alternariol-monomethyl-ether and alternariol

Natural dibenzo-α-pyrones (DAPs) can be viewed from two opposite angles. From one angle, the gastrointestinal metabolites urolithins are regarded as beneficial, while from the other, the emerging mycotoxin alternariol and related fungal metabolites are evaluated critically with regards to potential hazardous effects. Thus, the important question is: can the structural characteristics of DAP subgroups be held responsible for distinct bioactivity patterns? If not, certain toxicological and/or pharmacological aspects of natural DAPs might yet await elucidation. Thus, this review focuses on comparing published data on the two groups of natural DAPs regarding both adverse and beneficial effects on human health. Literature on genotoxic, estrogenic, endocrine-disruptive effects, as well as on the induction of the cellular anti-oxidative defense system, anti-inflammatory properties, the inhibition of kinases, the activation of mitophagy and the induction of autophagy, is gathered and critically reviewed. Indeed, comparing published data suggests similar bioactivity profiles of alternariol and urolithin A. Thus, the current stratification into hazardous

Topics: Alternaria; Animals; Anti-Inflammatory Agents, Non-Steroidal; Coumarins; Estrogens; Gastrointestinal Microbiome; Humans; Lactones; Mitophagy; Mutagenicity Tests; Mycotoxins

Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) are the three major Alternaria toxin contaminants in food. In the present study, we conducted their single and combined toxicity analyses using human gastric epithelial cell line (GES-1) that was first exposed to the toxins when they entered the human body. By comparing the cytotoxicity IC50, we found that compared to several other mycotoxins with limit standards there was cytotoxicity DON > OTA > AME > AOH > ZEN > TeA. Further, we obtained combination index (CI)-isobologram equation by the Chou-Talalay method according to a toxin ratio of 1:1:2 and carried out the combined toxicity analysis of the three binary and ternary compounds, and the results showed that AOH + AME + TeA showed synergistic toxic effects. Based on the co-occurring status, we also carried out the combined toxicity analysis of AME and AOH at different ratios and found antagonistic effects at low cytotoxic concentrations as well as synergistic and additive effects at high concentrations. Also, we found that all three and their combinations caused apoptosis, activation of caspase-3 cleavage, activation of DNA damage pathways ATR-Chk1-P53 and ATM-Chk2-P53. In conclusion, we used GES-1 cells to inform the risk of coaction of AOH, AME, and TeA in dietary exposure.

Topics: Alternaria; Epithelial Cells; Food Contamination; Humans; Lactones; Mycotoxins; Tenuazonic Acid; Tumor Suppressor Protein p53

Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus Physalis as a source of anti-inflammatory constituents, the present study aimed at isolation for the first time some endophytic fungi from the medicinal plant Physalis pruinosa.. The endophytic fungi were isolated from the fresh leaves of P. pruinosa then purified and identified by both morphological and molecular methods. Comparative evaluation of the cytotoxic and ex vivo anti-inflammatory activity in addition to gene expression of the three pro-inflammatory indicators (TNF-α, IL-1β and INF-γ) was performed in WBCs treated with lipopolysaccharide (LPS) for the identified endophytes, isolated compounds and the standard anti-inflammatory drug (piroxicam). For prediction of the binding mode of the top-scoring constituents-targets complexes, the Schrödinger Maestro 11.8 package (LLC, New York, NY) was employed in the docking study.. A total of 50 endophytic fungal isolates were separated from P. pruinosa leaves. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters, which were then identified as Stemphylium simmonsii MN401378, Stemphylium sp. MT084051, Alternaria infectoria MT573465, Alternaria alternata MZ066724, Alternaria alternata MN615420 and Fusarium equiseti MK968015. It could be observed that A. alternata MN615420 extract was the most potent anti-inflammatory candidate with a significant downregulation of TNF-α. Moreover, six secondary metabolites, alternariol monomethyl ether (1), 3'-hydroxyalternariol monomethyl ether (2), alternariol (3), α-acetylorcinol (4), tenuazonic acid (5) and allo-tenuazonic acid (6) were isolated from the most potent candidate (A. alternata MN615420). Among the tested isolated compounds, 3'-hydroxyalternariol monomethyl ether showed the highest anti-inflammatory potential with the most considerable reductions in the level of INF-γ and IL-1β. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein (IL-1β, TNF-α and INF-γ)-ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis.. The results obtained suggested alternariol derivatives may serve as naturally occurring potent anti-inflammatory candidates. This study opens new avenues for the design and development of innovative anti-inflammatory drugs that specifically target INF-γ, IL-1β and INF-γ.

Topics: Anti-Inflammatory Agents; Endophytes; Ethers; Molecular Docking Simulation; Physalis; Tenuazonic Acid; Tumor Necrosis Factor-alpha

Mycotoxins are frequent toxic contaminants in foods and beverages, causing a significant health threat. Interactions of mycotoxins with biotransformation enzymes (e.g., cytochrome P450 enzymes, sulfotransferases, and uridine 5'-diphospho-glucuronosyltransferases) may be important due to their possible detoxification or toxic activation during enzymatic processes. Furthermore, mycotoxin-induced enzyme inhibition may affect the biotransformation of other molecules. A recent study described the strong inhibitory effects of alternariol and alternariol-9-methylether on the xanthine oxidase (XO) enzyme. Therefore, we aimed to test the impacts of 31 mycotoxins (including the masked/modified derivatives of alternariol and alternariol-9-methylether) on XO-catalyzed uric acid formation. Besides the in vitro enzyme incubation assays, mycotoxin depletion experiments and modeling studies were performed. Among the mycotoxins tested, alternariol, alternariol-3-sulfate, and α-zearalenol showed moderate inhibitory actions on the enzyme, representing more than tenfold weaker impacts compared with the positive control inhibitor allopurinol. In mycotoxin depletion assays, XO did not affect the concentrations of alternariol, alternariol-3-sulfate, and α-zearalenol in the incubates; thus, these compounds are inhibitors but not substrates of the enzyme. Experimental data and modeling studies suggest the reversible, allosteric inhibition of XO by these three mycotoxins. Our results help the better understanding of the toxicokinetic interactions of mycotoxins.

Topics: Enzyme Inhibitors; Mycotoxins; Sulfates; Xanthine Oxidase

A quick and selective analytical method was developed via LC-MS/MS for the simultaneous quantitation of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) which belong to the large group of secondary metabolites produced by fungi of the genus Alternaria. Cocoa is susceptible to a number of toxin-producing microorganisms, including Aspergillus and Penicillium species. The method relies on a single-step extraction, followed by an easy clean up, dilution of the raw extract and direct analysis. To assess whether cocoa and chocolate products can be a source of Alternaria toxins, a monitoring of cocoa and chocolate products (N = 99) as well as cocoa raw and semi-finished materials (cocoa shells, cocoa masses; N = 10) was performed. As the results, cocoa and products made from cocoa (without other ingredients) are no source of the Alternaria toxins considered here.

Topics: Alternaria; Chocolate; Chromatography, Liquid; Food Contamination; Lactones; Mycotoxins; Tandem Mass Spectrometry; Tenuazonic Acid

Wheat is one of three major food crops in China. Alternaria species can cause spoilage of wheat with consequent mycotoxin accumulation. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are the most common and frequently studied mycotoxins. There are limited regulations placed on Alternaria mycotoxin concentrations worldwide due to the lack of toxicity data available. More data on the levels of mycotoxin contamination are also needed. It is also important to reduce the risks of Alternaria mycotoxins.. One hundred and thirty-two wheat samples were collected from Hebei Province, China, and analyzed for AOH, AME, and TeA. Tenuazonic acid was found to be the predominant Alternaria mycotoxin, especially in flour samples. Studying Alternaria species that cause black-point disease of wheat indicated that Alternaria alternata and Alternaria tenuissima were the dominant species. Most of the Alternaria strains studied produced more than one mycotoxin and TeA was produced at the highest concentration, which may have resulted in the high level of TeA contamination in the wheat samples. Furthermore, magnolol displayed obvious antifungal and antimycotoxigenic activity against Alternaria. This is the first report on the antimycotoxigenic activity of magnolol against Alternaria species.. The Alternaria mycotoxin contamination levels in wheat and wheat products from Hebei Province, China, were correlated with the toxigenic capacity of the Alternaria strains colonizing the wheat. Considering its safety, magnolol could be developed as a natural fungicide in wheat, or as a natural alternative food preservative based on its strong antifungal and antimycotoxigenic activity against Alternaria strains. © 2020 Society of Chemical Industry.

Topics: Alternaria; Biphenyl Compounds; China; Flour; Food Contamination; Fungicides, Industrial; Lactones; Lignans; Plant Diseases; Tenuazonic Acid; Triticum

Alternariol (AOH) and Alternariol monomethyl ether (AME) mycotoxins are found to be present naturally in various food commodities, such as barley, oats, pepper, rye, sorghum, sunflower seeds, tomatoes, and wheat. A few epidemiological studies have correlated the consumption of Alternaria-contaminated cereal grains with higher occurrence of esophageal cancer in Chinese populations. In addition, several studies have reported the toxicological properties of Alternaria mycotoxins. However, surveillance data on AOH and AME occurrence are still limited. Therefore, the goal of this study was to determine the presence of AOH and AME in various commonly consumed, edible oils using HPLC-FLD method. Thirty four percent of samples were found positive for AOH and 35% for AME. Moreover, AOH retained 80% stability, while AME retained 84% stability, after deep frying for 25 min, which is an important factor with respect to Indian cooking style. To the best of our knowledge, this is the first report on the presence of Alternaria mycotoxins in edible oils and their probable dietary intake in Indian population. This surveillance study may help in formulating guidelines for Alternaria mycotoxin levels in India, which are not yet implemented by Food Safety and Standards Authority of India. PRACTICAL APPLICATIONS: At present, no safety guidelines exist for Alternaria mycotoxins in any part of the world. This study will help the regulatory bodies to set permissible levels of Alternaria mycotoxins to safeguard the health of consumers. This study shows that Alternaria mycotoxins are heat stable even after deep frying for 25 min. The data will also help to issue guidelines against exposure of these mycotoxins, keeping in the mind the heat stability factor.

Topics: Dietary Exposure; Edible Grain; Food Contamination; Humans; India; Lactones; Mycotoxins; Plant Oils; Risk Assessment; Temperature

Barley (Hordeum vulgare L.) is one of the most important cereals worldwide, and its quality is affected by fungal contamination such as species of the genus Alternaria. No information is available about the occurrence of Alternaria mycotoxins in Argentinean barley grains, which is of concern, because they can be transferred into malt and beer. The aim of this study was to analyze the occurrence of alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) in malting barley grains from the main producing region of Argentina during the 2014 and 2015 growing seasons.. The most frequent mycotoxin was AOH (64%), which was detected at higher levels (712 μg kg. The results obtained in the present work represent a tool for risk assessment of exposition to these mycotoxins and could be used by food safety authorities to determine the need for their regulation. Furthermore, the establishment of a hazard analysis and critical control point (HACCP) system to minimize fungal and mycotoxin contamination in barley from farm to processing could be apply to ensure food safety. © 2019 Society of Chemical Industry.

Topics: Alternaria; Argentina; Food Contamination; Food Handling; Hordeum; Lactones; Mycotoxins; Seeds

Topics: Alternaria; Animals; Consumer Product Safety; Dose-Response Relationship, Drug; Food Microbiology; Humans; Lactones; Mycotoxins; Risk Assessment; Risk Factors; Structure-Activity Relationship; Tenuazonic Acid; Toxicity Tests

Alternaria mycotoxins, such as tenuazonic acid (TeA), altenuene (ALT), alternariol (AOH), tentoxin (TEN) and alternariol monomethyl ether (AME) are frequently found in foods and may pose a potential risk to human health. Human biomonitoring can help measure our exposure to these mycotoxins, and help us determine if the exposure is changing over time. In this study, a simple liquid-liquid extraction sample preparation procedure followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of five Alternaria mycotoxins in human urine. High recoveries (92.7-103.2%) were obtained for all the tested mycotoxins with relative standard deviations (RSDs, %) of less than 6.4%. The limits of quantification (LOQs) for the analytes in urine ranged from 0.001 to 0.05 ng/mL. The method was successfully applied to investigate the levels of five Alternaria mycotoxins from 135 volunteers. In all of the samples, at least one Alternaria mycotoxin was detected. TeA, AME and AOH were the predominant Alternaria mycotoxins, and the detection rates were 85.9%, 96.3% and 51.9%, respectively.

