Deoxynivalenol Induces Local Inflammation and Lesions in Tissues at Doses Recommended by the EU.

The mycotoxin deoxynivalenol (DON) is frequently present in cereals at low levels, resulting in its occurrence in food and feed. DON has been proven to alter the immune response and induce inflammation in all species, with pigs exhibiting heightened sensitivity and exposure. However, no study has yet evaluated the effects of exposure to DON at the recommended levels in pig feed. In two separate trials, piglets were subjected to control feed or feed contaminated with a low level of purified DON (0.83 mg/kg feed in trial 1 and 0.85 mg/kg feed in trial 2) for either three weeks (trial 1) or two weeks (trial 2). Additionally, a group of animals exposed to 2.85 mg/kg feed of DON was included as a positive control in Trial 1. The impact of DON on porcine tissues (intestine, liver, and spleen) was evaluated through histological and qPCR analyses of immune-related genes. Additionally, biochemical analyses and acute-phase proteins were examined in plasma samples. Lesions were identified in the intestine (jejunum and ileum), the liver, and the spleen of pigs receiving diets contaminated with low and high concentrations of DON. The low level of DON also resulted in impaired expression of genes associated with intestinal barrier integrity, intestinal immune responses, and liver function. In conclusion, the results of the two trials demonstrate the impact of DON exposure even at doses below the recommended level of 0.9 mg/kg feed set by the European Union. This suggests that the current recommended level should be reconsidered to ensure the optimal health and well-being of pigs.

From ribosome to ribotoxins: understanding the toxicity of deoxynivalenol and Shiga toxin, two food borne toxins.

Ribosomes that synthesize proteins are among the most central and evolutionarily conserved organelles. Given the key role of proteins in cellular functions, prokaryotic and eukaryotic pathogens have evolved potent toxins to inhibit ribosomal functions and weaken their host. Many of these ribotoxin-producing pathogens are associated with food. For example, food can be contaminated with bacterial pathogens that produce the ribotoxin Shiga toxin, but also with the fungal ribotoxin deoxynivalenol. Shiga toxin cleaves ribosomal RNA, while deoxynivalenol binds to and inhibits the peptidyl transferase center. Despite their distinct modes of action, both groups of ribotoxins hinder protein translation, but also trigger other comparable toxic effects, which depend or not on the activation of the ribotoxic stress response. Ribotoxic stress response-dependent effects include inflammation and apoptosis, whereas ribotoxic stress response-independent effects include endoplasmic reticulum stress, oxidative stress, and autophagy. For other effects, such as cell cycle arrest and cytoskeleton modulation, the involvement of the ribotoxic stress response is still controversial. Ribotoxins affect one organelle yet induce multiple toxic effects with multiple consequences for the cell. The ribosome can therefore be considered as the cellular "Achilles heel" targeted by food borne ribotoxins. Considering the high toxicity of ribotoxins, they pose a substantial health risk, as humans are highly susceptible to widespread exposure to these toxins through contaminated food sources.

Overview and Comparison of Intestinal Organotypic Models, Intestinal Cells, and Intestinal Explants Used for Toxicity Studies.

The intestine is a complex organ formed of different types of cell distributed in different layers of tissue. To minimize animal experiments, for decades, researchers have been trying to develop in vitro/ex vivo systems able to mimic the cellular diversity naturally found in the gut. Such models not only help our understanding of the gut physiology but also of intestinal toxicity. This review describes the different systems used to evaluate the effects of drugs/contaminants on intestinal functions and compares their advantages and limitations. The comparison showed that the organotypic model is the best available model to perform intestinal toxicity studies, including on human tissues.

Exposure of intestinal explants to NX, but not to DON, enriches the secretome in mitochondrial proteins.

NX is a type A trichothecene produced by Fusarium graminearum with limited information on its toxicity. NX is structurally similar to deoxynivalenol (DON), only differing by the lacking keto group at C8. Because of the structural similarity of the two toxins as well as their potential co-occurrence in food and feed, it is of interest to determine the toxicity of this new compound. In this study, we compared the protein composition of the extracellular media of pig intestinal explants (secretome) exposed to 10 µM of DON or NX for 4 h compared with controls. The combination of two complementary quantitative proteomic approaches (a gel-based and a gel-free approach) identified 18 and 23 differentially abundant proteins (DAPs) for DON and NX, respectively, compared to controls. Functional analysis suggested that, whereas DON toxicity was associated with decreased cell viability and cell destruction, NX toxicity was associated with an enrichment of mitochondrial proteins in the secretome. The presence of these proteins may be associated with the already known ability of NX to induce an intestinal inflammation. Overall, our results indicated that DON- and NX-induced changes in the extracellular proteome of intestinal explants are different. The increased leakage/secretion of mitochondrial proteins by NX may be a feature of NX toxicity.

