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Mycotoxins, toxic compounds produced by fungi, pose significant risks to food safety and human health. This study investigates the bio-detoxification potential of 238 strains of lactic acid bacteria (LAB) and Bacillus spp., previously isolated from cereals (including mycotoxin-contaminated grains), against the emerging mycotoxin, enniatin B (ENB), and the prevalent mycotoxin, deoxynivalenol (DON). Out of the tested strains, 26 demonstrated notable mycotoxin reduction capabilities, including 2 Bacillus pumilus and 24 Bacillus licheniformis strains. B. licheniformis strains MA572, MA695, MA696, TR174a, TR284, TR363, and TR466a degraded ENB to levels below the detection limit, and six strains reduced DON by 30–35%; B. licheniformis TR251b and TR374 showed the highest DON reduction with 35.7%. The most promising strains for bio-detoxification were B. licheniformis TR284, which achieved a 100% reduction in ENB and a 28.6% reduction in DON and B. licheniformis TR388 with a 97.5% reduction in ENB and a 31.9% reduction in DON. None of the tested LAB strains significantly reduced either mycotoxin. These findings highlight the promising potential of B. licheniformis strains in bio-detoxifying mycotoxin-contaminated cereal products. Further research into the underlying detoxification mechanisms and safety aspects is essential to develop effective bio-detoxification strategies for enhancing food safety. [ABSTRACT FROM AUTHOR]

Mycotoxins, toxic compounds produced by fungi, pose significant risks to food safety and human health. This study investigates the bio-detoxification potential of 238 strains of lactic acid bacteria (LAB) and Bacillus spp., previously isolated from cereals (including mycotoxin-contaminated grains), against the emerging mycotoxin, enniatin B (ENB), and the prevalent mycotoxin, deoxynivalenol (DON). Out of the tested strains, 26 demonstrated notable mycotoxin reduction capabilities, including 2 Bacillus pumilus and 24 Bacillus licheniformis strains. B. licheniformis strains MA572, MA695, MA696, TR174a, TR284, TR363, and TR466a degraded ENB to levels below the detection limit, and six strains reduced DON by 30–35%; B. licheniformis TR251b and TR374 showed the highest DON reduction with 35.7%. The most promising strains for bio-detoxification were B. licheniformis TR284, which achieved a 100% reduction in ENB and a 28.6% reduction in DON and B. licheniformis TR388 with a 97.5% reduction in ENB and a 31.9% reduction in DON. None of the tested LAB strains significantly reduced either mycotoxin. These findings highlight the promising potential of B. licheniformis strains in bio-detoxifying mycotoxin-contaminated cereal products. Further research into the underlying detoxification mechanisms and safety aspects is essential to develop effective bio-detoxification strategies for enhancing food safety.

Food and feed contamination by nonlegislated mycotoxins beauvericin (BEA) and enniatin B (ENB) is a worldwide health concern in the present. The principal objective of this work is to assess some of the existing protocols to discover the single nucleotide variants (SNVs) in transcriptomic data obtained by RNA-seq from Jurkat cells in vitro samples individually exposed to BEA and ENB at three concentration levels (1.5, 3 and 5 µM). Moreover, previous transcriptomic results will be compared with new findings obtained using a different protocol. SNVs rs201003509 in BEA exposed cells and the rs36045790 in ENB were found in the differentially expressed genes in all doses compared to controls by means of the Genome Analysis Toolkit (GATK) Best Practices workflow. SNV-RNA-seq complementary pipeline did not show any SNV. Concerning gene expression, discrepant results were found for 1.5 µM BEA exposed cells compared with previous findings. However, 354 overlapped differentially expressed genes (DEGs) were identified in the three ENB concentrations used, with 147 matches with respect to the 245 DEGs found in the previous results. In conclusion, the two discovery SNVs protocols based on variant calling from RNA-seq used in this work displayed very different results and there were SNVs found manually not identified by any pipeline. Additionally, the new gene expression analysis reported comparable but non identical DEGs to the previous transcriptomic results obtained from these RNA-seq data. Single-nucleotide variants discovery protocols in transcriptomic data by RNA-seq from Jurkat cells exposed to mycotoxins for the first time [ABSTRACT FROM AUTHOR]

Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl (2S, 3S)-epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway. [ABSTRACT FROM AUTHOR]

