A Review of the Mycotoxin Enniatin B

Mycotoxin enniatin B (ENN B) is a secondary metabolism product by Fusarium fungi. It is a well-known antibacterial, antihelmintic, antifungal, herbicidal, and insecticidal compound. It has been found as a contaminant in several food commodities, particularly in cereal grains, co-occurring also with other mycotoxins. The primary mechanism of action of ENN B is mainly due to its ionophoric characteristics, but the exact mechanism is still unclear. In the last two decades, it has been a topic of great interest since its potent mammalian cytotoxic activity was demonstrated in several mammalian cell lines. Moreover, the co-exposure in vitro with other mycotoxins enhances its toxic potential through synergic effects, depending on the concentrations tested. Despite its clear cytotoxic effect, European Food Safety Authority stated that acute exposure to ENNs, such as ENN B, does not indicate concern for human health, but a concern might be the chronic exposure. However, given the lack of relevant toxicity data, no firm conclusion could be drawn and a risk assessment was not possible. In fact, very few studies have been carried out in vivo and, in these studies, no adverse effects were observed. So, research on toxicological effects induced by ENN B is still on-going. Recently, some studies are dealing with new advances regarding ENN B. This review summarizes the information on biochemical and biological activity of ENN B, focusing on toxicological aspects and on the latest advances in research on ENN B.

Moreover, some food processes including cooking, baking, frying, roasting, etc. do not affect their chemical structure; so, detoxification strategies to mitigate the risks of ENNs presence in foods and feed may be difficult (8,9). Structurally, ENNs are cyclohexadepsipeptides composed of alternating residues of three N-methyl amino acids, commonly valine, leucine, and isoleucine, and three hydroxy acids, typically hydroxyisovaleric acid. Several ENNs analogs (A, A1, B, B1, B2, B3, B4, D, E, F, and G) have been identified. Among them, the most prevalent ENNs reported as natural contaminants in cereals in Europe are ENN A, A1, B, and B1 (10). Their chemical structure is reported in Figure 1.
The lipophilic nature of ENNs allows them to be incorporated into lipid bilayers of cell membranes and creates cation selective pores that cause an increase in the permeability for cations, resulting in disturbances of the physiological cation level in the cell (11). Their ionophoric 3 behavior seems to be related to their wide range of biological activity. ENNs are known to be insecticidal, antifungal, antibacterial, and antihelmintic (12). Moreover, they exerted a potent cytotoxic effect in several human and animal cell lines at very low micromolar range (10,(13)(14)(15)(16)(17)(18). Despite the strong cytotoxicity in vitro, a few studies carried out in vivo did not show relevant toxicity (19)(20)(21)(22)(23).
Unlike other Fusarium mycotoxins, such as deoxynivalenol (DON), T-2, HT-2, fumonisins (FB), and zearalenone (ZEA), whose presence in food and feed has been regulated by authorities, no limits have been set for ENNs, up to now. However, an increasing number of studies are proving their presence in several food and feed commodities and also their toxicity (2). This fact may constitute a great concern for human and animal health, since their toxicity could be also enhanced by the presence of other mycotoxins at the same time. The European Commission asked the European Food Safety Authority (EFSA) for a scientific opinion on the risks to human and animal health related to the presence of ENNs in food and feed. EFSA concluded that acute exposure to ENNs does not indicate concern for human health. There might be a concern with respect to chronic exposure, but no firm conclusion could be drawn and a risk assessment was 3 Ionophores are molecules that facilitate ion passage in or out of cell membranes. not possible for dietary exposure to ENNs, due to the overall lack of toxicity data (24). At the moment, EFSA is still collecting occurrence data for a future risk assessment.
Among the four ENNs above-mentioned, ENN B is currently the most studied since it has been the most-often detected in unprocessed and processed grains from European countries. Concentrations of ENN B in grains range from a few μg/kg to over mg/kg (12). In a multi-mycotoxins analysis of maize silage in NW Spain, Dagnac et al. (25) found that ENN B was the most prevalent mycotoxin detected in 51% of the samples (average concentration: 157 µg/kg). Similar ENN B concentrations (195.5 ± 47.0 µg/kg) were observed in cereal samples collected from European and African countries (26). Svingen et al. (27) demonstrated the ENN B presence in all of the samples of Danish grain collected during the 2010 and 2011 harvests, with the highest value of 3,900 µg/kg detected in rye sample. A survey in Finland showed that ENNs were frequently detected in unprocessed grains including wheat, barley, rye, and oats, and that the maximum concentration was found for ENN B (10,280 µg/kg) in a barley sample (28).
Regarding grain-based products, in pasta samples bought from Dutch shops, de Nijs et al. (8) found the highest incidence for ENN B with concentrations ranging from 7.0 to 175 µg/kg. Higher concentrations of ENN B (up to 1,100 µg/kg) was detected in pasta and baby food from Italian supermarkets by Juan et al. (2). Zinedine et al. (7) demonstrated that wheat couscous semolina has a higher ENN B incidence and concentration (592 ng/g) than barley (50 ng/g) or corn (57 ng/g) semolina couscous. In beer samples from Germany, ENN B was the only ENN detected (0.9 µg/L) showing increased incidence than other mycotoxins (29).
Therefore, the attention on ENN B toxicological aspect is still highly concerning, considering that its potential toxicity may be enhanced by co-occurrence with other ENNs or other mycotoxins (15,30,31).
The objective of this review is to compile the effects produced by the Fusarium mycotoxin ENN B, focusing on its biological properties, biochemical activity and in vitro toxicological effects including the latest research on ENN B, in terms of biological properties, biochemical activity, and toxicity.

