Enniatin B1 Research Articles

Further data on the levels of emerging Fusarium mycotoxins in cereals collected from Tianjin, China

ABSTRACT A number of 344 samples were collected from Tianjin, China and were analysed for the occurrence of emerging Fusarium mycotoxins including enniatin A (ENN A), enniatin A1 (ENN A1), enniatin B (ENN B), enniatin B1 (ENN B1) and beauvericin (BEA) by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The frequencies of mycotoxins studied were 69.0% (40/58), 69.8% (37/53), 85.9% (67/78), 78.9% (75/95), and 30.0% (18/60) for rice, wheat, corn, wheat flour, and corn flour, respectively. BEA was the predominant toxin in rice (average = 37.2 μg/kg) and wheat (average = 58.4 μg/kg), followed by ENN B and ENN B1, while less ENN A and ENN A1 were detected. ENN A was most common in corns with an average level of 28.1 μg/kg, while BEA had a higher average of 62.8 μg/kg. The levels of ENNs and BEA significantly decreased in wheat flours and corn flours, presumably due to the production process. The co-occurrences of ENNs and BEA in cereal samples were common in the combination of two and three mycotoxins and the significant positive correlations in concentrations were also obtained among them. Besides, agroclimate was considered as an important factor for Fusarium production and the mycotoxin contamination was found more serious in Jizhou district, which had more rainfall and less sunshine, than the other agriculture regions. These results suggested the necessity of carrying out in-depth and large-scale monitoring of mycotoxins in cereals and their products nationwide.

A 28-day repeated oral dose toxicity study of enniatin complex in mice.

Enniatins are so-called "emerging mycotoxins" that commonly occur in milligrams per kilogram levels in grains and their derived products, as well as in fish, dried fruits, nuts, spices, cocoa, and coffee. The present study investigated the 28-day repeated oral dose toxicity of enniatin complex in CD1(ICR) mice. Enniatin B, enniatin B1, and enniatin A1 at a ratio of 4:4:1 were administered to male and female mice at doses of 0 (vehicle controls), 0.8, 4, and 20 mg/kg body weight/day. In life parameters did not change during the study period, with the exception of slight reductions in food consumption in male mice administered 4 and 20 mg/kg and in female mice administered 20 mg/kg. Body and organ weights did not change, and no alterations in hematology, blood biochemistry, or histopathology parameters were observed at the end of the administration period. Thus, we determined that the no-observed-adverse-effect level of enniatin complex was 20 mg/kg/day for both sexes under the present experimental conditions.

Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC30 and IC50 values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.

Presence of Mycotoxins in Milk Thistle (Silybum marianum) Food Supplements: A Review.

The consumption of herbal-based supplements, which are believed to have beneficial effects on human health with no side effects, has become popular around the world and this trend is still increasing. Silybum marianum (L.) Gaertn, commonly known as milk thistle (MT), is the most commonly studied herb associated with the treatment of liver diseases. The hepatoprotective effects of active substances in silymarin, with silybin being the main compound, have been demonstrated in many studies. However, MT can be affected by toxigenic micro-fungi and contaminated by mycotoxins with adverse effects. The beneficial effect of silymarin can thus be reduced or totally antagonized by mycotoxins. MT has proven to be affected by micro-fungi of the Fusarium and Alternaria genera, in particular, and their mycotoxins. Alternariol-methyl-ether (AME), alternariol (AOH), beauvericin (BEA), deoxynivalenol (DON), enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1), HT-2 toxin (HT-2), T-2 toxin (T-2), tentoxin (TEN), and zearalenone (ZEA) seem to be most significant in MT-based dietary supplements. This review focuses on summarizing cases of mycotoxins in MT to emphasize the need for strict monitoring and regulation, as mycotoxins in relation with MT-based dietary supplements are not covered by European Union legislation.

