Oxidative stress, glutathione, and gene expression as key indicators in SH-SY5Y cells exposed to zearalenone metabolites and beauvericin.

The co-presence of mycotoxins from fungi of the genus Fusarium is a common fact in raw food and food products, as trace levels of them or their metabolites can be detected, unless safety practices during manufacturing are carried out. Zearalenone (ZEA), its metabolites α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) and, beauvericin (BEA) are co/present in cereals, fruits or their products which is a mixture that consumer are exposed and never evaluated in neuronal cells. In this study the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation and glutathione (GSH) ratio activity in a human neuroblastoma cell line, SH-SY5…

Occurrence of ochratoxin A in bread consumed in Morocco

Abstract One hundred samples of commercial bread purchased from January to October (2006) from retail bakeshops in five different cities (Rabat, Temara, Sale, Casablanca and Meknes) in Morocco were surveyed for the presence of ochratoxin A (OTA) using liquid chromatography coupled to fluorescence detection. The identification of OTA in positive bread samples was confirmed by methyl ester derivatization. Analytical results showed that forty eight (48%) samples were positive with OTA greater than the limit of quantification (LOQ = 0.051 ng/g). Levels of OTA in positive samples ranged between 0.14 and 149 ng/g. The average contamination of bread samples with OTA was 13 ± 1.5 ng/g. The highest …

Aflatoxins levels in dried fruits and nuts from Rabat-Salé area, Morocco

Abstract The occurrence of aflatoxins (AFs) in dried fruits and nuts available in Rabat-Sale area (Morocco) was surveyed in this study. One hundred samples of dried fruits and nuts, purchased from retail shops and local markets from January to October 2006, were analyzed for AFs content by immunoaffinity (IAC) clean-up with liquid chromatography and fluorescence detection. Results showed that the incidences of total aflatoxins (AFT) and aflatoxin B1 (AFB1) in peanut, dried raisins, dried figs, walnut, and pistachio were 5%, 20%, 30%, 30% and 45% and 5%, 20%, 5%, 30% and 45%, respectively. The highest contamination levels of AFB1 were found in one walnut sample (2500 μg/kg) and one pistachio…

Neurotoxicity of zearalenone’s metabolites and beauvericin mycotoxins via apoptosis and cell cycle disruption

Cell cycle progression and programmed cell death are imposed by pathological stimuli of extrinsic or intrinsic including the exposure to neurotoxins, oxidative stress and DNA damage. All can cause abrupt or delayed cell death, inactivate normal cell survival or cell death networks. Nevertheless, the mechanisms of the neuronal cell death are unresolved. One of the cell deaths triggers which have been wildly studied, correspond to mycotoxins produced by Fusarium species, which have been demonstrated cytotoxicity and neurotoxicity through impairing cell proliferation, gene expression and induction of oxidative stress. The aim of present study was to analyze the cell cycle progression and cell …

Multi-mycotoxin contamination of green tea infusion and dietary exposure assessment in Moroccan population

Green tea infusion is one of the most widely drunk beverages worldwide due to its health benefits associated with microelements, essential oils, and polyphenols, etc. Several studies have reported that green tea is subjected to contamination by various toxigenic fungi. Thus, this work aims to investigate the co-occurrence of 15 mycotoxins [four aflatoxins (AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), beauvericin (BEA), four enniatins (ENA, ENA1, ENB, ENB1), zearalenone (ZEN), alternariol (AOH), tentoxin (TENT), T-2 and HT-2 toxins] in green tea samples available in Morocco by liquid chromatography tandem mass spectrometry method. Analytical and consumption data were then used to assess the …

In vitro biosynthesis of beauvericin and enniatins by Fusarium avenaceum and Fusarium poae strains isolated from Italian durum wheat grains

Nanoelectrospray with ion-trap mass spectrometry for the determination of beta-casomorphins in derived milk products.

Beta-casomorphins (b-CMs) are bioactive peptides derived from casein with opioid agonist effects similar to morphine. The use of electrospray (ESI) with quadrupole ion-trap mass spectrometry (QIT-MS) for these compounds in two matrices, cheese and milk, was examined. It was compared to a liquid chromatography (LC) coupled to mass spectrometry (LC-MS), and a "soft" ionisation technique, NanoMate, with selected ion monitoring (SIM), which are unreliable for the determination of trace casomorphins in derived milk products. b-CM mass fragmentation pathways were done for the four most common b-CMs: beta-casomorphin (1-5) bovine (b-CM-5), beta-casomorphin (1-7) bovine (b-CM-7), [D-Ala2, D-Pro4,Ty…

Presence of mycotoxins in Tunisian infant foods samples and subsequent risk assessment

