Effects of deoxynivalenol consumption on body weight and adiposity in the diet-induced obese mouse.

The potential for the obese state to alter sensitivity to toxic chemicals is poorly understood. In this study, dose-response effects of the trichothecene deoxynivalenol (DON), a common food-borne mycotoxin, were determined on body weight of diet-induced obese mice. In study 1, the effects of feeding adult female B6C3F1 mice a high-fat diet (HFD; 60% kcal from fat) containing 0, 2, 5, or 10 ppm DON for 10 wk on body weight and adiposity were compared. Mice consuming 5 or 10 ppm DON exhibited a 15 and 24% decrease in weight gain and a 50 and 83% reduction in periuterine fat, respectively. In study 2, mice were fed HFD for 8 wk to induce obesity and the effects of consuming HFD + 0, 2, 5, or 10 ppm DON for 8 wk were then determined. Mice fed 5 or 10 ppm DON exhibited a 16 and 23% weight reduction and a 0 and 40% periuterine fat reduction, respectively. In a follow-up experiment, food consumption was measured prior to and after the transition from HFD to HFD + 10 ppm DON. Exposure to DON was found to lower HFD consumption within 1 d, with significant weight loss in DON-fed mice evident after 6 d. In both studies 1 and 2, consumption of 5 or 10 ppm DON diminished circulating levels of insulin-like growth factor acid-labile subunit. Taken together, DON consumption lowered weight gain and produced weight loss in diet-induced obese mice at higher thresholds than that observed previously in normal B6C3F1 mice.

Mechanisms of deoxynivalenol-induced gene expression and apoptosis.

Fusarium infection of agricultural staples such as wheat, barley and corn with concurrent production of deoxynivalenol (DON) and other trichothecene mycotoxins is an increasingly common problem worldwide. In addition to its emetic effects, chronic dietary exposure to DON causes impaired weight gain, anorexia, decreased nutritional efficiency and immune dysregulation in experimental animals. Trichothecenes are both immunostimulatory or immunosuppressive depending on dose, frequency and duration of exposure as well as type of immune function assay. Monocytes, macrophages, as well as T- and B-lymphocytes of the immune system can be cellular targets of DON and other trichothecenes. In vitro exposure to low trichothecene concentrations upregulates expression both transcriptionally and post-transcriptionally of cytokines, chemokines and inflammatory genes with concurrent immune stimulation, whereas exposure to high concentrations 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 signalling events related to immune response and apoptosis. Using cloned macrophages, two critical upstream transducers of DON-induced MAPK activation have been identified. One transducer is double-stranded RNA (dsRNA)-activated protein kinase (PKR), a widely expressed serine/threonine protein kinase that can be activated by dsRNA, interferon and other agents. The other transducer is haematopoetic cell kinase (Hck), a non-receptor associated Src oncogene family kinase. Pharmacological inhibitors and gene suppression studies have revealed that Hck and PKR contribute to DON-induced gene expression and apoptosis. PKR, Hck and other kinases bind to the ribosome and are activated following DON interaction. Future studies will focus on the sequence of molecular events at the ribosome level that drive selective activation of these upstream kinases.

Deoxynivalenol and satratoxin G potentiate proinflammatory cytokine and macrophage inhibitory protein 2 induction by Listeria and Salmonella in the macrophage.

Health risks from microbial pathogens and toxins encountered in food and the environment continue to be of worldwide concern. The purpose of this research was to test the hypothesis that trichothecene mycotoxins amplify inflammatory responses to foodborne bacterial pathogens. We assessed the capacity of deoxynivalenol (DON) and satratoxin G (SG) to potentiate chemokine and proinflammatory cytokine production in RAW 264.7 murine macrophages induced by Listeria monocytogenes and Salmonella Typhimurium. When macrophage cultures were incubated with killed irradiated suspensions of the pathogens for 24 h, the minimum Listeria concentrations for induction of macrophage inhibitory protein 2 (MIP-2), interleukin-1beta (IL-beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were 0.01, 0.01, 1.0, and 1.0 microg/ml (P < 0.05) and the minimum Salmonella concentrations were 0.01, 0.01, 0.1, and 0.1 microg/ml, respectively (P < 0.05). Induction of all four mediators by both pathogens was potentiated by DON (at 100 and 250 ng/ml); observed responses were significantly higher than predicted additive responses (P < 0.05). SG (at 2 and 5 ng/ml) also significantly amplified induction of IL-1beta and TNF-alpha (P < 0.05) by both Listeria and Salmonella. These results indicate that DON encountered in Fusarium-contaminated food and SG from Stachybotrys-contaminated indoor environments could magnify innate inflammatory responses to foodborne bacterial pathogens.

