Introduction
Emerging and modified mycotoxins have historically received limited attention; however, they have attracted increasing interest in recent years following observations of clinical signs in animals that did not correlate with the mycotoxin levels detected in feed. Emerging mycotoxins include chemically diverse mycotoxins that are not routinely determined and for which there is no legislative regulation or recommendation.
In this scenario, it’s important to keep in mind that the natural contamination of raw materials and feed is often a “multi-contamination” of different mycotoxins, which may include these emerging mycotoxins that are generally not monitored in control analyses.
There is limited information regarding the toxicity of emerging mycotoxins. According to the latest publication by EFSA (European Food Safety Authority), focused on beauvericin and enniatins, few studies have evaluated the acute toxicity of these mycotoxins in vivo. Only one study in mice evaluating the effect of beauvericin is mentioned. However, different in vitro studies have investigated the toxicity of these mycotoxins in poultry.
Mechanisms of action of emerging mycotoxins
1. Oxidative stress
With respect to intracellular redox balance, multiple studies have reported elevated levels of reactive oxygen species (ROS) in various cell culture systems. Increased ROS has been accompanied by higher concentrations of lipid peroxidation products, including MDA (malondialdehyde) and TBARS (thiobarbituric acid–reactive substances), both widely recognised biomarkers of oxidative stress. Reductions in key antioxidant agents, such as GSH (glutathione), have also been observed. Collectively, these findings indicate that emerging mycotoxins are capable of inducing oxidative stress at the cellular level.
Notably, the European Food Safety Authority (EFSA) referenced a study in 2014 reporting reduced ROS levels in human cells treated with hydrogen peroxide. This observation highlights the need for further research into the relationship between redox balance and emerging mycotoxins across different cell lines and, potentially, across animal species (Dornetshuber et al., 2009).
2. Cytotoxicity
The cytotoxic effects of emerging mycotoxins have been investigated in multiple in vitro studies. In avian-derived cell systems, the study by Dombrink-Kurtzman (2003) is particularly noteworthy, as it evaluated the effects of BEA on turkey lymphocytes. DNA fragmentation followed by apoptosis was observed, providing in vitro evidence of the cytotoxic potential of emerging mycotoxins in poultry.
Emerging mycotoxins have been shown to exert marked cytotoxic effects, largely driven by apoptosis. According to EFSA, apoptosis occurs mainly through two pathways:
- An increase in intracellular calcium levels, leading to the activation of calcium-dependent endonucleases.
- Direct DNA intercalation, facilitating endonuclease activity.
Furthermore, the cytotoxicity of BEA and enniatins (ENNs) has often been attributed to their ionophoric properties.
3. Intestinal integrity
Given that cytotoxic effects have been observed in different cell lines and considering that the intestinal barrier represents the first site of contact between animals and mycotoxins following ingestion, understanding the effects of BEA and ENNs on intestinal cells is of particular interest. However, the impact of emerging mycotoxins on the intestinal barrier has been scarcely investigated to date.
Springler et al. (2016) evaluated the effects of BEA and ENNs on the transepithelial/transendothelial electrical resistance (TEER) of intestinal epithelial cells, specifically in the jejunum, a region known to play a key role in mycotoxin absorption. An increase in intestinal permeability was observed, associated with reduced expression of tight junctions. Among the tested compounds, enniatin B (ENN B) showed the strongest effect, followed by BEA, enniatin B1 (ENN B1), enniatin A (ENN A), and enniatin A1 (ENN A1). An additive effect was described among ENNs; however, this effect was not observed in combination with deoxynivalenol (DON).
By contrast, Albonico et al. (2017) did not report significant effects of BEA on intestinal permeability or pro-inflammatory cytokine production when the toxin was evaluated individually. In combination with fumonisin B1 (FB1) or DON, however, clear effects were observed. Differences between studies may be explained by the cell culture models employed and the mycotoxin concentrations tested.
Overall, the available evidence indicates that emerging mycotoxins can affect intestinal integrity and highlights the importance of considering multi-mycotoxin contamination.
4. Contextualization of in vivo studies
According to EFSA, the available studies on the toxicity of emerging mycotoxins in poultry were conducted in broiler chickens, laying hens, and turkeys exposed to multi-contamination with Fusarium mycotoxins, including BEA and ENNs. In most cases, the natural source of mycotoxins was maize, and no effects were observed on productive parameters, carcass yield, or the relative weight of organs such as the liver, spleen, bursa of Fabricius, or heart. Similarly, no concentrations of BEA or ENNs were detected in products intended for animal consumption (muscle tissue or eggs).
The following table presents the levels of emerging mycotoxin contamination that were not associated with adverse effects on production performance or avian physiology, according to EFSA (2014), under conditions of multi-contamination with other Fusarium mycotoxins:
| Fusarium Mycotoxins | Broilers chickens | Laying Hens | Turkeys |
| Beauvericin | 12600 ppb | 8930 ppb | 2480 ppb |
| Enniatin B | 12720 ppb | 11230 ppb | – |
| Enniatin B1 | 4060 ppb | 3060 ppb | – |
Table 1. Levels (ppb in feed) with no observed adverse effects.
