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Microbial fermentation with lactic acid bacteria (LAB) is a natural food biopreservation method. Yellow mustard and milk whey are optimum substrates for LAB fermentation. The aim of the present study was to evaluate the bioaccessibility and bioavailability of bioactive compounds from yellow mustard flour and milk whey both with and without LAB fermentation. All extracts were subjected to a simulated digestion process. Total polyphenols, DL-3-phenyllactic acid (PLA), lactic acid, and the antioxidant activity were determined in the studied matrices before and after simulated digestion. Yellow mustard flour was significantly richer in total polyphenols, whereas significantly higher concentrations of PLA and lactic acid were observed in milk whey. Similar antioxidant activity was determined in both ingredients being in all cases strongly reduced after in vitro digestion. Higher bioaccessibility was found for polyphenols and PLA in milk whey. Transepithelial transport of total polyphenols was higher in yellow mustard flour compared to milk whey, reaching bioavailability values between 3-7% and 1-2%, respectively. PLA transepithelial transport was only significant in both fermented matrices with bioavailability around 4-6%. Transepithelial transport of lactic acid reached values of 31-34% (bioavailability ∼ 22%) and 15-78% (bioavailability ∼ 3%) in milk whey and yellow mustard flour, respectively. LAB fermentation showed beneficial effects on enriching extracts with PLA, lactic acid, and antioxidant activity, as well as increasing bioaccessibility of these acids in yellow mustard flour and total polyphenol bioavailability in milk whey. Results pointed to yellow mustard flour and milk whey as natural preservative ingredients used in the food industry, especially when fermented with LAB.

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.

Background Scientific criteria to identify endocrine disruptors (ED) was recently implemented for plant protection products (PPP) and biocidal products (BP). A guidance document has been published by ECHA and EFSA in the context of ED criteria for PPPs and BPs. Methods In the present work, a case study was performed on Bisphenol AF (BPAF) to explore the application of the EU criteria and EFSA/ECHA guidance document for the ED assessment of a non-pesticide chemical regulated under REACH. A data dossier was built by a systematic literature search (Web of Science, Pubmed, Embase; n = 511), title/abstract screening (n = 124) and full text examination (n = 88). All the information was extracted and systematically reported for 309 parameters (100 for adversity; 209 for endocrine activity). The reliability of studies was assessed (SciRAP tool). Results Data were synthesized into 96 lines of evidence for adversity (n = 57), and endocrine activity (n = 39); and assessed by weight of evidence methodology. The initial analysis of the evidence indicated EATS-mediated adversity in mammals, therefore a mode of action (MoA) was postulated for both male and female adult exposure. Female MoA included estrogen receptor activation and altered steroidogenesis leading to ovarian dysfunction, altered estrous cycling and impaired female fertility. Male MoA was initiated by androgen receptor inhibition and altered steroidogenesis leading to dysfunction of male reproductive organs and impaired male fertility. Conclusions The overall conclusion of the ED assessment indicated that BPAF meets the ED criteria for human health. The steps described in the ED guidance document were successfully completed, resulting in a thorough, structured and transparent identification of BPAF as an ED. Advantages and limitations of applying the ED criteria and guidance for a REACH chemical are discussed.

A pilot study to investigate the occurrence of 10 mycotoxins (deoxynivalenol, DON, 3-acetyldeoxynivalenol, 3-ADON, 15-acetyldeoxynivalenol, 15-ADON, fusarenon-X, FUS-X, diacetoxyscirpenol, DAS, nivalenol, NIV, neosolaniol, NEO, zearalenone, ZON, zearalanone, ZAN, T-2 toxin, T-2, and HT-2 toxin, HT-2) in esophageal cancer patients was performed with the urinary biomarkers approach in Golestan, Iran. Urine multimycotoxin analysis was performed by dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry (GC–MS/MS) analysis, and values were normalized with urinary creatinine (μg/g). Four mycotoxins, namely NEO (40%), HT-2 (17.6%), DON (10%), and HT-2 (5.8%), were detected in the analyzed urine samples. DON was only detected in the control group (5.09 μg/g creatinine), while T-2 (44.70 μg/g creatinine) was only present in the esophageal cancer group. NEO and HT-2 were quantified in both control and case groups, showing average of positive samples of 9.09 and 10.45 μg/g creatinine for NEO and 16.81 and 29.09 μg/g creatinine for HT-2, respectively. Mycotoxin co-occurrence was observed in three samples as binary (NEO/HT-2 and T-2/HT-2) and ternary (DON/NEO/HT-2) combinations, reaching total concentrations of 44.58, 79.13, and 30.04 µg/g creatinine, respectively. Further investigations are needed to explore a causal association between mycotoxin contamination and esophageal cancer. For this pilot study in Golestan, the low sample size was a very limiting factor.

