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Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B

The common foodborne mycotoxin deoxynivalenol (DON, vomitoxin) can negatively impact animal and human health by causing food refusal and vomiting. Gut enteroendocrine cells (EECs) secrete hormones that mediate DON’s anorectic and emetic effects. In prior work utilizing a cloned EEC model, our laboratory discovered that DON-induced activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor (GPCR), and transient receptor ankyrin-1 (TRPA1), a transient receptor potential (TRP) channel, drives Ca2+-mediated hormone secretion. Consistent with these in vitro findings, CaSR and TRPA1 mediate DON-induced satiety hormone release and food refusal in the mouse, an animal model incapable of vomiting. However, the roles of this GPCR and TRP in DON’s emetic effects remain to be determined. To address this, we tested the hypothesis that DON triggers emesis in mink by activating CaSR and TRPA1. Oral gavage with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate; AITC) rapidly elicited emesis in the mink in dose-dependent fashion. Oral pretreatment of the animals with the CaSR antagonist NPS-2143 or the TRP antagonist ruthenium red (RR), respectively, inhibited these responses. Importantly, DON-induced emesis in mink was similarly inhibited by oral pretreatment with NPS-2143 or RR. In addition, these antagonists suppressed concurrent DON-induced elevations in plasma peptide YY3-36 and 5-hydroxytryptamine—hormones previously demonstrated to mediate the toxin’s emetic effects in mink. Furthermore, antagonist co-treatment additively suppressed DON-induced emesis and peptide YY 3-36 release. To summarize, the observations here strongly suggest that activation of CaSR and TRPA1 might have critical roles in DON-induced emesis.

Food refusal is a hallmark of exposure of experimental animals to the trichothecene mycotoxin deoxynivalenol (DON), a common foodborne contaminant. Although studies in the mouse suggest that DON suppresses food intake by aberrantly inducing the release of satiety hormones from enteroendocrine cells (EECs) found in the gut epithelium, the underlying mechanisms for this effect are not understood. To address this gap, we employed the murine neuroendocrine tumor STC-1 cell line, a widely used EEC model, to test the hypothesis that DON-induced hormone exocytosis is mediated by G protein-coupled receptor (GPCR)-mediated Ca(2+) signaling. The results indicate for the first time that DON elicits Ca(2)-dependent secretion of cholecystokinin (CCK) and glucagon-like peptide-1(7-36) amide (GLP-1), hormones that regulate food intake and energy homeostasis and that are products of 2 critical EEC populations--I cells of the small intestine and L cells of the large intestine, respectively. Furthermore, these effects were mediated by the GPCR Ca(2+)-sensing receptor (CaSR) and involved the following serial events: (1)PLC-mediated activation of the IP3 receptor and mobilization of intracellular Ca(2+) stores, (2) activation of transient receptor potential melastatin-5 ion channel and resultant L-type voltage-sensitive Ca(2+) channel-facilitated extracellular Ca(2+) entry, (3) amplification of extracellular Ca(2+) entry by transient receptor potential ankyrin-1 channel activation, and finally (4) Ca(2+)-driven CCK and GLP-1 excytosis. These in vitro findings provide a foundation for future investigation of mechanisms by which DON and other trichothecenes modulate EEC function in ex vivo and in vivo models.

Deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium sp. that frequently occurs in cereal grains, has been associated with human and animal food poisoning. Although a common hallmark of DON-induced toxicity is the rapid onset of emesis, the mechanisms for this adverse effect are not fully understood. Recently, our laboratory has demonstrated that the mink (Neovison vison) is a suitable small animal model for investigating trichothecene-induced emesis. The goal of this study was to use this model to determine the roles of two gut satiety hormones, peptide YY3-36 (PYY3-36) and cholecystokinin (CCK), and the neurotransmitter 5-hydroxytryptamine (5-HT) in DON-induced emesis. Following ip exposure to DON at 0.1 and 0.25mg/kg bw, emesis induction ensued within 15-30min and then persisted up to 120min. Plasma DON measurement revealed that this emesis period correlated with the rapid distribution and clearance of the toxin. Significant elevations in both plasma PYY3-36 (30-60min) and 5-HT (60min) but not CCK were observed during emesis. Pretreatment with the neuropeptide Y2 receptor antagonist JNJ-31020028 attenuated DON- and PYY-induced emesis, whereas the CCK1 receptor antagonist devezapide did not alter DON's emetic effects. The 5-HT3 receptor antagonist granisetron completely suppressed induction of vomiting by DON and the 5-HT inducer cisplatin. Granisetron pretreatment also partially blocked PYY3-36-induced emesis, suggesting a potential upstream role for this gut satiety hormone in 5-HT release. Taken together, the results suggest that both PYY3-36 and 5-HT play contributory roles in DON-induced emesis.

The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3.

Deoxynivalenol (DON) and other ribotoxic trichothecenes cause immune stimulation and suppression in leukocytes by upregulating gene expression and apoptosis, respectively. The purpose of this study was to test the hypothesis that MAPKs mediate both apoptosis and survival in DON-exposed macrophages. At concentrations which partially inhibit translation, DON induced phosphorylation of p38 and ERK 1/2 mitogen activated protein kinases within 15 min in RAW 264.7 macrophages and these effects lasted up to 3 h. DON-exposed cells exhibited marked caspase 3-dependent DNA fragmentation after 6 h which was suppressed and attenuated by the p38 inhibitor SB203580 and ERK inhibitor PD98059, respectively. DON readily induced the phosphorylation and activity of p53 and this was inhibitable by SB203580. DON exposure evoked BAX translocation to mitochondria and corresponding cytochrome C release but did not alter mitochondrial membrane potential. The p53 inhibitor PFTalpha reduced both DON-induced phosphorylation of p53 and p53 binding activity. Moreover, both PFTalpha and p53 siRNA transfection suppressed DON-induced caspase-3 activity and subsequent DNA fragmentation. Concurrent with p53 activation, DON activated two anti-apoptotic survival pathways as evidenced by both ERK-dependent p90 Rsk and AKT activation. Taken together, the results indicate that DON initiates competing apoptotic (p38/p53/Bax/Mitochondria/Caspase-3) and survival (ERK/AKT/p90Rsk/Bad) pathways in the macrophage.

