Novel oxidative in vitro metabolites of the mycotoxins alternariol and alternariol methyl ether

The Alternaria toxins alternariol (AOH; 3,7,9-trihydroxy-1-methyl-6H-benzo[c]chromen-6-one) and alternariol methyl ether (AME, 3,7-dihydroxy-9-methoxy-1-methyl-6H-benzo[c]chromen-6-one) are common contaminants of food and feed, but their oxidative metabolism in mammals is as yet unknown. We have therefore incubated AME and AOH with microsomes from rat, human, and porcine liver and analyzed the microsomal metabolites with HPLC and GC-MS/MS. Seven oxidative metabolites of AME and five of AOH were detected. Their chemical structures were derived from their mass spectra using deuterated trimethylsilyl (TMS) derivatives, and from the information obtained from enzymatic methylation. Several of the metabolites were identified by comparison with synthetic reference compounds. AME as well as AOH were monohydroxylated at each of the four possible aromatic carbon atoms and also at the methyl group. In addition, AME was demethylated to AOH and dihydroxylated to a small extent. As the four metabolites arising through aromatic hydroxylation of AME and AOH are either catechols or hydroquinones, the oxidative metabolism of these mycotoxins may be of toxicological significance.     

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds.     

Alternaria toxins in Argentinean wheat,bran, and flour

Alternaria species have been reported to infect a wide variety of vegetables, fruits, and cereal crops. Wheat is one of the most consumed cereal worldwide. A sensitive HPLC-DAD methodology was applied to quantify alternariol (AOH), alternariol methyl ether (AME) and tenuazonic acid (TeA) in 65 samples of whole wheat, bran, and flour. The extraction methodology allowed extracting the three toxins simultaneously. Limits of detection in wheat were 3.4, 4.5, and 0.5 µg kg^?1 for AOH, AME and TeA, respectively. For bran, these data were 3.1, 4.5, and 12 µg kg^?1 and for flour 50, 70, and 14 µg kg^?1, respectively. The studied recoveries were higher than 70% and RSD was below 10%. Wheat and bran samples showed low AOH and AME contamination compared to TeA. The averages levels found for TeA in wheat, bran and flour were 19,190, 16,760, and 7360 µg kg^?1, respectively.     

Rapid determination of Alternaria mycotoxins in tomato samples by pressurised liquid extraction coupled to liquid chromatography with fluorescence detection

ABSTRACT

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC–FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λ_exc = 258, λ_em = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20–72 µg· kg–1) with recoveries of 84–97% (RSD < 8%, n = 6) for AOH and 67–91% (RSD < 13%, n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg^–1, respectively. The ensuing PLE–MISPE–HPLC–FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.     

Multitoxin evaluation in fermented beverages and cork stoppers by liquid chromatography–tandem mass spectrometry

A total of twenty‐eight mycotoxins were surveyed in wine (red, white and rose), cider (white and rose) and their cork stoppers from eight countries. Toxins of different fungi genera were detected as follows: Alternaria (ATs: alternariol – AOH; alternariol methyl – AME) and Penicillium/Aspergillus (ochratoxin A – OTA; penicillic acid – PAC). Toxins and levels varied with the sample types and country of origin. Wine presented contamination of OTA, AOH and AME. OTA was detected in forty‐one wine samples with levels ranging from 0.01 to 0.86 μg L^?1, below EU legislation. AOH and AME were detected in thirty‐three and eight of wines samples, respectively, at levels from 0.2 to 13.3 μg L^?1, while no contamination was detected in ciders up to the method LOQs. Regarding the cork stoppers toxins detected, they were AOH, AME and PAC. Corks of red wine from different countries had levels of OAH and AME ranging from 5.0 to 101.0 and 2.5 to 5 μg g^?1, respectively. It is necessary to pay more attention on the corks processing and cork type used in the bottles as, different from the ordinary ones, the ground bark and compressed type did not have toxins detected.     