Enniatin B (ENNB) is a mycotoxin produced by moulds from the Fusarium genera and its toxic effects are still not fully elucidated, hence a safe reference exposure value has not been established yet. ENNB is the most prevalent emerging mycotoxin and is widely found in cereal-based products, nevertheless, there are no comprehensive exposure assessment studies. For that reason, the aim of this study was to characterise the occurrence of ENNB and estimate the exposure of the Spanish and Catalan populations. A total of 347 cereal-based products were collected in 2019 and were analysed using liquid chromatography-tandem mass spectrometry. Consumption data were obtained from the national food consumption surveys (ENALIA) and a regional survey conducted in Catalonia. The global exposure was estimated using deterministic and probabilistic methods. The results showed a high occurrence of close to 100% in all foodstuffs, with a range from 6 to 269 µg/kg, and a strong correlation with the levels of deoxynivalenol. Children aged one–nine years were the most exposed, showing mean estimates in the range 308–324 ng/kg bw/day and 95th percentiles 697–781 ng/kg bw/day. This study stresses the need for further toxicological data to establish reference doses and conclude formal risk assessment, accounting for the co-occurrence with deoxynivalenol. [ABSTRACT FROM AUTHOR]

Featured Application: The current investigation was part of a collaborative research project aiming at finding innovative decontamination strategies to prevent food waste and reintroduce safe whole wheat grain into the food value chain. Wheat is one of the world's key staple foods, but it is often contaminated with mycotoxin-producing microorganisms, resulting in a large amount of food waste every year. The contamination of wheat grains harvested in 2020 and 2021 in Switzerland, as well as of wheat flours bought in local stores with the two mycotoxins zearalenone (ZEA) and enniatin B (ENB) was investigated. The quantification was performed using LC–MS/MS. ZEA, the level in different cereals and food products of which is regulated by law, was detected in half of the grain samples at levels below 100 µg/kg, except for one sample contaminated with 147 µg/kg. No ZEA was detected in the commercial wheat flours. The emerging mycotoxin ENB was detected in all samples of wheat grains and flours, at levels between 3 and 938 µg/kg. The harvest year was shown to affect the ENB content (p value < 0.01), and in particular the humid weather conditions encountered in 2021 during the month of harvest. The refining grade of the flours showed no influence on the contamination by ENB, indicating that the contamination with ENB can occur not only on the surface layers but also on the inner layers on the wheat grain. As chronic exposure to ENB can therefore not be excluded, decontamination solutions are needed to prevent food waste and further improve the food safety of wheat-based products. [ABSTRACT FROM AUTHOR]

The current study has focused on the effects of enniatin B (ENN B, a major mycotoxin produced by Fusarium fungi) on early embryonic development. In in vitro analysis, mouse blastocysts were incubated in medium with ENN B (0–40 μM) or 0.5% DMSO (control group) for 24 hours. In an animal study, blastocysts were collected from mice which were intravenously injected with ENN B (1, 3, 5, and 7mg/kg body weight/day) for 4 days in order to analyze apoptosis and necrosis via Annexin V/PI staining assay; and proliferation using dual differential staining. Exposure to low ENN B concentration (10 μM in vitro and 3 mg/kg/day in vivo) promoted Reactive Oxygen Species (ROS) generation and apoptosis in the Inner Cell Mass (ICM), the mass of cells inside the blastocyst, impairing post-implantation development alone. On the other hand, exposure to a higher ENN B concentration (40 μM in vitro and 7 mg/kg/day in vivo) induced ROS generation and decreased in intracellular ATP which encouraged necrotic processes in both trophectoderm (TE) and ICM of blastocysts leading to impaired implantation and post-implantation development. Moreover, 5 and 7 mg/kg/day ENN B intraperitoneal injection to female mice for 4 days has caused downregulation of CXCL1, IL-1β and IL-8 expressions and increased ROS generation in the liver of newborn mice. Over all, ENN B can induce apoptosis and/or necrosis depending on the treatment dosage in mouse blastocysts. ENN B-induced necrosis in blastocysts may exert long-term harmful effects on next-generation newborns. [ABSTRACT FROM AUTHOR]

Wheat is one of the world’s key staple foods, but it is often contaminated with mycotoxin-producing microorganisms, resulting in a large amount of food waste every year. The contamination of wheat grains harvested in 2020 and 2021 in Switzerland, as well as of wheat flours bought in local stores with the two mycotoxins zearalenone (ZEA) and enniatin B (ENB) was investigated. The quantification was performed using LC–MS/MS. ZEA, the level in different cereals and food products of which is regulated by law, was detected in half of the grain samples at levels below 100 µg/kg, except for one sample contaminated with 147 µg/kg. No ZEA was detected in the commercial wheat flours. The emerging mycotoxin ENB was detected in all samples of wheat grains and flours, at levels between 3 and 938 µg/kg. The harvest year was shown to affect the ENB content (p value < 0.01), and in particular the humid weather conditions encountered in 2021 during the month of harvest. The refining grade of the flours showed no influence on the contamination by ENB, indicating that the contamination with ENB can occur not only on the surface layers but also on the inner layers on the wheat grain. As chronic exposure to ENB can therefore not be excluded, decontamination solutions are needed to prevent food waste and further improve the food safety of wheat-based products.

