High Sensitivity of Aged Mice to Deoxynivalenol (Vomitoxin)-Induced Anorexia Corresponds to Elevated Proinflammatory Cytokine and Satiety Hormone Responses

Deoxynivalenol (DON), a trichothecene mycotoxin that commonly contaminates cereal grains, is a public health concern because of its adverse effects on the gastrointestinal and immune systems. The objective of this study was to compare effects of DON on anorectic responses in aged (22 mos) and adult (3 mos) mice. Aged mice showed increased feed refusal with both acute i.p. (1 mg/kg and 5 mg/kg) and dietary (1, 2.5, 10 ppm) DON exposure in comparison to adult mice. In addition to greater suppression of food intake from dietary DON exposure, aged mice also exhibited greater but transient body weight suppression. When aged mice were acutely exposed to 1 mg/kg bw DON i.p., aged mice displayed elevated DON and DON3GlcA tissue levels and delayed clearance in comparison with adult mice. Acute DON exposure also elicited higher proinflammatory cytokine and satiety hormone responses in the plasma of the aged group compared with the adult group. Increased susceptibility to DON-induced anorexia in aged mice relative to adult mice suggests that advanced life stage could be a critical component in accurate human risk assessments for DON and other trichothecenes.     

Studies of immune function and host resistance in B6C3F1 mice exposed to formaldehyde

A series of immune function and host resistance parameters were examined in female B6C3F1 mice following a 21-day (6 hr/day) inhalation exposure to 15 ppm of formaldehyde (HCHO). Immune parameters examined included delayed hypersensitivity to keyhole limpet hemocyanin, antibody plaque-forming cell response to sheep erythrocytes (T-lymphocyte-dependent antigen) and TNP-Ficoll (T-lymphocyte-independent antigen), lymphoid organ weights and histopathology, routine hematology, bone marrow cellularity and CFU progenitor cell enumeration, lymphocyte subpopulation quantitation by cell surface markers, mitogen-induced lymphocyte blastogenesis, macrophage function parameters, and host resistance to challenge with the bacterium Listeria monocytogenes and transplantable tumor cells. Lymphoid organ weight, bone marrow cellularity, and hematology parameters were unchanged in HCHO exposed mice. Similarly, the percentage of T and B lymphocytes and their proliferative responses to mitogens were not significantly altered. Antibody (IgM) plaque-forming cell response following antigen challenge was unchanged. Macrophage function was normal although some evidence of enhanced H2O2 production associated with elevated bactericidal activity was observed in resident macrophages. Resistance to challenge with the bacteria Listeria monocytogenes was significantly enhanced, while resistance to tumor challenge remained unchanged. No evidence of immunosuppression following short-term exposure to HCHO was observed.     

Murine Anorectic Response to Deoxynivalenol (Vomitoxin) Is Sex-Dependent

Deoxynivalenol (DON, vomitoxin), a common trichothecene mycotoxin found in cereal foods, dysregulates immune function and maintenance of energy balance. The purpose of this study was to determine if sex differences are similarly evident in DON’s anorectic responses in mice. A bioassay for feed refusal, previously developed by our lab, was used to compare acute i.p. exposures of 1 and 5 mg/kg bw DON in C57BL6 mice. Greater anorectic responses were seen in male than female mice. Male mice had higher organ and plasma concentrations of DON upon acute exposure than their female counterparts. A significant increase in IL-6 plasma levels was also observed in males while cholecystokinin response was higher in females. When effects of sex on food intake and body weight changes were compared after subchronic dietary exposure to 1, 2.5, and 10 ppm DON, males were found again to be more sensitive. Demonstration of male predilection to DON-induced changes in food intake and weight gain might an important consideration in future risk assessment of DON and other trichothecenes.     

