Transcriptional changes after enniatins A, A1, B and B1 ingestion in rat stomach, liver, kidney and lower intestine

Enniatins (ENs) are depsipeptide mycotoxins produced by Fusarium fungi. They are known for their capacity to modulate cell membrane permeability and disruption of ionic gradients, affecting cell homeostasis and initiating oxidative stress mechanisms. The effect of the acute toxicity of ENs A, A1, B and B1 at two different concentrations after 8 h of exposure was analysed in Wistar rats by a transcriptional approach. The following key mitochondrial and nuclear codified genes related to the electron transport chain were considered for gene expression analysis in stomach, liver, kidney and lower intestine by quantitative Real-Time PCR: mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), mitochondrially encoded cytochrome c oxidase 1 (MT-COX1), succinate dehydrogenase flavoprotein subunit A and ATP synthase F1 subunit alpha, respectively. Moreover, the expression of markers involved in oxidative stresssuperoxide dismutase 1 (SOD1), glutathione peroxidase 1 (Gpx1), heme oxygenase 1, apoptosis B-cell lymphoma 2, Bcl2 Associated protein X (Bax), tumor suppressor protein (p53), inhibition of apoptosis nuclear factor kappa of activated B cells, immune system interleukin 1β and intestinal tight junction Occludin merely in lower intestine tissues have been investigated. For mitochondrial genes, the main differences were observed for MT-ND1 and MT-COX1, showing its deficiency in all selected organs. With regard to the intestinal barrier's cellular response to oxidative stress, the activity of the antioxidant gene SOD1 was decreased in a dose-dependent manner. Similarly, the catalytic enzyme GPx1 was also downregulated though merely at medium dose employed. On the contrary, the pro-apoptotic Bax and p53 regulators were activated after ENs exposure, reporting a significant increase in their expression. Furthermore, the alteration of intestinal permeability was assessed by the abnormal activity of the tight junction protein occludin. In summary, ENs may generate mitochondrial disorders and induce oxidative stress in intestinal barrier function

The soluble dietary fiber inulin can influence the bioaccessibility of enniatins

Enniatins (ENs) are bioactive compounds produced by the secondary metabolism of several Fusarium strains and are known to have various biological activities, such as acting as enzyme inhibitors, antifungal antibacterial agents, and immunomodulatory substances. This study investigated the bioaccessibility of the ENs in wheat crispy breads produced with three different inulin concentrations (1, 5 and 10%). The mean bioaccessibility data of the four ENs (A, A1, B and B1) ranged from 68.67% to 84.67 in the experiments carried out without inulin, whereas the data ranged from 51.00 to 74.00% in the experiments carried out with the wheat crispy bread produced with 5 and 10% of the inulin

Preliminary Estimation of Deoxynivalenol Excretion through a 24 h Pilot Study

A duplicate diet study was designed to explore the occurrence of 15 Fusarium mycotoxins in the 24 h-diet consumed by one volunteer as well as the levels of mycotoxins in his 24 h-collected urine. The employed methodology involved solvent extraction at high ionic strength followed by dispersive solid phase extraction and gas chromatography determination coupled to mass spectrometry in tandem. Satisfactory results in method performance were achieved. The method"s accuracy was in a range of 68%108%, with intra-day relative standard deviation and inter-day relative standard deviation lower than 12% and 15%, respectively. The limits of quantitation ranged from 0.1 to 8 μg/Kg. The matrix effect was evaluated and matrix-matched calibrations were used for quantitation. Only deoxynivalenol (DON) was quantified in both food and urine samples. A total DON daily intake amounted to 49.2 ± 5.6 μg whereas DON daily excretion of 35.2 ± 4.3 μg was determined. DON daily intake represented 68.3% of the established DON provisional maximum tolerable daily intake (PMTDI). Valuable preliminary information was obtained as regards DON excretion and needs to be confirmed in large-scale monitoring studie

Determination of deoxynivalenol in wheat-based snacks by gas chromatography-triple quadrupole tandem mass spectrometry

Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal and cereal-based products, and a continuous monitoring of this toxin in foodstuffs is highly desirable. In this sense,a QuEChERS based extraction and gas chromatography-tandem mass spectrometry detection is proposed to determine DON in an appetizer largely consumed, the wheat-based snacks. In this study, a total of 40 samples were analyzed. The samples were divided into two groups based on the composition. Extraction was carried out with acetonitrile followed by a dispersive solid phase extraction and analyzed for DON content by gas chromatography-tandem mass spectrometry (GC-QqQ-MS/MS) method. The overall occurrence of samples with DON was 67.5%, with maximum content of 61μg/kg. In spite of its high incidence, DON concentrations found in samples were much lower than the maximum limit established in the current European legislation (500 μg/kg) for the foodstuff evaluated. Data obtained indicated a low exposure to DON through the consumption of this food commodity. Resumen: Determinación de deoxinivalenol en rosquilletas mediante cromatografía de gases acoplada a espectrometría de masas en tándem. El deoxinivalenol (DON) es la micotoxina producida por hongos del género Fusarium que con más frecuencia se detecta en cereales y productos a base de cereales. Por ello, es recomendable realizar una continua monitorización de su incidencia en los alimentos. Este trabajo propone un procedimiento analítico basado en una extracción tipo QuEChERS seguido de una cromatografía de gases acoplada a un detector de triple cuadrupolo para la determinación de DON en rosquilletas. Se analizaron un total de 40 muestras las cuales se dividieron según su composición en dos grupos. El DON fue identificado en el 67,5% de las muestras analizadas con un contenido máximo de 61 μg/kg. A pesar de su incidencia elevada, los niveles de DON hallados fueron muy inferiores a los límites máximos legislados en la actual legislación europea (500 μg/kg). Los resultados obtenidos muestran una baja exposición a DON a través del consumo de esta matriz alimentaria

Assessment of Pesticide Residues in Honey Samples from Portugal and Spain

Fifty samples of honey collected from local markets of Portugal and Spain during year 2002 were analyzed for 42 organochlorine, carbamate, and organophosphorus pesticide residues. An analytical procedure based on solid-phase extraction with octadecyl sorbent followed by gas chromatography−mass spectrometry (GC−MS), for organochlorines, and by liquid chromatography−atmospheric pressure chemical ionization-mass spectrometry (LC−APCI-MS), for organophosphorus and carbamates, has been developed. Recoveries of spiked samples ranged from 73 to 98%, except for dimethoate (40%), with relative standard deviations from 3 to16% in terms of repeatability, and from 6 to 19% in terms of reproducibility. Limits of quantification were from 0.003 to 0.1 mg kg-1. Most of the pesticides found in honey were organochlorines. Among them, γ-HCH was the most frequently detected in 50% of the samples, followed by HCB in 32% of the samples and the other isomers of HCH (α-HCH and β-HCH) in 28 and 26% of the samples, respectively. Residues of DDT and their metabolites were detected in 20% of the samples. Of the studied carbamates, both methiocarb and carbofuran were detected in 10% of the samples, pirimicarb in 4% and carbaryl in 2%. The only organophosphorus pesticides found were heptenophos in 16%, methidathion in 4%, and parathion methyl in 2% of honey samples. Results indicate that Portuguese honeys were more contaminated than Spanish ones. However, honey consumers of both countries should not be concerned about the amounts of pesticide residues found in honeys available on the market

Bioaccessibility and decomposition of cylindrospermopsin in vegetablesmatrices after the application of anin vitrodigestion model

Research on the human exposure to Cylindrospermopsin (CYN) via consumption of contaminated food is of great interest for risk assessment purposes. The aim of this work is to evaluate for the first time the CYN bioaccessibility in contaminated vegetables (uncooked lettuce and spinach, and boiled spinach) after an in vitro digestion model, including the salivar, gastric and duodenal phases and, colonic fermentation under lactic acid bacteria. The results obtained showed that the digestion processes are able to diminish CYN levels, mainly in the colonic phase, especially in combination with the boiling treatment, decreasing CYN levels in a significant way. Moreover, the potential decomposition products in a pure CYN solution and in CYN-contaminated vegetables were evaluated using UHPLC-MS/MS Orbitrap. Under the conditions assayed, only two diastereoisomers of the same fragment with m/z 292.09617 have been detected in all the analysed samples, with the exception of digested vegetables. Therefore, in terms of risk assessment, the digestion seems to play an important role in reducing the final bioaccesibility of CYN, and the consumption of cooked vegetables (spinach) would be safer in comparison to raw vegetables.Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE

Uloga karotenoida iz ekstrakta mesa bundeve u zaštiti od oštećenja krvno-moždane barijere uzrokovane mikotoksinima in vitro

Some mycotoxins such as beauvericin (BEA), ochratoxin A (OTA), and zearalenone (ZEA) can cross the blood brain barrier, which is why we tested the anti-inflammatory action of a pumpkin carotenoid extract (from the pulp) against these mycotoxins and their combinations (OTA+ZEA and OTA+ZEA+BEA) on a blood brain barrier model with co-cultured ECV304 and C6 cells using an untargeted metabolomic approach. The cells were added with mycotoxins at a concentration of 100 nmol/L per mycotoxin and pumpkin carotenoid extract at 500 nmol/L. For control we used only vehicle solvent (cell control) or vehicle solvent with pumpkin extract (extract control). After two hours of exposure, samples were analysed with HPLC-ESI-QTOF-MS. Metabolites were identified against the Metlin database. The proinflammatory arachidonic acid metabolite eoxin (14,15-LTE4) showed lower abundance in ZEA and BEA+OTA+ZEA-treated cultures that also received the pumpkin extract than in cultures that were not treated with the extract. Another marker of inflammation, prostaglandin D2-glycerol ester, was only found in cultures treated with OTA+ZEA and BEA+OTA+ZEA but not in the ones that were also treated with the pumpkin extract. Furthermore, the concentration of the pumpkin extract metabolite dihydromorelloflavone significantly decreased in the presence of mycotoxins. In conclusion, the pumpkin extract showed protective activity against cellular inflammation triggered by mycotoxins thanks to the properties pertinent to flavonoids contained in the pulp.Pojedini mikotoksini poput bovericina (BEA), okratoksina A (OTA) i zearalenona (ZEA) prelaze krvno-moždanu barijeru, a to je i razlog zbog kojega smo istražili djelovanje ekstrakta karotenoida iz mesa bundeve protiv upalnih procesa izazvanih ovim mikotoksinima i njihovim kombinacijama (OTA+ZEA i OTA+ZEA+BEA) na modelu krvno-moždane barijere koji se sastojao od kultura stanica ECV304 i C6, oslanjajući se pritom na neciljani metabolomički pristup. Stanice su tretirane mikotoksinima u koncentraciji od 100 nmol/L po mikotoksinu odnosno ekstraktom karotenoida u koncentraciji od 500 nmol/L. Za kontrolu smo upotrijebili samo otapalo (stanična kontrola) odnosno otapalo s bundevinim ekstraktom (ekstraktna kontrola). Nakon dva sata tretmana uzorci su analizirani metodom tekućinske kromatografije / masene spektrometrije (HPLC-ESI-QTOF-MS), a dobiveni metaboliti identificirani su usporedbom s bazom podataka Metlin. Primjena ekstrakta značajno je smanjila količinu metabolita proupalne arahidonske kiseline eoksina (14,15-LTE4) u kulturama tretiranima samo zearalenonom odnosno kombinacijom BEA+OTA+ZEA. Drugi upalni biljeg, prostaglandin D2-glicerol ester, otkriven je samo u kulturama tretiranima kombinacijama OTA+ZEA odnosno BEA+OTA+ZEA, ali ne i u onima koje su usto tretirane bundevinim ekstraktom. Osim toga, u prisutnosti mikotoksina značajno je porasla koncentracija metabolita dihidromoreloflavona iz bundevina ekstrakta. Time je taj ekstrakt iskazao zaštitno djelovanje protiv stanične upale uzrokovane mikotoksinima zahvaljujući svojstvima flavonoida koji se nalaze u njezinu mesu

Quantitative determination of trichothecenes in breadsticks by gas chromatography-triple quadrupole tandem mass spectrometry

