[Alternaria toxins in tomato products marketed in the Russian Federation].

Tomatoes and tomato products are widely produced and consumed throughout the world. Alternaria spp. are the main cause of alternariosis (black mold disease) on fresh tomatoes, both in the field and after harvesting. Alternaria toxins are widespread contaminants of tomato products. The aim of the present study was to evaluate the contamination of tomato processing products from the domestic market with Alternaria toxins, as well as to assess their intake by humans through the consumption of tomato juices. Material and methods. The content of Alternaria toxins (alternatiol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid) was determined in 64 samples of tomato products (paste, ketchup, juice) by high-performance liquid chromatography coupled to tandem mass-spectrometric detection (HPLC-MS/MS). Results. The priority Alternaria toxins for tomato paste, ketchup and juice were tenuazonic acid (61% of 64 samples, in amounts from 20.0 to 1065.5 µg/kg), altenuene (52%, 8.9-200.1 µg/kg) and alternariol (27%, 12.2-561.6 µg/kg). Samples of tomato paste turned out to be the most contaminated with Alternaria toxins while tomato juice samples were the least contaminated. At the same time, several toxins were found in 91% of tomato paste samples, 35% of ketchups, and 23% of tomato juices. Conclusion. To the best of our knowledge, the present study is the first survey devoted to Alternaria toxins contamination of tomato paste, ketchup and tomato juice sold on the Russian market. The high frequency of their contamination with tenuazonic acid, altenuene and, to a lesser extent, alternariol has been established, which indicates a potential risk to human health when tomato processing products are consumed. This indicates the need for a hygienic assessment of contamination the above products with tenuazonic acid, altenuene and alternariol. When calculating the potential intake of Alternaria toxins for different age population groups, it was shown that high levels of alternariol (up to 56.77 ng/kg body weight per day) could be obtained under daily consumption of tomato juice by adults and children under three years of age, as well as tenuazonic acid when consuming tomato juice contaminated at the 95th percentile level as part of the diet in organized groups for orphans and children without parental care.

Competitiveness of reutericyclin producing and nonproducing Limosilactobacillus reuteri in food and intestinal ecosystems: a game of rock, paper, and scissors?

The ecological relationships among antimicrobial producing, resistant, and sensitive strains have been proposed to follow rock-paper-scissors dynamics, but evidence is mainly based on Gram-negative bacteriocins in vitro. The ecological relevance of antimicrobials in vivo or in situ has not been systematically studied. This study therefore aimed to analyze binary and ternary competitions among reutericyclin-producing strain Limosilactobacillus reuteri TMW1.656, its reutericyclin-resistant, nonproducing isogenic derivative L. reuteri TMW1.656∆rtcN, and the reutericyclin-sensitive, nonproducing L. reuteri TMW1.656∆rtcN∆rtcT in vitro (liquid culture and static plate), in situ (sourdough fermentation), and in vivo (gut of germ-free mice). In liquid culture, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Limosilactobacillus reuteri TMW1.656∆rtcN∆rtcT had a higher fitness than TMW1.656∆rtcN. On agar plates, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN∆rtcT. In situ, reutericyclin production and resistance had no influence on the fitness of the strains. In vivo, TMW1.656 had an advantage over TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Ternary competitions showed reutericyclin production was ecologically beneficial in all ecosystems. The findings support the ecological importance of reutericyclin in a variety of environments/niches, providing an explanation for the acquisition of the reutericyclin gene cluster in L. reuteri and its contribution to the ecological fitness of Streptococcus mutans.

Synergistic toxicity induced by the co-exposure of tenuazonic acid and patulin in Caenorhabditis elegans: Daf-16 plays an important regulatory role.