Macrophages, T cells, and B cells of the immune system are central targets of deoxynivalenol (DON) and other trichothecenes-mycotoxins that can be immunostimulatory or immunosuppressive depending on dose, exposure frequency and timing of functional immune assay. Notably, low dose trichothecene exposure transcriptionally and post-transcriptionally upregulates expression of cytokines, chemokines and inflammatory genes with concurrent immune stimulation, whereas high dose exposure promotes leukocyte apoptosis with concomitant immune suppression. DON and other trichothecenes, via a mechanism known as the ribotoxic stress response, bind to ribosomes and rapidly activate mitogen-activated protein kinases (MAPKs). The latter are important transducers of downstream signaling events related to immune response and apoptosis. Using cloned macrophages, our laboratory has identified two critical upstream transducers of DON-induced MAPK activation. One transducer is double-stranded RNA-(dsRNA)-activated protein kinase (PKR), a widely-expressed serine/theonine protein kinase that can be activated by dsRNA, interferon, and other agents. The second transducer is hematopoetic cell kinase (Hck), a non-receptor associated Src family kinase. Inhibitors and gene silencing studies have revealed that Hck and PKR play roles in DON induced gene expression and apoptosis. Future studies should focus on the molecular linkages between these kinases and trichothecene toxicity.

Deoxynivalenol (DON, vomitoxin) is a trichothecene mycotoxin that potentially mediates toxicity by upregulating proinflammatory cytokine gene expression in vitro and in vivo. The purpose of this study was to test the hypothesis that DON-induced activation of mitogen-activated protein kinases (MAPKs) mediates transcriptional and posttranscriptional upregulation of TNF-alpha gene expression. RNAse protection assay revealed that DON at 100 to 500 ng/ml induced mRNA expression of TNF-alpha as well as IL-6, IFN-gamma, TGFbeta-1, and TGFbeta-3 and that these effects were potentiated by 100 ng/ml lipopolysaccharide (LPS). DON was found to induce phosphorylation of p38 kinase, extracellular signal-regulated kinases (ERKs), and c-Jun amino terminal kinases (JNKs) in a dose-dependent manner in the RAW 264.7 murine macrophage model. A luciferase reporter gene driven by the murine TNF-alpha promoter was used to assess the role of various MAPKs on DON upregulation of TNF-alpha gene transcription. The p38 inhibitor SB203580 reduced induction of luciferase activity by DON, LPS, and DON + LPS. In addition, the ERK inhibitor PD 98059 blocked DON- and DON + LPS-induced luciferase activity whereas the JNK inhibitor impaired LPS- and DON + LPS-induced luciferase activity. To study the effects of MAPKs on DON-induced TNF-alpha mRNA stability, an asynchronous model was used whereby cells were pretreated with LPS for 4 h and the medium was removed. Following incubation with medium containing a transcription inhibitor, 5,6-dichloro-beta-D-ribofuranosyl-benzimidazole, MAPK inhibitors and/or DON (250 ng/ml) cultures were monitored for TNF-alpha mRNA expression. DON-induced TNF-alpha mRNA stabilization was abrogated in the presence of SB 203580, whereas the stabilization by DON was not affected by PD 98059 or SP 600125. To verify the role of MAPKs in DON + LPS-induced TNF-alpha production, cells were incubated with LPS, DON, or LPS + DON for 18 h in the presence of inhibitors. ELISA of supernatant indicated that induction of TNF-alpha production by DON alone was significantly reduced by SB 203580 and PD 98059, whereas all three inhibitors blocked LPS- and DON + LPS-induced TNF-alpha production. Taken together, these results suggest that relative to DON-induced TNF-alpha mRNA expression, p38 and ERK activation contribute to DON-induced transcriptional upregulation whereas p38 plays a role in increasing mRNA stability.

Trichothecene mycotoxins and other protein synthesis inhibitors activate mitogen-activated protein kinase (MAPKs) via a mechanism that has been termed the "ribotoxic stress response." MAPKs are believed to mediate the leukocyte apoptosis that is observed following experimental exposure to these chemical agents in vitro and in vivo. The purpose of this research was to test the hypothesis that double-stranded, RNA-activated protein kinase R (PKR) is a critical upstream mediator of the ribotoxic stress response induced by the trichothecene deoxynivalenol (DON) and other translational inhibitors. DON was found to readily induce phosphorylation of JNK 1/2, ERK 1/2, and p38 in the murine macrophage RAW 264.7 cell line, within 5 min of culture addition, in a concentration-dependent fashion. Effects were maximal from 15 to 30 min and lasted up to 6 h. The translational inhibitors anisomycin and emetine also had similar effects when added to cultures at equipotent concentrations to DON. DON rapidly activated PKR within 1 to 5 min, as evidenced by autophosphorylation and by phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Interestingly, the latter effect was associated with rapid degradation of eIF2alpha. Pretreatment of RAW 264.7 cells with two inhibitors of PKR, 2-aminopurine (2-AP) or adenine (Ad), markedly impaired MAPK phosphorylation in RAW 264.7 cells according to the following rank order JNKp38ERK. The capacity of DON to induce MAPK phosphorylation was also markedly suppressed in a stable transformant of the human promonocytic U-937 cell line containing an antisense PKR expression vector. This suppression followed a rank order of JNKp38ERK in this PKR-deficient cell line when compared to control cells transfected with vector only. Apoptosis induction by DON and two other translational inhibitors, anisomycin and emetine, was almost completely abrogated in PKR-deficient cells. Together, the results indicate that PKR plays a critical upstream role in the ribotoxic stress response inducible by translational inhibitors.

