Biomarkers are a tool to measure dietary exposure of human and animals to mycotoxins. In contrast, the industry looks at it from another perspective: as a cheaper, faster and a more reliable test of the effectiveness of an anti-mycotoxin additive.
Currently, there is a growing interest in mycotoxin biomarkers in the farming community based on the increasing number of farmers enquiring about this topic. The term ‘mycotoxin biomarker’ in that context is being associated with co-called exposure-related indicators (mycotoxins and their metabolites in body fluids, faeces or organs).
Many authors define 2 types of biomarkers:
Direct or exposure-based biomarkers are specific, while indirect or biomarkers of effect are generally nonspecific and represent structural or functional alterations produced in the body under exposure to certain drugs or toxins. However, these alterations in some cases may serve as biomarkers of exposure when both processes are directly linked (Groopman and Kensler, 1999; Perera and Weinstein, 2000; Silins and Högberg, 2011).
However, the suggestions would be to use 3 subtypes for mycotoxin biomarkers (Figure 1):
Exposure-based biomarkers characterise the presence of mycotoxins or their metabolites (e.g. glutathione or glucuronide conjugates). These compounds can be detected in easily-accessed biological fluids or tissues (Baldwin et al., 2011).
In contrast, mechanism-based biomarkers assess changes specific to the mycotoxin in specific proteins, cellular metabolites, or gene expression.
Effect-based biomarkers express the typical consequence of the mycotoxin on the performance and other health parameters such as gut integrity, antibody titre, typical lesions, level of serum liver enzymes or even weight gain and feed conversion. However, they are less specific to the mycotoxin when compared to mechanism-based biomarkers and more so when compared to exposure-based biomarkers.
Specific biomarkers (exposure- and mechanism-based) are measured in body fluids or tissues, as the molecule itself, a metabolite(s), or a product of a reaction with a biological molecule. The most common parameters in quantifying exposure to mycotoxins are measured in urine, serum and milk. However, there are other biological matrixes that can provide important information, like faeces or hair.
Several mycotoxins (aflatoxins, ochratoxin A, DON, zearalenone) are rapidly absorbed in the upper gut showing a sharp peak in blood within two hours of oral ingestion (Figure 2) in most animal species. In contrast, fumonisins have limited availability and their levels in blood are insignificant. Consequently, exposure-based biomarkers of mycotoxins with high bioavailability can only be detected in serum and plasma in high concentrations shortly after oral ingestion. They are also rapidly cleared from the bloodstream. This is crucial as the time of blood collection is key during in vivo study of blood biomarkers of exposure. The mechanism-based biomarkers such as protein adducts have longer half-lives in blood. Among all biomarkers they provide more information on their cumulative effects despite the fact that they could be less specific to a mycotoxin.
The portal vein conveys blood with mycotoxins absorbed in the stomach, pancreas, and intestines to the liver. Billiary excretion is a major route of elimination of mycotoxins with high or low absorption rates. In fact, in some cases, in which mycotoxins are fed at low concentrations, the mycotoxin can only be detected in bile (Armorini et al., 2015). As sampling bile requires animal euthanasia, bile biomarker substrates are unfortunately, only practical in field studies in poultry.
The main fraction of the rapidly absorbed mycotoxins passes through the liver and briefly circulates through the blood before it is excreted as its metabolites are unchanged in the urine. However, correlation between ingested mycotoxin and the level of mycotoxin in urine is lower when compared to blood due to food-related variations of urine amount. Several authors recommend using creatinine as an indicator of the amount of urine produced (Kraft and Dürr, 2005) as the excretion of creatinine is not food-related. This physiological substrate is not a suitable matrix for field studies in poultry.
Metabolites of aflatoxins, DON, zearalenone and ochratoxin A can be detected in various concentrations in the milk of non-ruminant mammals. In ruminants milk is an easy and widely used matrix for estimation of aflatoxin exposure only. There is currently no correlation between levels of toxins in milk compared to serum, suggesting that the transfer from blood to milk is not yet a fully understood process (Biasucci et al., 2010).
The gastric absorption rate of some mycotoxins (e.g. fumonisins) is very low and a large part is eliminated in faeces. Mycotoxins with high bioavailability are excreted in faeces at a very low rate (Turner et al., 2010). Quantification of biomarkers in faeces as an estimation of the mycotoxin binder has potential; however, this approach has many limits including the possible metabolisation of bound mycotoxin by microflora in the large intestine. Another worrying observation is that faecal concentrations of mycotoxins can decrease with increasing doses of adsorbent in the diet and it is suggested that bound toxins may be undetectable in faeces when using analytical procedures standard for other physiological substrates or for feed (Sulzberger et al., 2017).
Analysis of mycotoxin biomarkers in organs requires an invasive procedure such as biopsy or post-mortem study. The accumulation of several mycotoxins in tissues happens primarily in the liver and kidneys. Using organs as a biological matrix has several benefits such as the accumulation effect of the mycotoxin, and the lower variation linked to the feeding time and sample collection. Some mycotoxins even when fed at low concentrations are retained in the liver and kidneys in unmetabolised (reviewed in Voss et al., 2007) or metabolised form. Mycotoxins persist in kidneys much longer than in plasma or the liver, and the levels can be 10 times the amount in the liver (Martinez-Larranaga et al., 1999; Riley and Voss, 2006).
This is the easiest biological sample to collect in the biomarker assay. Sewram et al. (2003) showed for the first time that fumonisins can accumulate in hair of humans and primates. Unfortunately, there is no reliable information about accumulation of fumonisin or other mycotoxins in hairs of domestic animals.
Mycotoxins have diverse metabolic pathways in vivo that can be very specific to animal species. This is partly due to the setup of the digestive system (absence or presence of pre-stomachs) and the different intestinal cell metabolism in the gut epithelium or the species-specific gut microbiota may significantly influence the formation of biomarkers of different types. It is therefore important to choose the right representative substrate (biomarker) in each animal species. For example, the analysis of exposure-based biomarkers of DON, fumonisins and T-2 toxins in poultry blood has a lower practical value in field evaluation because of the low absorption levels of these mycotoxins. On the other hand, the rapid and moderate-to-high absorption of DON, aflatoxins and ochratoxin A in pigs makes the field study of exposure-based biomarkers in blood very dependent on the sampling time since they peak in blood within 15–30 min of ingestion (Prelusky et al., 1988). In ruminants, the only way to evaluate mycotoxin biomarkers in the field is the use of low- or non-invasive sampling methods. Milk is the substrate most preferred by dairy producers in monitoring [exposure-based] biomarker, aflatoxin M1.
Mycotoxin sequestrating agents (alternative names: deactivator, detoxifier, binder) are feed additives which aim to reduce mycotoxin toxicity by means of binding the toxin (mycotoxin binders), alterating the chemical structure of the mycotoxin in the gastro-intestinal tract to non-toxic metabolites (mycotoxin transforming agents) or diminishing the negative secondary effects of the mycotoxin exposure to animals (mycotoxin deactivators). While farmers seem excited about using exposure-based biomarkers as a practical tool to check the performance of mycotoxin sequestrants, is crucial to consider the several important limitations:
It could seem easier to evaluate efficacy of a product by measuring the level of mycotoxin or its metabolite in physiological fluids than to rely on animal performance data obtained in the long term. However, this approach depends a lot on several factors, including the specific toxin being measured, the availability of certain tissues or liquids, the specific purpose of the study and mode of action of the product. Currently, combining information about clinical indicators (indirect effect-based biomarkers), suspected or confirmed exposure (analysis of feed) and in vivo research data of the producer of the anti-mycotoxin additive is the best and the most economical solution for the evaluation of the product.
References are available on request.
Author: Olga Averkieva Phd, Business Development Manager Mycotoxins, Nutriad International