Endotoxin Translocation Is Increased in Broiler Chickens Fed a Fusarium Mycotoxin-Contaminated Diet.

Broiler chickens in livestock production face numerous challenges that can impact their health and welfare, including mycotoxin contamination and heat stress. In this study, we aimed to investigate the combined effects of two mycotoxins, deoxynivalenol (DON) and fumonisins (FBs), along with short-term heat stress conditions, on broiler gut health and endotoxin translocation. An experiment was conducted to assess the impacts of mycotoxin exposure on broilers, focusing on intestinal endotoxin activity, gene expression related to gut barrier function and inflammation, and the plasma concentration of the endotoxin marker 3-OH C14:0 either at thermoneutral conditions or short-term heat stress conditions. Independently of heat stress, broilers fed DON-contaminated diets exhibited reduced body weight gain during the starter phase (Day 1-12) compared to the control group, while broilers fed FB-contaminated diets experienced decreased body weight gain throughout the entire trial period (Day 1-24). Furthermore, under thermoneutral conditions, broilers fed DON-contaminated diets showed an increase in 3-OH C14:0 concentration in the plasma. Moreover, under heat stress conditions, the expression of genes related to gut barrier function (Claudin 5, Zonulin 1 and 2) and inflammation (Toll-like receptor 4, Interleukin-1 beta, Interleukin-6) was significantly affected by diets contaminated with mycotoxins, depending on the gut segment. This effect was particularly prominent in broilers fed diets contaminated with FBs. Notably, the plasma concentration of 3-OH C14:0 increased in broilers exposed to both DON- and FB-contaminated diets under heat stress conditions. These findings shed light on the intricate interactions between mycotoxins, heat stress, gut health, and endotoxin translocation in broiler chickens, highlighting the importance of understanding these interactions for the development of effective management strategies in livestock production to enhance broiler health and welfare.

Effects of a Curcumin/Silymarin/Yeast-Based Mycotoxin Detoxifier on Redox Status and Growth Performance of Weaned Piglets under Field Conditions.

The aim of this in vivo study was to investigate the effects of a novel mycotoxin detoxifier whose formulation includes clay (bentonite and sepiolite), phytogenic feed additives (curcumin and silymarin) and postbiotics (yeast products) on the health, performance and redox status of weaned piglets under the dietary challenge of fumonisins (FUMs). The study was conducted in duplicate in the course of two independent trials on two different farms. One hundred and fifty (150) weaned piglets per trial farm were allocated into two separate groups: (a) T1 (control group): 75 weaned piglets received FUM-contaminated feed and (b) T2 (experimental group): 75 weaned piglets received FUM-contaminated feed with the mycotoxin-detoxifying agent from the day of weaning (28 days) until 70 days of age. Thiobarbituric acid reactive substances (TBARSs), protein carbonyls (CARBs) and the overall antioxidant capacity (TAC) were assessed in plasma as indicators of redox status at 45 and 70 days of age. Furthermore, mortality and performance parameters were recorded at 28, 45 and 70 days of age, while histopathological examination was performed at the end of the trial period (day 70). The results of the present study reveal the beneficial effects of supplementing a novel mycotoxin detoxifier in the diets of weaners, including improved redox status, potential hepatoprotective properties and enhanced growth performance.

The Effect of Combined Exposure of Fusarium Mycotoxins on Lipid Peroxidation, Antioxidant Defense, Fatty Acid Profile, and Histopathology in Laying Hens' Liver.

