Biomedical subjects

James J Pestka

Publications and source records attributed to James J Pestka.

At least 19 recordsLinked to original sources

PubMed2003

ELISA for monitoring lipid oxidation in chicken myofibrils through quantification of hexanal-protein adducts.

The objectives of this study were to optimize a monoclonal competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) for hexanal detection, optimize solubilization and alkylation procedures for the formation of hexanal-protein adducts, and compare the ability the CI-ELISA, thiobarbituric acid reactive substances assay (TBARS), and a solid-phase microextraction-gas chromatography-mass spectrometry (GC/MS-SPME) method for monitoring lipid oxidation in freeze-dried chicken protein. Freeze-dried myofibrils with added methyl linoleate (0.6 mmol/g of protein) were stored at 50 degrees C at two water activities (a(w) = 0.30 and 0.75) for 5 days. Hexanal was measured by GC/MS-SPME and CI-ELISA, and malonaldehyde by TBARS. At an a(w) of 0.30, 34.7 and 39.7 microg of hexanal/g of myofibril were detected by GC/MS-SPME and CI-ELISA, respectively, after 4 days of storage. At an a(w) of 0.75, 39.8 and 61.1 microg of hexanal/g of myofibril were detected by GC/MS-SPME and CI-ELISA, respectively, after 4 days of storage. The CI-ELISA was well correlated with the GC/MS-SPME (r = 0.78) and TBARS (r = 0.87) methods. The correlation of the hexanal-specific CI-ELISA to both GC/MS-SPME and TBARS verified the ability of the CI-ELISA to be used as an index of lipid oxidation, offering the convenience for use in a kit to be utilized within a food-processing facility.

Aldehydes↗

PubMed2003

Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-alpha expression by deoxynivalenol (vomitoxin).

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.

Animals↗

PubMed2003

Comparative susceptibility of B cells with different lineages to cytotoxicity and apoptosis induction by translational inhibitors.

The trichothecene mycotoxins, Shiga toxins (STs), and ricin are potent translational inhibitors that exert diverse mechanisms of action but all have the capacity to induce death by apoptosis. Germinal centers containing actively dividing B cells are particularly sensitive to protein synthesis inhibition, and, of these, the immature B cell is reportedly most susceptible to apoptosis. The objective of this study was to test the hypothesis that immature and mature B-cell lineages were differentially susceptible to apoptosis and cytotoxicity induction by representative trichothecene mycotoxins, ST-1, and ricin, as well as cycloheximide (CHX), a prototypical protein synthesis inhibitor commonly used to study cell signal transduction. WEHI-231 and CH31 cells were used as representatives of phenotypically immature B cells, whereas CH12.LX cells were used to model mature B cells. Resultant data suggest that Type D and Type A trichothecenes, ricin, and ST-1 were more potent inducers of apoptosis than CHX, whereas Type B and Type A trichothecene metabolites were less. CHX and the trichothecenes affected immature and mature B cells equally, thus suggesting that toxicity due to these natural toxins was lineage independent. In contrast, mature B cells were more sensitive to ricin- and ST-1-induced cytotoxicity and apoptosis than immature B cells. Taken together, these results suggest that B cells are targets of a diverse array of naturally occurring translational inhibitors. Upregulation of apoptosis in B lymphocytes may contribute to the impairment of the immune response and other symptoms described following exposure to these toxins.

Apoptosis↗

PubMed2003

Potentiation of trichothecene-induced leukocyte cytotoxicity and apoptosis by TNF-alpha and Fas activation.

