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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.

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.

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.

Beauvericin (BEA) and zearalenone derivatives, α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL), are produced by several Fusarium species. Considering the impact of various mycotoxins on human’s health, this study determined and evaluated the cytotoxic effect of individual, binary, and tertiary mycotoxin treatments consisting of α-ZEL, β-ZEL, and BEA at different concentrations over 24, 48, and 72 h on SH-SY5Y neuronal cells, by using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide). Subsequently, the isobologram method was applied to elucidate if the mixtures produced synergism, antagonism, or additive effects. Ultimately, we determined the amount of mycotoxin recovered from the media after treatment using liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry (LC–ESI–qTOF-MS). The IC50 values detected at all assayed times ranged from 95 to 0.2 μM for the individual treatments. The result indicated that β-ZEL was the most cytotoxic mycotoxin when tested individually. The major effect detected for all combinations assayed was synergism. Among the combinations assayed, α-ZEL + β-ZEL + BEA and α-ZEL + BEA presented the highest cytotoxic potential with respect to the IC value. In individual treatment, α-ZEL was the most recovered mycotoxin; while, this was observed for BEA in binary combination α-ZEL + BEA.

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 studies.

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.

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 β -carotene. Quantification of target carotenoids was performed with external calibration through analytical standards. The concentration of lutein and β -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 β -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 β -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 β -carotene for both cultivars and for both cooking methods. Bioaccessibility values for lutein and β -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.

Simultaneous determination of twenty-seven mycotoxins in ready-to-eat food samples using “Quick Easy Cheap Rough and Safe” (QuEChERS) extraction and chromatographic methods coupled to mass spectrometry in tandem is described in this study. Mycotoxins included in this survey were aflatoxins (B1, B2, G1, G2), enniatins (A, A1, B, B1), beauvericin (BEA), fumonisins (FB1, FB2), sterigmatocystin (STG), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), 15-acetyl-deoxynivalenol (15-ADON), nivalenol (NIV), neosolaniol (NEO), diacetoxyscirpenol (DAS), fusarenon-X (FUS-X), zearalenone (ZEA), α-zearalanol (αZAL), β-zearalenone (βZAL), α-zearalenol (αZOL), β-zearalenol (βzol), T2, and HT-2 toxin. The method showed satisfactory extraction results with recoveries ranging from 63 to 119% for the different food matrix samples. Limits of detection (LODS) and quantification (LOQs) were between 0.15–1.5 µg/kg and 0.5–5 µg/kg, respectively. The method was successfully applied to the analysis of 25 ready-to-eat food samples. Results showed presence of deoxynivalenol at 36% of samples (2.61–21.59 µg/kg), enniatin B at 20% of samples (9.83–86.32 µg/kg), HT-2 toxin at 16% of samples (9.06–34.43 µg/kg), and aflatoxin G2 at 4% of samples (2.84 µg/kg). Mycotoxins were detected mainly in ready-to-eat food samples prepared with cereals, vegetables, and legumes, even at levels below those often obtained from raw food.

The lower mycotoxin levels detected in urine make the development of sensitive and accurate analytical methods essential. Three extraction methods, namely salting-out liquid–liquid extraction (SALLE), miniQuEChERS (quick, easy, cheap, effective, rugged, and safe), and dispersive liquid–liquid microextraction (DLLME), were evaluated and compared based on analytical parameters for the quantitative LC-MS/MS measurement of 11 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, ZEA, BEA, EN A, EN B, EN A1 and EN B1) in human urine. DLLME was selected as the most appropriate methodology, as it produced better validation results for recovery (79–113%), reproducibility (RSDs < 12%), and repeatability (RSDs < 15%) than miniQuEChERS (71–109%, RSDs

Mycotoxins are fungal secondary metabolites with bioaccumulation levels leading to their carry-over into animal fluids, organs, and tissues. As a consequence, mycotoxin determination in biological samples from humans and animals has been reported worldwide. Since most mycotoxins show toxic effects at low concentrations and considering the extremely low levels present in biological samples, the application of reliable detection methods is required. This review summarizes the information regarding the studies involving mycotoxin determination in biological samples over the last 10 years. Relevant data on extraction methodology, detection techniques, sample size, limits of detection, and quantitation are presented herein. Briefly, liquid-liquid extraction followed by LC-MS/MS determination was the most common technique. The most analyzed mycotoxin was ochratoxin A, followed by zearalenone and deoxynivalenol—including their metabolites, enniatins, fumonisins, aflatoxins, T-2 and HT-2 toxins. Moreover, the studies were classified by their purpose, mainly focused on the development of analytical methodologies, mycotoxin biomonitoring, and exposure assessment. The study of tissue distribution, bioaccumulation, carry-over, persistence and transference of mycotoxins, as well as, toxicokinetics and ADME (absorption, distribution, metabolism and excretion) were other proposed goals for biological sample analysis. Finally, an overview of risk assessment was discussed.