In vitro assessment of emerging mycotoxins co-occurring in cheese: a potential health hazard.

Some Penicillium strains used in cheese ripening produce emerging mycotoxins, notably roquefortine C (ROQC) and cyclopiazonic acid (CPA), as well as enniatins (ENNs) and beauvericin (BEA). Co-occurrence of these mycotoxins in natural samples has been reported worldwide, however, most studies focus on the toxicity of a single mycotoxin. In the present study, the effects of ROQC and CPA alone and in combination with BEA and ENNs A, A1, B, and B1 were analysed in human neuroblastoma cells. ROQC and CPA reduced cell viability, with IC50 values of 49.5 and 7.3 µM, respectively, and induced caspase-8-mediated apoptosis. When ROQC and CPA were binary combined with ENNs, an enhancement of their individual effects was observed. Furthermore, a clear synergism was produced when ROQC and CPA were mixed with the four ENNs. An additive effect was also described for the combination of CPA + ENNs (A, A1, B, B1) + BEA. Finally, the effects of commercial cheese extracts containing the mentioned mycotoxins were evaluated, finding a strong reduction in cell viability. These results suggest that the co-occurrence of emerging mycotoxins in natural matrices could pose a potential health risk.

Unexpected antagonism of deoxynivalenol and enniatins in intestinal toxicity through the Ras/PI3K/AKT signaling pathway.

Deoxynivalenol (DON) is a kind of widespread traditional Fusarium mycotoxins in the environment, and its intestinal toxicity has received considerable attention. Recently, the emerging Fusarium mycotoxin enniatins (ENNs) have also been shown to frequently coexist with DON in animal feed and food with large consumption. However, the mechanism of intestinal damage caused by the two mycotoxins co-exposure remains unclear. In this study, Caco-2 cell line was used to investigate the combined toxicity and potential mechanisms of four representative ENNs (ENA, ENA1, ENB, and ENB1) and DON. The results showed that almost all mixed groups showed antagonistic effects, particularly ENB at 1/4 IC50 (CI = 6.488). Co-incubation of ENNs mitigated the levels of signaling molecule levels disrupted by DON, including reactive oxygen species (ROS), calcium mobilization (Ca2+), adenosine triphosphate (ATP). The differentially expressed genes (DEGs) between the mixed and ENB groups were significantly enriched in the Ras/PI3K/Akt signaling pathway, including 28 up-regulated genes and 40 down-regulated genes. Quantitative real-time PCR further confirmed the lower expression of apoptotic gene in the mixed group, thereby reducing the cytotoxic effects caused by DON exposure. This study emphasizes that co-exposure of ENNs and DON reduces cytotoxicity by regulating the Ras/PI3K/Akt signaling pathway. Our results provide the first comprehensive evidence about the antagonistic toxicity of ENNs and DON on Caco-2 cells, and new insights into mechanisms investigated by transcriptomics.

Impact of enniatins and beauvericin on lipid metabolism: Insights from a 3D HepaRG spheroid model.

Emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) pose potential health risks to humans through dietary exposure. However, research into their mechanisms of toxicity is limited, with a lack of comprehensive toxicological data. This study investigates from a hepatic lipid metabolism perspective, establishing a more precise and reliable 3D HepaRG hepatocyte spheroid model as an alternative for toxicity assessment. Utilizing physiological indices, histopathological analyses, lipidomics, and molecular docking techniques, it comprehensively elucidates the effects of ENNs and BEA on hepatic lipid homeostasis and their molecular toxicological mechanisms. Our findings indicate that ENNs and BEA impact cellular viability and biochemical functions, significantly altering lipid metabolism pathways, particularly those involving glycerophospholipids and sphingolipids. Molecular docking has demonstrated strong binding affinity of ENNs and BEA with key enzymes in lipid metabolism such as Peroxisome Proliferator-Activated Receptor α (PPARα) and Cytosolic Phospholipase A2 (cPLA2), revealing the mechanistic basis for their hepatotoxic effects and potential to impair liver function and human health. These insights enhance our understanding of the potential hepatotoxicity of such fungal toxins and lay a foundation for the assessment of their health risks.

Incidence of fungal contamination in fresh ginseng samples and mycotoxigenic potential of representative fungal isolates.