Trichothecene mycotoxins cause immunosuppression by inducing apoptosis in lymphoid tissue. Trichothecene-induced leukocyte apoptosis can be augmented by bacterial lipopolysaccharide (LPS) but the mechanisms involved in this potentiating effect are not completely understood. The objective of this study was to test the hypothesis that the trichothecene deoxynivalenol (DON, vomitoxin) can interact with LPS directly and other mediators or agonists associated with immune/inflammatory responses to induce apoptosis in primary murine leukocyte cultures. Primary leukocyte suspensions were prepared from murine thymus (TH), spleen (SP), bone marrow (BM) and Peyer's patches (PP) and then cultured with DON in the absence or presence of LPS, prostaglandin E2 (PGE2), anti-immunoglobulin (as antigen mimic), dexamethasone, Fas ligand, or TNF-alpha. Cytotoxicity and apoptosis were evaluated by MTT assay and morphologic assays, respectively. DON was found to inhibit LPS-induced proliferation and dexamethasone-induced apoptosis in SP cultures. In contrast, potentiation of DON-induced apoptosis and cytotoxicity was observed in BM cultures treated with anti-Fas and in TH cultures treated with TNF-alpha. When potentiation of DON-induced apoptosis by TNF-alpha was assessed using pharmacological inhibitors, generation of ROS, intracellular Ca2+, p38/SAPK, and caspase-3 activation were found to play roles. Taken together, these data demonstrate that LPS and its downstream mediators can interact with trichothecenes to modulate proliferative, cytotoxic and apoptotic outcomes in leukocytes in a tissue-specific manner.

Animals↗

PubMed2003

Relationship of trichothecene structure to COX-2 induction in the macrophage: selective action of type B (8-keto) trichothecenes.

The trichothecene mycotoxin deoxynivalenol (DON, vomitoxin), when at partially cytotoxic concentrations, induces cyclooxygenase-2 (COX-2) expression by promoting transcriptional activity and mRNA stability via mitogen-activated protein kinase (MAPK) signaling pathways. The purpose of this study was to test the hypothesis that trichothecenes differentially affect COX-2 gene expression and that these effects were related to MAPK activation. Representative members of the three major trichothecene families (A, B, and D) were compared for their capacity to induce COX-2 in the RAW 264.7 murine macrophage cell line. When cells were treated with concentrations that inhibited the 3-(4,5-di-methylthizol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) viability response by 20% (IC20), Type B trichothecenes including DON, 15-acetyl-DON, 3-acetyl-DON, and fusarenon-X were found to be effective inducers of COX-2 mRNA expression, whereas equitoxic Type A and Type D trichothecenes had markedly less effects. To compare effects of COX-2 gene transactivation and mRNA stabilization, luciferase reporter vectors containing 5'-promoter or 3'-untranslated regions of the gene, respectively, were transfected into RAW 264.7 cells and the effects of various trichothecenes on luciferase activities were measured. Type B but not Type A or D toxins at concentrations up to the MTT IC50 enhanced luciferase activities, indicating preferential COX-2 transcriptional activation and mRNA stabilization by this trichothecene subset. At their respective IC20s, Type B trichothecenes also significantly activated the three major MAPK families, whereas Type A and D did not. Blocking ERK and p38 with chemical inhibitors significantly suppressed Type B-induced COX-2 expression. Although JNK reportedly contributes to COX-2 expression in the other signaling models, transfection with the dominant negative JNK vector did not diminish the COX-2 expression. Taken together, Type B trichothecenes selectively enhanced transcription and stabilization of the COX-2 gene, and this was mediated by the ERK 1/2 and p38 signaling pathways. Selective action on COX-2 might contribute to unique pathologic manifestations associated with Type B trichothecene-mediated immunotoxicity.

Animals↗

PubMed2003

Solid phase microextraction-gas chromatography for quantifying headspace hexanal above freeze-dried chicken myofibrils.

