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Mycotoxin research is facing unprecedented challenges, starting from the urgent need to cope with the consequences of climate change, the global shortage of grain due to unstable political scenarios, and the major transformation of the supply chains after the COVID-19 pandemic. In this scenario, the mycotoxin contamination of human and animal foods is still unavoidable, thus representing a major challenge to global food security. Next to this, the shift to sustainable and circular food production might be accompanied by an increase in food safety issues involving mycotoxins, e.g., when new technologies are applied to reuse side streams from the food industry, it is not known if and how mycotoxins accumulate in these by-products. MycoTWIN is an EU-funded Horizon 2020 project which fosters knowledge transfer and scientific cooperation within the Mediterranean area, involving worldwide experts, decision makers, and stakeholders in the field of mycotoxigenic fungi and mycotoxins. The MycoTWIN project hosted working group meetings, whose aim was to propose operational plans and/or scientific strategic plans to shape the future research directions to better cope with these challenges. In the working group cycle “Future proof approaches for the management of toxigenic fungi and associated mycotoxins along the food chain”, a multi-actor group was guided in co-creation exercises to elaborate on future research directions and propose relevant actions to be implemented for the present to long-term time periods. The discussion focused on three main topics relevant to the assessment and management of risks associated with mycotoxins and toxigenic fungi: (i) needs for the harmonization of molecular and chemical methods and data analysis, (ii) from lab research to marketable solutions: how to fill the gap, and (iii) gaps in data quality for risk assessment.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceous and various Penicillium species, which are known for contaminating agricultural products and posing significant health risks, which include immunotoxicity. This study aims to evaluate the potential of nanostructured lipid carriers (NLCs) loaded with a carotenoid-enriched extract from pumpkin peel (Cucurbita maxima L.) in mitigating the toxic effects of OTA. To address the poor bioavailability and instability of carotenoids, nanoencapsulation techniques were employed to enhance their delivery and efficacy. NLCs were formulated using hydrogenated sunflower oil, pumpkin oil, and soy lecithin using hot high-pressure homogenization. The in vitro study involved co-digesting OTA-contaminated bread with an NLC formulation and assessing the impact of the encapsulated carotenoid on OTA bioaccessibility, bioavailability, and cellular toxicity using Caco-2 and Jurkat T cells. Even though no significant influence was observed on the bioaccessibility and bioavailability of OTA, carotenoid-loaded NLCs exhibited cytoprotective effects by improving cell viability and mitigating OTA-induced toxicity in both Caco-2 and Jurkat T cells. Particularly, the flow cytometry analysis highlighted the ability of carotenoids to mitigate OTA-induced cellular damage by decreasing ROS production and limiting mitochondrial mass changes. The study suggests that the encapsulation of carotenoids in NLCs represents a promising strategy to enhance their protective effects against OTA toxicity, potentially offering a novel approach to food safety and public health protection. The study underscores the potential of nanotechnology in improving the bioavailability and efficacy of natural antioxidants to mitigate mycotoxin-induced damage.

The present work focuses on the evaluation of AFB1′s bioaccessibility and cytotoxicity in vitro using bread (naturally contaminated) enriched or not enriched with fresh Voghiera garlic (2%). Two different experiments were carried out: experiment 1 (E1), with low-AFB1-concentration breads (1.6–1.7 mg/kg); and experiment 2 (E2), with high-AFB1-concentration breads (96.4–102.7 mg/kg). Eight breads were prepared, four for E1 (experiment 1) and another four for E2 (experiment 2), with each experiment having a control group (C), a garlic-enriched group (2%) (G), an AFB1 group (A), and an AFB1 + garlic group (A + G). Simulated digestion was performed on each type of bread, and gastric and intestinal digests were obtained. AFB1 content in flours, baked bread, and gastric and intestinal digests was measured by High-Performance Liquid Chromatography coupled to Fluorescence Detection. The results demonstrate dose-dependent AFB1 bioaccessibility and that the presence of garlic contributed to its reduction in both doses (7–8%). Moreover, garlic’s presence in AFB1-contaminated bread increased cell viability (9–18%) in differentiated Caco-2 cells and mitigated the arrest of S and G2/M phases provoked by AFB1 on Jurkat T cells and reduced apoptosis/necrosis, cellular reactive oxygen species (ROS), and mitochondrial ROS by 16%, 71%, and 24% respectively. The inclusion of garlic as a functional ingredient helped relieve the presence and effects of AFB1.

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.

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.