Natural occurrence of alternariol and alternariol monomethyl ether in soya beans

The natural occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in soya beans harvested in Argentina was evaluated. Both toxins were simultaneously detected by using HPLC analysis coupled with a solid phase extraction column clean-up. Characteristics of this in-house method such as accuracy, precision and detection and quantification limits were defined by means of recovery test with spiked soya bean samples. Out of 50 soya bean samples, 60% showed contamination with the mycotoxins analyzed; among them, 16% were only contaminated with AOH and 14% just with AME. Fifteen of the positive samples showed co-occurrence of both mycotoxins analyzed. AOH was detected in concentrations ranging from 25 to 211?ng/g, whereas AME was found in concentrations ranging from 62 to 1,153?ng/g. Although a limited number of samples were evaluated, this is the first report on the natural occurrence of Alternaria toxins in soya beans and is relevant from the point of view of animal public health.     

Precise determination of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxins alternariol and alternariol monomethyl ether in cereal,fruit and vegetable products using stable isotope dilution assays

Cereal, fruit and vegetable products were analyzed for contamination with the Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) using stable isotope dilution assays (SIDAs). Both toxins were practically not detected in cereals and cereal products: AOH—one out of 13 samples at a content of 4.1 μg/kg; AME—two out of 13 samples at contents ranging between 0.2 and 0.6 μg/kg. However, if cereals for animal nutrition were analyzed, much higher values were found: AOH—five out of six samples (13–250 μg/kg); AME—six out of six samples (3–100 μg/kg). This finding may pose a potential problem concerning animal health. AOH and AME were frequently detected in vegetable products: AOH—5 out of 10 samples (2.6–25 μg/kg); AME—6 out of 10 samples (0.1–5 μg/kg). Tomato products were affected, especially. The highest content of AOH (25 μg/kg) and AME (5 μg/kg) were found in triple concentrated tomato paste. Special wines like “Trockenbeerenauslese” or “Spätlese” (affected by noble rot in the vineyard) contained AOH (4/6 samples; 1.2–4.9 μg/kg) and AME (4/6 samples; 0.1–0.3 μg/kg), but the values did not exceed the values of both toxins that were found generally in wines.     

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4 ng/mL, AOH 2 and 3.3 ng/mL; red wine: AME 0.1 and 0.2 ng/mL, AOH 4.5 and 7.5 ng/mL; apple juice: AME 1.7 and 2.8 ng/mL, AOH 5 and 9 ng/mL; other juices: AME 2.0 and 3.1 ng/mL, AOH 6 and 10 ng/mL). Recoveries in all cases were greater than 80 %. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18 ng/mL, 6 of 56 samples of red wine had a maximum of 13 ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225 ng/mL) did the toxin level exceed the LOQ.     

Production of alternarioi and alternariol monomethyl ether in natural substrates in comparison with semisynthetic culture medium

The comparison In toxins production and growth byAlternarla strains in liquid, solid culture media and natural substrates (rice and sunflower) was evaluated. Ground rice- corn steep liquor medium (GRCS) was the more suitable medium for production of alternariol (AOH) and alternariol monomethyl ether(AME). The maximum levels produced were 676 μg/50ml AOH and 1570/50ml AME. Rice was better than sunflower In supporting toxins production. Different ratios AOH/AME were found according to the substrate evaluated.     

Natural Occurrence of Alternaria Toxins in Wheat-Based Products and Their Dietary Exposure in China