The expedient application of microbial fermentation after whole-wheat milling and fractionation to mitigate mycotoxins in wheat-based products

The effect of whole milling and fractionation of wheat grains from the 2017 and 2018 harvests on the mycotoxin transfer into milling products and the influence of microbial fermentation of such products on mycotoxin levels were analysed. Additionally, the identification and occurrence of fungal contamination in grains with different mycotoxin contamination levels was evaluated. The mycotoxin contents were determined by chromatography (HPLC-TOF-HRMS). The distribution of mycotoxins in wheat wholemeal fractions depended on wheat kernel contamination level. The flour fractions contained zearalenone (ZEN) from 59 to 92%, and deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) at levels on average 60% of those in unprocessed wheat grains. The highest contents of enniatin B (ENNB) (84–86%) and ENNB1 (80–99%) were distributed within the flour fractions. Prolonged sourdough fermentation (48 h) allowed a reduction in DON content in the sourdough by 44–69% and a removal of 15-acetyldeoxynivalenol (15-AcDON), alternariol (AOH), deoxynivalenol-3-glucoside (D3G), toxins H-2 and HT-2, while the enniatin removal levels were in a range 5–70%, with the complete removal of only ENNB1. The study on the retention of mycotoxins during secondary grain processing (sourdough fermentation) is important for the risk assessment of wheat-based products.

Pulsed Electric Fields (PEF) to Mitigate Emerging Mycotoxins in Juices and Smoothies

The development of innovative food processing technologies has increased to answer the growing demand to supply of fresh-like products. The aim of the present study is to investigate the effect of pulsed electric fields (PEF) technology on reducing the emerging mycotoxins (enniatins (ENs) and beauvericin (BEA)) contents in juice and smoothie samples. The products of degradation obtained after PEF treatment were identified and their toxicological endpoint toxicities predicted by Pro Tox-II web. Mycotoxin reduction ranged from 43 to 70% in juices and smoothies, but in water the expected effect was lower. The acidified pH increased BEA reduction in water. The degradation products that were produced were the result of the loss of aminoacidic fragments of the original molecules, such as HyLv, Val, Ile, or Phe. Pro Tox-II server assigned a toxicity class I for enniatin B (ENB) degradation products with a predicted LD50 of 3 mg/Kgbw. The other degradation products were classified in toxicity class III and IV.

Fusarium Mycotoxins in Two Hulless Oat and Barley Cultivars Used for Food Purposes.

Hulless oats and hulless barley are highly valued for their excellent nutritional attributes and are increasingly being promoted in human nutrition. However, special attention should be paid to the risk of their contamination by Fusarium mycotoxins, as the rate of mycotoxin reduction during processing could be much lower than that for hulled cereals. In the present study, mycotoxin contamination of two cultivars, each of hulless oats and barley suitable for food purposes were studied in a 3-year field trial established in two contrasting environments. The contents of the mycotoxins regulated by law (deoxynivalenol and zearalenone) were low, and the present legal limits for their maximum content in unprocessed cereals were far from being exceeded. The mycotoxins most frequently occurring in hulless barley were enniatins (enniatin B, enniatin B1 and enniatin A1), beauvericin and nivalenol; hulless oats most frequently contained the HT-2 and T-2 toxins, beauvericin and enniatin B. The contents of enniatins and nivalenol were higher in barley than in oats. Close, positive relationships between the contents of the individual enniatins and between enniatins, beauvericin and nivalenol were observed, which implies that co-exposure could enhance the toxic potential of these mycotoxins through synergistic effects. The results highlight the need to pay more attention to the occurrence of enniatins, beauvericine and nivalenol in hulless oats and barley used for food purposes.

Biomonitoring of Enniatin B1 and Its Phase I Metabolites in Human Urine: First Large-Scale Study.

Enniatins (Enns) are mycotoxins produced by Fusarium spp. which are a fungus widely spread throughout cereals and cereal-based products. Among all the identified enniatins, Enn B1 stands as one of the most prevalent analogues in cereals in Europe. Hence, the aim of this study was to evaluate for the first time the presence of Enn B1 and its phase I metabolites in 300 human urine samples using an ultrahigh-performance liquid chromatography high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) methodology. Enn B1 was detected in 94.3% of samples ranging from 0.007 to 0.429 ng/mL (mean value: 0.065 ng/mL). In accordance with previous in vitro and in vivo analysis, hydroxylated metabolites (78.0% samples) and carbonylated metabolites (66.0% samples) were tentatively identified as the major products. Results from this biomonitoring study point to a frequent intake of Enn B1 in the studied population, suggesting that in-depth toxicological studies are needed in order to understand the potential effects in humans.

