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…

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 …

Determination of quinolone residues in chicken and fish by capillary electrophoresis-mass spectrometry

A specific pressure-assisted CE-MS method is described for the analysis of five quinolone residues. MS using a single quadrupole is compared with multiple-stage MS using a quadrupole IT (QIT-MS(n)). The procedure involves a common sample preparation by SPE on disposable cartridges. The most suitable electrolyte is 60 mM (NH(4))(2)CO(3) at pH 9.2. Single quadrupole does not provide enough fragmentation to confirm identities according to the current legislation. However, QIT-MS(n) achieves selective fragmentation. Using this method, danofloxacin, enrofloxacin, flumequine, ofloxacin, and pipemidic acid are analyzed in fortified samples of chicken and fish. Recoveries at levels of 50 ng/g were …

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 …

Micronucleus induction and cell cycle alterations produced by deoxynivalenol and its acetylated derivatives in individual and combined exposure on HepG2 cells.

Mycotoxins are produced by a number of fungal genera spp as e.g. Aspergillus, Penicillium, Alternaria, Fusarium and Claviceps. 3-Acetyl-Deoxynivalenol (3-A-DON) and 15-Acetyl-Deoxynivalenol (15-ADON) which are produced by Fusarium, chemically belong to trichothecenes and occur in significant amounts as modified forms of deoxynivalenol (DON) in various cereal crops and processed grains. This study aims to determine the cytotoxicity, cell cycle and genotoxicity of the mycotoxins DON, 3-A-DON and 15-A-DON on HepG2 cells. Cytotoxic concentration range studied was from 100 to 3.1 μM for DON and 12.5 to 0.04 μM for 3-A-DON and 15-A-DON by the Neutral Red (NR) assay, over 24, 48 and 72 h. Potentia…

Reactive oxygen species induced by beauvericin, patulin and zearalenone in CHO-K1 cells

The cytotoxic effects of mycotoxins, induction of reactive oxygen species (ROS) and generation of lipid peroxidation products in CHO-K1 cells were determined as function of increasing time of exposure and concentrations of beauvericin (BEA), patulin (PAT) and zearalenone (ZEA). The end points were evaluated after 24h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC(50) values obtained on the MTT and NR assays ranged from 0.69 to 79.40 microM and 4.40 to 108.76 microM, respectively. To determine the intracellular production of ROS, the intensity of fluorescence emitted from the probe H(2)-DCFDA was measured. The relative intensity of fluorescence from cells incu…

Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro.

Abstract Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health. In this study, the acute toxicity of individual and combined mycotoxins (BEA, DON and T-2) were tested in immortalized hamster ovarian cells (CHO-K1) at 24, 48 and 72 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC50 values obtained for all mycotoxins by the MTT and NR assays ranged from 0.017 to 12.08 μM and from 0.042 to 17.22 μM, respectively. Both, individual and combined mycotoxins demonstrated a significant cytotoxic effect in CHO-K1 cells in a dose-dependent manner. When mycotoxins were assayed individuall…

Pressurized liquid extraction combined with capillary electrophoresis–mass spectrometry as an improved methodology for the determination of sulfonamide residues in meat

A new analytical method, based on capillary electrophoresis and tandem mass spectrometry (CE-MS2), is proposed and validated for the identification and simultaneous quantification of 12 sulfonamides (SAs) in pork meat. The studied SAs include sulfathiazole, sulfadiazine, sulfamethoxypyridazine, sulfaguanidine, sulfanilamide, sulfadimethoxyne, sulfapyridine, sulfachloropyridazine, sulfisoxazole, sulfasalazine, sulfabenzamide and sulfadimidine. Different parameters (i.e. separation buffer, sheath liquid, electrospray conditions) were optimized to obtain an adequate CE separation and high MS sensitivity. MS2 experiments using an ion trap as analyzer, operating in the selected reaction monitori…

DNA damage and perturbation on cell cycle and mitochondrial membrane potential by alternariol mycotoxin in Caco-2 cells

Fluorescent probes on HepG2 cells treated with emergent Fusarium mycotoxins

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…

Introduction to the Toxins Special Issue on Toxicological Effects of Mycotoxin on Target Cells.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi from Fusarium, Alternaria and Penicillium spp [...]