Effects of tumor necrosis factor type 1 and 2 receptor deficiencies on anorexia, growth and IgA dysregulation in mice exposed to the trichothecene vomitoxin.

Dietary exposure of mice to vomitoxin (VT), a trichothecene mycotoxin, causes anorexia and impaired growth as well as inducing elevated serum IgA and kidney mesangial IgA deposition in a manner analogous to human IgA nephropathy. Based on the observations that TNF-alpha is induced by in vitro and in vivo VT exposure, it was hypothesized that this cytokine plays a role in the nutritional and immunological effects of this toxin. To test this hypothesis, the effects of dietary VT on feed intake, weight gain, serum IgA levels and kidney mesangial IgA deposition in mice homozygous for targeted disruption of the two known TNF-alpha cell surface receptors, TNFR1(p55) or TNFR2(p75), were compared to effects in corresponding C57BL/6J wild-type (WT) mice with normal receptor function. The capacity of VT to cause feed refusal or impair weight gain over a 12-week feeding period was not impaired in TNFR1 knockout (KO) or TNFR2-KO as compared to WT mice. Both WT and TNFR-KO mice fed VT exhibited reduced (P<0.05) feed conversion efficiency, but surprisingly, feed conversion efficiency was significantly higher (P<0.05) in TNFR1-KO and TNFR2-KO fed either control or VT diets than in corresponding WT mice. By week 12, serum IgA concentrations in all three mouse groups fed VT were significantly higher than those for corresponding mice fed control diets (P<0.05). Serum IgA levels in the VT-fed TNFR1-KO group were significantly less (P<0.05) than those for the VT-fed WT mice at 4, 8 and 12 weeks, whereas no differences in this parameter were found between the TNFR2-KO and WT groups. Serum IgA immune complex concentrations were measured at wk 12 and found to follow an identical pattern to IgA. Kidneys taken from VT-fed TNFR2-KO and WT mice after 12 weeks had significantly increased mesangial IgA deposition as compared to controls. While slight increases in mesangial IgA were also observed in VT-fed TNFR1-KO mice, these levels were significantly less (P<0.05) than that found in VT-fed TNFR2-KO and WT mice. Taken together, the data suggest that while VT-mediated anorexic and growth effects were largely independent of TNF-alpha, VT-induced dysregulation of IgA production was dependent, in part, on the interaction of TNF-alpha with TNFR1.

Elisa to quantify hexanal-protein adducts in a meat model system.

Monoclonal antibodies (MAb) were produced to hexanal-bovine serum albumin conjugates. An indirect competitive ELISA was developed with a detection range of 1-50 ng of hexanal/mL. Hexanal conjugated to three different proteins was recognized, whereas free hexanal and the native proteins were not detected. The antibody cross-reacted with pentanal, heptanal, and 2-trans-hexenal conjugated to chicken serum albumin (CSA) with cross-reactivities of 37.9, 76.6, and 45.0%, respectively. There was no cross-reactivity with propanal, butanal, octanal, and nonanal conjugated to CSA. The hexanal content of a meat model system was determined using MAb and polyclonal antibody-based ELISAs and compared with analysis by a dynamic headspace gas chromatographic (HS-GC) method and a thiobarbituric acid reactive substances (TBARS) assay. Both ELISAs showed strong correlations with the HS-GC and TBARS methods. ELISAs may be a fast and simple alternative to GC for monitoring lipid oxidation in meat.

Superinduction of TNF-alpha and IL-6 in macrophages by vomitoxin (deoxynivalenol) modulated by mRNA stabilization.