Topics: Alternaria; Arylsulfatases; Chromatography, High Pressure Liquid; Food Contamination; Glucuronidase; Humans; Lactones; Limit of Detection; Liquid-Liquid Extraction; Mycotoxins; Peptides, Cyclic; Tandem Mass Spectrometry; Tenuazonic Acid

Reference standards for

Topics: Alternaria; Biotransformation; Escherichia coli; Fragaria; Glucosides; Glycosyltransferases; Lactones; Mycotoxins; Plant Proteins

Alternariol (AOH) and alternariol monomethyl ether (AME), produced by Alternaria spp., are the two mycotoxins with the highest outbreak rates in food systems. The purpose of this study was to investigate the removal of AOH and AME from aqueous solutions by inactivated yeast cells. The effects of strains, yeast powder amount, temperature, and pH were evaluated. The kinetics of AOH and AME adsorption on inactivated yeast cells was fitted with four models and a release assay was carried out.. All three tested yeasts could remove AOH and AME. GIM 2.119 was the most effective strain. The reduction rate of both AOH and AME could be as much as 100% with 40 g‧L. The inactivated yeast cells could effectively remove AOH and AME. This was best fitted by the pseudo-second-order model. The release assay suggested that the adsorption of Alternaria mycotoxins was partially reversible. The results of this study provide a theoretical basis for the removal of Alternaria mycotoxins from food systems and are useful for the investigation of the mechanisms involved in mycotoxin adsorption by inactivated yeast cells. © 2020 Society of Chemical Industry.

Topics: Adsorption; Alternaria; Food Contamination; Lactones; Mycotoxins; Saccharomyces cerevisiae; Yeast, Dried

Molds of the genus Alternaria produce several mycotoxins, some of which may pose a threat for health due to their genotoxicity. Due to the lack of adequate toxicological and occurrence data, they are currently not regulated. Interactions between mycotoxins, gut microbiota and food constituents might occur after food ingestion, modifying the bioavailability and, therefore, overall toxicity of mycotoxins. The present work aimed to investigate the impact of in vitro short-term fecal incubation on the in vitro DNA-damaging effects exerted by 5 µg/mL of an Alternaria alternata extract, containing, among others, 15 nM alternariol, 12 nM alternariol monomethyl ether, 241 nM altertoxin II and 301 nM stemphyltoxin III, all of which are known as genotoxic. The involvement of microorganisms, undigested food constituents and soluble substances of human fecal samples in modifying the composition and the genotoxicity of the extract was investigated through the application of LC-MS/MS analysis and comet assays in HT-29 cells. Results showed that the potential of the mycotoxins to induce DNA strand breaks was almost completely quenched, even before anaerobic incubation, by contact with the different fractions of the fecal samples, while the potency to induce formamidopyrimidine DNA glycosylase (FPG)-sensitive sites was only slightly reduced. These effects were in line with a reduction of mycotoxin concentrations found in samples analyzed by LC-MS/MS. Although a direct correlation between the metabolic activity of the gut microbiota and modifications in mycotoxin contents was not clearly observed, adsorptive phenomena to bacterial cells and to undigested food constituents might explain the observed modifications.

Topics: Adult; Alternaria; Benz(a)Anthracenes; Chromatography, Liquid; Comet Assay; DNA Damage; Feces; Female; Food; Food Contamination; Gastrointestinal Contents; Gastrointestinal Microbiome; HT29 Cells; Humans; Lactones; Male; Mutagens; Mycotoxins; Perylene; Tandem Mass Spectrometry

Topics: Alternaria; Biphenyl Compounds; Fungal Proteins; Fungicides, Industrial; Gene Expression Regulation, Fungal; Gene Regulatory Networks; Lactones; Lignans; Mycotoxins; Transcriptome

Large amounts of tomato fruits and derived products are produced in China and may be contaminated by Alternaria mycotoxins, which may have the potential risks for human health. There is thus an increasing interest in reducing the mycotoxins. In the present study, 26 Alternaria strains isolated from tomato black rots were identified according to morphological and molecular grounds, and their mycotoxigenic abilities for alternariol (AOH), alternariol monomethyl-ether (AME) and tenuazonic acid (TeA) were also investigated. The results showed that A. alternate was the predominant species with incidence values of 65.4% (17/26), followed by A. brassicae (7/26) and A. tenuissima (2/26). A. alternate isolates showed the highest capacity for AOH, AME and TeA production among the studied isolates either in vitro or in vivo, suggested that A. alternata may be the most important mycotoxin-producing species in tomato fruits. Thus, UV-C irradiation was used to reduce the mycotoxin produced by A. alternata in our study. The results showed that low dose of UV-C irradiation (0.25 kJ/m

Topics: Alternaria; China; Coumaric Acids; Fruit; Lactones; Mycotoxins; Propionates; Solanum lycopersicum; Tenuazonic Acid; Ultraviolet Rays

To observe the variation in accumulation of Fusarium and Alternaria mycotoxins across a topographically heterogeneous field and tested biotic (fungal and bacterial abundance) and abiotic (microclimate) parameters as explanatory variables.. We selected a wheat field characterized by a diversified topography, to be responsible for variations in productivity and in canopy-driven microclimate. Fusarium and Alternaria mycotoxins where quantified in wheat ears at three sampling dates between flowering and harvest at 40 points. Tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), deoxynivalenol (DON), zearalenone (ZEN) and deoxynivalenol-3-Glucoside (DON.3G) were quantified. In canopy temperature, air and soil humidity were recorded for each point with data-loggers. Fusarium spp. as trichothecene producers, Alternaria spp. and fungal abundances were assessed using qPCR. Pseudomonas fluorescens bacteria were quantified with a culture based method. We only found DON, DON.3G, TeA and TEN to be ubiquitous across the whole field, while AME, AOH and ZEN were only occasionally detected. Fusarium was more abundant in spots with high soil humidity, while Alternaria in warmer and drier spots. Mycotoxins correlated differently to the observed explanatory variables: positive correlations between DON accumulation, tri 5 gene and Fusarium abundance were clearly detected. The correlations among the others observed variables, such as microclimatic conditions, varied among the sampling dates. The results of statistical model identification do not exclude that species coexistence could influence mycotoxin production.. Fusarium and Alternaria mycotoxins accumulation varies heavily across the field and the sampling dates, providing the realism of landscape-scale studies. Mycotoxin concentrations appear to be partially explained by biotic and abiotic variables.. We provide a useful experimental design and useful data for understanding the dynamics of mycotoxin biosynthesis in wheat.

Topics: Alternaria; Food Contamination; Fusarium; Glucosides; Lactones; Microclimate; Mycotoxins; Pseudomonas fluorescens; Secondary Metabolism; Soil Microbiology; Tenuazonic Acid; Trichothecenes; Triticum; Zearalenone

Alternaria mycotoxins frequently contaminate agricultural crops and may impact animal and human health. However, data on mammalian metabolism and potential biomarkers of exposure for human biomonitoring (HBM) are scarce. Here, we report the preliminary investigation with respect to metabolism and excretion of Alternaria toxins in Sprague Dawley rats. Four animals were housed in metabolic cages for 24 h after gavage administration of an Alternaria alternata culture extract containing ten known toxins. LC-MS/MS analysis of 17 Alternaria toxins in urine and fecal samples allowed to gain first insights regarding xenobiotic metabolism and excretion rates. Alternariol (6-10%), alternariol monomethyl ether (AME, 6-7%) and tenuazonic acid (up to 55%) were recovered in urine and fecal samples (9%, 87%, 0.3%, respectively), while perylene quinones administered at comparatively high levels, were either determined at very low levels (up to 0.5% altertoxin I in urine and 15% in feces; 0.2% alterperylenol in urine and 3% in feces) or not at all (altertoxin II, stemphyltoxin III). AME-3-sulfate, which was not present in the administered extract, was determined in urine, representing up to 23% of the AME intake. Critical evaluation of the applied sample preparation protocol and LC-MS/MS analysis revealed interesting preliminary results and information crucial for improving follow-up experiments.

Topics: Alternaria; Animals; Benz(a)Anthracenes; Chromatography, Liquid; Feces; Lactones; Limit of Detection; Male; Mycotoxins; Perylene; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry; Tenuazonic Acid

The group of the small-spored Alternaria species is particularly relevant in foods due to its high frequency and wide distribution in different crops. These species are responsible for the accumulation of mycotoxins and bioactive secondary metabolites in food. The taxonomy of the genus has been recently revised with particular attention on them; several morphospecies within this group cannot be segregated by phylogenetic methods, and the most recent classifications proposed to elevate several phylogenetic species-groups to the taxonomic status of section. The purpose of the present study was to compare the new taxonomic revisions in Alternaria with secondary metabolite profiles with special focus on sections Alternaria and Infectoriae and food safety. A total of 360 small-spored Alternaria isolates from Argentinean food crops (tomato fruit, pepper fruit, blueberry, apple, wheat grain, walnut, pear, and plum) was morphologically identified to species-group according to Simmons (2007), and their secondary metabolite profile was determined. The isolates belonged to A. infectoria sp.-grp. (19), A. tenuissima sp.-grp. (262), A. arborescens sp.-grp. (40), and A. alternata sp.-grp. (7); 32 isolates, presenting characteristics overlapping between the last three groups, were classified as Alternaria sp. A high chemical diversity was observed; 78 different metabolites were detected, 31 of them of known chemical structure. The isolates from A. infectoria sp.-grp. (=Alternaria section Infectoriae) presented a specific secondary metabolite profile, different from the other species-groups. Infectopyrones, novae-zelandins and phomapyrones were the most frequent metabolites produced by section Infectoriae. Altertoxin-I and alterperylenol were the only compounds that these isolates produced in common with members of section Alternaria. None of the well-known Alternaria toxins, considered relevant in foods, namely alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), tentoxin (TEN) or altenuene (ALT), were produced by isolates of this section. On the other hand, strains from section Alternaria (A. tenuissima, A. arborescens, and A. alternata sp.-grps.) shared a common metabolite profile, indistinguishable from each other. AOH, AME, ALT, TEN, and TeA were the most frequently mycotoxins produced, together with pyrenochaetic acid A and altechromone A. Alternaria section Alternaria represents a substantial risk in food, since their members in all types

Topics: Alternaria; Argentina; Blueberry Plants; Crops, Agricultural; Food Contamination; Food Microbiology; Fruit; Juglans; Lactones; Malus; Mycotoxins; Peptides, Cyclic; Phylogeny; Piper nigrum; Prunus domestica; Pyrus; Secondary Metabolism; Solanum lycopersicum; Tenuazonic Acid; Triticum

The filamentous fungus Alternaria alternata is a potent producer of many toxic secondary metabolites, which contaminate food and feed. The most prominent one is the polyketide-derived alternariol (AOH) and its derivative alternariol monomethyl ether (AME). Here, we identified the gene cluster for the biosynthesis of AOH and AME by CRISPR/Cas9-mediated gene inactivation of several biosynthesis genes in A. alternata and heterologous expression of the gene cluster in Aspergillus oryzae. The 15 kb-spanning gene cluster consists of a polyketide synthase gene, pksI, an O-methyltransferase, omtI, a FAD-dependent monooxygenase, moxI, a short chain dehydrogenase, sdrI, a putative extradiol dioxygenase, doxI and a transcription factor gene, aohR. Heterologous expression of PksI in A. oryzae was sufficient for AOH biosynthesis. Co-expression of PksI with different tailoring enzymes resulted in AME, 4-hydroxy-alternariol monomethyl ether (4-OH-AME), altenusin (ALN) and altenuene (ALT). Hence, the AOH cluster is responsible for the production of at least five different compounds. Deletion of the transcription factor gene aohR led to reduced expression of pksI and delayed AOH production, while overexpression led to increased expression of pksI and production of AOH. The pksI-deletion strain displayed reduced virulence on tomato, citrus and apple suggesting AOH and the derivatives as virulence and colonization factors.