Considering body weight (BW) and feed intake, the levels are expressed on a body-weight basis (BW/day).
| Fusarium Mycotoxins | Broilers chickens | Laying Hens | Turkeys |
| Beauvericin | 1220 µg/kg BW/day | 536 µg/kg BW/day | 136 µg/kg BW/day |
| Enniatin B | 763 µg/kg BW/day | 674 µg/kg BW/day | – |
| Enniatin B1 | 244 µg/kg BW/day | 216 µg/kg BW/day | – |
Table 2. Levels (µg/kg BW/day) not associated with observed adverse effects.
These values correspond to the highest mycotoxin levels evaluated in the reviewed studies. The absence of adverse effects at these doses may be related to the low bioavailability and rapid elimination of these mycotoxins in poultry (Fraeyman et al., 2018).
Recent studies have shown that emerging mycotoxins can affect intestinal barrier function and production performance. The reduction in crypt depth observed in broiler chickens exposed to enniatin may be attributed to the mycotoxin’s inhibitory effect on enterocyte proliferation (Fraeyman et al., 2018).
Similarly, Santos et al. (2021) reported an increased villus height to crypt depth ratio (VH:CD) in the ileum of 14-day-old broiler chickens, which was attributed to reduced intestinal cell proliferation, without an immediate effect on villus height. By day 28, a reduction in villus height in the jejunum and a decreased VH:CD ratio in both the jejunum and ileum (associated with increased crypt depth) were observed. The resulting reduction in the nutrient absorption surface may help explain the poorer production performance recorded.
In addition, growth and feed efficiency may be compromised, as energy is redirected toward epithelial restoration. These effects have been observed in broiler chickens challenged after consuming diets multi-contaminated with mycotoxins, including BEA, ENNs, DON, and their metabolites.
The following table summarizes selected in vitro toxicity studies of these mycotoxins.
| Reference | Study | Model | Mycotoxins | Effects |
| Albonico et al. (2017) | In vitro | Caco-2 cells | FB1 + β-ZEL (5 µg/mL each) | Cytotoxicity |
| BEA (>6 µM) | Direct cytotoxicity | |||
| BEA (>6 µM) | ↓ Progesterone and estradiol | |||
| α-ZEL / β-ZEL (dose-dependent) | ↓ Progesterone | |||
| Dornetshuber et al. (2009) | In vitro | Human promyelocytic leukemia cells (HL60) and human cervical carcinoma cells (KB-3-1) | BEA (1-10 µM) ENN (1-10 µM) | Cytotoxicity Apoptosis ↓ ERO |
| Dombrik-Kurtzmann (2003) | In vitro | Turkey lymphocytes | BEA (8-50 µM) | DNA fragmentation Apoptosis |
| Fraeyman et al. (2018) | In vitro | Porcine intestinal epithelial cells from the jejunum (IPEC-J2) | BEA (10 µM) | Total loss of cell viability |
| ENN A (10 µM) ENN A1 (10 µM) ENN B1 (10 µM) | Cytotoxicity | |||
| Mallebrera et al. (2014) | In vitro | CHO-K1 cells | BEA (5 µM) | ↑ Lipid peroxidation ↓ GSH |
| Prosperini et al. (2013) | In vitro | Caco-2 cells | BEA (5-10 µM) | ↑ ROS, MDA, GSSG ↓ GSH |
| BEA (>12 µM) | Apoptosis | |||
| Prosperini et al. (2014) | In vitro | Caco-2 cells | ENN A (0.9-15 µM) ENN A1 (0.9-15 µM) ENN B (0.9-15 µM) ENN B1 (0.9-15 µM) | Cytotoxicity Synergistic effect between enniatins |
| Springler et al. (2016) | In vitro | Porcine intestinal epithelial cells from the jejunum (IPEC-J2) | ENN A (5 µM) ENN A1 (5 µM) ENN B (2.5-5 µM) ENN B1 (5 µM) BEA (5-10 µM) | ↓ TEER Additive effect between enniatins Toxicity: ENN B > BEA > ENN B1 > ENN A > ENN A1 |
Table 3. Compilation of in vitro studies on the effects of emerging mycotoxins in poultry¹.
1 ROS: reactive oxygen species; GSSG: glutathione disulfide; GSH: reduced glutathione; MDA: malondialdehyde; TEER: transepithelial/transendothelial electrical resistance.
Conclusion
Although the effects of common mycotoxins have been widely studied, emerging mycotoxins still represent a challenge. Their mechanism of action is not yet fully understood, and their effects on animal health, losses in agricultural production, and their possible interaction with factors like climate change are under investigation.