The food contaminant, deoxynivalenol, modulates the Thelper/Treg balance and increases inflammatory bowel diseases.

The incidence of inflammatory bowel diseases (IBD) is increasing in both Western and developing countries. IBD are multifactorial disorders involving complex interactions between genetic, immune, and environmental factors such as exposure to food contaminants. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food and induces intestinal breakdown and inflammatory response. To delineate the role of DON oral exposure in IBD, we used a Dextran sulfate sodium (DSS) colitis model in rats fed with a DON-contaminated diet or a control diet for 4 weeks. Colitis was induced in the 4th week by increasing concentrations of DSS in the drinking water (0, 2, 3 or 5%). DON exacerbated body weight loss and accelerated the appearance of symptoms in animals treated with DSS. DON increased morphological damage, pro-inflammatory markers (myeloperoxidase, CXCL-1 and IL-1ß) and immune cell responses. In lamina propria of the rat with colitis, DON increased adaptive and innate immune responses after anti-CD3/28 or LPS stimulation, respectively. In the spleen, DON increased IFNγ secretion and reduced Treg populations. Interestingly, De-epoxy-DON (DOM-1) a detoxified form of DON did not have any consequences on colitis. These results suggest that DON is a risk factor in the onset of IBD.

The brlA Gene Deletion Reveals That Patulin Biosynthesis Is Not Related to Conidiation in Penicillium expansum.

Dissemination and survival of ascomycetes is through asexual spores. The brlA gene encodes a C2H2-type zinc-finger transcription factor, which is essential for asexual development. Penicillium expansum causes blue mold disease and is the main source of patulin, a mycotoxin that contaminates apple-based food. A P. expansum PeΔbrlA deficient strain was generated by homologous recombination. In vivo, suppression of brlA completely blocked the development of conidiophores that takes place after the formation of coremia/synnemata, a required step for the perforation of the apple epicarp. Metabolome analysis displayed that patulin production was enhanced by brlA suppression, explaining a higher in vivo aggressiveness compared to the wild type (WT) strain. No patulin was detected in the synnemata, suggesting that patulin biosynthesis stopped when the fungus exited the apple. In vitro transcriptome analysis of PeΔbrlA unveiled an up-regulated biosynthetic gene cluster (PEXP_073960-PEXP_074060) that shares high similarity with the chaetoglobosin gene cluster of Chaetomium globosum. Metabolome analysis of PeΔbrlA confirmed these observations by unveiling a greater diversity of chaetoglobosin derivatives. We observed that chaetoglobosins A and C were found only in the synnemata, located outside of the apple, whereas other chaetoglobosins were detected in apple flesh, suggesting a spatial-temporal organization of the chaetoglobosin biosynthesis pathway.

Regulation of Secondary Metabolism in the Penicillium Genus.

Penicillium, one of the most common fungi occurring in a diverse range of habitats, has a worldwide distribution and a large economic impact on human health. Hundreds of the species belonging to this genus cause disastrous decay in food crops and are able to produce a varied range of secondary metabolites, from which we can distinguish harmful mycotoxins. Some Penicillium species are considered to be important producers of patulin and ochratoxin A, two well-known mycotoxins. The production of these mycotoxins and other secondary metabolites is controlled and regulated by different mechanisms. The aim of this review is to highlight the different levels of regulation of secondary metabolites in the Penicillium genus.

Combination of Isotope Labeling and Molecular Networking of Tandem Mass Spectrometry Data To Reveal 69 Unknown Metabolites Produced by Penicillium nordicum.