A guidance document for the identification of endocrine disruptors (EDs) in the regulatory assessment of plant protection products (PPP) and biocidal products (BP) has been published by the European Chemical Agency (ECHA) and the European Food Safety Authority (EFSA). The ECHA/EFSA guidance, mainly addressing EATS (estrogen, androgen, thyroid, steroidogenesis) modalities, is intended to guide applicants and assessors of the competent regulatory authorities on the implementation of the scientific criteria for the determination of ED properties pursuant to the recently implemented PPP ( EU 2018/605 ) and BP ( EU 2017/2100 ) EU Regulations. In this study, a search filter for targeted literature search in context of assessing if a substance can be identified as an ED relevant for human health was developed and validated. Development of the search filter was based on the search strategy presented in the ECHA/EFSA guidance and using the estrogenic chemical Bisphenol AF (BPAF) as a model substance. Information specialists from two independent institutions developed refined search filters based on the suggested original search strategy published (ECHA/EFSA guidance - Appendix F). Articles identified by a systematic literature search for BPAF were screened for relevance with inclusion and exclusion criteria by two independent reviewers obtaining positive (relevant) and negative (irrelevant) controls. The developed search filter was quantitatively evaluated in terms of sensitivity, specificity and precision based on the positive and negative controls. The developed filter was then validated for T modality by its application to the known thyroid-disruptor perchlorate. The result is a sensitive search filter with sufficient specificity, which can be applied for all chemicals where a targeted literature search is needed to assess and identify ED properties of chemicals with relevance for humans. Future application of the filter to a broader range of chemicals may identify further points of improvement.

Enniatin B is a ionophoric and lipophilic mycotoxin which reaches the bloodstream and has the ability to penetrate into cellular membranes. The purpose of this study was to reveal changes in the gene expression profile caused by enniatin B in human Jurkat lymphoblastic T-cells after 24 h of exposure at 1.5, 3 and 5 μM by next generation sequencing. It was found that up to 27% of human genome expression levels were significantly altered (5750 genes for both down-regulation and up-regulation). In the three enniatin B concentrations studied 245 differentially expressed genes were found to be overlapped, 83 were down and 162 up-regulated. ConsensusPathDB analysis of over-representation of differentially expressed genes provided a list of gene ontology terms in which several biological processes related to nucleoside monophosphate metabolic process, respiratory chain complex, electron transport chain, oxidative phosphorylation and cellular respiration were the most altered. Also, an interesting correlation was found between enniatin B toxicity and the up-regulation of the UCP protein complex. In summary, the transcriptomic analysis revealed that mitochondria are the organelles showing more related differentially expressed genes. Consequently, differentially expressed genes involved in biological processes, molecular functions and pathways related to mitochondrial metabolism and respiration were significantly changed.

Chicken meat and edible offal are included in a wide number of preparations in the worldwide cuisine. Among the offal, the liver is one of the most consumed in some countries like Egypt since it is rich in proteins and is an economical source of many essential nutrients for humans. Mycotoxins, secondary toxic metabolites produced by fungi, have the potential to enter human food chain not only with cereal consumption but also through edible tissues from animals fed with mycotoxin-contaminated feed. Accordingly, a rapid method for the determination of eight trichothecenes in chicken liver was developed based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) determination. The proposed method was successfully validated according to the European guideline recommendations with satisfactory trueness (recovery range from 85.1 to 108.4%), precision (relative standard deviation

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.

Beauvericin (BEA), an ionophoric cyclic hexadepsipeptide mycotoxin, is able to increase oxidative stress by altering membrane ion permeability and uncoupling oxidative phosphorylation. A toxicogenomic study was performed to investigate gene expression changes triggered by BEA exposure (1.5, 3 and 5 mu M; 24 h) in Jurkat cells through RNA-sequencing and differential gene expression analysis. Perturbed gene expression was observed in a concentration dependent manner, with 43 differentially expressed genes (DEGs) overlapped in the three studied concentrations. Gene ontology (GO) analysis showed several biological processes related to electron transport chain, oxidative phosphorylation, and cellular respiration significantly altered. Molecular functions linked to mitochondrial respiratory chain and oxidoreductase activity were over-represented (q-value < 0.01). Pathway analysis revealed oxidative phosphorylation and electron transport chain as the most significantly altered pathways in all studied doses (z-score > 1.96; adj p-value < 0.05). 77 genes involved in the respiratory chain were significantly down-regulated at least at one dose. Moreover, 21 genes related to apoptosis and programmed cell death, and 12 genes related to caspase activity were significantly altered, mainly affecting initiator caspases 8, 9 and 10. The results demonstrated BEA-induced mitochondrial damage affecting the respiratory chain, and pointing to apoptosis through the caspase cascade in human lymphoblastic T cells.