BACKGROUND: Fresh ginseng is typically accompanied by soil after harvest, leading to contamination with harmful fungi during storage and distribution. In this study, we investigated the incidence of fungal contamination in fresh ginseng (5-6 years old) purchased from 22 different stores in Geumsan, Korea. RESULTS: The incidence of fungal contamination in the samples was 67.4-111.5%. Fusarium solani was the most abundant species in the head (38.5%) and fine root (19.3%) parts of the ginseng samples, whereas F. oxysporum was the most abundant in the main root (22.0%) part. We isolated Aspergillus, Fusarium and Penicillium spp. (total number of isolates: 395) from the ginseng samples, and 138 isolates were identified using phylogenetic analysis. Polymerase chain reaction-based screening of 65 mycotoxin-producing species revealed that two P. expansum isolates were positive for citrinin and/or patulin, and five F. oxysporum isolates were positive for fumonisin biosynthesis gene. One P. expansum isolate produced 738.0 mg kg-1 patulin, and the other produced 10.4 mg kg-1 citrinin and 12.0 mg kg-1 patulin on potato dextrose agar (PDA) medium. Among the 47 representative F. oxysporum isolates, 43 (91.5%) produced beauvericin (0.1-15.4 mg kg-1) and four of them (8.5%) produced enniatin B and enniatin B1 (0.1-1.8 mg kg-1) as well. However, none of these toxins was detected in fresh ginseng samples. CONCLUSION: Fusarium solani and F. oxysporum were the most abundant species in fresh ginseng samples. Most F. oxysporum (43) and P. expansum (2) strains isolated from fresh ginseng produced beauvericin and enniatins (B and B1), and patulin or citrinin, respectively, on PDA medium. This is the first report of the mycotoxigenic potential of P. expansum and F. oxysporum strains isolated from fresh ginseng. © 2024 Society of Chemical Industry.

Emerging mycotoxin occurrence in chicken feed and eggs from Algeria.

Poultry farming has developed into one of Algeria's most productive industrial farming because of the growing demand for sources of protein among Algerian society. Laying hen feed consists mainly of cereals, which can be contaminated with molds and subsequently with their secondary metabolites known as mycotoxins. These later can pose a serious danger to the production and quality of eggs in the commercial layer industry. This work focuses on the detection of emerging mycotoxins, mainly enniatins (ENNs) and beauvericin (BEA), in poultry feed and eggs from different locations in Algeria. Two different QuEChERS-based extractions were established to extract ENNs and BEA from chicken feed and eggs. The determination of mycotoxin occurrence was achieved by a UHPLC-MS/MS method using 0.1% (v/v) formic acid in water and MeOH as mobile phase, an ESI interface operating in positive mode, and a triple quadrupole mass spectrometer operating in MRM for the detection. Matrix-matched calibration curves were carried out for both matrices, obtaining good linearity (R2 > 0.99). The method performance was assessed in terms of extraction recovery (from 87 to 107%), matrix effect (from - 47 to - 86%), precision (RSD < 15%), and limits of quantitation (≤ 1.1 µg/kg for feed and ≤ 0.8 µg/kg for eggs). The analysis of 10 chicken feed samples and 35 egg samples composed of a 10-egg pool each showed that ENN B1 was the most common mycotoxin (i.e., found in 9 feed samples) with contamination levels ranging from 3.6 to 41.5 µg/kg, while BEA was detected only in one feed sample (12 µg/kg). However, eggs were not found to be contaminated with any mycotoxin at the detection limit levels. Our findings indicate that the searched mycotoxins are present in traces in feed and absent in eggs. This can be explained by the application of a mycotoxin binder. However, this does not put a stop on the conduction of additional research and ultimately setting regulations to prevent the occurrence of emerging mycotoxins.

PI3K/Akt/FoxO Pathway Mediates Antagonistic Toxicity in HepG2 Cells Coexposed to Deoxynivalenol and Enniatins.

The emerging mycotoxins enniatins (ENNs) and the traditional mycotoxin deoxynivalenol (DON) often co-contaminate various grain raw materials and foods. While the liver is their common target organ, the mechanism of their combined effect remains unclear. In this study, the combined cytotoxic effects of four ENNs (ENA, ENA1, ENB, and ENB1) with DON and their mechanisms were investigated using the HepG2 cell line. Additionally, a population exposure risk assessment of these mycotoxins was performed by using in vitro experiments and computer simulations. The results showed that only ENA at 1/4 IC50 and ENB1 at 1/8 IC50 coexposed with DON showed an additive effect, while ENB showed the strongest antagonism at IC50 (CI = 3.890). Co-incubation of ENNs regulated the signaling molecule levels which were disrupted by DON. Transcriptome analysis showed that ENB (IC50) up-regulated the PI3K/Akt/FoxO signaling pathway and inhibited the expression of apoptotic genes (Bax, P53, Caspase 3, etc.) via phosphorylation of FoxO, thereby reducing the cytotoxic effects caused by DON. Both types of mycotoxins posed serious health risks, and the cumulative risk of coexposure was particularly important for emerging mycotoxins.