A method using solid phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MS) was developed and used to determine the oxidation of freeze-dried chicken myofibrils spiked with methyl linoleate. Freeze-dried chicken myofibrils were found to act as a significant reservoir for hexanal. Recovery of hexanal emissions from the headspace above spiked myofibrils was 95% using a 5 min sampling time, with a total analysis time of approximately 12 min/sample. The SPME-GC/MS working linear response was from 0.01 to 10 mg hexanal/L (r( 2) = 0.995). Freeze-dried chicken myofibrils with added methyl linoleate (0.6 mmol/g of protein) were stored at 50 degrees C at water activities of 0.30 and 0.75 for 0, 12, 27, and 50 h. Lipid oxidation was determined using SPME-GC/MS to measure headspace hexanal concentration, the thiobarbituric acid reactive substances assay (TBARS) to quantify malonaldehyde, and a conjugated diene assay. Lipid oxidation was influenced by storage time and water activity. A strong correlation (r = 0.938) existed between SPME-GC/MS and TBARS. The use of SPME-GC/MS was a sensitive and rapid method for detecting hexanal as an indicator of lipid oxidation in chicken myofibrils.

Aldehydes↗

PubMed2003

Role of IL-1(beta) in endotoxin potentiation of deoxynivalenol-induced corticosterone response and leukocyte apoptosis in mice.

Endotoxin (lipopolysaccharide, LPS) and the trichothecenes are microbial toxins that are frequently encountered in food and the environment. Coexposure to LPS and the trichothecene deoxynivalenol (DON, vomitoxin) induces corticosterone-dependent apoptosis in thymus, Peyer's patches, and bone marrow in mice. The purpose of this study was to test the hypothesis that interleukin-1beta (IL-1beta) plays a central role in corticosterone induction and subsequent leukocyte apoptosis in this model. Coexposure to LPS (0.1 mg/kg, ip) plus DON (12.5 mg/kg, po) was found to significantly upregulate splenic IL-1beta mRNA and IL-1beta protein expression in B6C3F1 mice, as compared to treatments with vehicle or either of the toxins alone. B6.129S7-IL1r1tm1Imx mice, which are functionally deficient for the IL-1 receptor 1, produced significantly less corticosterone upon coexposure to LPS plus DON than did corresponding wild-type (WT) C57BL/6J mice. Consistent with these findings, IL-1 receptor 1-deficient mice were recalcitrant to apoptosis induction in leukocytes as determined by assessment of DNA fragmentation assay and flow cytometry. Furthermore, intraperitoneal injection of IL-1 receptor antagonist (100 microgram/mouse, twice at 3 h intervals) in B6C3F1 mice significantly inhibited LPS plus DON-induced increases in plasma corticosterone, as well as apoptosis in thymus, Peyer's patches, and bone marrow. To confirm IL-1beta's capacity to induce apoptosis, B6C3F1 mice were injected with the cytokine (500 ng/mouse, ip) three times at 2 h intervals, and then corticosterone and apoptosis were monitored. Plasma corticosterone levels and thymus and Peyer's patch apoptosis in IL-1beta-injected mice were significantly higher at 12 h than in control mice. Plasma adrenocorticotropic hormone (ACTH) levels in LPS plus DON-treated B6C3F1 mice did not correlate with the induction of plasma corticosterone or leukocyte apoptosis. Taken together, the results indicate that IL-1beta is an important mediator of LPS plus DON-induced corticosterone and subsequent leukocyte apoptosis and, furthermore, this cytokine possibly acts through an ACTH-independent mechanism.

Adrenocorticotropic Hormone↗

PubMed2003

Role of double-stranded RNA-activated protein kinase R (PKR) in deoxynivalenol-induced ribotoxic stress response.

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 JNK>p38>ERK. 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 JNK>p38>ERK 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.

Animals↗

PubMed2003

Up-regulation of macrophage inflammatory protein-2 and complement 3A receptor by the trichothecenes deoxynivalenol and satratoxin G.