Characterization of deoxynivalenol-induced anorexia using mouse bioassay

A short-term mouse model was devised to investigate induction of food refusal by the common foodborne trichothecene deoxynivalenol (DON). DON dose-dependently induced anorexia within 2 h of exposure when administered either by intraperitoneal (ip.) injection or by oral gavage. The no observed adverse effect and lowest observed adverse effect levels in this assay were 0.5 and 1 mg/kg bw for ip. exposure and 1 and 2.5 mg/kg bw for oral exposure, respectively. DON’s effects on food intake were transient, lasting up to 3 h at 1 mg/kg bw and up to 6 h at 5 mg/kg bw. Interestingly, a dose-dependent orexigenic response was observed in the 14 h following the initial 2 h food intake measurement. Toxin-treated mice exhibited partial resistance to feed refusal when exposed to DON subsequently after 2 d, but not after 7 d suggesting that this modest tolerance was reversible. The short-term mouse bioassay described here was useful in characterizing DON-induced anorexia and should be applicable to elucidating mechanisms underlying this adverse nutritional effect.     

Immunotoxicity of bis(tri-n-butyltin) oxide in the rat

Previous studies in the rat have shown that bis(tri-n-butyltin) oxide (TBTO), used as a biocide, was immunotoxic at dose levels that did not affect other organs. In order to determine a no-effect level, weanling rats were treated for at least 28 consecutive days with TBTO at 0, 0.5, 2, 5, or 50 mg/kg of diet. Studies on clinical chemistry, hematology, pathology, and immune function, that is, plaque-forming cell (PFC) assay, delayed-type hypersensitivity (DTH) response, and the splenic clearance of Listeria monocytogenes, were performed at the end of treatment. No treatment-related effects were noted on clinical chemistry and hematology parameters and on PFC and DTH response, whereas thymic atrophy and impaired clearance of L. monocytogenes were noted only at a dietary concentration of 50 mg/kg. These results confirm the thymus as a target organ of TBTO immunotoxicity. Under the conditions of these experiments the dietary concentration of 5 mg/kg, equivalent to a dose of 0.5 mg/kg body weight, represents a no observed effect level (NOEL) for immunotoxicity in the Sprague-Dawley rat.     

Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone

Weanling female B6C3F1 mice were fed semi-purified diets containing 0, 0.5, 2.0, 5.0, 10.0 or 25.0 ppm (mg/kg) deoxynivalenol (DON) over 8 wk and were assessed for effects on feed intake, body-weight gain, terminal organ weights, histopathology, haematology and serum immunoglobulin levels. To determine whether DON effects were potentiated by the oestrogen zearalenone (ZEA), a mycotoxin frequently found to occur with DON in cereals, two additional groups of mice were fed diets containing either 10 ppm ZEA or 10 ppm ZEA plus 5 ppm DON. The rate of body-weight gain was significantly reduced (P less than 0.01) for all mice consuming feed containing 2.0 ppm or more of DON, whereas only the mice ingesting the diet containing 25 ppm DON showed a significantly decreased (P less than 0.01) rate of feed consumption. Gross and histopathological evaluation of thymus, spleen, liver, kidney, uterus, small intestine, colon, heart, brain, lungs and bone marrow from control and all mycotoxin-exposed mice revealed that these tissues were normal in appearance and in histological architecture. DON-amended diets did however, cause dose-dependent decreases in the terminal organ weights recorded (thymus, spleen, liver, kidney and brain). In the DON-treated groups, statistically significant dose-dependent decreases in the counts of total circulating white blood cells were associated with an increase in polymorphonuclear neutrophils and a decrease in lymphocytes and monocytes. Dietary DON caused a dose-dependent decrease in serum IgM but, in contrast, a dose-dependent increase in serum IgA. In none of the above instances was 10 ppm ZEA shown to act synergistically or antagonistically with 5 ppm DON. Since dietary DON at levels as low as 2.0 ppm exerted significant effects on the growing B6C3F1 female mouse, future approaches should include studies of the mechanisms by which this mycotoxin affects nutrient utilization and modifies the normal immune response.     