Topics: Alternaria; Infections; Lactones; Methyltransferases; Multigene Family; Mycotoxins; Polyketide Synthases; Solanum lycopersicum; Virulence

Topics: Alternaria; Beijing; Chromatography, High Pressure Liquid; Chromatography, Liquid; Drinking Water; Lactones; Limit of Detection; Mycotoxins; Solid Phase Extraction; Tandem Mass Spectrometry; Water Pollutants

The stability of two Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), has been investigated during the food processing of tomato products simulating commercial processing conditions. The production stages assessed were the storage of raw fruits, fruit washing, and thermal processing. It was observed that time of storage significantly reduced the initial concentration of AOH, but only if tomatoes were stored at 35 °C. For AME, 12 h were sufficient to reduce the initial concentration, regardless of the temperature at which samples were stored (25, 30 and 35 °C). The washing step achieved the highest reduction of AOH and AME. This reduction was even more efficient when using sodium hypochlorite solutions. Finally, during the heat treatment (80-110 °C), results showed that heating tomato samples at 100 and 110 °C, significantly affected AOH stability, though AME seemed to not be affected by these thermal processes.

Topics: Alternaria; Food Contamination; Food Handling; Fruit; Hot Temperature; Lactones; Mycotoxins; Solanum lycopersicum

Xenoestrogenic mycotoxins may contaminate food and feed posing a public health issue. Besides the zearalenone group, the Alternaria toxin alternariol (AOH) has been described as a potential mycoestrogen. However, the estrogenicity of Alternaria toxins is still largely overlooked and further data are needed to better describe the group toxicity. In the frame of risk assessment, mixed in silico/in vitro approaches already proved to be effective first-line analytical tools. An integrated in silico/in vitro approach was used to investigate the effects of metabolic and chemical modifications on the estrogenicity of AOH. Among the considered modifications, methylation was found critical for enhancing estrogenicity (as seen for alternariol monomethyl ether (AME)) while hydroxylation and glucuronidation had the opposite effect (as seen for 4-hydroxy AOH and 4-hydroxy AME). The structure-activity relationship analysis provided the structural rationale. Our results provide insights to design more efficient risk assessment studies expanding knowledge over the group toxicity.

Topics: Alternaria; Computer Simulation; Estrogens; Humans; Hydroxylation; Lactones; Methylation; Mycotoxins; Receptors, Estrogen; Risk Assessment; Structure-Activity Relationship; Xenobiotics

Topics: Alternaria; Chromatography, Reverse-Phase; Food Analysis; Food Microbiology; Fruit; Lactones; Mycotoxins; Peptides, Cyclic; Solanum lycopersicum; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tenuazonic Acid

Interest in endophytes as natural sources for new medicines was inspired by the discovery of paclitaxel-producing endophytic fungi. This study investigated the anti-cancer activity of extracts of endophytes isolated from two Australian plants, Eremophila longifolia (EL) and Eremophila maculata (EM). Endophytes were isolated from surface-sterilised leaf tissue, grown as pure cultures and identified by sequencing of Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. To determine cytotoxicity, two leukaemic (MOLT-4, T-cell leukaemia; PreB-697, Pre-B leukaemia), a lung cancer cell line (A549) and a normal human fibroblast cell line were treated with endophyte extracts to assess cytotoxicity in relation to alternariol monomethyl ether (AME) and alternariol (AOH). Endophyte extracts that showed cell cytotoxicity were analysed by UV-HPLC to determine the metabolites. Pure AME and AOH, three extracts form Alternaria sp. (EM-6, EM-7 and EM-9) and one from Preussia minima (EL-14) were cytotoxic to the cancer cell lines. All cytotoxic endophytes contained AME and AOH, the most cytotoxic endophyte EM-6 also contained two unique peaks. These data indicate that these four endophyte extracts may have anti-cancer properties due to the presence of AME and AOH; however, the unique compounds found in the EM-6 extract may be exclusively cytotoxic and warrant further investigation.

Topics: Antineoplastic Agents; Australia; Cell Line, Tumor; Cell Survival; Endophytes; Eremophila Plant; Fungi; Humans; Lactones; Phylogeny

Topics: Alternaria; Animals; Cell Cycle Checkpoints; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Epithelial Cells; Host-Pathogen Interactions; Humans; Immunity, Innate; Immunomodulation; Inflammation; Lactones; Lipopolysaccharides; Macrophages; Mice; Mycotoxins

Topics: Alternaria; Benz(a)Anthracenes; Chromatography, High Pressure Liquid; Food Contamination; Helianthus; Lactones; Mycotoxins; Seeds; South Africa; Tandem Mass Spectrometry; Tenuazonic Acid

Members of the Alternaria genus produce various toxins whose occurrence in agricultural commodities is a major concern for humans and the environment. The present study developed a simple and efficient matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the rapid detection of Alternaria toxins.. A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) by MALDI-TOF MS was developed. Different solid phase extraction (SPE) clean-up methods were tried to optimize the purification of wheat matrix, and an optimal extraction method was designed to recover the three Alternaria toxins. In addition, various MALDI matrices were examined and α-cyano-4-hydroxycinnamic acid (CHCA) matrix gave good repeatability for all three Alternaria toxins.. This is the first study to report the detection of three important Alternaria toxins concurrently using MALDI-TOF MS and opens up the possibility of rapid screening of Alternaria toxins in several other cereals and food products. © 2016 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2016 Society of Chemical Industry.

Topics: Alternaria; Edible Grain; Lactones; Mycotoxins; Peptides, Cyclic; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Studies on the genotoxicity of Alternaria mycotoxins focus primarily on the native compounds. Alternariol (AOH) and its methyl ether (AME) have been reported to represent substrates for cytochrome P450 enzymes, generating hydroxylated metabolites. The impact of these phase I metabolites on genotoxicity remains unknown. In the present study, the synthesis and the toxicological effects of the metabolites 4-hydroxy alternariol (4-OH-AOH) and 4-hydroxy alternariol monomethyl ether (4-OH-AME) are presented and compared to the effects of the parent molecules. Although the two phase I metabolites contain a catecholic structure, which is expected to be involved in redox cycling, only 4-OH-AOH increased reactive oxygen species (ROS) in human esophageal cells (KYSE510), 4 times more pronounced than AOH. No ROS induction was observed for 4-OH-AME, although the parent compound showed some minor impact. Under cell-free conditions, both metabolites inhibited topoisomerase II activity comparable to their parent compounds. In KYSE510 cells, both metabolites were found to enhance the level of transient DNA-topoisomerase complexes in the ICE assay. Although the level of ROS was significantly increased by 4-OH-AOH, neither DNA strand breaks nor enhanced levels of formamidopyrimidine-DNA-glycosylase (FPG)-sensitive sites were observed. In contrast, AOH induced significant DNA damage in KYSE510 cells. Less pronounced or even absent effects of hydroxylated metabolites compared to the parent compounds might at least partly be explained by their poor cellular uptake. Glucuronidation as well as sulfation appear to have only a minor influence. Instead, methylation of 4-OH-AOH seems to be the preferred way of metabolism in KYSE510 cells, whereby the toxicological relevance of the methylation product remains to be clarified.

Topics: Antigens, Neoplasm; Cell Line, Tumor; Cell-Free System; DNA Damage; DNA Topoisomerases, Type II; DNA-Binding Proteins; Esophageal Neoplasms; Humans; Hydroxylation; Lactones; Mitochondria; Mutagenicity Tests; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species

To study the effects of water activity (a. Interacting conditions of a. The optimum and marginal conditions for growth and mycotoxin production by A. alternata on a SN media similar to grape composition were in agreement, but certain stressful conditions for growth evaluated also promote mycotoxin production.. Temperature and a

Topics: Alternaria; Argentina; Lactones; Mycotoxins; Temperature; Tenuazonic Acid; Vitis; Water; Wine

Six new dibenzo-α-pyrones, rhizopycnolides A (1) and B (2) and rhizopycnins A-D (3-6), together with eight known congeners (7-14), were isolated from the endophytic fungus Rhizopycnis vagum Nitaf22 obtained from Nicotiana tabacum. The structures of the new compounds were unambiguously elucidated using NMR, HRESIMS, TDDFT ECD calculation, and X-ray crystallography data. Rhizopycnolides A (1) and B (2) feature an uncommon γ-butyrolactone-fused dibenzo-α-pyrone tetracyclic skeleton (6/6/6/5), while rhizopycnin B (4) was the first amino group containing dibenzo-α-pyrone. Rhizopycnolides A (1) and B (2) are proposed to be biosynthesized from polyketide and tricarboxylic acid cycle pathways. The isolated compounds were tested for their antibacterial, antifungal, and cytotoxic activities. Among them, rhizopycnolide A (1), rhizopycnins C (5) and D (6), TMC-264 (8), penicilliumolide D (11), and alternariol (12) were active against the tested pathogenic bacteria Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas lachrymans, Ralstonia solanacearum, Staphylococcus hemolyticus, and Xanthomonas vesicatoria with MIC values in the range 25-100 μg/mL. Rhizopycnin D (6) and TMC-264 (8) strongly inhibited the spore germination of Magnaporthe oryzae with IC50 values of 9.9 and 12.0 μg/mL, respectively. TMC-264 (8) showed potent cytotoxicity against five human cancer cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780) with IC50 values of 3.2-7.8 μM.

Topics: 4-Butyrolactone; Agrobacterium tumefaciens; Anti-Bacterial Agents; Antifungal Agents; Ascomycota; Bacillus subtilis; Chromones; Crystallography, X-Ray; Drug Screening Assays, Antitumor; HCT116 Cells; Hep G2 Cells; Heterocyclic Compounds, 3-Ring; Humans; Lactones; Microbial Sensitivity Tests; Molecular Structure; Nicotiana; Polyketides; Pseudomonas; Ralstonia solanacearum; Staphylococcus; Xanthomonas vesicatoria

The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) have been reported previously to act as activators of the aryl hydrocarbon receptor (AhR) in murine hepatoma cells, thus enhancing the expression of cytochrome P450 (CYP) 1A monooxygenases. Concomitantly, both benzopyrones represent substrates of CYP1A, giving rise to catecholic metabolites. The impact of AOH and AME on CYP1A expression in human cells of different tissue origin colon (HT29), esophagus (KYSE510), liver (HepG2) and their effects on cell viability, generation of reactive oxygen species (ROS) and DNA integrity were investigated. ROS production was induced by both mycotoxins in all cell lines with AOH exhibiting the highest potency in esophageal cells concomitant with the most prominent CYP1A induction level. Of note, altertoxin-II (ATX-II), the more potent DNA-damaging mutagen formed by Alternaria alternata, induces CYP1A even at significant lower concentrations. AhR-siRNA knockdown in human esophageal cells supported the hypothesis of AhR-mediated CYP1A1 induction by AOH. However, DNA damage was minor at CYP1A1-inducing AOH concentrations. AhR-depletion did not affect the DNA-damaging properties of AOH indicating no substantial impact of AhR in this regard. However, in combination with xenobiotics prone to metabolic activation by CYP1A the induction of CYP1A by Alternaria toxins deserves further attention.

Topics: Alternaria; Benz(a)Anthracenes; Cell Line, Tumor; Cell Survival; Comet Assay; Cytochrome P-450 CYP1A1; DNA Damage; Gene Expression Regulation; Hep G2 Cells; HT29 Cells; Humans; Lactones; Mycotoxins; Reactive Oxygen Species; Receptors, Aryl Hydrocarbon

Alternaria arborescens has been reported as a common fungal species invading tomatoes and is capable of producing several mycotoxins in infected plants, fruits and in agricultural commodities. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are some of the main Alternaria mycotoxins that can be found as contaminants of food. This species can produce these toxic metabolites together with AAL toxins (Alternaria alternata f. sp. lycopersicum toxins), which can act as inhibitors of sphingolipid biosynthesis. The objective of this study was to determine the effect of water activity (aw, 0.995, 0.975, 0.950) and temperature (6, 15, 20, 25 and 30 °C) on mycotoxin production by A. arborescens on a synthetic tomato medium. The optimum production of AOH and AME occurred at 0.975 aw after 40 days of incubation at 30 °C. The maximum TeA accumulation was observed at 0.975 aw and 25 °C and at 0.950 aw and 30 °C. AAL TA was produced in higher quantities at 0.995 aw and 30 °C. At 6 °C no quantifiable levels of AOH or AME were detected, but significant amounts of TeA were produced at 0.975 aw. In general, high aw levels and high temperatures were favorable for mycotoxin production. The greatest accumulation of all four toxins occurred at 0.975 aw and 30 °C. The results obtained here could be extrapolated to evaluate the risk of tomato fruits and tomato products contamination caused by these toxins.

