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From a fresh root of Trema guineensis (Ulmaceae), endophytic fungi were isolated, among which a taxon belonging to the new species Diaporthe cameroonensis . This strain was fermented in shake flask batch cultures and the broth was extracted with ethyl acetate. From the crude extract, a hemiketal polyketide 1 , and an acetylated alternariol 2 were isolated, along with fifteen known secondary metabolites. Their structures were established by extensive NMR spectroscopy and mass spectrometry analyses, as well as by comparison with literature data of their analogs., (Copyright © 2023, Mountessou et al.)

Alternariol monomethyl ether (AME) is one of the major Alternaria mycotoxins present in a wide range of fruits, vegetables, grains, and their products, and possesses the properties of mutagenicity and carcinogenicity. In this study, a simple, rapid, and highly sensitive colorimetric immunosensor based on magnetic nanoparticles (MNPs) was firstly developed for the detection of AME in fruit by nonaggregated gold nanoparticles (GNPs). AME-BSA-Fe3O4 MNP conjugates and free AME molecules in samples competitively bind with monoclonal antibody (mAb)-GNP conjugates. After magnetic separation, the UV absorbance of the nonaggregated GNP supernatant was measured directly. The absorption intensity was proportional to the concentration of AME in the sample. Carboxyl-group-modified AME, AME-bovine serum albumin (BSA) conjugates, anti-AME mAbs, AME-BSA-Fe3O4 MNP conjugates, and mAb-GNP conjugates were prepared and characterized. The effect of GNP sizes (16, 24, and 40 nm) on the colorimetric determination of AME was studied. Under optimized conditions, the limit of detection and the linear range for AME were 0.16 ng/mL and 0.08-0.48 ng/mL, respectively. Moreover, the colorimetric immunosensor developed has lower cross-reactivity with AME analogues. The recoveries of spiked fruits ranged from 80.6% to 90.7%. The colorimetric immunosensor developed provides a promising method for simple, rapid, highly sensitive, and highly specific detection of other mycotoxins in the field of food safety.Competitive colorimetric immunosensor based on MNPs for the detection of AME by non-aggregated GNPs [ABSTRACT FROM AUTHOR]

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

A new hybrid of dehydroergosterol and nitrogenous alternariol derivative, pestauvicomorpholine A ( 1 ), and three alternariol analogues ( 2 – 4 ) including a new aminated one, pestauvicolactone A ( 2 ), were isolated from the fermentation product of the fungus Pestalotiopsis uvicola on rice media. Compounds 1 and 2 represent the first example of aza-alternariol and aza-alternariol-steroid derived from transamination followed by intermolecular hetero-Diels-Alder reaction. [ABSTRACT FROM AUTHOR]

This study aimed to explore the antioxidant potential and antiviral activity of endophytic fungi which were isolated from healthy living tissues of medicinal plants. Endophytic strains (29 different taxa) were isolated from 18 Egyptian medicinal plants collected from Saint Katherine Protectorate, Egypt. The fungal endophytes were identified based on morphological characters. All isolates were identified as ascomycetes, except two Zygomycetes strains (Absidia corymbifera and Mucor fuscus). Isolated endophytes were cultivated on potato dextrose media. The fungal metabolites were extracted by ethyl acetate and examined for their biological activities. Among 99 total extracts, only Chaetomium globosum, which was isolated from Adiantum capillus, showed a promising DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity (99% at 100 g/mL). Fifteen extracts prohibited the reproduction of HSV-2 virus. On the other hand, the reproduction of VSV-virus was inhibited by sixteen endophytic extracts. The promising anti-(HSV-2 and VSV) extract of endophytic Pleospora tarda strain; that was originally isolated from the medicinal plant Ephedra aphylla, showed viral inhibitory activity of 40.7% and 15.2%, respectively. Two compounds, for which antiviral activates could be attributed, were isolated and identified as alternariol and alternariol-(9)-methyl ether using different NMR techniques from P. tarda extract. For the first time, we report here the ability of the endophytic fungus P. tarda to produce alternariol and alternariol-(9)-methyl ether. The results indicate that the endophytic fungi from medicinal plants are promising sources of bioactive compounds. [ABSTRACT FROM AUTHOR]

Consumption of mycotoxin contaminated foodstuffs is common in regions where foods are not adequately controlled and routinely monitored, and this could have adverse effects on the health of consumers. In this study, 100 samples of roasted nuts (50 cashew nuts and 50 peanuts) vended within two cities of Sierra Leone were analysed for mycotoxins and other microbial metabolites by a LC-MS/MS method. The peanut samples contained detectable levels of 17 microbial metabolites, including aflatoxins B1, B2, G1 and G2 and alternariol, while none of these metabolites were found in the cashew samples. Aflatoxins (max: 5,729 μg/kg; mean: 487.8 μg/kg) and alternariol (3 μg/kg) were found in 24% and 2% of the peanut samples, respectively. One-third of the aflatoxin-contaminated peanut samples contained aflatoxins at levels exceeding the total aflatoxin limit of 4 μg/kg set by the European Union. Aflatoxin contamination of Sierra Leonean peanuts is high and requires urgent intervention to reduce consequent exposure. [ABSTRACT FROM AUTHOR]