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…

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…

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…

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…

Mechanisms of beauvericin toxicity and antioxidant cellular defense

Beauvericin (BEA) is a secondary metabolite produced by many species of fungus Fusarium. This study determines the injury (cell viability, cell proliferation, mitochondrial membrane potential, cell death and DNA damage) and the intracellular defense mechanisms (catalase and superoxide dismutase) in Chinese Hamster ovary (CHO-K1) cells after BEA exposure. The results obtained in this study demonstrated that BEA induces cytotoxicity in a dose- and time-dependent manner in CHO-K1 cells. Moreover, disruption in mitochondrial enzymatic activity and cell proliferation has been observed after BEA exposure, which can lead or be consequence of cell death. BEA inhibits cell proliferation by arresting…

On-line preconcentration strategies for analyzing pesticides in fruits and vegetables by micellar electrokinetic chromatography.

Five pesticides (fludioxonil, procymidone, pyriproxyfen, dinoseb and carbendazim) were separated in reversed migration micellar electrokinetic chromatography (RM-MEKC) using 20 mmol l(-1) phosphate buffer at pH 2.3, containing 25 mmol l(-1) sodium dodecylsulfate and 10% methanol. Three on-line concentration strategies, sweeping (SW), normal stacking with reversed migration and a water plug (SRW) and stacking with reverse migration and removal of sample matrix using polarity switching (SRMM), were compared. About 10-, 30- and 50-fold increases in detection sensitivity, compared with standard hydrodynamic injection (5 s at 0.5 psi), were observed with SW, SRW and SRMM, respectively. Limits of…

Does low concentration mycotoxin exposure induce toxicity in HepG2 cells through oxidative stress?

The purpose of this study was to determine whether exposure to low concentrations of deoxynivalenol (DON), T-2 toxin (T-2) and patulin (PAT) in a human hepatocellular carcinoma cell line (HepG2) exerts toxic effects through mechanisms related to oxidative stress, and how cells deal with such exposure. Cell viability was determined by the MTT and protein content (PC) assays over 24, 48 and 72 h. The IC

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…

Beauvericin-induced cytotoxicity via ROS production and mitochondrial damage in Caco-2 cells.

The cytotoxicity of beauvericin (BEA) on human colon adenocarcinoma (Caco-2) cells was studied as a function of time. Moreover, the oxidative damage and cell death endpoints were monitored after 24, 48 and 72 h. After BEA exposure, the IC₅₀ values ranged from 1.9 ± 0.7 to 20.6 ± 6.9 μM. A decrease in reduced glutathione (GSH; 31%) levels, as well as an increase in oxidized glutathione (GSSG, 20%) was observed. In the presence of BEA, reactive oxygen species (ROS) level was highly increased at an early stage with the highest production of 2.0-fold higher than the control that was observed at 120 min. BEA induced cell death by mitochondria-dependent apoptotic process with loss of the mitochon…

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…

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…

Evaluation of solid-phase extraction and stir-bar sorptive extraction for the determination of fungicide residues at low-microg kg(-1) levels in grapes by liquid chromatography-mass spectrometry.

Abstract A liquid chromatography–mass spectrometry method has been developed for determining bitertanol, carboxin, flutriafol, pyrimethanil, tebuconazole and triadimefon. The evaluation of both atmospheric pressure interfaces (API), atmospheric pressure chemical ionization (APCI) and electrospray (ESI) using positive and negative ionization modes, clearly shows that the studied pesticides are more sensitive using APCI in positive mode. Two procedures based on solid-phase extraction (SPE) and stir-bar sorptive extraction (SBSE) have been assessed for extracting these compounds in grape. The recoveries obtained by SPE in samples spiked at the limit of quantification (LOQ) level ranged from 60…

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…

Simultaneous determination of different classes of antibiotics in fish and livestock by CE-MS

A specific CE-MS method was developed for the simultaneous determination of 12 antibacterial residues (four sulfonamides: sulfamethazine, sulfathiazole, sulfadiazine, and sulfachlorpyridazine; four beta-lactams: amoxicillin, ampicillin, oxacillin, and penicillin V, and four quinolones: danofloxacin, enrofloxacin, ofloxacin, and flumequine) in fish and livestock. Separation conditions, sheath liquid composition and electrospray parameters were optimized to obtain adequate CE separation and a high sensitivity. CE employed a 75 cm long fused-silica capillary (50 cm thermostated plus 25 cm at room temperature) 75 microm id and a 60 mM ammonium acetate separation buffer at pH 8 with 10% of metha…

Capillary electrophoresis for analyzing pesticides in fruits and vegetables using solid-phase extraction and stir-bar sorptive extraction.