Fumonisin B1, T-2 toxin, and deoxynivalenol are frequently detected in feed materials. The mycotoxins induce free radical formation and, thereby, lipid peroxidation. The effects of mycotoxin exposure at the EU recommended limit (T-2/HT-2 toxin: 0.25 mg/kg; DON = 3AcDON/15-AScDON: 5 mg/kg; fumonisin B1: 20 mg/kg) and double dose (T-2/HT-2 toxin: 0.5 mg/kg, DON/3-AcDON/15-AcDON: 10 mg, and FB1: 40 mg/kg feed) were investigated during short-term (3 days) per os exposure in the liver of laying hens. On day 1 higher while on day 3 lower MDA concentrations were found in the low-dose group compared to the control. Fatty acid composition also changed: the proportion of monounsaturated fatty acids increased (p < 0.05) and the proportion of polyunsaturated fatty acids decreased by day 3. These alterations resulted in a decrease in the index of unsaturation and average fatty acid chain length. Histopathological alterations suggested that the incidence and severity of liver lesions were higher in the mycotoxin-treated laying hens, and the symptoms correlated with the fatty acid profile of total phospholipids. Overall, the findings revealed that mycotoxin exposure, even at the EU-recommended limits, induced lipid peroxidation in the liver, which led to changes in fatty acid composition, matched with tissue damage.

Edodin: A New Type of Toxin from Shiitake Mushroom (Lentinula edodes) That Inactivates Mammalian Ribosomes.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.

Evaluating the human neurotoxicity and toxicological interactions impact of co-occurring regulated and emerging mycotoxins.

Mycotoxins can inflict harmful effects on diverse organs, and mounting evidence indicates their potential involvement in human neurodegenerative diseases. Given the common occurrence of these toxins in food, there is an increasing demand for a comprehensive assessment of their combined toxicity to enhance our understanding of their potential hazards. This research investigates mycotoxin exposure from widely consumed cereal-based products, including enniatin B (ENNB), sterigmatocystin (STG), aflatoxin B1 (AFB1), cyclopiazonic acid (CPZ), citrinin (CIT), and ochratoxin A (OTA). Employing the median-effect equation based on Chou and Talalay's mass-action law, we assessed their cytotoxicity in human SH-SY5Y neuronal cells. Notably, ENNB displayed the highest neurotoxicity (IC50 = 3.72 µM), followed by OTA (9.10 µM) and STG (9.99 µM). The combination of OTA + STG exhibited the highest toxicity (IC50 = 3.77 µM), while CPZ + CIT showed the least detrimental effect. Approximately 70 % of tested binary combinations displayed synergistic or additive effects, except for ENNB + STG, ENNB + AFB1, and CPZ + CIT, which showed antagonistic interactions. Intriguingly, the senary combination displayed moderate antagonism at the lowest exposure and moderate synergism at higher doses. OTA exhibited predominantly synergistic interactions, comprising approximately 90 %, a noteworthy finding considering its prevalence in food. Conversely, ENNB interactions tended to be antagonistic. The most remarkable synergy occurred in the STG and CIT combination, enabling a 50-fold reduction in CIT dosage for an equivalent toxic effect. These findings highlight the biological relevance of robust synergistic interactions, emphasizing the need to assess human exposure hazards accurately, particularly considering frequent mycotoxin co-occurrence in environmental and food settings.

PI3K/Akt/FoxO Pathway Mediates Antagonistic Toxicity in HepG2 Cells Coexposed to Deoxynivalenol and Enniatins.

The emerging mycotoxins enniatins (ENNs) and the traditional mycotoxin deoxynivalenol (DON) often co-contaminate various grain raw materials and foods. While the liver is their common target organ, the mechanism of their combined effect remains unclear. In this study, the combined cytotoxic effects of four ENNs (ENA, ENA1, ENB, and ENB1) with DON and their mechanisms were investigated using the HepG2 cell line. Additionally, a population exposure risk assessment of these mycotoxins was performed by using in vitro experiments and computer simulations. The results showed that only ENA at 1/4 IC50 and ENB1 at 1/8 IC50 coexposed with DON showed an additive effect, while ENB showed the strongest antagonism at IC50 (CI = 3.890). Co-incubation of ENNs regulated the signaling molecule levels which were disrupted by DON. Transcriptome analysis showed that ENB (IC50) up-regulated the PI3K/Akt/FoxO signaling pathway and inhibited the expression of apoptotic genes (Bax, P53, Caspase 3, etc.) via phosphorylation of FoxO, thereby reducing the cytotoxic effects caused by DON. Both types of mycotoxins posed serious health risks, and the cumulative risk of coexposure was particularly important for emerging mycotoxins.