Vomitoxin (VT or deoxynivalenol), a trichothecene, superinduces proinflammatory cytokine gene expression in vitro and in vivo. To better understand the underlying molecular mechanisms for this observation, post-transcriptional effects of VT on TNF-alpha and IL-6 gene expression were studied in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. VT was found to enhance both TNF-alpha and IL-6 protein secretion in the presence of LPS. Upon addition of the transcriptional inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), secretion of both cytokines was inhibited. Using Northern analysis, the mRNA stabilities of TNF-alpha and IL-6 were studied in DRB-treated cells exposed to VT and LPS in both asynchronous and delayed synchronous modes. In the asynchronous model, cells were first incubated with LPS for 2 h, and then the medium was removed and replaced with medium containing DRB and VT. In the delayed synchronous model, cells were pretreated with LPS for 2 h and then DRB and VT were added to the culture. TNF-alpha and IL-6 mRNA were rapidly stabilized by VT (100 and 250 ng/ml) in both asynchronous and delayed synchronous models. In the asynchronous model, TNF-alpha mRNA half-life was 25 min but this was extended in the presence of 100 and 250 ng/ml of VT to >3 h. VT also extended half-lives of IL-6 mRNA from 60 min to >3 h. In the delayed synchronous model, the half-lives for TNF-alpha and IL-6 mRNA of 1.3 and 1.5 h, respectively, were extended to >3 h upon incubation with 100 and 250 ng/ml VT. These results suggest that post-transcriptional control via enhancement of mRNA stability is likely to contribute to proinflammatory cytokine superinduction in macrophages by VT and other trichothecenes.

Effects of ingestion of yogurts containing Bifidobacterium and Lactobacillus acidophilus on spleen and Peyer's patch lymphocyte populations in the mouse.

Certain probiotic lactic acid bacteria have been reported to improve immune system function. Here, the effects of ingesting yogurts on lymphocyte populations in the spleens and Peyer's patches were determined in mice. Three probiotic-supplemented yogurts containing Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium, and Lactobacillus acidophilus and one conventional yogurt containing only S. thermophilus and L. bulgaricus were prepared from commercial starter cultures and used in the study. B6C3F1 female mice were fed the four different types of yogurts mixed with an AIN-93G diet in a 50:50 (wt/wt) ratio. Nonfat dry milk mixed at a 50:50 (wt/wt) ratio with AIN-93G diet was used as the control. After a 14-day feeding period, spleen and Peyer's patches were removed and lymphocytes subjected to phenotype analysis by flow cytometry. Ingestion of the four yogurts had no effect on percentages of CD8+ (cytotoxic T cells), B220+ (B cells), IgA+, or IgM+ cells in spleen or Peyer's patches. The percentage of CD4+ (T helper) cells was significantly increased in the spleens from one group of mice fed a yogurt containing Bifidobacterium and L. acidophilus, and a similar trend was found in the remaining two probiotic-supplemented yogurts. Effects on CD4+ populations were not observed in spleens of mice fed conventional yogurt or in the Peyer's patches of any of the four yogurt groups. In total, the results suggested that ingestion of conventional or probiotic-supplemented yogurts for 2 weeks had very little effect on lymphocyte distribution in the systemic or mucosal immune compartments.

Differential upregulation of TNF-alpha, IL-6, and IL-8 production by deoxynivalenol (vomitoxin) and other 8-ketotrichothecenes in a human macrophage model.

The effects of deoxynivalenol (DON or vomitoxin) and four closely related 8-ketotrichothecenes on proinflammatory cytokine and chemokine production were evaluated in a clonal human macrophage model. U-937 cells, which represent a human monocytelike histocytic lymphoma, were differentiated into macrophages by preincubation with phorbol 12-myristate 13-acetate (PMA). Differentiated macrophages were incubated with DON in the absence or presence of lipopolysaccharide (LPS), and supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and for the chemokine interleukin-8 (IL-8). In the absence of LPS, DON at 500 or 1,000 ng/ml upregulated TNF-alpha production as early as 3 h and up to 6 h, whereas 100 to 1,000 ng/ml of DON significantly increased production of IL-6 from 3 to 24 h and IL-8 from 6 to 48 h. In cells costimulated with 0.2 microg/ml LPS, DON at 500 or 1000 ng/ml markedly superinduced TNF-alpha and IL-8 production. Although 100 ng/ml of DON also potentiated LPS-induced IL-6 production, 500 or 1,000 ng/ ml of the toxin suppressed the LPS-induced IL-6 response. Four other 8-ketotrichothecenes, fusarenon X, nivalenol, 3-acetyl DON, and 15-acetyl DON, were also capable of upregulating or suppressing TNF-alpha, IL-6, and IL-8 production at concentrations similar to that of DON. In total, the results suggest that DON and other 8-ketotrichothecenes have the potential to both directly induce and superinduce proinflammatory cytokine and chemokine expression in human macrophages, even at toxin concentrations that are cytotoxic.

