Johanna Fink-Gremmels

Trichothecene type A mycotoxins, such as T-2, HT-2, and diacetoxyscirpenol (DAS), are known to induce cytotoxicity and apoptosis in different cell types. As all three Fusarium toxins may occur concomitantly in a given food or feed commodity, there is growing interest in the effect of such mycotoxin mixtures. This study aimed to identify the toxic interactions among T-2, HT-2, and DAS in a human Jurkat cell model. As a first step, an MTT assay was used to assess cytotoxicity after 24 h of cell exposure to individual mycotoxins and their mixtures. The results were used to calculate the combination index (CI), which indicates the nature of the mycotoxin interactions. In Jurkat T cells, the toxicity ranking for the individual mycotoxins was T-2 > HT-2 > DAS. The CI values of the dual and triple mycotoxin combinations calculated from the results of the MTT and reactive oxygen species assays showed synergistic effects at low concentrations and an apparent antagonism at very high concentrations for all combinations. The additional cytometric analyses confirmed the synergistic effects, as expected, following co-exposure to the three tested trichothecenes. As the lower toxin concentrations investigated reflect natural contamination levels in food and feeds, the synergistic effects identified should be considered in risk characterization for trichothecene exposure in humans and animals.

Ensuring animal health and well-being requires animal-based measures for early and direct intervention at the point of care. Insight into the pathophysiology of Swine Inflammation and Necrosis Syndrome (SINS) has led to a non-invasive, clinical scoring system that can be used in daily practice. This provides information on the cause of observed lesions for direct intervention in affected herds. The aim of the current study was to re-evaluate the practicality of the SINS scoring system under field conditions. In addition, this is the first study to provide insight into the prevalence of SINS on Dutch farms. This study involved the scoring of 5958 piglets from 20 visits on 13 farms randomly selected by a veterinary practice in the Netherlands. The results showed that up to 64.1% of the piglets had visible inflammatory alterations in different body parts within the first seven days of life. Sow rectal temperature, signs of coprostasis and water intake during pregnancy were significantly (p ≤ 0.05) correlated with the prevalence of SINS in piglets. In conclusion, the SINS scoring system is an easy-to-use, non-invasive diagnostic tool that summarises animal-based observations at the point of care, providing a valuable communication tool between farmers, nutritionists and veterinarians in their efforts to improve animal health and welfare.

The study focused on the examination of the different fungal species isolated from commercial rice samples, applying conventional culture techniques, as well as different molecular and phylogenic analyses to confirm phenotypic identification. Additionally, the mycotoxin production and contamination were analyzed using validated liquid chromatography–tandem mass spectrometry (LC-MS/MS). In total, 40 rice samples were obtained covering rice berry, red jasmine rice, brown rice, germinated brown rice, and white rice. The blotting paper technique applied on the 5 different types of rice samples detected 4285 seed-borne fungal infections (26.8%) for 16,000 rice grains. Gross morphological data revealed that 19 fungal isolates belonged to the genera Penicillium/Talaromyces (18 of 90 isolates; 20%) and Aspergillus (72 of 90 isolates; 80%). To check their morphologies, molecular data (fungal sequence-based BLAST results and a phylogenetic tree of the combined ITS, BenA, CaM, and RPB2 datasets) confirmed the initial classification. The phylogenic analysis revealed that eight isolates belonged to P. citrinum and, additionally, one isolate each belonged to P. chermesinum, A. niger, A. fumigatus, and A. tubingensis. Furthermore, four isolates of T. pinophilus and one isolate of each taxon were identified as Talaromyces (T. radicus, T. purpureogenum, and T. islandicus). The results showed that A. niger and T. pinophilus were two commonly occurring fungal species in rice samples. After subculturing, ochratoxin A (OTA), generated by T. pinophilus code W3-04, was discovered using LC-MS/MS. In addition, the Fusarium toxin beauvericin was detected in one of the samples. Aflatoxin B1 or other mycotoxins, such as citrinin, trichothecenes, and fumonisins, were detected. These preliminary findings should provide valuable guidance for hazard analysis critical control point concepts used by commercial food suppliers, including the analysis of multiple mycotoxins. Based on the current findings, mycotoxin analyses should focus on A. niger toxins, including OTA and metabolites of T. pinophilus (recently considered a producer of emerging mycotoxins) to exclude health hazards related to the traditionally high consumption of rice by Thai people.

Zearalenone (ZEN) is produced by Fusarium species contaminating various agriculture crops. In this study, the effects of ZEN and its metabolites α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL) on the formation of carcinogenic oestrogen-catechols in MCF-7 cells were investigated. To assess the effects of mycoestrogens on the activity of cytochrome P450 1A1 and CYP1B1, the rate of ethoxyresorufin O-deethylation (EROD-assay) was measured. The effects of mycoestrogens on the expression of CYP 1A1, CYP 1B1, aryl-hydrocarbon receptor (AhR), and oestrogen receptor alpha (ERα) were determined by qPCR. The catechol-O-methyltransferase (COMT) activity was measured as the ratio of the methoxy metabolites of oestradiol. Results show that mycoestrogens inhibited significantly the CYP1-dependent EROD activities. In the presence of selective inhibitors, mycoestrogens reduced CYP 1A1 and enhanced CYP 1B1 activity. Quantitative PCR analyses demonstrated the upregulation of AhR and confirmed the selective effect of mycoestrogens on CYP1 expression levels and the decline of the CYP 1A1/CYP 1B1 ratio. Mycoestrogens increased the ratio of 4-MeOE to 2-MeOE2 formation significantly (P < 0.05). Our results suggest that the tested mycoestrogens increase the production of CYP1B1-mediated oestrogen catechol metabolites, directing the biotransformation of E2 towards 4-OHE2, which has been identified earlier as a crucial factor in oestrogen-induced tumour initiation.

