exposure can serve as a biomarker of effect, with an increased Sa/So ratio in serum and tissues observed in a dose-dependent manner. For AFB1, the concentration of AFB1-lysine in serum is indicative of long-term exposure. Additionally, AFB1-N7-guanine and AFM1 in urine have been suggested as biomarkers for AFB1 exposure, with AFB1, AFM1, and AFB2 identified as biomarkers in the urine of pigs. OTA concentrations in blood and urine are also reported as good markers for OTA contamination in various organs.

Future prospects

Several issues need further research in the field of mycotoxicosis. One key area is understanding the occurrence and fate of modified or masked mycotoxins. Research is needed to clarify how these altered forms of mycotoxins behave and degrade in contaminated feed. Additionally, the interactions among mycotoxins, particularly when present as mixtures, require further investigation. Understanding how different mycotoxins interact with each other and their combined effects on pigs is crucial for assessing overall toxicity and managing contaminated feed effectively.

• The use of biomonitoring and the assessment of respective biomarkers seems a promising tool for understanding complex procedures and their effects on the 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 to enable proper and timely counteracting measures regarding contaminated feed and animals.
• Accurate and sensitive methods (at reasonable cost) for quick biomonitoring testing in biological matrices, with minimal animal restraint, should be the focus of future research efforts in mycotoxicosis diagnostics, especially in cases of combined mycotoxicosis exposure.
• The complexity of sampling preparation and the presence of multiple mycotoxins with different toxicokinetic properties make the development of such technology challenging.