Tenuazonic acid (TeA) and patulin (PAT), as the naturally occurring mycotoxins with various toxic effects, are often detected in environment and food chain, has attracted more and more attention due to their widespread and high contaminations as well as the coexistence, which leads to potential human and animals' risks. However, their combined toxicity has not been reported yet. In our study, C. elegans was used to evaluate the type of combined toxicity caused by TeA+PAT and its related mechanisms. The results showed that TeA and PAT can induce synergistic toxic effects based on Combination Index (CI) evaluation model (Chou-Talalay method), that is, the body length, brood size as well as the levels of ROS, CAT and ATP were significantly affected in TeA+PAT-treated group compared with those in TeA- or PAT-treated group. Besides, the expressions of oxidative (daf-2, daf-16, cyp-35a2, ctl-1, ctl-3, pmk-1, jnk-1, skn-1) and intestinal (fat-5, pod-2, egl-8, pkc-3, ajm-1, nhx-2) stress-related genes were disrupted, among which daf-16 displayed the most significant alternation. Further study on daf-16 gene defective C. elegans showed that the damages to the mutant nematodes were significantly attenuated. Since daf-2, daf-16, jnk-1 and pmk-1 are evolutionarily conserved, our findings could hint synergistic toxic effects of TeA+PAT on higher organisms.

Unraveling biomarkers of exposure for tenuazonic acid through urinary metabolomics.

Mycotoxins are secondary metabolites produced by fungi such as Aspergillus, Alternaria, and Penicillium, affecting nearly 80% of global food crops. Tenuazonic acid (TeA) is the major mycotoxin produced by Alternaria alternata, a prevalent pathogen affecting plants, fruits, and vegetables. TeA is notably prevalent in European diets, however, TeA biomarkers of exposure and metabolites remain unknown. This research aims to bridge this knowledge-gap by gaining insights about human TeA exposure and metabolization. Nine subjects were divided into two groups. The first group received a single bolus of TeA at the Threshold of Toxicological Concern (TTC) to investigate the presence of TeA urinary biomarkers, while the second group served as a control. Sixty-nine urinary samples were prepared and analyzed using UPLC-Xevo TQ-XS for TeA quantification and UPLC-Orbitrap Exploris for polar metabolome acquisition. TeA was rapidly excreted during the first 13 h and the fraction extracted was 0.39 ± 0.22. The polar metabolome compounds effectively discriminating the two groups were filtered using Orthogonal Partial Least Squares-Discriminant Analysis and subsequently annotated (n = 122) at confidence level 4. Finally, the urinary metabolome was compared to in silico predicted TeA metabolites. Nine metabolites, including oxidized, N-alkylated, desaturated, glucuronidated, and sulfonated forms of TeA were detected.

The fate of mycotoxins in oranges during storage and processing.

To evaluate the safety of orange consumption induced by mycotoxins, 'Newhall' navel oranges were artificially inoculated with P. expansum and A. tenuissima, followed by an evaluation of the distribution and migration patterns of corresponding mycotoxins (patulin [PAT], tentoxin [Ten], altenuene [ALT], alternariol monomethyl ether [AME], alternariol [AOH] and tenuazonic acid [TeA]) during orange storage and processing. The concentration of mycotoxins decreased as the increase of distance from the lesion, and mycotoxins could be detected throughout the orange when the lesion extended to 8 mm in diameter. AOH and AME pose the primary source of dietary risk with high concentrations and low thresholds of toxicological concern. Orange juice and pectin processing could remove 43.4-98.7% of mycotoxins, while tangerine peelprocessing might lead to significant enrichment of mycotoxins with the processing factors (PFs) of 2.8-3.5. The findings may offer scientific insights into mitigating the dietary risk of mycotoxin exposure from oranges and their derivatives.

Natural Occurrence of Regulated and Emerging Mycotoxins in Wheat Grains and Assessment of the Risks from Dietary Mycotoxins Exposure in China.