Augmentation of mercury-induced nephrotoxicity by endotoxin in the mouse.

Endotoxin (lipopolysaccharide; LPS) and mercury are compounds of food safety concern. Endotoxin is a product of cell walls of gram negative bacteria. Humans are constantly exposed to LPS through infection plus translocation into circulation from the gastrointestinal tract. Food is the major source of mercury in humans. The toxic interaction between LPS and mercury has not been well investigated. In a previous study, we demonstrated that LPS potentiated mercury-induced nephrotoxicity in the rat. Whether this observation was species specific was not clear. In this study we tested the hypothesis that LPS enhances mercuric chloride (HgCl(2))-induced nephrotoxicity in mice. In a 2x2 factorial design, mice received either Escherichia coli 0128:B12 endotoxin (2.0 mg/kg body weight) or 200 microliter of 0.9% sodium chloride (saline), and this was followed 4 h later by either mercury (1.75 mg mercuric chloride per kg body weight) or 200 microliter of saline. Mice were monitored for 48 h. Monitored end-points included body and renal weights, urine volume, renal histology and ultrastructural pathology, serum urea nitrogen and creatinine, selected serum and urine cytokines, and renal mercury concentrations. Endotoxin by itself was not nephrotoxic at the dose used in this study. Overall, mice given LPS plus mercury were the most severely affected. Mice given LPS and mercury also had significantly greater renal mercury concentration than those given mercury alone (P

Interleukin-6-deficient mice refractory to IgA dysregulation but not anorexia induction by vomitoxin (deoxynivalenol) ingestion.

Dietary exposure to the trichothecene vomitoxin (VT) causes feed refusal and elevates IgA production in the mouse. Based on the observations that IL-6 can cause anorexia and promote IgA production and that gene expression of this cytokine is increased in vivo and ex vivo on VT exposure, we hypothesized that IL-6 is an essential cytokine in VT-induced feed refusal and IgA dysregulation. To test this hypothesis, the effects of dietary VT on feed intake, weight gain, serum IgA levels and kidney mesangial IgA deposition in an IL-6-"knockout" mouse (B6129-IL6(tmi Kopf)) were compared to those in both a corresponding "wildtype" (B6129F2) and a previously characterized "sentinel" strain (B6C3F1) that possess the intact gene for this cytokine. IL-6 deficiency did not alter the capacity of VT to cause feed refusal or impair weight gain. VT-fed B6129F2 and B6C3F1 mice had significantly higher serum IgA concentrations than did their corresponding controls fed clean diet, whereas significant differences were not observed between IL-6 KO mice fed VT or control diets. Kidneys taken from VT-fed wild-type and sentinel mice had significantly increased mesangial IgA deposition as compared to controls. While slight increases in mesangial IgA were observed in VT-fed IL-6 KO mice, mean fluorescence intensities were significantly less than that found in the corresponding wild-type and sentinel strains. IL-6 KO mice appeared to be less prone to the development of microscopic haematuria following VT exposure than were the corresponding wild-type and sentinel strains. In total, the results suggested that IL-6-deficient mice were refractory to VT-induced dysregulation of IgA production and development of IgA nephropathy, whereas chronic VT-mediated nutritional effects related to feed intake and weight gain were unaffected.

Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants.

The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S(2). After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.

Immunomodulation by fungal toxins.

