Daphnia magna model for the study of mycotoxins present in food: Gliotoxin, ochratoxin A and its combination.

Mycotoxins are low molecular weight compounds present in food and feed. Although their effects on human health have been widely described, their mechanisms of action are still undefined. Gliotoxin (GTX) and ochratoxin A (OTA) are among the most dangerous mycotoxins produced by Aspergillus spp. Therefore, their toxicity was studied in the Daphnia magna model, which has high capacity to predict cytotoxicity and assess ecotoxicity, comparable to mammalian models. The study consisted of a series of tests to evaluate the effects of mycotoxins GTX, OTA and their combinations at different dilutions on Daphnia magna that were conducted according to standardized OECD 202 and 211 guidelines. The following assays were carried out: acute toxicity test, heartbeat, delayed toxicity test, reproduction, growth rate test. Reproducibility was determined by observing the offspring after 21 days of GTX exposure. In acute and delayed toxicity transcript levels of genes involved in xenobiotic metabolism (mox, gst, abcb1, and abcc5), and oxidative stress (vtg-SOD) were analyzed by qPCR. GTX showed acute toxicity and decreased heart rate in D. magna compared to OTA. On the other hand, OTA showed a delayed effect as evidenced by the immobility test. Both mycotoxins showed to increase genes involved in xenobiotic metabolism, while only the mycotoxin mixture increased oxidative stress. These results suggest that the mycotoxins tested could have negative impact on the environment and human health.

Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure.

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

Cell cycle and enzymatic activity alterations induced by ROS production in human neuroblastoma cells SH-SY5Y exposed to Fumonisin B1, Ochratoxin A and their combination.

The presence of mycotoxins such as Fumonisin B1(FB1) and Ochratoxin A (OTA) in food and feed has become a threat to human and animal health since they can produce several afflictions. Different mechanisms of action by which they exercise their cytotoxic activity have been attributed to them, including the production of reactive oxygen species (ROS). For this reason, a measurement of the production of ROS species, and an evaluation of the intrinsic cell enzymatic antioxidant activity, including glutathione peroxidase (GPx), glutathione transferase (GTS), and catalase (CAT) together with a cytotoxicity and cell cycle assay have been performed in undifferentiated SH-SY5Y cells exposed to FB1, OTA and [FB1 + OTA] after 24 h and 48 h. FB1 and OTA. Monitoring of intracellular ROS production was carried out by the H2-DCFDA probe; while spectrometry analysis of absorbances was used for measuring GPx, GST and CAT activity. Finally, cell proliferation and cell cycle distribution were studied by flow cytometry. When cells were treated with OTA, an increase in GPx and GST activity was observed compared to FB1 and [FB1 + OTA]; conversely, a decrease in CAT activity was observed when cells were exposed to OTA coinciding with the results observed for ROS measurement. Regarding the cell cycle, when cells were exposed to OTA, a decrease in G0/G1 was detected, revealing an arrest of cell division for SH-SY5Y cells at the concentrations studied.

Effectiveness of beetroot extract in SH-SY5Y neuronal cell protection against Fumonisin B1, Ochratoxin A and its combination.

Fumonisin B1 (FB1) and ochratoxin A (OTA) are fungal metabolites of worldwide concern because of their effect on human and animal health, as both have been classified by IARC as possible carcinogens (Group 2B). Beetroot is a source of dietary fiber, folic acid, and vitamin C, and some studies have demonstrated their antioxidant activity. Therefore, this work presents the cytoprotective effect of beetroot extract (BRE) on a neuroblastoma cell line (SH-SY5Y cells) exposed to FB1, OTA, and its combination. Cytotoxicity was studied by the MTT ([3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, for 24 h and 48 h. Simultaneous treatment and pre-treatment strategies were tested with 1:512-1:2 and 1:0 dilutions of BRE, with a concentration range from 0.4 to 100 µM of FB1 and from 0.19 to 50 µM of OTA. IC50 values of 5.8 µM and 9.1 µM at 24 h and 48 h, respectively were obtained for OTA while no cytotoxic effect was detected at the concentrations tested for FB1. Cytoprotection with increased viability was obtained when the simultaneous BRE + OTA strategy was performed. Finally, better protection was observed in the pretreatment strategy in which cells were exposed 24 h previously to BRE, compared to that shown in the simultaneous assay.

Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing.

Given the increasing importance of establishing better risk assessments for mycotoxins, novel in vitro tools for the evaluation of their toxicity are mandatory. In this study, an in vitro 3D spheroid model from SH-SY5Y cells, a human neuroblastoma cell line, was developed, optimized and characterized to test the cytotoxic effects caused by the mycotoxin sterigmatocystin (STE). STE induced a concentration- and time-dependent cell viability decrease in spheroids. Spheroids displayed cell disaggregation after STE exposure, increasing in a dose-dependent manner and over time. STE also induced apoptosis as confirmed by immunofluorescence staining and Western blot. Following the decreased proliferation and increased apoptosis, STE cytostasis effects were observed by migration assays both in 2D and 3D cell culture. Increased ROS generation, as well as DNA damage were also observed. Taken together, these data highlight the cytotoxic properties of STE and suggest that cell culture models play a pivotal role in the toxicological risk assessment of mycotoxins. The evaluation of cytotoxicity in spheroids (3D) rather than monolayer cultures (2D) is expected to more accurately reflect in vivo-like cell behaviour.

Role of quercetin on sterigmatocystin-induced oxidative stress-mediated toxicity.

Oxidative stress appears to be a common trigger for many of the effects associated with the exposure to various mycotoxins, including sterigmatocystin (STE). However, studies to alleviate STE toxicity through the use of natural antioxidants are sparsely reported in literature. In the present study, the cytoprotective effect of quercetin (QUE) was tested in SH-SY5Y cells against STE-induced oxidative stress and cytotoxicity. The MTT assay revealed that STE decreased cell viability, whereas pre-treatment of cells with QUE restored it. The QUE was also found to counteract STE-induced ROS generation and decrease STE-induced up-regulation of the expression of the stress-inducible enzymes HO-1 and NOS-2. Pre-treatment with QUE also prevented STE-induced nuclear translocation of NF-κB, as measured by immunofluorescence. Finally, considering the key role played by NF-κB in the regulation of inflammation, the effect of STE on the pro-inflammatory cytokines TNF-α and IL-6 expression was evaluated. Our results showed the down-regulation of TNF-α and IL-6 following STE exposure, suggesting a negative immunomodulatory effect of STE. In QUE pre-treated samples, TNF-α and IL-6 were significantly further reduced, indicating the anti-inflammatory role of QUE. The results of the present study demonstrate for the first time that QUE exerts a cytoprotective role in STE-induced toxicity.

Dietary Exposure to Mycotoxins through Alcoholic and Non-Alcoholic Beverages in Valencia, Spain.

The present study investigated the presence of 30 mycotoxins in 110 beverage samples of beer, wine, cava, and cider purchased in Valencia (Spain). A validated method based on dispersive liquid-liquid microextraction and chromatographic methods coupled with tandem mass spectrometry was applied. The method showed satisfactory recoveries ranging from 61 to 116% for the different beverages studied. The detection and quantification limits ranged from 0.03 to 2.34 µg/L and 0.1 to 7.81 µg/L, respectively. The results showed that beer samples were the most contaminated, even with concentrations ranging from 0.24 to 54.76 µg/L. A significant presence of alternariol was found in wine, which reached concentrations up to 26.86 µg/L. Patulin and ochratoxin A were the most frequently detected mycotoxins in cava and cider samples, with incidences of 40% and 26%, respectively. Ochratoxin A exceeded the maximum level set by the EU in one wine sample. The results obtained were statistically validated. The combined exposure was assessed by the sum of mycotoxin concentrations contaminating the same samples to provide information on the extent of dietary exposure to mycotoxins. No significant health risk to consumers was associated with the mycotoxin levels detected in the beverages tested.

Sterigmatocystin-induced DNA damage triggers cell-cycle arrest via MAPK in human neuroblastoma cells.

