The numerous pores in the basement membrane (BM) of the intestinal villi are essential for the communication of enterocytes with cells in the lamina propria, an important mechanism for the induction of intestinal immune responses. The intestinal epithelial barrier is affected by the mycotoxin deoxynivalenol (DON) from both the apical (luminal) and basolateral (serosal) side. The pig is the most susceptible species to the anorectic and immune-modulating effects of DON, which is most prevalent in crops. We analysed in pigs the effect of DON-contaminated feed on the composition and perforation of the BM and the presence of CD16+ cells or their dendrites in the epithelium. In addition to in vivo experiments, in vitro studies were carried out. Using microarray analyses, the effects of DON on IPEC-J2 cells were studied with the focus on the BM. Our in vivo results showed in the control pigs: (1) a significant increased pore number (p ≤ 0.001) in the jejunum in comparison to ileum, (2) no difference in the pore size, and (3) comparable frequency of intraepithelial CD16+ cells/dendrites in the jejunum and ileum. There was a marked trend that DON feeding increases: (1) the pore number in jejunum, and (2) the number of CD16+ cells/dendrites in the epithelium (Tukey–Kramer; p = 0.055 and p = 0.067, respectively). The in vivo results were extended with microarray analyses of epithelial cell (IPEC-J2 cells). The down-regulation of genes like syndecan, fibulin 6 and BM-40 was observed. These proteins are important factors in the BM composition and in formation of pores. Our results provide evidence that already low basolateral concentrations of DON (50 ng/mL) influence the production of the BM protein laminin by epithelial cells. Thus, DON affects the composition of the BM.

The complete extraction of analytes is of utmost importance when analyzing matrix samples for mycotoxins. Mycotoxins consist of substances with widely different physicochemical properties; therefore, the loss of toxins that occurs in multi-mycotoxin methods due to compromises in the extraction solvent is currently a topic under discussion. With regard to fumonisins, several extractants from recently published multi-mycotoxin methods were investigated when analyzing unprocessed and processed maize matrices. All extractants were tested in a validated on-site method and the extraction yields were compared to those of an HPLC-FLD reference method (EN 14352). Most of the compared multi-mycotoxin methods that have been published were only for analyzing fumonisins in maize or maize-meal; we have applied the extractants of these methods to processed, complex maize matrices for the first time. Our results show that, for extractions with aqueous acetonitrile mixtures with the addition of acid, e.g. MeCN/H2O/acetic acid (79/20/1, v/v/v), higher extraction yields are obtained than with MeCN/H2O (80/20, v/v), in both spiked and naturally contaminated maize matrices. But compared to the results of the reference method EN 14352, the two extractants did not show a similar extraction efficiency. Overall, the extractant MeCN/MeOH/H2O (1/1/2, v/v/v) turned out to be the most appropriate extractant applied in all experiments, obtaining the best and most comparable extraction yields and recoveries. Furthermore, our investigations showed that, with some of the tested extraction solvents, e.g. MeCN/H2O (75/25) containing 50 mmol/l formic acid, stark differences occur when analyzing spiked and naturally contaminated matrices. With spiked matrices, recoveries of approximately 80-110% were obtained, but with naturally contaminated matrices no results comparable to the EN method have been achieved. In contrast, a double extraction with MeCN/H2O/formic acid (80/19,9/0,1, v/v/v), followed by a second polar extraction step with MeCN/H2O/formic acid (20/79,9/0,1, v/v/v), led, for most naturally contaminated samples, to comparable results with the EN method. However, for spiked samples, the same extractant led to raised recoveries of between 120 and 140 %. For some processed matrices, like taco-chips, all tested extractants showed a poor extraction efficiency for fumonisins. By extending the extraction time from 1 to 15 min, a result comparable to that of the reference method could also be obtained for the extractant using MeCN/MeOH/H2O (1/1/2, v/v/v). As this extractant has been used in our recently published method (Trebstein et al. Mycotoxin Res 25:201, 2009), this work also presents an update on this method with respect to the extended extraction time.

The genotoxic potential of fumonisin B(1) (FB(1)) in vivo in BALB/c mice (male and female) was assessed by induction of micronuclei (MN) formation in bone marrow polychromatic erythrocytes. The ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) was also determined. Mice were injected intraperitoneally (i.p.) with FB(1) at a low dose (0.1mg kg(-1) body mass), middle dose (1.0mg kg(-1) body mass) and high dose (10mg kg(-1) body mass) as single and multiple doses in normal saline to test the genotoxicity. Mitomycin C, a known clastogen, was used as positive control. The frequency of MN and the PCE/NCE value in animals treated with FB(1) at low, middle, and high doses in single dose studies, and the frequency of MN in multiple dose studies, were statistically non-significant from that of the controls injected with saline only. The multiple dose studies at all doses revealed that the PCE/NCE value was found to be reduced upon exposure to FB(1) as compared to the controls. In animals injected with multiple low doses of FB(1), the PCE/NCE value was found to be 0.66 in males and 0.82 in the females; at multiple middle doses the value was 0.30 in males and 0.41 in the females and was statistically significant (p < 0.001); however, at multiple high doses, the ratio was found to be 0.36 in both males and females. The present study confirms that FB(1) is non-genotoxic in nature while the reduced ratio of PCE/NCE suggests the cytotoxic nature of FB(1).