The secondary metabolome of Penicillium nordicum is poorly documented despite its frequent detection on contaminated food and its capacity to produce toxic metabolites such as ochratoxin A. To characterize metabolites produced by this fungi, we combined a full stable isotopes labeling with the dereplication of tandem mass spectrometry (MS/MS) data by molecular networking. First, the untargeted metabolomic analysis by high-resolution mass spectrometry of a double stable isotope labeling of P. nordicum enabled the specific detection of its metabolites and the unambiguous determination of their elemental composition. Analyses showed that infection of substrate by P. nordicum lead to the production of at least 92 metabolites and that 69 of them were completely unknown. Then, curated molecular networks of MS/MS data were generated with GNPS and MetGem, specifically on the features of interest, which allowed highlighting 13 fungisporin-related metabolites that had not previously been identified in this fungus and 8 that had never been observed in any fungus. The structures of the unknown compounds, namely, a native fungisporin and seven linear peptides, were characterized by tandem mass spectrometry experiments. The analysis of P. nordicum growing on its natural substrates, i.e. pork ham, turkey ham, and cheese, demonstrated that 10 of the known fungisporin-related metabolites and three of the new metabolites were also synthesized. Thus, the curation of data for molecular networking using a specific detection of metabolites of interest with stable isotopes labeling allowed the discovery of new metabolites produced by the food contaminant P. nordicum.

Deoxynivalenol inhibits the expression of trefoil factors (TFF) by intestinal human and porcine goblet cells.

Trefoil factors (TFFs) are bioactive peptides expressed by several epithelia, including the intestine, where they regulate key functions such as tissue regeneration, barrier function and inflammation. Although food-associated mycotoxins, including deoxynivalenol (DON), are known to impact many intestinal functions, modulation of TFFs during mycotoxicosis has never been investigated. Here, we analyzed the effect of DON on TFFs expression using both human goblet cells (HT29-16E cells) and porcine intestinal explants. Results showed that very low doses of DON (nanomolar range) inhibit the secretion of TFFs by human goblet cells (IC50 of 361, 387 and 243 nM for TFF1, 2 and 3, respectively) and prevent wound healing. RT-qPCR analysis demonstrated that the inhibitory effect of DON is related to a suppression of TFFs mRNA expression. Experiments conducted on porcine intestinal explants confirmed the results obtained on cells. Finally, the use of specific inhibitors of signal pathways demonstrated that DON-mediated suppression of TFFs expression mainly involved Protein Kinase R and the MAP kinases (MAPK) p38 and ERK1/2. Taken together, our results show for the first time that at very low doses, DON suppresses the expression and production of intestinal TFFs and alters wound healing. Given the critical role of TFFs in tissue repair, our results suggest that DON-mediated suppression of TFFs contributes to the alterations of intestinal integrity the caused by this toxin.

The protective role of liver X receptor (LXR) during fumonisin B1-induced hepatotoxicity.

Fumonisin B1 (FB1), a congener of fumonisins produced by Fusarium species, is the most abundant and most toxicologically active fumonisin. FB1 causes severe mycotoxicosis in animals, including nephrotoxicity, hepatotoxicity, and disruption of the intestinal barrier. However, mechanisms associated with FB1 toxicity are still unclear. Preliminary studies have highlighted the role of liver X receptors (LXRs) during FB1 exposure. LXRs belong to the nuclear receptor family and control the expression of genes involved in cholesterol and lipid homeostasis. In this context, the toxicity of FB1 was compared in female wild-type (LXR+/+) and LXRα,ß double knockout (LXR-/-) mice in the absence or presence of FB1 (10 mg/kg body weight/day) for 28 days. Exposure to FB1 supplemented in the mice's drinking water resulted in more pronounced hepatotoxicity in LXR-/- mice compared to LXR+/+ mice, as indicated by hepatic transaminase levels (ALT, AST) and hepatic inflammatory and fibrotic lesions. Next, the effect of FB1 exposure on the liver transcriptome was investigated. FB1 exposure led to a specific transcriptional response in LXR-/- mice that included altered cholesterol and bile acid homeostasis. ELISA showed that these effects were associated with an elevated FB1 concentration in the plasma of LXR-/- mice, suggesting that LXRs participate in intestinal absorption and/or clearance of the toxin. In summary, this study demonstrates an important role of LXRs in protecting the liver against FB1-induced toxicity, suggesting an alternative mechanism not related to the inhibition of sphingolipid synthesis for mycotoxin toxicity.

Porcine Small and Large Intestinal Microbiota Rapidly Hydrolyze the Masked Mycotoxin Deoxynivalenol-3-Glucoside and Release Deoxynivalenol in Spiked Batch Cultures In Vitro.