Bacterial lipopolysaccharide (LPS) elicits inflammation and endotoxic shock by inducing proinflammatory cytokine gene expression. The purpose of this study was to test the hypothesis that differential activation of transcription factor binding in the spleen correlates with proinflammatory cytokine gene expression in mice exposed to LPS. When proinflammatory cytokine expression in spleen was evaluated in mice injected ip with 4 mg/kg LPS over an 8-h period, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 mRNAs were elevated up to 5-, 6-, and 300-fold, respectively, over vehicle controls. Both TNF- alpha and IL-6 mRNA peaked at 2 h and begin to decline thereafter, whereas IL-1beta mRNA remained elevated from 2 to 8 h. The capacities of splenic nuclear proteins to bind to six different consensus transcriptional control motifs associated with proinflammatory cytokine promoters were also measured over 8 h. Electrophoretic mobility shift assay (EMSA) revealed that binding activity was markedly increased at 0.5 to 8 h for activator protein-1 (AP-1) as were CCAAT enhancer-binding protein (C/EBP) and nuclear factor kappaB (NF-kappaB) at 0.5 to 1.5 h. At 0.5 h, cyclic AMP response element (CRE)-binding protein (CREB) and binding was slightly elevated, whereas activator protein- 2 (AP-2) and specificity protein 1 (Sp1) binding were not affected. Antibody supershift EMSA and Western blot analysis confirmed that increased binding of these factors correlated with LPS-induced increases in nuclear concentrations of AP-1 (c-Jun, phosphorylated c-Jun, Jun D, and Jun B), C/EBPbeta, NF-kappaB (p50, p65, and c-Rel), CREB (CREB-1, CREB-2, and ATF-2), and AP-2alpha proteins. Remarkably, after 8 h, C/EBP, CREB, AP-2, and Sp1 binding activities were greatly depleted relative to both naive and corresponding vehicle controls. When mice were exposed to a second dose of LPS, 8 h after a 4 mg/kg priming dose, TNF-alpha and IL-6 mRNA responses were markedly impaired, suggesting that the mice were endotoxin tolerant at this time point. Taken together, the quiescent, active, and suppressive phases of transcription factor binding observed in this model were highly consistent with the rapid transient nature of LPS-induced proinflammatory cytokine expression in vivo as well as tolerance to secondary LPS exposure.

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.

Dietary fish oil (FO) supplementation reportedly retards the progression of renal disease in patients with immunoglobulin (Ig)A nephropathy (IgAN), the most common glomerulonephritis worldwide. Using an experimental mouse model in which early immunopathological hallmarks of IgAN are induced by the mycotoxin vomitoxin (VT), the ameliorative effects of FO ingestion on this disease were evaluated in two studies. In Study 1, the capacity of VT to induce IgAN was evaluated in mice fed for 12 wk AIN-76A diets containing 50 g/kg corn oil (CO), 50 g/kg CO plus 9 mg/kg tert butylhydroquinone (TBHQ), or 5 g/kg CO plus 45 g/kg menhaden FO that contained 200 mg/kg TBHQ. Serum IgA, serum IgA immune complexes and kidney mesangial IgA deposition were greater in mice fed VT + CO compared with the CO control group, whereas all three variables were significantly attenuated in mice fed VT + FO. Although TBHQ also had attenuating effects, these were significantly less than those for the VT + FO group. In Study 2, the effects of feeding modified AIN 93G diets containing either 70 g/kg CO or 10 g/kg CO plus 60 g/kg FO for 20 wk on VT-induced IgAN were compared. Again, consumption of FO attenuated all three immunopathological variables. In addition, spleen cell cultures from the VT + FO group produced markedly less IgA than those cultures from mice fed VT + CO. Taken together, the results suggested that diets containing FO may impair early immunopathogenesis in VT-induced IgAN and that this was not totally dependent on the presence of the antioxidant TBHQ.

Trichothecene mycotoxins are a family of potent translational inhibitors that are associated with foodborne outbreaks of human and animal gastroenteritis in which vomiting is a clinical hallmark. Deoxynivalenol (DON, vomitoxin) and other Type B trichothecenes have been previously demonstrated to cause emesis in the mink (Neovison vison), and this response has been directly linked to secretion of both the satiety hormone peptide YY3–36 (PYY3–36) and neurotransmitter 5-hydroxytryptamine (5-HT). Here, we characterized the emetic responses in the mink to T-2 toxin (T-2) and HT-2 toxin (HT-2), two highly toxic Type A trichothecenes that contaminate cereals, and further compared these effects to those of emetine, a natural alkaloid that is used medicinally and also well known to block translation and cause vomiting. Following intraperitoneal (IP) and oral exposure, all three agents caused vomiting with evident dose-dependent increases in both duration and number of emetic events as well as decreases in latency to emesis. T-2 and HT-2 doses causing emesis in 50 % of treated animals (ED50s) were 0.05 and 0.02 mg/kg BW following IP and oral administration, respectively, whereas the ED50s for emetine were 2.0 and 1.0 mg/kg BW for IP and oral exposure, respectively. Importantly, oral administration of all three toxins elicited marked elevations in plasma concentrations of PYY3–36 and 5-HT that corresponded to emesis. Taken together, the results suggest that T-2 and HT-2 were much more potent than emetine and that emesis induction by all three translational inhibitors co-occurred with increases in circulating levels of PYY3–36 and 5-HT.

To assess the potential for ingestion of yogurt to modulate immunity, its effects on basal gene expression of cytokines in systemic and mucosal sites were determined in mice. Yogurts were manufactured from pasteurized nonfat dry milk using five commercial starter cultures with or without Bifidobacterium sp. and Lactobacillus acidophilus. Treatment mice were fed the AIN-93G diet mixed 1:1 with unheated yogurt or heat-treated yogurt (wt/wt) for 2 and 4 weeks, and control mice were fed the AIN-93G diet mixed 1:1 (wt/wt) with nonfat dry milk. The viability of the various bacterial groups in unheated yogurts was maintained above 10(6) CFU/g throughout the feeding period. The yogurt-feeding regimens did not significantly affect weight gain. Relative mRNA levels in spleen, mesenteric lymph nodes, or Peyer's patches for the cytokines interferon-gamma, tumor necrosis factor-alpha, interleukin-2, -4, and -6, and the "housekeeping gene" beta2-microglobulin were determined by reverse transcriptase-polymerase chain reaction in conjunction with hybridization analysis. Prolonged feeding of some yogurts decreased expression of several cytokine mRNAs, the depression of tumor necrosis factor-alpha mRNA in the spleen being the most prominent effect. Heat-treated yogurts were more effective in altering cytokine mRNA expression than were unheated yogurts containing viable organisms. Generally, yogurts either had no effect or decreased specific cytokine mRNA in the test organs, regardless of whether they contained Bifidobacterium sp. and L. acidophilus. These results suggest that, in contrast with previous studies in vitro, some yogurt formulations may reduce rather than stimulate basal cytokine expression and that these effects are most prominent in the systemic compartment.