Compound feed is particularly vulnerable to multi-mycotoxin contamination. A method for the determination of 12 mycotoxins; enniatins A, A1, B, B1; aflatoxins B1, B2, G1, G2; OTA; ZEA; T-2 and HT-2 by liquid chromatography-tandem mass spectrometry has been developed and applied for the analysis of laboratory rat commercial feeds. The method trueness was checked by recovery assays at three different spiked levels (n = 9). Recoveries ranged from 73% to 112%, and the intra-day and inter-day precision were lower than 9% and 13%, respectively. Limits of quantitation were lower than 15 μg/kg. Twenty-seven laboratory rats feed samples showed multi-contamination by at least three up to six different mycotoxins. ENNs B and B1, followed by ZEA were the most prevalent mycotoxins. T-2, HT-2, and OTA were not detected. ZEA showed the highest concentration levels reaching 492 μg/kg. The results underline the importance of implementing mycotoxin regular surveillance programs for laboratory animal feeds.

Aphids display life cycles largely determined by the photoperiod. During the warm long-day seasons, most aphid species reproduce by viviparous parthenogenesis. The shortening of the photoperiod in autumn induces a switch to sexual reproduction. Males and sexual females mate to produce overwintering resistant eggs. In addition to this full life cycle (holocycle), there are anholocyclic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis. The molecular or hormonal events that trigger the seasonal response (i.e., induction of the sexual phenotypes) are still unknown. Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism, the involvement of the circadian clock and/or of the hormone melatonin in insect seasonal responses is not so well established. Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclic aphids reared under short days than under long days, while no differences were found between anholocyclic aphids under the same conditions. We also found that melatonin is localized in the aphid suboesophageal ganglion (SOG) and in the thoracic ganglionic mass (TGM). In analogy to vertebrates, insect-type arylalkylamine N-acetyltransferases (i-AANATs) are thought to play a key role in melatonin synthesis. We measured the expression of four i-AANAT genes identified in A. pisum and localized two of them in situ in the insect central nervous systems (CNS). Levels of expression of these genes were compatible with the quantities of melatonin observed. Moreover, like melatonin, expression of these genes was found in the SOG and the TGM.

A method for the determination of seven trichothecenes, neosolaniol (NEO), diacetoxyscirpenol (DAS), deoxynivalenol (DON), nivalenol (NIV), fusarenon-X (FUS-X), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON), in laboratory rat feed by GC-MS/MS was developed. Sample extraction and purification was performed by an acidified mixture of acetonitrile/water (80-20% v/v). Limits of quantitation (LOQs) were between 1 and 10 μg kg(-1) for all studied trichothecenes. Eight concentration levels between the LOQ and 100 × LOQ were used for the calibration curves. Matrix-matched calibration was used for quantitation purposes to compensate the detector signal enhancement obtained for all the analytes. The method accuracy was evaluated by recovery assays at three concentration levels, 25, 50 and 100 μg kg(-1) (n = 9). Recoveries ranged from 62% to 97% and precision, expressed as intra- and inter-day relative standard deviations, was evaluated for all compounds. The validated method was successfully applied to the analysis of 35 laboratory rat feed samples showing mycotoxin contamination in 66% of the samples. DON was the most prevalent trichothecene followed by 15-ADON, NIV and 3-ADON. The maximum DON concentration reached in real samples was 2156 ± 4.3 μg kg(-1), while NEO, DAS and FUS-X were not detected in any sample. Multi-contamination by at least two mycotoxins was observed in 17% of the analysed feed samples.

Alternariais one of the major mycotoxigenic fungal genera with more than 70 reported metabolites.Alternariamycotoxins showed notably toxicity, such as mutagenicity, carcinogenicity, induction of DNA strand break, sphingolipid metabolism disruption, or inhibition of enzymes activity and photophosphorylation. This review reports on the toxicity, stability, metabolism, current analytical methods, and prevalence ofAlternariamycotoxins in food and feed through the most recent published research. Half of the publications were focused on fruits, vegetables, and derived products—mainly tomato and apples—while cereals and cereal by-products represented 38%. The most studied compounds were alternariol, alternariol methyl ether, tentoxin, and tenuazonic acid, but altenuene, altertoxins (I, II, and III), and macrosporin have been gaining importance in recent years. Solid-liquid extraction (50%) with acetonitrile or ethyl acetate was the most common extraction methodology, followed by QuEChERS and dilution-direct injection (both 14%). High- and ultraperformance liquid chromatography coupled with tandem mass spectrometry was the predominant determination technique (80%). The highest levels of alternariol and alternariol methyl ether were found in lentils, oilseeds, tomatoes, carrots, juices, wines, and cereals. Tenuazonic acid highest levels were detected in cereals followed by beer, while alternariol, alternariol methyl ether, tenuazonic acid, and tentoxin were found in legumes, nuts, and oilseeds.