Breadsticks are pencil-sized sticks of dry bread widely consumed as a pre-meal appetiser. They are basically wheat-based snacks, which makes them a good matrix to evaluate mycotoxin contamination, since wheat is very susceptible to fungal attack. In this sense, the fast, selective and sensitive gas chromatography-triple quadrupole tandem mass spectrometry (GCQqQ- MS/MS) method proposed here allows simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, nivalenol, neosolaniol, HT-2 and T-2 toxin in breadsticks after QuEChERS extraction and clean-up. The performance of the method was assessed with respect to European Commission Regulations by studying the selectivity and specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, matrix effect, accuracy, precision and trueness. Satisfactory results in terms of validation parameters were obtained for all selected mycotoxins (recovery range of 70110%, RSD 1). The trueness of the method was supported by using certified reference material (DON 1062 ± 110 μg kg1). The method was successfully used to evaluate the occurrence of the studied Fusarium toxins in 61 breadstick samples. A total of 64% of the samples showed mycotoxin contamination, DON being the most frequently detected toxin. Nonetheless, mean levels obtained were far below the maximum levels permitted by European Union legislation. An additional goal was to carry out a risk-characterisation approach to DON by comparing probable daily intake and provisional maximum tolerable daily intake (PMTDI)

Effect of daily consumption of Lactobacillus reuteri CRL 1098 on cholesterol reduction in hypercholesterolemic subjects

The effect of daily consumption of a yogurt containing Lactobacillus reuteri CRL 1098 on the lipid profile of hypercholesterolemic subjects was evaluated by performing a prospective, randomized, double-blind, cross-over placebo Controlled clinical study. Participants consumed daily a yogurt containing L. reuteri CRL 1098 or a placebo for four weeks, separated by a wash-out period. Total cholesterol, triacylglycerol, high-density (HDL) and low-density (LDL) lipoprotein levels were assessed at the beginning and at the end of each period. We found a statistically significant reduction of total (-7.86 g/dl) and LDL (-7.02 g/dl) cholesterol in absolute changes (before-after) as well as a decreasing trend in the group receiving the yogurt containing L. reuteri with respect to the placebo group, without detecting changes in HDL-cholesterol and triacylglycerol levels. Our results suggest that low amounts of yogurt (125 g/day) and low doses of the CRL 1098 strain (106 CFU) are sufficient to reduce total and LDL-cholesterol levels in hypercholesterolemic subjects.Fil: Malpeli, Agustina. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; ArgentinaFil: Taranto, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Cravero, Ricardo C.. Sancor Cooperativas Unidas Limitada.; ArgentinaFil: Tavella, Marina. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; ArgentinaFil: Fasano, Maria Victoria. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; ArgentinaFil: Vicentin, Dimas. Sancor Cooperativas Unidas Limitada.; ArgentinaFil: Ferrari, Guillermina. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; ArgentinaFil: Magrini, Graciano. Sancor Cooperativas Unidas Limitada.; ArgentinaFil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Font, Graciela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Varea, Ana María. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; ArgentinaFil: Tavella, Julio Marcelo. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; ArgentinaFil: González, Horacio F.. Ministerio de Salud. Comisión de Investigaciones Científicas de la Provincia de Buenos Aires. Instituto de Desarrollo e Investigaciones Pediátricas “Prof. Dr. Fernando E. Viteri”. Hospital de Niños de La Plata; Argentin

Effect of Daily Consumption of Lactobacillus reuteri CRL 1098 on Cholesterol Reduction in Hypercholesterolemic Subjects

The effect of daily consumption of a yogurt containing Lactobacillus reuteri CRL 1098 on the lipid profile of hypercholesterolemic subjects was evaluated by performing a prospective, randomized, double-blind, cross-over placebo controlled clinical study. Participants consumed daily a yogurt containing L. reuteri CRL 1098 or a placebo for four weeks, separated by a wash-out period. Total cholesterol, triacylglycerol, high-density (HDL) and low-density (LDL) lipoprotein levels were assessed at the beginning and at the end of each period. We found a statistically significant reduction of total (−7.86 g/dl) and LDL (−7.02 g/dl) cholesterol in absolute changes (before-after) as well as a decreasing trend in the group receiving the yogurt containing L. reuteri with respect to the placebo group, without detecting changes in HDL-cholesterol and triacylglycerol levels. Our results suggest that low amounts of yogurt (125 g/day) and low doses of the CRL 1098 strain (106 CFU) are sufficient to reduce total and LDL-cholesterol levels in hypercholesterolemic subjects.Facultad de Ciencias Médica