Studies of Mycotoxins in Medicinal Plants Conducted Worldwide over the Last Decade: A Systematic Review, Meta-Analysis, and Exposure Risk Assessment.

BACKGROUND: Mycotoxins have been reported to be present in medicinal plants. With the growing usage of medicinal plants, contamination of mycotoxins has emerged as one of the biggest threats to global food hygiene and ecological environment, posing a severe threat to human health. PURPOSE: This study aimed to determine the mycotoxin prevalence and levels in medicinal plants and conduct a risk assessment by conducting a systematic review and meta-analysis. METHODS: A thorough search on Web of Science and PubMed was conducted for the last decade, resulting in 54 studies (meeting the inclusion criteria) with 2829 data items that were included in the meta-analysis. RESULTS: The combined prevalence of mycotoxins in medicinal plants was 1.7% (95% confidence interval, CI = 1.1% - 2.4%), with a mean mycotoxin concentration in medicinal plants of 3.551 µg/kg (95% CI = 3.461 - 3.641 µg/kg). Risk assessment results indicated that aflatoxins and ochratoxin A found in several medicinal plants posed a health risk to humans; additionally, emerging enniatins exhibited possible health risks. CONCLUSION: Therefore, the study underlines the need for establishing stringent control measures to reduce the severity of mycotoxin contamination in medicinal plants.

Cytotoxic Effects of Major and Emerging Mycotoxins on HepaRG Cells and Transcriptomic Response after Exposure of Spheroids to Enniatins B and B1.

Mycotoxins, produced by fungi, frequently occur at different stages in the food supply chain between pre- and postharvest. Globally produced cereal crops are known to be highly susceptible to contamination, thus constituting a major public health concern. Among the encountered mycotoxigenic fungi in cereals, Fusarium spp. are the most frequent and produce both regulated (i.e., T-2 toxin, deoxynivalenol -DON-, zearalenone -ZEA-) and emerging (i.e., enniatins -ENNs-, beauvericin -BEA-) mycotoxins. In this study, we investigated the in vitro cytotoxic effects of regulated and emerging fusariotoxins on HepaRG cells in 2D and 3D models using undifferentiated and differentiated cells. We also studied the impact of ENN B1 and ENN B exposure on gene expression of HepaRG spheroids. Gene expression profiling pinpointed the differentially expressed genes (DEGs) and overall similar pathways were involved in responses to mycotoxin exposure. Complement cascades, metabolism, steroid hormones, bile secretion, and cholesterol pathways were all negatively impacted by both ENNs. For cholesterol biosynthesis, 23/27 genes were significantly down-regulated and could be correlated to a 30% reduction in cholesterol levels. Our results show the impact of ENNs on the cholesterol biosynthesis pathway for the first time. This finding suggests a potential negative effect on human health due to the essential role this pathway plays.

Evaluation of distribution of emerging mycotoxins in human tissues: applications of dispersive liquid-liquid microextraction and liquid chromatography-mass spectrometry.

In this work, a complete study of the distribution of emerging mycotoxins in the human body has been carried out. Specifically, the presence of enniatins (A, A1, B, B1) and beauvericin has been monitored in brain, lung, kidney, fat, liver, and heart samples. A unique methodology based on solid-liquid extraction (SLE) followed by dispersive liquid-liquid microextraction (DLLME) was proposed for the six different matrices. Mycotoxin isolation was performed by adding ultrapure water, acetonitrile, and sodium chloride to the tissue sample for SLE, while the DLLME step was performed using chloroform as extraction solvent. Subsequently, the analysis was carried out by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The proposed method allowed limits of quantification (LOQs) to be obtained in a range of 0.001-0.150 ng g-1, depending on the tissue and mycotoxin. The precision was investigated intraday and interday, not exceeding of 9.8% of relative standard deviation. In addition, trueness studies achieved 75 to 115% at a mycotoxin concentration of 25 ng g-1 and from 82 to 118% at 5 ng g-1. The application of this methodology to 26 forensic autopsies demonstrated the bioaccumulation of emerging mycotoxins in the human body since all mycotoxins were detected in tissues. Enniatin B (ENNB) showed a high occurrence, being detected in 100% of liver (7 ± 13 ng g-1) and fat samples (0.2 ± 0.8 ng g-1). The lung had a high incidence of all emerging mycotoxins at low concentrations, while ENNB, ENNB1, and ENNA1 were not quantifiable in heart samples. Co-occurrence of mycotoxins was also investigated, and statistical tests were applied to evaluate the distribution of these mycotoxins in the human body.