Mycotoxins produced by Alternaria fungi are ubiquitous food contaminants, but analytical methods for generating comprehensive exposure data are rare. We describe the development of an LC-MS/MS method covering 17 toxins for investigating the natural occurrence of free and modified Alternaria toxins in tomato sauce, sunflower seed oil, and wheat flour. Target analytes included alternariol (AOH), AOH-3-glucoside, AOH-9-glucoside, AOH-3-sulfate, alternariol monomethyl ether (AME), AME-3-glucoside, AME-3-sulfate, altenuene, isoaltenuene, tenuazonic acid (TeA), tentoxin (TEN), altertoxin I and II, alterperylenol, stemphyltoxin III, altenusin, and altenuic acid III. Extensive optimization resulted in a time- and cost-effective sample preparation protocol and a chromatographic baseline separation of included isomers. Overall, adequate limits of detection (0.03-9 ng/g) and quantitation (0.6-18 ng/g), intermediate precision (9-44%), and relative recovery values (75-100%) were achieved. However, stemphyltoxin III, AOH-3-sulfate, AME-3-sulfate, altenusin, and altenuic acid III showed recoveries in wheat flour below 70%, while their performance was stable and reproducible. Our pilot study with samples from the Austrian retail market demonstrated that tomato sauces (n = 12) contained AOH, AME, TeA, and TEN in concentrations up to 20, 4, 322, and 0.6 ng/g, while sunflower seed oil (n = 7) and wheat flour samples (n = 9) were contaminated at comparatively lower levels. Interestingly and of relevance for risk assessment, AOH-9-glucoside, discovered for the first time in naturally contaminated food items, and AME-3-sulfate were found in concentrations similar to their parent toxins. In conclusion, the established multi-analyte method proved to be fit for purpose for generating comprehensive Alternaria toxin occurrence data in different food matrices.ᅟ [ABSTRACT FROM AUTHOR]

Alternariol (AOH) is one of the major mycotoxins produced by various species of Alternaria fungi. Natural occurrences of AOH have been reported in various foods, including fruits; processed fruit products such as apple juice, tomato products; wheat and other grains; oilseeds and products thereof, such as sunflower seeds, oilseed rape meal, and flax seed/linseed; and pecans. In this study, AOH-specific polyclonal antibodies were generated and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for monitoring AOH in bread and bran samples. The assay was very sensitive with a limit of detection (LOD) of 2.4 ± 0.6 ng/g and a half maximal inhibitory concentration (IC50) of 15.2 ± 2.6 ng/g in bread and a LOD of 8.4 ± 1.2 ng/g and IC50 of 52.8 ± 10.8 ng/g in bran extract. The assay was very specific to AOH and showed no cross-reactivity to alternariol monomethyl ether, altertoxin, altenuene, tentoxin, or tenuazonic acid. The effect of organic solvents on the assay was tested. The ELISA system tolerated methanol and acetonitrile as co-solvents at up to 5% content without significant loss of IC50 value. Recoveries in all cases were greater than 75%, and the results using this method were comparable to those obtained from mass spectrometry methods. We conclude that this method is suitable for rapid detection of AOH in bread and bran samples, without expensive analytical equipment or time-consuming sample preparation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Molecularly imprinted porous polymer microspheres selective to Alternaria mycotoxins, alternariol (AOH) and alternariol monomethyl ether (AME), were synthesized and applied to the extraction of both mycotoxins in food samples. The polymer was prepared using 4-vinylpiridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as cross-linker and 3,8,9-trihydroxy-6 H -dibenzo[ b,d ]pyran-6-one (S2) as AOH surrogate template. A molecularly imprinted solid phase extraction (MISPE) method has been optimized for the selective isolation of the mycotoxins from aqueous samples coupled to HPLC with fluorescence (λ ex = 258 nm; λ em = 440 nm) or MS/MS analysis. The MISPE method was validated by UPLC–MS/MS for the determination of AOH and AME in tomato juice and sesame oil based on the European Commission Decision 2002/657/EC. Method performance was satisfactory with recoveries from 92.5% to 106.2% and limits of quantification within the 1.1–2.8 µg kg −1 range in both samples. [ABSTRACT FROM AUTHOR]

