Fermented whey modulated AFB1 and OTA-induced hepatotoxicity and nephrotoxicity in vivo. A relative and absolute quantification about sex differences.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are well-known to promote hepatotoxicity and nephrotoxicity in vivo, which may be counteracted by natural compounds like fermented whey (FW). Carbamoyl phosphate synthetase 1 (CPS1) and kidney injury molecule 1 (KIM-1) are typical biomarkers used to detect liver and kidney damage, respectively. Thus, RT-qPCR and droplet digital PCR (ddPCR) analysis were performed to assess the potential beneficial effect of FW against AFB1 and OTA hepatotoxicity and nephrotoxicity in male and female Wistar rats by analyzing the altered gene expression of hepatic CPS1 and renal KIM-1 after 28 days of oral exposure. In male livers, the most damaging treatment was AFB1 by reducing CPS1 expression, which was totally reversed by FW-administration. This bioactive compound also improved gene expression changes induced by OTA and mycotoxins mixture. In female livers, a significant CPS1 overexpression was observed for each exposure performed, in which FW-supplementation reported no remarkable differences compared with mycotoxins exposure. Conversely, in the kidneys of male and female rats, exposure to mycotoxins promoted renal damage by altering KIM-1 gene expression, being OTA-exposure the most harmful condition. In both sexes, ddPCR analysis demonstrated that FW-addition modulated mycotoxins induced KIM-1 gene expression changes, thus reducing kidney damage. In this organ, sex-related responses were not clearly observed. Therefore, these findings confirmed that AFB1 and OTA-promoted hepatotoxicity and nephrotoxicity in vivo, which could be modulated by dietary FW supplementation.

Mycotoxins occurrence in medicinal herbs dietary supplements and exposure assessment.

The multimycotoxin analysis of aflatoxins (AFs), zearalenone (ZEA), ochratoxin A (OTA), enniatins (ENNs) and beauvericin (BEA) was performed in 85 samples of medicinal herbs dietary supplements. The samples were classified in 64 samples of one herbal ingredient and 21 mixed samples. The extraction was performed by QuEChERS method and the determination by liquid chromatography coupled to ion-trap tandem mass spectrometry (LC-MS/MS-IT). Then, the risk characterization to mycotoxins through the consumption of medicinal herbs dietary supplements was assessed. The results showed that ZEA, OTA, ENNs and BEA showed in the samples with incidences between 1 and 34%, being ENNB the most detected mycotoxin. Mycotoxins contents ranged from LOQ to 3850.5 µg/kg while the mean of positives samples were 65.5 µg/kg (ENNA), 82.7 µg/kg (ENNA1), 88.7 µg/kg (ENNB), 324.9 µg/kg (ENNB1), 137.9 µg/kg (BEA) and 1340.11 µg/kg (ZEA), respectively. OTA was detected in one herbal mix tablet for insomnia at concentration of 799 µg/kg. In herbal drugs the European Pharmacopoeia Commission has implemented limits of 2 µg/kg for AFB1 and 4 µg/kg for total AFs. In the present study AFs have not been detected in the analyzed medicinal herbs dietary supplements. The Estimated Daily Intakes (EDIs) values were calculated using a deterministic method, considering two exposure scenarios (lower bound (LB) and upper bound (UB)). The values obtained were in general far below the Tolerable Daily Intakes (TDIs) established. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05306-y.

Mycotoxin Incidence in Some Fish Products: QuEChERS Methodology and Liquid Chromatography Linear Ion Trap Tandem Mass Spectrometry Approach.

The inclusion of vegetal raw materials in feed for fish farming has increased the risk of mycotoxin occurrence in feed, as well as in edible tissues from fish fed with contaminated feed, due to the carry-over to muscle portions. Therefore, the objective of this study was to evaluate the occurrence of 15 mycotoxins in processed fish products, which are commonly consumed, such as smoked salmon and trout, different types of sushi, and gula substitutes. A QuEChERS method was employed to perform the mycotoxin extraction from fish samples. For mycotoxin identification and quantitation, the selected technique was the liquid chromatography-tandem mass spectrometry linear ion trap (LC-MS/MS-LIT). Smoked fish and sushi samples results were negative regarding the presence of all 15 mycotoxins studied. In contrast, small amounts of fusarenon-X and enniatin B were found in gula substitute samples.