Sterigmatocystin (STE) is a common mycotoxin found in food and feed. Many studies showed that STE is genotoxic. However, up to now, the potential genotoxicity of STE on human neuronal system remains unknown. In this study, we explored the effect of STE on DNA damage and cell-cycle progression on human neuroblastoma SH-SY5Y cells exposed to various concentrations of STE (0.78, 1.56 and 3.12 µM) for 24 h. The results indicated that STE exposure induced DNA damage, as evidenced by DNA comet tails formation and increased γH2AX foci. Additionally, genotoxicity was confirmed by micronuclei (MN) analysis. Furthermore, we found that STE exposure led to cell-cycle arrest at the S and the G2/M phase. Considering the important role played by MAPK and p53 signaling pathways in cell-cycle arrest, we explored their potential involvement in STE-induced cell-cycle arrest by using specific inhibitors. The inhibition of JNK and ERK resulted to attenuate S and G2/M arrest, whereas the inhibition of p38 and p53 attenuated only STE-induced S phase arrest. In conclusion, the present study demonstrates that STE induced DNA damage and triggered MAPK and p53 pathways activation, resulting in cell-cycle arrest at the S and the G2/M phase.

Occurrence, mitigation and in vitro cytotoxicity of nivalenol, a type B trichothecene mycotoxin - Updates from the last decade (2010-2020).

The present review aims to give an overview of the literature of the last decade (2010-2020) concerning the occurrence of the type B trichothecene mycotoxin nivalenol (NIV) and its in vitro toxicity, with the purpose of updating information regarding last researches on this mycotoxin. The most recent studies on the possible methods for preventing Fusarium spp. growth and NIV production are also discussed. Recently, various environmental factors have been shown to influence strongly NIV occurrence. However, Fusarium spp. of the NIV genotype have been found almost worldwide. With regard to NIV cytotoxicity, NIV has been reported to cause a marked decrease in cell proliferation in different mammalian cells. In particular, the recent data suggest that organs containing actively proliferating cells represent the main targets of NIV. Moreover, NIV resulted to cause immunosuppression, gastrointestinal toxicity and genotoxicity. However, sufficient evidence of carcinogenicity in humans is currently lacking, and the International Agency for Research on Cancer (IARC) classifies it as a group 3 carcinogen. Further researches and the discovery of effective treatment strategies to prevent NIV contamination and to counteract its toxicity are urgently required against this common food-borne threat to human health and livestock.

Sterigmatocystin: Occurrence, toxicity and molecular mechanisms of action - A review.

The mycotoxin sterigmatocystin (STE) is produced mainly by Aspergillus fungi. It has been reported to occur in grains and grain-based products, cheese, coffee, spices and beer. The STE is a known biogenic precursor of aflatoxin B1, sharing with it several structural and biological similarities. The STE has been shown to be hepatotoxic and nephrotoxic in animals and it has been classified as possible human carcinogen (group 2B) by IARC. The STE has been reported to cause a marked decrease in cell proliferation in different mammalian cells. Data available on literature suggest that the cellular mechanisms underlying STE-induced toxicity include the induction of oxidative stress, mitochondrial dysfunction, apoptosis, cell cycle arrest, as well as alteration of immune system function and activation of different signalling pathways. Moreover, STE resulted to be genotoxic, being able to form DNA-adducts and induce DNA damage. Despite its strong cytotoxicity, no risk assessments have been still carried out by authorities due to the lack of toxicity data, so research on STE toxicological impact is still going on. This review reports information available regarding STE toxicity and its related mechanisms of action with the aim of updating information regarding last researches on this mycotoxin.

The role of mitochondria in sterigmatocystin-induced apoptosis on SH-SY5Y cells.

Mitochondria are cellular organelles involved in many crucial functions, such as generation of energy (ATP) and initiation of apoptosis. The aim of the present study was to evaluate the role of mitochondria in the toxicity induced by sterigmatocystin (STE), a mycotoxin produced by fungi of the genus Aspergillus, on SH-SY5Y cells. Our results showed that STE exposure decreased cell viability in a time- and concentration-dependent manner by MTT assay and caused mitochondrial dysfunction, as highlighted by the increase of STE cytotoxicity in cells forced to rely on mitochondrial oxidative phosphorylation. Furthermore, intracellular ATP depletion and increased mitochondrial reactive oxygen species were also observed. Since mitochondria play a pivotal role in apoptosis, the induction of this process in response to STE exposure was decided to study. Our results showed an increase in apoptotic cell population by flow cytometry, further confirmed by the up-regulation of the expression levels of the pro-apoptotic genes Bax and Casp-3 and the down-regulation of the anti-apoptotic gene Bcl-2 by qPCR technique. Taken together, our results provide novel insights in the signalling pathways of the cell death process induced by STE in SH-SY5Y cells, highlighting the key role played by mitochondria in STE toxicity.

