The mycotoxin deoxynivalenol affects nutrient absorption in human intestinal epithelial cells. (33/447)

Deoxynivalenol (DON) is a mycotoxin belonging to the tricothecene family that has many toxic effects in animals, including diarrhea and weight loss. Using the human epithelial intestinal cell line HT-29-D4 as an in vitro model, we studied the effect of DON on the uptake of different classes of nutrients, including sugars, amino acids and lipids. At low concentrations (below 10 micro mol/L), DON selectively modulated the activities of intestinal transporters: the D-glucose/D-galactose sodium-dependent transporter (SGLT1) was strongly inhibited by the mycotoxin (50% inhibition at 10 micro mol DON, P < 0.05), followed by the D-fructose transporter GLUT5 (42% inhibition at 10 micro mol/L, P < 0.001), active and passive L-serine transporters (30 and 38% inhibition, respectively, at 10 micro mol/L, P < 0.05). The passive transporters of D-glucose (GLUT) were slightly inhibited by DON (15% inhibition at 1 micro mol/L, P < 0.01), whereas the transport of palmitate was increased by 35% at 10 micro mol/L DON (P < 0.001). In contrast, the uptake of cholesterol was not affected by the mycotoxin. At high concentrations (100 micro mol/L), SGLT1 activity was inhibited by 76% (P < 0.01), whereas the activities of all other transporters were increased. The selective effects of DON on intestinal transporters were mimicked by cycloheximide and deoxycholate, suggesting that inhibition of protein synthesis and induction of apoptosis are the main mechanisms of DON toxicity in intestinal cells.  (+info)

Feed refusal factors in pure cultures of Fusarium roseum 'graminearum'. (34/447)

Isolations from 1972 Wisconsin feed refusal corn yielded predominantly cultures of Fusarium roseum 'graminearum.' With one possible exception, none of the selected isolates of this fungus induced emesis in pigeons, whereas six of nine isolates produced feed refusal responses in all test animals. A single isolate of F. roseum 'equiseti' also induced a severe refusal response and possibly slight emesis. None of the other fungi isolated from this corn (F. moniliforme, Acremoniella atra) or controls caused either emesis or feed refusal. Zearalenone was detected in all isolates and was shown to be partially responsible for refusal activity. The remaining activity was ascribed to one or more nonvolatile, neutral, relatively polar molecules. T-2 toxin, although not detected in these isolates, was shown to have dramatic refusal activity in rats.  (+info)

Vomitoxin-induced cyclooxygenase-2 gene expression in macrophages mediated by activation of ERK and p38 but not JNK mitogen-activated protein kinases. (35/447)

Vomitoxin (VT) and other trichothecene mycotoxins mediate a broad range of immunotoxic effects via the induction of inflammation-associated genes in leukocytes. The purpose of this study was to test the hypothesis that VT induces cyclooxygenase-2 (COX-2) gene expression in macrophages and that this is regulated at the level of mitogen-activated protein kinases (MAPKs). Exposure of the murine macrophage cell line RAW 264.7 to 50-250 ng/ml VT for 24 h markedly enhanced the production of prostaglandin E(2) (PGE(2)), a major COX-2 metabolite. PGE(2) elevation was preceded by increases in COX-2 mRNA (2 h) and COX-2 protein (15 h) in VT-treated cells. VT induced rapid (15 min) and persistent (up to 240 min) phosphorylation of extracellular, signal regulated protein kinases 1 and 2 (ERK1/2) and p38 MAPK as well as a rapid (15 min) but transient (up to 60 min) phosphorylation of c-Jun N-terminal kinases 1 and 2 (JNK1/2). The ERK inhibitor PD98059 and p38 inhibitor SB203580 suppressed VT-induced PGE(2) and COX-2 protein expression, whereas impairment of JNK function by transient transfection with a dominant negative (dn) JNK vector had no effect on COX-2 protein expression. Relatedly, in cells transfected with a COX-2 promoter-luciferase construct, PD98059- and SB203580-, but not dnJNK-treatment, suppressed VT-induced luciferase transcription. VT also increased COX-2 mRNA stability, and this was inhibited by PD98059 but not by SB203580. Taken together, these results indicate that VT-induced PGE(2) production and COX-2 expression by elevating transcriptional activity and mRNA stability. Enhanced transcriptional activity was modulated by ERK and p38 signaling pathways, whereas mRNA stability was promoted exclusively by VT-activated p38 phosphorylation. These data provide insight into possible general mechanisms by which VT and other trichlothecenes upregulate proinflammatory genes and impart immunotoxicity.  (+info)

Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol. (36/447)

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.  (+info)

Rapid, sequential activation of mitogen-activated protein kinases and transcription factors precedes proinflammatory cytokine mRNA expression in spleens of mice exposed to the trichothecene vomitoxin. (37/447)

Since proinflammatory cytokine mRNA expression is induced within lymphoid tissue in vivo by the trichothecene vomitoxin (VT) in a rapid (1-2 h) and transient (4-8 h) fashion, it was hypothesized that mitogen-activated protein kinases (MAPKs) and transcription factors associated upstream with gene transcription of these cytokines are activated prior to or within these time windows. To test this hypothesis, mice were first treated with a single oral dose of VT and then analyzed for MAPK phosphorylation in the spleen. As little as 1 mg/kg of VT induced JNK 1/2, ERK 1/2, and p38 phosphorylation with maximal effects being observed at 5 to 100 mg/kg of VT. VT transiently induced JNK and p38 phosphorylation over a 60-min time period with peak effects being observed at 15 and 30 min, respectively. In contrast, ERK remained phosphorylated from 15 to 120 min. Next, the binding of activating protein 1 (AP-1), CCAAT enhancer-binding protein (C/EBP), CRE-binding protein (CREB), and nuclear factor-kappaB (NF-kappaB) was measured by electrophoretic mobility shift assay (EMSA) using four different consensus transcriptional control motifs at 0, 0.5, 1.5, 4, and 8 h after oral exposure to 25 mg/kg of VT. AP-1 binding activity was differentially elevated from 0.5 h to 8 h, whereas C/EBP binding was elevated only at 0.5 h. CREB binding decreased slightly at 0.5 h but gradually increased, reaching a maximum at 4 h. NF-kappaB binding was increased only slightly at 4 and 8 h. The specificities of AP-1, C/EBP, CREB, and NF-kappaB for relevant DNA motifs were verified by competition assays, using an excess of unlabeled consensus and mutant oligonucleotides. Supershift EMSAs and Western blot analysis identified specific VT-inducible DNA binding proteins for AP-1 (cJun, phospho c-jun, JunB, and JunD), C/EBP (C/EBPbeta), CREB (CREB-1 and ATF-2), and NF-kappaB (p50 and cRel). Finally, when the effects of oral VT exposure on proinflammatory gene expression were assessed at 3, 6, and 9 h, splenic TNF-alpha, IL-1beta, and IL-6 mRNA were found to peak at 3 h and were still significantly elevated at 6 h but not at 9 h. Taken together, VT first activated MAPKs in vivo and either concurrently (AP-1, C/EBP) or subsequently (AP-1, CREB, NF-kappaB) modulated binding activities of transcription factors specific for potential regulatory motifs in cytokine promoters. The timing of these events was highly consistent with the kinetics of proinflammatory gene expression in the spleens of mice exposed to VT. This study provides a novel model for studying the interrelationship of MAPK phosphorylation, transcription factor activation, and cytokine gene expression in an intact animal exposed to a toxic compound.  (+info)

Fusarium graminearum and deoxynivalenol contamination in the durum wheat area of Argentina. (38/447)

Fusarium graminearum head blight of wheat is a destructive disease of the world's wheat-growing areas. This work was performed to analyze the distribution and contamination of deoxynivalenol (DON) and its relationship with F. graminearum kernel invasion in Argentina durum wheat area during two consecutive harvests. A total of 147 samples (cultivars and lines) of durum wheat from 5 locations of the major cropping area (Southern Buenos Aires Province) were analyzed. Percentage of F. graminearum kernel infection was evaluated following the blotter test (ISTA method) and fusarotoxins were analyzed by thin layer chromatography. None of the varieties and lines were free of F. graminearum infection. In the first harvest fungal invasion was very low. From 40 samples, 55% showed DON contamination but only 4 samples (10%) were higher than 2 ppm. In the second harvest, a crop year conducive to scab development, the highest level of F. graminearum kernel invasion observed was 42% on a sample from the humid area (eastern Buenos Aires Province) DON was detected in 47 (78.2%) of 60 samples analyzed and 19 (31.6%) showed levels of DON higher than those established in the guidelines in Canada and USA for food and feedstuff. In both years all locations situated in the humid area showed levels ranging from 0 to > 8 ppm. Within the durum wheat area differences among locations were found. This analysis indicates the need for more information on the problem and distribution of Fusarium mycotoxins in durum wheat grown in Argentina.  (+info)

Trichothecene nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes. (39/447)

The trichothecene 3-O-acetyltransferase gene (FgTri101) required for trichothecene production by Fusarium graminearum is located between the phosphate permease gene (pho5) and the UTP-ammonia ligase gene (ura7). We have cloned and sequenced the pho5-to-ura7 regions from three trichothecene nonproducing Fusarium (i.e., F. oxysporum, F. moniliforme, and Fusarium species IFO 7772) that belong to the teleomorph genus Gibberella. BLASTX analysis of these sequences revealed portions of predicted polypeptides with high similarities to the TRI101 polypeptide. While FspTri101 (Fusarium species Tri101) coded for a functional 3-O-acetyltransferase, FoTri101 (F. oxysporum Tri101) and FmTri101 (F. moniliforme Tri101) were pseudogenes. Nevertheless, F. oxysporum and F. moniliforme were able to acetylate C-3 of trichothecenes, indicating that these nonproducers possess another as yet unidentified 3-O-acetyltransferase gene. By means of cDNA expression cloning using fission yeast, we isolated the responsible FoTri201 gene from F. oxysporum; on the basis of this sequence, FmTri201 has been cloned from F. moniliforme by PCR techniques. Both Tri201 showed only a limited level of nucleotide sequence similarity to FgTri101 and FspTri101. The existence of Tri101 in a trichothecene nonproducer suggests that this gene existed in the fungal genome before the divergence of producers from nonproducers in the evolution of Fusarium species.  (+info)

Tri1 encodes the cytochrome P450 monooxygenase for C-8 hydroxylation during trichothecene biosynthesis in Fusarium sporotrichioides and resides upstream of another new Tri gene. (40/447)

Many Fusarium species produce one or more agriculturally important trichothecene mycotoxins, and the relative level of toxicity of these compounds is determined by the pattern of oxygenations and acetylations or esterifications on the core trichothecene structure. Previous studies with UV-induced Fusarium sporotrichioides NRRL 3299 trichothecene mutants defined the Tri1 gene and demonstrated that it was required for addition of the oxygen at the C-8 position during trichothecene biosynthesis. We have cloned and characterized the Tri1 gene from NRRL 3299 and found that it encodes a cytochrome P450 monooxygenase. The disruption of Tri1 blocks production of C-8-oxygenated trichothecenes and leads to the accumulation of 4,15-diacetoxyscirpenol, the same phenotype observed in the tri1 UV-induced mutants MB1716 and MB1370. The Tri1 disruptants and the tri1 UV-induced mutants do not complement one another when coinoculated, and the Tri1 gene sequence restores T-2 toxin production in both MB1716 and MB1370. The DNA sequence flanking Tri1 contains another new Tri gene. Thus, Tri1 encodes a C-8 hydroxylase and is located either in a new distal portion of the trichothecene gene cluster or in a second separate trichothecene gene cluster.  (+info)