Topics: Alternaria; Food Contamination; Food Microbiology; Fruit; Lactones; Mycotoxins; Solanum lycopersicum; Sphingolipids; Temperature; Tenuazonic Acid; Water

Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are some of the main Alternaria mycotoxins that can be found as contaminants in food materials. The objective of this study was to develop a pretreatment method with countercurrent chromatography (CCC) for enrichment and cleanup of trace Alternaria mycotoxins in food samples prior to high-performance liquid chromatography (HPLC) analysis. An Analytical CCC instrument with a column volume 22.5mL was used, and a two-phase solvent system composed of ethyl acetate and water modified with 6% [HOOMIM][Cl] in mass to volume ratio was selected. Under the optimized CCC operation conditions, trace amounts of AOH, AME, and TeA in large volume of liquid sample were efficiently extracted and enriched in the stationary phase, and then eluted out just by reversing the stationary phase as mobile phase in the opposite flowing direction tail-to-head. The enrichment and elution strategies are unique and can be fulfilled online with high enrichment factors (87-114) and high recoveries (81.14-110.94%). The method has been successively applied to the determination of Alternaria mycotoxins in real apple juice and wine samples with the limits of detection (LOD) in the range of 0.03-0.14μgL(-1). Totally 12 wine samples and 15 apple juice samples from the local market were analyzed. The detection rate of AOH and AME in both kinds of the samples were more than 50%, while TeA was found in relatively high level of 1.75-49.61μgL(-1) in some of the apple juice samples. The proposed method is simple, rapid, and sensitive and could also be used for the analysis and monitoring of Alternaria mycotoxin in other food samples.

Topics: Alternaria; Chromatography, High Pressure Liquid; Chromatography, Liquid; Countercurrent Distribution; Fruit and Vegetable Juices; Ionic Liquids; Lactones; Malus; Mycotoxins; Tenuazonic Acid; Wine

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.

Topics: Alternaria; Chromatography, High Pressure Liquid; Food Contamination; Food Inspection; Food Preservation; Food Storage; Fragaria; Fruit; Hydrogen-Ion Concentration; Lactones; Liquid-Liquid Extraction; Mutagens; Mycotoxins; Peptides, Cyclic; Refrigeration; Spain; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Teratogens

It is well known that the type and the availability of nitrogen have a great influence on the biosynthesis of certain mycotoxins. Here it is shown that some amino acids have no influence, some others strongly support and a third group inhibits the biosynthesis of ochratoxin (OTA) by Penicillium nordicum even in a complex medium, such as PDA. Arginine (Arg) is one of the strong OTA inhibiting amino acids. It was shown that Arg not only inhibits OTA in Penicillium but also citrinin (CIT) biosynthesis in Penicillium verrucosum, Penicillium expansum and Penicillium citrinum and alternariol (AOH), alternariol monomethylether (AME) and tenuazonic acid (TeA) biosynthesis in Alternaria alternata. The minimal inhibitory concentration of Arg differs depending on the mycotoxin and the species analysed. However, the OTA biosynthesis by P. verrucosum and P. nordicum was most sensitive. Growth, on the other hand, was much less affected by Arg. Urea, a metabolite of Arg catabolism, shows a similar inhibitory activity. In wheat medium containing 50mM Arg almost no OTA was produced by Penicillium, in contrast to plain wheat medium.

Topics: Alternaria; Arginine; Citrinin; Lactones; Mycotoxins; Ochratoxins; Penicillium; Tenuazonic Acid; Triticum; Urea

This study represents the first report about possibility of reduction of Alternaria toxins in wheat using the extrusion process. Effects of extrusion processing parameters - moisture content (w=16, 20, 24g/100g), feeding rate (q=15, 20, 25kg/h), and screw speed (v=300, 390, 480rpm), on reduction rate of tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME), in whole wheat flour were investigated. Temperature ranged between 111.1 and 160.8°C, while the absolute pressure was from 0.17 to 0.23MPa. The simultaneous influence of w and v was the most important for TeA reduction (p<0.05), while v and q were the most influential for AOH reduction (p<0.01). Level of AME reduction was mostly influenced by w and v (p<0.10). Optimal parameters for reduction of all three Alternaria toxins were as follows: w=24g/100g, q=25kg/h, v=390rpm, with a reduction of 65.6% for TeA, 87.9% for AOH and 94.5% for AME.

Topics: Alternaria; Flour; Food Contamination; Food Handling; Food Microbiology; Lactones; Mycotoxins; Pilot Projects; Principal Component Analysis; Reproducibility of Results; Tandem Mass Spectrometry; Temperature; Tenuazonic Acid; Triticum; Whole Grains

Ethylene is a naturally produced plant regulator involved in several plant functions, such as regulation of fruit ripening. Inhibition of ethylene perception by using 1-methylcyclopropene (1-MCP) slows down the ripening of the fruit maintaining its quality and freshness. The use of 1-MCP is a commercial strategy commonly used in the food industry to extend the postharvest life of several fruits, including tomatoes. To assess how 1-MCP affected infection by Alternaria alternata on tomatoes, three different cultivars were artificially inoculated with 5μL of an A. alternata conidial suspension (10(5)conidia/mL). Tomatoes were treated with 0.6μL/L of 1-MCP for 24h. Spiked but untreated tomatoes were considered controls. Then, fruit were stored 6days at 10°C and one more week at 20°C to simulate shelf-life. Fungal growth development and mycotoxin production (alternariol, AOH and alternariol monomethyl ether, AME) were assessed both on the first and on the second week. After the first 6days at 10°C, in just one variety the black mold disease was higher in the 1-MCP treated samples. However, after two weeks of storage, in all cases, tomatoes treated with 1-MCP showed more significant fungal growth disease. Regarding mycotoxin production, no large differences were observed among different treatments, which was corroborated with gene expression analysis of pksJ, a gene related to AOH and AME biosynthesis.

Topics: Alternaria; Cyclopropanes; Food Microbiology; Food Preservation; Humans; Lactones; Solanum lycopersicum

Topics: Alternaria; Lactones; Molecular Structure; Mycotoxins; Nicotiana

The glycogen-synthase-kinase 3 (GSK-3) is an important target in drug discovery. This enzyme is involved in the signaling pathways of type 2 diabetes, neurological disorders, cancer, and other diseases. Therefore, inhibitors of GSK-3 are promising drug candidates for the treatment of a broad range of diseases. Here we report pannorin (

Topics: Coumarins; Drug Discovery; Enzyme Inhibitors; Fungi; Glycogen Synthase Kinase 3 beta; Lactones; Naphthols; Protein Kinase Inhibitors; Pyrones; Signal Transduction; Thiadiazoles

Mycotoxins are naturally occurring contaminants encountered at high levels in a wide variety of agricultural products intended for human and animal consumptions. Various Alternaria mycotoxins may occur simultaneously in small grain cereals. Considering the concomitant production of alternariol (AOH) and alternariol monomethyl ether (AME), it is expected that humans and animals are exposed to the mixture rather than to individual compounds. Therefore, we studied the interactive effects of binary mixture of alternariols (AOH and AME) on the human intestinal cell line, HCT116 cells. Exposure of HCT116 cells to low cytotoxic alternariols doses, resulted in a moderate cytotoxicity manifested by a loss in the cell viability mediated by an activation of the mitochondrial apoptotic process, associated with the opening of mitochondrial permeability transition pore (PTP) and the loss of the mitochondrial transmembrane potential (ΔΨm). However, when combined, they exert a significant increase in their toxic potential. Altogether, our study showed that AOH and AME combination is obviously additive.

Topics: Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; HCT116 Cells; Humans; Lactones; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oxidative Stress; Reactive Oxygen Species; Risk Assessment; Time Factors

Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece. The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressiveness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequencies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial inoculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation at two different temperatures (2 and 25°C) showed that fruit of Fuji variety were the most susceptible and fruit of Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species. Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially inoculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD). The results showed that most of the isolates of both species were able to produce all the 3 metabolites both in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of apple by-products.

Topics: Alternaria; Chromatography, High Pressure Liquid; Fruit; Greece; Lactones; Malus; Mycotoxins; Peptides, Cyclic

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.

Topics: Alternaria; Chromatography, High Pressure Liquid; Food Contamination; Lactones; Mycotoxins; Serbia; Tandem Mass Spectrometry; Tenuazonic Acid; Triticum

Fungi belonging to the genus Alternaria are common pathogens of fruit and vegetables with some species able to produce secondary metabolites dangerous to human health. Twenty-eight Alternaria isolates from rocket and cabbage were investigated for their mycotoxin production. Five different Alternaria toxins were extracted from synthetic liquid media and from plant material (cabbage, cultivated rocket, cauliflower). A modified Czapek-Dox medium was used for the in vitro assay. Under these conditions, more than 80% of the isolates showed the ability to produce at least one mycotoxin, generally with higher levels for tenuazonic acid. However, the same isolates analyzed in vivo seemed to lose their ability to produce tenuazonic acid. For the other mycotoxins; alternariol, alternariol monomethyl ether, altenuene and tentoxin a good correlation between in vitro and in vivo production was observed. In vitro assay is a useful tool to predict the possible mycotoxin contamination under field and greenhouse conditions.

Topics: Alternaria; Brassica; Chromatography, High Pressure Liquid; Food Contamination; Food Microbiology; Lactones; Limit of Detection; Mycotoxins; Peptides, Cyclic; Tandem Mass Spectrometry; Tenuazonic Acid; Vegetables

The mycotoxins alternariol (AOH) and alternariol-9-O-methyl ether (AME) carry three and two phenolic hydroxyl groups, respectively, which makes them candidates for the formation of conjugated metabolites in plants. Such conjugates may escape routine methods of analysis and have therefore been termed masked or, more recently, modified mycotoxins. We report now that AOH and AME are extensively conjugated in suspension cultures of tobacco BY-2 cells. Five conjugates of AOH were identified by MS and NMR spectroscopy as β-D-glucopyranosides (attached in AOH 3- or 9-position) as well as their 6'-malonyl derivatives, and as a gentiobiose conjugate. For AME, conjugation resulted in the d-glucopyranoside (mostly attached in the AME 3-position) and its 6'- and 4'-malonyl derivatives. Pronounced differences were noted for the quantitative pattern of AOH and AME conjugates as well as for their phytotoxicity. Our in vitro study demonstrates for the first time that masked mycotoxins of AOH and AME can be formed in plant cells.

Topics: Alternaria; Cells, Cultured; Lactones; Mycotoxins; Nicotiana

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.