Exposure to Mycotoxin-Mixtures via Breast Milk: An Ultra-Sensitive LC-MS/MS Biomonitoring Approach.

Exposure to natural food contaminants during infancy may influence health consequences later in life. Hence, breast milk may serve as a vehicle to transport these contaminants, including mycotoxins, from mothers to their infants. Analytical methods mostly focused on single exposures in the past, thus neglecting co-occurrences and mixture effects. Here, we present a highly sensitive multi-biomarker approach by a sophisticated combination of steps during sample preparation including a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction followed by a solid phase extraction (SPE) cleanup and utilizing stable isotopes for compensating challenging matrix effects. The assay was validated in-house, reaching limits of detection (LOD) for all 34 analytes in the range of 0.1 to 300 ng/L with satisfying extraction efficiencies (75–109%) and stable intermediate precisions (1–18%) for most analytes. Compared to a similar multi-mycotoxin assay for breast milk, LOD values were decreased by a factor of 2–60x enabling the assessment of chronic low-dose exposures. The new method was applied to a small set of Nigerian breast milk samples (n = 3) to compare results with already published data. Concentration levels of samples that were found to be contaminated before could be confirmed. In addition, other mycotoxins were determined in all three samples, for example the newly investigated alternariol monomethyl ether (AME) was found for the first time in this biological fluid at concentrations up to 25 ng/L. Moreover, in a pooled Austrian sample obtained from a milk bank, trace amounts of multiple mycotoxins including AME (1.9 ng/L), beauvericin (5.4 ng/L), enniatin B (4.7 ng/L), enniatin B1 (<LOQ), ochratoxin A (<LOQ) and the estrogenic zearalenone (<LOQ) confirmed co-occurrence and exposure even in a country with high food safety standards. In conclusion, the method facilitates the determination of mycotoxins at ultra-trace levels in breast milk, enabling the generation of occurrence data necessary for comprehensive co-exposure assessment.

Risk Assessment and Mitigation of the Mycotoxin Content in Medicinal Plants by the Infusion Process.

Medicinal plants are often consumed as infusions with boiled water. Scarce information is available in the literature about the migration of mycotoxins into the resulting beverage and/or the effects of the infusion procedure on the final mycotoxin contents. The aim of the present study was to investigate the impact of the infusion process on mycotoxin contents during medicinal plant preparation. For this purpose, the contents of aflatoxins (AFs) [aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2)], zearalenone (ZEA), enniatins (ENNs) [enniatin B (ENNB), enniatin B1 (ENNB1), enniatin A (ENNA), enniatin A1 (ENNA1)] and beauvericin (BEA) were analyzed in 224 samples of medicinal plants and in their resulting beverages. The quick, easy, cheap, effective, rugged and safe extraction method (QuEChERS) was applied to the medicinal plants while the dispersive liquid-liquid microextraction procedure (DLLME) was applied to their infusions, and the mycotoxins were determined by liquid chromatography coupled to ion trap tandem mass spectrometry (LC-MS/MS-IT). The results revealed that ZEA, ENNB, ENNB1, AFB2, AFG1 and AFG2 were detected in the beverages with incidences of ≤6% and at concentrations from less than the limit of quantification (LOQ) to 82.2μg/L. Mycotoxins reduction ranged from 74 to 100% after the infusion process. The risk assessment revealed that the estimated daily intakes (EDIs) obtained for ZEA, ENNB and ENNB1 were far below the tolerable daily intakes (TDIs) established.

Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Orbitrap High-Resolution Mass Spectrometry for Multi-Residue Analysis of Mycotoxins and Pesticides in Botanical Nutraceuticals.