Cytotoxic effects of individual and combined sterigmatocystin and nivalenol on liver hepatocellular carcinoma cells.

Since humans are exposed to different mycotoxins through daily intake, there is increasing concern about the adverse effects of the interactions between them. Cytotoxicity of sterigmatocystin (STE) and nivalenol (NIV) alone and in combination in human hepatocarcinoma (HepG2) cells was evaluated by MTT assay. Furthermore, ROS production and alteration of ΔΨm as mechanisms of action were assessed. Cells were treated with concentrations ranging from 0.15 to 5 µM for NIV and from 0.78 to 50 µM for STE individually and in binary combinations. The combination ratio between the mixture STE + NIV was 10:1. The IC50 values of NIV ranged from 0.96 to 0.66 µM, whereas no IC50 values were obtained for STE at any time tested. For the combinations studied, synergistic, antagonistic and addictive effects were obtained with the two type of analyses performed, the isobologram analysis and the Combenefit method. No relevant effects on ROS and ΔΨm were observed. In conclusion, predictive models based on combination data could help to better understand the interactions between mycotoxins and their implications in food safety assessment. However, a further analysis of the molecular mechanism underlying these interactive effects is required.

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

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

Sterigmatocystin-induced cytotoxicity via oxidative stress induction in human neuroblastoma cells.

Sterigmatocystin (STE) is a mycotoxin produced by fungi of the genus Aspergillus. Considering that the effect of STE on neuronal system has not been well studied, the aim of the present study consists to investigate the cytotoxic effects of STE in human neuroblastoma (SH-SY5Y) cells. Moreover, the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and antioxidant no-enzymatic (GSH) levels and enzymatic (GPx, GST, CAT and SOD) activity. Our results revealed that STE decreased cell viability in a dose and time-dependent manner. Furthermore, after 24 h of exposure, STE induced an increase in ROS generation and LPO at all concentrations tested (0.78, 1.56 and 3.12 µM), as well as a depletion of GSH levels, an increase in GSSG content and a decrease in GSH/GSSG ratio at the highest concentrations. The activity of all antioxidant enzymes resulted to be also decreased. Additionally, an enhance of the oxidative damage was caused by BSO, a GSH depletor, while NAC, a GSH precursor, showed a scavenger activity. Our findings suggest that STE could injure SH-SY5Y cells via oxidative stress and highlight the antioxidant role of the glutathione system.

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

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

Multi-Occurrence of Twenty Mycotoxinsin Pasta and a Risk Assessment in the Moroccan Population.

In the present study, the multi-occurrence of twenty (20) mycotoxins in pasta samples consumed in Morocco was assessed. For this, a modified Quick, Easy, Cheap Effective, Rugged, and Safe method was validated. The mycotoxins studied were identified and quantified by liquid chromatography⁻tandem mass spectrometry (LC⁻MS/MS) and gas chromatography⁻tandem mass spectrometry (GC-MS/MS). The validated method was applied to one hundred and six (n = 106) pasta samples purchased from several areas in the country. The analytical results showed that 99 out of 106 total samples (93.4%) were contaminated with at least one mycotoxin. Nine mycotoxins (Aflatoxin B1, Enniatin B, Enniatin B1, Enniatin A1, Zearalenone, Deoxynivalenol, 3-Acetyl-Deoxynivalenol, T-2, and HT-2 toxins) were present in the pasta samples. Enniatin B and Enniatin B1 were the predominant mycotoxins. The Zearalenone, Deoxynivalenol, HT-2, and T-2 toxins were present in 51.8%, 43.5%, 34.9%, and 16% of samples, respectively. Aflatoxin B1 was detected in only 2 samples. Risk exposure assessment concluded that mycotoxin levels found in pasta do not pose a significant human health risk for the Moroccan population. This is the first paper drafted on the multi-occurrence of mycotoxins in pasta from this country.