Meta-Analysis of the Effects of Yeast Cell Wall Extract Supple-Mentation during Mycotoxin Challenges on the Performance of Laying Hens.

A random-effects meta-analysis was conducted to investigate the effect of mycotoxins (MT) without or with the inclusion of yeast cell wall extract (YCWE, Mycosorb®, Alltech, Inc., Nicholasville, KY, USA) on laying hen performance. A total of 25 trials were collected from a literature search, and data were extracted from 8 of these that met inclusion criteria, for a total of 12 treatments and 1774 birds. Laying hens fed MT had lower (p < 0.05) body weight (BW) by -50 g, egg production by -6.3 percentage points, and egg weight by -1.95 g than control fed hens (CTRL). Inclusion of YCWE during the mycotoxin challenges (YCWE + MT) resulted in numerically greater (p = 0.441) BW by 12.5 g, while egg production and egg weight were significantly (p < 0.0001) higher by 4.2 percentage points and 1.37 g, respectively. Furthermore, economic assessment calculations indicated that YCWE may not only support hen performance but also resulted in a positive return on investment. In conclusion, mycotoxins can play a role in negatively impacting laying hen performance and profitability. Inclusion of YCWE in feed with mycotoxin challenges provided benefits to egg production and egg weight and may support profitability. As such, the inclusion of YCWE could play an important role in minimizing mycotoxin effects and in turn aid farm efficiency and profitability.

New insight into mycotoxins and bacterial toxins: Toxicity assessment, molecular mechanism and food safety (preface to the special issue of food and chemical toxicology on the outcomes of Myco & bacterial toxin).

The special issue "New Insight into Mycotoxins and Bacterial Toxins: Toxicity Assessment, Molecular Mechanism and Food Safety" in Food and Chemical Toxicology contains 19 articles on current hot topics in mycotoxins and bacterial toxins. Dietary exposure to mycotoxins and risk assessments are reported in this issue. Molecular mechanisms of multiple mycotoxins and emerging mechanisms of toxicity are especially concerned by researchers. Moreover, mycotoxin-detoxifying substances and antimicrobial agents are also fully investigated in the context. This special issue will help to further understand the mycotoxins and bacterial toxins, casting new light for the control of food safety.

Computational methods meet in vitro techniques: A case study on fusaric acid and its possible detoxification through cytochrome P450 enzymes.

Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants.

Beauvericin and enniatin B mycotoxins alter aquatic ecosystems: Effects on green algae.

Myxotoxins can contaminate algal-based products and arrive to the food chain to consumers producing chronic toxicity effects. Here, we studied phytotoxicity of mycotoxins, beauvericin (BEA) and ennaitin B (ENN B) in four phytoplankton strains: Acutodesmus sp., Chlamydomonas reinhardtii, Haematococcus pluvialis, and Monoraphidium griffithii, which are all green algae. It was tested the capacity of clearing the media of BEA and ENN B at different concentrations by comparing nominal and measured quantifications. Results revealed that Acutodesmus sp. and C. reinhardtii tended to flow up and down growth rate without reaching values below 50% or 60%, respectively. On the other hand, for H. pluvialis and M. griffith, IC50 values were reached. Regarding the clearance of media, in individual treatment a decrease of the quantified mycotoxin between nominal and measured values was observed; while in binary treatment, differences among both values were higher and more noted for BEA than for ENN B.

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.

Mechanistic insight into human androgen receptor-mediated endocrine disrupting potential of cyclic depsipeptide mycotoxin, beauvericin, and influencing environmental factors for its biosynthesis in Fusarium oxysporum KFCC 11363P on rice cereal.

