Mycotoxin biomarkers: A tool for early detection

The main exposure route to mycotoxins is oral, by contaminated feed ingestion; however it can also be absorbed through skin and airway. Currently chronic mycotoxicosis, exposure to low or moderate mycotoxins concentration over a long period of time, is the most important and causative of large economic losses. Furthermore, the symptoms are hard to identify and relate to the presence of mycotoxins. In general the diseases that these substances produce on organisms are physiological changes that result in a decrease in growth, development and production. The unspecificity of clinical signs makes that they can be confused with other diseases and even it is common that the mycotoxicosis is confused with nutritional or management deficiencies.

Four stages

The toxicity level will depend on the mycotoxins presence in the feed, dose, exposure time, as well as the animal species, age, hormonal and nutritional status, among other factors (Bryden, 2012). Pigs are considered one of the most sensitive to mycotoxins (Richard, 2007) (Figure 1).

Figure 1 – Sensitivity of animal species to the main important mycotoxins in animal feed.

Once mycotoxins enter the body, as any xenobiotic, their toxicokinetics can be studied through the application of ADME system. According to this model we distinguish four stages: Absorption, Distribution, Metabolism or biotransformation and Excretion, which explain the phenomena experimented by a substance from entering the body until disposal (Figure 2). The particularities of these stages for each mycotoxin justify the different toxicity degrees and sensibility of various animal species.

Figure 2 – ADME model applied to mycotoxins.

Absorption by passive diffusion

Absorption governs the entry of mycotoxins, the passage to blood and their distribution in the body. It takes place primarily at the digestive tract by passive diffusion, mycotoxins must cross the lipid bilayer (hydrophobic), and therefore the kinetic absorption rate depends on the physicochemical characteristics of the mycotoxins and the concentration gradient between outside and inside. The more lipophilic the molecule is the easier will be to transport. In the case of aflatoxin B1 
absorption is around 80%, however for fumonisin B1 does not exceed 3%.

Distribution mainly via blood

The main distribution channel is the bloodstream, which is largely affected by the binding capacity of certain mycotoxins to plasma proteins (e.g. aflatoxin and ochratoxin A to serum albumin). On the other hand, can be given reabsorption processes through the enterohepatic circulation (via secretion biliary) or renal reabsorption (in the case of ochratoxin A). These processes extend the mycotoxins permanence in blood, favouring its absorption and therefore toxic development processes.

Metabolism and excretion

The metabolism itself or biotransformation is the modifying process which suffers mycotoxins in order to facilitate their elimination from the body. This modification may be performed by microorganisms at the gastrointestinal level or enzymes at the target organs level, such as liver and kidney. In the case of ruminants, the rumen is the first line of defence against mycotoxins. Some rumen microorganisms play an important role because they are able to metabolize mycotoxins in less toxic compounds (OTA-α, de-epoxy-deonivalenol, aflatoxicol, etc) or less permeable compounds and therefore less bioavailable (α-zeralenone). Aflatoxin B1 is metabolised primarily in the liver by hepatic microsomal enzymes (enzymes of the family of cytochrome P-450). These enzymes convert AFB1 in their toxic form, carcinogenic form, AFB-8,9-epoxy, which can be covalently bound to DNA bases and serum albumin. AFB1 can also be oxidised giving rise to other metabolites, primarily AFM1 and AFQ1 hydroxylated forms, the AFP1 demethylated form and the aflatoxicol reduced (Figure 3). Finally, the efficacy in mycotoxins excretion, via urine and faeces, depends on the animal species and is directly related with the mycotoxins metabolisms.

Figure 3 – Biotransformation of aflatoxin B1.

3 classes of biomarkers

The study of biological markers is especially useful to provide information on exposure to xenobiotics, the effects produced or individual susceptibility. Biomarkers are defined as the cellular changes or alteration, biological or molecular that takes place in the tissues in response to a xenobiotic, in this case mycotoxins (Turner et al., 1999; Mayeux, 2004; Garban et al, 2005; Baldwin et al, 2011; Silins and Högberg, 2011). To establish a biomarker requires a lot toxicology studies and an extensive validation in order to establish, for example, a relationship between a biomarker and the external dose or the disease degree. Based on the sequence of events that is produced from exposure to development of the disease, the biomarkers can be classified into three classes: biomarkers of exposure, effect or susceptibility (Committe on Biological Markers of the National Research Council, 1987) (Figure 4).

Figure 4 – Stages in the development of a disease and classification of biomarker (adapted from Groopman and Kensler, 1999).

Biomarkers of exposure are specific, can be the mycotoxins itself or any of its metabolites and represent the internal dose (absorbed mycotoxins), while biomarkers of effect are generally nonspecific and represent structural or functional alterations produced in the body. However, these alterations may serve as biomarkers of exposure when both processes are directly linked (Groopman and Kensler, 1999; Perera and Weinstein, 2000; Silins and Högberg, 2011). The sphingolipids are directly related to regulating cellular processes. Given the high structural similarity between fumonisin and sphinganine (Sa) or sphingosine (So), the mycotoxin inhibits the acetylation causing an increase in the concentration of free sphinganine and in less degree free sphingosine, and a consequent decrease of sphingolipids, which triggers a series of processes that result in disease.

Early detection

Biomarkers are beneficial when you want to study the development of the disease and to be able to monitor and control of mycotoxins better. This is because its early detection can provide management options to limit diseases and accumulation of mycotoxins (Table 1).

Mycotoxins are also difficult to detect in raw materials and feed, due to heterogeneous dispersion of mycotoxins in these substrates. Legislation (Regulation nº 401/2006) requires taking a high number of samples, in order to “guarantee” their detection (Figure 5). This is not always feasible.

Adiveter has developed a sensible and effective technique, which consists of analysing mycotoxins and its metabolites in target organs (liver and kidney), obtained by necropsy in farm or at slaughterhouse. The reliability of the method is more than proven, existing even legislation in some European countries on maximum limits of mycotoxins in organs (FAO, 2004). The result is a “radiography”, showing which mycotoxins are present on farms. This allows to have an effective and specific control of every situation, identifying trends and preventing problems. It also enables to assess the effectiveness of the mycotoxins adsorbent used.

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