Topics: Alternaria; China; Chromatography, High Pressure Liquid; Chromatography, Liquid; Food Contamination; Lactones; Mycotoxins; Tandem Mass Spectrometry; Tenuazonic Acid; Triticum

Chemical investigation of an acetonitrile fraction from the endophytic fungus Phomopsis sp. led to the isolation of the new natural product 2-hydroxy-alternariol (7) together with the known compounds cytochalasins J (1) and H (2), 5'-epialtenuene (3) and the mycotoxins alternariol monomethyl ether (AME, 4), alternariol (AOH, 5) and cytosporone C (6). The structure of the new compound was elucidated by using 1-D and 2-D NMR (nuclear magnetic resonance) and high resolution mass spectrometry. The cytochalasins J (1) and H (2) and AOH (5) exhibited potent inhibition of the total ROS (reactive oxygen species) produced by stimulated human neutrophils and acted as potent potential anti-inflammatory agents. Moreover, cytochalasin H (2) demonstrated antifungal and acetylcholinesterase enzyme (AChE) inhibition in vitro.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antifungal Agents; Antioxidants; Ascomycota; Cholinesterase Inhibitors; Cytochalasins; Drug Evaluation, Preclinical; Endophytes; Humans; Lactones; Magnetic Resonance Spectroscopy; Molecular Structure; Mycotoxins; Neutrophils; Reactive Oxygen Species; Secondary Metabolism; Senna Plant; Sesquiterpenes

The mycotoxin alternariol (AOH) has been reported to possess genotoxic properties, inducing enhanced levels of DNA damage after only 1 h of incubation. In the present study we addressed the question whether the induction of oxidative stress might contribute to the genotoxic effects of AOH or its naturally occurring monomethylether (AME). In the dichlorofluorescein (DCF) assay, treatment of HT29 cells for 1 h enhanced the formation of dichlorofluorescein, indicative for ROS formation. The total glutathione (tGSH) was transiently decreased. In accordance with the results of the DCF assay, AOH and AME enhanced the proportion of the transcription factor Nrf2 in the nucleus. Concomitantly, the Nrf2/ARE-dependent genes γ-glutamylcysteine ligase (γ-GCL) and glutathione-S-transferase (GSTA1/2) showed enhanced transcript levels. After 24 h of incubation this effect was also reflected on the protein level by an increase of GST activity. However, in spite of the positive DCF assay and the activation of the redox-sensitive Nrf2/ARE-pathway, the level of oxidative DNA damage, measured in the comet assay by the addition of formamidopyrimidine-DNA-glycosylase (fpg) remained unaffected. Of note, after 3 h of incubation no significant DNA damaging potential of AOH and AME was detectable, indicating either inactivation of the compounds or enhanced DNA repair. In summary, the mycotoxins AOH and AME were found to modulate the redox balance of HT29 cells but without apparent negative effect on DNA integrity.

Topics: Alternaria; Comet Assay; Culture Media, Serum-Free; DNA Damage; Gene Expression Regulation; Glutathione; HT29 Cells; Humans; Lactones; MAP Kinase Signaling System; Molecular Structure; Mycotoxins; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress

Since there is an increasing demand on the world market for alternative crops suitable for organic production, spelt wheat (Triticum aestivum spp. spelta L.) is a highly attractive farming option. Alternaria species are widespread and infect a great variety of economically important crops. Certain species are known producers of mycotoxins. The aim of this study was to assess the protective effect of hulls covering the spelt kernels on Alternaria toxins.. Alternariol (AOH) and alternariol monomethyl ether (AME) were evaluated in hulls and dehulled kernels after plant inoculation with one A. alternata and two different A. tenuissima isolates. Mycotoxins were determinated using high-performance liquid chromatography with dioade array detection. The detected levels of AOH and AME were four times higher in hulls compared to kernels in inoculation treatments. AOH was registered at levels ranging from 227 to 331 µg kg(-1) in dehulled kernels and from 433 up to 1647 µg kg(-1) in hulls. AME was predominant toxin detected in the range of 277 to 398 µg kg(-1) in dehulled kernels and from 1844 to 2183 µg kg(-1) in hulls, with highly significant difference to water control treatment.. Obtained results indicate the significantly higher concentrations of Alternaria toxins in hulls than in dehulled kernels which implicate the possible protective effect of spelt wheat hulls.

Topics: Alternaria; Food Contamination; Lactones; Mycotoxins; Seeds; Triticum

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.

Topics: Argentina; Beverages; Chromatography, High Pressure Liquid; Lactones; Sensitivity and Specificity; Specimen Handling

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.

Topics: Alternaria; Argentina; Chromatography, High Pressure Liquid; Food Contamination; Glycine max; Lactones; Mycotoxins

The Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME) are potential carcinogens. As planar compounds, AOH and AME are preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore, we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa-1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa-1c1c7 cells but not in cells with inactivated AhR (Hepa-1c1c12) or ARNT (Hepa-1c1c4). AOH and AME did not increase the production of reactive oxygen species but reduced cell counts in Hepa-1c1c7 cells after 24 and 48 h. This effect, however, was independent of AhR/ARNT. At 48 h, AOH and AME increased apoptosis dependent on AhR and ARNT. In conclusion, AOH and AME are novel inducers of the AhR/ARNT pathway, which mediates induction of CYP1A1 and apoptosis and might thereby contribute to the toxicity of these mycotoxins.

Topics: Animals; Aryl Hydrocarbon Receptor Nuclear Translocator; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Cytochrome P-450 CYP1A1; Enzyme Induction; Hepatocytes; Lactones; Mice; Mycotoxins; Receptors, Aryl Hydrocarbon

For studies on the aryl hydrocarbon receptor (AhR)-dependent toxicity of the mycotoxins alternariol (AOH) and alternariol methyl ether (AME), three mouse hepatoma (Hepa-1) cell lines with intact and with compromised AhR signaling were compared with respect to their activities for hydroxylation, methylation, and glucuronidation. Whereas the activities of cytochrome P450-mediated monooxygenase and catechol-O-methyl transferase were very low and did not differ between the three cell lines, a pronounced difference was observed for UDP-glucuronosyl transferase activity, which was much higher in Hepa-1c1c4 than in c1c7 and c1c12 cells. In all three cell types, the rate of glucuronidation of AOH was about four times higher than that of AME. Whereas AME caused a concentration-dependent G2/M arrest in each cell line, AOH arrested Hepa-1c1c7 and c1c12 cells but not c1c4 cells. However, Hepa-1c1c4 cells were arrested by AOH when β-glucuronidase was added to the incubation medium in order to reverse the formation of AOH glucuronides. We conclude that the failure of AOH to cause cell cycle inhibition in Hepa-1c1c4 cells is due to its efficient glucuronidation. The considerable UDP-glucuronosyl transferase activity of Hepa-1c1c4 cells should be taken into account when other compounds are studied in this cell line. Moreover, we demonstrate that differences in glucuronide formation between cell types can be overcome by the addition of β-glucuronidase to the cell culture medium.

Topics: Animals; Carcinoma, Hepatocellular; Catechol O-Methyltransferase; Cell Cycle; Cell Line, Tumor; Drug Therapy, Combination; Glucuronidase; Glucuronides; Glucuronosyltransferase; Hepatocytes; Lactones; Liver Neoplasms; Mice; Mycotoxins; Receptors, Aryl Hydrocarbon; Signal Transduction

Two new heptaketides, (+)-(2S,3S,4aS)-altenuene (1a) and (-)-(2S,3S,4aR)-isoaltenuene (2a), together with six known compounds, (-)-(2R,3R,4aR)-altenuene (1b), (+)-(2R,3R,4aS)-isoaltenuene (2b), 5'-methoxy-6-methyl-biphenyl-3,4,3'-triol (3), alternariol (4), alternariol-9-methyl ether (5), and 4-hydroxyalternariol-9-methyl ether (6) were isolated from the EtOAc extract of an endolichenic fungal strain Nigrospora sphaerica (No.83-1-1-2). Compounds 1a and 1b were separated from enantiomers 1 by chiral HPLC, and so were 2a and 2b from enantiomers 2. Interestingly, 1-6 were also obtained from other two endolichenic fungal strains Alternaria alternata (No.58-8-4-1) and Phialophora sp. (No.96-1-8-1). The structures of 1-6 were elucidated by means of MS, HR-MS, NMR, and X-ray diffraction. Furthermore, the absolute configurations of 1a-2b were determined by CD experiments and CD calculation. Of these compounds, 4 and 5 showed antiviral activity against herpes simplex virus (HSV) in vitro, with IC(50) values of 13.5 and 21.3 μM, and with selective index (SI) values of 26.5 and 17.1, respectively.

Topics: Alternaria; Antiviral Agents; Biological Products; Chromatography, High Pressure Liquid; Lactones; Lichens; Mitosporic Fungi; Molecular Structure; Phialophora; Polyketides; Simplexvirus; Stereoisomerism

Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH) and alternariol-9-methyl ether (AME) are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS). In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.

Topics: Alternaria; Gene Expression Profiling; Gene Expression Regulation, Fungal; Lactones; Metabolome; Multigene Family; Polyketide Synthases; RNA Interference

Alternaria spp. are known to form a spectrum of secondary metabolites with alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TA) as the major mycotoxins with respect to quantity. In the present study we investigated the contribution of these compounds for the DNA damaging properties of complex extracts of Alternaria spp. infested rice. Five different Alternaria strains were cultured on rice and analyzed for their production of AOH, AME, ALT and TA. The extracts of two strains with distinctly different toxin profiles were selected for further toxicological analysis. An extract from A. alternata DSM 1102 infested rice, found to contain predominantly TA, exhibited substantial DNA strand breaking properties in cultured human colon carcinoma cells in the comet assay, whereas TA as a single compound did not affect DNA integrity up to 200μM. An extract of A. alternata DSM 12633 infested rice, containing in comparable proportions AOH, AME and TA, exceeded by far the DNA damaging properties of the single compounds. In contrast to AOH, AME and TA, both selected extracts induced an increase of DNA modifications sensitive to the bacterial repair enzyme formamidopyrimidine DNA glycosylase (FPG) in the comet assay, indicative for oxidative DNA damage. Toxicity-guided fractionation of the DSM 12633 extract confirmed that these effects were not caused by AOH, AME or TA. Taken together, the mycotoxins AOH, AME and TA, representing the major mycotoxins with respect to quantity in A. alternata infested food, play only a subordinate role for the genotoxic properties of complex extracts and appear not to be involved in the induction of FPG sensitive sites.

Topics: Alternaria; Animals; Cell Cycle; Cell Line; Cricetinae; Dose-Response Relationship, Drug; Lactones; Molecular Structure; Mutagenicity Tests; Mutagens; Oryza; Tenuazonic Acid

Alternaria alternata has been reported to be the most common fungus on Canadian Western wheat. The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) are mutagenic in vitro and there is also limited evidence for carcinogenic properties. They have been found in wheat from Europe, Argentina, China and Australia, but they have not been looked for in Canadian grains or grain foods. In the present study, 83 samples of grain-based food sold in Canada, including flour, bran, breakfast cereals, infant cereals and bread, were analysed for AOH and AME using extraction with methanol, clean-up on combined aminopropyl/C18 solid phase extraction (SPE) columns, and liquid chromatography (LC) with tandem mass spectrometric (MS/MS) determination. The overall average recoveries of AOH and AME from a variety of spiked cereal foods (n = 13) were 45 ± 9% and 53 ± 9%, which could be attributed mainly to MS matrix effects The instrumental limits of detection (LOD) were 0.34 ng/g and 0.13 ng/g for AOH and AME, respectively, and the instrumental limits of quantitation (LOQ) were 1.1 and 0.43 ng/g. Of 83 samples analysed, 70 were positive for AOH (up to 63 ng/g, in a soft wheat bran) and 64 contained AME (up to 12 ng/g in a bran-based breakfast cereal). Of particular interest was the presence of AOH and/or AME in 27 out of 30 infant foods (up to 4.4 ng/g and 9.0 ng/g, respectively, in a sample of multigrain cereal).

Topics: Alternaria; Canada; Chromatography, Liquid; Edible Grain; Food Contamination; Humans; Infant; Infant Food; Lactones; Limit of Detection; Methanol; Solid Phase Extraction; Tandem Mass Spectrometry

Large amounts of tomato fruits and derived products are produced in Argentina and may be contaminated by Alternaria toxins. Limited information is available on the genetic variability, toxigenicity, and pathogenicity of Alternaria strains occurring on tomato. We analyzed 65 Alternaria strains isolated in Argentina from tomato fruits affected by black mould and from tomato puree, using amplified fragment length polymorphisms (AFLPs) technique. AFLP analysis resolved the set of strains in 3 main clusters (DICE similarity values of 58 and 60%) corresponding to A. alternata/tenuissima (44 strains), A. arborescens (15 strains) and to an unknown group (6 strains). Most of the representative strains, belonging to each AFLP cluster, when cultured on rice, produced tenuazonic acid (up to 46,760 mg/kg), alternariol monomethyl ether (AME, up to 1860 mg/kg), and alternariol (up to 70 mg/kg). The toxin profile related to the strains was not related to any AFLP cluster, except for AME which was produced at lower level by A. arborescens. Most of strains were pathogenic on two types of commonly cultivated tomato fruits. These findings provide new information on the variability within the Alternaria species complex associated with tomato disease.