The exposure to mycotoxins of Swedish adolescents is currently unknown. The aim of the present study was to investigate the exposure to mycotoxins and their association with food intake, and background characteristics in adolescents of a national dietary survey. About 3000 school students (1000 from the 5th, 8th and 11th school years) were recruited for the survey. The participants completed Web-based questionnaires on food propensity, sociodemography and health, and a Web-based dietary recall. Spot urine and blood samples were collected from 1105 of the participants for mycotoxin biomarker analysis. Mycotoxins were analysed with multibiomarker methods in urine (HPLC-MS/MS) and serum (HPLC-MS/MS). Of the 35 different analytes in urine, the frequency of positive samples were the following: deoxynivalenol (DON, 4.8%), DON-15-β-D-O-glucuronide (DON-15GlcA, 9.1%), dihydro-citrinone (DH-CIT, 0.5%), HT-2-glucuronide (HT-2-3-GlcA, 0.1%) and ochratoxin A (OTA, 0.1%). Of the 27 different analytes in serum, OTA was detected in all samples, while 2'R-ochratoxin A (2'R-OTA) was found in 8.3% and enniatin B (EnB) in 99.2% of the samples. Exposure assessment calculations were performed on OTA from the serum concentration and on DON equivalents (DON eqv) from the urine concentration. All probable daily intake (PDI) estimates were below tolerable daily intakes, except for 1.6% of the participants for DON. The maximum PDI was 4.3 μg DON eqv/kg body weight and day. Consumption of cereal grain commodities was associated with levels of DON, EnB or OTA in biofluids. Serum OTA was also associated with intakes of raisins and coffee. Furthermore, coffee consumption correlated well with 2'R-OTA concentration in serum. In conclusion, exposure to mycotoxins in Swedish adolescents is common, but fortunately, high exposure was rare. [ABSTRACT FROM AUTHOR]

Cheese represents a dairy product extremely inclined to fungal growth and mycotoxin production. The growth of fungi belonging to Aspergillus, Penicillium, Fusarium, Claviceps, Alternaria, and Trichoderma genera in or on cheese leads to undesirable changes able to affect the quality of the final products. In the present investigation, a total of 68 types of commercial and traditional Slovak cheeses were analyzed to investigate the occurrence of fungal metabolites. Altogether, 13 fungal metabolites were identified and quantified. Aflatoxin M1, the only mycotoxin regulated in milk and dairy products, was not detected in any case. However, the presence of metabolites that have never been reported in cheeses, such as tryptophol at a maximum concentration level from 13.4 to 7930 µg/kg (average: 490 µg/kg), was recorded. Out of all detected metabolites, enniatin B represents the most frequently detected mycotoxin (0.06–0.71 µg/kg) in the analyzed samples. Attention is drawn to the lack of data on mycotoxins’ origin from Slovak cheeses; in fact, this is the first reported investigation. Our results indicate the presence of fungal mycotoxin contamination for which maximum permissible levels are not established, highlighting the importance of monitoring the source and producers of contamination in order to protect consumers’ health.

Abstract Background Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. Results In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. Conclusions The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.

Simultaneous mycotoxins toxicity is complex and non-predictable based on their individual toxicities. Beauvericin and Enniatins are emerging mycotoxins highly co-occurrent in food and feed, and their cytotoxicity has been reported in several human cell lines. RNA-seq studies of individual exposure in Jurkat cells demonstrated human genome perturbation mainly affecting mitochondrial pathways, however, both mycotoxins showed differences between their toxic responses. This study investigates the transcriptional effects of combined exposure to Beauvericin and Enniatin B (1:1) (0.1, 0.5, 1.5 μM; 24 h) in Jurkat cells by qPCR on 30 selected target genes (10 mitochondrial, 20 nuclear). Gene expression after combined and individual exposures were compared and functional data analysis (ToxPi) on the most relevant biological processes (cycle and apoptosis regulation; cholesterol metabolism and transport; cellular signaling transduction; cellular stress responses; immune regulation; protein metabolism; retinoic acid metabolism; transcription regulation) was applied to RNA-seq data from individual exposure (1.5, 3, 5 μM; 24 h; Jurkat cells). Transcriptional changes, especially at mitochondrial level, were observed after Beauvericin-Enniatin B co-exposure including down-regulation of antioxidant activity related genes. Different expression patterns between combined and individual exposures were identified. ToxPi analysis confirmed different dose-dependent relationship profiles between these two mycotoxins after individual exposure. • BEA:EN B co-exposure triggered mitochondrial transcriptional changes, mainly a slight up-regulation. • BEA:EN B co-exposure slenderly down-regulated antioxidant activity related genes. • Different expression patterns were identified between combined and individual exposures. • Different dose-dependent gene expression was found between BEA and EN B individual exposure. [ABSTRACT FROM AUTHOR]