Cannabidiol (CBD) food supplements made of Cannabis sativa L. extracts have quickly become popular products due to their health-promoting effects. However, potential contaminants, such as mycotoxins and pesticides, can be coextracted during the manufacturing process and placed into the final product. Accordingly, a novel methodology using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was developed to quantify 16 mycotoxins produced by major C. sativa fungi, followed by a post-target screening of 283 pesticides based on a comprehensive spectral library. The validated procedure was applied to ten CBD-based products. Up to six different Fusarium mycotoxins were found in seven samples, the most prevalent being zearalenone (60%) and enniatin B1 (30%), both found at a maximum level of 11.6 ng/g. Co-occurrence was observed in four samples, including one with enniatin B1, enniatin A and enniatin A1. On the other hand, 46 different pesticides were detected after retrospective analysis. Ethoxyquin (50%), piperonyl butoxide (40%), simazine (30%) and cyanazine (30%) were the major residues found. These results highlight the necessity of monitoring contaminants in food supplements in order to ensure a safe consumption, even more considering the increase trend in their use. Furthermore, the developed procedure is proposed as a powerful analytical tool to evaluate the potential mycotoxin profile of these particular products.

Mycotoxins in blood and urine of Swedish adolescents\u2014possible associations to food intake and other background characteristics

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.

Co-occurrence of DON and Emerging Mycotoxins in Worldwide Finished Pig Feed and Their Combined Toxicity in Intestinal Cells.

Food and feed can be naturally contaminated by several mycotoxins, and concern about the hazard of exposure to mycotoxin mixtures is increasing. In this study, more than 800 metabolites were analyzed in 524 finished pig feed samples collected worldwide. Eighty-eight percent of the samples were co-contaminated with deoxynivalenol (DON) and other regulated/emerging mycotoxins. The Top 60 emerging/regulated mycotoxins co-occurring with DON in pig feed shows that 48%, 13%, 8% and 12% are produced by Fusarium, Aspergillus, Penicillium and Alternaria species, respectively. Then, the individual and combined toxicity of DON and the 10 most prevalent emerging mycotoxins (brevianamide F, cyclo-(L-Pro-L-Tyr), tryptophol, enniatins A1, B, B1, emodin, aurofusarin, beauvericin and apicidin) was measured at three ratios corresponding to pig feed contamination. Toxicity was assessed by measuring the viability of intestinal porcine epithelial cells, IPEC-1, at 48-h. BRV-F, Cyclo and TRPT did not alter cell viability. The other metabolites were ranked in the following order of toxicity: apicidin > enniatin A1 > DON > beauvericin > enniatin B > enniatin B1 > emodin > aurofusarin. In most of the mixtures, combined toxicity was similar to the toxicity of DON alone. In terms of pig health, these results demonstrate that the co-occurrence of emerging mycotoxins that we tested with DON does not exacerbate toxicity.

Mycotoxin Dietary Exposure Assessment through Fruit Juices Consumption in Children and Adult Population.

Consumption of fruit juice is becoming trendy for consumers seeking freshness and high vitamin and low caloric intake. Mycotoxigenic moulds may infect fruits during crop growth, harvest, and storage leading to mycotoxin production. Many mycotoxins are resistant to food processing, which make their presence in the final juice product very likely expected. In this way, the presence of 30 mycotoxins including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), alternariol (AOH), alternariol monomethyl ether (AME), Ochratoxin A (OTA), fumonisin B1 (FB1), fumonisin B2 (FB2), enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1), beauvericin (BEA), sterigmatocystin (STG), zearalenone (ZEA), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FUS-X), neosolaniol (NEO), patulin (PAT), T-2 toxin and HT-2 toxin was evaluated in 80 juice samples collected from Valencia retail Market. An efficient Dispersive Liquid-Liquid Microextraction method (DLLME) was carried out before their trace level determination by chromatographic techniques coupled to tandem mass spectrometry. The results obtained revealed the presence of nine mycotoxins namely AOH, AME, PAT, OTA, AFB1, AFB2, AFG2, β-ZAL, and HT2 in the analyzed samples, with incidences ranging from 3 to 29% and mean contents between 0.14 and 59.52 µg/L. Considerable percentages of TDIs were reached by children when 200 mL was considered as daily fruit juice intake.

Mycotoxin Occurrence in Maize Silage-A Neglected Risk for Bovine Gut Health?