In current study, Fusarium mycotoxin, beauvericin (BEA), has endocrine disrupting potential through suppressing the exogenous androgen receptor (AR)-mediated transcriptional activation. BEA was classified as an AR antagonist, with IC30 and IC50 values indicating that it suppressed AR dimerization in the cytosol. BEA suppress the translocation of cytosolic activated ARs to the nucleus via exogenous androgens. Furthermore, we investigated the impact of environmental conditions for BEA production on rice cereal using response surface methodology. The environmental factors affecting the production of BEA, namely temperature, initial moisture content, and growth time were optimized at 20.28 °C, 42.79 % (w/w), and 17.31 days, respectively. To the best of our knowledge, this is the first report showing that BEA has endocrine disrupting potential through suppressing translocation of cytosolic ARs to nucleus, and temperature, initial moisture content, and growth time are important influencing environmental factors for its biosynthesis in Fusarium strains on cereal.

Natural compounds mitigate mycotoxins-induced neurotoxicity by modulating oxidative tonus: in vitro and in vivo insights - a review.

This review explores the repercussions of mycotoxin contamination in food and feed, emphasising potential threats to agriculture, animal husbandry and public health. The primary objective is to make a comprehensive assessment of the neurotoxic consequences of mycotoxin exposure, an aspect less explored in current literature. Emphasis is placed on prominent mycotoxins, including aflatoxins, fumonisins, zearalenone (ZEA) and ochratoxins, known for inducing acute and chronic diseases such as liver damage, genetic mutation and cancer. To elucidate the effects, animal studies were conducted, revealing an association between mycotoxin exposure and neurological damage. This encompasses impairments in learning and memory, motor alterations, anxiety and depression. The underlying mechanisms involve oxidative stress, disrupting the balance between reactive oxygen species (ROS) and antioxidant capacity. This oxidative stress is linked to neuronal damage, brain inflammation, neurochemical imbalance, and subsequent behavioural changes. The review underscores the need for preventive measures against mycotoxin exposure. While complete avoidance is ideal, exploration into the potential use of antioxidants as a viable solution is discussed, given the widespread contamination of many food products. Specifically, the protective role of natural compounds, such as polyphenols, is highlighted, showcasing their efficacy in mitigating mycotoxicosis in the central nervous system (CNS), as evidenced by findings in various animal models. In summary, countering mycotoxin-induced neurotoxicity requires a multifaceted approach. The identified natural compounds show promise, but their practical use hinges on factors like bioavailability, toxicity and understanding their mechanisms of action. Extensive research is crucial, considering the diverse responses to different mycotoxins and neurological conditions. Successful implementation relies on factors such as the specific mycotoxin(s) involved and achievable effective concentrations. Further research and clinical trials are imperative to establish the safety and efficacy of these compounds in practical applications.

Combined in vitro and in silico mechanistic approach to explore the potential of Alternaria mycotoxins alternariol and altertoxin II to hamper γH2AX formation in DNA damage signaling pathways.

Risk assessment of food and environmental contaminants is faced by substantial data gaps and novel strategies are needed to support science-based regulatory actions. The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATXII) have garnered attention for their possible genotoxic effects. Nevertheless, data currently available are rather scattered, hindering a comprehensive hazard characterization. This study combined in vitro/in silico approaches to elucidate the potential of AOH and ATXII to induce double-strand breaks (DSBs) in HepG2 cells. Furthermore, it examines the impact of co-exposure to AOH and the DSB-inducing drug doxorubicin (Doxo) on γH2AX expression. AOH slightly increased γH2AX expression, whereas ATXII did not elicit this response. Interestingly, AOH suppressed Doxo-induced γH2AX expression, despite evidence of increased DNA damage in the comet assay. Building on these observations, AOH was postulated to inhibit γH2AX-forming kinases. Along this line, in silico analysis supported AOH potential interaction with the ATP-binding sites of these kinases and immunofluorescence experiments showed decreased intracellular phosphorylation events. Similarly, in silico results suggested that ATXII might also interact with these kinases. This study emphasizes the importance of understanding the implications of AOH-induced γH2AX expression inhibition on DNA repair processes and underscores the need for caution when interpreting γH2AX assay results.