Enniatins (Enns) are mycotoxins produced by Fusarium spp. and are widely distributed contaminants of cereals and derivate products. Among the different identified enniatins, Enn B is the most relevant analogue in cereals in Europe. Therefore, the aim of this study was to investigate for the first time the occurrence of Enn B and Enn B phase I metabolites in 300 human urine samples throughout an ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) methodology. Three different sample preparation procedures were evaluated and salting-out liquid-liquid extraction showed satisfactory validation results. Enn B was quantified in 83.7% of samples ranging from 0.006 to 0.391 ng/mL (average content: 0.016 ng/mL). In line with the in vitro observations with human liver microsomes, in the here analyzed samples the Enn B monooxygenated, N -demethylated and dioxygenated metabolites were tentatively found in 87.7%, 96.3% and 6.7% of samples. The data of this pilot biomonitoring survey indicate a frequent intake of enniatins in Italy, supporting further toxicological studies to provide better basis for understanding their potential effects in humans. [ABSTRACT FROM AUTHOR]

Several mycotoxins exert their effect on the immunological system; some are classified as immunotoxic. Jurkat T-cells were used to study toxic effects of beauvericin (BEA) and enniatin B (ENN B). Both are not legislated mycotoxins with increasing presence in feed and food. Concentrations studied were from 1 to 15 μM at 24, 48 and 72 h. Cell death by increasing the percentage of apoptotic/necrotic cells was: BEA > ENN B. IC50 values ranged from 3 to 7.5 μM for BEA. ENN B 15 μM decreased viability (21-29%). The percentage of apoptotic/necrotic cells was BEA > ENN B at 24 h but not at 48 h. Caspase-3&7 activation profile varied, although both mycotoxins increased this activation. No difference in ROS production for any mycotoxin was observed. Arrest in S phase for both mycotoxins was obtained. BEA increased the percentage of DNA in the tail (18% and 20%) with respect to the control, whereas not for ENN B. In summary, cytotoxicity of BEA involved mitochondrial alterations; while ENN B only at highest concentrations and time assayed. BEA had cell cycle disturbances and apoptotic and apoptotic/necrotic cells increased; for ENN B these were not evident. Different toxic responses in Jurkat T-cells may be involved in BEA and ENN B toxicity. [ABSTRACT FROM AUTHOR]

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.

The Fusarium mycotoxin enniatin B (ENN B) is a so-called emerging mycotoxin frequently contaminating poultry feed. To investigate the impact of chronic ENN B exposure on animal health, broiler chickens were fed either a diet naturally contaminated with ENN B (2352 μg/kg) or a control diet (135 μg/kg) for 2, 7, 14, or 21 days. ENN B concentrations were determined in plasma and liver using a validated ultra-high performance liquid chromatography--tandem mass spectrometry UHPLC-MS/MS method. Liver was evaluated histologically, and the villus length and crypt depth of the duodenum, jejunum, and ileum were measured. Histopathology of the livers did not reveal major abnormalities. Feeding an ENN B-contaminated diet could possibly inhibit the proliferation of enterocytes in the duodenal crypts, but did not affect villus length, crypt depth, or villus length-crypt depth ratio of the jejunum and ileum. ENN B levels in plasma and liver were significantly higher in the ENN B-fed group and ranged between <25-264 pg/mL and <0.05-0.85 ng/g, respectively. ENN B carry-over rates from feed to liver tissue were 0.005-0.014% and 0.034-0.109% in the ENN B and control group, respectively. Carry-over rates were low and indicated a limited contribution of poultry tissue-derived products to the total dietary ENN B intake for humans. The above results support the opinion of the European Food Safety Authority stating that adverse health effects from ENN B in broiler chickens are unlikely. [ABSTRACT FROM AUTHOR]

Multidrug transporters are often responsible for failure of medical treatment, since they expel a variety of structurally and functionally unrelated drugs out of the cell. We found that the fluorescent probe diS-C3(3) is a substrate of not only Pdr5p of Saccharomyces cerevisiae (ScPdr5p) but also of its less-explored Kluyveromyces lactis homologue (KlPdr5p). This enabled us to compare the ability of azoles to competitively inhibit the Pdr5p-mediated probe efflux in the two species. In K. lactis, these azoles completely inhibit probe transport by KlPdr5p and also compete with each other for transport. This indicates that the probe and the azoles are bound by the same site(s) of the KlPdr5p binding pocket. On the other hand, the azoles' capacity to inhibit the probe transport by ScPdr5p is limited, as a result of their partial cotransport with the probe. While the azoles bind to only one or two separate binding sites, the probe is able to bind to all three of them. Moreover, the bulky ScPdr5p substrate enniatin B, which effectively inhibits both probe and azole transport by the pump, has negligible effect on KlPdr5p. Our data point to a tighter arrangement of the KlPdr5p binding pocket compared to that of ScPdr5p. [ABSTRACT FROM AUTHOR]