The availability of immunotoxicity data for fungal toxins varies considerably for different toxins. The following is a comprehensive review of the most recent literature on the immunotoxicity of aflatoxins, fumonisins, gliotoxin, ochratoxins, patulin, and trichothecenes. Aflatoxin is an immunomodulating agent that acts primarily on cell-mediated immunity and phagocytic cell function. In addition to further characterization of aflatoxin-induced immunotoxicity in various species, some recent studies have focused on ameliorating the effects of aflatoxin by supplementing or amending the diet. The immunomodulatory effects of ochratoxins have also been considered for many years. Notably, recent studies have examined immune function in the offspring of rats and mice exposed to ochratoxin pre- and perinatally. Fumonisin toxicity has been characterized relatively recently in comparison to aflatoxin and ochratoxin, and fumonisin-induced immunotoxicity is an area of active research. As these studies progress, they may also clarify the role of sphingolipid metabolism in immune function. The most recent study of patulin immunotoxicity in mice indicates that exposure to levels found in foods and feeds would not likely result in immunotoxicity. Exposure to gliotoxin would most likely be by infection with gliotoxin-producing fungi. Although the toxin is immunosuppressive in vitro, the link between immunosuppression and the presence of gliotoxin in infected tissues in vivo has yet to be made. The trichothecenes can both suppress and stimulate immune function. By comparison, more information is available on the molecular events associated with trichothecene-induced immunomodulation than for any other fungal toxins. The molecular basis of immune function modulation by fungal toxins remains a frontier for future research.

Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation.

The satratoxins are members of the trichothecene mycotoxin family that are produced by the fungus Stachybotrys and that have been etiologically associated with building-related health problems. The purpose of this study was to relate cytotoxic and apoptotic capacities of satratoxins and other trichothecenes to the activation of three groups of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated protein kinase (ERK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)). Two myeloid models, RAW 264.7 murine macrophage and U937 human leukemic cells were used. Upon evaluating representative trichothecenes in the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) cleavage assay, cytotoxicity was evident according to the following rank order: satratoxin G, roridin A, and verrucarin A > T-2 toxin, satratoxin F, H > nivalenol, and vomitoxin. Comparable results were found when measuring trichothecene-mediated apoptosis using DNA fragmentation and fluorescence microscopy assays, thus suggesting that cytotoxicity was mediated through an apoptotic process. Assessment of MAPK activation using Western blot analysis revealed that trichothecenes activated not only SAPK/JNK and p38 MAPK but also ERK. Activation of MAPKs by satratoxins and other trichothecenes correlated with and preceded apoptosis. The concentration of satratoxin G sufficient for protein synthesis inhibition correlated with that required for apoptosis and activation of all three MAPKs. Cycloheximide had similar effects to trichothecenes, suggesting that ribosome binding or protein synthesis inhibition may play roles in MAPK activation and apoptosis induction. Apoptosis induction by satratoxin G and vomitoxin was markedly enhanced when ERK activation was selectively inhibited by ERK-specific inhibitor PD98059, thus indicating a negative role for ERK. Inhibition of p38 MAPK activity with the p38-specific inhibitor SB203580 had no effect on apoptosis induction by the highly toxic satratoxin G. However, SB203580 moderately inhibited apoptosis induction by the less toxic trichothecene vomitoxin, thus implying a partial role of p38 MAPK in trichothecene-induced apoptosis. The results suggest that the satratoxins are among the most potent trichothecenes and that MAPKs may play integral roles in the diverse toxic manifestations of these mycotoxins.

Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs.

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<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Viability of bifidobacteria in commercial dairy products during refrigerated storage.

Commercial milk and two brands of yogurt containing bifidobacteria were obtained from retail outlets. All products were evaluated for viability of bifidobacteria and lactic acid bacteria during refrigerated storage at 4 degrees C. Milk was evaluated at 9, 6, and 3 days prior and past its expiration date. The yogurts were evaluated at 3, 2, and 1 week prior and past their expiration. Viability of bifidobacteria and lactic acid bacteria in milk and yogurt remained above 10(6) CFU/ml or g until the expiration date of the respective products. This microbial concentration is the recommended minimum dose to receive the health benefits of these organisms.

Modulation of transcription factor AP-1 activity in murine EL-4 thymoma cells by vomitoxin (deoxynivalenol).