Topics: Alternaria; Amplified Fragment Length Polymorphism Analysis; Argentina; DNA, Fungal; Fruit; Genetic Variation; Lactones; Mycotoxins; Plant Diseases; Sequence Analysis, DNA; Solanum lycopersicum; Tenuazonic Acid

Monohydroxylation of alternariol (AOH) and alternariol-9-methyl ether (AME) has previously been reported as a prominent metabolic route under cell-free conditions. This pathway gives rise to several catechol metabolites and may therefore be of toxicological relevance.. To clarify whether hydroxylation of AOH and AME occurs under in vivo-like conditions in the presence of conjugation reactions, the metabolism of the Alternaria toxins has now been studied in precision-cut rat liver slices. Four catechol metabolites of AOH and two of AME, together with several of their O-methylation products, as catalyzed by catechol-O-methyl transferase, were clearly identified after incubation of the liver slices with AOH and AME. These metabolites were predominantly present as conjugates with glucuronic acid and/or sulfate. In preliminary studies with bile duct-cannulated male rats dosed with AOH by gavage, the four monohydroxylated metabolites of AOH could also be demonstrated in the bile either as catechols or as O-methyl ethers.. These experiments clearly show that AOH and AME undergo catechol formation in vivo and warrant closer examination of the toxicological significance of this metabolic pathway.

Topics: Animals; Bile Ducts; Catechol O-Methyltransferase; Catechols; Catheterization; Glucuronic Acid; Hydroxylation; In Vitro Techniques; Lactones; Liver; Male; Oxidation-Reduction; Rats; Rats, Sprague-Dawley

The aim of this study was to determine the effects of water activity (a(w)) (0.99-0.90), temperature (15, 25 and 30°C) and their interactions on growth and alternariol (AOH) and alternariol monomethyl ether (AME) production by Alternaria alternata on irradiated soya beans. Maximum growth rates were obtained at 0.980 a(w) and 25°C. Minimum a(w) level for growth was dependent on temperature. Both strains were able to grow at the lowest a(w) assayed (0.90). Maximum amount of AOH was produced at 0.98 a(w) but at different temperatures, 15 and 25°C, for the strains RC 21 and RC 39 respectively. Maximum AME production was obtained at 0.98 a(w) and 30°C for both strains. The concentration range of both toxins varied considerably depending on a(w) and temperature interactions. The two metabolites were produced over the temperature range 15 to 30°C and a(w) range 0.99 to 0.96. The limiting a(w) for detectable mycotoxin production is slightly greater than that for growth. Two-dimensional profiles of a(w)× temperature were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soya bean. Knowledge of AOH and AME production under marginal or sub-optimal temperature and a(w) conditions for growth can be important since improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality. This could present a hazard if the grain is used for human consumption or animal feedstuff.

Topics: Alternaria; Food Irradiation; Food Microbiology; Glycine max; Humans; Lactones; Mycotoxins; Temperature; Water

The objective of this study was to determine the effect of water activity (a(w); 0.995, 0.98, 0.96, 0.94, 0.92, and 0.90), temperature (5, 18, 25, and 30 degrees C), incubation time (7 to 35 days), and their interactions on mycelial growth and alternariol (AOH) and alternariol monomethyl ether (AME) production. Two Alternaria alternata strains isolated from soybeans in Argentina were grown on 2% soybean extract agar. Maximum growth rates were obtained at the highest a(w) (0.995) and 25 degrees C, with growth decreasing as the water availability of the medium was reduced. Maximum amount of AOH was produced at 0.98 a(w) and 25 degrees C for both strains. Maximum AME production was obtained for both strains at 30 degrees C but different a(w) values, 0.92 and 0.94, for the strains RC 21 and RC 39, respectively. The concentrations of both toxins varied considerably depending on the a(w) and temperature interactions assayed. The two metabolites were produced from 5 to 30 degrees C and at a(w) values of 0.92 to 0.995. Although at 5 and 18 degrees C little mycotoxin was produced at a(w) lower than 0.94. Two-dimensional profiles of a(w) by temperature interactions were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soybeans. All the conditions of a(w) and temperature that resulted in maximum production of both toxins are those found during soybean development in the field. Thus, field conditions are likely to be conducive to optimum A. alternata growth and toxin production.

Topics: Alternaria; Colony Count, Microbial; Consumer Product Safety; Food Contamination; Glycine max; Lactones; Mycotoxins; Temperature; Time Factors; Water

Alternaria spp. have been reported to be the most frequent fungal species invading tomatoes. Certain species, in particular the most common one, A. alternata, are capable of producing several mycotoxins in infected plants and in agricultural commodities. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA) are some of the main Alternaria mycotoxins that can be found as contaminants of food. The objective of this study was to determine the effect of water activity (a(w), 0.904, 0.922, 0.954, and 0.982) and temperature (6, 15, 21 and 35 degrees C) on mycotoxin production on a synthetic tomato medium of a cocktail inoculum of five strains of A. alternata isolated from tomato fruits affected by Blackmould. The optimum AOH production occurred at 0.954 a(w) after 28days of incubation at 21 degrees C. A temperature of 21 degrees C was the most favourable for AOH synthesis at all a(w) levels. The maximum concentration of AME was determined at 0.954 a(w) and 35 degrees C. The optimum conditions for TA accumulation were 0.982 a(w) and 21 degrees C. At the 0.904 a(w) no growth or germination was registered at 6 degrees C and 15 degrees C over the whole incubation period. At 21 degrees C and 35 degrees C growth occurred slowly but none of the toxins were detected at this a(w) level. In general, high a(w) levels were favourable for mycotoxin production. None of the other toxins was detected at quantifiable levels at 6 degrees C after the whole incubation period. A storage temperature of 6 degrees C or below could be considered as safe for tomato fruits and high moisture tomato products (a(w)>0.95), in relation with Alternaria toxins. The results obtained here could be extrapolated to evaluate the risk of spoilage in tomato fruits and tomato products caused by this pathogen.

Topics: Alternaria; Colony Count, Microbial; Consumer Product Safety; Culture Media; Food Contamination; Food Preservation; Lactones; Mycotoxins; Solanum lycopersicum; Temperature; Water

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was developed for the determination of altenuene (ALT), alternariol (AOH), tentoxin and alternariol monomethyl ether (AME) in apple juice concentrate (AJC). The sample was diluted with water, and then cleaned up with a PS DVB column. The quantification was carried out using an external standard method. The UPLC was performed on a BEH C18 column (50 mm x 2.1 mm, 1.7 microm) using a gradient elution of acetonitrile and water. The MS/MS was performed with multiple reaction monitoring (MRM) mode. The limits of quantification of the four Alternaria toxins were between 1.0 and 5.0 microg/L. The recoveries were 77.8%-117.2% with the relative standard deviations less than 9.7%. The method is sensitive, stable and reliable. It's suitable for the quantitative and qualitative analyses of the four Alternaria toxins in AJC.

Topics: Alternaria; Beverages; Chromatography, High Pressure Liquid; Food Contamination; Lactones; Malus; Mycotoxins; Peptides, Cyclic; Tandem Mass Spectrometry

Alternariol (AOH), a mycotoxin formed by Alternaria alternata, has been reported to possess genotoxic properties. However, the underlying mechanism of action is unclear. Here, we tested the hypothesis that interactions with DNA-topoisomerases play a role in the DNA-damaging properties of AOH. First we compared DNA-damaging properties of AOH with other Alternaria mycotoxins such as AOH monomethyl ether (AME), altenuene and isoaltenuene. AOH and AME significantly increased the rate of DNA strand breaks in human carcinoma cells (HT29, A431) at micromolar concentrations, whereas altenuene and isoaltenuene did not affect DNA integrity up to 100 microM. Next, we selected AOH as the most DNA-damaging Alternaria metabolite for further studies of interactions with DNA topoisomerases. In cell-free assays, AOH potently inhibited DNA relaxation and stimulated DNA cleavage activities of topoisomerase I, IIalpha and IIbeta. Stabilisation of covalent topoisomerase II-DNA intermediates by AOH was also detectable in cell culture, and here, the IIalpha isoform was preferentially targeted. AOH is thus characterised as a poison of topoisomerase I and II with a certain selectivity for the IIalpha isoform. Since topoisomerase poisoning and DNA strand breakage occurred within the same concentration range, poisoning of topoisomerase I and II might at least contribute to the genotoxic properties of AOH.

Topics: Antigens, Neoplasm; Bisbenzimidazole; Cell Line, Tumor; DNA Breaks; DNA Topoisomerases, Type II; DNA-Binding Proteins; Ethidium; Humans; Lactones; Mycotoxins; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors

Mycotoxins as contaminants of animal food can impair fertility in farm animals. In the regulation of female fertility the ovarian steroid hormone progesterone (P(4)) plays an important role. In the present study we have investigated the influence of the mycotoxins alternariol (AOH), alternariol mono-methyl ether (AME), and tenuazonic acid (TeA) on cell viability, P(4) synthesis, abundance of the key enzymes of P(4) synthesis, P450 cholesterol side-chain cleavage enzyme (P450SCC) and 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD), and of the corresponding Cyp11a1 and Hsd3b transcripts in cultured pig granulosa cells. Already 0.8 microM, AOH and AME inhibited P(4) secretion and 1.6 microM also significantly reduced cell viability. The abundance of P450scc protein but not of Cyp11a1 or Hsd3b transcripts was already significantly reduced by 0.8 microM AOH and AME. 1.6 microM AOH but not AME significantly reduced the abundance of alpha-tubulin and also clearly affected actin protein concentrations. TeA neither impaired viability nor P(4) secretion. Also mycotoxin extracts isolated from naturally occurring Alternaria strains by HPLC purification inhibited cell viability and P(4) synthesis, however at higher concentrations compared to AOH and AME. In conclusion, AOH and AME, but not TeA specifically inhibited P(4) secretion in cultured porcine granulosa cells. Alternaria toxin contaminated food may therefore affect reproductive performance in pig and other mammalian species.

Topics: Actins; Alternaria; Animals; Cell Count; Cell Survival; Cells, Cultured; Cholinesterase Inhibitors; Cytoskeleton; Female; Granulosa Cells; Lactones; Mycotoxins; Oryza; Progesterone; RNA; Swine; Tubulin

Stable isotope dilution assays (SIDAs) for the determination of the most important mycotoxins of the black mold Alternaria, namely, alternariol and alternariol monomethyl ether, have been developed. For this purpose, deuterated alternariol and alternariol methyl ether were synthesized by palladium catalyzed protium-deuterium exchange from the unlabeled toxins. Reaction conditions were chosen in such a manner that the formation of the [(2)H(4)]-isotopologues was favored. The synthesized products were characterized by LC-MS, NMR, and UV-spectroscopy. On the basis of the use of [(2)H(4)]-alternariol and [(2)H(4)]-alternariol methyl ether as internal standards, SIDAs were developed and applied to the determination of alternariol and alternariol methyl ether in beverages using LC-MS/MS. Method validation revealed a high sensitivity, i.e., low limits of detection (alternariol, 0.03 microg/kg; alternariol methyl ether, 0.01 microg/kg) and limits of quantitation (alternariol, 0.09 microg/kg; alternariol methyl ether, 0.03 microg/kg), respectively. Recovery from spiked apple juice was 100.5 +/- 3.4% for alternariol (range 0.1-1 microg/kg) and 107.3 +/- 1.6% for alternariol methyl ether (range 0.05-0.5 microg/kg). Interassay precision (expressed as coefficient of variation, CEV) for alternariol was 4.0% (7.82 +/- 0.31 microg/kg; vegetable juice, naturally contaminated) and 4.6% (1.04 +/- 0.05 microg/kg; grape juice, naturally contaminated). For alternariol methyl ether, a CEV of 2.3% (0.79 +/- 0.02 microg/kg; vegetable juice, naturally contaminated) was obtained. Analysis of fruit juices showed low contamination with alternariol and alternariol methyl ether in general, but higher values of both toxins were found in wine and vegetable juices. The values for alternariol were higher than those for alternariol methyl ether in nearly any case. However, the developed SIDA has proven to be optimally suited for further studies on alternariol and alternariol methyl ether content in food samples to obtain further insight into possible health hazards for the consumer.