Abstract A study on a set of cereal and cereal-based products (n = 117) intended for infant consumption was performed to determine the natural presence of twenty different mycotoxins by both liquid chromatography and gas chromatography coupled to tandem mass spectrometry (MS/MS). Analytical data showed that 67% of analyzed samples were contaminated by at least one mycotoxin at variable levels. Deoxynivalenol presented the highest incidence (38%) and reached a maximum level of 240 ng/g in a bsissa sample (oat product), higher than the maximum level set for DON in cereal products (200 ng/g). While, enniatin B was found in 25 samples with a maximum level of 316 ng/g found also in a bsissa samp…

Occurrence and co-occurrence of Fusarium mycotoxins in wheat grains and wheat flour from Romania

Abstract In this study, the presence of fourteen Fusarium mycotoxins, legislated by the European Union – deoxynivalenol, zearalenone, HT-2 and T-2 toxins (EC/1881/2006; 2013/165/EU), or non-legislated (five trichothecens and five “emerging” mycotoxins), was evaluated in 31 whole unprocessed wheat samples and 35 white wheat flour samples from different areas of Romania. For this purpose, a validated multi-mycotoxins liquid chromatography tandem mass spectrometry method was applied. Seventy three percent of the analyzed samples contained at least one mycotoxin. The highest occurrence was for enniatin B, 71% of the analyzed samples being positive (21–407 μg kg−1). Regarding the legislated myco…

Biosynthesis of beauvericin and enniatins in vitro by wheat Fusarium species and natural grain contamination in an area of central Italy

Contamination of wheat grain by beauvericin (BEA) and enniatins (ENs) is a global emerging mycotoxicological food problem. In this study, strains of Fusarium avenaceum (FA), Fusarium poae (FP), Fusarium equiseti and Fusarium sporotrichioides, all potential BEA and EN producers, isolated from 162 grain samples of durum and soft wheat harvested in 2009 and 2010 collected in an area of central Italy, were preliminarily screened for the presence of the esyn1 gene, encoding the multifunctional enzyme enniatin-synthetase for the detection of potential hexadepsipeptide-producing isolates. All positive isolates were tested for their ability to biosynthesize BEA and ENs in vitro. In addition, all wh…

Presence of mycotoxin in commercial infant formulas and baby foods from Italian market

In this study a total of 75 commercially Italian samples of baby foods, including 13 infant formula milks (infant formula powders, ready-to-use preparation), 11 dairy products (cheese and yogurt), 25 cereal-based baby foods, 16 fruit and vegetables compotes, and 10 fruit and vegetables purees (composed of pear, peach, banana and for apple), were analyzed to provide an overview on mycotoxin presence. The presence was carried out by evaluating of 23 mycotoxins: ochratoxin-A (OTA), patulin (PAT), two aflatoxins (AFM1, AFB1), three zearalenones (ZONs), which include zearalenone (ZON) and its metabolites (α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL)), nine trichothecenes: deoxynivalenol (DON), …

Study of enzymatic activity in human neuroblastoma cells SH-SY5Y exposed to zearalenone's derivates and beauvericin.

Abstract Beauvericin (BEA), α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), are produced by several Fusarium species that contaminate cereal grains. These mycotoxins can cause cytotoxicity and neurotoxicity in various cell lines and they are also capable of produce oxidative stress at molecular level. However, mammalian cells are equipped with a protective endogenous antioxidant system formed by no-enzymatic antioxidant and enzymatic protective systems such as glutathione peroxidase (GPx), glutathione S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD). The aim of this study was evaluating the effects of α-ZEL, β-ZEL and BEA, on enzymatic GPx, GST, CAT and SOD activity in huma…

Incidence of ochratoxin A in rice and dried fruits from Rabat and Salé area, Morocco.

One hundred samples of dried fruits (20 dried raisins, 20 walnuts, 20 peanuts, 20 dried figs and 20 pistachios) and 20 samples of rice purchased from retail shops in the Rabat and Salé area in Morocco were analysed for ochratoxin A (OTA) by immunoaffinity clean-up (IAC) and liquid chromatography (LC) with fluorescence detection. The limit of quantification (LOQ) (S/N = 10:1) of OTA was 0.02 ng g(-1) in rice, 0.03 ng g(-1) in pistachio, peanut and walnut, and 0.03 ng g(-1) in dried raisins and dried figs. The incidences of occurrence of OTA in dried raisins, walnuts, peanuts, dried figs and rice were 30, 35, 25, 65 and 90%, respectively. Analytical results showed that pistachio samples conta…

Multimycotoxin Determination in Tunisian Farm Animal Feed

Mycotoxins presence was evaluated in animal feed marketed in Tunisia for the first time ever. A QuEChERS method was performed to analyze the natural copresence of 22 mycotoxins (enniatins, beauvericin, ochratoxin A, aflatoxins, alternariol monomethyl ether, alternariol, tentoxin, zearalenone, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, neosolaniol, diacetoxyscirpenol, T-2 toxin, and HT-2 toxin) in 122 Tunisian marketed feed samples, intended for poultry (n = 43), cattle (n = 35), rabbit (n = 12), sheep (n = 16), and horse (n = 16). Analytes detection and quantification were done using both liquid chromatography and gas chromatography coupled to tandem mass sp…