Forages are important components of dairy cattle rations but might harbor a plethora of mycotoxins. Ruminants are considered to be less susceptible to the adverse health effects of mycotoxins, mainly because the ruminal microflora degrades certain mycotoxins. Yet, impairment of the ruminal degradation capacity or high ruminal stability of toxins can entail that the intestinal epithelium is exposed to significant mycotoxin amounts. The aims of our study were to assess (i) the mycotoxin occurrence in maize silage and (ii) the cytotoxicity of relevant mycotoxins on bovine intestinal cells. In total, 158 maize silage samples were collected from European dairy cattle farms. LC-MS/MS-based analysis of 61 mycotoxins revealed the presence of emerging mycotoxins (e.g., emodin, culmorin, enniatin B1, enniatin B, and beauvericin) in more than 70% of samples. Among the regulated mycotoxins, deoxynivalenol and zearalenone were most frequently detected (67.7%). Overall, 87% of maize silages contained more than five mycotoxins. Using an in vitro model with calf small intestinal epithelial cells B, the cytotoxicity of deoxynivalenol, nivalenol, fumonisin B1 and enniatin B was evaluated (0–200 µM). Absolute IC50 values varied in dependence of employed assay and were 1.2–3.6 µM, 0.8–1.0 µM, 8.6–18.3 µM, and 4.0–6.7 µM for deoxynivalenol, nivalenol, fumonisin B1, and enniatin B, respectively. Results highlight the potential relevance of mycotoxins for bovine gut health, a previously neglected target in ruminants.

Twenty-Eight Fungal Secondary Metabolites Detected in Pig Feed Samples: Their Occurrence, Relevance and Cytotoxic Effects In Vitro.

Feed samples are frequently contaminated by a wide range of chemically diverse natural products, which can be determined using highly sensitive analytical techniques. Next to already well-investigated mycotoxins, unknown or unregulated fungal secondary metabolites have also been found, some of which at significant concentrations. In our study, 1141 pig feed samples were analyzed for more than 800 secondary fungal metabolites using the same LC-MS/MS method and ranked according to their prevalence. Effects on the viability of the 28 most relevant were tested on an intestinal porcine epithelial cell line (IPEC-J2). The most frequently occurring compounds were determined as being cyclo-(L-Pro-L-Tyr), moniliformin, and enniatin B, followed by enniatin B1, aurofusarin, culmorin, and enniatin A1. The main mycotoxins, deoxynivalenol and zearalenone, were found only at ranks 8 and 10. Regarding cytotoxicity, apicidin, gliotoxin, bikaverin, and beauvericin led to lower IC50 values, between 0.52 and 2.43 µM, compared to deoxynivalenol (IC50 = 2.55 µM). Significant cytotoxic effects were also seen for the group of enniatins, which occurred in up to 82.2% of the feed samples. Our study gives an overall insight into the amount of fungal secondary metabolites found in pig feed samples compared to their cytotoxic effects in vitro.

Biotransformation of the Mycotoxin Enniatin B1 by CYP P450 3A4 and Potential for Drug-Drug Interactions.

Enniatins (ENNs) are fungal secondary metabolites that frequently occur in grain in temperate climates. Their toxic potency is connected to their ionophoric character and lipophilicity. The biotransformation of ENNs predominantly takes place via cytochrome P450 3A (CYP 3A)-dependent oxidation reactions. Possible interaction with ENNs is relevant since CYP3A4 is the main metabolic enzyme for numerous drugs and contaminants. In the present study, we have determined the kinetic characteristics and inhibitory potential of ENNB1 in human liver microsomes (HLM) and CYP3A4-containing nanodiscs (ND). We showed in both in vitro systems that ENNB1 is mainly metabolised by CYP3A4, producing at least eleven metabolites. Moreover, ENNB1 significantly decreased the hydroxylation rates of the typical CYP3A4-substrate midazolam (MDZ). Deoxynivalenol (DON), which is the most prevalent mycotoxin in grain and usually co-occurrs with the ENNs, was not metabolised by CYP3A4 or binding to its active site. Nevertheless, DON affected the efficiency of this biotransformation pathway both in HLM and ND. The metabolite formation rates of ENNB1 and the frequently used drugs progesterone (PGS) and atorvastatin (ARVS) lactone were noticeably reduced, which indicated a certain affinity of DON to the enzyme with subsequent conformational changes. Our results emphasise the importance of drug–drug interaction studies, also with regard to natural toxins.