Wheat grains are susceptible to contamination with various natural mycotoxins including regulated and emerging mycotoxins. This study surveyed the natural presence of regulated mycotoxins, such as deoxynivalenol (DON) and zearalenone (ZEN), and emerging mycotoxins such as beauvericin (BEA), enniatins (ENNs such as ENA, ENA1, ENB, ENB1) and Alternaria mycotoxins (i.e., alternariol monomethyl ether (AME), alternariol (AOH), tenuazonic acid (TeA), tentoxin (TEN), and altenuene (ALT)) in wheat grains randomly collected from eight provinces across China in 2021. The results revealed that each wheat grain sample was detected with at least one type of mycotoxin. The detection rates of these mycotoxins ranged from 7.1% to 100%, with the average occurrence level ranging from 1.11 to 921.8 µg/kg. DON and TeA were the predominant mycotoxins with respect to both prevalence and concentration. Approximately 99.7% of samples were found to contain more than one toxin, and the co-occurrence of ten toxins (DON + ZEN + ENA + ENA1 + ENB + ENB1 + AME + AOH + TeA + TEN) was the most frequently detected combination. The dietary exposure to different mycotoxins among Chinese consumers aged 4-70 years was as follows: 0.592-0.992 µg/kg b.w./day for DON, 0.007-0.012 µg/kg b.w./day for ZEN, 0.0003-0.007 µg/kg b.w./day for BEA and ENNs, 0.223-0.373 µg/kg b.w./day for TeA, and 0.025-0.041 µg/kg b.w./day for TEN, which were lower than the health-based guidance values for each mycotoxin, with the corresponding hazard quotient (HQ) being far lower than 1, implying a tolerable health risk for Chinese consumers. However, the estimated dietary exposure to AME and AOH was in the range of 0.003-0.007 µg/kg b.w./day, exceeding the Threshold of Toxicological Concern (TTC) value of 0.0025 µg/kg b.w./day, demonstrating potential dietary risks for Chinese consumers. Therefore, developing practical control and management strategies is essential for controlling mycotoxins contamination in the agricultural systems, thereby ensuring public health.

Saturated brine dissolution and liquid-liquid extraction combined with UPLC-MS/MS for the detection of typical Alternaria toxins in pear paste.

BACKGROUND: Alternaria can infest pears to produce metabolites, which can contaminate pears and their processed products. Pear paste, one of the most important pear-based products, is popular among Chinese consumers especially for its cough relieving and phlegm removal properties. Although people are concerned about the risk of Alternaria toxins in many agro-foods and their products, little is known about the toxins in pear paste. RESULTS: A method was developed for the determination of tenuazonic acid, alternariol, alternariol menomethyl ether, altenuene and tentoxin in pear paste by ultra-performance liquid chromatography tandem mass spectrometry with saturated sodium sulphate dissolution and acidified acetonitrile extraction. The mean recoveries of the five toxins were 75.3-113.8% with relative standard deviations of 2.8-12.2% at spiked levels of 1.0-100 µg kg-1 . Alternaria toxins were detected in 53 out of 76 samples, with a detection rate of 71.4%. Tenuazonic acid (67.1%), alternariol (35.5%), tentoxin (23.7%) and alternariol monomethyl ether (7.9%) were detected in all samples at concentrations of < limit of quantification (LOQ)-105.0 µg kg-1 , < LOQ-32.1 µg kg-1 , < LOQ-74.2 µg kg-1 and < LOQ-15.1 µg kg-1 , respectively. Altenuene was never found in pear paste samples. Tenuazonic acid, alternariol, tentoxin and alternariol menomethyl ether should be focused on due to their toxicity and detection rates. CONCLUSION: To the best of our knowledge, this is the first report on the detection method and residue levels of Alternaria toxins in pear paste. The proposed method and research data can provide technical support for the Chinese government to continuously monitor and control Alternaria toxins in pear paste, especially tenuazonic acid. It can also provide a useful reference for related researchers. © 2023 Society of Chemical Industry.

Natural Occurrence of Alternaria Toxins in Citrus-Based Products Collected from China in 2021.