The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1β, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expressions. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1β mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528's effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity.

Cereal grain contamination by trichothecene mycotoxins is known to negatively impact human and animal health with adverse effects on food intake and growth being of particular concern. The head blight fungus Fusarium graminearum elaborates five closely related 8-ketotrichothecene congeners: (1) deoxynivalenol (DON), (2) 3-acetyldeoxynivalenol (3-ADON), (3) 15-acetyldeoxynivalenol (15-ADON), (4) fusarenon X (FX), and (5) nivalenol (NIV). While anorexia induction in mice exposed intraperitoneally to DON has been linked to plasma elevation of the satiety hormones cholecystokinin (CCK) and peptide YY3–36 (PYY3–36), the effects of oral gavage of DON or of other 8-keotrichothecenes on release of these gut peptides have not been established. The purpose of this study was to (1) compare the anorectic responses to the aforementioned 8-ketotrichothecenes following oral gavage at a common dose (2.5 mg/kg bw) and (2) relate these effects to changes plasma CCK and PYY3–36 concentrations. Elevation of plasma CCK markedly corresponded to anorexia induction by DON and all other 8-ketotrichothecenes tested. Furthermore, the CCK1 receptor antagonist SR 27897 and the CCK2 receptor antagonist L-365,260 dose-dependently attenuated both CCK- and DON-induced anorexia, which was consistent with this gut satiety hormone being an important mediator of 8-ketotrichothecene-induced food refusal. In contrast to CCK, PYY3–36 was moderately elevated by oral gavage with DON and NIV but not by 3-ADON, 15-ADON, or FX. Taken together, the results suggest that CCK plays a major role in anorexia induction following oral exposure to 8-ketotrichothecenes, whereas PYY3–36 might play a lesser, congener-dependent role in this response.

Dietary exposure to vomitoxin (VT) results in hyperelevated serum IgA and IgA nephropathy in mice. To assess the possible role of cytokines in this IgA dysregulation, the effects of a single oral exposure in B6C3F1 male mice to 0, 5 or 25 mg/kg BW VT on production of IgA and cytokines in Peyer's patch (PP) and spleen cell cultures were evaluated. IgA levels were increased significantly in PP cell cultures prepared from mice at 2 or 24 h after oral exposure to VT and subsequently stimulated with phorbol myristate acetate (PMA) and ionomycin (ION) or with lipopolysaccharide (LPS). Significant effects on IgA production were not observed in spleen cell cultures. Since cytokines such as IL-2, IL-4, IL-5 and IL-6 have been shown to promote IgA production, the effect of the same VT exposure regimen on secretion of these mediators was determined in PP and spleen cultures. Supernatant IL-2 and IL-4 levels were unaffected by the prior treatment of animals with VT. In contrast, IL-5 levels were increased significantly in 7-day PP cell cultures obtained 2 h after VT exposure both with and without PMA + ION exposure but not in other cultures. IL-6 levels were increased significantly in LPS-treated cultures prepared from PP at 2 and 24 h following exposure to VT. IL-6 levels were also elevated significantly in both PMA + ION or LPS treated cultures from spleen isolated at 2 h but not 24 h post VT exposure. To determine whether IL-5 or IL-6 play a role in IgA hyperelevation in vitro, PP and spleen cells from mice obtained 2 h after exposure to 25 mg/kg VT were cultured in the presence of neutralizing cytokine antibodies (Abs) and IgA production was monitored. Consistent with IL-5's previously documented role in IgA production, anti-IL-5 decreased IgA levels to background in cultures of both control and VT-exposed PP or spleen cells in the presence of either PMA + ION or LPS. Similar results were seen with addition of anti-IL-6. IgA levels were decreased to a lesser extent in PP cells cultured with LPS and in spleen cells cultured with PMA + ION from VT-exposed mice to which anti-IL-2 Ab was added. Thus, the potential for enhanced IgA production exists in lymphocytes as early as 2 h and as late as 24 h after a single oral exposure to VT and this may be related to the increased capacity to secrete helper cytokines of T cell and macrophage origin. Taken together, the results suggest that the superinduction of cytokine expression may, in part, be responsible for upregulation of IgA secretion in mice exposed orally to VT.

Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B 1 (FB 1 )-specific antibodies, composed of a single chain containing a variable heavy (V H ) and light (V L ) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB 1 -BSA conjugate or from an existing hybridoma cell line that produces anti-FB 1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of V H and V L cDNAs, (iii) ligation of V L and V H chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB 1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB 1 , the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB 2 or FB 3 . The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other foodborne disease hazards.

Polychlorinated biphenyls (PCBs) possess a variety of biological effects, including alterations in growth, development and metabolism, that may be dependent on insulin. However, no reports on the action of PCBs on cells which produce and secrete insulin are available. The current study examined the ability of a commercial mixture of PCBs (Aroclor 1254) and three specific PCB congeners, to alter the release of insulin using the hormone producing cell line RINm5F. Exposure of cells to Aroclor 1254 (A-1254) produced a concentration-dependent increase in media insulin reaching a peak, when expressed as percent of control, at 30 min. In spite of continued exposure, media insulin relative to control declined and no treatment-related difference was observed at 48 hrs. Cellular levels of the hormone declined as much as 50% by that time. The insulin releasing action of A-1254 was mimicked by each of the non-coplanar congeners 2,2′,4,4′-tetrachlorobiphenyl (TCB) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (HCB) but the coplanar congener 3,3′,4,4′-TCB showed no significant activity. These results indicate that PCBs are capable of producing a release of insulin from RINm5F cells, an effect that is unlikely to be associated with coplanar congeners that initiate their action by binding to the Ah-receptor.

The trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin produced by toxigenic Fusarium sp. that commonly contaminates cereal-based foods, has the potential to adversely affect humans and animals and therefore represents an important public health concern (Pestka, 2010). Primary targets of this mycotoxin are monocytes and macrophages of the innate immune system. Both in vitro and in vivo studies have demonstrated that DON rapidly activates mitogen-activated protein kinases which drive upregulated expression of mRNAs and proteins for inflammation-related genes such as the cytokines, chemokines, and cyclooxygenase-2 (Chung et al., 2003; Islam et al., 2006; Moon and Pestka, 2002; Shifrin and Anderson, 1999; Zhou et al., 2003a). DON-induced increases in cellular pools of inflammation-associated mRNAs have been linked to both transcriptional activation and stabilization of mRNA through AUUUA motif in the 3′-untranslated region (UTR; Choi et al., 2009; Chung et al., 2003; Moon and Pestka, 2002) indicating that these two mechanisms contribute to upregulated gene expression. It is critical to note that the overall rate of translation in a cell depends not wholly on available mRNA but also on the capacity for and efficiency of translation (Proud, 2007; Sonenberg and Hinnebusch, 2009). Capacity relates to the availability and abundance of ribosomal subunits and other translational components, whereas efficiency is regulated by the rate of translational initiation and peptide chain elongation. Individual mRNAs are subject to additional levels of translational regulation, and elements in their 5′- and 3′-UTRs may interact with regulatory RNAs (e.g., antisense sequences and microRNAs) or RNA-binding proteins (RBPs) to modulate ribosomal association. As yet, it is not known whether ribotoxins such as DON might also selectively modulate translation through these mechanisms. In addition, DON’s capability of inhibiting translation at high concentrations (Zhou et al., 2003b) makes this question more complicated and interesting. One strategy for monitoring changes in protein expression in stressed cells is proteomic analysis, but such an approach is time-consuming, expensive, and relatively insensitive compared with transcriptomic approaches employing highly sensitive PCR (Cheeseman et al., 2011; Kuny et al., 2012). Additionally, measures of protein levels are affected by protein degradation and hence do not fully reflect translation. An alternative approach for identifying and quantitating genes being translated in cells under a specific set of conditions is to first isolate their polysomes and then profile the associated mRNAs. This “translatome” strategy has successfully been used in fungal, plant, and animal cells (Halbeisen and Gerber, 2009; Markou et al., 2010; Mustroph and Bailey-Serres, 2010; Preiss et al., 2003; Shenton et al., 2006). For example, yeasts exposed to different stresses, such as amino acid depletion and fusel alcohol addition, show distinct translational profiles (Smirnova et al., 2005), suggesting there is a role of translational regulation in rapidly responding environmental stress. Further studies employing additional high-throughput array analysis have revealed that translatome does not correlate with the transcriptome under mild stresses but does so under severe stresses such as amino acid deprivation (Halbeisen and Gerber, 2009), suggesting that coordination of translatome and transcriptome is stress dependent. Uncoupling of transcriptome and translatome has also been documented in human cells in response to various stimuli and stresses (Grolleau et al., 2002; Mikulits et al., 2000; Tebaldi et al., 2012). The purpose of this study was to test the hypothesis that DON selectively modulates translation of inflammation-associated genes in the RAW 264.7 murine macrophage cell model. Specifically, we employed a focused inflammation/autoimmune PCR array to compare the DON-induced inflammation-associated translatome and transcriptome. The results revealed that DON’s capacity to modulate translation of most inflammation-associated genes is predominantly driven by transcription and mRNA stabilization; however, a small subset of these genes appeared to be regulated, in part, by selective translation.

The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10ng/ml) and ribosome-inactivating protein ricin (≥300ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism.

Specific polyclonal antibodies were generated in BALB/c mice ascitic fluid against retinoic acid with all-transretinoic acid-oxime-chicken immunoglobulin G as immunogen. Mice were pretreated with pristane, immunized three times with immunogen, then injected with mouse myeloma cell line NS-1 to induce ascites formation. Antibody titration of ascitic fluid was 6400 to 12800, and was similar to that obtained with serum. Fifty percent inhibition was obtained with all-trans-retinoic acid, 0.2 ug/ML. Indirect competitive enzyme linked immunosorbent assay indicated considerable specificity (75%) towards both all-trans-and 13-cis-retinoic acids; retinal exhibited 37% competition, retinol, 17%; retinyl palmitate and B-carotene exhibited low (9–11%) competition for retinoic acid. Crossreactivity of the antibody with the synthetic hapten, 4-oxoretinoic acid oxime and with 4-oxoretinoic acid was high (127 and 106%, respectively), while retinoic acid derivatives with modified B-ionone ring or a modified carboxyl group on polyene side chain, exhibited low cross reactivity (13–27%, respectively). Similarly, retinol, retinyl palmitate and B-carotene had relatively low cross reactivity (23, 15 and 12%, respectively).

The mycotoxin deoxynivalenol (DON) induces IgA nephropathy in mice by upregulating IL-6 expression, which is suppressed by (n-3) PUFA consumption. The purpose of this study was to test the hypothesis that consumption of the (n-3) PUFA docosahexaenoic acid (DHA) interferes with DON-induced transcriptional and post-transcriptional upregulation of IL-6 mRNA in murine macrophages. DON evoked expression of IL-6 mRNA and IL-6 heterogenous nuclear RNA (hnRNA), an indicator of ongoing IL-6 transcription, in macrophages elicited from mice fed control AIN-93G diet for 4 wk, whereas expression of both RNA species was suppressed in macrophages from mice fed AIN-93G modified to contain 30 g DHA/kg diet for the same time period. DON enhanced IL-6 mRNA stability similarly in macrophages from control and DHA-fed mice suggesting that (n-3) PUFA effects were not post-transcriptional. DON upregulated binding activity of cAMP response element binding protein (CREB) and activator protein (AP-1) to their respective consensus sequences in nuclear extracts from control-fed mice, whereas both activities were suppressed in nuclear extracts from DHA-fed mice. DON induced phosphorylation of CREB at Ser-133 and ATF1 at Ser-63 as well as intranuclear binding of phospho-CREB/ATF1 to the cis element of the IL-6 promoter in control macrophages, whereas both activities were inhibited in macrophages from DHA-fed mice. DHA consumption blocked DON-induced phosphorylation of the CREB kinase AKT. Inhibition of AKT suppressed both CREB/ATF1 phosphorylation and IL-6 transcription. These data suggest that DHA consumption suppresses DON-induced IL-6 transcription in macrophages in part by interfering with AKT-dependent phosphorylation and subsequent binding of CREB/ATF1 to the IL-6 promoter.