Enniatin B1-induced lysosomal membrane permeabilization in mouse embryonic fibroblasts

The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24h exposure to ENN B1 at levels of 2.5-10μmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7μmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.

Fusarium species and enniatin mycotoxins in wheat, durum wheat, triticale and barley harvested in France.

Contamination with enniatins A, A1, B and B1 was investigated in 1240 samples of small grain cereals (470 wheat, 260 durum wheat, 282 spring barley, 172 triticale and 56 winter barley) from the French harvests of 2012 to 2014. Associations with Fusarium avenaceum, F. tricinctum and F. poae were assessed, with the identification of Fusarium species by real-time PCR and mycotoxin quantification by LC-MS/MS. Enniatins were common in the fields sampled. Enniatin concentrations varied between years but were consistently highest on spring barley (mean values of 199 to 1316μg/kg) and triticale (mean values from 131 to 1218μg/kg), and lower on wheat (mean values from 47 to 142μg/kg) and durum wheat (mean values from 55 to 596μg/kg). The concentrations of the various enniatins were strongly correlated with each other (Pearson's correlation coefficient of 0.61 to 0.98). Enniatin B was the most frequent (68% of the total enniatin content), followed by enniatin B1 (22%), enniatin A1 (7%) and enniatin A (3%). Fusarium species were quantified by calculating arithmetic mean total DNA levels. F. tricinctum was the most abundant (0.177pg/ng total DNA), followed by F. avenaceum (0.141pg/ng total DNA) and F. poae (0.091pg/ng total DNA). Total DNA levels for each species, and the predominant species varied between years and crops. Small grain cereal species (p value < 0.001), harvest year (p value = < 0.001) and the presence of F. avenaceum (p value < 0.001), F. tricinctum (p value < 0.001) or F. poae (p value = 0.017) affected enniatin content. F. tricinctum was the leading enniatin producer on durum wheat (29% to 45%) and spring barley (23 to 37%). F. avenaceum produced large amounts of enniatins on durum wheat (13% to 17%) and wheat (1% to 18%) and was the leading producer on triticale (30% to 55%). F. poae made a minor contribution on all crops, never accounting for more than 2% of total enniatin content. Enniatins are, thus, highly prevalent in French small grain cereals and are mostly produced by F. avenaceum and F. tricinctum.

Development of Sensitive and Reliable UPLC-MS/MS Methods for Food Analysis of Emerging Mycotoxins in China Total Diet Study.

With the climatic changes that have taken place during the last decade, the spectrum of fungal pathogens as well as mycotoxins has considerably changed. As a result, some emerging mycotoxins have been shown to occur frequently in agricultural products. In this study, a sensitive and reliable method for the determination of 10 emerging mycotoxins (beauvericin, enniatin A, enniatin A1, enniatin B, enniatin B1, alternariol, alternariol monomethyl ether, altenuene, tentoxin, and tenuazonic acid) in 12 different food matrices (cereals, legumes, potatoes, meats, eggs, aquatic foods, dairy products, vegetables, fruits, sugars, beverages, and alcohol beverages) was developed and validated. After a simple extraction, a one-step sample clean-up by a HLB solid phase extraction (SPE) column was sufficient for all 12 food matrices prior to analysis with ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Isotope internal standards 13C-TeA, TEN-d3, and 13C-AFB2 were used for accurate quantification. Validation in terms of linearity, selectivity, sensitivity, accuracy, and precision (intra and inter-day variability) were evaluated for the 10 mycotoxins in all selected matrices. The sensitivity varied from 0.0004 to 0.3 ng mL−1 (limits of detection) and from 0.002 to 0.9 ng mL−1 (limits of quantitation). The recoveries of 10 mycotoxins in fortified samples were from 60.6% to 164% including very low spiking levels in all 12 food matrices, with relative standard deviations (RSDs) less than 12%. The proposed methodology was applied to the analysis of 60 samples collected from five provinces within the 6th China Total Diet Study with the results discussed in detail. The advantages of sensitivity, accuracy, and robustness made it a powerful tool for emerging mycotoxin monitoring and dietary exposure assessment.