The current climate changes have increased the prevalence and intensity of heat stress (HS) conditions. One of the initial consequences of HS is the impairment of the intestinal epithelial barrier integrity due to hyperthermia and hypoxia following blood repartition, which often results in a leaky gut followed by penetration and transfer of luminal antigens, endotoxins, and pathogenic bacteria. Under extreme conditions, HS may culminate in the onset of “heat stroke”, a potential lethal condition if remaining untreated. HS-induced alterations of the gastrointestinal epithelium, which is associated with a leaky gut, are due to cellular oxidative stress, disruption of intestinal integrity, and increased production of pro-inflammatory cytokines. This review summarizes the possible resilience mechanisms based on in vitro and in vivo data and the potential interventions with a group of nutritional supplements, which may increase the resilience to HS-induced intestinal integrity disruption and maintain intestinal homeostasis.

The potential effect of difructose anhydride III (DFA III) supplementation in cattle feed was evaluated using a previously developed urinary-zearalenone (ZEN) monitoring system. Japanese Black cattle from two beef herds aged 9–10 months were used. DFA III was supplemented for two weeks. ZEN concentrations in feed were similar in both herds (0.27 and 0.22 mg/kg in roughage and concentrates, respectively), and below the maximum allowance in Japan. ZEN, α-zearalenol (α-ZOL), and β-ZOL concentrations in urine were measured using LC/MS/MS the day before DFA III administration, 9 and 14 days thereafter, and 9 days after supplementation ceased. Significant differences in ZEN, α-ZOL, β-ZOL, and total ZEN were recorded on different sampling dates. The concentration of inorganic phosphate in DFA III-supplemented animals was significantly higher than in controls on day 23 (8.4 vs. 7.7 mg/dL), suggesting a possible role of DFA III in tight junction of intestinal epithelial cells. This is the first evidence that DFA III reduces mycotoxin levels reaching the systemic circulation and excreted in urine. This preventive effect may involve an improved tight-junction-dependent intestinal barrier function. Additionally, our practical approach confirmed that monitoring of urinary mycotoxin is useful for evaluating the effects of dietary supplements to prevent mycotoxin adsorption.

The objective of this study was to evaluate the metabolic profile of cattle fed with or without zearalenone (ZEN) and sterigmatocystin (STC)-contaminated diets using a gas chromatography-mass spectrometry metabolomics approach. Urinary samples were collected from individual animals (n = 6 per herd) from fattening female Japanese Black (JB) cattle herds (23 months old, 550–600 kg). Herd 1 had persistently high urinary ZEN and STC concentrations due to the presence of contaminated rice straw. Herd 2, the second female JB fattening herd (23 months old, 550–600 kg), received the same dietary feed as Herd 1, with non-contaminated rice straw. Urine samples were collected from Herd 1, two weeks after the contaminated rice straw was replaced with uncontaminated rice straw (Herd 1N). Identified metabolites were subjected to principal component analysis (PCA) and ANOVA. The PCA revealed that the effects on cattle metabolites depended on ZEN and STC concentrations. The contamination of cattle feed with multiple mycotoxins may alter systemic metabolic processes, including metabolites associated with ATP generation, amino acids, glycine-conjugates, organic acids, and purine bases. The results obtained from Herd 1N indicate that a two-week remedy period was not sufficient to improve the levels of urinary metabolites, suggesting that chronic contamination with mycotoxins may have long-term harmful effects on the systemic metabolism of cattle.

Considering the diverse toxic effects of the Fusarium toxin deoxynivalenol (DON), its common occurrence in wheat-based products, and its stability during processing, DON constitutes an increasing health concern for humans and animals. In addition to the parent compound DON, human and animal exposure encompasses the acetylated fungal metabolites 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON) as well as the plant-derived DON-glucoside (DON3G) and the bacterial product de-epoxy-DON (DOM-1). In the current study we used the well-established Caco-2 cell model to compare the effects of these naturally occurring forms of DON on cell viability and markers of barrier integrity, as well as on the release of the pro-inflammatory chemokine chemokine CXC motif ligand (CXCL8). Results show that 3ADON is less potent in inducing adverse effects on barrier integrity when compared to DON, whereas 15ADON appears to be slightly more potent than DON. In contrast, DON3G and DOM-1 exerted no measurable adverse effects on the intestinal barrier. It was also demonstrated that galacto-oligosaccharides (GOS) are able to protect epithelial cells against DON and its acetylated forms, which suggests that GOS are beneficial food additives in the protection of vulnerable segments of the human population against adverse effects of DON and its derivatives.

Deoxynivalenol (DON) is one of the major mycotoxins produced by Fusarium fungi, and exposure to this mycotoxin requires an assessment of the potential adverse effects, even at low toxin levels. The aim of this study was to investigate the effects of a short-term, low-dose DON exposure on various gut health parameters in pigs. Piglets received a commercial feed or the same feed contaminated with DON (0.9 mg/kg feed) for 10 days, and two hours after a DON bolus (0.28 mg/kg BW), weight gain was determined and samples of different segments of the intestine were collected. Even the selected low dose of DON in the diet negatively affected weight gain and induced histomorphological alterations in the duodenum and jejunum. The mRNA expression of different tight junction (TJ) proteins, especially occludin, of inflammatory markers, like interleukin-1 beta and interleukin-10 and the oxidative stress marker heme-oxigenase1, were affected along the intestine by low levels of DON in the diet. Taken together, our results indicate that even after low-level exposure to DON, which has been generally considered as acceptable in animal feeds, clinically-relevant changes are measurable in markers of gut health and integrity.