A total of 181 wheat flour and 142 wheat-based foods including dried noodle, steamed bread and bread collected in China were analyzed for alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN) and tenuazonic acid (TeA) by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. TeA was the predominant toxin found in 99.4% wheat flour samples at levels ranging from 1.76 μg/kg to 520 μg/kg. TEN was another Alternaria toxin frequently detected in wheat flour samples (97.2%) at levels between 2.72 μg/kg and 129 μg/kg. AOH and AME were detected in 11 (6.1%) samples at levels ranging from 16.0 μg/kg to 98.7 μg/kg (AOH) and in 165 (91.2%) samples with a range between 0.320 μg/kg and 61.8 μg/kg (AME). AOH was quantified at higher levels than AME with the ratio of AOH/AME ranging from 1.0 to 3.7. Significant linear regressions of correlation in toxin concentrations were observed between AOH and AME, AME and TeA, TEN and TeA, AOH+AME and TeA. At an average and 95th percentile, dietary exposure to AOH and AME in the Chinese general population and different age subgroups exceeded the relevant threshold value of toxicological concern (TTC), with the highest exposure found in children which deserves human health concern. TEN and TeA seem unlikely to be health concerns for the Chinese via wheat-based products but attention should be paid to synergistic or additive effects of TeA with AOH, AME, TEN and a further assessment will be performed once more data on toxicity-guided fractionation of the four toxins are available. It is necessary to conduct a systemic surveillance of Alternaria toxins in raw and processed foods in order to provide the scientific basis for making regulations on these toxins in China.     

Analysis of wines, grape juices and cranberry juices forAlternaria toxins

Sixty six samples of red and white wine from Ontario (VQA), British Columbia (VQA), Québec (“vins artisanaux”), imported wines (from Italy, South America and USA) and Canadian and US grape and cranberry juices were analysed for theAlternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME). After cleanup on aminopropyl SPE columns, AOH and AME were initially determined by reversed phase LC with UV detection. Positive sample extracts were re-analysed by LC-tandem negative ion electrospray mass spectrometry (MS/MS) in multiple reaction mode. Overall mean method recoveries measured by LC-UV were 93% for AOH and 81% for AME. Limits of detection in wine (and juice) by LC-UV for AOH were 0.8 (0.4) ng/ml and for AME were 0.5 (0.4) ng/ml; they were below 0.01 ng/ml by LC-MS/MS. As determined by LC-MS/MS, AOH was found in 13/17 Canadian red wines at levels of 0.03 to 5.02 ng/ml and in 7/7 imported red wines at 0.27–19.4 ng/ml, usually accompanied by lower concentrations of AME. Red grape juices (5 positive/10 samples) contained only sub ng/ml levels of AOH or AME except for one sample (39 ng AME/ml). White wines (3/23 samples), white grape juices (0/4 samples) and cranberry juices (1/5 samples) contained little AOH/AME (≤1.5 ng/ml). Presented at the World Mycotoxin Forum, Noordwijk, The Netherlands, November 10–11, 2005     

Occurrence of alternariol, alternariol monomethyl ether and tenuazonic acid in Argentinean tomato puree

The occurrence ofAlternaria mycotoxins was investigated in 80 samples of tomato puree processed and sold in Argentina. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) were searched for by liquid chromatography. Thirty-nine of the 80 samples showed mycotoxin contamination. TA was found in 23 samples (39-4021 μg/kg), AOH in 5 samples (187-8756 μg/kg), and AME in 21 samples (84-1734 μg/kg). Co-occurrence of two of these toxins was detected in 10 samples. This is the first report of natural occurrence of AOH, AME and TA in tomato products in Argentina.     

Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay.

The effects in the chicken embryo assay of four Alternaria metabolites (alternariol [AOH], alternariol methyl ether [AME], altenuene [ALT], and tenuazonic acid [TA]) were investigated. Administered to 7-day-old chicken embryos by yolk sac injection, AOH, AME, and ALT caused no mortality or teratogenic effect at doses up to 1,000, 500, and 1,000 micrograms per egg, respectively. TA exhibited a calculated 50% lethal dose of 548 micrograms per egg, with no teratogenic effect observed at either lethal or sublethal doses.     

Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay

The effects in the chicken embryo assay of four Alternaria metabolites (alternariol [AOH], alternariol methyl ether [AME], altenuene [ALT], and tenuazonic acid [TA]) were investigated. Administered to 7-day-old chicken embryos by yolk sac injection, AOH, AME, and ALT caused no mortality or teratogenic effect at doses up to 1,000, 500, and 1,000 micrograms per egg, respectively. TA exhibited a calculated 50% lethal dose of 548 micrograms per egg, with no teratogenic effect observed at either lethal or sublethal doses.     