Survey of the enniatins and beauvericin in raw and UHT cow's milk in Poland.

Introduction: The enniatins A, A1, B and B1 (ENNs) and beauvericin (BEA) are structurally related compounds produced by Fusarium species. They occur as contaminants in cereals, such as wheat, barley and maize. They are called "emerging mycotoxins", because they have been reported in feed and food and their toxic effects are not fully known. Data on their levels in food (especially in milk) are limited. The study aimed to evaluate the occurrence of ENNs and BEA in milk. Material and Methods: A total of 103 bovine milk samples (76 of raw milk and 27 of UHT milk) were collected from different parts of Poland and analysed using liquid chromatography-tandem mass spectrometry. Results: Among the 76 raw milk samples, 31 (41%) and 15 (20%) samples were contaminated with ENN B and with BEA, respectively. No contamination with other enniatins was found. The highest concentration of BEA was found in raw milk and was 6.17 µg kg-1. Out of the 27 samples of UHT milk, 16 (59%) were contaminated with ENN B at concentrations ranging from 0.157 µg kg-1 to 0.587 µg kg-1 (limit of quantification (LOQ) 0.098 µg kg-1). Beauvericin was detected in 9 UHT milk samples (33%) at concentrations ranging from 0.101 µg kg-1 to 1.934 µg kg-1 (LOQ 0.095 µg kg-1). Conclusion: This study demonstrated constant but low milk contamination in Poland with ENN B and BEA. The analysis of milk samples revealed that the emerging mycotoxins ENN B and BEA were measured in trace amounts. It does not suggest any immediate risk to milk consumers; however, it is unknown whether long-term exposure to low levels of toxins may be harmful.

Antibacterial Metabolites from Kiwi Endophytic Fungus Fusarium tricinctum, a Potential Biocontrol Strain for Kiwi Canker Disease.

Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium causing the kiwifruit canker disease, resulting in serious economic losses to the kiwifruit industry. This study investigated the use of an endophytic fungus, Fusarium tricinctum, obtained from the kiwi plant (Actinidia chinesis) as a potential biocontrol strain against the Psa. F. tricinctum showed an inhibition rate of 59.5% in vitro against Psa. Bioassay-guided isolation was conducted on the cultural broth of F. tricinctum and seven new imidazole alkaloids, (±)-fusaritricine J ((±)-1) and fusaritricines K-P (2-7), and four enniatins (8-11) were identified. Their absolute configurations were established through extensive spectroscopic methods, quantum chemical calculations, and X-ray single crystal diffraction. Compounds 1, 4, 5, and 8-11 showed comparable anti-bacterial activities against Psa as positive control, with MIC values of 25-50 µg/mL. Further cell membrane permeability assay suggested that the most active compound 4 could destroy the bacterial cell wall structure. Hence, F. tricinctum metabolites could be applied as potential anti-Psa agents, and F. tricinctum could be considered a biocontrol strain for the control of the kiwifruit canker disease.

Impact of Combined Processes Involving Ultrasound and Pulsed Electric Fields on ENNs, and OTA Mitigation of an Orange Juice-Milk Based Beverage.

In recent years, several innovative food processing technologies such as ultrasound (USN) and pulsed electric fields (PEF) have emerged in the market, showing a great potential both alone and in combination for the preservation of fresh and processed products. Recently, these technologies have also shown promising applications to reduce mycotoxin levels in food products. Therefore, the objective of this study is to investigate the potential of the combined treatments USN + PEF and PEF + USN on the reduction of Ochratoxin A (OTA) and Enniatins (ENNs) of an orange juice mixed with milk beverage. For this purpose, the beverages were elaborated in the laboratory and individually spiked with mycotoxins at a concentration of 100 µg/L. They were then treated by PEF (30 kV, 500 kJ/Kg) and USN (20 kHz, 100 W, at a maximum power for 30 min). Finally, mycotoxins were extracted using dispersive liquid-liquid microextraction (DLLME), and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS-IT) was employed to determine them. The results showed promising applications, with reductions up to 50% for OTA and up to 47% for Enniatin B (ENNB) after the PEF + USN treatment combination. Lower reduction rates, up to 37%, were obtained with the USN + PEF combination. In conclusion, the combination of USN and PEF technologies could be a useful tool to reduce mycotoxins in fruit juices mixed with milk.

