Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells.

The T-2 toxin is a mycotoxin produced by molds belonging to Fusarium. Among the Fusarium mycotoxins, trichothecenes are frequently reported in food and feed, being the T-2 toxin (T-2) the mycotoxin which possesses the highest toxicity. According to EFSA, T-2 is found in various cereal grains used in food and feed products, mainly in oats, and it has a high environmental impact due to its mechanisms of toxicity. However, recent information on its genotoxic and mutagenic effects is lacking. This work aimed to evaluate the genotoxic and mutagenic potential of T-2 in vitro. For this purpose, HepG2 cells were exposed to 15, 30, and 60 nM T-2 for 24 h, then the DNA damage was evaluated by the micronucleus and the comet assays. In addition, point mutation analysis was performed by the bacterial reverse mutation test using 0.15-60 nM of T-2 concentrations. The results showed chromosomal damage at 60 nM T-2 since significantly more MN appeared at this concentration than in the control samples. Regarding the comet assay, DNA double helix breaks appeared at all concentrations tested and, in a concentration-dependent manner. However, no mutagenic effects were observed at any of the concentrations tested for the Salmonella typhimurium (S. Typhimurium) strains TA98, TA100, TA1535, TA1537, or the Escherichia coli (E. Coli) WP2 strain in the absence or presence of a metabolic activation system. Therefore, these results showed that T-2 mycotoxin produced genotoxic effects by MN and comet assay, while no mutagenicity was observed. However, further research simulating different metabolic activation pathways and the combined exposure of this mycotoxin with other mutagenic chemicals that could be present in the diet is necessary to discard the mutagenic potential of T-2 fully. These results highlight the carcinogenic potential and danger associated with T-2 exposure and should be considered to prevent associated food risks for the human population.

Evaluation of cytotoxicity, analysis of metals and cumulative risk assessment in microalgae.

Microalgae are one promising source for the production of bioactive compounds. However, microalgae can accumulate harmful substances. So, our objectives were (i) to evaluate cell viability after Phaeodactylum triconutum (0% and 65% cell disruption, DR) and Tetraselmis chuii (0% and 67% DR) freeze-dried exposure in HepG2 cells by MTT assay; (ii) to evaluate cell viability after P. triconutum and T. chuii extract exposure; (iii) to assess the effect in cell viability when they were simultaneously exposed to T-2 toxin and, (iv) to evaluate if inflammatory response is related to the mechanism of toxicity of these microalgae by qPCR assays. Results demonstrated that cell viability did not increase after freeze-dried microalgae exposure in HepG2 cells. And, no IC50 values were observed. However, an increase in HepG2 cell viability after exposure of T. chuii 0% DR extract at 5, 25 and 100 µg/mL was observed. Additionally, 1:64 diluted T. chuii 0% DR with IC50/4 T-2 and with IC50/2 T-2 and 1:32 diluted T. chuii 0% DR with IC50/4 T-2 showed an increase in cell viability. Both microalgae increased the relative TNF-α, IL-1ß and IL-6 mRNA expression. Concluding, no cytotoxic effect was evidenced but, it was noted up-regulation of inflammatory genes after T. chuii exposure in HepG2 cell. Thus, more studies related the mechanistic toxicity of microalgae are needed to evaluate the potential toxicological risk of inflammation of these novel foods. .

Citrinin Dietary Exposure Assessment Approach through Human Biomonitoring High-Resolution Mass Spectrometry-Based Data.

Citrinin (CIT) is a scarcely studied mycotoxin within foodstuffs, so the biomonitoring of this toxin and its metabolite dihydrocitrinone (DH-CIT) in biological samples represents the main alternative to estimate the exposure. Hence, this study aimed to evaluate the presence of CIT and DH-CIT in 300 urine samples from Italian individuals in order to assess the exposure. Quantification was performed through an ultrahigh-performance liquid chromatography high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS)-based methodology. CIT was quantified in 47% of samples (n = 300) up to 4.0 ng/mg Crea (mean = 0.29 ng/mg Crea), whereas DH-CIT was quantified in 21% of samples up to 2.5 ng/mg Crea (mean = 0.39 ng/mg Crea). Considering different age groups, average exposure ranged from 8% to 40% of the provisional tolerable daily intake, whereas four individuals surpassed the limits suggested by the European Food Safety Authority. These results revealed non-negligible exposure levels to CIT, encouraging further investigation in foodstuffs monitoring studies.

Mycotoxin Occurrence and Risk Assessment in Gluten-Free Pasta through UHPLC-Q-Exactive Orbitrap MS.