Covering: 1893 to 2017 Harold Raistrick was involved in the discovery of many of the most important classes of fungal metabolites during the 20th century. This review focusses on how these discoveries led to developments in isotopic labelling, biomimetic chemistry and the discovery, analysis and exploitation of biosynthetic gene clusters for major classes of fungal metabolites including: alternariol; geodin and metabolites of the emodin pathway; maleidrides; citrinin and the azaphilones; dehydrocurvularin; mycophenolic acid; and the tropolones. Key recent advances in the molecular understanding of these important pathways, including the discovery of biosynthetic gene clusters, the investigation of the molecular and chemical aspects of key biosynthetic steps, and the reengineering of key components of the pathways are reviewed and compared. Finally, discussion of key relationships between metabolites and pathways and the most important recent advances and opportunities for future research directions are given. [ABSTRACT FROM AUTHOR]

• A mixture of two MIPs is used for the simultaneous determination of Zearalenone and Alternariol mycotoxins. • The experimental parameters of the MISPE method are optimized through a CCD Experimental design. • The developed analytical method is applied the analysis vegetable oils. This work reports the optimization of a method using Molecularly Imprinted Polymers (MIPs) for the simultaneous determination of zearalenone and alternariol mycotoxins. The method was optimized using a chemometric approach where in the optimized conditions, the cartridges with a mixture (50:50, w/w) of both MIPs, were loaded with 30 mL of sample, washed with 2 mL of ACN/water (20/80, v/v) and eluted with 2.5 mL of trifluoroacetic acid/MeOH (3/97, v/v). The extracts were analyzed by HPLC coupled to a fluorescence detector (FLD). The optimized method has been applied and validated to the analysis of the mycotoxins in maize, sunflower and olive oils samples with a limit of detection of 5 and 2 µg kg−1, respectively. Recoveries were in the range of 94 % to 108 % (RSD < 6 %) for zearalenone and 92 % to 113 % (RSD < 5 %) for alternariol. The results were confirmed by HPLC-MS/MS. [ABSTRACT FROM AUTHOR]

Abstract Alternaria toxins are secondary metabolites produced by fungi that can contaminate cereals, oilseeds, fruits and vegetables. The chronic dietary exposure to four individual Alternaria toxins was estimated using 15,563 analytical results/4,249 samples (3,648 on alternariol (AOH), 3,654 on alternariol monomethyl ether (AME), 4,168 on tenuazonic acid (TeA) and 4,093 on tentoxin (TEN)). Most of the analytical results were left‐censored data (92%), with only 1,290 quantified results (mainly on ‘Oilseeds’ and ‘Grain milling products’). The highest exposure to AOH was estimated in ‘Toddlers’, with the mean exposure between 3.8 and 71.6 ng/kg body weight (bw) per day (minimum lower bound–maximum upper bound, (LB–UB)) and the 95th percentile exposure between 11.4 and 270.5 ng/kg bw per day (LB–UB). Overall, ‘Fruit and fruit products’ were the most important contributors to the dietary exposure to AOH. The highest exposure to AME was estimated in ‘Toddlers’, with mean exposure between 3.4 and 38.8 ng/kg bw per day (LB–UB) and 95th percentile exposure between 10.3 and 97.3 ng/kg bw per day (LB–UB). Overall, the main contributors to the dietary exposure to AME were ‘Vegetable oil’ and ‘Pome fruits’ (pears). The highest exposure to TeA was estimated in ‘Toddlers’ with mean exposure between 100 and 1,614 ng/kg bw per day (LB–UB), and in ‘Infants’ for the 95th percentile exposure estimations (98–3,603 ng/kg bw per day, LB–UB). ‘Cereal‐based food for infants and young children’ was the main contributor in ‘Infants’ and ‘Toddlers’, and tomatoes and tomato‐based products in other age classes. The highest exposure to TEN was estimated in ‘Toddlers’, with the mean exposure between 1.6 and 33.4 ng/kg bw per day (LB–UB), and the 95th percentile exposure around 55 ng/kg bw per day (UB) in different age classes. ‘Fruiting vegetables’ (tomatoes) were the major contributor to the dietary exposure to TEN. Although based on limited data, vegetarians seem to have higher dietary exposure to Alternaria toxins than the general population.

Alternaria toxins are emerging mycotoxins that gained considerable interest with increasing evidence of their existence and toxicological properties. There is limited research and insufficient data about their in vivo hazardous effects. We designed this study to evaluate histopathological and genotoxic in vivo impacts of alternariol (AOH) on the parotid gland as well as to assess the competency of gallic acid (GA) in reversing these effects. Forty healthy adult male Wister rats were utilized and assigned equally on control, GA, alternariol and AOH+ gallic treated groups. Parotid gland samples from experimental groups were collected and then examined for histopathological, ultrastructural and immunohistochemical examination for 4-hydroxynonenal “4-HNE as lipid peroxidation marker” as well as Comet assay for DNA damage. Additionally, parotid tissue homogenates were tested for catalase “CAT”, superoxide dismutase “SOD” and malondialdehyde “MDA” levels. Our data proved that alternariol produced various histopathological and ultrastructural alterations of parotid acini as well as significant DNA damage, significant reduction of CAT and SOD enzymatic activity and significant boosting of 4-HNE immunohistochemical expression and MDA levels as compared to control group. On the other hand, gallic acid administration almost restored histological and ultrastructural parotid architecture, 4-HNE immune-expression and biochemical levels. Ultimately, we demonstrated alternariol-induced histopathological and genotoxic alterations on parotid gland as well as the competency of gallic acid in reversing these effects.