Two procedures based on solid-phase extraction (SPE) and stir-bar sorptive extraction (SBSE) in combination with micellar electrokinetic chromatography (MEKC)--diode array detection (DAD) were compared for the simultaneous extraction of acrinathrin, bitertanol, cyproconazole, fludioxonil, flutriafol, myclobutanil, pyriproxyfen, and tebuconazole in lettuce, tomato, grape, and strawberry. Selectivity and resolution of the MEKC procedure were studied changing the pH and the molarity of the buffer, the type and the concentration of surfactant, and the methanol content in the mobile phase. A buffer consisting of 6 mM sodium tetraborate decahydrate with 75 mM of cholic acid sodium solution (pH 9.…

Blood, breast milk and urine: potential biomarkers of exposure and estimated daily intake of ochratoxin A: a review.

The purposes of this review are to study potential biomarkers of exposure for ochratoxin A (OTA) in biological fluids (blood, urine and breast milk) for the period 2005-14, calculate the estimated daily intake (EDI) of OTA by using database consumption for the Spanish population, and, finally, to correlate OTA levels detected in blood and EDI values calculated from food products. The values of OTA detected in potential biomarkers of exposure for blood, breast milk and urine ranged from 0.15 to 18.0, from 0.002 to 13.1, and from 0.013 to 0.2 ng ml(-1), respectively. The calculated EDI for OTA in plasma ranged from 0.15 to 26 ng kg(-1) bw day(-1), higher than that obtained in urine (0.017-0.4…

In vitro effect of organophosphate pesticides, Malathion and chlorpyriphos, on lipid peroxidation and antioxidant enzymes

Determination of organic contaminants in food by capillary electrophoresis

This review addresses recent advances in the analysis of organic contaminants, such as antibiotics, pesticides, biological toxins, and food-borne pathogens, in foods by capillary electrophoresis (CE). Special attention is paid to those aspects that increase sensitivity and/or selectivity, such as sample extraction and concentration, on-line preconcentration techniques (stacking), affinity capillaries or/and specific detectors (laser induced fluorescence (LIF), mass spectrometry (MS)). The various CE modes used to separate the compounds and the quantification strategies are also examined. As a result, this work presents an updated overview on the principal applications of CE, together with a…

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…

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…

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 …

Determination of Alternaria mycotoxins in strawberries and storage conditions

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…

Beauvericin and enniatin B effects on a human lymphoblastoid Jurkat T-cell model

Abstract 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 d…

Enniatin A1, enniatin B1 and beauvericin on HepG2: Evaluation of toxic effects

Hepatotoxicity of three Fusarium mycotoxins, beauvericin (BEA) and two enniatins (ENNs) ENN A1 and ENN B1, in hepatocarcinoma cells (HepG2) were evaluated and compared. Concentrations used were 1.5 and 3 μM at 24, 48 and 72 h for each mycotoxin. Flow cytometry was used to examine enniatins effects on cell proliferation, to characterize the cell cycle phase where the cells blocked and to study the mitochondria role in ENNs-induced apoptosis. ENN B1 treated cells showed a time dependent G1 blockade at both concentrations used. ENN A1 and BEA decreased the apoptotic-necrotic percentage of cells comparing to control and disrupted the MMP as observed by TMRM and ToPro-3 fluorochromes signal. It …

Beauvericin and ochratoxin A mycotoxins individually and combined in HepG2 cells alter lipid peroxidation, levels of reactive oxygen species and glutathione

The co-presence of more than one mycotoxin in food is being evidenced in last food surveys as reported in the literature. Beauvericin (BEA) is a non-legislated emergent mycotoxin while Ochratoxin A (OTA) has been widely studied and legislated. Concentration range individually studied was from 2.5 to 0.3 μM for BEA and from 25 to 3.1 μM for OTA; binary mixture [BEA + OTA] comprised concentrations of 1:10 ratio from [2.5 + 25] to [3.1 + 0.3] μM. Potential of toxicity of BEA in HepG2 cells was the highest at all times assayed (24, 48 and 72h). LPO was performed through malondyaldehyde (MDA) detection denoting in the binary mixture for [1.25 + 12.5] μM and at 24 and 72h the highest disturbance …

Larval zebrafish as an in vitro model for evaluating toxicological effects of mycotoxins.