Trichothecene mycotoxins and other translational inhibitors activate mitogen-activated protein kinase (MAPKs) by a mechanism called the "ribotoxic stress response," which drives both cytokine gene expression and apoptosis in macrophages. The purpose of this study was to identify upstream kinases involved in the ribotoxic stress response using the trichothecene deoxynivalenol (DON) and the RAW 264.7 macrophage as models. DON (100 to 1000 ng/ml) dose-dependently induced phosphorylation of c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs. MAPK phosphorylation in response to DON exposure occurred as early as 5 min, was maximal from 15 to 30 min, and lasted up to 8 h. Preincubation with inhibitors of protein kinase C, protein kinase A, or phospholipase C had no effect on DON-induced MAPK phosphorylation. In contrast, the Src family tyrosine kinase inhibitors, PP1 (4-amino-5-[4-methylphenyl)]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) and, PP2 (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]-pyrimidine) concentration-dependently impaired phosphorylation of all three MAPK families. PP1 suppressed DON-induced phosphorylation of the MAPK substrates c-jun, ATF-2, and p90(Rsk). MAPK phosphorylation by two other translational inhibitors, anisomycin and emetine, were similarly Src-dependent. PP1 reduced DON-induced increases in nuclear levels and binding activities of several transcription factors (NF-kappaB, AP-1, and C/EBP), which corresponded to decreases in TNF-alpha production, caspase-3 activation, and apoptosis. Tyrosine phosphorylation of hematopoeitic cell kinase (Hck), a Src found in macrophages, was detectable within 1 to 5 min after DON addition, and this was suppressed by PP1. Knockdown of Hck expression with siRNAs confirmed involvement of this Src in DON-induced TNF-alpha production and caspase activation. Taken together, activation of Hck and possibly other Src family tyrosine kinases are likely to be critical signals that precede both MAPK activation and induction of resultant downstream sequelae by DON and other ribotoxic stressors.

The purpose of this investigation was to evaluate the dose-dependent effects of docosahexaenoic acid (DHA) on deoxynivalenol (DON)-induced IgA nephropathy in mice and their relation to proinflammatory gene expression and mitogen-activated protein kinase (MAPK) activation. Consumption of a modified AIN-93G diet containing 1, 5, and 30 g/kg DHA resulted in dose-dependent increases of DHA in liver phospholipids with concomitant decreases in arachidonic acid compared with control diets. DHA dose dependently inhibited increases in serum IgA and IgA immune complexes (IC) as well as IgA deposition in the kidney in DON-fed mice; the 30 g/kg DHA diet had the earliest detectable effects and maximal efficacy. Both splenic interleukin-6 (IL-6) mRNA and heterogeneous nuclear RNA (hnRNA), an indicator of IL-6 transcription, were significantly reduced in DON-fed mice that consumed 5 and 30 g/kg DHA; a similar reduction was observed for cyclooxygenase (COX-2) mRNA. In a subsequent study, acute DON exposure (25 mg/kg body weight) induced splenic IL-6 mRNA and hnRNA as well as COX-2 mRNA in mice fed the control diet, whereas induction of both RNA species was significantly inhibited in mice fed 30 g/kg DHA. These latter inhibitory effects corresponded to a reduction in DON-induced phosphorylation of p38, extracellular-signal related kinase 1/2, and c-Jun N-terminal kinase 1/2 MAPKs in the spleen. Taken together, the results indicate that DHA dose-dependently inhibited DON-induced IgA dysregulation and nephropathy, and that impairment of MAPK activation and expression of COX-2 and IL-6 are potential critical upstream mechanisms.

The effects of vomitoxin (VT) on the binding activity of three transcription factors critical to pro-inflammatory cytokine regulation were assessed in the RAW 264.7 murine macrophage model by electrophoretic mobility shift assay (EMSA). When cells were treated with 100 to 250 ng/ml of VT, activator protein-1 (AP-1 binding) was increased after 2 and 8 h. This effect was potentiated when cells were coincubated with lipopolysaccharide (LPS) (synchronous model) but not when preincubated with LPS (delayed synchronous model). Supershift EMSA revealed that VT preferentially induced JunB, JunD, phosphorylated c-Jun, c-Fos, and Fra-2 binding activities of the AP-1 family. Nuclear factor kappaB (NF-kappaB) binding was increased at 2 and 8 h in cells subjected to synchronous and delayed synchronous VT exposure in the presence of LPS. Supershift EMSA indicated that the p-50 and c-Rel subunits of NF-kappaB/ Rel were specifically affected. Nuclear factor-IL6 (NF-IL6) binding was increased at 2 and 8 h with or without LPS in synchronous and delayed synchronous VT-exposure models. Here, the C/EBPbeta subunit was primarily involved in enhanced NF-IL6 binding. The capacity of VT to elevate binding of AP-1, NF-kappaB, and NF-IL6 may contribute to the VT-mediated cytokine up-regulation in vitro and in vivo. The observations that VT was active in synchronous and delayed synchronous models suggest that macrophages activated simultaneously or prior to toxin exposure were vulnerable to the effects of this trichothecene.