Background: Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. Results: In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. Conclusions: The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to coexpress any genes of interest in equimolar amounts. [ABSTRACT FROM AUTHOR]

The fusariotoxins Enniatin B (Enn B) and Beauvericin (Bea) have recently aroused interest as food contaminants and as potential anticancer drugs. However, limited data are available about their toxic profile. Aim of this study was to investigate their pharmacological behavior in vivo and their persistence in mice. Therefore, liquid chromatography tandem mass spectrometry (LC–MS/MS) was used to analyze the distribution of Enn B and Bea in selected tissue samples and biological fluids originating from mice treated intraperitoneally with these cyclohexadepsipeptides. Overall, no toxicological signs during life time or pathological changes were observed. Moreover, both fusariotoxins were found in all tissues and serum but not in urine. Highest amounts were measured in liver and fat demonstrating the moleculeś tendency to bioaccumulate in lipophilic tissues. While for Bea no metabolites could be detected, for Enn B three phase I metabolites (dioxygenated-Enn B, mono- and di-demethylated-Enn B) were found in liver and colon, with dioxygenated-Enn B being most prominent. Consequently, contribution of hepatic as well as intestinal metabolism seems to be involved in the overall metabolism of Enn B. Thus, despite their structural similarity, the metabolism of Enn B and Bea shows distinct discrepancies which might affect long-term effects and tolerability in humans. [ABSTRACT FROM AUTHOR]

• Angelica root rot can be suppressed by P. polymyxa YF and B. tequilensis SY89. • P. polymyxa YF and B. tequilensis SY89 can function by inhibiting and degrading ENN B. • The GFP-tagged strains of YF and SY89 can colonize in Angelica root. • Antagonistic bacteria have significant disease control effect. Fusarium avenaceum is the predominant pathogen associated with Angelica sinensis root rot, which results in mycotoxin contamination of Angelica , most prominently Enniatin B (ENN B). This study aimed to isolate bacteria capable of combating various phytopathogenic fungi and degrading ENN B to reduce Angelica root rot. Through co-culture with F. avenaceum , the bacterial strains YF and SY89 were isolated from Angelica rhizosphere soil for their antifungal activities. They were identified as Paenibacillus polymyxa and Bacillus tequilensis based on their morphological characteristics and phylogenetic trees constructed using the 16 S rDNA genes sequence. The strains YF and SY89 could produce antimicrobial substance such as surfactin, fengycin, iturin, polyketide synthases and non ribosomal polypeptide synthase detected by Polymerase Chain Reaction (PCR). In addition, strain P. polymyxa YF and B. tequilensis SY89 showed a prominent ability to inhibit synthesis and degrade ENN B in F. avenaceum suspensions and standard samples. The inhibition rate reached 61.93% and 77.64%, respectively, and the degradation rate reached 60.32% and 76.03%, respectively. Angelica pot experiments were conducted to further evaluate the strains YF and SY89 culture ability to promote plant growth and control Angelica root rot to assess its potential agricultural use. The results indicated that strains YF and SY89 could produce IAA and siderophores, which significantly promoted Angelica roots growth. In addition, strains YF and SY89 have the potential to increase the activity of resistant enzymes, thereby inhibiting F. avenaceum infection. The disease index of Angelica roots treated by strain YF and SY89 decreased by 65.38% and 61.54%, respectively. To further elucidate the antagonistic mechanism of YF and SY89, we examined their colonization pattern in the Angelica root using a green fluorescent protein (GFP) marker. The results indicated that YF and SY89 mainly colonized the root surface before migrating into the roots interior part. The present study demonstrated that P. polymyxa YF and B. tequilensis SY89 showed promising prospects for use as a biological control agent against Angelica root rot and ENN B inhibition under field conditions. [ABSTRACT FROM AUTHOR]

Reasons for mammalian cell toxicity observed in barley and spring wheat grains were sought. Streptomyces sp. isolates from wheat and barley produced heat-stable methanol-soluble substances which inhibited the motility of exposed porcine spermatozoa used as a toxicity indicator. Several barley isolates produced antimycin A (2 to 5 ng/mg wet wt of biomass), a macrolide antibiotic known to block oxygen utilization in mitochondria. The antimycin-producing isolates were members of the Streptomyces albidoflavus group. In in vitro assays with porcine kidney tubular epithelial cells, the specific toxicity of antimycin A towards mitochondria was higher than that of the mycotoxin enniatin B but lower than that of the mitochondriotoxins cereulide and paenilide, produced by food-related Bacillus cereus and Paenibacillus tundrae , respectively. The toxic wheat isolates, related to Streptomyces sedi , did not produce antimycin A and or any other known toxin. Our results suggest that the presence of toxin-producing streptomycetes in stored cereal grains may pose a thus far unrecognized threat for food and feed safety. [ABSTRACT FROM AUTHOR]