The presence of mycotoxins in food has created concern. Mycotoxin prevalence in our environment has changed in the last few years maybe due to climatic and other environmental changes. Evidence has emerged from in vitro and in vivo models: some mycotoxins have been found to be potentially carcinogenic, embryogenically harmful, teratogenic, and to generate nephrotoxicity. The risk assessment of exposures to mycotoxins at early life stages became mandatory. In this regard, the effects of toxic compounds on zebrafish have been widely studied, and more recently, mycotoxins have been tested with respect to their effects on developmental and teratogenic effects in this model system, which offers …

Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A1, B and B1

The cytotoxic effects, the generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) as well as the cell cycle disruption, the induction of apoptosis and changes in mitochondrial membrane potential (ΔΨm) as a function of increasing time have been determined in human colorectal adenocarcinoma (Caco-2) cells after exposure to enniatins (ENs) A, A₁, B and B₁. IC₅₀ values obtained by the MTT and Neutral Red assay, after 24, 48 and 72 h of exposure ranged from 0.5±0.1 to >15 μM. A significant increase (p≤0.05) in ROS generation and LPO production, as determined by the fluorescent probe H2-DCFDA and TBARS method respectively, was observed for all mycotoxins tested at 3.0 μM concent…

Comparison of toxic effects on CHO-KL and VERO cells after individual and combined treatment with Fusarium mycotoxins, beauvericin, deoxynivalenol and the T-2 toxin

Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, t…

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…

Quantitative analysis of six pesticides in fruits by capillary electrophoresis-electrospray-mass spectrometry.

A method to identify and quantify six pesticide residues - dinoseb, pirimicarb, procymidone, pyrifenox, pyrimethanil, and thiabendazole - in peaches and nectarines using capillary electrophoresis-electrospray ionization-quadrupole ion trap-tandem mass spectrometry (CE-ESI-MS/MS) is described. Separation was carried out using a buffer of 0.3 M ammonium acetate at pH 4 with 10% methanol. Pesticide residues present in peach and nectarine samples were preconcentrated by solid-phase extraction using C(18), eluted with CH(2)Cl(2), concentrated to dryness, and redissolved in buffer to obtain lower detection limits. The recoveries of the analytes ranged from 58 to 99% and the relative standard devi…

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…

Cytotoxic effects of mycotoxin combinations in mammalian kidney cells

The cytotoxicity of three Fusarium mycotoxins (beauvericin, deoxynivalenol and T-2 toxin) has been investigated using the NR assay, after 24, 48 and 72h of incubation. The IC(50) values ranged from 6.77 to 11.08, 3.30 to 10.00 and 0.004 to 0.005 for beauvericin, deoxynivalenol and T-2 toxin, respectively. Once the potential interaction has been detected, a quantitative assessment is necessary to ensure and characterize these interactions, that is, each mycotoxin contributes to the toxic effect in accord with its own potency. Combination of mycotoxins was determined in Vero cells after 24, 48 and 72h of exposure. Isobolograms and median effect method of Chou and Talalay were used to assess t…

Presence of ochratoxin A (OTA) mycotoxin in alcoholic drinks from southern European countries: wine and beer.

The main filamentous fungi producers of mycotoxins are Aspergillus spp., Penicillium spp., and Fusarium spp. Their effect can provoke a broad range of toxic properties including carcinogenicity and neurotoxicity, as well as reproductive and developmental toxicities. Ochratoxin A (OTA) is produced by Aspergillus and Penicillium spp. The purpose of this review was to evaluate the risk assessment of OTA in alcoholic drinks (beer and wine) by compiling the results obtained from studies and reviews related to the presence of OTA in these two drinks from southern European countries in the period 2005–2013 and comparing those results with the legislation available in the European Union.

Applications of flow cytometry to toxicological mycotoxin effects in cultured mammalian cells: a review.

This review gives an overview of flow cytometry applications to toxicological studies of several physiological target sites of mycotoxins on different mammalian cell lines. Mycotoxins are secondary metabolites of fungi that may be present in food, feed, air and water. The increasing presence of mycotoxins in crops, their wide distribution in the food chain, and their potential for toxicity demonstrate the need for further knowledge. Flow cytometry has become a valuable tool in mycotoxin studies in recent years for the rapid analysis of single cells in a mixture. In toxicology, the power of these methods lies in the possibility of determining a wide range of cell parameters, providing valuab…

Does low concentration mycotoxin exposure induce toxicity in HepG2 cells through oxidative stress?

The purpose of this study was to determine whether exposure to low concentrations of deoxynivalenol (DON), T-2 toxin (T-2) and patulin (PAT) in a human hepatocellular carcinoma cell line (HepG2) exerts toxic effects through mechanisms related to oxidative stress, and how cells deal with such exposure. Cell viability was determined by the MTT and protein content (PC) assays over 24, 48 and 72 h. The IC50 values detected ranged from >10 to 2.53 ± 0.21 μM (DON), 0.050 ± 0.025 to 0.034 ± 0.007 μM (T-2) and 2.66 ± 0.66 to 1.17 ± 0.21 µM (PAT). The key players in oxidative stress are the generation of reactive oxygen species (ROS), lipid peroxidation (LPO) and mitochondrial membrane potential …