Topics: Alternaria; Beverages; Chemistry Techniques, Analytical; Deuterium; Food Contamination; Lactones; Mycotoxins; Sensitivity and Specificity

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.

Topics: Alternaria; Colony Count, Microbial; Consumer Product Safety; Dose-Response Relationship, Radiation; Food Contamination; Food Irradiation; Gamma Rays; Helianthus; Humans; Lactones; Seeds

From the Egyptian medicinal plant Polygonum senegalense the fungal endophyte Alternaria sp. was isolated. Extracts of the fungus grown either in liquid culture or on solid rice media exhibited cytotoxic activity when tested in vitro against L5178Y cells. Chromatographic separation of the extracts yielded 15 natural products, out of which seven were new compounds, with both fungal extracts differing considerably with regard to their secondary metabolites. Compounds 1, 2, 3, 6, and 7 showed cytotoxic activity with EC 50 values ranging from 1.7 to 7.8 microg/mL. When analyzed in vitro for their inhibitory potential against 24 different protein kinases, compounds 1- 3, 5- 8, and 15 inhibited several of these enzymes (IC 50 values 0.22-9.8 microg/mL). Interestingly, compounds 1, 3, and 6 were also identified as constituents of an extract derived from healthy leaves of the host plant P. senegalense, thereby indicating that the production of natural products by the endophyte proceeds also under in situ conditions within the plant host.

Topics: Alternaria; Animals; Antineoplastic Agents; Biphenyl Compounds; Drug Screening Assays, Antitumor; Heterocyclic Compounds, 3-Ring; Humans; Lactones; Mice; Plant Leaves; Polygonum

The natural occurrence of Alternaria mycotoxins in Argentinean wheat from the zone 5 South during the 2004 to 2005 harvest was investigated in 64 wheat samples. All samples were highly contaminated with a wide range of fungal species. Alternaria was found as the main component of the mycota, with an infection percentage of 100%. Three mycotoxins produced by species of Alternaria were determined in wheat: alternariol, alternariol monomethyl ether, and tenuazonic acid. Alternariol was detected in 4 (6%) of 64 samples, with a range of 645 to 1,388 microg/kg (mean of 1,054 microg/kg); alternariol monomethyl ether, with a range of 566 to 7,451 microg/kg (mean of 2,118 microg/kg) in 15 (23%) of 64 samples; and tenuazonic acid in 12 (19%) of 64 samples, with a range of 1,001 to 8,814 microg/kg (mean, 2,313 microg/kg). Alternariol monomethyl ether was the predominant toxin, but tenuazonic acid was detected in higher concentrations. Alternariol was present in fewer samples and in lower levels than were the other toxins. Tenuazonic acid and alternariol monomethyl ether occurred together in four samples, while tenuazonic acid and alternariol co-occurred in one sample. This the first report of the natural occurrence of Alternaria mycotoxins in Argentinean wheat. Toxin levels were high, probably due to the heavy infection with Alternaria species found in the samples.

Topics: Alternaria; Argentina; Colony Count, Microbial; Consumer Product Safety; Food Contamination; Food Microbiology; Humans; Lactones; Mycotoxins; Prevalence; Species Specificity; Tenuazonic Acid; Triticum

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.

Topics: Animals; Chromatography, High Pressure Liquid; Food Contamination; Gas Chromatography-Mass Spectrometry; Humans; Hydroxylation; In Vitro Techniques; Lactones; Male; Microsomes, Liver; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Swine

A method was developed for the quantification of alternariol and alternariol monomethyl ether on tangerines with and without symptoms of Alternaria brown spot disease. The method employs solid-phase extraction for cleanup, followed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for detection. This method was validated on flavedo (exocarp or epicarp, exterior yellow peel) and on albedo tissue (mesocarp, interior white peel). An excellent linearity over a range of 0.50-20.0 mg/kg was achieved, with r2 >or= 0.997. The limits of detection (LOD) and quantification (LOQ) were fewer than 0.13 and 0.50 microg/kg, respectively. The relative standard deviations (RSDs) were

Topics: Alternaria; Chromatography, High Pressure Liquid; Citrus; Fruit; Lactones; Mass Spectrometry; Mycotoxins; Plant Diseases

Fungi contaminant of alpataco (Prosopis flexuosa) fruits from La Pampa province (Argentina) were identified. Alternaria alternata and Sphaeropsis sapinea were the dominant species. Phoma sp., Nigrospora sp., Preussia minima, Cladosporium sp., Pithomyces chartarum, Epicoccum nigrum, Aspergillus niger and Aspergillus speluneus were also isolated but with less frequency. Twelve strains of Alternaria alternata, the toxigenic species with higher incidence, were screened for alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production. Since one isolate was able to produce AME, six isolates produced AOH and AME and two isolates produced AOH, AME and TA, these results indicate a potential risk of contamination with Alternaria toxins in this substrate.

Topics: Alternaria; Argentina; Food Contamination; Food Microbiology; Fruit; Fungi; Lactones; Mitosporic Fungi; Mycotoxins; Prosopis; Species Specificity; Tenuazonic Acid

The toxigenic potential of Alternaria strains isolated from Argentinean wheat was investigated. A total of 123 strains were assayed for the production of tenuazonic acid (TA), alternariol (AOH) and alternariol monomethyl ether (AME). All but one of the isolates were able to produce at least one of the three mycotoxins. TA was produced by 72% of the strains (1-14782 mg/kg), AOH by 87% (4-622 mg/kg) and AME by 91% (7-2625 mg/kg). The average level of TA detected for all strains (1757 mg/kg) was higher than the average level of both alternariols (162 mg/kg for AOH and 620 mg/kg for AME). TA was the toxin produced at the highest concentration but in lower frequency. Most of the strains were able to synthesize more than one toxin: 74 isolates (60%) were positive for all three toxins, 30 (24%) for both AOH and AME, 5 (4%) for both TA and AME, and 2 (2%) for TA and AOH. The widespread occurrence of Alternaria in wheat and its ability to produce mycotoxins suggests the possible occurrence of its toxins in wheat naturally infected with this fungus.

Topics: Alternaria; Argentina; Chromatography, High Pressure Liquid; Food Contamination; Food Microbiology; Lactones; Mycotoxins; Prevalence; Species Specificity; Tenuazonic Acid; Time Factors; Triticum

Some Alternaria species are able to produce plant pathogenic as well as toxic metabolites. In both agriculture and the food industry it is important know if toxigenic Alternaria are present to rapidly employ the correct corrective actions. The purpose of this work was to establish a real-time PCR method, which can detect and quantify apple pathogenic and toxigenic Alternaria. An AM-toxin I primer set, which could recognize Alternaria DNA only, was designed by using primers complementary to the AM-toxin I gene. The method could detect small amounts of DNA (4 pg) and still obtain a large dynamic range (4 decades) without interference from apple material. Eight Alternaria isolates were analyzed for the presence of AM-toxin I gene and their production of secondary metabolites. Then analyses showed that all eight isolates contained the AM toxin gene and were able to produce the plant pathogenic tentoxin in addition to AM toxin I. The analyses also showed the production of tenuazonic acid, alternariols, Altenuene, altenusin and/or altertoxin I in pure culture. Analyses of inoculated apples showed that both the AM-toxin gene and alternariol monomethyl ether could be detected. Morphological analyses suggested that the eight Alternaria strains, though they all carried the AM toxin genes, probably belong to different but closely related un-described Alternaria taxa in the A. tenuissima species-group based on morphological and chemical differences.

Topics: Alternaria; Chromatography, High Pressure Liquid; Lactones; Malus; Mycotoxins; Polymerase Chain Reaction; Sensitivity and Specificity; Tenuazonic Acid

The present study aimed to analyze the mycoflora and the occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in grain samples of sunflower during different stages of plant development in Nova Odessa, State of São Paulo, Brazil. The data obtained were correlated with the presence of fungi in soil, wind-dispersed fungi, and the predominant climatic conditions of the region where the experiment was carried out. Analysis of the mycoflora revealed the presence of Fusarium verticillioides and Alternaria alternata in 70% and 46% of the samples, respectively. The profile of wind-dispersed fungi also showed F. verticillioides as the most frequently isolated fungus (68%), although A. alternata was detected in 28% of samples. In soil, Penicillium was the most frequent species (49.9%), followed by F. verticillioides (47.7%) and A. alternata (10.9%). Regarding water activity, sunflower grains presenting a high frequency of isolation of F. verticillioides and A. alternatahad a water activity ranging from 0.92 to 0.96, and statistical analysis revealed a negative linear correlation between the isolation of fungi and water activity. HPLC analysis showed that 18% of the sunflower grains were contaminated with alternariol (24.9-170.9 ng/g) and 10% with alternariol monomethyl ether (14.1-108.6 ng/g). The contamination of sunflower grains with AOH and AME in the field was low when compared to the LD50 necessary to cause toxicity to animals. However, the contamination with other toxigenic fungi such as F. verticillioides may indicate the presence of other mycotoxins in sunflower grains and a possible synergistic effect between them. This is the first report of the natural occurrence of alternariol and alternariol monomethyl ether in sunflower grains in Brazil.

Topics: Alternaria; Brazil; Cladosporium; Fusarium; Helianthus; Lactones; Seeds

Alternariol (AOH) and alternariol monomethyl ether (AME) are among the main mycotoxins formed in apples and other fruits infected by Alternaria alternata. For determination of AOH and AME by LC, apple juice and other fruit beverages were cleaned up on C18 and aminopropyl solid-phase extraction columns. Positive and negative ion mass spectra of AOH and AME under electrospray (ESI) and atmospheric pressure chemical ionization (APCI) conditions were obtained. Collision-induced dissociation of the [M+H]+ and [M-H]- ions for both compounds were also studied. The phenolic anions of both compounds are more stable with less fragmentation. In quantitative analysis, negative ion detection also offers lower background and better sensitivity. Sensitive LC-MS and LC-MS-MS confirmatory procedures based on APCI with negative ion detection were applied to confirm the natural occurrence of AOH in nine samples of apple juice and in single samples of some other clear fruit beverages--grape juice, cranberry nectar, raspberry juice, red wine, and prune nectar (which also contained 1.4 ng AME/ml)--at levels of up to 6 ng AOH/ml. Electrospray LC-MS-MS with negative ion detection and in multiple reaction monitoring mode offers higher sensitivity and specificity. Absolute detection was better than 4 pg per injection for both compounds.

Topics: Beverages; Chromatography, Liquid; Fruit; Lactones; Mycotoxins; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

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.

Topics: Alternaria; Cholinesterase Inhibitors; Chromatography, Liquid; Food Microbiology; Fruit; Lactones; Temperature; Time Factors

Molds of the genus Alternaria are common food pathogens responsible for the spoilage of fruits, vegetables, grains, and nuts. Although consumption of Alternaria alternata-contaminated foodstuffs has been implicated in an elevated incidence of esophageal carcinogenesis, the mutagenic potencies of several A. alternata toxins seem unable to account for the levels of activity found using crude mycelial extracts. In this study, the mutagenic effects of nitrosylation were examined with the major Alternaria metabolites Altenuene (ALT), Alternariol (AOH), Alternariol Monomethyl Ether (AME), Altertoxin I (ATX I), Tentoxin (TENT), Tenuazonic Acid (TA), and Radicinin (RAD) using the Ames Salmonella strains TA98 and TA100. In the absence of nitrosylation, ATX I was mutagenic when tested from 1 to 100 microg/plate in TA98 with rat liver S9 for activation, while AOH and ATX I were weakly mutagenic +/- S9 in TA100. Incubation with nitrite generally increased mutagenic potencies with ATX I strongly mutagenic +/- S9 in both TA98 and TA100, while ALT, AOH, AME, and RAD responses were enhanced in TA100 + S9. However, subsequent examination of three extracts made from A. alternata culture broth, acetone-washed mycelia, and the acetone washes showed a different mutagenic response with both broth and acetone washes directly mutagenic in TA98 and TA100 but with a reduced response + S9. The acetone-washed mycelial extract was found to have the lowest mutagenic activity of the three extracts tested. Nitrosylation had little effect on the mutagenicity of any of the extracts. Thus, while nitrosylation increases the mutagenicity of ATX I, and to a lesser extent that of several other Alternaria toxins, the results demonstrate that Alternaria produces a major mutagenic activity with a S. typhimurium response different from that found with the purified toxins. Efforts are currently underway to chemically identify this mutagenic species. Published 2001 Wiley-Liss, Inc.

