Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay

A colorimetric MTT (tetrazolium salt) cleavage test was used to evaluate cytotoxicity of twenty-three Fusarium mycotoxins on two cultured human cell lines (K-562 and MIN-GL1) as well as their inhibitory effect on proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes. The values of 50% inhibition of lymphocyte blastogenesis were very close to the 50% cytotoxic doses observed with the more sensitive cell line (MIN-GL1). T-2 toxin was the most cytotoxic with CD₅₀ and ID₅₀ values less than 1 ng/ml. Type A trichothecenes were the most cytotoxic followed by the type B trichothecenes; the non-trichothecenes were the least cytotoxic. The MTT cleavage test, in conjunction with cell culture, is a simple and rapid bioassay to evaluate cytotoxicity and immunotoxicity of Fusarium mycotoxins.Abbreviations Ac acetyl - ACU acuminatin - DAS diacetoxyscirpenol - DON deoxynivalenol - FUS fusarenon-X - HT-2 HT-2 toxin - MC mononuclear cell - MTT 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide - NEO neosolaniol - NIV nivalenol - NT-1 4,8-diacetoxy T-2 tetraol - PBS phosphate buffered saline - TAT-2T tetraacetoxy T-2 tetraol - T-2 T-2 toxin     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 µg and 100 µg/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.     

The cytotoxic effect of fumonisin B<Subscript>1</Subscript> and ochratoxin A on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay

Lymphocytes cell obtained from healthy human donors and pigs were exposed to fumonisin B₁ (FB₁) and ochratoxin A (OTA), which have been found to be immunosuppressive, carcinogenic and mutagenic, to ascertain their single and combined cytotoxic effects with time and to assess the suitability of animal lymphocytes as test agents in comparison to human cells. The main objectives of this work were to assess the use of animal lymphocytes, particularly pig lymphocytes, for their use in the Methyl Thiazol Tetrazolium (MTT) cytotoxicity test, making them more accessible to animal research-based institutes in comparison to human lymphocytes previously used, and to study the cytotoxic and synergism or antagonistic effects of FB1 and OTA. The MTT assay, which measures cell viability and proliferation based on reduction of MTT to a blue dye, also used the addition of phytohaemagglutinin (PHA) to stimulate the blood cells. The results showed a progressive decrease in lymphocytes viability with time of exposure to the toxins. It was also noted that FB1, as compared to OTA, had a lower cytotoxicity on both human and pig lymphocytes cells. In addition, when the two mycotoxins were combined, a synergistic decrease of cell viability in both human and pig lymphocytes was observed, with pig lymphocytes showing a greater sensitivity. This study has shown that the MTT assay can be used for the determination of cytotoxicity of mycotoxins using animal, and in particular pig, lymphocytes, which eliminates the use of human donors and other cell cultures.     

The effects of four mycotoxins on the mitogen stimulated proliferation of bovine peripheral blood mononuclear cells in vitro

The effects of four mycotoxins, T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA) and fumonisin B1 (FB1) on the response of bovine peripheral blood mononuclear cells (PBM) in vitro to the mitogens concanavalin A (Con A), phytohaemagglutinin A (PHA) and pokeweed mitogen (PWM) were assayed after 4 days' incubation using 3H-thymidine uptake and the MTT bioassay. The concentrations of mycotoxin required to reduce the proliferative response of PBM by 50% for Con A, PHA and PWM as measured by 3H-thymidine incorporation was for T-2 toxin 0.30, 0.40 and 0.18 ng ml-1; for DON 0.07, 0.09 and 0.04 μg ml-1; for OTA 0.10, 0.20 and 0.15 μg ml- 1, and for FB1 35, 18 and 11 μg ml-1M by 50% for Con A, PHA and PWM as measured by the MTT bioassay were for T-2 toxin 2.0, 2.0 and 1.0 mg ml-1; for DON 0.70, 0.50 and 0.50 μg ml-1; OTA 1.5, 1,5 and 1.5 μg ml- 1; and FB1 >50, >50 and 20 μg ml-1 respectively. Further cytotoxicity assays including the LDH bioassay and Trypan blue exclusion were performed only on Con A-stimulated PBM cells after 72 h incubation. With the LDH-bioassay the 50% inhibition levels were T-2 toxin 0.3 ng ml-1, DON 0.4 μg ml- 1, OTA 1.4 μg ml-1 and FB1 3.5 μg ml-1; for Trypan blue uptake the 50% inhibition levels were T-2 toxin 5 ng ml-1, DON 2.3 μg ml-1 and OTA 4 μg ml-1 respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased cytotoxicity of food-borne mycotoxins toward human cell lines in vitro via enhanced cytochrome p450 expression using the MTT bioassay