HPLC-UV/Vis-APCI-MS/MS Determination of Major Carotenoids and Their Bioaccessibility from "Delica" (Cucurbita maxima) and "Violina" (Cucurbita moschata) Pumpkins as Food Traceability Markers.

Carotenoids are a widespread group of fat-soluble pigments, and their major nutritional importance comes from their pro-vitamin A activity and their antioxidant capacity. In this study, two different pumpkin cultivars (Cucurbita maxima, also named `Delica' and Cucurbita moschata, also known as `Violina') from the southern Po Delta area were investigated in terms of carotenoid content and the influence of food processing on compositional changes and carotenoid bioaccessibility. Quali- and quantitative determination of carotenoids in sample extracts were performed on a C30 column by means of an online coupled HPLC-UV/Vis-APCI-MS/MS technique. The identification of separated compounds was tentatively achieved by merging (i) chromatographic data, (ii) UV-Vis spectra, and (iii) MS/MS fragmentation spectra. The chromatographic profiles for the two cultivars showed qualitative differences. Two major carotenoids were considered for quantification purposes and further investigations: lutein and ß -carotene. Quantification of target carotenoids was performed with external calibration through analytical standards. The concentration of lutein and ß -carotene was higher in C. maxima than in the other variety, C. moschata. Carotenoids are susceptible to degradation (isomerization and oxidation) during food processing (i.e., cooking), and the concentration of lutein and ß -carotene were monitored in oven-cooked and steam-cooked pumpkins. The steam-cooking process was superior in terms of limiting carotenoid loss. A complete functional profile of pumpkins as a source of carotenoids was gained with the evaluation of their in vitro bioaccessibility and their bioavailability after intake during human digestion. Bioaccessibility of lutein and ß -carotene were estimated by an in vitro static digestion model that involved salivary, gastric, and duodenal phases. Bioaccessibility values progressively increased from the salivary to the duodenal phase for both pumpkin varieties and cooking methods. Bioaccessibility of lutein was always lower than ß -carotene for both cultivars and for both cooking methods. Bioaccessibility values for lutein and ß -carotene changed from 1.93% to 2.34% vs. 4.94% and 8.83% in the salivary phase, from 2.7% to 4.63% vs. 7.83% and 15.60% in the gastric phase, and from 10.04% to 13.42% vs. 25.81% and 35.32% in the duodenal phase. For both target compounds, bioaccessibility in the duodenal phase was more than twice the gastric values, and it underlined that the type of cooking did not influence release from the initial matrix.

Effects of technological processes on enniatin levels in pasta.

BACKGROUND: Potential human health risks posed by enniatins (ENs) require their control primarily from cereal products, creating a demand for harvesting, food processing and storage techniques capable to prevent, reduce and/or eliminate the contamination. In this study, different methodologies to pasta processing simulating traditional and industrial processes were developed in order to know the fate of the mycotoxin ENs. The levels of ENs were studied at different steps of pasta processing. The effect of the temperature during processing was evaluated in two types of pasta (white and whole-grain pasta). Mycotoxin analysis was performed by LC-MS/MS. RESULTS: High reductions (up to 50% and 80%) were achieved during drying pasta at 45-55°C and 70-90°C, respectively. The treatments at low temperature (25°C) did not change EN levels. The effect of pasta composition did not cause a significant effect on the stability of ENs. The effect of the temperature allowed a marked mycotoxin reduction during pasta processing. Generally, ENA1 and ENB showed higher thermal stability than did ENA and ENB1 . CONCLUSIONS: The findings from the present study suggested that pasta processing at medium-high temperatures is a potential tool to remove an important fraction of ENs from the initial durum wheat semolina.

Mycotoxin contamination in laboratory rat feeds and their implications in animal research.

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

Quantitation of enniatins in biological samples of Wistar rats after oral administration by LC-MS/MS.

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

A preliminary study in Wistar rats with enniatin A contaminated feed.