Alternaria alternata is a critical phytopathogen that causes foodborne spoilage and produces a polyketide mycotoxin, alternariol (AOH), and its derivative, alternariol monomethyl ether (AME). In this study, the inhibitory effects of the essential oil citral on the fungal growth and mycotoxin production of A. alternata were evaluated. Our findings indicated that 0.25 μL/mL (222.5 μg/mL) of citral completely suppressed mycelial growth as the minimum inhibitory concentration (MIC). Moreover, the 1/2MIC of citral could inhibit more than 97% of the mycotoxin amount. Transcriptomic profiling was performed by comparative RNA-Seq analysis of A. alternata with or without citral treatment. Out of a total of 1334 differentially expressed genes (DEGs), 621 up-regulated and 713 down-regulated genes were identified under citral stress conditions. Numerous DEGs for cell survival, involved in ribosome and nucleolus biogenesis, RNA processing and metabolic processes, and protein processing, were highly expressed in response to citral. However, a number of DEGs responsible for the metabolism of several carbohydrates and amino acids, sulfate and glutathione metabolism, the metabolism of xenobiotics and transporter activity were significantly more likely to be down-regulated. Citral induced the disturbance of cell integrity through the disorder of gene expression, which was further confirmed by the fact that exposure to citral caused irreversibly deleterious disruption of fungal spores and the inhibition of ergosterol biosynthesis. Citral perturbed the balance of oxidative stress, which was likewise verified by a reduction of total antioxidative capacity. In addition, citral was able to modulate the down-regulation of mycotoxin biosynthetic genes, including pksI and omtI. The results provide new insights for exploring inhibitory mechanisms and indicate citral as a potential antifungal and antimytoxigenic alternative for cereal storage.

Alternariol is an Alternaria mycotoxin that appears in fruits, tomatoes, oilseeds, and corresponding products. Chronic exposure to it can induce carcinogenic and xenoestrogenic effects. Cyclodextrins (CDs) are ring-shaped molecules built up by glucose units, which form host−guest type complexes with some mycotoxins. Furthermore, insoluble CD polymers seem suitable for the extraction/removal of mycotoxins from aqueous solutions. In this study, the interactions of alternariol with β- and γ-CDs were tested by employing fluorescence spectroscopic and modeling studies. Moreover, the removal of alternariol from aqueous solutions by insoluble β-CD bead polymer (BBP) was examined. Our major observations/conclusions are the following: (1) CDs strongly increased the fluorescence of alternariol, the strongest enhancement was induced by the native γ-CD at pH 7.4. (2) Alternariol formed the most stable complexes with the native γ-CD (logK = 3.2) and the quaternary ammonium derivatives (logK = 3.4−3.6) at acidic/physiological pH and at pH 10.0, respectively. (3) BBP effectively removed alternariol from aqueous solution. (4) The alternariol-binding ability of β-CD polymers was significantly higher than was expected based on their β-CD content. (5) CD technology seems a promising tool to improve the fluorescence detection of alternariol and/or to develop new mycotoxin binders to decrease alternariol exposure.

Alternaria toxins are mycotoxins produced by various Alternaria species which, besides the Fusarium species, represent the principal contaminants of wheat worldwide. As currently, only limited information on the behaviour of Alternaria toxins during processing of cereals is available, the objective of this study was to investigate the effect of the dry milling process of wheat on Alternaria toxins distribution. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) content were analysed by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in all milling fractions of untreated (control), fungicide-treated, Alternaria tenuissima inoculated and commercial wheat sample. After dry milling process, in last break and milling flows and by-products, increased concentration of examined Alternaria toxins was detected. TeA was quantified in almost all milling fractions in all tested wheat samples, while AOH and AME were detectable mostly in last break and milling flows and by-products. In respect to the contamination with Alternaria toxins, white flour can be considered as relatively safe product. Since Alternaria toxins are concentrated mainly in the peripheral parts of the kernel, a special attention should be given to their content in low-grade flours and milling by-products.