Deoxynivalenol-induced weight loss in the diet-induced obese mouse is reversible and PKR-independent

The trichothecene deoxynivalenol (DON), a potent ribotoxic mycotoxin produced by the cereal blight fungus Fusarium graminearum, commonly contaminates grain-based foods. Oral exposure to DON causes decreased food intake, reduced weight gain and body weight loss in experimental animals – effects that have been linked to dysregulation of hormones responsible for mediating satiety at the central nervous system level. When diet-induced obese (DIO) mice are fed DON, they consume less food, eventually achieving body weights of control diet-fed mice. Here, we extended these findings by characterizing: (1) reversibility of DON-induced body weight loss and anorexia in DIO mice and (2) the role of double-stranded RNA-activated protein kinase (PKR) which has been previously linked to initiation of the ribotoxic stress response. The results demonstrated that DON-induced weight loss was reversible in DIO mice and this effect corresponded to initiation of a robust hyperphagic response. When DIO mice deficient in PKR were exposed to DON, they exhibited weight suppression similar to DIO wild-type fed the toxin, suggesting the toxin's weight effects were not dependent on PKR. Taken together, DON's effects on food consumption and body weight are not permanent and, furthermore, PKR is not an essential signaling molecule for DON's anorectic and weight effects.     

Immunomodulatory effects of individual and combined mycotoxins in the THP-1 cell line

Mycotoxins commonly contaminate food and may pose a risk for disease in humans and animals. As they frequently co-occur, mixed exposures often take place. Monocyte function, including differentiation into active macrophages, is a central part of the immune response. Here we studied effects of naturally co-occurring mycotoxins in grain on monocyte function, and effects of individual and combined exposure on the differentiation process from monocytes into macrophages. The THP-1 cell line was used as a model system. The mycotoxins 2-amino-14,16-dimethyloctadecan-3-ol (AOD), alternariol (AOH), enniatin B (ENNB), deoxynivalenol (DON), sterigmatocystin (ST) and zearalenone (ZEA) differently affected cell viability in THP-1 monocytes, with DON as the most potent. AOH, ZEA and DON inhibited differentiation from monocytes into macrophages. Using this differentiation model, combined exposure of AOH, ZEA and DON were mainly found to be additive. However, the combination AOH + ZEA had somewhat synergistic effect at lower concentrations. Furthermore, alterations in macrophage functionality were found, as single exposure of AOH and ZEA inhibited lipopolysaccharide (LPS) induced TNF-α secretion, while DON increased this response. Overall, the mycotoxins affected monocyte viability and differentiation into macrophages differently. Combined exposures affected the differentiation process mainly additively.     

Deoxynivalenol-induced proinflammatory gene expression: mechanisms and pathological sequelae

The trichothecene mycotoxin deoxynivalenol (DON) is commonly encountered in human cereal foods throughout the world as a result of infestation of grains in the field and in storage by the fungus Fusarium. Significant questions remain regarding the risks posed to humans from acute and chronic DON ingestion, and how to manage these risks without imperiling access to nutritionally important food commodities. Modulation of the innate immune system appears particularly critical to DON's toxic effects. Specifically, DON induces activation of mitogen-activated protein kinases (MAPKs) in macrophages and monocytes, which mediate robust induction of proinflammatory gene expression-effects that can be recapitulated in intact animals. The initiating mechanisms for DON-induced ribotoxic stress response appear to involve the (1) activation of constitutive protein kinases on the damaged ribosome and (2) autophagy of the chaperone GRP78 with consequent activation of the ER stress response. Pathological sequelae resulting from chronic low dose exposure include anorexia, impaired weight gain, growth hormone dysregulation and aberrant IgA production whereas acute high dose exposure evokes gastroenteritis, emesis and a shock-like syndrome. Taken together, the capacity of DON to evoke ribotoxic stress in mononuclear phagocytes contributes significantly to its acute and chronic toxic effects in vivo. It is anticipated that these investigations will enable the identification of robust biomarkers of effect that will be applicable to epidemiological studies of the human health effects of this common mycotoxin.     