Aromatic hydroxylation is a major metabolic pathway of the mycotoxin zearalenone in vitro

Zearalenone (ZEN) is a common mycotoxin, for which only reductive metabolites have been identified so far. We now report that ZEN is extensively monohydroxylated by microsomes from human liver in vitro. Two of the major oxidative metabolites arise through aromatic hydroxylation and are catechols. Their chemical structures have been unambiguously determined by using deuterium‐labeled ZEN and by comparison with authentic reference compounds. Moreover, both catechol metabolites of ZEN were substrates of the enzyme catechol‐O‐methyl transferase. One of the monomethyl ethers represented the major metabolite when ZEN was incubated with rat liver slices, thus demonstrating that catechol formation also takes place under in vivo‐like conditions. Out of ten major human cytochrome P450 (hCYP) isoforms only hCYP1A2 was able to hydroxylate ZEN to its catechols with high activity. Catechol formation represents a novel pathway in the metabolism of ZEN and may be of toxicological relevance.     

Production of alternariol and alternariol methyl ether by Alternaria alternata grown on fruits at various temperatures

Two toxigenic strains of the fungus Alternaria alternata (ATCC 56836 and ATCC 66868) were grown on surface-disinfected, fresh, ripe fruits and tested for the production of alternariol (AOH) and alternariol methyl ether (AME). Examined fruits included strawberries; red and green seedless grapes; concord grapes; red delicious, golden delicious, and gala apples; and blueberries. After inoculation, fruits were incubated at 4, 10 degrees C, or room temperature (approximately 21 degrees C) for up to 3 weeks. At weekly intervals, duplicate samples were analyzed for AOH and AME by using liquid chromatography. Results indicated that A. alternata and its metabolites were not a major problem in strawberries due to the presence of fast-growing molds like Rhizopus and Botrytis that outgrew and possibly inhibited Alternaria. Both Alternaria strains showed limited growth on apples, although fast-growing molds were not present after surface disinfection; AOH and AME were produced only by the ATCC 56836 strain on the golden delicious and gala varieties, (ranging from <0.1 to 5 microg/g and <0.1 to 14 microg/g for AOH and AME, respectively). Restricted growth of both strains without toxin production occurred in blueberries, whereas moderate growth and AOH (<0.1 to 3,336 microg/g) and AME (<0.1 to 1,716 microg/g) production took place in grapes.     

PRODUCTION OF ALTERNARIOL AND ALTERNARIOL METHYL ETHER BY ALTERNARIA SPP.

Five isolates of Alternaria alternata, five isolates of A. tenuis, one isolate of A. hemicota and three isolates of unidentified Alternaria spp. were examined for their production of alternariol methyl ether (AME) and alternariol (AOH) in complex liquid media. The data were compared with the production of these two toxins on solid substrate rice cultures. A new high-performance liquid chromatography method with minimum detection levels for AME and AOH of 20 ng and 40 ng, respectively, was used to quantitate the toxins. All isolates produced AME and AOH, but the amount varied considerably with the isolates and media tested. Of the three different liquid media tested, the highest quantity of AME and AOH was produced in a modified Czapek-Dox broth. The highest yield of toxins was obtained in the solid rice cultures. As much as 125 mg of AME and 16 mg of AOH were produced by one A. tenuis isolate on 300 g of rice after 14 days of incubation at 25°C in darkness.     

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer’s storage conditions. Sample extraction was performed using a liquid–liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g^–1. AME levels ranged between 11 and 137 ng g^–1, which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.     

Optimization of the Method of Detection of Metabolites Produced by the Alternaria Genus: Alternariol, Alternariol Monomethyl Ether, Altenuene, Altertoxin I and Tentoxin

ABSTRACT: A simple method has been developed in this work for the detection of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), tenuazonic acid (TA), and tentoxin by means of thin-layer chromatography from cultures of several strains of Alternaria. Strains were incubated in 2% malt extract broth for 7 d at 25 °C. For extraction of metabolites, chloroform was used. The solvent system benzene/methanol/acetic acid (92:6:2) was chosen. TLC plates were observed under ultraviolet light (254/366 nm). Results show that A. alternata IMI 354942 produced AOH, AME, ALT, ATX-I, tentoxin, and TA; A. alternata IMI 354943, AOH, AME, and TA; A. triticina IMI 289962 tentoxin; and A. triticina IMI 289680 AME, ATX-I, and TA.     

