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.     

Effects of several mycotoxins on specific growth rate of Butyrivibrio fibrisolvens and toxin degradation in vitro

Four strains of Butyrivibrio fibrisolvens did not degrade aflatoxin B1. Acetyl T-2 toxin, T-2 toxin, HT-2 toxin, deoxynivalenol, diacetoxyscirpenol, verrucarin A, zearalenone, and ochratoxin A did not affect the specific growth rate of B. fibrisolvens CE51 significantly, but all were degraded to greater or lesser extents. Breakdown products were produced as a result of deacetylation reactions.     

Effects of several mycotoxins on specific growth rate of Butyrivibrio fibrisolvens and toxin degradation in vitro.

Four strains of Butyrivibrio fibrisolvens did not degrade aflatoxin B1. Acetyl T-2 toxin, T-2 toxin, HT-2 toxin, deoxynivalenol, diacetoxyscirpenol, verrucarin A, zearalenone, and ochratoxin A did not affect the specific growth rate of B. fibrisolvens CE51 significantly, but all were degraded to greater or lesser extents. Breakdown products were produced as a result of deacetylation reactions.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Natural contamination with mycotoxins in forage maize and green coffee in Nayarit State (Mexico)

The presence of mycotoxins in forage maize (zearalenone, fumonisin B1, T-2 toxin and diacetoxyscirpenol) and green coffee (ochratoxin A) from Nayarit State (Mexico) has been studied. All maize samples analyzed showed fumonisin B1 contamination, with an average concentration of 2,541 microg/kg. Fifteen percent of the samples contained zearalenone, with an average concentration of 1,610 microg/kg. Only one sample showed T-2 toxin contamination (7 microg/kg), and no diacetoxyscirpenol was detected. Sixty-seven per cent of green coffee samples were contaminated with ochratoxin A, with an average concentration of 30.1 microg/kg. This is the first study about mycotoxins developed in Nayarit and it has shown that mycotoxin contamination is a real problem in both foodstuffs studied.     

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.     

Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry

Liquid chromatographic/tandem mass spectrometric methods using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for determination of 18 mycotoxins and metabolites-ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol (zeranol), beta-zearalanol (taleranol), fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deepoxy-deoxynivalenol (DOM-1) and aflatoxin M1--in milk. The mycotoxins were extracted and cleaned up simultaneously. Extraction and removal of lipophilic compounds was performed at pH 2 using a two-phase mixture of acetonitrile and hexane. The acetonitrile concentration of the aqueous phase was reduced and the pH was adjusted to 8.5 before clean up by solid phase extraction (SPE) on Oasis HLB. The toxins DON, DOM-1, 3-AcDON, 15-AcDON, ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol were detected in negative ion mode after separation on a Hypersil ENV analytical column, while the toxins T-2 toxin, HT-2 toxin, T-2 triol, DAS, MAS, fumonisin B1, fumonisin B2 and aflatoxin M1 were detected in positive ion mode after separation on a Luna C18 column. Two transition products were monitored for each compound. The extraction and SPE conditions were optimised to obtain maximum recovery and minimum signal suppression/enhancement. The detection capabilities related to the transition products of lowest abundance were in the range 0.020-0.15 microg/l. The mean true recoveries were in the range 76-108% at levels of 0.2-10 microg/l.     

In Vitro Detoxification of Aflatoxin B1, Deoxynivalenol,Fumonisins, T-2 Toxin and Zearalenone by Probiotic Bacteria from Genus Lactobacillus and Saccharomyces cerevisiae Yeast

The aim of the following research was to determine the detoxification properties of probiotic Lactobacillus sp. bacteria (12 strains) and S. cerevisiae yeast (6 strains) towards mycotoxins, such as aflatoxin B₁, deoxynivalenol, fumonisins, T-2 toxin and zearalenone, which pose as frequent feed contamination. The experiment involved analysing changes in concentration of mycotoxins in PBS solutions, after 6, 12 and 24 h of incubation with monocultures of tested microorganisms, measured by high-performance liquid chromatography (HPLC). We found that all strains detoxified the mycotoxins, with the highest reduction in concentration observed for the fumonisin B₁ and B₂ mixture, ranging between 62 and 77% for bacterial strains and 67–74% for yeast. By contrast, deoxynivalenol was the most resistant mycotoxin: its concentration was reduced by 19–39% by Lactobacillus sp. strains and 22–43% by yeast after 24 h of incubation. High detoxification rates for aflatoxin B₁, T-2 toxin and zearalenone were also observed, with concentration reduced on average by 60%, 61% and 57% by Lactobacillus, respectively, and 65%, 69% and 52% by yeast, respectively. The greatest extent of reduction in the concentration for all mycotoxins was observed after 6 h of incubation; however, a decrease in concentration was noted even after 24 h of incubation. Thus, the tested microorganisms can potentially be used as additives to decrease the concentrations of toxins in animal feed.