Assessment of Human Exposure to Deoxynivalenol,Ochratoxin A,Zearalenone and Their Metabolites Biomarker in Urine Samples Using LC-ESI-qTOF

Human are exposed to a wide range of mycotoxins through dietary food intake, including processed food. Even most of the mycotoxin exposure assessment studies are based on analysis of foodstuffs, and evaluation of dietary intake through food consumption patterns and human biomonitoring methods are rising as a reliable alternative to approach the individual exposures, overcoming the limitations of the indirect dietary assessment. In this study, human urine samples were analyzed, seeking the presence of deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEA), and their metabolites. For this purpose, 40 urine samples from female and male adult residents in the city of Valencia (Spain) were evaluated by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF) after salting-out liquid–liquid extraction. Analytical data showed that 72.5% of analyzed samples were contaminated by at least one mycotoxin at variable levels. The most prevalent mycotoxins were de-epoxy DON (DOM-1) (53%), ZEA (40%), and α-zearalenol (αZOL) (43%), while OTA was only detected in one sample. The mean concentrations in positive samples were DON (9.07 ng/mL), DOM-1 (20.28 ng/mL), ZEA (6.70 ng/mL), ZEA-14 glucoside (ZEA-14-Glc) (12.43 ng/mL), αZOL (27.44 ng/mL), αZOL-14 glucoside (αZOL-14-Glc) (12.84 ng/mL), and OTA (11.73 ng/mL). Finally, probable daily intakes (PDIs) were calculated and compared with the established tolerable daily intakes (TDIs) to estimate the potential risk of exposure to the studied mycotoxins. The calculated PDI was below the TDI value established for DON in both female and male adults, reaching a percentage up to 30%; however, this percentage increased up to 92% considering total DON (DON + DOM-1). On the other hand, the PDI obtained for ZEA and its metabolites were higher than the TDI value fixed, but the low urine excretion rate (10%) considered should be highlighted. Finally, the PDI calculated in the detected positive sample for OTA exceeded the TDI value. The findings of the present study confirm the presence of the studied mycotoxins and their metabolites as some of the most prevalent in urine.     

Development of a LC–MS/MS Method for the Simultaneous Determination of the Mycotoxins Deoxynivalenol (DON) and Zearalenone (ZEA) in Soil Matrix

Mycotoxins, toxins of fungal origin, can directly or indirectly contaminate food and feed and are poisonous to livestock and humans. While a large amount is known about their occurrence in crops, food, and feeds, little is known about mycotoxin amounts in soil. However, soil is known as a major fungal habitat and a potential sink for mycotoxins in the environment. Furthermore, there is neither a reliable detection nor an extraction method for mycotoxins testing in different soil textures or for potential deficits due to aging processes. Therefore, the aim of the present study was to present a reliable extraction and detection method for the simultaneous quantification of the most common mycotoxins, deoxynivalenol (DON) and zearalenone (ZEA), via liquid chromatography-tandem mass spectrometry (LC–MS/MS). This method was validated with six different samples with different textures and different soil organic matter (SOM). Deuterated standards were used to overcome possible matrix effects. This extraction method could eliminate potential aging processes. The recovery rate was always >80% for DON and >82% for ZEA. The quantification limits were 1 ng per g soil for DON and 0.5 ng per g soil for ZEA.     

Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells.

We studied the interactive effects of either binary or tertiary mixtures of Fusarium mycotoxins, deoxynivalenol (DON), zearalenone (ZEA), and fumonisin B1 (FB1) on the human intestinal cell line, Caco-2, using the endpoints including malonedialdehyde (MDA) production, inhibition of protein and DNA syntheses, DNA methylation, DNA fragmentation, and cell viability as measured by the neutral red (NR) test. The mixtures of mycotoxins reduce cellular viability in increasing order: [FB1+ZEA]<[FB1+DON]<[ZEA+DON]<[FB1+DON+ZEA] in NR test. Because FB1 antagonizes the effects of estrogenic Zearalenone, FB1 was assayed against estradiol. In NR assay, mixture of FB1 and estradiol and/or ZEA improves Caco-2 cells viability in contrast to individual effects. Mixtures of ZEA or FB1 and DON, display synergistic effects in lipid peroxidation. The ability of the toxins to inhibit DNA synthesis is 45%, 70%, and 43% for 10 microM of ZEA, DON, and FBI, respectively. Their binary mixtures (at 10 microM each), inhibit DNA synthesis by 35%, 62%, and 65%, far less than additive effects. Surprisingly, the tertiary mixture (10 microM each) only inhibits DNA synthesis by 25%. ZEA, DON, and FB1 induce DNA fragmentation individually. However, mixtures of these mycotoxins always damage DNA to a greater extent. Each individual mycotoxin (10 microM) raises the percentage of 5-methylcytosine (m5dC) in DNA from 4.5% to 9%, while the combination does not increase this rate any further. Altogether, the data indicate that mixtures of Fusarium toxins are able to induce lipid peroxidation, DNA damage, DNA fragmentation, DNA methylation, and cytotoxicity in Caco-2 cells, and suggest a potential promoter effect in human intestinal cells.     