Rapid Quantification Method of Three <Emphasis Type="Italic">Alternaria</Emphasis> Mycotoxins in Strawberries

Validation of simple and rapid method for three Alternaria mycotoxins determination including alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) in strawberries is described. The extraction procedure was based on a simple liquid–liquid extraction with ethyl acetate, which provided the highest recoveries and the lowest matrix effect. Analytes were determinated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The obtained relative recoveries were higher than 63 % for the studied mycotoxins in strawberries at the limit of quantification (LQ). Good linearity (r ²?>?0.993) and quantification limits (3–14 ng/g) were obtained. Repeatability, expressed as relative standard deviation, was always lower than 6 %, whereas interday precision was lower than 13 % for the developed method. The method was applied to analyse 24 strawberry samples commercialized in markets of the Valencia city (Spain). Analysed samples were only contaminated with AOH and AME.     

Alternaria toxins in wheat from the Autonomous Province of Vojvodina,Serbia: a preliminary survey

Although Fusarium species remain a main source of mycotoxin contamination of wheat, in recent years, due to the evident climatic changes, other mycotoxigenic fungi have been recognised as important wheat contaminants. Alternaria species, especially A. alternata, have been found as contaminants of wheat as well as wheat-based products. Under favourable conditions A. alternata very often produce alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and others Alternaria toxins. The aim of the present study was to examine the presence of three Alternaria toxins (AOH, AME and TeA) in wheat samples harvested during three years (2011–13). To this end, 92 samples were collected during wheat harvesting from different growing regions of the Autonomous Province of Vojvodina, which represents the most important wheat-growing area in Serbia. The presence of Alternaria toxins was analysed by HPLC with electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-MS/MS). Among all the analysed wheat samples, 63 (68.5%) were contaminated with TeA, 11 (12.0%) with AOH and 6 (6.5%) with AME. Furthermore, the maximum and mean toxin concentrations were 2676 and 92.4 µg kg^?1, 48.9 and 18.6 µg kg^?1, and 70.2 and 39.0 µg kg^?1 for TeA, AOH and AME, respectively. Co-occurrence of three Alternaria toxins in wheat samples was detected in six samples; a combination of two toxins was found in two samples; and 64 samples contained one toxin. The results showed that among 92 analysed wheat samples, only 20 (21.7%) samples were without Alternaria toxins. The presence of Alternaria toxins was also investigated in terms of weather conditions recorded during the period of investigation, as well as with the sampling region. This study represents the first preliminary report of the natural occurrence of Alternaria toxins in wheat (Triticum aestivum) from Serbia.     

The influence of different nitrogen and carbon sources on mycotoxin production in Alternaria alternata

The aim of this study was to determine the influence of different carbon and nitrogen sources on the production of the mycotoxins alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by Alternaria alternata at 28 °C using a semi-synthetic medium (modified Czapek-Dox broth) supplemented with nitrogen and carbon sources. Additionally the effect of shaken and static cultivation on mycotoxin production was tested. Initial experiments showed a clear dependency between nitrogen depletion and mycotoxin production. To assess whether nitrogen limitation in general or the type of nitrogen source triggers the production, various nitrogen sources including several ammonium/nitrate salts and amino acids were tested. In static culture the production of AOH/AME can be enhanced greatly with phenylalanine whereas some nitrogen sources seem to inhibit the AOH/AME production completely. TA was not significantly affected by the choice of nitrogen source. In shaken culture the overall production of all mycotoxins was lower compared to static cultivation. Furthermore tests with a wide variety of carbon sources including monosaccharides, disaccharides, complex saccharides such as starch as well as glycerol and acetate were performed. In shaken culture AOH was produced when glucose, fructose, sucrose, acetate or mixtures of glucose/sucrose and glucose/acetate were used as carbon sources. AME production was not detected. The use of sodium acetate resulted in the highest AOH production. In static culture AOH production was also stimulated by acetate and the amount is comparable to shaken conditions. Under static conditions production of AOH was lower except when cultivated with acetate. In static cultivation 9 of 14 tested carbon sources induced mycotoxin production compared to 4 in shaken culture. This is the first study which analyses the influence of carbon and nitrogen sources in a semi-synthetic medium and assesses the effects of culture conditions on mycotoxin production by A. alternata.     