Multi-mycotoxin contamination of couscous semolina commercialized in Morocco.

The multi-mycotoxin contamination of ninety-eight (98) couscous semolina samples collected from various areas in Morocco was investigated in this study. Samples were surveyed for the presence of 22 mycotoxins (four aflatoxins, ochratoxin A, diacetoxiscyrpenol (DAS), three fumonisins, beauvericin (BEA), deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15-ADON), 3-acetyl-deoxynivalenol (3-ADON), nivalenol (NIV), sterigmatocystin (STG), zearalenone (ZEA), four enniatins, T-2 and HT-2 toxins). Results showed that 96 out of 98 total couscous samples (98%) were contaminated by at least one mycotoxin. Enniatin B (ENB), Enniatin B1 (ENB1), Enniatin A1 (ENA1) and zearalenone (ZEA) have shown the highest incidences in contaminated samples. The dietary exposure was estimated to be 1.02, 0.57, 0.06, 0.57 and 0.3µg/kgbw/day for the sum of (DON+3-ADON+15-ADON), fumonisins (FB1+FB2+FB3), the sum of (T2+HT-2), NIV and ZEA, respectively.

Occurrence of mycotoxins in refrigerated pizza dough and risk assessment of exposure for the Spanish population.

Mycotoxins are toxic metabolites produced by filamentous fungi, as Aspergillus, Penicillium and Fusarium. The first objective of this research was to study the presence of mycotoxins in 60 samples of refrigerated pizza dough, by extraction with methanol and determination by liquid chromatography associated with tandem mass spectrometry (LC-MS/MS). Then, the estimated dietary intakes (EDIs) of these mycotoxins, among the Spanish population, was calculated and the health risk assessment was performed, comparing the EDIs data with the tolerable daily intake values (TDIs). The mycotoxins detected in the analyzed samples were aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), zearalenone (ZEA), enniatin A (ENA), enniatin A1 (ENA1), enniatin (ENB), enniatin B1 (ENB1) and BEA (beauvericin) with average concentration of the positive samples of 4.09 µg/kg, 0.50 µg/kg, 0.79 µg/kg, 77.78 µg/kg, 14.96 µg/kg, 4.54 µg/kg, 3.37 µg/kg, 1.69 µg/kg and 22.39 µg/kg, respectively. The presence of ZEA, ENA1, ENB and ENB1 was detected in 100% of the samples, AFB2 in 32%, AFB1 in 23%, ENA in 8% and BEA in 3%. Twelve percent of the samples contaminated with AFB1 and 12% of the doughs contaminated with ZEA exceeded the EU legislated maximum limits. The dietary intakes were estimated considering three different age groups of population, and the EDIs calculated for the mycotoxins detected in the samples were all below the established TDI.

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.

Isolation, purification, LC-MS/MS characterization and reactive oxygen species induced by fumonisin B1 in VERO cells.

Fumonisins are mycotoxins produced by Fusarium verticillioides that commonly contaminate maize and maize products. The present work shows the results of a comparative study of three different fermentation's techniques (solid and liquid medium of corn and a solid agarized medium) for the production of fumonisins B(1), B(2) and B(3) with strains of F. verticillioides. The solid medium of corn was the most effective in the production of fumonisins, being Fumonisin B(1) the one produced with higher concentration, so the extract obtained by solid fermentation process was used for FB(1) purification. Fumonisins characterization and quantification were performed with reversed-phase high-performance liquid chromatography with electrospray ionization triple quadrupole tandem mass spectrometry. The role of production of reactive oxygen species (ROS) in Fumonisin B(1) mediated toxicology has not been fully addresses in studies exploring FB(1) toxicity. It is evaluated the level of ROS production in kidney cell line (VERO) exposed to 1, 5 and 10 µM of FB(1) for 0.5-100 min. The ROS level was detected using a fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS. Significant increase of ROS products was observed in VERO cells at 10 µM dose. These results indicate that ROS production by FB(1) on renal cells is a mechanism of fumonisin mediated toxicity.