Chemoprotective effect of carotenoids from Lycium barbarum L. on SH-SY5Y neuroblastoma cells treated with beauvericin

Goji berry has recently been introduced in Mediterranean diet and its consumption is increasing. This study aims to determine cytoprotection of lutein (LUT), zeaxanthin (ZEAX) and goji berry extract (GBE) rich in carotenoids against Beauvericin (BEA)-induced cytotoxicity on SH-SY5Y neuroblastoma cells. Both carotenoids and GBE showed cytoprotective effects. Cytoprotection was evaluated by simultaneous combination of the two xanthophylls LUT and ZEAX with BEA, as well as using pre-treatment assays. The highest protective effect occurred in 16%, 24% and 12% respectively for LUT, ZEAX and LUT + ZEAX incubating simultaneously with BEA, while by pre-treatment assay LUT showed a cytoprotection ef…

Occurrence of Fusarium mycotoxins in Italian cereal and cereal products from organic farming.

In the present study, the occurrence of eighteen mycotoxins, nine trichothecenes (deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, neosolaniol, diacetoxyscirpenol, fusarenon-X, T-2 toxin and HT-2 toxin), three zearalenones (zearalenone, α-zearalenol and β-zearalenol), and six emergent mycotoxins, beauvericin and five enniatins (A, A1, B, B1 and B4), was monitored in different Italian organic cereals and cereal products by using a liquid chromatography coupled to triple quadrupole mass spectrometry method. A total of 93 organic cereal samples (wheat, barley, rye and oat) were collected from Italy. Limits of quantification ranged from 5 to 15 μg/kg. 80% of analyze…

Cytoprotection assessment against mycotoxins on HepG2 cells by extracts from Allium sativum L

Abstract Cytoprotection effects of Allium sativum L garlic extract from a local garlic ecotype from Ferrara (Italy) on hepatocarcinoma cells, HepG2 cells, is presented in this study. This garlic type is known as Voghiera garlic and has been characterized as PDO (Protected designation of Origin) product. Voghiera garlic extract (VGE) was evaluated against beauvericin (BEA) and two zearalenone (ZEA) metabolites (α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL))-induced cytotoxicity on HepG2 cells by the MTT (3–4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, over 24 h and 48 h. Direct treatment, simultaneous treatment and pre-treatment strategies at the dilution 1:16–1:00 for VG…

Ochratoxin A in rice on the Moroccan retail market

One hundred (100) samples of rice purchased from retail markets in five different cities (Rabat, Témara, Salé, Casablanca and Méknès) in Morocco from January to October 2006 were surveyed for the presence of ochratoxin A (OTA) using Accelerated Solvent Extraction (ASE) coupled to liquid chromatography with fluorescence detection. The identification of OTA in positive rice samples was confirmed by methyl ester derivatization. Analytical results showed a frequency of contamination of 26% of total analyzed rice samples. The percentage of contamination of samples was 24, 26.6, 16.6, 27.7 and 30% in Rabat, Témara, Méknès, Salé and Casablanca respectively. Levels of OTA in positive samples ranged…

Simultaneous analysis of twenty-six mycotoxins in durum wheat grain from Italy

Abstract A multi-mycotoxin analysis method based on liquid chromatography coupled to triple quadrupole mass spectrometry was validated and applied for the determination of twenty-six mycotoxins, including eight trichothecenes, three fumonisins, sterigmatocystin, ochratoxin A, four aflatoxins, zearalenone, five “emerging” mycotoxins and three Alternaria mycotoxins in 74 durum wheat samples from central Italy. Eighty-eight percent of the analysed samples contained one or more mycotoxins, with enniatin B showing the highest contamination levels (78% of samples with levels ranging from 23 to 1826 ng/g), followed by enniatin B1 (10–1384 ng/g) and deoxynivalenol (48–2267 ng/g). This work gives an…

Pressurised liquid extraction and capillary electrophoresis–mass spectrometry for the analysis of pesticide residues in fruits from Valencian markets, Spain

Abstract A procedure based in capillary electrophoresis–mass spectrometry (CE–MS) for the analysis of seven pesticides (flutolanil, simazine, haloxyfop, acifluorfen, dinoseb, picloram, and ioxynil) in four Mediterranean summer fruits with high water content (peaches, melon, watermelon, and apricot) is developed. Several conditions were studied for the optimisation of both, the separation and the electrospray (ESI) connection. The best results were obtained using 35 mM ammonium formiate (pH 9.7) as separation buffer, 20 °C as capillary temperature and 23 kV as applied voltage in an uncoated fused-silica capillary with 50 cm total length, 25 cm thermostated length, 25 cm at room temperature l…

Binary and tertiary combinations of 3-ADON, 15-ADON and AOH mycotoxins on HepG2 cells: Evaluation of cytotoxic effects and detection of metabolite products

Transfer of Fusarium mycotoxins from malt to boiled wort

Malting was simulated using two different batches of barley as raw material: a naturally contaminated batch and laboratory inoculated (with a deoxynivalenol (DON) and zearalenone (ZEA) producing Fusarium graminearum strain) one. Up to three contamination levels were prepared, every process being carried out in triplicate. A significant washout effect on DON was observed by the end of the first water phase (between 22.4 and 34 % reduction) with an even more pronounced reduction (up to 75 % decrease) by the end of the steeping process. ZEA content remained almost unchanged (no significant difference between the initial and the final concentration). Germination was characterized by an increase…

Study of locomotion response and development in zebrafish (Danio rerio) embryos and larvae exposed to enniatin A, enniatin B, and beauvericin.