A total of 181 citrus-based products, including dried fruits, canned fruits, and fruit juices, collected from China and from abroad in 2021 were analyzed for the four Alternaria toxins (ALTs): alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA) via ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS). Although the concentrations of the four ALTs varied by product and geographically, TeA was the predominant toxin followed by AOH, AME, and TEN. Products made in China showed higher levels of ALTs than those made abroad. Maximum levels of TeA, AOH, and AME in analyzed domestic samples were 4.9-fold, 1.3-fold, and 1.2-fold, respectively, higher than those in imported products. Furthermore, 83.4% (151/181) of the analyzed citrus-based products were contaminated with at least two or more ALTs. There were significant positive correlations between AOH and AME, AME and TeA, and TeA and TEN in all analyzed samples. More importantly, the solid and the condensed liquid products had higher concentrations of ALTs than the semi-solid product samples, as well as tangerines, pummelos, and grapefruits compared to the other kinds of citrus-based products. In conclusion, co-contamination with ALTs in commercially available Chinese citrus-based products was universal. Extensive and systematic surveillance of ALTs in citrus-based products, both domestic and imported, is required to obtain more scientific data for the determination of the maximum allowable concentrations of ALTs in China.

Bio-guided isolation of potential anti-inflammatory constituents of some endophytes isolated from the leaves of ground cherry (Physalis pruinosa L.) via ex-vivo and in-silico studies.

BACKGROUND: 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. METHODS: 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-1ß 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. RESULTS: 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-1ß. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein (IL-1ß, TNF-α and INF-γ)-ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis. CONCLUSIONS: 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-1ß and INF-γ.

Tenuazonic acid alters immune and physiological reactions and susceptibility to pathogens in Galleria mellonella larvae.

Tenuazonic acid (TeA) is synthesized by phytopathogenic and opportunistic fungi and is detected in a broad range of foods. This natural compound is of interest in terms of toxicity to animals, but its mechanisms of action on insects are poorly understood. We administered TeA orally at different concentrations (0.2-5.0 mg/[gram of a growth medium]) to the model insect Galleria mellonella, with subsequent estimation of physiological, histological, and immunological parameters in different tissues (midgut, fat body, and hemolymph). Susceptibility of the TeA-treated larvae to pathogenic microorganisms Beauveria bassiana and Bacillus thuringiensis was also analyzed. The feeding of TeA to the larvae led to a substation delay of larval growth, apoptosis-like changes in midgut cells, and an increase in midgut bacterial load. A decrease in activities of detoxification enzymes and downregulation of genes Nox, lysozyme, and cecropin in the midgut and/or hemocoel tissues were detected. By contrast, genes gloverin, gallerimycin, and galiomycin and phenoloxidase activity proved to be upregulated in the studied tissues. Hemocyte density did not change under the influence of TeA. TeA administration increased susceptibility of the larvae to B. bassiana but diminished their susceptibility to B. thuringiensis. The results indicate that TeA disturbs wax moth gut physiology and immunity and also exerts a systemic action on this insect. Mechanisms underlying the observed changes in wax moth susceptibility to the pathogens are discussed.

Metabolite Analysis of Alternaria Mycotoxins by LC-MS/MS and Multiple Tools.

Alternaria fungi are widely distributed plant pathogens that invade crop products, causing significant economic damage. In addition, toxic secondary metabolites produced by the fungi can also endanger consumers. Many of these secondary metabolites are chemically characterized as mycotoxins. In this study, Q Exactive Orbitrap mass spectrometry was used for the non-targeted analysis of the metabolome of seven Alternaria isolates cultured on Potato Carrot Agar (PCA), Potato Dextrose Agar (PDA) and Potato Sucrose Agar (PSA) medium. Due to the difficulty of detecting modified toxins, an analytical strategy with multiple visual analysis tools was also used to determine the presence of sulfate conjugated toxins, as well as to visualize the molecular network of Alternaria toxins. The results show that PSA medium exhibits more advantageous properties for the culture of Alternaria, with more toxigenic species and quantities and more obvious metabolic pathways. Based on high-resolution tandem mass spectrometry (MS/MS) data, the mycotoxins and their metabolites were mainly clustered into four groups: alternariol (AOH)/alternariol monomethyl ether (AME)/altenusin (ALU)/altenuene (ALT)/dehydroaltenusin (DHA)/Desmethyldehydroaltenusin (DMDA) families, Altertoxin-I (ATX-I) family, tentoxin (TEN) family and tenuazonic acid (TeA) family. Moreover, the PSA medium is more suitable for the accumulation of AOH, AME, ALU, ALT, DHA and DMDA, while the PDA medium is more suitable for the accumulation of ATX-I, TEN and TeA. This research may provide theoretical support for the metabolomics study of Alternaria.