Alternaria brown spot, caused by Alternaria alternata (Fr.) Keissl, is an important disease in tangerines and their hybrids, affecting leaves, twigs, and immature fruit. Differences in susceptibility to this pathogenic fungus have been described for different Citrus species. In this paper, the expression of the mycotoxins alternariol and alternariol monomethyl ether in different A. alternata isolates was analyzed by HPLC-MS. A correlation was observed between the mycotoxins content and the pathogenicity of each isolated of A. alternata used, suggesting that the mycotoxins may be involved in the evolution of brown spot in ‘Fortune’ fruits caused by this fungus. The increased expression of the above mycotoxins was associated with the end of mycelia growth, high sporulation, and an increase in hyphal melanization in the fungus. On the other hand, the presence of laccase activity in the xylem of ‘Fortune’ fruits inoculated with A. alternata suggests that this is the way the fungus propagates in the plant. These results add to our knowledge of the pathogenesis of A. alternata in Citrus.

An immunochromatographic strip was developed for the determination of alternariol(AOH) using both semi-quantitative and quantitative methods based on a sensitive monoclonal antibody we produced. With AOH-spiked samples, the visual limit of detection for cereal and juice were 160 ng/g and 25 ng/mL, and the cut-off values were 640 ng/g and 100 ng/mL, respectively. For quantitative analysis based on a strip scan reader, the calculated limit of detection was 15.5–19.4 ng/g for cereal and 2.8–3.5 ng/mL for fruit juice. The recovery rates ranged from 116% to 125%, and 113% to 123%, respectively. Therefore, this method was effective for AOH detection and suitable for on-site detection and rapid screening of samples. [ABSTRACT FROM PUBLISHER]

Alternariol (AOH) is a toxic metabolite of phytopathogenic fungi of the Alternaria spp. and important contaminant of agricultural commodities. According to the recent studies, AOH has a potential to modulate the endocrine system of humans and animals. In the view of these reports, our study addressed the effects of AOH on human estrogen receptor (hERα) and androgen receptor (hAR) signaling with the use of the yeast estrogen and androgen reporter bioassays. Our results show that, apart from a weak estrogenic response, AOH induces full androgenic response of the bioassay with the EC50 of 269.4 μM. The androgenic potency of AOH relative to testosterone (T) is 0.046%. Moreover, in the presence of T, AOH at 5 μM acts as a weak antiandrogen, whereas at higher concentrations AOH sum up with the androgenic activity of T in a dose-dependent manner, suggesting additive effect. To our knowledge it is the first report of the androgenic potency of natural, nonsteroidal substance and may have the impact on the direction of the further studies. Further research is warranted to clarify the role of AOH in disruption of AR signaling in humans and animals. [ABSTRACT FROM AUTHOR]

Polyamines are essential for all living organisms as they are involved in several vital cell functions. The biosynthetic pathway of polyamines and its regulation is well established and, in this sense, the ornithine descarboxylase (ODC) enzyme acts as one of the controlling factors of the entire pathway. In this work we assessed the inhibition of the ODC with D, l -α-difluoromethylornithine (DFMO) on Alternaria alternata and we observed that fungal growth and mycotoxin production were reduced. This inhibition was not completely restored by the addition of exogenous putrescine. Actually, increasing concentrations of putrescine on the growth media negatively affected mycotoxin production, which was corroborated by the downregulation of pksJ and altR , both genes involved in mycotoxin biosynthesis. We also studied the polyamine metabolism of A. alternata with the goal of finding new targets that compromise its growth and its mycotoxin production capacity. In this sense, we tested two different polyamine analogs, AMXT-2455 and AMXT-3016, and we observed that they partially controlled A. alternata ’s viability in vitro and in vivo using tomato plants. Finding strategies to design new fungicide substances is becoming a matter of interest as resistance problems are emerging. [ABSTRACT FROM AUTHOR]

The Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been shown to possess genotoxic and cytotoxic properties. In this study, the ability of AOH and AME to modulate innate immunity in the human bronchial epithelial cell line (BEAS-2B) and mouse macrophage cell line (RAW264.7) were investigated. During these studies, it was discovered that AOH and to a lesser extent AME potently suppressed lipopolysaccharide (LPS)-induced innate immune responses in a dose-dependent manner. Treatment of BEAS-2B cells with AOH resulted in morphological changes including a detached pattern of growth as well as elongated arms. AOH/AME-related immune suppression and morphological changes were linked to the ability of these mycotoxins to cause cell cycle arrest at the G2/M phase. This model was also used to investigate the AOH/AME mechanism of immune suppression in relation to aryl hydrocarbon receptor (AhR). AhR was not found to be important for the immunosuppressive properties of AOH/AME, but appeared important for the low levels of cell death observed in BEAS-2B cells. [ABSTRACT FROM AUTHOR]