In current climate change scenarios, mean air temperatures and summer droughts are expected to increase over the long-term average in large parts of Europe. These changes will strongly affect the growth and health of cultivated plants. In a field experiment in 2009 and 2010 in rain-out shelters, the consequences of plant water availability under three water regimes on the severity of Fusarium ear rot, deoxynivalenol (DON) contamination and yield of maize were investigated. Water was provided exclusively to the plants by a mobile sprinkler system installed in the rain-out shelter. Three maize cultivars were supplied with 50, 75, and 125% of the long-term average monthly precipitation of the experimental site. In 2009, Fusarium graminearum-infected oat kernels were placed on the soil surrounding maize plants to promote ear infection, whereas in 2010, the maize silks were directly inoculated with suspensions of F. graminearum conidia. Heavy drought stress in the 50% water regime was reflected in the average ear yield of the three maize cultivars of 75 dt ha(-1) compared with 192 dt ha(-1) at 125% water. In this comparison, the DON concentrations in the kernels were fivefold higher at 50% water than at the high water regime: 380 compared with 75 μg DON kg(-1) DM. In 2010, the drought stress symptoms were less pronounced than in 2009, and a much lower ear yield loss from 128 to 108 dt DM ha(-1) was observed with decreasing water supply. The DON contamination of the kernels was at a higher level than in 2009, but a similar upward trend from 330 μg kg(-1) DM at 125% water supply to about 3.5-fold higher DON levels at 75 and 50% water supply was observed. These different yield responses presumably resulted from variable climatic conditions at the experimental site in the 2 years. The results of this study suggest that the risk of DON contamination of maize kernels increases when plants are grown under conditions of long-term water deficit.

Leucocytes are susceptible to the toxic effects of deoxynivalenol (DON), which is a trichothecene mycotoxin produced by a number of fungi including Fusarium species. One mechanism of action is mediated by reactive oxygen species (ROS). The liver is an important target for toxicity caused by foreign compounds including mycotoxins. On the other hand, little is known about the influence of the redox state on hepatocytes treated with DON. The present study investigated the effect of DON on the cytosolic redox state and antioxidative system in the human hepatoma cell line HepG2. The cell viability of human monocyte cell line THP-1 or leukemia cell line KU812 treated with 2.5 and 5μmol/l DON were significantly reduced. However, HepG2 cells showed no toxic effects under the same conditions and did not exhibit an increased oxidative state. Further experiments showed that thioredoxin-1 (Trx-1) protein levels but not glutathione increased in the cells treated with 10μmol/l DON. In addition, the enhancement of Trx-1 content was repressed by antioxidants. These results suggest that DON-induced accumulation of Trx-1 in HepG2 cells plays one of the key roles in protection against cytotoxicity caused by DON and that the mechanism may be mediated by the antioxidant properties of Trx-1.

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB1 and CPA. The most toxigenic one was CA28 which produced 164μg AFB1 per 5ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400μm). The other aflatoxigenic strains produce AFB1 ranging from 1.2μg to 80μg per 5ml fungal culture. Twenty-one percent of the CA isolates produced AFB1, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.

Trichothecene mycotoxins are toxic secondary metabolites produced by a number of fungi including Fusarium species, which adversely affect lymphocytes. Deoxynivalenol (DON) and HT-2 toxin (HT-2) belong to the trichothecene group of mycotoxins and the occurrence of cereals and foodstuffs with these compounds are serious health problems. The aim of this study was to examine the effect of (-)-epigallocatechin gallate (EGCG), one of the main components in green tea catechins, on DON- or HT-2-induced cytotoxicity in mouse macrophages. EGCG had protective effects against the trichothecene-induced cytotoxicities of both mycotoxins. Additionally, EGCG suppressed the DON-induced activation of caspase-3/7, which is an indicator of apoptosis. These results indicate that EGCG might be useful in protection against DON- or HT-2-induced cell death, suggesting that EGCG could contribute to reducing the toxicities of trichothecenes.

Deoxynivalenol (DON) is a trichothecene mycotoxin commonly produced by Fusarium graminearum and F. culmorum during infection of cereal plants, such as wheat and barley. This toxin is a fungal virulence factor that facilitates the development of Fusarium head blight (FHB) disease. Wheat cultivar (cv.) Remus is susceptible to DON; the toxin causes premature bleaching of spikelets and inhibits root growth. This study used custom-made wheat cDNA arrays to analyse the effect of DON on the transcriptome of heads of the toxin-sensitive wheat cv. Remus at both 4 and 24h post-toxin treatment. DON-induced transcripts encoded an array of proteins collectively associated with a range of cellular functions, such as metabolite transformation and detoxification, the ubiquitin-proteasome proteolytic pathway, jasmonate biosynthesis and signalling, carbohydrate metabolism, and phenylpropanoid biosynthesis. This study is the first to demonstrate that the fungal virulence factor DON modulates jasmonate biosynthesis and signalling. It also highlights the fact that the toxin-mediated accumulation of transcripts associated with metabolite transformation and detoxification, proteolysis and phenylpropanoid accumulation is not unique to DON-resistant wheat genotypes. Therefore, the respective encoded proteins are likely part of the general wheat defence against DON. Comparative analysis of the results of this and other studies suggests that it is likely to be the rapidity and magnitude rather than the components of the response that are critical in determining resistance to DON and thus the spread of FHB disease in wheat heads.

Aflatoxin B1 (AFB1) was detected in 57% of the nuts and nut products marketed in Penang, Malaysia using the liquid chromatography tandem mass spectrometry. The contamination levels ranged from 0.40 to 222μg/kg and 17 out of 128 samples (13.3%) contained AFB1 above the European Commission permitted level (2μg/kg). Estimated dietary exposure of AFB1 in nuts and nut products were 0.36ng per kg body weight and day and 8.89ng per kg body weight and day, representing the low and high-level of exposure, respectively. Dose-response modelling resulted in benchmark dose lower confidence limit (BMDL10) values of 0.305ng per kg body weight and day, with the best fitted from the log-logistic model. The derived margin of exposure (MoE) values ranged from 34 to 847 suggested that AFB1 would be of public health concern and might reasonably be considered as a high priority for risk management actions.