Encapsulation of Ammoides pusila Essential Oil into Mesoporous Silica Particles for the Enhancement of Their Activity against Fusarium avenaceum and Its Enniatins Production.

Essential oils (EOs) that have antifungal activity and mycotoxin reduction ability are candidates to develop bioactive alternatives and environmentally friendly treatment against Fusarium species in cereals. However, their practical use is facing limitations such as high volatility, UV sensitivity, and fast oxidation. Encapsulation techniques are supposed to provide protection to the EOs and control their release into the environment. Ammoides pusilla essential oil (AP-EO) proved to be an efficient inhibitor of Fusarium avenaceum growth and its enniatins (ENNs) production. In the present work, AP-EO was encapsulated, using the impregnation method, into mesoporous silica particles (MSPs) with narrow slit pores (average diameter = 3.1 nm) and coated with chitosan. In contact assays using an agar medium, the antifungal activity of AP-EO at 0.1 µL mL-1 improved by three times when encapsulated into MSPs without chitosan and the ENNs production was significantly inhibited both in coated and non-coated MSPs. Controls of MSPs also inhibited the ENNs production without affecting the mycelial growth. In fumigation experiments assessing the activity of the EO volatile compounds, encapsulation into MSPs improved significantly both the antifungal activity and ENNs inhibition. Moreover, coating with chitosan stopped the release of EO. Thus, encapsulation of an EO into MSPs improving its antifungal and antimycotoxin properties is a promising tool for the formulation of a natural fungicide that could be used in the agriculture or food industry to protect plant or food products from the contamination by toxigenic fungi such as Fusarium sp. and their potential mycotoxins.

Aggressiveness and mycotoxin profile of Fusarium avenaceum isolates causing Fusarium seedling blight and Fusarium head blight in UK malting barley.

Introduction: Fusarium avenaceum causing Fusarium seedling blight (FSB) and Fusarium head blight (FHB) on barley is associated with economic losses of crop yield and quality, and the accumulation of mycotoxins including the enniatins (ENNs) A, A1, B and B1. Although F. avenaceum is the main producer of ENNs, studies on the ability of isolates to cause severe Fusarium diseases or produce mycotoxins in barley are limited. Methods: In this work, we investigated the aggressiveness of nine isolates of F. avenaceum to two cultivars of malting barley, Moonshine and Quench, and defined their ENN mycotoxin profiles in in vitro and in planta experiments. We assessed and compared the severity of FSB and FHB caused by these isolates to disease severity by F. graminearum, F. tricinctum and F. poae. Quantitative real-time polymerase chain reaction and Liquid Chromatography Tandem Mass Spectrometry assays were used to quantify pathogen DNA and mycotoxin accumulation, respectively, in barley heads. Results: Isolates of F. avenaceum were equally aggressive to barley stems and heads and caused the most severe FSB symptoms resulting in up to 55% reductions of stem and root length. Fusarium graminearum caused the most severe FHB disease, followed by the isolates of F. avenaceum with the most aggressive F. avenaceum isolates capable of causing similar bleaching of barley heads as F. avenaceum. Fusarium avenaceum isolates produced ENN B as the predominant mycotoxin, followed by ENN B1 and A1 in vitro. However, only the most aggressive isolates produced ENN A1 in planta and none produced ENN A or beauvericin (BEA) either in planta or in vitro. Discussion: The capacity of F. avenaceum isolates to produce ENNs was related to the accumulation of pathogen DNA in barley heads, whilst FHB severity was related to the synthesis and accumulation of ENN A1 in planta. Cv. Moonshine was significantly more resistant than Quench to FSB or FHB, caused by any Fusarium isolate, and to the accumulation of pathogen DNA, ENNs or BEA. In conclusion, aggressive F. avenaceum isolates are potent ENN producers causing severe FSB and FHB with ENN A1 requiring further investigation as potential virulence factor for F. avenaceum in cereals.