Development of Acid Hydrolysis-Based UPLC-MS/MS Method for Determination of Alternaria Toxins and Its Application in the Occurrence Assessment in Solanaceous Vegetables and Their Products.

In this work, we proposed an acid hydrolysis-based analytical method for the detection of Alternaria toxins (ATs) in solanaceous vegetables and their products with solid-phase extraction (SPE) and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This study was the first to reveal that some compounds in the eggplant matrix bind to altenusin (ALS). Validation under optimal sample preparation conditions showed that the method met the EU criteria, exhibiting good linearity (R2 > 0.99), matrix effects (-66.6--20.5%), satisfying recovery (72.0-107.4%), acceptable precision (1.5-15.5%), and satisfactory sensitivity (0.05-2 µg/kg for limit of detection, 2-5 µg/kg for limit of quantification). Out of 393 marketed samples, only 47 samples were detected, ranging from 0.54-806 µg/kg. Though the occurrence ratio (2.72%) in solanaceous vegetables could be negligible, the pollution status in solanaceous vegetable products was much more serious, and the incidences were 41.1%. In the 47 contaminated samples, the incidences were 4.26% for alternariol monomethyl ether (AME), 6.38% for alternariol (AOH) and altenuene (ALT), 42.6% for tentoxin (TEN), and 55.3% for tenuazonic acid (TeA).

Development of a Novel LC-MS/MS Multi-Method for the Determination of Regulated and Emerging Food Contaminants Including Tenuazonic Acid, a Chromatographically Challenging Alternaria Toxin.

The regulation of food contaminants in the European Union (EU) is comprehensive, and there are several compounds in the register or being added to the recommendation list. Recently, European standard methods for analysis have also been issued. The quick analysis of different groups of analytes in one sample requires a number of methods and the simultaneous use of various instruments. The aim of the present study was to develop a method that could analyze several groups of food contaminants: in this case, 266 pesticides, 12 mycotoxins, 14 alkaloid toxins, and 3 Alternaria toxins. The main advantage of the herein described approach over other methods is the simultaneous analysis of tenuazonic acid (TEA) and other relevant food contaminants. The developed method unites the newly published standard methods such as EN 15662:2018, EN 17194:2019, EN 17256:2019, EN 17425:2021, EN 17521:2021, which describes the analysis of both regulated and emerging contaminants. The developed method is based on a QuEChERS sample preparation, followed by LC-MS/MS analysis under alkaline mobile phase conditions. The pH of the aqueous eluent was set to 8.3, which resulted in baseline separation among ergot alkaloids and their corresponding epimers, a symmetric chromatographic peak shape for analyzing TEA and fit-for-purpose sensitivity for MS/MS detection in both positive and negative ionization modes. Those compounds, which possess the corresponding isotopically labeled internal standards (ISTD), allowed for direct quantification by the developed method and no further confirmation was necessary. This was proven by satisfactory analyses of a number of quality control (QC), proficiency test (PT), and validation samples.

Effects of Bacillus amyloliquefaciens XJ-BV2007 on Growth of Alternaria alternata and Production of Tenuazonic Acid.