Influence of subchronic exposure to low dietary deoxynivalenol, a trichothecene mycotoxin, on intestinal absorption of nutrients in mice

during a 6-wk feeding trial, effects of low dietary deoxynivalenol (DON; 0, 0.1, 1 and 10 ppm) on food consumption and weight gain were investigated in male mice. Food intake was similar in all four dietary groups. Weight gain in the group receiving 10 ppm DON was significantly (P < 0.01) reduced. At the end of the feeding period, test animals were sacrificed and absorption of water, -glucose, -leucine, -tryptophan, 5-methyltetrahydrofolic acid and iron was measured in isolated perfused jejunal segments in vitro. No effects were observed on absorption of water, leucine, tryptophan and iron. However, at a dietary DON concentration of 10 ppm, a slightly but significantly (P < 0.05) reduced transfer of glucose was measured. Furthermore, transfer as well as tissue accumulation of 5-methyltetrahydrofolic acid in the jejunal segment were both significantly decreased up to 50%. Heavy metal and trace element content was determined in liver, kidney and small intestine. Manganese and molybdenum content in liver tissue was reduced with a DON concentration of 10 ppm in the diet. The findings indicate that subchronic ingestion of DON, in concentrations occurring in contaminated food and feed, results in an impairment of intestinal transfer and uptake of nutrients such as glucose and 5-methyltetrahydrofolic acid.     

The effect of dimethylnitrosamine on host resistance and immunity

Adult female B6C3F1 mice were injected ip with 0.2 ml phosphate-buffered saline (PBS) only or PBS containing 1.5, 3, or 5 mg dimethylnitrosamine (DMN)/kg body wt daily for 14 days. On Day 16, mice were evaluated for changes in immune status as measured by the antibody response to sheep red blood cells (SRBCs), blastogenesis to T- and B-cell mitogens, natural killer (NK) cell function, delayed hypersensitivity, and alveolar macrophage (AM) bactericidal activity; and for changes in host resistance following challenge with various microorganisms or tumor cells. DMN-exposed animals exhibited reduced humoral antibody responses, T-cell mitogenesis, and AM bactericidal activity. B-cell mitogenesis, NK cell activity, and delayed hypersensitivity were increased. Resistance to challenge with Listeria monocytogenes, Trichinella spiralis, or Herpes simplex types 1 or 2 virus (HSV-1, HSV-2) was not significantly impaired, while that to Streptococcus zooepidemicus and influenza virus was significantly reduced. Resistance to B16-F10 tumor challenge was enhanced following DMN exposure. The data show that DMN treatment altered humoral immunity and antibody-mediated host defense mechanisms. Increased NK cell activity may account for the increased resistance to challenge with Herpes virus and B16-F10 tumor cells.     