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate food and feed resources, and are capable of inducing a wide range of toxicity. Here, we studied the developmental and behavioral toxicity in zebrafish (Danio rerio) embryos and larvae exposed to three mycotoxins: beauvericin (BEA), Enniatin A (ENN A), and Ennitain B (ENN B). Zebrafish embryos were collected after fertilization, treated individually from 1 to 6 dpf with BEA at 8, 16, 32 and, 64 μM and for both enniatins at 3.12, 6.25, 12.5 and, 25 μM. Mixture of mycotoxins were assayed as follows: i) for BEA + ENN A and BEA + ENN B at [32 + 12.5] μM and [16 + 6.25] μM; ii) for ENN A + ENN B at [12.5 + 12.5] …

Occurrence of Free and Conjugated Mycotoxins in Aromatic and Medicinal Plants and Dietary Exposure Assessment in the Moroccan Population.

Aromatic and medicinal plants (AMPs), as herbal material, are subjected to contamination by various mycotoxin-producing fungi, either free and conjugated. Such a problem is associated with poor storage practices, and lack of adopting good agricultural practices and good harvesting practices. Nevertheless, AMPs are poorly investigated. The purpose of this study was to investigate the co-occurrence of 15 mycotoxins (four aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), beauvericin (BEA), four enniatins (ENA, ENA1, ENB, and ENB1), zearalenone (ZEN), alternariol (AOH), tentoxin (TENT), T-2, and HT-2 toxins) in 40 samples of AMPs frequently consumed in Morocco by using liquid chromat…

Protective Effects of the Hydroethanolic Extract of Fridericia chica on Undifferentiated Human Neuroblastoma Cells Exposed to α-Zearalenol (α-ZEL) and β-Zearalenol (β-ZEL)

Fridericia chica (Bignoniaceae) is a traditional medicinal plant. The aim of this research was to determine the protective effects of the hydroethanolic extract from the F. chica leaves (HEFc) against the cytotoxicity of zearalenone (α-ZEL) and β-ZEL on SH-SY5Y cells. Free radical scavenging activity of HEFc was evaluated using the DPPH method. The cytotoxicity of both zearalenone metabolites and HEFc was examined using MTT test, as was the cytoprotective effects of the HEFc on cells treated with these mycotoxins. The chemical composition of HEFc was determined using UPLC-QTOF-MS/MS. HEFc elicited good DPPH radical scavenging activity following a concentration-dependent relationship. Cells …

Coffee Silverskin and Spent Coffee Suitable as Neuroprotectors against Cell Death by Beauvericin and α-Zearalenol: Evaluating Strategies of Treatment

Coffee silverskin and spent coffee have been evaluated in a neuroblastoma cell line (SH-SY5Y cells) against beauvericin (BEA) and α-zearalenol (α-ZEL)-induced cytotoxicity with different strategies of treatment. First, the direct treatment of mycotoxins and coffee by-products extracts in SH-SY5Y cells was assayed. IC50 values for α-ZEL were 20.8 and 14.0 µM for 48 h and 72 h, respectively and, for BEA only at 72 h, it was 2.5 µM. Afterwards, the pre-treatment with spent coffee obtained by boiling water increased cell viability for α-ZEL at 24 h and 48 h from 10% to 16% and from 25% to 30%, respectively

In silico methods for metabolomic and toxicity prediction of zearalenone, α-zearalenone and β-zearalenone.

Zearalenone (ZEA), α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) (ZEA's metabolites) are co/present in cereals, fruits or their products. All three with other compounds, constitute a cocktail-mixture that consumers (and also animals) are exposed and never entirely evaluated, nor in vitro nor in vivo. Effect of ZEA has been correlated to endocrine disruptor alterations as well as its metabolites (α-ZEL and β-ZEL); however, toxic effects associated to metabolites generated once ingested are unknown and difficult to study. The present study defines the metabolomics profile of all three mycotoxins (ZEA, α-ZEL and β-ZEL) and explores the prediction of their toxic effects proposing an in silico w…

Presence of Enniatins and Beauvericin in Romanian Wheat Samples: From Raw Material to Products for Direct Human Consumption