Mycotoxin contamination of cereal grains causes well-recognized toxicities in animals and humans, but the fate of plant-bound masked mycotoxins in the gut is less well understood. Masked mycotoxins have been found to be stable under conditions prevailing in the small intestine but are rapidly hydrolyzed by fecal microbiota. This study aims to assess the hydrolysis of the masked mycotoxin deoxynivalenol-3-glucoside (DON3Glc) by the microbiota of different regions of the porcine intestinal tract. Intestinal digesta samples were collected from the jejunum, ileum, cecum, colon, and feces of 5 pigs and immediately frozen under anaerobic conditions. Sample slurries were prepared in M2 culture medium, spiked with DON3Glc or free deoxynivalenol (DON; 2 nmol/ml), and incubated anaerobically for up to 72 h. Mycotoxin concentrations were determined using liquid chromatography-tandem mass spectrometry, and the microbiota composition was determined using a quantitative PCR methodology. The jejunal microbiota hydrolyzed DON3Glc very slowly, while samples from the ileum, cecum, colon, and feces rapidly and efficiently hydrolyzed DON3Glc. No further metabolism of DON was observed in any sample. The microbial load and microbiota composition in the ileum were significantly different from those in the distal intestinal regions, whereas those in the cecum, colon and feces did not differ.IMPORTANCE Results from this study clearly demonstrate that the masked mycotoxin DON3Glc is hydrolyzed efficiently in the distal small intestine and large intestine of pigs. Once DON is released, toxicity and absorption in the distal intestinal tract likely occur in vivo This study further supports the need to include masked metabolites in mycotoxin risk assessments and regulatory actions for feed and food.

Secondary metabolism in Penicillium expansum: Emphasis on recent advances in patulin research.

The plant pathogenic fungus Penicillium expansum is a major concern of the global food industry due to its wide occurrence and ability to produce various mycotoxins, of which the most significant is patulin. Relatively less highlighted in the literature, in comparison with the other food-borne mycotoxins, patulin is one of the main factors in economic losses of vegetables and fruits. Otherwise, patulin is a health hazard which results in both short-term and long-term risks. This review includes knowledge on the biosynthetic mechanisms used for secondary metabolite production in P. expansum, with special emphasis on patulin biosynthesis. The abiotic factors triggering the production of patulin and the strategies developed to reduce or prevent the contamination by this mycotoxin are comprehensively discussed. The database presented in this review would be useful for the prioritization and development of future research.

Deepoxy-deoxynivalenol retains some immune-modulatory properties of the parent molecule deoxynivalenol in piglets.

Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) was compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg b.w./day for 2 and 3 weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the 3-week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased E-cadherin expression and modified the intestinal expression of cytokines. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody response. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immune-modulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.

Intestinal toxicity of deoxynivalenol is limited by Lactobacillus rhamnosus RC007 in pig jejunum explants.

Probiotics have been explored to stimulate gut health in weaned pigs, when they started to consume solid diet where mycotoxins could be present. The aim of this study was to evaluate the effect of Lactobacillus rhamnosus RC007 on the intestinal toxicity of deoxynivalenol (DON) in an ex vivo model. Jejunal explants, obtained from 5-week-old crossbred castrated male piglets, were kept as control, exposed for 3 h to 10 µM DON, incubated for 4 h with 109 CFU/mL L. rhamnosus, or pre-incubated 1 h with 109 L. rhamnosus and exposed to DON. Histological lesions were observed, para- and transcellular intestinal permeability was measured in Ussing chambers. The expression levels of mRNA encoding six inflammatory cytokines (CCL20, IL-10, IL-1ß, TNFα, IL-8 and IL-22) were determined by RT-PCR. The expressions of the phosphorylated MAP kinases p42/p44 and p38 were assessed by immunoblotting. Exposure to DON induced histological changes, significantly increased the expression of CCL20, IL-1ß, TNFα, IL-8, IL-22 and IL-10, increased the intestinal paracellular permeability and activated MAP kinases. Incubation with L. rhamnosus alone did not have any significant effect. By contrast, the pre-incubation with L. rhamnosus reduced all the effects of DON: the histological alterations, the pro-inflammatory response, the paracellular permeability and the phosphorylation of MAP kinases. Of note, L. rhamnosus did not adsorb DON and only slightly degrade the toxin. In conclusion, L. rhamnosus RC007 is a promising probiotic which, included as feed additive, can decrease the intestinal toxicity of DON.

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response.