From extraction experiments and γ -activity measurements, the extraction constant corresponding to the equilibrium NH 4 + (aq) + 1 ·Na + (nb) ⇆ 1 ·NH 4 + (nb) + Na + (aq) taking place in the two-phase water–nitrobenzene system ( 1 = enniatin B; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K ex (NH 4 + , 1 ·Na + ) = 1.9 ± 0.1. Further, the stability constant of the 1 ·NH 4 + complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β nb ( 1 ·NH 4 + ) = 6.4 ± 0.2. Finally, applying quantum mechanical DFT calculations, the most probable structure of the cationic complex species 1 ·NH 4 + was derived. In the resulting 1 ·NH 4 + complex, the “central” cation NH 4 + is bound by three relatively strong hydrogen bonds to the corresponding three carbonyl oxygens of the parent enniatin B ligand. The interaction energy, E (int), of the considered complex 1 ·NH 4 + was found to be − 305.5 kJ/mol, confirming also the formation of this investigated complex. [ABSTRACT FROM AUTHOR]

By using extraction experiments and γ -activity measurements, the extraction constant corresponding to the equilibrium Cs + (aq) + A − (aq) + 1 (nb) ⇆ 1 ⋅Cs + (nb) + A − (nb) taking place in the two-phase water–nitrobenzene system (A − = picrate, 1 = enniatin B; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K ex ( 1⋅ Cs + , A − ) = 2.3 ± 0.1. Further, the stability constant of the 1⋅ Cs + complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β nb ( 1⋅ Cs + ) = 4.2 ± 0.1. Finally, applying quantum mechanical DFT calculations, the most probable structure of the cationic complex species 1⋅ Cs + was derived. In the resulting 1⋅ Cs + complex, which is most energetically favored, the “central” cation Cs + is bound by nine bonding interactions to the corresponding nine oxygen atoms of the parent enniatin B ligand. The interaction energy of the considered complex 1⋅ Cs + was found to be −228.3 kJ/mol, confirming the formation of this investigated complex as well. [ABSTRACT FROM AUTHOR]

With aim to provide information on chemical contaminants in byproducts in animal feed, the data from an official control by the Danish Plant Directorate during 1998–2009, were reviewed and several samples of citrus pulp and dried distillers grains with solubles (DDGS) were additionally collected for analysis and risk assessment. The levels of contaminants in the samples from the official control were below maximum limits from EU regulations with only a few exceptions in the following groups; dioxins and dioxin-like polychlorobiphenyls (PCBs) in fish-containing byproducts and dioxins in vegetable and animal fat, hydrogen cyanide in linseed, and cadmium in sunflowers. The levels of pesticides and mycotoxins in the additionally collected samples were below maximum limits. Enniatin B (ENN B) was present in all DDGS samples. The hypothetical cases of carry-over of contamination from these byproducts were designed assuming total absorption and accumulation of the ingested contaminant in meat and milk and high exposure (a byproduct formed 15–20% of the feed ration depending on the species). The risk assessment was refined based on literature data on metabolism in relevant animal species. Risk assessment of contaminants in byproducts is generally based on a worst-case approach, as data on carry-over of a contaminant are sparse. This may lead to erroneous estimation of health hazards. The presence of ENN B in all samples of DDGS indicates that potential impact of this emerging mycotoxin on feed and food safety deserves attention. A challenge for the future is to fill up gaps in toxicological databases and improve models for carry-over of contaminants. [ABSTRACT FROM AUTHOR]