While Highly Active Antiretroviral Therapy (HAART) has significantly decreased the mortality of human immunodeficiency virus (HIV)-infected patients, emerging drug resistance to approved HIV-1 integrase inhibitors highlights the need to develop new antivirals with novel mechanisms of action. In this study, we screened a library of microbial natural compounds from endophytic fungus Colletotrichum sp. and identified alternariol 5-O-methyl ether (AME) as a compound that inhibits HIV-1 pre-integration steps. Time-of addition analysis, quantitative real-time PCR, confocal microscopy, and WT viral replication assay were used to elucidate the mechanism. As opposed to the approved integrase inhibitor Raltegravir, AME reduced both the integrated viral DNA and the 2-long terminal repeat (2-LTR) circular DNA, which suggests that AME impairs the nuclear import of viral DNA. Further confocal microscopy studies showed that AME specifically blocks the nuclear import of HIV-1 integrase and pre-integration complex without any adverse effects on the importin α/β and importin β-mediated nuclear import pathway in general. Importantly, AME inhibited Raltegravir-resistant HIV-1 strains and exhibited a broad anti-HIV-1 activity in diverse cell lines. These data collectively demonstrate the potential of AME for further development into a new HIV inhibitor, and suggest the utility of viral DNA nuclear import as a target for anti-HIV drug discovery. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study isolated and identified the fungus that causes postharvest disease in dragon fruit ( Hylocereus undatus Haw.). The in vitro and in vivo antifungal activity of some essential oils were evaluated against the fungus. Morphophysiological and molecular identification confirmed the fungus was Alternaria alternata . The essential oils of Cinnamomum zeylanicum , Cymbopogon flexuosus , Eucalyptus globulus , Eugenia caryophyllus , and Rosmarinus officinalis were evaluated by the microdilution broth technique, disc diffusion, scanning electron microscopy, and fluorescence microscopy. Evaluation of the composition of the essential oils by gas chromatography/mass spectrometry revealed substantial amounts of eugenol as a major constituent of E. caryophyllus and C. zeylanicum (90.50% and 80.70%, respectively). The other essential oils of R. officinalis contained α-pinene (24.5%) and camphor (22.0%) as major components. E. globulus contained 1,8-cineole (78.9%). C. flexuosus contained neral (35.1%) and geranial (42.6%). C. zeylanicum and E. caryophyllus were the most active against the isolated fungi at minimum inhibitory concentrations of 250 and 500 μg/ml, respectively, causing morphological changes in the hyphae. The in vivo assay indicated that the fruits that were treated with E. caryophyllus at concentrations of 500 and 1000 μg/ml exhibited a 31% reduction of mycelial growth compared with the control. These results suggest that the essential oils of C. zeylanicum and E. caryophyllus are active against A. alternata both in vitro and in vivo , which may be promising for control of the microorganism. [ABSTRACT FROM AUTHOR]

Alternariol (AOH) and altertoxin II (ATX II) are mycotoxins formed by Alternaria spp . Since they are expected to co-occur in Alternaria -infested food and feed, we addressed the question of combinatory effects. In addition, potentially involved regulatory microRNAs were surveyed in an exploratory approach. Cytotoxicity measurements in constant ratio combinations of 1:10 or 1:1 (ATX II: AOH) mainly revealed additive effects in HepG2, HT29 and HCEC-1CT cells. Yet, in specific high doses antagonism was found. Microarray analysis of miRNA expression profiles in HepG2 cells indicated different patterns of miRNA regulation by AOH and ATX II, including several miRNA species for which no distinct functions are currently known. Among others, miR-4654, miR-4715_3p and miR-6720_3p were up-regulated by AOH and miR-5583_5p was down-regulated by ATX II. Additionally, miR-1323, involved in hindering DNA repair mechanisms, was decreased by ATX II. Digital droplet PCR (ddPCR) analysis of selected miRNAs indicated regulation of miR-29a by AOH, which might play a role in AOH-induced apoptosis. miR-192 and miR-224 regulation was associated with antagonistic cytotoxic effects of AOH and ATX II combinations. Our study represents the first evaluation on combinatory effects of AOH and ATX II. [ABSTRACT FROM AUTHOR]

BACKGROUND 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. RESULTS 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. CONCLUSION 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 [ABSTRACT FROM AUTHOR]

Effect of induced drought on plant defense response to Alternaria blight infection and subsequent impact on yield and oil quality was investigated in leading Indian mustard varieties viz. RSPR-69, Pusa Mahak and RH-30 with reference to variety PC-5 as control. Alternaria blight incidence in induced drought was found to be reduced significantly in all mustard varieties. Foliar levels of antioxidant ascorbic acid and total phenol, detoxifying/cell protecting enzymes viz. superoxide dismutase (SOD), peroxidase (POX), phenylalanine ammonia ayase (PAL) and polyphenol oxidase (PPO) and related biochemical parameters viz. proline, total amino acid and total soluble protein increased in infected leaves and their level further elevated under induced drought as well as Alternaria brassicae inoculation. However, remarkable rise in total sugar level and further loss in chlorophyll content and relative water content (RWC) were observed under induced drought conditions. Moreover, losses due to Alternaria blight infection i.e. seed/oil yield, decline in iodine number and rise in peroxide and saponification number were recovered when drought was artificially induced to the mustard varieties. The Alternaria blight sensitive but drought tolerant RSPR-69 variety got maximum recovery out of induced drought treatment. [ABSTRACT FROM AUTHOR]