Aspergillus flavus, a soil-borne pathogen, represents a danger for humans and animals since it produces the carcinogenic mycotoxin Aflatoxin B1 (AFB1). Approaches aiming the reduction of this fungal contaminant mainly involve chemicals that may also be toxic. Therefore, identification and characterization of natural anti-aflatoxigenic products represents a sustainable alternative strategy. Piperine, a major component of black and long peppers, has been previously demonstrated asan AFB1-inhibitor; nevertheless its mechanism of action was yet to be elucidated. The aim of the present study was to evaluate piperine's molecular mechanism of action in A. flavus with a special focus on oxidative stress response. For that, the entire AFB1 gene cluster as well asa targeted gene-network coding for fungal stress response factors and cellular receptors were analyzed. In addition to this, fungal enzymatic activities were also characterized. We demonstrated that piperine inhibits aflatoxin production and fungal growth in a dose-dependent manner. Analysis of the gene cluster demonstrated that almost all genes participating in aflatoxin's biosynthetic pathway were down regulated. Exposure to piperine also resulted in decreased transcript levels of the global regulator veA together with an over-expression of genes coding for several basic leucine zipper (bZIP) transcription factors such as atfA, atfB and ap-1 and genes belonging to superoxide dismutase and catalase's families. Furthermore, this gene response was accompanied by a significant enhancement of catalase enzymatic activity. In conclusion, these data demonstrated that piperine inhibits AFB1 production while positively modulating fungal antioxidant status in A. flavus.

Aerosolization of Mycotoxins after Growth of Toxinogenic Fungi on Wallpaper.

Many fungi can develop on building material in indoor environments if the moisture level is high enough. Among species that are frequently observed, some are known to be potent mycotoxin producers. This presence of toxinogenic fungi in indoor environments raises the question of the possible exposure of occupants to these toxic compounds by inhalation after aerosolization. This study investigated mycotoxin production by Penicillium brevicompactum, Aspergillus versicolor, and Stachybotrys chartarum during their growth on wallpaper and the possible subsequent aerosolization of produced mycotoxins from contaminated substrates. We demonstrated that mycophenolic acid, sterigmatocystin, and macrocyclic trichothecenes (sum of 4 major compounds) could be produced at levels of 1.8, 112.1, and 27.8 mg/m2, respectively, on wallpaper. Moreover, part of the produced toxins could be aerosolized from the substrate. The propensity for aerosolization differed according to the fungal species. Thus, particles were aerosolized from wallpaper contaminated with P. brevicompactum when an air velocity of just 0.3 m/s was applied, whereas S. chartarum required an air velocity of 5.9 m/s. A. versicolor was intermediate, since aerosolization occurred under an air velocity of 2 m/s. Quantification of the toxic content revealed that toxic load was mostly associated with particles of size ≥3 µm, which may correspond to spores. However, some macrocyclic trichothecenes (especially satratoxin H and verrucarin J) can also be found on smaller particles that can deeply penetrate the respiratory tract upon inhalation. These elements are important for risk assessment related to moldy environments.IMPORTANCE The possible colonization of building material by toxinogenic fungi in cases of moistening raises the question of the subsequent exposure of occupants to aerosolized mycotoxins. In this study, we demonstrated that three different toxinogenic species produce mycotoxins during their development on wallpaper. These toxins can subsequently be aerosolized, at least partly, from moldy material. This transfer to air requires air velocities that can be encountered under real-life conditions in buildings. Most of the aerosolized toxic load is found in particles whose size corresponds to spores or mycelium fragments. However, some toxins were also found on particles smaller than spores that are easily respirable and can deeply penetrate the human respiratory tract. All of these data are important for risk assessment related to fungal contamination of indoor environments.

Impact of mycotoxins on the intestine: are mucus and microbiota new targets?

There is an increasing awareness of the deleterious effects attributed to mycotoxins during their fate within the gut, particularly for deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), and patulin (PAT). Evidence indicates that disruption of the epithelial barrier is well established. However, intestinal barrier function on its luminal side involves two other partners, mucus and microbiota, which have rarely been considered in the context of mycotoxin exposure. The current review aimed at providing a summary of DON, ZEN, OTA, FB1, AFB1, and PAT effects on intestinal barrier function, with special focus on mucus and microbiota. DON, ZEN, OTA, FB1, AFB1, and PAT are known to markedly affect epithelial cell integrity and functions. Regarding mucus, DON is the most documentated mycotoxin. In vivo, toxicological impact of DON generally has only been assessed through goblet cell number. Evaluation of the mycotoxins/mucus interplay considering other indicators such as composition, thickness, and penetrability of mucus, mucin O-glycosylation thus warrants further attention. With respect to microbiota, few short-term studies to date have been reported indicating deleterious effects. However, long-term exposure to mycotoxins may also produce significant changes in microbiota composition and metabolic activity, which requires further experimentation. In conclusion, mucus and microbiota are key targets for dietary mycotoxins although assessment of induced effects is preliminary. A significant research effort is now underway to determine the adverse consequences of mycotoxins on mucus and microbiota considered as individual but also as tightly connected gut players.