     

Natural occurrence of Fusarium toxins in feedstuff.

The mycotoxins diacetoxyscirpenol, deoxynivalenol, and zearalenone, produced by Fusarium roseum, were found naturally occuring in mixed feed samples. In all cases analyzed, deoxynivalenol occurred together with zearalenone. The natural occurrence of zearalenone in sesame seed is reported for the first time. Strains of F. roseum isolated in various parts of the world form feed implicated in animal mycotoxicosis produced monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and zearalenone.     

Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were determined by enzyme-linked immunosorbent assay. The following mycotoxins were found in these lichens in a broad concentration range with a frequency of 70–100%: sterigmatocystin (7–2090 ng/g), alternariol (20–6460 ng/g), and emodin (45–94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B₁ was detected in only six species with a frequency of 2–42%, whereas roridin A was present in 10% of Hypogymnia physodes samples.     

Mycotoxins in laboratory rodent feed

Twenty-one batches of fixed-formula rodent diets from three feed manufacturers were tested for the presence of five mycotoxins: deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin, T-2 toxin and ochratoxin A (OTA). Five batches were also tested for the presence of zearalenone (ZEN) and six batches for aflatoxins. Detectable levels of DON (up to 298 microg/kg), NIV (up to 118 microg/kg), OTA (up to 3.1 microg/kg) or ZEN (up to 26.7 microg/kg) were found in samples from all manufacturers. Three batches contained two (DON or NIV and OTA or ZEN) and one batch contained three (DON, OTA and ZEN) different mycotoxins. Aflatoxins, T-2 and HT-2 were not detected in any of the batches. The concentrations of mycotoxins detected in the feed were low, but indicated that feed ingredients, probably the cereal ingredients, were contaminated by mycotoxins. Since mycotoxins are known to have toxic and/or immunosuppressive effects, non-contaminated ingredients should be used for production of laboratory animal feed. The results imply that an improved quality control of ingredients used for laboratory rodent feed should be implemented.     

Lack of mutagenicity to Salmonella typhimurium of some Fusarium mycotoxins.

The mutagenicity of eight Fusarium toxins (mono-, di-, and triacetoxyscirpenol, T-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, zearalenone, and moniliformin) and of two positive controls (aflatoxin B1 and sterigmatocystin) to histidine-requiring strains TA 98, 100, 1535, and 1537 of Salmonella typhimurium was tested both with and without metabolic activation. Both aflatoxin B1 and sterigmatocystin, but none of the eight Fusarium toxins, were mutagenic to S. typhimurium. The lack of mutagenic activity of T-2 toxin and diacetoxyscirpenol supports the negative results that have been obtained with in vivo carcinogenicity tests. The negative mutagenicity of the four other 12,13-epoxytrichothecenes tested, and of zearalenone and moniliformin, could not be correlated with in vivo tests because published accounts of their chronic toxicity were not available.     

Lack of mutagenicity to Salmonella typhimurium of some Fusarium mycotoxins.

The mutagenicity of eight Fusarium toxins (mono-, di-, and triacetoxyscirpenol, T-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, zearalenone, and moniliformin) and of two positive controls (aflatoxin B1 and sterigmatocystin) to histidine-requiring strains TA 98, 100, 1535, and 1537 of Salmonella typhimurium was tested both with and without metabolic activation. Both aflatoxin B1 and sterigmatocystin, but none of the eight Fusarium toxins, were mutagenic to S. typhimurium. The lack of mutagenic activity of T-2 toxin and diacetoxyscirpenol supports the negative results that have been obtained with in vivo carcinogenicity tests. The negative mutagenicity of the four other 12,13-epoxytrichothecenes tested, and of zearalenone and moniliformin, could not be correlated with in vivo tests because published accounts of their chronic toxicity were not available.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States.