Topics: Alternaria; Animals; Benz(a)Anthracenes; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Lactones; Microsomes, Liver; Models, Chemical; Mutagenicity Tests; Mutagens; Peptides, Cyclic; Perylene; Pyrones; Rats; Salmonella typhimurium; Sodium Nitrite; Tenuazonic Acid

Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TEA) are secondary metabolites of Alternaria species. Cyclopiazonic acid (CPA) is produced by fungi belonging to the ubiquitous genera of Penicillium and Aspergillus and has been found in a wide range of foods. These toxins were searched for by liquid chromatography with diode-array detection in tomato products processed and sold in Brazil. Eighty samples of tomato products were examined (juice 11, pulp 22, purée 22, paste 24 and whole stewed tomato 1). Eleven brands sold nationwide were covered. TEA was found in seven samples of tomato pulp (39-111 ng/g) and four samples of tomato puree (29-76 ng/g). CPA was found in six samples of pulp (64-178 ng/g) and two samples of purée (36-117 ng/g). Co-occurrence of TEA and CPA was found in two samples of purée and one of pulp. This is the first time that the presence of CPA has been reported in tomato products. Neither AME nor AOH were detected in the samples.

Topics: Alternaria; Brazil; Chromatography, High Pressure Liquid; Humans; Indoles; Lactones; Mycotoxins; Solanum lycopersicum; Tenuazonic Acid

The production of mycotoxins by Alternaria alternata in cellulosic ceiling tiles was examined with thin-layer chromatography and high-performance liquid chromatography procedures. Alternariol and alternariol monomethyl ether were found in ceiling tile extracts, whereas extracts of control rice cultures of all three isolates produced these mycotoxins plus altenuene and altertoxin I. Extensive fungal growth and mycotoxin production occurred in the ceiling tiles at relative humidities of 84-89% and 97%.

Topics: Air Microbiology; Alternaria; Benz(a)Anthracenes; Cellulose; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Lactones; Mutagens; Mycotoxins; Perylene; Sick Building Syndrome

Alternaria cultures (87 isolates of Alternaria alternata, four of A. tenuissima, two of A. radicina, and three of Alternaria state of Pleospora infectoria respectively, from maize) were screened to determine their ability to produce alternariol (AOH) and alternariol monomethyl ether (AME) on maize and rice. Only 28 A. alternata stains had toxigenic capacity. When maize was used as substrate 21 of 28 isolates produced AOH and AME, and 23 of 28 strains produced AOH and 22 of 28 produced AME when rice was used. The level of AOH produced by the isolates ranged from 0.3 to 2.1 mg/kg on maize and from 0.4 to 9.9 mg/kg on rice. The AME production by the stains ranged between 0.3 and 3.3 mg/kg both on maize and on rice. These results could indicate a low probability of AOH and AME occurring naturally on maize in Argentina.

Topics: Alternaria; Argentina; Cholinesterase Inhibitors; Food Contamination; Lactones; Mycotoxins; Oryza; Zea mays

A limited survey of 32 samples of apple juice concentrates, destined for the production of commercial juices, was carried out in order to evaluate the natural occurrence of the Alternaria metabolites alternariol and alternariol methyl ether. A high-performance liquid chromatographic method based on solid-phase extraction columns for extraction and purification of the toxins was used. Both mycotoxins were found as natural contaminants in 50% of the samples analyzed. Levels of alternariol were in the range 1.35-5.42 ng/ml. Alternariol methyl ether was present in most cases only at trace levels, and the highest amount detected was 1.71 ng/ml in one sample.

Topics: Beverages; Chromatography, High Pressure Liquid; Lactones; Mycotoxins; Rosales; Spain

Sunflower (Heliantus annuus) is an important crop in the economy of Argentina due to its high production and demand from domestic and export markets. The aim of this study was to evaluate the presence of Alternaria species and its mycotoxins in ensiled sunflower seeds. The sampling was carried out in three periods: at the beginning of ensiling, on the second month and finally on the fourth month. The mycological analysis was made with disinfected seeds, cultured on Dichloran-Rose Bengal-Chloramphenicol (DRBC) and Dichloran-Chloramphenicol-Malt Extract-Agar (DCMA). The toxins were analyzed by thin layer chromatography (TLC). A alternata was the main fungal species isolated. The incidence of Alternaria species and the levels of alternariol and tenuazonic acid decreased as the time of ensiling increased. Alternariol monomethyl ether was detected in two samples from the second and third sampling periods.

Topics: Alternaria; Chromatography, Thin Layer; Helianthus; Lactones; Mycotoxins; Seeds; Silage; Tenuazonic Acid; Time Factors

The present work describes a new method for determination of alternariol (AOH) and alternariol methyl ether (AME) in apple juice using solid-phase extraction (SPE) columns for extraction and cleanup of samples for high-performance liquid chromatography (HPLC). Chromatograms of spiked samples show that both toxins can be easily detected without interferences, and good recoveries for AOH (82.8 +/- 7.4%) and AME (91.9 +/- 6.1%) with detection limits as low as 1.6 and 0.7 micrograms/l, respectively, were obtained.

Topics: Beverages; Chromatography, High Pressure Liquid; Fruit; Lactones; Mycotoxins; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

Eleven Alternaria alternata strains isolated from Red Delicious apples in cold storage in Argentina, were tested for alternariol and alternariol methyl ether production in laboratory media and in whole fresh fruits. Most of them were able to produce both toxins in all media. They were detected also in mycelium free filtrates from liquid cultures and in asymptomatic tissues from inoculated fruit. Thus, in the evaluation of mouldy core incidence in apples, the presence of A. alternata toxins in tissues should be considered even in the absence of mycelia.

Topics: Alternaria; Argentina; Fruit; Lactones

Alternariol and alternariol methyl ether were tested in the Ames Salmonella typhimurium assay, and both were shown, with and without metabolic activation, to be nonmutagenic to strains TA98 and TA100. The finding of other investigators that alternariol methyl ether is weakly mutagenic to TA98 without metabolic activation could have resulted from the presence of a small amount of one of the highly mutagenic altertoxins in the alternariol methyl ether originally tested.

Topics: Animals; Biotransformation; In Vitro Techniques; Lactones; Male; Microsomes, Liver; Mutagens; Mycotoxins; Rats; Rats, Sprague-Dawley; Salmonella typhimurium

In this paper, the mutagenicity and carcinogenicity of alternariol monomethyl ether (AME), alternariol (AOH), and their relevance to the etiology of human esophageal cancer were studied. These mycotoxins were produced by Alternaria alternata which was the main contaminating fungi isolated from the grain in Linxian County, an area with high incidence of esophageal cancer. This study demonstrated that: 1. AME and AOH might cause cell mutagenicity and transformation; 2. AME and AOH could combine with the DNA isolated from human fetal esophageal epithelium, activate the oncogens, c-H-ras and c-mys in it, and promote proliferation of human fetal esophageal epithelium in vitro; 3. squamous cell carcinoma of the fetal esophagus could be induced by AOH. According to the results of the studies of AME and AOH mentioned above, we consider that Alternaria alternata plays an important role in the etiology of human esophageal cancer.

Topics: Alternaria; Animals; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cells, Cultured; Esophageal Neoplasms; Humans; Lactones; Mice; Mice, Inbred BALB C; Mutagenicity Tests; Mycotoxins

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.

Topics: Alternaria; Cerulenin; Fatty Acids; Lactones; Multienzyme Complexes

Analytical methods are described for detection of the Alternaria mycotoxins alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TeA) in natural and semisynthetic laboratory cultures. After extraction and purification of the crude extract by column chromatography on silica gel the qualitative and quantitative analyses were carried out by thin layer (TLC)- and high performance liquid chromatography (HPLC). HPLC separations were achieved using a Hypersil ODS column with methanol/water containing a complexing agent as eluent. Detection at 340 nm (AOH, AME, ALT) and 280 nm (TeA), respectively, has proved to be favourably. AME and TeA were produced in high purity and high yields as standard substances by two Alternaria strains. The identity of the toxins could be confirmed by EI-, CI- and FAB-mass spectrometry.

Topics: Alternaria; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Lactones; Mass Spectrometry; Molecular Structure; Mycotoxins; Spectrometry, Mass, Fast Atom Bombardment; Tenuazonic Acid

The ability to produce the Alternaria mycotoxins alternariol (AOH), alternariol monomethyl ether (AME) and altenuene (ALT) were studied on 87 Alternaria strains. 50 isolates were studied to produce tenuazonic acid (TeA). The strains were isolated from CCM-maize and stored hay. They were cultured under optimum conditions and analysed by various chromatographic methods for the 4 mycotoxins. All Alternaria strains studied produced AME and TeA, 77 per cent also AOH. A production of ALT was detected only in 18 per cent of the isolates. The concentrations of toxins produced were 0.08-482 (162) ppm for AME, 0.05-1862 (121) ppm for AOH, 0.1-34 (9.1) ppm for ALT and 0.02-42 (11.3) mg/100 ml liquid medium for TeA. The species Alternaria alternata (Fries) Keissler and Alternaria tenuissima (Kunze:Fr.) Wiltshire produced the greatest amounts of all mycotoxins studied.

Topics: Alternaria; Lactones; Mycotoxins; Poaceae; Tenuazonic Acid; Zea mays

Alternaria alternata produces the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase. Addition of 12 mM NaNO3 to the cultures before initiation of polyketide production reduced the AOH and AME content to 5 to 10% of that of controls. Glutamate and urea also reduced AOH and AME accumulation, whereas increasing the ionic strength did not affect the polyketide content. Adding NaNO3 after polyketide production had started did not inhibit further AOH accumulation, although over 90% of the added NO3- disappeared from the medium within 24 h. Activity of an AME-synthesizing enzyme, alternariol-O-methyltransferase (AOH-MT), appeared in control mycelia during the early stationary growth phase. No AOH-MT activity appeared in mycelia blocked in polyketide synthesis by addition of NaNO3. Later addition of NaNO3 reduced the AOH-MT specific activity to 50% of that of the control, whereas the total of activity per mycelium was the same. The AOH-MT activity in vitro was not affected by 100 mM NaNO3. The results suggest that nitrogen in some way inhibited the formation of active enzymes in the polyketide-synthesizing pathway in A. alternata when it was added before these enzymes were formed.

Topics: Alternaria; Fumarates; Glutamates; Glutamic Acid; Hydrogen-Ion Concentration; Lactones; Mitosporic Fungi; Mycotoxins; Nitrates; Nitrogen; Urea

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.

Topics: Alternaria; Aspergillus; Culture Media; Lactones; Mitosporic Fungi; Mycotoxins; Penicillium; Plant Extracts; Temperature; Tenuazonic Acid; Time Factors; Triticum; Water

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.

Topics: Alternaria; Animals; Antibiotics, Antineoplastic; Chick Embryo; Food Contamination; Lactones; Lethal Dose 50; Mitosporic Fungi; Tenuazonic Acid

Alternaria alternata (Fr.) Keissler, grown in drop culture, produced alternariol and alternariol monomethyl ether in late growth phase. Production was almost completely inhibited when the fungal cultures were exposed to white light (180 W/m2), although mycelial dry weight was not significantly affected. The fungus was most sensitive to light during the exponential growth phase. Twelve hours of light exposure was sufficient to decrease significantly the production of the secondary metabolites. In light the fungus produced a red-brown pigment of unknown nature.

Topics: Alternaria; Lactones; Light; Methyl Ethers; Mitosporic Fungi; Mycotoxins; Pigments, Biological

Topics: Alternaria; Biphenyl Compounds; Fungi; Lactones