In this study, a total of 244 wheat and wheat-based products collected from Romania were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in order to evaluate the presence of four enniatins (ENs; i.e., ENA, ENA1, ENB, and ENB1) and beauvericin (BEA). For the wheat samples, the influence of agricultural practices was assessed, whereas the results for the wheat-based products were used to calculate the estimated daily intake of emerging mycotoxins through wheat consumption for the Romanian population. ENB presented the highest incidence (41% in wheat and 32% in wheat-based products), with its maximum levels of 815 μg kg−1 and 170 μg kg−1 in wheat and wheat-based products,…

Cytotoxic effects and degradation products of three mycotoxins: Alternariol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in liver hepatocellular carcinoma cells

This work is focused in studying the cytotoxic effects on HepG2 cells of the mycotoxins alternariol (AOH), 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) by the MTT assay, as well as in the identification of the degradation products and/or metabolites originated after treatment by liquid chromatography tandem mass spectrometry (LC-MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72 h. The IC50 values were from 65 to 96 μM, from 3.6 to 6.2 μM and from 5.2 to 8.1 μM for AOH, 3-ADON and 15-ADON, respectively. Among all three mycotoxins assayed, deoxynivalenol (DON) derivated presented the highest to…

Occurrence and daily intake of ochratoxin A of organic and non-organic rice and rice products

Abstract Ochratoxin A (OTA) was extracted from 84 rice samples and rice products by using accelerated solvent extraction (ASE) and analysed with liquid chromatography coupled with fluorescence detection. Samples were collected from rice cultivars, local markets and supermarkets; 64 were of non-organic and 20 of organic production. 7.8% of non-organic samples had OTA levels from 4.3 to 27.3 μg/kg and in 30% of organic samples was detected the presence of this mycotoxin varying from 1.0 to 7.1 μg/kg. OTA presence was confirmed by methyl-ester derivatization. Rice and rice products labelled with denomination of origin (DO) were not detected OTA due to the fact that its production has implement…

Cytoprotective effects of carotenoids-rich extract from Lycium barbarum L. on the beauvericin-induced cytotoxicity on Caco-2 cells.

Abstract In this work, the cytotoxicity of Beauvericin (BEA), lutein (LUT), zeaxanthin (ZEAX) and goji berries extract (GBE) rich in carotenoids, was investigated, as well as cytoprotective effects of these carotenoids against BEA induced-cytotoxicity on Caco-2 cells. Cytotoxicity was carried out using MTT and protein content (PC) assays during 24 and 48 h of exposure. Only BEA showed cytotoxic effect obtaining a reduction in cell proliferation range from 6.5 to 92.8%. Simultaneous combination of LUT and ZEAX with BEA slightly increased cell proliferation compared to BEA tested alone. LUT, ZEAX and GBE showed cytoprotective effects against cytotoxicity induced by BEA on Caco-2 cells. Pre-tr…

Determination of trichothecenes and zearalenones in grain cereal, flour and bread by liquid chromatography tandem mass spectrometry

Although analytical methods have been already reported for legislated mycotoxins as trichothecenes and zearalenone (ZON) separately, we describe the optimization of a simple and rapid multimycotoxin method for the determination of a total of 12 mycotoxins simultaneously, nine trichothecenes (NIV, DON, FUS-X, DAS, 15-AcDON, 3-AcDON, NEO, HT-2, T-2 T2), and zearalenone and its metabolites (ZON, a-ZOL, b-ZOL), of different origin (wheat, oat, barley and spelt) and in three different products where these substance can be present (grain, flour and bread) reach the food chain and cause toxic effect either in humans or animals. The extraction procedure was based on a mixture of acetonitrile/water …

Analysis of enniatins and beauvericin by LC-MS/MS in wheat-based products

Due to the matrix complexity for wheat-based products, a comparative study of different rapid extraction procedures was performed for the extraction of enniatins (ENA, ENA1, ENB, ENB1) and beauvericin in flour, pasta, breakfast cereals, and biscuits. Three different approaches were studied during the extraction and purification steps (shaker, Ultra-Turrax, and QuEChERS) for each matrix. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray source working in a positive mode was used. For the analysis of the five mycotoxins, the three methods were tested in terms of recovery, matrix effect, and sensibility, concluding that Ultra-Turrax extraction was the most …

Alterations in Daphnia magna exposed to enniatin B and beauvericin provide additional value as environmental indicators

Mycotoxins beauvericin (BEA) and enniatin B (ENN B) affect negatively several systems and demand more studies as the mechanisms are still unclear. The simultaneous presence of contaminants in the environment manifests consequences of exposure for both animals and flora. Daphnia magna is considered an ideal invertebrate to detect effects of toxic compounds and environmental alterations. In this study, the potential toxicity and the basic mechanism of BEA and ENN B individually and combined were studied in D. magna. Acute and delayed toxicity were evaluated, and transcript levels of genes involved in xenobiotic metabolism (mox, gst, abcb1, and abcc5), reproduction, and oxidative stress (vtg-S…

