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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.

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.

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.

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.

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.

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.

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.

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

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.

Background Leptin-deficient ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in ob/ob mice. Methods Using leptin deficient ob/ob mice, we investigated whether leptin injection into ob/ob mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells. Results We report here that adipose stem cell number is significantly increased following leptin injection in ob/ob mice and with treatment of isolated stem cells with leptin in vitro. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells. Conclusions Taken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies.