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Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are legislated mycotoxins considered a major food safety risk. Dietary exposure to AFB1 and OTA has been associated with several human and animal diseases, including hepatotoxicity and nephrotoxicity. Functional ingredients ingestion has been proposed as a strategy to decrease mycotoxin toxicity. Fermented whey (FW), derived from milk whey fermentation, contains several bioactive compounds such as organic and phenolic acids. Pumpkin is rich in carotenoids and fiber. In order to evaluate in vivo the preventive effect of functional ingredients against mycotoxins, different feeds were prepared in the lab from fungal contaminated flour. A 28 days study was performed using Wistar rats divided in 12 groups fed with: 1) control diet; 2) AFB1 (5±0.6 mg/kg); 3) OTA (10.2±1.1 mg/kg); 4) AFB1 and OTA (8.8±1.5 and 10.9 mg/kg respectively); 5) FW feed; 6) FW (1%) and AFB1 (6.1±1.4 mg/kg); 7) FW (1%) and OTA (6.1±0.3 mg/kg); 8) FW (1%) with AFB1 and OTA (8.4±0.3 mg/kg and 8.4±0.4 mg/kg respectively); 9) FW and pumpkin (1%) feed; 10) FW and pumpkin feed and OTA (6.1 mg/kg); 11) FW and pumpkin feed and AFB1 (7.0 mg/kg) and 12) FW and pumpkin feed, OTA and AFB1 (9.0 and 7.7 mg/kg respectively). Each group had 10 rats, 5 males and 5 females. Urine from 24 h was collected every week in a metabolic cage for metabolomics study. Gastrointestinal tract organs, liver, kidneys and blood were collected at sacrifice. Biochemical parameters were evaluated in blood. Gene expression of carbamoyl phosphate synthetase 1 (CPS1) and kidney injury molecule 1 (KIM-1), biomarkers of liver and kidney damage, was measured by droplet digital PCR. Overall, results show a protection against mycotoxin oral exposure by FW and pumpkin ingestion. Acknowledgments: This work was supported by the Spanish Ministry of Science and Innovation (PID2019-108070RB-I00-ALI). [ABSTRACT FROM AUTHOR]

Mycotoxin contamination of cereals and cereal-based products is a serious problem for food safety. Antioxidant-rich ingredients such as bilberries ( Vaccinium myrtillus L., VM) may mitigate their harmful effects. Firstly, total phenolic content, antioxidant activity, and analytical phytochemical composition (hydroxycinnamic and hydroxybenzoic acids, flavanols, flavonols, and anthocyanins) were assessed in lyophilized wild bilberries from Romania. Secondly, this study evaluated bilberries' effects on reducing ochratoxin A (OTA) bioaccessibility and cytotoxicity. An in vitro digestion model was developed and applied to four different types of bread: Control, VM (2%), OTA (15.89 ± 0.13 mg/kg), and OTA (16.79 ± 0.55 mg/kg)-VM (2%). The results indicated that VM decreased OTA bioaccessibility by 15% at the intestinal level. OTA-VM digests showed improved Caco-2 cell viability in comparison to OTA digests across different exposure times. Regarding the alterations in Jurkat cell line cell cycle phases and apoptosis/necrosis, significant increases in cell death were observed using OTA digests (11%), while VM addition demonstrated a protective effect (1%). Reactive oxygen species (ROS) analysis confirmed these findings, with OTA-VM digests showing significantly lower ROS levels compared to OTA digests, resulting in a 3.7-fold decrease. Thus, bilberries exhibit high potential as a functional ingredient, demonstrating protection in OTA mitigation effects.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mycotoxins, secondary metabolites produced by molds, pose significant health risk through contamination of globally consumed cereals. Ochratoxin A (OTA), a prevalent mycotoxin in cereals, is associated with various health hazards, including immunotoxicity. This study explores the bioaccessibility of OTA in bread and its impact on the gastrointestinal barrier. A focus is placed on grape pomace (GP), a by-product of the wine industry, as a potential mitigator of OTA toxicity. Results demonstrate that GP reduces OTA bioaccessibility in the human gastrointestinal system from 94% to 81% at intestinal level, showing promise in limiting the absorption of the harmful toxin. Additionally, GP exhibits cytoprotective effects, enhancing cell viability and mitigating OTA-induced toxicity in both Caco-2 and Jurkat T cells. In view of the above, to understand the mechanisms by which OTA exhibits its toxic effects, flow cytometry was chosen as the main technique for the analysis of cell cycle, reactive oxygen species levels and mitochondrial parameters. Cytofluorimetric evaluation indicates GP's potential in limiting OTA-induced damage at cellular level. The study suggests that GP could serve as functional ingredient to reduce mycotoxin bioaccessibility and toxicity in cereal-based foods, offering a novel and promising approach to enhance food safety and protect public health. The finding highlights the potential of utilizing grape pomace in food formulations to mitigate mycotoxin contamination, providing a valuable contribution to the ongoing efforts to ensure the safety of globally consumed cereal products., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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 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.

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., (© 2023 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

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.

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 βIII-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 G 0 /G 1 . 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Due to the globalization, mycotoxins have been considered a major risk to human health being the main contaminants of foodstuffs. Among them, AFB1 and OTA are the most toxic and studied. Therefore, the goal of this review is to deepen the knowledge about the toxicological effects that AFB1 and OTA can induce on human health by using flow cytometry and immunofluorescence techniques in vitro and in vivo models. The examination of the selected reports shows that the majority of them are focused on immunotoxicity while the rest are concerned about nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, neurotoxicity, embryotoxicity, reproductive system, breast, esophageal and lung toxicity. In relation to immunofluorescence analysis, biological processes related to AFB1- and OTA-toxicity were evaluated such as inflammation, neuronal differentiation, DNA damage, oxidative stress and cell death. In flow cytometry analysis, a wide range of assays have been performed across the reviewed studies being apoptosis assay, cell cycle analysis and intracellular ROS measurement the most employed. Although, the toxic effects of AFB1 and OTA have been reported, further research is needed to clarify AFB1 and OTA-mechanism of action on human health., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.