Co-exposure to low doses of the food contaminants deoxynivalenol and nivalenol has a synergistic inflammatory effect on intestinal explants.

The global incidence of Fusarium head blight and attendant cereal grains multi-contamination by the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are increasing as a possible result of climate change and inadequate agricultural practices. At the molecular level, these mycotoxins bind to the ribosome, activate the mitogen-activated protein kinase and induce a local and systemic inflammation. DON is of public health concern owing to the narrow margin between exposure and tolerable daily intake. The intestinal inflammatory response to DON, NIV and their mixture was analyzed to determine thresholds for their intestinal pro-inflammatory effects and characterize the type and magnitude of their interaction. Fully differentiated three-dimensional porcine jejunal explants were exposed to increasing doses of DON and NIV alone or in combination; the expression levels of IL-1α, IL-1ß, IL-8, IL-17A and IL-22 were measured by RT-PCR. Doses as low as 0.16 µM DON or 0.73 µM NIV significantly increase the intestinal expression levels of the tested inflammation-related genes. These doses are lower than those previously reported for other intestinal toxicity endpoints. The combined pro-inflammatory activity of DON and NIV was synergistic for all the tested genes with combination index value range of 0.23-0.8. Our results indicate that (1) inflammation is a very sensitive endpoint for the intestinal toxicity of the trichothecenes and (2) co-exposure to DON and NIV has a greater inflammatory effect than induced by mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of DON and NIV in the diet.

Patulin transformation products and last intermediates in its biosynthetic pathway, E- and Z-ascladiol, are not toxic to human cells.

Patulin is the main mycotoxin contaminating apples. During the brewing of alcoholic beverages, this mycotoxin is degraded to ascladiol, which is also the last precursor of patulin. The present study aims (1) to characterize the last step of the patulin biosynthetic pathway and (2) to describe the toxicity of ascladiol. A patE deletion mutant was generated in Penicillium expansum. In contrast to the wild strain, this mutant does not produce patulin but accumulates high levels of E-ascladiol with few traces of Z-ascladiol. This confirms that patE encodes the patulin synthase involved in the conversion of E-ascladiol to patulin. After purification, cytotoxicities of patulin and E- and Z-ascladiol were investigated on human cell lines from liver, kidney, intestine, and immune system. Patulin was cytotoxic for these four cell lines in a dose-dependent manner. By contrast, both E- and Z-ascladiol were devoid of cytotoxicity. Microarray analyses on human intestinal cells treated with patulin and E-ascladiol showed that the latter, unlike patulin, did not alter the whole human transcription. These results demonstrate that E- and Z-ascladiol are not toxic and therefore patulin detoxification strategies leading to the accumulation of ascladiol are good approaches to limit the patulin risk.

Toxicology of deoxynivalenol and its acetylated and modified forms.

Mycotoxins are the most frequently occurring natural contaminants in human and animal diet. Among them, deoxynivalenol (DON), produced by Fusarium, is one of the most prevalent and thus represents an important health risk. Recent detection methods revealed new mycotoxins and new molecules derivated from the "native" mycotoxins. The main derivates of DON are the acetylated forms produced by the fungi (3- and 15-acetyl-DON), the biologically "modified" forms produced by the plant (deoxynivalenol-3-ß-D-glucopyranoside), or after bacteria transformation (de-epoxy DON, 3-epi-DON and 3-keto-DON) as well as the chemically "modified" forms (norDON A-C and DON-sulfonates). High proportions of acetylated and modified forms of DON co-occur with DON, increasing the exposure and the health risk. DON and its acetylated and modified forms are rapidly absorbed following ingestion. At the molecular level, DON binds to the ribosome, induces a ribotoxic stress leading to the activation of MAP kinases, cellular cell-cycle arrest and apoptosis. The toxic effects of DON include emesis and anorexia, alteration of intestinal and immune functions, reduced absorption of the nutrients as well as increased susceptibility to infection and chronic diseases. In contrast to DON, very little information exists concerning the acetylated and modified forms; some can be converted back to DON, their ability to bind to the ribosome and to induce cellular effects varies according to the toxin. Except for the acetylated forms, their toxicity and impact on human and animal health are poorly documented.