The trichothecenes are a group of mycotoxins that target leukocytes and have a wide range of immunomodulatory effects. Differential display analysis was applied to assess the effects of the trichothecenes deoxynivalenol (vomitoxin, DON) and satratoxin G (SG), on mRNA in the RAW 264.7 macrophage cell line. Cells were incubated with DON (1 microg/ml) or SG (5 ng/ml) for 2 h and total RNA then subjected to RT-PCR with a set of oligo(dT) primers. Resultant cDNA was amplified using an oligo (dT) downstream primer and an arbitrary decanucleotide upstream primer to make 35S-labeled PCR products. After separation of the products in denaturing polyacrylamide gel, 23 differentially expressed cDNA fragments were isolated and sequenced. Two of these were identified as known genes, namely, macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant involved in tissue injury and inflammation, and complement 3a receptor (C3aR), a proinflammatory mediator. Both MIP-2 and C3aR mRNAs were up-regulated by DON while only MIP-2 mRNA was induced by SG. Using commercially available antibodies, MIP-2 protein was also found to be induced by both DON and SG in RAW 264.7 cell cultures. When mice were treated with DON (12.5 mg/kg), splenic MIP-2 mRNA and serum MIP-2 levels were increased. MIP-2 mRNA and serum MIP-2 levels were synergistically increased when mice were co-treated with DON and LPS. Up-regulation of MIP-2 and C3aR are consistent with previous reports of trichothecene-induced inflammatory gene up-regulation and suggest that the specific genes affected may depend on trichothecene structures.

Animals↗

PubMed2003

Deoxynivalenol-induced IgA production and IgA nephropathy-aberrant mucosal immune response with systemic repercussions.

Dietary exposure to the common foodborne mycotoxin deoxynivalenol (DON) selectively upregulates serum immunoglobulin A (IgA) in the mouse, most of which is polymeric, thus suggesting that the mucosal immune system is a primary target. When ingested, DON has no adjuvant or antigen properties but, rather, induces polyclonal IgA synthesis and serum elevation in an isotype-specific fashion. Resultant hyperelevated IgA is polyspecific, autoreactive and is likely to be involved in immune complex formation as well as kidney mesangial deposition. These latter effects mimic IgA nephropathy, the most common human glomerulonephritis. At the cellular level, DON upregulates production of T helper cytokines and enhances T cell help for IgA secretion. Analogous effects are observed in the macrophage with IL-6 being of particular importance based on ex vivo reconstitution and antibody ablation studies as well as experiments with IL-6 deficient mice. Upregulation of cytokines by DON involves both increased transcriptional activation and mRNA stability which are mediated by activation of mitogen-activated protein kinases. Interestingly, dietary omega-3 fatty acids can downregulate these processes and ameliorate DON-induced IgA nephropathy. From the perspective of gut mucosal immunotoxicology, these studies demonstrate that the capacity of a chemical to affect mucosal immune response can have systemic repercussions and, further, that these effects can be modulated by an appropriate nutritional intervention.

Animals↗

PubMed2003

Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen.

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.

Active Transport, Cell Nucleus↗

PubMed2003

Differential induction of glucocorticoid-dependent apoptosis in murine lymphoid subpopulations in vivo following coexposure to lipopolysaccharide and vomitoxin (deoxynivalenol).

Lipopolysaccharide (LPS) and vomitoxin (VT) synergistically induce glucocorticoid- mediated apoptotic cell death in lymphoid tissues of the mouse. Based on the known effects of glucocorticoids, it was hypothesized that the combined exposure to LPS and VT targets immature lymphocyte populations. To test this hypothesis, we quantified the effects of VT and LPS on apoptosis induction in T lymphocyte subsets in thymus and B lymphocyte subsets in Peyer's patches and bone marrow. Flow cytometry revealed that a single dose of LPS (0.1 mg/kg body wt ip) together with VT (12.5 mg/kg body wt po) promoted apoptosis of immature (CD4(-)CD8(-), CD4(+)CD8(+)) and mature (CD4(-)CD8(+)) thymocytes at 12 h with a subsequent reduction of these populations being detectable at 24 h. RU 486, a glucocorticoid receptor antagonist, significantly abrogated apoptosis in CD4(-)CD8(-), CD4(+)CD8(+), and CD4(-)CD8(+) subsets and also prevented loss in cell numbers. In Peyer's patches, mature-B lymphocytes (B220(+)IgM(-)IgD(+)) underwent apoptosis and, in bone marrow, pro/pre-B lymphocytes (B220(+)IgM(-)IgD(-)) and mature-B lymphocytes (B220(+)IgM(-)IgD(+)) underwent apoptosis at 12 h after toxin co- exposure. RU 486 blocked LPS + VT-induced apoptosis of the aforementioned subsets in Peyer patches and bone marrow at 12 h. Taken together, these data suggest that LPS can interact with VT in mice to induce the glucocorticoid-driven apoptotic loss of immature thymocytes and cytotoxic T lymphocytes in thymus, mature-B lymphocytes in Peyer's patch, and pro/pre-B lymphocytes and mature-B lymphocytes in bone marrow in mice.