A 28-day repeated dose preliminary assay, using enniatin A naturally contaminated feed through microbial fermentation by a Fusarium tricinctum strain, was carried out employing 2-month-old female Wistar rats as in vivo experimental model. In order to simulate a physiological test of a toxic compound naturally produced by fungi, five treated animals were fed during 28 days with fermented feed. As control group, five rats were fed with standard feed. At the 28th day, blood samples were collected for biochemical analysis and the gastrointestinal tract, liver and kidneys were removed from each rat for enniatin A detection and quantitation. Digesta were collected from stomach, duodenum, jejunum, ileum and colon. Enniatin A present in organs and in biological fluids was analyzed by liquid chromatography-diode array detector (LC-DAD) and confirmed by LC-mass spectrometry linear ion trap (MS-LIT); also several serum biochemical parameters and a histological analysis of the duodenal tract were performed. No adverse effects were found in any treated rat at the enniatin A concentration (20.91 mg/kg bw/day) tested during the 28-day experiment. Enniatin A quantitation in biological fluids ranged from 1.50 to 9.00 mg/kg, whereas in the gastrointestinal organs the enniatin A concentration ranged from 2.50 to 23.00 mg/kg. The high enniatin A concentration found in jejunum liquid and tissue points to them as an absorption area. Finally, two enniatin A degradation products were identified in duodenum, jejunum and colon content, probably produced by gut microflora.

In vitro and in vivo assessment of AFB1 and OTA toxic effects and the beneficial role of bioactive compounds. A systematic review.

The purpose of this review was to investigate the current knowledge about aflatoxin B1 (AFB1) and ochratoxin A (OTA) toxicity and the possible beneficial role of bioactive compounds by using in vitro and in vivo models. Although AFB1 and OTA were tested in a similar percentage, the majority of studies focused on nephrotoxicity, hepatotoxicity, immune toxicity and neurotoxicity in which oxidative stress, inflammation, structural damage and apoptosis were the main mechanisms of action reported. Conversely, several biological compounds were assayed in order to modulate mycotoxins damage mainly in the liver, brain, kidney and immune system. Among them, pumpkin, curcumin and fermented whey were the most employed. Although a clear progress has been made by using in vivo models, further research is needed to assess not only the toxicity of multiple mycotoxins contamination but also the effect of functional compounds mixture, thereby reproducing more realistic situations for human health risk assessment.

The Protective Effect of Pumpkin and Fermented Whey Mixture against AFB1 and OTA Immune Toxicity In Vitro. A Transcriptomic Approach.

SCOPE: The aim of the study is to investigate in Jurkat cells the possible beneficial effect of pumpkin (P) and fermented milk whey (FW) mixture against aflatoxin B1 (AFB1) and ochratoxin A (OTA) induced alterations in gene expression profile. METHODS AND RESULTS: Human T cells are exposed for 7 days to digested bread extracts containing P-FW mixture along with AFB1 and OTA, individually and in combination. The results of RNA sequencing show that AFB1 P-FW exposure resulted in 34 differentially expressed genes (DEGs) while 3450 DEGs are found in OTA P-FW exposure and 3264 DEGs in AFB1-OTA P-FW treatment. Gene ontology analysis reveals biological processes and molecular functions related to immune system and inflammatory response. Moreover, PathVisio analysis points to eicosanoid signaling via lipoxygenase as the main pathway altered by AFB1 P-FW exposure whereas interferon signaling is the most affected pathway after OTA P-FW and AFB1-OTA P-FW treatments. CONCLUSIONS: The mitigation of genes and inherent pathways typically associated with the inflammatory response suggest not only the anti-inflammatory and protective role of P-FW mixture but also their possible application in food industry to counteract AFB1 and OTA toxic effects on human and animal health.

AFB1 and OTA Promote Immune Toxicity in Human LymphoBlastic T Cells at Transcriptomic Level.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are typical contaminants of food and feed, which have serious implications for human and animal health, even at low concentrations. Therefore, a transcriptomic study was carried out to analyze gene expression changes triggered by low doses of AFB1 and OTA (100 nM; 7 days), individually and combined, in human lymphoblastic T cells. RNA-sequencing analysis showed that AFB1-exposure resulted in 99 differential gene expressions (DEGs), while 77 DEGs were obtained in OTA-exposure and 3236 DEGs in the combined one. Overall, 16% of human genome expression was altered. Gene ontology analysis revealed, for all studied conditions, biological processes and molecular functions typically associated with the immune system. PathVisio analysis pointed to ataxia telangiectasia mutated signaling as the most significantly altered pathway in AFB1-exposure, glycolysis in OTA-exposure, and ferroptosis in the mixed condition (Z-score > 1.96; adjusted p-value ≤ 0.05). Thus, the results demonstrated the potential DNA damage caused by AFB1, the possible metabolic reprogramming promoted by OTA, and the plausible cell death with oxidative stress prompted by the mixed exposure. They may be considered viable mechanisms of action to promote immune toxicity in vitro.

Comparison of three solid-phase extraction processes in quantification of ciprofloxacin and enrofloxacin in pork meat.

Due to strong implications for food safety, control of fluoroquinolones residues in swine meat should be undertaken to verify compliance of the contamination levels with the maximum residue limits recently updated by Commission Regulation (EU) No. 37/2010 of 22 December 2009. Solid-phase extraction is widely used in antibiotic analysis in food of animal origin. In this study, the results of a comparative study using different types of solid-phase extraction columns, HLB, MCX, and MAX, for ciprofloxacin and enrofloxacin analysis, in pork meat, are presented. In addition, diverse sample treatments for defatting, precipitate proteins, eliminate cations, and increase the ionic strength, were used to obtain the most suitable method of analysis. Only the MCX's use followed by liquid chromatography with fluorescence detection resulted in chromatograms that allow the quantification at maximum residue limits. The validation method, in terms of CCα and CCß, recovery and precision determination, was according to the EU Decision 2002/657/EC. This procedure was used in the analysis of 50 samples of pork meat of Portuguese origin. Only two samples presented residues of enrofloxacin at 30 and 42 µg/kg, values under the legal maximum residue limit.

Pumpkin extract and fermented whey individually and in combination alleviated AFB1- and OTA-induced alterations on neuronal differentiation invitro.

Food and feed are daily exposed to mycotoxin contamination which effects may be counteracted by functional compounds like carotenoids and fermented whey. Among mycotoxins, the most toxic and studied are aflatoxin B1 (AFB1) and ochratoxin A (OTA), which neurotoxicity is not well reported. Therefore, SH-SY5Y human neuroblastoma cells ongoing differentiation were exposed during 7 days to digested bread extracts contained pumpkin and fermented whey, individually and in combination, along with AFB1 and OTA and their combination, in order to evaluate their presumed effects on neuronal differentiation. The immunofluorescence analysis of ßIII-tubulin and dopamine markers pointed to OTA as the most damaging treatment for cell differentiation. Cell cycle analysis reported the highest significant differences for OTA-contained bread compared to the control in phase G0/G1. Lastly, RNA extraction was performed and gene expression was analyzed by qPCR. The selected genes were related to neuronal differentiation and cell cycle. The addition of functional ingredients in breads not only enhancing the expression of neuronal markers, but also induced an overall improvement of gene expression compromised by mycotoxins activity. These data confirm that in vitro neuronal differentiation may be impaired by AFB1 and OTA-exposure, which could be modulated by bioactive compounds naturally found in diet.

Effects of Voghiera garlic extracts in neuronal human cell line against zearalenone's derivates and beauvericin.

The Fusarium toxins constitute one of the largest groups of mycotoxins produced by Fusarium species, which are major pathogens of cereal plants. In the present study neuroprotection effect of Allium sativum L garlic extract which is known as Voghiera garlic, from a local garlic ecotype of Ferrara (Italy) was examined on an undifferentiated SH-SY5Y neuronal cells against ZEA's metabolites (α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL)) and beauvericin (BEA) mycotoxins which are considered as the most reported Fusarium mycotoxins, via MTT (3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, over 24 h and 48 h through direct treatment, simultaneous treatment and pre-treatment strategies. The results demonstrated remarkable improvement in cells viability in simultaneous and pre-treatment strategy with Voghiera garlic extract (VGE); specifically, for simultaneous treatment of VGE with ß-ZEL which viability increased significantly up to 56%, and subsequently with α-ZEL and BEA by up to 38% and 37% respectively, compared to each mycotoxin tested alone for their highest concentrations assayed, while direct treatments for each mycotoxins individually decreased significantly (for α-ZEL up to 69%, for ß-ZEL 82% and for BEA up to 43%). It is proposed by the present study that VGE extract found to be effective in reducing the cytotoxicity/neurotoxicity of α-ZEL, ß-ZEL and BEA mycotoxins encountered in food and feed commodity.

Development and Validation of LC-Q-TOF-MS Methodology to Determine Mycotoxin Biomarkers in Human Urine.