Trichothecene mycotoxins have been reported to suppress or superinduce cytokine mRNA expression by leukocytes both in vitro and in vivo. Modulation of transcription factor activities may be critical for these observations. Here, the effect of trichothecene vomitoxin (VT, deoxynivalenol) on activator protein-1 (AP-1) activity was determined in the murine EL-4 thymoma. Electrophoretic mobility shift assay (EMSA) revealed that VT modulated AP-1 binding activity in a concentration- and time-dependent manner when using a synchronous model in which VT was added concurrently with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) to EL-4 cells. Induction of AP-1 binding activity by PMA/ION was suppressed in the presence of VT for a short period (1 to 12 h), but was enhanced upon prolonged VT exposure (48 to 72 h). VT also enhanced AP-1 binding activity when added to the cell culture 12 h after PMA/ION activation (delayed synchronous model). Using specific antibodies against AP-1 complex proteins, it was demonstrated by gel supershift assay that VT preferentially affected phosphorylated c-Jun, Jun B, c-Fos, and Fra-2 binding activities, whereas it did not alter Jun D and Fra-1 binding. A transient transfection assay demonstrated that these increased binding activities are associated with enhanced AP-1 transactivation potential. Elevation of AP-1 activity may contribute to cytokine dysregulation and immunotoxic effects associated with exposure to trichothecene mycotoxins such as VT.

Down-regulation of the endoplasmic reticulum chaperone GRP78/BiP by vomitoxin (Deoxynivalenol).

The mechanisms by which trichothecene mycotoxins cause immunological effects in leukocytes such as cytokine up-regulation, aberrant IgA production, or apoptotic cell death are not fully understood. In the present study, mRNA differential display analysis was used to evaluate changes in gene expression induced by the trichothecene vomitoxin (VT or deoxynivalenol) in a T-cell model, the murine EL-4 thymoma, that was stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION). Ten differentially expressed fragments of cDNA were isolated and sequenced and three of these were identified as the known genes GRP78/BiP, P58(IPK), and RAD17. Most notably, expression of GRP78/BiP (a 78-kDa glucose-regulated protein), a stress-response gene induced by agents or conditions that adversely affect endoplasmic reticulum (ER) function, was found to decrease in VT-exposed cells. Competitive RT-PCR analysis revealed that 250 ng/ml VT decreased GRP78/BiP mRNA expression in both unstimulated and PMA/ION-stimulated EL-4 cells at 6 and 24 h after VT treatment. Western blotting confirmed that VT (50 to 1000 ng/ml) also significantly diminished GRP/BiP protein levels in a dose-response manner in PMA/ION-stimulated cells. GRP78/BiP has been shown to play a role in regulation of protein folding and secretion, and to protect cells from apoptosis. When PMA/ION-stimulated cells were incubated with 50 to 1000 ng/ml VT for 24 h, 200-bp DNA laddering, a hallmark of apoptosis, increased in a dose-dependent manner. In addition to GRP78, mRNA expression of the cochaperone P58(IPK), which is the 58-kDa cellular inhibitor of the double-stranded RNA-regulated protein kinase (PKR), was also shown to be suppressed by VT-treatment. GRP78 and P58(IPK) are critical for maintenance of cell homeostasis and prevention of apoptosis. The down-regulation of these molecular chaperones by VT represent a novel observation and has the potential to impact immune function at multiple levels.

Proinflammatory cytokine and nitric oxide induction in murine macrophages by cell wall and cytoplasmic extracts of lactic acid bacteria.

Cells from a number of bacterial genera have been shown to possess mitogenic and polyclonal activating properties when cultured with cells of the immune system. Based on previously reported health immune-enhancing effects of fermented dairy products, we tested the potentiating effects of representative lactic acid bacteria and their extracts on leukocyte function. Specifically, the effects of in vitro exposure to heat-killed cells of Bifidobacterium, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. gasseri, L. helveticus, L. reuteri, and Streptococcus thermophilus, their cell walls, and their cytoplasmic extracts on proliferation as well as cytokine and nitric oxide (NO) production were examined in the RAW 264.7 macrophage cell line. A similar strategy was applied to murine cultures composed of peritoneal, spleen, and Peyer's patch cells. Both the cell wall and cytoplasmic fractions of lactic acid bacteria were able to stimulate cloned macrophages to produce significant amounts of tumor necrosis factor-alpha, (interleukin) IL-6, and NO. Pronounced enhancement of IL-6 production by peritoneal cells was observed when cultured with those extracts, whereas, effects were not noted in spleen and Peyer's patch cell cultures from mice. Based on the results, it appears that, as a group, the lactic acid bacteria were capable of stimulating macrophages and possibly other immune cells to produce cytokines and NO, and both their cell walls and cytoplasm contributed to these capacities.

Production and specificity of polyclonal antibodies to hexanal-lysine adducts.

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.