BiOLOGiCAL PROPeRTieS OF eNN B
Enniatin B exhibits a wide array of biological activities. Several studies investigated the insecticidal activity of ENN B individually 4 ACAT is an intracellular enzyme located in the endoplasmic reticulum that transfer fatty acyl groups from one molecule to another. 5 Oxidative stress is defined as a disturbance in the balance between the production of reactive oxygen species (free radicals) and antioxidant defenses. and in complex with other ENNs (38)(39)(40)(41)(42). This activity has been confirmed in the blowfly Calliphora erythrocephala, in the mosquito larvae (Aedes aegypti), in the spruce budworm (Choristoneura fumiferana) and against the plant-parasitic nematode Meloidogyne javanica (38)(39)(40). Moreover, ENN B partially inhibited spore germination of B. cinerea (42). However, no insecticidal activity of ENN B was found by Mulè et al. (43) against larvae of Galleria mellonella.
Enniatin B exhibits antibacterial activity against some pathogens of humans, such as Escherichia coli ( (49) and ENN B on knapweed leaves (Centaurea maculosa) when exposed with acetamido-butenolide (50). Combination of ENN A + ENN B showed decreased leaf and root development, wilting of shoots, necrosis of leaves, and loss of turgor (51,52).

BiOCHeMiCAL ACTiviTY OF eNN B ionophoric Properties
The ionophoric property of ENNs allows them to be capable of promoting the transport of mono-and divalent cations through membranes leading to toxic actions via disturbances in their normal physiological concentrations (1). The primary action is the ionophoric property, which enables ENNs to form stable complexes with cations, and transport them into the lipophilic phase (1) evoking changes in intracellular ion concentration, disrupting cell functions (Figure 2) (53).
The ability of the ENNs to form complexes with alkali metal ions and increase the cationic permeability of membranes has been previously documented (54,55). In particular, cations transported by ENNs in liposome seems to involve a mobile carrier mechanism which is selective for K + versus Na + , requiring two ENN molecules, and it is realized by a "sandwich" model (56). ENNs form both 1:1 and 2:1 ENN:cation complexes with alkali, alkaline earth, and various transition metal ions. The probability of the 3:2 conformations is much less than the two other conformations (57). It has been suggested that electronic, inductive, or steric effects could indirectly stabilize the 2:1 complex. Cation selectivity was ranked as follows: (56). In addition, the transport efficiency appears to be related to the hydrophobic trait of the ENN molecules. The largest conductivity was shown for ENN B, followed by ENN A1 and B1 (56). The mitochondriotoxic properties of ENNs have been demonstrated in isolated rat mitochondria (11). The mitochondrial effects were strongly connected with the K + ionophoric activity with ENNs inducing K + uptake by mitochondria. Moreover, they decreased the calcium retention capacity of the mitochondrion matrix leading to the mitochondrial membrane potential (MMP) collapse via permeability transition pore (PTP) opening (11,58).

enzyme inhibitor
The inhibition of the activity of ACAT by ENN B has been demonstrated (32). Such inhibition could be significant in the treatment and prevention of atherosclerosis and hypercholesterolemia. Trenin et al. (59) showed strong hypolipidemic activity of ENN B in human hepatoma HepG2 cells as a result of the inhibition of ACAT activity, triglyceride biosynthesis, and diminished pool of free fatty acids in the cells.

Other Biochemical Properties
Enniatin B was the most effective inhibitor of one of the major multidrug efflux pumps such as Pdr5p 6 in Saccharomyces cerevisiae at non-toxic concentrations (60). The inhibition mechanism is clearly different from its function as an ionophore (60). This ENN B property may be important for the clinical use in combination with chemotherapeutic drugs.
Enniatins interact with membrane-located ATP-binding cassette (ABC) transporters 7 , especially with ABCB1 and ABCG2 6 Yeast multidrug resistance protein that belongs to the family of ABC transporters. Pdr5p has been shown to confer resistance to a wide range of compounds and metal ions. 7 The ABC transporter superfamily is the largest transporter gene family. These proteins translocate a wide variety of substrates including sugars, amino acids, transporters, suggesting potential influences on bioavailability of xenobiotics and pharmaceuticals (61).

TOXiCiTY OF eNN B
Few toxicological studies of ENN B have been performed in vivo. Table 1 illustrates in vivo studies carried out with ENN B alone and in combination with other ENNs. In vivo toxicokinetic trials using pigs demonstrated a higher bioavailability of 91% for ENN B (62). Interestingly, Rodríguez-Carrasco et al. (22) found no acute toxicity in mice after intraperitoneal administration, although ENN B bioaccumulation in the lipophilic tissues was observed. According to Fraeyman et al. (63), ENN B was readily distributed to broiler chicken tissues, with mean volumes of distribution of 33.91 L/kg.
Comparing to in vitro studies, the number of studies in vivo is very low. In vitro cytotoxicity studies have been carried out for individual ENN B as well as for mixtures of ENNs, since mycotoxins, either from the same or from different fungal species, occur simultaneously in plant and food products (12). A scheme of in vitro studies on ENN B is shown in Figure 3.

In Vitro Cytotoxicity
Cytotoxicity Studies of Individual ENN B Different cell lines and assays have been chosen to determine ENN B cytotoxicity. Table 2 collects the cytotoxic activity studies performed in several cell lines exposed to ENN B tested individually and in complex with other ENNs (ENN A, A1, and B1) according to the type of cells, the toxicity endpoint, and the time of exposure. Data from literature show that human colon intestinal Caco-2 cells have been the most studied cell line when ENN B is applied alone (not in complex mixture), followed by HepG2 and CHO-K1 cells ( Table 2) (30) in CHO-K1 cells. Both studies aimed to investigate the type of interaction that occurs when ENNs appear in combination such as: synergism, antagonism, or additive effect by using isobologram method (70). The analysis was performed by testing binary and ternary combination in CHO-K1 and binary, ternary, and quaternary combination in Caco-2 cells. Lu et al. (30) found that the binary combinations ENNs A + B1, ENNs A1 + B, and ENNs B + B1 showed additive effects with all concentrations tested in CHO-K1 cells. Synergistic effect of combined ENNs A + A1, A + B, A1 + B1, A + A1 + B, A + A1 + B1, A + B + B1, and A1 + B + B1 at higher concentrations occurred. Synergism effect was observed at higher concentrations with binary and tertiary combinations of ENN A, while antagonism effects were obtained at lower concentrations for ENNs A + A1 + B1 and ENNs A1 + B + B1.
In addition, studies about the combination of ENNs with other mycotoxins have been carried out (15,65,71). Briefly, ENN B, tested with beauvericin, had additive cytotoxic effect on Human hematopoietic progenitors (71). Binary mixtures of ENN B with ZEA, DON, and nivalenol showed antagonistic and strong antagonistic effects on Caco-2 cell viability (65). As Fernandez-Blanco et al. (15) reported, mixtures of ENN B + DON and ENN B + Alternariol (AOH) were found to be synergic, depending on the concentrations tested.