Mycotoxin contamination of foodstuffs is a health concern worldwide and monitoring human exposure to mycotoxins is a key concern. Most mycotoxins and their metabolites are excreted in urine, but a reliable detection method is required, considering the low levels present in this biological sample. The aim of this work is to validate a sensitive methodology capable of simultaneously determining ten targeted mycotoxins as well as detecting untargeted ones by using Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry (LC-Q-TOF-MS). The targeted mycotoxins were: enniatin A, B, A1, and B1, beauvericine, aflatoxin B1, B2, G1 and G2, and ochratoxin A. Several extraction procedures such as liquid-liquid extraction, dilute and shoot, and QuEChERS were assessed. Finally, a modified simple QuEChERS extraction method was selected. Creatinine adjustment and matrix-matched calibration curves are required. The limit of detection and limit of quantification values ranged from 0.1 to 1.5 and from 0.3 to 5 ng/mL, respectively. Recoveries achieved were higher than 65% for all mycotoxins. Later, the method was applied to 100 samples of women's urine to confirm the applicability and determine their internal exposure. The untargeted mycotoxins most found were trichothecenes, zearalenones, and ochratoxins.

Antibacterial activity of the enniatin B, produced by Fusarium tricinctum in liquid culture, and cytotoxic effects on Caco-2 cells.

The enniatins (ENs) are bioactive compounds of hexadepsipeptidic structure produced by several strains of Fusarium sp. The EN B was purified from extracts of Fusarium tricinctum growth on liquid culture of potato dextrose broth (PDB), using a semipreparative liquid chromatography (LC) followed by an analytical LC. The purity and the structure of the isolated compound were confirmed by the determination of the extinction coefficient and with electrospray ionization-mass spectrometry (ESI-MS) study. The pure fraction of EN B was utilized to determine the antibiotic effects on several bacterial strains that are considered normally pathogens of the intestinal tract: Escherichia coli, Enterococcus faecium, Salmonella enterica, Shigella dysenteriae, Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens, Pseudomonas aeruginosa, and Staphylococcus aureus, and to study the cytotoxic effects on Caco-2 differentiated and undifferentiated cells. The results obtained demonstrated that in several antibiograms, EN B induced the inhibition of the grown microorganisms tested and no significant differences over control were detected when Caco-2 cells were exposed to EN B, at any of the concentrations used.

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 death pathway by flow cytometry in undifferentiated SH-SY5Y neuronal cells exposed to α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL) and beauvericin (BEA) over 24 h and 48 h individually and combined at the following concentration ranges: from 1.56 to 12.5 µM for α-ZEL and ß-ZEL, from 0.39 to 2.5 µM for BEA, from 1.87 to 25 µM for binary combinations and from 3.43 to 27.5 µM for tertiary combination. Alterations in cell cycle were observed remarkably for ß-ZEL at the highest concentration in all treatments where engaged (ß-ZEL, ß-ZEL + BEA and ß-ZEL + α-ZEL), for both 24 h and 48 h. by activating the cell proliferation in G0/G1 phase (up to 43.6 %) and causing delays or arrests in S and G2/M phases (up to 19.6 %). Tertiary mixtures revealed increases of cell proliferation in subG0 phase by 4-folds versus control. Similarly, for cell death among individual treatments ß-ZEL showed a significant growth in early apoptotic cells population at the highest concentration assayed as well as for all combination treatments where ß-ZEL was involved, in both early apoptotic and apoptotic/necrotic cell death pathways.

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 1ß 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.

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

Beauvericin (BEA), α-zearalenol (α-ZEL) and ß-zearalenol (ß-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, ß-ZEL and BEA, on enzymatic GPx, GST, CAT and SOD activity in human neuroblastoma cells using the SH-SY5Y cell line, over 24 h and 48 h with different treatments at the following concentration range: from 1.56 to 12.5 µM for α-ZEL and ß-ZEL, from 0.39 to 2.5 µM for BEA, from 1.87 to 25 µM for binary combinations and from 3.43 to 27.5 µM for tertiary combination. SH-SY5Y cells exposed to α-ZEL, ß-ZEL and BEA revealed an overall increase in the activity of i) GPx, after 24 h of exposure up to 24-fold in individual treatments and 15-fold in binary combination; ii) GST after 24 h of exposure up to 10-fold (only in combination forms), and iii) SOD up to 3.5- and 5-fold in individual and combined treatment, respectively after 48 h of exposure. On the other hand, CAT activity decreased significantly in all treatments up to 92% after 24 h except for ß-ZEL + BEA, which revealed the opposite.

The role of pumpkin pulp extract carotenoids against mycotoxin damage in the blood brain barrier 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.