Eight food-borne mycotoxins epidemiologically implicated in human disease were tested for their cytotoxic effects on human cells previously immortalised and transfected to introduce human cytochrome p450 (CYP 450) genes. Such cells retain many characteristics of normal cell growth and differentiation while simultaneously having the potential of either increasing or decreasing the metabolic activity (cytotoxicity) of the challenging mycotoxins. The MTT assay provided an indication of cytotoxicity. Of the nine CYP450s introduced CYP1A2 was most effective, rendering the cells 540 times more sensitive than the control cells to aflatoxin B1, 28 times more sensitive to aflatoxin G1 and 8-fold more sensitive to ochratoxin A. CYP3A4 resulted in the cells being 211 times more toxic to aflatoxin B1 and 8-fold more toxic to aflatoxin G1 while CYP 2A6, CYP 3A5 and CYP 2E1 also produced observable effects. No increase in metabolic activity was found using cyclopiazonic acid, deoxynivalenol, fumonisin B1, patulin or T-2 toxin. CD5Os were calculated for the mycotoxins against the non-CYP-introduced control cells. There was almost a five order of magnitude difference between the most toxic, T-2 toxin (CD50 0.0057 microgram/ml) and the least toxic, fumonisin B1 (CD50 476.2 micrograms/ml). In vitro biological assays thus provide an excellent system for quantifying the often low CD50s expressed by mycotoxins in foods.     

Airborne mycotoxins in dust from grain elevators

Workers in grain elevators are exposed to grain dust and may therefore have an increased risk of inhalatory contact with mycotoxins. To study the mycotoxin burden of such environments, settled grain dust samples (n=35) were collected from several locations of a total of 13 grain elevators in Germany, and analysed for ochratoxin A (OTA, detection limit 0.01 ng/g), deoxynivalenol (DON, detection limit 15 ng/g), and zearalenone (ZEA, detection limit 6 ng/g), respectively. Cytotoxicity of these samples was assessed by a MTT bioassay with a swine kidney target cell line. Additionally, the airborne dust concentration of these locations was determined. Nearly all settled dust samples contained OTA (96%), DON (100%), and ZEA (100%) with median concentrations of 0.4 ng/g, 416 ng/g, and 126 ng/g, respectively. Cytotoxic effects in varying degrees from weakly to highly toxic were caused by crude extracts of 86% of the dust samples. However, cytotoxicity did not correlate with mycotoxin levels in these samples and thus indicated the presence of cytotoxic compounds of unknown origin. Based on the mycotoxin findings in settled dust samples and the airborne dust concentrations, the average airborne mycotoxin concentrations were estimated to be 0.002 ng/m³ (OTA), 2 ng/m³ (DON), and 1 ng/m³ (ZEA), respectively. The relevance of these findings for occupational health was assessed by comparison with WHO recommendations for the maximum tolerable daily (oral) intake (TDI). Even in a worst case scenario, the calculated inhalatory intake was far below the TDI values. However, considering the uncertainties resulting from different exposure pathways, namely oral ingestion versus inhalation, further research should primarily address the problem of how adequate assessment criteria for airborne exposure to mycotoxins could be established. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

A triggering structure recognized by G7 monoclonal antibody on porcine lymphocytes and granulocytes.

Monoclonal antibody (mAb) G7 has been developed and appears to recognize a triggering structure on porcine natural killer (NK) cells and granulocytes. G7 mAb binds to approximately 13% of lymphocytes, 70% of monocytes, and greater than 95% of granulocytes. G7 mAb does not react with B cells. G7 mAb immunoprecipitates a heterodispersed molecule of approximately 40 kDa. Functionally, whole but not F(ab')2 fragments of G7 mAb enhance NK killing of Fc receptor positive K562, U937, and MOLT-4 targets but not Fc receptor negative CEM, WEHI-164, or YAC-1 targets. Both whole and F(ab')2 fragments of G7 mAb inhibit lymphocyte-mediated antibody-dependent cellular cytotoxicity. Interestingly, G7 mAb induces dramatic levels of granulocyte killing against nucleated K562 targets. These results suggest that G7 mAb recognizes a trigger molecule involved in porcine cellular cytotoxicity.     