Mycotoxins presence in pre- and post-fermented silage from Tunisia

Abstract Silage represents a major part of the feed ration for livestock, being a potential cause of mycotoxicosis as it can be contaminated with toxigenic fungi capable of producing mycotoxins in suitable environmental conditions. In the present work, the presence of natural mycotoxins in samples collected of silage from the main producing areas in Tunisia (Ariana, Bizerte, Beja and Jendouba) was assessed based on different plant material (oat, barley, triticale, artichoke, sulla or raygrass). Mycotoxins were evaluated during three periods in green forage (P1) and subsequently in ensiled products after 60 days (P2) and 100 days (P3) of fermentation. Samples were extracted by a QuEChERS pro…

Limited survey for the occurrence of aflatoxins in cereals and poultry feeds from Rabat, Morocco

Fifty eight (58) samples of cereals for human consumption (20 corn flour, 17 wheat flour) and poultry feeds (n=21) purchased from popular markets of Rabat in Morocco were analyzed for aflatoxins (AF) by HPLC with immunoaffinity column (IAC) clean-up and fluorimetric detection. The incidence of AF in corn, wheat flour and poultry feeds was about 80, 17.6 and 66.6% respectively. High contamination levels were found in corn and poultry feeds samples. Levels of contamination of analyzed samples with AFB1 ranged from 0.23 to 11.2, 0.03 to 0.15 and 0.05 to 5.38 ng/g for corn, wheat and poultry feeds respectively. Results showed also that the contamination of 10% of samples of corn was higher than…

Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats.

Abstract Study of dietary supplementation with ENN A mycotoxin during 28 days of exposure time on Wistar rats to determinate its levels in serum, urine and feces and, to evaluate the immunologic effect in peripheral blood lymphocytes (PBL) is presented. The first method for ENN A extraction, determination and detection by LC–MS/MS in serum, urine and feces samples is reported. ENN A food dose administrated was detected in serum samples and influenced lymphocyte phenotyping. Levels in serum were founded from the second week of the experiment; reaching values of 4.76 μg/ml on the fourth week, which corresponds to 3.24 μg/ml in blood. PBL as T helper (CD4 + ) were presented in greater percenta…

Multi-mycotoxin determination in barley and derived products from Tunisia and estimation of their dietary intake.

A study on raw barley and derived products (barley soup and beers) was carried out to determine the natural presence of twenty-four mycotoxins by both liquid chromatography and gas chromatography coupled to tandem mass spectrometry (MS/MS). The developed multi-mycotoxin procedure was based on both SLE and QuEChERS extraction steps. 66% of analyzed samples presented mycotoxin contamination and only one sample, which was soup of barley (6 ng/g), exceeded the maximum level (ML) established by EU for OTA (5 ng/g). Raw barley was the most contaminated matrix (62%), which concentrations ranged from 1.70 to 287.13 ng/g) and type of detected mycotoxins (DON, 15AcDON, NEO, NIV, HT2, FB1, OTA, ENA, E…

Cytotoxicity, Genotoxicity and Disturbance of Cell Cycle in HepG2 Cells Exposed to OTA and BEA: Single and Combined Actions

Mycotoxins are produced by a number of fungal genera spp., for example, Aspergillus, Penicillium, Alternaria, Fusarium, and Claviceps. Beauvericin (BEA) and Ochratoxin A (OTA) are present in various cereal crops and processed grains. This goal of this study was to determine their combination effect in HepG2 cells, presented for the first time. In this study, the type of interaction among BEA and OTA through an isobologram method, cell cycle disturbance by flow cytometry, and genotoxic potential by in vitro micronucleus (MN) assay following the TG 487 (OECD, 2016) of BEA and OTA individually and combined in HepG2 cells are presented. Cytotoxic concentration ranges studied by the MTT assay ov…

Alternariol induce toxicity via cell death and mitochondrial damage on Caco-2 cells

Alternariol (AOH), a mycotoxin produced by Alternaria sp, appears as food contaminant in fruit, vegetables and cereal products. Its toxicity has been demonstrated, but the mechanisms involved have not been elucidated yet. In this study, the pathways triggered by AOH and degradation products generated on Caco-2 cells were evaluated. Cells were exposed to AOH sub-cytotoxic concentrations of 15, 30 and 60 μM. Cell cycle disruption, the induction of apoptosis/necrosis and changes in mitochondrial membrane potential (Δψm) after 24 and 48 h was asses by flow cytometry. Also, AOH and its degradation products were evaluated after 24 and 48 h by high-performance liquid chromatography with tandem mas…

Reducing the effect of beauvericin on neuroblastoma SH-SY5Y cell line by natural products

Abstract In the present work, different natural compounds from coffee by-product extracts (coffee silverskin and spent coffee) rich in polyphenols, was investigated against beauvericin (BEA) induced-cytotoxicity on SH-SY5Y cells. Spent coffee arise as waste products through the production of instant coffee and coffee brewing; while the silverskin is a tegument which is removed and eliminated with toasting coffee grains. First of all, polyphenol extraction methods, measurement of total polyphenols content and its identification were carried out. Afterwards evaluating in vitro effects with MTT assay on SH-SY5Y cells of coffee by-product extracts and mycotoxins at different concentrations and …