By using extraction experiments and γ-activity measurements, the extraction constant corresponding to the equilibrium Sr2+(aq) + 2A-(aq) + 1(nb) = 1.Sr2+(nb) + 2A-(nb) occurring in the two-phase water-nitrobenzene system (A- = picrate, 1 = enniatin B; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K ex (1·Sr2+, 2A-) = 3.4 ± 0.1. Further, the stability constant of the 1·Sr2+ complex in nitrobenzene saturated with water was calculated for a temperature of 25°C: logβnb (1.Sr2+) = 12.5 ± 0.1. Finally, applying quantum mechanical DFT calculations, the most probable structure of the proved 1.Sr2+ cationic complex was derived. In the resulting complex, which is most energetically favored, the "central" cation Sr2+ is bound by nine bonding interactions to the corresponding nine oxygen atoms of the parent enniatin B ligand. The interaction energy of the considered 1.Sr2+ complex was found to be -877.4 kJ/mol, confirming also the formation of this complex. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to assess the in vitro effects of emerging mycotoxins beauvericin, enniatin B and moniliformin on human dendritic cells and macrophages. Beauvericin and enniatin B were cytotoxic on these cells. IC50 were equal to 1.0 μM, 2.9 μM and 2.5 μM beauvericin for immature dendritic cells, mature dendritic cells and macrophages, respectively. IC50 were equal to 1.6 μM, 2.6 μM and 2.5 μM for immature dendritic cells, mature dendritic cells and macrophages exposed to enniatin B, respectively. Effects on the differentiation process of monocytes into macrophages or into immature dendritic cells as well as effects on dendritic cells maturation have been studied. The differentiation process of monocytes into immature dendritic cells was not disturbed in the presence of beauvericin. Dendritic cells exposed to beauvericin during the maturation process presented a decrease of CCR7 expression and an increase of IL-10 secretion. Monocytes exposed to beauvericin during the differentiation process into macrophages presented a decrease of endocytosis ability. The differentiation process of monocytes into immature dendritic cells was not disturbed in the presence of enniatin B. Dendritic cells exposed to enniatin B during the maturation process presented a decrease of expression of the maturation makers CD80, CD86 and CCR7 and an increase of IL-10 secretion. Monocytes exposed to enniatin B during the differentiation process into macrophages presented a decrease of endocytosis ability and an increase of CD71. CD1a expression and endocytosis capacity were decreased on immature dendritic cells exposed to moniliformin. Monocytes-derived macrophages exposed to moniliformin during the differentiation process presented a decrease of endocytosis ability, and a decrease of CD71 and HLA-DR expression. According to these results, immunological disorders could be observed on human after ingestion of these alimentary toxins. [Copyright &y& Elsevier]

Abstract: The aim of this study was to screen potential myelotoxicity of the emerging mycotoxins Beauvericin, Enniatin b and Moniliformin using human hematopoietic progenitor clonogenic assays. Depending on mycotoxins, inhibitory effects on proliferation of white blood cells progenitors (CFU-GM), platelet progenitors (CFU-MK) and red blood cells progenitors (BFU-E) have been detected at various concentrations. Beauvericin was cytotoxic at 32μM, 3.2μM and 6.4μM, had no effect on proliferation in the presence of 0.032μM, 0.16μM and 0.064μM, and the IC50 was equal to 3.4μM, 0.7μM and 3.7μM for CFU-GM, CFU-MK and BFU-E, respectively. Enniatin b was cytotoxic at 6μM, 1.8μM and 5μM, had no effect on proliferation in the presence of 1μM, 1.1μM and 1.2μM and the IC50 was equal to 4.4μM, 1.3μM and 3.3μM for CFU-GM, CFU-MK and BFU-E, respectively. Moniliformin was not cytotoxic at tested concentrations for CFU-GM and CFU-MK and cytotoxic at 10μM for BFU-E, had no effect on proliferation in the presence of 5μM, 0.1μM and 0.1μM and the IC50 was equal to 31μM, 39μM and 4.1μM for CFU-GM, CFU-MK and BFU-E, respectively. Inhibition of the BFU-E differentiation has been observed in the presence of Enniatin b or Moniliformin. For the three mycotoxins, variation of distribution of CFU-MK colonies according to their size has been observed. These in vitro effects may be responsible for in vivo hematological troubles in case of consumption of contaminated commodities. In vivo studies have to be performed to test this hypothesis. [Copyright &y& Elsevier]

The enniatins (ENs) are bioactive compounds of hexadepsipeptidic structure produced by several strains of Fusarium sp. The EN B was purified from extracts of Fusarium tricinctum growth on liquid culture of potato dextrose broth (PDB), using a semipreparative liquid chromatography (LC) followed by an analytical LC. The purity and the structure of the isolated compound were confirmed by the determination of the extinction coefficient and with electrospray ionization--mass spectrometry (ESI-MS) study. The pure fraction of EN B was utilized to determine the antibiotic effects on several bacterial strains that are considered normally pathogens of the intestinal tract: Escherichia coli, Enterococcus faecium, Salmonella enterica, Shigella dysenteriae, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens, Pseudomonas aeruginosa, and Staphylococcus aureus, and to study the cytotoxic effects on Caco-2 differentiated and undifferentiated cells. The results obtained demonstrated that in several antibiograms, EN B induced the inhibition of the grown microorganisms tested and no significant differences over control were detected when Caco-2 cells were exposed to EN B, at any of the concentrations used. [ABSTRACT FROM AUTHOR]

Abstract: A pathogenic fungus, Fusarium sporotrichioides Sherb., was isolated from Hypericum barbatum Jacq. The volatile compounds of broth and mycelium were analyzed using GC–MS and three cyclodidepsipeptides (dioxomorpholines), 3,6-di(propan-2-yl)-4-methyl-morpholine-2,5-dione, 3-(2-methylpropyl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione and 3-(butan-2-yl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione, were found for the first time in the natural products. The structures of the compounds were confirmed by comparison of the analytical data for the natural products with samples obtained via synthetic methods. The conformational features and vibrational spectra of the three cyclodidepsipeptides were characterized by density functional theory (DFT) calculations and IR spectroscopy. The cyclic hexadepsipeptide enniatin B was identified by a LC–MS/MS analysis of the non-volatile products of broth and mycelium. The above-mentioned three cyclodidepsipeptides are probably synthesized using similar biosynthetic ways to enniatin B involving a nonribosomal mechanism. [Copyright &y& Elsevier]