Animals↗

PubMed2003

Cyclooxygenase-2 mediates interleukin-6 upregulation by vomitoxin (deoxynivalenol) in vitro and in vivo.

Interleukin-6 (IL-6) is a central mediator of immunotoxicity that is associated with exposure to the trichothecene vomitoxin (VT). The purpose of this investigation was to test the hypothesis that the inducible cyclooxygenase-2 (COX-2) and its metabolites contribute to VT-induced IL-6 upregulation. VT at 100 to 250 ng/ml readily induced COX-2 protein expression in the RAW 264.7 murine macrophage cell line. Superinduction of lipopolysaccharide (LPS)-mediated IL-6 production by VT in these cells was significantly reduced by the COX inhibitors indomethacin and NS-398, whereas the inhibitors did not affect direct induction of IL-6 by LPS alone. Mice that had been gavaged orally with 5 and 25 mg/kg VT exhibited elevated COX-2 mRNA expression in Peyer's patches and spleen with peak induction occurring 2 h after VT exposure. IL-6 mRNA was also induced by VT in vivo, however, peak expression occurred from 2 to 4 h after toxin exposure, suggesting that maximal COX-2 gene upregulation preceded or was concurrent with that for IL-6. Also consistent with a putative contributory role for COX-2 was the finding that both induction of splenic IL-6 mRNA and serum IL-6 by VT were significantly reduced by pretreating mice with the COX inhibitors indomethacin or NS-398. Finally, COX-2 knockout mice showed significantly reduced splenic IL-6 mRNA and serum IL-6 responses to oral VT exposure compared to their parental wild type. Taken together, these in vitro and in vivo data suggest that VT-induced COX-2 gene expression and resultant COX-2 metabolites contributed, in part, to subsequent upregulation of IL-6 gene expression, which has been previously shown to be a hallmark of VT-mediated immunotoxicity.

Animals↗

PubMed2003

An improved method for the purification of the trichothecene deoxynivalenol (vomitoxin) from Fusarium graminearum culture.

It was hypothesized that a simplified and efficient strategy could be developed for large-scale production and purification of the mycotoxin deoxynivalenol from Fusarium graminearum rice cultures for toxicological studies. F. graminearum R6576 was cultured on rice and extracted with methanol, and the extract was concentrated and subjected to silica gel low-pressure liquid chromatography (LPLC) under a hexane-acetone gradient system. Deoxynivalenol isolation was monitored by thin-layer chromatography, and fractions containing deoxynivalenol were pooled, concentrated, and applied to a second LPLC column under the same conditions. An enriched deoxynivalenol fraction was obtained, which yielded a crystalline material. Repeated crystallization yielded spectroscopically pure deoxynivalenol. The identity of this compound was confirmed by HPLC comparison to an authentic deoxynivalenol standard, FABMS analysis, and comparison of the (1)H and (13)C NMR spectra with published data. This simplified purification scheme eliminated many laborious steps and equipment previously required to obtain gram quantities of crystalline deoxynivalenol for biological testing in animal models.