Exposure to bacterial endotoxin (lipopolysaccharide, LPS) is quite common and may increase human susceptibility to chemical-induced tissue injury. The purpose of this study was to identify mechanisms by which LPS potentiates lymphoid tissue depletion in B6C3F1 mice exposed to the common food-borne trichothecene mycotoxin, vomitoxin (VT). As demonstrated by DNA fragmentation and flow cytometric analysis, apoptosis in thymus, Peyer's patches, and bone marrow was marked in mice 12 h after administering Escherichia coli LPS (0.1 mg/kg body wt ip) concurrently with VT (12.5 mg/kg body wt po), whereas apoptosis in control mice or mice treated with either toxin alone was minimal. Based on observed increases in tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 serum concentrations following LPS and VT cotreatment, the roles of these cytokines in apoptosis potentiation were assessed. Injection with rolipram, an inhibitor of TNF-alpha expression, or use of IL-6 knockout mice was ineffective at impairing thymic apoptosis induction by the toxin cotreatment, suggesting that these cytokines did not mediate LPS potentiation. Toxin cotreatment increased splenic cyclooxygenase-2 mRNA expression, suggesting possible involvement of prostaglandins in apoptosis. However, indomethacin, a broad spectrum inhibitor of cyclooxygenases, failed to block thymus apoptosis. Toxin cotreatment increased serum corticosterone and, furthermore, RU 486, a glucocorticoid receptor antagonist, significantly abrogated apoptosis in thymus, Peyer's patches, and bone marrow following LPS + VT exposure. The results presented herein and the known capacity of glucocorticoids to cause apoptosis indicate that hypothalamic-pituitary-adrenal axis plays a key role in LPS potentiation of trichothecene-induced lymphocyte apoptosis.

Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p

A single oral exposure to the trichothecene vomitoxin (VT) has been previously shown in the mouse to increase splenic mRNA levels for several cytokines in as little as 2 h. Since one underlying mechanism for these effects likely involves superinduction of transiently expressed cytokine genes, VT may also potentially amplify cytokine responses to inflammatory stimuli. To test this possibility, the effects of oral VT exposure on tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta expression were measured in mice that were intraperitoneally injected with lipopolysaccharide (LPS), a prototypic inflammatory agent. As anticipated, VT alone at 1, 5, and 25 mg/kg body weight increased splenic mRNA expression of all three cytokines after 3 h in a dose-response fashion. LPS injection at 1 and 5 mg/kg body weight also induced proinflammatory cytokine mRNA expression. There was a synergistic increase in TNF-alpha splenic mRNA levels in mice treated with both VT and LPS as compared to mice treated with either toxin alone, whereas the effects were additive for IL-6 and IL-1beta mRNA expression. When relative mRNA levels were examined over a 12-h period in mice given LPS (1 mg/kg) and/or VT (5 mg/kg), significant enhancement was observed up to 6, 12, and 3 h for TNF-alpha, IL-6, and IL-1beta, respectively. When plasma cytokine concentrations were measured, TNF-alpha was found to peak at 1 h and was significantly increased at 1, 3, and 6 h if mice were given LPS and VT, whereas LPS or VT alone caused much smaller increases in plasma TNF-alpha Plasma IL-6 peaked at 3 h in LPS, VT, and LPS/VT groups, with the combined toxin group exhibiting additive effects. Plasma IL-1beta was not detectable. The potential for VT and LPS to enhance toxicity was examined in a subsequent study. Mortality was not observed up to 72 h in mice exposed to a single oral dose of VT at 25 mg/kg body weight or to an intraperitoneal dose of LPS at 1 or 5 mg/kg body weight; however, all mice receiving VT and either LPS dose became moribund in less than 40 h. The principal histologic lesions in the moribund mice treated with VT and LPS were marked cell death and loss in thymus, Peyer's patches, spleen, and bone marrow. In all of these lymphoid tissues, treatment-induced cell death had characteristic histologic features of apoptosis causing lymphoid atrophy. These results suggest that LPS exposure may markedly increase the toxicity of trichothecenes and that the immune system was a primary target of these interactive effects.

A single oral exposure to vomitoxin (VT) in mice has been previously shown to induce in lymphoid tissues the rapid expression of cytokine mRNAs that are produced by both macrophages and T cells. To determine whether prior VT exposures positively or negatively modulate the cytokine response to the toxin in this model, we evaluated the effects of short-term oral (two to seven consecutive daily doses) and subchronic dietary (4 weeks) exposure to VT on expression of a panel of cytokine mRNAs. Effects of a single oral exposure to 0, 5, and 25 mg/kg body wt of VT or of two such daily consecutive doses on splenic cytokine mRNA abundance were compared 2 h after the last toxin administration using RT-PCR in combination with hybridization analysis. While robust cytokine mRNA responses occurred after a single VT exposure, attenuated but significant induction of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-12p40 mRNA was observed after a second VT dose. Similar but insignificant trends occurred with interferon (IFN)-gamma, IL-2, IL-4, and IL-10 mRNAs. Serum TNF-alpha and IL-6 proteins mimicked cytokine mRNA responses although attenuation responses were less marked. Mice were also dosed with VT at 0, 0.5, 2, or 5 mg/kg body wt consecutively for 2, 4, or 7 days and cytokine mRNAs were assessed 2 h after the last treatment in spleen and Peyer's patches. Upon exposure to 2 and 5 mg/kg body wt VT, the relative abundance of IL-1beta, IL-6, TNF-alpha, IL-12 p35, IL-12p40, IL-2, and IL-10 mRNAs increased with dose frequency whereas IFN-gamma and IL-4 mRNAs were unaffected. When mice were fed 0, 10, and 25 ppm VT for 4 weeks, increased expression of mRNAs for TNF-alpha, IL-2, IFN-gamma, and IL-10 was most prominent. However, when VT-fed mice were also challenged with an oral dose of VT equivalent to daily intake at 2 h prior to RNA isolation, vigorous mRNA responses were observed for IL-1beta, IL-6, TNF-alpha, IL-12p40, IL-12p35, IL-2, IFN-gamma, IL-4, and IL-10. In general, spleens were more responsive to the above effects than Peyer's patches. The results indicate that, following a single prior VT exposure, a significant but attenuated cytokine mRNA response occurred upon a second VT treatment. This hyporesponsiveness was overcome upon repeated exposures to the toxin. These data further support the contention that elevated cytokine expression may play a contributory role in the pathophysiologic and immunologic effects of VT and other trichothecene mycotoxins.

Oral vomitoxin (VT) exposure in mice results in elevated cytokine gene expression, increased production of IgA, and IgA nephropathy. To determine the potential role of macrophages (Mphi) in these effects, an ex vivo model was devised whereby Peyer's patch (PP) and spleen cells were prepared from mice 2 h after oral exposure to 0 or 25 mg/kg body wt VT, cultured, and then evaluated for IgA and cytokine IL-6 production. Both PP and, to a lesser extent, spleen cells from treatment mice produced more IgA over a 7-day period than did corresponding control cells when cultured without a costimulus or in the presence of either phorbol myristate acetate plus ionomycin (PMA + ION) or lipopolysaccharide (LPS); IgA elevation was most marked in LPS-treated cultures. The VT effect was completely ablated in PP cultures that were depleted of Mphi but not in Mphi-depleted spleen cultures. VT exposure similarly increased production of IL-6, an important helper factor for IgA secretion, in LPS-stimulated PP and spleen cell cultures. IL-6 production was also ablated by Mphi depletion. A potential costimulatory role for Mphi was further suggested because both IgA and IL-6 production increased when Mphi-depleted PP cells from VT-treated animals were cocultured with peritoneal Mphi from VT-treated animals. Similar effects were observed when an analogous ex vivo approach was used with purified PP B cells and peritoneal Mphi. PP B cells from control animals also secreted elevated levels of IgA when cocultured with splenic CD4(+) cells from VT-treated animals, thus confirming previous studies showing that T cell help also contributes to increased IgA production. Potential roles for soluble mediators and cell contact in this process were suggested when IgA production was measured in cultures of PP cells separated from VT-treated Mphi by a semipermeable membrane. Taken together, these and previous results suggest that Mphi may play a key mechanistic role in elevated IgA production and IgA nephropathy in VT-exposed mice.

Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to the detection of microbial agents and their toxins in foods. Recombinant single chain variable region fragments (scFv) specific to either fumonisin B1 (FB1) or zearalenone were displayed on the surface of recombinant phage, or as soluble protein after infection of E. coli HB2151. Compared to polyclonal or monoclonal antibodies for FB1, the recombinant anti-FB1 scFv proteins were 10 to 100 times less sensitive in a competitive indirect enzyme linked immunosorbent assay (CI-ELISA). High affinity anti-zearalenone scFv phage were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. Sensitivity of the anti-zearalenone scFv phage displayed protein or soluble protein was similar to the parent monoclonal antibody (IC50 of 14 ng/ml vs 17 ng/ml), but exhibited higher cross reactivity with zearalenone analogs. Affinity selection via a competitive elution was necessary for the isolation of the variable regions of anti-zearalenone scFv with the desired affinity.

Acute oral exposure of B6C3F1 mice to vomitoxin (VT) has been previously shown to induce expression of mRNAs for cytokines that are characteristically produced in lymphoid tissues by macrophage and T cells. The purpose of this study was to evaluate the effects of VT dose on the expression of mRNAs for a cytokine profile consisting of TNF-alpha, IL-1beta, IL-6, IL-12, IFN-gamma, IL-2, IL-4, IL-5, IL-10, IL-12, and TGF-beta and to measure the kinetics of these responses. The effects of a single oral exposure to 0, 0.1, 0.5, 1, 5, and 25 mg/kg BW of VT on cytokine mRNA abundance in spleen and Peyer's patches (indicators of systemic and mucosal immune compartments, respectively) were assessed at 2 hr postexposure using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with hybridization analysis. Both 5 and 25 mg/kg VT significantly induced the mRNAs for the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha; the T helper 1 cytokines IFN-gamma and IL-2; and the T helper 2 cytokines IL-4 and IL-10, whereas lower doses had no effect. IL-12p40 mRNA was also induced but not IL-12p35 mRNA. The effects were more pronounced in spleen than in the Peyer's patches. IL-5 and TGF-beta mRNAs were expressed constitutively in spleen and Peyer's patches but were not affected by VT. When cytokine mRNA levels were measured at 1, 2, 4, 8, and 24 hr after oral exposure to 25 mg/kg BW of VT, mRNA expression kinetics varied among cytokines or between spleen and Peyer's patches. The duration of elevated mRNA expression in spleen was 1-8 hr for TNF-alpha, IL-6, IFN-gamma, and IL-10 and was 1-4 hr for IL-1beta, IL-12p40, IL-2, and IL-4. In Peyer's patches, duration periods were 1-8 hr for IL-6, IL-2, and IL-10; 1-4 hr for IL-1beta, IL-12p40, and TNF-alpha; and 1-2 hr for IFN-gamma. Serum levels of TNF-alpha, IL-6, and IFN-gamma were elevated 3 hr after exposure to 25 mg/kg VT, thus suggesting that elevation of splenic and Peyer's patch mRNA abundance correlated with increases in systemic production of these cytokines. Taken together, the results indicate that a single VT exposure rapidly induces gene expression in vivo for a wide range of cytokines with apparently complete recovery occurring after 24 hr. Elevated cytokine expression may play an important role in the pathophysiologic effects of VT and other trichothecenes.

The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene VK23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.

A stabilized hybridoma cell line secreting anti-retinoic acid monoclonal antibodies of subclass IgG1 with kappa chains was produced by fusing NS-1 myeloma cells with the spleen cells from BALB/c female mice immunized with all-trans-4-oxoretinoic acid-oxime-chicken IgG conjugate. The antibody titer of mice ascitic fluid ranged from 1/12,800 to 1/25,600, as determined by competitive indirect enzyme-linked immunosorbent assay (ELISA). 50% inhibition dosage of all-trans-retinoic acid at a 1/20,000 dilution of mice ascitic fluid was 6.6 ng/ml, as determined by ELISA. The anti-retinoic acid monoclonal antibody was generated in mice ascitic fluid and purified by protein G affinity chromatography. Cross-reactivity of the monoclonal antibody was determined at 0.1 microgram/ml concentration of retinoids and indicated high specificity to both all-trans-retinoic acid (86% inhibition) and 13-cis-retinoic acid (87% inhibition), and strong cross-reactivity with 4-oxoretinoic acid (77%) and 4-oxoretinoic acid oxime (109%). Specificity was confirmed by the horseradish peroxidase-linked immunostaining method and immunoradioassay. The affinity constant of the monoclonal antibody, K, was determined to be 3.6 X 10(9) l/mol. A calibration curve for retinoic acid using the monoclonal antibody to retinoic acid was developed; the detection limit for all-trans-retinoic acid is 1 ng/ml in the competitive indirect ELISA. The antibody counteracts the effect of retinoic acid on growth inhibition and differentiation in HL-60 cells.