Ribotoxic mycotoxin deoxynivalenol induces G2/M cell cycle arrest via p21Cip/WAF1 mRNA stabilization in human epithelial cells

Deoxynivalenol (DON) and other trichothecene mycotoxins mediate a broad range of epithelial injury including atrophic growth inhibition and inflammation in the human gastrointestinal and respiratory tracts. The purpose of this study was to test the hypothesis that DON alters the cell cycle progress linked to the pathogenesis in the human epithelium. We demonstrated that human epithelial cells underwent G(2)/M phase arrest in response to DON treatment without significant increase in apoptotic cell death. Moreover, cells deficient in p21 or p53 gene expression showed the attenuated response of G(2)/M phase arrest by DON. Gene expression of p21 was also induced by DON treatment in a dose-dependent manner with no increase in p53 protein levels, suggesting p53-independent p21 induction. Signaling pathways associated with DON-induced p21 gene expression included PI3 kinase and ERK1/2 MAP kinase cascade. Particularly, ERK1/2 signal was associated with DON-induced p21 mRNA stabilization in the human epithelial cells. Taken together, deoxynivalenol arrested epithelial cell cycle at G(2)/M phase via elevated p21 gene expression.     

Induction of apoptosis and cytokine production in the Jurkat human T cells by deoxynivalenol: role of mitogen-activated protein kinases and comparison to other 8-ketotrichothecenes

The Jurkat T-cell line was used to study potential impact of deoxynivalenol (DON) and related 8-ketotrichothecenes on human immune function. DON (250-1000 ng/ml) readily induced caspase-3 and apoptosis in Jurkat cells. DON (62.5-500 ng/ml) also significantly upregulated IL-2 and IL-8 production following prestimulation with phorbol myristate acetate and ionomycin. DON markedly induced phosphorylation of p38, JNK 1/2, and ERK2. SB203580, a specific inhibitor of p38, reduced DON-induced apoptosis. The MEK1 inhibitor PD98059 which blocks ERK activation had only a small inhibitory effect on DON-induced apoptosis while the JNK inhibitor SP600125 was without effect. Inhibition of p38 attenuated DON-induced upregulation of IL-2 while all three MAPK inhibitors suppressed IL-8 upregulation. When effects of DON were compared to other 8-ketotrichothecenes, the concentrations of DON, 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon X (FX) causing 50% apoptosis were 0.6, 0.5, 0.5, 0.5 and 7.5 microg/ml, respectively. Relative to IL-2 upregulation, FX was suppressive whereas 3-ADON, 15-ADON and NIV had no effect at concentrations of 62.5-500 ng/ml. In contrast, 15-ADON at 62.5-500 ng/ml and 3-ADON at 625-5000 ng/ml upregulated IL-8 production but FX and NIV had no effect. Taken together, these data suggest that DON's effects on apoptosis and cytokine production were differentially regulated by MAPKs. Although DON shared its capacity to induce apoptosis and potentiate IL-8 production with other 8-ketotrichothecenes, it appeared to be unique in its capacity to upregulate IL-2.     

The role and regulatory mechanism of autophagy in hippocampal nerve cells of piglet damaged by deoxynivalenol

Deoxynivalenol (DON), a type B trichothecene mycotoxin mainly affects the health status of pigs and reduced their growth. This study aimed to determine the effects of PI3K/Akt/mTOR pathway on DON-induced autophagy of piglet hippocampal nerve cells (PHNCs), and the relationship between autophagy and apoptosis. The effects of DON on autophagy of PHNCs were examined by cell morphology, cell viability, apoptosis rate, electron microscopy, transient transfection of GFP-LC3 plasmid, immunofluorescence and expression of autophagy-related genes and proteins. The relationship between autophagy and cell apoptosis was analyzed by western blotting, CCK-8 and flow cytometry. The results indicated that, DON inhibited the proliferation of PHNCs and significantly changed cell morphology, and induced apoptosis and autophagy. The expression levels of LC3 protein and gene increased, while the expression levels of PI3K/Akt/mTOR pathway-related genes and proteins decreased, when the concentration of DON increased. Activation of autophagy significantly increased cell viability, reduced apoptosis rate, inhibits autophagy significantly, reduced cell activity and increased apoptosis rate. This data demonstrated that DON exerts certain toxic effect on PHNCs, induced apoptosis and autophagy. PI3K/Akt/mTOR signaling pathway plays a negative regulatory role in DON-induced autophagy of PHNCs. At the same time, autophagy plays a protective role in DON-induced PHNCs injury.