Effect of heat treatments on stability of altemariol, alternariol monomethyl ether and tenuazonic acid in sunflower flour

A study was carried out to evaluate the effect of heat treatment on the stability of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) in sunflower flour and the effectiveness of this treatment by a biological assay in rats. The concentrations of AOH and AME remained constant during heating at 100°C for up to 90 minutes while TeA concentration decreased with time to 50% after 90 minutes. The most effective treatment in reducing AOH and AME levels was heating at 121°C for 60 minutes. Histopathological evaluation in the biological assay in rats fed withAlternaria toxins showed marked atrophy and fusion of villi in the intestines and liver cell damage; these lesions were less severe in rats fed heat-treated sunflower flour in line with the reduced toxin content. However, a lower weight gain and a noticeable renal damage in rats were produced when they fed decontaminated flour.     

Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

Effect of water activity and temperature on mycotoxin production by Alternaria alternata in culture and on wheat grain.

Both water activity (aW) and temperature affected the production of altenuene (AE), alternariol (AOH), and alternariol monomethyl ether (AME) by Alternaria alternata on wheat extract agar and wheat grain. Greatest production of all three mycotoxins occurred at 0.98 aW and 25 degrees C on both substrates. At 0.98 aW and 25 degrees C, a single colony of A. alternata grown on wheat extract agar produced 807 micrograms of AOH, 603 micrograms of AME, and 169 micrograms of AE ml in 30 days. However, production of all three mycotoxins at 0.95 aW was less than 40% of these amounts. Little toxin was produced at 0.90 aW. Changing temperature and aW altered the relative amounts of the different toxins produced on agar. At 15 degrees C and 0.98 aW, maxima of 52 micrograms of AOH and 25 micrograms of AME per ml were produced after 15 and 30 days, respectively, whereas AE continued to increase and reached 57 micrograms/ml after 40 days. At 15 degrees C and 0.95 aW, production was, respectively, 62, 10, and 5 micrograms/ml after 40 days. All three metabolites were produced at 5 degrees C and 0.98 to 0.95 aW and at 30 degrees C and 0.98 to 0.90 aW. On wheat grain at 25 degrees C and 0.98 to 0.95 aW, more AME was produced than AOH or AE, but at 15 degrees C there was less AME than AOH or AE. Only trace amounts of AE, AOH, and AME were found at 15 to 25 degrees C and 0.90 aW, but production of AME was inhibited at 30 degrees C and 0.95 aW or less.     

Inhibition of polyketide synthesis in Alternaria alternata by the fatty acid synthesis inhibitor cerulenin.

The fatty acid synthase inhibitor cerulenin (50 to 100 micrograms/ml) inhibited production of the polyketide mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) by the mold Alternaria alternata. The results suggested that AOH synthesis was inhibited by a direct mechanism by cerulenin, whereas production of AME was probably limited by a shortage of the precursor AOH.     

Inhibition of polyketide synthesis in Alternaria alternata by the fatty acid synthesis inhibitor cerulenin.

The fatty acid synthase inhibitor cerulenin (50 to 100 micrograms/ml) inhibited production of the polyketide mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) by the mold Alternaria alternata. The results suggested that AOH synthesis was inhibited by a direct mechanism by cerulenin, whereas production of AME was probably limited by a shortage of the precursor AOH.     