Toxin production by Alternaria alternata in tomatoes and apples stored under various conditions and quantitation of the toxins by high-performance liquid chromatography

Alternaria alternata strain 8442-3 was inoculated into tomatoes (Lycopersicon esculentum Mill.) and Red Delicious cultivar apples (Malus domestica Borkh.). Half of the lots of each fruit were shrink-wrapped in high-density polyethylene film. Wrapped and unwrapped fruits were incubated under darkness at 4, 15 and 25 degrees C for up to 5 weeks. A high-performance liquid chromatography method was developed to quantitate tenuazonic acid (TeA), alternariol (AOH), alternariol methyl ether (AME), and altenuene (ALT). Shrink-wrapping retarded, but did not completely inhibit growth in tomatoes for 3-7 days. Concentrations of up to 120.6 mg of AOH and 63.7 mg of AME per 100 g of tissue were produced in unwrapped tomatoes stored at 15 degrees C for 4 weeks; 19.0 mg of ALT per 100 g of tomato tissue was produced after 3 weeks at 25 degrees C. AOH, AME and ALT were also produced in unwrapped tomatoes stored at 4 degrees C; however, no TeA was detected in decayed tomatoes, regardless of type of wrapping or storage temperature. Shrink-wrapping resulted in decreased production of AOH, AME, and ALT. Alternaria toxins were not detected in apples stored at 4 and 15 degrees C. The highest concentration of AOH produced (48.8 mg per 100 g of tissue) was in unwrapped apples stored at 25 degrees C for 2 weeks; 12.3 mg per 100 g of tissue of shrink-wrapped apples was detected after 5 weeks of storage at 25 degrees C, while ALT reached 5.7 mg per 100 g after 4 weeks. TeA was not detected in apples infected with A. alternata.     

Contamination of fresh and dried tomato by Alternaria toxins in southern Italy

In the present investigation, fresh and dried tomato samples from markets and packinghouses located in Apulia region (southern Italy) were analysed for Alternaria toxins. All samples proved to be contaminated by tenuazonic acid (TeA); in particular, dried tomatoes were contaminated in the range 425–81,592 µg/kg, while fresh tomatoes were in the range 11–4560 µg/kg. The second most abundant toxin was alternariol monomethyl ether (AME), followed by tentoxin (TEN) and alternariol (AOH). Overall dried tomatoes were more contaminated than fresh ones, although this seemed not directly related to the presence of sodium chloride, utilized in the drying process. Five representative Alternaria isolates within those collected from samples proved to be one Alternaria arborescens (A215) and four Alternaria alternata. Within the latter species, one strain belonged to morphotype tenuissima (A216), and three to alternata (A214, A217 and A218). They were confirmed to produce TeA, AOH, and AME in vitro. This study demonstrates the possible risk for consumers’ health related to the consumption of contaminated fresh and dried tomatoes, and thus the need for suitable control strategies.     

Production of alternaria mycotoxins by Alternaria alternata isolated from weather-damaged wheat

The production of Alternaria mycotoxins by Alternaria alternata isolated from Chinese weathered wheat kernels were first investigated on polished rice and durum wheat grains. These mycotoxins included alternariol (AOH) and its monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), and tenuazonic acid (TA). Of 25 isolates tested, all were AOH and AME producers, 21 (84%) coproduced ALT and ATX-I, and 8 (32%) produced TA in rice culture. TA was the most abundant toxin produced at a level ranging from 1,369 to 3,563 mg/kg. Much smaller amounts of AOH, AME, ALT, and ATX-I were present with average concentrations of 54, 40, 44, and 8 mg/kg, respectively. There were linear correlations between the level of AOH and AME (r = 0.846), alternariols (AOH plus AME) and ALT (r = 0.785), and ATX-I and TA (r = 0.553). Polished rice medium seems to support a bit more production of Alternaria metabolites than wheat but with an insignificant difference in concentrations (P > 0.05). A study of the time-course of toxin production by A. alternata isolates indicated that AOH production began faster than any other toxins monitored, and ALT production exhibited a progressive increase throughout the experiment. TA producers might reveal their considerably higher ability to produce toxin in the field despite their low frequency.     