In Vitro Assessment of Ozone-Treated Deoxynivalenol by Measuring Cytotoxicity and Wheat Quality.

Deoxynivalenol (DON), a trichothecene mycotoxin, could lead to cytotoxicity in both animal bodies and plant seed cells. Ozone degradation technology has been applied to DON control. However, the safety and quality of the contaminated grain after DON degradation are largely obscured. In this work, we evaluated the cytotoxicity of ozone-treated DON through seed germination experiments and cytotoxicity tests. Cell experiments showed that the inhibition rate of HepG2 viability gradually increased within the concentrations of 1-10 mg/L of DON, alongside which an IC50 (half maximal inhibitory concentration) of 9.1 mg/L was determined. In contrast, degrading DON had no significant inhibitory effect on cell growth. Moreover, a 1-10 mg/L concentration of DON increased production of a large amount of reactive oxygen radicals in HepG2, with obvious fluorescence color development. However, fluorescence intensity decreased after DON degradation. Further, DON at a concentration of >1 mg/L significantly inhibited the germination of mung bean seeds, whereas no significant inhibition of their germination or growth were observed if DON degraded. Changes in total protein content, fatty acid value, and starch content were insignificant in wheat samples suffering ozone degradation, compared to the untreated group. Lastly, the ozone-treated wheat samples exhibited higher tenacity and whiteness. Together, our study indicated that the toxicity of DON-contaminated wheat was significantly reduced after ozone degradation.

Effect of Acrylamide and Mycotoxins in SH-SY5Y Cells: A Review.

Thermal processes induce the formation of undesired toxic components, such as acrylamide (AA), which has been shown to induce brain toxicity in humans and classified as Group 2A by the International Agency of Research in Cancer (IARC), as well as some mycotoxins. AA and mycotoxins' toxicity is studied in several in vitro models, including the neuroblastoma cell line model SH-SY5Y cells. Both AA and mycotoxins occur together in the same food matrix cereal base (bread, pasta, potatoes, coffee roasting, etc.). Therefore, the goal of this review is to deepen the knowledge about the neurological effects that AA and mycotoxins can induce on the in vitro model SH-SY5Y and its mechanism of action (MoA) focusing on the experimental assays reported in publications of the last 10 years. The analysis of the latest publications shows that most of them are focused on cytotoxicity, apoptosis, and alteration in protein expression, while others are interested in oxidative stress, axonopathy, and the disruption of neurite outgrowth. While both AA and mycotoxins have been studied in SH-SY5Y cells separately, the mixture of them is starting to draw the interest of the scientific community. This highlights a new and interesting field to explore due to the findings reported in several publications that can be compared and the implications in human health that both could cause. In relation to the assays used, the most employed were the MTT, axonopathy, and qPCR assays. The concentration dose range studied was 0.1-10 mM for AA and 2 fM to 200 µM depending on the toxicity and time of exposure for mycotoxins. A healthy and varied diet allows the incorporation of a large family of bioactive compounds that can mitigate the toxic effects associated with contaminants present in food. Although this has been reported in some publications for mycotoxins, there is still a big gap for AA which evidences that more investigations are needed to better explore the risks for human health when exposed to AA and mycotoxins.

Trichothecenes and Fumonisins: Key Players in Fusarium-Cereal Ecosystem Interactions.