Celiac disease (CD) is a genetic-based autoimmune disorder which is characterized by inflammation in the small intestinal mucosa due to the intolerance to gluten. Celiac people should consume products without gluten, which are elaborated mainly with maize or other cereals. Contamination of cereals with mycotoxins, such as fumonisins (FBs) and aflatoxins (AFs) is frequently reported worldwide. Therefore, food ingestion is the main source of mycotoxin exposure. A new analytical method was developed and validated for simultaneous analysis of 21 mycotoxins in gluten-free pasta, commonly consumed by celiac population as an alternative to conventional pasta. Ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) was used for analyte separation and detection. The mycotoxins included in this work were those widely reported to occur in cereal samples, namely, ochratoxin-A (OTA), aflatoxins (AFB1, AFB2, AFG1 and AFG2), zearalenone (ZON), deoxynivalenol (DON), 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol (3-AcDON and 15-AcDON, respectively), nivalenol (NIV), neosolaniol (NEO), fusarenone-X, (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2), fumonisin B1 and B2 (FB1 and FB2, respectively), enniatins (ENN A, ENN A1, ENN B and ENN B1) and beauvericin (BEA). The validated method was successfully applied to 84 gluten-free pasta samples collected from several local markets of Campania region (Italy) during September to November 2020 to monitor the occurrence of mycotoxins and to assess the exposure to these food contaminants. A significant number of samples (95%) showed mycotoxin contamination, being Fusarium mycotoxins (FB1, ZON and DON) the most commonly detected ones. Regarding the risk assessment, the higher exposures were obtained for NIV, DON and FB1 for children and teenagers age group which can be explained due to their lower body weight.

Occurrence and Exposure Assessment of Mycotoxins in Ready-to-Eat Tree Nut Products through Ultra-High Performance Liquid Chromatography Coupled with High Resolution Q-Orbitrap Mass Spectrometry.

Tree nuts have become popular snacks due to their attributed benefits in the health state. Nevertheless, their susceptibility to fungal contamination lead to the occurrence of potentially dangerous mycotoxins. Hence, the aim of this work was to evaluate the presence of mycotoxins in ready-to-eat almonds, walnuts, and pistachios from Italian markets. The most relevant mycotoxin found in almonds was α-zearalanol in 18% of samples (n = 17) ranging from 3.70 to 4.54 µg/kg. Walnut samples showed frequent contamination with alternariol, present in 53% of samples (n = 22) at levels from 0.29 to 1.65 µg/kg. Pistachios (n = 15) were the most contaminated commodity, with ß-zearalenol as the most prevalent toxin present in 59% of samples ranging from 0.96 to 8.60 µg/kg. In the worst-case scenario, the exposure to zearalenone-derived forms accounted for 15.6% of the tolerable daily intake, whereas it meant 12.4% and 21.2% of the threshold of toxicological concern for alternariol and alternariol monomethyl-ether, respectively. The results highlighted the extensive presence of Alternaria toxins and zearalenone-derived forms, scarcely studied in ready-to-eat tree nut products, highlighting the necessity to include these mycotoxins in analytical methods to perform more realistic risk assessments.

Target analysis and retrospective screening of mycotoxins and pharmacologically active substances in milk using an ultra-high-performance liquid chromatography/high-resolution mass spectrometry approach.

Milk is a nutritious food suitable for infants and adults, and it plays an important role in the human diet. However, it may also be a vehicle for food contaminants. In this report, we developed a method using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS; Thermo Fisher Scientific, Waltham, MA) for simultaneous identification of target pharmacologically active substances and mycotoxins in milk. We also used the Q-Orbitrap operating in full scan mode to identify other possible drugs and microbial metabolites that occurred in samples. Fifty-six commercially available milk samples from the Italian market were analyzed. Investigated analytes were extracted using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach. Method detection and quantification limits and performance criteria set by European regulations were fulfilled. Pharmacologically active substances were detected in 49% of samples (range 0.007-4.53 ng/mL), including nontarget mycotoxins. Retrospective analysis allowed us to identify other antibiotics and pharmacologically active substances, as well as nonregulated fungal/bacterial metabolites at a relatively high incidence. From the obtained values, the need for continuous monitoring of contaminants in the milk production chain is clear. This is the first study to assess the presence of pharmacologically active substances, mycotoxins, and other microbial metabolites in Italian milk samples using the UHPLC-Q-Orbitrap HRMS system.

Exposure assessment approach through mycotoxin/creatinine ratio evaluation in urine by GC-MS/MS.

In this pilot survey human urine samples were analyzed for presence of 15 mycotoxins and some of their metabolites using a novel urinary multi-mycotoxin GC-MS/MS method following salting-out liquid-liquid extraction. Fifty-four urine samples from children and adults residents in Valencia were analyzed for presence of urinary mycotoxin and expressed in gram of creatinine. Three out of 15 mycotoxins were detected namely, HT-2 toxin, nivalenol and deoxynivalenol (DON). 37 samples showed quantifiable values of mycotoxins. Co-occurrence of these contaminants was also observed in 20.4% of assayed samples. DON was the most frequently detected mycotoxin (68.5%) with mean levels of 23.3 µg/g creatinine (range: 2.8-69.1 µg/g creatinine). The levels of urinary DON were used to carry out an exposure assessment approach. 8.1% of total subjects were estimated to exceed the DON provisional maximum tolerable daily intake (PMTDI) (1 µg/kg b.w.). Two out of 9 exposed children exceeded the DON PMTDI thus, making them the most exposed based on the urinary results.