Intestinal transport of deoxynivalenol across porcine small intestines.

Deoxynivalenol (DON) is one of the most important trichothecenes, due to its worldwide distribution and common contamination of animal feed. It mainly affects the gastrointestinal tract and the immune system with a high susceptibility for swine. Lipopolysaccharides (LPS) are endotoxins and are part of the outer membrane of most gram-negative bacteria. They induce inflammatory responses under systemic application. We hypothesised that dietary DON as well as LPS challenge could affect the transport of DON in vitro. For assessment of this hypothesis, a total of 16 pigs were divided into two groups, Control and DON-feeding. In each group, four animals were injected intraperitoneally with LPS (5 µg/kg BW). Jejunal preparations were mounted on the Ussing chambers, and after luminal addition of DON at two different concentrations (4000 and 8000 ng/ml), buffer samples were collected at different time points to measure the concentration of DON using LC-MS/MS analysis. Our findings revealed a significant interaction effect between dietary DON and DON in vitro represented by higher mucosal uptake of DON in DON-fed animals. Animals challenged with LPS showed higher mucosal uptake but without significant effect of LPS. We concluded that the transport of DON was proportional to its concentration and DON in feed could have an effect on the transport of DON across porcine jejunal mucosa. LPS challenge induced no apparent significant effect on DON transport, although induction of acute phase reaction was present.

Tissue distribution and proinflammatory cytokine induction by the trichothecene deoxynivalenol in the mouse: comparison of nasal vs. oral exposure.

Oral exposure to the trichothecene deoxynivalenol (DON), a common cereal grain contaminant, adversely affects growth and immune function in experimental animals. Besides foodborne exposure, the potential exists for DON to become airborne during the harvest and handling of grains and therefore pose a risk to agricultural workers. The purpose of this study was to compare the effects of oral and intranasal exposure to DON (5mg/kg bw) on tissue distribution and proinflammatory cytokine induction in the adult female mouse. Competitive direct ELISA revealed that, regardless of exposure route, DON concentrations in plasma, spleen, liver, lung and kidney were maximal within 15-30 min and declined by 75-90% after 120 min. However, plasma and tissue DON concentrations were 1.5-3 times higher following intranasal exposure as compared to oral exposure. The functional significance of elevated DON tissue concentrations was assessed by measuring IL-1beta, IL-6, and TNF-alpha mRNA responses in spleen, liver and lung. Oral exposure to DON-induced robust proinflammatory cytokine gene expression after 60 and 120 min. In contrast, inductions of IL-1beta, IL-6 and TNF-alpha mRNAs in nasally exposed mice were 2-10, 2-5 and 2-4 times greater, respectively, than those in the tissues of orally exposed mice. Taken together, these data suggest that DON was more toxic to the mouse when nasally exposed than when orally exposed, and that this might relate to greater tissue burden of the toxin.

The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro.

The trichothecene mycotoxin deoxynivalenol (DON) causes systemic immuno-suppression in pigs and possibly also in humans after chronic dietary exposure. Since the outcome of every immune response is largely controlled by dendritic cells (DC), we hypothesised that a direct influence of DON on DC function might play a role in mediating DON immunotoxicity. To test this hypothesis, a 2x2 factorial design study was performed. Pigs were fed a control diet or a diet containing DON (DON-diet); monocyte-derived DC (MoDC) from these pigs were then treated with DON in vitro or left untreated. Phenotype and function of the MoDC were analysed. In vitro DON-treatment of MoDC from pigs fed the control diet resulted in a down-regulation of CD80/86 and CD40. This was associated with an activation of the mitogen-associated protein kinases ERK1/2 and JNK. The endocytic activity of MoDC was decreased after in vitro DON-exposure while their T cell stimulatory capacity was not altered. MoDC derived from pigs that had been fed the DON-diet failed to up-regulate MHC-II in response to LPS/TNFalpha. Dietary exposure of pigs to DON inhibited endocytosis of FITC-dextran by MoDC, but did not influence T cell stimulatory capacity. ERK1/2 and JNK were constitutively activated in MoDC from pigs fed the DON-diet. If MoDC derived from pigs fed the DON-diet were exposed to DON in vitro, this resulted in an up-regulation of MHC-II and CD80/86, but not CD40. In comparison to untreated MoDC from pigs fed DON-diet, endocytic capacity was further down-regulated, whereas mitogen-activated protein kinase activation was increased. In summary, DON disrupts porcine DC function in vitro and in vivo, which might contribute to the immunosuppressive effects of this mycotoxin.