Oxidative Stress
One of the key players in the production of oxidative stress is reactive oxygen species (ROS). Moreover, intracellular ROS generation in the hydrophobic compartment of a cell can induce lipid peroxidation (LPO). 8 ROS generation and LPO have been observed in mammalian cells exposed to ENN B (10,16). In addition, Ivanova et al. (10) found that ENN B-induced ROS production after 3 h exposure to ENN B in Caco-2 cells that were generated downstream the ENN B-induced cytotoxic events by the mitochondria. On the contrary, Dornetshuber (68) demonstrated that genotoxic potential and cytotoxicity of ENNs is independent of ROS generation. Further research is needed in this area.

impairment of Cell Cycle Distribution
Cell cycle is the entire process by which a cell undergoes cell division. Cell cycle phases are: the G1 phase, where cells are preparing for DNA, RNA, and protein synthesis, the S phase where DNA is synthesized, the G2 phase, where cells are preparing for mitosis, and finally the M phase (mitosis) where two daughter cells are generated. Cells can remain in a quiescent phase (G0 phase) and they need growth factors to enter the G1 phase. It has been demonstrated that mycotoxins can disturb the normal cell cycle distribution due to their anti-proliferative effects on several cell types, with an accumulation of cells in one or more phases of the cell cycle (14)  A noticeable increase of cells in the G2/M phase in Caco-2 cells after ENN B treatment was also observed by Ivanova et al. (10) confirming the impairment of mitosis. This type of arrest has been described as a possible consequence of external stimuli leading to apoptosis by activation of the caspase pathway or to non-apoptotic mitotic death.
Juan-García et al. (72) found that both ENN B and ENN A (1.5 and 3.0 µM) caused apoptosis in HepG2 cells, after 48 h of exposure, identifying ENN B more toxic than ENN A. Necrotic pathway was not observed. Similar results were obtained in mouse embryo fibroblast (Balb 3T3) cells (from 11 to 45 µM) (13), in murine monocyte (RAW 267.4) macrophages (35) and in human adrenocortical carcinoma cell line H295R (36). 9 The natural cell cycle includes a number of checkpoints that allow the cell to determine whether to proceed with division or stop. These halts can also be induced by external factors like chemicals. Research on cell cycle arrest provides important information about how cells regulate themselves and what happens when these processes go wrong. 10 Two different ways of cells can die are apoptosis or necrosis. Apoptosis is described as an active, programmed process of autonomous cellular dismantling that avoids eliciting inflammation. Necrosis has been characterized as passive, accidental cell death resulting from environmental perturbations with uncontrolled release of inflammatory cellular contents.