Einfluß des Rohproteinniveaus in Maisfutterrationen bei verschiedenen Harnstoffgaben in der Jungbullenmast

Mycotoxins are fungal secondary metabolites that elicit a wide spectrum of toxicological effects, including the alteration of normal immune function. In the present study we investigated the independent effect of four mycotoxins, aflatoxin B₁ (AFB₁), fumonisin B₁ (FB₁), deoxynivalenol (DON) and nivalenol (NIV), on lymphocyte proliferation using human and porcine lymphocytes. Human and porcine peripheral blood mononuclear cells and porcine splenocytes were cultured with increasing concentrations of mycotoxins for 72 hours and labelled in the last 24 hours with [methyl-³H]-thymidine. The results showed that increased concentrations of AFB₁, DON and NIV affected the [methyl-³H]-thymidine cellular proliferation following mitogen stimulation in both species and cell types. Lower concentrations of mycotoxins enhanced cellular proliferation, which was more pronounced in human than in porcine cells, while higher concentrations caused a dose-dependent decrease. DON and NIV were the most potent mycotoxin in both species and both cell types. Based on the results of this in vitro study, high correlations were found between proliferation of human and porcine lymphocytes after mycotoxin exposure, especially for DON and NIV.     

Comparative aspects of in vitro proliferation of human and porcine lymphocytes exposed to mycotoxins

Mycotoxins are fungal secondary metabolites that elicit a wide spectrum of toxicological effects, including the alteration of normal immune function. In the present study we investigated the independent effect of four mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1), deoxynivalenol (DON) and nivalenol (NIV), on lymphocyte proliferation using human and porcine lymphocytes. Human and porcine peripheral blood mononuclear cells and porcine splenocytes were cultured with increasing concentrations of mycotoxins for 72 hours and labelled in the last 24 hours with [methyl-3H]-thymidine. The results showed that increased concentrations of AFB1, DON and NIV affected the [methyl-3H]-thymidine cellular proliferation following mitogen stimulation in both species and cell types. Lower concentrations of mycotoxins enhanced cellular proliferation, which was more pronounced in human than in porcine cells, while higher concentrations caused a dose-dependent decrease. DON and NIV were the most potent mycotoxin in both species and both cell types. Based on the results of this in vitro study, high correlations were found between proliferation of human and porcine lymphocytes after mycotoxin exposure, especially for DON and NIV.     

A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B₁, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B₁ and M₁ be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.     

The emerging <Emphasis Type="Italic">Fusarium</Emphasis> toxin enniatin B: in-vitro studies on its genotoxic potential and cytotoxicity in V79 cells in relation to other mycotoxins

The Fusarium metabolite enniatin B is now recognized as a frequent contaminant of grains used for human foods and animal feeds. Yet, so far very limited data are available on its toxicity and that of other emerging Fusarium mycotoxins (Jestoi M, 2008, Crit Rev Food Sci Nutr 48:21-49). Thus, the mutagenic/genotoxic potential of enniatin B was investigated in a battery of short-term tests, and its cytotoxicity compared with that of several other mycotoxins. No mutagenicity was detected in the Ames assay with four Salmonella typhimurium strains, and in the HPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 cells, in either the presence or absence of an external metabolizing enzyme system (rat liver S9). For other types of genotoxicity, i.e., clastogenicity and chromosomal damage, studied in V79 cells by means of alkaline single-cell gel electrophoresis (Comet) assay and micronucleus assay, no significant genotoxic potential of enniatin B was revealed. However, the Fusarium metabolite exerts pronounced time- and concentration-dependent cytotoxic effects in V79 cells as determined by Alamar Blue reduction and by neutral red uptake assays. For instance, IC₂₀ and IC₅₀ values determined for enniatin B by neutral red assay for 48-h exposure are 1.5 μM and 4 μM. These values are higher than those of the more potent Fusarium toxin deoxynivalenol (IC₂₀ 0.7 μM, IC₅₀ of 0.8 μM), but clearly lower than the IC values of several other mycotoxins tested in parallel. Their ranking of cytotoxicity in V79 cells was as follows: deoxynivalenol > enniatin B > patulin > ochratoxin A > zearalenone > citrinin. Moreover, enniatin B was found to induce nuclear fragmentation, a sign of apoptosis, already at low submicromolar concentrations. In summary, despite an apparent lack of mutagenic and genotoxic activity, enniatin B can cause pronounced cytotoxicity in mammalian cells, detectable at low micromolar concentrations.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

Increased cytotoxicity of food-borne mycotoxins toward human cell lines in vitro via enhanced cytochrome p450 expression using the MTT bioassay