On the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from sows to their fetuses during days 35-70 of gestation.

Eleven pregnant sows with a body weight between 153 and 197 kg were fed a control diet (CON, 0.15 mg DON and 0.0035 mg ZON/kg diet) or a diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.42 mg DON and 0.048 mg ZON/kg diet) in the period of day 35 and 70 of gestation. The indirect effect of feed intake was separated from the direct effects of the Fusarium toxins by the restricted feeding regimen where all sows were fed the same amount of feed (2000 g/d) over the whole study. At the end of experiment, fetuses were delivered by Caesarian section and samples of serum, bile, urine, liver, kidney and spleen of euthanatized sows and fetuses were taken to analyze the concentrations of DON, ZON and their metabolites. Feeding the Fusarium toxin contaminated diet to pregnant sows caused neither adverse effects on performance, organ weights and maintenance of pregnancy of sows nor on fetus weight and length. Furthermore, no teratogenic or embryolethal effects could be observed in the MYCO group. Hematological and clinical-chemical parameters of sows and fetuses were not affected by feeding, with the exception of significantly lower GLDH (glutamate dehydrogenase) serum activities in MYCO sows. The carry over of DON and ZON from the diet to the sow or fetus tissues was calculated by the diet ratio (sum of concentrations of all metabolites in the physiological specimen divided by the dietary toxin concentration), while the fetus ratio was evaluated by the sum of concentrations of all metabolites in the physiological specimen of the fetus divided by that of the sows. DON and deepoxy-DON were found in urine, bile, serum, liver, kidney and spleen of sows of the MYCO group, but not in the bile of fetuses (spleen not analyzed). ZON and its metabolite alpha-zearalenol (alpha-ZOL) were detected in urine and bile of sows, while all specimens of fetuses as well as serum and liver of sows were negative for ZON metabolites. The maximum diet ratios for urine and bile in sows of the MYCO group were 0.84 and 0.05 for DON metabolites and 1.2 and 3.8 for ZON metabolites, underscoring the differences in metabolism and excretion of both toxins. The maximum diet ratio of DON and deepoxy-DON into liver, kidney and spleen of MYCO sows were 0.003, 0.007 and 0.003, respectively. The maximum fetus ratio of DON and deepoxy-DON into urine, bile, serum, liver and kidney of fetuses were 0.006, 0, 0.5, 0.88, and 0.33, while the maximum placental ratio (sum of toxin concentrations in the physiological specimen of the fetus divided by the toxin serum concentration of the sow) were 0.64, 0, 0.50, 0.70 and 0.52, respectively. Therefore, it can be concluded that the developing fetus is exposed to DON between the gestation days 35 and 70 when the sows are fed a Fusarium toxin contaminated diet. ZON concentration in the MYCO diet was too low to get reliable results for fetus or placental ratios.

The analysis of trichothecenes in wheat and human plasma samples by chemical ionization tandem mass spectrometry.