Large amounts of processing tomato are grown in Xinjiang, China. Tomato black spot disease, caused by Alternaria spp., and the produced alternaria toxins in tomato products are posing risks to human health. In this study, we isolated a rhizospheric bacterium, XJ-BV2007, from tomato (Solanum lycopersicum) fields, which we identified as Bacillus amyloliquefaciens. We found that this bacterium has a strong antagonistic effect against Alternaria alternata and reduces the accumulation of alternaria toxins in tomatoes. According to the antifungal activity of the bacteria-free filtrate, we revealed that B. amyloliquefaciens XJ-BV2007 suppresses A. alternata by the production of antifungal metabolites. Combining semi-preparative high-performance liquid chromatography, we employed UPLC-QTOF-MS analysis and the Oxford cup experiment to find that fengycin plays an important role in inhibiting A. alternata. This paper firstly reported that B. amyloliquefaciens efficiently controls tomato black spot disease and mycotoxins caused by A. alternata. B. amyloliquefaciens XJ-BV2007 may provide an alternative biocontrol strain for the prevention of tomato black spot disease.

Temperature-dependent mycotoxins production investigation in Alternaria infected cherry by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry.

For temperature-dependent Alternaria mycotoxins production analysis, cherry samples were inoculated with Alternaria sp. and incubated at two different temperatures (4 °C and 25 °C). Six Alternaria mycotoxins, including altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), altertoxin-I (ATX-I), tenuazonic acid (TeA), and tentoxin (TEN), in cherries were detected with integrated visible data-processing tools. Maximum concentration of these mycotoxins reached 71,862.2 µg/kg at 25 °C. Notably, considerable amount of TeA (290.4 µg/kg) was detected at 4 °C, which indicated that low temperature is not a safe storage condition for fruits. A total of 102 compounds were detected with a neutral loss of 162.0528 Da, and TeA-glucose was identified in this work. Based on MS/MS cosine similarity, products were verified and annotated with feature based molecular networking (FBMN) in global natural products social networking (GNPS). The results showed Alternaria mycotoxins in cherry samples were mainly demethylation, hydrogenation, and dehydration. This work revealed the production of Alternaria mycotoxins in cherries under different storage temperature, which will provide theoretical basis for the control of mycotoxin contamination in food commodities.

Cytotoxicities of Co-occurring alternariol, alternariol monomethyl ether and tenuazonic acid on human gastric epithelial cells.

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.

Natural Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods Marketed in South Korea by LC-MS/MS.

Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA), are ubiquitous contaminants in agricultural products. A method for the simultaneous determination of these six toxins by ultrahigh performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) with solid phase extraction (SPE) was validated in rice, sesame, tomato, and apple juice matrices. The performance of the method was evaluated in terms of linearity (R2 > 0.999), the limit of detection (0.04-1.67 µg/kg), the limit of quantification (0.12-5.06 µg/kg), recovery (80.0-114.7%), and precision (<17.7%). The validated method was applied to monitor 152 marketed food samples in South Korea, as well as to investigate the co-occurrence and correlation between Alternaria toxins. The mean occurrence levels were 2.77 µg/kg for AOH, 4.36 µg/kg for AME, 0.14 µg/kg for ALT, 0.11 µg/kg for ATX-I, 0.43 µg/kg for TEN, and 104.56 µg/kg for TeA. Mean and extreme (95th percentile) daily dietary exposures of South Koreans to Alternaria toxins were estimated to be 22.93 ng/kg b.w./day and 86.07 ng/kg b.w./day, respectively.

Anti-Epstein-Barr Viral Agents from the Medicinal Herb-Derived Fungus Alternaria alstroemeriae Km2286.

Chromatographic separation on the liquid-state fermented products produced by the fungal strain Alternaria alstroemeriae Km2286 isolated from the littoral medicinal herb Atriplex maximowicziana Makino resulted in the isolation of compounds 1-9. Structures were determined by spectroscopic analysis as four undescribed perylenequinones, altertromins A-D (1-4), along with altertoxin IV (5), altertoxin VIII (6), stemphyperylenol (7), tenuazonic acid (8), and allo-tenuazonic acid (9). Compounds 1-6 exhibited antiviral activities against Epstein-Barr virus (EBV) with EC50 values ranging from 0.17 ± 0.07 to 3.13 ± 0.31 µM and selectivity indices higher than 10. In an anti-neuroinflammatory assay, compounds 1-4, 6, and 7 showed inhibitory activity of nitric oxide production in lipopolysaccharide-induced microglial BV-2 cells, with IC50 values ranging from 0.33 ± 0.04 to 4.08 ± 0.53 µM without significant cytotoxicity. This is the first report to describe perylenequinone-type compounds with potent anti-EBV and anti-neuroinflammatory activities.