The genus Alternaria comprises ubiquitous pathogens and saprophytes. They can even grow at low temperature, so they are the main fungi responsible for the spoilage of various fruits, vegetables, grains and their products during long-distance transport and refrigerated storage. Alternaria mycotoxins are the secondary metabolite of the genus Alternaria. They can be divided into five main classes according to their chemical structures, including dibenzopyrone derivatives, tetramic acid derivatives, perylene derivatives, AAL toxins and miscellaneous structures. Alternaria mycotoxins are associated with many health effects because of their mutagenicity, teratogenicity and carcinogenicity, which can cause economic losses to agriculture and serious diseases in humans and animals. So far, there is still a lack of monitoring data on these contaminants of Alternaria mycotoxins. Moreover, there are still no statutory or guideline limits set for Alternaria mycotoxins in food and feed by regulatory authorities worldwide. Until now, many analytical methods have been developed for the detection and quantification of Alternaria mycotoxins. On the basis of briefly introducing the chemical structures and toxicities of Alternaria mycotoxins, this article provides an overview of the progress achieved in the detection techniques for Alternaria mycotoxins, focusing on the analytical methods of thin layer chromatography (TLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA) and so on. Finally, the problems of these analytical methods and future development trends are discussed. [ABSTRACT FROM AUTHOR]

Herein, rhenium disulfide/polydopamine/Ag(ReS 2 /PDA/Ag) plasmonic composites assembled surface-enhanced Raman scattering(SERS) membrane substrates were fabricated for rapid analysis of 6-benzylaminopurine(6-BAP) and alternariol(AOH). The ReS 2 /PDA/Ag composites including ReS 2 /PDA/AgNPs and ReS 2 /PDA/AgNPs@thioglycolic acid(ReS 2 /PDA/AgNPs@TGA) were prepared via surface modification strategy and assembling method. With PDA modification, the ReS 2 /PDA/AgNPs improved the affinity of substrate to 6-BAP. With further TGA modification, the ReS 2 /PDA/AgNPs@TGA advanced the affinity between substrate and AOH. ReS 2 /PDA/Ag composites were assembled to SERS membrane substrate, which integrated SERS enhancement, fast separation/adsorption, and detection all-in-one. The membrane substrates exhibited good stability with relative standard deviations less than 5.5% and 6.5% for one batch and different batches, respectively. SERS methods based on the membrane substrates were developed for analysis of 6-BAP and AOH with detection limit of 24.4 and 23.6 μg/L, respectively. The methods were applied for determination of 6-BAP in bean sprout with recoveries of 87.1%− 107.0% and RSDs of 2.5%− 6.6%, AOH in pear with recoveries of 86.0%− 102.0% and RSDs of 2.9%− 8.1%, respectively. The results were confirmed by chromatographic method with relative error less than 5.5%. The analytical process completed within 35 min shows great potential in rapid food safety analysis. [Display omitted] • ReS 2 /PDA/AgNPs and ReS 2 /PDA/AgNPs@TGA SERS membrane substrates prepared via modification strategy and assembling method. • SERS enhancement and membrane separation/adsorption coupled with detection all-in-one. • SERS methods for 6-benzylaminopurine and alternariol analysis. • Rapid quantifications of 6-benzylaminopurine and alternariol in food. [ABSTRACT FROM AUTHOR]

In the present study, CotA laccase was cloned from Bacillus licheniformis ZOM-1 and expressed in Escherichia coli. In addition to the oxidative degradation of zearalenone (ZEN) and aflatoxin B1 (AFB1), CotA laccase also has the ability to degrade the Alternaria toxin alternariol (AOH). This is the first report of an Alternaria toxin-degrading enzyme. The optimal reaction temperature for CotA laccase is 80 °C, and the optimal reaction pH is approximately 9.0. By high-resolution mass spectrometry, we identified the oxidative degradation products. Additionally, toxicological analysis indicated that the degradation products had a significantly reduced effect on cell viability relative to the original toxins. In addition, we found that B. licheniformis ZOM-1 has the ability to degrade three mycotoxins, ZEN, AOH and alternariol monomethyl ether (AME). These findings provide possible application prospects for CotA laccase and B. licheniformis ZOM-1 as mycotoxin degradation products in food or feed. • First report on the biodegradation of AOH and AME. • Simultaneous decomposition of ZEN, AOH, and AME by Bacillus licheniformis ZOM-1. • Simultaneous decomposition of ZEN, AFB1, and AOH by CotA laccase. [ABSTRACT FROM AUTHOR]