Ammonia desorption chemical ionization (D/CI) tandem mass spectrometry (method A), isobutane D/CI tandem mass spectrometry using reactive collisions with ammonia (method B), and gas chromatography negative CI (GC-NCI) tandem mass spectrometry (method C) were compared for the detection and quantitation of trichothecenes in spiked human plasma and wheat samples. The trichothecenes were analyzed as their heptafluorobutyrate (HFB) esters in method C and without derivatization using the direct exposure probe in methods A and B. The instrument was operated in the multiple reaction mode. With standard solutions, the detection limits were about two orders of magnitude better with method C (0.1-2 pg) than with methods A and B. The trichothecenes could be detected in the spiked samples at lower concentration levels by method C (0.001 microgram g-1) than by methods A and B (0.01-0.1 microgram g-1), since the sensitivity and selectivity of method C were better than those of methods A and B. Because of the need of derivatization, method C was more time-consuming than methods A and B. Iso-T-2 was used as an internal standard in method C and a steroid nandrolone in methods A and B. The linearities and reproducibilities of the quantitation were better with methods A and C than with method B.

Analysis of trichothecene mycotoxins in human blood by capillary column gas chromatography-ammonia chemical ionization mass spectrometry.

Capillary column gas chromatography-ammonia chemical ionization mass spectrometry was found to be an excellent technique for the trace detection and identification of underivatized trichothecene mycotoxins. Abundant (M + H)+ and/or (M + NH4)+ pseudo-molecular ions were observed for T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol, deoxynivalenol and verrucarol under the conditions developed. This method was successfully applied to the analysis of human blood samples spiked with mycotoxins in the 0-500 ng/g range during a recent interlaboratory exercise. T-2 toxin and diacetoxyscirpenol were detected in these samples in the 2-180 ng/g range. Detection limits of 0.7 and 3.6 ng/g for T-2 toxin and diacetoxyscirpenol, respectively, were possible owing to the specificity of the method.

Detection of trace levels of trichothecenes in human blood using capillary gas chromatography-electron-capture negative ion chemical ionisation mass spectrometry.

A sensitive and selective method has been developed for the simultaneous detection in blood of eleven trichothecenes of widely varying polarity. The procedure involved precipitation of blood proteins with acetone followed by a clean-up using reversed-phase Sep-Pak C18 cartridges. The extracted trichothecenes were derivatised as their pentafluoropropionyl esters, separated using capillary gas chromatography and detected using electron-capture negative ion chemical ionisation with methane reagent gas and selected-ion monitoring. Optimum sensitivity and selectivity were obtained using low source temperatures (60 degrees C indicated) and high source pressures (1 Torr indicated). Detection limits on 1-ml blood samples were in the range 0.1-5 ppb. The method was readily adaptable to the detection of other trichothecenes. A protocol was used which minimised the risk of cross-contamination. The method was validated in collaborative studies by the successful analysis of 42 blood samples spiked and submitted blind by two independent laboratories for analysis.

The acute toxicopathy of intravenous diacetoxyscirpenol (anguidine) administration in swine.

Diacetoxyscirpenol (DAS, anguidine) was given intravenously to swine at 0.0, 0.5, and 1.0 mg/kg body wt. In mitotically and metabolically active tissues such as gastrointestinal epithelium and lymphoid aggregates the effects of DAS mimicked radiation poisoning. A quadratic dose-response relationship between the cytotoxicity of DAS and damage to enterocytes was found. Enterocytes in different anatomical regions of the bowel had differing susceptibilities to the toxic effects of DAS. In lymphoid tissues, DAS was preferentially cytotoxic to B-lymphocyte-rich tissues as compared to T-lymphocyte-rich tissues. In all pigs dosed with DAS the bone marrow was void of hemopoietic elements. DAS was cytotoxic to cells with specialized ion pumps, namely, renal tubular, gastric parietal, and salivary ducts. Cell damage in the exocrine and endocrine pancreas and adrenal gland accounted for changes in blood glucose. Endothelial necrosis and hemorrhage were observed in the brain. These findings were compared with those reported for other 12,13-epoxytrichothecenes and ionizing radiation and we concluded that a similar mechanism of cytotoxicity could exist.