Mycotoxin Production and the Relationship between Microbial Diversity and Mycotoxins in Pyrus bretschneideri Rehd cv. Huangguan Pear.

Mycotoxins are generated by a series of fungal pathogens in postharvest fruit, resulting in serious health threat to consumers and great economic loss to the fruit storage industry. The microbial differences between rotten and healthy fruit during storage and their relationship with mycotoxin production have not been fully studied. In this study, differences in microbial diversity between rotten and healthy fruit after 30 days of storage at ambient temperature were investigated using high-throughput sequencing technology in 'Huangguan' pear (Pyrus bretschneideri Rehd cv. Huangguan) harvested from five different producing regions of Hebei province, China. The bacterial genus Gluconobacter was much more abundant in rotten fruit (76.24%) than that in healthy fruit (32.36%). In addition, Komagataeibacter and Acetobacter were also relatively higher in abundance in rotten fruit. In contrast, bacterial genera Pantoea, Alistipes, Muribaculaceae, Lactobacillus, and Ruminococcaceae_UCG were found to be more abundant in healthy fruit. Fungal genera including Botryosphaeria, Colletotrichum, Valsa, Alternaria, Rosellinia, Fusarium, and Trichothecium were found to be abundant in rotten fruit. The results of principal coordinate analysis (PCoA) showed that there were significant differences in the microbial diversity of different regions. PAT (patulin) was detected in all rotten fruit samples, while tenuazonic acid (TeA), alternariol (AOH), and alternariolmonomethyl ether (AME) were only detected in samples collected from one region (Weixian). Canonical correlation analysis (CCA) and Pearson correlation analysis showed that the abundance of Alistipes and Pantoea were negatively correlated with the contents of PAT, suggesting that bacterial genera Alistipes and Pantoea have potential in reducing mycotoxin production in 'Huangguan' pear.

Structure-based ligand design and discovery of novel tenuazonic acid derivatives with high herbicidal activity.

INTRODUCTION: Computer-aided design has become an important tool to develop novel pesticides based on natural lead compounds. Tenuazonic acid (TeA), a typical representative of the natural tetramic acid family, was patented as a potential bioherbicide. However, its herbicidal efficacy is still not up to the ideal standard of commercial products. OBJECTIVES: We aim to find new TeA's derivatives with improved potency. METHODS: Molecular docking was used to build ligand-acceptor interaction models, design and screen new derivatives. Phytotoxicity, oxygen evolution rate, chlorophyll fluorescence and herbicidal efficacy were determined to estimate biological activity of compounds. RESULTS: With the aid of a constructed molecular model of natural lead molecule TeA binding to the QB site in Arabidopsis D1 protein, a series of derivatives differing in the alkyl side chain were designed and ranked according to free energies. All compounds are stabilized by hydrogen bonding interactions between their carbonyl oxygen O2 and D1-Gly256 residue; moreover, hydrogen bond distance is the most important factor for maintaining high binding affinity. Among 54 newly designed derivatives, D6, D13 and D27 with better affinities than TeA were screened out and synthesized to evaluate their photosynthetic inhibitory activity and herbicidal efficacy. Analysis of structure-activity relationship indicated that D6 and D13 with sec-pentyl and sec-hexyl side chains, respectively, were about twice more inhibitory of PSII activity and effective as herbicide than TeA with a sec-butyl side chain. CONCLUSION: D6 and D13 are promising compounds to develop TeA-derived novel PSII herbicides with superior performance.