MMP Disruption
Mitochondria have been recognized for their role in mediating physiological processes and their involvement in signal transduction and regulation of cell proliferation and differentiation. They are involved also in cell death regulation, i.e., necrosis and apoptosis. Due to this role, mitochondria are vulnerable to the toxic effects of xenobiotics that interfere in cellular energy production. Apoptosis and necrosis induced cell death by cytotoxic agents involve similar metabolic disturbances and above all, mitochondrial permeability transition (MPT). Mitochondrial events of apoptosis and necrosis involve opening of a pore in the inner mitochondrial membrane, referred as mitochondrial PTP (MPTP) and the consequent dissipation of membrane potential (73). The dissipation of the MMP results from the unequal distribution of ions (mainly protons) on the inner mitochondrial membrane. The MMP disruption suggests that the protonmoving force and/or the inner membrane permeability has been affected during cell damage. The dissipation of MMP is a general feature of both cell death types (16).
Measurements of MMP are carried out by using lipophylic dyes, which pass through cell membranes and accumulate according to their charge. The alteration of fluorescent intensity can be determined by flow cytometry. Among these dyes, the tetramethylrhodamine methyl ester (TMRM), coupled with the carbocyanine monomer nucleic acid (To-Pro-3), has been used to determine the mitochondrial starting depolarization and cells progressing to death through apoptosis (72).
The disruption of MMP has been demonstrated in KB-3-1 cells exposed to ENNs mixture (33), and to ENN B in Caco-2 and HepG2 cells (10,16,72). The exact mechanism by which pro-oxidant mycotoxins induced pore opening is still not fully understood. At least two molecular sites of the complex contribute to this effect. The first site is a redox sensitive membrane dithiol group that can be oxidized by ROS (produced by mycotoxins), the second one remains undetermined.
Mitochondrial membrane potential was measured also by Svingen et al. (27) by the Hep-G2 quadroprobe multi-parametric liver toxicity assay. The strongest effect was seen for plasma membrane integrity, with concomitant effects on mitochondrial area/ mass and mitochondrial potential, confirming the involvement of mitochondria in ENNs toxicity (11).

estrogenic Activity
Recently, an investigation to evaluate possible endocrine disruptor effects 11 of ENN B was conducted by Kalayou et al. (36) demonstrating that in the human adrenocortical carcinoma cell line H295R, ENN B (10 µM) was able to reduce progesterone, testosterone, and cortisol production at a non-cytotoxic concentration. Higher concentrations (>10 μM) reduced both estradiol and testosterone levels in Leydig cells (36). Additional research 11 Chemicals that mimic or antagonize the in vitro and/or in vivo actions of naturally occurring estrogens are typically defined as having estrogenic activity or antiestrogenic activity. Effects on estrogen signaling represent the most common and best studied endocrine disruptor activity. eMeRGiNG FiNDiNGS OF eNN B Some researchers are underlying the anticancer potential of ENN B (33,34,61,68). Apoptosis with the involvement of p53 and p21 genes was found by Dornetshuber et al. (33), which tested a mixture of ENNs against several human cancer cells, promoting ENNs as anticancer drugs, according also to Wätjen et al. (34) and Dornetshuber-Fleiss et al. (37). In these surveys, ENNs caused caspase 3/7 activation in hepatoma H4IIE cells and caspase-7 activation in the KB-3-1 cell line, respectively, as well as nuclear fragmentation. Enniatin B is capable of resisting expulsion by the ABC transporters, and also naturally targets tumor cells more specifically than other chemotherapeutic agents. The action is synergic with the clinically approved multi-kinase inhibitor sorafenib (Sora) showing profound synergistic in vitro and in vivo anticancer effects against cervical cancer (37).

CONCLUDiNG ReMARKS
Mycotoxins constitute a serious health concern both for animals and for humans, besides economic problems. Productive and nutritive values of food and feed can be compromised by mold and mycotoxin contamination, and toxicological risk derived by ingestion is constantly under Authorities control. Regarding ENNs, a risk assessment is still not available, despite its clear toxicity in vitro and its presence in food and feed.
Indeed, several in vitro and in vivo studies have revealed that ENN B interacts with primary target molecules, induce signaling pathways and effector mechanisms, affects the biological response of cell defenses, promotes cell damage, produces potential interactions between food contaminants (particularly other mycotoxins) leading to abnormally high response, and other molecular events underlying ENN B toxicity. Nevertheless, regulatory limits have not yet been defined, due to a lack of complete toxicity data.
However, in the last decade, novel findings about a potential therapeutic action of ENNB have been proven. These promising findings introduce a new aspect of this toxic compound. Future research focused on elucidating the toxic mechanism of ENN B as well as its anticancer activity could better clarify the real potential of ENN B. These research findings could contribute to establish emerging therapeutic strategy to chronic health problems.
This review wanted to collect all available data regarding toxicological aspect and emerging findings on ENN B in order to underlying the need to continue to study toxic/emerging effects of this compound to finally protect and improve both animal and human well-being.