Melatonin is a hormone mainly involved in the regulation of circadian and seasonal rhythms in both invertebrates and vertebrates. Despite the identification of melatonin in many insects, its involvement in the insect seasonal response remains unclear. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for melatonin analysis in aphids (Acyrthosiphon pisum) for the first time. After comparing two different procedures and five extraction solvents, a sample preparation procedure with a mixture of methanol/water (50:50) was selected for melatonin extraction. The method was validated by analyzing melatonin recovery at three spiked concentrations (5, 50 and 100 pg/mg) and showed satisfactory recoveries (75-110%), and good repeatability, expressed as relative standard deviation (

The emerging Fusarium mycotoxins enniatins (ENNs) have diverse biological properties, mainly due to their ionophoric activity, and represent a potential risk to human and animal health since they are commonly found in food and feed. In vivo toxicity studies are scarce and limited to the major mycotoxins. Until now, any method for the simultaneous analysis of these compounds in plasma, serum and feces from rat has been reported. A method for the extraction and determination of ENNs A, A1, B and B1 from Wistar rat samples by liquid chromatography tandem mass spectrometry has been developed. The method was successfully validated with satisfactory recoveries (70-106%), good intraday (10%) and interday (20%) precision, expressed as relative standard deviation, and good linearity between limits of quantitation (LOQ) and 100 times LOQ. Limits of detection (LOD) and LOQ were ≤1 and ≤10 ng/ml, respectively. The validated method was applied for the analysis of biological Wistar rat samples that were administered a mixture of ENNs containing 1.19, 2.16, 1.03 and 1.41 mg/kg body weight of ENN A, A1, B and B1, respectively. Blood, urine and feces samples collected every 2 h during the 8-h duration of the experiment were analyzed. The administered dose of the mixture of ENNs did not cause observable adverse effects on the animals. ENNs concentrations detected in serum and urine were below LOQs. The four ENNs were detected in feces reaching the maximum concentration at 6 h after administration.

This review summarizes the information regarding the in vivo studies of Fusarium mycotoxins in the last decade. The most common studies are classified as subacute toxicity, subchronic toxicity, acute toxicity, toxicokinetic studies and teratogenicity in order of importance. The most used animals in in vivo studies are pigs, rats, chickens and mice. Fumonisin B1, deoxynivalenol, zearalenone, nivalenol and T-2 toxin are the most studied fusarotoxins. Studies with combinations of mycotoxins are also frequent, deoxynivalenol generally being one of them. The predominant route of administration is oral, administered mostly in the form of naturally contaminated feed. Other administration routes also used are intraperitoneal, intravenous and subcutaneous. In vivo research on Fusarium mycotoxins has increased since 2010 highlighting the need for such studies in the field of food and feed safety.

A 28-day repeated dose preliminary assay, using enniatin A naturally contaminated feed through microbial fermentation by a Fusarium tricinctum strain, was carried out employing two months-old female Wistar rats as in vivo experimental model. In order to simulate a physiological test of a toxic compound naturally produced by fungi, five treated animals were fed during twenty-eight days with fermented feed. As control group, five rats were fed with standard feed. At the 28th day, blood samples were collected for biochemical analysis and the gastrointestinal tract, liver and kidneys were removed from each rat for enniatin A detection and quantitation. Digesta were collected from stomach, duodenum, jejunum, ileum and colon. Enniatin A present in organs and in biological fluids was analyzed by liquid chromatography-diode array detector (LC-DAD) and confirmed by LC-mass spectrometry linear ion trap (MS-LIT); also several serum biochemical parameters and a histological analysis of the duodenal tract were performed. No adverse effects were found in any treated rat at the enniatin A concentration (20.91 mg/kg bw/day) tested during the 28-day experiment. Enniatin A quantitation in biological fluids ranged from 1.50 to 9.00 mg/kg, whereas in the gastrointestinal organs the enniatin A concentration ranged from 2.50 to 23.00 mg/kg. The high enniatin A concentration found in jejunum liquid and tissue points to them as an absorption area. Finally, two enniatin A degradation products were identified in duodenum, jejunum and colon content, probably produced by gut microflora.