Chromatography, High Pressure Liquid↗

PubMed2003

Deoxynivalenol-induced mitogen-activated protein kinase phosphorylation and IL-6 expression in mice suppressed by fish oil.

The trichothecene mycotoxin deoxynivalenol (DON) induces IgA hyperelevation and mesangial IgA deposition in mice that mimics the early stages of human IgA nephropathy (IgAN). Among potential mediators of this disease, interleukin-6 (IL-6) is likely to play a particularly critical role in IgA elevation and disease exacerbation. Based on previous findings that dietary fish oil (FO) suppresses DON-induced IgAN, we hypothesized that FO inhibits the induction of IL-6 expression by this mycotoxin in vivo and in vitro. Mice were fed modified AIN 93G diet amended with 7% corn oil (CO) or with 1% corn oil plus 6% menhaden fish oil (FO) for up to 8 weeks and then exposed acutely to DON by oral gavage. DON-induced plasma IL-6 and splenic mRNA elevation in FO-fed mice were significantly suppressed after 8 weeks when compared to the CO-fed group. The effects of FO on phosphorylation of mitogen-activated protein kinases (MAPKs), critical upstream transducers of IL-6 up-regulation, were also assessed. DON-induced phosphorylation of extracellular signal regulated protein kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2) was significantly suppressed in spleens of mice fed with FO, whereas p38 was not. Splenic COX-2 mRNA expression, which has been previously shown to enhance DON-induced IL-6, was also significantly decreased by FO, whereas plasma levels of the COX-2 metabolite, prostaglandin E2, were not affected. To confirm in vivo findings, the effects of pretreatment with the two primary n-3 PUFAs in FO, eicosapentaenoic acid (20:5[n-3]; EPA) and docosahexaenoic acid, (22:6[n-3]; DHA), on DON-induced IL-6 expression were assessed in LPS-treated RAW 264.7 macrophage cells. Consistent with the in vivo findings, both EPA and DHA significantly suppressed IL-6 superinduction by DON, as well as impaired DON-induced ERK1/2 and JNK1/2 phosphorylation. In contrast, the n-6 PUFA arachidonic acid (20:4[n-3]) had markedly less effects on these MAPKs. Taken together, the capacity of FO and its component n-3 PUFAs to suppress IL-6 expression as well as ERK 1/2 and JNK 1/2 activation might explain, in part, the reported suppressive effects of these lipids on DON-induced IgA nephropathy.

Animals↗

PubMed2003

Modulation of lipopolysaccharide-induced proinflammatory cytokine production in vitro and in vivo by the herbal constituents apigenin (chamomile), ginsenoside Rb(1) (ginseng) and parthenolide (feverfew).

Dietary supplements are not subject to the same pre-market approval as conventional drugs, thus the true efficacy and, in cases, safety of these products is not known. The objective of this study was to evaluate the potential anti-inflammatory properties of three herbal constituents, apigenin (chamomile), ginsenoside Rb(1) (ginseng) and parthenolide (feverfew) on lipopolysaccaharide (LPS)-induced proinflammatory cytokine production, and to determine if effects in cell culture could predict results in an intact animal model. Murine macrophage cells and mice were treated with the stimulant LPS and herbal constituents, and resultant culture supernatant and serum, respectively, were evaluated for interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha by ELISA. All three constituents inhibited LPS-induced IL-6 and/or TNF-alpha production in culture. Inhibition of these two cytokines was observed in mice, but did not display the same patterns of inhibition as cell culture data. The results suggest that all three constituents possessed anti-inflammatory properties, but that cell culture data can only be used to approximate potential effects in animals, and must be confirmed using appropriate animal models.

Animals↗

PubMed2003

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

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.

Administration, Oral↗

PubMed2002

Effects of vomitoxin (deoxynivalenol) on the binding of transcription factors AP-1, NF-kappaB, and NF-IL6 in raw 264.7 macrophage cells.

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.

Animals↗