Fusarium species, chemotype characterisation and trichothecene contamination of durum and soft wheat in an area of central Italy

BACKGROUND Fusarium head blight (FHB) of wheat is an important disease causing yield losses and mycotoxin contamination. The aim of the work was to detect and characterise trichothecene producing Fusarium species in durum and soft wheat cultivated in an area of central Italy in 2009 and 2010 and to determine trichothecene contamination by LC-MS/MS in the grain. RESULTS F. graminearum s. str. was the most frequent species. In 2009, the occurrence of F. avenaceum and F. poae was higher than in 2010. Among F. graminearum strains, the 15-acetyl deoxynivalenol (15-ADON) chemotype could be found more frequently, followed by nivalenol (NIV) and 3-ADON chemotypes, while all F. culmorum isolates bel…

Determination of Alternaria mycotoxins in strawberries and storage conditions

Development and Validation of a LC-ESI-MS/MS Method for the Determination of Alternaria Toxins Alternariol, Alternariol Methyl-Ether and Tentoxin in Tomato and Tomato-Based Products

Alternaria species are capable of producing several secondary toxic metabolites in infected plants and in agricultural commodities, which play important roles in food safety. Alternaria alternata turn out to be the most frequent fungal species invading tomatoes. Alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) are some of the main Alternaria mycotoxins that can be found as contaminants in food. In this work, an analytical method based on liquid chromatography (LC) tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of AOH, AME, and TEN in tomato and tomato-based products was developed. Mycotoxin analysis was performed by dispersive liquid-…

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer's storage conditions. Sample extraction was performed using a liquid-liquid extraction method prior to LC-MS/MS analysis…

Individual and Combined Effect of Zearalenone Derivates and Beauvericin Mycotoxins on SH-SY5Y Cells

Beauvericin (BEA) and zearalenone derivatives, &alpha

Evaluation of Mycotoxins in Infant Breast Milk and Infant Food, Reviewing the Literature Data

In this review, an analysis focusing on mycotoxin determination in infant breast milk and infant food has been summarised for the last fifteen years of research focused on the intended population group of 1–9 months. The objective was to know the level of exposure of the child population to an estimated daily intake (EDI) of mycotoxins from the consumption of habitual foods. The EDI was compared with the tolerable daily intake (TDI) established by EFSA to estimate risk. In breast milk, the high prevalence and levels were for samples from Africa (Egypt and Tanzania) with aflatoxin M1 (1.9 μg/L and 10%), and Asia (Iran) with ochratoxin-A (7.3 μg/L and 100%). In infant formulas, high incidence…

Determination of ochratoxin A in maize bread samples by LC with fluorescence detection.

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus, A. carbonarius, A. niger and by Penicillium verrucosum. The present work shows the results of comparative studies using different procedures for the analysis of OTA in maize bread samples. The studied analytical methods involved extraction with different volumes of PBS/methanol, different extraction apparatus, and clean-up through immunoaffinity columns. The separation and identification were carried out by high-performance liquid chromatography with fluorescence detection. The optimized method for analysis of OTA in maize bread involved extraction with PBS:methanol (50:50), and …

Extraction of Phenolic Compounds from Fresh Apple Pomace by Different Non-Conventional Techniques

Red Delicious apple pomace was produced at laboratory scale with a domestic blender and different non-conventional extraction techniques were performed to isolate phenolic compounds, such as ultrasound-assisted extraction (UAE), ultraturrax extraction (UTE), accelerated solvent extraction (ASE) and pulsed electric field (PEF) extraction pre-treatment. Total phenolic content (TPC) was determined by Folin–Ciocalteu assay. Phloridzin, the main phenolic compound in apples, was determined by chromatographic analysis Q-TOF-LC/MS. The results obtained with these techniques were compared in order to identify the most efficient method to recover polyphenols. The highest value of TPC (1062.92 ± 59.80…

Comparative cytotoxicity effect of zearalenone and its metabolites on the CHO-K1 cells

First study on trichothecene and zearalenone exposure of the Romanian population through wheat-based products consumption

Abstract In this study, a dietary exposure assessment of mycotoxins was conducted for the Romanian population using the contamination data of a various categories of wheat-based products for direct human consumption. Wheat-based foods (n = 181) commercialized in Romania, including flour, bread, biscuits, breakfast cereals and pasta, were evaluated by GC-QqQ-MS/MS for the occurrence of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3AcDON), 15-acetyldeoxynivalenol (15AcDON), fusarenon-X, nivalenol, HT-2 and T-2 toxins, diacetoxyscirpenol, neosolaniol and zearalenone (ZEA). DON and 15AcDON were detected in 63 and 5% of all the analyzed samples, whereas 13-AcDON, HT-2, T-2, NIV and ZEA were not…

Study on Trichothecene and Zearalenone Presence in Romanian Wheat Relative to Weather Conditions

To evaluate the influence of weather conditions on mycotoxin presence in wheat, deoxynivalenol (DON), 3-acetyldeoxynivalenol (3AcDON), 15-acetyldeoxynivalenol (15AcDON), fusarenon-X (FUS-X), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), diacetoxyscirpenol (DAS), neosolaniol (NEO) and zearalenone (ZEN) were evaluated in 102 Romanian wheat samples coming from five wheat growing areas during 2015. Only six mycotoxins were detected, while FUS-X, DAS, NEO and NIV were not present in the wheat samples. Mycotoxin concentrations were correlated with precipitation and temperature values during anthesis and the preharvest period. Overall, the highest frequency was registered for DON, while the…

Proteomics evaluation of enniatins acute toxicity in rat liver

Abstract Enniatins (ENs) are emerging mycotoxins produced by Fusarium fungi which are cytotoxic also at low concentrations due to its ionophoric properties. The aim of this study was to evaluate the hepatic toxicity of ENs exposure at different concentrations in Wistar rats through a proteomic approach. Animals were intoxicated by oral gavage with medium (EN A 256, ENA1 353, ENB 540, ENB1 296 μg/mL) and high concentrations (ENA 513, ENA1 706, ENB 1021, ENB1 593 μg/mL) of an ENs mixture and sacrificed after 8 h. Protein extraction was performed using powdered liver. Peptides were analyzed using a liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Proteins were …

Zearalenone presence in wheat field samples from Romania

Climatic conditions influence emerging mycotoxin presence in wheat grown in Romania – A 2-year survey

Abstract The correlation between the occurrence of four enniatins (ENA, ENA1, ENB, and ENB1) and beauvericin (BEA) and the weather parameters during anthesis and preharvest period was studied in 97 wheat samples collected in 2014 and 2015 across three counties from central and south Romania (Brasov, Dâmboviţa, and Teleorman). The highest mean values of ENA (16.1 μg kg −1 ) and ENB (147.1 μg kg −1 ) were measured in the samples from Brasov county in the harvest year 2015, whereas for ENA1 and ENB1 the highest means (55.2 μg kg −1 , and 108.0 μg kg −1 , respectively) were noted in samples from Teleorman county in 2014. Statistically significant differences ( P r s  = 0.8745 and r s  = 0.9326;…

Binary and tertiary combination of alternariol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on HepG2 cells: Toxic effects and evaluation of degradation products.

Fungi producers of mycotoxins are able to synthesize more than one toxin. Alternariol (AOH) is one of the mycotoxins produced by several Alternaria species, the most common one being Alternaria alternata. The toxins 3-Acetyl-deoxynivalenol (3-ADON) and 15-Acetyl-deoxynivalenol (15-ADON) are acetylated forms of deoxynivalenol (DON) produced by Fusarium graminearum. In the present work it is determined and evaluated the toxic effects of binary and tertiary combination treatment of HepG2 cells with AOH, 3-ADON and 15-ADON, by using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), to subsequently apply the isobologram method and elucidate if the mixtures of these m…

Determination of mycotoxins in fruit berry by-products using QuEChERS extraction method

Abstract Fruit berries by-products are mainly elaborated with berry but most of them mixed with other fruits, grapes, plums or apples. These present favourable properties for the growth of a great range of mycotoxins mould producers. Consequently, alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), aflatoxins (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) are the main mycotoxins that should be present in berries by-products. To establish a reliable analytical method of these mycotoxins on two fruit berries by-products (jam and juice), four different QuEChERS extraction method were evaluated. Recoveries obtained were higher than 74% and 76% in berries juice and jam berrie…

Accelerated solvent extraction of ochratoxin a from rice samples.

In this paper, accelerated solvent extraction (ASE) for the analysis of ochratoxin A (OTA) is applied for the first time. Optimization of the method is given for the extraction of OTA from rice samples. Several parameters such as type of solvent, temperature, pressure, static time, and cell size were investigated thoroughly to find the optimal extraction conditions. The optimum ASE operating conditions were methanol as extraction solvent, 1500 psi, 40 degrees C, 5 min of static time, 50% flush volume, 60 s of purge, 1 cycle, and 11 mL cell size. The total extraction time was approximately 15 min. OTA was determined by liquid chromatography coupled with a fluorescence detector and confirmed …

Evaluation of beauvericin and enniatins in Italian cereal products and multicereal food by liquid chromatography coupled to triple quadrupole mass spectrometry

In this study, 48 multicereal baby foods samples including 25 of pasta and 23 of multicereal baby foods from supermarkets of Campania region (Italy) were analysed for evaluating the presence of beauvericin (BEA) and enniatins (ENs) A, A1, B, B1 and B4. Subsequently to evaluate the risk exposure of Italian population and infant population over the consumption of pasta or multicereal baby food, was, respectively, evaluated. For the above mentioned evaluation, a method developed in our laboratory by liquid chromatography tandem mass spectrometry was used. A liquid phase dispersion procedure was optimised for the simultaneous extraction of BEA and the five ENs from multicereal baby food samples…