Fusarium fungi produce a diverse array of mycotoxic metabolites during the pathogenesis of cereals. Some, such as the trichothecenes and fumonisins, are phytotoxic, acting as non-proteinaceous effectors that facilitate disease development in cereals. Over the last few decades, we have gained some depth of understanding as to how trichothecenes and fumonisins interact with plant cells and how plants deploy mycotoxin detoxification and resistance strategies to defend themselves against the producer fungi. The cereal-mycotoxin interaction is part of a co-evolutionary dance between Fusarium and cereals, as evidenced by a trichothecene-responsive, taxonomically restricted, cereal gene competing with a fungal effector protein and enhancing tolerance to the trichothecene and resistance to DON-producing F. graminearum. But the binary fungal-plant interaction is part of a bigger ecosystem wherein other microbes and insects have been shown to interact with fungal mycotoxins, directly or indirectly through host plants. We are only beginning to unravel the extent to which trichothecenes, fumonisins and other mycotoxins play a role in fungal-ecosystem interactions. We now have tools to determine how, when and where mycotoxins impact and are impacted by the microbiome and microfauna. As more mycotoxins are described, research into their individual and synergistic toxicity and their interactions with the crop ecosystem will give insights into how we can holistically breed for and cultivate healthy crops.

Identification of the hub genes linked to zearalenone-induced hepatotoxicity in broiler chickens.

Zearalenone (ZEN) is a mycotoxin found in food and feed that impairs the function of multiple organs, especially the liver. However, the specific mechanisms through which ZEN induces liver damage in broiler chickens are not well understood. Therefore, this study aimed to identify the key genes linked to the hepatotoxicity induced by ZEN exposure in broiler chickens. Gene expression data from ZEN-treated and control chicken embryo primary hepatocytes (CEPHs) were used to implement differential expression analysis. Totally, 436 differentially expressed genes (DEGs) were detected, in which 223 and 213 genes were up- and down-regulated in ZEN-treated CEPHs, respectively. Gene ontology analysis suggested that these DEGs were involved in various biological processes, including chromosome segregation, mitotic cytokinesis, mitotic cell cycle, cell division, and mitotic spindle organization. Pathway analysis showed that the DEGs were associated with p53, FoxO, ubiquitin-mediated proteolysis, cell cycle, and mismatch repair signaling pathways. Furthermore, the hub genes, including BRCA1, CDC45, CDCA3, CDKN3, CENPE, CENPF, CENPI, CENPM, CENPU, and CEP55, potentially contributed to ZEN-induced hepatotoxicity. In conclusion, our study provides the valuable insight into the mechanism underlying ZEN-induced hepatotoxicity in broiler chickens.

Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure.

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

Cytotoxic Effects of Major and Emerging Mycotoxins on HepaRG Cells and Transcriptomic Response after Exposure of Spheroids to Enniatins B and B1.

Mycotoxins, produced by fungi, frequently occur at different stages in the food supply chain between pre- and postharvest. Globally produced cereal crops are known to be highly susceptible to contamination, thus constituting a major public health concern. Among the encountered mycotoxigenic fungi in cereals, Fusarium spp. are the most frequent and produce both regulated (i.e., T-2 toxin, deoxynivalenol -DON-, zearalenone -ZEA-) and emerging (i.e., enniatins -ENNs-, beauvericin -BEA-) mycotoxins. In this study, we investigated the in vitro cytotoxic effects of regulated and emerging fusariotoxins on HepaRG cells in 2D and 3D models using undifferentiated and differentiated cells. We also studied the impact of ENN B1 and ENN B exposure on gene expression of HepaRG spheroids. Gene expression profiling pinpointed the differentially expressed genes (DEGs) and overall similar pathways were involved in responses to mycotoxin exposure. Complement cascades, metabolism, steroid hormones, bile secretion, and cholesterol pathways were all negatively impacted by both ENNs. For cholesterol biosynthesis, 23/27 genes were significantly down-regulated and could be correlated to a 30% reduction in cholesterol levels. Our results show the impact of ENNs on the cholesterol biosynthesis pathway for the first time. This finding suggests a potential negative effect on human health due to the essential role this pathway plays.