A study was carried out to determine the efficacy of Alternaria alternate as biological control agent against the weed Pteridium aquilinum. The study was undertaken throughout grasslands of Trabzon province in 2009-2010. Symptoms of the disease were observed on the leaves of P. aquilinum in many locations. Host selectivity and biological efficacy tests were performed to determine biological control potential of A. alternata. Fourteen cultural plants from them seven forage plants were used in these tests. None of the tested plants was infected by A. alternata but detected only on P. aquilinum. P. aquilinum plants with 3-4 leaves were sprayed with a conidial suspension of A. alternata. Biocontrol effectiveness of A. alternata on P. aquilinum expressed 72.25% disease incidence at the end of one-month incubation period of the sprayed plants. This study is the first step against this host, and further studies should be done to improve effectiveness of the present A. alternate isolate and its effect on other economic cultivated crops. [ABSTRACT FROM AUTHOR]

The mycotoxin alternariol ( AOH) is produced by Alternaria fungi. It occurs naturally in foodstuffs and is frequently found as contaminant in fruit and grain products. Most information regarding AOH toxicity and the potential mechanisms involved comes from in vitro studies, as only very limited in vivo studies have been performed. AOH forms reactive oxygen species ( ROS) and interacts with DNA topoisomerase, thereby generating both single ( SSB)- and double-strand DNA beaks ( DSB). This triggers various DNA damage response pathways. AOH causes a marked reduction in proliferation in mammalian cells due to cell cycle arrest often in the G2/M-phase. After an additional inhibition of cytokinesis, cells with abnormal nuclei as well as polyploidy are reported. In macrophages, AOH may increase autophagic activity and induce senescence. Furthermore, AOH is found to change the morphology and phenotype of various human macrophage cell models. Studies so far indicate that the AOH-induced effects are primarily a result of DSB via its effects on topoisomerase activity. Thus, most probably there will be a threshold for the AOH-induced effects, typically seen in the 5-10 μM range. These in vitro mechanistic studies further support the in vivo studies suggesting low acute toxicity. However, a decreased immune response to infections and/or a disturbed balance of the adaptive immune system when exposed together with other mycotoxins cannot be excluded. This hypothesis needs to be further explored with proper in vivo studies. [ABSTRACT FROM AUTHOR]

Alternaria toxins are secondary metabolites produced by fungi that can contaminate cereals, oilseeds, fruits and vegetables. The chronic dietary exposure to four individual Alternaria toxins was estimated using 15,563 analytical results/4,249 samples (3,648 on alternariol (AOH), 3,654 on alternariol monomethyl ether (AME), 4,168 on tenuazonic acid (TeA) and 4,093 on tentoxin (TEN)). Most of the analytical results were left-censored data (92%), with only 1,290 quantified results (mainly on 'Oilseeds' and 'Grain milling products'). The highest exposure to AOH was estimated in 'Toddlers', with the mean exposure between 3.8 and 71.6 ng/kg body weight (bw) per day (minimum lower bound-maximum upper bound, (LB-UB)) and the 95th percentile exposure between 11.4 and 270.5 ng/kg bw per day (LB-UB). Overall, 'Fruit and fruit products' were the most important contributors to the dietary exposure to AOH. The highest exposure to AME was estimated in 'Toddlers', with mean exposure between 3.4 and 38.8 ng/kg bw per day (LB-UB) and 95th percentile exposure between 10.3 and 97.3 ng/kg bw per day (LB-UB). Overall, the main contributors to the dietary exposure to AME were 'Vegetable oil' and 'Pome fruits' (pears). The highest exposure to TeA was estimated in 'Toddlers' with mean exposure between 100 and 1,614 ng/kg bw per day (LB-UB), and in 'Infants' for the 95th percentile exposure estimations (98-3,603 ng/kg bw per day, LB-UB). 'Cereal-based food for infants and young children' was the main contributor in 'Infants' and 'Toddlers', and tomatoes and tomato-based products in other age classes. The highest exposure to TEN was estimated in 'Toddlers', with the mean exposure between 1.6 and 33.4 ng/kg bw per day (LB-UB), and the 95th percentile exposure around 55 ng/kg bw per day (UB) in different age classes. 'Fruiting vegetables' (tomatoes) were the major contributor to the dietary exposure to TEN. Although based on limited data, vegetarians seem to have higher dietary exposure to Alternaria toxins than the general population. [ABSTRACT FROM AUTHOR]

This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC50 values which range from 0.11 (±0.02) to 4.69 (±0.47) μM showed the high cytotoxicity of these compounds.