Enniatins from a marine-derived fungus Fusarium sp. inhibit biofilm formation by the pathogenic fungus Candida albicans.

Candidemia is a life-threatening disease common in immunocompromised patients, and is generally caused by the pathogenic fungus Candida albicans. C. albicans can change morphology from yeast to hyphae, forming biofilms on medical devices. Biofilm formation contributes to the virulence and drug tolerance of C. albicans, and thus compounds that suppress this morphological change and biofilm formation are effective for treating and preventing candidemia. Marine organisms produce biologically active and structurally diverse secondary metabolites that are promising lead compounds for treating numerous diseases. In this study, we explored marine-derived fungus metabolites that can inhibit morphological change and biofilm formation by C. albicans. Enniatin B (1), B1 (2), A1 (3), D (4), and E (5), visoltricin (6), ergosterol peroxide (7), 9,11-dehydroergosterol peroxide (8), and 3ß,5α,9α-trihydroxyergosta-7,22-dien-6-one (9) were isolated from the marine-derived fungus Fusarium sp. Compounds 1-5 and 8 exhibited inhibitory activity against hyphal formation by C. albicans, and compounds 1-3 and 8 inhibited biofilm formation by C. albicans. Furthermore, compounds 1-3 decreased cell surface hydrophobicity and expression of the hypha-specific gene HWP1 in C. albicans. Compound 1 was obtained in the highest yield. An in vivo evaluation system using silkworms pierced with polyurethane fibers (a medical device substrate) showed that compound 1 inhibited biofilm formation by C. albicans in vivo. These results indicate that enniatins could be lead compounds for therapeutic agents for biofilm infections by C. albicans.

Non-aqueous capillary electrophoresis-time of flight mass spectrometry method to determine emerging mycotoxins.

Enniatins (ENN) and beauvericin (BEA) are emerging mycotoxins that have been traditionally determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). However, to the best of our knowledge, no analytical methods based on capillary electrophoresis (CE)-MS/MS have been reported so far. Due to their non-polar nature, in this work, a non-aqueous CE (NACE) method coupled to quadrupole time-of-flight-MS is proposed for the first time to identify and quantify these mycotoxins. Determination was achieved in 4 min under optimum conditions: 40 mM ammonium acetate in 80:20 (v/v) acetonitrile-methanol (buffer), 30 kV (voltage), 80 cm (capillary length), 20 °C (capillary temperature) and 50 mbar × 30 s (injection). Higher selectivity can be achieved when compared with LC due to the formation of exclusive CE adducts such as [M + CH3CH2NH3]+. "All Ions" acquisition mode was selected as it allows the quantification of the usual ENNs, as well as the identity confirmation of less common ENNs. The method was validated for wheat samples, obtaining limits of quantification from 4.0 to 8.3 µg/kg depending on the emerging mycotoxin, recovery values higher than 87.4%, and intra- and inter-day precision values (RSDs) lower than 15.1% in all cases. Finally, 29 wheat samples were analyzed, finding 26 samples with concentrations of enniatin B higher than the limit of quantification (7.5-1480 µg/kg), 20 for enniatin B1 (5.2-550 µg/kg), 7 for enniatin A (10-55 µg/kg), 4 for enniatin A1 (12.6-77 µg/kg) and 5 for BEA (9.2-16.4 µg/kg). Moreover, two other ENNs were tentatively identified.

Enniatins A1 and B1 alter calcium homeostasis of neuronal cells leading to apoptotic death.

Enniatins (ENNs) A1 and B1 are non-regulated mycotoxins produced by Fusarium spp. that commonly occur in different types of food. These toxins are cytotoxic in several cell lines, but their mechanism of action is unclear. In this study, the cytotoxic effects of ENNs A1 and B1 in SH-SY5Y human neuroblastoma cells were analysed. Moreover, to better understand their mechanism of action, mitochondrial function, reactive oxygen species (ROS) levels and calcium fluxes were monitored. ENNs A1 and B1 reduced cell viability, presenting IC50 values of 2.0 and 2.7 µM, respectively. Both toxins induced caspase-dependent apoptosis, but only ENN A1 increased ROS production. Apoptotic cell death seems to be triggered by the increase in cytosolic calcium produced by both ENNs, since the toxins altered Ca2+ homeostasis by depleting intracellular reservoirs. Finally, binary combinations of ENN A1, ENN B1, ENN A and ENN B were tested. All mixtures resulted in an antagonistic effect, with the exception of ENN A and ENN B1 combination, which produced an additive effect. The results presented in this study provide the first evidence of ENNs A1 and B1 effects on calcium fluxes, providing new insights into the mechanism of action of these mycotoxins.