Alternaria toxins—Still emerging?

Alternaria molds are known to cause the contamination of food with their secondary metabolites, a chemically very heterogeneous group of compounds. Yet, after decades of research on the occurrence and the toxicity of Alternaria toxins in academia, no regulation has been implemented yet, thus leaving these potential food contaminants in the status of so-called “emerging mycotoxins”. However, research on this topic has been far from static, leading to the European Food Safety Authority repeatedly calling for more data on the occurrence and toxicity of genotoxic metabolites such as alternariol (AOH) and its monomethyl ether (AME). To give an overview on recent developments in the field, this comprehensive review summarizes published data and addresses current challenges arising from the chemical complexity of Alternaria’s metabolome, mixture effects and the emergence of novel biological targets like cell membranes or the interaction with different receptors. Besides toxicodynamics, we review recent research on toxicokinetics, including the first in vivo studies which incorporated the rarely investigated—but highly genotoxic—perylene quinones. Furthermore, a particular focus lies on the advances of liquid chromatography/tandem mass spectrometry (LC-MS/MS)-based analytical tools for determining a broader spectrum of Alternaria toxins including modified/masked forms and assessing exposure via human biomonitoring (HBM).     

肉桂醛对链格孢菌生长及非寄主选择性毒素合成的影响

目的 初步探究肉桂醛对葡萄采后链格孢菌菌丝体生长及非寄主选择性毒素合成的影响。方法 采用扫描电镜(scanning electron microscopy, SEM)观察肉桂醛处理后链格孢菌菌丝体的形态结构; 测定处理后菌丝体脂质和麦角固醇含量, 胞外电导率、OD260值和菌丝体荧光强度; 高效液相色谱(high performance liquid chromatography, HPLC)测定处理后链格孢酚(alternariol, AOH)、交链格孢酚单甲醚(alternariol monomethyl ether, AME)含量的变化。结果 扫描电镜结果显示肉桂醛处理后链格孢菌菌丝体形态结构明显被破坏; 脂质和麦角固醇含量显著下降; 胞外电导率和OD260值以及菌丝体荧光强度显著增加; AOH、AME的含量显著下降。 结论 肉桂醛通过破坏细胞膜通透性和完整性来抑制链格孢菌菌丝体生长, 同时体内主要非寄主选择性毒素合成受到抑制。     

Natural occurrence of Alternaria mycotoxins in olives--their production and possible transfer into the oil

A limited survey of the natural occurrence of the major Alternaria mycotoxins, i.e. alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), and tenuazonic acid (TA) has been carried out on olives and related processing products (oil and husks). The toxigenicity of Alternaria strains isolated from olives and the possible mycotoxin transfer into the oil have also been investigated. Four out of 13 olive samples were contaminated by 2 to 4 Alternaria mycotoxins. The highest contamination was found in a badly damaged sample containing 2.9, 2.3, 1.4 and 0.3 mg/kg of AME, AOH, ALT and TA, respectively. No mycotoxins were detected in olive-oil destined for human consumption (6 samples) or olive-husks (3 samples) collected from oil-mills after the first pressing of olives. An oil sample produced in our laboratory by processing the most contaminated olive sample contained AME (0.79 mg/kg) and AOH (0.29 mg/kg). The estimated mycotoxin amount transferred into the oil was 4% for AME, 1.8% for AOH, and zero for ALT and TA (considering 15% the oil yield). Although Alternaria species, mostly A. alternata (Fr.) Keissler, were present at various extent on all the examined olive samples, mycotoxins were only detected in samples of physically damaged olives. The production of mycotoxins by A. alternata isolated from olives was much higher (up to 3 order of magnitude for TA) on rice cultures than on olive cultures.