Eight food-borne mycotoxins epidemiologically implicated in human disease were tested for their cytotoxic effects on human cells previously immortalised and transfected to introduce human cytochrome p450(CYP 450) genes. Such cells retain many characteristics of normal cell growth and differentiation while simultaneously having the potential of either increasing or decreasing the metabolic activity (cytotoxicity) of the challenging mycotoxins. The MTT assay provided an indication of cytotoxicity. Of the nine CYP 450s introduced CYP1A2 was most effective,rendering the cells 540 times more sensitive than the control cells to aflatoxin B₁, 28 times more sensitive to aflatoxin G₁ and 8-fold more sensitive to ochratoxin A. CYP3A4 resulted in the cells being 211 times more toxic to aflatoxin B₁ and 8-fold more toxic to aflatoxin G₁ while CYP 2A6, CYP 3A5 and CYP 2E1 also produced observable effects. No increase in metabolic activity was found using cyclopiazonic acid, deoxynivalenol,fumonisin B₁, patulin or T-2 toxin. CD5Os were calculated for the mycotoxins against the non-CYP-introduced control cells. There was almost a five order of magnitude difference between the most toxic,T-2 toxin (CD50 0.0057 g/ml) and the least toxic, fumonisin ₁(CD50 476.2 g/ml). In vitro biological assays thus provide an excellent system for quantifying the often low CD50s expressed bymycotoxins in foods.This revised version was published online in October 2005 with corrections to the Cover Date.     

Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

We evaluated the inhibitory effect of 3,7-dini-trodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K562 cell by trypan blue exclusion test and MTT colorimetric assay.The degree of DNA damage in K562 cells treated with cBr,was detected by isotopic tracer method (3H-TdR).The morphological changes of these K562 cells were examined by fluorescence and electron microscopy.Biochemical characteristics of K562 cells were detected by flow cytome-try and 3H-thymidine incorporation assay.Findings indi-cated that cBr could significantly inhibit cell proliferation and result in DNA damage of K562 cells,cBr is a new type of immunostimulant and can induce cell apoptosis.     

Simultaneous and rapid detection of six different mycotoxins using an immunochip

Mycotoxins are highly toxic contaminants in food, animal feed, and commodities. The study has developed an immunochip for quantifying the concentrations of six mycotoxins: aflatoxin B1, aflatoxin M1, deoxynivalenol, ochratoxin A, T-2 toxin, and zearalenone, which were added to drinking water. The complete antigens (Ags) of the mycotoxins were contact printed and immobilized onto agarose-modified glass slides with 12 physically isolated subarrays, based on the reaction of both diffusion and covalent bond. The optimal concentration of each antigen and antibody (Ab) was obtained using an Ag-Ab immunoassay. Based on the indirect competitive immunoassay for the simultaneous detection of six mycotoxins in one single chip, six standard curves with good logistic correlation (R(2)>0.97) were respectively plotted. The working ranges (0.04-1.69, 0.45-3.90, 20.20-69.23, 35.68-363.18, 0.11-1.81, and 0.08-7.47 ng/mL, respectively) were calculated, as well as the median inhibitory concentrations (0.31±0.04, 1.49±0.21, 34.54±1.30, 134.06±11.75, 0.49±0.05, and 1.54±0.22 ng/mL, respectively), when six mycotoxins were detected simultaneously. Finally, the recovery rates in drinking water generally ranged from 80% to 120% on the same chip, with an intra-assay coefficient of variation lower than 15%. We successfully established an immunochip for simultaneous detection of six mycotoxins within 4h, with advantages of using minimal samples and being visually semiquantitative with our naked eyes. In summary, the method could be developed on one single chip for detecting multiple contaminants in actual samples.     

Gliotoxin inhibits transformation and its cytotoxic to turkey peripheral blood lymphocytes

Gliotoxin, an epipolythiodioxopiperizine mycotoxin, has been shown to be produced by, among other fungi,Aspergillus fumigatus Fresenius. This organism is the major causative agent of the respiratory disease aspergillosis in avian species, especially turkeys. Because gliotoxin has been shown to be immunosuppressive and has the potential for being involved in the pathogenesis of aspergillosis, the in vitro activity of this compound with avian lymphocytes was investigated. Immunosuppression was investigated using peripheral blood lymphocytes from turkeys in a lymphoblastogenesis assay and a cytotoxicity assay using conversion of the tetrazolium salt MTT to MTT formazan by the mitochondrial succinate dehydrogenase enzyme elaborated only by living cells. Gliotoxin appeared to have a threshold level in both tests because little or no response or stimulation was evident when cells were exposed to concentrations of the toxin below 100 ng/ml, but at 100 ng/ml, all cells appeared to be dead. Using T-2 mycotoxin as a known cytotoxic agent, the response in the MTT bioassay using turkey peripheral lymphocytes was linear with increasing concentrations of toxin. Gliotoxin may potentially cause immunosuppression in turkey poults through action on the lymphocytes or if this toxin were present in low concentrations stimulation could possibly occur.     

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines--relationship between structure and biological activity

The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3相似文献