Simultaneous Determination of 15 Mycotoxins in Aquaculture Feed by Liquid Chromatography-Tandem Mass Spectrometry.

The use of plant-based fish feed may increase the risk of contamination by mycotoxins. The multiresidue analysis of mycotoxins in fish feed presents many difficulties due to the complexity of the matrix, the different characteristics of the compounds, and their presence in highly different concentration levels. The aim of this study was to develop a selective, sensitive, and efficient analytical method for the simultaneous determination of 15 mycotoxins (regulated and emerging mycotoxins) in aquaculture feed by LC-MS/MS. Sample extraction was performed with ultrasonic assistance, and different cleanup strategies were evaluated. The optimized method was composed by ultrasound-assisted extraction (two cycles, 55 °C, 20 min), followed by cleanup using a Captiva EMR Lipid cartridge. Then, nine commercial samples of aquaculture fish feed were analyzed. Eight of the 15 target mycotoxins were detected in the samples. Results showed that two enniatins (EENB and ENNB1), beauvericin, and fumonisin B2 were detected in all samples. These results show the multi-mycotoxin contamination of fish feed, highlighting the need to improve current knowledge on the occurrence and toxicity of mycotoxins in fish feed, mainly the emerging ones.

Host Genotype and Weather Effects on Fusarium Head Blight Severity and Mycotoxin Load in Spring Barley.

Epidemiology of Fusarium Head Blight (FHB) of spring barley is relatively little understood. In a five-year study, we assessed quantitative resistance to FHB in an assortment of 17 spring barley genotypes in the field in southern Germany. To this end, we used soil and spray inoculation of plants with F. culmorum and F. avenaceum. This increased disease pressure and provoked genotypic differentiation. To normalize effects of variable weather conditions across consecutive seasons, we used a disease ranking of the genotypes based on quantification of fungal DNA contents and multiple Fusarium toxins in harvested grain. Together, this allowed for assessment of stable quantitative FHB resistance of barley in several genotypes. Fungal DNA contents were positively associated with species-specific Fusarium toxins in single years and over several years in plots with soil inoculation. In those plots, plant height limited FHB; however, this was not observed after spray inoculation. A multiple linear regression model of recorded weather parameter and fungal DNA contents over five years identified time periods during the reproductive phase of barley, in which weather strongly influenced fungal colonization measured in mature barley grain. Environmental conditions before heading and late after anthesis showed strongest associations with F. culmorum DNA in all genotypes, whereas for F. avenaceum, this was less consistent where we observed weather-dependent associations, depending on the genotype. Based on this study, we discuss aspects of practical resistance breeding in barley relevant to improve quantitative resistance to FHB and associated mycotoxin contaminations.

High Pressure Processing Impact on Emerging Mycotoxins (ENNA, ENNA1, ENNB, ENNB1) Mitigation in Different Juice and Juice-Milk Matrices.

The aim of the present study was to investigate the potential of high-pressure processing (HPP) (600 MPa during 5 min) on emerging mycotoxins, enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1) reduction in different juice/milk models, and to compare it with the effect of a traditional thermal treatment (HT) (90 °C during 21 s). For this purpose, different juice models (orange juice, orange juice/milk beverage, strawberry juice, strawberry juice/milk beverage, grape juice and grape juice/milk beverage) were prepared and spiked individually with ENNA, ENNA1, ENNB and ENNB1 at a concentration of 100 µg/L. After HPP and HT treatments, ENNs were extracted from treated samples and controls employing dispersive liquid-liquid microextraction methodology (DLLME) and determined by liquid chromatography coupled to ion-trap tandem mass spectrometry (HPLC-MS/MS-IT). The results obtained revealed higher reduction percentages (11% to 75.4%) when the samples were treated under HPP technology. Thermal treatment allowed reduction percentages varying from 2.6% to 24.3%, at best, being ENNA1 the only enniatin that was reduced in all juice models. In general, no significant differences (p > 0.05) were observed when the reductions obtained for each enniatin were evaluated according to the kind of juice model, so no matrix effects were observed for most cases. HPP technology can constitute an effective tool in mycotoxins removal from juices.