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.     

Simultaneous occurrence of deoxynivalenol, zearalenone, and aflatoxin in 1982 scabby wheat from the midwestern United States

Thirty-three samples of wheat of the 1982 crop year from Kansas and Nebraska were analyzed for deoxynivalenol, T-2 toxin, zearalenone, and aflatoxin. Deoxynivalenol was identified in 31 of 33 samples, zearalenone was identified in 3 of 33 samples, and aflatoxin B1 was identified in 23 of 31 samples. One 1982 wheat sample from Illinois and one from Texas were also contaminated with deoxynivalenol at 1,200 and 600 ng/g, respectively. None of the samples contained detectable T-2 toxin. The mean concentration of deoxynivalenol was 1,782 +/- 262 ng/g, and the concentrations of aflatoxin B1 ranged from 0.8 to 17.0 ng/g, with a mean of 3.37 +/- 0.7. Zearalenone concentrations of the three positive samples were 35, 90, and 115 ng/g. However, density segregation of two other samples which tested negative yielded light fractions, comprising less than 2% of the samples, contaminated at 230 and 254 ng of zearalenone per g; calculated zearalenone concentrations for these two samples were below the limit of detection of the method. The high frequency of aflatoxin B1 and deoxynivalenol in wheat from the 1982 crop is unprecedented, as is the simultaneous contamination of some samples with deoxynivalenol, zearalenone, and aflatoxin B1.     

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.     

Spontaneous regression of pulmonary paracoccidioidomycosis

The Fusarium and Myrothecium mycotoxins roridin A, diacetoxyscirpenol, verrucarin A, T-2 toxin and zearalenone (10(-2) and 10(-3) mg/ml) inhibit the unspecific dehydrogenase activity of baker's yeast (Saccharomyces cerevisiae) in vivo. The action of these toxins is in the same order as that of aflatoxin B1. It is suggested that at least the trichothecenes decrease the dehydrogenase activity by an interaction with thiol groups of the active center of the enzymes.     

Simultaneous determination of type A-& B-trichothecenes and zearalenone in cereals by High Performance Liquid Chromatography — Tandem Mass Spectrometry

A rapid quantitative method for the simultaneous determination of the majorFusarium mycotoxins nivalenol, deoxynivalenol, fusarenon-X, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, T-2 toxin and zearalenone in maize and wheat was developed. Raw extracts (acetonitrile/water 84/16) are cleaned-up with MycoSep^® columns., Chromatographic separation and end determination is carried out by HPLC-APCI-MS/MS.HPLC run times of 10 minutes considerably increases sample throughput and make this method suitable for routine analysis. The use of a triple quadrupole mass spectrometer allows the selective detection of the mycotoxins and their quantification in the low μg/kg-range.     

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.     

Mycotoxins and fungi in wheat harvested during 1990 in test plots in the state of São Paulo,Brazil

Wheat from two cultivars with contrasting characteristics were harvested in ten experimental plots located in wheat producing areas of the State of São Paulo, Brazil. The samples (10 of each cultivar) were analyzed by a gaschromatographic method for deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), toxins T-2 (T-2) and HT-2, T-2 tetraol, T-2 triol, and by a thin-layer chromatographic method for zearalenone (ZEN), aflatoxins B₁, B₂, G₁, G₂, ochratoxin A and sterigmatocystin. No mycotoxins were detected in 13 samples. DON was found in four samples (0.47–0.59 µg/g), NIV in three samples (0.16–0.40 µg/g), T-2 in two samples (0.40, 0.80 µg/g), DAS in one sample (0.60 µg/g), and ZEN in three samples (0.04–0.21 µg/g). The wheat samples were also examined for the incidence of fungi.Alternaria, Drechslera, Epicoccum andCladosporium were the prevailing genera. Among theFusarium spp.,F. semitectum was present in 19 samples andF. moniliforme in 18 samples. NoF. graminearum was isolated in the samples.Abbreviations DAS diacetoxyscirpenol - DON deoxynivalenol - NIV nivalenol - T-2 T-2 toxin - ZEN zearalenone