链格孢霉醇和链格孢霉醇单甲醚产生菌株的筛选

本文对分离自小麦、马铃薯、番茄和茄子上链格孢霉属(Alternaria)2个种(链格孢和茄链格孢)的96个菌株,用枯草杆菌生长抑制试验筛选链格孢霉醇(AOH)和链格孢霉醇单甲醚(AME)的产生菌株,有48株产生毒性作用(占所测菌株的50％)。18株产强、中毒性菌用高效液相色谱分析,有13株产AOH和AME(占所测菌株的72.2％)。链格孢的产毒素菌株率比茄链格孢低。但产毒素含量却是前者明显高于后者。其中产AOH和AME的最高含量,链格孢菌株XA-8分别为280和5140mg/kg,而茄链格孢菌株SA-10分别为95.9和94.3mg/kg。     

Determinación de perfiles de producción de metabolitos secundarios característicos de especies del género Alternaria aisladas de tomate

BackgroundMany Alternaria species have been studied for their ability to produce bioactive secondary metabolites, such as tentoxin (TEN), some of which have toxic properties. The main food contaminant toxins are tenuazonic acid, alternariol (AOH), alternariol monomethyl ether (AME), altenuene, and altertoxins i, ii and iii.AimsTo determine the profiles of secondary metabolites characteristic of Alternaria strains isolated from tomato for their chemotaxonomic classification.MethodsThe profiles of secondary metabolites were determined by HPLC MS.ResultsThe Alternaria isolates obtained from spoiled tomatoes belong, according to their morphological characteristics, to the species groups Alternaria alternata, Alternaria tenuissima and Alternaria arborescens, with A. tenuissima being the most frequent. The most frequent profiles of secondary metabolites belonging to the species groups A. alternata (AOH, AME, TEN), A. tenuissima (AOH, AME, TEN, tenuazonic acid) and A. arborescens (AOH, AME, TEN, tenuazonic acid) were determined, with some isolates of the latter being able to synthesize AAL toxins.ConclusionsSecondary metabolite profiles are a useful tool for the differentiation of small spored Alternaria isolates not easily identifiable by their morphological characteristics.     

Absorption and metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in Caco-2 cells in vitro

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria. In order to simulate their in vivo intestinal absorption and metabolism, AOH and AME have been studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro. AOH was found to be readily conjugated to two glucuronides and one sulfate, whereas AME gave rise to one major glucuronide and one sulfate. Whereas the glucuronides of AOH and AME were sequestered about equally well into the basolateral and the apical compartment, the sulfates of both toxins were preferentially released to the apical side. Unconjugated AOH but not AME aglycone reached the basolateral chamber. The apparent permeability coefficients (P_app values) were calculated for the aglycones as well as total mycotoxin-associated compounds using an initial apical concentration of 20 µmol/l AOH or AME. Based on these P_app values, AOH must be expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycone and as glucuronide and sulfate. In contrast, intestinal absorption of AME appears to be poor and sluggish, with no AME agylcone and only AME conjugates reaching the portal blood.     

Toxicity of mycotoxins produced by four Alternaria species toArtemia salina larvae

22 isolates ofAlternaria alternata, A raphani, A consortiale, andA chartarum were examined for the production of alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin I (ATX I), and tenuazonic acid (TA) on wheat grain and for toxicity of culture extracts toArtemia salina larvae. The total amount of 5 toxins produced under laboratory conditions ranged from 5 mg/kg to 11.112mg/kg. The toxic extracts showed EC50 values in the range of 3.3 to 144.5 mg/mL. There was no correlation between toxicity of extracts toArtemia salina and the amount of mentioned mycotoxins in culture.     

Influence of dietary fibre on plasma and urinary levels of zearalenone and metabolites in swine

The genusAlternaria is responsible for different plant diseases such as tobacco brown spot, tomato blight, and citrus seedling chlorosis but can also be present during storage of grain. The objective of the present paper is to summarize the knowledge concerning regulation of secondary metabolism inAlternaria, particularA alternata (A tenuis). The paper mainly deals with regulation of polyketide biosynthesis, one of the major pathways leading to the biosynthesis of mycotoxins inAlternaria. The mostly studiedAlternaria mycotoxins are dibenzopyrones such as alternariol (AOH) and alternariol monomethyl ether (AME) and altenuene along with the tetramic acid tenuazonic acid. The biosynthesis ofAlternaria mycotoxins has been reviewed by Stinson (12). Most information is available for the biosynthesis of the polyketides AOH / AME while a few biosynthetic studies have been accomplished for tenuazonic acid (11).