Beauvericin (BEA) and enniatin B (ENNB) are emerging mycotoxins frequently detected in plant-based fish feed. With ionophoric properties, they have shown cytotoxic potential in mammalian models. Sensitivity in fish is still largely unknown. Primary hepatocytes isolated from Atlantic salmon (Salmo salar) were used as a model and exposed to BEA and ENNB (0.05–10 μM) for 48 h. Microscopy, evaluation of cell viability, total ATP, total H 2 O 2 , total iron content, total Gpx enzyme activity, and RNA sequencing were used to characterize the toxicodynamics of BEA and ENNB. Both mycotoxins became cytotoxic at ≥ 5 μM, causing condensation of the hepatocytes followed by formation of blister-like protrusions on the cell's membrane. RNA sequencing analysis at sub-cytotoxic levels indicated BEA and ENNB exposed hepatocytes to experience increased energy expenditure, elevated oxidative stress, and iron homeostasis disturbances sensitizing the hepatocytes to ferroptosis. The present study provides valuable knowledge disclosing the toxic action of these mycotoxins in Atlantic salmon primary hepatocytes. • BEA and ENNB-induced impairment of mitochondria, culminating in reduced ATP levels. • BEA and ENNB-induced impairment of lysosomes. • A dose-dependent increase in the GSH-dependent Gpx enzyme activity was observed following BEA and ENNB exposure. • Cytotoxic levels of BEA significantly increased the intracellular iron content. [ABSTRACT FROM AUTHOR]

The Fusarium metabolite enniatin B is now recognized as a frequent contaminant of grains used for human foods and animal feeds. Yet, so far very limited data are available on its toxicity and that of other emerging Fusarium mycotoxins (Jestoi M, 2008, Crit Rev Food Sci Nutr 48:21-49). Thus, the mutagenic/genotoxic potential of enniatin B was investigated in a battery of short-term tests, and its cytotoxicity compared with that of several other mycotoxins. No mutagenicity was detected in the Ames assay with four Salmonella typhimurium strains, and in the HPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 cells, in either the presence or absence of an external metabolizing enzyme system (rat liver S9). For other types of genotoxicity, i.e., clastogenicity and chromosomal damage, studied in V79 cells by means of alkaline single-cell gel electrophoresis (Comet) assay and micronucleus assay, no significant genotoxic potential of enniatin B was revealed. However, the Fusarium metabolite exerts pronounced time- and concentration-dependent cytotoxic effects in V79 cells as determined by Alamar Blue reduction and by neutral red uptake assays. For instance, IC20 and IC50 values determined for enniatin B by neutral red assay for 48-h exposure are 1.5 μM and 4 μM. These values are higher than those of the more potent Fusarium toxin deoxynivalenol (IC20 0.7 μM, IC50 of 0.8 μM), but clearly lower than the IC values of several other mycotoxins tested in parallel. Their ranking of cytotoxicity in V79 cells was as follows: deoxynivalenol > enniatin B > patulin > ochratoxin A > zearalenone > citrinin. Moreover, enniatin B was found to induce nuclear fragmentation, a sign of apoptosis, already at low submicromolar concentrations. In summary, despite an apparent lack of mutagenic and genotoxic activity, enniatin B can cause pronounced cytotoxicity in mammalian cells, detectable at low micromolar concentrations. [ABSTRACT FROM AUTHOR]

The known metabolite, enniatin B, and a cyclic tetrapeptide, JM47, which is a new natural product, were extracted from brown rice cultures of a marine fungus, identified as a Fusarium species, isolated from the marine alga Codium fragile. NMR studies, including 15N HMQC and 15N HMBC, established the structure of JM47 as cyclo(Ala-Ala-Aoh-Pro), where Aoh is the amino acid, (2S,9S)-2-amino-8-oxo-9-hydroxydecanoic acid. The absolute stereochemistry of the Aoh side chain carbinol centre was determined using Mosher ester methodology. Analysis of NOESY data assisted by molecular modelling revealed an alternating l-, d-, l-, d-configuration for the tetrapeptide core. The absolute stereochemistry of the core was determined by acidic hydrolysis and chiral TLC analysis of the proline residue. JM47 belongs to the HC-toxin family of cyclic tetrapeptides which possess a 2-amino-8-oxo-9,10-epoxydecanoic acid residue in place of the Aoh unit. This is the first report of an analogue of HC-toxin from a marine Fusarium species. [Copyright &y& Elsevier]